High affinity human antibodies to tumor antigens

This invention provides novel human antibodies that specifically bind to human c-erbB-2. In one embodiment, the antibodies are single chain antibodies initially developed by phage display against a c-erbB-2 target. The resulting antibodies (designated C6 antibodies) show improved specificity and affinity for c-erbB-2. In addition, since the C6 antibodies are both relatively small and fully human they are less immunogenic in humans than other (e.g., full-size or chimeric) anti-c-erbB-2 antibodies. The C6 antibodies may be used alone or as components of chimeric molecules that specifically target and deliver effector molecules to cells overexpressing c-erbB-2.

BACKGROUND OF THE INVENTION 
This invention pertains to the fields of immunodiagnostics and 
immunotherapeutics. In particular, this invention pertains to the 
discovery of novel human antibodies that specifically bind to c-erbB-2, 
and to chimeric molecules containing these antibodies. 
Conventional cancer chemotherapeutic agents cannot distinguish between 
normal cells and tumor cells and hence damage and kill normal 
proliferating tissues. One approach to reduce this toxic side effect is to 
specifically target the chemotherapeutic agent to the tumor. This is the 
rationale behind the development of immunotoxins, chimeric molecules 
composed of an antibody either chemically conjugated or fused to a toxin 
that binds specifically to antigens on the surface of a tumor cell thereby 
killing or inhibiting the growth of the cell (Frankel et al. Ann. Rev. 
Med., 37: 127 (1986)). The majority of immunotoxins prepared to date, have 
been made using murine monoclonal antibodies (Mabs) that exhibit 
specificity for tumor cells. Immunotoxins made from Mabs demonstrate 
relatively selective killing of tumor cells in vitro and tumor regression 
in animal models (id.). 
Despite these promising results, the use of immunotoxins in humans has been 
limited by toxicity, immunogenicity and a failure to identify highly 
specific tumor antigens (Byers et al. Cancer Res., 49: 6153). Nonspecific 
toxicity results from the failure of the monoclonal antibody to bind 
specifically and with high affinity to tumor cells. As a result, 
nonspecific cell killing occurs. In addition, the foreign immunotoxin 
molecule elicits a strong immune response in humans. The immunogenicity of 
the toxin portion of the immunotoxin has recently been overcome by using 
the human analog of RNase (Rybak et al. Proc. Nat. Acad. Sci., USA, 89: 
3165 (1992)). The murine antibody portion, however, is still significantly 
immunogenic (Sawler et al., J. Immunol., 135: 1530 (1985)). 
Immunogenicity could be avoided and toxicity reduced if high affinity tumor 
specific human antibodies were available. However, the production of human 
monoclonal antibodies using conventional hybridoma technology has proven 
extremely difficult (James et al, J. Immunol Meth., 100: 5 (1987)). 
Furthermore, the paucity of purified tumor-specific antigens makes it 
necessary to immunize with intact tumor cells or partially purified 
antigen. Most of the antibodies produced react with antigens which are 
also common to normal cells and are therefore unsuitable for use as 
tumor-specific targeting molecules. 
SUMMARY OF THE INVENTION 
This invention provides novel human antibodies that specifically bind to 
the extracellular domain of the c-erbB-2 protein product of the HER2/neu 
oncogene. This antigen (marker) is overexpressed on many cancers (e.g. 
carcinomas) and thus the antibodies of the present invention specifically 
bind to tumor cells that express c-erbB-2. 
In a preferred embodiment, the antibody is a C6 antibody derived from the 
sFv antibody C6.5. The antibody may contain a variable heavy chain, a 
variable light chain, or both a variable heavy and variable light chain of 
C6.5 or its derivatives. In addition the antibody may contain a variable 
heavy chain, a variable light chain or both a variable heavy and variable 
light chain of C6.5 in which one or more of the variable heavy or variable 
light complementarity determining regions (CDR1, CDR2 or CDR3) has been 
altered (e.g., mutated). Particularly preferred CDR variants are listed in 
the specification and in Examples 1, 2 and 3. Particularly preferred C6 
antibodies include C6.5, C6ML3-14, C6L-1 and C6MH3-B1. In various 
preferred embodiments, these antibodies are single chain antibodies (sFv 
also known as scFv) comprising a variable heavy chain joined to a variable 
light chain either directly or through a peptide linker. Other preferred 
embodiments of the C6 antibodies and C6.5, C6ML3-14, C6L1, and C6MH3-B1, 
in particular, include Fab, the dimer (Fab').sub.2, and the dimer 
(sFv').sub.2. Particularly preferred (sFv').sub.2 dimers are fusion 
proteins where the Sfv' components are joined through a peptide linkage or 
through a peptide (G.sub.4 S). Still other preferred C6 antibodies include 
an antibody selected from the group consisting of an antibody having a 
V.sub.L domain with one of the amino acid sequences shown in Table 10, an 
antibody having a V.sub.H domain with one of the amino acid sequences 
shown in Table 12, an antibody having a V.sub.L CDR3 domain having one of 
the amino acid sequences shown in Tables 4, 15, and 16, and an antibody 
having a V.sub.H CDR3 domain having one of the amino acid sequences shown 
in Tables 13 and 14. Other preferred embodiments are to be found replete 
throughout the specification. 
In a particularly preferred embodiment, the C6 antibody has a K.sub.d 
ranging from about 1.6.times.10.sup.-8 to about 1.times.10.sup.-12 M in 
SK-BR-3 cells using Scatchard analysis or as measured against purified 
c-erbB-2 by surface plasmon resonance in a BIAcore. 
In another embodiment the present invention provides for nucleic acids that 
encode any of the above-described C6 antibodies. The invention also 
provides for nucleic acids that encode the amino acid sequences of C6.5, 
C6ML3-14, C6L1, C6MH3-B1, or any of the other amino acid sequences 
encoding C6 antibodies and described in Example 1, 2 or 3. In addition 
this invention provides for nucleic acid sequences encoding any of these 
amino acid sequences having conservative amino acid substitutions. 
In still another embodiment, this invention provides for proteins 
comprising one or more complementarity determining regions selected from 
the group consisting of the complementarity determining regions of Tables 
10, 12, 13, 14, 15, and 16 and of any of the examples, in particular of 
Examples 1, 2 or 3. Other particularly preferred antibodies include any of 
the antibodies expressed by the clones described herein. 
In still yet another embodiment, this invention provides for cells 
comprising a recombinant nucleic acid which is any of the above described 
nucleic acids. 
This invention also provides for chimeric molecules that specifically bind 
a tumor cell bearing c-erbB-2. The chimeric molecule comprises an effector 
molecule joined to any of the above-described C6 antibodies. In a 
preferred embodiment, the effector molecule is selected from the group 
consisting of a cytotoxin (e.g. PE, DT, Ricin A, etc.), a label, a 
radionuclide, a drug, a liposome, a ligand, an antibody, and an antigen 
binding domaine). The C6 antibody may be chemically conjugated to the 
effector molecule or the chimeric molecule may be expressed as a fusion 
protein. 
This invention provides for methods of making C6 antibodies. One method 
proceeds by i) providing a phage library presenting a C6.5 variable heavy 
chain and a multiplicity of human variable light chains; ii) panning the 
phage library on c-erbB-2; and iii) isolating phage that specifically bind 
c-erbB-2. This method optionally further includes iv) providing a phage 
library presenting the variable light chain of the phage isolated in step 
iii and a multiplicity of human variable heavy chains; v) panning the 
phage library on c-erbB-2; and vi) isolating phage that specifically bind 
c-erbB-2. 
Another method for making a C6 antibody proceeds by i) providing a phage 
library presenting a C6.5 variable light chain and a multiplicity of human 
variable heavy chains; ii) panning the phage library on c-erbB-2; and iii) 
isolating phage that specifically bind c-erbB-2. 
Yet another method for making a C6 antibody involves i) providing a phage 
library presenting a C6.5 variable light and a C6.5 variable heavy chain 
encoded by a nucleic acid variable in the sequence encoding CDR1, CDR2 or 
CDR3 such that each phage displays a different CDR; ii) panning the phage 
library on c-erbB-2; and isolating the phage that specifically bind 
c-erbB-2. 
This invention also provides a method for impairing growth of tumor cells 
bearing c-erbB-2. This method involves contacting the tumor with a 
chimeric molecule comprising a cytotoxin attached to a human C6 antibody 
that specifically binds c-erbB-2. 
Finally, this invention provides a method for detecting tumor cells bearing 
c-erbB-2. This method involves contacting the biological samples derived 
from a tumor with a chimeric molecule comprising a label attached to a 
human C6 antibody that specifically binds c-erbB-2. 
Definitions 
The following abbreviations are used herein: AMP, ampicillin; c-erbB-2 ECD, 
extracellular domain of c-erbB-2; CDR, complementarity determining region; 
ELISA, enzyme linked immunosorbent assay; FACS, fluorescence activated 
cell sorter; FR, framework region; Glu, glucose; HBS, hepes buffered 
saline, 10 mM hepes, 150 mM NaCl, pH 7.4; IMAC, immobilized metal affinity 
chromatography; k.sub.on, association rate constant; k.sub.off, 
dissociation rate constant; MPBS, skimmed milk powder in PBS; MTPBS, 
skimmed milk powder in TPBS; PBS, phosphate buffered saline, 25 mM 
NaH.sub.2 PO.sub.4, 125 mM NaCl, pH 7.0; PCR, polymerase chain reaction; 
RU, resonance units; scFv or sFv, single-chain Fv fragment; sFv': Fv 
containing cysteine; TPBS, 0.05% v/v Tween 20 in PBS; SPR, surface plasmon 
resonance; V.sub.k, immunoglobulin kappa light chain variable region; 
V.sub.1, immunoglobulin lambda light chain variable region; V.sub.L, 
immunoglobulin light chain variable region; V.sub.H, immunoglobulin heavy 
chain variable region; wt, wild type. 
As used herein, an "antibody" refers to a protein consisting of one or more 
polypeptides substantially encoded by immunoglobulin genes or fragments of 
immunoglobulin genes. The recognized immunoglobulin genes include the 
kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, 
as well as myriad immunoglobulin variable region genes. Light chains are 
classified as either kappa or lambda. Heavy chains are classified as 
gamma, mu, alpha, delta, or epsilon, which in turn define the 
immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. 
A typical immunoglobulin (antibody) structural unit is known to comprise a 
tetramer. Each tetramer is composed of two identical pairs of polypeptide 
chains, each pair having one "light" (about 25 kD) and one "heavy" chain 
(about 50-70 kD). The N-terminus of each chain defines a variable region 
of about 100 to 110 or more amino acids primarily responsible for antigen 
recognition. The terms variable light chain (V.sub.L) and variable heavy 
chain (V.sub.H) refer to these light and heavy chains respectively. 
Antibodies exist as intact immunoglobulins or as a number of well 
characterized fragments produced by digestion with various peptidases. 
Thus, for example, pepsin digests an antibody below the disulfide linkages 
in the hinge region to produce F(ab)'.sub.2, a dimer of Fab which itself 
is a light chain joined to V.sub.H -C.sub.H 1 by a disulfide bond. The 
F(ab)'.sub.2 may be reduced under mild conditions to break the disulfide 
linkage in the hinge region thereby converting the (Fab').sub.2 dimer into 
an Fab' monomer. The Fab' monomer is essentially an Fab with part of the 
hinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press, 
N.Y. (1993), for a more detailed description of other antibody fragments). 
While various antibody fragments are defined in terms of the digestion of 
an intact antibody, one of skill will appreciate that such Fab' fragments 
may be synthesized de novo either chemically or by utilizing recombinant 
DNA methodology. Thus, the term antibody, as used herein also includes 
antibody fragments either produced by the modification of whole antibodies 
or synthesized de novo using recombinant DNA methodologies. Preferred 
antibodies include single chain antibodies, more preferably single chain 
Fv (sFv) antibodies in which a variable heavy and a variable light chain 
are joined together (directly or through a peptide linker) to form a 
continuous polypeptide. 
An "antigen-binding site" or "binding portion" refers to the part of an 
immunoglobulin molecule that participates in antigen binding. The antigen 
binding site is formed by amino acid residues of the N-terminal variable 
("V") regions of the heavy ("H") and light ("L") chains. Three highly 
divergent stretches within the V regions of the heavy and light chains are 
referred to as "hypervariable regions" which are interposed between more 
conserved flanking stretches known as "framework regions" or "FRs". Thus, 
the term "FR" refers to amino acid sequences which are naturally found 
between and adjacent to hypervariable regions in immunoglobulins. In an 
antibody molecule, the three hypervariable regions of a light chain and 
the three hypervariable regions of a heavy chain are disposed relative to 
each other in three dimensional space to form an antigen binding 
"surface". This surface mediates recognition and binding of the target 
antigen. The three hypervariable regions of each of the heavy and light 
chains are referred to as "complementarity determining regions" or "CDRs" 
and are characterized, for example by Kabat et al. Sequences of proteins 
of immunological interest, 4th ed. U.S. Dept. Health and Human Services, 
Public Health Services, Bethesda, Md. (1987). 
As used herein, the terms "immunological binding" and "immunological 
binding properties" refer to the non-covalent interactions of the type 
which occur between an immunoglobulin molecule and an antigen for which 
the immunoglobulin is specific. The strength or affinity of immunological 
binding interactions can be expressed in terms of the dissociation 
constant (K.sub.d) of the interaction, wherein a smaller Kd represents a 
greater affinity. Immunological binding properties of selected 
polypeptides can be quantified using methods well known in the art. One 
such method entails measuring the rates of antigen-binding site/antigen 
complex formation and dissociation, wherein those rates depend on the 
concentrations of the complex partners, the affinity of the interaction, 
and on geometric parameters that equally influence the rate in both 
directions. Thus, both the "on rate constant" (k.sub.on) and the "off rate 
constant" (k.sub.off) can be determined by calculation of the 
concentrations and the actual rates of association and dissociation. The 
ratio of k.sub.off /k.sub.on enables cancellation of all parameters not 
related to affinity and is thus equal to the dissociation constant 
K.sub.d. See, generally, Davies et al. Ann. Rev. Biochem., 59: 439-473 
(1990). 
The term "C6 antibody", as used herein refers to antibodies derived from 
C6.5 whose sequence is expressly provided herein. C6 antibodies preferably 
have a binding affinity of about 1.6.times.10.sup.8 or better and are 
preferably derived by screening (for affinity to c-erbB-2) a phage display 
library in which a known C6 variable heavy (V.sub.H) chain is expressed in 
combination with a multiplicity of variable light (V.sub.L) chains or 
conversely a known C6 variable light chain is expressed in combination 
with a multiplicity of variable heavy (V.sub.H) chains. C6 antibodies also 
include those antibodies produced by the introduction of mutations into 
the variable heavy or variable light complementarity determining regions 
(CDR1, CDR2 or CDR3) as described herein. Finally C6 antibodies include 
those antibodies produced by any combination of these modification methods 
as applied to C6.5 and its derivatives. 
A single chain Fv ("sFv" or "scFv") polypeptide is a covalently linked 
V.sub.H ::V.sub.L heterodimer which may be expressed from a nucleic acid 
including V.sub.H - and V.sub.L -encoding sequences either joined directly 
or joined by a peptide-encoding linker. Huston, et al. Proc. Nat. Acad. 
Sci. USA, 85: 5879-5883 (1988). A number of structures for converting the 
naturally aggregated--but chemically separated light and heavy polypeptide 
chains from an antibody V region into an sFv molecule which will fold into 
a three dimensional structure substantially similar to the structure of an 
antigen-binding site. See, e.g. U.S. Pat. Nos. 5,091,513 and 5,132,405 and 
4,956,778. 
In one class of embodiments, recombinant design methods can be used to 
develop suitable chemical structures (linkers) for converting two 
naturally associated--but chemically separate--heavy and light polypeptide 
chains from an antibody variable region into a sFv molecule which will 
fold into a three-dimensional structure that is substantially similar to 
native antibody structure. 
Design criteria include determination of the appropriate length to span the 
distance between the C-terminal of one chain and the N-terminal of the 
other, wherein the linker is generally formed from small hydrophilic amino 
acid residues that do not tend to coil or form secondary structures. Such 
methods have been described in the art. See, e.g., U.S. Pat. Nos. 
5,091,513 and 5,132,405 to Huston et al.; and U.S. Pat. No. 4,946,778 to 
Ladner et al. 
In this regard, the first general step of linker design involves 
identification of plausible sites to be linked. Appropriate linkage sites 
on each of the V.sub.H and V.sub.L polypeptide domains include those which 
will result in the minimum loss of residues from the polypeptide domains, 
and which will necessitate a linker comprising a minimum number of 
residues consistent with the need for molecule stability. A pair of sites 
defines a "gap" to be linked. Linkers connecting the C-terminus of one 
domain to the N-terminus of the next generally comprise hydrophilic amino 
acids which assume an unstructured configuration in physiological 
solutions and preferably are free of residues having large side groups 
which might interfere with proper folding of the V.sub.H and V.sub.L 
chains. Thus, suitable linkers under the invention generally comprise 
polypeptide chains of alternating sets of glycine and serine residues, and 
may include glutamic acid and lysine residues inserted to enhance 
solubility. One particular linker under the invention has the amino acid 
sequence [(Gly).sub.4 Ser].sub.3 (SEQ ID NO:1). Another particularly 
preferred linker has the amino acid sequence comprising 2 or 3 repeats of 
[(Ser).sub.4 Gly] (SEQ ID NO:2) such as [(Ser).sub.4 Gly].sub.3 (SEQ ID 
NO:3). Nucleotide sequences encoding such linker moieties can be readily 
provided using various oligonucleotide synthesis techniques known in the 
art. See, e.g., Sambrook, supra. 
The phrase "specifically binds to a protein" or "specifically 
immunoreactive with", when referring to an antibody refers to a binding 
reaction which is determinative of the presence of the protein in the 
presence of a heterogeneous population of proteins and other biologics. 
Thus, under designated immunoassay conditions, the specified antibodies 
bind to a particular protein and do not bind in a significant amount to 
other proteins present in the sample. Specific binding to a protein under 
such conditions may require an antibody that is selected for its 
specificity for a particular protein. For example, C6 antibodies can be 
raised to the c-erbB-2 protein that bind c-erbB-2 and not to other 
proteins present in a tissue sample. A variety of immunoassay formats may 
be used to select antibodies specifically immunoreactive with a particular 
protein. For example, solid-phase ELISA immunoassays are routinely used to 
select monoclonal antibodies specifically immunoreactive with a protein. 
See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring 
Harbor Publications, New York, for a description of immunoassay formats 
and conditions that can be used to determine specific immunoreactivity. 
A chimeric molecule is a molecule in which two or more molecules that exist 
separately in their native state are joined together to form a single 
molecule having the desired functionality of all of its constituent 
molecules. While the chimeric molecule may be prepared by covalently 
linking two molecules each synthesized separately, one of skill in the art 
will appreciate that where the chimeric molecule is a fusion protein, the 
chimera may be prepared de novo as a single "joined" molecule. 
The term "conservative substitution" is used in reference to proteins or 
peptides to reflect amino acid substitutions that do not substantially 
alter the activity (specificity or binding affinity) of the molecule. 
Typically conservative amino acid substitutions involve substitution one 
amino acid for another amino acid with similar chemical properties (e.g. 
charge or hydrophobicity). The following six groups each contain amino 
acids that are typical conservative substitutions for one another: 
1) Alanine (A), Serine (S), Threonine (T); 
2) Aspartic acid (D), Glutamic acid (E); 
3) Asparagine (N), Glutamine (Q); 
4) Arginine (R), Lysine (K); 
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

DETAILED DESCRIPTION 
This invention provides for novel human antibodies that specifically bind 
to the extracellular domain of the c-erbB-2 protein product of the 
HER2/neu oncogene. The c-erbB-2 marker is overexpressed by 30-50% of 
breast carcinomas and other adenocarcinomas and thus provides a suitable 
cell surface marker for specifically targeting tumor cells such as 
carcinomas. In contrast to previous known anti-cerbB-2 antibodies, the 
antibodies of the present invention (designated herein as C6 antibodies) 
are fully human antibodies. Thus, administration of these antibodies to a 
human host elicits a little or no immunogenic response. 
This invention additionally provides for chimeric molecules comprising the 
C6 antibodies of the present invention joined to an effector molecule. The 
C6 antibodies act as a "targeting molecule" that serves to specifically 
bind the chimeric molecule to cells bearing the c-erbB-2 marker thereby 
delivering the effector molecule to the target cell. 
An effector molecule typically has a characteristic activity that is 
desired to be delivered to the target cell (e.g. a tumor overexpressing 
c-erbB-2). Effector molecules include cytotoxins, labels, radionuclides, 
ligands, antibodies, drugs, liposomes, and viral coat proteins that render 
the virus capable of infecting a c-erbB-2 expressing cell. Once delivered 
to the target, the effector molecule exerts its characteristic activity. 
For example, in one embodiment, where the effector molecule is a cytotoxin, 
the chimeric molecule acts as a potent cell-killing agent specifically 
targeting the cytotoxin to tumor cells bearing the c-erbB-2 marker. 
Chimeric cytotoxins that specifically target tumor cells are well known to 
those of skill in the art (see, for example, Pastan et al., Ann. Rev. 
Biochem., 61: 331-354 (1992)). 
In another embodiment, the chimeric molecule may be used for detecting the 
presence or absence of tumor cells in vivo or in vitro or for localizing 
tumor cells in vivo. These methods involve providing a chimeric molecule 
comprising an effector molecule, that is a detectable label attached to 
the C6 antibody. The C6 antibody specifically binds the chimeric molecule 
to tumor cells expressing the c-erbB-2 marker which are then marked by 
their association with the detectable label. Subsequent detection of the 
cell-associated label indicates the presence and/or location of a tumor 
cell. 
In yet another embodiment, the effector molecule may be another specific 
binding moiety including, but not limited to an antibody, an antigen 
binding domain, a growth factor, or a ligand. The chimeric molecule will 
then act as a highly specific bifunctional linker. This linker may act to 
bind and enhance the interaction between cells or cellular components to 
which the chimeric protein binds. Thus, for example, where the "effector" 
component is an anti-receptor antibody or antibody fragment, the C6 
antibody component specifically binds c-erbB-2 bearing cancer cells, while 
the effector component binds receptors (e.g., IL-2, IL-4, Fc.gamma.I, 
Fc.gamma.II and Fc.gamma.III receptors) on the surface of immune cells. 
The chimeric molecule may thus act to enhance and direct an immune 
response toward target cancer cells. 
In still yet another embodiment the effector molecule may be a 
pharmacological agent (e.g. a drug) or a vehicle containing a 
pharmacological agent. This is particularly suitable where it is merely 
desired to invoke a non-lethal biological response. Thus the C6 antibody 
receptor may be conjugated to a drug such as vinblastine, vindesine, 
melphalan, N-Acetylmelphalan, methotrexate, aminopterin, doxirubicin, 
daunorubicin, genistein (a tyrosine kinase inhibitor), an antisense 
molecule, and other pharmacological agents known to those of skill in the 
art, thereby specifically targeting the pharmacological agent to tumor 
cells expressing c-erbB-2. 
Alternatively, the C6 antibody may be bound to a vehicle containing the 
therapeutic composition. Such vehicles include, but are not limited to 
liposomes, micelles, various synthetic beads, and the like. 
One of skill in the art will appreciate that the chimeric molecules of the 
present invention optionally includes multiple targeting moieties bound to 
a single effector or conversely, multiple effector molecules bound to a 
single targeting moiety. In still other embodiment, the chimeric molecules 
includes both multiple targeting moieties and multiple effector molecules. 
Thus, for example, this invention provides for "dual targeted" cytotoxic 
chimeric molecules in which the C6 antibody is attached to a cytotoxic 
molecule while another molecule (e.g. an antibody, or another ligand) is 
attached to the other terminus of the toxin. Such a dual-targeted 
cytotoxin might comprise, e.g. a C6 antibody substituted for domain Ia at 
the amino terminus of a PE and anti-TAC(Fv) inserted in domain III. Other 
antibodies may also be suitable effector molecules. 
I. Preparation of C6 Antibodies. 
The C6 antibodies of this invention are prepared using standard techniques 
well known to those of skill in the art in combination with the 
polypeptide and nucleic acid sequences provided herein. The polypeptide 
sequences may be used to determine appropriate nucleic acid sequences 
encoding the particular C6 antibody disclosed thereby. The nucleic acid 
sequence may be optimized to reflect particular codon "preferences" for 
various expression systems according to standard methods well known to 
those of skill in the art. Alternatively, the nucleic acid sequences 
provided herein may also be used to express C6 antibodies. 
Using the sequence information provided, the nucleic acids may be 
synthesized according to a number of standard methods known to those of 
skill in the art. Oligonucleotide synthesis, is preferably carried out on 
commercially available solid phase oligonucleotide synthesis machines 
(Needham-VanDevanter et al. (1984) Nucleic Acids Res. 12:6159-6168) or 
manually synthesized using the solid phase phosphoramidite triester method 
described by Beaucage et. al. (Beaucage et. al. (1981) Tetrahedron Letts. 
22(20): 1859-1862). 
Once a nucleic acid encoding a C6 antibody is synthesized it may be 
amplified and/or cloned according to standard methods. Molecular cloning 
techniques to achieve these ends are known in the art. A wide variety of 
cloning and in vitro amplification methods suitable for the construction 
of recombinant nucleic acids, e.g., encoding C6 antibody genes, are known 
to persons of skill. Examples of these techniques and instructions 
sufficient to direct persons of skill through many cloning exercises are 
found in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods 
in Enzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger); 
Sambrook et al. (1989) Molecular Cloning--A Laboratory Manual (2nd ed.) 
Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, 
(Sambrook); and Current Protocols in Molecular Biology, F. M. Ausubel et 
al., eds., Current Protocols, a joint venture between Greene Publishing 
Associates, Inc. and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel). 
Methods of producing recombinant immunoglobulins are also known in the 
art. See, Cabilly, U.S. Pat. No. 4,816,567; and Queen et al. (1989) Proc. 
Nat'l Acad. Sci. USA 86: 10029-10033. 
Examples of techniques sufficient to direct persons of skill through in 
vitro amplification methods, including the polymerase chain reaction (PCR) 
the ligase chain reaction (LCR), Q.beta.-replicase amplification and other 
RNA polymerase mediated techniques are found in Berger, Sambrook, and 
Ausubel, as well as Mullis et al., (1987) U.S. Pat. No. 4,683,202; PCR 
Protocols A Guide to Methods and Applications (Innis et al. eds) Academic 
Press Inc. San Diego, Calif. (1990) (Innis); Arnheim & Levinson (Oct. 1, 
1990) C&EN 36-47; The Journal Of NIH Research (1991) 3, 81-94; (Kwoh et 
al. (1989) Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli et al. (1990) 
Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al. (1989) J. Clin. Chem 
35, 1826; Landegren et al., (1988) Science 241, 1077-1080; Van Brunt 
(1990) Biotechnology 8, 291-294; Wu and Wallace, (1989) Gene 4, 560; and 
Barringer et al. (1990) Gene 89, 117. Improved methods of cloning in vitro 
amplified nucleic acids are described in Wallace et al., U.S. Pat. No. 
5,426,039. 
Once the nucleic acid for a C6 antibody is isolated and cloned, one may 
express the gene in a variety of recombinantly engineered cells known to 
those of skill in the art. Examples of such cells include bacteria, yeast, 
filamentous fungi, insect (especially employing baculoviral vectors), and 
mammalian cells. It is expected that those of skill in the art are 
knowledgeable in the numerous expression systems available for expression 
of C6 antibodies. 
In brief summary, the expression of natural or synthetic nucleic acids 
encoding C6 antibodies will typically be achieved by operably linking a 
nucleic acid encoding the antibody to a promoter (which is either 
constitutive or inducible), and incorporating the construct into an 
expression vector. The vectors can be suitable for replication and 
integration in prokaryotes, eukaryotes, or both. Typical cloning vectors 
contain transcription and translation terminators, initiation sequences, 
and promoters useful for regulation of the expression of the nucleic acid 
encoding the C6 antibody. The vectors optionally comprise generic 
expression cassettes containing at least one independent terminator 
sequence, sequences permitting replication of the cassette in both 
eukaryotes and prokaryotes, i.e., shuttle vectors, and selection markers 
for both prokaryotic and eukaryotic systems. See Sambrook. 
To obtain high levels of expression of a cloned nucleic acid it is common 
to construct expression plasmids which typically contain a strong promoter 
to direct transcription, a ribosome binding site for translational 
initiation, and a transcription/translation terminator. Examples of 
regulatory regions suitable for this purpose in E. coli are the promoter 
and operator region of the E. coli tryptophan biosynthetic pathway as 
described by Yanofsky, C., 1984, J. Bacteriol., 158:1018-1024 and the 
leftward promoter of phage lambda (P.sub.L) as described by Herskowitz and 
Hagen, 1980, Ann. Rev. Genet., 14:399-445. The inclusion of selection 
markers in DNA vectors transformed in E. coli is also useful. Examples of 
such markers include genes specifying resistance to ampicillin, 
tetracycline, or chloramphenicol. See Sambrook for details concerning 
selection markers, e.g., for use in E. coli. 
Expression systems for expressing C6 antibodies are available using E. 
coli, Bacillus sp. (Palva et al. (1983) Gene 22:229-235; Mosbach et al., 
Nature, 302:543-545 and Salmonella. E. coli systems are preferred. 
The C6 antibodies produced by prokaryotic cells may require exposure to 
chaotropic agents for proper folding. During purification from, e.g., E. 
coli, the expressed protein is optionally denatured and then renatured. 
This is accomplished, e.g., by solubilizing the bacterially produced 
antibodies in a chaotropic agent such as guanidine HCl. The antibody is 
then renatured, either by slow dialysis or by gel filtration. See, U.S. 
Pat. No. 4,511,503. 
Methods of transfecting and expressing genes in mammalian cells are known 
in the art. Transducing cells with nucleic acids can involve, for example, 
incubating viral vectors containing C6 nucleic acids with cells within the 
host range of the vector. See, e.g., Methods in Enzymology, vol. 185, 
Academic Press, Inc., San Diego, Calif. (D. V. Goeddel, ed.) (1990) or M. 
Krieger, Gene Transfer and Expression--A Laboratory Manual, Stockton 
Press, New York, N.Y., (1990) and the references cited therein. 
The culture of cells used in the present invention, including cell lines 
and cultured cells from tissue or blood samples is well known in the art. 
Freshney (Culture of Animal Cells, a Manual of Basic Technique, third 
edition Wiley-Liss, New York (1994)) and the references cited therein 
provides a general guide to the culture of cells. 
Techniques for using and manipulating antibodies are found in Coligan 
(1991) Current Protocols in Immunology Wiley/Greene, NY; Harlow and Lane 
(1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY; 
Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical 
Publications, Los Altos, Calif., and references cited therein; Goding 
(1986) Monoclonal Antibodies: Principles and Practice (2d ed.) Academic 
Press, New York, NY; and Kohler and Milstein (1975) Nature 256: 495-497. 
C6 antibodies which are specific for c-erbB-2 bind c-erbB-2 and have a 
K.sub.D of 1 .mu.M or better, with preferred embodiments having a K.sub.D 
of 1 nM or better and most preferred embodiments having a K.sub.D of 0.1 
nM or better. 
In a preferred embodiment the C6 antibody gene (e.g. C6.5 sFv gene) is 
subcloned into the expression vector pUC119Sfi/NotHismyc, which is 
identical to the vector described by Griffiths et al., EMBO J., 13: 
3245-3260 (1994), (except for the elimination of an XBaI restriction 
site). This results in the addition of a hexa-histidine tag at the 
C-terminal end of the sFv. A pHEN-1 vector DNA containing the C6.5 sFv DNA 
is prepared by alkaline lysis miniprep, digested with NcoI and NotI, and 
the sFv DNA purified on a 1.5% agarose gel. The C6 sFv DNA is ligated into 
pUC119Sfi1/Not1Hismyc digested with NcoI and NotI and the ligation mixture 
used to transform electrocompetent E.coli HB2151. For expression, 200 ml 
of 2.times.TY media containing 100 mg/ml ampicillin and 0.1% glucose is 
inoculated with E.coli HB2151 harboring the C6 gene in 
pUC119Sfi1/Not1Hismyc. The culture is grown at 37.degree. C. to an A600 nm 
of 0.8. Soluble sFv is expression induced by the addition of IPTG to a 
final concentration of 1 mM, and the culture is grown at 30.degree. C. in 
a shaker flask overnight. 
The C6 sFv may then be harvested from the periplasm using the following 
protocol: Cells are harvested by centrifugation at 4000 g for 15 min, 
resuspended in 10 ml of ice cold 30 mM Tris-HCl pH 8.0, 1 mM EDTA, 20% 
sucrose, and incubated on ice for 20 minutes. The bacteria are then 
pelleted by centrifugation at 6000 g for 15 min. and the "periplasmic 
fraction" cleared by centrifugation at 30,000 g for 20 min. The 
supernatant is then dialyzed overnight at 4.degree. C. against 8 L of IMAC 
loading buffer (50 mM sodium phosphate pH 7.5, 500 mM NaCl, 20 mM 
imidazole) and then filtered through a 0.2 micron filter. 
In a preferred embodiment, the C6 sFv is purified by IMAC. All steps are 
performed at 4.degree. C. A column containing 2 ml of Ni-NTA resin 
(Qiagen) is washed with 20 ml IMAC column wash buffer (50 mM sodium 
phosphate pH 7.5, 500 mM NaCl, 250 mM imidazole) and 20 ml of IMAC loading 
buffer. The periplasmic preparation is then loaded onto the column and the 
column washed sequentially with 50 ml IMAC loading buffer and 50 ml IMAC 
washing buffer (50 mM sodium phosphate pH 7.5, 500 mM NaCl, 25 mM 
imidazole). Protein was eluted with 25 ml IMAC elution buffer (50 mM 
sodium phosphate pH 7.5, 500 mM NaCl, 100 mM imidazole) and 4 ml fractions 
collected. The C6 antibody may be detected by absorbance at 280 nm and sFv 
fraction eluted. To remove dimeric and aggregated sFv, samples can be 
concentrated to a volume &lt;1 ml in a Centricon 10 (Amicon) and fractionated 
on a Superdex 75 column using a running buffer of HBS (10 mM Hepes, 150 mM 
NaCl, pH 7.4). 
The purity of the final preparation may be evaluated by assaying an aliquot 
by SDS-PAGE. The protein bands can be detected by Coomassie staining. The 
concentration can then be determined spectrophotometrically, assuming that 
an A.sub.280 nm of 1.0 corresponds to an sFv concentration of 0.7 mg/ml. 
II. Modification of C6 Antibodies. 
A) Display of antibody fragments on the surface of bacteriophage (phage 
display). 
Display of antibody fragments on the surface of viruses which infect 
bacteria (bacteriophage or phage) makes it possible to produce human sFvs 
with a wide range of affinities and kinetic characteristics. To display 
antibody fragments on the surface of phage (phage display), an antibody 
fragment gene is inserted into the gene encoding a phage surface protein 
(pIII) and the antibody fragment-pIII fusion protein is expressed on the 
phage surface (McCafferty et al. (1990) Nature, 348: 552-554; Hoogenboom 
et al. (1991) Nucleic Acids Res., 19: 4133-4137). For example, a sFv gene 
coding for the V.sub.H and V.sub.L domains of an anti-lysozyme antibody 
(D1.3) was inserted into the phage gene III resulting in the production of 
phage with the DI.3 sFv joined to the N-terminus of pIII thereby producing 
a "fusion" phage capable of binding lysozyme (McCafferty et al (1990) 
Nature, 348: 552-554). 
Since the antibody fragments on the surface of the phage are functional, 
phage bearing antigen binding antibody fragments can be separated from 
non-binding or lower affinity phage by antigen affinity chromatography 
(McCafferty et al. (1990) Nature, 348: 552-554). Mixtures of phage are 
allowed to bind to the affinity matrix, non-binding or lower affinity 
phage are removed by washing, and bound phage are eluted by treatment with 
acid or alkali. Depending on the affinity of the antibody fragment, 
enrichment factors of 20 fold-1,000,000 fold are obtained by single round 
of affinity selection. By infecting bacteria with the eluted phage, 
however, more phage can be grown and subjected to another round of 
selection. In this way, an enrichment of 1000 fold in one round becomes 
1,000,000 fold in two rounds of selection (McCafferty et al. (1990) 
Nature, 348: 552-554). Thus, even when enrichments in each round are low 
(Marks et al. (1991) J. Mol. Biol, 222: 581-597), multiple rounds of 
affinity selection leads to the isolation of rare phage and the genetic 
material contained within which encodes the sequence of the binding 
antibody. The physical link between genotype and phenotype provided by 
phage display makes it possible to test every member of an antibody 
fragment library for binding to antigen, even with libraries as large as 
100,000,000 clones. For example, after multiple rounds of selection on 
antigen, a binding sFv that occurred with a frequency of only 1/30,000,000 
clones was recovered (Marks et al (1991) J. Mol. BioL, 222: 581-597). 
Analysis of binding is simplified by including an amber codon between the 
antibody fragment gene and gene III. This makes it possible to easily 
switch between displayed and soluble antibody fragments simply by changing 
the host bacterial strain. When phage are grown in a supE suppresser 
strain of E. coli, the amber stop codon between the antibody gene and gene 
III is read as glutamine and the antibody fragment is displayed on the 
surface of the phage. When eluted phage are used to infect a non-supressor 
strain, the amber codon is read as a stop codon and soluble antibody is 
secreted from the bacteria into the periplasm and culture media 
(Hoogenboom et al. (1991) Nucleic Acids Res., 19: 4133-4137). Binding of 
soluble sFv to antigen can be detected, e.g., by ELISA using a murine IgG 
monoclonal antibody (e.g., 9E1O) which recognizes a C-terminal myc peptide 
tag on the sFv (Evan et al. (1985) Mol. Cell Biol., 5: 3610-3616; Munro et 
al. (1986) Cell, 46: 291-300), e.g., followed by incubation with 
polyclonal anti-mouse Fc conjugated to horseradish peroxidase. 
B) Phage display can be used to increase antibody affinity. 
To create higher affinity antibodies, mutant sFv gene repertories, based on 
the sequence of a binding sFv, are created and expressed on the surface of 
phage. Higher affinity sFvs are selected on antigen as described above and 
in Examples 1 and 2. One approach for creating mutant sFv gene repertoires 
has been to replace either the V.sub.H or V.sub.L gene from a binding sFv 
with a repertoire of nonimmune V.sub.H or V.sub.L genes (chain shuffling) 
(Clackson et al. (1991) Nature, 352: 624-628). Such gene repertoires 
contain numerous variable genes derived from the same germline gene as the 
binding sFv, but with point mutations (Marks et al. (1992) Bio/Technology, 
10: 779-783). Using light chain shuffling and phage display, the binding 
avidities of a human sFv antibody fragment can be dramatiaclly increased. 
See, e.g., Marks et al Bio/Technology, 10: 779-785 (1992) in which the 
affinity of a human sFv antibody fragment which bound the hapten 
phenyloxazolone (phox) was increased from 300 nM to 15 nM (20 fold) (Marks 
et al. (1992) Bio/Technology, 10: 779-783). 
C) Isolation and characterization of C6.5. a human sFv which binds 
c-erbB-2. 
Isolation and characterization of C6.5 is described in detail in the 
Examples below. Human sFvs which bound to c-erbB-2 were isolated by 
selecting the nonimmune human sFv phage antibody library (described in 
Example 1) on c-erbB-2 extracellular domain peptide immobilized on 
polystyrene. After five rounds of selection, 45 of 96 clones analyzed 
(45/96) produced sFv which bound c-erb-B2 by ELISA. Restriction fragments 
analysis and DNA sequencing revealed the presence of two unique human 
sFvs, C4 and C6.5. Both of these sFvs bound only to c-erbB-2 and not to a 
panel of 10 irrelevant antigens. Cell binding assays, however, indicated 
that only C6.5 bound c-erb-B2 expressed on cells, and thus this sFv was 
selected for further characterization. 
D) Purification of C6.5. 
To facilitate purification, the C6.5 sFv gene was subcloned into the 
expression vector pUC119 Sfi-NotmycHIS which results in the addition of 
the myc peptide tag followed by a hexahistidine tag at the C-terminal end 
of the sFv. The vector also encodes the pectate lyase leader sequence 
which directs expression of the sFv into the bacterial periplasm where the 
leader sequence is cleaved. This makes it possible to harvest native 
properly folded sFv directly from the bacterial periplasm. Native C6.5 sFv 
was expressed and purified from the bacterial supernatant using 
immobilized metal affinity chromatography. The yield after purification 
and gel filtration on a Superdex 75 column was 10.5 mg/L. Other C6 
antibodies may be purified in a similar manner. 
E) Measurement of C6.5 affinity for c-erbB-2. 
As explained above, selection for increased avidity involves measuring the 
affinity of a C6 antibody (e.g. a modified C6.5) for c-erbB-2. Methods of 
making such measurements are described in detail in Examples 1 and 2. 
Briefly, for example, the Kd of C6.5 and the kinetics of binding to 
c-erbB-2 were determined in a BIAcore, a biosensor based on surface 
plasmon resonance. For this technique, antigen is coupled to a derivatized 
sensor chip capable of detecting changes in mass. When antibody is passed 
over the sensor chip, antibody binds to the antigen resulting in an 
increase in mass which is quantifiable. Measurement of the rate of 
association as a function of antibody concentration can be used to 
calculate the association rate constant (k.sub.on). After the association 
phase, buffer is passed over the chip and the rate of dissociation of 
antibody (k.sub.off) determined. Rate constant k.sub.on is typically 
measured in the range 1.0.times.10.sup.2 to 5.0.times.10.sup.6 and 
k.sub.off in the range 1.0.times.10.sup.-1 to 1.0.times.10.sup.-6. The 
equilibrium constant K.sub.d is often calculated as k.sub.off /k.sub.on 
and thus is typically measured in the range 10.sup.-5 to 10.sup.-12. 
Affinities measured in this manner correlate well with affinities measured 
in solution by fluorescence quench titration. 
F) Affinity of C6.5 for c-erbB-2. 
The kinetics of binding and affinity of purified C6.5 were determined by 
BIAcore and the results are shown in Table 2. The K.sub.d of 
1.6.times.10.sup.-8 M determined by BIAcore is in close agreement to the 
K.sub.d determined by Scatchard analysis after radioiodination 
(2.0.times.10.sup.-8 M). C6.5 has a rapid k.sub.on, and a relatively rapid 
k.sub.off. The rapid k.sub.off correlates with the in vitro measurement 
that only 22% of an injected dose is retained on the surface of SK-OV-3 
cells after 30 minutes. Biodistribution of C6.5 was determined and the 
percent injected dose/gm tumor at 24 hours was 1.1% with tumor/organ 
ratios of 5.6 for kidney and 103 for bone. These values compare favorably 
to values obtained for 741F8 sFv. 741F8 is a monoclonal antibody capable 
of binding c-erbB-2 (see, e.g., U.S. Pat. No. 5,169,774). The K.sub.d of 
741F8 was also measured by BIAcore and agreed with the value determined by 
scatchard analysis (Table 1). 
TABLE 1 
______________________________________ 
Characterization of anti-cerbB-2 sFv species. Characteristics of the 
murine anti-cerbB-2 sFv, 741F8, and the human sFv C6.5 are 
compared. The affinity and dissociation constants were determined 
by Scatchard plot analysis, unless otherwise stated. Dissociation from 
c-erbB-2 positive (SK-OV-3) cells was measured in an in vitro live cell 
assay. The percentage of injected dose per gram (% ID/g) tumor and 
tumor to organ ratios were determined in biodistribution studies 
performed in separate groups of scid mice (n = 10-14) bearing 
SK-OV-3 tumors overexpressing c-erbB-2. SEM are &lt;35% of the 
associated values. a = significantly improved (p &lt; 0.05) 
compared to 741F8 sFv. 
Parameter 741F8 C6.5 
______________________________________ 
K.sub.d (BIAcore) 
2.6 .times. 10.sup.-8 M 
1.6 .times. 10.sup.-8 M 
K.sub.d (Scatchard) 5.4 .times. 10.sup.-8 M 2.l .times. 10.sup.-8 M 
k.sub.on (BIAcore) 2.4 .times. 10.sup.5 
M.sup.-1 s.sup.-1 4.0 .times. 10.sup.5 
M.sup.-1 s.sup.-1 
k.sub.off (BIAcore) 6.4 .times. 10.sup.-3 s.sup.-1 6.3 .times. 
10.sup.-3 s.sup.-1 
% associated with cell 32.7% 60.6% 
surface at 15 min 
% associated with cell 8.6% 22.2% 
surface at 30 min 
% ID/g Tumor 0.8 1.0 
T:Blood 14.7 22.9 
T:Kidney 2.8 5.6a 
T:Liver 14.2 22.3 
T:Spleen 10.3 34.1 
T:Intestine 25.0 29.7 
T:Lung 9.4 15.8 
T:Stomach 8.9 11.1 
T:Muscle 78.8 158.7 
T:Bone 30.0 102.7 
______________________________________ 
These results show that a human sFv which binds specifically to c-erbB-2 
with moderate affinity was been produced. The sFv expresses at high level 
in E. coli as native sFv, and can be easily purified in high yield in two 
steps. Techniques are known for the rapid and efficiently purification of 
sFv from the bacterial periplasm and to measure affinity without the need 
for labeling. 
G) Estimating the affinity of unpurified sFv for c-erbB2. 
Phage display and selection generally results in the selection of higher 
affinity mutant sfvs (Marks et al. (1992) Bio/Technology, 10: 779-783; 
Hawlins et al. (1992) J. Mol. Biol. 226: 889-896; Riechmann et al. (1993) 
Biochemistry, 32: 8848-8855; Clackson et al. (1991) Nature, 352: 624-628), 
but probably does not result in the separation of mutants with less than a 
6 fold difference in affinity (Riechmann et al. (1993) Biochemistry, 32: 
8848-8855). Thus a rapid method is needed to estimate the relative 
affinities of mutant sfvs isolated after selection. Since increased 
affinity results primarily from a reduction in the k.sub.off, measurement 
of k.sub.off should identify higher affinity sFv. k.sub.off can be 
measured in the BLkcore on unpurified sFv in bacterial periplasm, since 
expression levels are high enough to give an adequate binding signal and 
k.sub.off is independent of concentration. The value of k.sub.off for 
periplasmic and purified sFv is in close agreement (Table 2). 
TABLE 2 
______________________________________ 
Comparison of k.sub.off determined on sFv in bacterial periplasm and 
after 
purification by IMAC and gel filtration. 
sFv k.sub.off (s.sup.-1) 
______________________________________ 
C6-5 periplasm 5.7 .times. 10.sup.-3 
C6-5 purified 6.3 .times. 10.sup.-3 
C6-5ala3 periplasm 9.3 .times. 10.sup.-3 
C6-5ala3 purified 1.5 .times. 10.sup.-3 
C6-5ala10 periplasm 3.7 .times. 10.sup.-3 
C6-5ala10 purified 4.1 .times. 10.sup.-3 
______________________________________ 
Ranking of sFv by k.sub.off, and hence relative affinity, can be determined 
without purification. Determination of relative affinity without 
purification significantly increases the rate at which mutant sFv are 
characterized, and reduces the number of mutant sFv subcloned and purified 
which do not show improved binding characteristics over C6.5 (see results 
of light chain shuffling and randomization below). 
H) Increasing the affinity of C6.5 by chain shuffling. 
To alter the affinity of C6.5, a mutant sFv gene repertoire was created 
containing the VH gene of C6.5 and a human VL gene repertoire (light chain 
shuffling). The sFv gene repertoire was cloned into the phage display 
vector pHEN-1 (Hoogenboom et al. (1991) Nucleic Acids Res., 19: 4133-4137) 
and after transformation a library of 2.times.10.sup.5 transformants was 
obtained. Phage were prepared and concentrated as described in Example 1 
or 2. 
Selections were performed by incubating the phage with biotinylated 
c-erbB-2 in solution. The antigen concentration was decreased each round, 
reaching a concentration less than the desired K.sub.d by the final rounds 
of selection. This results in the selection of phage on the basis of 
affinity (Hawkins et al. (1992) J. Mol. Biol. 226: 889-896). After four 
rounds of selection, 62/90 clones analyzed produced sFv which bound 
c-erbB-2 by ELISA. Single chain Fv was expressed from 48 ELISA positive 
clones (24 from the 3rd round of selection and 24 from the 4th round of 
selection), the periplasm harvested, and the sFv k.sub.off determined by 
BIAcore. Single-chain Fvs were identified with a k.sub.off three times 
slower than C6.5. The light chain gene of 10 of these sFvs was sequenced. 
One unique light chain was identified, C6L1. This sFv was subcloned into 
the hexahistidine vector, and expressed sFv purified by IMAC and gel 
filtration. Affinity was determined by BIAcore (Table 3). 
TABLE 3 
______________________________________ 
Affinity and kinetics of binding of C6.5 light and heavy chain 
shuffled mutant sFv. 
sFv clone K.sub.d (M) 
k.sub.on k.sub.off (s.sup.-1) 
______________________________________ 
C6.5 1.6 .times. 10.sup.-8 
4.0 .times. 10.sup.5 
6.3 .times. 10.sup.-3 
C6L1 (light chain shuffle) 2.6 .times. 10.sup.-9 7.8 .times. 10.sup.5 
2.0 .times. 10.sup.-3 
C6VHB-4 (heavy chain shuffle) 4.8 .times. 10.sup.-9 1.25 .times. 
10.sup.6 6.0 .times. 10.sup.-3 
C6VHC (heavy chain shuffle) 3.1 
.times. 10.sup.-9 8.4 .times. 
10.sup.5 2.6 .times. 10.sup.-3 
______________________________________ 
For heavy chain shuffling, the C6.5 VH CDR3 and light chain were cloned 
into a vector containing a human VH gene repertoire to create a phage 
antibody library of 1.times.10.sup.6 transformants. Selections were 
performed on biotinylated c-erbB-2 and after four rounds of selection, 
82/90 clones analyzed produced sFv which bound c-erbB-2 by ELISA- sFv was 
expressed from 24 ELISA positive clones (24 from the 3rd round of 
selection and 24 from the 4th round of selection), the periplasm 
harvested, and the sFv k.sub.off determined by BIAcore. Two clones were 
identified which had slower k.sub.off than C6.5 (C6VHB-4 and C6VHC-4). 
Both of these were subcloned, purified, and affinities determined by 
BIAcore (Table 3). The affinity of C6.5 was increased 5 fold by heavy 
chain shuffling and 6 fold by light chain shuffling. 
I) Increasing the affinity of C6-5 by site directed mutagenesis of the 
third CDR of the light chain. 
The majority of antigen contacting amino acid side chains are located in 
the complementarity determining regions (CDRs), three in the V.sub.H 
(CDR1, CDR2, and CDR3) and three in the V.sub.L (CDR1, CDR2, and CDR3) 
(Chothia et al. (1987) J. Mol. Biol., 196: 901-917; Chothia et al. (1986) 
Science, 233: 755-8; Nhan et al. (1991) J. Mol. Biol., 217: 133-151). 
These residues contribute the majority of binding energetics responsible 
for antibody affinity for antigen. In other molecules, mutating amino 
acids which contact ligand has been shown to be an effective means of 
increasing the affinity of one protein molecule for its binding partner 
(Lowman et al. (1993) J. Mol. Biol., 234: 564-578; Wells (1990) 
Biochemistry, 29: 8509-8516). Thus mutation (randomization) of the CDRs 
and screening against c-erbB-2 may be used to generate C6 antibodies 
having improved binding affinity. 
For example, to increase the affinity of C6.5 for c-erbB-2, nine amino acid 
residues located in VL CDR3 (residues 89-95b, numbering according to Kabat 
et al. (1987) supra.; Table 2). were partially randomized by synthesizing 
a `doped` oligonucleotide in which the wild type nucleotide occurred with 
a frequency of 49%. The oligonucleotide was used to amplify the remainder 
of the C6.5 sFv gene using PCR. The resulting sFv gene repertoire was 
cloned into pCANTAB5E (Pharmacia) to create a phage antibody library of 
1.times.10.sup.7 transformants. The mutant phage antibody library was 
designated C6VLCDR3. 
Selection of the C6.5 mutant VL CDR3 library (C6VLCDR3) was performed on 
biotinylated c-erbB-2 as described above for light chain shuffling. After 
three rounds of selection 82/92 clones analyzed produced sFv which bound 
c-erbB-2 by ELISA and after 4 rounds of selection, 92/92 clones analyzed 
produced sFv which bound c-erbB-2. Single-chain Fv was expressed from 24 
ELISA positive clones from the 3rd and 4th rounds of selection, the 
periplasm harvested, and the k.sub.off determined by BlAcore. The best 
clones had a k.sub.off approximately 5 to 10 times slower than that of 
C6.5. The light chain genes of 12 sFvs with the slowest k.sub.off times 
from the 3rd and fourth round of selection were sequenced and each unique 
sFv subcloned into pUC119 Sfi-NotmycHis. Single-chain Fv was expressed, 
purified by IMAC and gel filtration, and sFv affinity and binding kinetics 
determined by BlAcore (Table 4). Mutant sFv were identified with 16 fold 
increased affinity for c-erbB-2. 
TABLE 4 
__________________________________________________________________________ 
Amino acid sequence, affinity, and binding kinetics of sFv isolated from 
a library of C6.5 
mutants. Table identified mutants isolated after the third and fourth 
rounds of selection. 
The entire VL CDR3 of C6.5 is shown with the residues subjected to 
mutagenesis (89-95b) 
underlined. Rate constants k.sub.on and k.sub.off were measured on 
purified and gel 
filtered sFv by SPR in a BIAcore and the Kd calculated. A hyphen 
"-"indicates 
that there is no change from the C6.5 V.sub.L CDR3 sequence at that 
position. 
sFv clone 
V.sub.L CDR3 sequence 
k.sub.d (M) 
k.sub.on (M.sup.-1 s.sup.-1) 
k.sub.off (s.sup.-1) 
SEQ ID NO 
__________________________________________________________________________ 
C6.5 8 9 9 1.6 .times. 10.sup.-8 
4.0 .times. 10.sup.5 
6.3 .times. 10.sup.-3 
6 
9 5ab 7 
AAWDDSLSGWV 
3rd Round of selection: 
C6ML3-5 
--------Y------------ 
3.2 .times. 10.sup.-9 
5.9 .times. 10.sup.5 
1.9 .times. 10.sup.-3 
7 
C6ML3-2 --------H------------ 2.8 .times. 10.sup.-9 7.1 .times. 
10.sup.5 2.0 .times. 10.sup.-3 8 
C6ML3-6 --S----Y------------ 3.2 
.times. 10.sup.-9 5.9 .times. 10.sup.5 
1.9 .times. 10.sup.-3 9 
C6ML3-1 --------Y----W------ 6.7 .times. 10.sup.-9 3.0 .times. 10.sup.5 
2.0 .times. 10.sup.-3 10 
C6ML3-3 --T----YA---------- 4.3 .times. 10.sup.-9 4.6 .times. 10.sup.5 
2.0 .times. 10.sup.-3 11 
C6ML3-7 --------YAV-------- 2.6 .times. 10.sup.-9 6.5 .times. 10.sup.5 
1.7 .times. 10.sup.-3 12 
C6ML3-4 --S--EY----W------ 3.5 .times. 10.sup.-9 4.0 .times. 10.sup.5 
1.4 .times. 10.sup.-3 13 
4th Round of selection: 
C6ML3-12 
--------Y--R-------- 
1.6 .times. 10.sup.-9 
4.5 .times. 10.sup.5 
7.2 .times. 10.sup.-4 
14 
C6ML3-9 --S----YT---------- 1.0 .times. 10.sup.-9 6.1 .times. 10.sup.5 
9.2 .times. 10.sup.-4 15 
C6ML3-10 ------E--PWY------ 2.3 .times. 10.sup.-9 6.1 .times. 10.sup.5 
1.4 .times. 10.sup.-3 16 
C6ML3-11 --------YA--W------ 3.6 .times. 10.sup.-9 6.1 .times. 10.sup.5 
2.2 .times. 10.sup.-3 17 
C6ML3-13 --------AT--W------ 2.4 .times. 10.sup.-9 8.7 .times. 10.sup.5 
2.1 .times. 10.sup.-3 18 
C6ML3-8 --------HLRW------ 2.6 .times. 10.sup.-9 6.5 .times. 10.sup.5 
1.7 .times. 10.sup.-3 19 
C6ML3-23 --S----H----W------ 1.5 .times. 10.sup.-9 6.7 .times. 10.sup.5 
1.7 .times. 10.sup.-3 20 
C6ML3-19 --S----RP--W------ 1.5 .times. 10.sup.-9 6.7 .times. 10.sup.5 
1.0 .times. 10.sup.-3 21 
C6ML3-29 --------GT--W------ 2.7 .times. 10.sup.-9 12.9 .times. 
10.sup.5 2.2 .times. 10.sup.-3 22 
C6ML3-15 --------RP--W------ 2.2 
.times. 10.sup.-9 5.9 .times. 10.sup.5 
1.3 .times. 10.sup.-3 23 
C6ML3-14 ----------P--W------ 1.0 .times. 10.sup.-9 7.7 .times. 
10.sup.5 7.7 .times. 10.sup.-4 
__________________________________________________________________________ 
24 
Partial randomization of a single CDR (V.sub.L CDR3) resulted in the 
creation of mutant sFvs with 16 fold higher affinity for c-erbB-2, 
indicating that CDR randomization is an effective means of creating higher 
affinity sFv. The results also show that the method of selecting and 
identifying higher affinity sFv by reducing soluble antigen concentration 
during selections and screening periplasms by BIAcore prior to sequencing, 
subcloning and purification provides an effective way to isolate high 
affinity antibodies. 
J) Creation of C6.5 (sFv').sub.2 and (sFv).sub.2 homodimers and effect on 
affinity and binding kinetics for cerbB-2. 
To create C6 (sFv').sub.2 antibodies, two C6 sFvs are joined through a 
disulfide bond, or linker (e.g., a carbon linker) between the two 
cysteines. To create C6 (sFv).sub.2, two C6 sFv are joined directly 
through a peptide bond or through a peptide linker. Thus, for example, to 
create disulfide linked C6.5 sFv', a cysteine residue was introduced by 
site directed mutagenesis between the myc tag and hexahistidine tag at the 
carboxy-terminus of C6.5. Introduction of the correct sequence was 
verified by DNA sequencing. The construct is in pUC119, the pelB leader 
directs expressed sFv' to the periplasm and cloning sites (Ncol and Notl) 
exist to introduce C6.5 mutant sFv'. This vector is called pUC119/C6.5 
mycCysHis. Expressed sFv' has the myc tag at the C-terminus, followed by 2 
glycines, a cysteine, and then 6 histidines to facilitate purification by 
IMAC. After disulfide bond formation between the two cysteine residues, 
the two sFv' are separated from each other by 26 amino acids (two 11 amino 
acid myc tags and 4 glycines). An sFv' was expressed from this construct, 
purified by IMAC, and analyzed by gel filtration. The majority of the sFv' 
was monomeric. To produce (sFv').sub.2 dimers, the cysteine was reduced by 
incubation with 1 MM beta-mercaptoethanol, and half of the sFv' blocked by 
the addition of DTNB. Blocked and unblocked sFv's were incubated together 
to form (sFv').sub.2 and the resulting material analyzed by gel 
filtration. 50% of the monomer was converted to (sFv').sub.2 homodimer as 
determined by gel filtration and nonreducing polyacrylamide gel 
electrophoresis. The affinity of the C6.5 sFv' monomer and (sFv')2 dimer 
were determined by BIAcore (Table 5). The apparent affinity (avidity) of 
C6.5 increases 40 fold when converted to an (sFv').sub.2 homodimer. 
TABLE 5 
______________________________________ 
Affinities and binding kinetics of C6.5 sFv and C6.5 (sFv').sub.2. 
Clone K.sub.d (M) 
k.sub.on k.sub.off (s.sup.-1) 
______________________________________ 
C6.5 monomer 
1.6 .times. 10.sup.-8 
4.0 .times. 10.sup.5 
6.3 .times. 10.sup.-3 
C6.5 dimer 4.0 .times. 10.sup.-10 6.7 .times. 10.sup.5 2.7 .times. 
10.sup.-4 
______________________________________ 
The C6.5 (sFv').sub.2 exhibits a significant avidity effect compared to the 
sFv. Thus, this approach increases antibody fragment affinity, while 
remaining below the renal threshold for excretion. 
In a particularly preferred embodiment, the (sFv).sub.2 dimer is expressed 
as a diabody (Holliger et al. (1993) Proc. Natl. Acad. Sci. USA, 90: 
6444-6448; and WO 94/13804). This yields a bivalent molecule consisting of 
two C6.5 sFv polypeptide chains, since the VH and VL on the same peptide 
chain cannot pair. The production of a peptide linked C6.5 diabody is 
described in Example 5, below. In this example, the peptide linker 
sequence between the VH and VL domains was shortened from 15 amino acids 
to 5 amino acids. Synthetic oligonucleotides encoding the 5 amino acid 
linker (Gly.sub.4 Ser) (SEQ ID NO:25) were used to PCR amplify the C6.5 
V.sub.H and V.sub.L genes which were then spliced together to create the 
C6.5 diabody gene. The gene was then cloned into an appropriate vector, 
expressed, and purified according to standard methods well known to those 
of skill in the art. In another preferred embodiment, the (sFv).sub.2 
dimer is produced using a longer peptide liner that permits the Vh and Vl 
to pair, yielding a single polypeptide chain with two C6 binding sites. 
K) Effect of sFv affinity on in vitro cell binding and in vivo 
biodistribution. 
As described in the preceding section, chain-shuffled and point-mutation 
variants of C6.5 have been prepared with K.sub.d ranging from 
1.0.times.10-.sup.6 M to 1.0.times.10-.sup.9 M. The mutant sFv have been 
used to examine the effects of binding affinity and kinetics on in vitro 
cell binding and on in vivo biodistribution. Cell surface retention assays 
demonstrate that higher affinity sFv are retained to a much greater extent 
than lower affinity sFv. For sFv of approximately the same affinity, sFv 
with slower k.sub.off are better retained on the cell surface. In 
competitive binding assays, all of the molecules compete in a dose 
dependent fashion with biotinylated C6.5 for c-erbB-2 on the surface of 
SK-BR-3 cells. 
Twenty four hour biodistribution studies were performed in scid micebearing 
s.c. SK-OV-3 tumors to examine the role of affinity in the specificity and 
degree of tumorretention. These assays employed .sup.125 I-labeled forms 
of C6.5, C6G98A, C6ML3-9 and a negative control sFv at a dose of 25 mg. 
The c-erbB-2-specific sFv were selected to provide the following stepwise 
increase in affinity; C6G98A (3.2.times.10.sup.-7), C6.5 
(1.6.times.10.sup.-8) and C6ML3-9 (1.0.times.10.sup.-9). The 
biodistribution studies revealed a close correlation between the affinity 
and the % ID/g of the radioiodinated sFv retained in tumor. The greatest 
degree of tumor retention was observed with .sup.125 I-C6ML3-9 
(1.42.+-.0.23% ID/g). Significantly less tumor retention was achieved with 
.sup.125 I-C6.5 (0.80.+-.0.07% ID/g) (p=0.0306). Finally, the tumor 
retention of the lowest affinity clone .sup.125 I-C6G98A (0.19.+-.0.04% 
ID/g) was significantly less than that of C6.5 (p=0.00001) and was 
identical to that of the negative control .sup.125 I-26-10. The T:O ratios 
also reflected the greater retention of higher-affinity species in tumor. 
For example, tumor:blood ratios of 17.2, 13.3, 3.5 and 2.6, and tumor to 
liver ratios of 26.2, 19.8, 4.0 and 3.1 were observed for C6ML3-9, C6.5, 
C6G98A and 26-10, respectively. 
These results demonstrate that selective tumor retention of sFv molecules 
correlates with their affinity properties. With further increases in 
affinity, additional improvements in tumor retention are observed. 
L) Approach to produce higher affinity human sFv. 
As described above and in Examples 1 and 2, a C6 antibody (e.g. C6.5 sFv), 
which binds specifically to c-erbB-2, is expressed at high level in E. 
coli as native protein, and can be simply purified in high yield. 
Optimized techniques for creating large C6.5 mutant phage antibody 
libraries and developed techniques for efficiently selecting higher 
affinity mutants from these libraries are provided. These techniques were 
used to increase C6.5 affinity 16 fold, to 1.0.times.10.sup.-8 M, by 
randomizing VL CDR3, and 5 and 6 fold by heavy and light chain shuffling 
respectively. 
To further increase affinity, mutant C6.5 phage antibody libraries can be 
created where the other CDRs are randomized (V.sub.L CDR1 and CDR2 and 
V.sub.H CDR1, CDR2 and CDR3). Each CDR is randomized in a separate 
library, using, for example, C6ML3-9 as a template (K.sub.d 
=1.0.times.10.sup.-9 M). In a preferred embodiment, CDRs can be 
sequentially randomized, using the highest affinity sFv as the template 
for the next round of mutagenesis. This approach would be preferred when 
mutating CDRs that pack on each other, for example VL and VH CDR3. In 
another embodiment, CDRs could be mutated in parallel, and mutations 
combined to achieve an additive effect on affinity. This approach has been 
used to increase the affinity of human growth hormone (hGH) for the growth 
hormone receptor over 1500 fold from 3.4.times.10.sup.-10 to 
9.0.times.10.sup.-13 M (Lowman et al. (1993) J. Mol. Biol., 234: 564-578. 
V.sub.H CDR3 occupies the center of the binding pocket, and thus mutations 
in this region are likely to result in an increase in affinity (Clackson 
et al. (1995) Science, 267: 383-386). In one embodiment, four V.sub.H CDR3 
residues at a time are randomized using the nucleotides NNS. To create the 
library, an oligonucleotide is synthesized which anneals to the C6.5 
V.sub.H framework 3 and encodes V.sub.H CDR3 and a portion of framework 4. 
At the four positions to be randomized, the sequence NNS is used, where 
N=any of the 4 nucleotides, and S=C or T. The oligonucleotide are used to 
amplify the C6.5 V.sub.H gene using PCR, creating a mutant C6.5 VH gene 
repertoire. PCR is used to splice the VH gene repertoire with the C6ML3-9 
light chain gene, and the resulting sFv gene repertoire cloned into the 
phage display vector pHEN-1. Ligated vector DNA is used to transform 
electrocompetent E. coli to produce a phage antibody library of 
&gt;1.0.times.10.sup.7 clones. 
To select higher affinity mutant sFv, each round of selection of the phage 
antibody libraries is conducted on decreasing amounts of biotinylated 
c-erbB-2, as described in the Examples. Typically, 96 clones from the 
third and fourth round of selection are screened for binding to c-erbB-2 
by ELISA on 96 well plates. Single-chain Fv from twenty to forty ELISA 
positive clones are expressed in 10 ml cultures, the periplasm harvested, 
and the sFv k.sub.off determined by BIAcore. Clones with the slowest 
k.sub.off are sequenced, and each unique sFv subcloned into pUC119 
SfiNotmycHis. Single chain Fv is expressed in 1 L cultures, and purified 
as described supra. Affinities of purified sFv are determined by BIAcore. 
Randomization of one four amino acid segment of V.sub.H CDR3 produces a C6 
mutant with a K.sub.D of 1.6.times.10.sup.-10 M (see Example 3). 
M) In vitro cell binding assays, in vivo pharmacokinetic and 
biodistribution studies. 
Once higher affinity sFv's are identified, production is scaled up to 
provide adequate material for in vitro cell binding assays and in vivo 
pharmacokinetic and biodistribution studies. Techniques for scaling up 
production are known. Briefly, in one embodiment, sFv is expressed in E. 
coli cultures grown in 2 liter shaker flasks. Single-chain Fv is purified 
from the periplasm as described above and in Examples 1 and 2. Mutant sFv 
of higher affinity are tested using the cell retention assay described in 
Examples 1 and 2. Since the t.sub.1/2 of retention should be approximately 
two hours when k.sub.off is less than 10.sup.-4, the assay is done at 30, 
60, 120, 240 minutes and 18 hour incubations. Scatchard analyses may be 
performed on selected samples. 
These studies show that affinities measured in the BlAcore on immobilized 
antigen correspond to improved cell binding. The pharmacokinetic and 
biodistribution properties of sFv molecules with broadly different 
affinity characteristics are screened using labeled sFv and scid mice 
bearing human SK-OV-3 tumors. This serves to identify molecules with in 
vivo properties that make them unsuitable for use as therepeutics i.e., 
unexpected aggregation, or unacceptable normal organ retention properties. 
Twenty four hour biodistribution results are convenient indicators of 
overall biodistribution properties. C6 antibodies, for example C6.5 
mutants, with affinities between 1.6.times.10.sup.-8 M and 
1.0.times.10.sup.-11 M, and which differ at least 3 to 4 fold in affinity, 
are screened. Mutants with similar K.sub.d but with dissimilar k.sub.off 
are also studied. A number of C6.5 series affinity variants are tested and 
more extensive biodistribution studies performed on molecules that differ 
significantly from C6.5 or the nearest affinity variant in 24 hour 
biodistribution characteristics. These data are used to generate 
tissue-specific AUC determinations, as well as tumor:normal organ AUC 
ratios and MIRD estimates. 
Sample molecules associated with favorable predicted human dosimetry (e.g., 
based upon the MIRD formulation) are assayed for their in vivo therapeutic 
efficacy in mice. 
An affinity of 1.0.times.10.sup.-11 can be chosen as an endpoint in this 
preferred embodiment because the associated k.sub.off (10.sup.-5) results 
in a t.sub.1/2 for dissociation from tumor of greater than 20 hours. 
Higher affinity endpoints can be selected and result in even longer 
retention. The t.sub.1/2 for dissociation of C6.5 is approximately 3 
minutes. This invention provides optimized techniques for creating large 
C6.5 mutant phage antibody libraries and techniques for efficiently 
selecting higher affinity mutants from these libraries. A number of C6.5 
mutants with affinities between 1.6.times.10.sup.-8 M to 
1.0.times.10.sup.-10 M are provided. Combining these mutations into the 
same sFv produces sFv mutants with K.sub.d between 1.6.times.10.sup.-10 M 
and 3.3.times.10.sup.-11 M. 
N) Preparation of C6 (sFv).sub.2,(sFv').sub.2 Fab, and (Fab').sub.2 
conjugates and diabodies. 
C6 antibodies such as C6.5 sFv, or a variant with higher affinity, are 
suitable templates for creating size and valency variants. For example, a 
C6.5 (sFv').sub.2 is created from the parent sFv as described above and in 
Example 1. An sFv' can be created by excising teh sFv gene, e.g., with 
Ncol and Notl from pHEN-1 or pUC119 Sfi-NotmycHis and cloned into 
pUC119C6.5mycCysHis, cut with Ncol and Notl. In one embodiment, expressed 
sFv' has a myc tag at the C-terminus, followed by 2 glycines, a cysteine, 
and 6 histidines to facilitate purification. After disulfide bond 
formation between the two cysteine residues, the two sFv should be 
separated from each other by 26 amino acids (e.g., two 11 amino acid myc 
tags and 4 glycines). SFv is expressed from this construct and purified. 
To produce (sFv')2 dimers, the cysteine is reduced by incubation with 1 Mm 
beta-mercaptoethanol, and half of the sFv blocked by the addition of DTNB. 
Blocked and unblocked sFv are incubated together to form (sFv')2, which is 
purified. This approach was used to produce C6.5 (sFv')2 dimer, which 
demonstrates a 40 fold higher affinity than C6.5. A (sFv')2 may be 
constructed for example, from C6L1 (K.sub.d =2.5.times.10.sup.-9 M) and 
C6ML3-9 (K.sub.d =1.0.times.10.sup.-9 M). As higher affinity sFv become 
available, their genes are similarly used to construct (sFv').sub.2. 
Alternatively, C6 (sFv).sub.2 can be produced by linking the two sFv by a 
peptide, as described in Example 5. As higher affinity sFv become 
available their genes can be used to construct higher affinity 
(sFv).sub.2. 
C6.5 based Fab are expressed in E. coli using an expression vector similar 
to the one described by Better et. al. (Better et al. (1988) Science, 240: 
1041-1043). To create a C6.5 based Fab, the VH and VL genes are amplified 
from the sFv using PCR. The VH gene is cloned into a PUC119 based 
bacterial expression vector which provides the human IgG CH1 domain 
downstream from, and in frame with, the V.sub.H gene. The vector also 
contains the lac promoter, a pelb leader sequence to direct expressed 
V.sub.H -CH1 domain into the periplasm, a gene 3 leader sequence to direct 
expressed light chain into the periplasm, and cloning sites for the light 
chain gene. Clones containing the correct VH gene are identified, e.g., by 
PCR fingerprinting. The VL gene is spliced to the C.sub.L gene using PCR 
and cloned into the vector containing the V.sub.H CH1 gene. 
III. Preparation of Chineric Molecules. 
In another embodiment this invention provides for chimeric molecules 
comprising a C6 antibody attached to an effector molecule. As explained 
above, the effector molecule component of the chimeric molecules of this 
invention may be any molecule whose activity it is desired to deliver to 
cells that express c-erbB-2. Suitable effector molecules include 
cytotoxins such as PE, Ricin, Abrin or DT, radionuclides, ligands such as 
growth factors, antibodies, detectable labels such as fluorescent or 
radioactive labels, and therapeutic compositions such as liposomes and 
various drugs. 
A) Cytotoxins. 
Particularly preferred cytotoxins include Pseudomonas exotoxins, Diphtheria 
toxins, ricin, and abrin. Pseudomonas exotoxin and Dipthteria toxin, in 
particular, are frequently used in chimeric cytotoxins. 
i) Pseudomonas exotoxin (PE). 
Pseudomonas exotoxin A (PE) is an extremely active monomeric protein 
(molecular weight 66 kD), secreted by Pseudomonas aeruginosa, which 
inhibits protein synthesis in eukaryotic cells through the inactivation of 
elongation factor 2 (EF-2) by catalyzing its ADP-ribosylation (catalyzing 
the transfer of the ADP ribosyl moiety of oxidized NAD onto EF-2). 
The toxin contains three structural domains that act in concert to cause 
cytotoxicity. Domain Ia (amino acids 1-252) mediates cell binding. Domain 
II (amino acids 253-364) is responsible for translocation into the cytosol 
and domain III (amino acids 400-613) mediates ADP ribosylation of 
elongation factor 2, which inactivates the protein and causes cell death. 
The function of domain Ib (amino acids 365-399) remains undefined, 
although a large part of it, amino acids 365-380, can be deleted without 
loss of cytotoxicity. See Siegall et al., J. Biol. Chem. 264: 14256-14261 
(1989). 
For maximum cytotoxic properties of a preferred PE molecule, several 
modifications to the molecule are recommended. An appropriate carboxyl 
terminal sequence to the recombinant molecule is preferred to translocate 
the molecule into the cytosol of target cells. Amino acid sequences which 
have been found to be effective include, REDLK (SEQ ID NO:26) (as in 
native PE), REDL (SEQ ID NO:27), RDEL (SEQ ID NO:28), or KDEL (SEQ ID 
NO:29), repeats of those, or other sequences that function to maintain or 
recycle proteins into the endoplasmic reticulum, referred to here as 
"endoplasmic retention sequences". See, for example, Chaudhary et al, 
Proc. Natl. Acad. Sci. USA 87:308-312 and Seetharam et al, J. Biol. Chem. 
266: 17376-17381 (1991). 
The targeting molecule can be inserted in replacement for domain Ia. A 
similar insertion has been accomplished in what is known as the 
TGF.alpha.-PE40 molecule (also referred to as TP40) described in Heimbrook 
et al., Proc. Natl. Acad. Sci., USA, 87: 4697-4701 (1990). See also, 
Debinski et al. Bioconj. Chem., 5: 40 (1994) for other PE variants). 
The PE molecules can be fused to the C6 antibody by recombinant means. The 
genes encoding protein chains may be cloned in cDNA or in genomic form by 
any cloning procedure known to those skilled in the art. See for example 
Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring 
Harbor Laboratory, (1989). Methods of cloning genes encoding PE fused to 
various ligands are well known to those of skill in the art. See, for 
example, Siegall et al., FASEB J., 3: 2647-2652 (1989); Chaudhary et al. 
Proc. Natl. Acad. Sci. USA, 84: 4538-4542 (1987). 
Those skilled in the art will realize that additional modifications, 
deletions, insertions and the like may be made to the chimeric molecules 
of the present invention or to the nucleic acid sequences encoding the C6 
chimeric molecules. Especially, deletions or changes may be made in PE or 
in a linker connecting an antibody gene to PE, in order to increase 
cytotoxicity of the fusion protein toward target cells or to decrease 
nonspecific cytotoxicity toward cells without antigen for the antibody. 
All such constructions may be made by methods of genetic engineering well 
known to those skilled in the art (see, generally, Sambrook et al., supra) 
and may produce proteins that have differing properties of affinity, 
specificity, stability and toxicity that make them particularly suitable 
for various clinical or biological applications. 
ii) Diphtheria toxin (DT). 
Like PE, diphtheria toxin (DT) kills cells by ADP-ribosylating elongation 
factor 2 (EF-2) thereby inhibiting protein synthesis. Diphtheria toxin, 
however, is divided into two chains, A and B, linked by a disulfide 
bridge. In contrast to PE, chain B of DT, which is on the carboxyl end, is 
responsible for receptor binding and chain A, which is present on the 
amino end, contains the enzymatic activity (Uchida et al., Science, 175: 
901-903 (1972); Uchida et al. J. Biol. Chem., 248: 3838-3844 (1973)). 
The targeting molecule-Diphtheria toxin fusion proteins of this invention 
may have the native receptor-binding domain removed by truncation of the 
Diphtheria toxin B chain. DT388, a DT in which the carboxyl terminal 
sequence beginning at residue 389 is removed is illustrated in Chaudhary, 
et al., Bioch. Biophys. Res. Comm., 180: 545-551 (1991). 
Like the PE chimeric cytotoxins, the DT molecules may be chemically 
conjugated to the C6 antibody but, may also be prepared as fusion proteins 
by recombinant means. The genes encoding protein chains may be cloned in 
cDNA or in genomic form by any cloning procedure known to those skilled in 
the art. Methods of cloning genes encoding DT fused to various ligands are 
also well known to those of skill in the art. See, for example, Williams 
et al. J. Biol. Chem. 265: 11885-11889 (1990) which describes the 
expression of growth-factor-DT fusion proteins. 
The term "Diphtheria toxin" (DT) as used herein refers to full length 
native DT or to a DT that has been modified. Modifications typically 
include removal of the targeting domain in the B chain and, more 
specifically, involve truncations of the carboxyl region of the B chain. 
B) Detectable labels. 
Detectable labels suitable for use as the effector molecule component of 
the chimeric molecules of this invention include any composition 
detectable by spectroscopic, photochemical, biochemical, immunochemical, 
electrical, optical or chemical means. Useful labels in the present 
invention include magnetic beads (e.g. Dynabeads.TM.), fluorescent dyes 
(e.g., fluorescein isothiocyanate, texas red, rhodamine, green fluorescent 
protein, and the like), radiolabels (e.g., .sup.3 H, .sup.125 I, .sup.35 
S, .sup.14 C, or 32P), enzymes (e.g., horse radish peroxidase, alkaline 
phosphatase and others commonly used in an ELISA), and colorimetric labels 
such as colloidal gold or colored glass or plastic (e.g. polystyrene, 
polypropylene, latex, etc.) beads. Patents teaching the use of such labels 
include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 
4,277,437; 4,275,149; and 4,366,241. 
Means of detecting such labels are well known to those of skill in the art. 
Thus, for example, radiolabels may be detected using photographic film or 
scintillation counters, fluorescent markers may be detected using a 
photodetector to detect emitted illumination. Enzymatic labels are 
typically detected by providing the enzyme with a substrate and detecting 
the reaction product produced by the action of the enzyme on the 
substrate, and colorimetric labels are detected by simply visualizing the 
colored label. 
C) Ligands. 
As explained above, the effector molecule may also be a ligand or an 
antibody. Particularly preferred ligand and antibodies are those that bind 
to surface markers of immune cells. Chimeric molecules utilizing such 
antibodies as effector molecules act as bifunctional linkers establishing 
an association between the immune cells bearing binding partner for the 
ligand or antibody and the tumor cells expressing the c-erbB-2. Suitable 
antibodies and growth factors are known to those of skill in the art and 
include, but are not limited to, IL-2, IL-4, IL-6, IL-7, tumor necrosis 
factor (TNF), anti-Tac, TGF.alpha., and the like. 
D) Other therapeutic moieties. 
Other suitable effector molecules include pharmacological agents or 
encapsulation systems containing various pharmacological agents. Thus, the 
C6 antibody may be attached directly to a drug that is to be delivered 
directly to the tumor. Such drugs are well known to those of sldll in the 
art and include, but are not limited to, doxirubicin, vinblastine, 
genistein, antisense molecules, ribozymes and the like. 
Alternatively, the effector molecule may comprise an encapsulation system, 
such as a liposome or micelle that contains a therapeutic composition such 
as a drug, a nucleic acid (e.g. an antisense nucleic acid), or another 
therapeutic moiety that is preferably shielded from direct exposure to the 
circulatory system. Means of preparing liposomes attached to antibodies 
are well known to those of skill in the art. See, for example, U.S. Pat. 
No. 4,957,735 and Connor et al., Pharm. Ther., 28: 341-365 (1985). 
E) Attachment of the c6 antibody to the effector molecule. 
One of skill will appreciate that the C6 antibody and the effector molecule 
may be joined together in any order. Thus the effector molecule may be 
joined to either the amino or carboxy termini of the C6 antibody. The C6 
antibody may also be joined to an internal region of the effector 
molecule, or conversely, the effector molecule may be joined to an 
internal location of the C6 antibody as long as the attachment does not 
interfere with the respective activities of the molecules. 
The C6 antibody and the effector molecule may be attached by any of a 
number of means well known to those of skill in the art. Typically the 
effector molecule is conjugated, either directly or through a linker 
(spacer), to the C6 antibody. However, where the effector molecule is a 
polypeptide it is preferable to recombinantly express the chimeric 
molecule as a single-chain fusion protein. 
i) Conjugation of the effector molecule to the targeting molecule. 
In one embodiment, the targeting molecule C6 antibody is chemically 
conjugated to the effector molecule (e.g. a cytotoxin, a label, a ligand, 
or a drug or liposome). Means of chemically conjugating molecules are well 
known to those of skill (see, for example, Chapter 4 in Monoclonal 
Antibodies: Principles and Applications, Birch and Lennox, eds. John Wiley 
& Sons, Inc. N.Y. (1995) which describes conjugation of antibodies to 
anticancer drugs, labels including radio labels, enzymes, and the like). 
The procedure for attaching an agent to an antibody or other polypeptide 
targeting molecule will vary according to the chemical structure of the 
agent. Polypeptides typically contain variety of functional groups; e.g., 
carboxylic acid (COOH) or free amine (--NH.sub.2) groups, which are 
available for reaction with a suitable functional group on an effector 
molecule to bind the effector thereto. 
Alternatively, the targeting molecule and/or effector molecule may be 
derivatized to expose or attach additional reactive functional groups. The 
derivatization may involve attachment of any of a number of linker 
molecules such as those available from Pierce Chemical Company, Rockford 
Ill. 
A "linker", as used herein, is a molecule that is used to join the 
targeting molecule to the effector molecule. The linker is capable of 
forming covalent bonds to both the targeting molecule and to the effector 
molecule. Suitable linkers are well known to those of skill in the art and 
include, but are not limited to, straight or branched-chain carbon 
linkers, heterocyclic carbon linkers, or peptide linkers. Where the 
targeting molecule and the effector molecule are polypeptides, the linkers 
may be joined to the constituent amino acids through their side groups 
(e.g., through a disulfide linkage to cysteine). However, in a preferred 
embodiment, the linkers will be joined to the alpha carbon amino and 
carboxyl groups of the terminal amino acids. 
A bifunctional linker having one functional group reactive with a group on 
a particular agent, and another group reactive with an antibody, may be 
used to form the desired immunoconjugate. Alternatively, derivatization 
may involve chemical treatment of the targeting molecule, e.g., glycol 
cleavage of a sugar moiety attached to the protein antibody with periodate 
to generate free aldehyde groups. The free aldehyde groups on the antibody 
may be reacted with free amine or hydrazine groups on an agent to bind the 
agent thereto. (See U.S. Pat. No. 4,671,958). Procedures for generation of 
free sulfhydryl groups on polypeptide, such as antibodies or antibody 
fragments, are also known (See U.S. Pat. No. 4,659,839). 
Many procedure and linker molecules for attachment of various compounds 
including radionuclide metal chelates, toxins and drugs to proteins such 
as antibodies are known. See, for example, European Patent Application No. 
188,256; U.S. Pat. Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784; 
4,680,338; 4,569,789; and 4,589,071; and Borlinghaus et al. Cancer Res. 
47: 4071-4075 (1987) which are incorporated herein by reference. In 
particular, production of various immunotoxins is well-known within the 
art and can be found, for example in "Monoclonal Antibody-Toxin 
Conjugates: Aiming the Magic Bullet," Thorpe et al., Monoclonal Antibodies 
in Clinical Medicine, Academic Press, pp. 168-190 (1982), Waldmann, 
Science, 252: 1657 (1991), U.S. Pat. Nos. 4,545,985 and 4,894,443. 
In some circumstances, it is desirable to free the effector molecule from 
the targeting molecule when the chimeric molecule has reached its target 
site. Therefore, chimeric conjugates comprising linkages which are 
cleavable in the vicinity of the target site may be used when the effector 
is to be released at the target site. Cleaving of the linkage to release 
the agent from the antibody may be prompted by enzymatic activity or 
conditions to which the immunoconjugate is subjected either inside the 
target cell or in the vicinity of the target site. When the target site is 
a tumor, a linker which is cleavable under conditions present at the tumor 
site (e.g. when exposed to tumor-associated enzymes or acidic pH) may be 
used. 
A number of different cleavable linkers are known to those of skill in the 
art. See U.S. Pat. Nos. 4,618,492; 4,542,225, and 4,625,014. The 
mechanisms for release of an agent from these linker groups include, for 
example, irradiation of a photolabile bond and acid-catalyzed hydrolysis. 
U.S. Pat. No. 4,671,958, for example, includes a description of 
immunoconjugates comprising linkers which are cleaved at the target site 
in vivo by the proteolytic enzymes of the patient's complement system. In 
view of the large number of methods that have been reported for attaching 
a variety of radiodiagnostic compounds, radiotherapeutic compounds, drugs, 
toxins, and other agents to antibodies one skilled in the art will be able 
to determine a suitable method for attaching a given agent to an antibody 
or other polypeptide. 
ii) Production of fusion proteins. 
Where the C6 antibody and/or the effector molecule are relatively short 
(i.e., less than about 50 amino acids) they may be synthesized using 
standard chemical peptide synthesis techniques. Where both molecules are 
relatively short the chimeric molecule may be synthesized as a single 
contiguous polypeptide. Alternatively the C6 antibody and the effector 
molecule may be synthesized separately and then fused by condensation of 
the amino terminus of one molecule with the carboxyl terminus of the other 
molecule thereby forming a peptide bond. Alternatively, the targeting and 
effector molecules may each be condensed with one end of a peptide spacer 
molecule thereby forming a contiguous fusion protein. 
Solid phase synthesis in which the C-terminal amino acid of the sequence is 
attached to an insoluble support followed by sequential addition of the 
remaining amino acids in the sequence is the preferred method for the 
chemical synthesis of the polypeptides of this invention. Techniques for 
solid phase synthesis are described by Barany and Merrifield, Solid-Phase 
Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, 
Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A., 
Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156 (1963), and Stewart et 
al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, 
Ill. (1984). 
In a preferred embodiment, the chimeric fusion proteins of the present 
invention are synthesized using recombinant DNA methodology. Generally 
this involves creating a DNA sequence that encodes the fusion protein, 
placing the DNA in an expression cassette under the control of a 
particular promoter, expressing the protein in a host, isolating the 
expressed protein and, if required, renaturing the protein. 
DNA encoding the fusion proteins (e.g. C6.5Ab-PE) of this invention may be 
prepared by any suitable method, including, for example, cloning and 
restriction of appropriate sequences or direct chemical synthesis by 
methods such as the phosphotriester method of Narang et al. Meth. Enzymol. 
68: 90-99 (1979); the phosphodiester method of Brown et al., Meth. Enzmol. 
68: 109-151 (1979); the diethylphosphoramidite method of Beaucage et al., 
Tetra. Lett., 22: 1859-1862 (1981); and the solid support method of U.S. 
Pat. No. 4,458,066. 
Chemical synthesis produces a single stranded oligonucleotide. This may be 
converted into double stranded DNA by hybridization with a complementary 
sequence, or by polymerization with a DNA polymerase using the single 
strand as a template. One of sldll would recognize that while chemical 
synthesis of DNA is limited to sequences of about 100 bases, longer 
sequences may be obtained by the ligation of shorter sequences. 
Alternatively, subsequences may be cloned and the appropriate subsequences 
cleaved using appropriate restriction enzymes. The fragments may then be 
ligated to produce the desired DNA sequence. 
In a preferred embodiment, DNA encoding fusion proteins of the present 
invention may be cloned using DNA amplification methods such as polymerase 
chain reaction (PCR). Thus, for example, the gene for the C6 antibody may 
be amplified from a nucleic acid template (clone) using a sense primer 
containing a first restriction site and an antisense primer containing a 
second restriction site. This produces a nucleic acid encoding the mature 
C6 antibody sequence and having terminal restriction sites. A cytotoxin 
(or other polypeptide effector) may be cut out of a plasmid encoding that 
effector using restriction enzymes to produce cut ends suitable for 
annealing to the C6 antibody. Ligation of the sequences and introduction 
of the construct into a vector produces a vector encoding the C6-effector 
molecule fusion protein. Such PCR cloning methods are well known to those 
of skill in the art (see, for example, Debinski et al. Int. J. Cancer, 58: 
744-748 (1994), for an example of the preparation of a PE fusion protein). 
While the two molecules may be directly joined together, one of skill will 
appreciate that the molecules may be separated by a peptide spacer 
consisting of one or more amino acids. Generally the spacer will have no 
specific biological activity other than to join the proteins or to 
preserve some minimum distance or other spatial relationship between them. 
However, the constituent amino acids of the spacer may be selected to 
influence some property of the molecule such as the folding, net charge, 
or hydrophobicity. One of skill will appreciate that PCR primers may be 
selected to introduce an amino acid linker or spacer between the C6 
antibody and the effector molecule if desired. 
The nucleic acid sequences encoding the fusion proteins may be expressed in 
a variety of host cells, including E. coli, other bacterial hosts, yeast, 
and various higher eukaryotic cells such as the COS, CHO and HeLa cells 
lines and myeloma cell lines. The recombinant protein gene will be 
operably linked to appropriate expression control sequences for each host. 
For E. coil this includes a promoter such as the 17, trp, or lambda 
promoters, a ribosome binding site and preferably a transcription 
termination signal. For eukaryotic cells, the control sequences will 
include a promoter and preferably an enhancer derived from immunoglobulin 
genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence, and 
may include splice donor and acceptor sequences. 
The plasmids of the invention can be transferred into the chosen host cell 
by well-known methods such as calcium chloride transformation for E. coli 
and calcium phosphate treatment or electroporation for mammalian cells. 
Cells transformed by the plasmids can be selected by resistance to 
antibiotics conferred by genes contained on the plasmids, such as the amp, 
gpt, neo and hyg genes. 
Once expressed, the recombinant fusion proteins can be purified according 
to standard procedures of the art, including ammonium sulfate 
precipitation, affinity columns, column chromatography, gel 
electrophoresis and the like (see, generally, R. Scopes, Protein 
Purification, Springer-Verlag, N.Y. (1982), Deutscher, Methods in 
Enzymology Vol. 182: Guide to Protein Purification., Academic Press, Inc. 
N.Y. (1990)). In a preferred embodiment, the fusion proteins are purified 
using affinity purification methods as described in Examples 1 and 2. 
Substantially pure compositions of at least about 90 to 95% homogeneity 
are preferred, and 98 to 99% or more homogeneity are most preferred for 
pharmaceutical uses. Once purified, partially or to homogeneity as 
desired, the polypeptides may then be used therapeutically. 
One of skill in the art would recognize that after chemical synthesis, 
biological expression, or purification, the C6 antibody-effector fusion 
protein may possess a conformation substantially different than the native 
conformations of the constituent polypeptides. In this case, it may be 
necessary to denature and reduce the polypeptide and then to cause the 
polypeptide to re-fold into the preferred conformation. Methods of 
reducing and denaturing proteins and inducing re-folding are well known to 
those of skill in the art. (See, Debinski et al. J. Biol. Chem., 268: 
14065-14070 (1993); Kreitman and Pastan, Bioconjug. Chem., 4: 581-585 
(1993); and Buchner, et al., Anal. Biochem., 205: 263-270 (1992). Debinski 
et al., for example, describe the denaturation and reduction of inclusion 
body proteins in guanidine-DTE. The protein is then refolded in a redox 
buffer containing oxidized glutathione and L-arginine. 
One of skill would recognize that modifications can be made to the C6 
antibody-effector fusion proteins without diminishing their biological 
activity. Some modifications may be made to facilitate the cloning, 
expression, or incorporation of the targeting molecule into a fusion 
protein. Such modifications are well known to those of skill in the art 
and include, for example, a methionine added at the amino terminus to 
provide an initiation site, or additional amino acids placed on either 
terminus to create conveniently located restriction sites or termination 
codons. 
IV. Diagnostic Assays. 
As explained above, the C6 antibodies may be used for the in vivo or in 
vitro detection of c-erbB-2 and thus, in the diagnosis and/or localization 
of cancers characterized by the expression of c-erbB-2. 
A) In Vivo Detection of c-erbB-2. 
The C6 antibodies and/or chimeric molecules of the present invention may be 
used for in vivo detection and localization of cells (e.g. c-erbB-2 
positive carcinoma) bearing c-erbB-2. Such detection involves 
administering to an organism a chimeric molecule comprising a C6 joined to 
a label detectable in vivo. Such labels are well known to those of skill 
in the art and include, but are not limited to, electron dense labels such 
as gold or barium which may be detected by X-ray or CAT scan, various 
radioactive labels that may be detected using scintillography, and various 
magnetic and paramagnetic materials that may be detected using positron 
emission tomography (PET) and magnetic resonance imaging (MRI). The C6 
antibody associates the label with the c-erbB-2 bearing cell which is then 
detected and localized using the appropriate detection method. 
B) In Vitro Detection of c-erbB-2. 
The C6 antibodies of this invention are also useful for the detection of 
c-erbB-2 in vitro e.g., in biological samples obtained from an organism. 
The detection and/or quantification of c-erbB-2 in such a sample is 
indicative the presence or absence or quantity of cells (e.g., tumor 
cells) overexpressing c-erbB-2. 
The c-erbB-2 antigen may be quantified in a biological sample derived from 
a patient such as a cell, or a tissue sample derived from a patient. As 
used herein, a biological sample is a sample of biological tissue or fluid 
that contains a c-erbB-2 antigen concentration that may be correlated with 
and indicative of cells overexpressing c-erbB-2. Preferred biological 
samples include blood, urine, and tissue biopsies. 
In a particularly preferred embodiment, erbB-2 is quantified in breast 
tissue cells derived from normal or malignant breast tissue samples. 
Although the sample is typically taken from a human patient, the assays 
can be used to detect erbB-2 in cells from mammals in general, such as 
dogs, cats, sheep, cattle and pigs, and most particularly primates such as 
humans, chimpanzees, gorillas, macaques, and baboons, and rodents such as 
mice, rats, and guinea pigs. 
Tissue or fluid samples are isolated from a patient according to standard 
methods well known to those of skill in the art, most typically by biopsy 
or venipuncture. The sample is optionally pretreated as necessary by 
dilution in an appropriate buffer solution or concentrated, if desired. 
Any of a number of standard aqueous buffer solutions, employing one of a 
variety of buffers, such as phosphate, Tris, or the like, at physiological 
pH can be used. 
C) Assay Formats (Detection or Quantification of c-erbB2). 
i) Immunological Binding Assavs 
The c-erbB-2 peptide (analyte) or an anti-c-erb-2 antibody is preferably 
detected in an immunoassay utilizing a C6 antibody as a capture agent that 
specifically binds to a c-erbB-2 peptide. 
As used herein, an immunoassay is an assay that utilizes an antibody (e.g. 
a C6 antibody) to specifically bind an analyte (e.g., c-erb-2). The 
immunoassay is characterized by the use of specific binding to a C6 
antibody as opposed to other physical or chemical properties to isolate, 
target, and quantify the c-erbB-2 analyte. 
The c-erbB-2 marker may be detected and quantified using any of a number of 
well recognized immunological binding assays. (See for example, U.S. Pat. 
Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168, which are hereby 
incorporated by reference.) For a review of the general immunoassays, see 
also Methods in Cell Biology Volume 37: Antibodies in Cell Biology, Asai, 
ed. Academic Press, Inc. New York (1993); Basic and Clinical Immunology 
7th Edition, Stites & Terr, eds. (1991)). 
The immunoassays of the present invention are performed in any of several 
configurations, e.g., those reviewed in Maggio (ed.) (1980) Enzyme 
Immunoassay CRC Press, Boca Raton, Fla.; Tijan (1985) "Practice and Theory 
of Enzyme Immunoassays," Laboratory Techniques in Biochemistry and 
Molecular Biology, Elsevier Science Publishers B. V., Amsterdam; Harlow 
and Lane, supra; Chan (ed.) (1987) Immunoassay: A Practical Guide Academic 
Press, Orlando, Fla.; Price and Newman (eds.) (1991) Principles and 
Practice of Immunoassays Stockton Press, NY; and Ngo (ed.) (1988) Non 
isotopic Immunoassays Plenum Press, NY. 
Immunoassays often utilize a labeling agent to specifically bind to and 
label the binding complex formed by the capture agent and the analyte 
(i.e., a C6 antibody-erbB-2 complex). The labeling agent may itself be one 
of the moieties comprising the antibody/analyte complex. Thus, the 
labeling agent may be a labeled c-erbB-2 peptide or a labeled C6 antibody. 
Alternatively, the labeling agent is optionally a third moiety, such as 
another antibody, that specifically binds to the C6 antibody, the c-erbB-2 
peptide, the anti-c-erbB-2 antibody/c-erbB-2 peptide complex, or to a 
modified capture group (e.g., biotin) which is covalently linked to 
c-erbB-2 or the C6 antibody. 
In one embodiment, the labeling agent is an antibody that specifically 
binds to the C6 antibody. Such agents are well known to those of skill in 
the art, and most typically comprise labeled antibodies that specifically 
bind antibodies of the particular animal species from which the C6 
antibody is derived (e.g., an anti-species antibody). Thus, for example, 
where the capture agent is a human derived C6 antibody, the label agent 
may be a mouse anti-human IgG, i.e., an antibody specific to the constant 
region of the human antibody. 
Other proteins capable of specifically binding immunoglobulin constant 
regions, such as streptococcal protein A or protein G are also used as the 
labeling agent. These proteins are normal constituents of the cell walls 
of streptococcal bacteria. They exhibit a strong non immunogenic 
reactivity with immunoglobulin constant regions from a variety of species. 
See, generally Kronval, et al., (1973) J. Immunol., 111: 1401-1406, and 
Akerstrom, et al., (1985) J. Immunol., 135:2589-2542. 
Throughout the assays, incubation and/or washing steps may be required 
after each combination of reagents. Incubation steps can vary from about 5 
seconds to several hours, preferably from about 5 minutes to about 24 
hours. However, the incubation time will depend upon the assay format, 
analyte, volume of solution, concentrations, and the like. Usually, the 
assays are carried out at ambient temperature, although they can be 
conducted over a range of temperatures, such as 5.degree. C. to 45.degree. 
C. 
(a) Non competitive assay fonnats. 
Immunoassays for detecting c-erb-2 are typically either competitive or 
noncompetitive. Noncompetitive immunoassays are assays in which the amount 
of captured analyte (in this case, c-erb-2) is directly measured. In one 
preferred "sandwich" assay, for example, the capture agent (e.g., C6 
antibody) is bound directly or indirectly to a solid substrate where it is 
immobilized. These immobilized C6 antibodies capture c-erb-2 present in a 
test sample (e.g., a biological sample derived from breast tumor tissue). 
The c-erb-2 thus immobilized is then bound by a labeling agent, such as a 
second c-erb-2 antibody bearing a label. Alternatively, the second 
antibody may lack a label, but it may, in turn, be bound by a labeled 
third antibody specific to antibodies of the species from which the second 
antibody is derived. Free labeled antibody is washed away and the 
remaining bound labeled antibody is detected (e.g., using a gamma detector 
where the label is radioactive). One of skill will appreciate that the 
analyte and capture agent is optionally reversed in the above assay, e.g., 
when the presence, quantity or avidity of a C6 antibody in a sample is to 
be measured by its binding to an immobilized c-erb-2 peptide. 
(b) Competitive assay formats. 
In competitive assays, the amount of analyte (e.g., c-erbB-2) present in 
the sample is measured indirectly by measuring the amount of an added 
(exogenous) analyte displaced (or competed away) from a capture agent 
(e.g., C6 antibody) by the analyte present in the sample. In one 
competitive assay, a known amount of c-erb-2 is added to a test sample 
with an unquantified amount of c-erbB-2, and the sample is contacted with 
a capture agent, e.g., a C6 antibody that specifically binds c-erb-2. The 
amount of added c-erbB-2 which binds to the C6 antibody is inversely 
proportional to the concentration of c-erbB-2 present in the test sample. 
The C6 antibody can be immobilized on a solid substrate. The amount of 
erbB2 bound to the C6 antibody is determined either by measuring the 
amount of erbB-2 present in an erbB-2-C6 antibody complex, or 
alternatively by measuring the amount of remaining uncomplexed erbB-2. 
Similarly, in certain embodiments where the amount of erbB-2 in a sample 
is known, and the amount or avidity of a C6 antibody in a sample is to be 
determined, erbB-2 becomes the capture agent (e.g., is fixed to a solid 
substrate) and the C-6 antibody becomes the analyte. 
(c) Reduction of Non Specific Binding. 
One of skill will appreciate that it is often desirable to reduce non 
specific binding in immunoassays and during analyte purification. Where 
the assay involves c-erbB-2, C6 antibody, or other capture agent 
immobilized on a solid substrate, it is desirable to minimize the amount 
of non specific binding to the substrate. Means of reducing such non 
specific binding are well known to those of skill in the art. Typically, 
this involves coating the substrate with a proteinaceous composition. In 
particular, protein compositions such as bovine serum albumin (BSA), 
nonfat powdered milk, and gelatin are widely used. 
(d) Substrates. 
As mentioned above, depending upon the assay, various components, including 
the erbB-2, C6 or antibodies to erbB-2 or C6, are optionally bound to a 
solid surface. Many methods for immobilizing biomolecules to a variety of 
solid surfaces are known in the art. For instance, the solid surface may 
be a membrane (e.g., nitrocellulose), a microtiter dish (e.g., PVC, 
polypropylene, or polystyrene), a test tube (glass or plastic), a dipstick 
(e.g. glass, PVC, polypropylene, polystyrene, latex, and the like), a 
microcentrifuge tube, or a glass, silica, plastic, metallic or polymer 
bead. The desired component may be covalently bound, or noncovalently 
attached through nonspecific bonding. 
A wide variety of organic and inorganic polymers, both natural and 
synthetic may be employed as the material for the solid surface. 
Illustrative polymers include polyethylene, polypropylene, 
poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene 
terephthalate), rayon, nylon, poly(vinyl butyrate), polyvinylidene 
difluoride (PVDF), silicones, polyformaldehyde, cellulose, cellulose 
acetate, nitrocellulose, and the like. Other materials which may be 
employed, include paper, glassess, ceramics, metals, metalloids, 
semiconductive materials, cements or the like. In addition, substances 
that form gels, such as proteins (e.g., gelatins), lipopolysaccharides, 
silicates, agarose and polyacrylamides can be used. Polymers which form 
several aqueous phases, such as dextrans, polyalkylene glycols or 
surfactants, such as phospholipids, long chain (12-24 carbon atoms) alkyl 
ammonium salts and the like are also suitable. Where the solid surface is 
porous, various pore sizes may be employed depending upon the nature of 
the system. 
In preparing the surface, a plurality of different materials may be 
employed, e.g., as laminates, to obtain various properties. For example, 
protein coatings, such as gelatin can be used to avoid non specific 
binding, simplify covalent conjugation, enhance signal detection or the 
like. 
If covalent bonding between a compound and the surface is desired, the 
surface will usually be polyfunctional or be capable of being 
polyfunctionalized. Functional groups which may be present on the surface 
and used for linking can include carboxylic acids, aldehydes, amino 
groups, cyano groups, ethylenic groups, hydroxyl groups, mercapto groups 
and the like. The manner of linking a wide variety of compounds to various 
surfaces is well known and is amply illustrated in the literature. See, 
for example, Immobilized Enzymes, Ichiro Chibata, Halsted Press, New York, 
1978, and Cuatrecasas, J. Biol. Chem. 245 3059 (1970). 
In addition to covalent bonding, various methods for noncovalently binding 
an assay component can be used. Noncovalent binding is typically 
nonspecific absorption of a compound to the surface. Typically, the 
surface is blocked with a second compound to prevent nonspecific binding 
of labeled assay components. Alternatively, the surface is designed such 
that it nonspecifically binds one component but does not significantly 
bind another. For example, a surface bearing a lectin such as Concanavalin 
A will bind a carbohydrate containing compound but not a labeled protein 
that lacks glycosylation. Various solid surfaces for use in noncovalent 
attachment of assay components are reviewed in U.S. Pat. Nos. 4,447,576 
and 4,254,082. 
ii) Other Assay Formats 
C-erbB-2 polypeptides or C6 antibodies and can also be detected and 
quantified by any of a number of other means well known to those of skill 
in the art. These include analytic biochemical methods such as 
spectrophotometry, radiography, electrophoresis, capillary 
electrophoresis, high performance liquid chromatography (HPLC), thin layer 
chromatography (TLC), hyperdiffusion chromatography, and the like, and 
various immunological methods such as fluid or gel precipitin reactions, 
immunodiffusion (single or double), immunoelectrophoresis, 
radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), 
immunofluorescent assays, and the like. 
Western blot analysis and related methods can also be used to detect and 
quantify the presence of erbB-2 peptides and C6 antibodies in a sample. 
The technique generally comprises separating sample products by gel 
electrophoresis on the basis of molecular weight, transferring the 
separated products to a suitable solid support, (such as a nitrocellulose 
filter, a nylon filter, or derivatized nylon filter), and incubating the 
sample with the antibodies that specifically bind either the erbB-2 
peptide or the anti-erbB-2 antibody. The antibodies specifically bind to 
the biological agent of interest on the solid support. These antibodies 
are directly labeled or alternatively are subsequently detected using 
labeled antibodies (e.g., labeled sheep anti-human antibodies where the 
antibody to a marker gene is a human antibody) which specifically bind to 
the antibody which binds either anti-erbB-2 or erbB-2 as appropriate. 
Other assay formats include liposome immunoassays (LIAs), which use 
liposomes designed to bind specific molecules (e.g., antibodies) and 
release encapsulated reagents or markers. The released chemicals are then 
detected according to standard techniques (see, Monroe et al., (1986) 
Amer. Clin. Prod. Rev. 5:34-41). 
iii) Labeling of C6 antibodies. 
The labeling agent can be, e.g., a monoclonal antibody, a polyclonal 
antibody, a protein or complex such as those described herein, or a 
polymer such as an affinity matrix, carbohydrate or lipid. Detection 
proceeds by any known method, including immunoblotting, western analysis, 
gel-mobility shift assays, tracking of radioactive or bioluminescent 
markers, nuclear magnetic resonance, electron paramagnetic resonance, 
stopped-flow spectroscopy, column chromatography, capillary 
electrophoresis, or other methods which track a molecule based upon an 
alteration in size and/or charge. The particular label or detectable group 
used in the assay is not a critical aspect of the invention. The 
detectable group can be any material having a detectable physical or 
chemical property. Such detectable labels have been well-developed in the 
field of immunoassays and, in general, any label useful in such methods 
can be applied to the present invention. Thus, a label is any composition 
detectable by spectroscopic, photochemical, biochemical, immunochemical, 
electrical, optical or chemical means. Useful labels in the present 
invention include magnetic beads (e.g. Dynabeads.TM.), fluorescent dyes 
(e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), 
radiolabels (e.g., .sup.3 H, .sup.125 I, .sup.35 S, .sup.14 C, or .sup.32 
P), enzymes (e.g., LacZ, CAT, horse radish peroxidase, alkaline 
phosphatase and others, commonly used as detectable enzymes, either as 
marker gene products or in an ELISA), and colorimetric labels such as 
colloidal gold or colored glass or plastic (e.g. polystyrene, 
polypropylene, latex, etc.) beads. 
The label may be coupled directly or indirectly to the desired component of 
the assay according to methods well known in the art. As indicated above, 
a wide variety of labels may be used, with the choice of label depending 
on the sensitivity required, ease of conjugation of the compound, 
stability requirements, available instrumentation, and disposal 
provisions. 
Non radioactive labels are often attached by indirect means. Generally, a 
ligand molecule (e.g., biotin) is covalently bound to the molecule. The 
ligand then binds to an anti-ligand (e.g., streptavidin) molecule which is 
either inherently detectable or covalently bound to a signal system, such 
as a detectable enzyme, a fluorescent compound, or a chemiluminescent 
compound. A number of ligands and anti-ligands can be used. Where a ligand 
has a natural anti-ligand, for example, biotin, thyroxine, and cortisol, 
it can be used in conjunction with the labeled, naturally occurring 
anti-ligands. Alternatively, any haptenic or antigenic compound can be 
used in combination with an antibody. 
The molecules can also be conjugated directly to signal generating 
compounds, e.g., by conjugation with an enzyme or fluorophore. Enzymes of 
interest as labels will primarily be hydrolases, particularly 
phosphatases, esterases and glycosidases, or oxidoreductases, particularly 
peroxidases. Fluorescent compounds include fluorescein and its 
derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. 
Chemiluminescent compounds include luciferin, and 
2,3-dihydrophthalazinediones, e.g., luminol. For a review of various 
labelling or signal producing systems which may be used, see, U.S. Pat. 
No. 4,391,904, which is incorporated herein by reference. 
Means of detecting labels are well known to those of skill in the art. 
Thus, for example, where the label is a radioactive label, means for 
detection include a scintillation counter or photographic film as in 
autoradiography. Where the label is a fluorescent label, it may be 
detected by exciting the fluorochrome with the appropriate wavelength of 
light and detecting the resulting fluorescence, e.g., by microscopy, 
visual inspection, via photographic film, by the use of electronic 
detectors such as charge coupled devices (CCDs) or photomultipliers and 
the Like. Similarly, enzymatic labels may be detected by providing 
appropriate substrates for the enzyme and detecting the resulting reaction 
product. Finally, simple colorimetric labels may be detected simply by 
observing the color associated with the label. Thus, in various dipstick 
assays, conjugated gold often appears pink, while various conjugated beads 
appear the color of the bead. 
Some assay formats do not require the use of labeled components. For 
instance, agglutination assays can be used to detect the presence of C6 
antibodies and C6 antibody-erbB-2 peptides. In this case, antigen-coated 
particles are agglutinated by samples comprising the target antibodies. In 
this format, none of the components need be labeled and the presence of 
the target antibody is detected by simple visual inspection. 
V. Pharmaceutical Compositions. 
The chimeric molecules of this invention are useful for parenteral, 
topical, oral, or local administration, such as by aerosol or 
transdermally, for prophylactic and/or therapeutic treatment. The 
pharmaceutical compositions can be administered in a variety of unit 
dosage forms depending upon the method of administration. For example, 
unit dosage forms suitable for oral administration include powder, 
tablets, pills, capsules and lozenges. It is recognized that the fusion 
proteins and pharmaceutical compositions of this invention, when 
administered orally, must be protected from digestion. This is typically 
accomplished either by complexing the protein with a composition to render 
it resistant to acidic and enzymatic hydrolysis or by packaging the 
protein in an appropriately resistant carrier such as a liposome. Means of 
protecting proteins from digestion are well known in the art. 
The pharmaceutical compositions of this invention are particularly useful 
for parenteral administration, such as intravenous administration or 
administration into a body cavity or lumen of an organ. The compositions 
for administration will commonly comprise a solution of the chimeric 
molecule dissolved in a pharmaceutically acceptable carrier, preferably an 
aqueous carrier. A variety of aqueous carriers can be used, e.g., buffered 
saline and the like. These solutions are sterile and generally free of 
undesirable matter. These compositions may be sterilized by conventional, 
well known sterilization techniques. The compositions may contain 
pharmaceutically acceptable auxiliary substances as required to 
approximate physiological conditions such as pH adjusting and buffering 
agents, toxicity adjusting agents and the like, for example, sodium 
acetate, sodium chloride, potassium chloride, calcium chloride, sodium 
lactate and the like. The concentration of chimeric molecule in these 
formulations can vary widely, and will be selected primarily based on 
fluid volumes, viscosities, body weight and the like in accordance with 
the particular mode of administration selected and the patient's needs. 
Thus, a typical pharmaceutical composition for intravenous administration 
would be about 0.1 to 10 mg per patient per day. Dosages from 0.1 up to 
about 100 mg per patient per day may be used, particularly when the drug 
is administered to a secluded site and not into the blood stream, such as 
into a body cavity or into a lumen of an organ. Methods for preparing 
parenterally administrable compositions will be known or apparent to those 
skilled in the art and are described in more detail in such publications 
as Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, 
Easton, Pa. (1980). 
The compositions containing the present fusion proteins or a cocktail 
thereof (i.e., with other proteins) can be administered for therapeutic 
treatments. In therapeutic applications, compositions are administered to 
a patient suffering from a disease, typically a c-erbB-2 positive 
carcinoma, in an amount sufficient to cure or at least partially arrest 
the disease and its complications. An amount adequate to accomplish this 
is defmed as a "therapeutically effective dose." Amounts effective for 
this use will depend upon the severity of the disease and the general 
state of the patient's health. 
Single or multiple administrations of the compositions may be administered 
depending on the dosage and frequency as required and tolerated by the 
patient. In any event, the composition should provide a sufficient 
quantity of the proteins of this invention to effectively treat the 
patient. 
Among various uses of the cytotoxic fusion proteins of the present 
invention are included a variety of disease conditions caused by specific 
human cells that may be eliminated by the toxic action of the protein. One 
application is the treatment of cancer, such as by the use of a C6 
antibody attached to a cytotoxin. 
Another approach involves using a ligand that binds a cell surface marker 
(receptor) so the chimeric associates cells bearing the ligand substrate 
are associated with the c-erbB-2 overexpressing tumor cell. The ligand 
portion of the molecule is chosen according to the intended use. Proteins 
on the membranes of T cells that may serve as targets for the ligand 
includes Fc.gamma.I, Fc.gamma.II and Fc.gamma.III, CD2 (T11), CD3, CD4 and 
CD8. Proteins found predominantly on B cells that might serve as targets 
include CD10 (CALLA antigen), CD19 and CD20. CD45 is a possible target 
that occurs broadly on lymphoid cells. These and other possible target 
lymphocyte target molecules for the chimeric molecules bearing a ligand 
effector are described in Leukocyte Typing III, A. J. McMichael, ed., 
Oxford University Press (1987). Those skilled in the art will realize 
ligand effectors may be chosen that bind to receptors expressed on still 
other types of cells as described above, for example, membrane 
glycoproteins or ligand or hormone receptors such as epidermal growth 
factor receptor and the like. 
VI. Kits For Diagnosis or Treatment. 
In another embodiment, this invention provides for kits for the treatment 
of tumors or for the detection of cells overexpressing c-erbB-2. Kits will 
typically comprise a chimeric molecule of the present invention (e.g. C6 
antibody-label, C6 antibody-cytotoxin, C6 antibody-ligand, etc.). In 
addition the kits will typically include instructional materials 
disclosing means of use of chimeric molecule (e.g. as a cytotoxin, for 
detection of tumor cells, to augment an immune response, etc.). The kits 
may also include additional components to facilitate the particular 
application for which the kit is designed. Thus, for example, where a kit 
contains a chimeric molecule in which the effector molecule is a 
detectable label, the kit may additionally contain means of detecting the 
label (e.g. enzyme substrates for enzymatic labels, filter sets to detect 
fluorescent labels, appropriate secondary labels such as a sheep 
anti-human antibodies, or the like). The kits may additionally include 
buffers and other reagents routinely used for the practice of a particular 
method. Such kits and appropriate contents are well known to those of 
skill in the art. 
EXAMPLES 
The following examples are provided by way of illustration only and not by 
way of limitation. Those of skill will readily recognize a variety of 
noncritical parameters which could be changed or modified to yield 
essentially similar results. 
Example 1 
Isolation and Characterization of Human Single-chain Fvs Binding C-erbb-2 
Materials and Methods: 
Preparation of c-erbB-2 ECD 
The antigen c-erbB-2 ECD with a Ser-Gly-His.sub.6 C-terminal fusion was 
expressed from Chinese Hamster Ovary cells and purified by immobilized 
metal affinity chromatography (IMAC). 
Phage preparation 
Phage were prepared from a phagemid library (3.times.10.sup.7 members) 
expressing sFv as pill fusions on the phage surface (Marks et al. (1991) 
J. Mol. Biol. 222:581-597). The library was created from a repertoire of 
sFv genes consisting of human heavy and light chain variable region 
(V.sub.H and V.sub.L) genes isolated from the peripheral blood lymphocytes 
of unimmunized human volunteers. To rescue phagemid particles from the 
library, 50 ml of 2.times.TY media containing 100 .mu.g/ml ampicillin and 
1% glucose were inoculated with 10.sup.8 bacteria taken from the frozen 
library glycerol stock. The culture was grown at 37.degree. C. with 
shaking to an A.sub.600 nm of 0.8, 7.0.times.10.sup.11 colony forming 
units of VCS-MI3 (Stratgene) added, and incubation continued at 37.degree. 
C. for 1 h without shaldng followed by 1 h with shaking. The cells were 
pelleted by centrifugation at 4500g for 10 min, resuspended in 200 ml of 
2.times.TY media containing 100 .mu.g/ml ampicillin and 2.5 .mu.g/ml 
kanamycin and grown overnight at 37.degree. C. Phage particles were 
purified and concentrated by 2 polyethylene glycol precipitations and 
resuspended in PBS (25 mM NaH.sub.2 PO.sub.4, 125 mM NaCl, pH 7.0) to 
approximately 10.sup.13 transducing units/ml ampicillin resistant clones. 
Selection of binding phage antibodies 
Phage expressing sFv which bound c-erbB-2 were selected by panning the 
phage library on immobilized c-erbB-2 ECD (Marks et al. (1991) supra.). 
Briefly, immunotubes (Nunc, Maxisorb) were coated with 2 ml (100 .mu.g/ml) 
c-erbB-2 ECD in PBS overnight at 20.degree. C. and blocked with 2% milk 
powder in PBS for 2 h at 37.degree. C. 1 ml of the phage solution 
(approximately 10.sup.13 phage) was added to the tubes and incubated at 
20.degree. C. with tumbling on an over and under turntable for 2 h. 
Nonbinding phage were eliminated by sequential washing (15 times with PBS 
containing 0.05% Tween followed by 15 times with PBS). Binding phage were 
then eluted from the immunotubes by adding 1 ml of 100 mM triethylamine, 
incubating for 10 min at 20.degree. C., transferring the solution to a new 
tube, and neutralizing with 0.5 ml 1M Tris HCl, PH 7.4. Half of the eluted 
phage solution was used to infect 10 ml of E.coli TGl (Gibson, T.J. (1984) 
Studies on the Epstein-Barr virus genome, Cambridge University Ph.D. 
thesis; Carter et aL (1985) Nucleic Acids Res., 13: 4431-4443) grown to an 
A.sub.600 nm of 0.8-0.9. After incubation for 30 min at 37.degree. C., 
bacteria were plated on TYE plates containing 100 .mu.g/ml ampicillin and 
1 % glucose and grown overnight at 37.degree. C. Phage were rescued and 
concentrated as described above and used for the next selection round. The 
selection process was repeated for a total of 5 rounds. 
Screening for binders 
After each round of selection, 10 ml of E.coli HB2151 (Carter et al. (1985) 
Nucleic Adds Res., 13: 4431-43) (A.sub.600 run.about.0.8) were infected 
with 100 .mu.l of the phage eluate in order to prepare soluble sFv. In 
this strain, the amber codon between the sFv gene and gene III is read as 
a stop codon and native soluble sFv secreted into the periplasm and media 
(Hoogenboom et al. (1991) Nucleic Acids Res. 19: 4133-4137). Single 
ampicillin resistant colonies were used to inoculate microtitre plate 
wells containing 150 .mu.l of 2.times.TY containing 100 .mu.g/ml 
ampicillin and 0.1% glucose. The bacteria were grown to an A.sub.600 
nm.about.1.0, and sFv expression induced by the addition of IPTG to a 
final concentration of 1 mM (De Bellis et al., (1990) Nucleic Acids Res., 
18:1311). Bacteria were grown overnight at 30.degree. C., the cells 
removed by centrifugation, and the supernatant containing sFv used 
directly. 
To screen for binding, 96-well microtiter plates (Falcon 3912) were coated 
overnight at 4.degree. C. with 10 .mu.g/ml c-erbB-2 ECD in PBS, blocked 
for 2 h at 37.degree. C. with 2% milk powder in PBS, and incubated for 1.5 
hours at 20.degree. C. with 50 .mu.l of the E. coli supernatant containing 
sFv. Binding of soluble sFv to antigen was detected with a mouse 
monoclonal antibody (9E1O) which recognizes the C-terminal myc peptide tag 
(Munro, S. et al., (1986) Cell, 46:291-300) and peroxidase conjugated 
anti-mouse Fc antibody (Sigma) using ABTS as substrate (Ward et al. (1989) 
Nature, 341: 544-546). The reaction was stopped after 30 min with NaF (3.2 
mg/ml) and the A.sub.405 nm measured. Unique clones were identified by PCR 
fingerprinting (Marks, J. D. et al., (1991) J. Mol. Biol., 222:581-597) 
and DNA sequencing. The specificity of each unique sFv was determined by 
ELISA performed as described above with wells coated with 10 .mu.g/ml of 
bovine serum albumin, hen egg white lysozyme, bovine 
glutamyltranspeptidase, c-erbB-2 ECD, VCS M13 (3.5.times.10.sup.12 /ml) 
and casein (0.5%). For ELISA with biotinylated c-erbB-2 ECD, microtiter 
plates (Immunolon 4, Dynatech) were coated with 50 .mu.l immunopure avidin 
(Pierce; 10 .mu.g/ml in PBS) overnight at 4.degree. C., blocked with 1% 
bovine serum albumin in PBS for 1 h at 37.degree. C. and incubated with 50 
.mu.l biotinylated c-erbB-2 extracellular domain (5 .mu.g/ml) for 30 min 
at 20.degree. C. To prepare biotinylated antigen, 0.2 ml c-erbB-2 ECD (1 
mg/ml in PBS) was incubated with 0.5 mM NHS-LC-biotin (Pierce) overnight 
at 4.degree. C. and then purified on a presto desalting column (Pierce). 
Subcloning, expression and purification. 
To facilitate purification, the C6.5 sFv gene was subcloned into the 
expression vector pUC119Sfi/NotHismyc (Griffiths, et al. (1994) EMBO J., 
13: 3245-3260) which results in the addition of a hexa-histidine tag at 
the C-terminal end of the sFv. Briefly, pHEN-1 vector DNA containing the 
C6.5 sFv DNA was prepared by alline lysis milliprep, digested with NcoI 
and NotI, and the sFv DNA purified on a 1.5% agarose gel. C6.5 sFv DNA was 
ligated into pUC119Sfil/NotIHismyc digested with NcoI and NotI and the 
legation mixture used to transform electrocompetent E. coli HB2151. For 
expression, 200 ml of 2.times.TY media containing 100 .mu.g/ml ampicillin 
and 0.1% glucose was inoculated with E. coli HB2151 harboring the C6.5 
gene in pUC119Sfil/NotIHismyc. The culture was grown at 37.degree. C. to 
an A.sub.600 nm of 0.8, soluble sFv expression induced by the addition of 
IPTG to a final concentration of 1 mM, and the culture grown at 30.degree. 
C. in a shaker flask overnight. Single-chain Fv was harvested from the 
periplasm using the following protocol. Cells were harvested by 
centrifugation at 4000 g for 15 min, resuspended in 10 ml of ice cold 30 
mM Tris-HCl pH 8.0, 1 mM EDTA, 20% sucrose, and incubated on ice for 20 
min. The bacteria were pelleted by centrifugation at 6000 g for 15 min. 
and the "periplasmic fraction" cleared by centrifugation at 30,000 g for 
20 min. The supernatant was dialyzed overnight at 4.degree. C. against 8 L 
of IMAC loading buffer (30 mM sodium phosphate pH 7.5, 500 mM NaCl, 20 mM 
imidazole) and then filtered through a 0.2 micron filter. 
The sFv was purified by IMAC. All steps were performed at 4.degree. C. on a 
Perceptive Biosystems BIOCAD Sprint. A column containing 2 ml of Ni-NTA 
resin (Qiagen) was washed with 20 ml IMAC column wash buffer (50 mM sodium 
phosphate pH 7.5, 500 mM NaCl, 2.50 mM imidazole) and 20 ml of IMAC 
loading buffer. The periplasmic preparation was loaded onto the column by 
pump and the column washed sequentially with 50 ml IMAC loading buffer and 
50 ml IMAC washing buffer (50 mM sodium phosphate pH 7.5, 500 mM NaCl, 23 
mM imidazole). Protein was eluted with 2.5 ml IMAC elution buffer (50 mM 
sodium phosphate pH 7.5, 300 mM NaCl, 100 mM imidazole) and 4 ml fractions 
collected. Protein was detected by absorbance at 280 nm and sFv typically 
eluted between fractions 6 and 8. To remove dimeric and aggregated sFv, 
samples were concentrated to a volume &lt;1 ml in a Centricon 10 (Amicon) and 
fractionated on a Superdex 75 column using a running buffer of HBS (10 mM 
Hepes, 150 mM NaCl, pH 7.4). The purity of the final preparation was 
evaluated by assaying an aliquot by SDS-PGE. Protein bands were detected 
by Coomassie staining. The concentration was determined 
spectrophotometrically, assuming an A.sub.280 run of 1.0 corresponds to an 
sFv concentration of 0.7 mg/ml. 
Affinity and kinetic measurements 
The K.sub.d of C6.5 and 74IF8 sFv were determined using surface plasmon 
resonance in a BIAcore (Pharmacia) and by Scatchard analysis. In a BlAcore 
flow cell, 1400 resonance units (RU) of c-erbB-2 ECD (25 .mu.g/ml in 10 mM 
sodium acetate, pH 4.5) was coupled to a CM5 sensor chip (Johnsson, B. et 
al., (1991) Anal. Biochem., 198:268-277). Association and dissociation of 
C6.5 and 741F8 sFv (100 nM-600 nM) were measured under continuous flow of 
5 .mu.l/min. Rate constant k.sub.on was determined from a plot of (1 n 
(dR/dt))/t vs concentration (Karlsson et al. (1991) J. Immunol. Meth., 
145: 229-240). Rate constant k.sub.off was determined from the 
dissociation part of the sensorgram at the highest concentration of sFv 
analyzed (Johnsson et al. (1991) Anal. Biochem., 198: 268-277). The 
K.sub.d of C6.5 was also determined by Scatchard analysis (Scatchard 
(1949) Annal. N.Y. Acad. Sci., 51: 660). All assays were performed in 
triplicate. Briefly, 50 .mu.g of radioiodinated sFv was added to 
5.times.10.sup.6 SK-OV-3 cells in the presence of increasing 
concentrations of unlabeled sFv from the same preparation. After a 30 
minute incubation at 20.degree. C., the samples were washed with PBS at 
40.degree. C. and centrifuged at 500 g. The amount of labeled sFv bound to 
the cells was determined by counting the pellets in a gamma counter and 
the K.sub.a and K.sub.d were calculated using the EBDA program (V 2.0, G. 
A. McPherson, 1983). 
Radiolabeling 
The C6.5 sFv was labeled with radioiodine using the CT method (DeNardo et 
al. (1986) Nud. Med. Biol., 13: 303-310). Briefly, 1.0 mg of protein was 
combined with .sup.125 I (14-17 mCi/mg) (Amersham, Arlington Heights, 
Ill.), or .sup.131 I (9.25 mCi/mg) (DuPont NEN, Wilmington, Del.) at an 
iodine to protein ratio of 1:10. 10 .mu.g of CT (Sigma, St. Louis, Mo.) 
was added per 100 .mu.g of protein and the resulting mixture was incubated 
for three minutes at room temperature. The reaction was quenched by the 
addition of 10 .mu.g of sodium metabisulfite (Sigma) per 100 .mu.g of 
protein. Unincorporated radioiodine was separated from the labeled protein 
by gel filtration using the G-50-80 centrifuged-column method (Adams et 
al. (1993) Cancer Res. 53: 4026-4034). The fmal specific activity of the 
CT labelling was 1.4 mCi/mg for the .sup.131 I-C6.5 sFv and typically 
about 1.0 mCi/mg for the .sup.125 I-C6.5 sFv. 
Quality Control 
The quality of the radiopharmaceuticals was evaluated by HPLC, SDS-PAGE, 
and a live cell binding assay as previously described (Adams et al. (1993) 
Cancer Res. 53: 4026-4034). The HPLC elution profiles from a Spherogel 
TSK-3000 molecular sieving column consistently demonstrated that greater 
than 99% of the radioactivity was associated with the protein peak. 
Greater than 98% of the nonreduced .sup.125 I-C6.5 sFv preparations 
migrated on SDS-PAGE as approximately 26 D.sub.d proteins while the 
remaining activity migrated as a dimer. The immunoreactivity of the 
radiopharmaceuticals was determined in a live cell binding assay utilizing 
c-erbB-2 overexpressing SK-OV-3 cells (#HTB 77; American Type Culture 
Collection, Rockville, Md.) and c-erbB-2 negative CEM cells (#119; 
American Type Culture Collection) (Adams, G. P. et al., (1993) Cancer Res. 
53:4026-4034). Live cell binding assays revealed 49% of the activity 
associated with the positive cell pelleted less than 3% bound to the 
negative control cells; these results were lower than those typically seen 
with 741F8 sFv (60-80% bound) (Adams et al., (1993) supra.). 
Cell Surface Dissociation Studies 
Cell surface retention of biotinylated forms of the sFv molecules were 
measured by incubating 2 .mu.g of either sFv with 2.times.10.sup.6 SK-BR-3 
cells (#HTB 30; American Type Culture Collection) in triplicate in 20 ml 
of FACS buffer, with 0.01% azide for 15 min at 4.degree. C. The cells were 
washed twice with FACS buffer (4.degree. C.) and resuspended in 2 ml of 
FACS buffer. 0.5 ml of the cell suspension were removed and placed in 
three separate tubes for incubations under differing conditions; 0 min at 
4.degree. C., 15 min at 37.degree. C., and 30 min at 37.degree. C. After 
the incubations, the cells were centrifuged at 300 g, the supenatants were 
removed, the cell pellets were washed 2.times. (4.degree. C.) and the 
degree of retention of sFv on the cell surface at 37.degree. C. (for 15 or 
30 min) was compared to retention at 0 min at 4.degree. C. 
Biodistribution and Radioimmunoimaging Studies 
Four to six week old C.BI71Icr-scid mice were obtained from the Fox Chase 
Cancer Center Laboratory Animal Facility. 2.5.times.10.sup.6 SK-OV-3 cells 
in log phase were implanted s.c. on the abdomens of the mice. After about 
7 weeks the tumors had achieved sizes of 100-200 mg and Lugol's solution 
was placed in the drinking water to block thyroid accumulation of 
radioiodine. Three days later, biodistribution studies were initiated. 
.sup.125 I-C6.5 sFv was diluted in PBS to a concentration of 0.2 mg/ml and 
each mouse was given 100 .mu.L, containing 20 .mu.g of 
radiopharmaceutical, by tail vein injection. Total injected doses were 
determined by counting each animal on a Series 30 multichannel 
analyzer/probe system (probe model #2007, Canaberra, Meridian, Conn.). 
Blood samples and whole body counts of the mice were obtained at regular 
intervals. Groups of 8 mice were sacrificed at 24 h after injection and 
the tumors and organs removed, weighed and counted in a gamma counter to 
determine the % ID/g (Adams et al. (1993) supra.; Adams et al. (1992) 
Antibody Inmmunoconj. and Radiopharm., 5: 81-95). The mean and standard 
error of the mean (SEM) for each group of data were calculated, and T:O 
ratios determined. Significance levels were determined using Students 
t-test. 
For the radioimmunoimaging studies, tumor-bearing scid mice were injected 
with 100 .mu.g (100 .mu.l) of .sup.131 I-C6.5. At 24 hours after 
injection, the mice were euthanized by asphyxiation with CO.sub.2 and 
images were acquired on a Prism 2000XP gamma camera (Picker, Highland 
Heights, Ohio 44142). Preset acquisitions of 100 k counts were used. 
TABLE 6 
__________________________________________________________________________ 
Deduced Amino Acid Sequence of C4.1 and C6.5 Heavy and Light Chain. 
Sequences 
are aligned to the most homologous human germline gene. Dashes indicate 
sequence indentity, GL = germline sequence. DP58 and DP73 (ref. 22), 
IGLV3S1 
(ref. 23), HUMLV122 and DPL 5. 
SEQ ID Nos. 
__________________________________________________________________________ 
Heavy Chains 
Framework 1 CDR1 Framework 2 CDR2 
- C4.1QVQLVESGGGLVQPGGSLRLSCAASGFTFS SYEMN WVRQAPGKGLEWVS YISSSGSTIYYAD 
SVKGSeq ID 30 
- DP58E------------------------------ ---- -------------- ------------- 
----Seq ID 31 
- C6.5QVQLLQSGAELKKPGESLKISCKGSGYSFT SYWIA WVRQMPGKGLEYMG LIYPGDSDTKYSP 
SFQGSeq ID 32 
- DP73E---V-----V------------------- ----G -----------W-- I--------R--- 
----Seq ID 33 
- Framework 3 CDR3 Framework 4 
- C4.1RFTISRDNAKNSLYLQMNS 
LRAEDTAVYYCAR DLGGYSYGYVGLD 
Y WGQGTLVTVSSSeq ID 
30 
- DP58 
Seq ID 31 
- C6.5QVTISVDKSVSTAYLQWSSLKPSDSAVYFCAR HDVGYCSSSNCAKWPEYFQH WGQGTLVTVSS 
Seq ID 32 
- DB73 
A---I-----------A--T-M-Y---Seq ID 33 
- 
Light Chains 
Framework 1 CDR1 Framework 2 CDR2 
- C4.1SELTQDPAVSVALGTVRITC QGDSLRSYYAS WYQQKPGQAPVLVIY GKNNRPSSeq ID 
34 
- IGLV3S1 
S------------------ ----------- --------------- -------Seq ID 35 
- C6.5QSVLTQPPSVSAAPGQKVT 
ISC SGSSSNIGNNYVS WYQQLPGTA 
PKLLIY GHTNRPASeq ID 36 
- HUMLV122 
------------- --------------- DNKK--SSeq ID 37 
- DPL5 
A-GT---R----- --------S---Y --------------- RNNQ--SSeq ID 38 
- Framework 3 CDR3 Framework 4 
- C4.1GIPDRFSGSSSGNIASLTITGAQAEDEADYYC NSRDSSGNPYWV FGGGTKVTVLGSeq ID 
34 
- IGLV3S1 
T------------------ --------H V-Seq ID 35 
- C6.5GVPDRFSGSKSGTSASLAISGFRSEDEADYYC AAWDDSLSG WV FGGGTKLTVLGSeq ID 
36 
- humlv122 
I-------------T-G-T-LQTG------- GT--S---ASeq ID 37 
- dp15 
L---------- ---------Seq ID 38 
__________________________________________________________________________ 
Results 
After four rounds of selection, 9/190 clones analyzed by ELISA expressed 
sFv which bound c-erbB-2 ECD (ELISA signals greater than 0.4, 6 times 
higher than background). After five rounds of selection, 33/190 clones 
expressed c-erbB-2 binding sFv. PCR fingerprinting of the 42 positive 
clones identified two unique restriction patterns and DNA sequencing of 6 
clones from each pattern revealed two unique human sFv sequences, C4. 1 
and C6.5 (Table 6). The V.sub.H gene of C6.5 is from the human V.sub.H 5 
gene family, and the V.sub.L gene from the human V.sub..lambda. family 
(Table 6). The V.sub.L gene appears to be derived from two different 
germline genes (HUMLV122 and DPL 5) suggesting the occurrence of PCR 
crossover (Table 6). The VH gene of C4. 1 is from the human V.sub.H 3 
family, and the V.sub.L gene from the human V.sub..lambda. 3 family (Table 
6). C4. 1 and C6.5 both bound c-erbB-2 specifically, as determined by 
ELISA against the relevant antigen and a panel of irrelevant antigens. 
However, when biotinylated c-erbB-2 ECD was bound to avidin coated plates 
and used in ELISA assays, the signal obtained with C6.5 was 6 times higher 
than observed when c-erbB-2 ECD was absorbed to polystyrene (1.5 vs 0.25). 
In contrast, C4.1 was not capable of binding to biotinylated c-erbB-2 ECD 
captured on avidin microtitre plates. Additionally, biotinylated and 
iodinated C6.5, but not C4.1, bound SK-BR-3 cells overexpressing c-erbB-2. 
These results indicate that C6.5 binds the native c-erbB-2 expressed on 
cells, but C4 binds a denatured epitope that appears when the antigen is 
adsorbed to polystyrene. 
C6.5 was purified in yields of 10 mg/L of E. coli grown in shake flasks and 
gel filtration analysis indicated a single peak of approximately 27 
K.sub.d. The K.sub.d of purified C6.3 was determined using both surface 
plasmon resonance in a BIAcore and by Scatchard. The K.sub.d determined by 
BIAcore (1.6.times.10.sup.-8 M) agreed closely to the value determined by 
Scatchard (2.0.times.10.sup.-8 M) (Table 7). Kinetic analysis by BIAcore 
indicated that C6.5 had a rapid on-rate (k.sub.on 4.0.times.10.sup.5 
M.sup.-1 s.sup.-1) and a rapid off-rate (k.sub.off 6.3.times.10.sup.-3 
s.sup.-1) (Table 2). Cell retention assay confirmed that C6.5 dissociated 
rapidly from the cell surface (Table 2). 
After injection of .sup.125 I-C6.5 into scid mice bearing SK-OV-3 tumors, 
1.47% ID/gm of tumor was retained after 24 hours (Table 7). Tumor:normal 
organ values ranged from 8.9 (tumor:kidney) to 283 (tumor:muscle). These 
values were higher than values observed for 741F8 sFv, produced from a 
murine monoclonal antibody (K.sub.d =2.6.times.10.sup.-8 M. The high T:O 
ratios resulted in the highly specific visualization of the tumor by gamma 
scintigraphy using .sup.131 I-labelled C6.5. 
TABLE 7 
______________________________________ 
Characterization of anti-cerbB-2 sFv species. Characteristics of the 
murine anti-c-erbB-2 sFv, 741F8, and the human sFv C6.5 are compared. 
The affinity and dissociation constants were determined by Scatchard 
plot analysis, unless otherwise stated. Dissociation from c-erbB-2 
positive (SK-OV-3) cells was measured in an in vitro live cell assay. 
The percentage of injected dose per gram (% ID/g) tumor M and tumor 
to organ ratios were determined in biodistribution studies performed 
in separate groups of scid mice (n = 10-14) bearing SK-OV-3 
tumors overexpressing c-erbB-2. SEM are &lt;35% of the associated 
values a = significantly unproved (p &lt; 0.05) compared to 741F8 sFv. 
Parameter 741F8 C6.5 
______________________________________ 
K.sub.d (BIAcore) 
2.6 .times. 10.sup.-8 M 
1.6 .times. 10.sup.-8 M 
K.sub.d (Scatchard) 5.4 .times. 10.sup.-8 M 2.1 .times. 10.sup.-8 M 
k.sub.on (BIAcore) 2.4 .times. 10.sup.5 
M.sup.-1 s.sup.-1 4.0 .times. 10.sup.5 
M.sup.-1 s.sup.-1 
k.sub.off (BIAcore) 6.4 .times. 10.sup.-3 s.sup.-1 6.3 .times. 
10.sup.-3 s.sup.-1 
% associated with cell 32.7% 60.6% 
surface at 15 min 
% associated with cell 8.6% 22.2% 
surface at 15 min 
% ID/g Tumor 0.8 1.0 
T:Blood 14.7 22.9 
T:Kidney 2.8 5.6a 
T:Liver 14.2 22.3 
T:Spleen 10.3 34.1 
T:Intestine 25.0 29.7 
T:Lung 9.4 15.8 
T:Stomach 8.9 11.1 
T:Muscle 78.8 158.7 
T:Bone 30.0 102.7 
______________________________________ 
Example 2 
Isolation of High Affinity Monomeric Human Anti-cerb-2 Single Chain Fv 
Using Affinity Driven Selection 
Materials and Methods 
Construction of heavy chain shuffled libraries 
To facilitate heavy chain shuffling, libraries were constructed in pHEN-1 
(Hoogenboom et al. (1991) Nucleic Acids Res. 19, 4133-4137) containing 
human V.sub.H gene repertoires (FR1 to FR3) and a cloning site at the end 
of V.sub.H FR3 for inserting the V.sub.H CDR3, V.sub.H FR4, linker DNA and 
light chain from binding sFv as a BssHII-NotI fragment. To create the 
libraries three V.sub.H gene repertoires enriched for human V.sub.H 1, 
V.sub.H 3, and V.sub.H 5 gene were amplified by PCR using as a template 
single stranded DNA prepared from a 1.8.times.10.sup.8 member sFv phage 
antibody library pHEN-1 (Marks et al. (1991) J. Mol. Biol. 222: 581-597). 
For PCR, 50 .mu.l reactions were prepared containing 10 ng template, 25 
pmol back primer (LMB3), 25 pmol forward primer (PV.sub.H 1FOR1, PV.sub.H 
3FOR1, or PV.sub.H 5FOR1), 250 uM-dNTPs, 1 mM MgCl.sub.2, and 0.5 .mu.l (2 
units) Taq DNA polymerase (Promega) in the manufacturer's buffer. Primers 
PV.sub.H 1For1, PV.sub.H 3For1, and PV.sub.H 5For1 were designed to anneal 
to the consensus V.sub.H 1, V.sub.H 3, or, V.sub.H 5 3' FR3 sequence 
respectively (Tomlinson et al. (1992) J. Mol. Biol. 227, 776-798; see 
Table 18). The reaction mixture was subjected to 25 cycles of 
amplification (94.degree. C. for 30 sec, 55.degree. C. for 30 sec and 
72.degree. C. for 30 sec) using a Hybaid OmniGene cycler. The products 
were gel purified, isolated from the gel using DEAE membranes, eluted from 
the membranes with high salt buffer, ethanol precipitated, and resuspended 
in 20 .mu.l of water (Sambrook et al. (1990)). 
The DNA fragments from the first PCR were used as templates for a second 
PCR to introduce a BssHII site at the 3'-end of FR3 followed by a NotI 
site. The BssHII site corresponds to amino acid residue 93 and 94 (Kabat 
numbering (Kabat et al (1987) Sequences ofproteins of inununological 
interest, 4th ed., US Department of Health and Human Services, Public 
Health Service, Bethesda, Md.; see, Table 5 in this reference) does not 
change the amino acid sequence (alanine-arginine). PCR was performed as 
described above using 200 ng purified first PCR product as template and 
the back primers PV.sub.H 1For2, PV.sub.H 3For2, and PV.sub.H 5For2. The 
PCR products were purified by extraction with phenol/chloroform, 
precipitated with ethanol, resuspended in 50 .mu.l water and 5 .mu.g 
digested with NotI and NcoI. The digested fragments were gel purified and 
each V.sub.H gene repertoire ligated separately into pHEN-1 (Hoogenboom et 
al. 1991 supra.) digested with Noti and NcoI. The ligation mix was 
purified by extraction with phenol/chloroform, ethanol precipitated, 
resuspended in 20 .mu.l water, and 2.5 .mu.l samples electroporated (Dower 
et al. (1988) Nucleic Acids Res. 16, 6127-6145) into 50 .mu.l E. coli TG1 
(Gibson et al (1984) Ph.D. Thesis, University of Cambridge). Cells were 
grown in 1 ml SOC (Sambrook et al. 1990) for 3 min and then plated on TYE 
(Miller (1972) Experiments in Molecular Genetics Cold Springs Harbor Lab 
Press, Cold Springs Harbor, N.Y.) media containing 100 .mu.g ampicillin/ml 
and 1% (w/v) glucose (TYE-AMP-GLU). Colonies were scraped off the plates 
into 5 ml of 2.times.TY broth (Miller (1972), supra) containing 100 .mu.g 
ampicillin/ml, 1% glucose (2.times.TY-AMP-GLU) and 15 (v/v) glycerol for 
storage at -70.degree. C. The cloning efficiency and diversity of the 
libraries were determined by PCR screening (Gussow and Clackson (1989) 
Nucleic Acids Res. 17, 4000) as described (Marks et al. (1991), supra). 
The resulting phage libraries were termed pHEN-1-V.sub.H 1rep, 
pHEN-1-V.sub.H 3rep and pHEN-1-V.sub.H 5rep. 
Three separate C6.5 heavy chain shuffled phage antibody libraries were made 
from the pHEN-1-V.sub.H 1rep, pHEN-1-V.sub.H 3rep, and pHEN-1-V.sub.H 5rep 
phage libraries. The C6.5 light chain gene, linker DNA, and V.sub.H CDR 
and FR4 were amplified by PCR from pHEN-1-C6.5 plasmid DNA using the 
primers PC6VL1Back and fdSEQ1. The PCR reaction mixtures were digested 
with BssHII and NotI and ligated intpHEN-1-V.sub.H 1rep, pHEN-1-V.sub.H 
3rep, and pHEN-1-V.sub.H 5rep digested with NotI and BssHII. 
Transformation and creation of library stocks was as described above. 
Construction of light chain shuffled libraries 
To facilitate light chain shuffling, a library was constructed in PHEN-1 
containing human V.sub.k and V.sub..lambda. gene repertoires, linker DNA, 
and cloning sites for inserting a V.sub.H gene as an NcoI-XhoI fragment. 
An XhoI can be encoded at the end of FR4 without changing the amino acid 
sequence of residues 102 and 103 (serine-serine) (Kabat et al. Sequences 
ofproteins of immunological interest, 4th ed. U.S. Dept. Health and Human 
Services, Public Health Services, Bethesda, Md. (1987)). To create the 
library, a V.sub.k and V.sub..lambda. gene repertoire was amplified by PCR 
from a 1.8.times.10.sup.8 member sFv phage antibody library in pHEN-1 
(Marks et al. (1991), supra). PCR was performed as described above using 
10 ng template, 25 pmol Back primer (RJH1/2/6Xho, RJH3Xho, oRJH4/5Xho) and 
25 pmol forward primer (fdSEQ1). The back primers were designed to anneal 
to the first 6 nucleotides of the (Gly.sub.4 Ser) (SEQ ID NO:25) linker 
and either the J.sub.H 1, 2, 6, J.sub.H 3, or J.sub.H 4,5 segments 
respectively. The PCR reaction mixture was purified as described above, 
digested with XhoI and NotI, gel purified and ligated into 
pHEN-V.sub..lambda. 3S1 (Hoogenboom and Winter (1992) J. Mol. BioL 227, 
381-388) digested with XhoI and NotI. Transformation of E. coli, TG1, PCR 
screening, and creation of library stocks was as described above. The 
resulting phage library was termed pHEN-1-V.sub.L rep. 
The light chain shuffled phage antibody library was made for pHEN-1-V.sub.L 
rep. The C6.5 V.sub.H gene was amplified by PCR from pHEN-1-C6.5 plasmid 
DNA using the primers PC6V.sub.H 1For and LMB3. The PCR reaction mixture 
was purified, digested with XhoI and Ncol, gel purified and ligated into 
pHEN-1-V.sub.L rep digested with Xho and NcoI. Transformation of E. coli 
TG1, PCR screening, and creation of library stocks was as described above. 
Construction of sFv containing highest affinity V.sub.H and V.sub.L gene 
obtained by chain shuffling 
Two new sFv were made by combining the V.sub.L gene of the highest affinity 
light chain shuffled sFv (C6L1) with the V.sub.H gene of the highest 
affinity heavy chain shuffled sFv (C6H1 or C6H2). The C6L1 plasmid was 
digested with NcoI and XhoI to remove the C6.5 V.sub.H gene and gel 
purified. The VH gene of C6H1 or C6H2 was amplified by PCR using the 
primers LMB3 and PC6V.sub.H 1For, digested with NcoI and XhoI and ligated 
into the previously digested C6L1 vector. Clones were screened for the 
presence of the correct insert by PCR fingerprinting and confirmed by DNA 
sequencing. 
Preparation of phage 
To rescue phagemid particles from the libraries, 10 ml of 2 TY-AMP-GLU were 
inoculated with an appropriate volume of bacteria (approximately 50 to 100 
.mu.l) from the library stocks to give an A.sub.600 of 0.3 to 0.5 and 
grown for 30 min, shaking at 37.degree. C. About 1.times.10.sup.12 
plaque-forming units of VCS-M13 (Stratagene) particles were added and the 
mixture incubated at 37.degree. C. for 30 min without shaking followed by 
incubation at 37.degree. C. for 30 min with shaking. Cells were spun down, 
resuspended in 50 ml 2.times.TY broth containing 100 .mu.g ampicillin/ml 
and 50 .mu.g kanamycin/ml (2.times.TY-AMP-KAN), and grown overnight, 
shaking at 25.degree. C. Phage particles were purified and concentrated by 
two PEG-precipitations (Sambrook et al., 1990), resuspended in 5 ml 
phosphate buffered saline (25 mM NaH.sub.2 PO.sub.4, 125 mM NaCl, pH 7.0, 
PBS) and filtered through a 0.45 u filter. The phage preparation 
consistently resulted in a titre of approximately 1013 transducing 
units/ml ampicillin-resistant clones. 
Selection of phage antibody libraries 
The light chain shuffled library was selected using immunotubes (Nunc; 
Maxisorb) coated with 2 ml c-erbB-2 ECD (25 .mu.g/ml) in PBS overnight at 
room temperature (Marks et al. (1991) supra). The tube was blocked for 1 h 
at 37.degree. C. with 2% skimmed milk powder in PBS (2% MPBS) and the 
selection, washing, and elution were performed as described (Marks et al. 
(1991), supra) using phage at a concentration of 5.0.times.10.sup.12 /ml. 
One third of the eluted phage was used to infect 1 ml log phase E. coli 
TG1, which were plated on TYE-AMP-GLU plates and described above. The 
rescue-selection-plating cycle was repeated 3 times, after which clones 
were analyzed for binding by ELISA. 
All libraries were also selected using biotinylated c-erbB-2 ECD and 
streptavidin-coated paramagnetic beads as described (Hawkin et al. (1992) 
J. Mol. Biol. 226, 889-896) with some modifications. To prepare 
biotinylated antigen, 0.2 ml c-erbB-2 ECD (1 mg/ml) was incubated with 5 
mM NHS-LC-Biotin (Pierce) overnight at 4.degree. C. and then purified on a 
presto desalting column. For each round of selection, 1 ml of phage 
(approximately 10.sup.13 t.u.) were mixed with 1 ml PBS containing 4% 
skimmed milk powder, 0.05% Tween 20, and biotinylated c-erbB-2 ECD. 
Affinity-driven selections were performed by decreasing the amount of 
biotinylated c-erbB-2 ECD used for selection. Two selection schemes were 
used. 
In selection scheme 1 (S1) antigen concentrations of 10 nM, 50 nM, 10 nM, 
and 1 nM were used for selection rounds 1, 2, 3, and 4 respectively. In 
selection scheme 2 (S2) antigen concentrations of 40 nM, 1 nM, 100 pM, and 
10 pM were used for selection rounds 1, 2, 3, and 4 respectively. The 
mixture of phage and antigen was gently rotated on an 
under-and-over-turntable for 1 hour at room temperature. To capture phage 
binding biotinylated antigen, streptavidin coated M280 magnetic beads 
(Dynabeads, Dynal) were blocked with 2% MPBS for 1 h at 37.degree. C., and 
then added to the mixture of phage and antigen. In S1, 200 .mu.l (round 
1), 100 .mu.l (round 2) or 50 .mu.l (rounds 3 and 4) of beads were 
incubated with the phage-antigen mixture for 15 min, rotating on an 
under-and-over-turntable at room temperature. In S2, 100 .mu.l (round 1) 
or 50 .mu.l (rounds 2, 3, and 4) of beads were incubated with the 
phage-antigen mixture for 15 min (round 1), 10 min (round 2), or 5 min 
(rounds 3 and 4). After capture of phage, Dynabeads were washed a total of 
10 times (3.times.PBS containing 0.05% Tween 20 (TPBS), 2.times.TPBS 
containing 2% skimmed milk powder, x PBS, 1.times.2% MPBS, and 
2.times.PBS) using a Dynal magnetic particle concentrator. The Dynabeads 
were resuspended in 1 ml PBS, and 300 .mu.l were used to infect 10 ml log 
phase E. coli TG1 which were plated on TYE-AMP-GLU plates. 
Initial sFv characterization 
Initial analysis of chain shuffled sFv clones for binding tc-erbB-2 was 
performed by ELISA using bacterial supernatant containing expressed sFv. 
Expression of sFv (De Bellis and Schwartz (1990) Nucleic Acids Res. 18, 
1311) was performed in 96 well microtitre plates as described (Marks et 
al. (1991), supra) with the following exception. After overnight growth 
and expression at 30.degree. C., 50 .mu.l 0.5% Tween 20 was added to each 
well and the plates incubated for 4 h at 37.degree. C. with shaking to 
induce bacterial lysis and increase the concentration of sFv in the 
bacterial supernatant. For selection performed on Immunotubes, ELISA 
plates (Falcon 3912) were incubated with c-erbB-ECD (2.5 .mu.g/ml) in PBS 
at 4.degree. C. overnight. For selections performed with biotinylated 
protein, Immunolon 4 plates (Dynatech) were incubated overnight at 
4.degree. C. with Immunopure avidin (10 .mu.g/ml in PBS; Pierce). After 
washing 3 times with PBS to remove unbound avidin, wells were incubated 
with biotinylated c-erbB-2 ECD as in Example 1. In both cases, binding of 
sFv to c-erbB-2 ECD was detected with the mouse monoclonal antibody 9E10 
(1 .mu.g/ml), which recognizes the C-terminal peptide tag (Munro and 
Pelham (1986), Cell 46, 291-300) and peroxidase-conjugated anti-mouse Fc 
antibody (Sigma), as described (Marks et al., 1991, supra). Selected 
binders were further characterized by sequencing of the V.sub.H and 
V.sub.L genes (Sanger et al. (1977) Proc. Nati. Acad. Sci. USA, 74: 
5463-5467). Sequence data has been deposited with the GenBank Data 
Library. 
Screening of sFv for relative affinity was performed essentially as 
described (Friguet et al. (1985) J. Immunol. Meth. 77: 305-319). Immunolon 
4 ELISA plates (Dynatech) were coated with avidin in PBS (10 .mu.g/ml) at 
4.degree. C. overnight. Biotinylated c-erbB-2 ECD (5 .mu.g/ml) was added 
to the wells and incubated for 30 min at room temperature. Bacterial 
supernatant containing sFv was incubated with varying concentrations of 
c-erbB-2 (0 to 100 nM) at 4.degree. C. for 1 h. The amount of free sFv was 
then determined by transferring 100 .mu.l of each mixture into the wells 
of the previously prepared ELISA plate and incubating for lh at 4.degree. 
C. Binding of sFv was detected as under ELISA screening and the IC50 
calculated as described (Friguet et al. (1985), supra) 
Screening of sFv by dissociation rate constant (k.sub.off) was performed 
using real-time biospecific interaction analysis based on surface plasmon 
resonance (SPR) in a BIAcore (Pharmacia). Typically 24 ELISA positive 
clones from each of the final two rounds of selection were screened. A 10 
ml culture of E. coli TG1 containing the appropriate phagemid was grown 
and expression of sFv induced with IPTG (De Bellis and Schwartz, 1990). 
Cultures were grown overnight at 25.degree. C., sFv harvested from the 
periplasm (Breitling et al. (1991) Gene 104, 147-153), and the periplasmic 
fraction dialyzed for 24 h against HEPES buffered saline (10 mM Hepes, 150 
mM NaCl, pH 7.4, HBS). In a BlAcore flow cell, approximately 1400 
resonance units (RU) of c-erbB-ECD (25 .mu.g/ml) in 10 mM acetate buffer 
pH 4.5 were coupled to a CM5 sensor chip (Johnsson et al. (1991) Anal. 
Biochem. 198, 268-277). Association and dissociation of undiluted sFv in 
the periplasmic fraction was measured under a constant flow of 5 
.mu.l/min. An apparent dissociation rate constant (k.sub.off) was 
determined from the dissociation part of the sensorgram for each sFv 
analyzed (Karlsson et al. (1991) J. Immunol. Methods 145, 229-240). 
Typically 30 to 40 samples were measured during a single BIAcore run, with 
C6.5 periplasmic preparations analyzed as the first and final samples to 
ensure stability during the run. The flow cell was regenerated between 
samples using 2.6 M MgCl.sub.2 in 10 mM glycine, pH 9.5 without 
significant change in the sensorgram baseline after analysis of more than 
100 samples. 
Subcloning, expression and purification of Single-chain Fv. 
To facilitate purification, shuffled sFv genes were subcloned (Example 1) 
into the expression vector pUC11Sfi-NotmycHis, which results in the 
addition of a hexa-histidine tag at the C-terminal end of the sFv. 200 ml 
cultures of E.coli TG1 harboring one of the C6.5 mutant phagemids were 
grown, expression of sFv induced with IPTG (De Bellis and Schwartz (1990), 
supra) and the culture grown at 25.degree. C. overnight. Single-chain Fv 
was harvested from the periplasm (Breitling et al. (1991), supra) dialyzed 
overnight at 4.degree. C. against 8 L of IMAC loading buffer (50 mM sodium 
phosphate, pH 7.5, 500 mNaCl, 20 mM imidazole) and then filtered through a 
0.2 micron filter. 
Single-chain Fv was purified by immobilized metal affinity chromatography 
(IMAC) (Hochuli et al. (1988) Bio/Technology, 6, 1321-1325) as described 
in Example 1. To remove dimeric and aggregated sFv, samples were 
concentrated to a volume &lt;1 ml in a Centricon 10 (Amicon) and fractionated 
on a Superdex 75 column using a running buffer of HBS. The purity of the 
final preparation was evaluated by assaying an aliquot by SDS-PAGE. 
Protein bands were detected by Coomassie staining. The concentration was 
determined spectrophotometrically assuming an A.sub.280 nm of 1.0 
corresponds to an sFv concentration of 0.7 mg/ml. 
Measurement of affinity, kinetics, and cell suface retention 
The K.sub.d of light chain shuffled C6.5 mutants isolated from phage 
selection using Immunotubes (Nunc) were determined by Scatchard analysis. 
All assays were performed in triplicate. Briefly, 50 mg of radioiodinated 
sFv was added to 5.times.10.sup.6 SK-OV-cells in the presence of 
increasing concentrations of unlabeled sFv from the same preparation. 
After a 30 minute incubation at 20.degree. C., the samples were washed 
with PBS at 4.degree. C. and centrifuged at 500 g. The amount of labeled 
sFv bound to the cells was determined by counting the pellets in a gamma 
counter and the K.sub.a and K.sub.d were calculated using the EBDA program 
(V 2.0, G. A. McPherson, 1983). The K.sub.d of all the other isolated sFv 
were determined using surface plasmon resonance in a BIAcore (Pharmacia). 
In a BIAcore flow cell, approximately 1400 resonance units (RU) of 
c-erbB-2 ECD (25 .mu.g/ml in 10 mM sodium acetate, pH 4.5) was coupled to 
a CM5 sensor chip (Johnsson et al. (1991), supra). Association and 
dissociation-rates were measured under continuous flow of 5 ml/min using a 
concentration range from 50 to 800 nM. Rate constant k.sub.on was 
determined from a plot of (1(dR/dt))/t vs concentration (Karlsson et al. 
(1991), supra). Rate constant k.sub.off was determined from the 
dissociation part of the sensorgram at the highest concentration of sFv 
analyzed. Cell surface retention of C6.5 and C6L1 was determined as 
described in Example 1. 
Modeling of location of mutations 
The location of mutations in shuffled sFv was modeled on the structure of 
the Fab KOL (Marquart et al. (1980) J. Mol. Biol. 141, 369-391) using 
Maclmdad v5.0 (Molecular Applications Group, Palo Alto, Calif.) running on 
an Apple MacIntosh Quadra 650. 
Results 
Construction of shuffled phage antibody libraries 
To facilitate heavy chain shuffling, libraries were constructed in pHEN-1 
(Hoogenboom et al. (1991), supra) containing human VH gene repertoires 
(FR1 to FR3) and cloning sites for inserting the V.sub.H CDR3FR4, single 
chain linker, and light chain gene from a binding sFv as a BssHII-NotI 
fragment. Three heavy chain shuffling libraries were created 
(pHEN-1-V.sub.H 1rep, pHEN-1-V.sub.H 3rep, and pHEN-1-V.sub.H 5rep), each 
enriched for V.sub.H 1, V.sub.H 3, or V.sub.H 5 genes by using PCR primers 
designed to anneal to the consensus sequence of the 3' end of V.sub.H 1, 
V.sub.H 3, or V.sub.H FR3 (Tomlinson et al. (1992), supra). These primers 
also introduced a BssHII site at the end of FR3, without changing the 
amino acid sequence typically observed at these residues. Libraries of 
5.0.times.10.sup.5 clones for pHEN-1-V.sub.H 1rep, 1.0.times.10.sup.6 
clones for pHEN-1-V.sub.H 3rep and 1.5.times.10.sup.6 clones for 
pHEN-1-V.sub.H 5rep were obtained. Analysis of 50 clones from each library 
indicated that greater than 80% of the clones had inserts, and the 
libraries were diverse as shown by the BstNI restriction pattern (Marks et 
al. (1991), supra). Three heavy chain shuffled libraries were made by 
cloning the C6.5 V.sub.H CDR3, FR4, linker, and light chain genes into the 
previously created V.sub.H 1. V.sub.H 3, or V.sub.H 5 repertoire using the 
BssHII and NotI restriction sites. After transformation, libraries of 
1.0-2.0.times.10.sup.6 clones were obtained. PCR screening revealed that 
100% of clones analyzed had full length insert and diverse BstNI 
restriction pattern. Prior to selection, 20/92 clones selected at random 
from the V.sub.H 5 library expressed sFv which bound c-erbB-2. 0/92 clones 
selected at random from the V.sub.H 1 or V.sub.H repertoire expressed sFv 
which bound c-erbB-2. 
To facilitate light chain shuffling, a library was constructed in pHEN-1 
containing human V.sub.k and V.sub.l gene repertoires, single chain linker 
DNA, and cloning sites for inserting the V.sub.H gene from binding sFv as 
an NcoI-XhoI fragment. The resulting library (pHEN-1-V.sub.1 rep) 
consisted of 4.5.times.10.sup.6 clones. PCR screening revealed that 95% of 
clones analyzed had full length insert and a diverse BstNI restriction 
pattern. A light chain shuffled library was made by cloning the C6.5 
V.sub.H gene into pHEN-1-V.sub.1 rep. After transformation a library of 
2.0.times.10.sup.6 clones was obtained. PCR screening revealed that 100% 
of clones analyzed had full length insert and a diverse BstNI restriction 
pattern. Prior to selection, 0/92 clones selected at random expressed sFv 
which bound c-erbB-2. 
Isolation and characterization of higher affinity light chain shuffled scF 
In a first approach to increase affinity, c-erbB-2 ECD coated polystyrene 
tubes were used for selecting the light chain shuffled library. Phage were 
subjected to three rounds of the rescue-selection-infection cycle. One 
hundred and eighty clones from the 2nd and the 3rd round of selection were 
analyzed for binding to recombinant c-erbB-2 ECD by ELISA. After the 3rd 
round of selection, greater than 50% of the clones were positive by ELISA 
(Table 8). 
TABLE 8 
______________________________________ 
Frequency of binding sFv and percent of binding sFv with slower k.sub.off 
than C6.5. binding was determined by ELISA. Rate constant k.sub.off was 
determined by BIAcore on unpurified sFv in bacterial periplasm. 
sFv with slower k.sub.off 
than C6.5 (parental 
ELISA sFv) 
Library and Round of Selection Round of Selection 
method of selection 
2 3 4 2 3 4 
______________________________________ 
V.sub.L -shuffling, 
41/180 97/180 ND ND ND ND 
selected on: 
antigen coated 
immunotubes 
soluble antigen 74/90 22/90 13/90 ND 0% 42% 
(rd 1, 100 nM; rd 2, 
50 nM; rd 3 10 nM; 
rd 4, 1 nM). 
soluble antigen ND 65/90 62/90 ND 25% 84% 
(rd 1, 40 nM; rd 2, 
1 nM,; rd 3, 0.1 nM; 
rd 4, 0.01 nM) 
V.sub.H -shuffling, ND 43/90 56/90 ND 0% 0% 
selected on: 
soluble antigen; 
(rd 1, 100 nM; rd 2, 
50 nM; rd 3, 10 nM; 
rd 4, 1 nM) 
soluble antigen ND 90/90 82/90 ND 0% 12% 
(rd 1, 40 nM; rd 2, 
1 nM; rd 3, 0.1 nM; 
rd 4, 0.01 nM) 
______________________________________ 
rd = round, ND = not determined, nM = 1.0 .times. 10.sup.-9 M 
TABLE 9 
______________________________________ 
IC.sub.50 and K.sub.d of C6.5sFv and 4 chain shuffled mutant sFvs. 
IC.sub.50 was 
determined by competition ELISA and K.sub.d by Scatchard 
after radioiodination. 
sFv IC.sub.50 (M) 
K.sub.d (M) 
______________________________________ 
C6.5 2.0 .times. 10.sup.-8 
2.0 .times. 10.sup.-8 
C6VLB 1.0 .times. 10.sup.-8 3.0 .times. 10.sup.-8 
C6VLD 5.8 .times. 10.sup.-9 2.6 .times. 10.sup.-8 
C6VLE 2.8 .times. 10.sup.-9 7.1 .times. 10.sup.-8 
C6VLF 7.5 .times. 10.sup.-9 7.9 .times. 10.sup.-8 
______________________________________ 
TABLE 10 
__________________________________________________________________________ 
Deduced protein sequences of light chain variable region 
genes of C6.5 and chain shuffled mutants. 
SEQ ID NO: 
__________________________________________________________________________ 
Framework 1 CDR1 Framework 2 CDR2 
10 20 30 35 40 50 
C6.5QSVLTQPPSVSAAPGQKVTISC SGSSSNIGNNYVS WYOOLPGTAPKLLIY GHTNRPA 36 
- 
Light chain shuffled mutants selected on polystyrene adsorbed antigen: 
C6VLB 
------------ --------------- SDNQ--S 
39 
- C6VLD 
--TN--------- --------------- TNDQ--S 40 
- C6VLE 
A------- -----S--R---- -N------ -----W RNNQ--S 41 
- C6VLF 
M------------ ------------- ----F---------H DNNK--S 42 
Light chain shuffled mutant selected on biotinylated antigen: 
C6L1 
G----W----- 
------------- --------------- DNNK--S 
43 
__________________________________________________________________________ 
SEQ ID NO: 
__________________________________________________________________________ 
Framework 3 CDR3 Framework 4 
60 70 80 
90 100 
C6.5 GVPDRFSGSKSGTSASLAISGFRSEDEADYYC AAWDDSLSG WV FGGGTKLTVLG 36 
- 
Light chain shuffled mutants selected on polystyrene adsorbed antigen: 
C6VLB 
L---------- 
--------H-- ------------ 
39 
- C6VLD 
LQ--------- -------N- -M ------------40 
- C6VLE 
V---------- -S--N---- -- ------------41 
- C6VLF 
I---I---------------LQ-D------- -------N- -- ------------42 
- 
Light chain shuffled mutant selected on biotinylated antigen: 
C6L1 
Q-----------L---------- 
--------- -- ------------ 
43 
__________________________________________________________________________ 
CDR, complementarity determining region; dashes indicate sequence 
identity. Numbering is according to Kabat et al., 1987, supra.. Underline 
residues are those that form the sheet interface that packs on the VH 
domain (Chothia et al., 1985 supra). 
Positive clones were ranked by IC.sub.50 as determined by competition ELISA 
(Table 9). Sixteen sFv with IC.sub.50 s less than the IC.sub.50 of the 
parental sfv were sequenced and four unique DNA sequences identified 
(Table 10). These clones were purified by IMAC after subcloning into 
PUC119SFI/NotmycHis, and the affinity determined by Scatchard analysis. 
Despite their lower IC.sub.5 0s, none of these 4 sfv had a higher affinity 
for c-erbB-2 (Table 9). Gel filtration analysis of the four purified sfv 
demonstrated the presence of two species, with size consistent for 
monomeric and dimeric sfv. In contrast, the parental sFv existed only as 
monomer. 
As a result of these observations, it was hypothesized that selection on 
immobilized antigen favored the isolation of lower affinity dimeric sFv 
which could achieve a higher apparent affinity due to avidity. In 
addition, determination of IC.sub.50 by inhibition ELISA using native sFv 
in periplasm did not successfully screen for sFv of higher affinity. To 
avoid the selection of lower affinity dimeric sFv, subsequent selections 
were performed in solution by incubating the phage with biotinylated 
c-erbB-2 ECD, followed by capture on streptavidin coated magnetic beads. 
To select phage on the basis of affinity, the antigen concentration was 
reduced each round of selection to below the range of the desired sFv 
K.sub.d (Hawkins et al. (1992), supra). To screen ELISA positive sFv for 
improved binding to c-erbB-2, a BIAcore was used. Periplasm preparations 
containing unpurified native sFv can be applied directly to a c-erbB-2 
coated BIAcore flow cell, and the k.sub.off determined from the 
dissociation portion of the sensorgram. This permitted ranking the chain 
shuffled clones by k.sub.off. Moreover, by plotting In (Rn/R.sub.0) vs t, 
the presence of multiple k.sub.off can be detected, indicating the 
presence of mixtures of monomeric and dimeric sFv. This strategy of 
selecting on antigen in solution, followed by BIAcore screening of ELISA 
positive sFv, was used to isolate higher affinity chain shuffle mutants. 
The light chain shuffled library was subjected to four rounds of selection 
on decreasing soluble antigen concentration (100 Nm, 50 Nm, 10 Nm, and, 1 
Nm). In a separate set of experiments, the 4 rounds of selection were 
performed using 40 nM, 1 nM, 0.1 nM, and 0.01 nM antigen concentration. 
Using the higher set of antigen concentrations for selection, 13/90 clones 
were positive for c-erbB-binding by ELISA after the 4th round of 
selection. In the BIAcore, 42% of these clones had a slower k.sub.off than 
the parental sFv. Using the lower set of antigen concentrations for 
selection, more clones were positive for c-erbB-2 binding by ELISA (62/90) 
after the 4th round of selection, and 84% had a slower k.sub.off than the 
parental sFv. Sequencing of the V.sub.L gene of ten of these sFv revealed 
one unique sFv (C6L1) (Table 10). The V.sub..lambda. gene of C6L1 was 
derived from the same germline gene as the parental sFv, but had 9 amino 
acid substitutions. The C6L1 gene was subcloned and the sFv purified by 
IMAC and gel filtration. C6L1 sFv was monomeric as determined by gel 
filtration and had an affinity 6 times higher than parental (Table 11). 
The increased affinity was due to both a faster k.sub.on and a slower 
k.sub.off (Table 11). The slower k.sub.off was associated with a three 
fold increase in the retention of sFv on the surface of SK-OV-3 cells (28% 
at 30 minutes for C6L1 compared to 10% at 3 minutes for the parental sFv). 
TABLE 11 
______________________________________ 
Affinities and binding kinetics of c-erbB-2 binding Single-chain Fv, 
K.sub.d, k.sub.on and k.sub.off were determined by surface plasmon 
resonance 
in a BIAcore. Combined Single-chain Fv result from combining the 
V.sub.L 
of C6L1 with the V.sub.H of either C6H1 or C6H2. 
sFv source and clone name 
K.sub.d (M) 
k.sub.on (M.sup.-1 s.sup.-1) 
k.sub.off (M.sup.-1 s.sup.-1) 
______________________________________ 
Parental C6.5 1.6 .times. 10.sup.-8 
4.0 .times. 10.sup.-5 
6.3 .times. 10.sup.-3 
Light Chain Shuffled C6L 2.6 .times. 10.sup.-9 7.8 .times. 10.sup.-5 
2.0 .times. 10.sup.-3 
Heavy Chain Shuffled 
C6H1 5.9 .times. 10.sup.-9 1.1 .times. 10.sup.-6 6.2 .times. 10.sup.-3 
C6H2 3.1 .times. 10.sup.-9 8.4 
.times. 10.sup.-5 2.6 .times. 10.sup.-3 
Combined sFv 
C6H1L1 1.5 .times. 10.sup.-8 4.1 .times. 10.sup.-5 6.2 .times. 10.sup.-3 
C6H2L1 6.0 .times. 10.sup.-9 3.0 .times. 10.sup.-5 1.8 .times. 10.sup.-3 
______________________________________ 
Isolation and characterization of higher affinity heavy chain shuffled scf. 
The VH5 heavy chain shuffled library was subjected to four rounds of 
selection on decreasing soluble antigen concentration (100 Nm, 5 nM, 10 
Nm, and, 1 Nm). In a separate set of experiments, the rounds of selection 
were performed using 40 Nm, 1 Nm, 0.1 Nm, and 0.01 Nm antigen 
concentration. Using the higher set of antigen concentrations for 
selection, 56/90 clones were positive for c-erbB-binding by ELISA after 
the 4th round of selection. None of these clones, however, had a slower 
k.sub.off than the parental sFv. Using the lower set of antigen 
concentrations for selection, more clones were positive for c-erbB-2 
binding by ELISA (82/90) after the 4th round of selection, and 12% had a 
slower k.sub.off than the parental sFv. No binders were isolated from 
either the V.sub.H 1 or V.sub.H 3 shuffled libraries. Sequencing of the 
V.sub.H gene of all slower k.sub.off clones revealed two unique sFv, C6H1 
and C6H2 (Table 12). The V.sub.H genes of C6H1 and C6H2 were derived from 
the same germline gene as the parental sFv, but differed by 7 and 9 amino 
acids respectively. C6H1 also had a stop codon in the heavy chain CDR1 and 
was expressed as a PIII fusion due to read through, albeit at very low 
levels. The two sFv were subcloned and purified by IMAC and gel 
filtration. Both sFv were monomeric as determined by gel filtration C6H1 
had 3 fold higher affinity for c-erbB2 than C6.5 and C6H2 had 5 fold 
higher affinity than C6.5 (Table 11). The increased affinity of 
C6H(5.9.times.10.sup.-9 M) was due to a faster k.sub.on, whereas the 
increased affinity of C6H2 (3.1.times.10.sup.-9 M) was due to both a 
faster k.sub.on and slower k.sub.off (Table 11). 
Location of mutations in chain shuffled scf 
Mutations in chain shuffled sFv were modeled on the Fv fragment of the 
immunoglobulin KOL (Marquart et al. (1980), supra) (FIGS. 2 and 3). KOL 
was selected as the model because it has a V.sub..lambda. gene derived 
from the same family as C6.5, and a V.sub.H gene with the same length 
CDR2. Mutations in higher affinity sFv were located both in surface 
residues at the antigen combining site, as well as residues located far 
from the binding site (FIG. 2). Except for two conservative mutations in 
V.sub.H framework 3 (V89M and F91Y), no mutations were located in residues 
which form the two 5 stranded B-sheets that form the V.sub.H -V.sub.L 
interface (Chothia et al. (1985) J. Mol. Biol. 186, 651-663) (FIG. 2 and 
Tables 10 and 12). In contrast, all 4 light chain shuffled sFv which 
formed mixtures of monomer and dimer had mutations in residues which 
formed the .beta.-sheet that packs on the V.sub.H domain (Table 4 and FIG. 
3). 
3 TABLE 12 
- Deduced protein sequences of heavy chain variable region genes of 
C6.5 and chain shuffled mutants. 
SEQ ID NO: 
Framework 1 CDR1 Framework 2 CDR2 
Framework 3 CDR3 Framework 4 
10 20 30 40 50 60 
70 80 abc 90 
100 C6.5 QVQLLQSGAELKKPGESLKISCKGSGYSFT 
SYWIA WVROMPGKGLEYMG LIYPGDSDTKYSPSFQG QVTISVDKSVSTAYLQWSSLKPSDSAVYFCAR 
HDVGYCSSSNCAKWPEYFQH 
WGOGTLV44 
Heavy chain shuffled mutants selected on high 
concentration biotinylated antigen: 
C6VHA2 
V---G-M-------------L--D-- T---- -------------- ----------------- 
--------------------R---T-M-Y--- -------------------- -------- 45 
C6VHB2 
Q---G-M-------------L--D-- T---- -------------- ----------------- 
-----A-E-I-----E-----A--T-M-Y--- -------------------- --------46 
C6VHC2 
Q---G-M--------------E---S T---- -------------- ----------------- 
------------T-------R---T---Y--- -------------------- --------47 
C6VHD2 
VE----M-------------F--D-S T---- -------------- ----------------- 
------------------------T-M-Y--- -------------------- --------48 
C6VHE2 
VE--G-M---R---------L--D-- T---- -------------- ----------------- 
--------------------R---T-M-Y--- -------------------- --------49 
C6VHF2 
VE--G-M-------------L--D-S T---- -------------- ----------------- 
--------------------R---T-M-Y--- -------------------- --------50 
C6VHG2 
VE--G-M-------------L--D-- T---- -------------- ----------------- 
--------------------R---T-M-Y--- -------------------- --------51 
C6VHH2 
VE----M-------------F--D-S T---- -------------- ----------------- 
--------------------R---T-M-Y--- -------------------- --------52 
C6VHA3 
V---G-M-------------L--D-- T---- -------------- ----------------- 
--------------------R---T-M-Y--- -------------------- --------45 
C6VHB3 
V---G-M-------------L--D-S T---- -------------- ----------------- 
-----A-E-I-----E-----A--T-M-Y--- -------------------- --------53 
C6VHC3 
V---G-M-------------L--D-- T---- -------------- ----------------- 
--------------------R---T-M-Y--- -------------------- --------45 
C6VHD3 
V---G-M-------------L--D-- T---- -------------- --------IR-----E- 
-------R-I-------T---A--T-M-Y--- -------------------- --------54 
C6VHE3 
VE--G-M---R---------L--D-- T---- -------------- ----------------- 
--------------------R---T-M-Y--- -------------------- --------49 
C6VHF3 
M-------------F--D-S T---- -------------- ----------------- 
------------------------T-M-Y--- -------------------- --------55 
C6VHG3 
V-------Q--------D-- T-Y-- -------------- I------R-I------- 
--R--A---------------A--T-M-Y--- -------------------- --------56 
C6VHH3 
E----V-E--Q--------F--D-S T---- -------------- ----------------- 
------------------------T-M-Y--- -------------------- --------57 
Heavy chain shuffled mutants selected on lower concentration 
biotinylated antigen: 
(E) 
C6H1 
VE----v---R--------------- --*-- -------------- ----------------- 
------------------------T-M-Y--- -------------------- --------58 
C6H2 
V-----v---R--------------- ----- -------------- ----A--K-I------- 
---------------------T--T-M-Y--- -------------------- 
--------59 
CDR, complementarity determining region; dashes indicate sequence 
identity. Numbering is according to Kabat et al. 1987, supra. Underlined 
residues are those that form the sheet interface that packs on the V.sub. 
domain (Chothia et al. 1985 supra.). 
Affinities of sFv resulting from combining higher affinity V.sub.H and 
V.sub.L genes obtained by chain shuffling. 
In an attempt to further increase affinity, shuffled V.sub.H and V.sub.L 
genes from higher affinity sFv were combined into the same sFv. Combining 
the V.sub.L gene from C6L1 with the V.sub.H gene from C6H1 resulted in an 
sFv (C6H1L1) with lower affinity than either C6L1 or C6H2 (Table 11). No 
additional reduction in k.sub.off was achieved, and the k.sub.on was 
reduced approximately 2 fold. Similarly, combining the V.sub.L gene from 
C6L1 with the V.sub.H gene from C6H2 resulted in an sFv (C6H2L1) with 
lower affinity than C6L1 or C6H2 (Table 11). No additional reduction in 
k.sub.off was achieved, and the k.sub.on was reduced approximately 2 fold. 
Thus, in both instances, combining the independently isolated higher 
affinity V.sub.H and V.sub.1 genes had a negative effect on affinity. 
Example 3 
Production of Higher Affinity Mutants 
In order to prepare higher affinity mutants derived from C6.5 part of the 
light chain and heavy chain CDR3 were sequentially randomized. The C6.5 VL 
CDR3 was modified by randomizing the sequence AAWDDSLSG (SEQ ID NO:60). 
The heavy chain CDR3 domain was randomized. The variable heavy chain CDR3 
was randomized 4 amino acids at a time: In other words, the CDR3 sequence 
of HDVGYCSSSNCAKWPEYFQH (SEQ ID NO:61) was modified by randomizing SSSN 
(SEQ ID NO:62) (library B), DVGY (SEQ ID NO:63) (library A), AKPE (SEQ ID 
NO:64) (library C) and YFQH (SEQ ID NO:65) (library D) respectively as 
described below. 
I. Materials and Methods 
Construction of phage antibody libraries 
As explained above, mutant sFv phage antibody libraries were constructed 
based on the sequence of C6.5, a human sFv isolated from a non-immune 
phage antibody library which binds to the tumor antigen c-erbB-2 with a Kd 
of 1.6.times.10.sup.-8 M (see Example 1). For construction of a library 
containing V.sub.L CDR3 mutants, an oligonucleotide (VL1; Table 18) was 
designed which partially randomized nine amino acid residues located in 
V.sub.L CDR3 (Table 4, above). For the nine amino acids randomized, the 
ratio of nucleotides was chosen so that the frequency of wild-type (wt) 
amino acid was 49%. 
To create the library, C6.5 sFv DNA (10 ng) was amplified by PCR in 50 
.mu.l reactions containing 25 pmol LMB3 (Marks et al., 1991 J. Mol. Biol. 
222: 581-597), 25 pmol VL1, 250 .mu.M dNTPs, 1.5 mM MgCl.sub.2, and 1 
.mu.l (5 units) Taq DNA polymerase (Promega) in the manufacturers buffer. 
The reaction mixture was subjected to 30 cycles of amplification 
(94.degree. C. for 30 s, 50.degree. C. for 30 s and 72.degree. C. for 1 
min) using a Hybaid OmniGene cycler. 
To introduce a NotI restriction site at the 3' end of the sFv gene 
repertoire, the PCR fragment (850 bp) was gel purified and reamplified 
using the primers LMB3 and VL2 (Table 18). The PCR product was purified, 
digested with SfiI and NotI, and ligated into pCANTABSE (Pharmacia) 
digested with SfiI and NotI. 
Ligation mixtures were purified as previously described above and aliquots 
electroporated (Dower et al. (1988) Nucleic Acids Res., 16: 6127-45) into 
50 .mu.l E. coli TG1 (Gibson (1984) Studies on the Epstein-Barr virus 
genome. PhD thesis, University of Cambridge). Cells were grown in 1 ml SOC 
(Sambrook et al., (1990) supra.) for 30 min and then plated on TYE 
(Miller, J. H. (1972) Experiments in Molecular Genetics, Cold Springs 
Harbor Lab Press, Cold Springs Harbor, N.Y.) media containing 100 .mu.g 
ampicillin/ml and 1 % (w/v) glucose (TYE-AMP-Glu). Colonies were scraped 
off the plates into 5 ml of 2.times.TY broth (Miller (1972) supra.) 
containing 100 .mu.g ampicillin/ml, 1% glucose (2.times.TY-AMP-Glu) and 
15% (v/v) glycerol for storage at -70.degree. C. The cloning efficiency 
and diversity of libraries was determined by PCR screening (Gussow & 
Clackson (1989) Nucleic Acids Res. 17) exactly as described in (Marks et 
al., (1991) supra.) and by DNA sequencing (Sanger et al. (1977) Proc. 
Natl. Acad. Sci. USA, 74: 5463-7). The mutant phage antibody library was 
designated C6VLCDR3. 
Four libraries of V.sub.H CDR3 mutants were constructed. For construction 
of each V.sub.H CDR3 library, oligonucleotides (VHA, VHB, VHC, and VHD; 
Table 18) were designed which completely randomized four amino acid 
residues located in V.sub.H CDR3 (amino acid residues 96 to 99, library A; 
residues 100a to 100d, library B; residues 100f, 100g, 100i, and 100j, 
library C; and residues 100k to H102, library D; Table 13). To create the 
libraries, DNA encoding the V.sub.H gene of C6.5 sFv DNA (10 ng) was 
amplified by PCR in 50 .mu.l reactions containing 25 pmol LMB3 (Marks et 
al., 1991) and 25 pmol of either VHA, VHB, VHC, or VHD exactly as 
described above. The resulting PCR fragments were designated VHA1, VHB1, 
VHC1, and VHD1, based on the mutagenic oligonucleotide used for 
amplification. In four separate PCR reactions, DNA encoding the light 
chain, sFv linker, V.sub.H framework 4 (FR4), and a portion of V.sub.H 
CDR3 of C6ML3-9 was amplified by PCR as described above using the primers 
C6hisnot and either RVHA, RVHB, RVHC, or RVHD (Table 18). 
These amplifications yielded PCR fragments VHA2, VHB2, VHC2, and VHD2. The 
5' end of primers RVHA, RVHB, RVHC, and RVHD were designed to be 
complementary to the 5' ends of primers VHA, VHB, VHC, and VHD 
respectively. This complementarity permits joining of the VH1 and VH2 PCR 
fragments together to create a full length sFv gene repertoire using 
splicing by overlap extension. To create the mutant sFv gene repertoires, 
200 ng of each PCR fragment (VHA1 and VHA2, VHB1 and VHB2, VHC1 and VHC2, 
or VHD1 and VHD2) were combined in 50 ml PCR reaction mixtures (as 
described above) and cycled seven times to join the fragments (94.degree. 
C. for 30 s, 60.degree. C. for 5 s, 40.degree. C. for 5 s (RAMP: 5 s), 
72.degree. C. for 1 min). After seven cycles, outer primers LMB3 and 
C6hisnot were added and the mixtures amplified for 30 cycles (94.degree. 
C. for 30 s, 50.degree. C. for 30 s, 72.degree. C. for 1 min). The PCR 
products were purified as described above, digested with SfiI and NotI, 
and separately ligated into pCANTAB5E (Pharmacia) digested with SfiI and 
NotI. The four ligation mixtures were purified as described above and 
electroporated into 50 .mu.l E. coli TG1. Transformed cells were grown and 
plated, and libraries characterized and stored, as described above. The 
mutant phage antibody libraries were designated C6VHCDR3A, C6VHCDR3B, 
C6VHCDR3C, and C6VHCDR3D. 
Preparation of phage and selection of phage antibody libraries. 
Preparation of phage for selection was performed exactly as described in 
Examples 1 and 2. Phage particles were purified and concentrated by two 
PEG-precipitations (Sambrook et al., 1990), resuspended in 5 ml phosphate 
buffered saline (25 mM NaH.sub.2 PO.sub.4, 125 mM NaCl, pH 7.0, PBS) and 
filtered through a 0.45 .mu. filter. All libraries were selected using 
biotinylated c-erbB-2 ECD and streptavidin-coated paramagnetic beads M280 
(Dynal) as described above. For selection of the C6VLCDR3 library, 
c-erbB-2 ECD concentrations of 4.0.times.10.sup.-8 M, 1.0.times.10hu -9 M, 
1.0.times.10.sup.-10 M, and 1.0.times.10.sup.-11 M were used for selection 
rounds 1, 2, 3, and 4 respectively. The mixture of phage and antigen was 
gently rotated for 1 h at room temperature and phage bound to biotinylated 
antigen captured using 100 .mu.l (round 1) or 50 .mu.l (rounds 2, 3, and 
4) of streptavidin-coated M280 magnetic beads. After capture of phage, 
Dynabeads were washed a total of ten times (three times in PBS containing 
0.05% Tween 20 (TPBS), twice in TPBS containing 2% skimmed milk powder (2% 
MTPBS), twice in PBS, once in 2% MPBS, and twice in PBS) using a Dynal 
magnetic particle concentrator. The Dynabeads were resuspended in 1 ml 
PBS, and 300 .mu.l were used to infect 10 ml log phase E. coli TG1 which 
were plated on TYE-AMP-Glu plates. For selection of the C6VHCDR3 
libraries, c-erbB-2 ECD concentrations of 5.0.times.10.sup.-9 M, 
5.0.times.10.sup.-11 M, 5.0.times.10.sup.-12 M, and 5.0.times.10.sup.-13 M 
were used for selection rounds 1, 2, 3, and 4 respectively and the phage 
captured by incubating with 50 .mu.l of Dynabeads for 5 min. 
The washing protocol was altered to select for sFv with the lowest 
k.sub.off (Hawkins et al. (1992) J. Mol. Biol. 226: 889-896). Dynabeads 
with bound phage were initially subjected to five rapid washes 
(4.times.TPBS, 1.times.MPBS) followed by six 30 min incubations in one of 
three washing buffer (2.times.TPBS, 2.times.MPBS, 2.times.PBS) containing 
1.0.times.10.sup.-7 M c-erbB-2 ECD. Bound phage were eluted from the 
Dynabeads by sequential incubation with 100 .mu.l of 4 M MgCl.sub.2 for 15 
min followed by 100 .mu.l of 100 mM HCl for 5 min. Eluates were combined 
and neutralized with 1.5 ml of 1 M Tris HCl, pH 7.5 and one third of the 
eluate used to infect log phase E. coli TG1. 
Initial sFv characterization. 
Initial analysis of selected sFv clones for binding to c-erbB-2 ECD was 
determined by phage ELISA. To prepare phage for ELISA, single ampicillin 
resistant colonies were transferred into microtitre plate wells containing 
100 .mu.l 2.times.TY-AMP-0.1% glucose and grown for three hours at 
37.degree. C. to an A.sub.600 of approximately 0.5. VCSM13 helper phage 
(2.5.times.10.sup.8 phage) were added to each well, and the cells 
incubated for 1 hour at 37.degree. C. 
Kanamycin was then added to each well to a final concentration of 25 
.mu.g/ml and the bacteria grown overnight at 37.degree. C. Supernatants 
containing phage were used for ELISA. For ELISA, Immunolon 4 plates 
(Dynatech) were incubated overnight at 4.degree. C. with ImmunoPure avidin 
(10 .mu.g/ml in PBS; Pierce). After washing three times with PBS to remove 
unbound avidin, wells were incubated with biotinylated c-erbB-2 ECD as 
described above. 
Binding of sFv phage to c-erbB-2 ECD was detected with 
peroxidase-conjugated anti-M13 antibody (Pharmacia) and ABTS (Sigma) as 
substrate. Selected binders were further characterized by DNA sequencing 
of the V.sub.H and V.sub.L genes. 
Ranking of sFv by k.sub.off was performed using SPR in a BIAcore as 
described above. Briefly, 10 ml cultures of 24 ELISA positive clones from 
the third and fourth round of selection were grown to an A.sub.600 of 
approximately 0.8, sFv expression induced (De Bellis et al. (1990). 
Nucleic Acids Res., 18: 1311) and the culture grown overnight at 
25.degree. C. Single-chain Fv were harvested from the periplasm (Breitling 
et al. (1991) Gene, 104: 147-153), and the periplasmic fraction dialyzed 
for 48 h against hepes buffered saline (10 mM hepes, 150 mM NaCl, pH 7.4, 
HBS). In a BIAcore flow cell, approximately 1400 resonance units (RU) of 
c-erbB-2 ECD were coupled to a CM5 sensor chip using NHS-EDC chemistry 
(Johnsson et al. (1991) Anal. Biochem. 198: 268-277). Association and 
dissociation of undiluted sFv in the periplasmic fraction were measured 
under a constant flow of 5 .mu.l/min and HBS as running buffer. An 
apparent k.sub.off was determined from the dissociation part of the 
sensorgram for each sFv analyzed (Karlsson et al. (1993) J. Immunol. Meth. 
166: 75-84). The flow cell was regenerated between samples using 
sequential injections of 4 M MgCl.sub.2 and 100 mM triethylamine without 
significant change in the sensorgram baseline after analysis of more than 
100 samples. 
Subvloning, expression and purification of sFv. 
To facilitate purification of sFv selected from the C6VLCDR3 library, the 
sFv genes were subcloned into the expression vector pUC119 Sfi-NotmycHis, 
which results in the addition of a hexa-histidine tag at the C-terminal 
end of the sFv. The sFv selected from the C6VHCDR3 library already have a 
C-terminal hexa-histidine tag and therefore could be purified without 
subcloning. 500 ml cultures of E. coli TG1 harboring one of the C6.5 
mutant phagemids were grown, expression of sFv induced (De Bellis et al. 
(1990) supra.), and the culture grown at 25.degree. C. overnight. 
Single-chain Fv were harvested from the periplasm (Breitling et al. (1991) 
supra.), dialyzed overnight at 4.degree. C. against 8 L of IMAC loading 
buffer (50 mM sodium phosphate, pH 7.5, 500 mM NaCl, 20 mM imidazole) and 
then filtered through a 0.2 micron filter. Single-chain Fv was purified by 
IMAC (Hochuli et al. (1988) Bio/Technology, 6: 1321-1325) as described 
above. 
To separate monomeric, dimeric and aggregated sFv, samples were 
concentrated to a volume &lt;1 ml in a Centricon 10 (Amicon) and fractionated 
on a Superdex 75 column using a running buffer of HBS. The purity of the 
final preparation was evaluated by assaying an aliquot by SDS-PAGE. 
Protein bands were detected by Coomassie staining. The concentration was 
determined spectrophotometrically, assuming an A.sub.280 nm of 1.0 
corresponds to an sFv concentration of 0.7 mg/ml. 
Measurement of affinity and binding kinetics. 
The K.sub.d of sFv were determined using SPR in a BlAcore. In a BIAcore 
flow cell, approximately 1400 RU of c-erbB-2 ECD (90 kDa, McCartney et al. 
(1995) Protein Eng. 8: 301-314) were coupled to a CM5 sensor chip 
(Johnsson et al. (1991) supra.). Association rates were measured under 
continuous flow of 5 ml/min using concentrations ranging from 
5.0.times.10.sup.-8 to 8.0.times.10.sup.-7 M. Rate constant k.sub.on was 
determined from a plot of (1n (dR/dt))/t vs concentration (Karlsson et 
al., 1991). 
To verify that differences in k.sub.on were not due to differences in 
immunoreactivity, the relative concentrations of functional sFv was 
determined using SPR in a BIAcore (Karisson et al. (1993) supra.). 
Briefly, 4000 RU of c-erbB-2 ECD were coupled to a CM5 sensor chip and the 
rate of binding of C6.5 (RU/s) determined under a constant flow of 30 
ml/min. Over the concentration range of 1.0.times.10.sup.-9 to 
1.0.times.10.sup.-7 M, the rate of binding was proportional to the log of 
the sFv concentration. Purified sFv were diluted to the same concentration 
(1.0.times.10.sup.-8 M and 2.0.times.10.sup.-8 M) as determined by 
A.sub.280. The rate of binding to c-erbB-2 ECD was measured and used to 
calculate the concentration based on the standard curve constructed from 
C6.5. Dissociation rates were measured using a constant flow of 25 
.mu.l/min and a sFv concentration of 1.0.times.10.sup.-6 M. k.sub.off was 
determined during the first 2 min of dissociation for sFv mutated in 
V.sub.L CDR3 (Karlsson et al. (1991) supra.) and during the first 15 to 60 
min for clones with k.sub.off below 5.times.10.sup.-4 s.sup.-1 (sFv 
mutated in V.sub.H CDR3 and combined sFv). To exclude rebinding, k.sub.off 
was determined in the presence and absence of 5.0.times.10.sup.-7 M 
c-erbB-2 ECD as described above in Examples 1 and 2. 
Cell surface retention assay. 
The cell surface retention of selected sFv was determined on live SK-OV-3 
cells using a fluorescence activated cell sorter (FACS). Purified sFv were 
labeled with NHS-LC-Biotin (Pierce) using the manufacturers instructions. 
The concentration of immunoreactive biotinylated sFv was calculated using 
SPR as described above. The efficiency of biotinylation was also 
determined in a BIAcore using a flow cell to which 5000 RU of streptavidin 
was coupled. The total responses after association were compared between 
samples and concentrations of sFv were adjusted using the results obtained 
from the BIAcore. For the assay, aliquots of SK-OV-3 cells 
(1.2.times.10.sup.7 c-erbB-2 positive cells) were incubated with 14 .mu.g 
biotinylated sFv in a total volume of 0.5 ml (1 .mu.M sFv) FACS buffer 
(PBS containing 1% BSA and 0.1 % NaN.sub.3) for 1 h at 37.degree. C. Cells 
were washed twice with 10 ml FACS buffer (4.degree. C.) and resuspended in 
12 ml FACS buffer and further incubated at 37.degree. C. Aliquots of cells 
(0.5 ml from 12 ml containing 5.times.10.sup.5 cells) were taken after 5 
min, every 15 min for the first hour and after two hours repeating the 
wash and resuspension cycle. Washed cell aliquots were fixed with 1% 
paraformaldehyde, washed twice with FACS buffer, and incubated for 15 min 
at 4.degree. C. with a 1:800 dilution of phycoerythrine-labeled 
streptavidin (Pierce). Fluorescence was measured by FACS and the percent 
retained fluorescence on the cell surface plotted versus the time points. 
Single-chain Fv used for the cell surface retention assay were C6.5 
(K.sub.d =1.6.times.10.sup.-8 M), C6ML3-9 (K.sub.d =1.0.times.10.sup.-9 
M), C6MH3-B1 (K.sub.d =1.2.times.10.sup.-10 M), and the anti-digoxin sFv 
26-10 (Huston et al. (1988) Proc. Natl. Acad. Sci. USA, 85: 5879-83) as 
negative control. 
High resolution functional scan of C6. 5 V.sub.H CDR3. 
A high resolution functional scan of the C6.5 V.sub.H CDR3 was performed by 
individually mutating residues 95-99, 100a-100d, and 100g-102 to alanine. 
The pair of cysteine r esidues (100 and 100e) were sim u ltaneously 
mutated to serine. Residue 100f (alanine) was not studied. Mutations were 
introduced by oligonucleotide directed mutagenesis using the method of 
Kunkel et al. (1987)Meth. Enzymol., 154: 367-82. 
Insertion of the correct mutation was verified by DNA sequencing, and sFv 
was expressed (De Bellis et al. (1990) supra.; Breitling et al. (1991) 
supra.) and purified by IMAC (Hochuli et al. (1988) supra.). Affinities 
were determined by SPR as described above and compared to C6.5 sFv. 
Modeling of location of mutations. 
The location of mutations in mutated sFv was modeled on the structure of 
the Fab KOL (Marquart et al. (1980) J. Mol. Biol., 141: 369-391) using the 
program O (Jones et al. (1991). Acta Cryst., A47: 110-119) on a Silicon 
Graphics workstation. 
II. Results 
1) Mutation of C6.5 sFv V.sub.L CDR3 
Library construction and selection. 
As explained above, 9 amino acids in V.sub.L CDR3 were partially randomized 
by synthesizing a "doped" oligonucleotide in which the wild-type 
nucleotide occurred with a frequency of 49%. After transformation, a 
library of 1.0.times.10.sup.7 clones was obtained. The mutant phage 
antibody library was designated C6VLCDR3. 
Polymerase chain reaction (PCR) screening revealed that 100% of clones 
analyzed had full length insert and diversity was confirmed by sequencing 
the V.sub.L CDR3 of ten clones from the unselected library. Prior to 
selection, 5/92 clones selected at random expressed sFv which bound 
c-erbB-2 ECD by enzyme linked immunosorbent assay (ELISA). 
The C6VLCDR3 library was subjected to four rounds of selection using 
decreasing concentrations of biotinylated c-erbB-2 ECD. A relatively high 
antigen concentration (4.0.times.10.sup.-8 M) was used for the first round 
to capture rare or poorly expressed phage antibodies. The concentration 
was decreased 40 fold for the second round (1.0.times.10.sup.-9 M), and 
decreased a further tenfold each of the subsequent two rounds 
(1.0.times.10.sup.-10 M, 3rd round; 1.0.times.10.sup.-11 M, 4th round). 
After each round of selection, the concentration of binding phage in the 
polyclonal phage preparation was determined by measuring the rate of 
binding of polyclonal phage to c-erbB-2 ECD under mass transport limited 
conditions using surface plasmon resonance (SPR) in a BIAcore. The results 
were used to guide the antigen concentration for the subsequent round of 
selection. After both the third and fourth rounds of selection, 92/92 
clones bound c-erbB-2 ECD by ELISA. 
Characterization of mutant sFv. 
To identify sFv with a lower K.sub.d than wild-type sFv, apparent k.sub.off 
was determined by SPR in a BIAcore on unpurified native sFv in bacterial 
periplasm. Twenty-four sFv from the third and fourth rounds of selection 
were ranked by k.sub.off. After the third round of selection, 80% of sFv 
had a lower k.sub.off than wt and after four rounds, 100% of sFv had a 
lower k.sub.off than wild-type sFv. The twelve sFv with the lowest 
k.sub.off from each of these rounds of selection were sequenced and each 
unique sFv gene was subcloned for purification. Single-chain Fv were 
purified by immobilized metal affinity chromatography (IMAC), followed by 
gel filtration to remove any dimeric or aggregated sFv. 
The k.sub.on, and k.sub.off were determined by BIAcore, and the K.sub.d 
calculated. After the third round of selection, seven unique sFv were 
identified, all with higher affinity than wild-type sFv. Single-chain Fv 
had on average 1.8 amino acid substitutions/sFv, with a single 
substitution at residue 92 the most frequently observed mutation. These 
single amino acid substitutions would have occurred with a frequency of 
1/12,000 in the original library, assuming equal nucleotide coupling 
efficiency. The average sFv affinity was 3.6.times.10.sup.-9 M (4.4 fold 
increase), with the highest affinity 2.6.times.10.sup.-9 M (sixfold 
increase). 
After four rounds of selection, six sFv were identified, and none of these 
sequences were observed in the sFv sequenced from the third round. 
Single-chain Fv from the fourth round had on average 2.9 amino acid 
substitutions/sFv, with expected frequencies of between 1/590,000 and 
1/24,000,000 in the original library. The average sFv affinity after the 
fourth round was 1.9.times.10.sup.-9 M (8.4 fold increase), with the 
highest affinity 1.0.times.10.sup.-9 M (16 fold increase). The results 
demonstrate the efficiency of the selection technique for isolating very 
rare high affinity clones from a library. Additional high affinity sFv 
(Table 14; C6ML3-14, -15, -19, -23, and -29) were isolated from the 
C6VLCDR3 library by using a different elution solution after capture of 
antigen bound phage. 
Location of mutations in higher affinity sFv. 
Significant sequence variability (six different amino acids) was observed 
at residues 93, and 94, with less variability (three different amino 
acids) at residues 95 and 95a. Thus a subset of the randomized residues 
appear to be more important in modulating affinity. All but one of these 
four residues (V.sub.1 L95) appear to have solvent accessible side chains 
in the C6.5 model. Three of the residues randomized (A89, W91, and G96) 
were 100% conserved in all mutants sequenced. Two additional residues 
(A90S and D92E) showed only a single conservative substitution. These 
conserved residues appear to have a structural role in the variable 
domain, either in maintaining the main chain conformation of the loop, or 
in packing on the V.sub.H domain. Residues A89, W91, and D92 are identical 
in both C6.5 and KOL, with conservative substitutions A90S and G96A 
observed at the other two positions in KOL, consistent with a structural 
role. 
In the model of C6.5 indicated by this invention, G95b is in a turn and 
A89, A90, and W91 pack against the V.sub.H domain at the V.sub.H -V.sub.L 
interface. Hydrogen bonds between V.sub.1 D92 and V.sub.1 S27a and V.sub.1 
N27b bridge L3 and L1 to stabilize the L3 and L1 conformations. 
2) Mutation of C6ML3-9 sFv V.sub.H CDR3 
Library construction and selection. 
To further increase the affinity of C6.5, we chose to mutate the V.sub.H 
CDR3 of the highest affinity sFv (C6ML3-9, K.sub.d =1.0.times.10.sup.-9 M) 
isolated from the C6VLCDR3 library, rather than mutate C6.5 V.sub.H CDR3 
independently and combine mutants. This sequential approach was taken 
since the kinetic effects of independently isolated antibody fragment 
mutations are frequently not additive (Yang et al. (1995) J. Mol. Biol., 
254: 392-403). 
Due to the length of the C6.5 V.sub.H CDR3 (20 amino acids), a high 
resolution functional scan was performed on C6.5 sFv in an attempt to 
reduce the number of amino acids subjected to mutation. Residues 95-99, 
100a-100d, and 100g-102 were separately mutated to alanine, and the 
K.sub.d of the mutated sFv determined. Residue 100f (alanine) was not 
studied. Residues 100 and 100e are a pair of cysteines separated by four 
amino acids. A homologous sequence in KOL (Marquardt et al. (1980) supra.) 
results in a disulfide bond between the two cysteines and a four residue 
miniloop. Therefore the two cysteines were simultaneously mutated to 
serine. 
Results of the alanine scan are shown in Table 13. No detectable binding to 
c-erbB-2 ECD could be measured by BIAcore for C6.5H95A, C6.5W100hA, and 
C6.5E100jA. Three additional alanine mutants (G98A, Y100kA, and F1001A) 
yielded sFv with 20 fold to 100 fold higher K.sub.d than wt sFv. 
Substitution of the two cysteines by alanine (100, 100e) yielded an sFv 
with an 17.5 fold higher K.sub.d, and a much faster k.sub.off 
(1.38.times.10.sup.-1 s.sup.-1) than wt C6.5. The remainder of the alanine 
substitutions yielded only minor (0.5 to 3.7 fold) increases or decreases 
in K.sub.d. 
Based on the results of the alanine scan and a model of C6.5 based on the 
Fab KOL (Marquardt et al., 1980), residues H95A, C100, and C100e were not 
mutated due to their probability of having an important structural role. 
H95 is likely to be buried at the V.sub.H -V.sub.L interface where it 
makes critical packing contacts with the V.sub.L domain. The two cysteine 
residues also are likely to have a structural role in maintaining the 
miniloop conformation. W100h was also not mutated given the unique 
features of tryptophan in antibody combining sites (Mian et al. (1991) J. 
Mol. Biol., 217: 133-151). 
The remaining 16 amino acids were completely randomized four residues at a 
time in four separate C6VHCDR3 libraries (96-99, library A; 100a-100d, 
library B; 100f, 100g, 100i, and 100j, library C, and 100k-102, library D; 
see Table 14). After transformation, libraries were obtained with sizes 
1.7.times.10.sup.7 (library A), 1.3.times.10.sup.7 (library B), 
3.0.times.10.sup.6 (library C), and 2.4.times.10.sup.7 (library D). The 
mutant phage antibody libraries were designated C6VHCDR3 libraries A, B, 
C, and D. PCR screening and DNA sequencing 
TABLE 13 
______________________________________ 
Binding kinetics of C6.5 V.sub.H CDR3 mutants obtained by high 
resolutions functional scan. Amino acid residues 95-99, 100a-100d, 
and 100g-102 of C6.5 V.sub.H CDR3 were mutated to alanine using site 
directed mutagenesis. Cysteine residues, C100 and C100e, were 
simultaneously mutated to serine. k.sub.on and k.sub.off were measured 
by SPR in a BIAcore, and the K.sub.d calculated. Numbering is according 
to 
Kabat et al. (1987). NB = no binding. 
k.sub.on 
Kd (mutant) 
K.sub.d [10.sup.5 M.sup.-1 
k.sub.off 
sFv clone Kd (C6.5) [10.sup.-8 M] s.sup.-1 ] [10.sup.-2 s.sup.-1 
______________________________________ 
] 
C6.5H95A NB NB NB NB 
C6.5D96A 2.8 4.5 2.2 .+-. 0.34 1.0 .+-. 0.02 
C6.5V97A 3.0 4.8 3.1 .+-. 0.62 1.5 .+-. 0.02 
C6.5G98A 19.8 31.7 4.1 .+-. 0.71 13 .+-. 0.55 
C6.5Y99A 3.7 5.9 9.0 .+-. 0.17 5.3 .+-. 0.07 
C6.5C100S/ 17.5 28.0 5.0 .+-. 0.25 13.8 .+-. 0.71 
C100eS 
C6.5S100aA 1.8 2.8 4.7 .+-. 0.55 1.3 .+-. 0.04 
C6.5S100bA 2.9 4.7 3.4 .+-. 0.49 1.6 .+-. 0.07 
C6.5S100cA 1.5 2.4 4.5 .+-. 0.62 1.1 .+-. 0.03 
C6.5N100dA 1.8 2.9 4.1 .+-. 0.34 1.2 .+-. 0.05 
C6.5K100gA 0.6 0.98 4.3 .+-. 0.31 0.42 .+-. 0.01 
C6.5W100hA NB NB NB NB 
C6.5P100iA 0.6 1.0 10.5 .+-. 0.12 1.1 .+-. 0.02 
C6.5E100jA NB NB NB NB 
C6.5Y100kA 101.0 161.6 0.73 .+-. 0.07 11.8 .+-. 0.25 
C6.5F100lA 28.4 45.4 1.1 .+-. 0.13 5.0 .+-. 0.06 
C6.5Q101A 0.5 0.82 12.0 .+-. 0.02 0.98 .+-. 0.02 
C6.5H102A 1.2 1.9 5.9 .+-. 0.57 1.1 .+-. 0.02 
______________________________________ 
revealed that 100% of clones from all four libraries had full length insert 
and that the sequences were diverse (results not shown). Prior to 
selection, the percent of clones expressing sFv which bound c-erbB-2 ECD 
by ELISA was 1% for C6VHCDR3 library A, 57%, library B, 2% library C, and 
3% library D. The C6VHCDR3 libraries A, B, C, and D were selected on 
biotinylated c-erbB-2 ECD as described above, but using lower antigen 
concentration. The first round of selection was performed using 
5.0.times.10.sup.-9 M c-erbB-2 ECD, tenfold lower than for the first round 
of selection of the C6VLCDR3 library. This concen tration was ch osen 
because the parental sFv for these libraries (C6ML3-9) had a greater than 
tenfold lower K.sub.d than the parental clone for the C6VLCDR3 library 
(C6.5). Biotinylated c-erbB-2 ECD concentration was then decreased 100 
fold for the second round of selection (5.0.times.10.sup.-11 M) and 
tenfold for the third and fourth rounds (5.0.times.10.sup.-12 M and 
5.0.times.10.sup.-13 M). As for the C6VLCDR3 library, the rate of binding 
of polyclonal phage was measured in a BIAcore to determin e the antigen 
concentration used for the subsequent round of selection as discussed 
below. 
Characterization of mutant sFv. 
After four rounds of selection, positive clones were identified by ELISA 
and at least 24 sFv from the fourth round of selection were ranked by 
k.sub.off using SPR in a BIAcore. The ten sFv with the lowest k.sub.off 
from C6VHCDR3 libraries A, C, and D were sequenced. 
TABLE 14 
__________________________________________________________________________ 
Sequences, affinities and binding kinetics of scFv isolated from heavy 
chain CDR3 libraries A, B, C, 
and D. k.sub.on and k.sub.off were determined in a BIAcore using 
purified scFv, and k.sub.d calculated. 
Dashes indicate sequence identity. Mutations arising from PCR error and 
located outside V.sub.H CDR3 
are listed under the heading "other mutations". F = frequency of 
isolated scFv. *k.sub.off determined 
from unpurified scFv samples. Underline indicates mutated residue. 
Clone Name 
VH CDR3 sequence Other Mutations 
K.sub.d (10.sup.-10 M) 
K.sub.off (10.sup.-4 s.sup.-1) 
SEQ ID NO 
__________________________________________________________________________ 
C6.5 HDVGYCSSSNCAKWPEYFQH 1.60 63.0 61 
VH CDR3 library A: 
C6ML3-9 (wt) 
--DVGY-------------------------------- 61 
C6ML3-A2 HDVGFCSSSNCAKWPEYFQH 66 
C6ML3-A3 HDVGYCSSSDCAKWPEYFQH 160.0 63.0 67 
VH CDR3 library B: 
C6ML3-9 (wt) 
------------SSSN---------------------- 
10.0 7.6 61 
C6MH3-B1 HDVGYCTDRTCAKWPEYFQH 1.6 0.67 68 
C6MH3-B15 HDVGYCESSRCAKWPEYFQH 7.7 2.9 69 
C6MH3-B11 HDVGYCSDRSCAKWPEYFQH 2.2 2.3 70 
C6MH3-B9 HDVGYCKTAACAKWPEYFQH 8.7 3.3 71 
C6MH3-B8 HDVGYC*TERCAKWPEYFQH 7.2 2.9 72 
C6MH3-B2 HDVGYCTDPRCAKWPEYFQH 3.1 3.1 73 
C6MH3-B39 HDVGYCTDPTCAKWPEYFQH 3.2 1.9 74 
C6MH3-B25 HDVGYCLTTRCAKWPEYFQH 3.6 1.9 75 
C6MH3-B21 HDVGYCTTPLCAKWPEYFQH 7.3 2.4 76 
C6MH3-B20 HDVGYCSCAKWPEYFQH 8.7 1.6 77 
C6MH3-B16 HDVGYCADVRCAKWPEYFQH 3.1 2.8 78 
C6MH3-B47 HDVGYCTDRSCAKWPEYFQH 1.1 0.75 79 
C6MH3-B48 HDVGYCTDPSCAKWPEYFQH 2.3 1.3 80 
C6MH3-B5 HDVGYCTDATCAKWPEYFQH 3.4 2.3 81 
C6MH3-B41 HDVGYCTDRPCAKWPEYFQH 5.3 2.7 82 
C6MH3-B2 HDVGYCTDPRCAKWPEYFQH 5.8 3.2 73 
C6MH3-B27 HDVGYCKNSRCAKWPEYFQH 4.7 4.0 83 
C6MH3-B34 HDVGYCQDTRCAKWPEYFQH VL Q1R ND ND 84 
C6MH3-B43 HDVGYCEDYTCAKWPEYFQH ND ND 85 
C6MH3-B46 HDVGYCTTPRCAKWPEYFQH VH K23Q ND ND 86 
VH V76G 
C6MH3-B33 HDVGYCSDQTTCAKWPEYFQH ND ND 87 
C6MH3-B31 HDVGYCDDYTCAKWPEYFQH VL P7L ND ND 88 
VH CDR3 library C: 
C6ML3-9 (wt) 
----------------------AKWPE-------- 
10.0 7.6 61 
C6MH3-C4 HDVGYCSSSNCAVWPEYFQH 3.7 2.0 89 
C6MH3-C3 HDVGYCSSSNCAKWPEYFQH VH G15E 6.5 3.2 61 
VH CDR3 library D: 
C6ML3-9 (wt) 
HDVGYCSSSNCAKWPEYFQH 10.0 7.6 61 
C6MH3-D2 HDVGYCSSSNCAKWPEWLGV 1.6 2.0 90 
C6MH3-D3 HDVGYCSSSNCAKWPEWLDN 2.7 2.5 91 
C6MH3-D6 HDVGYCSSSNCAKWPEWMYP 3.5 1.8 92 
C6MH3-D5 HDVGYCSSSNCAKWPEWMQM 3.8 2.1 93 
C6MH3-D1 HDVGYCSSSNCAKWPEWLHV 3.1 1.1 94 
C6MH3-D7 HDVGYCSSSNCAKWPEWQDP ND 3.1 95 
__________________________________________________________________________ 
Due to the diversity of isolated sFv in C6VHCDR3 library B, 48 sFv were 
ranked by k.sub.off using SPR, and 22 clones with the lowest k.sub.off 
were sequenced. Single-chain Fv were purified by IMAC, followed by gel 
filtration to remove any dimeric or aggregated sFv. The K.sub.on, and 
k.sub.off were determined by BIAcore and the K.sub.d calculated. 
Very different results were obtained from the four libraries with respect 
to the number of higher affinity sFv isolated, and the value of the 
highest affinity sFv. The best results were obtained from library B (Table 
14). Fifteen sFv were isolated with a K.sub.d lower than wt C6ML3-9 and no 
wt sequences were observed (Table 14). The best sFv (C6MH3-B47) had a 
K.sub.d =1.1.times.10.sup.-10 M, ninefold lower than C6ML3-9 and 145 fold 
lower than C6.5. The k.sub.off of this sFv was 7.5.times.10.sup.-5 
s.sup.-1, tenfold lower than C6ML3-9 and 84 fold lower than C6.5. While a 
wide range of sequences was observed (Table 14, library B), a subset of 
sFv had the consensus sequence TDRT (SEQ ID NO:96) (first eight sFv, Table 
14). The consensus sequence is identical with the sequence of C6MH3-B1, 
which is the sFv with the lowest k.sub.off (6.0.times.10.sup.-5 s.sup.-1). 
Five sFv were isolated that had a k.sub.off 2.5 to 3.75 fold lower than 
C6ML3-9, however expression levels were too low to obtain adequate 
purified sFv for measurement of the K.sub.d (last five sequences, Table 
14, library B). The next best results were obtained from library D (Table 
14). Five higher affinity sFv were isolated, with the best having a 
K.sub.d sevenfold higher than wt C6ML3-9. An additional sFv was isolated 
that had a k.sub.off lower than wt sFv, however the expression level was 
too low to obtain adequate purified sFv for measurement of the K.sub.d 
(last sequence, Table 14, library D). There was selection for a consensus 
mutation of Y100kW and replacement of F1001 with hydrophobic methionine or 
leucine. No higher affinity sFv were isolated from either the A or C 
libraries. From library A, 8/10 sFv were wild-type, with one higher 
affinity sFv, a contaminant from library B. A single mutant sFv with the 
conservative replacement of Y99F had an apparent k.sub.off 2.5 times lower 
than wt, but expression levels were too low to obtain adequate purified 
sFv to measure the K.sub.d. From library C, 8/10 sFv were wt sFv, with one 
higher affinity sFv having mutations located in the V.sub.H and V.sub.L 
genes, but not in the region intentionally mutated. The isolated mutant 
sFv K100gV had a K.sub.d 2.7 fold lower than wt (k.sub.off 3.8 fold lower 
than C6ML3-9), correlating with the data of the alanine scan, in which 
K100gA was the only sFv with decreased k.sub.off. 
Ability of alanine scanning to identijy residues which modulated affinity. 
Residue E100j, the only residue that when converted to alanine had no 
detectable binding, was 100% conserved. Otherwise, there was no 
correlation between the frequency with which the wt amino acid was 
recovered and the extent to which binding was reduced by substitution to 
alanine. Similarly, there was no correlation between residues shown to 
modulate affinity by alanine scanning and mutations exhibiting improved 
binding. This is clear when comparing the results obtained from library B 
(where no alanine mutant had more than a 2.9 fold increase in K.sub.d) and 
library D (where K.sub.d was markedly increased for two alanine mutants, 
Y100kA and F1001A). Despite the different alanine scan results, both 
libraries yielded similar nine and sevenfold increases in affinity. This 
result appears to be different than the results of Lowman et al. (1993) J. 
Mol. Biol., 234: 564-578, who found a mild (R.sup.2 =0.71) positive 
correlation between the frequency with which the wt amino acid was 
recovered from a phage library of human growth hormone mutants and the 
extent to which binding was reduced by alanine scanning. In addition, 
their largest improvements in affinity were for those residues shown by 
alanine scanning to significantly affect binding. 
The reason for the different results is unclear, however in two of the 
V.sub.H CDR3 libraries where alanine scanning indicated a significant 
effect on binding (libraries A and C), expression levels of mutants were 
generally low. This could have affected the selection results. 
3) Correlation between affinity and cell surface retention of sFv. 
The retention of biotinylated C6.5, C6ML3-9, and C6MH3-B1 sFv on the 
surface of SK-OV-3 cells expressing c-erbB-2 was determined, both to 
verify the observed differences in k.sub.off, and to confirm that the 
antigen as presented in the BIAcore had biologic significance. The half 
life (t.sub.1/2 of the sFv on the cell surface was much less than 5 min 
for C6.5, 11 min for C6ML3-9, and 102 min for C6MH3-B1. These values agree 
closely with the t.sub.1/2 calculated from the k.sub.off as determined by 
SPR in a BIAcore (1.6 min for C6.5, 13 min for C6ML3-9, and 135 min for 
C6MH3-B1). The anti-digoxin sFv 26-10 (Huston et al. (1988) supra.) was 
used as negative control, and no binding to c-erbB-2 ECD in a BIAcore or 
to c-erbB-2 on SK-OV-3 cells was observed. 
Example 4 
Elution of Antibodies 
As higher affinity phage antibodies are generated, it becomes more 
difficult to elute them from c-erbB-2. Selection of the highest affinity 
mutants is enhanced when elution conditions are optimized. To determine 
optimal elution conditions, the C6.5 V1 CDR3 mutant library was selected 
on c-erbB-2, and a number of different elution conditions studied 
(infecting directly off of magnetic beads, 10 mM HCl, 50 mM HCl, 100 mM 
HCl, 2.6 M MgCl.sub.2, 4 M MgCl.sub.2, 100 mM TDA, and with 1 .mu.M 
c-erbB-2). The greatest percentage of clones with a k.sub.off slower than 
C6.5 was obtained when eluting with 50 mM HCl, 100 mM HCl, or 4 M 
MgCl.sub.2. Even after the eluted clones were screened by BIAcore to 
identify those with the slowest k.sub.off, the highest affinity clones 
resulted from elutions performed with 100 mM HCl as shown in Table 15 (in 
this experiment 4 mM MgCl.sub.2 was not examined). 
These results correlated with the amount of phage antibody library that 
remained bound in the BIAcore after using one of the different elution 
conditions. For the V.sub.H CDR3 elutions phage were eluted sequentially 
with 4 mM MgCl.sub.2 and 100 mM HCl. As affinity increases urther more 
stringent elution conditions may be required. This can be determined by 
analyzing phage libraries in the BIAcore. 
TABLE 15 
__________________________________________________________________________ 
Results of C6.5 L3 randomization 4th round off-rate selection and 
elution. Underlines 
indicate mutated amino acids. 
Clones F V.sub.H CDR3 Sequence 
K.sub.d (M) 
K.sub.off (s.sup.-1) 
SEQ ID NO: 
__________________________________________________________________________ 
C6.5 AAWDDSLSGWV 
1.6 .times. 10.sup.-8 
6.3 .times. 10.sup.-3 
6 
Elution with 100 mM HCl: 
C6ML3-5 
4 AAWDYSLSGWV 
3.7 .times. 10.sup.-9 
6.3 .times. 10.sup.-3 
7 
C6ML3-9 ASWDYTLSGWV 1.0 .times. 10.sup.-9 1.9 .times. 10.sup.-4 15 
C6ML3-14 2 AAWDDPLWGWV 1.1 .times. 
10.sup.-9 7.6 .times. 10.sup.-4 24 
C6ML3-15 AAWDRPLWGWV 2.2 .times. 
10.sup.-9 7.7 .times. 10.sup.-3 23 
Elutionwith 2.6 M MgCl.sub.2 : 
C6ML3-5 
2 AAWDYSLSGWV 
3.7 .times. 10.sup.-9 
1.9 .times. 10.sup.-3 
7 
C6ML3-7 2 AAWDYAVSGWV 2.6 .times. 10.sup.-9 1.7 .times. 10.sup.-3 12 
C6ML3-12 AAWDYSRSGWV 1.6 .times. 
10.sup.-9 7.2 .times. 10.sup.-4 14 
C6ML3-16 2 ASWDYYRSGWV 5.0 .times. 
10.sup.-9 1.7 .times. 10.sup.-3 97 
C6ML3-15 AAWDRPLWGWV 2.2 .times. 
10.sup.-9 1.3 .times. 10.sup.-3 23 
Elution with 100 mM triethylamine: 
C6ML3-5 
3 AAWDYSLSGWV 
3.7 .times. 10.sup.-9 
1.9 .times. 10.sup.-3 
7 
C6ML3-12 2 AAWDYSRSGWV 1.6 .times. 10.sup.-9 7.2 .times. 10.sup.-4 14 
C6ML3-18 ASWDASLWDWV 2.4 .times. 
10.sup.-9 6.2 .times. 10.sup.-4 98 
C6ML3-19 ASWDRPLWGWV 1.5 .times. 
10.sup.-9 1.0 .times. 10.sup.-3 21 
C6ML3-20 AAWEQSLWGWV 3.0 .times. 
10.sup.-9 1.4 .times. 10.sup.-3 99 
Elution with 10 mM HCl: 
C6ML3-5 AAWDYSLSGWV 
3.7 .times. 10.sup.-9 
1.9 .times. 10.sup.-3 
7 
C6ML3-7 AAWDYAVSGWV 2.6 .times. 10.sup.-9 1.7 .times. 10.sup.-3 12 
C6ML3-21 AAWDYSQSGWV 4.5 .times. 
10.sup.-9 2.2 .times. 10.sup.-3 100 
C6ML3-22 AAWDASLSGWV 8.3 .times. 
10.sup.-9 3.6 .times. 10.sup.-3 101 
C6ML3-23 ASWDHSLWGWV 1.5 .times. 
10.sup.-9 1.0 .times. 10.sup.-3 20 
C6ML3-24 AAWDEQIFGWV 12.4 .times. 
10.sup.-9 7.9 .times. 10.sup.-3 102 
C6ML3-25 AAWDNRHSGWV 7.4 .times. 
10.sup.-9 4.4 .times. 10.sup.-3 103 
C6ML3-26 AAWDDSRSGWV 8.3 .times. 
10.sup.-9 5.0 .times. 10.sup.-3 104 
Elution with 50 mM HCl: 
C6ML3-6 ASWDYSLSGWV 
3.2 .times. 10.sup.-9 
1.9 .times. 10.sup.-3 
9 
C6ML3-7 AAWDYAVSGWV 2.6 .times. 10.sup.-9 1.7 .times. 10.sup.-3 12 
C6ML3-12 AAWDYSRSGWV 1.6 .times. 
10.sup.-9 7.2 .times. 10.sup.-4 14 
C6ML3-17 ASWDYYRSGWV 5.0 .times. 
10.sup.-9 1.7 .times. 10.sup.-3 105 
C6ML3-27 TAWDYSLSGWV no expression 
106 
C6ML3-28 ASWDYALSGWV 2.5 .times. 10.sup.-9 1.7 .times. 10.sup.-3 107 
C6ML3-29 AAWDGTLWGWV 1.7 .times. 
10.sup.-9 2.2 .times. 10.sup.-3 22 
Elution with 1 .mu.M c-erbB-2 ECD for 30 minutes 
C6ML3-5 
5 AAWDYSLSGWV 
3.7 .times. 10.sup.-9 
1.9 .times. 10.sup.-3 
7 
C6ML3-17 AAWDYALSGWV no expression 108 
C6ML3-30 3 ASWDYYLIGWV no expression 109 
__________________________________________________________________________ 
For example, in a second experiment, polyclonal phage were prepared after 
three rounds of selection of the C6VLCDR3 library and studied using SPR in 
a BIAcore. After an initial bulk refractive index change, binding of phage 
to immobilized c-erbB-2 ECD was observed, resulting in an average of 189 
RU bound. Phage were then allowed to either spontaneously dissociate from 
c-erbB-2 ECD using hepes buffered saline (HBS) as running buffer, or were 
eluted with either 100 mM HCl, 50 mM HCl, 10 mM HCl, 2.6 M MgCl.sub.2, or 
100 mM TEA. 
Major differences were observed between eluents in their ability to remove 
bound phage. The most effective solutions in removing bound phage 
antibodies were 100 mM HCl and 50 mM HCl, followed by 100 mM TEA. 2.6 M 
MgCl.sub.2 (which removes 100% of wild type C6.5) and 10 mM HCl were only 
minimally more effective than the running buffer in removing bound phage. 
These results demonstrate the important effect of eluent choice on the 
affinities of selected antibodies, even when using limiting antigen 
concentration and BIAcore screening to identify the highest affinity sFv. 
Two previously described elution regimens were found to be the least 
effective for selecting higher affinity antibodies; infecting without 
elution by adding magnetic beads with antigen-bound phage directly to E. 
coli cultures (Figini et al. (1994) J. Mol. Biol., 239: 68) and 
competitive elution of sFv with soluble antigen (Hawkins et al. (1992) J. 
Mol. Biol., 226: 889; Clackson et al. (1991) Nature, 352: 624; Riechmann 
et al. (1993) Biochemistry, 32: 8848). 
When eluting by incubating phage bound to antigen with E. coli, it is 
believed the phage must dissociate from antigen for infection to occur. 
Steric hindrance, due to the size of paramagnetic beads, blocks the 
attachment of pill on antigen bound phage to the f-pilus on E. coli. This 
would result in preferential selection of sFv with rapid k.sub.off, 
consistent with the present results. Since a reduction in k.sub.off is the 
major mechanism for decreases in K.sub.d, this results in the selection of 
lower affinity sFv. 
Eluting with soluble antigen has a similar effect on the kinetics of 
selected sFv. The phage must first dissociate from immobilized antigen, 
then rebinding is blocked by binding of the phage to soluble antigen. 
Phage antibodies with the lowest k.sub.off will remain bound to 
immobilized antigen and therefore are not available for infection of E. 
coli. 
The optimal type of eluent (acidic, basic, chaotropic) and concentration 
required will depend on the phage antibody affinity (Lewis et al. (1985) 
J. Steroid. Biochem. 22: 387; Parini et al. (1995) Analyst, 120: 1153) and 
the type of bonds that need to be interrupted. This will vary considerably 
between libraries, depending on the nature of the antigen-antibody 
interaction. 
In this example, significantly higher affinity sFv were obtained eluting 
with HCl, pH 1.3 compared to HCl, pH 2.0. In fact, the affinities of sFv 
isolated after elution with HCl, pH 2.0 were no different than results 
obtained without eluting. Similarly, 2.6 M MgCl.sub.2 was studied because 
it was previously determined (see above) that it would remove 100% of 
bound wild type C6.5. This concentration of MgCl.sub.2, however, was 
ineffective in eluting C6.5 V.sub.L CDR3 mutants. Eluting with higher 
concentrations of MgCl.sub.2 would have resulted in the selection of 
higher affinity sFv. For example, 3 M MgCl.sub.2 was required to elute 
100% of C6L1 sFv (K.sub.d =2.5.times.10.sup.-9 M) from a c-erbB-2 ECD 
BIAcore sensor chip and 4 M MgCl.sub.2 was required to elute 100% of 
C6ML3-9 (K.sub.d =1.0.times.10.sup.-9 M). 
A convenient way to predict the optimal eluent is to analyze polyclonal 
phage in a BIAcore. The results can then be used to design elution 
conditions to achieve optimal enrichment for high affinity clones. One 
approach is to elute sequentially, using a less stringent eluent to remove 
low affinity binders, followed by a more stringent eluent to remove high 
affinity binders. Thus the BIAcore information is used to select `washing` 
reagents which remove low affinity phage antibodies more effectively than 
PBS. This will reduce the number of selection rounds and amount of 
screening required to select and identify the highest affinity binders. 
This strategy is also be useful to isolate antibodies to low density 
antigens on intact cells or tissue. A mild eluent could be used to remove 
low affinity phage antibodies, which are preferentially selected due to 
high density antigen present on the cell surface, as well as 
non-specifically bound phage. Phage specific for lower density antigens 
would then be removed using a more stringent solution. 
An alternative to eluting with stringent solutions is to use antigen 
biotinylated with NHS-SS-Biotin (Pierce) (Griffiths et al. (1994) EMBO J., 
13: 3245). All of the bound phage can be released from the magnetic beads 
by reducing the disulfide bond between antigen and biotin. One advantage 
of this approach is that elution of all phage is guaranteed. Use of 
NHS-SS-Biotin could be combined with use of a milder eluent for washing 
(determined by BIAcore analysis) to increase enrichment for higher 
affinity phage antibodies. 
The present experiments suggest, however, that use of stringent eluents 
that are chemically different (acidic, basic, or chaotropic) results in 
the selection of sFv of equally high affinity, but of different sequence. 
Isolation of sFv of different sequences has a number of advantages. Single 
amino acid changes can affect expression levels in E. coli dramatically. 
For example, expression level of C6ML3-5 (100 .mu.g/L) was 100 times less 
than for wild type C6.5 (10 mg/L). Furthermore, different sFv might have 
different physicochemical characteristics (dimerization, stability, or 
immunoreactivity) or even different effects in vivo (specificity, 
biodistribution, or clearance). Thus parallel selections using different 
stringent eluents should result in a greater number of high affinity 
binders than use of a single eluent. 
Example 5 
Production of Antibodies Combining C6MH3-B1 or C6MH3-B47 with D Library 
(YFQH) (SEQ Id NO:65) Mutations 
I. Methods. 
Construction of sFv combining higher affinity V.sub.H and V.sub.L genes. 
The V.sub.L CDR3 gene sequences of the two highest affinity sFv isolated 
from the C6VLCDR3 library (C6ML3-9 or C6ML3-12) were combined with the 
highest affinity sFv previously obtained from light chain shuffling (C6L1, 
K.sub.d =2.5.times.10.sup.-9 M). The C6L1 plasmid (10 ng/.mu.l) was used 
as a template for PCR amplification using primers LMB3 and either PML3-9 
or PML3-12 (Table 18). The gel purified PCR fragments were reamplified 
using primers LMB3 and HUJ1 2-3ForNot (Marks et al. (1991) supra.) to 
introduce a NotI restriction site at the 3'-end of the sFv. The gel 
purified PCR fragments were digested with NcoI and NotI and ligated into 
pUC119 Sfi-NotmycHis digested with NcoI and NotI. The resulting sFv were 
designated C6-9L1 and C6-12L1. The V.sub.L genes of C6-9L1 and C6-12L1 
were combined with the V.sub.H genes of the two highest affinity sFv from 
the C6VHCDR3 libraries (C6MH3-B1 and C6MH3-B47). The rearranged V.sub.H 
genes of C6MH3-B1 and -B47 were amplified by PCR using the primer LMB3 and 
PC6VHlFOR, digested with NcoI and XhoI (located in FR4 of the heavy chain) 
and ligated into C6-9L1 or C6-12L1 digested with NcoI and XhoI to create 
C6-B1L1 and C6-B47L1. The heavy chain of C6MH3-B1 or C6MH3-B47 was 
amplified by PCR using LMB3 and one of the PCD primer (PCD1, PCD2, PCD3, 
PCD5, or PCD6; Table 18) to construct combinations of sFv from the 
C6VHCDR3B and D libraries. The purified PCR fragments were spliced with 
the V.sub.L fragment of C6ML3-9 (VHD2) that was used to create the 
C6VHCDR3D library exactly as described above. The full length sFv gene was 
digested with NcoI and NotI and ligated into pUC119 Sfi-NotmycHis. Clones 
were termed C6-B1D1, -B1D2, -B1D3, -B1D5, -B1D6, -B47D1, -B47D2, -B47D3, 
-B47D5, and -B47D6. Colonies were screened for the presence of the correct 
insert by PCR fingerprinting and confirmed by DNA sequencing. Single-chain 
Fv were expressed, purified, and affinities determined by SPR, as 
described above. 
II. Results. 
Effects on binding kinetics by combining mutations from high affinity sFv. 
As described above, to further increase affinity, the sequences of the two 
highest affinity sFv obtained from the VH CDR3B library (C6MH3-B1 or 
C6MH3-B47) were combined with the sequences of sFv isolated from the 
C6VHCDR3D library (C6MH3-D1, -D2, -D3, -D5, or -D6). An increase in 
affinity from wild-type was obtained for all these combinations, yielding 
an sFv (C6-B1D3) that had a 1230 fold lower K.sub.d than wt C6.5 (Table 
16). The extent of additivity varied considerably, however, and could not 
be predicted from the parental k.sub.on, k.sub.off, or K.sub.d. In some 
combinations, cooperativity was observed, with a negative 
.DELTA..DELTA.G.sub.I. Additional combinations were made between a 
previously described light chain shuffled C6.5 mutant (C6L1, sixfold 
decreased K.sub.d) and one of two V.sub.L CDR3 mutants (C6ML3-9 and 
C6ML3-12). These combinations yielded sFv with 49 and 84 fold improved 
affinity (Table 16). Introducing the same rearranged V.sub.L gene into the 
highest affinity V.sub.H CDR3 mutants (C6MH3-B1 or C6MH3-B47) resulted in 
decreased affinity compared to C6MH3-B1 (Table 5). 
TABLE 16 
______________________________________ 
Binding kinetics of sFv derived from C6.5 V.sub.L CDR3 and V.sub.H CDR3 
mutants. Mutants obtained by combining mutations of C6MH3-B1 or 
C6MH3-B47 with mutations from D library clones (D1, D2, D3, D5, D6). 
Rate constants k.sub.on, and k.sub.off were measured by SPR in a 
BIAcore, and the K.sub.d calculated. 
K.sub.d K.sub.d 
k.sub.on (parent) (C6.5) 
.DELTA..DELTA.G.sub.I 
K.sub.d [10.sup.5 s.sup.-1 
k.sub.off 
K.sub.d 
K.sub.d 
[kcal/ 
Clone [10.sup.-10 M] M.sup.-1] [10.sup.-4 s.sup.-1 ] (mut) (mut) 
______________________________________ 
mol] 
A. Combined mutants: C6ML3-9 or C6ML3-12 with 
light chain shuffled C6L1: 
C6-9L1 3.3 9.2 .+-. 
3.0 .+-. 
3.0 49 +0.42 
0.20 0.40 
C6-12L1 1.9 6.7 .+-. 1.3 .+-. 8.4 84 -0.18 
0.12 0.32 
B. Combined mutants: C6MH3-B1 or C6MH3-B47 
with light chain shuffled C6L1: 
C6-B1L1 6.3 3.8 .+-. 
2.4 .+-. 
0.19 25 +0.43 
0.19 0.01 
C6-B47L1 6.0 3.0 .+-. 1.8 .+-. 0.18 27 +0.45 
0.16 0.01 
C. Combined mutants: C6MH3-B1 or C6MH3-B47 
with D library mutants: 
C6-B1D1 0.32 4.7 .+-. 
0.15 .+-. 
3.8 500 -0.61 
0.31 0.005 
C6-B1D2 0.15 6.9 .+-. 0.10 .+-. 8.0 1067 -0.07 
0.42 0.014 
C6-B1D3 0.13 6.4 .+-. 0.08 .+-. 9.2 1231 -0.53 
0.20 0.002 
C6-B1D5 0.35 5.1 .+-. 0.18 .+-. 3.4 457 -0.40 
0.36 0.001 
C6-B1D6 0.32 4.1 .+-. 0.13 .+-. 3.8 500 -0.16 
0.17 0.002 
C6-B47D1 0.68 7.1 .+-. 0.48 .+-. 1.6 235 -0.11 
0.95 0.001 
C6-B47D2 0.44 9.8 .+-. 0.43 .+-. 2.5 364 +0.62 
0.72 0.001 
C6-B47D3 0.48 6.6 .+-. 0.32 .+-. 2.3 333 +0.29 
0.26 0.001 
C6-B47D5 0.63 6.2 .+-. 0.39 .+-. 1.7 254 -0.01 
0.31 0.002 
C6-B47D6 0.51 5.9 .+-. 0.30 .+-. 2.2 314 +0.17 
0.30 0.001 
______________________________________ 
Example 6 
Production of C6.5-Based Diabodies. 
To improve tumor retention sFv dimers (sFv').sub.2 were created as 
described above by introducing a free cysteine at the C-terminus of the 
sFv. The dimer had a 40 fold improved affinity compared to the monomer 
(K.sub.d =4.0.times.10.sup.-10 M). However, evaluation of the C6.5 
(sFv').sub.2 in vivo, showed no significantly improved tumor retention at 
24 hours. Without being bound to a theory, it is believed that the 
disulfide bond is being reduced in vivo, yielding monomeric sFv. 
To obtain a stable molecule for evaluation in vivo, a C6.5 diabody (also a 
(sFv).sub.2) was produced without introducing a cysteine and crosslinking. 
Instead, the diabody was produced as described in Holliger et al., Proc. 
Natl. Acad. Sci. USA., 90: 6444-6448 (1993) (see also WO 94/13804). To 
produce the C6.5 diabody, the peptide linker sequence between the V.sub.H 
and V.sub.L domains was shortened from 15 amino acids to 5 amino acids. 
This was done at the genetic level. Synthetic oligonucleotides encoding 
the 5 amino acid linker (Gly.sub.4 Ser) (SEQ ID NO:25) were used to PCR 
amplify the C6.5 V.sub.H and V.sub.L genes, which were then spliced 
together to create the C6.5 diabody gene. The diabody gene was cloned into 
pUC119mycHis, the diabody expressed, and purified by IMAC followed by gel 
filtration as described above. 
The affinity of the diabody was measured using surface plasmon resonance in 
a BIAcore and found to be 4.2.times.10.sup.-10 M, with a k.sub.off of 
3.2.times.10.sup.-4 s.sup.-1. The retention of the FITC labeled diabody on 
the surface of c-erbB-2 expressing cells was determined by FACS. After 180 
minutes, 77% was still retained on the cell surface. Assuming an 
exponential decay for binding, this value for cell surface retention 
correlates with a k.sub.off of 7.times.10.sup.-5 s.sup.-1. This is 
significantly slower than the k.sub.off measured on the BIAcore, and 
suggests that c-erbB-2 density is higher on the cell surface than the 
density used for the BIAcore measurements. 
The retention of the C6.5 diabody in scid mice bearing subcutaneous SK-OV-3 
tumors was compared to C6.5. Single chain Fv were radio-iodinated using 
the chloramine-T method, and 25 .mu.g injected into mice. Values are shown 
in Table 17 and plotted in FIG. 4. At 24 hours, tumor retention was 6.48% 
of the injected dose/gm of tumor, compared to 0.98% for C6.5. Tumor:blood 
ratios were 9.7:1 for the diabody and 19.6:1 for the C6.5 sFv. Significant 
amounts (1.41%) of the diabody was retained at 72 hours. The total area 
under the curve (AUC) for tumor: blood was 2.3:1. 
The ability of the C6.5 diabody to be internalized into c-erbB-2 expressing 
cells was compared to C6.5 sFv and higher affinity C6.5 mutants. Only the 
diabody was internalized, consistent with studies using monoclonal 
antibodies to c-erbB-2 which show that crosslinking of c-erbB-2 results in 
internalization. This does not occur with all anti-c-erbB-2 antibodies, 
but rather is epitope dependent. Thus C6.5 recognizes an internalizing 
epitope, but internalization only results when the receptor is crosslinked 
by the diabody. This opens up the possibility of creating diabody-toxin 
fusions (since toxins must be internalized to be active). It is believed 
that C6.5 also causes signalling through c-erbB-2 via cross-linking of the 
receptor and activation of the tyrosine kinase activity. It has been shown 
that activation of the cell through c-erbB-2 signalling increases the 
sensitivity of the cell to conventional cancer chemotherapeutics. Through 
activation of the kinase, C6.5 is expected to have therapeutic properties 
when combined with a conventional cancer chemotherapeutic. 
TABLE 17 
______________________________________ 
Tissue distribution of diabody as a function of time. 
C6.5 
Time Tumor Blood Tumor Blood 
(Hrs) mean .+-. se mean .+-. se mean .+-. se mean .+-. se 
______________________________________ 
0.08 42.08 .+-. 0.77 
1 6.93 .+-. 0.39 21.47 .+-. 1.67 
4 10.06 .+-. 0.63 6.73 .+-. 0.29 0.98 .+-. 0.08 0.05 .+-. 0.001 
24 6.48 .+-. 0.77 0.67 .+-. 0.05 
48 2.42 .+-. 0.18 0.11 .+-. 0.01 
72 1.41 .+-. 0.13 0.06 .+-. 0 
______________________________________ 
TABLE 18 
__________________________________________________________________________ 
Sequences of primers used in the foregoing examples. Nucleotide mixtures 
used, molar fraction: 
1: A (0.7), C, G, and T (0.1); 2: C (0.7), A, G, and T (0.1); 3: G 
(0.7), A, T, and C (0.1); 4: T (0.7), 
A, C, and G (0.1); 5: C and G (0.5); 6: C (0.7) and G (0.3); 7: C (0.3) 
and 3: G (0.7), A, T, and C 
(0.1); 8: A, C, G, and T (0.25). 
SEQ 
ID 
PrimerSequenc 
e NO. 
__________________________________________________________________________ 
LMB3 5'-CAGGAAACAGCTATGAC-3' 110 
- fd-seq15'-GAATTTTCTGTATGAGG-3'111 
- PHEN=1seq5'-CTATGCGGCCCCATTCA-3'112 
- Linkseq5'-CGATCCGCCACCGCCAGAG-3'113 
- PVH1For15'-TCGCGCGCAGTAATACACGGCCGTGTC-3'114 
- PVH3For15'-TCGCGCGCAGTAATACACAGCCGTGTCCTC-3'115 
- PVH5For15'-TCGCGCGCAGTAATACATGGCGGTGTCCGA-3'116 
- PVH1For25'-GAGTCATTCTCGACTTGCGGCCGCTCGCGCGCAGTAATACACGGCCGTGTC-3'117 
- PVH3For25'-GAGTCATTCTCGACTTGCGGCCGCTCGCGCGCAGTAATACACAGCCGTGTCCTC-3'1 
18 
- PVH5For25'-GAGTCATTCTCGACTTGCGGCCGCTCGCGCGCAGTAATACATGGCGGTGTCCGA-3'1 
19 
- PC6VL1back5'-AGCGCCGTGTATTTTTGCGCGCGACATGACGTGGGATATTGC-3'120 
- RJH1/2/6Xh 
o5'-ACCCTGGTCA 
CCGTCTCGAGTGGT 
GGA-3'121 
- RJH3Xho5'- 
ACAATGGTCACCGT 
CTCGAGTGGTGGA- 
3'122 
- RJH4/5Xho5 
'-ACCCTGGTCACC 
GTCTCGAGTGGTGG 
A-3'121 
- PC6VH1For5 
'-GAGTCATTCTCG 
TCTCGAGACGGTGA 
CCAGGGTGCC-3'1 
23 
- VL15'-GTCCCTCCGCCGAACACCCA,5,2,2,5,3,1,6,1,3,5,3,1,7,4,2,7,4,2,2,2,1, 
5,3,2,5,3,2,AC 
AGTAAT124 
- AATCAGCCTC 
AT-3' 
- VL25'-GAGT 
CATTCTCGACTTGC 
GGCCGCACCTAGGA 
CGGTCAGCTTGGTC 
CCTCCGCCGAACAC 
CGA-3'125 
- VHA5'-GCGC 
AGTTGGAACTACTG 
CA,5,8,8,5,8,8 
,5,8,8,5,8,8,A 
TGTCTCGCACAAAA 
ATACACGGC-3'12 
6 
- RVHA5'-TGCAGTAGTTCCAACTGCGC-3'127 
- VHB5'-GTATTCAGGCCACTTTGCGCA,5,8,8,5,8,8,5,8,8,5,8,8,8,GCAATATCCCACGTC 
ATGTC-3'128 
- RVHB5'-TGC 
GCAAAGTGGCCTGA 
ATAC-3'129 
- VHC5'-CTGG 
CCCCAATGCTGGAA 
GTA,5,8,8,5,8, 
8,CCA,5,8,8,5, 
8,8,GCAGTTGGAA 
CTACTGCAATATCC 
-3'130 
- RVHC5'-TAC 
TTCCAGCATTGGGG 
CCAG-3'131 
- VHD5'-GACC 
AGGGTGCCCTGGCC 
CCA,5,8,8,5,8, 
8,5,8,8,5,8,8, 
TTCAGGCCACTTTG 
CGCAGTTGG-3'13 
2 
- RVHD5'-TGGGGCCAGGGCACCCTGGTC-3'133 
- C6hisnot5'-GATACGGCACCGGCGCACCTGCGGCCGCATGGTGATGATGGTGATGTGCGGCACCTAG 
GACGGTCAGCTTGG 
-3'134 
- PML3-95'-C 
CTAGGACGGTCAGC 
TTGGTCCCTCCGCC 
GAACACCCAACCAC 
TCAGGGTGTAATCC 
CAGGATGCACAGTA 
ATAATCAGC-3'13 
5 
- PML3-125'-CCTAGGACGGTCAGCTTGGTCCCTCCGCCGAACACCCAACCACTCCGGCTGTAATCCCA 
TGCTGCACAG-3'1 
36 
- PCD15'-GACGGTGACCAGGGTGCCCTGGCCCCAAACGTGCAGCCATTCAGGCCACTTTGCGCA-3'13 
7 
- PCD25'-GACGGTGACCAGGGTGCCCTGGCCCCATACGCCCAGCCATTCAGGCCACTTTGCGCA-3'13 
8 
- PCD35'-GACGGTGACCAGGGTGCCCTGGCCCCAGTTGTCCAACCATTCAGGCCACTTTGCGCA-3'13 
9 
- PCD55'-GACGGTGACCAGGGTGCCCTGGCCCCACATCTGCATCCATTCAGGCCACTTTGCGCA-3'14 
0 
- PCD65'-GACGGTGACCAGGGTGCCCTGGCCCCAGGGGTACATCCATTCAGGCCACTTTGCGCA-3'14 
1 
__________________________________________________________________________ 
It is understood that the examples and embodiments described herein are for 
illustrative purposes only and that various modifications or changes in 
light thereof will be suggested to persons skilled in the art and are to 
be included within the spirit and purview of this application and scope of 
the appended claims. All publications, patents, and patent applications 
cited herein are hereby incorporated by reference. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - (1) GENERAL INFORMATION: 
- - (iii) NUMBER OF SEQUENCES: 141 
- - - - (2) INFORMATION FOR SEQ ID NO:1: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:1: 
- - Gly Gly Gly Gly Ser Gly Gly Gly - # Gly Ser Gly Gly Gly Gly 
Ser 
1 - # 5 - # 10 - # 
15 
- - - - (2) INFORMATION FOR SEQ ID NO:2: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:2: 
- - Ser Ser Ser Ser Gly 
1 - # 5 
- - - - (2) INFORMATION FOR SEQ ID NO:3: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:3: 
- - Ser Ser Ser Ser Gly Ser Ser Ser - # Ser Gly Ser Ser Ser Ser 
Gly 
1 - # 5 - # 10 - # 
15 
- - - - (2) INFORMATION FOR SEQ ID NO:4: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 774 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..774 
(D) OTHER INFORMATION: - #/note= "sequence of C6 sFv 
antibody - #C6.5" 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:4: 
- - CAG GTG CAG CTG TTG CAG TCT GGG GCA GAG TT - #G AAA AAA CCC GGG 
GAG 48 
Gln Val Gln Leu Leu Gln Ser Gly Ala Glu Le - #u Lys Lys Pro Gly Glu 
1 5 - # 10 - # 15 
- - TCT CTG AAG ATC TCC TGT AAG GGT TCT GGA TA - #C AGC TTT ACC AGC TAC 
96 
Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Ty - #r Ser Phe Thr Ser Tyr 
20 - # 25 - # 30 
- - TGG ATC GCC TGG GTG CGC CAG ATG CCC GGG AA - #A GGC CTG GAG TAC ATG 
144 
Trp Ile Ala Trp Val Arg Gln Met Pro Gly Ly - #s Gly Leu Glu Tyr Met 
35 - # 40 - # 45 
- - GGG CTC ATC TAT CCT GGT GAC TCT GAC ACC AA - #A TAC AGC CCG TCC TTC 
192 
Gly Leu Ile Tyr Pro Gly Asp Ser Asp Thr Ly - #s Tyr Ser Pro Ser Phe 
50 - # 55 - # 60 
- - CAA GGC CAG GTC ACC ATC TCA GTC GAC AAG TC - #C GTC AGC ACT GCC TAC 
240 
Gln Gly Gln Val Thr Ile Ser Val Asp Lys Se - #r Val Ser Thr Ala Tyr 
65 - # 70 - # 75 - # 80 
- - TTG CAA TGG AGC AGT CTG AAG CCC TCG GAC AG - #C GCC GTG TAT TTT TGT 
288 
Leu Gln Trp Ser Ser Leu Lys Pro Ser Asp Se - #r Ala Val Tyr Phe Cys 
85 - # 90 - # 95 
- - GCG AGA CAT GAC GTG GGA TAT TGC AGT AGT TC - #C AAC TGC GCA AAG TGG 
336 
Ala Arg His Asp Val Gly Tyr Cys Ser Ser Se - #r Asn Cys Ala Lys Trp 
100 - # 105 - # 110 
- - CCT GAA TAC TTC CAG CAT TGG GGC CAG GGC AC - #C CTG GTC ACC GTC TCC 
384 
Pro Glu Tyr Phe Gln His Trp Gly Gln Gly Th - #r Leu Val Thr Val Ser 
115 - # 120 - # 125 
- - TCA GGT GGA GGC GGT TCA GGC GGA GGT GGC TC - #T GGC GGT GGC GGA TCG 
432 
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Se - #r Gly Gly Gly Gly Ser 
130 - # 135 - # 140 
- - CAG TCT GTG TTG ACG CAG CCG CCC TCA GTG TC - #T GCG GCC CCA GGA CAG 
480 
Gln Ser Val Leu Thr Gln Pro Pro Ser Val Se - #r Ala Ala Pro Gly Gln 
145 1 - #50 1 - #55 1 - 
#60 
- - AAG GTC ACC ATC TCC TGC TCT GGA AGC AGC TC - #C AAC ATT GGG AAT 
AAT 528 
Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Se - #r Asn Ile Gly Asn Asn 
165 - # 170 - # 175 
- - TAT GTA TCC TGG TAC CAG CAG CTC CCA GGA AC - #A GCC CCC AAA CTC CTC 
576 
Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Th - #r Ala Pro Lys Leu Leu 
180 - # 185 - # 190 
- - ATC TAT GGT CAC ACC AAT CGG CCC GCA GGG GT - #C CCT GAC CGA TTC TCT 
624 
Ile Tyr Gly His Thr Asn Arg Pro Ala Gly Va - #l Pro Asp Arg Phe Ser 
195 - # 200 - # 205 
- - GGC TCC AAG TCT GGC ACC TCA GCC TCC CTG GC - #C ATC AGT GGG TTC CGG 
672 
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Al - #a Ile Ser Gly Phe Arg 
210 - # 215 - # 220 
- - TCC GAG GAT GAG GCT GAT TAT TAC TGT GCA GC - #A TGG GAT GAC AGC CTG 
720 
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Al - #a Trp Asp Asp Ser Leu 
225 2 - #30 2 - #35 2 - 
#40 
- - AGT GGT TGG GTG TTC GGC GGA GGG ACC AAG CT - #G ACC GTC CTA GGT 
GCG 768 
Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Le - #u Thr Val Leu Gly Ala 
245 - # 250 - # 255 
- - GCC GCA - # - # - 
# 774 
Ala Ala 
- - - - (2) INFORMATION FOR SEQ ID NO:5: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 258 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:5: 
- - Gln Val Gln Leu Leu Gln Ser Gly Ala Glu Le - #u Lys Lys Pro Gly 
Glu 
1 5 - # 10 - # 15 
- - Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Ty - #r Ser Phe Thr Ser Tyr 
20 - # 25 - # 30 
- - Trp Ile Ala Trp Val Arg Gln Met Pro Gly Ly - #s Gly Leu Glu Tyr Met 
35 - # 40 - # 45 
- - Gly Leu Ile Tyr Pro Gly Asp Ser Asp Thr Ly - #s Tyr Ser Pro Ser Phe 
50 - # 55 - # 60 
- - Gln Gly Gln Val Thr Ile Ser Val Asp Lys Se - #r Val Ser Thr Ala Tyr 
65 - # 70 - # 75 - # 80 
- - Leu Gln Trp Ser Ser Leu Lys Pro Ser Asp Se - #r Ala Val Tyr Phe Cys 
85 - # 90 - # 95 
- - Ala Arg His Asp Val Gly Tyr Cys Ser Ser Se - #r Asn Cys Ala Lys Trp 
100 - # 105 - # 110 
- - Pro Glu Tyr Phe Gln His Trp Gly Gln Gly Th - #r Leu Val Thr Val Ser 
115 - # 120 - # 125 
- - Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Se - #r Gly Gly Gly Gly Ser 
130 - # 135 - # 140 
- - Gln Ser Val Leu Thr Gln Pro Pro Ser Val Se - #r Ala Ala Pro Gly Gln 
145 1 - #50 1 - #55 1 - 
#60 
- - Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Se - #r Asn Ile Gly Asn 
Asn 
165 - # 170 - # 175 
- - Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Th - #r Ala Pro Lys Leu Leu 
180 - # 185 - # 190 
- - Ile Tyr Gly His Thr Asn Arg Pro Ala Gly Va - #l Pro Asp Arg Phe Ser 
195 - # 200 - # 205 
- - Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Al - #a Ile Ser Gly Phe Arg 
210 - # 215 - # 220 
- - Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Al - #a Trp Asp Asp Ser Leu 
225 2 - #30 2 - #35 2 - 
#40 
- - Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Le - #u Thr Val Leu Gly 
Ala 
245 - # 250 - # 255 
- - Ala Ala 
- - - - (2) INFORMATION FOR SEQ ID NO:6: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:6: 
- - Ala Ala Trp Asp Asp Ser Leu Ser - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:7: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:7: 
- - Ala Ala Trp Asp Tyr Ser Leu Ser - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:8: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:8: 
- - Ala Ala Trp Asp His Ser Leu Ser - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:9: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:9: 
- - Ala Ser Trp Asp Tyr Ser Leu Ser - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:10: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:10: 
- - Ala Ala Trp Asp Tyr Ser Leu Trp - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:11: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:11: 
- - Ala Thr Trp Asp Tyr Ala Leu Ser - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:12: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:12: 
- - Ala Ala Trp Asp Tyr Ala Val Ser - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:13: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:13: 
- - Ala Ser Trp Glu Tyr Ser Leu Trp - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:14: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:14: 
- - Ala Ala Trp Asp Tyr Ser Arg Ser - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:15: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:15: 
- - Ala Ser Trp Asp Tyr Thr Leu Ser - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:16: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:16: 
- - Ala Ala Trp Glu Asp Pro Trp Tyr - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:17: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:17: 
- - Ala Ala Trp Asp Tyr Ala Leu Trp - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:18: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:18: 
- - Ala Ala Trp Asp Ala Thr Leu Trp - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:19: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:19: 
- - Ala Ala Trp Asp His Leu Arg Trp - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:20: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:20: 
- - Ala Ser Trp Asp His Ser Leu Trp - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:21: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:21: 
- - Ala Ser Trp Asp Arg Pro Leu Trp - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:22: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:22: 
- - Ala Ala Trp Asp Gly Thr Leu Trp - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:23: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:23: 
- - Ala Ala Trp Asp Arg Pro Leu Trp - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:24: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:24: 
- - Ala Ala Trp Asp Asp Pro Leu Trp - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:25: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:25: 
- - Gly Gly Gly Gly Ser 
1 - # 5 
- - - - (2) INFORMATION FOR SEQ ID NO:26: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:26: 
- - Arg Glu Asp Leu Lys 
1 - # 5 
- - - - (2) INFORMATION FOR SEQ ID NO:27: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:27: 
- - Arg Glu Asp Leu 
1 
- - - - (2) INFORMATION FOR SEQ ID NO:28: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:28: 
- - Arg Asp Glu Leu 
1 
- - - - (2) INFORMATION FOR SEQ ID NO:29: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:29: 
- - Lys Asp Glu Leu 
1 
- - - - (2) INFORMATION FOR SEQ ID NO:30: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 123 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:30: 
- - Gln Val Gln Leu Val Glu Ser Gly - # Gly Gly Leu Val Gln Pro 
Gly Gly 
1 - # 5 - # 10 - # 
15 
- - Ser Leu Arg Leu Ser Cys Ala Ala - # Ser Gly Phe Thr Phe Ser 
Ser Tyr 
20 - # 25 - # 30 
- - Glu Met Asn Trp Val Arg Gln Ala - # Pro Gly Lys Gly Leu Glu 
Trp Val 
35 - # 40 - # 45 
- - Ser Tyr Ile Ser Ser Ser Gly Ser - # Thr Ile Tyr Tyr Ala Asp 
Ser Val 
50 - # 55 - # 60 
- - Lys Gly Arg Phe Thr Ile Ser Arg - # Asp Asn Ala Lys Asn Ser 
Leu Tyr 
65 - # 70 - # 75 - # 
80 
- - Leu Gln Met Asn Ser Leu Arg Ala - # Glu Asp Thr Ala Val Tyr 
Tyr Cys 
- # 85 - # 90 - # 
95 
- - Ala Arg Asp Leu Gly Gly Tyr Ser - # Tyr Gly Tyr Val Gly Leu 
Asp Tyr 
100 - # 105 - # 110 
- - Trp Gly Gln Gly Thr Leu Val Thr - # Val Ser Ser 
115 - # 120 
- - - - (2) INFORMATION FOR SEQ ID NO:31: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 98 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:31: 
- - Glu Val Gln Leu Val Glu Ser Gly - # Gly Gly Leu Val Gln Pro 
Gly Gly 
1 - # 5 - # 10 - # 
15 
- - Ser Leu Arg Leu Ser Cys Ala Ala - # Ser Gly Phe Thr Phe Ser 
Ser Tyr 
20 - # 25 - # 30 
- - Glu Met Asn Trp Val Arg Gln Ala - # Pro Gly Lys Gly Leu Glu 
Trp Val 
35 - # 40 - # 45 
- - Ser Tyr Ile Ser Ser Ser Gly Ser - # Thr Ile Tyr Tyr Ala Asp 
Ser Val 
50 - # 55 - # 60 
- - Lys Gly Arg Phe Thr Ile Ser Arg - # Asp Asn Ala Lys Asn Ser 
Leu Tyr 
65 - # 70 - # 75 - # 
80 
- - Leu Gln Met Asn Ser Leu Arg Ala - # Glu Asp Thr Ala Val Tyr 
Tyr Cys 
- # 85 - # 90 - # 
95 
- - Ala Arg 
- - - - (2) INFORMATION FOR SEQ ID NO:32: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 129 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:32: 
- - Gln Val Gln Leu Leu Gln Ser Gly - # Ala Glu Leu Lys Lys Pro 
Gly Glu 
1 - # 5 - # 10 - # 
15 
- - Ser Leu Lys Ile Ser Cys Lys Gly - # Ser Gly Tyr Ser Phe Thr 
Ser Tyr 
20 - # 25 - # 30 
- - Trp Ile Ala Trp Val Arg Gln Met - # Pro Gly Lys Gly Leu Glu 
Tyr Met 
35 - # 40 - # 45 
- - Gly Leu Ile Tyr Pro Gly Asp Ser - # Asp Thr Lys Tyr Ser Pro 
Ser Phe 
50 - # 55 - # 60 
- - Gln Gly Gln Val Thr Ile Ser Val - # Asp Lys Ser Val Ser Thr 
Ala Tyr 
65 - # 70 - # 75 - # 
80 
- - Leu Gln Trp Ser Ser Leu Lys Pro - # Ser Asp Ser Ala Val Tyr 
Phe Cys 
- # 85 - # 90 - # 
95 
- - Ala Arg His Asp Val Gly Tyr Cys - # Ser Ser Ser Asn Cys Ala 
Lys Trp 
100 - # 105 - # 110 
- - Pro Glu Tyr Phe Gln His Trp Gly - # Gln Gly Thr Leu Val Thr 
Val Ser 
115 - # 120 - # 125 
- - Ser 
- - - - (2) INFORMATION FOR SEQ ID NO:33: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 98 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:33: 
- - Glu Val Gln Leu Val Gln Ser Gly - # Ala Glu Val Lys Lys Pro 
Gly Glu 
1 - # 5 - # 10 - # 
15 
- - Ser Leu Lys Ile Ser Cys Lys Gly - # Ser Gly Tyr Ser Phe Thr 
Ser Tyr 
20 - # 25 - # 30 
- - Trp Ile Gly Trp Val Arg Gln Met - # Pro Gly Lys Gly Leu Glu 
Trp Met 
35 - # 40 - # 45 
- - Gly Ile Ile Tyr Pro Gly Asp Ser - # Asp Thr Arg Tyr Ser Pro 
Ser Phe 
50 - # 55 - # 60 
- - Gln Gly Gln Val Thr Ile Ser Ala - # Asp Lys Ser Ile Ser Thr 
Ala Tyr 
65 - # 70 - # 75 - # 
80 
- - Leu Gln Trp Ser Ser Leu Lys Ala - # Ser Asp Thr Ala Met Tyr 
Tyr Cys 
- # 85 - # 90 - # 
95 
- - Ala Arg 
- - - - (2) INFORMATION FOR SEQ ID NO:34: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 109 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:34: 
- - Ser Glu Leu Thr Gln Asp Pro Ala - # Val Ser Val Ala Leu Gly 
Gln Thr 
1 - # 5 - # 10 - # 
15 
- - Val Arg Ile Thr Cys Gln Gly Asp - # Ser Leu Arg Ser Tyr Tyr 
Ala Ser 
20 - # 25 - # 30 
- - Trp Tyr Gln Gln Lys Pro Gly Gln - # Ala Pro Val Leu Val Ile 
Tyr Gly 
35 - # 40 - # 45 
- - Lys Asn Asn Arg Pro Ser Gly Ile - # Pro Asp Arg Phe Ser Gly 
Ser Ser 
50 - # 55 - # 60 
- - Ser Gly Asn Ile Ala Ser Leu Thr - # Ile Thr Gly Ala Gln Ala 
Glu Asp 
65 - # 70 - # 75 - # 
80 
- - Glu Ala Asp Tyr Tyr Cys Asn Ser - # Arg Asp Ser Ser Gly Asn 
Pro Tyr 
- # 85 - # 90 - # 
95 
- - Trp Val Phe Gly Gly Gly Thr Lys - # Val Thr Val Leu Gly 
100 - # 105 
- - - - (2) INFORMATION FOR SEQ ID NO:35: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 97 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:35: 
- - Ser Ser Leu Thr Gln Asp Pro Ala - # Val Ser Val Ala Leu Gly 
Gln Thr 
1 - # 5 - # 10 - # 
15 
- - Val Arg Ile Thr Cys Gln Gly Asp - # Ser Leu Arg Ser Tyr Tyr 
Ala Ser 
20 - # 25 - # 30 
- - Trp Tyr Gln Gln Lys Pro Gly Gln - # Ala Pro Val Leu Val Ile 
Tyr Gly 
35 - # 40 - # 45 
- - Lys Asn Asn Arg Pro Ser Gly Ile - # Pro Asp Arg Phe Ser Gly 
Ser Ser 
50 - # 55 - # 60 
- - Ser Gly Asn Thr Ala Ser Leu Thr - # Ile Thr Gly Ala Gln Ala 
Glu Asp 
65 - # 70 - # 75 - # 
80 
- - Glu Ala Asp Tyr Tyr Cys Asn Ser - # Arg Asp Ser Ser Gly Asn 
His Val 
- # 85 - # 90 - # 
95 
- - Val 
- - - - (2) INFORMATION FOR SEQ ID NO:36: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 111 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:36: 
- - Gln Ser Val Leu Thr Gln Pro Pro - # Ser Val Ser Ala Ala Pro 
Gly Gln 
1 - # 5 - # 10 - # 
15 
- - Lys Val Thr Ile Ser Cys Ser Gly - # Ser Ser Ser Asn Ile Gly 
Asn Asn 
20 - # 25 - # 30 
- - Tyr Val Ser Trp Tyr Gln Gln Leu - # Pro Gly Thr Ala Pro Lys 
Leu Leu 
35 - # 40 - # 45 
- - Ile Tyr Gly His Thr Asn Arg Pro - # Ala Gly Val Pro Asp Arg 
Phe Ser 
50 - # 55 - # 60 
- - Gly Ser Lys Ser Gly Thr Ser Ala - # Ser Leu Ala Ile Ser Gly 
Phe Arg 
65 - # 70 - # 75 - # 
80 
- - Ser Glu Asp Glu Ala Asp Tyr Tyr - # Cys Ala Ala Trp Asp Asp 
Ser Leu 
- # 85 - # 90 - # 
95 
- - Ser Gly Trp Val Phe Gly Gly Gly - # Thr Lys Leu Thr Val Leu 
Gly 
100 - # 105 - # 110 
- - - - (2) INFORMATION FOR SEQ ID NO:37: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 98 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:37: 
- - Gln Ser Val Leu Thr Gln Pro Pro - # Ser Val Ser Ala Ala Pro 
Gly Gln 
1 - # 5 - # 10 - # 
15 
- - Lys Val Thr Ile Ser Cys Ser Gly - # Ser Ser Ser Asn Ile Gly 
Asn Asn 
20 - # 25 - # 30 
- - Tyr Val Ser Trp Tyr Gln Gln Leu - # Pro Gly Thr Ala Pro Lys 
Leu Leu 
35 - # 40 - # 45 
- - Ile Tyr Asp Asn Lys Lys Arg Pro - # Ser Gly Ile Pro Asp Arg 
Phe Ser 
50 - # 55 - # 60 
- - Gly Ser Lys Ser Gly Thr Ser Ala - # Thr Leu Gly Ile Thr Gly 
Leu Gln 
65 - # 70 - # 75 - # 
80 
- - Thr Gly Asp Glu Ala Asp Tyr Tyr - # Cys Gly Thr Trp Asp Ser 
Ser Leu 
- # 85 - # 90 - # 
95 
- - Ser Ala 
- - - - (2) INFORMATION FOR SEQ ID NO:38: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 98 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:38: 
- - Gln Ser Val Leu Thr Gln Pro Pro - # Ser Ala Ser Gly Thr Pro 
Gly Gln 
1 - # 5 - # 10 - # 
15 
- - Arg Val Thr Ile Ser Cys Ser Gly - # Ser Ser Ser Asn Ile Gly 
Ser Asn 
20 - # 25 - # 30 
- - Tyr Val Tyr Trp Tyr Gln Gln Leu - # Pro Gly Thr Ala Pro Lys 
Leu Leu 
35 - # 40 - # 45 
- - Ile Tyr Arg Asn Asn Gln Arg Pro - # Ser Gly Val Pro Asp Arg 
Phe Ser 
50 - # 55 - # 60 
- - Gly Ser Lys Ser Gly Thr Ser Ala - # Ser Leu Ala Ile Ser Gly 
Leu Arg 
65 - # 70 - # 75 - # 
80 
- - Ser Glu Asp Glu Ala Asp Tyr Tyr - # Cys Ala Ala Trp Asp Asp 
Ser Leu 
- # 85 - # 90 - # 
95 
- - Ser Gly 
- - - - (2) INFORMATION FOR SEQ ID NO:39: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 112 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:39: 
- - Gln Ser Val Leu Thr Gln Pro Pro - # Ser Val Ser Ala Ala Pro 
Gly Gln 
1 - # 5 - # 10 - # 
15 
- - Lys Val Thr Ile Ser Cys Ser Gly - # Ser Ser Ser Asn Ile Gly 
Asn Asn 
20 - # 25 - # 30 
- - Tyr Val Ser Trp Tyr Gln Gln Leu - # Pro Gly Thr Ala Pro Lys 
Leu Leu 
35 - # 40 - # 45 
- - Ile Tyr Ser Asp Asn Gln Arg Pro - # Ser Gly Val Pro Asp Arg 
Phe Ser 
50 - # 55 - # 60 
- - Gly Ser Lys Ser Gly Thr Ser Ala - # Ser Leu Ala Ile Ser Gly 
Leu Arg 
65 - # 70 - # 75 - # 
80 
- - Ser Glu Asp Glu Ala Asp Tyr Tyr - # Cys Ala Ala Trp Asp Asp 
Ser Leu 
- # 85 - # 90 - # 
95 
- - Ser Gly His Trp Val Phe Gly Gly - # Gly Thr Lys Leu Thr Val 
Leu Gly 
100 - # 105 - # 110 
- - - - (2) INFORMATION FOR SEQ ID NO:40: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 111 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:40: 
- - Gln Ser Val Leu Thr Gln Pro Pro - # Ser Val Ser Ala Ala Pro 
Gly Gln 
1 - # 5 - # 10 - # 
15 
- - Lys Val Thr Ile Ser Cys Ser Gly - # Thr Asn Ser Asn Ile Gly 
Asn Asn 
20 - # 25 - # 30 
- - Tyr Val Ser Trp Tyr Gln Gln Leu - # Pro Gly Thr Ala Pro Lys 
Leu Leu 
35 - # 40 - # 45 
- - Ile Tyr Thr Asn Asp Gln Arg Pro - # Ser Gly Val Pro Asp Arg 
Phe Ser 
50 - # 55 - # 60 
- - Gly Ser Lys Ser Gly Thr Ser Ala - # Ser Leu Ala Ile Ser Gly 
Leu Gln 
65 - # 70 - # 75 - # 
80 
- - Ser Glu Asp Glu Ala Asp Tyr Tyr - # Cys Ala Ala Trp Asp Asp 
Ser Leu 
- # 85 - # 90 - # 
95 
- - Asn Gly Trp Met Phe Gly Gly Gly - # Thr Lys Leu Thr Val Leu 
Gly 
100 - # 105 - # 110 
- - - - (2) INFORMATION FOR SEQ ID NO:41: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 111 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:41: 
- - Gln Ser Val Leu Thr Gln Pro Pro - # Ser Val Ser Ala Ala Ala 
Gly Gln 
1 - # 5 - # 10 - # 
15 
- - Lys Val Thr Ile Ser Cys Ser Gly - # Ser Ser Ser Ser Ile Gly 
Arg Asn 
20 - # 25 - # 30 
- - Tyr Val Ser Trp Asn Gln Gln Leu - # Pro Gly Thr Ala Pro Lys 
Leu Leu 
35 - # 40 - # 45 
- - Ile Trp Arg Asn Asn Gln Arg Pro - # Ser Gly Val Pro Asp Arg 
Phe Ser 
50 - # 55 - # 60 
- - Gly Ser Lys Ser Gly Thr Ser Ala - # Ser Leu Ala Ile Ser Gly 
Val Arg 
65 - # 70 - # 75 - # 
80 
- - Ser Glu Asp Glu Ala Asp Tyr Tyr - # Cys Ala Ser Trp Asp Asn 
Ser Leu 
- # 85 - # 90 - # 
95 
- - Ser Gly Trp Val Phe Gly Gly Gly - # Thr Lys Leu Thr Val Leu 
Gly 
100 - # 105 - # 110 
- - - - (2) INFORMATION FOR SEQ ID NO:42: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 111 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:42: 
- - Gln Ser Val Leu Thr Gln Pro Pro - # Ser Met Ser Ala Ala Pro 
Gly Gln 
1 - # 5 - # 10 - # 
15 
- - Lys Val Thr Ile Ser Cys Ser Gly - # Ser Ser Ser Asn Ile Gly 
Asn Asn 
20 - # 25 - # 30 
- - Tyr Val Ser Trp Tyr Gln Gln Phe - # Pro Gly Thr Ala Pro Lys 
Leu Leu 
35 - # 40 - # 45 
- - Ile His Asp Asn Asn Lys Arg Pro - # Ser Gly Ile Pro Asp Arg 
Ile Ser 
50 - # 55 - # 60 
- - Gly Ser Lys Ser Gly Thr Ser Ala - # Ser Leu Ala Ile Ser Gly 
Leu Gln 
65 - # 70 - # 75 - # 
80 
- - Ser Asp Asp Glu Ala Asp Tyr Tyr - # Cys Ala Ala Trp Asp Asp 
Ser Leu 
- # 85 - # 90 - # 
95 
- - Asn Gly Trp Val Phe Gly Gly Gly - # Thr Lys Leu Thr Val Leu 
Gly 
100 - # 105 - # 110 
- - - - (2) INFORMATION FOR SEQ ID NO:43: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 111 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:43: 
- - Gln Ser Val Leu Thr Gln Pro Pro - # Ser Val Ser Gly Ala Pro 
Gly Gln 
1 - # 5 - # 10 - # 
15 
- - Trp Val Thr Ile Ser Cys Ser Gly - # Ser Ser Ser Asn Ile Gly 
Asn Asn 
20 - # 25 - # 30 
- - Tyr Val Ser Trp Tyr Gln Gln Leu - # Pro Gly Thr Ala Pro Lys 
Leu Leu 
35 - # 40 - # 45 
- - Ile Tyr Asp Asn Asn Lys Arg Pro - # Ser Gly Val Pro Asp Arg 
Phe Ser 
50 - # 55 - # 60 
- - Gly Ser Gln Ser Gly Thr Ser Ala - # Ser Leu Ala Ile Ser Gly 
Leu Arg 
65 - # 70 - # 75 - # 
80 
- - Ser Glu Asp Glu Ala Asp Tyr Tyr - # Cys Ala Ala Trp Asp Asp 
Ser Leu 
- # 85 - # 90 - # 
95 
- - Ser Gly Trp Val Phe Gly Gly Gly - # Thr Lys Leu Thr Val Leu 
Gly 
100 - # 105 - # 110 
- - - - (2) INFORMATION FOR SEQ ID NO:44: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 125 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:44: 
- - Gln Val Gln Leu Leu Gln Ser Gly - # Ala Glu Leu Lys Lys Pro 
Gly Glu 
1 - # 5 - # 10 - # 
15 
- - Ser Leu Lys Ile Ser Cys Lys Gly - # Ser Gly Tyr Ser Phe Thr 
Ser Tyr 
20 - # 25 - # 30 
- - Trp Ile Ala Trp Val Arg Gln Met - # Pro Gly Lys Gly Leu Glu 
Tyr Met 
35 - # 40 - # 45 
- - Gly Leu Ile Tyr Pro Gly Asp Ser - # Asp Thr Lys Tyr Ser Pro 
Ser Phe 
50 - # 55 - # 60 
- - Gln Gly Gln Val Thr Ile Ser Val - # Asp Lys Ser Val Ser Thr 
Ala Tyr 
65 - # 70 - # 75 - # 
80 
- - Leu Gln Trp Ser Ser Leu Lys Pro - # Ser Asp Ser Ala Val Tyr 
Phe Cys 
- # 85 - # 90 - # 
95 
- - Ala Arg His Asp Val Gly Tyr Cys - # Ser Ser Ser Asn Cys Ala 
Lys Trp 
100 - # 105 - # 110 
- - Pro Glu Tyr Phe Gln His Trp Gly - # Gln Gly Thr Leu Val 
115 - # 120 - # 125 
- - - - (2) INFORMATION FOR SEQ ID NO:45: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 125 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:45: 
- - Gln Val Gln Leu Val Gln Ser Gly - # Gly Glu Met Lys Lys Pro 
Gly Glu 
1 - # 5 - # 10 - # 
15 
- - Ser Leu Lys Ile Ser Cys Lys Gly - # Leu Gly Tyr Asp Phe Thr 
Thr Tyr 
20 - # 25 - # 30 
- - Trp Ile Ala Trp Val Arg Gln Met - # Pro Gly Lys Gly Leu Glu 
Tyr Met 
35 - # 40 - # 45 
- - Gly Leu Ile Tyr Pro Gly Asp Ser - # Asp Thr Lys Tyr Ser Pro 
Ser Phe 
50 - # 55 - # 60 
- - Gln Gly Gln Val Thr Ile Ser Val - # Asp Lys Ser Val Ser Thr 
Ala Tyr 
65 - # 70 - # 75 - # 
80 
- - Leu Gln Trp Ser Ser Leu Arg Pro - # Ser Asp Thr Ala Met Tyr 
Tyr Cys 
- # 85 - # 90 - # 
95 
- - Ala Arg His Asp Val Gly Tyr Cys - # Ser Ser Ser Asn Cys Ala 
Lys Trp 
100 - # 105 - # 110 
- - Pro Glu Tyr Phe Gln His Trp Gly - # Gln Gly Thr Leu Val 
115 - # 120 - # 125 
- - - - (2) INFORMATION FOR SEQ ID NO:46: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 125 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:46: 
- - Gln Val Gln Leu Gln Gln Ser Gly - # Gly Glu Met Lys Lys Pro 
Gly Glu 
1 - # 5 - # 10 - # 
15 
- - Ser Leu Lys Ile Ser Cys Lys Gly - # Leu Gly Tyr Asp Phe Thr 
Thr Tyr 
20 - # 25 - # 30 
- - Trp Ile Ala Trp Val Arg Gln Met - # Pro Gly Lys Gly Leu Glu 
Tyr Met 
35 - # 40 - # 45 
- - Gly Leu Ile Tyr Pro Gly Asp Ser - # Asp Thr Lys Tyr Ser Pro 
Ser Phe 
50 - # 55 - # 60 
- - Gln Gly Gln Val Thr Ile Ser Ala - # Asp Glu Ser Ile Ser Thr 
Ala Tyr 
65 - # 70 - # 75 - # 
80 
- - Leu Glu Trp Ser Ser Leu Lys Ala - # Ser Asp Thr Ala Met Tyr 
Tyr Cys 
- # 85 - # 90 - # 
95 
- - Ala Arg His Asp Val Gly Tyr Cys - # Ser Ser Ser Asn Cys Ala 
Lys Trp 
100 - # 105 - # 110 
- - Pro Glu Tyr Phe Gln His Trp Gly - # Gln Gly Thr Leu Val 
115 - # 120 - # 125 
- - - - (2) INFORMATION FOR SEQ ID NO:47: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 125 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:47: 
- - Gln Val Gln Leu Gln Gln Ser Gly - # Gly Glu Met Lys Lys Pro 
Gly Glu 
1 - # 5 - # 10 - # 
15 
- - Ser Leu Lys Ile Ser Cys Lys Gly - # Ser Glu Tyr Ser Phe Ser 
Thr Tyr 
20 - # 25 - # 30 
- - Trp Ile Ala Trp Val Arg Gln Met - # Pro Gly Lys Gly Leu Glu 
Tyr Met 
35 - # 40 - # 45 
- - Gly Leu Ile Tyr Pro Gly Asp Ser - # Asp Thr Lys Tyr Ser Pro 
Ser Phe 
50 - # 55 - # 60 
- - Gln Gly Gln Val Thr Ile Ser Val - # Asp Lys Ser Val Ser Thr 
Thr Tyr 
65 - # 70 - # 75 - # 
80 
- - Leu Gln Trp Ser Ser Leu Arg Pro - # Ser Asp Thr Ala Val Tyr 
Tyr Cys 
- # 85 - # 90 - # 
95 
- - Ala Arg His Asp Val Gly Tyr Cys - # Ser Ser Ser Asn Cys Ala 
Lys Trp 
100 - # 105 - # 110 
- - Pro Glu Tyr Phe Gln His Trp Gly - # Gln Gly Thr Leu Val 
115 - # 120 - # 125 
- - - - (2) INFORMATION FOR SEQ ID NO:48: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 125 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:48: 
- - Gln Val Gln Leu Val Glu Ser Gly - # Ala Glu Met Lys Lys Pro 
Gly Glu 
1 - # 5 - # 10 - # 
15 
- - Ser Leu Lys Ile Ser Cys Lys Gly - # Phe Gly Tyr Asp Phe Ser 
Thr Tyr 
20 - # 25 - # 30 
- - Trp Ile Ala Trp Val Arg Gln Met - # Pro Gly Lys Gly Leu Glu 
Tyr Met 
35 - # 40 - # 45 
- - Gly Leu Ile Tyr Pro Gly Asp Ser - # Asp Thr Lys Tyr Ser Pro 
Ser Phe 
50 - # 55 - # 60 
- - Gln Gly Gln Val Thr Ile Ser Val - # Asp Lys Ser Val Ser Thr 
Ala Tyr 
65 - # 70 - # 75 - # 
80 
- - Leu Gln Trp Ser Ser Leu Lys Pro - # Ser Asp Thr Ala Met Tyr 
Tyr Cys 
- # 85 - # 90 - # 
95 
- - Ala Arg His Asp Val Gly Tyr Cys - # Ser Ser Ser Asn Cys Ala 
Lys Trp 
100 - # 105 - # 110 
- - Pro Glu Tyr Phe Gln His Trp Gly - # Gln Gly Thr Leu Val 
115 - # 120 - # 125 
- - - - (2) INFORMATION FOR SEQ ID NO:49: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 125 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:49: 
- - Gln Val Gln Leu Val Glu Ser Gly - # Gly Glu Met Lys Lys Pro 
Arg Glu 
1 - # 5 - # 10 - # 
15 
- - Ser Leu Lys Ile Ser Cys Lys Gly - # Leu Gly Tyr Asp Phe Thr 
Thr Tyr 
20 - # 25 - # 30 
- - Trp Ile Ala Trp Val Arg Gln Met - # Pro Gly Lys Gly Leu Glu 
Tyr Met 
35 - # 40 - # 45 
- - Gly Leu Ile Tyr Pro Gly Asp Ser - # Asp Thr Lys Tyr Ser Pro 
Ser Phe 
50 - # 55 - # 60 
- - Gln Gly Gln Val Thr Ile Ser Val - # Asp Lys Ser Val Ser Thr 
Ala Tyr 
65 - # 70 - # 75 - # 
80 
- - Leu Gln Trp Ser Ser Leu Arg Pro - # Ser Asp Thr Ala Met Tyr 
Tyr Cys 
- # 85 - # 90 - # 
95 
- - Ala Arg His Asp Val Gly Tyr Cys - # Ser Ser Ser Asn Cys Ala 
Lys Trp 
100 - # 105 - # 110 
- - Pro Glu Tyr Phe Gln His Trp Gly - # Gln Gly Thr Leu Val 
115 - # 120 - # 125 
- - - - (2) INFORMATION FOR SEQ ID NO:50: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 125 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:50: 
- - Gln Val Gln Leu Val Glu Ser Gly - # Gly Glu Met Lys Lys Pro 
Gly Glu 
1 - # 5 - # 10 - # 
15 
- - Ser Leu Lys Ile Ser Cys Lys Gly - # Leu Gly Tyr Asp Phe Ser 
Thr Tyr 
20 - # 25 - # 30 
- - Trp Ile Ala Trp Val Arg Gln Met - # Pro Gly Lys Gly Leu Glu 
Tyr Met 
35 - # 40 - # 45 
- - Gly Leu Ile Tyr Pro Gly Asp Ser - # Asp Thr Lys Tyr Ser Pro 
Ser Phe 
50 - # 55 - # 60 
- - Gln Gly Gln Val Thr Ile Ser Val - # Asp Lys Ser Val Ser Thr 
Ala Tyr 
65 - # 70 - # 75 - # 
80 
- - Leu Gln Trp Ser Ser Leu Arg Pro - # Ser Asp Thr Ala Met Tyr 
Tyr Cys 
- # 85 - # 90 - # 
95 
- - Ala Arg His Asp Val Gly Tyr Cys - # Ser Ser Ser Asn Cys Ala 
Lys Trp 
100 - # 105 - # 110 
- - Pro Glu Tyr Phe Gln His Trp Gly - # Gln Gly Thr Leu Val 
115 - # 120 - # 125 
- - - - (2) INFORMATION FOR SEQ ID NO:51: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 125 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:51: 
- - Gln Val Gln Leu Val Glu Ser Gly - # Gly Glu Met Lys Lys Pro 
Gly Glu 
1 - # 5 - # 10 - # 
15 
- - Ser Leu Lys Ile Ser Cys Lys Gly - # Leu Gly Tyr Asp Phe Thr 
Thr Tyr 
20 - # 25 - # 30 
- - Trp Ile Ala Trp Val Arg Gln Met - # Pro Gly Lys Gly Leu Glu 
Tyr Met 
35 - # 40 - # 45 
- - Gly Leu Ile Tyr Pro Gly Asp Ser - # Asp Thr Lys Tyr Ser Pro 
Ser Phe 
50 - # 55 - # 60 
- - Gln Gly Gln Val Thr Ile Ser Val - # Asp Lys Ser Val Ser Thr 
Ala Tyr 
65 - # 70 - # 75 - # 
80 
- - Leu Gln Trp Ser Ser Leu Arg Pro - # Ser Asp Thr Ala Met Tyr 
Tyr Cys 
- # 85 - # 90 - # 
95 
- - Ala Arg His Asp Val Gly Tyr Cys - # Ser Ser Ser Asn Cys Ala 
Lys Trp 
100 - # 105 - # 110 
- - Pro Glu Tyr Phe Gln His Trp Gly - # Gln Gly Thr Leu Val 
115 - # 120 - # 125 
- - - - (2) INFORMATION FOR SEQ ID NO:52: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 125 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:52: 
- - Gln Val Gln Leu Val Glu Ser Gly - # Ala Glu Met Lys Lys Pro 
Gly Glu 
1 - # 5 - # 10 - # 
15 
- - Ser Leu Lys Ile Ser Cys Lys Gly - # Phe Gly Tyr Asp Phe Ser 
Thr Tyr 
20 - # 25 - # 30 
- - Trp Ile Ala Trp Val Arg Gln Met - # Pro Gly Lys Gly Leu Glu 
Tyr Met 
35 - # 40 - # 45 
- - Gly Leu Ile Tyr Pro Gly Asp Ser - # Asp Thr Lys Tyr Ser Pro 
Ser Phe 
50 - # 55 - # 60 
- - Gln Gly Gln Val Thr Ile Ser Val - # Asp Lys Ser Val Ser Thr 
Ala Tyr 
65 - # 70 - # 75 - # 
80 
- - Leu Gln Trp Ser Ser Leu Arg Pro - # Ser Asp Thr Ala Met Tyr 
Tyr Cys 
- # 85 - # 90 - # 
95 
- - Ala Arg His Asp Val Gly Tyr Cys - # Ser Ser Ser Asn Cys Ala 
Lys Trp 
100 - # 105 - # 110 
- - Pro Glu Tyr Phe Gln His Trp Gly - # Gln Gly Thr Leu Val 
115 - # 120 - # 125 
- - - - (2) INFORMATION FOR SEQ ID NO:53: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 125 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:53: 
- - Gln Val Gln Leu Val Gln Ser Gly - # Gly Glu Met Lys Lys Pro 
Gly Glu 
1 - # 5 - # 10 - # 
15 
- - Ser Leu Lys Ile Ser Cys Lys Gly - # Leu Gly Tyr Asp Phe Ser 
Thr Tyr 
20 - # 25 - # 30 
- - Trp Ile Ala Trp Val Arg Gln Met - # Pro Gly Lys Gly Leu Glu 
Tyr Met 
35 - # 40 - # 45 
- - Gly Leu Ile Tyr Pro Gly Asp Ser - # Asp Thr Lys Tyr Ser Pro 
Ser Phe 
50 - # 55 - # 60 
- - Gln Gly Gln Val Thr Ile Ser Ala - # Asp Glu Ser Ile Ser Thr 
Ala Tyr 
65 - # 70 - # 75 - # 
80 
- - Leu Glu Trp Ser Ser Leu Lys Ala - # Ser Asp Thr Ala Met Tyr 
Tyr Cys 
- # 85 - # 90 - # 
95 
- - Ala Arg His Asp Val Gly Tyr Cys - # Ser Ser Ser Asn Cys Ala 
Lys Trp 
100 - # 105 - # 110 
- - Pro Glu Tyr Phe Gln His Trp Gly - # Gln Gly Thr Leu Val 
115 - # 120 - # 125 
- - - - (2) INFORMATION FOR SEQ ID NO:54: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 125 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:54: 
- - Gln Val Gln Leu Val Gln Ser Gly - # Gly Glu Met Lys Lys Pro 
Gly Glu 
1 - # 5 - # 10 - # 
15 
- - Ser Leu Lys Ile Ser Cys Lys Gly - # Leu Gly Tyr Asp Phe Ser 
Thr Tyr 
20 - # 25 - # 30 
- - Trp Ile Ala Trp Val Arg Gln Met - # Pro Gly Lys Gly Leu Glu 
Tyr Met 
35 - # 40 - # 45 
- - Gly Leu Ile Tyr Pro Gly Asp Ser - # Asp Thr Lys Tyr Ser Pro 
Ser Phe 
50 - # 55 - # 60 
- - Gln Gly Gln Val Thr Ile Ser Ala - # Asp Glu Ser Ile Ser Thr 
Ala Tyr 
65 - # 70 - # 75 - # 
80 
- - Leu Glu Trp Ser Ser Leu Lys Ala - # Ser Asp Thr Ala Met Tyr 
Tyr Cys 
- # 85 - # 90 - # 
95 
- - Ala Arg His Asp Val Gly Tyr Cys - # Ser Ser Ser Asn Cys Ala 
Lys Trp 
100 - # 105 - # 110 
- - Pro Glu Tyr Phe Gln His Trp Gly - # Gln Gly Thr Leu Val 
115 - # 120 - # 125 
- - - - (2) INFORMATION FOR SEQ ID NO:55: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 125 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:55: 
- - Gln Val Gln Leu Leu Gln Ser Gly - # Ala Glu Met Lys Lys Pro 
Gly Glu 
1 - # 5 - # 10 - # 
15 
- - Ser Leu Lys Ile Ser Cys Lys Gly - # Phe Gly Tyr Asp Phe Ser 
Thr Tyr 
20 - # 25 - # 30 
- - Trp Ile Ala Trp Val Arg Gln Met - # Pro Gly Lys Gly Leu Glu 
Tyr Met 
35 - # 40 - # 45 
- - Gly Leu Ile Tyr Pro Gly Asp Ser - # Asp Thr Lys Tyr Ser Pro 
Ser Phe 
50 - # 55 - # 60 
- - Gln Gly Gln Val Thr Ile Ser Val - # Asp Lys Ser Val Ser Thr 
Ala Tyr 
65 - # 70 - # 75 - # 
80 
- - Leu Gln Trp Ser Ser Leu Lys Pro - # Ser Asp Thr Ala Met Tyr 
Tyr Cys 
- # 85 - # 90 - # 
95 
- - Ala Arg His Asp Val Gly Tyr Cys - # Ser Ser Ser Asn Cys Ala 
Lys Trp 
100 - # 105 - # 110 
- - Pro Glu Tyr Phe Gln His Trp Gly - # Gln Gly Thr Leu Val 
115 - # 120 - # 125 
- - - - (2) INFORMATION FOR SEQ ID NO:56: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 125 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:56: 
- - Gln Val Gln Leu Leu Gln Ser Gly - # Ala Glu Val Lys Lys Pro 
Gly Glu 
1 - # 5 - # 10 - # 
15 
- - Ser Leu Gln Ile Ser Cys Lys Gly - # Ser Gly Tyr Asp Phe Thr 
Thr Tyr 
20 - # 25 - # 30 
- - Tyr Ile Ala Trp Val Arg Gln Met - # Pro Gly Lys Gly Leu Glu 
Tyr Met 
35 - # 40 - # 45 
- - Gly Ile Ile Tyr Pro Gly Asp Ser - # Arg Thr Ile Tyr Ser Pro 
Ser Phe 
50 - # 55 - # 60 
- - Gln Gly Gln Val Arg Ile Ser Ala - # Asp Lys Ser Val Ser Thr 
Ala Tyr 
65 - # 70 - # 75 - # 
80 
- - Leu Gln Trp Ser Ser Leu Lys Ala - # Ser Asp Thr Ala Met Tyr 
Tyr Cys 
- # 85 - # 90 - # 
95 
- - Ala Arg His Asp Val Gly Tyr Cys - # Ser Ser Ser Asn Cys Ala 
Lys Trp 
100 - # 105 - # 110 
- - Pro Glu Tyr Phe Gln His Trp Gly - # Gln Gly Thr Leu Val 
115 - # 120 - # 125 
- - - - (2) INFORMATION FOR SEQ ID NO:57: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 125 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:57: 
- - Gln Val Gln Leu Leu Glu Ser Gly - # Ala Glu Val Lys Glu Pro 
Gly Gln 
1 - # 5 - # 10 - # 
15 
- - Ser Leu Lys Ile Ser Cys Lys Gly - # Phe Gly Tyr Asp Phe Ser 
Thr Tyr 
20 - # 25 - # 30 
- - Trp Ile Ala Trp Val Arg Gln Met - # Pro Gly Lys Gly Leu Glu 
Tyr Met 
35 - # 40 - # 45 
- - Gly Leu Ile Tyr Pro Gly Asp Ser - # Asp Thr Lys Tyr Ser Pro 
Ser Phe 
50 - # 55 - # 60 
- - Gln Gly Gln Val Thr Ile Ser Val - # Asp Lys Ser Val Ser Thr 
Ala Tyr 
65 - # 70 - # 75 - # 
80 
- - Leu Gln Trp Ser Ser Leu Lys Pro - # Ser Asp Thr Ala Met Tyr 
Tyr Cys 
- # 85 - # 90 - # 
95 
- - Ala Arg His Asp Val Gly Tyr Cys - # Ser Ser Ser Asn Cys Ala 
Lys Trp 
100 - # 105 - # 110 
- - Pro Glu Tyr Phe Gln His Trp Gly - # Gln Gly Thr Leu Val 
115 - # 120 - # 125 
- - - - (2) INFORMATION FOR SEQ ID NO:58: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 125 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (ix) FEATURE: 
(A) NAME/KEY: Modified-sit - #e 
(B) LOCATION: 33 
(D) OTHER INFORMATION: - #/product= "OTHER" 
/note= - #"Xaa = unknown (possibly Glu)" 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:58: 
- - Gln Val Gln Leu Val Glu Ser Gly - # Ala Glu Val Lys Lys Pro 
Arg Glu 
1 - # 5 - # 10 - # 
15 
- - Ser Leu Lys Ile Ser Cys Lys Gly - # Ser Gly Tyr Ser Phe Thr 
Ser Tyr 
20 - # 25 - # 30 
- - Xaa Ile Ala Trp Val Arg Gln Met - # Pro Gly Lys Gly Leu Glu 
Tyr Met 
35 - # 40 - # 45 
- - Gly Leu Ile Tyr Pro Gly Asp Ser - # Asp Thr Lys Tyr Ser Pro 
Ser Phe 
50 - # 55 - # 60 
- - Gln Gly Gln Val Thr Ile Ser Val - # Asp Lys Ser Val Ser Thr 
Ala Tyr 
65 - # 70 - # 75 - # 
80 
- - Leu Gln Trp Ser Ser Leu Lys Pro - # Ser Asp Thr Ala Met Tyr 
Tyr Cys 
- # 85 - # 90 - # 
95 
- - Ala Arg His Asp Val Gly Tyr Cys - # Ser Ser Ser Asn Cys Ala 
Lys Trp 
100 - # 105 - # 110 
- - Pro Glu Tyr Phe Gln His Trp Gly - # Gln Gly Thr Leu Val 
115 - # 120 - # 125 
- - - - (2) INFORMATION FOR SEQ ID NO:59: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 125 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:59: 
- - Gln Val Gln Leu Val Gln Ser Gly - # Ala Glu Val Lys Lys Pro 
Arg Glu 
1 - # 5 - # 10 - # 
15 
- - Ser Leu Lys Ile Ser Cys Lys Gly - # Ser Gly Tyr Ser Phe Thr 
Ser Tyr 
20 - # 25 - # 30 
- - Trp Ile Ala Trp Val Arg Gln Met - # Pro Gly Lys Gly Leu Glu 
Tyr Met 
35 - # 40 - # 45 
- - Gly Leu Ile Tyr Pro Ala Asp Ser - # Lys Thr Ile Tyr Ser Pro 
Ser Phe 
50 - # 55 - # 60 
- - Gln Gly Gln Val Thr Ile Ser Val - # Asp Lys Ser Val Ser Thr 
Ala Tyr 
65 - # 70 - # 75 - # 
80 
- - Leu Gln Trp Ser Ser Leu Lys Thr - # Ser Asp Thr Ala Met Tyr 
Tyr Cys 
- # 85 - # 90 - # 
95 
- - Ala Arg His Asp Val Gly Tyr Cys - # Ser Ser Ser Asn Cys Ala 
Lys Trp 
100 - # 105 - # 110 
- - Pro Glu Tyr Phe Gln His Trp Gly - # Gln Gly Thr Leu Val 
115 - # 120 - # 125 
- - - - (2) INFORMATION FOR SEQ ID NO:60: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:60: 
- - Ala Ala Trp Asp Asp Ser Leu Ser - # Gly 
1 - # 5 
- - - - (2) INFORMATION FOR SEQ ID NO:61: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:61: 
- - His Asp Val Gly Tyr Cys Ser Ser - # Ser Asn Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Tyr Phe Gln His 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:62: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:62: 
- - Ser Ser Ser Asn 
1 
- - - - (2) INFORMATION FOR SEQ ID NO:63: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:63: 
- - Asp Val Gly Tyr 
1 
- - - - (2) INFORMATION FOR SEQ ID NO:64: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:64: 
- - Ala Lys Pro Glu 
1 
- - - - (2) INFORMATION FOR SEQ ID NO:65: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:65: 
- - Tyr Phe Gln His 
1 
- - - - (2) INFORMATION FOR SEQ ID NO:66: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:66: 
- - His Asp Val Gly Phe Cys Ser Ser - # Ser Asn Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Tyr Phe Gln His 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:67: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:67: 
- - His Asp Val Gly Tyr Cys Ser Ser - # Ser Asp Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Tyr Phe Gln His 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:68: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:68: 
- - His Asp Val Gly Tyr Cys Thr Asp - # Arg Thr Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Tyr Phe Gln His 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:69: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:69: 
- - His Asp Val Gly Tyr Cys Glu Ser - # Ser Arg Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Tyr Phe Gln His 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:70: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:70: 
- - His Asp Val Gly Tyr Cys Ser Asp - # Arg Ser Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Tyr Phe Gln His 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:71: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:71: 
- - His Asp Val Gly Tyr Cys Lys Thr - # Ala Ala Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Tyr Phe Gln His 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:72: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (ix) FEATURE: 
(A) NAME/KEY: Modified-sit - #e 
(B) LOCATION: 7 
(D) OTHER INFORMATION: - #/product= "OTHER" 
/note= - #"Xaa = unknown" 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:72: 
- - His Asp Val Gly Tyr Cys Xaa Thr - # Glu Arg Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Tyr Phe Gln His 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:73: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:73: 
- - His Asp Val Gly Tyr Cys Thr Asp - # Pro Arg Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Tyr Phe Gln His 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:74: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:74: 
- - His Asp Val Gly Tyr Cys Thr Asp - # Pro Thr Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Tyr Phe Gln His 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:75: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:75: 
- - His Asp Val Gly Tyr Cys Leu Thr - # Thr Arg Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Tyr Phe Gln His 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:76: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:76: 
- - His Asp Val Gly Tyr Cys Thr Thr - # Pro Leu Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Tyr Phe Gln His 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:77: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:77: 
- - His Asp Val Gly Tyr Cys Ser Pro - # Ala Arg Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Tyr Phe Gln His 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:78: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:78: 
- - His Asp Val Gly Tyr Cys Ala Asp - # Val Arg Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Tyr Phe Gln His 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:79: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:79: 
- - His Asp Val Gly Tyr Cys Thr Asp - # Arg Ser Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Tyr Phe Gln His 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:80: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:80: 
- - His Asp Val Gly Tyr Cys Thr Asp - # Pro Ser Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Tyr Phe Gln His 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:81: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:81: 
- - His Asp Val Gly Tyr Cys Thr Asp - # Ala Thr Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Tyr Phe Gln His 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:82: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:82: 
- - His Asp Val Gly Tyr Cys Thr Asp - # Arg Pro Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Tyr Phe Gln His 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:83: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:83: 
- - His Asp Val Gly Tyr Cys Lys Asn - # Ser Arg Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Tyr Phe Gln His 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:84: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:84: 
- - His Asp Val Gly Tyr Cys Gln Asp - # Thr Arg Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Tyr Phe Gln His 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:85: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:85: 
- - His Asp Val Gly Tyr Cys Glu Asp - # Tyr Thr Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Tyr Phe Gln His 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:86: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:86: 
- - His Asp Val Gly Tyr Cys Thr Thr - # Pro Arg Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Tyr Phe Gln His 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:87: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:87: 
- - His Asp Val Gly Tyr Cys Ser Asp - # Gln Thr Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Tyr Phe Gln His 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:88: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:88: 
- - His Asp Val Gly Tyr Cys Asp Asp - # Tyr Thr Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Tyr Phe Gln His 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:89: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:89: 
- - His Asp Val Gly Tyr Cys Ser Ser - # Ser Asn Cys Ala Val Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Tyr Phe Gln His 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:90: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:90: 
- - His Asp Val Gly Tyr Cys Ser Ser - # Ser Asn Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Trp Leu Gly Val 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:91: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:91: 
- - His Asp Val Gly Tyr Cys Ser Ser - # Ser Asn Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Trp Leu Asp Asn 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:92: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:92: 
- - His Asp Val Gly Tyr Cys Ser Ser - # Ser Asn Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Trp Met Tyr Pro 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:93: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:93: 
- - His Asp Val Gly Tyr Cys Ser Ser - # Ser Asn Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Trp Met Gln Met 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:94: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:94: 
- - His Asp Val Gly Tyr Cys Ser Ser - # Ser Asn Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Trp Leu His Val 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:95: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:95: 
- - His Asp Val Gly Tyr Cys Ser Ser - # Ser Asn Cys Ala Lys Trp 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Trp Gln Asp Pro 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:96: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:96: 
- - Thr Asp Arg Thr 
1 
- - - - (2) INFORMATION FOR SEQ ID NO:97: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:97: 
- - Ala Ser Trp Asp Tyr Tyr Arg Ser - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:98: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:98: 
- - Ala Ser Trp Asp Ala Ser Leu Trp - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:99: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:99: 
- - Ala Ala Trp Glu Gln Ser Leu Trp - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:100: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:100: 
- - Ala Ala Trp Asp Tyr Ser Gln Ser - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:101: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:101: 
- - Ala Ala Trp Asp Ala Ser Leu Ser - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:102: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:102: 
- - Ala Ala Trp Asp Glu Gln Ile Phe - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:103: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:103: 
- - Ala Ala Trp Asp Asn Arg His Ser - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:104: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:104: 
- - Ala Ala Trp Asp Asp Ser Arg Ser - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:105: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:105: 
- - Ala Ser Trp Asp Tyr Tyr Arg Ser - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:106: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:106: 
- - Thr Ala Trp Asp Tyr Ser Leu Ser - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:107: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:107: 
- - Ala Ser Trp Asp Tyr Ala Leu Ser - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:108: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:108: 
- - Ala Ala Trp Asp Tyr Ala Leu Ser - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:109: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:109: 
- - Ala Ser Trp Asp Tyr Tyr Leu Ile - # Gly Trp Val 
1 - # 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:110: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:110: 
- - CAGGAAACAG CTATGAC - # - # 
- # 17 
- - - - (2) INFORMATION FOR SEQ ID NO:111: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:111: 
- - GAATTTTCTG TATGAGG - # - # 
- # 17 
- - - - (2) INFORMATION FOR SEQ ID NO:112: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:112: 
- - CTATGCGGCC CCATTCA - # - # 
- # 17 
- - - - (2) INFORMATION FOR SEQ ID NO:113: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:113: 
- - CGATCCGCCA CCGCCAGAG - # - # 
- # 19 
- - - - (2) INFORMATION FOR SEQ ID NO:114: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:114: 
- - TCGCGCGCAG TAATACACGG CCGTGTC - # - # 
27 
- - - - (2) INFORMATION FOR SEQ ID NO:115: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:115: 
- - TCGCGCGCAG TAATACACAG CCGTGTCCTC - # - # 
30 
- - - - (2) INFORMATION FOR SEQ ID NO:116: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:116: 
- - TCGCGCGCAG TAATACATGG CGGTGTCCGA - # - # 
30 
- - - - (2) INFORMATION FOR SEQ ID NO:117: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 51 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:117: 
- - GAGTCATTCT CGACTTGCGG CCGCTCGCGC GCAGTAATAC ACGGCCGTGT C - # 
51 
- - - - (2) INFORMATION FOR SEQ ID NO:118: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 54 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:118: 
- - GAGTCATTCT CGACTTGCGG CCGCTCGCGC GCAGTAATAC ACAGCCGTGT CC - #TC 
54 
- - - - (2) INFORMATION FOR SEQ ID NO:119: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 54 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:119: 
- - GAGTCATTCT CGACTTGCGG CCGCTCGCGC GCAGTAATAC ATGGCGGTGT CC - #GA 
54 
- - - - (2) INFORMATION FOR SEQ ID NO:120: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 42 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:120: 
- - AGCGCCGTGT ATTTTTGCGC GCGACATGAC GTGGGATATT GC - # 
- # 42 
- - - - (2) INFORMATION FOR SEQ ID NO:121: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:121: 
- - ACCCTGGTCA CCGTCTCGAG TGGTGGA - # - # 
27 
- - - - (2) INFORMATION FOR SEQ ID NO:122: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:122: 
- - ACAATGGTCA CCGTCTCGAG TGGTGGA - # - # 
27 
- - - - (2) INFORMATION FOR SEQ ID NO:123: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:123: 
- - GAGTCATTCT CGTCTCGAGA CGGTGACCAG GGTGCC - # - 
# 36 
- - - - (2) INFORMATION FOR SEQ ID NO:124: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 67 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:124: 
- - GTCCCTCCGC CGAACACCCA SNNSNNSNNS NNSNNSNNNN NSNNSNNACA GT - 
#AATAATCA 60 
- - GCCTCAT - # - # 
- # 67 
- - - - (2) INFORMATION FOR SEQ ID NO:125: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 63 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:125: 
- - GAGTCATTCT CGACTTGCGG CCGCACCTAG GACGGTCAGC TTGGTCCCTC CG - 
#CCGAACAC 60 
- - CCA - # - # - # 
63 
- - - - (2) INFORMATION FOR SEQ ID NO:126: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 56 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:126: 
- - GCGCAGTTGG AACTACTGCA SSSSSSSSSS SSATGTCTCG CACAAAAATA CA - #CGGC 
56 
- - - - (2) INFORMATION FOR SEQ ID NO:127: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:127: 
- - TGCAGTAGTT CCAACTGCGC - # - # 
- # 20 
- - - - (2) INFORMATION FOR SEQ ID NO:128: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 53 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:128: 
- - GTATTCAGGC CACTTTGCGC ASSSSSSSSS SSSGCAATAT CCCACGTCAT GT - #C 
53 
- - - - (2) INFORMATION FOR SEQ ID NO:129: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:129: 
- - TGCGCAAAGT GGCCTGAATA C - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:130: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 60 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:130: 
- - CTGGCCCCAA TGCTGGAAGT ASSSSSSCCA SSSSSSGCAG TTGGAACTAC TG - 
#CAATATCC 60 
- - - - (2) INFORMATION FOR SEQ ID NO:131: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:131: 
- - TACTTCCAGC ATTGGGGCCA G - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:132: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 56 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:132: 
- - GACCAGGGTG CCCTGGCCCC ASSSSSSSSS SSSTTCAGGC CACTTTGCGC AG - #TTGG 
56 
- - - - (2) INFORMATION FOR SEQ ID NO:133: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:133: 
- - TGGGGCCAGG GCACCCTGGT C - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:134: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 72 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:134: 
- - GATACGGCAC CGGCGCACCT GCGGCCGCAT GGTGATGATG GTGATGTGCG GC - 
#ACCTAGGA 60 
- - CGGTCAGCTT GG - # - # 
- # 72 
- - - - (2) INFORMATION FOR SEQ ID NO:135: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 80 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:135: 
- - CCTAGGACGG TCAGCTTGGT CCCTCCGCCG AACACCCAAC CACTCAGGGT GT - 
#AATCCCAG 60 
- - GATGCACAGT AATAATCAGC - # - # 
- # 80 
- - - - (2) INFORMATION FOR SEQ ID NO:136: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 69 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:136: 
- - CCTAGGACGG TCAGCTTGGT CCCTCCGCCG AACACCCAAC CACTCAGGGT GT - 
#AATCCCAT 60 
- - GCTGCACAG - # - # 
- # 69 
- - - - (2) INFORMATION FOR SEQ ID NO:137: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 57 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:137: 
- - GACGGTGACC AGGGTGCCCT GGCCCCAAAC GTGCAGCCAT TCAGGCCACT TT - #GCGCA 
57 
- - - - (2) INFORMATION FOR SEQ ID NO:138: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 57 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:138: 
- - GACGGTGACC AGGGTGCCCT GGCCCCATAC GCCCAGCCAT TCAGGCCACT TT - #GCGCA 
57 
- - - - (2) INFORMATION FOR SEQ ID NO:139: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 57 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:139: 
- - GACGGTGACC AGGGTGCCCT GGCCCCAGTT GTCCAACCAT TCAGGCCACT TT - #GCGCA 
57 
- - - - (2) INFORMATION FOR SEQ ID NO:140: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 57 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:140: 
- - GACGGTGACC AGGGTGCCCT GGCCCCACAT CTGCATCCAT TCAGGCCACT TT - #GCGCA 
57 
- - - - (2) INFORMATION FOR SEQ ID NO:141: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 57 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:141: 
- - GACGGTGACC AGGGTGCCCT GGCCCCAGGG GTACATCCAT TCAGGCCACT TT - #GCGCA 
57 
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