Note: Descriptions are shown in the official language in which they were submitted.
WO 95/03~8 ~ 1 6 ~ ~ 9 ~ PCT/US94/08423
B 7 - 2: CTL A4/CD 28 COUNTER RECEPTOR
Govern~ent Fnndi~
Work described herein was supported under CA-40216-08 awarded by the National
5 Institutes of Health. The U.S. government therefore may have certain rights in this invention.
Rarl~round of the Inv~ntion
To induce antigen-specific T cell activation and clonal expansion, two signals
provided by antigen-presentin~ cells (APCs) must be delivered to the surface of resting T
lymphocytes (Jenkins, M. and Schwartz, R. (1987)J. Exp. Med. 165, 302-319; Mueller, D.L.,
et al. (1990) J. Immunol. 144, 3701-3709; Williams, I.R. and Unanue, E.R. (1990) J.
Immunol. 145, 85-93). The first signal, which confers specificity to the imm~1ne response, is
mediated ~ia the T cell receptor (TCR) following recognition of foreign antigenic peptide
presented in the context of the major histocompatibility complex (MHC). The second signal,
termed costim~ tion, induces T cells to proliferate and become functional (Schwartz, R.H.
(1990) Science 2~, 1349-1356). Costimulation is neither antigen-specific, nor MHC
restricted and is thought to be provided by one or more distinct cell surface molecules
expressed by APCs (Jenkins, M.K., et al. (1988) J. Immunol. 140, 3324-3330; Linsley, P.S.,
etal. (l991)J. ~p. Med. 173, 721-730; Gimmi, C.D., etal., (1991)Proc. Natl. Acad. Sci.
USA. 88, 6575-6579; Young, J.W., et al. (1992) J. Clin. Invest. 90, 229-237; Koulova, L., et
al. (1991) J. Exp. Med. 173, 759-762; Reiser, H., et al. (1992) Proc. Natl. Acad. Sci. USA. 89,
271-275; van-Seventer, G.A., et al. (1990) J. Immunol. 144, 4579-4586; LaSalle, J.M., et al.,
(1991) J. Immunol. 147, 774-80; Dustin, M.I., et al., (1989) J. E~cp. Med. 169, 503; Armitage,
R.J., et al. (1992) Nature ~1, 80-82; Liu, Y., et al. (1992) J. E~p. Med. ~ 75, 437-445).
Considerable evidence suggests that the B7 protein, expressed on APCs, is one such
critical costimulatory molecule (Linsley, P.S., et al., (1991) J. Exp. Med. 173, 721-730;
Gimmi, C.D., et al., (1991) Proc. Natl. Acad. Sci USA. $8, 6575-6579; Koulova, L., et al.,
(1991) J. E~cp. Med. L73, 759-762; Reiser, H., et al. (1992) Proc. Natl. Acad. Sci. USA. 89,
271 -275; Linsley, P.S. et al. (1990) Proc. Natl. Acad. Sci. USA. 87, 5031 -5035; Freeman, G.J.
et al. (1991) J. ~p. Med. 174,625-631.). B7 is the counter-receptor for two ligands
expressed on T lymphocytes. The first ligand, terrned CD28, is constitutively expressed on
resting T cells and increases after activation. After ~ign~linp through the T cell receptor,
ligation of CD28 induces T cells to proliferate and secrete IL-2 (Linsley, P.S., et al. (1991) J
E~p. Med. 173, 721-730; Girnmi, C.D., et al. (1991) Proc. Nafl. ,4cad. Sci. USA. 88, 6575-
6579; Thompson, C.B., et al. (1989) Proc. Natl. Acad. Sci. USA. 86, 1333-1337; June, C.H.,
et al. (1990) Immunol. Today. ~1, 211-6; Harding, F.A., et al. (1992) Nature. 356, 607-609.).
The second ligand, termed CTLA4 is homologous to CD28 but is not expressed on resting T
cells and appears following T cell activation (Brunet, J.F., et al., (1987) Nature ~, 267-
270). DNA sequences encoding the human and murine CTLA4 protein are described in
WO 9~/03408 PCT/US94/08423
~67~91
--2 -
Dariavich, et al. (1988) Eur. J. Immunol. 18(12), 1901-1905; Brunet, J.F., et al. (1987) supra;
Brunet, J.F. et al. (1988) Immunol. Rev. 103:21-36; and Freeman, G.J.? et al. (1992) J.
Immunol. 149, 3795-3801. Although B7 has a higher affinity for CTLA4 than for CD28
(Linsley, P.S., et al., (1991) J. Exp. Med 174, 561-569), the function of CTL~;4 is still
5 unknown.
The importance of the B7:CD28/CTLA4 costimulatory pathway has been
demonstrated in vitro and in several in vivo model systems. Blockade of this costimulatory
pathway results in the development of antigen specific tolerance in murine and hl-m~n~
systems (Harding, F.A., et al. (1992) Nature. ~, 607-609; Lenschow, D.J., et al. (1992)
Science. ~, 789-792, Turka, L.A., et al. (1992) Proc. Natl. Acad. Sci. USA. 89, 11102-
11105; Gimmi, C.D., et al. (1993) Proc. Natl. Acad. Sci USA ~, 6586-6590; Boussiotis, V.,
et al. (1993) J: Exp. Med. 178, 1753-1763). Conversely, expression of B7 by B7 negative
murine tumor cells induces T-cell mediated specific immunity accompanied by tumor
rejection and long lasting protection to tumor challenge (Chen, L., et al. (1992) Cell 71,
1093-1102; Townsend, S.E. and Allison, J.P. (1993) Science ~,~, 368-370; Baskar, S., et al.
(1993) Proc. Natl. Acad. Sci. 9Q, 5687-5690.). Therefore, manipulation of the
B7:CD28/CTLA4 pathway offers great potential to stim~ te or suppress immune responses
in hllm~n~
20 ~I-mm~ry of the Inver tion
This invention pertains to isolated nucleic acids encoding novel molecules whichcostimlll~te T cell activation. Preferred cosfim~ tQry molecules include antigens on the
surface of B lymphocytes, professional antigen ples~ in~ cells (e.g., monocytes, dendritic
cells, Langerhan cells) and other cells (e.g., keratinocytes, endothelial cells, astrocytes,
25 fibroblasts, oligodendrocytes) which present antigen to immune cells, and which bind either
CTLA4, CD28, both CTLA4 and CD28 or other known or as yet undefined receptors onimmune cells. Such costim~ tQry molecules are referred to herein as CTLA4/CD28 binding
counter-receptors or B lymphocyte antigens, and are capable of providing costimulation to
activated T cells to thereby induce T cell proliferation and/or cytokine secretion. Preferred B
30 lymphocyte antigens include B7-2 and B7-3 and soluble fragments or derivatives thereof
which bind CTLA4 and/or CD28 and have the ability to inhibit or induce costimulation of
immllne cells. In one embodiment, an isolated nucleic acid which encodes a peptide having
the activity of the hDan B7-2 B Iymphocyte antigen is provided. Preferably, the nucleic
acid is a cDNA molecule having a nucleotide sequence encoding human B7-2, as shown in
35 Figure 8 (SEQ ID NO: 1). In another embodiment, the nucleic acid is a cDNA molecule
having a nucleotide sequence encoding murine B7-2, as shown in Figure 14 (SEQ IDNO:22).
wo 95,03408 ~ ~ ~ 7 0 ~ :L PCT/US94/08423
-3 -
The invention also features nucleic acids which encode a peptide having B7-2 activity
and at least about 50%, more preferably at least about 60% and most preferably at least about
70% homologous with an amino acid sequence shown in Figure 8 (SEQ ID NO:2) or anamino acid sequence shown in Figure 14 (SEQ ID NO:23). Nucleic acids which encode
peptides having B7-2 activity and at least about 80%, more preferably at least about 90%,
more preferably at least about 95% and most preferably at least about 98% or at least about
99% homologous with an amino acid sequence shown in Figure 8 (SEQ ID NO:2) or anamino acid sequence shown in Figure 14 (SEQ ID NO:23) are also within the scope of the
invention. In another embodiment, the peptide having B7-2 activity is encoded by a nucleic
acid which hybridizes under high or low stringency conditions to a nucleic acid which
encodes a peptide having an amino acid sequence of Figure 8 (SEQ ID NO:2) or a peptide
having an amino acid sequence shown in Figure 14 (SEQ ID NO:23).
The invention further pertains to an isolated nucleic acid comprising a nucleotide
sequence encoding a peptide having B7-2 activity and having a length of at least 20 amino
acid residues. Peptides having B7-2 activity and con~i~tin~ of at least 40 amino acid residues
in length, at least 60 amino acid residues in length, at least 80 amino acid residues in length,
at least 100 amino acid residues in length or at least 200 or more amino acid residues in
length are also ~,vithin the scope of this invention. Particularly preferred nucleic acids encode
a peptide having B7-2 activity, a length of at least 20 amino acid residues or more and at least
50% or greater homology (preferably at least 70%) with a sequence shown in Figure 8 (SEQ
ID NO:2).
In one preferred embodiment, the invention features an isolated DNA encoding a
peptide having B7-2 activity and an amino acid sequence represented by a formula:
Xn~Y~Zm
In the formula, Y consists e~enti~lly of amino acid residues 24-245 of the sequence shown in
Figure 8 (SEQ ID NO:2). Xn and Zm are additional amino acid residue(s) linked to Y by an
amide bond. Xn and Zm are arnino acid residues selected from amino acid residuescontiguous to Y in the amino acid sequence shown in Figure 8 (SEQ ID NO:2). Xn is amino
acid residue(s) selected from amino acids contiguous to the amino terminus of Y in the
" sequence shown in Figure 8 (SEQ ID NO:2), i.e., selected from arnino acid residue 23 to 1.
Zm is amino acid residue(s) selected from amino acids contiguous to the carboxy terminus of
Y in the sequence shown in Figure 8 (SEQ ID NO:2), i.e., selected from amino acid residue
246 to 329. According to the formula, n is a number from 0 to 23 (n=0-23) and m is a
number from 0 to 84 (m=0-84). A particularly preferred DNA encodes a peptide having an
WO 95/03408 PCT/US94/08423
9 ~ ~
amino acid sequence represented by the formula Xn-Y-Zm, where Y is amino acid residues
24-245 of the sequence shown in Figure 8 (SEQ ID NO:2) and n=0 and m=0.
The invention also features an isolated DNA encoding a B7-2 fusion protein whichincludes a nucleotide sequence encoding a first peptide having B7-2 activity and a nucleotide
5 sequence encoding a second peptide corresponding to a moiety that alters the solubility,
binding affinity, stability or valency of the f1rst peptide. Preferably, the first peptide having
B7-2 activity includes an extracellular domain portion of the B7-2 protein (e.g., about amino
acid residues 24-245 of the sequence shown in Figure 8 (SEQ ID NO:2)) and the second
peptide is an immunoglobulin constant region, for example, a human Cyl or C~4 domain,
10 including the hinge, CH2 and CH3 region, to produce a B7-2 imml-noglobulin fusion protein
(B7-2Ig)(see Capon et al. (1989) Nature ~1, 525-531 and Capon U.S. 5,116,964).
The nucleic acids obtained in accordance with the present invention can be inserted
into various expression vectors, which in turn direct the synthesis of the corresponding
protein or peptides in a variety of hosts, particularly eucaryotic cells, such as m~mm~ n and
15 insect cell culture, and procaryotic cells such as E. coli. Expression vectors within the scope
of the invention comprise a nucleic acid encoding at least one peptide having the activity of a
novel B Iymphocyte antigen as described herein, and a promoter operably linked to the
nucleic acid sequence. In one embodiment, the t;~ s~ion vector contains a DNA encoding a
peptide having the activity of the B7-2 antigen and a DNA encoding a peptide having the
20 activity of another B Iymphocyte antigen, such as the previously characterized B7 activation
antigen, referred to herein as B7-1. Such t;~ ;s~ion vectors can be used to transfect host
cells to thereby produce proteins and peptides, including fusion proteins, encoded by nucleic
acids as described herein.
Nucleic acid probes useful for assaying a biological sample for the presence of B cells
25 e~les~ing the B Iymphocyte antigens B7-2 and B7-3 are also within the scope of the
invention.
The invention further pertains to isolated peptides having the activity of a novel B
Iymphocyte antigen, including the B7-2 and B7-3 protein antigens. A preferred peptide
having B7-2 activity is produced by recombinant expression and comprises an amino acid
30 sequence shown in Figure 8 (SEQ ID NO: 2). Another preferred peptide having B7-2 activity
comprises an amino acid sequence shown in Figure 14 (SEQ ID NO:23). A particularly
preferred peptide having the activity of the B7-2 antigen includes at least a portion of the
mature form of the protein, such as an extracellular domain portion (e.g., about amino acid
residues 24-245 of SEQ ID NO:2) which can be used to enhance or suppress T-cell mediated
35 immune responses in a subject. Other preferred peptides having B7-2 activity include
peptides having an amino acid sequence represented by a formula:
.
wo gs/03408 2 ~ 6 7 ~ 9 1 PCT/US94/08423
Xn~Y~Zm
In the formula, Y is amino acid residues selected from the group consisting of: amino acid
residues 55-68 ofthe sequence shown in Figure 8 (SEQ ID NO:2); amino acid residues 81-89
ofthe sequence shown in Figure 8 (SEQ ID NO:2); amino acid residues 128-142 ofthe
sequence shown in Figure 8 (SEQ ID NO:2); amino acid residues 160-169 ofthe sequence
shown in Figure 8 (SEQ ID NO:2); amino acid residues 188-200 of the sequence shown in
Figure 8 (SEQ ID NO:2); and amino acid residues 269-282 of the sequence shown in Figure
8 (SEQ ID NO:2). In the formula Xn and Zm are additional amino acid residue(s) lin~ed to
10 Y by an amide bond and are selected from amino acid residues contiguous to Y in the amino
acid sequence shown in Figure 8 (SEQ ID NO:2). Xn is amino acid residue(s) selected from
amino acids contiguous to the amino terminl-~ of Y in the sequence shown in Figure 8 (SEQ
ID NO:2). Zm is arnino acid residue(s) selected from amino acids contiguous to the carboxy
terminus of Y in the sequence shown in Figure 8 (SEQ ID NO:2). According to the formula,
15 n is a number from 0 to 30 (n=0-30) and m is a number from 0 to 30 (m=0-30).
Fusion proteins or hybrid fusion proteins including a peptide having the activity of a
novel B lymphocyte antigen (e.g., B7-2, B7-3) are also featured. For example, a fusion
protein comprising a first peptide which includes an extracellular domain portion of a novel B
lymphocyte antigen fused to second peptide, such as an immunoglobulin constant region, that
alters the solubility, binding affinity, stability and/or valency of the first peptide are provided.
In one embodiment, a fusion protein is produced comprising a first peptide which includes
amino acid residues of an extracellular domain portion of the B7-2 protein joined to a second
pepide which includes amino acid residues of a sequence corresponding to the hinge, CH2
and CH3 regions of C~1 or C~4 to form a B7-2Ig fusion protein. In another embodiment, a
hybrid fusion protein is produced compri~in~ a first peptide which includes an extracellular
domain portion of the B7-1 antigen and an extracellular domain portion of the B7-2 antigen
and a second peptide which includes amino acid residues corresponding to the hinge, CH2
and CH3 of C~1 (see e.g., Linsley et al. (1991) ~ E xp. Med. 1783 :721 -730; Capon et al.
(1989) Nature ~, 525-531, and Capon U.S. 5,116,964).
Isolated peptides and fusion proteins of the invention can be a-lmini~tered to a subject
to either upregulate or inhibit the expression of one or more B Iymphocyte antigens or block
the ligation of one or more B Iymphocyte antigens to their natural ligand on immune cells,
such as T cells, to thereby provide enhancement or su~up~es~ion of cell-mediated immune
responses in vivo.
Another embodiment of the invention provides antibodies, preferably monoclonal
antibodies, specifically reactive with a peptide of a novel B lymphocyte antigen or fusion
protein as described herein. Preferred antibodies are anti-human B7-2 monoclonal antibodies
'
WO 95/03408 7 ~ 6- PCT/US94/08423
produced by hybridoma cells HF2.3D1, HA5.2B7 and HA3.1F9. These hybridoma cells
have been deposited with the American Type Culture Collection at ATCC Accession
No._ (EIF2.3D1), ATCC Accession No. (HA5.2B7)7 and ATCC Accession No.
(HA3.1F9).
A still further aspect of the invention involves the use of the nucleic acids of the r
invention, especially the cDNAs, to enhance the immlln~genicity of a m~mm~ n cell. In
preferred embodiments, the m~mm~ n cell is a tumor cell, such as a sarcoma, a lymphoma,
a melanoma, a neuroblastoma, a leukemia or a carcinoma, or an antigen presenting cell, such
as a macrophage, which is transfected to allow expression of a peptide having the activity of a
novel B lymphocyte antigen of the invention on the surface of the cell. Macrophages that
express a peptide having the activity of a B lymphocyte antigen, such as the B7-2 antigen,
can be used as antigen presentin~ cells, which, when pulsed with an ~ iate pathogen-
related antigen or tumor antigen, enhance T cell activation and immllne stimulation.
~mm~ n cells can be transfected with a suitable expression vector cont~inin~ a
nucleic acid encoding a peptide having the activity of a novel B Iymphocyte antigen, such as
the B7-2 antigen, ex vivo and then introduced into the host m~mm~l, or alternatively, cells
can be transfected with the gene in vivo via gene therapy techniques. For example, the
nucleic acid encoding a peptide having B7-2 activity can be transfected alone, or in
combination with nucleic acids encoding other costimlll~tQry molecules. In enhancing the
immlmogenicity of tumors which do not express Class I or Class II MHC molecules, it may
be beneficial to additionally transfect a~prop,iate class I or II genes into the ms~mm~ n cells
to be transfected with a nucleic acid encoding a peptide having the activity of a B lymphocyte
antigen, as described herein.
The invention also provides methods for inducing both general immunosuppression
and antigen-specific tolerance in a subject by,-for example, blocking the functional
interaction ofthe novel B lymphocyte antigens ofthe invention, e.g., B7-2 and B7-3, to their
natural ligand(s) on T cells or other immllne system cells, to thereby block co-stim~ tion
through the receptor-ligand pair. In one embodiment, inhibitory molecules that can be used
to block the interaction of the natural human B7-2 antigen to its natural ligands (e.g., CTLA4
and CD28) include a soluble peptide having B7-2 binding activity but lacking the ability to
costimlll~te immllne cells, antibodies that block the binding of B7-2 to its ligands and fail to
deliver a co-stimnlsltQry signal (so called "blocking antibodies", such as blocking anti-B7-2
antibodies), B7-2-Ig fusion proteins, which can be produced in accordance with the te~c.hing.
of the present invention, as well as soluble forms of B7-2 receptors~ such as CTLA4Ig or
CD28Ig. Such blocking agents can be used alone or in combination with agents which block
interaction of other costimul~tory molecules with their natural ligands (e.g., anti-B7
antibody). Inhibition of T cell responses and induction of T cell tolerance according to the
WO 95/03408 2 16 7 0 91 PCT/US94/08423
..
-7-
methods described herein may be useful prophylactically, in preventing transplantation
rejection (solid organ, skin and bone marrow) and graft versus host disease, especially in
allogeneic bone marrow transplantation. The methods of the invention may also be useful
fherapeutically, in the treatment of autoimmllne ~ e~es, allergy and allergic reactions,
transplantation rejection, and established graft versus host disease in a subject.
Another aspect of the invention features methods for upregulating immune responses
by delivery of a costim~ tory signal to T cells through use of a stimulatory form of B7-2
antigen, which include soluble, multivalent forms of B7-2 protein, such as a peptide having
B7-2 activity and B7-2 fusion proteins. Delivery of a stimul~tory form of B7-2 in
conjunction with antigen may be useful prophylactically to enhance the efficacy of
vaccination against a variety of pathogens and may also be useful therapeutically to
upregulate an immune response against a particular pathogen during an infection or against a
tumor in a tumor-bearing host.
The invention also features methods of identifying molecules which can inhibit either
the interaction of B Iymphocyte antigens, e.g., B7-2, B7-3, with their receptors or ~ r~le
with intracellular si~n~llin~ through their receptors. Methods for identifying molecules
which can modulate the expression of B lymphocyte antigens on cells are also provided. In
addition, methods for identifying cytokines produced in response to costimlll~tion of T cells
by novel B Iymphocyte antigens are within the scope of the invention.
Brief Description of the nr.~
Figure lA-B are graphic le~l`eSr~ 1 ions of the responses of CD28+ T cells, as
~e~ce~l by 3H-thymidine incorporation or IL-2 secretion, to costim~ tion provided by
either B7 (B7-1) transfected CHO cells (panel a) or syngeneic activated B Iymphocytes
(panel b) cultured in media, anti-CD3 alone, or anti-CD3 in the presence of the following
monoclonal antibodies or recombinant proteins: aB7 (133, anti-B7-1); CTLA4Ig; Fab a
CD28; control Ig fusion protein (isotype control for CTLA4Ig); or aB5 (anti-B5, the isotype
control for anti-B7-1).
Figure 2A-C are graphs of log fluorescence intensity of cell surface expression of
B7-1 on splenic B cells activated with surface immunoglobulin (sIg) crocslinkin~;. The total
(panel a), B7-1 positive (B7-1+, panel b) and B7-1 negative (B7-1-, panel c) activated B cells
" were stained with anti-B7-1 monoclonal antibody (133) and fluoroscein isothiocyanate
(FITC) labeled goat anti-mouse immunoglobulin and analyzed by flow cytometry.
Figure 3A-B are graphic representations of the responses of CD28+ T cells, as
35 assessed by 3H-thymidine incorporation and IL-2 secretion, to costimulation provided by
B7-1+ (panel a) or B7-1- (panel b) activated syngeneic B Iymphocytes cultured in media,
anti-CD3 alone, or anti-CD3 in the presence of the following monoclonal antibodies or
WO 9s/03408 ~16 ~ ~ 91 PCT/USg4/08423
-8-
recombinant proteins: aBB-I (133, anti-B7-1 and anti-B7-3); aB7 (anti-B7-1); CTLA4Ig;
Fab aCD28, control Ig fusion protein or aB5 (anti-B5).
Figure 4 is a graphic lel)lest:lltalion of the cell surface expression of B7-1, B7-3 and
total CTLA4 counter-receptors on fractionated B7-1+ and B7-1- activated B Iymphocytes.
- 5 Figure S is a graphic representation of temporal surface expression of B7-1
(CTLA4Ig and mAbs BB-l and 133), B7-3 (CTLA4Ig and mAb BBl) and B7-2 (CTLA4Ig)
counter-receptors on splenic B cells activated by sIg cro~linking.
Figure 6is a graphic representation of temporal surface expression of B7- 1
- (CTLA4Ig and mAbs BB-l and 133), B7-3 (CTLA4Ig and mAb BBl) and B7-2 (CTLA4Ig)
counter-receptors on splenic B cells activated by MHC class II cros~linkinf~.
Figure 7A-B are graphic representations of the response of CD28+ T cells, as assessed
by 3H-thymidine incorporation and IL-2 secretion, to costimulation provided by syngeneic B
lymphocytes activated by sIg cro~.~linking for 24 hours (panel a) or 48 hours (panel b) and
cultured in media, anti-CD3 alone, or anti-CD3 in the presence of the following monoclonal
antibodies or recombinant protein: aB7(133, anti-B7-1); aBBl (anti-B7-1, anti-B7-3)
CTLA4Ig; Fab aCD28; and aB5(anti-BS).
Figure 8 is the nucleotide and ~le~ cecl amino acid sequence of the human B
lymphocyte antigen B7-2 (hB7-2-clone29).
Figure 9 is a graphic representation of COS cells transfected with control plasmid
(pCDNAI), plasmid expressing B7-1 (B7-1), or plasmid ~;x~ules~ g B7-2 (B7-2) stained with
either control mAb (IgM), anti-B7-1 (mAbs 133 and BB-l), recombinant protein CTLA4Ig,
or isotype matched control Ig protein followed by the applopliate second FITC labelled
immllnoglobulin and analyzed by flow cytometry.
Figure lOA-B show RNA blot analyses of B7-2 ~ lession in unstimulated and anti-
Ig activated human spenic B cells and cell lines (panel a) and human myelomas (panel b).
Figure 11 is a graphic representation of the proliferation of CD28+ T cells, as
~se~ecl by 3H-thymidine incorporation or IL-2 secretion, to submitogenic stiml-l~tion with
phorbol myristic acid (PMA) and COS cells transfected with vector alone or vectors directing
the expression of either B7- 1 or B7-2.
Figure 12 is a graphic representation of the inhibition by mAbs and recombinant
proteins of the proliferation of CD28+ T cells, as assessed by 3H-thymidine incorporation
and IL-2 secretion, to stimulation by PMA and COS cells transfected with vector alone
(vector), or with a vector expressing B7-1 (B7-1) or B7-2 (B7-2). Inhibition studies were
performed with the addition of either no antibody (no mAb), anti-B7 mAb 133 (133), anti-B7
mAb BB-1 (BB1), anti-BS mAb (BS), Fab fragment of anti-CD28 (CD28 Fab), CTLA4Ig
(CTLA4Ig), or Ig control protein (control Ig) to the PMA stimulated COS cell admixed
CD28+ T cells.
wo 95/0340~ 2 ~ 6 7 ~ ~1 PCT/US94/08423
Figure 13 shows the sequence homology between the human B7-2 protein (h B7-2)
deduced amino acid sequence (SEQ ID NO: 2) and the amino acid sequence of both the
human B7-1 protein (h B7-1) (SEQ ID NO: 28 and 29) and the murine B7-1 protein (m B7)
(SEQ ID NO: 30 and 31).
~igure 14 is the nucleotide and deduced amino acid sequence of the murine B7-2
antigen (mB7-2) (SEQ ID NO: 22 and 23).
Figure 15 is a graphic representation of the competitive inhibition of binding of
biotinylated-CTLA4Ig to immobilized B7-2 Ig by B7 family-Ig fusion proteins. The Ig
fusion proteins e~mined as competitors were: full-length B7-2 (hB7.2), full-length B7-1
(hB7.1), the variable region-like domain of B7-2 (hB7.2V) or the constant region-like domain
of B7-2 (hB7.2C).
Figure 16A-B are graphic representations of the competitive inhibition of binding of
biotinylated-B7-1-Ig (panel A) or B7-2-Ig (panel B) to immobilized CTLA4-Ig by increasing
concentrations of unlabelled B7-1-Ig (panel A) or B7-2-Ig (panel B). The experimentally
determined ICso values are indicated in the upper right corner of the panels.
Figure 17 depicts flow cytometric profiles of cells stained with an anti-hB7-2
monoclonal antibody, HA3. lF9. Cells stained with the antibody were CHO cells transfected
to express human B7-2 (CHO-hB7.2), NIH 3T3 cells transfected to express human B7-2
(3T3-hB7.2) and conkol transfected NIH 3T3 cells (3T3-neo). The anti-hB7.2 antibody B70
was used as a positive control.
Figure 18 depicts flow cytometric profiles of cells stained with an anti-hB7-2
monoclonal antibody, HA5.2B7. Cells stained with the antibody were CHO cells transfected
to express human B7-2 (CHO-hB7.2), NIH 3T3 cells transfected to express human B7-2
(3T3-hB7.2) and control transfected NIH 3T3 cells (3T3-neo). The anti-hB7.2 antibody B70
was used as a positive control.
Figure 19 depicts flow cytometric profiles of cells stained with an anti-hB7-2
monoclonal antibody, H~2.3D1. Cells stained with the antibody were CHO cells transfected
to express human B7-2 (CHO-hB7.2), NIH 3T3 cells transfected to express human B7-2
(3T3-hB7.2) and control transfected NIH 3T3 cells (3T3-neo). The anti-hB7.2 antibody B70
was used as a positive control.
Figure 20 is a graphic representation of tumor cell growth (as measured by tumorsize) in mice following transplantation of J558 plasmacytoma cells or J558 plasmacytoma
cells transfected to express B7-1 (J558-B7.1) or B7-2 (JS58-B7.2).
I)etailed Description of the Inv*ntion
In addition to the previously characterized B lymphocyte activation antigen B7
(referred to herein as B7-1), human B lymphocytes express other novel molecules which
WO 95/03408 2 ~ ~ 7 ~ 9 ~ PCT/US94/08423
-10-
costim~ te T cell activation. These costimulatory molecules include antigens on the surface
of B lymphocytes, professional antigen presenting cells (e.g., monocytes, dendritic cells,
Langerhan cells) and other cells (e.g.7 keratinocytes, endothelial cells, astrocytes, fibroblasts,
oligodendrocytes) which present antigen to imml-ne cells, and which bind either CTLA4,
5 CD28, both CTLA4 and CD28 or other known or as yet undefined receptors on immllne
cells. Costim~ tory molecules within the scope of the invention are referred to herein as
CTLA4/CD28 ligands (counter-receptors) or B lymphocyte antigens. Novel B lymphocyte
antigens which provide cotimulation to activated T cells to thereby induce T cell proliferation
and/or cytokine secretion include the B7-2 (human and murine) and the B7-3 antigens
10 described and characterized herein.
The B Iymphocyte antigen B7-2 is expressed by human B cells at about 24 hours
following stimlll~tion with either anti-immunoglobulin or anti-MHC class II monoclonal
antibody. The B7-2 antigen induces detectable IL-2 secretion and T cell proliferation. At
about 48 to 72 hours post activation, human B cells express both B7-1 and a third CTLA4
15 counter-receptor, B7-3, identified by a monoclonal antibody BB-l, which also binds B7-1
(Yokochi, T., et al. (1982) J. Immunol. 128, 823-827). The B7-3 antigen is also expressed on
B7-1 negative activated B cells and can costimlll~te T cell proliferation without detectable
IL-2 production, indicating that the B7-1 and B7-3 molecules are distinct. B7-3 is expressed
on a wide variety of cells including activated B cells, activated monocytes, dendritic cells,
20 Langerhan cells and keratinocytes. At 72 hours post B cell activation, the expression of B7-1
and B7-3 begins to decline. The presence of these costimlll~tory molecules on the surface of
activated B lymphocytes indicates that T cell costimulation is regulated, in part, by the
temporal expression of these molecules following B cell activation.
Accordingly, one aspect of this invention pertains to isolated nucleic acids comprising
25 a nucleotide sequence encoding a novel costiml~ Qry molecule, such as the B lymphocyte
antigen, B7-2, fragments of such nucleic acids, or equivalents thereo The term "nucleic
acid" as used herein is intended to include such fragments or equivalents. The term
"equivalent" is int~ncle~l to include nucleotide sequences encoding functionally equivalent B
lymphocyte antigens or functionally equivalent peptides having an activity of a novel B
30 lymphocyte antigen, i.e., the ability to bind to the natural ligand(s) of the B lymphocyte
antigen on immllne cells, such as CTLA4 and/or CD28 on T cells, and inhibit (e.g., block) or
stimulate (e.g., enhance) immune cell costimulation. Such nucleic acids are considered
equivalents ofthe human B7-2 nucleotide sequence provided in Figure 8 (SEQ ID NO:l) and
the murine B7-2 nucleotide sequence provided in Figure 14 (SEQ ID NO:22) and are within ..
35 the scope of this invention.
In one embodiment, the nucleic acid is a cDNA encoding a peptide having an activity
of the B7-2 B Iymphocyte antigen. Preferably, the nucleic acid is a cDNA molecule
WO 95/03408 ~ 9 L PCT/US94/08423
consisting of at least a portion of a nucleotide sequence encoding human B7-2~ as shown in
Figure 8 (SEQ ID NO: 1) or at least a portion of a nucleotide sequence encoding murine B7-2,
as shown in Figure 14 (SEQ ID NO:22). A preferred portion of the cDNA molecule of
Figure 8 (SEQ ID NO: 1) or Figure 14 (SEQ ID NO:22) includes the coding region of the
molecule.
In another embodiment, the nucleic acid of the invention encodes a peptide having an
activity of B7-2 and comprising an amino acid sequence shown in Figure 8 (SEQ ID NO:2)
or Figure 14 (SEQ ID NO:23). Preferred nucleic acids encode a peptide having B7-2 activity
and at least about 50% homology, more preferably at least about 60% homology and most
preferably at least about 70% homology with an amino acid sequence shown in Figure 8
(SEQ ID NO:2). Nucleic acids which encode peptides having B7-2 activity and at least about
90%, more preferably at least about 95%, and most preferably at least about 98-99%
homologous with a sequence set forth in Figure 8 (SEQ ID NO:2) are also within the scope of
the invention. Homology refers to sequence similarity between two peptides having the
activity of a novel B lymphocyte antigen, such as B7-2, or between two nucleic acid
molecules. Homology can be rlett~rmined by comp~ring a position in each sequence which
may be aligned for purposes of comparison. When a position in the compared sequences is
occupied by the same nucleotide base or amino acid, then the molecules are homologous at
that position. A degree (or percentage) of homology between sequences is a function of the
number of m~tc.hing or homologous positions shared by the sequences.
Another aspect of the invention provides a nucleic acid which hybridizes under high
or low skingency conditions to a nucleic acid which encodes a peptide having all or a portion
of an amino acid sequence shown in Figure 8 (SEQ ID NO:2) or a peptide having all or a
portion of an amino acid sequence shown in Figure 14 (SEQ ID NO:23). Appropriatestringency conditions which promote DNA hybridization, for example, 6.0 x sodiumchloride/sodium cikate (SSC) at about 45C, followed by a wash of 2.0 x SSC at 50C are
known to those skilled in the art or can be found in Current Protocols in Molecular Biology,
John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the salt concenkation in the
wash step can be selected from a low stringency of about 2.0 x SSC at 50C to a high
stringency of about 0.2 x SSC at 50C. In addition, the temperature in the wash step can be
increased from low skingency conditions at room temperature, about 22C to high stringency
conditions, at about 65C.
Isolated nucleic acids encoding a peptide having an activity of a novel B lymphocyte
antigen, as described herein, and having a sequence which differs from nucleotide sequence
shown in Figure 8 (SEQ ID NO:l) or Figure 14 (SEQ ID NO:22) due to degeneracy in the
genetic code are also within the scope of the invention. Such nucleic acids encode
functionally equivalent peptides (e.g., a peptide having B7-2 activity) but differ in sequence
WO 95/03408 PCTIUS94/08423
~16709`1 ~
-12-
from the sequence of Figure 8 or Figure 14 due to degeneracy in the genetic code. For
example, a number of amino acids are clecign~ted by more than one triplet. Codons that
specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for
histidine) may occur due to degeneracy in the genetic code. As one example, bNA sequence
5 polymorphisms within the nucleotide sequence of a B7-2 (especially those within the third
base of a codon) may result in "silent" mutations in the DNA which do not affect the amino
acid encoded. However, it is expected that DNA sequence polymorphisms that do lead to
changes in the amino acid sequences of the B7-2 antigen will exist within a population. It
will be appreciated by one skilled in the art that these variations in one or more nucleotides
10 (up to about 3-4% of the nucleotides) of the nucleic acids encoding peptides having the
activity of a novel B lymphocyte antigen may exist among individuals within a population
due to natural allelic variation. Any and all such nucleotide variations and resnltin~ amino
acid polymorphisms are within the scope of the invention. Furthermore, there may be one or
more isoforms or related, cross-reacting family members of the novel B Iymphocyte antigens
15 described herein. Such isoforms or family members are defined as proteins related in
- function and amino acid sequence to a B lymphocyte antigen (e.g., the B7-2 antigen), but
encoded by genes at dirrelenl loci.
A "fragment" of a nucleic acid encoding a novel B lymphocyte antigen is defined as a
nucleotide sequence having fewer nucleotides than the nucleotide sequence encoding the
20 entire amino acid sequence of the B lymphocyte antigen and which encodes a peptide having
an activity of the B Iymphocyte antigen (i.e., the ability to bind to the natural ligand(s) of the
B lymphocyte antigen on immllne cells, such as CTLA4 and/or CD28 on T cells and either
stim--l~te or inhibit immllne cell costimnl~tion). Thus, a peptide having B7-2 activity binds
CTLA4 and/or CD28 and stimulates or inhibits a T cell mediated immune response, as
25 evidenced by, for example, cytokine production and/or T cell proliferation by T cells that
- have received a primary activation signal. In one embodiment, the nucleic acid fragment
encodes a peptide of the B7-2 antigen which retains the ability of the antigen to bind CTLA4
and/or CD28 and deliver a costim~ tory signal to T Iymphocytes. In another embodiment,
the nucleic acid fragment encodes a peptide including an extracellular portion of the human
30 B7-2 antigen (e.g., approximately amino acid residues 24-245 of the sequence provided in
Figure 8 (SEQ ID NO:2)) which can be used to bind CTLA4 and/or CD28 and, in
monovalent form, inhibit costimulation, or in multivalent form, induce or enhance
costim~ tion.
Preferred nucleic acid fragments encode peptides of at least 20 amino acid residues in
35 length, preferably at least 40 amino acid residues and length, and more preferably at least 60
amino acid residues in length. Nucleic acid fragments which encode peptides of at least 80
amino acid residues in length, at least 100 amino acid residues in length, and at least 200 or
WO 9~/03408 2 ~ 6 7 0 91 PCT/US94/08423
-13-
more amino acids in length are also within the scope of the invention. Particularly plef~ d
nucleic acid fragments encode a peptide having the activity of human B7-2 and an amino acid
sequence represented by a formula:
Xn-Y-Zm
In the fomula, Y comprises amino acid residues 24-245 of the sequence shown in Figure 8
(SEQ ID NO:2). Xn and Zm are additional amino acid residue(s) linked to Y by an amide
bond. Xn and Zm are selected from amino acid residues contiguous to Y in the amino acid
sequence shown in Figure 8 (SEQ ID NO:2). In the formula, Xn is amino acid residue(s)
selected from amino acids contiguous to the amino terminus of Y in the sequence shown in
Figure 8 (SEQ ID NO:2), i.e., from amino acid residue 23 to 1. Zm is amino acid residue(s)
selected from amino acids contiguous to the carboxy terminlls of Y in the sequence shown in
Figure 8 (SEQ ID NO:2), i.e., from amino acid residue 246 to 329. In addition, in the
formula, n is a number from 0 to 23 (n=0-23) and m is a number from 0 to 84 (m=0-84). A
particularly ~rer~lled peptide has an amino acid sequence represented by the formula Xn-Y-
Zm as above, where n=0 and m=0.
Nucleic acid fragments within the scope of the invention include those capable of
hybridizing with nucleic acid from other animal species for use in screening protocols to
detect novel proteins that are cross-reactive with the B lymphocyte antigens described herein.
These and other fr~gment~ are described in detail herein. Generally, the nucleic acid
encoding a fragment of a B lymphocyte antigen will be selected from the bases coding for the
mature protein, however, in some instances it may be desirable to select all or part of a
fragment or fragments from the leader sequence or non-coding portion of a nucleotide
sequence. Nucleic acids within the scope of the invention may also contain linker sequences,
modified restriction endonuclease sites and other sequences useful for molecular cloning,
t;x~l~ssion or purification of recombinant protein or fragments thereof. These and other
modifications of nucleic acid sequences are described in further detail herein.
A nucleic acid encoding a peptide having an activity of a novel B lymphocyte antigen,
such as the B7-2 antigen, may be obtained from mRNA present in activated B lymphocytes.
It should also be possible to obtain nucleic acid sequences encoding B lymphocyte antigens
from B cell genomic DNA. For example, the gene encoding the B7-2 antigen can be cloned
from either a cDNA or a genomic library in accordance with protocols herein described. A
, cDNA encoding the B7-2 antigen can be obtained by isolating total mRNA from an
35 ~propl,ate cell line. Double stranded cDNAs can then prepared from the total mRNA.
Subsequently, the cDNAs can be inserted into a suitable plasmid or viral (e.g., bacteriophage)
vector using any one of a number of known techniques. Genes encoding novel B lymphocyte
WO 95/03408 ~ 7 ~ ~ ~ PCT/US94/08423
-14-
antigens can also be cloned using established polymerase chain reaction techniques in
accordance with the nucleotide sequence information provided by the invention. The nucleic
acids of the invention can be DNA or RNA. A p,~erell~ed nucleic acid is a cDNA encoding the
human B7-2 antigen having the sequence depicted in Figure 8 (SEQ ID NO:1). Another
5 preferred nucleic acid is a cDNA encoding the murine B7-2 antigen having the sequence
shown on Figure 14 (SEQ ID NO:22).
This invention further pertains to ~x~les~ion vectors cont~ininp a nucleic acid
encoding at least one peptide having the activity of a novel B lymphocyte antigen, as
described herein, operably linked to at least one regulatory sequence. "Operably linked" is
10 inten~le~l to mean that the nucleotide acid sequence is linked to a regulatory sequence in a
manner which allows t;~,c;s~ion of the nucleotide sequence (e.g., in cis or trans). Regulatory
sequences are art-recognized and are selected to direct expression of the desired protein in an
appropriate host cell. Accordingly, the term regulatory sequence includes promoters,
enhancers and other expression control elements. Such regulatory sequences are known to
15 those skilled in the art or one described in Goeddel, Gene Expression Technology: Methods
in Enzymology 185, ~c~ tnic Press, San Diego, CA (1990). It should be understood that the
design of the expression vector may depend on such factors as the choice of the host cell to
be transfected and/or the type of protein desired to be expressed. In one embodiment, the
.lession vector includes a nucleic acid encoding at least a portion of the B7-2 protein, such
20 as an extracellular domain portion. In another embodiment, the expression vector includes a
DNA encoding a peptide having an activity of the B7-2 antigen and a DNA encoding a
peptide having an activity of another B lymphocyte antigen, such as B7-1. cDNAs encoding
the human B7-1 and mouse B7-1 antigens are shown in SEQ ID NO:28 and SEQ ID NO:30,
respectively. The ~iedl~cecl amino acid sequences of these antigens are also shown in SEQ ID
25 NO:29 and SEQ ID NO:3 1, respectively. Such expression vectors can be used to transfect
cells to thereby produce proteins or peptides, including fusion proteins or peptides encoded
by nucleic acid sequences as described herein. These and other embodiments are described in
further detail herein.
The invention also features methods of producing peptides having an activity of a
30 novel B lymphocyte antigen. For example, a host cell transfected with a nucleic acid vector
directing ~ ,ession of a nucleotide sequence encoding a peptide having an activity of the
B7-2 protein can be cultured in a medium under applopliate conditions to allow expression of
the peptide to occur. In addition, one or more expression vectors cont~inin~ DNA encoding a
- peptide having an activity of B7-2 and DNA encoding another peptide, such as a peptide
35 having an activity of a second B Iymphocyte antigen (e.g., B7-1, B7-3) can be used to
transfect a host cell to coexpress these peptides or produce fusion proteins or peptides. In one
embodiment, a recombinant ~ ssion vector cont~ininp DNA encoding a B7-2 fusion
WO 95/03408 PCT/US94/08423
7 0 9 1
-15-
protein is produced. A B7-2 fusion protein can be produced by recombinant expression of a
nucleotide sequence encoding a first peptide having B7-2 activity and a nucleotide sequence
encoding second peptide corresponding to a moiety that alters the solubility, affinity, stability
or valency of the first peptide, for example, an immunoglobulin constant region. Preferably,
- 5 the first peptide consists of a portion of the extracellular domain of the human B7-2 antigen
(e.g., approximately amino acid residues 24-245 of the sequence shown in Figure 8 (SEQ ID
NO:2)). The second peptide can include an immnnoglobulin constant region, for example, a
human C~1 domain or C~4 domain (e.g., the hinge, CH2 and CH3 regions of human Ig~
or human IgC~4, see e.g., Capon et al. US 5,116,964, incol~o~led herein by reference). A
reslllting B7-2Ig fusion protein may have altered B7-2 solubility, binding affinity, stability
and/or valency (i.e., the number of binding sites available per molecule) and may increase the
efficiency of protein purification. Fusion proteins and peptides produced by recombinant
technique may be secreted and isolated from a mixture of cells and medium cont~ining the
protein or peptide. Alternatively, the protein or peptide may be retained cytoplasmically and
the cells harvested, lysed and the protein isolated. A cell culture typically includes host cells,
media and other byproducts. Suitable mediums for cell culture are well known in the art.
Protein and peptides can be isolated from cell culture medium, host cells, or both using
techniques known in the art for purifying proteins and peptides. Techniques for transfecting
host cells and purifying proteins and peptides are described in further detail herein.
Particularly preferred human B7-2Ig fusion proteins include the extracellular domain
portion or variable region-like domain of human B7-2 coupled to an immunoglobulin
constant region. The immllnoglobulin constant region may contain genetic modifications
which reduce or elimin~te effector activity inherent in the immlmnglobulin structure. For
example, DNA encoding the extracellular portion of human B7-2 (hB7-2), as well as DNA
encoding the variable region-like domain of human B7-2 (hB7.2V) or the constant region-
like domain of human B7-2 (hB7.2C) can be joined to DNA encoding the hinge, CH2 and
CH3 regions of human IgC~1 and/or IgCy4 modified by site directed mutagenesis. The
Lion and chara~;le. ;,~ ion of these fusion proteins is described in detail in Example 7.
Transfected cells which express peptides having an activity of one or more B
lymphocyte antigens (e.g., B7-2, B7-3) on the surface of the cell are also within the scope of
this invention. In one embodiment, a host cell such as a COS cell is transfected with an
~x~lts~ion vector directing the ~ c;s~ion of a peptide having B7-2 activity on the surface of
the cell. Such a transfected host cell can be used in methods of identifying molecules which
inhibit binding of B7-2 to its counter-receptor on T cells or which interfere with intracellular
~i~n~ling of costim~ tion to T cells in response to B7-2 interaction. In another embodiment,
a tumor cell such as a sarcoma, a melanoma, a lellkemi~ a lymphoma, a carcinoma or a
neuroblastoma is transfected with an e~ ion vector directing the expression of at least one
WO 95/03408 21~ PCT/US94/08423
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peptide having the activity of a novel B lymphocyte antigen on the surface of the tumor cell.
In some instances, it may be beneficial to transfect a tumor cell to coexpress major
histocompatibility complex (MHC) proteins, for example MHC class II a and ,B chain
proteins or an MHC class I a chain protein, and, if necessary, a ~2 microglobulin protein.
5 Such transfected tumor cells can be used to induce tumor immllnity in a subject. These and
other embo-liment~ are described in further detail herein.
The nucleic acid sequences of the invention can also be chemically synthesi7.?d using
standard techniques. Various methods of chemically synthesizing polydeoxynucleotides are
known, including solid-phase synthesis which, like peptide synthesis, has been fully
10 automated in commercially available DNA synthesi7~rs (See e.g., Itakura et al. U.S. Patent
No. 4,598,049; Caruthers et al. U.S. Patent No. 4,458,066, and Itakura U.S. Patent Nos.
4,401,796 and 4,373,071, incorporated by reference herein).
Another aspect of the invention pertains to isolated peptides having an activity of a
novel B lymphocyte antigen (e.g., B7-2, B7-3). A peptide having an activity of a B
15 Iymphocyte antigen may differ in amino acid sequence from the B Iymphocyte antigen, such
as the human B7-2 sequence depicted in Figure 8 (SEQ ID NO:2), or murine B7-2 sequence
depicted in Figure 14 (SEQ ID NO:22), but such differences result in a peptide which
functions in the same or similar manner as the B Iymphocyte antigen or which has the same
or similar characteristics of the B Iymphocyte antigen. For example, a peptide having an
20 activity of the B7-2 protein is defined herein as a peptide having the ability to bind to the
natural ligand(s) of the B7-2 protein on immllne cells, such as CLTA4 and/or CD28 on T
cells and either stimulate or inhibit immune cell costimlll~tion. Thus, a peptide having B7-2
activity binds CTLA4 and/or CD28 and stimlll~tes or inhibits a T cell mediated immune
response (as evidenced by, for example, cytokine production andlor proliferation by T cells
25 that have received a primary activation signal). One embodiment provides a peptide having
B7-2 binding activity, but lacking the ability to deliver a costim~ tory signal to T cells.
Such a peptide can be used to inhibit or block T cell proliferation and/or cytokine secretion in
a subject. Alternatively, a peptide having both B7-2 binding activity and the ability to deliver
a costimlll~tory signal to T cells is used to stimulate or enhance T cell proliferation and/or
30 cytokine secretion in a subject. Various modifications of the B7-2 protein to produce these
and other functionally equivalent peptides are described in detail herein. The term "peptide"
as used herein, refers to peptides, proteins and polypeptides.
A peptide can be produced by modification of the amino acid sequence of the human
B7-2 protein shown in Figure 8 (SEQ ID NO:2) or the murine B7-2 protein shown in Figure
35 14 (SEQ ID NO:23), such as a substitution, addition or deletion of an arnino acid residue
which is not directly involved in the function of B7-2 (i.e., the ability of B7-2 to bind CTLA4
and/or CD28 and/or stim~ te or inhibit T cell costimlll~tion). Peptides of the invention are
WO 95/03408 21 6 ~ 0 91 PCT/US94/084Z3
-17-
typically at least 20 amino acid residues in length, preferably at least 40 amino acid residues
in length, and most preferably 60 amino acid residues in length. Peptides having B7-2
activity and including at least 80 amino acid residues in length, at least 100 arnino acid
residues in length, or at least 200 or more amino acid residues in length are also within the
- 5 scope of the invention. A pl~f~lled peptide includes an extracellular domain portion of the
human B7-2 antigen (e.g., about amino acid residues 24-245 of the sequence shown in Figure
8 (SEQ ID NO:2). Other preferred peptides have an amino acid sequence represented by a
formula:
1 0 Xn~Y~Zm
where Y is amino acid residues selected from the group consisting of: amino acid residues
55-68 of the sequence shown in Figure 8 (SEQ ID NO:2); amino acid residues 81 -89 of the
sequence shown in Figure 8 (SEQ ID NO:2), amino acid residues 128-142 of the sequence
shown in Figure 8 (SEQ ID NO:2), amino acid residues 160-169 of the sequence shown in
Figure 8 (SEQ ID NO:2); arnino acid residues 188-200 of the sequence shown in Figure 8
(SEQ ID NO:2); and amino acid residues 269-282 of the sequence shown in Figure 8 (SEQ
ID NO:2). In the formula, Xn and Zm are additional amino acid residues linked to Y by an
amide bond. Xn and Zm are amino acid residues selected from amino acids contiguous to Y
in the amino acid sequence shown in Figure 8 (SEQ ID NO:2). Xn is amino acid residues
selected from amino acids contiguous to the amino terminus of Y in the sequence shown in
Figure 8 (SEQ ID NO:2). Zm is amino acid residues selected from amino acids contiguous to
the carboxy terminl~c of Y in the sequence shown in Figure 8 (SEQ ID NO:2). According to
the formula, n is a number from 0 to 30 (n=0-30) and m is a number from 0 to 30 (m=0-30).
A particularly preferred peptide has an amino acid sequence represented by the formula Xn-
Y~Zm~ where n=0 and m=0.
Another embodiment of the invention provides a subst~nti~lly pure ~ep~Lion of a
peptide having an activity of a novel B lymphocyte antigen such as B7-2 or B7-3. Such a
,~l~dldLion is subst~nti~lly free of proteins and peptides with which the peptide naturally
occurs in a cell or with which it naturally occurs when secreted by a cell.
The term "isolated" as used throughout this application refers to a nucleic acid,
protein or peptide having an activity of a novel B Iymphocyte antigen, such as B7-2,
subst~nti~lly free of cellular material or culture medium when produced by recombinant
DNA techniques, or chemical precursors or other chemicals when chemic~lly synthesized.
An isolated nucleic acid is also free of sequences which naturally flank the nucleic acid (i.e.,
sequences located at the 5' and 3' ends of the nucleic acid) in the organism from which the
nucleic acid is derived.
WO 9',/03408 ~ 9 1 PCT/US94/08423
-18-
These and other aspects of this invention are described in detail in the following
- subsections.
k Isolation of Nucleic Acid From Cell T inec
S Suitable cells for use in isolating nucleic acids encoding peptides having an activity of
a novel B lymphocyte antigen include cells capable of producing mRNA coding for B
lymphocyte antigens (e.g., B7-1, B7-2, B7-3) and ~ Jpliately translating the mRNA into
the corresponding protein. One source of mRNA is normal human splenic B cells, either
resting or activated by treatment with an anti-immunoglobulin antibody or an anti-MHC class
II antibody, or from subsets of neoplastic B cells. Expression ofthe human B7-2 antigen is
cletect~hle in resting B cells and in activated B cells, with mRNA levels increasing 4-fold
from resting levels following stim~ tion. Total cellular RNA can be obtained using standard
techniques from resting or activated B cells during these intervals and utilized in the
construction of a cDNA library.
In addition, various subsets of neoplastic B cells may express B7-2 and B7-3 and can
~ltern~tively serve as a source of the mRNA for construction of a cDNA library. For
example, tumor cells isolated from patients with non-Hodgkins Iymphoma express B7-1
mRNA. B cells from nodular, poorly differenti~tt-d lymphoma (NPDL), diffuse large cell
lymphoma (LCL) and Burkitt's lymphoma cell lines are also suitable sources of human B7-1
mRNA and, potentially B7-2 and B7-3 mRNA. Myelomas generally express B7-2, but not
B7-1 mRNA, and, thus can provide a source of B7-2 mRNA. The Burkitt's Iymphoma cell
line Raji is one source of B Iymphocyte antigen mRNA. Preferably, B7-2 mRNA is obtained
from a population of both resting and activated normal human B cells. Activated B cells can
be obtained by stim~ tion over a broad spectrum of time (e.g., from minlltes to days) with,
for example, an anti-immllnoglobulin antibody or an anti-MCH class II antibody.
Tr. T~olation of mRNA ~nd Con~truction of cDNA L ibr~ry
Total cellular mRNA can be isolated by a variety of techniques, e.g., by using the
guanidinium-thiocyanate extraction procedure of Chirgwin et al., Biochemistry 18, 5294-
5299 (1979). According to this method, Poly (A+) mRNA is prepared and purified for use in
a cDNA library construction using oligo (dT) cellulose selection. cDNA is then synthe~i7~d
from the poly(A+) RNA using oligo(dT) priming and reverse transcriptase. Moloney MLV
reverse transcriptase (available from Gibco/BRL, Bethesda, MD) or AMV reverse
transcriptase (available from Seikagaku America, Inc., St. Petersburg, FL) are preferably
employed.
Following reverse transcription, the mRNA/DNA hybrid molecule is converted to
double stranded DNA using conventional techniques and incorporated into a suitable vector.
~WO 95/03408 21~ ~ ~ 9 ~ PCTIUS94/08423
-19-
The ex~ llents herein employed E. coli DNA polymerase I and ribonuclease H in the
conversion to double stranded cDNA.
Cloning of the cDNAs can be accomplished using any of the conventional techniques
for joining double stranded DNA with an appropriate vector. The use of synthetic adaptors is
particularly preferred, since it alleviates the possibility of cleavage of the cDNA with
restriction enzyme prior to cloning. Using this method, non-self complementary~ kin~efl
adaptors are added to the DNA prior to ligation with the vector. Virtually any adaptor can be
employed. As set forth in more detail in the examples below, non-self complementary BstXI
adaptors are preferably added to the cDNA for cloning, for ligation into a pCDM8 vector
prepared for cloning by digestion with BstXI.
Eucaryotic cDNA can be e~ ,ssed when placed in the sense orientation in a vectorthat supplies an a~plo~l;ate eucaryotic promoter and origin of replication and other elements
including enhancers, splice acceptors and/or donor sequences and polyadenylation signals.
The cDNAs of the present invention are placed in suitable vectors cont~ininp: a eucaryotic
promoter, an origin of replication functional in E. coli, an SV40 origin of replication which
allows growth in COS cells, and a cDNA insertion site. Suitable vectors include ~H3 (Seed
and ~ruffo, Proc. Natl. Acad. Sci., 84:3365-3369 (1987)),7~H3m (Aruffo and Seed, Proc.
Natl. Acad. Sci., 84:8573-8577 (1987)), pCDM7 and pCDM8 (Seed, Nature, 329:840-841
(1987), with the pCDM8 vector being particularly ~lere,-ed (available commercially from
Invitrogen, San Diego, CA).
TTT Tr~n~fection of Host Cells and Screenin~ for Novel B T,ymphocyte Activation Anti~ens
The thus prepared cDNA library is then used to clone the gene of interest by
t;x~essiorl cloning techniques. A basic expression cloning technique has been described by
Seed and Aruffo, Proc. Natl. Acad. Sci. USA, 84:3365-3369 (1987) and Aruffo and Seed,
Proc. Natl. Acad. Sci. USA, 84:8573-8577 (1987), although modifications to this technique
may be n~cess~ry.
According to one embodiment, plasmid DNA is introduced into a simian COS cell
line (Gluzman, Cell 23: 175 (1981)) by known methods of transfection (e.g., DEAE-Dextran)
and allowed to replicate and express the cDNA inserts. The transfectants expressing B7-1
antigen are depleted with an anti-B7-1 monoclonal antibody (e.g., 133 and B1.1) and anti-
murine IgG and IgM coated immunomagnetic beads. Transfectants expressing human B7-2
antigen can be positively selected by reacting the transfectants with the fusion proteins
CTLA4Ig and CD28Ig, followed by panning with anti-human Ig antibody coated plates.
Although human CTLA4Ig and CD28Ig fusion proteins were used in the examples described
herein, given the cross-species reactivity between B7-1 and, for example murine B7-1, it can
be expected that other fusion proteins reactive with another cross-reactive species could be
WO 95/03408 PCT/US94/08423
2 ~
-20-
used. After p~nning! episomal DNA is recovered from the panned cells and transformed into
a competent bacterial host, preferably E. coli. Plasmid DNA is subsequently reintroduced
into COS cells and the cycle of expression and panning repeated at least two times. After the
final cycle, plasmid DNA is prepared from individual colonies, transfected into COS cells
and analyzed for expression of novel B Iymphocyte antigens by indirect immunofluorescence
with, for example, CTLA4Ig and CD28Ig.
IV. Sequencin~ of Novel R T~y~hoc,vte ~nti~ens
Plasmids are prepared from those clones which are strongly reactive with the
CTLA4Ig and/or CD28Ig. These plasmids are then sequenced. Any of the conventional
sequencing techniques suitable for sequencing tracts of DNA about 1.0 kb or larger can be
employed.
As described in Example 4, a human B7-2 clone (clone29) was obtained cont~inin~ an
insert of 1,120 base pairs with a single long open reading frame of 987 nucleotides and
- 15 approximately 27 nucleotides of 3' noncoding sequences (Figure 8, SEQ ID NO: 1). The
predicted amino acid sequence encoded by the open reading frame of the protein is shown
below the nucleotide sequence in Figure 8. The encoded human B7-2 protein, is predicted to
be 329 amino acid residues in length (SEQ ID NO:2). This protein sequence exhibits many
features common to other type I Ig ~u~clr~llily membrane proteins. Protein translation is
predicted to begin at the methionine codon (ATG, nucleotides 107 to 109) based on the DNA
homology in this region with the consensus eucaryotic translation initiation site (see Kozak,
M. (1987) Nucl. ~4cids Res. 15:8125-8148). The amino terminll~ ofthe B7-2 protein (amino
acids 1 to 23) has the characteristics of a secretory signal peptide with a predicted cleavage
between the ~l~nin~s at positions 23 and 24 (von Heijne (1987) Nucl. Acids Res. 14:4683).
Processing at this site would result in a B7-2 membrane bound protein of 306 amino acids
having an unmodified molecular weight of approximately 34 kDa. This protein would
consist of an approximate extracellular Ig superfamily V and C like domains of from about
amino acid residue 24 to 245, a hydrophobic tr~n~m~.mbrane domain of from about amino
acid residue 246 to 268, and a long cytoplasmic domain of from about amino acid residue
269 to 329. The homologies to the Ig ~u~lr~llily are due to the two contiguous Ig-like
~lom~in~ in the extracellular region bound by the cysteines at positions 40 to 110 and 157 to
218. The extracellular domain also contains eight potential N-linked glycosylation sites and,
like B7-1, is probably glycosylated. Glycosylation of the human B7-2 protein may increase
the molecular weight to about 50-70 kDa. The cytoplasmic domain of human B7-2, while
somewhat longer than B7-1, contains a common region of multiple cysteines followed by
positively charged amino acids which pl~ulnably function as sign~ling or regulatory
domains within an antigen-presenting cell (APC). Comparison of both the nucleotide and
WO 9~/0340~ 21~ i~ O ~ 3 PCT/USg4/08423
-21-
amino acid sequences of the human B7-2 with the GenBank and EMBL ~l~t~b~ces yielded
significant homology (about 26% amino acid sequence identity) with human B7-1. Since
human B7-1, human B7-2 and murine B7-1 all bind to human CTLA4 and CD28, the
homologous amino acids probably represent those necessary to comprise a CTLA4 or CD28
- 5 binding sequence. Æ. coli transfected with a vector cont~ining a cDNA insert encoding
human B7-2 (clone 29) was deposited with the American Type Culture Collection (ATCC)
on July 26, 1993 as Accession No. 69357.
V. Clo~in~ Novel P~ Lyrr~rhocyte ~nt~gens from Other M~mm~lian Species
The present invention is not limited to human nucleic acid molecules and
con~ plates that novel B lymphocyte antigen homologues from other m~mm~ n species
that express B lymphocyte antigens can be cloned and sequenced using the techniques
described herein. B lymphocyte antigens isolated for one species (e.g., hllm~n~) which
exhibit cross-species reactivity may be used to modify T cell mediated immune responses in a
different species (e.g., mice). Isolation of cDNA clones from other species can also be
accomplished using human cDNA inserts, such as human B7-2 cDNA, as hybridizationprobes.
As described in Example 6, a murine B7-2 clone (mB7-2, clone 4) was obtained
cont~ining an insert of 1,163 base pairs with a single long open reading frame of 927
nucleotides and approximately 126 nucleotides of 3' noncoding sequences (Figure 14, SEQ
ID NO:22). The predicted amino acid sequence encoded by the open reading frame of the
protein is shown below the nucleotide sequence in ~igure 14. The encoded murine B7-2
protein, is predicted to be 309 amino acid residues in length (SEQ ID NO:23). This protein
sequence exhibits many features common to other type I Ig superfamily membrane proteins.
Protein translation is predicted to begin at the methionine codon (ATG, nucleotides 111 to
113) based on the DNA homology in this region with the consensus eucaryotic translation
initiation site (see Kozak, M. (1987) Nucl. Acids Res. 15:8125-8148). The amino terminus of
the murine B7-2 protein (amino acids 1 to 23) has the characteristics of a secretory signal
peptide with a predicted cleavage between the alanine at position 23 and the valine at position
24 (von Heijne (1987) Nucl. Acids Res. 14:4683). Processing at this site would result in a
murine B7-2 membrane bound protein of 286 amino acids having an unmodified molecular
weight of approximately 32 kDa. This protein would consist of an approximate extracellular
Ig superfamily V and C like domains of from about amino acid residue 24 to 246, a
hydrophobic transmembrane domain of from about amino acid residue 247 to 265, and a long
cytoplasmic domain of from about amino acid residue 266 to 309. The homologies to the Ig
superfarnily are due to the two contiguous Ig-like domains in the extracellular region bound
by the cysteines at positions 40 to 110 and 157 to 216. The extracellular domain also
WO 95/03408 ~ PCTIUSs4/08423
-22-
contains nine potential N-linked glycosylation sites and, like murine B7-1, is probably
glycosylated. Glycosylation of the murine B7-2 protein may increase the molecular weight to
about 50-70 kDa. The cytoplasmic domain of murine B7-2 contains a common region which
has a cysteine followed by positively charged amino acids which presumably functions as
S ~i~n~linf~ or regulatory domain within an APC. Comparison of both the nucleotide and
amino acid sequences of murine B7-2 with the GenBank and EMBL cl~t~h~ees yieldedsignificant homology (about 26% amino acid sequence identity) with human and murine B7-
1. Murine B7-2 exhibits about 50% identity and 67% similarity with its human homologue,
hB7-2. E. coli (DH106/p3) transfected with a vector (plasmid pmBx4) cont~inin~ a cDNA
10 insert encoding murine B7-2 (clone 4) was deposited with the American Type Culture
Collection (ATCC) on August 18, 1993 as Accession No. 69388.
Nucleic acids which encode novel B lymphocyte antigens from other species, such as
the murine B7-2, can be used to generate either transgenic ~nim~l.c or "knock out" ~nim~l~
which, in turn, are useful in the development and screening of therapeutically useful reagents.
15 A tr~n~genic animal (e.g., a mouse) is an animal having cells that contain a transgene, which
transgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an
embryonic stage. A transgene is a DNA which is integrated into the genome of a cell from
which a tr~n~genic animal develops. In one embodiment, murine B7-2 cDNA or an
al)~.o~.liate sequence thereof can be used to clone genomic B7-2 in accordance with
20 established techniques and the genomic sequences used to generate transgenic ~nim~ls that
contain cells which express B7-2 protein. Methods for generating transgenic ~nim~
particularly ~nim~ls such as mice, have become conventional in the art and are described, for
example, in U.S. Patent Nos. 4,736,866 and 4,870,009. Typically, particular cells would be
targeted for B7-2 transgene incorporation with tissue specific enhancers, which could result
25 in T cell costimulation and enh~n~e~l T cell proliferation and autoimmllnity. Transgenic
~nim~l~ that include a copy of a B7-2 transgene introduced into the germ line of the animal at
an embryonic stage can be used to examine the effect of increased B7 t;x~ ssion. Such
~nim~l~ can be used as tester ~nim~l~ for reagents thought to confer protection from, for
example, autoilll,llulle disease. In accordance with this facet of the invention, an animal is
30 keated with the reagent and a reduced incidence of the fli~eZI'~Ç7 compared to untreated
~nim~l~ bearing the transgene, would indicate a potential therapeutic intervention for the
dlsease.
Alternatively, the non-human homologues of B7-2 can be used to construct a B7-2
"knock out" animal which has a defective or altered B7-2 gene as a result of homologous
35 recombination between the endogenous B7-2 gene and altered B7-2 genomic DNA
introduced into an embryonic cell of the animal. For example, murine B7-2 cDNA can be
used to clone genomic B7-2 in accordance with established techniques. A portion of the
WO 95/03408 ~ ~ 6 7 0 91 PCT/US94/08423
. .
-23-
genomic B7-2 DNA (e.g., such as an exon which encodes an extracellular domain) can be
deleted or replaced with another gene, such as a gene encoding a selectable marker which can
be used to monitor integration. Typically, several kilobases of unaltered fl~nkin~ DNA (both
at the 5' and 3' ends) are included in the vector (see e.g., Thomas, K.R. and Capecchi, M. R.
(1987) Cell ~1:503 for a description of homologous recombination vectors). The vector is
introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the
introduced DNA has homologously recombined with the endogçnous DNA are selected (see
e.g., Li, E. et al. (1992) Cell 69:915). The selected cells are then injected into a blastocyst of
an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. inTeratocarcinomas and ~mbryonic Stem Cells: A Practical ~pproach, E.J. Robertson, ed.
(IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted into a suitable
pseudopregnant female foster animal and the embryo brought to term to create a "knock out"
animal. Progeny harbouring the homologously recombined DNA in their germ cells can be
identified by standard techniques and used to breed ~nim~l~ in which all cells of the animal
contain the homologously recombined DNA. Knockout ~nim~l~ can be characterized for
their ability to accept grafts, reject tumors and defend against infectious ~ e~es and can be
used in the study of basic immllnobiology.
VI. Fxpression of B l.ym,~hocyte ~nt~el7~
Host cells transfected to express peptides having the activity of a novel B lymphocyte
antigen are also within the scope of the invention. The host cell may be any procaryotic or
eucaryotic cell. For exarnple, a peptide having B7-2 activity may be expressed in bacterial
cells such as E. coli, insect cells (baculovirus), yeast, or m~mm~ n cells such as Chinese
harnster ovary cells (CHO) and NS0 cells. Other suitable host cells may be found in
Goeddel, (1990) supra or are known to those skilled in the art.
FOI exarnple, expression in eucaryotic cells such as m~mm~ n, yeast, or insect cells
can lead to partial or complete glycosylation and/or formation of relevant inter- or intra-chain
~lixlllfi~le bonds of recombinant protein. Exarnples of vectors for e~pression in yeast
5. cerivisae include pYepSec l (Baldari. et ~L, (1987) Embo J. 6:229-234), pMFa (Kurjan and
Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and
pYES2 (Invitrogen Corporation, San Diego, CA). Baculovirus vectors available forexpression of proteins in cultured insect cells (SF 9 cells) include the pAc series (Smith et al.,
(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow, V.A., and Surnrners,
M.D., (198g) Virology 170:31-39). Generally, COS cells (Gluzman, Y., (1981) Cell ;~:175-
182) are used in conjunction with such vectors as pCDM8 (Seed, B., (1987) Nature ~2:840)
for transient arnplification/e~les~ion in m~mm?~ n cells, while CHO (dhfr~ Chinese
~arnster Ovary~ cells are used with vectors such as pMT2PC (~llfm~n et ~L (1987),
WO 95/03408 ; PCT/US94/08423
7 ~ 24-
EMBO J. 6:187-195) for stable amplification/~x~ ion in m~mm~ n cells. A preferred
cell line for production of recombinant protein is the NS0 myeloma cell line available from
the ECACC (catalog #85110503) and described in Galfre, G. and Milstein, C. ((1981)
Methods in Enzymology Z~(13):3-46; and Preparation of Monoclonal Antibodies: Strategies
5 and Procedures, Academic Press, N.Y., N.Y). Vector DNA can be introduced into
m~mm~ n cells via conventional techniques such as calcium phosphate or calcium chloride
co-precipitation, DEAE-dextran-mediated transfection, lipofectin, or electroporation.
Suitable methods for transforming host cells can be found in Sambrook et ~L (Molecular
Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)),
10 and other laboratory textbooks. When used in m~mm~ n cells, the expression vector's
control functions are often provided by viral material. For example, commonly used
promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and most frequently,
Simian Virus 40.
It is known that a small faction of cells (about I out of 105) typically integrate DN~
15 into their genomes. In order to identify these integrants, a gene that contains a selectable
marker (i.e., resistance to antibiotics) is generally introduced into the host cells along with the
gene of interest. Preferred selectable markers include those which confer resistance to drugs,
such as G418, hygromycin and methollc;xal~. Selectable markers may be introduced on the
same plasmid as the gene of interest or may be introduced on a separate plasmid. Cells
20 cont~ining the gene of interest can be identified by drug selection; cells that have
incorporated the selectable marker gene will survive, while the other cells die. The surviving
cells can then be screened for production of novel B lymphocyte antigens by cell surface
staining with ligands to the B cell antigens (e.g., CTLA4Ig and CD28Ig). Alternatively, the
protein can be metabolically radiolabeled with a labeled amino acid and immlln~precipitated
25 from cell s~c;~ t with an anti-B lymphocyte antigen monoclonal antibody or a fusion
protein such as CTLA4Ig or CD28Ig.
Expression in procaryotes is most often carried out in E coli with vectors cont~ining
constitutive or inducible promotors directing the e~res~ion of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids usually to the amino terminus of the
30 expressed target gene. Such fusion vectors typically serve three purposes: 1) to increase
sion of recombinant protein; 2) to increase the solubility of the target recombinant
protein; and 3) to aid in the purification of the target recombinant protein by acting as a
ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site
is introduced at the junction of the fusion moiety and the target recombinant protein to enable
35 separation of the target recombinant protein from the fusion moiety subsequent to purification
of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor
Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Amrad
WO 95/03408 2 I G ~ O 91 PCT/US94/08423
-25-
Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, MA) and pRIT5
(Pharrnacia, Piscataway, NJ) which fuse glutathione S-tranferase, maltose E binding protein,
or protein A, respectively, to the target recombinant protein.
E coli ~x~ ssion systems include the inducible ~x~.~ssion vectors pTrc (Amann et 5 ~L, (1988) Gene 69:301 -315) and pET 11 (Studier et ~L, Gene Expression Technology.
Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89;
commercially available from Novagen). In the pTrc vector system, the inserted gene is
expressed with a pelB signal sequence by host RNA polymerase transcription from a hybrid
trp-lac fusion promoter. After induction, the recombinant protein can be purified from the
periplasmic fraction. In the pET 11 vector system, the target gene is expressed as non-fusion
protein by transcription from the T7 gnlO-lac 0 fusion promoter mediated by a coexpressed
viral RNA polymerase (T7 gnl ). This viral polymerase is supplied by host E. coli strains
BL21 (DE3) or HMS 174(DE3) from a resident ~ prophage harboring a T7 gnl under the
transcriptional control of the lacUV S promoter. In this system, the recombinant protein can
be purified from inclusion bodies in a denatured form and, if desired, renatured by step
gradient dialysis to remove denaturants.
One strategy to maximize recombinant B7-2 expression in E. coli is to express the
protein in a host bacteria ~,vith an impaired capacity to proteolytically cleave the recombinant
protein (Gotte~m~n, S., Gene Expression Technology: Methods in En~ymology 18$,
~c~lemic Press, San Diego, California (1990) 119-128). Another strategy would be to alter
the nucleic acid sequence of the B7-2 gene or other DNA to be inserted into an expression
vector so that the individual codons for each amino acid would be those preferentially utilized
in highly expressed E. coli proteins (Wada et ~LL, (1992) Nuc. ~cids Res. ~Q:2111-2118).
Such alteration of nucleic acid sequences of the invention could be carried out by standard
DNA synthesis techniques.
Novel B lymphocyte antigens and portions thereof, expressed in m~mm~ n cells or
otherwise, can be purified according to standard procedures of the art, including ammonium
sulfate ~lecipiL~Iion, fractionation column chromatography (e.g. ion exchange, gel filtration,
electrophoresis, affinity chromatography, etc.) and ultimately, crystallization (see generally,
"Enzyme Purification and Related Techniques", Methods in Enzymolo~y, 22:233-577
(1971)). Orlce purified, partially or to homogeneity, the recombinantly produced B
lymphocyte antigens or portions thereof can be utilized in compositions suitable for
ph~rm~ceutical ~fimini.~tration as described in detail herein.
WO 95/03408 ~ ~ 6 ~ ~ 91 PCT/US94/08423
-26-
VIT. Motlifications of Nuçleic Acid and Amino Acid Sequences of the Invention
~n~l Assays for B7 J ~y~hocvte Anti~en Activity
It will be appreciated by those skilled in the art that other nucleic acids encoding
peptides having the activity of a novel B lymphocyte antigen can be isolated by the above
5 process. Different cell lines can be expected to yield DNA molecules having different
sequences of bases. Additionally, variations may exist due to genetic polymorphisms or cell-
mediated modifications of the genetic material. Furthermore, the DNA sequence of a B
lymphocyte antigen can be modified by genetic techniques to produce proteins or peptidçs
with altered amino acid sequences. Such sequences are considered within the scope of the
10 present invention, where the expressed peptide is capable of either inducing or inhibiting
activated T cell mediated immune responses and imml-ne function.
A number of processes can be used to generate equivalents or fragments of an isolated
DNA sequence. Small subregions or fr~gment~ of the nucleic acid encoding the B7-2
protein, for example 1-30 bases in length, can be prepared by standard, synthetic organic
15 chemical means. The technique is also useful for ~ .~dtion of antisense oligonucleotides
and primers for use in the generation of larger synthetic fr~gment~ of B7-2 DNA.Larger subregions or fragments of the genes encoding B lymphocyte antigens can be
expressed as peptides by syntht?~i7inE the relevant piece of DNA using the polymerase chain
reaction (PCR) (Sambrook, Fritsch and ~ni~ti~, 2 Molecular Cloning; A Laboratory20 Manual, Cold Spring Harbor, N.Y., (1989)), and lig~ting the thus obtained DNA into an
a~,v~l;ate expression vector. Using PCR, specific sequences ofthe cloned double stranded
DNA are generated. cloned into an expression vector, and then assayed for CTLA4/CD28
binding activity. For example, to express a secreted (soluble) form of the human B7-2
protein, using PCR, a DNA can be synthesized which does not encode the transmembrane
25 and cytoplasmic regions of the protein. This DNA molecule can be ligated into an
a~ ,;ate expression vector and introduced into a host cell such as CHO, where the B7-2
protein fragment is synthesized and secreted. The B7-2 protein fragment can then readily be
obtained from the culture media.
In another embodiment, mutations can be introduced into a DNA by any one of a
30 number of methods, including those for producing simple deletions or insertions, systematic
deletions, insertions or substitutions of clusters of bases or substitutions of single bases, to
generate variants or modified equivalents of B lymphocyte antigen DNA. For example,
changes in the human B7-2 cDNA sequence shown in Figure 8 (SEQ ID NO:1) or murine
B7-2 cDNA sequence shown in Figure 14 (SEQ ID NO:22) such as amino acid substitutions
35 or deletions are preferably obtained by site-directed mutagenesis. Site directed mutagenesis
systems are well known in the art. Protocols and reagents can be obtained commercially
from Amersham Tntem~tional PLC, Amersham, U.K.
WO 95/03403 ~ 1 6 7 0 91 PCTIUS94/08423
-27-
Peptides having an activity of a novel B lymphocyte antigen, i.e., the ability to bind to
the natural ligand(s) of a B lymphocyte antigen on T cells and either stim~ te (amplify) or
inhibit (block) activated T cell mediated immlme responses, as evidenced by, for example,
cytokine production and/or T cell proliferation by T cells that have received a primary
5 activation signal are considered within the scope of the invention. More specifically,
peptides that bind to T lymphocytes, for example CD28+ cells, may be capable of delivering
a costim~ tory signal to the T lymphocytes, which, when transmitted in the presence of
antigen and class II MHC, or other material capable of tr~n~mit~in~ a primary signal to the T
cell, results in activation of cytokine genes within the T cell. Alternatively, such a peptide
10 can be used in conjunction with class I MHC to thereby activate CD8+ cytolytic T cells. In
addition, soluble, monomeric forms of the B7-2 protein, may retain the ability to bind to their
natural ligand(s) on CD28+ T cells but, perhaps because of insufficient cross-linking with the
ligand, fail to deliver the secondary signal essential for enhanced cytokine production and cell
division. Such peptides, which provide a means to induce a state of anergy or tolerance in the
15 cells, are also considered within the scope of the invention.
Screening the peptides for those which retain a characteristic B lymphocyte antigen
activity as described herein can be accomplished using one or more of several different
assays. For example, the peptides can be screened for specific reactivity with an anti-B7-2
monoclonal antibody reactive with cell surface B7-2 or with a fusion protein, such as
20 CTLA4Ig or CD28Ig. Specifically, appropriate cells, such as COS cells, can be transfected
with a B7-2 DNA encoding a peptide and then analyzed for cell surface phenotype by indirect
immunofluorescence and flow cytometry to determine whether the peptide has B7-2 activity.
Cell surface e~ es~ion of the transfected cells is evaluated using a monoclonal antibody
specifically reactive with cell surface B7-2 or with a CTLA4Ig or CD28Ig fusion protein.
25 Production of secreted forms of B7-2 is evaluated using anti-B7-2 monoclonal antibody or
CTLA4Ig or CD28 fusion protein for immllnoprecipitation.
Other, more ~,le~lled, assays take advantage of the functional characteristics of the
B7-2 antigen. As previously set forth, the ability of T cells to synthesi7~ cytokines depends
not only on occllp~nGy or cross-linking of the T cell receptor for antigen (the "prima~y
30 activation signal" provided by, for example anti-CD3, or phorbol ester to produce an
"activated T cell"), but also on the induction of a costimnl~tt ry signal, in this case, by
interaction with a B lymphocyte antigen, such as B7-2, B7-1 or B7-3. The binding of B7-2 to
its natural ligand(s) on, for example, CD28+ T cells, has the effect of transmitting a signal to
the T cell that induces the production of increased levels of cytokines, particularly of
35 interleukin-2, which in turn ~tim~ tt-s the proliferation of the T lymphocytes. Other assays
for B7-2 function thus involve assaying for the synthesis of cytokines, such as interleukin-2,
WO 95/03408 PCT/US94/08423
2; 1 6 ~ 28-
interleukin-4 or other known or unknown novel cytokines? and/or assaying for T cell
proliferation by CD28+ T cells which have received a primary activation signal.
In vitro, T cells can be provided with a first or primary activation signal by anti-T3
monoclonal antibody (e.g. anti-CD3) or phorbol ester or, more preferably, by antigen in
5 association with class II MHC. T cells which have received a primary activation signal are
referred to herein as activated T cells. B7-2 function is assayed by adding a source of B7-2
(e.g., cells expressing a peptide having B7-2 activity or a secreted form of B7-2) and a
~1;111~.~ activation signal such as antigen in association with Class II MHC to a T cell culture
and assaying the culture supernatant for interleukin-2, garnma interferon, or other known or
10 unknown cytokine. For example, any one of several conventional assays for interleukin-2
can be employed, such as the assay described in Proc. Natl. Acad Sci. USA, 86: 1333 (1989)
the pertinent portions of which are incorporated herein by reference. A kit for an assay for
the production of interferon is also available from Genzyme Corporation (Cambridge, MA.).
T cell proliferation can also be measured as described in the Examples below. Peptides that
15 retain the characteristics of the B7-2 antigen as described herein may result in increased per
cell production of cytokines, such as IL-2, by T cells and may also result in enhanced T cell
proliferation when colllp~d to a negative control in which a costim~ tory signal is lacking.
The same basic functional assays can also be used to screen for peptides having B7-2
activity, but which lack the ability to deliver a costimulatory signal, but in the case of such
20 peptides, addition of the B7-2 protein will not result in a marked increase in proliferation or
cytokine secretion by the T cells. The ability of such proteins to inhibit or completely block
the normal B7-2 costim~ tory signal and induce a state of anergy can be determined using
subsequent attempts at stimlll~tion of the T cells with antigen pres~nting cells that express
cell surface B7-2 and present antigen. If the T cells are unresponsive to the subsequent
25 activation attempts, as determined by IL-2 synthesis and T cell proliferation~ a state of anergy
has been in~ ce~l See, e.g., Gimmi, C.D. et al. (1993) Proc. Natl. Acad. Sci. USA ~Q, 6586-
6590, and Schwartz (1990) Science, 248, 1349-1356, for assay systems that can used as the
basis for an assay in accordance with the present invention.
It is possible to modify the structure of a peptide having the activity of a novel B
30 lymphocyte antigen for such purposes as increasing solubility, enhancing therapeutic or
prophylactic efficacy, or stability (e.g., shelf life ex vivo and resistance to proteolytic
degradation in vivo). Such modified peptides are considered functional equivalents of the B
lymphocyte antigens as defined herein. For example, a peptide having B7-2 activity can be
modified so that it m~int~in~ the ability to co-stimulate T cell proliferation and/or produce
35 cytokines. Those residues shown to be ess~nti~l to interact with the CTLA4/CD28 receptors
on T cells can be modified by replacing the essenti~l amino acid with another, preferably
similar amino acid residue (a conse, v~liv~ substitution) whose presence is shown to enhance,
WO 95/03408 ~ ~ fi ~ ~ ~1 PCT/US94/08423
-29-
fiimini~h, but not elimin~te or not effect receptor interaction. In addition. those amino acid
residues which are not essential for receptor interaction can be modified by being replaced by
another amino acid whose incorporation may enhance, ~imini~h, or not effect reactivity.
Another example of modification of a peptide having the activity of a novel B
- S lymphocyte antigen is substitution of cysteine residues preferably with ~l~nine, serine,
threonine, leucine or glutamic acid residues to minimi7~ dimerization via ~ llfide linkages.
In addition, amino acid side chains of a peptide having B7-2 activity can be chemically
modified. Another modification is cyclization of the peptide.
In order to enhance stability and/or reactivity, peptides having B7-2 activity can be
modified to incorporate one or more polymorphisms in the amino acid sequence of the
antigen resnlting from any natural allelic variation. Additionally, D-arnino acids, non-natural
amino acids, or non-amino acid analogs can be substituted or added to produce a modified
protein within the scope of this invention. Furthermore, the peptides can be modified using
polyethylene glycol (PEG) according to the method of A. Sehon and co-workers (Wie et ~L,
supra) to produce a peptide conjugated with PEG. In addition, PEG can be added during
chemical synthesis of the peptide. Other modifications of the peptides include
reduction/alkylation (Tarr in: Methods of Protein Microcharacterization, J. E. Silver ed.,
Hurnana Press, Clifton NJ 155-194 (1986)); acylation (Tarr, supra); chemical coupling to an
a~plo~l;ate carrier (Mishell and Shiigi, eds, ~electe~Methods in Cellular Immunology, WH
Freeman, San Francisco, CA (1980), U.S. Patent 4,939,239; or mild formalin tre~tment
(Marsh (1971), lnt. Arch. of Aller~ andAppl. Immunol. 41:199-215).
To facilitate purification and potentially increase solubility of a peptide, it is possible
to add an amino acid fusion moiety to the protein backbone. For example, hexa-hi~ticiine can
be added to the peptide for purification by immobilized metal ion affinity chromatography
(Hochuli, E. et ~L, (1988) Bio/Technology 6:1321-1325). In addition, to facilitate isolation of
a B lymphocyte antigen free of irrelevant sequences, specific endoprotease cleavage sites can
be introduced between the sequences of a fusion moiety and the peptide. It may be necessary
to increase the solubility of a peptide by adding functional groups to the peptide, or by
omitting hydrophobic regions of the peptide.
VIT. Uses of Nucleic Acid Sequçnces Fnco~ B T ~n~l~hocyte Anti~e~ ~nd Peptides
Hav;~ B7-2 Activity
A. MolecularProbes
The nucleic acids of this invention are useful diagnostically, for tracking the progress
of tli~e~e, by measuring the activation status of B lymphocytes in biological samples or for
assaying the effect of a molecule on the ~ esssion of a B Iymphocyte antigen (e.g.,
WO 95/03408 PCT/US94/08423
-30-
~letecting cellular mRNA levels). In accordance with these diagnostic assays, the nucleic acid
sequences are labeled with a detectable marker, e.g., a radioactive, fluorescent, or biotinylated
marker and used in a conventional dot blot or Northern hybridization procedure to probe
mRNA molecules of total or poly(A+) RNAs from a biological sample.
.
R. Antibody Production
The peptides and fusion proteins produced from the nucleic acid molecules of thepresent invention can also be used to produce antibodies specifically reactive with B
lymphocyte antigens. For example, by using a full-length B7-2 protein, or a peptide fragment
thereof, having an amino acid sequence based on the predicted amino acid sequence of B7-2,
anti-protein/anti-peptide polyclonal antisera or monoclonal antibodies can be made using
standard methods. A m~mm~l, (e.g., a mouse, h~met~r, or rabbit) can be immunized with an
immlln~genic form of the protein or peptide which elicits an antibody response in the
m~mm~l The immunogen can be, for example, a recombinant B7-2 protein, or fragment
thereof, a synthetic peptide fragment or a cell that expresses a B lymphocyte antigen on its
surface. The cell can be for example, a splenic B cell or a cell transfected with a nucleic acid
encoding a B Iymphocyte antigen of the invention (e.g., a B7-2 cDNA) such that the B
lymphocyte antigen is expressed on the cell surface. The immllnogen can be modified to
increase its immllnogenicity. For example, techniques for conferring immunogenicity on a
peptide include conjugation to carriers or other techniques well known in the art. For
example, the peptide can be ~lminietered in the presence of adjuvant. The progress of
imml~ni7~tion can be monitored by detection of antibody titers in plasma or serum. Standard
ELISA or other immllno~ee~y can be used with the immunogen as antigen to assess the levels
of antibodies.
Following immllni7~tion, antisera can be obtained and, if desired, polyclonal
antibodies isolated from the sera. To produce monoclonal antibodies, antibody producing
cells (lymphocytes) can be harvested from an immlmi7e-1 animal and fused with myeloma
cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding
hybridoma cells. Such techniques are well known in the art. For example, the hybridoma
technique originally developed by Kohler and Milstein (Nature (1975) ~:495-497) as well
as other techniques such as the human B-cell hybridoma technique (Kozbar et al.~ Immunol.
Today (19~3) 4:72), the EBV-hybridoma technique to produce human monoclonal antibodies
(Cole et al. Monoclonal Antibodies in Cancer Therapy (1985) (Allen R. Bliss, Inc., pages 77-
96), and screening of combinatorial antibody libraries (Huse et al., Science ( l 989) ~:1275).
Hybridoma cells can be screened immunochemically for production of antibodies specifically
reactive with the peptide and monoclonal antibodies isolated.
WO 95/03401~ 21 ~ 1 PCT/US94/08423
-31-
The term antibody as used herein is intended to include fragments thereof which are
also specifically reactive with a peptide having the activity of a novel B Iymphocyte antigen
or fusion protein as described herein. Antibodies can be fragmented using conventional
techniques and the fragments screened for utility in the same manner as described above for
5 whole antibodies. For exarnple, F(ab')2 fragments can be generated by treating antibody with
pepsin. The resulting F(ab')2 fragment can be treated to reduce tli~ulfi~le bridges to produce
Fab' frAgment~. The antibody of the present invention is further intended to include
bispecific and chimeric molecules having an anti-B Iymphocyte antigen (i.e., B7-2, B7-3)
portion.
Particularly preferred antibodies are anti-human B7-2 monoclonal antibodies
produced by hybridomas HA3.1 F9, HA5.2B7 and HF2.3D 1. The p~cl)al~Lion and
characterization of these antibodies is described in detail in Example 8. Monoclonal antibody
HA3.1F9 was determined to be ofthe IgG1 isotype; monoclonal antibody HA5.2B7 wasdetermined to be of the IgG2b isotype; and monoclonal anibody HF2.3D I was determined to
be of the IgG2a isotype. Hybidoma cells were deposited with the American Type Culture
Collection, which meets the requirements of the Budapest Treaty, on July 19, 1994 as ATCC
AccessionNo. (hybridomaHA3.1F9),ATCCAccessionNo. (HA5.2B7)and
ATCC Accession No. (HF2.3Dl).
When antibodies produced in non-human subjects are used therapeutically in h~lm~n~,
they are recognized to varying degrees as foreign and an immune response may be generated
in the patient. One approach for minimi7ing or elimin~ting this problem, which is preferable
to general immlm~suppression~ is to produce chimeric antibody derivatives, i.e., antibody
molecules that combine a non-human animal variable region and a human constant region.
Chimeric antibody molecules can include, for example, the antigen binding domain from an
antibody of a mouse, rat, or other species, with human constant regions. A variety of
approaches for m~king chimeric antibodies have been described and can be used to make
chimeric antibodies cont~ining the imml-noglobulin variable region which recognizes the
gene product of the novel B Iymphocyte antigens of the invention. See, for example,
Morrison et al., Proc. Natl. Acad. Sci. U.S.A. 81 :6851 (1985); Takeda et al., Nature 314:452
(1985), Cabilly et al., U.S. Patent No. 4,816,567; Boss et al., U.S. Patent No. 4,816,397;
Tanaguchi et al., European Patent Publication EP171496; European Patent Publication
0173494, United Kingdom Patent GB 2177096B. It is expected that such chimeric antibodies
would be less immllnogenic in a human subject than the corresponding non-chimeric
antibody.
For human therapeutic purposes, the monoclonal or chimeric antibodies specifically
reactive with a peptide having the activity of a B lymphocyte antigen as described herein can
be further hl-m~ni7e~1 by producing human variable region chimeras, in which parts of the
WO 95/03408 . PCT/US94/08423
~1~7~9~ ~
-32-
variable regions, especially the conserved framework regions of the antigen-binding domain,
are of human origin and only the hypervariable regions are of non-human origin. General
reviews of "hllm~ni7to~1" chimeric antibodies are provided by Morrison, S. L. (1985) Science
~2:1202-1207 and by Oi et al. (1986) BioTechniques _:214. Such altered immunoglobulin
molecules may be made by any of several techniques known in the art? (e.g., Teng et al.,
Proc. Natl. Acad. Sci. U.S.A., 80:7308-7312 (1983); Kozbor et al., Immunology Today,
~:7279 (1983); Olsson et al., Meth. Enzymol., 92:3-16 (1982)), and are preferably made
according to the te~chin~ of PCT Publication WO92/06193 or EP 0239400. Hllm~ni7~d
antibodies can be commercially produced by, for example, Scotgen Limited, 2 Holly Road,
Twickenham, Middlesex, Great Britain. Suitable "hllm~ni7~1" antibodies can be
alternatively produced by CDR or CEA substitution (see U.S. Patent 5,225,539 to Winter;
Jones et al. (1986) Nature ~1:552-525; Verhoeyan et al. (1988) Science 239:1534; and
Beidler et al. (1988) J. Immunol. 141 :4053-4060). Hllm~ni7ecl antibodies which have
reduced immun~genicity are preferred for immlln~therapy in human subjects.
Immunotherapy with a hl-m~ni7~-1 antibody will likely reduce the necessity for any
concomitant imm--nosuppression and may result in increased long term effectiveness for the
tre~tment of chronic disease situations or situations requiring repeated antibody tre~tment.~.
As an alterntive to hl-m~ni7ing a monoclonal antibody from a mouse or other species,
a human monoclonal antibody directed against a human protein can be generated. Transgenic
mice carrying human antibody repertoires have been created which can be immunized with a
human B lymphocyte antigen, such as B7-2. Splenocytes from these immunized transgenic
mice can then be used to create hybridomas that secrete human monoclonal antibodies
specifically reactive with a human B lymphocyte antigen (see, e.g., Wood et al. PCT
publication WO 91/00906, Kucherlapati et al. PCT publication WO 91/10741; Lonberg et al.
PCT publication WO 92/03918; Kay et al. PCT publication 92/03917; Lonberg, N. et al.
(1994) Nature ~:856-859; Green, L.L. et al. (1994) Nature Genet. 1:13-21; Morrison, S.L.
et al. (1994) Proc. Natl. Acad. Sci. USA 81 :6851-6855; Bruggeman et al. (1993) Year
Immunol 1:33-40; Tuaillon et al. (1993) PNAS 90:3720-3724; and Bruggeman et al. (1991)
~ur JImmunol ~1:1323-1326).
Monoclonal antibody compositions of the invention can also be produced by other
methods well known to those skilled in the art of recombinant DNA technology. Analternative method, referred to as the "combinatorial antibody display" method, has been
developed to identify and isolate antibody fragments having a particular antigen specificity,
and can be utilized to produce monoclonal antibodies that bind a B lymphocyte antigen of the
invention (for descriptions of combinatorial antibody display see e.g., Sastry et al. ~1989)
PNAS~:5728; Huse et al. (1989) Science 246:1275; and Orlandi et al. (1989) PNAS
86:3833). After immunizing an animal with a B lymphocyte antigen, the antibody repertoire
~WO ~5/D3408 33 PCTIU594/08423
of the resulting B-cell pool is cloned. Methods are generally known for directly obtaining the
DNA sequence of the variable regions of a diverse population of immunoglobulin molecules
by using a mixture of oligomer primers and PCR. For instance, mixed oligonucleotide
primers corresponding to the S' leader (signal peptide) sequences and/or framework 1 (FRl )
5 sequences, as well as primer to a conserved 3' constant region primer can be used for PCR
amplification of the heavy and light chain variable regions from a number of murine
antibodies (Larrick et al. (1991) Biotechniques 11:152-156). A similar strategy can also been
used to amplify human heavy and light chain variable regions from human antibodies
(Larrick et al. (1991) Methods: Companion fo Methods in Enzymology ~: 106- 110).In an illustrative embodiment, RNA is isolated from activated B cells of, for example,
peripheral blood cells, bone marrow, or spleen ~lel)aldlions, using standard protocols (e.g.,
U.S. Patent No. 4,683,`202; Orlandi, et al. PNAS (1989) 86:3833-3837; Sastry et al., PNAS
(1989) 86:5728-5732; and Huse et al. (1989) Science ~:1275-1281.) First-strand cDNA is
synthesi7P~l using primers specific for the constant region of the heavy chain(s) and each of
15 the K and ~ light chains, as well as primers for the signal sequence. Using variable region
PCR primers, the variable regions of both heavy and light chains are amplified, each alone or
in combinantion, and ligated into al)~ro~l;ate vectors for further manipulation in generating
the display packages. Oligonucleotide primers useful in amplification protocols may be
unique or degenerate or incorporate inosine at degenerate positions. Restriction endonuclease
20 recognition sequences may also be incorporated into the primers to allow for the cloning of
the amplified fragment into a vector in a predetermined reading frame for expression.
The V-gene library cloned from the il,,,..l..li7~tion-derived antibody repertoire can be
expressed by a population of display packages, preferably derived from filamentous phage, to
form an antibody display library. Ideally, the display package comprises a system that allows
the sampling of very large diverse antibody display libraries, rapid sorting after each affinity
separation round, and easy isolation of the antibody gene from purified display packages. In
addition to cornmercially available kits for gen~r~tin?~ phage display libraries (e.g., the
Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; and the Stratagene
Sur~ZAPTM phage display kit, catalog no. 240612), exarnples of methods and reagents
particularly arnenable for use in generating a diverse antibody display library can be found in,
for exarnple, Ladner et al. U.S. Patent No. 5,223,409; Kang et al. International Publication
No. WO 92/18619; Dower et al. International Publication No. WO 91/17271; Winter et al.
International Publication WO 92/20791; Markland et al. International Publication No. WO
92/15679; Breitling et al. International Publication WO 93/01288, McCafferty et al.
International Publication No. WO 92/01047; Garrard et al. Tntetn~tional Publication No. WO
92/09690; Ladner et al. International Publication No. WO 90/02809; Fuchs et al. (1991)
Bio/Technology 2: 1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3 :81 -85; Huse et
WO 95/03408 PCT/US94l08423
2 ~ 9 ~ -34-
al. (1989) Science ~:1275-1281; Griffths et al. (1993) EMBO J12:725-734; Hawkins et al.
(1992) JMol Biol ~:889-896; Clackson et al. (1991) Nature ~:624-628; Gram et al.(1992) PNAS ~2:3576-3580; Garrad et al. (1991) Bio/Technology 2:1373-1377; Hoogenboom
et al. (1991) Nuc Acid Res 12:4133-4137; and Barbas et al. (1991) PN,45 88:7978-7982.
In certain embodiments, the V region domains of heavy and light chains can be
e~ essed on the same polypeptide, joined by a flexible linker to form a single-chain Fv
fragment, and the scFV gene subsequently cloned into the desired expression vector or phage
genome. As generally described in McCafferty et al., Nature (1990) ~:552-554, complete
VH and VL domains of an antibody, joined by a flexible (Gly4-Ser)3 linker can be used to
produce a single chain antibody which can render the display package separable based on
antigen affinity. Isolated scFV antibodies immunoreactive with a peptide having activity of a
- B lymphocyte antigen can subsequently be formulated into a pharm~entical p~ ua,dlion for
use in the subject method.
Once displayed on the surface of a display package (e.g., filamentous phage), the
antibody library is screened with a B lymphocyte antigen protein, or peptide fragment
thereof, to identify and isolate packages that express an antibody having specificity for the B
lymphocyte antigen. Nucleic acid encoding the selected antibody can be recovered from the
display package (e.g., from the phage genome) and subcloned into other exples~ion vectors
by standard recombinant DNA techniques.
The antibodies of the current invention can be used therapeutically to inhibit T cell
activation through blocking receptor:ligand interactions necess~ry for costimulation of the T
cell. These so-called "blocking antibodies" can be identified by their ability to inhibit T cell
proliferation and/or cytokine production when added to an in vitro costimulation assay as
described herein. The ability of blocking antibodies to inhibit T cell functions may result in
immunosuppression and/or tolerance when these antibodies are ~lmini~tered in vivo.
C. Protein Purification
The polyclonal or monoclonal antibodies of the current invention, such as an antibody
specifically reactive with a recombinant or synthetic peptide having B7-2 activity or B7-3
activity can also be used to isolate the native B lymphocyte antigen from cells. For example,
antibodies reactive with the peptide can be used to isolate the naturally-occurring or native
form of B7-2 from activated B lymphocytes by immllno~ffinity chromatography. In addition,
the native form of B7-3 can be isolated from B cells by immunoaffinity chromatography with
monoclonal antibody BB-l.
WO 95/03408 2 ~ ~ 7 ~ 91 PCT/US94/08423
-35-
D. Other Therapeutic Reagents
The nucleic acid sequences and novel B Iymphocyte antigens described herein can be
used in the development of therapeutic reagents having the ability to either upregulate (e.g.,
amplify) or downregulate (e.g., suppress or tolerize) T cell mediated immune responses. For
5 example, peptides having B7-2 activity, including soluble, monomeric forms of the B7-2
antigen or a B7-2 fusion protein, e.g., B7-2Ig, and anti-B7-2 antibodies that fail to deliver a
costimulatory signal to T cells that have received a primary activation signal, can be used to
block the B7-2 ligand(s) on T cells and thereby provide a specific means by which to cause
immunosuppression and/or induce tolerance in a subject. Such blocking or inhibitory forms
10 of B lymphocyte antigens and fusion proteins and blocking antibodies can be identified by
their ability to inhibit T cell proliferation and/or cytokine production when added to an in
vitro costimulation assay as previously described herein. In contrast to the monomeric form,
stimulatory forms of B7-2, such as an intact cell surface B7-2, retain the ability to transmit
the costimulatory signal to the T cells, resulting in an increased secretion of cytokines when
15 compared to activated T cells that have not received the secondary signal.
In addition, fusion proteins compri~ing a first peptide having an activity of B7-2 fused
to a second peptide having an activity of another B lymphocyte antigen (e.g., B7-1) can be
used to modify T cell mediated immllne responses. ~ ely, two separate peptides
having an activity of B lymphocyte antigens, for example, B7-2 and B7- 1, or a combination
20 of blocking antibodies (e.g., anti-B7-2 and anti-B7- 1 monoclonal antibodies) can be
combined as a single composition or ~(lmini~tered st;~ ely (simultaneously or
sequentially), to upregulate or downregulate T cell mediated immune responses in a subject.
Furthermore, a therapeutically active amount of one or more peptides having B7-2 activity
and or B7-1 activity can be used in conjunction with other immunomod-~l~tin~ reagents to
25 influence immune responses. Exarnples of other immllnomo~ ting reagents include
blocking antibodies, e.g., against CD28 or CTLA4, against other T cell markers or against
cytokines, fusion proteins, e.g., CTLA4Ig, or immunosuppressive drugs, e.g., cyclosporine A
or FK506.
The peptides produced from the nucleic acid molecules of the present invention may
30 also be useful in the construction of therapeutic agents which block T cell function by
destruction of the T cell. For example, as described, secreted forms of a B lymphocyte
antigen can be constructed by standard genetic engineering techniques. By linking a soluble
form of B7- 1, B7-2 or B7-3 to a toxin such as ricin, an agent capable of preventing T cell
activation can be made. Infusion of one or a combination of immunotoxins, e.g., B7-2-ricin,
35 B7-1-ricin, into a patient may result in the death of T cells, particularly of activated T cells
that express higher amounts of CD28 and CTLA4. Soluble forms of B7-2 in a monovalent
WO 95/03408 : PCT/US94/08423
~16~
-36-
form alone may be useful in blocking B7-2 function, as described above~ in which case a
carrier molecule may also be employed.
Another method of preventing the function of a B Iymphocyte antigen is through the
use of an antisense or triplex oligonucleotide. For example, an oligonucleotide
S complement~ry to the area around the B7-1, B7-2 or B7-3 translation initiation site, (e.g., for
B7-1, TGGCCCATGGCTTCAGA, (SEQ ID NO:20) nucleotides 326-309 and for B7-2,
GCCAAAATGGATCCCCA (SEQ ID NO:21)), can be synthesized. One or more antisense
oligonucleotides can be added to cell media, typically at 200 llg/ml, or ~Amini~tered to a
patient to prevent the synthesis of B7-1, B7-2 and/or B7-3. The antisense oligonucleotide is
10 taken up by cells and hybridizes to the appro~liate B lymphocyte antigen mRNA to prevent
translation. Alternatively, an oligonucleotide which binds double-stranded DNA to form a
triplex construct to prevent DNA unwinding and transcription can be used. As a result of
either, synthesis of one or more B lymphocyte antigens is blocked.
15 F Therapeutic Uses by Dow~re~ulation of Immune Respo~es
Given the structure and function of the novel B lymphocyte antigens disclosed herein,
it is possible to downregulate the function of a B lymphocyte antigen, and thereby
downregulate immllne responses, in a number of ways. Downregulation may be in the form
of inhibiting or blocking an immllne response already in progress or may involve preventing
20 the induction of an immllne response. The functions of activated T cells may be inhibited by
~u~ S~ g T cell responses or by inducing specific tolerance in T cells, or both.Immunosuppression of T cell responses is generally an active, non-antigen-specific, process
which requires continuous exposure of the T cells to the s~l~s~ive agent. Tolerance, which
involves inducing non-responsiveness or anergy in T cells, is distinguishable from
25 immunosuppression in that it is generally antigen-specific and persists after exposure to the
tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T
cell response upon reexposure to specific antigen in the absence of the tolerizing agent.
Downregulating or preventing one or more B lymphocyte antigen functions, e.g.,
preventing high level lymphokine synthesis by activated T cells, will be useful in situations
30 of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD). For
example, blockage of T cell function should result in reduced tissue destruction in tissue
transplantation. Typically, in tissue transplants, rejection of the kansplant is initiated through
its recognition as foreign by T cells, followed by an immlme reaction that destroys the
kansplant. The ~lmini~kation of a molecule which inhibits or blocks interaction of a B7
35 lymphocyte antigen with its natural ligand(s) on immllne cells (such as a soluble, monomeric
form of a peptide having B7-2 activity alone or in conjunction with a monomeric form of a
peptide having an activity of another B lymphocyte antigen (e.g., B7-1, B7-3) or blocking
WO 95/03408 2 1 ~ 7 ~ ~ 1 PCT/US94/08423
-37-
antibody), prior to transplantation can lead to the binding of the molecule to the natural
ligand(s) on the immune cells without tr~n~mitting the corresponding costimulatory signal.
Blocking B Iymphocyte antigen function in this manner prevents cytokine synthesis by
imml-ne cells, such as T cells~ and thus acts as an immunosuppressant. Moreover, the lack of
5 costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a
subject. Induction of long-term tolerance by B Iymphocyte antigen-blocking reagents may
avoid the necessity of repeated ~flmini~tration of these blocking reagents. To acheive
sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the
function of a combination of B lymphocyte antigens. For example, it may be desirable to
block the function of B7-2 and B7-1, B7-2 and B7-3, B7-1 and B7-3 or B7-2, B7-1 and B7-3
by ~-lmini~tering a soluble form of a combination of peptides having an activity of each of
these antigens or a blocking antibody (separately or together in a single composition) prior to
transplantation. Alternatively, inhibitory forms of B lymphocyte antigens can be used with
other suppressive agents such as blocking antibodies against other T cell markers or against
cytokines, other fusion proteins, e.g., CTLA41g, or immunosuppressive drugs.
The efficacy of particular blocking reagents in preventing organ transplant rejection or
GVHD can be ~esesse~l using animal models that are predictive of efficacy in hllm~n~. The
functionally important aspects of B7-1 are conserved structurally between species and it is
therefore likely that other B lymphocyte antigens can function across species, thereby
allowing use of reagents composed of human proteins in animal systems. Examples of
~I)rop.;ate systems which can be used include allogeneic cardiac grafts in rats and
xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the
immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et
al., Science, 257: 789-792 (1992) and Turka et al., Proc. Natl. Acad. Sci. USA, 89: 11102-
11105 (1992). In addition, murine models of GVHD (see Paul ed., Fundamental
Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect
of blocking B Iymphocyte antigen function in vivo on the development of that disease.
Blocking B Iymphocyte antigen function, e.g., by use of a peptide having B7-2
activity alone or in combination with a peptide having B7-1 activity and/or a peptide having
B7-3 activity, may also be therapeutically useful for treating autoimmune diseases. Many
autoimmune disorders are the result of inapl)lol~liate activation of T cells that are reactive
against self tissue and which promote the production of cytokines and autoantibodies
involved in the pathology of the diseases. Preventing the activation of autoreactive T cells
may reduce or elimin~te disease symptoms. Administration of reagents which blockcostimulation of T cells by disrupting receptor:ligand interactions of B Iymphocyte antigens
can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-
derived cytokines which may be involved in the disease process. Additionally, blocking
-
wo 95/03408 ~ 91 - PCT/US94/08423
-38-
reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to
long-term relief from the disease. The efficacy of blocking reagents in preventing or
alleviating autoimml-ne disorders can be determined using a number of well-characterized
animal models of human autoimmnne ~ e~ees Examples include murine ~ue~ .ental
S autoimmune encephalitis, systemic lupus erythmatosis in MRl llpr/lpr mice or NZB hybrid
mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and
murine experimental myaetheni~ gravis (see Paul ed., Fundamental Immunology, Raven
Press, New York, 1989, pp. 840-856).
The IgE antibody response in atopic allergy is highly T cell dependent and, thus,
inhibition of B lymphocyte antigen in~ e-1 T cell activation may be useful
theld~tulically in the tre~tment of allergy and allergic reactions. An inhibitory form of
B7-2 protein, such as a peptide having B7-2 activity alone or in combination with a
peptide having the activity of another B lymphocyte antigen, such as B7-1, can be
~mini~tered to an allergic subject to inhibit T cell me~ te-l allergic responses in the
subject. Inhibition of B lymphocyte antigen costim~llation of T cells may be
accompagnied by exposure to allergen in conjunction with a~rop,iate MHC molecules.
Allergic reactions may be systemic or local in nature, depending on the route of entry of
the allergen and the pattern of deposition of IgE on mast cells or basophils. Thus, it may
be necessary to inhibit T cell me~liattod allergic responses locally or systemically by
proper ~lmini~tration of an inhibitory form of B7-2 protein.
Inhibition of T cell activation through blockage of B lymphocyte antigen function
may also be important therapeutically in viral infections of T cells. For example, in the
acquired immune deficiency syndrome (AIDS), viral replication is stim~ tçA by T cell
activation. Blocking B7-2 function could lead to a lower level of viral replication and
thereby ameliorate the course of AIDS. In addition, it may also be nt~cess~ry to block the
function of a combination of B lymphocyte antigens i.e., B7-1, B7-2 and B7-3. Surprisingly,
HTLV-I infected T cells express B7-1 and B7-2. This expression may be important in the
growth of HTLV-I infected T cells and the blockage of B7- 1 function together with the
function of B7-2 and/or B7-3 may slow the growth of HTLV-I inc~l~cec~ lellk~mi~
Alternatively, stimlll~tion of viral replication by T cell activation may be in~ ced by contact
with a stimnl~tQry form of B7-2 protein, for such purposes as generating retroviruses (e.g.,
various HIV isolates) in sufficient quantities for isolatation and use.
F. Therapeutic Uses by Upre~ulation of Tmmllne ~esponses
Upregulation of a B lymphocyte antigen function, as a means of upregulating immune
responses, may also be useful in therapy. Upregulation of immllne responses may be in the
form of enhancing an existing immune response or eliciting an initial immune response. For
WOg5/03~8 ~16 ~ O 91 PCT~S94/08423
-39-
example, enhancing an immune response through stimul~ting B lymphocyte antigen function
may be useful in cases of viral infection. Viral infections are cleared primarily by cytolytic T
cells. In accordance with the present invention, it is believed that B7-2 and thus, B7-1 and
B7-3 with their natural ligand(s) on T cells may result in an increase in the cytolytic activity
S of at least some T cells. It is also believed that B7-2,B7-1, and B7-3 are involved in the
initial activation and generation of CD8~ cytotoxic T cells. The addition of a soluble peptide
having B7-2 activity, alone, or in combination with a peptide having the activity of another B
lymphocyte antigen, in a multi-valent form, to stim~ te T cell activity through the
costimulation pathway would thus be therapeutically useful in situations where more rapid or
10 thorough clearance of virus would be beneficial. These would include viral skin diseases
such as Herpes or shingles, in which cases the multi-valent soluble peptide having B7-2
activity or combination of such peptide and/or a peptide having B7-1 activity and/or a peptide
having B7-3 activity is delivered topically to the skin. In addition, systemic viral diseases
such as influenza, the common cold, and encephalitis might be alleviated by the
15 ~lmini.etration of stiml]l~tQry forms of B lymphocyte antigens systemically.
Altern~tively, anti-viral imml~ne responses may be enhanced in an infected patient by
removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed
APCs either ex~lc;ssillg a peptide having B7-2 activity (alone or in combination with a
peptide having B7-1 activity and/or a peptide having B7-3 activity) or together with a
20 stim~ tory form of a soluble peptide having B7-2 activity (alone or in combination with a
peptide having B7-1 activity and/or a peptide having B7-3 activity) and reintroducing the in
vitro activated T cells into the patient. Another method of enhancing anti-viral immune
responses would be to isolate infected cells from a patient, transfect them with a nucleic acid
encoding a peptide having the activity of a B Iymphocyte antigen as described herein such
that the cells express all or a portion of a B lymphocyte antigen on their surface, e.g., B7-2 or
B7-3, and reintroduce the transfected cells into the patient. The infected cells would now be
capable of delivering a costimlll~tory signal to, and thereby activate, T cells in vivo.
Stim~ tory forms of B lymphocyte antigens may also be used prophylactically in
vaccines against various pathogens. Immunity against a pathogen, e.g., a virus, could be
induced by vaccinating with a viral protein along with a stimulatory form of a peptide having
B7-2 activity or another peptide having the activity of B lymphocyte antigen in an
a~lupliate adjuvant. Alternately, an expression vector which encodes genes for both a
pathogenic antigen and a peptide having the activity of a B lymphocyte antigen, e.g., a
vaccinia virus expression vector engineered to express a nucleic acid encoding a viral protein
and a nucleic acid encoding a peptide having B7-2 activity as described herein, can be used
for vaccination. Present~tion of B7-2 with class I MHC proteins by, for example, a cell
transfected to coexpress a peptide having B7-2 activity and MHC class I a chain protein and
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~2 microglobulin may also result in activation of cytolytic CD8+ T cells and provide
immunity from viral infection. Pathogens for which vaccines may be useful include hepatitis
B, hepatitis C, Epstein-Barr virus, cytomegalovirus, HIV-1, HIV-2, tuberculosis, malaria and
schistosomiasis.
In another aspect, a stimulatory form of one or more soluble peptides having an
activity of a B lymphocyte antigen can be ~tlmini~tered to a tumor-bearing patient to provide
a costim~ tQry signal to T cells in order to induce anti-tumor immllnity.
G. Modification of a Tumor Cell to Fxpress a Costimulatory Molecule
The inability of a tumor cell to trigger a costimulatory signal in T cells may be due to
a lack of e~s~,ei,~ion of a costiml-l~tory molecule, failure to express a costim~ tory molecule
even though the tumor cell is capable of t;x~r~s~ lg such a molecule, insufficient expression
of a costimulatory molecule on the tumor cell surface or lack of ex~r~s~ion of an appropriate
costiml-l~t--ry molecule (e.g. ex~,les~ion of B7 but not B7-2 and/or B7-3). Thus, according to
one aspect of the invention, a tumor cell is modified to express B7-2 and/or B7-3 by
transfection of the tumor cell with a nucleic acid encoding B7-2 and/or B7-3 in a form
suitable for ~,es~ion of B7-2 and/or B7-3 on the tumor cell surface. Alternatively, the
tumor cell is modified by contact with an agent which induces or increases expression of B7-
2 and/or B7-3 on the tumor cell surface. In yet another embodiment, B7-2 and/or B7-3is
coupled to the surface of the tumor cell to produce a modified tumor cell. These and other
emodiments are described in further detail in the following subsections.
(1). Tr~n~fection of a Tumor Cell with a Nucleic Acid Fn~oding a Costimulatory
Molecule
Tumor cells can be modified ex vivo to express B7-2 or B7-3, alone or in combination
or in combination with B7-1 by transfection of isolated tumor cells with a nucleic acid
encoding B7-2 and/or B7-3 and B7-1 in a form suitable for ~ s~.ion of the molecule on the
surface of the tumor cell. The terms "transfection" or "transfected with" refers to the
introduction of exogenous nucleic acid into a m~mm~ n cell and encompass a variety of
techniques useful for introduction of nucleic acids into m~nnm~ n cells including
electroporation, calciurn-phosphate precipitation, DEAE-dextran treatment, lipofection,
microinjection and infection with viral vectors. Suitable methods for transfecting
m~mm~ n cells can be found in Sarnbrook et al. (Molec~ r Clonin~: A T ~horatory ~anuaL
~nd F.rlition, Cold Spring Harbor Laboratory press (1989)) and other laboratory textbooks.
The nucleic acid to be introduced may be, for example, DNA encompassing the gene(s)
encoding B7-2 and/or B7-3, sense strand RNA encoding B7-2 and/or B7-3 or a recombinant
WO 95lO3408 21 6 7 0 91 pcTluss4los423
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expression vector containing a cDNA encoding B7-2 and/or B7-3. The nucleotide sequence
of a cDNA encoding human B7-2 is shown in the Sequence Listing.
A plefe,l~d approach for introducing nucleic acid encoding B7-2 and/or B7-3 intotumor cells is by use of a viral vector cont~ining nucleic acid, e.g. a cDNA, encoding B7-2
and/or B7-3. Examples of viral vectors which can be used include retroviral vectors (Eglitis,
M.A., et al., Science 230, 1395-1398 (1985); Danos, O. and Mulligan, R., Proc. Natl. Acad.
Sci. USA 85, 6460-6464 (1988); Markowitz, D., et al., J. Virol. 62, 1120-1124 (1988)),
adenoviral vectors (Rosenfeld, M.A., et al., Cell 68, 143-155 (1992)) and adeno-associated
viral vectors (Tratschin, J.D., et al., Mol. Cell. Biol. 5, 3251-3260 (1985)). Infection of tumor
cells with a viral vector has the advantage that a large proportion of cells will receive nucleic
acid, thereby obviating a need for selection of cells which have received nucleic acid, and
molecules encoded within the viral vector, e.g. by a cDNA contained in the viral vector, are
expressed efficiently in cells which have taken up viral vector nucleic acid.
Alternatively, B7-2 and/or B7-3 can be expressed on a tumor cell using a plasmidexl ,es~ion vector which contains nucleic acid, e.g. a cDNA, encoding B7-2 and/or B7-3.
Suitable plasmid ~x~ ssion vectors include CDM8 (Seed, B., Nature 329, 840 (1987)) and
pMT2PC (~ n, et al., EMBO J. 6, 187- 195 (1987)). Suitable vectors and methods for
~x~le3~ing nucleic acids in host cells, such as tumor cells are described in further detail
herein.
When transfection of tumor cells leads to modification of a large proportion of the
tumor cells and efficient expression of B7-2 and/or B7-3 on the surface of tumor cells, e.g.
when using a viral ex~leSSiOn vector, tumor cells may be used without further isolation or
subcloning. Alternatively, a homogenous population of transfected tumor cells can be
prepared by isolating a single transfected tumor cell by limitin~ dilution cloning followed by
expansion of the single tumor cell into a clonal population of cells by standard techniques.
(2). Tn~ tion or Jncr~ed F~ression of a Costimulatory Molecule on a T-lmor
Cell Surface
A tumor cell can be modified to trigger a costim--l~tory signal in T cells by inducing
or increasing the level of expression of B7-2 and/or B7-3 on a tumor cell which is capable of
expressing B7-2 and/or B7-3 but fails to do so or which expresses insufficient amounts of
B7-2 and/or B7-3 to activate T cells. An agent which stimulates expression of B7-2 and/or
B7-3 can be used in order to induce or increase expression of B7-2 and/or B7-3 on the tumor
cell surface. ~or example, tumor cells can be contacted with the agent in vitro in a culture
medium. The agent which stimulates expression of B7-2 and/or B7-3 may act, for instance,
by increasing transcription of B7-2 and/or B7-3 gene, by increasing translation of B7-2
and/or B7-3 mRNA or by increasing stability or transport of B7-2 and/or B7-3 to the cell
WO 95t03408 ~16 ~ ~ ~1 PCT/US94/08423
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s-lrf~ce For example, it is known that expression of B7 can be upregulated in a cell by a
second messenger pathway involving cAMP. Nabavi, N., et al. Nature 360, 266-268 (1992).
B7-2 and B7-3 may likewise be inducible by cAMP. Thus, a tumor cell can be contacted
with an agent, which increases intracellular cAMP levels or which mimics cAMP, such as a
5 cAMP analogue, e.g. dibutyryl cAMP, to stim~ te expression of B7-2 and/or B7-3 on the
tumor cell surface. It is also known that expression of B7 can be in(l~lcecl on normal resting B
cells by cro.s~linking cell-surface MHC class II molecules on the B cells with an antibody
against the MHC class II molecules. Kuolova, L., et al., J. Exp. Med 173, 759-762 (1991).
Similarly, B7-2 and B7-3 can be in~ ecl on resting B cells by crosslinking cell-surface MHC
10 class II molecules on the B cells. Accordingly, a tumor cell which expresses MHC class Il
molecules on its surface can be treated with anti-MHC class II antibodies to induce or
increase B7-2 and or B7-3 ex~les~ion on the tumor cell surface. In addition, interleukin-4
(IL-4) which has been found to induce expression of B7-2 on B cells, may be used to
upregulate expression of B7-2 on tumor cells (Stack R.M., et al., J. Cell. Biochem. Suppl
1(18):434 (1994).
Another agent which can be used to induce or increase expression of B7-2 and/or B7-
3 on a tumor cell surface is a nucleic acid encoding a transcription factor which upregulates
transcription of the gene encoding the costimulatory molecule. This nucleic acid can be
transfected into the tumor cell to cause increased transcription of the costim~ tory molecule
20 gene, resulting in increased cell-surface levels of the costimulatory molecule.
(3). Couplir~ of a Costimulatory Molecule to the Sllrface of a Tllmor Cell
In another embodiment, a tumor cell is modified to be capable of triggering a
costim~ tory signal in T cells by coupling B7-2 and/or B7-3 to the surface of the tumor cell.
25 For example, B7-2 and/or B7-3 molecules can be obtained using standard recombinant DNA
technology and ~ s~ion systems which allow for production and isolation of the
costim~ tory molecule(s). Altern~tively, B7-2 and/or B7-3 can be isolated from cells which
express the costimlll~tory molecule(s) using standard protein purification techniques. For
example, B7-3 protein can be isolated from activated B cells by immunoprecipitation with an
30 anti-B7-3 antibody such as the BB1 monoclonal antibody. The isolated costimlll~tory
molecule is then coupled to the tumor cell. The terms "coupled" or "coupling" refer to a
chemical, enzymatic or other means (e.g., antibody) by which B7-2 and/or B7-3 is linked to a
tumor cell such that the costimulatory molecule is present on the surface of the tumor cell and
is capable of triggering a costimulatory signal in T cells. For example, B7-2 and/or B7-3 can
35 be chemically crosslinked to the tumor cell surface using commercially available cro~slinking
reagents (Pierce, Rockford IL). Another approach to coupling B7-2 and/or B7-3 to a tumor
cell is to use a bispecific antibody which binds both the costim~ tory molecule and a cell-
WO 95/0340~ 2 1 6 7 0 9 ~ PCT/US94/08423
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surface molecule on the tumor cell. Fragments, mutants or variants of B7-2 and/or B7-3
which retain the ability to trigger a costim~ tQry signal in T cells when coupled to the
surface of a tumor cell can also be used.
(4). Mo~lificatio~ of T-lmor Cell~ to Fxpress Multiple Costimulatory Molecules
Another aspect of the invention is a tumor cell modified to express multiple
costimlll~tory molecules. The temporal ~ s~ion of costim~ tory molecules on activated
B cells is different for B7, B7-2 and B7-3. For example, B7-2 is expressed early following B
cell activation, whereas B7-3 is ~ essed later. The different costim~ tory molecules may
thus serve distinct functions during the course of an immllne response. An effective T cell
response may require that the T cell receive costim~ tQry signals from multiple
costimulatory molecules. Accordingly, the invention encompasses a tumor cell which is
modified to express more than one costimlll~tQry molecule. For example, a tumor cell can be
modified to express both B7-2 and B7-3. ~It~rn~tively, a tumor cell modified to express B7-
2 can be further modified to express B7-1. Similarly, a tumor cell modified to express B7-3
can be further modified to express B7-1. A tumor cell can also be modified to express B7-1,
B7-2 and B7-3. A tumor cell can be modified to express multiple costimulatory molecules
(e.g., B7- 1 and B7-2) by any of the techniques described herein.
Before modification, a tumor cell may not express any costimulatory molecules, or
may express certain costimulatory molecules but not others. As described herein, tumor cells
can be modified by transfecting the tumor cell with nucleic acid encoding a costimulatory
molecule(s), by inducing the c;~s~les~ion of a costim~ tory molecule(s) or by coupling a
costimulatory molecule(s) to the tumor cell. For example, a tumor cell transfected with
nucleic acid encoding B7-2 can be further transfected with nucleic acid encoding B7-1. The
cDNA sequence and d~ ce~1 amino acid sequence of human B7-1 is shown in the Sequence
T .i~ting Alten ~tively, more than one type of modification can be used. For example, a
tumor cell transfected with a nucleic acid encoding B7-2 can be stimulated with an agent
which in~ ces t;~ s~ion of B7-1.
(5) Additio~l Motlifiçation of a Tllmor Cell to F~ress MHC Molec--les
Another aspect of this invention features modified tumor cells which express a
costimulatory molecule and which express one or more MHC molecules on their surface to
trigger both a costimulatory signal and a primary, antigen-specific, signal in T cells. Before
modification, tumor cells may be unable to express MHC molecules, may fail to express
MHC molecules although they are capable of e~les~ing such molecules, or may express
insufficient amounts of MHC molecules on the tumor cell surface to cause T cell activation.
Tumor cells can be modified to express either MHC class I or MHC class II molecules, or
WO 95/03408 PCT/US94/08423
2~09i ~
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both. One approach to modifying tumor cells to express MHC molecules is to transfect the
tumor cell with one or more nucleic acids encoding one or more MHC molecules.
Alternatively, an agent which induces or increases expression of one or more MHCmolecules on tumor cells can be used to modify tumor cells. Inducing or increasing
5 ~ s~ion of MHC class II molecules on a tumor cell can be particularly beneficial for
activating CD4+ T cells against the tumor since the ability of MHC class II+ tumor cells to
directly present tumor peptides to CD4+ T cells bypasses the need for professional MHC
class II+ APCs. This can improve tumor immunogenicity because soluble tumor antigen (in
the form of tumor cell debris or secreted protein) may not be available for uptake by
10 professional MHC class II + APCs.
One embodiment of the invention is a modified tumor cell which expresses B7-2
and/or B7-3 and one or more MHC class II molecules on their cell surface. MHC class II
molecules are cell-surface al~ heterodimers which structurally contain a cleft into which
antigenic peptides bind and which function to present bound peptides to the antigen-specific
15 TcR. Multiple, different MHC class II proteins are e~les~ed on professional APCs and
different MHC class II proteins bind different antigenic peptides. Expression of multiple
MHC class II molecules, therefore, increases the spectrum of antigenic peptides that can be
presented by an APC or by a modified tumor cell. The a and ,B chains of MHC class II
molecules are encoded by dirrt l~llL genes. For instance, the hurnan MHC class II protein
20 HLA-DR is encoded by the HLA-DRa and HLA-DR,~ genes. Additionally, many
polymorphic alleles of MHC class II genes exist in human and other species. T cells of a
particular individual respond to stimulation by antigenic peptides in conjunction with self
MHC molecules, a phenomenon termed MHC restriction. In addition, certain T cells can also
respond to stim~ tion by polymorphic alleles of MHC molecules found on the cells of other
25 individuals, a phenomenon termed allogenicity. For a review of MHC class II structure and
function, see Germain and Margulies, Ann. Rev. Immunol. 1 1: 403-450, 1993.
Another embodiment of the invention is a modified tumor cell which expresses B7-2
and/or B7-3 and one or more MHC class I molecules on the cell surface. Similar to MHC
class II genes, there are multiple MHC class I genes and many polymorphic alleles of these
30 genes are found in human and other species. Like MHC class II proteins, class I proteins
bind peptide fr~gment~ of antigens for presentation to T cells. A functional cell-surface class
I molecule is composed of an MHC class I a chain protein associated with a ~2-
microglobulin protein.
(6). Tran~fection of a Tumor Cell with Nucleic Acid F.nco~lin~ M~IC Molecules
Tumor cells can be modified ex vivo to express one or more MHC class II molecules
by transfection of isolated tumor cells with one or more nucleic acids encoding one or more
~WO 95/03408 21 6 ~ O ~1 PCT/US94/08423
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MHC class II a chains and one or more MHC class II ~ chains in a form suitable for
~x~ ssion of the MHC class II molecules(s) on the surface of the tumor cell. Both an a and
a ,B chain protein must be present in the tumor cell to form a surface heterodimer and neither
chain will be expressed on the cell surface alone. The nucleic acid sequences of many murine
and human class II genes are known. For examples see Hood, L., et al. Ann. Rev. Immunol. 1,
529-568 (1983) and Auffray, C. and Strominger, J.L., Advances in Human Genetics 15, 197-
247 (1987). Preferably, the introduced MHC class II molecule is a selfMHC class II
molecule. Alternatively, the MHC class II molecule could be a foreign, allogeneic, MHC
class II molecule. A particular foreign MHC class II molecule to be introduced into tumor
cells can be selected by its ability to induce T cells from a tumor-bearing subject to
proliferate and/or secrete cytokines when stimulated by cells expressing the foreign MHC
class II molecule (i.e. by its ability to induce an allogeneic response). The tumor cells to be
transfected may not express MHC class II molecules on their surface prior to transfection or
may express amounts insufficient to stim~ t~ a T cell response. Alternatively, tumor cells
which express MHC class II molecules prior to transfection can be further transfected with
additional, different MHC class II genes or with other polymorphic alleles of MHC class II
genes to increase the spectrum of antigenic fr~gment~ that the tumor cells can present to T
cells.
Fr~ment.~, mutants or variants of MHC class II molecules that retain the ability to
bind peptide antigens and activate T cell responses, as evidenced by proliferation and/or
lymphokine production by T cells, are considered within the scope of the invention. A
preferred variant is an MHC class II molecule in which the cytoplasmic domain of either one
or both of the a and ~ chains is tr~lnc~t~A It is known that truncation of the cytoplasmic
domains allows peptide binding by and cell surface ex~le3~ion of MHC class II molecules but
prevents the induction of endogenous B7 ~x~les~ion, which is triggered by an intracellular
signal generated by the cytoplasmic domains of the MHC class II protein chains upon
crosslinking of cell surface MHC class II molecules. Kuolova. L., et al., J. Exp. Med. 173,
759-762 (1991), Nabavi, N., et al. Nature 360, 266-268 (1992). Expression of B7-2 and B7-3
is also in~ recl by crosslinking surface MHC class II molecules, and thus truncation of MHC
class II molecules may also prevent induction of B7-2 and/or B7-3. In tumor cells transfected
to constitutively express B7-2 and/or B7-3, it may be desirable to inhibit the expression of
- endogenous costimulatory molecules, for instance to restrain potential downregulatory
fee~lb~ck mech~ni~m~ Transfection of a tumor cell with a nucleic acid(s) encoding a
cytoplasmic domain-trllnr~tt~l form of MHC class II a and ,B chain proteins would inhibit
endogenous B7-1 expression and possibly also endogenous B7-2 and B7-3 expression. Such
variants can be produced by, for example, introducing a stop codon in the MHC class II chain
gene(s) after the nucleotides encoding the transmembrane spanning region. The cytoplasmic
WO 95/03408 PCT/US94/08423
2~0~1 ~
-46-
domain of either the a chain or the ,B chain protein can be truncated, or~ for more complete
inhibition of B7 (and possibly B7-2 and/or B7-3) induction, both the a and ,B chains can be
kuncated. See e.g. Griffith et al., Proc. Natl. Acad. Sci US~l 85: 4847-4852, (1988), Nabavi
et al., J. Immunol. 142: 1444-1447, (1989).
S Turnor cells can be modified to express an MHC class I molecule by kansfection with
a nucleic acid encoding an MHC class I a chain protein. For examples of nucleic acids see
Hood, L., et al. Ann. Rev. ImmunoL 1, 529-568 (1983) and Auffray, C. and Strominger, J.L.,
Advances in Human Genetics 15, 197-247 (1987). Optionally, if the tumor cell does not
express ,~-2 microglobulin, it can also be kansfected with a nucleic acid encoding the ~-2
microglobulin protein. For examples of nucleic acids see Gussow, D., et al., J. Immunol. 139,
3132-3138 (1987) and Parnes, J.R., et al., Proc. Natl. Acad. Sci. USA 78, 2253-2257 (1981).
As for MHC class II molecules, increasing the number of different MHC class I genes or
polymorphic alleles of MHC class I genes expressed in a tumor cell can increase the spectrum
of antigenic fr~gment~ that the turnor cells can present to T cells.
When a tumor cell is kansfected with nucleic acid which encodes more than one
molecule, for example a B7-2 and/or B7-3 molecule(s), an MHC class II a chain protein and
an MHC class II ,B chain protein, the transfections can be performed simultaneously or
sequentially. If the transfections are performed ~iml-lt~neously, the molecules can be
introduced on the same nucleic acid, so long as the encoded sequences do not exceed a
carrying capacity for a particular vector used. Alternatively, the molecules can be encoded by
separate nucleic acids. If the kansfections are con~ cte~l sequentially and tumor cells are
selected using a selectable marker, one selectable marker can be used in conjunction with the
first inkoduced nucleic acid while a dirr~ l selectable marker can be used in conjunction
with the next introduced nucleic acid.
The expression of MHC molecules (class I or class II) on the cell surface of a turnor
cell can be determined, for example, by immllnoflourescence of tumor cells usingfluorescently labeled monoclonal antibodies directed against different MHC molecules.
Monoclonal antibodies which recognize either non-polymorphic regions of a particular MHC
molecule (non-allele specific) or polymorphic regions of a particular MHC molecule (allele-
specific) can be used and are known to those skilled in the art.
(7). In~ tion or Tncreased Fxpression of MHC Molecules on a Tumor Cell
Another approach to modifying a tumor cell ex vivo to express MHC molecules on the
surface of a tumor cell is to use an agent which stimulates expression of MHC molecules in
order to induce or increase expression of MHC molecules on the tumor cell surface. For
example, tumor cells can be contacted with the agent in vitro in a culture medium. An agent
which stimul~tes expression of MHC molecules may act, for instance, by increasing
WO 95/03408 PCT/US94/08423
2l~a~l
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transcription of MHC class I and/or class II genes, by increasing translation of MHC class I
and/or class II mRNAs or by increasing stability or transport of MHC class I and/or class Il
proteins to the cell surface. A nurnber of agents have been shown to increase the level of
cell-surface expression of MHC class II molecules. See for example Cockfield, S.M. et al., J.
5 Immunol. 144, 2967-2974 (1990); Noelle, R.J. et al. J. ImmunoL 137, 1718-1723 (1986);
Mond, J.J., et al., J. lmmunol. 127, 881-888 (1981); Willman, C.L., et al. J. Exp. Med., 170,
1559-1567 (1989); Celada, A.and Maki, R. J. Immunol. 146, 114-120 (1991) and Glimcher,
L.H. and Kara, C.J. Ann. Rev. Immunol. 10, 13-49 (1992) and references therein. These
agents include cytokines, antibodies to other cell surface molecules and phorbol esters. One
agent which upregulates MHC class I and class II molecules on a wide variety of cell types is
the cytokine interferon-~. Thus, for example, tumor cells modif1ed to express B7-2 and/or
B7-3 and B7- 1 can be further modified to increase ~ cssion of MHC molecules by contact
with interferon-~.
Another agent which can be used to induce or increase ex~l~ssion of an MHC
molecule on a tumor cell surface is a nucleic acid encoding a transcription factor which
upregulates transcription of MHC class I or class II genes. Such a nucleic acid can be
transfected into the tumor cell to cause increased transcription of MHC genes, resulting in
increased cell-surface levels of MHC proteins. MHC class I and class II genes are regulated
by different transcription factors. However, the multiple MHC class I genes are regulated
coordinately, as are the multiple MHC class II genes. Therefore, transfection of a tumor cell
with a nucleic acid encoding a transcription factor which regulates MHC gene expression
may increase e~res~ion of several different MHC molecules on the tumor cell surface.
Several transcription factors which regulate the expression of MHC genes have been
identified, cloned and characterized. For example, see Reith, W. et al., Genes Dev. 4, 1528-
1540, (1990); Liou, H.-C., et al., Science 247, 1581-1584 (1988), Didier, D.K., et al., Proc.
Natl. Acad. Sci. US~ 85, 7322-7326 (1988).
(8). Inhibition of Invari~nt Ch~in Fxpression in Tllmor Cell~
Another embodiment of the invention provides a tumor cell modified to express a T
cell costimulatory molecule (e.g., B7-2 and/or B7-3 and B7-1) and in which expression of an
MHC class II-associated protein, the invariant chain, is inhibited. Invariant chain expression
is inhibited to promote association of endogenously-derived TAA peptides with MHC class II
molecules to create an antigen-MHC complex. This complex can trigger an antigen-specific
signal in T cells to induce activation of T cells in conjunction with a costimulatory signal.
MHC class II molecules have been shown to be capable of pres~ontinP endogenously-derived
peptides. Nuchtern, J.G., et al. Nature 343, 74-76 (1990); Weiss, S. and Bogen, B. Cell 767-
776 (1991). However, in cells which naturally express MHC class II molecules, the a and ,B
WO 95/03408 ;,~ Q 9 l PCT/US94/08423
-48 -
chain proteins are associated with the invariant chain (hereafter Ii) during intracellular
transport of the proteins from the endoplasmic reticulum. It is believed that Ii functions in
part by preventing the association of endogenously-derived peptides with MHC class II
molecules. Elliott, W., et al. J. Immunol. 138, 2949-2952 (1987); Stockinger, B., et al. Cell
56, 683-689 (1989); Guagliardi, L., et al. Nature (London) 343, 133-139 (1990); Bakke, O.,
et al. Cell 63, 707-716 (1990); Lottreau, V., et al. Nature 348,600-605 (1990); Peters, J., et
al. Nature 349, 669-676 (1991); Roche, P., et al.Nature 345, 615-618 (1990); Teyton, L., et
al. Nature 348, 39-44 (1990). Since TAAs are synthesized endogenously in tumor cells,
peptides derived from them are likely to be available intracellularly. Accordingly, inhibiting
the ex~,~s~ion of Ii in tumor cells which express Ii may increase the likelihood that TAA
peptides will associate with MHC class II molecules. Consistent with this mech~ni~m, it was
shown that supertransfection of an MHC class II+, Ii- tumor cell with the Ii gene prevented
stim~ tion of tumor-specific immllnity by the tumor cell. Clements, V.K., et al. J. Immunol.
149, 2391-2396 (1992).
Prior to modification, the ~ rt;s~ion of Ii in a tumor cell can be assessed by detecting
the presence or absence of Ii mRNA by Northern blotting or by detecting the presence or
~bsence of Ii protein by imml-noprecipitation. A preferred approach for inhibiting ex~ ssion
of Ii is by introducing into the tumor cells a nucleic acid which is antisense to a coding or
regulatory region of the Ii gene, which have been previously described. Koch, N., et al.,
EMBO J. 6, 1677-1683, (1987). For example7 an oligonucleotide complement~ry to
nucleotides near the translation initiation site of the Ii mRNA can be synth~si7P-l One or
more antisense oligonucleotides can be added to media cont~inin~ tumor cells, typically at a
concentration of oligonucleotides of 200 ,~Lg/ml. The ~ntisçn~e oligonucleotide is taken up by
tumor cells and hybridizes to Ii mRNA to prevent translation. In another embodiment, a
recombinant expression vector is used in which a nucleic acid encoding sequences of the Ii
gene in an orientation such that mRNA which is ~nti~çn~e to a coding or regulatory region of
the Ii gene is produced. Tumor cells transfected with this recombinant expression vector thus
contain a continuous source of Ii ~nfi~çn~e nucleic acid to prevent production of Ii protein.
~ltern~tively, Ii expression in a tumor cell can be inhibited by treating the tumor cell with an
agent which interferes with Ii expression. For example, a ph~ ceutical agent which
inhibits Ii gene c~ es~ion, Ii mRNA translation or Ii protein stability or intracellular
transport can be used.
(9). Types of Tl-mor Cells to be Modified
The tumor cells to be modified as described herein include tumor cells which can be
transfected or treated by one or more of the approaches encompassed by the present invention
to express B7-2 and/or B7-3, alone or in combination with B7-1. If necessary, the tumor
wo 9~,03408 ~ 9 ~ PCT/US94108423
-49-
cells can be further modified to express MHC molecules or an inhibitor of Ii expression. A
tumor from which tumor cells are obtained can be one that has arisen spontaneously, e.g in a
human subject, or may be experimentally derived or induced, e.g. in an animal subject. The
tumor cells can be obtained, for example, from a solid tumor of an organ, such as a tumor of
5 the lung, liver, breast, colon, bone etc. ~lign~ncies of solid organs include carcinomas,
sarcomas, melanomas and neuroblastomas. The tumor cells can also be obtained from a
blood-borne (ie. dispersed) m~ n~ncy such as a lymphoma, a myeloma or a leukemia.
The tumor cells to be modified include those that express MHC molecules on theircell surface prior to transfection and those that express no or low levels of MHC class I
and/or class II molecules. A minority of normal cell types express MHC class II molecules.
It is therefore expected that many tumor cells will not express MHC class II molecules
naturally. These tumors can be modified to express B7-2 and/or B7-3 and MHC class II
molecules. Several types of tumors have been found to naturally express surface MHC class
II molecules, such as melanomas (van Duinen et al., Cancer Res. 48, 1019-1025, 1988),
diffuse large cell lymphomas (O'Keane et al., Cancer 66, 1147-1153, 1990), squamous cell
carcinomas of the head and neck (Mattijssen et al., Int. J. Cancer 6, 95- l O0, 1991) and
colorectal carcinomas (Moller et al., Int. J. Cancer 6, 155-162, 1991). Tumor cells which
naturally express class II molecules can be modified to express B7-2 and/or B7-3, and, in
addition, other class II molecules which can increase the spectrum of TAA peptides which
can be presented by the tumor cell. Most non-m~ n~nt cell types express MHC class I
molecules. However, m~lign~nt transformation is often accompanied by downregulation of
expression of MHC class I molecules on the surface of tumor cells. Csiba, A., et al., Brit. J.
Cancer 50, 699-709 (1984). Importantly, loss of expression of MHC class I antigens by
tumor cells is associated with a greater aggressiveness and/or metastatic potential of the
tumor cells. Schrier, P.I., et al. Nature 305, 771-775 (1983); Holden, C.A., et al. J. Am. Acad.
Dermatol. 9., 867-871 (1983); Baniyash, M., et al. J Immunol. 129, 1318-1323 (1982).
Types of tumors in which MHC class I expression has been shown to be inhibited include
melarlomas, colorectal carcinomas and squarnous cell carcinomas. van Duinen et al., Cancer
Res. 48, 1019-1025, (1988); Moller et al., Int. J. Cancer 6, 155-162, (1991), Csiba, A., et al.,
Brit. J. Cancer 50, 699-709 (1984); Holden, C.A., et al. J. Am. Acad. Dermatol. 9., 867-871
(1983). A tumor cell which fails to express class I molecules or which expresses only low
levels of MHC class I molecules can be modified by one or more of the techniques described
herein to induce or increase expression of MHC class I molecules on the tumor cell surface to
enhance tumor cell immlmngenicity.
WO 95/03408 PCTtUS94/08423
2~7~ 50
(10). Modification of Tllmor Cells In Vivo
Another aspect of the invention provides methods for increasing the immunogenicity
of a tumor cell by modification of the tumor cell in vivo to express B7-2 and/or B7-3 and B7- ~
1 to trigger a costimulatory signal in T cells. In addition, tumor cells can be further modified
in vivo to express MHC molecules to trigger a primary, antigen-specific, signal in T cells.
Tumor cells can be modified in vivo by introducing a nucleic acid encoding B7-2 and/or B7-3
and B7-1 into the tumor cells in a form suitable for expression of the costimnl~t-)ry
molecule(s) on the surface of the tumor cells. Likewise, nucleic acids encoding MHC class I
or class II molecules or an ~nti~Pn~e sequence of the Ii gene can be introduced into tumor
cells in vivo. In one embodiment, a recombinant ~x~les~ion vector is used to deliver nucleic
acid encoding B7-2 and/or B7-3 and B7-1 to tumor cells in vivo as a form of gene therapy.
Vectors useful for in vivo gene therapy have been previously described and include retroviral
vectors, adenoviral vectors and adeno-associated viral vectors. See e.g. Rosenfeld, M.A.,
Cell 68, 143-155 (1992); Anderson, W.F., Science 226, 401-409 (1984); Friedman, T.,
Science 244, 1275-1281 (1989). Alternatively, nucleic acid can be delivered to tumor cells in
vivo by direct injection of naked nucleic acid into tumor cells. See e.g. Acsadi, G., et al.,
Nafure 332, 815-818 (1991). A delivery ayy~lus is commercially available (BioRad).
Optionally, to be suitable for injection, the nucleic acid can be complexed with a carrier such
as a liposome. Nucleic acid encoding an MHC class I molecule complexed with a liposome
has been directly injected into tumors of melanoma patients. Hoffman, M., Science 256, 305-
309 (1992)-
Tumor cells can also be modified in vivo by use of an agent which induces or
increases ~;x~,c;s~ion of B7-2 and/or B7-3 and B7-1 (and, if necessary, MHC molecules) as
described herein. The agent may be ~lmini.~tered systemically, e.g. by inll~v~llous injection,
or, preferably, locally to the tumor cells.
(11). The Fffector Ph~ of the Anti-TIlmor T Cell-Mediated ~mmune Response
The modified tumor cells of the invention are useful for stimnl~ting an anti-tumor T
cell-mediated immune response by triggering an antigen-specific signal and a costimlll~tory
signal in tumor-specific T cells. Following this inductive, or afferent, phase of an immlm~
response, effector populations of T cells are generated. These effector T cell populations can
include both CD4+ T cells and CD8+ T cell. The effector populations are responsible for
elimin~tion of tumors cell, by, for example, cytolysis of the tumor cells. Once T cells are
activated, ~;xylt;;s~ion of a costim~ tory molecule is not required on a target cell for
recognition of the target cell by effector T cells or for the effector functions of the T cells.
Harding, F.A. and Allison, J.P. J. ~xp. Med. 177, 1791-1796 (1993). Therefore, the anti-
tumor T cell-mç~i~tecl immune response inclll~ecl by the modified tumor cells of the invention
WO 95/03408 2 ~ 6 ~ ~ ~1 PCT/tJS94/08423
-51-
is effective against both the modified tumor cells and unrnodified tumor cells which do not
express a costim~ tory molecule.
- Additionally, the density and/or type of MHC molecules on the cell surface required
for the afferent and efferent phases of a T cell-mediated immune response can differ. Fewer
S MHC molecules, or only certain types of MHC molecules (e.g. MHC class I but not MHC
class II) may be needed on a tumor cell for recognition by effector T cells than is needed for
the initial activation of T cells. Therefore, tumor cells which naturally express low amounts
of MHC molecules but are modified to express increased amounts of MHC molecules can
induce a T cell-mediated imm~lne response which is effective against the unmodified tumor
10 cells. Alt~rn~tively, tumor cells which naturally express MHC class I molecules but not
MHC class II molecules which are then modified to express MHC class II molecules can
induce a T cell-mediated imm~lne response which includes effector T cell populations which
can elimin~te the parental MHC class I+, class II- tumor cells.
(12). Therapeutic Co~ )osilions of Tllmor Cells
Another aspect of the invention is a composition of modified tumor cells in a
biologically compatible form suitable for ph~rm~ce-ltical ~-lminictration to a subject in vivo.
This composition compri~es an amount of modified tumor cells and a physiologically
acceptable carrier. The amount of modified tumor cells is selected to be therapeutically
20 effective. The term "biologically compatible form suitable for ph~rm~ceutical ~tlmini~tration
in vivo" means that any toxic effects of the tumor cells are outweighed by the therapeutic
effects of the tumor cells. A "physiologically acceptable carrier" is one which is biologically
compatible with the subject. Exarnples of acceptable carriers include saline and aqueous
buffer solutions. In all cases, the compositions must be sterile and must be fluid to the extent
25 that easy syringability exists. The term "subject" is inten~le~l to include living org~ni~m~ in
which tumors can arise or be experiment~lly in~ e~1 Examples of subjects include hllm~n~
dogs, cats, mice, rats, and transgenic species thereof.
A~lmini~tration of the therapeutic compositions of the present invention can be carried
out using known procedures, at dosages and for periods of time effective to achieve the
30 desired result. For example, a therapeutically effective dose of modified tumor cells may
vary according to such factors as age, sex and weight of the individual, the type of tumor cell
and degree of tumor burden, and the immunological competency of the subject. Dosage
regimens may be adjusted to provide optimum therapeutic responses. For instance, a single
dose of modified tumor cells may be ~lmini~tt?red or several doses may be ~-~mini~tered over
35 time. Admini~tration may be by injection, including intravenous, intramuscular,
a~e~;Loneal and subcutaneous injections.
W095/03~8 PCT~S94/08423
9~ ~
-52-
(13). Activation of Tllmor-specific T J,ymphocytes In Vitro
Another approach to inducing or enhancing an anti-tumor T cell-mediated immune
response by triggering a costimulatory signal in T cells is to obtain T lymphocytes from a
tumor-bearing subject and activate them in vitro by stimulating them with tumor cells and a
stim~ tory form of B7-2 and/or B7-3, alone or in combination with B7-1. T cells can be
obtained from a subject, for example, from peripheral blood. Peripheral blood can be further
fractionated to remove red blood cells and enrich for or isolate T lymophocytes or T
lymphocyte subpopulations. T cells can be activated in vitro by culturing the T cells with
tumor cells obtained from the subject (e.g. from a biopsy or from peripheral blood in the case
of blood-borne m~lign~ncies) together with a stimulatory form of B7-2 and/or B7-3 or,
t~rn~tively, by exposure to a modified tumor cell as described herein. The term
"stim~ tory form" means that the costimulatory molecule is capable of cro~linking its
receptor on a T cell and triggering a costimtll~tory signal in T cells. The stim~ tory form of
the costimulatory molecule can be, for example, a soluble multivalent molecule or an
immobilized form of the costimulatory molecule, for instance coupled to a solid support.
Fr~gment~, mllt~nt~ or variants (e.g. fusion proteins) of B7-2 and/or B7-3 which retain the
ability to trigger a costimlll~tory signal in T cells can also be used. In a plefellcd
embodiment, a soluble extracellular portion of B7-2 and/or B7-3is used to provide
costimlll~tion to the T cells. Following culturing of the T cells in vitro with tumor cells and
B7-2 and/or B7-3, or a modified tumor cell, to activate tumor-specific T cells, the T cells can
be ~lmini~t~red to the subject, for example by inkavenous injection.
(~4). Therapeutic Uses of Mo-1ified Tllmor Cells
The modified tumor cells of the present invention can be used to increase tumor
immunogenicity, and therefore can be used therapeutically for inducing or enhancing T
lymphocyte-me(li~te-l anti-tumor immunity in a subject with a tumor or at risk of developing
a tumor. A method for keating a subject with a tumor involves obtaining tumor cells from
the subject, modifying the tumor cells ex vivo to express a T cell costimlll~tory molecule, for
example by transfecting them with an al,plu~,;ate nucleic acid, and ~lmini~tering a
therapeutically effective dose of the modified tumor cells to the subject. Appropriate nucleic
acids to be inkoduced into a tumor cell include nucleic acids encoding B7-2 and/or B7-3,
alone or together with nucleic acids encoding B7-l,MHC molecules (class I or class II) or Ii
antisense sequences as described herein. Alternatively, after tumor cells are obtained from a
subject, they can be modified ex vivo using an agent which induces or increases expression of
B7-2 and/or B7-3 (and possibly also using agent(s) which induce or increase B7-1 or MHC
molecules).
WO 95/03408 2 1 6 7 0 9 1 PCT/US94/08423
-53-
Tumor cells can be obtained from a subject by, for example, surgical removal of
tumor cells, e.g. a biopsy of the tumor, or from a blood sample from the subject in cases of
blood-borne m~lign~ncies. In the case of an experimentally in-luced tumor, the cells used to
induce the tumor can be used, e.g. cells of a tumor cell line. Samples of solid tumors may be
5 treated prior to modification to produce a single-cell suspension of tumor cells for m~xim~l
efficiency of transfection. Possible tre~tment~ include manual dispersion of cells or
enzymat;c digestion of connective tissue fibers, e.g. by collagenase.
Tumor cells can be transfected immediately after being obtained from the subject or
can be cultured in vitro prior to transfection to allow for further ch~r~clel;~aLion of the tumor
cells (e.g. determination ofthe ~,ession of cell surface molecules). The nucleic acids
chosen for transfection can be cl~ i .ed following characterization of the proteins
expressed by the tumor cell. ~or instance, ~,res~ion of MHC proteins on the cell surface of
the tumor cells and/or expression of the Ii protein in the tumor cell can be assessed. Tumors
which express no, or limited amounts of or types of MHC molecules (class I or class II) can
be transfected with nucleic acids encoding MHC proteins; tumors which express Ii protein
can be transfected with Ii ~nti~en~e sequences. If necessary, following transfection, tumor
cells can be screened for introduction of the nucleic acid by using a selectable marker (e.g.
drug resistance) which is introduced into the tumor cells together with the nucleic acid of
interest.
Prior to ~tlmini~tration to the subject, the modified tumor cells can be treated to render
them incapable of further proliferation in the subject, thereby preventing any possible
outgrowth of the modified tumor cells. Possible tre~tment~ include irradiation or mitomycin
C treatment, which abrogate the proliferative capacity ofthe tumor cells while m~inl~i,li,lp;
the ability of the tumor cells to trigger antigen-specific and costimulatory signals in T cells
and thus to stim~ te an hlllllune response.
The modified tumor cells can be ~lmini~t?red to the subject by injection of the tumor
cells into the subject. The route of injection can be, for example, intravenous, intramuscular,
intraperitoneal or subcutaneous. ~tlminictration of the modified tumor cells at the site of the
original tumor may be beneficial for inducing local T cell-mediated immune responses
against the original tumor. Aclmini~tration of the modified tumor cells in a dissemin~tecl
manner, e.g. by intravenous injection, may provide systemic anti-tumor immunity and,
furthermore, may protect against metastatic spread of tumor cells from the original site. The
modified tumor cells can be ~-lmini~tered to a subject prior to or in conjunction with other
forms of therapy or can be ~mini~tered after other treatments such as chemotherapy or
surgical intervention.
Additionally, more than one type of modified tumor cell can be ~tlministered to a
subject. For example, an effective T cell response may require exposure of the T cell to more
-
W095/03~8 - PCT~S94/084~
2~70~ ~
-54-
than one type of costim~ t--ry molecule. Furthermore, the temporal sequence of exposure of
the T cell to different costimlll~tory mocules may be important for generating an effective
response. For example, it is known that upon activation, a B cell expresses B7-2 early in its
response (about 24 hours after stimulation). Subsequently, B7-1 and B7-3 are expressed by
the B cell (about 48-72 hours after stim~ tion). Thus, a T cell may require exposure to B7-2
early in the induction of an immlme response by exposure to B7-1 and/or B7-3 in the immune
response. Accordingly, different types of modified tumor cells can be ~tlminictered at
dirrele~ll times to a subject to generate an effective immune response against the tumor ce~lls.
For example, tumor cells modified to express B7-2 can be ~lmini~tered to a subject.
Following this ~-lmini~tration, a tumor cell from the same tumor but modified to express B7-
3 (alone or in conjunction with B7-1) can be ~-lmini~t~red to the subject.
Another method for treating a subject with a tumor is to modify tumor cells in vivo to
express B7-2 and/or B7-3, alone or in conjunction with B7-1, MHC molecules and/or an
inhibitor of Ii expression. This method can involve modifying tumor cells in vivo by
providing nucleic acid encoding the protein(s) to be expressed using vectors and delivery
methods effective for in vivo gene therapy as described in a previous section herein.
ltPrn~tively, one or more agents which induce or increase t;x~ies~ion of B7-2 and/or B7-3,
and possibly B7-1 or MHC molecules, can be ~rlmini.~t~ted to a subject with a tumor.
The modified tumor cells of the current invention may also be used in a method for
preventing or treating metastatic spread of a tumor or ~l~vt;llLing or treating recurrence of a
tumor. As demonstrated in detail in one of the following examples, anti-tumor immlmity
inclll~e~l by B7-1-~;x~les~ g turnor cells is effective against subsequent challenge by tumor
cells, regardless of whether the tumor cells of the re-exposure express B7-1 or not. Thus,
?rltnini~tr~tion of modified tumor cells or modification of tumor cells in vivo as described
herein can provide tumor immllnity against cells of the original, unmodified tumor as well as
mPt~t~es of the original tumor or possible l~,lOw~l of the original tumor.
The current invention also provides a composition and a method for specifically
inducing an anti-tumor response in CD4+ T cells. CD4+ T cells are activated by antigen in
conjunction with MHC class II molecules. Association of peptidic fragments of TAAs with
MHC class II molecules results in recognition of these antigenic peptides by CD4+ T cells.
Providing a subject with tumor cells which have been modified to express MHC class II
molecules along with B7-2 and/or B7-3, or modified in vivo to express MHC class II
molecules along with B7-2 and/or B7-3, can be useful for directing tumor antigenpresentation to the MHC class II pathway and thereby result in antigen recognition by and
activation of CD4+ T cells specific for the tumor cells. Depletion of either CD4+ or CD8+ T
cells in vivo, by ?~mini~tration of anti-CD4 or anti-CD8 antibodies, can be used to
Wo 95/03408 ~16 71) ~ 1 PCT/US94/08423
-55-
demonstrate that specific anti-tumor immllnity is mediated by a particular (e.g. CD4+) T cell
subpopulation.
Subjects initially exposed to modified tumor cells develop an anti-tumor specific T
cell response which is effective against subsequent exposure to unmodified tumor cells. Thus
r 5 the subject develops anti-tumor specific imm~lnity. The generalized use of modified tumor
cells of the invention from one human subject as an immunogen to induce anti-tumor
immlmity in another human subject is prohibited by histocompatibility dirr~ lel1ces between
unrelated hnm~n~ However, use of modified tumor cells from one individual to induce anti-
turnor immunity in another individual to protect against possible future occurrence of a tumor
may be useful in cases of f~mili~l m~ n~ncies. In this situation, the tumor-bearing donor of
tumor cells to be modified is closely related to the (non-tumor bearing) recipient of the
modified tumor cells and therefore the donor and recipient share MHC antigens. A strong
hereditary component has been identified for certain types of m~lign~ncies, for example
certain breast and colon cancers. In families with a known susceptibility to a particular
m~ n~ncy and in which one individual presently has a tumor, tumor cells from that
individual could be modified to express B7-2 and/or B7-3, alone or in combination with B7-1
and ~lmini~tered to susceptible, histocompatible family members to induce an anti-tumor
response in the recipient against the type of turnor to which the family is susceptible. This
anti-tumor response could provide protective immllnity to subsequent development of a
tumor in the immunized recipient.
(15). Tl]mor-Specific T Cell Toler~nce
In the case of an experimentally in~ cecl tumor, a subject (e.g. a mouse) can beexposed to the modified tumor cells of the invention before being challenged with
unrnodified tumor cells. Thus, the subject is initially exposed to TAA peptides on tumor
cells together with B7-2 and/or B7-3, and B7-1 which activates TAA-specific T cells. The
activated T cells are then effective against subsequent challenge with unmodified tumor cells.
In the case of a spontaneously arising tumor, as is the case with human subjects, the subject's
immune system will be exposed to unmodified tumor cells before exposure to the modified
turnor cells of the invention. Thus the subject is initially exposed to TAA peptides on tumor
cells in the absence of a costimlll~tory signal. This situation is likely to induce TAA-specific
T cell tolerance in those T cells which are exposed to and are in contact with the unmodified
tumor cells. Secondary exposure of the subject to modified tumor cells which can trigger a
costimulatory signal may not be sufficient to overcome tolerance in TAA-specific T cells
which were anergized by primary exposure to the tumor. Use of modified tumor cells to
induce anti-tumor immllnity in a subject already exposed to unmodified tumor cells may
therefore be most effective in early diagnosed patients with small tumor burdens~ for instance
WO 95/03408 PCT/US94/08423
2~67~
-56-
a small localized tumor which has not met~t~i7P~l In this situation, the tumor cells are
confined to a limited area of the body and thus only a portion of the T cell repertoire may be
exposed to tumor antigens and become anergized. A(lmini~tration of modified tumor cells in
a systemic manner, for instance after surgical removal of the localized tumor and
5 modification of isolated tumor cells, may expose non-anergized T cells to tumor antigens
together with B7-2 and/or B7-3 alone, or in combination with B7-1 thereby inducing an anti-
tumor response in the non-anergized T cells. The anti-tumor response may be effective
against possible regrowth of the tumor or against micrometastases of the original tumor
which may not have been detected. To overcome widespread peripheral T cell tolerance to
10 tumor cells in a subject, additional signals, such as a cytokine, may need to be provided to the
subject together with the modified tumor cells. A cytokine which functions as a T cell
growth factor, such as IL-2, could be provided to the subject together with the modified
tumor cells. IL-2 has been shown to be capable of restoring the alloantigen-specific
responses of previously anergized T cells in an in vitro system when exogenous IL-2 is added
atthetimeofsecondaryalloantigenicstim~ tion. Tan,P.,etal.J. Exp. Med. 177,165-173
(1993).
Another approach to ge~G~ g an anti-tumor T cell response in a subject despite
tolerance of the subject's T cells to the tumor is to stim~ te an anti-tumor response in T cells
from another subject who has not been exposed to the tumor (referred to as a naive donor)
20 and transfer the stimlll~te~l T cells from the naive donor back into the tumor-bearing subject
so that the transferred T cells can mount an immune response against the tumor cells. An
anti-tumor response is in~ çecl in the T cells from the naive donor by stimulating the T cells
in vitro with the modified tumor cells of the invention. Such an adoptive transfer approach is
generally prohibited in outbred populations because of histocolllp~libity differences between
25 the transferred T cells and the tumor-bearing recipient. However, advances in allogeneic
bone marrow transplantation can be applied to this situation to allow for acceptance by the
recipient of the adoptively transferred cells and prevention of graft versus host disease. First,
a tumor-bearing subject (referred to as the host) is prepared for and receives an allogeneic
bone marrow transplant from a naive donor by a known procedure. Preparation of the host
30 involves whole body irradiation, which destroys the host's immune system, including T cells
tolerized to the tumor, as well as the tumor cells themselves. Bone marrow transplantation is
accompanied by treatment(s) to prevent graft versus host disease such as depletion of mature
T cells from the bone marrow graft, treatment of the host with immlln-~suppressive drugs or
treatment of the host with an agent, such as CTLA4Ig, to induce donor T cell tolerance to
35 host tissues. Next, to provide anti-tumor specific T cells to the host which can respond
against residual tumor cells in the host or regrowth or met~t~ces of the original tumor in the
host, T cells from the naive donor are ~timlll~te~l in vitro with tumor cells from the host
WO 95/03408 21 ~ 7 0 91 PCT/US94/08423
-57-
which have been modified, as described herein, to express B7-2 and/or B7-3. Thus, the
donor T cells are initially exposed to tumor cells together with a costim~ tory signal and
therefore are activated to respond to the tumor cells. These activated anti-tumor specific T
cells are then transferred to the host where they are reactive against unmodified tumor cells.
5 Since the host has been reconstituted with the donor's immllne system, the host will not reject
the transferred T cells and, additionally, the tre~tm~nt of the host to prevent graft versus host
disease will prevent reactivity of the transferred T cells with normal host tissues.
H. Admini~.tration of Thera~eutic Forrn~ of R ~ ~ymphocyte Anti~en~
The peptides ofthe invention are allmini~ered to subjects in a biologically compatible
form suitable for ph~rm~reutical atlministration in vivo to either enhance or suppress T cell
mediated immlme response. By "biologically compatible form suitable for a-lministration in
vivo" is meant a form of the protein to be ~lministered in which any toxic effects are
outweighed by the therapeutic effects of the protein. The term subject is intended to include
15 living org~ni~ms in which an imml~ne response can be elicited, e.g., m~mm~l~. Examples of
subjects include hnm~n~, dogs, cats, mice, rats, and transgenic species thereof.A-lministration of a peptide having the activity of a novel B lymphocyte antigen as described
herein can be in any ph~rm~rological form including a thc.d~ulically active amount of
peptide alone or in combination with a peptide having the activity of another B lymphocyte
20 antigen and a ph~rm~c~eutically acceptable carrier. A-lministration of a therapeutically active
amount of the therapeutic compositions of the present invention is defined as an amount
effective, at dosages and for periods of time n~ces~ay to achieve the desired result. For
example, a therapeutically active arnount of a peptide having B7-2 activity may vary
according to factors such as the disease state, age, sex, and weight of the individual, and the
25 ability of peptide to elicit a desired response in the individual. Dosage regima may be
adjusted to provide the optimum th~l~eulic response. For example, several divided doses
may be atlminiet~?red daily or the dose may be ~.ropolLionally reduced as indicated by the
exigencies of the therapeutic situation.
The active compound (e.g., peptide) may be ~tlministered in a convenient manner
30 such as by imjection (subcutaneous, intravenous, etc.), oral ~rlministration, inhalation,
transdermal application, or rectal a~lministration. Depending on the route of atlministration,
the active compound may be coated in a material to protect the compound from the action of
enzymes, acids and other natural conditions which may inactivate the compound.
To admini~ter a peptide having B7-2 activity by other than parenteral a~lministration,
35 it may be necessary to coat the peptide with, or co-a~lminister the peptide with, a material to
prevent its inactivation. For example, a peptide hving B7-2 activity may be a-lmini~tered to
an individual in an ~ ;ate carrier, diluent or adjuvant, co-a~lministered with enzyme
wo 95,03408 ~ ~ 6 '~ O ~ ~ PCT/US94/08423
-58-
inhibitors or in an ~propl;ate carrier such as liposomes. Ph~rm~ceutically acceptable
diluents include saline and aqueous buffer solutions. Adjuvant is used in its broadest sense
and includes any immllne stimulating compound such as interferon. Adjuvants contemplated
herein include resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether and n-
S he~ cyl polyethylene ether. Enzyme inhibitors include pancreatic trypsin inhibitor,diisopropylfluorophosphate (DEP) and trasylol. Liposomes include water-in-oil-in-water
emulsions as well as conventional liposomes (Strejan et ~L, (1984) J. Neuroimmunol 1:27).
The active compound may also be ~imini~tered parenterally or intraperitoneally.
Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures
l 0 thereof and in oils. Under ordinary conditions of storage and use, these preparations may
contain a preservative to prevent the growth of microorg~ni~m~
Pharmaceutical compositions suitable for injectable use include sterile aqueous
solutions (where water soluble) or dispersions and sterile powders for the extemporaneous
aldlion of sterile injectable solutions or dispersion. In all cases, the composition must be
l 5 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 cont~min~ting
action of microor~ni~m~ such as bacteria and fungi. The carrier can be a solvent or
dispersion medium cont~inin~, for example, water, ethanol, polyol (for example, glycerol,
propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures
20 thereof. The proper fluidity can be ~ ed, for example, by the use of a coating such as
lecithin, by the m~inlen~nce of the required particle size in the case of dispersion and by the
use of sllrf~ct~nt~ Prevention of the action of microorg~ni~m~ can be achieved by various
antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, asorbic
acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents,
25 for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions can be brought about by
including in the composition an agent which delays absorption, for example, alnminllm
monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating active compound (e.g.,
30 peptide having B7-2 activity) in the required amount in an appr~,pliate solvent with one or a
combination of ingredients enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active compound into a sterile
vehicle which contains a basic dispersion medium and the required other ingredients from
those enumerated above. In the case of sterile powders for the pl~d~ion of sterile
35 injectable solutions, the preferred methods of ~l~paldLion are vacuum drying and freeze-
drying which yields a powder of the active ingredient (e.g., peptide) plus any additional
desired ingredient from a previously sterile-filtered solution thereof.
9~/0340~ ~16 ~ O 91 PCTIUS94l08423
-59-
When the active compound is suitably protected? as described above, the protein may
be orally ~tlmini~tered, for example, with an inert diluent or an ~imil~ble edible carrier. As
used herein "ph~rrn~eutically acceptable carrier" includes any and all solvents? dispersion
media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents?
and the like. The use of such media and agents for ph~rm~ceutically active substances is well
known in the art. Except insofar as any conventional media or agent is incompatible with the
active compound, use thereof in the therapeutic compositions is contemplated.
Supplement~ry active compounds can also be incorporated into the compositions.
It is especially advantageous to formulate palel1teldl compositions in dosage unit form
for ease of ~-lmini~tration and uniformity of dosage. Dosage unit form as used herein refers
to physically discrete units suited as unitary dosages for the m~mm~ n subjects to be
treated; each unit cont~inin~ a pre.letermined quantity of active compound calculated to
produce the desired therapeutic effect in association with the required ph~rm~reutical carrier.
The specification for the dosage unit forms of the invention are dictated by and directly
dependent on (a) the unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding
such an active compound for the tre~tment of sensitivity in individuals.
I. Identification of Cytokines ~nduced by Costimulation
The nucleic acid sequences encoding peptides having the activity of novel B
Iymphocyte antigens as described herein can be used to identify cytokines which are
produced by T cells in response to stimulation by a form of B lymphocyte antigen, e.g., B7-2.
T cells can be suboptimally stim~ te~i in vitro with a primary activation signal, such as
phorbol ester, anti-CD3 antibody or preferably antigen in association with an MHC class II
molecule, and given a costimulatory signal by a stim~ tQry form of B7-2 antigen, for
in.~t~n~e by a cell transfected with nucleic acid encoding a peptide having B7-2 activity and
expressing the peptide on its surface or by a soluble, stimulatory form of the peptide. Known
cytokines released into the media can be identified by ELISA or by the ability of an antibody
which blocks the cytokine to inhibit T cell proliferation or proliferation of other cell types
that is in~llced by the cytokine. An IL-4 ELISA kit is available from Genzyme (Cambridge
MA~, as is an IL-7 blocking antibody. Blocking antibodies against IL-9 and IL-12 are
available from Genetics Institute (Cambridge, MA).
An in vitro T cell costimulation assay as described above can also be used in a method
for identifying novel cytokines which may be in~ ced by costim~ tion. If a particular
activity in~ ce~l upon costimlll~tion, e.g., T cell proliferation, cannot be inhibited by addition
of blocking antibodies to known cytokines, the activity may result from the action of an
wo 95,03408 2~6 ~ PCT/US94/08423
-60-
unkown cytokine. Following costimulation, this cytokine could be purified from the media
by conventional methods and its activity measured by its ability to induce T cell proliferation.
To identify cytokines which prevent the induction of tolerance, an in vitro T cell
costimulation assay as described above can be used. In this case, T cells would be given the
primary activation signal and contacted with a selected cytokine, but would not be given the
cosfim~ tory signal. After washing and resting the T cells, the cells would be rechallenged
with both a primary activation signal and a costim~ tory signal. If the T cells do not respond
(e.g., proliferate or produce IL-2) they have become tolerized and the cytokine has not
prevented the induction of tolerance. However, if the T cells respond, induction of tolerance
has been prevented by the cytokine. Those cytokines which are capable of preventing the
induction of tolerance can be targeted for blockage in vivo in conjunction with reagents which
block B lymphocyte antigens as a more efficient means to induce tolerance in transplant
recipients or subjects with autoimml-ne diseases. For example, one could ~mini~ter a B7-2
blocking reagent together with a cytokine blocking antibody to a subject.
J. Identification of Molecules which Inhihit Costimulation
Another application of the peptide having the activity of a novel B lymphocyte
antigen of the invention (e.g., B7-2 and B7-3) is the use of one or more of these peptides in
screening assays to discover as yet undefined molecules which are inhibitors of costimulatory
ligand binding and/or of intracellular ~i~n~lin~ through T cells following costiml-l~tion. For
example, a solid-phase binding assay using a peptide having the activity of a B lymphocyte
antigen, such as B7-2, could be used to identify molecules which inhibit binding of the
antigen with the a~plopliate T cell ligand (e.g., CTLA4, CD28). In addition, an in vitro T
cell costim~ tion assay as described above could be used to identify molecules which
interfere with intracellular ~ipn~ling through the T cells following costimlll~tion as
cleterminPcl by the ability of these molecules to inhibit T cell proliferation and/or cytokine
production (yet which do not prevent binding of B lymphocyte antigens to their receptors).
For example, the compound cyclosporine A inhibits T cell activation through stimlll~tion via
the T cell receptor pathway but not via the CD28/CTLA4 pathway. Therefore, a different
intracellular si~n~ling pathway is involved in costimulation. Molecules which interfere with
intracellular sign~lin~ via the CD28/CTLA4 pathway may be effective as immunosuppressive
agents in vivo (similar to the effects of cyclosporine A).
K. Identification of Molecules which Modulate B Lymphocyte Anti~en l~xpression
The monoclonal antibodies produced using the proteins and peptides of the current
invention can be used in a screening assay for molecules which modulate the expression of B
lymphocyte antigens on cells. For example, molecules which effect intracellular sign~ling
WO 95/03408 ~ ~. 6 ~ ~ ~1 PCT/US94/08423
.
-61 -
which leads to induction of B Iymphocyte antigens, e.g. B7-2 or B7-3, can be identified by
assaying e~lession of one or more B lymphocyte antigens on the cell surface. ~çcl~lce~l
- imml]n~fluorescent staining by an anti-B7-2 antibody in the presence of the molecule would
indicate that the molecule inhibits intracellular signals. Molecules which upregulate B
lymphocyte antigen expression result in an increased immunofluorescent st~ining.Alternatively, the effect of a molecule on expression of a B lymphocyte antigen, such as B7-
2, can be determined by detecting cellular B7-2 mRNA levels using a B7-2 cDNA as a probe
For example, a cell which expresses a peptide having B7-2 activity can be contacted with a
molecule to be tested, and an increase or decrease in B7-2 mRNA levels in the cell detected
by standard technique, such as Northern hybridization analysis or conventional dot blot of
mRNA or total poly(A+)RNAs using a B7-2 cDNA probe labeled with a detectable marker.
Molecules which modulate B lymphocyte antigen expression may be useful therapeutically
for either upregulating or downregulating immune responses alone or in conjunction with
soluble blocking or stimulating reagents. F~r instance, a molecule which inhibits expression
of B7-2 could be ~lmini~tered together with a B7-2 blocking reagent for immunosuppressive
purposes. Molecules which can be tested in the above-described assays include cytokines
such as IL-4, yINF, IL-10, IL-12, GM-CSF and prost~gl~lin~
This invention is further illustrated by the following examples which should not be
construed as limiting. The contents of all references and published patent applications cited
throughout this application are hereby incorporated by reference.
The following methodology was used in Examples 1, 2 and 3.
METHODS AND MATERIALS
A Cells
Mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation
from single cell suspensions of normal human spleens and were separated into E- and E+
fractions by rosetting with sheep red blood cells (Boyd, A.W., et al. (1985) J. Immunol. 134,
1516). B cells were purified from the E- fraction by adherence of monocytes on plastic and
depletion of residual T, natural killer cells (NK) and residual monocytes by two treatments
with anti-MsIgG and anti-MsIgM coated magnetic beads (Advanced Magnetics, Cambridge,
MA), using monoclonal antibodies: anti-CD4, -CD8, -CDl lb, -CD14 and -CD16. CD4+ T
cells were isolated from the E+ fraction of the same spleens after adherence on plastic and
depletion of NK, B cells and residual monocytes with magnetic beads and monoclonal
antibodies: anti-CD20, -CDl lb, -CD8 and -CD16. CD28+ T cells were identically isolated
from the E+ fraction using anti-CD20, -CD 1 l b, -CD 14 and -CD 16 monoclonal antibodies.
The efficiency of the purification was analyzed by indirect immunofluorescence and flow
WO 95/03408 PCT/US94/08423
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-62 -
cytometry using an EPICS flow cytometer (Coulter). B cell ~ ualdliOnS were >95% CD20+,
<2% CD3+, <1% CD14+. CD4+ T cell preparations were >98% CD3+, >98% CD4+.<1%
CD8+, <1% CD20+, <1% CD14+. CD28+ T cell preparations were >98% CD3+, >98%
CD28+, <1% CD20+, <1% CD14+.
R. Monoclonal Antibodies and Fusion Proteins
Monoclonal antibodies were used as purified Ig unless in~lir~ted otherwise: anti-
B7:133, IgM is a blocking antibody and has been previously described (FreeAm~n, A.S. et al.
(1987) Immunol. 137, 3260-3267); anti-B7:Bl.l, IgGl (RepliGen Corp., Cambridge, MA)
(Nickoloff, B., et al (1993) Am. J. Pathol. 142, 1029-1040) is a non-blocking monoclonal
antibody; BB-1: IgM is a blocking antibody (Dr. E. Clark, University of Washington, Seattle,
WA) (Yokochi, T., et al. (1982) J. Immunol. 128, 823-827); anti-CD20: B1, IgG2a
(St~henk~, P., et al.(1980) J. Immunol. L~, 1678-1685); anti-BS: IgM (Freerln~n, A., et al.
(1985) J. Immunol. 134, 2228-2235); anti-CD8: 7PT 3F9, IgG2a; anti-CD4: l9ThySD7,
IgG2a; anti-CDl lb: Mol, IgM and anti-CD14: Mo2, IgM (Todd, R, et al. (1981) J. Immunol.
126, 1435-1442); anti-MHC class II: 9-49, IgG2a (Dr R. Todd, University of Michigan, Ann
Arbor) (Todd, R.I., et al. (1984) Hum Immunol. 10, 23-40; anti-CD28: 9.3, IgG2a (Dr. C.
June, Naval Research Institute, Bethesda) (Hansen, J.A., et al. (1980) Immunogenetics. 10,
247-260); anti-CD16: 3G8, IgGl (used as ascites) (Dr. J. Ritz, Dana-Farber Cancer Tn~titllte,
Boston); anti-CD3: OKT3, IgG2a hybridoma was obtained from the American Type Culture
Collection and the purified monoclonal antibody was adhered on plastic plates at a
concentration of 1,ug/ml; anti-CD28 Fab fr~gment~ were generated from the 9.3 monoclonal
antibody, by papain digestion and purification on a protein A column, according to the
manufacturer's instructions (Pierce, Rockford, IL). Human CTLA4 fusion protein
(CTLA4Ig) and control fusion protein (control-Ig) were prepared as previously described
(Gimmi, C.D., et al. (1993) Proc. Natl. Acad. Sci USA ~1:6586-6590); Boussiotis, V., et al J.
Exp. Med. (accepted for publication)).
C. CHO Cell Tr~n~fection
B7-1 transfectants (CHO-B7) were prepared from the B7-1 negative chinese hamsterovary (CHO) cell line, fixed with paraformaldehyde and used as previously described
(Gimmi, C.D., et al. Proc. Natl. Acad. Sci USA ~, 6575-6579).
D. In Vitro B Cell Activation and Selection of B7+ and B7- Cells
Splenic B cells were cultured at 2X106 cells/ml in complete culture media, {RPMI1640 with 10% heat inactivated fetal calf serum (FCS), 2mM gh1t~nnin~, 1 mM sodium
pyruvate, penicillin (100 units/ml), streptomycin sulfate (100~1g/ml) and gentamycin sulfate
-
~ o 95,03408 2 l 6 ~ O 9 1 PCT/US94/08423
-63 -
(5~1g/ml)}, in tissue culture flasks and were activated by cro.~.~linking of sIg with affinity
purif1ed rabbit anti-human IgM coupled to Affi-Gel 702 beads (Bio-Rad), Richmond, CA)
(Boyd, A.W., et al., (1985) J: Immunol. 134,1516) or by cro.s.~linking of MHC class II with 9-
~9 antibody coupled to Affi-Gel 702 beads. B cells activated for 72 hours, were used as total
- 5 activated B cell populations or were indirectly stained with anti-B7 (B 1.1) monoclonal
antibody and fluorscein isothiocyanate (FITC) labeled goat anti-mouse immunoglobulin
(Fisher, Pittsburgh, PA), and fractionated into B7-1+ and B7-1- populations by flow
cytometric cell sorting (EPICS Elite flow cytometer, Coulter).
F. Tmmlmoflouoresce~ce ~ntl Flow Cytornetrv
For surface phenotype analysis populations of B cells activated by either slg or MHC
class II cro.~slinking for 6, 12, 24, 48, 72 and 96 hours were stained with either anti-B7 (133),
BB-1 monoclonal antibodies, control IgM antibody, CTLA4Ig or control-Ig. Cell
suspensions were stained by two step indirect membrane st~inin~ with l O~lg/ml of primary
monoclonal antibody followed by the a~l)ropliate secondary reagents. Specifically,
immunoreactivity with anti-B7 (133) and BB-1 monoclonal antibodies was studied by
indirect staining using goat anti-mouse Ig or immlmc)globulin FITC (Fisher) as secondary
reagent and immlm~reactivity with fusion proteins was studied using biotinylated CTLA4Ig
or biotinylated control-Ig and streptavidin-phycoerythrin as secondary reagent. PBS
cont~ining 10% AB serum was used as diluent and wash media. Cells were fixed with 0.1 %
p~dru,..laldehyde and analyzed on a flow cytometer (EPICS Elite Coulter).
F. Prolifer~tion A~
T cells were cultured at a concentration of lxl O5 cells per well in 96-well flat bottom
microtiter plate at 37C for 3 days in 5% CO2. Syngeneic activated B cells (total B cell
population or B7+ and B7- fractions) were irr~ te-l (2500 rad) and added into the cultures at
a concentration of 1 x 105 cells per well. Factors under study were added to the required
concentration for a total final volume of 200 Ill per well. When indicated, T cells were
incubated with anti-CD28 Fab (final concentration of lO,ug/ml), for 30 minutes at 4C, prior
to addition in c;~ nental plates. Similarly, CHO-B7 or B cells were incubated with
CTL~4Ig or control-Ig (lO~lg/ml) for 30 minllt~s at 4C. Thymidine incorporation as an
index of mitogenic activity, was assessed after incubation with 1 ~lCi (37kBq) of {methyl-3H}
thymidine (Du Pont, Boston, MA) for the last 15 hours of the culture. The cells were
harvested onto filters and the radioactivity on the dried filters was measured in a Pharrnacia
beta plate liquid scintilation counter.
WO 95/03408 PCT/US94/08423
~16~
-64-
G. JT -2 ~n(l IL-4 A~ay
IL-2 and IL-4 concenkations were assayed by ELISA (R&D Systems, Minneapolis,
MN and BioSource, Camarillo, CA) in culture supern~t~nt~ collected at 24 hours after
initiation of the culture.
EXAMPLE 1
li ~pre~sion of a Novel CTT ,~4 ~.~and on Activated B Cells
Whi-`h Induces T Cell Proliferation
Since crosslinking surface Ig in~l~ce~ human resting B cells to express B7-l
maximally (50-80%) at 72 hours, the ability of activated human B lymphocytes to induce
submitogenically activated T cells to proliferate and secrete IL-2 was determined. Figure 1
depicts the costimulatory response of human splenic CD28+ T cells, submitogenically
activated with anti-CD3 monoclonal antibody, to either B7 (B7-1) transfected CHO cells
(CHO-B7) or syngeneic splenic B cells activated with anti-Ig for 72 hours. 3H-Thymidine
incorporation was ~sessed for the last 15 hours of a 72 hours culture. IL-2 was assessed by
ELISA in supern~t~nt~ after 24 hours of culture (Detection limits of the assay: 31-2000
pg/ml). Figure 1 is .~;~.ese~ e of seventeen experiments.
Submitogenically activated CD28+ T cells proliferated and secreted high levels of IL-
2 in response to B7-1 costim~ ion provided by CHO-B7 (Figure 1, panel a). Both
proliferation and IL-2 secretion were totally inhibited by blocking the B7-1 molecule on
CHO cells with either anti-B7- 1 monoclonal antibody or by a fusion protein for its high
affinity receptor, CTLA4. Similarly, proliferation and IL-2 secretion were abrogated by
blocking B7-1 ~ign~lling via CD28 with Fab anti-CD28 monoclonal antibody. Control
monoclonal antibody or control fusion protein had no effect. Nearly identical costimlll~tion
of proliferation and IL-2 secretion was provided by splenic B cells activated with anti-Ig for
72 hours (panel b). Though anti-B7-1 monoclonal antibody could completely abrogate both
proliferation and IL-2 secretion delivered by CHO-B7, anti-B7-1 monoclonal antibody
con~i~tently inhibited proliferation in~ ce~l by activated B cells by only 50% whereas IL-2
secretion was totally inhibited. In contrast to the partial blockage of proliferation in~l~lcecl by
anti-B7-1 monoclonal antibody, both CTLA4Ig and Fab anti-CD28 monoclonal antibody
completely blocked proliferation and IL-2 secretion. These results are consistent with the
hypothesis that activated human B cells express one or more additional CTLA4/CD28
ligands which can induce T cell proliferation and IL-2 secretion.
WO g~/~3408 21~ 7 ~ 91 PCTIUS94/08423
.
-65 -
EXAMPLE 2
aled ~um~n Splenic B Celle F.~press CT~ ~4 T ~nd(s) I)ietinct from n7-1
In light of the above observations, whether other CTLA4 binding cour;ter-receptors
- 5 were ex~ressed on activated B cells was determined. To this end, human splenic B cells were
activated for 72 hours with anti-Ig and then stained with an anti-B7-1 monoclonal antibody
(Bl.1) which does not inhibit B7-1 mediated costim~ tion. Fluoroscein isothiocyanate
(FITC) and mAb B 1.1 were used with flow cytometric cell sorting to isolate B7- 1 + and B7- 1 ~
fractions. The resulting post-sort positive population was 99% B7-1+ and the post-sort
negative population was 98% B7- 1 ~ (Figure 2).
To ~mine the costimulatory potential of each population, human splenic CD28+ T
cells were submitogenically stimulated with anti-CD3 monoclonal antibody in the presence
of irradiated B7-1+ or B7-1- anti-Ig activated (72 hours) splenic B cells. 3H-Thymidine
incorporation was assessed for the last 15 hours of a 72 hours culture. IL-2 was assessed by
ELISA in supernatants after 24 hours of culture (Detection limits of the assay: 31 -2000
pg/ml). The results of Figure 3 are representative of ten experiments. B7-1+ B cells in~ recl
anti-CD3 activated T cells to proliferate and secrete IL-2 (Figure 3a) but not IL-4. As was
observed with the unfractionated activated B cell population, anti-B7-1 monoclonal antibody
(133) inhibited proliferation only 50% but con.ei.ett?ntly abrogated IL-2 secretion. As above,
CTLA4Ig binding or blockade of CD28 with Fab anti-CD28 monoclonal antibody completely
inhibited both proliferation and IL-2 secretion. Control monoclonal antibody and control-Ig
were not inhibitory. In an attempt to identify other potential CTLA4/CD28 binding
costimulatory ligand(s) which might account for the residual, non-B7 mediated proliferation
delivered by B7+ B cells, the effect of BB-1 monoclonal antibody on proliferation and IL-2
secretion was exarnined. As seen, BB-l monoclonal antibody completely inhibited both
proliferation and IL-2 secretion (Figure 3a). Figure 3b displays the costimulatory potential of
B7-1- activated human splenic B cells. Irradiated B7-1- activated (72 hr) B cells could also
deliver a significant costimulatory signal to submitogenically activated CD4+ lymphocytes.
This costim~ tion was not accompanied by detectable IL-2 (Figure 3b) or IL-4 accumulation
and anti-B7-1 monoclonal antibody did not inhibit proliferation. However, CTLA4Ig, Fab
anti-CD28 monoclonal antibody, and BB-l monoclonal antibody all completely inhibited
proliferation.
Phenotypic analysis ofthe B7-1+ and B7-1- activated splenic B cells confirmed the
above functional results. Figure 4 shows the cell surface expression of B7-1, B7-2 and B7-3
on fractionated B7-1+ and B7-1- activated B cell. As seen in Figure 4, B7-1+ activated
splenic B cells stained with anti-B7-1 (133) monoclonal antibody, BB-1 monoclonal
antibody, and bound CTLA4-Ig. In contrast, B7- activated splenic B cells did not stain with
WO 95/03408 PCTIUS94/08423
æ~6~9~ -
- -66-
- anti-B7-1 (133) monoclonal antibody but did stain with BB-l monoclonal antibody and
CTLA4Ig. These phenotypic and functional results demonstrate that both B7-1+ and B7-1-
activated (72 hours) human B Iymphocytes express CTLA4 binding counter-receptor(s)
which: 1) can induce submitogenically activated T cells to proliferate without detectable IL-
2 secretion; and 2) are identified by the BB-l monoclonal antibody but not anti-B7-1
monoclonal antibody. Thus, these CTLA4/CD28 ligands can be distinguished on the basis of
their temporal expression after B cell activation and their reactivity with CTLA41g and anti-
B7 monoclonal antibodies. The results of Figure 4 are representative of five experiments.
EXAMPLE 3
Three I)istinct CT~,~4/CD28 1 i~nds Are Fx~ressed
Followin~ Human B CellActivation
To fletçrmine the sequential ~x~les~ion of CTLA4 binding counter-receptors
following activation, human splenic B cells were activated by crosslinking of either surface
Ig or MHC class II and the expression of B7-1, B7-3 and B7-2 binding proteins were
examined by flow cytometric analysis. Ig or MHC class II cro~linkinp in~ ce~l a similar
pattern of CTLA4Ig binding (Figures 5 and 6). Figure S is representative of the results of 25
experiments for anti-B7-1 and BB-l binding and 5 experiments for CTLA4Ig binding.
Figure 6 is ~ ,resellL~Li~e of 25 experiments for anti-B7-1 binding and 5 experiments for
CTLA4Ig binding. The results of these experiments indictes that prior to 24 hours, none of
these molecules are expressed. At 24 hours post-activation, the majority of cells express a
protein that binds CTLA4Ig (B7-2), however, fewer than 20% express either B7-1 or B7-3.
Crosslinkin~ of MHC class II induces m~im~l ~x~l~s~.ion and intensity of B7-1 and B7-3 at
48 hours whereas cros~linkin~ of Ig induces maximal ~ ression at 72 hours and ex~les~ion
declines thereafter. These results suggest that an additional CTLA4 binding counter-receptor
is expressed by 24 hours and that the temporal expression of the distinct B7- 1 and B7-3
proteins appears to coincide.
A series of experiments was conducted to determine whether the temporal expression
of CTLA4 binding counter-receptors differentially correlated with their ability to costimnl~te
T cell proliferation and/or IL-2 secretion. Human splenic CD28+ T cells submitogenically
~tim~ te~l with anti-CD3 were cultured for 72 hours in the presence of irradiated hurnan
splenic B cells that had been previously activated in vitro by sIg crosslinkin~ for 24, 48, or 72
hours. IL-2 secretion was ~es~ed by ELISA in supernatants after 24 hours and T cell
proliferation as ~sesse~l by 3H-thymidine incorporation for the last 15 hours of a 72 hour
culture. The results of Figure 7 are representative of 5 experiments. As seen in Figure 7a, 24
hour activated B cells provided a costim~ tory signal which was accompanied by modest
~ - -
WO 95to3~ 7 Q ~1 PCT/US94/08423
-67-
levels o:~ IL-2 production, although the m~itn-le of proliferation was significantly less than
observed with 48 and 72 hours activated human B cells (note differences in scale for
3H-Thymidine incorporation). Neither proliferation nor IL-2 accurnulation was inhibited by
anti-B7-1 (133) or BB-l. In contrast, with CTLA4Ig and anti-CD28 Fab monoclonal
5 antibody totally abrogated proliferation and IL-2 accumulation. B cells activated for 48
hours, provided costimulation which resulted in nearly maximal proliferation and IL-2
secretion (Figure 7b). Here, anti-B7-1 (133) monoclonal antibody, inhibited proliferation
approximately 50% but totally blocked IL-2 accu~nulation. BB-l monoclonal antibody
totally inhibited both proliferation and IL-2 secretion. As above, CTLA4Ig and Fab
10 anti-CDZ8 also totally blocked proliferation and IL-2 production. Finally, 72 hour activated
B cells intluced T cell response identical to that in(ll1ce~1 by 48 hour activated B cells. Similar
results are observed if the submitogenic signal is delivered by phorbol myristic acid (PMA)
and if the human splenic B cells are activated by MHC class II rather than Ig cro.~linking.
These results indicate that there are three CTLA4 binding molecules that are temporarily
15 ~ ssed on activated B cells and each can induce submitogenically stimulated T cells to
proliferate. Two of these molecules, the early CTLA4 binding counter-receptor (B7-2) and
B7-1 (133) induce IB-2 production whereas B7-3 inf31l~çs proliferation without detectable
IL-2 production.
Previous studies provided conflicting evidence whether the anti-B7 monoclonal
20 antibody,l33 and monoclonal antibody BB-l identified the same molecule (Free~lm~n, A.S.
et al. (1987) Immunol. ~, 3260-3267; Yokochi, T., et al. (1982) J: Immunol. 128, 823-827;
Freeman, G.J., et al. (1989) ~ Immunol. 1~, 2714-2722.). Although both monoclonal
antibodies identified molecules expressed 48 hours following human B-cell activation,
several reports suggested that B7 (B7-1) and the molecule identified by monoclonal antibody
25 BB-1 were distinct since they were differentially expressed on cell lines and B cell neoplasms
(Free~im~n, A.S. et al. (1987) Immunol. 137, 3260-3267; Yokochi, T., et al. (1982) J.
Immunol. 128, 823-827; Freeman, G.J., et al. (1989) J. Immunol. 143, 2714-2722; Clark, E
and Yokochi, T. (1984) Leulcocyte Typing, Ist International References Workshop. 339-346;
Clark, E., et al. (1984) Leukocyte Typing, 1stInternational References Workshop. 740). In
30 addition, immllnf~precipitation and Western Blotting with these IgM monoclonal antibodies
suggested that they identified different molecules (Clark, E and Yokochi, T. (1984)
Leukocyte Typing, Ist International References Workshop. 339-346; Clark, E., et al. (1984)
Leukocyte Typing, I st International References Workshop. 740). The original anti-B7
monoclonal antibody, 133, was generated by immlmi7~tion with anti-immunoglobulin35 activated human B lymphocytes whereas the BB-l monoclonal antibody was generated by
imml-ni7~tif~n with a baboon cell line. Thus, the BB-I monoclonal antibody must identify an
epitope on human cells that is conserved between baboons and hllm~n~
WO 95/03408 PCT/US94/08423
21~7~ 68-
Following the molecular cloning and expression of the human B7 gene (B7-1), B7
transfected COS cells were found to be identically stained with the anti-B7 (133) and BB-1
monoclonal antibodies and that they both ple~ led the identical broad molecular band
(44-54kD) strongly suggesting that they identified the same molecule (Freeman, G.J., et al.
(1989) J. Immunol. 143, 2714-2722). This observation was unexpected since the gene
encoding the molecule identified by the BB-1 monoclonal antibody had been previously
mapped to chromosome 12 (Katz, ~.E., et al. (1985) Eur. J. Immunol. 103-6), whereas the
B7 gene was located by two groups on chromosome 3 (Freeman, G.J., et al. (1992) Blood. 79,
489-494; Selvakumar, A., et al. (1992) Immunogenetics 36, 175-181.). Subsequently,
additional discrepancies between the phenotypic expression of B7 (B7-1) and the molecule
identified by the BB-1 monclonal antibody were noted. BB-l monoclonal antibody stained
thymic epithelial cells (Turka, L.A., et al. (1991) J. Immunol. 1~, 1428-36; Munro, J.M., et
al. Blood submitted.) and keratinocytes (Nickoloff, B., et al (1993) ~m. J. Pathol. 142, 1029-
1040; Augustin, M., et al. (1993) J. Invest. Dermatol. 100, 275-281.) whereas anti-B7 did
not. Recently, Nickoloff et al. (1993) ~lm. J. Pathol. ~, 1029-1040, reported discordant
expression of the molecule identified by the BB-l monoclonal antibody and B7 on
keratinocytes using a BB-1 and anti-B7 (B 1.1 and 133) monoclonal antibodies. Nickoloff et
al. also demonstrated that these BB-l positive cells did not express B7 mRNA yet bound
CD28 transfected COS cells providing further support for the existence of a distinct protein
which binds monoclonal antibody BB-l.
The present finrlin~ confirm that there is an additional CTLA4 counter-receptor
identified by the BB-l monoclonal antibody, B7-3, and that this protein appears to be
functionally distinct from B7-1 (133). Although the ~ ression of B7-1 and B7-3 following
B cell activation appears to be concordant on B7 positive B cells, these studies demonstrate
that the B7-3 molecule is also expressed on B7 negative activated B cells. More importantly,
the B7-3 molecule appears to be capable of inducing T cell proliferation without detectable
IL-2 or IL-4 production. This result is similar to the previous observation that ICAM-I could
costim~ t~ T cell proliferation without detectable IL-2 or IL-4 production (Boussiotis, V., et
al J. E~p. Med. (accepted for publication)). These data indicate that the BB-l monoclonal
antibody recognizes an epitope on the B7-1 protein and that this epitope is also found on a
distinct B7-3 protein, which also has costimulatory function. Phenotypic and blocking
studies demonstrate that the BB-1 monoclonal antibody could detect one (on B7 negative
cells) or both (on B7 positive cells) of these proteins. In conkast, the anti-B7 monoclonal
antibodies, 133 and Bl.l detect only the B7-1 protein. Taken together, these results suggest
that by 48 hours post B-cell activation by cros~linking of surface immlmt~globulin or MHC
class II, B cells express at least two distinct CTLA4 binding counter-receptors, one identified
~ O 95/03408 21 6 7 ~ 9 ~ PCT/US94/08423
-69-
by both anti-B7 and BB-1 monclonal antibodies and the other identified only by BB-l
monoclonal antibody.
The B7-2 antigen is not detectable on activated B cells after 12 hours, but by 24 hours
it is strongly expressed and functional. This molecule appears to signal via CD28 since
- 5 proliferation and IL-2 production are completely blocked by Fab anti-CD28 monoclonal
antibody. At 48 hours post activation, IL-2 secretion seems to be accounted for by B7-1
costimulation, since anti-B7 monoclonal antibody completely inhibits IL-2 production.
Previous studies and results presented here demonstrate that B7 (B7-1) is neither
expressed (Free~lm~n, A.S. et al. (1987) Immunol. ~1, 3260-3267; Free~lm~n, A.S., et al.
(1991) Cell. Immunol. 137, 429-437) nor capable of costimulating T cell proliferation or IL-2
secretion until 48 hours post B-cell activation. Previous studies have shown that activation of
T cells via the TCR in the absence of cos~im~ tion (Gimmi, C.D., et al. (1993) Proc. Natl.
Acad. Sci USA 90:6586-6590; Schwartz, R.H., et al. (1989) Cold Spring Harb. Symp. Quant.
Biol 54, 605-10; Beverly, B., et al. (1992) Int. Immunol. 4. 661-671.) and lack of IL-2
(Boussiotis, V., et al J. ~p. Med. (submitted); Beverly, B., et al. (1992) Int. Immunol. _, 661-
671; Wood, M., et al. (1993) J. Exp. Med. ~11, 597-603) results in anergy. If B7-1 were the
only costimulatory molecule capable of inducing IL-2 secretion, T cells would be anergized
within the first 24 hours following activation since there is no B7-1 present to costimulate
IL-2 production. Therefore, the existence of another, early inducible costimulatory molecule,
which can costim~ te IL-2 secretion during the first 24 hours would be necessary to induce
an effective immune response rather than anergy. The appearance of the early CTLA4
binding counter-receptor, B7-2, between 12 and 24 hours post B cell activation, fulfills this
function.
Two observations shed light on the biologic and potential clinical significance of
these two additional CTLA4 binding counter-receptors. First, B7 (B7-1) deficient mouse has
been developed and its antigen ~lese~ g cells were found to still bind CTLA4Ig (Freeman
and Sharpe manuscript in plc;î,aldlion). This mouse is viable and isolated mononuclear cells
induce ~etect~hle levels of IL-2 when cultured with T cells in vitro. Therefore, an alternative
CD28 co~im~ tory counter-receptor or an alternative IL-2 producing pathway must be
functional. Second, thus far the most effective reagents to induce antigen specific anergy in
murine and human systems are CTLA4Ig and Fab anti-CD28, whereas anti-B7 monoclonal
- antibodies have been much less effective (Harding, F.A., et al. (1992) Nature. ~, 607-609;
Lenschow, D.J., et al. (1992) Science. ~1, 789-792, Chen, L., et al. (1992) Cell. 71, 1093-
1102; Tan, P., et al. (1993) J. Exp. Med. 177, 165-173.). These observations are also
consistent with the hypothesis that alternative CTLA4/CD28 ligands capable of inducing IL-2
exist, and taken together with the results p.~sellled herein, suggest that all three CTLA4
binding counter-receptors may be critical for the induction of T cell immllnity. Furthermore,
WO 95/03408 PCT/US94/08423
21~7~ 70
their blockade will likely be required for the induction of T cell anergy. Identical results
have been observed in the murine system with the identification of two CTLA4 binding
lig~n~lc, corresponding to the human B7-1 and B7-2 molecules. APCs in the B7 deficient
mouse bind to the CTLA4 and can induce IL-2 secretion. Taken together, these observations
indicate that multiple CTLA-4 binding counter-receptors exist and sequentially costimulate T
cell activation in the murine system.
EXAMPLE 4
Cloni~ Sequenrin~ and F~pression of the R7-2 An~i~en
A. Col ~truction of cDN~ T ibrarv
A cDNA library was constructed in the pCDM8 vector (Seed, Nature, 329:840
(1987)) using poly (A)+ RNA from the human anti-IgM activated B cells as described
(Aruffo et al, Proc. Natl. Acad. Sci. USA, 84:3365 (1987)). Splenic B cells were cultured at
2X106 cells/ml in complete culture media, {RPMI 1640 with 10% heat inactivated fetal calf
serum (FCS), 2mM gll-t~mine7 1 mM sodium pyruvate, penicillin (100 units/ml),
streptomycin sulfate ( l OO,ug/ml) and ge~ ycin sulfate (5,ug/ml) }, i~ tissue culture flasks
and were activated by cro~linking of sIg with affinity purified rabbit anti-human IgM
coupled to Affi-Gel 702 beads (Bio-Rad), Richmond, CA) (Boyd, A.W., et al., (1985) J.
Immunol. 134,1516). Activated B cells were harvested after 1/6, 1/2, 4, 8 12, 24, 48, 72 and
96 hours.
RNA was prepared by homogenizing activated B cells in a solution of 4M guanidinethiocyanate, 0.5% sarkosyl, 25mM EDTA, pH 7.5, 0.13% Sigma anti-foam A, and 0.7%mercaptoethanol. RNA was purified from the homogenate by centrifugation for 24 hour at
32,000 rpm through a solution of 5.7M CsCl, lOmM EDTA, 25mM Na acetate, pH 7. The
pellet of RNA was dissolved in 5% sarkosyl, lmM EDTA, lOmM Tris, pH 7.5 and extracted
with two volumes of 50% phenol, 49% chloroform, 1% isoamyl alcohol. RNA was ethanol
precipitated twice. Poly (A)+ RNA used in cDNA library construction was purified by two
cycles of oligo (dT)-cellulose selection.
Complement~ry DNA was synthesized from 5.5,ug of anti-IgM activated human B
cell poly(A)+ RNA in a reaction cont~ining 50mM Tris, pH 8.3, 75mM KCl, 3mM MgC12,
lOmM dithiothreitol, 500,uM dATP, dCTP, dGTP, dTTP, 50,ug/ml oligo(dT)12 18, 180units/ml RNasin, and 10,000 units/ml Moloney-MLV reverse transcriptase in a total volume
of 55,u1 at 37 for 1 hr. Following reverse transcription, the cDNA was converted to double-
stranded DNA by adjusting the solution to 25mM Tris, pH 8.3, lOOmM KCl, SmM MgCl2,
250~M each dATP, dCTP, dGTP, dTTP, 5mM dithiothreitol, 250 units/ml DNA polymerase
I, 8.5 units/ml ribonuclease H and incubating at 16 for 2 hr. EDTA was added to 18mM and
WO 95/03408 PCT/US94/08423
~1670~1
-71 -
the solution was extracted with an equal volume of 50% phenol, 49% chloroform, 1 %
isoamyl alcohol. DNA was precipitated with two volumes of ethanol in the presence of 2.5M
ammonium acetate and with 4 micrograms of linear polyacrylamide as carrier. In addition,
cDNA was synthesi7~d from 4~Lg of anti-IgM activated human B cell poly(A)+ RNA in a
- 5 reaction cont~ining 50mM Tris, pH 8.8, 50,ug/ml oligo(dT)12 18, 327 units/ml RNasin, and
952 units/ml AMV reverse transcriptase in a total volume of lOO,ul at 42 for 0.67 hr.
Following reverse transcription, the reverse transcriptase was inactivated by heating at 70
for 10 min. The cDNA was converted to double-stranded DNA by adding 320,u1 H20 and
80~11 of a solution of 0. lM Tris, pH 7.5, 25mM MgC12, 0.5M KCl, 250,ug/ml bovine serum
albumin, and 50mM dithiothreitol, and adjusting the solution to 200~M each dATP, dCTP,
dGTP, dTTP, 50 units/ml DNA polymerase I, 8 units/ml ribonuclease H and incubating at
16, C for 2 hours. EDTA was added to 18 mM and the solution was extracted with an equal
volume of 50 % phenol, 49 % chloroform, 1 % isoamyl alcohol. DNA was precipitated with
two volumes of ethanol in the presence of 2.5M ammonium acetate and with 4 micrograms of
linear polyacrylamide as carrier.
The DNA from 4,ug of AMV reverse transcription and 2,ug of Moloney MLV reverse
transcription was combined. Non-selfcomplement~ry BstXI adaptors were added to the DNA
as follows: The double-stranded cDNA from 6,ug of poly(A)+ RNA was incubated with 3.6,u
g of a kin~ee~l oligonucleotide ofthe sequence CTTTAGAGCACA (SEQ ID NO:15) and 2.4
~Lg of a kin~ce~l oligonucleotide of the sequence CTCTAAAG (SEQ ID NO: 16) in a solution
cont~ining 6mM Tris, pH 7.5, 6mM MgC12, SmM NaCl, 35011g/ml bovine serum albumin,
7mM mercaptoethanol, O.lmM ATP, 2mM dithiothreitol, lmM spermicline~ and 600 units T4
DNA ligase in a total volume of 0.45ml at 15 C for 16 hours. EDTA was added to 34mM
and the solution was extracted with an equal volume of 50% phenol, 49% chloroform, 1 %
isoamyl alcohol. DNA was precipitated with two volumes of ethanol in the presence of 2.5M
ammonium acetate.
DNA larger than 600bp was selected as follows: The adaptored DNA was redissolvedin lOmM Tris, pH 8, lmM EDTA, 600mM NaCl, 0.1% sarkosyl and chromatographed on aSepharose CL-4B column in the same buffer. DNA in the void volume of the column
(cont~ining DNA greater than 600bp) was pooled and ethanol precipitated.
The pCDM8 vector was prepared for cDNA cloning by digestion with BstXI and
purification on an agarose gel. Adaptored DNA from 6~1g of poly(A)+RNA was ligated to
2.25~1g of BstXI cut pCDM8 in a solution cont~ining 6mM Tris, pH 7.5, 6mM MgC12, SmM
NaCl, 350~Lg/ml bovine serum albumin, 7mM mercaptoethanol, O.lmM ATP, 2mM
dithiothreitol, lmM spermidine, and 600 units T4 DNA ligase in a total volume of 1.5ml at
15 for 24 hr. The ligation reaction mixture was transformed into competent E.coli
MC 1061/P3 and a total of 4,290,000 independent cDNA clones were obtained.
WO 9s/03408 2~ 1 PCTIUS94/08423
-72-
Plasmid DNA was prepared from a 500 ml culture of the original transformation ofthe cDNA library. Plasmid DNA was purified by the ~lk~line Iysis procedure followed by
twice banding in CsCl equilibrium gradients (Maniatis et al, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor, NY (1987)).
S
P~. Clonin~ Procedure
In the first round of screening, thirty 100 mm dishes of 50% confluent COS cells were
transfected with O.O5~1g/ml anti-IgM activated human B cells library DNA using the DEAE-
Dextran method (Seed et al, Proc. Nafl. Acad. Sci USA, 84:3365 (1987)). The cells were
10 Lly~.si~ l and re-plated after 24 hours. After 47 hours, the cells were ~let~t~.h~d by
in~ub~tion in PBS/0.5 mM EDTA, pH 7.4/0.02% Na azide at 37C for 30 min. The let~ched
cells were treated with 10 ~g/ml/CTLA4Ig and CD28Ig for 45 minntes at 4C. Cells were
washed and distributed into panning dishes coated with affinity-purified Goat anti-human
IgG antibody and allowed to attach at room le~ e~ lt;. After 3 hours, the plates were
gently washed twice with PBS/O.SmM EDTA, pH 7.4/0.02% Na azide, 5% FCS and once
with O.l5M NaCl, 0.01 M Hepes, pH 7.4, 5% FCS. Episomal DNA was recovered from the
panned cells and transformed into E. coli DHlOB/P3. The plasmid DNA was re-introduced
into COS cells via spheroplast fusion as described (Seed et al, Proc. Natl. Acad. Sci. USA,
84:3365 (1987)) and the cycle of ~A~l~s~ion and panning was repeated twice. In the second
and third rounds of selection, after 47 hours, the det~hecl COS cells were first incubated with
a-B7-1 mAbs (133 and Bl.1, 10 ~g/ml~, and COS cells ~A~ S~illg B7-1 were removed by a-
mouse IgG and IgM coated magnetic beads. COS cells were then treated with 10 ,ug/ml of
human CTLA4Ig (hCTLA4Ig) and human CD28Ig (hCD28Ig) and hurnan B7-2 ~A~les~ing
COS cells were selected by p~nning on dishes with goat anti-human IgG antibody plates.
After the third round, plasmid DNA was ple~aled from individual colonies and kansfected
into COS cells by the DEAE-Dexkan method. Expression of B7-2 on transfected COS cells
was analyzed by indirect immunt~fluorescence with CTLA4Ig.
After the final round of selection, plasmid DNA was prepared from individual
colonies. A total of 4 of 48 ç~n~ te clones contained a cDNA insert of approximately 1.2
kb. Plasmid DNA from these four clones was kansfected into COS cells. All four clones
were skongly positive for B7-2 expression by indirect immunofluorescence using CTLA4Ig
and flow cytometric analysis.
C. Sequencin~
The B7-2 cDNA insert in clone29 was sequenced in the pCDM8 expression vector
employing the following skategy. Initial sequencing was performed using sequencing
primers T7, CDM8R (Invikogen) homologous to pCDM8 vector sequences adjacent to the
WO 95/03408 PCT/US94/08423
' 2~6~0~
-73-
cloned B7-2 cDNA (see Table I). Sequencing was performed using dye terminator chemistr~
and an ABI automated DNA sequencer. (ABI, Foster City, CA). DNA sequence obtained
using these primers was used to design additional sequencing primers (see Table I). This
cycle of sequencing and selection of additional primers was continued until the B7-2 cDNA
5 was completely sequenced on both strands.
TABLE I
T7(F) (SE~Q ID NO:3) S'drTAATACGACTCACTATAGGG]3'
10 CDM8(R) (SEQ ID NO:4) 5'd[TAAGGTTCCTTCACAAAG]3'
CDM8 RGV(2) (SEQ ID NO:5) S'd[ACTGGTAGGTATGGAAGATCC]3'
HBX29-5P (2R) (SEQ ID NO:6) 5'd~ATGCGAATCATTCCTGTGGGC]3'
HBX29-5P (2F) (SEQ ID NO:7) 5'd[AAAGCCCACAGGAATGATTCG]3'
HBX29-5P (SEQ ID NO:8) 5'd[CTCTCAAAACCAAABCCTGAG]3'
SPA (SEQ ID NO:9) 5'd[TTAGGTCACAGCAGAAGCAGC]3'
5PA (3FA) (SEQ ID NO:10) 5'd[TCTGGAAACTGACAAGACGCG~3'
HBX29-SP(lR) (SEQ ID NO:11) 5'd[CTCAGGCTTTGGTTTTGAGAG]3'
HBX29-3P(lR) (SEQ ID NO: 12) 5'd[CACTCTCTTCCCTCTCCATTG]3'
HBX29-5P(3R) (SEQ ID NO:13) 5'd[GACAAGCTGATGGAAACGTCG]3'
HBX29-3P(lP) (SEQ ID NO:14) 5'd~CAATGGAGAGGGAAGAGAGTG]3'
The human B7-2 clone 29 contained an insert of 1,120 base pairs with a single long
open reading frame of 987 nucleotides and approximately 27 nucleotides of 3' noncoding
sequences (Figure 8 (SEQ ID NO: 1)). The predicted amino acid sequence encoded by the
open reading frarne of the protein is shown below the nucleotide sequence in Figure 8. The
encoded protein, human B7-2, is predicted to be 329 amino acids in length (SEQ ID NO:2).
This protein sequence exhibits many features common to other type 1 Ig ~u~ r~llily
embrane proteins. Protein translation is predicted to begin at the ATG codon (nucleotide
107-109) based on DNA homology in this region with the consensus eukaryotic translation
initiation site (Kozalc, M. (1987) ~ucl. Acids Res. 1~:8125-8148)- The amino terminus of the
human B7-2 protein (amino acids 1 to 23) has the characteristics of a secretory signal peptide
with a predicted cleavage between the ~l~nines at positions 23 and 24 (von Heiine (1986)
Nucl. Acids Res. 14:4683). Processing at this site would result in a human B7-2 membrane
bound protein of 306 amino acid with an unmodifed molecular weight of approximately 34
kDa. This protein would consist of an extracellular Ig superfamily V and C like domains, of
from about amino acid residue 24^245, a hydrophobic tr~n.~membrane domain of from about
wo 95,03408 2 ~ PCT/US94/08423
-74-
amino acid residue 246-268 and a long cytoplasmic domain of from about amino acid residue
269-329. The homologies to the Ig superfamily are due to the two contiguous Ig-like
domains in the extracellular region bound by the cysteines at positions 40 to 110 and 157 to
218. The extracellular domain also contains eight potential N-linked glycosylation sites. E.
5 coli transfected with a vector cont~inin~ the cDNA insert of clone 29, encoding the human
B7-2 protein, was deposited with the American Type Culture Collection (ATCC) on July 26,
1993 as Accession No. 69357.
Comparison of both the nucleotide and amino acid sequences of human B7-2 with the
GenBank and EMBL databases showed that only the human and murine B7-1 proteins are
related. Ali~nment of the three B7 protein sequences (see Figure 13) shows that human B7-2
has approximately 26% amino acid identity with human B7-1. Figure 13 represents the
comparison of the amino acid sequences for human B7-2 (hB7-2) (SEQ ID NO:2), human
B7-1 (hB7-1) (SEQ ID NO: 28 and 29) and murine B7 (mB7) (SEQ ID NO: 30 and 31). The
amino acid sequences for the human B7-1 and murine B7 (referred to herein as murine B7-1)
can be found in Genbank at Accession #M27533 and X60958 respectively. Vertical lines in
Figure 13 show iclentir~l amino acids between the hB7-2 and hB7-1 or mB7. Identical amino
acids between hB7-1 and mB7 are not shown. The hB7-2 protein exhibits the same general
structure as hB7-1 as defined by the common cysteines (positions 40 and 110, IgV domains;
positions 157 and 217, IgC domain) which the Ig ~u~elr~llily domains and by many other
20 common amino acids. Since both hB7-1 and mB7 have been shown to bind to both human
CTLA4 and human CD28, the amino acids in common between these two related proteins
will be those necessary to comprise a CTLA4 or CD28 binding sequence. An example of
such a sequence would be the KYMGRTSFD (position 81-89, hB7-2) (SEQ ID NO:17) orKSQDNVTELYDVS (position 188-200, hB7-2) (SEQ ID NO: 18). Additional related
25 sequences are evident from the sequence comparison and others can be inferred by
con~ rin~ homologous related amino acids such as aspartic acid and glutamic acid, alanine
and glycine and other recognized functionally related amino acids. The B7 sequences share a
highly positive charged domain with the cytoplasmic portion WKWKKKKRPRNSYKC
(position 269-282, hB7-2) (SEQ ID NO:19) which is probably involved in intracellular
30 ~i~n~lin~
EXAMPLE 5
Ch~racteri~ on of the Recombin~nt B7-2 Ant~en
~ B7-~ R;nds CT~ ~4I~ and Not Anti-R7-1 and Anti-n7-3 Monoclonal Antibodies
COS cells transfected with either vector DNA (pCDNAI), or an expression plasmid
cont~inin~ B7-1 (B7-1) or B7-2 (B7-2) were prepared. After 72 hours, the transfected COS
WO 95/03408 ~ ~ 6 7 0 9 1 PCT/US94/08423
-75 -
cells were detached by incubation in PBS cont~ining 0.5 mM EDTA and 0.02% Na azide for
30 min. at 37C. Cells were analyzed for cell surface e~-~ssion by indirect
imml-nofluorescence and flow cytometric analysis using fluoroscein isothiocyanate
conjugated (FITC) goat-anti-mouse Ig or goat-anti-human IgG FITC (Figure 9). Cell surface
- 5 t;~yLc;ssion of B7-1 was detected with mAbs 133 (anti-B7-1) and BB-1 (anti-B7-1 and anti-
B7-3) and with CTLA4Ig, whereas B7-2 reacted only with CTLA4Ig. Neither of the B7
transfectants showed any staining with the isotype controls (IgM or control Ig). The vector
transfected COS cells showed no st~ining with any of the detection reagents. In addition,
none of the cells showed any staining with the FITC labeled detection reagents and alone.
This demonstrates that B7-2 encodes a protein that is a CTLA4 counter-receptor but is
distinct from B7-1 and B7-3.
R. RNA Rlot ~n~lysiS of P~7-2 Fxpre~ion in Un~timulated ~nd Activated Hl-m~n R Cells~
C~ell T ines ~ntl Myelom~
Human splenic B cells were isolated by removing T cells and monocytes as
previously described (Free~lm~n, A.S., Freeman, G.J., Horowitz, J.C., Daley, J., Nadler, L.M.,
J. Immunol. (1987) 1;~:3260-3267). Splenic B cells were activated using anti-Ig beads and
cells were harvested at the indicated times (Free-lm~n et al., (1987), cited supra). Human
myelomas from bone marrow specimens were enriched by removing T cells and monocytes
using E rosettes and adherence as previously described (Freeman, G.J., et al., J. Immunol.
(1989) 143:2714-2722). RNA was prepared by gl~ni(1ine thiocyanate homogenization and
cesium chloride centrifugation. Equal amounts of RNA (2011g) were electrophoresed on an
agarose gel, blotted, and hybridized to 32P-labelled B7-2 cDNA. Figure 10, panel a, shows
RNA blot analysis of Im~timl7l~ted and anti-Ig activated human splenic B cells and of cell
lines including Raji (B cell Burkitts lymphoma), Daudi (B cell Burkitt's lymphoma), RPMI
8226 (myeloma), K562 (erythrole-lk~mi~), and REX (T cell acute lymphoblastic leukemia).
Figure 10, panel b shows RNA blot analysis of human myeloma specimens.
Three mRNA transcripts of 1.35, 1.65 and 3.0 kb were identified by hybridization to
the B7-2 cDNA (Figure 10, panel b). RNA blot analysis demonstrated that B7-2 mRNA is
expressed in lln~tim~ ted human splenic B cells and increases 4-fold following activation
(Figure 10, panel a). B7-2 mRNA was expressed in B cell neoplastic lines (Raji, Daudi) and
a myeloma (RPMI 8226) but not in the erythroleukemia K562 and the T cell line REX. In
contrast, we have previously shown that B7-1 mRNA is not expressed in resting B cells and
is transiently expressed following activation (G.J. Freeman et al. (1989) supra). F~min~tion
of mRNA isolated from human myelomas demonstrates that B7-2 mRNA is expressed in 6 of
6 p~ti~nt~, whereas B7-1 was found in only 1 ofthese 6 (G.J. Freeman et al. (1989) supra).
Thus, B7-1 and B7-2 e~ s~ion appears to be independently regulated.
Wo 95/03408 PCT/US94/08423
76-
C. Costimulation
Human CD28+ T cells were isolated by immunomagnetic bead depletion using
monoclonal antibodies directed against B cells, natural killer cells and macrophages as
previously described (Gimmi, C.D., et al. (1993) Proc. Natl. Acad. Sci. USA ~, 6586-6590).
B7-1, B7-2 and vector transfected COS cells were harvested 72 hours after transfection,
incubated with 2511g/ml of mitomycin-C for 1 hour, and then extensively washed. 105
CD28+ and T cells were incubated with 1 ng/ml of phorbol myristic acid (PMA) and the
in~lic~ted number of COS transfectants (Figure 11). As shown in Figure 11, panel a, T cell
proliferation was measured by 3H-thymidine (1 ~Ci) incorporated for the last 12 hours of a
72 hour incubation. Panel b of Figure 11 shows IL-2 production by T cells as measured by
ELISA (Biosource, CA) using supernatants harvested 24 hours after the initiation of culture.
n. P,7-2 Costimulation i~ n~t Blocked by Anti-B7-1 and Anti-P~7-3 mAbs but is Blocked by
CTT ~4-I~ and Anti-CD28 Fab
Human CD28+ T cells were isolated by immunomagnetic bead depletion using mAbs
directed against B cells, natural killer cells, and macrophages as previously described
(Gimmi, C.D., Freeman, G.J., Gribben, J.G., Gray, G., Nadler, L.M. (1993) Proc. Natl. Acad.
Sci USA 90, 6586-6590). B7-1, B7-2, and vector transfected COS cells were harvested 72
hours after transfection, incubated with 25,ug/ml of mitomycin-C for I hour, and then
extensively washed. 105 CD28+ T cells were incubated with 1 ng/ml of phorbol myristic
acetate (PMA) and 2 x 104 COS transfectants. Blocking agents (lO~g/ml) are indicated on
the left side of Figure 12 and include: 1) no monoclonal antibody (no blocking agents), 2)
mAb 133 (anti-B7-1 mAb), 3) rnAb BBl (anti-B7-1 and anti-B7-3 mAb), 4) mAb B5
(control IgM mAb), 5) anti-CD28 Fab (mAb 9.3), 6) CTLA-Ig, and 7) control Ig. Panel a of
Figure 12 shows proliferation measured by 3H-thymidine (l,uCi) incorporation for the last 12
hours of a 72 hour incubation. Figure 12, panel b, shows IL-2 production æ measured by
ELISA (Biosource~ CA) using supern~t~nt~ harvested 24 hours after the initiation of culture.
B7-1 and B7-2 transfected COS cells costim~ te~l equivalent levels of T cell
proliferation when tested at various stimulator to responder ratios (Figure 11). Like B7-1,
B7-2 transfected COS cell costimulation resulted in the production of IL-2 over a wide range
of stimul~tor to responder cell ratios (Figure 11). In contrast, vector transfected COS cells
did not costimulate T cell proliferation or IL-2 production.
Wo 95/03408 ~16 7 ~ 91 PCT/US94/08423
-77-
F. P~7-2 Costimulation is not Blocked by Anti-B7-1 and Anti-~7-3 mAbs but is Blocked by
CTT ~4-I~ ~nd ~nti-CD28 Fab
Human CD28+ T cells were isolated by immunomagnetic bead depletion using mAbs
directed against B cells, natural killer cells, and macrophages as previously described
- 5 (Gimmi, C.D., Freeman, G.J., Gribben, J.G., Gray, G., Nadler, L.M. (1993) Proc. Natl. Acad
Sci USA ~, 6586-6590). B7-1, B7-2, and vector transfected COS cells were harvested 72
hours after transfection, incubated with 25~1g/ml of mitomycin-C for 1 hour, and then
extensively washed. 105 CD28+ T cells were incubated with 1 ng/ml of phorbol myristic
acetate (PMA) and 2 x 104 COS transfectants. Blocking agents (lOIlg/ml) are indicated on
the left side of Figure 12 and include: 1) no monoclonal antibody (no blocking agents), 2)
mAb 133 (anti-B7-1 mAb), 3) mAb BB1 (anti-B7-1 and anti-B7-3 mAb), 4) mAb B5
(control IgM mAb), 5) anti-CD28 Fab (mAb 9.3), 6) CTLA-Ig, and 7) control Ig. Panel a of
Figure 12 shows proliferation measured by 3H-thymidine (l~Ci) incorporation for the last 12
hours of a 72 hour incubation. Figure 12, panel b, shows IL-2 production as measured by
ELISA (Biosource, CA) using sUpern~t~nt~ harvested 24 hours after the initiation of culture.
To distinguish B7-2 from B7-1 and B7-3, mAbs directed against B7-1 and B7-3 wereused to inhibit proliferation and IL-2 production of submitogenically activated human CD28+
T cells. Both B7-1 and B7-2 COS tran~r~ck~ costimulated T cell proliferation and IL-2
production (Figure 12). MAbs 133 (Free~lm~n, A.S. et al. (1987) supra) (anti-B7-1) and BBl
(Boussiotis, V.A., et al., (in review) Proc. Natl. Acad. Sci. USA; Yokochi, T., Holly, R.D.,
Clark, E.A. (1982) J. Immunol. 128, 823-827) (anti-B7-1 and anti-B7-3) completely inhibited
proliferation and IL-2 secretion induced by B7-1 but had no effect upon costim~ ion by B7-
2 transfected COS cells. Isotype m~trhP~l control B5 mAb had no effect. To ~letermine
whether B7-2 signals via the CD28/CTLA4 pathway, anti-CD28 Fab and CTLA4-Ig fusion
protein were tested to cl~t~rmine whether they inhibited B7-2 costimlll~tion. Both anti-CD28
Fab and CTLA4-Ig inhibited proliferation and IL-2 production intlll~e~l by either B7-1 or B7-
2 COS transfectants whereas control Ig fusion protein had no effect (Figure 12). While
CTLA4-Ig inhibited B7-2 costimlll~tion of proliferation by only 90%, in other experiments
inhibition was more pronounced (98-100%). None of the blocking agents inhibited T cell
proliferation or IL-2 production induced by the combination of PMA and
phytohem~g~lutinin.
Like B7-1, B7-2 is a counter-receptor for the CD28 and CTLA4 T cell surface
molecules. Both proteins are similar in that they are: 1) expressed on the surface of APCs;
2) structurally related to the Ig supergene family with an IgV and IgC domain which share
26% arnino acid identity, and 3) capable of costimulating T cells to produce IL-2 and
proliferate. However, B7-1 and B7-2 differ in several fimc1~ment~l ways. First, B7-2 mRNA
is co~ iLulively expressed in unstimulated B cells, whereas B7-1 mRNA does not appear
WO 95/03408 PCT/US94/08423
21~9 1 -78-
until 4 hours and cell surface protein is not detected until 24 hours (Free~1m~n? A.S., et al.
(1987) supra; Freeman, G.J., et al. (1989) supra). Unstim~ te~l hurnan B cells do not express
CTLA4 counter-receptors on the cell surface and do not costim~ te T cell proliferation
(Boussiotis, V.A., et al. supra). Therefore, expression of B7-2 mRNA in unstimulated B cells
would allow rapid expression of B7-2 protein on the cell surface following activation,
presurnably from stored mRNA or protein. Costimlll~tion by B7-2 transfectants is partially
sensitive to paraformaldehyde fixation, whereas B7-2 costimlll~tion is resistant (Gimmi,
C.D., et al. (1991) Proc. Natl. Acad. Sci USA 88, 6575-6579). Second, ~xl~les~ion of B7-1
and B7-2 in cell lines and human B cell neoplasms substantially differs. Third, B7-2 protein
contains a longer cytoplasmic domain than B7-1 and this could play a role in ~i~n~lin~ B-cell
di~,ellliation. These phenotypic and functional differences suggest that these homologous
molecules may have biologically distinct functions.
EXAMPLE 6
Clor~i~ and Sequenl~in~ of the Murine B7-2 Al~tu~en
A. Construction of cDNA T ibr~ly
A cDNA library was constructed in the pCDM8 vector (Seed, Nature, 329:840
(1987)) using poly (A)+ RNA from dibutryl cyclic AMP (cAMP) activated M12 cells (a
murine B cell tumor line) as described (Aruffo et al, Proc. Natl. Acad. Sci USA, 84:3365
(1987)).
M12 cells were cultured at 1x106 cells/ml in complete culture media, {RPMI 1640
with 10% heat inactivated fetal calf serum (FCS), 2mM glllt~mine, 1 mM sodium pyruvate,
penicillin (100 unitslml), ~Llc;~Loll~ycin sulfate (lOO~g/ml) and gen~..ycin sulfate (5,ug/ml)3,
25 in tissue culture flasks and were activated by 300~1g/ml dibutryl cAMP (Nabavi, N., et al.
(1992) Nature ~Q., 266-268). Activated M12 cells were harvested after 0, 6, 12, 18, 24 and
30 hours.
RNA was prepared by homogenizing activated M12 cells in a solution of 4M
gll~niclin~ thiocyanate, 0.5% sarkosyl, 25mM EDTA, pH 7.5, 0.13% Sigma anti-foam A, and
30 0.7% mercaptoethanol. RNA was purified from the homogenate by centrifugation for 24
hour at 32,000 rpm through a solution of 5.7M CsCI, lOmM EDTA, 25mM Na acetate, pH 7.
The pellet of RNA was dissolved in 5% sarkosyl, lmM EDTA, lOmM Tris, pH 7.5 and
extracted with two volumes of 50% phenol, 49% chloroform, 1% isoamyl alcohol. RNA was
ethanol precipitated twice. Poly (A)+ RNA used in cDNA library construction was purified
35 by two cycles of oligo (dT)-cellulose selection
Complement~ry DNA was synthesi7ecl from S.S,ug of dibutryl cAMP activated
murine M12 cell poly(A)+ RNA in a reaction co"~ i"~ SOmM Tris, pH 8.3, 75mM KCl,
WO 95/0340~ 2 ~ 6 7 ~ 91 PCT/US94tO8423
.~
-79-
3mM MgC12, 1 OmM dithiothreitol, SOO~lM dATP, dCTP, dGTP, dTTP, 50~g/ml
oligo(dT)12 18, 180 units/ml RNasin, and 10,000 units/ml Moloney-MLV reverse
transcriptase in a total volume of SS~ll at 37 C for 1 hr. Following reverse transcription, the
cDNA was converted to double-stranded DNA by adjusting the solution to 25mM Tris, pH
8.3, l OOmM KCl, SmM MgC12, 250~1M each dATP, dCTP, dGTP, dTTP, SmM
dithiothreitol, 250 units/ml DNA polymerase I, 8.5 units/ml ribonuclease H and incubating at
16 C for 2 hr. EDTA was added to 18mM and the solution was extracted with an equal
volume of 50% phenol, 49% chloroform, 1 % isoamyl alcohol. DNA was precipitated with
two volurnes of ethanol in the presence of 2.5M ammonium acetate and with 4 micrograms of
linear polyacrylamide as carrier. Following reverse transcription, the reverse transcriptase
was inactivated by heating at 70 C for 10 min. The cDNA was converted to double-stranded
DNA by adding 320~L1 H20 and 80,u1 of a solution of O.lM Tris, pH 7.5, 25mM MgC12,
O.SM KCI, 250~1g/ml bovine serum albumin, and 50mM dithiothreitol, and adjusting the
solution to 200~M each dATP, dCTP, dGTP, dTTP, SO units/ml DNA polymerase I, 8
units/ml ribonuclease H and incllbating at 16 C for 2 hours. EDTA was added to 18 mM and
the solution was extracted with an equal volume of 50% phenol, 49% chloroform, 1%
isoamyl alcohol. DNA was precipitated with two volumes of ethanol in the presence of 2.5M
ammonium acetate and with 4 micrograms of linear polyacrylamide as carrier.
2~Lg of non-selfcomplçment~ry BstXI adaptors were added to the DNA as follows:
The double-stranded cDNA from 5.5~1g of poly(A)+ RNA was incubated with 3.6,~Lg of a
kin~t~ecl oligonucleotide of the sequence Cl-l~AGAGCACA (SEQ ID NO: 15) and 2.4~g of a
kin~eed oligonucleotide ofthe sequence CTCTAAAG (SEQ ID NO:16) in a solution
co..l~t;~ 6mM Tris, pH 7.5, 6mM MgC12, 5mM NaCl, 350,ug/ml bovine serum albumin,
7mM mercaptoethanol, O.lmM ATP, 2mM dithiothreitol, lmM spermi(line, and 600 units T4
DNA ligase in a total volume of 0.45ml at 15 for 16 hours. EDTA was added to 34mM and
the solution was extracted with an equal volume of 50% phenol, 49% chloroform, 1%
isoamyl alcohol. DNA was precipitated with two volumes of ethanol in the presence of 2.5M
ammoniD acetate.
DNA larger than 600bp was selected as follows: The adaptored DNA was redissolvedin lOmM Tris, pH 8, lmM EDTA, 600mM NaCl, 0.1% sarkosyl and chromatographed on aSepharose CL-4B column in the same buffer. DNA in the void volume of the column
(co--l~i.,;t~f~ DNA greater than 600bp) was pooled and ethanol precipitated.
The pCDM8 vector was prepared for cDNA cloning by digestion with BstXI and
purification on an agarose gel. Adaptored DNA from 5.5,ug of poly(A)+RNA was ligated to
2.25~1g of BstXI cut pCDM8 in a solution co~ 6mM Tris, p~J 7.5, 6mM MgC12, SmM
NaCl, 350~Lg/ml bovine serum albumin, 7mM mercaptoethanol, O.lmM ATP, 2mM
dithiothreitol, lmM sperrni~line, and 600 units T4 DNA ligase in a total volume of 1.5ml at
wo 95,03408 ~ ~ 6 7 ~ ~ ~ PCT/US94/08423 ~
-80-
15 for 24 hr. The ligation reaction mixture was transformed into competent E.coli
MC1061/P3 and a total of 200 x 106 independent cDNA clones were obtained.
Plasmid DNA was prepared from a 500 ml culture of the original transformation ofthe cDNA library. Plasmid DNA was purified by the ~lk~line Iysis procedure followed by
5 twice banding in CsCl equilibrium gradients (Maniatis et al, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor, NY (1987)).
R. Clonin~ Proce~lllre
In the first round of screening, thirty 100 mm dishes of 50% confluent COS cells were
transfected with 0.05~1g/ml activated M12 murine B cell library DNA using the DEAE-
Dextran method (Seed et al, Proc. Natl. Acad. Sci. US~, 84:3365 (1987)). The cells were
trypsini7~cl and re-plated after 24 hours. After 47 hours, the cells were detached by
incubation in PBS/0.5 mM EDTA, pH 7.4/0.02% Na azide at 37C for 30 min. The detached
cells were treated with 10 ~g/ml/human CTLA4Ig and murine CD28Ig for 45 minutes at 4C.
Cells were washed and distributed into p~nning dishes coated with affinity-purified Goat anti-
human IgG antibody and allowed to attach at room temperature. After 3 hours, the plates
were gently washed twice with PBS/O.SmM EDTA, pH 7.4/0.02% Na azide, 5% FCS and
once with 0.15M NaCl, 0.01 M Hepes, pH 7.4, 5% FCS. Episomal DNA was recovered from
the panned cells and transformed into E. coli DHlOB/P3. The plasmid DNA was re-
introduced into COS cells via spheroplast fusion as described (Seed et al, Proc. Natl. Acad.
Sci USA, 84:3365 (1987)) and the cycle of expression and p~nnin~ was repeated twice. In
the second and third rounds of selection, after 47 hours, the detached COS cells were first
incubated with a-murine B7-1 mAb (16-lOA1, 10 ,ug/ml), and COS cells expressing B7-1
were removed by a-mouse IgG and IgM coated magnetic beads. COS cells were then treated
with lO~Lg/ml of human CTLA4Ig and murine CD28Ig and murine B7-2 expressing COS
cells were selected by p~nning on dishes coated with goat anti-human IgG antibody. After
the third round, plasmid DNA was prepared from individual colonies and transfected into
COS cells by the DEAE-Dextran method. Expression of B7-2 on transfected COS cells was
analyzed by indirect immllnnfluorescence with CTLA4Ig.
After the final round of selection, plasmid DNA was prepared from individual
colonies. A total of 6 of 8 candidate clones contained a cDNA insert of approximately 1.2
kb. Plasmid DNA from these eight clones was transfected into COS cells. All six clones
with the 1.2 Kb cDNA insert were strongly positive for B7-2 ~iession by indirectimmunofluorescence using CTLA4Ig and flow cytometric analysis.
9 ~
~YO 95/03408 PCT/US94/08423
-81-
C. Se~uencin~
The B7-2 cDNA insert in clone4 was sequenced in the pCDM8 expression vector
employing the following strategy. Initial sequencing was performed using sequencing
primers T7, CDM8R (Invitrogen) homologous to pCDM8 vector sequences adjacent to the
5 cloned B7-2 cDNA (see Table II). Sequencing was performed using dye termin~torchPmi~try and an ABI automated DNA sequencer. (ABI, Foster City, CA). DNA sequence
obtained using these primers was used to design additional sequencing primers (see Table II).
This cycle of sequencing and selection of additional primers was continl1c-1 until the murine
B7-2 cDNA was completely sequenced on both strands.
TABLE II
T7(F) (SEQ ID NO:3) 5'd[TAATACGACTCACTATAGGG]3'
CDM8(R) (SEQ ID NO:4) 5'd[TAAGGTTCCTTCACAAAG]3'
MBX4-lF (SEQ ID NO:24) 5'd[ACATAAGCCTGAGTGAGCTGG]3'
MBX4-2R (SEQ ID NO:25) 5'd[ATGATGAGCAGCATCACAAGG]3'
MBX4-14 (SEQ ID NO:26) 5'd[TGGTCGAGTGAGTCCGAATAC]3'
MBX4-2F (SEQ ID NO:27) 5'd[GACGAGTAGTAACATACAGTG]3'
A murine B7-2 clone (mB7-2, clone 4) was obtained cont~inin~ an insert of 1,163
base pairs with a single long open reading frame of 927 nucleotides and approximately 126
nucleotides of 3' noncoding sequences (Figure 14, SEQ ID NO:22). The predicted amino
acid sequence encoded by the open reading frame of the protein is shown below the
nucleotide sequence in Figure 14. The encoded murine B7-2 protein, is predicted to be 309
amino acid residues in length (SEQ ID NO:23). This protein sequence exhibits many
features common to other type I Ig superfamily membrane proteins. Protein translation is
predicted to begin at the methionine codon (ATG, nucleotides 111 to 113) based on the DNA
homology in this region with the consensus eucaryotic translation initiation site (see Kozak,
M. (1987) Nucl. Acids Res. 15:8125-8148). The amino terminus ofthe murine B7-2 protein
(amino acids 1 to 23) has the characteristics of a secretory signal peptide with a predicted
cleavage between the alanine at position 23 and the valine at position 24 (von Heijne (1987)
Nucl. ~cids Res. 14:4683). Processing at this site would result in a murine B7-2 membrane
bound protein of 286 amino acids having an unmodified molecular weight of approximately
- 32 kDa. This protein would consist of an approximate extracellular Ig superfamily V and C
like domains of from about amino acid residue 24 to 246, a hydrophobic transmembrane
domain of from about amino acid residue 247 to 265, and a long cytoplasmic domain of from
about amino acid residue 266 to 309. The homologies to the Ig superfamily are due to the
WO 95/03408 ~ PCT/US94/08423
-82-
two contiguous Ig-like domains in the extracellular region bound by the cysteines at positions
40 to 110 and 157 to 216. The extracellular domain also contains nine potential N-linked
glycosylation sites and~ like murine B7-1, is probably glycosylated. Glycosylation ofthe
murine B7-2 protein may increase the molecular weight to about 50-70 kDa. The
cytoplasmic domain of murine B7-2 contains a common region which has a cysteine
followed by positively charged amino acids which presumably functions as ~ign~ling or
regulatory domain within an APC. Comparison of both the nucleotide and amino acid
sequences of mur~ne B7-2 with the GenBank and EMBL ~l~t~b~es yielded significanthomology (about 26% amino acid sequence identity) with human and murine B7-1. Murine
B7-2 exhibits about 50% identity and 67% similarity with its human homologue, hB7-2. E.
coli (DH106/p3) transfected with a vector (plasmid pmBx4) con~ining a cDNA insert
encoding murine B7-2 (clone 4) was deposited with the American Type Culture Collection
(ATCC) on August 18, 1993 as Accession No. 69388.
n. Costimulation
CD4+ murine T cells were purified by first depleting red blood cells by tre~tment
with Tris-NH4Cl. T cells were enriched by passage over a nylon wool column. CD4+ T
cells were purified by two-fold tre~tment with a lllixlu~e of anti-MHC class II and anti-CD28
mAbs and rabbit complement. Murine B7-1 (obtained from Dr. Gordon Freeman, Dana-Farber Cancer Institute, Boston, MA; see also, Freeman, G.J. et al (1991) J. Exp. Med. 174,
625-631) murine B7-2, and vector transfected COS cells were harvested 72 hours after
trnasfection, incubated with 25,ug/ml mitomycin-C for one hour, and then extensively
washed. 105 murine CD4+ T cells were incubated with lng/ml of phorbol myristic acid
(PMA) and 2 x 104 COS transfectants (Table III). T cell proliferation was measured by 3H-
thymidine (l~lCi) incorporated for the last 12 hours of a 72 hour incubation.
TABLE III
3H-Thymidine Tncorporation (cpm)
CD4+ T cells 175
CD4+ T cells + lng/ml PMA 49
CD4+ T cells + COS-vector 1750
CD4+ T cells + COS-B7-1 4400
CD4+ T cells + COS-B7-2 2236
CD4+ T cells + lng/ml PMA + COS-vector 2354
CD4+ T cells + lng/ml PMA + COS-B7-l67935
CD4+ T cells + lng/ml PMA + COS-B7-243847
O 95/03408 ~ 1 6 7 0 9 ~L PCT/US94/08423
-83 -
EXAMPLE 7
Con~tru~tion and Ch~r~cteri7~tion of H~lman B7-2 Imlnuno~Jobulin Fusion Proteins
,; ..
A. PreparationOfH-Im~nF~7-2IgFusionProtein~
The extracellular portion of human B7-2 was prepared as a fusion protein coupled to
an immllnoglobulin constant region. The immnnc)globulin constant region may contain
genetic modifications including those which reduce or elimin~te effector activity inherent in
the immunoglobulin skucture. Briefly, DNA encoding the extracellular portion of hB7-2 was
joined to DNA encoding the hinge, CH2 and CH3 regions of human IgC~l or IgC~4 modified
by directed mutagenesis. This was accomplished as described in the following subsections.
B. Preparation of Gene Fusions
DNA fragments corresponding to the DNA sequences of interest were prepared by
polymerase chain reaction (PCR) using primer pairs described below. In general, PCR
reactions were prepared in 100 1ll final volume composed of Taq, polymerase buffer (Gene
Amp PCR Kit, Perkin-Elmer/Cetus, Norwalk, CT) cont~ining primers (1 ,uM each), dNTPs
(200 ~lM each) 1 ng oftemplate DNA, and Taq, polymerase (Saiki, R.K., et al. (1988)
Science 239:487-491). PCR DNA amplifications were run on a thermocycler (Ericomp, San
Diego, CA) for 25 to 30 cycles each composed of a denaturation step (1 minute at 94C), a
renaturation step (30 seconds at 54C), and a chain elongation step (1 minute at 72C). The
skucture of each hB7-2 Ig genetic fusion consisted of a signal sequence to facilitate secretion
coupled to the exkacellular domain of B7-2 and the hinge, CH2 and CH3 domains of human
IgC~l or IgCy4. The IgC gamma 1 and IgC gamma 4 sequences contained nucleotide
changes within the hinge region to replace cysteine residues available for disulfide bond
formation with serine residues and may contain nucleotide changes to replace amino acids
within the CH2 domain thought to be required for IgC binding to Fc receptors andcomplement activation.
Sequence analysis confirmed structures of both m~4 and ~1 clones, and each construct
was used to kansfect 293 cells to test transient expression. hIgG ELISA measured/confirmed
transient ex~)lc~sion levels approximately equal to 100 ng protein/ml cell supernatant for both
constructs. NSO cell lines were transfected for permanent ~ sion the the fusion proteins.
- C. Genf~tic Constructioll of h~7-2Ig Fusion Prote;ns
(1). Ple~.dlion of Si~n~l Se~uence
PCR amplification was used to generate an immunoglobulin signal sequence suitable
for secretion of the B7-2Ig fusion protein from m~rnm~ n cells. The Ig signal sequence was
WO 95t03408 2 ~ PCT/US94/08423
-84-
d from a plasmid cont~ining the murine IgG heavy chain gene (Orlandi. R. et al.
(1989) Proc. Natl. Acad. Sci. USA. 86:38333837) using the oligonucleotide 5'-
GGCACTAGGTCTCCAGCTTGAGATCACAGTTCTCTCTAC-3' (#01) (SEQ ID NO: ) as
the forward primer and the oligonucleotide 5'-
GCTTGAATCTTCAGAGGAGCGGAGTGGACACCTGTGG-3' (#02) (SEQ ID NO: ) as
the reverse PCR primer. The forward PCR primer (SEQ ID NO: ) contains recognition
sequences for restriction enzymes BsaI and is homologous to sequences 5' to the initiating
methionine of the Ig signal sequence. The reverse PCR primer (SEQ ID NO: ) is composed
of sequences derived from the 5' end of the extracellular domain of hB7-2 and the 3' end of
the Ig signal sequence. PCR amplification of the murine Ig signal template DNA using these
primers resulted in a 224 bp product which is composed of BsaI restriction sites followed by
the sequence of the Ig signal region fused to the first 20 nucleotides of the coding sequence of
the extracellular domain of hB7-2. The junction between the signal sequence and hB7-2 is
such that protein translation beginning at the signal sequence will continue into and through
hB7-2 in the correct reading frame.
(2). Pl~a~dlion of thf? hP~7-2 Gene St~ ment
The extracellular domain of the hB7.2 gene was prepared by PCR amplification of
plasmid cont~inin~ the hB7-2 cDNA inserted into t;~lession vector pCDNAI (Freeman et
al., Science 262:909-11 (1994)):
The extracellular domain of hB7-2 was prepared by PCR amplification using
oligonucleotide 5'-GCTCCTCTGAAGATTCAAGC-3' (#03) (SEQ ID NO: ) as the fonvard
primer and oligonucleotide 5'-GGCACTATGATCAGGGGGAGGCTGAGGTCC-3' (#04)
(SEQ ID NO: ) as the reverse prirner. The forward PCR primer contained sequencescorresponding to the first 20 nucleotides of the B7-2 extracellular domain and the reverse
PCR primer contained sequences corresponding to the last 22 nucleotides of the B7-2
extracellular domain followed by a Bcl I restriction site and 7 noncoding nucleotides. PCR
amplification with primer #03 and #04 yields a 673 bp product corresponding to the
extracellular IgV and IgC like domains of hB7-2 followed by a unique Bcl I restriction site.
The signal sequence was attached to the extracellular portion of hB7-2 by PCR asfollows. DNA-PCR products obtained above corresponding to the signal sequence and the
hB7-2 extracellular domain were mixed in equimolar amounts, denatured by heating to
100C, held at 54C for 30C to allow the complementary ends to anneal and the strands
were filled in using dNTPs and Toq polymerase. PCR primers #01 and #04 were added and
the entire fragment produced by PCR amplification to yield a ~880 fragment composed of a
BsaI restriction site followed by the signal sequence fused to the extracellular domain of hB7-
2, followed by a Bcl I restriction site.
WO 95/03408 PCT/US94/08423
9 ~
-85-
(3) Clonin~ ~nd Mo(lification of ~mmlmo~loblllin Fusion Do~in
Plasmid pSP72 lgGI was prepared by cloning the 2000 bp segment of human IgGI
heavy chain genomic DNA (Ellison, J.W., et al. (1982) Nucl. Acids. Res. 10:4071-4079) into
5 the multiple cloning site of cloning vector pSP72 (Promega, Madison, Wl). Plasmid
pSP721 gGI contained genomic DNA encoding the CHI, hinge, CH2 and CH3 domains of the
heavy chain human IgC~1 gene. PCR primers designed to amplify the hinge-CH2-CH3
portion of the heavy chain along with the intervening DNA were pl~ed as follows. The
forward PCR primer 5'-GCATTTTAAG( l~l-l l l CCTGATCAGGAGCCCAAATCTTCT
10 GACAAAACTCACACATCTCCACCGTCTCCAGGTAAGCC-3' (SEQ ID NO: )
contained HindIII and Bcl I restriction sites and was homologous to the hinge domain
sequence except for five nucleotide substitutions which would change the three cysteine
residues to serines. The reverse PCR primer 5'TAATACGACTCACTATAGGG-3' (SEQ ID
NO: ) was identical to the commercially available T7 primer (Promega, Madison, Wl).
Amplification with these primers yielded a 1050 bp fiagment bounded on the 5' end by
HindIII and BclI restriction sites and on the 3' end by BamH1, Smal, Kpnl, Sacl, EcoR1,
Clal, EcoR5 and Bglll restriction sites. This fragment contained the IgC hinge domain in
which the three cysteine codons had been replaced by serine codons followed by an intron,
the CH2 domain, an intron, the CH3 domain and additional 3' sequences. After PCR20 amplification, the DNA fragment was digested with Hindlll and EcoRl and cloned into
c ~,es~ion vector pNRDSH digested with the same restriction enzymes. This created plasmid
pNRDSH/IgG 1.
A similar PCR based strategy was used to clone the hinge-CH2-CH3 domains of
hurnan IgCgamrna4 constant regions. A plasmid, p428D (Medical Research Council,
25 London, F.npl~n~) cont~ining the complete IgCgamma4 heavy chain genomic sequence
(Ellison, J. Buxb~llnn, J. and Hood, L.E. (1981) DNA 1: 11 -18) was used as atemplate for
PCR amplification using oligonucleotide 5'GAGCATTTTCCTGATCAGGA
GTCCAAATATGGTCCCCCATCCCATCATCCCCAGGTAAGCCAACCC-3' (SEQ ID
NO: ) as the forward PCR primer and oligonucleotide
30 S'GCAGAGGAATCGAGCTCGGTACCCGGGGATCCCCAGTGTGGGGACAGTGGGA
CCGCTCTGCCTCCC-3' (SEQ ID NO: ) as the reverse PCR primer. The forward PCR
- primer (SEQ ID NO: ) contains a Bcl l restriction site followed by the coding sequence for
the hinge domain of IgCgamma4. Nucleotide substitutions have been made in the hinge
region to replace the cysteines residues with serines. The reverse PCR primer (SEQ ID NO. )
35 contains a PspAI restriction site (5'CCCGGG-3'). PCR amplification with these primers
results in a 1179 bp DNA fragment. The PCR product was digested with Bcll and PspAI and
ligated to pNRDSH/IgGl digested with the same restriction enzymes to yield plasmid
wo g~/03408 ,~ 9 ~ PCTIUS94/08423 ~
;, -86-
pNRDSH/IgG4. In this reaction, the IgCr 4 domain replaced the IgCyl domain present in
pNRDSH/IgGl .
Modification of the CH2 domain in IgC to replace amino acids thought to be involved
in binding to Fc receptor was accomplished as follows. Plasmid pNRDSH/IgGl served as
template for modifications of the IgCrl CH2 domain and plasmid pNRDSH/IgG4 served as
template for modifications of the IgC~ 4 CH2 domain. Plasmid pNRDSH/IgGl was PCRamplified using a fol~d PCR primer (SEQ ID NO: ) and oligonucleotide 5'-GGGTTTT
GGGGGGAAGAGGAAGACTGACGGTGCCCCC TCGGCTTCAGGTGCTGAGGAAG-3'
(SEQ ID NO: ) as the reverse PCR primer. The forward PCR primer (SEQ ID NO: ) has
been previously described and the reverse PCR primer (SEQ ID NO: ) was homologous to
the amino termin~l portion of the CH2 domain of IgGl except for five nucleotide
substitutions ~le~ignecl to change amino acids 234, 235, and 237 (Canfield, S. M. and
Morrison, S. L. (1991) J. ~cp. Med. 173: 1483-1491.) from Leu to Ala, Leu to Glu, and Gly
to Ala, respectively. Amplification with these PCR primers will yield a 239 bp DNA
fragment con~i~ting of a modified hinge domain, an intron and modified portion of the CH2
domain. Plasmid pNRDSHlIgGl was also PCR amplified with the oligonucleotide 5'-
CATCTCTTCCTCAGCACCTGAAGCCGAGGGGGCACCGTCAGTCTTCCTCTTCCC
CC-3' (SEQ ID NO: ) as the forward primer and oligonucleotide (SEQ ID NO: ) as the
reverse PCR primer. The forward PCR primer (SEQ ID NO: ) is complemf?nt~ry to primer
(SEQ ID NO: ) and contains the five complement~ry nucleotide changes necessary for the
CH2 amino acid repl~ement~ The reverse PCR primer (SEQ ID NO: ) has been previously
described. Amplification with these primes yields a 875 bp fragment con~i~ting of the
modified portion of the CH2 domain, an intron, the CH3 domain, and 3' additional sequences.
The complete IgC~l segment consisting of modified hinge domain, modified CH2 domain
and CH3 domain was prepared by an additional PCR reaction. The purified products of the
two PCR reactions above were mixed, denatured (95C,1 minute) and then renatured (54C,
30 seconds) to allow complementary ends of the two fr~gment~ to anneal. The strands were
filled in using dNTP and Taq polymerase and the entire fragment arnplified using forward
PCR primer (SEQ ID NO: ) and reverse PCR primer (SEQ ID NO: ). The resulting fragment
of 1050 bp was purified, digested with HindIII and EcoR1 and ligated to pNRDSH
previously digested with the same restriction enzymes to yield plasmid pNRDSHIgGl m.
Two amino acids at immllnoglobulin positions 235 and 237 were changed from Leu
to Glu and Gly to Ala, respectively, within the IgCr4 CH2 domain to elimin~te Fc receptor
binding. Plasmid pNRDSH/IgG4 was PCR amplified using the forward primer (SEQ ID NO:
) and the oligonucleotide 5'-
CGCACGTGACCTCAGGGGTCCGGGAGATCATGAGAGTGTCCTTGGGTTTTGGGG
GGAACAGGAAGACTGATGGTGCCCCCTCGAACTCAGGTGCTGAGG-3 ' (SEQ ID
~0 95/03408 ~ l 6 ~ O 9 1 PCT/US94tO8423
-87-
NO: ) as the reverse primer. The forward primer has been previously described and the
reverse primer was homologous to the amino terminal portion of the CH2 domain, except for
three nucleotide substitutions designed to replace the amino acids described above. This
primer also contained a Pmll restriction site for subsequent cloning. Amplification with these
5 primers yields a 265 bp fragment composed of the modified hinge region, and intron, and the
modified 5' portion of the CH2 domain.
Plasmid pNRDSH/lgG4 was also PCR amplified with the oligonucleotide S
'-CCTCAGCACCTGAGTTCGAGGGGGCACCATCAGTCTCCTGTTCCCCCC
AAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCG-3 '
10 (SEQ ID NO: ) as the forward primer and oligonucleotide (SEQ ID NO: ) as the reverse PCR
primer. The forward PCR primer (SEQ ID NO: ) is complement~ry to primer (SEQ ID NO: )
and contains the three complementary nucleotide changes necessary for the CH2 amino acid
replacements. The reverse PCR primer (SEQ ID NO: ) has been previously described.
Amplification with these primes yields a 1012 bp fragment consisting of the modified portion
15 of the CH2 domain, an intron, the CH3 ~lom~in, and 3' additional sequences. The complete
IgC~4 segment consisting of modified hinge domain, modified CH2 domain and CH3 domain
was prepared by an additional PCR reaction. The purified products of the two PCR reactions
above were mixed, denatured (95C,1 minute) and then renatured (54C, 30 seconds) to allow
complementary ends of the two fragments to anneal. The strands were filled in using dNTP
20 and Taq polymerase and the entire fragment amplified using forward PCR primer (SEQ ID
NO: ) and reverse PCR primer (SEQ ID NO: ). The resulting fragment of 1179 bp was
purified, digested with Bcll and PspAI and ligated to pNRDSH previously digested with the
same restriction enzymes to yield plasmid pNRDSH/IgG4m.
(4). A~sernhly of Fin~l hP~7~ Genes
The PCR fragment corresponding to the Ig signal-hB7-2 gene fusion prepared abovewas digested with BsaI and Bcl 1 restriction enzymes and ligated to pNRDSH/IgGl,pNRDSH/lgGlm, pNRDSH/IgG4, and pNRDSH/IgG4m previously digested with Hind III
and BclI. The ligated plasmids were transformed into E. coli JMI09 using CaC12 competent
cells and transformants were selected on L-agar cont~ining ampicillin (50 ,ug/ml; Molecular
Cloning: A Laboratory Manual (1982) Eds. M~ni~ti~, T., Fritsch, E. E., and Sambrook, J.
Cold Spring Harbor Laboratory). Plasmids isolated from the transformed E coli were
analyzed by restriction enzyme digestion. Plasmids with the expected restriction plasmid
were sequenced to verify all portions of the signal-hB7-2-IgG gene fusion segments.
wo 95/03408 ~ 7 ~ ~ ~ PCTIUS94/08423
-88-
n. Fxpression Clonin~ of h~7-2V-I~G 1 and hB7-2C I~G1
The variable and constant domains of human B7-2 were separately cloned into
pNRDSH/IgG1. These clonings were accomplished using PCR. The portions of hB7-2
corresponding to the variable and constant regions were determined from intron/exon
5 mapping and previously published gene structure analysis.
Human B7-2 Variable Domain
5'GCTCCTCTGAAGATT......... GAACTGTCAGTGCTT3' (SEQ ID NO: )
A P L K I E L S V L (SEQ ID NO: )
Human B7-2 Constant Domain
5'GCTAACTTCAGTCAA......... CCTTTCTCTATAGAG3' (SEQ ID NO: )
A N F S Q P F S I E (SEQ ID NO: )
(1). ~sçnlbly of hB7-2VI~
The hB7-2V domain Ig sequence was assembled using a PCR strategy similar to thatshown above. The signal sequence was derived from the onco M gene by PCR amplification
of a plasmid cont~inin~ the onco M gene using oligonucleotide 5'-
GCAACCGGAAGCTTGCCACCATGGGGGTACTGCTCACACAGAGGACG-3' (#05)
20 (SEQ ID NO: ) as the forward PCR primer and 5'-
AGTCTCATTGAAATAAGCTTGAATCTTCAGAGGAGCCATGCTGGCCATGCTTGGA
AACAGGAG-3' (#06) (SWQ ID NO: ) as the reverse primer. The forward PCR primer
(#05) contains a Hind III restriction site and the amino t~rmin~l portion of the onco M signal
sequence. The reverse PCR (#06) contains the sequence corresponding to the 3' portion of
25 the onco M signal sequence fused to the 5' end of the hB7-2 IgV like domain.
The hB7-2 IgV like domain was obtained by PCR amplification of the hB7-2 cDNA
using oligonucleotide 5'-CTCCTGTTTCCAAGCATGGCCAGCATGGCTCCTCTGAA
GATTCAGGCTTATTTCAATGAGAC-3' (#07) (SEQ ID NO: ) as the forward and
oligonucleotide 5'-
30 TGTGTGTGGAATTCTCATTACTGATCAAGCACTGACAGTTCAGAATTCATC-3'
(#08) (SEQ ID NO: ) as the reverse PCR primer. PCR amplification with these primers
yields the hB7-2 IgV domain with a portion of the 3' end of the onco M signal sequence on
the 5' end and a Bcl I restriction site on the 3' end. The signal and IgV domain were linked
together in a PCR reaction in which equimolar amounts of the onco M signal and IgV domain
35 DNA fragments were mixed, denatured, annealed, and the strands filled in. Subsequent PCR
amplification using forward primer #05 and reverse primer #08 yielded a DNA fragment
co~ i"i"g a Hind III restriction site, followed by the onco M signal fused to the B7-2 IgV
~O 95l03408 1 ~ 7 0 9 1 PCT/US94/08423
-89-
domain followed by a Bcl I restriction site. This PCR fragment was digested with Hind II
and Bcl I and cloned into expression vector pNRDSH/IgG1 digested with the same restriction
enzymes to yield pNRDSH/B7-2CIg.
(2). A~mbly of hR7-~CI~
The ~ s~ion plasmid for hB7-2IgC domain was prepared as described above for
the IgV domain except for using PCR primers specific for the IgC domain. The onco M
signal sequence was prepared using oligonucleotide #05 as the forward PCR primer and
oligonucleotide 5'-
1 0 AGAAATTGGTACTATTTCAGGTTGACTGAAGTTAGCCATGCTGGCCATGCTTGGA
AACAGGAG-3' (#09) (SEQ ID NO: ) as the reverse PCR primer. The hB7-2 IgC domain
was prepared using oligonucleotide 5'-
CTCCTGTTTCCAAGCATGGCCAGCATGGCTAACTTCAGTC
AACCTGAAATAGTACCAATTTC-3' (#11) (SEQ ID NO: ) as the reverse PCR primer.
The two PCR products were mixed and amplified with primers #05 and #1 1 to assemble the
onco M signal sequence with the hB7-2IgC domain. The PCR product was subsequently
digested with Hind III and BclI and ligated to pNRDSH/IgG1 digested with similarrestriction enzymes to yield the final ex~r~ssion plasmid pNRDSH~B7-2CIgG1.
F Cornr-etition Rintli~ Ac~ys With Hl~m~n ~7-~Tg Fusion Prote;n~
The ability of various B7 farnily-Ig fusion proteins to competitively inhibit the
binding of biotinylated-CTLA4Ig to immobilized B7-2Ig was determinç-1 Competition
binding assays were done as follows and analysed according to McPherson (McPherson,
G.A. (1985) J. Pharmacol. Methods 14:213-228). Soluble hCTLA4Ig was labelled with 125I
to a specific activity of approximately 2 x 1 o6 cpm/pmol. hB7-2-Ig fusion protein was
coated overnight onto microtiter plates at l0~Lg/ml in 10 mM Tris-HCl, pH8.0, 50 ,ul /well.
The wells were blocked with binding buffer (DMEM cont~ining 10% heat-inactivated FBS,
0.1% BSA, and 50 mM BES, pH 6.8) for 2 h at room temperature. The labeled CTLA4-Ig
(4nM) was added to each well in the presence or absence of unlabeled competing Ig fusion
proteins, including full-length B7-2 (hB7-2Ig), full-length B7-1 (hB7-lIg), the variable
region-like domain of B7-2 (hB7-2VIg) and the constant region-like domain of B7-2 (hB7-
- 2~Ig) and allowed to bind for 2.5 h at room temperature. The wells were washed once with
ice-cold binding buffer and then four times with ice-cold PBS. Bound radioactivity was
recovered by treatment of the wells with 0.5 N NaOH for 5 min and the solubilized material
removed and counted in a gamma counter.
The results of these assays are shown in Figure 15 in which both hB7-2Ig (10-20 nM)
and hB7-2VIg (30-40 nM) competitively inhibit the binding of CTLA4Ig to immobilized B7-
WO 95/03408 PCTIUS94/08423 ~
~7~
so-
2 protein. hB7-2CIg is unable to compete with soluble CTLA4, indicating that the B7-2
binding region is in found in the variable-region like domain.
F. Competitive bindin~ Assays for B7-1 and B7-2 fusion proteins
The ability of the various recombinant CTLA4 forms to bind to hB7-1 or hB7-2 was~s~essed in a competitive binding ELISA assay as follows. Purified recombinant hB7-Ig (20
~Lg/ml in PBS) was bound to a Costar EIAIRIA 96 well microtiter dish (Costar Corp,
Cambridge MA, USA) in 50 ~L overnight at room temperature. The wells were washed three
times with 200 ~lL of PBS and the unbound sites blocked by the addition of 1 % BSA in PBS
(200/well) for 1 hour at room temperature. The wells were washed as above. Biotinylated
hCTLA4IgG1 (ref, MFGR;1 ~Lg/ml serially diluted in twofold steps to 15.6 ng/mL; 50 ,uL)
was added to each well and incubated for 2.5 hours at room tc;lllp~ld~-lre. The wells were
washed as above. The bound biotinylated CTLA4Ig was detected by the addition of 50 1/l of
a 1 :2000 dilution of streptavidin-HRP (Pierce Chemical Co., Rockford, IL) for 30 minutes at
room temperature. The wells were washed as above and 50 ,uL of ABTS (Zymed, California)
added and the developing blue color monitored at 405 nm after 30 min. A graphic
representation of a typical binding assay is shown in Figure 16. The ability of the various
forms of CTLA4 to compete with biotinylated CTLA4IgG1 was ~se~ecl by mixing varying
amounts of the competing protein with a quantity of biotinylated CTLA4IgGl shown to be
non-saturating (i.e., 70 ng/mL; 1.5nM) and perfor~ning the binding assays as described above
(Figure 15). A reduction in the signal (Abs 405 nm) expected for biotinylated CTLA4IgGl
indicated a competition for binding to hB7-1.
Considering the previous evidence that CTLA4 was the high affinity receptor for B7-
1, the avidity of binding of CTLA4 and CD28 to B7-1 and B7-2 was compared. B7-1-Ig or
B7-2-Ig was labelled with biotin and bound to immobilized CTLA4-Ig in the presence or
absence of increasing concentrations of unlabeled B7-1-Ig or B7-2-Ig. The experiment was
repeated with 125-I-labeled B7-1-Ig or B7-2-Ig. Using this solid phase binding assay, the
avidity of B7-2 (2.7 nM) for CTLA4 was dete~nined to be approximately two-fold greater
~an that observed for B7-1 (4.6 nM). The experimentally determined ICso values are
indicated in the upper right corner of the panels. The affinity of both B7-1 and B7-2 for
CD28 was lower and was difficult to confidently determine.
?~ ~7~gl
~0 95/03408 ~ PCT/US94/08423
-91-
EXAMPLE 8
Production ~nd Characteri~ion of Monoclonal Antibo~lies to Hllm~n n7-2
~. Tmmllni7~tion~ ~nd Cell Fusio}l~
Balb/c female mice (obtained from Taconic Labs, Germantown, NY) were immllni7~d
hlll~p~liLoneally with 50 ,~Lg human B7.2-Ig emulsified in complete Freund's adjuvant (Sigma
Chemical Co., St. Louis, MO) or 106 CHO-human B7.2 cells per mouse. The mice were
given two booster immlmi7~tions with 10-25 ~lg human B7.2-Ig emulsified in incomplete
Freund's adjuvant (Sigma Chemical Co., St. Louis, MO) or CHO-human B7.2 cells at10 fourteen, day intervals following the initial i~mmunization for the next two months. The mice
were bled by retro-orbital bleed and the sera assayed for the presence of antibodies reactive to
the immllnogen by ELISA against human B7.2-Ig. ELISA against hCTLA4-Ig was also used
to control for Ig tail directed antibody responses. Mice showing a strong serological response
were boosted intravenously via the tail vein with 25 ~g human hB7.2-Ig diluted in phosphate-
15 buffered saline (PBS), pH 7.2 (GIBCO, Grand Island, NY). Three to four days following this
boost, the spleens from these mice were fused 5:1 with SP 2/0 myeloma cells (American
Type Culture Collection, Rockville, MD, No. CRL8006), which are inc~p~kle of secreting
both heavy and light immunoglobulin chains (Kearney et al. (1979) J. Immunol. l ~3: 1548).
Standard methods based upon those developed by Kohler and Milstein (Nature (1975)
20 ~:495) were used.
F3. Antibo~y Screenir~
After 10-21 days, supern~t~nt~ from wells cont~ining hybridoma colonies from thefusion were screened for the presence of antibodies reactive to human B7.2 as follows: Each
25 well of a 96 well flat bottomed plate (Costar Corp., Cat.3~3590) was coated with 50 ~11 per
well of a I ,ug/ml hurnan B7.2-Ig solution or S x 104 3T3-hB7.2 cells on Iysine coated plates
in phosphate-buffered saline, pH 7.2, overnight at 4 C. The hurnan B7.2-Ig solution was
aspirated off, or the cells were cross-linked to the plates with glutaraldehyde, and the wells
were washed three times with PBS, then blocked with 1% BSA solution (in PBS) (1001l
30 l/well) for one hour at room temperature. Following this blocking incubation, the wells were
washed three times with PBS and 50 1ll of hybridoma supernatant was added per well and
incubated for 1.5 hours at room temperature. Following this incubation, the wells were
washed three times with PBS and then incubated for 1.5 hours at room temperature with 50
~Ll per well of a I :4000 dilution of horseradish peroxidase-conjugated, affinity purified, goat
35 anti-mouse IgG or IgM heavy and light chain-specific antibodies (HRP; Zymed Laboratories,
San Francisco, CA). The wells were then washed three times with PBS, followed by a 30
minute incubation in 50 ,ul per well of I mM 2,2-azino-bis-3-ethylben7t~ 7Oline-6-sulfonic
wO g~/03408 2~ 9~ PCT/US94/08423
-92-
acid (ABTS) in 0.1 M Na-Citrate, pH 4.2 to which a 1:1000 dilution of 30 % hydrogen
peroxide had been added as a substrate for HRP to detect bound antibody. The absorbence
was then deterrnined at OD410 on a spectrophotometric autoreader (Dynatech, Virginia).
Three hybridomas, HA3.1F9, HA5.2B7 and HF~.3Dl, were identified that produced
antibodies to human B7.2-Ig. HA3.1F9 was determined to be of the IgGl isotype, HA5.2B7
was determined to be of the IgG2b isotype and HF2.3Dl as deterrnined to be of the IgG2a
isotype. Each of these hybridomas were subcloned two additional times to insure that they
were monoclonal. Hybidoma cells were deposited with the American Type Culture
Collection, which meets the requirements of the Budapest Treaty, on July 19, 1994 as ATCC
Accession No. (hybridoma HA3.1F9), ATCC Accession No. (HA5.2B7) and
ATCC Accession No. (HF2.3D1).
C. Colnpetitive FT ISA
Supçrn~t~nt~ from the hybridomas HA3.1F9, HA5.2B7 and HF2.3Dl were further
characterized by competitive ELISA, in which the ability of the monoclonal antibodies to
inhibit the binding of biotinylated hCTLA4Ig to immobilized hB7-2 immlln~globulin fusion
proteins was e~r~mined. Biotinylation of hCTLA4Ig was perforrned using Pierce
~mml-nopure NHS-LC Biotin (Cat. No. 21335). B7-2 immunoglobulin fusion proteins used
were: hB7.2-Ig (full-length hB7-2), hB7.2-VIg (hB7-2 variable domain only) and hB7.2-CIg
(B7-2 constant domain only). ~ hB7.1 -Ig fusion protein was used as a control. For the
ELISA, 96 well plates were coated with the Ig fusion protein (50 ,~Ll/well of a 20 ~Lg/ml
solution) overnight at room tc~ c~ . The wells were washed three times with PBS,blocked with 10 % fetal bovine serum (FBS), 0.1 % bovine serum albumin (BSA) in PBS for
1 hour at room temperature, and washed again three times with PBS. To each well was
added 50 ~1 of Bio-hCTLA4-Ig (70 ng/ml) and 50 ,ul of competitor monoclonal antibody
supernatant. Control antibodies were an anti-B7.1 mAb (EW3.5D12) and the anti-hB7-2
mAb B70 (IgG2bK, obtained from Ph~rmin~en). The wells were washed again and
streptavidin-conjugated horse radish peroxidase (from Pierce, Cat. No. 21126; 1 :2000
dilution, 50 ,ul/well) was added and incubated for 30 minutes at room tem~c,alllre. The wells
were washed again, followed by a 30 minute incubation in 50 ~11 per well of ABTS in 0.1 M
Na-Citrate, pH 4.2 to which a 1:1000 dilution of 30 % hydrogen peroxide had been added as
a substrate for HRP to detect bound antibody. The absorbence was then determined at
OD410 on a spectrophotometric autoreader (Dynatech, Virginia). The results, sho~-vn in
Table IV below, demonstrate that each of the mAbs produced by the hybridomas HA3.1 F9,
HA5.2B7 and HF2.3D1 are able to co,,l~clilively inhibit the binding of hCLTA4Ig to full-
length hB7.2-Ig or hB7.2-VIg (hCTLA4Ig does not bind to hB7.2CIg).
2 ~ 9 1
~tO 95/03408 l'CT/US9~/08423
-93 -
TART ~ IV
Blocking of F3in~
hR7.1 -1~ hR7.2-I~ hR7.2-VIg hR7.2-CIg
EW3.5Dl2 (anti-hB7.1 mAb) Yes No No No
B70 (anti-hB7-2) No Yes Yes No
HA3.1F9 (anti-hB7-2) No Yes Yes No
HA5.2B7 (anti-hB7-2) No Yes Yes No
HF2.3D1 (anti-hB7-2) No Yes Yes No
5 r). Flow Cytometry
Supernatants from the hybridomas HA3.1F9, HA5.2B7 and HF2.3D1 were also
characterized by flow cytometry. Supern~t~ntc collected from the clones were screened by
flow cytometry on CHO and 3T3 cells transfected to express hB7.2 (CHO-hB7.2 and 3T3-
h~7.2, respectively) or control transfected 3T3 cells (3T3-Neo). Flow cytometry was
performed as follows: 1 x 106 cells were washed three times in 1 % BSA in PBS, then the
cells were incubated in 50,ul hybridoma supernatant or culture media per 1 x I o6 cells for 30
minutes at 4 C. Following the incubation, the cells were washed three times with l % BSA
in PBS, then incubated in 50 ,ul fluorescein-conjugated goat anti-mouse IgG or IgM
antibodies (Zymed Laboratories, San Francisco, CA) at 1 :50 dilution per 1 x 1 o6 cells for 30
15 rninlltes at 4 C. The cells were then washed three times in 1 % BSA in PBS and fixed with 1
% p~aro..naldehyde solution. The cell sarnples were then analyzed on a FACScan flow
cytometer (Becton Dickinson, San Jose CA). The results, shown in Figures 17, 18 and 19,
demonstrate the monoclonal antibodies produced by the hybridomas HA3.1F9, HA5.2B7 and
HF2.3Dl each bind to hB7-2 on the surface of cells.
F Inhibition of Prolifer~tio~ of H--m~n T Cell~ by Anti-hR7-2 rn~bs
Hybridoma supern~t~nt~ cont~ining anti-hurnan B7-2 mAbs were tested for their
ability to inhibit hB7-2 costimulation of human T cells. In this assay, purified CD28+ human
T cells were treated with submitogenic amounts of PMA (lng/ml) to deliver the primary
signal and with CHO cells expressing hB7-2 on their surface to deliver the costimulatory
signal. Proliferation of the T cells was measured after three days in culture by the addition of
3H-thymidine for the rem~inin~ 18 hours. As shown in Table V, resting T cells show little
proliferation as measured by 3H-thymidine incorporation (510 pm). Delivery of signal 1 by
PMA results in some proliferation (3800 pm) and T cells receiving both the primary (PMA)
and costimulatory (CHO/hB7-2) signals proliferate m~im~lly (9020 cpm). All three anti-
WO95/03408 ~ PCT/US94/08423
-94-
hB7-2 mAbs tested reduce the costimulatory signal intlllcecl proliferation to that found for
PMA treated cells alone showing that these mAbs can inhibit T cell proliferation by blocking
the B7/CD28 costimulatory pathway.
TARTFV
Addition to CD28+ T Cells hB7-2 mAb CPM
510
+PMA --- 3800
+PMA + CHOlhB7-2 --- 9020
+PMA + CHO/hB7-2 HF2.301 3030
--- HA5.2B7 1460
--- HA3.1F9 2980
EXAMPLE 9
10Re~ressior~ of I~planted T--mor Cells Transfected to F.~?ress ~7-2
In this example, untransfected or B7-2 transfected J558 plasmacytoma cells were used
in turnor regression studies to exAmine the effect of ~ cssion of B7-2 on the surface of
tumor cells on the growth of the tumor cells when transplanted into ~nimAI~
15J558 plasmacytoma cells (obtained from the American Type Culture Collection,
Rockville, MD; # TIB 6) were transfected with an expression vector cont~ining cDNA
encoding either mouse B7-2 (pAWNE03) or B7-1 (pNRDSH or pAWNE03) and a neomycin-resi~tAnce gene. Stable trAn~fectAnt~ were selected based upon their neomycin resictAnce and
cell surface expression of B7-2 or B7-1 on the tumor cells was confirme~l by FACS analysis
20 using either an anti-B7-2 or anti-B7-1 antibody.
Syngeneic Balb/c mice, in groups of 5-10 mice/set, were used in experiments
~lesi~n~d to determine whether cell-surface expression of B7-2 on tumor cells would result in
regression of the implanted tumor cells. Untransfected and transfected J558 cells were
cultured in vitro, collected, washed and resuspended in Hank's buffered salt solution
25 (GIBCO, Grand Island, New York) at a concentration of 10~ cells/ml. A patch of skin on the
right flank of each mouse was removed of hair with a depilatory and, 24 hours later, 5 x 106
tumor cells/mouse were implanted intradermally or subdermally. Measurements of tumor
volume (by linear measurements in three perpendicular directions) were made every two to
three days using calipers and a ruler. A typical experiment lasted 18-21 days, after which
~O 95/0340~ ~ 1 6 7 0 91 PCTIUS94/08423
time the tumor size exceeded 10 % of the body mass of mice transplanted with untransfected,
control J558 cells. As shown in Figure 20, J558 cells transfected to express B7-2 on their
surface were rejected by the mice. No tumor growth was observed even after three weeks.
Similar results were observed with J558 cells transfected to express B7-1 on their surface. In
5 contrast, the untransfected (wild-type) J558 cells produced massive tumors in as little as 12
days, requiring the animal to be enth~ni7~cl This example demonstrates that cell-surface
expression of B7-2 on tumor cells, such as by transfection of the tumor cells with a B7-2
cDNA, induces an anti-tumor response in naive ~nim~l~ that is sufficient to cause rejection of
the tumor cells.
FQUIVAT .FNTS
Those skilled in the art will recognize, or be able to ascertain using no more than
routine experimentation, many equivalents to the specific embodiments of the invention
described herein. Such equivalents are intended to be encompassed by the following claims.
WO95/03408 2~6~9~ PCTIUS94/08423 ~
-96-
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: DANA-FARBER CANCER lNS'l'l'l'U'l'~
(B) STREET: 44 BINNEY STREET
(C) CITY: BOSTON
(D) STATE: MASSA~u~LlS
~ (E) COUNTRY: USA
= (F) POSTAL CODE (ZIP): 02115
(G) TELEPHONE: (617) 632-4016
(H) TELEFAX: (617) 632-4012
(A) NAME: REPLIGEN CORPORATION
(B) STREET: ONE KENDALL SQUARE, BLDG 700
(C) CITY: CAMBRIDGE
(D) STATE: MASSA~US~llS
(E) COUNTRY: USA
(F) POSTAL CODE (ZIP): 02139
(G) TELEPHONE: (617) 225-6000
(H) TELEFAX: (617) 494-1975
(ii) TITLE OF INVENTION: Novel CTLA4/CD28 Ligands and
Uses Therefor
(iii) NUMBER OF SEQUENCES: 31
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: LA~IVE ~ COCKFIELD
(B) STREET: 60 State Street, Suite 510
(C) CITY: Boston
(D) STATE: Massachusetts
(E) COUNL~Y: USA
(F) ZIP: 02109
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(c) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version ~1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
4~ (B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US08/101,624; US08/109,393; US08/147,773
(B) FILING DATE: 26-JUL-1993; 19-AUG-1993; 03-NOV-1993
(viii) ArlloKN~y/AGENT INFORMATION:
(A) NAME: Mandragourasl Amy E.
(B) REGISTRATION NUMBER: 36,207
(C) REFERENCE/DOCKET NUMBER: RPI-004CP2PC
~vo 95~03408 2 ~ 1 PCT/US94/08423
-97-
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617) 227-7400
(B) TELEFAX: (617) 227-5941
- (2) INFORMATION FOR SEQ ID NO:1:
~ Q~ ~ CHARACTERISTICS:
(A) LENGTH: 1120 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
( ii ) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 107..1093
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
CACAGGGTGA AAGCTTTGCT TCTCTGCTGC TGTAACAGGG ACTAGCACAG ACACACGGAT 60
GAGTGGGGTC ATTTCCAGAT ATTAGGTCAC AG Q GAAGCA GCCAAA ATG GAT CCC 115
Met Asp Pro
30 CAG TGC ACT ATG GGA CTG AGT AAC ATT CTC TTT GTG ATG GCC TTC CTG 163
Gln Cys Thr Met Gly Leu Ser Asn Ile Leu Phe Val Met Ala Phe Leu
5 10 15
CTC TCT GGT GCT GCT CCT CTG AAG ATT CAA GCT TAT TTC AAT GAG ACT 211
35 Leu Ser Gly Ala Ala Pro heu Lys Ile Gln Ala Tyr Phe Asn Glu Thr
20 25 30 35
GCA GAC CTG CCA TGC CAA TTT GCA AAC TCT CAA AAC CAA AGC CTG AGT 259
Ala Asp Leu Pro Cys Gln Phe Ala Asn Ser Gln Asn Gln Ser Leu Ser
40 45 50
GAG CTA GTA GTA TTT TGG CAG GAC CAG GAA AAC TTG GTT CTG AAT GAG 307
Glu Leu Val Val Phe Trp Gln Asp Gln Glu Asn Leu Val Leu Asn Glu
55 60 65
GTA TAC TTA GGC AAA GAG AAA TTT GAC AGT GTT CAT TCC AAG TAT ATG 355
Val Tyr Leu Gly Lys Glu Lys Phe Asp Ser Val His Ser Lys Tyr Met
70 75 80
50 GGC CGC ACA AGT TTT GAT TCG GAC AGT TGG ACC CTG AGA CTT CAC AAT 403
Gly Arg Thr Ser Phe Asp Ser Asp Ser Trp Thr Leu Arg Leu His Asn
WO 95/03408 2 ~ 1 PCT/US94/08423
-98-
CTT CAG ATC AAG GAC AAG GGC TTG TAT CAA TGT ATC ATC CAT CAC A~A 451
Leu Gln Ile Lys Asp Lys Gly Leu Tyr Gln Cys Ile Ile His His Lys
100 105 110 115
AAG CCC ACA GGA ATG ATT CGC ATC CAC CAG ATG A~T TCT GAA CTG TCA 499
Lys Pro Thr Gly Met Ile Arg Ile His Gln Met Asn Ser Glu Leu Ser
120 125 130
GTG CTT GCT AAC TTC AGT CAA CCT GAA ATA GTA CCA ATT TCT AAT ATA 547
0 Val Leu Ala Asn Phe Ser Gln Pro Glu Ile Val Pro Ile Ser Asn Ile
135 140 145
ACA GAA AAT GTG TAC ATA AAT TTG ACC TGC TCA TCT ATA CAC GGT TAC 595
Thr Glu Asn Val Tyr Ile Asn Leu Thr Cys Ser Ser Ile His Gly Tyr
150 155 160
CCA GAA CCT AAG AAG ATG AGT GTT TTG CTA AGA ACC AAG AAT TCA ACT 643
Pro Glu Pro Lys Lys Met Ser Val Leu Leu Arg Thr Lys Asn Ser Thr
165 170 175
ATC GAG TAT GAT GGT ATT ATG QG A~A TCT CAA GAT AAT GTC ACA GAA 691
Ile Glu Tyr Asp Gly Ile Met Gln Lys Ser Gln Asp Asn Val Thr Glu
180 185 190 195
CTG TAC GAC GTT TCC ATC AGC TTG TCT GTT TCA TTC CCT GAT GTT ACG 739
Leu Tyr Asp Val Ser Ile Ser Leu Ser Val Ser Phe Pro Asp Val Thr
200 205 210
AGC AAT ATG ACC ATC TTC TGT ATT CTG GAA ACT GAC AAG ACG CGG CTT 787
Ser Asn Met Thr Ile Phe Cy5 Ile Leu Glu Thr Asp Lys Thr Arg Leu
215 220 225
TTA TCT TCA CCT TTC TCT ATA GAG CTT GAG GAC CCT CAG CCT CCC CCA 835
Leu Ser Ser Pro Phe Ser Ile Glu Leu Glu Asp Pro Gln Pro Pro Pro
230 235 240
GAC CAC ATT CCT TGG ATT ACA GCT GTA CTT CCA ACA GTT ATT ATA TGT 883
Asp His Ile Pro Trp Ile Thr Ala Val Leu Pro Thr Val Ile Ile Cys
245 250 255
GTG ATG GTT TTC TGT CTA ATT CTA TGG A;~A TGG AAG AAG AAG AAG CGG 931
Val Met Val Phe Cys Leu Ile Leu Trp Lys Trp Lys Lys Lys Lys Arg
260 265 270 275
CCT CGC AAC TCT TAT A~A TGT GGA ACC AAC ACA ATG GAG AGG GAA GAG 979
Pro Arg Asn Ser Tyr Lys Cys Gly Thr Asn Thr Met Glu Arg Glu Glu
280 285 290
AGT GAA CAG ACC AAG AAA AGA GAA AAA ATC CAT ATA CCT GAA AGA TCT 1027
Ser Glu Gln Thr Lys Lys Arg Glu Lys Ile His Ile Pro Glu Arg Ser
295 300 305
GAT GAA GCC CAG CGT GTT TTT A~A AGT TCG AAG ACA TCT TCA TGC GAC 1075
Asp Glu Ala Gln Arg Val Phe Lys Ser Ser Lys Thr Ser Ser Cys Asp
SS 310 315 320
*VO 95/03408 21 ~ ~ O 91 PCTIUS94/08423
_99
AAA AGT GAT ACA TGT TTT TAATTAAAGA GTAAAGCCCA AAAAAAA 1120
Lys Ser Asp Thr Cys Phe
325
s
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTBRISTICS:
(A) LENGTH: 329 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Asp Pro Gln Cys Thr Met Gly Leu Ser Asn Ile Leu Phe Val Met
1 5 10 15
Ala Phe Leu Leu Ser Gly Ala Ala Pro Leu Lys Ile Gln Ala Tyr Phe
20 25 30
Asn Glu Thr Ala Asp Leu Pro Cys Gln Phe Ala Asn Ser Gln Asn Gln
35 40 45
Ser Leu Ser Glu Leu Val Val Phe Trp Gln Asp Gln Glu Asn Leu Val
Leu Asn Glu Val Tyr Leu Gly Lys Glu Lys Phe Asp Ser Val His Ser
65 70 75 80
Lys Tyr Met Gly Arg Thr Ser Phe Asp Ser Asp Ser Trp Thr Leu Arg
85 90 95
Leu His Asn Leu Gln Ile Lys Asp Lys Gly Leu Tyr Gln Cys Ile Ile
100 105 110
His His Lys Lys Pro Thr Gly Met Ile Arg Ile His Gln Met Asn Ser
115 120 125
Glu Leu Ser Val Leu Ala Asn Phe Ser Gln Pro Glu Ile Val Pro Ile
130 135 140
Ser Asn Ile Thr Glu Asn Val Tyr Ile Asn Leu Thr Cys Ser Ser Ile
145 150 155 160
His Gly Tyr Pro Glu Pro Lys Lys Met Ser Val Leu Leu Arg Thr Lys
165 170 175
Asn Ser Thr Ile Glu Tyr Asp Gly Ile Met Gln Lys Ser Gln Asp Asn
180 185 190
Val Thr Glu Leu Tyr Asp Val Ser Ile Ser Leu Ser Val Ser Phe Pro
195 200 205
WO 95/03408 2~ PCT/US94/08423 ~
-100-
Asp Val Thr Ser Asn Met Thr Ile Phe Cys Ile Leu Glu Thr Asp Lys
210 215 220
5 Thr Arg Leu Leu Ser Ser Pro Phe Ser Ile Glu Leu Glu Asp Pro Gln
225 230 235 240
Pro Pro Pro Asp His Ile Pro Trp Ile Thr Ala Val Leu Pro Thr Val
245 250 255
Ile Ile Cys Val Met Val Phe Cys Leu Ile Leu Trp Lys Trp Lys Lys
260 265 270
Lys Lys Arg Pro Arg Asn Ser Tyr Lys Cys Gly Thr Asn Thr Met Glu
275 280 285
Arg Glu Glu Ser Glu Gln Thr Lys Lys Arg Glu Lys Ile His Ile Pro
290 295 300
Glu Arg Ser Asp Glu Ala Gln Arg Val Phe Lys Ser Ser Lys Thr Ser
305 310 315 320
Ser Cys Asp Lys Ser Asp Thr Cys Phe
325
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
tB) TYPE: nucleic acid
(C) sTRANn~nN~s single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
40 TAATACGACT CACTATAGGG 20
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) sTR~Nn~n~s single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
o 9 ~
, ~ 0 95/03408 PCTrUS94/08423
-101 -
TAAGGTTCCT TCACA~AG 18
~2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) sTRp~n~n~cs single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(xi) ~.Qu~ DESCRIPTION: SEQ ID NO:5:
ACTGGTAGGT ATGGAAGATC C 21
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRPNn~nN~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
ATGCGA~TCA TTCCTGTGGG C 21
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRA~N~:SS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
AAAGCCCACA GGAATGATTC G 21
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
WO 95/03408 ~ . PCT/US94/08423
-102-
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
S
(xi) ~u~N~ DESCRIPTION: SEQ ID NO:8:
10 CTCTCAAAAC CA~AGCCTGA G 21
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
~ (C) sTR~N~n~s single
= (D) TOPOLOGY: linear
- 20 (ii) MOLECULE TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
TTAGGT QCA G Q GAAG Q G C 21
(2) INFORMATION FOR SEQ ID NO:10:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STR~NDEDNESS: 8 ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(xi) ~U~N~ DESCRIPTION: SEQ ID NO:10:
TCTGGA~ACT GACAAGACGC G 21
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
~VO 95/03408 ~ PCT/US94/08423
-103-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
CTCAGGCTTT GGTTTTGAGA G 21
5 (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
CA~l-lcllC CCTCTCCATT G 21
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANu~uN~SS: 5 ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
35 GACAAGCTGA TGGAAACGTC G 21
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(xi) ShQU~. DESCRIPTION: SEQ ID NO:14:
CAATGGAGAG GGAAGAGAGT G 21
(2) INFORMATION FOR SEQ ID NO:15:
( i ) S~u~N~ CHARACTERISTICS:
WO 95/03408 21~ 7 0 ~ ~ PCT/US94/08423 ~
-104-
~A) LENGTH: 12 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 5 ingle
(D) TOPOLOGY: linear
tii) MOLECULE TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
CTTTAGAGCA CA 12
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 base pairs
(B) TYPE: nucleic acid
(C) STR~ S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) ~u~N~ DESCRIPTION: SEQ ID NO:16:
CTCTA~AG 8
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Lys Tyr Met Gly Arg Thr Ser Phe Asp
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
~WO 95t03408 ~ 16 ~ ~ 91 PCTtUS94tO8423
-105-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Lys Ser Gln Asp Asn Val Thr Glu Lys Tyr Asp Val Ser
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Trp Lys Trp Lys Lys Lys Lys Arg Pro Arg Asn Ser Tyr Lys Cys
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) sTRANn~nN~s single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(Xi) ~QU~N~'~ DESCRIPTION: SEQ ID NO:20:
TGGCCCATGG CTTCAGA 17
(2) INFORMATION FOR SEQ ID NO:21:
( i ) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(c) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleo~ide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
GCCAAAATGG ATCCCCA 17
WO 95/03408 PCTIUS94/08423 ~
2 ~
-106-
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1163 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
10 (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 111.. 1040
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
~ 20 CCCACGCGTC CGGGAGCAAG CAGACGCGTA AGAGTGGCTC CTGTAGGCAG CACGGACTTG 60
- AACAACCAGA CTCCTGTAGA C~L~llC~AG AACTTACGGA AGCACCCACG ATG GAC 116
Met Asp
CCC AGA TGC ACC ATG GGC TTG GCA ATC CTT ATC TTT GTG ACA GTC TTG 164
Pro Arg Cys Thr Met Gly Leu Ala Ile Leu Ile Phe Val Thr Val Leu
5 10 15
30 CTG ATC TCA GAT GCT GTT TCC GTG GAG ACG CAA GCT TAT TTC AAT GGG 212
Leu Ile Ser Asp Ala Val Ser Val G1U Thr Gln Ala Tyr Phe Asn Gly
20 25 30
ACT GCA TAT CTG CCG TGC CCA TTT ACA AAG GCT CAA AAC ATA AGC CTG 260
35 Thr Ala Tyr Leu Pro Cys Pro Phe Thr Lys Ala Gln Asn Ile Ser Leu
35 40 45 50
AGT GAG CTG GTA GTA TTT TGG CAG GAC CAG CAA AAG TTG GTT CTG TAC 308
Ser Glu Leu Val Val Phe Trp Gln Asp Gln Gln Lys Leu Val Leu Tyr
55- 60 65
GAG CAC TAT TTG GGC ACA GAG A~A CTT GAT AGT GTG AAT GCC AAG TAC 356
Glu His Tyr Leu Gly Thr Glu Lys Leu Asp Ser Val Asn Ala Lys Tyr
70 75 80
CTG GGC CGC ACG AGC TTT GAC AGG AAC AAC TGG ACT CTA CGA CTT CAC 404
Leu Gly Arg Thr Ser Phe Asp Arg Asn Asn Trp Thr Leu Arg Leu His
85 90 95
50 AAT GTT CAG ATC AAG GAC ATG GGC TCG TAT GAT TGT TTT ATA CAA A~A 452
Asn Val Gln Ile Lys Asp Met Gly Ser Tyr Asp Cys Phe Ile Gln Lys
100 105 110
~WO 9~;/03408 ~ ~ 6 ~ 9 ~1 PCT/US94/08423
-107-
AAG CCA CCC ACA GGA TCA ATT ATC CTC CAA CAG ACA TTA ACA GAA CTG 500
Lys Pro Pro Thr Gly Ser Ile Ile Leu Gln Gln Thr Leu Thr Glu ~eu
115 120 125 130
5 TCA GTG ATC GCC AAC TTC AGT GAA CCT GAA ATA AAA CTG GCT CAG AAT 548
Ser Val Ile Ala Asn Phe Ser Glu Pro Glu Ile Lys Leu Ala Gln Asn
135 140 145
GTA ACA GGA AAT TCT GGC ATA AAT TTG ACC TGC ACG TCT AAG CAA GGT 596
0 Val Thr Gly Asn Ser Gly Ile Asn Leu Thr Cys Thr Ser Lys Gln Gly
150 155 160
CAC CCG AAA CCT AAG AAG ATG TAT TTT CTG ATA ACT AAT TCA ACT AAT 644
His Pro Lys Pro Lys Lys Met Tyr Phe Leu Ile Thr Asn Ser Thr Asn
165 170 175
GAG TAT GGT GAT AAC ATG CAG ATA TCA CAA GAT AAT GTC ACA GAA CTG 692
Glu Tyr Gly Asp Asn Met Gln Ile Ser Gln Asp Asn Val Thr Glu Leu
180 185 190
TTC AGT ATC TCC AAC AGC CTC TCT CTT TCA TTC CCG GAT GGT GTG TGG 740
Phe Ser Ile Ser Asn Ser Leu Ser Leu Ser Phe Pro Asp Gly Val Trp
195 200 205 210
25 CAT ATG ACC GTT GTG TGT GTT CTG GAA ACG GAG TCA ATG AAG ATT TCC 788
His Met Thr Val Val Cys Val Leu Glu Thr Glu Ser Met Lys Ile Ser
215 220 225
TCC AAA CCT CTC AAT TTC ACT CAA GAG TTT CCA TCT CCT CAA ACG TAT 836
30 Ser Lys Pro Leu Asn Phe Thr Gln Glu Phe Pro Ser Pro Gln Thr Tyr
230 235 240
TGG AAG GAG ATT ACA GCT TCA GTT ACT GTG GCC CTC CTC CTT GTG ATG 884
Trp Lys Glu Ile Thr Ala Ser Val Thr Val Ala Leu Leu Leu Val Met
245 250 255
CTG CTC ATC ATT GTA TGT CAC AAG AAG CCG AAT CAG CCT AGC AGG CCC 932
Leu Leu Ile Ile Val Cys His Lys Lys Pro Asn Gln Pro Ser Arg Pro
260 265 270
AGC AAC ACA GCC TCT AAG TTA GAG CGG GAT AGT AAC GCT GAC AGA GAG 980
Ser Asn Thr Ala Ser Lys Leu Glu Arg Asp Ser Asn Ala Asp Arg Glu
275 280 285 290
45 ACT ATC AAC CTG AAG GAA CTT GAA CCC CAA ATT GCT TCA GCA AAA CCA 1028
Thr Ile Asn Leu Lys GlU Leu Glu Pro Gln Ile Ala Ser Ala Lys Pro
295 300 305
AAT GCA GAG TGAAGGCAGT GAGAGCCTGA GGA~AGAGTT AAAAATTGCT 1077
50 Asn Ala Glu
TTGCCTGAAA TAAGAAGTGC AGAGTTTCTC AGAATTCAAA AATGTTCTCA GCTGATTGGA 1137
55 ATTCTACAGT TGAATAATTA AAGAAC 1163
WO 95/03408 PCT/US94/08423
-108-
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 309 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) S~Qu~N~ DESCRIPTION: SEQ ID NO:23:
Met Asp Pro Arg Cys Thr Met Gly Leu Ala Ile Leu Ile Phe Val Thr
1 5 10 15
Val Leu Leu Ile Ser Asp Ala Val Ser Val Glu Thr Gln Ala Tyr Phe
20 Asn Gly Thr Ala Tyr Leu Pro Cys Pro Phe Thr Lys Ala Gln Asn Ile
35 40 45
Ser Leu Ser Glu Leu Val Val Phe Trp Gln Asp Gln Gln Lys Leu Val
50 55 60
Leu Tyr Glu His Tyr Leu Gly Thr Glu Lys Leu Asp Ser Val Asn Ala
65 70 75 80
Lys Tyr Leu Gly Arg Thr Ser Phe Asp Arg Asn Asn Trp Thr Leu Arg
85 90 95
Leu His Asn Val Gln Ile Lys Asp Met Gly Ser Tyr Asp Cys Phe Ile
100 105 110
Gln Lys Lys Pro Pro Thr Gly Ser Ile Ile Leu Gln Gln Thr Leu Thr
115 120 125
Glu Leu Ser Val Ile Ala Asn Phe Ser Glu Pro Glu Ile Lys Leu Ala
= 130 135 140
Gln Asn Val Thr Gly Asn Ser Gly Ile Asn Leu Thr Cys Thr Ser Lys
145 150 155 160
Gln Gly His Pro Lys Pro Lys Lys Met Tyr Phe Leu Ile Thr Asn Ser
165 170 175
Thr Asn Glu Tyr Gly Asp Asn Met Gln Ile Ser Gln Asp Asn Val Thr
180 185 190
Glu Leu Phe Ser Ile Ser Asn Ser Leu Ser Leu Ser Phe Pro Asp Gly
195 200 205
Val Trp His Met Thr Val Val Cys Val Leu Glu Thr Glu Ser Met Lys
210 215 220
~WO 95/03408 ~16 7 ~ 91 PCTIUS94/08423
-109-
Ile Ser Ser Lys Pro Leu Asn Phe Thr Gln Glu Phe Pro Ser Pro Gln
225 230 235 240
Thr Tyr Trp Lys Glu Ile Thr Ala Ser Val Thr Val Ala Leu Leu Leu
245 250 255
Val Met Leu Leu Ile Ile Val Cys His Lys Lys Pro Asn Gln Pro Ser
260 265 270
Ary Pro Ser Asn Thr Ala Ser Lys Leu Glu Arg Asp Ser Asn Ala Asp
275 280 285
Arg Glu Thr Ile Asn Leu Lys Glu Leu Glu Pro Gln Ile Ala Ser Ala
290 295 300
Lys Pro Asn Ala Glu
305
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(c) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
ACATAAGCCT GAGTGAGCTG G 21
(2) INFORMATION FOR SEQ ID NO:25:
(i) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRAN~N~SS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
ATGATGAGCA GCATCACAAG G 21
(2) INFORMATION FOR SEQ ID NO:26:
W O 9S/03408 PCTrUS94/08423 _
2~709~ --
-1 10-
(i) S~U~N~ CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: s ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
TGGTCGAGTG AGTCCGAATA C 21
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULB TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
GACGAGTAGT AACATACAGT G 21
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1491 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: no
(iv) ANTI-SENSE: no
(vi) ORIGINAL SOURCE:
(A) ORGANISM: HomQ sapi~n
(F) TISSUE TYPE: lymphoid
(G) CELL TYPE: B cell
(H) CELL LINE: Raji
~WO 95/03408 2 ~ S ~ O 9 I PCTlUS94108423
(vii~ IMMEDIATE SOURCE:
(A) LIBRARY: cDNA in pCDM8 vector
(B) CLONE: B7, Raji clone #13
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT: 3
(ix) FEATURE:
(A) NAME/KEY: Open reading frame (translated region)
(B) LOCATION: 318 to 1181 bp
(C) IDENTIFICATION METHOD: similarity to other pattern
(ix) FEATURE:
(A) NAME/KEY: Alternate polyadenylation signal
(B) LOCATION: 1474 to 1479 bp
(C) IDENTIFICATION METHOD: similarity to other pattern
(x) PUBLICATION INFORMATION:
(A) AUTHORS: FREEMAN, GORDON J.
FREEDMAN, ARNOLD S.
SEGIL, JEFFREY M.
LEE, GRACE
WHITMAN, JAMES F.
NADLER, LEE M.
(B) TITLE: B7, A New Member Of The Ig Superfamily With
Unique Expression On Activated And Neoplastic B Cells
(c) JOURNAL: The Journal of Immunology
(D) VOLUME: 143
(E) ISSUE: 8
(F) PAGES: 2714-2722
(G) DATE: 15-OCT-1989
(H) RELEVANT RESIDUES In SEQ ID NO:28: FROM 1 TO 1491
(xi) ~Uu~ DESCRIPTION: SEQ ID NO:28:
CCAAAGAAAA AGTGATTTGT CATTGCTTTA TAGACTGTAA GAAGAGAACA TCTCAGAAGT 60
GGAGTCTTAC CCTGAAATCA AAGGATTTAA AGAAAAAGTG GAALLlLl~l~ TCAGCAAGCT 120
GTGAAACTAA ATCCACAACC TTTGGAGACC CAGGAACACC CTCCAATCTC 'l'~'l'~'L~'l"L-l"l' 180
GTAAACATCA CTGGAGGGTC TTCTACGTGA GCAATTGGAT TGTCATCAGC CCTGCCTGTT 240
TTGCACCTGG GAAGTGCCCT GGTCTTACTT GGGTCCA~AT TGTTGGCTTT CACTTTTGAC 300
wo 95,03408 ~ ~ ~ 7 o ~ ~ PCT/US94/08423 ~
-112-
CCTAAGCATC TGAAGCC ATG GGC CAC ACA CGG AGG CAG GGA ACA TCA CCA TCC 353
Met Gly His Thr Arg Arg Gln Gly Thr Ser Pro Ser
-30 -25
S
AAG TGT CCA TAC CTG AAT TTC TTT CAG CTC TTG GTG CTG GCT GGT CTT 401
Lys Cys Pro Tyr Leu Asn Phe Phe Gln Leu Leu Val Leu Ala Gly Leu
-20 -15 -10
TCT CAC TTC TGT TCA GGT GTT ATC CAC GTG ACC AAG GAA GTG A~A GAA 449
Ser His Phe Cys Ser Gly Val Ile His Val Thr Lys Glu Val Lys Glu
-5 1 5 10
GTG GCA ACG CTG TCC TGT GGT CAC AAT GTT TCT GTT GAA GAG CTG GCA 497
Val Ala Thr Leu Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala
15 20 25
CAA ACT CGC ATC TAC TGG CAA AAG GAG AAG A~A ATG GTG CTG ACT ATG 545
Gln Thr Arg Ile Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met
30 35 40
ATG TCT GGG GAC ATG AAT ATA TGG CCC GAG TAC AAG AAC CGG ACC ATC 593
Met Ser Gly Asp Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile
45 50 55
TTT GAT ATC ACT AAT AAC CTC TCC ATT GTG ATC CTG GCT CTG CGC CCA 641
Phe Asp Ile Thr Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro
60 65 70
TCT GAC GAG GGC ACA TAC GAG TGT GTT GTT CTG AAG TAT GAA A~A GAC 689
Ser Asp Glu Gly Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp
75 80 85 90
GCT TTC AAG CGG GAA CAC CTG GCT GAA GTG ACG TTA TCA GTC A~A GCT 737
Ala Phe Lys Arg Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala
95 100 105
GAC TTC CCT ACA CCT AGT ATA TCT GAC TTT GAA ATT CCA ACT TCT AAT 785
Asp Phe Pro Thr Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn
110 115 120
ATT AGA AGG ATA ATT TGC TCA ACC TCT GGA GGT TTT CCA GAG CCT CAC 833
Ile Arg Arg Ile Ile Cys Ser Thr Ser Gly Gly Phe Pro Glu Pro His
125 130 135
~1 67~91
~WO 95/03408 PCT/US94/08423
-1 13-
CTC TCC TGG TTG GAA AAT GGA GAA GAA TTA AAT GCC ATC AAC ACA ACA 881
Leu Ser Trp Leu Glu Asn Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr
140 145 150
GTT TCC CAA GAT CCT GAA ACT GAG CTC TAT GCT GTT AGC AGC AAA CTG 929
Val Ser Gln Asp Pro Glu Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu
155 160 165 170
GAT TTC AAT ATG ACA ACC AAC CAC AGC TTC ATG TGT CTC ATC AAG TAT 977
Asp Phe Asn Met Thr Thr Asn His Ser Phe Met Cys Leu Ile Lys Tyr
175 180 185
GGA CAT TTA AGA GTG AAT CAG ACC TTC AAC TGG AAT ACA ACC AAG CAA 1025
Gly His Leu Arg Val Asn Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln
190 195 200
GAG CAT TTT CCT GAT AAC CTG CTC CCA TCC TGG GCC ATT ACC TTA ATC 1073
Glu His Phe Pro Asp Asn Leu Leu Pro Ser Trp Ala Ile Thr Leu Ile
205 210 215
TCA GTA AAT GGA ATT TTT GTG ATA TGC TGC CTG ACC TAC TGC TTT GCC 1121
Ser Val Asn Gly Ile Phe Val Ile Cy8 Cy8 Leu Thr Tyr Cy8 Phe Ala
220 225 230
30 CCA AGA TGC AGA GAG AGA AGG AGG AAT GAG AGA TTG AGA AGG GAA AGT 1169
Pro Ary Cy8 Arg Glu Arg Arg Arg Asn Glu Arg Leu Arg Arg Glu Ser
235 240 245 250
3 5 GTA CGC CCT GTA TAACAGTGTC CGCAGAAGCA AGGGGCTGAA AAGATCTGAA 1221
Val Arg Pro Val
GGTAGCCTCC GTCATCTCTT CTGGGATACA TGGATCGTGG GGATCATGAG GCATTCTTCC 1281
CTTAACAAAT TTAAGCTGTT TTACCCACTA CCTCACCTTC TTAAAAACCT CTTTCAGATT 1341
45 AAGCTGAACA GTTACAAGAT GGCTGGCATC CCTCTCCTTT CTCCCCATAT GCAATTTGCT 1401
TAATGTAACC l~llLlLllG CCATGTTTCC ATTCTGCCAT CTTGAATTGT ~ll~l~AGCC 1461
- AATTCATTAT CTATTAAACA CTAATTTGAG 1491
55 (3) INFORMATION FOR SEQ ID NO:29:
WO 95/03408 PCT/US94/08423 ~
~t~ 114-
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 288 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(A) DESCRIPTION: B cell activation antigen; natural ligand
for CD28 T cell surface antigen; tr~n~m~mhrane protein
(ix) FEATURE:
(A) NAME/KEY: signal æequence
(B) LOCATION: -34 to -1
(C) IDENTIFICATION METHOD: amino terminal sequencing of
soluble protein
(D) OTHER INFORMATION: hydrophobic
(ix) FEATURE:
(A) NAME/KEY: extracellular domain
(B) LOCATION: 1 to 208
(c) lV~NllrlCATION METHOD: similarity with known
sequence
(ix) FEATURE:
(A) NAME/KEY: tr~n~m~mhrane domain
(B) LOCATION: 209 to 235
(C) IDENTIFICATION METHOD: similarity with known
sequence
(ix) FEATURE:
(A) NAME/KEY: intracellular domain
(B) LOCATION: 236 to 254
(C) lv~Nll~lCATION METHOD: similarity with known
sequence
45 ( ix) FEATURE:
(A) NAME/KEY: N-linked glycosylation
(B) LOCATION: 19 to 21
(C) IDENTIFICATION METHOD: similarity with known
sequence
(ix) FEATURE:
(A) NAME/KEY: N-linked glycosylation
~O 95/03408 21 ~ 7 0 9 I PCT/US94/08423
- 1 1 5-
(B) LOCATION: 55 to 57
(C) IDENTIFICATION METHOD: similarity with known
æequence
(ix) FEATURE:
(A) NAME/ Æ Y: N-linked glycosylation
(B) LOCATION: 64 to 66
(C) lv~NLl~lCATION METHOD: similarity with known
sequence
(ix) FEATURE:
(A) NAME/ Æ Y: N-linked glycosylation
(B) LOCATION: 152 to 154
(C) IDENTIFICATION METHOD: similarity with known
sequence
(ix) FEATURE:
(A) NAME/ Æ Y: N-linked glycosylation
(B) LOCATION: 173 to 175
(C) l~Nll~lCATION METHOD: similarity with known
sequence
(ix) FEATURE:
(A) NAME/KEY: N-linked glycosylation
(B) LOCATION: 177 to 179
(C) lv~Nll~lCATION METHOD: similarity with known
sequence
(ix) FEATURE:
(A) NAME/ Æ Y: N-linked glycosylation
(B) LOCATION: 192 to 194
(C) IDENTIFICATION METHOD: similarity with known
sequence
(ix) FEATURE:
(A) NAME/ Æ Y: N-linked glycosylation
(B) LOCATION: 198 to 200
(C) IDENTIFICATION METHOD: similarity with known
sequence
WO 95/03408 ~ ~ ~ 7 ~ 91 PCT/US94/084~3
-1 16-
tix) FEATURE:
(A) NAME/KEY: Ig V-set domain
(B) LOCATION: 1 to 104
(c) IDENTIFICATION METHOD: similarity with known
sequence
(ix) FEATURE:
(A) NAME/KEY: Ig C-set domain
(B) LOCATION: 105 to 202
(C) IDENTIFICATION METHOD: similarity with known
sequence
(x) PUBLICATION INFORMATION:
(A) AUTHORS: FREEMAN, GORDON J.
FREEDMAN, ARNOLD S.
SEGIL, JEFFREY M.
LEE, GRACE
WHITMAN, JAMES F.
NADLER, LEE M.
(B) TITLE: B7, A New Member Of The Ig Superfamily With
Unique Expression On Activated And Neoplastic B Cells
(C) JOURNAL: The Journal of Immunology
(D) VOLUME: 143
(E) ISSUE: 8
(F) PAGES: 2714-2722
(G) DATE: 15-OCT-1989
(H) RELEVANT RESIDUES IN SEQUENCE ID NO:29, From -26 to 262
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
Met Gly His Thr Arg Arg Gln Gly Thr Ser Pro Ser Lys Cys Pro Tyr
-30 -25 -20
Leu Asn Phe Phe Gln Leu Leu Val Leu Ala Gly Leu Ser His Phe Cys
-15 -10 -5
Ser Gly Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu
-1 1 5 10
Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile
50 Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp
Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr
6~
~WO 95/03408 PCT/US94/08423
-1 17-
Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly
65 70 75
Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg
80 85 9o
Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr
100 105 110
Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile
115 120 125
Ile Cys Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu
130 135 140
Glu Asn Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp
145 150 155
Pro Glu Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met
160 165 170
Thr Thr Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg
175 180 185 190
Val Asn Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro
195 200 205
Asp Asn Leu Leu Pro Ser Trp Ala Ile Thr Leu Ile Ser Val Asn Gly
210 215 220
Ile Phe Val Ile Cys Cys Leu Thr Tyr Cys Phe Ala Pro Arg Cys Arg
225 230 235
35 Glu Arg Arg Arg Asn Glu Arg Leu Arg Arg Glu Ser Val Arg Pro Val
240 245 250
40 (4) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTBRISTICS:
(A) LENGTH: 1716 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULAR TYPE: cDNA to mRNA
- (iii) HYPOTHETICAL: no
(vi) ORIGINAL SOURCE:
(A) ORGANISM: ~a m
W 0 95/03408 PCTrUS94/08423
~ a ~ 18-
(D) DEVELOPMENTAL STAGE: germ line
(F) TISSUE TYPE: lymphoid
(G) CELL TYPE: B lymphocyte
(H) CELL LINE: 70Z and A20
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: cDNA in pCDM8 vector
(B) CLONE: B7 #'s 1 and 29
(ix) FEATURE:
(A) NAME/KEY: translated region
(B) LOCATION: 249 to 1166 bp
(C) IDENTIFICATION METHOD: similarity to other pattern
(ix) FEATURE:
(A) NAME/REY: Alternate ATG initiation codons
(B) LOCATION: 225 to 227 and 270 to 272
(C) IDENTIFICATION METHOD: similarity to other pattern
(Xi ) ~QU~'N~ DESCRIPTION: SEQ ID NO:30:
GAGTTTTATA CCTCAATAGA CTCTTACTAG ~ l TCAGGTTGTG AAACTCAACC 60
TTCAAAGACA ~l~l~lLCCA lll~l~lGGA CTAATAGGAT CATCTTTAGC ATCTGCCGGG 120
TGGATGCCAT CCAGGCTTCT llll~LACAT ~1~l~lll~l CGALllll~l~ GAGCCTAGGA 180
GGTGCCTAAG CTCCATTGGC TCTAGATTCC TGGCTTTCCC CATCATGTTC TCCAAAGCAT 240
5 CTGAAGCT ATG GCT TGC AAT TGT CAG TTG ATG CAG GAT ACA CCA CTC CTC 290
Met Ala Cys Asn Cys Gln Leu Met Gln Asp Thr Pro Leu Leu
-35 -30 -25
AAG TTT CCA TGT CCA AGG CTC AAT CTT CTC TTT GTG CTG CTG ATT CGT 338
Lys Phe Pro Cys Pro Arg Leu Ile Leu Leu Phe Val Leu Leu Ile Arg
-20 -15 -10
CTT TCA CAA GTG TCT TCA GAT GTT GAT GAA CAA CTG TCC AAG TCA GTG 386
Leu Ser Gln Val Ser Ser Asp Val Asp Glu Gln Leu Ser Lys Ser Val
-5 -1 1 5
AAA GAT AAG GTA TTG CTG CCT TGC CGT TAC AAC TCT CCT CAT GAA GAT 434
Lys Asp Lys Val Leu Leu Pro Cys Arg Tyr Asn Ser Pro His Glu Asp
GAG TCT GAA GAC CGA ATC TAC TGG CAA A~A CAT GAC A~A GTG GTG CTG 482
Glu Ser Glu Asp Arg Ile Tyr Trp Gln Lys His Asp Lys Val Val Leu
~WO 95/03408 21 6 7 0 91 PCT/US94/08423
-119-
TCT GTC ATT GCT GGG AAA CTA AAA GTG TGG CCC GAG TAT AAG AAC CGG 530
Ser Val Ile Ala Gly Lys Leu Lys Val Trp Pro Glu Tyr Lys Asn Arg
5 ACT TTA TAT GAC AAC ACT ACC TAC TCT CTT ATC ATC CTG GGC CTG GTC 578
Thr Leu Tyr Asp Asn Thr Thr Tyr Ser Leu Ile Ile Leu Gly Leu Val
CTT TCA GAC CGG GGC ACA TAC AGC TGT GTC GTT CAA AAG AAG GAA AGA 626
0 Leu Ser Asp Arg Gly Thr Tyr Ser Cy8 Val Val Gln Lys Lys Glu Arg
75 80 85
GGA ACG TAT GAA GTT AAA CAC TTG GCT TTA GTA AAG TTG TCC ATC AAA 674
Gly Thr Tyr Glu Val Lys His Leu Ala Leu Val Lys Leu Ser Ile Lys
90 95 100 105
GCT GAC TTC TCT ACC CCC AAC ATA ACT GAG TCT GGA AAC CCA TCT GCA 722
Ala Asp Phe Ser Thr Pro Asn Ile Thr Glu Ser Gly Asn Pro Ser Ala
ilO 115 120
GAC ACT AAA AGG ATT ACC TGC TTT GCT TCC GGG GGT TTC CCA AAG CCT 770
Asp Thr Lys Arg Ile Thr Cys Phe Ala Ser Gly Gly Phe Pro Lys Pro
125 130 135
25 CGC TTC TCT TGG TTG GAA AAT GGA AGA GAA TTA CCT GGC ATC AAT ACG 818
Arg Phe Ser Trp Leu Glu Asn Gly Arg Glu Leu Pro Gly Ile Asn Thr
140 145 150
ACA ATT TCC CAG GAT CCT GAA TCT GAA TTG TAC ACC ATT AGT AGC CAA 866
Thr Ile Ser Gln Asp Pro Glu Ser Glu Leu Tyr Thr Ile Ser Ser Gln
155 160 165
CTA GAT TTC AAT ACG ACT CGC AAC CAC ACC ATT AAG TGT CTC ATT AAA 914
Leu Asp Phe Asn Thr Thr Arg Asn His Thr Ile Lys Cys Leu Ile Lys
35 170 175 180 185
TAT GGA GAT GCT CAC GTG TCA GAG GAC TTC ACC TGG GAA AAA CCC CCA 962
Tyr Gly Asp Ala His Val Ser Glu Asp Phe Thr Trp Glu Lys Pro Pro
190 195 200
GAA GAC CCT CCT GAT AGC AAG AAC ACA CTT GTG CTC TTT GGG GCA GGA lOlo
Glu Asp Pro Pro Asp Ser Lys Asn Thr Leu Val Leu Phe Gly Ala Gly
205 210 215
45 TTC GGC GCA GTA ATA ACA GTC GTC GTC ATC GTT GTC ATC ATC AAA TGC 1058
Phe Gly Ala Val Ile Thr Val Val Val Ile Val Val Ile Ile Lys Cys
220 225 230
TTC TGT AAG CAC AGA AGC TGT TTC AGA AGA AAT GAG GCA AGC AGA GAA 1106
50 Phe Cys Lys His Arg Ser Cys Phe Arg Arg Asn Glu Ala Ser Arg Glu
235 240 245
ACA AAC AAC AGC CTT ACC TTC GGG CCT GAA GAA GCA TTA GCT GAA CAG 1154
Thr Asn Asn Ser Leu Thr Phe Gly Pro Glu Glu Ala Leu Ala Glu Gln
250 255 260 265
WO 9~/03408 PCT/US94/08423 ~
~1~7091
-120-
ACC GTC TTC CTT TAGTTCTTCT CTGTCCATGT GGGATACATG GTATTATGTG 1206
Thr Val Phe Leu
GCTCATGAGG TACAATCTTT CTTTCAGCAC CGTGCTAGCT GATCTTTCGG ACAACTTGAC 1266
ACAAGATAGA GTTAACTGGG AAGAGA~AGC CTTGAATGAG GATTTCTTTC CATCAGGAAG 1326
CTACGGGCAA GTTTGCTGGG CCTTTGATTG CTTGATGACT GAAGTGGAAA GGCTGAGCCC 1386
ACTGTGGGTG GTGCTAGCCC TGGGCAGGGG CAGGTGACCC TGGGTGGTAT AAGAAAAAGA 1446
GCTGTCACTA A~AGGAGAGG TGCCTAGTCT TACTGCAACT TGATATGTCA TGTTTGGTTG 1506
15 GTGTCTGTGG GAGGCCTGCC ~Ll~ AA GAGAAGTGGT GGGAGAGTGG ATGGGGTGGG 1566
GGCAGAGGAA AAGTGGGGGA GAGGGCCTGG GAGGAGAGGA GGGAGGGGGA CGGGGTGGGG 1626
GTGGGGA~AA CTATGGTTGG GATGTA~AAA CGGATAATAA TATAAATATT A~ATAAAAAG 1686
AGAGTATTGA GC~AAAAA AAA~L~aAA 1716
(5) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 306 amino acids
(B) TYPE: amino acid
(c) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(A) DESCRIPTION: B lymphocyte activation antigen; Ig
superfamily member; T cell costimulatory signal
via activation of CD28 pathways, binds to CD28
T cells, tr~n~m~mhrane protein
(ix) FEATURE:
(A) NAME/KEY: signal sequence
(B) LOCATION: -37 to -1
(C) IDENTIFICATION METHOD: similarity with known
sequence
(D) OTHER INFORMATION: hydrophobic
(ix) FEATURE:
(A) NAME/KEY: extracellular domain
(B) LOCATION: 1 to 210
(C) l~NLl~lCATION METHOD: similarity with known
sequence
~0 95/03408 ~ 1 6 ~ ~ ~1 PCT/US94/08423
-121-
(ix) FEATURE:
(A) NAME/KEY: transmembrane domain
(B) LOCATION: 211 to 235
(C) l~Nll~lCATION METHOD: similarity with known
sequence
(ix) FEATURE:
(A) NAME/KEY: intracellular (cytoplasmic) domain
(B) LOCATION: 236 to 269
(C) IDENTIFICATION METHOD: similarity with known
sequence
(ix) FEATURE:
(A) NAME/KEY: Ig V-set domain
(B) LOCATION: 1 to 105
(c) IDENTIFICATION METHOD: similarity with known
sequence
(ix) FEATURE:
(A) NAME/KEY: Ig C-set domain
(B) LOCATION: 106 to 199
(C) l~NLl~lCATION METHOD: similarity with known
sequence
(x) P B LICATION INFORMATION:
(A) AUTHORS: FREEMAN, GORDON J.
3 5 GRAY, GARY S.
GIMMI, CLAUDE D.
LOMBARD, DAVID B.
ZHOU, LIANG-JI
WHITE, MICHAEL
FINGEROTH, JOYCE D.
~RTRR~N, JOHN G.
NADLER, LEE M.
(B) TITLE: Structure, Expression, and T Cell Costimulatory
Activity Of The Murine Homologue O~ The Human B
Lymphocyte Activation Antigen B7
(C) JOURNAL: Journal of Experimental Medicine
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
- (G) DATE: IN PRESS
(H) RELEVANT RESIDUES IN SEQUENCE ID NO:31: From -37 to 269
(xi) ~Qu~ DESCRIPTION: SEQ ID NO:31:
WO 95/03408 PCT/US94/08423
-122-
Met Ala Cys Asn Cys Gln Leu Met Gln Asp Thr Pro Leu Leu Lys Phe
-35 -30 -25
Pro Cys Pro Arg Leu Ile Leu Leu Phe Val Leu Leu Ile Arg Leu Ser
-20 -15 -10
Gln Val Ser Ser Asp Val Asp Glu Gln Leu Ser Lys Ser Val Lys Asp
0 -5 -1 1 5 10
Ly~ Val Leu Leu Pro Cys Arg Tyr Asn Ser Pro His Glu Asp Glu Ser
15 20 25
15 Glu Asp Arg Ile Tyr Trp Gln Lys His Asp Lys Val Val Leu Ser Val
30 35 40
Ile Ala Gly Lys Leu Lys Val Trp Pro Glu Tyr Lys Asn Arg Thr Leu
45 50 55
Tyr Asp Asn Thr Thr Tyr Ser Leu Ile Ile Leu Gly Leu Val Leu Ser
60 65 70 75
Asp Arg Gly Thr Tyr Ser Cys Val Val Gln Lys Lys Glu Arg Gly Thr
80 85 90
Tyr Gly Val Lys His Leu Ala Leu Val Lys Leu Ser Ile Lys Ala Asp
95 100 105
30 Phe Ser Thr Pro Asn Ile Thr Glu Ser Gly Asn Pro Ser Ala Asp Thr
110 115 120
Lys Arg Ile Thr Cys Phe Ala Ser Gly Gly Phe Pro Lys Pro Arg Phe
125 130 135
Ser Trp Leu Glu Asn Gly Arg Glu Leu Pro Gly Ile Asn Thr Thr Ile
140 145 lS0 155
Ser Gln Asp Pro Glu Ser Glu Leu Tyr Thr Ile Ser Ser Gln Leu Asp
160 165 170
Phe Asn Thr Thr Arg Asn His Thr Ile Lys Cys Leu Ile Lys Tyr Gly
175 180 185
5 Asp Ala His Val Ser Glu Asp Phe Thr Trp Glu Lys Pro Pro Glu Asp
190 195 200
Pro Pro Asp Ser Lys Asn Thr Leu Val Leu Phe Gly Ala Gly Phe Gly
205 210 215
Ala Val Ile Thr Val Val Val Ile Val Val Ile Ile Lys Cys Phe Cys
220 225 230 235
Lys His Arg Ser Cys Phe Arg Arg Asn Glu Ala Ser Arg Glu Thr Asn
240 245 250
~VO 95/03408 2 t 6 ~ Q 91 PCT/US94/08423
-123-
Asn Ser Leu Thr Phe Gly Pro Glu Glu Ala Leu Ala Glu Gln Thr Val
255 260 265
Phe Leu
