U.S. patent number 7,569,673 [Application Number 10/519,580] was granted by the patent office on 2009-08-04 for humanized anti-tag 72 cc49 for diagnosis and therapy of human tumors.
This patent grant is currently assigned to N/A, The United States of America as represented by the Department of Health and Human Services. Invention is credited to Syed V. S. Kashmiri, Eduardo A. Padlan, Jeffrey Schlom.
United States Patent |
7,569,673 |
Kashmiri , et al. |
August 4, 2009 |
**Please see images for:
( Certificate of Correction ) ** |
Humanized anti-tag 72 cc49 for diagnosis and therapy of human
tumors
Abstract
The present disclosure provides humanized CC49 monoclonal
antibodies that bind TAG-72 with high binding affinity and that are
minimally immunogenic. In one embodiment, a humanized CC49 antibody
includes a non-conservative amino acid substitution in a light
chain complementarity determining region 3 of the CC49 antibody. In
a further embodiment, the humanized CC49 antibody includes a
non-conservative substitution of a first residue in a light chain
complementarity determining region 3 and a substitution of a second
residue in a complementarity determining region of the humanized
CC49 antibody. In several of the embodiments, methods are disclosed
for the use of a humanized CC49 antibody in the detection or
treatment of a tumor in a subject. Also disclosed is a kit
including the humanized CC49 antibody described herein.
Inventors: |
Kashmiri; Syed V. S.
(Gaithersburg, MD), Schlom; Jeffrey (Potomac, MD),
Padlan; Eduardo A. (Kensington, MD) |
Assignee: |
The United States of America as
represented by the Department of Health and Human Services
(Washington, DC)
N/A (N/A)
|
Family
ID: |
30000969 |
Appl.
No.: |
10/519,580 |
Filed: |
June 26, 2003 |
PCT
Filed: |
June 26, 2003 |
PCT No.: |
PCT/US03/20367 |
371(c)(1),(2),(4) Date: |
July 11, 2005 |
PCT
Pub. No.: |
WO2004/003155 |
PCT
Pub. Date: |
January 08, 2004 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20060165680 A1 |
Jul 27, 2006 |
|
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
60393077 |
Jun 28, 2002 |
|
|
|
|
Current U.S.
Class: |
530/387.3;
530/388.8; 530/388.1; 435/70.21; 435/7.23; 435/69.6; 424/178.1;
424/155.1; 424/141.1; 424/133.1; 530/391.3 |
Current CPC
Class: |
C07K
16/30 (20130101); A61P 35/00 (20180101); A61K
51/1045 (20130101); A61K 2039/505 (20130101); C07K
2317/92 (20130101); C07K 2317/24 (20130101); C07K
2317/55 (20130101); C07K 2317/622 (20130101) |
Current International
Class: |
C12P
21/08 (20060101); C07K 16/00 (20060101); G01N
33/574 (20060101); A61K 39/395 (20060101); C07H
21/04 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
2131355 |
|
Apr 2001 |
|
CA |
|
2068593 |
|
Jul 2003 |
|
CA |
|
0239400 |
|
Sep 1987 |
|
EP |
|
0365997 |
|
May 1990 |
|
EP |
|
WO 89/00692 |
|
Jan 1989 |
|
WO |
|
WO 89/01783 |
|
May 1989 |
|
WO |
|
WO 90/04410 |
|
May 1990 |
|
WO |
|
WO 91/00295 |
|
Jan 1991 |
|
WO |
|
WO 93/12231 |
|
Jun 1993 |
|
WO |
|
WO 96/13594 |
|
May 1996 |
|
WO |
|
WO 97/26010 |
|
Jul 1997 |
|
WO |
|
WO 98/18809 |
|
May 1998 |
|
WO |
|
WO 99/43816 |
|
Feb 1999 |
|
WO |
|
WO 00/26394 |
|
May 2000 |
|
WO |
|
Other References
Iwahashi et al. Molecular Immunology, 1999. 36:1079-1091. cited by
examiner .
Paul, Fundamental Immunology, 3rd Edition, 1993, pp. 292-295. cited
by examiner .
Rudikoff et al. Proc. Natl. Acad. Sci. USA, 79:1979-1983, Mar.
1982. cited by examiner .
Colman. Research in Immunology, 145:33-36, 1994. cited by examiner
.
Bendig M. M. Methods: A Companion to Methods in Enzymology, 1995;
8:83-93. cited by examiner .
MacCallum et al. J. Mol. Biol., 262, 732-745, 1996. cited by
examiner .
Casset et al. Biochemical and Biophysical Research Communications,
307:198-205, 2003. cited by examiner .
Abergel et al., "Crystallographic Studies and Primary Structure of
the Antitumor Monoclonal CC49 Fab'," Proteins: Structure, Function,
and Genetics 17:438-443, 1993. cited by other .
Colcher et al., "Radioimmunolocalization of Human Carcinoma
Xenografts with B72.3 Second Generation Monoclonal Antibodies,"
Cancer Research 48:4597-4603, Aug. 15, 1988. cited by other .
De Pascalis et al., "Grafting of "abbreviated"
complementarity-determining regions containing
specificity-determining residues essential for ligand contact to
engineer a less immunogenic humanized monoclonal antibody," J.
Immunol. 169:3076-3084, 2002. cited by other .
De Pascalis et al., "In vitro affinity maturation of a
specificity-determining region-grafted humanized anticarcinoma
antibody: isolation and characterization of minimally immunogenic
high-affinity variants," Clin. Can. Res. 9:5521-5531, 2003. cited
by other .
Divgi et al., "Clinical Comparison of Radiolocalization of Two
Monoclonal Antibodies (mAbs) Against the TAG-72 Antigen," Nucl.
Med. Biol. 21(1):9-15, 1994. cited by other .
Gonzales et al., "Minimizing immunogenicity of the SDR-grafted
humanized antibody CC49 by genetic manipulation of the framework
residues," Mol. Immunol. 40:337-349, 2003. cited by other .
Hakimi et al., "Reduced immunogenicity and improved
pharmacokinetics of humanized anti-Tac in cynomolgus monkeys," J.
Immunol. 147:1352-1359, 1991. cited by other .
Hand et al., "Potential for Recombinant Immunoglobulin Constructs
in the Management of Carcinoma," Cancer Supplement 73(3):1105-1113,
Feb. 1, 1994. cited by other .
Iwahashi et al., "CDR substitutions of a humanized monoclonal
antibody (CC49): contributions of individual CDRs to antigen
binding and immunogenicity," Mol. Immunol. 36:1079-1091:1999. cited
by other .
Johnson et al., "Analysis of a Human Tumor-associated Glycoprotein
(TAG-72) Identified by Monoclonal Antibody B72.3," Cancer Research
46:850-857, Feb. 1986. cited by other .
Jones et al., "Replacing the Complementarity-determining Regions in
a Human Antibody with those from a Mouse," Nature 321:522-525, May
29, 1986. cited by other .
Kashmiri et al., "Generation, characterization, and in vivo studies
of humanized anticarcinoma antibody CC49," Hybridoma 14(5):461-473,
1995. cited by other .
Kashmiri et al., "Development of a minimally immunogenic variant of
humanized anti-carcinoma monoclonal antibody CC49," Crit. Rev.
Oncol. Hematol. 38:3-16, 2001. cited by other .
Kashmiri et al., Chapter 21 in Methods in Molecular Biology, vol.
248: Antibody Engineering: Methods and Protocols, p. 361-376; Lo
(ed.), Humana Press, Inc., Tolowa, NJ, 2003. cited by other .
Mulligan et al., "Phase I Study of Intravenous .sup.177Lu-labeled
CC49 Murine Monoclonal Antibody in Patients with Advanced
Adenocarcinoma," Clinical Cancer Research 1:1447-1454, Dec. 1995.
cited by other .
Muraro et al., "Generation and Characterization of B72.3 Second
Generation Monoclonal Antibodies Reactive with the Tumor-associated
Glycoprotein 72 Antigen," Cancer Research 48:4588-4596, Aug. 15,
1988. cited by other .
Padlan, "A Possible Procedure for Reducing the Immunogenicity of
Antibody Variable Domains while Preserving their Ligand-binding
Properties," Molecular Immunology 28(4/5):489-498, 1991. cited by
other .
Padlan, "Anatomy of the antibody molecule," Mol. Immunol.
31:169-217, 1994. cited by other .
Padlan et al., "Identification of Specificity-determining Residues
in Antibodies," The FASEB Journal 9:133-139, Jan. 1995. cited by
other .
Paul, Fundamental Immunology, Raven Press, NY, Ch. 8, p. 242, 1993.
cited by other .
Reichman et al., "Reshaping human antibodies for therapy," Nature
(London) 332:323-327, 1988. cited by other .
Rixon et al., "Preferential Use of a H Chain V Region in
Antitumor-associated Glycoprotein-72 Monoclonal Antibodies," The
Journal of Immunology 151(11):6559-6568, Dec. 1, 1993. cited by
other .
Rudikoff et al., "Single amino acid substitution altering
antigen-binding specificity," Proc Natl Acad Sci U S A. March;
79(6): 1979-1983, 1982. cited by other .
Saldanha et al., "A single backmutation in the human kIV framework
of a previously unsuccessfully humanized antibody restores the
binding activity and increases the secretion in cos cells," Mol.
Immunol. 36:709-719, 1999. cited by other .
Schier et al., "Isolation of picomolar affinity anti-c-erbB-2
single-chain Fv by molecular evolution of the complementarity
determining regions in the center of the antibody binding site," J.
Mol. Biol. 263:551-567, 1996. cited by other .
Sha et al., "A heavy-chain grafted antibody that recognizes the
tumor-associated TAG72 antigen," Cancer Biother. 9(4):341-349,
1994. cited by other .
Sharkey et al., "Evaluation of a complementarity-determining
region-grafted (humanized) anti-carcinoembryonic antigen monoclonal
antibody in preclinical and clinical studies," Cancer Res.
55:5935s-5945s, 1995. cited by other .
Slavin-Chiorini et al., "Biological properties of chimeric
domain-deleted anticarcinoma immunoglobulins," Cancer Res. 55(23
Suppl.):5957s-5967s, 1995. cited by other .
Slavin-Chiorini et al., "A CDR-grafted (humanized) domain-deleted
antitumor antibody," Cancer Biother. Radiopharm. 12:305-316, 1997.
cited by other .
Tamura et al., "Structural correlates of an anticarcinoma antibody:
identification of specificity-determining residues (SDRs) and
development of a minimally immunogenic antibody variant by
retention of SDRs only," J. Immunol. 164:1432-1441, 2000. cited by
other .
Wu et al., "Humanization of a murine monoclonal antibody by
simultaneous optimization of framework and CDR residues," J. Mol.
Biol. 294:151-162, 1999. cited by other .
Xiang et al., "The tyrosine residue at position 97 in the VH CDR3
region of a mouse/human chimeric anti-colorectal carcinoma antibody
contributes hydrogen bonding to the TAG72 antigen," Cancer Biother.
8:253-262, 1993. cited by other .
Xiang et al., "Complementarity determining region residues aspartic
acid at H55, serine at H95 and tyrosines at H97 and L96 play
important roles in the B72.3 antibody-TAG-72 antigen interaction,"
Protein Eng. 9:539-543, 1996. cited by other .
Xiang et al., "Light-chain framework region residue Tyr71 of
chimeric B72.3 antibody plays an important role in influencing the
TAG72 antigen binding," Protein Eng. 12:417-421, 1999. cited by
other .
Adams and Schier, "Generating improved single-chain Fv molecules
for tumor targeting," Journal of Immunological Methods,
231(1-2):249-260, 1999. cited by other .
De Pascalis et al., "In Vitro Affinity Maturation of a
Specificity-Determining Region-Grafted Humanized Anticarcinoma
Antibody: Isolation and Characterization of Minimally Immunogenic
High-Affinity Variants," Clinical Cancer Research, 9(15):5521-5531,
2003. cited by other .
Osbourn et al., "Generation of a panel of related human scFv
antibodies with high affinities for human CEA," Immunotechnology,
2(3):181-196, 1996. cited by other .
Supplementary Partial European Search Report issued on Feb. 27,
2006, for European Patent Application No. EP03762161.2. cited by
other.
|
Primary Examiner: Blanchard; David J.
Attorney, Agent or Firm: Klarquist Sparkman, LLP.
Parent Case Text
PRIORITY CLAIM
This is the .sctn. 371 U.S. National Stage of International
Application No. PCT/US2003/020367, filed Jun. 26, 2003, which was
published in English under PCT Article 21(2), which in turn claims
the benefit of U.S. Provisional Application No. 60/393,077, filed
Jun. 28, 2003, which is incorporated herein by reference.
Claims
We claim:
1. A humanized CC49 antibody, deposited as ATCC Accession number
PTA-4182 or ATCC Accession number PTA-4183.
2. A humanized CC49 antibody, comprising: four variable light
framework regions and four variable heavy framework regions of a
human antibody; a light chain complementarity determining region
(L-CDR)1, a L-CDR2, a L-CDR3, a heavy chain complementarity
determining region (H-CDR)1, a H-CDR2, and a H-CDR3; a
non-conservative substitution of a first residue, wherein the first
residue is in the L-CDR 3 of the antibody; and a substitution of a
second residue, wherein the second residue is in any L-CDR or H-CDR
of the antibody; wherein the non-conservative substitution of the
first residue at position 91 is a tyrosine to proline substitution,
the substitution of the second residue at position 27b is a valine
to leucine substitution, the L-CDR1, L-CDR2, L-CDR3, H-CDR1,
H-CDR2, and H-CDR3 are the parent HuCC49V10 antibody L-CDR1,
L-CDR2, L-CDR3, H-CDR1, H-CDR2, and H-CDR3, respectively, and the
humanized CC49 antibody has a high binding affinity for TAG-72 and
is minimally immunogenic, compared to HuCC49V10, deposited as ATCC
Accession No. PTA-5416.
3. A humanized CC49 antibody, comprising: a light chain
complementarity determining region (L-CDR)1, a L-CDR2, and a
L-CDR3, a heavy chain complementarity determining region (H-CDR)1,
a H-CDR2, and a H-CDR3, all of a parent CC49 antibody, wherein the
L-CDR3 of the humanized CC49 antibody or of an antigen binding
fragment of the humanized CC49 antibody comprises a
non-conservative amino acid substitution at position 91 and has a
high binding affinity for TAG-72, compared to the parent CC49
antibody, wherein the parent CC49 antibody is HuCC49V10, deposited
as ATCC Accession No. PTA-5416.
4. The humanized CC49 antibody of claim 3, wherein the
non-conservative substitution is a tyrosine to proline
substitution.
5. A humanized CC49 antibody, comprising: a light chain
complementarity determining region (L-CDR)1, a L-CDR2, and a
L-CDR3, a heavy chain complementarity determining region (H-CDR)1,
a H-CDR2, and a H-CDR3, all of a parent CC49 antibody, wherein the
L-CDR3 of the humanized CC49 antibody or of an antigen binding
fragment of the humanized CC49 antibody comprises a tyrosine to
proline substitution at position 91 and has a high binding affinity
for TAG-72, compared to the parent CC49 antibody, wherein the
parent CC49 antibody is HuCC49V10, deposited as ATCC Accession No.
PTA-5416.
6. The antibody of claim 5, wherein the high binding affinity is at
least about 1.2.times.10.sup.-8M.
7. The antibody of claim 5, wherein the humanized CC49 antibody is
minimally immunogenic.
8. The antibody of claim 5, wherein the humanized CC49 antibody
further comprises an effector molecule.
9. The antibody of claim 8, wherein the effector molecule is a
detectable label.
10. A pharmaceutical composition comprising a therapeutically
effective amount of the antibody of claim 5 in a pharmaceutically
acceptable carrier.
11. A humanized CC49 antibody, comprising: four variable light
framework regions and four variable heavy framework regions of a
human antibody; a light chain complementarity determining region
(L-CDR)1, a L-CDR2, and a L-CDR3 of the parent HuCC49V10 antibody,
a heavy chain complementarity determining region (H-CDR)1, a
H-CDR2, and a H-CDR3 of the parent HuCC49V10 antibody; a
non-conservative substitution of a residue at position 91 in the
L-CDR3 of the antibody; and a substitution of a residue at position
27b of L-CDR1 of the antibody; wherein the humanized CC49 antibody
has a high binding affinity for TAG-72 and is minimally
immunogenic, compared to a parent HuCC49V10 antibody, deposited as
ATCC Accession No. PTA-5416.
12. The humanized CC49 antibody of claim 11, wherein the
substitution at position 91 is a proline to tyrosine substitution
and the substitution at position 27b is a valine to leucine
substitution.
13. A humanized CC49 antibody, comprising: four variable light
framework regions and four variable heavy framework regions of a
human antibody; a light chain complementarity determining region
(L-CDR)1, a L-CDR2, and a L-CDR3 of the parent HuCC49V10antibody, a
heavy chain complementarity determining region (H-CDR)1, a H-CDR2,
and a H-CDR3 of the parent HuCC49V10 antibody; a tyrosine to
proline substitution at position 91 in the L-CDR3 of the antibody;
and a valine to leucine substitution at position 27b of L-CDR1 of
the antibody; wherein the humanized CC49 antibody has a high
binding affinity for TAG-72 and is minimally immunogenic, compared
to a parent HuCC49V10 antibody, deposited as ATCC Accession No.
PTA-5416.
14. The antibody of claim 13, wherein the humanized CC49 antibody
further comprises an effector molecule.
15. The antibody of claim 14, wherein the effector molecule is a
detectable label.
16. A pharmaceutical composition comprising a therapeutically
effective amount of the antibody of claim 13 in a pharmaceutically
acceptable carrier.
17. A method of detecting a TAG-72-expressing tumor in a subject,
comprising: contacting a sample from the subject in vivo or in
vitro with the antibody of claim 5 for a sufficient amount of time
to form an immune complex; and detecting the presence of the immune
complex, wherein the presence of the immune complex demonstrates
the presence of the TAG-72-expressing tumor.
18. The method of claim 17, wherein the tumor is a colorectal
tumor, a gastric tumor, a pancreatic tumor, a breast tumor, a lung
tumor, an adenocarcinoma, or an ovarian tumor.
19. The method of claim 17, wherein the antibody further comprises
an effector molecule.
20. The method of claim 19, wherein the effector molecule is a
detectable label.
21. A method of detecting a TAG-72-expressing tumor in a subject,
comprising: contacting a sample from the subject in vivo or in
vitro with the antibody of claim 13 for a sufficient amount of time
to form an immune complex; and detecting the presence of the immune
complex, wherein the presence of the immune complex demonstrates
the presence of the TAG-72-expressing tumor.
22. The method of claim 21, wherein the tumor is a colorectal
tumor, a gastric tumor, a pancreatic tumor, a breast tumor, a lung
tumor, an adenocarcinoma, or an ovarian tumor.
23. The method of claim 21, wherein the antibody further comprises
an effector molecule.
24. The method of claim 23, wherein the effector molecule is a
detectable label.
25. A method of treating a subject having a tumor that expresses
TAG-72, comprising administering to the subject a therapeutically
effective amount of the antibody of claim 1, wherein administering
the therapeutically effective amount of the antibody inhibits the
growth of the tumor or reduces the size of the tumor, thereby
treating the subject.
26. The method of claim 25, wherein the administration of a
therapeutically effective amount of the antibody does not elicit a
human anti-murine antibody response in a subject.
27. The method of claim 25, wherein the antibody further comprises
an effector molecule.
28. The method of claim 27, wherein the effector molecule is a
toxin or a radioactive isotope.
29. A method of treating a subject having a tumor that expresses
TAG-72, comprising administering to the subject a therapeutically
effective amount of the antibody of claim 2, wherein administering
the therapeutically effective amount of the antibody inhibits the
growth of the tumor or reduces the size of the tumor, thereby
treating the subject.
30. A method of treating a subject having a tumor that expresses
TAG-72, comprising administering to the subject a therapeutically
effective amount of the antibody of claim 3, wherein administering
the therapeutically effective amount of the antibody inhibits the
growth of the tumor or reduces the size of the tumor, thereby
treating the subject.
31. A method of treating a subject having a tumor that expresses
TAG-72, comprising administering to the subject a therapeutically
effective amount of the antibody of claim 5, wherein administering
the therapeutically effective amount of the antibody inhibits the
growth of the tumor or reduces the size of the tumor, thereby
treating the subject.
32. A method of treating a subject having a tumor that expresses
TAG-72, comprising administering to the subject a therapeutically
effective amount of the antibody of claim 11, wherein administering
the therapeutically effective amount of the antibody inhibits the
growth of the tumor or reduces the size of the tumor, thereby
treating the subject.
33. A method of treating a subject having a tumor that expresses
TAG-72, comprising administering to the subject a therapeutically
effective amount of the antibody of claim 13, wherein administering
the therapeutically effective amount of the antibody inhibits the
growth of the tumor or reduces the size of the tumor, thereby
treating the subject.
34. A pharmaceutical composition comprising a therapeutically
effective amount of the antibody of claim 1 in a pharmaceutically
acceptable carrier.
35. A pharmaceutical composition comprising a therapeutically
effective amount of the antibody of claim 2 in a pharmaceutically
acceptable carrier.
36. A pharmaceutical composition comprising a therapeutically
effective amount of the antibody of claim 3 in a pharmaceutically
acceptable carrier.
37. A pharmaceutical composition comprising a therapeutically
effective amount of the antibody of claim 11 in a pharmaceutically
acceptable carrier.
38. The antibody of claim 1, wherein the humanized CC49 antibody
further comprises an effector molecule.
39. The antibody of claim 38, wherein the effector molecule is a
detectable label, a toxin, or a radioactive isotope.
40. The antibody of claim 2, wherein the humanized CC49 antibody
further comprises an effector molecule.
41. The antibody of claim 40, wherein the effector molecule is a
detectable label, a toxin, or a radioactive isotope.
42. The antibody of claim 3, wherein the humanized CC49 antibody
further comprises an effector molecule.
43. The antibody of claim 42, wherein the effector molecule is a
detectable label, a toxin, or a radioactive isotope.
44. The antibody of claim 11, wherein the humanized CC49 antibody
further comprises an effector molecule.
45. The antibody of claim 44, wherein the effector molecule is a
detectable label, a toxin, or a radioactive isotope.
46. A method of detecting a TAG-72-expressing tumor in a subject,
comprising: contacting a sample from the subject in vivo or in
vitro with the antibody of claim 1 for a sufficient amount of time
to form an immune complex; and detecting the presence of the immune
complex, wherein the presence of the immune complex demonstrates
the presence of the TAG-72-expressing tumor.
47. A method of detecting a TAG-72-expressing tumor in a subject,
comprising: contacting a sample from the subject in vivo or in
vitro with the antibody of claim 2 for a sufficient amount of time
to form an immune complex; and detecting the presence of the immune
complex, wherein the presence of the immune complex demonstrates
the presence of the TAG-72-expressing tumor.
48. A method of detecting a TAG-72-expressing tumor in a subject,
comprising: contacting a sample from the subject in vivo or in
vitro with the antibody of claim 3 for a sufficient amount of time
to form an immune complex; and detecting the presence of the immune
complex, wherein the presence of the immune complex demonstrates
the presence of the TAG-72-expressing tumor.
49. A method of detecting a TAG-72-expressing tumor in a subject,
comprising: contacting a sample from the subject in vivo or in
vitro with the antibody of claim 11 for a sufficient amount of time
to form an immune complex; and detecting the presence of the immune
complex, wherein the presence of the immune complex demonstrates
the presence of the TAG-72-expressing tumor.
Description
FIELD
The present disclosure relates to humanized monoclonal antibodies
that bind a tumor antigen. More specifically, the present
disclosure relates to humanized monoclonal antibodies with
non-conservative amino acid substitutions that have a high binding
affinity for tumor-associated glycoprotein (TAG)-72 and minimal
immunogenicity.
BACKGROUND
The use of murine monoclonal antibodies in medicine has significant
potential especially in the diagnosis and treatment of various
diseases, including cancer. The advantage of using monoclonal
antibodies resides in their specificity for a single antigen. A
monoclonal antibody raised against a specific tumor cell surface
antigen can be coupled to therapeutic agents, such as radioisotopes
and chemotherapeutic drugs, and these immunoconjugates can be used
clinically to specifically target, for example, a tumor cell of
interest.
A major limitation in the clinical use of monoclonal antibodies is
the development of a human anti-murine antibody (HAMA) response in
the patients receiving the treatments. The HAMA response can
involve allergic reactions and an increased rate of clearance of
the administered antibody from the serum. Various types of modified
monoclonal antibodies have been developed to minimize the HAMA
response while trying to maintain the antigen binding affinity of
the parent monoclonal antibody. One type of modified monoclonal
antibody is a human-mouse chimera in which a murine antigen-binding
variable region is coupled to a human constant domain (Morrison and
Schlom, Important Advances in Oncology, Rosenberg, S. A. (Ed.),
1989). A second type of modified monoclonal antibody is the
complementarity determining region (CDR)-grafted, or humanized,
monoclonal antibody (Winter and Harris, Immunol. Today 14:243-246,
1993).
The tumor-associated glycoprotein (TAG)-72, is expressed on the
cells of a majority of human carcinomas, including adenocarcinoma,
colorectal, gastric, pancreatic, breast, lung and ovarian
carcinomas. Murine monoclonal antibodies have been disclosed that
specifically bind TAG-72. One of these antibodies, CC49, has been
shown to efficiently target and reduce the size of human colon
carcinoma xenografts in nude mice, and has been targeted to a
variety of carcinomas in a number of clinical trials.
Unfortunately, the clinical utility of the CC49 monoclonal antibody
has been limited because of its murine origin. Thus, there clearly
exists a need to develop a humanized CC49 antibody with both high
antigen binding affinity and low immunogenicity for use in human
subjects.
SUMMARY
The present disclosure relates to humanized CC49 monoclonal
antibodies that bind TAG-72 with high binding affinity and that are
minimally immunogenic.
In one embodiment of the disclosure, a humanized CC49 antibody
includes a non-conservative amino acid substitution in a light
chain complementarity determining region 3 of the CC49 antibody, or
functional fragment thereof, and has a high binding affinity for
TAG-72.
In another embodiment, a humanized CC49 antibody includes a nucleic
acid sequence encoding the antibody that is deposited as ATCC
Accession number PTA-4182 or ATCC Accession number PTA-4183. ATCC
Accession numbers PTA-4182 and PTA-4183 were deposited in the
American Type Culture Collection (10801 University Boulevard,
Manassas, VA 20110-2209) on Mar. 26, 2002.
In one embodiment, a humanized CC49 antibody with high binding
affinity for TAG-72 and minimal immunogenicity includes a variable
light framework region and a variable heavy framework region of a
human antibody. The humanized CC49 antibody has at least one
complementarity determining region from a human antibody and the
remaining complementarity determining regions from a murine CC49
antibody. The humanized CC49 antibody also includes a
non-conservative substitution of a first residue in a light chain
complementarity determining region 3 and a substitution of a second
residue in a complementarity determining region of the human CC49
antibody.
Methods are disclosed herein for the use of a humanized CC49
antibody in the detection or treatment of a tumor in a subject. In
one specific embodiment, a method is disclosed for detecting a
tumor. The method includes contacting a sample obtained from the
subject with a humanized CC49 antibody for a sufficient amount of
time to form an immune complex, and then detecting the presence of
the immune complex. Another method is disclosed for detecting a
tumor in a subject that includes administering a humanized CC49
antibody to the subject for a sufficient amount of time to form an
immune complex and then detecting the presence of the immune
complex. In a further embodiment, a method is disclosed for
treating a subject having a tumor that expresses TAG-72. The method
includes administering to the subject a therapeutically effective
amount of a humanized CC49 antibody, for example, such as an
antibody conjugated to a drug or toxin.
A kit is disclosed herein that includes a container with the
humanized CC49 antibody described herein.
The foregoing and other features and advantages will become more
apparent from the following detailed description of several
embodiments, which proceeds with reference to the accompanying
figures.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a schematic drawing comparing amino acid substitutions in
CC49, HuCC49V10, HuCC49V10-14 and HuCC49V10-15. Amino acid residue
number is shown at the top of the figure. CDR region is indicated
at the bottom of the figure.
FIG. 2 is a schematic representation of the phage display vector,
PComb3H-SS. Only those restriction endonuclease sites are shown
that are relevant to cloning of the target genes or converting the
expression construct for soluble Fab expression. Gene III indicates
a sequence encoding the carboxyl-terminal domain of the gene III
protein of phage M13. Lac Z p represents the lac Z promoter of E.
coli. Omp A and pel B are the prokaryotic leader sequences; RBS
shows ribosomal binding site for protein translation. SS I and SS
II are the stuffer sequences, and stop denotes the termination
codon for protein synthesis.
FIG. 3 is a schematic representation of the vectors pIZ/V5-His
(FIG. 3A) and ppIB/V5-His (FIG. 3B) for the expression of proteins
in insect cells.
FIG. 4 is a set of digital images demonstrating SDS-PAGE analysis
of purified HuCC49 and the variant antibodies derived from it under
non-reducing (FIG. 4A) and reducing (FIG. 4B) conditions. Lane 1,
HuCC49; lane 2, HuCC49V10; lane 3, HuCC49V10-7; lane 4,
HuCC49V10-12, lane 5, HuCC49V10-14, lane 6, HuCC49V10-15 (lane
designations are the same for FIGS. 4A and 4B).
FIG. 5 is a graph demonstrating the reactivity of CC49 antibodies
(identified by their symbols in the inset) in a competition RIA.
Increasing concentrations of different antibodies were used to
compete for the binding of .sup.125I-labeled HuCC49 to the TAG-72
positive BSM.
FIG. 6 is a series of tables of flow cytometric analysis of the
binding of CC49 derived recombinant antibodies to Jurkat cells that
express TAG-72 antigen on their cell surface. The percent of gated
cells for different antibodies are tabulated.
FIG. 7 is a set of graphs demonstrating the reactivity of HuCC49
and its variants to sera from patient EA (FIG. 7A) and DS (FIG.
7B), measured by surface plasma resonance (SPR). Increasing
concentrations of the antibodies tested were used to compete with
the sera anti-idiotypic (anti variable region) antibodies for
binding to HuCC49 immobilized on sensor chip. Percent binding of
the sera to HuCC49 was calculated from the sensogram and plotted as
a function of the concentration of the competitor.
DETAILED DESCRIPTION
I. Abbreviations
BSM bovine submaxillary mucin C constant CH constant heavy CL
constant light CDR complementarity determining region Fab fragment
antigen binding F(ab').sub.2 Fab with additional amino acids,
including cysteines necessary for disulfide bonds FACS fluorescence
activated cell sort FR framework region Fv fragment variable H
heavy HAMA human antimurine antibody HuIgG human immunoglobulin G
Ig immunoglobulin Ka relative affinity constant L light PCR
polymerase chain reaction scFv single chain Fv SDR specificity
determining residue SPR surface plasmon resonance TAG-72 tumor
associated glycoprotein-72 V variable VH variable heavy VL variable
light II. Terms
Unless otherwise noted, technical terms are used according to
conventional usage. Definitions of common terms in molecular
biology may be found in Benjamin Lewin, Genes V, published by
Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: A Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8).
In order to facilitate review of the various embodiments of the
invention, the following explanations of specific terms are
provided:
Animal: Living multi-cellular vertebrate organisms, a category that
includes, for example, mammals and birds. The term mammal includes
both human and non-human mammals. Similarly, the term "subject"
includes both human and veterinary subjects.
Antibody: Immunoglobulin (Ig) molecules and immunologically active
portions of Ig molecules, i.e., molecules that contain an antigen
binding site which specifically binds (immunoreacts with) an
antigen. In one embodiment the antigen is tumor-associated
glycoprotein (TAG-72). Monoclonal, and humanized immunoglobulins
are encompassed by the disclosure. In one embodiment, a murine
monoclonal antibody that recognizes the TAG-72 antigen is CC49. In
another embodiment, a humanized CC49 antibody is HuCC49. In other
embodiments, variant humanized CC49 antibodies are HuCC49V10-14 or
HuCC49V10-15. The disclosure also includes synthetic and
genetically engineered variants of these immunoglobulins.
A naturally occurring antibody (e.g., IgG) includes four
polypeptide chains, two heavy (H) chains and two light (L) chains
inter-connected by disulfide bonds. However, it has been shown that
the antigen-binding function of an antibody can be performed by
fragments of a naturally occurring antibody. Thus, these
antigen-binding fragments are also intended to be designated by the
term "antibody." Examples of binding fragments encompassed within
the term antibody include (i) an Fab fragment consisting of the VL,
VH, CL and CH1 domains; (ii) an Fd fragment consisting of the VH
and CH1 domains; (iii) an Fv fragment consisting of the VL and VH
domains of a single arm of an antibody, (iv) a dAb fragment (Ward
et al., (1989) Nature 341:544-546) which consists of a VH domain;
and (v) an F(ab').sub.2 fragment, a bivalent fragment comprising
two Fab fragments linked by a disulfide bridge at the hinge region.
Furthermore, although the two domains of the Fv fragment are coded
for by separate genes, a synthetic linker can be made that enables
them to be made as a single protein chain (known as single chain Fv
(scFv); Bird et al. (1988) Science 242:423-426; and Huston et al.
(1988) Proc. Natl. Acad. Sci. 85:5879-5883) by recombinant methods.
Such single chain antibodies, as well as dsFv, a disulfide
stabilized Fv (Bera et al. (1998) J. Mol. Biol. 281:475-483), and
dimeric Fvs (diabodies), that are generated by pairing different
polypeptide chains (Holliger et al. (1993) Proc. Natl. Acad. Sci.
90:6444-6448), are also included.
In one embodiment, antibody fragments for use in this disclosure
are those which are capable of cross-linking their target antigen,
e.g., bivalent fragments such as F(ab').sub.2 fragments.
Alternatively, an antibody fragment which does not itself
cross-link its target antigen (e.g., a Fab fragment) can be used in
conjunction with a secondary antibody which serves to cross-link
the antibody fragment, thereby cross-linking the target antigen.
Antibodies can be fragmented using conventional techniques and the
fragments screened for utility in the same manner as described for
whole antibodies. An antibody is further intended to include
humanized monoclonal molecules that specifically bind the target
antigen.
"Specifically binds" refers to the ability of individual antibodies
to specifically immunoreact with an antigen. This binding is a
non-random binding reaction between an antibody molecule and the
antigen. In one embodiment, the antigen is TAG-72. Binding
specificity is typically determined from the reference point of the
ability of the antibody to differentially bind the antigen of
interest and an unrelated antigen, and therefore distinguish
between two different antigens, particularly where the two antigens
have unique epitopes. An antibody that specifically binds to a
particular epitope is referred to as a "specific antibody."
A variety of methods for linking effector molecules to antibodies
are well known in the art. Detectable labels useful for such
purposes are also well known in the art, and include radioactive
isotopes such as .sup.32P, fluorophores, chemiluminescent agents,
and enzymes. Also encompassed in the disclosure are the chemical or
biochemical modifications that incorporate toxins in the antibody.
In one embodiment, the toxin is chemically conjugated to the
antibody. In another embodiment, a fusion protein is genetically
engineered to include the antibody and the toxin. Specific,
non-limiting examples of toxins are radioactive isotopes,
chemotherapeutic agents, bacterial toxins, viral toxins, or venom
proteins. The disclosure also includes chemical or genetically
engineered modifications that link a cytokine to an antibody (such
as by a covalent linkage). Specific, non-limiting examples of
cytokines are interleukin (IL)-2, IL-4, IL-10, tumor necrosis
factor (TNF)-alpha and interferon (IFN)-gamma.
Antigen: Any molecule that can bind specifically with an antibody.
An antigen is also a substance that antagonizes or stimulates the
immune system to produce antibodies. Antigens are often foreign
substances such as allergens, bacteria or viruses that invade the
body.
CC49 monoclonal antibody: A murine monoclonal antibody of the
IgG.sub.1 isotype that specifically binds TAG-72 (deposited as ATCC
Accession No. HB 9459). This monoclonal antibody is a second
generation monoclonal antibody prepared by immunizing mice with
TAG-72 that was purified using the first generation antibody B72.3
(Colcher et al., Proc. Natl. Acad. Sci. USA 78:3199-3203, 1981).
The CC49 monoclonal antibody efficiently targets human colon
carcinoma xenografts in athymic mice and reduces or eliminates
their growth (Colcher et al., Cancer Res. 48:4597-4603, 1988).
Radiolabeled CC49 has been shown to successfully target a number of
human tumors including adenocarcinoma, colorectal, breast, prostate
and ovarian (Liu et al., Cancer Biotherap Radiopharm. 12:79-87,
1997; Macey et al., Clin. Cancer Res. 3:1547-1555, 1997; Meredith
et al. J. Nucl. Med., 37:1491-1496, 1996.)
cDNA (complementary DNA): A piece of DNA lacking internal,
non-coding segments (introns) and regulatory sequences that
determine transcription. cDNA is synthesized in the laboratory by
reverse transcription from messenger RNA extracted from cells.
Chimeric antibody: An antibody which includes sequences derived
from two different antibodies, which typically are of different
species. Most typically, chimeric antibodies include human and
murine antibody domains, generally human constant and murine
variable regions.
Complementarity Determining Region (CDR): Amino acid sequences
which together define the binding affinity and specificity of the
natural Fv region of a native Ig binding site. The light and heavy
chains of an Ig each have three CDRs, designated L-CDR1, L-CDR2,
L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. By definition, the
CDRs of the light chain are bounded by the residues at positions 24
and 34 (L-CDR1), 50 and 56 (L-CDR2), 89 and 97 (L-CDR3); the CDRs
of the heavy chain are bounded by the residues at positions 31 and
35b (H-CDR1), 50 and 65 (H-CDR2), 95 and 102 (H-CDR3), using the
numbering convention delineated by Kabat et al, (1991) Sequences of
Proteins of Immunological Interest, 5.sup.th Edition, Department of
Health and Human Services, Public Health Service, National
Institutes of Health, Bethesda (NIH Publication No. 91-3242).
Constant Region: The portion of the antibody molecule which confers
effector functions. In the present disclosure, the variant
antibodies include constant regions derived from human
immunoglobulins. The heavy chain constant region can be selected
from any of five isotypes: alpha, delta, epsilon, gamma or mu.
Heavy chains of various subclasses (such as the IgG subclass of
heavy chains) are responsible for different effector functions.
Thus, by choosing the desired heavy chain constant region,
humanized antibodies with the desired effector function can be
produced. The light chain constant region can be of the kappa or
lambda type.
Cytotoxin: An agent that is toxic for cells. Examples of cytotoxins
include radioactive isotopes, chemotherapeutic drugs, bacterial
toxins, viral toxins, and proteins contained in venom (e.g. insect,
reptile, or amphibian venom). A cytokine, such as interleukin-2 or
interferon, can also be a cytotoxin.
DNA: Deoxyribonucleic acid. DNA is a long chain polymer which
constitutes the genetic material of most living organisms (some
viruses have genes composed of ribonucleic acid (RNA)). The
repeating units in DNA polymers are four different nucleotides,
each of which contains one of the four bases, adenine, guanine,
cytosine and thymine bound to a deoxyribose sugar to which a
phosphate group is attached. Triplets of nucleotides (referred to
as codons) code for each amino acid in a polypeptide. The term
codon is also used for the corresponding (and complementary)
sequence of three nucleotides in the mRNA that is transcribed from
the DNA.
Effector Molecule: Therapeutic, diagnostic or detection moieties
linked to an antibody, using any number of means known to those of
skill in the art. Both covalent and noncovalent linkage means may
be used. The procedure for linking an effector molecule to an
antibody varies according to the chemical structure of the
effector. Polypeptides typically contain a variety of functional
groups; e.g., carboxylic acid (COOH), free amine (--NH.sub.2) or
sulfhydryl (--SH) groups, which are available for reaction with a
suitable functional group on an antibody to result in the linkage
of the effector molecule. Alternatively, the antibody is
derivatized to expose or link additional reactive functional
groups. The derivatization may involve linkage of any of a number
of linker molecules such as those available from Pierce Chemical
Company, Rockford Ill. The linker can be any molecule used to join
the antibody to the effector molecule. The linker is capable of
forming covalent bonds to both the antibody and to the effector
molecule. Suitable linkers are well known to those of skill in the
art and include, but are not limited to, straight or branched-chain
carbon linkers, heterocyclic carbon linkers, or peptide linkers.
Where the antibody and the effector molecule are polypeptides, the
linkers may be joined to the constituent amino acids through their
side groups (e.g., through a disulfide linkage to cysteine) or to
the alpha carbon amino and carboxyl groups of the terminal amino
acids.
In some circumstances, it is desirable to free the effector
molecule from the antibody when the immunoconjugate has reached its
target site. Therefore, in these circumstances, immunoconjugates
will comprise linkages that are cleavable in the vicinity of the
target site. Cleavage of the linker to release the effector
molecule from the antibody may be prompted by enzymatic activity or
conditions to which the immunoconjugate is subjected either inside
the target cell or in the vicinity of the target site. When the
target site is a tumor, a linker which is cleavable under
conditions present at the tumor site (e.g. when exposed to
tumor-associated enzymes or acidic pH) may be used.
In view of the large number of methods that have been reported for
linking a variety of radiodiagnostic compounds, radiotherapeutic
compounds, label (e.g. enzymes or fluorescent molecules) drugs,
toxins, and other agents to antibodies one skilled in the art will
be able to determine a suitable method for linking a given agent to
an antibody.
Encode: A polynucleotide is said to "encode" a polypeptide if, in
its native state or when manipulated by methods well known to those
skilled in the art, it can be transcribed and/or translated to
produce the mRNA for and/or the polypeptide or a fragment thereof.
The anti-sense strand is the complement of such a nucleic acid, and
the encoding sequence can be deduced therefrom.
Epitope: A site on an antigen recognized by an antibody, as
determined by the specificity of the antibody amino acid sequence.
Epitopes are also called antigenic determinants.
Framework Region: Amino acid sequences interposed between CDRs.
Includes variable light and variable heavy framework regions. The
framework regions serve to hold the CDRs in an appropriate
orientation for antigen binding.
High binding affinity: Affinity of an antibody for an antigen where
the relative affinity of the humanized CC49 antibody is
significantly greater than that of a parent CC49 antibody, for
example HuCC49V10 (deposited in the American Type Culture
Collection, 10801 University Boulevard, Manassas, Va. 20110-2209,
on Aug. 28, 2003 as ATCC Accession No. PTA-5416). In one
embodiment, affinity is calculated by a modification of the
Scatchard method described by Frankel et al. (1979) Mol. Immunol.,
16:101-106. One of skill in the art can readily identify a
statistical test that determines a statistically significant result
for example, the Student's t-test, the Wilcoxon two sample test, or
the Median test. In one embodiment, a high binding affinity is at
least about 1.2.times.10.sup.-8 M. In other embodiments, a high
binding affinity is at least about 1.5.times.10.sup.-8, at least
about 2.0.times.10.sup.-8, at least about 2.5.times.10.sup.-8, at
least about 3.0.times.10.sup.-8, at least about
3.5.times.10.sup.-8, at least about 4.0.times.10.sup.-8, at least
about 4.5.times.10.sup.-8, or at least about 5.0.times.10.sup.-8
M.
In another embodiment, a high binding affinity is measured by an
antigen/antibody dissociation rate of a humanized CC49 antibody
that is significantly lower than the parent CC49 antibody. In yet
another embodiment, a high binding affinity is measured by a
competition radioimmunoassay, where the amount of antibody needed
for 50% inhibition of the binding of .sup.125I-labeled HuCC49
antibody to BSM is less than that required by the parent CC49
antibody. In another embodiment, a high binding affinity is
measured by flow cytometry as an increased number of gated cells
labeled with humanized CC49 antibody compared to the number of
cells labeled by the parent CC49 antibody.
HAMA (Human anti-murine antibody) response: An immune response in a
human subject to the variable and constant regions of a murine
antibody that has been administered to the patient. Repeated
antibody administration may lead to an increased rate of clearance
of the antibody from the patient's serum and may also elicit
allergic reactions in the patient.
Humanized antibody: A human antibody genetically engineered to
include mouse hypervariable regions. In one embodiment, the DNA
encoding hypervariable loops of mouse monoclonal antibodies or
variable regions selected in phage display libraries is inserted
into the framework regions of human Ig genes. Antibodies can be
"customized" to have a desired binding affinity or to be minimally
immunogenic in the humans treated with them.
Humanized CC49 antibodies: CC49 antibodies humanized by grafting
CC49 CDRs onto the frameworks of the relevant human antibodies
(Kashmiri et al., Hybridoma, 14: 461-473, 1995). The murine CDRs in
the resultant humanized CC49 (HuCC49) could evoke an anti-idiotypic
response when administered in human subjects. CC49 can be humanized
by grafting only CC49 CDRs that are important for antigen binding
onto the variable light and variable heavy framework regions of,
for example, LEN and 21/28'CL human antibodies (Tamura et al., J.
Immunol. 164:1432-1441, 2000; WO 00/26394). In addition,
non-specificity determining residues (SDRs) in the murine CDRs can
be substituted with the corresponding residue in the human
antibody. One specific, non-limiting example of a humanized CC49
monoclonal antibody is HuCC49V10 (see published PCT patent
application PCT/US99/25552, herein incorporated by reference). In
one embodiment, HuCC49V10 has minimal immunogenicity (compared to
the parental HuCC49 antibody, at least 16-fold higher molar
concentration of HuCC49V10 was required to attain 25% inhibition of
HuCC49 binding to patient serum) and a partial loss in
antigen-binding affinity (1.15.times.10.sup.-8 M) compared to the
parent HuCC49 antibody (3.20.times.10.sup.-8 M). In one embodiment,
a humanized CC49 antibody is HuCC49V10-14 (ATCC Accession Number
PTA-4182, deposited in the American Type Culture Collection (10801
University Boulevard, Manassas, Va. 20110-2209) on Mar. 26, 2002,
see FIG. 1; also termed HuCC49V14 in the deposit). In another
embodiment, a humanized CC49 antibody is HuCC49V10-15 (ATCC
Accession Number PTA-4183, deposited in the American Type Culture
Collection (10801 University Boulevard, Manassas, Va. 20110-2209)
on Mar. 26, 2002, see FIG. 1; also termed HuCC49V15 in the
deposit).
Idiotype: the property of a group of antibodies or T cell receptors
defined by their sharing a particular idiotope (an antigenic
determinant on the variable region); i.e., antibodies that share a
particular idiotope belong to the same idiotype. "Idiotype" may be
used to describe the collection of idiotopes expressed by an Ig
molecule. An "anti-idiotype" antibody may be prepared to a
monoclonal antibody by methods known to those of skill in the art
and may be used to prepare pharmaceutical compositions.
Immune cell: Any cell involved in a host defense mechanism. These
can include, for example, T cells, B cells, natural killer cells,
neutrophils, mast cells, macrophages, antigen-presenting cells,
basophils, eosinophils, and neutrophils.
Immune response: A response of a cell of the immune system, such as
a neutrophil, a B cell, or a T cell, to a stimulus. In one
embodiment, the response is specific for a particular antigen (an
"antigen-specific response"). In another embodiment, the response
is against an antibody, such as HAMA response, including an
anti-variable region response.
Immunoconjugate: A covalent linkage of an effector molecule to an
antibody. The effector molecule can be a toxin or a detectable
label. Specific, non-limiting examples of toxins include, but are
not limited to, abrin, ricin, Pseudomonas exotoxin (such as PE35,
PE37, PE38, and PE40), diphtheria toxin, anthrax toxin, botulinum
toxin, or modified toxins thereof. For example, Pseudomonas
exotoxin and diphtheria toxin are highly toxic compounds that
typically bring about death through liver toxicity. Pseudomonas
exotoxin and diphtheria toxin, however, can be modified into a form
for use as an immunotoxin by removing the native targeting
component of the toxin (e.g., domain Ia of Pseudomonas exotoxin and
the B chain of diphtheria toxin) and replacing it with a different
targeting moiety, such as an antibody. Other toxic agents, that
directly or indirectly inhibit cell growth or kill cells, include
chemotherapeutic drugs, cytokines, for example interleukin-2 or
interferon, radioactive isotopes, viral toxins, or proteins
contained within, for example, insect, reptile, or amphibian venom.
Specific, non-limiting examples of detectable labels include, but
are not limited to, radioactive isotopes, enzyme substrates,
co-factors, ligands, chemiluminescent agents, fluorescent agents,
haptens, or enzymes. A "chimeric molecule" is a targeting moiety,
such as a ligand or an antibody, conjugated (attached or coupled)
to an effector molecule. The term "conjugated" or "linked" refers
to making two polypeptides into one contiguous polypeptide
molecule. In one embodiment, an antibody is joined to an effector
molecule. In another embodiment, an antibody joined to an effector
molecule is further joined to a lipid or other molecule to a
protein or peptide to increase its half-life in the antibody. The
linkage can be, for example, either by chemical or recombinant
means. In one embodiment, the linkage is chemical, wherein a
reaction between the antibody moiety and the effector molecule has
produced a covalent bond formed between the two molecules to form
one molecule. A peptide linker (short peptide sequence) can
optionally be included between the antibody and the effector
molecule.
Immunogenicity: A measure of the ability of a targeting protein or
therapeutic moiety to elicit an immune response (humoral or
cellular) when administered to a subject.
Immunoreactivity: A measure of the ability of an Ig to recognize
and bind to a specific antigen.
Isolated: An biological component (such as a nucleic acid, peptide
or protein) that has been substantially separated, produced apart
from, or purified away from other biological components in the cell
of the organism in which the component naturally occurs, i.e.,
other chromosomal and extrachromosomal DNA and, RNA, and proteins.
Nucleic acids, peptides and proteins that have been "isolated" thus
include nucleic acids and proteins purified by standard
purification methods. The term also embraces nucleic acids,
peptides and proteins prepared by recombinant expression in a host
cell as well as chemically synthesized nucleic acids.
Label: A detectable compound or composition that is conjugated
directly or indirectly to another molecule to facilitate detection
of that molecule. Specific, non-limiting examples of labels include
fluorescent tags, chemiluminescent tags, haptens, enzymatic
linkages, and radioactive isotopes.
Ligand contact residue: A residue within a CDR that is involved in
contact with a ligand or antigen. A ligand contact residue is also
known as a specificity determining residue (SDR). A non-ligand
contact residue is a residue in a CDR that does not contact a
ligand. A non-ligand contact residue can also be a framework
residue.
Lymphocytes: A type of white blood cell that is involved in the
immune defenses of the body. There are two main types of
lymphocytes: B-cells and T-cells.
Mammal: This term includes both human and non-human mammals.
Similarly, the term "subject" includes both human and veterinary
subjects.
Minimally immunogenic: An antibody that generates a reduced, for
example low, immune response when administered to a subject, such
as a human subject. In one embodiment, immunogenicity is measured
in a competitive binding assay. In one specific, non-limiting
example, immunogenicity is the ability of a variant HuCC49 antibody
to prevent a parental HuCC49 antibody from binding to CC49
anti-idiotypic antibodies in a patient's serum. If a variant HuCC49
antibody competes with an equal molar amount of the parental HuCC49
antibody (i.e. elicits greater than about 50% inhibition of
parental HuCC49 binding to anti-idiotypic antibodies in a patient's
serum) then the variant HuCC49 antibody is immunogenic. If a
variant HuCC49 antibody competes poorly with an equal molar or less
amount of the parental HuCC49 antibody (i.e. elicits about 50% or
less inhibition of parental HuCC49 binding to anti-idiotypic
antibodies in a patient's serum) then the variant HuCC49 antibody
is minimally immunogenic. In another embodiment, if a five-fold or
greater molar concentration of a variant HuCC49 antibody is
required to achieve about 50% inhibition of binding of the parental
antibody to its cognate anti-idiotypic antibodies present in a
subject's sera, then the variant antibody is minimally
immunogenic.
Monoclonal antibody: An antibody produced by a single clone of
B-lymphocytes. Monoclonal antibodies are produced by methods known
to those of skill in the art, for instance by making hybrid
antibody-forming cells from a fusion of myeloma cells with immune
spleen cells.
Nucleic acid: A deoxyribonucleotide or ribonucleotide polymer in
either single or double stranded form, and unless otherwise
limited, encompasses known analogues of natural nucleotides that
hybridize to nucleic acids in a manner similar to naturally
occurring nucleotides.
Oligonucleotide: A linear single-stranded polynucleotide sequence
of up to about 200 nucleotide bases in length, for example a
polymer of deoxyribonucleotides or ribonucleotides which is at
least 6 nucleotides, for example at least 15, 50, 100 or even 200
nucleotides long.
Operably linked: A first nucleic acid sequence is operably linked
with a second nucleic acid sequence when the first nucleic acid
sequence is placed in a functional relationship with the second
nucleic acid sequence. For instance, a promoter is operably linked
to a coding sequence if the promoter affects the transcription or
expression of the coding sequence. Generally, operably linked DNA
sequences are contiguous and, where necessary to join two protein
coding regions, in the same reading frame.
Phage display: A technique wherein DNA sequences are amplified and
cloned into phage vector to create a "phage library," in which the
phage present on their surface the proteins encoded by the DNA. In
one embodiment, a phage library is produced that expresses
HuCC49V10 variant immunoglobulins. From the rescued phages, the
individual phage clones are selected through interaction of the
displayed protein with a ligand, and the specific phage is
amplified by infection of bacteria. Antigen specific
immunoglobulins can then be expressed and characterized for their
antigen binding and sera reactivity (potential immunogenicity).
Pharmaceutical agent: A chemical compound or composition capable of
inducing a desired therapeutic or prophylactic effect when properly
administered to a subject or a cell. "Incubating" includes a
sufficient amount of time for a drug to interact with a cell.
"Contacting" includes incubating a drug in solid or in liquid form
with a cell.
A "therapeutically effective amount" is a quantity of a specific
substance sufficient to achieve a desired effect in a subject being
treated. For instance, this can be the amount necessary to inhibit
or suppress growth of a tumor or to decrease a sign or symptom of
the tumor in the subject. In one embodiment, a therapeutically
effective amount is the amount necessary to eliminate a tumor. When
administered to a subject, a dosage will generally be used that
will achieve target tissue concentrations (for example, in tumors)
that has been shown to achieve a desired in vitro effect.
Pharmaceutically acceptable carriers: The pharmaceutically
acceptable carriers useful in this disclosure are conventional.
Remington's Pharmaceutical Sciences, by E. W. Martin, Mack
Publishing Co., Easton, Pa., 15.sup.th Edition (1975), describes
compositions and formulations suitable for pharmaceutical delivery
of humanized CC49 monoclonal antibodies disclosed herein.
In general, the nature of the carrier will depend on the particular
mode of administration employed. For instance, parenteral
formulations usually comprise injectable fluids that include
pharmaceutically and physiologically acceptable fluids such as
water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol or the like as a vehicle. For solid compositions
(e.g., powder, pill, tablet, or capsule forms), conventional
non-toxic solid carriers can include, for example, pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. In
addition to biologically-neutral carriers, pharmaceutical
compositions to be administered can contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate.
Polynucleotide: A single-stranded linear nucleotide sequence,
including sequences of greater than 100 nucleotide bases in
length.
Polypeptide: A polymer in which the monomers are amino acid
residues that are joined together through amide bonds. When the
amino acids are alpha-amino acids, either the L-optical isomer or
the D-optical isomer can be used, the L-isomers being preferred in
nature. The term polypeptide or protein as used herein encompasses
any amino acid sequence and includes, but may not be limited to,
modified sequences such as glycoproteins. The term polypeptide is
specifically intended to cover naturally occurring proteins, as
well as those that are recombinantly or synthetically produced.
Substantially purified polypeptide as used herein refers to a
polypeptide that is substantially free of other proteins, lipids,
carbohydrates or other materials with which it is naturally
associated. In one embodiment, the polypeptide is at least 50%, for
example at least 80% free of other proteins, lipids, carbohydrates
or other materials with which it is naturally associated. In
another embodiment, the polypeptide is at least 90% free of other
proteins, lipids, carbohydrates or other materials with which it is
naturally associated. In yet another embodiment, the polypeptide is
at least 95% free of other proteins, lipids, carbohydrates or other
materials with which it is naturally associated.
Conservative amino acid substitution tables providing functionally
similar amino acids are well known to one of ordinary skill in the
art. The following six groups are examples of amino acids that are
considered to be conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (1), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
A non-conservative amino acid substitution can result from changes
in: (a) the structure of the amino acid backbone in the area of the
substitution; (b) the charge or hydrophobicity of the amino acid;
or (c) the bulk of an amino acid side chain. Substitutions
generally expected to produce the greatest changes in protein
properties are those in which: (a) a hydrophilic residue is
substituted for (or by) a hydrophobic residue; (b) a proline is
substituted for (or by) any other residue; (c) a residue having a
bulky side chain, e.g., phenylalanine, is substituted for (or by)
one not having a side chain, e.g., glycine; or (d) a residue having
an electropositive side chain, e.g., lysyl, arginyl, or histadyl,
is substituted for (or by) an electronegative residue, e.g.,
glutamyl or aspartyl.
Variant amino acid sequences may, for example, be 80, 90 or even 95
or 98% identical to the native amino acid sequence. Programs and
algorithms for determining percentage identity can be found at the
NCBI website.
Preventing or treating a disease: Preventing a disease refers to
inhibiting completely or in part the development or progression of
a disease, for example in a person who is known to have a
predisposition to a disease. An example of a person with a known
predisposition is someone with a history of cancer in the family,
or who has been exposed to factors that predispose the subject to
the development of a tumor. Treating a disease refers to a
therapeutic intervention that inhibits, or suppressed the growth of
a tumor, eliminates a tumor, ameliorates at least one sign or
symptom of a disease or pathological condition, or interferes with
a pathophysiological process, after the disease or pathological
condition has begun to develop.
Protein: A biological molecule encoded by a gene and comprised of
amino acids.
Recombinant: A recombinant nucleic acid is one that has a sequence
that is not naturally occurring or was made artificially.
Artificial combination is often accomplished by chemical synthesis
or, more commonly, by the artificial manipulation of isolated
segments of nucleic acids, e.g., by genetic engineering techniques.
Similarly, a recombinant protein is one encoded by a recombinant
nucleic acid molecule.
Subject: Living multi-cellular vertebrate organisms, a category
that includes both human and non-human mammals.
TAG (Tumor-Associated Glycoprotein)-72: A cell-surface glycoprotein
that is expressed on human carcinomas, including adenocarcinoma,
colorectal, gastric, pancreatic, breast, lung and ovarian
carcinomas. TAG-72 has a high molecular weight (greater than
1.times.10.sup.6) as measured by size-exclusion chromatography, a
density of 1.45 g/ml, is resistant to Chondroitinase digestion,
expresses blood group-related oligosaccharides, and is heavily
sialylated with O-glycosidically linked oligosaccharides
characteristic of mucins. These characteristics suggest that TAG-72
is a mucin-like molecule (Johnson et al., Cancer Res. 46:850-857,
1986, incorporated herein by reference).
Therapeutically effective amount: A quantity of a specific
substance sufficient to achieve a desired effect in a subject being
treated. For instance, this can be the amount necessary to inhibit
or suppress growth of a tumor. In one embodiment, a therapeutically
effective amount is the amount necessary to eliminate a tumor. When
administered to a subject, a dosage will generally be used that
will achieve target tissue concentrations (for example, in tumors)
that has been shown to achieve a desired in vitro effect.
Treatment: Refers to both prophylactic inhibition of initial
infection or disease, and therapeutic interventions to alter the
natural course of an untreated infection or disease process, such
as a tumor growth or an infection with a bacteria.
Tumor: A neoplasm that may be either malignant or non-malignant.
Tumors of the same tissue type are primary tumors originating in a
particular organ (such as breast, prostate, bladder or lung).
Tumors of the same tissue type may be divided into tumor of
different sub-types (a classic example being bronchogenic
carcinomas (lung tumors) which can be an adenocarcinoma, small
cell, squamous cell, or large cell tumor). Breast cancers can be
divided histologically into scirrhous, infiltrative, papillary,
ductal, medullary and lobular. In one embodiment, cells in a tumor
express TAG-72.
Variable region (also variable domain or V domain): The regions of
both the light-chain and the heavy-chain on an Ig that contain
antigen-binding sites. The regions are composed of polypeptide
chains containing four relatively invariant "framework regions"
(FRs) and three highly variant "hypervariable regions" (HVs).
Because the HVs constitute the binding site for antigen(s) and
determine specificity by forming a surface complementarity to the
antigen, they are more commonly termed the
"complementarity-determining regions," or CDRs, and are denoted
CDR1, CDR2, and CDR3. Because both of the CDRs from the heavy- and
light-chain domains contribute to the antigen-binding site, it is
the three-dimensional combination of the heavy and the light chain
that determines the final antigen specificity.
Within the heavy- and light-chain, the framework regions surround
the CDRs. Proceeding from the N-terminus of a heavy or light chain,
the order of regions is: FR1-CDR1-FR2--CDR2-FR3--CDR3-FR4. As used
herein, the term "variable region" is intended to encompass a
complete set of four framework regions and three
complementarity-determining regions. Thus, a sequence encoding a
"variable region" would provide the sequence of a complete set of
four framework regions and three complementarity-determining
regions.
Unless otherwise explained, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. The
singular terms "a," "an," and "the" include plural referents unless
context clearly indicates otherwise. Similarly, the word "or" is
intended to include "and" unless the context clearly indicates
otherwise. Hence "comprising A or B" means "including A or B, or A
and B." It is further to be understood that all base sizes or amino
acid sizes, and all molecular weight or molecular mass values,
given for nucleic acids or polypeptides are approximate, and are
provided for description. Although methods and materials similar or
equivalent to those described herein can be used in the practice or
testing of the present invention, suitable methods and materials
are described below. All publications, patent applications,
patents, and other references mentioned herein are incorporated by
reference in their entirety. In case of conflict, the present
specification, including explanations of terms, will control. In
addition, the materials, methods, and examples are illustrative
only and not intended to be limiting.
Humanized CC49 Antibodies
Disclosed herein are humanized monoclonal CC49 antibodies that have
a non-conservative amino acid substitution in the light chain
complementarity determining region (LCDR) 3 of the CC49 antibody
grafted onto a human antibody framework. In one embodiment, the
humanized CC49 antibody has a non-conservative amino acid
substitution of a ligand contact residue in LCDR3. In several
examples, the CC49 antibody has a non-conservative substitution of
a ligand contact residue at position 89, 90, 91, 92, 93, 94, 95 or
96 of LCDR3 (Table 1).
TABLE-US-00001 TABLE 1 HuCC49V10 CDR sequences 89 90 91 92 93 94 95
96 97 LCDR3 Gln Gln Tyr Tyr Ser Tyr Pro Leu Ser 50 51 52 a 53 54 55
56 57 HCDR2 Tyr Phe Ser Pro Gly Asn Asp Asp Phe 58 59 60 61 62 63
64 65 HCDR2 Lys Tyr Ser Gln Lys Phe Gln Gly In bold: The SDRs that
are targeted for mutation. In italic: The residues of HuCC49
already modified to generate HuCC49V10.
In one embodiment, the humanized CC49 antibody has a
non-conservative amino acid substitution at position 91 of LCDR3.
In one specific, non-limiting example, the humanized CC49 antibody
has a tyrosine to proline substitution at position 91 of LCDR3 (see
HuCC49V10-14 in FIG. 1 and in Table 2).
TABLE-US-00002 TABLE 2 Mutations in variants* isolated by phage
display LCDR1 LCDR3 27b 89 90 91 92 93 94 95 96 97 HuCC49V10 Val
Gln Gln Tyr Tyr Ser Tyr Pro Leu Ser HuCC49V10-7 Leu -- -- -- -- Leu
-- -- -- -- HuCC49V10-10 -- -- -- Ser -- -- -- -- -- --
HuCC49V10-12 -- -- -- -- -- Leu -- -- -- -- HuCC49V10-13 -- -- --
-- -- -- Thr -- -- -- HuCC49V10-14 -- -- -- Pro -- -- -- -- -- --
HuCC49V10-15 Leu -- -- Pro -- -- -- -- -- -- HCDR2 50 51 52 a 53 54
55 56 57 HuCC49V10 Tyr Phe Ser Pro Gly Asn Asp Asp Phe HuCC49V10-13
-- -- Asn -- -- -- -- -- -- HuCC49V10-7, -10, -12, -14, -15 -- --
-- -- -- -- -- -- -- HCDR2 58 59 60 61 62 63 64 65 HuCC49V10 Lys
Tyr Ser Gln Lys Phe Gln Gly HuCC49V10-13 Gln -- -- -- -- -- -- --
HuCC49V10-7, -10, -12, -14, -15 -- -- -- -- -- -- -- -- Bold: The
SDRs targeted for mutation to generate a phage display library.
Italic: The residues of HuCC49 already modified to generate
HuCC49V10. *Variants HuCC49V10-7, -10, -12, -13, -14, and -15 can
be derived from HuCC49V10 as described in Examples 1-4.
In one embodiment, the humanized CC49 antibody has no more than one
non-conservative amino acid substitution in LCDR3. However, the
humanized CC49 antibody can include no more than two, no more than
three, no more than four, no more than five, or no more than nine
non-conservative amino acid substitutions in LCDR3. In some
embodiments, the CC49 antibody has a non-conservative amino acid
substitution at position 91, an additional non-conservative
substitution of a ligand contact residue at position 89, 90, 91,
92, 93, 94, 95 or 96 of LCDR3 (Table 1), and has a high binding
affinity for TAG-72. In another embodiment, the humanized CC49
antibody has a non-conservative amino acid substitution at position
91, and has an additional non-conservative amino acid substitution
of a non-ligand contact residue of LCDR3.
A humanized CC49 monoclonal antibody including a non-conservative
amino acid substitution in LCDR3 can also include an additional
non-conservative amino acid substitution in a region other than
LCDR3. For example, an additional non-conservative amino acid
substitution can be a non-conservative substitution in another LCDR
or in an HCDR. Specific, non-limiting examples of a humanized CC49
monoclonal antibody that includes more than one non-conservative
substitution are a humanized CC49 monoclonal antibody with a
non-conservative substitution in an LCDR3 ligand contact residue
and a non-conservative substitution in an LCDR non-ligand contact
residue, or a non-conservative substitution in LCDR3 and a
non-conservative substitution in HCDR2. In several embodiments, the
CC49 antibody has a non-conservative substitution of a ligand
contact residue at position 50, 51, 52, 52a, 53, 54, 56, 57, or 58
of HCDR2 (Table 1) in addition to an LCDR3 non-conservative
substitution. In another embodiment, the humanized CC49 antibody
has an additional non-conservative amino acid substitution in a
framework residue.
In one embodiment, the humanized CC49 antibody has a conservative
amino acid substitution in addition to an LCDR3 non-conservative
substitution, such as, but not limited to, a conservative
substitution in a CDR in addition to an LCDR3 non-conservative
substitution. In other specific non-limiting examples, the
humanized CC49 antibody has a conservative substitution in an LCDR
or in an HCDR in addition to an LCDR3 non-conservative
substitution. In other specific non-limiting examples, the
humanized CC49 antibody has a conservative substitution in LCDR1,
LCDR2, LCDR3, HCDR1, HCDR2, or HCDR3 in addition to an LCDR3
non-conservative substitution. Thus in another specific,
non-limiting example, the humanized CC49 antibody has a
conservative amino acid substitution at position 27b of LCDR1 and a
non-conservative amino acid substitution at position 91 of LCDR3. A
specific, non-limiting example of a conservative amino acid
substitution is a valine to leucine substitution at position 27b of
LCDR1 and specific, non-limiting example of a non-conservative
amino acid substitution is a tyrosine to proline substitution at
position 91 of LCDR3 (see HuCC49V10-15 in FIG. 1 and in Table
2).
In one embodiment, the humanized CC49 antibody has a CH2 domain
deletion. In one specific embodiment, a humanized CC49 antibody
with a CH2 domain deletion is cleared more quickly from the plasma
compared to the parent CC49 antibody. In another specific
embodiment, a humanized CC49 antibody with a CH2 domain deletion
has reduced immunogenicity compared to the parent CC49
antibody.
The humanized monoclonal antibodies disclosed herein bind TAG-72
with high binding affinity. In one embodiment, the humanized CC49
antibody has a high binding affinity for TAG-72 that is at least
about 1.2.times.10.sup.-8 M. In other embodiments, the humanized
CC49 antibody has a high binding affinity for TAG-72 that is at
least about 1.2, 10.sup.-8, about 1.5.times.10.sup.-8, about
2.0.times.10.sup.-8, about 2.5.times.10.sup.-8, about
3.0.times.10.sup.-8, about 3.5.times.10.sup.-8, about
4.0.times.10.sup.-8, about 4.5.times.10.sup.-8, or about
5.0.times.10.sup.-8 M. In one embodiment, the humanized CC49
antibody has a high binding affinity if it has a significantly
lower antigen/antibody dissociation rate compared to that of the
parent CC49 antibody. In another embodiment, the humanized CC49
antibody has a high binding affinity if less antibody is required
for a 50% inhibition of the binding of .sup.125I-labeled-HuCC49 to
BSM compared to the parent CC49 antibody. In yet another
embodiment, the humanized CC49 antibody has a high binding affinity
when the number of cells labeled with humanized CC49 antibody is
significantly greater than the number of cells labeled by the
parent CC49 antibody, as measured by flow cytometry.
Immunogenicity of variant HuCC49 antibodies can be measured in a
competitive binding assay as the ability of a variant HuCC49
antibody to prevent a parent CC49 or HuCC49 antibody from binding
to anti-idiotypic antibodies in a human subject's serum. In one
embodiment, a variant humanized CC49 antibody with a
non-conservative amino acid substitution in LCDR3 is minimally
immunogenic in a subject. In other embodiments, the variant
humanized CC49 antibody with an additional amino acid substitution
is minimally immunogenic. In one embodiment, at least about
five-fold higher molar concentration of the variant humanized CC49
antibody, than that of the parental HuCC49 antibody, is required to
elicit 50% inhibition of the parental HuCC49 binding to its cognate
anti-idiotypic antibodies in a subject's sera. In other
embodiments, at least about ten-fold, at least about twenty
five-fold, at least about fifty-fold, at least about seventy-fold,
or at least about one hundred-fold higher molar concentration of
the variant humanized CC49 antibody, than that of the parental
antibody, is required to elicit 50% inhibition of the parental
HuCC49 binding to its cognate anti-idiotypic antibodies in a
subject's sera.
Effector molecules, e.g., therapeutic, diagnostic, or detection
moieties, can be linked to a humanized CC49 antibody that
specifically binds TAG-72, using any number of means known to those
of skill in the art. Thus, a humanized CC49 antibody with a
non-conservative amino acid substitution can have any one of a
number of different types of effector molecules linked to it. In
one embodiment, the humanized CC49 antibody is linked to a
detectable label. In some embodiments, the humanized CC49 antibody
is linked to a radioactive isotope, an enzyme substrate, a
co-factor, a ligand, a chemiluminescent agent, a fluorescent agent,
a hapten, or an enzyme. In another embodiment, the humanized CC49
antibody is linked to a cytotoxin. In other embodiments, the
humanized CC49 antibody is linked to a chemotherapeutic drug, a
radioactive isotope, a bacterially-expressed toxin, a
virally-expressed toxin, or a venom protein. In yet other
embodiments, the humanized CC49 antibody is linked to a cytokine.
Specific, non-limiting examples of cytokines are IL-2, IL-4, IL-10,
TNF-alpha and IFN-gamma. In some embodiments, the humanized CC49
antibody is linked to an effector molecule by a covalent or
non-covalent means.
Pharmaceutical Compositions and Therapeutic Methods
Pharmaceutical compositions are disclosed herein that include a
humanized CC49 monoclonal antibody, such as HuCC49V10-14 or
HuCC49V10-15, and can be formulated with an appropriate solid or
liquid carrier, depending upon the particular mode of
administration chosen. In addition, a humanized CC49 monoclonal
antibody linked to an effector molecule (i.e., toxin,
chemotherapeutic drug, or detectable label) can be prepared in
pharmaceutical compositions.
The pharmaceutically acceptable carriers and excipients useful in
this disclosure are conventional. For instance, parenteral
formulations usually comprise injectable fluids that are
pharmaceutically and physiologically acceptable fluid vehicles such
as water, physiological saline, other balanced salt solutions,
aqueous dextrose, glycerol or the like. Excipients that can be
included are, for instance, other proteins, such as human serum
albumin or plasma preparations. If desired, the pharmaceutical
composition to be administered can also contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate.
The dosage form of the pharmaceutical composition will be
determined by the mode of administration chosen. For instance, in
addition to injectable fluids, topical, inhalation, oral and
suppository formulations can be employed. Topical preparations can
include eye drops, ointments, sprays and the like. Inhalation
preparations can be liquid (e.g., solutions or suspensions) and
include mists, sprays and the like. Oral formulations can be liquid
(e.g., syrups, solutions or suspensions), or solid (e.g., powders,
pills, tablets, or capsules). Suppository preparations can also be
solid, gel, or in a suspension form. For solid compositions,
conventional non-toxic solid carriers can include pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. Actual
methods of preparing such dosage forms are known, or will be
apparent, to those skilled in the art.
The pharmaceutical compositions that include a humanized CC49
monoclonal antibody can be formulated in unit dosage form suitable
for individual administration of precise dosages. In addition, the
pharmaceutical compositions may be administered as an
immunoprophylactic in a single dose schedule or as an immunotherapy
in a multiple dose schedule. A multiple dose schedule is one in
which a primary course of treatment may be with more than one
separate dose, for instance 1-10 doses, followed by other doses
given at subsequent time intervals as needed to maintain or
reinforce the action of the compositions. Treatment can involve
daily or multi-daily doses of compound(s) over a period of a few
days to months, or even years. Thus, the dosage regime will also,
at least in part, be determined based on the particular needs of
the subject to be treated and will be dependent upon the judgement
of the administering practitioner. In one specific, non-limiting
example, a unit dosage can be about 0.1 to about 10 mg per patient
per day. Dosages from about 0.1 up to about 100 mg per patient per
day may be used, particularly if the agent is administered to a
secluded site and not into the circulatory or lymph system, such as
into a body cavity, into a lumen of an organ, or directly into a
tumor. In one embodiment, about 10 mCi of a radiolabeled humanized
CC49 monoclonal antibody is administered to a subject. In other
embodiments, about 15 mCi, about 20 mCi, about 50 mCi, about 75 mCi
or about 100 mCi of a radiolabeled humanized CC49 monoclonal
antibody is administered to a subject. The amount of active
compound(s) administered will be dependent on the subject being
treated, the severity of the affliction, and the manner of
administration, and is best left to the judgment of the prescribing
clinician. Within these bounds, the formulation to be administered
will contain a quantity of the active component(s) in amounts
effective to achieve the desired effect in the subject being
treated.
The compounds of this disclosure can be administered to humans on
whose tissues they are effective in various manners such as
topically, orally, intravenously, intramuscularly,
intraperitoneally, intranasally, intradermally, intrathecally,
subcutaneously, via inhalation or via suppository. The particular
mode of administration and the dosage regimen will be selected by
the attending clinician, taking into account the particulars of the
case (e.g. the subject, the disease, the disease state involved,
and whether the treatment is prophylactic).
In one embodiment, a therapeutically effective amount of a
humanized CC49 antibody, such as HuCC49V10-14 or HuCC49V10-15, is
the amount of humanized CC49 antibody necessary to inhibit further
growth of a TAG-72-expressing tumor or suppress the growth of a
TAG-72-expressing tumor, without eliciting a HAMA response in the
patient receiving the treatment. In other embodiments, a
therapeutically effective amount of humanized CC49 antibody is the
amount of humanized CC49 antibody necessary to eliminate or reduce
the size of a TAG-72-expressing tumor, without eliciting a HAMA
response. Specific, non-limiting examples of TAG-72-expressing
tumors are adenocarcinoma, colorectal, gastric, pancreatic, breast,
lung, and ovarian tumors. In yet another embodiment, a
therapeutically effective amount of humanized CC49 antibody is an
amount of humanized CC49 antibody that is effective at reducing a
sign or a symptom of the tumor and induces a minimal immune
response.
A therapeutically effective amount of a humanized CC49 monoclonal
antibody, such as HuCC49V10-14 or HuCC49V10-15, can be administered
in a single dose, or in several doses, for example daily, during a
course of treatment. In one embodiment, treatment continues until a
therapeutic result is achieved. However, the effective amount of
humanized CC49 antibody will be dependent on the subject being
treated, the severity and type of the affliction, and the manner of
administration of the therapeutic(s).
Controlled release parenteral formulations of a humanized CC49
monoclonal antibody can be made as implants, oily injections, or as
particulate systems. For a broad overview of protein delivery
systems (see Banga, A. J., Therapeutic Peptides and Proteins:
Formulation, Processing, and Delivery Systems, Technomic Publishing
Company, Inc., Lancaster, Pa., 1995). Particulate systems include
microspheres, microparticles, microcapsules, nanocapsules,
nanospheres, and nanoparticles. Microcapsules contain the
therapeutic protein as a central core. In microspheres the
therapeutic is dispersed throughout the particle. Particles,
microspheres, and microcapsules smaller than about 1 .mu.m are
generally referred to as nanoparticles, nanospheres, and
nanocapsules, respectively. Capillaries have a diameter of
approximately 5 .mu.m so that only nanoparticles are administered
intravenously. Microparticles are typically around 100 .mu.m in
diameter and are administered subcutaneously or intramuscularly
(see Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed.,
Marcel Dekker, Inc., New York, N.Y., pp. 219-342, 1994; Tice &
Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed.,
Marcel Dekker, Inc. New York, N.Y., pp. 315-339, 1992).
Polymers can be used for ion-controlled release. Various degradable
and nondegradable polymeric matrices for use in controlled drug
delivery are known in the art (Langer, R., Accounts Chem. Res.
26:537, 1993). For example, the block copolymer, polaxamer 407
exists as a viscous yet mobile liquid at low temperatures but forms
a semisolid gel at body temperature. It has shown to be an
effective vehicle for formulation and sustained delivery of
recombinant interleukin-2 and urease (Johnston et al., Pharm. Res.
9:425, 1992; and Pec et al., J. Parent. Sci. Tech. 44:58, 1990).
Alternatively, hydroxyapatite has been used as a microcarrier for
controlled release of proteins (Ijntema et al., Int. J. Pharm.
112:215, 1994). In yet another aspect, liposomes are used for
controlled release as well as drug targeting of the
lipid-capsulated drug (Betageri, et al., Liposome Drug Delivery
Systems, Technomic Publishing Co., Inc., Lancaster, Pa., 1993).
Numerous additional systems for controlled delivery of therapeutic
proteins are known (e.g., U.S. Pat. Nos. 5,055,303, 5,188,837,
4,235,871, 4,501,728, 4,837,028 4,957,735 and 5,019,369, 5,055,303;
5,514,670; 5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206,
5,271,961; 5,254,342 and 5,534,496).
Site-specific administration of the disclosed compounds can be
used, for instance by applying the humanized CC49 antibody to a
pre-cancerous region, a region of tissue from which a tumor has
been removed, or a region suspected of being prone to tumor
development. In some embodiments, sustained intra-tumoral (or
near-tumoral) release of the pharmaceutical preparation that
includes a therapeutically effective amount of the humanized CC49
antibody may be beneficial.
The present disclosure also includes therapeutic uses of humanized
CC49 monoclonal antibodies that are non-covalently or covalently
linked to effector molecules. In one specific embodiment, the
humanized CC49 monoclonal antibody is covalently linked to an
effector molecule that is toxic to a tumor or cell expressing
TAG-72. In one specific, non-limiting example, the effector
molecule is a cytotoxin. In other specific, non-limiting examples
the effector molecule is a radioactive isotope, a chemotherapeutic
drug, a bacterially-expressed toxin, a virally-expressed toxin, a
venom protein, or a cytokine. Humanized CC49 monoclonal antibodies
covalently linked to an effector molecule have a variety of uses.
For example, a humanized CC49 antibody linked to a radioactive
isotope us if use in immunotherapy. A humanized CC49 antibody
covalently linked to a radioactive isotope is of use to localize a
tumor in radioimmunoguided surgery, such that the tumor can be
removed.
The present disclosure also includes combinations of a humanized
CC49 monoclonal antibody, such as HuCC49V10-14 or HuCC49V10-15,
with one or more other agents useful in the treatment of tumors.
For example, the compounds of this disclosure can be administered
in combination with effective doses of immunostimulants,
anti-cancer agents, anti-inflammatory agents, anti-infectives,
and/or vaccines. The term "administration in combination" or
"co-administration" refers to both concurrent and sequential
administration of the active agents. A subject that is suffering
from a tumor, or is predisposed to the development of a tumor, will
be a candidate for treatment using the therapeutic methods
disclosed herein.
Diagnostic Methods and Kits
A method is provided herein for the in vivo or in vitro detection
of TAG-72-expressing tumors or cells. An in vivo detection method
can localize any tumor or cell that expresses TAG-72 in a subject.
In one embodiment, a humanized CC49 antibody is administered to the
subject for a sufficient amount of time for the antibody to
localize to the tumor or cell in the subject and to form an immune
complex with TAG-72. The immune complex can then be detected. In
one specific, non-limiting example detection of an immune complex
is performed by immunoscintography. Other specific, non-limiting
examples of immune complex detection include radiolocalization,
radioimaging, or fluorescence imaging. In another embodiment, the
antibody is linked to an effector molecule. In one specific,
non-limiting embodiment, the effector molecule is a detectable
label. Specific, non-limiting examples of detectable labels include
a radioactive isotope, an enzyme substrate, a co-factor, a ligand,
a chemiluminescent agent, a fluorescent agent, a hapten, or an
enzyme.
A method of detecting tumors in a subject includes the
administration of a humanized CC49 antibody complexed to an
effector molecule, such as a radioactive isotope. In one
embodiment, a humanized CC49 antibody complexed to an effector
molecule, such as a radioactive isotope, is administered to a
subject prior to surgery or treatment. In another embodiment, a
humanized CC49 antibody complexed to an effector molecule, such as
a radioactive isotope, is administered to a subject following
surgery or treatment. After a sufficient amount of time has elapsed
to allow for the administered radiolabeled antibody to localize to
the tumor, the tumor is detected. In one specific embodiment, the
detection step is performed prior to surgery. In another
embodiment, the detection step is performed during surgery, for
example to detect the location of the tumor prior to removing it,
as in radioimmunoguided surgery. In yet another embodiment, the
detection step is performed after surgery to ensure the complete
removal of the tumor, or to detect a recurrence of the tumor. In
one specific, non-limiting example, a radiolabeled immune complex
is detected using a hand held gamma detection probe. Primary
tumors, metastasized tumors or cells expressing TAG-72 can be
detected.
In another embodiment, a humanized CC49 antibody and a secondary
antibody are administered to the subject for a sufficient amount of
time for the humanized CC49 antibody to form an immune complex with
TAG-72 on a tumor or cell, and for the secondary antibody to form
an immune complex with the humanized CC49 antibody. In one
embodiment, the humanized CC49 antibody is complexed with the
secondary antibody prior to their administration to the subject. In
one specific, non-limiting embodiment, the secondary antibody is
linked to a detectable label. In one embodiment, the immune
complex, which includes TAG-72, the humanized CC49 antibody, and
the secondary antibody linked to a detectable label, is detected as
described above.
An in vitro detection method can screen any biological sample
containing any tumor or cell that expresses TAG-72. Such samples
include, but are not limited to, tissue from biopsies, autopsies,
and pathology specimens. Biological samples also include sections
of tissues, such as frozen sections taken for histological
purposes. Biological samples further include body fluids, such as
blood, serum, saliva, or urine. A biological sample is typically
obtained from a mammal, such as a human. In one embodiment the
subject has a colorectal tumor. In other embodiments, the subject
has a gastric tumor, a pancreatic tumor, a breast tumor, a lung
tumor, an adenocarcinoma, or an ovarian tumor. Other biological
samples that can be detected by the in vitro detection method
include samples of cultured cells that express TAG-72.
In one embodiment, a method is provided for detecting a
TAG-72-expressing tumor or cell. Kits for detecting a
TAG-72-expressing tumor or cell will typically comprise a humanized
CC49 antibody that specifically binds TAG-72. In some embodiments,
an antibody fragment, such as an Fv fragment is included in the
kit. In a further embodiment the antibody is an immunoconjugate. In
some embodiments, the antibody is conjugated to a detectable label
(e.g. radioactive isotope, enzyme substrate, co-factor, ligand,
fluorescent agent, hapten, enzyme, or chemiluminescent agent).
The kit can include instructional materials disclosing means of use
of an antibody that specifically binds TAG-72 or fragment thereof
(e.g. for detection of TAG-72-expressing cells in a sample). The
instructional materials may be written, in an electronic form (e.g.
computer diskette or compact disk) or may be visual (e.g. video
files). The kits may also include additional components to
facilitate the particular application for which the kit is
designed. Thus, for example, the kit may additionally contain means
of detecting a label (e.g. enzyme substrates for enzymatic labels,
filter sets to detect fluorescent labels, appropriate secondary
labels such as a secondary antibody, or the like). In one
embodiment, the kit contains a secondary antibody that is
conjugated to a detectable label. The kits may additionally include
buffers and other reagents, such as an antigen (e.g. purified
TAG-72) routinely used for the practice of a particular method.
Such kits and appropriate contents are well known to those of skill
in the art.
In one embodiment of the present invention, the diagnostic kit
comprises an immunoassay. Although the details of the immunoassays
may vary with the particular format employed, the method of
detecting TAG-72 or fragment thereof in a biological sample
generally includes the steps of contacting the biological sample
with an antibody which specifically reacts, under immunologically
reactive conditions, to TAG-72. The antibody is allowed to
specifically bind under immunologically reactive conditions to form
an immune complex, and the presence of the immune complex (bound
antibody) is detected directly or indirectly.
The invention is illustrated by the following non-limiting
Examples.
EXAMPLES
Example 1
Construction of the Variant HuCC49V10 Antibody Phage Library
A phage display library of isolates was derived from the humanized
CC49 variant HuCC49V10 (Tamura et al., J. Immunol., 164: 1432,
2000) by the mutagenesis of the LCDR3 and the HCDR2, the two CDRs
that were earlier shown to be the targets of the patient's
anti-variable region response. Primer-induced mutagenesis was used
to replace the targeted SDRs of the two CDRs (Table 1) with all
possible residues located at the corresponding positions in human
antibodies (Table 3). A dual step PCR (Landt et al., Gene 96: 125,
1990) was used for DNA amplification.
TABLE-US-00003 TABLE 3 Residue substitutions in the library Encoded
aa L-CDR3 Position 89: R,L,N,K,M,H,Q,I,S Position 91:
S,Y,R,A,G,H,T,C,P,D,N Positions 92, 93, 94, 96:
S,R,L,V,A,G,T,P,Y,F,D,N, I,W,H,E,L,Q,M,C H-CDR2 Positions 50, 52,
53, 58: S,R,L,V,A,G,T,P,Y,F,D,N, I,W,H,E,L,Q,M,C Position 54:
S,T,D,G,F,N,R,L,I,A,V,Y, C,H,P Position 56:
S,T,A,G,V,L,R,I,D,E,Y,C, W,F,L,N,M,
DNA Amplification
For the first step of DNA amplification, a primer derived from the
leader sequence of the light (SEQ ID NO: 1) or heavy (SEQ ID NO: 9)
chain was used as the 5' primer, while the degenerate mutagenic
primers for each of the light (SEQ ID NO: 2-7) or heavy (SEQ ID NO:
10-15) chain were mixed together and used as 3' primers (see Table
4 for primer sequences of SEQ ID NO: 1-12). The degenerate primers
were mixed in a ratio that made all the 3' primers equimolar in
concentration. For the second step of the amplification, the gel
purified product of the first round of PCR served as the 5' primer,
while the 3' primer was derived from the 3'-end of the light chain
(SEQ ID NO: 8) or the 3'-end of the CH1 region of the heavy chain
(SEQ ID NO: 16).
TABLE-US-00004 TABLE 4 Oligonucleotide primers used to generate the
library of genes encoding light chains and Fd regions of the
variants of HuCC49V10 SEQ ID Name Sequence NO. 5' V.sub.L
5'-TGAGCGGCACAGAGCTCGACATCGTGATGAG-3' 1 3' 89 V.sub.L
5'-AGCTATAATACTGSHKACAATAATAG-3' 2 3' 91 V.sub.L
5'-GGGGATAGCTATAGBNCTGCTGACAA-3' 3 3' 92 V.sub.L
5'-TGAGGGGATAGCTSNNATACTGCTGA-3' 4 3' 93 V.sub.L
5'-AGCTGAGGGGATASNNATAATACTGC-3' 5 3' 94 V.sub.L
5'-CGAAGCTGAGGGGSNNGCTATAATAC-3' 6 3' 96 V.sub.L
5'-CAGCGCCGAAGCTSNNGGGATAGCTA-3' 7 3' V.sub.L
5'-GCGCCGTCTAGAATTAACACTCTCCCCTGTTGAAGC 8 TCTTTGTGACGGGCGAACTCAG-3'
5' V.sub.H 5'-GCCCGTACCATGGCCCAGGTCCAGCTGGTGCA-3' 9 3' 50 V.sub.H
5'-CGGGGAGAGAASNNTCCAATCCACT-3' 10 3' 52 V.sub.H
5'-CGTTTCCGGGSNNGAAATATCCAA-3' 11 3' 53 V.sub.H
5'-AATCATCGTTSNNGGGAGAGAAAT-3' 12 3' 54 V.sub.H
5'-AAAAATCATCGNNTCCGGGAGAGA-3' 13 3' 56 V.sub.H
5'-AGTACTTAAASNHATCGTTTCCGG-3' 14 3' 58 V.sub.H
5'-TCTGTGAGTASNNAAAATCATCGT-3' 15 3' V.sub.H
5'-GCATGTACTAGTTTTGCACAAGATTTGG-3' 16 S = G/C H = A/C/T K = G/T B =
G/T/C N = A/G/C/T
The oligonucleotide primers used for DNA amplification, listed in
Table 4, were supplied by Biosynthesis Inc. (Lewisville, Tex.).
They were purified by polyacrylamide gel electrophoresis. Each of
the 5' primers used for the first step PCR and the 3' primers used
for the second step PCR carry a unique restriction endonuclease
site at its flank. The 5' VL (SEQ ID NO: 1) carried a Sac I site,
while the 3' VL (SEQ ID NO: 8) had Xba I site. The 5' VH (SEQ ID
NO: 9) and the 3' VH primers carried Nco I and Spe I sites,
respectively. To eliminate an existing Sac I site from the constant
region of the kappa chain, a point mutated Sac I site was
incorporated into the 3' VL primer.
The first PCR was carried out in a final volume of 50 .mu.l
containing 10 ng of template, 200 .mu.M dNTPs and 5 units of Taq
polymerase (Gibco BRL, Gaithersburg, Md.). The PCR mix contained
200 pmol of each of the 5' and 3' primers; the latter being a
mixture of degenerate primers. Thirty cycles of a denaturing step
at 94.degree. C. for 30 seconds, a primer annealing step at
55.degree. C. for 50 seconds, and a polymerization step at
72.degree. C. for 60 seconds were followed by a final primer
extension step for 10 minutes at 72.degree. C. The second PCR
consisted of 30 cycles of denaturation (94.degree. C. for 30
seconds), primer annealing (55.degree. C. for 90 seconds) and
polymerization (72.degree. C. for 90 seconds) followed by a final
extension for 10 minutes at 72.degree. C.
Phagemid Vector
A phagemid vector pComb3H-SS (Barbas, C. F. and Burton, D. R. Cold
Spring Harbor Laboratory Course on Monoclonal Antibodies From
Combinatorial Libraries, Cold Spring Harbor, N.Y., 1994) was used
to generate a combinatorial library of the mutated HuCC49V10 Fabs
displayed on the surface of the filamentous phage M13. The SS
designation is used for pComb3H vector when it carries a 1200 bp
stuffer sequence in place of Ig light chain (SS I), and a 300 bp
sequence as a stuffer in place of Ig heavy chain (SS II).
PComb3H-SS, a modified version of the original pComb3 vector
(Barbas et al., Proc. Natl. Acad. Sci. USA 88: 7978, 1991), was
obtained from Dr. Carlos Barbas of Scripps Research Institute,
LaJolla, Calif.
pComb3 contains both the origin of replication of the plasmid ColE1
and origin of replication of the filamentous bacteriophage f1. The
plasmid contains the gene for ampicillin (carbenicillin)
resistance. More importantly, it is designed to carry a number of
unique and convenient restriction endonuclease sites (FIG. 2). The
restriction fragment EcoR I/Sac I located downstream from lac Z
promoter carries a ribosomal binding site and the outer membrane
(omp A) leader sequence of E. coli. A sequence encoding an Ig light
chain can be inserted as a Sac I/Xba I fragment, immediately
downstream from the omp A. Located 3' to omp A is a Xba I/Nco I
fragment that contains a stop codon for the translation of the
light chain, a ribosomal binding site for the translation of the
second protein from the bicistronic message, and a pel B leader
peptide of E. carotovora. A sequence encoding the Fd fragment can
be inserted as a Nco I/Spe I fragment, immediately downstream from
the pel B leader. This is followed by a Spe I/Not I fragment that
contains the carboxy-terminal part of the gene III protein of phage
M13 and two tandem stop codons. The carboxy-terminal part of gene
III, which is fused to the Fd through a flexible linker, is located
on a Spe I/Nhe I fragment. This fragment can be removed by cleavage
with Spe I/Nhe I, followed by re-ligation which is possible because
cleavage with the two enzymes generates identical cohesive ends.
The leader peptides facilitate transport of the expressed light
chain and Fd fragments to the periplasm, where the leader peptides
are proteolytically cleaved and the free light chains and Fd
fragments assemble into Fabs. The carboxyl-terminal half of gene
III serves a capping role in the morphogenesis of filamentous
phages. In the presence of the helper phage, one Fab-gene III
fusion protein molecule along with 3-4 molecules of the native gene
III protein are displayed on the phage surface. The helper phage is
essential for replication and assembly of phage particles, because
pComb3 is devoid of all these genes. The helper phage, VCSM13, used
here for the rescue of the phage particles carries a kanamycin
resistance gene that facilitates selection of bacteria infected
with the helper virus. A recombination-deficient strain of E. coli,
XL1-Blue, carrying a transposon 10 in its F' factor has been used
to develop the phage display libraries. The F' factor is essential
for the susceptibility of bacteria to the male-specific phages.
Transposon 10 carries a tetracycline resistance gene.
Library Construction
To clone the light chain PCR products in pComb3H vector, the
PComb3H-SS plasmid was simultaneously treated with 5 units of Sac I
and 9 units of Xba I for each microgram of DNA in a reaction
mixture of appropriate volume containing the desired buffer. The
reaction mixture was incubated for 3 hours at 37.degree. C. The DNA
was ethanol precipitated, pelleted, washed with 70% ethanol, air
dried and suspended in 100 .mu.l of Tris-EDTA (TE) buffer (pH 8.0).
To isolate the linearized vector, the DNA was electrophoresed
through 0.6% agarose gel. The gel containing a band of
approximately 3.7 Kb DNA, visualized by ethidium bromide staining,
was excised and the DNA was recovered from the agarose slice by
electroelution. The recovered DNA was ethanol precipitated,
pelleted, washed, dried and resuspended in TE buffer as before.
Similarly, the PCR products generated by using the light chain
primers were treated with Sac I/Xba I, and the purified Sac I/Xba I
fragments were electrophoresed through a 2% agarose gel. The 750 bp
DNA band was excised, and the DNA fragments were recovered from the
gel by electroelution, as described earlier. Multiple reactions
were set up to ligate the Sac I/Xba I linearized vector and the Sac
I/Xba I digested PCR products. Ligation was performed for 1 hour at
room temperature, using a commercially available ligation kit
(Gibco BRL, Gaithersberg, Md.). The ligation reactions were pooled
and the DNA was ethanol precipitated, pelleted, washed, dried and
resuspended in 15 .mu.l of water. Electroporation-competent
XL-1Blue cells (Stratagene, La Jolla, Calif.) were transformed with
the ligated DNA by electroporation. Using an electroporator
(BioRad, Hercules, Calif.), a pulse of 1700 volts at the field
strength of 17 kV/cm was applied for 5 milliseconds. A series of
transformations were performed and pooled together. After adding
SOC medium (Gibco BRL), the transformation mix was incubated at
37.degree. C. for 1 hour. Subsequently, Super Broth (Gibco BRL)
containing 50 .mu.g/ml of carbenicillin and 10 .mu.g/ml of
tetracyclin was added and the culture was incubated at 37.degree.
C. on a shaker, overnight. The plasmid was isolated from the
transformed cells, using a maxiprep kit (Qiagen, Valencia, Calif.).
The isolated plasmid was linearized by digestion with 3 units of
Spe I and 9 units of of Nco I per .mu.g of the DNA. A procedure
similar to that described for preparing Sac I/Xba I linearized
vector was used to prepare Nco I/Spe I linearized plasmid.
Similarly, the PCR products that were generated by using the heavy
chain primers were digested with Nco I/Spe I and purified. The
purified PCR products were inserted into the vector by ligation,
and the ligation mixture was used to transform XL-1 Blue cells, by
electroporation. After adding tetracyclin- and
carbenicillin-supplemented Super Broth to the culture and
incubating for 1 hour at 37.degree. C., 1 ml of VCSM13 helper phage
(Stratagene) containing approximately 10.sup.12 pfus (plaque
forming units) was added to the culture. After incubation for 2
hour, kanamycin (70 .mu.g/ml) was added and the culture was shaken
overnight at 37.degree. C. Next morning, cells were spun down
(4,000 rpm, 15 minutes, 4.degree. C.) and the phage was
precipitated from the supernatant by adding PEG-8000and NaCl to the
final concentration of 4% and 3%, respectively, and incubating on
ice for 30 minutes. The precipitate was collected by centrifugation
(9,000 rpm, 20 minutes, 4.degree. C.). The pellet was suspended in
2 ml of Tris borate saline (TBS) containing 1% BSA. The phage was
titered by infecting XL-1Blue cells (OD.sub.600=0.5) with serial
dilutions of phage suspension, and plating the infected cells on
LB/carbenicillin plates.
Example 2
Selection of TAG-72-Binding HuCC49V10 Variants and Production of
Soluble Fabs
The phage library was screened and enriched for isolates binding to
TAG-72. To that end, the library was subjected to multiple (7)
rounds of panning. During each round, variants that specifically
bind to TAG-72 were selected and amplified. The selected variants
were used as a source of phagemids that were isolated and
genetically manipulated en mass to express soluble Fab
molecules.
Panning
For panning, a modification of the procedure that has been
described earlier (Parmley and Smith Gene 73:305, 1988; Barbas et
al., Proc. Natl. Acad. Sci. USA 88: 7978, 1991) was used. During
each round of panning, variants that specifically bind to TAG-72
were selected and amplified. ELISA plates (Nalgene Nunc
International, Rochester, N.Y.) were coated overnight at 4.degree.
C. with 50 .mu.l of TAG-72 positive bovine submaxillary mucin (BSM)
(Type 1-S; Sigma, St Louis, Mo.) in D-PBS with calcium and
magnesium chloride (Gibco BRL). The amount of BSM was progressively
reduced from 1.0 .mu.g to 0.01 .mu.g/well, with increasing rounds
of panning. The wells were washed with water and blocked by
incubating with milk blocking solution (KPL, Gaithersburg, Md.) at
37.degree. C. for 1 hour. Fifty .mu.l of phage
(0.15-5.times.10.sup.12 pfus) suspended in 50 .mu.l of milk diluent
solution (KPL) was pre-incubated at room temperature for 30
minutes, before it was added to the wells and incubated at
4.degree. C. overnight. After removing the milk/phage solution, the
wells were washed by pipetting TBS/0.5% Tween 20 vigorously up and
down. The washing cycles were progressively increased with
increasing rounds of panning. Finally the phage was eluted by
adding 50 .mu.l of elution buffer (0.1 M HCl, pH 2.2/BSA 1 mg/ml)
and incubating for 10 minutes at room temperature. The eluate was
removed and neutralized with 43 .mu.l of 1 M Tris base. The eluted
phage was used to infect growing XL-1 Blue cells, at room
temperature for 15 minutes, and the virus was replicated in the
presence of helper phage VCSM13. Super Broth (SB) medium containing
carbenicillin and tetracycline was added, and the culture was
shaken at 37.degree. C. for 1 hour. Phage preparations and pannings
were repeated, as described earlier. After the final round of
panning, the virus was harvested, precipitated and re-suspended in
TBS/1% BSA.
The stringent condition of panning (decreasing amounts of BSM on
the plate and increasing number of washing cycles with the higher
rounds of panning) reduced the titer of the eluted phage from
1.2.times.10.sup.9 in the first round to 1.3.times.10.sup.6 in the
seventh round. The phage eluted from the seventh round was
amplified to a titer of approximately 2.times.10.sup.13. Thus, a
significant enrichment of TAG-72 binding variants was achieved.
Preparation of Soluble Fab: Genetic Manipulation of Phagemid for
Soluble Fab Expression
The phage eluted from the last round of panning was used to infect
logarithmically growing XL-1Blue cells. The culture was grown in SB
medium containing carbenicillin (20 .mu.g/ml) and tetracycline (10
.mu.g/ml), overnight at 37.degree. C. Cells were collected by
pelleting and phagemid DNA was isolated. The DNA was digested by
treatment with Nhe I/Spe I to remove DNA fragment that encodes gene
III. The enzyme treated DNA was electrophoresed on a 0.6% agarose
gel. The large DNA fragment was gel purified, self-ligated and used
to transform competent XL-1Blue cells. Transformation mixture was
streaked on LB/carbenicillin plates. After incubating the plates
o/n at 37.degree. C., fifty individual colonies were inoculated in
10 ml of SB medium containing 20 mM MgCl.sub.2 and 50 .mu.g/ml
carbenicillin. Cultures were grown at 37.degree. C. for 6 hours,
before isopropyl-.beta.-D thiogalactopyranoside (IPTG) was added to
a final concentration of 1 mM for the induction of the Fab
expression. The culture was then shifted to 30.degree. C. and
shaken overnight. Cells were recovered by centrifugation. A part of
the cell pellet was saved to isolate the phagemid for the
subsequent sequence analysis of the variants. The rest of the cell
pellet was resuspended in 1 ml of PBS and lysed by four cycles of
freezing in a dry ice-ethanol bath for 5 minutes and thawing in a
37.degree. C. water bath. The cell debris was pelleted by
centrifugation at 15,000 rpm, and the supernatant was collected for
the Fab assay.
Example 3
Screening for High-Binding-Affinity Variants of HuCC49V10
Cell lysates from all 48 cultures of the XL-1Blue cells transformed
by the Fab constructs of pComb3H lacking the gene III fragment were
tested for the presence of Fab by ELISA and Western Blot analysis.
The cell lysates that were found positive were then screened for
binding to the TAG-72 positive BSM by Surface Plasmon Resonance
(SPR).
ELISA
Supernatants from each of the 48 cell lysates were screened to
detect Fab by ELISA assay that has earlier been described (Tamura
et al., J. Immunol. 164:1432, 2000; Bei et al., J. Immunol. Methods
186: 245, 1995). Individual wells of the 96-well polyvinyl
microtiter plates were coated with 0.1 .mu.g (50 .mu.l) of goat
anti-human kappa (Southern Biotechnology Associate, Inc., Bringham,
Ala.). The plates were blocked with 5% BSA in PBS for 1 hour at
37.degree. C. and then washed with 1% BSA in PBS. Fifty .mu.l of
the supernatant to be tested was loaded in each well. After a 1
hour incubation at 37.degree. C., plates were washed with 1% BSA in
PBS. This was followed by adding 100 .mu.l of 1:5,000 dilution (in
1% BSA in PBS) of peroxidase-conjugated goat anti-human IgG
(Fab).sub.2 fragment specific antibody (Jackson Immuno Research
Lab, West Grove, Pa.). The plates were incubated for another hour
at 37.degree. C. They were washed prior to the addition of 100
.mu.l of freshly prepared substrate (H.sub.2O.sub.2) mixed with
o-phenylene diamine hydrochloride as a chromogen (Sigma, St. Louis,
Mo.). The colorimetric reaction was allowed to proceed for 10 min
at room temperature in the dark, before it was terminated by the
addition of 50 .mu.l of 4N H.sub.2SO.sub.4 per well. The absorbance
was read at 490 nm.
Of the 48 lysates that were tested in duplicate, a great majority
of them were positive, and many among them were strongly positive
for the Fab.
Western Blot Analysis
The presence of soluble Fab in cell lysates was also tested by
Western Blot Forty .mu.l of the supernatant from each of the 48
cell lysates was electrophoresed on a 4-20% pre-cast
SDS-polyacrylamide gel. Two sets of SDS-PAGE were carried out, one
under reducing and the other under non-reducing conditions. The
proteins were electro-blotted on to the Immobilon-P membrane
(Millipore, Bedford, Mass.) according to the instructions of the
manufacturer. The blotted membrane was saturated with 5% milk for 1
hour, washed, dried and then probed with peroxidase-conjugated goat
anti-human IgG (Fab).sub.2 fragment specific antibody (Jackson
ImmunoResearch Lab, West Grove, Pa.). The membrane was washed
several times with TBS/0.05% Tween 20, before ECL Western blotting
detection kit (Amersham Pharmacia Biotech UK Limited,
Buckinghamshire, UK) was used to detect the protein band.
Western blots that were done after electrophoresis under
non-reducing conditions showed a band of approximately 55 kD, a
size in conformity with that of a Fab molecule. Similarly, Western
blotting done following SDS-PAGE under reducing conditions yielded
a band of 27-28 kD, a size expected of the Fd fragment. The Fab
could be detected in 47 of the 48 cell lysates that were tested,
albeit the degree of the intensity of the band varied.
Screening of Fabs for Their TAG-72 Binding Affinity by SPR
The expressed Fab molecules were screened for their
immunoreactivity to the TAG-72 positive BSM, using BIAcore X
instrument (Biacore, Piscataway, N.J.) for SPR measurements. All
samples were run in duplicate over a sensor chip immobilized with
500 Resonance Units (RU) of BSM. Another sensor chip immobilized
with 500 RU of BSA was used as a reference. Proteins were
immobilized on carboxymethylated dextran CM5 chips (Biacore) by
amine coupling using standard procedures (Johnsson et al., Anal.
Biochem., 198: 268, 1991; Schuck et al., 1999 Measuring protein
interactions by optical biosensors. In: J. E. Coligan, B. M. Dunn,
H. L. Ploegh, D. W. Speicher, and P. T. Wingfield (Eds.) Current
Protocols in Protein Science. John Wiley & Sons, New York, Vol.
2, p. 20.2.1.) One hundred .mu.l of each sample was applied at a
flow rate of 20 .mu.l/min and the dissociation was observed for 300
seconds. After the samples were washed with the running buffer, the
surfaces were regenerated with 1 M CAPS buffer. The BIAeval 3.0.2
program was used to analyze the data. The BSA sensorgram was
substracted from the corresponding BSM sensorgram and the Langmuir
dissociation model was used to evaluate the off rate (k off).
Supernatants from all 48 cell lysates were analyzed for their
reactivity to BSM. Since the Fabs were not purified, they were
evaluated for their dissociation rates only. Fabs derived from the
murine CC49, HuCC49V10 and human IgG (HuIgG) were included as
controls. The dissociation rates of only 6 isolates were lower than
that of HuCC49V10 (Table 5). They were characterized further,
because these isolates are likely to have higher affinity for
TAG-72.
TABLE-US-00005 TABLE 5 Dissociation rates of Fabs by Surface
Plasmon Resonance Isolate K.sub.off (1/s) HuCC49V10-12 2.20 .times.
10.sup.-4 HuCC49V10-7 2.58 .times. 10.sup.-4 HuCC49V10-14 9.14
.times. 10.sup.-4 HuCC49V10-15 1.21 .times. 10.sup.-4 HuCC49V10-10
2.50 .times. 10.sup.-3 HuCC49V10-13 4.34 .times. 10.sup.-4 mCC49
1.04 .times. 10.sup.-4 HuCC49V10 1.07 .times. 10.sup.-3 HuIgG 1.38
.times. 10.sup.-2
The phagemids prepared from the cell pellets of the six isolates
were used for the sequencing of the inserts encoding the variable
regions of the light and heavy chain. DNA sequencing was carried
out by the method of dideoxy-mediated chain termination. Amino acid
sequences deduced from the nucleotide sequences showed
substitutions in LCDR3 of all the six variants. These substitutions
were limited to the positions 91, 93 and 94. Whereas, only one
variant showed substitutions in HCDR2 (positions 52 and 58). Two
variants showed inadvertent mutation in position 27b of the LCDR1
(Table 2).
Example 4
Expression of HuCC49V10 Variants in Insect Cells; Purification and
Characterization of the Expressed Antibodies
For further characterization of the variants, they were expressed
in insect cells as whole antibodies, rather than as Fab fragments.
To that end, expression constructs of the genes encoding the heavy
and light chains of the variants were made in vectors containing
promoters that are functional in insect cells. The variant
antibodies were purified and studied for their relative
antigen-binding affinity and their ability to bind to a cell
surface antigen.
Expression Constructs of the Heavy and Light Chain Genes of the
Variant Antibodies
Two different vectors, pIZ/V5-His (FIG. 3A) and pIB/V5-His (FIG.
3B) (Invitrogen, Carlsbad, Calif.), carry the baculovirus immediate
early promoter OpIE2 that drives the expression of heterologous
proteins in lepidopteran insect cells, without requiring viral
factors for its activation. A multiple cloning site located
downstream from the promoter, in both vectors, facilitates cloning
of the gene of interest to be expressed constitutively. A
polyadenylation sequence placed immediately 3' to the multiple
cloning site ensures efficient transcription termination and
polyadenylation of mRNA. One vector, pIZ/V5-His, carries the Zeocin
resistance gene, while the other, pIB/V5-His, carries the
blasticidin resistance gene. These genes are used for selecting
stable transfectants of the insect cell line.
The sequences encoding the light chain of the variants were
assembled in the pIZ/V5-His vector. A series of genetic
manipulations were required to provide a leader sequence for each
of the genes encoding the light chain. Essentially, a 538 bp BsmA
I/Blp I DNA fragment carrying the LCDR3 and/or LCDR1 mutation(s)
and extending into the constant region was PCR amplified from each
of the variants. This DNA fragment replaced the corresponding
sequence from a pBluescript construct of the variant HuCC49V10-5,
which carried the light chain gene of HuCC49V10 along with its
leader sequence. The construct was digested with Sma I, for which a
unique site was located immediately 3' to the insert, an Xba I
linker was ligated to the DNA ends, and the insert was lifted as a
Hind III/Xba I fragment of approximately 1 Kb. The insert was
cloned downstream from the OpIE2 promoter at the Hind III/Xba I
site.
The heavy chain expression constructs were made in the p13/V5-His
vector. Since only one (varaint HuCC49V10-13) of the six variants
showed any mutations in the HCDR2, the heavy chain construct of the
variant HuCC49V10-8, which carried the heavy chain gene of the
HuCC49V10 along with its leader sequence, was paired with the light
chain constructs of the variants (variants HuCC49V10-7,
HuCC49V10-10, HuCC49V10-12, HuCC49V10-14 and HuCC49V10-15) for the
production of the variant antibodies. The gene encoding the heavy
chain along with its leader was excised from the pBSc construct of
the variant HuCC49V10-8 as a Hind III/Xho I fragment. The insert
was cloned at the Hind III/Xho I site, downstream of the OpIE2
promoter in pIB/V5-His vector.
Construction of the expression vector containing the heavy chain of
variant HuCC49V10-13 in the pIB/V5-His vector, while incorporating
the eukaryotic leader sequence between the promoter and the gene,
required some genetic manipulation. Essentially, an 85 bp Bcl I/Sca
I fragment of variant HuCC49V10-8 was replaced with the
corresponding DNA fragment of variant HuCC49V10-13 that carried the
mutations in HCDR2. The manipulated insert was lifted from the
construct as a Hind III/Xho I fragment and cloned in pIB/V5-His
vector, downstream from the promoter.
Production of the Variant Antibodies and Their Immunoreactivity
To develop transfectomas secreting the HuCC49 variants,
serum-free-adapted Sf9 insect cells (Gibco BRL) were used. Two
million insect cells plated in each well of a 6-well plate were
co-transfected with 10 .mu.g each of the pIZ/V5-His-light chain and
pIB/V5-His-heavy chain constructs, using Insectin-Plus Liposomes
(Invitrogen) to mediate the transfections. After four days, the
culture supernatants were harvested and tested, by ELISA, for Ig
secretion and the reactivity of the secreted antibody to TAG-72.
For isolating stable transfectants, selection medium containing 200
.mu.g/ml of zeocin and 50 .mu.g/1 ml of blasticidin was used. For
ELISA assays, four-day harvests of culture supernatants were
collected and frozen at -20.degree. C., prior to use.
The ELISA assay for monitoring Ig production was carried out by a
procedure described earlier (Example 3). To test the reactivity of
the secreted antibody to TAG-72, 1 .mu.g/well of BSM was coated on
the individual wells of the 96-well polyvinyl microtiter plates.
After saturation of the plates for 1 hour at 37.degree. C. with
milk blocking solution (KPL), 50 .mu.l of diluted culture
supernatants were added to the wells, in duplicate, followed by
incubation at 37.degree. C. for 1 hour. After a cycle of washings
with the washing solution (KPL), 100 .mu.l of peroxidase-conjugated
anti-human IgG (Fc.gamma.-fragment specific), diluted 1:3000 in
milk diluent solution (KPL), was added to the plates and the
incubation continued for an additional hour at 37.degree. C. They
were washed prior to the addition of 100 .mu.l of TMB peroxidase
substrate (KPL). The colorimetric reaction proceeded for 10 minutes
at room temperature, before the addition of the stop solution
(KPL). The absorbance was read at 450 nm.
Results of the ELISA of the culture supernatants for IgG showed
that all the variants produced antibody. When the culture
supernatants were assayed for their reactivity to TAG-72, it became
evident that the antibodies produced by the variants were specific
to TAG-72. When the ELISA was carried out using serially diluted
supernatants, the results of the assay suggested that the
antigen-binding reactivity of, at least two variant antibodies,
HuCC49V10-14 and HuCC49V10-15, was either comparable to or exceeded
that of the parental HuCC49V10, while the reactivity of variant
HuCC49V10-13 was convincingly lower than that of HuCC49V10. Variant
HuCC49V10-13 was not included in further studies.
Purification of the Variant Antibodies and Their SDS-PAGE
Analysis
The supernatants collected from the cultures of the transfectomas
producing the variant antibodies were centrifuged at 2,000.times.g
for 10 minutes to remove cellular debris and loaded on a protein G
agarose column (Gibco BRL). 0.1 M glycine hydrochloride pH 2.5 was
used to elute the proteins bound to the column. The pH of the
eluted material was immediately adjusted to 7.4 with 1.0 M Tris pH
8.0. The proteins were concentrated using a Centricon 30 (Amicon,
Beverly, Mass.) and dialyzed in PBS buffer using Slide-A-Lyzer
cassette (Pierce, Rockford, Ill.). The protein concentration was
determined by the method of Lowry (Lowry et al., J. Biol. Chem.193:
265, 1951). The concentration of the variant antibodies ranged
between 2-5 .mu.g/ml of the culture supernatant. The purity of the
eluted proteins was evaluated by SDS-PAGE, under reducing and
non-reducing condition, using pre-cast 4-20% Tris-glycine gel
(Novex, San Diego, Calif.) and Coomassie blue staining (Novex)
visualization. Under non-reducing conditions (FIG. 4A) a protein
band of approximately 160 kD was seen, while the reducing condition
(FIG. 4B) yielded two protein bands of approximately 50-55 kD and
approximately 25-28 kD, the sizes expected of the heavy and light
chains of an IgG molecule.
Competition Radioimmunoassay (RIA)
The relative antigen-binding affinity of the variant antibodies was
determined using competition RIA, as described earlier (Iwahashi et
al., Mol. Immunol. 36:1079, 1999; Tamura et al., J. Immunol.
164:1432, 2000). Murine CC49, HuCC49 and HuIgG were included in the
assay as positive and negative controls. Twenty five .mu.l of
serial dilutions of the antibodies, re-suspended in 1% BSA in PBS,
were added to microtiter plates containing 10 ng of BSM saturated
with 5% BSA in PBS. .sup.125I-labeled HuCC49 (100,000 cpm in 25
.mu.l of 1% BSA in PBS) was then added to each well. The assay was
set up in triplicate. After an overnight incubation at 4.degree.
C., the plates were washed and counted in a .gamma.-scintillation
counter.
The results of the competition assay (FIG. 5) show that all the
antibodies, except the HuIgG control, were able to completely
inhibit the binding of .sup.125I-labeled HuCC49 to BSM. The
competition profiles of the variants HuCC49V10-7 and HuCC49V10-12
were shifted to the right, while the profile of HuCC49V10-14 was
shifted only slightly and that of HuCC49V 10-15 considerably to the
left of the competition profile of the parental antibody HuCC49V10.
Competition profiles, shown in FIG. 5, were used to calculate the
amount of each unlabeled competitor required for 50% inhibition of
the binding of .sup.125I-labeled HuCC49 to BSM (Table 6). Compared
to 150 ng of HuCC49V10, 220 ng and 350 ng of variants HuCC49V10-7
and HuCC49V10-12, respectively, were needed. In contrast, 92 ng of
HuCC49V10-14 and only 58 ng of HuCC49V10-15 were required for 50%
inhibition of the binding of .sup.125I labeled HuCC49 to BSM. Thus,
HuCC49V10-15 is approximately 3-fold better than that of HuCC49V10
antigen binding. The relative affinity constants (K.sub.a) were
calculated, from a range of competition experiments, using a
modification of the Scatchard methods (Frankel and Gerhard, Gene
16:101, 1979). The K.sub.a values for HuCC49V10-14 were only
comparable, while those for HuCC49V10-15 were approximately 50%
higher than that of HuCC49V10.
TABLE-US-00006 TABLE 6 Relative affinity binding of CC49 antibodies
Amount needed for 50% inhibition of the binding CC49 antibody of
.sup.125I-labeled HuCC49 to BSM (ng) mCC49: 25 HuCC49: 78
HuCC49V10: 150 HuCC49V10-7: 220 HuCC49V10-12: 350 HuCC49V10-14: 92
HuCC49V10-15: 58
Flow Cytometric Analysis
Flow cytometric analysis was used to measure the binding of the
HuCC49V10 variants to the TAG-72 expressed on the cell surface of a
T cell line, Jurkat. The procedure for FACS analysis has been
described (Guadagni et al., Cancer Res. 50:6248, 1990). In addition
to the isotype matched antibody, HuIgG, used as a negative control,
HuCC49 and chimeric CC49 were included as positive controls. To
evaluate the ability of the variants to bind to cell-surface
TAG-72, 1.times.10.sup.6 Jurkat cells were resuspended in cold
Ca.sup.++ and Mg.sup.++ free Dulbecco's PBS and incubated with the
antibody to be tested for 30 minutes on ice. After one washing
cycle, the cell suspension was stained with FITC-conjugated mouse
anti-human antibody (Pharmingen) for 30 minutes on ice. A second
washing cycle was performed before the samples were analyzed with a
FACScan (Becton Dickinson, Mountain View, Calif.) using CellQuest
for Macintosh. Data from the analysis of 10,000 cells were
obtained.
Different concentrations of antibodies were used to compare their
binding to the Jurkat cells expressing cell surface TAG-72. When
1.0 .mu.g of each antibody was used, the percentage of gated cells,
calculated after exclusion of irrelevant binding, was 27.8 for
HuCC49V10, while for the variants HuCC49V10-14 and HuCC49V10-15, it
was 43.7 and 68.1, respectively. Thus, the two variants show
significantly better binding to the cells displaying TAG-72 on
their surface. In contrast, the binding of the variants HuCC49V10-7
and HuCC49V10-12 was comparable to that of the parental HuCC49V10
(FIG. 6).
Example 5
Sera Reactivity of HuCC49V10 Variants
To assess the potential immunogenicity of the HuCC49V10 variants in
patients, the variants were tested for their reactivity to sera
stored from the adenocarcinoma patients in a phase I clinical trial
(Mulligan et al., Clin. Cancer Res. 1: 1447, 1995). Patients in
this clinical trial were administered .sup.177Lu-labeled murine
CC49 and were found to have anti variable region, including
anti-idiotypic, antibodies to CC49 (Iwahashi et al., Mol. Immunol.
36: 1079, 1999; Tamura et al., J. Immunol. 164: 1432, 2000). Sera
reactivity was determined by a highly sensitive Surface Plasmon
Resonance (SPR)-based competition assay. This assay involves the
use of a device (BIAcore X instrument) that monitors binding of the
sera anti-idiotypic (or anti variable region) antibodies to HuCC49,
and the inhibition of this binding by the variants. IC.sub.50, the
concentration of the competitor antibody required for 50%
inhibition of the binding of the HuCC49 to the patient's serum was
calculated by plotting the percent inhibition as a function of the
competitor concentration. A higher IC.sub.50 indicates a decreased
reactivity to the serum suggesting potentially reduced
immunogenicity of the competitor antibody (HuCC49 variant) in
patients.
Immunoadsorption of Patients Sera
Sera from patient EA and DS from the phase I clinical trial were
used to compare the reactivity of the variants HuCC49V10-14 and
HuCC49V10-15 to that of the parental HuCC49V10. The sera, however,
contain circulating TAG-72 antigen and anti-murine Fc antibodies
that might interfere with the binding of the HuCC49 and its
variants to the sera anti-idiotypic (anti variable) antibodies. To
overcome this difficulty, TAG-72 and antibodies to murine Fc were
removed from the sera by immunoadsorption prior to checking the
sera reactivity. The procedure for immunoadsorption has been
described (Iwahashi et al., Mol. Immunol. 36: 1079, 1999; Tamura et
al., J. Immunol. 164: 1432, 2000). Essentially, a murine antibody,
CC92, which reacts with an epitope of TAG-72 distinct from the one
recognized by CC49 (Kuroki et al., Cancer Res. 50: 4872, 1990) was
coupled to Reactigel (HW65F; Pierce, Rockford. IL) (Hearn et al.,
J. Chomatogr. 185: 463, 1979). Serum was added to an equivalent
volume of the CC92 gel (wet-packed volume) and incubated overnight
at 4.degree. C. with end-over-end rotation. The samples were
centrifuged at 1,000.times.g for 5 minutes and the supernatant was
saved and stored.
SPR-Based Competition Assay
To test the sera reactivities of antibodies, SPR measurements were
done with the BIAcore X instrument (described in Example 3) using
carboxymethylated dextran chips CM5 (BIAcore, Piscataway, N.J.).
Proteins were immobilized on the CM5 chips by amine coupling
(Johnsson et al., Anal. Biochem. 198: 268, 1991). The dextran layer
of the sensor chip was activated by injecting 35 .mu.l of a mixture
of N-ethyl-N'-(3 dimethylaminopropyl)carbodiimide hydrochloride and
N-hydroxysuccinimide at a flow rate of 5 .mu.l/min. Proteins
diluted in 10 mM sodium acetate buffer (pH 5.0) at a concentration
of 100 .mu.g/ml were then injected until surfaces of 5000 resonance
units (RUs) were obtained. The remaining reactive groups on the
surfaces were blocked by injecting 35 .mu.l of 1 M ethanolamine (pH
8.5).
To compare the sera reactivity of HuCC49, HuCC49V10 and its
variants HuCC49V10-14 and HuCC49V10-15, competition experiments
were done at 25.degree. C. on a sensor chip containing HuCC49 in
flow cell 1 and rabbit gamma globulin (BioRad, Hercules, Calif.) in
flow cell 2 as a reference. A recently developed sample application
technique was used (Abrantes et al., Anal. Chem. 73: 2828, 2001),
in which the microfluidics control of the instrument was replaced
by an externally installed computer-controlled syringe pump with
stepping motor (model 402 from Gilson Inc., Middleton, Wis.). A
tubing was inserted into the open port of the connector block in
order to serve as an inlet port through which the sample can be
aspirated, and the port previously designated as running buffer
inlet was connected to the syringe pump (Abrantes et al., Anal.
Chem. 73: 2828, 2001). The computer-controlled aspiration made it
possible to use small sample volumes. Typically, the microfluidics
system was rinsed and filled with running buffer (10 mM HEPES (pH
7.4), 150 mM NaCl, 3 mM EDTA and 0.005% Tween 20). This was
followed by sequential aspiration of 2 .mu.l of air, 0.3 .mu.l of
sample (preadsorbed serum.+-.antibodies as competitors), 2 .mu.l of
air, 5-6 .mu.l of sample, 2 .mu.l of air, 0.3 .mu.l of sample, and
2 .mu.l of air into the inlet tubing at a rate of 20 .mu.l/min. The
sample was centered across the sensor surface and an oscillatory
flow was applied at a rate of 20 .mu.l/min. This flow ensures
efficient mass transfer of the sample to the surface and allows for
a very long contact time without net displacement of the sample.
The binding was measured for 1000 seconds. After the unbound
samples were removed from the surfaces by washing with running
buffer using a flow rate of 100 .mu.l/min, the surfaces were
regenerated with a one-minute injection of 10 mM glycine (pH 2.0).
The percent binding at each antibody concentration was calculated
as follows: % binding=[slope of the signal obtained with competitor
(serum+antibody)/slope of the signal obtained without competitor
(serum only)].times.100.
FIG. 7 shows the competition profiles generated by HuCC49 and
different variants when they were used to compete with the HuCC49
immobilized on the sensor chip for binding to the anti-idiotypic
(anti variable) antibodies to CC49 present in the sera of the
patients EA and DS. For serum from patient DS, approximately one
micromole of the variant HuCC49V10 is required for 50% inhibition
of the binding of HuCC49 to the serum, while the variants
HuCC49V10-14 and HuCC49V10-15 do not show any significant
inhibition at this concentration. For serum from patient EA,
approximately 20 nanomoles of the HuCC49V10 is needed for 50%
inhibition of the binding of HuCC49 to the serum, whereas one
micromole of each of the HuCC49V10-14 and HuCC49V10-15 variants
cause less than 40% inhibition. Thus, the two mutants showed not
only significantly higher antigen binding affinity than that of
HuCC49V10, but they also showed much lower reactivity to sera from
patients who showed an anti-idiotypic response to the parental CC49
antibody.
The improved affinity and the minimal sera reactivity of the
variants HuCC49V10-14 and HuCC49V10-15 make them potentially much
better clinical reagents than the variant HuCC49V 10.
Example 6
HuCC49V10-14 and HuCC46V10-15 Testing in Patients
Patients and Sample Collection
Patients with recurrent metastatic adenocarcinoma are assessed to
determine the maximum tolerated dose of intravenously administered
.sup.177Lutetium radiolabeled HuCC49V10-14 and .sup.177Lutetium
radiolabeled HuCC49V10-15 (Mulligan, (1995) Clin. Cancer Res. 1:
1447-1454). Adenocarcinoma patients are given a test dose of 0.1 mg
(intravenous bolus) of HuCC49V10-14 or HuCC49V10-15 and are
observed for 30 minutes prior to administration of the
.sup.177Lu-labeled HuCC49V10-14 or .sup.177Lu-labeled HuCC49V10-15.
The radiolabeled antibodies are given as an intravenous infusion
over the course of a one hour time interval. Blood samples are
collected prior to and at the end of the infusion, as well as 0.5,
1 and 2 hours following the completion of the infusion. In
addition, blood samples are collected daily over the subsequent 7
days. Patients return for a follow-up examination at 3, 6 or 8
weeks. Blood samples are again collected during these visits. Sera
are separated and stored at -20.degree. C.
Determination of Patient Humoral Response
The sera from the patients are evaluated for the presence of human
anti-murine antibodies (HAMA) in response to radiolabeled
HuCC49V10-14 or HuCC49V10-15 using the SPR-based assay described in
Example 5, above. The sera is pre-absorbed with a CC92 monoclonal
antibody that recognizes an epitope of TAG-72 which is different
from the epitope recognized by the humanized CC49 monoclonal
antibody. Pre-absorption using the CC92 antibody removes
circulating TAG-72 from the sera. To monitor the sera-reactivity of
the anti-variable antibodies in the pre-absorbed sera, HuCC49V10-14
or HuCC49V10-15 is coated on the surface of flow cell 1 and a
reference protein (HuIgG1, bovine serum albumin, or rabbit gamma
globulin) is immobilized on the surface of flow cell 2. A small,
known volume of a patient serum sample us applied to each flow cell
using the recently developed sample application technique described
in Example 5 (Abrantes et al., Anal. Chem. 73:2828, 2001).
Sensograms to flow cell 1 and flow cell 2 are generated and the
response difference between the two cells is plotted for each serum
sample, thus providing a measure of the anti-variable region
response against HuCC49V10-14 or HuCC49V10-15 in each particular
serum sample. Results indicate that the patients' sera have a
minimal anti-variable region response against the HuCC49V10-14 and
HuCC49V10-15 antibodies.
This disclosure provides humanized CC49 monoclonal antibodies. The
disclosure further provides methods of diagnosing and treating
tumors using these humanized CC49 antibodies. It will be apparent
that the precise details of the methods described may be varied or
modified without departing from the spirit of the described
invention. We claim all such modifications and variations that fall
within the scope and spirit of the claims below.
SEQUENCE LISTINGS
1
16 1 31 DNA Artificial sequence primer 1 tgagcggcac agagctcgac
atcgtgatga g 31 2 26 DNA Artificial sequence primer 2 agctataata
ctgshkacaa taatag 26 3 26 DNA Artificial sequence primer 3
ggggatagct atagbnctgc tgacaa 26 4 26 DNA Artificial sequence primer
4 tgaggggata gctsnnatac tgctga 26 5 26 DNA Artificial sequence
primer 5 agctgagggg atasnnataa tactgc 26 6 26 DNA Artificial
sequence primer 6 cgaagctgag gggsnngcta taatac 26 7 26 DNA
Artificial sequence primer 7 cagcgccgaa gctsnnggga tagcta 26 8 58
DNA Artificial sequence primer 8 gcgccgtcta gaattaacac tctcccctgt
tgaagctctt tgtgacgggc gaactcag 58 9 32 DNA Artificial sequence
primer 9 gcccgtacca tggcccaggt ccagctggtg ca 32 10 25 DNA
Artificial sequence primer 10 cggggagaga asnntccaat ccact 25 11 24
DNA Artificial sequence primer 11 cgtttccggg snngaaatat ccaa 24 12
24 DNA Artificial sequence primer 12 aatcatcgtt snngggagag aaat 24
13 24 DNA Artificial sequence primer 13 aaaaatcatc gnntccggga gaga
24 14 24 DNA Artificial sequence primer 14 agtacttaaa snhatcgttt
ccgg 24 15 24 DNA Artificial sequence primer 15 tctgtgagta
snnaaaatca tcgt 24 16 28 DNA Artificial sequence primer 16
gcatgtacta gttttgcaca agatttgg 28
* * * * *