IE84214B1 - Heterodimeric receptor libraries using phagemids - Google Patents
Heterodimeric receptor libraries using phagemidsInfo
- Publication number
- IE84214B1 IE84214B1 IE1992/1169A IE921169A IE84214B1 IE 84214 B1 IE84214 B1 IE 84214B1 IE 1992/1169 A IE1992/1169 A IE 1992/1169A IE 921169 A IE921169 A IE 921169A IE 84214 B1 IE84214 B1 IE 84214B1
- Authority
- IE
- Ireland
- Prior art keywords
- dna
- polypeptide
- phage
- vector
- sequence
- Prior art date
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Description
PATENTS ACT, 1992
1992/1169
HETERODIMERIC RECEPTOR LIBRARIES USING PHAGEMIDS
THE SCRIPPS RESEARCH INSTITUTE
HETERODIMERIC RECEPTOR LIBRARIES USING PHAGEMIDS
Technical Field
The present invention relates to cloning vectors
and methods for producing a library of DNA molecules
capable of expressing a fusion polypeptide on the
surface of a filamentous phage particle.
Background
Filamentous bacteriophages are a group of related
viruses that infect bacteria. They are termed
filamentous because they are long and thin particles
comprised of an elongated capsule that envelopes the
deoxyribonucleic acid (DNA) that forms the
bacteriophage genome. The F pili filamentous
bacteriophage (Ff phage) infect only gram-negative
bacteria by specifically adsorbing to the tip of F
pili, and include fd, fl and M13.
The mature capsule of Ff phage is comprised of a
coat of five phage-encoded gene products: cpVIII, the
major coat protein product of gene VIII that forms the
bulk of the capsule; and four minor coat proteins,
cpIII and cpIV at one end of the capsule and cpVII and
cpIX at the other end of the capsule. The length of
the capsule is formed by 2500 to 3000 copies of cpVIII
in an ordered helix array that forms the
characteristic filament structure. About five copies
each of the minor coat proteins are present at the
ends of the capsule. The gene III-encoded protein
(cpIII) is typically present in 4 to 6 copies at one
end of the capsule and serves as the receptor for
binding of the phage to its bacterial host in the
initial phase of infection. For detailed reviews of
Ff phage structure, see Rasched et al., Microbiol.
ggyy, 50:401-427 (1986); and Model et al., in "The
Bacteriophages, Volume 2", R. Calendar, Ed., Plenum
Press, pp. 375-456 (1988).
The assembly of a Ff phage particle involves
highly complex mechanics. No phage particles are‘
assembled within a host cell; rather, they are
assembled during extrusion of the viral genome through
the host cell's membrane. Prior to extrusion, the
major coat protein cpVIII and the minor coat protein
cpIII are synthesized and transported to the host
cell's membrane. Both cpVIII and cpIII are anchored
in the host cell membrane prior to their incorporation
into the mature particle. In addition, the viral
genome is produced and coated with cpV protein.
During the extrusion process, cpV-coated genomic DNA
is stripped of the cpV coat and simultaneously re-
coated with the mature coat proteins. The assembly
mechanisms that control transferral of these proteins
from the membrane to the particle is not presently
known.
Both cpIII and cpVIII proteins include two
domains that provide signals for assembly of the
mature phage particle. The first domain is a
secretion signal that directs the newly synthesized
protein to the host cell membrane. The secretion
signal is located at the amino terminus of the
polypeptide and targets the polypeptide at least to
the cell membrane. The second domain is a membrane
anchor domain that provides signals for association
with the host cell membrane and for association with
the phage particle during assembly. This second signal
for both cpVIII and cpIII comprises at least a
hydrophobic region for spanning the membrane.
cpVIII has been extensively studied as a model
membrane protein because it can integrate into lipid
bilayers such as the cell membrane in an asymmetric
orientation with the acidic amino terminus toward the
outside and the basic carboxy terminus toward the
inside of the membrane. The mature protein is about
50 amino acid residues in length of which 11 residues
provide the carboxy terminus, 19 residues provide the
hydrophobic transmembrane region, and the remaining
residues comprise the amino terminus. Considerable
research has been done on the secretion signal region
of cpVIII to advance the study of membrane protein
synthesis and targeting to membranes. However, little
is known about the changes that are tolerated in the
structure of the cpVIII membrane anchor region that
would allow for assembly of phage particles.
Manipulation of the sequence of cpIII shows that
the C—terminal 23 amino acid residue stretch of
hydrophobic amino acids normally responsible for a
membrane anchor function can be altered in a variety
of ways and retain the capacity to associate with
membranes. However, those anchor-modified cpIII
proteins lost their ability to genetically complement
gene III mutants indicating that the requirements of a
membrane anchor for functional assembly have not been
elucidated.
Ff phage-based expression vectors have been
described in which the entire cpIII amino acid residue
sequence was modified by insertion of short
polypeptide "epitopes" [Parmely et al., Gene, 73:305-
3l8 (1988); and Cwirla et al., Proc. Natl. Acad. Sci.
ggg, 87:6378-6382 (1990)] or an amino acid residue
sequence defining a single chain antibody domain.
Mccafferty et al., Science, 348:552-554 (1990). These
hybrid proteins were synthesized and assembled onto
phage particles in amounts of about 5 copies per
particle, a density at which normal cpIII is usually
found. However, these expressed fusion proteins
include the entire cpIII amino acid residue sequence
and do not suggest fusion proteins that utilize only
the carboxy terminal membrane anchor domain of cpIII.
In addition, no expression system has been
described in which a phage coat protein has been
engineered to allow assembly of a heterodimeric
molecule that is functional and capable of
incorporation into the coat of a phage particle.
Brief Summary of the Invention
A new surface-integration technology has been
discovered for expressing a heterodimeric recombinant
gene product on the surface of a filamentous phage
containing the recombinant gene. The invention uses a
filamentous phage coat protein membrane anchor domain
as a means for linking gene—product and gene during
the assembly stage of filamentous phage replication.
That is, during filamentous phage replication,
coat proteins assemble into a matrix which
encapsulates the phage genome. It has now been
discovered that (1) phage assembly is not disrupted
when recombinant filamentous phage coat proteins are
present, (2) recombinant filamentous phage coat
proteins can be integrated into the assembling matrix,
and (3) integration into the matrix can be directed to
occur in a surface-accessible orientation.
The present invention can be advantageously
applied to the production of heterodimeric receptors
of predetermined specificity, i.e., it can be used to
produce antibodies, T—cell receptors and the like that
bind a preselected ligand.
Thus, the present invention provides for linking
the functions of heterodimeric receptor recognition
and filamentous phage replication in a method for
isolating a heterodimeric receptor and the gene that
encodes that receptor. The method produces a filamentous
phage comprised of a matrix of gene VIII-encoded
proteins that encapsulate a recombinant genome. The
recombinant genome contains genes encoding the
heterodimeric receptor polypeptides. The
heterodimeric receptor is surface-integrated into the
encapsulating matrix via a filamentous phage coat
protein's membrane anchor domain that is fused by a
peptide bond during translation to one of the
heterodimeric receptor polypeptides. The
heterodimeric receptor polypeptides and the genes
which encode the polypeptides are physically linked
during the assembly stage of the phage replication
cycle. Specifically binding the receptor-coated phage
to a solid-support advantageously provides a means for
isolating a recombinant genome that encodes a desired
heterodimeric receptor from a diverse library of
recombinant genomes.
In one embodiment, the present invention
contemplates an antibody molecule comprising heavy-
and light-chain polypeptides, said heavy-chain
polypeptide comprising a V,-domain flanked by an
amino-terminal prokaryotic secretion signal domain and
a carboxy-terminal filamentous phage membrane anchor
domain, said light chain polypeptide comprising a VF-
domain fused to an amino-terminal prokaryotic
secretion signal domain.
In another embodiment, the present invention
contemplates a vector for expressing a fusion
polypeptide, said vector comprising upstream and
downstream translatable DNA sequences operatively
linked via a sequence of nucleotides adapted for
directional ligation of an insert DNA, said upstream
sequence encoding a prokaryotic secretion signal, said
downstream sequence encoding a filamentous phage
membrane anchor, said translatable DNA sequences
operatively linked to a set of DNA expression signals
for expression of said translatable DNA sequences as
portions of said fusion polypeptide.
Brief Description of the Drawings
In the drawings forming a portion of this
disclosure:
Figure 1 illustrates a schematic diagram of the
immunoglobulin molecule showing the principal
structural features. The circled area on the heavy
chain represents the variable region (VH), a
polypeptide containing a biologically active (ligand
binding) portion of that region, and a gene coding for
that polypeptide, are produced by the methods of the
present invention.
Figure 2A is a diagrammatic sketch of a heavy (H)
chain of human IgG (IgG1 subclass). Numbering is from
the N-terminus on the left to the C—terminus on the
right. Note the presence of four domains, each
containing an intrachain disulfide bond (S-S) spanning
approximately 60 amino acid residues. The symbol CHO
stands for carbohydrate. The V region of the heavy
(H) chain (VH) resembles V, in having three
hypervariable CDR (not shown).
Figure 2B is a diagrammatic sketch of a human
light (Kappa) chain (Panel 1). Numbering is from the
N-terminus on the left to the C—terminus on the right.
Note the intrachain disulfide bond (S-S) spanning
about the same number of amino acid residues in the VL
and CL domains. Panel 2 shows the locations of the
complementarity—determining regions (CDR) in the VL
domain. Segments outside the CDR are the framework
segments (FR).
Figure 3 illustrates the sequence of the double-
stranded synthetic DNA inserted into Lambda Zap to
produce a Lambda Hcz expression vector. The
preparation of the double—stranded synthetic DNA
insert is described in Example 1a(ii). The various
features required for this vector to express the V[-
coding DNA homologs include the Shine—Da1garno
ribosome binding site, a leader sequence to direct the
expressed protein to the periplasm as described by
Mouva et al., J. Biol. Chem., 255:27, 1980, and
various restriction enzyme sites used to operatively
link the V‘ homologs to the expression vector. The V"
expression vector sequence also contains a short
nucleic acid sequence that codes for amino acids
typically found in variable regions heavy chain (VH
backbone). This VH backbone is just upstream and in
the proper reading as the V“ DNA homologs that are
operatively linked into the Xho I and Spe I cloning
sites. The sequences of the top and bottom strands of
the double—stranded synthetic DNA insert are listed
respectively as SEQ ID NO 1 and SEQ ID NO 2. The
synthetic DNA insert is directionally ligated into
Lambda Zap II digested with the restriction enzymes
Not 1 and Xho I to form Lambda Hcz expression vector.
Figure 4 illustrates the major features of the
bacterial expression vector Lambda Hc2 (VH expression
vector). The synthetic DNA sequence from Figure 3 is
shown at the top along with the T3 polymerase promoter
from Lambda Zap II. The orientation of the insert in
Lambda Zap II is shown. The V” DNA homologs are
inserted into the Xho I and Spe I cloning sites. The
read through transcription produces the decapeptide
epitope (tag) that is located just 3' of the cloning
site.
Figure 5 illustrates the sequence of the double-
stranded synthetic DNA inserted into Lambda Zap to
produce a Lambda Lc2 expression vector. The various
features required for this vector to express the VE-
coding DNA homologs are described in Figure 3. The
Vi-coding DNA homologs are operatively linked into the
Lc2 sequence at the Sac I and Xho I restriction sites.
The sequences of the top and bottom strands of the
double-stranded synthetic DNA insert are listed
respectively as SEQ ID NO 3 and SEQ ID NO 4. The
synthetic DNA insert is directionally ligated into
Lambda Zap II digested with the restriction enzymes
Sac I and Not I to form Lambda Lc2 expression vector.
Figure 6 illustrates the major features of the
bacterial expression vector Lc2 (VL expression
vector). The synthetic DNA sequence from Figure 5 is
shown at the top along with the T3 polymerase promoter
from Lambda Zap II. The orientation of the insert in
Lambda Zap II is shown. The VL DNA homologs are
inserted into the Sac I and Xho I cloning sites.
Figure 7 illustrates the dicistronic expression
vector, pcomb, in the form of a phagemid expression
vector. To produce pComb, phagemids were first
excised from the expression vectors, Lambda Hc2 and
Lambda Lc2, using an in vivo excision protocol
according to manufacturers instructions (Stratagene,
La Jolla, California). The pcomb expression vector is
prepared from Lambda Hc2 and Lambda Lc2 which do not
contain Vi-coding or VL—coding DNA homologs. The in
yiyg excision protocol moved the cloned insert from
the Lambda Hc2 and Lc2 vectors into a phagemid vector.
The resultant phagemids contained the same nucleotide
sequences for antibody fragment cloning and expression
as did the parent vectors. Hcz and Lc2 phagemid
expression vectors were separately restriction
digested with Sca I and EcoR I. The linearized
phagemids were ligated via the Sca I and EcoR I
cohesive termini to form the dicistronic
(combinatorial) vector, pcomb.
Figure 8 illustrates a schematic diagram of the
composition of pCBAK phagemid vector, the pathway
for Fab assembly and incorporation in phage coat. The
vector carries the chloramphenicol acetyl transferase
(CAT) marker gene in addition to the nucleotide
residue sequences encoding the Fd—cpVIII fusion
polypeptide and the kappa chain. The f1 phage origin
of replication facilitates the generation of single
stranded phagemid. The isopropyl
thiogalactopyranoside (IPTG) induced expression of a
dicistronic message encoding the Fd-cpVIII fusion (VH,
Cm, cpVIII) and the light chain (V1, CL) leads to the
formation of heavy and light chains. Each chain is
delivered to the periplasmic space by the pelB target
sequence, which is subsequently cleaved. The heavy
chain is anchored in the membrane by cpVIII fusion
while the light chain is secreted into the periplasm.
The heavy chain in the presence of light chain
assembles to form Fab molecules. The Fabs are
incorporated into phage particles via cpVIII (black
dots).
Figure 9 illustrates the electron micrographic
localization of 5-7 nm colloidal gold particles coated
with NPN-BSA conjugate along the surface of
filamentous phage, and from phage emerging from a
bacterial cell. Panel 9A shows filamentous phage
emerging from the surface of the bacterial cell
specifically labelled with the colloidal gold
particles coated with BSA-NPN antigen. Panel 9B shows
a portion of a mature filamentous phage on the length
of which is exhibited the labelling of antigen binding
sites.
Figure 10 illustrates the results of a two-site
ELISA for assaying for the presence and function of
Fab antibody attached to the surface of bacteriophage
particles as described in Example 4b. For expression
of Fab antibody on phage surfaces, XL1-Blue cells were
transformed with the phagemid expression vector,
pCBAK8-2b. The inducer, isopropyl
thiogalactopyranoside (IPTG), was admixed with the
bacterial suspension at a final concentration of 1 mM
for one hour. Helper phage was then admixed with the
bacterial suspension to initiate the generation of
copies of the sense strand of the phagemid DNA. After
a two hour maintenance period, bacterial supernatants
containing bacteriophage particles were collected for
assaying in ELISA.
Specific titratable binding of NPN-Fab-expressing
bacteriophage particles to NPN-coated plates was
exhibited. No binding was detected with helper phage
alone.
Figure 11 illustrates the inhibition of NPN-Fab
expressing bacteriophage to NPN antigen-coated plates
with the addition of increasing amounts of free
hapten. The assays were performed as described in
Figure 10. Complete inhibition of binding was
observed with 5 ng of added free NPN hapten.
Figure 12 illustrates schematically the process
of mutagenizing the CDR3 region of a heavy chain
fragment resulting in an alteration of binding
specificity. The oligonucleotide primers are
indicated by black bars. The process is described in
Example 6.
Figure 13 illustrates the amino acid sequences
and corresponding SEQ ID N0 of the heavy chain of the
CDR3 region of the starting and selected clones, and
the affinities of the clones for free fluorescein and
FI-BSA. The asterisk indicates the approximate Kd as
determined by competitive ELISA.
Figure 14 illustrates the amino acid sequences
and corresponding SEQ ID N0 of the light chain of the
CDR3 region of the starting and selected clones, and
the affinities of the clones for free fluorescein and
FI-BSA. The asterisk indicates the approximate Kd as
determined by competitive ELISA.
Detailed Description of the Invention
A. Definitions
Amino Acid Residue: An amino acid formed
upon chemical digestion (hydrolysis) of a polypeptide
at its peptide linkages. The amino acid residues
described herein are preferably in the "L" isomeric
form. However, residues in the "D" isomeric form can
be substituted for any L—amino acid residue, as long
as the desired functional property is retained by the
polypeptide. 1&5 refers to the free amino group
present at the amino terminus of a polypeptide. COOH
refers to the free carboxy group present at the
carboxy terminus of a polypeptide. In keeping with
standard polypeptide nomenclature (described in Q;
Biol. Chem., 243:3552-59 (1969) and adopted at 37
C.F.R. 1.822(b)(2)), abbreviations for amino acid
residues are shown in the following Table of
Correspondence:
TABLE OF CORRESPONDENCE
SYMBOL AMINO ACID
1-Letter 3-Letter
Y Tyr tyrosine
G Gly glycine
F Phe phenylalanine
M Met methionine
A Ala alanine
S Ser serine
I Ile isoleucine
L Leu leucine
T Thr threonine
V Val valine
P Pro proline
K Lys lysine
H His histidine
Q Gln glutamine
E Glu glutamic acid
Z Glx Glu and/or Gln
W Trp tryptophan
R Arg arginine
D Asp aspartic acid
N Asn asparagine
B Asx Asn and/or Asp
C Cys cysteine
J Xaa Unknown or other
It should be noted that all amino acid residue
sequences represented herein by formulae have a left-
to-right orientation in the conventional direction of
amino terminus to carboxy terminus. In addition, the
phrase "amino acid residue" is broadly defined to
include the amino acids listed in the Table of
Correspondence and modified and unusual amino acids,
such as those listed in 37 CFR 1.822(b)(4L
Furthermore, it
should be noted that a dash at the beginning or end of
an amino acid residue sequence indicates a peptide
bond to a further sequence of one or more amino acid
residues or a covalent bond to an amino-terminal group
such as NH2 or acetyl or to a carboxy—terminal group
such as COOH.
Nucleotide: A monomeric unit of DNA or RNA
consisting of a sugar moiety (pentose), a phosphate,
and a nitrogenous heterocyclic base. The base is
linked to the sugar moiety via the glycosidic carbon
(1' carbon of the pentose) and that combination of
base and sugar is a nucleoside. When the nucleoside
contains a phosphate group bonded to the 3' or 5'
position of the pentose it is referred to as a
nucleotide. A sequence of operatively linked
nucleotides is typically referred to herein as a "base
sequence" or "nucleotide sequence", and their
grammatical equivalents, and is represented herein by
a formula whose left to right orientation is in the
conventional direction of 5'—terminus to 3'-terminus.
Base Pair (bp): A partnership of adenine (A)
with thymine (T), or of cytosine (C) with guanine (G)
in a double stranded DNA molecule. In RNA, uracil (U)
is substituted for thymine.
Nucleic Acid: A polymer of nucleotides, either
single or double stranded.
Polynucleotide: a polymer of single or double
stranded nucleotides. As used herein "polynucleotide"
and its grammatical equivalents will include the full
range of nucleic acids. A polynucleotide will
typically refer to a nucleic acid molecule comprised
of a linear strand of two or more deoxyribonucleotides
and/or ribonucleotides. The exact size will depend on
many factors, which in turn depends on the ultimate
conditions of use, as is well known in the art. The
polynucleotides of the present invention include
primers, probes, RNA/DNA segments, oligonucleotides or
"oligos" (relatively short polynucleotides), genes,
vectors, plasmids, and the like.
gene: A nucleic acid whose nucleotide sequence
codes for an RNA or polypeptide. A gene can be either
RNA or DNA.
-14..
Duplex DNA: a double-stranded nucleic acid
molecule comprising two strands of substantially
complementary polynucleotides held together by one or
more hydrogen bonds between each of the complementary
bases present in a base pair of the duplex. Because
the nucleotides that form a base pair can be either a
ribonucleotide base or a deoxyribonucleotide base, the
phrase "duplex DNA" refers to either a DNA-DNA duplex
comprising two DNA strands (ds DNA), or an RNA—DNA
duplex comprising one DNA and one RNA strand.
Complementary Bases: Nucleotides that normally
pair up when DNA or RNA adopts a double stranded
configuration.
Complementary Nucleotide Seggence: A sequence of
nucleotides in a single—stranded molecule of DNA or
RNA that is sufficiently complementary to that on
another single strand to specifically hybridize to it
with consequent hydrogen bonding.
Conserved: A nucleotide sequence is conserved
with respect to a preselected (reference) sequence if
it non—randomly hybridizes to an exact complement of
the preselected sequence.
Hybridization: The pairing of substantially
complementary nucleotide sequences (strands of nucleic
acid) to form a duplex or heteroduplex by the
establishment of hydrogen bonds between complementary
base pairs. It is a specific, i.e. non-random,
interaction between two complementary polynucleotides
that can be competitively inhibited.
Nucleotide Analog: A purine or pyrimidine
nucleotide that differs structurally from A, T, G, C,
or U, but is sufficiently similar to substitute for
the normal nucleotide in a nucleic acid molecule.
DNA Homolog: Is a nucleic acid having a
preselected conserved nucleotide sequence and a
-15..
sequence coding for a receptor capable of binding a
preselected ligand.
Recombinant DNA (rDNA) molecule: a DNA molecule
produced by operatively linking two DNA segments.
Thus, a recombinant DNA molecule is a hybrid DNA
molecule comprising at least two nucleotide sequences
not normally found together in nature. rDNA's not
having a common biological origin, i.e.,
evolutionarily different, are said to be
"heterologous".
Vector: a rDNA molecule capable of autonomous
replication in a cell and to which a DNA segment,
e.g., gene or polynucleotide, can be operatively
linked so as to bring about replication of the
attached segment. Vectors capable of directing the
expression of genes encoding for one or more
polypeptides are referred to herein as "expression
vectors". Particularly important vectors allow
cloning of cDNA (complementary DNA) from mRNAs
produced using reverse transcriptase.
Receptor: A receptor is a molecule, such as a
protein, glycoprotein and the like, that can
specifically (non-randomly) bind to another molecule.
Antibody: The term antibody in its various
grammatical forms is used herein to refer to
immunoglobulin molecules and immunologically active
portions of immunoglobulin molecules, i.e., molecules
that contain an antibody combining site or paratope.
Exemplary antibody molecules are intact immunoglobulin
molecules, substantially intact immunoglobulin
molecules and portions of an immunoglobulin molecule,
including those portions known in the art as Fab,
Fab‘, F(ab')2 and F(v).
Antibody Combining Site: An antibody combining
site is that structural portion of an antibody
molecule comprised of a heavy and light chain variable
and hypervariable regions that specifically binds
(immunoreacts with) an antigen. The term immunoreact
in its various forms means specific binding between an
antigenic determinant—containing molecule and a
molecule containing an antibody combining site such as
a whole antibody molecule or a portion thereof.
Monoclonal Antibody: The phrase monoclonal
antibody in its various grammatical forms refers to a
population of antibody molecules that contains only
one species of antibody combining site capable of
immunoreacting with a particular antigen. A
monoclonal antibody thus typically displays a single
binding affinity for any antigen with which it
immunoreacts. A monoclonal antibody may therefore
contain an antibody molecule having a plurality of
antibody combining sites, each immunospecific for a
different antigen, e.g., a bispecific monoclonal
antibody.
Fusion Polypeptide: A polypeptide comprised of at
least two polypeptides and a linking sequence to
operatively link the two polypeptides into one
continuous polypeptide. The two polypeptides linked
in a fusion polypeptide are typically derived from two
independent sources, and therefore a fusion
polypeptide comprises two linked polypeptides not
normally found linked in nature.
Upstream: In the direction opposite to the
direction of DNA transcription, and therefore going
from 5' to 3' on the non-coding strand, or 3' to 5' on
the mRNA.
Downstream: Further along a DNA sequence in the
direction of sequence transcription or read out, that
is traveling in a 3'- to 5'—direction along the non-
coding strand of the DNA or 5'— to 3'-direction along
the RNA transcript.
Cistron: Sequence of nucleotides in a DNA
molecule coding for an amino acid residue sequence and
including upstream and downstream DNA expression
control elements.
Stop Codon: Any of three codons that do not code
for an amino acid, but instead cause termination of
protein synthesis. They are UAG, UAA and UGA and are
also referred to as a nonsense or termination codon.
Leader Polypeptide: A short length of amino acid
sequence at the amino end of a polypeptide, which
carries or directs the polypeptide through the inner
membrane and so ensures its eventual secretion into
the periplasmic space and perhaps beyond. The leader
sequence peptide is commonly removed before the
polypeptide becomes active.
Reading Frame: Particular sequence of contiguous
nucleotide triplets (codons) employed in translation.
The reading frame depends on the location of the
translation initiation codon.
B. Filamentous Phage
The present invention contemplates a
filamentous phage comprising a matrix of proteins
encapsulating a genome encoding first and second
polypeptides capable of forming a heterodimeric
receptor. The phage further contains a heterodimeric
receptor comprised of the first and second
polypeptides surface-integrated into the matrix via a
filamentous phage membrane anchor domain fused to at
least one of the first or second polypeptides. The
heterodimeric receptor has the capacity to bind ligand
and therefor is referred to as a ligand—binding
heterodimeric receptor. Thus, in one aspect of the
invention, there is provided a filamentous phage as claimed
in claim 1.
The heterodimeric receptor in a preferred
embodiment is an epitope-binding complex. That is, a
complex of first and second polypeptides capable of
binding an epitope. Preferably, the first and second
polypeptides are antibody heavy chain and light chain
polypeptides.
The first and second polypeptides are capable of
autogenous assembly into a functional epitope-binding
complex (heterodimeric receptor), which is then
expressed on the outer surface of the phage in a
manner accessible to ligand, i.e., they are surface-
integrated into the phage. Thus, an epitope-binding
complex is typically present on the surface of a phage
of this invention. Typically, the epitope-binding
complex is comprised of a linking polypeptide that
contains a filamentous phage membrane anchor domain,
such as a polypeptide described in Section C, and a
non—linking polypeptide(s).
Because the receptor is linked to the phage in a
surface accessible manner, the phage can be advantageously
used as a solid—phase affinity sorbent. In methods which
make use of the present invention, the phage can be linked,
preferably removably linked, to a solid (aqueous insoluble)
matrix such as agarose, cellulose, synthetic resins,
polysaccharides and the like. For example, transformants
shedding the phage can be applied to and retained in a
column and maintained under conditions that support
shedding of the phage. An aqueous composition containing a
ligand that binds to the receptor expressed by the phage is
then passed through the column at a predetermined rate and
under receptor—binding conditions to form a solid—phase
receptor—ligand complex. The column is then washed to
remove unbound material, leaving the ligand bound to
the solid-phase phage. The ligand can then be removed
and recovered by washing the column with a buffer that
promotes dissociation of the receptor-ligand complex.
Alternatively, purified phage can be admixed with
a aqueous solution containing the ligand to be
affinity purified. The receptor/ligand binding
reaction admixture thus formed is maintained for a
time period and under binding conditions sufficient
for a phage-linked receptor-ligand complex to form.
The phage—bound ligand (ligand-bearing phage) are then
separated and recovered from the unbound materials,
such as by centrifugation, electrophoresis,
precipitation, and the like.
The present invention also provides a filamentous
phage as claimed in claim 3.
Phage of this invention can be labeled when used
in a diagnostic method of this invention. Preferred
labels include radioactively labeled nucleic acids
incorporated into the phage genome, or radioactively
labeled amino acids incorporated into protein
components of the phage particle. Preparation of
labeled phage can be routinely prepared by growing
phage as described herein, but including radiolabeled
nucleotides or radiolabeled amino acids in the culture
medium for incorporation into nucleic acids or
polypeptides of the phage, respectively.
labels are 3H-thymidine or “S-methionine.
Exemplary
other
isotopic labels and other nucleotide or amino acid
precursors are readily available to one skilled in the
art. The labeled phage preferably contains sufficient
label to be detectable in a ligand binding assay of
this invention, i.e., the phage is detectably labeled.
C. DNA Expression Vectors
A vector of the present invention is a
recombinant DNA (rDNA) molecule adapted for receiving
..20_
and expressing translatable DNA sequences in the form
of a fusion polypeptide containing a filamentous phage
membrane anchor domain and a prokaryotic secretion
signal domain. The vector comprises a cassette that
includes upstream and downstream translatable DNA
sequences operatively linked via a sequence of
nucleotides adapted for directional ligation to an
insert DNA. The upstream translatable sequence
encodes the secretion signal as defined herein. The
downstream translatable sequence encodes the
filamentous phage membrane anchor as defined herein.
The cassette preferably includes DNA expression
control sequences for expressing the fusion
polypeptide that is produced when an insert
translatable DNA sequence (insert DNA) is
directionally inserted into the cassette via the
sequence of nucleotides adapted for directional
ligation.
The DNA expression vector provides a system for
independently cloning (inserting) two translatable DNA
sequences into two separate cassettes present in the
vector, to form two separate 'cistrons‘, (see below) for
expressing both polypeptides of a heterodimeric receptor,
or the ligand binding portions of the polypeptides that
comprise a heterodimeric receptor. The DNA expression
vector for expressing two cistrons is referred to as a
dicistronic expression vector.
Thus, the DNA expression vector of this invention
-20a-
comprises, in addition to the above cassette , a second
cassette for expressing a second fusion polypeptide. The
second cassette includes a second translatable DNA sequence
that encodes a secretion signal, as defined herein
operatively linked at its 3' terminus via a sequence of
nucleotides adapted for directional ligation to a
downstream DNA sequence of the vector that typically
defines at least one stop codon in the reading frame of the
cassette. The second translatable DNA sequence is
operatively linked at its 5' terminus to DNA expression
control sequences forming the 5' elements defined above.
The second cassette is capable, upon insertion of a
translatable DNA sequence (insert DNA), of expressing the
second fusion polypeptide comprising a fusion of the
secretion signal with a polypeptide coded by the insert
DNA.
The filamentous phage membrane anchor is
preferably a domain of the cpIII or cpVIII coat
protein capable of binding the,matrix of a filamentous
phage particle, thereby incorporating the fusion
polypeptide onto the phage surface.
An expression vector is characterized as being
capable of expressing, in a compatible host, a
structural gene product such as a fusion polypeptide
of the present invention. I
As used herein, the term "vector" refers to a
nucleic acid molecule capable of transporting between
different genetic environments another nucleic acid to
which it has been operatively linked. Preferred
vectors are those capable of autonomous replication
and expression of structural gene products present in
the DNA segments to which they are operatively linked.
As used herein with regard to DNA sequences or
segments, the phrase "operatively linked" means the
sequences or segments have been covalently joined,
preferably by conventional phosphodiester bonds, into
one strand of DNA, whether in single or double
stranded form.
The choice of vector to which the cassettes of this
invention are operatively linked depends directly, as
is well known in the art, on the functional properties
desired, e.g., vector replication and protein
expression, and the host cell to be transformed, these
being limitations inherent in the art of constructing
recombinant DNA molecules.
In preferred embodiments, the vector utilized
includes a prokaryotic replicon i.e., a DNA sequence
having the ability to direct autonomous replication
and maintenance of the recombinant DNA molecule extra
chromosomally in a prokaryotic host cell, such as a
bacterial host cell, transformed therewith. Such
replicons are well known in the art. In addition,
those embodiments that include a prokaryotic replicon
also include a gene whose expression confers a
selective advantage, such as drug resistance, to a
bacterial host transformed therewith. Typical
bacterial drug resistance genes are those that confer
resistance to ampicillin or tetracycline. Vectors
typically also contain convenient restriction sites
for insertion of translatable DNA sequences.
Exemplary vectors are the plasmids pUC8, pUC9, pBR322,
and pBR329 available from BioRad Laboratories,
(Richmond, CA) and pPL and pKK223 available from
Pharmacia, (Piscataway, NJ).
A sequence of nucleotides adapted for directional
ligation, i.e., a polylinker, is a region of the DNA
expression vector that (1) operatively links for
replication and transport the upstream and downstream
translatable DNA sequences and (2) provides a site or
means for directional ligation of a DNA sequence into
the vector. Typically, a directional polylinker is a
sequence of nucleotides that defines two or more
restriction endonuclease recognition sequences, or
restriction sites. Upon restriction cleavage, the two
sites yield cohesive termini to which a translatable
DNA sequence can be ligated to the DNA expression
vector. Preferably, the two restriction sites
provide, upon restriction cleavage, cohesive termini
that are non-complementary and thereby permit
directional insertion of a translatable DNA sequence
into the cassette. In one embodiment, the directional
ligation means is provided by nucleotides present in
the upstream translatable DNA sequence, downstream
translatable DNA sequence, or both. In another
embodiment, the sequence of nucleotides adapted for
directional ligation comprises a sequence of
nucleotides that defines multiple directional cloning
means. Where the sequence of nucleotides adapted for
directional ligation defines numerous restriction
sites, it is referred to as a multiple cloning site.
A translatable DNA sequence is a linear series of
nucleotides that provide an uninterrupted series of at
least 8 codons that encode a polypeptide in one
reading frame.
An upstream translatable DNA sequence encodes a
prokaryotic secretion signal. The secretion signal is
a leader peptide domain of protein that targets the
protein to the periplasmic membrane of gram negative
bacteria.
A preferred secretion signal is a pelB secretion
signal. The predicted amino acid residue sequences of
the secretion signal domain from two pelB gene product
variants from Erwinia carotova are shown in Table 1 as
described by Lei, et al., Nature, 33l:543-546 (1988).
...23_
A particularly preferred pelB secretion signal is also
shown in Table 1.
The leader sequence of the pelB protein has
previously been used as a secretion signal for fusion
proteins. Better et al., Science, 240:1041-1043
(1988); Sastry et al., Proc. Natl. Acad. Sci. USA,
86:5728—5732 (1989); and Mullinax et al., Proc. Natl.
Acad. Sci. USA, 8728095-8099 (1990).
Amino acid residue sequences for other secretion
signal polypeptide domains from E. coli useful in this
invention are also listed in Table 1. Oliver, In
Neidhard, F.C. (ed.), Escherichia coli and Salmonella
T himurium, American Society for Microbiology,
Washington, D.C., 1:56-69 (1987).
Thus, the present invention provides a vector as
claimed in claim 15 and claim 16.
A translatable DNA sequence encoding the pelB
secretion signal having the amino acid residue
sequence shown in SEQ. ID. NO. 5 is a preferred DNA
sequence for inclusion in a DNA expression vector of
this invention.
A downstream translatable DNA sequence encodes
a filamentous phage membrane anchor. Preferred
membrane anchors are obtainable from filamentous phage
M13, f1, fd, and the like equivalent filamentous
phage. Preferred membrane anchor domains are found in
the coat proteins encoded by gene III and gene VIII.
Thus, a downstream translatable DNA sequence encodes
an amino acid residue sequence that corresponds, and
preferably is identical, to the membrane anchor domain
of either a filamentous phage gene III or gene VIII
coat polypeptide.
The membrane anchor domain of a filamentous phage
coat protein is a portion of the carboxy terminal
region of the coat protein and includes a region of
hydrophobic amino acid residues for spanning a lipid
bilayer membrane, and a region of charged amino acid
ID NO
(10)
(11)
(12)
(13)
(14)
(15)
Pe1B‘
pe1B2
pe1B3
Ma1E‘
ompF‘
PhoA4
B1a4
LamB
Lpp‘
cpvIII5
cpIII‘
Table 1
Leader Sequences
Amino Acid Residue Sequence
MetLysTyrLeuLeuProThrA1aAlaAlaGlyLeuLeu
LeuLeuA1aA1aG1nProA1aMet
MetLysTyrLeuLeuProThrA1aA1aAlaGlyLeuLeu
LeuLeuAlaA1aG1nProA1aGlnProAlaMetA1a
MetLysSerLeuI1eThrProIleA1aAlaG1yLeuLeu
LeuAlaPheSerG1nTyrSerLeuA1a
MetLysI1eLysThrG1yAlaArgIleLeuAlaLeuSer
A1aLeuThrThrMetMetPheSerAlaSerAlaLeuA1a
LysI1e
MetMetLysArgAsnIleLeuA1aValIleValProA1a
LeuLeuValAlaG1yThrA1aAsnAlaAlaGlu
MetLysG1nSerThrI1eAlaLeuAlaLeuLeuProLeu
LeuPheThrProVa1ThrLysA1aArgThr
MetSerI1eG1nHisPheArgVa1AlaLeuIleProPhe
PheA1aA1aPheCysLeuProVa1PheAlaHisPro
MetMetI1eThrLeuArgLysLeuProLeuA1aValAla
Va1AlaAlaGlyValMetSerA1aG1nAlaMetAlaVa1
Asp
MetLysA1aThrLysLeuValLeuG1yA1aValI1eLeu
G1ySerThrLeuLeuAlaGlyCysSer
MetLysLysSerLeuValLeuLysA1aSerValAlaVal
A1aThrLeuVa1ProMetLeuSerPheA1a
MetLysLysLeuLeuPheAlaIleProLeuValValPro
PheTyrSerHisSer
pelB used in this invention
pe1B from Erwinia carotovora gene
pelB from Erwinia carotovora EC 16 gene
leader sequences from E. coli
leader sequence for cpVIII
leader sequence for cpIII
-25.-
residues normally found at the cytoplasmic face of the
membrane and extending away from the membrane.
In the phage f1, gene VIII coat protein's
membrane spanning region comprises residue Trp-26
through Lys-40, and the cytoplasmic region comprises
the carboxy—terminal 11 residues from 41 to 52.
Ohkawa et al., J. Biol. Chem., 256:9951—9958 (1981).
An exemplary membrane anchor would consist of residues
26 to 40 of cpVIII.
Thus, the amino acid residue sequence of a
preferred membrane anchor domain is derived from the
M13 filamentous phage gene III coat protein (also
designated cpIII). A preferred cpIII-derived membrane
anchor has a sequence shown in SEQ ID NO. 16 from
residue 1 to residue 211. Gene III coat protein is
present on a mature filamentous phage at one end of
the phage particle with typically about 4 to 6 copies
of the coat protein.
The amino acid residue sequence of another
preferred membrane anchor domain is derived from the
M13 filamentous phage gene VIII coat protein (also
designated cpVIII). A preferred cpVIII—derived
membrane anchor has a sequence shown in SEQ ID NO. 17
from residue 1 to residue 50. Gene VIII coat protein
is present on a mature filamentous phage over the
majority of the phage particle with typically about
2500 to 3000 copies of the coat protein.
In another embodiment, the membrane anchor has the amino
acid sequence shown in SEQ ID NO 113 from base 28 to base
663.
For detailed descriptions of the structure of
filamentous phage particles, their coat proteins and
particle assembly, see the reviews by Rached et al.,
Microbiol. Rev., 50:401-427 (1986); and Model et al.,
in "The Bacteriophages: Vol. 2", R. Calendar, ed.
Plenum Publishing Co., pp. 375-456, (1988).
A cassette in a DNA expression vector of this
invention is the region of the vector that forms, upon
insertion of a translatable DNA sequence (insert DNA),
a sequence of nucleotides capable of expressing, in an
appropriate host, a fusion polypeptide of this
invention. The expression-competent sequence of
nucleotides is referred to as a cistron. Thus, the
cassette comprises DNA expression control elements
operatively linked to the upstream and downstream
translatable DNA sequences. A cistron is formed when
a translatable DNA sequence is directionally inserted
(directionally ligated) between the upstream and
downstream sequences via the sequence of nucleotides
adapted for that purpose. The resulting three
translatable DNA sequences, namely the upstream, the
inserted and the downstream sequences, are all
operatively linked in the same reading frame.
DNA expression control sequences comprise a set
of DNA expression signals for expressing a structural
gene product and include both 5' and 3' elements, as
is well known, operatively linked to the cistron such
that the cistron is able to express a structural gene
product. The 5' control sequences define a promoter
for initiating transcription and a ribosome binding
site operatively linked at the 5' terminus of the
upstream translatable DNA sequence.
To achieve high levels of gene expression in E.
coli, it is necessary to use not only strong promoters
to generate large quantities of mRNA, but also
ribosome binding sites to ensure that the mRNA is
efficiently translated. In E. coli, the ribosome
binding site includes an initiation codon (AUG) and a
sequence 3-9 nucleotides long located 3-11 nucleotides
upstream from the initiation codon [Shine et al.,
Nature, 254:34 (1975)). The sequence, AGGAGGU, which
is called the Shine—Dalgarno (SD) sequence, is
complementary to the 3' end of E. coli 16S mRNA.
-27..
Binding of the ribosome to mRNA and the sequence at
the 3' end of the mRNA can be affected by several
factors:
(i) The degree of complementarity between
the SD sequence and 3' end of the 16S tRNA.
(ii) The spacing and possibly the DNA
sequence lying between the SD sequence and the AUG
[Roberts et al., Proc. Natl. Acad. Sci. USA, 76:760
(1979a); Roberts et al., Proc. Natl. Acad. Sci. USA,
76:5596 (1979b); Guarente et al., Science, 209:1428
(1980); and Guarente et al., Qgll, 20:543 (1980).]
Optimization is achieved by measuring the level of
expression of genes in plasmids in which this spacing
is systematically altered. Comparison of different
mRNAs shows that there are statistically preferred
sequences from positions -20 to +13 (where the A of
the AUG is position 0) [Gold et al., Annu. Rev.
Microbiol., 35:365 (1981)]. Leader sequences have
been shown to influence translation dramatically
(Roberts et al., 1979 a, b gupgg).
(iii) The nucleotide sequence following the
AUG, which affects ribosome binding [Taniguchi et al.,
J. Mol. Biol., 113:533 (l978)].
Useful ribosome binding sites are shown in Table
2 below.
Table 2
Ribosome Binding Sites*
SEQ.
ID. NO.
(18) 5' AAUCUUQQAQQCUUUUUUAQQGUUCGUUCU
(19) 5' UAACUAAGGAUGAAAUGCAQQUCUAAGACA
3. (20) 5' UCCUAGGAGGUUUGACCUAQQCGAGCUUUU
4. (21) 5' AUGUACUAAGGAGGUUGUAQQGAACAACGC
* . . . . . .
Sequences of initiation regions for protein
synthesis in four phage mRNA molecules are
underlined.
AUG = initiation codon (double underlined)
. = Phage ¢X174 gene-A protein
. = Phage QB replicase
. = Phage R17 gene-A protein
. = Phage lambda gene-cro protein
The 3' control sequences define at least one
termination (stop) codon in frame with and operatively
linked to the downstream translatable DNA sequence.
Thus, a DNA expression vector of this invention
provides a system for cloning translatable DNA
sequences into the cassette portion of the vector to
produce cistrons capable of expressing the fusion
polypeptides of this invention.
In a preferred embodiment, a DNA expression
vector is designed for convenient manipulation in the
form of a filamentous phage particle encapsulating a
genome according to the teachings of the present
invention. In this embodiment, a DNA expression
vector further contains a nucleotide sequence that
defines a filamentous phage origin of replication such
that the vector, upon presentation of the appropriate
genetic complementation, can replicate as a
filamentous phage in single stranded replicative form
and be packaged into filamentous phage particles.
This feature provides the ability of the DNA
expression vector to be packaged into phage particles
for subsequent segregation of the particle, and vector
contained therein, away from other particles that
The present invention also provides a vector as
claimed in claims 20-22.
Insofar as a vector of this invention may be
manipulated to contain an insert DNA, thereby having the
capacity to express a fusion polypeptide, one embodiment
contemplates the previously described vectors containing
insert DNA. Particularly preferred vectors containing
antibody genes are described in the Examples.
D. Polypeptides
In another embodiment, the present invention provides
a polypeptide as claimed in claim 23.
The polypeptide is a fusion polypeptide having a
receptor domain comprised of an amino acid residue sequence
that defines the ligand (epitope) binding domain of a
receptor protein positioned adjacent to a filamentous phage
membrane anchor domain. That is, the insert domain in the
fusion polypeptide is the ligand—binding domain of a
receptor and is also referred to as a ligand—binding
receptor polypeptide.
Insofar as the polypeptide has a receptor domain, it
is also referred to herein as a receptor. In other related
polypeptides, the polypeptide may additionally comprise a
secretion signal domain, preferably a pelB secretion signal
as described herein.
In preferred embodiments, the receptor protein is a
polypeptide chain of a ligand—binding heterodimeric
receptor. More preferably the heterodimeric receptor is an
epitope—binding complex.
Preferred heterodimeric receptors include
immunoglobulins, major histocompatibility antigens of class
I or II,
lymphocyte receptors, integrins and the like
heterodimeric receptors. Immunoglobulins (antibody
molecules) can be in the form of Fab or Fv fragments, or
other portions of an antibody molecule that contain regions
of the variable domain of the heavy and light chains.
A polypeptide precursor of this invention may have an
amino acid residue sequence that can be represented by the
formula, shown in the direction of amino— to carboxy
terminus:
(Fl) NH2-O-(U)m—V-(X)n-Z-COOH,
where 0 represents an amino acid residue sequence defining
a secretion signal, U represents a first spacer
polypeptide, V represents an amino acid residue sequence
defining a receptor domain (a ligand—binding receptor
polypeptide), X represents a second spacer polypeptide, and
Z represents an amino acid residue sequence defining a
filamentous phage coat protein membrane anchor, with the
proviso that m is the integer O or 1 such that when m is O,
U is not present, and when m is l, U is present, and n is O
or 1 such that when n is O, X is not present and when n is
l, X is present.
In the formula (E1), the secretion signal and the
filamentous phage coat protein membrane anchor are as
defined herein above. Thus, some such polypeptides may
comprise an antibody variable chain domain—derived
polypeptide operatively linked at its amino—terminus to the
_32_
secretion signal and operatively linked at its carboxy—
terminus to the membrane anchor.
A preferred polypeptide of this embodiment consists
essentially of an antibody heavy chain polypeptide as the
variable domain. In this regard "consists essentially of"
means that the polypeptide does not contain an antibody
light chain polypeptide, or portion thereof. For example,
such a polypeptide may be a polypeptide according to
formula (Fl) where Z defines the cpIII membrane anchor as
described herein.
As used herein with regard to polypeptides, the phrase
"operatively linked" means that polypeptide fragments, or
protein domains represented by polypeptides, have been
covalently joined into a single polypeptide polymer,
preferably by conventional amide bonds between the adjacent
amino acids being linked in the polypeptide.
In one possible arrangement, V is an amino acid
residue sequence that defines the ligand binding domain of
a chain of a heterodimeric receptor molecule, and
preferably is an immunoglobulin variable region
polypeptide. For example V may be a VH or VL polypeptide.
The present invention provides a polypeptide as claimed in
claim 26. In a preferred possibility V is an immunoglobulin
VH polypeptide (antibody heavy chain polypeptide), and m
and H are both zero.
U or X can define a proteolytic cleavage site, such as
the sequence of amino acids found in a precursor protein,
such as prothrombin, factor X and the like, that defines
the site of cleavage of the polypeptide. A fusion
polypeptide having a cleavage site provides a means to
purify the polypeptide away from the phage particle to
which it is attached.
The polypeptide spacers U and X can each have any
sequence of amino acid residues of from about 1 to 6 amino
acid residues in length. Typically the spacer residues are
present in a polypeptide to accommodate the continuous
reading frame that is required when a polypeptide is
produced by the methods disclosed herein using a DNA
expression vector of this invention.
A receptor of the present invention assumes a
conformation having a binding site specific for, as
evidenced by its ability to be competitively inhibited, a
preselected or predetermined ligand such as an antigen,
hapten, enzymatic substrate and the like.
In one possible
use of the present invention,
a receptor of this invention
may be a ligand—binding polypeptide that forms an antigen
binding site which specifically binds to a preselected
antigen to form a complex having a sufficiently strong
binding between the antigen and the binding site for the
complex to be isolated. When the receptor is an antigen-
binding polypeptide its affinity or avidity is generally
greater than lO5 Mi more usually greater than lO6 and
preferably greater than 108 M4.
In another possible use of the present invention, a
receptor of the subject invention may bind a substrate and
catalyzes the formation of a product from the substrate.
While the topology of the ligand binding site of a
catalytic receptor is probably more important for its
preselected activity than its affinity (association
constant or pKa)
for the substrate, the subject catalytic
receptors have an association constant for the preselected
substrate generally greater than 103 M”
, more usually
greater than 105 M* or 106 M4 and preferably greater than
107 M”.
Preferably the receptor produced by the subject
invention is heterodimeric and is therefore normally
comprised of two different polypeptide chains, which
together assume a conformation having a binding affinity,
or association constant for the preselected ligand that is
different, preferably higher, than the affinity or
association constant of either of the polypeptides alone,
i.e., as monomers. The heterodimeric receptor is referred
to as a ligand—binding heterodimeric receptor to connote
its ability to bind ligand.
Thus, a preferred embodiment provides a ligand-binding
heterodimeric receptor comprising first and second
polypeptides as claimed in claim 5. The first polypeptide
may be flanked by an amino—terminal prokaryotic secretion
signal domain in addition to the carboxy-terminal
filamentous phage membrane anchor domain. The second
polypeptide may be fused to an amino—terminal prokaryotic
secretion signal domain. A particularly preferred ligand-
binding heterodimeric receptor utilizes a prokaryotic
secretion signal as described herein.
A ligand—binding heterodimeric receptor is referred to
as an epitope—binding complex to connote that the complex
has a capacity to bind an epitope present on a ligand, and
to connote that the heterodimeric receptor is formed by the
association
(complexation) of two polypeptides as described
herein. Thus, the present invention provides a receptor as
claimed in claim 6.
One or both of the different polypeptide chains is
preferably derived from the variable region of the light
and heavy chains of an immunoglobulin. Typically,
polypeptides comprising the light (VL) (Va
variable regions are employed together for binding the
and heavy
preselected ligand.
Thus, one embodiment contemplates a ligand—binding
heterodimeric receptor in which the first polypeptide is an
antibody heavy chain polypeptide and the second polypeptide
is a light chain polypeptide. An alternative embodiment
contemplates a ligand—binding heterodimeric receptor in
which the first polypeptide is an antibody light chain
polypeptide and the second is an antibody heavy chain
polypeptide. Thus, the present invention provides a
receptor as claimed in claims 7 and 8.
A receptor produced by the subject invention can be
active in monomeric as well as multimeric forms,
either
homomeric or heteromeric, preferably heterodimeric. For
example, V3 and VL ligand binding polypeptide produced by
the present invention can be advantageously combined in the
heterodimer to modulate the activity of either or to
produce an activity unique to the heterodimer.
Where the individual ligand polypeptides are referred
to as VH and VL the heterodimer can be referred to as a Fv.
However, it should be understood that a VH may contain in
addition to the VH, substantially all or a portion of the
heavy chain constant region. Similarly, a VL may contain,
in addition to the VL, substantially all or a portion of
I\)
U:
the light chain constant region. A heterodimer comprised of
a VB containing a portion of the heavy chain constant
region and a V; containing substantially all of the light
chain constant region is termed a Fab fragment. The
production of Fab can be advantageous in some situations
because the additional constant region sequences contained
in a Fab as compared to a Fv can stabilize the vg and VL
interaction. Such stabilization can cause the Fab to have
higher affinity for antigen. In addition the Fab is more
commonly used in the art and thus there are more commercial
antibodies available to specifically recognize a Fab in
screening procedures.
The individual Vg and VL polypeptides can be produced
in lengths equal to or substantially equal to their
naturally occurring lengths. However, in preferred
embodiments, the VH and VL polypeptides will generally have
fewer than l25 amino acid residues, more usually fewer than
about l2O amino acid residues, while normally having
greater than 60 amino acid residues, usually greater than
about 95 amino acid residues, more usually greater than
about lOO amino acid residues. Preferably, the V3 will be
from about llO to about 230 amino acid residues in length
while VL will be from about 95 to about 214 amino acid
residues in length. VH and VL chains sufficiently long to
form Fabs are preferred. Thus, the present invention
provides a receptor as claimed in claims 9-13.
The amino acid residue sequences will vary widely,
depending upon the particular idiotype involved. Usually,
there will be at least two cysteines separated by from
about 60 to 75 amino acid residues and joined by a
l\)
U:
disulfide bond. The polypeptides produced by the subject
invention will normally be substantial copies of idiotypes
of the variable regions of the heavy and/or light chains of
immunoglobulins, but in some situations a polypeptide may
contain random mutations in amino acid residue sequences in
order to advantageously improve the desired activity.
In some situations, it is desirable to provide for
covalent cross linking of the VH and V; polypeptides, which
can be accomplished by providing cysteine resides at the
carboxyl termini. The polypeptide will normally be prepared
free of the immunoglobulin constant regions, however a
small portion of the J region may be included as a result
of the advantageous selection of DNA synthesis primers. The
D region will normally be included in the transcript of the
VB.
Typically the C terminus region of the VB and VL
polypeptides will have a greater variety of sequences than
the N terminus and, based on the present strategy, can be
further modified to permit a variation of the normally
occurring vg and V¢ chains. A synthetic polynucleotide can
be employed to vary one or more amino acid in a
hypervariable region.
E. Methods for Producing a Library
. General Rationale
The present invention may also be of use in a system for
the simultaneous cloning and screening of preselected
ligand—binding specificities from gene repertoires using a
single vector system. This system may provide linkage of
cloning and screening methodologies and has two
requirements. First, that expression of the polypeptide
chains of a heterodimeric receptor in an in vitro
expression host such as E. coli requires coexpression of
the two polypeptide chains in order that a functional
heterodimeric receptor can assemble to produce a receptor
that binds ligand. Second, that screening of isolated
members of the library for a preselected ligand-binding
capacity requires a means to correlate (a linkage) the
binding capacity of an expressed receptor molecule with a
convenient means to isolate the gene that encodes the
member from the library.
Linkage of expression and screening is accomplished by
the combination of targeting of a fusion polypeptide into
the periplasm of a bacterial cell to allow assembly of a
functional receptor, and the targeting of a fusion
polypeptide onto the coat of a filamentous phage particle
during phage assembly to allow for convenient screening of
the library member of interest. Periplasmic targeting is
provided by the presence of a secretion signal domain in a
fusion polypeptide of this invention. Targeting to a phage
particle is provided by the presence of a filamentous phage
coat protein membrane anchor domain (i.e., a cpIII— or
cpVIII—derived membrane anchor domain) in a fusion
polypeptide of this invention.
The present invention provides in one embodiment a
method for producing a library of dicistronic DNA
molecules, as claimed in claim 31.
In this embodiment, the repertoire of polypeptide
encoding genes are in the form of double-stranded (ds)
DNA and each member of the repertoire has cohesive
termini adapted for directional ligation. In
addition, the plurality of DNA expression vectors are
each linear DNA molecules having upstream and
downstream cohesive termini that are (a) adapted for
directionally receiving the polypeptide genes in a
common reading frame, and (b) operatively linked to
respective upstream and downstream translatable DNA
sequences. The upstream translatable DNA sequence
encodes a secretion signal, preferably a pelB
secretion signal, and the downstream translatable DNA
sequence encodes a filamentous phage coat protein
membrane anchor as described herein for a polypeptide
of this invention. The translatable DNA sequences are
also operatively linked to respective upstream and
downstream DNA expression control sequences as defined
for a DNA expression vector described herein.
The library so produced can be utilized for
expression and screening of the fusion polypeptides
encoded by the resulting library of cistrons
represented in the library by the expression and
screening methods described herein.
. Production of Gene Repertoires
A gene repertoire is a collection of
different genes, preferably polypeptide-encoding genes
(polypeptide genes), and may be isolated from natural
sources or can be generated artificially. Preferred
gene repertoires are comprised of conserved genes.
Particularly preferred gene repertoires comprise
either or both genes that code for the members of a
dimeric receptor molecule.
A gene repertoire useful in practicing the
present invention contains at least 103, preferably at
least 10‘, more preferably at least 105, and most
preferably at least 107 different genes. Methods for
evaluating the diversity of a repertoire of genes is
well known to one skilled in the art.
Thus, in one embodiment, the present invention
contemplates a method of isolating a pair of genes
coding for a dimeric receptor having a preselected
activity from a repertoire of conserved genes.
Additionally, expressing the cloned pair of genes and
isolating the resulting expressed dimeric receptor
protein is also described. Preferably, the receptor
will be a heterodimeric polypeptide capable of binding
a ligand, such as an antibody molecule or
immunologically active portion thereof, a cellular
receptor, or a cellular adhesion protein coded for by
one of the members of a family of conserved genes,
i.e., genes containing a conserved nucleotide sequence
of at least about 10 nucleotides in length.
Exemplary conserved gene families encoding
different polypeptide chains of a dimeric receptor are
those coding for immunoglobulins, major
histocompatibility complex antigens of class I or II,
lymphocyte receptors, integrins and the like.
A gene can be identified as belonging to a
repertoire of conserved genes using several methods.
For example, an isolated gene may be used as a
hybridization probe under low stringency conditions to
detect other members of the repertoire of conserved
genes present in genomic DNA using the methods
described by Southern, J. Mol. Biol., 98:503 (1975).
If the gene used as a hybridization probe hybridizes
to multiple restriction endonuclease fragments of the
genome, that gene is a member of a repertoire of
conserved genes .
Immunoglobulins
The immunoglobulins, or antibody molecules, are a
large family of molecules that include several types
of molecules, such as IgD, IgG, IgA, IgM and IgE. The
antibody molecule is typically comprised of two heavy
(H) and light (L) chains with both a variable (V) and
constant (C) region present on each chain as shown in
Figure 1. Schematic diagrams of human IgG heavy chain
and human kappa light chain are shown in Figures 2A
and 2B, respectively. Several different regions of an
immunoglobulin contain conserved sequences useful for
isolating an immunoglobulin repertoire. Extensive
amino acid and nucleic acid sequence data displaying
exemplary conserved sequences is compiled for
immunoglobulin molecules by Kabat et al., in Sequences
of Proteins of Immunological Interest, National
Institutes of Health, Bethesda, MD, 1987.
The C region of the H chain defines the
particular immunoglobulin type. Therefore the
selection of conserved sequences as defined herein
from the C region of the H chain results in the
preparation of a repertoire of immunoglobulin genes
having members of the immunoglobulin type of the
selected C region.
The V region of the H or L chain typically
comprises four framework (FR) regions each containing
relatively lower degrees of variability that includes
lengths of conserved sequences. The use of conserved
sequences from the FR1 and FR4 (J region) framework
regions of the V" chain is a preferred exemplary
embodiment and is described herein in the Examples.
Framework regions are typically conserved across
several or all immunoglobulin types and thus conserved
sequences contained therein are particularly suited
for preparing repertoires having several
immunoglobulin types.
Major Histocompatibility Complex
The major histocompatibility complex (MHC) is a
large genetic locus that encodes an extensive family
of proteins that include several classes of molecules
referred to as class I, class II or class III MHC
molecules. Paul et al., in Fundamental Immunolo ,
Raven Press, NY, pp. 303-378 (1984).
Class I MHC molecules are a polymorphic group of
transplantation antigens representing a conserved
family in which the antigen is comprised of a heavy
chain and a non-MHC encoded light chain. The heavy
chain includes several regions, termed the N, C1, C2,
membrane and cytoplasmic regions. Conserved sequences
useful in the present invention are found primarily in
the N, C1 and C2 regions and are identified as
continuous sequences of "invariant residues" in Kabat
et al., ggpgg.
Class II MHC molecules comprise a conserved
family of polymorphic antigens that participate in
immune responsiveness and are comprised of an alpha
and a beta chain. The genes coding for the alpha and
beta chain each include several regions that contain
conserved sequences suitable for producing MHC class
II alpha or beta chain repertoires. Exemplary
conserved nucleotide sequences include those coding
for amino acid residues 26-30 of the A1 region,
residues 161-170 of the A2 region and residues 195-206
of the membrane region, all of the alpha chain.
Conserved sequences are also present in the B1, B2 and
membrane regions of the beta chain at nucleotide
sequences coding for amino acid residues 41-45, 150-
and 200-209, respectively.
Lymphocyte Receptors and Cell Surface Antiqens
Lymphocytes contain several families of proteins
on their cell surfaces including the T-cell receptor,
Thy-1 antigen and numerous T-cell surface antigens
including the antigens defined by the monoclonal
antibodies OKT4 (leu3), OKTS/8 (leu2), OKT3, OKT1
(leul), OKT 11 (leu5) OKT6 and OKT9. Paul, guprg at
pp. 453-479.
The T-cell receptor is a term used for a family
of antigen binding molecules found on the surface of
T-cells. The T-cell receptor as a family exhibits
polymorphic binding specificity similar to
immunoglobulins in its diversity. The mature T-cell
receptor is comprised of alpha and beta chains each
having a variable (V) and constant (C) region. The
similarities that the T-cell receptor has to
immunoglobulins in genetic organization and function
shows that T-cell receptor contains regions of
conserved sequence. Lai et al., Nature, 331:543-546
(1988).
Exemplary conserved sequences include those
coding for amino acid residues 84-90 of alpha chain,
amino acid residues 107-115 of beta chain, and amino
acid residues 91-95 and 111-116 of the gamma chain.
Kabat et a1., su ra, p. 279.
Integrins And Adhesions
Adhesive proteins involved in cell attachment are
members of a large family of related proteins termed
integrins. Integrins are heterodimers comprised of a
beta and an alpha subunit. Members of the integrin
family include the cell surface glycoproteins platelet
receptor GpIIb-IIIa, vitronectin receptor (VnR),
-44..
fibronectin receptor (FnR) and the leukocyte adhesion
receptors LFA-1, Mac-1, Mo-1 and 60.3. Rouslahti et
al., Science, 238:491-497 (1987). Nucleic acid and
protein sequence data demonstrates regions of
conserved sequences exist in the members of these
families, particularly between the beta chain of
GpIIb-IIIa, VnR and FnR, and between the alpha subunit
of VnR, Mac-1, LFA-1, FnR and GpIIb-IIIa. Suzuki et
al., Proc. Natl. Acad. Sci. USA, 83:8614-8618, 1986;
Ginsberg et al., J. Biol. Chem., 262:5437-5440, 1987.
Various well known methods can be employed to
produce a useful gene repertoire. For instance, V"
and VL gene repertoires can be produced by isolating
VH- and Vi-coding mRNA from a heterogeneous population
of antibody producing cells, i.e., B lymphocytes (B
cells), preferably rearranged B cells such as those
found in the circulation or spleen of a vertebrate.
Rearranged B cells are those in which immunoglobulin
gene translocation, i.e., rearrangement, has occurred
as evidenced by the presence in the cell of mRNA with
the immunoglobulin gene V, D and J region transcripts
adjacently located thereon. Typically, the B cells
are collected in a 1-100 ml sample of blood which
usually contains 106 B cells/ml.
In some cases, it is desirable to bias a
repertoire for a preselected activity, such as by
using as a source of nucleic acid cells (source cells)
from vertebrates in any one of various stages of age,
health and immune response. For example, repeated
immunization of a healthy animal prior to collecting
rearranged B cells results in obtaining a repertoire
enriched for genetic material producing a receptor of
high affinity. Mullinax et al., Proc. Natl. Acad.
Sci. USA, 87:8095-8099 (1990). Conversely, collecting
-45..
rearranged B cells from a healthy animal whose immune
system has not been recently challenged (i.e., a naive
immune system) results in producing a repertoire that
is not biased towards the production of high affinity
V” and/or V1 polypeptides.
It should be noted the greater the genetic
heterogeneity of the population of cells for which the
nucleic acids are obtained, the greater the diversity
of the immunological repertoire (comprising Vh- and
Vi-coding genes) that will be made available for
screening according to the method of the present
invention. Thus, cells from different individuals,
particularly those having an immunologically
significant age difference, and cells from individuals
of different strains, races or species can be
advantageously combined to increase the heterogeneity
(diversity) of a repertoire.
Thus, in one preferred embodiment, the source
cells are obtained from a vertebrate, preferably a
mammal, which has been immunized or partially
immunized with an antigenic ligand (antigen) against
which activity is sought, i.e., a preselected antigen.
The immunization can be carried out conventionally.
Antibody titer in the animal can be monitored to
determine the stage of immunization desired, which
stage corresponds to the amount of enrichment or
biasing of the repertoire desired. Partially
immunized animals typically receive only one
immunization and cells are collected from those
animals shortly after a response is detected. Fully
immunized animals display a peak titer, which is
achieved with one or more repeated injections of the
antigen into the host mammal, normally at 2 to 3 week
intervals. Usually three to five days after the last
challenge, the spleen is removed and the genetic
repertoire of the splenocytes, about 90% of which are
rearranged B cells, is isolated using standard
procedures. See, Current Protocols in Molecular
Biolo , Ausubel et al., eds., John Wiley & Sons, NY.
Nucleic acids coding for V“ and Vi polypeptides can be
derived from cells producing IgA, IgD, IgE, IgG or
IgM, most preferably from IgM and IgG, producing
cells.
Methods for preparing fragments of genomic DNA
from which immunoglobulin variable region genes can be
cloned as a diverse population are well known in the
art. See for example Herrmann et al., Methods In
Enz ol., 152:180—183, (1987); Frischauf, Methods In
Enzymo1., 152:183—190 (1987); Frischauf, Methods In
Enzymol., 152:190-199 (1987); and DiLel1a et al.,
Methods In Enzymo1., 152:199-212 (1987).
The desired gene repertoire can be isolated from
either genomic material containing the gene expressing
the variable region or the messenger RNA (mRNA) which
represents a transcript of the variable region. The
difficulty in using the genomic DNA from other than
non-rearranged B lymphocytes is in juxtaposing the
sequences coding for the variable region, where the
sequences are separated by introns. The DNA
fragment(s) containing the proper exons must be
isolated, the introns excised, and the exons then
spliced in the proper order and in the proper
orientation. For the most part, this will be
difficult, so that the alternative technique employing
rearranged B cells will be the method of choice
because the V, D and J immunoglobulin gene regions
have translocated to become adjacent, so that the
sequence is continuous (free of introns) for the
entire variable regions.
Where mRNA is utilized the cells will be lysed
under RNase inhibiting conditions. In one embodiment,
the first step is to isolate the total cellular mRNA.
Poly A+ mRNA can then be selected by hybridization to
an oligo—dT cellulose column. The presence of mRNAs
coding for the heavy and/or light chain polypeptides
can then be assayed by hybridization with DNA single
strands of the appropriate genes. Conveniently, the
sequences coding for the constant portion of the V"
and V, can be used as polynucleotide probes, which
sequences can be obtained from available sources. See
for example, Early and Hood, Genetic Engineering,
Setlow and Hollaender, eds., Vol. 3, Plenum Publishing
Corporation, NY, (1981), pages 157-188; and Kabat et
al., Sequences of Immunological Interest, National
Institutes of Health, Bethesda, MD, (1987).
In preferred embodiments, the preparation
containing the total cellular mRNA is first enriched
for the presence of V” and/or VL coding mRNA.
Enrichment is typically accomplished by subjecting the
total mRNA preparation or partially purified mRNA
product thereof to a primer extension reaction
employing a polynucleotide synthesis primer as
described herein. Exemplary methods for producing V”
and V, gene repertoires using polynucleotide synthesis
primers are described in PCT Application No. PCT/US
90/02836 (International Publication No. W0 90/14430).
Particularly preferred methods for producing a gene
repertoire rely on the use of preselected
oligonucleotides as primers in a polymerase chain
reaction (PCR) to form PCR reaction products as
described herein.
In preferred embodiments, isolated B cells are
immunized in vitro against a preselected antigen. In
vitro immunization is defined as the clonal expansion
of epitope-specific B cells in culture, in response to
antigen stimulation. The end result is to increase
the frequency of antigen—specific B cells in the
immunoglobulin repertoire, and thereby decrease the
number of clones in an expression library that must be
screened to identify a clone expressing an antibody of
the desired specificity. The advantage of in vitro
immunization is that human monoclonal antibodies can
be generated against a limitless number of
therapeutically valuable antigens, including toxic or
weak immunogens. For example, antibodies specific for
the polymorphic determinants of tumor-associated
antigens, rheumatoid factors, and histocompatibility
antigens can be produced, which can not be elicited in
immunized animals. In addition, it may be possible to
generate immune responses which are normally
suppressed in vivo.
In vitro immunization can be used to give rise to
either a primary or secondary immune response. A
primary immune response, resulting from first time
exposure of a B cell to an antigen, results in clonal
expansion of epitope-specific cells and the secretion
of IgM antibodies with low to moderate apparent
affinity constants (106-108M4). Primary immunization
of human splenic and tonsillar lymphocytes in culture
can be used to produce monoclonal antibodies against a
variety of antigens, including cells, peptides,
macromolecule, haptens, and tumor-associated antigens.
Memory B cells from immunized donors can also be
stimulated in culture to give rise to a secondary
immune response characterized by clonal expansion and
the production of high affinity antibodies (>109 M4)
of the IgG isotype, particularly against viral
antigens by clonally expanding sensitized lymphocytes
derived from seropositive individuals.
In one embodiment, peripheral blood lymphocytes
are depleted of various cytolytic cells that appear to
down-modulate antigen—specific B cell activation.
When lysosome-rich subpopulations (natural killer
cells, cytotoxic and suppressor T cells, monocytes)
are first removed by treatment with the lysosmotropic
methyl ester of leucine, the remaining cells
(including B cells, T helper cells, accessory cells)
respond antigen-specifically during in vitro
immunization. The lymphokine requirements for
inducing antibody production in culture are satisfied
by a culture supernatant from activated, irradiated T
cells.
In addition to in vitro immunization, cell
panning (immunoaffinity absorption) can be used to
further increase the frequency of antigen-specific B
cells. Techniques for selecting B cell subpopulations
via solid-phase antigen binding are well established.
Panning conditions can be optimized to selectively
enrich for B cells which bind with high affinity to a
variety of antigens, including cell surface proteins.
Panning can be used alone, or in combination with in
yitrg immunization to increase the frequency of
antigen—specific cells above the levels which can be
obtained with either technique alone. Immunoglobulin
expression libraries constructed from enriched
populations of B cells are biased in favor of antigen-
specific antibody clones, and thus, enabling
identification of clones with the desired
specificities from smaller, less complex libraries.
In one embodiment, donor peripheral blood
lymphocytes (PBL) can be transferred into severe
combined immunodeficiency (SCID) mice and then boosted
in vivo in the SCID mice to increase the immune
response prior to harvesting the heavy and light chain
coding nucleic acids from the SCID mouse's B cells.
See for example, Duchosal et al, Nature, 355:258—262
(1992). In that report, human PBLs from a donor who
had anti-tetanus toxoid (TT) titers were boosted while
in the SCID mouse host. The resulting library of
370,000 clones yielded 2 phage particles expressing a
surface Fab able to bind TT.
. Preparation of Polvnucleotide Primers
The term "polynucleotide" as used
herein in reference to primers, probes and nucleic
acid fragments or segments to be synthesized by primer
extension is defined as a molecule comprised of two or
more deoxyribonucleotide or ribonucleotides,
preferably more than 3. Its exact size will depend on
many factors, which in turn depends on the ultimate
conditions of use.
The term "primer" as used herein refers to a
polynucleotide whether purified from a nucleic acid
restriction digest or produced synthetically, which is
capable of acting as a point of initiation of nucleic
acid synthesis when placed under conditions in which
synthesis of a primer extension product which is
complementary to a nucleic acid strand is induced,
i.e., in the presence of nucleotides and an agent for
polymerization such as DNA polymerase, reverse
transcriptase and the like, and at a suitable
temperature and pH. The primer is preferably single
stranded for maximum efficiency, but may alternatively
be in double stranded form. If double stranded, the
primer is first treated to separate it from its
complementary strand before being used to prepare
extension products. Preferably, the primer is a
polydeoxyribonucleotide. The primer must be
sufficiently long to prime the synthesis of extension
products in the presence of the agents for
polymerization. The exact lengths of the primers will
depend on many factors, including temperature and the
source of primer. For example, depending on the
complexity of the target sequence, a polynucleotide
primer typically contains 15 to 25 or more
nucleotides, although it can contain fewer
nucleotides. Short primer molecules generally require
cooler temperatures to form sufficiently stable hybrid
complexes with template.
The primers used herein are selected to be
"substantially" complementary to the different strands
of each specific sequence to be synthesized or
amplified. This means that the primer must be
sufficiently complementary to non-randomly hybridize
with its respective template strand. Therefore, the
primer sequence may or may not reflect the exact
sequence of the template. For example, a non-
complementary nucleotide fragment can be attached to
the 5' end of the primer, with the remainder of the
primer sequence being substantially complementary to
the strand. Such non-complementary fragments
typically code for an endonuclease restriction site.
Alternatively, non—complementary bases or longer
sequences can be interspersed into the primer,
provided the primer sequence has sufficient
complementarily with the sequence of the strand to be
synthesized or amplified to non-randomly hybridize
therewith and thereby form an extension product under
polynucleotide synthesizing conditions.
Primers of the present invention may also contain
a DNA-dependent RNA polymerase promoter sequence or
its complement. See for example, Krieg et al., figgl;
Acids Res., 12:7057—70 (1984); Studier et al., Q;_flgl;
When a primer containing a DNA—dependent RNA
polymerase promoter is used the primer is hybridized
to the polynucleotide strand to be amplified and the
second polynucleotide strand of the DNA-dependent RNA
polymerase promoter is completed using an inducing
agent such as E. coli DNA polymerase I, or the Klenow
fragment of E. coli DNA polymerase. The starting
polynucleotide is amplified by alternating between the
production of an RNA polynucleotide and DNA
polynucleotide.
Primers may also contain a template sequence or
replication initiation site for a RNA-directed RNA
polymerase. Typical RNA-directed RNA polymerase
include the QB replicase described by Lizardi et al.,
Biotechnology, 6:1197-1202 (1988). RNA-directed
polymerases produce large numbers of RNA strands from
a small number of template RNA strands that contain a
template sequence or replication initiation site.
These polymerases typically give a one million-fold
amplification of the template strand as has been
described by Kramer et al., J. Mol. Biol., 89:719-736
(1974).
The polynucleotide primers can be prepared using
any suitable method, such as, for example, the
phosphotriester or phosphodiester methods see Narang
et al., Meth. Enz ol., 68:90, (1979); U.S. Patent No.
,356,270; and Brown et al., Meth. Enzymol., 68:109,
(1979).
The choice of a primer's nucleotide sequence
depends on factors such as the distance on the nucleic
acid from the region coding for the desired receptor,
its hybridization site on the nucleic acid relative to
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any second primer to be used, the number of genes in
the repertoire it is to hybridize to, and the like.
a. Primers for Producing
Immunoqlobulin Gene Repertoires
V“ and Vi gene repertoires can be
separately prepared prior to their utilization in the
present invention. Repertoire preparation is
typically accomplished by primer extension, preferably
by primer extension in a polymerase chain reaction
(PCR) format.
To produce a repertoire of Vi-coding DNA homologs
by primer extension, the nucleotide sequence of a
primer is selected to hybridize with a plurality of
immunoglobulin heavy chain genes at a site
substantially adjacent to the Vi-coding region so that
a nucleotide sequence coding for a functional (capable
of binding) polypeptide is obtained. To hybridize to
a plurality of different Vh-coding nucleic acid
strands, the primer must be a substantial complement
of a nucleotide sequence conserved among the different
strands. Such sites include nucleotide sequences in
the constant region, any of the variable region
framework regions, preferably the third framework
region, leader region, promoter region, J region and
the like .
If the repertoires of Vh-coding and Vl—coding DNA
homologs are to be produced by (PCR) amplification,
two primers, i.e., a PCR primer pair, must be used for
each coding strand of nucleic acid to be amplified.
The first primer becomes part of the nonsense (minus
or complementary) strand and hybridizes to a
nucleotide sequence conserved among V" (plus or
coding) strands within the repertoire. To produce V”
coding DNA homologs, first primers are therefore
chosen to hybridize to (i.e. be complementary to)
conserved regions within the J region, CH1 region,
hinge region, CH2 region, or CH3 region of
immunoglobulin genes and the like. To produce a VL
coding DNA homolog, first primers are chosen to
hybridize with (i.e. be complementary to) a conserved
region within the J region or constant region of
immunoglobulin light chain genes and the like. Second
primers become part of the coding (plus) strand and
hybridize to a nucleotide sequence conserved among
minus strands. To produce the V}-coding DNA homologs,
second primers are therefore chosen to hybridize with
a conserved nucleotide sequence at the 5' end of the
Vfi-coding immunoglobulin gene such as in that area
coding for the leader or first framework region. It
should be noted that in the amplification of both VW-
and Vi-coding DNA homologs the conserved 5' nucleotide
sequence of the second primer can be complementary to
a sequence exogenously added using terminal
deoxynucleotidyl transferase as described by Loh et
al., Science, 243:217-220 (1989). One or both of the
first and second primers can contain a nucleotide
sequence defining an endonuclease recognition site.
The site can be heterologous to the immunoglobulin
gene being amplified and typically appears at or near
the 5' end of the primer.
When present, the restriction site—defining
portion is typically located in a 5'—terminal non-
priming portion of the primer. The restriction site
defined by the first primer is typically chosen to be
one recognized by a restriction enzyme that does not
recognize the restriction site defined by the second
primer, the objective being to be able to produce a
DNA molecule having cohesive termini that are non-
complementary to each other and thus allow directional
insertion into a vector.
In one embodiment, the present invention utilizes
a set of polynucleotides that form primers having a
priming region located at the 3'—terminus of the
primer. The priming region is typically the 3'—most
(3'-terminal) 15 to 30 nucleotide bases. The 3'-
terminal priming portion of each primer is capable of
acting as a primer to catalyze nucleic acid synthesis,
i.e., initiate a primer extension reaction off its 3'
terminus. one or both of the primers can additionally
contain a 5'-terminal (5'-most) non-priming portion,
i.e., a region that does not participate in
hybridization to repertoire template.
In PCR, each primer works in combination with a
second primer to amplify a target nucleic acid
sequence. The choice of PCR primer pairs for use in
PCR is governed by considerations as discussed herein
for producing gene repertoires. That is, the primers
have a nucleotide sequence that is complementary to a
sequence conserved in the repertoire. Useful V“ and
Vi priming sequences are shown in Tables 5 and 6,
herein below.
. Polymerase Chain Reaction to Produce
Gene Repertoires
The strategy used for cloning the V“
and Vi genes contained within a repertoire will
depend, as is well known in the art, on the type,
complexity, and purity of the nucleic acids making up
the repertoire. other factors include whether or not
the genes are contained in one or a plurality of
repertoires and whether or not they are to be
amplified and/or mutagenized.
The Vh- and Vi-coding gene repertoires are
comprised of polynucleotide coding strands, such as
mRNA and/or the sense strand of genomic DNA. If the
repertoire is in the form of double stranded genomic
DNA, it is usually first denatured, typically by
melting, into single strands. A repertoire is
subjected to a PCR reaction by treating (contacting)
the repertoire with a PCR primer pair, each member of
the pair having a preselected nucleotide sequence.
The PCR primer pair is capable of initiating primer
extension reactions by hybridizing to nucleotide
sequences, preferably at least about 10 nucleotides in
length and more preferably at least about 20
nucleotides in length, conserved within the
repertoire. The first primer of a PCR primer pair is
sometimes referred to herein as the "sense primer"
because it hybridizes to the coding or sense strand of
a nucleic acid. In addition, the second primer of a
PCR primer pair is sometimes referred to herein as the
"anti-sense primer" because it hybridizes to a non-
coding or anti-sense strand of a nucleic acid, i.e., a
strand complementary to a coding strand.
The PCR reaction is performed by mixing the PCR
primer pair, preferably a predetermined amount
thereof, with the nucleic acids of the repertoire,
preferably a predetermined amount thereof, in a PCR
buffer to form a PCR reaction admixture. The
admixture is maintained under polynucleotide
synthesizing conditions for a time period, which is
typically predetermined, sufficient for the formation
of a PCR reaction product, thereby producing a
plurality of different Vh-coding and/or V,-coding DNA
homologs.
A plurality of first primer and/or a plurality of
second primers can be used in each amplification,
e.g., one species of first primer can be paired with a
number of different second primers to form several
different primer pairs. Alternatively, an individual
-57..
pair of first and second primers can be used. In any
case, the amplification products of amplifications
using the same or different combinations of first and
second primers can be combined to increase the
diversity of the gene library.
In another strategy, the object is to clone the
Vfi— and/or V1-coding genes from a repertoire by
providing a polynucleotide complement of the
repertoire, such as the anti—sense strand of genomic
dsDNA or the polynucleotide produced by subjecting
mRNA to a reverse transcriptase reaction. Methods for
producing such complements are well known in the art.
The PCR reaction is performed using any suitable
method. Generally it occurs in a buffered aqueous
solution, i.e., a PCR buffer, preferably at a pH of 7-
9, most preferably about 8. Preferably, a molar
excess (for genomic nucleic acid, usually about 106:1
primer:template) of the primer is admixed to the
buffer containing the template strand. A large molar
excess is preferred to improve the efficiency of the
process.
The PCR buffer also contains the deoxyribo—
nucleotide triphosphates dATP, dCTP, dGTP, and dTTP
and a polymerase, typically thermostable, all in
adequate amounts for primer extension (polynucleotide
synthesis) reaction. The resulting solution (PCR
admixture) is heated to about 90°C - 100°C for about 1
to 10 minutes, preferably from 1 to 4 minutes. After
this heating period the solution is allowed to cool to
54°C, which is preferable for primer hybridization.
The synthesis reaction may occur at from room
temperature up to a temperature above which the
polymerase (inducing agent) no longer functions
efficiently. Thus, for example, if DNA polymerase is
used as inducing agent, the temperature is generally
-58..
no greater than about 40°C. An exemplary PCR buffer
comprises the following: 50 mM Kcl; 10 mM Tris—HCl;
pH 8.3; 1.5 mM MgCl2; 0.001% (wt/vol) gelatin, 200 uM
dATP; 200 MM dTTP; 200 MM dCTP; 200 MM dGTP; and 2.5
units Thermus aguaticus DNA polymerase I (U.s. Patent
No. 4,889,818) per 100 microliters of buffer.
The inducing agent may be any compound or system
which will function to accomplish the synthesis of
primer extension products, including enzymes.
Suitable enzymes for this purpose include, for
example, E. coli DNA polymerase I, Klenow fragment of
E. coli DNA polymerase I, T4 DNA polymerase, other
available DNA polymerases, reverse transcriptase, and
other enzymes, including heat-stable enzymes, which
will facilitate combination of the nucleotides in the
proper manner to form the primer extension products
which are complementary to each nucleic acid strand.
Generally, the synthesis will be initiated at the 3'
end of each primer and proceed in the 5' direction
along the template strand, until synthesis terminates,
producing molecules of different lengths. There may
be inducing agents, however, which initiate synthesis
at the 5' end and proceed in the above direction,
using the same process as described above.
The inducing agent also may be a compound or
system which will function to accomplish the synthesis
of RNA primer extension products, including enzymes.
In preferred embodiments, the inducing agent may be a
DNA-dependent RNA polymerase such as T7 RNA
polymerase, T3 RNA polymerase or SP6 RNA polymerase.
These polymerases produce a complementary RNA
polynucleotide. The high turn over rate of the RNA
polymerase amplifies the starting polynucleotide as
has been described by Chamberlin et al., The Enz es,
ed. P. Boyer, PP. 87-108, Academic Press, New York
(1982). Another advantage of T7 RNA polymerase is
that mutations can be introduced into the
polynucleotide synthesis by replacing a portion of
cDNA with one or more mutagenic oligodeoxynucleotides
(polynucleotides) and transcribing the partially-
mismatched template directly as has been previously
described by Joyce et al., Nuc. Acid Res., 17:711—722
(1989). Amplification systems based on transcription
have been described by Gingeras et al., in ggg
Protocols. A Guide to Methods and Applications. pp
245-252, Academic Press, Inc., San Diego, CA (1990).
If the inducing agent is a DNA-dependent RNA
polymerase and therefore incorporates ribonucleotide
triphosphates, sufficient amounts of ATP, CTP, GTP and
UTP are admixed to the primer extension reaction
admixture and the resulting solution is treated as
described above.
The newly synthesized strand and its
complementary nucleic acid strand form a double-
stranded molecule which can be used in the succeeding
steps of the process.
The first and/or second PCR reactions discussed
above can advantageously be used to incorporate into
the receptor a preselected epitope useful in
immunologically detecting and/or isolating a receptor.
This is accomplished by utilizing a first and/or
second polynucleotide synthesis primer or expression
vector to incorporate a predetermined amino acid
residue sequence into the amino acid residue sequence
of the receptor.
After producing Vfi- and Vi-coding DNA homologs
for a plurality of different Vh- and Vt-coding genes
within the repertoires, the DNA molecules are
typically further amplified. While the DNA molecules
can be amplified by classic techniques such as
-60..
incorporation into an autonomously replicating vector,
it is preferred to first amplify the molecules by
subjecting them to a polymerase chain reaction (PCR)
prior to inserting them into a vector. PCR is
typically carried out by thermocycling i.e.,
repeatedly increasing and decreasing the temperature
of a PCR reaction admixture within a temperature range
whose lower limit is about 10°C to about 40°C and
whose upper limit is about 90°C to about 100°C. The
increasing and decreasing can be continuous, but is
preferably phasic with time periods of relative
temperature stability at each of temperatures favoring
polynucleotide synthesis, denaturation and
hybridization.
PCR amplification methods are described in detail
in U.S. Patent Nos. 4,683,195, 4,683,202, 4,800,159,
and 4,965,188, and at least in several texts including
"PCR Technology: Principles and Applications for DNA
Amplification", H. Erlich, ed., Stockton Press, New
York (1989); and "PCR Protocols: A Guide to Methods
and Applications", Innis et al., eds., Academic Press,
San Diego, California (1990).
In preferred embodiments only one pair of first
and second primers is used per amplification reaction.
The amplification reaction products obtained from a
plurality of different amplifications, each using a
plurality of different primer pairs, are then
combined.
However, the present invention also contemplates
DNA homolog production via co-amplification (using two
pairs of primers), and multiplex amplification (using
up to about 8, 9 or 10 primer pairs).
In preferred embodiments, the PCR process is used
not only to produce a library of DNA molecules, but
also to induce mutations within the library or to
create diversity from a single parental clone and
thereby provide a library having a greater
heterogeneity. First, it should be noted that the PCR
process itself is inherently mutagenic due to a
variety of factors well known in the art. Second, in
addition to the mutation inducing variations described
in the above referenced U.S. Patent No. 4,683,195,
other mutation inducing PCR variations can be
employed. For example, the PCR reaction admixture,
can be formed with different amounts of one or more of
the nucleotides to be incorporated into the extension
product. Under such conditions, the PCR reaction
proceeds to produce nucleotide substitutions within
the extension product as a result of the scarcity of a
particular base. Similarly, approximately equal molar
amounts of the nucleotides can be incorporated into
the initial PCR reaction admixture in an amount to
efficiently perform X number of cycles, and then
cycling the admixture through a number of cycles in
excess of X, such as, for instance, 2X.
Alternatively, mutations can be induced during the PCR
reaction by incorporating into the reaction admixture
nucleotide derivatives such as inosine, not normally
found in the nucleic acids of the repertoire being
amplified. During subsequent in vivo DNA synthesis
and replication of the nucleic acids in a host cell,
the nucleotide derivative will be replaced with a
substitute nucleotide thereby inducing a point
mutation.
. Linear DNA Expression Vectors
A DNA expression vector for use in a
method of the invention for producing a library of DNA
molecules is a linearized DNA molecule as described
before having two (upstream and downstream) cohesive
-.62-
termini adapted for directional ligation to a
polypeptide gene.
A linear DNA expression vector is typically
prepared by restriction endonuclease digestion of a
circular DNA expression vector of this invention to
cut at two preselected restriction sites within the
sequence of nucleotides of the vector adapted for
directional ligation to produce a linear DNA molecule
having the required cohesive termini that are adapted
for direction ligation. Directional ligation refers
to the presence of two (a first and second) cohesive
termini on a vector, or on the insert DNA molecule to
be ligated into the vector selected, so that the
termini on a single molecule are not complementary. A
first terminus of the vector is complementary to a
first terminus of the insert, and the second terminus
of the vector is complementary to the second terminus
of the insert.
. Ligation Reactions to Produce Gene
Libraries
In preparing a library of DNA molecules
of this invention, a ligation admixture is prepared as
described above, and the admixture is subjected to
ligation conditions for a time period sufficient for
the admixed repertoire of polypeptide genes to ligate
(become operatively linked) to the plurality of DNA
expression vectors to form the library.
Ligation conditions are conditions selected to
favor a ligation reaction wherein a phosphodiester
bond is formed between adjacent 3' hydroxyl and 5'
phosphoryl termini of DNA. The ligation reaction is
preferably catalyzed by the enzyme T4 DNA ligase.
Ligation conditions can vary in time, temperature,
concentration of buffers, quantities of DNA molecules
.
to be ligated, and amounts of ligase, as is well
known. Preferred ligation conditions involve
maintaining the ligation admixture at 4 degrees
Centigrade (4°C) to 12°C for 1 to 24 hours in the
presence of 1 to 10 units of T4 DNA ligase per
milliliter (ml) and about 1 to 2 micrograms (ug) of
DNA. Ligation buffer in a ligation admixture
typically contains 0.5 M Tris-HCl (pH 7.4), 0.01 M
Mgclz, 0.01 M dithiothrietol, 1 mM spermidine, 1 mM
ATP and 0.1 mg/ml bovine serum albumin (BSA). other
ligation buffers can also be used.
Exemplary ligation reactions are described in
Example 2.
. Preparation of Dicistronic Gene
Libraries
As described above the
present invention provides methods for the
preparation of a library of dicistronic DNA molecules.
A dicistronic DNA molecule is a single DNA molecule
having the capacity to express two separate
polypeptides from two separate cistrons. In preferred
embodiments, the two cistrons are operatively linked
at relative locations on the
DNA molecule such that both cistrons are under the
transcriptional control of a single promoter. Each
dicistronic molecule is capable of expressing first
and second polypeptides from first and second
cistrons, respectively, that can form, in a suitable
host, a heterodimeric receptor on the surface of a
filamentous phage particle.
The method for producing a library of dicistronic
DNA molecules comprises the steps of:
(a) Forming a first ligation admixture
by combining in a ligation buffer:
(i) a repertoire of first
polypeptide genes in the form of dsDNA, each having
cohesive termini adapted for directional ligation, and
(ii) a plurality of DNA expression
vectors in linear form, each having upstream and
downstream first cohesive termini that are (a) adapted
for directionally receiving the first polypeptide
genes in a common reading frame, and (b) operatively
linked to respective upstream and downstream
translatable DNA sequences. The upstream translatable
DNA sequence encodes a pelB secretion signal, the
downstream translatable DNA sequence encodes a
filamentous phage coat protein membrane anchor, and
translatable DNA sequences are operatively linked to
respective upstream and downstream DNA expression
control sequences.
(b) subjecting the admixture to
ligation conditions for a time period sufficient to
operatively link the first polypeptide genes to the
vectors and produce a plurality of circular DNA
molecules each having a first cistron for expressing
the first polypeptide.
(c) Treating the plurality of circular
DNA molecules under DNA cleavage conditions to produce
a plurality of DNA expression vectors in linear form
that each have upstream and downstream second cohesive
termini that are (i) adapted for directionally
receiving a repertoire of second polypeptide genes in
a common reading frame, and (ii) operatively linked to
respective upstream and downstream DNA sequences. The
upstream DNA sequence is a translatable sequence
encoding a secretion signal, the downstream DNA
sequence has at least one stop codon in the reading
frame, and the translatable DNA sequence is
operatively linked to a DNA expression control
sequence.
(d) Forming a second ligation
admixture by combining in a ligation buffer:
(i) the plurality of DNA
expression vectors formed in step (c), and
(ii) the repertoire of second
polypeptide genes in the form of dsDNA, each having
cohesive termini adapted for directional ligation to
the plurality of DNA expression vectors; and
(e) subjecting the second admixture to
ligation conditions for a time period sufficient to
operatively link the second polypeptide genes to said
vectors and produce a plurality of circular DNA
molecules each having the second cistron for
expressing the second polypeptide, thereby forming the
library.
In preferred embodiments a secretion signal is a
pe1B secretion signal. Also preferred is the use of a
filamentous phage membrane anchor that is derived from
cpIII or cpVIII as described herein.
The present invention also provides a method as claimed
in claim 32 and a library of vectors as claimed in claim 33.
DNA expression vectors useful for practicing the
above method are the dicistronic expression vectors
described in greater detail before.
In practicing the method of producing a library
of dicistronic DNA molecules, it is preferred that the
upstream and downstream first cohesive termini do not
have the same nucleotide sequences as the upstream and
downstream second cohesive termini. In this
embodiment, the treating step (c) to linearize the
circular DNA molecules typically involves the use of
restriction endonucleases that are specific for
producing said second termini, but do not cleave the
circular DNA molecule at the sites that formed the
first termini. Exemplary and preferred first and
second termini are the termini defined by cleavage of
pCBAK8 with Xho I and spe I to form the upstream and
downstream first termini, and defined by cleavage of
pCBAK8 with Sac I and Xba I to form the upstream and
downstream second termini. In this embodiment, other
pairs of cohesive termini can be utilized at the
respective pairs of first and second termini, so long
as the four termini are each distinct, non-
complementary termini. Exemplary are the termini
found on the vectors pCOMB3, pCOMB2-3, pCOMB2-3',
pCOMB8 and pCOMB2-8 described herein.
Methods of treating the plurality of circular DNA
molecules under DNA cleavage conditions to form linear
DNA molecules are generally well known and depend on
the nucleotide sequence to be cleaved and the
mechanism for cleavage. Preferred treatments involve
admixing the DNA molecules with a restriction
endonuclease specific for a endonuclease recognition
site at the desired cleavage location in an amount
sufficient for the restriction endonuclease to cleave
the DNA molecule. Buffers, cleavage conditions, and
substrate concentrations for restriction endonuclease
cleavage are well known and depend on the particular
enzyme utilized. Exemplary restriction enzyme
cleavage conditions are described in Example 2.
. Methods for Changing the Diversity of a Library
The present invention may be used with methods
for changing the diversity of a library of filamentous
phage library of this invention. These methods
generally increase the diversity of the library,
thereby increasing the pool of possible epitope-
binding complexes from which to screen for a desired
binding activity. Alternatively, the methods can be
directed at enriching for a class of epitope—binding
complexes. The class is typically defined by the
ability to bind a particular epitope or family of
epitopes present on a preselected antigen or group of
antigens.
a. Increasing Library Diversity by
Mutation
A suitable method
for increasing diversity is to alter the amino acid
residue sequence of one or more polypeptides of the
epitope—binding complex encoded by the genome of a
phage of this invention. Alterations can be
conveniently introduced at the nucleic acid level by
mutation of the nucleic acid. The method can be
practiced on a single species of nucleic acid coding a
polypeptide of this invention, or can be practiced on
a library of nucleic acids present in a library of
phage of this invention.
Mutation of nucleic acid can be conducted by a
variety of means, but is most conveniently conducted
in a PCR reaction during a PCR process of the present
invention. PCR mutagenesis can be random or directed
to specific nucleotide sequences, as is generally well
known. Conducting PCR under conditions favorable to
random mutagenesis has been described previously, and
is referred to as "error prone PCR". Similarly,
directed mutagenesis involves the use of PCR primers
designed to target a specific type of mutation into a
specific type of mutation into a specific region of
nucleotide sequence.
The invention may be of use in increasing the diversity
of one or more epitope—binding complexes by PCR—directed
mutation of a complementarity determining region (CDR) of an
antibody variable domain present in an epitope—binding
complex polypeptide of this invention. CDR mutagenesis has
been previously described in general terms for "humanizing"
an antibody by introducing human sequences into the CDR
region of a murine antibody. See European Application No. EP
239400.
For example, the invention may be of use in a
mutagenesis method for altering the immunological specificity
of a cloned immunoglobulin gene present in a DNA vector of
this invention. The method provides directed mutagenesis in a
preselected CDR of an immunoglobulin gene which comprises
subjecting a recombinant DNA molecule (rDNA) containing the
cloned immunoglobulin gene having a target CDR to PCR
conditions suitable for amplifying a preselected region of
the CDR. In the method, the rDNA molecule is subjected to PCR
conditions that include a PCR primer oligonucleotide as
described below constituting the first primer in a PCR primer
pair as is well known to produce an amplified PCR product
that is derived from the preselected CDR region but that
includes the nucleotide sequences of the PCR primer. The
second oligonucleotide in the PCR amplifying conditions can
be any PCR primer derived from the immunoglobulin gene to be
mutagenized, as described herein.
Preferred methods would be those an oligonucleotide of
this invention as described below.
In such methods, therefore, an oligonucleotide
is contemplated that is useful as a primer in
for
a polymerase chain reaction (PCR)
inducing mutagenesis in a complementarity determining
region (CDR) of an immunoglobulin gene. The
oligonucleotide has 3' and 5' termini and comprises
(1) a nucleotide sequence at its 3' terminus capable
of hybridizing to a first framework region of an
immunoglobulin gene, (2) a nucleotide sequence at its
' terminus capable of hybridizing to a second
framework region of an immunoglobulin gene, and (3) a
nucleotide sequence between the 3' and 5' termini
adapted for introducing mutations during a PCR into
the CDR region between the first and second framework
regions of the immunoglobulin gene, thereby
mutagenizing the CDR region.
Insofar as immunoglobulin genes have three CDR
regions on both the heavy chain and the light chain of
an immunoglobulin, each separated by a distinctive
framework region, it is to be understood that the
above example is readily applicable to introducing
mutations into a specific CDR by selection of the
above 5' and 3' nucleotide sequences as to hybridize
to the framework regions flanking the targeted CDR.
Thus the above first and second framework sequences
can be the conserved sequences flanking CDR1, CDR2 or
CDR3 on either the heavy or light chain. Exemplary
and preferred is the CDR3 of the human immunoglobulin
heavy chain.
The length of the 3' and 5' terminal nucleotide
sequences of a subject mutagenizing oligonucleotide
can vary in length as is well known, so long as the
length provides a stretch of nucleotides complementary
to the target framework sequences as to hybridize
thereto. In the case of the 3' terminal nucleotide
sequence, it must be of sufficient length and
complementarity to the target framework region located
3' to the CDR region to be mutagenized as to hybridize
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and provide a 3'hydroxyl terminus for initiating a
primer extension reaction. In the case of the 5'
terminal nucleotide sequence, it must be of sufficient
length and complementarity to the target framework
region located 5' to the CDR region to be mutagenized
as to provide a means for hybridizing in a PCR overlap
extension reaction as described above to assemble the
complete immunoglobulin heavy or light chain.
Framework regions flanking a CDR are well
characterized in the immunological arts, and include
known nucleotide sequences or consensus sequences as
described elsewhere herein. Where a single,
preselected immunoglobulin gene is to be mutagenized,
the framework-defined sequences flanking a particular
CDR are known, or can be readily determined by
nucleotide sequencing protocols. Where a repertoire
of immunoglobulin genes are to be mutagenized, the
framework-derived sequences are preferably conserved,
as described elsewhere herein.
Preferably, the length of the 3' and 5' terminal
nucleotide sequences are each at least 6 nucleotides
in length, and can be up to 50 or more nucleotides in
length, although these lengths are unnecessary to
assure accurate and reproducible hybridization.
Preferred are lengths in the range of 12 to 30
nucleotides, and typically are about 18 nucleotides.
A particularly preferred framework-defined
nucleotide sequence for use as a 3' terminus
nucleotide sequence has the nucleotide sequence 5'-
TGGGGCCAAGGGACCACG-3’ (SEQ ID NO 122).
A particularly preferred framework-defined
nucleotide sequence for use as a 5' terminus
nucleotide sequence has the nucleotide sequence 5'-
GTGTATTATTGTGCGAGA-3‘ (SEQ ID NO 123).
The nucleotide sequence located between the 3'
and 5' termini adapted for mutagenizing a CDR can be
any nucleotide sequence, insofar as the novel sequence
will be incorporated by the above methods. However,
the present approach provides a means to produce a
large population of mutagenized CDR's in a single PCR
reaction by the use of a population of redundant
sequences defining randomized or nearly randomized
nucleotides in the CDR region to be mutagenized.
A preferred oligonucleotide comprises a
nucleotide sequence between the above described 3' and
' termini that is represented by the formula:
[NNR]n, wherein N can independently be any
nucleotide, R can be S, K or analogs thereof, where S
is G or C, K is G or T, and where n is from 3 to about
24. In preferred methods, the oligonucleotide has
the formula:
'-GTGTATTATTGTGCGAGA[NNR]nTGGGGCCAAGGGACCACG-3' (SEQ
ID NO 124).
Exemplary and particularly preferred is the
oligonucleotide where R is S and n is 16, such that
the oligonucleotide represents a large population of
redundant oligonucleotide sequences.
Thus, the invention may be of use in a method for
increasing the diversity of a library of filamentous
phage particles comprising the steps of: a) providing
a library of filamentous phage particles according to
the present invention, and b) mutating the
immunoglobulin variable domain-coding nucleotide
sequence present in each DNA expression vector in the
library to form a library of phage particles each
containing a mutated immunoglobulin variable domain
nucleotide sequence.
The providing can include manipulating the
genomes of the phage particles in the library in order
to isolate the nucleic acids in preparation for a
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mutagenizing PCR reaction. Manipulations of a phage
library to isolate the phage genome for use in a PCR
reaction is described elsewhere herein.
In one method, the mutating comprises
subjecting the immunoglobulin variable domain—coding
nucleotide sequence to an error-prone polymerase chain
reaction. In another method, the mutating
comprises subjecting the immunoglobulin variable
domain-coding nucleotide sequence to a method for
mutating a CDR of the immunoglobulin variable domain-
coding nucleotide sequence using a CDR—directed
oligonucleotide as described herein.
Exemplary methods of mutating the CDR region of a
particular epitope-binding complex coding nucleic acid
using the above CDR—directing oligonucleotide or using
error-prone PCR to produce a large library of diverse
complexes is described in the Examples.
b. Enrichment of a Library
The invention may also be of use in conjunction with a
method to change the diversity of the library by enriching the
library for a preselected class of epitope-binding
complexes. The process generally involves affinity
selection of those phage particles in a library that
are capable of binding a preselected antigen. The
process of affinity selection, or panning, is
described in detail in the Examples.
Thus the invention may be of use in a method for
changing the diversity of a library of filamentous
phage particles comprising the steps of a) providing a
library of filamentous phage particles according to
the present invention, b) contacting the provided
library with a preselected ligand under conditions
sufficient for members of the library to bind to the
ligand and form a ligand—phage particle complex, and
c) isolating phage particles in the complex away from
non-bound library members to form a ligand-enriched
library comprising phage particles having binding
specificity for the preselected ligand.
In preferred methods, the preselected ligand is
affixed to a solid support, and the ligand—phage
particle complex is formed in the solid phase. This
method further comprises the steps of i) washing
the solid support after the contacting step to rinse
non-bound library members from the solid support; and
ii) eluting any solid-phase bound phage particles off
of the solid support. The eluted phage particles are
collected, thereby forming isolated phage particles
that comprise an enriched library.
Elution can be conducted under a variety of
conditions that disrupt the ligand—epitope-binding
complex interaction. Typical conditions include high
salt or low pH buffers. Particularly preferred are
buffers of about pH 1 to 5, preferably about pH 2 to
3. Alternatively, the interaction can be disrupted by
competition with an excess amount of the preselected
ligand in the elution buffer. Both elution procedures
are described in the Examples.
A related method combines the features of
both increasing diversity of a library by mutation and
enriching the library by panning to "mature" epitope-
binding complex affinities for a preselected ligand.
Thus it is possible to evolve new binding
specificities, and more potent binding specificities,
using these methods for changing library
diversity.
The combination of these methods can be
configured in a variety of ways, as will be apparent
to a skilled practitioner. For example, one can
isolate a library, mutagenize (diversify), and then
screen (enrich) for a particular binding activity.
Alternatively, one can enrich for a particular
activity from a library, mutagenize the specific
epitope-binding complex and further enrich the library
produced by the mutagenesis.
In another permutation on this theme, one can
utilize the differences between libraries based on
cpIII- and cpVIII-derived membrane anchors due to
their inherent differences in valency. Because a
library of phage having the cpIII-derived membrane
anchor will typically contain only 1 to 4 copies of
the epitope-binding complex on the surface of each
phage particle, the phage presents a binding complex
of relatively "low" valency, approaching one. In
contrast, a library of phage having a cpVIII-derived
membrane anchor will typically contain 20 to 1000
copies of the epitope-binding complex on the surface
of each phage particle, the particle presents a
relatively "high" valency. Thus, cpIII-based
libraries are referred to as monovalent and cpVIII-
based libraries are referred to as multivalent.
Applying the well-known principles of antibody
affinity and valence, it is understood that a cpIII~
based library can be enriched upon screening for
generally higher affinity binding interactions
(binding constants of 10° to 109lf1) as compared to
the broader range of affinities (binding constants of
‘ to l09‘M4) isolatable using a multivalent reagent
found in the cpVIII—based library. Therefore, a
cpVIII—based library is useful to isolate a broad
range of affinities of epitope-binding complexes from
low to high, whereas a cpIII-based library is useful
to isolate a narrower range of higher affinity
epitope-binding complexes.
Thus the invention may be of use in producing a first
enriched library by enrichment of a cpVIII-based
library. Thereafter the genes for encoding the
epitope-binding complex polypeptides are transferred
into a cpIII-based vector, and subsequently enriched
for a high affinity binding interaction. In one
method, a mutation step can be utilized prior to the
transfer into the cpIII-based vector.
In another method, the ability to mature a
novel affinity is shown by an example herein in which
a cloned V“/VL heterodimer—coding gene capable of
expressing a heterodimer that binds the ligand tetanus
toxoid (TT) is mutagenized using CDR-directed PCR
mutagenesis, and the mutagenized nucleic acid
population resulting therefrom is inserted into a
cpIII-based library and screened for binding to a
different ligand, fluorescein. A high affinity
epitope-binding complex was identified that binds
fluorescein.
In a related method, a naive (non—immunized)
library was cloned into a cpVIII-based library and
screened for binding to the antigen progesterone. Low
affinity binders were cloned into a cpIII-based
library, and three high affinity binding clones
identified that bind progesterone. The three clones
were pooled, and the pool was subjected to error-prone
PCR mutagenesis, and the resulting library of mutated
nucleic acids were cloned into a cpIII-based vector
and screened against progesterone to yield a high
affinity epitope-binding complex that binds
progesterone. Thus a high affinity complex was
"matured" from a naive library.
Thus, the present invention may also be of use in a
method for maturing the affinity of an epitope-binding
complex encoded by a filamemtous phage of this
invention comprising the steps of a) providing the
genome of a filamentous phage, b) mutating the
immunoglobulin variable domain-coding nucleotide
sequence present in the provided genome to form a
library of phage particles containing a mutated
immunoglobin variable domain nucleotide sequence, c)
contacting the library formed in step (b) with a
preselected ligand under conditions sufficient for
members of the library to bind to the ligand and form
a ligand-phage particle complex, and d) isolating
phage particles in said complex away from non—bound
library members to form a ligand—enriched library
comprising phage particles having binding specificity
for the preselected ligand.
F. Phage Libraries
The present invention contemplates a library
of DNA molecules that each encode a fusion polypeptide
of this invention where the library is in the form of
a population of different filamentous phage particles
each containing a different rDNA molecule of this
invention. By different rDNA molecule is meant a rDNA
molecule differing in nucleotide base sequence
encoding a polypeptide of this invention when compared
in nucleotide sequence to another rDNA molecule in the
library.
Thus, a phage library is a population of
filamentous phage, preferably f1, fd or M13
filamentous phage, each phage having packaged inside
the particle a rDNA expression vector of this
invention, the rDNA is encapsulated in the phage
particle by the matrix proteins of the phage. Stated
differently, a phage library contains a plurality of
filamentous phage particles, each different phage
particle containing at least one epitope-binding
complex on its surface as described herein. Thus,
the present invention provides a library of
phage particles as claimed in claim 27. A
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preferred library is comprised of phage particles
containing DNA molecules that encode at least 106,
preferably 107 and more preferably 10&9 different
fusion polypeptides of this invention. By different
fusion polypeptides is meant fusion polypeptides
differing in amino acid residue sequence. Even higher
library diversities are available when the methods of
random combination or mutagenesis are utilized as
described herein to increase library diversity.
Where the packaged expression vector encodes
first and second polypeptides of an autogenously
assembling receptor, e.g. V" and Vi polypeptides that
form a Fab, the library can also be characterized as
containing or expressing a multiplicity of receptor
specificities. Thus, libraries express at least 105,
preferably at least 106 and more preferably at least
107 different receptors, such as different antibodies,
T cell receptors, integrins and the like.
The size of the library can vary depending on a
number of factors, particularly the method in which
the library is produced. As used herein, size
connotes the complexity or diversity of the library,
that is the number of different species making up the
library, rather than the absolute number of particles
in the library.
Thus, where a library is produced by first
separately cloning two repertoires of genes,
corresponding to the first and second polypeptides,
the resulting library size after randomly combining
the two repertoires in the form of a dicistronic
vector is greatly increased. For example, consider
light chain and heavy chain variable antibody gene
repertoires, each having 106 different members.
Combining the two repertoires theoretically yields a
library of 10” possible different dicistronic vector
species.
An experimental system was designed to evaluate
the ability to "shuffle" two repertoires as described
above in order to generate greater diversity. The
system utilized a combinatorial Fab library derived
from a mouse immunized with the hapten para-
nitrophenyl phosphonamidate (NPN). Twenty-two
different clones were isolated, and the heavy and
light chain coding nucleic acids were isolated and
sequenced to determine that 21 of the 22 pairs were
different at the level of nucleic acid sequence. The
22 NPN ligand-binding clones were randomly recombined
(shuffled), and rescreened for binding to NPN.
Assuming that the heavy and light chains can only
form ligand-binding heterodimeric receptor molecules
if the original pairs are rejoined, the model predicts
that 4.6 percent of the total combinations would
provide ligand-binding combinations. Any higher
percentage demonstrates that pairings other than the
original pairs are also capable of binding NPN. The
results showed that 27 percent of the clones isolated
bound NPN, indicating a 5.8—fold increase in the
library size of receptors able to bind NPN upon
shuffling. This demonstrated increase is limited to
those clones that bind NPN. other members of the
randomly shuffled library have the capacity to bind
diverse, non-NPN, ligands. Thus, shuffling was shown
to increase diversity.
Library complexity can also be increased using the
methods described herein for mutating nucleotide sequences
in a pre—existing library of sequences. Stated in
terms of amino acid residue differences for an
expressed fusion polypeptide, there can be potentially
a twenty-fold increase in library size for each amino
acid residue position that is targeted for random
mutation.
For example, using the complementarity
determining region (CDR)-directed mutagenesis of
antibody genes as described in the Examples, a linear
region of 16 amino acid residues was targeted for
random mutation. Starting with a single species and
mutating all 16 residue positions through all possible
combinations with a choice of 20 different amino acids
would theoretically produce a library of 20“
different species, or 6 x 10” different species.
As described herein, a particular advantage of a
filamentous phage in the present invention is that the
DNA molecule present in the phage particle and
encoding one or both of the members of the
heterodimeric receptor can be segregated from other
DNA molecules present in the library on the basis of
the presence of the particular expressed fusion
polypeptide the surface of the phage particle.
Isolation (segregation) of a DNA molecule
encoding the members of a heterodimeric
receptor is conducted by segregation of the
filamentous phage particle containing the gene or
genes of interest away from the population of other
phage particles comprising the library. Segregation
of phage particles involves the physical separation
and propagation of individual phage particles away
from other particles in the library. Methods for
physical separation of filamentous phage particles to
produce individual particles, and the propagation of
the individual particles to form populations of
progeny phage derived from the individual segregated
particle are well known in the filamentous phage arts.
A preferred separation method involves the
identification of the expressed heterodimer on the
surface of the phage particle by means of a ligand
binding specificity between the phage particle and a
preselected ligand. Exemplary and preferred is the
use of "panning" methods whereby a suspension of phage
particles is contacted with a solid phase ligand
(antigen) and allowed to specifically bind (or
immunoreact where the heterodimer includes an
immunoglobulin variable domain). After binding, non-
bound particles are washed off the solid phase, and
the bound phage particles are those that contain
ligand-specific heterodimeric receptor (heterodimer)
on their surface. The bound particles can then be
recovered by elution of the bound particle from the
solid phase, typically by the use of aqueous solvents
that interfere with the ligand-receptor interaction.
Typical solvent include buffers having high ionic
strength, low pH, or an amount of soluble competing
ligand sufficient to disrupt the receptor-ligand
binding interaction.
An alternate method for separating a phage
particle based on the ligand specificity of the
surface-expressed heterodimer from a population of
particles is to precipitate the phage particles from
the solution phase by crosslinkage with the ligand.
An exemplary and preferred crosslinking and
precipitation method is described in detail in Example
4c.
The use of the above particle segregation methods
provides a means for screening a population of
filamentous phage particles present in a phage library
of this invention. As applied to a phage library,
screening can be utilized to enrich the library for
one or more particles that express a heterodimer
having a preselected ligand binding specificity.
Where the library is designed to contain multiple
species of heterodimers that all have some detectable
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measure of ligand binding activity, but differ in
protein structure, antigenicity, ligand binding
affinity or avidity, and the like, the screening
methods can be utilized sequentially to first produce
a library enriched for a preselected binding
specificity, and then to produce a second library
further enriched by further screening comprising one
or more isolated phage particles. Methods for
measuring ligand binding activities, antigenicity and
the like interactions between a ligand and a receptor
are generally well known and are not discussed further
as they are not essential features of the present
invention.
Thus, a phage library may be a population of
particles enriched for a preselected ligand binding
specificity.
A phage library may also comprise
a population of particles wherein each particle
contains at least one fusion polypeptide of this
invention on the surface of the phage particle. The
actual amount of fusion polypeptide present on the
surface of a phage particle depends, in part, on the
choice of coat protein membrane anchor present in the
fusion polypeptide.
Where the anchor is derived from cpIII, there are
typically about 1 to 4 fusion polypeptides per phage
particle. Where the anchor is derived from the more
preferred cpVIII, there is the potential for hundreds
of fusion polypeptides on the particle surface
depending on the growth conditions and other factors
as discussed herein. The actual amount of fusion
polypeptides present on a phage particle can be
adjusted by controlling the amount "captured" by the
phage particle as it is being synthesized in a host
cell.
Typically, a phage particle in a library of this
invention contains from about 10 to about 500 cpVIII—
derived fusion polypeptides on the surface of each
particle, and more preferably about 20 to 50 fusion
polypeptides per particle. Exemplary amounts of
surface fusion polypeptide are shown by the electron
micrographs described in Example 4a that describe
particles having about 20 to 24 cpVIII-derived fusion
polypeptides per particle.
In some instances, the present invention may be used
to create a population of phage particles that are
the progeny of a single particle, and therefor all
express the same heterodimer on the particle surface.
Such a population of phage are homogeneous and
clonally derived, and therefore provide a source for
expressing large quantities of a particular fusion
polypeptide. An exemplary clonally homogeneous phage
population is described in Example 4.
A filamentous phage particle in a library of this
invention is produced by standard filamentous phage
particle preparation methods and depends on the
presence in a DNA expression vector of this invention
of a filamentous phage origin of replication as
described herein to provide the signals necessary for
(1) production of a single-stranded filamentous phage
replicative form and (2) packaging of the replicative
form into a filamentous phage particle. Such a DNA
molecule can be packaged when present in a bacterial
cell host upon introduction of genetic complementation
to provide the filamentous phage proteins required for
production of infectious phage particles. A typical
and preferred method for genetic complementation is to
infect a bacterial host cell containing a DNA
expression vector of this invention with a helper
filamentous phage, thereby providing the genetic
elements required for phage particle assembly.
Exemplary helper rescue methods are described herein
at Example 2, and described by Short et al., Hug;
Acids Res., 16:7583-7600 (1988).
The level of heterodimeric receptor captured on
the surface of a filamentous phage particle during the
process of phage particle extrusion from the host cell
can be controlled by a variety of means. In one
method, the levels of fusion polypeptides are
controlled by the use of strong promoters in the first
and second cistrons for expressing the polypeptides,
such that transcription of the fusion polypeptide
cistrons occurs at a relative rate greater than the
rate of transcription of the cpVIII gene on the helper
phage. In another method, the helper phage can
have an amber mutation in the gene for expressing
cpVIII, such that less wild-type cpVIII is transcribed
in the host cell than fusion polypeptides, thereby
leading to increased ratios of fusion polypeptide
compared to cpVIII during the extrusion process.
In another method, the amount of
heterodimeric receptor on the phage particle surface
can be controlled by controlling the timing between
expression of fusion polypeptides and the
superinfection by helper phage. After introduction of
the expression vector into the host cell, longer delay
times before the addition of helper phage will allow
for increased accumulation of the fusion polypeptides
in the host cell, thereby increasing the amount of
fusion polypeptide captured by the extruding phage
particle.
The present invention provides a library as claimed in
claims 28-30.
G. Diagnostic Methods
The present invention may also be of use in a
diagnostic system, preferably in kit form, for
assaying for the presence of a preselected ligand, or
antigen, in a sample where it is desirable to detect
the presence, and preferably the amount, of the ligand
'or antigen in a sample according to the diagnostic
methods described herein.
The sample can be a tissue, tissue extract, fluid
sample or body fluid sample, such as blood, plasma or
serum.
The diagnostic system may include, in an amount
sufficient to perform at least one assay, a
filamentous phage or ligand-binding heterodimeric
receptor according to the present invention, as a
separately packaged reagent.
Exemplary diagnostic systems for detecting a
preselected ligand in the solid phase and utilizing a
filamentous phage of this invention are described in
the Examples.
Instructions for use of the packaged reagent(s)
are also typically included.
As used herein, the term "package" refers to a
solid matrix or material such as glass, plastic (e.g.,
polyethylene, polypropylene or polycarbonate), paper,
foil and the like capable of holding within fixed
limits a heterodimeric receptor, filamentous phage or
library of phage of the present invention. Thus, for
example, a package can be a glass vial used to contain
milligram quantities of a contemplated labeled phage
preparation, or it can be a microtiter plate well to
which microgram quantities of a contemplated receptor
or phage particle(s) have been operatively affixed,
i.e., linked so as to be capable of binding a ligand.
"Instructions for use" typically include a
tangible expression describing the reagent
concentration or at least one assay method parameter
such as the relative amounts of reagent and sample to
be admixed, maintenance time periods for reagent/
sample admixtures, temperature, buffer conditions and
the like.
A diagnostic system for use with the present invention
may preferably also includes a label or indicating means
capable of signaling the formation of a binding
reaction complex containing a ligand—binding
heterodimeric receptor or phage complexed with the
preselected ligand.
The word "complex" as used herein refers to the
product of a specific binding reaction such as an
phage—1igand or receptor—ligand reaction. Exemplary
complexes are immunoreaction products.
As used herein, the terms "label" and "indicating
means" in their various grammatical forms refer to
single atoms and molecules that are either directly or
indirectly involved in the production of a detectable
signal to indicate the presence of a complex. Any
label or indicating means can be linked to or
incorporated in an expressed polypeptide, or phage
particle that is used in a diagnostic method. Such
labels are themselves well-known in clinical
diagnostic chemistry and constitute a part of this
invention only insofar as they are utilized with
otherwise novel proteins methods and/or systems.
The labeling means can be a fluorescent labeling
agent that chemically binds to antibodies or antigens
without denaturing them to form a fluorochrome (dye)
that is a useful immunofluorescent tracer. Suitable
fluorescent labeling agents are fluorochromes such as
fluorescein isocyanate (FIC), fluorescein
isothiocyante (FITC), 5-dimethylamine
naphthalenesulfonyl chloride (DANSC),
tetramethylrhodamine isothiocyanate (TRITC),
lissamine, rhodamine 8200 sulphonyl chloride (RB 2
SC) and the like. A description of immunofluorescence
analysis techniques is found in DeLuca,
"lmmunofluorescence Analysis", in Antibody As a Tool,
Marchalonis, et al., eds., John Wiley & Sons, Ltd.,
pp. 139-231 (l982)_
In preferred methods, the indicating group is
an enzyme, such as horseradish peroxidase (HRP),
glucose oxidase, or the like. In such cases where the
principal indicating group is an enzyme such as HRP or
glucose oxidase, additional reagents are required to
visualize the fact that a receptor-ligand complex
(immunoreactant) has formed. Such additional reagents
for HRP include hydrogen peroxide and an oxidation dye
precursor such as diaminobenzidine. An additional
reagent useful with glucose oxidase is 2,2'-amino-di-
(3-ethyl-benzthiazoline—G-sulfonic acid) (ABTS).
Radioactive elements are also useful labeling
agents and are used illustratively herein. An
exemplary radiolabeling agent is a radioactive element
that produces gamma ray emissions. Elements which
themselves emit gamma rays, such as 1“I, 131 1”I, BZI
I
and “Cr represent one class of gamma ray emission-
producing radioactive element indicating groups.
Particularly preferred is 15I. Another group of
useful labeling means are those elements such as “C
I
18
F, 50 and “N which themselves emit positrons. The
positrons so emitted produce gamma rays upon
encounters with electrons present in the animal's
body. Also useful is a beta emitter, such "1 indium
of 3H.
The linking of labels, i.e., labeling of,
polypeptides and proteins is well known in the art.
For instance, proteins or phage can be labeled by
metabolic incorporation of radioisotope-containing
amino acids provided as a component in the culture
medium. See, for example, Galfre et al., Math;
Enzymol., 73:3-46 (1981). The techniques of protein
conjugation or coupling through activated functional
groups are particularly applicable. See, for example,
Aurameas, et al., Scand. J. Immunol., Vol. 8 Suppl.
7:7-23 (1978), Rodwell et al., Biotech., 3:889—894
(1984), and U.S. Pat. No. 4,493,795.
The diagnostic systems can also include,
preferably as a separate package, a specific binding
agent. A "specific binding agent" is a molecular
entity capable of selectively binding a reagent
species of the present invention or a complex
containing such a species, but is not itself a
polypeptide or phage of the present invention.
Exemplary specific binding agents are antibody
molecules, complement proteins or fragments thereof,
S. aureus protein A, and the like. Preferably the
specific binding agent binds the reagent species when
that species is present as part of a complex.
In preferred systems, the specific binding agent is
labeled. However, when the diagnostic system includes a
specific binding agent that is not labeled, the agent is
typically used as an amplifying means or reagent. In these
systems, the labeled specific binding agent is capable of
specifically binding the amplifying means when the
amplifying means is bound to a reagent species—containing
complex.
The diagnostic kits useful in relation to the present
invention can be used in an "ELISA" format to detect the
quantity of a preselected ligand in a fluid sample. "ELISA"
refers to an enzyme—linked immunosorbent assay that
employs an antibody or antigen bound to a solid phase
and an enzyme-antigen or enzyme-antibody conjugate to
detect and quantify the amount of an antigen present
in a sample and is readily applicable to the present
methods. A description of the ELISA technique is
found in Chapter 22 of the 4th Edition of Basic and
Clinical Immunology by D.P. Sites et al., published by
Lange Medical Publications of Los Altos, CA in 1982
and in U.S. Patents No. 3,554,090; No. 3,850,752;
No. 4,015,043.
Thus, in some diagnostic kits, a polypeptide, or a
phage of the present invention can be affixed to a
solid matrix to form a solid support that comprises a
package in the subject diagnostic systems.
A reagent is typically affixed to a solid matrix
by adsorption from an aqueous medium although other
modes of affixation applicable to proteins and
polypeptides can be used that are well known to those
skilled in the art. Exemplary adsorption methods are
described herein.
Useful solid matrices are also well known in the
art. Such materials are water insoluble and include
the cross-linked dextran available under the trademark
SEPHADEX from Pharmacia Fine Chemicals (Piscataway,
NJ); agarose; beads of polystyrene beads about 1
micron to about 5 millimeters in diameter available
from Abbott Laboratories of North Chicago, IL;
polyvinyl chloride, polystyrene, cross-linked
polyacrylamide, nitrocellulose— or nylon—based webs
such as sheets, strips or paddles; or tubes, plates or
the wells of a microtiter plate such as those made
from polystyrene or polyvinylchloride.
The reagent species, labeled specific binding
agent or amplifying reagent of any diagnostic system
described herein can be provided in solution, as a
liquid dispersion or as a substantially dry power,
e.g., in lyophilized form. Where the indicating means
is an enzyme, the enzyme's substrate can also be
provided in a separate package of a system. A solid
support such as the before-described microtiter plate
and one or more buffers can also be included as
separately packaged elements in this diagnostic assay
system.
The packaging materials discussed herein in
relation to diagnostic systems are those customarily
utilized in diagnostic systems.
H. Assay Methods
The present invention may also be applied to various
assay methods for determining the presence, and
preferably amount, of a preselected ligand, typically
present in an aqueous composition such as a biological
fluid sample using a heterodimeric receptor or phage
of this invention as an ligand-binding reagent to form
a binding reaction product whose amount relates,
either directly or indirectly, to the amount of the
preselected ligand in the sample.
Those skilled in the art will understand that
there are numerous well known clinical diagnostic
chemistry procedures in which a binding reagent of
this invention can be used to form an binding reaction
product whose amount relates to the amount of the
ligand in a sample. Thus, exemplary assay methods are
described herein.
Various heterogenous and homogeneous protocols, either
competitive or noncompetitive, can be employed in
performing an assay method of this invention.
In one method, the invention might be applied to
a direct binding assay using a ligand—binding
heterodimeric receptor of this invention as a binding
reagent to detect the presence of a preselected ligand
with which the receptor binds. The method comprises
the steps of a) admixing a sample suspected to contain
a preselected antigen with a ligand—binding
heterodimeric receptor of this invention that binds to
the preselected ligand under binding conditions
sufficient for the ligand-binding heterodimeric
receptor to bind the ligand and form a ligand-receptor
complex; and b) detecting the presence of the ligand-
receptor complex or the ligand—binding heterodimeric
receptor in the complex.
Binding conditions are those that maintain the
ligand—binding activity of the receptor. Those
conditions include a temperature range of about 4 to
50 degrees Centigrade, a pH value range of about 5 to
9 and an ionic strength varying from about that of
distilled water to that of about one molar sodium
chloride.
The detecting step can be directed, as is well
known in the immunological arts, to either the complex
or the binding reagent (the receptor component of the
complex). Thus, a secondary binding reagent such as
an antibody specific for the receptor may be utilized.
Alternatively, the complex may be detectable by
virtue of having used a labeled receptor molecule,
thereby making the complex labeled. Detection in this
case comprises detecting the label present in the
complex.
In a preferred method, the ligand—binding
heterodimeric receptor is provided as an attachment on
a filamentous phage particle, i.e., on the surface of
the phage. An exemplary assay using a filamentous
phage of this invention is described in an ELISA
format in the Examples.
In another method, a filamentous phage
particle is detectably labeled, such as by a
-9 1-
radioisotope incorporated in a protein or nucleic acid of
the phage as described herein. In this method, detection
comprises detecting the label in the complex, and thereby
detecting the presence of the ligand in the complex.
A further possible diagnostic method utilizes the
multivalency of a filamentous phage particle to crosslink
ligand, thereby forming an aggregation of multiple ligands
and phage particles, producing a precipitable aggregate.
This method is comparable to the well known methods of
immune precipitation. This method comprises the steps of
admixing a sample with a plurality of phage particle of
this invention to form a binding admixture under binding
conditions, followed by a separation step to isolate the
formed binding complexes. Typically, isolation is
accomplished by centrifugation or filtration to remove the
aggregate from the admixture. The presence of binding
complexes indicates the presence of the preselected ligand
to be detected.
Examples
The following examples are intended to
illustrate, but not limit, the scope of the invention.
. Construction of a Dicistronic Expression Vector
for Producing a Heterodimeric Receptor on Phage
Particles
To obtain a vector system for generating a large
number of Fab antibody fragments that can be screened
directly, expression libraries in bacteriophage Lambda
have previously been constructed as described in Huse
et al., Science, 246:1275-1281 (1989). These systems
did not contain design features that provide for the
expressed Fab to be targeted to the surface of a
filamentous phage particle.
The main criterion used in choosing a vector
system was the necessity of generating the largest
number of Fab fragments which could be screened
directly. Bacteriophage Lambda was selected as the
starting point to develop an expression vector for
three reasons. First, in yitgg packaging of phage DNA
was the most efficient method of reintroducing DNA
into host cells. Second, it was possible to detect
protein expression at the level of single phage
plaques. Finally, the screening of phage libraries
typically involved less difficulty with nonspecific
binding. The alternative, plasmid cloning vectors,
are only advantageous in the analysis of clones after
they have been identified. This advantage was not
lost in the present system because of the use of a
dicistronic expression vector such as pCombVIII,
thereby permitting a plasmid containing the heavy
chain, light chain, or Fab expressing inserts to be
excised.
a. Construction of Dicistronic Expression
Vector QCOMB
(i) Preparation of Lambda Zap “II
Lambda Zap” II is a derivative of the
original Lambda Zap (ATCC # 40,298) that maintains all
of the characteristics of the original Lambda Zap
including 6 unique cloning sites, fusion protein
expression, and the ability to rapidly excise the
insert in the form of a phagemid (Bluescript SK-), but
lacks the SAM 100 mutation, allowing growth on many
Non-Sup F strains, including XL1—B1ue. The Lambda
Zap” II was constructed as described in Short et al.,
Nuc. Acids Res., l6:7583—7600, 1988, by replacing the
Lambda S gene contained in a 4254 base pair (bp) DNA
fragment produced by digesting Lambda Zap with the
-93..
restriction enzyme Nco I. This 4254 bp DNA fragment
was replaced with the 4254 bp DNA fragment containing
the Lambda S gene isolated from Lambda gt10 (ATCC #
40,179) after digesting the vector with the
restriction enzyme Nco I. The 4254 bp DNA fragment
isolated from lambda gt10 was ligated into the
original Lambda Zap vector using T4 DNA ligase and
standard protocols such as those described in Current
Protocols in Molecular Biologv, Ausubel et al., eds.,
John Wiley and Sons, NY, 1987, to form Lambda Zapm
II.
(ii) Preparation of Lambda Hc2
To express a plurality of Vh-coding DNA
homologs in an E. coli host cell, a vector designated
Lambda Hc2 was constructed. The vector provided the
following: the capacity to place the Vh—coding DNA
homologs in the proper reading frame; a ribosome
binding site as described by Shine et al., Nature,
254:34, 1975; a leader sequence directing the
expressed protein to the periplasmic space designated
the pelB secretion signal; a polynucleotide sequence
that coded for a known epitope (epitope tag); and also
a polynucleotide that coded for a spacer protein
between the V}-coding DNA homolog and the
polynucleotide coding for the epitope tag. Lambda Hcz
has been previously described by Huse et al., Science,
246:l275-1281 (1989).
To prepare Lambda Hc2, a synthetic DNA sequence
containing all of the above features was constructed
by designing single stranded polynucleotide segments
of 20-40 bases that would hybridize to each other and
form the double stranded synthetic DNA sequence shown
in Figure 3. The individual single-stranded
polynucleotide segments are shown in Table 3.
Polynucleotides N2, N3, N9-4, N11, N10—5, N6, N7
and N8 (Table 3) were kinased by adding 1 pl of each
polynucleotide 0.1 micrograms/microliter (pg/pl) and
units of T4 polynucleotide kinase to a solution
containing 70 mM Tris—HC1, pH 7.6, 10 mM MgCl2, 5 mM
dithiothreitol (DTT), 10 mM beta-mercaptoethanol, 500
micrograms per milliliter (pg/ml) bovine serum albumin
(BSA). The solution was maintained at 37 degrees
Centigrade (37°C) for 30 minutes and the reaction
stopped by maintaining the solution at 65°C for 10
minutes. The two end polynucleotides, 20 ng of
polynucleotides N1 and polynucleotides N12, were added
to the above kinasing reaction solution together with
1/10 volume of a solution containing 20.0 mM Tris—HCl,
pH 7.4, 2.0 mM MgCl2 and 50.0 mM NaCl. This solution
was heated to 70°C for 5 minutes and allowed to cool
to room temperature, approximately 25°C, over 1.5
hours in a 500 ml beaker of water. During this time
period all 10 polynucleotides annealed to form the
double stranded synthetic DNA insert shown in Figure
3. The individual polynucleotides were covalently
linked to each other to stabilize the synthetic DNA
insert by adding 40 pl of the above reaction to a
solution containing 50 mM Tris-HC1, pH 7.5, 7 mM
MgCl2, 1 mM DTT, 1 mM adenosine triphosphate (ATP) and
units of T4 DNA ligase. This solution was
maintained at 37°C for 30 minutes and then the T4 DNA
ligase was inactivated by maintaining the solution at
65°C for 10 minutes. The end polynucleotides were
kinased by mixing 52 pl of the above reaction, 4 pl of
a solution containing 10 mM ATP and 5 units of T4
polynucleotide kinase. This solution was maintained
at 37°C for 30 minutes and then the T4 polynucleotide
kinase was inactivated by maintaining the solution at
65°C for 10 minutes.
-95..
Table 3
SEQ.
ID. NO.
(22) N1) 5' GGCCGCAAATTCTATTTCAAGGAGACAGTCAT 3'
(23) N2) 5' AATGAAATACCTATTGCCTACGGCAGCCGCTGGATT 3'
(24) N3) 5' GTTATTACTCGCTGCCCAACCAGCCATGGCCC 3'
(25) N6) 5' CAGTTTCACCTGGGCCATGGCTGGTTGGG 3'
(26) N7) 5' CAGCGAGTAATAACAATCCAGCGGCTGCCGTAGGCAATAG 3'
(27) N8) 5' GTATTTCATTATGACTGTCTCCTTGAAATAGAATTTGC 3'
(28) N9-4) 5' AGGTGAAACTGCTCGAGATTTCTAGACTAGTTACCCGTAC 3'
(29) N10-5) 5' CGGAACGTCGTACGGGTAACTAGTCTAGAAATCTCGAG 3'
(30) N11) 5' GACGTTCCGGACTACGGTTCTTAATAGAATTCG 3'
(31) N12) 5' TCGACGAATTCTATTAAGAACCGTAGTC 3'
The completed synthetic DNA insert was ligated
directly into the Lambda Zap” II vector described in
Example 1a(i) that had been previously digested with
the restriction enzymes, Not I and Xho I. The
ligation mixture was packaged according to the
manufacture's instructions using Gigapack II Gold
packing extract available from Stratagene, La Jolla,
California. The packaged ligation mixture was plated
on XL1-Blue cells (Stratagene). Individual lambda
plaques were cored and the inserts excised according
to the in vivo excision protocol for Lambda Zap” II
provided by the manufacturer (Stratagene). This in
yiyg excision protocol moved the cloned insert from
the Lambda Hc2 vector into a phagemid vector to allow
easy for manipulation and sequencing. The accuracy of
the above cloning steps was confirmed by sequencing
the insert using the Sanger dideoxy method described
in by Sanger et al., Proc. Natl. Acad. Sci. USA,
74:5463-5467, (1977) and using the manufacture's
instructions in the AMV Reverse Transcriptase ”S—ATP
sequencing kit (Stratagene). The sequence of the
resulting double-stranded synthetic DNA insert in the
V“ expression vector (Lambda Hcz) is shown in Figure
3. The sequence of each strand (top and bottom) of
Lambda Hcz is listed in the sequence listing as SEQ ID
NO 1 and SEQ ID NO 2, respectively. The resultant
Lambda Hc2 expression vector is shown in Figure 4.
(iii) Preparation of Lambda Lc2
To express a plurality of V1-coding
DNA homologs in an E. coli host cell, a vector
designated Lambda Lc2 was constructed having the
capacity to place the Vi-coding DNA homologs in the
proper reading frame, provided a ribosome binding site
as described by Shine et al., Nature, 254:34 (1975),
provided the pe1B gene leader sequence secretion
signal that has been previously used to successfully
secrete Fab fragments in E. coli by Lei et al., Q;
gggy, 169:4379 (1987) and Better et al., Science,
240:1041 (1988), and also provided a polynucleotide
containing a restriction endonuclease site for
cloning. Lambda Lc2 has been previously described by
Huse et al., Science, 246:1275-1281 (1989).
A synthetic DNA sequence containing all of the
above features was constructed by designing single
stranded polynucleotide segments of 20-60 bases that
would hybridize to each other and form the double
stranded synthetic DNA sequence shown in Figure 5.
The sequence of each individual single—stranded
polynucleotide segment (01-08) within the double
stranded synthetic DNA sequence is shown in Table 4.
Polynucleotides O2, 03, 04, 05, 06 and 07 (Table
4) were kinased by adding 1 ul (0.1 ug/pl) of each
polynucleotide and 20 units of T2 polynucleotide
kinase to a solution containing 70 mM Tris-HCl, pH
7.6, 10 mM Mgcl, 5 mM DTT, 10 mM beta-mercaptoethanol,
-97.-
mg/ml of BSA. The solution was maintained at 37°C
for 30 minutes and the reaction stopped by maintaining
the solution at 65°C for 10 minutes. The 20 ng each
of the two end polynucleotides, 01 and 08, were added
to the above kinasing reaction solution together with
1/10 volume of a solution containing 20.0 mM Tris-Hcl,
pH 7.4, 2.0 mM Mgcl and 15.0 mM sodium chloride
(Nacl). This solution was heated to 70°C for 5
minutes and allowed to cool to room temperature,
approximately 25°C, over 1.5 hours in a 500 ml beaker
of water. During this time period all 8
polynucleotides annealed to form the double stranded
synthetic DNA insert shown in Figure 5. The
individual polynucleotides were covalently linked to
each other to stabilize the synthetic DNA insert by
adding 40 pl of the above reaction to a solution
containing 50 ml Tris-Hcl, pH 7.5, 7 ml Mgcl, 1 mm
DTT, 1 mm ATP and 10 units of T4 DNA ligase. This
solution was maintained at 37°C for 30 minutes and
then the T4 DNA ligase was inactivated by maintaining
the solution at 65°C for 10 minutes. The end
polynucleotides were kinased by mixing 52 ul of the
above reaction, 4 pl of a solution containing 10 mM
ATP and 5 units of T4 polynucleotide kinase. This
solution was maintained at 37°C for 30 minutes and
then the T4 polynucleotide kinase was inactivated by
maintaining the solution at 65°C for 10 minutes.
TABLE 4
SEQ.
ID. NO.
(32) 01) 5' TGAATTCTAAACTAGTCGCCAAGGAGACAGTCAT 3'
(33) 02) 5' AATGAAATACCTATTGCCTACGGCAGCCGCTGGATT 3'
(34) 03) 5' GTTATTACTCGCTGCCCAACCAGCCATGGCC 3'
(35) 04) 5' GAGCTCGTCAGTTCTAGAGTTAAGCGGCCG 3'
(36) 05) 5' GTATTTCATTATGACTGTCTCCTTGGCGACTAGTTTAGAA-
TTCAAGCT 3'
(37) O6) 5' CAGCGAGTAATAACAATCCAGCGGCTGCCGTAGGCAATAG
3:
(38) 07) 5' TGACGAGCTCGGCCATGGCTGGTTGGG 3'
(39) 08) 5' TCGACGGCCGCTTAACTCTAGAAC 3'
The completed synthetic DNA insert was ligated
directly into the Lambda Zap” II vector described in
Example 1(a)(i) that had been previously digested with
the restriction enzymes Sac I and Xho I. The ligation
mixture was packaged according to the manufacture's
instructions using Gigapack II Gold packing extract
(Stratagene). The packaged ligation mixture was
plated on XL1-Blue cells (Stratagene). Individual
lambda plaques were cored and the inserts excised
according to the in vivo excision protocol for Lambda
Zap” II provided by the manufacturer (Stratagene).
This in vivo excision protocol moved the cloned insert
from the Lambda Lc2 vector into a plasmid phagemid
vector allow for easy manipulation and sequencing.
The accuracy of the above cloning steps was confirmed
by sequencing the insert using the manufacture's
instructions in the AMV Reverse Transcriptase “S-dATP
sequencing kit (Stratagene). The sequence of the
resulting Lc2 expression vector (Lambda Lc2) is shown
in Figure 5. Each strand is separately listed in the
Sequence Listing as SEQ ID NO 3 and SEQ ID NO 4. The
resultant Lc2 vector is schematically diagrammed in
Figure 6.
A preferred vector for use in this invention,
designated Lambda Lc3, is a derivative of Lambda Lc2
prepared above. Lambda Lc2 contains a Spe I
restriction site (ACTAGT) located 3' to the EcoR I
restriction site and 5' to the Shine-Dalgarno ribosome
binding site as shown in the sequence in Figure 5 and
in SEQ ID NO 3. A Spe I restriction site is also
present in Lambda Hc2 as shown in Figures 3 and 4 and
in SEQ ID NO 1. A combinatorial vector, designated
pcomb, was constructed by combining portions of Lambda
Hc2 and Lc2 together as described in Example 1a(iv)
below. The resultant combinatorial pcomb vector
contained two Spe I restriction sites, one provided by
Lambda Hcz and one provided by Lambda Lcz, with an
EcoR I site in between. Despite the presence of two
Spe I restriction sites, DNA homologs having Spe I and
EcoR I cohesive termini were successfully
directionally ligated into a pcomb expression vector
previously digested with Spe I and EcoR I as described
in Example 1b below. The proximity of the EcoR I
restriction site to the 3' Spe I site, provided by the
Lc2 vector, inhibited the complete digestion of the 3'
Spe I site. Thus, digesting pcomb with Spe I and EcoR
I did not result in removal of the EcoR I site between
the two Spe I sites.
The presence of a second Spe I restriction site
may be undesirable for ligations into a pcomb vector
digested only with Spe I as the region between the two
sites would be eliminated. Therefore, a derivative of
Lambda Lc2 lacking the second or 3' Spe I site,
designated Lambda Lc3, is produced by first digesting
Lambda Lc2 with Spe I to form a linearized vector.
The ends are filled in to form blunt ends which are
ligated together to result in Lambda Lc3 lacking a Spe
I site. Lambda Lc3 is a preferred vector for use in
constructing a combinatorial vector as described
below.
(iv) Preparation of pcomb
Phagemids were excised from the
expression vectors Lambda Hcz or Lambda Lc2 using an
in vivo excision protocol described above. Double
stranded DNA was prepared from the phagemid-containing
cells according to the methods described by Holmes et
al., Anal. Biochem., 114:193 (1981). The phagemids
resulting from in vivo excision contained the same
nucleotide sequences for antibody fragment cloning and
expression as did the parent vectors, and are
designated phagemid Hc2 and Lc2, corresponding to
Lambda Hc2 and Lc2, respectively.
For the construction of combinatorial phagemid
vector pcomb, produced by combining portions of
phagemid Hc2 and phagemid Lc2, phagemid Hc2 was first
digested with Sac I to remove the restriction site
located 5' to the LacZ promoter. The linearized
phagemid was then blunt ended with T4 polymerase and
ligated to result in a Hc2 phagemid lacking a Sac I
site. The modified Hcz phagemid and the Lc2 phagemid
were then separately restriction digested with Sca I
and EcoR I to result in a Hcz fragment having from 5'
to 3' Sea I, Not I Xho I, Spe I and EcoR I restriction
sites and a Lc2 fragment having from 5' to 3' EcoR I,
Sac I, Xba I and Sac I restriction sites. The
linearized phagemids were then ligated together at
their respective cohesive ends to form pcomb, a
circularized phagemid having a linear arrangement of
restriction sites of Not I, Xho I, Spe I, EcoR I, Sac
I, Xba I, Not I, Apa I and Sca I. The ligated
phagemid vector was then inserted into an appropriate
bacterial host and transformants were selected on the
antibiotic ampicillin.
Selected ampicillin resistant transformants were
screened for the presence of two Not I sites. The
resulting ampicillin resistant combinatorial phagemid
vector was designated pcomb, the schematic
organization of which is shown in Figure 7. The
resultant combinatorial vector, pcomb, consisted of a
DNA molecule having two cassettes to express two
fusion proteins and having nucleotide residue
sequences for the following operatively linked
elements listed in a 5' to 3' direction: a first
cassette consisting of an inducible Lacz promoter
upstream from the Lacz gene; a Not I restriction site;
a ribosome binding site; a pelB leader; a spacer; a
cloning region bordered by a 5' Xho and 3' Spe I
restriction site; a decapeptide tag followed by
expression control stop sequences; an EcoR I
restriction site located 5' to a second cassette
consisting of an expression control ribosome binding
site; a pelB leader; a spacer region; a cloning region
bordered by a 5' Sac I and a 3' Xba I restriction site
followed by expression control stop sequences and a
second Not I restriction site.
A preferred combinatorial vector for use in this
invention, designated pcombz, is constructed by
combining portions of phagemid Hc2 and phagemid Lc3 as
described above for preparing pcomb. The resultant
combinatorial vector, pComb2, consists of a DNA
molecule having two cassettes identical to pcomb to
express two fusion proteins identically to pcomb
except that a second Spe I restriction site in the
second cassette is eliminated.
b. Construction of Vectors pCombVIII and
pCombIII for Expressing Fusion Proteins
Having a Bacteriophage Coat Protein Membrane
Anchor
Because of the multiple endonuclease
restriction cloning sites, the pcomb phagemid
expression vector prepared above is a useful cloning
vehicle for modification for the preparation of an
expression vector of this invention. To that end,
pcomb is digested with EcoR I and Spe I followed by
phosphatase treatment to produce linearized pcomb.
(i) Preparation of pCombVIII
A PCR product produced in Example 2g
and having a nucleotide sequence that defines a
filamentous bacteriophage coat protein VIII (cpVIII)
membrane anchor domain and cohesive Spe I and EcoR I
termini was admixed with the linearized pcomb to form
a ligation admixture. The cpVIII—membrane anchor-
encoding PCR fragment was directionally ligated into
the pcomb phagemid expression vector at corresponding
cohesive termini, that resulted in forming pCombVIII
(also designated pComb8). pCombVIII contains a
cassette defined by the nucleotide sequence shown in
SEQ ID NO 116 from nucleotide base 1 to base 259, and
contains a pelB secretion signal operatively linked to
the cpVIII membrane anchor.
A preferred phagemid expression vector for use in
this invention, designated either pComb2-VIII or
pComb2-8, was prepared as described above by
directionally ligating the cpVIII membrane anchor-
encoding PCR fragment into a pComb2 phagemid
expression vector via Spe I and EcoR I cohesive
termini. The pcombz-8 had only one Spe I restriction
site.
(ii) Preparation of pCombIII
A separate phagemid expression vector
was constructed using sequences encoding bacteriophage
cpIII membrane anchor domain. A PCR product defining
the cpIII membrane anchor containing a LacZ promoter
region sequence 3' to the membrane anchor for
expression of the light chain and Spe I and EcoR I
cohesive termini was prepared as described for cpVIII,
the details of which are described in Example 2g. The
cpIII-derived PCR product was then ligated into
linearized pcombz vector having only one Spe I site to
form the vector pComb2-3 (also designated pComb2-III).
c. Construction of pCBAK Vectors Having a
chloramphenicol Resistance Marker
In order to utilize a different selectable
marker gene, such as chloramphenicol acetyl
transferase (CAT), for the selection of bacteria
transformed with a vector of this invention,
expression vectors based on pcomb were developed
having a gene encoding CAT and are designated pCBAK
vectors. The pCBAK vectors are prepared by combining
portions of pCB and pcomb.
(i) Preparation of DCB
pBluescript phagemid vectors, pBC SK(-)
and pBS SK(-), (Stratagene), were modified and
combined to generate a third vector designated pCB as
described below.
pBC SK(—), which contains a chloramphenicol
resistance selectable marker gene, was digested with
Bst BI and blunt ended with T4 polymerase. A second
digestion with Pvu I allowed for the removal of a 1
kilobase (kb) fragment leaving a 2.4 kb linearized
vector which retained the CAT selectable resistance
marker gene, an inducible Lacz promoter upstream from
the Lacz gene and a Co1E1 origin region. The 2.4 kb
fragment was recovered. The pBS SK(—) vector was
digested with Aat II and blunt ended with T4
polymerase. A second digestion with Pvu I allowed for
the isolation of an 800 base pair (bp) fragment
containing the f1 origin of replication. Ligation of
the pBS derived 800 bp fl fragment with the 2.4 kb pBC
fragment created a pCB precursor vector containing a
Sac I site, an fl origin of replication, a CAT
selectable resistance marker gene, Co1E1 origin, a
multiple cloning site (MCS) flanked by T3 and T7
promoters, and an inducible Lacz promoter upstream
from Lacz gene.
The pCB precursor vector was then digested with
Sac I and blunt-ended with T4 polymerase. The T4
polymerase-treated pCB vector was then religated to
form pCB vector and is lacking a Sac I site.
(ii) Preparation of DCBAKO
The pCB vector containing the CAT
selectable resistance marker gene was digested with
Sac II and Apa I and treated with phosphatase to
prevent religation and to form linearized pCB vector.
The pcomb vector prepared in Example 1(a)(iv) was
restriction digested with Sac II and Apa I to release
a fragment containing nucleotide residue sequences
starting 5' to the Lacz promoter and extending past
the 3' end of the second Not I site. The Sac II and
Apa I pComb DNA fragment was then directionally
-105*
ligated into the similarly digested pCB vector to form
phagemid expression vector pCBAKO. Preferred pCBAK
expression vectors are constructed with pComb2. The
resultant pCBAK expression vector contained only one
Spe I restriction site.
(iii) Preparation of pCBAK8
To prepare a pCBAK-based phagemid
expression vector which encodes a bacteriophage coat
protein membrane anchor domain in the expressed fusion
protein, pCB phagemid cloning vector prepared in
Example 1c(ii) was linearized by digestion with Sac II
and Apa I. The pCombVIII phagemid expression vector,
prepared in Example 1b(i), was restriction digested
with Sac II and Apa I to form a fragment containing a
nucleotide residue sequence starting 5' to the Lacz
promoter and extending past the 3' end of the second
Not 1 site. The fragment was directionally ligated
into the linearized pCB cloning vector to form
phagemid expression vector pCBAK8.
(iv) Preparation of pCBAK3
The phagemid expression vector,
pCBAK3, for the expression of fusion protein having
cpIII membrane anchor domains, was similarly
constructed by directionally ligating the Sac II and
Apa I restriction digested fragment from pCombIII with
Sac II and Apa I linearized pCB cloning vector.
. Construction of Dicistronic Expression Vectors
for Expressing Anti-NPN Heterodimer on Phage
Surfaces
In practicing this invention, the heavy (Fd
consisting of V“ and CH1) and light (kappa) chains (VL,
CL) of antibodies are first targeted to the periplasm
of E. coli for the assembly of heterodimeric Fab
molecules. In order to obtain expression of antibody
Fab libraries on a phage surface, the nucleotide
residue sequences encoding either the Fd or light
chains must be operatively linked to the nucleotide
residue sequence encoding a filamentous bacteriophage
coat protein membrane anchor. Two preferred coat
proteins for use in this invention in providing a
membrane anchor are VIII and III (cpVIII and cpIII,
respectively). In the Examples described herein,
methods for operatively linking a nucleotide residue
sequence encoding a Fd chain to either cpVIII or cpIII
membrane anchors in a fusion protein of this invention
are described.
In a phagemid vector, a first and second cistron
consisting of translatable DNA sequences are
operatively linked to form a dicistronic DNA molecule.
Each cistron in the dicistronic DNA molecule is linked
to DNA expression control sequences for the coordinate
expression of a fusion protein, Fd-cpVIII or Fd-cpIII,
and a kappa light chain.
The first cistron encodes a periplasmic secretion
signal (pelB leader) operatively linked to the fusion
protein, either Fd-cpVIII or Fd-cpIII. The second
cistron encodes a second pelB leader operatively
linked to a kappa light chain. The presence of the
pelB leader facilitates the coordinated but separate
secretion of both the fusion protein and light chain
from the bacterial cytoplasm into the periplasmic
space.
The process described above is schematically
diagrammed in Figure 8. Briefly, the phagemid
expression vector carries a chloramphenicol acetyl
transferase (CAT) selectable resistance marker gene in
addition to the Fd-cpVIII fusion and the kappa chain.
The f1 phage origin of replication facilitates the
generation of single stranded phagemid. The isopropyl
thiogalactopyranoside (IPTG) induced expression of a
dicistronic message encoding the Fd-cpVIII fusion (Vw
Cm, cpVIII) and the light chain (Vi, CL) leads to the
formation of heavy and light chains. Each chain is
delivered to the periplasmic space by the pelB leader
sequence, which is subsequently cleaved. The heavy
chain is anchored in the membrane by the cpVIII
membrane anchor domain while the light chain is
secreted into the periplasm. The heavy chain in the
presence of light chain assembles to form Fab
molecules. This same result can be achieved if, in
the alternative, the light chain is anchored in the
membrane via a light chain fusion protein having a
membrane anchor and heavy chain is secreted via a pe1B
leader into the periplasm.
With subsequent infection of E. coli with a
helper phage, as the assembly of the filamentous
bacteriophage progresses, the coat protein VIII is
incorporated along the entire length of the
filamentous phage particles as shown in Figures 8 and
9. If cpIII is used, the accumulation occurs on the
tail of the bacteriophage. The advantage of the
utilization of membrane anchors from cpVIII over cpIII
is two fold. Firstly, a multiplicity of binding
sites, consisting of approximately 2700 cpVIII
monomers assembled in a tubular array, exist along the
particle surface. Secondly, the construct does not
interfere with phage infectivity.
a. Polynucleotide Selection
The nucleotide sequences encoding the
immunoglobulin protein CDR's are highly variable.
However, there are several regions of conserved
sequences that flank the V region domains of either
the light or heavy chain, for instance, and that
contain substantially conserved nucleotide sequences,
i.e., sequences that will hybridize to the same primer
sequence. Therefore, polynucleotide synthesis
(amplification) primers that hybridize to the
conserved sequences and incorporate restriction sites
into the DNA homolog produced that are suitable for
operatively linking the synthesized DNA fragments to a
vector were constructed. More specifically, the
primers are designed so that the resulting DNA
homologs produced can be inserted into an expression
vector of this invention in reading frame with the
upstream translatable DNA sequence at the region of
the vector containing the directional ligation means.
(i) E; Primers
For amplification of the V“ domains,
primers are designed to introduce cohesive termini
compatible with directional ligation into the unique
Xho I and Spe I sites of the phagemid Hc2 expression
vector. For example, the 3' primer (primer 12A in
Table 5), was designed to be complementary to the mRNA
in the J“ region. In all cases, the 5' primers
(primers 1-10, Table 5) were chosen to be
complementary to the first strand cDNA in the
conserved N—terminus region (antisense strand).
Initially amplification was performed with a mixture
of 32 primers (primer 1, Table 5) that were degenerate
at five positions. Hybridoma mRNA could be amplified
with mixed primers, but initial attempts to amplify
mRNA from spleen yielded variable results. Therefore,
several alternatives to amplification using the mixed
' primers were compared.
The first alternative was to construct multiple
unique primers, eight of which are shown in Table 5,
corresponding to individual members of the mixed
primer pool. The individual primers 2-9 of Table 5
were constructed by incorporating either of the two
possible nucleotides at three of the five degenerate
positions.
The second alternative was to construct a primer
containing inosine (primer 10, Table 5) at four of the
variable positions based on the published work of
Takahashi, et al., Proc. Natl. Acad. Sci. (USA),
82:1931-1935, (1985) and Ohtsuka et al., J. Biol.
ghgmp, 260: 2605-2608, (1985). This primer has the
advantage that it is not degenerate and, at the same
time minimizes the negative effects of mismatches at
the unconserved positions as discussed by Martin et
al., Nuc. Acids Res., 13:8927 (1985). However, it was
not known if the presence of inosine nucleotides would
result in incorporation of unwanted sequences in the
cloned V" regions. Therefore, inosine was not
included at the one position that remains in the
amplified fragments after the cleavage of the
restriction sites. As a result, inosine was not in
the cloned insert.
Additional V” amplification primers including the
unique 3' primer were designed to be complementary to
a portion of the first constant region domain of the
gamma 1 heavy chain mRNA (primers 16 and 17, Table 5).
These primers will produce DNA homologs containing
polynucleotides coding for amino acids from the V" and
the first constant region domains of the heavy chain.
These DNA homologs can therefore be used to produce
Fab fragments rather than Fv.
Additional unique 3' primers designed to
hybridize to similar regions of another class of
immunoglobulin heavy chain such as IgM, IgE and IgA
are contemplated. other 3' primers that hybridize to
a specific region of a specific class of CH1 constant
region and are adapted for transferring the V" domains
amplified using this primer to an expression vector
capable of expressing those V, domains with a
different class of heavy or light chain constant
region are also contemplated.
As a control for amplification from spleen or
hybridoma mRNA, a set of primers hybridizing to a
highly conserved region within the constant region
IgG, heavy chain gene were constructed. The 5' primer
(primer 11, Table 5) is complementary to the cDNA in
the CH2 region whereas the 3' primer (primer 13, Table
) is complementary to the mRNA in the CH3 region. It
is believed that no mismatches were present between
these primers and their templates.
The primers used for amplification of heavy chain
Fd fragments for construction of Fabs are shown at
least in Table 5. Amplification was performed in
eight separate reactions, each containing one of the
' primers (primers 2-9) and one of the 3' primers
(primer 16). The remaining 5' primers that have been
used for amplification in a single reaction are either
a degenerate primer (primer 1) or a primer that
incorporates inosine at four degenerate positions
(primer 10, Table 5, and primers 17 and 18, Table 6).
The remaining 3' primer (primer 14, Table 6) has been
used to construct FV fragments. Many of the 5'
primers incorporate a Xho I site, and the 3' primers
incorporate a Spe I restriction site for insertion of
the V“ DNA homolog into the phagemid Hc2 expression
vector (Figure 4).
V" amplification primers designed to amplify
human heavy chain variable regions are shown in Table
6. one of the 5' heavy chain primer contains inosine
residues at degenerate nucleotide positions allowing a
single primer to hybridize to a large number of
variable region sequences. Primers designed to
hybridize to the constant region sequences of various
IgG mRNAs are also shown in Table 6.
(ii) yL Primers
The nucleotide sequences encoding the VL
CDRs are highly variable. However, there are several
regions of conserved sequences that flank the VL CDR
domains including the J1, VL framework regions and VL
leader/promotor. Therefore, amplification primers
were constructed that hybridized to the conserved
sequences and incorporate restriction sites that allow
cloning the amplified fragments into the phagemid Lc2
vector cut with Sac I and Xba I.
For amplification of the VL CDR domains, the 5'
primers (primers 1-8 in Table 6) were designed to be
complementary to the first strand cDNA in the
conserved N-terminus region. These primers also
introduced a Sac I restriction endonuclease site to
allow the VLDNA homolog to be cloned into the
phagemid LC2 expression vector. The 3' VL
amplification primer (primer 9 in Table 6) was
designed to be complementary to the mRNA in the JL
regions and to introduce the Xba I restriction
endonuclease site required to insert the VL DNA
homolog into the phagemid Lc2 expression vector
(Figure 6).
Additional 3' VL amplification primers were
designed to hybridize to the constant region of either
kappa or lambda mRNA (primers 10 and 11 in Table 6).
These primers allow a DNA homolog to be produced
containing polynucleotide sequences coding for
constant region amino acids of either kappa or lambda
chain. These primers make it possible to produce an
Fab fragment rather than an Fv.
The primers used for amplification of kappa light
chain sequences for construction of Fabs are shown at
least in Table 6. Amplification with these primers
was performed in 5 separate reactions, each containing
one of the 5' primers (primers 3-6, and 12) and one of
the 3' primers (primer 13). The remaining 3' primer
(primer 9) has been used to construct Fv fragments.
The 5' primers contain a Sac I restriction site and
the 3' primers contain a Xba I restriction site.
VL amplification primers designed to amplify
human light chain variable regions of both the lambda
and kappa isotypes are also shown in Table 6.
All primers and synthetic polynucleotides
described herein, including those shown in Tables 3-7
were either purchased from Research Genetics in
Huntsville, Alabama or synthesized on an Applied
Biosystems DNA synthesizer, model 381A, using the
manufacturer's instruction.
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The 19 primers listed in Table 5 have been listed
in the Sequence Listing and have been assigned the
following SEQ ID NO:
(1) = SEQ ID NO 40
(2) = SEQ ID NO 41
(3) = SEQ ID NO 42
(4) = SEQ ID NO 43
(5) = SEQ ID NO 44
(6) = SEQ ID NO 45
(7) = SEQ ID NO 46
(8) = SEQ ID NO 47
(9) = SEQ ID NO 43
(10) = SEQ ID NO 49
(11) = SEQ ID NO 50
(12) = SEQ ID NO 51
(12A)= SEQ ID NO 52
(13) = SEQ ID NO 53
(14) = SEQ ID NO 54
(15) = SEQ ID NO 55
(15) = SEQ ID NO 55
(17) = SEQ ID NO 57
(18) = SEQ ID NO 58
The 40 primers listed as "(1)" through "(40)" in
Table 6 have also been individually and sequentially
listed in the Sequence Listing beginning with SEQ ID
NO 59 through SEQ ID NO 98, respectively.
b. Preparation of a Repertoire of Genes
Encoding Immunoglobulin Variable Domain
Nitrophenylphosphonamidate (NPN) was
selected as the ligand for receptor binding in
preparing a heterodimeric receptor according to the
methods of the invention.
Keyhole limpet hemocyanin (KLH) was conjugated to
NPN to form a NPN-KLH conjugate used for immunizing a
mouse to produce an anti-NPN immune response and
thereby provide a source of ligand specific
heterodimeric receptor genes.
The NPN-KLH conjugate was prepared by admixing
250 pl of a solution containing 2.5 mg of NPN in
dimethylformamide with 750 pl of a solution containing
2 mg of KLH in 0.01 Molar (M) sodium phosphate buffer
(pH 7.2). The two solutions were admixed by slow
addition of the NPN solution to the KLH solution while
the KLH solution was being agitated by a rotating
stirring bar. Thereafter the admixture was maintained
at 4°C for 1 hour with the same agitation to allow
conjugation to proceed. The conjugated NPN-KLH was
isolated from the nonconjugated NPN and KLH by gel
filtration through Sephadex G-25. The isolated NPN-
KLH conjugate was injected into mice as described
below.
The NPN—KLH conjugate was prepared for injection
into mice by adding 100 pg of the conjugate to 250 pl
of phosphate buffered saline (PBS). An equal volume
of complete Freund's adjuvant was added and emulsified
the entire solution for 5 minutes. A 129 G”+ mouse
was injected with 300 pl of the emulsion. Injections
were given subcutaneously at several sites using a 21
gauge needle. A second immunization with NPN—KLH was
given two weeks later. This injection was prepared as
follows: 50 micrograms (pg) of NPN—KLH were diluted
in 250 pl of PBS and an equal volume of alum was
admixed to the NPN-KLH solution. The mouse was
injected intraperitoneally with 500 pl of the solution
using a 23 gauge needle. one month later the mice
were given a final injection of 50 pg of the NPN-KLH
conjugate diluted to 200 pl in PBS. This injection
was given intravenously in the lateral tail vein using
a 30 gauge needle. Five days after this final
injection the mice were sacrificed and total cellular
RNA was isolated from their spleens.
Total cellular RNA was prepared from the spleen
of a single mouse immunized with KLH-NPN as described
above using the RNA preparation methods described by
Chomczynski et al., Anal Biochem., 162:156-159 (1987)
and using the RNA isolation kit (Stratagene) according
to the manufacturer's instructions. Briefly,
immediately after removing the spleen from the
immunized mouse, the tissue was homogenized in 10 ml
of a denaturing solution containing 4.0 M guanine
isothiocyanate, 0.25 M sodium citrate at pH 7.0, and
0.1 M beta-mercaptoethanol using a glass homogenizer.
One ml of sodium acetate at a concentration of 2 M at
pH 4.0 was admixed with the homogenized spleen. One
ml of phenol that had been previously saturated with
I50 was also admixed to the denaturing solution
containing the homogenized spleen. Two ml of a
chloroformzisoamyl alcohol (24:1 v/v) mixture was
added to this homogenate. The homogenate was mixed
vigorously for ten seconds and maintained on ice for
minutes. The homogenate was then transferred to a
thick—walled 50 ml polypropylene centrifuged tube
(Fisher Scientific Company, Pittsburg, PA). The
solution was centrifuged at 10,000 x g for 20 minutes
at 4°C. The upper RNA-containing aqueous layer was
transferred to a fresh 50 ml polypropylene centrifuge
tube and mixed with an equal volume of isopropyl
alcohol. This solution was maintained at —20°C for at
least one hour to precipitate the RNA. The solution
containing the precipitated RNA was centrifuged at
,000 x g for twenty minutes at 4°C. The pelleted
total cellular RNA was collected and dissolved in 3 ml
of the denaturing solution described above. Three ml
of isopropyl alcohol was added to the re—suspended
total cellular RNA and vigorously mixed. This
solution was maintained at -20°C for at least 1 hour
to precipitate the RNA. The solution containing the
precipitated RNA was centrifuged at 10,000 x g for ten
minutes at 4°C. The pelleted RNA was washed once with
a solution containing 75% ethanol. The pelleted RNA
was dried under vacuum for 15 minutes and then re-
suspended in dimethyl pyrocarbonate (DEPC) treated
(DEPC-H20) H20 .
salt loading buffer consisting of 50 mM Tris-HCl, pH
.5, 500 mM sodium chloride, 1 mM EDTA at pH 7.5 and
0.1% SDS. The oligo dT column was then washed with 2
ml of 1 X medium salt buffer consisting of 50 mM Tris-
HCl, pH 7.5, 100 mM, 1 mM EDTA and 0.1% SDS. The
messenger RNA was eluted from the oligo-dT column with
1 ml of buffer consisting of 10 mM Tris—HCl, pH 7.5, 1
mM EDTA, at pH 7.5, and 0.05% SDS. The messenger RNA
was purified by extracting this solution with phenol]
chloroform followed by a single extraction with 100%
chloroform. The messenger RNA was concentrated by
ethanol precipitation and resuspended in DEPC Hgx
The messenger RNA (mRNA) isolated by the above
process contains a plurality of different V“ coding
polynucleotides, i.e., greater than about 10‘
different Vi-coding genes, and contains a similar
number of V1-coding genes. Thus, the mRNA population
represents a repertoire of variable region—coding
genes.
c. Preparation of DNA Homoloqs
In preparation for PCR amplification, mRNA
prepared above is used as a template for cDNA
synthesis by a primer extension reaction. In a
typical 50 pl transcription reaction, 5-10 pg of
spleen mRNA in water is first hybridized (annealed)
with 500 ng (50.0 pmol) of the 3' V, primer (primer
12A, Table 5),at 65°C for five minutes. subsequently,
the mixture is adjusted to 1.5 mM dATP, dCTP, dGTP and
dTTP, 40 mM Tris-HCl, pH 8.0, 8 mM MgC12, 50 mM NaCl,
and 2 mM spermidine. Moloney-Murine Leukemia virus
Reverse transcriptase (stratagene), 26 units, is added
and the solution is maintained for 1 hour at 37°C.
PCR amplification is performed in a 100 pl
reaction containing the products of the reverse
transcription reaction (approximately 5 ug of the
cDNA/RNA hybrid), 300 ng of 3' V“ primer (primer 12A
of Table 5), 300 ng each of the 5' V" primers
(primers 2-10 of Table 5) 200 mM of a mixture of
dNTP's, 50 mM KCl, 10 mM Tris-HCl pH 8.3, 15 mM MgC1y
0.1% gelatin and 2 units of Thermus aguaticus (Taq)
DNA polymerase (Perkin-Elmer—Cetus, Emeryville,
California). The reaction mixture is overlaid with
mineral oil and subjected to 40 cycles of
amplification. Each amplification cycle includes
denaturation at 92°C for 1 minute, annealing at 52°C
for 2 minutes and polynucleotide synthesis by primer
extension (elongation) at 72°C for 1.5 minutes. The
amplified V}-coding DNA homolog containing samples are
then extracted twice with phenol/chloroform, once with
chloroform, ethanol precipitated and are stored at —
70°C in 10 mM Tris-Hcl, pH 7.5, and 1 mM EDTA.
Using unique 5' primers (2-9, Table 5), efficient
Vh—coding DNA homolog synthesis and amplification from
the spleen mRNA is achieved as shown by agarose gel
electrophoresis. The amplified cDNA (Vh-coding DNA
homolog) was seen as a major band of the expected size
(360 bp). The amount the amplified Vfi—coding
polynucleotide fragment in each reaction is similar,
indicating that all of these primers were about
equally efficient in initiating amplification. The
yield and quality of the amplification with these
primers is reproducible.
The primer containing inosine also synthesizes
amplified Vfi—coding DNA homologs from spleen mRNA
reproducibly, leading to the production of the
expected sized fragment, of an intensity similar to
that of the other amplified cDNAs. The presence of
inosine also permits efficient DNA homolog synthesis
and amplification, clearly indicating that such
primers are useful in generating a plurality of Vf-
coding DNA homologs. Amplification products obtained
from the constant region primers (primers 11 and 13,
Table 5) are more intense indicating that
amplification was more efficient, possibly because of
a higher degree of homology between the template and
primers. Following the above procedures, a Vh-coding
gene library is constructed from the products of eight
amplifications, each performed with a different 5'
primer. Equal portions of the products from each
primer extension reaction are mixed and the mixed
product is then used to generate a library of Vf-
coding DNA homolog—containing vectors.
DNA homologs of the VL are also prepared from the
purified mRNA prepared as described above. In
preparation for PCR amplification, mRNA prepared
according to the above examples is used as a template
for CDNA synthesis. In a typical 50 pl transcription
reaction, 5-10 pg of spleen mRNA in water is first
annealed with 300 ng (50.0 pmol) of the 3' Vi primer
(primer 14, Table 5), at 65°C for five minutes.
Subsequently, the mixture is adjusted to 1.5 mM dATP,
dCTP, dGTP, and dTTP, 40 mM Tris-HCl, pH 8.0, 8 mM
Mgclz, 50 mM Nacl, and 2 mM spermidine. Moloney-
Murine Leukemia virus reverse transcriptase
(Stratagene), 26 units, is added and the solution is
maintained for 1 hour at 37°C. The PCR amplification
is performed in a 100 pl reaction containing
approximately 5 pg of the cDNA/RNA hybrid produced as
described above, 300 ng of the 3' VL primer (primer 14
of Table 5), 300 ng of the 5' VL primer (primer 16 of
Table 5), 200 mM of a mixture of dNTP's, 50 mM KCl, 10
mM Tris-HCl, pH 8.3, 15 mM MgCl2, 0.1% gelatin and 2
units of Taq DNA polymerase. The reaction mixture is
overlaid with mineral oil and subjected to 40 cycles
of amplification. Each amplification cycle includes
denaturation at 92°C for 1 minute, annealing at 52°C
for 2 minutes and elongation at 72°C for 1.5 minutes.
The amplified samples are then extracted twice with
phenol/chloroform, once with chloroform, ethanol
precipitated and are stored at -70°C in 10 mM Tris-
Hcl, 7.5 and 1 mM EDTA.
d. Insertion of DNA Homoloqs into a DNA
Expression Vector
To prepare an expression library enriched in
V" sequences, DNA homologs enriched in V" sequences
are prepared according to Example 2c using the same
set of 5' primers but with primer 12A (Table 5) as the
3' primer. The resulting PCR amplified products (2.5
pg/30 pl of 150 mM Nacl, 8 mM Tris—HCl, pH 7.5, 6 mM
MgSO4, 1 mM DTT, 200 pg/ml BSA) are digested at 37°C
with restriction enzymes Xho I (125 units) and Spe I
(125 units). In cloning experiments which required a
mixture of the products of the amplification
reactions, equal volumes (50 pl, 1-10 pg
concentration) of each reaction mixture are combined
after amplification but before restriction digestion.
The V" homologs are purified on a 1% agarose gel using
the standard electroelution technique described in
Molecular Cloning A Laboratory Manual, Maniatis et
al., eds., Cold Spring Harbor, NY, (1982). After gel
electrophoresis of the digested PCR amplified spleen
mRNA, the region of the gel containing DNA fragments
of approximate 350 bp is excised, electroeluted into a
dialysis membrane, ethanol precipitated and
resuspended in a TE solution containing 10 mM Tris-
HCl, pH 7.5 and 1 mM EDTA to a final concentration of
50 ng/pl. The resulting V“ DNA homologs represent a
repertoire of polypeptide genes having cohesive
termini adapted for directional ligation to the vector
Lambda Hc2. These prepared V" DNA homologs are then
directly inserted by directional ligation into
linearized Lambda Hcz expression vector prepared as
described below.
The Lambda Hc2 expression DNA vector is prepared
for inserting a DNA homolog by admixing 100 pg of this
DNA to a solution containing 250 units each of the
restriction endonucleases Xho I and Spe I (both from
Boehringer Mannheim, Indianapolis, IN) and a buffer
recommended by the manufacturer. This solution is
maintained at 37 from 1.5 hours. The solution is
heated at 65°C for 15 minutes top inactivate the
restriction endonucleases. The solution is chilled to
°C and 25 units of heat—killable (HK) phosphatase
(Epicenter, Madison, WI) and CaCl2 is admixed to it
according to the manufacturer's specifications. This
solution is maintained at 30°C for 1 hour. The DNA is
purified by extracting the solution with a mixture of
phenol and chloroform followed by ethanol
precipitation. The Lambda Hc2 expression vector is
now ready for ligation to the V“ DNA homologs prepared
in the above examples. These prepared V" DNA homologs
are then directly inserted into the Xho I and Spe I
restriction digested Lambda Hcz expression vector that
prepared above by ligating 3 moles of V“ DNA homolog
inserts with each mole of the Hc2 expression vector
overnight at 5°C. Approximately 3.0 x 105 plague
forming units are obtained after packaging the DNA
with Gigapack II Bold (Stratagene) of which 50% are
recombinants. The ligation mixture containing the V"
DNA homologs are packaged according to the
manufacturers specifications using Gigapack Gold II
Packing Extract (Stratagene). The resulting Lambda
Hc2 expression libraries are then transformed into
XL1—Blue cells.
To prepare a library enriched in V, sequences,
PCR amplified products enriched in V, sequences are
prepared according to Example 2c. These VL DNA
homologs are digested with restriction enzymes Sac I
and Xba I and the digested Vi DNA homologs are
purified on a 1% agarose gel as described above for
the V" DNA homologs to form a repertoire of VL-
polypeptide genes adapted for directional ligation.
The prepared VL DNA homologs are then directionally
ligated into the Lambda Lcz expression vector
previously digested with the restriction enzymes, sac
I and Xba I as described for Lambda Hc2. The ligation
mixture containing the VL DNA homologs is packaged to
form a Lambda Lc2 expression library as described
above and is ready to be plated on XL1-Blue cells.
e. Randomly Combining V" and V} DNA Homologs on
the Same Expression Vector
The construction of a library containing
vectors for expressing two cistrons that express heavy
and light chains is accomplished in two steps. In the
first step, separate heavy and light chain libraries
are constructed in the expression vectors Lambda Hc2
and Lambda Lc2, respectively, as described using gene
repertoires obtained from a mouse immunized with NPN—
KLH. In the second step, these two libraries are
combined at the antisymmetric EcoR I sites present in
each vector. This resulted in a library of clones
each of which potentially co-expresses a heavy and a
light chain. The actual combinations are random and
do not necessarily reflect the combinations present in
the B-cell population in the parent animal.
The spleen mRNA resulting from the above
immunizations (Example 2b) is isolated and used to
create a primary library of Vh gene sequences using
the Lambda Hc2 expression vector. The primary library
contains 1.3 x 106 plaque-forming units (pfu) and can
be screened for the expression of the decapeptide tag
to determine the percentage of clones expressing V“
and CH1 (Fd) sequences. The sequence for this peptide
is only in frame for expression following the cloning
of a Fd (or V") fragment into the vector. At least
80% of the clones in the library express Fd fragments
based on immunodetection of the decapeptide tag.
The light chain library is constructed in the
same way as the heavy chain and contains 2.5 x 106
members. Plaque screening, using an anti-kappa chain
antibody, indicates that 60% of the library contained
_express light chain inserts. A small percentage of
inserts results from incomplete dephosphorylation of
vector after cleavage with Sac I and Xba I.
Once obtained, the two libraries are used to
construct a combinatorial library by crossing them at
the EcoR I site. To accomplish the cross, DNA is
first purified from each library.
The Lambda Lc2 library prepared in Example 2d is
amplified and 500 pg of Lambda Lc2 expression library
phage DNA is prepared from the amplified phage stock
using the procedures described in Molecular Cloning:
A Laboratory Manual, Maniatis et al., eds., Cold
Spring Harbor, NY (1982). Fifty pg of this amplified
expression library phage DNA is maintained in a
solution containing 100 units of MLu I restriction
endonuclease (Boehringer Mannheim, Indianapolis, IN)
in 200 pl of a buffer supplied by the endonuclease
manufacturer for 1.5 hours at 37°C. The solution is
then extracted with a mixture of phenol and
chloroform. The DNA is then ethanol precipitated and
resuspended in 100 pl of water. This solution is
admixed with 100 units of the restriction endonuclease
EcoR I (Boehringer) in a final volume of 200 pl of
buffer containing the components specified by the
manufacturer. This solution is maintained at 37°C for
.5 hours and the solution is then extracted with a
mixture of phenol and chloroform. The DNA was ethanol
precipitated and the DNA resuspended in TE.
The Lambda Hc2 expression library prepared in
Example 2d is amplified and 500 pg of Lambda Hcz
expression library phage DNA is prepared using the
methods detailed above. 50 pg of this amplified
library phage DNA is maintained in a solution
containing 100 units of Hind III restriction
endonuclease (Boehringer) in 200 pl of a buffer
supplied by the endonuclease manufacturer for 1.5
hours at 37°C. The solution is then extracted with a
mixture of phenol and chloroform saturated with 0.1 M
Tris-HCl, pH 7.5. The DNA is then ethanol
precipitated and re—suspended in 100 pl of water.
This solution is admixed with 100 units of the
restriction endonuclease EcoR I (Boehringer) in a
final volume of 200 pl of buffer containing the
components specified by the manufacturer. This
solution is maintained at 37°C for 1.5 hours and the
solution is then extracted with a mixture of phenol
and chloroform. The DNA is ethanol precipitated and
the DNA re-suspended in TE.
The restriction digested H02 and Lc2 expression
libraries are ligated together. To that end, a DNA
admixture consists of 1 pg of Hc2 and 1 pg of Lc2
phage library DNA is prepared in a 10 pl reaction
using the reagents supplied in a ligation kit
(Stratagene). The DNA admixture is warmed to 45°C for
minutes to melt any cohesive termini that may have
reannealed. The admixture is then chilled to 0°C to
prevent religation. Bacteriophage T4 DNA ligase (0.1
Weiss units which is equivalent to 0.02 units as
determined in an exonuclease resistance assay) is
admixed into the chilled DNA solution along with 1 pl
of 5 mM ATP and 1 pl 10X bacteriophage T4 DNA ligase
buffer (10X buffer is prepared by admixing 200 mM
Tris-HCl, pH 7.6, 50 mM MgCl2, 50 mM DTT, and 500
pg/ml BSA) to form a ligation admixture. After
ligation for 16 hr at 4°C, 1 pl of the ligated the
phage DNA is packaged with Gigapack Gold II packaging
extract and plated on XL1—Blue cells prepared
according to the manufacturers instructions to form a
Lambda phage library of dicistronic expression vectors
capable of expressing heavy and light chains derived
from the NPN-immunized mouse. A portion of the clones
obtained are used to determine the effectiveness of
the combination.
f. Selection of Anti-NPN Reactive Heterodimer-
Producing Dicistronic Vectors
The combinatorial Fab expression library
prepared above in Example 2a was screened to identify
clones having affinity for NPN. To determine the
frequency of the phage clones which co—expressed the
light and heavy chain fragments, duplicate lifts of
the light chain, heavy chain and combinatorial
libraries were screened as above for light and heavy
chain expression. In this study of approximately 500
recombinant phage, approximately 60% co-expressed
light and heavy chain proteins.
All three libraries, the light chain, the heavy
chain and the combinatorial, were screened to
determine if they contained recombinant phage that
expressed antibody fragments which bound NPN. In a
typical procedure 30,000 phage were plated on XL1-Blue
cells and duplicate lifts with nitrocellulose were
screened for binding to NPN coupled to ‘El labeled
BSA. The BSA was iodinated following the Ch1oramine—T
method as described by Bolton et a1., Biochem.,
:529-534 (1973). Duplicate screens of 80,0
recombinant phage from the light chain library and a
similar number from the heavy chain library did not
identify any clones which bound the antigen. In
contrast, the screen of a similar number of clones
from the Fab expression library identified many phage
plaques that bound NPN. This observation indicates
that under conditions where many heavy chains in
combination with light chains bind to antigen the same
heavy or light chains alone do not. Therefore, in the
case of NPN, it is believed that there are many heavy
and light chains that only bind antigen when they are
combined with specific light and heavy chains,
respectively.
To assess the ability to screen large numbers of
clones and obtain a more quantitative estimate of the
frequency of antigen binding clones in the
combinatorial library, one million phage plaques were
screened and approximately 100 clones which bound to
antigen were identified. For six clones which were
believed to bind NPN, a region of the plate containing
the six positive and approximately 20 surrounding
bacteriophage plaques was selected and each plaque was
cored, replated, and screened with duplicate lifts.
As expected, approximately one in twenty of the phage
specifically bound to antigen. Cores of regions of
the plated phage believed to be negative did not give
positives on replating.
Clone 2b, one of the plaques which reacted with
NPN, was excised according to an in vivo excision
protocol where 200 pl of phage stock and 200 pl of a
F+ derivative of XLl-Blue (Afim = 1.00) (Stratagene)
were admixed with 1 pl of M13mp8 helper phage (1 X
101°pfu/milliliter (m1)) and maintained at 37°C for 15
minutes. After a four hour maintenance in Luria-
Bertani (LB) medium and heating at 70°C for 20 minutes
to heat kill the XL1-Blue cells, the phagemids were
re-infected into XL1-Blue cells and plated onto LB
plates containing ampicillin. This procedure
converted the cloned insert from the Lambda Zap II
vector into a plasmid vector to allow easy
manipulation and sequencing (stratagene). The
phagemid DNA encoding the V" and part of the V1 was
then determined by DNA sequencing using the Sanger
dideoxy method described in Sanger et al., Proc. Natl.
Acad. Sci., 74:5463-5467 (1977) using a Sequenase kit
according to manufacturer's instructions (US
Biochemical Corp., Cleveland, Ohio). The nucleotide
residue sequence of Clone 2b Fd chain is listed in the
Sequence Listing as SEQ ID NO 99. The nucleotide
residue sequences of the kappa light chain variable
and constant regions are listed in the Sequence
Listing as SEQ ID NO 100 and SEQ ID NO 101,
respectively.
g. Preparation of a DNA Sequence Encoding a
Filamentous Phaqe Coat Protein Membrane
Anchor
cpVIII Membrane Anchor: M13mp18, a
commercially available bacteriophage vector
(Pharmacia, Piscataway, New Jersey), was used as a
source for isolating the gene encoding cpVIII. The
sequence of the gene encoding the membrane anchor
domain of cpVIII listed in Sequence Listing as SEQ ID
NO 102, was modified through PCR amplification to
incorporate the restriction endonuclease sites, Spe I
and EcoR I, and two stop codons prior to the EcoR I
site. The corresponding amino acid residue sequence
of the membrane anchor domain of cpVIII is listed as
SEQ ID N0 17.
To prepare a modified cpVIII, replicative form
DNA from M13mp18 was first isolated. Briefly, into 2
ml of LB (Luria—Bertani medium), 50 pl of a culture of
a bacterial strain carrying an F‘ episome (JM107,
JM109 or TG1) was admixed with a one tenth suspension
of bacteriophage particles derived from a single
plaque. The admixture was incubated for 4 to 5 hours
at 37°C with constant agitation. The admixture was
then centrifuged at 12,000 x g for 5 minutes to pellet
the infected bacteria. After the supernatant was
removed, the pellet was resuspended by vigorous
vortexing in 100 pl of ice-cold solution I. Solution
I was prepared by admixing 50 mM glucose, 10 mM EDTA
and 25 mM Tris—HCl, pH 8.0, and autoclaving for 15
minutes.
To the bacterial suspension, 200 pl of freshly
prepared Solution II was admixed and the tube was
rapidly inverted five times. Solution II was prepared
by admixing 0.2 N NaOH and 1% SDS. To the bacterial
suspension, 150 pl of ice-cold Solution III was
admixed and the tube was vortexed gently in an
inverted position for 10 seconds to disperse Solution
III through the viscous bacterial lysate. Solution
III was prepared by admixing 60 ml of 5 M potassium
acetate, 11.5 ml of glacial acetic acid and 28.5 ml of
water. The resultant bacterial lysate was then stored
on ice for 5 minutes followed by centrifugation at
12,000 x g for 5 minutes at 4°C in a microfuge. The
resultant supernatant was recovered and transferred to
a new tube. To the supernatant was added an equal
volume of phenolchloroform and the admixture was
vortexed. The admixture was then centrifuged at
12,000 x g for 2 minutes in a microfuge. The
resultant supernatant was transferred to a new tube
and the double-stranded bacteriophage DNA was
precipitated with 2 volumes of ethanol at room
temperature. After allowing the admixture to stand at
room temperature for 2 minutes, the admixture was
centrifuged to pellet the DNA. The supernatant was
removed and the pelleted replicative form DNA was
resuspended in 25 pl of Tris-HCl, pH 7.6, and 10 mM
EDTA (TE) .
The double-stranded M13mp18 replicative form DNA
was then used as a template for PCR. Primers, AK 5
(SEQ ID NO 103) and AK 6 (SEQ ID NO 104), the
sequences of which are listed in Table 7 below, were
used in the PCR reaction to amplify the mature gene
for cpVIII member anchor domain and incorporate the
two cloning sites, Spe I and EcoR I. For the PCR
reaction, 2 pl containing 1 ng of M13mp18 replicative
form DNA was admixed with 10 pl of 10X PCR buffer
purchased commercially (Promega Biotech, Madison,
Wisconsin) in a 0.5 ml microfuge tube. To the DNA
admixture, 8 pl of a 2.5 mM solution of dNTPs (dATP,
dCTP, dGTP, dTTP) was admixed to result in a final
concentration of 200 micromolar (uM). Three pl
(equivalent to 60 picomoles (pM)) of the 5' forward AK
primer and 3 pl (60 pM) of the 3' backward AK 6
primer was admixed into the DNA solution. To the
admixture, 73 pl of sterile water and 1 pl/5 units of
polymerase (Promega Biotech) was added. Two drops of
mineral oil were placed on top of the admixture and 40
rounds of PCR amplification in a thermocycler were
performed. The amplification cycle consisted of 52°C
for 2 minutes, 72°C for 1.5 minutes and 91°C for 2
minutes. The resultant PCR modified cpVIII membrane
anchor domain DNA fragment from M13mp18 containing
samples were then purified with Gene Clean (BIO101, La
Jolla, California), extracted twice with
phenol/chloroform, once with chloroform followed by
ethanol precipitation and were stored at —70°C in 10
mM Tris—HCl, pH 7.5, and 1 mM EDTA.
Table 7
SEQ.
ID. NO. Primer
(103)‘ AK 5 (F) 5' AI 3'
(104)? AK 6 (B) 5' ACTCGAATTCTATCAGCTTGCTTTCGAGGTGAA 3'
(105)-3 Hc3 (F) 5' AGGTCCAGCTTCTCGAGTCTGG 3'
me)‘ AK 7 (B) 5' mmg 3'
<1os)° 6-3 (B) 5' TTAcTA
GAAQTQG 3'
(1o9)’ LAC-F 5' CACGACAGGTTTCCCGAC
TGG 3'
(no)“ LAC-B 5' ACCGAGCTCGAATTCGTAATCATGGTC 3'
(125)9 LAC-B‘ 5' AGCTGTTGAATTCGTGAAATTGTTATCCGCT 3'
F Forward Primer
Backward Primer
From 5' to 3': the overlapping sequence for CH1 3'
end is double underlined; the Spe I restriction
site sequence is single underlined; the overlapping
sequence for cpVIII is double underlined.
EcoR I restriction site sequence is single
underlined
Xho I restriction site sequence is underlined
From 5' to 3': the overlapping sequence for cpVIII
is double underlined; the Spe I restriction site
sequence is single underlined; the overlapping
sequence for CH1 3' end is double underlined.
From 5' to 3': Spe I restriction site sequence is
single underlined; the overlapping sequence with
the 5' end of cpIII is double underlined
From 5' to 3': Nhe I restriction site sequence is
single underlined; the overlapping sequence with 3'
end of cpIII is double underlined.
From 5' to 3': overlapping sequence with the 3'
end of cpIII is double underlined; Nhe I
restriction sequence begins with the nucleotide
residue "G" at position 4 and extends 5 more
residues = GCTAGC.
EcoR I restriction site sequence is single
underlined.
. Alternative backwards primer for amplifying Lacz;
EcoR I restriction site sequence is single
underlined.
To verify amplification of the modified cpVIII
membrane anchor domain, the PCR purified DNA products
were electrophoresed in a 1% agarose gel. The
expected size of the cpVIII was approximately 150 base
pairs. The area in the agarose containing the
modified cpVIII DNA fragment was isolated from the
agarose as described above. The sequence of the
isolated modified cpVIII DNA fragment is listed as SEQ
ID NO 111. The isolated cpVIII DNA fragment was then
admixed with a similarly prepared fragment of modified
Fd as described below in Example 2i in order to form a
DNA segment encoding the fusion protein Fd-cpVIII.
cpIII Membrane Anchor: M13mp18 was also used as
a source for isolating the gene encoding the membrane
anchor domain at cpIII, the sequence of which is
listed in the Sequence Listing as SEQ ID NO 112. The
amino acid residue sequence of membrane anchor domain
cpIII is listed in SEQ ID NO 16. M13mp18 replicative
form DNA was prepared as described above and used as a
template for two PCR amplifications for construction
of a DNA fragment consisting of the mature gene for
cpIII membrane anchor domain located 5' to a sequence
encoding the LacZ promoter, operator and cap-binding
site for controlling light chain expression. The
restriction sites, Spe I and EcoR I, were created in
the amplification reactions and were located at the 5'
and 3' ends of the fragment respectively. The
procedure for creating this fragment by combining the
products of two separate PCR amplifications is
described below.
The primer pair, G—3(F) (SEQ ID NO 107) and G-
3(B) (SEQ ID NO 108) listed in Table 7, was used in
the first PCR reaction as performed above to amplify
the cpIII membrane anchor gene and incorporate Spe I
and Nhe I restriction sites into the fragment. The
amplified PCR fragment also contained nucleotide
sequences for encoding a five amino acid tether
composed of four glycine residues and one serine
juxtaposed between the heavy chain and cpIII encoding
domains. Once expressed, the five amino acid sequence
lacking an orderly secondary structure served to
minimize the interaction between the Fab and cpIII
domains. The resultant PCR modified cpIII DNA
fragment having Spe I and Nhe I sites in the 5' and 3'
ends, respectively, of the fragment was verified and
purified as described above. The sequence of the PCR
modified cpIII membrane anchor domain DNA fragment is
listed in the Sequence Listing as SEQ ID NO 113. A
second PCR amplification using the primer pairs, Lac-F
(SEQ ID NO 109) and Lac-B (SEQ ID NO 110) listed in
Table 7, was performed on a separate aliquot of
M13mp18 replicative form template DNA to amplify the
LacZ promoter, operator and Cap-binding site having a
' Nhe I site and a 3' EcoR I site. The primers used
for this amplification were designed to incorporate a
Nhe I site on the 5' end of the amplified fragment to
overlap with a portion of the 3' end of the cpIII gene
fragment and of the Nhe I site 3' to the amplified
cpIII fragment. The reaction and purification of the
PCR product was performed as described above. The
sequence of the resultant PCR modified cpIII DNA
fragment having a 5' Nhe I and 3' EcoR I restriction
site is listed in the Sequence Listing as SEQ ID NO
114.
An alternative Lac-B primer for use in
constructing the cpIII membrane anchor and Lacz
promotor region was Lac-B‘ as shown in Table 7. The
amplification reactions were performed as described
above with the exception that in the second PCR
amplification, Lac-B‘ was used with Lac-F instead of
Lac-B. The product from the amplification reaction is
listed in the sequence listing as SEQ ID NO 114 from
nucleotide position 1 to nucleotide position 172. The
use of Lac-B’ resulted in a LacZ region lacking 29
nucleotides on the 3' end but was functionally
equivalent to the longer fragment produced with the
Lac-F and Lac-B primers.
The products of the first and second PCR
amplifications using the primer pairs 6—3(F) and 6-
3(B) and Lac-F and Lac-B were then recombined at the
nucleotides corresponding to cpIII membrane anchor
overlap and Nhe I restriction site and subjected to a
second round of PCR using the G3-F (SEQ ID NO 107) and
Lac-B (SEQ ID NO 110) primer pair to form a recombined
PCR DNA fragment product consisting of the following:
a 5' Spe I restriction site; a cpIII DNA membrane
anchor domain beginning at the nucleotide residue
sequence which corresponds to the amino acid residue
198 of the entire mature cpIII protein; an endogenous
stop site provided by the membrane anchor at amino
acid residue number 112; a Nhe I restriction site, a
Lacz promoter, operator and Cap-binding site sequence;
and a 3' EcoR I restriction site. The recombined PCR
modified cpIII membrane anchor domain DNA fragment was
then restriction digested with Spe I and EcoR I to
produce a DNA fragment for directional ligation into a
pComb2 phagemid expression vector having only one Spe
I site prepared in Example 1a(iv) to form a pComb2—III
(also referred to as pcombz-III) phagemid expression
vector as described in Example 1b(ii).
h. Isolation of Anti-NPN Coding V" DNA Segment
To prepare modified Fd fragments for
recombination with the PCR modified cpVIII membrane
anchor domain fragment to form a Fd-cpVIII DNA fusion
product, PCR amplification as described above was
performed using Clone 2b, prepared in Example 2f, as a
template. The primers, HC3 (SEQ ID NO 105) and AK 7
(SEQ ID NO 106), the sequences of which are listed in
Table 7, were used in PCR to amplify the Fd portion of
the Clone 2b and incorporate Xho I and Spe I cloning
sites along with a cpVIII overlapping sequence. The
amplified PCR modified Fd product was purified,
electrophoresed and isolated from 1% agarose gels as
described above. The size of the Fd fragment was 680
base pairs.
i. Preparation of a DNA Segment Encoding a
Portion of the Fusion Protein Fd—cpVIII
The purified PCR modified Fd DNA fragment
containing cpVIII overlapping nucleotide sequences
prepared above was then admixed with the PCR modified
cpVIII membrane anchor domain fragment to form an
admixture. The fragments in the admixture were
allowed to recombine at their complementary regions.
The admixture containing the recombined PCR fragments
was then subjected to a second round of PCR
amplification as described above using the end primer
pair AK 6 (SEQ ID NO 104) and HC3 (SEQ ID NO 105)
(Table 7). The corresponding product of the PCR
amplification was purified and electrophoresed on
agarose gels as described above. The PCR product was
determined to be approximately 830 base pairs (Fd =
680 + 150) confirming the fusion of Fd with cpVIII.
The sequence of the PCR product linking the Fd
sequence with the cpVIII sequence in frame in a 5' to
3' direction is listed as SEQ ID NO 115. The Fd-
cpVIII fusion product was then used in directional
ligations described in Example 2j for the construction
of a pCBAK8—2b dicistronic phagemid expression vector.
j. Construction of pCBAK8-2b Dicistronic
Expression Vector
To construct a phagemid vector for the
coordinate expression of a Fd-cpVIII fusion protein
with kappa light chain, the PCR amplified Fd-cpVIII
fusion product prepared in above in Example 2i was
first ligated into Clone 2b phagemid expression vector
isolated from the NPN combinatorial library prepared
in Example 2f. For the ligation, the Fd-cpVIII PCR
fusion product was first restriction digested with Xho
I and EcoR I. Clone 2b phagemid vector was similarly
digested resulting in the removal of the cloning and
decapeptide regions. The digested Fd-cpVIII fragment
was admixed and ligated into the digested Clone 2b at
the cohesive termini generated by Xho I and EcoR I
restriction digestion. The ligation resulted in
operatively linking the nucleotide residue sequence
encoding the Fd-cpVIII polypeptide fusion protein to a
second cassette having the nucleotide residue
sequences encoding the ribosome binding site, a pelB
leader sequence and the kappa light chain already
present in Clone 2b to form a dicistronic DNA molecule
in the original Clone 2b phagemid expression vector.
E. coli, strain TG1, was then transformed with
the phagemid containing the dicistronic DNA molecule
and transformants were selected on ampicillin as the
original Clone 2b contained an ampicillin selectable
resistance marker gene. For high efficiency electro-
transformation of E. coli, a 1:100 volume of an
overnight culture of TG1 cells was inoculated into one
liter of L-broth (1% Bacto tryptone, 0.5% Bacto yeast
extract, 0.5% Nacl). The cell suspension was
maintained atilriiwith vigorous shaking to a
absorbance at 600 nm of 0.5 to 1.0. The cell
suspension in log phase growth was then harvested by
first chilling the flask on ice for 15 to 30 minutes
followed by centrifugation in a cold rotor at 4000 x g
for 15 minutes to pellet the bacteria. The resultant
supernatant was removed and the bacterial cell pellet
was resuspended in a total of one liter of cold water
to form a cell suspension. The centrifugation and
resuspension procedure was repeated two more times and
after the final centrifugation, the cell pellet was
resuspended in 20 ml of cold 10% glycerol. The
resuspended cell suspension was then centrifuged to
form a cell pellet. The resultant cell pellet was
resuspended to a final volume of 2 to 3 ml in cold 10%
glycerol resulting in a cell concentration of 1 to 3 X
” cells/ml. For the electro-transformation
procedure, 40 pl of the prepared cell suspension was
admixed with 1 to 2 ul of phagemid DNA to form a cell-
phagemid DNA admixture. The resultant admixture was
mixed and allowed to sit on ice for one minute. An
electroporation apparatus, for example a Gene Pulsar,
was set a 25 uF and 2.5 kV. The pulse controller was
set to 200 ohms. The cell—DNA admixture was
transferred to a cold 0.2 cm electroporation cuvette.
The cuvette was then placed in the chilled safety
chamber and pulsed once at the above settings. To the
pulsed admixture, 1 ml of SOC medium was then admixed
and the cells were resuspended with a Pasteur pipette
(SOC medium was prepared by admixing 2% Bacto
tryptone, 0.5% Bacto yeast extract, 10 mM Nacl, 2.5 mM
Kcl, 10 mM MgCl2, 10 mM MgSo,, and 20 mM glucose).
The cells suspension was then transferred to a 17 X
100 mm polypropylene tube and maintained at 37°C for
one hour. After the maintenance period, the
transformed TG1 cells were then plated on ampicillin
LB plates for selection of ampicillin resistant
colonies containing the phagemid which provided the
selectable marker gene.
Ampicillin resistant colonies were selected and
analyzed for the correct insert size and expression of
Fab. Briefly, DNA minipreps of selected colonies were
prepared for the isolation of phagemid DNA. The
isolated phagemid DNA from each miniprep was
restriction digested with Xho I and EcoR I and the
digests were electrophoresed on a 1% agarose gel.
Clone AK16 was selected as an 830 bp fragment was
visualized on the gels confirming the insertion of the
Fd—cpVIII PCR fusion product into digested Clone 2b.
Clone AK16 phagemid was then restriction digested
with Xho I and Xba I and the nucleotide residue
sequence of the dicistronic DNA molecule encoding the
Fd—cpVIII fusion protein, the ribosome binding site
and pelB leader sequence for expression of the light
chain, a spacer region and the 2b kappa light chain
was isolated by agarose gel electrophoresis. The
isolated dicistronic DNA fragment was then ligated
into a Xho I and Xba I restriction digested pCBAKO
expression vector prepared in Example lc(ii) to form a
dicistronic phagemid expression vector designated
pCBAK8-2b.
The resultant pCBAK8—2b expression vector
consisted of nucleotide residue sequences encoding the
following elements: fl filamentous phage origin of
replication; a chloramphenicol acetyl transferase
selectable resistance marker gene; an inducible Lacz
promoter upstream from the Lacz gene; a multiple
cloning site flanked by T3 and T7 polymerase
promoters; and the dicistronic DNA molecule (a first
cassette consisting of a ribosome binding site, a pelB
leader, and a Fd-cpVIII DNA fusion product operatively
linked to a second cassette consisting of a second
ribosome binding site, a second pelB leader, and a
kappa light chain).
k. Construction of QCBAK3-2b Dicistronic
Expression Vector
To construct a phagemid vector for the
coordinate expression of a Fd-cpIII fusion protein
with kappa light chain, the PCR amplified and
recombined cpIII membrane anchor and Lacz promoter
region fragment prepared in Example 2g having a 5' Spe
I and 3' EcoR I restriction site was first
directionally ligated into a pComb2 phagemid
expression vector previously digested with Spe I and
EcoR I prepared in Example 1a(iv) to form a pComb2—3
(also called pComb2-III) phagemid vector. See Example
1b(ii) for details of vector construction. This
vector was used in this invention when ampicillin
resistant vectors were preferred. Thus, the resultant
pcombz-3 vector, having only one Spe I restriction
site, contained separate Lacz promoter/operator
sequences for directing the separate expression of the
heavy chain (Fd)—cpIII fusion product and the light
chain protein. The expressed proteins were directed
to the periplasmic space by pelB leader sequences for
functional assembly on the membrane. Inclusion of the
phage F1 intergenic region in the vector allowed for
packaging of single stranded phagemid with the aid of
helper phage. The use of helper phage superinfection
lead to expression of two form of cpIII. Thus, normal
phage morphogenesis was perturbed by competition
between the Fab-cpIII fusion and the native cpIII of
the helper phage for incorporation into the virion as
schematically shown in Figure 8 for Fab-cpVIII
fusions.
For producing chloramphenicol resistant vectors
for use in this invention, the resultant pcombz-3
phagemid vector was then restriction digested with Sac
II and Apa I to form an isolated fragment. The
resultant isolated fragment containing the expression
control sequences and the cpIII sequence was then
directionally ligated into a similarly digested pCBAKO
phagemid vector prepared in Example 1c(ii) to form a
pCBAK3 phagemid expression vector. This vector lacked
Fd and kappa light chain sequences.
A chloramphenicol-resistant phagemid expression
vector, pCBAK3-2b, for the expression of a fusion
protein and kappa light chain was then constructed.
Briefly, the pCBAK3 phagemid expression vector
prepared above was first digested with Xho I and Spe I
to form a linearized pCBAK3 phagemid expression
vector. PCR amplified and modified Fd fragment,
prepared in Example 2h containing Xho I and Spe I
sites, was subsequently restriction digested with Xho
I and Spe I. The resultant Fd fragment was then
directionally ligated via cohesive termini into the
Xho I and Spe I restriction digested pCBAK3 phagemid
expression vector to form a second phagemid expression
vector in which the PCR modified Fd fragment was
operatively linked in—frame to nucleotide residue
sequences encoding cpIII. E. coli strain XL1-Blue
(Stratagene) was then transformed with the above
phagemid vector containing Fd-cpIII. Transformants
containing the Fd-cpIII encoding phagemid were
selected on chloramphenicol. Phagemid DNA was
isolated from chloramphenicol resistant clones and was
restriction digested with Sac I and Xba I to form a
linearized phagemid expression vector into which a Sac
I and Xba I light chain fragment prepared below was
directionally ligated.
Phagemid Clone 2b, isolated from the original
combinatorial library as described in Example 2a, was
restriction digested with Sac I and Xba I to isolate
the nucleotide residue sequence encoding the kappa
light chain. The isolated kappa light chain sequence
was then directionally ligated into the Sac I and Xba
I restriction digested phagemid expression vector
prepared above containing Fd-cpIII to form the
phagemid expression vector, pCBAK3-2b. The resultant
vector contained the nucleotide residue sequence of a
dicistronic DNA molecule for the coordinate expression
of a Fd-cpIII fusion protein with kappa light chain.
The resultant phagemid expression vector consisted of
nucleotide residue sequences encoding the following
elements: fl filamentous phage origin of replication;
a chloramphenicol acetyl transferase selectable
resistance marker gene; an inducible Lacz promoter
upstream from the Lacz gene; a multiple cloning site
flanked by T3 and T7 polymerase promoters; and the
dicistronic molecule (a first cassette consisting of a
first ribosome binding site and pelB leader
operatively linked to Fd-cpIII operatively linked to a
second cassette consisting of a second Lacz, a second
ribosome binding site, and a second pelB leader
operatively linked to a kappa light chain).
XL1—Blue cells were then transformed with the
phagemid expression vector pCBAK3—2b. Transformed
colonies containing the chloramphenicol resistant
phagemids were selected as described above and
analyzed for the correct size insert and expression of
Fab as described in Example 2j. Following
verification of the insert and expression of Fab in
the pCBAK3-2b phagemid vector, XL1-Blue cells were
then transformed and induced for the expression of Fab
antibodies as described in Examples 3 and 4.
The results of the expression, selection and
screening of the Fab—cpIII fusions revealed an
advantage of monovalent display provided by Fab—cpIII
fusions over multivalent displays provided by
Fab—cpVIII fusions as it allowed for the sorting of
clones based on affinity as well as specificity, as
does the immune system. A 253-fold enrichment of the
tight binding clone 10C over the weaker binding clone
7E using the pComb3 system as described in Barbas et
al., Proc. Natl. Acad. Sci.. USA, 88:7978-7982 (1991).
Studies with peptide libraries on phage that displayed
four to five copies of the peptide on the phage
surface have shown that multivalency prevented the
separation of phage displaying moderate affinity
peptides (10% M) from those displaying high affinity
peptides (1o‘ M). Cwirla et al., Proc. Natl. Acad.
Sci., USA, 87:6378-6382 (1990). Multivalency lead to
a chelation effect that reduces the ability to
discriminate between phage-bearing high affinity Fabs
from those bearing low affinity Fabs.
The use of the system was further demonstrated by
sorting a previously characterized (one binder per
5000 clones) human combinatorial antitetanus toxoid
Fab library as described in Example 6 and by Persson
et al., Proc. Natl. Acad. Sci.. USA, 88:2432-2436
(1991). The library, originally in a lambda phage
vector system, was reconstructed in pComb2-3 retaining
the original pairings of heavy and light chains. The
library size, 107 clones was 10-fold larger than the
original lambda phage library. After a single round
of panning, 13 or 57 clones picked were determined to
be tetanus toxoid binders which represented a 103—fold
enrichment. Following the third panning, the phage
yield had increased 200-fold, indicating enrichment of
specific phage. All the clones were thus
antigen-specific binders. Large combinatorial
libraries of 108 members are thus accessible using
this system. Even larger libraries are achieved by
mutagenesis as described in Examples 6-8.
. Expression of Anti-NPN Heterodimer on Phage
Surfaces
For expression of antibody Fab directed against
NPN on phage surfaces, XL1-Blue cells were separately
transformed with the phagemid vectors, pCBAK8-2b and
pCBAK3-2b, prepared in Examples 2j and 2k,
respectively. The transformants were selected on LB
plates containing 30 pg/ml chloramphenicol.
Antibiotic resistant colonies were selected for each
phagemid transformation and grown in liquid cultures
at 37°C in super broth (super broth was prepared by
admixing the following: 20 g 3 [N—Morpholino] propane-
sulfonic acid (MOPS); 60 g tryptone; 40 g yeast
extract; and 2 liter of water; adjust pH to 7.0 with
m NaOH) containing 30 pg/ml chloramphenicol and
12.5 pg/ml tetracycline for the respective antibiotic
selection of the phagemid and the F’ episome. The
antibiotic resistant transformed XL1-Blue cells were
diluted to an optical density (ODwmm) of 0.4 in super
broth. The inducer, isopropyl thiogalactopyranoside
(IPTG), was admixed to the bacterial suspension for a
final concentration of 1 mM and the admixture was
maintained at 37°C for 1 hour to induce the expression
of the fusion protein and kappa light chain from the
Lacz promoter. Helper phage, either R408 or VCS M13
(Stratagene), was then admixed to the induced
bacterial suspension at a ratio of 10-20 helper phage
to 1 transformed bacterial cell to initiate the
generation of copies of the sense strand of the
phagemid DNA. The admixture containing the helper
phage was then maintained for an additional two hours
at 37°C to allow for filamentous bacteriophage
assembly wherein the expressed anti-NPN Fab antibodies
fused to either bacteriophage membrane anchor domains
of cpVIII or cpIII were incorporated into surface of
the bacteriophage particles. The bacterial suspension
was then centrifuged resulting in a bacterial cell
pellet and a supernatant containing phage. The
supernatant was removed, collected and assayed as
described below for the presence of functional anti-
NPN Fab molecules anchored to the phage particles by
either cpVIII or cpIII.
. Assays for Verifying the Presence and Function of
Anti-NPN Heterodimer on the Surface of
Filamentous Phage
a. Electron Microscopy
To localize functional Fab molecules, the
binding to antigen labelled with colloidal gold was
studied. Phage containing supernatants and bacterial
cells prepared in Example 3 were spotted on formvar
(Polysciences, Inc., Warrington, Pennsylvania) coated
grids affixed onto a solid phase. In some experiments
grids were coated with cells and infected with phage
in situ. Subsequently grids were blocked with 1%
bovine serum albumin (BSA) in PBS at pH 7.2, washed
and incubated with 2-7 nanometer (nm) colloidal gold
particles coated with BSA-NPN hapten conjugate for a
time period sufficient to form a labeled
immunoreaction complex. The grids were washed to
remove excess gold particles and negatively stained in
uranylacetate and visualized by electron microscopy.
Examination of filamentous phage and permeabilized
cells producing phage revealed specific labelling of
phage or exposed bacterial membranes. Phage were
observed to contain 1 to 24 copies of antigen binding
sites per particle. Neither helper phage alone nor
intact E. coli labelled with antigen. Background
nonspecific binding was very low. Filamentous phage
particles emerging from the E. coli surfaces were
labelled with antigen as shown in Figure 9.
The generation of a related phage surface
expression vector utilizing cpIII as a fusion partner
with Clone 2b, pCBAK3-2b, revealed specific antigen
labelling to the phage head but not the column.
Additionally human anti—tetanus Fab expressed as a
cpIII fusion did not bind to BSA—NPN antigen.
b. Phage Elisa
Microtitration plates were coated with NPN-
BSA conjugate (0.1 ml, 1 pg/ml in 0.1 M Tris-HCl, pH
9.2), and blocked with 1% BSA in PBS. Serial two fold
dilutions of pCBAK8—2b derived phage (0.1 ml),
prepared in Example 3, were added to the pre-coated
microtitration plate and incubated for 3 hours at
ambient temperature or 16 hours at 4°C. The plates
were washed with PBS and goat anti-kappa alkaline
phosphatase conjugate (Fisher Biotech, Pittsburgh,
Pennsylvania) added (0.1 ml diluted 1/1000 in PBS
containing 0.1% BSA) and incubated for 2 hours at room
temperature. The plates were washed in PBS and
substrate added (0.1 ml, 1 mg/ml 9-
nitrophenylphosphate in 0.1 M Tris-HC1, pH 9.5,
containing 50 mM Mgclz). After incubation at 37°C for
signal development, the optical densities at 400 nm
were determined. Competition assays were performed
with the addition of increasing amounts of free NPN
hapten ranging from zero up to 5 mg/well.
The ELISA assays confirmed the presence of
functional antibody Fab. In a two site ELISA on NPN
antigen coated plates when probed with anti—mouse
kappa chain enzyme conjugate, phage supernatant
generated from helper phage infection of cells
carrying the pCBAK8-2b construct exhibited expected
titration curves with serial two fold dilutions of
phage containing antibody. The results of the two-
site ELISA are shown in Figure 10. For a signal to be
generated in this assay, the phage particle must (i)
have functionally associated Fd and kappa chains and
(ii) be multivalent. Specificity of the particle was
assessed by inhibiting binding to the plate in the
presence of increasing concentrations free hapten.
The generated phage particles exhibited binding to
solid phase of the ELISA and could be inhibited by
addition of hapten as shown in Figure 11. Complete
inhibition was achieved when 5 ng of free NPN hapten
was used in the assay. Helper phage did not give a
signal in the ELISA. These results show the
functional assembly of antibody heavy and light chain
polypeptides to form an epitope-binding complex that
is present on the surface of the phage particle and
able to bind the preselected ligand chapter containing
an epitope.
c. Antigen Specific Precipitation of Phage
Phage supernatant from XL1-Blue was
transformed with the pCBAK8-2b dicistronic expression
vector prepared in Example 3 (1 ml) was incubated with
BSA—NPN conjugate (10 pl, 2 mg/ml) for 18 hours at
4°C. The mixture was then pelleted by centrifugation
at 3000 rpm on a bench top centrifuge and the
appearance of precipitate noted. Helper phage was
used as a control. The pellet was washed repeatedly
in cold PBS (5 x 3 ml/wash) and then resuspended in LB
(0.5 ml). The solubilized precipitates were added to
fresh XL1-Blue cells (0.5 ml of overnight culture),
incubated for 1 hour at 37°C and aliquots plated out
on LB agar containing chloramphenicol (30 pg/ml).
Colonies were selected randomly. Colony lifts on
nitrocellulose were treated with lysozyme to digest
the cell wall, briefly treated with chloroform to
breakdown the outer membrane, blocked in BSA 1% in PBS
and incubated with E51 labelled BSA—NPN antigen.
After several washes in PBS (containing 0.05% Tween-
), film was exposed to the washed and dried filter
overnight at -70°C and the autoradiographs were then
developed.
Precipitates were obtained with antibody
containing phage but not helper phage in the presence
of BSA-NPN. In addition, the particles retained
infectivity on subsequent incubation with bacterial
cells carrying the F‘ episome and generated 4 X 105
colonies from a single solubilized precipitate.
Additionally, DNA restriction analysis was
carried out to determine the presence of heavy and
light chain inserts. DNA restriction analysis of the
clones revealed the presence of a Xho I and Xba I
fragment of 1.4 kb as expected for Fd-cpVIII fusion
construct and kappa chain insert.
These results give additional evidence for
antigen specificity and multivalency. In addition to
providing immunological parameters, this precipitation
offers possibilities for facile enrichment of antigen
specific phage particles. In principle, phage
containing specific antibodies can be highly enriched
by precipitation with antigens (which may be cell
surface markers, viral, bacterial as well as synthetic
molecules). The washed antigen-antibody precipitates
can be solubilized by the addition of excess antigen
and viable phage recovered. For the recovery of rare
species an immobilized antigen may be used which opens
the way for differential affinity elution.
In order to demonstrate the utility of
immobilized antigen for the enrichment of clones of
defined binding specificity, a panning experiment was
performed. An ampicillin resistant phagemid
expressing an anti-tetanus Fab as a cpVIII fusion was
constructed. Rescue of this clone with helper phage
produced phage encoding the ampicillin resistant
phagemid which displayed the anti-tetanus Fab on their
coat. These phage encoding tetanus specificity were
admixed with NPN hapten encoding phage (1:100) and
allowed to bind to a microtitration plate coated with
tetanus toxoid. Following a one hour maintenance
period, the plate was washed extensively and phage
were then eluted with a low pH buffer. Infection of
XL1—Blue cells in log phase growth and subsequent
plating of aliquots on ampicillin and chloramphenicol
allowed for direct quantitation of enrichment.
Examination of over 1,000 colonies showed that
ampicillin resistant colonies derived from the eluted
phage exceeded chloramphenicol resistant colonies by
27 to 1. Therefore, panning enriched the phage
displaying the anti-tetanus Fab by 2700 fold. This
result suggests that a clone of defined specificity
present at one part per million will dominate over
nonspecific clones following two rounds of panning.
. Advantages of Assembling Combinatorial Antibody
Fab Libraries Along Phage Surfaces
A powerful technique for generating libraries
with 10&9 members and selecting from the library of
combinatorial Fabs with preselected binding
activities, , is presented. In the vector described
herein, the restriction cloning sites for inserting
PCR generated antibody fragments have been retained as
previously reported for the lambda vector. The rescue
of the genes encoding the antibody Fd and kappa chains
is mediated through the utilization of the fl origin
of replication leading to the synthesis and packaging
of the positive strand of the vector on co—infection
with helper phage. Since the 'mature' virus particle
assembles by incorporating the major coat protein
around the single stranded DNA as it passes through
the inner membrane into the periplasmic space, not
only does it capture the genetic information carried
on the phagemid vector but also incorporates several
copies of functional Fab along the length of the
particle. On subsequent infection of hosts cells
carrying the F‘ episome the phagemid confers
resistance allowing selection of colonies on the
appropriate antibiotic. In essence, the antigen
recognition unit has been linked to instructions for
its production.
The full power of the earlier combinatorial
system could not be fully utilized since screening
allowed ready access to only about 0.1-1% of the
members. In the phagemid/M13 system similar size
libraries are generated and all the members are
accessed via affinity selection. Furthermore, unlike
the lambda vector which generated monovalent Fabs,
this system generates multivalent particles, thus
allowing the capture of a wider range of affinities.
The unique phagemid restriction sites permit the
recombination of Fd and kappa chains allowing chain
replacement or shuffling. The rescue of filamentous
single stranded DNA allows rapid sequencing and
analysis of the genetic make up of the clone of
interest. Indeed it can be envisaged that phage
encoding antibody specificity may be enriched by
antigen selection prior to DNA sequencing or
mutagenesis. The option to further develop an
iterative process of mutation followed by selection
may allow the rapid generation of high affinity
antibodies from germ line sequences. The process may
be automated. Setting aside the potential of the
system to mimic nature, the phagemid/M13 system would
allow a more complete dissection of the antibody
response in humans which may yield useful therapeutic
and diagnostic reagents.
The membrane anchoring of the heavy chain and the
compartmentalization of the kappa chain in the
periplasm is the key to expressing this functional
dimeric protein. The potential of this system is by
no means limited to antibodies and may be extended to
any protein recognition system or combination of
systems containing multiple members. For example
coupling of ligand and effector systems in a high
avidity matrix is now possible. In a similar vein a
library of ligands can be sorted against a library of
receptors.
. Randomized Mutaqenesis of the CDR3 Region of a
Heayy Chain Encoding Anti—Tetanus Toxoid
a. PCR Mutagenesis with Degenerate
Oligonucleotides
To obtain a mutagenized heterodimer of this
invention of altered specificity that would no longer
recognize a tetanus toxoid antigen (TT) but would
recognize and specifically bind to a new antigen, a
method was developed to randomize only the CDR3 region
of a heavy chain fragment encoded by a known
nucleotide sequence. This approach is schematically
diagrammed in Figure 12 where a representative heavy
chain fragment within a phagemid clone, consisting of
alternating framework regions (1 through 4) shown by
white blocks and complementarity determining regions
(CDR) (1 through 3) shown by cross-hatched blocks and
the first constant region (CH1), is subjected to two
separate rounds of PCR. In the first PCR
amplification reaction, the 5' end of the heavy chain
beginning at framework 1 and extending to the 3’ end
of framework 3 is amplified. In the second PCR
amplification reaction, the CDR3 region is randomly
mutagenized shown by the black box. This is
accomplished through the use of a pool of
oligonucleotide primers synthesized with a degenerate
region sandwiched between and contiguous with
conserved framework region 3 and 4 sequences. The
resulting amplification products from the second
amplification, each having a randomized CDR3 region,
have their 5' end at the 3' end of framework 3 and the
3' end of the product extends to the 3' end of the CH1
region.
The pool of degenerate oligonucleotide primers
have been designed to result in the amplification of
products having a 5' end that is complementary to and
will overlap with the 3' end of the products of the
first PCR reaction product. Thus, the two separate
PCR reaction products are pooled and subjected to a
third PCR reaction in which the overlapping region
between the two products is extended to result in
heavy chain having a randomized CDR3 region.
A heavy chain DNA template for use in this
invention was available in a clone (a phagemid vector
designated 7E containing heavy and light chain
fragments) from a human combinatorial anti-tetanus
toxoid (TT) Fab library derived from lambda Hc2 and
Lc2 libraries as described by Persson et al., gggg;
Natl. Acad. Sci., USA, 88:2432—2436 (1991). To create
a semi-synthetic combinatorial library, clone 7E was
constructed in pComb2-3' dicistronic expression vector
for the expression of a heavy chain-cpIII membrane
anchor fusion protein (Fd—cpIII) and a soluble light
chain as described for anti-NPN in Example 2k by
Barbas et al., Proc. Natl. Acad. Sci. USA,
88:7978-7982 (1991).
The phagemid vector pComb2-3' was prepared as
described in Example lb(ii). The original pairing of
the heavy and light chains from the 7E lambda clone
was maintained by ligation of the Xho I - Xba I
fragment into a Xho I - Xba I digested pComb2-3'
vector. To replace the cpIII membrane anchor
sequence, Lacz promoter sequence and pel B leader
deleted by the Xho I - Xba I digestion, a Spe I — Sac
I fragment from pComb3, as prepared in Example 1b(fi),
was ligated into the pComb2-3' vector containing the
heavy and light chain sequences from clone 7E. The
resultant phagemid clone, hereinafter referred to as
pC3—TT7E, was first expressed as described for
anti-NPN heterodimers on phage surfaces in Example 3
and subsequently screened by panning on TT—coated
plates as described for anti-NPN in Example 4c. Clone
pC3-TT7E exhibited a Kd towards TT on the order of 104
M and was enriched over nonspecific phage by 103-fold
as described by Barbas et al., su ra.
Clone pC3-TT7E, having both heavy and light chain
sequences, was used as the template DNA for the
randomized mutagenesis of the CDR3 region of the heavy
chain to alter antigen binding specificity as
described herein. The sequence of the heavy chain was
determined as described in Example 1a(ii). Two
separate PCR reactions were performed as illustrated
in Figure 12.
The first PCR reaction resulted in the
amplification of the region of the heavy chain
fragment in the pC3-TT7E clone beginning of framework
region 1 and extending to the end of framework region
3 which is located 5' to CDR3 which is approximately
400 base pairs in length. To amplify this region, the
following primer pairs were used. The 5' anti-sense
oligonucleotide primer, FT3X, having the nucleotide
sequence 5'-G-CAA-TAA-ACC-CTC-ACT—AAA-GGG—3' (SEQ ID
NO 118), hybridized to the non-coding strand of the
heavy chain corresponding to the region 5' of and
including the beginning of framework 1. The 3' sense
oligonucleotide primer, B7EFR3, having the nucleotide
sequence 5'-TCT-CGC-ACA-ATA-ATA—CAC-GGC-3' (SEQ ID NO
119), hybridized to the coding strand of the heavy
chain corresponding to the 3' end of the framework 3
region. The oligonucleotide primers were synthesized
by Research Genetics (Hunstville, AL). The PCR
reaction was performed in a 100 pl reaction containing
one pg of each of oligonucleotide primers FTX3 and
B7EFR3, 8 #1 2.5 mM dNTP's (dATP, dCTP, dGTP, dTTP), 1
pl Taq polymerase, 10 ng of template pCE-TT7E, and
10ul of 10X PCR buffer purchased commercially (Promega
Biotech). Two drops of mineral oil were placed on top
of the admixture and 35 rounds of PCR amplification in
a thermocycler were performed. The amplification
cycle consisted of denaturing at 94°C for one minute,
annealing at 50°C for one minute, followed by
extension at 72°C for two minutes. The resultant PCR
amplification products were then gel purified as
described in Example 1d and used in an overlap
extension PCR reaction with the products of the second
PCR reaction, both as described below, to recombine
the two products into reconstructed heavy chains
containing mutagenized CDR3 regions as illustrated in
Figure 12. The total yield of DNA from this
amplification was approximately 3ug/looul.
The second PCR reaction resulted in the
amplification of the heavy chain from the 3' end of
framework region 3 extending to the end of CH1 region
which is approximately 390 base pairs in length. To
amplify this region, the following primers were used.
The 5' anti-sense oligonucleotide primer pool,
designated 7ECDR3, had the nucleotide sequence
represented by the formula,
'-GTG-TAT-TAT-TGT-GCG-AGA-NNS-NNS-NNS-NNS-NNS-NNS-NNS
-NNS-NNS-NNS-NNS-NNS-NNS-NNS-NNS-NNS-TGG—GGC-CAA-GGG-A
CC-ACG-3' where N can be A, C, G, or T and S is either
C or G (SEQ ID NO 120), wherein the 5' end of the
primer pool was complementary to the 3' end of
framework 3 represented by the complementary
nucleotide sequence of the oligonucleotide primer
B73FR3 and the 3' end of the primer pool was
complementary to the 5' end of framework 4. The
region between the two specified ends of the primer
pool was represented by a 48-mer NNS degeneracy which
ultimately encoded a diverse population of mutagenized
CDR3 regions of 16 amino acid residues in length. The
3' sense oligonucleotide primer, CG1Z, as described by
Persson et al., ggpra, having the nucleotide sequence
'-GCATGTACTAGTTTTGTCACAAGATTTGGG-3' (SEQ ID NO 121),
hybridized to the coding strand of the heavy chain
corresponding to the 3' end of the CH1. The second
PCR reaction was performed on the pC3-TT7E in a 100 pl
reaction as described above containing one pg of each
of oligonucleotide primers 7ECDR3 and CG1Z. The
resultant PCR amplification products were then gel
purified as described above. The total yield of DNA
from this second PCR mutagenesis amplification was
approximately 3ug/100ul.
one hundred nanograms of gel purified products
from the first and second PCR reactions were then
admixed with 1 pg each of FTX3 and CG1Z
oligonucleotide primers as a primer pair in a final
PCR reaction to form a complete heavy chain fragment
by overlap extension as illustrated in Figure 12. The
PCR reaction admixture also contained 10 pl 10X PCR
buffer, 1 pl Taq polymerase and 8 ul 2.5 mM dNTP's as
described above. The PCR reaction was performed as
described above. To obtain sufficient quantities of
amplification product, 15 identical PCR reactions were
performed. The resulting heavy chain fragments
beginning at framework 1 and extending to the end of
CH1 and having randomly mutagenized CDR3 regions were
approximately 790 base pairs in length. The heavy
chain fragment amplification products from the 15
reactions were first pooled and then gel purified as
described above prior to their incorporation into a
phagemid library. The total yield of DNA from each
amplification was approximately 3ug/loopl thus the
total pooled yield contained approximately 45 pg of
amplified mutagenized heavy chain.
b. Phagemid Library Construction
The resultant gel purified heavy chain
fragments prepared in Example 6a were then digested
with the restriction enzymes, Xho I and Spe I, as
described in Example 2d. The resultant digested heavy
chain fragments were subsequently gel purified prior
to insertion into the pC3-TT7E phagemid vector clone
which was previously digested with the same
restriction enzymes to remove the non-mutagenized
heavy chain fragment and form a linear vector.
Ligation of 640 ng of the heavy chain Xho I Spe I
fragments having mutagenized CDR3 regions into two pg
of the linearized pC3-TT7E phagemid vector to form
circularized vectors having mutagenized CDR3 regions
was performed overnight at room temperature using 10
units of BRL ligase (Gaithersburg, MD) in BRL ligase
buffer in a reaction volume of 150 pl. Five separate
ligation reactions were performed to increase the size
of the phage library having mutagenized CDR3 regions.
Thus, the total amount of amplified mutagenized heavy
chain for five ligation reactions was 3.2 pg.
Following the ligation reactions, the circularized DNA
was precipitated at —20°C for two hours by the
admixture of 2 pl of 20 mg/ml glycogen, 15 pl of 3 M
sodium acetate at pH 5.2 and 300 pl of ethanol. DNA
was then pelleted by microcentrifugation at 4°C for 15
minutes. The DNA pellet was washed with cold 70%
ethanol and dried under vacuum. The pellet was
resuspended in 10 pl of water and transformed by
electroporation into 300 pl of E. coli XL1-Blue cells
as described in Example 2k to form a phage library.
The total yield from the mutagenesis and
transformation procedure described herein was
approximately 5 X 107 transformants. XL1-Blue cells
from E. coli were selected as the hosts as the single
step codon TAG is suppressed.
After transformation, to isolate phage on which
heterodimer expression has been induced, for
subsequent panning on target antigens such as
fluorescein, 3 ml of SOC medium (SOC was prepared by
admixture of 20 g bacto-tryptone, 5 g yeast extract
and 0.5 g NaCl in one liter of water, adjusting the pH
to 7.5 and admixing 20 ml of glucose just before use
to induce the expression of the Fd-cpIII and light
chain heterodimer) was admixed and the culture was
shaken at 220 rpm for one hour at 37°C, after which
time 10 ml of SB (SB was prepared by admixing 30 g
tryptone, 20 g yeast extract, and 10 g Mops buffer per
liter with pH adjusted to 7) containing 20 ug/ml
carbenicillin and 10 ug/ml tetracycline and the
admixture was shaken at 300 rpm for an additional
hour. This resultant admixture was admixed to 100 ml
SB containing 50 ug/ml carbenicillin and 10 ug/ml
tetracycline and shaken for one hour, after which time
helper phage VCSM13 (1o” pfu) were admixed and the
admixture was shaken for an additional two hours.
After this time, 70 ug/ml kanamycin was admixed and
maintained at 30°C overnight. The lower temperature
resulted in better heterodimer incorporation on the
surface of the phage. The supernatant was cleared by
centrifugation (4000 rpm for 15 minutes in a JA10
rotor at 4°C). Phage were precipitated by admixture
of 4% (w/v) polyethylene glycol 8000 and 3% (w/v) Nacl
and maintained on ice for 30 minutes, followed by
centrifugation (9000 rpm for 20 minutes in a JA10
rotor at 4°C). Phage pellets were resuspended in 2 ml
of PBS and microcentrifuged for three minutes to
pellet debris, transferred to fresh tubes and stored
at -20°C for subsequent screening as described below.
For determining the titering colony forming units
(cfu), phage (packaged phagemid) were diluted in SB
and 1 pl was used to infect 50 pl of fresh (OD600 = 1)
E. coli XLI-Blue cells grown in SE containing 10 ug/ml
tetracycline. Phage and cells were maintained at room
temperature for 15 minutes and then directly plated on
LB/carbenicillin plates.
c. Selection of Anti-Fluorescein Heterodimers
on Phage Surfaces
1) Multiple Pannings of the Phage Library
Having Mutagenized CDR3 Regions
The phage library produced in Example
6b having heavy chain fragments with mutagenized CDR3
regions was panned as described herein on a microtiter
plate coated with a 50 pg/ml fluorescein—BSA conjugate
to screen for anti-fluorescein heterodimers.
Fluorescein was conjugated to BSA according to the
methods described in "Antibodies: A Laboratory
Manual", eds Harlow et al., Cold Spring Harbor
Laboratory, 1988.
The panning procedure used was a modification of
that originally described by Parmley and Smith
(Parmley et al., Qggg, 73:30—5-318). Two to four
wells of a microtiter plate (Costar 3690) were coated
overnight at 4°C with 25 pl of 50 pg/ml TT antigen
prepared above in 0.1 M bicarbonate, pH 8.6. The
wells were washed twice with water and blocked by
completely filling the well with 3% (w/v) BSA in PBS
and maintaining the plate at 37°C for one hour. After
the blocking solution was shaken out, 50 pl of the
phage library prepared above (typically 10” cfu) were
admixed to each well, and the plate was maintained for
two hours at 37°C.
Phage were removed and the plate was washed once
with water. Each well was then washed ten times with
TBS/Tween (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5%
Tween 20) over a period of one hour at room
temperature where the washing consisted of pipetting
up and down to wash the well, each time allowing the
well to remain completely filled with TBS/Tween
between washings. The plate was washed once more with
distilled water and adherent phage were eluted by the
addition of 50 pl of elution buffer (0.1 M HCl,
adjusted to pH 2.2 with solid glycine, containing 1
mg/ml BSA) to each well followed by maintenance at
room temperature for 10 minutes. The elution buffer
was pipetted up and down several times, removed, and
neutralized with 3 pl of 2 M Tris base per 50 pl of
elution buffer used.
Eluted phage were used to infect 2 ml of fresh
(OD“m = 1) E. coli XL1-Blue cells for 15 minutes at
room temperature, after which time 10 ml of SB
containing 20 ug/ml carbenicillin and 10 ug/ml
tetracycline was admixed. Aliquots of 20, 10, and
1/10 ul were removed from the culture for plating to
determine the number of phage (packaged phagemids)
that were eluted from the plate. The culture was
shaken for one hour at 37°C, after which it was added
to 100 ml of SB containing 50 pg/ml carbenicillin and
pg/ml tetracycline and shaken for one hour. Helper
phage VCSM13 (1o” pfu) were then added and the
culture was shaken for an additional two hours. After
this time, 70 pg/ml kanamycin was added and the
culture was incubated at 37°C overnight. Phage
preparation and further panning were repeated as
described above.
Following each round of panning, the percentage
yield of phage were determined, where % yield -
(number of phage eluted/number of phage applied) X
100. The initial phage input ratio was determined by
titering on selective plates, as described in Example
6b, to be approximately 10" cfu for each round of
panning. The final phage output ratio was determined
by infecting two ml of logarithmic phase XL1—B1ue
cells as described above and plating aliquots on
selective plates.
As an alternative to elution with acid, phage
bound to the wells of the microtiter plate were eluted
by admixing 50 pl of a solution of 1045M fluorescein
diluted in PBS followed by a maintenance period of one
hour at 37°C. The solution was then pipetted up and
down to wash the wells. The resultant eluate was
transferred to 2 ml of fresh E. coli XLI—Blue cells
for infection as described above for preparing phage
and further panning. In subsequent rounds of panning,
phage were eluted with 10* M fluorescein.
The results of the amount of phage that were
specifically bound to fluorescein-coated wells over
four consecutive rounds of panning and elution with
acid or with fluorescein alone are shown below in
Table 8. Comparable yields of phage on which
heterodimers were expressed that bound specifically to
fluorescein were achieved with either elution
protocol. These data confirm that mutagenesis of the
CDR3 region as described in this invention resulted in
the altering of a heterodimer which initially
specifically bound to TT to one that specifically
bound fluorescein.
Table 8
Phage Eluted
Acid Elution Fluorescein Elution
round 1 5.6 x 105/well 4.7 x 105/well
round 2 4.6 X 106/well 5.6 x 105/well
round 3 3.8 x 105/well 1.4 x 10°/well
round 4 1.3 x 106/well 4.0 x 106/well
Nonspecific binding to this surface with control
phase varied between 10‘ and 105 phage per well.
Production of soluble Fab and verification of binding
to fluorescein by ELISA as described in 2) below
revealed 8 reactive clones of 60 randomly selected
from the transformed colonies and 38 reactive clones
of 40 randomly selected from the transformed colonies
for the acid eluted and fluorescein eluted libraries,
respectively.
) Preparation of Soluble Heterodimers for
Characterizing Binding Specificitv to
Fluorescein
In order to further characterize the
specificity of the mutagenized heterodimers expressed
on the surface of phage as described above, soluble
Fab heterodimers from both acid eluted and fluorescein
eluted phage were prepared and analyzed in ELISA
assays on fluorescein—coated plates, by competitive
ELISA with increasing concentrations of soluble
fluorescein-BSA and also by fluorescence quenching
assays. The latter assays were performed as described
in "Fluorescein Hapten: An Immunological Probe", ed
E. w. Voss, CRC Press, Inc. pp 52-54, 1984.
To prepare soluble heterodimers, phagemid DNA
from positive clones was isolated and digested with
Spe I and Nhe I. Digestion with these enzymes
produced compatible cohesive ends. The 4.7-kb DNA
fragment lacking the gene III portion was gel-purified
(0.6% agarose) and self-ligated. Transformation of E;
coli XL1-Blue afforded the isolation of recombinants
lacking the cpIII fragment. Clones were examined for
removal of the cpIII fragment by Xho I - Xba I
digestion, which should yield an 1.6-kb fragment.
Clones were grown in 100 ml SB containing 50 pg/ml
carbenicillin and 20 mM Mgclz at 37°C until an 0Dwo of
0.2 was achieved. IPTG (1 mM) was added and the
culture grown overnight at 30°C (growth at 37°C
provides only a light reduction in heterodimer yield).
Cells were pelleted by centrifugation at 4000 rpm for
minutes in a JA10 rotor at 4°C. Cells were
resuspended in 4 ml PBS containing 34 pg/ml
phenylmethylsulfonyl fluoride (PMSF) and lysed by
sonication on ice (2-4 minutes at 50% duty). Debris
was pelleted by centrifugation at 14,000 rpm in a JA20
rotor at 4°C for 15 minutes. The supernatant was used
directly for ELISA analysis as described below and was
stored at -20°C. For the study of a large number of
clones, 10ml cultures provided sufficient heterodimer
for analysis. In this case, sonications were
performed in 2 ml of buffer.
The soluble heterodimers prepared above were
assayed by ELISA. For this assay, 1 pg/well of
fluorescein—BSA solution was admixed to individual
wells of a microtiter plate and maintained at 4°C
overnight to allow the protein solution to adhere to
the walls of the well. After the maintenance period,
the wells were washed one time with PBS and thereafter
maintained with a solution of 3% BSA to block
nonspecific sites on the wells. The plates were
maintained at 37°C for one hour after which time the
plates were inverted and shaken to remove the BSA
solution. Soluble heterodimers prepared above were
then admixed to each well and maintained at 37°C for
one hour to form immunoreaction products. Following
the maintenance period, the wells were washed ten
times with PBS to remove unbound soluble antibody and
then maintained with a secondary goat anti-human FAB
conjugated to alkaline phosphatase diluted in PBS
containing 1% BSA. The wells were maintained at 37°C
for one hour after which the wells were washed ten
times with PBS followed by development with
p-nitrophenyl phosphate (PNPP).
Immunoreactive heterodimers as determined in the
above ELISA were then analyzed by competition ELISA to
determine the affinity of the mutagenized
heterodimers. The ELISA was performed as described
above with increasing concentrations of soluble
fluorescein—BSA ranging in concentration from 104 M
up to 105 M in concentration admixed in the presence
of the soluble heterodimers. Maximal inhibition of
binding was achieved at a concentration of 10* M free
antigen with a half-maximal inhibition obtained with
approximately 104 M free antigen. Antibodies
expressed from all clones had approximate dissociation
constants (Kd) in the range of 104 to 10% M for
fluorescein-BSA conjugates for both the fluorescein
and acid elutions. True Kd's were determined in
fluorescence quenching assays. Antibodies expressed
on phage from clones following fluorescein elution had
higher affinities for fluorescein, 104 M versus 10* M
for the acid eluted antibodies. The parent clone, 7E,
showed no quenching within detectable limits of the
assay suggesting an affinity for free fluorescein less
than 105 M. The affinities of the tightest binders
of antibodies to fluorescein (104 M) approached the
average Kd of the secondary response of immunized mice
for free fluorescein (104 M) as shown by Kranz et
al., Mol. Immunol., 20:1313—1322 (1983).
Thus, the mutagenized heterodimers of this
invention specifically recognized and bound to
fluorescein. Additional experiments were performed to
confirm that the mutagenized heterodimers no longer
recognized the TT to which the nonmutagenized
heterodimer originally bound. Fluorescence quenching
assays were also performed to confirm the specificity
of binding of the mutagenized heterodimers. Soluble
heterodimers prepared from phage that were either
eluted with acid or with fluorescein alone were
equally effective at binding fluorescein by any of the
aforementioned approaches. The invention of
mutagenesis of the CDR3 region of the heavy chain of a
heterodimer described herein thus resulted in the
alteration of binding specificity from TT to
fluorescein.
d. Sequence Analysis of Selected
Anti—Fluorescein Heterodimers
The complete nucleotide sequence of the
mutated heavy chain of a representative number of
fluorescein—BSA binding clones was determined. No PCR
induced mutations outside of the CDR3 region were
observed. The predicted amino acid sequences of the
heavy chain CDR3 region are shown in Figure 13 with
the corresponding SEQ ID NO shown in parenthesis.
Seven clones recovered from the acid elution regimen
showed no consensus sequence. The lack of consensus
behavior in the acid eluted clones may be contributed
by their recognition of a more complex epitope
consisting of fluorescein and BSA and is reflected in
the more disparate affinities to fluorescein and
fluorescein-BSA.
Conversely, the clones isolated by fluorescein
elution showed a high selection of consensus
sequences. Of ten clones sequenced, only three
different sequences were observed. All of the clones
had a glycine residue at amino acid residue position
95 and an aspartic acid residue at position 101. The
amino acid residue positions are based on the Kabat
numbering system as described by Kabat et al., in
"Sequences of Proteins of Immunological Interest, U.S.
Department of Health and Human Services, (1987). Both
possible codons for encoding the glycine residue
provided by the synthesis protocol in which all 32
possible codons were produced were used. In natural
antibodies, the aspartic acid residue at position 101
usually plays a structural role by forming a salt
bridge with an arginine residue at position 94 in
framework region 3 (FR3). Thus, the artificial
selection process had recapitulated an interaction of
structural significance which mirrors that seen in the
animal.
In addition, nine of the semi-synthetic
antibodies expressed from ten clones contained a
serine-arginine-proline triplet sequence near the
center of the loop directly adjacent or one residue
removed on the amino~terminal side of an arginine
residue, though the codon usage for two of these
residues was different. Clone F31 lacked this central
motif. All sequences were rich in arginine residues
which was encoded by three of the 32 possible codons.
Comparison of the occurrence within the ten difference
CDR3 sequences of arginine with leucine and serine,
which were also encoded by three possible codons in
the synthesis, revealed a arginine-leucine-serine
ratio of 29:16:15. This bias towards selection of
arginine may be the result of the dianionic character
of fluorescein.
The finding that different codons were used
support the proposition that clonal selection occurred
at the level of antigen—antibody union and not because
of some unexpected bias of nucleotide incorporation
into DNA.
. Randomized Mutagenesis of the CDR3 Region of a
Light Chain Encoding Anti-Tetanus Toxoid
a. PCR Mutagenesis with Degenerate
Oligonucleotides
Following a similar procedure to the random
mutagenesis of the heavy chain as described in Example
6, the CDR3 region of a light chain fragment of the
anti-tetanus toxoid specific phagemid clone pC3-TT7E
was randomized to produce antibodies having a
specificity for fluorescein. The PCR amplifications
were performed as described in Example 6a with the
exception of the oligonucleotides primers used in the
reactions.
The first PCR reaction resulted in the
amplification of the region of the light chain
fragment in the pC3-TT7E clone from the 5' EcoR V site
of the vector extending into the 5' end of the CDR3
region. To amplify this region, the following primers
were used: The 5' anti-sense oligonucleotide primer,
KEF, having the nucleotide sequence
'-GAATTCTAAACTAGCTAGTCG-3' (SEQ ID NO 126),
hybridized to the non-coding strand of the light chain
corresponding to the EcoR V site in the vector; The
3' sense oligonucleotide primer, KV12B, having the
nucleotide sequence 5'-ATACTGCTGACAGTAATACAC-3' (SEQ
ID NO 127), hybridized to the coding strand of the
heavy chain corresponding to the 5‘ end of CDR3. The
PCR amplification was performed as described in
Example 6a. The resultant PCR products were then gel
purified as described in Example 1d and used in an
overlap extension PCR reaction with the products of
the second PCR reaction, both as described below, to
recombine the two products into reconstructed heavy
chains containing mutagenized CDR3 regions as
illustrated in Figure 12.
The second PCR reaction resulted in the
amplification of the light chain from the 5' end of
the CDR3 region extending to the end of the CH1
region. To amplify this region, the following primers
were used. The 5' anti-sense oligonucleotide primer
pool, designated KV5R, had the nucleotide sequence
represented by the formula,
'-TATTACTGTCAGCAGTATNNKNNKNNKNNKNNKACTTTCGGCGGAGGGAC
C-3' (SEQ ID NO 128) where N can be A, C, G or T and
where K is either G or T, wherein the 5' end of the
primer pool was complementary to the 5' end of CDR3
and the 3' end of the primer pool was complementary to
the 3' end of CDR3 and the 5' end of framework 4. The
region between the two specified ends of the primer
pool was represented by a 15-mer degeneracy which
ultimately encoded a diverse population of internal
mutagenized CDR3 regions of 5 amino acids in length
bordered by non-mutagenized 5' and 3' ends of the CDR3
regions. The 3' sense oligonucleotide primer, T7B,
having the nucleotide sequence
'-AATACGACTCACTATAGGGCG-3" (SEQ ID NO 129),
hybridized to the coding strand of the light chain
corresponding to the T7 region in the vector. The
second PCR reaction was performed on the pC3-TT7E as
described in Example 6a with the KVSR and T7B primers.
The resultant PCR amplification products were then gel
purified as described above.
Five hundred nanograms of gel purified products
from the first and second PCR reactions were then
admixed with 1 pg each of KEF and T7B oligonucleotide
primers as a primer pair in a final PCR reaction to
form a complete light chain fragment by overlap
extension as illustrated in Figure 12. The PCR
amplification was performed as described in Example ‘
6a. To obtain sufficient quantities of amplification
product, 5 identical PCR reactions were performed.
The resulting light chain fragments, that began at the
' EcoR V site and extended to the T7 region, had five
internal amino acids randomly mutagenized CDR3.
b. Phagemid Library Construction
The resultant gel purified light chain
fragments prepared in Example 7a were then digested
with the restriction enzymes, Sac I and Xba I, as
described in Example 2d. The resultant light chain
fragments were subsequently gel purified prior to
ligation into the pC3—TT7E phagemid vector clone which
was previously digested with the same restriction
enzymes to remove the non—mutagenized light chain
fragment and form a linearized vector. Ligation of
450 ng of light chain amplification products into 1.4
pg linearized pC3—TT7E phagemid vector to form
circularized vectors having mutagenized CDR3 regions
was performed as described in Example 6b. Five
separate ligation reactions were performed to increase
the size of the phage library having internally
mutagenized CDR3 regions. Following the ligation
reactions, the circularized DNA was precipitated and
transformed into E. coli XLI-Blue as described in
Example 6b to form a phage library. The total yield
from the mutagenesis and transformation procedure
described herein was approximately 2 X 107
transformants. Phage were isolated as described for
the heavy chain mutagenesis transformants.
c. Selection of Anti—Fluorescein Heterodimers
on Phage Surfaces and Seggence Analysis of
Selected Antibodies
The phage library produced in Example 7b
having light chain fragments with five internally
mutagenized amino acids in the CDR3 region was panned
as described in Example 6c. The numbers of phage that
were specifically bound to fluorescein-coated wells
over three consecutive rounds of panning and hapten
elution with a phage input of 10” were 0.75 X 106, 1 X
106 and 2.4 X 107. The repeated cycles of
transformation, phage preparation, panning and elution
thus resulted in a significant enrichment of
heterodimers that specifically bound to fluorescein.
Soluble Fabs were prepared as described in Example
6b2) for characterizing binding specificity to
fluorescein. Seven clones were selected for sequence
analysis as described in Example 6d. The results of
the sequence analysis are shown in Figure 14. The
mutated region of the light chain CDR3 spans Kabat
amino acid immunoglobulin light chain positions from
92 to 96. The sequence of this region from the
starting clone, pC3-TT7E, was Gly-Ser-Ser-Leu-Trp (SEQ
ID NO 148). of the seven antibodies mutated and
selected on fluorescein, five of them from clones P2,
P21, P23, P28 and P19 had the amino acid sequence
Thr—Arg—Pro-Gly-Val (SEQ ID NO 149) but each were the
result of translations from unique nucleotide
sequences. The two remaining antibodies from P15 and
P11 clones, derived from unique nucleotide sequences,
also had unique amino acid sequences. Thus, since
most of the mutagenized light chains had the same
amino acid sequence encoded by the possible codons in
the synthesis, the artificial selection process has
recapitulated an interaction of structural
significance which in the animal resulted from the
process of natural selection.
Mutagenesis of the CDR regions is not limited to
the CDR3 region as primers can be designed to result
in the random mutagenesis of CDR1 and CDR2 of both the
heavy and the light chain. Mutating all six CDR
regions would result in an exceptionally diverse
library beyond that which can be obtained in the
animal. To obtain randomization in all the CDRs, the
heavy chain CDRs could first be randomized from a
starting clone followed by selection of the best
antibody binders. A second mutagenesis step on the
CDRs of the light chain could be performed and the
resultant library can be mixed with the selected heavy
chain binders. Alternatively, all CDRs could be
simultaneously randomized resulting in heavy and light
chain libraries which are then combined and subjected
to selection against a preselected antigen.
Thus, the Examples 6 and 7 illustrate a method
of relevance to the present invention for mutagenizing
the heavy and light complementarity determining
regions (CDR) of an immunoglobulin gene, and also
illustrates oligonucleotides useful therefor.
. In Vitro Selection and Affinity Maturation of
Antibodies from a Naive Combinatorial
Immunoqlobulin Library
A combinatorial immunoglobulin library approach
has been used to obtain monoclonal antibodies from
non-immune adult mice, thereby establishing the
principles of (i) accessing naive combinatorial
antibody libraries for predetermined specificities and
(ii) increasing the affinity of the selected antibody
binding sites by random mutagenesis. A combinatorial
Fab library expressing Igp and k light chain fragments
on the surface of filamentous phage was prepared from
bone marrow of non-immunized, adult Balb/c mice with
the multivalent display vector pComb8 prepared in
Example lb(i). Phage displaying low affinity Fab's
having binding constants of approximately 10‘ to 105
M” specific for progesterone were isolated from the
library by their ability to bind the hapten. Random
mutagenesis of the heavy and light chain variable
regions expressed in the monovalent phage display
vector pComb3 was performed by error-prone PCR.
Clones with improved affinity for progesterone were
subsequently selected. Thus, as described herein,
antibodies with desirable characteristics from a non-
immune source were selected and affinity maturation
achieved by using the twin vectors pComb8 and pComb3,
thus opening the route to obtaining specific
antibodies from a generic library and bypassing
immunization.
The invention described herein has three
essential features: (i) the ability to initially
access low affinity Fabs from a naive library by the
use of multivalent phage expression systems, (ii)
subsequent affinity maturation by error-prone PCR and
(iii) the use of a single chain construct during the
maturation process to avoid a high background of
artifactual binding due to loss of the light chain.
When used in concert, these methods allowed for the
selection and affinity maturation of antibodies from a
naive library.
a. RNA Isolation and cDNA Svnthesis
Three non-immunized adult male (6 months)
Balb/cByJ mice (Scripps breeding colony) were used to
prepare 5 x 107 bone marrow cells in 4% fetal calf
serum in PBS. To deplete for surface IgG positive
cells, the preparation was maintained with rat-anti
mouse IgG% (0.1 ml), goat anti-mouse IgG (0.1 ml),
and rabbit anti-mouse IgG% (0.1 ml) for 30 minutes at
ambient temperature. The cells were pelleted, washed
with PBS and resuspended in 9 ml PBS. Rabbit
complement was added (1 ml) and maintained at 37°C for
minutes. The cells were pelleted and total RNA
isolated as described in Example 2b. The total RNA
was used as a template for the cDNA synthesis for p-
and k-chains with the following primers: Igp, 5'-
ATTGGGACTAGTTTCTGCGACAGCTGGAAT-3' (SEQ ID NO 151) (the
Spe I restriction site sequence is underlined) and k,
'-GCGCCGTCTAGAATTAACACTCATTCCTGTTGAA-3' (SEQ ID NO
152) (the Xba I restriction site is underlined)
respectively, using Superscript Kit (BRL).
Briefly, 7 pg of total RNA was admixed with 60
pmol of primer, heated to 70°C for 10 minutes and
immediately cooled on ice. Two pl of RNase inhibitor,
pl of 5x synthesis buffer, 8 pl of dNTP mix (to
give final concentration of 200 pM of each NTP), 5 pl
of 0.1 M DTT, and 1 pl of BRL Superscript RT (200
U/pl) were admixed, and the reaction was made up to 50
pl with DEPC treated water. The reaction was allowed
to proceed at room temperature for 10 minutes and then
at 42°C for 50 minutes. The reaction was terminated
by maintaining at 90°C for 5 minutes and then placing
on ice for 10 minutes followed by admixing 1 pl of
RNase H and maintaining at 37°C for 20 minutes. PCR
amplification was performed in a 100 pl reaction
mixture as described in Example 2, using V“ 1-9 and
the p chain primer for the heavy chains and VL3-7 and
the k chain primers for the light chains as shown in
Table 5.
b. Naive Immunoglobulin p[k Library
Construction
The PCR amplified p-chain and k-chain DNA
were cleaved with Xho I-Spe I and Sac I-Xba I,
respectively. The resulting p chain Xho I—Spe I
fragments were inserted into the pComb8 phagemid
vector prepared in Example 1b(i) to generate a u
chain-cp VIII fusion library. Transformation into E;
ggli XL1-Blue and phage production was carried out
essentially as described in Example 6. Subsequently,
the k light chain Sac I-Xba I fragments were cloned
into the heavy chain Fd p-cpVIII fusion library.
A combinatorial library of 5 x 105 members was
established by subsequently cloning the Igu Fd and k
light chain fragments into the pComb8 vector which
allowed the fusion of the heavy chain Pd fragment to
cpVIII. Since the Fab antibody fragments were
displayed at a high copy number on the phage surface,
this vector was selected for accessing low affinity
antibodies which are expected to be found in an
unselected and unprimed repertoire.
c. Selection of Low Affinity Antibodies
Specific for Progesterone
The recombinant phagemids prepared above
were packaged into M13 phage particles and five rounds
of panning on progesterone(O-Carboxymethyl)-oxime—
BSA coated ELISA wells was performed as described in
Example 6. ’Briefly, wells of a microtitration plate
were coated at 4°C with 50 pl of 100 ug/ml
progesterone(0—Carboxymethyl)-oxime BSA conjugate
(Sigma #P4778) in PBS. The wells were washed twice
with water and blocked by completely filling with 1%
w/v BSA in PBS and maintaining the plates at 37°C for
one hour. Blocking solution was flicked out and 50 ul
of the phage library (typically 10” cfu) in PBS-BSA
(0.1% w/v) were admixed to each well and the plates
maintained for two hours at 37°C. The washing steps,
elution, and multiplication of the phage were done
essentially as described in Example 6a1).
Phage eluted after the first and third round were
analyzed for expression of anti-progesterone Fab's by
bacterial colony lifts as described in Example 2f.
The colonies were probed for progesterone binding with
a progesterone(O—Carboxymethyl)-oxime HRP
conjugate. The filters were developed using 4-
chloronaphthol. Care was taken to exclude artifacts
caused by phagemids expressing the Igp Fd cpVIII
fusion without a corresponding light chain. These
heavy chain-only phages reacted nonspecifically to
unrelated antigens such as BSA, HRP, hen egg lysozyme,
presumably due to the hydrophobic patch displayed on
an unpaired heavy chain.
Those colonies producing the strongest signal in
the western blot were further examined, and three
clones, PgA11, PgB6, and PgF1, were isolated for
subsequent analysis. The first two emerged from the
first round of panning, and the latter was isolated
after the third round of selection. All three Fab's,
produced in their soluble form as described in Example
6c2) bound specifically to progesterone(O-
Carboxymethyl)-oxime-BSA and progesterone-11a-
hemisuccinyl—BSA. Additionally, all three Fab's
displayed a significant crossreactivity against an
epitope on cytochrome C. Their apparent binding
constants for progesterone—3—(0-carboxymethyl)-oxime-
BSA were determined as 10‘ M4 for PgA11, and 3 x 10‘
M4 and 105 M” for PgF1 and PgB6, respectively. These
binding constants were much below that reported for
anti-progesterone monoclonal antibodies with
affinities of 2 to 5 x 108 M4. Clones PgB6 and PgF1
utilized the same combination of closely related V"
and VL genes. Both of their V" genes were identical
to two closely related but distinct germline genes
with no evidence of somatic mutation, as one would
expect for a naive repertoire. The true germline
gene(s) for their closely related VL genes are not yet
known. Since both the V, genes have joined to
different D and J segments, the clones PgB6 and PgF1
cannot be of the same origin, but must have been
selected from two independent cloning events, pointing
towards a possible importance of this particular
combination for progesterone binding. The VL and V"
genes used by PgA1l are not closely related to the
other two clones.
Thus, this demonstrated that by using a
multivalent display vector, Fab's can be isolated from
naive combinatorial libraries with affinities
comparable to those observed for the primary immune
response to haptens, such as phosphorylcholine and
nitrophenol. Further, the combinatorial library
approach can yield V genes or V gene combinations
which would not have been selected in vivo.
d. Affinitv Maturation bv PCR Directed
Mutagenesis
To mimic the process of somatic mutation
which leads to the selection of antibodies with a
higher affinity, random mutations were created in both
the V, and V, regions and antibodies were subsequently
selected with an increased affinity to the hapten
progesterone. In order to target the mutations by
error-prone PCR specifically and only to the V
regions, a single chain fusion plasmid Fv-cpIII was
constructed in a pComb2-3 phagemid vector which
contained in frame fusions of the following elements:
the pelB leader sequence for secretion, the V“ and VL
reading frames linked with a synthetic oligonucleotide
coding for a flexible peptide as discussed in Example
6a, and the cpIII moiety. The use of a single chain
vector further overcomes the difficulties due to
unwanted selection for nonspecific binding by phage
which express Ig heavy chains only.
For preparing a single—chain heavy and light
chain (designated FQ fusion with the membrane anchor
cpIII, the plasmid pComb2-3 prepared in Example 1b(ii)
having only one Spe I restriction site, was digested
with the restriction endonucleases Xba I and Nhe I and
religated, thus eliminating the light chain cloning
cassette. A synthetic DNA linker which encodes a 15
amino acid linker sequence consisting of two
oligonucleotides, a 5' anti-sense primer having the
nucleotide sequence 5'-
TCGAGAAAGTCTCTAGAGGTAAATCTTCTGGTTCTGGTTCCGAATCTAAATCTA
CTGAGCTCAAAGTCA-3' (SEQ ID NO 153) and a 3' sense
primer having the nucleotide sequence 5'-
CTAGTGACTTTGAGCTCAGTAGATTTAGATTCGGAACCAGAACCAGAAGATTTA
CCTCTAGAGACTTTC-3 (SEQ ID NO 154) was inserted into
the Xho I-Spe I digested, truncated pComb2-3 vector
forming the phagemid Scpcombz-3. The internal
recognition sequence for restriction endonucleases Xba
I (TCTAGA) and Sac I (GAGCTC) are underlined. The V"
and VL segments of the progesterone binders were then
amplified by PCR as described in Example 2g in two
separate reactions.
In the first PCR amplification, the primer
corresponding to SEQ ID NO 153 listed above and the
oligonucleotide having the nucleotide sequence 5'-
ATTTGGGAAGGACTGTCTAGATGMRGAGAC-3', (SEQ ID NO 155),
where M is either A or C and R is either A or G, were
used to amplify the heavy chain fragment. The light
chain fragment was separately amplified with the
primer corresponding SEQ ID NO 154 listed above and
the oligonucleotide having the nucleotide sequence 5'-
GAGGACTAGTTACAGTTGGTGCAGCATCAG-3‘ (SEQ ID NO 156. The
internal recognition sequences for Xba I (TCTAGA) and
Spe I (ACTAGT) are underlined. The V“ and VL PCR
fragments were digested with Xho I-Xba I and Sac I-Spe
I, respectively, and subsequently inserted into Scp-
Comb2-3.
e. Expression and Detection of Soluble Fabs and
Single Chain Fusion Antibodies Specific for
Progesterone
For Fab production, the gene VIII moiety in
the phagemids encoding the progesterone binders PgA11,
PgB6, and PgF1 was excised with restriction
endonucleases Spe I and EcoR I and subsequently
replaced by a synthetic linker encoding a TAA stop
codon (underlined). The linker was formed by the
oligonucleotides 5'—CTAGT1AACTGAGTAAG-3' (SEQ ID NO
157) and 5'AATTCTTACTCAGIIAA-3' (SEQ ID NO 158). The
production and detection of antibody Fab fragment was
performed essentially as described in Example 6c2),
except that the E. coli cells were disrupted by three
freeze thaw cycles. For producing soluble antibody
fragments, the Vw—linker-VL fusions were excised from
the ScpComb2—3 phagemid with Xho I and Spe I and
subcloned into the expression vector pTAC01
(Pharmacia) which is a derivative of pF1260. The
pTAC01 vector has the inducible tac promoter, the pelB
leader sequence for secretion and allowed for in-frame
fusion of the inserted single chain fusion construct
with a decapeptide sequence as described in Example 1a
as a tag for immunochemical detection. Expression and
detection of the single chain fusion antibody
fragments was as described above, except that an anti-
decapeptide antibody conjugated to alkaline
phosphatase was used for the ELISA.
Three single chain fusion clones were selected by
the screening protocol and designated ScpComb2PgFl,
-PgB6 and -PgA11. These resultant plasmids were
subjected to error—prone PCR mutagenesis as described
below.
f. Targeted Mutagenesis of the Heayy and Light
chains by Error—Prone PCR
Equal amounts of undigested PgF1, PgB6, and
PgA11 Scpcombz-3 plasmids prepared above were admixed
and serially diluted. Aliquots of 100 ng, 10 ng, 1
ng, 0.1 ng, and 0.01 ng of the admixtures were
subjected separately to 35 cycles (1 minute at 94°C, 2
minutes at 50°C, 1 minute at 72°C) of amplification
under the following reaction conditions: 50 mM KCl, 10
mM Tris-Hcl (pH 9.0), 6.5 mM MgCl2, 0.5 mM MnClZ,
0.01% gelatin, 0.1% Triton X-100, 1 mM each dCTP,
dGTP, dTTP, 0.2 mM dATP, 0.1 mM dITP, using the M 13
reverse sequencing primer, 5'—AACAGCTATGACCATG-3' (SEQ
ID No 159), and a backward primer complementary to the
cpIII moiety, 5'-GACAGGAGGTTGAGGCAGGT-3' (SEQ ID NO
160) at 100 uM. The basic method of error-prone PCR
was as originally described by Leung et al.,
J. Methods Cell. Mol. Biol., 1:11-15 (1989). The
DNA region to be mutagenized was PCR
amplified under conditions that
reduced the fidelity of DNA synthesis by Taq DNA
polymerase. As shown by Leung et al., su ra, the
concentrations of the reagents Muclz and dATP used in
the PCR amplifications resulted in 1.0% and 1.4%
mutation frequency, respectively. Thus, the frequency
of mutation increased as the dATP concentration
decreased.
production and panning were carried out as described
in Example 6, except that the phage were panned in the
absence of BSA.
Thus, a library of mutated anti—progesterone
single chain phagemid antibodies was established and
panned on progesterone—3-(0-Carboxymethyl)-oxime-BSA
and the number of eluted cfu was taken as a measure
for the relative affinity of the displayed single
chain antibodies to the hapten. After the third round
of panning a 50 to 100-fold increase in the yield of
eluted phagemids relative to the non-mutated
population was noted. Individual mutants showed a 10
to 300—fold increase in yield after panning as
compared to the parent clones, indicating that the
mutants encoded antibody binding sites with an
increased affinity. The four best mutants, designated
ScPgB6-1, -2, -3 and -4, were chosen for determination
of their affinity for the hapten-conjugate and
sequence analysis.
g. Determination of Affinity of Mutagenized
Single Chain Fusion Anti-Progesterone
Antibodies
The binding constants of the soluble
antibody fragments prepared form the four best
mutants, ScPgB6—1, -2, -3 and -4 chosen above were
determined by competitive ELISA as described in
Example 6c2). Briefly, wells of a microtitration
plate were coated at 4°C with 50 pl of 100 pg/ml
progesterone(O—Carboxymethyl)-oxime-BSA conjugate
in PBS. The wells were washed twice with water and
blocked with 1% w/v BSA in PBS at 37°C for one hour.
Fab or single chain fusion supernatants were mixed
with progesterone(O-Carboxymethyl)-oxime-BSA in
PBS—BSA (0.1% w/v), and maintained in the wells at
37°C for two hours. The plates were washed with PBS-
Tween (0.05% V/V), and goat anti-mouse k-chain
alkaline phosphatase conjugate (Southern Biotech) or
mouse anti-decapeptide monoclonal antibodies
conjugated to alkaline phosphatase was admixed and
maintained for one hour at 37°C. The plates were
washed as before and substrate was admixed (0.1 ml, p-
nitrophenyl phosphate at 1 mg/ml in 0.1 M Tris, pH 9.4
containing 50 mM MgCl2). After maintenance at 25°C
for 60-180 minutes, the absorbance was read at 405 nm.
Apparent affinities were determined as the reciprocal
of the hapten concentration required to inhibit 50% of
the maximal binding in a competitive ELISA. This was
a close approximation to the affinity and permitted
the ranking of the binding activities.
The affinity of the mutated Sc antibodies to
progesterone(0-carboxymethyl)-oxime-BSA as
determined by competitive ELISA had increased over the
parent ScPgB6 antibody by 30-fold for ScPgB6-1, and
approximately 13-fold for both ScPgB6-3 and ScPgB6-4.
Interestingly, the clone with the least mutations
exhibited the highest affinity. The crossreactivity
pattern for the mutant Sc antibodies did not change,
except that ScPgB6—1 had lost most of its reactivity
to cytochrome C. In extensive studies of immune
responses to haptens, an increase in affinity by one
order of magnitude could be assigned to specific
single amino acid substitutions, which implies that
only one or two amino acid exchanges in the combining
site of the Sc anti-progesterone antibodies may have
accounted for their increased affinity to the hapten-
conjugate. Since a mutant with only a single amino
acid exchange was not recovered, the critical
residue(s) could not be identified that caused the
enhanced affinity to the hapten-conjugate. Further,
amino acid substitutions common to all three of the
mutants were not observed which suggested that
changing different residues can account for the
increased affinity to 3-(O-carboxymethyl)-
progesterone. A Ser“ to Pro“ substitution in the V"
CDR2 is common to the mutants ScPgB6-3 and ScPgB6-4.
Although both mutants showed a similar affinity to the
hapten-conjugate, assessing the importance of that
residue for antigen binding cannot unequivocally be
done, since multiple amino acid exchanges occurred in
the VL and V" regions of the two mutants.
h. Nucleic Acid Sequencing
The complete nucleotide sequences of the
variable regions of the heavy and light chains were
determined from double stranded DNA using Sequenase
2.0 (United States Biochemical). DNA sequencing
revealed that all four mutant clones, ScPgB6—1, -2, -3
and -4, had arisen from ScPgB6, with ScPgB6—1 and
ScPgB6-2 being identical. The predominant type of
mutation obtained by this PCR protocol was an A-G/T—C
nucleotide exchange (68%), while T-G/A-C, G-A/C-T, T-
A/A-T, G-C/C-G, or C-A/G—T exchanges occurred at
approximately the same frequency. DNA sequences with
a higher than average mutation frequency, i.e.
mutational hot spots, were observed. In addition, the
three mutant clones differed in the number of basepair
changes. The mutation frequencies for both the V" and
VL regions were found to be 1.5% for ScPgB6-1, 2.1%
for ScPgB6-3, and 4.1% for ScPgB6-4, which led to
multiple amino acid substitutions in the CDR's and
framework regions of the mutants.
In summary, this invention is useful for the
selection and affinity maturation of
specific antibodies to a hapten from a naive library.
Although in vitro selection and mutagenesis system is
quite simple compared to the complexity of the immune
system, some common features exist: (i) The affinity
of antibodies selected from a naive combinatorial
library can reach the same order of magnitude as
antibodies from a primary immune response to haptens;
(ii) Although the mechanisms generating mutations in
yiyg or in vitro were different, mutational hot spots
were observed; (iii) The increase in affinity after
one round of mutation and selection in vitro is in the
same order of magnitude as observed for the transition
from primary to secondary immune responses to haptens;
and (iv) A mutated antibody combining site with an
altered crossreactivity was recovered as is
occasionally observed in vivo.
When the combinatorial antibody approach was
first described, whether it could be used to
efficiently tap into the vast antibody repertoire in
yiyg was questionable. The present invention shows
that antibody chain combinations can be accessed and
evolved which may never be selected in vivo. Thus, it
now seems as if it is possible to exceed the diversity
of the antibody response in vivo by molecular cloning
techniques.
SEQUENCES NOT INSERTED 187
IN THE SPECIFICATION
SEQ. ID NO 17
!
Ala Glu Gly Asp Asp Pro Ala Lys Ala Ala Phe Asn ser Len
Gln Ala Ser Ala Thr Glu Tyr Ile Gly Tyr Ala Trp Ala Met
val val Val Ile Val Gly Ala Thr Ile Gly Ile Lys Leu Phe
Lys Lys Phe Thr Ser Lys Ala Ser
SEQ ID NO 99
GGCCGCAAATTCTATTTCAAGGAGACAGTCATAATGAAATACCTATTGCCTACG
GCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCCAGGTGAAA
CTGCTCGAGTCAGGACCTGGCCTCGTGAAACCTTCTCAGTCTCTGTCTCTCACC
TGCTCTGTCACTGACTACTCCATCACCAGTGCTTATTACTGGAACTGGATCCGG
CAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATAAGCTACGACGGTGTC
AATAAGTATGATCCATCTCTCAAGAATCGAATCTCCATCACTCGTGACACATCT,
AACAATCAGTTTTTCCAGAAGTTGATTTCTGTGACTTCTGAGGACACAGGAACA
TATGACTGTTCAAGA¢GGACTAGGGCCTCTGCTATGGACTACTGGGGTCAAGGA
ATTTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTG.
GCCCCTGéATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTC
AAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCC
AGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGC
AGCTCAGTGACTGTCCCCTCCAGCCCTCGGCCCAGCGAGACCGTCACCTGCAAC
GTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGAT
TGTACTAGTTACCCGTACGACGTTCCGGACTACGGTTCTTAA
SEQ ID NO 100
|
TGAATTCTAAACTAGTCGCCAAGGAGACAGTCATAATGAAATACCTATTGCCTACG
GCAGCCGCTGGATTGTTACTCGCTGCCCAACCAGCCATGGCCGAGCTCCAGATGAC
CCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTC
GATCAAGTGAGAATATTACAATTACT
SEQ ID NO 101
CTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCT
GGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACTACAATGT
CAAGGGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTG
ATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAG
GACGAGTATGAACGACATAACAGCTATACCTGTGATGCCACTCACAAGACATCAAC
TTCACCCATTGTCAAGAGCTTCAACACGAATGAGTGTTAATTCTAGACGGCGC
SEQ ID NO 102
I
GCTGAGGGTGACGATCCCGCAAAAGCGGCCTTTAACTCCCTGCAAGCCTCAGCGAC
.GTATCAAGCTGTTTAAGAAATTCACCTCGAAAGCAAGC
SEQ ID NO 111
GTGCCCAGGGATTGTACTAGTGCTGAGGGTGACGATCCCGCAAAAGCGGCCTTTAA
WTFCCTGCAAGCCTCAGCGACCGAATATATCGGTTATGCGTGGGCGATGGTTGTTG
TCATTGTCGGCGCAACTATCGGTATCAAGCTGTTTAAGAAATTCACCTCGAAAGCA
AGCTGATAGAATTCGAGT
SEQ ID NO 112
.
CCATTCGTTTGTGAATATCAAGGCCAAGGCCAATCGTCTGACCTGCCTCAA
CCTCCTGTCAATGCTGG¢GGCGGCTCTGGTGGTGGTTCTGGTGGCGGCTCT
GAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGCTCTGAGGGA
GGCGGTTCCGGTGGTGGCTCTGGTTCCGGTGATTTTGATTATGAAAAGATG
GCAAACGCTAATAAGGGGGCTATGACCGAAAATGCCGATGAAAACGCGCTA
CAGTCTGACGCTAAAGGCAgACTTGATTCTGTCGCTACTGATTACGGTGCT
GCTATCGATGGTTTCATTGé&GACGTTTCCGGCCTTGCTAATGGTAATGGT
GCTACTGGTGATTTTGCTGGCTCTAATTCCCAAATGGCTCAAGTCGGTGAC
GGTGATAATTCACCTTTAATGAATAATTTCCGTCAATATTTACCTTCCCTC
CCTCAATCGGTTGAATGTCGCCCTTTTGTCTTTAGCGCTQGTAAACCATAT
GAATTTTCTATTGATTGTGACAAAATAAACTTATTCGGTGTCTTTGCGTTT
CTTTTATATGTTGCCACCTTTATGTATGTATTTTCTACGTTTGCTAACATA
CTGCGTAATAAGGAGTCTTAATCATGCCAGTTCTTTTGGGTATTCCGTTAT
SEQ ID NO 113
.
GAGACGACTAGTGGTGGCGGTGGCTCTCCATTCGTTTGTGAATATCAAGGC
CAAGGCCAATCGTCTGACCTGCCTCAACCTCCTGTCAATGCTGGCGGCGGC
TCTGGTGGTGGTTCTGGTGGCGGCTCTGAGGGTGGTGGCTCTGAGGGTGGC
GGTTCTGAGGGTGGCGGCTCTGAGGGAGGCGGTTCCGGTGGTGGCTCTGGT
TCCGGTGATTTTGATTATGAAAAGATGGCAAACGCTAATAAGGGGGCTATG
ACCGAAAATGCCGATGAAAACGCG¢TACAGTCTGACGCTAAAGGCAAACTT
GATTCTGTCGCTACTGATTACGGTG&TGCTATCGATGGTTTCATTGGTGAC
GTTTCCGGCCTTGCTAATGGTAATGGTGCTACTGGTGATTTTGCTGGCTCT
AATTCCCAAATGGCTCAAGTCGGTGACGGTGATAATTCACCTTTAATGAAT
’AATTTCCGTCAATATTTACCTTCCCTCCCTCAATCGGTTGAATGTCGCCCT
TTTGTCTTTAGCGCTGGTAAACCATATGAATTTTCTATTGATTGTGACAAA
ATAAACTTATTCCGTGGTGTCTTTGCGTTTCTTTTATATGTTGCCACCTTT
ATGTATGTATTTTCTACGTTTGCTAACATACTGCGTAATAAGGAGTCTTAA
TCATGCCAGTTCTTTTGGGTATTCCGTTATTATGCTAGCTAGTAA
SEQ ID NO 114
TATGCTAGCTAGTAACACGACAGGTTTCCCGACTGG
AAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGT
TAGCTCACTCATTAGGCACCCCAGGCTTTACACTTT
ATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGC
GGATAACAATTTCACACAGGAAACAGCTATGACCAT
GATTACGAATTCGAGCTCGGT
SEQ ID NO 115
AGGTCCAGCTTCTCGAGTCTGGACCTGGCCTCGTGAAACCTTCTCAGTCTCTGTCT
CTCACCTGCTCTGTCACTGACTACTCCATCACCAGTGCTTATTACTGGAACTGGAT
CCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATAAGCTACGACGGTG
TCAATAAGTATGATCCATCTCTCAAEAATCGAATCTCCATCACTCGTGACACATCT
AACAATCAGTTTTTCCAGAAGTTGATTTCTGTGACTTCTGAGGACACAGGAACATA
TGACTGTTCAAGAGGGACTAGGGCCTCTGCTATGGACTACTGGGGTCXAGGAATTT
CAGTCACCGTCTCCTCAGCCAAAACGAEACCCCCATCTGTCTATCCACTGGCCCCT
GGATCTGCT¢CCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTA
TTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGC
ACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACT
GTCCCCTCCAGCCCTCGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGC
CAGCAGCACCAAGGTGGACAAGAAAATTGTGCbCAGGGATTGTACTAGTGCTGAGG
GTGACGATCCCGCAAAAGCGGCCTTTAACTCCCTGCAAGCCTCAGCGACCGAATAT
ATCGGTTATGCGTGGGCGATGGTTGTTGTCATTGTCGGCGCAACTATCGGTATCAA
GCTGTTTAAGAAATTCACCTCGAAAGCAAGCTGATAGAATTCGAGT
SEQ ID NO 116
:
ATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCC
AACCAGCCATGGCCCAGGTGAAACTGCTCGAGATTTCTAGACTAGTGCTGAG
GGTGACGATCCCGCAAAAGCGGCCTTTAACTCCCTGCAAGCCTCAGCGACCG
AATATATCGGTTATGCGTGGGCGATGGTTGTTGTCATTGTCGGCGCAACTAT
CGGTATCAAGCTGTTTAAGAAATTCACCTCGAAAGCAAGCTGATAGAATTCG
SEQ ID NO 117
GTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCG
CAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTTGCTTT
CTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAA
TCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCC
CAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATA
GACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACT
CTTGTTCCAAACTGGAACAAéACTCAACCCTATCTCGGTCTATTCTTTTGA
TTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGAT
TTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTA
Claims (1)
- CLAIMS A filamentous phage encapsulating a genome encoding first and second polypeptides capable of forming a ligand—binding heterodimeric receptor, wherein said first polypeptide is flanked by an amino—terminal prokaryotic secretion signal domain and a carboxy— terminal filamentous phage cpIII membrane anchor domain, and the second polypeptide is fused to an amino-terminal prokaryotic secretion signal domain. The filamentous phage of claim 1 wherein said receptor is an epitope—binding complex. A filamentous phage according to claim 1, wherein said first polypeptide is a VH polypeptide having greater than 60 amino acid residues and fewer than 125 amino acid residues, and said second polypeptide is a V; polypeptide having greater than 60 amino acid residues and fewer than 125 amino acid residues. The filamentous phage of claim 1 or claim 3 wherein said phage is detectably labeled. A ligand—binding heterodimeric receptor comprising first and second polypeptides, said first polypeptide being fused to a carboxy—terminal filamentous phage cpIII membrane anchor domain. The receptor of claim 5 wherein said receptor is an epitope—binding complex. The receptor of claim 6 wherein said first polypeptide is an antibody heavy chain polypeptide and said second polypeptide is an antibody light chain polypeptide. The receptor of claim 6 wherein said first polypeptide is an antibody light chain polypeptide and said second polypeptide is an antibody heavy chain polypeptide. A ligand—binding heterodimeric receptor comprising first and second polypeptides, wherein one of said first and second polypeptides is a V5 polypeptide having greater than 60 amino acid residues and fewer than 125 amino acid residues, and the other of said first and second polypeptides is a VL polypeptide having greater than 60 amino acid residues and fewer than 125 amino acid residues, and wherein one of said first and second polypeptides is fused to a carboxy-terminal filamentous phage membrane anchor domain. The receptor of claim 9 wherein said filamentous phage membrane anchor is the cpVIII membrane anchor. The receptor of claim 10 wherein said filamentous phage membrane anchor has an amino acid residue sequence represented by the formula in SEQ ID NO. 17 from residue 1 to residue 50. The receptor of claim 9 wherein said filamentous phage membrane anchor is the cplll membrane anchor. The receptor of claim 5 or claim 12 wherein said filamentous phage membrane anchor has an amino acid residue sequence encoded by the nucleotide sequence shown in SEQ ID NO. 113 from residues 28 to 663. A vector for expressing a fusion polypeptide, said vector comprising upstream and downstream translatable DNA sequences operatively linked via a sequence of nucleotides adapted for directional ligation of an insert DNA, said upstream sequence encoding a prokaryotic secretion signal, said downstream sequence encoding a filamentous phage membrane anchor, said translatable DNA sequences operatively linked to a set of DNA expression signals for expression of said translatable DNA sequences as portions of said fusion polypeptide, and said vector further comprising a second upstream translatable DNA sequence encoding a prokaryotic secretion signal operatively linked via a sequence of nucleotides adapted for directional ligation of a second insert DNA, said second translatable DNA sequence operatively linked to a set of DNA expression signals for expression of said second translatable DNA sequences as a portion of a second fusion polypeptide. The vector of claim 14 wherein said prokaryotic secretion signal is a pelB secretion signal. The vector of claim 15 wherein said pelB secretion signal has an amino acid residue sequence represented by a formula selected from the group consisting of: (a) SEQ ID NO 5, (b) SEQ ID NO 6, and (C) SEQ ID NO 7. The vector of claim 14 wherein said filamentous phage membrane anchor is as recited in any of claims 10 to 12. The vector of claim 17 wherein the filamentous phage membrane anchor has the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO 113 from base 28 to base 663. The vector of claim 14 further comprising a filamentous phage origin of replication. The Vector of claim 14 wherein said set of expression signals includes a promoter, a ribosome binding site, and at least one stop codon in frame with said downstream translatable DNA sequence. The vector of claim 14 wherein said vector has a nucleotide sequence shown in SEQ. ID. NO. 116 from base 1 to base 259. The vector of claim 14 wherein said vector has a nucleotide sequence shown in SEQ ID NO 3 from base 36 to base 118. A polypeptide consisting of a ligand—binding receptor polypeptide operatively linked at the carboxy—terminus to a filamentous phage cpIII membrane anchor domain. The polypeptide of claim 23 wherein the polypeptide is an antibody variable chain polypeptide. The polypeptide of claim 24 wherein the variable chain is an antibody heavy chain polypeptide. A VH or V¢ polypeptide having greater than 60 amino acid residues and fewer than 125 amino acid residues, operatively linked at the carboxy—terminus to a filamentous phage membrane anchor domain. A library of filamentous phage particles wherein each phage particle contains a DNA expression Vector according to claim 14. The library of claim 27 wherein said library contains at least 107 different species of said DNA expression vector. A library of filamentous phage particles wherein each phage particle contains at least one ligand- binding heterodimeric receptor according to claim 5. A library of filamentous phage particles wherein each phage particle contains at least one ligand- binding heterodimeric receptor comprising first and second polypeptides, said first polypeptide being fused to a carboxy—terminal filamentous phage membrane anchor domain, wherein said library expresses at least 105 different receptors. A method of producing a library of dicistronic DNA molecules, each dicistronic DNA molecule comprising first and second cistrons for expressing first and second polypeptides of a heterodimeric receptor on the.surface of a filamentous phage, which method comprises: (a) forming a first ligation admixture by combining in a ligation buffer: (i) a repertoire of first polypeptide genes in the form of linear dsDNA, each having cohesive termini adapted for directional ligation, and (ii) a plurality of DNA expression vectors in linear form, each vector having upstream and downstream first cohesive termini that are (a) adapted for directionally receiving one of said first polypeptide genes in a common reading frame, and (b) operatively linked to respective upstream an downstream translatable DNA sequences, said upstream translatable DNA sequence encoding a prokaryotic secretion signal, said downstream translatable DNA - 204 — sequence encoding a filamentous phage membrane anchor, and said translatable DNA sequences operatively linked to respective upstream and downstream DNA expression control sequences; and (b) subjecting said admixture to ligation conditions for a time period sufficient to operatively link said first polypeptide genes to said vectors and produce a plurality of circular DNA molecules each having said first cistron for expressing said first polypeptide; (c) producing a plurality of DNA expression vectors in linear form, each linear vector having second upstream and downstream cohesive termini (i) adapted for directionally receiving one of a repertoire of second polypeptide genes in a common reading frame, and (ii) operatively linked to respective upstream and downstream DNA sequences, said upstream DNA sequence being a translatable sequence encoding a prokaryotic secretion signal, said downstream DNA sequence having at least one stop codon in said reading frame, and said translatable DNA sequence operatively linked to a DNA expression control sequence, by treating said plurality of circular DNA molecules to restriction endonucleolytic conditions sufficient to cleave said circular DNA molecules and form said second cohesive termini; (d) forming a second ligation admixture by combining in a ligation buffer: (i) said plurality of DNA expression vectors formed in step (C), and (ii) said repertoire of second polypeptide genes in the form of dsDNA, each having cohesive termini adapted for directional ligation to said plurality of linear DNA vectors; and (e) subjecting said second admixture to ligation conditions for a time period sufficient to operatively link said second polypeptide genes to said vectors and produce a plurality of circular DNA molecules each having said second cistron for expressing said second polypeptide, thereby forming said library. The method of claim 31 wherein said heterodimeric receptor is an epitope—binding complex. A library of dicistronic DNA molecules each comprising first and second cistrons capable of expressing first and second polypeptides of a heterodimeric receptor on the surface of a filamentous phage, said library produced according to the method of claim 31.
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US68360291A | 1991-04-10 | 1991-04-10 | |
US82662392A | 1992-01-27 | 1992-01-27 |
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