U.S. patent number 6,054,297 [Application Number 08/437,642] was granted by the patent office on 2000-04-25 for humanized antibodies and methods for making them.
This patent grant is currently assigned to Genentech, Inc.. Invention is credited to Paul J. Carter, Leonard G. Presta.
United States Patent |
6,054,297 |
Carter , et al. |
April 25, 2000 |
Humanized antibodies and methods for making them
Abstract
Variant immunoglobulins, particularly humanized antibody
polypeptides are provided, along with methods for their preparation
and use. Consensus immunoglobulin sequences and structural models
are also provided.
Inventors: |
Carter; Paul J. (San Francisco,
CA), Presta; Leonard G. (San Francisco, CA) |
Assignee: |
Genentech, Inc. (South San
Francisco, CA)
|
Family
ID: |
27377060 |
Appl.
No.: |
08/437,642 |
Filed: |
May 9, 1995 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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934373 |
Aug 21, 1992 |
5821337 |
Oct 13, 1998 |
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PCTUS9205126 |
Jun 15, 1992 |
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715272 |
Jun 14, 1991 |
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Current U.S.
Class: |
435/69.6;
424/155.1; 424/130.1; 424/174.1; 530/387.3; 530/389.7; 435/328;
530/387.7; 530/388.8; 435/69.7; 435/344; 424/138.1; 424/133.1 |
Current CPC
Class: |
G06F
15/00 (20130101); C07K 16/465 (20130101); A61P
37/02 (20180101); C07K 16/28 (20130101); C07K
16/32 (20130101); A61P 35/00 (20180101); C07K
16/2809 (20130101); C07K 2317/54 (20130101); C07K
2317/24 (20130101); C07K 2317/31 (20130101); A61K
38/00 (20130101); C07K 2317/732 (20130101); C07K
2319/55 (20130101); C07K 2319/30 (20130101); C07K
2317/55 (20130101) |
Current International
Class: |
C07K
16/18 (20060101); C07K 16/32 (20060101); C07K
16/28 (20060101); C07K 16/46 (20060101); G06F
15/00 (20060101); A61K 38/00 (20060101); A61K
039/395 (); C12N 015/13 (); C07K 016/00 (); C07K
016/30 () |
Field of
Search: |
;530/387.3,387.7,388.8,389.7 ;424/130.1,133.1,138.1,155.1,174.1
;435/328,344,69.6,69.7 |
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WO 94/12214 |
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|
Primary Examiner: Hutzell; Paula K.
Assistant Examiner: Reeyes; Julie E
Attorney, Agent or Firm: Lee; Wendy M.
Parent Case Text
This application is a continuation of U.S. Ser. No. 07/934,373
filed Aug. 21, 1992 (now U.S. Pat. No. 5,821,337 issued Oct. 13,
1998), which is a continuation-in-part of PCT/US92/05126 filed Jun.
15, 1992, which is a continuation-in-part of U.S. Ser. No.
07/715,272 filed Jun. 14, 1991 (now abandoned).
Claims
We claim:
1. A method for making a humanized antibody comprising non-human,
import Complementarity Determining Region (CDR) amino acid residues
and human Framework Region (FR) amino acid residues, comprising the
steps of:
(a) obtaining the amino acid sequences of an import variable domain
and of a V.sub.H subgroup III consensus human variable domain;
(b) identifying CDR amino acid sequences in the import and the
human variable domain sequences;
(c) substituting import CDRs for the corresponding human CDRs;
(d) aligning the amino acid sequences of a FR of the import
antibody and the corresponding FR of the consensus variable
domain;
(e) identifying import antibody FR residues in the aligned FR
sequences that are non-homologous to the corresponding consensus
variable domain residues;
(f) determining if the non-homologous import amino acid residue is
expected to have at least one of the following effects:
(1) non-covalently binds antigen directly;
(2) interacts with a CDR; or
(3) participates in the V.sub.L -V.sub.H interface;
(g) for any non-homologous import antibody amino acid residue which
is expected to have at least one of these effects, substituting
that residue for the corresponding amino acid residue in the
consensus variable domain FR sequence; and
(h) preparing a humanized antibody which binds antigen, wherein the
humanized antibody comprises an amino acid sequence determined
according to the above steps.
2. The method of claim 1, having an additional step of determining
if any such non-homologous residues are exposed on the surface of
the domain or buried within it, and if the residue is exposed,
retaining the consensus residue.
3. The method of claim 1, having the additional steps of searching
the import variable domain sequence for glycosylation sites,
determining if any such glycosylation site is expected to affect
the antigen binding or affinity of the antibody, and if so,
substituting the glycosylation site into the consensus
sequence.
4. The method of claim 1, having the additional steps of searching
the consensus variable domain sequence for glycosylation sites
which are not present at the corresponding amino acid in the import
sequence, and if the glycosylation site is not present in the
import sequence, substituting the import amino acid residues for
the amino acid residues comprising the consensus glycosylation
site.
5. The method of claim 1, having an additional step which comprises
aligning import antibody and consensus variable domain FR
sequences, identifying import antibody FR residues which are
non-homologous with the aligned consensus FR sequence, and for each
such non-homologous import antibody FR residue, determining if the
corresponding consensus variable domain residue represents a
residue which is highly conserved across all species at that site,
and if it is so conserved, preparing a humanized antibody which
comprises the consensus amino acid residue at that site.
6. The method of claim 1, wherein the corresponding consensus
residues are selected from the group consisting of 4L, 35L, 36L,
38L, 43L, 44L, 46L, 58L, 62L, 63L, 64L, 65L, 66L, 67L, 68L, 69L,
70L, 71L, 73L, 85L 87L, 98L, 2H, 4H, 24H, 36H, 37H, 39H, 43H, 45H,
49H, 58H, 60H, 67H, 68H, 69H, 70H, 73H, 74H, 75H, 76H, 78H, 91H,
92H, 93H, and 103H.
7. A method for making a humanized antibody comprising non-human
Complementarity Determining Region (CDR) amino acid residues and
human Framework Region (FR) amino acid residues, comprising
providing an import, non-human antibody variable domain amino acid
sequence having CDR amino acid residues and FR amino acid residues;
obtaining the amino acid sequence of a V.sub.H subgroup III
consensus human antibody variable domain having CDR amino acid
residues and FR amino acid residues; substituting non-human CDR
amino acid residues for human CDR amino acid residues in the
consensus human antibody variable domain; substituting an amino
acid residue for the consensus amino acid residue at at least one
of the following sites:
4L, 35L, 36L, 38L, 43L, 44L, 46L, 58L, 62L, 63L, 64L, 65L, 66L,
67L, 68L, 69L, 70L, 71L, 73L, 85L, 87L, 98L, 2H, 4H, 24H, 36H, 37H,
39H, 43H, 45H, 49H, 58H, 60H, 67H, 68H, 69H, 70H, 73H, 74H, 75H,
76H, 78H, 91H, 92H, 93H. and 103H; and preparing a humanized
antibody which binds an antigen, wherein the humanized antibody
comprises an amino acid sequence determined according to the above
steps.
8. The method of claim 7, wherein the substituted residue is the
residue found at the corresponding location of the non-human
antibody.
9. A humanized antibody variable domain having a functional antigen
binding region, said humanized antibody variable domain comprising
non-human Complementarity Determining Region (CDR) amino acid
residues incorporated into a V.sub.H subgroup III consensus human
antibody variable domain, and further comprising an amino acid
substitution at a site selected from the group consisting of:
4L, 35L, 36L, 38L, 43L, 44L, 46L, 58L, 62L, 64L, 65L, 66L, 67L,
68L, 69L, 70L, 71L, 73L, 85L, 87L, 98L, 2H, 4H, 24H, 36H, 37H, 39H,
43H, 45H, 49H, 58H, 60H, 68H, 69H, 70H, 73H, 74H, 75H, 76H, 78H,
92H, and 93H.
10. The humanized antibody variable domain of claim 9, wherein the
substituted residue is the residue found at the corresponding
location of the non-human antibody from which the non-human CDR
amino acid residues are obtained.
11. The humanized antibody variable domain of claim 9, wherein no
human (FR) Framework Region (FR) residue other than those set forth
in the group has been substituted.
12. A method for making a humanized antibody comprising introducing
Complementarity Determining Region (CDR) amino acid residues from
an import antibody variable domain into a V.sub.H subgroup III
consensus human antibody variable domain.
13. A humanized antibody variable domain having a functional
antigen binding region, said humanized antibody variable domain
comprising non-human Complementarity Determining Region (CDR) amino
acid residues incorporated into a V.sub.H subgroup III consensus
human antibody variable domain and further comprising a non-human
import Framework Region (FR) residue, wherein the non-human import
FR residue introduces a glycosylation site which affects the
antigen binding or affinity of the humanized antibody variable
domain.
14. A humanized antibody which binds the HER2 receptor with an
affinity of about 4.7 nM K.sub.d or better affinity and comprises a
heavy chain variable domain which comprises non-human import
antibody Complementarity Determining Region (CDR) amino acid
residues incorporated into a V.sub.H subgroup III consensus human
variable domain.
15. A humanized variant of a non-human parent antibody, wherein the
humanized variant comprises non-human Complementarity Determining
Region (CDR) amino acid residues and human Framework Region (FR)
amino acid residues, and:
(a) binds the HER2 receptor with an affinity of about 4.7 nM
K.sub.d or better affinity:
(b) mediates specific cell lysis of SK-BR-3 calls in the presence
of IL-2 activated human peripheral blood lymphocytes at least about
four fold more effectively than the non-human parent antibody;
and
(c) mediates Antibody Dependent Cellular Cytotoxicity (ADCC)
selective for cell types which overexpress p185.sup.HER2 at least
about two fold more effectively than for cell types which express
low levels of p185.sup.HER2.
16. The humanized variant of claim 15 which binds the HER2 receptor
with an affinity of about 0.82 nM K.sub.d or better affinity.
17. The humanized variant of claim 16 which binds the HER2 receptor
with an affinity of about 0.10 nM K.sub.d.
18. The humanized variant of claim 15 which inhibits proliferation
of SK-BR-3 cells incubated for 96 hr with the antibody.
19. The humanized variant of claim 18 wherein the antibody inhibits
proliferation of SK-BR-3 cells to about 66% of untreated control or
greater inhibition.
20. A humanized variant of a non-human parent antibody, wherein the
humanized variant comprises non-human Complementarity Determining
Region (CDR) amino acid residues and human Framework Region (FR)
amino acid residues, and;
(a) binds the HER2 receptor with an affinity of about 4.7 nM
K.sub.d or better affinity; and
(b) comprises a Framework Region (FR) amino acid substitution at a
site selected from the group consisting of 73H, 78H, 93H and 66L,
utilizing the numbering system set forth in Kabat.
21. The humanized variant of claim 20 which consists of about 1 to
about 5 FR substitutions.
22. The humanized variant of claim 20 which comprises a FR
substitution at site 73H.
23. The humanized variant of claim 20 which comprises a FR
substitution at site 78H.
24. The humanized variant of claim 20 which comprises a FR
substitution at site 93H.
25. The humanized variant of claim 20 which comprises a FR
substitution at site 66L.
26. The humanized variant of claim 20 which further comprises a FR
substitution at site 71H.
27. The humanized variant of claim 26 which comprises FR
substitutions at sites 71H, 73H, 78H, 93H and 66L.
28. A humanized variant of a non-human parent antibody, wherein the
humanized variant comprises non-human Complementarity Determining
Region (CDR) amino acid residues and human Framework Region (FR)
amino acid residues; binds the HER2 receptor with better affinity
than the non-human parent antibody; and comprises Framework Region
(FR) amino acid substitutions at sites 71H, 73H, 78H, 93H and 66L,
utilizing the numbering system set forth in Kabat.
29. An antibody which binds an antigen and comprises non-human
heavy chain variable domain Complementarity Determining Region
(CDR) amino acid residues which bind said antigen and V.sub.H
subgroup III consensus human variable domain Framework Region (FR)
amino acid residues; and further comprises non-human light chain
variable domain CDR amino acid residues which bind said
antigen.
30. The antibody of claim 29, further comprising V.sub.L .kappa.
subgroup I consensus human variable domain FR amino acid residues.
Description
FIELD OF THE INVENTION
This invention relates to methods for the preparation and use of
variant antibodies and finds application particularly in the fields
of immunology and cancer diagnosis and therapy.
BACKGROUND OF THE INVENTION
Naturally occurring antibodies (immunoglobulins) comprise two heavy
chains linked together by disulfide bonds and two light chains, one
light chain being linked to each of the heavy chains by disulfide
bonds. Each heavy chain has at one end a variable domain (V.sub.H)
followed by a number of constant domains. Each light chain has a
variable domain (V.sub.L) at one end and a constant domain at its
other end; the constant domain of the light chain is aligned with
the first constant domain of the heavy chain, and the light chain
variable domain is aligned with the variable domain of the heavy
chain. Particular amino acid residues are believed to form an
interface between the light and heavy chain variable domains, see
e.g. Chothia et al., J. Mol. Biol. 186:651-663 (1985); Novotny and
Haber, Proc. Natl. Acad. Sci. USA 82:4592-4596 (1985).
The constant domains are not involved directly in binding the
antibody to an antigen, but are involved in various effector
functions, such as participation of the antibody in
antibody-dependent cellular cytotoxicity. The variable domains of
each pair of light and heavy chains are involved directly in
binding the antibody to the antigen. The domains of natural light
and heavy chains have the same general structure, and each domain
comprises four framework (FR) regions, whose sequences are somewhat
conserved, connected by three hyper-variable or complementarity
determining regions (CDRs) (see Kabat, E. A. et al., Sequences of
Proteins of Immunological Interest, National Institutes of Health,
Bethesda, Md., (1987)). The four framework regions largely adopt a
.beta.-sheet conformation and the CDRs form loops connecting, and
in some cases forming part of, the .beta.-sheet structure. The CDRs
in each chain are held in close proximity by the framework regions
and, with the CDRs from the other chain, contribute to the
formation of the antigen binding site.
Widespread use has been made of monoclonal antibodies, particularly
those derived from rodents including mice, however they are
frequently antigenic in human clinical use. For example, a major
limitation in the clinical use of rodent monoclonal antibodies is
an anti-globulin response during therapy (Miller, R. A. et al.,
Blood 62:988-995 (1983); Schroff, R. W. et al., Cancer Res.
45:879-885 (1985)).
The art has attempted to overcome this problem by constructing
"chimeric" antibodies in which an animal antigen-binding variable
domain is coupled to a human constant domain (Cabilly et al., U.S.
Pat. No. 4,816,567; Morrison, S. L. et al., Proc. Natl. Acad. Sci.
USA 81:6851-6855 (1984); Boulianne, G. L. et al., Nature
312:643-646 (1984); Neuberger, M. S. et al., Nature 314:268-270
(1985)). The term "chimeric" antibody is used herein to describe a
polypeptide comprising at least the antigen binding portion of an
antibody molecule linked to at least part of another protein
(typically an immunoglobulin constant domain).
The isotype of the human constant domain may be selected to tailor
the chimeric antibody for participation in antibody-dependent
cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity
(see e.g. Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987);
Riechmann, L. et al., Nature 332:323-327 (1988); Love et al.,
Methods in Enzymology 178:515-527 (1989); Bindon et al., J. Exp.
Med. 168:127-142 (1988).
In the typical embodiment, such chimeric antibodies contain about
one third rodent (or other non-human species) sequence and thus are
capable of eliciting a significant anti-globulin response in
humans. For example, in the case of the murine anti-CD3 antibody,
OKT3, much of the resulting anti-globulin response is directed
against the variable region rather than the constant region
(Jaffers, G. J. et al., Transplantation 41:572-578 (1986)).
In a further effort to resolve the antigen binding functions of
antibodies and to minimize the use of heterologous sequences in
human antibodies, Winter and colleagues (Jones, P. T. et al.,
Nature 321:522-525 (1986); Riechmann, L. et al., Nature 332:323-327
(1988); Verhoeyen, M. et al., Science 239:1534-1536 (1988)) have
substituted rodent CDRs or CDR sequences for the corresponding
segments of a human antibody. As used herein, the term "humanized"
antibody is an embodiment of chimeric antibodies wherein
substantially less than an intact human variable domain has been
substituted by the corresponding sequence from a non-human species.
In practice, humanized antibodies are typically human antibodies in
which some CDR residues and possibly some FR residues are
substituted by residues from analogous sites in rodent
antibodies.
The therapeutic promise of this approach is supported by the
clinical efficacy of a humanized antibody specific for the
CAMPATH-1 antigen with two non-Hodgkin lymphoma patients, one of
whom had previously developed an anti-globulin response to the
parental rat antibody (Riechmann, L. et al., Nature 332:323-327
(1988); Hale, G. et al., Lancet i:1394-1399 (1988)). A murine
antibody to the interleukin 2 receptor has also recently been
humanized (Queen, C. et al., Proc. Natl. Acad. Sci. USA
86:10029-10033 (1989)) as a potential immunosuppressive reagent.
Additional references related to humanization of antibodies include
Co et al., Proc. Natl. Acad. Sci. USA 88:2869-2873 (1991); Gorman
et al., Proc. Natl. Acad. Sci. USA 88:4181-4185 (1991); Daugherty
et al., Nucleic Acids Research 19(9):2471-2476 (1991); Brown et
al., Proc. Natl. Acad. Sci. USA 88:2663-2667 (1991); Junghans et
al., Cancer Research 50:1495-1502 (1990).
In some cases, substituting CDRs from rodent antibodies for the
human CDRs in human frameworks is sufficient to transfer high
antigen binding affinity (Jones, P. T. et al., Nature 321:522-525
(1986); Verhoeyen, M. et al., Science 239:1534-1536 (1988)),
whereas in other cases it has been necessary to additionally
replace one (Riechmann, L. et al., Nature 332:323-327 (1988)) or
several (Queen, C. et al., Proc. Natl. Acad. Sci. USA
86:10029-10033 (1989)) framework region (FR) residues. See also Co
et al., supra.
For a given antibody a small number of FR residues are anticipated
to be important for antigen binding. Firstly for example, certain
antibodies have been shown to contain a few FR residues which
directly contact antigen in crystal structures of antibody-antigen
complexes (e.g., reviewed in Davies, D. R. et al.,Ann Rev. Biochem.
59:439-473 (1990)). Secondly, a number of FR residues have been
proposed by Chothia, Lesk and colleagues (Chothia, C. & Lesk,
A. M., J. Mol. Biol. 196:901-917 (1987); Chothia, C. et al., Nature
342:877-883 (1989); Tramontano, A. et al., J. Mol. Biol. 215:175-1
82 (1990)) as critically affecting the conformation of particular
CDRs and thus their contribution to antigen binding. See also
Margolies et al., Proc. Natl. Acad. Sci. USA 72:2180-2184
(1975).
It is also known that, in a few instances, an antibody variable
domain (either V.sub.H or V.sub.L) may contain glycosylation sites,
and that this glycosylation may improve or abolish antigen binding,
Pluckthun, Biotechnology 9:545-51 (1991); Spiegelberg et al.,
Biochemistry 9:4217-4223 (1970); Wallic et al., J. Exp. Med.
168:1099-1109 (1988); Sox et al., Proc. Natl. Acad. Sci. USA
66:975-982 (1970); Margni et al., Ann Rev. Immunol. 6:535-554
(1988). Ordinarily, however, glycosylation has no influence on the
antigen-binding properties of an antibody, Pluckthun, supra, (1
991).
The three-dimensional structure of immunoglobulin chains has been
studied, and crystal structures for intact immunoglobulins, for a
variety of immunoglobulin fragments, and for antibody-antigen
complexes have been published (see e.g., Saul et al., Journal of
Biological Chemistry 25:585-97 (1978); Sheriff et al., Proc. Natl.
Acad. Sci. USA 84:8075-79 (1987); Segal et al., Proc. Natl. Acad.
Sci. USA 71:4298-4302 (1974); Epp et al.,Biochemistry 1
4(22):4943-4952 (1975); Marquart et al., J. Mol. Biol 141:369-391
(1980); Furey et al., J. Mol Biol. 167:661-692 (1983); Snow and
Amzel, Protein: Structure, Function, and Genetics 1:267-279, Alan
R. Liss, Inc. pubs. (1986); Chothia and Lesk, J. Mol Biol.
196:901-917 (1987); Chothia et al., Nature 342:877-883 (1989);
Chothia et al., Science 233:755-58 (1986); Huber et al., Nature
264:415-420 (1976); Bruccoleri et al., Nature 335:564-568 (1988)
and Nature 336:266 (1988); Sherman et al., Journal of Biological
Chemistry 263:4064-4074 (1988); Amzel and Poljak, Ann Rev. Biochem.
48:961-67 (1979); Silverton et al., Proc. Natl. Acad. Sci. USA
74:5140-5144 (1977); and Gregory et al., Molecular Immunology
24:821-829 (1987). It is known that the function of an antibody is
dependent on its three dimensional structure, and that amino acid
substitutions can change the three-dimensional structure of an
antibody, Snow and Amzel, supra. It has previously been shown that
the antigen binding affinity of a humanized antibody can be
increased by mutagenesis based upon molecular modelling (Riechmann,
L. et al., Nature 332:323-327 (1988); Queen, C. et al., Proc. Natl.
Acad. Sci. USA 86:10029-10033 (1989)).
Humanizing an antibody with retention of high affinity for antigen
and other desired biological activities is at present difficult to
achieve using currently available procedures. Methods are needed
for rationalizing the selection of sites for substitution in
preparing such antibodies and thereby increasing the efficiency of
antibody humanization.
The proto-oncogene HER2 (human epidermal growth factor receptor 2)
encodes a protein tyrosine kinase (p185.sup.HER2) that is related
to and somewhat homologous to the human epidermal growth factor
receptor (see Coussens, L. et al., Science 230:1132-1139 (1985);
Yamamoto, T. et al., Nature 319:230-234 (1986); King, C. R. et al.,
Science 229:974-976 (1985)). HER2 is also known in the field as
c-erbB-2, and sometimes by the name of the rat homolog, neu.
Amplification and/or overexpression of HER2 is associated with
multiple human malignancies and appears to be integrally involved
in progression of 25-30% of human breast and ovarian cancers
(Slamon, D. J. et al., Science 235:177-182 (1987), Slamon, D. J. et
al., Science 244:707-712 (1989)). Furthermore, the extent of
amplification is inversely correlated with the observed median
patient survival time (Slamon, supra, Science 1989).
The murine monoclonal antibody known as muMAb4D5 (Fendly, B. M. et
al., Cancer Res. 50:1550-1558 (1990)), directed against the
extracellular domain (ECD) of p185.sup.HER2, specifically inhibits
the growth of tumor cell lines overexpressing p185.sup.HER2 in
monolayer culture or in soft agar (Hudziak, R. M. et al., Molec.
Cell. Biol. 9:1165-1172 (1989); Lupu, R. et al., Science
249:1552-1555 (1990)). MuMAb4D5 also has the potential of enhancing
tumor cell sensitivity to tumor necrosis factor, an important
effector molecule in macrophage-mediated tumor cell cytotoxicity
(Hudziak, supra, 1989; Shepard, H. M. and Lewis, G. D. J. Clinical
Immunology 8:333-395 (1988)). Thus muMAb4D5 has potential for
clinical intervention in and imaging of carcinomas in which
p185.sup.HER2 is overexpressed. The muMAb4D5 and its uses are
described in PCT application WO 89/06692 published Jul. 27, 1989.
This murine antibody was deposited with the ATCC and designated
ATCC CRL 10463. However, this antibody may be immunogenic in
humans.
It is therefore an object of this invention to provide methods for
the preparation of antibodies which are less antigenic in humans
than non-human antibodies but have desired antigen binding and
other characteristics and activities.
It is a further object of this invention to provide methods for the
efficient humanization of antibodies, i.e. selecting non-human
amino acid residues for importation into a human antibody
background sequence in such a fashion as to retain or improve the
affinity of the non-human donor antibody for a given antigen.
It is another object of this invention to provide humanized
antibodies capable of binding p185.sup.HER2.
Other objects, features, and characteristics of the present
invention will become apparent upon consideration of the following
description and the appended claims.
SUMMARY OF THE INVENTION
The objects of this invention are accomplished by a method for
making a humanized antibody comprising amino acid sequence of an
import, non-human antibody and a human antibody, comprising the
steps of:
a. obtaining the amino acid sequences of at least a portion of an
import antibody variable domain and of a consensus variable
domain;
b. identifying Complementarity Determining Region (CDR) amino acid
sequences in the import and the human variable domain
sequences;
c. substituting an import CDR amino acid sequence for the
corresponding human CDR amino acid sequence;
d. aligning the amino acid sequences of a Framework Region (FR) of
the import antibody and the corresponding FR of the consensus
antibody;
e. identifying import antibody FR residues in the aligned FR
sequences that are non-homologous to the corresponding consensus
antibody residues;
f. determining if the non-homologous import amino acid residue is
reasonably expected to have at least one of the following
effects:
1. non-covalently binds antigen directly,
2. interacts with a CDR; or
3. participates in the V.sub.L -V.sub.H interface; and
g. for any non-homologous import antibody amino acid residue which
is reasonably expected to have at least one of these effects,
substituting that residue for the corresponding amino acid residue
in the consensus antibody FR sequence.
Optionally, the method of this invention comprises the additional
steps of determining if any non-homologous residues identified in
step (e) are exposed on the surface of the domain or buried within
it, and if the residue is exposed but has none of the effects
identified in step (f), retaining the consensus residue.
Additionally, in certain embodiments the method of this invention
comprises the feature wherein the corresponding consensus antibody
residues identified in step (e) above are selected from the group
consisting of 4L, 35L, 36L, 38L, 43L, 44L, 46L, 58L, 62L, 63L, 64L,
65L, 66L, 67L, 68L, 69L, 70L, 71L, 73L, 85L, 87L, 98L, 2H, 4H, 24H,
36H, 37H, 39H, 43H, 45H, 49H, 58H, 60H, 67H, 68H, 69H, 70H, 73H,
74H, 75H, 76H, 78H, 91H, 92H, 93H, and 103H (utilizing the
numbering system set forth in Kabat, E. A. et al., Sequences of
Proteins of Immunological Interest (National Institutes of Health,
Bethesda, Md., 1987)).
In certain embodiments, the method of this invention comprises the
additional steps of searching either or both of the import,
non-human and the consensus variable domain sequences for
glycosylation sites, determining if the glycosylation is reasonably
expected to be important for the desired antigen binding and
biological activity of the antibody (i.e., determining if the
glycosylation site binds to antigen or changes a side chain of an
amino acid residue that binds to antigen, or if the glycosylation
enhances or weakens antigen binding, or is important for
maintaining antibody affinity). If the import sequence bears the
glycosylation site, it is preferred to substitute that site for the
corresponding residues in the consensus human if the glycosylation
site is reasonably expected to be important. If only the consensus
sequence, and not the import, bears the glycosylation site, it is
preferred to eliminate that glycosylation site or substitute
therefor the corresponding amino acid residues from the import
sequence.
Another embodiment of this invention comprises aligning import
antibody and the consensus antibody FR sequences, identifying
import antibody FR residues which are non-homologous with the
aligned consensus FR sequence, and for each such non-homologous
import antibody FR residue, determining if the corresponding
consensus antibody residue represents a residue which is highly
conserved across all species at that site, and if it is so
conserved, preparing a humanized antibody which comprises the
consensus antibody amino acid residue at that site.
Certain alternate embodiments of the methods of this invention
comprise obtaining the amino acid sequence of at least a portion of
an import, non-human antibody variable domain having a CDR and a
FR, obtaining the amino acid sequence of at least a portion of a
consensus antibody variable domain having a CDR and a FR,
substituting the non-human CDR for the human CDR in the consensus
antibody variable domain, and then substituting an amino acid
residue for the consensus amino acid residue at at least one of the
following sites:
a. (in the FR of the variable domain of the light chain) 4L, 35L,
36L, 38L, 43L, 44L, 58L, 46L, 62L, 63L, 64L, 65L, 66L, 67L, 68L,
69L, 70L, 71L, 73L, 85L, 87L, 98L, or
b. (in the FR of the variable domain of the heavy chain) 2H, 4H,
24H, 36H, 37H, 39H, 43H, 45H, 49H, 58H, 60H, 67H, 68H, 69H, 70H,
73H, 74H, 75H, 76H, 78H, 91H, 92H, 93H, and 103H.
In preferred embodiments, the non-CDR residue substituted at the
consensus FR site is the residue found at the corresponding
location of the non-human antibody.
Optionally, this just-recited embodiment comprises the additional
steps of following the method steps appearing at the beginning of
this summary and determining whether a particular amino acid
residue can reasonably be expected to have undesirable effects.
This invention also relates to a humanized antibody comprising the
CDR sequence of an import, non-human antibody and the FR sequence
of a human antibody, wherein an amino acid residue within the human
FR sequence located at any one of the sites 4L, 35L, 36L, 38L, 43L,
44L, 46L, 58L, 62L, 63L, 64L, 65L, 66L, 67L, 68L, 69L, 70L, 71 L,
73L, 85L, 87L, 98L, 2H, 4H, 24H, 36H, 37H, 39H, 43H, 45H, 49H, 58H,
60H, 67H, 68H, 69H, 70H, 73H, 74H, 75H, 76H, 78H, 91H, 92H, 93H,
and 103H has been substituted by another residue. In preferred
embodiments, the residue substituted at the human FR site is the
residue found at the corresponding location of the non-human
antibody from which the non-human CDR was obtained. In other
embodiments, no human FR residue other than those set forth in this
group has been substituted.
This invention also encompasses specific humanized antibody
variable domains, and isolated polypeptides having homology with
the following sequences.
1. SEQ. ID NO. 1, which is the light chain variable domain of a
humanized version of muMAb4D5:
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSA -
SFLESGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGT - KVEIKRT
2. SEQ. ID NO. 2, which is the heavy chain variable domain of a
humanized version of muMAb4D5):
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARI -
YPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGG -
DGFYAMDVWGQGTLVTVSS
In another aspect, this invention provides a consensus antibody
variable domain amino acid sequence for use in the preparation of
humanized antibodies, methods for obtaining, using, and storing a
computer representation of such a consensus sequence, and computers
comprising the sequence data of such a sequence. In one embodiment,
the following consensus antibody variable domain amino acid
sequences are provided:
SEQ. ID NO. 3 (light chain):
DIQMTQSPSSLSASVGDRVTITCRASQDVSSYLAWYQQKPGKAPKLLIYAA -
SSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSLPYTFGQGT - KVEIKRT,
and
SEQ. ID NO. 4 (heavy chain):
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGLEWVAV -
ISENGSDTYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDR -
GGAVSYFDVWGQGTLVTVSS
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows the comparison of the V.sub.L domain amino acid
residues of muMAb4D5, huMAb4D5, and a consensus sequence (FIG. 1A,
SEQ. ID NO. 5, SEQ. ID NO. 1 and SEQ. ID NO. 3, respectively). FIG.
1B shows the comparison between the V.sub.H domain amino acid
residues of the muMAb4D5, huMAb4D5, and a consensus sequence (FIG.
1B, SEQ. ID NO. 6, SEQ. ID NO. 2 and SEQ. ID NO. 4, respectively).
Both FIGS. 1A and 1B use the generally accepted numbering scheme
from Kabat, E. A., et al., Sequences of Proteins of Immunological
Interest (National Institutes of Health, Bethesda, Md. (1987)). In
both FIG. 1A and FIG. 1B, the CDR residues determined according to
a standard sequence definition (as in Kabat, E. A. et al.,
Sequences of Proteins of Immunological Interest (National
Institutes of Health, Bethesda, Md., 1987)) are indicated by the
first underlining beneath the sequences, and the CDR residues
determined according to a structural definition (as in Chothia, C.
& Lesk, A. M., J. Mol. Biol. 196:901-917 (1987)) are indicated
by the second, lower underlines. The mismatches between genes are
shown by the vertical lines.
FIG. 2 shows a scheme for humanization of muMAb4D5 V.sub.L and
V.sub.H by gene conversion mutagenesis.
FIG. 3 shows the inhibition of SK-BR-3 proliferation by MAb4D5
variants. Relative cell proliferation was determined as described
(Hudziak, R. M. et al., Molec. Cell. Biol. 9:1165-1172 (1989)) and
data (average of triplicate determinations) are presented as a
percentage of results with untreated cultures for muMAb4D5 (I),
huMAb4D5-8 (n) and huMAb4D5-1 (I).
FIG. 4 shows a stereo view of a-carbon tracing for a model of
huMAb4D5-8 V.sub.L and V.sub.H. The CDR residues (Kabat, E. A. et
al., Sequences of Proteins of Immunological Interest (National
Institutes of Health, Bethesda, Md., 1987)) are shown in bold and
side chains of V.sub.H residues A71, T73, A78, S93, Y102 and
V.sub.L residues Y55 plus R66 (see Table 3) are shown.
FIG. 5 shows an amino acid sequence comparison of V.sub.L (top
panel) and V.sub.H (lower panel) domains of the murine anti-CD3
monoclonal Ab UCHT1 (muxCD3, Shalaby et al., J. Exp. Med. 175,
217-225 (1992) with a humanized variant of this antibody
(huxCD3v1). Also shown are consensus sequences (most commonly
occurring residue or pair of residues) of the most abundant human
subgroups, namely V.sub.L .kappa.1 and V.sub.H III upon which the
humanized sequences are based (Kabat, E. A. et al., Sequences of
Proteins of Immunological Interest, 5.sup.th edition, National
Institutes of Health, Bethesda, Md., U.S.A. (1991)). The light
chain sequences--muxCD3, huxCD3v1 and huIII--correspond to SEQ. ID.
NOs 16, 17, and 18, respectively. The heavy chain
sequences--muxCD3, huxCD3v1 and hu.kappa.I--correspond to SEQ. ID.
NOs 19, 45, and 21, respectively. Residues which differ between
muxCD3 and huxCD3v1 are identified by an asterisk (*), whereas
those which differ between humanized and consensus sequences are
identified by a sharp sign (#). A bullet (.smallcircle.) denotes
that a residue at this position has been found to contact antigen
in one or more crystallographic structures of antibody/antigen
complexes (Kabat et al., 1991; Mian, I. S. et al., J. Mol. Biol.
217, 133-151 (1991)). The location of CDR residues according to a
sequence definition (Kabat et al., 1991) and a structural
definition (Chothia and Lesk, supra 1987) are shown by a line and
carats (A) beneath the sequences, respectively.
FIGS. 6A-1 and 6A-2 compare murine and humanized amino acid
sequences for the heavy chain of an anti-CD1 8 antibody. H52H4-160
(SEQ. ID. NO.22) is the murine sequence, and pH52-8.0 (SEQ. ID. NO.
23) is the humanized heavy chain sequence. pH52-8.0 residue 143S is
the final amino acid in the variable heavy chain domain V.sub.H
(SEQ ID NO:46) and residue 144A is the first amino acid in the
constant heavy chain domain C.sub.H1.
FIG. 6B compares murine and humanized amino acid sequences for the
light chain of an anti-CD18 antibody. H52L6-158 (SEQ. ID. NO. 24)
is the murine sequence, and pH52-9.0 (SEQ. ID. NO. 25) is the
humanized light chain sequence. pH52-9.0 residue 128T is the final
amino acid in the light chain variable domain V.sub.L (SEQ ID
NO:47), and residue 129V is the first amino acid in the light chain
constant domain C.sub.L.
FIGS. 7A-1 and 7A-2 show an amino acid sequence alignment of the
sequences of the heavy chains of thirteen humanized anti-CD18 (H52)
variants (SEQ. ID. NOs 26-38).
FIG. 7B shows an amino acid sequence alignment of two humanized
anti-CD18 (H52) light chain variants (SEQ. ID. NOs 39-40).
DETAILED DESCRIPTION OF THE INVENTION
Definitions
In general, the following words or phrases have the indicated
definitions when used in the description, examples, and claims:
The murine monoclonal antibody known as muMAb4D5 (Fendly, B. M. et
al., Cancer Res. 50:1550-1558 (1990)) is directed against the
extracellular domain (ECD) of p185.sup.HER2. The muMAb4D5 and its
uses are described in PCT application WO 89/06692 published Jul.
27, 1989. This murine antibody was deposited with the ATCC and
designated ATCC CRL 10463. In this description and claims, the
terms muMAb4D5, chMAb4D5 and huMAb4D5 represent murine, chimerized
and humanized versions of the monoclonal antibody 4D5,
respectively.
A humanized antibody for the purposes herein is an immunoglobulin
amino acid sequence variant or fragment thereof which is capable of
binding to a predetermined antigen and which comprises a FR region
having substantially the amino acid sequence of a human
immunoglobulin and a CDR having substantially the amino acid
sequence of a non-human immunoglobulin.
Generally, a humanized antibody has one or more amino acid residues
introduced into it from a source which is non-human. These
non-human amino acid residues are referred to herein as "import"
residues, which are typically taken from an "import" antibody
domain, particularly a variable domain. An import residue,
sequence, or antibody has a desired affinity and/or specificity, or
other desirable antibody biological activity as discussed
herein.
In general, the humanized antibody will comprise substantially all
of at least one, and typically two, variable domains (Fab, Fab',
F(ab').sub.2, Fabc, Fv) in which all or substantially all of the
CDR regions correspond to those of a non-human immunoglobulin and
all or substantially all of the FR regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin. Ordinarily,
the antibody will contain both the light chain as well as at least
the variable domain of a heavy chain. The antibody also may include
the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain.
The humanized antibody will be selected from any class of
immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any
isotype, including IgG1, IgG2, IgG3 and IgG4. Usually the constant
domain is a complement fixing constant domain where it is desired
that the humanized antibody exhibit cytotoxic activity, and the
class is typically IgG.sub.1. Where such cytotoxic activity is not
desirable, the constant domain may be of the IgG.sub.2 class. The
humanized antibody may comprise sequences from more than one class
or isotype, and selecting particular constant domains to optimize
desired effector functions is within the ordinary skill in the
art.
The FR and CDR regions of the humanized antibody need not
correspond precisely to the parental sequences, e.g., the import
CDR or the consensus FR may be mutagenized by substitution,
insertion or deletion of at least one residue so that the CDR or FR
residue at that site does not correspond to either the consensus or
the import antibody. Such mutations, however, will not be
extensive. Usually, at least 75% of the humanized antibody residues
will correspond to those of the parental FR and CDR sequences, more
often 90%, and most preferably greater than 95%.
In general, humanized antibodies prepared by the method of this
invention are produced by a process of analysis of the parental
sequences and various conceptual humanized products using three
dimensional models of the parental and humanized sequences. Three
dimensional immunoglobulin models are commonly available and are
familiar to those skilled in the art. Computer programs are
available which illustrate and display probable three dimensional
conformational structures of selected candidate immunoglobulin
sequences. Inspection of these displays permits analysis of the
likely role of the residues in the functioning of the candidate
immunoglobulin sequence, i.e., the analysis of residues that
influence the ability of the candidate immunoglobulin to bind its
antigen.
Residues that influence antigen binding are defined to be residues
that are substantially responsible for the antigen affinity or
antigen specificity of a candidate immunoglobulin, in a positive or
a negative sense. The invention is directed to the selection and
combination of FR residues from the consensus and import sequence
so that the desired immunoglobulin characteristic is achieved. Such
desired characteristics include increases in affinity and greater
specificity for the target antigen, although it is conceivable that
in some circumstances the opposite effects might be desired. In
general, the CDR residues are directly and most substantially
involved in influencing antigen binding (although not all CDR
residues are so involved and therefore need not be substituted into
the consensus sequence). However, FR residues also have a
significant effect and can exert their influence in at least three
ways: They may noncovalently directly bind to antigen, they may
interact with CDR residues and they may affect the interface
between the heavy and light chains.
A residue that noncovalently directly binds to antigen is one that,
by three dimensional analysis, is reasonably expected to
noncovalently directly bind to antigen. Typically, it is necessary
to impute the position of antigen from the spatial location of
neighboring CDRs and the dimensions and structure of the target
antigen. In general, only those humanized antibody residues that
are capable of forming salt bridges, hydrogen bonds, or hydrophobic
interactions are likely to be involved in non-covalent antigen
binding, however residues which have atoms which are separated from
antigen spatially by 3.2 Angstroms or less may also non-covalently
interact with antigen. Such residues typically are the relatively
larger amino acids having the side chains with the greatest bulk,
such as tyrosine, arginine, and lysine. Antigen-binding FR residues
also typically will have side chains that are oriented into an
envelope surrounding the solvent oriented face of a CDR which
extends about 7 Angstroms into the solvent from the CDR domain and
about 7 Angstroms on either side of the CDR domain, again as
visualized by three dimensional modeling.
A residue that interacts with a CDR generally is a residue that
either affects the conformation of the CDR polypeptide backbone or
forms a noncovalent bond with a CDR residue side chain.
Conformation-affecting residues ordinarily are those that change
the spatial position of any CDR backbone atom (N, C.alpha., C, O,
C.beta.) by more than about 0.2 Angstroms. Backbone atoms of CDR
sequences are displaced for example by residues that interrupt or
modify organized structures such as beta sheets, helices or loops.
Residues that can exert a profound affect on the conformation of
neighboring sequences include proline and glycine, both of which
are capable of introducing bends into the backbone. Other residues
that can displace backbone atoms are those that are capable of
participating in salt bridges and hydrogen bonds.
A residue that interacts with a CDR side chain is one that is
reasonably expected to form a noncovalent bond with a CDR side
chain, generally either a salt bridge or hydrogen bond. Such
residues are identified by three dimensional positioning of their
side chains. A salt or ion bridge could be expected to form between
two side chains positioned within about 2.5-3.2 Angstroms of one
another that bear opposite charges, for example a lysinyl and a
glutamyl pairing. A hydrogen bond could be expected to form between
the side chains of residue pairs such as seryl or threonyl with
aspartyl or glutamyl (or other hydrogen accepting residues). Such
pairings are well known in the protein chemistry art and will be
apparent to the artisan upon three dimensional modeling of the
candidate immunoglobulin.
Immunoglobulin residues that affect the interface between heavy and
light chain variable regions ("the V.sub.L - V.sub.H interface")
are those that affect the proximity or orientation of the two
chains with respect to one another. Certain residues involved in
interchain interactions are already known and include V.sub.L
residues 34, 36, 38, 44, 46, 87, 89, 91, 96, and 98 and V.sub.H
residues 35, 37, 39, 45, 47, 91, 93, 95, 100, and 103 (utilizing
the nomenclature set forth in Kabat et al., Sequences of Proteins
of Immunological Interest (National Institutes of Health, Bethesda,
Md., 1987)). Additional residues are newly identified by the
inventors herein, and include 43L, 85L, 43H and 60H. While these
residues are indicated for IgG only, they are applicable across
species. In the practice of this invention, import antibody
residues that are reasonably expected to be involved in interchain
interactions are selected for substitution into the consensus
sequence. It is believed that heretofore no humanized antibody has
been prepared with an intrachain-affecting residue selected from an
import antibody sequence.
Since it is not entirely possible to predict in advance what the
exact impact of a given substitution will be it may be necessary to
make the substitution and assay the candidate antibody for the
desired characteristic. These steps, however, are per se routine
and well within the ordinary skill of the art.
CDR and FR residues are determined according to a standard sequence
definition (Kabat et al., Sequences of Proteins of Immunological
Interest, National Institutes of Health, Bethesda Md. (1987), and a
structural definition (as in Chothia and Lesk, J. Mol Biol.
196:901-917 (1987). Where these two methods result in slightly
different identifications of a CDR, the structural definition is
preferred, but the residues identified by the sequence definition
method are considered important FR residues for determination of
which framework residues to import into a consensus sequence.
Throughout this description, reference is made to the numbering
scheme from Kabat, E. A., et al., Sequences of Proteins of
Immunological Interest (National Institutes of Health, Bethesda,
Md. (1987) and (1991). In these compendiums, Kabat lists many amino
acid sequences for antibodies for each subclass, and lists the most
commonly occurring amino acid for each residue position in that
subclass. Kabat uses a method for assigning a residue number to
each amino acid in a listed sequence, and this method for assigning
residue numbers has become standard in the field. The Kabat
numbering scheme is followed in this description. For purposes of
this invention, to assign residue numbers to a candidate antibody
amino acid sequence which is not included in the Kabat compendium,
one follows the following steps. Generally, the candidate sequence
is aligned with any immunoglobulin sequence or any consensus
sequence in Kabat. Alignment may be done by hand, or by computer
using commonly accepted computer programs; an example of such a
program is the Align 2 program discussed in this description.
Alignment may be facilitated by using some amino acid residues
which are common to most Fab sequences. For example, the light and
heavy chains each typically have two cysteines which have the same
residue numbers; in V.sub.L domain the two cysteines are typically
at residue numbers 23 and 88, and in the V.sub.H domain the two
cysteine residues are typically numbered 22 and 92. Framework
residues generally, but not always, have approximately the same
number of residues, however the CDRs will vary in size. For
example, in the case of a CDR from a candidate sequence which is
longer than the CDR in the sequence in Kabat to which it is
aligned, typically suffixes are added to the residue number to
indicate the insertion of additional residues (see, e.g. residues
100abcde in FIG. 5). For candidate sequences which, for example,
align with a Kabat sequence for residues 34 and 36 but have no
residue between them to align with residue 35, the number 35 is
simply not assigned to a residue.
Thus, in humanization of an import variable sequence, where one
cuts out an entire human or consensus CDR and replaces it with an
import CDR sequence, (a) the exact number of residues may be
swapped, leaving the numbering the same, (b) fewer import amino
acid residues may be introduced than are cut, in which case there
will be a gap in the residue numbers, or (c) a larger number of
amino acid residues may be introduced then were cut, in which case
the numbering will involve the use of suffixes such as
100abcde.
The terms "consensus sequence" and "consensus antibody" as used
herein refers to an amino acid sequence which comprises the most
frequently occurring amino acid residues at each location in all
immunoglobulins of any particular subclass or subunit structure.
The consensus sequence may be based on immunoglobulins of a
particular species or of many species. A "consensus" sequence,
structure, or antibody is understood to encompass a consensus human
sequence as described in certain embodiments of this invention, and
to refer to an amino acid sequence which comprises the most
frequently occurring amino acid residues at each location in all
human immunoglobulins of any particular subclass or subunit
structure. This invention provides consensus human structures and
consensus structures which consider other species in addition to
human.
The subunit structures of the five immunoglobulin classes in humans
are as follows:
______________________________________ Class Heavy Chain Subclasses
Light Chain Molecular Formula
______________________________________ IgG .gamma. .gamma.1,
.gamma.2, .gamma.3, .gamma.4 .kappa. or (.gamma..sub.2
.kappa..sub.2), (.gamma.. sub.2 .sub.2) IgA .alpha. .alpha.1,
.alpha.2 .kappa. or (.alpha..sub.2 .kappa..sub.2 ).sub.n .sup.a,
(.alpha..sub.2 .sub.2).s ub.n .sup.a IgM .mu. none .kappa. or
(.mu..sub.2 .kappa..sub.2).sub.5, (.mu..sub.2 .sub.2).sub.5 IgD
.delta. none .kappa. or (.delta..sub.2 .kappa..sub.2), (.delta..su
b.2 .sub.2) IgE .epsilon. none .kappa. or (.epsilon..sub.2
.kappa..sub.2), (.epsilon..sub.2 .sub.2)
______________________________________ (.sup.a .sub.n may equal 1,
2, or 3)
In preferred embodiments of an IgG.gamma.1 human consensus
sequence, the consensus variable domain sequences are derived from
the most abundant subclasses in the sequence compilation of Kabat
et al., Sequences of Proteins of Immunological Interest, National
Institutes of Health, Bethesda Md. (1987), namely V.sub.L .kappa.
subgroup I and V.sub.H group III. In such preferred embodiments,
the V.sub.L consensus domain has the amino acid sequence:
the V.sub.H consensus domain has the amino acid sequence:
These sequences include consensus CDRs as well as consensus FR
residues (see for example in FIG. 1).
While not wishing to be limited to any particular theories, it may
be that these preferred embodiments are less likely to be
immunogenic in an individual than less abundant subclasses.
However, in other embodiments, the consensus sequence is derived
from other subclasses of human immunoglobulin variable domains. In
yet other embodiments, the consensus sequence is derived from human
constant domains.
Identity or homology with respect to a specified amino acid
sequence of this invention is defined herein as the percentage of
amino acid residues in a candidate sequence that are identical with
the specified residues, after aligning the sequences and
introducing gaps, if necessary, to achieve the maximum percent
homology, and not considering any conservative substitutions as
part of the sequence identity. None of N-terminal, C-terminal or
internal extensions, deletions, or insertions into the specified
sequence shall be construed as affecting homology. All sequence
alignments called for in this invention are such maximal homology
alignments. While such alignments may be done by hand using
conventional methods, a suitable computer program is the "Align 2"
program for which protection is being sought from the U.S. Register
of Copyrights (Align 2, by Genentech, Inc., application filed Dec.
9, 1991).
"Non-homologous" import antibody residues are those residues which
are not identical to the amino acid residue at the analogous or
corresponding location in a consensus sequence, after the import
and consensus sequences are aligned.
The term "computer representation" refers to information which is
in a form that can be manipulated by a computer. The act of storing
a computer representation refers to the act of placing the
information in a form suitable for manipulation by a computer.
This invention is also directed to novel polypeptides, and in
certain aspects, isolated novel humanized anti-p185.sup.HER2
antibodies are provided. These novel anti-p185.sup.HER2 antibodies
are sometimes collectively referred to herein as huMAb4D5, and also
sometimes as the light or heavy chain variable domains of huMAb4D5,
and are defined herein to be any polypeptide sequence which
possesses a biological property of a polypeptide comprising the
following polypeptide sequence:
"Biological property", as relates for example to
anti-p185.sup.HER2, for the purposes herein means an in vivo
effector or antigen-binding function or activity that is directly
or indirectly performed by huMAb4D5 (whether in its native or
denatured conformation). Effector functions include p185.sup.HER2
binding, any hormonal or hormonal antagonist activity, any
mitogenic or agonist or antagonist activity, any cytotoxic
activity. An antigenic function means possession of an epitope or
antigenic site that is capable of cross-reacting with antibodies
raised against the polypeptide sequence of huMAb4D5.
Biologically active huMAb4D5 is defined herein as a polypeptide
that shares an effector function of huMAb4D5. A principal known
effector function of huMAb4D5 is its ability to bind to
p185.sup.HER2.
Thus, the biologically active and antigenically active huMAb4D5
polypeptides that are the subject of certain embodiments of this
invention include the sequence of the entire translated nucleotide
sequence of huMAb4D5; mature huMAb4D5; fragments thereof having a
consecutive sequence of at least 5, 10, 15, 20, 25, 30 or 40 amino
acid residues comprising sequences from muMAb4D5 plus residues from
the human FR of huMAb4D5; amino acid sequence variants of huMAb4D5
wherein an amino acid residue has been inserted N- or C-terminal
to, or within, huMAb4D5 or its fragment as defined above; amino
acid sequence variants of huMAb4D5 or its fragment as defined above
wherein an amino acid residue of huMAb4D5 or its fragment as
defined above has been substituted by another residue, including
predetermined mutations by, e.g., site-directed or PCR mutagenesis;
derivatives of huMAb4D5 or its fragments as defined above wherein
huMAb4D5 or its fragments have been covalent modified, by
substitution, chemical, enzymatic, or other appropriate means, with
a moiety other than a naturally occurring amino acid; and
glycosylation variants of huMAb4D5 (insertion of a glycosylation
site or deletion of any glycosylation site by deletion, insertion
or substitution of suitable residues). Such fragments and variants
exclude any polypeptide heretofore identified, including muMAb4D5
or any known polypeptides fragment, which are anticipatory order 35
U.S.C. 102 as well as polypeptides obvious thereover under 35
U.S.C. 103.
An "isolated" polypeptide means polypeptide which has been
identified and separated and/or recovered from a component of its
natural environment. Contaminant components of its natural
environment are materials which would interfere with diagnostic or
therapeutic uses for the polypeptide, and may include enzymes,
hormones, and other proteinaceous or nonproteinaceous solutes. In
preferred embodiments, for example, a polypeptide product
comprising huMAb4D5 will be purified from a cell culture or other
synthetic environment (1) to greater than 95% by weight of protein
as determined by the Lowry method, and most preferably more than
99% by weight, (2) to a degree sufficient to obtain at least 15
residues of N-terminal or internal amino acid sequence by use of a
gas- or liquid-phase sequenator (such as a commercially available
Applied Biosystems sequenator Model 470, 477, or 473), or (3) to
homogeneity by SDS-PAGE under reducing or nonreducing conditions
using Coomassie blue or, preferably, silver stain. Isolated
huMAb4D5 includes huMAb4D5 in situ within recombinant cells since
at least one component of the huMAb4D5 natural environment will not
be present. Ordinarily, however, isolated huMAb4D5 will be prepared
by at least one purification step.
In accordance with this invention, huMAb4D5 nucleic acid is RNA or
DNA containing greater than ten bases that encodes a biologically
or antigenically active huMAb4D5, is complementary to nucleic acid
sequence encoding such huMAb4D5, or hybridizes to nucleic acid
sequence encoding such huMAb4D5 and remains stably bound to it
under stringent conditions, and comprises nucleic acid from a
muMAb4D5 CDR and a human FR region.
Preferably, the huMAb4D5 nucleic acid encodes a polypeptide sharing
at least 75% sequence identity, more preferably at least 80%, still
more preferably at least 85%, even more preferably at 90%, and most
preferably 95%, with the huMAb4D5 amino acid sequence. Preferably,
a nucleic acid molecule that hybridizes to the huMAb4D5 nucleic
acid contains at least 20, more preferably 40, and most preferably
90 bases. Such hybridizing or complementary nucleic acid, however,
is further defined as being novel under 35 U.S.C. 102 and unobvious
under 35 U.S.C. 103 over any prior art nucleic acid.
Stringent conditions are those that (1) employ low ionic strength
and high temperature for washing, for example, 0.015 M NaCl/0.0015
M sodium citrate/O/1% NaDodSO.sub.4 at 50.degree. C.; (2) employ
during hybridization a denaturing agent such as formamide, for
example, 50% (vol/vol) formamide with 0.1% bovine serum
albumin/O/1% Ficoll/O/1% polyvinylpyrrolidone/50 mM sodium
phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate
at 42.degree. C.; or (3) employ 50% formamide, 5.times.SSC (0.75 M
NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8),
0.1% sodium pyrophosphate, 5.times.Denhardt's solution, sonicated
salmon sperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfate at 42
C., with washes at 42 C. in 0.2.times.SSC and 0.1% SDS.
The term "control sequences" refers to DNA sequences necessary for
the expression of an operably linked coding sequence in a
particular host organism. The control sequences that are suitable
for prokaryotes, for example, include a promoter, optionally an
operator sequence, a ribosome binding site, and possibly, other as
yet poorly understood sequences. Eukaryotic cells are known to
utilize promoters, polyadenylation signals, and enhancers.
Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous and, in the case of a
secretory leader, contiguous and in reading phase. However
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, then synthetic oligonucleotide adaptors or linkers are used
in accord with conventional practice.
An "exogenous" element is defined herein to mean nucleic acid
sequence that is foreign to the cell, or homologous to the cell but
in a position within the host cell nucleic acid in which the
element is ordinarily not found.
As used herein, the expressions "cell," "cell line," and "cell
culture" are used interchangeably and all such designations include
progeny. Thus, the words "transformants" and "transformed cells"
include the primary subject cell and cultures derived therefrom
without regard for the number of transfers. It is also understood
that all progeny may not be precisely identical in DNA content, due
to deliberate or inadvertent mutations. Mutant progeny that have
the same function or biological activity as screened for in the
originally transformed cell are included. Where distinct
designations are intended, it will be clear from the context.
"Oligonucleotides" are short-length, single- or double-stranded
polydeoxynucleotides that are chemically synthesized by known
methods (such as phosphotriester, phosphite, or phosphoramidite
chemistry, using solid phase techniques such as described in EP
266,032 published May 4, 1988, or via deoxynucleoside H-phosphonate
intermediates as described by Froehler et al., Nucl. Acids Res.,
14: 5399-5407 [19861). They are then purified on polyacrylamide
gels.
The technique of "polymerase chain reaction," or "PCR," as used
herein generally refers to a procedure wherein minute amounts of a
specific piece of nucleic acid, RNA and/or DNA, are amplified as
described in U.S. Pat. No.4,683,195 issued Jul. 28, 1987.
Generally, sequence information from the ends of the region of
interest or beyond needs to be available, such that oligonucleotide
primers can be designed; these primers will be identical or similar
in sequence to opposite strands of the template to be amplified.
The 5' terminal nucleotides of the two primers may coincide with
the ends of the amplified material. PCR can be used to amplify
specific RNA sequences, specific DNA sequences from total genomic
DNA, and cDNA transcribed from total cellular RNA, bacteriophage or
plasmid sequences, etc. See generally Mullis et al., Cold Spring
Harbor Symp. Quant. Biol., 51: 263 (1987); Erlich, ed., PCR
Technology, (Stockton Press, NY, 1989). As used herein, PCR is
considered to be one, but not the only, example of a nucleic acid
polymerase reaction method for amplifying a nucleic acid test
sample, comprising the use of a known nucleic acid (DNA or RNA) as
a primer and utilizes a nucleic acid polymerase to amplify or
generate a specific piece of nucleic acid or to amplify or generate
a specific piece of nucleic acid which is complementary to a
particular nucleic acid.
Suitable Methods for Practicing the Invention
Some aspects of this invention include obtaining an import,
non-human antibody variable domain, producing a desired humanized
antibody sequence and for humanizing an antibody gene sequence are
described below. A particularly preferred method of changing a gene
sequence, such as gene conversion from a non-human or consensus
sequence into a humanized nucleic acid sequence, is the cassette
mutagenesis procedure described in Example 1. Additionally, methods
are given for obtaining and producing antibodies generally, which
apply equally to native non-human antibodies as well as to
humanized antibodies.
Generally, the antibodies and antibody variable domains of this
invention are conventionally prepared in recombinant cell culture,
as described in more detail below. Recombinant synthesis is
preferred for reasons of safety and economy, but it is known to
prepare peptides by chemical synthesis and to purify them from
natural sources; such preparations are included within the
definition of antibodies herein.
Molecular Modeling
An integral step in our approach to antibody humanization is
construction of computer graphics models of the import and
humanized antibodies. These models are used to determine if the six
complementarity-determining regions (CDRs) can be successfully
transplanted from the import framework to a human one and to
determine which framework residues from the import antibody, if
any, need to be incorporated into the humanized antibody in order
to maintain CDR conformation. In addition, analysis of the
sequences of the import and humanized antibodies and reference to
the models can help to discern which framework residues are unusual
and thereby might be involved in antigen binding or maintenance of
proper antibody structure.
All of the humanized antibody models of this invention are based on
a single three-dimensional computer graphics structure hereafter
referred to as the consensus structure. This consensus structure is
a key distinction from the approach of previous workers in the
field, who typically begin by selecting a human antibody structure
which has an amino acid sequence which is similar to the sequence
of their import antibody.
The consensus structure of one embodiment of this invention was
built in five steps as described below.
Step 1: Seven Fab X-ray crystal structures from the Brookhaven
Protein Data Bank were used (entries 2FB4, 2RHE, 3FAB, and 1 REI
which are human structures, and 2MCP, 1FBJ, and 2HFL which are
murine structures). For each structure, protein mainchain geometry
and hydrogen bonding patterns were used to assign each residue to
one of three secondary structure types: alpha-helix, beta-strand or
other (i.e. non-helix and non-strand). The immunoglobulin residues
used in superpositioning and those included in the consensus
structure are shown in Table I.
TABLE I
__________________________________________________________________________
Immunoglobulin Residues Used in Superpositioning and Those Included
in the Consensus Structure
__________________________________________________________________________
V.sub.L K domain Ig.sup.a 2FB4 2RHE 2MCP 3FAB 1FBJ 2HFL 1REI
Consensus.sup.b
__________________________________________________________________________
2-11 18-24 18-24 19-25 18-24 19-25 19-25 19-25 16-27 32-37 34-39
39-44 32-37 32-37 32-37 33-38 33-39 41-49 60-66 62-68 67-72 53-66
60-65 60-65 61-66 59-77 69-74 71-76 76-81 69-74 69-74 69-74 70-75
84-88 86-90 91-95 84-88 84-88 84-88 85-89 82-91 101-105 RMS.sup.c
0.40 0.60 0.53 0.54 0.48 0.50
__________________________________________________________________________
V.sub.H domain Ig.sup.a 2FB4 2MCP 3FAB 1FBJ 2HFL Consensus.sup.b
__________________________________________________________________________
3-8 18-25 18-25 18-25 18-25 18-25 17-23 34-39 34-39 34-39 34-39
34-39 33-41 46-52 46-52 46-52 46-52 46-52 45-51 57-61 59-63 56-60
57-61 57-61 57-61 68-71 70-73 67-70 68-71 68-71 66-71 78-84 80-86
77-83 78-84 78-84 75-82 92-99 94-101 91-98 92-99 92-99 88-94
102-108 RMS.sup.c 0.43 0.85 0.62 0.91 RMS.sup.d 0.91 0.73 0.77 0.92
__________________________________________________________________________
.sup.a Four-letter code for Protein Data Bank file. .sup.b Residue
numbers for the crystal structures are taken from the Protein Data
Bank files. Residue numbers for the consensus structure are
according to Kabat et al. .sup.c Root-mean-square deviation in
.ANG. for (N,C.alpha.,C) atoms superimposed on 2FB4. .sup.d
Root-mean-square deviation in .ANG. for (N,C.alpha.,C) atoms
superimposed on 2HFL.
Step 2: Having identified the alpha-helices and beta-strands in
each of the seven structures, the structures were superimposed on
one another using the INSIGHT computer program (Biosym
Technologies, San Diego, Calif.) as follows: The 2FB4 structure was
arbitrarily chosen as the template (or reference) structure. The
2FB4 was held fixed in space and the other six structures rotated
and translated in space so that their common secondary structural
elements (i.e. alpha-helices and beta-strands) were oriented such
that these common elements were as close in position to one another
as possible. (This superpositioning was performed using accepted
mathematical formulae rather than actually physically moving the
structures by hand.)
Step 3: With the seven structures thus superimposed, for each
residue in the template (2FB4) Fab one calculates the distance from
the template alpha-carbon atom (C.alpha.) to the analogous C.alpha.
atom in each of the other six superimposed structures. This results
in a table of C.alpha.--C.alpha. distances for each residue
position in the sequence. Such a table is necessary in order to
determine which residue positions will be included in the consensus
model. Generally, if all C.alpha.--C.alpha. distances for a given
residue position were .ltoreq.1.0 .ANG., that position was included
in the consensus structure. If for a given position only one Fab
crystal structure was >1.0 .ANG., the position was included but
the outlying crystal structure was not included in the next step
(for this position only). In general, the seven .beta.-strands were
included in the consensus structure while some of the loops
connecting the .beta.-strands, e.g. complementarity-determining
regions (CDRs), were not included in view of C.alpha.
divergence.
Step 4: For each residue which was included in the consensus
structure after step 3, the average of the coordinates for
individual mainchain N, C.alpha., C, O and C.beta. atoms were
calculated. Due to the averaging procedure, as well as variation in
bond length, bond angle and dihedral angle among the crystal
structures, this "average" structure contained some bond lengths
and angles which deviated from standard geometry. For purposes of
this invention, "standard geometry" is understood to include
geometries commonly accepted as typical, such as the compilation of
bond lengths and angles from small molecule structures in Weiner,
S. J. et. al., J. Amer. Chem. Soc., 106: 765-784 (1984).
Step 5: In order to correct these deviations, the final step was to
subject the "average" structure to 50 cycles of energy minimization
(DISCOVER program, Biosym Technologies) using the AMBER (Weiner, S.
J. et. al., J. Amer. Chem. Soc., 106: 765-784 (1984)) parameter set
with only the C.alpha. coordinates fixed (i.e. all other atoms are
allowed to move) (energy minimization is described below). This
allowed any deviant bond lengths and angles to assume a standard
(chemically acceptable) geometry. See Table II.
TABLE II ______________________________________ Average Bond
Lengths and Angles for "Average" (Before) and Energy-Minimized
Consensus (After 50 Cycles) Structures V.sub.L K V.sub.L K V.sub.H
V.sub.H Standard before after before after Geometry (.ANG.) (.ANG.)
(.ANG.) (.ANG.) (.ANG.) ______________________________________
N--C.alpha. 1.459 1.451(0.004) 1.451 1.452(0.004) 1.449 (0.012)
(0.023) Ca-C 1.515 1.523(0.005) 1.507 1.542(0.005) 1.522 (0.012)
(0.033) O.dbd.C 1.208 1.229(0.003) 1.160 1.231(0.003) 1.229 (0.062)
(0.177) C--N 1.288 1.337(0.002) 1.282 1.335(0.004) 1.335 (0.049)
(0.065) C.alpha.-C.beta. 1.508 1.530(0.002) 1.499 1.530(0.002)
1.526 (0.026) (0.039) ______________________________________
(.degree.) (.degree.) (.degree.) (.degree.) (.degree.)
______________________________________ C--N--C.alpha. 123.5
123.8(1.1) 125.3 124.0(1.1) 121.9 (4.2) (4.6) N--C.alpha.-C 110.0
109.5(1.9) 110.3 109.5(1.6) 110.1 (4.0) (2.8) C.alpha.-C--N 116.6
116.6(1.2) 117.6 116.6(0.8) 116.6 (4.0) (5.2) O.dbd.C--N 123.1
123.4(0.6) 122.2 123.3(0.4) 122.9 (4.1) (4.9) N--C.alpha.-C.beta.
110.3 109.8(0.7) 110.6 109.8(0.6) 109.5 (2.1) (2.5)
C.beta.-C.alpha.-C 111.4 111.1(0.7) 111.2 111.1(0.6) 111.1 (2.4)
(2.2) ______________________________________ Values in parentheses
are standard deviations. Note that while some bond length and angle
averages did not change appreciably after energyminimization, the
corresponding standard deviations are reduced due to deviant
geometries assuming standard values after energyminimization.
Standard geometry values are from the AMBER forcefield as
implemented in DISCOVER (Biosym Technologies).
The consensus structure might conceivably be dependent upon which
crystal structure was chosen as the template on which the others
were superimposed. As a test, the entire procedure was repeated
using the crystal structure with the worst superposition versus
2FB4, i.e. the 2HFL Fab structure, as the new template (reference).
The two consensus structures compare favorably (root-mean-squared
deviation of 0.11 .ANG. for all N, C.alpha. and C atoms).
Note that the consensus structure only includes mainchain (N,
C.alpha., C, O, C.beta. atoms) coordinates for only those residues
which are part of a conformation common to all seven X-ray crystal
structures. For the Fab structures, these include the common
.beta.-strands (which comprise two .beta.-sheets) and a few non-CDR
loops which connect these .beta.-strands. The consensus structure
does not include CDRs or sidechains, both of which vary in their
conformation among the seven structures. Also, note that the
consensus structure includes only the VL and VH domains.
This consensus structure is used as the archetype. It is not
particular to any species, and has only the basic shape without
side chains. Starting with this consensus structure the model of
any import, human, or humanized Fab can be constructed as follows.
Using the amino acid sequence of the particular antibody VL and VH
domains of interest, a computer graphics program (such as INSIGHT,
Biosym Technologies) is used to add sidechains and CDRs to the
consensus structure. When a sidechain is added, its conformation is
chosen on the basis of known Fab crystal structures (see the
Background section for publications of such crystal structures) and
rotamer libraries (Ponder, J. W. & Richards, F. M., J. Mol.
Biol. 193: 775-791 (1987)). The model also is constructed so that
the atoms of the sidechain are positioned so as to not collide with
other atoms in the Fab.
CDRs are added to the model (now having the backbone plus side
chains) as follows. The size (i.e. number of amino acids) of each
import CDR is compared to canonical CDR structures tabulated by
Chothia et al., Nature, 342:877-883 (1989)) and which were derived
from Fab crystals. Each CDR sequence is also reviewed for the
presence or absence of certain specific amino acid residues which
are identified by Chothia as structurally important: e.g. light
chain residues 29 (CDR1) and 95 (CDR3), and heavy chain residues
26, 27, 29 (CDR1) and 55 (CDR2). For light chain CDR2, and heavy
chain CDR3, only the size of the CDR is compared to the Chothia
canonical structure. If the size and sequence (i.e. inclusion of
the specific, structurally important residues as denoted by Chothia
et al.) of the import CDR agrees in size and has the same
structurally important residues as those of a canonical CDR, then
the mainchain conformation of the import CDR in the model is taken
to be the same as that of the canonical CDR. This means that the
import sequence is assigned the structural configuration of the
canonical CDR, which is then incorporated in the evolving
model.
However, if no matching canonical CDR can be assigned for the
import CDR, then one of two options can be exercised. First, using
a program such as INSIGHT (Biosym Technologies), the Brookhaven
Protein Data Bank can be searched for loops with a similar size to
that of the import CDR and these loops can be evaluated as possible
conformations for the import CDR in the model. Minimally, such
loops must exhibit a conformation in which no loop atom overlaps
with other protein atoms. Second, one can use available programs
which calculate possible loop conformations, assuming a given loop
size, using methods such as described by Bruccoleri et al., Nature
335: 564-568 (1988).
When all CDRs and sidechains have been added to the consensus
structure to give the final model (import, human or humanized), the
model is preferably subjected to energy minimization using programs
which are available commercially (e.g. DISCOVER, Biosym
Technologies). This technique uses complex mathematical formulae to
refine the model by performing such tasks as checking that all
atoms are within appropriate distances from one another and
checking that bond lengths and angles are within chemically
acceptable limits.
Models of a humanized, import or human antibody sequence are used
in the practice of this invention to understand the impact of
selected amino acid residues of the activity of the sequence being
modeled. For example, such a model can show residues which may be
important in antigen binding, or for maintaining the conformation
of the antibody, as discussed in more detail below. Modeling can
also be used to explore the potential impact of changing any amino
acid residue in the antibody sequence.
Methods for Obtaining a Humanized Antibody Sequence
In the practice of this invention, the first step in humanizing an
import antibody is deriving a consensus amino acid sequence into
which to incorporate the import sequences. Next a model is
generated for these sequences using the methods described above. In
certain embodiments of this invention, the consensus human
sequences are derived from the most abundant subclasses in the
sequence compilation of Kabat et al. (Kabat, E. A. et al.,
Sequences of Proteins of Immunological Interest (National
Institutes of Health, Bethesda, Md., 1987)), namely V.sub.L .kappa.
subgroup I and V.sub.H group III, and have the sequences indicated
in the definitions above.
While these steps may be taken in different order, typically a
structure for the candidate humanized antibody is created by
transferring the at least one CDR from the non-human, import
sequence into the consensus human structure, after the entire
corresponding human CDR has been removed. The humanized antibody
may contain human replacements of the non-human import residues at
positions within CDRs as defined by sequence variability (Kabat, E.
A. et al., Sequences of Proteins of Immunological Interest
(National Institutes of Health, Bethesda, Md., 1987)) or as defined
by structural variability (Chothia, C. & Lesk, A. M., J. Mol.
Biol. 196:901-917 (1987)). For example, huMAb4D5 contains human
replacements of the muMAb4D5 residues at three positions within
CDRs as defined by sequence variability (Kabat, E. A. et al.,
Sequences of Proteins of Immunological Interest (National
Institutes of Health, Bethesda, Md., 1987)) but not as defined by
structural variability (Chothia, C. & Lesk, A. M., J. Mol Biol.
196:901-917 (1987)): V.sub.L -CDR1 K24R, V.sub.L -CDR2 R54L and
V.sub.L -CDR2 T56S.
Differences between the non-human import and the human consensus
framework residues are individually investigated to determine their
possible influence on CDR conformation and/or binding to antigen.
Investigation of such possible influences is desirably performed
through modeling, by examination of the characteristics of the
amino acids at particular locations, or determined experimentally
through evaluating the effects of substitution or mutagenesis of
particular amino acids.
In certain preferred embodiments of this invention, a humanized
antibody is made comprising amino acid sequence of an import,
non-human antibody and a human antibody, utilizing the steps
of:
a. obtaining the amino acid sequences of at least a portion of an
import antibody variable domain and of a consensus human variable
domain;
b. identifying Complementarity Determining Region (CDR) amino acid
sequences in the import and the human variable domain
sequences;
c. substituting an import CDR amino acid sequence for the
corresponding human CDR amino acid sequence;
d. aligning the amino acid sequences of a Framework Region (FR) of
the import antibody and the corresponding FR of the consensus
antibody;
e. identifying import antibody FR residues in the aligned FR
sequences that are non-homologous to the corresponding consensus
antibody residues;
f. determining if the non-homologous import amino acid residue is
reasonably expected to have at least one of the following
effects:
1. non-covalently binds antigen directly,
2. interacts with a CDR; or
3. participates in the V.sub.L -V.sub.H interface; and
g. for any non-homologous import antibody amino acid residue which
is reasonably expected to have at least one of these effects,
substituting that residue for the corresponding amino acid residue
in the consensus antibody FR sequence.
Optionally, one determines if any non-homologous residues
identified in step (e) are exposed on the surface of the domain or
buried within it, and if the residue is exposed but has none of the
effects identified in step (f), one may retain the consensus
residue.
Additionally, in certain embodiments the corresponding consensus
antibody residues identified in step (e) above are selected from
the group consisting of 4L, 35L, 36L, 38L, 43L, 44L, 46L, 58L, 62L,
63L, 64L, 65L, 66L, 67L, 68L, 68L, 69L, 70L, 71 L, 73L, 85L, 87L,
98L, 2H, 4H, 24H, 36H, 37H, 39H, 43H, 45H, 49H, 58H, 60H, 67H, 68H,
69H, 70H, 73H, 74H, 75H, 76H, 78H, 91H, 92H, 93H, and 103H
(utilizing the numbering system set forth in Kabat, E. A. et al.,
Sequences of Proteins of Immunological Interest (National
Institutes of Health, Bethesda, Md., 1987)).
In preferred embodiments, the method of this invention comprises
the additional steps of searching either or both of the import,
non-human and the consensus variable domain sequences for
glycosylation sites, determining if the glycosylation is reasonably
expected to be important for the desired antigen binding and
biological activity of the antibody (i.e., determining if the
glycosylation site binds to antigen or changes a side chain of an
amino acid residue that binds to antigen, or if the glycosylation
enhances or weakens antigen binding, or is important for
maintaining antibody affinity). If the import sequence bears the
glycosylation site, it is preferred to substitute that site for the
corresponding residues in the consensus human sequence if the
glycosylation site is reasonably expected to be important. If only
the consensus sequence, and not the import, bears the glycosylation
site, it is preferred to eliminate that glycosylation site or
substitute therefor the corresponding amino acid residues from the
import sequence.
Another preferred embodiment of the methods of this invention
comprises aligning import antibody and the consensus antibody FR
sequences, identifying import antibody FR residues which are
non-homologous with the aligned consensus FR sequence, and for each
such non-homologous import antibody FR residue, determining if the
corresponding consensus antibody residue represents a residue which
is highly conserved across all species at that site, and if it is
so conserved, preparing a humanized antibody which comprises the
consensus antibody amino acid residue at that site.
In certain alternate embodiments, one need not utilize the modeling
and evaluation steps described above, and may instead proceed with
the steps of obtaining the amino acid sequence of at least a
portion of an import, non-human antibody variable domain having a
CDR and a FR, obtaining the amino acid sequence of at least a
portion of a consensus human antibody variable domain having a CDR
and a FR, substituting the non-human CDR for the human CDR in the
consensus human antibody variable domain, and then substituting an
amino acid residue for the consensus amino acid residue at at least
one of the following sites:
a. (in the FR of the variable domain of the light chain) 4L, 35L,
36L, 38L, 43L, 44L, 58L, 46L, 62L, 63L, 64L, 65L, 66L, 67L, 68L,
69L, 70L, 71L, 73L, 85L, 87L, 98L, or
b. (in the FR of the variable domain of the heavy chain) 2H, 4H,
24H, 36H, 37H, 39H, 43H, 45H, 49H, 58H, 60H, 67H, 68H, 69H, 70H,
73H, 74H, 75H, 76H, 78H, 91H, 92H, 93H, and 103H.
Preferably, the non-CDR residue substituted at the consensus FR
site is the residue found at the corresponding location of the
non-human antibody. If desired, one may utilize the other method
steps described above for determining whether a particular amino
acid residue can reasonably be expected to have undesirable
effects, and remedying those effects.
If after making a humanized antibody according to the steps above
and testing its activity one is not satisfied with the humanized
antibody, one preferably reexamines the potential effects of the
amino acids at the specific locations recited above. Additionally,
it is desirable to reinvestigate any buried residues which are
reasonably expected to affect the V.sub.L -V.sub.H interface but
may not directly affect CDR conformation. It is also desirable to
reevaluate the humanized antibody utilizing the steps of the
methods claimed herein.
In certain embodiments of this invention, amino acid residues in
the consensus human sequence are substituted for by other amino
acid residues. In preferred embodiments, residues from a particular
non-human import sequence are substituted, however there are
circumstances where it is desired to evaluate the effects of other
amino acids. For example, if after making a humanized antibody
according to the steps above and testing its activity one is not
satisfied with the humanized antibody, one may compare the
sequences of other classes or subgroups of human antibodies, or
classes or subgroups of antibodies from the particular non-human
species, and determine which other amino acid side chains and amino
acid residues are found at particular locations and substituting
such other residues.
Antibodies
Certain aspects of this invention are directed to natural
antibodies and to monoclonal antibodies, as illustrated in the
Examples below and by antibody hybridomas deposited with the ATCC
(as described below). Thus, the references throughout this
description to the use of monoclonal antibodies are intended to
include the use of natural or native antibodies as well as
humanized and chimeric antibodies. As used herein, the term
"antibody" includes the antibody variable domain and other
separable antibody domains unless specifically excluded.
In accordance with certain aspects of this invention, antibodies to
be humanized (import antibodies) are isolated from continuous
hybrid cell lines formed by the fusion of antigen-primed immune
lymphocytes with myeloma cells.
In certain embodiments, the antibodies of this invention are
obtained by routine screening. Polyclonal antibodies to an antigen
generally are raised in animals by multiple subcutaneous (sc) or
intraperitoneal (ip) injections of the antigen and an adjuvant. It
may be useful to conjugate the antigen or a fragment containing the
target amino acid sequence to a protein that is immunogenic in the
species to be immunized, e.g., keyhole limpet hemocyanin, serum
albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a
bifunctional or derivatizing agent, for example, maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxysuccinimide (through lysine residues), glutaraldehyde,
succinic anhydride, SOCl.sub.2, or R.sup.1 N=C=NR, where R and
R.sup.1 are different alkyl groups.
The route and schedule of the host animal or cultured
antibody-producing cells therefrom are generally in keeping with
established and conventional techniques for antibody stimulation
and production. While mice are frequently employed as the test
model, it is contemplated that any mammalian subject including
human subjects or antibody-producing cells obtained therefrom can
be manipulated according to the processes of this invention to
serve as the basis for production of mammalian, including human,
hybrid cell lines.
Animals are typically immunized against the immunogenic conjugates
or derivatives by combining 1 mg or 1 .mu.g of conjugate (for
rabbits or mice, respectively) with 3 volumes of Freund's complete
adjuvant and injecting the solution intradermally at multiple
sites. One month later the animals are boosted with 1/5 to 1/10 the
original amount of conjugate in Freund's complete adjuvant (or
other suitable adjuvant) by subcutaneous injection at multiple
sites. 7 to 14 days later animals are bled and the serum is assayed
for antigen titer. Animals are boosted until the titer plateaus.
Preferably, the animal is boosted with the conjugate of the same
antigen, but conjugated to a different protein and/or through a
different cross-linking agent. Conjugates also can be made in
recombinant cell culture as protein fusions. Also, aggregating
agents such as alum are used to enhance the immune response.
After immunization, monoclonal antibodies are prepared by
recovering immune lymphoid cells--typically spleen cells or
lymphocytes from lymph node tissue--from immunized animals and
immortalizing the cells in conventional fashion, e.g. by fusion
with myeloma cells or by Epstein-Barr (EB)-virus transformation and
screening for clones expressing the desired antibody. The hybridoma
technique described originally by Kohler and Milstein, Eur. J.
Immunol. 6:511 (1976) has been widely applied to produce hybrid
cell lines that secrete high levels of monoclonal antibodies
against many specific antigens.
It is possible to fuse cells of one species with another. However,
it is preferable that the source of the immunized antibody
producing cells and the myeloma be from the same species.
The hybrid cell lines can be maintained in culture in vitro in cell
culture media. The cell lines of this invention can be selected
and/or maintained in a composition comprising the continuous cell
line in hypoxanthine-aminopterin thymidine (HAT) medium. In fact,
once the hybridoma cell line is established, it can be maintained
on a variety of nutritionally adequate media. Moreover, the hybrid
cell lines can be stored and preserved in any number of
conventional ways, including freezing and storage under liquid
nitrogen. Frozen cell lines can be revived and cultured
indefinitely with resumed synthesis and secretion of monoclonal
antibody. The secreted antibody is recovered from tissue culture
supernatant by conventional methods such as precipitation, Ion
exchange chromatography, affinity chromatography, or the like. The
antibodies described herein are also recovered from hybridoma cell
cultures by conventional methods for purification of IgG or IgM as
the case may be that heretofore have been used to purify these
immunoglobulins from pooled plasma, e.g. ethanol or polyethylene
glycol precipitation procedures. The purified antibodies are
sterile filtered, and optionally are conjugated to a detectable
marker such as an enzyme or spin label for use in diagnostic assays
of the antigen in test samples.
While routinely rodent monoclonal antibodies are used as the source
of the import antibody, the invention is not limited to any
species. Additionally, techniques developed for the production of
chimeric antibodies (Morrison et al., Proc. Natl. Acad. Sci.,
81:6851 (1984); Neuberger et al., Nature 312:604 (1984); Takeda et
al., Nature 314:452 (1985)) by splicing the genes from a mouse
antibody molecule of appropriate antigen specificity together with
genes from a human antibody molecule of appropriate biological
activity (such as ability to activate human complement and mediate
ADCC) can be used; such antibodies are within the scope of this
invention.
Techniques for creating recombinant DNA versions of the
antigen-binding regions of antibody molecules (known as Fab
fragments) which bypass the generation of monoclonal antibodies are
encompassed within the practice of this invention. One extracts
antibody-specific messenger RNA molecules from immune system cells
taken from an immunized animal, transcribes these into
complementary DNA (cDNA), and clones the cDNA into a bacterial
expressions system. One example of such a technique suitable for
the practice of this invention was developed by researchers at
Scripps/Stratagene, and incorporates a proprietary bacteriophage
lambda vector system which contains a leader sequence that causes
the expressed Fab protein to migrate to the periplasmic space
(between the bacterial cell membrane and the cell wall) or to be
secreted. One can rapidly generate and screen great numbers of
functional FAb fragments for those which bind the antigen. Such FAb
fragments with specificity for the antigen are specifically
encompassed within the term "antibody" as it is defined, discussed,
and claimed herein.
Amino Acid Sequence Variants
Amino acid sequence variants of the antibodies and polypeptides of
this invention (referred to in herein as the target polypeptide)
are prepared by introducing appropriate nucleotide changes into the
DNA encoding the target polypeptide, or by in vitro synthesis of
the desired target polypeptide. Such variants include, for example,
humanized variants of non-human antibodies, as well as deletions
from, or insertions or substitutions of, residues within particular
amino acid sequences. Any combination of deletion, insertion, and
substitution can be made to arrive at the final construct, provided
that the final construct possesses the desired characteristics. The
amino acid changes also may alter post-translational processes of
the target polypeptide, such as changing the number or position of
glycosylation sites, altering any membrane anchoring
characteristics, and/or altering the intra-cellular location of the
target polypeptide by inserting, deleting, or otherwise affecting
any leader sequence of the native target polypeptide.
In designing amino acid sequence variants of target polypeptides,
the location of the mutation site and the nature of the mutation
will depend on the target polypeptide characteristic(s) to be
modified. The sites for mutation can be modified individually or in
series, e.g., by (1) substituting first with conservative amino
acid choices and then with more radical selections depending upon
the results achieved, (2) deleting the target residue, or (3)
inserting residues of the same or a different class adjacent to the
located site, or combinations of options 1-3. In certain
embodiments, these choices are guided by the methods for creating
humanized sequences set forth above.
A useful method for identification of certain residues or regions
of the target polypeptide that are preferred locations for
mutagenesis is called "alanine scanning mutagenesis" as described
by Cunningham and Wells (Science, 244: 1081-1085 [1989]). Here, a
residue or group of target residues are identified (e.g., charged
residues such as arg, asp, his, lys, and glu) and replaced by a
neutral or negatively charged amino acid (most preferably alanine
or polyalanine) to affect the interaction of the amino acids with
the surrounding aqueous environment in or outside the cell. Those
domains demonstrating functional sensitivity to the substitutions
then are refined by introducing further or other variants at or for
the sites of substitution. Thus, while the site for introducing an
amino acid sequence variation is predetermined, the nature of the
mutation per se need not be predetermined. For example, to optimize
the performance of a mutation at a given site, ala scanning or
random mutagenesis may be conducted at the target codon or region
and the expressed target polypeptide variants are screened for the
optimal combination of desired activity.
There are two principal variables in the construction of amino acid
sequence variants: the location of the mutation site and the nature
of the mutation. In general, the location and nature of the
mutation chosen will depend upon the target polypeptide
characteristic to be modified.
Amino acid sequence deletions of antibodies are generally not
preferred, as maintaining the generally configuration of an
antibody is believed to be necessary for its activity. Any
deletions will be selected so as to preserve the structure of the
target antibody.
Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Intrasequence insertions (i.e., insertions within the target
polypeptide sequence) may range generally from about 1 to 10
residues, more preferably 1 to 5, most preferably 1 to 3. Examples
of terminal insertions include the target polypeptide with an
N-terminal methionyl residue, an artifact of the direct expression
of target polypeptide in bacterial recombinant cell culture, and
fusion of a heterologous N-terminal signal sequence to the
N-terminus of the target polypeptide molecule to facilitate the
secretion of the mature target polypeptide from recombinant host
cells. Such signal sequences generally will be obtained from, and
thus homologous to, the intended host cell species. Suitable
sequences include STII or Ipp for E. coil, alpha factor for yeast,
and viral signals such as herpes gD for mammalian cells.
Other insertional variants of the target polypeptide include the
fusion to the N- or C-terminus of the target polypeptide of
immunogenic polypeptides, e.g., bacterial polypeptides such as
beta-lactamase or an enzyme encoded by the E. coli trp locus, or
yeast protein, and C-terminal fusions with proteins having a long
half-life such as immunoglobulin constant regions (or other
immunoglobulin regions), albumin, or ferritin, as described in WO
89/02922 published Apr. 6, 1989.
Another group of variants are amino acid substitution variants.
These variants have at least one amino acid residue in the target
polypeptide molecule removed and a different residue inserted in
its place. The sites of greatest interest for substitutional
mutagenesis include sites identified as the active site(s) of the
target polypeptide, and sites where the amino acids found in the
target polypeptide from various species are substantially different
in terms of side-chain bulk, charge, and/or hydrophobicity. Other
sites for substitution are described infra, considering the effect
of the substitution of the antigen binding, affinity and other
characteristics of a particular target antibody.
Other sites of interest are those in which particular residues of
the target polypeptides obtained from various species are
identical. These positions may be important for the biological
activity of the target polypeptide. These sites, especially those
falling within a sequence of at least three other identically
conserved sites, are substituted in a relatively conservative
manner. If such substitutions result in a change in biological
activity, then other changes are introduced and the products
screened until the desired effect is obtained.
Substantial modifications in function or immunological identity of
the target polypeptide are accomplished by selecting substitutions
that differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Naturally occurring residues are
divided into groups based on common side chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;
(4) basic: asn, gin, his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and
(6) aromatic: trp, tyr, phe.
Non-conservative substitutions will entail exchanging a member of
one of these classes for another. Such substituted residues may be
introduced into regions of the target polypeptide that are
homologous with other antibodies of the same class or subclass, or,
more preferably, into the non-homologous regions of the
molecule.
Any cysteine residues not involved in maintaining the proper
conformation of target polypeptide also may be substituted,
generally with serine, to improve the oxidative stability of the
molecule and prevent aberrant crosslinking.
DNA encoding amino acid sequence variants of the target polypeptide
is prepared by a variety of methods known in the art. These methods
include, but are not limited to, isolation from a natural source
(in the case of naturally occurring amino acid sequence variants)
or preparation by oligonucleotide-mediated (or site-directed)
mutagenesis, PCR mutagenesis, and cassette mutagenesis of an
earlier prepared variant or a non-variant version of the target
polypeptide. A particularly preferred method of gene conversion
mutagenesis is described below in Example 1. These techniques may
utilized target polypeptide nucleic acid (DNA or RNA), or nucleic
acid complementary to the target polypeptide nucleic acid.
Oligonucleotide-mediated mutagenesis is a preferred method for
preparing substitution, deletion, and insertion variants of target
polypeptide DNA. This technique is well known in the art as
described by Adelman et al., DNA, 2: 183 (1983). Briefly, the
target polypeptide DNA is altered by hybridizing an oligonucleotide
encoding the desired mutation to a DNA template, where the template
is the single-stranded form of a plasmid or bacteriophage
containing the unaltered or native DNA sequence of the target
polypeptide. After hybridization, a DNA polymerase is used to
synthesize an entire second complementary strand of the template
that will thus incorporate the oligonucleotide primer, and will
code for the selected alteration in the target polypeptide DNA.
Generally, oligonucleotides of at least 25 nucleotides in length
are used. An optimal oligonucleotide will have 12 to 15 nucleotides
that are completely complementary to the template on either side of
the nucleotide(s) coding for the mutation. This ensures that the
oligonucleotide will hybridize properly to the single-stranded DNA
template molecule. The oligonucleotides are readily synthesized
using techniques known in the art such as that described by Crea et
al. (Proc. Natl. Acad. Sci. USA, 75: 5765 [1978]).
Single-stranded DNA template may also be generated by denaturing
double-stranded plasmid (or other) DNA using standard
techniques.
For alteration of the native DNA sequence (to generate amino acid
sequence variants, for example), the oligonucleotide is hybridized
to the single-stranded template under suitable hybridization
conditions. A DNA polymerizing enzyme, usually the Klenow fragment
of DNA polymerase 1, is then added to synthesize the complementary
strand of the template using the oligonucleotide as a primer for
synthesis. A heteroduplex molecule is thus formed such that one
strand of DNA encodes the mutated form of the target polypeptide,
and the other strand (the original template) encodes the native,
unaltered sequence of the target polypeptide. This heteroduplex
molecule is then transformed into a suitable host cell, usually a
prokaryote such as E. coli JM101. After the cells are grown, they
are plated onto agarose plates and screened using the
oligonucleotide primer radiolabeled with 32-phosphate to identify
the bacterial colonies that contain the mutated DNA. The mutated
region is then removed and placed in an appropriate vector for
protein production, generally an expression vector of the type
typically employed for transformation of an appropriate host.
The method described immediately above may be modified such that a
homoduplex molecule is created wherein both strands of the plasmid
contain the mutation(s). The modifications are as follows: The
single-stranded oligonucleotide is annealed to the single-stranded
template as described above. A mixture of three
deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine
(dGTP), and deoxyribothymidine (dTTP), is combined with a modified
thio-deoxyribocytosine called dCTP-(aS) (which can be obtained from
Amersham Corporation). This mixture is added to the
template-oligonucleotide complex. Upon addition of DNA polymerase
to this mixture, a strand of DNA identical to the template except
for the mutated bases is generated. In addition, this new strand of
DNA will contain dCTP-(aS) instead of dCTP, which serves to protect
it from restriction endonuclease digestion.
After the template strand of the double-stranded heteroduplex is
nicked with an appropriate restriction enzyme, the template strand
can be digested with ExoIII nuclease or another appropriate
nuclease past the region that contains the site(s) to be
mutagenized. The reaction is then stopped to leave a molecule that
is only partially single-stranded. A complete double-stranded DNA
homoduplex is then formed using DNA polymerase in the presence of
all four deoxyribonucleotide triphosphates, ATP, and DNA ligase.
This homoduplex molecule can then be transformed into a suitable
host cell such as E. coli JM101, as described above.
DNA encoding target polypeptide variants with more than one amino
acid to be substituted may be generated in one of several ways. If
the amino acids are located close together in the polypeptide
chain, they may be mutated simultaneously using one oligonucleotide
that codes for all of the desired amino acid substitutions. If,
however, the amino acids are located some distance from each other
(separated by more than about ten amino acids), it is more
difficult to generate a single oligonucleotide that encodes all of
the desired changes. Instead, one of two alternative methods may be
employed.
In the first method, a separate oligonucleotide is generated for
each amino acid to be substituted. The oligonucleotides are then
annealed to the single-stranded template DNA simultaneously, and
the second strand of DNA that is synthesized from the template will
encode all of the desired amino acid substitutions.
The alternative method involves two or more rounds of mutagenesis
to produce the desired mutant. The first round is as described for
the single mutants: wild-type DNA is used for the template, an
oligonucleotide encoding the first desired amino acid
substitutions) is annealed to this template, and the heteroduplex
DNA molecule is then generated. The second round of mutagenesis
utilizes the mutated DNA produced in the first round of mutagenesis
as the template. Thus, this template already contains one or more
mutations. The oligonucleotide encoding the additional desired
amino acid substitution(s) is then annealed to this template, and
the resulting strand of DNA now encodes mutations from both the
first and second rounds of mutagenesis. This resultant DNA can be
used as a template in a third round of mutagenesis, and so on.
PCR mutagenesis is also suitable for making amino acid variants of
target polypeptide. While the following discussion refers to DNA,
it is understood that the technique also finds application with
RNA. The PCR technique generally refers to the following procedure
(see Erlich, supra, the chapter by R. Higuchi, p. 61-70): When
small amounts of template DNA are used as starting material in a
PCR, primers that differ slightly in sequence from the
corresponding region in a template DNA can be used to generate
relatively large quantities of a specific DNA fragment that differs
from the template sequence only at the positions where the primers
differ from the template. For introduction of a mutation into a
plasmid DNA, one of the primers is designed to overlap the position
of the mutation and to contain the mutation; the sequence of the
other primer must be identical to a stretch of sequence of the
opposite strand of the plasmid, but this sequence can be located
anywhere along the plasmid DNA. It is preferred, however, that the
sequence of the second primer is located within 200 nucleotides
from that of the first, such that in the end the entire amplified
region of DNA bounded by the primers can be easily sequenced. PCR
amplification using a primer pair like the one just described
results in a population of DNA fragments that differ at the
position of the mutation specified by the primer, and possibly at
other positions, as template copying is somewhat error-prone.
If the ratio of template to product material is extremely low, the
vast majority of product DNA fragments incorporate the desired
mutation(s). This product material is used to replace the
corresponding region in the plasmid that served as PCR template
using standard DNA technology. Mutations at separate positions can
be introduced simultaneously by either using a mutant second
primer, or performing a second PCR with different mutant primers
and ligating the two resulting PCR fragments simultaneously to the
vector fragment in a three (or more)-part ligation.
In a specific example of PCR mutagenesis, template plasmid DNA (1
.mu.g) is linearized by digestion with a restriction endonuclease
that has a unique recognition site in the plasmid DNA outside of
the region to be amplified. Of this material, 100 ng is added to a
PCR mixture containing PCR buffer, which contains the four
deoxynucleotide tri-phosphates and is included in the GeneAmp.RTM.
kits (obtained from Perkin-Elmer Cetus, Norwalk, Conn. and
Emeryville, Calif.), and 25 pmole of each oligonucleotide primer,
to a final volume of 50 .mu.l. The reaction mixture is overlayed
with 35 .mu.l mineral oil. The reaction is denatured for 5 minutes
at 100.degree. C., placed briefly on ice, and then 1 .mu.l Thermus
aquaticus (Taq) DNA polymerase (5 units/.mu.l, purchased from
Perkin-Elmer Cetus, Norwalk, CT and Emeryville, Calif.) is added
below the mineral oil layer. The reaction mixture is then inserted
into a DNA Thermal Cycler (purchased from Perkin-Elmer Cetus)
programmed as follows: 2 min. at 55.degree. C., then 30 sec. at
72.degree. C., then 19 cycles of the following: 30 sec. at
94.degree. C., 30 sec. at 55.degree. C., and 30 sec. at 72.degree.
C.
At the end of the program, the reaction vial is removed from the
thermal cycler and the aqueous phase transferred to a new vial,
extracted with phenol/chloroform (50:50:vol), and ethanol
precipitated, and the DNA is recovered by standard procedures. This
material is subsequently subjected to the appropriate treatments
for insertion into a vector.
Another method for preparing variants, cassette mutagenesis, is
based on the technique described by Wells et al. (Gene, 34: 315
[1985]). The starting material is the plasmid (or other vector)
comprising the target polypeptide DNA to be mutated. The codon(s)
in the target polypeptide DNA to be mutated are identified. There
must be a unique restriction endonuclease site on each side of the
identified mutation site(s). If no such restriction sites exist,
they may be generated using the above-described
oligonucleotide-mediated mutagenesis method to introduce them at
appropriate locations in the target polypeptide DNA. After the
restriction sites have been introduced into the plasmid, the
plasmid is cut at these sites to linearize it. A double-stranded
oligonucleotide encoding the sequence of the DNA between the
restriction sites but containing the desired mutation(s) is
synthesized using standard procedures. The two strands are
synthesized separately and then hybridized together using standard
techniques. This double-stranded oligonucleotide is referred to as
the cassette. This cassette is designed to have 3' and 5' ends that
are compatible with the ends of the linearized plasmid, such that
it can be directly ligated to the plasmid. This plasmid now
contains the mutated target polypeptide DNA sequence.
Insertion of DNA into a Cloning Vehicle
The cDNA or genomic DNA encoding the target polypeptide is inserted
into a replicable vector for further cloning (amplification of the
DNA) or for expression. Many vectors are available, and selection
of the appropriate vector will depend on 1) whether it is to be
used for DNA amplification or for DNA expression, 2) the size of
the DNA to be inserted into the vector, and 3) the host cell to be
transformed with the vector. Each vector contains various
components depending on its function (amplification of DNA or
expression of DNA) and the host cell for which it is compatible.
The vector components generally include, but are not limited to,
one or more of the following: a signal sequence, an origin of
replication, one or more marker genes, an enhancer element, a
promoter, and a transcription termination sequence.
(a) Signal Sequence Component
In general, the signal sequence may be a component of the vector,
or it may be a part of the target polypeptide DNA that is inserted
into the vector.
The target polypeptides of this invention may be expressed not only
directly, but also as a fusion with a heterologous polypeptide,
preferably a signal sequence or other polypeptide having a specific
cleavage site at the N-terminus of the mature protein or
polypeptide. In general, the signal sequence may be a component of
the vector, or it may be a part of the target polypeptide DNA that
is inserted into the vector. Included within the scope of this
invention are target polypeptides with any native signal sequence
deleted and replaced with a heterologous signal sequence. The
heterologous signal sequence selected should be one that is
recognized and processed (i.e. cleaved by a signal peptidase) by
the host cell. For prokaryotic host cells that do not recognize and
process the native target polypeptide signal sequence, the signal
sequence is substituted by a prokaryotic signal sequence selected,
for example, from the group of the alkaline phosphatase,
penicillinase, Ipp, or heat-stable enterotoxin II leaders. For
yeast secretion the native target polypeptide signal sequence may
be substituted by the yeast invertase, alpha factor, or acid
phosphatase leaders. In mammalian cell expression the native signal
sequence is satisfactory, although other mammalian signal sequences
may be suitable.
(b) Origin of Replication Component
Both expression and cloning vectors contain a nucleic acid sequence
that enables the vector to replicate in one or more selected host
cells. Generally, in cloning vectors this sequence is one that
enables the vector to replicate independently of the host
chromosomal DNA, and includes origins of replication or
autonomously replicating sequences. Such sequences are well known
for a variety of bacteria, yeast, and viruses. The origin of
replication from the plasmid pBR322 is suitable for most
Gram-negative bacteria, the 2.mu. plasmid origin is suitable for
yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or
BPV) are useful for cloning vectors in mammalian cells. Generally,
the origin of replication component is not needed for mammalian
expression vectors (the SV40 origin may typically be used only
because it contains the early promoter).
Most expression vectors are "shuttle" vectors, i.e. they are
capable of replication in at least one class of organisms but can
be transfected into another organism for expression. For example, a
vector is cloned in E. coli and then the same vector is transfected
into yeast or mammalian cells for expression even though it is not
capable of replicating independently of the host cell
chromosome.
DNA may also be amplified by insertion into the host genome. This
is readily accomplished using Bacillus species as hosts, for
example, by including in the vector a DNA sequence that is
complementary to a sequence found in Bacillus genomic DNA.
Transfection of Bacillus with this vector results in homologous
recombination with the genome and insertion of the target
polypeptide DNA. However, the recovery of genomic DNA encoding the
target polypeptide is more complex than that of an exogenously
replicated vector because restriction enzyme digestion is required
to excise the target polypeptide DNA.
(c) Selection Gene Component
Expression and cloning vectors should contain a selection gene,
also termed a selectable marker. This gene encodes a protein
necessary for the survival or growth of transformed host cells
grown in a selective culture medium. Host cells not transformed
with the vector containing the selection gene will not survive in
the culture medium. Typical selection genes encode proteins that
(a) confer resistance to antibiotics or other toxins, e.g.
ampicillin, neomycin, methotrexate, or tetracycline, (b) complement
auxotrophic deficiencies, or (c) supply critical nutrients not
available from complex media, e.g. the gene encoding D-alanine
racemase for Bacilli.
One example of a selection scheme utilizes a drug to arrest growth
of a host cell. Those cells that are successfully transformed with
a heterologous gene express a protein conferring drug resistance
and thus survive the selection regimen. Examples of such dominant
selection use the drugs neomycin (Southern et al., J. Molec. Appl.
Genet., 1: 327 [1982]), mycophenolic acid (Mulligan et al.,
Science, 209: 1422 [1980]) or hygromycin (Sugden et al., Mol. Cell.
Biol., 5: 410-413 [1985]). The three examples given above employ
bacterial genes under eukaryotic control to convey resistance to
the appropriate drug G418 or neomycin (geneticin), xgpt
(mycophenolic acid), or hygromycin, respectively.
Another example of suitable selectable markers for mammalian cells
are those that enable the identification of cells competent to take
up the target polypeptide nucleic acid, such as dihydrofolate
reductase (DHFR) or thymidine kinase. The mammalian cell
transformants are placed under selection pressure which only the
transformants are uniquely adapted to survive by virtue of having
taken up the marker. Selection pressure is imposed by culturing the
transformants under conditions in which the concentration of
selection agent in the medium is successively changed, thereby
leading to amplification of both the selection gene and the DNA
that encodes the target polypeptide. Amplification is the process
by which genes in greater demand for the production of a protein
critical for growth are reiterated in tandem within the chromosomes
of successive generations of recombinant cells. Increased
quantities of the target polypeptide are synthesized from the
amplified DNA.
For example, cells transformed with the DHFR selection gene are
first identified by culturing all of the transformants in a culture
medium that contains methotrexate (Mtx), a competitive antagonist
of DHFR. An appropriate host cell when wild-type DHFR is employed
is the Chinese hamster ovary (CHO) cell line deficient in DHFR
activity, prepared and propagated as described by Urlaub and
Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216 [1980]. The
transformed cells are then exposed to increased levels of
methotrexate. This leads to the synthesis of multiple copies of the
DHFR gene, and, concomitantly, multiple copies of other DNA
comprising the expression vectors, such as the DNA encoding the
target polypeptide. This amplification technique can be used with
any otherwise suitable host, e.g., ATCC No. CCL61 CHO-K1,
notwithstanding the presence of endogenous DHFR if, for example, a
mutant DHFR gene that is highly resistant to Mtx is employed (EP
117,060). Alternatively, host cells (particularly wild-type hosts
that contain endogenous DHFR) transformed or co-transformed with
DNA sequences encoding the target polypeptide, wild-type DHFR
protein, and another selectable marker such as aminoglycoside 3'
phosphotransferase (APH) can be selected by cell growth in medium
containing a selection agent for the selectable marker such as an
aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See
U.S. Pat. No. 4,965,199.
A suitable selection gene for use in yeast is the trp1 gene present
in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282: 39
[1979]; Kingsman et al., Gene, 7: 141 [1979]; or Tschemper et al.,
Gene, 10: 157 [1980]). The trp1 gene provides a selection marker
for a mutant strain of yeast lacking the ability to grow in
tryptophan, for example, ATCC No. 44076 or PEP4-1 (Jones, Genetics,
85: 12 [1977]). The presence of the trp1 lesion in the yeast host
cell genome then provides an effective environment for detecting
transformation by growth in the absence of tryptophan. Similarly,
Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are
complemented by known plasmids bearing the Leu2 gene.
(d) Promoter Component
Expression and cloning vectors usually contain a promoter that is
recognized by the host organism and is operably linked to the
target polypeptide nucleic acid. Promoters are untranslated
sequences located upstream (5') to the start codon of a structural
gene (generally within about 100 to 1000 bp) that control the
transcription and translation of a particular nucleic acid
sequence, such as that encoding the target polypeptide, to which
they are operably linked. Such promoters typically fall into two
classes, inducible and constitutive. Inducible promoters are
promoters that initiate increased levels of transcription from DNA
under their control in response to some change in culture
conditions, e.g. the presence or absence of a nutrient or a change
in temperature. At this time a large number of promoters recognized
by a variety of potential host cells are well known. These
promoters are operably linked to DNA encoding the target
polypeptide by removing the promoter from the source DNA by
restriction enzyme digestion and inserting the isolated promoter
sequence into the vector. Both the native target polypeptide
promoter sequence and many heterologous promoters may be used to
direct amplification and/or expression of the target polypeptide
DNA. However, heterologous promoters are preferred, as they
generally permit greater transcription and higher yields of
expressed target polypeptide as compared to the native target
polypeptide promoter.
Promoters suitable for use with prokaryotic hosts include the
.beta.-lactamase and lactose promoter systems (Chang et al.,
Nature, 275: 615 [1978]; and Goeddel et al., Nature, 281: 544
[1979]), alkaline phosphatase, a tryptophan (trp) promoter system
(Goeddel, Nucleic Acids Res., 8: 4057 [1980] and EP 36,776) and
hybrid promoters such as the tac promoter (deBoer et al., Proc.
Natl. Acad. Sci. USA, 80: 21-25 [1983]). However, other known
bacterial promoters are suitable. Their nucleotide sequences have
been published, thereby enabling a skilled worker operably to
ligate them to DNA encoding the target polypeptide (Siebenlist et
al., Cell, 20: 269 [1980]) using linkers or adaptors to supply any
required restriction sites. Promoters for use in bacterial systems
also generally will contain a Shine-Dalgarno (S.D.) sequence
operably linked to the DNA encoding the target polypeptide.
Suitable promoting sequences for use with yeast hosts include the
promoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol.
Chem., 255: 2073 [1980]) or other glycolytic enzymes (Hess et al.,
J. Adv. Enzyme Reg., 7: 149 [1968]; and Holland, Biochemistry, 17:
4900 [1978]), such as enolase, glyceraldehyde-3-phosphate
dehydrogenase, hexokinase, pyruvate decarboxylase,
phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase.
Other yeast promoters, which are inducible promoters having the
additional advantage of transcription controlled by growth
conditions, are the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, degradative enzymes associated
with nitrogen metabolism, metallothionein,
glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible
for maltose and galactose utilization. Suitable vectors and
promoters for use in yeast expression are further described in
Hitzeman et al., EP 73,657A. Yeast enhancers also are
advantageously used with yeast promoters.
Promoter sequences are known for eukaryotes. Virtually all
eukaryotic genes have an AT-rich region located approximately 25 to
30 bases upstream from the site where transcription is initiated.
Another sequence found 70 to 80 bases upstream from the start of
transcription of many genes is a CXCAAT region where X may be any
nucleotide. At the 3' end of most eukaryotic genes is an AATAAA
sequence that may be the signal for addition of the poly A tail to
the 3' end of the coding sequence. All of these sequences are
suitably inserted into mammalian expression vectors.
Target polypeptide transcription from vectors in mammalian host
cells is controlled by promoters obtained from the genomes of
viruses such as polyoma virus, fowlpox virus (UK 2,211,504
published Jul. 5, 1989), adenovirus (such as Adenovirus 2), bovine
papilloma virus, avian sarcoma virus, cytomegalovirus, a
retrovirus, hepatitis-B virus and most preferably Simian Virus 40
(SV40), from heterologous mammalian promoters, e.g. the actin
promoter or an immunoglobulin promoter, from heat-shock promoters,
and from the promoter normally associated with the target
polypeptide sequence, provided such promoters are compatible with
the host cell systems.
The early and late promoters of the SV40 virus are conveniently
obtained as an SV40 restriction fragment that also contains the
SV40 viral origin of replication. Fiers et al., Nature, 273:113
(1978); Mulligan and Berg, Science, 209: 1422-1427 (1980); Pavlakis
et al., Proc. Natl. Acad. Sci. USA, 78: 7398-7402 (1981). The
immediate early promoter of the human cytomegalovirus is
conveniently obtained as a HindIII E restriction fragment.
Greenaway et al., Gene, 18: 355-360 (1982). A system for expressing
DNA in mammalian hosts using the bovine papilloma virus as a vector
is disclosed in U.S. Pat. No. 4,419,446. A modification of this
system is described in U.S. Pat. No. 4,601,978. See also Gray et
al., Nature, 295: 503-508 (1982) on expressing cDNA encoding immune
interferon in monkey cells; , Reyes et al., Nature, 297: 598-601
(1982) on expression of human .beta.-interferon cDNA in mouse cells
under the control of a thymidine kinase promoter from herpes
simplex virus, Canaani and Berg, Proc. Natl. Acad. Sci. USA, 79:
5166-5170 (1982) on expression of the human interferon .beta.1 gene
in cultured mouse and rabbit cells, and Gorman et al., Proc. Natl.
Acad. Sci. USA, 79: 6777-6781 (1982) on expression of bacterial CAT
sequences in CV-1 monkey kidney cells, chicken embryo fibroblasts,
Chinese hamster ovary cells, HeLa cells, and mouse NIH-3T3 cells
using the Rous sarcoma virus long terminal repeat as a
promoter.
(e) Enhancer Element Component
Transcription of DNA encoding the target polypeptide of this
invention by higher eukaryotes is often increased by inserting an
enhancer sequence into the vector. Enhancers are cis-acting
elements of DNA, usually about from 10-300 bp, that act on a
promoter to increase its transcription. Enhancers are relatively
orientation and position independent having been found 5' (Laimins
et al., Proc. Natl. Acad. Sci. USA, 78: 993 [1981]) and 3' (Lusky
et al., Mol. Cell Bio., 3: 1108 [1983]) to the transcription unit,
within an intron (Banerji et al., Cell, 33: 729 [1983]) as well as
within the coding sequence itself (Osborne et al., Mol. Cell Bio.,
4:1293 [1984]). Many enhancer sequences are now known from
mammalian genes (globin, elastase, albumin, .alpha.-fetoprotein and
insulin). Typically, however, one will use an enhancer from a
eukaryotic cell virus. Examples include the SV40 enhancer on the
late side of the replication origin (bp 100-270), the
cytomegalovirus early promoter enhancer, the polyoma enhancer on
the late side of the replication origin, and adenovirus enhancers.
See also Yaniv, Nature, 297: 17-18 (1982) on enhancing elements for
activation of eukaryotic promoters. The enhancer may be spliced
into the vector at a position 5' or 3' to the target polypeptide
DNA, but is preferably located at a site 5' from the promoter.
(f) Transcription Termination Component
Expression vectors used in eukaryotic host cells (yeast, fungi,
insect, plant, animal, human, or nucleated cells from other
multicellular organisms) will also contain sequences necessary for
the termination of transcription and for stabilizing the mRNA. Such
sequences are commonly available from the 5' and, occasionally 3'
untranslated regions of eukaryotic or viral DNAs or cDNAs. These
regions contain nucleotide segments transcribed as polyadenylated
fragments in the untranslated portion of the mRNA encoding the
target polypeptide. The 3' untranslated regions also include
transcription termination sites.
Construction of suitable vectors containing one or more of the
above listed components the desired coding and control sequences
employs standard ligation techniques. Isolated plasmids or DNA
fragments are cleaved, tailored, and religated in the form desired
to generate the plasmids required.
For analysis to confirm correct sequences in plasmids constructed,
the ligation mixtures are used to transform E. coli K12 strain 294
(ATCC 31,446) and successful transformants selected by ampicillin
or tetracycline resistance where appropriate. Plasmids from the
transformants are prepared, analyzed by restriction endonuclease
digestion, and/or sequenced by the method of Messing et al.,
Nucleic Acids Res., 9: 309 (1981) or by the method of Maxam et al.,
Methods in Enzymology, 65: 499 (1980).
Particularly useful in the practice of this invention are
expression vectors that provide for the transient expression in
mammalian cells of DNA encoding the target polypeptide. In general,
transient expression involves the use of an expression vector that
is able to replicate efficiently in a host cell, such that the host
cell accumulates many copies of the expression vector and, in turn,
synthesizes high levels of a desired polypeptide encoded by the
expression vector. Transient expression systems, comprising a
suitable expression vector and a host cell, allow for the
convenient positive identification of polypeptides encoded by
cloned DNAs, as well as for the rapid screening of such
polypeptides for desired biological or physiological properties.
Thus, transient expression systems are particularly useful in the
invention for purposes of identifying analogs and variants of the
target polypeptide that have target polypeptide-like activity.
Other methods, vectors, and host cells suitable for adaptation to
the synthesis of the target polypeptide in recombinant vertebrate
cell culture are described in Gething et al., Nature, 293: 620-625
[1981]; Mantei et al., Nature, 281: 40-46 [1979]; Levinson et al.;
EP 117,060; and EP 117,058. A particularly useful plasmid for
mammalian cell culture expression of the target polypeptide is pRK5
(EP pub. no. 307,247) or pSVI6B.
Selection and Transformation of Host Cells
Suitable host cells for cloning or expressing the vectors herein
are the prokaryote, yeast, or higher eukaryote cells described
above. Suitable prokaryotes include eubacteria, such as
Gram-negative or Gram-positive organisms, for example, E. coli,
Bacilli such as B. subtilis, Pseudomonas species such as P.
aeruginosa, Salmonella typhimurium, or Serratia marcescans. One
preferred E. coli cloning host is E. coli 294 (ATCC 31,446),
although other strains such as E. coli B, E. coli .chi.1776 (ATCC
31,537), and E. coli W3110 (ATCC 27,325) are suitable. These
examples are illustrative rather than limiting. Preferably the host
cell should secrete minimal amounts of proteolytic enzymes.
Alternatively, in vitro methods of cloning, e.g. PCR or other
nucleic acid polymerase reactions, are suitable.
In addition to prokaryotes, eukaryotic microbes such as filamentous
fungi or yeast are suitable hosts for target polypeptide-encoding
vectors. Saccharomyces cerevisiae, or common baker's yeast, is the
most commonly used among lower eukaryotic host microorganisms.
However, a number of other genera, species, and strains are
commonly available and useful herein, such as Schizosaccharomyces
pombe [Beach and Nurse, Nature, 290: 140 (1981); EP 139,383
published May 2, 1985], Kluyveromyces hosts (U.S. Pat. No.
4,943,529) such as, e.g., K. lactis [Louvencourt et al., J.
Bacteriol., 737 (1983)], K. fragilis, K. bulgaricus, K.
thermotolerans, and K. marxianus, yarrowia [EP 402,226], Pichia
pastoris [EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:
265-278 (1988)], Candida, Trichoderma reesia [EP 244,234],
Neurospora crassa [Case et al., Proc. Natl. Acad. Sci. USA, 76:
5259-5263 (1979)], and filamentous fungi such as, e.g, Neurospora,
Penicillium, Tolypocladium [WO 91/00357 published Jan. 10, 1991],
and Aspergillus hosts such as A. nidulans [Ballance et al.,
Biochem. Biophys. Res. Commun., 112: 284-289 (1983); Tilburn et
al., Gene, 26: 205-221 (1983); Yelton et al., Proc. Natl. Acad.
Sci. USA, 81: 1470-1474 (1984)] and A. niger [Kelly and Hynes, EMBO
J. 4: 475-479 (1985)].
Suitable host cells for the expression of glycosylated target
polypeptide are derived from multicellular organisms. Such host
cells are capable of complex processing and glycosylation
activities. In principle, any higher eukaryotic cell culture is
workable, whether from vertebrate or invertebrate culture. Examples
of invertebrate cells include plant and insect cells. Numerous
baculoviral strains and variants and corresponding permissive
insect host cells from hosts such as Spodoptera frugiperda
(caterpillar), Aedes aegypti (mosquito), Aedes albopictus
(mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori
host cells have been identified. See, e.g., Luckow et al.,
Bio/Technology 6: 47-55 (1988); Miller et al., in Genetic
Engineering, Setlow, J. K. et al., eds., Vol. 8 (Plenum Publishing,
1986), pp. 277-279; and Maeda et al., Nature, 315: 592-594 (1985).
A variety of such viral strains are publicly available, e.g., the
L-1 variant of Autographa californica NPV and the Bm-5 strain of
Bombyx mori NPV, and such viruses may be used as the virus herein
according to the present invention, particularly for transfection
of Spodoptera frugiperda cells. Plant cell cultures of cotton,
corn, potato, soybean, petunia, tomato, and tobacco can be utilized
as hosts. Typically, plant cells are transfected by incubation with
certain strains of the bacterium Agrobacterium tumefaciens, which
has been previously manipulated to contain the target polypeptide
DNA. During incubation of the plant cell culture with A.
tumefaciens, the DNA encoding target polypeptide is transferred to
the plant cell host such that it is transfected, and will, under
appropriate conditions, express the target polypeptide DNA. In
addition, regulatory and signal sequences compatible with plant
cells are available, such as the nopaline synthase promoter and
polyadenylation signal sequences. Depicker et al., J. Mol. Appl.
Gen., 1: 561 (1982). In addition, DNA segments isolated from the
upstream region of the T-DNA 780 gene are capable of activating or
increasing transcription levels of plant-expressible genes in
recombinant DNA-containing plant tissue. See EP 321,196 published
Jun. 21, 1989.
However, interest has been greatest in vertebrate cells, and
propagation of vertebrate cells in culture (tissue culture) has
become a routine procedure in recent years [Tissue Culture,
Academic Press, Kruse and Patterson, editors (1973)]. Examples of
useful mammalian host cell lines are monkey kidney CV1 line
transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney
line (293 or 293 cells subcloned for growth in suspension culture,
Graham et al., J. Gen Virol., 36: 59 [1977]); baby hamster kidney
cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO,
Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216 [1980]);
mouse sertoli cells (TM4, Mather, Biol. Reprod., 23: 243-251
[1980]); monkey kidney cells (CV1 ATCC CCL 70); African green
monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical
carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC
CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human
lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB
8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells
(Mather et al., Annals N.Y. Acad. Sci., 383: 44-68 [1982]); MRC 5
cells; FS4 cells; and a human hepatoma cell line (Hep G2).
Preferred host cells are human embryonic kidney 293 and Chinese
hamster ovary cells.
Host cells are transfected and preferably transformed with the
above-described expression or cloning vectors of this invention and
cultured in conventional nutrient media modified as appropriate for
inducing promoters, selecting transformants, or amplifying the
genes encoding the desired sequences.
Transfection refers to the taking up of an expression vector by a
host cell whether or not any coding sequences are in fact
expressed. Numerous methods of transfection are known to the
ordinarily skilled artisan, for example, CaPO.sub.4 and
electroporation. Successful transfection is generally recognized
when any indication of the operation of this vector occurs within
the host cell.
Transformation means introducing DNA into an organism so that the
DNA is replicable, either as an extrachromosomal element or by
chromosomal integrant. Depending on the host cell used,
transformation is done using standard techniques appropriate to
such cells. The calcium treatment employing calcium chloride, as
described in section 1.82 of Sambrook et al., supra, is generally
used for prokaryotes or other cells that contain substantial
cell-wall barriers. Infection with Agrobacterium tumefaciens is
used for transformation of certain plant cells, as described by
Shaw et al., Gene, 23: 315 (1983) and WO 89/05859 published Jun.
29, 1989. For mammalian cells without such cell walls, the calcium
phosphate precipitation method described in sections 16.30-16.37 of
Sambrook et al, supra, is preferred. General aspects of mammalian
cell host system transformations have been described by Axel in
U.S. Pat. No. 4,399,216 issued Aug. 16, 1983. Transformations into
yeast are typically carried out according to the method of Van
Solingen et al., J. Bact., 130: 946 (1977) and Hsiao et al., Proc.
Natl. Acad. Sci. (USA), 76: 3829 (1979). However, other methods for
introducing DNA into cells such as by nuclear injection,
electroporation, or protoplast fusion may also be used.
Culturing the Host Cells
Prokaryotic cells used to produce the target polypeptide of this
invention are cultured in suitable media as described generally in
Sambrook et al., supra.
The mammalian host cells used to produce the target polypeptide of
this invention may be cultured in a variety of media. Commercially
available media such as Ham's F10 (Sigma), Minimal Essential Medium
([MEM], Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's
Medium ([DMEM], Sigma) are suitable for culturing the host cells.
In addition, any of the media described in Ham and Wallace, Meth.
Enz., 58: 44 (1979), Barnes and Sato, Anal. Biochem., 102: 255
(1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; or
4,560,655; WO 90/03430; WO 87/00195; U.S. Pat. No. Re. 30,985, may
be used as culture media for the host cells. Any of these media may
be supplemented as necessary with hormones and/or other growth
factors (such as insulin, transferrin, or epidermal growth factor),
salts (such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleosides (such as adenosine and
thymidine), antibiotics (such as Gentamycin.TM. drug), trace
elements (defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art. The culture conditions, such as
temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
The host cells referred to in this disclosure encompass cells in in
vitro culture as well as cells that are within a host animal.
It is further envisioned that the target polypeptides of this
invention may be produced by homologous recombination, or with
recombinant production methods utilizing control elements
introduced into cells already containing DNA encoding the target
polypeptide currently in use in the field. For example, a powerful
promoter/enhancer element, a suppressor, or an exogenous
transcription modulatory element is inserted in the genome of the
intended host cell in proximity and orientation sufficient to
influence the transcription of DNA encoding the desired target
polypeptide. The control element does not encode the target
polypeptide of this invention, but the DNA is present in the host
cell genome. One next screens for cells making the target
polypeptide of this invention, or increased or decreased levels of
expression, as desired.
Detecting Gene Amplification/Expression
Gene amplification and/or expression may be measured in a sample
directly, for example, by conventional Southern blotting, northern
blotting to quantitate the transcription of mRNA (Thomas, Proc.
Natl. Acad. Sci. USA, 77: 5201-5205 [1980]), dot blotting (DNA
analysis), or in situ hybridization, using an appropriately labeled
probe, based on the sequences provided herein. Various labels may
be employed, most commonly radioisotopes, particularly .sup.32 P.
However, other techniques may also be employed, such as using
biotin-modified nucleotides for introduction into a polynucleotide.
The biotin then serves as the site for binding to avidin or
antibodies, which may be labeled with a wide variety of labels,
such as radionuclides, fluorescers, enzymes, or the like.
Alternatively, antibodies may be employed that can recognize
specific duplexes, including DNA duplexes, RNA duplexes, and
DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in
turn may be labeled and the assay may be carried out where the
duplex is bound to a surface, so that upon the formation of duplex
on the surface, the presence of antibody bound to the duplex can be
detected.
Gene expression, alternatively, may be measured by immunological
methods, such as immunohistochemical staining of tissue sections
and assay of cell culture or body fluids, to quantitate directly
the expression of gene product. With immunohistochemical staining
techniques, a cell sample is prepared, typically by dehydration and
fixation, followed by reaction with labeled antibodies specific for
the gene product coupled, where the labels are usually visually
detectable, such as enzymatic labels, fluorescent labels,
luminescent labels, and the like. A particularly sensitive staining
technique suitable for use in the present invention is described by
Hsu et al., Am. J. Clin. Path., 75: 734-738 (1980).
Antibodies useful for immunohistochemical staining and/or assay of
sample fluids may be either monoclonal or polyclonal, and may be
prepared in any mammal. Conveniently, the antibodies may be
prepared against a native target polypeptide or against a synthetic
peptide based on the DNA sequences provided herein as described
further in Section 4 below.
Purification of the Target Polypeptide
The target polypeptide preferably is recovered from the culture
medium as a secreted polypeptide, although it also may be recovered
from host cell lysates when directly expressed without a secretory
signal.
When the target polypeptide is expressed in a recombinant cell
other than one of human origin, the target polypeptide is
completely free of proteins or polypeptides of human origin.
However, it is necessary to purify the target polypeptide from
recombinant cell proteins or polypeptides to obtain preparations
that are substantially homogeneous as to the target polypeptide. As
a first step, the culture medium or lysate is centrifuged to remove
particulate cell debris. The membrane and soluble protein fractions
are then separated. The target polypeptide may then be purified
from the soluble protein fraction and from the membrane fraction of
the culture lysate, depending on whether the target polypeptide is
membrane bound. The following procedures are exemplary of suitable
purification procedures: fractionation on immunoaffinity or
ion-exchange columns; ethanol precipitation; reverse phase HPLC;
chromatography on silica or on a cation exchange resin such as
DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation;
gel filtration using, for example, Sephadex G-75; and protein A
Sepharose columns to remove contaminants such as IgG.
Target polypeptide variants in which residues have been deleted,
inserted or substituted are recovered in the same fashion, taking
account of any substantial changes in properties occasioned by the
variation. For example, preparation of a target polypeptide fusion
with another protein or polypeptide, e.g. a bacterial or viral
antigen, facilitates purification; an immunoaffinity column
containing antibody to the antigen (or containing antigen, where
the target polypeptide is an antibody) can be used to adsorb the
fusion. Immunoaffinity columns such as a rabbit polyclonal
anti-target polypeptide column can be employed to absorb the target
polypeptide variant by binding it to at least one remaining immune
epitope. A protease inhibitor such as phenyl methyl sulfonyl
fluoride (PMSF) also may be useful to inhibit proteolytic
degradation during purification, and antibiotics may be included to
prevent the growth of adventitious contaminants. One skilled in the
art will appreciate that purification methods suitable for native
target polypeptide may require modification to account for changes
in the character of the target polypeptide or its variants upon
expression in recombinant cell culture.
Covalent Modifications of Target Polypeptides
Covalent modifications of target polypeptides are included within
the scope of this invention. One type of covalent modification
included within the scope of this invention is a target polypeptide
fragment. Target polypeptide fragments having up to about 40 amino
acid residues may be conveniently prepared by chemical synthesis,
or by enzymatic or chemical cleavage of the full-length target
polypeptide or variant target polypeptide. Other types of covalent
modifications of the target polypeptide or fragments thereof are
introduced into the molecule by reacting specific amino acid
residues of the target polypeptide or fragments thereof with an
organic derivatizing agent that is capable of reacting with
selected side chains or the N- or C-terminal residues.
Cysteinyl residues most commonly are reacted with
.alpha.-haloacetates (and corresponding amines), such as
chloroacetic acid or chloroacetamide, to give carboxymethyl or
carboxyamidomethyl derivatives. Cysteinyl residues also are
derivatized by reaction with bromotrifluoroacetone,
.alpha.-bromo-.beta.-(5-imidozoyl)propionic acid, chloroacetyl
phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl
2-pyridyl disulfide, p-chloromercuribenzoate,
2-chloromercuri-4-nitrophenol, or
chloro-7-nitrobenzo-2-oxa-1,3-diazole.
Histidyl residues are derivatized by reaction with
diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively
specific for the histidyl side chain. Parabromophenacyl bromide
also is useful; the reaction is preferably performed in 0.1 M
sodium cacodylate at pH 6.0.
Lysinyl and amino terminal residues are reacted with succinic or
other carboxylic acid anhydrides. Derivatization with these agents
has the effect of reversing the charge of the lysinyl residues.
Other suitable reagents for derivatizing .alpha.-amino-containing
residues include imidoesters such as methyl picolinimidate;
pyridoxal phosphate; pyridoxal; chloroborohydride;
trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione;
and transaminase-catalyzed reaction with glyoxylate.
Arginyl residues are modified by reaction with one or several
conventional reagents, among them phenylglyoxal, 2,3-butanedione,
1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine
residues requires that the reaction be performed in alkaline
conditions because of the high pK.sub.a of the guanidine functional
group. Furthermore, these reagents may react with the groups of
lysine as well as the arginine epsilon-amino group.
The specific modification of tyrosyl residues may be made, with
particular interest in introducing spectral labels into tyrosyl
residues by reaction with aromatic diazonium compounds or
tetranitromethane. Most commonly, N-acetylimidizole and
tetranitromethane are used to form O-acetyl tyrosyl species and
3-nitro derivatives, respectively. Tyrosyl residues are iodinated
using .sup.125 I or .sup.131 I to prepare labeled proteins for use
in radioimmunoassay, the chloramine T method described above being
suitable.
Carboxyl side groups (aspartyl or glutamyl) are selectively
modified by reaction with carbodiimides (R'--N.dbd.C.dbd.N--R'),
where R and R' are different alkyl groups, such as
1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or
1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore,
aspartyl and glutamyl residues are converted to asparaginyl and
glutaminyl residues by reaction with ammonium ions.
Derivatization with bifunctional agents is useful for crosslinking
target polypeptide to a water-insoluble support matrix or surface
for use in the method for purifying anti-target polypeptide
antibodies, and vice versa. Commonly used crosslinking agents
include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxysuccinimide esters, for example, esters with
4-azido-salicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), and bifunctional maleimides
such as bis-N-maleimido-1,8-octane. Derivatizing agents such as
methyl-3-[(p-azidophenyl)-dithio]propioimidate yield
photoactivatable intermediates that are capable of forming
crosslinks in the presence of light. Alternatively, reactive
water-insoluble matrices such as cyanogen bromide-activated
carbohydrates and the reactive substrates described in U.S. Pat.
Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and
4,330,440 are employed for protein immobilization.
Glutaminyl and asparaginyl residues are frequently deamidated to
the corresponding glutamyl and aspartyl residues, respectively.
Alternatively, these residues are deamidated under mildly acidic
conditions. Either form of these residues falls within the scope of
this invention.
Other modifications include hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the .alpha.-amino groups of lysine, arginine, and
histidine side chains (T. E. Creighton, Proteins: Structure and
Molecular Properties, W. H. Freeman & Co., San Francisco, pp.
79-86 [1983]), acetylation of the N-terminal amine, and amidation
of any C-terminal carboxyl group.
Another type of covalent modification of the target polypeptide
included within the scope of this invention comprises altering the
native glycosylation pattern of the polypeptide. By altering is
meant deleting one or more carbohydrate moieties found in the
native target polypeptide, and/or adding one or more glycosylation
sites that are not present in the native target polypeptide.
Glycosylation of polypeptides is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tri-peptide
sequences asparagine-X-serine and asparagine-X-threonine, where X
is any amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tri-peptide
sequences in a polypeptide creates a potential glycosylation site.
0-linked glycosylation refers to the attachment of one of the
sugars N-acetylgalactosamine, galactose, or xylose, to a
hydroxyamino acid, most commonly serine or threonine, although
5-hydroxyproline or 5-hydroxylysine may also be used.
Addition of glycosylation sites to the target polypeptide is
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tri-peptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the native target polypeptide
sequence (for O-linked glycosylation sites). For ease, the target
polypeptide amino acid sequence is preferably altered through
changes at the DNA level, particularly by mutating the DNA encoding
the target polypeptide at preselected bases such that codons are
generated that will translate into the desired amino acids. The DNA
mutation(s) may be made using methods described above under the
heading of "Amino Acid Sequence Variants of Target
Polypeptide".
Another means of increasing the number of carbohydrate moieties on
the target polypeptide is by chemical or enzymatic coupling of
glycosides to the polypeptide. These procedures are advantageous in
that they do not require production of the polypeptide in a host
cell that has glycosylation capabilities for N-and O-linked
glycosylation. Depending on the coupling mode used, the sugar(s)
may be attached to (a) arginine and histidine, (b) free carboxyl
groups, (c) free sulfhydryl groups such as those of cysteine, (d)
free hydroxyl groups such as those of serine, threonine, or
hydroxyproline, (e) aromatic residues such as those of
phenylalanine, tyrosine, or tryptophan, or (f) the amide group of
glutamine. These methods are described in WO 87/05330 published
Sep. 11, 1987, and in Aplin and Wriston (CRC Crit. Rev. Biochem.,
pp. 259-306 [1981]).
Removal of carbohydrate moieties present on the native target
polypeptide may be accomplished chemically or enzymatically.
Chemical deglycosylation requires exposure of the polypeptide to
the compound trifluoromethanesulfonic acid, or an equivalent
compound. This treatment results in the cleavage of most or all
sugars except the linking sugar (N-acetylglucosamine or
N-acetylgalactosamine), while leaving the polypeptide intact.
Chemical deglycosylation is described by Hakimuddin et al. (Arch.
Biochem. Biophys., 259:52 [1987]) and by Edge et al. (Anal.
Biochem., 118:131 [1981]). Enzymatic cleavage of carbohydrate
moieties on polypeptides can be achieved by the use of a variety of
endo- and exo-glycosidases as described by Thotakura et al. (Meth.
Enzymol. 138:350 [1987]).
Glycosylation at potential glycosylation sites may be prevented by
the use of the compound tunicamycin as described by Duskin et al.
(J. Biol. Chem., 257:3105 [1982]). Tunicamycin blocks the formation
of protein-N-glycoside linkages.
Another type of covalent modification of the target polypeptide
comprises linking the target polypeptide to various
nonproteinaceous polymers, e.g. polyethylene glycol, polypropylene
glycol or polyoxyalkylenes, in the manner set forth in U.S. Pat.
Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or
4,179,337.
The target polypeptide also may be entrapped in microcapsules
prepared, for example, by coacervation techniques or by interfacial
polymerization (for example, hydroxymethylcellulose or
gelatin-microcapsules and poly-[methylmethacylate]microcapsules,
respectively), in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules), or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences, 16th edition, Osol, A.,
Ed., (1980).
Target polypeptide preparations are also useful in generating
antibodies, for screening for binding partners, as standards in
assays for the target polypeptide (e.g. by labeling the target
polypeptide for use as a standard in a radioimmunoassay,
enzyme-linked immunoassay, or radioreceptor assay), in affinity
purification techniques, and in competitive-type receptor binding
assays when labeled with radioiodine, enzymes, fluorophores, spin
labels, and the like.
Since it is often difficult to predict in advance the
characteristics of a variant target polypeptide, it will be
appreciated that some screening of the recovered variant will be
needed to select the optimal variant. For example, a change in this
immunological character of the target polypeptide molecule, such as
affinity for a given antigen or antibody, is measured by a
competitive-type immunoassay. The variant is assayed for changes in
the suppression or enhancement of its activity bad comparison to
the activity observed for the target polypeptide in the same assay.
Other potential modifications of protein or polypeptide properties
such as redox or thermal stability, hydrophobicity, susceptibility
to proteolytic degradation, stability in recombinant cell culture
or in plasma, or the tendency to aggregate with carriers or into
multimers are assayed by methods well known in the art.
Diagnostic and Related Uses of the Antibodies
The antibodies of this invention are useful in diagnostic assays
for antigen expression in specific cells or tissues. The antibodies
are detectably labeled and/or are immobilized on an insoluble
matrix.
The antibodies of this invention find further use for the affinity
purification of the antigen from recombinant cell culture or
natural sources. Suitable diagnostic assays for the antigen and its
antibodies depend on the particular antigen or antibody. Generally,
such assays include competitive and sandwich assays, and steric
inhibition assays. Competitive and sandwich methods employ a
phase-separation step as an integral part of the method while
steric inhibition assays are conducted in a single reaction
mixture. Fundamentally, the same procedures are used for the assay
of the antigen and for substances that bind the antigen, although
certain methods will be favored depending upon the molecular weight
of the substance being assayed. Therefore, the substance to be
tested is referred to herein as an analyte, irrespective of its
status otherwise as an antigen or antibody, and proteins that bind
to the analyte are denominated binding partners, whether they be
antibodies, cell surface receptors, or antigens.
Analytical methods for the antigen or its antibodies all use one or
more of the following reagents: labeled analyte analogue,
immobilized analyte analogue, labeled binding partner, immobilized
binding partner and steric conjugates. The labeled reagents also
are known as "tracers."
The label used (and this is also useful to label antigen nucleic
acid for use as a probe) is any detectable functionality that does
not interfere with the binding of analyte and its binding partner.
Numerous labels are known for use in immunoassay, examples
including moieties that may be detected directly, such as
fluorochrome, chemiluminescent, and radioactive labels, as well as
moieties, such as enzymes, that must be reacted or derivatized to
be detected. Examples of such labels include the radioisotopes
.sup.32 P, .sup.14 C, .sup.125 I, .sup.3 H, and .sup.131 I,
fluorophores such as rare earth chelates or fluorescein and its
derivatives, rhodamine and its derivatives, dansyl, umbelliferone,
luceriferases, e.g., firefly luciferase and bacterial luciferase
(U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,
horseradish peroxidase (HRP), alkaline phosphatase,
.beta.-galactosidase, glucoamylase, lysozyme, saccharide oxidases,
e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate
dehydrogenase, heterocyclic oxidases such as uricase and xanthine
oxidase, coupled with an enzyme that employs hydrogen peroxide to
oxidize a dye precursor such as HRP, lactoperoxidase, or
microperoxidase, biotin/avidin, spin labels, bacteriophage labels,
stable free radicals, and the like.
Conventional methods are available to bind these labels covalently
to proteins or polypeptides. For instance, coupling agents such as
dialdehydes, carbodiimides, dimaleimides, bis-imidates,
bis-diazotized benzidine, and the like may be used to tag the
antibodies with the above-described fluorescent, chemiluminescent,
and enzyme labels. See, for example, U.S. Pat. No. 3,940,475
(fluorimetry) and U.S. Pat. No. 3,645,090 (enzymes); Hunter et al.,
Nature, 144: 945 (1962); David et al., Biochemistry, 13: 1014-1021
(1974); Pain et al., J. Immunol. Methods. 40: 219-230 (1981); and
Nygren, J. Histochem. and Cytochem., 30: 407-412 (1982). Preferred
labels herein are enzymes such as horseradish peroxidase and
alkaline phosphatase. The conjugation of such label, including the
enzymes, to the antibody is a standard manipulative procedure for
one of ordinary skill in immunoassay techniques. See, for example,
O'Sullivan et al., "Methods for the Preparation of Enzyme-antibody
Conjugates for Use in Enzyme Immunoassay," in Methods in Enzymology
ed. J. J. Langone and H. Van Vunakis, Vol. 73 (Academic Press, New
York, N.Y., 1981), pp.147-166. Such bonding methods are suitable
for use with the antibodies and polypeptides of this invention.
Immobilization of reagents is required for certain assay methods.
Immobilization entails separating the binding partner from any
analyte that remains free in solution. This conventionally is
accomplished by either insolubilizing the binding partner or
analyte analogue before the assay procedure, as by adsorption to a
water-insoluble matrix or surface (Bennich et al., U.S. Pat. No.
3,720,760), by covalent coupling (for example, using glutaraldehyde
cross-linking), or by insolubilizing the partner or analogue
afterward, e.g., by immunoprecipitation.
Other assay methods, known as competitive or sandwich assays, are
well established and widely used in the commercial diagnostics
industry.
Competitive assays rely on the ability of a tracer analogue to
compete with the test sample analyte for a limited number of
binding sites on a common binding partner. The binding partner
generally is insolubilized before or after the competition and then
the tracer and analyte bound to the binding partner are separated
from the unbound tracer and analyte. This separation is
accomplished by decanting (where the binding partner was
preinsolubilized) or by centrifuging (where the binding partner was
precipitated after the competitive reaction). The amount of test
sample analyte is inversely proportional to the amount of bound
tracer as measured by the amount of marker substance. Dose-response
curves with known amounts of analyte are prepared and compared with
the test results to quantitatively determine the amount of analyte
present in the test sample. These assays are called ELISA systems
when enzymes are used as the detectable markers.
Another species of competitive assay, called a "homogeneous" assay,
does not require a phase separation. Here, a conjugate of an enzyme
with the analyte is prepared and used such that when anti-analyte
binds to the analyte the presence of the anti-analyte modifies the
enzyme activity. In this case, the antigen or its immunologically
active fragments are conjugated with a bifunctional organic bridge
to an enzyme such as peroxidase. Conjugates are selected for use
with antibody so that binding of the antibody inhibits or
potentiates the enzyme activity of the label. This method per se is
widely practiced under the name of EMIT.
Steric conjugates are used in steric hindrance methods for
homogeneous assay. These conjugates are synthesized by covalently
linking a low-molecular-weight hapten to a small analyte so that
antibody to hapten substantially is unable to bind the conjugate at
the same time as anti-analyte. Under this assay procedure the
analyte present in the test sample will bind anti-analyte, thereby
allowing anti-hapten to bind the conjugate, resulting in a change
in the character of the conjugate hapten, e.g., a change in
fluorescence when the hapten is a fluorophore.
Sandwich assays particularly are useful for the determination of
antigen or antibodies. In sequential sandwich assays an immobilized
binding partner is used to adsorb test sample analyte, the test
sample is removed as by washing, the bound analyte is used to
adsorb labeled binding partner, and bound material is then
separated from residual tracer. The amount of bound tracer is
directly proportional to test sample analyte. In "simultaneous"
sandwich assays the test sample is not separated before adding the
labeled binding partner. A sequential sandwich assay using an
anti-antigen monoclonal antibody as one antibody and a polyclonal
anti-antigen antibody as the other is useful in testing samples for
particular antigen activity.
The foregoing are merely exemplary diagnostic assays for the import
and humanized antibodies of this invention. Other methods now or
hereafter developed for the determination of these analytes are
included within the scope hereof, including the bioassays described
above.
Immunotoxins
This invention is also directed to immunochemical derivatives of
the antibodies of this invention such as immunotoxins (conjugates
of the antibody and a cytotoxic moiety). Antibodies which carry the
appropriate effector functions, such as with their constant
domains, are also used to induce lysis through the natural
complement process, and to interact with antibody dependent
cytotoxic cells normally present.
For example, purified, sterile filtered antibodies are optionally
conjugated to a cytotoxin such as ricin for use in AIDS therapy.
U.S. patent application Ser. No. 07/350,895 illustrates methods for
making and using immunotoxins for the treatment of HIV infection.
The methods of this invention, for example, are suitable for
obtaining humanized antibodies for use as immunotoxins for use in
AIDS therapy.
The cytotoxic moiety of the immunotoxin may be a cytotoxic drug or
an enzymatically active toxin of bacterial, fungal, plant or animal
origin, or an enzymatically active fragment of such a toxin.
Enzymatically active toxins and fragments thereof used are
diphtheria A chain, nonbinding active fragments of diphtheria
toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A
chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites
fordii proteins, dianthin proteins, Phytolaca americana proteins
(PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin,
crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin,
restrictocin, phenomycin, enomycin and the tricothecenes. In
another embodiment, the antibodies are conjugated to small molecule
anticancer drugs such as cis-platin or 5FU. Conjugates of the
monoclonal antibody and such cytotoxic moieties are made using a
variety of bifunctional protein coupling agents. Examples of such
reagents are SPDP, IT, bifunctional derivatives of imidoesters such
as dimethyl adipimidate HCl, active esters such as disuccinimidyl
suberate, aldehydes such as; glutaraldehyde, bis-azido compounds
such as bis(p-azidobenzoyl)hexanediamine, bis-diazonium derivatives
such as bis-(p-diazoniumbenzoyl)-ethylenediamine, diisocyanates
such as tolylene 2,6-diisocyanate and bis-active fluorine compounds
such as 1,5-difluoro-2,4-dinitrobenzene. The lysing portion of a
toxin may be joined to the Fab fragment of the antibodies.
Immunotoxins can be made in a variety of ways, as discussed herein.
Commonly known crosslinking reagents can be used to yield stable
conjugates.
Advantageously, monoclonal antibodies specifically binding the
domain of the antigen which is exposed on the infected cell
surface, are conjugated to ricin A chain. Most advantageously the
ricin A chain is deglycosylated and produced through recombinant
means. An advantageous method of making the ricin immunotoxin is
described in Vitetta et al., Science 238:1098 (1987).
When used to kill infected human cells in vitro for diagnostic
purposes, the conjugates will typically be added to the cell
culture medium at a concentration of at least about 10 nM. The
formulation and mode of administration for in vitro use are not
critical. Aqueous formulations that are compatible with the culture
or perfusion medium will normally be used. Cytotoxicity may be read
by conventional techniques.
Cytotoxic radiopharmaceuticals for treating infected cells may be
made by conjugating radioactive isotopes (e.g. I, Y, Pr) to the
antibodies. Advantageously alpha particle-emitting isotopes are
used. The term "cytotoxic moiety" as used herein is intended to
include such isotopes.
In a preferred embodiment, ricin A chain is deglycosylated or
produced without oligosaccharides, to decrease its clearance by
irrelevant clearance mechanisms (e.g., the liver). In another
embodiment, whole ricin (A chain plus B chain) is conjugated to
antibody if the galactose binding property of B-chain can be
blocked ("blocked ricin").
In a further embodiment toxin-conjugates are made with Fab or
F(ab').sub.2 fragments. Because of their relatively small size
these fragments can better penetrate tissue to reach infected
cells.
In another embodiment, fusogenic liposomes are filled with a
cytotoxic drug and the liposomes are coated with antibodies
specifically binding the particular antigen.
Antibody Dependent Cellular Cytotoxicity
Certain aspects of this invention involve antibodies which are (a)
directed against a particular antigen and (b) belong to a subclass
or isotype that is capable of mediating the lysis of cells to which
the antibody molecule binds. More specifically, these antibodies
should belong to a subclass or isotype that, upon complexing with
cell surface proteins, activates serum complement and/or mediates
antibody dependent cellular cytotoxicity (ADCC) by activating
effector cells such as natural killer cells or macrophages.
Biological activity of antibodies is known to be determined, to a
large extent, by the constant domains or Fc region of the antibody
molecule (Uananue and Benacerraf, Textbook of Immunology, 2nd
Edition, Williams & Wilkins, p. 218 (1984)). This includes
their ability to activate complement and to mediate
antibody-dependent cellular cytotoxicity (ADCC) as effected by
leukocytes. Antibodies of different classes and subclasses differ
in this respect, as do antibodies from the same subclass but
different species; according to the present invention, antibodies
of those classes having the desired biological activity are
prepared. Preparation of these antibodies involves the selection of
antibody constant domains are their incorporation in the humanized
antibody by known technique. For example, mouse immunoglobulins of
the IgG3 and IgG2a class are capable of activating serum complement
upon binding to the target cells which express the cognate antigen,
and therefore humanized antibodies which incorporate IgG3 and IgG2a
effector functions are desirable for certain therapeutic
applications.
In general, mouse antibodies of the IgG2a and IgG3 subclass and
occasionally IgG1 can mediate ADCC, and antibodies of the IgG3,
IgG2a, and IgM subclasses bind and activate serum complement.
Complement activation generally requires the binding of at least
two IgG molecules in close proximity on the target cell. However,
the binding of only one IgM molecule activates serum
complement.
The ability of any particular antibody to mediate lysis of the
target cell by complement activation and/or ADCC can be assayed.
The cells of interest are grown and labeled in vitro; the antibody
is added to the cell culture in combination with either serum
complement or immune cells which may be activated by the antigen
antibody complexes. Cytolysis of the target cells is detected by
the release of label from the lysed cells. In fact, antibodies can
be screened using the patient's own serum as a source of complement
and/or immune cells. The antibody that is capable of activating
complement or mediating ADCC in the in vitro test can then be used
therapeutically in that particular patient.
This invention specifically encompasses consensus Fc antibody
domains prepared and used according to the teachings of this
invention.
Therapeutic and Other Uses of the Antibodies
When used in vivo for therapy, the antibodies of the subject
invention are administered to the patient in therapeutically
effective amounts (i.e. amounts that have desired therapeutic
effect). They will normally be administered parenterally. The dose
and dosage regimen will depend upon the degree of the infection,
the characteristics of the particular antibody or immunotoxin used,
e.g., its therapeutic index, the patient, and the patient's
history. Advantageously the antibody or immunotoxin is administered
continuously over a period of 1-2 weeks, intravenously to treat
cells in the vasculature and subcutaneously and intraperitoneally
to treat regional lymph nodes. Optionally, the administration is
made during the course of adjunct therapy such as combined cycles
of radiation, chemotherapeutic treatment, or administration of
tumor necrosis factor, interferon or other cytoprotective or
immunomodulatory agent.
For parenteral administration the antibodies will be formulated in
a unit dosage injectable form (solution, suspension, emulsion) in
association with a pharmaceutically acceptable parenteral vehicle.
Such vehicles are inherently nontoxic, and non-therapeutic.
Examples of such vehicles are water, saline, Ringer's solution,
dextrose solution, and 5% human serum albumin. Nonaqueous vehicles
such as fixed oils and ethyl oleate can also be used. Liposomes may
be used as carriers. The vehicle may contain minor amounts of
additives such as substances that enhance isotonicity and chemical
stability, e.g., buffers and preservatives. The antibodies will
typically be formulated in such vehicles at concentrations of about
1 mg/ml to 10 mg/ml.
Use of IgM antibodies may be preferred for certain applications,
however IgG molecules by being smaller may be more able than IgM
molecules to localize to certain types of infected cells.
There is evidence that complement activation in vivo leads to a
variety of biological effects, including the induction of an
inflammatory response and the activation of macrophages (Uananue
and Benecerraf, Textbook of Immunology, 2nd Edition, Williams &
Wilkins, p. 218 (1984)). The increased vasodilation accompanying
inflammation may increase the ability of various agents to localize
in infected cells. Therefore, antigen-antibody combinations of the
type specified by this invention can be used therapeutically in
many ways. Additionally, purified antigens (Hakomori, Ann Rev.
Immunol 2:103 (1984)) or anti-idiotypic antibodies (Nepom et al.,
Proc. Natl. Acad. Sci. 81:2864 (1985); Koprowski et al., Proc.
Natl. Acad. Sci. 81:216 (1984)) relating to such antigens could be
used to induce an active immune response in human patients. Such a
response includes the formation of antibodies capable of activating
human complement and mediating ADCC and by such mechanisms cause
infected cell destruction.
Optionally, the antibodies of this invention are useful in
passively immunizing patients, as exemplified by the administration
of humanized anti-HIV antibodies.
The antibody compositions used in therapy are formulated and
dosages established in a fashion consistent with good medical
practice taking into account the disorder to be treated, the
condition of the individual patient, the site of delivery of the
composition, the method of administration and other factors known
to practitioners. The antibody compositions are prepared for
administration according to the description of preparation of
polypeptides for administration, infra.
Deposit of Materials
As described above, cultures of the muMAb4D5 have been deposited
with the American Type Culture Collection, 10801 University Blvd.,
Manassas, Va., U.S.A. (ATCC).
This deposit was made under the provisions of the Budapest Treaty
on the International Recognition of the Deposit of Microorganisms
for the Purpose of Patent Procedure and the Regulations thereunder
(Budapest Treaty). This assures maintenance of viable cultures for
30 years from the date of the deposit. The organisms will be made
available by ATCC under the terms of the Budapest Treaty, and
subject to an agreement between Genentech, Inc. and ATCC, which
assures permanent and unrestricted availability of the progeny of
the cultures to the public upon issuance of the pertinent U.S.
patent or upon laying open to the public of any U.S. or foreign
patent application, whichever comes first, and assures availability
of the progeny to one determined by the U.S. Commissioner of
Patents and Trademarks to be entitled thereto according to 35 USC
.sctn.122 and the Commissioner's rules pursuant thereto (including
37 CFR .sctn.1.12 with particular reference to 886 OG 638).
In respect of those designations in which a European patent is
sought, a sample of the deposited microorganism will be made
available until the publication of the mention of the grant of the
European patent or until the date on which the application has been
refused or withdrawn or is deemed to be withdrawn, only by the
issue of such a sample to an expert nominated by the person
requesting the sample. (Rule 28(4) EPC)
The assignee of the present application has agreed that if the
cultures on deposit should die or be lost or destroyed when
cultivated under suitable conditions, they will be promptly
replaced on notification with a viable specimen of the same
culture. Availability of the deposited strain is not to be
construed as a license to practice the invention in contravention
of the rights granted under the authority of any government in
accordance with its patent laws.
The foregoing written specification is considered to be sufficient
to enable one skilled in the art to practice the invention. The
present invention is not to be limited in scope by the constructs
deposited, since the deposited embodiments are intended to
illustrate only certain aspects of the invention and any constructs
that are functionally equivalent are within the scope of this
invention. The deposit of material herein does not constitute an
admission that the written description herein contained is
inadequate to enable the practice of any aspect of the invention,
including the best mode thereof, nor is it to be construed as
limiting the scope of the claims to the specific illustrations that
they represent. Indeed, various modifications of the invention in
addition to those shown and described herein will become apparent
to those skilled in the art from the foregoing description and fall
within the scope of the appended claims.
It is understood that the application of the teachings of the
present invention to a specific problem or situation will be within
the capabilities of one having ordinary skill in the art in light
of the teachings contained herein. Examples of the products of the
present invention and representative processes for their isolation,
use, and manufacture appear below, but should not be construed to
limit the invention.
EXAMPLES
Example 1
Humanization of muMAb4D5
Here we report the chimerization of muMAb4D5 (chMAb4D5) and the
rapid and simultaneous humanization of heavy (V.sub.H) and light
(V.sub.L) chain variable region genes using a novel "gene
conversion mutagenesis" strategy. Eight humanized variants
(huMAb4D5) were constructed to probe the importance of several FR
residues identified by our molecular modeling or previously
proposed to be critical to the conformation of particular CDRs (see
Chothia, C. & Lesk, A. M., J. Mol. Biol. 196:901-917 (1987);
Chothia, C. et al., Nature 342:877-883 (1989); Tramontano, A. et
al., J. Mol. Biol. 215:175-182 (1990)). Efficient transient
expression of humanized variants in non-myeloma cells allowed us to
rapidly investigate the relationship between binding affinity for
p185.sup.HER2 ECD and anti-proliferative activity against
p185.sup.HER2 overexpressing carcinoma cells.
Materials and Methods
Cloning of Variable Region Genes. The muMAb4D5 V.sub.H and V.sub.L
genes were isolated by polymerase chain reaction (PCR)
amplification of mRNA from the corresponding hybridoma (Fendly, B.
M. et al., Cancer Res. 50:1550-1558 (1990)) as described by Orlandi
et al. (Orlandi, R. et al., Proc. Natl. Acad. Sci. USA 86:3833-3837
(1989)). Amino terminal sequencing of muMAb4D5 V.sub.L and V.sub.H
was used to design the sense strand PCR primers, whereas the
anti-sense PCR primers were based upon consensus sequences of
murine framework residues (Orlandi, R. et al., Proc. Natl. Acad.
Sci. USA 86:3833-3837 (1989); Kabat, E. A. et al., Sequences of
Proteins of Immunological Interest (National Institutes of Health,
Bethesda, Md., 1987)) incorporating restriction sites for
directional cloning shown by underlining and listed after the
sequences: V.sub.L sense, 5'-TCCGATATCCAGCTGACCCAGTCTCCA-3' (SEQ.
ID NO. 7), EcoRV; V.sub.L anti-sense,
5'-GTTTGATCTCCAGCTTGGTACCHSCDCCGAA-3' (SEQ. ID NO. 8), Asp718;
V.sub.H sense, 5'-AGGTSMARCTGCAGSAGTCWGG-3' (SEQ. ID NO. 9), PstI
and V.sub.H anti-sense, 5'-TGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAG-3'
(SEQ. ID NO. 10), BstEII; where H=A or C or T, S=C or G, D=A or G
or T, M=A or C, R=A or G and W=A or T. The PCR products were cloned
into pUC119 (Vieira, J. & Messing, J., Methods Enzymol.
153:3-11 (1987)) and five clones for each variable domain sequenced
by the dideoxy method (Sanger, F. et al., Proc. Natl. Acad. Sci.
USA 74:5463-5467 (1977)).
Molecular Modelling. Models for muMAb4D5 V.sub.H and V.sub.L
domains were constructed separately from consensus coordinates
based upon seven Fab structures from the Brookhaven protein data
bank (entries 1FB4, 2RHE, 2MCP, 3FAB, 1FBJ, 2HFL and 1REI). The Fab
fragment KOL (Marquart, M. et al., J. Mol. Biol. 141:369-391
(1980)) was first chosen as a template for V.sub.L and V.sub.H
domains and additional structures were then superimposed upon this
structure using their main chain atom coordinates (INSIGHT program,
Biosym Technologies). The distance from the template C.alpha. to
the analogous C.alpha. in each of the superimposed structures was
calculated for each residue position. If all (or nearly all)
C.alpha.--C.alpha. distances for a given residue were .ltoreq.1
.ANG., then that position was included in the consensus structure.
In most cases the .beta.-sheet framework residues satisfied these
criteria whereas the CDR loops did not. For each of these selected
residues the average coordinates for individual N, C.alpha., C, O
and C.beta. atoms were calculated and then corrected for resultant
deviations from non-standard bond geometry by 50 cycles of energy
minimization using the DISCOVER program (Biosym Technologies) with
the AMBER forcefield (Weiner, S. J. et al., J. Amer. Chem. Soc.
106:765-784 (1984)) and C.alpha. coordinates fixed. The side chains
of highly conserved residues, such as the disulfide-bridged
cysteine residues, were then incorporated into the resultant
consensus structure. Next the sequences of muMAb4D5 V.sub.L and
V.sub.H were incorporated starting with the CDR residues and using
the tabulations of CDR conformations from Chothia et al. (Chothia,
C. et al., Nature 342:877-883 (1989)) as a guide. Side-chain
conformations were chosen on the basis of Fab crystal structures,
rotamer libraries (Ponder, J. W. & Richards, F. M., J. Mol.
Biol. 193:775-791 (1987)) and packing considerations. Since V.sub.H
-CDR3 could not be assigned a definite backbone conformation from
these criteria, two models were created from a search of similar
sized loops using the INSIGHT program. A third model was derived
using packing and solvent exposure considerations. Each model was
then subjected to 5000 cycles of energy minimization.
In humanizing muMAb4D5, consensus human sequences were first
derived from the most abundant subclasses in the sequence
compilation of Kabat et al. (Kabat, E. A. et al., Sequences of
Proteins of Immunological Interest (National Institutes of Health,
Bethesda, Md., 1987)), namely V.sub.L .kappa. subgroup I and
V.sub.H group III, and a molecular model generated for these
sequences using the methods described above. A structure for
huMAb4D5 was created by transferring the CDRs from the muMAb4D5
model into the consensus human structure. All huMAb4D5 variants
contain human replacements of muMAb4D5 residues at three positions
within CDRs as defined by sequence variability (Kabat, E. A. et
al., Sequences of Proteins of Immunological Interest (National
Institutes of Health, Bethesda, Md., 1987)) but not as defined by
structural variability (Chothia, C. & Lesk, A. M., J. Mol Biol.
196:901-917 (1987)): V.sub.L -CDR1 K24R, V.sub.L -CDR2 R54L and
V.sub.L -CDR2 T56S. Differences between muMAb4D5 and the human
consensus framework residues (FIG. 1) were individually modeled to
investigate their possible influence on CDR conformation and/or
binding to the p185.sup.HER2 ECD.
Construction of Chimeric Genes. Genes encoding chMAb4D5 light and
heavy chains were separately assembled in previously described
phagemid vectors containing the human cytomegalovirus enhancer and
promoter, a 5' intron and SV40 polyadenylation signal (Gorman, C.
M. et al., DNA & Prot. Engin. Tech. 2:3-10 (1990)). Briefly,
gene segments encoding muMAb4D5 V.sub.L (FIG. 1A) and REI human
.kappa..sub.1 light chain C.sub.L (Palm, W. & Hilschmann, N.,
Z. Physiol. Chem. 356:167-191 (1975)) were precisely joined as were
genes for muMAb4D5 V.sub.H (FIG. 1B) and human .gamma.1 constant
region (Capon, D. J. et al., Nature 337:525-531 (1989)) by simple
subcloning (Boyle, A., in Current Protocols in Molecular Biology,
Chapter 3 (F. A. Ausubel et al., eds., Greene Publishing &
Wiley-Interscience, New York, 1990)) and site-directed mutagenesis
(Carter, P., in Mutagenesis: A Practical Approach, Chapter 1 (IRL
Press, Oxford, UK 1991)). The .gamma.1 isotype was chosen as it has
been found to be the preferred human isotype for supporting ADCC
and complement dependent cytotoxicity using matched sets of
chimeric (Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987))
or humanized antibodies (Riechmann, L. et at., Nature 332:323-327
(1988)). The PCR-generated V.sub.L and V.sub.H fragments (FIG. 1)
were subsequently mutagenized so that they faithfully represent the
sequence of muMAb4D5 determined at the protein level: V.sub.H Q1E,
V.sub.L V104L and T109A (variants are denoted by the amino acid
residue and number followed by the replacement amino acid). The
human .gamma.1 constant regions are identical to those reported by
Ellison et al. (Ellison, J. W. et al., Nucleic Acids Res.
13:4071-4079 (1982)) except for the mutations E359D and M361 L (Eu
numbering, as in Kabat, E. A. et al., Sequences of Proteins of
Immunological Interest (National Institutes of Health, Bethesda,
Md., 1987)) which we installed to convert the antibody from the
naturally rare A allotype to the much more common non-A allotype
(Tramontano, A. et a., J. Mol. Biol. 215:175-182 (1990)). This was
an attempt to reduce the risk of anti-allotype antibodies
interfering with therapy.
Construction of Humanized Genes. Genes encoding chMAb4D5 light
chain and heavy chain Fd fragment (V.sub.H and C.sub.H 1 domains)
were subcloned together into pUC119 (Vieira, J. & Messing, J.,
Methods Enzymol. 153:3-11 (1987)) to create pAK1 and simultaneously
humanized in a single step (FIG. 2). Briefly, sets of 6 contiguous
oligonucleotides were designed to humanize V.sub.H and V.sub.L
(FIG. 1). These oligonucleotides are 28 to 83 nucleotides in
length, contain zero to 19 mismatches to the murine antibody
template and are constrained to have 8 or 9 perfectly matched
residues at each end to promote efficient annealing and ligation of
adjacent oligonucleotides. The sets of V.sub.H and V.sub.L
humanization oligonucleotides (5 pmol each) were phosphorylated
with either ATP or .gamma.-.sup.32 P-ATP (Carter, P. Methods
Enzymol. 154:382-403 (1987)) and separately annealed with 3.7 pmol
of pAK1 template in 40 .mu.l 10 mM Tris-HCl (pH 8.0) and 10 mM
MgCl.sub.2 by cooling from 100 .degree. C. to room temperature over
.about.30 min. The annealed oligonucleotides were joined by
incubation with T4 DNA ligase (12 units; New England Biolabs) in
the presence of 2 .mu.l 5 mM ATP and 2 .mu.l 0.1 M DTT for 10 min
at 14.degree. C. After electrophoresis on a 6% acrylamide
sequencing gel the assembled oligonucleotides were located by
autoradiography and recovered by electroelution. The assembled
oligonucleotides (.about.0.3 pmol each) were simultaneously
annealed to 0.15 pmol single-stranded deoxyuridine-containing pAK1
prepared according to Kunkel et al. (Kunkel, T. A. et al., Methods
Enzymol. 154:367-382 (1987)) in 10 .mu.l 40 mM Tris-HCl (pH 7.5)
and 16 mM MgCl.sub.2 as above. Heteroduplex DNA was constructed by
extending the primers with T7 DNA polymerase and transformed into
E. coli BMH 71-18 mutL as previously described (Carter, P., in
Mutagenesis: A Practical Approach, Chapter 1 (IRL Press, Oxford, UK
1991)). The resultant phagemid DNA pool was enriched first for
huV.sub.L by restriction purification using XhoI and then for
huV.sub.H by restriction selection using StuI as described in
Carter, P., in Mutagenesis: A Practical Approach, Chapter 1 (IRL
Press, Oxford, UK 1991); and in Wells, J. A. et al., Phil. Trans.
R. Soc. Lond. A 317:415-423 (1986). Resultant clones containing
both huV.sub.L and huV.sub.H genes were identified by nucleotide
sequencing (Sanger, F. et al., Proc. Natl. Acad. Sci. USA
74:5463-5467 (1977)) and designated pAK2. Additional humanized
variants were generated by site-directed mutagenesis (Carter, P.,
in Mutagenesis: A Practical Approach, Chapter 1 (IRL Press, Oxford,
UK 1991)). The muMAb4D5 V.sub.L and V.sub.H gene segments in the
transient expression vectors described above were then precisely
replaced with their humanized versions.
Expression and Purification of MAb4D5 Variants. Appropriate MAb4D5
light and heavy chain cDNA expression vectors were co-transfected
into an adenovirus transformed human embryonic kidney cell line,
293 (Graham, F. L. et al., J. Gen. Virol. 36:59-72 (1977)) using a
high efficiency procedure (Gorman, C. M. et al., DNA & Prot.
Engin. Tech. 2:3-10 (1990); Gorman, C., in DNA Cloning, vol II, pp
143-190 (D. M. Glover, ed., IRL Press, Oxford, UK 1985)). Media
were harvested daily for up to 5 days and the cells re-fed with
serum free media. Antibodies were recovered from the media and
affinity purified on protein A sepharose CL-4B (Pharmacia) as
described by the manufacturer. The eluted antibody was
buffer-exchanged into phosphate-buffered saline by G25 gel
filtration, concentrated by ultrafiltration (Centriprep-30 or
Centricon-100, Amicon),sterile-filtered (Millex-GV, Millipore) and
stored at 4.degree. C. The concentration of antibody was determined
by using both total immunoglobulin and antigen binding ELISAs. The
standard used was huMAb4D5-5, whose concentration had been
determined by amino acid composition analysis.
Cell Proliferation Assay. The effect of MAb4D5 variants upon
proliferation of the human mammary adenocarcinoma cell line,
SK-BR-3, was investigated as previously described (Fendly, B. M. et
al, Cancer Res. 50:1550-1558 (1990)) using saturating MAb4D5
concentrations.
Affinity Measurements. The antigen binding affinity of MAb4D5
variants was determined using a secreted form of the p185.sup.HER2
ECD prepared as described in Fendly, B. M. et al., J. Biol Resp.
Mod. 9:449-455 (1990). Briefly, antibody and p185.sup.HER2 ECD were
incubated in solution until equilibrium was found to be reached.
The concentration of free antibody was then determined by ELISA
using immobilized p185.sup.HER2 ECD and used to calculate affinity
(K.sub.d) according to Friguet et al. (Friguet, B. et al., J.
Immunol Methods 77:305-319 (1985)).
Results
Humanization of muMAb4D5. The muMAb4D5 V.sub.L and V.sub.H gene
segments were first cloned by PCR and sequenced (FIG. 1). The
variable genes were then simultaneously humanized by gene
conversion mutagenesis using preassembled oligonucleotides (FIG.
2). A 311-mer oligonucleotide containing 39 mismatches to the
template directed 24 simultaneous amino acid changes required to
humanize muMAb4D5 V.sub.L. Humanization of muMAb4D5 V.sub.H
required 32 amino acid changes which were installed with a 361-mer
containing 59 mismatches to the muMAb4D5 template. Two out of 8
clones sequenced precisely encode huMAb4D5-5, although one of these
clones contained a single nucleotide imperfection. The 6 other
clones were essentially humanized but contained a small number of
errors: <3 nucleotide changes and <1 single nucleotide
deletion per kilobase. Additional humanized variants (Table 3) were
constructed by site-directed mutagenesis of huMAb4D5-5.
Expression levels of huMAb4D5 variants were in the range of 7 to 15
.mu.g/ml as judged by ELISA using immobilized p185.sup.HER2 ECD.
Successive harvests of five 10 cm plates allowed 200 .mu.g to 500
mg of each variant to be produced in a week. Antibodies affinity
purified on protein A gave a single band on a Coomassie blue
stained SDS polyacrylamide gel of mobility consistent with the
expected M.sub.r of .about.150 kDa. Electrophoresis under reducing
conditions gave 2 bands consistent with the expected M.sub.r of
free heavy (48 kDa) and light (23 kDa) chains (not shown). Amino
terminal sequence analysis (10-cycles) gave the mixed sequence
expected (see FIG. 1) from an equimolar combination of light and
heavy chains (not shown). huMAb4D5 Variants. In general, the FR
residues were chosen from consensus human sequences (Kabat, E. A.
et al., Sequences of Proteins of Immunological Interest (National
Institutes of Health, Bethesda, Md., 1987)) and CDR residues from
muMAb4D5. Additional variants were constructed by replacing
selected human residues in huMAb4D5-1 with their muMAb4D5
counterparts. These are V.sub.H residues 71, 73, 78, 93 plus 102
and V.sub.L residues 55 plus 66 identified by our molecular
modeling. V.sub.H residue 71 has previously been proposed by others
(Tramontano, A. et al., J. Mol. Biol. 215:175-182 (1990)) to be
critical to the conformation of V.sub.H -CDR2. Amino acid sequence
differences between huMAb4D5 variant molecules are shown in Table
3, together with their p185.sup.HER2 ECD binding affinity and
maximal anti-proliferative activities against SK-BR-3 cells. Very
similar K.sub.d values were obtained for binding of MAb4D5 variants
to either SK-BR-3 cells or to p185.sup.HER2 ECD (Table 3). However,
K.sub.d estimates derived from binding of MAb4D5 variants to
p185.sup.HER2 ECD were more reproducible with smaller standard
errors and consumed much smaller quantities of antibody than
binding measurements with whole cells.
The most potent humanized variant designed by molecular modeling,
huMAb4D5-8, contains 5 FR residues from muMAb4D5. This antibody
binds the p185.sup.HER2 ECD 3-fold more tightly than does muMAb4D5
itself (Table 3) and has comparable anti-proliferative activity
with SK-BR-3 cells (FIG. 3). In contrast, huMAb4D5-1 is the most
humanized but least potent muMAb4D5 variant, created by simply
installing the muMAb4D5 CDRs into the consensus human sequences.
huMAb4D5-1 binds the p185.sup.HER2 ECD 80-fold less tightly than
does the murine antibody and has no detectable anti-proliferative
activity at the highest antibody concentration investigated (16
.mu.g/ml).
The anti-proliferative activity of huMAb4D5 variants against
p185.sup.HER2 overexpressing SK-BR-3 cells is not simply correlated
with their binding affinity for the p185.sup.HER2 ECD. For example,
installation of three murine residues into the V.sub.H domain of
huMAb4D5-2 (D73T, L78A and A93S) to create huMAb4D5-3 does not
change the antigen binding affinity but does confer significant
anti-proliferative activity (Table 3).
The importance of V.sub.H residue 71 (Tramontano, A. et al., J.
Mol. Biol. 215:175-182 (1990)) is supported by the observed 5-fold
increase in affinity for p185.sup.HER2 ECD on replacement of R71 in
huMAb4D5-1 with the corresponding murine residue, alanine
(huMAb4D5-2). In contrast, replacing V.sub.H L78 in huMAb4D5-4 with
the murine residue, alanine (huMAb4D5-5), does not significantly
change the affinity for the p185.sup.HER2 ECD or change
anti-proliferative activity, suggesting that residue 78 is not of
critical functional significance to huMAb4D5 and its ability to
interact properly with the extracellular domain of
p185.sup.HER2.
V.sub.L residue 66 is usually a glycine in human and murine .kappa.
chain sequences (Kabat, E. A. et al., Sequences of Proteins of
Immunological Interest (National Institutes of Health, Bethesda,
Md., 1987)) but an arginine occupies this position in the muMAb4D5
k light chain. The side chain of residue 66 is likely to affect the
conformation of V.sub.L -CDR1 and V.sub.L -CDR2 and the hairpin
turn at 68-69 (FIG. 4). Consistent with the importance of this
residue, the mutation V.sub.L G66R (huMAb4D5-3.fwdarw.huMAb4D5-5)
increases the affinity for the p185.sup.HER2 ECD by 4-fold with a
concomitant increase in anti-proliferative activity.
From molecular modeling it appears that the tyrosyl side chain of
muMAb4D5 V.sub.L residue 55 may either stabilize the conformation
of V.sub.H -CDR3 or provide an interaction at the V.sub.L -V.sub.H
interface. The latter function may be dependent upon the presence
of V.sub.H Y102. In the context of huMAb4D5-5 the mutations V.sub.L
E55Y (huMAb4D5-6) and V.sub.H V102Y (huMAb4D5-7) individually
increase the affinity for p185.sup.HER2 ECD by 5-fold and 2-fold
respectively, whereas together (huMAb4D5-8) they increase the
affinity by 11-fold. This is consistent with either proposed role
of V.sub.L Y55 and V.sub.H Y102.
Secondary Immune Function of huMAb4D5-8. MuMAb4D5 inhibits the
growth of human breast tumor cells which overexpress p185.sup.HER2
(Hudziak, R. M. et al., Molec. Cell. Biol. 9:1165-1172 (1989)). The
antibody, however, does not offer the possibility of direct tumor
cytotoxic effects. This possibility does arise in huMAb4D5-8 as a
result of its high affinity (K.sub.d =0.1 .mu.M) and its human IgG1
subtype. Table 4 compares the ADCC mediated by huMAb4D5-8 with
muMAb4D5 on a normal lung epithelial cell line, WI-38, which
expresses a low level of p185.sup.HER2 and on SK-BR-3, which
expresses a high level of p185.sup.HER2. The results demonstrate
that: (1) huMAb4D5 has a greatly enhanced ability to carry out ADCC
as compared with its murine parent; and (2) that this activity may
be selective for cell types which overexpress p185.sup.HER2.
Discussion
MuMAb4D5 is potentially useful for human therapy since it is
cytostatic towards human breast and ovarian tumor lines
overexpressing the HER2-encoded p185.sup.HER2 receptor-like
tyrosine kinase. Since both breast and ovarian carcinomas are
chronic diseases it is anticipated that the optimal MAb4D5 variant
molecule for therapy will have low immunogenicity and will be
cytotoxic rather than solely cytostatic in effect. Humanization of
muMAb4D5 should accomplish these goals. We have identified 5
different huMAb4D5 variants which bind tightly to p185.sup.HER2 ECD
(K.sub.d .ltoreq.1 nM) and which have significant
anti-proliferative activity (Table 3). Furthermore huMAb4D5-8 but
not muMAb4D5 mediates ADCC against human tumor cell lines
overexpressing p185.sup.HER2 in the presence of human effector
cells (Table 4) as anticipated for a human .gamma.1 isotype
(Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987);
Riechmann, L. et al., Nature 332:323-327 (1988)).
Rapid humanization of huMAb4D5 was facilitated by the gene
conversion mutagenesis strategy developed here using long
preassembled oligonucleotides. This method requires less than half
the amount of synthetic DNA as does total gene synthesis and does
not require convenient restriction sites in the target DNA. Our
method appears to be simpler and more reliable than a variant
protocol recently reported (Rostapshov, V. M. et al., FEBS Lett.
249:379-382 (1989)). Transient expression of huMAb4D5 in human
embryonic kidney 293 cells permitted the isolation of a few hundred
micrograms of huMAb4D5 variants for rapid characterization by
growth inhibition and antigen binding affinity assays. Furthermore,
different combinations of light and heavy chain were readily tested
by co-transfection of corresponding cDNA expression vectors.
The crucial role of molecular modeling in the humanization of
muMAb4D5 is, illustrated by the designed variant huMAb4D5-8 which
binds the p185.sup.HER2 ECD 250-fold more tightly than the simple
CDR loop swap variant, huMAb4D5-1. It has previously been shown
that the antigen binding affinity of a humanized antibody can be
increased by mutagenesis based upon molecular modelling (Riechmann,
L. et al., Nature 332:323-327 (1988); Queen, C. et al., Proc. Natl.
Acad. Sci. USA 86:10029-10033 (1989)). Here we have extended this
earlier work by others with a designed humanized antibody which
binds its antigen 3-fold more tightly than the parent rodent
antibody. While this result is gratifying, assessment of the
success of the molecular modeling must await the outcome of X-ray
structure determination. From analysis of huMAb4D5 variants (Table
3) it is apparent that their anti-proliferative activity is not a
simple function of their binding affinity for p185.sup.HER2 ECD.
For example the huMAb4D5-8 variant binds p185.sup.HER2 3-fold more
tightly than muMAb4D5 but the humanized variant is slightly less
potent in blocking the proliferation of SK-BR-3 cells. Additional
huMAb4D5 variants are currently being constructed in an attempt to
identify residues triggering the anti-proliferative activity and in
an attempt to enhance this activity.
In addition to retaining tight receptor binding and the ability to
inhibit cell growth, the huMAb4D5-8 also confers a secondary immune
function (ADCC). This allows for direct cytotoxic activity of the
humanized molecule in the presence of human effector cells. The
apparent selectivity of the cytotoxic activity for cell types which
overexpress p185.sup.HER2 allows for the evolution of a
straightforward clinic approach to those human cancers
characterized by overexpression of the HER2 protooncogene.
TABLE 3
__________________________________________________________________________
p185.sup.HER2 ECD binding affinity and anti-proliferative
activities of MAb4D5 variants V.sub.H Residue* V.sub.L Residue*
MAb4D5 71 73 78 93 102 55 66 K.sub.d.sup..dagger. Relative cell
Variant FR3 FR3 FR3 FR3 CDR3 CDR2 FR3 nM
proliferation.sup..dagger-dbl.
__________________________________________________________________________
huMAb4D5-1 R D L A V E G 25 102 huMAb4D5-2 Ala D L A V E G 4.7 101
huMAb4D5-3 Ala Thr Ala Ser V E G 4.4 66 huMAb4D5-4 Ala Thr L Ser V
E Arg 0.82 56 huMAb4D5-5 Ala Thr Ala Ser V E Arg 1.1 48 huMAb4D5-6
Ala Thr Ala Ser V Tyr Arg 0.22 51 huMAb4D5-7 Ala Thr Ala Ser Tyr E
Arg 0.62 53 huMAb4D5-8 Ala Thr Ala Ser Tyr Tyr Arg 0.10 54 muMAb4D5
Ala Thr Ala Ser Tyr Tyr Arg 0.30 37
__________________________________________________________________________
*Human and murine residues are shown in one letter and three letter
amino acid code respectively. .sup..dagger. K.sub.d values for the
p185.sup.HER2 ECD were determined using the method of Friguet et
al. (43) and the standard error of each estimate is .ltoreq.
.+-.10%. .sup..dagger-dbl. Proliferation of SKBR-3 cells incubated
for 96 hr with MAb4D5 variants shown as a percentage of the
untreated control as described (Hudziak, R. M. et al., Molec. Cell.
Biol. 9:1165-1172(1989)). Data represent the maximal
antiproliferative effect for each variant # (see FIG. 3A)
calculated as the mean of triplicate determinations at a MAb4D5
concentration of 8 .mu.g/ml. Data are all taken from the same
experiment with an estimated standard error of .ltoreq.
.+-.15%.
TABLE 4
__________________________________________________________________________
Selectivity of antibody dependent tumor cell cytotoxicity mediated
by huMAb4D5-8 Effector:Target WI-38* SK-BR-3 ratio.dagger. muMAb4D5
huMAb4D5-8 muMAb4D5 huMAb4D5-8
__________________________________________________________________________
A..dagger-dbl. 25:1 <1.0 9.3 7.5 40.6 12.5:1 <1.0 11.1 4.7
36.8 6.25:1 <1.0 8.9 0.9 35.2 3.13:1 <1.0 8.5 4.6 19.6 B.
25:1 <1.0 3.1 6.1 33.4 12.5:1 <1.0 1.7 5.5 26.2 6.25:1 1.3
2.2 2.0 21.0 3.13:1 <1.0 0.8 2.4 13.4
__________________________________________________________________________
*Sensitivity to ADCC of two human cell lines (WI38, normal lung
epithelium; and SKBR-3, human breast tumor cell line) are compared.
WI38 expresses a low level of p185.sup.HER2 (0.6 pg per .mu.g cell
protein) and SKBR-3 expresses a high level of p185.sup.HER2 (64 pg
p185.sup.HER2 per .mu.g cell protein), as determined by ELISA
(Fendly et al., J. Biol. Resp. Mod. 9:449455 (1990)). .dagger.ADCC
assays were carried out as described in Bruggemann et al., J Exp.
Med. 166:13511361 (1987). Effector to target ratios were of IL2
activated human peripheral blood lymphocytes to either WI38
fibroblasts o SKBR-3 tumor cells in 96well microtiter plates for 4
hours at 37.degree. C. Values given represent percent specific cell
lysis as determined by .sup.51 Cr release. Estimated standard error
in these quadruplicate determinations was .ltoreq..+-.10%.
.dagger-dbl.Monoclonal antibody concentrations used were 0.1
.mu.g/ml (A) and 0.1 .mu.g/ml (B).
Example 2
Schematic Method for Humanizing an Antibody Sequence
This example illustrates one stepwise elaboration of the methods
for creating a humanized sequence described above. It will be
understood that not all of these steps are essential to the claimed
invention, and that steps may be taken in different order.
1. ascertain a consensus human variable domain amino acid sequence
and prepare from it a consensus structural model.
2. prepare model of import (the non-human domain to be humanized)
variable domain sequences and note structural differences with
respect to consensus human model.
3. identify CDR sequences in human and in import, both by using
Kabat (supra, 1987) and crystal structure criteria. If there is any
difference in CDR identity from the different criteria, use of
crystal structure definition of the CDR, but retain the Kabat
residues as important framework residues to import.
4. substitute import CDR sequences for human CDR sequences to
obtain initial "humanized" sequence.
5. compare import non-CDR variable domain sequence to the humanized
sequence and note divergences.
6. Proceed through the following analysis for each amino acid
residue where the import diverges from the humanized.
a. If the humanized residue represents a residue which is generally
highly conserved across all species, use the residue in the
humanized sequence. If the residue is not conserved across all
species, proceed with the analysis described in 6b.
b. If the residue is not generally conserved across all species,
ask if the residue is generally conserved in humans.
i. If the residue is generally conserved in humans but the import
residue differs, examine the structural models of the import and
human sequences and determine if the import residue would be likely
to affect the binding or biological activity of the CDRs by
considering 1) could it bind antigen directly and 2) could it
affect the conformation of the CDR. If the conclusion is that an
affect on the CDRs is likely, substitute the import residue. If the
conclusion is that a CDR affect is unlikely, leave the humanized
residue unchanged.
ii. If the residue is also not generally conserved in humans,
examine the structural models of the import and human sequences and
determine if the import residue would be likely to affect the
binding or biological activity of the CDRs be considering 1) could
it bind antigen directly and 2) could it affect the conformation of
the CDR. If the conclusion is that an affect on the CDRs is likely,
substitute the import residue. If the conclusion is that a CDR
affect is unlikely, proceed to the next step.
a) Examine the structural models of the import and human sequences
and determine if the residue is exposed on the surface of the
domain or is buried within. If the residue is exposed, use the
residue in the humanized sequence. If the residue is buried,
proceed to the next step.
(i) Examine the structural models of the import and human sequences
and determine if the residue is likely to affect the V.sub.L
-V.sub.H interface. Residues involved with the interface include:
34L, 36L, 38L, 43L, 33L, 36L, 85L, 87L, 89L, 91L, 96L, 98L, 35H,
37H, 39H, 43H, 45H, 47H, 60H, 91H, 93H, 95H, 100H, and 103H. If no
effect is likely, use the residue in the humanized sequence. If
some affect is likely, substitute the import residue.
7. Search the import sequence, the consensus sequence and the
humanized sequence for glycosylation sites outside the CDRs, and
determine if this glycosylation site is likely to have any affect
on antigen binding and/or biological activity. If no effect is
likely, use the human sequence at that site; if some affect is
likely, eliminate the glycosylation site or use the import sequence
at that site.
8. After completing the above analysis, determine the planned
humanized sequence and prepare and test a sample. If the sample
does not bind well to the target antigen, examine the particular
residues listed below, regardless of the question of residue
identity between the import and humanized residues.
a. Examine particular peripheral (non-CDR) variable domain residues
that may, due to their position, possibly interact directly with a
macromolecular antigen, including the following residues (where the
* indicates residues which have been found to interact with antigen
based on crystal structures):
i. Variable light domain: 36, 46, 49*, 63-70
ii. Variable heavy domain: 2, 47*, 68, 70, 73-76.
b. Examine particular variable domain residues which could interact
with, or otherwise affect, the conformation of variable domain
CDRs, including the following (not including CDR residues
themselves, since it is assumed that, because the CDRs interact
with one another, any residue in one CDR could potentially affect
the conformation of another CDR residue) (L=LIGHT, H=HEAVY,
residues appearing in bold are indicated to be structurally
important according the Chothia et al., Nature 342:877 (1989), and
residues appearing in italic were altered during humanization by
Queen et al. (PDL), Proc. Natl. Acad. Sci. U.S.A. 86:10029 (1989)
and Proc. Natl. Acad. Sci. U.S.A. 88:2869 (1991).):
i. Variable light domain:
a) CDR-1 (residues 24L-34L): 2L, 4L, 66L-69L, 71L
b) CDR-2 (residues 50L-56L): 35L, 46L, 47L, 48L, 49L, 58L, 62L,
64L-66L, 71 L, 73L
c) CDR-3 (residues 89L-97L): 2L, 4L, 36L, 98L, 37H, 45H, 47H, 58H,
60H
ii. Variable heavy domain:
a) CDR-1 (residues 26H-35H): 2H, 4H, 24H, 36H, 71H, 73H, 76H, 78H,
92H, 94H
b) CDR-2 (residues 50H-55H): 49H, 69H, 69H, 71H, 73H, 78H
c) CDR-3 (residues 95H-102H): examine all residues as possible
interaction partners with this loop, because this loop varies in
size and conformation much more than the other CDRs.
9. If after step 8 the humanized variable domain still is lacking
in desired binding, repeat step 8. In addition, re-investigate any
buried residues which might affect the V.sub.L -V.sub.H interface
(but which would not directly affect CDR conformation).
Additionally, evaluate the accessibility of non-CDR residues to
solvent.
Example 3
Engineering a Humanized Bispecific F(ab').sub.2 Fragment
This example demonstrates the construction of a humanized
bispecific antibody (BsF(ab').sub.2 v1 by separate E. coli
expression of each Fab' arm followed by directed chemical coupling
in vitro. BsF(ab').sub.2 v1 (anti-CD3/anti-p185.sup.HER2) was
demonstrated to retarget the cytotoxic activity of human CD3.sup.+
CTL in vitro against the human breast tumor cell line, SK-BR-3,
which overexpresses the p185.sup.HER2 product of the protooncogene
HER2. This example demonstrates the minimalistic humanization
strategy of installing as few murine residues as possible into a
human antibody in order to recruit antigen-binding affinity and
biological properties comparable to that of the murine parent
antibody. This strategy proved very successful for the
anti-p185.sup.HER2 arm of BsF(ab').sub.2 v1. In contrast
BsF(ab').sub.2 v1 binds to T cells via its anti-CD3 arm much less
efficiently than does the chimeric BsF(ab').sub.2 which contains
the variable domains of the murine parent anti-CD3 antibody. Here
we have constructed additional BsF(ab').sub.2 fragments containing
variant anti-CD3 arms with selected murine residues restored in an
attempt to improve antibody binding to T cells. One such variant,
BsF(ab').sub.2 v9, was created by replacing six residues in the
second hypervariable loop of the anti-CD3 heavy chain variable
domain of BsF(ab').sub.2 v1 with their counterparts from the murine
parent anti-CD3 antibody. BsF(ab').sub.2 v9 binds to T cells
(Jurkat) much more efficiently than does BsF(ab').sub.2 v1 and
almost as efficiently as the chimeric BsF(ab').sub.2. This
improvement in the efficiency of T cell binding of the humanized
BsF(ab').sub.2 is an important step in its development as a
potential therapeutic agent for the treatment of p185.sup.HER2
-overexpressing cancers.
Bispecific antibodies (BsAbs) with specificities for
tumor-associated antigens and surface markers on immune effector
cells have proved effective for retargeting effector cells to kill
tumor targets both in vitro and in vivo (reviewed by Fanger, M. W.
et al., Immunol. Today 10: 92-99 (1989); Fanger, M. W. et al.,
Immunol. Today 12: 51-54 (1991); and Nelson, H., Cancer Cells 3:
163-172 (1991)). BsF(ab').sub.2 fragments have often been used in
preference to intact BsAbs in retargeted cellular cytotoxicity to
avoid the risk of killing innocent bystander cells binding to the
Fc region of the antibody. An additional advantage of
BsF(ab').sub.2 over intact BsAbs is that they are generally much
simpler to prepare free of contaminating monospecific molecules
(reviewed by Songsivilai, S. and Lachmann, P. J., Clin. Exp.
Immunol. 79: 315-321 (1990) and Nolan, O. and O'Kennedy, R.,
Biochim. Biophys. Acta 1040: 1-11 (1990)).
BsF(ab').sub.2 fragments are traditionally constructed by directed
chemical coupling of Fab' fragments obtained by limited proteolysis
plus mild reduction of the parent rodent monoclonal Ab (Brennan, M.
et al., Science 229, 81-83 (1985) and Glennie, M. J. et al., J.
Immunol. 139: 2367-2375 (1987)). One such BsF(ab').sub.2 fragment
(anti-glioma associated antigen/anti-CD3) was found to have
clinical efficacy in glioma patients (Nitta, T. et al., Lancet 335:
368-371 (1990) and another BsF(ab').sub.2 (anti-indium
chelate/anti-carcinoembryonic antigen) allowed clinical imaging of
colorectal carcinoma (Stickney, D. R. et al., Antibody, Immunoconj.
Radiopharm. 2: 1-13 (1989)). Future BsF(ab').sub.2 destined for
clinical applications are likely to be constructed from antibodies
which are either human or at least "humanized" (Riechmann, L. et
al., Nature 332: 323-327 (1988) to reduce their immunogenicity
(Hale, G. et al., Lancet i: 1394-1399 (1988)).
Recently a facile route to a fully humanized BsF(ab').sub.2
fragment designed for tumor immunotherapy has been demonstrated
(Shalaby, M. R. et al., J. Exp. Med. 175: 217-225 (1992)). This
approach involves separate E. coli expression of each Fab' arm
followed by traditional directed chemical coupling in vitro to form
the BsF(ab').sub.2. One arm of the BsF(ab').sub.2 was a humanized
version (Carter, P. et al., Proc. Natl. Acad. Sci. USA (1992a) and
Carter, P., et al., Bio/Technology 10: 163-167 (1992b)) of the
murine monoclonal Ab 4D5 which is directed against the
p185.sup.HER2 product of the protooncogene HER2 (c-erbB-2) (Fendly,
B. M. et al. Cancer Res. 50: 1550-1558 (1989)). The humanization of
the antibody 4D5 is shown in Example 1 of this application. The
second arm was a minimalistically humanized anti-CD3 antibody
(Shalaby et al. supra) which was created by installing the CDR
loops from the variable domains of the murine parent monoclonal Ab
UCHT1 (Beverley, P. C. L. and Callard, R. E., Eur. J. Immunol. 11:
329-334 (1981)) into the humanized anti-p185.sup.HER2 antibody. The
BsF(ab').sub.2 fragment containing the most potent humanized
anti-CD3 variant (v1) was demonstrated by flow cytometry to bind
specifically to a tumor target overexpressing p185.sup.HER2 and to
human peripheral blood mononuclear cells carrying CD3. In addition,
BsF(ab')2 v1 enhanced the cytotoxic effects of activated human CTL
4-fold against SK-BR-3 tumor cells overexpressing p185.sup.HER2.
The example describes efforts to improve the antigen binding
affinity of the humanized anti-CD3 arm by the judicious recruitment
of a small number of additional murine residues into the
minimalistically humanized anti-CD3 variable domains.
Materials and Methods
Construction of Mutations in the Anti-CD3 Variable Region Genes
The construction of genes encoding humanized anti-CD3 variant 1
(v1) variable light (V.sub.L) and heavy (V.sub.H) chain domains in
phagemid pUC119 has been described (Shalaby et al. supra).
Additional anti-CD3 variants were generated using an efficient
site-directed mutagenesis method (Carter, P., Mutagenesis: a
practical approach, (M. J. McPherson, Ed.), Chapter 1, IRL Press,
Oxford, UK (1991)) using mismatched oligonucleotides which either
install or remove unique restriction sites. Oligonucleotides used
are listed below using lowercase to indicate the targeted
mutations. Corresponding coding changes are denoted by the starting
amino acid in one letter code followed by the residue numbered
according to Kabat, E. A. et al., Sequences of Proteins of
Immunological Interest, 5.sup.th edition, National Institutes of
Health, Bethesda, Md., U.S.A. (1991), then the replacement amino
acid and finally the identity of the anti-CD3 variant:
Oligonucleotides HX11, HX12 and HX13 each remove a site for BspMI,
whereas LX6 removes a site for XhoI and HX14 installs a site for
EcoRV (bold). Anti-CD3 variant v10 was constructed from v9 by
site-directed mutagenesis using oligonucleotide HX13. Mutants were
verified by dideoxynucleotide sequencing (Sanger, F. et al., Proc.
Natl. Acad. Sci. USA 74: 5463-5467 (1977)).
E. coli Expression of Fab' Fragments
The expression plasmid, pAK19, for the co-secretion of light chain
and heavy chain Fd' fragment of the most preferred humanized
anti-p185.sup.HER2 variant, HuMAb4D5-8, is described in Carter et
al., 1992b, supra. Briefly, the Fab' expression unit is bicistronic
with both chains under the transcriptional control of the phoA
promoter. Genes encoding humanized V.sub.L and V.sub.H domains are
precisely fused on their 5' side to a gene segment encoding the
heat-stable enterotoxin II signal sequence and on their 3' side to
human k.sub.1 C.sub.L and IgG1 C.sub.H 1 constant domain genes,
respectively. The C.sub.H 1 gene is immediately followed by a
sequence encoding the hinge sequence CysAlaAla and followed by a
bacteriophage .lambda. t.sub.o transcriptional terminator. Fab'
expression plasmids for chimeric and humanized anti-CD3 variants
(v1 to v4, Shalaby et al.,supra; v6 to v12, this study) were
created from pAK19 by precisely replacing anti-p185.sup.HER2
V.sub.L and V.sub.H gene segments with those encoding murine and
corresponding humanized variants of the anti-CD3 antibody,
respectively, by sub-cloning and site-directed mutagenesis. The
Fab' expression plasmid for the most potent humanized anti-CD3
variant identified in this study (v9) is designated pAK22. The
anti-p185.sup.HER2 Fab' fragment was secreted from E. coli K12
strain 25F2 containing plasmid pAK19 grown for 32 to 40 hr at
37.degree. C. in an aerated 10 liter fermentor. The final cell
density was 120-150 OD.sub.550 and the titer of soluble and
functional anti-p185.sup.HER2 Fab' was 1-2 g/liter as judged by
antigen binding ELISA (Carter et al., 1992b, supra). Anti-CD3 Fab'
variants were secreted from E. coli containing corresponding
expression plasmids using very similar fermentation protocols. The
highest expression titers of chimeric and humanized anti-CD3
variants were 200 mg/liter and 700 mg/liter, respectively, as
judged by total immunoglobulin ELISA.
Construction of BsF(ab').sub.2 Fragments
Fab' fragments were directly recovered from E. coli fermentation
pastes in the free thiol form (Fab'-SH) by affinity purification on
Streptococcal protein G at pH 5 in the presence of EDTA (Carter et
al, 1992b supra). Thioether linked BsF(ab').sub.2 fragments
(anti-p185.sup.HER2 /anti-CD3) were constructed by the procedure of
Glennie et al. supra with the following modifications.
Anti-p185.sup.HER2 Fab'-SH in 100 mM Tris acetate, 5 mM EDTA (pH
5.0) was reacted with 0.1 vol of 40 mM N,N'-1,2-phenylenedimalemide
(o-PDM) in dimethyl formamide for .about.1.5 hr at 20.degree. C.
Excess o-PDM was removed by protein G purification of the Fab'
maleimide derivative (Fab'-mal) followed by buffer exchange into 20
mM sodium acetate, 5 mM EDTA (pH 5.3) (coupling buffer) using
centriprep-30 concentrators (Amicon). The total concentration of
Fab' variants was estimated from the measured absorbance at 280 nm
(HuMAb4D5-8 Fab' e.sup.0.1% =1.56, Carter et al., 1992b, supra).
The free thiol content of Fab' preparations was estimated by
reaction with 5,5'-dithiobis(2-nitrobenzoic acid) as described by
Creighton, T. E., Protein structure: a practical approach, (T. E.
Creighton, Ed.), Chapter 7, IRL Press, Oxford, UK (1990). Equimolar
amounts of anti-p185.sup.HER2 Fab'-mal (assuming quantitative
reaction of Fab'-SH with o-PDM) and each anti-CD3 Fab'-SH variant
were coupled together at a combined concentration of 1 to 2.5 mg/ml
in the coupling buffer for 14 to 48 hr at 4.degree. C. The coupling
reaction was adjusted to 4 mM cysteine at pH 7.0 and incubated for
15 min at 20.degree. C. to reduce any unwanted disulfide-linked
F(ab').sub.2 formed. These reduction conditions are sufficient to
reduce inter-heavy chain disulfide bonds with virtually no
reduction of the disulfide between light and heavy chains. Any free
thiols generated were then blocked with 50 mM iodoacetamide.
BsF(ab').sub.2 was isolated from the coupling reaction by S100-HR
(Pharmacia) size exclusion chromatography (2.5 cm.times.100 cm) in
the presence of PBS. The BsF(ab').sub.2 samples were passed through
a 0.2 mm filter flash frozen in liquid nitrogen and stored at
-70.degree. C.
Flow Cytometric Analysis of F(ab').sub.2 Binding to Jurkat
Cells
The Jurkat human acute T cell leukemia cell line was purchased from
the American Type Culture Collection (10801 University Blvd.,
Manassas, Va.) (ATCC TIB 152) and grown as recommended by the ATCC.
Aliquots of 10.sup.6 Jurkat cells were incubated with appropriate
concentrations of BsF(ab').sub.2 (anti-p185.sup.HER2 /anti-CD3
variant) or control mono-specific anti-p185.sup.HER2 F(ab').sub.2
in PBS plus 0.1% (w/v) bovine serum albumin and 10 mM sodium azide
for 45 min at 4.degree. C. The cells were washed and then incubated
with fluorescein-conjugated goat anti-human F(ab').sub.2 (Organon
Teknika, West Chester, Pa.) for 45 min at 4.degree. C. Cells were
washed and analyzed on a FACScan (Becton Dickinson and Co.,
Mountain View, Calif.). Cells (8.times.10.sup.3) were acquired by
list mode and gated by forward light scatter versus side light
scatter excluding dead cells and debris.
Results
Design of Humanized Anti-CD3 Variants
The most potent humanized anti-CD3 variant previously identified,
v1, differs from the murine parent antibody, UCHT1 at 19 out of 107
amino acid residues within V.sub.L and at 37 out of 122 positions
within V.sub.H (Shalaby et al,supra) 1992). Here we recruited back
additional murine residues into anti-CD3 v1 in an attempt to
improve the binding affinity for CD3. The strategy chosen was a
compromise between minimizing both the number of additional murine
residues recruited and the number of anti-CD3 variants to be
analyzed. We focused our attentions on a few CDR residues which
were originally kept as human sequences in our minimalistic
humanization regime. Thus human residues in V.sub.H CDR2 of
anti-CD3 v1 were replaced en bloc with their murine counterparts to
give anti-CD3 v9: T57S:A60N:D61Q:S62K:V63F:G65D (SEQ ID NO: 20)
(FIG. 5). Similarly, the human residue E55 in V.sub.L CDR2 of
anti-CD3 v1 was replaced with histidine from the murine anti-CD3
antibody to generate anti-CD3 v11. In addition, V.sub.H framework
region (FR) residues 75 and 76 in anti-CD3 v1 were also replaced
with their murine counterparts to create anti-CD3 v8: K75S:N76S.
V.sub.H residues 75 and 76 are located in a loop close to V.sub.H
CDR1 and CDR2 and therefore might influence antigen binding.
Additional variants created by combining mutations at these three
sites are described below.
Preparation of BsF(ab').sub.2 Fragments
Soluble and functional anti-p185.sup.HER2 and anti-CD3 Fab'
fragments were recovered directly from corresponding E. coli
fermentation pastes with the single hinge cysteine predominantly in
the free thiol form (75-100% Fab'-SH) by affinity purification on
Streptococcal protein G at pH 5 in the presence of EDTA (Carter et
al., 1992b, supra). Thioether-linked BsF(ab').sub.2 fragments were
then constructed by directed coupling using o-PDM as described by
Glennie et al., supra. One arm was always the most potent humanized
anti-p185.sup.HER2 variant, HuMAb4D5-8 (Carter et al., 1992a,
supra) and the other either a chimeric or humanized variant of the
anti-CD3 antibody. Anti-p185.sup.HER2 Fab'-SH was reacted with
o-PDM to form the maleimide derivative (Fab'-mal) and then coupled
to the Fab'-SH for each anti-CD3 variant. F(ab').sub.2 was then
purified away from unreacted Fab' by size exclusion chromatography
as shown for a representative preparation (BsF(ab').sub.2 v8) in
data not shown. The F(ab').sub.2 fragment represents .about.54% of
the total amount of antibody fragments (by mass) as judged by
integration of the chromatograph peaks.
SDS-PAGE analysis of this BsF(ab').sub.2 v8 preparation under
non-reducing conditions gave one major band with the expected
mobility (M.sub.r .about.96 kD) as well as several very minor bands
(data not shown). Amino-terminal sequence analysis of the major
band after electroblotting on to polyvinylidene difluoride membrane
Matsudaira, P., J. Biol. Chem. 262:10035-10038 (1987) gave the
expected mixed sequence from a stoichiometric 1:1 mixture of light
and heavy chains (V.sub.L /V.sub.H : D/E, I/V, Q/Q, M/L, TN, Q/E,
S/S) expected for BsF(ab').sub.2. The amino terminal region of both
light chains are identical as are both heavy chains and correspond
to consensus human FR sequences. We have previously demonstrated
that F(ab').sub.2 constructed by directed chemical coupling carry
both anti-p185.sup.HER2 and anti-CD3 antigen specificities (Shalaby
et al., supra). The level of contamination of the BsF(ab').sub.2
with monospecific F(ab').sub.2 is likely to be very low since mock
coupling reactions with either anti-p185.sup.HER2 Fab'-mal or
anti-CD3 Fab'-SH alone did not yield detectable quantities of
F(ab').sub.2. Furthermore the coupling reaction was subjected to a
mild reduction step followed by alkylation to remove trace amounts
of disulfide-linked F(ab').sub.2 that might be present. SDS-PAGE of
the purified F(ab').sub.2 under reducing conditions gave two major
bands with electrophoretic mobility and amino terminal sequence
anticipated for free light chain and thioether-linked heavy chain
dimers.
Scanning LASER densitometry of a o-PDM coupled F(ab').sub.2
preparation suggest that the minor species together represent
.about.10% of the protein. These minor contaminants were
characterized by amino terminal sequence analysis and were
tentatively identified on the basis of stoichiometry of light and
heavy chain sequences and their electrophoretic mobility (data not
shown). These data are consistent with the minor contaminants
including imperfect F(ab').sub.2 in which the disulfide bond
between light and heavy chains is missing in one or both arms,
trace amounts of Fab' and heavy chain thioether-linked to light
chain.
Binding of BsF(ab').sub.2 to Jurkat Cells
Binding of BsF(ab').sub.2 containing different anti-CD3 variants to
Jurkat cells (human acute T cell leukemia) was investigated by flow
cytometry (data not shown). BsF(ab').sub.2 v9 binds much more
efficiently to Jurkat cells than does our starting molecule,
BsF(ab').sub.2 v1, and almost as efficiently as the chimeric
BsF(ab').sub.2. Installation of additional murine residues into
anti-CD3 v9 to create v10 (V.sub.H K75S:N76S) and v12 (V.sub.H
K75S:N76S plus V.sub.L E55H) did not further improve binding of
corresponding BsF(ab').sub.2 to Jurkat cells. Nor did recruitment
of these murine residues into anti-CD3 v1improve Jurkat binding:
V.sub.H K75S (v6), V.sub.H N76S (v7), V.sub.H K75S:N76S (v8),
V.sub.L E55H (v11) (not shown). BsF(ab').sub.2 v9 was chosen for
future study since it is amongst the most efficient variants in
binding to Jurkat cells and contains fewest murine residues in the
humanized anti-CD3 arm. A monospecific anti-p185.sup.HER2
F(ab').sub.2 did not show significant binding to Jurkat cells
consistent with the interaction being mediated through the anti-CD3
arm.
Discussion
A minimalistic strategy was chosen to humanize the
anti-p185.sup.HER2 (Carter et al., 1992a, supra) and anti-CD3 arms
(Shalaby et al., supra) of the BsF(ab').sub.2 in this study in an
attempt to minimize the potential immunogenicity of the resulting
humanized antibody in the clinic. Thus we tried to install the
minimum number of murine CDR and FR residues into the context of
consensus human variable domain sequences as required to recruit
antigen-binding affinity and biological properties comparable to
the murine parent antibody. Molecular modeling was used firstly to
predict the murine FR residues which might be important to antigen
binding and secondly to predict the murine CDR residues that might
not be required. A small number of humanized variants were then
constructed to test these predictions.
Our humanization strategy was very successful for the
anti-p185.sup.HER2 antibody where one out of eight humanized
variants (HuMAb4D5-8, IgG 1) was identified that bound the
p185.sup.HER2 antigen .about.3-fold more tightly than the parent
murine antibody (Carter et al., 1992a, supra). HuMAb4D5-8 contains
a total of five murine FR residues and nine murine CDR residues,
including V.sub.H CDR2 residues 60-65, were discarded in favor of
human counterparts. In contrast, BsF(ab').sub.2 v1 containing the
most potent humanized anti-CD3 variant out of four originally
constructed (Shalaby et al., supra) binds J6 cells with an affinity
(K.sub.d) of 140 nM which is .about.70-fold weaker than that of the
corresponding chimeric BsF(ab').sub.2.
Here we have restored T cell binding of the humanized anti-CD3
close to that of the chimeric variant by replacing six human
residues in V.sub.H CDR2 with their murine counterparts:
T57S:A60N:D61Q:S62K:V63F:G65D (anti-CD3 v9, FIG. 5). It appears
more likely that these murine residues enhance antigen binding
indirectly by influencing the conformation of residues in the
N-terminal part of V.sub.H CDR2 rather than by directly contacting
antigen. Firstly, only N-terminal residues in V.sub.H CDR2 (50-58)
have been found to contact antigen in one or more of eight
crystallographic structures of antibody/antigen complexes (Kabat et
al., supra; and Mian, I. S. et al., J. Mol. Biol. 217: 133-151
(1991), FIG. 5). Secondly, molecular modeling suggests that
residues in the C-terminal part of V.sub.H CDR2 are at least
partially buried (FIG. 5). BsF(ab').sub.2 v9 binds to SK-BR-3
breast tumor cells with equal efficiency to BsF(ab').sub.2 v1 and
chimeric BsF(ab').sub.2 as anticipated since the anti-p185.sup.HER2
arm is identical in all of these molecules (Shalaby et al., supra,
not shown).
Our novel approach to the construction of BsF(ab').sub.2 fragments
exploits an E. coli expression system which secretes humanized Fab'
fragments at gram per liter titers and permits their direct
recovery as Fab'-SH (Carter et al., 1992b, supra). Traditional
directed chemical coupling of Fab'-SH fragments is then used to
form BsF(ab').sub.2 in vitro (Brennan et al.,supra; and Glennie et
al., supra). This route to Fab'-SH obviates problems which are
inherent in their generation from intact antibodies: differences in
susceptibility to proteolysis and nonspecific cleavage resulting in
heterogeneity, low yield as well as partial reduction that is not
completely selective for the hinge disulfide bonds. The strategy of
using E. coli-derived Fab'-SH containing a single hinge cysteine
abolishes some sources of heterogeneity in BsF(ab').sub.2
preparation such as intra-hinge disulfide formation and
contamination with intact parent antibody whilst greatly diminishes
others, eg. formation of F(ab').sub.3 fragments.
BsF(ab').sub.2 fragments constructed here were thioether-linked as
originally described by Glennie et al., supra with future in vivo
testing of these molecules in mind. Thioether bonds, unlike
disulfide bonds, are not susceptible to cleavage by trace amounts
of thiol, which led to the proposal that thioether-linked
F(ab').sub.2 may be more stable than disulfide-linked F(ab').sub.2
in vivo (Glennie et al., supra). This hypothesis is supported by
our preliminary pharmacokinetic experiments in normal mice which
suggest that thioether-linked BsF(ab').sub.2 v1has a 3- fold longer
plasma residence time than BsF(ab').sub.2 v1 linked by a single
disulfide bond. Disulfide and thioether-linked chimeric
BsF(ab').sub.2 were found to be indistinguishable in their
efficiency of cell binding and in their retargeting of CTL
cytotoxicity, which suggests that o-PDM directed coupling does not
compromise binding of the BsF(ab').sub.2 to either antigen (not
shown). Nevertheless the nature of the linkage appears not to be
critical since a disulfide-linked BsF(ab').sub.2 (murine anti-p
185.sup.HER2 /murine anti-CD3) was recently shown by others
(Nishimura et al., Int. J. Cancer 50: 800-804 (1992) to have potent
anti-tumor activity in nude mice. Our previous study (Shalaby et
al., supra) together with this one and that of Nishimura, T. et
al., supra improve the potential for using BsF(ab').sub.2 in
targeted immunotherapy of p185.sup.HER2 -overexpressing cancers in
humans.
Example 4
Humanization of an Anti-CD18 Antibody
A murine antibody directed against the leukocyte adhesion receptor
.beta.-chain (known as the H52 antibody) was humanized and several
versions were created following the methods described above. FIGS.
6A and 6B provide amino acid sequence comparisons for light chains
and heavy chains of the murine and a preferred humanized antibody.
FIG. 7A shows an amino acid sequence alignment of the sequences of
thirteen humanized H52 variants (SEQ. ID. NOs 26-38); FIG. 7B shows
an amino acid sequence alignment of two humanized H52 light chain
variants (SEQ. ID. NOs 39-40). A specific light chain amino acid
sequence may be paired with a selected heavy chain amino acid
sequence to obtain antibody or antibody fragment molecules with
desired binding characteristics.
__________________________________________________________________________
# SEQUENCE LISTING - - - - (1) GENERAL INFORMATION: - - (iii)
NUMBER OF SEQUENCES: 47 - - - - (2) INFORMATION FOR SEQ ID NO:1: -
- (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 109 amino - #acids (B)
TYPE: Amino Acid (D) TOPOLOGY: Linear - - (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:1: - - Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser - #Leu Ser Ala Ser Val 1 - #5 - #10 - #15 - - Gly Asp Arg Val
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- Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro - #Gly Lys Ala Pro Lys -
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Gly Val Pro Ser - #50 - #55 - #60 - - Arg Phe Ser Gly Ser Arg Ser
Gly Thr Asp - #Phe Thr Leu Thr Ile - #65 - #70 - #75 - - Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr - #Tyr Tyr Cys Gln Gln - #80 - #85
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INFORMATION FOR SEQ ID NO:2: - - (i) SEQUENCE CHARACTERISTICS: (A)
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- Leu Leu Ile Tyr Ala Ala Ser Ser Leu Glu - #Ser Gly Val Pro Ser -
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Ser Leu Pro Tyr Thr Phe Gly Gln - #Gly Thr Lys Val Glu - #95 1 -
#00 1 - #05 - - Ile Lys Arg Thr 109 - - - - (2) INFORMATION FOR SEQ
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#acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear - - (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:4: - - Glu Val Gln Leu Val Glu Ser Gly Gly
Gly - #Leu Val Gln Pro Gly 1 - #5 - #10 - #15 - - Gly Ser Leu Arg
Leu Ser Cys Ala Ala Ser - #Gly Phe Thr Phe Ser - #20 - #25 - #30 -
- Asp Tyr Ala Met Ser Trp Val Arg Gln Ala - #Pro Gly Lys Gly Leu -
#35 - #40 - #45 - - Glu Trp Val Ala Val Ile Ser Glu Asn Gly - #Ser
Asp Thr Tyr Tyr - #50 - #55 - #60 - - Ala Asp Ser Val Lys Gly Arg
Phe Thr Ile - #Ser Arg Asp Asp Ser - #65 - #70 - #75 - - Lys Asn
Thr Leu Tyr Leu Gln Met Asn Ser - #Leu Arg Ala Glu Asp - #80 - #85
- #90 - - Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg - #Gly Gly Ala
Val Ser - #95 1 - #00 1 - #05 - - Tyr Phe Asp Val Trp Gly Gln Gly
Thr Leu - #Val Thr Val Ser Ser 110 - # 115 - # 120 - - - - (2)
INFORMATION FOR SEQ ID NO:5: - - (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 109 amino - #acids (B) TYPE: Amino Acid (D) TOPOLOGY:
Linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: - - Asp Ile Val
Met Thr Gln Ser His Lys Phe - #Met Ser Thr Ser Val 1 - #5 - #10 -
#15 - - Gly Asp Arg Val Ser Ile Thr Cys Lys Ala - #Ser Gln Asp Val
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#Gly His Ser Pro Lys - #35 - #40 - #45 - - Leu Leu Ile Tyr Ser Ala
Ser Phe Arg Tyr - #Thr Gly Val Pro Asp - #50 - #55 - #60 - - Arg
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#70 - #75 - - Ser Ser Val Gln Ala Glu Asp Leu Ala Val - #Tyr Tyr
Cys Gln Gln - #80 - #85 - #90 - - His Tyr Thr Thr Pro Pro Thr Phe
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Ser - #Gly Phe Asn Ile Lys - #20 - #25 - #30 - - Asp Thr Tyr Ile
His Trp Val Lys Gln Arg - #Pro Glu Gln Gly Leu - #35 - #40 - #45 -
- Glu Trp Ile Gly Arg Ile Tyr Pro Thr Asn - #Gly Tyr Thr Arg Tyr -
#50 - #55 - #60 - - Asp Pro Lys Phe Gln Asp Lys Ala Thr Ile - #Thr
Ala Asp Thr Ser - #65 - #70 - #75 - - Ser Asn Thr Ala Tyr Leu Gln
Val Ser Arg - #Leu Thr Ser Glu Asp - #80 - #85 - #90 - - Thr Ala
Val Tyr Tyr Cys Ser Arg Trp Gly - #Gly Asp Gly Phe Tyr - #95 1 -
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Val Ser Ser 110 - # 115 - # 120 - - - - (2) INFORMATION FOR SEQ ID
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INFORMATION FOR SEQ ID NO:9: - - (i) SEQUENCE CHARACTERISTICS: (A)
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SEQUENCE DESCRIPTION: SEQ ID NO:16: - - Asp Ile Gln Met Thr Gln Thr
Thr Ser Ser - #Leu Ser Ala Ser Leu 1 - #5 - #10 - #15 - - Gly Asp
Arg Val Thr Ile Ser Cys Arg Ala - #Ser Gln Asp Ile Arg - #20 - #25
- #30 - - Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro - #Asp Gly Thr
Val Lys - #35 - #40 - #45 - - Leu Leu Ile Tyr Tyr Thr Ser Arg Leu
His - #Ser Gly Val Pro Ser - #50 - #55 - #60 - - Lys Phe Ser Gly
Ser Gly Ser Gly Thr Asp - #Tyr Ser Leu Thr Ile - #65 - #70 - #75 -
- Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr - #Tyr Phe Cys Gln Gln -
#80 - #85 - #90 - - Gly Asn Thr Leu Pro Trp Thr Phe Ala Gly - #Gly
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Linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: - - Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser - #Leu Ser Ala Ser Val 1 - #5 - #10 -
#15 - - Gly Asp Arg Val Thr Ile Thr Cys Arg Ala - #Ser Gln Asp Ile
Arg - #20 - #25 - #30
- - Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro - #Gly Lys Ala Pro Lys
- #35 - #40 - #45 - - Leu Leu Ile Tyr Tyr Thr Ser Arg Leu Glu -
#Ser Gly Val Pro Ser - #50 - #55 - #60 - - Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp - #Tyr Thr Leu Thr Ile - #65 - #70 - #75 - - Ser
Ser Leu Gln Pro Glu Asp Phe Ala Thr - #Tyr Tyr Cys Gln Gln - #80 -
#85 - #90 - - Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln - #Gly Thr
Lys Val Glu - #95 1 - #00 1 - #05 - - Ile Lys 107 - - - - (2)
INFORMATION FOR SEQ ID NO:18: - - (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 107 amino - #acids (B) TYPE: Amino Acid (D) TOPOLOGY:
Linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: - - Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser - #Leu Ser Ala Ser Val 1 - #5 - #10 -
#15 - - Gly Asp Arg Val Thr Ile Thr Cys Arg Ala - #Ser Gln Ser Ile
Ser - #20 - #25 - #30 - - Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro -
#Gly Lys Ala Pro Lys - #35 - #40 - #45 - - Leu Leu Ile Tyr Ala Ala
Ser Ser Leu Glu - #Ser Gly Val Pro Ser - #50 - #55 - #60 - - Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp - #Phe Thr Leu Thr Ile - #65 -
#70 - #75 - - Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr - #Tyr Tyr
Cys Gln Gln - #80 - #85 - #90 - - Tyr Asn Ser Leu Pro Trp Thr Phe
Gly Gln - #Gly Thr Lys Val Glu - #95 1 - #00 1 - #05 - - Ile Lys
107 - - - - (2) INFORMATION FOR SEQ ID NO:19: - - (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 122 amino - #acids (B) TYPE: Amino
Acid (D) TOPOLOGY: Linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:19: - - Glu Val Gln Leu Gln Gln Ser Gly Pro Glu - #Leu Val Lys
Pro Gly 1 - #5 - #10 - #15 - - Ala Ser Met Lys Ile Ser Cys Lys Ala
Ser - #Gly Tyr Ser Phe Thr - #20 - #25 - #30 - - Gly Tyr Thr Met
Asn Trp Val Lys Gln Ser - #His Gly Lys Asn Leu - #35 - #40 - #45 -
- Glu Trp Met Gly Leu Ile Asn Pro Tyr Lys - #Gly Val Ser Thr Tyr -
#50 - #55 - #60 - - Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu - #Thr
Val Asp Lys Ser - #65 - #70 - #75 - - Ser Ser Thr Ala Tyr Met Glu
Leu Leu Ser - #Leu Thr Ser Glu Asp - #80 - #85 - #90 - - Ser Ala
Val Tyr Tyr Cys Ala Arg Ser Gly - #Tyr Tyr Gly Asp Ser - #95 1 -
#00 1 - #05 - - Asp Trp Tyr Phe Asp Val Trp Gly Ala Gly - #Thr Thr
Val Thr Val 110 - # 115 - # 120 - - Ser Ser 122 - - - - (2)
INFORMATION FOR SEQ ID NO:20: - - (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 122 amino - #acids (B) TYPE: Amino Acid (D) TOPOLOGY:
Linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: - - Glu Val Gln
Leu Val Glu Ser Gly Gly Gly - #Leu Val Gln Pro Gly 1 - #5 - #10 -
#15 - - Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser - #Gly Tyr Ser Phe
Thr - #20 - #25 - #30 - - Gly Tyr Thr Met Asn Trp Val Arg Gln Ala -
#Pro Gly Lys Gly Leu - #35 - #40 - #45 - - Glu Trp Val Ala Leu Ile
Asn Pro Tyr Lys - #Gly Val Ser Thr Tyr - #50 - #55 - #60 - - Asn
Gln Lys Phe Lys Asp Arg Phe Thr Ile - #Ser Val Asp Lys Ser - #65 -
#70 - #75 - - Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser - #Leu Arg
Ala Glu Asp - #80 - #85 - #90 - - Thr Ala Val Tyr Tyr Cys Ala Arg
Ser Gly - #Tyr Tyr Gly Asp Ser - #95 1 - #00 1 - #05 - - Asp Trp
Tyr Phe Asp Val Trp Gly Gln Gly - #Thr Leu Val Thr Val 110 - # 115
- # 120 - - Ser Ser 122 - - - - (2) INFORMATION FOR SEQ ID NO:21: -
- (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 122 amino - #acids (B)
TYPE: Amino Acid (D) TOPOLOGY: Linear - - (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:21: - - Glu Val Gln Leu Val Glu Ser Gly Gly
Gly - #Leu Val Gln Pro Gly 1 - #5 - #10 - #15 - - Gly Ser Leu Arg
Leu Ser Cys Ala Ala Ser - #Gly Phe Thr Phe Ser - #20 - #25 - #30 -
- Ser Tyr Ala Met Ser Trp Val Arg Gln Ala - #Pro Gly Lys Gly Leu -
#35 - #40 - #45 - - Glu Trp Val Ser Val Ile Ser Gly Asp Gly - #Gly
Ser Thr Tyr Tyr - #50 - #55 - #60 - - Ala Asp Ser Val Lys Gly Arg
Phe Thr Ile - #Ser Arg Asp Asn Ser - #65 - #70 - #75 - - Lys Asn
Thr Leu Tyr Leu Gln Met Asn Ser - #Leu Arg Ala Glu Asp - #80 - #85
- #90 - - Thr Ala Val Tyr Tyr Cys Ala Arg Gly Arg - #Val Gly Tyr
Ser Leu - #95 1 - #00 1 - #05 - - Ser Gly Leu Tyr Asp Tyr Trp Gly
Gln Gly - #Thr Leu Val Thr Val 110 - # 115 - # 120 - - Ser Ser 122
- - - - (2) INFORMATION FOR SEQ ID NO:22: - - (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 454 amino - #acids (B) TYPE: Amino
Acid (D) TOPOLOGY: Linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:22: - - Gln Val Gln Leu Gln Gln Ser Gly Pro Glu - #Leu Val Lys
Pro Gly 1 - #5 - #10 - #15 - - Ala Ser Val Lys Ile Ser Cys Lys Thr
Ser - #Gly Tyr Thr Phe Thr - #20 - #25 - #30 - - Glu Tyr Thr Met
His Trp Met Lys Gln Ser - #His Gly Lys Ser Leu - #35 - #40 - #45 -
- Glu Trp Ile Gly Gly Phe Asn Pro Lys Asn - #Gly Gly Ser Ser His -
#50 - #55 - #60 - - Asn Gln Arg Phe Met Asp Lys Ala Thr Leu - #Ala
Val Asp Lys Ser - #65 - #70 - #75 - - Thr Ser Thr Ala Tyr Met Glu
Leu Arg Ser - #Leu Thr Ser Glu Asp - #80 - #85 - #90 - - Ser Gly
Ile Tyr Tyr Cys Ala Arg Trp Arg - #Gly Leu Asn Tyr Gly - #95 1 -
#00 1 - #05 - - Phe Asp Val Arg Tyr Phe Asp Val Trp Gly - #Ala Gly
Thr Thr Val 110 - # 115 - # 120 - - Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro - #Ser Val Phe Pro Leu 125 - # 130 - # 135 - - Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly - #Thr Ala Ala Leu Gly 140 - # 145 - #
150 - - Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro - #Val Thr Val Ser
Trp 155 - # 160 - # 165 - - Asn Ser Gly Ala Leu Thr Ser Gly Val His
- #Thr Phe Pro Ala Val 170 - # 175 - # 180 - - Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser - #Ser Val Val Thr Val 185 - # 190 - # 195 - -
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr - #Ile Cys Asn Val Asn 200
- # 205 - # 210 - - His Lys Pro Ser Asn Thr Lys Val Asp Lys - #Lys
Val Glu Pro Lys 215 - # 220 - # 225 - - Ser Cys Asp Lys Thr His Thr
Cys Pro Pro - #Cys Pro Ala Pro Glu 230 - # 235 - # 240 - - Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe - #Pro Pro Lys Pro Lys 245 - # 250
- # 255 - - Asp Thr Leu Met Ile Ser Arg Thr Pro Glu - #Val Thr Cys
Val Val 260 - # 265 - # 270 - - Val Asp Val Ser His Glu Asp Pro Glu
Val - #Lys Phe Asn Trp Tyr 275 - # 280 - # 285 - - Val Asp Gly Val
Glu Val His Asn Ala Lys - #Thr Lys Pro Arg Glu 290 - # 295 - # 300
- - Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val - #Ser Val Leu Thr Val
305 - # 310 - # 315 - - Leu His Gln Asp Trp Leu Asn Gly Lys Glu -
#Tyr Lys Cys Lys Val 320 - # 325 - # 330 - - Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu - #Lys Thr Ile Ser Lys 335 - # 340 - # 345 - -
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val - #Tyr Thr Leu Pro Pro 350
- # 355 - # 360 - - Ser Arg Glu Glu Met Thr Lys Asn Gln Val - #Ser
Leu Thr Cys Leu 365 - # 370 - # 375 - - Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala - #Val Glu Trp Glu Ser 380 - # 385 - # 390 - - Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr - #Thr Pro Pro Val Leu 395 - # 400
- # 405 - - Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser - #Lys Leu Thr
Val Asp 410 - # 415 - # 420 - - Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe - #Ser Cys Ser Val Met 425 - # 430 - # 435 - - His Glu Ala Leu
His Asn His Tyr Thr Gln - #Lys Ser Leu Ser Leu 440 - # 445 - # 450
- - Ser Pro Gly Lys 454 - - - - (2) INFORMATION FOR SEQ ID NO:23: -
- (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 469 amino - #acids (B)
TYPE: Amino Acid (D) TOPOLOGY: Linear - - (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:23: - - Met Gly Trp Ser Cys Ile Ile Leu Phe
Leu - #Val Ala Thr Ala Thr 1 - #5 - #10 - #15 - - Gly Val His Ser
Glu Val Gln Leu Val Glu - #Ser Gly Gly Gly Leu - #20 - #25 - #30 -
- Val Gln Pro Gly Gly Ser Leu Arg Leu Ser - #Cys Ala Thr Ser Gly -
#35 - #40 - #45 - - Tyr Thr Phe Thr Glu Tyr Thr Met His Trp - #Met
Arg Gln Ala Pro - #50 - #55 - #60 - - Gly Lys Gly Leu Glu Trp Val
Ala Gly Ile - #Asn Pro Lys Asn Gly - #65 - #70 - #75 - - Gly Thr
Ser His Asn Gln Arg Phe Met Asp - #Arg Phe Thr Ile Ser - #80 - #85
- #90 - - Val Asp Lys Ser Thr Ser Thr Ala Tyr Met - #Gln Met Asn
Ser Leu - #95 1 - #00 1 - #05 - - Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys - #Ala Arg Trp Arg Gly 110 - # 115 - # 120 - - Leu Asn Tyr
Gly Phe Asp Val Arg Tyr Phe - #Asp Val Trp Gly Gln 125 - # 130 - #
135 - - Gly Thr Leu Val Thr Val Ser Ser Ala Ser - #Thr Lys Gly Pro
Ser 140 - # 145 - # 150 - - Val Phe Pro Leu Ala Pro Cys Ser Arg Ser
- #Thr Ser Glu Ser Thr 155 - # 160 - # 165 - - Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr - #Phe Pro Glu Pro Val 170 - # 175 - # 180 - -
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr - #Ser Gly Val His Thr 185
- # 190 - # 195 - - Phe Pro Ala Val Leu Gln Ser Ser Gly Leu - #Tyr
Ser Leu Ser Ser 200 - # 205 - # 210 - - Val Val Thr Val Thr Ser Ser
Asn Phe Gly - #Thr Gln Thr Tyr Thr 215 - # 220 - # 225 - - Cys Asn
Val Asp His Lys Pro Ser Asn Thr - #Lys Val Asp Lys Thr 230 - # 235
- # 240 - - Val Glu Arg Lys Cys Cys Val Glu Cys Pro - #Pro Cys Pro
Ala Pro 245 - # 250 - # 255 - - Pro Val Ala Gly Pro Ser Val Phe Leu
Phe - #Pro Pro Lys Pro Lys 260 - # 265 - # 270 - - Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu - #Val Thr Cys Val Val 275 - # 280 - # 285
- - Val Asp Val Ser His Glu Asp Pro Glu Val - #Gln Phe Asn Trp Tyr
290 - # 295 - # 300 - - Val Asp Gly Met Glu Val His Asn Ala Lys -
#Thr Lys Pro Arg Glu 305 - # 310 - # 315 - - Glu Gln Phe Asn Ser
Thr Phe Arg Val Val - #Ser Val Leu Thr Val 320 - # 325 - # 330 - -
Val His Gln Asp Trp Leu Asn Gly Lys Glu - #Tyr Lys Cys Lys Val 335
- # 340 - # 345 - - Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu - #Lys
Thr Ile Ser Lys 350 - # 355 - # 360 - - Thr Lys Gly Gln Pro Arg Glu
Pro Gln Val - #Tyr Thr Leu Pro Pro 365 - # 370 - # 375 - - Ser Arg
Glu Glu Met Thr Lys Asn Gln Val - #Ser Leu Thr Cys Leu 380 - # 385
- # 390 - - Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala - #Val Glu Trp
Glu Ser 395 - # 400 - # 405 - - Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr - #Thr Pro Pro Met Leu 410 - # 415 - # 420 - - Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser - #Lys Leu Thr Val Asp 425 - # 430 - # 435
- - Lys Ser Arg Trp Gln Gln Gly Asn Val Phe - #Ser Cys Ser Val Met
440 - # 445 - # 450 - - His Glu Ala Leu His Asn His Tyr Thr Gln -
#Lys Ser Leu Ser Leu 455 - # 460 - # 465
- - Ser Pro Gly Lys 469 - - - - (2) INFORMATION FOR SEQ ID NO:24: -
- (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 214 amino - #acids (B)
TYPE: Amino Acid (D) TOPOLOGY: Linear - - (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:24: - - Asp Val Gln Met Thr Gln Thr Thr Ser
Ser - #Leu Ser Ala Ser Leu 1 - #5 - #10 - #15 - - Gly Asp Arg Val
Thr Ile Asn Cys Arg Ala - #Ser Gln Asp Ile Asn - #20 - #25 - #30 -
- Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro - #Asn Gly Thr Val Lys -
#35 - #40 - #45 - - Leu Leu Ile Tyr Tyr Thr Ser Thr Leu His - #Ser
Gly Val Pro Ser - #50 - #55 - #60 - - Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp - #Tyr Ser Leu Thr Ile - #65 - #70 - #75 - - Ser Asn
Leu Asp Gln Glu Asp Ile Ala Thr - #Tyr Phe Cys Gln Gln - #80 - #85
- #90 - - Gly Asn Thr Leu Pro Pro Thr Phe Gly Gly - #Gly Thr Lys
Val Glu - #95 1 - #00 1 - #05 - - Ile Lys Arg Thr Val Ala Ala Pro
Ser Val - #Phe Ile Phe Pro Pro 110 - # 115 - # 120 - - Ser Asp Glu
Gln Leu Lys Ser Gly Thr Ala - #Ser Val Val Cys Leu 125 - # 130 - #
135 - - Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys - #Val Gln Trp Lys
Val 140 - # 145 - # 150 - - Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
- #Glu Ser Val Thr Glu 155 - # 160 - # 165 - - Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Leu - #Ser Ser Thr Leu Thr 170 - # 175 - # 180 - -
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys - #Val Tyr Ala Cys Glu 185
- # 190 - # 195 - - Val Thr His Gln Gly Leu Ser Ser Pro Val - #Thr
Lys Ser Phe Asn 200 - # 205 - # 210 - - Arg Gly Glu Cys 214 - - - -
(2) INFORMATION FOR SEQ ID NO:25: - - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 233 amino - #acids (B) TYPE: Amino Acid (D) TOPOLOGY:
Linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: - - Met Gly Trp
Ser Cys Ile Ile Leu Phe Leu - #Val Ala Thr Ala Thr 1 - #5 - #10 -
#15 - - Gly Val His Ser Asp Ile Gln Met Thr Gln - #Ser Pro Ser Ser
Leu - #20 - #25 - #30 - - Ser Ala Ser Val Gly Asp Arg Val Thr Ile -
#Thr Cys Arg Ala Ser - #35 - #40 - #45 - - Gln Asp Ile Asn Asn Tyr
Leu Asn Trp Tyr - #Gln Gln Lys Pro Gly - #50 - #55 - #60 - - Lys
Ala Pro Lys Leu Leu Ile Tyr Tyr Thr - #Ser Thr Leu His Ser - #65 -
#70 - #75 - - Gly Val Pro Ser Arg Phe Ser Gly Ser Gly - #Ser Gly
Thr Asp Tyr - #80 - #85 - #90 - - Thr Leu Thr Ile Ser Ser Leu Gln
Pro Glu - #Asp Phe Ala Thr Tyr - #95 1 - #00 1 - #05 - - Tyr Cys
Gln Gln Gly Asn Thr Leu Pro Pro - #Thr Phe Gly Gln Gly 110 - # 115
- # 120 - - Thr Lys Val Glu Ile Lys Arg Thr Val Ala - #Ala Pro Ser
Val Phe 125 - # 130 - # 135 - - Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys - #Ser Gly Thr Ala Ser 140 - # 145 - # 150 - - Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro - #Arg Glu Ala Lys Val 155 - # 160 - # 165
- - Gln Trp Lys Val Asp Asn Ala Leu Gln Ser - #Gly Asn Ser Gln Glu
170 - # 175 - # 180 - - Ser Val Thr Glu Gln Asp Ser Lys Asp Ser -
#Thr Tyr Ser Leu Ser 185 - # 190 - # 195 - - Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr - #Glu Lys His Lys Val 200 - # 205 - # 210 - -
Tyr Ala Cys Glu Val Thr His Gln Gly Leu - #Ser Ser Pro Val Thr 215
- # 220 - # 225 - - Lys Ser Phe Asn Arg Gly Glu Cys 230 - # 233 - -
- - (2) INFORMATION FOR SEQ ID NO:26: - - (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 232 amino - #acids (B) TYPE: Amino
Acid (D) TOPOLOGY: Linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:26: - - Glu Val Gln Leu Val Glu Ser Gly Gly Gly - #Leu Val Gln
Pro Gly 1 - #5 - #10 - #15 - - Gly Ser Leu Arg Leu Ser Cys Ala Thr
Ser - #Gly Tyr Thr Phe Thr - #20 - #25 - #30 - - Glu Tyr Thr Met
His Trp Met Arg Gln Ala - #Pro Gly Lys Gly Leu - #35 - #40 - #45 -
- Glu Trp Val Ala Gly Ile Asn Pro Lys Asn - #Gly Gly Thr Ser Tyr -
#50 - #55 - #60 - - Ala Asp Ser Val Lys Gly Arg Phe Thr Ile - #Ser
Val Asp Lys Ser - #65 - #70 - #75 - - Lys Asn Thr Leu Tyr Leu Gln
Met Asn Ser - #Leu Arg Ala Glu Asp - #80 - #85 - #90 - - Thr Ala
Val Tyr Tyr Cys Ala Arg Trp Arg - #Gly Leu Asn Tyr Gly - #95 1 -
#00 1 - #05 - - Phe Asp Val Arg Tyr Phe Asp Val Trp Gly - #Gln Gly
Thr Leu Val 110 - # 115 - # 120 - - Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro - #Ser Val Phe Pro Leu 125 - # 130 - # 135 - - Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly - #Thr Ala Ala Leu Gly 140 - # 145 - #
150 - - Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro - #Val Thr Val Ser
Trp 155 - # 160 - # 165 - - Asn Ser Gly Ala Leu Thr Ser Gly Val His
- #Thr Phe Pro Ala Val 170 - # 175 - # 180 - - Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser - #Ser Val Val Thr Val 185 - # 190 - # 195 - -
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr - #Ile Cys Asn Val Asn 200
- # 205 - # 210 - - His Lys Pro Ser Asn Thr Lys Val Asp Lys - #Lys
Val Glu Pro Lys 215 - # 220 - # 225 - - Ser Cys Asp Lys Thr His Thr
230 - # 232 - - - - (2) INFORMATION FOR SEQ ID NO:27: - - (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 232 amino - #acids (B) TYPE:
Amino Acid (D) TOPOLOGY: Linear - - (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:27: - - Glu Val Gln Leu Val Glu Ser Gly Gly Gly - #Leu Val
Gln Pro Gly 1 - #5 - #10 - #15 - - Gly Ser Leu Arg Leu Ser Cys Ala
Thr Ser - #Gly Tyr Thr Phe Thr - #20 - #25 - #30 - - Glu Tyr Thr
Met His Trp Met Arg Gln Ala - #Pro Gly Lys Gly Leu - #35 - #40 -
#45 - - Glu Trp Val Ala Gly Ile Asn Pro Lys Asn - #Gly Gly Thr Ser
His - #50 - #55 - #60 - - Asn Gln Arg Phe Met Asp Arg Phe Thr Ile -
#Ser Val Asp Lys Ser - #65 - #70 - #75 - - Lys Asn Thr Leu Tyr Leu
Gln Met Asn Ser - #Leu Arg Ala Glu Asp - #80 - #85 - #90 - - Thr
Ala Val Tyr Tyr Cys Ala Arg Trp Arg - #Gly Leu Asn Tyr Gly - #95 1
- #00 1 - #05 - - Phe Asp Val Arg Tyr Phe Asp Val Trp Gly - #Gln
Gly Thr Leu Val 110 - # 115 - # 120 - - Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro - #Ser Val Phe Pro Leu 125 - # 130 - # 135 - - Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly - #Thr Ala Ala Leu Gly 140 - # 145
- # 150 - - Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro - #Val Thr Val
Ser Trp 155 - # 160 - # 165 - - Asn Ser Gly Ala Leu Thr Ser Gly Val
His - #Thr Phe Pro Ala Val 170 - # 175 - # 180 - - Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser - #Ser Val Val Thr Val 185 - # 190 - # 195
- - Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr - #Ile Cys Asn Val Asn
200 - # 205 - # 210 - - His Lys Pro Ser Asn Thr Lys Val Asp Lys -
#Lys Val Glu Pro Lys 215 - # 220 - # 225 - - Ser Cys Asp Lys Thr
His Thr 230 - # 232 - - - - (2) INFORMATION FOR SEQ ID NO:28: - -
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 232 amino - #acids (B)
TYPE: Amino Acid (D) TOPOLOGY: Linear - - (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:28: - - Glu Val Gln Leu Val Glu Ser Gly Gly
Gly - #Leu Val Gln Pro Gly 1 - #5 - #10 - #15 - - Gly Ser Leu Arg
Leu Ser Cys Ala Thr Ser - #Gly Tyr Thr Phe Thr - #20 - #25 - #30 -
- Glu Tyr Thr Met His Trp Met Arg Gln Ala - #Pro Gly Lys Gly Leu -
#35 - #40 - #45 - - Glu Trp Val Ala Gly Ile Asn Pro Lys Asn - #Gly
Gly Thr Ser His - #50 - #55 - #60 - - Asn Gln Arg Phe Met Asp Arg
Phe Thr Leu - #Ala Val Asp Lys Ser - #65 - #70 - #75 - - Lys Asn
Thr Leu Tyr Leu Gln Met Asn Ser - #Leu Arg Ala Glu Asp - #80 - #85
- #90 - - Thr Ala Val Tyr Tyr Cys Ala Arg Trp Arg - #Gly Leu Asn
Tyr Gly - #95 1 - #00 1 - #05 - - Phe Asp Val Arg Tyr Phe Asp Val
Trp Gly - #Gln Gly Thr Leu Val 110 - # 115 - # 120 - - Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro - #Ser Val Phe Pro Leu 125 - # 130 - #
135 - - Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly - #Thr Ala Ala Leu
Gly 140 - # 145 - # 150 - - Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
- #Val Thr Val Ser Trp 155 - # 160 - # 165 - - Asn Ser Gly Ala Leu
Thr Ser Gly Val His - #Thr Phe Pro Ala Val 170 - # 175 - # 180 - -
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser - #Ser Val Val Thr Val 185
- # 190 - # 195 - - Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr - #Ile
Cys Asn Val Asn 200 - # 205 - # 210 - - His Lys Pro Ser Asn Thr Lys
Val Asp Lys - #Lys Val Glu Pro Lys 215 - # 220 - # 225 - - Ser Cys
Asp Lys Thr His Thr 230 - # 232 - - - - (2) INFORMATION FOR SEQ ID
NO:29: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 232 amino -
#acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear - - (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:29: - - Glu Val Gln Leu Val Glu Ser Gly Gly
Gly - #Leu Val Gln Pro Gly 1 - #5 - #10 - #15 - - Gly Ser Leu Arg
Leu Ser Cys Ala Thr Ser - #Gly Tyr Thr Phe Thr - #20 - #25 - #30 -
- Glu Tyr Thr Met His Trp Met Arg Gln Ala - #Pro Gly Lys Gly Leu -
#35 - #40 - #45 - - Glu Trp Val Ala Gly Ile Asn Pro Lys Asn - #Gly
Gly Thr Ser His - #50 - #55 - #60 - - Asn Gln Arg Phe Met Asp Arg
Phe Thr Ile - #Ser Val Asp Lys Ser - #65 - #70 - #75 - - Thr Ser
Thr Ala Tyr Met Gln Met Asn Ser - #Leu Arg Ala Glu Asp - #80 - #85
- #90 - - Thr Ala Val Tyr Tyr Cys Ala Arg Trp Arg - #Gly Leu Asn
Tyr Gly - #95 1 - #00 1 - #05 - - Phe Asp Val Arg Tyr Phe Asp Val
Trp Gly - #Gln Gly Thr Leu Val 110 - # 115 - # 120 - - Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro - #Ser Val Phe Pro Leu 125 - # 130 - #
135 - - Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly - #Thr Ala Ala Leu
Gly 140 - # 145 - # 150 - - Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
- #Val Thr Val Ser Trp 155 - # 160 - # 165 - - Asn Ser Gly Ala Leu
Thr Ser Gly Val His - #Thr Phe Pro Ala Val 170 - # 175 - # 180 - -
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser - #Ser Val Val Thr Val 185
- # 190 - # 195 - - Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr - #Ile
Cys Asn Val Asn 200 - # 205 - # 210 - - His Lys Pro Ser Asn Thr Lys
Val Asp Lys - #Lys Val Glu Pro Lys 215 - # 220 - # 225 - - Ser Cys
Asp Lys Thr His Thr 230 - # 232 - - - - (2) INFORMATION FOR SEQ ID
NO:30: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 235 amino -
#acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear - - (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:30: - - Glu Val Gln Leu Val Glu Ser Gly Gly
Gly - #Leu Val Gln Pro Gly 1 - #5 - #10 - #15 - - Gly Ser Leu Arg
Leu Ser Cys Ala Thr Ser - #Gly Tyr Thr Phe Thr - #20 - #25 - #30 -
- Glu Tyr Thr Met His Trp Met Arg Gln Ala - #Pro Gly Lys Gly Leu -
#35 - #40 - #45 - - Glu Trp Val Ala Gly Ile Asn Pro Lys Asn - #Gly
Gly Thr Ser His - #50 - #55 - #60 - - Asn Gln Arg Phe Met Asp Arg
Phe Thr Ile - #Ser Val Asp Lys Ser - #65 - #70 - #75 - - Thr Ser
Thr Ala Tyr Met Gln Met Asn Ser - #Leu Arg Ala Glu Asp - #80 - #85
- #90 - - Thr Ala Val Tyr Tyr Cys Ala Arg Trp Arg - #Gly Leu Asn
Tyr Gly - #95 1 - #00 1 - #05 - - Phe Asp Val Arg Tyr Phe Asp Val
Trp Gly - #Gln Gly Thr Leu Val 110 - # 115 - # 120 - - Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro - #Ser Val Phe Pro Leu
125 - # 130 - # 135 - - Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly -
#Thr Ala Ala Leu Gly 140 - # 145 - # 150 - - Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro - #Val Thr Val Ser Trp 155 - # 160 - # 165 - -
Asn Ser Gly Ala Leu Thr Ser Gly Val His - #Thr Phe Pro Ala Val 170
- # 175 - # 180 - - Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser - #Ser
Val Val Thr Val 185 - # 190 - # 195 - - Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr - #Ile Cys Asn Val Asn 200 - # 205 - # 210 - - His Lys
Pro Ser Asn Thr Lys Val Asp Lys - #Lys Val Glu Pro Lys 215 - # 220
- # 225 - - Ser Cys Asp Lys Thr His Thr Cys Pro Pro 230 - # 235 - -
- - (2) INFORMATION FOR SEQ ID NO:31: - - (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 232 amino - #acids (B) TYPE: Amino
Acid (D) TOPOLOGY: Linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:31: - - Glu Val Gln Leu Val Glu Ser Gly Gly Gly - #Leu Val Gln
Pro Gly 1 - #5 - #10 - #15 - - Gly Ser Leu Arg Leu Ser Cys Ala Thr
Ser - #Gly Tyr Thr Phe Thr - #20 - #25 - #30 - - Glu Tyr Thr Met
His Trp Met Arg Gln Ala - #Pro Gly Lys Gly Leu - #35 - #40 - #45 -
- Glu Trp Val Ala Gly Ile Asn Pro Lys Asn - #Gly Gly Thr Ser His -
#50 - #55 - #60 - - Asn Gln Arg Phe Met Asp Arg Phe Thr Ile - #Ser
Val Asp Lys Ser - #65 - #70 - #75 - - Lys Asn Thr Leu Tyr Met Glu
Leu Arg Ser - #Leu Arg Ala Glu Asp - #80 - #85 - #90 - - Thr Ala
Val Tyr Tyr Cys Ala Arg Trp Arg - #Gly Leu Asn Tyr Gly - #95 1 -
#00 1 - #05 - - Phe Asp Val Arg Tyr Phe Asp Val Trp Gly - #Gln Gly
Thr Leu Val 110 - # 115 - # 120 - - Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro - #Ser Val Phe Pro Leu 125 - # 130 - # 135 - - Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly - #Thr Ala Ala Leu Gly 140 - # 145 - #
150 - - Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro - #Val Thr Val Ser
Trp 155 - # 160 - # 165 - - Asn Ser Gly Ala Leu Thr Ser Gly Val His
- #Thr Phe Pro Ala Val 170 - # 175 - # 180 - - Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser - #Ser Val Val Thr Val 185 - # 190 - # 195 - -
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr - #Ile Cys Asn Val Asn 200
- # 205 - # 210 - - His Lys Pro Ser Asn Thr Lys Val Asp Lys - #Lys
Val Glu Pro Lys 215 - # 220 - # 225 - - Ser Cys Asp Lys Thr His Thr
230 - # 232 - - - - (2) INFORMATION FOR SEQ ID NO:32: - - (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 232 amino - #acids (B) TYPE:
Amino Acid (D) TOPOLOGY: Linear - - (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:32: - - Glu Val Gln Leu Val Glu Ser Gly Gly Gly - #Leu Val
Gln Pro Gly 1 - #5 - #10 - #15 - - Gly Ser Leu Arg Leu Ser Cys Ala
Thr Ser - #Gly Tyr Thr Phe Thr - #20 - #25 - #30 - - Glu Tyr Thr
Met His Trp Met Arg Gln Ala - #Pro Gly Lys Gly Leu - #35 - #40 -
#45 - - Glu Trp Val Ala Gly Ile Asn Pro Lys Asn - #Gly Gly Thr Ser
His - #50 - #55 - #60 - - Asn Gln Arg Phe Met Asp Arg Phe Thr Ile -
#Ser Val Asp Lys Ser - #65 - #70 - #75 - - Lys Asn Thr Leu Tyr Leu
Gln Met Asn Ser - #Leu Thr Ser Glu Asp - #80 - #85 - #90 - - Ser
Gly Ile Tyr Tyr Cys Ala Arg Trp Arg - #Gly Leu Asn Tyr Gly - #95 1
- #00 1 - #05 - - Phe Asp Val Arg Tyr Phe Asp Val Trp Gly - #Gln
Gly Thr Leu Val 110 - # 115 - # 120 - - Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro - #Ser Val Phe Pro Leu 125 - # 130 - # 135 - - Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly - #Thr Ala Ala Leu Gly 140 - # 145
- # 150 - - Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro - #Val Thr Val
Ser Trp 155 - # 160 - # 165 - - Asn Ser Gly Ala Leu Thr Ser Gly Val
His - #Thr Phe Pro Ala Val 170 - # 175 - # 180 - - Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser - #Ser Val Val Thr Val 185 - # 190 - # 195
- - Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr - #Ile Cys Asn Val Asn
200 - # 205 - # 210 - - His Lys Pro Ser Asn Thr Lys Val Asp Lys -
#Lys Val Glu Pro Lys 215 - # 220 - # 225 - - Ser Cys Asp Lys Thr
His Thr 230 - # 232 - - - - (2) INFORMATION FOR SEQ ID NO:33: - -
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 232 amino - #acids (B)
TYPE: Amino Acid (D) TOPOLOGY: Linear - - (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:33: - - Glu Val Gln Leu Val Glu Ser Gly Gly
Gly - #Leu Val Gln Pro Gly 1 - #5 - #10 - #15 - - Gly Ser Leu Arg
Leu Ser Cys Ala Thr Ser - #Gly Tyr Thr Phe Thr - #20 - #25 - #30 -
- Glu Tyr Thr Met His Trp Met Arg Gln Ala - #Pro Gly Lys Gly Leu -
#35 - #40 - #45 - - Glu Trp Val Ala Gly Ile Asn Pro Lys Asn - #Gly
Gly Thr Ser His - #50 - #55 - #60 - - Asn Gln Arg Phe Met Asp Arg
Phe Thr Ile - #Ser Val Asp Lys Ser - #65 - #70 - #75 - - Lys Asn
Thr Leu Tyr Leu Gln Met Asn Ser - #Leu Arg Ala Glu Asp - #80 - #85
- #90 - - Thr Ala Val Tyr Tyr Cys Ala Arg Trp Arg - #Gly Leu Asn
Tyr Gly - #95 1 - #00 1 - #05 - - Phe Asp Val Arg Tyr Phe Asp Val
Trp Gly - #Ala Gly Thr Thr Val 110 - # 115 - # 120 - - Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro - #Ser Val Phe Pro Leu 125 - # 130 - #
135 - - Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly - #Thr Ala Ala Leu
Gly 140 - # 145 - # 150 - - Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
- #Val Thr Val Ser Trp 155 - # 160 - # 165 - - Asn Ser Gly Ala Leu
Thr Ser Gly Val His - #Thr Phe Pro Ala Val 170 - # 175 - # 180 - -
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser - #Ser Val Val Thr Val 185
- # 190 - # 195 - - Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr - #Ile
Cys Asn Val Asn 200 - # 205 - # 210 - - His Lys Pro Ser Asn Thr Lys
Val Asp Lys - #Lys Val Glu Pro Lys 215 - # 220 - # 225 - - Ser Cys
Asp Lys Thr His Thr 230 - # 232 - - - - (2) INFORMATION FOR SEQ ID
NO:34: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 232 amino -
#acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear - - (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:34: - - Glu Val Gln Leu Gln Gln Ser Gly Pro
Glu - #Leu Val Gln Pro Gly 1 - #5 - #10 - #15 - - Gly Ser Leu Arg
Leu Ser Cys Ala Thr Ser - #Gly Tyr Thr Phe Thr - #20 - #25 - #30 -
- Glu Tyr Thr Met His Trp Met Arg Gln Ala - #Pro Gly Lys Gly Leu -
#35 - #40 - #45 - - Glu Trp Val Ala Gly Ile Asn Pro Lys Asn - #Gly
Gly Thr Ser His - #50 - #55 - #60 - - Asn Gln Arg Phe Met Asp Arg
Phe Thr Ile - #Ser Val Asp Lys Ser - #65 - #70 - #75 - - Lys Asn
Thr Leu Tyr Leu Gln Met Asn Ser - #Leu Arg Ala Glu Asp - #80 - #85
- #90 - - Thr Ala Val Tyr Tyr Cys Ala Arg Trp Arg - #Gly Leu Asn
Tyr Gly - #95 1 - #00 1 - #05 - - Phe Asp Val Arg Tyr Phe Asp Val
Trp Gly - #Gln Gly Thr Leu Val 110 - # 115 - # 120 - - Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro - #Ser Val Phe Pro Leu 125 - # 130 - #
135 - - Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly - #Thr Ala Ala Leu
Gly 140 - # 145 - # 150 - - Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
- #Val Thr Val Ser Trp 155 - # 160 - # 165 - - Asn Ser Gly Ala Leu
Thr Ser Gly Val His - #Thr Phe Pro Ala Val 170 - # 175 - # 180 - -
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser - #Ser Val Val Thr Val 185
- # 190 - # 195 - - Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr - #Ile
Cys Asn Val Asn 200 - # 205 - # 210 - - His Lys Pro Ser Asn Thr Lys
Val Asp Lys - #Lys Val Glu Pro Lys 215 - # 220 - # 225 - - Ser Cys
Asp Lys Thr His Thr 230 - # 232 - - - - (2) INFORMATION FOR SEQ ID
NO:35: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 232 amino -
#acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear - - (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:35: - - Glu Val Gln Leu Val Glu Ser Gly Gly
Gly - #Leu Val Lys Pro Gly 1 - #5 - #10 - #15 - - Ala Ser Leu Arg
Leu Ser Cys Ala Thr Ser - #Gly Tyr Thr Phe Thr - #20 - #25 - #30 -
- Glu Tyr Thr Met His Trp Met Arg Gln Ala - #Pro Gly Lys Gly Leu -
#35 - #40 - #45 - - Glu Trp Val Ala Gly Ile Asn Pro Lys Asn - #Gly
Gly Thr Ser His - #50 - #55 - #60 - - Asn Gln Arg Phe Met Asp Arg
Phe Thr Ile - #Ser Val Asp Lys Ser - #65 - #70 - #75 - - Lys Asn
Thr Leu Tyr Leu Gln Met Asn Ser - #Leu Arg Ala Glu Asp - #80 - #85
- #90 - - Thr Ala Val Tyr Tyr Cys Ala Arg Trp Arg - #Gly Leu Asn
Tyr Gly - #95 1 - #00 1 - #05 - - Phe Asp Val Arg Tyr Phe Asp Val
Trp Gly - #Gln Gly Thr Leu Val 110 - # 115 - # 120 - - Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro - #Ser Val Phe Pro Leu 125 - # 130 - #
135 - - Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly - #Thr Ala Ala Leu
Gly 140 - # 145 - # 150 - - Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
- #Val Thr Val Ser Trp 155 - # 160 - # 165 - - Asn Ser Gly Ala Leu
Thr Ser Gly Val His - #Thr Phe Pro Ala Val 170 - # 175 - # 180 - -
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser - #Ser Val Val Thr Val 185
- # 190 - # 195 - - Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr - #Ile
Cys Asn Val Asn 200 - # 205 - # 210 - - His Lys Pro Ser Asn Thr Lys
Val Asp Lys - #Lys Val Glu Pro Lys 215 - # 220 - # 225 - - Ser Cys
Asp Lys Thr His Thr 230 - # 232 - - - - (2) INFORMATION FOR SEQ ID
NO:36: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 232 amino -
#acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear - - (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:36: - - Glu Val Gln Leu Val Glu Ser Gly Gly
Gly - #Leu Val Gln Pro Gly 1 - #5 - #10 - #15 - - Gly Ser Leu Lys
Ile Ser Cys Lys Thr Ser - #Gly Tyr Thr Phe Thr - #20 - #25 - #30 -
- Glu Tyr Thr Met His Trp Met Arg Gln Ala - #Pro Gly Lys Gly Leu -
#35 - #40 - #45 - - Glu Trp Val Ala Gly Ile Asn Pro Lys Asn - #Gly
Gly Thr Ser His - #50 - #55 - #60 - - Asn Gln Arg Phe Met Asp Arg
Phe Thr Ile - #Ser Val Asp Lys Ser - #65 - #70 - #75 - - Lys Asn
Thr Leu Tyr Leu Gln Met Asn Ser - #Leu Arg Ala Glu Asp - #80 - #85
- #90 - - Thr Ala Val Tyr Tyr Cys Ala Arg Trp Arg - #Gly Leu Asn
Tyr Gly - #95 1 - #00 1 - #05 - - Phe Asp Val Arg Tyr Phe Asp Val
Trp Gly - #Gln Gly Thr Leu Val 110 - # 115 - # 120 - - Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro - #Ser Val Phe Pro Leu 125 - # 130 - #
135 - - Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly - #Thr Ala Ala Leu
Gly 140 - # 145 - # 150 - - Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
- #Val Thr Val Ser Trp 155 - # 160 - # 165 - - Asn Ser Gly Ala Leu
Thr Ser Gly Val His - #Thr Phe Pro Ala Val 170 - # 175 - # 180 - -
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser - #Ser Val Val Thr Val 185
- # 190 - # 195 - - Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr - #Ile
Cys Asn Val Asn 200 - # 205 - # 210 - - His Lys Pro Ser Asn Thr Lys
Val Asp Lys - #Lys Val Glu Pro Lys 215 - # 220 - # 225 - - Ser Cys
Asp Lys Thr His Thr 230 - # 232
- - - - (2) INFORMATION FOR SEQ ID NO:37: - - (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 232 amino - #acids (B) TYPE: Amino
Acid (D) TOPOLOGY: Linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:37: - - Glu Val Gln Leu Val Glu Ser Gly Gly Gly - #Leu Val Gln
Pro Gly 1 - #5 - #10 - #15 - - Gly Ser Leu Arg Leu Ser Cys Ala Thr
Ser - #Gly Tyr Thr Phe Thr - #20 - #25 - #30 - - Glu Tyr Thr Met
His Trp Met Lys Gln Ser - #His Gly Lys Ser Leu - #35 - #40 - #45 -
- Glu Trp Val Ala Gly Ile Asn Pro Lys Asn - #Gly Gly Thr Ser His -
#50 - #55 - #60 - - Asn Gln Arg Phe Met Asp Arg Phe Thr Ile - #Ser
Val Asp Lys Ser - #65 - #70 - #75 - - Lys Asn Thr Leu Tyr Leu Gln
Met Asn Ser - #Leu Arg Ala Glu Asp - #80 - #85 - #90 - - Thr Ala
Val Tyr Tyr Cys Ala Arg Trp Arg - #Gly Leu Asn Tyr Gly - #95 1 -
#00 1 - #05 - - Phe Asp Val Arg Tyr Phe Asp Val Trp Gly - #Gln Gly
Thr Leu Val 110 - # 115 - # 120 - - Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro - #Ser Val Phe Pro Leu 125 - # 130 - # 135 - - Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly - #Thr Ala Ala Leu Gly 140 - # 145 - #
150 - - Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro - #Val Thr Val Ser
Trp 155 - # 160 - # 165 - - Asn Ser Gly Ala Leu Thr Ser Gly Val His
- #Thr Phe Pro Ala Val 170 - # 175 - # 180 - - Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser - #Ser Val Val Thr Val 185 - # 190 - # 195 - -
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr - #Ile Cys Asn Val Asn 200
- # 205 - # 210 - - His Lys Pro Ser Asn Thr Lys Val Asp Lys - #Lys
Val Glu Pro Lys 215 - # 220 - # 225 - - Ser Cys Asp Lys Thr His Thr
230 - # 232 - - - - (2) INFORMATION FOR SEQ ID NO:38: - - (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 232 amino - #acids (B) TYPE:
Amino Acid (D) TOPOLOGY: Linear - - (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:38: - - Glu Val Gln Leu Val Glu Ser Gly Gly Gly - #Leu Val
Gln Pro Gly 1 - #5 - #10 - #15 - - Gly Ser Leu Arg Leu Ser Cys Ala
Thr Ser - #Gly Tyr Thr Phe Thr - #20 - #25 - #30 - - Glu Tyr Thr
Met His Trp Met Arg Gln Ala - #Pro Gly Lys Gly Leu - #35 - #40 -
#45 - - Glu Trp Ile Gly Gly Phe Asn Pro Lys Asn - #Gly Gly Thr Ser
His - #50 - #55 - #60 - - Asn Gln Arg Phe Met Asp Arg Phe Thr Ile -
#Ser Val Asp Lys Ser - #65 - #70 - #75 - - Lys Asn Thr Leu Tyr Leu
Gln Met Asn Ser - #Leu Arg Ala Glu Asp - #80 - #85 - #90 - - Thr
Ala Val Tyr Tyr Cys Ala Arg Trp Arg - #Gly Leu Asn Tyr Gly - #95 1
- #00 1 - #05 - - Phe Asp Val Arg Tyr Phe Asp Val Trp Gly - #Gln
Gly Thr Leu Val 110 - # 115 - # 120 - - Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro - #Ser Val Phe Pro Leu 125 - # 130 - # 135 - - Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly - #Thr Ala Ala Leu Gly 140 - # 145
- # 150 - - Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro - #Val Thr Val
Ser Trp 155 - # 160 - # 165 - - Asn Ser Gly Ala Leu Thr Ser Gly Val
His - #Thr Phe Pro Ala Val 170 - # 175 - # 180 - - Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser - #Ser Val Val Thr Val 185 - # 190 - # 195
- - Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr - #Ile Cys Asn Val Asn
200 - # 205 - # 210 - - His Lys Pro Ser Asn Thr Lys Val Asp Lys -
#Lys Val Glu Pro Lys 215 - # 220 - # 225 - - Ser Cys Asp Lys Thr
His Thr 230 - # 232 - - - - (2) INFORMATION FOR SEQ ID NO:39: - -
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 214 amino - #acids (B)
TYPE: Amino Acid (D) TOPOLOGY: Linear - - (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:39: - - Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser - #Leu Ser Ala Ser Val 1 - #5 - #10 - #15 - - Gly Asp Arg Val
Thr Ile Thr Cys Arg Ala - #Ser Gln Asp Ile Asn - #20 - #25 - #30 -
- Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro - #Gly Lys Ala Pro Lys -
#35 - #40 - #45 - - Leu Leu Ile Tyr Tyr Thr Ser Thr Leu Glu - #Ser
Gly Val Pro Ser - #50 - #55 - #60 - - Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp - #Tyr Thr Leu Thr Ile - #65 - #70 - #75 - - Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr - #Tyr Tyr Cys Gln Gln - #80 - #85
- #90 - - Gly Asn Thr Leu Pro Pro Thr Phe Gly Gln - #Gly Thr Lys
Val Glu - #95 1 - #00 1 - #05 - - Ile Lys Arg Thr Val Ala Ala Pro
Ser Val - #Phe Ile Phe Pro Pro 110 - # 115 - # 120 - - Ser Asp Glu
Gln Leu Lys Ser Gly Thr Ala - #Ser Val Val Cys Leu 125 - # 130 - #
135 - - Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys - #Val Gln Trp Lys
Val 140 - # 145 - # 150 - - Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
- #Glu Ser Val Thr Glu 155 - # 160 - # 165 - - Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Leu - #Ser Ser Thr Leu Thr 170 - # 175 - # 180 - -
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys - #Val Tyr Ala Cys Glu 185
- # 190 - # 195 - - Val Thr His Gln Gly Leu Ser Ser Pro Val - #Thr
Lys Ser Phe Asn 200 - # 205 - # 210 - - Arg Gly Glu Cys 214 - - - -
(2) INFORMATION FOR SEQ ID NO:40: - - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 214 amino - #acids (B) TYPE: Amino Acid (D) TOPOLOGY:
Linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: - - Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser - #Leu Ser Ala Ser Val 1 - #5 - #10 -
#15 - - Gly Asp Arg Val Thr Ile Thr Cys Arg Ala - #Ser Gln Asp Ile
Asn - #20 - #25 - #30 - - Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro -
#Gly Lys Ala Pro Lys - #35 - #40 - #45 - - Leu Leu Ile Tyr Tyr Thr
Ser Thr Leu His - #Ser Gly Val Pro Ser - #50 - #55 - #60 - - Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp - #Tyr Thr Leu Thr Ile - #65 -
#70 - #75 - - Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr - #Tyr Tyr
Cys Gln Gln - #80 - #85 - #90 - - Gly Asn Thr Leu Pro Pro Thr Phe
Gly Gln - #Gly Thr Lys Val Glu - #95 1 - #00 1 - #05 - - Ile Lys
Arg Thr Val Ala Ala Pro Ser Val - #Phe Ile Phe Pro Pro 110 - # 115
- # 120 - - Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala - #Ser Val Val
Cys Leu 125 - # 130 - # 135 - - Leu Asn Asn Phe Tyr Pro Arg Glu Ala
Lys - #Val Gln Trp Lys Val 140 - # 145 - # 150 - - Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln - #Glu Ser Val Thr Glu 155 - # 160 - # 165
- - Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu - #Ser Ser Thr Leu Thr
170 - # 175 - # 180 - - Leu Ser Lys Ala Asp Tyr Glu Lys His Lys -
#Val Tyr Ala Cys Glu 185 - # 190 - # 195 - - Val Thr His Gln Gly
Leu Ser Ser Pro Val - #Thr Lys Ser Phe Asn 200 - # 205 - # 210 - -
Arg Gly Glu Cys 214 - - - - (2) INFORMATION FOR SEQ ID NO:41: - -
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 107 amino - #acids (B)
TYPE: Amino Acid (D) TOPOLOGY: Linear - - (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:41: - - Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser - #Leu Ser Ala Ser Val 1 - #5 - #10 - #15 - - Gly Asp Arg Val
Thr Ile Thr Cys Arg Ala - #Ser Gln Asp Val Asn - #20 - #25 - #30 -
- Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro - #Gly Lys Ala Pro Lys -
#35 - #40 - #45 - - Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr - #Ser
Gly Val Pro Ser - #50 - #55 - #60 - - Arg Phe Ser Gly Ser Arg Ser
Gly Thr Asp - #Phe Thr Leu Thr Ile - #65 - #70 - #75 - - Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr - #Tyr Tyr Cys Gln Gln - #80 - #85
- #90 - - His Tyr Thr Thr Pro Pro Thr Phe Gly Gln - #Gly Thr Lys
Val Glu - #95 1 - #00 1 - #05 - - Ile Lys 107 - - - - (2)
INFORMATION FOR SEQ ID NO:42: - - (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 120 amino - #acids (B) TYPE: Amino Acid (D) TOPOLOGY:
Linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: - - Glu Val Gln
Leu Val Glu Ser Gly Gly Gly - #Leu Val Gln Pro Gly 1 - #5 - #10 -
#15 - - Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser - #Gly Phe Asn Ile
Lys - #20 - #25 - #30 - - Asp Thr Tyr Ile His Trp Val Arg Gln Ala -
#Pro Gly Lys Gly Leu - #35 - #40 - #45 - - Glu Trp Val Ala Arg Ile
Tyr Pro Thr Asn - #Gly Tyr Thr Arg Tyr - #50 - #55 - #60 - - Ala
Asp Ser Val Lys Gly Arg Phe Thr Ile - #Ser Ala Asp Thr Ser - #65 -
#70 - #75 - - Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser - #Leu Arg
Ala Glu Asp - #80 - #85 - #90 - - Thr Ala Val Tyr Tyr Cys Ser Arg
Trp Gly - #Gly Asp Gly Phe Tyr - #95 1 - #00 1 - #05 - - Ala Met
Asp Tyr Trp Gly Gln Gly Thr Leu - #Val Thr Val Ser Ser 110 - # 115
- # 120 - - - - (2) INFORMATION FOR SEQ ID NO:43: - - (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 107 amino - #acids (B) TYPE: Amino
Acid (D) TOPOLOGY: Linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:43: - - Asp Ile Gln Met Thr Gln Ser Pro Ser Ser - #Leu Ser Ala
Ser Val 1 - #5 - #10 - #15 - - Gly Asp Arg Val Thr Ile Thr Cys Arg
Ala - #Ser Gln Asp Val Asn - #20 - #25 - #30 - - Thr Ala Val Ala
Trp Tyr Gln Gln Lys Pro - #Gly Lys Ala Pro Lys - #35 - #40 - #45 -
- Leu Leu Ile Tyr Ser Ala Ser Phe Leu Xaa - #Ser Gly Val Pro Ser -
#50 - #55 - #60 - - Arg Phe Ser Gly Ser Xaa Ser Gly Thr Asp - #Phe
Thr Leu Thr Ile - #65 - #70 - #75 - - Ser Ser Leu Gln Pro Glu Asp
Phe Ala Thr - #Tyr Tyr Cys Gln Gln - #80 - #85 - #90 - - His Tyr
Thr Thr Pro Pro Thr Phe Gly Gln - #Gly Thr Lys Val Glu - #95 1 -
#00 1 - #05 - - Ile Lys 107 - - - - (2) INFORMATION FOR SEQ ID
NO:44: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 120 amino -
#acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear - - (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:44: - - Glu Val Gln Leu Val Glu Ser Gly Gly
Gly - #Leu Val Gln Pro Gly 1 - #5 - #10 - #15 - - Gly Ser Leu Arg
Leu Ser Cys Ala Ala Ser - #Gly Phe Asn Ile Lys - #20 - #25 - #30 -
- Asp Thr Tyr Ile His Trp Val Arg Gln Ala - #Pro Gly Lys Gly Leu -
#35 - #40 - #45 - - Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn - #Gly
Tyr Thr Arg Tyr - #50 - #55 - #60 - - Ala Asp Ser Val Lys Gly Arg
Phe Thr Ile - #Ser Xaa Asp Xaa Ser - #65 - #70 - #75 - - Lys Asn
Thr Xaa Tyr Leu Gln Met Asn Ser - #Leu Arg Ala Glu Asp - #80 - #85
- #90 - - Thr Ala Val Tyr Tyr Cys Xaa Arg Trp Gly - #Gly Asp Gly
Phe Tyr - #95 1 - #00 1 - #05 - - Ala Met Asp Xaa Trp Gly Gln Gly
Thr Leu - #Val Thr Val Ser Ser 110 - # 115 - # 120 - - - - (2)
INFORMATION FOR SEQ ID NO:45: - - (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 122 amino - #acids (B) TYPE: Amino Acid (D) TOPOLOGY:
Linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: - - Glu Val Gln
Leu Val Glu Ser Gly Gly Gly - #Leu Val Gln Pro Gly 1 - #5 - #10 -
#15 - - Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser - #Gly Tyr Ser Phe
Thr - #20 - #25 - #30 - - Gly Tyr Thr Met Asn Trp Val Arg Gln Ala -
#Pro Gly Lys Gly Leu - #35 - #40 - #45 - - Glu Trp Val Ala Leu Ile
Asn Pro Tyr Lys - #Gly Val Thr Thr Tyr - #50 - #55 - #60 - - Ala
Asp Ser Val Lys Gly Arg Phe Thr Ile - #Ser Val Asp Lys Ser
- #65 - #70 - #75 - - Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser -
#Leu Arg Ala Glu Asp - #80 - #85 - #90 - - Thr Ala Val Tyr Tyr Cys
Ala Arg Ser Gly - #Tyr Tyr Gly Asp Ser - #95 1 - #00 1 - #05 - -
Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly - #Thr Leu Val Thr Val 110
- # 115 - # 120 - - Ser Ser 122 - - - - (2) INFORMATION FOR SEQ ID
NO:46: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 124 amino -
#acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear - - (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:46: - - Glu Val Gln Leu Val Glu Ser Gly Gly
Gly - #Leu Val Gln Pro Gly 1 - #5 - #10 - #15 - - Gly Ser Leu Arg
Leu Ser Cys Ala Thr Ser - #Gly Tyr Thr Phe Thr - #20 - #25 - #30 -
- Glu Tyr Thr Met His Trp Met Arg Gln Ala - #Pro Gly Lys Gly Leu -
#35 - #40 - #45 - - Glu Trp Val Ala Gly Ile Asn Pro Lys Asn - #Gly
Gly Thr Ser His - #50 - #55 - #60 - - Asn Gln Arg Phe Met Asp Arg
Phe Thr Ile - #Ser Val Asp Lys Ser - #65 - #70 - #75 - - Thr Ser
Thr Ala Tyr Met Gln Met Asn Ser - #Leu Arg Ala Glu Asp - #80 - #85
- #90 - - Thr Ala Val Tyr Tyr Cys Ala Arg Trp Arg - #Gly Leu Asn
Tyr Gly - #95 1 - #00 1 - #05 - - Phe Asp Val Arg Tyr Phe Asp Val
Trp Gly - #Gln Gly Thr Leu Val 110 - # 115 - # 120 - - Thr Val Ser
Ser 124 - - - - (2) INFORMATION FOR SEQ ID NO:47: - - (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 109 amino - #acids (B) TYPE: Amino
Acid (D) TOPOLOGY: Linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:47: - - Asp Ile Gln Met Thr Gln Ser Pro Ser Ser - #Leu Ser Ala
Ser Val 1 - #5 - #10 - #15 - - Gly Asp Arg Val Thr Ile Thr Cys Arg
Ala - #Ser Gln Asp Ile Asn - #20 - #25 - #30 - - Asn Tyr Leu Asn
Trp Tyr Gln Gln Lys Pro - #Gly Lys Ala Pro Lys - #35 - #40 - #45 -
- Leu Leu Ile Tyr Tyr Thr Ser Thr Leu His - #Ser Gly Val Pro Ser -
#50 - #55 - #60 - - Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp - #Tyr
Thr Leu Thr Ile - #65 - #70 - #75 - - Ser Ser Leu Gln Pro Glu Asp
Phe Ala Thr - #Tyr Tyr Cys Gln Gln - #80 - #85 - #90 - - Gly Asn
Thr Leu Pro Pro Thr Phe Gly Gln - #Gly Thr Lys Val Glu - #95 1 -
#00 1 - #05 - - Ile Lys Arg Thr 109
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