U.S. patent application number 10/835392 was filed with the patent office on 2004-11-18 for antigen binding peptides (abtides) from peptide libraries.
This patent application is currently assigned to Cytogen Corporation. Invention is credited to Alvarez, Vernon L..
Application Number | 20040229289 10/835392 |
Document ID | / |
Family ID | 23201382 |
Filed Date | 2004-11-18 |
United States Patent
Application |
20040229289 |
Kind Code |
A1 |
Alvarez, Vernon L. |
November 18, 2004 |
Antigen binding peptides (abtides) from peptide libraries
Abstract
Abtides are provided. Abtides are peptides identified by a
two-step process of screening random peptide libraries. In the
first step, the target ligand is an antibody or receptor (or
derivative thereof). The peptides identified in the first screening
step are used as target ligands in the second screening step. The
peptides identified in the second screening step are abtides.
Abtides possess binding specificities that are similar to the
binding specificities of the antibodies or receptors that are used
in the first screening step. Abtides may be used in place of
antibodies in many assays or therapeutic applications. Abtides
binding to polymorphic epithelial mucin (PEM) are provided. Also
provided are methods of obtaining abtides as well as diagnostic and
therapeutic compounds containing abtides.
Inventors: |
Alvarez, Vernon L.;
(Morrisville, PA) |
Correspondence
Address: |
JONES DAY
222 EAST 41ST ST
NEW YORK
NY
10017
US
|
Assignee: |
Cytogen Corporation
|
Family ID: |
23201382 |
Appl. No.: |
10/835392 |
Filed: |
April 28, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10835392 |
Apr 28, 2004 |
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09484879 |
Jan 18, 2000 |
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09484879 |
Jan 18, 2000 |
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09273685 |
Mar 22, 1999 |
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6015561 |
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09273685 |
Mar 22, 1999 |
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08488161 |
Jun 7, 1995 |
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5885577 |
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08488161 |
Jun 7, 1995 |
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08310192 |
Sep 21, 1994 |
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Current U.S.
Class: |
435/7.1 ; 506/18;
506/9; 514/19.4; 514/19.5; 530/324; 530/387.1; 530/388.22 |
Current CPC
Class: |
C40B 30/04 20130101;
C40B 40/02 20130101; C12N 15/1037 20130101; C07K 14/4727 20130101;
C07K 14/001 20130101; C07K 1/047 20130101; G01N 33/6854 20130101;
G01N 33/6845 20130101; A61K 38/00 20130101; C07K 2319/00
20130101 |
Class at
Publication: |
435/007.1 ;
530/387.1 |
International
Class: |
G01N 033/53; C07K
016/18 |
Claims
What is claimed is:
1. A molecule comprising a peptide which binds to a substance of
interest, which peptide is identified by a method comprising: (a)
screening a first random peptide library with a first ligand, said
first ligand being a specific binding partner of said substance of
interest, to identify a first peptide that specifically binds to
said first ligand; and (b) screening a second random peptide
library with a second ligand comprising said first peptide
identified in step (a), to identify a second peptide which binds to
said second ligand and which binds to said substance of
interest.
2. A molecule comprising a peptide which binds to an antigen of
interest, which peptide is identified by a method comprising: (a)
screening a first random peptide library with an antibody or
antigen-binding derivative thereof that specifically binds to an
antigen of interest, to identify a first peptide that specifically
binds to said antibody or antigen-binding derivative thereof; and
(b) screening a second random peptide library with a compound
comprising said first peptide identified in step (a), to identify a
second peptide which binds to said compound and which binds to said
antigen of interest.
3. The molecule of claim 2, in which said first random peptide
library is a different library from said second random peptide
library.
4. The molecule of claim 2, in which said first random peptide
library is the same library as said second random peptide
library.
5. The molecule of claim 1, in which said method further comprises
comparing the sequences of a plurality of different first peptides
identified as binding said first ligand in step (a.), to identify a
consensus binding sequence, in which said second ligand of step (b)
comprises said consensus binding sequence.
6. The molecule of claim 2, in which said method further comprises
comparing the sequences of a plurality of different first peptides
identified as binding said antibody or antigen-binding derivative
thereof in step (a), to identify a consensus binding sequence, in
which said compound of step (b) comprises said consensus binding
sequence.
7. The molecule of claim 1 in which the first ligand comprises a
receptor.
8. The molecule of claim 2 in which the antibody is the monoclonal
antibody 7E11-C5.
9. The molecule of claim 1 in which the library of step (a) or step
(b) is a library of recombinant vectors that express a plurality of
heterofunctional fusion proteins, said fusion proteins comprising a
binding domain encoded by an oligonucleotide comprising
unpredictable nucleotides in which the unpredictable nucleotides
are arranged in one or more contiguous sequences, wherein the total
number of unpredictable nucleotides is greater than or equal to
about 15 and less than or equal to about 600.
10. The molecule of claim 2 in which the library of step (a) or
step (b) is a library of recombinant vectors that express a
plurality of heterofunctional fusion proteins, said fusion proteins
comprising a binding domain encoded by an oligonucleotide
comprising unpredictable nucleotides in which the unpredictable
nucleotides are arranged in one or more contiguous sequences,
wherein the total number of unpredictable nucleotides is greater
than or equal to about 15 and less than or equal to about 600.
11. The molecule of claim 1 in which the library of step (a) or
step (b) is a chemically synthesized library.
12. A molecule comprising: an amino acid sequence 10 selected from
the group consisting of:
9 (SEQ ID NO: 1) GIINANDPLPFWFMSPYTPGPAPIDINASRALVSNESG, (SEQ ID
NO: 2) CGRAYCLSGNYNIFGALFPGVSTPYADVGHDDA- QSWRR, (SEQ ID NO: 3)
DLSRNLDFGRFLLYNAYVPGFTPTFI- SLTAEHLSSPKG, (SEQ ID NO: 4)
RCSPIWGISYPFGLLSSNPGVCHSSDAETNIRNDILTT, and (SEQ ID NO: 5)
GHSNYCFVSTLGMPIVGFPSINARGLIHYGGSDPRLAA;
or a binding portion thereof.
13. A peptide in-which the amino acid sequence of said peptide
consists of the sequence selected from the group consisting of:
10 (SEQ ID NO: 1) GIINANDPLPFWFMSPYTPGPAPIDINASRALVSNESG, (SEQ ID
NO: 2) CGPAYCLSGNYNIFGALFPGVSTPYADVGHDD- AQSWRR, (SEQ ID NO: 3)
DLSRNLDFGRFLLYNAYVPGFTPTF- ISLTAEHLSSPKG, (SEQ ID NO: 4)
RCSPIWGISYPFGLLSSNPGVCHSSDAETNIRNDILTT, and (SEQ ID NO: 5)
GHSNYCFVSTLGMPIVGFPSINARGLIHYGGSDPRLAA;
or a binding portion thereof.
14. A method of identifying a peptide which binds to a substance of
interest, comprising: (a) screening a first random peptide library
with a ligand, said ligand being a specific binding partner of said
substance of interest, to identify a first peptide that
specifically binds to said ligand; and (b) screening a second
random peptide library with a compound comprising said first
peptide identified in step (a), to identify a second peptide which
binds to said compound and which binds to said substance of
interest.
15. A method of identifying a peptide which binds to an antigen of
interest comprising: (a) screening a first random peptide library
with an antibody or antigen-binding derivative thereof that
specifically binds to an antigen of interest, to identify a first
peptide that specifically binds to said antibody or antigen-binding
derivative thereof; and (b) screening a second random peptide
library with a molecule comprising said first peptide identified in
step (a), to identify a second peptide sequence which binds to said
molecule and which binds to said antigen of interest.
16. The method of claim 14, in which said first random peptide
library is a different library from said second random peptide
library.
17. The method of claim 14, in which said first random peptide
library is the same library as said second random peptide
library.
18. The method of claim 14 in which the ligand is a receptor.
19. The method of claim 15 in which the antibody is the monoclonal
antibody 7E11-C5.
20. The method of claim 14 in which the library of step (a) or step
(b) is a library of recombinant vectors that express a plurality of
heterofunctional fusion proteins, said fusion proteins comprising a
binding domain encoded by an oligonucleotide comprising
unpredictable nucleotides in which the unpredictable nucleotides
are arranged in one or more contiguous sequences, wherein the total
number of unpredictable nucleotides is greater than or equal to
about 15 and less than or equal to about 600.
21. The method of claim 15 in which the library of step (a) or step
(b) is a library of recombinant vectors that express a plurality of
heterofunctional fusion proteins, said fusion proteins comprising a
binding domain encoded by an oligonucleotide comprising
unpredictable nucleotides in which the unpredictable nucleotides
are arranged in one or more contiguous sequences, wherein the total
number of unpredictable nucleotides is greater than or equal to
about 15 and less than or equal to about 600.
22. The method of claim 14 where the library of step (a) or step
(b) is a chemically synthesized library.
23. A method of detecting or measuring an analyte of interest in a
sample, comprising: (a) contacting a sample with a molecule
comprising a peptide capable of specifically binding said analyte
of interest under conditions such that specific binding between
said molecule and said analyte can occur; and (b) detecting or
measuring the amount of said binding in which the presence and
amount of said binding indicates the presence and amount,
respectively, of said analyte in the sample; in which said peptide
is identified by the method of claim 14.
24. The method of claim 23, in which said molecule is immobilized
on a solid substratum.
25. A method of determining the location in a patient of a tumor
comprising: (a) introducing a molecule comprising a peptide that
specifically binds to a tumor antigen into the patient; and (b)
determining the location in the patient of the molecule; in which
the molecule is detectably labeled; and in which said peptide is
identified by a method comprising: (i) screening a first random
peptide library with an antibody or antigen-binding derivative
thereof that specifically binds to said tumor antigen, to identify
a first peptide that specifically binds to said antibody or
antigen-binding derivative thereof; and (ii) screening a second
random peptide library with a molecule comprising said first
peptide identified in (i), to identify a second peptide which binds
to said molecule and which binds to said tumor antigen.
26. A therapeutic or diagnostic composition comprising the molecule
of claim 1; and a pharmaceutically acceptable carrier.
27. A therapeutic or diagnostic composition comprising the molecule
of claim 2; and a pharmaceutically acceptable carrier.
28. A therapeutic or diagnostic composition comprising the molecule
of claim 5; and a pharmaceutically acceptable carrier.
29. A therapeutic or diagnostic composition comprising the molecule
of claim 7; and a pharmaceutically acceptable carrier.
30. A therapeutic or diagnostic composition comprising the molecule
of claim 8; and a pharmaceutically acceptable carrier.
31. A therapeutic or diagnostic composition comprising the molecule
of claim 12; and a pharmaceutically acceptable carrier.
32. A composition comprising a plurality of molecules of claim 1,
in which said peptide sequences of said molecules differ.
33. A molecule comprising a peptide or a binding portion thereof
which binds to a ligand of interest, which peptide is identified by
a method comprising: screening a random peptide library with a
ligand of interest, said ligand of interest being a peptide having
a length of between 5 and 40 amino acids, to identify a peptide
that specifically binds to the ligand of interest, in which the
ligand of interest is also specifically bound by an antibody or a
receptor.
34. The molecule of claim 31 in which the ligand is a peptide
having a length of between 10 and 20 amino acids.
35. A method of obtaining an image of an internal region of a
subject comprising administering to said subject an effective
amount of the molecule of claim 1 in which said molecule is
radiolabeled with a radioactive metal, and recording the
scintigraphic image obtained from the decay of said radioactive
metal.
36. A molecule comprising a peptide which binds to a substance of
interest, which peptide is identified by a method comprising:
screening a random peptide library with a ligand, said ligand being
a peptide of 36 amino acids or fewer, in which the ligand is an
epitope of an antigen that is specifically bound by an antibody or
in which the ligand represents the portion of a receptor-ligand
that is responsible for the specific binding of the receptor to the
receptor-ligand.
37. A peptide comprising the amino acid sequence WQGTHF (SEQ ID NO:
23) and the amino acid sequence LVSKNDSG (SEQ ID NO: 24) that
specifically binds to an antigen of human prostate carcinoma
cells.
38. A molecule comprising an amino acid sequence selected from the
group consisting of:
11 (SEQ ID NO:26) SFMDYFFHTPEPKPAGYPNAYTDPKHPA, (SEQ ID NO:27)
SSSIFDYAPFSWGSAGLSNSSINVFERS, (SEQ ID NO:28)
SASLWDALGGWTTSAVPSYPRFHQTPGR, (SEQ ID NO:29)
SLGLPWIDVFGRSSAEPWPFGRTNLPRS, (SEQ ID NO:30)
SVHGAFLDSFFPWAADGPHGRGRLTSF, (SEQ ID NO:31)
EEKQGGRWSTMMPRPWCHEGGCGFLYYDAMTKPKTPPIMRTAA, (SEQ ID NO:32)
LPRPFDDASWKLRAVKESPDGCGFGSPLLFPPYSGLPT- FSSCD, (SEQ ID NO:33)
GSFESARGVTCIGNHSIGAHGCGPLR- SYASFNRGSGRRH, (SEQ ID NO:34)
DQIGSRPQTTSRSISGSWWENAKTLWQQDYAFSAPNAA, (SEQ ID NO:35)
LSDAWGNFTTSYRDSAGFPSHAMTTSQGGKRNHASRFP, (SEQ ID NO:36)
VQLDDTSPRASGQETSQSEYDARPLLSKFAIPRPWSR, (SEQ ID NO:37)
IDSSKNRISGTGYLSFPHIRHANRRHMADDSNLAPGPS, (SEQ ID NO:38)
WSIGTHTGPEGKFRIPCDRSGCGGTTLTHGGLNSSPTGQHER- P, (SEQ ID NO:39)
DPCEDGYWLSSVGRAGASIRGCGAIRRSSR- TLTAEYSTRASNH, (SEQ ID NO:40)
GSKRSCWGTTISNYFRPVPEGCGSASSINPNTNTGRLPSLHRQ, (SEQ ID NO:41)
SSASSGCLGRAEHLDLDSVWGCGSQADMSRRYSPWYGRPRTGV, (SEQ ID NO:42)
NVMWSSSKAGIRDCSQVPPGGCGPVNRHRASPPLTPFRHGSIR, (SEQ ID NO:43)
PLTSGSSSEYRNRDDCPVYKYATNCPRLNFSPS- RYSPF, (SEQ ID NO:44)
GDAYGGIFSRPRQGLADSYIHASYTG- KHFFRGPRPPTR, (SEQ ID NO:45)
STCIGAEGEWKSFHNFLQCRDATSTSSSTLDPTALRFG, (SEQ ID NO:46)
YSATLWDQFGSRQVELWSNRHASSALPFASRASVLGSR, (SEQ ID NO:47)
ILGWPFLTGLGDSTVHPRGRKGTDPS, (SEQ ID NO:48)
SIPSFSMWLNQLGSAALPSKGNSQDRSD, (SEQ ID NO:49)
SRDDIFTGGPLVLFRGSKTSNHDVHSMR, (SEQ ID NO:50)
RAELVNWYEWFHVTAEAETPVINSHNMT, (SEQ ID NO:51)
GAPVWRGNPRWRGPGGFKWPGCGNGPMCNTFTPARGGSRNNGP, (SEQ ID NO:52)
GSASSCFPNFTARGVTVGFFGCGSPAHPAAPRVLNPATDFPAP, (SEQ ID NO:53)
VFRRTARSSRPIGATVFPWYGCGNSNDETLPHH- DSPPSFFLGA, (SEQ ID NO:54)
NTCWTDLFWHGLPGGDLPRDGCGLPSELTTHPSRERRDASEN, (SEQ ID NO:55)
IDWNWLERGQHNRGYLHSFPDAKSQPTRGPRVAPNGND and (SEQ ID NO:56)
GRGSDMREHWPWSMPLILDQHANDPSPRA- QSHYYSHPF.
39. A molecule comprising an amino acid sequence selected from the
group consisting of:
12 (SEQ ID NO:57) VSTGWSGTPRWCAPGGKQGSGCGNGPRWTTLTPDLGGTR- KYGP,
(SEQ ID NO:57) GAPLWCEKLSGTGSGGFKWPGCGSGPT- YNTFTPARVGSDNKWP, (SEQ
ID NO:57) GPPVWSAKSRWTGTGVLNWPGCGKVPSCSTYTPSRDRSRKSDP, (SEQ ID
NO:57) GSALLTSKGCVRGPGGLMRPGCGNDRLGKSSTYAHGGWIKTGP, (SEQ ID NO:57)
GSPVWSGDNRWRGSSPLKRPGCGNGAKCNTLKDNRKDSRKTKH, (SEQ ID NO:57) G
PLLPGEAAVHGARGLMRSGCGNGPTWNRLTA- ACRDSRNKGP, (SEQ ID NO:57)
GSPVWMGSTRWTGHGWFRSQGCGNVPRTNSCAPAGKDSQNKGP, (SEQ ID NO:57)
GAPVWRGNRWCSDNGELERPGCGYGPRFNILPPGRGNSRKPSP, (SEQ ID NO:57)
GSSGWKVKHRCGGPGTLQRPGCGNLPLGHTFPPTRGGSHMEGA, (SEQ ID NO:57)
GPRSWMGQPRGSDAGSCKWAGCGDAPMWRASTP- GHGGPPNRGS, (SEQ ID NO:57)
EALVCRGKPPWSGPAGLLWQGCGTGPVSRTFTSAQGRSRNKTS, (SEQ ID NO:57)
GAPVVGDILWCSGARGAKWPGCGKGPTNKTFSHSRGGTQKSGL, (SEQ ID NO:57)
GAPVSRCKPACGGFWGVNWPGCGNASMCKTFTNGHGVSSDNGH, (SEQ ID NO:57)
GAHGYKNGSTCTGLGGWRCRGCGKGAMCNNPSP- AGGAYHNQGP. (SEQ ID NO:57) G
PQGSEHQCCSGHWGLKFPGCGNGPICNNFTALRGASRKNGP, (SEQ ID NO:57)
GEPVWCRHSGGRVQGGLDWLGCGDGPLRYTVTPARGGPSKHGP, (SEQ ID NO:57)
GLSLVRGDSWGSGAGGWKRHGCGHGPMYNPQTPARGGSCTRNT, (SEQ ID NO:57)
VSRAWSGKPRLMGSHGLNCPGCGKGHSGIMFIP- DPAGSANTPP, (SEQ ID NO:57)
CAPMWSGKPPWCVGGGVKFRGCGNRPDCNIITPRLVESRDKAL, and (SEQ ID NO:57)
ADPVCSRKPDGGGLRGLRWPGCGKGPILYNVTATTGGSRNN- GP.
40. The molecule which binds to a ligand of interest of claim 33 in
which said ligand comprises VTSAPDTRPAPGSTAPPAHGVTSAPDTR (SEQ ID
NO: 9) or a portion thereof.
41. A therapeutic or diagnostic composition comprising a molecule
chosen from the group of molecules of claim 38 and a
pharmaceutically acceptable carrier.
42. A therapeutic or diagnostic composition comprising a molecule
chosen from the group of molecules of claim 39 and a
pharmaceutically acceptable carrier.
43. A molecule that binds to polymorphic epithelial mucin,
comprising an amino acid sequence represented by the formula:
13
R.sub.1R.sub.2R.sub.3R.sub.4R.sub.5R.sub.6R.sub.7R.sub.8R.sub.9R-
.sub.9R.sub.10R.sub.11R.sub.12R.sub.13R.sub.14R.sub.15R.sub.16R.sub.17R.su-
b.18R.sub.19R.sub.20R.sub.21R.sub.22R.sub.23R.sub.24R.sub.25R.sub.26
(SEQ ID NO: 88) R.sub.27R.sub.28R.sub.29R.sub.30R.sub.31R.sub-
.32R.sub.33R.sub.34R.sub.35R.sub.36R.sub.37R.sub.38R.sub.39R.sub.40R.sub.4-
1R.sub.42R.sub.43
wherein: R.sub.1=G, C, E, or V; R.sub.2=A, S, P, or L; R.sub.3=P,
T, H, or L; R.sub.4=L, M, Q, G, A, or S; R.sub.5=W or Y; R.sub.6=S,
C, K or T; R.sub.7=E, S, C, D, V, or R; R.sub.8=N, H, K, S, or E;
R.sub.9=L, H, R, N, Q, T, or G; R.sub.10=W, P, R, T, or D;
R.sub.11=W, C, V, L, or G; R.sub.12=S, T, M, or H; R.sub.13=G;
R.sub.14=S, A, G, N, Q, or H; R.sub.15=W, H, G, A, or R;
R.sub.16=G, T, E, P, V, or W; R.sub.17=V, F, W, K, or A;
R.sub.18=K, Q, D, E, R, or L; R.sub.19=R, F, or S; R.sub.20=P, S,
or H; R.sub.23=G; R.sub.22=C; R.sub.23=G; R.sub.24=D, S, T, N;
R.sub.25=G, D, L; R.sub.26=P or S; R.sub.27=M, S, D, I, L, or R;
R.sub.28=G, W, C, L, F, Y, or T; R.sub.29=S, N, V, F, H, or R;
R.sub.30=N, A, S, M, or R; R.sub.31=F, Q, P, or V; R.sub.32=S, V,
I, K, A, or S; R.sub.33=P, A, N, or Y; R.sub.34=G, N, or L;
R.sub.35=K, R, C, Q or L; R.sub.36=V, K, R, or A; R.sub.37=G, D, A,
or E; R.sub.38=S, T, P, Y or W; R.sub.39=R, I, L, P, A or S;
R.sub.40=N, K, or M; R.sub.41=S, R, T, E, Q, P, Y or H; R.sub.42=G,
A, S, D, N, P, Y, or K; R.sub.43=P, H or A.
44. The molecule of claim 43 wherein: R.sub.1=G; R.sub.2=A;
R.sub.3=P; R.sub.5=W; R.sub.6=S; R.sub.10=W; R.sub.11=w; R.sub.12=S
or T; R.sub.14=S; R.sub.16=G; R.sub.18=K; R.sub.19=R; R.sub.20=P;
R.sub.26=P; R.sub.28=G or W; R.sub.30=N; R.sub.31=F; R.sub.33=P;
R.sub.35=K or R; R.sub.3=G; R.sub.38=S; R.sub.40=N or K;
R.sub.42=G;
45. The molecule of claim 2 in which the antibody or
antigen-binding derivative thereof is capable of specifically
binding to a human tumor antigen.
Description
[0001] This application is a continuation-in-part of co-pending
U.S. patent application Ser. No. 08/310,192 filed Sep. 21, 1994,
the entire contents of which are incorporated herein by
reference.
1. FIELD OF THE INVENTION
[0002] The present invention relates generally to peptides capable
of specific binding to ligands of interest. The present invention
also relates to peptides capable of mimicking the specific binding
of a receptor to its ligand, an antibody to its antigen, and the
like. Such peptides are known as "abtides." Abtides are identified
by first and second screening steps of peptide libraries. The first
screening step uses an antibody or receptor as a first target
ligand and identifies peptide sequences ("mimetopes") which
specifically bind to the antibody or receptor. The mimetopes are
then incorporated into a second target ligand in a second screening
step to identify abtides that bind the mimetope. Abtides mimic the
binding specificity of the antibody (to its antigen) or the
receptor (to its ligand) that was used as the first target ligand
in the first screening step. The invention further relates to the
use of abtides in the place of antibodies in assays. The invention
also provides abtide compositions for use in therapy and diagnosis
of disease.
2. BACKGROUND OF THE INVENTION
2.1. Peptide Libraries
[0003] The use of peptide libraries is well known in the art. Such
peptide libraries have generally been constructed by one of two
approaches. According to one approach, peptides have been
chemically synthesized in vitro in several formats. For example,
Fodor et al., 1991, Science 251: 767-773, describes use of complex
instrumentation, photochemistry and computerized inventory control
to synthesize a known array of short peptides on an individual
microscopic slide. Houghten et al., 1991, Nature 354: 84-86,
describes mixtures of free hexapeptides in which the first and
second residues in each peptide were individually and specifically
defined. Lam et al., 1991, Nature 354: 82-84, describes a "one
bead, one peptide" approach in which a solid phase split synthesis
scheme produced a library of peptides in which each bead in the
collection had immobilized thereon a single, random sequence of
amino acid residues. For the most part, the chemical synthetic
systems have been directed to generation of arrays of short length
peptides, generally fewer than about 10 amino acids or so, more
particularly about 6-8 amino acids. Direct amino acid sequencing,
alone or in combination with complex record keeping of the peptide
synthesis schemes, is required to use these libraries.
[0004] According to a second approach using recombinant DNA
techniques, peptides have been expressed in biological systems as
either soluble fusion proteins or viral capsid fusion proteins.
[0005] A number of peptide libraries according to the second
approach have used the M13 phage. M13 is a filamentous
bacteriophage that has been a workhorse in molecular biology
laboratories for the past 20 years. M13 viral particles consist of
six different capsid proteins and one copy of the viral genome, as
a single-stranded circular DNA molecule. Once the M13 DNA has been
introduced into a host cell such as E. coli, it is converted into
double-stranded, circular DNA. The viral DNA carries a second
origin of replication that is used to generate the single-stranded
DNA found in the viral particles. During viral morphogenesis, there
is an ordered assembly of the single-stranded DNA and the viral
proteins, and the viral particles are extruded from cells in a
process much like secretion. The M13 virus is neither lysogenic nor
lytic like other bacteriophage (e.g., .lambda.); cells, once
infected, chronically release virus. This feature leads to high
titers of virus in infected cultures, i.e., 10.sup.12 pfu/ml.
[0006] The genome of the M13 phage is .about.8000 nucleotides in
length and has been completely sequenced. The viral capsid protein,
protein III (pIII) is responsible for infection of bacteria. In E.
coli, the pillin protein encoded by the F factor interacts with
pIII protein and is responsible for phage uptake. Hence, all E.
coli hosts for M13 virus are considered male because they carry the
F factor. Several investigators have determined from mutational
analysis that the 406 amino acid long pIII capsid protein has two
domains. The C-terminus anchors the protein to the viral coat,
while portions of the N-terminus of pIII are essential for
interaction with the E. coli pillin protein (Crissman and Smith,
1984, Virology 132: 445-455). Although the N-terminus of the pIII
protein has been shown to be necessary for viral infection, the
extreme N-terminus of the mature protein does tolerate alterations.
In 1985, George Smith published experiments reporting the use of
the pIII protein of bacteriophage M13 as an experimental system for
expressing a heterologous protein on the viral coat surface (Smith,
1985, Science 228: 1315-1317). It was later recognized,
independently by two groups, that the M13 phage pIII gene display
system could be a useful one for mapping antibody epitopes. De la
Cruz et al., 1988, J. Biol. Chem. 263: 4318-4322 cloned and
expressed segments of the cDNA encoding the Plasmodium falciparum
surface coat protein into the pIII gene, and recombinant phage were
tested for immunoreactivity with a polyclonal antibody. Parmley and
Smith, 1988, Gene 73: 305-318 cloned and expressed segments of the
E. coli .beta.-galactosidase gene in the pIII gene and identified
recombinants carrying the epitope of an anti-.beta.-galactosidase
monoclonal antibody. The latter authors also described a process
termed "biopanning", in which mixtures of recombinant phage were
incubated with biotinylated monoclonal antibodies, and
phage-antibody complexes could be specifically recovered with
streptavidin-coated plastic plates.
[0007] In 1989, Parmley and Smith, 1989, Adv. Exp. Med. Biol.
251:215-218 suggested that short, synthetic DNA segments cloned
into the pIII gene might represent a library of epitopes. These
authors reasoned that since linear epitopes were often .about.6
amino acids in length, it should be possible to use a random
recombinant DNA library to express all possible hexapeptides to
isolate epitopes that bind to antibodies.
[0008] Scott and Smith, 1990, Science 249:386-390 describe
construction and expression of an "epitope library" of hexapeptides
on the surface of M13. The library was made by inserting a 33 base
pair Bgl I digested oligonucleotide sequence into an Sfi I digested
phage fd-tet, i.e., fUSE5 RF. The 33 base pair fragment contains a
random or "degenerate" coding sequence (NNK).sub.6 where N
represents G, A, T or C and K represents G or T. The authors stated
that the library consisted of 2.times.10.sup.8 recombinants
expressing 4.times.10.sup.7 different hexapeptides; theoretically,
this library expressed 69% of the 6.4.times.10.sup.7 possible
peptides (20.sup.6). Cwirla et al., 1990, Proc. Natl. Acad. Sci.
USA 87: 6378-6382 also described a somewhat similar library of
hexapeptides expressed as pIII gene fusions of M13 fd phage. PCT
publication WO 91/19818 dated Dec. 26, 1991 by Dower and Cwirla
describes a similar library of pentameric to octameric random amino
acid sequences.
[0009] Devlin et al., 1990, Science, 249:404-406, describes a
peptide library of about 15 residues generated using an (NNS)
coding scheme for oligonucleotide synthesis in which S is G or
C.
[0010] Christian and colleagues have described a phage display
library, expressing decapeptides (Christian et al., 1992, J. Mol.
Biol. 227:711-718). The starting. DNA was generated by means of an
oligonucleotide comprising the degenerate codons [NN(G/T)].sub.10
with a self-complementary 3' terminus. This sequence, in forming a
hairpin; creates a self-priming replication site which could be
used by T4 DNA polymerase to generate the complementary strand. The
double-stranded DNA was cleaved at the Sfi I sites at the 5'
terminus and hairpin for cloning into the fUSE5 vector described by
Scott and Smith, supra.
[0011] Other investigators have used other viral capsid proteins
for expression of non-viral DNA on the surface of phage particles.
The protein pVIII is a major M13 viral capsid protein and interacts
with the single stranded DNA of M13 viral particles at its
C-terminus. It is 50 amino acids long and exists in approximately
2,700 copies per particle. The N-terminus of the protein is exposed
and will tolerate insertions, although large inserts have been
reported to disrupt the assembly of pVIII fusion proteins into
viral particles (Cesareni, 1992, FEBS Lett. 307:66-70). To minimize
the negative effect of pVIII fusion proteins, a phagemid system has
been utilized. Bacterial cells carrying the phagemid are infected
with helper phage and secrete viral particles that have a mixture
of both wild-type and pVIII fusion capsid molecules. pVIII has also
served as a site for expressing peptides on the surface of M13
viral particles. Four and six amino acid sequences corresponding to
different segments of the Plasmodium falciparum major surface
antigen have been cloned and expressed in the comparable gene of
the filamentous bacteriophage fd (Greenwood et al., 1991, J. Mol.
Biol. 220:821-827).
[0012] Lenstra, 1992, J. Immunol. Meth. 152:149-157 described
construction of a library by a laborious process encompassing
annealing oligonucleotides of about 17 or 23 degenerate bases with
an 8 nucleotide long palindromic sequence at their 3' ends. This
resulted in the expression of random hexa- or octa-peptides as
fusion proteins with the .beta.-galactosidase protein in a
bacterial expression vector. The DNA was then converted into a
double-stranded form with Klenow DNA polymerase, blunt-end ligated
into a vector, and then released as Hind III fragments. These
fragments were then cloned into an expression vector at the
C-terminus of a truncated .beta.-galactosidase to generate 10.sup.7
recombinants. Colonies were then lysed, blotted on nitrocellulose
filters (10.sup.4/filter) and screened for immunoreactivity with
several different monoclonal antibodies. A number of clones were
isolated by repeated rounds of screening and were sequenced.
[0013] Cull et al., 1992, Proc. Natl. Acad. Sci. USA 89:1865-1869
described a system in which random peptides were fused to the
carboxy terminus of the lac repressor. The fusion proteins
contained an intact lac amino terminus (which is responsible for
specific binding of the lac repressor to the DNA sequences
constituting the lac operator sites). The nucleotide sequences
encoding the fusion protein were cloned into a plasmid containing
copies of the lac operator site. Thus, when the fusion protein was
expressed in bacteria, it became bound to the operator sites of the
plasmid encoding it. This provided a physical linkage between the
fusion protein and the gene encoding it. When bacteria containing
the plasmid were screened with ligands for which it was desired to
isolate binding partners, the fusion proteins comprising peptides
that specifically bound to the ligand were isolated, carrying along
the genes that encoded those fusion proteins.
[0014] A comprehensive reView of various types of peptide libraries
can be found in GaTlop et al., 1994, J. Med. Chem.
37:1233-1251.
2.2. Ligands Used to Screen Peptide Libraries
[0015] Screening of peptide libraries has generally been confined
to the use of a restricted number of ligands. Most commonly, the
ligand has been an antibody (Parmley and Smith, 1989, Adv. Exp.
Med. Biol. 251:215-218; Scott and Smith, 1990, Science
249:386-390). In many cases, the aim of the screening is to
identify peptides from the library that mimic the epitopes to which
the antibodies are directed. Thus, given an available antibody,
peptide libraries are excellent sources for identifying epitopes or
epitope-like molecules of that antibody (Yayon et al., 1993, Proc.
Natl. Acad. Sci. USA. 90:10643-10647).
[0016] While previous studies have succeeded in identifying
epitopes and epitope-like molecules: from peptide libraries, it has
not been realized in the prior art that this approach could be
extended by using the identified epitopes in a further round of
screening of a peptide library to identify antibody-like
molecules.
[0017] When it has been desired to obtain antibody-like molecules,
the prior art has employed peptide libraries that contain naturally
occurring antibody sequences. This has probably been due to the
fact that specific binding by antibodies is known to depend upon a
complex structure involving various complementarity determining
regions (CDRs), often from both heavy and light antibody chains.
Short peptides would not be expected to mimic such structures and
longer peptides were thought to be unsuitable for display in the
most commonly used libraries.
[0018] McCafferty et al., 1.990, Nature 348:552-554 used PCR to
amplify immunoglobulin variable (V) region genes and cloned those
genes into phage expression vectors. The authors suggested that
phage libraries of V, diversity (D), and joining (J) regions could
be screened with antigen. The phage that bound to antigen could
then be mutated in the antigen-binding loops of the antibody genes
and rescreened. The process could be repeated several times,
ultimately giving rise to phage which bind the antigen
strongly.
[0019] Marks et al., 1991, J. Mol. Biol. 222:581-597 also used PCR
to amplify immunoglobulin variable (V) region genes and cloned
those genes into phage expression vectors.
[0020] Kang et al., 1991, Proc., Natl. Acad. Sci. USA 88:4363-4366
created a phagemid vector that could be used to express the V and
constant (C) regions of the heavy and light chains of an antibody
specific for an antigen. The heavy and light chain V-C regions were
engineered to combine in the periplasm to produce an antibody-like
molecule with a functional antigen binding site. Infection of cells
harboring this phagemid with helper phage resulted in the
incorporation of the antibody-like molecule on the surface of phage
that carried the phagemid DNA. This allowed for identification and
enrichment of these phage by screening with the antigen. It was
suggested that the enriched phage could be subject to mutation and
further rounds of screening, leading to the isolation of
antibody-like molecules that were capable of even stronger binding
to the antigen.
[0021] Hoogenboom et al., 1991, Nucleic Acids Res. 19:4133-4137
suggested that naive antibody genes might be cloned into phage
display libraries. This would be followed by random mutation of the
cloned antibody genes to generate high affinity variants.
[0022] In the prior art, peptide libraries have been screened with
receptors to identify receptor ligand-like peptides, but peptide
libraries have not been considered useful for identifying such
ligand-binding peptides as those that mimic receptors.
[0023] Bass et al., 1990, Proteins: Struct. Func. Genet. 8:309-314
fused human growth hormone (hGH) to the carboxy terminus of the
gene III protein of phage fd. This fusion protein was built into a
phagemid vector. When cells carrying the phagemid were infected
with a helper phage, about 10% of the phage particles produced
displayed the fusion protein on their surfaces. These phage
particles were enriched by screening with hGH receptor-coated
beads. It was suggested that this system could be used to develop
mutants of hGH with altered receptor binding characteristics.
[0024] Lowman et al., 1991, Biochemistry 30:10832-10838 used an
improved version of the system of Bass et al. described above to
select for mutant hGH proteins with exceptionally high affinity for
the hGH receptor. The authors randomly mutagenized the hGH-pIII
fusion proteins at sites near the vicinity of 12 amino acids of hGH
that had previously been identified as being important in receptor
binding.
[0025] Balass et al., 1993, Proc. Natl. Acad. Sci. USA
90:10638-10642 used a phage display library to isolate
linear-peptides that mimicked a conformationally dependent epitope
of the nicotinic acetylcholine receptor. This was done by-screening
the library with a monoclonal antibody specific for the
conformationally dependent epitope. The monoclonal antibody used
was thought to be specific to the acetylcholine receptor's binding
site for its natural ligand, acetylcholine.
[0026] Citation or identification of any reference herein shall not
be construed as an admission that such reference is available as
prior art to the present invention.
3. SUMMARY OF THE INVENTION
[0027] The present invention relates to abtides. As used herein,
the term "abtides" refers to peptides that mimic the binding
specificity of a larger molecule such as an antibody or receptor.
Abtides specifically bind to a ligand of interest, in which the
ligand is a specific binding partner of the larger molecule (e.g.
antibody or receptor). To identify the abtides of the present
invention, peptide libraries are screened in a two-step process.
The first screening step uses an antibody (or antigen-binding
derivative thereof) or receptor (or ligand-binding derivative
thereof) as a first target ligand. This step identifies peptide
sequences termed "epitopes" or "mimetopes" which specifically bind
the first target ligand. In the case where an antibody or
derivative thereof is used as the first target ligand, a mimetope
will often resemble, either functionally in terms of its binding
capability and/or structurally in terms of its amino acid sequence,
the epitope recognized by the antibody used as the first ligand. An
epitope or mimetope is then used as a second target ligand in a
second screening step to identify a peptide sequence that
specifically binds the epitope or mimetope. Such peptides are known
as "abtides." Surprisingly, it was found by the current inventors
and is demonstrated herein that abtides possess binding
specificities strikingly similar to those possessed by the first
target ligands (usually antibodies or receptors) described
above.
[0028] Abtides are useful since they mimic the binding
specificities of antibodies or receptors. Thus, they may be used in
many instances where antibodies or receptors may be used. The
present invention further relates to the use of abtides in the
place of antibodies in assays such as the many types of
immunoassays known in the art. Abtides may take the place of
antibodies in such assays as, for example, enzyme-linked
immunosorbent assays (ELISAs) or sandwich immunoassays. The
invention also provides abtide compositions for use in therapy and
diagnosis. In a specific example, abtides have been discovered and
demonstrated to be useful in place of antibodies in enzyme-linked
immunosorbent assays and in in vivo localization to prostate
carcinoma in a xenograft model.
[0029] The use of abtides has many potential advantages over the
use of antibodies or receptors: the smaller size of abtides allows
their easier production at lower cost, reduced immunogenicity, and
may facilitate their in vivo delivery if such is desired;
biological reactions and functions mediated by constant domains of
antibodies, and cross-linking of antibodies/receptors and resulting
biological effects can be avoided if desired.
4. FIGURE LEGENDS
[0030] The present invention may be understood more fully by
reference to the following detailed description of the invention,
examples of specific embodiments of the invention and the appended
figures in which:
[0031] FIG. 1 shows in schematic diagram a general method for
identifying an abtide by a two-step screening process. See Section
5.3 for a discussion of this method.
[0032] FIG. 2 shows the binding of biotinylated monoclonal antibody
7E11-C5 to immobilized mimetope peptide 7E11-9.5. See Section
6.1.2.2 for details.
[0033] FIG. 3 shows similarities in the amino acid sequences of the
CDR2L and CDR3L regions of monoclonal antibody 7E11-C5 and the
7E11-C5 abtides of Table 2. The number of amino acids in the
abtides that are similar to the CDRs is indicated in parentheses,
along with the percent homology. Dashes indicate gaps which have
been added to improve the homology. In the case of clones 13 and
16, the homology with CDR2L was greatest if the sequence of CDR2L
was reversed. The sequence shown for clone 14 is SEQ ID NO: 1; the
sequence shown for clone 17 is SEQ ID NO: 2; The sequence shown for
clone 15 is SEQ ID NO: 3; the sequence shown for clone 13 is SEQ ID
NO: 4; the sequence shown for clone 16 is SEQ ID NO: 5; the
sequence shown for CDR3L is SEQ ID NO: 6; the sequence shown for
CDR2L is SEQ ID NO: 7; the sequence shown for CDR2L(rev) is SEQ ID
NO: 8.
[0034] FIG. 4 shows binding of abtides to the 7E11-9.5 mimetope
peptide in a dot blot assay as described in Section 6.1.2.1.
Numbers along the left side of the figure refer to the 7E11-C5
abtide that was spotted in the indicated position. The number 351
refers to the monoclonal antibody 7E11-C5, used as a positive
control. The numbers along the top of the figure refer to the
various dilutions of the abtide or the monoclonal antibody that
were used.
[0035] FIG. 5 shows the binding of biotinylated mimetopes to
immobilized abtides. .quadrature. represents binding of mimetope
peptide Biotin-LYANPGMYSRLHSPA-NH.sub.2 to 7E11-C5 abtide clone 14;
.largecircle. represents binding of mimetope peptide
Biotin-LYANPGMYSRLHSPA-NH.sub.2 to 7E11-C5 abtide clone 17;
.diamond. represents binding of mimetope peptide
Biotin-GMYSRLH-NH.sub.2 to 7E11-C5 abtide clone 14; .DELTA.
represents binding of mimetope peptide Biotin-GMYSRLH-NH.sub.2 to
7E11-C5 abtide clone 17. See Section 6.1.2.2 for details.
[0036] FIG. 6 shows the capture of an antigen from a lysate of
LNCaP tumor cells by the monoclonal antibody 7E11-C5 and the
7E11-C5 abtide clone 14. See Section 6.1.3 for details.
[0037] FIG. 7 shows the biodistribution of abtide clone
14-DPTA-.sup.111In in SCID mice bearing human prostate carcinoma
LNCaP xenograft tumors 2 hours (, bar on the left for each pair of
bars) or 4 hours (, bar on the right for each pair of bars)
post-injection of 2 .mu.g of peptide, specific activity 32
.mu.Ci/.mu.g. See Section 6.1.4 for details.
[0038] FIG. 8 shows the biodistribution of abtide clone.
17-DPTA-.sup.111-In in four SCID mice bearing human prostate LNCaP
carcinoma xenograft tumors 2 hours (, leftmost bar for each group
of four bars, mouse 1; second bar from left for each group of four
bars, mouse 6) or five hours (, third bar from left for each group
of four bars, mouse 2; , rightmost bar for each group of four bars,
mouse 4) post-injection of 0.02 .mu.g of peptide, specific activity
2.4 .mu.Ci/ng. See Section 6.1.4 for details.
[0039] FIG. 9 shows the biodistribution of .sup.111-In labeled
control irrelevant peptide in SCID mice bearing human prostate
carcinoma LNCaP xenograft tumors 2 hours (, leftmost bar for each
group of five bars; , second bar from left for each group of five
bars) or 5 hours (, third bar from left for each group of five
bars; , fourth bar from left for each group of five bars; ,
rightmost bar for each group of five bars) post-injection of 1.5
.mu.g of peptide, specific activity 30 .mu.Ci/.mu.g. See Section
6.1.4 for details.
[0040] FIG. 10 schematically illustrates the construction of the
R26 TSAR library. The R26 expression library was constructed
essentially as described for the TSAR-9 library that is described
in PCT publication WO 94/18318, dated Aug. 18, 1994; except for the
modifications depicted in FIG. 10. The oligonucleotide assembly
process depicted in FIG. 10 results in expression of peptides with
the following amino acid sequence:
[0041] S(S/R)X.sub.12.pi.A.delta.X.sub.12SR (SEQ ID NO: 89), where
.pi.=S, P, T or A; and .delta.=V, A, D, E OR G.
[0042] FIG. 11 schematically illustrates the construction of the
D38 TSAR library. The D38 expression library was constructed
essentially as described for the TSAR-9 library that is described
in PCT publication WO 94/18318, dated Aug. 18, 1994, except for the
modifications depicted in FIG. 11.
[0043] FIG. 12 schematically illustrates the construction of the
DC43 TSAR library. The DC43 expression library was constructed
essentially as described for the TSAR-9 library that is described
in PCT publication WO 94/18318, dated Aug. 18, 1994, except for the
modifications depicted in FIG. 12.
[0044] FIG. 13 schematically illustrates the oligonucleotides used
to construct the polymorphic epithelial mucin (PEM) abtide
saturation mutagenesis TSAR library (See Section 6.2.2).
5. DETAILED DESCRIPTION OF THE INVENTION
[0045] The present invention relates generally to abtides. As used
herein, the term "abtides" refers to peptides that mimic the
binding specificity of a larger molecule such as an antibody or
receptor. Abtides specifically bind to a ligand of interest, in
which the ligand is a specific binding partner of the mimicked
larger molecule (e.g. antibody or receptor) To identify the abtides
of the present invention, peptide libraries are typically screened
in a two-step process (see FIG. 1). The first screening step uses
an antibody (or antigen-binding derivative thereof) or receptor (or
ligand-binding derivative thereof) as a first target ligand. This
step identifies peptide sequences termed "epitopes" or "mimetopes"
which specifically bind the first target ligand. If the first
screening step uses an antibody and the peptide identified contains
the amino acid sequence of the natural antigen that is responsible
for the specific binding of the antigen to the antibody, then the
identified peptide is said to be an epitope; if the identified
peptide does not contain the sequence of the natural antigen, then
the identified peptide is said to be a mimetope. In the case where
an antibody or derivative thereof is used as the first target
ligand, a mimetope will often resemble, either functionally in
terms of its binding capability and/or structurally in terms of its
amino acid sequence, the epitope recognized by the antibody used as
the first ligand.
[0046] A mimetope is then used as a second target ligand in a
second screening step to identify a peptide sequence that
specifically binds the epitope or mimetope. Such peptides are known
as "abtides." Abtides possess binding specificities similar to
those possessed by the first target ligands (usually antibodies or
receptors) described above.
[0047] In a specific embodiment, the antibody or derivative thereof
used in the first screening step recognizes a tumor antigen,
preferably a human tumor antigen, most preferably of a malignant
tumor.
[0048] The present invention provides a method to successfully
screen against very small peptide or protein targets, e.g. 5 to 40
amino acids, preferably 10 to 20 amino acids. To date, screening
against such targets has not been successful. The methods of the
present invention increase the likelihood that the abtide obtained
will bind its target in a complex or structurally dependent
fashion.
[0049] Abtides are useful since they mimic the binding
specificities of antibodies or receptors. Thus, they may be used in
many instances where antibodies or receptors may be used. The
present invention further relates to the use of abtides in the
place of antibodies in assays such as the many types of
immunoassays known in the art. Abtides may take the lace of
antibodies in such assays as, for example, enzyme-linked
immunosorbent assays (ELISAs) or sandwich immunoassays. The
invention also provides abtide compositions for use in therapy and
diagnosis of disease. In a specific example, abtides have been
produced and demonstrated to be useful in place of antibodies in
enzyme-linked immunosorbent assays and in in vivo localization to
prostate carcinoma in xenograft models.
[0050] The use of abtides has many potential advantages over the
use of antibodies or receptors: the smaller size of abtides allows
their easier production at lower cost, reduced immunogenicity, and
facilitates their in vivo delivery if such is desired; biological
reactions and functions mediated by constant domains of antibodies,
and cross-linking of antibodies/receptors and resulting biological
effects can be avoided if desired.
5.1. Peptide Libraries for Use in Identifying Abtides
[0051] The abtides of the present invention can be identified from
a chemically synthesized peptide library or a biologically
expressed peptide library. If a biological peptide expression
library is used, the nucleic acid which encodes the peptide which
binds to the ligand of choice can be recovered, and then sequenced
to determine its nucleotide sequence and hence deduce the amino
acid sequence that mediates binding. Alternatively, the amino acid
sequence of an appropriate binding domain can be determined by
direct determination of the amino acid sequence of a peptide
selected from a peptide library containing chemically synthesized
peptides. In a less preferred aspect, direct amino acid sequencing
of a binding peptide selected from a biological peptide expression
library can also be performed.
[0052] In a preferred embodiment of the present invention, the
abtides are advantageously identified from random peptide
libraries. Typically, random peptide libraries will be encoded by
synthetic oligonucleotides with a plurality of variant nucleotide
positions having the potential to encode all 20 naturally occurring
amino acids. The sequence of amino acids encoded by the variant
nucleotides is unpredictable and substantially random. The terms
"unpredicted", "unpredictable" and "substantially random" are used
interchangeably with respect to the amino acids encoded and are
intended to mean that the variant nucleotides at any given position
are such that it cannot be predicted which of the 20 naturally
occurring amino acids will appear at that position. These variant
nucleotides are the product of random chemical synthesis. The
biological random peptide libraries envisioned for use include
those in which a bias has been introduced into the random sequence,
e.g., to disfavor stop codon usage.
5.1.1. Chemically Synthesized Peptide Libraries
[0053] The peptide libraries used in the present invention may be
libraries that are chemically synthesized in vitro. Examples of
such libraries are given in Fodor et al., 1991, Science
251:767-773, which describes the synthesis of a known array of
short peptides on an individual microscopic slide; Houghten et al.,
1991, Nature 354:84-86, which describes mixtures of free
hexapeptides in which the first and second residues in each peptide
were individually and specifically defined. Lam et al., 1991,
Nature 354:82-84, which describes a split synthesis scheme;
Medynski, 1994, Bio/Technology 12:709-710, describes split
synthesis and T-bag synthesis methods as well. See also Gallop et
al., 1994, J. Medicinal Chemistry 37:1233-1251.
[0054] PCT publication WO 91/05058, dated Apr. 18, 1991, is
directed to random libraries containing semi-random nucleotide
sequences. The semi-random nucleotide sequences are transcribed in
vitro under conditions such that polysomes are produced. The
polysomes are screened for binding to a substance of interest.
Those polysomes that bind to the substance of interest are
recovered. The RNA from those polysomes is used to construct cDNA,
which is expressed to produce polypeptides.
[0055] Screening to identify peptides which bind to a ligand of
choice can be carried out by methods well known in the art.
[0056] In a specific embodiment, the total number of unpredictable
amino acids in the peptides of the chemical library used for
screening is greater than or equal to 5 and less than or equal to
25; in other embodiments the total is in the range of 5-15 or 5-10
amino acids, preferably contiguous amino acids.
[0057] While a binding domain can be identified from chemically
synthesized peptide libraries, such a domain would be small (i.e.
less than 10 amino acids, and most probably. 5-6 amino acids, in
length). Therefore, use of a chemically synthesized peptide library
is less preferred for the second screening step involved in
isolating abtides than is use in the second screening step of
biological peptide libraries containing unpredictable sequences of
greater length, described below.
5.1.2. Biological Peptide Libraries
[0058] In another embodiment, biological peptide libraries are used
to identify abtides. Many suitable biological peptide libraries are
known in the art and can be used.
[0059] According to this second approach, involving recombinant DNA
techniques, peptides have been expressed in biological systems as
either soluble fusion proteins or viral capsid fusion proteins.
[0060] A number of peptide libraries according to this approach
have used the M13 phage. Although the N-terminus of the viral
capsid protein, protein III (pIII), has been shown to be necessary
for viral infection, the extreme N-terminus of the mature protein
does tolerate alterations such as insertions. Accordingly, various
peptide libraries, in which the diverse peptides are expressed as
pIII fusion proteins, are known in the art; these libraries can be
used to identify abtides. Examples of such libraries are described
below.
[0061] Scott and Smith, 1990, Science 249:386-390 describe
construction and expression of an "epitope library" of hexapeptides
on the surface of M13. The library was made by inserting a 33 base
pair Bgl .sup..about.I digested oligonucleotide sequence into an
Sfi I digested phage fd-tet, i.e., fUSES RF. The 33 base pair
fragment contains a random or "degenerate" coding sequence
(NNK).sub.6 where N represents G, A, T or C and K represents G or
T. The authors stated that the library consisted of
2.times.10.sup.8 recombinants expressing 4.times.10.sup.7 different
hekapeptides; theoretically, this library expressed 69% of the
6.4.times.10.sup.7 possible peptides (20.sup.6). Cwirla et al.,
1990, Proc. Natl. Acad. Sci. USA 87: 6378-6382 also described a
somewhat similar library of hexapeptides expressed as pIII gene
fusions of M13 fd phage. PCT publication WO 91/19818 dated Dec. 26,
1991 by Dower and Cwirla describes a similar library of pentameric
to octameric random amino acid sequences.
[0062] Devlin et al. 1990, Science, 249:404-406, describes a
peptide library of about 15 residues generated using an (NNS)
coding scheme for oligonucleotide synthesis in which S is G or
C.
[0063] Christian and colleagues have described a phage display
library, expressing decapeptides (Christian et al., 1992, J. Mol.
Biol. 227:711-718). The starting DNA was generated by means of an
oligonucleotide comprising the degenerate codons [NN(G/T)].sub.10
with a self-complementary 3' terminus. This sequence, in forming a
hairpin, creates a self-priming replication site which could be
used by T4 DNA polymerase to generate the complementary strand. The
double-stranded DNA was cleaved at the SfiI sites at the 5'
terminus and hairpin for cloning into the fUSE5 vector described by
Scott and Smith, supra.
[0064] Lenstra, 1992, J. Immunol. Meth. 152:149-157 describes
construction of a library by a laborious process encompassing
annealing oligonucleotides of about 17 or 23 degenerate bases with
an 8 nucleotide long palindromic sequence at their 3' ends. This
resulted in the expression of random hexa- or octa-peptides as
fusion proteins with the .beta.-galactosidase protein in a
bacterial expression vector. The DNA was then converted into a
double-stranded form with Klenow DNA polymerase, blunt-end ligated
into a vector, and then released as Hind III fragments. These
fragments were then cloned into an expression vector at the
sequence encoding the C-terminus of a truncated
.beta.-galactosidase to generate 10' recombinants.
[0065] Other biological peptide libraries which can be used include
those described in U.S. Pat. No. 5,270,170 dated Dec. 14, 1993 and
PCT Publication No. WO 91/19818 dated Dec. 26, 1991. Also suitable
are those in U.S. Pat. No. 5,096,815, U.S. Pat. No. 5,198,346, and
U.S. Pat. No. 5,223,409, all to Ladner et al.
[0066] The biological peptide libraries discussed above are meant
to be illustrative and not limiting. It will be recognized by one
of skill in the art that many other biological peptide libraries
disclosed in various publications may be suitable for use in the
practice of the present invention.
[0067] The protein pVIII is a major M13 viral capsid protein, which
can also serve as a site for expressing peptides on the surface of
M13 viral particles, in the construction of random peptide
libraries.
[0068] While it would be understood by one skilled in the art that
as few as 5 amino acids can constitute a binding domain, the
average functional domain within a natural protein is considered to
be about 40 amino acids. Thus, the random peptide libraries from
which the abtides of the present invention are preferably
identified encode peptides having in the range of 5 to 200 total
variant amino acids. Although it is contemplated that biologically
expressed random peptide libraries displaying short random inserts
(i.e. less than 20 amino acids in length) could be used to identify
abtides of the invention, the most preferred biologically expressed
random peptide libraries for use in the invention are those in
which the displayed peptide has 20 or greater unpredictable amino
acids i.e. preferably in the range of 20 to 100, and most
preferably 20 to 50 amino acids, as exemplified by the TSAR
libraries described in PCT publication WO 91/12328, dated Aug. 22,
1991, and PCT publication WO 94/18318, dated Aug. 18, 1994.
[0069] To identify abtides, particularly in the second screening
step, the invention preferably uses libraries of greater complexity
than are commonly employed in the art. The conventional teaching in
the random peptide library art is that the length of inserted
oligonucleotides should be kept short, encoding preferably fewer
than 15 and most preferably about 6-8 amino acids. However, not
only can libraries encoding more than about 20 amino acids be
constructed, but such libraries can be advantageously screened to
identify peptides having binding specificity for a variety of
ligands. Such libraries with longer length inserts are exemplified
by the TSAR libraries, described in PCT publication WO 91/12328,
dated Aug. 22, 1991, and PCT publication WO 94/18318, dated Aug.
18, 1994.
[0070] These. PCT publications disclose that the use of libraries
composed of longer length oligonucleotides has many advantages.
[0071] Libraries composed of longer length oligonucleotides afford
the ability to identify peptides in which a short sequence of amino
acids is common to or shared by a number of peptides binding a
given ligand, i.e., library members having shared binding motifs.
The use of longer length libraries also affords the ability to
identify peptides which do not have any shared sequences with other
peptides but which nevertheless have binding specificity for the
same ligand.
[0072] When screened by the method of the present invention,
libraries having large inserted oligonucleotide sequences provide
the opportunity to identify or map binding sites which encompass
not only a few contiguous amino acid residues, i.e., simple binding
sites, but also those which encompass discontinuous amino acids,
i.e., complex binding sites, and may afford the complex binding
characteristic of antibodies and receptor-like molecules.
[0073] Additionally, the large size of the inserted synthesized
oligonucleotides of certain libraries provides the opportunity for
the development of secondary and/or tertiary structure in the
potential bending peptides and in sequences flanking the actual
binding site in the binding domain. Secondary and tertiary
structure often significantly affect the ability of a sequence to
mediate binding, as well as the strength and specificity of any
binding which occurs. Such complex structural effects are not
possible when only small length oligonucleotides are used in
libraries. It may be that secondary and tertiary structures are
especially important in the identification of abtides since abtides
mimic the binding of large molecules such as antibodies. It is well
known that the antigen binding properties of antibodies depend in
most instances upon several different regions of the heavy chain
(complementarity determining regions) and upon regions contributed
by the light chain as well.
[0074] Therefore, it is contemplated that the most preferred
binding domains for identifying the abtides of the present
invention will be those from biologically expressed random peptide
libraries in which the displayed peptide is 20 or greater amino
acids in length. Examples of such random peptide libraries are the
TSAR libraries, described in in PCT publication WO 91/12328, dated
Aug. 22, 1991, and PCT publication WO 94/18318, dated Aug. 18,
1994.
[0075] In one embodiment, the library utilized in the present
invention is a linear, non-constrained library. As would be
understood by one in the art having considered the present
disclosure, in another specific embodiment, "constrained",
"structured" or "semi-rigid" random peptide libraries could also be
used in the present methods to identify abtides. Typically, these
libraries express peptides that are substantially random but
contain a small percentage of fixed residues within or flanking the
random sequences that have the result of conferring structure or
some degree of conformational rigidity to the peptide. In a
semirigid peptide library, the plurality of synthetic
oligonucleotides express peptides that are each able to adopt only
one or a small number of different conformations that are
constrained by the positioning of codons encoding certain structure
conferring amino acids in or flanking the synthesized variant or
unpredicted oligonucleotides. Unlike linear, unconstrained
libraries in which the plurality of proteins expressed potentially
adopt thousands of short-lived different conformations, in a
semirigid peptide library, the plurality of proteins expressed can
adopt only a single or a small number of conformations. Such
libraries are exemplified by the TSAR-13 and TSAR-14 libraries
described in PCT publication WO 94/18318, dated Aug. 18, 1994; by a
library of random 6 amino acid sequences, each flanked by invariant
cysteine residues (O'Neil et al., 1992, Proteins 14:509-515); and
by those libraries disclosed in PCT Publication No. WO 94/11496,
dated May 26, 1994 (Huse).
[0076] In a preferred embodiment, a biological peptide library that
is a random peptide "TSAR" library is screened to identify an
abtide. TSARs is an acronym for "Totally Synthetic Affinity
Reagents" as described in PCT publication WO 91/12328, dated Aug.
22, 1991, and PCT publication WO 94/18318, dated Aug. 18, 1994.
TSAR libraries, their construction and use, and specific examples
of TSAR libraries, are described in detail in those PCT
publications. Nucleic acids encoding TSARs or a TSAR portion which
mediates binding to the ligand used for screening can be used to
identify the abtides of the present invention.
[0077] A TSAR may be a heterofunctional fusion protein, said fusion
protein comprising (a) a binding domain encoded by an
oligonucleotide comprising unpredictable nucleotides in which the
unpredictable nucleotides are arranged in one or more contiguous
sequences, wherein the total number of unpredictable nucleotides is
greater than or equal to about 60 and less than or equal to about
600, and optionally, (b) an effector domain encoded by an
oligonucleotide sequence which is a protein or peptide that
enhances expression or detection of the binding domain.
[0078] Alternatively, a TSAR may be a heterofunctional fusion
protein as described above but in which the contiguous sequences
are flanked by invariant residues designed to encode amino acids
that confer a desired structure to the binding domain of the
expressed heterofunctional fusion protein.
[0079] In addition to TSAR libraries, other libraries for use in
the present invention may be those wherein the library is a library
of recombinant vectors that express a plurality of heterofunctional
fusion proteins, said fusion proteins comprising a binding domain
encoded by an oligonucleotide comprising unpredictable nucleotides
in which the unpredictable nucleotides are arranged in one or more
contiguous sequences, wherein the total number of unpredictable
nucleotides is greater than or equal to about 15 and less than or
equal to about 600.
5.2. Abtides
[0080] An abtide is typically a peptide that mimics, with respect
to binding specificity, and possibly other characteristics (e.g.,
binding affinity, sequence, etc.) a large molecule such as an
antibody or receptor. However, an abtide is generally much smaller
than an antibody or receptor. An abtide is generally a peptide of
about 5 to 200 amino acids. Preferably, an abtide is a peptide of
about 10 to 100 amino acids. Most preferably, an abtide is a
peptide of about 20 to 50 amino acids. In addition to the amino
acid sequences which are responsible for the abtide's specific
binding properties, an abtide may be linked to additional amino
acid sequences or additional non-amino acid sequences. Such
additional sequences may aid in the identification or isolation of
the abtide. Or, they may aid in the biodistribution, stability, or
diagnostic or therapeutic effectiveness of the abtide when the
abtide is used diagnostically or therapeutically.
[0081] The abtides may be linked to a variety of non-peptide
moieties. Such moieties might include toxins; drugs;
polysaccharides; nucleotides; oligonucleotides; labels such as
radioactive substances (e.g. .sup.111In, .sup.125I, .sup.131I,
.sup.99mTc, .sup.212B, .sup.90Y, .sup.186Rh); biotin; fluorescent
tags; imaging reagents (e.g. those described in U.S. Pat. No.
4,741,900 and U.S. Pat. No. 5,326,856); hydrocarbon linkers (e.g.,
an alkyl group or derivative thereof) conjugated to a moiety
providing for attachment to a solid substratum, or to a moiety
providing for easy separation (e.g., a hapten recognized by an
antibody bound to a magnetic bead), etc. Linkage of the peptide to
the non-peptide moiety may be by any of several well-known methods
in the art.
[0082] In addition, in an embodiment in which an abtide has a free
amino- or carboxy-terminus, such termini can be modified by known
methods, e.g., to provide greater resistance to degradation,
greater cell permeability, greater solubility, etc., e.g., by
acetylation, biotinylation, fatty acylation, etc. at the
amino-terminus; amidation at the carboxy-terminus; or the abtide
can be stabilized by inclusion of D amino acids, normatural amino
acids or glycosyl amino acids, etc.
[0083] The abtides of the invention are preferably made by commonly
known methods of chemical synthesis, e.g., as described by way of
example in Section 5.6 and its subsections.
[0084] Alternatively, abtides (or portions thereof) can be obtained
by recombinant expression of a nucleic acid encoding the desired
abtide in an appropriate host, by well-known methods.
[0085] Occasionally, it may happen that not all of the amino acids
in the identified peptide will be necessary for the binding
function of an abtide. Where it is desired to decrease the size of
the abtide, methods can be used to identify portions of the
determined synthetic amino acid or nucleotide sequences which
respectively mediate binding, or encode the sequences which mediate
binding, as described in Section 5.4 below.
5.3. Screening of Peptide Libraries to Identify Abtides
[0086] The process of identifying abtides from a peptide library
comprises two distinct screening steps. The first step is designed
to identify epitopes or ligands with binding specificity for the
larger molecule of interest, e.g. antigen mimics, receptor-ligand
mimics, and the like. In that first step, a peptide library is
screened with a ligand that possesses a specific, often complex,
binding site of interest. Those peptides in the library that are
specific binding partners of the ligand bind to the ligand and are
readily recoverable because of this specific binding. An example of
a ligand suitable for use in this first screening step would be an
antibody, inherently possessing an antigen binding site, or an
antigen-binding derivative thereof. Another example would be a
receptor e.g. the epidermal growth factor receptor or the platelet
derived growth factor receptor. These particular receptors possess
specific binding sites for epidermal growth factor and platelet
derived growth factor, respectively. Other receptors are known in
the art and are also potential ligands, for example, the estrogen
receptor, the various acetylcholine receptors, the human growth
hormone receptor, etc.
[0087] Molecules comprising those peptide sequences in the library
that are identified by the first screening step will be referred to
herein as epitopes or mimetopes. For example, in the case where the
first ligand is an antibody, the eptides in the library that are
identified as specific inding partners of the antibody would be
known as antigen epitopes or mimetopes.
[0088] The peptides that are isolated in the first screening step
are then preferably analyzed, as, for example, by DNA sequencing of
the binding domain of the phage that encode the peptides if the
library used was a phage library. The DNA sequence of the binding
domain encodes the-amino acid sequence of the epitope or mimetope
peptide. Due to the known relationship between DNA sequences and
their encoded amino acid sequences, obtaining the DNA sequence of
the epitope or mimetope allows the determination of the amino acid
sequence of the epitope or mimetope. Alternatively, if the library
used was a chemical library, direct amino acid sequencing of the
peptide epitope or mimetope can be carried out by well known
methods in the art.
[0089] In a specific embodiment, sequences of different peptide
mimetopes identified in the first screening step can be compared to
determine a consensus mimetope sequence.
[0090] Once the amino acid sequence of the epitope or mimetope is
known (or a portion thereof, which mediates binding), a molecule,
preferably a peptide, is produced comprising that amino acid
sequence which mediates binding. This peptide may be synthesized
chemically, or, alternatively, may be produced by methods involving
recombinant DNA. This peptide may contain only the amino acids of
the epitope or mimetope or, preferably, it may contain additional
amino acids or non-amino acid moieties to aid in identifying or
recovering the epitope or mimetope peptide and any new peptide
binders found in the subsequent screening step discussed below.
[0091] In the second screening step, the epitope or mimetope that
was identified in the first screening step is used as a ligand for
the second screening step. The second screening step identifies
peptides with binding specificity for the epitope or mimetope and
that surprisingly mimic the binding specificity of the antibody or
receptor that was used as ligand in the first screening step. In
other words, the second screening step yields peptides with
antibody or receptor-like binding activity for antigens or receptor
ligands that are known as abtides.
[0092] FIG. 1 is a schematic representation of an exemplary
two-step screening process used to identify abtides.
[0093] In a particular embodiment of the invention, it may be that
the epitope to which an antibody specifically binds is known. For
example, the monoclonal antibody SM-3 that specifically binds the
polymorphic epithelial mucin (PEM) found on human breast carcinoma
cells has been shown to be specific for the epitope defined by the
amino acid sequence VTSAPDTRPAPGSTAPPAHGVTSAPDTR (SEQ ID NO: 9)
(Burchell et al., 1989, Int. J. Cancer 44:691-696). In such cases,
the first screening step described above may be dispensed with. A
peptide comprising the sequence of the epitope for which the
antibody is specific can be synthesized and used in the second
screening step described above in order to identify abtides of the
antibody.
[0094] It may also be that the portion of a "receptor-ligand"
(i.e., a ligand which specifically binds to a receptor) to which a
receptor specifically binds is known. For example, it has been
shown that granulocyte/macrophage colony stimulating factor
(GM-CSF) binds to the GM-CSF receptor through amino acids 88-121
(HCPPTPETSCATQTITFESFKENLKDFLL- VIPFDC [SEQ ID NO: 22]) of GM-CSF.
It should be possible to synthesize a peptide Corresponding to the
portion of the receptor-ligand that has been shown to be
responsible for specific binding to the receptor and to use such a
peptide in the second screening step of the methods of the present
invention in order to identify an abtide of the receptor.
[0095] As used in the present invention, a ligand is a substance
for which it is desired to isolate a specific binding partner from
a peptide library. A ligand can function as a lock, i.e., a large
polypeptide or protein analogous to a lock into which a smaller
specific binding partner fits as a key; or a ligand can function as
a key which fits into and specifically binds a larger binding
partner or lock.
[0096] In this invention, an epitope or mimetope is typically a
peptide that acts as a key; it is identified by screening a peptide
library for peptides that fit into and bind the specific binding
site of a larger molecule which acts as a lock, e.g. antibody or
receptor. If the larger molecule is an antibody and the peptide
identified contains the portion of the amino acid sequence of the
natural antigen that is responsible for the specific binding of the
antigen to the antibody, then the identified peptide is said to be
an epitope; if the identified peptide does not contain the sequence
of the natural antigen, then the identified peptide is said to be a
mimetope.
[0097] In a specific embodiment, a mimetope is identified by
screening a peptide library with an antibody or antibody fragment.
Mimetopes thus identified functionally mimic the antigen to which
the antibody binds in that the mimetopes also specifically bind
with the antibody. In some cases, if the antigen is a protein or
polypeptide, the mimetopes may share amino acid sequence motifs
with the antigen. In another embodiment, a mimetope is identified
by screening a peptide library with a receptor or receptor
fragment. Mimetopes thus identified functionally mimic the natural
ligand of the receptor.
[0098] The peptide libraries that are used in the first and second
screening steps may be the same or different. In one embodiment, a
peptide library containing small random inserts (from about 6 to
about 15 amino acids) is used in the first screening step.
[0099] In the second screening step, it may be desirable to use a
larger peptide library. Such larger libraries preferably express
peptides of about 20 to 200 random amino acids. Examples of such
larger libraries are the TSAR libraries described in PCT
publication WO 91/12328, dated Aug. 22, 1991, and in PCT
Publication WO 94/18318, dated Aug. 18, 1994.
[0100] Biological or chemically synthesized peptide libraries can
be used in either the first or second screenings. The peptide
libraries used in the present invention may have a plurality of
residues that are random, i.e. residues for which the amino acid
occupying that residue cannot be predicted in advance. Such
libraries are said to be random peptide libraries.
[0101] A preferred method for identifying abtides comprises
screening a library of recombinant vectors that express a plurality
of heterofunctional fusion proteins, said fusion proteins
comprising (a) a binding domain encoded by an oligonucleotide
comprising unpredictable nucleotides in which the unpredictable
nucleotides are arranged in one or more contiguous sequences,
wherein the total number of unpredictable nucleotides is greater
than or equal to about 15 and less than or equal to about 600, and
optionally, (b) an effector domain encoded by an oligonucleotide
sequence which is a protein or peptide that enhances expression or
detection of the binding domain. Screening is done by contacting
the plurality of heterofunctional fusion proteins with a ligand
under conditions conducive to ligand binding and then isolating the
fusion proteins which bind to the ligand. The methods of the
invention further preferably comprise determining the nucleotide
sequence encoding the binding domain of the heterofunctional fusion
protein identified to determine the DNA sequence that encodes the
binding domain and simultaneously to deduce the amino acid sequence
of the mimetope used in the second screen. Nucleotide sequence
analysis can be carried out by any method known in the art,
including but not limited to the method of Maxam and Gilbert (1980,
Meth. Enzymol. 65:499-560), the Sanger dideoxy method (Sanger et
al., 1977, Proc. Natl, Acad. Sci. U.S.A. 74:5463), the use of T7
DNA polymerase (Tabor and Richardson, U.S. Pat. No. 4,795,699;
Sequenase.TM., U.S. Biochemical Corp.), or Taq polymerase, or use
of an automated DNA sequenator (e.g., Applied Biosystems, Foster
City, Calif.).
[0102] Alternatively, the libraries used to screen for mimetopes
and abtides of the invention have unpredictable nucleotides
arranged in one or more contiguous sequences that are flanked by
invariant residues designed to encode amino acids that confer a
desired structure to the binding domain of the expressed
heterofunctional fusion protein.
[0103] Once a suitable peptide library has been constructed (or
otherwise obtained), the library is screened to identify peptides
having binding affinity for a ligand of choice. Screening the
libraries can be accomplished by any of a variety of methods known
to those of skill in the art. See, e.g., the following references,
which disclose screening of peptide libraries: Parmley and Smith,
1989, Adv. Exp. Med. Biol. 251:215-218; Scott and Smith, 1990,
Science 249:386-390; Fowlkes et al., 1992; BioTechniques
13:422-427; Oldenburg et al., 1992, Proc. Natl. Acad. Sci. USA
89:5393-5397; Yu et al., 1994, Cell 76:933-945; Staudt et al.,
1988, Science 241:577-580; Bock et al., 1992, Nature 355:564-566;
Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA 89:6988-6992;
Ellington et al., 1992, Nature 355:850-852; U.S. Pat. No.
5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No. 5,198,346,
all to Ladner et al.; and Rebar and Pabo, 1993, Science
263:671-673. See also PCT publication WO 94/18318, dated Aug. 18,
1994.
[0104] If the libraries are expressed as fusion proteins with a
cell surface molecule, then screening is advantageously achieved by
contacting the vectors with an immobilized target ligand and
harvesting those vectors that bind to said ligand. Such useful
screening methods, designated "panning"-techniques are described in
Fowlkes et al., 1992, BioTechniques 13:422-427. In panning methods
useful to screen the libraries, the target ligand can be
immobilized on plates, beads, such as magnetic beads, sepharose,
etc., or on beads used in columns. In particular embodiments, the
immobilized target ligand can be "tagged", e.g., using such as
biotin, 2-fluorochrome, e.g. for FACS sorting.
[0105] In one embodiment, presented by way of example but not
limitation, screening a library of phage expressing random peptides
on phage and phagemid vectors can be achieved by using magnetic
beads as described in PCT publication WO 94/18318, dated Aug. 18,
1994.
[0106] Alternatively, as yet another non-limiting example,
screening a library of phage expressing random peptides can be
achieved by panning using microtiter plates. In a preferred method
for recovering the phage bound to the wells of the microtiter
plates, a pH change is used.
[0107] By way of another example, the libraries expressing random
peptides as a surface protein of either a vector or a host cell,
e.g., phage or bacterial cell, can be screened by passing a
solution of the library over a column of a ligand immobilized to a
solid matrix, such as sepharose, silica, etc., and recovering those
phage that bind to the column after extensive washing and
elution.
[0108] By way of yet another example, weak binding library members
can be isolated based on retarded chromatographic properties.
According to one mode of this embodiment for screening, fractions
are collected as they come off the column, saving the trailing
fractions (i.e., those members that are retarded in mobility
relative to the peak fraction are saved). These members are then
concentrated and passed over the column a second time, again saving
the retarded fractions. Through successive rounds of
chromatography, it is possible to isolate those that have some
affinity, albeit weak, to the immobilized ligand. These library
members are retarded in their mobility because of the millions of
possible ligand interactions as the member passes down the column.
In addition, this methodology selects those members that have
modest affinity to the target, and which also have a rapid
dissociation time.
[0109] If desired, the oligonucleotides encoding the binding domain
selected in this manner can be mutagenized, expressed and
rechromatographed (or screened by another method) to discover
improved binding activity. In particular, saturation mutagenesis
can be carried out using synthetic oligonucleotides synthesized
from "doped" nucleotide reservoirs. The doping is carried out such
that the original peptide sequence is represented only once in 106
unique clones of the mutagenized oligonucleotide. The assembled
oligonucleotides are cloned into a parental TSAR vector.
Preferably, the vector is m663 (Fowlkes et al., 1992, BioTechniques
13:422-427). m663 is able to make blue plaques when grown in E.
coli stain JM101 or DH5.alpha.F'. A library of greater than
10.sup.6 is preferred; however a library of 10.sup.5 is sufficient
to isolate TSAR phage displaying peptide domains with increased
selectivity for binding to the target ligand.
[0110] According to another alternative method, screening a library
of can be achieved using a method comprising an "enrichment" step
and a filter lift step as follows.
[0111] Random peptides from an expressed library capable of binding
to a given ligand ("positives") are initially enriched by one or
two cycles of panning or affinity chromatography, as described
above. The goal is to enrich the positives to a frequency of about
>1/10.sup.5. Following enrichment, a filter lift assay is
conducted. For example, approximately 1-2.times.10.sup.5 phage,
enriched for binders, are added to 500 .mu.l of log phase E. coli
and plated on a large LB-agarose plate with 0.7w agarose in broth.
The agarose is allowed to solidify, and a nitrocellulose filter
(e.g., 0.45.mu.) is placed on the agarose surface. A series of
registration marks is made with a sterile needle to allow
re-alignment of the filter and plate following development as
described below. Phage plaques are allowed to develop by overnight
incubation at 37.degree. C. (the presence of the filter does not
inhibit this process). The filter is then removed from the plate
with phage from each individual plaque adhered in situ. The filter
is then exposed to a solution of BSA or other blocking agent for
1-2 hours to prevent non-specific binding of the ligand (or
"probe").
[0112] The probe itself is labeled, for example, either by
biotinylation (using commercial NHS-biotin) or direct enzyme
labeling, e.g., with horse radish peroxidase (HRP) or alkaline
phosphatase. Probes labeled in this manner are indefinitely stable
and can be re-used several times. The blocked filter is exposed to
a solution of probe for several hours to allow the probe to bind in
situ to any phage on the filter displaying a peptide with
significant affinity to the probe. The filter is then washed to
remove unbound probe, and then developed by exposure to enzyme
substrate solution (in the case of directly labeled probe) or
further exposed to a solution of enzyme-labeled avidin (in the case
of biotinylated probe). In a preferred method, an HRP-labeled probe
is detected by ECL western blotting methods (Amersham, Arlington
Heights, Ill.), which involves using luminol in the presence of
phenol to yield enhanced chemiluminescence detectable by brief
exposure of film by autoradiographyl in which the exposed areas of
film correspond to positive plaques on the original plate. Where an
enzyme substrate is used, positive phage plaques are identified by
localized deposition of colored enzymatic cleavage product on the
filter which corresponds to plaques on the original plate. The
developed filter or film, as the case may be, is simply realigned
with the plate using the registration marks, and the "positive"
plaques are cored from the agarose to recover the phage. Because of
the high density of plaques on the original plate, it is usually
impossible to isolate a single plaque from the plate on the first
pass. Accordingly, phage recovered from the initial core are
re-plated at low density and the process is repeated to allow
isolation of individual plaques and hence single clones of
phage.
[0113] Successful screening experiments are optimally conducted
using 3 rounds of serial screening. The recovered cells are then
plated at a low density to yield isolated colonies for individual
analysis. The individual colonies are selected and used to
inoculate LB culture medium containing ampicillin. After overnight
culture at 37.degree. C., the cultures are then spun down by
centrifugation. Individual cell aliquots are then retested for
binding to the target ligand attached to the beads. Binding to
other beads, having attached thereto a non-relevant ligand, can be
used as a negative control.
[0114] One important aspect of screening the libraries is that of
elution. For clarity of explanation, the following is discussed in
terms of TSAR expression by phage; however, it is readily
understood that such discussion is applicable to any system where
the random peptide is expressed on a surface fusion molecule. It is
conceivable that the conditions that disrupt the peptide-target
interactions during recovery of the phage are specific for every
given peptide sequence from a plurality of proteins expressed on
phage. For example, certain interactions may be disrupted by acid
pH's but not by basic pH's, and vice versa. Thus, it may be
desirable to test a variety of elution conditions (including but
not limited to pH 2-3, pH 12-13, excess target in competition,
detergents, mild protein denaturants, urea, varying temperature,
light, presence or absence of metal ions, chelators, etc.) and
compare the primary structures of the TSAR proteins expressed on
the phage recovered for each set of conditions to determine the
appropriate elution conditions for each ligand/TSAR combination.
Some of these elution conditions may be incompatible with phage
infection because they are bactericidal and will need to be removed
by dialysis (i.e., dialysis bag, Centricon/Amicon
microconcentrators).
[0115] The ability of different expressed proteins to be eluted
under different condition's may not only be due to the denaturation
of the specific peptide region involved in binding to the target
but also may be due to conformational changes in the flanking
regions. These flanking sequences may also be denatured in
combination with the actual binding sequence; these flanking
regions may also change their secondary or tertiary structure in
response to exposure to the elution conditions (i.e., pH 2-3, pH
12-13, excess target in competition, detergents, mild protein
denaturants, urea, heat, cold, light, metal ions, chelators, etc.)
which in turn leads to the conformational deformation of the
peptide responsible for binding to the target.
[0116] According to another alternative method in which the TSARs
contain a linker region between the binding domain and the effector
domain, particular TSAR libraries can be prepared and screened by:
(1) engineering a vector, preferably a phage vector, so that a DNA
sequence encodes a segment of Factor Xa (or Factor Xa protease
cleavable peptide) and is present adjacent to the gene encoding the
effector domain, e.g., the pIII coat protein gene; (2) construct
and assemble the double stranded synthetic oligonucleotides as
described above and insert into the engineered vector; (3) express
the plurality of vectors in a suitable host to form a library of
vectors; (4) screen for binding to an immobilized ligand; (5) wash
away excess phage; and (6) treat the immobilized phage with Factor
Xa protease. The particle will be uncoupled from the peptide-ligand
complex and can then be used to infect bacteria to regenerate the
particle with its full-length pIII molecule for additional rounds
of screening. This alternative embodiment advantageously allows the
use of universally effective elution conditions and thus allows
identification of phage expressing TSARs that otherwise might not
be recovered using other known methods for elution. To illustrate,
using this embodiment, exceptionally tight binding TSARs could be
recovered. If desired, the oligonucleotides encoding the binding
domain selected in this manner can be mutagenized, expressed and
rechromatographed (or screened by another method) to discover
improved binding activity. In particular, saturation mutagenesis
can be carried out using synthetic oligonucleotides synthesized
from "doped" nucleotide reservoirs. The doping is carried out such
that the original peptide sequence is represented only once in
10.sup.6 unique clones of the mutagenized oligonucleotide. The
assembled oligonucleotides are cloned into a parental TSAR vector.
Preferably, the vector is m663 (Fowlkes et al., 1992, BioTechniques
13:422-427). m663 is able to make blue plaques when grown in E.
coli stain JM101 or DH5.alpha.F'. A library of greater than
10.sup.6 is preferred; however a library of 10.sup.5 is sufficient
to isolate TSAR phage displaying peptide domains with increased
selectivity for binding to the target ligand.
[0117] For the examples in Section 6 herein, a TSAR library is
utilized; however, to those skilled in the art, it will be apparent
that other peptide libraries may be used. An example of a TSAR
library is the TSAR-9 library disclosed in Kay et al., 1993, Gene
128:59-65. TSAR-9 constructs display a peptide of about 38 amino
acids in length having 36 totally random positions.
5.3.1. Antibodies and Derivatives Thereof for Use in Screening
[0118] Antibodies can be produced which recognize an antigen of
interest. Such antibodies can be polyclonal or monoclonal. Such
antibodies may be used as ligands in the first screening step of
the present invention.
[0119] Various procedures known in the art may be used for the
production of polyclonal antibodies to an antigen of interest. For
the production of antibody, various host animals can be immunized
by injection with an antigen of interest or derivative thereof,
including but not limited to rabbits, mice, rats, etc. Various
adjuvants may be used to increase the immunological response,
depending on the host species, and including but not limited to
Freund's (complete and incomplete), mineral gels such as aluminum
hydroxide, surface active substances such as lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet,
hemocyanins, dinitrophenol, and potentially useful human adjuvants
such as BCG (bacille Calmette-Guerin) and corynebacterium
parvum.
[0120] A monocloinal antibody to an antigen of interest can be
prepared by using any technique which provides for the production
of antibody molecules by continuous cell lines in culture. These
include but are not limited to the hybridoma technique originally
described by Kohler and Milstein (1975, Nature 256:495-497), and
the more recent human B cell hybridoma technique (Kozbor et al.,
1983, Immunology Today 4:72) and EBV-hybridoma technique (Cole et
al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,
Inc., pp. 77-96).
[0121] The monoclonal antibodies may be human monoclonal antibodies
or chimeric human-mouse (or other species) monoclonal antibodies.
Human monoclonal antibodies may be made by any of numerous
techniques known in the art (e.g., Teng et al., 1983, Proc. Natl.
Acad. Sci. U.S.A. 80:7308-7312; Kozbor et al., 1983, Immunology
Today 4:72-79; Olsson et al., 1982, Meth. Enzymol. 92:3-16).
Chimeric antibody molecules may be prepared containing a mouse
antigen-binding domain with human constant regions (Morrison et
al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851, Takeda et al.,
1985, Nature 314:452).
[0122] A molecular clone of an antibody to an antigen of interest
can be prepared by known techniques. Recombinant DNA methodology
(see e.g., Maniatis et al., 1982, Molecular Cloning, A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.)
may be used to construct nucleic acid sequences which encode a
monoclonal antibody molecule, or antigen binding region
thereof.
[0123] Antibody molecules may be purified by known techniques,
e.g., immunoabsorption or: immunoaffinity chromatography,
chromatographic methods such as HPLC (high performance liquid
chromatography), or a combination thereof, etc.
[0124] Antibody fragments which contain the idiotype or antigen
binding region of the molecule can be generated by known
techniques. For example, such fragments include but are not limited
to: the F(ab').sub.2 fragment which can be produced by pepsin
digestion of the antibody molecule; the Fab' fragments which can be
generated by reducing the disulfide bridges of the F(ab').sub.2
fragment, and the 2 Fab or Fab fragments which can be generated by
treating the antibody molecule with papain and a reducing
agent.
5.4. Identification of Synthetic Sequences Which Mediate
Binding
[0125] When a peptide from a peptide library has been identified as
an abtide or mimetope for a particular target ligand of interest
according to the method of the invention (in either the first or
second screening step), it may be useful to determine what
region(s) of the expressed peptide sequence is (are) responsible
for binding to the target ligand. Such analysis can be conducted at
two different levels, i.e., the nucleotide sequence and amino acid
sequence levels.
[0126] By molecular biological techniques it is possible to verify
and further analyze a ligand binding peptide at the level of the
oligonucleotides. First, the inserted oligonucleotides can be
cleaved using appropriate restriction enzymes and religated into
the original expression vector and the expression product of such
vector screened for ligand binding to identify the oligonucleotides
that encode the binding region of the abtide or mimetope. Second,
the oligonucleotides can be transferred into another vector, e.g.,
from phage to phagemid. The newly expressed fusion proteins should
acquire the same binding activity if the domain is necessary and
sufficient for binding to the ligand. This last approach also
assesses whether or not flanking amino acid residues encoded by the
original vector influence peptide binding in any fashion. Third,
the oligonucleotides can be synthesized, based on the nucleotide
sequence determined for the phage in the library that encodes the
binding peptide, amplified by cloning or PCR amplification using
internal and flanking primers, cleaved into two pieces and cloned
as two half-binding domain fragments. In the foregoing manner, the
inserted oligonucleotides are subdivided into two equal halves. If
the peptide domain important for binding is small, then one
recombinant clone would demonstrate binding and the other would
not. If neither have binding, then either both are important or the
essential portion of the domain spans the middle (which can be
tested by expressing just the central region).
[0127] Alternatively, by synthesizing peptides corresponding to the
deduced sequence of the abtide or mimetope, the binding domains can
be analyzed. First, the entire peptide should be synthesized and
assessed for binding to the target ligand to verify that the
peptide is necessary and sufficient for binding. Second, short
peptide fragments, for example, overlapping 10-mers, can be
synthesized, based on the amino acid sequence of the random peptide
binding domain, and tested to identify those binding the
ligand.
[0128] In addition, in certain instances, linear motifs (consensus
motifs) may become apparent after comparing the primary structures
of different binding peptides from the library having binding
affinity for a target ligand. The contribution of these motifs to
binding can be verified with synthesized peptides in competition
experiments (i.e., determine the concentration of peptide capable
of inhibiting 50% of the binding of the phage to its target;
IC.sub.50). Conversely, the motif or any region suspected to be
important for binding can be removed from or mutated within the DNA
encoding the random peptide insert and the altered displayed
peptide can be retested for binding.
[0129] Furthermore, once the binding domain of a peptide has been
identified, the binding characteristics of that peptide can be
modified by varying the binding domain sequence to produce a
related family of peptides with differing properties for a specific
ligand.
[0130] Moreover, in a method of directed evolution, the identified
peptides can be improved by additional rounds of random
mutagenesis, selection, and amplification of the nucleotide
sequences encoding the binding domains. Mutagenesis can be
accomplished by creating and cloning a new set of oligonucleotides
that differ slightly from the parent sequence, e.g., by 1-10%.
Selection and amplification are achieved as described above. By way
of example, to verify that the isolated peptides have improved
binding characteristics, mutants and the parent phage, differing in
their lacZ expression, can be processed together during the
screening experiments. Alteration of the original blue-white color
ratios during the course of the screening experiment ill serve as a
visual means to assess the successful selection of enhanced
binders. This process can go through numerous cycles.
5.5. Uses of Abtides
5.5.1. Assays Using Abtides
[0131] The abtides of the present invention possess binding
specificities that are similar to those of the ligands (e.g.
antibodies, receptors) that are used in the first screening step of
the process by which the abtides are identified. Consequently, the
abtides may be used in many of the same instances where the ligand
of the first screening step might be used. For example, if the
ligand used in the first screening step is an antibody, the abtide
that is identified after the second screening step will bind
specifically to the same antigen to which the antibody specifically
binds. Therefore, the abtide may be used as a substitute for the
antibody in many of the reactions or assays that the antibody could
be used in. For example, the abtide could be used in immunoassays
known in the art, e.g., those designed to detect or measure the
amount of the antigen. Of course, such immunoassays may have to be
suitably modified. For example, many immunoassays make use of a
step in which a second antibody, labeled with a radioactive moiety
or an enzyme such as alkaline phosphatase, specifically binds to
the first antibody. Such a second antibody would not be expected to
specifically bind to the abtide. However, it would be well within
the competence of one of ordinary skill in the art to fabricate
another labelling moiety, perhaps a third antibody, that was able
to specifically bind to the abtide, or to label the abtide with a
detectable marker prior to use.
[0132] The immunoassays which can be used include but are not
limited to competitive and non-competitive assay systems using
techniques such as western blots, radioimmunoassays, ELISA (enzyme
linked immunosorbent assays), "sandwich" immunoassays, dot
immunoblot assays, immunoprecipitation assays, precipitin
reactions, gel diffusion precipitin reactions, immunodiffusion
assays, agglutination assays, complement-fixation assays,
immunoradiometric assays, fluorescent immunoassays, protein A
immunoassays, immunoaffinity chromatography, and flow dipstick
assays to name but a few. For examples of exemplary procedures
which can be used in immunoassays, see generally Kricka, 1985,
Clinical and Biochemical Analysis 17:1-15; Armbruster, 1993, Clin.
Chem. 39/2:181-195; Birnbaum et al., 1992, Anal. Biochem.
206:168-171; Miyai, 1985, Adv. Clin. Chem. 24:61-110; and
references cited therein.
[0133] The samples to be assayed in the immunoassays can be any
sample that may contain the antigen or ligand desired to be
assayed. For example, these samples can be body fluids such as
plasma, blood, serum, saliva, cerebrospinal fluid, synovial fluid,
etc.
[0134] The detectable label to be used in the immunoassays can be
any detectable label known in the art. Such labels include
radioisotopes, fluorescent dyes, enzymes (for example horseradish
peroxidase or alkaline phosphatase), chemiluminescent molecules,
metal atoms, or phosphorescent dyes, colored particles, metal and
dye colloids.
5.5.1.1. Sandwich ELISA
[0135] In a particular embodiment, the abtides can be used in a
sandwich enzyme immunoassay. One description of such an embodiment,
presented by way of example and not limitation, follows: A molecule
comprising an abtide is affixed to a solid substratum. The molecule
comprising the abtide may be linked to a substance that will
provide for greater attachment of the molecule to the substratum.
The sample to be assayed is contacted with the molecule comprising
the abtide that is bound to the substratum. The substances in the
sample that are specific binding partners of the abtide (analyte)
will bind to the abtide, and non-binding sample components are
removed by washing. An enzyme-conjugated monoclonal antibody
directed against the analyte is added. This enzyme-conjugated
monoclonal antibody binds to the part of the analyte it is specific
for and completes the sandwich. After removal of unbound
enzyme-conjugated monoclonal antibody by washing, a substrate
solution is added to the wells. A colored product is formed in
proportion to the amount of analyte present in the sample. The
reaction may be terminated by addition of stop solution and
absorbance is measured spectrophotometrically. The following
illustrates these steps in more detail.
[0136] (a) Polystyrene microtiter wells (Flow Laboratory) are
coated overnight at room temperature with 100 .mu.l of a solution
of a molecule comprising an abtide at a concentration of 1 mg/ml in
phosphate buffered saline (PBS).
[0137] (b) Coating solution is discarded and wells are blocked for
1-2 hours at room temperature with 300 .mu.l of 1% bovine serum
albumin (BSA) in phosphate-buffered saline (PBS) with 0.05% of
Tween 20 (PBS-Tween buffer).
[0138] (c) 150 .mu.l of sample (suspected of containing an analyte
the presence or amount of which it is desired to measure) diluted
in 1% BSA-PBS is added per well. Wells are incubated 1 hour at room
temperature.
[0139] (d) Wells are washed 4 times with PBS-Tween buffer.
[0140] (e) 100 .mu.l of horseradish peroxidase conjugated
monoclonal antibody specific for the analyte in 1% BSA-PBS is added
per well. The concentration of the monoclonal antibody can be from
about 10 ng/ml to 10 mg/ml. Wells are incubated 1 hour at room
temperature.
[0141] (f) Wells are washed 6 times with PBS-Tween buffer.
[0142] (g) 100 .mu.l of ABTS.RTM. Boehringer Mannheim
(2,2'-Azino-di-[3-ethylbenzthiazdine sulfonate (6)] crystallized
diammonium salt working solution is added per well. ABTS.RTM. stock
solution is prepared at 15 mg/ml in dH.sub.2O. To make the working
solution, 200 .mu.l of this ABTS.RTM. stock is diluted into 10 ml
of citrate phosphate buffer (17 mm citric acid, 65 mm dibasic
sodium phosphate) and 10 .mu.l 30% H.sub.2O.sub.2.
[0143] (h) The absorbance of each well is measured at 405 nm in a
microtiter plate reader (Dynatech MR600, Dynatech Corp.,
Alexandria, Va.).
5.5.2. Pharmaeceutical Compositions
[0144] The invention provides methods of treatment by
administration to a subject of an effective amount of a
pharmaceutical (therapeutic or diagnostic) composition comprising
an abtide. Such an abtide envisioned for therapeutic or diagnostic
use is referred to hereinafter as a "Therapeutic" or "Therapeutic
of the invention." Such therapeutics are abtides that specifically
bind to a molecule in vivo, to exert a therapeutic or diagnostic
effect. In a preferred aspect, the Therapeutic is substantially
purified. The subject is preferably an animal, including but not
limited to animals such as cows, pigs, horses, chickens, cats,
dogs, etc., and is preferably a mammal, and most preferably
human.
[0145] Formulations and methods of administration that can be
employed are known in the art and can be selected from among those
described hereinbelow.
[0146] Various delivery systems are known and can be used to
administer a Therapeutic of the invention, e.g., encapsulation in
liposomes, microparticles, microcapsules, recombinant cells
containing the Therapeutic, receptor-mediated endocytosis (see,
e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), etc. Methods
of introduction include but are not limited to intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, epidural, and oral routes as well as transdermal and
subcutaneous time-release implants. The Therapeutics may be
administered by any convenient route, for example by infusion or
bolus injection, by absorption through epithelial or mucocutaneous
linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and
may be administered together with other biologically active agents.
Administration can be systemic or local. In addition, it may be
desirable to introduce the pharmaceutical compositions of the
invention into the central nervous system by any suitable route,
including intraventricular and intrathecal injection;
intraventricular injection may be facilitated by an
intraventricular catheter, for example, attached to a reservoir,
such as an Ommaya reservoir. In a specific embodiment, it may be
desirable to utilize liposomes targeted via abtides to specific
identifiable cell surface antigens.
[0147] In a specific embodiment, it may be desirable to administer
the Therapeutics of the invention locally to the area in need of
treatment; this may be achieved by, for example, and not by way of
limitation, local infusion during surgery, topical application,
e.g., in conjunction with a wound dressing after surgery, by
injection, by means of a catheter, by means of a suppository, or by
means of an implant, said implant being of a porous, non-porous, or
gelatinous material, including membranes, such as sialastic
membranes, or fibers.
[0148] The present invention provides pharmaceutical compositions.
Such compositions comprise a therapeutically effective amount of a
Therapeutic, and a pharmaceutically acceptable carrier or
excipient. Such a carrier includes but is not limited to saline,
buffered saline, dextrose, water, glycerol, ethanol, and
combinations thereof. The carrier and composition can be sterile.
The formulation should suit the mode of administration.
[0149] The composition, if desired, can also contain minor amounts
of wetting or emulsifying agents, or pH buffering agents. The
composition can be a liquid solution, suspension, emulsion, tablet,
pill, capsule, sustained release formulation, or powder. The
composition can be formulated as a suppository, with traditional
binders and carriers such as triglycerides. Oral formulation can
include standard carriers such as pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharine,
cellulose, magnesium carbonate, etc.
[0150] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lignocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0151] The Therapeutics of the invention can be formulated as
neutral or salt forms. Pharmaceutically acceptable salts include
those formed with free amino groups such as those derived from
hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and
those formed with free carboxyl groups such as those derived from
sodium, potassium, ammonium, calcium, ferric hydroxides,
isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
[0152] The amount of the Therapeutic of the invention which will be
effective in the treatment of a particular disorder or condition
will depend on the nature of the disorder or condition, and can be
determined by standard clinical techniques. In addition, in vitro
assays may optionally be employed to help identify optimal dosage
ranges. The precise dose to be employed in the formulation will
also depend on the route of administration, and the seriousness of
the disease or disorder, and should be decided according to the
judgment of the practitioner and each patient's circumstances.
[0153] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration.
5.5.3. In Vivo Diagnostic and Therapeutic Uses of Abtides
[0154] Another area where abtides can be used in place of
antibodies is in the imaging, detection, or treatment of disease.
Current diagnostic and therapeutic methods make use of antibodies
to target imaging agents or therapeutic substances, e.g. to tumors.
Since abtides possess the same specificity of binding as
antibodies, abtides can be used in place of antibodies in such
diagnostic and therapeutic methods.
[0155] Abtides may be linked to chelators such as those described
in U.S. Pat. No. 4,741,900 or U.S. Pat. No. 5,326,856. The
abtide-chelator complex may then be radiolabeled to provide an
imaging agent for diagnosis or treatment of disease. The abtide may
also be used in the methods that are disclosed in co-pending U.S.
patent application Ser. No. 08/127,351 for creating a radiolabeled
peptide for use in imaging or radiotherapy. This application
contains a review of methods of using peptides in imaging
agents.
[0156] In in vivo diagnostic applications, specific tissues or even
specific cellular disorders may be imaged by administration of a
sufficient amount of a labeled abtide of the instant invention.
[0157] A wide variety of metal ions suitable for in vivo tissue
imaging have been tested and utilized clinically. For imaging with
radioisotopes, the following characteristics are generally
desirable: (a) low radiation dose to the patient; (b) high photon
yield which permits a nuclear medicine procedure to be performed in
a short time period; (c) ability to be produced in sufficient
quantities; (d) acceptable cost; (e) simple preparation for
administration; and (f) no requirement that the patient be
sequestered subsequently. These characteristics generally translate
into the following: (a) the radiation exposure to the most critical
organ is less than 5 rad; (b) a single image can be obtained
within-several hours after infusion; (c) the radioisotope does not
decay by emission of a particle; (d) the isotope can be readily
detected; and (e) the half-life is less than four days (Lamb and
Kramer, "Commercial Production of Radioisotopes for Nuclear
Medicine", In Radiotracers For Medical Applications, Vol. 1, Rayudu
(Ed.), CRC Press, Inc., Boca Raton, pp. 17-62). Preferably, the
metal is technetium-99m.
[0158] By way of illustration, the targets that one may image
include any solid neoplasm, certain organs such a lymph nodes,
parathyroids, spleen and kidney, sites of inflammation or infection
(e.g., macrophages at such sites)., myocardial infarction or
thromboses (neoantigenic determinants on fibrin or platelets), and
the like evident to one of ordinary skill in the art. Furthermore,
the neoplastic tissue may be present in bone, internal organs,
connective tissue, or skin.
[0159] As is also apparent to one of ordinary skill in the art, one
may use the present invention in in vivo therapeutics (e.g., using
radiotherapeutic metal complexes), especially after having
diagnosed a diseased condition via the in vivo diagnostic method
described above, or in in vitro diagnostic application (e.g., using
a radiometal or a fluorescent metal complex).
[0160] Accordingly, a method of obtaining an image of an internal
region of a subject is contemplated in the instant invention which
comprises administering to a subject an effective amount of an
abtide composition containing a metal in which the metal is
radioactive, and recording the scintigraphic image obtained from
the decay of the radioactive metal. Likewise, a method is
contemplated of enhancing an MR image of an internal region of a
subject which comprises administering to a subject an effective
amount of an abtide composition containing a metal in which the
metal is paramagnetic, and recording the MR image of an internal
region of the subject.
[0161] Other methods include a method of enhancing a sonographic
image of an internal region of a subject comprising administering
to a subject an effective amount of an abtide composition
containing a metal and recording the sonographic image of an
internal region of the subject. In this latter application, the
metal is preferably any non-toxic heavy metal ion. A method of
enhancing an X-ray image of an internal region of a subject is also
provided which comprises administering to a subject an abtide
composition containing a metal, and recording the X-ray image of an
internal region of the subject. A radioactive, non-toxic heavy
metal ion is preferred.
[0162] The use of abtides in place of antibodies in such methods
has certain advantages. Because abtides are peptides rather than
large proteins such as antibodies, the kinetics of their
distribution in the body and clearance from the bloodstream differ
from that of large proteins such as antibodies. For example, as
demonstrated in Section 6.1.4, abtides can be used for in vivo
imaging of disease states in about 2 to 5 hours. Current methods of
tumor imaging using antibodies require approximately 24 to 48
hours.
[0163] Because abtides are peptides, they are cleared from the
blood faster than antibodies. This means that there will be less
background signal in the bloodstream when using abtides to image
disease states than there is when using antibodies.
[0164] Peptides most likely will provoke less of an immune response
in patients than do large proteins such as antibodies. This
consideration is especially important when diagnosis or treatment
is required to be done repeatedly or over a long period of
time.
[0165] Abtides, because they are generally small proteins, can
remain soluble in physiological fluids under conditions where
antibodies cannot.
[0166] Abtides, again because they are generally peptides, can be
produced synthetically or by recombinant methods and therefore may
be less costlylto produce than antibodies.
[0167] Abtides may be used individually. Alternatively, abtides may
be used as compositions of abtides in which the peptide sequences
of the abtides differ.
5.6. Synthesis of Peptides
5.6.1. Procedure for Solid Phase Synthesis
[0168] Abtide or mimetope peptides may be prepared by methods that
are known in the art. For example, in brief, solid phase peptide
synthesis consists of coupling the carboxyl group of the C-terminal
amino acid to a resin and successively adding N-alpha protected
amino acids. The protecting groups may be any known in the art.
Before each new amino acid is added to the growing chain, the
protecting group of the previous amino acid added to the chain is
removed. The coupling of amino acids to appropriate resins is
described by Rivier et al., U.S. Pat. No. 4,244,946. Such solid
phase syntheses have been described, for example, by Merrifield,
1964, J. Am. Chem. Soc. 85:2149; Vale et al., 1981, Science
213:1394-1397; Marki et al., 1981, J. Am. Chem. Soc. 103:3178 and
in U.S. Pat. Nos. 4,305,872 and 4,316,891. In a preferred aspect,
an automated peptide synthesizer is employed.
[0169] By way of example but not limitation, peptides can be
synthesized on an Applied Biosystems Inc. ("ABI".) model 431A
automated peptide synthesizer using the "Fastmo" synthesis protocol
supplied by ABI, which uses
2-(1H-Benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium
hexafluorophosphate ("HBTU") (R. Knorr et al., 1989, Tet. Lett.,
30:1927) as coupling agent. Syntheses can be carried out on 0.25
mmol of commercially available
4-(2',4'-dimethoxyphenyl-(9-fluorenyl-methoxycarbo-
nyl)-aminomethyl)-phenoxy polystyrene resin ("Rink resin" from
Advanced ChemTech) (H. Rink, 1987, Tet. Lett. 28:3787). Fmoc amino
acids (1 mmol) are coupled according to the Fastmoc protocol. The
following side chain protected Fmoc amino acid derivatives are
used: FmocArg(Pmc)OH; FmocAsn(Mbh)OH; FmocAsp(.sup.tBu)OH;
FmocCys(Acm)OH; FmocGlu(tBu)OH; FmocGln(Mbh)OH; FmocHis(Tr)OH;
FmocLys(Boc)OH; FmocSer(.sup.tBu)OH; FmocThr(.sup.tBu)OH;
FmocTyr(.sup.tBu)OH. [Abbreviations: Acm, acetamidomethyl; Boc,
tert-butoxycarbonyl; .sup.tBu, tert-butyl; Fmoc,
9-fluorenylmethoxycarbonyl; Mbh, 4,4'-dimethoxybenzhydryl; Pmc,
2,2,5,7,8-pentamethylchroman-6-sulfonyl; Tr, trityl].
[0170] Synthesis is carried out using N-methylpyrrolidone (NMP) as
solvent, with HBTU dissolved in N,N-dimethylformamide (DMF).
Deprotection of the Fmoc group is effected using ca. 20% piperidine
in NMP. At the end of each synthesis the amount of peptide present
is assayed by ultraviolet spectroscopy. A sample of dry peptide
resin (ca. 3-10 mg)-is weighed, then 20% piperidine in DMA (10 mL)
is added. After 30 min sonication, the UV (ultraviolet) absorbance
of the dibenzofulvene-piperidine adduct (formed by cleavage of the
N-terminal Fmoc group) is recorded at 301 nm. Peptide substitution
(in mmol g.sup.-1) can be calculated according to the equation: 1
substitution = A .times. v 7800 .times. w .times. 1000
[0171] where A is the absorbance at 301 nm, v is the volume of 20%
piperidine in DMA (in mL), 7800 is the extinction coefficient (in
mol.sup.-1dm.sup.3 cm.sup.-1) of the dibenzofulvene-piperidine
adduct, and w is the weight of the peptide-resin sample (in
mg).
[0172] Finally, the N-terminal Fmoc group is cleaved using 20%
piperidine in DMA, then acetylated using acetic anhydride and
pyridine in DMA. The peptide resin is thoroughly washed with DMA,
CH.sub.2Cl.sub.2 and finally diethyl ether.
5.6.2. Cleavage and Deprotection
[0173] By way of example but not limitation, cleavage and
deprotection can be carried out as follows: The air-dried peptide
resin is treated with ethylmethyl-sulfide (EtSMe), ethanedithiol
(EDT), and thioanisole (PhSMe) for approximately 20 min. prior to
addition of-95% aqueous trifluoracetic acid (TFA). A total volume
of ca. 50 mL of these reagents are used per gram of peptide-resin.
The following ratio is used: TFA:EtSMe:EDT:PhSme (10:0.5:0.5:0.5).
The mixture is stirred for 3 h at room temperature under an
atmosphere of N.sub.2. The mixture is filtered and the resin washed
with TFA (2.times.3 mL). The combined filtrate is evaporated in
vacuo, and anhydrous diethyl ether added to the yellow/orange
residue. The resulting white precipitate is isolated by filtration.
See King et al., 1990, Int. J. Peptide Protein Res. 36:255-266
regarding various cleavage methods.
5.6.3. Purification of the Peptides
[0174] Purification of the synthesized peptides can be carried out
by standard methods including chromatography (e.g., ion exchange,
affinity, and sizing column chromatography, high performance liquid
chromatography (HPLC)), centrifugation, differential solubility, or
by any other standard technique.
5.6.4. Conjugation of Peptides to Other Molecules
[0175] The abtides of the present invention may be linked to other
molecules (e.g., a detectable label, a molecule facilitating
adsorption to a solid substratum, or a toxin, according to various
embodiments of the invention) by methods that are well known in the
art. Such methods include the use of homobifunctional and
heterobifunctional cross-linking molecules.
[0176] The homobifunctional molecules have at least two reactive
functional groups, which are the same. The reactive functional
groups on a homobifunctional molecule include, for example,
aldehyde groups and active ester groups. Homobifunctional molecules
having aldehyde groups include, for example, glutaraldehyde and
subaraldehyde. The use of glutaraldehyde as a cross-linking agent
was disclosed by Poznansky et al., 1984, Science 223:1304-1306.
[0177] Homobifunctional molecules having at least two active ester
units include esters of dicarboxylic acids and
N-hydroxysuccinimide. Some examples of such N-succinimidyl esters
include disuccinimidyl suberate and dithio-bis-(succinimidyl
propionate), and their soluble bis-sulfonic acid and bis-sulfonate
salts such as their sodium and potassium salts. These
homobifunctional reagents are available from Pierce, Rockford,
Ill.
[0178] The heterobifunctional molecules have at least two different
reactive groups. Some examples of heterobifunctional reagents
containing reactive disulfide bonds include N-succinimidyl
3-(2-pyridyl-dithio)propi- onate (Carlsson et al., 1978, Biochem J.
173:723-737), sodium
S-4-succinimidyloxycarbonyl-alpha-methylbenzylthiosulfate, and
4-succinimidyloxycarbonyl-alpha-methyl-(2-pyridyldithio)toluene.
N-succinimidyl 3-(2-pyridyldithio)propionate is preferred. Some
examples of heterobifunctional reagents comprising reactive groups
having a double bond that reacts with a thiol group include
succinimidyl 4-(N-maleimidomethyl)cyclohexahe-1-carboxylate and
succinimidyl m-maleimidobenzoate.
[0179] Other heterobifunctional molecules include succinimidyl
3-(maleimido)propionate, sulfosuccinimidyl
4-(p-maleimido-phenyl)butyrate- , sulfosuccinimidyl
4-(N-maleimidomethyl-cyclohexane)-1-carboxylate,
maleimidobenzoyl-N-hydroxy-succinimide ester. The sodium sulfonate
salt of succinimidyl m-maleimidobenzoate is preferred. Many of the
above-mentioned heterobifunctional reagents and their sulfonate
salts are available from Pierce.
[0180] Additional information regarding how to make and use these
as well as other polyfunctional reagents may be obtained from the
following publications or others available in the art:
[0181] Carlsson et al., 1978, Biochem. J. 173:723-737.
[0182] Cumber et al., 1985, Methods in Enzymology 112:207-224.
[0183] Jue et al., 1978, Biochem 17:539.9-5405.
[0184] Sun et al., 1974, Biochem. 13:2334-2340.
[0185] Blattler et al., 1985, Biochem. 24:1517-152.
[0186] Liu et al., 1979, Biochem. 18:690-697.
[0187] Youle and Neville, 1980, Proc. Natl. Acad. Sci. USA
77:5483-5486.
[0188] Lerner et al., 1981, Proc. Natl. Acad. Sci. USA
78:3403-3407.
[0189] Jung and Moroi, 1983, Biochem. Biophys. Acta 761:162.
[0190] Caulfield et al., 1984, Biochem. 81:7772-7776.
[0191] Staros, 1982, Biochem. 21:3950-3955.
[0192] Yoshitake et al., 1979, Eur. J. Biochem. 101:395-399.
[0193] Yoshitake et al., 1982, J. Biochem. 92:1413-1424.
[0194] Pilch and Czech, 1979, J. Biol. Chem. 254:3375-3381.
[0195] Novick et al., 1987, J. Biol. Chem. 262:8483-8487.
[0196] Lomant and Fairbanks, 1976, J. Mol. Biol. 104:243-261.
[0197] Hamada and Tsuruo, 1987, Anal. Biochem. 160:483-488.
[0198] Hashida et al., 1984, J. Applied Biochem. 6:56-63.
[0199] Additionally, methods of cross-linking are reviewed by Means
and Feeney, 1990, Bioconjugate Chem. 1:2-12.
5.6.4.1. Biotinylation of Peptides
[0200] Methods of biotinylating peptides are well known in the art.
Any convenient method may be employed in the practice of the
invention. For example, the following procedure was used:
[0201] (1) dissolve 10 mg of peptide in 100 .mu.L of 0.1% acetic
acid;
[0202] (2) add 900 .mu.L of PBS;
[0203] (3) add 3.3 mg of biotin-LC-NHS (Pierce, Rockford,
Ill.);
[0204] (4) incubate for 30 minutes at room temperature;
[0205] (5) purify over a Superose 12 column (Pharmacia, Piscataway,
N.J.).
6. EXAMPLES
6.1. Abtides Mimicking the Binding Specificity of Monoclonal
Antibody 7E11-C5
6.1.1. Identification and Isolation of Abtides Mimicking the
Binding Specificity of Monoclonal Antibody 7E11-C5
[0206] 7E11-C5 is a murine IgG1 monoclonal antibody specific for an
antigen of a human prostate carcinoma, LNCaP. 7E11-C5 binds
strongly to malignant prostatic epithelium but only weakly to
normal prostatic epithelium. It does not bind to non-prostatic
tumors or to most normal organs. See Horoszewicz et al., 1987,
Anticancer Res. 7:927-936. 7E11-C5 is also described in U.S. Pat.
No. 5,162,504 issued Nov. 10, 1992. Hybridomas producing monoclonal
antibody 7E11-C5 were grown as ascites in mice and 7E11-C5 was
purified from ascites fluid by Protein A affinity chromatography to
over 90% purity as judged by sodium dodecylsulfate polyacrylamide
gel electrophoresis.
[0207] In order to identify abtides mimicking binding specificity
of monoclonal antibody 7E11-C5, monoclonal antibody 7E11-C5 was
used as the target ligand in a first screening of the TSAR-9
library (see Kay et al., 1993, Gene 128:59-65 and PCT publication
WO 94/18318, dated Aug. 18, 1994). The following-screening
procedure was used. First, 7E11-CS was bound to a well of a
microtiter plate. 7E11-C5 at a concentration of 11.2 mg/mL in
phosphate buffered saline (PBS), pH 6.0, was diluted to 100 .mu.g
per mL in 0.1.times.PBS pH 7.2. One hundred microliters (100 .mu.L)
of this dilution was added to one well of a microtiter plate, and
allowed to incubate for 1-6 hours at room temperature or overnight
at 40 C. After incubation, the well was washed at least 4 times
with a blocking buffer which consisted of either 1% bovine serum
albumin (BSA) in PBS, 1% non-fat dry milk (NFDM) in PBS, or 0.1
Tween.RTM. in either 1% BSA in PBS or 1% NFDM in PBS. Two hundred
microliters of the blocking buffer was then added to the well and
allowed to incubate for at least an hour at room temperature.
[0208] Next, an aliquot of the TSAR-9 library was added to the well
containing bound 7E11-C5. An aliquot of the library containing
10.sup.10 phage particles was added to the well and allowed to
incubate for at least 1 hour at room temperature. This resulted in
the binding to the plate of those phage containing binding domains
that bind to 7 .mu.l-C5. After an hour, the well was washed
extensively with either 1% bovine serum albumin (BSA) in PBS, 1%
non-fat dry milk (NFDM) in PBS, or 0.1% Tween.RTM. in either 1% BSA
in PBS or 1% NFDM in PBS.
[0209] After washing, phage bound to the 7E11-C5 antibody in the
well were eluted by adding 100 .mu.L of an acid solution of 0.2 M
glycine-HCl, pH 2.0. After incubation from 15 minutes to 1 hour,
the acid solution containing eluted phage was transferred to a 1.5
mL microfuge tube, and an equal volume of 0.2 M Tris-HCl, pH 7.5
added to neutralize the acid solution. In some cases, the
neutralized phage solution was immediately added to a second well
containing bound 7E11-CS antibody, and the binding and elution
procedure repeated.
[0210] If it was desired that the level of enrichment be monitored
during the-above steps, an irrelevant phage that does not bind
7E11-C5 but that expresses the .beta.-galactosidase gene was added
to the aliquot from the TSAR-9 library. This phage gives rise to
blue plaques when plated in the presence of X-Gal and IPTG.
Following a screening step, the eluted phage were plated in X-gal
and IPTG. An aliquot of unscreened phage were plated as well. The
ratio of white to blue plaques was measured for both phage samples.
The increase in the proportion of white plaques (from the TSAR-9
phage that bind to 7E11-C5) to blue plaques (from the irrelevant
phage) indicated the degree to which the screening process enriched
the population of phage for those phage that bind 7E11-C5.
[0211] If it was desired that the specificity of binding be
monitored during the above screening steps, screening was done
against an irrelevant target (either BSA, mouse IgG, or plastic) in
addition to being done against 7E11-C5. The enrichment of white
plaques over blue plaques when panning was done against 7E11-C5
rather than an irrelevant target indicated the level of specificity
of binding.
[0212] After screening, the phage were amplified by adding an
aliquot of the eluted phage to a solution containing LB broth and
competent DH5.alpha.F' E. coli cells (GIBCO BRL, Gaithersburg,
Md.). Typically a 2-5 .mu.L aliquot of the phage solution was added
to 125 .mu.L of LB broth containing a 1:50 dilution of DH5.alpha.F'
E. Coli cells (about 1.times.10.sup.9 cells/ml). 3.3 mL of top agar
was added to this solution, and the mixture was plated out onto a
Petri dish containing agar. Often the phage were titered by making
several serial 1:10 dilutions, and plating out the dilution as
described above. After incubation overnight at 37.degree. C., the
plates were evaluated for growth of plaques, and counted if
desired. The plates were eluted by adding 3-5 mL of 100 mM NaCl, 10
mM MgCl.sub.2, 10 mM Tris-HCl, pH 7.5 (SM buffer) to each plate and
incubating for 1-5 hours with gentle rocking. The solution was then
removed from the plate, centrifuged, and either stored, amplified
further, or analyzed.
[0213] In some cases, the phage were amplified in solution by
adding 1-5 .mu.L of the phage solution to 1-5 mL of LB broth
containing a 1:50 or 1:100 dilution of competent DH5.alpha.F' E.
coli cells (about 1.times.10.sup.9 cells/ml. After incubation for
either 6 hours or overnight, the solution was centrifuged and the
supernatant collected. In some cases, the phage particles were
precipitated with polyethylene glycol (PEG) by adding a 1/5 volume
of PEG to the clarified phage solution, and incubating for 1 hour
on ice. After centrifugation, the phage were usually reconstituted
with 100 .mu.L of SM buffer.
[0214] Using the above procedures, nine different phage were
isolated that expressed peptides containing binding domains that
were capable of binding monoclonal antibody 7E11-C5. Molecules
comprising these binding domains are thus mimetopes of the antigen
recognized by the monoclonal antibody 7E11-C5. The binding domains
of the peptides expressed by the nine phage were sequenced
according to standard methods of DNA sequencing (Sequenase.TM.,
U.S. Biochemical Corp., Cleveland, Ohio). The determination of
those DNA sequences allowed the determination of the amino acid
sequences of these mimetopes. These sequences are shown in Table 1.
Examination of these amino acid sequences showed that they shared a
common motif of MYxxLH (SEQ ID NO. 10).
1TABLE 1 SCVSHMLDTSRVYTAYANPG MYSRLH SPAVRPLTQSSA (SEQ ID No.: 11)
SVQFKSISSRSMDDVVKDPGPKPA MYNRLH SKNPFTLS (SEQ ID No.: 12)
YFDHTYSGPVVKNGGLVSPGVLS MYNRLH SDGGPSLAS (SEQ ID No.: 13) TVAT
MHDRLH SAPGSGNLPGSYDIKPIFKAQSGALHS (SEQ ID No.: 14) T IDMPQTAST
MYNMLH RNEPGGRKLSPPANDMPPALLKR (SEQ ID No.: 15) RLGNHVWREGGG MYQQLH
HNFP (SEQ ID No.: 16) RDSAVENPSVGGEIP MYRYLH QR (SEQ ID No.: 17)
PVQKEYGFGMSGAS MIRLLR ETP (SEQ ID No.: 18) QKGGPGLLLYGGDS MYITLH
EPG (SEQ ID No.: 19) MYxxLH (SEQ ID No.: 10) LYANPGMYSRLHSPA (SEQ
ID No: 20)
[0215] In order to use the mimetopes to identify abtides, peptides
corresponding to the mimetope sequences were synthesized, and then
dissolved in either water or PBS to give a final concentration of 5
.mu.g/mL. Specifically, a peptide called 7E11-9.5, with the
sequence LYANPGMYSRLHSPA (SEQ ID NO: 20) and a peptide with the
sequence GMYSRLHSPA (SEQ ID NO: 21) were synthesized.
[0216] First the mimetope peptide 7 .mu.l-9.5 was tested for its
ability to bind to the monoclonal antibody 7E11-C5. Ninety-six well
plates (Immunlon 4, Dynatech, Alexandria, Va.) were coated with 50
.mu.L of a 5 .mu.g/mL solution of the mimetope peptide 7 .mu.l-9.5
and incubated overnight at room temperature. Following incubation,
the wells were washed 4 times with 1% BSA in PBS. Biotinylated
monoclonal antibody 7E11-C5 was serially diluted with PBS beginning
with a concentration of 29 .mu.g/mL and various amounts of the
monoclonal antibody were added to the wells that had been coated
with the mimetope peptide. After incubating for 1 hour at room
temperature, the wells were washed four times with 1% BSA in PBS
and 2 times with PBS. Then, 100 AL of a 1:2000 dilution of
Extravidin-Alkaline Phosphatase (4,250 units/mL) (Sigma, St. Louis,
Mo.) in PBS was added to each well. After an hour, the plate was
again washed 4 times with PBS and 100 .mu.L of a 1 mg/mL solution
of the enzyme substrate p-nitrophenyl phosphate was added to each
well. Color was allowed to develop for 15 minutes to 1 hour and
absorbance was read at 405 nm. FIG. 2 shows the results. It can be
seen from FIG. 2 that monoclonal antibody 7E11-C5 binds to the
synthesized mimetope 7E11-9.5 in a concentration dependent
manner.
[0217] Next, the mimetope peptide was used to isolate abtides from
a peptide library. Fifty to one hundred microliters of the solution
of this peptide was used to coat the wells of a 96-well plate, the
wells were blocked with 0.5% BSA in PBS, and the wells were used
for screening. An aliquot of the TSAR-9 random phage library
(containing approximately 3.times.10.sup.10 phage particles) was
used as in the initial screening, and 4 rounds of screening were
performed. After the first two rounds, the phage were amplified.
Two more rounds of screening were then performed. By this
procedure, phage from the TSAR-9 library that expressed peptides
capable of binding to the 7E11-9.5 mimetope peptide were identified
and isolated. The peptides containing the binding domains of these
phage are abtides and were discovered to mimic the binding
specificities of monoclonal antibody 7E11-C5. These abtides are
termed "7E11-C5 abtides."
[0218] Phage encoding the 7E11-C5 abtides were subjected to DNA
sequencing of the nucleotide sequences encoding their binding
peptides in order to obtain the DNA and amino acid sequences of the
7E11-C5 abtides. Table 2 shows the amino acid sequence of five-of
the 7E11-C5 abtides that had relatively high affinity for the
mimetope.
2TABLE 2 Clone Sequence 14 GIINANDPLPFWFMSPYTPGPAPIDINASRALVSNESG
17 DLSRNLDFGRFLLYNAYVPGFTPTFISLTAEHLSSPKG 15
CGRAYCLSGNYNIFGALFPGVSTPYADVGHDDAQSWRR 13
RCSPIWGISYPFGLLSSNPGVCHSSDAETNIRNDILTT 16
GHSNYCFVSTLGMPIVGFPSINARGLIHYGGSDPRLAA
[0219] The amino acid sequence shown in Table 2 for clone 14 is SEQ
ID NO: 1. The amino acid sequence shown in Table 2 for clone 17 is
SEQ ID NO: 2. The amino acid sequence shown in Table 2 for clone 15
is SEQ ID NO: 3. The amino acid sequence shown in Table 2 for clone
13 is SEQ ID NO: 4. The amino acid sequence shown in Table 2 for
clone 16 is SEQ ID NO: 5.
[0220] It was of interest to determine whether there might be some
structural basis for the similarity in binding characteristics
between the monoclonal antibody 7E11-C5 and the 7E11-C5 abtides.
The amino acid sequences of the complementarity determining regions
(CDRs) of the monoclonal antibody 7E11-C5 were determined by
sequencing cDNA clones of the genes encoding the variable regions
of the antibody. These CDRs are responsible for the specific
binding of monoclonal antibody 7E11-C5 to its antigen. FIG. 3
presents a comparison of the amino acid sequences of the abtides of
Table 2 and portions of the amino acid sequences of some of the
CDRs of monoclonal antibody 7E11-C5. Surprisingly, it can be seen
that there are similarities in the sequences of these abtides and
the sequences of the CDRs of the monoclonal antibody.
6.1.2. Characterization of 7E11'-C5 Abtides
[0221] 7E11-C5 abtides were tested for their ability to bind to the
0.7 .mu.l-C5 mimetopes that were used as target ligands in the
second screening step above. The DNA sequences of the regions of
the phage DNA encoding the abtides were determined. This allowed
the determination of the amino acid sequences of the abtides. Based
upon these determined amino acid sequences, synthetic peptides
corresponding to these sequences were made. These synthetic
peptides (7E11-C5 abtides) were 38 amino acids in length.
6.1.2.1. Dot Blots Using 7E11-C5 Abtides
[0222] In some cases, these abtides were used in a dot blot
experiment. In those cases, 1 .mu.L of a 1 mg/mL solution of the
38-residue abtides was spotted onto nitrocellulose (0.2 .mu.m or
0.45 .mu.m, Schleicher & Schuell, Keene, NH) strips or circles.
After drying (about {fraction (1/2)} hour), the nitrocellulose was
blocked for 1 hour in a solution of 1% BSA in PBS. The
nitrocellulose was then allowed to incubate in approximately 5 mL
of a solution of 0.1 mg/mL of a biotinylated 7E11-9.5 mimetope
peptide (biotin-LYANPGMYSRLHSPA). This mimetope peptide was one of
those described in Section 6.1.1 above that were synthesized based
upon the nine peptides that were identified in the screening of
Section 6.1.1 above. After an hour, the nitrocellulose was washed
approximately 5 times with a solution of 1% BSA in PBS. A 1:2000
dilution of Extravidin-Alkaline Phosphatase (4,250 units/mL)
(Sigma, St. Louis, Mo.) in PBS was then added and allowed to
incubate for 1 hour, after which the nitrocellulose was again
washed extensively. Finally, a solution of
5-bromo-4-chloro-3-indolyl phosphate (0.15 mg/mL) and nitro blue
tetrazolium (0.3 mg/mL) (Sigma, St Louis, Mo.) (BCIP/NBT) was added
as an enzyme substrate. Color was allowed to develop and the
absorbance at 405 nm was read.
[0223] An example of such a dot blot assay is shown in FIG. 4. In
FIG. 4, the 7E11-C5 abtides known as clone 14, clone 17, clone 15,
clone 16, and clone 13 were tested for their ability to bind the
biotinylated 7E11-9.5 mimetope peptide. Also tested, as a positive
control, was the monoclonal antibody 7E11-C5. 7E11-C5 was spotted
onto the region marked 351 in FIG. 4. Inspection of FIG. 4 shows
that at least three of the abtides (clone 14, clone. 17, and clone
15) bound the mimetope. This shows that these abtides are capable
of mimicking the specific binding exhibited by the monoclonal
antibody 7E11-C5.
6.1.2.2. 7E11-C5 Abtides in Place of Antibodies in Immunoassays
[0224] The ability of abtides synthesized having the amino acid
sequence encoded by the random inserts of the phage that bound the
7E11-9.5 mimetope was further evaluated by ELISA assay methods.
[0225] The 7E11-C5 abtides clone 14 and clone 17 (See Table 2) were
each dissolved in 0.1.times.PBS to give a solution of 5 .mu.g/mL.
Fifty microliters of each of these solutions was used to coat the
wells of a 96-well microtiter plate (Immulon 4, Dynatech,
Alexandria, Va.) by overnight incubation at 4.degree. C. Following
this incubation, the abtide solutions were removed and the wells
were blocked with 200 .mu.L .mu.l of a solution of 1% BSA in PBS.
Mimetope peptides 7E11-9.5 (LYANPGMYSRLHSPA [SEQ IN NO: 20]) and
GMYSRLHSPA (SEQ ID NO: 21) were biotinylated as described in
Section 5.6.4.1 and dissolved in H.sub.2O to give 1 mg/mL
solutions. Serial 1:2 dilutions were made of these solutions and
these dilutions were added to the wells of the microtiter plate
containing the bound abtides. After incubation for 1 hour at room
temperature, the wells were washed four times with 1% BSA in PBS.
Then a 1:2000 dilution of Extravidin-Alkaline Phosphatase (4,250
units/mL) (Sigma, St. Louis, Mo.) in PBS was added to each well and
incubated for 1 hour at room temperature. Following incubation, the
wells were washed four times with 1% BSA in PBS and then twice in
PBS. One hundred microliters of a 1 mg/mL solution of p-nitrophenyl
phosphate in diethanol amine (DEA) buffer (both from Kirkegaard
& Perry Laboratories, Gaithersburg, Md.) was then added and,
after incubation for 15-30 minutes at room temperature, the
absorbance of the solutions in the wells was read at 405 nm. The
results are shown in FIG. 5.
[0226] FIG. 5 shows that, except for the non-linear effect at high
concentrations of mimetope, there is a good correlation between the
amount of mimetope added to the wells and the absorbance at 405 nm.
The use of antibodies in assays such as enzyme-linked immunosorbent
assays (ELISAs) to measure the concentration of a substance is well
known in the art. The ability of antibodies to specifically bind
their antigens is crucial to the success of such assays. Since
abtides also specifically bind to antigens, it was of interest to
determine if abtides can be used in place of antibodies in
immunoassays such as ELISA-like assays to measure the concentration
of a substance. The results of FIG. 5 show that the abtides of the
present invention can be used in assays in much the same way that
antibodies can be used in immunoassays such as ELISAs.
6.1.2.3. Biotinylation of Antibodies
[0227] Methods of biotinylating antibodies are well known in the
art. Any convenient method may be employed in the practice of the
invention. For example, the following procedure was used:
[0228] (1) dissolve 10 mg of antibody in 1 mL of PBS;
[0229] (2) add 0.44 mg of biotin-LC-NHS (Pierce, Rockford,
Ill.);
[0230] (3) incubate for 30 minutes at room temperature;
[0231] (4.) purify over a Superose 12 column (Pharmacia,
Piscataway, N.J.).
6.1.3. 7E11-C5 Abtides and Monoclonal Antibody 7E11-C5 Recognize an
LNSaP Antigen
[0232] The monoclonal antibody 7E11-CS recognizes a prostate
specific mucin antigen of the human prostate cancer cell line LNCaP
(Horoszewicz et al., 1987, Anticancer. Res. 7:927-936). To
determine if 7E11-C5 abtides recognize and bind the native antigen,
the following experiment was done.
[0233] A sandwich assay using the LNCaP antigen was performed. The
wells of a microtiter plate were coated with either monoclonal
antibody 7E11-C5, the clone 14 7E11-C5 abtide, or, as a negative
control, BSA. Coating and washing was as for-the assay described in
Section 6.1.2.2. One hundred microliters of a lysate of LNCaP cells
was added to the wells. The LNCaP lysate was prepared as described
in PCT publication WO 94/18318, dated Aug. 18, 1994. Following
capture of the lysate on the plate, 100 .mu.L of a 5 .mu.g/mL
solution of biotinylated 7E11-CS monoclonal antibody was added to
each well. Following incubation and washing as in Section 6.1.2.2,
a 1:2000 dilution of Extravidin-Alkaline Phosphatase (4,250
units/mL) (Sigma, St. Louis, Mo.) in PBS was added to each well and
incubated for 1 hour at room temperature. Following incubation, the
wells were washed four times with 1% BSA in PBS and then twice in
PBS. One hundred microliters of a 1 mg/mL solution of p-nitrophenyl
phosphate in diethanol amine (DEA) buffer (both from Kirkegaard
& Perry Laboratories, Gaithersburg, Md.) was then added and,
after incubation at room temperature for 15-30 minutes, the
absorbance of the solutions in the wells was read at 405 nm.
[0234] The results are shown in FIG. 6. FIG. 6 shows that the
7E11-C5 abtide is capable of recognizing the native 7E11-C5 antigen
in LNCaP lysates. This was a surprising discovery and would not
have been predicted by those skilled in the art. It was generally
felt that screening a library with a mimetope could yield a binder
to that mimetope. Whether such a binder could also bind the native
epitope that the mimetope mimics was unknown. The mimetope could
have represented the epitope in a loose fashion, e.g. its primary
sequence could be slightly modified; its secondary structure or
factors influencing the presentation of the mimetope could be
different from those presenting the native epitope. In such a case,
the binder to the mimetope would not be specific for the native
epitope. The foregoing is presented as possible explanation and not
as a limitation of the present invention.
6.1.4. Use of 7E11-C5 Abtides in Biodistribution Studies
[0235] The 7E11 abtides described in Section 6.1 and its
subsections above were used in biodistribution studies to assess
their ability to target human prostate carcinoma LNCaP xenograft
tumors that had been transplanted into mice.
[0236] Male SCID mice (C.B-17/Icr Tac--SCID mice) were purchased
from Fox Chase (Philadelphia, Pa.) or. Taconic Farms (Germantown,
N.Y.), were housed in sterilized cages with filter bonnets, and
were given autoclaved laboratory rodent chow (Purina, St. Louis,
Mo.), and filtered tap water ad libitum.
[0237] 1.times.10.sup.7 cells of the human prostate tumor line
LNCaP (Horoszewicz et al., 1987, Anticancer Res. 7:927-936) were
injected subcutaneously (s.c.) into the left rear flank of the
mice. The cells were growing in exponential phase before harvesting
and had been resuspended in 0.2 mL of sterile saline. Tumors were
grown in the mice for 2-3 months before abtides were injected into
the mice.
[0238] For biodistribution studies, abtides were modified at their
amino termini with the chelator diethylene-triamine-pentaacetic
acid anhydride (DTPA-A) (Sigma, St. Louis, Mo.). Approximately 2 mg
of each abtide was initially dissolved in an appropriate volume of
0.1% acetic acid and then 1 mL of 0.1 M sodium bicarbonate, pH 8.0,
was added. Two mg of DTPA-A was suspended in 100 AL of
dimethylsulfoxide (DMSO), and 10 .mu.L of the abtide solution added
to this DTPA-A suspension. After 5 min incubation at room
temperature, the suspension was filtered through a 0.2 .mu.m
polyvinylidene difluoride (PVDF) sample filter (Acrodisc, Gelman
Sciences, Inc., Ann Arbor, Mich.), and purified using a Superose-12
FPLC column (Pharmacia, Piscataway, N.J.) with PBS as the running
buffer. Modified peptides were stored-frozen at -20.degree. C. or
-70.degree. C.
[0239] Abtides modified with DTPA were labeled with
.sup.111InCl.sub.2 as follows. 0.1 to 0.5 mCi of .sup.111InCl.sub.2
(Amersham, Chicago, Ill.) were first neutralized by adding an equal
volume of 0.1 M NaOAc, and then added to 100 to 200 .mu.g of the
DTPA-A-modified abtide. After incubation for one half hour, the
labeled peptide was purified using a Superose-12 FPLC column
(Pharmacia, Piscataway, N.J.) with PBS as the running buffer.
Labeled fractions were collected in a fraction collector. Tubes
containing the labeled peptide ere pooled and used to prepare
syringes for injection into mice.
[0240] In one experiment, abtide clone 14-DPTA-.sup.111In (see
Table 2) was injected intravenously (i.v.) into two groups of mice
bearing measurable LNCaP xenografts. About 0.2 mL of a 10 .mu.g/mL
solution of the radioactively labeled abtide in sterile saline was
used. The specific activity of the abtide was about 32
.mu.Ci/.mu.g. Thus, the total injected dose of radioactivity was
about 120-140.times.10.sup.6 cpm.
[0241] The first group of mice was sacrificed 2 hours after
injection of the abtide and tissues were dissected for analysis.
The second group was sacrificed 4 hours after injection. Dissected
tissues were weighed and the amount of .sup.111In in them was
determined by gamma counting. The cpm per gram of each tissue was
calculated by dividing the cpm of .sup.111In found in the tissue by
the weight in grams of the tissue. The data are presented as the
ratio of the cpm/g in each organ to the cpm/g in blood (organ to
blood ratio). This gave the ratios that are shown in Table 3 and
FIG. 7.
3TABLE 3 BIODISTRIBUTION OF ABTIDE CLONE 14-DPTA-.sup.111In IN
LNCaP XENOGRAFT BEARING MICE Group 1.sup.a Group 2.sup.b
Tissue.sup.c AVG s.e.m. AVG s.e.m. Blood 1.00 0.00 1.00 0.00 Lung
0.81 0.04 1.06 0.26 Spleen 0.83 0.16 1.42 0.74 Liver 0.95 0.04 2.12
0.69 Kidney-R 55.85 10.22 171.73 77.97 Kidney-L 53.03 11.97 182.79
86.38 Tumor 1 0.80 2 2.85 Muscle 0.33 0.03 0.42 0.01 Testes-R 0.54
0.02 0.94 0.25 Testes-L 0.68 0.24 0.88 0.24 .sup.aGroup 1:
sacrificed at 2 hours; n = 2. .sup.bGroup 2: sacrificed at 4 hours;
n = 2. .sup.cValue shown is the organ to blood ratio.
[0242] Table 3 and FIG. 7 show that, with the exception of kidney,
the highest organ to blood ratio is found in the tumor, both at 2
hours and at 4 hours post-injection of abtide. This result shows
that abtides with the binding specificity of antibodies, e.g. that
are specific for tumor antigens, can be used to localize to those
tumors.
[0243] No unusual localization was seen to any non-tumor tissue or
organ except kidney. The ratio for kidney is extremely high due to
the well known tendency of injected peptides to localize to the
kidneys prior to being cleared from the body.
[0244] In another experiment, abtide clone 17-DPTA-.sup.111In (see
Table 2) was injected intravenously into four SCID mice bearing
measurable LNCaP xenografts. Administration of xenografts was as
above. About 0.2 mL of a 0.1 .mu.g/mL solution of the radioactively
labeled clone 17 abtide in sterile saline was injected. The
specific activity of the abtide was about 2.4 .mu.Ci/ng. Thus, the
total injected dose of radioactivity was about
100-110.times.10.sup.6 cpm.
[0245] In this experiment, mice were sacrificed at either 2 or 5
hours post-injection with labeled abtide. Again, as above, the data
are presented as organ to blood ratios. As shown in Table 4 and
FIG. 8, abtide clone 17-DPTA-.sup.111In localized to LNCaP
xenograft tumors in mice.
4TABLE 4 BIODISTRIBUTION OF ABTIDE CLONE 17-DPTA-.sup.111In IN
LNC.alpha.P-XENOGRAFT BEARING MICE GROUP 1 GROUP 2 MOUSE # MOUSE #
TISSUE.sup.a 1 6 2 3 BLOOD 1.00 1.00 1.00 1.00 LUNG 2.52 2.57 4.33
2.47 SPLEEN 5.82 3.00 5.53 3.70 LIVER-S 5.42 5.58 8.37 4.03
KIDNEY-R 235.63 234.88 321.09 106.74 KIDNEY-L 563.71 220.69 424.64
104.09 TUMOR-S 10.60 15.01 8.36 2.90 MUSCLE 0.93 2.96 1.16 3.04
TESTES-R 2.71 2.19 2.31 3.15 TESTES-L 1.64 1.14 5.36 3.41
.sup.aValue shown is the organ to blood ratio.
[0246] Table 4 and FIG. 8, like Table 3 and FIG. 7, show that the
injected abtide localized to the tumor. This indicates that abtides
can be useful in the localization of tumors.
[0247] In contrast to the results of the two experiments described
above, in which radioactively labeled abtides were shown to
localize to tumors, when the same experiments were done with a
radioactively labeled control (non-abtide) peptide (the tripeptide
GYK-DPTA), no specific localization to tumors was observed. This
can be seen in FIG. 9, which shows the biodistribution results for
experiments using the .sup.111In-labeled control peptide.
[0248] The radiolabeled peptide conjugate GYK-DPTA-.sup.111In was
injectd intravenously into 5 SCID mice bearing measurable LNCaP
xenografts. Mice were dissected 2 hours (n=2) and 5 hours (n=3)
after injection of 1.5 .mu.g of control peptide having a specific
activity of 30 .mu.Ci/g. The organ to blood ratios are presented in
Table 5 and FIG. 9. As shown, the control peptide did not
selectively localize to the tumor. While the tumor to blood ratio
in one mouse was 3.26, the control peptide distributed equally well
to other organs (e.g. lung 3.52, spleen 3.27, liver 21.70, etc.).
These results show that there was non-specific uptake of the
control peptide in these organs. While abtide clone
14-DPTA-.sup.111In demonstrated a tumor to blood ratio of only 1.85
at 2 hours (which appears lower than that obtained with the control
peptide), clone 14-DPTA-.sup.111In demonstrated specific
localization to the tumor as the organ to blood ratios in the other
organs were much lower (e.g. lung 0.81, spleen 0.83, liver 0.95,
etc.).
5TABLE 5 BIODISTRIBUTION OF GYK-DPTA-.sup.111In IN LNCaP XENOGRAFT
BEARING MICE GROUP 1 GROUP 2 MOUSE # MOUSE # ORGAN/BLOOD 1 2 3 4 5
BLOOD 1.00 1.00 1.00 1.00 1.00 LUNG 2.96 3.52 0.95 2.84 2.39 SPLEEN
1.74 3.27 0.97 2.21 4.04 LIVER-S 31.79 21.70 21.56 25.38 17.78
KIDNEY-R 563.87 406.27 273.82 509.53 269.30 KIDNEY-L 584.69 417.98
297.65 433.97 280.50 TUMOR-S 3 4 5 6 7 MUSCLE 1.04 1.02 0.29 4.34
2.51 TESTES-R 603.01 58.49 0.82 2.05 2.25 TESTES-L 44.65 2.08 0.93
2.11 2.32
[0249] 6.2. Abtides Binding to a Breast Cancer Antigen
[0250] 6.2.1. Identification and Isolation of Abtides Binding To
Polymorphic Epithelial Mucin (PEM)
[0251] The monoclonal antibody SM-3 that specifically binds the
polymorphic epithelial mucin (PEM) tumor antigen found on human
breast cancer cells has been shown to be specific for the epitope
defined by the amino acid sequence VTSAPDTRPAPGSTAPPAHGVTSAPDTR
(SEQ ID NO: 9) (Bruchell et al., 1989, Int. J. Cancer 44:691-696).
A peptide comprising this sequence was synthesized and used to
isolate abtides from TSAR peptide libraries by methods analogous to
those described above. In these experiments, the specific TSAR
libraries used were R26, D38 and DC43. See FIGS. 10-12 for
description of these libraries. Phage bound to PEM were eluted by
either standard acid elution methods, stringent acid elution
methods where phage were incubated with the PEM peptide for only 10
minutes prior to washing and elution, or were eluted using excess
PEM peptide. Phage from each library were isolated that express
peptides capable of binding to PEM. The amino acid sequences of PEM
binding phage are shown in Table 6.
6TABLE 6 Sequences of PEM Binding Phage Acid Eluted R26 Library A15
SFMDYFFHTPEPKPAGYPNAYTDPKHPA (SEQ ID NO: 26) A54
SSSIFDYAPFSWGSAGLSNSSINVFERS (SEQ ID NO: 27) A5
SASLWDALGGWTTSAVPSYPRPHQTPGR (SEQ ID NO: 28) A39
SLGLPWIDVFGRSSAEPWPFGRTNLPRS (SEQ ID NO: 29) A16
SVHGAFLDSFFPWAADGPHGRGRL-TSF (SEQ ID NO: 30) DC43 Library MA-8
EEKQGGRWSTMMPRPWCHEGGCGFLYYDAMTKPKTPPIMRTAA (SEQ ID NO: 31) MA-21
LPRPFDDASWKLRAVKESPDGCGFGSPLLFPPYSGLPTFSSCD (SEQ ID NO: 32) V22
GSFESARGVTCIGNHSIGAHGCGPLRSYASFNRGSGRRH (SEQ ID NO: 33) D38 Library
MA-32 DQIGSRPQTTSRSISGSWWENAKTLWQQDYAFSAPNAA (SEQ ID NO: 34) V23
LSDAWGNFTTSYRDSAGFPSHAMTTSQGGKRNHASRFP (SEQ ID NO: 35) V21
VQLDDTSPRASGQETSQSEYDARPLLSKFAIPRPWSR (SEQ ID NO: 36) V1
IDSSKNRISGTGYLSFPHIRHANRRHMADDSNLAPGPS (SEQ ID NO: 37) Acid Eluted:
"Stringent" DC43 Library V44
WSIGTHTGPEGKFRIPCDRSGCGGTTLTHGGLNSSPTGQHERP (SEQ ID NO: 38) V39
DPCEDGYWLSSVGRAGASIRGCGAIRRSSRTLTAEYSTRASNH (SEQ ID NO: 39) V10
GSKRSCWGTTISNYFRPVPEGCGSASSINPNTNTGRLPSLHRQ (SEQ ID NO: 40) V7
SSASSGCLGRAEHLDLDSVWGCGSQADMSRRYSPWYGRPRTGV (SEQ ID NO: 41) V4
NVMWSSSKAGIRDCSQVPPGGCGPVNRHRASPPLTPFRHGSIR (SEQ ID NO: 42) D38
Library V45 PLTSGSSSEYRNRDDCPVYKYATNCPRLNFSPSRYSPF (SEQ ID NO: 43)
V32 GDAYGGIFSRPRQGLADSYIHASYTGKHFFRGPRPPTR (SEQ ID NO: 44) V27
STCIGAEGEWKSFHNFLQCRDATSTSSSTLDPTALRFG (SEQ ID NO: 45) V40
YSATLWDQFGSRQVELWSNRHASSAlPFASRASVLGSR (SEQ ID NO: 46) Peptide
Eluted R26 Library P24 ILGWPFLTGLGDSTVHPRGRKGTD- PS (SEQ ID NO: 47)
P49 SIPSFSMWLNQLGSAALPSKGNSQDRSD (SEQ ID NO: 48) P26
SRDDIFTGGPLVLFRGSKTSNHDVHSMR (SEQ ID NO: 49) P6
RAELVNWYEWFHVTAEAETPVINSHNMT (SEQ ID NO: 50) DC43 Library MP-1
GAPVWRGNPRWRGPGGFKWPGCGNGPMCNTFTPARGG- SRNNGP (SEQ ID NO: 51) MP-2
GSASSCFPNFTARGVTVGFFGCGSPAHPA- APRVLNPATDFPAP (SEQ ID NO: 52) MP-22
VFRRTARSSRPIGATVFPWYGCGNSNDETLPHHDSPPSFFLGA (SEQ ID NO: 53) MA-13
NTCWTDLFWHGLPGGDLPRDGCGLPSELTTHPSRERRDASEN (SEQ ID NO: 54) D38
Library MP-20 IDWNWLERGQHNRGYLHSFPDAKSQPTRGPRVAPNGND (SEQ ID NO:
55) MP-30 GRGSDMREHWPWSMPLILDQHANDPSPRAQSHYY- SHPF (SEQ ID NO:
56)
[0252] 6.2.2. Saturation Mutagenesis of MP-1 and Identification of
Additional PEM Binding Abtides
[0253] A saturation mutagenesis library based on one of the PEM
abtides, MP-1, was constructed. Nucleotide sequences encoding the
MP-1 abtide were synthesized using a doping scheme similar to that
described in Section 5.3 whereby each nucleotide was contaminated
with 9% of each of the other 3 nucleotides (e.g. G=73% G, 9% A, 9%
T, 9.degree. C.). The resulting mutagenic oligonucleotides were
used to construct a library by TSAR library methods described above
(see FIG. 13).
[0254] The resulting library was screened to identify phage
expressing abtides capable of binding to PEM. The binding of
isolated phage to PEM was confirmed by an ELISA assay. Phage that
were shown to bind to PEM as well as phage that did not bind to PEM
were sequenced to determine the amino acid sequences of the
expressed abtides. Table 7 shows the amino acid sequences of these
positive and negative binding phage.
7TABLE 7 Sequence Comparison: MP-1 Binding Motif Positive Binding
Sequences MP1 GAPVWRGNPRWRGPGGFKWPGCGNGPMCNTFTPARGGSRNNGP (SEQ ID
NO: 51) E4 VSTGWSGTPRWCAPGGKQGSGCGNGPRWTTLTPDLGGTRKYGP (SEQ ID NO:
57) E7 GAPLWCEKLSGTGSGGFKWPGCGSGPTYNTFTPARVGSDNKWP (SEQ ID NO: 58)
E16 GPPVWSAKSRWTGTGVLNWPGCGKVPSCSTYTPSRDRSRKSDP (SEQ ID NO: 59) E21
GSALLTSKGCVRGPGGLMRPGCGNDRLGKSSTYAHGGWIKTGP (SEQ ID NO: 60) E33
GSPVWSGDNRWRGSSPLKRPGCGNGAKCNTLKDNRKDSRKTK- H (SEQ ID NO: 61) E44 G
PLLPGEAAVHGARGLMRSGCGNGPTWNRLTAACR- DSRNKGP (SEQ ID NO: 62) E65
GSPVWMGSTRWTGHGWFRSQGCGNVPRTNS- CAPAGKDSQNKGP (SEQ ID NO: 63) E73
GAPVWRGNRWCSDNGELERPGCGY- GPRFNILPPGRGNSRKPSP (SEQ ID NO: 64) E84
GSSGWKVKHRCGGPGTLQRPGCGNLPLGHTFPPTRGGSHMEGA (SEQ ID NO: 65) E85
GPRSWMGQPRGSDAGSCKWAGCGDAPMWRASTPGHGGPPNRGS (SEQ ID NO: 66) E88
EALVCRGKPPWSGPAGLLWQGCGTGPVSRTFTSAQGRSRNKTS (SEQ ID NO: 67) E90
GAPVVGDILWCSGARGAKWPGCGKGPTNKTFSHSRGGTQKSGL (SEQ ID NO: 68) E22
GAPVSRCKPACGGFWGVNWPGCGNASMCKTFTNGHGVSSDNGH (SEQ ID NO: 69) E29
GAHGYKNGSTCTGLGGWRCRGCGKGAMCNNPSPAGGAYHNQG- P (SEQ ID NO: 70) E62 G
PQGSEHQCCSGHWGLKFPGCGNGPICNNFTALRG- ASRKNGP (SEQ ID NO: 71) E64
GEPVWCRHSGGRVQGGLDWLGCGDGPLRYT- VTPARGGPSKHGP (SEQ ID NO: 72) E66
GLSLVRGDSWGSGAGGWKRHGCGH- GPMYNPQTPARGGSCTRNT (SEQ ID NO: 73) E67
VSRAWSGKPRLMGSHGLNCPGCGKGHSGIMFIPDPAGSANTPP (SEQ ID NO: 74) E68
CAPMWSGKPPWCVGGGVKFRGCGNRPDCNIITPRLVESRDKAL (SEQ ID NO: 75) E70
ADPVCSRKPDGGGLRGLRWPGCGKGPILYNVTAARGGSRNNGP (SEQ ID NO: 76)
Negative Binding Sequences MPI
GAPVWRGNPRWRGPGGFKWPGCGNGPMCNTFTPARGGSRNNGP (SEQ ID NO: 51) E3
GTRVPPGFALRGGRDGLSWAGCGKAPISKTYTSARGRSRKKGS (SEQ ID NO: 77) E15
RSAVSEGKPREIVPGGCMWPGCGNGRKSNTLTHGPEQFQEIEP (SEQ ID NO: 78) E24
SSGVGNGKPRSWAPDALNGGCGNIQFANTITPDRGGSCNQTL (SEQ ID NO: 79) E27
GSSVCGGQPSGRGFGGLPGPGCGNGPTSNTLTSARGGFPNKGL (SEQ ID NO: 80) E37
GAPLWQGDPADEVLGGSMIPGCGIGALSQTFTPTPGGSRKNV- T (SEQ ID NO: 81) E43
AGRELRQDEGEGGAGADVARLREGPICSTFTPARGG- SCPSGL (SEQ ID NO: 82) E49
QARVSMAISCRSGPSDLMHQGCGYGPRCNPD- TTDSGGSHTNTP (SEQ ID NO: 83) E60
GDPECRGKPRGRWTGSLACTGCGNG- PNSKICTRARGVSRNKGP (SEQ ID NO: 84) E72
STPGCSGYSGSGDPRCLTCTACGNGHTRKTLTPAHGRSTHKEP (SEQ ID NO: 85) E34
GQPECRITSGCCGTDGNKWLGCGKVDMCNTLNPAVGCHGTNGS (SEQ ID NO: 86) E83
REPVVGGKPWCRGPGGLRWRGCGKSQFDKIITLSRDNRRDKRP (SEQ ID NO: 87)
[0255] When the sequences shown in Table 7 are compared (see
particularly the amino acid residues marked in boldface type), it
is possible to determine the influence of particular amino acid
residues at specific positions in the sequence on a peptide's
ability to bind to PEM. Abtides that bind to PEM can be
characterized by the formula:
8
R.sub.1R.sub.2R.sub.3R.sub.4R.sub.5R.sub.6R.sub.7R.sub.8R.sub.9R.-
sub.9R.sub.10R.sub.11R.sub.12R.sub.13R.sub.14R.sub.15R.sub.16R.sub.17R.sub-
.18R.sub.19R.sub.20R.sub.21R.sub.22R.sub.23R.sub.24R.sub.25R.sub.26R.sub.2-
7R.sub.28R.sub.29 (SEQ ID NO: 88) R.sub.30R.sub.31R.sub.3-
2R.sub.33R.sub.34R.sub.35R.sub.36R.sub.37R.sub.38R.sub.39R.sub.40R.sub.41R-
.sub.42R.sub.43
[0256] where:
[0257] R.sub.1=G, C, E, or V, preferably G;
[0258] R.sub.2=A, S, P, or L, preferably A;
[0259] R.sub.3=P, T, H, or L, preferably P;
[0260] R.sub.4=L, M, Q, G, A, or S;
[0261] R.sub.5=W or Y, preferably W;
[0262] R.sub.6=S, C, K or T, preferably S;
[0263] R.sub.7=E, S, C, D, V, or R;
[0264] R.sub.8=N, H, K, S, or E;
[0265] R.sub.9=L, H, R, N, Q, T, or G;
[0266] R.sub.10=W, P, R, T, or D, preferably W;
[0267] R.sub.11=W, C, V, L, or G, preferably W;
[0268] R.sub.12=S, T, M, or H, preferably S or T;
[0269] R.sub.13=G;
[0270] R.sub.14=S, A, G, N, Q, or H, preferably S;
[0271] R.sub.15=W, H, G, A, or R;
[0272] R.sub.16=G, T, E, P, V, or W, preferably G;
[0273] R.sub.17=V, F, W, K, or A;
[0274] R.sub.18=K, Q, D, E, R, or L, preferably K;
[0275] R.sub.19=R, F, or S, preferably R;
[0276] R.sub.20=P, S, I or H, preferably P;
[0277] R.sub.21=G;
[0278] R.sub.22=C;
[0279] R.sub.23=G;
[0280] R.sub.24=D, S, T, N, or H;
[0281] R.sub.25=G, D, L, or R;
[0282] R.sub.26=P or S, preferably P;
[0283] R.sub.27=M, S, D, I, L, or R;
[0284] R.sub.28=G, W, C, L, F, Y, or T, preferably G or W;
[0285] R.sub.29=S, N, V, F, H, or R;
[0286] R.sub.30=N, A, S, M, or R, preferably N;
[0287] R.sub.31=F, Q, P, or V, preferably F;
[0288] R.sub.32=S, V, I, K, A, or S;
[0289] R.sub.33=P, A, N, or Y, preferably P;
[0290] R.sub.34=G, N, or L;
[0291] R.sub.35=K, R, C, Q or L, preferably K or R;
[0292] R.sub.36=V, K, R, or A;
[0293] R.sub.37=G, D, A, or E, preferably G;
[0294] R.sub.38=S, T, P, Y or W; preferably S;
[0295] R.sub.39=R, I, L, P, A or S;
[0296] R.sub.40=N, K, or M, preferably N or K;
[0297] R.sub.41=S, R, T, E, Q, P, Y or H;
[0298] R.sub.42=G, A, S, D, N, P, Y, or K, preferably G;
[0299] R.sub.43=P, H or A.
[0300] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and accompanying figures. Such modifications
are intended to fall within the scope of the appended claims.
[0301] Various publications are cited herein, the disclosures of
which are incorporated by reference in their entireties.
Sequence CWU 1
1
* * * * *