Binding peptides for carcinoembryonic antigen (CEA)

Details Binding peptides for carcinoembryonic antigen (CEA) Binding peptides for carcinoembryonic antigen (CEA)

2000-04-03

2004-08-10

Canella, Karen A. (Department: 1642)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

C530S326000, C530S350000

Reexamination Certificate

active

06774209

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to CEA binding polypeptides and compositions for detection and treatment of cancer. More particularly, the invention relates to materials useful for and methods of detecting, imaging, localizing, and targeting tumors exhibiting CEA. The invention provides binding polypeptides capable of associating specifically with CEA and of distinguishing between CEA and known cross-reactive antigens, such as NCA (non-specific cross-reacting antigen). Such binding polypeptides are useful for the detection, imaging, localization, and targeting of CEA-containing tissues or solutions, e.g., by radioimaging, magnetic resonance imaging, or x-ray imaging, and are also useful in the diagnosis and treatment of cancers associated with CEA.
BACKGROUND OF THE INVENTION
Carcinoembryonic antigen, or CEA, is a complex immunoreactive glycoprotein with a molecular weight of 180,000 found in adenocarcinomas of endodermally derived digestive system epithelia and fetal colon. Tumor cells at many sites, including colon, breast, lung, cervix, ovary, stomach, bladder, pancreas and esophagus express large amounts of carcinoembryonic antigen and/or the closely related immunoglobulin supergene family member, nonspecific cross-reactive antigen, or NCA, on their surfaces. The expression of these glycoproteins, especially CEA, in normal cells is very limited in mature individuals (as opposed to prenatal infants), and this antigen has been used as a target in immunoassays for diagnosis and for serially monitoring cancer patients for recurrent disease or response to therapy. (See, Mach et al.,
Immun. Today,
2:239, 1981; Berche et al.,
Br. Med. J.,
285: 1447, 1982.) Anti-CEA antibodies also have been proposed for cancer therapy and for use in forming immunoconjugates which in turn can be used in cancer therapy. (See, e.g., Buchegger et al., U.S. Pat. No. 5,047,507 (1991); Osbourne et al. U.S. Pat. No. 5,872,215 (1999).)
CEA was as first described by Gold and Freedman,
J. Exp. Med.,
121: 439, 1965, and has now been completely sequenced and characterized (see, Beauchemin et al.,
Mol. Cell. Biol.,
7:3221-30, 1987; WO 95/06067). CEA has a domain structure of N-A1-B1-A2-B2-A3-B3-GPI where GPI is a glycophosphatidylinositol membrane anchor. A significant degree of sequence homology exists between the domains of CEA and other members of the immunoglobulin supergene family, and immunological cross-reactivity between CEA and as many as sixteen other homologous antigens, such as NCA and biliary glycoprotein-1 (BGP-1), has been reported.
One of the major drawbacks of the use of anti-CEA antibodies for clinical purposes has been the cross-reactivity of these antibodies with some apparently normal adult tissues. Previous studies have shown that most conventional hyperimmune antisera raised against different immunogenic forms of CEA cross-react with CEA-related antigens found in normal colonic mucosa, spleen, liver, lung, sweat glands, polymorphonuclear leukocytes and monocytes of normal individuals, as well as many different types of carcinomas.
Accordingly, there is a great need for binding moieties that bind to CEA but do not cross-react with other antigens such as NCA. This and other objects are accomplished herein with the discovery of novel peptide binders of CEA.
SUMMARY OF THE INVENTION
The present invention addresses the need for improved materials and methods for detecting, localizing, measuring and treating CEA-expressing cells by providing a group of non-naturally occurring polypeptides that bind specifically to CEA. Appropriate labeling of such polypeptides provides detectable imaging agents that bind at high concentration to a CEA-expressing tumor, providing excellent tumor-specific imaging agents. Conjugation or fusion of such polypeptides with effective agents such as cytokines, chemotherapeutic agents, radionuclides or other cancer therapeutics produce conjugates that can be used for cancer therapy, i.e., by causing the conjugate to target the site of a tumor that is producing CEA. Recombinant baeteriophage displaying the CEA-binding polypeptides of the invention have been identified and isolated, and such phage products are also valuable reagents for effective detection and diagnosis of cancers associated with the expression of CEA in cells and tissues. The CEA binding moieties of the instant invention can be used in the detection, diagnosis, and therapy of such CEA-related disorders.
This invention pertains to CEA binding moieties. Binding moieties according to this invention are useful in any application where binding, detecting or isolating CEA or its fragments is advantageous. A particularly advantageous use of the binding moieties disclosed herein is in a method of imaging cells or tissues expressing CEA in vivo. The method entails the use of CEA specific binding moieties according to the invention for detecting CEA-expressing cells, where the binding moieties have been detectably labeled for use as imaging agents, including magnetic resonance imaging (MRI) contrast agents, x-ray imaging agents, radiopharmaceutical imaging agents, ultrasound imaging agents. and optical imaging agents.
Preferred CEA binding moieties according to the invention are isolated synthetic polypeptides having a high affinity for CEA. This invention provides a new class of CEA binding polypeptides having an amino acid sequence comprising:
X
1
-X
2
-X
3
-Cys-X
4
-X
5
-X
6
-X
7
X
8
-X
9
-X
10
-X
11
-Cys-X
12
-X
13
-X
14
  (SEQ ID NO:1),
wherein
X
1
is Asn, Asp, or is absent;
X
2
is Trp;
X
3
is Asp, Phe, or Val;
X
4
is Asn, Glu, or Met;
X
5
is Asn, Leu, Met or Phe;
X
6
is Asp, Gly, Ile, Lys, Phe or Thr;
X
7
is Ala, Gin, Gly, Lys, or Thr;
X
8
is Arg, Asn, Asp, Glu, or Gly;
X
9
is Gln, Gly, or Leu;
X
10
is Ala, Trp or Tyr;
X
11
is Ala, Gly, His, Phe, Thr, or Val;
X
12
is Asn, Gln, Phe, Ser or Val;
X
13
is Arg, Leu, Pro or Ser; and
X
14
is Leu, Ser, Trp, or Tyr;
and wherein said polypeptide has the ability to bind CEA. Said polypeptide may have additional amino acids attached at either end. Peptides having a serine at the N-terminus (before X
1
) are preferred embodiments.
Preferred CEA binding polypeptides of the above formula will have the amino acid sequence:
X
1
-Trp-Val-Cys-Glu-X
5
-X
6
-Lys-X
8
-Gln-Trp-X
11
-Cys Asn-X
13
-X
14
  (SEQ ID NO:2),
wherein
X
1
is Asn or Asp;
X
5
is Asn, Leu, Met or Phe;
X
H
is Asp, Gly, Ile, Lys, Phe or Thr;
X
5
is Arg, Asn. Asp, Glu, Gly or Trp;
X
11
is Ala, Gly, His, Phe, Thr, Tyr or Val;
X
13
is Arg, Leu, Pro or Ser; and
X
14
is Leu, Ser, Trp or Tyr;
In particular, a stable binding loop having a high affinity for CEA is disclosed, having the formula:
Cys-X
4
-X
5
-X
6
-X
7
-X
8
-X
9
-X
10
-X
11
-Cys  (SEQ ID NO: 3),
wherein
X
4
is Asn, Glu, or Met;
X
5
is Asn, Leu, Met or Phe;
X
6
is Asp, Gly, Ile, Lys, Phe or Thr;
X
7
is Ala, Gln, Gly, Lys, or Thr;
X
8
is Arg, Asn, Asp, Glu, or Gly;
X
9
is Gln, Gly, or Leu;
X
10
is Ala, Trp or Tyr;
X
11
is Ala, Gly, His, Phe, Thr, or Val;
and wherein it is preferred that
X
4
is Glu;
X
5
is Asn, Leu, Met or Phe;
X
6
is Asp, Gly, Ile, Lys, Phe or Thr;
X
7
is Lys;
X
8
is Arg, Asn, Asp, Glu, or Gly;
X
9
is Gln:
X
10
is Trp; and
X
11
is Ala, Gly, His, Phe, Thr, or Val.
Preferred polypeptides according to the invention comprise an amino acid sequence:
Asn-Trp-Val-Cys-Asn-Leu-Phe-Lys-Asn-Gln-Trp-Phe-Cys-Asn-Ser-Tyr  (SFQ ID NO:4)(TN 10/9-G08),
Asp-Trp-Val-Cys-Glu-Asn-Lys-Lys-Asp-Gln-Trp-Thr-Cys-Asn-Leu-Leu  (SEQ ID NO:5)(TN10/9-A07),
 Asn-Trp-Asp-Cys-Met-Phe-Gly-Ala-Glu-Gly-Trp-Ala-Cys-Ser-Pro-Trp  (SEQ ID NO:6)(TN10/9-E01),
Asp-Trp-Val-Cys-Glu-Lys-Thr-Thr-Gly Gly-Tyr-Val-Cys-Gln-Pro-Leu  (SEQ ID NO:7)(TN10/9-B09),
Asn-Trp Phe-Cys-Glu-Met-Ile-Gly-Arg-Gln Trp-Gly-Cys-Val-Pro-Ser  (SEQ ID NO:8)(TN10/9-F11),
and
Asp-Trp-Val-Cys-Asn-Phe-Asp-Gln-Gly-Leu-Ala-His-Cys-Phe-Pro-Ser  (SEQ ID NO:9)(TN10/9-D04).
The most preferred CEA binding moieties according to the invention are isolated, synthetic

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