Compositions and methods for the therapy and diagnosis of cancer,
such as breast cancer, are disclosed. Compositions may comprise
one or more breast tumor proteins, immunogenic portions thereof,
or polynucleotides that encode such portions. Alternatively, a therapeutic
composition may comprise an antigen presenting cell that expresses
a breast tumor protein, or a T cell that is specific for cells expressing
such a protein. Such compositions may be used, for example, for
the prevention and treatment of diseases such as breast cancer.
Diagnostic methods based on detecting a breast tumor protein, or
mRNA encoding such a protein, in a sample are also provided.
What is claimed is:
1. An isolated polypeptide comprising SEQ ID NO:475.
2. A fusion protein comprising at least one polypeptide according
to claim 1.
3. A fusion protein according to claim 2, wherein the fusion protein
comprises an expression enhancer that increases expression of the
fusion protein in a host cell transfected with a polynucleotide
encoding the fusion protein.
4. A fusion protein according to claim 2, wherein the fusion protein
comprises a T helper epitope that is not present within the polypeptide
of claim 1.
5. A fusion protein according to claim 2, wherein the fusion protein
comprises an affinity tag.
6. The isolated polypeptide according to claim 1, wherein the polypeptide
comprises an amino acid encoded by SEQ ID NO:474.
TECHNICAL FIELD OF THE INVENTION
The present invention relates generally to therapy and diagnosis
of cancer, such as breast cancer. The invention is more specifically
related to polypeptides comprising at least a portion of a breast
tumor protein, and to polynucleotides encoding such polypeptides.
Such polypeptides and polynucleotides may be used in compositions
for prevention and treatment of breast cancer, and for the diagnosis
and monitoring of such cancers.
BACKGROUND OF THE INVENTION
Breast cancer is a significant health problem for women in the
United States and throughout the world. Although advances have been
made in detection and treatment of the disease, breast cancer remains
the second leading cause of cancer-related deaths in women, affecting
more than 180,000 women in the United States each year. For women
in North America, the life-time odds of getting breast cancer are
one in eight.
No vaccine or other universally successful method for the prevention
or treatment of breast cancer is currently available. Management
of the disease currently relies on a combination of early diagnosis
(through routine breast screening procedures) and aggressive treatment,
which may include one or more of a variety of treatments such as
surgery, radiotherapy, chemotherapy and hormone therapy. The course
of treatment for a particular breast cancer is often selected based
on a variety of prognostic parameters, including an analysis of
specific tumor markers. See, e.g., Porter-Jordan and Lippman, Breast
Cancer 8:73-100 (1994). However, the use of established markers
often leads to a result that is difficult to interpret, and the
high mortality observed in breast cancer patients indicates that
improvements are needed in the treatment, diagnosis and prevention
of the disease.
Accordingly, there is a need in the art for improved methods for
the treatment and diagnosis of breast cancer. The present invention
fulfills these needs and further provides other related advantages.
SUMMARY OF THE INVENTION
Briefly stated, the present invention provides compositions and
methods for the diagnosis and therapy of cancer, such as breast
cancer. In one aspect, the present invention provides polypeptides
comprising at least a portion of a breast tumor protein, or a variant
thereof. Certain portions and other variants are immunogenic, such
that the ability of the variant to react with antigen-specific antisera
is not substantially diminished. Within certain embodiments, the
polypeptide comprises a sequence that is encoded by a polynucleotide
sequence selected from the group consisting of: (a) sequences recited
in SEQ ID NO:1-175, 178, 180, 182-468, 474, 476, 477 479, 484, 486
and 489; (b) variants of a sequence recited in SEQ ID NO:1-175,
178, 180, 182-468, 474, 476, 477, 479, 484, 486 and 489; and (c)
complements of a sequence of (a) or (b). In specific embodiments,
the polypeptides of the present invention comprise at least a portion
of a tumor protein that includes an amino acid sequence selected
from the group consisting of sequences recited in SEQ ID NO:176,
179, 181, 469-473, 475, 485, 487 and 488, and variants thereof.
The present invention further provides polynucleotides that encode
a polypeptide as described above, or a portion thereof (such as
a portion encoding at least 15 amino acid residues of a breast tumor
protein), expression vectors comprising such polynucleotides and
host cells transformed or transfected with such expression vectors.
Within other aspects, the present invention provides pharmaceutical
compositions comprising a polypeptide or polynucleotide as described
above and a physiologically acceptable carrier.
Within a related aspect of the present invention, immunogenic compositions,
or vaccines for prophylactic or therapeutic use are provided. Such
compositions comprise a polypeptide or polynucleotide as described
above and an immunostimulant.
The present invention further provides pharmaceutical compositions
that comprise: (a) an antibody or antigen-binding fragment thereof
that specifically binds to a breast tumor protein; and (b) a physiologically
Within further aspects, the present invention provides pharmaceutical
compositions comprising: (a) an antigen presenting cell that expresses
a polypeptide as described above and (b) a pharmaceutically acceptable
carrier or excipient. Antigen presenting cells include dendritic
cells, macrophages, monocytes, fibroblasts and B cells.
Within related aspects, immunogenic compositions, or vaccines,
are provided that comprise: (a) an antigen presenting cell that
expresses a polypeptide as described above and (b) an immunostimulant.
The present invention further provides, in other aspects, fusion
proteins that comprise at least one polypeptide as described above,
as well as polynucleotides encoding such fusion proteins.
Within related aspects, pharmaceutical compositions comprising
a fusion protein, or a polynucleotide encoding a fusion protein,
in combination with a physiologically acceptable carrier are provided.
Compositions are further provided, within other aspects, that comprise
a fusion protein, or a polynucleotide encoding a fusion protein,
in combination with an immunostimulant.
Within further aspects, the present invention provides methods
for inhibiting the development of a cancer in a patient, comprising
administering to a patient a composition as recited above. The patient
may be afflicted with breast cancer, in which case the methods provide
treatment for the disease, or patient considered at risk for such
a disease may be treated prophylactically.
The present invention further provides, within other aspects, methods
for removing tumor cells from a biological sample, comprising contacting
a biological sample with T cells that specifically react with a
breast tumor protein, wherein the step of contacting is performed
under conditions and for a time sufficient to permit the removal
of cells expressing the protein from the sample.
Within related aspects, methods are provided for inhibiting the
development of a cancer in a patient, comprising administering to
a patient a biological sample treated as described above.
Methods are further provided, within other aspects, for stimulating
and/or expanding T cells specific for a breast tumor protein, comprising
contacting T cells with one or more of: (i) a polypeptide as described
above; (ii) a polynucleotide encoding such a polypeptide; and/or
(iii) an antigen presenting cell that expresses such a polypeptide;
under conditions and for a time sufficient to permit the stimulation
and/or expansion of T cells. Isolated T cell populations comprising
T cells prepared as described above are also provided.
Within further aspects, the present invention provides methods
for inhibiting the development of a cancer in a patient, comprising
administering to a patient an effective amount of a T cell population
as described above.
The present invention further provides methods for inhibiting the
development of a cancer in a patient, comprising the steps of: (a)
incubating CD4.sup.+ and/or CD8.sup.+ T cells isolated from a patient
with one or more of: (i) a polypeptide comprising at least an immunogenic
portion of a breast tumor protein; (ii) a polynucleotide encoding
such a polypeptide; and (iii) an antigen-presenting cell that expressed
such a polypeptide; and (b) administering to the patient an effective
amount of the proliferated T cells, and thereby inhibiting the development
of a cancer in the patient. Proliferated cells may, but need not,
be cloned prior to administration to the patient.
Within further aspects, the present invention provides methods
for determining the presence or absence of a cancer in a patient,
comprising: (a) contacting a biological sample obtained from a patient
with a binding agent that binds to a polypeptide as recited above;
(b) detecting in the sample an amount of polypeptide that binds
to the binding agent; and (c) comparing the amount of polypeptide
with a predetermined cut-off value, and therefrom determining the
presence or absence of a cancer in the patient. Within preferred
embodiments, the binding agent is an antibody, more preferably a
monoclonal antibody. The cancer may be breast cancer.
The present invention also provides, within other aspects, methods
for monitoring the progression of a cancer in a patient. Such methods
comprise the steps of: (a) contacting a biological sample obtained
from a patient at a first point in time with a binding agent that
binds to a polypeptide as recited above; (b) detecting in the sample
an amount of polypeptide that binds to the binding agent; (c) repeating
steps (a) and (b) using a biological sample obtained from the patient
at a subsequent point in time; and (d) comparing the amount of polypeptide
detected in step (c) with the amount detected in step (b) and therefrom
monitoring the progression of the cancer in the patient.
The present invention further provides, within other aspects, methods
for determining the presence or absence of a cancer in a patient,
comprising the steps of: (a) contacting a biological sample obtained
from a patient with an oligonucleotide that hybridizes to a polynucleotide
that encodes a breast tumor protein; (b) detecting in the sample
a level of a polynucleotide, preferably mRNA, that hybridizes to
the oligonucleotide; and (c) comparing the level of polynucleotide
that hybridizes to the oligonucleotide with a predetermined cut-off
value, and therefrom determining the presence or absence of a cancer
in the patient. Within certain embodiments, the amount of mRNA is
detected via polymerase chain reaction using, for example, at least
one oligonucleotide primer that hybridizes to a polynucleotide encoding
a polypeptide as recited above, or a complement of such a polynucleotide.
Within other embodiments, the amount of mRNA is detected using a
hybridization technique, employing an oligonucleotide probe that
hybridizes to a polynucleotide that encodes a polypeptide as recited
above, or a complement of such a polynucleotide.
In related aspects, methods are provided for monitoring the progression
of a cancer in a patient, comprising the steps of: (a) contacting
a biological sample obtained from a patient with an oligonucleotide
that hybridizes to a polynucleotide that encodes a breast tumor
protein; (b) detecting in the sample an amount of a polynucleotide
that hybridizes to the oligonucleotide; (c) repeating steps (a)
and (b) using a biological sample obtained from the patient at a
subsequent point in time; and (d) comparing the amount of polynucleotide
detected in step (c) with the amount detected in step (b) and therefrom
monitoring the progression of the cancer in the patient.
Within further aspects, the present invention provides antibodies,
such as monoclonal antibodies, that bind to a polypeptide as described
above, as well as diagnostic kits comprising such antibodies. Diagnostic
kits comprising one or more oligonucleotide probes or primers as
described above are also provided.
These and other aspects of the present invention will become apparent
upon reference to the following detailed description and attached
drawings. All references disclosed herein are hereby incorporated
by reference in their entirety as if each was incorporated individually.
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE IDENTIFIERS
FIG. 1 shows the results of a Northern blot of the clone SYN18C6
(SEQ ID NO:40).
SEQ ID NO:1 is the determined cDNA sequence of JBT2.
SEQ ID NO:2 is the determined cDNA sequence of JBT6.
SEQ ID NO:3 is the determined cDNA sequence of JBT7.
SEQ ID NO:4 is the determined cDNA sequence of JBT10.
SEQ ID NO:5 is the determined cDNA sequence of JBT13.
SEQ ID NO:6 is the determined cDNA sequence of JBT14.
SEQ ID NO:7 is the determined cDNA sequence of JBT15.
SEQ ID NO:8 is the determined cDNA sequence of JBT16.
SEQ ID NO:9 is the determined cDNA sequence of JBT17.
SEQ ID NO:10 is the determined cDNA sequence of JBT22.
SEQ ID NO:11 is the determined cDNA sequence of JBT25.
SEQ ID NO:12 is the determined cDNA sequence of JBT28.
SEQ ID NO:13 is the determined cDNA sequence of JBT32.
SEQ ID NO:14 is the determined cDNA sequence of JBT33.
SEQ ID NO:15 is the determined cDNA sequence of JBT34.
SEQ ID NO:16 is the determined cDNA sequence of JBT36.
SEQ ID NO:17 is the determined cDNA sequence of JBT37.
SEQ ID NO:18 is the determined cDNA sequence of JBT51.
SEQ ID NO:19 is the determined cDNA sequence of JBTT1.
SEQ ID NO:20 is the determined cDNA sequence of JBTT7.
SEQ ID NO:21 is the determined cDNA sequence of JBTT11.
SEQ ID NO:22 is the determined cDNA sequence of JBTT14.
SEQ ID NO:23 is the determined cDNA sequence of JBTT18.
SEQ ID NO:24 is the determined cDNA sequence of JBTT19.
SEQ ID NO:25 is the determined cDNA sequence of JBTT20.
SEQ ID NO:26 is the determined cDNA sequence of JBTT21.
SEQ ID NO:27 is the determined cDNA sequence of JBTT22.
SEQ ID NO:28 is the determined cDNA sequence of JBTT28.
SEQ ID NO:29 is the determined cDNA sequence of JBTT29.
SEQ ID NO:30 is the determined cDNA sequence of JBTT33.
SEQ ID NO:31 is the determined cDNA sequence of JBTT37.
SEQ ID NO:32 is the determined cDNA sequence of JBTT38.
SEQ ID NO:33 is the determined cDNA sequence of JBTT47.
SEQ ID NO:34 is the determined cDNA sequence of JBTT48.
SEQ ID NO:35 is the determined cDNA sequence of JBTT50.
SEQ ID NO:36 is the determined cDNA sequence of JBTT51.
SEQ ID NO:37 is the determined cDNA sequence of JBTT52.
SEQ ID NO:38 is the determined cDNA sequence of JBTT54.
SEQ ID NO:39 is the determined cDNA sequence of SYN17F4.
SEQ ID NO:40 is the determined cDNA sequence of SYN18C6 (also known
SEQ ID NO:41 is the determined cDNA sequence of SYN19A2.
SEQ ID NO:42 is the determined cDNA sequence of SYN19C8.
SEQ ID NO:43 is the determined cDNA sequence of SYN20A12.
SEQ ID NO:44 is the determined cDNA sequence of SYN20G6.
SEQ ID NO:45 is the determined cDNA sequence of SYN20G6-2.
SEQ ID NO:46 is the determined cDNA sequence of SYN21B9.
SEQ ID NO:47 is the determined cDNA sequence of SYN21B9-2.
SEQ ID NO:48 is the determined cDNA sequence of SYN21C10.
SEQ ID NO:49 is the determined cDNA sequence of SYN21G10.
SEQ ID NO:50 is the determined cDNA sequence of SYN21G10-2.
SEQ ID NO:51 is the determined cDNA sequence of SYN21G11.
SEQ ID NO:52 is the determined cDNA sequence of SYN21G11-2.
SEQ ID NO:53 is the determined cDNA sequence of SYN21H8.
SEQ ID NO:54 is the determined cDNA sequence of SYN22A10.
SEQ ID NO:55 is the determined cDNA sequence of SYN22A10-2.
SEQ ID NO:56 is the determined cDNA sequence of SYN22A12.
SEQ ID NO:57 is the determined cDNA sequence of SYN22A2.
SEQ ID NO:58 is the determined cDNA sequence of SYN22B4.
SEQ ID NO:59 is the determined cDNA sequence of SYN22C2.
SEQ ID NO:60 is the determined cDNA sequence of SYN22E10.
SEQ ID NO:61 is the determined cDNA sequence of SYN22F2.
SEQ ID NO:62 is a predicted amino acid sequence for SYN18C6 (also
known as B709P).
SEQ ID NO:63 is the determined cDNA sequence of B723P.
SEQ ID NO:64 is the determined cDNA sequence for B724P.
SEQ ID NO:65 is the determined cDNA sequence of B770P.
SEQ ID NO:66 is the determined cDNA sequence of B716P.
SEQ ID NO:67 is the determined cDNA sequence of B725P.
SEQ ID NO:68 is the determined cDNA sequence of B717P.
SEQ ID NO:69 is the determined cDNA sequence of B771P.
SEQ ID NO:70 is the determined cDNA sequence of B722P.
SEQ ID NO:71 is the determined cDNA sequence of B726P.
SEQ ID NO:72 is the determined cDNA sequence of B727P.
SEQ ID NO:73 is the determined cDNA sequence of B728P.
SEQ ID NO:74-87 are the determined cDNA sequences of isolated clones
which show homology to known sequences.
SEQ ID NO:88 is the determined cDNA sequence of 13053.
SEQ ID NO:89 is the determined cDNA sequence of 13057.
SEQ ID NO:90 is the determined cDNA sequence of 13059.
SEQ ID NO:91 is the determined cDNA sequence of 13065.
SEQ ID NO:92 is the determined cDNA sequence of 13067.
SEQ ID NO:93 is the determined cDNA sequence of 13068.
SEQ ID NO:94 is the determined cDNA sequence of 13071.
SEQ ID NO:95 is the determined cDNA sequence of 13072.
SEQ ID NO:96 is the determined cDNA sequence of 13073.
SEQ ID NO:97 is the determined cDNA sequence of 13075.
SEQ ID NO:98 is the determined cDNA sequence of 13078.
SEQ ID NO:99 is the determined cDNA sequence of 13079.
SEQ ID NO:100 is the determined cDNA sequence of 13081.
SEQ ID NO:101 is the determined cDNA sequence of 13082.
SEQ ID NO:102 is the determined cDNA sequence of 13092.
SEQ ID NO:103 is the determined cDNA sequence of 13097.
SEQ ID NO:104 is the determined cDNA sequence of 13101.
SEQ ID NO:105 is the determined cDNA sequence of 13102.
SEQ ID NO:106 is the determined cDNA sequence of 13119.
SEQ ID NO:107 is the determined cDNA sequence of 13131.
SEQ ID NO:108 is the determined cDNA sequence of 13133.
SEQ ID NO:109 is the determined cDNA sequence of 13135.
SEQ ID NO:110 is the determined cDNA sequence of 13139.
SEQ ID NO:111 is the determined cDNA sequence of 13140.
SEQ ID NO:112 is the determined cDNA sequence of 13146.
SEQ ID NO:113 is the determined cDNA sequence of 13147.
SEQ ID NO:114 is the determined cDNA sequence of 13148.
SEQ ID NO:115 is the determined cDNA sequence of 13149.
SEQ ID NO:116 is the determined cDNA sequence of 13151.
SEQ ID NO:117 is the determined cDNA sequence of 13051
SEQ ID NO:118 is the determined cDNA sequence of 13052
SEQ ID NO:119 is the determined cDNA sequence of 13055
SEQ ID NO:120 is the determined cDNA sequence of 13058
SEQ ID NO:121 is the determined cDNA sequence of 13062
SEQ ID NO:122 is the determined cDNA sequence of 13064
SEQ ID NO:123 is the determined cDNA sequence of 13080
SEQ ID NO:124 is the determined cDNA sequence of 13093
SEQ ID NO:125 is the determined cDNA sequence of 13094
SEQ ID NO:126 is the determined cDNA sequence of 13095
SEQ ID NO:127 is the determined cDNA sequence of 13096
SEQ ID NO:128 is the determined cDNA sequence of 13099
SEQ ID NO:129 is the determined cDNA sequence of 13100
SEQ ID NO:130 is the determined cDNA sequence of 13103
SEQ ID NO:131 is the determined cDNA sequence of 13106
SEQ ID NO:132 is the determined cDNA sequence of 13107
SEQ ID NO:133 is the determined cDNA sequence of 13108
SEQ ID NO:134 is the determined cDNA sequence of 13121
SEQ ID NO:135 is the determined cDNA sequence of 13126
SEQ ID NO:136 is the determined cDNA sequence of 13129
SEQ ID NO:137 is the determined cDNA sequence of 13130
SEQ ID NO:138 is the determined cDNA sequence of 13134
SEQ ID NO:139 is the determined cDNA sequence of 13141
SEQ ID NO:140 is the determined cDNA sequence of 13142
SEQ ID NO:141 is the determined cDNA sequence of 14376
SEQ ID NO:142 is the determined cDNA sequence of 14377
SEQ ID NO:143 is the determined cDNA sequence of 14383
SEQ ID NO:144 is the determined cDNA sequence of 14384
SEQ ID NO:145 is the determined cDNA sequence of 14387
SEQ ID NO:146 is the determined cDNA sequence of 14392
SEQ ID NO:147 is the determined cDNA sequence of 14394
SEQ ID NO:148 is the determined cDNA sequence of 14398
SEQ ID NO:149 is the determined cDNA sequence of 14401
SEQ ID NO:150 is the determined cDNA sequence of 14402
SEQ ID NO:151 is the determined cDNA sequence of 14405
SEQ ID NO:152 is the determined cDNA sequence of 14409
SEQ ID NO:153 is the determined cDNA sequence of 14412
SEQ ID NO:154 is the determined cDNA sequence of 14414
SEQ ID NO:155 is the determined cDNA sequence of 14415
SEQ ID NO:156 is the determined cDNA sequence of 14416
SEQ ID NO:157 is the determined cDNA sequence of 14419
SEQ ID NO:158 is the determined cDNA sequence of 14426
SEQ ID NO:159 is the determined cDNA sequence of 14427
SEQ ID NO:160 is the determined cDNA sequence of 14375
SEQ ID NO:161 is the determined cDNA sequence of 14378
SEQ ID NO:162 is the determined cDNA sequence of 14379
SEQ ID NO:163 is the determined cDNA sequence of 14380
SEQ ID NO:164 is the determined cDNA sequence of 14381
SEQ ID NO:165 is the determined cDNA sequence of 14382
SEQ ID NO:166 is the determined cDNA sequence of 14388
SEQ ID NO:167 is the determined cDNA sequence of 14399
SEQ ID NO:168 is the determined cDNA sequence of 14406
SEQ ID NO:169 is the determined cDNA sequence of 14407
SEQ ID NO:170 is the determined cDNA sequence of 14408
SEQ ID NO:171 is the determined cDNA sequence of 14417
SEQ ID NO:172 is the determined cDNA sequence of 14418
SEQ ID NO:173 is the determined cDNA sequence of 14423
SEQ ID NO:174 is the determined cDNA sequence of 14424
SEQ ID NO:175 is the determined cDNA sequence of B726P-20
SEQ ID NO:176 is the predicted amino acid sequence of B726P-20
SEQ ID NO:177 is a PCR primer
SEQ ID NO:178 is the determined cDNA sequence of B726P-74
SEQ ID NO:179 is the predicted amino acid sequence of B726P-74
SEQ ID NO:180 is the determined cDNA sequence of B726P-79
SEQ ID NO:181 is the predicted amino acid sequence of B726P-79
SEQ ID NO:182 is the determined cDNA sequence of 19439.1, showing
homology to the mammaglobin gene
SEQ ID NO:183 is the determined cDNA sequence of 19407.1, showing
homology to the human keratin gene
SEQ ID NO:184 is the determined cDNA sequence of 19428.1, showing
homology to human chromosome 17 clone
SEQ ID NO:185 is the determined cDNA sequence of B808P (19408),
showing no significant homology to any known gene
SEQ ID NO:186 is the determined cDNA sequence of 19460.1, showing
no significant homology to any known gene
SEQ ID NO:187 is the determined cDNA sequence of 19419.1, showing
homology to Ig kappa light chain
SEQ ID NO:188 is the determined cDNA sequence of 19411.1, showing
homology to human alpha-1 collagen
SEQ ID NO:189 is the determined cDNA sequence of 19420.1, showing
homology to mus musculus proteinase-3
SEQ ID NO:190 is the determined cDNA sequence of 19432.1, showing
homology to human high motility group box
SEQ ID NO:191 is the determined cDNA sequence of 19412.1, showing
homology to the human plasminogen activator gene
SEQ ID NO:192 is the determined cDNA sequence of 19415.1, showing
homology to mitogen activated protein kinase
SEQ ID NO:193 is the determined cDNA sequence of 19409.1, showing
homology to the chondroitin sulfate proteoglycan protein
SEQ ID NO:194 is the determined cDNA sequence of 19406.1, showing
no significant homology to any known gene
SEQ ID NO:195 is the determined cDNA sequence of 19421.1, showing
homology to human fibronectin
SEQ ID NO:196 is the determined cDNA sequence of 19426.1, showing
homology to the retinoic acid receptor responder 3
SEQ ID NO:197 is the determined cDNA sequence of 19425.1, showing
homology to MyD88 mRNA
SEQ ID NO:198 is the determined cDNA sequence of 19424.1, showing
homology to peptide transporter (TAP-1) mRNA
SEQ ID NO:199 is the determined cDNA sequence of 19429.1, showing
no significant homology to any known gene
SEQ ID NO:200 is the determined cDNA sequence of 19435.1, showing
homology to human polymorphic epithelial mucin
SEQ ID NO:201 is the determined cDNA sequence of B813P (19434.1),
showing homology to human GATA-3 transcription factor
SEQ ID NO:202 is the determined cDNA sequence of 19461.1, showing
homology to the human AP-2 gene
SEQ ID NO:203 is the determined cDNA sequence of 19450.1, showing
homology to DNA binding regulatory factor
SEQ ID NO:204 is the determined cDNA sequence of 19451.1, showing
homology to Na/H exchange regulatory co-factor
SEQ ID NO:205 is the determined cDNA sequence of 19462.1, showing
no significant homology to any known gene
SEQ ID NO:206 is the determined cDNA sequence of 19455.1, showing
homology to human mRNA for histone HAS.Z
SEQ ID NO:207 is the determined cDNA sequence of 19459.1, showing
homology to PAC clone 179N16
SEQ ID NO:208 is the determined cDNA sequence of 19464.1, showing
no significant homology to any known gene
SEQ ID NO:209 is the determined cDNA sequence of 19414.1, showing
homology to lipophilin B
SEQ ID NO:210 is the determined cDNA sequence of 19413.1, showing
homology to chromosome 17 clone hRPK.209_J.sub.-- 20
SEQ ID NO:211 is the determined cDNA sequence of 19416.1, showing
no significant homology to any known gene
SEQ ID NO:212 is the determined cDNA sequence of 19437.1, showing
homology to human clone 24976 mRNA
SEQ ID NO:213 is the determined cDNA sequence of 19449.1, showing
homology to mouse DNA for PG-M core protein
SEQ ID NO:214 is the determined cDNA sequence of 19446.1, showing
no significant homology to any known gene
SEQ ID NO:215 is the determined cDNA sequence of 19452.1, showing
no significant homology to any known gene
SEQ ID NO:216 is the determined cDNA sequence of 19483.1, showing
no significant homology to any known gene
SEQ ID NO:217 is the determined cDNA sequence of 19526.1, showing
homology to human lipophilin C
SEQ ID NO:218 is the determined cDNA sequence of 19484.1, showing
homology to the secreted cement gland protein XAG-2
SEQ ID NO:219 is the determined cDNA sequence of 19470.1, showing
no significant homology to any known gene
SEQ ID NO:220 is the determined cDNA sequence of 19469.1, showing
homology to the human HLA-DM gene
SEQ ID NO:221 is the determined cDNA sequence of 19482.1, showing
homology to the human pS2 protein gene
SEQ ID NO:222 is the determined cDNA sequence of B805P (19468.1),
showing no significant homology to any known gene
SEQ ID NO:223 is the determined cDNA sequence of 19467.1, showing
homology to human thrombospondin mRNA
SEQ ID NO:224 is the determined cDNA sequence of 19498.1, showing
homology to the CDC2 gene involved in cell cycle control
SEQ ID NO:225 is the determined cDNA sequence of 19506.1, showing
homology to human cDNA for TREB protein
SEQ ID NO:226 is the determined EDNA sequence of B806P (19505.1),
showing no significant homology to any known gene
SEQ ID NO:227 is the determined EDNA sequence of 19486.1, showing
homology to type I epidermal keratin
SEQ ID NO:228 is the determined cDNA sequence of 19510.1, showing
homology to glucose transporter for glycoprotein
SEQ ID NO:229 is the determined cDNA sequence of 19512.1, showing
homology to the human lysyl hydroxylase gene
SEQ ID NO:230 is the determined cDNA sequence of 19511.1, showing
homology to human palimotoyl-protein thioesterase
SEQ ID NO:231 is the determined cDNA sequence of 19508.1, showing
homology to human alpha enolase
SEQ ID NO:232 is the determined cDNA sequence of B807P (19509.1),
showing no significant homology to any known gene
SEQ ID NO:233 is the determined cDNA sequence of B809P (19520.1),
showing homology to clone 102D24 on chromosome 11q13.31
SEQ ID NO:234 is the determined cDNA sequence of 19507.1, showing
homology toprosome beta-subunit
SEQ ID NO:235 is the determined cDNA sequence of 19525.1, showing
homology to human pro-urokinase precursor
SEQ ID NO:236 is the determined cDNA sequence of 19513.1, showing
no significant homology to any known gene
SEQ ID NO:237 is the determined cDNA sequence of 19517.1, showing
homology to human PAC 128Ml9 clone
SEQ ID NO:238 is the determined cDNA sequence of 19564.1, showing
homology to human cytochrome P450-IIB
SEQ ID NO:239 is the determined cDNA sequence of 19553.1, showing
homology to human GABA-A receptor pi subunit
SEQ ID NO:240 is the determined cDNA sequence of B811P (19575.1),
showing no significant homology to any known gene
SEQ ID NO:241 is the determined cDNA sequence of B810P (19560.1),
showing no significant homology to any known gene
SEQ ID NO:242 is the determined cDNA sequence of 19588.1, showing
homology to aortic carboxypetidase-like protein
SEQ ID NO:243 is the determined cDNA sequence of 19551.1, showing
homology to human BCL-1 gene
SEQ ID NO:244 is the determined cDNA sequence of 19567.1, showing
homology to human proteasome-related mRNA
SEQ ID NO:245 is the determined cDNA sequence of B803P (19583.1),
showing no significant homology to any known gene
SEQ ID NO:246 is the determined cDNA sequence of B812P (19587.1),
showing no significant homology to any known gene
SEQ ID NO:247 is the determined cDNA sequence of B802P (19392.2),
showing homology to human chromosome 17
SEQ ID NO:248 is the determined cDNA sequence of 19393.2, showing
homology to human nicein B2 chain
SEQ ID NO:249 is the determined cDNA sequence of 19398.2, human
MHC class II DQ alpha mRNA
SEQ ID NO:250 is the determined cDNA sequence of B804P (19399.2),
showing homology to human Xp22 BAC GSHB-184P14
SEQ ID NO:251 is the determined cDNA sequence of 19401.2, showing
homology to human ikB kinase-b gene
SEQ ID NO:252 is the determined cDNA sequence of 20266, showing
no significant homology to any known gene
SEQ ID NO:253 is the determined cDNA sequence of B826P (20270),
showing no significant homology to any known gene
SEQ ID NO:254 is the determined cDNA sequence of 20274, showing
no significant homology to any known gene
SEQ ID NO:255 is the determined cDNA sequence of 20276, showing
no significant homology to any known gene
SEQ ID NO:256 is the determined cDNA sequence of 20277, showing
no significant homology to any known gene
SEQ ID NO:257 is the determined cDNA sequence of B823P (20280),
showing no significant homology to any known gene
SEQ ID NO:258 is the determined cDNA sequence of B821P (20281),
showing no significant homology to any known gene
SEQ ID NO:259 is the determined cDNA sequence of B824P (20294),
showing no significant homology to any known gene
SEQ ID NO:260 is the determined cDNA sequence of 20303, showing
no significant homology to any known gene
SEQ ID NO:261 is the determined cDNA sequence of B820P (20310),
showing no significant homology to any known gene
SEQ ID NO:262 is the determined cDNA sequence of B825P (20336),
showing no significant homology to any known gene
SEQ ID NO:263 is the determined cDNA sequence of B827P (20341),
showing no significant homology to any known gene
SEQ ID NO:264 is the determined cDNA sequence of 20941, showing
no significant homology to any known gene
SEQ ID NO:265 is the determined cDNA sequence of 20954, showing
no significant homology to any known gene
SEQ ID NO:266 is the determined cDNA sequence of 20961, showing
no significant homology to any known gene
SEQ ID NO:267 is the determined cDNA sequence of 20965, showing
no significant homology to any known gene
SEQ ID NO:268 is the determined cDNA sequence of 20975, showing
no significant homology to any known gene
SEQ ID NO:269 is the determined cDNA sequence of 20261, showing
homology to Human p120 catenin
SEQ ID NO:270 is the determined cDNA sequence of B822P (20262),
showing homology to Human membrane glycoprotein 4F2
SEQ ID NO:271 is the determined cDNA sequence of 20265, showing
homology to Human Na, K-ATPase Alpha 1
SEQ ID NO:272 is the determined cDNA sequence of 20267, showing
homology to Human heart HS 90, partial cds
SEQ ID NO:273 is the determined cDNA sequence of 20268, showing
homology to Human mRNA GPI-anchored protein p137
SEQ ID NO:274 is the determined cDNA sequence of 20271, showing
homology to Human cleavage stimulation factor 77 kDa subunit
SEQ ID NO:275 is the determined cDNA sequence of 20272, showing
homology to Human p190-B
SEQ ID NO:276 is the determined cDNA sequence of 20273, showing
homology to Human ribophorin
SEQ ID NO:277 is the determined cDNA sequence of 20278, showing
homology to Human omithine amino transferase
SEQ ID NO:278 is the determined cDNA sequence of 20279, showing
homology to Human S-adenosylmethionine synthetase
SEQ ID NO:279 is the determined cDNA sequence of 20293, showing
homology to Human x inactivation transcript
SEQ ID NO:280 is the determined cDNA sequence of 20300, showing
homology to Human cytochrome p450
SEQ ID NO:281 is the determined cDNA sequence of 20305, showing
homology to Human elongation factor-1 alpha
SEQ ID NO:282 is the determined cDNA sequence of 20306, showing
homology to Human epithelial ets protein
SEQ ID NO:283 is the determined cDNA sequence of 20307, showing
homology to Human signal transducer mRNA
SEQ ID NO:284 is the determined cDNA sequence of 20313, showing
homology to Human GABA-A receptor pi subunit mRNA
SEQ ID NO:285 is the determined cDNA sequence of 20317, showing
homology to Human tyrosine phosphatase
SEQ ID NO:286 is the determined cDNA sequence of 20318, showing
homology to Human cathepsine B proteinase
SEQ ID NO:287 is the determined cDNA sequence of 20320, showing
homology to Human 2-phosphopyruvate-hydratase-alpha-enolase
SEQ ID NO:288 is the determined cDNA sequence of 20321, showing
homology to Human E-cadherin
SEQ ID NO:289 is the determined cDNA sequence of 20322, showing
homology to Human hsp86
SEQ ID NO:290 is the determined cDNA sequence of B828P (20326),
showing homology to Human x inactivation transcript
SEQ ID NO:291 is the determined cDNA sequence of 20333, showing
homology to Human chromatin regulator, SMARCA5
SEQ ID NO:292 is the determined cDNA sequence of 20335, showing
homology to Human sphingolipid activator protein 1
SEQ ID NO:293 is the determined cDNA sequence of 20337, showing
homology to Human hepatocyte growth factor activator inhibitor type
SEQ ID NO:294 is the determined cDNA sequence of 20338, showing
homology to Human cell adhesion molecule CD44
SEQ ID NO:295 is the determined cDNA sequence of 20340, showing
homology to Human nuclear factor (erythroid-derived)-like 1
SEQ ID NO:296 is the determined cDNA sequence of 20938, showing
homology to Human vinculin mRNA
SEQ ID NO:297 is the determined cDNA sequence of 20939, showing
homology to Human elongation factor EF-1-alpha
SEQ ID NO:298 is the determined cDNA sequence of 20940, showing
homology to Human nestin gene
SEQ ID NO:299 is the determined cDNA sequence of 20942, showing
homology to Human pancreatic ribonuclease
SEQ ID NO:300 is the determined cDNA sequence of 20943, showing
homology to Human transcobalamin I
SEQ ID NO:301 is the determined cDNA sequence of 20944, showing
homology to Human beta-tubulin
SEQ ID NO:302 is the determined cDNA sequence of 20946, showing
homology to Human HS1 protein
SEQ ID NO:303 is the determined cDNA sequence of 20947, showing
homology to Human cathepsin B
SEQ ID NO:304 is the determined cDNA sequence of 20948, showing
homology to Human testis enhanced gene transcript
SEQ ID NO:305 is the determined cDNA sequence of 20949, showing
homology to Human elongation factor EF-1-alpha
SEQ ID NO:306 is the determined cDNA sequence of 20950, showing
homology to Human ADP-ribosylation factor 3
SEQ ID NO:307 is the determined cDNA sequence of 20951, showing
homology to Human IFP53 or WRS for tryptophanyl-tRNA synthetase
SEQ ID NO:308 is the determined cDNA sequence of 20952, showing
homology to Human cyclin-dependent protein kinase
SEQ ID NO:309 is the determined cDNA sequence of 20957, showing
homology to Human alpha-tubulin isoform 1
SEQ ID NO:310 is the determined cDNA sequence of 20959, showing
homology to Human tyrosine phosphatase-61bp deletion
SEQ ID NO:311 is the determined cDNA sequence of 20966, showing
homology to Human tyrosine phosphatase
SEQ ID NO:312 is the determined cDNA sequence of B830P (20976),
showing homology to Human nuclear factor NF 45
SEQ ID NO:313 is the determined cDNA sequence of B829P (20977),
showing homology to Human delta-6 fatty acid desaturase
SEQ ID NO:314 is the determined cDNA sequence of 20978, showing
homology to Human nuclear aconitase
SEQ ID NO:315 is the determined cDNA sequence of clone 23176.
SEQ ID NO:316 is the determined cDNA sequence of clone 23140.
SEQ ID NO:317 is the determined cDNA sequence of clone 23166.
SEQ ID NO:318 is the determined EDNA sequence of clone 23167.
SEQ ID NO:319 is the determined cDNA sequence of clone 23177.
SEQ ID NO:320 is the determined cDNA sequence of clone 23217.
SEQ ID NO:321 is the determined cDNA sequence of clone 23169.
SEQ ID NO:322 is the determined cDNA sequence of clone 23160.
SEQ ID NO:323 is the determined cDNA sequence of clone 23182.
SEQ ID NO:324 is the determined cDNA sequence of clone 23232.
SEQ ID NO:325 is the determined cDNA sequence of clone 23203.
SEQ ID NO:326 is the determined cDNA sequence of clone 23198.
SEQ ID NO:327 is the determined cDNA sequence of clone 23224.
SEQ ID NO:328 is the determined cDNA sequence of clone 23142.
SEQ ID NO:329 is the determined cDNA sequence of clone 23138.
SEQ ID NO:330 is the determined cDNA sequence of clone 23147.
SEQ ID NO:331 is the determined cDNA sequence of clone 23148.
SEQ ID NO:332 is the determined cDNA sequence of clone 23149.
SEQ ID NO:333 is the determined cDNA sequence of clone 23172.
SEQ ID NO:334 is the determined cDNA sequence of clone 23158.
SEQ ID NO:335 is the determined cDNA sequence of clone 23156.
SEQ ID NO:336 is the determined cDNA sequence of clone 23221.
SEQ ID NO:337 is the determined cDNA sequence of clone 23223.
SEQ ID NO:338 is the determined cDNA sequence of clone 23155.
SEQ ID NO:339 is the determined cDNA sequence of clone 23225.
SEQ ID NO:340 is the determined cDNA sequence of clone 23226.
SEQ ID NO:341 is the determined cDNA sequence of clone 23228.
SEQ ID NO:342 is the determined cDNA sequence of clone 23229.
SEQ ID NO:343 is the determined cDNA sequence of clone 23231.
SEQ ID NO:344 is the determined cDNA sequence of clone 23154.
SEQ ID NO:345 is the determined cDNA sequence of clone 23157.
SEQ ID NO:346 is the determined cDNA sequence of clone 23153.
SEQ ID NO:347 is the determined cDNA sequence of clone 23159.
SEQ ID NO:348 is the determined cDNA sequence of clone 23152.
SEQ ID NO:349 is the determined cDNA sequence of clone 23161.
SEQ ID NO:350 is the determined cDNA sequence of clone 23162.
SEQ ID NO:351 is the determined cDNA sequence of clone 23163.
SEQ ID NO:352 is the determined cDNA sequence of clone 23164.
SEQ ID NO:353 is the determined cDNA sequence of clone 23165.
SEQ ID NO:354 is the determined cDNA sequence of clone 23151.
SEQ ID NO:355 is the determined cDNA sequence of clone 23150.
SEQ ID NO:356 is the determined cDNA sequence of clone 23168.
SEQ ID NO:357 is the determined cDNA sequence of clone 23146.
SEQ ID NO:358 is the determined cDNA sequence of clone 23170.
SEQ ID NO:359 is the determined cDNA sequence of clone 23171.
SEQ ID NO:360 is the determined cDNA sequence of clone 23145.
SEQ ID NO:361 is the determined cDNA sequence of clone 23174.
SEQ ID NO:362 is the determined cDNA sequence of clone 23175.
SEQ ID NO:363 is the determined cDNA sequence of clone 23144.
SEQ ID NO:364 is the determined cDNA sequence of clone 23178.
SEQ ID NO:365 is the determined cDNA sequence of clone 23179.
SEQ ID NO:366 is the determined cDNA sequence of clone 23180.
SEQ ID NO:367 is the determined cDNA sequence of clone 23181.
SEQ ID NO:368 is the determined cDNA sequence of clone 23143
SEQ ID NO:369 is the determined cDNA sequence of clone 23183.
SEQ ID NO:370 is the determined cDNA sequence of clone 23184.
SEQ ID NO:371 is the determined cDNA sequence of clone 23185.
SEQ ID NO:372 is the determined cDNA sequence of clone 23186.
SEQ ID NO:373 is the determined cDNA sequence of clone 23187.
SEQ ID NO:374 is the determined cDNA sequence of clone 23190.
SEQ ID NO:375 is the determined cDNA sequence of clone 23189.
SEQ ID NO:376 is the determined cDNA sequence of clone 23202.
SEQ ID NO:378 is the determined cDNA sequence of clone 23191.
SEQ ID NO:379 is the determined cDNA sequence of clone 23188.
SEQ ID NO:380 is the determined cDNA sequence of clone 23194.
SEQ ID NO:381 is the determined cDNA sequence of clone 23196.
SEQ ID NO:382 is the determined cDNA sequence of clone 23195.
SEQ ID NO:383 is the determined cDNA sequence of clone 23193.
SEQ ID NO:384 is the determined cDNA sequence of clone 23199.
SEQ ID NO:385 is the determined cDNA sequence of clone 23200.
SEQ ID NO:386 is the determined cDNA sequence of clone 23192.
SEQ ID NO:387 is the determined cDNA sequence of clone 23201.
SEQ ID NO:388 is the determined cDNA sequence of clone 23141.
SEQ ID NO:389 is the determined cDNA sequence of clone 23139.
SEQ ID NO:390 is the determined cDNA sequence of clone 23204.
SEQ ID NO:391 is the determined cDNA sequence of clone 23205.
SEQ ID NO:392 is the determined cDNA sequence of clone 23206.
SEQ ID NO:393 is the determined cDNA sequence of clone 23207.
SEQ ID NO:394 is the determined cDNA sequence of clone 23208.
SEQ ID NO:395 is the determined cDNA sequence of clone 23209.
SEQ ID NO:396 is the determined cDNA sequence of clone 23210.
SEQ ID NO:397 is the determined cDNA sequence of clone 23211.
SEQ ID NO:398 is the determined cDNA sequence of clone 23212.
SEQ ID NO:399 is the determined cDNA sequence of clone 23214.
SEQ ID NO:400 is the determined cDNA sequence of clone 23215.
SEQ ID NO:401 is the determined cDNA sequence of clone 23216.
SEQ ID NO:402 is the determined cDNA sequence of clone 23137.
SEQ ID NO:403 is the determined cDNA sequence of clone 23218.
SEQ ID NO:404 is the determined cDNA sequence of clone 23220.
SEQ ID NO:405 is the determined cDNA sequence of clone 19462.
SEQ ID NO:406 is the determined cDNA sequence of clone 19430.
SEQ ID NO:407 is the determined cDNA sequence of clone 19407.
SEQ ID NO:408 is the determined cDNA sequence of clone 19448.
SEQ ID NO:409 is the determined cDNA sequence of clone 19447.
SEQ ID NO:410 is the determined cDNA sequence of clone 19426.
SEQ ID NO:411 is the determined cDNA sequence of clone 19441.
SEQ ID NO:412 is the determined cDNA sequence of clone 19454.
SEQ ID NO:413 is the determined cDNA sequence of clone 19463.
SEQ ID NO:414 is the determined cDNA sequence of clone 19419.
SEQ ID NO:415 is the determined cDNA sequence of clone 19434.
SEQ ID NO:416 is the determined extended cDNA sequence of B820P.
SEQ ID NO:417 is the determined extended cDNA sequence of B821P.
SEQ ID NO:418 is the determined extended cDNA sequence of B822P.
SEQ ID NO:419 is the determined extended cDNA sequence of B823P.
SEQ ID NO:420 is the determined extended cDNA sequence of B824P.
SEQ ID NO:421 is the determined extended cDNA sequence of B825P.
SEQ ID NO:422 is the determined extended cDNA sequence of B826P.
SEQ ID NO:423 is the determined extended cDNA sequence of B827P.
SEQ ID NO:424 is the determined extended cDNA sequence of B828P.
SEQ ID NO:425 is the determined extended cDNA sequence of B829P.
SEQ ID NO:426 is the determined extended cDNA sequence of B830P.
SEQ ID NO:427 is the determined cDNA sequence of clone 266B4.
SEQ ID NO:428 is the determined cDNA sequence of clone 22892.
SEQ ID NO:429 is the determined cDNA sequence of clone 266G3.
SEQ ID NO:430 is the determined cDNA sequence of clone 22890.
SEQ ID NO:431 is the determined cDNA sequence of clone 264B4.
SEQ ID NO:432 is the determined cDNA sequence of clone 22883.
SEQ ID NO:433 is the determined cDNA sequence of clone 22882.
SEQ ID NO:434 is the determined cDNA sequence of clone 22880.
SEQ ID NO:435 is the determined cDNA sequence of clone 263G1.
SEQ ID NO:436 is the determined cDNA sequence of clone 263G6.
SEQ ID NO:437 is the determined cDNA sequence of clone 262B2.
SEQ ID NO:438 is the determined cDNA sequence of clone 262B6.
SEQ ID NO:439 is the determined cDNA sequence of clone 22869.
SEQ ID NO:440 is the determined cDNA sequence of clone 21374.
SEQ ID NO:441 is the determined cDNA sequence of clone 21362.
SEQ ID NO:442 is the determined cDNA sequence of clone 21349.
SEQ ID NO:443 is the determined cDNA sequence of clone 21309.
SEQ ID NO:444 is the determined cDNA sequence of clone 21097.
SEQ ID NO:445 is the determined cDNA sequence of clone 21096.
SEQ ID NO:446 is the determined cDNA sequence of clone 21094.
SEQ ID NO:447 is the determined cDNA sequence of clone 21093.
SEQ ID NO:448 is the determined cDNA sequence of clone 21091.
SEQ ID NO:449 is the determined cDNA sequence of clone 21089.
SEQ ID NO:450 is the determined cDNA sequence of clone 21087.
SEQ ID NO:451 is the determined cDNA sequence of clone 21085.
SEQ ID NO:452 is the determined cDNA sequence of clone 21084.
SEQ ID NO:453 is a first partial cDNA sequence of clone 2BT1-40.
SEQ ID NO:454 is a second partial cDNA sequence of clone 2BT1-40.
SEQ ID NO:455 is the determined cDNA sequence of clone 21063.
SEQ ID NO:456 is the determined cDNA sequence of clone 21062.
SEQ ID NO:457 is the determined cDNA sequence of clone 21060.
SEQ ID NO:458 is the determined cDNA sequence of clone 21053.
SEQ ID NO:459 is the determined cDNA sequence of clone 21050.
SEQ ID NO:460 is the determined cDNA sequence of clone 21036.
SEQ ID NO:461 is the determined cDNA sequence of clone 21037.
SEQ ID NO:462 is the determined cDNA sequence of clone 21048.
SEQ ID NO:463 is a consensus DNA sequence of B726P (referred to
SEQ ID NO:464 is the determined cDNA sequence of a second splice
form of B726P (referred to as 27490.seq_B726P).
SEQ ID NO:465 is the determined cDNA sequence of a third splice
form of B726P (referred to as 27068.seq_B726P).
SEQ ID NO:466 is the determined cDNA sequence of a second splice
form of B726P (referred to as 23113.seq_B726P).
SEQ ID NO:467 is the determined cDNA sequence of a second splice
form of B726P (referred to as 23103.seq_B726P).
SEQ ID NO:468 is the determined cDNA sequence of a second splice
form of B726P (referred to as 19310.seq_B726P).
SEQ ID NO:469 is the predicted amino acid sequence encoded by the
upstream ORF of SEQ ID NO:463.
SEQ ID NO:470 is the predicted amino acid sequence encoded by SEQ
SEQ ID NO:471 is the predicted amino acid sequence encoded by SEQ
SEQ ID NO:472 is the predicted amino acid sequence encoded by SEQ
SEQ ID NO:473 is the predicted amino acid sequence encoded by SEQ
SEQ ID NO:474 is the determined cDNA sequence for an alternative
splice form of B726P.
SEQ ID NO:475 is the amino acid sequence encoded by SEQ ID NO:474.
SEQ ID NO:476 is the isolated cDNA sequence of B720P.
SEQ ID NO:477 is the cDNA sequence of a known keratin gene.
SEQ ID NO:478 is the amino acid sequence encoded by SEQ ID NO:477.
SEQ ID NO:479 is the determined cDNA sequence for clone 19465.
SEQ ID NO:480 and 481 are PCR primers.
SEQ ID NO:482 is the cDNA sequence for the expressed downstream
ORF of B726P.
SEQ ID NO:483 is the amino acid sequence for the expressed recombinant
downstream ORF of B726P.
SEQ ID NO:484 is the determined full-length cDNA sequence for B720P.
SEQ ID NO:485 is the amino acid sequence encoded by SEQ ID NO:484.
SEQ ID NO:486 is the determined cDNA sequence of a truncated form
of B720P, referred to as B720P-tr.
SEQ ID NO:487 is the amino acid sequence of B720P-tr.
SEQ ID NO:488 is the amino acid sequence of a naturally processed
epitope of B726P recognized by B726P-specific CTL.
SEQ ID NO:489 is a DNA sequence encoding the B726P epitope set
forth in SEQ ID NO:488.
DETAILED DESCRIPTION OF THE INVENTION
As noted above, the present invention is generally directed to
compositions and methods for using the compositions, for example
in the therapy and diagnosis of cancer, such as breast cancer. Certain
illustrative compositions described herein include breast tumor
polypeptides, polynucleotides encoding such polypeptides, binding
agents such as antibodies, antigen presenting cells (APCs) and/or
immune system cells (e.g., T cells). A "breast tumor protein,"
as the term is used herein, refers generally to a protein that is
expressed in breast tumor cells at a level that is at least two
fold, and preferably at least five fold, greater than the level
of expression in a normal tissue, as determined using a representative
assay provided herein. Certain breast tumor proteins are tumor proteins
that react detectably (within an immunoassay, such as an ELISA or
Western blot) with antisera of a patient afflicted with breast cancer.
Therefore, in accordance with the above, and as described further
below, the present invention provides illustrative polynucleotide
compositions having sequences set forth in SEQ ID NO:1-175, 178,
180, 182-468, 474, 476, 477, 479, 484, 486 and 489, illustrative
polypeptide compositions having amino acid sequences set forth in
SEQ ID NO:176, 179, 181, 469-473, 475, 485, 487 and 488, antibody
compositions capable of binding such polypeptides, and numerous
additional embodiments employing such compositions, for example
in the detection, diagnosis and/or therapy of human breast cancer.
As used herein, the terms "DNA segment" and "polynucleotide"
refer to a DNA molecule that has been isolated free of total genomic
DNA of a particular species. Therefore, a DNA segment encoding a
polypeptide refers to a DNA segment that contains one or more coding
sequences yet is substantially isolated away from, or purified free
from, total genomic DNA of the species from which the DNA segment
is obtained. Included within the terms "DNA segment" and
"polynucleotide" are DNA segments and smaller fragments
of such segments, and also recombinant vectors, including, for example,
plasmids, cosmids, phagemids, phage, viruses, and the like.
As will be understood by those skilled in the art, the DNA segments
of this invention can include genomic sequences, extra-genomic and
plasmid-encoded sequences and smaller engineered gene segments that
express, or may be adapted to express, proteins, polypeptides, peptides
and the like. Such segments may be naturally isolated, or modified
synthetically by the hand of man.
"Isolated," as used herein, means that a polynucleotide
is substantially away from other coding sequences, and that the
DNA segment does not contain large portions of unrelated coding
DNA, such as large chromosomal fragments or other functional genes
or polypeptide coding regions. Of course, this refers to the DNA
segment as originally isolated, and does not exclude genes or coding
regions later added to the segment by the hand of man.
As will be recognized by the skilled artisan, polynucleotides may
be single-stranded (coding or antisense) or double-stranded, and
may be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules
include HNRNA molecules, which contain introns and correspond to
a DNA molecule in a one-to-one manner, and mRNA molecules, which
do not contain introns. Additional coding or non-coding sequences
may, but need not, be present within a polynucleotide of the present
invention, and a polynucleotide may, but need not, be linked to
other molecules and/or support materials.
Polynucleotides may comprise a native sequence (i.e., an endogenous
sequence that encodes a breast tumor protein or a portion thereof)
or may comprise a variant, or a biological or antigenic functional
equivalent of such a sequence. Polynucleotide variants may contain
one or more substitutions, additions, deletions and/or insertions,
as further described below, preferably such that the immunogenicity
of the encoded polypeptide is not diminished, relative to a native
tumor protein. The effect on the immunogenicity of the encoded polypeptide
may generally be assessed as described herein. The term "variants"
also encompasses homologous genes of xenogenic origin.
When comparing polynucleotide or polypeptide sequences, two sequences
are said to be "identical" if the sequence of nucleotides
or amino acids in the two sequences is the same when aligned for
maximum correspondence, as described below. Comparisons between
two sequences are typically performed by comparing the sequences
over a comparison window to identify and compare local regions of
sequence similarity. A "comparison window" as used herein,
refers to a segment of at least about 20 contiguous positions, usually
30 to about 75, 40 to about 50, in which a sequence may be compared
to a reference sequence of the same number of contiguous positions
after the two sequences are optimally aligned.
Optimal alignment of sequences for comparison may be conducted
using the Megalign program in the Lasergene suite of bioinformatics
software (DNASTAR, Inc., Madison, Wis.), using default parameters.
This program embodies several alignment schemes described in the
following references: Dayhoff, M. O. (1978) A model of evolutionary
change in proteins--Matrices for detecting distant relationships.
In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure,
National Biomedical Research Foundation, Washington D.C. Vol. 5,
Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment
and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic
Press, Inc., San Diego, Calif.; Higgins, D. G. and Sharp, P. M.
(1989) CABIOS 5:151-153; Myers, E. W. and Muller W. (1988) CABIOS
4:11-17; Robinson, E. D. (1971) Comb. Theor 11:105; Santou, N. Nes,
M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P. H. A. and Sokal,
R. R. (1973) Numerical Taxonomy--the Principles and Practice of
Numerical Taxonomy, Freeman Press, San Francisco, Calif.; Wilbur,
W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA 80:726-730.
Alternatively, optimal alignment of sequences for comparison may
be conducted by the local identity algorithm of Smith and Waterman
(1981) Add. APL. Math 2:482, by the identity alignment algorithm
of Needleman and Wunsch (1970) J. Mol Biol. 48:443, by the search
for similarity methods of Pearson and Lipman (1988) Proc. Natl.
Acad. Sci. USA 85: 2444, by computerized implementations of these
algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group (GCG), 575 Science
Dr., Madison, Wis.), or by inspection.
One preferred example of algorithms that are suitable for determining
percent sequence identity and sequence similarity are the BLAST
and BLAST 2.0 algorithms, which are described in Altschul et al.
(1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990)
J. Mol Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can
be used, for example with the parameters described herein, to determine
percent sequence identity for the polynucleotides and polypeptides
of the invention. Software for performing BLAST analyses is publicly
available through the National Center for Biotechnology Information.
In one illustrative example, cumulative scores can be calculated
using, for nucleotide sequences, the parameters M (reward score
for a pair of matching residues; always >0) and N (penalty score
for mismatching residues; always <0). For amino acid sequences,
a scoring matrix can be used to calculate the cumulative score.
Extension of the word hits in each direction are halted when: the
cumulative alignment score falls off by the quantity X from its
maximum achieved value; the cumulative score goes to zero or below,
due to the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T and X determine the sensitivity and speed
of the alignment. The BLASTN program (for nucleotide sequences)
uses as defaults a wordlength (W) of 11, and expectation (E) of
10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989)
Proc. Natl Acad. Sci. USA 89:10915) alignments, (B) of 50, expectation
(E) of 10, M=5, N=-4 and a comparison of both strands.
Preferably, the "percentage of sequence identity" is
determined by comparing two optimally aligned sequences over a window
of comparison of at least 20 positions, wherein the portion of the
polynucleotide or polypeptide sequence in the comparison window
may comprise additions or deletions (i.e., gaps) of 20 percent or
less, usually 5 to 15 percent, or 10 to 12 percent, as compared
to the reference sequences (which does not comprise additions or
deletions) for optimal alignment of the two sequences. The percentage
is calculated by determining the number of positions at which the
identical nucleic acid bases or amino acid residue occurs in both
sequences to yield the number of matched positions, dividing the
number of matched positions by the total number of positions in
the reference sequence (i.e., the window size) and multiplying the
results by 100 to yield the percentage of sequence identity.
Therefore, the present invention encompasses polynucleotide and
polypeptide sequences having substantial identity to the sequences
disclosed herein, for example those comprising at least 50% sequence
identity, preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 96%, 97%, 98%, or 99% or higher, sequence identity compared
to a polynucleotide or polypeptide sequence of this invention using
the methods described herein, (e.g., BLAST analysis using standard
parameters, as described below). One skilled in this art will recognize
that these values can be appropriately adjusted to determine corresponding
identity of proteins encoded by two nucleotide sequences by taking
into account codon degeneracy, amino acid similarity, reading frame
positioning and the like.
In additional embodiments, the present invention provides isolated
polynucleotides and polypeptides comprising various lengths of contiguous
stretches of sequence identical to or complementary to one or more
of the sequences disclosed herein. For example, polynucleotides
are provided by this invention that comprise at least about 15,
20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500 or 1000 or more
contiguous nucleotides of one or more of the sequences disclosed
herein as well as all intermediate lengths there between. It will
be readily understood that "intermediate lengths", in
this context, means any length between the quoted values, such as
16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51,
52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.;
including all integers through 200-500; 500-1,000, and the like.
The polynucleotides of the present invention, or fragments thereof,
regardless of the length of the coding sequence itself, may be combined
with other DNA sequences, such as promoters, polyadenylation signals,
additional restriction enzyme sites, multiple cloning sites, other
coding segments, and the like, such that their overall length may
vary considerably. It is therefore contemplated that a nucleic acid
fragment of almost any length may be employed, with the total length
preferably being limited by the ease of preparation and use in the
intended recombinant DNA protocol. For example, illustrative DNA
segments with total lengths of about 10,000, about 5000, about 3000,
about 2,000, about 1,000, about 500, about 200, about 100, about
50 base pairs in length, and the like, (including all intermediate
lengths) are contemplated to be useful in many implementations of
In other embodiments, the present invention is directed to polynucleotides
that are capable of hybridizing under moderately stringent conditions
to a polynucleotide sequence provided herein, or a fragment thereof,
or a complementary sequence thereof. Hybridization techniques are
well known in the art of molecular biology. For purposes of illustration,
suitable moderately stringent conditions for testing the hybridization
of a polynucleotide of this invention with other polynucleotides
include prewashing in a solution of 5.times.SSC, 0.5% SDS, 1.0 mM
EDTA (pH 8.0); hybridizing at 50.degree. C.-65.degree. C., 5.times.SSC,
overnight; followed by washing twice at 65.degree. C. for 20 minutes
with each of 2.times., 0.5.times.and 0.2.times.SSC containing 0.1%
Moreover, it will be appreciated by those of ordinary skill in
the art that, as a result of the degeneracy of the genetic code,
there are many nucleotide sequences that encode a polypeptide as
described herein. Some of these polynucleotides bear minimal homology
to the nucleotide sequence of any native gene. Nonetheless, polynucleotides
that vary due to differences in codon usage are specifically contemplated
by the present invention. Further, alleles of the genes comprising
the polynucleotide sequences provided herein are within the scope
of the present invention. Alleles are endogenous genes that are
altered as a result of one or more mutations, such as deletions,
additions and/or substitutions of nucleotides. The resulting mRNA
and protein may, but need not, have an altered structure or function.
Alleles may be identified using standard techniques (such as hybridization,
amplification and/or database sequence comparison).
Probes and Primers
In other embodiments of the present invention, the polynucleotide
sequences provided herein can be advantageously used as probes or
primers for nucleic acid hybridization. As such, it is contemplated
that nucleic acid segments that comprise a sequence region of at
least about 15 nucleotide long contiguous sequence that has the
same sequence as, or is complementary to, a 15 nucleotide long contiguous
sequence disclosed herein will find particular utility. Longer contiguous
identical or complementary sequences, e.g., those of about 20, 30,
40, 50, 100, 200, 500, 1000 (including all intermediate lengths)
and even up to full length sequences will also be of use in certain
The ability of such nucleic acid probes to specifically hybridize
to a sequence of interest will enable them to be of use in detecting
the presence of complementary sequences in a given sample. However,
other uses are also envisioned, such as the use of the sequence
information for the preparation of mutant species primers, or primers
for use in preparing other genetic constructions.
Polynucleotide molecules having sequence regions consisting of
contiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even
of 100-200 nucleotides or so (including intermediate lengths as
well), identical or complementary to a polynucleotide sequence disclosed
herein, are particularly contemplated as hybridization probes for
use in, e.g., Southern and Northern blotting. This would allow a
gene product, or fragment thereof, to be analyzed, both in diverse
cell types and also in various bacterial cells. The total size of
fragment, as well as the size of the complementary stretch(es),
will ultimately depend on the intended use or application of the
particular nucleic acid segment. Smaller fragments will generally
find use in hybridization embodiments, wherein the length of the
contiguous complementary region may be varied, such as between about
15 and about 100 nucleotides, but larger contiguous complementarity
stretches may be used, according to the length complementary sequences
one wishes to detect.
The use of a hybridization probe of about 15-25 nucleotides in
length allows the formation of a duplex molecule that is both stable
and selective. Molecules having contiguous complementary sequences
over stretches greater than 15 bases in length are generally preferred,
though, in order to increase stability and selectivity of the hybrid,
and thereby improve the quality and degree of specific hybrid molecules
obtained. One will generally prefer to design nucleic acid molecules
having gene-complementary stretches of 15 to 25 contiguous nucleotides,
or even longer where desired.
Hybridization probes may be selected from any portion of any of
the sequences disclosed herein. All that is required is to review
the sequence set forth in SEQ ID NO:1-175, 178, 180, 182-468, 474,
476, 477 479, 484, 486 and 489, or to any continuous portion of
the sequence, from about 15-25 nucleotides in length up to and including
the full length sequence, that one wishes to utilize as a probe
or primer. The choice of probe and primer sequences may be governed
by various factors. For example, one may wish to employ primers
from towards the termini of the total sequence.
Small polynucleotide segments or fragments may be readily prepared
by, for example, directly synthesizing the fragment by chemical
means, as is commonly practiced using an automated oligonucleotide
synthesizer. Also, fragments may be obtained by application of nucleic
acid reproduction technology, such as the PCR.TM. technology of
U.S. Pat. No. 4,683,202 (incorporated herein by reference), by introducing
selected sequences into recombinant vectors for recombinant production,
and by other recombinant DNA techniques generally known to those
of skill in the art of molecular biology.
The nucleotide sequences of the invention may be used for their
ability to selectively form duplex molecules with complementary
stretches of the entire gene or gene fragments of interest. Depending
on the application envisioned, one will typically desire to employ
varying conditions of hybridization to achieve varying degrees of
selectivity of probe towards target sequence. For applications requiring
high selectivity, one will typically desire to employ relatively
stringent conditions to form the hybrids, e.g., one will select
relatively low salt and/or high temperature conditions, such as
provided by a salt concentration of from about 0.02 M to about 0.15
M salt at temperatures of from about 50.degree. C. to about 70.degree.
C. Such selective conditions tolerate little, if any, mismatch between
the probe and the template or target strand, and would be particularly
suitable for isolating related sequences.
Of course, for some applications, for example, where one desires
to prepare mutants employing a mutant primer strand hybridized to
an underlying template, less stringent (reduced stringency) hybridization
conditions will typically be needed in order to allow formation
of the heteroduplex. In these circumstances, one may desire to employ
salt conditions such as those of from about 0.15 M to about 0.9
M salt, at temperatures ranging from about 20.degree. C. to about
55.degree. C. Cross-hybridizing species can thereby be readily identified
as positively hybridizing signals with respect to control hybridizations.
In any case, it is generally appreciated that conditions can be
rendered more stringent by the addition of increasing amounts of
formamide, which serves to destabilize the hybrid duplex in the
same manner as increased temperature. Thus, hybridization conditions
can be readily manipulated, and thus will generally be a method
of choice depending on the desired results.
Polynucleotide Identification and Characterization
Polynucleotides may be identified, prepared and/or manipulated
using any of a variety of well established techniques. For example,
a polynucleotide may be identified, as described in more detail
below, by screening a microarray of cDNAs for tumor-associated expression
(i.e., expression that is at least two fold greater in a tumor than
in normal tissue, as determined using a representative assay provided
herein). Such screens may be performed, for example, using a Synteni
microarray (Palo Alto, Calif.) according to the manufacturer's instructions
(and essentially as described by Schena et al., Proc. Natl. Acad.
Sci. USA 93:10614-10619, 1996 and Heller et al., Proc. Natl. Acad.
Sci. USA 94:2150-2155, 1997). Alternatively, polynucleotides may
be amplified from cDNA prepared from cells expressing the proteins
described herein, such as breast tumor cells. Such polynucleotides
may be amplified via polymerase chain reaction (PCR). For this approach,
sequence-specific primers may be designed based on the sequences
provided herein, and may be purchased or synthesized.
An amplified portion of a polynucleotide of the present invention
may be used to isolate a full length gene from a suitable library
(e.g., a breast tumor cDNA library) using well known techniques.
Within such techniques, a library (cDNA or genomic) is screened
using one or more polynucleotide probes or primers suitable for
amplification. Preferably, a library is size-selected to include
larger molecules. Random primed libraries may also be preferred
for identifying 5' and upstream regions of genes. Genomic libraries
are preferred for obtaining introns and extending 5' sequences.
For hybridization techniques, a partial sequence may be labeled
(e.g., by nick-translation or end-labeling with .sup.32 p) using
well known techniques. A bacterial or bacteriophage library is then
generally screened by hybridizing filters containing denatured bacterial
colonies (or lawns containing phage plaques) with the labeled probe
(see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989). Hybridizing
colonies or plaques are selected and expanded, and the DNA is isolated
for further analysis. cDNA clones may be analyzed to determine the
amount of additional sequence by, for example, PCR using a primer
from the partial sequence and a primer from the vector. Restriction
maps and partial sequences may be generated to identify one or more
overlapping clones. The complete sequence may then be determined
using standard techniques, which may involve generating a series
of deletion clones. The resulting overlapping sequences can then
assembled into a single contiguous sequence. A full length cDNA
molecule can be generated by ligating suitable fragments, using
well known techniques.
Alternatively, there are numerous amplification techniques for
obtaining a full length coding sequence from a partial cDNA sequence.
Within such techniques, amplification is generally performed via
PCR. Any of a variety of commercially available kits may be used
to perform the amplification step. Primers may be designed using,
for example, software well known in the art. Primers are preferably
22-30 nucleotides in length, have a GC content of at least 50% and
anneal to the target sequence at temperatures of about 68.degree.
C. to 72.degree. C. The amplified region may be sequenced as described
above, and overlapping sequences assembled into a contiguous sequence.
One such amplification technique is inverse PCR (see Triglia et
al., Nucl. Acids Res. 16:8186, 1988), which uses restriction enzymes
to generate a fragment in the known region of the gene. The fragment
is then circularized by intramolecular ligation and used as a template
for PCR with divergent primers derived from the known region. Within
an alternative approach, sequences adjacent to a partial sequence
may be retrieved by amplification with a primer to a linker sequence
and a primer specific to a known region. The amplified sequences
are typically subjected to a second round of amplification with
the same linker primer and a second primer specific to the known
region. A variation on this procedure, which employs two primers
that initiate extension in opposite directions from the known sequence,
is described in WO 96/38591. Another such technique is known as
"rapid amplification of cDNA ends" or RACE. This technique
involves the use of an internal primer and an external primer, which
hybridizes to a polyA region or vector sequence, to identify sequences
that are 5' and 3' of a known sequence. Additional techniques include
capture PCR (Lagerstrom et al., PCR Methods Applic. 1:111-19, 1991)
and walking PCR (Parker et al., Nucl. Acids. Res. 19:3055-60, 1991).
Other methods employing amplification may also be employed to obtain
a full length cDNA sequence.
In certain instances, it is possible to obtain a full length cDNA
sequence by analysis of sequences provided in an expressed sequence
tag (EST) database, such as that available from GenBank. Searches
for overlapping ESTs may generally be performed using well known
programs (e.g., NCBI BLAST searches), and such ESTs may be used
to generate a contiguous full length sequence. Full length DNA sequences
may also be obtained by analysis of genomic fragments.
Polynucleotide Expression in Host Cells
In other embodiments of the invention, polynucleotide sequences
or fragments thereof which encode polypeptides of the invention,
or fusion proteins or functional equivalents thereof, may be used
in recombinant DNA molecules to direct expression of a polypeptide
in appropriate host cells. Due to the inherent degeneracy of the
genetic code, other DNA sequences that encode substantially the
same or a functionally equivalent amino acid sequence may be produced
and these sequences may be used to clone and express a given polypeptide.
As will be understood by those of skill in the art, it may be advantageous
in some instances to produce polypeptide-encoding nucleotide sequences
possessing non-naturally occurring codons. For example, codons preferred
by a particular prokaryotic or eukaryotic host can be selected to
increase the rate of protein expression or to produce a recombinant
RNA transcript having desirable properties, such as a half-life
which is longer than that of a transcript generated from the naturally
Moreover, the polynucleotide sequences of the present invention
can be engineered using methods generally known in the art in order
to alter polypeptide encoding sequences for a variety of reasons,
including but not limited to, alterations which modify the cloning,
processing, and/or expression of the gene product. For example,
DNA shuffling by random fragmentation and PCR reassembly of gene
fragments and synthetic oligonucleotides may be used to engineer
the nucleotide sequences. In addition, site-directed mutagenesis
may be used to insert new restriction sites, alter glycosylation
patterns, change codon preference, produce splice variants, or introduce
mutations, and so forth.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid sequences may be ligated to a heterologous sequence
to encode a fusion protein. For example, to screen peptide libraries
for inhibitors of polypeptide activity, it may be useful to encode
a chimeric protein that can be recognized by a commercially available
antibody. A fusion protein may also be engineered to contain a cleavage
site located between the polypeptide-encoding sequence and the heterologous
protein sequence, so that the polypeptide may be cleaved and purified
away from the heterologous moiety.
Sequences encoding a desired polypeptide may be synthesized, in
whole or in part, using chemical methods well known in the art (see
Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223,
Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232). Alternatively,
the protein itself may be produced using chemical methods to synthesize
the amino acid sequence of a polypeptide, or a portion thereof.
For example, peptide synthesis can be performed using various solid-phase
techniques (Roberge, J. Y. et al. (1995) Science 269:202-204) and
automated synthesis may be achieved, for example, using the ABI
431 A Peptide Synthesizer (Perkin E1 mer, Palo Alto, Calif.).
A newly synthesized peptide may be substantially purified by preparative
high performance liquid chromatography (e.g., Creighton, T. (1983)
Proteins, Structures and Molecular Principles, W H Freeman and Co.,
New York, N.Y.) or other comparable techniques available in the
art. The composition of the synthetic peptides may be confirmed
by amino acid analysis or sequencing (e.g., the Edman degradation
procedure). Additionally, the amino acid sequence of a polypeptide,
or any part thereof, may be altered during direct synthesis and/or
combined using chemical methods with sequences from other proteins,
or any part thereof, to produce a variant polypeptide.
In order to express a desired polypeptide, the nucleotide sequences
encoding the polypeptide, or functional equivalents, may be inserted
into appropriate expression vector, i.e., a vector which contains
the necessary elements for the transcription and translation of
the inserted coding sequence. Methods which are well known to those
skilled in the art may be used to construct expression vectors containing
sequences encoding a polypeptide of interest and appropriate transcriptional
and translational control elements. These methods include in vitro
recombinant DNA techniques, synthetic techniques, and in vivo genetic
recombination. Such techniques are described in Sambrook, J. et
al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current
Protocols in Molecular Biology, John Wiley & Sons, New York.
A variety of expression vector/host systems may be utilized to
contain and express polynucleotide sequences. These include, but
are not limited to, microorganisms such as bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast transformed with yeast expression vectors; insect
cell systems infected with virus expression vectors (e.g., baculovirus);
plant cell systems transformed with virus expression vectors (e.g.,
cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with
bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal
The "control elements" or "regulatory sequences"
present in an expression vector are those non--translated regions
of the vector--enhancers, promoters, 5' and 3' untranslated regions--which
interact with host cellular proteins to carry out transcription
and translation. Such elements may vary in their strength and specificity.
Depending on the vector system and host utilized, any number of
suitable transcription and translation elements, including constitutive
and inducible promoters, may be used. For example, when cloning
in bacterial systems, inducible promoters such as the hybrid lacZ
promoter of the PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.)
or PSPORT1 plasmid (Gibco BRL, Gaithersburg, Md.) and the like may
be used. In mammalian cell systems, promoters from mammalian genes
or from mammalian viruses are generally preferred. If it is necessary
to generate a cell line that contains multiple copies of the sequence
encoding a polypeptide, vectors based on SV40 or EBV may be advantageously
used with an appropriate selectable marker.
In bacterial systems, a number of expression vectors may be selected
depending upon the use intended for the expressed polypeptide. For
example, when large quantities are needed, for example for the induction
of antibodies, vectors which direct high level expression of fusion
proteins that are readily purified may be used. Such vectors include,
but are not limited to, the multifunctional E. coli cloning and
expression vectors such as BLUESCRIPT (Stratagene), in which the
sequence encoding the polypeptide of interest may be ligated into
the vector in frame with sequences for the amino-terminal Met and
the subsequent 7 residues of .beta.-galactosidase so that a hybrid
protein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster
(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX Vectors
(Promega, Madison, Wis.) may also be used to express foreign polypeptides
as fusion proteins with glutathione S-transferase (GST). In general,
such fusion proteins are soluble and can easily be purified from
lysed cells by adsorption to glutathione-agarose beads followed
by elution in the presence of free glutathione. Proteins made in
such systems may be designed to include heparin, thrombin, or factor
XA protease cleavage sites so that the cloned polypeptide of interest
can be released from the GST moiety at will.
In the yeast, Saccharomyces cerevisiae, a number of vectors containing
constitutive or inducible promoters such as alpha factor, alcohol
oxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)
and Grant et al. (1987) Methods Enzymol. 153:516-544.
In cases where plant expression vectors are used, the expression
of sequences encoding polypeptides may be driven by any of a number
of promoters. For example, viral promoters such as the 35S and 19S
promoters of CaMV may be used alone or in combination with the omega
leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311.
Alternatively, plant promoters such as the small subunit of RUBISCO
or heat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO
J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and
Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105).
These constructs can be introduced into plant cells by direct DNA
transformation or pathogen-mediated transfection. Such techniques
are described in a number of generally available reviews (see, for
example, Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science
and Technology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).
An insect system may also be used to express a polypeptide of interest.
For example, in one such system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
The sequences encoding the polypeptide may be cloned into a non-essential
region of the virus, such as the polyhedrin gene, and placed under
control of the polyhedrin promoter. Successful insertion of the
polypeptide-encoding sequence will render the polyhedrin gene inactive
and produce recombinant virus lacking coat protein. The recombinant
viruses may then be used to infect, for example, S. frugiperda cells
or Trichoplusia larvae in which the polypeptide of interest may
be expressed (Engelhard, E. K. et al. (1994) Proc. Natl. Acad. Sci.
In mammalian host cells, a number of viral-based expression systems
are generally available. For example, in cases where an adenovirus
is used as an expression vector, sequences encoding a polypeptide
of interest may be ligated into an adenovirus transcription/translation
complex consisting of the late promoter and tripartite leader sequence.
Insertion in a non-essential E1 or E3 region of the viral genome
may be used to obtain a viable virus which is capable of expressing
the polypeptide in infected host cells (Logan, J. and Shenk, T.
(1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition, transcription
enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be
used to increase expression in mammalian host cells.
Specific initiation signals may also be used to achieve more efficient
translation of sequences encoding a polypeptide of interest. Such
signals include the ATG initiation codon and adjacent sequences.
In cases where sequences encoding the polypeptide, its initiation
codon, and upstream sequences are inserted into the appropriate
expression vector, no additional transcriptional or translational
control signals may be needed. However, in cases where only coding
sequence, or a portion thereof, is inserted, exogenous translational
control signals including the ATG initiation codon should be provided.
Furthermore, the initiation codon should be in the correct reading
frame to ensure translation of the entire insert. Exogenous translational
elements and initiation codons may be of various origins, both natural
and synthetic. The efficiency of expression may be enhanced by the
inclusion of enhancers which are appropriate for the particular
cell system which is used, such as those described in the literature
(Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162).
In addition, a host cell strain may be chosen for its ability to
modulate the expression of the inserted sequences or to process
the expressed protein in the desired fashion. Such modifications
of the polypeptide include, but are not limited to, acetylation,
carboxylation. glycosylation, phosphorylation, lipidation, and acylation.
Post-translational processing which cleaves a "prepro"
form of the protein may also be used to facilitate correct insertion,
folding and/or function. Different host cells such as CHO, HeLa,
MDCK, HEK293, and W138, which have specific cellular machinery and
characteristic mechanisms for such post-translational activities,
may be chosen to ensure the correct modification and processing
of the foreign protein.
For long-term, high-yield production of recombinant proteins, stable
expression is generally preferred. For example, cell lines which
stably express a polynucleotide of interest may be transformed using
expression vectors which may contain viral origins of replication
and/or endogenous expression elements and a selectable marker gene
on the same or on a separate vector. Following the introduction
of the vector, cells may be allowed to grow for 1-2 days in an enriched
media before they are switched to selective media. The purpose of
the selectable marker is to confer resistance to selection, and
its presence allows growth and recovery of cells which successfully
express the introduced sequences. Resistant clones of stably transformed
cells may be proliferated using tissue culture techniques appropriate
to the cell type.
Any number of selection systems may be used to recover transformed
cell lines. These include, but are not limited to, the herpes simplex
virus thymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32)
and adenine phosphoribosyltransferase (Lowy, I. et al. (1990) Cell
22:817-23) genes which can be employed in tk.sup.- or aprt.sup.-
cells, respectively. Also, antimetabolite, antibiotic or herbicide
resistance can be used as the basis for selection; for example,
dhfr which confers resistance to methotrexate (Wigler, M. et al.
(1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which confers resistance
to the aminoglycosides, neomycin and G-418 (Colbere-Garapin, F.
et al (1981) J. Mol. Biol. 150:1-14); and als or pat, which confer
resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively (Murry, supra). Additional selectable genes have been
described, for example, trpB, which allows cells to utilize indole
in place of tryptophan, or hisD, which allows cells to utilize histinol
in place of histidine (Hartman, S. C. and R. C. Mulligan (1988)
Proc. Natl. Acad. Sci. 85:8047-51). Recently, the use of visible
markers has gained popularity with such markers as anthocyanins,
beta-glucuronidase and its substrate GUS, and luciferase and its
substrate luciferin, being widely used not only to identify transformants,
but also to quantify the amount of transient or stable protein expression
attributable to a specific vector system (Rhodes, C. A. et al. (1995)
Methods Mol. Biol. 55:121-131).
Although the presence/absence of marker gene expression suggests
that the gene of interest is also present, its presence and expression
may need to be confirmed. For example, if the sequence encoding
a polypeptide is inserted within a marker gene sequence, recombinant
cells containing sequences can be identified by the absence of marker
gene function. Alternatively, a marker gene can be placed in tandem
with a polypeptide-encoding sequence under the control of a single
promoter. Expression of the marker gene in response to induction
or selection usually indicates expression of the tandem gene as
Alternatively, host cells which contain and express a desired polynucleotide
sequence may be identified by a variety of procedures known to those
of skill in the art. These procedures include, but are not limited
to, DNA--DNA or DNA-RNA hybridizations and protein bioassay or immunoassay
techniques which include membrane, solution, or chip based technologies
for the detection and/or quantification of nucleic acid or protein.
A variety of protocols for detecting and measuring the expression
of polynucleotide-encoded products, using either polyclonal or monoclonal
antibodies specific for the product are known in the art. Examples
include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay
(RIA), and fluorescence activated cell sorting (FACS). A two-site,
monoclonal-based immunoassay utilizing monoclonal antibodies reactive
to two non-interfering epitopes on a given polypeptide may be preferred
for some applications, but a competitive binding assay may also
be employed. These and other assays are described, among other places,
in Hampton, R. et al. (1990; Serological Methods, a Laboratory Manual,
APS Press, St Paul. Minn.) and Maddox, D. E. et al. (1983; J. Exp.
A wide variety of labels and conjugation techniques are known by
those skilled in the art and may be used in various nucleic acid
and amino acid assays. Means for producing labeled hybridization
or PCR probes for detecting sequences related to polynucleotides
include oligolabeling, nick translation, end-labeling or PCR amplification
using a labeled nucleotide. Alternatively, the sequences, or any
portions thereof may be cloned into a vector for the production
of an mRNA probe. Such vectors are known in the art, are commercially
available, and may be used to synthesize RNA probes in vitro by
addition of an appropriate RNA polymerase such as T7, T3, or SP6
and labeled nucleotides. These procedures may be conducted using
a variety of commercially available kits. Suitable reporter molecules
or labels, which may be used include radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic agents as well as substrates, cofactors,
inhibitors, magnetic particles, and the like.
Host cells transformed with a polynucleotide sequence of interest
may be cultured under conditions suitable for the expression and
recovery of the protein from cell culture. The protein produced
by a recombinant cell may be secreted or contained intracellularly
depending on the sequence and/or the vector used. As will be understood
by those of skill in the art, expression vectors containing polynucleotides
of the invention may be designed to contain signal sequences which
direct secretion of the encoded polypeptide through a prokaryotic
or eukaryotic cell membrane. Other recombinant constructions may
be used to join sequences encoding a polypeptide of interest to
nucleotide sequence encoding a polypeptide domain which will facilitate
purification of soluble proteins. Such purification facilitating
domains include, but are not limited to, metal chelating peptides
such as histidine-tryptophan modules that allow purification on
immobilized metals, protein A domains that allow purification on
immobilized immunoglobulin, and the domain utilized in the FLAGS
extension/affinity purification system (Immunex Corp., Seattle,
Wash.). The inclusion of cleavable linker sequences such as those
specific for Factor XA or enterokinase (Invitrogen. San Diego, Calif.)
between the purification domain and the encoded polypeptide may
be used to facilitate purification. One such expression vector provides
for expression of a fusion protein containing a polypeptide of interest
and a nucleic acid encoding 6 histidine residues preceding a thioredoxin
or an enterokinase cleavage site. The histidine residues facilitate
purification on IMIAC (immobilized metal ion affinity chromatography)
as described in Porath, J. et al. (1992, Prot. Exp. Purif. 3:263
-281) while the enterokinase cleavage site provides a means for
purifying the desired polypeptide from the fusion protein. A discussion
of vectors which contain fusion proteins is provided in Kroll, D.
J. et al. (1993; DNA Cell Biol. 12:441-453).
In addition to recombinant production methods, polypeptides of
the invention, and fragments thereof, may be produced by direct
peptide synthesis using solid-phase techniques (Merrifield J. (1963)
J. Am. Chem. Soc. 85:2149-2154). Protein synthesis may be performed
using manual techniques or by automation. Automated synthesis may
be achieved, for example, using Applied Biosystems 431A Peptide
Synthesizer (Perkin E1 mer). Alternatively, various fragments may
be chemically synthesized separately and combined using chemical
methods to produce the full length molecule.
Site-specific mutagenesis is a technique useful in the preparation
of individual peptides, or biologically functional equivalent polypeptides,
through specific mutagenesis of the underlying polynucleotides that
encode them. The technique, well-known to those of skill in the
art, further provides a ready ability to prepare and test sequence
variants, for example, incorporating one or more of the foregoing
considerations, by introducing one or more nucleotide sequence changes
into the DNA. Site-specific mutagenesis allows the production of
mutants through the use of specific oligonucleotide sequences which
encode the DNA sequence of the desired mutation, as well as a sufficient
number of adjacent nucleotides, to provide a primer sequence of
sufficient size and sequence complexity to form a stable duplex
on both sides of the deletion junction being traversed. Mutations
may be employed in a selected polynucleotide sequence to improve,
alter, decrease, modify, or otherwise change the properties of the
polynucleotide itself, and/or alter the properties, activity, composition,
stability, or primary sequence of the encoded polypeptide.
In certain embodiments of the present invention, the inventors
contemplate the mutagenesis of the disclosed polynucleotide sequences
to alter one or more properties of the encoded polypeptide, such
as the antigenicity of a polypeptide vaccine. The techniques of
site-specific mutagenesis are well-known in the art, and are widely
used to create variants of both polypeptides and polynucleotides.
For example, site-specific mutagenesis is often used to alter a
specific portion of a DNA molecule. In such embodiments, a primer
comprising typically about 14 to about 25 nucleotides or so in length
is employed, with about 5 to about 10 residues on both sides of
the junction of the sequence being altered.
As will be appreciated by those of skill in the art, site-specific
mutagenesis techniques have often employed a phage vector that exists
in both a single stranded and double stranded form. Typical vectors
useful in site-directed mutagenesis include vectors such as the
M13 phage. These phage are readily commercially-available and their
use is generally well-known to those skilled in the art. Double-stranded
plasmids are also routinely employed in site directed mutagenesis
that eliminates the step of transferring the gene of interest from
a plasmid to a phage.
In general, site-directed mutagenesis in accordance herewith is
performed by first obtaining a single-stranded vector or melting
apart of two strands of a double-stranded vector that includes within
its sequence a DNA sequence that encodes the desired peptide. An
oligonucleotide primer bearing the desired mutated sequence is prepared,
generally synthetically. This primer is then annealed with the single-stranded
vector, and subjected to DNA polymerizing enzymes such as E. Coli
polymerase I Klenow fragment, in order to complete the synthesis
of the mutation-bearing strand. Thus, a heteroduplex is formed wherein
one strand encodes the original non-mutated sequence and the second
strand bears the desired mutation. This heteroduplex vector is then
used to transform appropriate cells, such as E. coli cells, and
clones are selected which include recombinant vectors bearing the
mutated sequence arrangement.
The preparation of sequence variants of the selected peptide-encoding
DNA segments using site-directed mutagenesis provides a means of
producing potentially useful species and is not meant to be limiting
as there are other ways in which sequence variants of peptides and
the DNA sequences encoding them may be obtained. For example, recombinant
vectors encoding the desired peptide sequence may be treated with
mutagenic agents, such as hydroxylamine, to obtain sequence variants.
Specific details regarding these methods and protocols are found
in the teachings of Maloy et al., 1994; Segal, 1976; Prokop and
Bajpai, 1991; Kuby, 1994; and Maniatis et al., 1982, each incorporated
herein by reference, for that purpose.
As used herein, the term "oligonucleotide directed mutagenesis
procedure" refers to template-dependent processes and vector-mediated
propagation which result in an increase in the concentration of
a specific nucleic acid molecule relative to its initial concentration,
or in an increase in the concentration of a detectable signal, such
as amplification. As used herein, the term "oligonucleotide
directed mutagenesis procedure" is intended to refer to a process
that involves the template-dependent extension of a primer molecule.
The term template dependent process refers to nucleic acid synthesis
of an RNA or a DNA molecule wherein the sequence of the newly synthesized
strand of nucleic acid is dictated by the well-known rules of complementary
base pairing (see, for example, Watson, 1987). Typically, vector
mediated methodologies involve the introduction of the nucleic acid
fragment into a DNA or RNA vector, the clonal amplification of the
vector, and the recovery of the amplified nucleic acid fragment.
Examples of such methodologies are provided by U.S. Pat. No. 4,237,224,
specifically incorporated herein by reference in its entirety.
Polynucleotide Amplification Techniques
A number of template dependent processes are available to amplify
the target sequences of interest present in a sample. One of the
best known amplification methods is the polymerase chain reaction
(PCR.TM.) which is described in detail in U.S. Pat. Nos. 4,683,195,
4,683,202 and 4,800,159, each of which is incorporated herein by
reference in its entirety. Briefly, in PCR.TM., two primer sequences
are prepared which are complementary to regions on opposite complementary
strands of the target sequence. An excess of deoxynucleoside triphosphates
is added to a reaction mixture along with a DNA polymerase (e.g.,
Taq polymerase). If the target sequence is present in a sample,
the primers will bind to the target and the polymerase will cause
the primers to be extended along the target sequence by adding on
nucleotides. By raising and lowering the temperature of the reaction
mixture, the extended primers will dissociate from the target to
form reaction products, excess primers will bind to the target and
to the reaction product and the process is repeated. Preferably
reverse transcription and PCR.TM. amplification procedure may be
performed in order to quantify the amount of mRNA amplified. Polymerase
chain reaction methodologies are well known in the art.
Another method for amplification is the ligase chain reaction (referred
to as LCR), disclosed in Eur. Pat. Appl. Publ. No. 320,308 (specifically
incorporated herein by reference in its entirety). In LCR, two complementary
probe pairs are prepared, and in the presence of the target sequence,
each pair will bind to opposite complementary strands of the target
such that they abut. In the presence of a ligase, the two probe
pairs will link to form a single unit. By temperature cycling, as
in PCR.TM., bound ligated units dissociate from the target and then
serve as "target sequences" for ligation of excess probe
pairs. U.S. Pat. No. 4,883,750, incorporated herein by reference
in its entirety, describes an alternative method of amplification
similar to LCR for binding probe pairs to a target sequence.
Qbeta Replicase, described in PCT Intl. Pat. Appl. Publ. No. PCT/US87/00880,
incorporated herein by reference in its entirety, may also be used
as still another amplification method in the present invention.
In this method, a replicative sequence of RNA that has a region
complementary to that of a target is added to a sample in the presence
of an RNA polymerase. The polymerase will copy the replicative sequence
that can then be detected.
An isothermal amplification method, in which restriction endonucleases
and ligases are used to achieve the amplification of target molecules
that contain nucleotide 5'-[.alpha.-thio]triphosphates in one strand
of a restriction site (Walker et al., 1992, incorporated herein
by reference in its entirety), may also be useful in the amplification
of nucleic acids in the present invention.
Strand Displacement Amplification (SDA) is another method of carrying
out isothermal amplification of nucleic acids which involves multiple
rounds of strand displacement and synthesis, i.e. nick translation.
A similar method, called Repair Chain Reaction (RCR) is another
method of amplification which may be useful in the present invention
and is involves annealing several probes throughout a region targeted
for amplification, followed by a repair reaction in which only two
of the four bases are present. The other two bases can be added
as biotinylated derivatives for easy detection. A similar approach
is used in SDA.
Sequences can also be detected using a cyclic probe reaction (CPR).
In CPR, a probe having a 3' and 5' sequences of non-target DNA and
an internal or "middle" sequence of the target protein
specific RNA is hybridized to DNA which is present in a sample.
Upon hybridization, the reaction is treated with RNaseH, and the
products of the probe are identified as distinctive products by
generating a signal that is released after digestion. The original
template is annealed to another cycling probe and the reaction is
repeated. Thus, CPR involves amplifying a signal generated by hybridization
of a probe to a target gene specific expressed nucleic acid.
Still other amplification methods described in Great Britain Pat.
Appl. No. 2 202 328, and in PCT Intl. Pat. Appl. Publ. No. PCT/US89/01025,
each of which is incorporated herein by reference in its entirety,
may be used in accordance with the present invention. In the former
application, "modified" primers are used in a PCR-like,
template and enzyme dependent synthesis. The primers may be modified
by labeling with a capture moiety (e.g., biotin) and/or a detector
moiety (e.g., enzyme). In the latter application, an excess of labeled
probes is added to a sample. In the presence of the target sequence,
the probe binds and is cleaved catalytically. After cleavage, the
target sequence is released intact to be bound by excess probe.
Cleavage of the labeled probe signals the presence of the target
Other nucleic acid amplification procedures include transcription-based
amplification systems (TAS) (Kwoh et al., 1989; PCT Intl. Pat. Appl.
Publ. No. WO 88/10315, incorporated herein by reference in its entirety),
including nucleic acid sequence based amplification (NASBA) and
3SR. In NASBA, the nucleic acids can be prepared for amplification
by standard phenol/chloroform extraction, heat denaturation of a
sample, treatment with lysis buffer and minispin columns for isolation
of DNA and RNA or guanidinium chloride extraction of RNA. These
amplification techniques involve annealing a primer that has sequences
specific to the target sequence. Following polymerization, DNA/RNA
hybrids are digested with RNase H while double stranded DNA molecules
are heat-denatured again. In either case the single stranded DNA
is made fully double stranded by addition of second target-specific
primer, followed by polymerization. The double stranded DNA molecules
are then multiply transcribed by a polymerase such as T7 or SP6.
In an isothermal cyclic reaction, the RNAs are reverse transcribed
into DNA, and transcribed once again with a polymerase such as T7
or SP6. The resulting products, whether truncated or complete, indicate
Eur. Pat. Appl. Publ. No. 329,822, incorporated herein by reference
in its entirety, disclose a nucleic acid amplification process involving
cyclically synthesizing single-stranded RNA ("ssRNA"),
ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance
with the present invention. The ssRNA is a first template for a
first primer oligonucleotide, which is elongated by reverse transcriptase
(RNA-dependent DNA polymerase). The RNA is then removed from resulting
DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase
specific for RNA in a duplex with either DNA or RNA). The resultant
ssDNA is a second template for a second primer, which also includes
the sequences of an RNA polymerase promoter (exemplified by T7 RNA
polymerase) 5' to its homology to its template. This primer is then
extended by DNA polymerase (exemplified by the large "Klenow"
fragment of E. coli DNA polymerase I), resulting as a double-stranded
DNA ("dsDNA") molecule, having a sequence identical to
that of the original RNA between the primers and having additionally,
at one end, a promoter sequence. This promoter sequence can be used
by the appropriate RNA polymerase to make many RNA copies of the
DNA. These copies can then re-enter the cycle leading to very swift
amplification. With proper choice of enzymes, this amplification
can be done isothermally without addition of enzymes at each cycle.
Because of the cyclical nature of this process, the starting sequence
can be chosen to be in the form of either DNA or RNA.
PCT Intl. Pat. Appl. Publ. No. WO 89/06700, incorporated herein
by reference in its entirety, disclose a nucleic acid sequence amplification
scheme based on the hybridization of a promoter/primer sequence
to a target single-stranded DNA ("ssDNA") followed by
transcription of many RNA copies of the sequence. This scheme is
not cyclic; i.e. new templates are not produced from the resultant
RNA transcripts. Other amplification methods include "RACE"
(Frohman, 1990), and "one-sided PCR" (Ohara, 1989) which
are well-known to those of skill in the art.
Methods based on ligation of two (or more) oligonucleotides in
the presence of nucleic acid having the sequence of the resulting
"di-oligonucleotide", thereby amplifying the di-oligonucleotide
(Wu and Dean, 1996, incorporated herein by reference in its entirety),
may also be used in the amplification of DNA sequences of the present
Biobogical Functional Equivalents
Modification and changes may be made in the structure of the polynucleotides
and polypeptides of the present invention and still obtain a functional
molecule that encodes a polypeptide with desirable characteristics.
As mentioned above, it is often desirable to introduce one or more
mutations into a specific polynucleotide sequence. In certain circumstances,
the resulting encoded polypeptide sequence is altered by this mutation,
or in other cases, the sequence of the polypeptide is unchanged
by one or more mutations in the encoding polynucleotide.
When it is desirable to alter the amino acid sequence of a polypeptide
to create an equivalent, or even an improved, second-generation
molecule, the amino acid changes may be achieved by changing one
or more of the codons of the encoding DNA sequence, according to
For example, certain amino acids may be substituted for other amino
acids in a protein structure without appreciable loss of interactive
binding capacity with structures such as, for example, antigen-binding
regions of antibodies or binding sites on substrate molecules. Since
it is the interactive capacity and nature of a protein that defines
that protein's biological functional activity, certain amino acid
sequence substitutions can be made in a protein sequence, and, of
course, its underlying DNA coding sequence, and nevertheless obtain
a protein with like properties. It is thus contemplated by the inventors
that various changes may be made in the peptide sequences of the
disclosed compositions, or corresponding DNA sequences which encode
said peptides without appreciable loss of their biological utility
TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine
Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA
GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine
His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG
Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine
Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA
CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU
UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA
GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU
In making such changes, the hydropathic index of amino acids may
be considered. The importance of the hydropathic amino acid index
in conferring interactive biologic function on a protein is generally
understood in the art (Kyte and Doolittle, 1982, incorporated herein
by reference). It is accepted that the relative hydropathic character
of the amino acid contributes to the secondary structure of the
resultant protein, which in turn defines the interaction of the
protein with other molecules, for example, enzymes, substrates,
receptors, DNA, antibodies, antigens, and the like. Each amino acid
has been assigned a hydropathic index on the basis of its hydrophobicity
and charge characteristics (Kyte and Doolittle, 1982). These values
are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine
(+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8);
glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9);
tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5);
glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9);
and arginine (-4.5).
It is known in the art that certain amino acids may be substituted
by other amino acids having a similar hydropathic index or score
and still result in a protein with similar biological activity,
i.e. still obtain a biological functionally equivalent protein.
In making such changes, the substitution of amino acids whose hydropathic
indices are within .+-.2 is preferred, those within .+-.1 are particularly
preferred, and those within .+-.0.5 are even more particularly preferred.
It is also understood in the art that the substitution of like amino
acids can be made effectively on the basis of hydrophilicity. U.S.
Pat. No. 4,554,101 (specifically incorporated herein by reference
in its entirety), states that the greatest local average hydrophilicity
of a protein, as governed by the hydrophilicity of its adjacent
amino acids, correlates with a biological property of the protein.
As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity
values have been assigned to amino acid residues: arginine (+3.0);
lysine (+3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine
(+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine
(-0.4); proline (-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine
(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine
(-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
It is understood that an amino acid can be substituted for another
having a similar hydrophilicity value and still obtain a biologically
equivalent, and in particular, an immunologically equivalent protein.
In such changes, the substitution of amino acids whose hydrophilicity
values are within .+-.2 is preferred, those within .+-.1 are particularly
preferred, and those within .+-.0.5 are even more particularly preferred.
As outlined above, amino acid substitutions are generally therefore
based on the relative similarity of the amino acid side-chain substituents,
for example, their hydrophobicity, hydrophilicity, charge, size,
and the like. Exemplary substitutions that take various of the foregoing
characteristics into consideration are well known to those of skill
in the art and include: arginine and lysine; glutamate and aspartate;
serine and threonine; glutamine and asparagine; and valine, leucine
In addition, any polynucleotide may be further modified to increase
stability in vivo. Possible modifications include, but are not limited
to, the addition of flanking sequences at the 5' and/or 3' ends;
the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase
linkages in the backbone; and/or the inclusion of nontraditional
bases such as inosine, queosine and wybutosine, as well as acetyl-
methyl-, thio- and other modified forms of adenine, cytidine, guanine,
thymine and uridine.
In vivo Polynucleotide Delivery Techniques
In additional embodiments, genetic constructs comprising one or
more of the polynucleotides of the invention are introduced into
cells in vivo. This may be achieved using any of a variety or well
known approaches, several of which are outlined below for the purpose
One of the preferred methods for in vivo delivery of one or more
nucleic acid sequences involves the use of an adenovirus expression
vector. "Adenovirus expression vector" is meant to include
those constructs containing adenovirus sequences sufficient to (a)
support packaging of the construct and (b) to express a polynucleotide
that has been cloned therein in a sense or antisense orientation.
Of course, in the context of an antisense construct, expression
does not require that the gene product be synthesized.
The expression vector comprises a genetically engineered form of
an adenovirus. Knowledge of the genetic organization of adenovirus,
a 36 kb, linear, double-stranded DNA virus, allows substitution
of large pieces of adenoviral DNA with foreign sequences up to 7
kb (Grunhaus and Horwitz, 1992). In contrast to retrovirus, the
adenoviral infection of host cells does not result in chromosomal
integration because adenoviral DNA can replicate in an episomal
manner without potential genotoxicity. Also, adenoviruses are structurally
stable, and no genome rearrangement has been detected after extensive
amplification. Adenovirus can infect virtually all epithelial cells
regardless of their cell cycle stage. So far, adenoviral infection
appears to be linked only to mild disease such as acute respiratory
disease in humans.
Adenovirus is particularly suitable for use as a gene transfer
vector because of its mid-sized genome, ease of manipulation, high
titer, wide target-cell range and high infectivity. Both ends of
the viral genome contain 100-200 base pair inverted repeats (ITRs),
which are cis elements necessary for viral DNA replication and packaging.
The early (E) and late (L) regions of the genome contain different
transcription units that are divided by the onset of viral DNA replication.
The E1 region (E1A and E1B) encodes proteins responsible for the
regulation of transcription of the viral genome and a few cellular
genes. The expression of the E2 region (E2A and E2B) results in
the synthesis of the proteins for viral DNA replication. These proteins
are involved in DNA replication, late gene expression and host cell
shut-off (Renan, 1990). The products of the late genes, including
the majority of the viral capsid proteins, are expressed only after
significant processing of a single primary transcript issued by
the major late promoter (MLP). The MLP, (located at 16.8 m.u.) is
particularly efficient during the late phase of infection, and all
the mRNA's issued from this promoter possess a 5'-tripartite leader
(TPL) sequence which makes them preferred mRNA's for translation.
In a current system, recombinant adenovirus is generated from homologous
recombination between shuttle vector and provirus vector. Due to
the possible recombination between two proviral vectors, wild-type
adenovirus may be generated from this process. Therefore, it is
critical to isolate a single clone of virus from an individual plaque
and examine its genomic structure.
Generation and propagation of the current adenovirus vectors, which
are replication deficient, depend on a unique helper cell line,
designated 293, which was transformed from human embryonic kidney
cells by Ad5 DNA fragments and constitutively expresses E1 proteins
(Graham et al., 1977). Since the E3 region is dispensable from the
adenovirus genome (Jones and Shenk, 1978), the current adenovirus
vectors, with the help of 293 cells, carry foreign DNA in either
the E1, the D3 or both regions (Graham and Prevec, 1991). In nature,
adenovirus can package approximately 105% of the wild-type genome
(Ghosh-Choudhury et al., 1987), providing capacity for about 2 extra
kB of DNA. Combined with the approximately 5.5 kB of DNA that is
replaceable in the E1 and E3 regions, the maximum capacity of the
current adenovirus vector is under 7.5 kB, or about 15% of the total
length of the vector. More than 80% of the adenovirus viral genome
remains in the vector backbone and is the source of vector-borne
cytotoxicity. Also, the replication deficiency of the E1-deleted
virus is incomplete. For example, leakage of viral gene expression
has been observed with the currently available vectors at high multiplicities
of infection (MOI) (Mulligan, 1993).
Helper cell lines may be derived from human cells such as human
embryonic kidney cells, muscle cells, hematopoietic cells or other
human embryonic mesenchymal or epithelial cells. Alternatively,
the helper cells may be derived from the cells of other mammalian
species that are permissive for human adenovirus. Such cells include,
e.g., Vero cells or other monkey embryonic mesenchymal or epithelial
cells. As stated above, the currently preferred helper cell line
Recently, Racher et al. (1995) disclosed improved methods for culturing
293 cells and propagating adenovirus. In one format, natural cell
aggregates are grown by inoculating individual cells into 1 liter
siliconized spinner flasks (Techne, Cambridge, UK) containing 100-200
ml of medium. Following stirring at 40 rpm, the cell viability is
estimated with trypan blue. In another format, Fibra-Cel microcarriers
(Bibby Sterlin, Stone, UK) (5 g/l) is employed as follows. A cell
inoculum, resuspended in 5 ml of medium, is added to the carrier
(50 ml) in a 250 ml Erlenmeyer flask and left stationary, with occasional
agitation, for 1 to 4 h. The medium is then replaced with 50 ml
of fresh medium and shaking initiated. For virus production, cells
are allowed to grow to about 80% confluence, after which time the
medium is replaced (to 25% of the final volume) and adenovirus added
at an MOI of 0.05. Cultures are left stationary overnight, following
which the volume is increased to 100% and shaking commenced for
another 72 h.
Other than the requirement that the adenovirus vector be replication
defective, or at least conditionally defective, the nature of the
adenovirus vector is not believed to be crucial to the successful
practice of the invention. The adenovirus may be of any of the 42
different known serotypes or subgroups A-F. Adenovirus type 5 of
subgroup C is the preferred starting material in order to obtain
a conditional replication-defective adenovirus vector for use in
the present invention, since Adenovirus type 5 is a human adenovirus
about which a great deal of biochemical and genetic information
is known, and it has historically been used for most constructions
employing adenovirus as a vector.
As stated above, the typical vector according to the present invention
is replication defective and will not have an adenovirus E1 region.
Thus, it will be most convenient to introduce the polynucleotide
encoding the gene of interest at the position from which the E1-coding
sequences have been removed. However, the position of insertion
of the construct within the adenovirus sequences is not critical
to the invention. The polynucleotide encoding the gene of interest
may also be inserted in lieu of the deleted E3 region in E3 replacement
vectors as described by Karlsson et al. (1986) or in the E4 region
where a helper cell line or helper virus complements the E4 defect.
Adenovirus is easy to grow and manipulate and exhibits broad host
range in vitro and in vivo. This group of viruses can be obtained
in high titers, e.g., 10.sup.9 -10.sup.11 plaque-forming units per
ml, and they are highly infective. The life cycle of adenovirus
does not require integration into the host cell genome. The foreign
genes delivered by adenovirus vectors are episomal and, therefore,
have low genotoxicity to host cells. No side effects have been reported
in studies of vaccination with wild-type adenovirus (Couch et al.,
1963; Top et al., 1971), demonstrating their safety and therapeutic
potential as in vivo gene transfer vectors.
Adenovirus vectors have been used in eukaryotic gene expression
(Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development
(Grunhaus and Horwitz, 1992; Graham and Prevec, 1992). Recently,
animal studies suggested that recombinant adenovirus could be used
for gene therapy (Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet
et al., 1990; Rich et al., 1993). Studies in administering recombinant
adenovirus to different tissues include trachea instillation (Rosenfeld
et al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et
al., 1993), peripheral intravenous injections (Herz and Gerard,
1993) and stereotactic inoculation into the brain (Le Gal La Salle
et al., 1993).
The retroviruses are a group of single-stranded RNA viruses characterized
by an ability to convert their RNA to double-stranded DNA in infected
cells by a process of reverse-transcription (Coffin, 1990). The
resulting DNA then stably integrates into cellular chromosomes as
a provirus and directs synthesis of viral proteins. The integration
results in the retention of the viral gene sequences in the recipient
cell and its descendants. The retroviral genome contains three genes,
gag, pol, and env that code for capsid proteins, polymerase enzyme,
and envelope components, respectively. A sequence found upstream
from the gag gene contains a signal for packaging of the genome
into virions. Two long terminal repeat (LTR) sequences are present
at the 5' and 3' ends of the viral genome. These contain strong
promoter and enhancer sequences and are also required for integration
in the host cell genome (Coffin, 1990).
In order to construct a retroviral vector, a nucleic acid encoding
one or more oligonucleotide or polynucleotide sequences of interest
is inserted into the viral genome in the place of certain viral
sequences to produce a virus that is replication-defective. In order
to produce virions, a packaging cell line containing the gag, pol,
and env genes but without the LTR and packaging components is constructed
(Mann et al., 1983). When a recombinant plasmid containing a cDNA,
together with the retroviral LTR and packaging sequences is introduced
into this cell line (by calcium phosphate precipitation for example),
the packaging sequence allows the RNA transcript of the recombinant
plasmid to be packaged into viral particles, which are then secreted
into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986;
Mann et al., 1983). The media containing the recombinant retroviruses
is then collected, optionally concentrated, and used for gene transfer.
Retroviral vectors are able to infect a broad variety of cell types.
However, integration and stable expression require the division
of host cells (Paskind et al., 1975).
A novel approach designed to allow specific targeting of retrovirus
vectors was recently developed based on the chemical modification
of a retrovirus by the chemical addition of lactose residues to
the viral envelope. This modification could permit the specific
infection of hepatocytes via sialoglycoprotein receptors.
A different approach to targeting of recombinant retroviruses was
designed in which biotinylated antibodies against a retroviral envelope
protein and against a specific cell receptor were used. The antibodies
were coupled via the biotin components by using streptavidin (Roux
et al., 1989). Using antibodies against major histocompatibility
complex class I and class II antigens, they demonstrated the infection
of a variety of human cells that bore those surface antigens with
an ecotropic virus in vitro (Roux et al., 1989).
3. Adeno-Associated Viruses
AAV (Ridgeway, 1988; Hermonat and Muzycska, 1984) is a parovirus,
discovered as a contamination of adenoviral stocks. It is a ubiquitous
virus (antibodies are present in 85% of the US human population)
that has not been linked to any disease. It is also classified as
a dependovirus, because its replications is dependent on the presence
of a helper virus, such as adenovirus. Five serotypes have been
isolated, of which AAV-2 is the best characterized. AAV has a single-stranded
linear DNA that is encapsidated into capsid proteins VP1, VP2 and
VP3 to form an icosahedral virion of 20 to 24 nm in diameter (Muzyczka
and McLaughlin, 1988).
The AAV DNA is approximately 4.7 kilobases long. It contains two
open reading frames and is flanked by two ITRs. There are two major
genes in the AAV genome: rep and cap. The rep gene codes for proteins
responsible for viral replications, whereas cap codes for capsid
protein VP1-3. Each ITR forms a T-shaped hairpin structure. These
terminal repeats are the only essential cis components of the AAV
for chromosomal integration. Therefore, the AAV can be used as a
vector with all viral coding sequences removed and replaced by the
cassette of genes for delivery. Three viral promoters have been
identified and named p5, p19, and p40, according to their map position.
Transcription from p5 and p19 results in production of rep proteins,
and transcription from p40 produces the capsid proteins (Hermonat
and Muzyczka, 1984).
There are several factors that prompted researchers to study the
possibility of using rAAV as an expression vector One is that the
requirements for delivering a gene to integrate into the host chromosome
are surprisingly few. It is necessary to have the 145-bp ITRs, which
are only 6% of the AAV genome. This leaves room in the vector to
assemble a 4.5-kb DNA insertion. While this carrying capacity may
prevent the AAV from delivering large genes, it is amply suited
for delivering the antisense constructs of the present invention.
AAV is also a good choice of delivery vehicles due to its safety.
There is a relatively complicated rescue mechanism: not only wild
type adenovirus but also AAV genes are required to mobilize rAAV.
Likewise, AAV is not pathogenic and not associated with any disease.
The removal of viral coding sequences minimizes immune reactions
to viral gene expression, and therefore, rAAV does not evoke an
4. Other Viral Vectors as Expression Constructs
Other viral vectors may be employed as expression constructs in
the present invention for the delivery of oligonucleotide or polynucleotide
sequences to a host cell. Vectors derived from viruses such as vaccinia
virus (Ridgeway, 1988; Coupar et al., 1988), lentiviruses, polio
viruses and herpes viruses may be employed. They offer several attractive
features for various mammalian cells (Friedmann, 1989; Ridgeway,
1988; Coupar et al., 1988; Horwich et al., 1990).
With the recent recognition of defective hepatitis B viruses, new
insight was gained into the structure-function relationship of different
viral sequences. In vitro studies showed that the virus could retain
the ability for helper-dependent packaging and reverse transcription
despite the deletion of up to 80% of its genome (Horwich et al.,
1990). This suggested that large portions of the genome could be
replaced with foreign genetic material. The hepatotropism and persistence
(integration) were particularly attractive properties for liver-directed
gene transfer. Chang et al. (1991) introduced the chloramphenicol
acetyltransferase (CAT) gene into duck hepatitis B virus genome
in the place of the polymerase, surface, and pre-surface coding
sequences. It was cotransfected with wild-type virus into an avian
hepatoma cell line. Culture media containing high titers of the
recombinant virus were used to infect primary duckling hepatocytes.
Stable CAT gene expression was detected for at least 24 days after
transfection (Chang et al., 1991).
5. Non-Viral Vectors
In order to effect expression of the oligonucleotide or polynucleotide
sequences of the present invention, the expression construct must
be delivered into a cell. This delivery may be accomplished in vitro,
as in laboratory procedures for transforming cells lines, or in
vivo or ex vivo, as in the treatment of certain disease states.
As described above, one preferred mechanism for delivery is via
viral infection where the expression construct is encapsulated in
an infectious viral particle.
Once the expression construct has been delivered into the cell
the nucleic acid encoding the desired oligonucleotide or polynucleotide
sequences may be positioned and expressed at different sites. In
certain embodiments, the nucleic acid encoding the construct may
be stably integrated into the genome of the cell. This integration
may be in the specific location and orientation via homologous recombination
(gene replacement) or it may be integrated in a random, non-specific
location (gene augmentation). In yet further embodiments, the nucleic
acid may be stably maintained in the cell as a separate, episomal
segment of DNA. Such nucleic acid segments or "episomes"
encode sequences sufficient to permit maintenance and replication
independent of or in synchronization with the host cell cycle. How
the expression construct is delivered to a cell and where in the
cell the nucleic acid remains is dependent on the type of expression
In certain embodiments of the invention, the expression construct
comprising one or more oligonucleotide or polynucleotide sequences
may simply consist of naked recombinant DNA or plasmids. Transfer
of the construct may be performed by any of the methods mentioned
above which physically or chemically permeabilize the cell membrane.
This is particularly applicable for transfer in vitro but it may
be applied to in vivo use as well. Dubensky et al. (1984) successfully
injected polyomaviras DNA in the form of calcium phosphate precipitates
into liver and spleen of adult and newborn mice demonstrating active
viral replication and acute infection. Benvenisty and Reshef (1986)
also demonstrated that direct intraperitoneal injection of calcium
phosphate-precipitated plasmids results in expression of the transfected
genes. It is envisioned that DNA encoding a gene of interest may
also be transferred in a similar manner in vivo and express the
Another embodiment of the invention for transferring a naked DNA
expression construct into cells may involve particle bombardment.
This method depends on the ability to accelerate DNA-coated microprojectiles
to a high velocity allowing them to pierce cell membranes and enter
cells without killing them (Klein et al., 1987). Several devices
for accelerating small particles have been developed. One such device
relies on a high voltage discharge to generate an electrical current,
which in turn provides the motive force (Yang et al., 1990). The
microprojectiles used have consisted of biologically inert substances
such as tungsten or gold beads.
Selected organs including the liver, skin, and muscle tissue of
rats and mice have been bombarded in vivo (Yang et al., 1990; Zelenin
et al., 1991). This may require surgical exposure of the tissue
or cells, to eliminate any intervening tissue between the gun and
the target organ, i.e. ex vivo treatment. Again, DNA encoding a
particular gene may be delivered via this method and still be incorporated
by the present invention.
The end result of the flow of genetic information is the synthesis
of protein. DNA is transcribed by polymerases into messenger RNA
and translated on the ribosome to yield a folded, functional protein.
Thus there are several steps along the route where protein synthesis
can be inhibited. The native DNA segment coding for a polypeptide
described herein, as all such mammalian DNA strands, has two strands:
a sense strand and an antisense strand held together by hydrogen
bonding. The messenger RNA coding for polypeptide has the same nucleotide
sequence as the sense DNA strand except that the DNA thymidine is
replaced by uridine. Thus, synthetic antisense nucleotide sequences
will bind to a mRNA and inhibit expression of the protein encoded
by that mRNA.
The targeting of antisense oligonucleotides to mRNA is thus one
mechanism to shut down protein synthesis, and, consequently, represents
a powerful and targeted therapeutic approach. For example, the synthesis
of polygalactauronase and the muscarine type 2 acetylcholine receptor
are inhibited by antisense oligonucleotides directed to their respective
mRNA sequences (U.S. Pat. Nos. 5,739,119 and 5,759,829, each specifically
incorporated herein by reference in its entirety). Further, examples
of antisense inhibition have been demonstrated with the nuclear
protein cyclin, the multiple drug resistance gene (MDG1), ICAM-1,
E-selectin, STK-1, striatal GABA.sub.A receptor and human EGF (Jaskulski
et al., 1988; Vasanthakumar and Amlued, 1989; Peris et al., 1998;
U.S. Pat. Nos. 5,801,154; 5,789,573; 5,718,709 and 5,610,288, each
specifically incorporated herein by reference in its entirety).
Antisense constructs have also been described that inhibit and can
be used to treat a variety of abnormal cellular proliferations,
e.g. cancer (U.S. Pat. Nos. 5,747,470; 5,591,317 and 5,783,683,
each specifically incorporated herein by reference in its entirety).
Therefore, in exemplary embodiments, the invention provides oligonucleotide
sequences that comprise all, or a portion of, any sequence that
is capable of specifically binding to polynucleotide sequence described
herein, or a complement thereof. In one embodiment, the antisense
oligonucleotides comprise DNA or derivatives thereof. In another
embodiment, the oligonucleotides comprise RNA or derivatives thereof.
In a third embodiment, the oligonucleotides are modified DNAs comprising
a phosphorothioated modified backbone. In a fourth embodiment, the
oligonucleotide sequences comprise peptide nucleic acids or derivatives
thereof. In each case, preferred compositions comprise a sequence
region that is complementary, and more preferably substantially-complementary,
and even more preferably, completely complementary to one or more
portions of polynucleotides disclosed herein.
Selection of antisense compositions specific for a given gene sequence
is based upon analysis of the chosen target sequence (i.e. in these
illustrative examples the rat and human sequences) and determination
of secondary structure, T.sub.m, binding energy, relative stability,
and antisense compositions were selected based upon their relative
inability to form dimers, hairpins, or other secondary structures
that would reduce or prohibit specific binding to the target mRNA
in a host cell.
Highly preferred target regions of the mRNA, are those which are
at or near the AUG translation initiation codon, and those sequences
which were substantially complementary to 5' regions of the mRNA.
These secondary structure analyses and target site selection considerations
were performed using v.4 of the OLIGO primer analysis software (Rychlik,
1997) and the BLASTN 2.0.5 algorithm software (Altschul et al.,
The use of an antisense delivery method employing a short peptide
vector, termed MPG (27 residues), is also contemplated. The MPG
peptide contains a hydrophobic domain derived from the fusion sequence
of HIV gp41 and a hydrophilic domain from the nuclear localization
sequence of SV40 T-antigen (Morris et al., 1997). It has been demonstrated
that several molecules of the MPG peptide coat the antisense oligonucleotides
and can be delivered into cultured mammalian cells in less than
1 hour with relatively high efficiency (90%). Further, the interaction
with MPG strongly increases both the stability of the oligonucleotide
to nuclease and the ability to cross the plasma membrane (Morris
et al., 1997).
Although proteins traditionally have been used for catalysis of
nucleic acids, another class of macromolecules has emerged as useful
in this endeavor. Ribozymes are RNA-protein complexes that cleave
nucleic acids in a site-specific fashion. Ribozymes have specific
catalytic domains that possess endonuclease activity (Kim and Cech,
1987; Gerlach et al., 1987; Forster and Symons, 1987). For example,
a large number of ribozymes accelerate phosphoester transfer reactions
with a high degree of specificity, often cleaving only one of several
phosphoesters in an oligonucleotide substrate (Cech et al., 1981;
Michel and Westhof, 1990; Reinhold-Hurek and Shub, 1992). This specificity
has been attributed to the requirement that the substrate bind via
specific base-pairing interactions to the internal guide sequence
("IGS") of the ribozyme prior to chemical reaction.
Ribozyme catalysis has primarily been observed as part of sequence-specific
cleavage/ligation reactions involving nucleic acids (Joyce, 1989;
Cech et al., 1981). For example, U.S. Pat. No. 5,354,855 (specifically
incorporated herein by reference) reports that certain ribozymes
can act as endonucleases with a sequence specificity greater than
that of known ribonucleases and approaching that of the DNA restriction
enzymes. Thus, sequence-specific ribozyme-mediated inhibition of
gene expression may be particularly suited to therapeutic applications
(Scanlon et al., 1991; Sarver et al., 1990). Recently, it was reported
that ribozymes elicited genetic changes in some cells lines to which
they were applied; the altered genes included the oncogenes H-ras,
c-fos and genes of HIV. Most of this work involved the modification
of a target mRNA, based on a specific mutant codon that is cleaved
by a specific ribozyme.
Six basic varieties of naturally-occurring enzymatic RNAs are known
presently. Each can catalyze the hydrolysis of RNA phosphodiester
bonds in trans (and thus can cleave other RNA molecules) under physiological
conditions. In general, enzymatic nucleic acids act by first binding
to a target RNA. Such binding occurs through the target binding
portion of a enzymatic nucleic acid which is held in close proximity
to an enzymatic portion of the molecule that acts to cleave the
target RNA. Thus, the enzymatic nucleic acid first recognizes and
then binds a target RNA through complementary base-pairing, and
once bound to the correct site, acts enzymatically to cut the target
RNA. Strategic cleavage of such a target RNA will destroy its ability
to direct synthesis of an encoded protein. After an enzymatic nucleic
acid has bound and cleaved its RNA target, it is released from that
RNA to search for another target and can repeatedly bind and cleave
The enzymatic nature of a ribozyme is advantageous over many technologies,
such as antisense technology (where a nucleic acid molecule simply
binds to a nucleic acid target to block its translation) since the
concentration of ribozyme necessary to affect a therapeutic treatment
is lower than that of an antisense oligonucleotide. This advantage
reflects the ability of the ribozyme to act enzymatically. Thus,
a single ribozyme molecule is able to cleave many molecules of target
RNA. In addition, the ribozyme is a highly specific inhibitor, with
the specificity of inhibition depending not only on the base pairing
mechanism of binding to the target RNA, but also on the mechanism
of target RNA cleavage. Single mismatches, or base-substitutions,
near the site of cleavage can completely eliminate catalytic activity
of a ribozyme. Similar mismatches in antisense molecules do not
prevent their action (Woolf et al., 1992). Thus, the specificity
of action of a ribozyme is greater than that of an antisense oligonucleotide
binding the same RNA site.
The enzymatic nucleic acid molecule may be formed in a hammerhead,
hairpin, a hepatitis .delta. virus, group I intron or RNaseP RNA
(in association with an RNA guide sequence) or Neurospora VS RNA
motif. Examples of hammerhead motifs are described by Rossi et al
(1992). Examples of hairpin motifs are described by Hampel et al.
(Eur. Pat. Appl. Publ. No. EP 0360257), Hampel and Tritz (1989),
Hampel et al. (1990) and U.S. Pat. No. 5,631,359 (specifically incorporated
herein by reference). An example of the hepatitis .delta. virus
motif is described by Perrotta and Been (1992); an example of the
RNaseP motif is described by Guerrier-Takada et al. (1983); Neurospora
VS RNA ribozyme motif is described by Collins (Saville and Collins,
1990; Saville and Collins, 1991; Collins and Olive, 1993); and an
example of the Group I intron is described in (U.S. Pat. No. 4,987,071,
specifically incorporated herein by reference). All that is important
in an enzymatic nucleic acid molecule of this invention is that
it has a specific substrate binding site which is complementary
to one or more of the target gene RNA regions, and that it have
nucleotide sequences within or surrounding that substrate binding
site which impart an RNA cleaving activity to the molecule. Thus
the ribozyme constructs need not be limited to specific motifs mentioned
In certain embodiments, it may be important to produce enzymatic
cleaving agents which exhibit a high degree of specificity for the
RNA of a desired target, such as one of the sequences disclosed
herein. The enzymatic nucleic acid molecule is preferably targeted
to a highly conserved sequence region of a target mRNA. Such enzymatic
nucleic acid molecules can be delivered exogenously to specific
cells as required. Alternatively, the ribozymes can be expressed
from DNA or RNA vectors that are delivered to specific cells.
Small enzymatic nucleic acid motifs (e.g., of the hammerhead or
the hairpin structure) may also be used for exogenous delivery.
The simple structure of these molecules increases the ability of
the enzymatic nucleic acid to invade targeted regions of the mRNA
structure. Alternatively, catalytic RNA molecules can be expressed
within cells from eukaryotic promoters (e.g., Scanlon et al., 1991;
Kashani-Sabet et al., 1992; Dropulic et al., 1992; Weerasinghe et
al., 1991; Ojwang et al., 1992; Chen et al., 1992; Sarver et al.,
1990). Those skilled in the art realize that any ribozyme can be
expressed in eukaryotic cells from the appropriate DNA vector. The
activity of such ribozymes can be augmented by their release from
the primary transcript by a second ribozyme (Int. Pat. Appl. Publ.
No. WO 93/23569, and Int. Pat. Appl. Publ. No. WO 94/02595, both
hereby incorporated by reference; Ohkawa et al., 1992; Taira et
al., 1991; and Ventura et al., 1993).
Ribozymes may be added directly, or can be complexed with cationic
lipids, lipid complexes, packaged within liposomes, or otherwise
delivered to target cells. The RNA or RNA complexes can be locally
administered to relevant tissues ex vivo, or in vivo through injection,
aerosol inhalation, infusion pump or stent, with or without their
incorporation in biopolymers.
Ribozymes may be designed as described in Int. Pat. Appl. Publ.
No. WO 93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595, each
specifically incorporated herein by reference) and synthesized to
be tested in vitro and in vivo, as described. Such ribozymes can
also be optimized for delivery. While specific examples are provided,
those in the art will recognize that equivalent RNA targets in other
species can be utilized when necessary.
Hammerhead or hairpin ribozymes may be individually analyzed by
computer folding (Jaeger et al., 1989) to assess whether the ribozyme
sequences fold into the appropriate secondary structure. Those ribozyrnes
with unfavorable intramolecular interactions between the binding
arms and the catalytic core are eliminated from consideration. Varying
binding arm lengths can be chosen to optimize activity. Generally,
at least 5 or so bases on each arm are able to bind to, or otherwise
interact with, the target RNA.
Ribozymes of the hammerhead or hairpin motif may be designed to
anneal to various sites in the mRNA message, and can be chemically
synthesized. The method of synthesis used follows the procedure
for normal RNA synthesis as described in Usman etal. (1987) and
in Scaringe etal. (1990) and makes use of common nucleic acid protecting
and coupling groups, such as dimethoxytrityl at the 5'-end, and
phosphoramidites at the 3'-end. Average stepwise coupling yields
are typically >98%. Hairpin ribozymes may be synthesized in two
parts and annealed to reconstruct an active ribozyme (Chowrira and
Burke, 1992). Ribozymes may be modified extensively to enhance stability
by modification with nuclease resistant groups, for example, 2'-amino,
2'-C-allyl, 2'-flouro, 2'-o-methyl, 2'-H (for a review see e.g.,
Usman and Cedergren, 1992). Ribozymes may be purified by gel electrophoresis
using general methods or by high pressure liquid chromatography
and resuspended in water.
Ribozyme activity can be optimized by altering the length of the
ribozyme binding arms, or chemically synthesizing ribozymes with
modifications that prevent their degradation by serum ribonucleases
(see e.g., Int. Pat. Appl. Publ. No. WO 92/07065; Perrault et al.,
1990; Pieken etal., 1991; Usman and Cedergren, 1992; Int. Pat. Appl.
Publ. No. WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur.
Pat. Appl. Publ. No. 92110298.4; U.S. Pat. No. 5,334,711; and Int.
Pat. Appl. Publ. No. WO 94/13688, which describe various chemical
modifications that can be made to the sugar moieties of enzymatic
RNA molecules), modifications which enhance their efficacy in cells,
and removal of stem II bases to shorten RNA synthesis times and
reduce chemical requirements.
Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595) describes
the general methods for delivery of enzymatic RNA molecules. Ribozymes
may be administered to cells by a variety of methods known to those
familiar to the art, including, but not restricted to, encapsulation
in liposomes, by iontophoresis, or by incorporation into other vehicles,
such as hydrogels, cyclodextrins, biodegradable nanocapsules, and
bioadhesive microspheres. For some indications, ribozymes may be
directly delivered ex vivo to cells or tissues with or without the
aforementioned vehicles. Alternatively, the RNA/vehicle combination
may be locally delivered by direct inhalation, by direct injection
or by use of a catheter, infusion pump or stent. Other routes of
delivery include, but are not limited to, intravascular, intramuscular,
subcutaneous or joint injection, aerosol inhalation, oral (tablet
or pill form), topical, systemic, ocular, intraperitoneal and/or
intrathecal delivery. More detailed descriptions of ribozyme delivery
and administration are provided in Int. Pat. Appl. Publ. No. WO
94/02595 and Int. Pat. Appl. Publ. No. WO 93/23569, each specifically
incorporated herein by reference.
Another means of accumulating high concentrations of a ribozyme(s)
within cells is to incorporate the ribozyme-encoding sequences into
a DNA expression vector. Transcription of the ribozyme sequences
are driven from a promoter for eukaryotic RNA polymerase I (pol
I), RNA polymerase II (pol II), or RNA polymerase III (pol III).
Transcripts from pol II or pol III promoters will be expressed at
high levels in all cells; the levels of a given pol II promoter
in a given cell type will depend on the nature of the gene regulatory
sequences (enhancers, silencers, etc.) present nearby. Prokaryotic
RNA polymerase promoters may also be used, providing that the prokaryotic
RNA polymerase enzyme is expressed in the appropriate cells (E1
roy-Stein and Moss, 1990; Gao and Huang, 1993; Lieber et al., 1993;
Zhou et al., 1990). Ribozymes expressed from such promoters can
function in mammalian cells (e.g. Kashani-Saber et al., 1992; Ojwang
et al., 1992; Chen et al., 1992; Yu et al., 1993; L'Huillier et
al., 1992; Lisziewicz et al., 1993). Such transcription units can
be incorporated into a variety of vectors for introduction into
mammalian cells, including but not restricted to, plasmid DNA vectors,
viral DNA vectors (such as adenovirus or adeno-associated vectors),
or viral RNA vectors (such as retroviral, semliki forest virus,
sindbis virus vectors).
Ribozymes may be used as diagnostic tools to examine genetic drift
and mutations within diseased cells. They can also be used to assess
levels of the target RNA molecule. The close relationship between
ribozyme activity and the structure of the target RNA allows the
detection of mutations in any region of the molecule which alters
the base-pairing and three-dimensional structure of the target RNA.
By using multiple ribozymes, one may map nucleotide changes which
are important to RNA structure and function in vitro, as well as
in cells and tissues. Cleavage of target RNAs with ribozymes may
be used to inhibit gene expression and define the role (essentially)
of specified gene products in the progression of disease. In this
manner, other genetic targets may be defined as important mediators
of the disease. These studies will lead to better treatment of the
disease progression by affording the possibility of combinational
therapies (e.g., multiple ribozymes targeted to different genes,
ribozymes coupled with known small molecule inhibitors, or intermittent
treatment with combinations of ribozymes and/or other chemical or
biological molecules). Other in vitro uses of ribozymes are well
known in the art, and include detection of the presence of mRNA
associated with an IL-5 related condition. Such RNA is detected
by determining the presence of a cleavage product after treatment
with a ribozyme using standard methodology.
Peptide Nucleic Acids
In certain embodiments, the inventors contemplate the use of peptide
nucleic acids (PNAs) in the practice of the methods of the invention.
PNA is a DNA mimic in which the nucleobases are attached to a pseudopeptide
backbone (Good and Nielsen, 1997). PNA is able to be utilized in
a number methods that traditionally have used RNA or DNA. Often
PNA sequences perform better in techniques than the corresponding
RNA or DNA sequences and have utilities that are not inherent to
RNA or DNA. A review of PNA including methods of making, characteristics
of, and methods of using, is provided by Corey (1997) and is incorporated
herein by reference. As such, in certain embodiments, one may prepare
PNA sequences that are complementary to one or more portions of
the ACE mRNA sequence, and such PNA compositions may be used to
regulate, alter, decrease, or reduce the translation of ACE-specific
mRNA, and thereby alter the level of ACE activity in a host cell
to which such PNA compositions have been administered.
PNAs have 2-aminoethyl-glycine linkages replacing the normal phosphodiester
backbone of DNA (Nielsen et al., 1991; Hanvey et al., 1992; Hyrup
and Nielsen, 1996; Neilsen, 1996). This chemistry has three important
consequences: firstly, in contrast to DNA or phosphorothioate oligonucleotides,
PNAs are neutral molecules; secondly, PNAs are achiral, which avoids
the need to develop a stereoselective synthesis; and thirdly, PNA
synthesis uses standard Boc (Dueholm et al., 1994) or Fmoc (Thomson
et al., 1995) protocols for solid-phase peptide synthesis, although
other methods, including a modified Merrifield method, have been
used (Christensen et al., 1995).
PNA monomers or ready-made oligomers are commercially available
from PerSeptive Biosystems (Framingham, Mass.). PNA syntheses by
either Boc or Fmoc protocols are straightforward using manual or
automated protocols (Norton et al., 1995). The manual protocol lends
itself to the production of chemically modified PNAs or the simultaneous
synthesis of families of closely related PNAs.
As with peptide synthesis, the success of a particular PNA synthesis
will depend on the properties of the chosen sequence. For example,
while in theory PNAs can incorporate any combination of nucleotide
bases, the presence of adjacent purines can lead to deletions of
one or more residues in the product. In expectation of this difficulty,
it is suggested that, in producing PNAs with adjacent purines, one
should repeat the coupling of residues likely to be added inefficiently.
This should be followed by the purification of PNAs by reverse-phase
high-pressure liquid chromatography (Norton et al., 1995) providing
yields and purity of product similar to those observed during the
synthesis of peptides.
Modifications of PNAs for a given application may be accomplished
by coupling amino acids during solid-phase synthesis or by attaching
compounds that contain a carboxylic acid group to the exposed N-terminal
amine. Alternatively, PNAs can be modified after synthesis by coupling
to an introduced lysine or cysteine. The ease with which PNAs can
be modified facilitates optimization for better solubility or for
specific functional requirements. Once synthesized, the identity
of PNAs and their derivatives can be confirmed by mass spectrometry.
Several studies have made and utilized modifications of PNAs (Norton
et al., 1995; Haaima et al., 1996; Stetsenko et al., 1996; Petersen
et al., 1995; Ulmann et al., 1996; Koch et al., 1995; Orum et al.,
1995; Footer et al., 1996; Griffith et al., 1995; Kremsky et al.,
1996; Pardridge et al., 1995; Boffa et al., 1995; Landsdorp et al.,
1996; Gambacorti-Passerini et al., 1996; Armitage et al., 1997;
Seeger et al., 1997; Ruskowski et al., 1997). U.S. Pat. No. 5,700,922
discusses PNA-DNA-PNA chimeric molecules and their uses in diagnostics,
modulating protein in organisms, and treatment of conditions susceptible
In contrast to DNA and RNA, which contain negatively charged linkages,
the PNA backbone is neutral. In spite of this dramatic alteration,
PNAs recognize complementary DNA and RNA by Watson-Crick pairing
(Egholm et al., 1993), validating the initial modeling by Nielsen
et al. (1991). PNAs lack 3' to 5' polarity and can bind in either
parallel or antiparallel fashion, with the antiparallel mode being
preferred (Egholm et al., 1993).
Hybridization of DNA oligonucleotides to DNA and RNA is destabilized
by electrostatic repulsion between the negatively charged phosphate
backbones of the complementary strands. By contrast, the absence
of charge repulsion in PNA-DNA or PNA-RNA duplexes increases the
melting temperature (T.sub.m) and reduces the dependence of T.sub.m
on the concentration of mono- or divalent cations (Nielsen et al.,
1991). The enhanced rate and affinity of hybridization are significant
because they are responsible for the surprising ability of PNAs
to perform strand invasion of complementary sequences within relaxed
double-stranded DNA. In addition, the efficient hybridization at
inverted repeats suggests that PNAs can recognize secondary structure
effectively within double-stranded DNA. Enhanced recognition also
occurs with PNAs immobilized on surfaces, and Wang et al. have shown
that support-bound PNAs can be used to detect hybridization events
(Wang et al., 1996).
One might expect that tight binding of PNAs to complementary sequences
would also increase binding to similar (but not identical) sequences,
reducing the sequence specificity of PNA recognition. As with DNA
hybridization, however, selective recognition can be achieved by
balancing oligomer length and incubation temperature. Moreover,
selective hybridization of PNAs is encouraged by PNA-DNA hybridization
being less tolerant of base mismatches than DNA--DNA hybridization.
For example, a single mismatch within a 16 bp PNA-DNA duplex can
reduce the T.sub.m by up to 15.degree. C. (Egholm et al., 1993).
This high level of discrimination has allowed the development of
several PNA-based strategies for the analysis of point mutations
(Wang et al., 1996; Carlsson et al., 1996; Thiede et al., 1996;
Webb and Hurskainen, 1996; Perry-O'Keefe et al., 1996).
High-affinity binding provides clear advantages for molecular recognition
and the development of new applications for PNAs. For example, 11-13
nucleotide PNAs inhibit the activity of telomerase, a ribonucleo-protein
that extends telomere ends using an essential RNA template, while
the analogous DNA oligomers do not (Norton et al., 1996).
Neutral PNAs are more hydrophobic than analogous DNA oligomers,
and this can lead to difficulty solubilizing them at neutral pH,
especially if the PNAs have a high purine content or if they have
the potential to form secondary structures. Their solubility can
be enhanced by attaching one or more positive charges to the PNA
termini (Nielsen et al., 1991).
Findings by Allfrey and colleagues suggest that strand invasion
will occur spontaneously at sequences within chromosomal DNA (Boffa
et al., 1995; Boffa et al., 1996). These studies targeted PNAs to
triplet repeats of the nucleotides CAG and used this recognition
to purify transcriptionally active DNA (Boffa et al., 1995) and
to inhibit transcription (Boffa et al., 1996). This result suggests
that if PNAs can be delivered within cells then they will have the
potential to be general sequence-specific regulators of gene expression.
Studies and reviews concerning the use of PNAs as antisense and
anti-gene agents include Nielsen et al. (1993b), Hanvey et al. (1992),
and Good and Nielsen (1997). Koppelhus et al. (1997) have used PNAs
to inhibit HIV-1 inverse transcription, showing that PNAs may be
used for antiviral therapies.
Methods of characterizing the antisense binding properties of PNAs
are discussed in Rose (1993) and Jensen et al. (1997). Rose uses
capillary gel electrophoresis to determine binding of PNAs to their
complementary oligonucleotide, measuring the relative binding kinetics
and stoichiometry. Similar types of measurements were made by Jensen
et al. using BIAcore.TM. technology.
Other applications of PNAs include use in DNA strand invasion (Nielsen
et al., 1991), antisense inhibition (Hanvey et al., 1992), mutational
analysis (Orum et al., 1993), enhancers of transcription (Mollegaard
et al., 1994), nucleic acid purification (Orum et al., 1995), isolation
of transcriptionally active genes (Boffa et al., 1995), blocking
of transcription factor binding (Vickers et al., 1995), genome cleavage
(Veselkov et al., 1996), biosensors (Wang et al., 1996), in situ
hybridization (Thisted et al., 1996), and in a alternative to Southern
blotting (Perry-O'Keefe, 1996).
The present invention, in other aspects, provides polypeptide compositions.
Generally, a polypeptide of the invention will be an isolated polypeptide
(or an epitope, variant, or active fragment thereof) derived from
a mammalian species. Preferably, the polypeptide is encoded by a
polynucleotide sequence disclosed herein or a sequence which hybridizes
under moderately stringent conditions to a polynucleotide sequence
disclosed herein. Alternatively, the polypeptide may be defined
as a polypeptide which comprises a contiguous amino acid sequence
from an amino acid sequence disclosed herein, or which polypeptide
comprises an entire amino acid sequence disclosed herein.
In the present invention, a polypeptide composition is also understood
to comprise one or more polypeptides that are immunologically reactive
with antibodies generated against a polypeptide of the invention,
particularly a polypeptide having the amino acid sequence disclosed
in SEQ ID NO:176, 179, 181, 469-473, 475, 485, 487 and 488, or to
active fragments, or to variants or biological functional equivalents
Likewise, a polypeptide composition of the present invention is
understood to comprise one or more polypeptides that are capable
of eliciting antibodies that are immunologically reactive with one
or more polypeptides encoded by one or more contiguous nucleic acid
sequences contained in SEQ ID NO:1-175, 178, 180, 182-468, 474,
476, 477 479, 484, 486 and 489, or to active fragments, or to variants
thereof, or to one or more nucleic acid sequences which hybridize
to one or more of these sequences under conditions of moderate to
high stringency. Particularly illustrative polypeptides include
the amino acid sequence disclosed in SEQ ID NO:176, 179, 181, 469-473,
475, 485, 487 and 488.
As used herein, an active fragment of a polypeptide includes a
whole or a portion of a polypeptide which is modified by conventional
techniques, e.g., mutagenesis, or by addition, deletion, or substitution,
but which active fragment exhibits substantially the same structure
function, antigenicity, etc., as a polypeptide as described herein.
In certain illustrative embodiments, the polypeptides of the invention
will comprise at least an immunogenic portion of a breast tumor
protein or a variant thereof, as described herein. As noted above,
a "breast tumor protein" is a protein that is expressed
by breast tumor cells. Proteins that are breast tumor proteins also
react detectably within an immunoassay (such as an ELISA) with antisera
from a patient with breast cancer. Polypeptides as described herein
may be of any length. Additional sequences derived from the native
protein and/or heterologous sequences may be present, and such sequences
may (but need not) possess further immunogenic or antigenic properties.
An "immunogenic portion," as used herein is a portion
of a protein that is recognized (i.e., specifically bound) by a
B-cell and/or T-cell surface antigen receptor. Such immunogenic
portions generally comprise at least 5 amino acid residues, more
preferably at least 10, and still more preferably at least 20 amino
acid residues of a breast tumor protein or a variant thereof. Certain
preferred immunogenic portions include peptides in which an N-terminal
leader sequence and/or transmembrane domain have been deleted. Other
preferred immunogenic portions may contain a small N- and/or C-terminal
deletion (e.g., 1-30 amino acids, preferably 5-15 amino acids),
relative to the mature protein.
Immunogenic portions may generally be identified using well known
techniques, such as those summarized in Paul, Fundamental Immunology,
3rd ed., 243-247 (Raven Press, 1993) and references cited therein.
Such techniques include screening polypeptides for the ability to
react with antigen-specific antibodies, antisera and/or T-cell lines
or clones. As used herein, antisera and antibodies are "antigen-specific"
if they specifically bind to an antigen (i.e., they react with the
protein in an ELISA or other immunoassay, and do not react detectably
with unrelated proteins). Such antisera and antibodies may be prepared
as described herein, and using well known techniques. An immunogenic
portion of a native breast tumor protein is a portion that reacts
with such antisera and/or T-cells at a level that is not substantially
less than the reactivity of the full length polypeptide (e.g., in
an ELISA and/or T-cell reactivity assay). Such immunogenic portions
may react within such assays at a level that is similar to or greater
than the reactivity of the full length polypeptide. Such screens
may generally be performed using methods well known to those of
ordinary skill in the art, such as those described in Harlow and
Lane, Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory,
1988. For example, a polypeptide may be immobilized on a solid support
and contacted with patient sera to allow binding of antibodies within
the sera to the immobilized polypeptide. Unbound sera may then be
removed and bound antibodies detected using, for example, .sup.125
I-labeled Protein A.
As noted above, a composition may comprise a variant of a native
breast tumor protein. A polypeptide "variant," as used
herein, is a polypeptide that differs from a native breast tumor
protein in one or more substitutions, deletions, additions and/or
insertions, such that the immunogenicity of the polypeptide is not
substantially diminished. In other words, the ability of a variant
to react with antigen-specific antisera may be enhanced or unchanged,
relative to the native protein, or may be diminished by less than
50%, and preferably less than 20%, relative to the native protein.
Such variants may generally be identified by modifying one of the
above polypeptide sequences and evaluating the reactivity of the
modified polypeptide with antigen-specific antibodies or antisera
as described herein. Preferred variants include those in which one
or more portions, such as an N-terminal leader sequence or transmembrane
domain, have been removed. Other preferred variants include variants
in which a small portion (e.g., 1-30 amino acids, preferably 5-15
amino acids) has been removed from the N- and/or C-terminal of the
Polypeptide variants encompassed by the present invention include
those exhibiting at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96% 97%, 98%, or 99% or more identity (determined
as described above) to the polypeptides disclosed herein.
Preferably, a variant contains conservative substitutions. A "conservative
substitution" is one in which an amino acid is substituted
for another amino acid that has similar properties, such that one
skilled in the art of peptide chemistry would expect the secondary
structure and hydropathic nature of the polypeptide to be substantially
unchanged. Amino acid substitutions may generally be made on the
basis of similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity and/or the amphipathic nature of the residues. For
example, negatively charged amino acids include aspartic acid and
glutamic acid; positively charged amino acids include lysine and
arginine; and amino acids with uncharged polar head groups having
similar hydrophilicity values include leucine, isoleucine and valine;
glycine and alanine; asparagine and glutamine; and serine, threonine,
phenylalanine and tyrosine. Other groups of amino acids that may
represent conservative changes include: (1) ala, pro, gly, glu,
asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu,
met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A
variant may also, or alternatively, contain nonconservative changes.
In a preferred embodiment, variant polypeptides differ from a native
sequence by substitution, deletion or addition of five amino acids
or fewer. Variants may also (or alternatively) be modified by, for
example, the deletion or addition of amino acids that have minimal
influence on the immunogenicity, secondary structure and hydropathic
nature of the polypeptide.
As noted above, polypeptides may comprise a signal (or leader)
sequence at the N-terminal end of the protein, which co-translationally
or post-translationally directs transfer of the protein. The polypeptide
may also be conjugated to a linker or other sequence for ease of
synthesis, purification or identification of the polypeptide (e.g.,
poly-His), or to enhance binding of the polypeptide to a solid support.
For example, a polypeptide may be conjugated to an immunoglobulin
Polypeptides may be prepared using any of a variety of well known
techniques. Recombinant polypeptides encoded by DNA sequences as
described above may be readily prepared from the DNA sequences using
any of a variety of expression vectors known to those of ordinary
skill in the art. Expression may be achieved in any appropriate
host cell that has been transformed or transfected with an expression
vector containing a DNA molecule that encodes a recombinant polypeptide.
Suitable host cells include prokaryotes, yeast, and higher eukaryotic
cells, such as mammalian cells and plant cells. Preferably, the
host cells employed are E. coli, yeast or a mammalian cell line
such as COS or CHO. Supernatants from suitable host/vector systems
which secrete recombinant protein or polypeptide into culture media
may be first concentrated using a commercially available filter.
Following concentration, the concentrate may be applied to a suitable
purification matrix such as an affinity matrix or an ion exchange
resin. Finally, one or more reverse phase HPLC steps can be employed
to further purify a recombinant polypeptide.
Portions and other variants having less than about 100 amino acids,
and generally less than about 50 amino acids, may also be generated
by synthetic means, using techniques well known to those of ordinary
skill in the art. For example, such polypeptides may be synthesized
using any of the commercially available solid-phase techniques,
such as the Merrifield solid-phase synthesis method, where amino
acids are sequentially added to a growing amino acid chain. See
Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963. Equipment for
automated synthesis of polypeptides is commercially available from
suppliers such as Perkin E1 mer/Applied BioSystems Division (Foster
City, Calif.), and may be operated according to the manufacturer's
Within certain specific embodiments, a polypeptide may be a fusion
protein that comprises multiple polypeptides as described herein,
or that comprises at least one polypeptide as described herein and
an unrelated sequence, such as a known tumor protein. A fusion partner
may, for example, assist in providing T helper epitopes (an immunological
fusion partner), preferably T helper epitopes recognized by humans,
or may assist in expressing the protein (an expression enhancer)
at higher yields than the native recombinant protein. Certain preferred
fusion partners are both immunological and expression enhancing
fusion partners. Other fusion partners may be selected so as to
increase the solubility of the protein or to enable the protein
to be targeted to desired intracellular compartments. Still further
fusion partners include affinity tags, which facilitate purification
of the protein.
Fusion proteins may generally be prepared using standard techniques,
including chemical conjugation. Preferably, a fusion protein is
expressed as a recombinant protein, allowing the production of increased
levels, relative to a non-fused protein, in an expression system.
Briefly, DNA sequences encoding the polypeptide components may be
assembled separately, and ligated into an appropriate expression
vector. The 3' end of the DNA sequence encoding one polypeptide
component is ligated, with or without a peptide linker, to the 5'
end of a DNA sequence encoding the second polypeptide component
so that the reading frames of the sequences are in phase. This permits
translation into a single fusion protein that retains the biological
activity of both component polypeptides.
A peptide linker sequence may be employed to separate the first
and second polypeptide components by a distance sufficient to ensure
that each polypeptide folds into its secondary and tertiary structures.
Such a peptide linker sequence is incorporated into the fusion protein
using standard techniques well known in the art. Suitable peptide
linker sequences may be chosen based on the following factors: (1)
their ability to adopt a flexible extended conformation; (2) their
inability to adopt a secondary structure that could interact with
functional epitopes on the first and second polypeptides; and (3)
the lack of hydrophobic or charged residues that might react with
the polypeptide functional epitopes. Preferred peptide linker sequences
contain Gly, Asn and Ser residues. Other near neutral amino acids,
such as Thr and Ala may also be used in the linker sequence. Amino
acid sequences which may be usefully employed as linkers include
those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et
al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Pat. Nos.
4,935,233 and 4,751,180. The linker sequence may generally be from
1 to about 50 amino acids in length. Linker sequences are not required
when the first and second polypeptides have non-essential N-terminal
amino acid regions that can be used to separate the functional domains
and prevent steric interference.
The ligated DNA sequences are operably linked to suitable transcriptional
or translational regulatory elements. The regulatory elements responsible
for expression of DNA are located only 5' to the DNA sequence encoding
the first polypeptides. Similarly, stop codons required to end translation
and transcription termination signals are only present 3' to the
DNA sequence encoding the second polypeptide.
Fusion proteins are also provided. Such proteins comprise a polypeptide
as described herein together with an unrelated immunogenic protein.
Preferably the immunogenic protein is capable of eliciting a recall
response. Examples of such proteins include tetanus, tuberculosis
and hepatitis proteins (see, for example, Stoute et al. New Engl.
J. Med., 336:86-91, 1997).
Within preferred embodiments, an immunological fusion partner is
derived from protein D, a surface protein of the gram-negative bacterium
Haemophilus influenza B (WO 91/18926). Preferably, a protein D derivative
comprises approximately the first third of the protein (e.g., the
first N-terminal 100-110 amino acids), and a protein D derivative
may be lipidated. Within certain preferred embodiments, the first
109 residues of a Lipoprotein D fusion partner is included on the
N-terminus to provide the polypeptide with additional exogenous
T-cell epitopes and to increase the expression level in E. coli
(thus functioning as an expression enhancer). The lipid tail ensures
optimal presentation of the antigen to antigen presenting cells.
Other fusion partners include the non-structural protein from influenzae
virus, NS1 (hemaglutinin). Typically, the N-terminal 81 amino acids
are used, although different fragments that include T-helper epitopes
may be used.
In another embodiment, the immunological fusion partner is the
protein known as LYTA, or a portion thereof (preferably a C-terminal
portion). LYTA is derived from Streptococcus pneumoniae, which synthesizes
an N-acetyl-L-alanine amidase known as amidase LYTA (encoded by
the LytA gene; Gene 43:265-292, 1986). LYTA is an autolysin that
specifically degrades certain bonds in the peptidoglycan backbone.
The C-terminal domain of the LYTA protein is responsible for the
affinity to the choline or to some choline analogues such as DEAE.
This property has been exploited for the development of E. coli
C-LYTA expressing plasmids useful for expression of fusion proteins.
Purification of hybrid proteins containing the C-LYTA fragment at
the amino terminus has been described (see Biotechnology 10:795-798,
1992). Within a preferred embodiment, a repeat portion of LYTA may
be incorporated into a fusion protein. A repeat portion is found
in the C-terminal region starting at residue 178. A particularly
preferred repeat portion incorporates residues 188-305.
In general, polypeptides (including fusion proteins) and polynucleotides
as described herein are isolated. An "isolated" polypeptide
or polynucleotide is one that is removed from its original environment.
For example, a naturally-occurring protein is isolated if it is
separated from some or all of the coexisting materials in the natural
system. Preferably, such polypeptides are at least about 90% pure,
more preferably at least about 95% pure and most preferably at least
about 99% pure. A polynucleotide is considered to be isolated if,
for example, it is cloned into a vector that is not a part of the
The present invention further provides agents, such as antibodies
and antigen-binding fragments thereof, that specifically bind to
a breast tumor protein. As used herein, an antibody, or antigen-binding
fragment thereof, is said to "specifically bind" to a
breast tumor protein if it reacts at a detectable level (within,
for example, an ELISA) with a breast tumor protein, and does not
react detectably with unrelated proteins under similar conditions.
As used herein, "binding" refers to a noncovalent association
between two separate molecules such that a complex is formed. The
ability to bind may be evaluated by, for example, determining a
binding constant for the formation of the complex. The binding constant
is the value obtained when the concentration of the complex is divided
by the product of the component concentrations. In general, two
compounds are said to "bind," in the context of the present
invention, when the binding constant for complex formation exceeds
about 10.sup.3 L/mol. The binding constant may be determined using
methods well known in the art.
Binding agents may be further capable of differentiating between
patients with and without a cancer, such as breast cancer, using
the representative assays provided herein. In other words, antibodies
or other binding agents that bind to a breast tumor protein will
generate a signal indicating the presence of a cancer in at least
about 20% of patients with the disease, and will generate a negative
signal indicating the absence of the disease in at least about 90%
of individuals without the cancer. To determine whether a binding
agent satisfies this requirement, biological samples (e.g., blood,
sera, sputum, urine and/or tumor biopsies) from patients with and
without a cancer (as determined using standard clinical tests) may
be assayed as described herein for the presence of polypeptides
that bind to the binding agent. It will be apparent that a statistically
significant number of samples with and without the disease should
be assayed. Each binding agent should satisfy the above criteria;
however, those of ordinary skill in the art will recognize that
binding agents may be used in combination to improve sensitivity.
Any agent that satisfies the above requirements may be a binding
agent. For example, a binding agent may be a ribosome, with or without
a peptide component, an RNA molecule or a polypeptide. In a preferred
embodiment, a binding agent is an antibody or an antigen-binding
fragment thereof. Antibodies may be prepared by any of a variety
of techniques known to those of ordinary skill in the art. See,
e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory, 1988. In general, antibodies can be produced
by cell culture techniques, including the generation of monoclonal
antibodies as described herein, or via transfection of antibody
genes into suitable bacterial or mammalian cell hosts, in order
to allow for the production of recombinant antibodies. In one technique,
an immunogen comprising the polypeptide is initially injected into
any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep
or goats). In this step, the polypeptides of this invention may
serve as the immunogen without modification. Alternatively, particularly
for relatively short polypeptides, a superior immune response may
be elicited if the polypeptide is joined to a carrier protein, such
as bovine serum albumin or keyhole limpet hemocyanin. The immunogen
is injected into the animal host, preferably according to a predetermined
schedule incorporating one or more booster immunizations, and the
animals are bled periodically. Polyclonal antibodies specific for
the polypeptide may then be purified from such antisera by, for
example, affinity chromatography using the polypeptide coupled to
a suitable solid support.
Monoclonal antibodies specific for an antigenic polypeptide of
interest may be prepared, for example, using the technique of Kohler
and Milstein, Eur. J. Immunol. 6:511-519, 1976, and improvements
thereto. Briefly, these methods involve the preparation of immortal
cell lines capable of producing antibodies having the desired specificity
(i.e., reactivity with the polypeptide of interest). Such cell lines
may be produced, for example, from spleen cells obtained from an
animal immunized as described above. The spleen cells are then immortalized
by, for example, fusion with a myeloma cell fusion partner, preferably
one that is syngeneic with the immunized animal. A variety of fusion
techniques may be employed. For example, the spleen cells and myeloma
cells may be combined with a nonionic detergent for a few minutes
and then plated at low density on a selective medium that supports
the growth of hybrid cells, but not myeloma cells. A preferred selection
technique uses HAT (hypoxanthine, aminopterin, thymidine) selection.
After a sufficient time, usually about 1 to 2 weeks, colonies of
hybrids are observed. Single colonies are selected and their culture
supernatants tested for binding activity against the polypeptide.
Hybridomas having high reactivity and specificity are preferred.
Monoclonal antibodies may be isolated from the supernatants of
growing hybridoma colonies. In addition, various techniques may
be employed to enhance the yield, such as injection of the hybridoma
cell line into the peritoneal cavity of a suitable vertebrate host,
such as a mouse. Monoclonal antibodies may then be harvested from
the ascites fluid or the blood. Contaminants may be removed from
the antibodies by conventional techniques, such as chromatography,
gel filtration, precipitation, and extraction. The polypeptides
of this invention may be used in the purification process in, for
example, an affinity chromatography step.
Within certain embodiments, the use of antigen-binding fragments
of antibodies may be preferred. Such fragments include Fab fragments,
which may be prepared using standard techniques. Briefly, immunoglobulins
may be purified from rabbit serum by affinity chromatography on
Protein A bead columns (Harlow and Lane, Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory, 1988) and digested by papain
to yield Fab and Fc fragments. The Fab and Fc fragments may be separated
by affinity chromatography on protein A bead columns.
Monoclonal antibodies of the present invention may be coupled to
one or more therapeutic agents. Suitable agents in this regard include
radionuclides, differentiation inducers, drugs, toxins, and derivatives
thereof. Preferred radionuclides include .sup.90 Y, .sup.123 I,
.sup.125 I, .sup.131 I, .sup.186 Re, .sup.188 Re, .sup.211 At, and
.sup.212 Bi. Preferred drugs include methotrexate, and pyrimidine
and purine analogs. Preferred differentiation inducers include phorbol
esters and butyric acid. Preferred toxins include ricin, abrin,
diptheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella
toxin, and pokeweed antiviral protein.
A therapeutic agent may be coupled (e.g., covalently bonded) to
a suitable monoclonal antibody either directly or indirectly (e.g.,
via a linker group). A direct reaction between an agent and an antibody
is possible when each possesses a substituent capable of reacting
with the other. For example, a nucleophilic group, such as an amino
or sulfhydryl group, on one may be capable of reacting with a carbonyl-containing
group, such as an anhydride or an acid halide, or with an alkyl
group containing a good leaving group (e.g., a halide) on the other.
Alternatively, it may be desirable to couple a therapeutic agent
and an antibody via a linker group. A linker group can function
as a spacer to distance an antibody from an agent in order to avoid
interference with binding capabilities. A linker group can also
serve to increase the chemical reactivity of a substituent on an
agent or an antibody, and thus increase the coupling efficiency.
An increase in chemical reactivity may also facilitate the use of
agents, or functional groups on agents, which otherwise would not
It will be evident to those skilled in the art that a variety of
bifunctional or polyfunctional reagents, both homo- and hetero-functional
(such as those described in the catalog of the Pierce Chemical Co.,
Rockford, Ill.), may be employed as the linker group. Coupling may
be effected, for example, through amino groups, carboxyl groups,
sulfhydryl groups or oxidized carbohydrate residues. There are numerous
references describing such methodology, e.g., U.S. Pat. No. 4,671,958,
to Rodwell et al.
Where a therapeutic agent is more potent when free from the antibody
portion of the immunoconjugates of the present invention, it may
be desirable to use a linker group which is cleavable during or
upon internalization into a cell. A number of different cleavable
linker groups have been described. The mechanisms for the intracellular
release of an agent from these linker groups include cleavage by
reduction of a disulfide bond (e.g., U.S. Pat. No. 4,489,710, to
Spitler), by irradiation of a photolabile bond (e.g., U.S. Pat.
No. 4,625,014, to Senter et al.), by hydrolysis of derivatized amino
acid side chains (e.g., U.S. Pat. No. 4,638,045, to Kohn et al.),
by serum complement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958,
to Rodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Pat.
No. 4,569,789, to Blattler et al.).
It may be desirable to couple more than one agent to an antibody.
In one embodiment, multiple molecules of an agent are coupled to
one antibody molecule. In another embodiment, more than one type
of agent may be coupled to one antibody. Regardless of the particular
embodiment, immunoconjugates with more than one agent may be prepared
in a variety of ways. For example, more than one agent may be coupled
directly to an antibody molecule, or linkers that provide multiple
sites for attachment can be used. Alternatively, a carrier can be
A carrier may bear the agents in a variety of ways, including covalent
bonding either directly or via a linker group. Suitable carriers
include proteins such as albumins (e.g., U.S. Pat. No. 4,507,234,
to Kato et al.), peptides and polysaccharides such as aminodextran
(e.g., U.S. Pat. No. 4,699,784, to Shih et al.). A carrier may also
bear an agent by noncovalent bonding or by encapsulation, such as
within a liposome vesicle (e.g., U.S. Pat. Nos. 4,429,008 and 4,873,088).
Carriers specific for radionuclide agents include radiohalogenated
small molecules and chelating compounds. For example, U.S. Pat.
No. 4,735,792 discloses representative radiohalogenated small molecules
and their synthesis. A radionuclide chelate may be formed from chelating
compounds that include those containing nitrogen and sulfur atoms
as the donor atoms for binding the metal, or metal oxide, radionuclide.
For example, U.S. Pat. No. 4,673,562, to Davison et al. discloses
representative chelating compounds and their synthesis.
A variety of routes of administration for the antibodies and immunoconjugates
may be used. Typically, administration will be intravenous, intramuscular,
subcutaneous or in the bed of a resected tumor. It will be evident
that the precise dose of the antibody/immunoconjugate will vary
depending upon the antibody used, the antigen density on the tumor,
and the rate of clearance of the antibody.
Immunotherapeutic compositions may also, or alternatively, comprise
T cells specific for a breast tumor protein. Such cells may generally
be prepared in vitro or ex vivo, using standard procedures. For
example, T cells may be isolated from bone marrow, peripheral blood,
or a fraction of bone marrow or peripheral blood of a patient, using
a commercially available cell separation system, such as the Isolex.TM.
System, available from Nexell Therapeutics, Inc. (Irvine, Calif.;
see also U.S. Pat. No. 5,240,856; U.S. Pat. No. 5,215,926; WO 89/06280;
WO 91/16116 and WO 92/07243). Alternatively, T cells may be derived
from related or unrelated humans, non-human mammals, cell lines
T cells may be stimulated with a breast tumor polypeptide, polynucleotide
encoding a breast tumor polypeptide and/or an antigen presenting
cell (APC) that expresses such a polypeptide. Such stimulation is
performed under conditions and for a time sufficient to permit the
generation of T cells that are specific for the polypeptide. Preferably,
a breast tumor polypeptide or polynucleotide is present within a
delivery vehicle, such as a microsphere, to facilitate the generation
of specific T cells.
T cells are considered to be specific for a breast tumor polypeptide
if the T cells specifically proliferate, secrete cytokines or kill
target cells coated with the polypeptide or expressing a gene encoding
the polypeptide. T cell specificity may be evaluated using any of
a variety of standard techniques. For example, within a chromium
release assay or proliferation assay, a stimulation index of more
than two fold increase in lysis and/or proliferation, compared to
negative controls, indicates T cell specificity. Such assays may
be performed, for example, as described in Chen et al., Cancer Res.
54:1065 -1070, 1994. Alternatively, detection of the proliferation
of T cells may be accomplished by a variety of known techniques.
For example, T cell proliferation can be detected by measuring an
increased rate of DNA synthesis (e.g., by pulse-labeling cultures
of T cells with tritiated thymidine and measuring the amount of
tritiated thymidine incorporated into DNA). Contact with a breast
tumor polypeptide (100 ng/ml -100 .mu.g/ml, preferably 200 ng/ml
25 .mu.g/ml) for 3-7 days should result in at least a two fold increase
in proliferation of the T cells. Contact as described above for
2-3 hours should result in activation of the T cells, as measured
using standard cytokine assays in which a two fold increase in the
level of cytokine release (e.g., TNF or IFN-.gamma.) is indicative
of T cell activation (see Coligan et al., Current Protocols in Immunology,
vol. 1, Wiley Interscience (Greene 1998)). T cells that have been
activated in response to a breast tumor polypeptide, polynucleotide
or polypeptide-expressing APC may be CD4.sup.+ and/or CD8.sup.+.
Breast tumor protein-specific T cells may be expanded using standard
techniques. Within preferred embodiments, the T cells are derived
from a patient, a related donor or an unrelated donor, and are administered
to the patient following stimulation and expansion.
For therapeutic purposes, CD4.sup.+ or CD8.sup.+ T cells that proliferate
in response to a breast tumor polypeptide, polynucleotide or APC
can be expanded in number either in vitro or in vivo. Proliferation
of such T cells in vitro may be accomplished in a variety of ways.
For example, the T cells can be re-exposed to a breast tumor polypeptide,
or a short peptide corresponding to an immunogenic portion of such
a polypeptide, with or without the addition of T cell growth factors,
such as interleukin-2, and/or stimulator cells that synthesize a
breast tumor polypeptide. Alternatively, one or more T cells that
proliferate in the presence of a breast tumor protein can be expanded
in number by cloning. Methods for cloning cells are well known in
the art, and include limiting dilution.
In additional embodiments, the present invention concerns formulation
of one or more of the polynucleotide, polypeptide, T-cell and/or
antibody compositions disclosed herein in pharmaceutically-acceptable
solutions for administration to a cell or an animal, either alone,
or in combination with one or more other modalities of therapy.
It will also be understood that, if desired, the nucleic acid segment,
RNA, DNA or PNA compositions that express a polypeptide as disclosed
herein may be administered in combination with other agents as well,
such as, e.g., other proteins or polypeptides or various pharmaceutically-active
agents. In fact, there is virtually no limit to other components
that may also be included, given that the additional agents do not
cause a significant adverse effect upon contact with the target
cells or host tissues. The compositions may thus be delivered along
with various other agents as required in the particular instance.
Such compositions may be purified from host cells or other biological
sources, or alternatively may be chemically synthesized as described
herein. Likewise, such compositions may further comprise substituted
or derivatized RNA or DNA compositions.
Formulation of pharmaceutically-acceptable excipients and carrier
solutions is well-known to those of skill in the art, as is the
development of suitable dosing and treatment regimens for using
the particular compositions described herein in a variety of treatment
regimens, including e.g., oral, parenteral, intravenous, intranasal,
and intramuscular administration and formulation.
1. Oral Delivery
In certain applications, the pharmaceutical compositions disclosed
herein may be delivered via oral administration to an animal. As
such, these compositions may be formulated with an inert diluent
or with an assimilable edible carrier, or they may be enclosed in
hard- or soft-shell gelatin capsule, or they may be compressed into
tablets, or they may be incorporated directly with the food of the
The active compounds may even be incorporated with excipients and
used in the form of ingestible tablets, buccal tables, troches,
capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz
et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515; 5,580,579
and 5,792,451, each specifically incorporated herein by reference
in its entirety). The tablets, troches, pills, capsules and the
like may also contain the following: a binder, as gum tragacanth,
acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate;
a disintegrating agent, such as corn starch, potato starch, alginic
acid and the like; a lubricant, such as magnesium stearate; and
a sweetening agent, such as sucrose, lactose or saccharin may be
added or a flavoring agent, such as peppermint, oil of wintergreen,
or cherry flavoring. When the dosage unit form is a capsule, it
may contain, in addition to materials of the above type, a liquid
carrier. Various other materials may be present as coatings or to
otherwise modify the physical form of the dosage unit. For instance,
tablets, pills, or capsules may be coated with shellac, sugar, or
both. A syrup of elixir may contain the active compound sucrose
as a sweetening agent methyl and propylparabens as preservatives,
a dye and flavoring, such as cherry or orange flavor. Of course,
any material used in preparing any dosage unit form should be pharmaceutically
pure and substantially non-toxic in the amounts employed. In addition,
the active compounds may be incorporated into sustained-release
preparation and formulations.
Typically, these formulations may contain at least about 0.1% of
the active compound or more, although the percentage of the active
ingredient(s) may, of course, be varied and may conveniently be
between about 1 or 2% and about 60% or 70% or more of the weight
or volume of the total formulation. Naturally, the amount of active
compound(s) in each therapeutically useful composition may be prepared
is such a way that a suitable dosage will be obtained in any given
unit dose of the compound. Factors such as solubility, bioavailability,
biological half-life, route of administration, product shelf life,
as well as other pharmacological considerations will be contemplated
by one skilled in the art of preparing such pharmaceutical formulations,
and as such, a variety of dosages and treatment regimens may be
For oral administration the compositions of the present invention
may alternatively be incorporated with one or more excipients in
the form of a mouthwash, dentifrice, buccal tablet, oral spray,
or sublingual orally-administered formulation. For example, a mouthwash
may be prepared incorporating the active ingredient in the required
amount in an appropriate solvent, such as a sodium borate solution
(Dobell's Solution). Alternatively, the active ingredient may be
incorporated into an oral solution such as one containing sodium
borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice,
or added in a therapeutically-effective amount to a composition
that may include water, binders, abrasives, flavoring agents, foaming
agents, and humectants. Alternatively the compositions may be fashioned
into a tablet or solution form that may be placed under the tongue
or otherwise dissolved in the mouth.
2. Injectable Delivery
In certain circumstances it will be desirable to deliver the pharmaceutical
compositions disclosed herein parenterally, intravenously, intramuscularly,
or even intraperitoneally as described in U.S. Pat. Nos. 5,543,158;
5,641,515 and 5,399,363 (each specifically incorporated herein by
reference in its entirety). Solutions of the active compounds as
free base or pharmacologically acceptable salts may be prepared
in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions may also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary conditions
of storage and use, these preparations contain a preservative to
prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile
aqueous solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersions (U.S.
Pat. No. 5,466,468, specifically incorporated herein by reference
in its entirety). In all cases the form must be sterile and must
be fluid to the extent that easy syringability exists. It must be
stable under the conditions of manufacture and storage and must
be preserved against the contaminating action of microorganisms,
such as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (e.g., glycerol,
propylene glycol, and liquid polyethylene glycol, and the like),
suitable mixtures thereof, and/or vegetable oils. Proper fluidity
may be maintained, for example, by the use of a coating, such as
lecithin, by the maintenance of the required particle size in the
case of dispersion and by the use of surfactants. The prevention
of the action of microorganisms can be facilitated by various antibacterial
and antifungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars or sodium
chloride. Prolonged absorption of the injectable compositions can
be brought about by the use in the compositions of agents delaying
absorption, for example, aluminum monostearate and gelatin.
For parenteral administration in an aqueous solution, for example,
the solution should be suitably buffered if necessary and the liquid
diluent first rendered isotonic with sufficient saline or glucose.
These particular aqueous solutions are especially suitable for intravenous,
intramuscular, subcutaneous and intraperitoneal administration.
In this connection, a sterile aqueous medium that can be employed
will be known to those of skill in the art in light of the present
disclosure. For example, one dosage may be dissolved in 1 ml of
isotonic NaCl solution and either added to 1000 ml of hypodermoclysis
fluid or injected at the proposed site of infusion, (see for example,
"Remington's Pharmaceutical Sciences" 15th Edition, pages
1035-1038 and 1570-1580). Some variation in dosage will necessarily
occur depending on the condition of the subject being treated. The
person responsible for administration will, in any event, determine
the appropriate dose for the individual subject. Moreover, for human
administration, preparations should meet sterility, pyrogenicity,
and the general safety and purity standards as required by FDA Office
of Biologics standards.
Sterile injectable solutions are prepared by incorporating the
active compounds in the required amount in the appropriate solvent
with various of the other ingredients enumerated above, as required,
followed by filtered sterilization. Generally, dispersions are prepared
by incorporating the various sterilized active ingredients into
a sterile vehicle which contains the basic dispersion medium and
the required other ingredients from those enumerated above. In the
case of sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum-drying
and freeze-drying techniques which yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
The compositions disclosed herein may be formulated in a neutral
or salt form. Pharmaceutically-acceptable salts, include the acid
addition salts (formed with the free amino groups of the protein)
and which are formed with inorganic acids such as, for example,
hydrochloric or phosphoric acids, or such organic acids as acetic,
oxalic, tartaric, mandelic, and the like. Salts formed with the
free carboxyl groups can also be derived from inorganic bases such
as, for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine, trimethylamine,
histidine, procaine and the like. Upon formulation, solutions will
be administered in a manner compatible with the dosage formulation
and in such amount as is therapeutically effective. The formulations
are easily administered in a variety of dosage forms such as injectable
solutions, drug-release capsules, and the like.
As used herein, "carrier" includes any and all solvents,
dispersion media, vehicles, coatings, diluents, antibacterial and
antifungal agents, isotonic and absorption delaying agents, buffers,
carrier solutions, suspensions, colloids, and the like. The use
of such media and agents for pharmaceutical active substances is
well known in the art. Except insofar as any conventional media
or agent is incompatible with the active ingredient, its use in
the therapeutic compositions is contemplated. Supplementary active
ingredients can also be incorporated into the compositions.
The phrase "pharmaceutically-acceptable" refers to molecular
entities and compositions that do not produce an allergic or similar
untoward reaction when administered to a human. The preparation
of an aqueous composition that contains a protein as an active ingredient
is well understood in the art. Typically, such compositions are
prepared as injectables, either as liquid solutions or suspensions;
solid forms suitable for solution in, or suspension in, liquid prior
to injection can also be prepared. The preparation can also be emulsified.
3. Nasal Delivery
In certain embodiments, the pharmaceutical compositions may be
delivered by intranasal sprays, inhalation, and/or other aerosol
delivery vehicles. Methods for delivering genes, nucleic acids,
and peptide compositions directly to the lungs via nasal aerosol
sprays has been described e.g., in U.S. Pat. Nos. 5,756,353 and
5,804,212 (each specifically incorporated herein by reference in
its entirety). Likewise, the delivery of drugs using intranasal
microparticle resins (Takenaga et al., 1998) and lysophosphatidyl-glycerol
compounds (U.S. Pat. No. 5,725,871, specifically incorporated herein
by reference in its entirety) are also well-known in the pharmaceutical
arts. Likewise, transmucosal drug delivery in the form of a polytetrafluoroetheylene
support matrix is described in U.S. Pat. No. 5,780,045 (specifically
incorporated herein by reference in its entirety).
4. Liposome-, Nanocapsule-, and Microparticle-Mediated Delivery
In certain embodiments, the inventors contemplate the use of liposomes,
nanocapsules, microparticles, microspheres, lipid particles, vesicles,
and the like, for the introduction of the compositions of the present
invention into suitable host cells. In particular, the compositions
of the present invention may be formulated for delivery either encapsulated
in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle
or the like.
Such formulations may be preferred for the introduction of pharmaceutically-acceptable
formulations of the nucleic acids or constructs disclosed herein.
The formation and use of liposomes is generally known to those of
skill in the art (see for example, Couvreur et al., 1977; Couvreur,
1988; Lasic, 1998; which describes the use of liposomes and nanocapsules
in the targeted antibiotic therapy for intracellular bacterial infections
and diseases). Recently, liposomes were developed with improved
serum stability and circulation half-times (Gabizon and Papahadjopoulos,
1988; Allen and Choun, 1987; U.S. Pat. No. 5,741,516, specifically
incorporated herein by reference in its entirety). Further, various
methods of liposome and liposome like preparations as potential
drug carriers have been reviewed (Takakura, 1998; Chandran et al.,
1997; Margalit, 1995; U.S. Pat. Nos. 5,567,434; 5,552,157; 5,565,213;
5,738,868 and 5,795,587, each specifically incorporated herein by
reference in its entirety).
Liposomes have been used successfully with a number of cell types
that are normally resistant to transfection by other procedures
including T cell suspensions, primary hepatocyte cultures and PC
12 cells (Renneisen et al., 1990; Muller et al., 1990). In addition,
liposomes are free of the DNA length constraints that are typical
of viral-based delivery systems. Liposomes have been used effectively
to introduce genes, drugs (Heath and Martin, 1986; Heath et al.,
1986; Balazsovits et al., 1989; Fresta and Puglisi, 1996), radiotherapeutic
agents (Pikul et al., 1987), enzymes (Imaizumi et al., 1990a; Imaizumi
et al., 1990b), viruses (Faller and Baltimore, 1984), transcription
factors and allosteric effectors (Nicolau and Gersonde, 1979) into
a variety of cultured cell lines and animals. In addition, several
successful clinical trails examining the effectiveness of liposome-mediated
drug delivery have been completed (Lopez-Berestein et al., 1985a;
1985b; Coune, 1988; Sculier et al., 1988). Furthermore, several
studies suggest that the use of liposomes is not associated with
autoimmune responses, toxicity or gonadal localization after systemic
delivery (Mori and Fukatsu, 1992).
Liposomes are formed from phospholipids that are dispersed in an
aqueous medium and spontaneously form multilamellar concentric bilayer
vesicles (also termed multilamellar vesicles (MLVs). MLVs generally
have diameters of from 25 nm to 4 .mu.m. Sonication of MLVs results
in the formation of small unilamellar vesicles (SUVs) with diameters
in the range of 200 to 500 .ANG., containing an aqueous solution
in the core.
Liposomes bear resemblance to cellular membranes and are contemplated
for use in connection with the present invention as carriers for
the peptide compositions. They are widely suitable as both water-
and lipid-soluble substances can be entrapped, i.e. in the aqueous
spaces and within the bilayer itself, respectively. It is possible
that the drug-bearing liposomes may even be employed for site-specific
delivery of active agents by selectively modifying the liposomal
In addition to the teachings of Couvreur et al. (1977; 1988), the
following information may be utilized in generating liposomal formulations.
Phospholipids can form a variety of structures other than liposomes
when dispersed in water, depending on the molar ratio of lipid to
water. At low ratios the liposome is the preferred structure. The
physical characteristics of liposomes depend on pH, ionic strength
and the presence of divalent cations. Liposomes can show low permeability
to ionic and polar substances, but at elevated temperatures undergo
a phase transition which markedly alters their permeability. The
phase transition involves a change from a closely packed, ordered
structure, known as the gel state, to a loosely packed, less-ordered
structure, known as the fluid state. This occurs at a characteristic
phase-transition temperature and results in an increase in permeability
to ions, sugars and drugs.
In addition to temperature, exposure to proteins can alter the
permeability of liposomes. Certain soluble proteins, such as cytochrome
c, bind, deform and penetrate the bilayer, thereby causing changes
in permeability. Cholesterol inhibits this penetration of proteins,
apparently by packing the phospholipids more tightly. It is contemplated
that the most useful liposome formations for antibiotic and inhibitor
delivery will contain cholesterol.
The ability to trap solutes varies between different types of liposomes.
For example, MLVs are moderately efficient at trapping solutes,
but SUVs are extremely inefficient. SUVs offer the advantage of
homogeneity and reproducibility in size distribution, however, and
a compromise between size and trapping efficiency is offered by
large unilamellar vesicles (LUVs). These are prepared by ether evaporation
and are three to four times more efficient at solute entrapment
In addition to liposome characteristics, an important determinant
in entrapping compounds is the physicochemical properties of the
compound itself. Polar compounds are trapped in the aqueous spaces
and nonpolar compounds bind to the lipid bilayer of the vesicle.
Polar compounds are released through permeation or when the bilayer
is broken, but nonpolar compounds remain affiliated with the bilayer
unless it is disrupted by temperature or exposure to lipoproteins.
Both types show maximum efflux rates at the phase transition temperature.
Liposomes interact with cells via four different mechanisms: endocytosis
by phagocytic cells of the reticuloendothelial system such as macrophages
and neutrophils; adsorption to the cell surface, either by nonspecific
weak hydrophobic or electrostatic forces, or by specific interactions
with cell-surface components; fusion with the plasma cell membrane
by insertion of the lipid bilayer of the liposome into the plasma
membrane, with simultaneous release of liposomal contents into the
cytoplasm; and by transfer of liposomal lipids to cellular or subcellular
membranes, or vice versa, without any association of the liposome
contents. It often is difficult to determine which mechanism is
operative and more than one may operate at the same time.
The fate and disposition of intravenously injected liposomes depend
on their physical properties, such as size, fluidity, and surface
charge. They may persist in tissues for h or days, depending on
their composition, and half lives in the blood range from min to
several h. Larger liposomes, such as MLVs and LUVs, are taken up
rapidly by phagocytic cells of the reticuloendothelial system, but
physiology of the circulatory system restrains the exit of such
large species at most sites. They can exit only in places where
large openings or pores exist in the capillary endothelium, such
as the sinusoids of the liver or spleen. Thus, these organs are
the predominate site of uptake. On the other hand, SUVs show a broader
tissue distribution but still are sequestered highly in the liver
and spleen. In general, this in vivo behavior limits the potential
targeting of liposomes to only those organs and tissues accessible
to their large size. These include the blood, liver, spleen, bone
marrow, and lymphoid organs.
Targeting is generally not a limitation in terms of the present
invention. However, should specific targeting be desired, methods
are available for this to be accomplished. Antibodies may be used
to bind to the liposome surface and to direct the antibody and its
drug contents to specific antigenic receptors located on a particular
cell-type surface. Carbohydrate determinants (glycoprotein or glycolipid
cell-surface components that play a role in cell--cell recognition,
interaction and adhesion) may also be used as recognition sites
as they have potential in directing liposomes to particular cell
types. Mostly, it is contemplated that intravenous injection of
liposomal preparations would be used, but other routes of administration
are also conceivable.
Alternatively, the invention provides for pharmaceutically-acceptable
nanocapsule formulations of the compositions of the present invention.
Nanocapsules can generally entrap compounds in a stable and reproducible
way (Henry-Michelland et al., 1987; Quintanar-Guerrero et al., 1998;
Douglas et al., 1987). To avoid side effects due to intracellular
polymeric overloading, such ultrafine particles (sized around 0.1
.mu.m) should be designed using polymers able to be degraded in
vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet
these requirements are contemplated for use in the present invention.
Such particles may be are easily made, as described (Couvreur et
al., 1980; 1988; zur Muhlen et al., 1998; Zambaux et al. 1998; Pinto-Alphandry
et al., 1995 and U.S. Pat. No. 5,145,684, specifically incorporated
herein by reference in its entirety).
In certain preferred embodiments of the present invention, immunogenic
compositions, or vaccines, are provided. The immunogenic compositions
will generally comprise one or more pharmaceutical compositions,
such as those discussed above, in combination with an immunostimulant.
An immunostimulant may be any substance that enhances or potentiates
an immune response (antibody and/or cell-mediated) to an exogenous
antigen. Examples of immunostimulants include adjuvants, biodegradable
microspheres (e.g., polylactic galactide) and liposomes (into which
the compound is incorporated; see e.g., Fullerton, U.S. Pat. No.
4,235,877). Vaccine preparation is generally described in, for example,
M. F. Powell and M. J. Newman, eds., "Vaccine Design (the subunit
and adjuvant approach)," Plenum Press (NY, 1995). Pharmaceutical
compositions and immunogenic compositions within the scope of the
present invention may also contain other compounds, which may be
biologically active or inactive. For example, one or more immunogenic
portions of other tumor antigens may be present, either incorporated
into a fusion polypeptide or as a separate compound, within the
Illustrative immunogenic compositions may contain DNA encoding
one or more of the polypeptides as described above, such that the
polypeptide is generated in situ. As noted above, the DNA may be
present within any of a variety of delivery systems known to those
of ordinary skill in the art, including nucleic acid expression
systems, bacteria and viral expression systems. Numerous gene delivery
techniques are well known in the art, such as those described by
Rolland, Crit. Rev. Therap. Drug Carrier Systems 15:143-198, 1998,
and references cited therein. Appropriate nucleic acid expression
systems contain the necessary DNA sequences for expression in the
patient (such as a suitable promoter and terminating signal). Bacterial
delivery systems involve the administration of a bacterium (such
as Bacillus-Calmette-Guerrin) that expresses an immunogenic portion
of the polypeptide on its cell surface or secretes such an epitope.
In a preferred embodiment, the DNA may be introduced using a viral
expression system (e.g., vaccinia or other pox virus, retrovirus,
or adenovirus), which may involve the use of a non-pathogenic (defective),
replication competent virus. Suitable systems are disclosed, for
example, in Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA 86:317-321,
1989; Flexner et al., Ann. N.Y Acad. Sci. 569:86-103, 1989; Flexner
et al., Vaccine 8:17-21, 1990; U.S. Pat. Nos. 4,603,112, 4,769,330,
and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651;
EP 0,345,242; WO 91/02805; Berkner, Biotechniques 6:616-627, 1988;
Rosenfeld et al., Science 252:431-434, 1991; Kolls et al., Proc.
Natl. Acad. Sci. USA 91:215-219, 1994; Kass-Eisler et al., Proc.
Natl. Acad. Sci. USA 90:11498-11502, 1993; Guzman et al., Circulation
88:2838-2848, 1993; and Guzman et al., Cir. Res. 73:1202-1207, 1993.
Techniques for incorporating DNA into such expression systems are
well known to those of ordinary skill in the art. The DNA may also
be "naked," as described, for example, in Ulmer et al.,
Science 259:1745-1749, 1993 and reviewed by Cohen, Science 259:1691-1692,
1993. The uptake of naked DNA may be increased by coating the DNA
onto biodegradable beads, which are efficiently transported into
the cells. It will be apparent that an immunogenic composition may
comprise both a polynucleotide and a polypeptide component. Such
immunogenic compositions may provide for an enhanced immune response.
It will be apparent that an immunogenic composition may contain
pharmaceutically acceptable salts of the polynucleotides and polypeptides
provided herein. Such salts may be prepared from pharmaceutically
acceptable non-toxic bases, including organic bases (e.g., salts
of primary, secondary and tertiary amines and basic amino acids)
and inorganic bases (e.g., sodium, potassium, lithium, ammonium,
calcium and magnesium salts).
While any suitable carrier known to those of ordinary skill in
the art may be employed in the compositions of this invention, the
type of carrier will vary depending on the mode of administration.
Compositions of the present invention may be formulated for any
appropriate manner of administration, including for example, topical,
oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous
or intramuscular administration. For parenteral administration,
such as subcutaneous injection, the carrier preferably comprises
water, saline, alcohol, a fat, a wax or a buffer. For oral administration,
any of the above carriers or a solid carrier, such as mannitol,
lactose, starch, magnesium stearate, sodium saccharine, talcum,
cellulose, glucose, sucrose, and magnesium carbonate, may be employed.
Biodegradable microspheres (e.g., polylactate polyglycolate) may
also be employed as carriers for the pharmaceutical compositions
of this invention. Suitable biodegradable microspheres are disclosed,
for example, in U.S. Pat. Nos. 4,897,268; 5,075,109; 5,928,647;
5,811,128; 5,820,883; 5,853,763; 5,814,344 and 5,942,252. One may
also employ a carrier comprising the particulate-protein complexes
described in U.S. Pat. No. 5,928,647, which are capable of inducing
a class I-restricted cytotoxic T lymphocyte responses in a host.
Such compositions may also comprise buffers (e.g., neutral buffered
saline or phosphate buffered saline), carbohydrates (e.g., glucose,
mannose, sucrose or dextrans), mannitol, proteins, polypeptides
or amino acids such as glycine, antioxidants, bacteriostats, chelating
agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide),
solutes that render the formulation isotonic, hypotonic or weakly
hypertonic with the blood of a recipient, suspending agents, thickening
agents and/or preservatives. Alternatively, compositions of the
present invention may be formulated as a lyophilizate. Compounds
may also be encapsulated within liposomes using well known technology.
Any of a variety of immunostimulants may be employed in the immunogenic
compositions of this invention. For example, an adjuvant may be
included. Most adjuvants contain a substance designed to protect
the antigen from rapid catabolism, such as aluminum hydroxide or
mineral oil, and a stimulator of immune responses, such as lipid
A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins.
Suitable adjuvants are commercially available as, for example, Freund's
Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit,
Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.);
AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminum salts such
as aluminum hydroxide gel (alum) or aluminum phosphate; salts of
calcium, iron or zinc; an insoluble suspension of acylated tyrosine;
acylated sugars; cationically or anionically derivatized polysaccharides;
polyphosphazenes; biodegradable microspheres; monophosphoryl lipid
A and quil A. Cytokines, such as GM-CSF or interleukin-2,-7, or
-12, may also be used as adjuvants.
Within the immunogenic compositions provided herein, the adjuvant
composition is preferably designed to induce an immune response
predominantly of the Th1 type. High levels of Th1-type cytokines
(e.g., IFN-.gamma., TNF.alpha., IL-2 and IL-12) tend to favor the
induction of cell mediated immune responses to an administered antigen.
In contrast, high levels of Th2-type cytokines (e.g., IL-4, IL-5,
IL-6 and IL-10) tend to favor the induction of humoral immune responses.
Following application of an immunogenic composition as provided
herein, a patient will support an immune response that includes
Th1- and Th2-type responses. Within a preferred embodiment, in which
a response is predominantly Th1type, the level of Th1-type cytokines
will increase to a greater extent than the level of Th2-type cytokines.
The levels of these cytokines may be readily assessed using standard
assays. For a review of the families of cytokines, see Mosmann and
Coffinan, Ann. Rev. Immunol. 7:145-173, 1989.
Preferred adjuvants for use in eliciting a predominantly Th1-type
response include, for example, a combination of monophosphoryl lipid
A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL), together
with an aluminum salt. MPL adjuvants are available from Corixa Corporation
(Seattle, Wash.; see U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034
and 4,912,094). CpG-containing oligonucleotides (in which the CpG
dinucleotide is unmethylated) also induce a predominantly Thl response.
Such oligonucleotides are well known and are described, for example,
in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and 5,856,462.
Immunostimulatory DNA sequences are also described, for example,
by Sato et al., Science 273:352, 1996. Another preferred adjuvant
is a saponin, preferably QS21 (Aquila Biopharmaceuticals Inc., Framingham,
Mass.), which may be used alone or in combination with other adjuvants.
For example, an enhanced system involves the combination of a monophosphoryl
lipid A and saponin derivative, such as the combination of QS21
and 3D-MPL as described in WO 94/00153, or a less reactogenic composition
where the QS21 is quenched with cholesterol, as described in WO
96/33739. Other preferred formulations comprise an oil-in-water
emulsion and tocopherol. A particularly potent adjuvant formulation
involving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion
is described in WO 95/17210.
Other preferred adjuvants include Montanide ISA 720 (Seppic, France),
SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59 (Chiron),
the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4, available
from SmithKline Beecham, Rixensart, Belgium), Detox (Corixa, Hamilton,
Mont.), RC-529 (Corixa, Hamilton, Mont.) and other aminoalkyl glucosaminide
4-phosphates (AGPs), such as those described in pending U.S. patent
application Ser. Nos. 08/853,826 and 09/074,720, the disclosures
of which are incorporated herein by reference in their entireties.
Any immunogenic composition provided herein may be prepared using
well known methods that result in a combination of antigen, immune
response enhancer and a suitable carrier or excipient. The compositions
described herein may be administered as part of a sustained release
formulation (i.e., a formulation such as a capsule, sponge or gel
(composed of polysaccharides, for example) that effects a slow release
of compound following administration). Such formulations may generally
be prepared using well known technology (see, e.g., Coombes et al.,
Vaccine 14:1429-1438, 1996) and administered by, for example, oral,
rectal or subcutaneous implantation, or by implantation at the desired
target site. Sustained-release formulations may contain a polypeptide,
polynucleotide or antibody dispersed in a carrier matrix and/or
contained within a reservoir surrounded by a rate controlling membrane.
Carriers for use within such formulations are biocompatible, and
may also be biodegradable; preferably the formulation provides a
relatively constant level of active component release. Such carriers
include microparticles of poly(lactide-co-glycolide), polyacrylate,
latex, starch, cellulose, dextran and the like. Other delayed-release
carriers include supramolecular biovectors, which comprise a non-liquid
hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide)
and, optionally, an external layer comprising an amphiphilic compound,
such as a phospholipid (see e.g., U.S. Pat. No. 5,151,254 and PCT
applications WO 94/20078, WO/94/23701 and WO 96/06638). The amount
of active compound contained within a sustained release formulation
depends upon the site of implantation, the rate and expected duration
of release and the nature of the condition to be treated or prevented.
Any of a variety of delivery vehicles may be employed within pharmaceutical
compositions and immunogenic compositions to facilitate production
of an antigen-specific immune response that targets tumor cells.
Delivery vehicles include antigen presenting cells (APCs), such
as dendritic cells, macrophages, B cells, monocytes and other cells
that may be engineered to be efficient APCs. Such cells may, but
need not, be genetically modified to increase the capacity for presenting
the antigen, to improve activation and/or maintenance of the T cell
response, to have anti-tumor effects per se and/or to be immunologically
compatible with the receiver (i.e., matched HLA haplotype). APCs
may generally be isolated from any of a variety of biological fluids
and organs, including tumor and peritumoral tissues, and may be
autologous, allogeneic, syngeneic or xenogeneic cells.
Certain preferred embodiments of the present invention use dendritic
cells or progenitors thereof as antigen-presenting cells. Dendritic
cells are highly potent APCs (Banchereau and Steinman, Nature 392:245-251,
1998) and have been shown to be effective as a physiological adjuvant
for eliciting prophylactic or therapeutic antitumor immunity (see
Timmernan and Levy, Ann. Rev. Med. 50:507-529, 1999). In general,
dendritic cells may be identified based on their typical shape (stellate
in situ, with marked cytoplasmic processes (dendrites) visible in
vitro), their ability to take up, process and present antigens with
high efficiency and their ability to activate naive T cell responses.
Dendritic cells may, of course, be engineered to express specific
cell-surface receptors or ligands that are not commonly found on
dendritic cells in vivo or ex vivo, and such modified dendritic
cells are contemplated by the present invention. As an alternative
to dendritic cells, secreted vesicles antigen-loaded dendritic cells
(called exosomes) may be used within an immunogenic composition
(see Zitvogel et al., Nature Med. 4:594-600, 1998).
Dendritic cells and progenitors may be obtained from peripheral
blood, bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltrating
cells, lymph nodes, spleen, skin, umbilical cord blood or any other
suitable tissue or fluid. For example, dendritic cells may be differentiated
ex vivo by adding a combination of cytokines such as GM-CSF, IL-4,
IL-13 and/or TNF.alpha. to cultures of monocytes harvested from
peripheral blood. Alternatively, CD34 positive cells harvested from
peripheral blood, umbilical cord blood or bone marrow may be differentiated
into dendritic cells by adding to the culture medium combinations
of GM-CSF, IL-3, TNF.alpha., CD40 ligand, LPS, flt3 ligand and/or
other compound(s) that induce differentiation, maturation and proliferation
of dendritic cells.
Dendritic cells are conveniently categorized as "immature"
and "mature" cells, which allows a simple way to discriminate
between two well characterized phenotypes. However, this nomenclature
should not be construed to exclude all possible intermediate stages
of differentiation. Immature dendritic cells are characterized as
APC with a high capacity for antigen uptake and processing, which
correlates with the high expression of Fcy receptor and mannose
receptor. The mature phenotype is typically characterized by a lower
expression of these markers, but a high expression of cell surface
molecules responsible for T cell activation such as class I and
class II MHC, adhesion molecules (e.g., CD54 and CD11) and costimulatory
molecules (e.g., CD40, CD80, CD86 and 4-1BB).
APCs may generally be transfected with a polynucleotide encoding
a breast tumor protein (or portion or other variant thereof) such
that the breast tumor polypeptide, or an immunogenic portion thereof,
is expressed on the cell surface. Such transfection may take place
ex vivo, and a composition comprising such transfected cells may
then be used for therapeutic purposes, as described herein. Alternatively,
a gene delivery vehicle that targets a dendritic or other antigen
presenting cell may be administered to a patient, resulting in transfection
that occurs in vivo. In vivo and ex vivo transfection of dendritic
cells, for example, may generally be performed using any methods
known in the art, such as those described in WO 97/24447, or the
gene gun approach described by Mahvi et al., Immunology and cell
Biology 75:456-460, 1997. Antigen loading of dendritic cells may
be achieved by incubating dendritic cells or progenitor cells with
the breast tumor polypeptide, DNA (naked or within a plasmid vector)
or RNA; or with antigen-expressing recombinant bacterium or viruses
(e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors). Prior
to loading, the polypeptide may be covalently conjugated to an immunological
partner that provides T cell help (e.g., a carrier molecule). Alternatively,
a dendritic cell may be pulsed with a non-conjugated immunological
partner, separately or in the presence of the polypeptide.
Immunogenic compositions and pharmaceutical compositions may be
presented in unit-dose or multi-dose containers, such as sealed
ampoules or vials. Such containers are preferably hermetically sealed
to preserve sterility of the formulation until use. In general,
formulations may be stored as suspensions, solutions or emulsions
in oily or aqueous vehicles. Alternatively, a immunogenic composition
or pharmaceutical composition may be stored in a freeze-dried condition
requiring only the addition of a sterile liquid carrier immediately
prior to use.
In further aspects of the present invention, the compositions described
herein may be used for immunotherapy of cancer, such as breast cancer.
Within such methods, pharmaceutical compositions and immunogenic
compositions are typically administered to a patient. As used herein,
a "patient" refers to any warm-blooded animal, preferably
a human. A patient may or may not be afflicted with cancer. Accordingly,
the above pharmaceutical compositions and immunogenic compositions
may be used to prevent the development of a cancer or to treat a
patient afflicted with a cancer. A cancer may be diagnosed using
criteria generally accepted in the art, including the presence of
a malignant tumor. Pharmaceutical compositions and immunogenic compositions
may be administered either prior to or following surgical removal
of primary tumors and/or treatment such as administration of radiotherapy
or conventional chemotherapeutic drugs. Administration may be by
any suitable method, including administration by intravenous, intraperitoneal,
intramuscular, subcutaneous, intranasal, intradermal, anal, vaginal,
topical and oral routes.
Within certain embodiments, immunotherapy may be active immunotherapy,
in which treatment relies on the in vivo stimulation of the endogenous
host immune system to react against tumors with the administration
of immune response-modifying agents (such as polypeptides and polynucleotides
as provided herein).
Within other embodiments, immunotherapy may be passive immunotherapy,
in which treatment involves the delivery of agents with established
tumor-immune reactivity (such as effector cells or antibodies) that
can directly or indirectly mediate antitumor effects and does not
necessarily depend on an intact host immune system. Examples of
effector cells include T cells as discussed above, T lymphocytes
(such as CD8.sup.+ cytotoxic T lymphocytes and CD4.sup.+ T-helper
tumor-infiltrating lymphocytes), killer cells (such as Natural Killer
cells and lymphokine-activated killer cells), B cells and antigen-presenting
cells (such as dendritic cells and macrophages) expressing a polypeptide
provided herein. T cell receptors and antibody receptors specific
for the polypeptides recited herein may be cloned, expressed and
transferred into other vectors or effector cells for adoptive immunotherapy.
The polypeptides provided herein may also be used to generate antibodies
or anti-idiotypic antibodies (as described above and in U.S. Pat.
No. 4,918,164) for passive immunotherapy.
Effector cells may generally be obtained in sufficient quantities
for adoptive immunotherapy by growth in vitro, as described herein.
Culture conditions for expanding single antigen-specific effector
cells to several billion in number with retention of antigen recognition
in vivo are well known in the art. Such in vitro culture conditions
typically use intermittent stimulation with antigen, often in the
presence of cytokines (such as IL-2) and non-dividing feeder cells.
As noted above, immunoreactive polypeptides as provided herein may
be used to rapidly expand antigen-specific T cell cultures in order
to generate a sufficient number of cells for immunotherapy. In particular,
antigen-presenting cells, such as dendritic, macrophage, monocyte,
fibroblast and/or B cells, may be pulsed with immunoreactive polypeptides
or transfected with one or more polynucleotides using standard techniques
well known in the art. For example, antigen-presenting cells can
be transfected with a polynucleotide having a promoter appropriate
for increasing expression in a recombinant virus or other expression
system. Cultured effector cells for use in therapy must be able
to grow and distribute widely, and to survive long term in vivo.
Studies have shown that cultured effector cells can be induced to
grow in vivo and to survive long term in substantial numbers by
repeated stimulation with antigen supplemented with IL-2 (see, for
example, Cheever et al., Immunological Reviews 157:177, 1997).
Alternatively, a vector expressing a polypeptide recited herein
may be introduced into antigen presenting cells taken from a patient
and clonally propagated ex vivo for transplant back into the same
patient. Transfected cells may be reintroduced into the patient
using any means known in the art, preferably in sterile form by
intravenous, intracavitary, intraperitoneal or intratumor administration.
Routes and frequency of administration of the therapeutic compositions
described herein, as well as dosage, will vary from individual to
individual, and may be readily established using standard techniques.
In general, the pharmaceutical compositions and immunogenic compositions
may be administered by injection (e.g., intracutaneous, intramuscular,
intravenous or subcutaneous), intranasally (e.g., by aspiration)
or orally. Preferably, between 1 and 10 doses may be administered
over a 52 week period. Preferably, 6 doses are administered, at
intervals of 1 month, and booster vaccinations may be given periodically
thereafter. Alternate protocols may be appropriate for individual
patients. A suitable dose is an amount of a compound that, when
administered as described above, is capable of promoting an anti-tumor
immune response, and is at least 10-50% above the basal (i.e., untreated)
level. Such response can be monitored by measuring the anti-tumor
antibodies in a patient or by vaccine-dependent generation of cytolytic
effector cells capable of killing the patient's tumor cells in vitro.
Such immunogenic compositions should also be capable of causing
an immune response that leads to an improved clinical outcome (e.g.,
more frequent remissions, complete or partial or longer disease-free
survival) in treated patients as compared to non-treated patients.
In general, for pharmaceutical compositions and immunogenic compositions
comprising one or more polypeptides, the amount of each polypeptide
present in a dose ranges from about 25 .mu.g to 5 mg per kg of host.
Suitable dose sizes will vary with the size of the patient, but
will typically range from about 0.1 mL to about 5 mL.
In general, an appropriate dosage and treatment regimen provides
the active compound(s) in an amount sufficient to provide therapeutic
and/or prophylactic benefit. Such a response can be monitored by
establishing an improved clinical outcome (e.g., more frequent remissions,
complete or partial, or longer disease-free survival) in treated
patients as compared to non-treated patients. Increases in preexisting
immune responses to a breast tumor protein generally correlate with
an improved clinical outcome. Such immune responses may generally
be evaluated using standard proliferation, cytotoxicity or cytokine
assays, which may be performed using samples obtained from a patient
before and after treatment.
Cancer Detection and Diagnosis
In general, a cancer may be detected in a patient based on the
presence of one or more breast tumor proteins and/or polynucleotides
encoding such proteins in a biological sample (for example, blood,
sera, sputum urine and/or tumor biopsies) obtained from the patient.
In other words, such proteins may be used as markers to indicate
the presence or absence of a cancer such as breast cancer. In addition,
such proteins may be useful for the detection of other cancers.
The binding agents provided herein generally permit detection of
the level of antigen that binds to the agent in the biological sample.
Polynucleotide primers and probes may be used to detect the level
of mRNA encoding a tumor protein, which is also indicative of the
presence or absence of a cancer. In general, a breast tumor sequence
should be present at a level that is at least three fold higher
in tumor tissue than in normal tissue
There are a variety of assay formats known to those of ordinary
skill in the art for using a binding agent to detect polypeptide
markers in a sample. See, e.g., Harlow and Lane, Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory, 1988. In general, the presence
or absence of a cancer in a patient may be determined by (a) contacting
a biological sample obtained from a patient with a binding agent;
(b) detecting in the sample a level of polypeptide that binds to
the binding agent; and (c) comparing the level of polypeptide with
a predetermined cut-off value.
In a preferred embodiment, the assay involves the use of binding
agent immobilized on a solid support to bind to and remove the polypeptide
from the remainder of the sample. The bound polypeptide may then
be detected using a detection reagent that contains a reporter group
and specifically binds to the binding agent/polypeptide complex.
Such detection reagents may comprise, for example, a binding agent
that specifically binds to the polypeptide or an antibody or other
agent that specifically binds to the binding agent, such as an anti-immunoglobulin,
protein G, protein A or a lectin. Alternatively, a competitive assay
may be utilized, in which a polypeptide is labeled with a reporter
group and allowed to bind to the immobilized binding agent after
incubation of the binding agent with the sample. The extent to which
components of the sample inhibit the binding of the labeled polypeptide
to the binding agent is indicative of the reactivity of the sample
with the immobilized binding agent. Suitable polypeptides for use
within such assays include full length breast tumor proteins and
portions thereof to which the binding agent binds, as described
The solid support may be any material known to those of ordinary
skill in the art to which the tumor protein may be attached. For
example, the solid support may be a test well in a microtiter plate
or a nitrocellulose or other suitable membrane. Alternatively, the
support may be a bead or disc, such as glass, fiberglass, latex
or a plastic material such as polystyrene or polyvinylchloride.
The support may also be a magnetic particle or a fiber optic sensor,
such as those disclosed, for example, in U.S. Pat. No. 5,359,681.
The binding agent may be immobilized on the solid support using
a variety of techniques known to those of skill in the art, which
are amply described in the patent and scientific literature. In
the context of the present invention, the term "immobilization"
refers to both noncovalent association, such as adsorption, and
covalent attachment (which may be a direct linkage between the agent
and functional groups on the support or may be a linkage by way
of a cross-linking agent). Immobilization by adsorption to a well
in a microtiter plate or to a membrane is preferred. In such cases,
adsorption may be achieved by contacting the binding agent, in a
suitable buffer, with the solid support for a suitable amount of
time. The contact time varies with temperature, but is typically
between about 1 hour and about 1 day. In general, contacting a well
of a plastic microtiter plate (such as polystyrene or polyvinylchloride)
with an amount of binding agent ranging from about 10 ng to about
10 .mu.g, and preferably about 100 ng to about 1 .mu.g, is sufficient
to immobilize an adequate amount of binding agent.
Covalent attachment of binding agent to a solid support may generally
be achieved by first reacting the support with a bifunctional reagent
that will react with both the support and a functional group, such
as a hydroxyl or amino group, on the binding agent. For example,
the binding agent may be covalently attached to supports having
an appropriate polymer coating using benzoquinone or by condensation
of an aldehyde group on the support with an amine and an active
hydrogen on the binding partner (see, e.g., Pierce Immunotechnology
Catalog and Handbook, 1991, at A12-A13).
In certain embodiments, the assay is a two-antibody sandwich assay.
This assay may be performed by first contacting an antibody that
has been immobilized on a solid support, commonly the well of a
microtiter plate, with the sample, such that polypeptides within
the sample are allowed to bind to the immobilized antibody. Unbound
sample is then removed from the immobilized polypeptide-antibody
complexes and a detection reagent (preferably a second antibody
capable of binding to a different site on the polypeptide) containing
a reporter group is added. The amount of detection reagent that
remains bound to the solid support is then determined using a method
appropriate for the specific reporter group.
More specifically, once the antibody is immobilized on the support
as described above, the remaining protein binding sites on the support
are typically blocked. Any suitable blocking agent known to those
of ordinary skill in the art, such as bovine serum albumin or Tween
20.TM. (Sigma Chemical Co., St. Louis, Mo.). The immobilized antibody
is then incubated with the sample, and polypeptide is allowed to
bind to the antibody. The sample may be diluted with a suitable
diluent, such as phosphate-buffered saline (PBS) prior to incubation.
In general, an appropriate contact time (i.e., incubation time)
is a period of time that is sufficient to detect the presence of
polypeptide within a sample obtained from an individual with breast
cancer. Preferably, the contact time is sufficient to achieve a
level of binding that is at least about 95% of that achieved at
eqlibrium between bound and unbound polypeptide. Those of ordinary
skill in the art will recognize that the time necessary to achieve
equilibrium may be readily determined by assaying the level of binding
that occurs over a period of time. At room temperature, an incubation
time of about 30 minutes is generally sufficient.
Unbound sample may then be removed by washing the solid support
with an appropriate buffer, such as PBS containing 0.1% Tween 20.TM..
The second antibody, which contains a reporter group, may then be
added to the solid support. Preferred reporter groups include those
groups recited above.
The detection reagent is then incubated with the immobilized antibody-polypeptide
complex for an amount of time sufficient to detect the bound polypeptide.
An appropriate amount of time may generally be determined by assaying
the level of binding that occurs over a period of time. Unbound
detection reagent is then removed and bound detection reagent is
detected using the reporter group. The method employed for detecting
the reporter group depends upon the nature of the reporter group.
For radioactive groups, scintillation counting or autoradiographic
methods are generally appropriate. Spectroscopic methods may be
used to detect dyes, luminescent groups and fluorescent groups.
Biotin may be detected using avidin, coupled to a different reporter
group (commonly a radioactive or fluorescent group or an enzyme).
Enzyme reporter groups may generally be detected by the addition
of substrate (generally for a specific period of time), followed
by spectroscopic or other analysis of the reaction products.
To determine the presence or absence of a cancer, such as breast
cancer, the signal detected from the reporter group that remains
bound to the solid support is generally compared to a signal that
corresponds to a predetermined cut-off value. In one preferred embodiment,
the cut-off value for the detection of a cancer is the average mean
signal obtained when the immobilized antibody is incubated with
samples from patients without the cancer. In general, a sample generating
a signal that is three standard deviations above the predetermined
cut-off value is considered positive for the cancer. In an alternate
preferred embodiment, the cut-off value is determined using a Receiver
Operator Curve, according to the method of Sackett et al., Clinical
Epidemiology: A Basic Science for Clinical Medicine, Little Brown
and Co., 1985, p. 106-7. Briefly, in this embodiment, the cut-off
value may be determined from a plot of pairs of true positive rates
(i.e., sensitivity) and false positive rates (100%-specificity)
that correspond to each possible cut-off value for the diagnostic
test result. The cut-off value on the plot that is the closest to
the upper left-hand corner (i.e., the value that encloses the largest
area) is the most accurate cut-off value, and a sample generating
a signal that is higher than the cut-off value determined by this
method may be considered positive. Alternatively, the cut-off value
may be shifted to the left along the plot, to minimize the false
positive rate, or to the right, to minimize the false negative rate.
In general, a sample generating a signal that is higher than the
cut-off value determined by this method is considered positive for
In a related embodiment, the assay is performed in a flow-through
or strip test format, wherein the binding agent is immobilized on
a membrane, such as nitrocellulose. In the flow-through test, polypeptides
within the sample bind to the immobilized binding agent as the sample
passes through the membrane. A second, labeled binding agent then
binds to the binding agent-polypeptide complex as a solution containing
the second binding agent flows through the membrane. The detection
of bound second binding agent may then be performed as described
above. In the strip test format, one end of the membrane to which
binding agent is bound is immersed in a solution containing the
sample. The sample migrates along the membrane through a region
containing second binding agent and to the area of immobilized binding
agent. Concentration of second binding agent at the area of immobilized
antibody indicates the presence of a cancer. Typically, the concentration
of second binding agent at that site generates a pattern, such as
a line, that can be read visually. The absence of such a pattern
indicates a negative result. In general, the amount of binding agent
immobilized on the membrane is selected to generate a visually discernible
pattern when the biological sample contains a level of polypeptide
that would be sufficient to generate a positive signal in the two-antibody
sandwich assay, in the format discussed above. Preferred binding
agents for use in such assays are antibodies and antigen-binding
fragments thereof. Preferably, the amount of antibody immobilized
on the membrane ranges from about 25 ng to about 1 .mu.g, and more
preferably from about 50 ng to about 500 ng. Such tests can typically
be performed with a very small amount of biological sample.
Of course, numerous other assay protocols exist that are suitable
for use with the tumor proteins or binding agents of the present
invention. The above descriptions are intended to be exemplary only.
For example, it will be apparent to those of ordinary skill in the
art that the above protocols may be readily modified to use breast
tumor polypeptides to detect antibodies that bind to such polypeptides
in a biological sample. The detection of such breast tumor protein
specific antibodies may correlate with the presence of a cancer.
A cancer may also, or alternatively, be detected based on the presence
of T cells that specifically react with a breast tumor protein in
a biological sample. Within certain methods, a biological sample
comprising CD4.sup.+ and/or CD8.sup.+ T cells isolated from a patient
is incubated with a breast tumor polypeptide, a polynucleotide encoding
such a polypeptide and/or an APC that expresses at least an immunogenic
portion of such a polypeptide, and the presence or absence of specific
activation of the T cells is detected. Suitable biological samples
include, but are not limited to, isolated T cells. For example,
T cells may be isolated from a patient by routine techniques (such
as by Ficoll/Hypaque density gradient centrifugation of peripheral
blood lymphocytes). T cells may be incubated in vitro for 2-9 days
(typically 4 days) at 37.degree. C. with polypeptide (e.g., 5-25
.mu.g/ml). It may be desirable to incubate another aliquot of a
T cell sample in the absence of breast tumor polypeptide to serve
as a control. For CD4.sup.+ T cells, activation is preferably detected
by evaluating proliferation of the T cells. For CD8.sup.+ T cells,
activation is preferably detected by evaluating cytolytic activity.
A level of proliferation that is at least two fold greater and/or
a level of cytolytic activity that is at least 20% greater than
in disease-free patients indicates the presence of a cancer in the
As noted above, a cancer may also, or alternatively, be detected
based on the level of mRNA encoding a breast tumor protein in a
biological sample. For example, at least two oligonucleotide primers
may be employed in a polymerase chain reaction (PCR) based assay
to amplify a portion of a breast tumor cDNA derived from a biological
sample, wherein at least one of the oligonucleotide primers is specific
for (i.e., hybridizes to) a polynucleotide encoding the breast tumor
protein. The amplified cDNA is then separated and detected using
techniques well known in the art, such as gel electrophoresis. Similarly,
oligonucleotide probes that specifically hybridize to a polynucleotide
encoding a breast tumor protein may be used in a hybridization assay
to detect the presence of polynucleotide encoding the tumor protein
in a biological sample.
To permit hybridization under assay conditions, oligonucleotide
primers and probes should comprise an oligonucleotide sequence that
has at least about 60%, preferably at least about 75% and more preferably
at least about 90%, identity to a portion of a polynucleotide encoding
a breast tumor protein that is at least 10 nucleotides, and preferably
at least 20 nucleotides, in length. Preferably, oligonucleotide
primers and/or probes hybridize to a polynucleotide encoding a polypeptide
described herein under moderately stringent conditions, as defined
above. Oligonucleotide primers and/or probes which may be usefully
employed in the diagnostic methods described herein preferably are
at least 10-40 nucleotides in length. In a preferred embodiment,
the oligonucleotide primers comprise at least 10 contiguous nucleotides,
more preferably at least 15 contiguous nucleotides, of a DNA molecule
having a sequence recited in SEQ ID NO:1-175, 178, 180, 182-468,
474, 476, 477 479, 484, 486 and 489. Techniques for both PCR based
assays and hybridization assays are well known in the art (see,
for example, Mullis et al., Cold Spring Harbor Symp. Quant. Biol.,
51:263, 1987; Erlich ed., PCR Technology, Stockton Press, NY, 1989).
One preferred assay employs RT-PCR, in which PCR is applied in
conjunction with reverse transcription. Typically, RNA is extracted
from a biological sample, such as biopsy tissue, and is reverse
transcribed to produce cDNA molecules. PCR amplification using at
least one specific primer generates a cDNA molecule, which may be
separated and visualized using, for example, gel electrophoresis.
Amplification may be performed on biological samples taken from
a test patient and from an individual who is not afflicted with
a cancer. The amplification reaction may be performed on several
dilutions of cDNA spanning two orders of magnitude. A two-fold or
greater increase in expression in several dilutions of the test
patient sample as compared to the same dilutions of the non-cancerous
sample is typically considered positive.
In another embodiment, the compositions described herein may be
used as markers for the progression of cancer. In this embodiment,
assays as described above for the diagnosis of a cancer may be performed
over time, and the change in the level of reactive polypeptide(s)
or polynucleotide(s) evaluated. For example, the assays may be performed
every 24-72 hours for a period of 6 months to 1 year, and thereafter
performed as needed. In general, a cancer is progressing in those
patients in whom the level of polypeptide or polynucleotide detected
increases over time. In contrast, the cancer is not progressing
when the level of reactive polypeptide or polynucleotide either
remains constant or decreases with time.
Certain in vivo diagnostic assays may be performed directly on
a tumor. One such assay involves contacting tumor cells with a binding
agent. The bound binding agent may then be detected directly or
indirectly via a reporter group. Such binding agents may also be
used in histological applications. Alternatively, polynucleotide
probes may be used within such applications.
As noted above, to improve sensitivity, multiple breast tumor protein
markers may be assayed within a given sample. It will be apparent
that binding agents specific for different proteins provided herein
may be combined within a single assay. Further, multiple primers
or probes may be used concurrently. The selection of tumor protein
markers may be based on routine experiments to determine combinations
that results in optimal sensitivity. In addition, or alternatively,
assays for tumor proteins provided herein may be combined with assays
for other known tumor antigens.
The present invention further provides kits for use within any
of the above diagnostic methods. Such kits typically comprise two
or more components necessary for performing a diagnostic assay.
Components may be compounds, reagents, containers and/or equipment.
For example, one container within a kit may contain a monoclonal
antibody or fragment thereof that specifically binds to a breast
tumor protein. Such antibodies or fragments may be provided attached
to a support material, as described above. One or more additional
containers may enclose elements, such as reagents or buffers, to
be used in the assay. Such kits may also, or alternatively, contain
a detection reagent as described above that contains a reporter
group suitable for direct or indirect detection of antibody binding.
Alternatively, a kit may be designed to detect the level of mRNA
encoding a breast tumor protein in a biological sample. Such kits
generally comprise at least one oligonucleotide probe or primer,
as described above, that hybridizes to a polynucleotide encoding
a breast tumor protein. Such an oligonucleotide may be used, for
example, within a PCR or hybridization assay. Additional components
that may be present within such kits include a second oligonucleotide
and/or a diagnostic reagent or container to facilitate the detection
of a polynucleotide encoding a breast tumor protein.
The following Examples are offered by way of illustration and not
by way of limitation.