Note: Descriptions are shown in the official language in which they were submitted.
CA 02492661 2000-02-04
DEMANDES OU BREVETS VOLUIVIINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.
CECI EST LE TOME _____________________ DE 3
NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des
Brevets.
JUMBO APPLICATIONS / PATENTS
THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE
THAN ONE VOLUME.
THIS IS VOLUME I OF 3
NOTE: For additional volumes please contact the Canadian Patent Office.
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NON-STOCHASTIC GENERATION
OF GENETIC VACCINES AND ENZYMES
CONTEWS
1. GENERAL
1.1. FIELD OF THE INVENTION
1.2. BACKGROUND
1.3. SUMMARY OF THE INVENTION
1.4. BRIEF DESCRIPTION OF THE DRAWINGS
2. DETAILED DESCRIPTION OF THE INVENTION
2.1. DEFINITIONS
2.2. GENERAL CONSIDERATIONS & FORMATS FOR RECOMBINATION
2.3. VECTORS USED IN GENETIC VACCINATION
23.1. VIRAL VECTORS
2.3.1.1. ADENO'VIRUSES
23.1.2. ADENO-ASSOCIATED VIRUS (AAV)
23.13. PAPILLOMA VIRUS
23.1.4. RETROVIRUSES
23.2. NON-VIRAL GENETIC VACCINE VECTORS
2.4. MULTICOMPONENT GENETIC VACCINES
2.4.1. VECTOR "AR ",DESIGNED TO PROVIDE OPTIMAL ANTIGEN
RELEASE
2.4.2. VECTOR COMPONENTS "CTL-DC", "CTL-LC" AND "CTL-
IVIM"I DESIGNED FOR OPTIMAL PRODUCTION OF CTLs
2.43. VECTORS "M" DESIGNED FOR OPTIMAL RELEASE OF
IMMUNE MODULATORS
2.4.4. VECTORS "CK", DESIGNED TO DIRECT RELEASE OF
CHEMOICINES
2.4.5. OTHER VECTORS
2.5. SCREENING METHODS
2.5.1. SCREENING FOR VECTOR LONGEVITY OR
TRANSLOCATION TO DESIRED TISSUE
2.5.1.1. SELECTION FOR EXPRESSION OF CELL SURFACE-
LOCALIZED ANTIGEN
2.5.1.2. SELECTION FOR EXPRESSION OF SECRETED
ANTIGEN/CYTOKINE/CHEMOICINE
2.5.2. FLOW CYTOMETRY
2.53. ADDITIONAL IN VITRO SCREENING METHODS
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2.5.4. ANTIGEN LIBRARY IMMUNIZATION
2.5.5. SCREENINGFOR OPTIMAL INDUCTION OF PROTECTIVE
IMMUNITY
2.5.6. SCREENING OF GENETIC VACCINE VECTORS THAT
ACTIVATE HUMAN ANTIGEN-SPECIFIC LYMPHOCYTE
RESPONSES
2.5.7. SCID-HUMAN SKIN MODEL FOR VACCINATION STUDIES
2.5.8. MOUSE MODEL FOR STUDYING THE EFFICIENCY
OFGENETIC VACCINES IN TRANSFECTING HUMAN MUSCLE
CELLS AND INDUCING HUMAN IMMUNE RESPONSES IN VIVO
2.5.9. SCFLEENINGFOR IMPROVED DELIVERY OF VACCINES
2.5.10. ENHANCED ENTRY OF GENETIC VACCINE VECTORS INTO
CELLS
2.6. OPTIMIZATION OF GENETIC VACCINE COMPONENTS
2.6.1. EPISOMAL VECTOR MAINTENANCE
2.6.2. EVOLUTION OF OPTEVIIZED PROMOTERS FOR EXPRESSION
OF AN ANTIGEN
2.6.2.1. CONSTITUTIVE PROMOTERS
2.6.2.2. CELL-SPECIFIC PROMOTERS
2.6.2.3. INDUCIBLE PROMOTERS
2.6.3. EVOLUTION OF BINDING POLYPEPTIDES THAT ENHANCE
SPECIFICITY AND EFFICIENCY OF GENETIC VACCINES
2.6.4. EVOLUTION OF BACTERIOPHAGE VECTORS
2.6.4.1. EVOLUTION OF EFFICIENT DELIVERY OF
BACTERIGPLIAGE VEHICLES BY INHALATION OR ORAL
DELIVE
2.6.4.2. EVOLUTION OF BACTERIOPHAGE VEHICLES FOR
EFFICIENT HOMING TO APCs
2.6.43. EVOLUTION OF BACTERIOPHAGE FOR INVASION
OF APCs
2.6.5. EVOLUTION OF IMPROVED IMMUNOMODULATORY
SEQUENCES
2.6.5.1. IMMUNOSTIMULATORY DNA SEQUENCES
2.6.5.2. CYTOICINES, CH1EMOKINES, AND ACCESSORY
MOLECULES
2.6.53. AGONISTS OR ANTAGONISTS OF CELLULAR
RECEPTORS
2.6.5.4. COSTIMULATORY MOLECULES CAPABLE OF
INHIBITING OR ENHANCING ACTIVATION,
DIFFERENTIATION, OR ANERGY OF ANTIGEN-SPECIFIC T
CELLS
2.6.6. EVOLUTION OF GENETIC VACCINE VECTORS FOR
INCREASED VACCINATION EFFICACY AND EASE OF
VACCINATION
2.6.6.1. TOPICAL APPLICATION OF GENETIC VACCINE
VECTORS
2.6.6.2. ENHANCED ABILITY TO ESCAPE HOST IMMUNE
SYSTEM
2.6.6.3. ENHANCED ANTIVIRAL ACTIVITY
2.6.6.4. EVOLUTION OF VECTORS HAVING INCREASED
COPY NUMBER IN PRODUCTION CELLS
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2.7. OPTIMIZATION OF TRANSPORT AND PRESENTATION OFANTIGENS
2.7.1. PROTEASOMES
2.7.2. ANTIGEN TRANSPORT
2.7.3. CYTOTOXIC T-CELL INDUCING SEQUENCES AND
IMMUNOGENIC AGONIST SEQUENCES
2.8. GENETIC VACCINE PHARMACEUTICAL COMPOSITIONS AND
METHODS OF ADMINISTRATION
2.9. USES OF GENETIC VACCINES
2.9.1. INFECTIOUS DISEASES
2.9.1.1. BACTERIAL PATHOGENS AND TOXINS
2.9.1.2. VIRAL PATHOGENS
2.9.2. INFLAMMATORY AND AUTOIMMUNE DISEASES
2.9.3. ALLERGY AND ASTHMA
2.9.4. CANCER
2.9.5. PARASITES
2.9.6. CONTRACEPTION
2.10. MALARIAL ANTIGENS AND VACCINES
2.10.1. MALARIAL POLYPEPTIDES
2.10.2. MALARIAL NUCLEIC ACIDS AND CELLS CAPABLE OF
EXPRESSING SAME
2.10.3. ANTIBODIES
2.10.4. METHODS OF USE
2.10.4.1. DIAGNOSTIC APPLICATIONS
2.10.4.2. SCREENING APPLICATIONS
2.10.4.3. THERAPEUTIC AND PROPHYLACTIC
APPLICATIONS
2.11. DIRECTED EVOLUTION METHODS
2.11.1. SATURATION MUTAGENESIS
2.11.2. CHIMERIZATIONS
2.11.2.1. "SHUFFLING"
2.11.2.2. EXONUCLEASE-MEDIATED REASSEMBLY
2.11.2.3. NON-STOCHASTIC LIGATION REASSEMBLY
2.11.2.4. END-SELECTION
2.11.3. ADDITIONAL SCREENING METHODS
3. LITERATURE CITED
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1. GENERAL
1.1. FIELD OF THE INVENTION
This invention pertains to the field of genetic vaccines. Specifically, the
invention provides multi-component genetic vaccines that contain components
that are optimized for a particular vaccination goal. In a particular aspect
this
invention provides methods for improving the efficacy of genetic vaccines by
providing materials that facilitate targeting of a genetic vaccine to a
particular
tissue or cell type of interest.
This invention also pertains to the field of modulation of immune
responses such as those induced by genetic vaccines and also pertains to the
field
of methods for developing immunogens that can induce efficient immune
responses against a broad range of antigens.
Thus, the present invention also relates generally to novel proteins, and
fragments thereof, as well as nucleic acids which encode these proteins, and
methods of making and using these proteins in diagnostic, prophylactic and
therapeutic applications. In a particular exemplification, the present
invention
relates to proteins from the Plasmodium falciparum erythrocyte membrane
protein
1 ("PffiMP1") gene family and fragments thereof which are derived from malaria
parasitized erythrocytes. In particular, these proteins are derived from the
erythrocyte membrane protein of Plasmodium falcipanun parasitized
erythrocytes, also termed "PfEMP1". The present invention also provides
nucleic
acids encoding these proteins, which proteins and nucleic acids are associated
with the pathology of malaria infections, and which may be used as vaccines or
other prophylactic treatments for the prevention of malaria infections, and/or
in
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diagnosing and treating the symptoms of patients who suffer from malaria and
associated diseases.
This invention also relates to the field of protein engineering. Specifically,
this invention relates to a directed evolution method for preparing a
polynucleotide encoding a polypeptide. More specifically, this invention
relates
to a method of using mutagenesis to generate a novel polynucleotide encoding a
novel polypeptide, which novel polypeptide is itself an improved biological
molecule &/or contributes to the generation of another improved biological
molecule. More specifically still, this invention relates to a method of
performing
both non-stochastic polynucleotide chimerization and non-stochastic site-
directed
point mutagenesis.
Thus, in one aspect, this invention relates to a method of generating a
progeny set of chimeric polynucleotide(s) by means that are synthetic and non-
stochastic, and where the design of the progeny polynucleotide(s) is derived
by
analysis of a parental set of polynucleotides &/or of the polypeptides
correspondingly encoded by the parental polynucleotides. In another aspect
this
invention relates to a method of performing site-directed mutagenesis using
means that are exhaustive, systematic, and non-stochastic.
Furthermore this invention relates to a step of selecting from among a
generated set of progeny molecules a subset comprised of particularly
desirable
species, including by a process termed end-selection, which subset may then be
screened further. This invention also relates to the step of screening a set
of
polynucleotides for the production of a polypeptide &/or of another expressed
biological molecule having a useful property.
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Novel biological molecules whose manufacture is taught by this invention
include genes, gene pathways, and any molecules whose expression is affected
thereby, including directly encoded polypetides &/or any molecules affected by
such polypeptides. Said novel biological molecules include those that contain
a
carbohydrate, a lipid, a nucleic acid, &/or a protein component, and specific
but
non-limiting examples of these include antibiotics, antibodies, enzymes, and
steroidal and non-steroidal hormones.
In a particular non-limiting aspect, the present invention relates to
enzymes, particularly to thermostable enzymes, and to their generation by
directed evolution. More particularly, the present invention relates to
thermostable enzymes which are stable at high temperatures and which have
improved activity at lower temperatures.
1.2. BACKGROUND
Providing protective immunity even in situations when the pathogens are
poorly characterized or cannotte isolated or cultured in laboratory
environment.
Genetic immunization represents a novel mechanism of inducing
protective Immoral and cellular immunity. Vectors for genetic vaccinations
generally consist of DNA that includes a promoter/enhancer sequence, the gene
of
interest and a polyadenylation/ transcriptional terminator sequence. After
intramuscular or intradennal injection, the gene of interest is expressed,
followed
by recognition of the resulting protein by the cells of the immune system.
Genetic
immunizations provide means to induce protective immunity even in situations
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CA 02492661 2000-02-04
when the pathogens are poorly characterized or cannot be isolated or cultured
in
laboratory environment.
Small improvement in the efficiency of genetic vaccine vectors can result in
dramatic increase if the leveLof immune response
The efficacy of genetic vaccination is often limited by inefficient uptake of
genetic vaccine vectors into cells. Generally, less than 1% of the muscle or
skin
cells at the sites of injections express the gene of interest. Even a small
improvement in the efficiency of genetic vaccine vectors to enter the cells
can
result in a dramatic increase in the level of immune response induced by
genetic
vaccination. A vector typically has to cross many barriers which can result in
only
a very minor fraction of the DNA ever being expressed.
Various limitations to immunogenicity
Limitations to immunogenicity include: loss of vector due to nucleases
present in blood and tissues; inefficient entry of DNA into a cell;
inefficient entry
of DNA into the nucleus of the cell and preference of DNA for other
compartments; lack of DNA stability in the nucleus (factor limiting nuclear
stability may differ from those affecting other cellular and extracellular
compartments), and, for vectors that integrate into the chromosome, the
efficiency
of integration and the site of integration. Moreover, for many applications of
genetic vaccines, it is preferable for the genetic vaccine to enter a
particular target
tissue or cell.
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Thus, a need exists for genetic vaccines that can be targeted to specific cell
and tissue types of interest, and which exhibit an increased ability to enter
the
target cells. The present invention fulfills these and other needs.
Pathways for immune responses induced by gentic vaccines
Elicitation of a desired in vivo response by a genetic vaccine generally
requires multiple cellular processes in a complex sequence. Several potential
pathways exist along which a genetic vaccine can exert its effect on the
mammalian immune system. In one pathway, the genetic vaccine vector enters
cells that are the predominant cell type in the tissue that receives vaccine
(e.g.,
muscle or epithelial cells). These cells express and release the antigen
encoded by
the vector. The vaccine vector can be engineered to have the antigen released
as
an intact protein from living transfected cells (i.e., via a secretion
process) or
directed to a membrane-bound form on the surface of these cells. Antigen can
also
be released from an intracellular compartment of such cells if those cells
die.
The antigen derived from vaccine vector internalization and antigen
expression within the_presinminant cell tnein_the tissue ends up within APC.
which then process the antigen internally to prime MHC Class I and or Class
11, essential steps in activation_of CD4+ T-helper cells and development of
potent specific immune responses.
Extracellular antigen derived from any of these situations interacts with
antigen presenting cells (APC) either by binding to the cell surface
(specifically
via IgM or via other non-immunoglobulin receptors) and subsequent endocytosis
of outer membrane, or by fluid phase micropinocytosis wherein the APC
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CA 02492661 2000-02-04
internalizes extracellular fluid and its contents into an endocytic
compartment.
Interaction with APC may occur before or after partial proteolytic cleavage in
the
extracellular environment. In any case, the antigen derived from vaccine
vector
internalization and antigen expression within the predominant cell type in the
tissue ends up within APC. The APC then process the antigen internally to
prime
MHC Class I and or Class II, essential steps in activation of CD4+ T-helper
cells
(TH1 and/or TH2) and development of potent specific immune responses.
The genetic vaccineplasmicl enters APC and antigen is proteolytically
cleaved in the cell cytoplasm,
In a parallel pathway, the genetic vaccine plasmid enters APC (or the
predominant cell type in the tissue) and, instead of antigen derived from
plasmid
expression being directed to extracellular export, antigen is proteolytically
cleaved in the cell cytoplasm (in a proteasome dependent or independent
process).
Often, intracellular processing in such cells occurs via proteasomal
degradation
into peptides that are recognized by the TAP-1 and TAP-2 proteins and
transported into the lumen of the rough endoplasmic reticulum (RER).
The peptide fragments are transportedinto the RER complex, expressed on
the cell surface; in the presencoof appropriate additional signals, can
differentiate into functional CTLs,
The peptide fragments transported into the RER complex with MHC Class
I. Such antigen fragments are then expressed on the cell surface in
association
with Class I. CD8+ cytotoxic T lymphocytes (CTL) bearing specific T cell
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receptor then recognize the complex and can, in the presence of appropriate
additional signals, differentiate into functional CTLs.
By virtue_of poorly characterized pathways for trafficking of
cytoplasmicallv generated peptides into endosumal compartments, a genetic
vaccine vector can lead to CD4+ T cell stimulation.
In addition, poorly characterized pathways, which are generally not
dominant, exist in APC for trafficking of cytoplasmically generated peptides
into
endosomal compartments where they can end up complexed with MHC Class II,
and thereby act to present antigen peptides to CD4+ TH1 and TH2 cells. Because
activation, proliferation, differentiation and immunoglobulin isotype
switching by
B lymphocytes requires help of CD4+ T cells, antigen presentation in the
context
of MHC Class II molecules is crucial for induction of antigen-specific
antibodies.
By virtue of this pathway, a genetic vaccine vector can lead to CD4+ T cell
stimulation in addition to the dominant CD8+ CTL activation process described
above. This alternative pathway is, however, of little consequence in muscle
cells
where levels of MHC Class II expression are very low or zero.
In this case cytokines are derived not only from processes intrinsic to the
interaction of DNA with cells, or amine cell responses to the antigen, but via
synthesis directed by the vaccine plasmid,
Genetic vaccination can also elicit cytokine release from cells that bind to
or take up DNA. So-called immunostimulatory or adjuvant properties of DNA are
derived from its interaction with cells that internalize DNA. Cytokines can be
released from cells that bind and/or internalize DNA in the absence of gene
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transcription. Separately, interaction of antigen with APC followed by
presentation and specific recognition also stimulates release of cytokines
that have
positive feedback effects on these cells and other immune cells. Chief among
these effects are the direction of CD4+ TH cells to differentiate/ proliferate
preferentially to TH1 or TH2 phenotypes. Furthermore, cytokines released at
the
site of DNA vaccination, regardless of the mechanism of their release,
contribute
to recruitment of other immune cells from the immediate local area and more
distant sites such as draining lymph nodes. In recognition of the importance
of
cytokines in elicitation of a potent immune response, some investigators have
included the genes for one or more cytokines in the DNA vaccine plasmid along
with the target antigen for immunization. In this case cytokines are derived
not
only from processes intrinsic to the interaction of DNA with cells, or
specific cell
responses to the antigen, but via synthesis directed by the vaccine plasmid.
Movement of immune cells from the blood stream and different sites to the
site of immunization and also from the site of immunization to other sites
Immune cells are recruited to the site of immunization from distant sites or
the bloodstream. Specific and non-specific immune responses are then greatly
amplified. Immune cells, including APC, bearing antigen fragments complexed
to MHC molecules or even expressing antigen from uptake of plasmid, also move
from the immunization site to other sites (blood, hence to all tissues; lymph
nodes; spleen) where additional immune recruitment and qualitative and
quantitative development of the immune response ensue.
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Current genetic vaccine vectors employ simple methods for expression of the
desired antiggn wfth_few if any design elements that control the precise
intracellular fate of tke_ antigen or the immunological conseauences of
antigen expression
While these pathways often compete, previously available genetic
vaccines have incorporated all components for influencing each of the pathways
into a single polynucleotide molecule. Because separate cell types are
involved in
the complex interactions required for a potent immune response to a genetic
vaccine vector, mutually incompatible consequences can arise from
administration of a genetic vaccine that is incorporated in a single vector
molecule. Current genetic vaccine vectors employ simple methods for expression
of the desired antigen with few if any design elements that control the
precise
intracellular fate of the antigen or the immunological consequences of antigen
expression. Thus, although genetic vaccines show great promise for vaccine
research and development, the need for major improvements and several severe
limitations of these technologies are apparent.
Existing genetic vaccine vectors have_not_been optimized for human tissue,
providing low and_short-lastin2 expression of the antigen of interest, with
insufficient stability. inducibility. or Levels otexpression in vivo, among
other
things
Largely due to the lack of suitable laboratory models, none of the existing
genetic vaccine vectors have been optimized for human tissues. The existing
genetic vaccine vectors typically provide low and short-lasting expression of
the
antigen of interest, and even large quantities of DNA do not always result in
sufficiently high expression levels to induce protective immune responses.
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Because the mechanisms of the vector entry into the cells and transfer into
the
nucleus are poorly understood, virtually no attempts have been made to improve
these key properties. Similarly, little is known about the mechanisms that
regulate
the maintenance of vector functions, including gene expression. Furthermore,
although there is increasing amount of data indicating that specific sequences
alter
the immunostimulatory properties of the DNA, rational engineering is a very
laborious and time- consuming approach when using this information to generate
vector backbones with improved immunomodulatory properties.
Moreover, presently available genetic vaccine vectors do not provide
sufficient stability, inducibility or levels of expression in vivo to satisfy
the desire
for vaccines which can deliver booster immunization without additional vaccine
administration. Booster immunizations are typically required 3-4 weeks after
the
primary injection with existing genetic vaccines.
Therefore a need exists for improved genetic vaccine vectors and
formulations, and methods for development of such vectors. The present
invention fulfills these and other needs.
The interactions between pathogens and hosts are results of millions of
years of evolution, during which the mammalian immune system has evolved
sophisticated means to counterattack pathogen invasions. However, bacterial
and
viral pathogens have simultaneously gained a number of mechanisms to improve
their virulence and survival in hosts, providing a major challenge for vaccine
research and development despite the powers of modem techniques of molecular
and cellular biology. Similar to the evolution of pathogen antigens, several
cancer
antigens are likely to have gained means to downregulate their immunogenicity
as
a mechanism to escape the host immune system.
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Efficient vaccine development is also hampered by the antigenic
heterogeneity of different strains of pathogens, driven in part by
evolutionary
forces as means for the pathogens to escape immune defenses. Pathogens also
reduce their immunogenicity by selecting antigens that are difficult to
express,
process and/or transport in host cells, thereby reducing the availability of
immunogenic peptides to the molecules initiating and modulating immune
responses. The mechanisms associated with these challenges are complex,
multivariate and rather poorly characterized. Accordingly, a need exists for
vaccines that can induce a protective immune response against bacterial and
viral
pathogens. The present invention fulfills this and other needs.
Antigen processing and presentation is only one factor which determines
the effectiveness of vaccination, whether performed with genetic vaccines or
more
classical methods. Other molecules involved in determining vaccine
effectiveness
include cytokines (interleuldns, interferons, chemokines, hematopoietic growth
factors, tumor necrosis factors and transforming growth factors), which are
small
molecular weight proteins that regulate maturation, activation, proliferation
and
differentiation of the cells of the immune system.
Characteristic features of cytokines are pleiotropy and redundancy; that is,
one cytokine often has several functions and a given function is often
mediated by
more than one cytokine. In addition, several cytokines have additive or
synergistic
effects with other cytokines, and a number of cytokines also share receptor
components.
Due to the complexity of the cytokine networks, studies on the
physiological significance of a given cytokine have been difficult, although
recent
studies using cytokine gene-deficient mice have significantly improved our
understanding on the functions of cytokines in vivo. In addition to soluble
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proteins, several membrane- bound costimulatory molecules play a fundamental
role in the regulation of immune responses. These molecules include CD40,
CD40 ligand, CD27, CD80, CD86 and CD150 (SLAM), and they are typically
expressed on lymphoid cells after activation via antigen recognition or
through
cell-cell interactions.
T helper (TH) cells, key regulators of the immune system, are capable of
producing a large number of different cytokines, and based on their cytokine
synthesis pattern TH cells are divided into two subsets (Paul and Seder (1994)
Cell
76: 241-251). TH1 cells produce high levels of IL-2 and IFN- and no or minimal
levels of IL-4, IL-5 and IL-13. In contrast, TH2 cells produce high levels of
IL-4,
IL-5 and IL-13, and IL-2 and IFN- production is minimal or absent. TH1 cells
activate macrophages, dendritic cells and augment the cytolytic activity of
CD8+
cytotoxic T lymphocytes and NK cells (Id.), whereas TH2 cells provide
efficient
help for B cells and they also mediate allergic responses due to the capacity
of
TH2 cells to induce IgE isotype switching and differentiation of B cells into
IgE
secreting cell (De Vries and Punnonen (1996) In Cytoldne regulation of humoral
immunity: basic and clinical aspects. Eds. Snapper, C.M., John Wiley & Sons,
Ltd., West Sussex, UK, p. 195- 215). The exact mechanisms that regulate the
differentiation of T helper cells are not fully understood, but cytokines are
believed to play a major role. IL-4 has been shown to direct TH2
differentiation,
whereas IL-12 induces development of TH1 cells (Paul and Seder, supra.). In
addition, it has been suggested that membrane bound costimulatory molecules,
such as CD80, CD86 and CD150, can direct TH1 and/or TH2 development, and
the same molecules that regulate TH cell differentiation also affect
activation,
proliferation and differentiation of B cells into Ig-secreting plasma cells
(Cocks et
al. (1995) Nature 376: 260-263; Lenschow etal. (1996) Immunity 5: 285-293;
Punnonen etal. (1993) Proc. Nat'l. Acad. Sci. USA 90: 3730-3734; Punnonen et
a/. (1997)J Exp. Med. 185: 993-1004).
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Studies in both man and mice have demonstrated that the cytokine
synthesis profile of T helper (TH) cells plays a crucial role in determining
the
outcome of several viral, bacterial and parasitic infections. High frequency
of TH1
cells generally protects from lethal infections, whereas dominant TH2
phenotype
often results in disseminated, chronic infections. For example, THI phenotype
is
observed in tuberculoid (resistant) form of leprosy and TH2 phenotype in
lepromatous, multibacillary (susceptible) lesions (Yamamura et al. (199 1)
Science 254: 277-279). Similarly, late-stage HIV patients have TH2-like
cytokine
synthesis profiles, and TH1 phenotype has been proposed to protect from AIDS
(Maggi et aL (1994) J Exp. Med. 180: 489-495). Furthermore, the survival from
meningococcal septicemia is genetically determined based on the capacity of
peripheral blood leukocytes to produce 'TNF- and IL- 10. Individuals from
families with high production of IL- 10 have increased risk of fatal
meningococcal disease, whereas members of families with high TNF-
production were more likely to survive the infection (Westaidotp et al. (1997)
Lancet 349: 170-173).
Cytokine treatments can dramatically influence TH1/TH2 cell
differentiation and macrophage activation, and thereby the outcome of
infectious
diseases. For example, BALB/c mice infected with Leishmania major generally
develop a disseminated fatal disease with a TH2 phenotype, but when treated
with
anti-IL-4 mAbs or IL- 12, the frequency of TH1 cells in the mice increases and
they are able to counteract the pathogen invasion (Chatelain et al. (1992) J
Immunol. 148: 1182-1187). Similarly, IFN- protects mice from lethal Herpes
Simplex Virus (HSV) infection, and MCP-1 prevents lethal infections by
Pseudomonas aeruginosa or Salmonella typhimurium. In addition, cytokine
treatments, such as recombinant IL-2, have shown beneficial effects in human
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CA 02492661 2000-02-04
common variable immunodeficiency (Cunningham-Rundles et al. (1994) N. EngL
J Med. 331: 918-921).
The administration of cytokines and other molecules to modulate immune
responses in a manner most appropriate for treating a particular disease can
provide a significant tool for the treatment of disease. However, presently
available immunomodulator treatments can have several disadvantages, such as
insufficient specific activity, induction of immune responses against, the
immunomodulator that is administered, and other potential problems. Thus, a
need exists for hrununomodulators that exhibit improved properties relative to
those currently available. The present invention fulfills this and other
needs.
Erythrocytes infected with the malaria parasite R falciparum disappear
from the peripheral circulation as they mature from the ring stage to
trophozoites
(Bignami and Bastianeli, Reforma Medica (1889) 6:1334-1335). This
phenomenon, known as sequestration, results from parasitized erythrocyte
("PE")
adherence to microvascular endothelial cells in diverse organs (Miller, Am. J.
Trop. Med. Hyg. (1969) 18:860-865). Sequestration is associated temporally
with
expression of knob protrusions (Leech et Biol.
(1984) 98:1256-1264),
expression of a very large antigenically variant surface protein, called
PfEMP1
(Aley et al., J. Exp. Med. (1984) 160:1585-1590; Leech etal., J. Exp. Med.
(1984)
159:1567-1575; Howard etal., Molec. Biochem. Parasitol. (1988) 27:207-223),
and expression of new receptor properties which mediate adherence to
endothelial
cells (Miller, supra; Udeinya et at., Science (1981) 213:555-557. Endothelial
cell
surface proteins such as CD36, thrombospondin (TSP) and ICAM-1 have been
identified as major host receptors for mature PE. See, e.g., Barnwell et al.,
J.
Immunol. (1985) 135:3494-3497; Roberts etal., Nature (1985) 318:64-66; and
Berendt etal., Nature (1989) 341:57-59.
-17-
CA 02492661 2000-02-04
WO 00/46344 PCT/US00/03086
PE sequestration confers unique advantages for P. falciparum parasites
(Howard and Gilladoga, Blood (1989) 74:2603-2618), but also contributes
directly to the acute pathology of P. falciparum (Miller et al., Science
(1994)
264:1878-1883). Of the four human malarias, only P. falciparum infection is
associated with neurological impairment and cerebral pathology seen
increasingly
in severe drug-resistant malaria (Howard and Gilladoga, supra).
Although the genesis of human cerebral malaria is likely due to a
combination of factors including particular parasite phenotypes (Berendt et
aL,
Parasitol. Today (1994) 10:412- 414), inappropriate immune responses and the
=
phenotype of endothelial cell surface molecules in the cerebral
microvasculature
(Pasloske and Howard, Ann. Rev. Med. (1994) :283-295), adherence of PE to
cerebral blood vessels and consequent local microvascular occlusion is a major
contributing factor. See, e.g., Berendt et al., supra; Patnaik et al., Am. J.
Trop.
Med. Hyg. (1994) 51:642-647.
The capacity of P. falciparum PE to express variant forms of PfEMP1
contributes to the special virulence of this parasite. Variant parasites can
evade
variant-specific antibodies elicited by earlier infections. The P. falciparum
variant
antigens have been defined in vitro using antiserum prepared in Aotus monkeys
infected with individual parasite strains (Howard et al., Molec. Biochem.
Parasitol. (1988) 27:207-223). Antibodies raised against a particular parasite
will
only react by PE agglutination, indirect immuno- fluorescence or
immunoelectronmicroscopy with PE from the same strain (van Schravendijk et
al., Blood (1991) 78:226-236).
Such studies with PE from malaria patients in diverse geographic locations
and sera from the same or different patients confirm that PE in natural
isolates
express variant surface antigens and that individual patients respond to
infection
- 18 -
CA 02492661 2000-02-04
by production of isolate-specific antibodies (Marsh and Howard, Science (1986)
231:150-153; Aguiar et al., Am. J. Trop. Med. Hyg. (1992) 47:621-632; Iqbal et
at., Trans. R. Soc. Trop. Med. Hyg. (1993) 87:583-588. Expression of a variant
antigen on PE has also been demonstrated in several simian, murine and human
5 malaria species, including .P knowlesi (Brown and Brown, Nature (1965)
208:1286-1288; Barnwell et at., Infect. Inunun. (1983) 40:985-994), P.
chabaudi
. .
(Gilks et al., Parasite Immunol. (1990) 12:45-64; Brannan et al., Proc. R.
Soc.
Lond. Biol. Sci. (1994) 256:71- 75), P. fragile (Handunnetti et al., J. Exp.
Mod.
(1987) 165:1269-1283) and P. vivax (Mendis et al., Am. J. Txop. Med. Hyg.
10 (1988) 38:42-46). Laboratory studies with P. lcnowlesi (Brown and Brown,
supra;
Barnwell et al., supra) or P. falciparum (Honunel et at., J. Exp. Med. (1983)
157:1137- 1148) in monkeys and P. chabaudi in mice (Gilks et al., supra)
confirmed that antigenic variation at the PE surface is associated with
prolonged
or chronic infection and the capacity to repeatedly re-establish blood
infection in
15 previously infected animals. Studies with cloned parasites demonstrated
that
antigenic variants can arise with extraordinary frequency, e.g., 2% per
generation
with P. falciparum (Roberts et al., Nature (1992) 357:689-692) and 1.6 % per
generation with P. chabaudi (Brannan et at., supra).
20 PfEMP1 was identified as a 125I-labeled, size diverse protein (200-
350 IcD)
on PE that is lacking from uninfected erythrocytes, and that is also labeled
by
biosynthetic incorporation of radiolabeled amino acids (Leech et at., J. Exp.
Med.
(1984) 159:1567-1575; Howard et al., Molec. Biochem. Parasitol. (1988) 27:207-
223). PfEMP1 is not extracted from PE by neutral detergents such as Triton-
25 100 but is extracted by SDS, suggesting that it is linked
to the erythrocyte
cytoskeleton (Aley et al., J. Med. Exp. (1984) 160:1585-1590). After addition
of
excess Triton-100, PfEMP1 is immunoreactive with appropriate serum
antibodies (Howard et at., (1988), supra). Mild trypsinization of intact PE
rapidly
cleaves PffiMP1 from the cell surface (Leech et al., J. Exp. Mod. (1984)
- 19 -
CA 02492661 2000-02-04
159:1567-1575). PfEMP1 bears antigenically diverse epitopes since it is
inununoprecipitated from particular strains of P. falciparum by antibodies
from
sera of Aotus monkeys infected with the same strain, but not by antibodies
from
animals infected with heterologous strains (Howard et al. (1988), supra).
Knobless PE derived from parasite passage in splenectomized Aotus monkeys
(Aley et al., supra) do not express surface PfEMP1 and are not agglutinated
with
sera from immune individuals or infected monkeys (Howard et al. (1988), supra;
Howard and Gilladoga, Blood (1989) 74:2603-2618). In general, sera that react
with the PE surface by indirect immunofluorescence and antibody-mediated PE
agglutination are the only sera to immunoprecipitate 1251-labeled PfEMP1 from
any particular strain (Howard et al., (1988), supra; van Schntvendijk et al.,
Blood
(1991) 78:226- 236; Biggs et al., J. Immunol. (1992) 149:2047-2054).
The adherence of parasitized erythrocytes to endothelial cells is mediated
by multiple receptor/counter- receptor interactions, including CD36,
thrombospondin and intracellular adhesion molecule-1 (ICAM_1) as the major
host cell receptors (Howard and Gilladoga, Blood (1989) 74:2603- 2618,
Pasloske
and Howard, Ann. Rev. Med. (1994) 45:283-295).
= 20 Vascular
cell adhesion molecule-1 (VCAM-1) and endothelial leukocyte
adhesion molecule-1 (ELAM-1) have also been implicated as additional
endothelial cell receptors that can mediate adherence of a minority of P.
falcipannn PE (Ockenhouse, et al., J. Exp. Med. (1992) 176:1183-1189, and
Howard and Paslaske, supra). The adherence receptors on the surface of PE has
not yet been conclusively identified, and several molecules, including AG 332
(Udomsangpetch, et al., Nature (1989) 338:763-765), modified band 3 (Crandall,
et al., Proc. Nat'l Acad. Sci. USA (1993) 90:4703-4707), Sequestrin
(Ockenhouse,
Proc. Narl Acad. Sci. USA (1991) 88:3175-3179), and PfEMP1 (Howard and
Gilladoga, supra, and Pasloske and Howard, supra), have been proposed as
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CA 02492661 2000-02-04
candidates. Several pieces of indirect evidence have linked expression of
PfEMP1
with the acquisition of new host protein receptor properties on the surface of
PE
(Howard and Gilladoga, supra; Pasloske and Howard, Ann. Rev. Med. (1994)
45:283-295). PE adherence is correlated with the expression of PfEMP1 on the
surface of mature stage PE (Leech, et al., J. Exp. Med. (1984) 159:1567-
1575).
Alterations in the adherence phenotype of the PE selected for in vitro are
usually
associated with the emergence of new forms of PfEMP1 (Biggs, et al., J.
hmnunol. (1992) 149:2047-2054; Roberts, et al., Nature (1992) 357:689- 692).
Mild trypsinization of intact mature PE cleaves the extracellular portion of
PfEMP1 and at the same time, reduces or eliminates PE cytoadherence (Leech, et
al, supra) Previously described antibody mediated blockade or reversal of
cytoadherence is strain specific and is correlated with the ability of the
reacting
sera to agglutinate the corresponding PE and to immunoprecipitate the surface
labeled 125I-PfEMP1 (Howard, et al., Molec. Biochem. Parasitol. (1988) 27:207-
224). Pfalhesin (modified band 3) have been shown to bind CD36 under non-
physiological conditions (Crandall, et al., Exp. Parasitol. (1994) 78:203-
209).
Sequestrin, which appears to be homologous to PfEMP1, extracted with TX100
from lmoble,ss PE, was shown to bind to immobilized CD36 (Ockenhouse, Proc.
Nat'l Acad. Sci. USA (1991) 88:3175-3179).
The complex nature and/or mechanism of malarial antigenic variation, and
its particular virulence has created a need for methods and compositions which
may be useful in the treatment diagnosis and prevention of malaria infections.
The
present invention meets these and other needs.
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CA 02492661 2000-02-04
=
General Overview of_Problems & Considerations in Directed Evolution
The approach, termed directed evolution, of experimentally modifying a
biological molecule towards a desirable property, can be achieved by
mutagenizing
one or more parental molecular templates and by identifying any desirable
molecules among the progeny molecules. Currently available technologies in
directed evolution include methods for achieving stochastic (i.e. random)
mutagenesis and methods for achieving non-stochastic (non-random) mutagenesis.
However, critical shortfalls in both types of methods are identified in the
instant
disclosure.
In prelude, it is noteworthy that it may be argued philosophically by some
that all mutagenesis ¨ if considered from an objective point of view ¨ is non-
stochastic; and furthermore that the entire universe is undergoing a process
that ¨ if
considered from an objective point of view ¨ is non-stochastic. Whether this
is true
is outside of the scope of the instant consideration. Accordingly, as used
herein, the
terms "randomness", "uncertainty", and "unpredictability" have subjective
meanings, and the knowledge, particularly the predictive knowledge, of the
designer
of an experimental process is a determinant of whether the process is
stochastic or
non-stochastic.
By way of illustration, stochastic or random mutagenesis is exemplified by a
situation in which a progenitor molecular template is mutated (modified or
changed)
to yield a set of progeny molecules having mutation(s) that are not
predetermined.
Thus, in an in vitro stochastic mutagenesis reaction, for example, there is
not a
particular predetermined product whose production is intended; rather there is
an
uncertainty ¨ hence randomness ¨ regarding the exact nature of the mutations
achieved, and thus also regarding the products generated. In contrast, non-
stochastic
- 22 -
CA 02492661 2000-02-04
or non-random mutagenesis is exemplified by a situation in which a progenitor
molecular template is mutated (modified or changed) to yield a progeny
molecule
having one or more predetermined mutations. It is appreciated that the
presence of
background products in some quantity is a reality in many reactions where
molecular
processing occurs, and the presence of these background products does not
detract
from the non-stochastic nature of a mutagenesis process having a predetermined
product.
Thus, as used herein, stochastic mutagenesis is manifested in processes such
as error-prone PCR and stochastic shuffling, where the mutation(s) achieved
are
random or not predetermined. In contrast, as used herein, non-stochastic
mutagenesis is manifested in instantly disclosed processes such as gene site-
saturation mutagenesis and synthetic ligation reassembly, where the exact
chemical
structure(s) of the intended product(s) are predetermined.
In brief, existing mutagenesis methods that are non-stochastic have been
serviceable in generating from one to only a very small number of
predetermined
mutations per method application, and thus produce per method application from
one to only a few progeny molecules that have predetermined molecular
structures.
Moreover, the types of mutations currently available by the application of
these non-
stochastic methods are also limited, and thus so are the types of progeny
mutant
molecules.
In contrast, existing methods for mutagenesis that are stochastic in nature
have been serviceable for generating somewhat larger numbers of mutations per
method application ¨ though in a random fashion & usually with a large but
unavoidable contingency of undesirable background products. Thus, these
existing
stochastic methods can produce per method application larger numbers of
progeny
molecules, but that have undetermined molecular structures. The types of
mutations
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CA 02492661 2000-02-04
that can be achieved by application of these current stochastic methods are
also
limited, and thus so are the types of progeny mutant molecules.
It is instantly appreciated that there is a need for the development of non-
stochastic mutagenesis methods that:
1) Can be used to generate large numbers of progeny molecules that have
predetermined molecular structures;
2) Can be used to readily generate more types of mutations;
3) Can produce a correspondingly larger variety of progeny mutant
molecules;
4) Produce decreased unwanted background products;
5) Can be used in a manner that is exhaustive of all possibilities; and
6) Can produce progeny molecules in a systematic & non-repetitive way.
The instant invention satisfies all of these needs.
Directed Evolution Supplements Natural Evolution: Natural
evolution
has been a springboard for directed or experimental evolution, serving both as
a
reservoir of methods to be mimicked and of molecular templates to be
mutagenized.
It is appreciated that, despite its intrinsic process-related limitations (in
the types of
favored &Jor allowed mutagenesis processes) and in its speed, natural
evolution has
had the advantage of having been in process for millions of years & and
throughout
a wide diversity of environments. Accordingly, natural evolution (molecular
mutagenesis and selection in nature) has resulted in the generation of a
wealth of
biological compounds that have shown usefulness in certain commercial
applications.
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CA 02492661 2000-02-04
However, it is instantly appreciated that many unmet commercial needs are
discordant with any evolutionary pressure &/or direction that can be found in
nature.
Moreover, it is often the case that when commercially useful mutations would
otherwise be favored at the molecular level in nature, natural evolution often
overrides the positive selection of such mutations, e.g. when there is a
concurrent
detriment to an organism as a whole (such as when a favorable mutation is
accompanied by a detrimental mutation). Additionally, natural evolution is
often
slow, and favors fidelity in many types of replication. Additionally still,
natural
evolution often favors a path paved mainly by consecutive beneficial mutations
while tending to avoid a plurality of successive negative mutations, even
though
such negative mutations may prove beneficial when combined, or may lead -
through a circuitous route - to final state that is beneficial.
Moreover, natural evolution advances through specific steps (e.g. specific
mutagenesis and selection processes), with avoidance of less favored steps.
For
example, many nucleic acids do not reach close enough proximity to each other
in a
operative environment to undergo chimerization or incorporation or other types
of
transfers from one species to another. Thus, e.g., when sexual intercourse
between 2
particular species is avoided in nature, the chimerization of nucleic acids
from these
2 species is likewise unlikely, with parasites common to the two species
serving as
an example of a very slow passageway for inter-molecular encounters and
exchanges
of DNA. For another example, the generation of a molecule causing self-
toxicity or
self-lethality or sexual sterility is avoided in nature. For yet another
example, the
propagation of a molecule having no particular immediate benefit to an
organism is
prone to vanish in subsequent generations of the organism. Furthermore, e.g.,
there
is no selection pressure for improving the performance of molecule under
conditions
other than those to which it is exposed in its endogenous environment; e.g. a
cytoplasmic molecule is not likely to acquire functional features extending
beyond
what is required of it in the cytoplasm. Furthermore still, the propagation of
a
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CA 02492661 2000-02-04
biological molecule is susceptible to any global detrimental effects ¨ whether
caused
by itself or not ¨ on its ecosystem. These and other characteristics greatly
limit the
types of mutations that can be propagated in nature.
On the other hand, directed (or experimental) evolution ¨ particularly as
provided herein ¨ can be performed much more rapidly and can be directed in a
more streamlined manner at evolving a predetermined molecular property that is
commercially desirable where nature does not provide one &/or is not likely to
provide. Moreover, the directed evolution invention provided herein can
provide
more wide-ranging possibilities in the types of steps that can be used in
mutagenesis
and selection processes. Accordingly, using templates harvested from nature,
the .
instant directed evolution invention provides more wide-ranging possibilities
in the
types of progeny molecules that can be generated and in the speed at which
they can
be generated than often nature itself might be expected to in the same length
of time.
In a particular exemplification, the instantly disclosed directed evolution
methods can be applied iteratively to produce a lineage of progeny molecules
(e.g.
comprising successive sets of progeny molecules) that would not likely be
propagated (i.e., generated &/or selected for) in nature, but that could lead
to the
generation of a desirable downstream mutagenesis product that is not
achievable by
natural evolution.
Previous Directed Evolution Methods Are Suboptimal:
Mutagenesis has been attempted in the past on many occasions, but by
methods that are inadequate for the purpose of this invention. For example,
previously described non-stochastic methods have been serviceable in the
generation
of only very small sets of progeny molecules (comprised often of merely a
solitary
progeny molecule). By way of illustration, a chimeric gene has been made by
joining 2 polynucleotide fragments using compatible sticky ends generated by
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CA 02492661 2000-02-04
restriction enzyme(s), where each fragment is derived from a separate
progenitor (or
parental) molecule. Another example might be the mutagenesis of a single codon
position (i.e. to achieve a codon substitution, addition, or deletion) in a
parental
polynucleotide to generate a single progeny polynucleotide encoding for a
single
site-mutagenized polypeptide.
Previous non-stochastic approaches have only been serviceable in the
generation of but one to a few mutations per method application. Thus, these
previously described non-stochastic methods thus fail to address one of the
central
goals of this invention, namely the exhaustive and non-stochastic
chimerization of
nucleic acids. Accordingly previous non-stochastic methods leave untapped the
vast
majority of the possible point mutations, chimerizations, and combinations
thereof,
which may lead to the generation of highly desirable progeny molecules.
In contrast, stochastic methods have been used to achieve larger numbers of
point mutations and/or chimerizations than non-stochastic methods; for this
reason,
stochastic methods have comprised the predominant approach for generating a
set of
progeny molecules that can be subjected to screening, and amongst which a
desirable molecular species might hopefully be found. However, a major
drawback
of these approaches is that ¨ because of their stochastic nature ¨ there is a
randomness to the exact components in each set of progeny molecules that is
produced. Accordingly, the experimentalist typically has little or no idea
what exact
progeny molecular species are represented in a particular reaction vessel
prior to
their generation. Thus, when a stochastic procedure is repeated (e.g. in a
continuation of a search for a desirable progeny molecule), the re-generation
and re-
screening of previously discarded undesirable molecular species becomes a
labor-
intensive obstruction to progress, causing a circuitous ¨ if not circular ¨
path to be
taken. The drawbacks of such a highly suboptimal path can be addressed by
subjecting a stochastically generated set of progeny molecules to a labor-
incurring
- 27 -
CA 02492661 2000-02-04
process, such as sequencing, in order to identify their molecular structures,
but even
this is an incomplete remedy.
Moreover, current stochastic approaches are highly unsuitable for
comprehensively or exhaustively generating all the molecular species within a
particular grouping of mutations, for attributing functionality to specific
structural
groups in a template molecule (e.g. a specific single amino acid position or a
sequence comprised of two or more amino acids positions), and for categorizing
and
comparing specific grouping of mutations. Accordingly, current stochastic
approaches do not inherently enable the systematic elimination'of unwanted
mutagenesis results, and are, in sum, burdened by too many inherently
shortcomings
to be optimal for directed evolution.
In a non-limiting aspect, the instant invention addresses these problems by
providing non-stochastic means for comprehensively and exhaustively generating
all
possible point mutations in a parental template. In another non-limiting
aspect, the
instant invention further provides means for exhaustively generating all
possible
chimerizations within a group of chimerizations. Thus, the aforementioned
problems are solved by the instant invention.
Specific shortfalls in the technological landscape addressed by this invention
include:
1) Site-directed mutagenesis technologies, such as sloppy or low-fidelity
PCR, are ineffective for systematically achieving at each position (site)
along a
polypeptide sequence the full (saturated) range of possible mutations (i.e.
all
possible amino acid substitutions).
2) There is no relatively easy systematic means for rapidly analyzing the
large amount of information that can be contained in a molecular sequence and
in the
- 28 -
CA 02492661 2000-02-04
potentially colossal number or progeny molecules that could be conceivably
obtained by the directed evolution of one or more molecular templates.
3) There is no relatively easy systematic means for providing
comprehensive empirical information relating structure to function for
molecular
positions.
4) There is no easy systematic means for incorporating internal controls,
such as positive controls, for key steps in certain mutagenesis (e.g.
chimerization)
procedures.
5) There is no easy systematic means to select for a specific group of
progeny molecules, such as full-length chimeras, from among smaller partial
sequences.
An exceedingly large number of possibilities exist for the purposeful and
random combination of amino acids within a protein to produce useful hybrid
proteins and their corresponding biological molecules encoding for these
hybrid
proteins, i.e., DNA, RNA. Accordingly, there is a need to produce and screen a
wide
variety of such hybrid proteins for a desirable utility, particularly widely
varying
random proteins.
The complexity of an active sequence of a biological macromolecule (e.g.,
polynucleotides, polypeptides, and molecules that are comprised of both
polynucleotide and polypeptide sequences) has been called its information
content
("IC"), which has been defined as the resistance of the active protein to
amino acid
sequence variation (calculated from the minimum number of invariable amino
acids
(bits) required to describe a family of related sequences with the same
function).
Proteins that are more sensitive to random mutagenesis have a high information
content.
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CA 02492661 2000-02-04
Molecular biology developments, such as molecular libraries, have
allowed the identification of quite a large number of variable bases, and even
provide ways to select functional sequences from random libraries. In such
libraries, most residues can be varied (although typically not all at the same
time)
depending on compensating changes in the context. Thus, while a 100 amino acid
protein can contain only 2,000 different mutations, 20100 sequence
combinations
are possible.
Information density is the IC per unit length of a sequence. Active sites of
enzymes tend to have a high information density. By contrast, flexible linkers
of
information in enzymes have a low information density.
Current methods in widespread use for creating alternative proteins in a
library format are error-prone polymerase chain reactions and cassette
mutagenesis, in which the specific region to be optimized is replaced with a
synthetically mutagenized oligonucleotide. In both cases, a substantial number
of
mutant sites are generated around certain sites in the original sequence.
Error-prone PCR uses low-fidelity polymerization conditions to introduce
a low level of point mutations randomly over a long sequence. In a mixture of
fragments of unknown sequence, error-prone PCR can be used to mutagenize the
mixture. The published error-prone PCR protocols suffer from a low
processivity
of the polymerase. Therefore, the protocol is unable to result in the random
mutagenesis of an average-sized gene. This inability limits the practical
application of error-prone PCR. Some computer simulations have suggested that
point mutagenesis alone may often be too gradual to allow the large-scale
block
changes that are required for continued and dramatic sequence evolution.
Further,
the published error-prone PCR protocols do not allow for amplification of DNA
fragments greater than 0.5 to 1.0 kb, limiting their practical application. In
-30-
CA 02492661 2000-02-04
addition, repeated cycles of error-prone PCR can lead to an accumulation of
neutral mutations with undesired results, such as affecting a protein's
immunogenicity but not its binding affinity.
In oligonucleotide-directed mutagenesis, a short sequence is replaced with
a synthetically mutagenized oligonucleotide. This approach does not generate
combinations of distant mutations and is thus not combinatorial. The limited
library size relative to the vast sequence length means that many rounds of
selection are unavoidable for protein optimization. Mutagenesis with synthetic
oligonucleotides requires sequencing of individual clones after each selection
round followed by grouping them into families, arbitrarily choosing a single
family, and reducing it to a consensus motif. Such motif is re-synthesized and
reinserted into a single gene followed by additional selection. This step
process
constitutes a statistical bottleneck, is labor intensive, and is not practical
for many
rounds of mutagenesis.
Error-prone PCR and oligonucleotide-directed mutagenesis are thus useful
for single cycles of sequence fine tuning, but rapidly become too limiting
when
they are apPlied for multiple cycles.
Another limitation of error-prone PCR is that the rate of down-mutations
grows with the information content of the sequence. As the information
content,
library size, and mutagenesis rate increase, the balance of down-mutations to
up-
mutations will statistically prevent the selection of further improvements
(statistical ceiling).
In cassette mutagenesis, a sequence block of a single template is typically
replaced by a (partially) randomized sequence. Therefore, the maximum
information content that can be obtained is statistically limited by the
number of
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______________________________________________________________________ _
CA 02492661 2000-02-04
random sequences (i.e., library size). This eliminates other sequence families
which are not currently best, but which may have greater long term potential.
Also, mutagenesis with synthetic oligonucleotides requires sequencing of
individual clones after each selection round. Thus, such an approach is
tedious
and impractical for many rounds of mutagenesis.
Thus, error-prone PCR and cassette mutagenesis are best suited, and have
been widely used, for fine-tuning areas of comparatively low information
content
One apparent exception is the selection of an RNA ligase ribozyme from a
random library using many rounds of amplification by error-prone PCR and
selection.
In nature, the evolution of most organisms occurs by natural selection and
sexual reproduction. Sexual reproduction ensures mixing and combining of the
genes in the offspring of the selected individuals. During meiosis, homologous
chromosomes from the parents line up with one another and cross-over part way
along their length, thus randomly swapping genetic material. Such swapping or
shuffling of the DNA allows organisms to evolve more rapidly.
In recombination, because the inserted sequences were of proven utility in
a homologous environment, the inserted sequences are likely to still have
substantial information content once they are inserted into the new sequence.
Theoretically there are 2,000 different single mutants of a 100 amino acid
protein. However, a protein of 100 amino acids has 20100 possible sequence
combinations, a number which is too large to exhaustively explore by
conventional methods. It would be advantageous to develop a system which
- 32
CA 02492661 2000-02-04
would allow generation and screening of all of these possible combination
mutations.
Some workers in the art have utilized an in vivo site specific
recombination system to generate hybrids of combine light chain antibody genes
with heavy chain antibody genes for expression in a phage system. However,
their system relies on specific sites of recombination and is limited
accordingly.
Simultaneous mutagenesis of antibody CDR regions in single chain antibodies
(scFv) by overlapping extension and PCR have been reported.
=
Others have described a method for generating a large population of
multiple hybrids using random in vivo recombination. This method requires the
recombination of two different libraries of plasmids, each library having a
different selectable marker. The method is limited to a finite number of
recombinations equal to the number of selectable markers existing, and
produces
a concomitant linear increase in the number of marker genes linked to the
selected
=
sequence(s).
In vivo recombination between two homologous, but truncated, insect-
toxin genes on a plasmid has been reported as a method of producing a hybrid
gene. The in vivo recombination of substantially mismatched DNA sequences in
a host cell having defective mismatch repair enzymes, resulting in hybrid
molecule formation has been reported.
-33-
"
CA 02492661 2000-02-04
13. SUMMARY OF THE INVENTION
Directing an immune response so as to achieve an optimal response to
vaccination,
The present invention provides multicomponent genetic vaccines that
include at least one, and preferably two or more genetic vaccine components
that
confer upon the vaccine the ability to direct an immune response so as to
achieve
an optimal response to vaccination. For example, the genetic vaccines can
include
a component that provides optimal antigen release; a component that provides
optimal production of cytotoxic T lymphocytes; a component that directs
release
of an inununomodulator; a component that directs release of a chemokine;
and/or
a component that facilitates binding to, or entry into, a desired turget cell
type. For
example, a component can confer improved binding to, and uptake of, ,
the genetic vaccine to target cells such as antigen- expressing cells or
antigen-
presenting cells.
Additional components include those that direct antigen peptides derived
= from uptake of an antigen into a cell to presentation on either Class I
or Class II
molecules. For example, one can include a component that directs antigen
peptides to presentation on Class I molecules and comprises a polynucleotide
that
encodes a protein such as tapasin, TAP-I and TAP-2, and/or a component that
directs antigen peptides to presentation on Class II molecules and comprises a
polynucleotide that encodes a protein such as an endosomal or lysosomal
protease.
In a particularly preferred aspect, this invention provides a method for
obtaining an immunomodulatory polynucleotide that has an optimized modulatory
effect on an immune response, or encodes a polypeptide that has an optimized
- 34 -
_________ _ _
CA 02492661 2014-08-06
modulatory effect on an immune response, the method comprising: creating a
library of non-stochastically generated progeny polynucleotides from a
parental
polynucleotide set; wherein optimization can thus be achieved using one or
more
of the directed evolution methods as described herein in any combination,
permutation and iterative manner, whereby these directed evolution methods
include the introduction of mutations by non-stochastic methods, including by
"gene site saturation mutagenesis" as described herein; and whereby these
directed evolution methods also include the introduction mutations by non-
stochastic polynucleotide reassembly methods as described herein; including by
synthetic ligation polynucleotide reassembly as described herein.
In a further particular preferred aspect, this invention provides a method of
producing a progeny library comprised of chimerized but pre-determined
polynucleotide sequences each of which is comprised of a pre-determined number
of building block sequences that are assembled in non-random order. The method
comprises: (a) generating a plurality of pre-determined nucleic acid building
block
sequences comprised of sequences delineated by demarcation points selected
from
aligned progenitor nucleic acid sequences, wherein each of said plurality of
pre-
determined nucleic acid building blocks is a double stranded building block
with
two overhangs generated by a method comprising the steps of (i) generating
overlapping blunt-ended amplification products; (ii) melting the blunt-ended
amplification products to produce single stranded nucleic acids; (iii)
annealing the
single stranded nucleic acids to produce a population of double stranded
nucleic
acids; and (iv) selecting for double stranded nucleic acids with two
overhangs;
and (b) non-stochastically assembling said nucleic acid building block
sequences
to produce said chimerized but pre-determined polynucleotide sequences, such
that a designed overall assembly order is achieved for each of said chimerized
but
pre-determined polynucleotide sequence.
In another particularly preferred aspect, this invention provides a method
for obtaining an immunomodulatory polynucleotide that has an optimized
modulatory effect on an immune response, or encodes a polypeptide that has an
optimized modulatory effect on an immune response, the method comprising:
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screening a library of non-stochastically generated progeny
polynucleotides to identify an optimized non-stochastically generated progeny
polynucleotide that has, or encodes a polypeptide that has, a modulatory
effect on
an immune response; wherein the optimized non-stochastically generated
polynucleotide or the polypeptide encoded by the non-stochastically generated
polynucleotide exhibits an enhanced ability to modulate an immune response
compared to a parental polynucleotide from which the library was created.
In another particularly preferred aspect, this invention provides a method
for obtaining an immunomodulatory polynucleotide that has an optimized
modulatory effect on an immune response, or encodes a polypeptide that has an
optimized modulatory effect on an immune response, the method comprising: a)
creating a library of non-stochastically generated progeny polynucleotides
from a
parental polynucleotide set; and b) screening the library to identify an
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CA 02492661 2000-02-04
optimized non-stochastically generated progeny polynucleotide that has, or
encodes a polypeptide that has, a modulatory effect on an immune response
induced by a genetic vaccine vector; wherein the optimized non-stochastically
generated polynucleotide or the polypeptide encoded by the non-stochastically
generated polynucleotide exhibits an enhanced ability to modulate an immune
response compared to a parental polynucleotide from which the library was
created; whereby optimization can thus be achieved using one or more of the
directed evolution methods as described herein in any combination,
permutation,
and iterative manner; whereby these directed evolution methods include the
introduction of point mutations by non-stochastic methods, including by "gene
site saturation mutagenesis" as described herein; and whereby these directed
evolution methods also include the introduction mutations by non-stochastic
polynucleotide reassembly methods as described herein; including by synthetic
ligation polynucleotide reassembly as described herein.
In another particularly preferred aspect, this invention provides a method
for obtaining an immunomodulatory polynucleotide that has, an optimized
expression in a recombinant expression host, the method comprising: creating a
library of non-stochastically generated progeny polynucleotides from a
parental
polynucleotide set; whereby optimization can thus be achieved using one or
more
of the directed evolution methods as described herein in any combination,
permutation and iterative manner; whereby these directed evolution methods
include the introduction of mutations by non-stochastic methods, including by
"gene site saturation mutagenesis" as described herein; and whereby these
directed evolution methods also include the introduction mutations by non-
stochastic polynucleotide reassembly methods as described herein; including by
synthetic ligation polynucleotide reassembly as described herein.
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In another particularly preferred aspect, this invention provides a method
for obtaining an inununomodulatory polynucleotide that has an optimized
expression in a recombinant expression host, the method comprising: screening
a
library of non-stochastically generated progeny polynucleotides to identify an
optimized non-stochastically generated progeny polynucleotide that has an
optimized expression in a recombinant expression host when compared to the
expression of a parental polynucleotide from which the library was created.
In another particularly preferred aspect, this invention provides a method
for obtaining an immunomodulatory polynucleotide that has an optimized
expression in a recombinant expression host, the method comprising: a)
creating a library of non-stochastically generated progeny polynucleotides
from a parental polynucleotide set; and b) screening a library of non-
stochastically generated progeny polynucleotides to identify an optimized non-
stochastically generated progeny polynucleotide that has an optimized
expression
in a recombinant expression host when compared to the expression of a parental
polynucleotide from which the library was created; whereby optimization can
thus
be achieved using one or more of the directed evolution methods as described
herein in any combination, permutation, and iterative manner, whereby these
directed evolution methods include the introduction of point mutations by non-
stochastic methods, including by "gene site saturation mutagenesis" as
described
herein; and whereby these directed evolution methods also include the
introduction mutations by non-stochastic polynucleotide reassembly methods as
described herein; including by synthetic ligation polynucleotide reassembly as
described herein.
In one aspect, this invention provides that the ability to a vaccine, for
example a genetic vaccine, or a component of a vaccine, for example a
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component of a genetic vaccine by optimizing its immunogenicity. Moreover, the
present invention provides for the modification of other properties, including
its:
= Catalysed reaction(s)
= Reaction type
= Natural substrate(s)
= Substrate spectrum
= Product spectrum
= Inhibitor(s)
= Cofactor(s)/prostetic group(s)
= Metal compounds/salts that affect it
= Turnover number
= Specific activity
= Km value
= pH optimum
= pH range
= Temperature optimum
= Temperature range
It is also instantly appreciated that the serviceability of amolecule with an
immunogenic effect can be affected by additional physical properties, which
can
likewise be modified by directed evolution as provided herein, such as how it
is
affected by subjection to:
= Isolation/Preparation
= Purification
= Renattwating conditions (reversibility or retention of activity upon:
heating
and cooling, urea, salts, detergents, pH extremes)
= Crystallization
= pH
= Temperature
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= Oxidation
= Organic solvent(s)
= Miscellaneous storage conditions
Moreover, the instant invention provides for the modification of
molecule's immunogenic properties properties such as
= Exposure to biological compartments (stomach acids, in vivo degradation)
= Expression (e.g.Transcription &/or Translation) level
= tnRNA stability
6 Any in vivo interactions with other cells or biologicals
Method for obtaining_the genetic components,
In some embodiments, one or more of the genetic vaccine components is
obtained by a method that involves: (1) reassembling (&/or subjecting to one
or
more directed evolution methods described herein) at least first and second
forms
of a nucleic acid which can confer a desired property upon a genetic vaccine,
wherein the first and second forms differ from each other in two or more
nucleotides, to produce a library of recombinant nucleic acids; and (2)
screening
the library to identify at least one optimized recombinant component that
exhibits
an enhanced capacity to confer the desired property upon the genetic vaccine.
If
further optimization of the component is desired, the following additional
steps
can be conducted: (3) reassembling (&/or subjecting to one or more directed
evolution methods described herein) at least one optimized recombinant
component with a further form of the nucleic acid, which is the same or
different
from the first and second forms, to produce a further library of recombinant
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CA 02492661 2000-02-04
nucleic acids; (4) screening the further library to identify at least one
further
optimized recombinant component that exhibits an enhanced capacity to confer
the desired property upon the genetic vaccine; and (5) repeating (3) and (4),
as
necessary, until the further optimized recombinant component exhibits a
further
enhanced capacity to confer the desired property upon the genetic vaccine.
Members of a gene family
In some embodiments of the invention, the first form of the nucleic acid is
a first member of a gene family and the second form of the nucleic acid
comprises
a second member of the gene family. Additional forms of the module nucleic
acid
can also be members of the gene family. As an example, the first member of the
gene family can be obtained from a first species of organism and the second
member of the gene family obtained from a second species of organism. If
desired, the optimized recombinant genetic vaccine component obtained by the
methods of the invention can be backcrossed by, for example, reassembling
(&/or
subjecting to one or more directed evolution methods described herein) the
optimized recombinant genetic vaccine component with a molar excess of one or
both of the first and second forms of the substrate nucleic acids to produce a
further library of recombinant genetic vaccine components; and screening the
further library to identify at least one optimized recombinant genetic vaccine
component that further enhances the capability of a genetic vaccine vector
that
includes the component to modulate the immune response.
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Methods of obtaining a genetic vaccine_component that confers upon a
genetic vaccine vector an enhanced ability to replicate in a host cell,
Additional embodiments of the invention provide methods of obtaining a
genetic vaccine component that confers upon a genetic vaccine vector an
enhanced ability to replicate in a host cell. These methods involve creating a
library of recombinant nucleic acids by subjecting to reassembly (&/or one or
more additonal directed evolution methods described herein) at least two forms
of
a polynucleotide that can confer episomal replication upon a vector that
contains
the polynucleotide; introducing into a population of host cells a library of
vectors,
each of which contains a member of the library of recombinant nucleic acids
and
a polynucleotide that encodes a cell surface antigen; propagating the
population of
host cells for multiple generations; and identifying cells which display the
cell
surface antigen on a surface of the cell, wherein cells which display the cell
surface antigen are likely to harbor a vector that contains a recombinant
vector
module which enhances the ability of the vector to replicate episomally.
Obtaining genetic vaccine components that confer upon a vector an enhanced
ability to replicate in a host cell,
Genetic vaccine components that confer upon a vector an enhanced ability
to replicate in a host cell can also be obtained by creating a library of
recombinant
nucleic acids by subjecting to reassembly (&/or one or more additonal directed
evolution methods described herein) at least two forms of a polynucleotide
derived from a human papillomavirus that can confer episomal replication upon
a
vector that contains the polynucleotide; introducing a library of vectors,
each of
which contains a member of the library of recombinant nucleic acids, into a
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CA 02492661 2000-02-04
population of host cells; propagating the host cells for a plurality of
generations;
and identifying cells that contain the vector.
In additional embodiments, the invention provides methods obtaining a
genetic vaccine component that confers upon a vector an enhanced ability to
replicate in a human host cell by creating a library of recombinant nucleic
acids
by subjecting to reassembly (8z/or one or more additonal directed evolution
methods described herein) at least two forms of a polynucleotide that can
confer
episomal replication upon a vector that contains the polynucleotide;
introducing a
library of genetic vaccine vectors, each of which comprises a member of the
library of recombinant nucleic acids, into a test system that mimics a human
immune response; and determining whether the genetic vaccine vector replicates
or induces an immune response in the test system. A suitable test system can
involve human skin cells present as a xenotransplant on skin of an
immunocompromised non-human host animal, for example, or a non-human
mammal that comprises a functional human immune system. Replication in these
systems can be detected by determining whether the animal exhibits an immune
response against the antigen.
The invention also provides methods of obtaining a genetic vaccine
component that confers upon a genetic vaccine an enhanced ability to enter an
antigen- presenting cell. These methods involve creating a library of
recombinant
nucleic acids by subjecting to reassembly (&/or one or more additonal directed
evolution methods described herein) at least two forms of a polynucleotide
that
can confer episomal replication upon a vector that contains the
polynucleotide;
introducing a library of genetic vaccine vectors, each of which comprises a
member of the library of recombinant nucleic acids, into a population of
antigen-
presenting or antigen-processing cells; and determining the percentage of
cells in
the population which contain the nucleic acid vector. Antigen- presenting or
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CA 02492661 2000-02-04
antigen-processing cells of interest include, for example, B cells,
monocytes/macrophages, dendritic cells, Langerhans cells, keratinocytes, and
muscle cells.
The present invention provides methods of obtaining a polynucleotide that
has a modulatory effect on an immune response that is induced by a genetic
vaccine, either directly (i.e., as an immunomodulatory polynucleotide) or
indirectly (i.e., upon translation of the polynucleotide to create an
iznmunomodulatory polypeptide. The methods of the invention involve: creating
a
library of experimentally generated (in vitro &/or in vivo) polynucleotides;
and
screening the library to identify at least one optimized experimentally
generated
(in vitro &/or in vivo) polynucleotide that exhibits, either by itself or
through the
encoded polypeptide, an enhanced ability to modulate an immune response than a
form of the nucleic acid from which the library was created. Examples include,
for example, CpG-rich polynucleotide sequences, polynucleotide sequences that
encode a costimulator (e.g., B7-1, B7-2, CD1, CD40, CD154 (ligand for CD40),
CD150 (SLAM), or a cytokine. The screening step used in these methods can
include, for example, introducing genetic vaccine vectors which comprise the
library of recombinant nucleic acids into a cell, and identifying cells which
exhibit an increased ability to modulate an immune response of interest or
increased ability to express an immunomodulatory molecule. For example, a
library of recombinant cytokine-encoding nucleic acids can be screened by
testing
the ability of cytokines encoded by the nucleic acids to activate cells which
contain a receptor for the cytokine. The receptor for the cytokine can be
native to
the cell, or can be expressed from a heterologous nucleic acid that encodes
the
cytokine receptor. For example, the optimized costimulators can be tested to
identify those for which the cells or culture medium are capable of inducing a
predominantly TH2 immune response, or a predominantly TH1 immune response.
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WO 00/46344
PCT/US00/03086
In some embodiments, the polynucleotide that has a modulatory effect on
an immune response is obtained by: (1) reassembling (&/or subjecting to one or
more directed evolution methods described herein) at least first and second
forms
of a nucleic acid that is, or encodes a molecule that is, involved in
modulating an
immune response, wherein the first and second forms differ from each other in
two or more nucleotides, to produce a library of experimentally generated (in
vitro &/or in vivo) polynucleotides; and (2) screening the library to identify
at
least one optimized experimentally generated (in vitro &/or in vivo)
polynucleotide that exhibits, either by itself or through the encoded
polypeptide,
an enhanced ability to modulate an immune response than a form of the nucleic
acid from which the library was created. If additional optimization is
desired, the
method can further involve: (3) reassembling (&/or subjecting to one or more
directed evolution methods described herein) at least one optimized
experimentally generated (in vitro &/or in vivo) polynucleotide with a further
form of the nucleic acid, which is the same or different from the first and
second
forms, to produce a further library of experimentally generated (in vitro &/or
in
vivo) polynucleotides; (4) screening, the further library to identify at least
one
further optimized experimentally generated (in vitro &/or in vivo)
polynucleotide
that exhibits an enhanced ability to modulate an immune response than a form
of
the nucleic acid from which the library was created.; and (5) repeating (3)
and (4),
as necessary, until the further optimized experimentally generated (in vitro
&/or in
vivo) polynucleotide exhibits an further enhanced ability to modulate an
immune
response than a form of the nucleic acid from which the library was created.
In some embodiments of the invention, the library of experimentally
generated (in vitro &/or in vivo) polynucleotides is screened by: expressing
the
experimentally generated (in vitro &/or in vivo) polynucleotides so that the
encoded peptides or polypeptide,s are produced as fusions with a protein
displayed
on the surface of a replicable genetic package; contacting the replicable
genetic
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CA 02492661 2000-02-04
packages with a plurality of cells that display the receptor; and identifying
cells
that exhibit a modulation of an immune response mediated by the receptor.
The invention also provides methods for obtaining a polynucleotide that
encodes an accessory molecule that improves the transport or presentation of
antigens by a cell. These methods involve creating a library of experimentally
generated (in vitro &/or in vivo) polynucleotides by subjecting to reassembly
(&/or one or more additonal directed evolution methods described herein)
nucleic
acids that encode all or part of the accessory molecule; and screening the
library
to identify an optimized experimentally generated (in vitro &lot in vivo)
polynucleotide that encodes a recombinant accessory molecule that confers upon
a cell an increased or decreased ability to transport or present an antigen on
a
surface of the cell compared to an accessory molecule encoded by the non-
recombinant nucleic acids. In some embodiments, the screening step involves:
introducing the library of experimentally generated (in vitro &/or in vivo)
polynucleotides into a genetic vaccine vector that encodes an antigen to form
a
library of vectors; introducing the library of vectors into mammalian cells;
and
identifying mammalian cells that exhibit increased or decreased immunogenicity
to the antigen.
In some embodiments of the invention, the cytokine that is optimized is
interleukin-12 and the screening is performed by growing mammalian cells which
contain the genetic vaccine vector in a culture medium, and detecting whether
T
cell proliferation or T cell differentiation is induced by contact with the
culture
medium. In another embodiment, the cytolcine is interferon- and the screening
is
performed by expressing the recombinant vector module as a fusion protein
which
is displayed on the surface of a bacteriophage to form a phage display
library, and
identifying phage library members which are capable of inhibiting
proliferation of
a B cell line. Another embodiment utilizes B7-1 (CD80) or B7-2 (CD86) as the
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CA 02492661 2000-02-04
costimulator and the cell or culture medium is tested for ability to modulate
an
immune response.
The invention provides methods of using stochastic (e.g. polynucleotide
shuffling & interrupted synthesis) and non-stochastic polynucleotide
reassembly
to obtain optimized recombinant vector modules that encode cytoldnes and other
costimulators that exhibit reduced immunogenicity compared to a corresponding
polypeptide encoded by anon-optimized vector module. The reduced
immunogenicity can be detected by introducing a cytokine or costimulator
encoded by the recombinant vector module into a mammal and determining
whether an immune response is induced against the cytokine.
The invention also provides methods of obtaining optimized
inununomodulatory sequences that encode a cytokine antagonist. For example,
suitable cytokine agonists include a soluble cytokine receptor and a
transmembrane cytokine receptor having, a defective signal sequence. Examples
include sIL-10R and sIL- 4R, and the like.
The present invention provides methods for obtaining a cell-specific
binding molecule that is useful for increasing uptake or specificity of a
genetic
vaccine to a target cell. The methods involve: creating a library of
experimentally
generated (in vitro &/or in vivo) polynucleotides that by reassembling (8/Jor
subjecting to one or more directed evolution methods described herein) a
nucleic
acid that encodes a polypeptide that comprises a nucleic acid binding domain
and
a nucleic acid that encodes a polypeptide that comprises a cell-specific
binding
domain; and screening the library to identify a experimentally generated (in
vitro
&/or in vivo) polynucleotide that encodes a binding molecule that can bind to
a
nucleic acid and to a cell-specific receptor. Target cells of particular
interest
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include antigen-presenting and antigen-processing cells, such as muscle cells,
monocytes, dendritic cells, B cells, Langerhans cells, keratinocytes, and M-
cells.
In some embodiments, the methods of the invention for obtaining a cell-
specific binding moiety useful for increasing uptake or specificity of a
genetic
vaccine to a target cell involve:
(1) reassembling (&/or subjecting to one or more directed evolution methods
described herein) at least first and second forms of a nucleic acid which
comprises a polynucleotide that encodes a nucleic acid binding domain and
at least first and second forms of a nucleic acid which comprises a cell-
specific ligand that specifically binds to a protein on the surface of a cell
of
interest, wherein the first and second forms differ from each other in two or
more nucleotides, to produce a library of recombinant binding moiety-
encoding nucleic acids;
(2) transfecting into a population of host cells a library of vectors, each of
which comprises: a) a binding site specific for the nucleic acid binding
domain and b) a member of the library of recombinant binding moiety-
encoding nucleic acids, wherein the recombinant binding moiety is
expressed and binds to the binding site to form a vector- binding moiety
complex;
(3) lysing the host cells under conditions that do not disrupt binding of the
vector-binding moiety complex;
(4) contacting the vector- binding moiety complex with a target cell of
interest; and
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(5) identifying target cells that contain a vector and isolating the optimized
recombinant cell-specific binding moiety nucleic acids from these target
cells.
If further optimization is desired, the methods can further involve:
(6) reassembling (8z/or subjecting to one or more directed evolution methods
described herein) at least one optimized recombinant binding moiety-
encoding nucleic acid with a further form of the polynucleotide that encodes
a nucleic acid binding domain and/or a further form of the polynucleotide
that encodes a cell-specific ligand, which are the same or different from the
first and second forms, to produce a further library of recombinant binding
moiety-encoding nucleic acids;
(7) transfecting into a population of host cells a library of vectors that
comprise: a) a binding site specific for the nucleic acid binding domain and
2) the recombinant binding moiety-encoding nucleic acids, wherein the
recombinant binding moiety is expressed and binds to the binding site to
form a vector- binding moiety complex;
(8) lysing the host cells under conditions that do not disrupt binding of the
vector-binding moiety complex;
(9) contacting the vector-binding moiety complex with a target cell of
interest
and identifying target cells that contain the vector; and
(10) isolating the optimized recombinant binding moiety nucleic acids from
the target cells which contain the vector; and
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(11) repeating (6) through (10), as necessary, to obtain a further optimized
cell-specific binding moiety useful for increasing uptake or specificity of a
genetic vaccine vector to a target cell.
The invention also provides cell-specific recombinant binding moieties
produced by expressing in a host cell an optimized recombinant binding moiety-
encoding nucleic acid obtained by the methods of the invention.
In another embodiment, the invention provides genetic vaccines that
include:
a) an optimized recombinant binding moiety that comprises a nucleic acid
binding
domain and a cell-specific ligand, and b) a polynticleotide sequence that
comprises a binding site, wherein the nucleic acid binding domain is capable
of
specifically binding to the binding site.
A further embodiment of the invention provides methods for obtaining an
optimized cell-specific binding moiety useful for increasing uptake, efficacy,
or
specificity of a genetic vaccine for a target cell by:
(1) reassembling (8:for subjecting to one or more directed evolution methods
described herein) at least first and second forms of a nucleic acid that
comprises a polynucleotide which encodes a non-toxic receptor binding
moiety-of an enterotoxin or other toxin, wherein the first and second forms
differ from each other in two or more nucleotides, to produce a library of
recombinant nucleic acids;
(2) transfecting vectors that contain the library of nucleic acids into a
population of host cells, wherein the nucleic acids are expressed to form
recombinant cell- specific binding moiety polypeptides;
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(3) contacting the recombinant cell-specific binding moiety polypeptides with
a cell surface receptor of a target cell; and
(4) determining which recombinant cell-specific binding moiety polypeptides
exhibit enhanced ability to bind to the target cell. Methods of enhancing
uptake of a genetic vaccine vector by a target cell by coating the genetic
vaccine vector with an optimized recombinant cell-specific binding moiety
produced by these methods are also provided by the invention.
The present invention also provides methods for evolving a vaccine
delivery vehicle, genetic vaccine vector, or a vector component to obtain an
optimized delivery vehicle or component that has, or confers upOn a vector,
enhanced ability to enter a selected mammalian tissue upon administration to a
mammal. These methods involve:
(I) reassembling (&/or subjecting to one or more directed evolution methods
described herein) members of a pool of polynucleotides to produce a library of
experimentally generated (in vitro &/or in vivo) polynucleotides;
(2) administering to a test animal a library of replicable genetic packages,
each of which comprises a member of the library of experimentally generated
(in vitro &/or in vivo) polynucleotides operably linked to a polynucleotide
that encodes a display polypeptide, wherein the experimentally generated (in
vitro &/or in vivo) polynucleotide and the display polypeptide are expressed
as a fusion protein which is displayed on the surface of the replicable
genetic package; and
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(3) recovering replicable genetic packages that are present in the selected
tissue of the test animal at a suitable time after administration, wherein
recovered replicable genetic packages have enhanced ability to enter the
selected mammalian tissue upon administration to the mammal.
If further optimization of the delivery vehicle is desired, the methods of
the invention further involve:
(4) reassembling (&/or subjecting to one or more directed evolution methods
described herein) a nucleic acid that comprises at least one experimentally
generated (in vitro Lidor in vivo) polynucleotide obtained from a replicable
genetic package recovered from the selected tissue with a further pool of
polynucleotides to produce a further library of experimentally generated (in
vitro 8,-/or in vivo) polynucleotides;
(5) administering to a test animal a library of replicable genetic packages,
each of which comprises a member of the further library of experimentally
generated (in vitro &/or in vivo) polynucleotides operably linked to a
polynucleotide that encodes a display polypeptide, wherein the experimentally
generated (in vitro &kr in vivo) polynucleotide and the display polypeptide
are expressed as a fusion protein which is displayed on the surface of
the replicable genetic package;
(6) recovering replicable genetic packages that are present in the selected
tissue of the test animal at a suitable time after administration; and
(7) repeating (4) through (6), as necessary, to obtain a further optimized
recombinant delivery vehicle that exhibits further enhanced ability to enter a
selected mammalian tissue upon administration to a mammal. Methods of
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administration that are of particular interest include, for example, oral,
topical,
and inhalation. Where the administration is intravenous, mammalian tissues of
interest include, for example, lymph node and spleen.
In another embodiment, the invention provides methods for evolving a
vaccine delivery vehicle, genetic vaccine vector, or a vector component to
obtain
an optimized delivery vehicle or component to obtain an optimized delivery
vehicle or vector component that has, or confers upon a vector containing the
component, enhanced specificity for antigen-presenting cells by:
(1) reassembling (&/or subjecting to one or more directed evolution methods
described herein) members of a pool of polynucleotides to produce a
library of experimentally generated (in vitro &/or in vivo) ),
polynucleotides;
(2) producing a library of replicable genetic packages, each of which
comprises a member of the library of experimentally generated (in vitro
8r.lor in vivo) polynucleotides operably linked to a polynucleotide that
encodes a display polypeptide, wherein the experimentally generated (in
vitro &for in vivo) polynucleotide and the display polypeptide are
expressed as a fusion protein which is displayed on the surface of
the replicable genetic package;
(3) contacting the library of recombinant replicable genetic packages with a
non-APC to remove replicable genetic packages that display non-APC-
specific fusion polypeptides; and
(4) contacting the recombinant replicable genetic packages that did not bind
to
the non-APC with an APC and recovering those that bind to the APC,
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wherein the recovered replicable genetic packages are capable of
specifically binding to APCs.
In an additional embodiment, the invention provides methods for evolving
a vaccine delivery vehicle, genetic vaccine vector, or a vector component to
obtain an optimized delivery vehicle or component to obtain an optimized
delivery vehicle or vector component that has, or confers upon a vector
containing
the component, an enhanced ability to enter a target cell by:
(1) reassembling (&/or subjecting to one or more directed evolution methods
described herein) at least first and second forms of a nucleic acid which
encodes an invasin polypeptide, wherein the first and second forms differ
from each other in two or more nucleotides, to produce a library of
recombinant invasin nucleic acids;
(2) producing a library of recombinant bacteriophage; each of which displays
on the bacteriophage surface a fusion polypeptide encoded by a chimeric
gene that comprises a recombinant invasin nucleic acid operably linked to a
polynucleotide that encodes a display polypeptide;
(3) contacting the library of recombinant bacteriophage with a population of
target cells;
(4) removing unbound phage and phage which is bound to the surface of the
target cells; and
(5) recovering phage which are present within the target cells, wherein the
recovered phage are enriched for phage that have enhanced ability to enter
the target cells.
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In some embodiments, the optimized recombinant genetic vaccine vectors,
delivery vehicles, or vector components obtained using these methods exhibit
improved ability to enter an antigen presenting cell. These methods can
involve
washing the cells after the transfection step to remove vectors which did not
enter
an antigen presenting cell.; culturing the cells for a predetermined time
after
transfection; lysing the antigen presenting cells; and isolating the optimized
recombinant genetic vaccine vector from the cell lysate.
Antigen presentinff cells that contain an optimized recombinant
genetic vaccine vectors_can_beidentified_by. for example. detecting expression
of a mnrker gene that is inclmledin_the vectors,
The invention also provides methods of evolving a bacteriophage-derived
vaccine delivery vehicle to obtain a delivery vehicle having enhanced ability
to
enter a target cell. These methods involve the steps of.
(1) reassembling (&/or subjecting to one or more directed evolution methods
described herein) at least first and second forms of a nucleic acid which
encodes an invasin polypeptide, wherein the first and second forms differ
from each other in two or more nucleotides, to produce a library of
recombinant invasin nucleic acids;
(2) producing a library of recombinant bacteriophage, each of which displays
on the bacteriophage surface a fusion polypeptide encoded by a chimeric
gene that comprises a recombinant invasin nucleic acid operably linked to a
polynucleotide that encodes a display polypeptide;
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(3) contacting the library of recombinant bacteriophage with a population of
target cells;
(4) removing unbound phage and phage which is bound to the surface of the
target cells; and
(5) recovering phage which are present within the target cells, wherein the
recovered phage are enriched for phage that have enhanced ability to enter
the target cells. Again, if further optimization is desired, the methods can
include the further steps of.
(6) reassembling (&/or subjecting to one or more directed evolution methods
described herein) a nucleic acid which comprises at least one recombinant
invasin nucleic acid obtained from a bacteriophage which is recovered from
a target cell with a further pool of polynucleotides to produce a further
library of recombinant invasin polynucleotides;
(7) producing a further library of recombinant bacteriophage, each of which
displays on the bacteriophage surface a fusion polypeptide encoded by a
chimeric gene that comprises a recombinant invasin nucleic acid operably
linked to a polynucleotide that encodes a display polypeptide;
(8) contacting the library of recombinant bacteriophage with a population of
target cells;
(9) removing unbound phage and phage which is bound to the surface of the
target cells; and
(10) recovering phage which are present within the target cells; and
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(11) repeating (6) through (10), as necessary, to obtain a further optimized
recombinant delivery vehicle which exhibits further have enhanced ability to
enter the target cells.
In some embodiments the methods of evolving a bacteriophage-derived
vaccine delivery vehicle to obtain a delivery vehicle having enhanced ability
to
enter a target cell can include the additional steps of.
(12) inserting into the optimized recombinant delivery vehiele a
polynucleotide which encodes an antigen of interest, wherein the antigen of
interest is expressed as a fusion polypeptide which comprises a second
display polypeptide;
(13) administering the delivery vehicle to a test animal; and (14) determining
whether the delivery vehicle is capable of inducing a CTL response in the
test animal.
Alternatively, the following steps can be employed:
(12) inserting into the optimized recombinant delivery vehicle a
polynucleotide which encodes an antigen of interest, wherein the antigen of
interest is expressed as a fusion polypeptide which comprises a second
display polypeptide;
(13) administering the delivery vehicle to a test animal; and
(14) determining whether the delivery vehicle is capable of inducing
neutralizing antibodies against a pathogen which comprises the antigen of
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interest. An example of a target cell of interest for these methods is an
antigen-presenting cell.
The present invention provides recombinant multivalent antigenic
polypeptides that include a first antigenic determinant from a first disease-
associated polypeptide and at least a second antigenic determinant from a
second
disease-associated polypeptide. The disease-associated polypeptides can be
selected from the group consisting of cancer antigens, antigens associated
with
autoinununity disorders, antigens associated with inflammatory conditions,
antigens associated with allergic reactions, antigens associated with
infectious =
agents, and other antigens that are associated with a disease condition.
In another embodiment, the invention provides a recombinant antigen
library that contains recombinant nucleic acids that encode antigenic
polypeptides. The libraries are typically obtained by reassembling (&/or
subjecting to one or more directed evolution methods described herein), at
least
first and second forms of a nucleic acid which includes a polynucleotide
sequence
that encodes a disease-associated antigenic polypeptide, wherein the first and
second forms differ from each other in two or more nucleotides, to produce a
library of recombinant nucleic acids.
Another embodiment of the invention provides methods of obtaining a
polynucleotide that encodes a recombinant antigen having improved ability to
induce an immune response to a disease condition. These methods involve:
(1) reassembling (&/or subjecting to one or more directed evolution methods
described herein) at least first and second forms of a nucleic acid which
comprises a polynucleotide sequence that encodes an antigenic polypeptide
that is associated with the disease condition, wherein the first and second
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forms differ from each other in two or more nucleotides, to produce a library
of recombinant nucleic acids; and
(2) screening the library to identify at least one optimized recombinant
nucleic
acid that encodes an optimized recombinant antigenic polypeptide that has
improved ability to induce an immune response to the disease condition.
These methods optionally further involve:
(3) reassembling (&/or subjecting to one or more directed evolution methods
described herein) at least one optimized recombinant nucleic acid with a
further form of the nucleic acid, which is the same or different from the
first
and second forms, to produce a further library of recombinant nucleic acids;
(4) screening the further library to identify at least one further optimized
recombinant nucleic acid that encodes a polypeptide that has improved
ability to induce an immune response to the disease condition; and
(5) repeating (3) and (4), as necessary, until the further optimized
recombinant
nucleic acid encodes a polypeptide that has improved ability to induce an
immune response to the disease condition.
In some embodiments, the optimized recombinant nucleic acid encodes a
multivalent antigenic polypeptide and the screening is accomplished by
expressing the library of recombinant nucleic acids in a phage display
expression
vector such that the recombinant antigen is expressed as a fusion protein with
a
phage polypeptide that is displayed on a phage particle surface; contacting
the
phage with a first antibody that is specific for a first serotype of the
pathogenic
agent and selecting those phage that bind to the first antibody; and
contacting
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those phage that bind to the first antibody with a second antibody that is
specific
for a second serotype of the pathogenic agent and selecting those phage that
bind
to the second antibody; wherein those phage that bind to the first antibody
and the
second antibody express a multivalent antigenic polypeptide.
The invention also providec methods of obtaining a recombinant viral
vector which has an enhancedLability to induce an antiviral response in a
cell,
10 Methods of obtainingn recombinant genetic vaccine component that confers
upon a genetic vaccine an enhanced ability to induce a desired immune
response in a mammal
In additional embodiments, the invention provides methods of obtaining a
15 recombinant genetic vaccine component that confers upon a genetic
vaccine an
enhanced ability to induce a desired immune response in a mammal. These
methods involve: (1) reassembling (&/or subjecting to one or more directed
evolution methods described herein) at least first and second forms of a
nucleic
acid which comprise a genetic vaccine vector, wherein the first and second
forms
20 differ from each other in two or more nucleotides, to produce a library
of
recombinant genetic vaccine vectors; (2) transfecting the library of
recombinant
vaccine vectors into a population of mammalian cells selected from the group
consisting of peripheral blood T cells, T cell clones, freshly isolated
monocytes/macrophages and dendritic cells; (3) staining the cells for the
presence
25 of one or more cytokines and identifying cells which exhibit
a cytokine staining
pattern indicative of the desired immune response; and (4) obtaining
recombinant
vaccine vector nucleic acid sequences from the cells which exhibit the desired
cytokine staining pattern.
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Methods of improrkg the ability of a genetic vaccine vector to modulate an
Immune response,
Also provided by the invention are methods of improving the ability of a
genetic vaccine vector to modulate an immune response by: (1) reassembling
(&/or subjecting to one or more directed evolution methods described herein)
at
least first and second forms of a nucleic acid which comprise a genetic
vaccine
vector, wherein the first and second forms differ from each other in two or
more
nucleotides, to produce a library of recombinant genetic vaccine vectors; (2)
transfecting the library of recombinant genetic vaccine vectors into a
population
of antigen presenting cells; and (3) isolating from the cells optimized
recombinant
genetic vaccine vectors which exhibit enhanced ability to modulate a desired
immune response.
Methods of obtaining a recombinant genetic vaccine vector thatlas an
enhanced ability to induce a desired immune response in a mammal upon
administration to the skin of the mammal,
Another embodiment of the invention provides methods of obtaining a
recombinant genetic vaccine vector that has an enhanced ability to induce a
desired immune response in a mammal upon administration to the skin of the
mammal. These methods involve: (1) reassembling (&/or subjecting to one or
more directed evolution methods described herein) at least first and second
forms
of a nucleic acid which comprise a genetic vaccine vector, wherein the first
and
second forms differ from each other in two or more nucleotides, to produce a
library of recombinant genetic vaccine vectors; (2) topically applying the
library
of recombinant genetic vaccine vectors to skin of a mammal; (3) identifying
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vectors that induce an immune response; and (4) recovering genetic vaccine
vectors from the skin cells which contain vectors that induce an immune
response.
Methods of inducing an immune response in a mammal by_tunicallv annlvinp
to skin of the mammaLa2enetic vaccine vector. wherein the genetic vaccine
vector is optimizedlorionical application through use of stochastic (e.g,
polynucleotide shuffline & interrupted miltfail) mid_ non-stochastic
polynucleotide reassembly,
The invention also provides methods of inducing an immune response in a
mammal by topically applying to skin of the mammal a genetic vaccine vector,
wherein the genetic vaccine vector is optimized for topical application
through
use of stochastic (e.g. polynucletide shuffling & interrupted synthesis) and
non-
stochastic polynucleotide reassembly. In some embodiments, the genetic vaccine
is administered as a formulation selected from the group consisting of a
transdermal patch, a cream, naked DNA, a mixture of DNA and a transfection-
enhancing agent. Suitable transfection-enhancing agents include one or more
agents selected from the group consisting of a lipid, a liposome, a protease,
and a
lipase.
Alternatively, or in addition, the genetic vaccine can be administered after
pretreatment of the skin by abrasion or hair removal.
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Methods of obtaining an optimizethEenetic vaccine component that confers
upon a genetic vaccine containing the component an enhanced ability to
Induce or inhibit apoptosis of a cell into which the vaccine is introduced,
In another embodiment, the invention provides methods of obtaining an
optimized genetic vaccine component that confers upon a genetic vaccine
containing the component an enhanced ability to induce or inhibit apoptosis of
a
cell into which the vaccine is introduced. These methods involve: (1)
reassembling (&/or subjecting to one or more directed evolution methods
described herein) at least first and second forms of a nucleic acid which
comprise
a nucleic acid that encodes an apoptosis- modulating polypeptide, wherein the
first and second forms differ from each other in two or more nucleotides, to
produce a library of recombinant nucleic acids; (2) transfecting the library
of
recombinant nucleic acids into a population of mammalian cells; (3) staining
the
cells for the presence of a cell membrane change which is indicative of
apoptosis
initiation; and (4) obtaining recombinant apoptosis-modulating genetic vaccine
components from the cells which exhibit the desired apoptotic membrane
changes.
Methods of obtaining a geneti vaccine component that confers upon a
genetic vaccine reduced susceptibility to a CTL immune response in a host
mammal.
Other embodiments of the invention provide methods of obtaining a
genetic vaccine component that confers upon a genetic vaccine reduced
susceptibility to a CTL immune response in a host mammal. These methods can
involve: (1) reassembling (&/or subjecting to one or more directed evolution
methods described herein) at least first and second forms of a nucleic acid
which
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comprises a gene that encodes an inhibitor of a CTL immune response, wherein
the first and second forms differ from each other in two or more nucleotides,
to
produce a library of recombinant CTL inhibitor nucleic acids; (2) introducing
genetic vaccine vectors which comprise the library of recombinant CTL
inhibitor
5 nucleic acids into a plurality of human cells; (3) selecting cells which
exhibit
reduced MHC class I molecule expression; and (4) obtaining optimized
recombinant CTL inhibitor nucleic acids from the selected cells.
10= Methods of obtainint a genetic vaccine component that c.onfers_upon a
genetic vaccine reduced susceptibility to a CTL imnume_response in a host
.mammaL
The invention also provides methods of obtaining a genetic vaccine
15 component that confers upon a genetic vaccine reduced susceptibility to
a CTL
immune response in a host mammal. These methods involve: (1) reassembling
(&/or subjecting to one or more directed evolution methods described herein)
at
least first and second forms of a nucleic acid which comprises a gene that
encodes
an inhibitor of a CTL immune response, wherein the first and second forms
differ
20 from each other in two or more nucleotides, to produce a library of
recombinant
CTL inhibitor nucleic acids; (2) introducing viral vectors which comprise the
library of recombinant CTL inhibitor nucleic acids into mammalian cells; (3)
identifying mammalian cells which express a marker gene included in the viral
vectors a predetermined time after introduction, wherein the identified cells
are
25 resistant to a CTL response; and (4) recovering as the genetic vaccine
component
the recombinant inhibitor nucleic acids from the identified
cells.
It is a general object of the invention to provide proteins and polypeptides
that are derived from PIEMP1 proteins, nucleic acids encoding these proteins
and
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antibodies that are specifically immunoreactive with these proteins. It is a
further
object to provide methods of using these various compositions in diagnosis,
treatment or prevention of the onset of symptoms of a malaria parasite
infection.
It is a further object to provide methods of screening compounds to identify
further compositions which may be used in these methods.
In one embodiment, the present invention provides substantially pure
polypeptides which have amino acid sequences substantially homologous to the
amino acid sequence of a PfEMP1 protein, or biologically active fragments
thereof.
In preferred aspects, the polypeptides of the present invention are
substantially homologous to the amino acid sequence shown, described &/or
referenced herein, . biologically active
fragments or analogues thereof. Also provided are pharmaceutical compositions
comprising these polypeptides.
In another embodiment, the present invention provides nucleic acids
= which encode the above-described polypeptides. Particularly preferred
nucleic
acids will be substantially homologous to a part or whole of the nucleic acid
sequence shown, described &for referenced herein,
or the nucleic acid encoding for the sequences shown, described &kr
referenced herein. The present invention
also provides expression vectors comprising these nucleic acid sequences and
cells capable of expressing same.
In an additional embodiment, the present invention provides antibodies
which recognize and bind PfEMP1 polypeptides or biologically active fragments
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thereof. More preferred are those peptides which recognize and bind PfEMP1
proteins associated with infection by more than one variant of P. falciparum.
In a further embodiment, the present invention provides methods of
In a related embodiment, the present invention provides methods of
inhibiting sequestration of erythrocytes in a patient suffering from a malaria
In still another embodiment, the present invention provides a method of
In an additional embodiment, the present invention provides a method of
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Summary of Directed Evolution Approaches
This invention also relates generally to the field of nucleic acid
engineering and correspondingly encoded recombinant protein engineering. More
particularly, the invention relates to the directed evolution of nucleic acids
and
screening of clones containing the evolved nucleic acids for resultant
activity(ies)
of interest, such nucleic acid activity(ies) &kr specified protein,
particularly
enzyme, activity(ies) of interest.
Mutagenized molecules provided by this invention may have chimeric
molecules and molecules with point mutations, including biological molecules
that
contain a carbohydrate, a lipid, a nucleic acid, &/or a protein component, and
specific
but non-limiting examples of these include antibiotics, antibodies, enzymes,
and
steroidal and non-steroidal hormones.
This invention relates generally to a method of: 1) preparing a progeny
generation of molecule(s) (including a molecule that is comprised of a
polynucleotide
sequence, a molecule that is comprised of a polypeptide sequence, and a
molecules
that is comprised in part of a polynucleotide sequence and in part of a
polypeptide
sequence), that is mutagenized to achieve at least one point mutation,
addition,
deletion, &for chimerization, from one or more ancestral or parental
generation
template(s); 2) screening the progeny generation molecule(s) - preferably
using a high
throughput method - for at least one property of interest (such as an
improvement in an
enzyme activity or an increase in stability or a novel chemotherapeutic
effect); 3)
optionally obtaining &/or cataloguing structural &/or and functional
information
regarding the parental Wor progeny generation molecules; and 4) optionally
repeating
any of steps 1) to 3).
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In a preferred embodiment, there is generated (e.g. from a parent
polynucleotide template) - in what is termed "codon site-saturation
mutagenesis" -
a progeny generation of polynucleotides, each having at least one set of up to
three contiguous point mutations (i.e. different bases comprising a new
codon),
Thus, in another aspect, this approach is also serviceable for generating
mutants containing - in addition to &/or in combination with the 20 naturally
encoded amino acids and amino acid derivatives. In yet another aspect, this
approach is also serviceable for generating mutants by the use of- in addition
to
&/or in combination with natural or unaltered codon recognition systems of
suitable hosts - altered, mutagenized, &/or designer codon recognition systems
In yet another aspect, this invention relates to recombination and more
specifically to a method for preparing polynucleotides encoding a polypeptide
by a
method of in vivo re-assortment of polynucleotide sequences containing regions
of
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partial homology, assembling the polynucleotides to form at least one
polynucleotide
and screening the polynucleotides for the production of polypeptide(s) having
a useful
property.
In yet another preferred embodiment, this invention is serviceable for
analyzing and cataloguing - with respect to any molecular property (e.g. an
enzymatic
activity) or combination of properties allowed by current technology - the
effects of
any mutational change achieved (including particularly saturation mutapmesis).
Thus,
a comprehensive method is provided for determining the effect of changing each
amino acid in a parental polypeptide into each of at least 19 possible
substitutions.
This allows each amino acid in a parental polypeptide to be characterized and
catalogued according to its spectrum of potential effects on a measurable
property of
the polypeptide.
In another aspect, the method of the present invention utilizes the natural
property of cells to recombine molecules and/or to mediate reductive processes
=
that reduce the complexity of sequences and extent of repeated or consecutive
sequences possessing regions of homology.
It is an object of the present invention to provide a method for generating
hybrid polynucleotides encoding biologically active hybrid polypeptides with
enhanced activities. In accomplishing these and other objects, there has been
provided, in accordance with one aspect of the invention, a method for
introducing polynucleotides into a suitable host cell and growing the host
cell
under conditions that produce a hybrid polynucleotide.
In another aspect of the invention, the invention provides a method for
screening for biologically active hybrid polypeptides encoded by hybrid
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pol)mucleotides. The present method allows for the identification of
biologically
active hybrid polypeptides with enhanced biological activities.
1.4. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Exonuclease Activity. Figure 1 shows the activity of the enzyme
exonuclease III. This is an exemplary enzyme that can be used to shuffle,
assemble, reassemble, recombine, and/or concatenate polynucleotide building
blocks. The asterisk indicates that the enzyme acts from the 3' direction
towards
the 5' direction of the polynucleotide substrate.
Figure 2. Generation of A Nucleic Acid Building Block by Polymerase-Based
Amplification. Figure 2 illustrates a method of generating a double-stranded
nucleic acid building block with two overhangs using a polymerase-based
amplification reaction (e.g., PCR). As illustrated, a first polymerase-based
amplification reaction using a first set of primers, F2 and RI, is used to
generate a
blunt-ended product (labeled Reaction 1, Product 1), which is essentially
identical
to Product A. A second polymerase-based amplification reaction using a second
set of primers, F1 and R2, is used to generate a blunt-ended product (labeled
Reaction 2, Product 2), which is essentially identical to Product B. These two
products are then mixed and allowed to melt and anneal, generating a
potentially
useful double-stranded nucleic acid building block with two overhangs. In the
example of Fig. 1, the product with the 3' overhangs (Product C) is selected
for
by nuclease-based degradation of the other 3 products using a 3' acting
exonuclease, such as exonuclease III. Alternate primers are shown in
parenthesis
to illustrate serviceable primers may overlap, and additionally that
serviceable
primers may be of different lengths, as shown.
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Figure 3. Unique Overhangs And Unique Couplings. Figure 3 illustrates the
point that the number of unique overhangs of each size (e.g. the total number
of
unique overhangs composed of 1 or 2 or 3, etc. nucleotides) exceeds the number
of unique couplings that can result from the use of all the unique overhangs
of
that size. For example, there are 4 unique 3' overhangs composed of a single
nucleotide, and 4 unique 5' overhangs composed of a single nucleotide. Yet the
total number of unique couplings that can be made using all the 8 unique
single-
nucleotide 3' overhangs and single-nucleotide 5' overhangs is 4.
Figure 4. Unique Overall Assembly Order Achieved by Sequentially
Coupling the Building Blocks
Figure 4 illustrates the fact that in order to assemble a total of "n" nucleic
acid
building blocks, "n-1" couplings are needed. Yet it is sometimes the case that
the
number of unique couplings available for use is fewer that the "n-1" value.
Under
these, and other, circumstances a stringent non-stochastic overall assembly
order
can still be achieved by performing the assembly process in sequential steps.
In
this example, 2 sequential steps are used to achieve a designed overall
assembly
order for five nucleic acid building blocks. In this illustration the designed
overall
assembly order for the five nucleic acid building blocks is: 5'-(#1-#2-#3-#4-
#5)-
3', where #1 represents building block number 1, etc.
Figure 5. Unique Couplings Available Using a Two-Nucleotide 3' Overhang.
Figure 5 further illustrates the point that the number of unique overhangs of
each
size (here, e.g. the total number of unique overhangs composed of 2
nucleotides)
exceeds the number of unique couplings that can result from the use of all the
unique overhangs of that size. For example, there are 16 unique 3' overhangs
composed of two nucleotides, and another 16 unique 5' overhangs composed of
two nucleotides, for a total of 32 as shown. Yet the total number of couplings
that
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are unique and not self-binding that can be made using all the 32 unique
double-
nucleotide 3' overhangs and double-nucleotide 5' overhangs is 12. Some
apparently unique couplings have "identical twins" (marked in the same
shading),
which are visually obvious in this illustration. Still other overhangs contain
Figure 6. Generation of an Exhaustive Set of Chimeric Combinations by
progenitor templates. Nucleic acid building blocks including their overhangs
(which are serviceable for the formation of ordered couplings) are then
designed
based on the sequence of each of the 8 progenitor templates, for a total of
144
nucleic acid building blocks (or double-stranded oligos). Performing the
ligation
synthesis procedure will then produce a library of progeny molecules comprised
of yield of 818 (or over 1.8 x 1016) chimeras.
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Figure 7. Synthetic genes from oligos:. According to one embodiment of this
invention, double-stranded nucleic acid building blocks are designed by
aligning a
plurality of progenitor nucleic acid templates. Preferably these templates
contain
some homology and some heterology. The nucleic acids may encode related
proteins, such as related enzymes, which relationship may be based on function
or
structure or both. Figure 7 shows the alignment of three polynucleotide
progenitor templates and the selection of demarcation points (boxed) shared by
all
the progenitor molecules. In this particular example, the nucleic acid
building
blocks derived from each of the progenitor templates were chosen to be
approximately 30 to 50 nucleotides in length.
Figure 8. Nucleic acid building blocks for synthetic ligation gene reassembly.
Figure 8 shows the nucleic acid building blocks from the example in Figure 7.
The nucleic acid building blocks are shown here in generic cartoon form, with
their compatible overhangs, including both 5' and 3' overhangs. There are 22
total nucleic acid building blocks derived from each of the 3 progenitor
templates.
Thus, the ligation synthesis procedure can produce a library of progeny
molecules
comprised of yield of 322 (or over 3.1 x 010) chimeras.
Figure 9. Addition of Introns by Synthetic Ligation Reassembly. Figure 9
shows in generic cartoon form that an intron may be introduced into a chimeric
progeny molecule by way of a nucleic acid building block. It is appreciated
that
introns often have consensus sequences at both termini in order to render them
operational. = It is also appreciated that, in addition to enabling gene
splicing,
introns may, serve an additional purpose by providing sites of homology to
other
nucleic acids to enable homologous recombination. For this purpose, and
potentially others, it may be sometimes desirable to generate a large nucleic
acid
building block for introducing an intron. If the size is overly large easily
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genrating by direct chemical synthesis of two single stranded oligos, such a
specialized nucleic acid building block may also be generated by direct
chemical
synthesis of more than two single stranded oligos or by using a polymerase-
based
amplification reaction as shown, described &/or referenced herein.
Figure 10. Ligation Reassembly Using Fewer Than All The Nucleotides Of
An Overhang. Figure 10 shows that coupling can occur in a manner that does
not make use of every nucleotide in a participating overhang. The coupling is
particularly lively to survive (e.g. in a transformed host) if the coupling
reinforced
by treatment with a ligase enzyme to form what may be referred to as a "gap
ligation" or a "gapped ligation". It is appreciated that, as shown, this type
of
coupling can contribute to generation of unwanted background prOduct(s), but
it
can also be used to advantageously increase the diversity of the progeny
library
generated by the designed ligation reassembly.
Figure 11. Avoidance of unwanted self-ligation in palindromic couplings. As
mentioned before and shown, described &/or referenced herein
, certain overhangs are able to undergo self-coupling to
= 20 form a palindromic coupling. A coupling is strengthened substantially
if it is
reinforced by treatment with a ligase enzyme. Accordingly, it is appreciated
that
the lack of 5' phosphates on these overhangs, as shown, can be used
advantageously to prevent this type of palindromic self-ligation. Accordingly,
this invention provides that nucleic acid building blocks can be chemically
made
(or ordered) that lack a 5' phosphate group (or alternatively they can be
remove ¨
e.g. by treatment with a phosphatase enzyme such as a calf intestinal alkaline
phosphatase (CIAP) ¨ in order to prevent palindromic self-ligations in
ligation
reassembly processes.
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=
Figure 12. Site-directed mutagenesis by polymerase-based extension. Panel
A. This figure shows one method of site-directed mutagenesis, among many
methods of site-directed mutagenesis, that are serviceable for performing site-
saturation mutagenesis. Section (1) shows the first and second mutagenic
primer
annealed to a circular closed double-stranded plasmid. The dot and the open-
sided triangle indicate the mutagenic sites in the mutagenic primers. The
arrows
indicate the direction of synthesis. Section (2)shows the newly synthesized
(mutagenized) DNA strands annealed to each other. The parental DNA can be
treated with a selection enzyme. The mutagenized DNA strands are shown as
being annealed to form a double-stranded mutagenized circular DNA
=
intermediate. The dot and the open-sided triangle indicate the mutagenic sites
in
the experimentally generated progeny (mutagenized) DNA strands. Note that the
staggered openings on the mutagenized DNA strands form "sticky" ends. Section
(3) shows the first and second mutagenic primer annealed to the mutagenized
DNA strands of Section (2). The arrows indicate the direction of synthesis.
Note the opening on each of the mutagenized DNA strands (i.e. they have not
been ligated). Section (4) shows a "Gapped Product", which is composed of
second generation mutagenized DNA strands, synthesized using the mutagenized
DNA stands (shown in Step (2)) as a template. The DNA strands of the "Gapped
Produce' are shown as being annealed to form a double-stranded mutagenized
circular DNA intermediate. The dot and the open-sided triangle indicate the
mutagenic sites in the mutagenized DNA strands. Note the large gap in each of
the mutagenized DNA strands. Section (5) shows the "Gapped Product" annealed
to the parental (non-mutated) plasmid, enabling polymerase-based synthesis to
occur. The arrows indicate the direction of synthesis. Section (6) shows the
newly synthesized DNA strands, as being annealed to form a double-stranded
mutagenized circular DNA product. The dot and the open-sided triangle indicate
the mutagenic sites in the mutagenized DNA strands. Note the staggered
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openings on the mutagenized DNA strands. Also note the presence of both
mutagenic sites on each of the mutagenized DNA strands.
Panel B. This figure shows two possible molecular structures produced from the
amplification steps of Figure 12A. Molecule (A) is shown also in Section (2)
of
Figure 12A. Molecule (B) is also shown in Section (6) of Figure 12A.
Figure 13. Site-directed mutagenesis by polymerase-based extension and
ligase-based ligation. Panel A. This figure shows one method of site-directed
mutagenesis, among many methods of site-directed mutagenesis, that are
serviceable for performing site-saturation mutagenesis. Section (1) shows the
first and second mutagenic primer annealed to a circular closed double-
stranded
plasmid. The dot and the open-sided triangle indicate the mutagenic sites in
the
mutagenic primers. The arrows indicate the direction of synthesis. Section (2)
shows the newly synthesized (mutagenized) DNA strands annealed to each other.
The parental DNA can be treated with a selection enzyme. The mutagenized
DNA strands are shown as being annealed to form a double-stranded mutagenized
circular DNA intermediate. The dot and the open-sided triangle indicate the
mutagenic sites in the experimentally generated progeny (mutagenized) DNA
strands. Note that the staggered openings on the mutagenized DNA strands form
"sticky" ends. Section (3) shows the resultant double-stranded mutagenized
circular DNA molecule produced after the double-stranded mutagenized circular
DNA intermediate of Section (2) is ligated (e.g. with T4 DNA ligase). Section
(4) shows the first and second mutagenic primer annealed to the mutagenized
DNA strands of Section (3). The arrows indicate the direction of synthesis.
Section (5) shows the recently generated (blue) mutagenized DNA strands as
being annealed to form a double-stranded mutagenized circular DNA
intermediate. The dot and the open-sided triangle indicate the mutagenic sites
in
the recently generated mutagenized DNA strands (blue). Note that the staggered
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openings on the mutagenized DNA strands form "sticky ends". Also note the
presence of both mutagenic sites on each of the two recently generated
mutagenized DNA strands (blue). Note the opening on each of the mutagenized
DNA strands (i.e. they have not been ligated). Section (6) shows the resultant
double-stranded mutagenized circular DNA molecule produced after the double-
stranded mutagenized circular DNA intermediate of Section (5) is ligated (e.g.
using T4 DNA ligase). The dot and the open-sided triangle indicate the
mutagenic sites in the mutagenized DNA molecules. Again, note the presence of
both mutagenic sites on each of the mutagenized DNA strands.
Panel B. This figure shows two molecular structures produced from the
amplification steps of Figure 13A. Molecule (A) is also shown in Section (3)
of
Figure 13A. Molecule (B) is produced in Section (6) of Figure 13A.
Figure 14: Strategy for Obtaining and Using Nucleic Acid Binding Proteins
that Facilitate Entry of Genetic Vaccines.
Shown here is a strategy for obtaining and using nucleic acid binding proteins
that
facilitate entry of genetic vaccines, in particular, naked DNA, into target
cells.
Members of a library obtained by the directed evolution methods described
herein
are linked to a coding region of M 13 protein VIII so that a fusion protein is
displayed on the surface of the phage particles. Phage that efficiently enter
the
desired target tissue are identified, and the fusion protein is then used to
coat a
genetic vaccine nucleic acid.
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Figure 15: A schematic representation of a method for generating a chimeric,
multivalent antigen that has immunogenic regions from multiple antigens.
Antibodies to each of the non-chimeric parental immunogenic polypeptides are
specific for the respective organisms (A, B, C). After carrying out the
directed
evolution and selection methods of the invention, however, a chimeric
immunogenic polypeptide is obtained that is recognized by antibodies raised
against each of the three parental immunogenic polypeptides.
Figure 16A and Figure 16B: Method for Obtaining Non-Stochastically
Generated Polypeptides that can induce a Broad-Spectrum Immune
Response.
Shown here is a schematic for a method by which one can obtain non-
stochastically generated polypeptides that can induce a broad-spectrum immune
response. In Figure 16A, wild-type immunogenic polypeptides from the
pathogens A, B, and C provide protection against the corresponding pathogen
from which the polypeptide is derived, but little or no cross-protection
against the
other pathogens (left panel). After evolving, an A/13/C chimeric polypeptide
is
obtained that can induce a protective immune response against all three
pathogen
types (right panel). In Figure I6B, directed evolution is used with substrate
nucleic acids from two pathogen strains (A, B), which encode polypeptides that
are protective only against the corresponding pathogen. After directed
evolution,
the resulting chimeric polypeptide can induce an immune response that is
effective against not only the two parental pathogen strains, but also against
a
third strain of pathogen (C).
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Figure 17: Possible factors for determining whether a particular
polynucleodde encodes an immunogenic polypeptide having a desired
property.
Shown here are some of the possible factors that can determine whether a
particular polynucleotide encodes an immunogenic polypeptide having a desired
property, such as enhanced immunogenicity and/or cross-reactivity. Those
sequence regions that positively affect a particular property are indicated as
plus
signs along the antigen gene, while those sequence regions that have a
negative
effect are shown as minus signs. A pool of related antigen genes are non-
stochastically generated using the methods described herein and screened to
obtain those evolved nucleic acids that have gained positive sequence regions
and
lost negative regions. No pre-existing knowledge as to which regions are
positive
or negative for a particular trait is required.
Figure 18: Screening strategy for antigen library screening.
Shown here is a schematic representation of the screening strategy for antigen
library screening.
Figure 19: Strategy for pooling and deconvolution as used in antigen library
screening.
Shown here is a schematic representation of a strategy for pooling and
deconvolution as used in antigen library screening.
Figure 20: Preferred Embodiments of Site-Saturation Mutagenesis.
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Figure 21. Schematic representation of a multimodule genetic vaccine vector.
Shown here is a schematic representation of a multimodule genetic vaccine
vector. A typical genetic vaccine vector will include one or more of the
components indicated, each of which can be native or optimized using the
directed evolution methods described herein. These directed evolution methods
can include the introduction of point mutations by stochastic methods &/or by
non-stochastic methods, including "gene site saturation mutagenesis" as
described
herein. These directed evolution methods can also include stochastic
polynucleotide reassembly methods, for example by interrupted synthesis (as
described in US5965408). These directed evolution methods can also include
non-stochastic polynucleotide reassembly methods as described herein,
including
synthetic ligation polynucleotide reassembly as described herein. The
components can be present on the same vaccine vector, or can be- included in a
genetic vaccine as separate molecules.
Figure 22A and Figure 22B. Generation of vectors with multiple T cell
epitopes. Shown here are two different strategies for generating vectors that
contain multiple T cell epitopes obtained, for example, by directed evolution.
In
Figure 22A, each individual non-stochastically generated epitope-encoding gene
is linked to a single promoter, and multiple promoter-epitope gene constructs
can
be placed in a single vector. The scheme shown, described &/or referenced
herein
involves linking multiple epitope-encoding
genes to a single promoter.
Figure 23. Generation of optimized genetic vaccines by directed evolution.
Shown here is a diagram of the application of directed evolution to the
generation
of optimized genetic vaccines. Different forms of polynucleotides having known
functional properties (e.g., regulatory, coding, and the like) are evolved and
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screened to identify variants that exhibit improved properties for use as
genetic
vaccines.
Figure 24. Recursive application of directed evolution and selection of
evolved promoter sequences as an example of flow cytometry-based
screening methods. Shown here is a diagram of flow cytometry-based screening
methods (FACS) for selection of optimized promoter sequences evolved using
recursive applications of the directed evolution methods as described herein.
A
cytomegalovirus (CMV) promoter is used for illustrative purposes.
Figure 25. An apparatus for microinjecdons of skin and muscle. Shown here
is an apparatus that is suitable for microinjection of genetic vaccines and
other
reagents into tissue such as skin and muscle. The apparatus is particularly
useful
for screening large numbers of agents in vivo, being based on a 96-well
format.
The tips of the apparatus are movable to allow adjustment so that the tips fit
into a
microtiter plate. After obtaining a reagent of interest is obtained from a
plate, the
tips are adjusted to a distance of about 2-3 min apart, enabling transfer of
96
different samples to an area of about 1.6 cm by 2.4 cm to about 2.4 cm by 3.6
cm.
If desired, the volume of each sample transferred can be electronically
controlled;
typically the volumes transferred range from about 2 ul to about 5 ul. Each
reagent can be mixed with a marker agent or dye to facilitate recognition of
the
=
injection site in the tissue. For example, gold particles of different sizes
and
shaped can be mixed with the reagent of interest, and microscopy and
immunohistochemistry used to identify each injection site and to study the
reaction induced by each reagent. When muscle tissue is injected, the
injection
site is first revealed by surgery.
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Figure 26. Polynucleotide reassembly. Shown in Panel A is an example of
directed evolution. n different strains of a virus are used in this
illustration, but the
technique is applicable to any single nucleic acid as well as to any nucleic
acid for
which different strains, species, or gene families have homologous nucleic
acids
that have one or more nucleotide changes compared to other homologous nucleic
acids. The different variant nucleic acids are experimentally generated,
preferably
non-stochastically, as described herein, and screened or selected to identify
those
variants that exhibit the desired property. The directed evolution method(s)
and
screening can be repeated one or more times to obtain further improvement.
Panel
B shows that successive rounds of directed evolution can produce progressively
enhanced properties, and that the combination of individual beneficial
mutations
can lead to an enhance improvement compared to the improvement achieved by
an individual beneficial mutation.
Figure 27. Vector for promoter evolution. Shown here is an example of a
vector that is useful for screening to identify improved promoters from a
library
of promoter nucleic acids evolved using the directed evolution methods as
described herein. Experimentally generated putative promoters are inserted
into
the vector upstream of a reporter gene for which expression is readily
detected.
For many applications, it is desirable that the product of the reporter gene
be a cell
surface protein so that cells which express high levels of the reporter gene
can be
sorted using flow cytometry-based cell sorting using the reporter gene
product.
Examples of suitable reporter genes include, for example, B7-2 and mAb179
epitopes. A polyadenylation region is typically placed downstream of the
reporter
gene (SV40 polyA is illustrated). The vector can also include a second
reporter
gene an internal control (GFP; green fluorescent protein); this gene is linked
to a
promoter (SR p). The vector also typically includes a selectable marker
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(canamycin/neomycin resistance is shown), and origins of replication that are
functional in mammalian (SV40 on) and/or bacterial (pUC on) cells.
Figure 28. Iterative evolution of inducible promoters using directed
evolution and flow cytometry-based selection. Shown here is a diagram of a
scheme for iterative evolution of inducible promoters using the directed
evolution
methods as described herein and flow cytometry-based selection. A library of
experimentally generated (i.e. produced by one or more directed evolution
methods as descried herein) promoter nucleic acids present in appropriate
vectors
is transfected into the cells, and those cells which exhibit the least
expression of =
marker antigen when grown under uninduced conditions are selected. The vectors
(&/or cells containing them) are recovered, and the vectors are introduced
into
cells (if not contained therein already), and grown under inducing conditions.
Those cells that express the highest level of marker antigen are selected.
Figure 29. Evolving a genetic vaccine vector for Oral, Intravenous,
Intramuscular, Intradermal, Anal, Vaginal, or Topical Delivery. Illustrated is
a strategy for screening of M13 libraries (e.g. generated experimentally using
directed evolution as descried herein) for desired targeting of various
tissues. The
particular example shown here is a schematic diagram of a method for evolving
a
genetic vaccine vector for improved oral delivery. This may comprise selecting
for stability under the acidic conditions of the stomach, and resistance to
other
degredatory factors of the digestive tract. The particular example illustrated
relates to screening for improved oral delivery, but the same principle
applies to
libraries administered by other routes, including intravenously,
intramuscularly,
intradermally, axially, vaginally, or topically. After delivery to a test
animal, the
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M13 phage (or a product thereof) is recovered from the tissue of interest. The
procedure can be repeated to obtain further optimization.
Figure 30. An alignment of the nucleotide sequences of two human CMV
strains and one monkey strain. Shown here is an alignment of the nucleotide
sequences of two human cytomegalovirus (CMV) strains and one monkey
(Rhesus) strains. This alignment is serviceable for performing non-stochastic
polynucleotide reassembly.
Figure 31. An alignment of IL-4 nucleotide sequences from 3 species
(human, primate, and canine). Shown here is an alignment of the IL-4
nucleotide sequences of human, dog and primate strains. This alignment is
serviceable for performing non-stochastic polynucleotide reassembly.
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Figure 32. Evoludon of polypeptides by synthesizing (in vivo or in vitro)
corresponding deduced polynucleotides and subjecting the deduced
polynucleotides to directed evolution and expression screening subsequently
expressed polypeptides.
Figure 33. Non-stochastic Reassembly of oligo-directed CpG knock-outs.
Shown here is a schematic representation of the use of the non-stochastic
methods
described herein to generate promoter sequences in which unnecessary CpG
10. sequences are deleted, potentially useful CpG sequences are added, and
non-
replaceable CpG sequences are identified. Additionally, other sequences (aside
from the CpG sequences) can be substituted into, added to, 8dor deleted from
working polynucleotides.
Figure 34. An Example of a cris obtained from HbsAg polypeptide (PreS2
plus S regions). Shown here is an example of a cytotoxic T-cell inducing
sequence (CTIS) obtained from HBsAg polypeptide (PreS2 plus S regions).
Figure 35. A CTIS Having Heterologous Epitopes Attached to the
Cytoplasmic Portion. Shown here is a CTIS having heterologous epitopes
attached to the cytoplasmic portion.
.25 Figure 36. Method for preparing immunogenic agonist sequences (IAS).
Shown here is a method for preparing immunogenic agonist sequences (IAS).
Wild-type (WT) and mutated forms of nucleic acids encoding a polypeptide of
. interest are assembled and subjected to non-stochastic reassembly to
obtain a
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nucleic acid encoding a poly-epitope region that contains potential agonist
sequences.
Figure 37. Improving Immunostimulatory Sequences (ISS) Using Directed
Evolution. Shown here is a scheme for improving inununostimulatory sequences
by the directed evolution methods described herein. Oligonucleotide building
blocks (e.g. synthetically generated), oligos with known ISS, CpG containing
hexamers &/or oligos containing CpG containing hexamers, poly A, C, G, T,
etc.. .can be assembled. The resultant molecule(s) can then by subjected to 1
or
more directed evolution methods as described herein.
Figure 38. Screening to identify IL-12 genes that encode recombinant IL-12
having an increased ability to induce T Cell proliferation. Shown here is a
diagram of a procedure by which experimentally generated molecules, e.g. non-
stochastically generated libraries of human IL-12 genes can be screened to
identify evolved IL-12 genes that encode evolved forms of IL-12 having
increased ability to induce T cell proliferation.
Figure 39. Model of induction of T cell activation or anergy by genetic
vaccine vectors encoding different CDS() and/or CD86 variants. Shown here
is a model of how T cell activation or anergy can be induced by genetic
vaccine
vectors that encode different B7-1 (CD80) and/or B7-2 (CD86) variants.
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Figure 40. Screening of CD80/CD86 variants that have improved capacity to
induce T cell activation or anergy. Shown here is a method for using directed
evolution as described herein to obtain CD80/CD86 variants that have improved
capacity to induce T cell activation or anergy.
Figure 41. An alignment of two CMV derived nucleotide sequences from
human and primate species. Shown here is an alignment of two CMV-derived
nucleotide sequences of human and primate strains. This alignment is
serviceable
for performing non-stochastic polynucleotide reassembly.
Figure 42. An alignment of the IFN-gamma nucleotide sequences from
human, cat, rodent species. Shown here is an alignment of the IFN-gamma
.= nucleotide sequences from human, cat, rodent species. This alignment is
serviceable for performing non-stochastic polynucleotide reassembly.
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2. DETAILED DESCRIPTION OF THE INVENTION
2.1. DEFINITIONS OF TERMS
In order to facilitate understanding of the examples provided herein,
certain frequently occurring methods and/or terms will be described.
The term "agent" is used herein to denote a chemical compound, a
mixture of chemical compounds, an array of spatially localized compounds
(e.g.,
a VLSWS peptide array, polynucleotide array, and/or combinatorial small
molecule array), biological macromolecule, a bacteriophage peptide display
library, a bacteriophage antibody (e.g., scFv) display library, a polysome
peptide
display library, or an extract made form biological materials such as
bacteria,
plants, fungi, or animal (particular mammalian) cells or tissues. Agents are
evaluated for potential activity as anti-neoplastics, anti-inflammatories or
apoptosis modulators by inclusion in screening assays described hereinbelow.
Agents are evaluated for potential activity as specific protein interaction
inhibitors
(i.e., an agent which selectively inhibits a binding interaction between two
predetermined polypeptides but which doe snot substantially interfere with
cell
viability) by inclusion in screening assays described hereinbelow.
An "ambiguous base requirement" in a restriction site refers to a
nucleotide base requirement that is not specified to the fullest extent, i.e.
that is
not a specific base (such as, in a non-limiting exemplification, a specific
base
selected from A, C, G, and T), but rather may be any one of at least two or
more
bases. Commonly accepted abbreviations that are used in the art as well as
herein
to represent ambiguity in bases include the following: R =0 or A; Y = C or T;
M
=AorC;K=GorT;S=GorC;W=AorT;H=AorCorT;B=GorTorC;
V=G or C or A; D = G or A or T; N = A or C or G or T.
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=
"Alignment" with respect to molecular sequences is a way to determine
similarity between 2 or more sequences. Optimal alignment of sequences for
comparison can be conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm
of Needleman & Wunsch, J Mol. Biol. 48:443 (1970), by the search for
similarity
method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by
computerized implementations of these algorithms (GAP, BESTFIT, PASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics Computer
Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally
Ausubel et al., infra).
One example of an algorithm that is suitable for determining percent
sequence identity and sequence similarity is the BLAST algorithm, which is
described in Altschul et al., J Mol. Biol. 215:403-410 (1990). Software for
performing BLAST analyses is publicly available through the National Center
for
Biotechnology Information. This algorithm
involves first identifying high scoring sequence pairs (HSPs) by identifying
short
words of length W in the query sequence, which either match or satisfy some
positive-valued threshold score T when aligned with a word of the same length
in
a database sequence. T is referred to as the neighborhood word score threshold
(Altschul et al., supra). These initial neighborhood word hits act as seeds
for
initiating searches to find longer HSPs containing them. The word hits are
then
extended in both directions along each sequence for as far as the cumulative
alignment score can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of matching
residues; always > 0) and N (penalty score for mismatching residues; always
<0).
For amino acid sequences, a scoring matrix is used to calculate the cumulative
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score. Extension of the word hits in each direction are halted when: the
cumulative alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to the
accumulation of one or more negative-scoring residue alignments; or the end of
either sequence is reached.
The BLAST algorithm parameters W, T, and X determine the sensitivity
and speed of the alignment. The BLASTN program (for nucleotide sequences)
uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of
100,
M=5, N=-4, and a comparison of both strands. For amino acid sequences, the
BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of
10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc.
Natl. Acad. Sci. USA 89:10915).
In addition to calculating percent sequence identity, the BLAST algorithm
also performs a statistical analysis of the similarity between two sequences
(see,
e.g., Karlin & Altschul (1993) Proc. Nat'l. Acad. Sci. USA 90:5873-5787). One
measure of similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability by which a
match between two nucleotide or amino acid sequences would occur by chance.
For example, a nucleic acid is considered similar to a reference sequence if
the
smallest sum probability in a comparison of the test nucleic acid to the
reference
nucleic acid is less than about 0. 1, more preferably less than about 0.0 1,
and
most preferably less than about 0.001.
Another indication that two nucleic acid sequences are substantially identical
is
that the two molecules hybridize to each other under stringent conditions. The
phrase "hybridizing specifically to", refers to the binding, duplexing, or
hybridizing of a molecule only to a particular nucleotide sequence under
stringent
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conditions when that sequence is present in a complex mixture (e.g., total
cellular)
DNA or RNA. "Bind(s) substantially" refers to complementary hybridization
between a probe nucleic acid and a target nucleic acid and embraces minor
mismatches that can be accommodated by reducing the stringency of the
hybridization media to achieve the desired detection of the target
polynucleotide
sequence.
The term "amino acid" as used herein refers to any organic compound
that contains an amino group (-NH2) and a carboxyl group (-COOH); preferably
either as free groups or alternatively after condensation as part of peptide
bonds.
The "twenty naturally encoded polypeptide-forming alpha-amino acids" are
understood in the art and refer to: alanine (ala or A), arginine (arg or R),
asparagine (asn or N), aspartic acid (asp or D), cysteine (cys or C),
gluatamic acid
(glu or E), glutamine (gin or Q), glycine (gly or G), histidine (his or H),
isoleucine
(ile or I), leucine (leu or L), lysine (lys or K), methionine (met or M),
phenylalanine (phe or F), proline (pro or P), serine (ser or S), threonine
(thr or T),
tryptophan (trp or W), tyrosine (tyr or Y), and valine (val or V).
The term "amplification" means that the number of copies of a
polynucleotide is increased.
The term "antibody", as used herein, refers to intact immunoglobulin
molecules, as well as fragments of imrnunoglobulin molecules, such as Fab,
Fab',
(Fab)2, Fv, and SCA fragments, that are capable of binding to an epitope of an
antigen. These antibody fragments, which retain some ability to selectively
bind
to an antigen (e.g., a polypeptide antigen) of the antibody from which they
are
derived, can be made using well known methods in the art (see, e.g., Harlow
and
Lane, supra), and are described further, as follows.
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(1) An Fab fragment consists of a monovalent antigen-binding fragment
of an antibody molecule, and can be produced by digestion of a whole
antibody molecule with the enzyme papain, to yield a fragment
consisting of an intact light chain and a portion of a heavy chain.
(2) An Fab' fragment of an antibody molecule can be obtained by treating
a whole antibody molecule with pepsin, followed by reduction, to
yield a molecule consisting of an intact light chain and a portion of a
heavy chain. Two Fab' fragments are obtained per antibody molecule
treated in this manner.
(3) An (Fab1)2 fragment of an antibody can be obtained by treating a
whole antibody molecule with the enzyme pepsin, without subsequent
reduction. A (Fab1)2 fragment is a dimer of two Fab' fragments, held
together by two disulfide bonds.
(4) An Fv fragment is defined as a genetically engineered fragment
containing the variable region of a light chain and the variable region
of a heavy chain expressed as two chains.
(5) An single chain antibody ("SCA") is a genetically engineered single
chain molecule containing the variable region of a light chain and the
variable region of a heavy chain, linked by a suitable, flexible
polypeptide linker.
The term "Applied Molecular Evolution" ("AME") means the
application of an evolutionary design algorithm to a specific, useful goal.
While
many different library formats for AME have been reported for polynucleotides,
peptides and proteins (phage, lad and polysomes), none of these formats have
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provided for recombination by random cross-overs to deliberately create a
combinatorial library.
A molecule that has a "chimeric property" is a molecule that is: 1) in part
homologous and in part heterologous to a first reference molecule; while 2) at
the
same time being in part homologous and in part heterologous to a second
reference molecule; without 3) precluding the possibility of being at the same
time in part homologous and in part heterologous to still one or more
additional
reference molecules. In a non-limiting embodiment, a chimeric molecule may be
prepared by assemblying a reassortment of partial molecular sequences. In a
non-
limiting aspect, a chimeric polynucleotide molecule may be prepared by
synthesizing the chimeric polynucleotide using plurality of molecular
templates,
such that the resultant chimeric polynucleotide has properties of a plurality
of
templates.
The term "cognate" as used herein refers to a gene sequence that is
evolutionarily and functionally related between species. For example, but not
limitation, in the human genome the human CD4 gene is the cognate gene to the
mouse 3d4 gene, since the sequences and structures of these two genes indicate
that they are highly homologous and both genes encode a protein which
functions
in signaling T cell activation through MHC class II-restricted antigen
recognition.
A "comparison window," as used herein, refers to a conceptual segment
of at least 20 contiguous nucleotide positions wherein a polynucleotide
sequence
may be compared to a reference sequence of at least 20 contiguous nucleotides
and wherein the portion of the polynucleotide sequence in the comparison
window may comprise additions or deletions (i.e., gaps) of 20 percent or less
as
compared to the reference sequence (which does not comprise additions or
deletions) for optimal alignment of the two sequences. Optimal alignment of
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sequences for aligning a comparison window may be conducted by the local
homology algorithm of Smith (Smith and Waterman, Adv App! Math, 1981;
Smith and Waterman, J Teor Biol, 1981; Smith and Waterman, J Mol Biol,
1981; Smith et al, J Mol Evol, 1981), by the homology alignment algorithm of
Needleman (Needleman and Wuncsch, 1970), by the search of similarity method
of Pearson (Pearson and Lipman, 1988), by computerized implementations of
these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science
Dr., Madison, WI), or by inspection, and the best alignment (i.e., resulting
in the
highest percentage of homology over the comparison window) generated by the
various methods is selected.
As used herein, the term "complementarity-determining region" and
"CDR" refer to the art-recognized term as exemplified by the Kabat and Chothia
CDR definitions also generally known as supervariable regions or hypervariable
loops (Chothia and Lesk, 1987; Clothia et al, 1989; Kabat et al, 1987; and
Tramontano et al, 1990). Variable region domains typically comprise the amino-
terminal approximately 105-115 amino acids of a naturally-occurring
irnmunoglobulin chain (e.g., amino acids 1-110), although variable domains
somewhat shorter or longer are also suitable for forming single-chain
antibodies.
"Conservative amino acid substitutions" refer to the interchangeability
of residues having similar side chains. For example, a group of amino acids
having aliphatic side chains is glycine, alanine, valine, leucine, and
isoleucine; a
group of amino acids having aliphatic-hydroxyl side chains is serine and
threonine; a group of amino acids having amide-containing side chains is
asparagine and glutamine; a group of amino acids having aromatic side chains
is
phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic
side
chains is lysine, arginine, and histidine; and a group of amino acids having
sulfur-
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containing side chains is cysteine and methionine. Preferred conservative
amino
acids substitution groups are : valine-leucine-isoleucine, phenylalanine-
tyrosine,
lysine-arginine, alanine-valine, and asparagine-glutamine.
"Conservatively modified variations" of a particular polynucleotide
sequence refers to those polynucleotides that encode identical or essentially
identical amino acid sequences, or where the polynucleotide does not encode an
amino acid sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally identical
nucleic
acids encode any given polypeptide. For instance, the codons CGU, CGC, CGA,
CGG, AGA, and AGG all encode the amino acid arginine.
Thus, at every position where an arginine is specified by a codon, the
codon can be altered to any of the corresponding codons described without
altering the encoded polypeptide. Such nucleic acid variations are "silent
variations," which are one species of "conservatively modified variations."
Every
polynucleotide sequence described herein which encodes a polypeptide also
describes every possible silent variation, except where otherwise noted.
One of skill will recognize that each codon in a nucleic acid (except AUG,
which is ordinarily the only codon for methionine) can be modified to yield a
functionally identical molecule by standard techniques. Accordingly, each
"silent
variation" of a nucleic acid which encodes a polypeptide is implicit in each
described sequence.
Furthermore, one of skill will recognize that individual substitutions,
deletions or additions which alter, add or delete a single amino acid or a
small
percentage of amino acids (typically less than 5%, more typically less than
1%) in
an encoded sequence are "conservatively modified variations" where the
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alterations result in the substitution of an amino acid with a chemically
similar
amino acid. Conservative substitution tables providing functionally similar
amino
acids are well known in the art. The following five groups each contain amino
acids that are conservative substitutions for one another:
Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine
(1);
Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
Sulfur-containing: Methionine (M), Cysteine (C);
Basic: Arginine (R), Lysine (K), Histidine (H);
Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine
(Q).
See also, Creighton (1984) Proteins, W.H. Freeman and Company, for
additional groupings of amino acids. In addition, individual substitutions,
deletions or additions which alter, add or delete a single amino acid or a
small
percentage of amino acids in an encoded sequence are also "conservatively
modified variations".
The term "corresponds to" is used herein to mean that a polynucleotide
sequence is homologous (i.e., is identical, not strictly evolutionarily
related) to all
or a portion of a reference polynucleotide sequence, or that a polypeptide
sequence is identical to a reference polypeptide sequence. In
contradistinction,
the term "complementary to" is used herein to mean that the complementary
sequence is homologous to all or a portion of a reference polynucleotide
sequence. For illustration, the nucleotide sequence "TATAC" corresponds to a
reference "TATAC" and is complementary to a reference sequence "GTATA."
The term "cytoldne" includes, for example, interleukins, interferons,
chemokines, hematopoietic growth factors, tumor necrosis factors and
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transforming growth factors. In general these are small molecular weight
proteins
that regulate maturation, activation, proliferation and differentiation of the
cells of
the immune system.
The term "degrading effective" amount refers to the amount of enzyme
which is required to process at least 50% of the substrate, as compared to
substrate not contacted with the enzyme. Preferably, at least 80% of the
substrate
is degraded.
As used herein, the term "defined sequence framework" refers to a set of
defined sequences that are selected on a non-random basis, generally on the
basis
of experimental data or structural data; for example, a defined sequence
framework may comprise a set of amino acid sequences that are predicted to
form
a 13-sheet structure or may comprise a leucine zipper heptad repeat motif, a
zinc-
finger domain, among other variations. A "defined sequence kernal" is a set of
sequences which encompass a limited scope of variability. Whereas (1) a
completely random 10-mer sequence of the 20 conventional amino acids can be
any of (20)10 sequences, and (2) a pseudorandom 10-mer sequence of the 20
conventional amino acids can be any of (20)10 sequences but will exhibit a
bias
for certain residues at certain positions and/or overall, (3) a defined
sequence
kemal is a subset of sequences if each residue position was allowed to be any
of
the allowable 20 conventional amino acids (and/or allowable unconventional
amino/imino acids). A defined sequence kemal generally comprises variant and
invariant residue positions and/or comprises variant residue positions which
can
comprise a residue selected from a defined subset of amino acid residues), and
the
like, either segmentally or over the entire length of the individual selected
library
member sequence. Defined sequence kernels can refer to either amino acid
sequences or polynucleotide sequences. Of illustration and not limitation, the
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sequences (NNK)10 and (NNM)io, wherein N represents A, T, G, or C; K
represents G or T; and M represents A or C, are defined sequence kernels.
"Digestion" of DNA refers to catalytic cleavage of the DNA with a
5 restriction enzyme that acts only at certain sequences in the DNA. The
various
restriction enzymes used herein are commercially available and their reaction
conditions, cofactors and other requirements were used as would be known to
the
ordinarily skilled artisan. For analytical purposes, typically 1 g of plasmid
or
DNA fragment is used with about 2 units of enzyme in about 20 I of buffer
10 solution. For the purpose of isolating DNA fragments for plasmid
construction,
typically 5 to 50 Lig of DNA are digested with 20 to 250 units of enzyme in a
larger volume. Appropriate buffers and substrate amounts for particular
restriction enzymes are specified by the manufacturer. Incubationitimes of
about 1
hour at 37 C are ordinarily used, but may vary in accordance with the
supplier's
15 instructions. After digestion the reaction is electrophoresed directly
on a gel to
isolate the desired fragment.
"Directional ligation" refers to a ligation in which a 5' end and a 3' end
of a polynucleotide are different enough to specify a preferred ligation
orientation.
20 For example, an otherwise untreated and undigested PCR product that has
two
blunt ends will typically not have a preferred ligation orientation when
ligated
into a cloning vector digested to produce blunt ends in its multiple cloning
site;
thus, directional ligation will typically not be displayed under these
circumstances. In contrast, directional ligation will typically displayed when
a
25 digested PCR product having a 5' EcoR I-treated end and a 3' BamH I-is
ligated
into a cloning vector that has a multiple cloning site digested with EcoR I
and
BamH I.
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=
The term "DNA shuffling" is used herein to indicate recombination
between substantially homologous but non-identical sequences, in some
embodiments DNA shuffling may involve crossover via non-homologous
recombination, such as via cer/lox and/or flp/frt systems and the like.
As used in this invention, the term "epitope" refers to an antigenic
determinant on an antigen, such as a phytase polypeptide, to which the
paratope
of an antibody, such as an phytase-specific antibody, binds. Antigenic
determinants usually consist of chemically active surface groupings of
molecules,
such as amino acids or sugar side chains, and can have specific three-
dimensional
structural characteristics, as well as specific charge characteristics. As
used
herein "epitope" refers to that portion of an antigen or other macromolecule
capable of forming a binding interaction that interacts with the variable
region
binding body of an antibody. Typically, such binding interaction is manifested
as
an intermolecular contact with one or more amino acid residues of a CDR.
An "exogenous DNA segment", "heterologous sequence" or a
"heterologous nucleic acid", as used herein, is one that originates from a
source
foreign to the particular host cell, or, if from the same source, is modified
from its
original form. Thus, a heterologous gene in a host cell includes a gene that
is
endogenous to the particular host cell, but has been modified. Modification of
a
heterologous sequence in the applications described herein typically occurs
through the use of stochastic (e.g. polynucleotide shuffling & interrupted
synthesis) and non-stochastic polynucleotide reassembly. Thus, the terms refer
to
a DNA segment which is foreign or heterologous to the cell, or homologous to
the
cell but in a position within the host cell nucleic acid in which the element
is not
ordinarily found.
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"Exogenous" DNA segments are expressed to yield exogenous
polypeptides.
The term "gene" is used broadly to refer to any segment of DNA
associated with a biological function. Thus, genes include coding sequences
and/or the regulatory sequences required for their expression. Genes also
include
nonexpressed DNA segments that, for example, form recognition sequences for
other proteins. Genes can be obtained from a variety of sources, including
cloning
from a source of interest or synthesizing from known or predicted sequence
information, and may include sequences designed to have desired parameters.
An "experimentally generated (in vitro &/or in vivo) polynucleotide"
(which term includes a "recombinant polynucleotide") or an "experimentally (in
vitro &/or in vivo) generated polypeptide" (which term includes a
"experimentally generated polypeptide") is a non-naturally occurring
polynucleotide or polypeptide that includes nucleic acid or amino acid
sequences,
respectively, from more than one source nucleic acid or polypeptide, which
source
nucleic acid or polypeptide can be a naturally occurring nucleic acid or
polypeptide, or can itself have been subjected to mutagenesis or other type of
modification. The source polynucleotides or polypeptides from which the
different nucleic acid or amino acid sequences are derived are sometimes
homologous (i.e., have, or encode a polypeptide that encodes, the same or a
similar structure and/or function), and are often from different isolates,
serotypes,
strains, species, of organism or from different disease states, for example.
The terms "fragment", "derivative" and "analog" when referring to a
reference polypeptide comprise a polypeptide which retains at least one
biological
function or activity that is at least essentially same as that of the
reference
polypeptide. Furthermore, the terms "fragment", "derivative" or "analog" are
exemplified by a "pro-form" molecule, such as a low activity proprotein that
can
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be modified by cleavage to produce a mature enzyme with significantly higher
activity.
A method is provided herein for producing from a template polypeptide a
set of progeny polypeptides in which a "full range of single amino acid
substitutions" is represented at each amino acid position. As used herein,
"full
range of single amino acid substitutions" is in reference to the naturally
encoded 20 naturally encoded polypeptide-forming alpha-amino acids, as
described herein.
The term "gene" means the segment of DNA involved in producing a
polypeptide chain; it includes regions preceding and following the coding
region
(leader and trailer) as well as intervening sequences (introns) between
individual
coding segments (exons).
"Genetic instability", as used herein, refers to the natural tendency of
highly repetitive sequences to be lost through a process of reductive events
generally involving sequence simplification through the loss of repeated
sequences. Deletions tend to involve the loss of one copy of a repeat and
everything between the repeats.
The term "beterologous" means that one single-stranded nucleic acid
sequence is unable to hybridize to another single-stranded nucleic acid
sequence
or its complement. Thus areas of heterology means that areas of
polynucleotides
or polynucleotides have areas or regions within their sequence which are
unable
to hybridize to another nucleic acid or polynucleotide. Such regions or areas
are
for example areas of mutations.
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The term "homologous" or "homeologous" means that one single-
stranded nucleic acid nucleic acid sequence may hybridize to a complementary
single-stranded nucleic acid sequence. The degree of hybridization may depend
on a number of factors including the amount of identity between the sequences
and the hybridization conditions such as temperature and salt concentrations
as
discussed later. Preferably the region of identity is greater than about 5 bp,
more
preferably the region of identity is greater than 10 bp.
An immunoglobulin light or heavy chain variable region consists of a
"framework" region interrupted by three hypervariable regions, also called
CDR's.
The extent of the framework region and CDR's have been precisely defmed; see
"Sequences of Proteins of Immunological Interest" (Kabat et al, 1987). The
sequences of the framework regions of different light or heavy chains are
relatively conserved within a specie. As used herein, a "human framework
region" is a framework region that is substantially identical (about 85 or
more,
usually 90-95 or more) to the framework region of a naturally occurring human
immunoglobulin. the framework region of an antibody, that is the combined
framework regions of the constituent light and heavy chains, serves to
position
and align the CDR's. The CDR's are primarily responsible for binding to an
epitope of an antigen.
The benefits of this invention extend to "commercial applications" (or
commercial processes), which term is used to include applications in
commercial
industry proper (or simply industry) as well as non-commercial commercial
applications (e.g. biomedical research at a non-profit institution). Relevant
applications include those in areas of diagnosis, medicine, agriculture,
manufacturing, and academia.
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The term "identical" or "identity" means that two nucleic acid sequences
have the same sequence or a complementary sequence. Thus, "areas of identity"
means that regions or areas of a polynucleotide or the overall polynucleotide
are
identical or complementary to areas of another polynucleotide or the
polynucleotide.
The terms "identical" or percent "identity," in the context of two or more
nucleic acid or polypeptide sequences, refer to two or more sequences or
subsequences that are the same or have a specified percentage of amino acid
residues or nucleotides that are the same, when compared and aligned for
maximum correspondence, as measured using one of the following sequence
comparison algorithms or by visual inspection.
For sequence comparison, typically one sequence acts as a reference
sequence to which test sequences are compared. When using a sequence
comparison algorithm, test and reference sequences are input into a computer,
subsequence coordinates are designated, if necessary, and sequence algorithm
program parameters are designated. The sequence comparison algorithm then
calculates the percent sequence identity for the test sequence(s) relative to
the
reference sequence, based on the designated program parameters.
A further indication that two nucleic acid sequences or polypeptides are
substantially "identical" is that the polypeptide encoded by the first nucleic
acid
is immunologically cross reactive with, or specifically binds to, the
polypeptide
encoded by the second nucleic acid. Thus, a polypeptide is typically
substantially
identical to a second polypeptide, for example, where the two peptides differ
only
by conservative substitutions.
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The term "isolated" means that the material is removed from its original
environment (e.g., the natural environment if it is naturally occurring). For
example, a naturally-occurring polynucleotide or enzyme present in a living
animal is not isolated, but the same polynucleotide or enzyme, separated from
some or all of the coexisting materials in the natural system, is isolated.
Such
polynucleotides could be part of a vector and/or such polynucleotides or
enzymes
could be part of a composition, and still be isolated in that such vector or
composition is not part of its natural environment.
The term "isolated", when applied to a nucleic acid or protein, denotes
that the nucleic acid or protein is essentially free of other cellular
components
with which it is associated in the natural state. It is preferably in a
homogeneous
state although it can be in either a dry or aqueous solution. Purity and
homogeneity are typically determined using analytical chemistry techniques
such
as polyacrylamide gel electrophoresis or high performance liquid
chromatography. A protein which is the predominant species present in a
preparation is substantially purified. In particular, an isolated gene is
separated
from open reading frames which flank the gene and encode a protein other than
the gene of interest.
By "isolated nucleic acid" is meant a nucleic acid, e.g., a DNA or RNA
molecule, that is not immediately contiguous with the 5' and 3' flanking
sequences
with which it normally is immediately contiguous when present in the naturally
occurring genome of the organism from which it is derived. The term thus
describes, for example, a nucleic acid that is incorporated into a vector,
such as a
plasmid or viral vector; a nucleic acid that is incorporated into the genome
of a
heterologous cell (or the genome of a homologous cell, but at a site different
from
that at which it naturally occurs); and a nucleic acid that exists as a
separate
molecule, e.g., a DNA fragment produced by PCR amplification or restriction
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enzyme digestion, or an RNA molecule produced by in vitro transcription. The
term also describes a recombinant nucleic acid that forms part of a hybrid
gene
encoding additional polypeptide sequences that can be used, for example, in
the
production of a fusion protein.
As used herein "ligand" refers to a molecule, such as a random peptide or
variable segment sequence, that is recognized by a particular receptor. As one
of
skill in the art will recognize, a molecule (or macromolecular complex) can be
both a receptor and a ligand. In general, the binding partner having a smaller
molecular weight is referred to as the ligand and the binding partner having a
greater molecular weight is referred to as a receptor.
"Ligation" refers to the process of forming phosphodiester bonds between
two double stranded nucleic acid fragments (Sambrook et al, 1982, p. 146;
Sambrook, 1989). Unless otherwise provided, ligation may be accomplished
using known buffers and conditions with 10 units of T4 DNA ligase ("ligase")
per
0.5 lig of approximately equimolar amounts of the DNA fragments to be ligated.
As used herein, "linker" or "spacer" refers to a molecule or group of
molecules that connects two molecules, such as a DNA binding protein and a
random peptide, and serves to place the two molecules in a preferred
configuration, e.g., so that the random peptide can bind to a receptor with
minimal
steric hindrance from the DNA binding protein.
As used herein, a "molecular property to be evolved" includes reference
to molecules comprised of a polynucleotide sequence, molecules comprised of a
polypeptide sequence, and molecules comprised in part of a polynucleotide
sequence and in part of a polypeptide sequence. Particularly relevant - but by
no
means limiting - examples of molecular properties to be evolved include
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enzymatic activities at specified conditions, such as related to temperature;
salinity; pressure; pH; and concentration of glycerol, DMSO, detergent, &/or
any
other molecular species with which contact is made in a reaction environment.
Additional particularly relevant - but by no means limiting - examples of
molecular properties to be evolved include stabilities - e.g. the amount of a
residual molecular property that is present after a specified exposure time to
a
specified environment, such as may be encountered during storage.
A "multivalent antigenic polypeptide" or a "recombinant multivalent
antigenic polypeptide" is a non-naturally occurring polypeptide that includes
amino acid sequences from more than one source polypeptide, which source
polypeptide is typically a naturally occurring polypeptide. At least some of
the
regions of different amino acid sequences constitute epitopes that are
recognized
by antibodies found in a mammal that has been injected with the source
polypeptide. The source polypeptides from which the different epitopes are
derived are usually homologous (i.e., have the same or a similar structure
and/or
function), and are often from different isolates, serotypes, strains, species,
of
organism or from different disease states, for example.
The term "mutations" includes changes in the sequence of a wild-type or
parental nucleic acid sequence or changes in the sequence of a peptide. Such
mutations may be point mutations such as transitions or transversions. The
mutations may be deletions, insertions or duplications. A mutation can also be
a
"chimerization", which is exemplified in a progeny molecule that is generated
to
contain part or all of a sequence of one parental molecule as well as part or
all of a
sequence of at least one other parental molecule. This invention provides for
both
chimeric polynucleotides and chimeric polypeptides.
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As used herein, the degenerate "N,N,G/T" nucleotide sequence represents
32 possible triplets, where "N" can be A, C, G or T.
The term "naturally-occurring" as used herein as applied to the object
refers to the fact that an object can be found in nature. For example, a
polypeptide or polynucleotide sequence that is present in an organism
(including
viruses bacteria, protozoa, insects, plants or mammalian tissue) that can be
isolated from a source in nature and which has not been intentionally modified
by
man in the laboratory is naturally occurring. Generally, the term naturally
occurring refers to an object as present in a non-pathological (un-diseased)
individual, such as would be typical for the species.
The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides
and polymers thereof in either single- or double-stranded form. Unless
specifically limited, the term encompasses nucleic acids containing known
analogues of natural nucleotides which have similar binding properties as the
reference nucleic acid and are metabolized in a manner similar to naturally
occurring nucleotides. Unless otherwise indicated, a particular nucleic acid
sequence also implicitly encompasses conservatively modified variants thereof
(e.g. degenerate codon substitutions) and complementary sequences and as well
as
the sequence explicitly indicated. Specifically, degenerate codon
substitutions
may be achieved by generating sequences in which the third position of one or
more selected (or all) codons is substituted with mixed-base and/or
deoxyinosine
residues (Batzer et al. (1991) Nucleic Acid Res. 19: 5081; Ohtsuka et al.
(1985) J
Biol. Chem. 260: 2605-2608; Cassol et al. (1992) Rossolini et al. (1994) Mol.
Cell. Probes 8: 91-98). The term nucleic acid is used interchangeably with
gene,
cDNA, and inRNA encoded by a gene.
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"Nucleic acid derived from a gene" refers to a nucleic acid for whose
synthesis the gene, or a subsequence thereof, has ultimately served as a
template.
Thus, an mRNA, a cDNA reverse transcribed from an mRNA, an RNA
transcribed from that cDNA, a DNA amplified from the cDNA, an RNA
transcribed from the amplified DNA, etc., are all derived from the gene and
detection of such derived products is indicative of the presence and/or
abundance
of the original gene and/or gene transcript in a sample.
As used herein, a "nucleic acid molecule" is comprised of at least one
base or one base pair, depending on whether it is single-stranded or double-
stranded, respectively. Furthermore, a nucleic acid molecule may belong
exclusively or chimerically to any group of nucleotide-containing molecules,
as
exemplified by, but not limited to, the following groups of nucleic acid
molecules:
RNA, DNA, genomic nucleic acids, non-genomic nucleic acids, naturally
occurring and not naturally occurring nucleic acids, and synthetic nucleic
acids.
This includes, by way of non-limiting example, nucleic acids associated with
any
organelle, such as the mitochondria, ribosomal RNA, and nucleic acid molecules
comprised chimerically of one or more components that are not naturally
occurring along with naturally occurring components.
Additionally, a "nucleic acid molecule" may contain in part one or more
non-nucleotide-based components as exemplified by, but not limited to, amino
acids and sugars. Thus, by way of example, but not limitation, a ribozyme that
is
in part nucleotide-based and in part protein-based is considered a "nucleic
acid
molecule".
In addition, by way of example, but not limitation, a nucleic acid molecule
that is labeled with a detectable moiety, such as a radioactive or
alternatively a
non-radioactive label, is likewise considered a "nucleic acid molecule".
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The terms "nucleic acid sequence coding for" or a "DNA coding
sequence of' or a "nucleotide sequence encoding" a particular enzyme ¨ as well
as other synonymous terms ¨ refer to a DNA sequence which is transcribed and
translated into an enzyme when placed under the control of appropriate
regulatory
sequences. A "promotor sequence" is a DNA regulatory region capable of
binding RNA polymerase in a cell and initiating transcription of a downstream
(3'
direction) coding sequence. The promoter is part of the DNA sequence. This
sequence region has a start codon at its 3' terminus. The promoter sequence
does
include the minimum number of bases where elements necessary to initiate
transcription at levels detectable above background. However, after the RNA
polymerase binds the sequence and transcription is initiated at the start
codon (3'
terminus with a promoter), transcription proceeds downstream in the 3'
direction.
Within the promotor sequence will be found a transcription initiation site
(conveniently defined by mapping with nuclease Si) as well as protein binding
domains (consensus sequences) responsible for the binding of RNA polymerase.
The terms "nucleic acid encoding an enzyme (protein)" or "DNA =
encoding an enzyme (protein)" or "polynucleotide encoding an enzyme
(protein)" and other synonymous terms encompasses a polynucleotide which
includes only coding sequence for the enzyme as well as a polynucleotide which
includes additional coding and/or non-coding sequence.
In one preferred embodiment, a "specific nucleic acid molecule species"
is defined by its chemical structure, as exemplified by, but not limited to,
its
primary sequence. In another preferred embodiment, a specific "nucleic acid
molecule species" is defined by a function of the nucleic acid species or by a
function of a product derived from the nucleic acid species. Thus, by way of
non-
limiting example, a "specific nucleic acid molecule species" may be defined by
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one or more activities or properties attributable to it, including activities
or
properties attributable its expressed product.
The instant defmition of "assembling a working nucleic acid sample
into a nucleic acid library" includes the process of incorporating a nucleic
acid
sample into a vector-based collection, such as by ligation into a vector and
transformation of a host. A description of relevant vectors, hosts, and other
reagents as well as specific non-limiting examples thereof are provided
hereinafter. The instant definition of "assembling a working nucleic acid
sample into a nucleic acid library" also includes the process of incorporating
a
nucleic acid sample into a non-vector-based collection, such as by ligation to
adaptors. Preferably the adaptors can anneal to PCR primers to facilitate
amplification by PCR.
Accordingly, in a non-limiting embodiment, a "nucleic acid library" is
comprised of a vector-based collection of one or more nucleic acid molecules.
In
another preferred embodiment a "nucleic acid library" is comprised of a non-
vector-based collection of nucleic acid molecules. In yet another preferred
embodiment a "nucleic acid library" is comprised of a combined collection of
nucleic acid molecules that is in part vector-based and in part non-vector-
based.
Preferably, the collection of molecules comprising a library is searchable and
separable according to individual nucleic acid molecule species.
The present invention provides a "nucleic acid construct" or alternatively
a "nucleotide construct" or alternatively a "DNA construct". The term
"construct" is used herein to describe a molecule, such as a polynucleotide
(e.g., a
phytase polynucleotide) may optionally be chemically bonded to one or more
additional molecular moieties, such as a vector, or parts of a vector. In a
specific -
but by no means limiting - aspect, a nucleotide construct is exemplified by a
DNA
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expression DNA expression constructs suitable for the transformation of a host
cell.
An "oligonucleotide" (or synonymously an "oligo") refers to either a
single stranded polydeoxynucleotide or two complementary polydeoxynucleotide
strands which may be chemically synthesized. Such synthetic oligonucleotides
may or may not have a 5' phosphate. Those that do not will not ligate to
another
oligonucleotide without adding a phosphate with an ATP in the presence of a
kinase. A synthetic oligonucleotide will ligate to a fragment that has not
been
dephosphorylated. To achieve polymerase-based amplification (such as with
PCR), a "32-fold degenerate oligonucleotide that is comprised of, in series,
at
least a first homologous sequence, a degenerate N,N,G/T sequence, and a
second homologous sequence" is mentioned. As used in this context,
"homologous" is in reference to homology between the oligo and the parental
polynucleotide that is subjected to the polymerase-based amplification.
A nucleic acid is "operably linked" when it is placed into a functional
relationship with another nucleic acid sequence. For instance, a promoter or
enhancer is operably linked to a coding sequence if it increases the
transcription
of the coding sequence.
As used herein, the term "operably linked" refers to a linkage of
polynucleotide elements in a functional relationship. A nucleic acid is
"operably
linked" when it is placed into a functional relationship with another nucleic
acid
sequence. For instance, a promoter or enhancer is operably linked to a coding
sequence if it affects the transcription of the coding sequence. Operably
linked
means that the DNA sequences being linked are typically contiguous and, where
necessary to join two protein coding regions, contiguous and in reading frame.
However, since enhancers generally function when separated from the promoter
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by several kilobases and intronic sequences may be of variable lengths, some
polynucleotide elements may be operably linked but not contiguous.
A coding sequence is "operably linked to" another coding sequence when
RNA polymerase will transcribe the two coding sequences into a single mRNA,
which is then translated into a single polypeptide having amino acids derived
from both coding sequences. The coding sequences need not be contiguous to
one another so long as the expressed sequences are ultimately processed to
produce the desired protein.
As used herein the term "parental polynucleotide set" is a set comprised
of one or more distinct polynucleotide species. Usually this term is used in
reference to a progeny polynucleotide set which is preferably obtained by
mutagenization of the parental set, in which case the terms "parental",
"starting"
and "template" are used interchangeably.
As used herein the term "physiological conditions" refers to temperature,
pH, ionic strength, viscosity, and like biochemical parameters which are
= compatible with a viable organism, and/or which typically exist
intracellularly in
a viable cultured yeast cell or mammalian cell. For example, the intracellular
conditions in a yeast cell grown under typical laboratory culture conditions
are
physiological conditions. Suitable in vitro reaction conditions for in vitro
transcription cocktails are generally physiological conditions. In general, in
vitro
physiological conditions comprise 50-200 mM NaCl or KC1, pH 6.5-8.5, 20-45 C
and 0.001-10 mM divalent cation (e.g., Mg, Ca); preferably about 150 mM
NaCI or KC1, pH 7.2-7.6, 5 mM divalent cation, and often include 0.01-1.0
NIP
percent nonspecific protein (e.g., BSA). A non-ionic detergent (Tween, -40,
Triton X-100) can often be present, usually at about 0.001 to 2%, typically
0.05-
0.2% (v/v). Particular aqueous conditions may be selected by the practitioner
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CA 02492661 2000-02-04
according to conventional methods. For general guidance, the following
buffered
aqueous conditions may be applicable: 10-250 rnM NaCl, 5-50 mM Iris HC1, pH
5-8, with optional addition of divalent cation(s) and/or metal chelators
and/or non-
ionic detergents and/or membrane fractions and/or anti-foam agents and/or
scintillants.
Standard convention (5' to 3') is used herein to describe the sequence of
double stranded polynucleotides.
The term "population" as used herein means a collection of components
such as polynucleotides, portions or polynucleotides or proteins. A "mixed
population: means a collection of components which belong to the same family
of
nucleic acids or proteins (i.e., are related) but which differ in their
sequence (i.e.,
are not identical) and hence in their biological activity.
A molecule having a "pro-form" refers to a molecule that undergoes any
combination of one or more covalent and noncovalent chemical modifications
(e.g. glycosylation, proteolytic cleavage, dimerization or oligomerization,
temperature-induced or pH-induced conformational change, association with a co-
factor, etc.) en route to attain a more mature molecular form having a
property
difference (e.g. an increase in activity) in comparison with the reference pro-
form
molecule. When two or more chemical modification (e.g. two proteolytic
cleavages, or a proteolytic cleavage and a deglycosylation) can be
distinguished
en route to the production of a mature molecule, the referemce precursor
molecule
may be termed a "pre-pro-form" molecule.
As used herein, the term "pseudorandom" refers to a set of sequences that
have limited variability, such that, for example, the degree of residue
variability at
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another position, but any pseudorandom position is allowed some degree of
residue variation, however circumscribed.
The term "purified" denotes that a nucleic acid or protein gives rise to
essentially one band in an electrophoretic gel. Particularly, it means that
the
nucleic acid or protein is at least about 50% pure, more preferably at least
about
85% pure, and most preferably at least about 99% pure.
"Quasi-repeated units", as used herein, refers to the repeats to be re-
assorted and are by definition not identical. Indeed the method is proposed
not
only for practically identical encoding units produced by mutagenesis of the
identical starting sequence, but also the reassortment of similar or related
sequences which may diverge significantly in some regions. Nevertheless, if
the
sequences contain sufficient homologies to be reassorted by this approach,
they
can be referred to as "quasi-repeated" units.
As used herein "random peptide library" refers to a set of polynucleotide
sequences that encodes a set of random peptides, and to the set of random
peptides encoded by those polynucleotide sequences, as well as the fusion
proteins contain those random peptides.
As used herein, "random peptide sequence" refers to an amino acid
sequence composed of two or more amino acid monomers and constructed by a
stochastic or random process. A random peptide can include framework or
scaffolding motifs, which may comprise invariant sequences.
As used herein, "receptor" refers to a molecule that has an affinity for a
given ligand. Receptors can be naturally occurring or synthetic molecules.
Receptors can be employed in an unaltered state or as aggregates with other
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species. Receptors can be attached, covalently or non-covalently, to a binding
member, either directly or via a specific binding substance. Examples of
receptors include, but are not limited to, antibodies, including monoclonal
antibodies and antisera reactive with specific antigenic determinants (such as
on
viruses, cells, or other materials), cell membrane receptors, complex
carbohydrates and glycoproteins, enzymes, and hormone receptors.
The term "recombinant" when used with reference to a cell indicates that
the cell replicates a heterologous nucleic acid, or expresses a peptide or
protein
encoded by a heterologous nucleic acid. Recombinant cells can contain genes
that are not found within the native (non-recombinant) form of the cell.
Recombinant cells can also contain genes found in the native form of the cell
wherein the genes are modified and re-introduced into the cell by artificial
means.
The term also encompasses cells that contain a nucleic acid endogenous to the
cell
that has been modified without removing the nucleic acid from the cell; such
modifications include those obtained by gene replacement, site-specific
mutation,
and related techniques.
"Recombinant enzymes" refer to enzymes produceil by recombinant
DNA techniques, i.e., produced from cells transformed by an exogenous DNA
construct encoding the desired enzyme. "Synthetic" enzymes are those prepared
by chemical synthesis.
A "recombinant expression cassette" or simply an "expression cassette"
is a nucleic acid construct, generated recombinantly or synthetically, with
nucleic
acid elements that are capable of effecting expression of a structural gene in
hosts
compatible with such sequences. Expression cassettes include at least
promoters
and optionally, transcription termination signals. Typically, the recombinant
expression cassette includes a nucleic acid to be transcribed (e.g., a nucleic
acid
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encoding a desired polypeptide), and a promoter. Additional factors necessary
or
helpful in effecting expression may also be used as described herein. For
example,
an expression cassette can also include nucleotide sequences that encode a
signal
sequence that directs secretion of an expressed protein from the host cell.
Transcription termination signals, enhancers, and other nucleic acid sequences
that influence gene expression, can also be included in an expression
cassette.
The term "related polynucleotides" means that regions or areas of the
polynucleotides are identical and regions or areas of the polynucleotides are
heterologous.
"Reductive reassortment", as used herein, refers to the increase in
molecular diversity that is accrued through deletion (and/or insertion) events
that
are mediated by repeated sequences.
The following terms are used to describe the sequence relationships
between two or more polynucleotides: "reference sequence," "comparison
window," "sequence identity," "percentage of sequence identity," and
"substantial identity."
A "reference sequence" is a defined sequence used as a basis for a
sequence comparison; a reference sequence may be a subset of a larger
sequence,
for example, as a segment of a full-length cDNA or gene sequence given in a
sequence listing, or may comprise a complete cDNA or gene sequence.
_25 Generally, a reference sequence is at least 20 nucleotides in length,
frequently at
least 25 nucleotides in length, and often at least 50 nucleotides in length.
Since
two polynucleotides may each (1) comprise a sequence (i.e., a portion of the
complete polynucleotide sequence) that is similar between the two
polynucleotides and (2) may further comprise a sequence that is divergent
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between the two polynucleotides, sequence comparisons between two (or more)
polynucleotides are typically performed by comparing sequences of the two
polynucleotides over a "comparison window" to identify and compare local
regions of sequence similarity.
"Repetitive Index (RI)", as used herein, is the average number of copies
of the quasi-repeated units contained in the cloning vector.
The term "restriction site" refers to a recognition sequence that is
necessary for the manifestation of the action of a restriction enzyme, and
includes
a site of catalytic cleavage. It is appreciated that a site of cleavage may or
may
not be contained within a portion of a restriction site that comprises a low
ambiguity sequence (i.e. a sequence containing the principal determinant of
the
frequency of occurrence of the restriction site). Thus, in many cases,
relevant
restriction sites contain only a low ambiguity sequence with an internal
cleavage
site (e.g. G/AATTC in the EcoR I site) or an immediately adjacent cleavage
site
(e.g. /CCWGG in the EcoR IT site). In other cases, relevant restriction
enzymes
[e.g. the Eco57 I site or CTGAAG(16/14)] contain a low ambiguity sequence
(e.g.
the CTGAAG sequence in the Eco57 I site) with an external cleavage site (e.g.
in
the N16 portion of the Eco57 I site). When an enzyme (e.g. a restriction
enzyme)
is said to "cleave" a polynucleotide, it is understood to mean that the
restriction
enzyme catalyzes or facilitates a cleavage of a polynucleotide.
The term "screening" describes, in general, a process that identifies
optimal antigens. Several properties of the antigen can be used in selection
and
screening including antigen expression, folding, stability, immunogenicity and
presence of epitopes from several related antigens. Selection is a form of
screening in which identification and physical separation are achieved
simultaneously by expression of a selection marker, which, in some genetic
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circumstances, allows cells expressing the marker to survive while other cells
die
(or vice versa). Screening markers include, for example, luciferase, beta-
galactosidase and green fluorescent protein. Selection markers include drug
and
toxin resistance genes, and the like. Because of limitations in studying
primary
immune responses in vitro, in vivo studies are particularly useful screening
methods. In these studies, the antigens are first introduced to test animals,
and the
immune responses are subsequently studied by analyzing protective immune
responses or by studying the quality or strength of the induced immune
response
using lymphoid cells derived from the immunized animal. Although spontaneous
selection can and does occur in the course of natural evolution, in the
present
methods selection is performed by man.
In a non-limiting aspect, a "selectable polynucleotide" is comprised of a
5' terminal region (or end region), an intermediate region (i.e. an internal
or
central region), and a 3' terminal region (or end region). As used in this
aspect, a
5' terminal region is a region that is located towards a 5' polynucleotide
terminus
(or a 5' polynucleotide end); thus it is either partially or entirely in a 5'
half of a
polynucleotide. Likewise, a 3' terminal region is a region that is located
towards
a 3' polynucleotide terminus (or a 3' polynucleotide end); thus it is either
partially
or entirely in a 3' half of a polynucleotide. As used in this non-limiting
exemplification, there may be sequence overlap between any two regions or even
among all three regions.
The term "sequence identity" means that two polynucleotide sequences
are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of
comparison. The term "percentage of sequence identity" is calculated by
comparing two optimally aligned sequences over the window of comparison,
determining the number of positions at which the identical nucleic acid base
(e.g.,
A, T, C, G, U, or I) occurs in both sequences to yield the number of matched
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positions, dividing the number of matched positions by the total number of
positions in the window of comparison (i.e., the window size), and multiplying
the result by 100 to yield the percentage of sequence identity. This
"substantial
identity", as used herein, denotes a characteristic of a polynucleotide
sequence,
wherein the polynucleotide comprises a sequence having at least 80 percent
sequence identity, preferably at least 85 percent identity, often 90 to 95
percent
sequence identity, and most commonly at least 99 percent sequence identity as
compared to a reference sequence of a comparison window of at least 25-50
nucleotides, wherein the percentage of sequence identity is calculated by
comparing the reference sequence to the polynucleotide sequence which may
include deletions or additions which total 20 percent or less of the reference
sequence over the window of comparison.
As known in the art "similarity" between two enzymes is determined by
comparing the amino acid sequence and its conserved amino acid substitutes of
one enzyme to the sequence of a second enzyme. Similarity may be determined
by procedures which are well-known in the art, for example, a BLAST program
(Basic Local Alignment Search Tool at the National Center for Biological
Information).
As used herein, the term "single-chain antibody" refers to a polypeptide
comprising a VH domain and a VI, domain in polypeptide linkage, generally
liked
via a spacer peptide (e.g., [Gly-Gly-Gly-Gly-Serb), and which may comprise
additional amino acid sequences at the amino- and/or carboxy- termini. For
example, a single-chain antibody may comprise a tether segment for linking to
the
encoding polynucleotide. As an example, a scFv is a single-chain antibody.
Single-chain antibodies are generally proteins consisting of one or more
polypeptide segments of at least 10 contiguous amino substantially encoded by
genes of the immunoglobulin superfamily (e.g., see Williams and Barclay, 1989,
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pp. 361-368), most
frequently encoded
by a rodent, non-human primate, avian, porcine bovine, ovine, goat, or human
heavy chain or light chain gene sequence. A functional single-chain antibody
generally contains a sufficient portion of an inununoglobulin superfamily gene
product so as to retain the property of binding to a specific target molecule,
typically a receptor or antigen (epitope).
The phrase "specifically (or selectively) binds to an antibody" or
"specifically (or selectively) immunoreactive with", when referring to a
protein
or peptide, refers to a binding reaction which is determinative of the
presence of
the protein, or an epitope from the protein, in the presence of a
heterogeneous
population of proteins and other biologics. Thus, under designated immunoassay
conditions, the specified antibodies bind to a particular protein and do not
bind in
a significant amount to other proteins present in the sample. The antibodies
raised
against a multivalent antigenic polypeptide will generally bind to the
proteins
from which one or more of the epitopes were obtained. Specific binding to an
antibody under such conditions may require an antibody that is selected for
its
specificity for a particular protein. A variety of immunoassay formats may be
used to select antibodies specifically irnmunoreactive with a particular
protein.
For example, solid-phase ELISA immunoassays, Western blots, or
inununohistochemistry are routinely used to select monoclonal antibodies
specifically imrnunoreactive with a protein. See Harlow and Lane (1988)
Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York
"Harlow and Lane"), for a description of immunoassay formats and conditions
that can be used to determine specific itrununoreactivity. Typically a
specific or
selective reaction will be at least twice background signal or noise and more
typically more than 10 to 100 times background.
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The members of a pair of molecules (e.g., an antibody-antigen pair or a
nucleic acid pair) are said to "specifically bind" to each other if they bind
to each
other with greater affinity than to other, non-specific molecules. For
example, an
antibody raised against an antigen to which it binds more efficiently than to
a non-
specific protein can be described as specifically binding to the antigen.
(Similarly, a nucleic acid probe can be described as specifically binding to a
nucleic acid target if it forms a specific duplex with the target by base
pairing
interactions (see above).)
A "specific binding affinity" between two molecules, for example, a
ligand and a receptor, means a preferential binding of one molecule for
another in
a mixture of molecules. The binding of the molecules can be considered
specific
if the binding affinity is about 1 X 104 M-1 to about I X I 06heorireater.
"Specific hybridization" is defined herein as the formation of hybrids
. between a first polynucleotide and a second polynucleotide (e.g., a
polynucleotide
having a distinct but substantially identical sequence to the first
polynucleotide),
wherein substantially unrelated polynucleotide sequences do not form hybrids
in
the mixture.
The term "specific polynucleotide" means a polynucleotide having certain
end points and having a certain nucleic acid sequence. Two polynucleotides
wherein one polynucleotide has the identical sequence as a portion of the
seCond
polynucleotide but different ends comprises two different specific
polynucleotides.
The T. is the temperature (under defined ionic strength and pH) at which
50% of the target sequence hybridizes to a perfectly matched probe. Very
stringent conditions are selected to be equal to the T., for a particular
probe. An
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example of stringent hybridization conditions for hybridization of
complementary
nucleic acids which have more than 100 complementary residues on a filter in a
Southern or northern blot is 50% formamide with I mg of heparin at 42'C, with
the hybridization being carried out overnight.
"Stringent hybridization conditions" means hybridization will occur
only if there is at least 90% identity, preferably at least 95% identity and
most
preferably at least 97% identity between the sequences. See Sambrook et al,
1989.
An example of highly "stringent" wash conditions is 0. 15M NaC1 at 72'C
for about 15 minutes. An example of stringent wash conditions is a 0.2x SSC
wash at 65'C for 15 minutes (see, Sambrook, infra., for a description of SSC
buffer). Often, a high stringency wash is preceded by a low stringency wash to
15 remove background probe signal. An example medium stringency wash for a
duplex of, e.g., more than 100 nucleotides, is lx SSC at 45 C for 15 minutes.
An
example low stringency wash for a duplex of, e.g., more than 100 nucleotides,
is
. 4-6x SSC at 40 C for 15 minutes. For short probes (e.g., about 10 to 50
nucleotides), stringent conditions typically involve salt concentrations of
less than
20 about 1.0 M Na ion, typically about 0.01 to 1.0 M Na+ ion concentration
(or
other salts) at pH 7.0 to 8.3, and the temperature is typically at least about
30 C.
Stringent conditions can also be achieved with the addition of destabilizing
agents
such as formamide. In general, a signal to noise ratio of 2x (or higher) than
that
observed for an unrelated probe in the particular hybridization assay
indicates
25 detection of a specific hybridization. Nucleic acids which do not
hybridize to each
other under stringent conditions are still substantially identical if the
polypeptides
which they encode are substantially identical. This occurs, e.g., when a copy
of a
nucleic acid is created using the maximum codon degeneracy permitted by the
genetic code.
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"Stringent hybridization conditions" and "stringent hybridization wash
conditions" in the context of nucleic acid hybridization experiments such as
Southern and northern hybridizations are sequence dependent, and are different
under different environmental parameters. Longer sequences hybridize
specifically at higher temperatures.
An extensive guide to the hybridization of nucleic acids is found in Tijssen
(1993) Laboratory Techniques in Biochemistry and Molecular Biology-
Hybridization with Nucleic Acid Probes part I chapter 2 "Overview of
principles
of hybridization and the strategy of nucleic acid probe assays", Elsevier, New
York. Generally, highly stringent hybridization and wash conditions are
selected
to be about 5 C lower than the thermal melting point (T.) for the specific
sequence at a defined ionic strength and pH. Typically, under "stringent
conditions" a probe will hybridize to its target subsequence, but to no other
sequences.
Also included in the invention are polypeptides having sequences that are
"substantially identical" to the sequence of a phytase polypepfide, such as
one of
SEQ 11)1. A "substantially identical" amino acid sequence is a sequence that
differs from a reference sequence only by conservative amino acid
substitutions,
for example, substitutions of one amino acid for another of the same class
(e.g.,
substitution of one hydrophobic amino acid, such as isoleucine, valine,
leucine, or
methionine, for another, or substitution of one polar amino acid for another,
such
as substitution of arginine for lysine, glutamic acid for aspartic acid, or
glutamine
for asparagine).
The phrase "substantially identical," in the context of two nucleic acids
or polypeptides, refers to two or more sequences or subsequences that have at
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least 60%, preferably 80%, most preferably 90-95% nucleotide or amino acid
residue identity, when compared and aligned for maximum correspondence, as
measured using one of the following sequence comparison algorithms or by
visual
inspection. Preferably, the substantial identity exists over a region of the
sequences that is at least about 50 residues in length, more preferably over a
region of at least about 100 residues, and most preferably the sequences are
substantially identical over at least about 150 residues. In some embodiments,
the
sequences are substantially identical over the entire length of the coding
regions.
A "subsequence" refers to a sequence of nucleic acids or amino acids that
comprise apart of a longer sequence of nucleic acids or amino acids (e. g.,
polypeptide) respectively.
Additionally a "substantially identical" amino acid sequence is a
sequence that differs from a reference sequence or by one or more non-
conservative substitutions, deletions, or insertions, particularly when such a
substitution occurs at a site that is not the active site the molecule, and
provided
that the polypeptide essentially retains its behavioural properties. For
example,
one or more amino acids can be deleted from a phytase polypeptide, resulting
in
modification of the structure of the polypeptide, without significantly
altering its
biological activity. For example, amino- or carboxyl-terminal amino acids that
are not required for phytase biological activity can be removed. Such
modifications can result in the development of smaller active phytase
polypeptides.
The present invention provides a "substantially pure enzyme". The term
"substantially pure enzyme" is used herein to describe a molecule, such as a
polypeptide (e.g., a phytase polypeptide, or a fragment thereof) that is
substantially free of other proteins, lipids, carbohydrates, nucleic acids,
and other
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biological materials with which it is naturally associated. For example, a
substantially pure molecule, such as a polypeptide, can be at least 60%, by
dry
weight, the molecule of interest. The purity of the polypeptides can be
determined using standard methods including, e.g., polyacrylamide gel
electrophoresis (e.g., SDS-PAGE), column chromatography (e.g., high
performance liquid chromatography (HPLC)), and amino-terminal amino acid
sequence analysis.
As used herein, "substantially pure" means an object species is the
predominant species present (i.e., on a molar basis it is more abundant than
any
other individual macromolecular species in the composition), and preferably
substantially purified fraction is a composition wherein the object species
comprises at least about 50 percent (on a molar basis) of all macromolecular
species present. Generally, a substantially pure composition will comprise
more
than about 80 to 90 percent of all macromolecular species present in the
composition. Most preferably, the object species is purified to essential
homogeneity (contaminant species cannot be detected in the composition by
conventional detection methods) wherein the composition consists essentially
of
a single macromolecular species. Solvent species, small molecules (<500
Daltons), and elemental ion species are not considered macromolecular species.
As used herein, the term "variable segment" refers to a portion of a
nascent peptide which comprises a random, pseudorandom, or defined kernal
sequence. A variable segment" refers to a portion of a nascent peptide which
comprises a random pseudorandom, or defined kernal sequence. A variable
segment can comprise both variant and invariant residue positions, and the
degree
of residue variation at a variant residue position may be limited: both
options are
selected at the discretion of the practitioner. Typically, variable segments
are
about 5 to 20 amino acid residues in length (e.g., 8 to 10), although variable
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segments may be longer and may comprise antibody portions or receptor
proteins,
such as an antibody fragment, a nucleic acid binding protein, a receptor
protein,
and the like.
The term "wild-type" mews that the polynucleotide does not comprise
any mutations. A "wild type" protein means that the protein will be active at
a
level of activity found in nature and will comprise the amino acid sequence
found
in nature.
The term "working", as in "working sample", for example, is simply a
sample with which one is working. Likewise, a "working molecule", for
example is a molecule with which one is working.
2.2. GENERAL CONSIDERATIONS & FORMATS FOR
RECOMBINATION
Component modules provides genetic vaccine with_th_e acquisition of or
improvement in a useful property or characteristic,
The present invention provides multicomponent genetic vaccines that
include one or more component modules, each of which provides the genetic
vaccine with the acquisition of or an improvement in a property or
characteristic
useful in genetic vaccination.
The invention provides significant advantages over previously used
genetic vaccines. Through use of a multicomponent vaccine, one can obtain an
immune response that is particularly effective for a particular application. A
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multicomponent genetic vaccine can, for example, contain a component that is
optimized for optimal antigen expression, as well as a component that confers
improved activation of cytotoxic T lymphocytes (CTLs) by enhancing the
presentation of the antigen on dendritic cell MHC Class I molecules.
Additional
examples are described herein.
The invention provides a new approach to vaccine development, which is
termed "antigen library immunization." No other technologies are available for
generating libraries of related antigens or optimizing known protective
antigens.
The most powerful previously existing methods for identification of vaccine
antigens, such as high throughput sequencing or expression library
immunization,
only explore the sequence space provided by the pathogen genome. These
approaches are likely to be insufficient, because they generally only target
single
pathogen strains, and because natural evolution has directed pathogens to
dOwnregulate their own immunogenicity. In contrast, the immunization protocols
of the invention, which use experimentally evolved (e.g. by polynucleotide
reassembly &/or polynucleotide site-saturation mutagenesis) antigen libraries,
provide a means to identify novel antigen sequences. Those antigens that are
most
protective can be selected from these pools by in vivo challenge models.
Antigen
library immpnization dramatically expands the diversity of available
irnmunogen
sequences, and therefore, these antigen chimera libraries can also provide
means
to defend against newly emerging pathogen variants of the future. The methods
of
the invention enable the identification of individual chimeric antigens that
provide
efficient protection against a variety of existing pathogens, providing
improved
vaccines for troops and civilian populations.
The methods of the invention provide an evolution-based approach, such
as stochastic (e.g. polynucleotide shuffling & interrupted synthesis) and non-
stochastic polynucleotide reassembly in particular, that is an optimal
approach to
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improve the irnmunogenicity of many types of antigens. For example, the
methods provide means of obtaining optimized cancer antigens useful for
preventing and treating malignant diseases. Furthermore, an increasing number
of
self-antigens, causing autoitnmune diseases, and allergens, causing atopy,
allergy
and asthma, have been characterized. The inununogenicity and manufacturing of
these antigens can likewise be improved with the methods of this invention.
The antigen library immunization methods of the invention provide a
means by which one can obtain a recombinant antigen that has improved ability
to
induce an immune response to a pathogenic agent. A "pathogenic agent" refers
to
an organism or virus that is capable of infecting a host cell. Pathogenic
agents
typically include and/or encode a molecule, usually a polypeptide, that is
immunogenic in that an immune response is raised against the immunogenic
polypeptide. Often, the immune response raised against an immunogenic
polypeptide from one serotype of the pathogenic agent is not capable of
recognizing, and thus protecting against, a different serotype of the
pathogenic
agent, or other related pathogenic agents. In other situations, the
polypeptide
produced by a pathogenic agent is not produced in sufficient amounts, or is
not
sufficiently immunogenic, for the infected host to raise an effective immune
response against the pathogenic agent.
These problems are overcome by the methods of the invention, which
typically involve reassembling (&/or subjecting to one or more directed
evolution
methods described herein) two or more forms of a nucleic acid that encode a
polypeptide of the pathogenic agent, or antigen involved in another disease or
condition. These reassembly methods, including stochastic (e.g. polynucleofide
shuffling & interrupted synthesis) and non-stochastic polynucleotide
reassembly,
use as substrates forms of the nucleic acid that differ from each other in two
or
more nucleotides, so a library of recombinant nucleic acids results. The
library is
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then screened to identify at least one Optimized recombinant nucleic acid that
encodes an optimized recombinant antigen that has improved ability to induce
an
immune response to the pathogenic agent or other condition.
The resulting recombinant antigens often are chimeric in that they are
recognized by antibodies (Abs) reacting against multiple pathogen strains, and
generally can also elicit broad spectrum immune responses. Specific
neutralizing
antibodies are known to mediate protection against several pathogens of
interest,
although additional mechanisms, such as cytotoxic T lymphocytes, are likely to
be involved. The concept of chimeric, multivalent antigens inducing broadly
reacting antibody responses is further illustrated herein.
In preferred embodiments, the different forms of the nucleic acids that
encode antigenic polypeptides are obtained from members of a family of related
pathogenic agents.
This scheme of performing stochastic (e.g. polynucleotide shuffling &
interrupted synthesis) and non-stochastic polynucleotide reassembly using
nucleic
acids from different organisms is shown schematically herein. Therefore, these
stochastic (e.g. polynucleotide shuffling & interrupted synthesis) and non-
stochastic polynucleotide reassembly methods provide an effective approach to
generate multivalent, crossprotective antigens. The methods are useful for
obtaining individual chimeras that effectively protect against most or all
pathogen
variants.
Moreover, immunizations using entire libraries or pools of experimentally
evolved (e.g. by polynucleotide reassembly &for polynucleotide site-saturation
mutagenesis) antigen chimeras can also result in identification of chimeric
antigens that protect against pathogen variants that were not included in the
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starting population of antigens (for example, protection against strain C by
experimentally evolved (e.g. by polynucleotide reassembly &/or polynucleotide
site-saturation mutagenesis) library of chimeras/mutants of strains A and B).
Accordingly, the antigen library immunization approach enables the
development of immunogenic polypeptides that can induce immune responses
against poorly characterized, newly emerging pathogen variants.
Sequence reassembly (&/or one or more additonal directed evolution
methods described herein) can be achieved in many different formats and
permutations of formats, as described in further detail below. These formats
share
some common principles. For example, the targets for modification vary in
different applications, as does the property sought to be acquired or
improved.
Examples of candidate targets for acquisition of a property or improvement in
a
property include genes that encode proteins which have immunogenic and/or
toxigenic activity when introduced into a host organism.
The methods use at least two variant forms of a starting target. The variant
forms of candidate substrates can show substantial sequence or secondary
structural similarity with each other, but they should also differ in at least
one and
preferably at least two positions. The initial diversity between forms can be
the
result of natural variation, e.g., the different variant forms (homologs) are
obtained from different individuals or strains of an organism, or constitute
related
sequences from the same organism (e.g. , allelic variations), or constitute
homologs from different organisms (interspecific variants).
Alternatively, initial diversity can be induced, e.g., the variant forms can
be generated by error-prone transcription, such as an error-prone PCR or use
of a
polymerase which lacks proof-reading activity (see, Liao (1990) Gene 88:107-
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111), of the first variant form, or, by replication of the first form in a
mutator
strain (mutator host cells are discussed in further detail below, and are
generally
well known). A mutator strain can include any mutants in any organism impaired
in the functions of mismatch repair. These include mutant gene products of
mutS,
mutT, muff!, mutL, ovrD, dcm, vsr, umuC, umuD, sbcB, recJ, etc. The
impairment is achieved by genetic mutation, allelic replacement, selective
inhibition by an added reagent such as a small compound or an expressed
antisense RNA, or other techniques. Impairment can be of the genes noted, or
of
homologous genes in any organism. Other methods of generating initial
diversity
include methods well known to those of skill in the art, including, for
example,
treatment of a nucleic acid with a chemical or other mutagen, through
spontaneous mutation, and by inducing an error-prone repair system (e.g., SOS)
in a cell that contains the nucleic acid. The initial diversity between
substrates is
greatly augmented in subsequent steps of reassembly (&/or one or more
additonal
directed evolution methods described herein) for library generation.
Properties jnvolved in immunogenicity
Polynucleotide sequences that can positively or negatively affect the
immunogenicity of an antigen encoded by the polynucleotide are often scattered
throughout the entire antigen gene. Several of these factors are shown
diagrammatically herein. By reassembling (&/or subjecting to one or more
directed evolution methods described herein) different forms of polynucleotide
that encode the antigen using stochastic (e.g. polynucleotide shuffling &
interrupted synthesis) and non-stochastic polynucleotide reassembly, followed
by
selection for those chimeric polynucleotides that encode an antigen that can
induce an improved immune response, one can obtain primarily sequences that
have a positive influence on antigen immunogenicity. Those sequences that
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negatively affect antigen immunogenicity are eliminated. One need not know the
particular sequences involved.
The present invention provides methods for obtaining polynucleotide
sequences that, either directly or indirectly (i.e., through encoding a
polypeptide),
can modulate an immune response when present on a genetic vaccine vector. In
another embodiment, the invention provides methods for optimizing the
transport
and presentation of antigens. The optimized immunomodulatory polynucleotides
obtained using the methods of the invention are particularly suited for use in
conjunction with vaccines, including genetic vaccines. One of the advantages
of =
genetic vaccines is that one can incorporate genes encoding immunomodulatory
molecules, such as cytokines, costimulatory molecules, and molecules that
improve antigen transport and presentation into the genetic vaccine vectors.
This
provides opportunities to modulate immune responses that are induced against
the
antigens expressed by the genetic vaccines.
Obtaining components for use bugnetic vaccines that are more effective
through the creation of a_library, the screening of the library, and the use
of
recombinant nucleic acids that exhibit improved properties,
In additional embodiments, the present invention provides methods of
obtaining components for use in genetic vaccines, including the multicomponent
vaccines, that are more effective in conferring a desired immune response
property upon a genetic vaccine. The methods involve creating a library of
recombinant nucleic acids and screening the library to identify those library
members that exhibits an enhanced capacity to confer a desired property upon a
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genetic vaccine. Those recombinant nucleic acids that exhibit improved
properties
can be used as components in a genetic vaccine, either directly as a
polynucleotide or as a protein that is obtained by expression of the component
nucleic acid.
Improvementgoals
The properties or characteristics that can be sought to be acquired or
improved vary widely, and, of course depend on the choice of substrate. For
genetic vaccines, improvement goals include higher titer, more stable
expression,
improved stability, higher specificity targeting, higher or lower frequency of
integration, reduced immunogenicity of the vector or an expression product
thereof, increased immunogenicity of the antigen, higher expression of gene
products, and the like. Other properties for which optimization is desired
include
the tailoring of an immune response to be most effective for a particular
application. Examples of genetic vaccine components are shown, described &/or
referenced herein. Two or
more components
can be included in a single vector molecule, or each component can be present
in
a genetic vaccine formulation as a separate molecule.
Sequence reassembtv (8c/or one orinoreadditonal directed evolution
methods described herein) can be achieved through different formats which
share some common principles
In the methods of the invention, at least two variant forms of a nucleic
acid are reassembled (&/or subjected to one or more directed evolution methods
described herein) to produce a library of recombinant nucleic acids, which is
then
screened to identify at least one recombinant component that is optimized for
the
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particular vaccine property. Often, improvements are achieved after one round
of
reassembly (&/or one or more additonal directed evolution methods described
herein) and selection. Sequence reassembly (&/or one or more additonal
directed
evolution methods described herein) can be achieved in many different formats
and permutations of formats, as described in further detail below. These
formats
share some common principles. A family of nucleic acid molecules that have
some sequence identity to each other, but differ in the presence of mutations,
is
typically used as a substrate for reassembly (&/or one or more additonal
directed
evolution methods described herein). In any given cycle, reassembly (&Jor one
or
more additonal directed evolution methods described herein) can occur in vivo
or
in vitro, intracellularly or extracellularly. Furthermore, diversity resulting
from
reassembly (&/or one or more additonal directed evolution methods described
herein) can be augmented in any cycle by applying prior methods of mutagenesis
'
(e.g., error-prone PCR or cassette mutagenesis) to either the substrates or
products
of reassembly (&/or one or more additonal directed evolution methods described
herein). In some instances, a new or improved property or characteristic can
be
achieved after only a single cycle of in vivo or in vitro reassembly (&/or one
or
more additonal directed evolution methods described herein), as when using
different, variant forms of the sequence, as homologs from different
individuals or
strains of an organism, or related sequences from the same organism, as
allelic
variations. However, recursive sequence reassembly (8r../or one or more
additonal
directed evolution methods described herein), which entails successive cycles
of
reassembly (&/or one or more additonal directed evolution methods described
herein), can also be employed to achieve still further improvements in a
desired
property, or to bring about new (or "distinct") properties, or to. generate
further
molecular diversity.
In a presently preferred embodiment, polynucleotides that encode
optimized recombinant antigens are subjected to molecular backcrossing, which
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provides a means to breed the experimentally evolved (e.g. by polynucleotide
reassembly &/or polynucleotide site-saturation mutagenesis) chimeras/mutants
back to a parental or wild-type sequence, while retaining the mutations that
are
critical to the phenotype that provides the optimized immune responses. In
addition to removing the neutral mutations, molecular backcrossing can also be
used to characterize which of the many mutations in an improved variant
contribute most to the improved phenotype. This cannot be accomplished in an
efficient library fashion by any other method. Backcrossing is performed by
reassembling (optionally in combination with other directed evolution methods
described herein) the improved sequence with a large molar excess of the
parental
sequences.
Stochastic (e.g-polynucleotide shuffling & interrupted synthesis) and non-
stochastic polynucleotide reassembly is used to obtain the library of
recombinant nucleic acids, usine_a_varietv of substrates to acquire or
improve various properties for _different applications.
Creation of Recombinant Libraries
The invention involves creating recombinant libraries of polynucleotides
that are then screened to identify those library members that exhibit a
desired
property. The recombinant libraries can be created using any of various
methods.
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Initial Diversity Between Substrates
The substrate nucleic acids used for the reassembly (&/or one or more
additonal directed evolution methods described herein) can vary depending upon
the particular application. For example, where a polynucleotide that encodes a
nucleic acid binding domain or a ligand for a cell-specific receptor is to be
optimized, different forms of nucleic acids that encode all or part of the
nucleic
acid binding domain or a ligand for a cell-specific receptor are subjected to
reassembly (&/or one or more additonal directed evolution methods described
herein).
In a presently preferred embodiment, stochastic (e.g. polynucleotide
shuffling & interrupted synthesis) and non-stochastic polynucleotide
reassembly
is used to obtain the library of recombinant nucleic acids. stochastic (e.g.
polynucleotide shuffling & interrupted synthesis) and non-stochastic
polynucleotide reassembly, which is described herein, can result in
optimization
of a desired property even in the absence of a detailed understanding of the
mechanism by which the particular property is mediated. The substrates for
this
modification, or evolution, vary in different applications, as does the
property
sought to be acquired or improved. Examples of candidate substrates for
acquisition of a property or improvement in a property include viral and
nonviral
vectors used in genetic vaccination, as well as nucleic acids that are
involved in
mediating a particular aspect of an immune response. The methods require at
least
two variant forms of a starting substrate. The variant forms of candidate
components can have substantial sequence or secondary structural similarity
with
each other, but they should also differ in at least two positions. The initial
diversity between forms can be the result of natural variation, e.g., the
different
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variant forms (homologs) are obtained from different individuals or strains of
an
organism (including geographic variants) or constitute related sequences from
the
same organism (e.g., allelic variations). Alternatively, the initial diversity
can be
induced, e.g., the second variant form can be generated by error-prone
transcription, such as an error- prone PCR or use of a polymerase which lacks
proof-reading activity (see, Liao (1990) Gene 88:107-111), of the first
variant
form, or, by replication of the first form in a mutator strain (mutator host
cells are
discussed in further detail below). The initial diversity between substrates
is
greatly augmented in subsequent steps of recursive sequence reassembly (&/or
one or more additonal directed evolution methods described herein).
Screening or selection after a reassembly (&/or one or more additonal
directed evolution methods described herein) cycle (screening after in vitro
and in
vivo reassembly (&/or one or more additonal directed evolution methods
described herein) cycles)
Once one has performed stochastic (e.g. polynucleotide shuffling &
interrupted synthesis) and non-stochastic polynucleotide reassembly to obtain
a
library of polynucleotides that encode recombinant antigens, the library is
subjected to selection and/or screening to identify those library members that
encode antigenic peptides that have improved ability to induce an immune
response to the pathogenic agent. Selection and screening of experimentally
generated polynucleotides that encode polypeptides having an improved ability
to
induce an immune response can involve either in vivo and in vitro methods, but
most often involves a combination of these methods. For example, in a typical
embodiment the members of a library of recombinant nucleic acids are picked,
either individually or as pools. The clones can be subjected to analysis
directly, or
can be expressed to produce the corresponding polypeptides. In a presently
preferred embodiment, an in vitro screen is performed to identify the best
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candidate sequences for the in vivo studies. Alternatively, the library can be
subjected to in vivo challenge studies directly. The analyses can employ
either the
nucleic acids themselves (e.g., as genetic vaccines), or the polypeptides
encoded
by the nucleic acids. A schematic diagram of a typical strategy shown,
described
&lor referenced herein. Both in vitro
and in
vivo methods are described in more detail below.
A cycle of reassembly (&/or one or more additonal directed evolution
methods described herein) is usually followed by at least one cycle of
screening
or selection for molecules having a desired property or characteristic. If a
cycle of
reassembly (&/or one or more additonal directed evolution methods described
herein) is performed in vitro, the products of reassembly (&/or one or more
additonal directed evolution methods described herein), i.e., recombinant
segments, are sometimes introduced into cells before the screening step.
Recombinant segments can also be linked to an appropriate vector or other
regulatory sequences before screening.
Alternatively, products of reassembly (&/or one or more additonal directed
evolution methods described herein) generated in vitro are sometimes packaged
as
viruses (in viruses- e.g., bacteriophage) before screening. If reassembly
(&/or one
or more additonal directed evolution methods described herein) is performed in
vivo, product of reassembly (&/or one or more additonal directed evolution
methods described herein) can sometimes be screened in the cells in which
reassembly (&/or one or more additional directed evolution methods described
herein) occurred. In other applications, recombinant segments are extracted
from
the cells, and optionally packaged as viruses, before screening.
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Component Rome = !mine different_ roles than the product of reassembly
(&/or one or _more additional ditectet1 evolution_methads described herein)
The nature of screening or selection depends on what property or
characteristic is to be acquired or the property or characteristic for which
improvement is sought, and many examples are discussed below. It is not
usually
necessary to understand the molecular basis by which particular products of
reassembly (&/or one or more additional directed evolution methods described
herein) (recombinant segments) have acquired new or improved properties or
characteristics relative to the starting substrates. For example, a genetic
vaccine
vector can have many component sequences each having a different intended role
(e.g., coding sequence, regulatory sequences, targeting sequences, stability-
conferring sequences, immunomodulatory sequences, sequences affecting antigen
presentation, and sequences affecting integration). Each of these component
sequences can be varied and reassembled (&/or subjected to one or more
directed
evolution methods described herein) simultaneously. Screening/selection can
then
be performed, for example, for recombinant segments that have increased
episomal maintenance in a target cell without the need to attribute such
improvement to any of the individual component sequences of the vector.
Initial screenings in bacteriaLcells vs later screening in memmallmsgth
Depending on the particular screening protocol used for a desired
property, initial round(s) of screening can sometimes be performed in
bacterial
cells due to high transfection efficiencies and ease of culture. . However,
especially for testing of immunogenic activity, test animals are used for
library
=
expression and screening. Later rounds, and other types of screening which are
not amenable to screening in bacterial cells, are generally performed (in
cells
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CA 02492661 2000-02-04
selected for use in an environment close to that of their intended use) in
mammalian cells to optimize recombinant segments for use in an environment
close to that of their intended use. Final rounds of screening can be
performed in
the cell type of intended use (e.g., a human antigen-presenting cell). In some
instances, this cell can be obtained from a patient to be treated with a view,
for
example, to minimizing problems of immunogenicity in this patient. In some
methods, use of a genetic vaccine vector in treatment can itself be used as a
round
of screening. That is, genetic vaccine vectors that are successively taken up
and/or
expressed by the intended target cells in one patient are recovered from those
target cells and used to treat another patient. The genetic vaccine vectors
that are
recovered from the intended target cells in one patient are enriched for
vectors
that have evolved, i.e., have been modified by recursive reassembly (&/or one
or
more additional directed evolution methods described herein), toward improved
or new properties or characteristics for specific uptake, immunogenicity,
stability,
and the like.
Identifying a subpopulation of recombinant segments
The screening or selection step identifies a subpopulation of recombinant
segments that have evolved toward acquisition of a new or improved desired
property or properties useful in genetic vaccination. Depending on the screen,
the
recombinant segments can be screened as components of cells, components of
viruses or other vectors, or in free form. More than one round of screening or
selection can be performed after each round of reassembly (&/or one or more
additional directed evolution methods described herein).
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The second round of reassembly (&/or one or more additignal directed
evolution methods described herein)
If further improvement in a property is desired, at least one and usually a
collection of recombinant segments surviving a first round of
screening/selection
are subject to a further round of reassembly (&/or one or more additional
directed
evolution methods described herein). These recombinant segments can be
reassembled (&/or subjected to one or more directed evolution methods
described
herein) with each other or with exogenous segments representing the original
substrates or further variants thereof. Again, reassembly (&/or one or more
additional directed evolution methods described herein) can proceed in vitro
or in
vivo. If the previous screening step identifies desired recombinant segments
as
components of cells, the components can be subjected to further reassembly
(&/or
one or more additional directed evolution methods described herein) in vivo,
or
can be subjected to further reassembly (&/or one or more additional directed
evolution methods described herein) in vitro, or can be isolated before
performing
a round of in vitro reassembly (&/or one or more additional directed evolution
methods described herein). Conversely, if the previous screening step
identifies
desired recombinant segments in naked form or as components of viruses or
other
vectors, these segments can be introduced into cells to perform a round of in
vivo
reassembly (&/or one or more additional directed evolution methods described
herein). The second round of reassembly (&/or one or more additional directed
evolution methods described herein), irrespective how performed, generates
further recombinant segments which encompass additional diversity compared to
recombinant segments resulting from previous rounds.
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Additionalrounds of reassembly ador one or more additional directed
evolution methods described_herelOscreenine to sufficiently evolve the
recombinant segments
The second round of reassembly (&/or one or more additional directed
evolution methods described herein) can be followed by a further round of
screening/selection according to the principles discussed above for the first
round.
The stringency of screening/selection can be increased between rounds. Also,
the
nature of the screen and the property being screened for can vary between
rounds
if improvement in more than one property is desired or if acquiring more than
one
new property is desired.
Additional rounds of reassembly (&/or one or more additional directed
evolution methods described herein) and screening can then be performed until
the recombinant segments have sufficiently evolved to acquire the desired new
or
improved property or function.
The practice of this invention involves the construction of recombinant
nucleic acids and the expression of genes in transfected host cells. Molecular
cloning techniques to achieve these ends are known in the art. A wide variety
of
cloning and in vitro amplification methods suitable for the construction of
recombinant nucleic acids such as expression vectors are well-known to persons
of skill. General texts which describe molecular biological techniques useful
herein, including mutagenesis, include Berger and Kimmel, Guide to Molecular
Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc.,
San Diego, CA (Berger); Sambrook et al., Molecular Cloning - A Laboratory
Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor,
New York, 1989 ("Sambrook") and Current Protocols in Molecular Biology, F.M.
Ausubel et al., eds., Current Protocols, a joint venture between Greene
Publishing
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CA 02492661 2000-02-04
Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 1998)
("Ausubel")).
Examples of techniques sufficient to direct persons of skill through in vitro
amplification methods, including the polymerase chain reaction (PCR) the
ligase
chain reaction (LCR), Q - replicase amplification and other RNA polymerase
mediated techniques (e. g., NASBA) are found in Berger, Sambrook, and
Ausubel, as well as Mullis et al. (1987) U.S. Patent No. 4,683,202; PCR
Protocols
A Guide to Methods and Applications (Innis et al. eds) Academic Press Inc. San
Diego, CA (1990) (Innis); Antheim & Levinson (October 1, 1990) C&EN 36-47;
The Journal Of NIH Research (1991) 3, 81-94; (Kwoh et al. (1989) Proc. Natl.
Acad Sci. USA 86, 1173; Guatelli el al. (1990) Proc. Natl. Acad Sci. USA 87,
1874; Lowell et al. (1989) J Clin. Chem 35, 1826; Landegren et al. (1988)
Science
241, 1077- 1080; Van Brunt (1990) Biotechnology 8,291-294; Wu and Wallace
(1989) Gene 4,560; Barringer et al. (1990) Gene 89, 117, and Sooknanan and
Malek (1995) Biotechnology 13: 563-564.
Improved methods of cloning in vitro amplified nucleic acids are
described in Wallace et al., U.S. Pat. No. 5,426,039. Improved methods of
amplifying large nucleic acids by PCR are summarized in Cheng et al. (1994)
Nature 369: 684-685 and the references therein, in which PCR amplicons of up
to
40kb are generated. One of skill will appreciate that essentially any RNA can
be
converted into a double stranded DNA suitable for restriction digestion, PCR
expansion and sequencing using reverse transcriptase and a polymerase. See,
Ausubel, Sambrook and Berger, all supra.
Oligonucleotides for use as probes, e.g., in in vitro amplification methods,
for use as gene probes, or as reassembly targets (e.g., synthetic genes or
gene
segments) are typically synthesized chemically according to the solid phase
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CA 02492661 2010-07-27
phosphoramidite triester method described by Beaucage and Caruthers (1981)
Tetrahedron Letts., 22(20):1859-1862, e.g., using an automated synthesizer, as
described in Needham- VanDevanter et al. (1984) Nucleic Acids Res., 12:6159-
6168. Oligonucleotides can also be custom made and ordered from a variety of
commercial sources known to persons of skill.
Indeed, essentially any nucleic acid with a known sequence can be
custom ordered from any of a variety of commercial sources, such as The
Midland Certified Reagent Company, The Great American Gene Company,
ExpressGen Inc., Operon Technologies Inc. (Alameda, CA) and many others.
Similarly, peptides and antibodies can be custom ordered from any of a variety
of sources, such as PeptidoGenic, HTI Bio-products, Inc., BMA Biomedicals
Ltd (U.K.), Bio-Synthesis, Inc., and many others.
Different formats are available for performing reassembly (&/or additional
directed evolution methods described herein) and screening/selection which
allow for large numbers of mutations in a minimum number of selection
cycles and does not require the extensive analysis and computation required
by conventional methods,
A number of different formats are available by which one can create a
library of recombinant nucleic acids for screening. In some embodiments, the
methods of the invention entail performing reassembly (&/or one or more
additional directed evolution methods described herein) and screening or
selection
to "evolve" individual genes, whole plasmids or viruses, multigene clusters,
or
even whole genomes (Stemmer (1995) Bio/Technology 13:549-553). Reiterative
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cycles of reassembly (&/or one or more additional directed evolution methods
described herein) and screening/selection can be performed to further evolve
the
nucleic acids of interest. Such techniques do not require the extensive
analysis
and computation required by conventional methods for polypeptide engineering.
Reassembly allows the combination of large numbers of mutations in a minimum
number of selection cycles, in contrast to traditional, pair wise
recombiantion
events (e.g., as occur during sexual replication). Thus, the directed
evolution
techniques described herein provide particular advantages in that they provide
reassembly (optionally in combination with one or more additional directed
evolution methods described herein) between any or all of the mutations,
thereby
providing a very fast way of exploring the manner in which different
combinations of mutations can affect a desired result. In some instances,
however,
structural and/or functional information is available which, although not
required
for sequence reassembly (&/or one or more additional directed evolution
methods
described herein), provides opportunities for modification of the technique.
Four different approaches to improve immunogenfc_activity_os w_eltas
broaden specificity: reassembly (optionally in_combination with other
directed evolution methoth_clescribed_herein) on single gene, sequence
comparison of homologous genes. whole genome reassembly, codon
modification of polypeptidencodine_genes,
The stochastic (e.g. polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly methods can involve one or more of at
least four different approaches to improve immunogenic activity as well as to
broaden specificity. First, stochastic (e.g. polynucleotide shuffling &
interrupted
synthesis) and non-stochastic polynucleotide reassembly can be performed on a
single gene. Secondly, several highly homologous genes can be identified by
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CA 02492661 2000-02-04
sequence comparison with known homologous genes. These genes can be
synthesized and experimentally evolved (e.g. by polynucleotide reassembly &/or
polynucleotide site-saturation mutagenesis) as a family of homologs, to select
recombinants with the desired activity. The experimentally evolved (e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation mutagenesis)
genes
can be introduced into appropriate host cells, which can include E. coli,
yeast,
plants, fungi, animal cells, and the like, and those having the desired
properties
can be identified by the methods described herein. Third, whole genome
reassembly can be performed to shuffle genes that can confer a desired
property
upon a genetic vaccine (along with other genomic nucleic acids): For whole
genome reassembly approaches, it is not even necessary to identify which genes
are being experimentally evolved (e.g. by polynucleotide reassembly &/or
polynucleotide site-saturation mutagenesis). Instead, e.g., bacterial cell or
viral
genomes are combined and experimentally evolved (e.g. by polynucleotide
reassembly &for polynucleotide site-saturation mutagenesis) to acquire
recombinant nucleic acids that, either itself or through encoding a
polypeptide,
have enhanced ability to induce an immune response, as measured in any of the
assays described herein. Fourth, polypeptide- encoding genes can be codon
modified to access mutational diversity not present in any naturally occurring
gene.
References hr formats and examples for sequence reassembly (&/or one or
more additional directed evolution methods described herein) and for other
methods
Exemplary formats and examples for polynucleotide reassembly, gene site
saturation mutagenesis, interrupted synthesis, and additional directed
evolution
methods described herein have been described by the present inventors and co-
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CA 02492661 2000-02-04
workers in issued and co-pending applications including USPN 5,965,408 (issued
10-12-99), USPN 5,830,696 (issued 11-03-98), and USPN 5939,250 (issued 08-
17-99).
Other methods for obtaining libraries of experimentally generated
polynucleotides and/or for obtaining diversity in nucleic acids used as the
substrates for directed evolution including stochastic (e.g. polynucleotide
shuffling & interrupted synthesis) and non-stochastic polynucleotide
reassembly
include, for example, W098/42727; Smith, Ann. Rev. Genet. 19: 423-462 (1985);
Botstein and Shortie, Science 229: 1193-1201 (1985); Carter, Biochem. J237: 1-
7
(1986); Kunkel, "The efficiency of oligonucleotide directed mutagenesis" in
Nucleic acids & Molecular Biology, Eckstein and Lilley, eds., Springer Verlag,
Berlin (1987)). Included among these methods are oligonucleotide-directed
mutagenesis (Zoller and Smith, Nucl. Acids Res. 10: 6487-6500 (1982), Methods
in Enzymol. 100: 468-500 (1983), and Methods in Enzymol. 154: 329-350
(1987)) phosphothioate-modified DNA mutagenesis (Taylor et al., Nucl. Acids
Res. 13: 8749-8764 (1985); Taylor et al., Nucl. Acids Res. 13: 8765-8787
(1985);
Nalcamaye and Eckstein, Nucl. Acids Res. 14: 9679-9698 (1986); Sayers et al.,
Nucl. Acids Res. 16: 791-802 (1988); Sayers et al., Nucl. Acids Res. 16: 803-
814
(1988)), mutagenesis using uracil-containing templates (Kunkel, Proc. Nat'l.
Acad. Sci. USA 82: 488- 492 (1985) and Kunkel et al., Methods in Enzymol. 154:
367-382)); mutagenesis using gapped duplex DNA (Kramer et al., Nucl. Acids
Res. 12: 9441-9456 (1984); Kramer and Fritz, Methods in Enzymol. 154: 350-367
(1987); Kramer et al., Nucl. Acids Res. 16: 7207 (1988)); and Fritz et al.,
Nucl.
Acids Res. 16: 6987-6999 (1988)). Additional suitable methods include point
mismatch repair (Kramer et al., Cell 38: 879-887 (1984)), mutagenesis using
repair-deficient host strains (Carter et al., Nucl. Acids Res. 13: 4431-4443
(1985);
Carter, Methods in Enzymol. 154: 382-403 (1987)), deletion mutagenesis
(Eghtedarzadeh and Henikoff, Nucl. Acids Res. 14: 5115 (1986)), restriction-
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CA 02492661 2000-02-04
selection and restriction-purification (Wells et al., Phil. Trans. R. Soc.
Lond. A
317: 415-423 (1986)), mutagenesis by total gene synthesis (Nambiar et al.,
Science 223: 1299- 1301 (1984); Salcamar and Khorana, Nucl. Acids Res. 14:
6361-6372 (1988); Wells et al., Gene 34: 315- 323 (1985); and Gnmdstr6m et
al.,
Nucl. Acids Res. 13: 3305-3316 (1985). Kits for mutagenesis are commercially
available (e.g., Bio-Rad, A,mersharn International, Anglian Biotechnology).
For reassembly (&/or one or more_additional directed evolution methods
described herein) to_ generate increased diversity relative to the starting
materials, the starting materials must differ from each other in at least two
nucleotide positions,
The reassembly procedure starts with at least two substrates that generally
show substantial sequence identity to each other (i.e., at least about 30%,
50%,
70%, 80% or 90% sequence identity), but differ from each other at certain
positions. The difference can be any type of mutation, for example,
substitutions,
insertions and deletions. Often, different segments differ from each other in
about
5-20 positions. For reassembly (&/or one or more additional directed evolution
methods described herein) to generate increased diversity relative to the
starting
materials, the starting materials must differ from each other in at least two
nucleotide positions. That is, if there are only two substrates, there should
be at
least two divergent positions. If there are three substrates, for example, one
substrate can differ from the second at a single position, and the second can
differ
from the third at a different single position. The starting DNA segments can
be
natural variants of each other, for example, allelic or species variants. The
segments can also be from nonallelic genes showing some degree of structural
and usually functional relatedness (e.g., different genes within a
superfamily, such
as the family of Yersinia V- antigens, for example). The starting DNA segments
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can also be induced variants of each other. For example, one DNA segment can
be
produced by error-prone PCR replication of the other, the nucleic acid can be
treated with a chemical or other mutagen, or by substitution of a mutagenic
cassette. Induced mutants can also be prepared by propagating one (or both) of
the
segments in a mutagenic strain, or by inducing an error-prone repair system in
the
cells.
The different segments forming the starting materials are related, and might
or might not be of similar length
In these situations, strictly speaking, the second DNA segment is not a
single segment but a large family of related segments. The different segments
forming the starting materials are often the same length or substantially the
same
length. However, this need not be the case; for example; one segment can be a
subsequence of another. The segments can be present as part of larger
molecules,
such as vectors, or can be in isolated form.
The starting DNA segments are reassembled (&/or subjected to one or more
directed evolution methods described herein) to generate a library of
recombinant DNA segments varying in size which will include full length
coding sequences and any essentiaLregulatory
The starting DNA segments are reassembled (&/or subjected to one or
more directed evolution methods described herein) by any of the sequence
reassembly (&/or one or more additional directed evolution methods described
herein) formats provided herein to generate a diverse library of recombinant
DNA
segments. Such a library can vary widely in size from having fewer than 10 to
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more than 105, 1 09, 1012 or more members. In some embodiments, the starting
segments and the recombinant libraries generated will include full-length
coding
sequences and any essential regulatory sequences, such as a promoter and
polyadenylation sequence, required for expression. In other embodiments, the
recombinant DNA segments in the library can be inserted into a common vector
providing sequences necessary for expression before performing
screening/selection.
Using reassembly PCR to assemble multiple segments that have been
'I II=
1.11 ,
A further technique for recombining mutations in a nucleic acid sequence
utilizes "reassembly PCR". This method can be used to assemble multiple
segments that have been separately evolved into a full length nucleic acid
template such as a gene. This technique is performed when a pool of
advantageous mutants is known from previous work or has been identified by
screening mutants that may have been created by any mutagenesis technique
known in the art, such as PCR mutagenesis, cassette mutagenesis, doped oligo
mutagenesis, chemical mutagenesis, or propagation of the DNA template in vivo
in mutator strains. Boundaries defining segments of a nucleic acid sequence of
interest preferably lie in intergenic regions, introns, or areas of a gene not
likely to
have mutations of interest.
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Oligos are_synthesize4 for PCR amplification of segments of the nucleic acid
sequence of interest so that the lips overlap the junctions of two segments
by, typically, about 10 to 100 nucleotides
Preferably, oligonucleotide primers (oligos) are synthesized for PCR
amplification of segments of the nucleic acid sequence of interest, such that
the
sequences of the oligonucleotides overlap the junctions of two segments. The
overlap region is typically about 10 to 100 nucleotides in length. Each of the
segments is amplified with a set of such primers. The PCR products are then
"reassembled" according to assembly protocols such as those discussed herein
to
assemble non-stochastically generated nucleic acid building blocks &/or
randomly fragmented genes. In brief, in an assembly protocol the PCR products
are first purified away from the primers, by, for example, gel electrophoresis
or
size exclusion chromatography. Purified products are mixed together and
subjected to about 1-10 cycles of denaturing, reannealing, and extension in
the
presence of polymerase and deoxynucleoside triphosphates (dNTP's) and
appropriate buffer salts in the absence of additional primers ("self-
priming").
Subsequent PCR with primers flanking the gene are used to amplify the yield of
the fully reassembled and experimentally evolved (e.g. by polynucleotide
reassembly &/or polynucleotide site-saturation mutagenesis) genes.
PCR primers are used to introduce variation into the gene of interest and the
mutations at sites of interest are screened or selected by sequencing
homologues of the nucleic acid sequence
In a further embodiment, PCR primers for amplification of segments of
the nucleic acid sequence of interest are used to introduce variation into the
gene
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of interest as follows. Mutations at sites of interest in a nucleic acid
sequence are
identified by screening or selection, by sequencing homologues of the nucleic
acid sequence, and so on.
Using nftgonncleotide PCR.primers Unfailing wild_typc or mutant
, 01III I l=
permutations of saidinfo. where the alternative screening or selection
process is expensive. cumbersome¨or impractical
Oligonucleotide PCR primers are then synthesized which encode wild
type or mutant information at sites of interest. These primers are then used
in PCR
mutagenesis to generate libraries of full length genes encoding permutations
of
wild type and mutant information at the designated positions. This technique
is
typically advantageous in cases where the screening or selection process is
expensive, cumbersome, or impractical relative to the cost of sequencing the
genes of mutants of interest and synthesizing mutagenic oligonucleotides.
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2.3. VECTORS USED IN GENETIC VACCINATION
Evolution of genet' vaccines and_components by stochastic (e u,õ
polynucleotide shuffling & interrupted synthesis) and non-stochastic
polynucleotide reassembly
The invention provides multicomponent genetic vaccines, and methods of
obtaining genetic vaccine components that improve the capability of the
genetic
vaccine for use in nucleic acid-mediated immunomodulation. A general approach
for evolution of genetic vaccines and components by stochastic (e.g.
polynucleotide shuffling & interrupted synthesis) and non-stochastic
polynucleotide reassembly is shown schematically herein.
Including an origin of replication is useful to obtain sufficient quantities
of
the vector prior to administration to a patient,, but might be undesirable if
the vector is designed t t integrate into_host chromosomal DNA or bind to
host mRNA or DNA,
Broadly speaking, a genetic vaccine vector is an exogenous polynucleotide
which produces a medically useful phenotypic effect upon the mammalian cell(s)
and organisms into which it is transferred. A vector may or may not have an
origin of replication. For example, it is useful to include an origin of
replication in
a vector to allow for propagation of the vector in order to obtain sufficient
quantities of the vector prior to administration to a patient. If the vector
is
designed to integrate into host chromosomal DNA or bind to host mRNA or
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DNA, or if replication in the host is otherwise undesirable, the origin of
replication can be removed before administration, or an origin can be used
that
functions in the cells used for vector production but not in the target cells.
However, in certain situations, including some of those discussed herein, it
is
desirable that the genetic vaccine vector be capable of replicating in
appropriate
host cells.
=
Incorporating nucleic ackis_that are modified by stochastic (e.g,
polyuucleodde shufflingii interrupted synthesis) and non-stochastic
polynucleotide reassembly Into viral vectors to be used in genetic vaccination
Vectors used in genetic vaccination can be viral or nonviral. Viral vectors
are usually introduced into a patient as components of a virus. Illustrative
viral
vectors into which one can incorporate nucleic acids that are modified by the
stochastic (e.g. polynucleotide shuffling & interrupted synthesis) and non-
stochastic polynucleotide reassemblyg methods of the invention include, for
example, adenovirus-based vectors (Cantwell (1996) Blood 88:4676-4683;
Ohashi (1997) Proc. Nat'l. Acad. Sci USA 94:1287-1292), Epstein-Barr virus-
based vectors (Mazda (1997) J. Immunol. Methods 204:143-15 1), adenovirus-
associated virus vectors, Sindbis virus vectors (Strong (1997) Gene Ther. 4:
624-
627), herpes simplex virus vectors (Kennedy (1997) Brain 120: 1245-1259) and
retroviral vectors (Schubert (1997) Curt Eye Res. 16:656-662).
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Techniques for transferrineDNA into a_cellusefulin vivo (naked DNA
delivered using liposomeafusing to cellular membrane or entering_through
endocytosis; permeabiliae the cells and use DNA binding protein to transport
into cell; and bombardment ofakin with_partia coated with DNA delivered
mechanically)
Nonviral vectors, typically dsDNA, can be transferred as naked DNA or
associated with a transfer-enhancing vehicle, such as a receptor- recognition
protein, liposome, lipoamine, or cationic lipid. This DNA can be transferred
into a
cell using a variety of techniques well known in the art. For example, naked
DNA
can be delivered by the use of liposomes which fuse with the cellular membrane
or are endocytosed, i.e., by employing ligands attached to the liposome, or
attached directly to the DNA, that bind to surface membrane protein receptors
of
the cell resulting in endocytosis. Alternatively, the cells may be
permeabilized to
enhance transport of the DNA into the cell, without injuring the host cells.
One
can use a DNA binding protein, e.g., HBGF-1, known to transport DNA into a
cell. Furthermore, DNA can be delivered by bombardment of the skin by gold or
other particles coated with DNA which are delivered by mechanical means, e.g.,
pressure. These procedures for delivering naked DNA to cells are useful in
vivo.
For example, by using liposomes, particularly where the liposome surface
carries
ligands specific for target cells, or are otherwise preferentially directed to
a
specific organ, one may provide for the introduction of the DNA into the
target
cells/organs in vivo.
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2.3.1. VIRAL VECTORS
Structure of viral victors often consist of a modified viral gnome and a coat
structure surrounding It. a structure which can changes' in many ways for
the viral nucleie acid in_a vector &sinned for genetic v_acsination,
Various viral vectors, such as retroviruses, adenoviruses, adenoassociated
viruses
and herpes viruses, are commonly used in genetic vaccination. They are often
made up of two components, a modified viral genome and a coat structure
surrounding it (see generally Smith (1995) Annu. Rev. Microbiol. 49, 807-83
8),
although sometimes viral vectors are introduced in naked form or coated with
proteins other than viral proteins. Most current viral vectors have coat
structures
similar to a wild type virus. This structure packages and protects the viral
nucleic
acid and provides the means to bind and enter target cells. In contrast, the
viral
nucleic acid in a vector designed for genetic vaccination can be changed in
many
ways. The goals of these changes can be, for example, to enhance or reduce
replication of the virus in target cells while maintaining its ability to grow
in
vector form in available packaging or helper cells, to incorporate new
sequences
that encode and enable appropriate expression of a gene of interest (e.g., an
antigen-encoding gene), and to alter the immunogenicity of the viral vector
itself
Viral vector nucleic acids generally comprise two components: essential cis-
acting viral sequences for replication and packaging in a helper line and a
transcription unit for the exogenous gene. Other viral functions can be
expressed
in trans in a specific packaging or helper cell line.
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2.3.1.1. ADENOVIRUSES
The normal life cycle_and production infection cycle of adenoviruses.
Adenoviruses comprise a large class of nonenveloped viruses that contain
linear
double-stranded DNA. The normal life cycle of the virus does not require
dividing cells and involves productive infection in permissive cells during
which
large amounts of virus accumulate. The productive infection cycle takes about
32-
36 hours in cell culture and comprises two phases, the early phase, prior to
viral
DNA synthesis, and the late phase, during which structural proteins and viral
DNA are synthesized and assembled into virions.
In general, adenovirus infections are associated with mild disease in humans.
E3-deletion vectors studied: replication in cells does not require E3
region, allowing insertion_of exogenous DNA sequences to yield vectors
capable of productive infection and the transient synthesis of relatively
large
amounts of encoded proteins
Adenovirus vectors are somewhat larger and more complex than retrovirus or
AAV vectors, partly because only a small fraction of the viral genome is
removed
from most current vectors. If additional genes are removed, they are provided
in
trans to produce the vector, which so far has proved difficult. Instead, two
general
types of adenovirus-based vectors have been studied, E3-deletion and El-
deletion
vectors. Some viruses in laboratory stocks of wild-type lack the E3 region and
can
grow in the absence of helper. This ability does not mean that the E3 gene
products are not necessary in the wild, only that replication in cultured
cells does
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not require them. Deletion of the E3 region allows insertion of exogenous DNA
sequences to yield vectors capable of productive infection and the transient
synthesis of relatively large amounts of encoded protein.
F1 replacement vectors grown in 293 cells utilized in most gene therapy
applications involving adenoviruses,
Deletion of the El region disables the adenovirus, but such vectors can still
be
grown because there exists an established human cell line (called "293") that
contain-3 the El region of Ad5 and that constitutively expresses the El
proteins.
Most recent gene-therapy applications involving adenovirus have utilized El
replacement vectors grown in 293 cells.
Adenovirus vectors capable of efficient episomal gene transfer, easy to grow,
can be topically applied Jo skin for antigen delivery, induction of antigen
specific immune responses can be observed, but host response limits duratign
of expression and ability to_tepea dosing in cases with high doses of first
generation vectors
The main advantages of adenovirus vectors are that they are capable of
efficient episomal gene transfer in a wide range of cells and tissues and that
they
are easy to grow in large amounts. Adenovirus-based vectors can also be used
to
deliver antigens after topical application onto the skin, and induction of
antigen-
specific immune responses can be observed following delivery to the skin (Tang
et al. (1997) Nature 388: 729-730). The main disadvantage is that the host
response to the virus appears to limit the duration of expression and the
ability to
repeat dosing, at least with high doses of first- generation vectors.
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This invention provides for the first time a phagemid system capable of
cloning large DNAInserts of_ever 111 kilobases and generating ssDNA in vitro
and in vivo corresponding to those large inserts,
In one embodiment, the directed evolution methods of the invention are used to
construct a novel adenovirus-phagemid capable of packaging DNA inserts over
kilobases in size. Incorporation of a phage origin in a plasmid using the
10 methods of the invention also generates a novel in vivo reassembly or
shuffling
format capable of evolving whole genomes of viruses, such as the 36 kb family
of
human adenoviruses. The widely used human adenovirus type 5 (Ad5) has a
genome size of 36 kb. It is difficult to shuffle this large genome in vitro
without
creating an excessive number of changes which may cause a high percentage of
nonviable recombinant variants. To minimize this problem and achieve whole
genome reassembly of Ad5, an adenovirus-phagemid was constructed. The Ad-
phagemid has been demonstrated to accept inserts as large as 15 and 24
kilobases
and to effectively generate ssDNA of that size. In a further embodiment,
larger
DNA inserts, as large as 50 to 100 kb are inserted into the Ad-phagemid of the
invention; with generation of full length ssDNA corresponding to those large
inserts. Generation of such large ssDNA non-stochastically generated nucleic
acid
building blocks &/or fragments provides a means to evolve, i.e. modify by the
recursive reassembly methods (&/or one or more additional recursive directed
evolution methods described herein) of the invention, entire viral genomes.
Thus,
this invention provides for the first time a unique phagemid system capable of
cloning large DNA inserts (>10 KB) and generating ssDNA in vitro and in vivo
corresponding to those large inserts.
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In vivo reassembly or shuffling of the genomes of related seroines of human
adenoviruses using system is useful for creation of recombinant adenovirus
variants with changes in multiple genes,
The genomes of related serotypes of human adenovirus are experimentally
evolved (e.g. by polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis) in vivo using this unique phagemid system, as described in
International Application No. PCT/US97/17302 (Publ. No. W098/13485). The
genomic DNA is first cloned into a phagemid vector, and the resulting plasmid,
designated an "Admid," can be used to produce single-stranded (ss) Admid phage
by using a helper M13 phage. To achieve in vivo reassembly (&/or one or more
additional directed evolution methods described herein), ssAdmid phages
containing the genome of homologous human adenoviruses are used to perform
high multiplicity of infection (MO!) on F+ MutS E. coil cells. The ssDNA is a
better substrate for reassembly (&/or one or more additional directed
evolution
methods described herein) enzymes such as RecA. The high MOI ensures that the
probability of having multiple cross-overs between copies of the infecting
ssAdmid DNA is high. The experimentally evolved (e.g. by polynucleotide
reassembly &/or polynucleotide site-saturation mutagenesis) adenovirus genome
is generated by purification of the double stranded Admid DNA from the
infected
cells and is introduction into a pemissive human cell line to produce the
adenovirus library. This genomic reassembly strategy is useful for creation of
recombinant adenovirus variants with changes in multiple genes. This allows
screening or selection of recombinant variant phenotypes resulting from
combinations of variations in multiple genes.
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2.3.1.2. ADENO-ASSOCIATED VIRUS (AAV)
AAV is a small, simple, nonautonomous virus containing linear single-
stranded DNA. See, Muzycka, Current Topics Microbiol. Irnmunol. 158, 97- 129
(1992). The virus requires co-infection with adenovirus or certain other
viruses in
order to replicate. AAV is widespread in the human population, as evidenced by
antibodies to the virus, but it is not associated with any known disease. AAV
genome organization is straightforward, comprising only two genes: rep and
cap.
The termini of the genome comprises terminal repeats (ITR) sequences of about
145 nucleotides.
Growth of AAV is cumbersome and helper virus such as adenovirus is often
=Ilia
AAV-based vectors typically contain only the ITR sequences flanking the
transcription unit of interest. The length of the vector DNA cannot greatly
exceed
the viral genome length of 4680 nucleotides. Currently, growth of AAV vectors
is
cumbersome and involves introducing into the host cell not only the vector
itself
but also a plasmid encoding rep and cap to provide helper functions. The
helper
plasmid lacks Ms and consequently cannot replicate and package. In addition,
helper virus such as adenovirus is often required.
Advantage: long-term expression in nondividing cells,
The potential advantage of AAV vectors is that they appear capable of
long-term expression in nondividing cells, possibly, though not necessarily,
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because the viral DNA integrates. The vectors are structurally simple, and
they
may therefore provoke less of a host-cell response than adenovirus.
2.3.13. PAPILLOMA VIRUS
Papillomaviruses are small, nonenveloped, icosahedral DNA viruses that
replicate in the nucleus of squamous epithelial cells. Papillomaviruses
consist of a
single molecule of double-stranded circular DNA about 8,000 bp in size within
a
spherical protein coat of 72 capsomeres. Such papillomaviruses are classified
by
the species they infect (e.g., bovine, human, rabbit) and by type within
species.
Over 50 distinct human papillomaviruses ("}{PV") have been described. See,
e.g.,
Fields Virology (3rd ed., eds. Fields et al., Lippincott-Raven, Philadelphia,
1996).
Cellular tropism for epithelial cells
Papillomaviruses display a marked degree of cellular tropism for epithelial
cells. Specific viral types have a preference for either cutaneous or mucosal
epithelial cells.
Benign. low-risk. intermedlate-risk,and high-risk HPVs,
All papillomaviruses have the capacity to induce cellular proliferation.
The most common clinical manifestation of proliferation is the production of
benign warts. However, many papillomaviruses have capacity to be oncogenic in
some individuals and some papillomaviruses are highly oncogenic. Based on the
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pathology of the associated lesions, most human papillomaviruses (HPVs) can be
classified in one of four major groups, benign, low-risk, intermediate-risk
and
high-risk (Fields Virology, (Fields et al., eds., Lippincott- Raven,
Philadelphia, 3d
ed. 1996); DNA Tumor Viruses: Papilloma in (Encyclopedia of Cancer, Academic
Press) Vol. 1, p 520-531). For example, viruses HPV-1, HPV-2, HPV-3, HPV-4,
and HPV-27 are associated with benign cutaneous lesions. Viruses HPV-6 and
HPV-11 are associated with vulva), penile, and laryngeal warts and are
considered
low-risk viruses as they are rarely associated with invasive carcinomas.
Viruses
HPV-16, HPV-18, HPV-31, and HPV-45 are considered high risk virus as they
are associated with a high frequency with adeno- and squamous carcinoma of the
cervix. Viruses HPV- 5 and HPV-8 are associated with benign cutaneous lesion
in
a multifactorial disease Epidermodysplasia Verruciformis (EV). Such lesions,
however, can progress into squamous cell carcinomas.
HPVssjassified for risk based on_fregueney_of cancerous legions relative to
previously classified HPVs,
These viruses do not fall under one of the four major risk groups. Newly
discovered HPVs can classified for risk based on the frequency of cancerous
lesions relative to that of HPVs that have already been classified for risk.
HPV vectors can be subjected to iterative cycles of reassembly (&/or one
or more additional directed evolution methods described herein) and screening
with a view to obtaining vectors with improved properties. Improved properties
= include increased tissue specificity, altered tissue specificity,
increased expression
level, prolonged expression, increased episomal copy number, increased or
decreased capacity for chromosomal integration, increased uptake capacity, and
other properties as discussed herein. The starting materials for reassembling
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(optionally in combination with other directed evolution methods described
herein) are typically vectors of the kind described above constructed from
different strains of human papillomaviruses, or segments or variants of such
generated by e.g., error-prone PCR or cassette mutagenesis. The human
papillomaviruses, or at least the El and E2 coding regions thereof are
preferably
human cutaneous papillomaviruses.
2.3.1.4. RETROVIRUSES
Normal virallifesysle_1111 viral genome organization,
Retroviruses comprise a large class of enveloped viruses that contain
single- stranded RNA as the viral genome. During the normal viral life cycle,
viral RNA is reverse- transcribed to yield double-stranded DNA that integrates
into the host genome and is expressed over extended periods. As a result,
infected
cells shed virus continuously without apparent harm to the host cell. The
viral
genome is small (approximately 10 kb), and its prototypical organization is
extremely simple, comprising three genes encoding gag, the group specific
antigens or core proteins; poi, the reverse transcriptase; and env, the viral
envelope protein. The termini of the RNA genome are called long terminal
repeats (LI'Rs) and include promoter and enhancer activities and sequences
involved in integration. The genome also includes a sequence required for
packaging viral RNA and splice acceptor and donor sites for generation of the
separate envelope mRNA. Most retroviruses can integrate only into replicating
cells, although human immunodeficiency virus (HIV) appears to be an exception.
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Providing the_missina_viraLfunctions to the_ retrovirus vector and
adding/removine additional features to render the vectors more efficacious
or reduce the possibility of contamination by helper virus,
Retrovirus vectors are relatively simple, containing the 5' and 3' LTRs, a
packaging sequence, and a transcription unit composed of the gene or genes of
interest, which is typically an expression cassette. To grow such a vector,
one
must provide the missing viral functions in trans using a so-called packaging
cell
line. Such a cell is engineered to contain integrated copies of gag, pol, and
env but
to lack a packaging signal so that no helper virus sequences become
encapsidated.
Additional features added to or removed from the vector and packaging cell
line
reflect attempts to render the vectors more efficacious or reduce the
possibility of
contamination by helper virus.
Potentially capable oflone-term expression, ean be grenalabrgt imollgta,
but must ensure the absence of helper virus,
For some genetic vaccine applications, retroviral vectors have the
advantage of being able integrate in the chromosome and therefore potentially
capable of long-term expression. They can be grown in relatively large
amounts,
but care is needed to ensure the absence of helper virus.
2.3.2. NON-VIRAL GENETIC VACCINE VECTORS
Nonviral nucleic acid vectors used in genetic vaccination include
plasmids, RNAs, polyamide nucleic acids, and yeast artificial chromosomes
(YACs), and the like.
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Vector organizationJusertion of enhancer sequence increases transcription,
Such vectors typically include an expression cassette for expressing a
polypeptide against which an immune response is induced. The promoter in such
an expression cassette can be constitutive, cell type-specific, stage-
specific, and/or
modulatable (e. g., by tetracycline ingestion; tetracycline-responsive
promoter).
Transcription can be increased by inserting an enhancer sequence into the
vector.
Enhancers are cis-acting sequences, typically between 10 to 300 base pairs in
length, that increase transcription by a promoter. Enhancers can effectively
increase transcription when either 5' or 3' to the transcription unit. They
are also
effective if located within an intron or within the coding sequence itself.
Typically, viral enhancers are used, including SV40 enhancers, cytomegalovirus
enhancers, polyoma enhancers, and adenovirus enhancers. Enhancer sequences
from mammalian systems are also commonly used, such as the mouse
inununoglobulin heavy chain enhancer.
Methods_for introduction oinonviral vectors into an animal,
Nonviral vectors encoding products useful in gene therapy can be
introduced into an animal by means such as lipofection, biolistics, virosomes,
liposomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA
injection, artificial virions, agent-enhanced uptake of DNA, ex vivo
transduction.
Lipofection is described in e.g., US Patent Nos. 5,049,386, 4,946,787; and
4,897,355) and lipofection reagents are sold commercially (e.g., TransfectamTm
and LipofectinTm). Cationic and neutral lipids that are suitable for efficient
receptor-recognition lipofection of polynucleotides include those of Feigner,
WO
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91/17424, WO 91/16024. Naked DNA genetic vaccines are described in, for
example, US Patent No. 5,589,486.
2.4. MULTICOMPONENT GENETIC VACCINES
Use of two or more_senarate_geneti vaccine comnonents for immunization,
providing_a_means for eliciting differentiated responses in different cell
types,
The invention provides multicomponent genetic vaccines that are designed
to obtain an optimal immune response upon administration to a mammal. In these
vaccines, two or more separate genetic vaccine components are used for
irnmnni7ation, preferably in the same formulation. Each component can be
optimized for particular functions that will occur in some cells and not in
others,
thus providing a means for eliciting differentiated responses in different
cell
types. When mutually incompatible consequences are derived from use of one
plasmid, those activities are separated into different vectors that will have
different fates and effects in vivo. Genetic vaccines are ideal for the
formulation
of several biologically active entities into one preparation. The vectors are
preferably all of the same chemical type so there is no incompatibility of
this
nature, and can all be manufactured by the same chemical and/or biological
processes. The vaccine preparation can consist of a defined molar ratio of the
separate vector components that can be formulated exactly and repeatedly.
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Developing vector components without knowledge of_mechanism by which
particular feature is controlled or property to be modified
5 Several genetic vaccine vector components that can be used as
components of a multicomponenfgenetic vaccine are described below. The
methods of the invention greatly simplify the development of such vector
components, because the mechanism by which a particular feature is controlled
and the properties of a molecule that, when modified, will enhance that
feature,
10 need not be known. Even in the absence of such knowledge, by carrying
out the
reassembly (&/or one or more additional directed evolution methods described
herein) and screening methods of the invention, one can obtain vector
components
that are improved for each of the properties listed.
2.4. VECTOR "AR ",DESIGNED TO PROVIDE OPTIMAL ANTIGEN
RELEASE
Genetic vaccine vector component "AR" is designed to provide optimal release
of
20 antigen in a form that will be recognized by antigen presenting cells
(APC) and
taken up by those cells for efficient intracellular processing and
presentation to T
helper (TH) cells. Cells transfected with AR plasmid can be considered as an
antigen factory for APC.
AR plasmids typically have one or more of the following properties, each of
which can be optimized using the stochastic (e.g. polynucleotide shuffling &
interrupted synthesis) and non-stochastic polynucleotide reassembly methods of
the invention.
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Optimal plasmid binding to and uptake by the chosen antigen expressing
cells (e.g., myocytes for intramuscular immunization or epithelial cells for
mucosa! immunization)
This is a critical property which differentiates AR from other vector
components in the multicomponent DNA vaccine. Optimal vector binding to the
target cell includes not only the concept of very avid binding and subsequent
internalization into target cells, but relative inability to bind to and enter
other
cells. Optimization of this ratio of desired binding to undesired binding will
significantly increase the number of target cells transfected. This property
can be
optimized using stochastic (e.g. polynucleotide shuffling & interrupted
synthesis)
and non-stochastic polynucleotide reassembly according to the present
invention
as described herein. For example, variant vector component sequences obtained
by stochastic (e.g. polynucleotide shuffling & interrupted synthesis) and non-
stochastic polynucleotide reassembly, combinatorial assembly of vector
components, insertion of random oligonucleotide sequences, and the like, can
first
be selected for those that bind to target cells, after which this population
of cells is
depleted for those that bind to other cells. Vector components for targeting
genetic
vaccine vectors to particular cell types, and methods of obtaining improved
targeting, are known in the art.
(a) optimal trafficking of the vector DNA to the nucleus.
Again, the present invention provides methods by which one can obtain genetic
vaccine components that are optimal for such properties.
(b) optimal transcription of the antigen gene(s).
This can involve, for example, the use of optimized promoters, enhancers,
introns,
and the like. In a preferred embodiment, cell-specific promoters are used that
only
allow transcription of the genes when the vector is within the nucleus of the
target
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cell type. In this case, specificity is derived not only from selective vector
entry
into target cells.
(c) optimal trafficking of mRNA to the cytoplasm and optimal
longevity of the mRNA in the cytoplasm.
To achieve this property, the methods of the invention are used to obtain
optimal
3' and 5' non-translated regions of the mRNA.
(d) optimal translation of the mRNA.
Again, the stochastic (e.g. polynucleotide shuffling & interrupted synthesis)
and
non-stochastic polynucleotide reassembly methods are used to obtain optimized
recombinant sequences which exhibit optimal ribosome binding and assembly of
translational machinery, plus optimal codon preference.
(e) optimal antigen structure for efficient uptake by APC.
Extracellular antigen is taken up by APC by at least five non-exclusive
mechanisms. One mechanism is sampling of the external fluid phase by
micropinocytosis and internalization of a vesicle.
Additional mechanistic considerations
The first mechanism has, as far as is presently known, no structural
requirements for an antigen in the fluid phase and is therefore not relevant
to
considerations of designing antigen structure. A second mechanism involves
binding of antigen to receptors on the APC surface; such binding occurs
according to rules that are only now being studied (these receptors are not
immunoglobulin family members and appear to represent several families of
proteins and glycoproteins capable of binding different classes of
extracellular
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proteins/glycoproteins). This type of binding is followed by receptor-
mediated
internalization, also in a vesicle. Because this mechanism is poorly
understood at
present, elements of antigen design cannot be incorporated in a rational
design
process. However, application of stochastic (e.g. polynucleotide shuffling &
interrupted synthesis) and non-stochastic polynucleotide reassembly methods,
an
empirical approach of selection of variant DNA molecules most successful at
entry into APC, can select for variants that are improved throughout this
mechanism.
The other three mechanisms all relate to specific antibody recognition of
the extracellular antigen. The first mechanism involves immunoglobulin-
mediated recognition of the specific antigen via IgG that is bound to Fc
receptors
on the cell surface. APC such as monocytes, macrophages and dendritic cells
can
be decorated with surface membrane IgG of diverse specificities. In a primary
response, this mechanism will not be operative. In previously immunized
animals,
IgG on the surface of APC can specifically bind extracellular antigen and
mediate
uptake of the bound antigen into an intracellular endosomal compartment.
Another mechanism involves binding to clonally-derived surface membrane
immunoglobulin which is present on each B cells (IgM in the case of primary B
cells and IgG when the animal has been previously exposed to the antigen). B
cells are efficient APC. Extracellular antigen can bind specifically to
surface Ig
and be internalized and processed in a membrane compartment for presentation
on the B cell surface. Finally, extracellular antigen can be recognized by
specific
soluble immunoglobulin (IgM in the case of a primary immunization and IgG in
the previously immunized animals). Complexing with Ig will elicit binding to
the
surface of APC (via Fc receptor recognition in the case of IgG) and
internalization.
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In each of these latter three mechanisms, the extent to which the
conformation of the antigen is the same as the recognition specificity of the
pre-
existing antibody is critical to the efficiency of the process of antigen
presentation. Antibodies can recognize linear protein epitopes as well as
conformational epitopes determined by the three dimensional structure of the
protein antigen. Protective antibodies that will recognize an extracellular
virus or
bacterial pathogen and by binding to its surface prevent infection or mediate
its
immune destruction (complement mediated lysis, immune complex formation and
phagocytosis) are almost exclusively generated against conformational
determinants on the proteins with native structure displayed on the surface of
the
pathogen. Hence, it is imperative for generation of host protective humoral
immunity, to have those naive B cells which bear antibody specific for
conformational epitopes present on the pathogen be stimulated by direct
contact
with T helper cells after intracellular processing of the antigen and
presentation of
degradation peptides in the context of MHC Class H. This T help will allow
selective proliferation of the relevant B cells with consequent mutation of
antibody and antigen driven selection for antibodies with increased
specificity, as
well as antibody class switching.
To summarize, optimal uptake of antigen by APC to elicit hwnoral
immunity, as well as specific CD4+ cytotoxic T cells, requires that the
antigen be
in native protein conformation (as presented subsequently to the immune system
upon natural infection) and recognized by naive B cells bearing the
appropriate
membrane antibody. Native protein conformation includes appropriate protein
folding, glycosylation and any other post- translational modifications
necessary
for optimal reactivity with the receptors (immunoglobulin and possibly non-
inununoglobulin) on APC. In addition to the three dimensional structure of the
expressed antigen required for recognition by specific antibody and
elicitation of
the required immune responses, the structure (and sequence) can be optimized
for
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increased protein stability outside the expressing cell, until the time when
it is
recognized by immune cells, including APCs. The reassembly (&/or one or more
additional directed evolution methods described herein) and screening methods
of
the invention can be used to optimize the antigen structure (and sequence) for
subsequent processing after uptake by APC so that intracellular processing
results
in derivation of the required peptide fragments for presentation on Class I or
Class
lion APC and desired immune responses.
(f) optimal partitioning of the nascent antigen into the desired
subcellular compartment or compartments.
This can be directed by signal and trafficking signals embodied in the antigen
sequence. It may be desirable for all of the antigen to be secreted from these
cells;
alternatively, all or part of the antigen could be directed to be expressed on
the
cell surface of these factory cells. Signals to direct vesicles containing the
antigen
to other subcellular compartments for post-translational modifications,
including
glycosylation, can be embodied in the antigen sequence.
(g) optimal display of the antigen on the cell surface or optimal
release of the antigen from the cells.
A variation on items (f) and (g) is to design the expression of the antigen
within
the cytoplasm of the factory cell followed by lysis of that cell to release
soluble
antigen. Cell death can be engineered by expression on the same genetic
vaccine
vector of an intracellular protein that will elicit apoptosis. In this case,
the timing
of cell death is balanced with the need for the cell to produce antigen, as
well as
the potential deleterious effect of killing some cells in a designed process.
In combination, items (a) -(h) lead to a variety of scenarios for the
optimizing the longevity and extent of antigen expression. It is not always
desirable that the antigen be expressed for the longest time at the highest
level. In
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certain clinical applications, it will be important to have antigen expression
that is
short time-low expression, short time-high expression, long time-low
expression,
long time-high expression or somewhere in between.
Plasmid AR can be designed to express one or more variants of a single antigen
gene or several quite different targets for immunization. Methods for
obtaining
optimized antigens for use in genetic vaccines are described herein. Multiple
antigens can be expressed from a monocistronic or multicistronic form of the
vector.
2.4.2. VECTOR COMPONENTS "CTL-DC", "CTL-LC" AND "CTL-
MM", DESIGNED FOR OPTIMAL PRODUCTION OF CTLs
Genetic vector components "CTL-DC", "CTL-LC" and "CTL-MM" are
designed to direct optimal production of cytotoxic CDS+ lymphocytes (CTLs) by
dendritic cells (CTL-DC), Langerhan's cells (CTL-LC), and monocytes and
macrophages (CTL-MM) These vector components direct presentation of optimal
antigen fragments in association with MHC Class I, thereby ensuring maximal
cytotoxic T cell immune responses. Cells transfected with CTL vector
components can be considered as the direct activators of this arm of specific
immunity that is usually critically important for protection against viral
diseases.
CTL vector components are typically designed to have one or more of the
following properties, each of which can be optimized using the stochastic
(e.g.
polynucleotide shuffling & interrupted synthesis) and non-stochastic
polynucleotide reassembly methods of the invention:
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(a) optimal vector binding to, and uptake by, the chosen antigen
presenting cells (e.g., dendritic cells, monocytes/macrophages, Langerhan's
cells).
This is a critical property to differentiate cri, series vectors from other
vectors in
the multicomponent DNA vaccine. CTL series vectors preferably do not bind to
or enter cells that are chosen to be the extracellular antigen expression host
via
AR vectors. This separation of functions is critical, as the intracellular
fate and
trafficking of antigen destined for stimulation of immune cells after release
from
an antigen expressing cell is quite different than the fate of antigen
destined to be
presented on the cell surface in association with MHC Class I. hi the former
case,
antigen is directed via a signal secretion sequence to be delivered intact to
the
lumen of the rough endoplasmic reticulum (RER) and then secreted. In the
latter
case, antigen is directed to remain in the cytoplasm and there be degraded
into
peptide fragments by the proteasomal system followed by delivery to the lumen
of
the RER for association with MHC Class I. These complexes of peptide and MHC
Class I are then delivered to the cell surface for specific interaction with
CD8
cytotoxic T cells. Vector components, and methods for obtaining optimized
vector
components, that are optimized for targeting to desired cell types are
described in
Optimizing transcription of the antigen gene(
This can be accomplished by optimizing promoters, enhancers, introns,
and the like, as discussed herein. Cell specific promoters are valuable in
such
vectors as an additional level of selectivity.
(b) optimal longevity of the mRNA.
Optimal 3' and 5' non-translated regions of the mRNA can be obtained using the
methods of the invention.
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=
(c) optimal translation of the mRNA.
Again, the stochastic (e.g. polynucleotide shuffling & interrupted synthesis)
and
non-stochastic polynucleotide reassembly and selection methods of the
invention
can be used to obtain polynucleotide sequences for optimal ribosome binding
and
assembly of translational machinery, as well as optimal codon preference.
(d) optimal protein conformation.
In this case, the optimal protein conformation yields appropriate cytoplasmic
proteolysis and production of the correct peptides for presentation on MHC
Class
I and elicitation of the desired specific CTL responses, rather than a
conformation
that will interact with specific antibody or other receptors on the surface of
APC.
(e) optimal proteolysis to generate the correct peptides.
The order of specific proteolytic cleavages will depend on the nature of
protein
folding and the nature of proteases either in the cytoplasm or in the
proteasome.
(f) optimal transport of the antigen peptides across the endoplasmic
reticulum membrane to be delivered into the RER lumen.
This may be mediated by recognition of the peptides by TAP proteins or by
other
membrane transporters.
(h) optimal association of the peptides with the Class 1-2 microglobulin
complex and trafficking to the cell surface via the secretory pathway.
(I) optimal display of the MHC-peptide complex with associated
accessory molecules for recognition by specific CTL.
Vector CTL can be designed to express one or more variants of a single antigen
gene or several different targets for immunization. Multiple optimized
antigens
can be expressed from a monocistronic or multicistronic form of the vector.
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=
2.4.3. VECTORS "M" DESIGNED FOR OPTIMAL RELEASE OF
IMMUNE MODULATORS
Vectors "M" are designed to direct optimal release of immune modulators,
such as cytokines and other growth factors, from target cells. Target cells
can be
either the predominant cell type in the immunized tissue or immune cells such
dendritic cells (M-DC), Langerhan's cells (M-LC), monocytes & macrophages
(M-MM)". These vectors direct simultaneous expression of optimal levels of
several immune cell "modulators" (cytokines, growth factors, and the like)
such
that the immune response is of the desired type, or combination of types, and
of
the desired level. Cells traxisfected with M vectors can be considered as the
directors of the nature of the vaccine immune response (CTL vs TH 1 VS TH2 vs
NK cell, etc.) and its magnitude. The properties of these vectors reflect the
nature
of the cell in which the vectors are designed to operate. For example, the
vectors
are designed to bind to and enter the desired cell type, and/or can have cell-
specific regulated promoters that drive transcription in the desired cell
type. The
vectors can also be engineered to direct maximal synthesis and release of the
cell
modulator proteins from the target cells in the desired ratio.
"M" genetic vaccine vectors are typically designed to have one or more of the
following properties, each of which can be optimized using the stochastic
(e.g.
polynucleotide shuffling & interrupted synthesis) and non-stochastic
polynucleotide reassembly methods of the invention:
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(a) optimal vector binding to and uptake by the chosen modulator
expressing cell.
Suitable expressing cells include, for example, muscle cells, epithelial cells
or
other dominant (by number) cell types in the target tissue, antigen presenting
cells
(e.g. dendritic cells, monocytes/macrophages, Langerhans cells). This is a
critical
property which differentiates M series vectors from those designed to bind to
and
enter other cells.
(b) optimal transcription of the immune modulator gene(s).
Again, promoters, enhancers, introns, and the like can be optimized according
to
the methods of the invention. Cell specific promoters are very valuable here
as an
additional level of selectivity.
(c) optimal longevity of the mRNA.
Optimal 3' and 5' non-translated regions of the mRNA can be obtained using the
methods of the invention.
(d) optimal translation of the mRNA.
Again, the stochastic (e.g. polynucleotide shuffling & interrupted synthesis)
and
non-stochastic polynucleotide reassembly and selection methods of the
invention
can be used to obtain polynucleotide sequences for optimal ribosome binding
and
assembly of translational machinery, as well as optimal codon preference.
(e) optimal trafficking of the modulator into the lumen of the RER
(via a signal secretion sequence).
An alternative strategy for modulation of the immune response uses membrane
anchored modulators rather than secretion of soluble modulator. Anchored
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modulator can be retained on the surface of the synthesizing cell by, for
example,
a hydrophobic tail and phosphoinositol glycan linkage.
(0 optimal protein conformation for each modulator.
In this case, the optimal protein conformation is that which allows
extracellular
modulator and/or cell membrane anchored modulator to interact with the
relevant
receptor.
(g) the ratio of modulators and their type can be determined
empirically.
One will test sets of modulators that are known to work in concert to direct
the
immune response in the direction of a TH response (e.g., production of IL-2
and/or
IFN ) or TH2 response (e.g., IL-4, IL-5, IL-13), for example. Vector M can be
designed to express one or more modulators. Optimized immunomodulators, and
methods for obtaining optimized immunomodulators, are described herein. These
optimized immunomodulatory sequences are particularly suitable for use as
components of the multicomponent genetic vaccines of the invention. Multiple
modulators can be expressed from a monocistronic or multicistronic form of the
vector.
2.4.4. VECTORS "CK", DESIGNED TO DIRECT RELEASE OF
CHEMOKINES
Genetic vaccine vectors designated "CK" are designed to direct optimal
release of chemokines from target cells. Target cells can be either the
predominant
cell type in the immunized tissue, or can be immune cells such as dendritic
cells
(CK-DC), Langerhan's cells (CK-LC), or monocytes and macrophages (CK-MM).
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These vectors typically direct simultaneous expression of optimal levels of
several
chemoldnes such that the recruitment of immune cells to the site of
immunization
is optimal. Cells transfected with CK vectors can be considered as the traffic
police, regulating the immune cells critical for the vaccine immune response.
The
properties of these vectors reflect the nature of the cell in which the
vectors are
designed to operate. For example, the vectors are designed to bind to and
enter the
desired cell type, and/or can have cell-specific regulated promoters that
drive
transcription in the desired cell type. The vectors are also engineered to
direct
maximal synthesis and release of the chemokines from the target cells in the
desired ratio. Genetic vaccine components, and methods for obtaining
components, that provide optimal release of chemoldnes are described herein.
CK vectors are typically designed to have one or more of the following
properties, each of which can be optimized using the stochastic (e.g.
polynucleotide shuffling & interrupted synthesis) and non-stochastic
polynucleotide reassembly methods of the invention:
(a) optimal vector binding to and uptake by the chosen chemokine
expressing cell.
Suitable cells include, for example, muscle cells, epithelial cells, or cell
types that
are dominant (by number) in the particular tissue of interest. Also suitable
are
antigen presenting cells (e.g. dendritic cells, monocytes and macrophages,
Langerhans cells). This is a critical property which differentiates CK series
vectors from those designed to bind to and enter other cells.
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(b) optimal transcription of the chemokine gene(s).
Again, promoters, enhancers, introns, and the like can be optimized according
to
the methods of the invention.
Cell specific promoters are very valuable here as an additional level of
selectivity.
(c) optimal longevity of the mRNA.
Optimal 3' and 5' non-translated regions of the mRNA can be obtained using the
methods of the invention.
10=
(d) optimal translation of the mRNA.
Again, the stochastic (e.g. polynucleotide shuffling & interrupted synthesis)
and
non-stochastic polynucleotide reassembly and selection methods of the
invention
can be used to obtain polynucleotide sequences for optimal ribosome binding
and
assembly of translational machinery, as well as optimal codon preference.
(e) optimal trafficking of the chemokine into the lumen of the RER
(via a signal secretion sequence).
An alternative strategy for modulation of the immune response via recruitment
of
cells will use membrane anchored chemokine rather than secretion of soluble
chemokine. Anchored chemokine will be retained on the surface of the
synthesizing cell by a hydrophobic tail and phosphoinositol glycan linkage.
(f) optimal protein conformation for each chemokine.
In this case, the optimal protein conformation is that which allows
extracellular
chemokine/cell membrane anchored chemokine to interact with the relevant
receptor.
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(g) the ratio of diverse chemokines can be determined empirically.
One can test sets of chemokines that are known to work in concert to direct
recruitment of CTL, TH cells, B cells, monocytes/macrophages, eosinophils,
and/or neutrophils as appropriate.
Vector CK can be designed to express one or more chemokines. Multiple
chemokines can be expressed from a monocistronic or multicistronic form of the
vector.
2.4.5. OTHER VECTORS
Genetic vaccines which contain one or more additional component vector
moieties are also provided by the invention. For example, the genetic vaccine
can
include a vector that is designed to specifically enter dendritic cells and
Langerhans cells, and will migrate to the draining lymph nodes.
This vector Is designed to prgyjils for expression of the target antigen(s),
as
well as a cocktail of cytakines and_ chemokines relevant to elicitation of the
desired immune response in the node
Depending on the clinical goals and nature of the antigen, the vector can
be optimized for relatively long lived expression of the target antigen so
that
stimulation of the immune system is prolonged at the node. Another example is
a
vector that specifically modulates MHC expression in B cells. Such vectors are
designed to specifically bind to and enter B cells, cells either resident in
the
injection site or attracted into the site. Within the B cell, this vector
directs the
association of antigen peptides derived from specific uptake of antigen into
the
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endocytic compartment of the cell to either association with Class I or Class
II,
hence directing the elicitation of specific immunity via CD4+ T helper cells
or
CD8+ cytotoxic lymphocytes. Numerous means exist for this intracellular
direction of the fate of processed peptide that are discussed herein.
Examples of molecules that direct Class I presentation include tapasin,
TAP-1 and TAP-2 (Koopman et al. (1997) Curr. Opin. Immunol. 9: 80-88), and
those affecting Class II presentation include, for example,
endosomal/lysosomal
proteases (Peters (1997) Curr. Opin. Immunol. 9: 89-96). Genetic vaccine
components, and methods for obtaining components, that provide optimized Class
I presentation are described herein. An optimal DNA vaccine could, for
example,
combine an AR vector (antigen release), a CTL-DC vector (CTL activation via
dendritic cell presentation of antigen peptide on MHC Class I), an M-MM vector
for release of IL- 12 and IFNg from resident tissue macrophages, and a CK
vector
for recruitment of TH cells into the immunization site.
Directed evolution aid the following DNA vaccination goals
DNA vaccination can be used for diverse goals that can include the following,
among others:
= stimulation of a CTL response and/or humoral response ready to
react rapidly and aggressively against an invading bacterial or viral pathogen
at
some time in the distant future
= a continuous but non-aggressive response to prevent inappropriate
responses to allergens
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= a continuous non-aggressive and tolerization of immunity to an
autoantigen in autoimmtme disease
= elicitation of an aggressive CTL response as rapidly as possible
against tumor cell antigens
= redirection of the immune response away from a strong but
inappropriate immune response to an on-going chronic infection in the
direction
of desired responses to clear the pathogen and/or prevent pathology.
These goals cannot always be met by the format of a single vector DNA
vaccine, particularly wherein competing goals are embodied within one DNA
sequence. A multicomponent format allows the generation of a portfolio of DNA
vaccine vectors, some of which will be reconstructed on each occasion (e.g.,
those
vectors containing antigen) while others will be used as well characterized
and
understood reagents for numerous different clinical applications (e.g., the
same
chemolcine-expressing vector can be used in different situations).
2.5. SCREENING METHODS
Screening assay varies depending of property for which improvement is
fitaight
Recombinant nucleic acid libraries that are obtained by the methods
described herein are screened to identify those DNA segments that have a
property which is desirable for genetic vaccination. The particular screening
assay
employed will vary, as described below, depending on the particular property
for
which improvement is sought. Typically, the experimentally evolved (e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation mutagenesis)
nucleic acid library is introduced into cells prior to screening. If the
stochastic
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(e.g. polynucleotide shuffling & interrupted synthesis) and non-stochastic
polynucleotide reassembly format employed is an in vivo format, the library of
recombinant DNA segments generated already exists in a cell. If the sequence
reassembly (8dor one or more additional directed evolution methods described
herein) is performed in vitro, the recombinant library is preferably
introduced into
the desired cell type before screening/selection. The members of the
recombinant
library can be linked to an episome or virus before introduction or can be
introduced directly.
Cell types
A wide variety of cell types can be used as a recipient of evolved genes.
Cells of particular interest include many bacterial cell types that are used
to
deliver vaccines or vaccine antigens (Courvaiin et al.(1995) C. R. Acad. Sci.
11118: 1207- 12), both gram- negative and gram-positive, such as salmonella
(Attridge et al. (1997) Vaccine 15: 155-62), clostridium. (Fox et al. (1996)
Gene
Ther. 3: 173-8), lactobacillus, shigella (Sizemore et al. (1995) Science 270:
299-
302), E. coli, streptococcus (Oggioni and Pozzi (1996) Gene 169: 85-90), as
well
as mammalian cells, including human cells. In some embodiments of the
invention, the library is amplified in a first host, and is then
recovered.from that
host and introduced to a second host more amenable to expression, selection,
or
screening, or any other desirable parameter. The manner in which the library
is
introduced into the cell type depends on the DNA-uptake characteristics of the
cell type, e.g., having viral receptors, being capable of conjugation, or
being
naturally competent. If the cell type is unsusceptible to natural and chemical-
induced competence, but susceptible to electroporation, one would usually
employ electroporation. If the cell type is unsusceptible to electroporation
as well,
one can employ biolistics. The biolistic PDS-1000 Gene Gun (Biorad, Hercules,
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CA 02492661 2000-02-04
CA) uses helium pressure to accelerate DNA-coated gold or tungsten
microcarriers toward target cells.
Competent or Potentially Competent Tissuft
The process is applicable to a wide range of tissues, including plants,
bacteria, fimgi, algae, intact animal tissues, tissue culture cells, and
animal
embryos. One can employ electronic pulse delivery, which is essentially a mild
electroporation format for live tissues in animals and patients (Zhao,
Advanced
Drug Delivery Reviews 17:257-262 (1995)). Novel methods for making cells
competent are described in International Patent Application PCT/US97/04494
(Publ. No. W097/35957). After introduction of the library of recombinant DNA
genes, the cells are optionally propagated to allow expression of genes to
occur.
Identifying cells that contain a vector through inclusion of a selectable
marker gene
In many assays, a means for identifying cells that contain a particular
vector is necessary. Genetic vaccine vectors of all kinds can include a
selectable
marker gene. Under selective conditions, only those cells that express the
selectable marker will survive.
Examples of Selectele Marker Genes
Examples of suitable markers include, the dihydrofolate reductase gene
(DHFR), the thymidine kinase gene (TK), or prokaryotic genes conferring drug
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resistance, gpt (xanthine- guanine phosphoribosyltransferase, which can be
selected for with mycophenolic acid; neo (neomycin phosphotransferase), which
can be selected for with 0418, hygromycin, or puromycin; and DHFR
(dihydrofolate reductase), which can be selected for with methotrexate
(Mulligan
identifying cells that contain a vector through inclusion of a screenable
marker gene
As an alternative to, or in addition to, a selectable marker, a genetic
vaccine
vector can include a screenable marker which, when expressed, confers upon a
cell containing the vector a readily identifiable phenotype. For example, gene
that
encodes a cell surface antigen that is not normally present on the host cell
is
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2.5.1. SCREENING FOR VECTOR LONGEVITY OR
TRANSLOCATION TO DESIRED TISSUE
For certain applications, it is desirable to identify those vectors with the
greatest longevity as DNA, or to identify vectors which end up in tissues
distant
from the injection site. This can be accomplished by administering to an
animal a
population of recombinant genetic vaccine vectors by the chosen route of
administration and, at various times thereafter excise the target tissue and
recover
vector from the tissue by standard molecular biology procedures. The recovered
vector molecules can be amplified in, for example, E. coil and/ or by PCR in
vitro.
The PCR amplification can involve further polynucleotide (e.g. gene, promoter,
enhancer, intron, & the like) reassembly (optionally in combination with other
directed evolution methods described herein), after which the derived selected
population used for readrninistration to animals and further improvement of
the
vector. After several rounds of this procedure, the selected vectors can be
tested
for their capacity to express the antigen in the correct conformation under
the
same conditions as the vector was selected in vivo.
Methods for in vitro Identification areas expressing the desired antigen
Because antigen expression is not part of the selection or screening
process described above, not all vectors obtained are capable of expressing
the
desired antigen. To overcome this drawback, the invention provides methods for
identifying those vectors in a genetic vaccine population that exhibit not
only the
desired tissue localization and longevity of DNA integrity in vivo, but
retention of
maximal antigen expression (or expression of other genes such as cytokines,
chemokines, cell surface accessory molecules, M3HC, and the like).
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The methods involve in vitro identification of cells which express the
desired molecule using cells purified from the tissue of choice, under
conditions
that allow recovery of very small numbers of cells and quantitative selection
of
those with different levels of antigen expression as desired.
Two embodiments of the invention are described, each of which uses a
library of genetic vaccine vectors as the starting point. The goal of each
method is
to identify those vectors that exhibit the desired biological properties in
vivo. The
recombinant library represents a population of vectors that differ in known
ways
(e.g., a combinatorial vector library of different functional modules), or has
randomly generated diversity generated either by insertion of random
nucleotide
stretches, or has been experimentally evolved (e.g. by polynucleotide
reassembly
&/or polynucleotide site-saturation mutagenesis) in vitro to introduce low
level
mutations across all or part of the vector.
2.5.1.1.SELECTION FOR EXPRESSION OF CELL SURFACE-
LOCALIZED ANTIGEN
In a first embodiment, the invention method involves selection for
expression of cell surface-localized antigen. The antigen gene is engineered
in the
vaccine vector library such that it has a region of amino acids which is
targeted to
the cell membrane. For example, the region can encode a hydrophobic stretch of
,
C-terminal amino acids which signals the attachment of a phosphoinositol-
glycan
(PIG) terminus on the expressed protein and directs the protein to be
expressed on
the surface of the transfected cell. With an antigen that is naturally a
soluble
protein, this method will likely not affect the three dimensional folding of
the
protein in this engineered fusion with a new C-terminus. With an antigen that
is
naturally a transmembrane protein (e.g., a surface membrane protein on
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pathogenic viruses, bacteria, protozoa or tumor cells) there are at least two
possibilities. First, the extracellular domain can be engineered to be in
fusion with
the C- terminal sequence for signaling PIG-linkage. Second, the protein can be
expressed in toto relying on the signaling of the host cell to direct it
efficiently to
the cell surface. In a minority of cases, the antigen for expression will have
an
endogenous PIG terminal linkage (e.g., some antigens of pathogenic protozoa).
Collection purification, identification and separation of target cells
The vector library is delivered in vivo and, after a suitable interval of time
tissue and/or cells from diverse target sites in the animal are collected.
Cells can
be purified from the tissue using standard cell biological procedures,
including the
use of cell specific surface reactive monoclonal antibodies as affinity
reagents. It
is relatively facile to purify isolated epithelial cells from mucosal sites
where
epithelium may have been inoculated or myoblasts from muscle. In some
embodiments, minimal physical purification is performed prior to analysis. It
is
sometimes desirable to identify and separate specific cell populations from
various tissues, such as spleen, liver, bone marrow, lymph node, and blood.
Blood
cells can be fractionated readily by FACS to separate B cells, CD4+ or CD8+ T
cells, dendritic cells, Langerhans cells, monocytes, and the like, using
diverse
fluorescent monoclonal antibody reagents.
Identification and purification of cells expressing the antigen
Those cells expressing the antigen can be identified with a fluorescent
monoclonal antibody specific for the C-terminal sequence on PIG-linked forms
of
the surface antigen. FACS analysis allows quantitative assessment of the level
of
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expression of the correct form of the antigen on the cell population. Cells
expressing the maximal level of antigen are sorted and standard molecular
biology methods used to recover the plasmid DNA vaccine vector that conferred
this reactivity. An alternative procedure that allows purification of all
those cells
expressing the antigen (and that may be useful prior to loading onto a cell
sorter
since antigen expressing cells may be a very small minority population), is to
rosette or pan-purify the cells expressing surface antigen. Rosettes can be
formed
between antigen expressing cells and erythrocytes bearing covalently coupled
antibody to the relevant antigen. These are readily purified by unit gravity
sedimentation. Panning of the cell population over petri dishes bearing
immobilized monoclonal antibody specific for the relevant antigen can also be
used to remove unwanted cells.
Cells expressing the required conformational structure of the target antigen
can be identified using specific conformationally-dependent monoclonal
antibodies that are known to react specifically with the same structure as
expressed on the target pathogen.
Using several monoclonal antibodies in the selection_process to minimize the
possibility of an antigen which_ reacts with_hiph affinity to the diagnostic
antibody but does not yield the correct conformation
Because one monoclonal antibody cannot define all aspects of correct
folding of the target antigen, one can minimize the possibility of an antigen
which
reacts with high affinity to the diagnostic antibody but does not yield the
correct
conformation as defined by that in which the antigen is found on the surface
of
the target pathogen or as secreted from the target pathogen. One way to
minimize
this possibility is to use several monoclonal antibodies, each known to react
with
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different conformational epitopes in the correctly folded protein, in the
selection
process. This can be achieved by secondary FACS sorting for example.
The enriched plasmid population that successfully expressed sufficient of
the antigen in the correct body site for the desired time is then used as the
starting
population for another round of selection, incorporating gene reassembling
(optionally in combination with other directed evolution methods described
herein) to expand the diversity. In this manner, one recovers the desired
biological
activity encoded by plasmid from tissues in DNA vaccine-immunized animals.
This. method can also provide the best in vivo selected vectors that express
immune accessory molecules that one may wish to incorporate into DNA vaccine
constructs. For example, if it is desired to express the accessory protein
B7.1 or
B7.2 in antigen- presenting-cells (APC) (to promote successful presentation of
antigen to T cells) one can sort APC isolated from different tissues (at or
different
to the inoculation site) using commercially available monoclonal antibodies
that
recognize functional B7 proteins.
2.5.1.2.SELECTION FOR EXPRESSION OF SECRETED
ANTIGEN/CYTOKINE/CHEMOICINE
Ssject vegtors that are optimal in_inducin2 secretion of soluble proteins that
can affect the qualitative and quantitative nature of lip elicited immune
response in vivo
The invention also provides methods to identify plasmids in a genetic
vaccine vector population that are optimal in secretion of soluble proteins
that can
affect the qualitative and quantitative nature of an elicited immune response.
For
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example, the methods are useful for selecting vectors that are optimal for
secretion of particular cytokines, growth factors and chemoldnes. The goal of
the
selection is to determine which particular combinations of cytokines,
chemokines
and growth factors, in combination with different promoters, enhancers, polyA
tracts, introns, and the like, elicits the required immune response in vivo.
Genes encoding the polypeptides are typicaly present in the vaccine vector
library in combination with optimal signalseeretion-sequences (proteins are
secreted from the cells.)
Combinations of the genes for the soluble proteins of interest can be
present in the vectors; transcription can be either from a single promoter, or
the
genes can be placed in multicistronic arrangements. Typically, the genes
encoding
the polypeptides are present in the vaccine vector library in combination with
optimal signal secretion sequences, such that the expressed proteins are
secreted
from the cells.
Generating vectors capable oLsecre.tine different combinations of soluble
factors in vitro and capable of expressing thoselletors for desired lengths of
time.
The first step in these methods is to generate vectors that are capable of
secreting high (or in some case low) levels of different combinations of
soluble
factors in vitro and that will express those factors for a short or long time
as
desired. This method allows one to select for and retain an inventory of
plasmids
which can be characterized by known patterns of soluble protein expression in
known tissues for a known time. These vectors can then be tested individually
for
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in vivo efficacy, after being placed in combination with the genetic vaccine
antigen in an appropriate expression construct.
Delivery of vector library and subsequent collection, testing, and
purification
using FACS sorting, affinity panning, rosetting, or magnetic bead separation
to separate cell populations prior to identification
The vector library is delivered to a test animal and, after a chosen interval
of time, tissue and/or cells from diverse sites on the animal are collected.
Cells are
purified from the tissue using standard cell biological procedures, which
often
include the use of cell specific surface reactive monoclonal antibodies as
affinity
reagents. As is the case for cell surface antigens described above, physical
purification of separate cell populations can be performed prior to
identification
of cells which express the desired protein. For these studies, the target
cells for
expression of cytokines will most usually be APC or B cells or T cells rather
than
muscle cells or epithelial cells. In such cases FACS sorting by established
methods will be preferred to separate the different cell types. The different
cell
types described above may also be separated into relatively pure fractions
using
affinity panning, rosetting or magnetic bead separation with panels of
existing
monoclonal antibodies known to define the surface membrane phenotype of
murine immune cells.
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Identifying and selecting purified cells through visual inspection or flow
cytometry for use in another round of selection incorporating gene
reassembling (optionally in combination with other directed evolution methods
described herein) to expand the diversity
Purified cells are plated onto agar plates under conditions that maintain
cell viability. Cells expressing the required conformational structure of the
target
antigen are identified using confonnationally-dependent monoclonal antibodies
that are known to react specifically with the same structure as expressed on
the
target pathogen. Release of the relevant soluble protein from the cells is
detected
by incubation with monoclonal antibody, followed by a secondary reagent that
gives a macroscopic signal (gold deposition, color development, fluorescence,
luminescence). Cells expressing the maximal level of antigen can be identified
by
visual inspection, the cell or cell colony picked and standard molecular
biology
methods used to recover the plasmid DNA vaccine vector that conferred this
reactivity. Alternatively, flow cytometry can be used to identify and select
cells
harboring plasmids that induce high levels of gene expression. The enriched
plasmid population that successfully expressed sufficient of the soluble
factor in
the correct body site for the desired time is then used as the starting
population for
another round of selection, incorporating gene reassembling (optionally in
combination with other directed evolution methods described herein) to expand
the diversity, if further improvement is desired. In this manner, one recovers
the
desired biological activity encoded by plasmid from tissues in DNA vaccine-
immunized animals.
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Using monoclonal antibody to confirm that the initial results from screening
still hold when several conformational epitopes are probed
Several monoclonal antibodies, each known to react with different
conformational epitopes in the correctly folded cytokine, chemokine or growth
factor, can be used to confirm that the initial results from screening with
one
monoclonal antibody reagent still hold when several conformational epitopes
are
probed. In some cases the primary probe for functional cytokine released from
the
cell/cell colony in agar could be a soluble domain of the cognate receptor.
2.5.2. FLOW CYTOMETRY
Most of the vector modulelibraries_can be assayed by flow cytometry to
select individual human tissue culture cells that contain the experimentally
generated nucleic acid sequences thathave the greatest improvement in the
desired property
Flow cytometry provides a means to efficiently analyze the functional
properties of millions of individual cells. The cells are passed through an
illumination zone, where they are hit by a laser beam; the scattered light and
fluorescence is analyzed by computer-linked detectors. Flow cytometry provides
several advantages over other methods of analyzing cell populations. Thousands
of cells can be analyzed per second, with a high degree of accuracy and
sensitivity. Gating of cell populations allows multiparameter analysis of each
sample. Cell size, viability, and morphology can be analyzed without the need
for
staining. When dyes and labeled antibodies are used, one can analyze DNA
content, cell surface and intracytoplasmic proteins, and identify cell type,
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activation state, cell cycle stage, and detect apoptosis. Up to four colors
(thus,
four separate antigens stained with different fluorescent labels) and light
scatter
characteristics can be analyzed simultaneously (four colors requires two-laser
instrument; one-laser instrument can analyze three colors). The expression
levels
of several genes can be analyzed simultaneously, and importantly, flow
cytometry-based cell sorting ("FACS sorting") allows selection of cells with
desired phenotypes. Most of the vector module libraries, including the
promoter,
enhancer, intron, episomal origin of replication, expression level aspect of
antigen, bacterial origin and bacterial marker, can be assayed by flow
cytometry
to select individual human tissue culture cells that contain the reassembled
(&/or
subjected to one or more directed evolution methods described herein) nucleic
acid sequences that have the greatest improvement in the desired property.
Typically the selection is for high level expression of a surface antigen or
surrogate marker protein, as diagrammed herein. The pool of the best
individual
sequences is recovered from the cells selected by flow cytometry-based
sorting.
An advantage of this approach is that very large numbers (>107) can be
evaluated
in a single vial experiment.
2.53. ADDITIONAL IN VITRO SCREENING METHODS
screening for improved vaccination properties using various in vitro testing
methods such as screening for improved adjuvant activity and
immunostimulatory properties,
Genetic vaccine vectors and vector modules can be screened for improved
vaccination properties using various in vitro testing methods that are known
to
those of skill in the art. For example, the optimized genetic vaccines can be
tested
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for their effect on induction of proliferation of the particular lymphocyte
type of
interest, e.g., B cells, T cells, T cell lines, and T cell clones. This type
of screening
for improved adjuvant activity and inununostimulatory properties can be
performed using, for example, human or mouse cells.
Screening for improved vaccination properties using various in vitro testing
methodsinch as screening for oytokine production (ELISA and/or
cytoplasmic cytokine staining and llow cyrometry) or for alterations in the
capacity of the vectors to direct TH1/ T112 differentiation
A library of genetic vaccine vectors, e.g. obtained either from
polynucleotide reassembly (optionally in combination with other directed
evolution methods described herein), or of vectors harboring genes encoding
cytokines, costimulatory molecules etc.) can be screened for cytokine
production
(e.g., IL-2, IL-4, IL-5, IL-6, IL- 10, IL- 12, IL- 13, IL- 15, IFN- , TNF- )
by B
cells, T cells, monocytes/macrophages, total human PBMC, or (diluted) whole
blood. Cytokines can be measured by ELISA or and cytoplasmic cytokine
staining and flow cytometry (single-cell analysis). Based on the cytokine
production profile, one can screen for alterations in the capacity of the
vectors to
direct TH1/ TH2 differentiation (as evidenced, for example, by changes in
ratios of
IL-4/ IFN- , IL-4/IL-2, IL-5/1FN- , IL-511L- 2, IL- I 3/ LF'N- , IL- 1311L-2).
Induction of APC activation can be detected based on changes in surface
expression levels of activation antigens, such as B7-1 (CD80), 137-2 (CD86),
MHC class I and II, CD14, CD23, and Fc receptors, and the like.
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Analyzing genetic yaccine vectors for their capacity to induce T cell
actjyation through isolating spleen cello' infected mice and studying the
capacity of cytotoliclUmpkocates to lyse infected, iwtologons target cells
In some embodiments, genetic vaccine vectors are analyzed for their
capacity to induce T cell activation. More specifically, spleen cells from
injected
mice can be isolated and the capacity of cytotoxic T lymphocytes to lyse
infected,
autologous target cells is studied. The spleen cells are reactivated with the
specific
antigen in vitro. In addition, T helper cell differentiation is analyzed by
measuring
proliferation or production of TH1 (IL-2 and IFN- ) and Ta (IL-4 and IL-5)
cytokines by ELISA and directly in CD4+ T cells by cytoplasmic cytolcine
staining and flow cytometry.
Testing for ability to induce humoralimmune responses with assays using,
for example,.peripheraLB lymphocytes from_ immunizedjudividnals or other
assays involving detection of antigen expression by_theiorgetsas
Genetic vaccines and vaccine components can also be tested for ability to
induce humoral immune responses, as evidenced, for example, by induction of B
cell production of antibodies specific for an antigen of interest. These
assays can
be conducted using, for example, peripheral B lymphocytes from immunized
individuals. Such assay methods are known to those of skill in the art. Other
assays involve detection of antigen expression by the target cells. For
example,
FACS selection provides the most efficient method of identifying cells which
produce a desired antigen on the cell surface. Another advantage of FACS
selection is that one can sort for different levels of expression; sometimes
lower
expression may be desired. Another method involves panning using monoclonal
antibodies on a plate. This method allows large numbers of cells to be handled
in
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a short time, but the method only selects for highest expression levels.
Capture by
magnetic beads coated with monoclonal antibodies provides another method of
identifying cells which express a particular antigen.
Screening forability to Inhibit groliferation of tumor cell lines in vitro
Genetic vaccines and vaccine components that are directed against cancer
cells can be screened for their ability to inhibit proliferation of tumor cell
lines in
vitro. Such assays are known in the art. An indication of the efficacy of a
genetic
vaccine against, for example, cancer or an autoimmune disorder, is the degree
of
skin inflammation when the vector is injected into the skin of a patient or
test
animal. Strong inflammation is correlated with strong activation of antigen-
specific T cells. Improved activation of tumor- specific T cells may lead to
enhanced killing of the tumors. In case of autoantigens, one can add
immunomodulators that skew the responses towards TH2. Skin biopsies can be
taken, enabling detailed studies of the type of immune response that occurs at
the
sites of each injection (in mice large numbers of injections/vectors can be
analyzed) Other suitable screening methods can involve detection of changes in
expression of cytokines, chemolcines, accessory molecules, and the like, by
cells
upon challenge by a library of genetic vaccine vectors.
Expressing the Recombinant Peptides_or Polypeptides as Fusions with a
Protein Displayed on the Surface of a Replicable Genetic Package
Various screening methods for particular applications are described herein. In
several instances, screening involves expressing the recombinant peptides or
polypeptides encoded by the experimentally generated polynucleotides of the
library as fusions with a protein that is displayed on the surface of a
replicable
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genetic package. For example, phage display can be used. See, e.g., Cwirla et
al.,
Proc. Natl. Acad. Sci. USA 87: 6378-6382 (1990); Devlin et al., Science 249:
404-406 (1990), Scott � Ladner et al., US 5,571,698. Other replicable
genetic packages include, for example, bacteria, eukaryotic viruses, yeast,
and
spores.
Purification and in pitro analysis ofrecombinantnucleic adds and
polypeptides
Once stochastic (e.g. polynucleotide shuffling & interrupted synthesis)
and/or non-stochastic polynucleotide reassembly has been performed, the
resulting library of experimentally generated polynucleotides can be subjected
to
purification and preliminary analysis in vitro, in order to identify the most
promising candidate recombinant nucleic acids. Advantageously, the assays can
be practiced in a high-throughput format. For example, to purify individual
experimentally evolved (e.g. by polynucleotide reassembly &/or polynucleotide
site-saturation mutagenesis) recombinant antigens, clones can robotically
picked
into 96- well formats, grown, and, if desired, frozen for storage.
Whole cell lysates (V-antigen), periplasmic extracts, or culture
supernatants (toxins) can be assayed directly by ELISA as described below, but
high throughput purification is sometimes also needed. Affinity chromatography
using immobilized antibodies or incorporation of a small nonimmunogenic
affinity tag such as a hexahistidine peptide with immobilized metal affinity
chromatography will allow rapid protein purification. High binding-capacity
reagents with 96-well filter bottom plates provide a high throughput
purification
process. The scale of culture and purification will depend on protein yield,
but
initial studies will require less than 50 micrograms of protein. Antigens
showing
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improved properties can be purified in larger scale by FPLC for re-assay and
animal challenge studies.
In some embodiments, the experimentally evolved (e.g. by polynucleotide
reassembly &/or polynucleotide site-saturation mutagenesis) antigen-encoding
polynucleotides are assayed as genetic vaccines. Genetic vaccine vectors
containing the experimentally evolved (e.g. by polynucleotide reassembly &/or
polynucleotide site-saturation mutagenesis) antigen sequences can be prepared
using robotic colony picking and subsequent robotic plasmid purification.
Robotic
plasmid purification protocols are available that allow purification of 600-
800
plasmids per day. The quantity and purity of the DNA can also be analyzed in
96-
well plates, for example. In a presently preferred embodiment, the amount of
DNA in each sample is robotically normalized, which can significantly reduce
the
variation between different batches of vectors.
Once the proteins and/or nucleic acids are picked and purified as desired,
they can be subjected to any of a number of in vitro analysis methods. Such
screenings include, for example, phage display, flow cytometry, and ELISA
assays to identify antigens that are efficiently expressed and have multiple
epitopes and a proper folding pattern. In the case of bacterial toxins, the
libraries
may also be screened for reduced toxicity in mammalian cells.
As one example, to identify recombinant antigens that are cross-reactive,
one can use a panel of monoclonal antibodies for screening. A hurnoral immune
response generally targets multiple regions of antigenic proteins.
Accordingly,
monoclonal antibodies can be raised against various regions of immunogenic
proteins (Alving et al. (1995) Immunol. Rev. 145: 5). In addition, there are
several
examples of monoclonal antibodies that only recognize one strain of a given
pathogen, and by definition, different serotypes of pathogens are recognized
by
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different sets of antibodies. For example, a panel of monoclonal antibodies
have
been raised against VEE envelope proteins, thus providing a means to recognize
different subtypes of the virus (Roehrig and Bolin (1997) J Clin. Microbiol.
35:
=
1887). Such antibodies, combined with phage display and ELISA screening, can
be used to enrich recombinant antigens that have epitopes from multiple
pathogen
strains. Flow cytometry based cell sorting will further allow for the
selection of
variants that are most efficiently expressed.
Phage display provides a powerful method for selecting proteins of
interest from large libraries (Bass et al. (1990) Proteins: Struct. Funct.
Genet. 8:
309; Lowman and Wells (1991) Methods: A Companion to Methods Enz.
3(3);205-216. Lowman and Wells (1993) J Mol. Biol. 234;564-578). Some recent
reviews on the phage display technique include, for example, McGregor (1996)
Mol Biotechnol. 6(2):15 5 -62; Dunn (1996) Curr. Opin. Biotechnol. 7(5):547-
53;
Hill et al. (1996) Mol Microbiol 20(4):685-92; Phage Display of Peptides and
Proteins: A Laboratory Manual. BK. Kay, J. Winter, J, McCafferty eds.,
Academic Press 1996; O'Neil et al. (1995) Cunr. Opin. Struct. Biol. 5(4):443-
9;
Phizicky et al. (1995) Microbiol Rev. 59(1):94-123; Clackson et al. (1994)
Trends
Biotechnol. 12(5):173-84; Felici et al. (1995) Biotechnol. Annu. Rev. 1: 149-
83;
Burton (1995) Inununotechnology 1(2):87-94.) See, also, Cwirla et al., Proc.
Natl.
Acad Sci. USA 87: 6378-6382 (1990); Devlin et al., Science 249: 404-406
(1990),
Scott & Smith, Science 249: 386-388 (1990); Ladner et al, US 5,571,698. Each
phage particle displays a unique variant protein on its surface and packages
the
gene encoding that particular variant. The experimentally evolved (e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation mutagenesis)
genes
for the antigens are fused to a protein that is expressed on the phage
surface, e.g.,
gene III of phage M 13, and cloned into phagemid vectors. In a presently
preferred embodiment, a suppressible stop codon (e.g., an amber stop codon)
separates the genes so that in a suppressing strain of E. coli, the antigen-
gUlp
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fusion is produced and becomes incorporated into phage particles upon
infection
with M 13 helper phage. The same vector can direct production of the unfused
antigen alone in a nonsuppressing E. coil for protein purification.
id I I=' = , ==11. = I =
The genetic packages most frequently used for display libraries are
bacteriophage, particularly filamentous phage, and especially phage M13, Fd
and
Fl. Most work has involved inserting libraries encoding polypeptides to be
displayed into either glII or gVIII of these phage forming a fusion protein.
See,
e.g., Dower, WO 91/19818; Devlin, WO 91/18989; MacCafferty, WO 92/01047
(gene III); Huse, WO 92/06204; Kang, WO 92/18619 (gene VIII). Such a fusion
protein comprises a signal sequence, usually but not necessarily, from the
phage
coat protein, a polypeptide to be displayed and either the gene III or gene
VIII
protein or a fragment thereof. Exogenous coding sequences are often inserted
at
or near the N-terminus of gene III or gene VIII although other insertion sites
are
possible.
Use of Eukaryotie Viruses to Display Po
Eukaryotic viruses can be used to display polypeptides in an analogous
manner. For example, display of human heregulin fused to gp70 of Moloney
murine leukemia virus has been reported by Han et al., Proc. Natl. Acad. Sci.
USA 92: 9747-9751 (1995). Spores can also be used as replicable genetic
packages. In this case, polypeptides are displayed from the outer surface of
the
spore. For example, spores from B. subtilis have been reported to be suitable.
Sequences of coat proteins of these spores are provided by Donovan et al., J.
Mol.
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Biol. 196, 1-10 (1987). Cells can also be used as replicable genetic packages.
Polypeptides to be displayed are inserted into a gene encoding a cell protein
that
is expressed on the cells surface. Bacterial cells including Salmonella
typhimurium, Bacillus subtilis, Pseudomonas aeruginosa, Vibrio cholerae,
Klebsiella pneumonia, Neisseria gonorrhoeae, Neisseria meningitidis,
Bacteroides nodosus, Moraxella bovis, and especially Escherichia coli are
preferred. Details of outer surface proteins are discussed by Ladner et al.,
US
5,571,698 and references cited therein. For example, the lamB, protein of E.
coli
is suitable.
Establishment of a Physical Association Between Polypeptides and Their
Genetic Material
A basic concept of display methods that use phage or other replicable
genetic package is the establishment of a physical association between DNA
encoding a polypeptide to be screened and the polypeptide. This physical
association is provided by the replicable genetic package, which displays a
polypeptide as part of a capsid enclosing the genome of the phage or other
package, wherein the polypeptide is encoded by the genome. The establishment
of
a physical association between polypeptides and their genetic material allows
simultaneous mass screening of very large numbers of phage bearing different
polypeptides. Phage displaying a polypeptide with affinity to a target, e.g.,
a
receptor, bind to the target and these phage are enriched by affinity
screening to
the target. The identity of polypeptides displayed from these phage can be
determined from their respective genomes.
Using these methods a polypeptide identified as having a binding affinity
for a desired target can then be synthesized in bulk by conventional means, or
the
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polynucleotide that encodes the peptide or polypeptide can be used as part of
a
genetic vaccine.
Variants with specific binding properties, in this case binding to family-
specific antibodies, are easily enriched by panning with immobilized
antibodies.
Antibodies specific for a single family are used in each round of panning to
rapidly select variants that have multiple epitopes from the antigen families.
For
example, A-family specific antibodies can be used to select those
experimentally
evolved (e.g. by polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis) clones that display A-specific epitopes in the first round of
panning.
A second round of panning with B-specific antibodies will select from the "A"
clones those that display both A- and B-specific epitopes. A third round of
panning with C- specific antibodies will select for variants with A, B, and C
epitopes. A continual selection exists during this process for clones that
express
well in E. coli and that are stable throughout the selection. Improvements in
factors such as transcription, translation, secretion, folding and stability
are often
observed and will enhance the utility of selected clones for use in vaccine
production.
Phage ELISA methods can be used to rapidly characterize individual
variants. These assays provide a rapid method for quantitation of variants
without
requiring purification of each protein. Individual clones are arrayed into 96-
well
plates, gown, and frozen for storage. Cells in duplicate plates are infected
with
helper phage, grown overnight and pelleted by centrifugation. The supernatants
containing phage displaying particular variants are incubated with immobilized
antibodies and bound clones are detected by anti- M13 antibody conjugates.
Titration series of phage particles, immobilized antigen, and/or soluble
antigen
competition binding studies are all highly effective means to quantitate
protein
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binding. Variant antigens displaying multiple epitopes will be further studied
in
appropriate animal challenge models.
Several groups have reported an in vitro ribosome display system for the
screening and selection of mutant proteins with desired properties from large
libraries. This technique can be used similarly to phage display to select or
enrich
for variant antigens with improved properties such as broad cross reactivity
to
antibodies and improved folding (see, e.g., Hanes et al. (1997) Proc. Nat'l. A
cad.
Sci. USA 94(10):493 7-42; Matthealds et al. (1994) Proc. Nat 7. Acad. Sci. USA
91(19):9022-6; He et al. (1997) Nucl. Acids Res. (24):5132-4; Nemoto et al.
(1997) FEBS Lett. 414(2):405-8)..
Other display methods exist to screen antigens for improved properties
such as increased expression levels, broad cross reactivity, enhanced folding
and
stability. These include, but are not limited to display of proteins on intact
E. coli
or other cells (e.g., Francisco et al. (1993) Proc. Nat'l. Acad. Sci. USA 90:
1044-
10448; Lu et al. (1995) BiolTechnology 13: 366-372). Fusions of experimentally
evolved (e.g. by polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis) antigens to DNA-binding proteins can link the antigen protein to
its
gene in an expression vector (Schatz et al. (1996) Methods Enzymol. 267: 171-
91;
Gates et al. (1996) J Mol. Biol. 255: 373-86.) The various display methods and
FJ.ISA assays can be used to screen for experimentally evolved (e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation mutagenesis)
antigens with improved properties such as presentation of multiple epitopes,
improved immunogenicity, increased expression levels, increased folding rates
and efficiency, increased stability to factors such as temperature, buffers,
solvents,
improved purification properties, etc. Selection of experimentally evolved
(e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation mutagenesis)
antigens with improved expression, folding, stability and purification profile
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under a variety of chromatographic conditions can be very important
improvements to incorporate for the vaccine manufacturing process.
To identify recombinant antigenic polypeptides that exhibit improved
expression
in a host cell, flow cytometry is a useful technique.
Flow cytometry provides a method to efficiently analyze the functional
properties of millions of individual cells. One can analyze the expression
levels of
several genes simultaneously, and flow cytometry-based cell sorting allows for
the selection of cells that display properly expressed antigen variants on the
cell
surface or in the cytoplasm. Very large numbers (> 107) of cells can be
evaluated
in a single vial experiment, and the pool of the best individual sequences can
be
recovered from the sorted cells. These methods are particularly useful in the
case
of, for example, Hantaan virus glycoproteins, which are generally very poorly
expressed in manunalian cells. This approach provides a general solution to
improve expression levels of pathogen antigens in mammalian cells, a
phenomenon that is critical for the function of genetic vaccines.
To use flow cytometry to analyze polypeptides that are not expressed on
the cell surface, one can engineer the experimentally generated
polynucleotides in
the library such that the polynucleotide is expressed as a fusion protein that
has a
region of amino acids which is targeted to the cell membrane. For example, the
region can encode a hydrophobic stretch of C-terminal amino acids which
signals
the attachment of a phosphoinositol- glycan (PIG) terminus on the expressed
protein and directs the protein to be expressed on the surface of the
transfected
cell (Whitehom etal. (1995) Biotechnology (N Y) 13:1215-9). With an antigen
that is naturally a soluble protein, this method will likely not affect the
three
dimensional folding of the protein in this engineered fusion with a new C-
terminus. With an antigen that is naturally a transmembrane protein (e.g., a
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surface membrane protein on pathogenic viruses, bacteria, protozoa or tumor
cells) there are at least two possibilities.
First, the extracellular domain can be engineered to be in fusion with the
C-terminal sequence for signaling PIG-linkage. Second, the protein can be
expressed in toto relying on the signaling of the host cell to direct it
efficiently to
the cell surface. In a minority of cases, the antigen for expression will have
an
endogenous PIG terminal linkage (e.g., some antigens of pathogenic protozoa).
Those cells expressing the antigen can be identified with a fluorescent
monoclonal antibody specific for the C-terminal sequence on PIG-linked forms
of
the surface antigen. FACS analysis allows quantitative assessment of the level
of
expression of the correct form of the antigen on the cell population. Cells
expressing the maximal level of antigen are sorted and standard molecular
biology methods are used to recover the plasmid DNA vaccine vector that
conferred this reactivity. An alternative procedure that allows purification
of all
those cells expressing the antigen (and that may be useful prior to loading
onto a
cell sorter since antigen expressing cells may be a very small minority
population), is to rosette or pan-purify the cells expressing surface antigen.
Rosettes can be formed between antigen expressing cells and erythrocytes
bearing
covalently coupled antibody to the relevant antigen. These are readily
purified by
unit gravity sedimentation. Panning of the cell population over petri dishes
bearing immobilized monoclonal antibody specific for the relevant antigen can
also be used to remove unwanted cells.
In the high throughput assays of the invention, it is possible to screen up to
several thousand different experimentally evolved (e.g. by polynucleotide
reassembly &/or polynucleotide site-saturation mutagenesis) variants in a
single
day. For example, each well of a microtiter plate can be used to run a
separate
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assay, or, if concentration or incubation time effects are to be observed,
every 5 -
wells can test a single variant. Thus, a single standard microliter plate can
assay about 100 (e.g., 96) reactions. If 1536 well plates are used, then a
single
plate can easily assay from about 100 to about 1500 different reactions. It is
5 possible to assay several different plates per day; assay screens for up
to about
6,000-20,000 different assays (i.e., involving different nucleic acids,
encoded
proteins, concentrations, etc.) is possible using the integrated systems of
the
invention. More recently, rnicrofluidic approaches to reagent manipulation
have
been developed, e.g., by Caliper Technologies (Palo Alto, CA).
In one aspect, library members, e.g., cells, viral plaques, or the like, are
separated on solid media to produce individual colonies (or plaques). Using an
automated colony picker (e.g., the Q-bot, Genetix, U.K.), colonies or plaques
are
identified, picked, and up to 10,000 different mutants inoculated into 96 well
microtiter dishes, optionally containing glass balls in the wells to prevent
aggregation. The Q-bot does not pick an entire colony but rather inserts a pin
through the center of the colony and exits with a small sampling of cells (or
viruses in plaque applications). The time the pin is in the colony, the number
of
dips to inoculate the culture medium, and the time the pin is in that medium
each
effect inoculum size, and each can be controlled and optimized. The uniform
process of the Q-bot decreases human handling error and increases the rate of
establishing cultures (roughly 10,000/4 hours). These cultures are then shaken
in a
temperature and humidity controlled incubator. The glass balls in the
microtiter
plates act to promote uniform aeration of cells dispersal of cells, or the
like,
similar to the blades of a fermentor. Clones from cultures of interest can be
cloned
by limiting dilution. Plaques or cells constituting libraries can also be
screened
directly for production of proteins, either by detecting hybridization,
protein
activity, protein binding to antibodies, or the like.
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The ability to detect a subtle increase in the performance of a
experimentally evolved (e.g. by polynucleotide reassembly &/or polynucleotide
site-saturation mutagenesis) library member over that of a parent strain
relies on
the sensitivity of the assay. The chance of finding the organisms having an
improvement in ability to induce an immune response is increased by the number
of individual mutants that can be screened by the assay. To increase the
chances of
identifying a pool of sufficient size, a prescreen that increases the number
of
mutants processed by I 0-fold can be used. The goal of the prescreen will be
to
quickly identify mutants having equal or better product titers than the parent
strain(s) and to move only these mutants forward to liquid cell culture for
subsequent analysis.
A number of well known robotic systems have also been developed for
solution phase chemistries useful in assay systems. These systems include
automated workstations like the automated synthesis apparatus developed by
Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems
utilizing robotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca,
Hewlett-Packard, Palo Alto, Calif.) which mimic the manual synthetic
operations
performed by a scientist. Any of the above devices are suitable for use with
the
present invention, e.g., for high- throughput screening of molecules encoded
by
codon-altered nucleic acids. The nature and implementation of modifications to
these devices (if any) so that they can operate as discussed herein with
reference
to the integrated system will be apparent to persons skilled in the relevant
art.
High throughput screening systems are commercially available (see, e.g.,
Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman
Instruments, Inc. Fullerton, CA; Precision Systems, Inc., Natick, MA, etc.).
These
systems typically automate entire procedures including all sample and reagent
pipetting, liquid dispensing, timed incubations, and final readings of the
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microplate in detector(s) appropriate for the assay. These configurable
systems
provide high throughput and rapid start up as well as a high degree of
flexibility
and customization.
The manufacturers of such systems provide detailed protocols the various
high throughput Thus, for example, Zymark Corp. provides technical bulletins
describing screening systems for detecting the modulation of gene
transcription,
ligand binding, and the like. Microfluidic approaches to reagent manipulation
have also been developed, e.g., by Caliper Technologies (Palo Alto, CA).
Optical images viewed (and, optionally, recorded) by a camera or other
recording device (e.g., a photodiode and data storage device) are optionally
further processed in any of the embodiments herein, e.g., by digitizing the
image
and/or storing and analyzing the image on a computer. As noted above, in some
applications, the signals resulting from assays are florescent, making optical
detection approaches appropriate in these instances. A variety of commercially
available peripheral equipment and software is available for digitizing,
storing
and analyzing a digitized video or digitized optical image, e.g., using PC
(Intel
x86 or Pentium chip- compatible DOS, 052 WINDOWS, WINDOWS NT or
VIMOWS95 based machines), MACINTOSH, or LTNDC based (e.g., SUN work
station) computers.
One conventional system carries light from the assay device to a cooled
charge-coupled device (CCD) camera, in common use in the art. A CCD camera
includes an array of picture elements (pixels). The light from the specimen is
imaged on the CCD. Particular pixels corresponding to regions of the specimen
(e.g., individual hybridization sites on an array of biological polymers) are
sampled to obtain light intensity readings for each position. Multiple pixels
are
processed in parallel to increase speed. The apparatus and methods of the
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invention are easily used for viewing any sample, e.g., by fluorescent or dark
field
microscopic techniques.
Integrated systems for analysis in the present invention typically include a
digital computer with high-throughput liquid control software, image analysis
software, data interpretation software, a robotic liquid control armature for
transferring solutions from a source to a destination operably linked to the
digital
computer, an input device (e.g., a computer keyboard) for entering data to the
digital computer to control high throughput liquid transfer by the robotic
liquid
control armature and, optionally, an image scanner for digitizing label
signals
from labeled assay component. The image scanner interfaces with the image
analysis software to provide a measurement of optical intensity. Typically,
the
intensity measurement is interpreted by the data interpretation software to
show
whether the optimized recombinant antigenic polypeptide products are produced.
2.5.4. ANTIGEN LIBRARY IMMUNIZATION
In a presently preferred embodiment, antigen library immunization (ALI)
is used to identify optimized recombinant antigens that have improved
immumagenicity. ALI involves introduction of the library of recombinant
antigen-
encoding nucleic acids, or the recombinant antigens encoded by the
experimentally evolved (e.g. by polynucleotide reassembly &/or polynucleotide
site-saturation mutagenesis) nucleic acids, into a test animal. The animals
are then
subjected to in vivo challenge using live pathogens. Neutralizing antibodies
and
cross-protective immune responses are studied after immunization with the
entire
libraries, pools and/or individual antigen variants.
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Methods of immunizing test animals are well known to those of skill in
the art. In presently preferred embodiments, test animals are immunized twice
or
three times at two week intervals. One week after the last immunization, the
animals are challenged with live pathogens (or mixtures of pathogens), and the
survival and symptoms of the animals is followed. Immunizations using test
animal challenge are described in, for example, Roggenkamp et al. (1997)
Infect.
Immun. 65: 446; Woody et al. (1997) Vaccine 2: 133; Agren et al. (1997) J
Irnmunol. 158: 3936; Konishi et al. (1992) Virology 190: 454; Kinney et al.
(1988) J Virol. 62: 4697; Iacono-Connors et al. (1996) Virus Res. 43: 125;
Kochel
et al. (1997) Vaccine 15: 547; and Chu et al. (1995) J Virol. 69: 6417.
The immunizations can be performed by injecting either the
experimentally generated polynucleotides themselves, i.e., as a genetic
vaccine, or
by immunizing the animals with polypeptides encoded by the experimentally
generated polynucleotides. Bacterial antigens are typically screened primarily
as
recombinant proteins, whereas viral antigens are preferably analyzed using
genetic vaccinations.
To dramatically reduce the number of experiments required to identify
individual antigens having improved immunogenic properties, one can use
pooling and deconvolution, as diagrammed herein. Pools of recombinant nucleic
acids, or polypeptides encoded by the recombinant nucleic acids, are used to
immunize test animals. Those pools that result in protection against pathogen
challenge are then subdivided and subjected to additional analysis. The high
throughput in vitro approaches described above can be used to identify the
best
candidate sequences for the in vivo studies.
The challenge models that can be used to screen for protective antigens
include pathogen and toxin models, such as Yersinia bacteria, bacterial toxins
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(such as Staphylococcal and Streptococcal enterotoxins, E. colifV. cholerae
enterotoxins), Venezuelan equine encephalitis virus (VEE), Flaviviruses
(Japanese
encephalitis virus, Tick-borne encephalitis virus, Dengue virus), Hantaan
virus,
Herpes simplex, influenza virus (e.g., Influenza A virus), Vesicular Steatites
Virus, Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli,
Klebsiella pneumoniae, Toxoplasma gondii, Plasmodium yoeliii, Herpes simplex,
influenza virus (e.g., Influenza A virus), and Vesicular Steatites Virus.
However,
the test animals can also be challenged with tumor cells to enable screening
of
antigens that efficiently protect against malignancies. Individual
experimentally
evolved (e.g. by polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis) antigens or pools of antigens are introduced into the animals
intradermally, intramuscularly, intravenously, intratracheally, anally,
vaginally,
orally, or intraperitoneally and antigens that can prevent the disease are
chosen,
when desired, for further rounds of reassembly (optionally in combination with
other directed evolution methods described herein) and selection. Eventually,
the
most potent antigens, based on in vivo data in test animals and comparative in
vitro studies in animals and man, are chosen for human trials, and their
capacity to
prevent and treat human diseases is investigated.
In some embodiments, antigen library immunization and pooling of
individual clones is used to immunize against a pathogen strain that was not
included in the sequences that were used to generate the library. The level of
crossprotection provided by different strains of a given pathogen can
significantly.
However, homologous titer is always higher than heterologous titer. Pooling
and
deconvolution is especially efficient in models where minimal protection is
provided by the wild-type antigens used as starting material for reassembly
(optionally in combination with other directed evolution methods described
herein). This approach can be taken, for example, when evolving the V-antigen
of Yersinae or Hantaan virus glycoproteins.
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In some embodiments, the desired screening involves analysis of the
immune response based on immunological assays known to those skilled in the
art. Typically, the test animals are first immunized and blood or tissue
samples are
collected for example one to two weeks after the last immunization. These
studies
enable one to one can measure immune parameters that correlate to protective
immunity, such as induction of specific antibodies (particularly IgG) and
induction of specific T lymphocyte responses, in addition to determining
whether
an antigen or pools of antigens provides protective immunity.
Spleen cells or peripheral blood mononuclear cells can be isolated from
immunized test animals and measured for the presence of antigen-specific T
cells
and induction of cytokine synthesis. ELISA, ELISPOT and cytoplasmic cytokine
staining, combined with flow cytometry, can provide such information on a
single-cell level.
Common immunological tests that can be used to identify the efficacy of
immunization include antibody measurements, neutralization assays and analysis
of activation levels or frequencies of antigen presenting cells or lymphocytes
that
are specific for the antigen or pathogen. The test animals that can be used in
such
studies include, but are not limited to, mice, rats, guinea pigs, hamsters,
rabbits,
cats, dogs, pigs and monkeys.
Monkey is a particularly useful test animal because the MHC molecules of
monkeys and humans are very similar. Virus neutralization assays are useful
for
detection of antibodies that not only specifically bind to the pathogen, but
also
neutralize the function of the virus. These assays are typically based on
detection
of antibodies in the sera of immunized animal and analysis of these antibodies
for
their capacity to inhibit viral growth in tissue culture cells. Such assays
are known
to those skilled in the art. One example of a virus neutralization assay is
described
=
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by Dolin R (J. Infect. Dis. 1995, 172:1175-83). Virus neutralization assays
provide means to screen for antigens that also provide protective immunity.
In some embodiments, experimentally evolved (e.g. by polynucleotide
reassembly &/or polynucleotide site-saturation mutagenesis) antigens are
screened for their capacity to induce T cell activation in vivo. More
specifically,
peripheral blood mononuclear cells or spleen cells from injected mice can be
isolated and the capacity of cytotoxic T lymphocytes to lyse infected,
autologous
target cells is studied. The spleen cells can be reactivated with the specific
antigen
in vitro. In addition, T helper cell activation and differentiation is
analyzed by
measuring cell proliferation or production of TH (IL-2 and IFN- ) and 112 (IL-
4
and IL-5) cytokines by ELISA and directly in CD4+ T cells by cytoplasmic
cytoldne staining and flow cytometry. Based on the cytokine production
profile,
one can also screen for alterations in the capacity of the antigens to direct
TH1/
TH2 differentiation (as evidenced, for example, by changes in ratios of IL-4/
IFN-
, IL-4/IL-2, IL-5/ IFN- , IL-5/IL-2, IL- 13/ IFN- , IL- I 3/IL-2). The
analysis
of the T cell activation induced by the antigen variants is a very useful
screening
method, because potent activation of specific T cells in vivo correlates to
induction of protective immunity.
The frequency of antigen-specific CD8+ T cells in vivo can also be
directly analyzed using tetramers of MHC class I molecules expressing specific
peptides derived from the corresponding pathogen antigens (Ogg and McMichael,
Curt Opin. Immunol. 1998, 10:393-6; Altman et al., Science 1996, 274:94-6).
The binding of the tetramers can be detected using flow cytometry, and will
provide information about the efficacy of the experimentally evolved (e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation mutagenesis)
antigens to induce activation of specific T cells. For example, flow cytometry
and
tetramer stainings provide an efficient method of identifying T cells that are
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specific to a given antigen or peptide. Another method involves panning using
plates coated with tetramers with the specific peptides. This method allows
large
numbers of cells to be handled in a short time, but the method only selects
for
highest expression levels. The higher the frequency of antigen-specific T
cells in
vivo is, the more efficient the immunization has been, enabling identification
of
the antigen variants that have the most potent capacity to induce protective
immune responses. These studies are particularly useful when conducted in
monkeys, or other primates, because the MEC class I molecules of humans mimic
those of other primates more closely than those of mice.
Measurement of the activation of antigen presenting cells (APC) in
response to immunization by antigen variants is another useful screening
method.
Induction of APC activation can be detected based on changes in surface
expression levels of activation antigens, such as 137-1 (CD80). 137-2 (CD86),
MHC class I and 11, CD14, CD23, and Fc receptors, and the like.
Experimentally evolved (e.g. by polynucleotide reassembly &/or
polynucleotide site-saturation mutagenesis) cancer antigens that induce
cytotoxic
T cells that have the capacity to kill cancer cells can be identified by
measuring
the capacity of T cells derived from immunized animals to kill cancer cells in
vitro. Typically the cancer cells are first labeled with radioactive isotopes
and the
release of radioactivity is an indication of tumor cell killing after
incubation in the
presence of T cells from immunized animals. Such cytotoxicity assays are known
in the art.
An indication of the efficacy of an antigen to activate T cells specific for,
for example, cancer antigens, allergens or autoantigens, is also the degree of
skin
inflammation when the antigen is injected into the skin of a patient or test
animal.
Strong inflammation is correlated with strong activation of antigen-specific T
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cells. Improved activation of tumor- specific T cells may lead to enhanced
killing
of the tumors. In case of autoantigens, one can add immunomodulators that skew
the responses towards TH2, whereas in the case of allergens a TH1 response is
desired. Skin biopsies can be taken, enabling detailed studies of the type of
immune response that occurs at the sites of each injection (in mice and
monkeys
large numbers of injections/antigens can be analyzed). Such studies include
detection of changes in expression of cytoldnes, chemoldnes, accessory
molecules, and the like, by cells upon injection of the antigen into the skin.
To screen for antigens that have optimal capacity to activate antigen-
specific T cells, peripheral blood mononuclear cells from previously infected
or
immunized humans individuals can be used. This is a particularly useful
method,
because the MHC molecules that will present the antigenic peptides are human
MHC molecules. Peripheral blood mononuclear cells or purified professional
antigen-presenting cells (APCs) can be isolated from previously vaccinated or
infected individuals or from patients with acute infection with the pathogen
of
interest. Because these individuals have increased frequencies of pathogen-
specific T cells in circulation, antigens expressed in PBMCs or purified APCs
of
these individuals will induce proliferation and cytokine production by antigen-
specific CD4+ and CD8+ T cells. Thus, antigens that simultaneously harbor
epitopes from several antigens can be recognized by their capacity to
stimulate T
cells from various patients infected or immunized with different pathogen
antigens, cancer antigens, autoantigens or allergens. One buffy coat derived
flow
a blood donor contains lymphocytes from 0.5 liters of blood, and up to 104
PBMC
can be obtained, enabling very large screening experiments using T cells from
one
donor.
When healthy vaccinated individuals (lab volunteers) are studied, one can
make EBV-transformed B cell lines from these individuals. These cell lines can
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be used as antigen presenting cells in subsequent experiments using blood from
the same donor; this reduces interassay and donor-to-donor variation. In
addition,
one can make antigen-specific T cell clones, after which antigen variants are
introduced to EBV transformed B cells. The efficiency with which the
transformed B cells induce proliferation of the specific T cell clones is then
studied. When working with specific T cell clones, the proliferation and
cytokine
synthesis responses are significantly higher than when using total PBMCs,
because the frequency of antigen-specific T cells among PBMC is very low.
CTL epitopes can be presented by most cells types since the class I major
histocompatibility complex (MHC) surface glycoproteins are widely expressed.
Therefore, transfection of cells in culture by libraries of experimentally
evolved
(e.g. by polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis) antigen sequences in appropriate expression vectors can lead to
class I epitope presentation. If specific CTLs directed to a given epitope
have
been isolated from an individual, then the co-culture of the transfected
presenting
cells and the CTLs can lead to release by the CTLs of cytolcines, such as IL-
2,
IFN- , or TNF, if the epitope is presented. Higher amounts of released TNT'
will
correspond to more efficient processing and presentation of the class I
epitope
from the experimentally evolved (e.g. by polynucleotide reassembly Wor
polynucleotide site-saturation mutagenesis), evolved sequence. Experimentally
evolved (e.g. by polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis) antigens that induce cytotoxic T cells that have the capacity to
kill
infected cells can also be identified by measuring the capacity of T cells
derived
from immunized animals to kill infected cells in vitro. Typically the target
cells
are first labeled with radioactive isotopes and the release of radioactivity
is an
indication of target cell killing after incubation in the presence of T cells
from
immunized animals. Such cytotoxicity assays are known in the art.
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A second method for identifying optimized cn, epitopes does not require
the isolation of CTLs reacting with the epitope. In this approach, cells
expressing
class I MHC surface glycoproteins are transfected with the library of evolved
sequences as above. After suitable incubation to allow for processing and
presentation, a detergent soluble extract is prepared from each cell culture
and
after a partial purification of the MHC-epitope complex (perhaps optional) the
products are submitted to mass spectrometry (Henderson et al. (1993) Proc.
Nat'l.
Acad. Sci. USA 90: 10275-10279). Since the sequence is known of the epitope
whose presentation to be increased, one can calibrate the mass spectrogram to
identify this peptide. In addition, a cellular protein can be used for
internal
calibration to obtain a quantitative result; the cellular protein used for
internal
calibration could be the MHC molecule itself. Thus one can measure the amount
of peptide epitope bound as a proportion of the N4HC molecules.
2.5.5. SCREENINGFOR OPTIMAL INDUCTION OF PROTECTIVE
IMMUNITY
Vectors that can provide_efficient.nrntedivlimmunibr are selected using
lethal jInfection models to choose vectors that can prevent the diseise for
further rounds of reassembly (optionally in combination with other directtd
evolution methods described herein) and selection
To select genetic vaccine vectors that provide efficient protective
immunity, one can screen the vector libraries in a test mammal using lethal
infection models, such as Pseudomonas aeruginosa, Salmonella typhimurium,
Escherichia coli, Klebsiella pneumoniae, Toxoplasma gondii, Plasmodium
yoeliii,
Herpes simplex, influenza virus (e.g., Influenza A virus), and Vesicular
Steatites
Virus. Pools of genetic vaccine vectors or individual vectors are introduced
into
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the animals intradermally, intramuscularly, intravenously, intratracheally,
anally,
vaginally, orally, or intraperitoneally and vectors that can prevent the
disease are
chosen for further rounds of reassembly (optionally in combination with other
directed evolution methods described herein) and selection.
Examples: anti-IL-4 mAbs or recombinant IL-12 recombinant IL-12
(advantage of latter model is that infection occurs_through lung. Common
route of human pathogen invasion)
As an example, optimal vectors can be screened in mice infected with
Leishmania major parasites. When injected into footpads of BALB/c mice, these
parasites cause a progressive infection later resulting in a disseminated
disease
with fatal outcome, which can be prevented by anti-IL-4 mAbs or recombinant
IL-12 (Chatelain et al. (1992) J. Immunol. 148: 1182-1187). Pools of plasmids
can
be injected intravenously, intraperitoneally or into footpads of these mice,
and
pools that can prevent the disease are chosen for further analysis and
screened for
vectors that can cure existing infections. The size of the footpad swelling
can be
followed visually providing simple yet precise monitoring of the disease
progression. Mice can be infected intratracheally with Klebsiella pneumoniae
resulting in lethal pneumonia, which can be prevented by recombinant IL-12
(Greenberger et al. (1996) J Immunol. 157: 3006-3012). The advantage of this
model is that the infection occurs through the lung, which is a common route
of
human pathogen invasion. The vectors can be given to the lung together with
the
pathogen or they can be administered after symptoms are evident in order to
screen for vectors that can cure established infections.
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Example: Influenza- provides a way to screen for vectors that provide
protection at very low gliantillea_ofDNA and/or high_virus concentrations,
and it also allows one to analyze the levels of antigen ipeciflc Abs and CTLs
Induced in vivo
In another example, the genetic vaccines are a mouse vaccination model
for Influenza A virus. Influenza was one of the first models in which the
efficacy
of genetic vaccines was demonstrated (Ulmer et al. (1993) Science 259: 1745-
1749). Several Influenza strains are lethal in mice providing an easy means to
screen for efficacy of genetic vaccines.
For example, Influenza virus strain A/PR/8/34, which is available through
the American Type Culture Collection (ATCC VR-95), causes lethal infection,
but
100% survival can be obtained when the mice are immunized with and influenza
hemag,glutinin (HA) genetic vaccine (Deck et al. (1997) Vaccine 15: 71-78).
This
model provides a way to screen for vectors that provide protection at very low
quantities of DNA and/or high virus concentrations, and it also allows one to
analyze the levels of antigen specific Abs and CTLs induced in vivo.
Prample: Mycobacterium tuberculosis (partial protection, requires major
improvements)
The genetic vaccine vectors can also be analyzed for their capacity to
provide protection against infections by Mycobacterium tuberculosis. This is
an
example of a situation where genetic vaccines have provided partial
protection,
and where major improvements are required.
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JAentification of candidate vectors followed by moreletting
Once a number of candidate vectors has been identified, these vectors can
be subjected to more detailed analysis in additional models. Testing in other
infectious disease models (such as HSV, Mycoplasma pulmonis, RSV and/or
rotavirus) will allow identification of vectors that are optimal in each
infectious
disease.
Optimal plasmids from the first round of screening are used as the starting
material for the next round. the_successful vectors are sequenced and the
corresponding human genes are cloned into genetic vaccine vectors which are
characterized in vitro for fink capacity tainduce differentiation of a dmired
trait.
In each case, the optimal plasmids from the first round of screening can be
used as the starting material for the next round of reassembly (optionally in
combination with other directed evolution methods described herein), assembly
and selection. Vectors that are successful in animal models are sequenced and
the
corresponding human genes are cloned into genetic vaccine vectors. These
vectors are then characterized in vitro for their capacity to induce
differentiation
of T11 1/ TH2 cells, activation of TH cells, cytotoxic T lymphocytes and
monocytes/macrophages, or other desired trait. Eventually, the most potent
vectors, based on in vivo data in mice and comparative in vitro studies in
mice and
man, are chosen for human trials, and their capacity to counteract various
human
infectious diseases is investigated.
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Methods for measurinOmmune parameters that correlate to protective
Immunity
In addition to determining whether a vector pool provides protective
immunity, one can measure immune parameters that correlate to protective
immunity, such as induction of specific antibodies (particularly IgG) and
induction of specific CTL responses. Spleen cells can be isolated from
vaccinated
mice and measured for the presence of antigen- specific T cells and induction
of
TH1 cytoldne synthesis profiles. ELISA and cytoplasmic cytokine staining,
combined with flow cytometry, can provide such information on a single-cell
level.
2.5.6. SCREENING OF GENETIC VACCINE VECTORS THAT
ACTIVATE HUMAN ANTIGEN-SPECIFIC LYMPHOCYTE
RESPONSES
Isolation _of_PBMCs or APCs to screen_for vectors with optimal
humunostimulatory properties for the humanimmune system
To screen for vectors with optimal inununostimulatory properties for the
human immune system, peripheral blood mononuclear cells (PBMCs) or purified
professional antigen-presenting cells (APCs) can be isolated from previously
vaccinated or infected individuals or from patients with acute infection with
the
pathogen of interest
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Genetic vaccine vectors encoding the antigen for which the individuals have
specific T cells can be transfected_into_PBMC and induction of T cell
proliferation and cytokine synthesis can be measured; also possible to sago
for spontaneous entry of_gcnetic vaccine vector into APCs
Because these individuals have increased frequencies of pathogen-
specific T cells in circulation, antigens expressed in PBMCs or purified APCs
of
these individuals will induce proliferation and cytokine production by antigen-
specific CD4+ and CD8+ T cells. Thus, genetic vaccine vectors encoding the
antigen for which the individuals have specific T cells can be transfected
into
=
PBMC of the individuals, after which induction of T cell proliferation and
cytokine synthesis can be measured. Alternatively, one can screen for
spontaneous
entry of the genetic vaccine vector into A-PCs, thus providing a means by
which
to screen simultaneously for improved transfection efficiency, improved
expression of antigen and improved induction of activation of specific T
cells.
Vectors with the most potent hnmunostimulatory properties can be screened
based
on their capacity to induce B cell proliferation and immunoglobulin synthesis.
One buffy coat derived from a blood donor contains PBMC lymphocytes from 0.5
liters of blood, and up to 104 PBMC can be obtained, enabling very large
screening experiments using T cells from one donor.
Making EBV-transformed B ceillines from healthy vaccinated individuals
for subsequent experiments
When healthy vaccinated individuals (lab volunteers) are studied, one can
make EBV-transformed B cell lines from these individuals. These cell lines can
be used as antigen presenting cells in subsequent experiments using blood from
the same donor; this reduces interassay and donor-to-donor variation). In
addition,
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one can make antigen-specific T cell clones, after which genetic vaccines are
transfected into EBV transformed B cells.
Efficiency with which_the transformed B cells induce proliferation of the
specific T cell clones
The efficiency with which the transformed B cells induce proliferation of
the specific T cell clones is then studied. When working with specific T cell
clones, the proliferation and cytokine synthesis responses are significantly
higher
than when using total PBMCs, because the frequency of antigen-specific T cells
among PBMC is very low.
Trausfection of cells in culture by libraries of experimentally evolved (e.g.
by
polynucleotide reassembly &Mr pnbEnucleotkle site-saturation mutagenesis)
DNA sequences in appropriate expression vectors can lead to class I epitope
presentation
cm epitopes can be presented by most cells types since the class I major
histocompatibility complex (MHC) surface glycoproteins are widely expressed.
Therefore, transfection of cells in culture by libraries of experimentally
evolved
(e.g. by polynucleotide reassembly 8/or polynucleotide site-saturation
mutagenesis) DNA sequences in appropriate expression vectors can lead to class
I
epitope presentation. If specific CTLs directed to a given epitope have been
isolated from an individual, then the co-culture of the transfected presenting
cells
and the CTLs can lead to release by the CTLs of cytokines, such as IL-2, IFN-
,
or 'INF , if the epitope is presented. Higher amounts of released TNF . will
correspond to more efficient processing and presentation of the class I
epitope
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from the experimentally evolved (e.g. by polynucleotide reassembly &/or
polynucleotide site-saturation mutagenesis), evolved sequence.
Transfeeting cells expressing class I MIIC surface glycoproteins with library
of evolvecisequences, preparing a detugentioluble extract, performing a
partial purification of_the MBC-epitope complex, and then submitting the
products to mass spectrometry
A second method for identifying optimized CTL epitopeS does not require
the isolation of CTLs reacting with the epitope. In this approach, cells
expressing
class I MHC surface glycoproteins are transfected with the library of evolved
sequences as above. After suitable incubation to allow for processing and
presentation,. a detergent soluble extract is prepared from each cell culture
and
after a partial purification of the MHC-epitope complex (perhaps optional) the
products are submitted to mass spectrometry (Henderson et al. (1993) Proc.
Nat'l.
Acad. Sci. USA 90: 10275-10279). Since the sequence is known of the epitope
whose presentation to be increased, one can calibrate the mass spectrogram to
identify this peptide. In addition, a cellular protein can be used for
internal
calibration to obtain a quantitative result; the cellular protein used for
internal
calibration could be the MHC molecule itself. Thus one can measure the amount
of peptide epitope bound as a proportion of the MHC molecules.
2.5.7. SCID-HUMAN SKIN MODEL FOR VACCINATION STUDIES
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Use of mouse models in vaccine studies limited in that the MIIC m9lecules
it II I Ik.1 Li 11 1 1, I II =I
that efficiently induce protect e immune responses in mice_ do not
necessarily function in humans
Successful genetic vaccinations require transfection of the target cells after
injection of the vector, expression of the desired antigen, processing the
antigen in
antigen presenting cells, presentation of the antigenic peptides in the
context of
MHC molecules, recognition of the peptide/MHC complex by T cell receptors,
interactions of T cells with B cells and professional APCs and induction of
specific T cell and B cell responses. All these events could be differentially
regulated in mouse and man. A limitation of mouse models in vaccine studies is
the fact that the MHC molecules of mice and man are substantially different.
Therefore, proteins and peptides that effectively induce protective immune
responses in mice do not necessarily function in humans.
,11.01111111, ,I I
of transfection_efficienev. transfer segilienees. antsnne expression leyek
To overcome these limitations mouse models can be used to study human
tissues in mice in vivo. Live pieces of human skin are xenotransplant onto the
back of immunodeficient mice, such as SC1D mice, allowing screening of the
vector libraries for optimal properties in human cells in vivo. Recursive
selection
of episomal vectors provides strong selection pressure for vectors that remain
episomal, yet provide high level of gene expression. These mice provide an
excellent model for studies on transfection efficiency, transfer sequences and
gene
expression levels. In addition, antigen presenting cells (APCs) derived from
these
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mice can also be used to assess the level of antigens delivered to
professional
APCs, and to study the capacity of these cells to present antigens and induce
activation of antigen-specific CD4+ and CD8+ T cells in vitro. Significantly,
although SCID mice have severely deficient T and B cell components, antigen
presenting cells (dendritic cells and monocytes) are relatively normal in
these
mice.
Rendering immunocomnetent mice immunodeficieut_in order to aid
transplautadunsaumanAzzlicailablin vaccine studies in human skin
zenotransplanted into mice with genetically normal immune systems as well,
due to the transient nature of the in vivo immunosuppressiou
In one embodiment of this model system, immunocompetent mice are rendered
immunodeficient in order to enable transplantation of human tissue. For
example,
blocking of CD28 and CD40 pathways promotes long-term survival of allogeneic
skin grafts in mice (Larsen et al. (1996) Nature 381: 434). Because the in
vivo
immunosuppression is transient, this model also enables vaccine studies in
human
skin xenotransplanted into mice with genetically normal immune systems.
Several
methods of blocking CD28- 137 interactions and CD4O-CD40 ligand interactions
are known to those of skill in the art, including, for example, administration
of
neutralizing anti-B7-1 and 137-2 antibodies, soluble CTLA-4, a soluble form of
the extracellular portion of CTLA-4, a fusion protein that includes CTLA-4 and
an Fe portion of an IgG molecule, and neutralizing anti-CD40 or anti- CD40
ligand antibodies. Additional methods by which one can improve transient
immunosuppression include administration of one or more of the following
reagents: cyclosporin A, anti-IL-2 receptor - chain Ab, soluble IL-2 receptor,
IL-
10, and combinations thereof.
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A model in which SCB3-mice transplanted with human skin are injected
with HLA-matched PBMC can be used to analyze vectors that provide long
lasting expression in vivo. In this model, the vectors are injected, or
topically
applied, into the human skin.
If the FILA-matched_PBMC Injected Into mice contains lymphocytes specific
for the vector the transfected cells wilLbe recomized, and eventually
destroyed. by these vector-ipecific lymphocytes. providing the possibility to
screen for vectors that efficiently escane destruction
Thereafter, HLA-matched PBMC are injected into these mice. If the
PBMC contains lymphocytes specific for the vector, the transfected cells will
be
recognized, and eventually destroyed, by these vector-specific lymphocytes.
Therefore, this model provides possibilities to screen for vectors that
efficiently
escape destruction by the immune cells. It has been shown that human PBLs
injected into mice with human skin transplants reject the organ, indicating
that the
CTLs reach the skin in this model. Obtaining HLA-matching skin and blood is
possible (e.g. blood sample and skin graft from a patient undergoing skin
removal
due to malignancy, or blood and foreskin from the same infant).
SCIDhu mouse model: additionally. transplanting human skin a1lo3y, studies
on the efficacy of genetic vaccine vectors following injection to the skin
An additional model that is suitable for screening as described herein is
the modified SCIDhu mouse model, in which pieces of human fetal thymus, liver
and bone marrow are transplanted into SCI]) mice providing functional human
immune system in mice (Roncarolo et al. (1996) Semin. Immunol. 8: 207).
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Functional human B and T cells, and APCs can be observed in these mice. When
additionally human skin is transplanted, it is likely to allow studies on the
efficacy
of genetic vaccine vectors following injection into the skin.
Cotransplantation of
skin is likely to improve the model because it will provide an additional
source of
2.5.8. MOUSE MODEL FOR STUDYING THE EFFICIENCY
OFGENETIC VACCINES IN TRANSFECTING HUMAN
MUSCLE CELLS AND INDUCING HUMAN IMMUNE
RESPONSES IN VIVO
There is a lack of suitable in vivo models for studies_ of the efficiency of
genetic vaccines and the vast majority 1st:tulles are 11 = rmedL on the mouse
obtained reliably predict similar vaccinations in humans because of the
complexity of events occurring after genetic vaccination
A lack of suitable in vivo models has hampered studies of the efficiency of
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recognition of the peptide/MHC complex by T cell receptors, interactions of T
cells with B cells and professional antigen presenting cells and finally
induction
of specific T cell and B cell responses. All these events are likely to be
somewhat
differentially regulated in mouse and man.
The invention provides an i n vivo model for human muscle cell transfection
Muscle tissue, obtained for example from cadavers, is transplanted
subcutaneously into immunodeficient mice, which can be transplanted with
tissues from other species without rejection. This model system is especially
valuable because there is no in vitro culture system available for normal
muscle
cells. Muscle tissue, obtained for example from cadavers, is transplanted
subcutaneously into immunodeficient mice. Immunodeficient mice can be
transplanted with tissues from other species without rejection. Mice suitable
for
xenotransplantations include, but are not limited to, SCID mice, nude mice and
mice rendered deficient in their genes encoding RAG1 or RAG2 genes. SCID
mice and RAG deficient mice lack functional T and B cells, and therefore are
severely inununocompromised and are unable to reject transplanted organs.
Previous studies indicate that these mice can be transplanted with human
tissues,
such as skin, spleen, liver, thymus or bone, without rejection (Roncarolo et
al.
(1996) Semin. hnrntmol. 8: 207). After transplantation of human fetal lymphoid
tissues into SCID mice, functional human immune system can be demonstrated in
these mice, a model generally referred to as SCID-hu mice. When human muscle
tissue is transplanted into SCID-hu mice, one can not only study transfection
efficiency and expression of the desired antigen, but one can also study
induction
of specific human immune responses induced by genetic vaccines in vivo. In
this
case, muscle and lymphoid organs from the same donor are used. Fetal muscle
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also has an advantage in that it contains few mature lymphocytes of donor
origin
decreasing likelihood of graft versus host reaction.
Genetic vaccine vectors are introduced into theluman muscle tissue to study
the expression of the antigen of interest
Once the human muscle tissue is established in the mouse, genetic vaccine
vectors are introduced into the human muscle tissue to study the expression of
the
antigen of interest. When studying transfection efficiency only, RAG deficient
mice are preferred, because these mice never have mature B or T cells in the
circulation, whereas "leakiness" of SCID phenotype has been demonstrated which
may cause variation in the transplantation efficiency.
Model provides an efficient means toittudv gene expression in human muscle
cells in vivo despite the limited survival of the tissue in mice
The survival of human muscle tissue in mice is likely to be limited even in
immuno-compromised mice. However, because expression studies can be
performed within one or two days, this model provides an efficient means to
study
gene expression in human muscle cells in vivo. A modified SCID-hu mouse
model with human muscle transplanted into these mice can be used to study
human immune responses in mice in vivo.
2.5.9. SCREENING FOR IMPROVED DELIVERY OF VACCINES
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Identifying genetic vaccine vectors that are capable of being administered iu
a particular manner
For certain applications, it is desirable to identify genetic vaccine vectors
that are capable of being administered in a particular manner, for example,
orally
or through the skin. The following screening methods provide suitable assays;
additional assays are also described herein in conjunction with particular
genetic
vaccine properties for which the assays are especially suitable.
Screening for oral delivery tither in vitro (based on Cam-2 cells) or in vivo
Screening for oral delivery can be performed either in vitro or in vivo. An
example of an in vitro method is based on Caco-2 (human colon adenocarcinoma)
cells which are grown in tissue culture. When grown on semipermeable filters,
these cells spontaneously differentiate into cells that resemble human small
intestine epithelium, both structurally and functionally. Genetic vaccine
libraries
and/or vectors can be placed on one side of the Caco-2 cell layer, and vectors
that
are able to move through the cell layer are detected on the opposite side of
the
layer.
Libraries can also be screened for amenability to oral delivery in vivo. For
example, a library of vectors can be administered orally, after which target
tissues
are assayed for presence of vectors. Intestinal epithelium, liver, and the
bloodstream are examples of tissues that can be tested for presence of library
members. Vectors that are successful in reaching the target tissue can be
recovered
and, if further improvement is desired, used in succeeding rounds of
reassembly
(optionally in combination with other directed evolution methods described
herein) and selection.
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Apparatuswhich permits lame numbers of vectors to be screened efficiently
and can be used to study the effect of large numbers of agents in vivo
For screening a library of genetic vaccine vectors for ability to transfect
cells upon injection into skin or muscle, the invention provides an apparatus
which permits large numbers of vectors to be screened efficiently. This
apparatus
is based on 96-well format and is designed to transfer small volumes (2-5 1)
from a microtiter plate to skin or muscle of laboratory animals, such as mice
and
rats. Moreover, human muscle or skin transplanted into immunodeficient mice
can be injected.
The apparatus is designed in such a way that the tips move to fit a
microtiter plate. After the reagent of interest has been obtained from the
plate, the
distance of the tips from each other is decreased to 2-3 mm, enabling transfer
of
96 reagents to an area of 1.6 cm x 2.4 cm to 2.4 cm x 3.6 cm. The volume of
each
sample transferred is electronically controlled. Each reagent is mixed with a
marker agent or dye to enable recognition of injection site in the tissue. For
example, gold particles of different sizes and shapes are mixed with the
reagent of
interest, and microscopy and immunohistochemistry can be used to identify each
injection site and to study the reaction induced by each reagent. When muscle
tissue is injected the injection site is first revealed by surgery.
This apparatus can be used to study the effects of large numbers of agents
in vivo. For example, this apparatus can be used to screen efficiency of large
numbers of different DNA vaccine vectors to transfect human skin or muscle
cells
transplanted into immunodeficient mice.
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2.5.10. ENHANCED ENTRY OF GENETIC VACCINE VECTORS INTO
CELLS
Using stochasticle.g polynucleotide shuffling & interrupted synthesis) and
eon-stochastic polynucleotide reassembly to efficiently improve the capacity
of DNA to enter the cylobsm and_subseouently the nucleus of human cells
The methods involve subjecting to stochastic (e.g. polynucleotide
shuffling & interrupted synthesis) and non-stochastic polynucleotide
reassembly
polynucleofides which are involved in cell entry. Such polynucleotides are
referred to herein as "transfer sequences" or "transfer modules." Transfer
modules
can be obtained which increase transfer in a cell- specific manner, or which
act in
a more general manner. Because the exact sequences that affect DNA binding and
transfer are not often known, stochastic (e.g. polynucleotide shuffling &
interrupted synthesis) and non-stochastic polynucleotide reassembly may be the
only efficient method to improve the capacity of DNA to enter the cytoplasm
and
subsequently the nucleus of human cells.
The stochastic (e.g. polynucleotide shuffling & intemmted synthesis) and
non-stochastic polynucleotide reassembly methods of the invention provide
means for optimizingiDNAiequences and the three-dimensional struclure of
the plasmids for ability to_amfer upon 2 vector the ability to enter a cell
even
In the absence of detailed information as to the_mechanism by which this
effect is achieved
The methods involve reassembling (&/or subjecting to one or more
directed evolution methods described herein) at least first and second forms
of a
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nucleic acid that comprises a transfer sequence. The first and second forms
differ
from each other in two or more nucleotides. Suitable substrates include, for
example, transcription factor binding sites, CpG sequences, poly A, C, G, T
oligonucleotides, non-stochastically generated nucleic acid building blocks
,and
random DNA fragments such as, for example, genomic DNA, from human or
other mammalian species. It has been suggested that cell surface proteins,
such as
the macrophage scavenger receptor, may act as receptors for specific DNA
binding (Pisetsky (1996) Immunity 5:303). It is not known whether these
receptors recognize specific DNA sequences or whether they bind DNA in a
sequence non-specific manner. However, GGGG tetrads have been shown to
enhance DNA binding to cell surfaces (Id.). In addition to the DNA sequence,
the
three-dimensional structure of the plasmids may play a role in the capacity of
these plasmids to enter cells. The stochastic (e.g. polynucleotide shuffling &
interrupted synthesis) and non-stochastic polynucleotide reassembly methods of
the invention provide means for optimizing such sequences for ability to
confer
upon a vector the ability to enter a cell even in the absence of detailed
information
as to the mechanism by which this effect is achieved.
Clonal isolates of vectors bearing recombinant segments are used to infect
separate cultures of cells_and the percentage of vectors which enter cells
then determined b_y,for example, counting cells expressing a marker
expressed by the vectors in the course of_transfection
The resulting library of recombinant transfer modules are screened to
identify at least one optimized recombinant transfer module that enhances the
capability of a vector comprising the transfer module to enter a cell of
interest.
For example, vectors that include a recombinant transfer module can be
contacted
with a population of cells under conditions conducive to entry of the vector
into
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the cells, after which the percentage of cells in the population which contain
the
nucleic acid vector is determined. Preferably, the vector will contain a
selectable
or screenable marker to facilitate identification of cells which contain the
vector.
In a preferred embodiment, clonal isolates of vectors bearing recombinant
segments are used to infect separate cultures of cells. The percentage of
vectors
which enter cells can then be determined by, for example, counting cells
expressing a marker expressed by the vectors in the course of transfection.
The reassembly (&/or one or more additional directed_ evolution methods
described herein) and_rescnenine process can be repeated as necessary, until
a transfer mothiluthathas sufficient ability tuenhanee_ transfer is obtained
Typically, the reassembly (&/or one or more additional directed evolution
methods described herein) process is repeated by reassembling (&/or subjecting
to
one or more directed evolution methods described herein) at least one
optimized
transfer sequence with a further form of the transfer sequence to produce a
further
library of recombinant transfer modules. The further form can be the same or
different from the first and second forms. The new library is screened to
identify
at least one further optimized recombinant vector module that exhibits an
enhancement of the ability of a genetic vaccine vector that includes the
optimized
transfer module to enter a cell of interest.
The reassembly (&/or one or more additional directed evolution methods
described herein) and rescreening process can be repeated as necessary, until
a
transfer module that has sufficient ability to enhance transfer is obtained.
After
one or more of reassembly (&/or one or more additional directed evolution
methods described herein) and screening, vector modules are obtained which are
capable of conferring upon a nucleic acid vector the ability to enter at least
about
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50 percent more target cells than a control vector which does not contain the
optimized module, more preferably at least about 75 percent more, and most
preferably at least about 95 or 99 percent more target cells than a control
vector.
For integration by homologauxemmbination. importantfactors are the
degree andlength of komologv_to chromosomaLsequences, the frequency of
sueLsequences in the genome. and the specific sequence mediating
homologous recombinationaor nonhomologous, illegitimate and site-specific
recombination¨recombinationismediatal by specific sites on the therapy
vector which interact with cell encoded recombination proteins
Although for vaccine purposes non-integrating vectors are generally
preferred, for some applications it may be desirable to use an integrating
vector;
for these applications DNA sequences that directly or indirectly affect the
efficiency of integration can be included in the genetic vaccine vector. For
integration by homologous recombination, important factors are the degree and
length of homology to chromosomal sequences, as well as the frequency of such
sequences in the genome (e.g., Alu repeats). The specific sequence mediating
homologous recombination is also important, since integration occurs much more
easily in transcriptionally active DNA. Methods and materials for constructing
homologous targeting constructs are described by e.g., Mansour (1988) Nature
336:348; Bradley (1992) Bio/Technology 10:534. For nonhomologous,
illegitimate and site-specific recombination, recombination is mediated by
specific sites on the therapy vector which interact with cell encoded
recombination proteins, e.g., Cre/Lox and FIp/Frt systems. See, e.g., Baubonis
(1993) Nucleic Acids Res. 21:2025-2029, which reports that a vector including
a
LoxP site becomes integrated at a LoxP site in chromosomal DNA in the presence
of Cre recombinase enzyme.
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2.6. OPTIMIZATION OF GENETIC VACCINE COMPONENTS
Optimizing properties that caninfluenee the efficacy of a genetic vaccine in
liii I! I 10IIIl t.1A1 IItIII'I 1!
Many factors can influence the efficacy of a genetic vaccine in modulating
an immune response. The ability of the vector to enter a cell, for.example,
has a
significant effect on the ability of the vector to modulate an immune
response.
The strength of an immune response is also mediated by the immimogenicity of
an antigen expressed by a genetic vaccine vector and the level at which the
antigen is expressed. The presence or absence of costimulatory molecules
produced by the genetic vaccine vector can affect not only the strength, but
also
the type of immune response that arises due to introduction of the vector into
a
mammal. An increase in the persistence of a vector in an organism can lengthen
the time of inununomodulation, and also makes feasible self-boosting vectors
which do not require multiple administrations to achieve long-lasting
protection.
The present invention provides methods for optimizing many of these
properties,
thus resulting in genetic vaccine vectors that exhibit improved ability to
elicit the
desired effect on a mammalian immune system.
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The selection from large libraries lisingxecursive _cycles of reassembly
(optionally in combination with other directed_evolution methods described
berein) to maximally access all the fortuitous but complex mechanisms that
cannot be approached rationally
Genetic vaccines can contain a variety of functional components, whose
preferred sequences are best determined by stochastic (e.g. polynucleotide
shuffling & interrupted synthesis) and non-stochastic polynucleotide
reassembly,
the empirical sequence evolution described in detail herein. The methods of
the
invention involve, in general, constructing a separate library for each of the
major
vector components by stochastic (e.g. polynucleotide shuffling & interrupted
synthesis) and non-stochastic polynucleotide reassembly of multiple homologous
starting sequences, or other methods of generating a population of
recombinants,
resulting in a complex mixture of chimeric sequences. The best sequences are
selected from these libraries using the high-throughput assays described
below.
After one or more cycles of selection from each of the single module
libraries, the
pools of the best sequences of different modules can be combined by stochastic
(e.g. polynucleotide shuffling & interrupted synthesis) and non-stochastic
polynucleotide reassembly as long as the screens are compatible. The screens
for
promoter, enhancer, intron, transfer sequences, mammalian ori, bacterial on
and
bacterial marker, and the like, can eventually be combined, resulting in co-
optimization of the context of each sequence. An important aspect in these
experiments is the selection from large libraries using recursive cycles of
reassembly (optionally in combination with other directed evolution methods
described herein) to maximally access all the fortuitous but complex
mechanisms
that cannot be approached rationally, such as DNA transfer into the cell.
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A library of different vectors can be generated by assembling vector modules
that provide promoters cytokines. Maine antagonists, chemokines,
immunostimulatory sequences. and costimulatory molecules using assembly
PCR and combinatorial molecular biology
Assembly PCR is a method for assembly of long DNA sequences, such as
genes, non-stochastically generated nucleic acid building blocks, and
fragments of
plasmids. In contrast to PCR, there is no distinction between primers and
template, because the non-stochastically generated nucleic acid building
blocks
&/or fragments to be assembled prime each other. The library of Vector modules
obtained as described herein can be fused with promoters, which can themselves
be optimized by the stochastic (e.g. polynucleotide shuffling & interrupted
synthesis) and non-stochastic polynucleotide reassembly methods of the
invention. The resulting genes can be assembled combinatorially into DNA
vaccine vectors, where each gene is expressed under a different promoter
(e.g., a
promoter derived from a library of experimentally evolved (e.g. by
polynucleotide
reassembly &/or polynucleotide site-saturation mutagenesis) CMV promoters),
and the vector library is screened as described herein to identify vectors
which
exhibit the desired effect on the immune system.
Properties that influence the efficacy or desirability of the vaccine
The methods of the invention are useful for obtaining genetic vaccines that
are optimized for one or more of many properties that influence the efficacy
or
desirability of the vaccine. These properties include, but are not limited to,
the
following.
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2.6.1. EPISOMAL VECTOR MAINTENANCE
Episomally replicating vectors are maintained in a cell for a longer period of
time and permit the development of self-boostingyaccines
One property that one can optimize using the sequence reassembly
methods of the invention is the ability of a genetic vaccine vector to
replicate
episomally in a mammalian cell. Episomal replication of a vaccine vector is
advantageous in many situations. For example, episomally replicating vectors
are
maintained in a cell for a longer period of time than non-replicating vectors,
thus
resulting in an increased length of immune response modulation or increased
delivery of a therapeutically useful protein. Episomal replication also
permits the
development of self-boosting vaccines which, unlike traditional vaccines, do
not
require multiple vaccine administrations. For example, a self-boosting vaccine
vector can include an antigen-encoding gene which is under the control of an
inducible control element which allows induction of antigen expression, and
the
corresponding immune response, in response to a specific stimulus. However,
screening for naturally occurring vector modules which result in enhanced
episomal maintenance using traditional approaches or attempts to rationally
design mutants with improved properties would require many person-years of
research. The invention provides methods for generating and screening orders
of
magnitude more diversity in a short time period.
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Using stochastic (kg, IMIYIllideolide shuffling & interrupted synthesis) and
Don-stochastic polxnucleotlde reassembly to recombine at least two forms of
a nucleic acid whichis capable of conferring upon a genetic vector the ability
to replicate autonomously in mammalian cells
The ability of a genetic vaccine vector to replicate episomally can be
optimized by using stochastic (e.g. polynucleotide shuffling & interrupted
synthesis) and non-stochastic polynucleotide reassembly to recombine at least
two
forms of a nucleic acid which is capable of conferring upon a genetic vector
the
ability to replicate autonomously in mammalian cells. The two or more forms of
the episomal replication vector module differ from each other in two or more
nucleotides. A library of recombinant episomal replication vector modules is
produced, and the library is screened to identify one or more optimized
replication
vector modules which, when placed in a genetic vaccine vector, confer upon the
vector an enhanced ability to replicate autonomously compared to a vector
which
contains a non-optimized episomal replication vector module.
Repetition of the stochastic (e.g. PidYlludeolide shuffling & interrupted
synthesis) and non-stochastic polygpcleotide reassembly process at least once
to identify modules which exhibit enitanced_ability to confer episomal
maintenance upon a vector containing the module
In one embodiment, the stochastic (e.g. polynucleotide shuffling &
interrupted synthesis) and non-stochastic polynucleotide reassembly process is
repeated at least once using as a substrate an optimized episomal replication
vector module obtained from a previous round of stochastic (e.g.
polynucleotide
shuffling & interrupted synthesis) and non-stochastic polynucleotide
reassembly.
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The optimized vector module obtained in the earlier round is reassembled (&/or
subjected to one or more directed evolution methods described herein) with a
further form of the vector module, which can be the same as one of the forms
used in the earlier round, or can be a different form of a nucleic acid that
functions
as an episomal replication element. Again, a library of recombinant episomal
replication vector modules is produced, and the screening process is repeated
to
identify those episomal replication modules which exhibit enhanced ability to
confer episomal maintenance upon a vector containing the module.
=
Ability to replicate autonomously in eukaryotic cells- examples
Nucleic acids which are useful as substrates for the use of stochastic (e.g.
polynucleotide shuffling & interrupted synthesis) and non-stochastic
polynucleotide reassembly to optimize episomal replication ability include any
nucleic acid that is involved in conferring upon a vector the ability to
replicate
autonomously in eulcaryotic cells. For example, papillomavirus sequences E I
and
E2, simian virus 40 (SV40) origin of replication, and the like.
Genes from human pagillamaxinues are exemplary episomal replication
vector modules
Exemplary episomal replication vector modules that can be optimized using the
methods of the invention are genes from human papillomaviruses (HPV) which
are involved in episomal replication. HPV are non-tumorigenic viruses which
replicate episomally in skin and are stably expressed in vivo for years.
Bernard
and Apt (1994) Arch. Dermatol. 130:210.
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CA 02492661 2000-02-04
'I. lit_ tia. =1 , = , I !=l= =
ye. = ,* _11
replication using directed_ evolution
5 Despite these in vivo properties, it has not been possible to
maintain HPV
episomally in tissue culture due to underreplication. The invention provides
methods by which HPV genes involved in episomal maintenance can be
optimized for use in genetic vaccine vectors. HPV genes involved in episomal
replication include, for example, the El and E2 genes. Thus, according to one
10 embodiment of the invention, either or both of the HPV E Land E2 genes
are
subjected to stochastic (e.g. polynucleotide shuffling & interrupted
synthesis) and
non-stochastic polynucleotide reassembly to obtain a recombinant episomal
replication module which, when placed in a nucleic acid vaccine Vector,
results in
increased maintenance of the vector in mammalian cells. In a preferred
15 embodiment, the HPV El and E2 genes from different, but closely related,
benign
HPVs are used in a polynucleotide reassembly procedure, as shown, described
&/or referenced herein.
For example,
polynucletide shuffling of HPV El and E2 genes from closely related strains of
HPV (such as, for example, HPV 2,27, and 57) can be used to obtain a library
of
20 recombinant El and E2 genes which are then subjected to an appropriate
screening
method to identify those that exhibit improved episomal maintenance
properties.
Identification, selection. enrichment of recombinant episomal replication
25 vector modules that exhibit improved ability to mediate episomal
maintenance
To identify recombinant episomal replication vector modules that exhibit
improved ability to mediate episomal maintenance, members of the library of
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CA 02492661 2000-02-04
recombinant vector modules are inserted into vectors which are introduced into
mammalian cells. The cells are propagated for at least several generations,
after
which cells that have maintained the vector are identified. Identification can
be
accomplished, for example, employing a vector that includes a selectable
marker.
Cells containing the library members are propagated in the absence of
selection
for the selectable marker for at least several generations, after which
selective
pressure is added. Cells which survive selection are enriched for cells that
harbor
vectors which contain a recombinant vector module which enhances the ability
of
the vector to replicate episomally. DNA is recovered from the selected cells
and
introduced into bacterial host cells, allowing recovery of episomal, non-
integrated vectors.
Screening by introducine to &vector containing a polynucleotide encoding an
antigen that is present on_the surface of the cell when expressed
In another embodiment of the invention, the screening step is
accomplished by introducing members of the library of recombinant episomal
replication vector modules into a vector that includes a polynucleotide that
encodes an antigen which, when expressed, is present on the surface of a cell.
The
library of vectors is introduced into mammalian cells which are propagated for
at
least several generations, after which cells which display the cell surface
antigen
on the surface of the cell are identified. Such cells most likely harbor a
genetic
vaccine vector which enhances the ability of the vector to replicate
autonomously.
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Use of optimized recombinant railainat replication vector module to
construct genetic vaccine vectors
Upon identifying cells which contain an episomally maintained vector, the
optimized recombinant episomal replication vector module is obtained and used
to construct genetic vaccine vectors. Cell surface antigens which are suitable
for
use in the screening methods are described above, and others are known to
those
of skill in the art. Preferably, an antigen is used for which a convenient
means Of
detection is available.
Preferred cells fpr use in the screening methods
Cells which are suitable for use in the screening methods include both
cultured mammalian cells and cells which are present in an animal. To screen
for
recombinant vector modules that are intended for use in humans, the preferred
cells for screening purposes are human cells. Generally, initial screening is
accomplished in cell culture, where processing of large libraries of
experimentally
evolved (e.g. by polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis) material is feasible. In a preferred embodiment, cells which
display
a vector-encoded cell surface antigen on the cell surface are identified by
flow
cytometry based cell sorting methods, such as fluorescence activated cell
sorting.
This approach allows very large numbers (> 107) cells to be evaluated in a
single
vial experiment.
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DEMANDES OU BREVETS VOLUIVIINEUX
LA PRESENTE PARTIE DE CETTE DEIVIANDE OU CE BREVETS
COIVIPREND PLUS D'UN TOME.
CECI EST LE TOME _____________________ DE 3
NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des
Brevets.
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THAN ONE VOLUME.
THIS IS VOLUME I OF 3
NOTE: For additional volumes please contact the Canadian Patent Office.
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