U.S. patent application number 10/854000 was filed with the patent office on 2005-11-10 for blockade of t lymphocyte down-regulation associated with ctla-4 signaling.
Invention is credited to Allison, James Patrick, Krummel, Matthew E., Leach, Dana R..
Application Number | 20050249700 10/854000 |
Document ID | / |
Family ID | 35239647 |
Filed Date | 2005-11-10 |
United States Patent
Application |
20050249700 |
Kind Code |
A1 |
Allison, James Patrick ; et
al. |
November 10, 2005 |
Blockade of T lymphocyte down-regulation associated with CTLA-4
signaling
Abstract
T cell activation in response to antigen is increased by the
administration of binding agents that block CTLA-4 signaling. When
CTLA-4 signaling is thus blocked, the T cell response to antigen is
released from inhibition. Such an enhanced response is useful for
the treatment of tumors, chronic viral infections, and as an
adjuvant during immunization.
Inventors: |
Allison, James Patrick;
(Berkeley, CA) ; Leach, Dana R.; (Albany, CA)
; Krummel, Matthew E.; (Berkeley, CA) |
Correspondence
Address: |
DORSEY & WHITNEY LLP
555 CALIFORNIA STREET, SUITE 1000
SUITE 1000
SAN FRANCISCO
CA
94104
US
|
Family ID: |
35239647 |
Appl. No.: |
10/854000 |
Filed: |
May 25, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10854000 |
May 25, 2004 |
|
|
|
09454851 |
Dec 7, 1999 |
|
|
|
09454851 |
Dec 7, 1999 |
|
|
|
08760288 |
Dec 4, 1996 |
|
|
|
6051227 |
|
|
|
|
08760288 |
Dec 4, 1996 |
|
|
|
08646605 |
May 8, 1996 |
|
|
|
5811097 |
|
|
|
|
08646605 |
May 8, 1996 |
|
|
|
08566853 |
Dec 4, 1995 |
|
|
|
5855887 |
|
|
|
|
08566853 |
Dec 4, 1995 |
|
|
|
08506666 |
Jul 25, 1995 |
|
|
|
Current U.S.
Class: |
424/85.1 ;
424/144.1; 424/85.2 |
Current CPC
Class: |
A61K 38/20 20130101;
A61K 39/39 20130101; A61K 39/3955 20130101; A61P 43/00 20180101;
C07K 16/2818 20130101; A61K 38/20 20130101; A61K 39/3955 20130101;
C07K 2319/00 20130101; A61P 13/00 20180101; A61P 13/12 20180101;
A61P 37/04 20180101; A61K 38/193 20130101; A61K 35/13 20130101;
C07K 2317/76 20130101; A61K 35/13 20130101; C07K 2317/55 20130101;
C07K 14/31 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2039/55522
20130101; A61P 31/00 20180101; A61P 31/12 20180101; C07K 14/70521
20130101; A61P 35/00 20180101; A61K 38/00 20130101; C07K 2317/74
20130101; A61K 38/193 20130101; A61K 2039/505 20130101; A61P 37/00
20180101; C07K 2317/73 20130101 |
Class at
Publication: |
424/085.1 ;
424/085.2; 424/144.1 |
International
Class: |
A61K 038/19; A61K
038/20; A61K 039/395 |
Goverment Interests
[0002] This invention was made with government support under
Contract Nos. CA 40041 and CA 09179 awarded by the National
Institutes of Health. The Government has certain rights in this
invention.
Claims
1. A CTLA-4 blocking agent characterized as specifically binding to
the extracellular domain of CTLA-4 and inhibitory of CTLA-4
signaling; wherein said blocking agent is other than an antibody to
the extracellular domain of CTLA-4 or an Fab or Fab' fragment
thereof and wherein said blocking agent is effective to increase
the response of mammalian T cells to antigenic stimulus or to
decrease the growth of tumor cells in a mammalian host.
2. (canceled)
3. (canceled)
4. (canceled)
5. (canceled)
6. (canceled)
7. (canceled)
8. (canceled)
9. (canceled)
10. (canceled)
11. (canceled)
12. (canceled)
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 09/454,851, filed Dec. 7, 1999, which is a continuation-in-part
of U.S. application Ser. No. 08/760,288, filed Dec. 4, 1996, U.S.
Pat. No. 6,051,227, which is a continuation-in-part of U.S.
application Ser. No. 08/646,605, filed May 8, 1996, U.S. Pat. No.
5,811,097, which is a continuation-in-part of U.S. patent
application Ser. No. 08/566,853, filed Dec. 4, 1995, U.S. Pat. No.
5,855,887, which is a continuation-in-part of U.S. application Ser.
No. 08/506,666, filed Jul. 25, 1995, abandoned.
BACKGROUND
[0003] Putting immunotherapy into practice is a highly desired goal
in the treatment of human disease. It promises a specificity of
action that is rarely found with the use of conventional drugs. The
basis for immunotherapy is the manipulation of the immune response,
particularly the responses of T cells. T cells possess complex and
subtle systems for controlling their interactions, utilizing
numerous receptors and soluble factors for the process. The effect
that any particular signal will have on the immune response may
vary, depending on the factors, receptors and counter-receptors
that are involved.
[0004] The pathways for down-regulating responses are as important
as those required for activation. Thymic education leading to
T-cell tolerance is one mechanism for preventing an immune response
to a particular antigen. Other mechanisms, such as secretion of
suppressive cytokines, are also known.
[0005] Activation of T cells requires not only stimulation through
the antigen receptor (TCR) but additional signaling through
co-stimulatory surface molecules such as CD28. The ligands for CD28
are the B7-1 (CD80) and B72 (CD86) proteins, which are expressed on
antigen-presenting cells such as dendritic cells, activated B-cells
or monocytes. The interaction between B7 and CD28 is one of several
co-stimulatory signaling pathways that appear to be sufficient to
trigger the maturation and proliferation of antigen specific
T-cells.
[0006] Lack of co-stimulation, and the concomitant inadequacy of
IL-2 production, prevent subsequent proliferation of the T cell and
induce a state of non-reactivity termed "anergy". This is
associated with a block in IL-2 gene transcription and a lack of
responsiveness of the affected T cells to IL-4. Anergy may be
overcome with prolonged IL-2 stimulation. A variety of viruses and
tumors may block T cell activation and proliferation through direct
or indirect means, thereby inducing a state of insufficient or
non-reactivity of the host's immune system to infected or
transformed cells. Among a number of functional T-cell
disturbances, anergy may be at least partially responsible for the
failure of the host to clear the pathogenic cells.
[0007] It would be advantageous if, in the treatment of infections
and tumors, one could activate a strong cellular immune response
through the manipulation of receptors involved in
co-stimulation.
[0008] The use of B7 protein in mediating anti-tumor immunity is
described in Chen et al. (1992) Cell 71:1093-1102 and Townsend and
Allison (1993) Science 259:368. Schwartz (1992) Cell 71:1065
reviews the role of CD28, CTLA-4 and B7 in IL-2 production and
immunotherapy. Harding et al. (1994) Nature 356:607-609
demonstrates that CD28 mediated signaling co-stimulates murine T
cells and prevents the induction of anergy in T cell clones.
[0009] CTLA-4 is a T cell surface molecule that was originally
identified by differential screening of a murine cytolytic T cell
cDNA library, Brunet et al. (1987) Nature 328:267-270. The role of
CTLA-4 as a second receptor for B7 is discussed in Linsley et al.
(1991) J. Exp. Med. 174:561-569. Freeman et al. (1993) Science
262:907-909 discusses CTLA-4 in B7 deficient mice. Ligands for
CTLA-4 are described in Lenschow et al. (1993) P.N.A.S.
90:11054-11058.
[0010] Linsley et al. (1992) Science 257:792-795 describes
immunosuppression in vivo by a soluble form of CTLA-4. Lenschow et
al. (1992) Science 257:789-792 discusses long term survival of
pancreatic islet grafts induced by CTLA-4Ig. It is suggested in
Walunas et al. (1994) Immunity 1:405-413, that CTLA-4 can function
as a negative regulator of T cell activation.
SUMMARY OF THE INVENTION
[0011] Methods and compositions are provided for increasing the
activation of T cells through a blockade of CTLA-4 signaling.
Binding molecules that specifically interact with the CTLA-4
antigen, but do not activate signaling (blocking agents), are
combined with T cells, in vitro or in vivo. The blocking agents can
also be combined with immune response stimulating agents such as
cytokines and antigens. When CTLA-4 signaling is thus blocked, the
T cell response to antigen is released from inhibition. Such an
enhanced response is useful for the treatment of tumors, chronic
viral infections, and as an adjuvant during immunization. In one
aspect of the invention, the blocking agent is other than an
antibody to the extracellular domain of CTLA-4 or fragment
thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1A is a graph illustrating the in vivo growth of the
tumor cell line V51Blim10 in the presence of absence of antibodies
directed against CTLA-4 or CD28. FIG. 1B is a graph illustrating
the average tumor size in mice injected with 2.times.10.sup.6
V51Blim10 cells and antibodies. FIG. 1C is a graph illustrating
individual tumor growth size in mice injected with V51Blim10
cells.
[0013] FIG. 2 is a graph showing the in vivo growth of B7-51BLim10
tumors in the presence of absence of antibodies directed against
CTLA-4 or CD28.
[0014] FIG. 3 shows the rejection of wild-type colon carcinoma
cells by mice previously treated with V51BLim10 cells and
anti-CTLA-4 antibody.
[0015] FIG. 4 shows the growth of established tumors after
treatment with anti-CTLA-4 antibody.
[0016] FIG. 5 shows the growth of the murine fibrosarcoma SA1N in
the absence of presence of anti-CTLA-4 antibodies.
[0017] FIGS. 6A to 6E illustrate the adjuvant effect of anti-CTLA-4
antibodies in the response of T cells to peptide antigens.
[0018] FIGS. 7A to 7F illustrate the effect of CTLA-4 blockade on
class switching.
[0019] FIGS. 8A to 8D present a kinetic analysis of CTLA-4/B7
blockade on the proliferation of purified CD4+ T cells. In FIG. 8B,
detection of IL-2 is shown. The kinetics of thymidine incorporation
are shown in FIG. 1C. Shown in FIG. 8D, a pronounced inhibition of
IL-2 production was observed when CTLA-4 was also engaged.
[0020] FIGS. 9A to 9E show propidium iodide staining of
permeabilized cells to measure DNA content at various stages
CTLA-4/B7 blockade in culture.
[0021] FIG. 10 shows the effect of delaying the CTLA-4 blockade on
a fibrosarcoma.
[0022] FIG. 11 shows the effect of treating a mammary carcinoma
with anti-CTLA-4 alone, GM-CSF transduced cells alone or a
combination thereof.
[0023] FIGS. 12A to 12B demonstrate the effect of delayed CTLA-4
blockade on a renal carcinoma.
[0024] FIG. 13 shows the effect of CTLA-4 blockade treatment alone
or in combination with immunization with irradiated B16 tumor cells
on B16 tumors.
[0025] FIG. 14 shows the effect of combining the CTLA-4 blockade
with irradiated B16 cells and/or cytokine treatment.
DATABASE REFERENCES FOR NUCLEOTIDE AND AMINO ACID SEQUENCES
[0026] The complete cDNA sequence of human CTLA-4 has the Genbank
accession number L15006. The region of amino acids 1-37 is the
leader peptide; 38-161 is the extracellular V-like domain; 162-187
is the transmembrane domain; and 188-223 is the cytoplasmic domain.
Variants of the nucleotide sequence have been reported, including a
G to A transition at position 49, a C to T transition at position
272, and an A to G transition at position 439. The complete DNA
sequence of mouse CTLA-4 has the EMBL accession number X05719
(Brunet et al. (1987) Nature 328:267-270). The region of amino
acids 1-35 is the leader peptide.
[0027] The complete DNA sequence of human B7-1 (CD80) has the
Genbank accession number X60958; the accession number for the mouse
sequence is X60958; the accession number for the rat sequence is
U05593. The complete cDNA sequence of human B7-2 (CD86) has the
Genbank accession number L25259; the accession number for the mouse
sequence is L25606.
[0028] The genes encoding CD28 have been extensively characterized.
The chicken mRNA sequence has the Genbank accession number X67915.
The rat mRNA sequence has the Genbank accession number X55288. The
human mRNA sequence has the Genbank accession number J02988. The
mouse mRNA sequence has the Genbank accession number M34536.
DETAILED DESCRIPTION OF THE INVENTION
[0029] Methods and compositions are provided for up-regulating the
response of T cells to antigenic stimulation. Binding molecules
that specifically interact with cell surface CTLA-4, but do not
activate CTLA-4 signaling (blocking agents), are combined with T
cells. The T cell response to antigen is increased in the presence
of the blocking agents. Such treatment is useful for increasing the
specific immune response against tumors, chronic pathogenic
infections, and as an adjuvant during immunization.
[0030] It is not necessary for the practice of the invention that
the mechanism of action be understood. The data indicate that the
subject therapy releases T cells from inhibitory signals mediated
through CTLA-4. CTLA-4 mediated signals apparently inhibit cell
cycle progression and IL-2 expression. The T cell response to
antigen and co-stimulatory CD28 signaling is thereby upregulated in
the presence of CTLA-4 blocking agents. The subject methods do not
promote a generalized proliferation of unstimulated T cells.
[0031] The subject methods are useful where there is an inadequate
T cell mediated response to an antigenic stimulus for an intended
purpose. In vivo T cell mediated responses include the generation
of cytolytic T cells, and the majority of antibody responses,
particularly those involving class switching of immunoglobulin
isotypes. The antigenic stimulus may be the presence of viral
antigens on infected cells; tumor cells that express proteins or
combinations of proteins in an unnatural context; parasitic or
bacterial infection; or an immunization, e.g. vaccination,
preparing monoclonal antibodies, etc. In vitro, the subject methods
are used to increase the response of cultured T cells to antigen.
Such activated T cells find use in adoptive immunotherapy, to study
the mechanisms of activation, in drug screening, etc.
[0032] CTLA-4 blocking agents are molecules that specifically bind
to the extracellular domain of CTLA-4 protein, and block the
binding of CTLA-4 to its counter-receptors, e.g. CD80, CD86, etc.
Usually the binding affinity of the blocking agent will be at least
about 100 .mu.M. The blocking agent will be substantially
unreactive with related molecules to CTLA-4, such as CD28 and other
members of the immunoglobulin superfamily. Molecules such as CD80
and CD86 are therefore excluded as blocking agents. Further,
blocking agents do not activate CTLA-4 signaling. Conveniently,
this is achieved by the use of monovalent or bivalent binding
molecules. It will be understood by one of skill in the art that
the following discussions of cross-reactivity and competition
between different molecules is intended to refer to molecules
having the same species of origin, e.g. human CTLA-4 binds human
CD80 and 86, etc.
[0033] Candidate blocking agents are screened for their ability to
meet this criteria. Assays to determine affinity and specificity of
binding are known in the art, including competitive and
non-competitive assays. Assays of interest include ELISA, RIA, flow
cytometry, etc. Binding assays may use purified or semi-purified
CTLA-4 protein, or alternatively may use T cells that express
CTLA-4, e.g. cells transfected with an expression construct for
CTLA-4; T cells that have been stimulated through cross-linking of
CD3 and CD28; the addition of irradiated allogeneic cells, etc. As
an example of a binding assay, purified CTLA-4 protein is bound to
an insoluble support, e.g. microtiter plate, magnetic beads, etc.
The candidate blocking agent and soluble, labeled CD80 or CD86 are
added to the cells, and the unbound components are then washed off.
The ability of the blocking agent to compete with CD80 and CD86 for
CTLA-4 binding is determined by quantitation of bound, labeled CD80
or CD86. Confirmation that the blocking agent does not cross-react
with CD28 may be performed with a similar assay, substituting CD28
for CTLA-4. Suitable molecules will have at least about 10.sup.3
less binding to CD28 than to CTLA-4, more usually at least about
10.sup.4 less binding.
[0034] Generally, a soluble monovalent or bivalent binding molecule
will not activate CTLA-4 signaling. A functional assay that detects
T cell activation may be used for confirmation. For example, a
population of T cells may be stimulated with irradiated allogeneic
cells expressing CD80 or CD86, in the presence or absence of the
candidate blocking agent. An agent that blocks CTLA-4 signaling
will cause an increase in the T cell activation, as measured by
proliferation and cell cycle progression, release of IL-2,
upregulation of CD25 and CD69, etc. It will be understood by one of
skill in the art that expression on the surface of a cell,
packaging in a liposome, adherence to a particle or well, etc. will
increase the effective valency of a molecule.
[0035] Blocking agents are peptides, small organic molecules,
peptidomimetics, soluble T cell receptors, antibodies, or the like.
Antibodies are a preferred blocking agent. Antibodies may be
polyclonal or monoclonal; intact or truncated, e.g. F(ab').sub.2,
Fab, Fv; xenogeneic, allogeneic, syngeneic, or modified forms
thereof, e.g. humanized, chimeric, etc.
[0036] In many cases, the blocking agent will be an oligopeptide,
e.g. antibody or fragment thereof, etc., but other molecules that
provide relatively high specificity and affinity may also be
employed. Combinatorial libraries provide compounds other than
oligopeptides that have the necessary binding characteristics.
Generally, the affinity will be at least about 10.sup.-6, more
usually about 10.sup.-8 M, i.e. binding affinities normally
observed with specific monoclonal antibodies.
[0037] A number of screening assays are available for blocking
agents. The components of such assays will typically include CTLA-4
protein; and optionally a CTLA-4 activating agent, e.g. CD80, CD86,
etc. The assay mixture will also comprise a candidate
pharmacological agent. Generally a plurality of assay mixtures are
run in parallel with different agent concentrations to obtain a
differential response to the various concentrations. Typically, one
of these concentrations serves as a negative control, i.e. at zero
concentration or below the level of detection.
[0038] Conveniently, in these assays one or more of the molecules
will be joined to a label, where the label can directly or
indirectly provide a detectable signal. Various labels include
radioisotopes, fluorescers, chemiluminescers, enzymes, specific
binding molecules, particles, e.g. magnetic particles, and the
like. Specific binding molecules include pairs, such as biotin and
streptavidin, digoxin and antidigoxin etc. For the specific binding
members, the complementary member would normally be labeled with a
molecule which provides for detection, in accordance with known
procedures.
[0039] One screening assay of interest is directed to agents that
interfere with the activation of CTLA-4 by its counter-receptors.
Quantitation of activation may achieved by a number of methods
known in the art. For example, the inhibition of T cell activation
may be determined by quantitating cell proliferation, release of
cytokines, etc.
[0040] Other assays of interest are directed to agents that block
the binding of CTLA-4 to its counter-receptors. The assay mixture
will comprise at least a portion of the natural counter-receptor,
or an oligopeptide that shares sufficient sequence similarity to
provide specific binding, and the candidate pharmacological agent.
The oligopeptide may be of any length amenable to the assay
conditions and requirements, usually at least about 8 aa in length,
and up to the full-length protein or fusion thereof. The CTLA-4 may
be bound to an insoluble substrate. The substrate may be made in a
wide variety of materials and shapes e.g. microtiter plate,
microbead, dipstick, resin particle, etc. The substrate is chosen
to minimize background and maximize signal to noise ratio. Binding
may be quantitated by a variety of methods known in the art. After
an incubation period sufficient to allow the binding to reach
equilibrium, the insoluble support is washed, and the remaining
label quantitated. Agents that interfere with binding will decrease
the detected label.
[0041] Candidate agents encompass numerous chemical classes, though
typically they are organic molecules, preferably small organic
compounds having a molecular weight of more than 50 and less than
about 2,500 daltons. Candidate agents comprise functional groups
necessary for structural interaction with proteins, particularly
hydrogen bonding, and typically include at least an amine,
carbonyl, hydroxyl, sulfhydryl or carboxyl group, preferably at
least two of the functional chemical groups. The candidate agents
often comprise cyclical carbon or heterocyclic structures and/or
aromatic or polyaromatic structures substituted with one or more of
the above functional groups. Candidate agents are also found among
biomolecules including peptides, saccharides, fatty acids,
steroids, purines, pyrimidines, derivatives, structural analogs or
combinations thereof.
[0042] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides. Alternatively, libraries
of natural compounds in the form of bacterial, fungal, plant and
animal extracts are available or readily produced. Additionally,
natural or synthetically produced libraries and compounds are
readily modified through conventional chemical, physical and
biochemical means. Known pharmacological agents may be subjected to
directed or random chemical modifications, such as acylation,
alkylation, esterification, amidification to produce structural
analogs.
[0043] A variety of other reagents may be included in the screening
assay. These include reagents like salts, neutral proteins, e.g.
albumin, detergents, etc which may be used to facilitate optimal
protein-DNA binding and/or reduce non-specific or background
interactions. Also reagents that otherwise improve the efficiency
of the assay, such as protease inhibitors, nuclease inhibitors,
anti-microbial agents, etc. may be used.
[0044] Suitable antibodies for use as blocking agents are obtained
by immunizing a host animal with peptides comprising all or a
portion of CTLA-4 protein. Suitable host animals include mouse, rat
sheep, goat, hamster, rabbit, etc. The origin of the protein
immunogen may be mouse, human, rat, monkey etc. The host animal
will generally be a different species than the immunogen, e.g.
mouse CTLA-4 used to immunize hamsters, human CTLA-4 to immunize
mice, etc. The human and mouse CTLA-4 contain highly conserved
stretches in the extracellular domain (Harper et al. (1991) J.
Immunol. 147:1037-1044). Peptides derived from such highly
conserved regions may be used as immunogens to generate
cross-specific antibodies.
[0045] The immunogen may comprise the complete protein, or
fragments and derivatives thereof. Preferred immunogens comprise
all or a part of the extracellular domain of human CTLA-4 (amino
acid residues 38-161), where these residues contain the
post-translation modifications, such as glycosylation, found on the
native CTLA-4. Immunogens comprising the extracellular domain are
produced in a variety of ways known in the art, e.g. expression of
cloned genes using conventional recombinant methods, isolation from
T cells, sorted cell populations expressing high levels of CTLA-4,
etc.
[0046] Where expression of a recombinant or modified protein is
desired, a vector encoding the desired portion of CTLA-4 will be
used. Generally, an expression vector will be designed so that the
extracellular domain of the CTLA-4 molecule is on the surface of a
transfected cell, or alternatively, the extracellular domain is
secreted from the cell. When the extracellular domain is to be
secreted, the coding sequence for the extracellular domain will be
fused, in frame, with sequences that permit secretion, including a
signal peptide. Signal peptides may be exogenous or native. A
fusion protein of interest for immunization joins the CTLA-4
extracellular domain to the constant region of an immunoglobulin.
For example, a fusion protein comprising the extracellular domain
of mouse CTLA-4 joined to the hinge region of human Cg1
(hinge-CH2-CH3) domain may be used to immunize hamsters.
[0047] When the CTLA-4 is to be expressed on the surface of the
cell, the coding sequence for the extracellular domain will be
fused, in frame, with sequences encoding a peptide that anchors the
extracellular domain into the membrane and a signal sequence. Such
anchor sequences include the native CTLA-4 transmembrane domain, or
transmembrane domains from other cell surface proteins, e.g. CD4,
CD8, sIg, etc. Mouse cells transfected with the human CTLA-4 gene
may be used to immunize mice and generate antibodies specific for
the human CTLA-4 protein.
[0048] Monoclonal antibodies are produced by conventional
techniques. Generally, the spleen and/or lymph nodes of an
immunized host animal provide a source of plasma cells. The plasma
cells are immortalized by fusion with myeloma cells to produce
hybridoma cells. Culture supernatant from individual hybridomas is
screened using standard techniques to identify those producing
antibodies with the desired specificity. Suitable animals for
production of monoclonal antibodies to the human protein include
mouse, rat, hamster, etc. To raise antibodies against the mouse
protein, the animal will generally be a hamster, guinea pig,
rabbit, etc. The antibody may be purified from the hybridoma cell
supernatants or ascites fluid by conventional techniques, e.g.
affinity chromatography using CTLA-4 bound to an insoluble support,
protein A sepharose, etc.
[0049] The antibody may be produced as a single chain, instead of
the normal multimeric structure. Single chain antibodies are
described in Jost et al. (1994) J.B.C. 269:26267-73, and others.
DNA sequences encoding the variable region of the heavy chain and
the variable region of the light chain are ligated to a spacer
encoding at least about 4 amino acids of small neutral amino acids,
including glycine and/or serine. The protein encoded by this fusion
allows assembly of a functional variable region that retains the
specificity and affinity of the original antibody.
[0050] For in vivo use, particularly for injection into humans, it
is desirable to decrease the antigenicity of the blocking agent. An
immune response of a recipient against the blocking agent will
potentially decrease the period of time that the therapy is
effective. Methods of humanizing antibodies are known in the art.
The humanized antibody may be the product of an animal having
transgenic human immunoglobulin constant region genes (see for
example International Patent Applications WO 90/10077 and WO
90/04036). Alternatively, the antibody of interest may be
engineered by recombinant DNA techniques to substitute the CH1,
CH2, CH3, hinge domains, and/or the framework domain with the
corresponding human sequence (see WO 92/02190).
[0051] The use of Ig cDNA for construction of chimeric
immunoglobulin genes is known in the art (Liu et al. (1987)
P.N.A.S. 84:3439 and (1987) J. Immunol. 139:3521). mRNA is isolated
from a hybridoma or other cell producing the antibody and used to
produce cDNA. The cDNA of interest may be amplified by the
polymerase chain reaction using specific primers (U.S. Pat. Nos.
4,683,195 and 4,683,202). Alternatively, a library is made and
screened to isolate the sequence of interest. The DNA sequence
encoding the variable region of the antibody is then fused to human
constant region sequences. The sequences of human constant regions
genes may be found in Kabat et al. (1991) Sequences of Proteins of
Immunological Interest, N.I.H. publication no. 91-3242. Human C
region genes are readily available from known clones. The choice of
isotype will be guided by the desired effector functions, such as
complement fixation, or activity in antibody-dependent cellular
cytotoxicity. Preferred isotypes are IgG1, IgG3 and IgG4. Either of
the human light chain constant regions, kappa or lambda, may be
used. The chimeric, humanized antibody is then expressed by
conventional methods.
[0052] Antibody fragments, such as Fv, F(ab').sub.2 and Fab may be
prepared by cleavage of the intact protein, e.g. by protease or
chemical cleavage. Alternatively, a truncated gene is designed. For
example, a chimeric gene encoding a portion of the F(ab').sub.2
fragment would include DNA sequences encoding the CH1 domain and
hinge region of the H chain, followed by a translational stop codon
to yield the truncated molecule.
[0053] Consensus sequences of H and L J regions may be used to
design oligonucleotides for use as primers to introduce useful
restriction sites into the J region for subsequent linkage of V
region segments to human C region segments. C region cDNA can be
modified by site directed mutagenesis to place a restriction site
at the analogous position in the human sequence.
[0054] Expression vectors include plasmids, retroviruses, YACs, EBV
derived episomes, and the like. A convenient vector is one that
encodes a functionally complete human CH or CL immunoglobulin
sequence, with appropriate restriction sites engineered so that any
VH or VL sequence can be easily inserted and expressed. In such
vectors, splicing usually occurs between the splice donor site in
the inserted J region and the splice acceptor site preceding the
human C region, and also at the splice regions that occur within
the human CH exons. Polyadenylation and transcription termination
occur at native chromosomal sites downstream of the coding regions.
The resulting chimeric antibody may be joined to any strong
promoter, including retroviral LTRs, e.g. SV-40 early promoter,
(Okayama et al. (1983) Mol. Cell. Bio. 3:280), Rous sarcoma virus
LTR (Gorman et al. (1982) P.N.A.S. 79:6777), and moloney murine
leukemia virus LTR (Grosschedl et al. (1985) Cell 41:885); native
Ig promoters, etc.
[0055] The CTLA-4 blocking agent can be used alone or in
combination with an immune response stimulating agent. As used
herein, an "immune response stimulating agent" refers to any agent
which directly or indirectly stimulates an immune response in
combination with a CTLA-4 blocking agent. For example, immune
response stimulating agents include cytokines as well as various
antigens including tumor antigens and antigens derived from
pathogens. In addition, immune response stimulating agents include
cytokine transduced tumor cells, e.g. tumor cells transduced with
GMCSF, as well as tumor cells which have been irradiated and/or
treated with a chemotherapeutic agent ex vivo or in vivo. In some
instances cellular debris from dead or dying tumor cells provides
immune response stimulation which can be combined in vivo or ex
vivo with a CTLA-4 blocking agent. The use of chemotherapeutic
agents is an example of production of an immune response
stimulating agent by indirect means. Use of a source to irradiate
tumor cells ex vivo or in vivo also constitutes a method which
indirectly produces immune response stimulating agents. Examples 9
through 12 demonstrate that immune response stimulating agents can
have a significant effect on tumor treatment when used in
combination with a CTLA-4 blocking agent.
[0056] The basis for use of chemotherapeutic agents with CTLA-4
blocking agents is as follows. As indicated in the examples, the
CTLA-4 blockade works better with established tumors and increases
immunogenicity of irradiated tumor cells. This suggests that the
CTLA-4 blockade can be combined with more conventional methods of
cancer treatment to produce a synergetic effect. For example, the
CTLA-4 blockade may be initiated shortly after treatment with
chemotherapeutic agent. The dose of the chemotherapeutic agent is
adjusted to a level that kills a reasonable amount of the tumor
mass and generates debris which act as an agent to stimulate an
immune response by T cells as a result of CTLA-4 blockade. This
allows the chemotherapeutic agent to be given at levels much below
those now used to obtain maximal killing of the tumor cells, since
the immune response facilitated by CTLA-4 eliminates the residual
tumor mass. This minimizes the often gruesome side effects,
including immunosuppression, associated with the conventional
application of chemotherapy. Similar considerations apply to
radiotherapy. The dose of chemotherapeutic agent or radiation if
used in conjunction with a CTLA-4 blocking agent is preferably
between 2-20%, more preferably between 5-10% of the dose usually
used.
[0057] When the CTLA-4 blocking agent is other than an antibody to
the extracellular domain of CTLA-4 or a fragment thereof, e.g. Fab'
fragment, such blocking agents can be used independently, i.e.,
without an immune response stimulating agent. However, CTLA-4
blocking agents, especially those which consist of an antibody to
the extracellular portion of the CTLA-4, are preferable used in
combination with one or more immune response stimulating agents.
CTLA-4 blocking agents may also be used in conjunction with
radiation and/or chemotherapeutic treatment which indirectly
produces immune response stimulating agents. Such combined use can
involve the simultaneous or sequential use of CTLA-4 blocking agent
and immune response stimulating agent and can occur at different
sites. For example, the CTLA-4 blocking agent can be administered
at a site away from a tumor after the tumor has been directly
irradiated. Alternatively, a chemotherapeutic agent can be used to
treat tumor cells either locally or systemically followed by use of
a CTLA-4 blocking agent.
[0058] Situations characterized by deficient host T cell response
to antigen include chronic infections, tumors, immunization with
peptide vaccines, and the like. Administration of the subject
CTLA-4 blockers to such hosts specifically changes the phenotype of
activated T cells, resulting in increased response to antigen
mediated activation. Treatment of primates, more particularly
humans is of interest, but other mammals may also benefit from
treatment, particularly domestic animals such as equine, bovine,
ovine, feline, canine, murine, lagomorpha, and the like.
[0059] The formulation is administered at a dose effective to
increase the response of T cells to antigenic stimulation. The
response of activated T cells will be affected by the subject
treatment to a greater extent than resting T cells. The
determination of the T cell response will vary with the condition
that is being treated. Useful measures of T cell activity are
proliferation, the release of cytokines, e.g. IL-2, IFNg, TNFa; T
cell expression of markers such as CD25 and CD69; and other
measures of T cell activity as known in the art.
[0060] The subject treatment may be performed in combination with
administration of cytokines that stimulate antigen presenting
cells, e.g. granulocyte-macrophage colony stimulating factor
(GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte
colony stimulating factor (G-CSF), interleukin 3 (IL-3),
interleukin 12 (IL-12), etc. Additional proteins and/or cytokines
known to enhance T cell proliferation and secretion, such as IL-1,
IL-2, B7, anti-CD3 and anti-CD28 can be employed simultaneously or
sequentially with the blocking agents to augment the immune
response. The subject therapy may be combined with the transfection
of tumor cells or tumor-infiltrating lymphocytes with genes
encoding for various cytokines or cell surface receptors (see
Ogasawara et al. (1993) Cancer Res. 53:3561-8; and Townsend et al.
(1993) Science 259:368-370). For example, it has been shown that
transfection of tumor cells with cDNA encoding CD80 leads to
rejection of transfected tumor cells, and can induce immunity to a
subsequent challenge by the non-transfected parent tumor cells
(Townsend et al. (1994) Cancer Res. 54:6477-6483). The subject
therapy enhances this effect.
[0061] Tumor-specific host T cells may be combined ex vivo with the
subject blocking agents, and tumor antigens or cells and reinfused
into the patient. When administered to a host, the stimulated cells
induce a tumoricidal reaction resulting in tumor regression. The
host cells may be isolated from a variety of sources, such as lymph
nodes, e.g. inguinal, mesenteric, superficial distal auxiliary,
etc.; bone marrow; spleen; or peripheral blood, as well as from the
tumor, e.g. tumor infiltrating lymphocytes. The cells may be
allogeneic or, preferably, autologous. For ex vivo stimulation, the
host cells are aseptically removed, and are suspended in any
suitable media, as known in the art. The cells are stimulated by
any of a variety of protocols, particularly combinations of B7,
anti-CD28, etc., in combination with the blocking agents. The
stimulated cells are reintroduced to the host by injection, e.g.
intravenous, intraperitoneal, etc. in a variety of pharmaceutical
formulations, including such additives as binder, fillers,
carriers, preservatives, stabilizing agents, emulsifiers and
buffers. Suitable diluents and excipients are water, saline,
glucose and the like.
[0062] Tumor cells whose growth may be decreased by administration
of the subject blocking agents include carcinomas e.g.
adenocarcinomas, which may have a primary tumor site in the breast,
ovary, endometrium, cervix, colon, lung, pancreas, eosophagus,
prostate, small bowel, rectum, uterus or stomach; and squamous cell
carcinomas, which may have a primary site in the lungs, oral
cavity, tongue, larynx, eosophagus, skin, bladder, cervix, eyelid,
conjunctiva, vagina, etc. Other classes of tumors that may be
treated include sarcomas, e.g. myogenic sarcomas; neuromas;
melanomas; leukemias, certain lymphomas, trophoblastic and germ
cell tumors; neuroendocrine and neuroectodermal tumors.
[0063] Tumors of particular interest are those that present
tumor-specific antigens. Such antigens may be present in an
abnormal context, at unusually high levels, or may be mutated
forms. The tumor antigen may be administered with the subject
blocking agents to increase the host T cell response against the
tumor cells. Such antigen preparations may comprise purified
protein, or lysates from tumor cells.
[0064] Examples of tumors antigens are cytokeratins, particularly
cytokeratin 8, 18 and 19, as an antigen for carcinomas. Epithelial
membrane antigen (EMA), human embryonic antigen (HEA-125); human
milk fat globules, MBr1, MBr8, Ber-EP4,17-1A, C26 and T16 are also
known carcinoma antigens. Desmin and muscle-specific actin are
antigens of myogenic sarcomas. Placental alkaline phosphatase,
beta-human chorionic gonadotropin, and alpha-fetoprotein are
antigens of trophoblastic and germ cell tumors. Prostate specific
antigen is an antigen of prostatic carcinomas, carcinoembryonic
antigen of colon adenocarcinomas. HMB-45 is an antigen of
melanomas. Chromagranin-A and synaptophysin are antigens of
neuroendocrine and neuroectodermal tumors. Of particular interest
are aggressive tumors that form solid tumor masses having necrotic
areas. The lysis of such necrotic cells is a rich source of
antigens for antigen-presenting cells.
[0065] Administration of the subject blocking agents may be
contra-indicated for certain lymphomas. In particular, T cell
lymphomas may not benefit from increased activation. CD80 antigen
is strongly expressed by the Reed-Sternberg cells in Hodgkin's
disease, which are frequently surrounded by CD28-expressing T cells
(Delabie et al. (1993) Blood 82:2845-52). It has been suggested
that the accessory cell function of Reed-Sternberg cells leads to T
cell activation, and contributes to the Hodgkin's syndrome.
[0066] Many conventional cancer therapies, such as chemotherapy and
radiation therapy, severely reduce lymphocyte populations. While
the subject therapy may alleviate this immunosuppression to some
extent, a preferred course of combined treatment will use such
lymphotoxic therapies before or after the subject therapy.
[0067] The subject blocking agents may be administered to increase
the response of T cells to pathogens. Infections with certain
viruses become chronic when the host anti-viral mechanisms fail.
Such infections can persist for many years or even the life-time of
the infected host, and often cause serious disease. Chronic
infections associated with significant morbidity and early death
include those with two human hepatitis viruses, hepatitis B virus
(HBV) and hepatitis C virus (HCC), which cause chronic hepatitis,
cirrhosis and liver cancer. Other chronic viral infections in man
include those with human retroviruses: human immunodeficiency
viruses (HIV-1 and HIV-2) which cause AIDS and human T lymphotropic
viruses (HTLV-1 and HTLV-2) which cause T cell leukemia and
myelopathies. Infections with human herpes viruses including herpes
simplex virus (HSV) types 1 and 2, Epstein Barr virus (EBV),
cytomegalovirus (CMV) varicella-zoster virus (VZV) and human herpes
virus 6 (HHV-6) are usually not eradicated by host mechanisms.
Infection with other agents that replicate intracellularly, such as
pathogenic protozoa, e.g. trypanosomes, malaria and toxoplasma
gondii; bacteria, e.g. mycobacteria, salmonella and listeria; and
fungi, e.g. candida; may also become chronic when host defense
mechanisms fail to eliminate them.
[0068] The subject blocking agents are administered to a patient
suffering from such a chronic pathogen infection. To increase the
immune response, it may be desirable to formulate the blocking
agent with antigens derived from the pathogen. A variety of such
antigens are known in the art, and available by isolation of the
pathogen or expression by recombinant methods. Examples include HIV
gp 120, HBV surface antigen, envelope and coat proteins of viruses,
etc.
[0069] Adjuvants potentiate the immune response to an antigen. The
CTLA-4 blocking agents are used as an adjuvant to increase the
activation of T cells, and to increase the class switching of
antibody producing cells, thereby increasing the concentration of
IgG class antibodies produced in response to the immunogen. The
blocking agents are combined with an immunogen in a physiologically
acceptable medium, in accordance with conventional techniques for
employing adjuvants. The immunogen may be combined in a single
formulation with the blocking agent, or may be administered
separately. Immunogens include polysaccharides, proteins, protein
fragments, haptens, etc. Of particular interest is the use with
peptide immunogens. Peptide immunogens may include tumor antigens
and viral antigens or fragments thereof, as described above.
[0070] The use of the subject blocking agents in conjunction with
genetic immunization is also of interest. A DNA expression vector
encoding a peptide or protein antigen of interest is injected into
the host animal, generally in the muscle or skin. The gene products
are correctly glycosylated, folded and expressed by the host cell.
The method is advantageous where the antigens are difficult to
obtain in the desired purity, amount or correctly glycosylated form
or when only the genetic sequences are known e.g. HCV. Typically,
DNA is injected into muscles or delivered coated onto gold
microparticles into the skin by a particle bombardment device, a
"gene gun". Genetic immunization has demonstrated induction of both
a specific humoral but also a more broadly reacting cellular immune
response in animal models of cancer, mycoplasma, TB, malaria, and
many virus infections including influenza and HIV. See, for
example, Mor et al. (1995) J Immunol 155:2039-46; Xu and Liew
(1995) Immunology 84:173-6; and Davis et al. (1994) Vaccine
12:1503-9.
[0071] The subject blocking agents are used during the immunization
of laboratory animals, e.g. mice, rats, hamsters, rabbits, etc. for
monoclonal antibody production. The administration increases the
level of response to the antigen, and increases the proportion of
plasma cells that undergo class switching.
[0072] CTLA-4 blockers are administered in vitro to increase the
activation of T cells in culture, including any in vitro cell
culture system, e.g. immortalized cell lines, primary cultures of
mixed or purified cell populations, non-transformed cells, etc. Of
particular interest are primary T cell cultures, where the cells
may be removed from a patient or allogeneic donor, stimulated ex
vivo, and reinfused into the patient.
[0073] Various methods for administration may be employed. The
CTLA-4 blocking agent formulation may be injected intravascularly,
subcutaneously, peritoneally, etc. The dosage of the therapeutic
formulation will vary widely, depending upon the nature of the
disease, the frequency of administration, the manner of
administration, the purpose of the administration, the clearance of
the agent from the host, and the like. The dosage administered will
vary depending on known factors, such as the pharmacodynamic
characteristics of the particular agent, mode and route of
administration, age, health and weight of the recipient, nature and
extent of symptoms, concurrent treatments, frequency of treatment
and effect desired. The dose may be administered as infrequently as
weekly or biweekly, or fractionated into smaller doses and
administered daily, semi-weekly, etc. to maintain an effective
dosage level. Generally, a daily dosage of active ingredient can be
about 0.1 to 100 mg/kg of body weight. Dosage forms suitable for
internal administration generally contain from about 0.1 mg to 500
mgs of active ingredient per unit. The active ingredient may vary
from 0.5 to 95% by weight based on the total weight of the
composition.
[0074] In some cases it may be desirable to limit the period of
treatment due to excessive T cell proliferation. The limitations
will be be empirically determined, depending on the response of the
patient to therapy, the number of T cells in the patient, etc. The
number of T cells may be monitored in a patient by methods known in
the art, including staining with T cell specific antibodies and
flow cytometry.
[0075] The subject CTLA-4 blockers are prepared as formulations at
an effective dose in pharmaceutically acceptable media, for example
normal saline, vegetable oils, mineral oil, PBS, etc. Therapeutic
preparations may include physiologically tolerable liquids, gel or
solid carriers, diluents, adjuvants and excipients. Additives may
include bactericidal agents, additives that maintain isotonicity,
e.g. NaCl, mannitol; and chemical stability, e.g. buffers and
preservatives. or the like. The CTLA-4 blockers may be administered
as a cocktail, or as a single agent. For parenteral administration,
the blocking agent may be formulated as a solution, suspension,
emulsion or lyophilized powder in association with a
pharmaceutically acceptable parenteral vehicle. Liposomes or
non-aqueous vehicles, such as fixed oils, may also be used. The
formulation is sterilized by techniques as known in the art.
[0076] The functional effect of CTLA-4 blockade may also be induced
by the administration of other agents that mimic the change in
intra-cellular signaling observed with the subject invention. For
example, it is known that specific cytoplasmic kinases may be
activated in response to binding of extracellular receptors. Agents
that block the kinase activity would have a similar physiological
effect as blocking receptor binding. Similarly, agents that
increase cyclic AMP, GTP concentrations and intracellular calcium
levels can produce physiological effects that are analagous to
those observed with extracellular receptor binding.
[0077] The following examples are offered by way of illustration
and not by way of limitation.
EXAMPLE 1
Generation of Monoclonal Antibodies Reactive with Mouse CTLA-4
[0078] (a) Preparation of a Mouse CTLA-4 Immunogen. A fusion
protein comprising the extracellular portions of the mouse CTLA-4
gene and the constant region of human IgGI, termed mCTLA4-Hgl, was
obtained from Drs. P. Lane and K. Karjalainen (Baser Institute for
Immunology, Basel, Switzerland). An expression vector capable of
expressing the mCTLA4-Hgl protein was constructed as described
(Lane, et al. Immunol. 80:56 (1993)). Briefly, sequences encoding
the extracellular portions of the mouse CTLA-4 molecule were
generated using PCR. The following primer pair was used to amplify
these CTLA-4 sequences from a plasmid containing mouse CTLA-4
sequences: 5'-TTACTCTACTCCCTGAGG AGCTCAGCACATTTGCC-3' (SEQ ID NO:1)
and 5'-TATACTTACCAGAATCCG GGCATGGTTCTGGATCA-3' (SEQ ID NO:2). The
amplified CTLA-4 sequences were then inserted into an expression
vector that permits the insertion of a gene of interest upstream of
sequences encoding the hinge, CH2 and CH3 domains of the human IgG1
protein (Traunecker, et al. Trends Biotech. 9:109 (1991)). Each
primer contained appropriate restriction sites for subcloning into
the human IgG1 expression vector, together with a 3' splice donor
site within the 3' primer to splice to the human g1 exons
correctly. The plasmid containing sequences encoding the
mCTLA-4-Hgl fusion protein was termed pH
.beta.-APr-1-neo-mCTLA4-Hgl. The amino acid sequence of the
mCTLA4-Hgl protein is listed in SEQ ID NO:3.
[0079] To express the mCTLA4-Hgl protein, the
pH.beta.APr-1-neo-mCTLA4-Hgl expression vector was transfected into
the mouse plasmacytoma line, J558L (J558L is identical to the J558
cell line which is available from ATCC (ATCC TIB 6)) using the
standard technique of protoplast fusion. J558L cells were cultured
at 5.times.10.sup.4 cells/ml. Transfected J558L cells were then
selected in the presence of medium containing xanthine (Sigma) and
mycophenolic acid (Calbiochem, LaJolla, Calif.) (selective medium).
The selective medium was applied 24 hr after transfection and
positive clones (ie., clones which grew in the selective medium)
were screened two weeks later. Clones that secreted the fusion
protein were identified using an ELISA for human IgG1. A good
secreting clone was identified and designated clone no. 15. Clone
no. 15 cells were metabolically labelled with (.sup.35S) methionine
and the secreted proteins were immunoprecipitated with protein A
and the precipitated proteins were resolved on an SDS
polyacrylamide gel. The mCTLA4-Hgl protein was found to migrate on
SDS-PAGE gels as a monomer of approximately 60,000 MW under
reducing conditions and as a dimer under non-reducing
conditions.
[0080] Purified preparations of mCTLA4-Hgl protein were obtained by
affinity chromatography of culture supernatants of clone no. 15
cells on a protein A-Sepharose (Zymed, South San Francisco, Calif.)
column. Briefly, J558 cells expressing the mCTLA4-Hgl protein were
grown in IMDM supplemented with 5% FCS, glutamine, 2ME and
antibiotics. Culture supernatants were collected from the cells and
centrifuged at 1500.times.g to remove any remaining cells and the
clarified supernatant was filtered through a 0.4 micron pore size.
The filtered supernatant was adjusted to pH 8.5 using 1N NaOH; the
supernatant was then passed over a 2 ml (packed volume) protein
A-Sepharose column at a flow rate of 2 ml/min. It is noted that the
J558 cell line produces an additional immunoglobulin (i.e., besides
the mouse CTLAIg fusion protein) that binds to protein G; therefore
the use of protein G resins is not recommended for the purification
of the mCTLA4-Hgl protein from transfected J558 cells.
[0081] The protein A column was washed with 20 to 30 column volumes
of PBS and the fusion protein was eluted with 50 mM diethylamine
(pH 11.0). Two milliliter fractions were collected into tubes
containing 0.2 ml 1M Tris-HC 1 to neutralize the pH of the sample.
The absorbance at 280 nm was determined and used to assess the
protein concentration of each fraction. Fractions containing
protein were combined and dialyzed overnight against 2 to 3 changes
of PBS (1 liter per change). The presence of mCTLA4-Hgl protein was
confirmed by SDS-PAGE, which showed a band of approximately 40 kD
(the predicted molecular weight of the fusion protein). In
addition, the purified mCTLA4-Hgl protein was tested in an ELISA
using an antihuman IgG1 antibody (HP6058; the HP6058 hybridoma
(ATCC CRL 1786) was used as the source of HP6058 antibodies).
[0082] (b) Immunization of Hamsters. To immunize hamsters with the
mouse CTLA-4 fusion protein, purified mCTLA4-Hgl protein (hereafter
referred to as CTLA-4Ig) was used to coat heat-killed
Staphylococcus aureus (StaphA) bacteria cells (Calbiochem, LaJolla,
Calif.). Six week old Golden Syrian hamsters (Harlan Sprague
Dawley, Indianapolis, Ind.) were injected in the footpad with 50
.mu.l (packed volume) of heat-killed StaphA bacteria coated with
approximately 100 .mu.g of CTLA-4Ig suspended in 0.2 ml of PBS. The
StaphA cells were coated as follows.
[0083] StaphA cells were prepared according to the manufacturer's
protocol to a concentration of 10% w/v in saline (0.9% NaCl). One
ml of the bacterial cell slurry was centrifuged at 1,400.times.g to
pellet the bacteria and the supernatant was removed. A 1 ml
solution containing approximately 100 .mu.g of purified CTLA-4Ig in
PBS was added to the pellet and the mixture was incubated at
37.degree. C. for 2 hours with agitation. The bacteria were then
pelleted by centrifugation as described above; the pellet was
washed twice with 1 ml of PBS/wash. The CTLA-4Ig-coated bacterial
cells were then resuspended in approximately 200 .mu.l of PBS; 50
.mu.l of this preparation was injected per footpad.
[0084] A total of five injections were given per hamster. On the
day of the final boost and prior to the injection, approximately
100 .mu.l of serum was obtained by intraocular bleeding performed
by the Office of Laboratory Animal Care staff (Univ. of Calif,
Berkeley). This serum was analyzed in comparison to serum obtained
by the identical methodology prior to the first injection.
[0085] A CTLA-4Ig binding ELISA was utilized to demonstrate the
presence of antibody that recognized the CTLA-4Ig fusion protein in
the post-immunization bleed. The CTLA-4Ig binding ELISA was
conducted as follows. CTLA-4Ig fusion protein or CD4Ig fusion
protein was used to coat the wells of 96 well modified flatbottom
ELISA plates (Corning, Corning, N.Y.).
[0086] CD4Ig is a fusion protein that consists of the extracellular
domain of mouse CD4 and the hinge, CH2 and CH3 domains of human
IgG1 (Traunecker et al., supra.); the CD4Ig protein was used as a
negative control in the ELISA assays. The CD4Ig fusion protein was
prepared from transfected J558 cells and purified by affinity
chromatography on protein A Sepharose as described for the
mCTLA4-H.mu.l (i.e., the CTLA-4Ig) fusion protein in section (a)
above.
[0087] Fifty microliters of the fusion proteins, at a concentration
of 1 .mu.g/ml in 0.4% gelatin in PBS were placed in the wells. The
plates were incubated at 37.degree. C. for 2-3 hours to allow the
proteins to absorb; the plates were then washed three times using
150 .mu.l of 0.9% NaCl containing 0.05% Tween-20. The remaining
protein binding sites in the wells were then blocked using 0.4%
gelatin in PBS (blocking buffer) for 30 min at 37.degree. C.;
following the blocking step, the plates were washed twice with 0.9%
NaCl containing 0.05% Tween-20. Fifty microliters of solution
containing antiCTLA-4 antibodies (i.e., serum from immunized
hamsters, purified antibodies or culture supernatants) were added
into triplicate wells and the plates were incubated for 2-3 hours
at 37.degree. C. To assess the amount of anti-CTLA-4 antibodies
present in the serum of immunized hamsters, the initial
post-immunization bleeds were tested using dilutions ranging from
1:1000 to 1:100 (diluted into PBS containing 0.4% gelatin).
[0088] The wells were then washed three times using 150 .mu.l of
0.9% NaCl containing 0.05% Tween-20. Fifty microliters of a
solution containing goat anti-hamster IgG polyclonal sera
conjugated to horseradish peroxidase (CalTag, South San Francisco,
Calif.) at a concentration of 1 .mu.g/ml in blocking buffer was
added to the wells and the plates were incubated for 1 hour at
37.degree. C. The plates were then washed four times with 0.9% NaCl
containing 0.05% Tween-20. A solution containing 0.55 mg/ml ABTS
2,2'-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)) in citrate
buffer (0.1 M citric acid (pH 4.35)) was added and the plates were
incubated for approximately 20 min at 37.degree. C. The plates were
then read at 405 nm using a BioTech plate reader (Beckman
Instruments, Palo Alto, Calif.) to assess the absorbance of the
green reaction product.
[0089] The results of the CTLA-4Ig binding ELISA demonstrated the
presence of antibody that recognized the CTLA-4Ig fusion protein in
the post-immunization bleed at serum dilutions 1000-fold greater
than the dilution at which background could be detected using the
pre-immune bleed.
[0090] (c) Isolation of Hybridoma Lines Secreting Anti-mouse CTLA-4
Antibodies. Three days following the final injection, draining
lymph nodes were removed from the hamsters. Lymphocytes were
isolated from the popliteal lymph nodes which drain the hind-limbs.
Cell suspensions were made from the isolated lymph nodes as
follows. The dissected nodes were placed in a tissue culture dish
(Falcon Plastics, Mountain View, Calif.) containing RPMI medium
(GibcoBRL, Gaithersburg, Md.) supplemented with 10% FCS
(BioWhittaker, Walkersville, Md.). Lymphocytes were released from
the nodes by gentle grinding of the nodes with frosted glass
slides; the lymphocyte suspensions were counted using a
hemocytometer.
[0091] The lymphocytes isolated from the immunized hamsters were
fused to the fusion cell partner, P3X3.Ag8.653 (ATCC CRL 1580).
P3X3.Ag8.653 cells were split 1:20 every 3 days prior to the fusion
in IMDM (Univ. of Calif., San Francisco Tissue Culture Facility)
containing 20% FCS (fetal calf serum) (BioWhittaker, Walkersville,
Md.), 50 .mu.M 2-ME, 50 .mu.M gentamicin.
[0092] The fusion with the myeloma line used a standard
polyethylene glycol fusion technique (McKeam et al., Immunol. Rev.
47:91 (1979)). Briefly, sterile lymphocyte cell suspensions were
prepared in serum free Iscove's Modified Dulbecco's Media (IMDM).
The lymphocytes were washed twice with IMDM and adjusted to a
density of 12.5.times.10.sup.6 cells/ml.
[0093] P3X3.Ag8.653 cells (grown as described above) were washed
twice with serum free IMDM (these cells were centrifuged for 5
minutes at 1000 r.p.m. in a TJ-6 centrifuge (Beckman Instruments,
Palo Alto, Calif.) at 25.degree. C. to pellet the cells) and the
P3X3.Ag8.653 cell density was adjusted to 5.times.10.sup.6
cells/ml.
[0094] Four milliliters of the lymphocyte cell suspension were
mixed with 1 ml of the washed P3X3.Ag8.653 cells in 60 mm tissue
culture dish (Falcon). The tissue culture dishes were placed in
microtiter plate carriers (Beckman Instruments, Palo Alto, Calif.)
and centrifuged at 250.times.g (1200 r.p.m.; TJ-6 centrifuge) for 5
minutes to generate an adherent monolayer of cells on the bottom of
the dish. The supernatant was aspirated from the dishes and the
dishes were neatly flooded with 1 ml of 50% polyethylene glycol
(PEG 1500, Boehringer Mannheim) in IMDM; the PEG solution was
prepared by warming 4 ml of PEG 1500 and 4 ml of IMDM separately in
60.degree. C. water bath and then combining by aspiration of the
PEG into a pipette followed by the IMDM and mixing thoroughly.
After 30 seconds at room temperature, the dishes were flooded with
5 ml of serum free IMDM.
[0095] Following the final wash on the day of the fusion, the cells
were left in the 60 mm dish with 5 ml of IMDM medium containing FCS
for 12 hours at 37.degree. C. with 5% CO.sub.2. On the following
day, the fused cells were diluted into 100 ml of IMDM containing
20% FCS and 1.times.HAT media (Boehringer Mannheim, N.J.) and 100
.mu.l was plated per well in 96 well flat bottom plates. After 5
and 9 days, an additional 50 .mu.l of media was added to each well.
Thereafter, 50 .mu.l of media was removed and fresh media added at
3 day intervals. Once cell numbers were within the 1000-5000 per
well range, hybridoma supernatants were tested for reactivity to
CTLA-4Ig and for a lack of reactivity to CD4Ig by ELISA as
described in section (b) above. Hybridoma supernatants were used
undiluted in the ELISA (50 .mu.l/well).
[0096] Hybridomas from positive wells were repetitively cloned by
limiting dilution in the presence of irradiated mouse thymocyte
feeder layers. A hybridoma line secreting a monoclonal antibody,
termed antibody 9H10, was selected by the following criteria: 1)
reactivity against CTLA-4Ig but not CD4Ig in ELISAs; 2) the ability
to block CTLA-4Ig binding to B7 transfectants; 3) the ability to
stain activated T cells but not freshly isolated T cells; and 4)
the ability to stain a CTLA-4 transfectant but not control
transfectants.
[0097] The ability of antibody 9H10 to block CTLA4Ig binding to B7
transfectants was demonstrated as follows. Approximately 10 .mu.l
of mAb 9H10 was incubated at 22.degree. C. for 30 min with 1 .mu.g
of CTLA-4Ig fusion protein in a final volume of 50 .mu.l of a
solution comprising PBS. To this mixture was added 2.times.10.sup.5
B7-EL-4 cells, suspended in 10 .mu.l ice-cold PBS containing 1%
calf serum and 0.05% sodium azide. B7-EL-4 cells are the
C57BL/6-derived EL4 thymoma cell line transfected with an
expression vector encoding the mouse B7 cell surface protein, as
described in Townsend et al. Cancer Res. 54:6477-83 (1994).
[0098] The resulting mixture was then incubated on ice for 30
minutes, followed by two washes with 4 ml/wash of PBS containing 1%
calf serum and 0.05% sodium azide. The cells were then stained with
fluorescein isothiocynate (FITC)-conjugated anti-human IgG (Caltag,
South San Francisco, Calif.). As a negative control for this
experiment, the CTLA-41g fusion protein was incubated with either a
control hamster IgG or the EL-4 parent cell line. The cells were
analyzed on a FACScan (BectonDickinson, Mountain View, Calif.); the
LYSIS II program (Becton Dickinson) was used to electronically gate
on relevant populations. In most experiments, 10,000 live gated
events were collected for analysis. The results showed that the
9H10 antibody blocked CTLA-4 binding to B7-EL-4 cells.
[0099] The ability of the 9H10 antibody to stain activated T cells
but not freshly isolated T cells was demonstrated as follows. Fresh
and activated splenocytes were generated. Spleens from 4-6 week
BALB/c mice were harvested and minced, and suspensions were treated
with hemolytic Gey's solution to remove the red blood cells, a
standard technique in the art (Mishell and Shiigi, Selected Methods
in Cellular Immunology, W.H. Freeman and Co., San Francisco (1980)
pp.23-24). The cells were cultured in RPMI containing 10% fetal
calf serum, with soluble anti-CD-3 antibody at 10 .mu.g/ml added to
activate one portion of the cell population. The other portion of
the splenocytes was not treated with anti-CD3 and represents fresh
(but not activated splenocytes). The two cell populations were then
stained with either 1) a combination of FITC-conjugated 9H10 (the
anti-CTLA-4 antibody; 5 .mu.g of antibody) and PE-conjugated Thy1.2
or 2) a combination of FITC-conjugated hamster Ig and PE-conjugated
Thy1.2. The data were analyzed on a FACScan and was electronically
gated for Thy1.2 positive cells to analyze only the relevant T cell
population. The results of this experiment demonstrated that the
9H10 antibody stained activated (i.e., CTLA-4 expressing) but not
freshly isolated T cells.
[0100] The ability of the 9H10 antibody to stain a CTLA-4
transfectant but not control transfectants was demonstrated as
follows. A parent CHO (Chinese Hamster Ovary, CHO-K1 cells) cell
line (ATCC CCL 61) was transfected with pSR1neo.CTLA-4.
pSR1neo.CTLA-4 contains the entire 1.9 kb cDNA encoding the mouse
CTLA-4 protein (Brunet et al., Nature 328:267 (1987)) inserted into
the pSR1neo expression vector. Cells transfected with the
pSR1neo.CTLA vector express the mouse CTLA-4 protein on the cell
surface.
[0101] The parent (i.e., CHO-K1 cells) and transfected cells were
stained either 1) a combination of FITC-conjugated 9H10 (the
anti-CTLA-4 antibody; 5 .mu.g of antibody) and PE-conjugated Thy1.2
or 2) a combination of FITC-conjugated hamster Ig and PE-conjugated
Thy1.2. The data was electronically gated for Thy1.2 positive cells
to analyze only the relevant T cell population. The results of this
experiment demonstrated that the 9H10 antibody stains CTLA-4
transfectants but not control transfectants.
[0102] The above results demonstrated that the 9H10 monoclonal
antibody reacts specifically with the mouse CTLA-4 protein.
EXAMPLE 2
Anti-CTLA-4 Monoclonal Antibodies Cause Rejection of V51BLim10
Tumors in Mice
[0103] The anti-mouse CTLA-4 monoclonal antibody, 9H10, was used to
treat mice that received injections of a colon carcinoma cell line.
The injection of the 9H10 mAb along with V51BLim10tumor cells
resulted in the complete rejection of the tumor cells in the
experimental animals. In contrast, mice injected with an anti-CD28
mAb and V51BLim10 cells or mice injected with V51BLim10 cells alone
both developed tumors which exhibited a steady increase in average
tumor size over a period of four weeks.
[0104] (a) Generation of the V51BLim10 Cell Line. The V51BLim10
cell line was generated by transfection of the SR1neo expression
vector into the 51BLim10 cell line. The 51BLim cell line is a colon
carcinoma cell line that provides an accurate animal model for
colon cancer metastasis in humans. Bresalier, et al., Cancer Res.
47:1398 (1987).
[0105] The V51BLim10 cell line used in the present experiments was
generated as follows. The murine colon cancer cell line 51B
established by Corbett et al., Cancer Res. 35:2434-9 (1975) was
injected into the cecal wall of BALB/c mice; the resulting colonic
tumors were found to spontaneously metastasize to the liver in a
minority of the injected mice. Bresalier et al., Cancer Res.
47:1398 (1987). Tumor cell lines having progressively increased
metastatic activity were developed by collecting cells from the
original metastases, which were then used for successive
reinjection into the ceca of additional mice. These cell lines were
termed 51 BLim-1 through 51 BLim-5 where the number following the
dash refers to the number of metastatic cycles.
[0106] A 51B metastatic derivative obtained from Dr. Warren at the
University of California San Francisco was designated 51BLim10; the
51BLim10 cell line corresponds to the 51BLiM5 cell line described
by Bresalier, et al., Cancer Res. 47:1398 (1987).
[0107] The SR1neo expression vector was transfected into the 51
BLiM-10 cell line to generate the V51BLim10 cell as described
(Townsend et al. Cancer Res. 54:6477-83 (1994)). The SR1neo
expression vector (obtained from L. Lanier at DNAX Research
Institute of Molecular and Cellular Biology, Palo Alto, Calif.)
allows the expression of a gene of interest under the
transcriptional control of the HTLV-1 LTR. The SR1neo vector also
contains the neo gene under the transcriptional control of the SV40
promoter/enhancer. The presence of the neo gene allows for the
selection of transfected cells containing the SR1neo vector.
[0108] The SR1neo expression vector was transfected into 51BLiM-10
cells by electroporation using a BTX T 800 electroporator (BTX,
Inc., San Diego, Calif.). Five pulses for 99 .mu.s each at 450 or
600 V were applied. The electroporation was carried out in a final
reaction volume of 750 .mu.l of a solution comprising 270 mM
sucrose, 7 mM NaPO.sub.4 (pH 7.4), 1 mM MgCl.sub.2,
5.times.10.sup.6 51B LiM-10 cells and 50 .mu.g of the SR1neo
expression vector. Following electroporation, the cells were
cultured for 24 hours in complete medium (Eagle's MEM (Univ. of
Calif. at San Francisco Cell Culture Facility, San Francisco,
Calif.) supplemented with 10% FCS (Sigma), nonessential amino
acids, MEM vitamin solution, L-glutamine, sodium pyruvate,
gentamicin (all from Irvine Scientific, Santa Ana, Calif.) and 7.5%
sodium bicarbonate (Sigma)) at 37.degree. C. Selection medium
(complete medium containing 1 mg/ml Geneticin (G418 sulfate, GIBCO,
Grand Island, N.Y.)). After 14 days of culture in the selection
medium, drug resistant cells were pooled and used in subsequent
experiments as a polyclonal population referred to as
V51BLim10.
[0109] V51BLim10 tumor cells were maintained in Eagle's MEM (Univ.
of Calif. at San Francisco Cell Culture Facility, San Francisco,
Calif.) supplemented with 10% FCS (Sigma), non-essential amino
acids, MEM vitamin solution, L-glutamine, sodium pyruvate,
gentarnicin, penicillin-streptomycin (all from Irvine Scientific,
Santa Ana, Calif.) and 1 mg/ml Geneticin. Cell cultures were
established from low passage (i.e, less than 10 passages) frozen
aliquots and maintained in culture for no more than 30 days prior
to use.
[0110] V51BLim10 cells and the parental 51BLim10 cells were found
to exhibit similar in vitro and in vivo growth rates. The
expression of the neomycin resistance gene in the V51BLim10 cells
and a variety of other tumor cell lines has had no effect on the
tumorigenicity or growth rate of tumors from the injected
cells.
[0111] (b) In ection of Mice with V51 BLim10 Tumor Cells and
Monoclonal Antibodies. The V51BLim10 tumor cells were harvested
from tissue culture plates with trypsin-EDTA (Sigma), washed three
times in serum-free media (Eagle's MEM) and suspended at a
concentration of 2.times.10.sup.7 cells/ml.
[0112] The mice used in this experiment were 6-8 week old female
BALB/c mice (Charles River Laboratories, Wilmington, Mass.). Groups
of five mice were anesthetized by methoxyflurane inhalation, ear
notched for identification, and injected with 200 .mu.l of the
V51BLim10 tumor cell suspension (4.times.10.sup.6) subcutaneously
in the left flank. Treated groups received 100 .mu.g
intraperitoneal injections of the antiCTLA-4 mAb 9H10 described
above, or alternatively the anti-CD28 mAb, 37.51, on the same day,
and additional 50 .mu.g i.p. injections on days 3 and 6 following
the injection of the tumor cells (designated by the darkened arrows
in FIG. 1). The monoclonal anti-CD28, 37.51, is directed against
the mouse CD28 protein (Gross et al., J. Immunol. 149:380 (1992))
and served as a negative control.
[0113] The mice were monitored for subcutaneous tumor growth and
the bisecting diameters of developing tumors were measured with
calipers. All of the mice left untreated, or treated with anti-CD28
antibody, developed progressively growing tumors and required
euthanasia by 35 days after inoculation. In contrast, all mice
treated with anti-CTLA-4 antibody completely rejected their tumors
after a short period of limited growth. As shown in FIG. 1A, the
average tumor area in mm2 (displayed along the y axis) decreased
gradually starting at approximately day 14 post-tumor injection
(displayed along the x axis), decreasing to zero at approximately
day 24. Anti-CTLA-4 treatment was less effective at at smaller
tumor doses. FIG. 1B shows the average tumor size in mice injected
with 2.times.10.sup.6 tumor cells and treated as described above
with anti-CTLA-4 antibody or an irrelevent hamster antibody.
Anti-CTLA-4 antibody treatment continued to have a dramatic effect
on tumor growth, but one mouse developed a tumor quickly, and
another much later. FIG. 1C illustrates the individual tumor growth
in mice injected with 2.times.10.sup.6 V51BLim10 cells. Three of
the mice remained tumor free beyond 80 days. It is clear that
CTLA-4 blockade significantly enhanced rejection of the B7 negative
tumor cells.
[0114] (c) Injection of Mice with B7-51BLim10 Tumor Cells and
Monoclonal Antibodies. 51BLim10 cells were transfected as described
above, with a plasmid containing the gene for murine B7-1, and
cloned by limiting dilution. The B7-51BLim10 tumor cells were
harvested from tissue culture plates with trypsin-EDTA (Sigma),
washed three times in serum-free media (Eagle's MEM) and suspended
at a concentration of 2.times.10.sup.7 cells/ml.
[0115] The mice used in this experiment were 6-8 week old female
BALB/c mice (Charles River Laboratories, Wilmington, Mass.). Groups
of five mice were anesthetized by methoxyflurane inhalation, ear
notched for identification, and injected with 100 .mu.l of the
B7-51BLim10 tumor cell suspension (4.times.10.sup.6) subcutaneously
in the left flank. Treated groups received 100 .mu.g
intraperitoneal injections of the antiCTLA-4 mAb 9H10 described
above, or alternatively the anti-CD28 mAb, 37.51. Injections of
100, 50 and 50 .mu.g were given on days 0.3 and 6, respectively
(injection days are designated by the darkened arrows in FIG. 2).
The monoclonal anti-CD28, 37.51, is directed against the mouse CD28
protein (Gross et al., J. Immunol. 149:380 (1992)) and served as a
negative control.
[0116] The mice were monitored for subcutaneous tumor growth and
the bisecting diameters of developing tumors were measured with
calipers. The data from this experiment is shown in FIG. 2.
Treatment with anti-CTLA-4 antibodies inhibited B7-51BLim10 tumor
growth as compared to the anti-CD28 and control groups. All mice in
the untreated and anti-CD28 treated groups developed small tumors
that grew progressively for five to ten days and then ultimately
regressed in eight of the ten mice by about day 23 post injection.
The two small tumors that did not regress remained static for over
90 days. In contrast, 3 of the 5 mice treated with anti-CTLA-4
antibody developed very small tumors, and all of these regressed
completely by day 17.
[0117] (d) Anti-CTLA-4 induced rejection of V51BLim10 tumor cells
results in protection against subsequent challenge with wild-type
colon carcinoma cells. Five anti-CTLA-4 treated mice that had
completely rejected V51BLim10 tumor cells were rechallenged 70 days
later with 4.times.10.sup.6 wild-type 51BLim10 tumor cells injected
sub-cutaneously in the opposite flank. Five naive mice were also
injected as controls. Tumor diameters were measured and reported as
described. Prior tumor rejection resulted in significant protection
against secondary challenge as compared to naive controls. All
control mice developed progressively growing tumors, developed
massive tumor burdens, and were euthanized on day 35
post-inoculation. 3 of 5 previously immunized mice remained tumor
free 70 days after challenge. Only one of the previously immunized
mice had a detectable tumor by day 14, and growth of this tumor was
very slow. Utimately, two more tumors developed in the immunized
mice 42 days after challenge. The data is shown in FIG. 3. These
results demonstrated that tumor rejection mediated by CTLA-4
blockade resulted in immunologic memory.
[0118] (e) Anti-CTLA-4 treatment reduces the growth of established
tumors. Groups of mice were injected s.c. with 2.times.10.sup.6
51BLim10 tumor cells. Control animals (n=10) were injected i.p.
with 100 .mu.g irrelevant hamster antibody on days 0, 3, 6 and 9,
as indicated by the upward pointing arrows in FIG. 4. One
anti-CTLA-4 treatment group received i.p. injections on the same
days. The other treated mice (n=5) were given i.p. injections of
anti-CTLA-4 antibody beginning on day 7 and subsequently on days
10, 13 and 16 (downward pointing arrows). Data is shown in FIG. 4.
Mice treated with anti-CTLA-4 antibodies at either time point had
significantly reduced tumor growth compared to untreated controls.
Delaying treatment appeared to be more effective, with 2 of 5 mice
remaining tumor free beyond thirty days after inoculation.
[0119] (f) Anti-CTLA-4 treatment reduces the growth of the murine
fibrosarcoma SA1N. The effects of anti-CTLA-4 treatment were not
limited to carcinoma cell lines. Similar results were obtained with
a rapidly growing fibrosarcoma cell line of A/JCr mice. Groups of
mice were injected s.c. in the flank with a suspension of
1.times.10.sup.6 SA1N fibrosarcoma cells. Treated groups were
injected i.p. with 100 .mu.g anti-CTLA-4 or irrelevant hamster
control antibody at days 0, 3 and 6, as indicated by the arrows in
FIG. 5. All control animals were killed by day 30. Two of five
anti-CTLA-4 treated animals remained tumor free at day 55. Data is
shown in FIG. 5.
EXAMPLE 3
Anti-CTLA-4 Monoclonal Antibodies Act as an Adjuvant
[0120] (a) Preparation of immunogen. DNP-KLH was obtained from
Calbiochem (san Diego, Calif.) and was suspended in deionized water
at 1 mg/ml, 100 ng/ml or 10 pg/ml. One ml of Freund's Complete
Adjuvant (Difco, Mich.) was added to each 1 ml of the DNP-KLH
preparations. These were then emulsified in two 5 ml syringes
connected by a double-ended luer lock connector by rapid passage
through the luer lock, as described in Current Protocols in
Immunology, Colligan et al., eds., section 2.4.
[0121] 30 minutes prior to injection of the immunogen, C57B1/6 mice
of 4-6 weeks in age were injected in the peritoneum using a 23
gauge syringe with 200 .mu.g of non-specific control hamster
antibody or with 200 .mu.g of anti-CTLA-4 antibody 9H10 (both in
200 .mu.l total volume). The mice were subsequently injected
subcutaneously using a 21 gauge syringe at two sites on the back,
with 200 .mu.l of the immunogen in the form described above, giving
a dose of 100 .mu.g, 10 ng or 1 pg/mouse, respectively. After three
days the antibody injections were repeated.
[0122] Ten days following the first treatment, the animals were
euthanized. Blood was obtained by heart puncture and removed to
eppendorf tubes. These samples were allowed to coagulate at
4.degree. C. overnight, and were then centrifuged to obtain
sera.
[0123] Sera was analyzed for isotype specific antibodies
recognizing DNP using a standard isotype ELISA, as described in
Current Protocols in Immunology (supra.) section 2.1. Briefly, DNP
was plated at 100 ng/ml in 50 .mu.l volume in each well of a 96
well Corning modified round-bottom ELISA plate. The wells are
blocking using buffers as described. Three-fold serial dilutions of
each sera, starting at 1:100 are added to each well. These are
incubated for one hour at 25.degree. C., and washed with wash
buffer. Isotypes are detected by using mouse specific antibodies as
detecting agents at 1 .mu.g/ml in 50 .mu.l of blocking buffer
incubated for one hour. The isotype antibodies are biotinylated,
and detection is achieved by incubating with avidin-horseradish
peroxidase, washing and addition of peroxidase substrate (ABTS,
Sigma, Mo.). Stop buffer is added, and the absorbance of each well
read with an ELISA reader at a wave length of 490-498 nm within 5-8
min of stopping the reaction.
[0124] The results are shown in FIG. 6. Each of the panels
illustrates the concentration of a different isotype in the serum
sample. The y axis shows the O.D. reading, where an increase in
O.D. indicates increased concentration of antibodies in the serum
having that isotype. The x axis shows the amount of antigen that
was injected, 100 .mu.g, 10 ng or 1 pg per animal, respectively. It
can be seen that anti-CTLA-4 antibody increases class switching to
IgG1, IgG2a and IgG2b at the higher dose of antigen.
[0125] Analysis of T cell function was performed as follows. Lymph
node cells were isolated and stimulated in vitro for 72 hours with
KLH. The axillary, inguinal, mesenteric, brachial, cervical and
popliteal lymph nodes were removed to a dish containing
RPMI-complete (10% FCS (Hyclone, Mont.), 2 mM glutamine, 50 .mu.M
b-mercaptoethanol, 50 .mu.g/ml gentamycin). The lymph nodes were
minced to obtain single cell suspensions, filtered through a nytex
mesh to remove particulate, and counted using a hemocytometer.
Cells were plated in 150 .mu.l of RPMI-complete in 96 well round
bottom cluster plates at either 5.times.10.sup.5,
2.5.times.10.sup.5, or 1.25.times.10.sup.5 cells/well. KLH
solutions in RPMI-complete were added to final concentrations of
100, 10, 1 or 0 g/ml and the plates were incubated at 37.degree. C.
for 64 hours in humidified incubators with 5% CO.sub.2. After 64
hours, 20 .mu.l of RPMI-complete containing 1 .mu.Ci of
.sup.3H-thymidine was added to each well, and the plates were
incubated an additional eight hours. At this time, cultures were
harvested onto glass fiber filters using an Inotech 96 well
harvester. Filters were dried and counted using a Packard Matrix
counter. Each condition was performed in triplicate, and data
represents the mean of triplicate values.
[0126] The results are shown in FIG. 7. The top row shows a
constant number of cells (5.times.10.sup.5 cells), with varying
concentrations of antigen (shown on the x axis). The y axis shows
incorporation of .sup.3H-thymidine, a measure of cell
proliferation. The lower panel shows a constant antigen
concentration (10 .mu.g/ml), with varying numbers of cells (shown
on the x axis). The data indicates that CTLA-4 blockade strongly
upregulates the T cell response to the higher doses of antigen.
EXAMPLE 4
Generation of Antibodies Directed Against Human CTLA-4 Proteins
[0127] Anti-human CTLA-4 antibodies are generated as follows.
[0128] (a) Human CTLA-4 Proteins for Immunization of Host Animals.
Immunogens comprising human CTLA-4 proteins contain all or a
portion of the extracellular domain of the human CTLA-4 protein.
The extracellular domain of the human CTLA-4 protein comprises
amino acid residues 38-161, as listed in the database
references.
[0129] The human CTLA-4 immunogen comprises the entire human CTLA-4
protein or a fusion protein comprising the extracellular domain of
human CTLA-4 and a fusion partner. The immunogen comprises the
entire human CTLA-4 protein inserted into the membrane of a cell;
the cell expressing human CTLA-4 on the surface is used to immunize
a host animal.
[0130] Immunogens comprising portions of the human CTLA-4 protein
are generated using the PCR to amplify DNA sequences encoding the
human CTLA-4 protein from mRNA from H38 cells, an HTLV
II-associated leukemia line (R. Gallo, National Cancer Institute).
The mRNA is reverse transcribed to generate first strand cDNA. The
cDNA is then amplified. These sequences are linked to sequences
that encode a fusion partner, as described in Linsley et al. (J.
Exp. Med. 174:561 (19991)). The expression vector encodes a fusion
protein termed CTLA4Ig, which comprises (from amino- to
carboxy-termini) the signal peptide from oncostatin M, the
extracellular domain of human CTLA-4 and the H, CH2 and CH3 domains
of human IgG1. The signal peptide from oncostatin M is used in
place of the naturally occurring human CTLA-4 signal peptide. The
cysteine residues found in the wild-type hinge domain of the human
IgG1 molecule were mutated to serines in the construction of the
vector encoding the CTLA41 g protein (Linsley et al., supra).
[0131] (b) Immunization of Host Animals With Human CTLA-4 Proteins.
To immunize animals with immunogens comprising human CTLA-4
proteins, non-human host animals are employed. The immunogen
comprising a human CTLA-4/IgG fusion protein (e.g., CTLA4Ig), is
used to coat heat-killed Staphylococcus A (StaphA) bacteria cells
as described in Example 1b. Six week old BALB/c mice are injected
in the footpad with 50 .mu.l (packed volume) of heat-killed StaphA
bacteria coated with approximately 100 .mu.g of CTLA-4Ig suspended
in 0.2 ml of PBS.
[0132] A total of five injections are given per mouse. On the day
of the final boost and prior to the injection, approximately 100
.mu.l of serum is obtained by intraocular bleeding as described in
Example 1b. The serum is analyzed in companion to serum obtained by
the identical methodology prior to the first injection (ie.,
pre-immune serum).
[0133] A human CTLA-4Ig binding ELISA is utilized to demonstrate
the presence of antibody that recognizes the human CTLA-4Ig fusion
protein in the post-immunization bleed. The human CTLA-4Ig binding
ELISA is conducted as described above in Example 1b with the
exception that the ELISA plates are coated with human CTLA-4
protein.
[0134] The serum and lymph nodes of the immunized mice containing
antibody that recognizes the human CTLA-4Ig fusion protein in the
post-immunization bleed at serum dilutions 1000-fold greater than
the dilution at which background could be detected are collected.
Lymphocytes are prepared from draining lymph nodes in the immunized
mice and are then used for the generation of monoclonal antibodies
directed against the human CTLA-4 protein as described above in
Example 1c.
[0135] Immunogens comprising transformed cells expressing the human
CTLA-4 protein on the cell surface are prepared as follows.
Expression vectors encoding the entire human CTLA-4 protein are
used to transfect the mouse lymphoma cell line EL4 (ATCC TIB 39).
Transfected EL4 cells are injected into mice using 1.times.10.sup.6
to 1.times.10.sup.7 transfected cells/injection. The transfected
cells are injected in a solution comprising PBS. The mice may be
injected either i.p. or in the hind footpad. When i.p. injections
are given, a total of approximately 4 injections are administered.
When the footpad is used as the site of injection, a total of
approximately 5 injections are administered. Serum is collected
from the immunized animals and tested for the presence of
antibodies directed against the human CTLA-4 protein using an ELISA
as described in Example 1b, with the exception that the plates are
coated with human CTLA-4 proteins.
[0136] (c) Isolation of Hybridoma Lines Secreting Anti-Human CTLA-4
Antibodies. Lymphocytes are isolated from draining lymph nodes or
the spleens of animals immunized with the human CTLA-4 immunogen
and fused to P3X3.Ag8.653 cells to generate hybridoma cell lines
using the PEG fusion protocol described in Example 1c. Culture
supernatant from wells containing 1000-5000 cells/well are tested
for reactivity to human CTLA-4 and for lack of reactivity to a
non-CTLA-4 protein such as human CD4 using an ELISA assay.
[0137] Hybridomas from positive wells are repetitively cloned by
limiting dilution as described in Example 1c. Hybridoma lines
secreting monoclonal antibodies that are reactive against human
CTLA-4 proteins but not irrelevant human proteins (e.g., human
CD4), and that have the ability to stain cells human CTLA-4
transfectants but not control transfectants are selected for
production of anti-human CTLA-4 monoclonal antibodies.
EXAMPLE 5
Ex Vivo Stimulation of Tumor Infiltrating Lymphocytes (TILs)
[0138] Host cells are stimulated ex vivo, allowing them to
differentiate into tumor-specific immune effector cells. The cells
are then reintroduced into the same host to mediate anticancer
therapeutic effects.
[0139] (a) Isolation of Tumor-Infiltrating Lymphocytes (TILs).
Tumor-infiltrating lymphocytes are obtained using standard
techniques. Solid tumors (freshly resected or cryopreserved) are
dispersed into single cell suspensions by overnight enzymatic
digestion (e.g., stirring overnight at room temperature in RPMI
1640 medium containing 0.01% hyaluronidase type V, 0.002% DNAse
type I, 0.1% collagenase type IV (Sigma, St. Louis), and
antibiotics). Tumor suspensions are then passed over Ficoll-Hypaque
gradients (Lymphocyte Separation Medium, Organon Teknika Corp.,
Durham, N.C.). The gradient interfaces contain viable tumor cells
and mononuclear cells are washed, adjusted to a total cell
concentration of 2.5 to 5.0.times.10.sup.5 cells/ml and cultured in
complete medium. Complete medium comprises RPMI 1640 with 10%
heat-inactivated type-compatible human serum, penicillin 50 IU/ml
and streptomycin 50 .mu.g/ml (Biofluids, Rockville, Md.),
gentamicin 50 .mu.g/ml (GIBCO Laboratories, Chagrin Falls, Ohio),
amphotericin 250 ng/ml (Funglzone, Squibb, Flow Laboratories,
McLean, Va.), HEPES buffer 10 mM (Biofluids), and L-glutamine 2 mM
(MA Bioproducts, Walkersville, Md.). Conditioned medium from 3- to
4-day autologous or allogeneic lymphokine-activated killer (LAK)
cell cultures (see below) is added at a final concentration of 20%
(v/v). Recombinant IL-2 is added at a final concentration of 1000
U/ml.
[0140] Cultures are maintained at 37.degree. C. in a 5% CO.sub.2
humidified atmosphere. Cultures are fed weekly by harvesting,
pelletting and resuspending cells at 2.5.times.10.sup.6 cells/ml in
fresh medium. Over an initial period (e.g., 2 to 3 weeks) of
culture, the lymphocytes selectively proliferate, while the
remaining tumor cells typically disappear completely.
[0141] To make LAK cell cultures, peripheral blood lymphocytes
(PBL) are obtained from patients or normal donors. After passage
over Ficoll-Hypaque gradients, cells are cultured at a
concentration of 1.times.10.sup.6/ml in RPMI 1640 medium with 2%
human serum, antibiotics, glutamme, and HEPES buffer. Recombinant
IL-2 is added at 1000 U/ml. Cultures are maintained for 3 to 7 days
in a humidified 5% CO.sub.2 atmosphere at 37.degree. C.
[0142] (b) Ex Vivo Stimulation of TILs. 4.times.10.sup.6 cells, in
2 ml of culture medium containing the anti-CTLA-4 mAbs, are
incubated in a well of 24-well plates at 37.degree. C. in a 5%
CO.sub.2 atmosphere for 2 days. The culture medium comprises RPMI
1640 medium supplemented with 10% heat inactivated fetal calf
serum, 0.1 mM nonessential amino acids, 1 .mu.M sodium pyruvate, 2
mM freshly prepared L-glutamine, 100 .mu.g/ml streptomycin, 100
U/ml penicillin, 50 llg/ml gentamicin, 0.5 .mu.g/ml fungizone (all
from GIBCO, Grand Island, N.Y.) and 5.times.10.sup.-5 M 2-ME
(Sigma). The cells are harvested and washed.
[0143] The initially stimulated cells are further cultured at
3.times.10.sup.5/well in 2 ml of culture media with recombinant
human IL-2 (available from Chiron Corp., Emeryville, Calif.;
specific activity of 6 to 8.times.10.sup.6 U/mg protein; units
equivalent to 2-3 International U). After 3 days incubation in
IL-2, the cells are collected, washed, counted to determine the
degree of proliferation, and resuspended in media suitable for
intravenous (i.v.) administration (e.g. physiological buffered
saline solutions). Bacterial cultures are performed to determine
the existence of bacterial contamination prior to reinfusion of the
activated cells.
[0144] After the activated TILs have been resuspended in a media
suitable for injection, IV access is obtained in the host and the
cell suspension is infused. Optionally, the host is treated with
agents to promote the in vivo function and survival of the
stimulated cells (e.g. IL-2).
EXAMPLE 6
[0145] In this study we investigated the effect of CD28 and CTLA-4
signals on the responses of T cell populations in response to the
superantigen Staphylococcus enterotoxin B (SEB) in vitro and in
vivo. The results indicate that CD28 provides an important
costimulus for the SEB response in vitro and that signals through
CTLA-4 inhibit the response. In vivo, blockade of CD28 by FAb
fragments or intact antibodies have the opposite effects upon
V.beta.8+expansion to a similar blockade with anti-CTLA-4 FAb
fragments or intact antibodies. Analysis of the kinetics of the
expansion imply that signals through CD28 promote T cell expansion,
whereas an opposing signal through CTLA-4 functions during T cell
expansion to attenuate the magnitude of the response to SEB.
[0146] Methods
[0147] Mice. BALB/c mice were purchased at four to five weeks of
age from Charles River and were used within three weeks.
[0148] Antibodies and Reagents. Hamster anti-mouse CD28 from clone
37.N.51.1 (Gross et al. (1992) J. Immunol. 149:380), hamster
anti-mouse CTLA-4 from clone 9H10.11D3 (Krummel and Allison (1995)
J. Exp. Med. 182:459) hamster anti-mouse B7-1 from clone 1610.A
(Razi-Wolf et al. (1992) J. Exp. Med. 89:4210), rat anti-mouse B7-2
(from clone GL-1 (Hathcock et al. (1993) Science 262:905) and
irrelevant hamster IgG from clone F560.31 were purified from
ascities fluid in our facility. FAb fragments were obtained by
digestion with immobilized papain (Pierce, Rockford Ill.) by
standard methodology and undigested antibody was removed by Protein
A adsorption. All FAb fragments were analyzed by SDS-PAGE prior to
use. Purity of anti-CD28 FAbs was further tested in functional
assays for the ability to block T cell proliferation in an
allo-MLR. Anti-V.sub..beta.8.1,8.2 FITC (clone MR5-2) was obtained
from Pharmingen (San Diego, Calif.).
[0149] In Vitro Assays. Spleens obtained from naive animals were
minced to obtain suspensions and RBCs were lysed by hypotonic
treatment with Geys solution followed by two washes with PBS.
2.times.10.sup.5 splenocytes were plated in 200 .mu.l RPMI
(containing 10% FCS, 50 .mu.M .beta.-mercaptoethanol, 2 mM
glutamine, and 50 .mu.g/ml gentamycin) in 96 well round bottom
plates. SEB was added at the indicated concentrations. Where
indicated, anti-CD28 was added at a 1:1000 dilution of ascites,
anti-B7-1 was added at 5 .mu.g/ml and anti-B7-2 was added at 20
.mu.g/ml, and equal quantities of non-specific control antibody
560.31 were added. For FAb experiments, anti-CD28, anti-CTLA-4 or
control FAb fragments were added at 100 .mu.g/ml. Cultures were
incubated for 60 hours at 37.degree. C., pulsed with 1 .mu.Ci of
.sup.3H thymidine and allowed to incubate for a further 12 hours
prior to harvesting.
[0150] In Vivo SEB Responses. Mice were injected intraperitoneally
with 200 .mu.l of PBS containing, where indicated, 200 .mu.g of
antibody. After 1-2 hours, mice were injected intravenously with 50
.mu.g per animal of SEB (Toxin Technologies, Sarasota Fl) in PBS or
PBS alone in 100 .mu.l total volume.
[0151] Flow Cytometry. To evaluate the population of V.sub..beta.8
expressing cells, spleens were minced to obtain suspensions and
RBCs were lysed using a hypotonic Geys solution. The resulting
cells were then resuspended in 5 ml of RPMI-10% FCS and triplicate
aliquots were counted using a hemocytometer. Standard error for
this was routinely within 10% of the mean. For staining, aliquots
were washed once in PBS/1% FCS with 0.01% NaN.sub.3 and resuspended
in PBS/FCS at a concentration of 10.sup.6 cells/50 .mu.l.
Antibodies were added and incubated on ice for 30 minutes. Cells
were washed and subsequently analyzed using a FACScan cytometer
utilizing the LysisII software (Becton-Dickinson, Mountain View,
Calif.). 10,000 live gated events were analyzed for the percentage
expressing V.sub..beta.8.sup.+ and was used to obtain the total
number of V.sub..beta.8 cells by applying the formula:
#V.beta.8=Total Cell Yield x % V.beta.8 in sample.
[0152] Results
[0153] Role of Costimulation in SEB Mediated Proliferation In
vitro. The proliferative response of splenocytes from BALB/c mice
to SEB was investigated to determine the role of B7/CD28
interactions. As SEB was added to splenocytes, dose-dependent
proliferation was observed in the cultures. B7 molecules on cells
in these cultures appear to supply costimulation, since addition of
anti-B7-1/B7-2 antibodies significantly inhibited the response.
Further, increased CD28 signaling via anti-CD28 antibodies enhanced
the proliferative response. This increase may have been mediated by
immobilization of antibody on FcR.sup.+ B cells or by the formation
of antibody microaggregates. Interestingly, the addition of
anti-CD28 and anti-B7-1/B7-2 induced a slight but reproducible
increase in proliferation compared to anti-CD28 by itself,
suggesting that another B7 ligand besides CD28 (i.e. CTLA-4) might
be important in downregulating the response of T cells to SEB.
[0154] To address the relative contibutions of CD28 and CTLA-4 on
the T cell response, antibody Fab fragments specific for these
molecules were added to SEB stimulated cultures. Addition of CD28
FAbs inhibited the SEB dependent proliferation. The magnitude of
the CD28 FAb blockade is similar to that observed using anti-B71/2
antibodies, implicating CD28/B7 interactions in providing some
costimulation for proliferation in the control cultures. However,
there was a two to three-fold augmentation of proliferation in the
presence of CTLA-4 FAb, implying that CTLA-4 signals plays an
important part in regulating the response. This further emphasizes
that B7 molecules on APC create an interplay of amplifying signals
through CD28 and attenuating signals through CTLA-4.
[0155] CD28 and CTLA-4 Signals Have Opposing Effects on In vivo
Expansion of V.sub..beta.8.sup.+T cells. The effects of anti-CD28
and anti-CTLA-4 antibody treatment on the T cell response to SEB
was examined. T cell expansion to superantigens in vivo typically
occurs within 2-3 days post-injection. 60 hours was chosen as a
convenient timepoint to initially analyze the affects of anti-CD28
and anti-CTLA-4 upon the response. Animals were injected with PBS
or SEB and the relevant mAbs or FAb fragments. After 0.60 hours,
the total number of V.beta.8-bearing TCRs was determined by
counting the spleen cellularity and antibody staining samples to
determine the percentage of V.beta.8+cells. The total number of
V.beta.8-bearing cells isolated from the spleen of animals injected
with SEB and control antibodies was approximately 2-3 times the
number present in control (PBS) injected animals. In contrast, the
injection of increasing doses of anti-CD28 in addition to SEB
decreased the number of V.beta.8-bearing cells observed at this
time point. The injection of 5 .mu.g of anti-CD28 modestly
decreased the number of recovered V.beta.8 and both 20 .mu.g and
200 .mu.g injections gave roughly identical two-fold reductions. To
address the discrepancy of this result and in vitro results showing
anti-CD28-mediated amplification of T cell responses, daily doses
of FAb fragments of CD28 antibody were injected during the SEB
response. In a similar manner to intact antibodies, these FAbs
blocked the expansion of V.beta.8+cells to SEB in a dose-dependent
manner. The inhibitory effects of intact antibodies was similar to
that observed using FAbs, implying that anti-CD28 antibodies and
FAb fragments in vivo both interfere with B7/CD28 signals. This may
be the result of inefficient signaling by bivalent antibody and
competition with native ligand by both antibody and FAb
fragments.
[0156] To compare the effects of CD28 versus CTLA-4, anti-CTLA-4
antibodies were co-injected with SEB. In contrast to what was
observed with anti-CD28 treatment, administration of anti-CTLA-4
resulted in a dose-dependent increase in accumulation of splenic
V.beta.8+cells. The highest dose of anti-CTLA-4 produced a 2-3 fold
increase in the number of V.beta.8+cells over that observed with
SEB alone. The daily injection of anti-CTLA-4 FAb fragements also
gave sizable increases in the number of V.beta.8+cells detected at
60 hours. The fact that both intact anti-CTLA-4 and its monovalent
FAb fragment produced the same result suggest that under these
conditions both forms of the antibody were blocking CTLA-4/B7
interactions. Further, the observation that an increase in
V.beta.8+cells was observed under these conditions is consistent
with the notion that the antibodies block an inhibitory signal.
[0157] Kinetic Analysis of SEB Responsive Populations. A kinetic
analysis was performed to address whether CD28 and CTLA-4 affect
the magnitude of the response or its timing. An antibody dose of
200 .mu.g/injection was utilized, as this dose was in the range
required for saturation of CD28, as determined by flow cytometry.
The response to SEB and control antibodies was as expected; the
expansion phase peaked at day 3, followed by a steady decline. In
contrast, mice treated with anti-CD28 and SEB showed only minimal
expansion with the peak at 72 hours being less than a third of
control levels. However, these cells appear to have undergone an
expansion and the cell numbers decay over the subsequent seven
days.
[0158] Mice receiving SEB and anti-CTLA-4 mAbs showed increased
cell numbers relative to control antibody treated animals
throughout the time course of the experiment. The number of cells
increased dramatically over the first three days with rapidly
decreasing cell numbers reaching levels similar to control/SEB
injected animals by day 10. At the peak of the response, CTLA-4
treated animals had approximately twice as many V.beta.8+T cells
relative to control antibody treated animals. Finally, to address
whether CTLA-4 or CD28 present a dominant signal, both antibodies
were added simultaneously. Throughout the time course, this
treatment produced results identical to those obtained with animals
treated with anti-CD28 alone.
[0159] B7/CD28/CTLA-4 Interactions Are Important for Regulating the
SEB Response In Vitro. The data presented here suggests an
important role for costimulatory signals in the response of murine
T cells to the superantigen SEB. Endogenous interactions of
B7-1/B7-2 with CD28 are important for promoting proliferation since
blocking with either anti-B7-1/2 antibodies or anti-CD28 FAb
fragments drastically reduced SEB-induced proliferation. In
contrast, engagement of CD28 by intact anti-CD28 antibodies
increases proliferation above the threshold provided by APC. This
increase is probably due to microaggregation or FcR-mediated
aggregation of anti-CD28 antibodies leading to efficient
crosslinking of CD28.
[0160] In contrast to CD28, CTLA-4 interactions with B7 molecules
dampens the T cell response to SEB. The observation that
anti-CTLA-4 FAb fragments enhance proliferation indicates that
CTLA-4/B7 interactions inhibit proliferative response of T cells to
SEB. Further, anti-B7-1/2 antibodies augment proliferation in the
presence of optimal stimulation with CD28 antibodies, providing
additional support for the notion that the inhibitory signals are
mediated through CTLA-4-B7 interactions.
[0161] CD28 and CTLA-4 Have Opposing Effects on the SEB Induced
Expansion of T cells In vivo. Manipulation of costimulation in SEB
treated mice by directly interfering with signals transduced
through CD28 or CTLA-4 have opposite effects on the expansion of
the V.beta.8+T cells. This result supports previous in vitro data
which suggests that these molecules might compete to determine the
proliferative outcome in the presence of a fixed level of TCR
signal. There appears to be a requirement for CD28 signals for
optimum responses to SEB; blocking with anti-CD28 FAb fragments or
intact anti-CD28 antibodies effectively diminishes the
proliferative expansion. The observation that CTLA-4 blockade
similarly allows increased expansion of responsive cells further
supports a similarity in costimulation requirements for
superantigen and peptide antigen responses in vivo. Further, the
kinetic analysis implies that competition between CD28 and CTLA-4
for B7-molecules determines a very early parameter of the T cell
response; in this experiment a CTLA-4-dependent change in expansion
occurred within the first two days. While it is clear that CTLA-4
blockade increases the response to SEB when CD28 engagement is
allowed, it has no effect upon the residual proliferation when CD28
is blocked.
[0162] The data demonstrate that CTLA-4 plays a role in dampening
the response to SEB by opposing the effects of CD28. Although this
may represent a mechanism for T cell tolerance, the inhibition may
also be involved in altering phenotype. For example, signals
generated by B7/CTLA-4 signals could induce memory cells or
alternative lymphokine expression and effector function.
EXAMPLE 7
[0163] Kinetic analysis of the effects of CTLA-4 ligation on
proliferation, IL-2 production, cell death, cell cycle progression,
and the appearance of T cell activation markers.
[0164] Materials and Methods
[0165] Antibodies and Reagents: Antibodies used for activation
were: anti-CD3 hybridoma 500A2 (Allison et al. (1987) in The T Cell
Receptor, UCLA Symposia on Molecular and Cellular Biology, New
Series. Alan R. Liss, Inc., New York. 33-45), anti-CD28 hybridoma
37.N.51.1 (Gross et al, supra.), anti-CTLA-4 hybridoma 9H10.11G3
(Krummel et al., supra.), and anti-Va3 hybridoma 536 (Havran et al.
(1989) P.N.A.S. 86:4185-4189). CTLA-4Ig is described in Lane et al.
(1994) Immunol. 80:56-61). APC and CD8 depletion was achieved using
anti-Class II MHC hybridomas 28-16-8s (Ozato and Sachs (1981) J.
Immunol. 126:317-323) and BP107 (Symington and Sprent (1981)
Immunogenetics 14:53-61), and anti-CD8 antibodies hybridoma 3.155
(Sarmiento et al. (1980) J. Immunol. 125:2665-2672). Sulfate
polystyrene latex microspheres of 5 .mu.M.+-.0.1 .mu.M mean
diameter were obtained from Interfacial Dynamics Corp. (Portland,
Or.).
[0166] Preparation of CD4+ T Lymphocytes: Lymph node cells were
isolated from 6-8 week old BALB/c mice obtained from NCI (Bethesda,
Md.). Isolated lymphocytes were obtained by mincing of tissue and
filtration of the resulting suspension through nytex. Enriched CD4+
T cell preparations were obtained by treatment with complement,
anti-Class II antibodies, and anti-CD8 antibodies. Typical
preparations were 95% CD4+ with less than 0.75% B220 positive
cells.
[0167] Activation of CD4+ T cells Using Immobilized anti-CD3: Round
bottom 96 well plates were coated with anti-CD3 at 0.1 .mu.g/ml in
50 .mu.l volumes for 2 hours at 37.degree. C., then washed
extensively and blocked for 30 minutes at 37.degree. C. with
complete RPMI-1640 (containing 10% FCS, 50 .mu.M
.beta.-mercaptoethanol, 2 mM glutamine, and 50 .mu.g/ml
gentamycin). T cells were added at 1.times.10.sup.5 per well in 200
.mu.l of complete RPMI-1640 and all cultures were incubated at
37.degree. C. in 5% CO.sub.2. Where indicated anti-CD28 was added
at 10 .mu.g/ml, CTLA-4Ig was added at 5 .mu.g/ml, and control or
anti-CTLA-4 FAb fragments were added at 50 .mu.g/ml. Twelve hours
prior to harvest, wells were pulsed with 20 .mu.l of complete RPMI
containing 1 .mu.Ci of .sup.3H thymidine. Plates were harvested to
glass filter mats and .sup.3H incorporation was measured using a
gas-phase counter (Packard, Meriden, Conn.).
[0168] Activation of T cells Using Latex Microspheres: Latex
microspheres (beads) were coated as described in Krummel et al.
(1995). Briefly, 1.times.10.sup.7 beads/ml were suspended in PBS
with the indicated antibodies and incubated for 1.5 hr at
37.degree. C., followed by washing with PBS and blocking with 10%
FCS. Anti-CD3 was added at 0.5 .mu.g/ml, anti-CD28 was added at 1
.mu.g/ml, anti-CTLA-4 was added at 4 .mu.g/ml, and binding
solutions were normalized with control antibody 536 to maintain a
constant total antibody concentration of 6 .mu.g/ml during binding.
T cells (1.times.10.sup.5/200 .mu.l) were cultured with
1.times.10.sup.5 beads in a total volume of 200 .mu.l/well. Round
bottom 96 well plates were used for all assays. Cultures were
incubated at 37.degree. C. in 5% CO.sub.2 and pulsed with 1 .mu.Ci
of .sup.3H-thymidine for the final 12 hours prior to harvesting.
The inhibitory action of CTLA-4 appears specific to anti-CTLA-4
antibodies as other T cell binding antibodies including anti-L
selectin (Mel-14), anti-Thy1.2 and irrelevant antibodies show
either no effect or augmentatory effects when co-immobilized with
anti-CD3 and anti-CD28.
[0169] Analysis of Cell Viability: T cells were cultured
identically as for proliferation assays. Cell viability was
assessed by the addition of one tenth volume of 0.4% trypan blue
(Sigma, St. Louis, Mo.) and cell numbers determined using a
hemocytometer. 10.sup.-4 ml of each culture was counted from
duplicate wells and the value for this volume was multiplied by 2
to obtain a value for the percent of input (50.times.10.sup.4
cells/ml was input). Standard deviations were always less than ten
percent.
[0170] Cell Cycle Analysis: Propidium iodide analysis of cell cycle
status was performed as previously described (Telford et al. (1992)
Cytometry 13:137-143). Briefly, cells were activated as described
using microspheres in 96 well plates. At the indicated times, three
identical wells (3.times.10.sup.5 input at the beginning of culture
per sample) were harvested, washed in PBS, and fixed with 1.0 ml of
80% ethanol. Cells were incubated on ice for 30 minutes, pelleted
by centrifugation and resuspended in 0.4 ml of an aqueous solution
containing 0.1% Triton X-100, 0.1 mM EDTA, 0.05 mg/ml RNase A
(50U/mg), and 50 .mu.g/ml propidium iodide. Samples were stored on
ice in the dark until analysis and each sample was analyzed at a
constant flow rate for two minutes. Data was analyzed using a
Coulter EPICS system.
[0171] IL-2 Determination An ELISA was utilized to detect IL-2 in
cell supernatants. Briefly, capture antibodies were coated at 1
ug/ml onto Corning (Corning, N.Y.) ELISA plates in Borate Buffer
(0.2 M Na Borate pH 8.0) for 2 hours at 37.degree. C. These plates
were then washed extensively, blocked with 0.4% Gelatin/PBS for 30
minutes and T cell culture supernatants (50 .mu.l) were added and
incubated for 2 hours at 37.degree. C. Plates were again washed and
biotinylated detection antibodies were added in PBS/0.5% Tween and
incubated for 1 hour at 37.degree. C. Plates were again washed and
a 50 .mu.l of a solution of 1 .mu.g/ml Streptavidin-HRPO in
PBS/Tween was added and incubated for 30 minutes at 37.degree. C.
50 .mu.l of developing reagent (0.55 mg/ml ABTS
(2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) in citrate
buffer (0.1M citric acid pH 4.35) was added, incubated at
25.degree. C. for 15 minutes, and absorbance at 405 nm determined.
Recombinant IL-2 was obtained from Boerringer Mannheim and was
diluted in series to develop a standard curve. Triplicate
absorbance values of test samples were thereby converted to
lymphokine quantities measured in nanograms per milliliter.
Antibodies (capture: JES6-1A12 and detection: biotinylated
JES6-5H4) were obtained from PharMingen (San Diego, Calif.).
[0172] Analysis of CD25 and CD69 Expression. 2.times.10.sup.5 cells
were suspended in 50 .mu.l ice-cold PBS/1% Calf Serum/0.05% Sodium
Azide. Anti-CD25.FITC, anti-CD69, or control RatIgG FITC antibodies
were added, incubated on ice for 30 minutes followed by two 4 ml
washes in PBS/Calf Serum/NaAzide. 5,000 live gated events were
acquired on a Becton-Dickinson FACScan and the LYSIS II program was
used to analyze relevant populations.
[0173] Results
[0174] CTLA-4 Engagement Inhibits Proliferation and IL-2
Production. It was previously shown that soluble antibodies to
CTLA-4 or B7 increased thymidine incorporation and IL-2 production
by T cells activated by immobilized anti-CD3 and anti-CD28 in
standard three day assays. These results indicated that blockade of
CTLA-4/B7 interactions between the T cells themselves augmented
responses by removing inhibitory signals. Since the cultures were
assayed at a single time point it was not possible to determine
when in the course of the cultures the effect occurred. A kinetic
analysis of the results of CTLA-4/B7 blockade on the proliferation
of purified CD4+ T cells is presented in FIG. 8A. Inclusion of
either CTLA-4Ig or Fab fragments of anti-CTLA-4 to cultures
stimulated with anti-CD3 and anti-CD28 resulted in an increase in
proliferation. The effect was slight at 26 hours, at which time
there was only marginal proliferation in any of the cultures. At
later time points CTLA-4/B7 blockade resulted in a 11/2 to 2 fold
increase in proliferation. The enhancing effect of this blockade
was even more apparent at the level of IL-2 production. As shown in
FIG. 8B, IL-2 was detectable, although at low levels, in
anti-CD3/CD28 stimulated cultures by 26 hours. The addition of
either anti-CTLA-4 Fab or CTLA-4Ig resulted in an increase of about
six fold in the amount of IL-2 accumulated by 26 hours, and nearly
ten fold by 40 hours.
[0175] The kinetics of the inhibition of proliferation and IL-2
production were examined by crosslinking CTLA-4 together with CD3
and CD28 using antibody coated microspheres. The kinetics of
thymidine incorporation are shown in FIG. 1C. Significant
incorporation was detectable by 26 hours in cultures stimulated by
anti-CD3 and anti-CD28. There was essentially no incorporation
detectable at 26 hours when CTLA-4 was also engaged, and
proliferation was 3-4 fold lower in these cultures throughout the
assay period. As shown in FIG. 8D, an even more pronounced
inhibition of IL-2 production was observed. IL-2 was readily
detectable in anti-CD3/CD28 stimulated cultures by 16 hours, and
increased up to 40 hours. When CTLA-4 was also engaged, IL-2 was
only barely detectable even after 30 hours, and reached a level of
only about 1/5 of that in the control cultures at its peak at 42
hours.
[0176] These results indicate that the inhibitory effects of
CTLA-4, whether mediated by its natural ligand or by antibody
crosslinking, can be detected early in the course of activation and
are not due to precipitous termination of responses at later stages
in the process.
[0177] CTLA-4 Engagement Does Not Induce Cell Death, But Prevents
Cell Cycle Progression. One mechanism which could account for the
inhibition of proliferation by CTLA-4 would be the induction or
enhancement of cell death. Since the inhibition was detectable
throughout the culture period, the kinetics of cell death occurring
in T cell cultures was assessed. Hematocytometric counting of cells
stained with the vital dye trypan blue showed that the total
recovery of cells from the cultures was essentially 100% of input,
even in those in which proliferation did not occur. In unstimulated
cultures, the number of non-viable cells increases over the culture
period, reaching 50% after 54 hours. There was a slight increase in
the number of dead cells recovered from cultures stimulated with
anti-CD3 alone, especially at the earlier time points. Consistent
with the proliferation data, cultures costimulated with anti-CD28
yielded an increase in viable cells after 42 hours, with a total
yield of over 300% at 78 hours. Stimulation with anti-CD3 plus
anti-CTLA-4 did not result in an increase in dead cells over that
observed in unstimulated cultures or in cultures stimulated with
anti-CD3 alone. There was also no increase in recovery of dead
cells from cultures stimulated with anti-CTLA-4 in the presence of
anti-CD3 and anti-CD28 over that of cultures stimulated by anti-CD3
and anti-CD28. Throughout the culture period the recovery of viable
cells was in fact higher than that from unstimulated cultures or
cultures stimulated with anti-CD3 alone. These data indicate that
crosslinking of CTLA-4 does not induce cell death as detectable at
the level of membrane permeability.
[0178] As a more direct and sensitive measure of cell death and
cell cycle status, propidium iodide staining of permeabilized cells
was used to measure DNA content at various stages in the cultures.
Each culture was started with identical numbers of cells, and equal
fractions of the cultures were analyzed in order to allow a
comparison of the absolute number of recovered cells in the G0/G1,
S/G2, and sub-diploid populations. The results are presented in
FIG. 9. Total cell recovery was essentially 100% of input or higher
under all stimulation conditions. Greater than 99% of input cells
were in G.sub.0/G.sub.1. In unstimulated cultures, the number of
cells with sub diploid amounts of DNA indicative of apoptosis
increased to slightly greater than 50% of the total over the course
of the culture period. A similar pattern was observed in cultures
stimulated with anti-CD3 alone, although slightly higher numbers of
cells in S/G.sub.2 were obtained. In cultures costimulated with
anti-CD28, there was a significant increase in the number of cells
in S/G.sub.2 as early as 20 hours, and this number increased
progressively over the assay period. The DNA profiles of cells
stimulated with anti-CD3 together with anti-CTLA-4 were essentially
the same as unstimulated or anti-CD3 stimulated cultures throughout
the assay period with no significant differences in the number of
apoptotic cells. However, there were significantly fewer cells in
S/G.sub.2 in cultures stimulated with anti-CD3 plus anti-CTLA-4
relative to stimulation with anti-CD3 alone. Cultures stimulated
with anti-CTLA-4 and anti-CD3 plus anti-CD28 and had similar
numbers or even fewer cells in the sub diploid population than any
of the other conditions throughout the culture period. Thus there
is no evidence of induction of apoptotic cell death by anti-CTLA-4
crosslinking at any time during the course of activation. The main
effect of crosslinking CTLA-4 on cells stimulated with anti-CD3 and
anti-CD28 is an inhibition of the increase in total viable cells,
especially those in S/G.sub.2. Together, these results indicate
that CTLA-4 engagement inhibits cell cycle progression, and an
arrest of cells in G.sub.0/G.sub.1.
[0179] CTLA-4 Engagement Partially Inhibits Induction of IL-2
Receptor Alpha Chain Expression. Another hallmark of T cell
activation is upregulation of expression of CD25, the IL-2 receptor
alpha chain. Flow cytometry was used to assess the expression of
CD25 on T cells under conditions of CD28 costimulation with and
without concomitant CTLA-4 ligation. Stimulation of T cells with
anti-CD3 alone resulted in the induction of expression of CD25 on
about 60% of T cells within 24 hours. Costimulation with anti-CD28
increased this expression with respect to both the number of
positive cells and the level of expression at 24 hours, and the
expression was further enhanced at 60 hours of culture. When CTLA-4
was also engaged, CD25 expression was expressed by a smaller
fraction of the cells (47% vs. 80%) and the mean level of
expression was much lower at 24 hours (mean fluorescence index 162
vs. 194) and at 60 hours (MFI 332 vs. 669) relative to cultures
costimulated with anti-CD28. This data demonstrate that CTLA-4
engagement inhibits the upregulation of CD25 throughout
activation.
[0180] CTLA-4 Engagement Partially Inhibits Expression of the Early
Activation Marker CD69. CD69 is a early and transient marker of T
cell activation. A kinetic analysis of the effects of CD28 and
CTLA-4 engagement on induction of CD69 expression was performed. At
12 hours, CD69 was expressed by greater than 50% of T cells
activated with CD3 alone or costimulated with anti-CD28, while
fewer than 15% of costimulated cells also subjected to CTLA-4
ligation were positive. At 24 hours, CD69 expression was
detectable, albeit in a heterogeneous pattern, on greater than 75%
of CD28 costimulated cells. At this point fewer than 45% of cells
from cultures in which CTLA-4 had also been engaged expressed CD69
and the level of expression was reduced. By 36 hours, CD69
expression had returned to essentially resting levels in all the
cultures. CD28 costimulation augments and prolongs CD69 expression,
whereas CTLA-4 ligation inhibits the initial upregulation of CD69.
This result is consistent with the observation that CD69 levels
were found to be constitutively elevated on T cells isolated from
CTLA-4 deficient mice and provides additional evidence suggesting a
role for CTLA-4 in preventing the early induction of T cell
activation.
[0181] These data demonstrate that CTLA-4 mediates inhibition of
proliferation and IL-2 production by resting T cells in the absence
of CTLA-4 mediated cell death. The recovery of viable and
non-viable cells from anti-CTLA-4 inhibited cultures is similar to
that observed in control antibody or anti-CD3 stimulated cultures.
There is no accumulation of cells with sub-diploid quantities of
DNA associated with apoptotic cell death even 1-2 days after
inhibitory effects of CTLA-4 crosslinking are first observed at the
level of proliferation and IL-2 production. Finally, CTLA-4
crosslinking arrests T cells in a G0/G1 phase of the cell cycle.
Taken together, these data clearly demonstrate that inhibition of T
cell proliferation and IL-2 secretion by CTLA-4 can occur in the
absence of cell death. An important implication of the data
presented here is that CTLA-4 may have a role in regulating T cell
responses at early stages in the process. Our data do not reveal a
precipitous termination of ongoing responses, but rather an
inhibition and delay of events associated with the progression of T
cell activation.
[0182] The above results demonstrate that the subject treatment
with CTLA-4 blocking agents increases the response of T cells to
antigenic stimulation. The growth of tumor cells in vivo is greatly
diminished in the presence of the the subject blocking agents. The
effects are observed against unmanipulated, wild-type tumors.
CTLA-4 blocking agents not only represent a novel approach to tumor
therapy, but, by removing potentially competing inhibitory signals,
may be a particularly useful adjunct to other therapeutic
approaches involving the co-stimulatory pathway. Class switching by
immunoglobulin producing cells, a measure of T cell help, is
greatly increased. The T cell response to immunization with peptide
antigens is also greatly increased by the treatment with the
subject agents.
EXAMPLE 8
Effectiveness Against Established Tumor
[0183] SA1 is a fibrosarcoma. As shown in FIG. 10 the CTLA-4
blockade using 10.degree. .mu.g of anti-CTLA-4 antibody per dose is
effective even when delayed 7 or 14 days after tumor implantation.
This indicates that CTLA-4 blockade can be effective in the
treatment of established tumors.
EXAMPLE 9
Synergy with Immune Response Stimulating Agent
[0184] SM1 is a mammary carcinoma that is poorly immunogenic. It is
resistant to rejection by transfection with B7. However, some
inhibition of growth using B7 and IFNg has been obtained. In the
experiment shown in FIG. 11, mice received s.c. implants of
unmodified SM1 tumor cells, and the indicated treatments on days 0,
3 and 6. As shown, treatment with anti-CTLA-4 (10.degree.
.mu.g/dose) by itself had no effect on growth of the tumor.
Immunization at a contralateral site with irradiated, GM-CSF
transduced cells also had no effect. However, the combination of
the two resulted in complete rejection in 4 of 5 mice. This clearly
demonstrates that CTLA-4 blockade can synergize with GM-CSF, and
probably other lymphokines, to obtain tumor rejection.
EXAMPLE 10
Delayed CTLA-4 Blockage
[0185] RENCA is a slow growing, poorly immunogenic tumor. As shown
in FIG. 12, the CTLA-4 blockade (100 .mu.g anti-CTLA-4 antibody per
dose) is only poorly effective when initiated at the time of tumor
implantation. However, it is quite effective if initiated 9 days
after tumor implantation. This suggests that generation of tumor
debris from a relatively large tumor mass is important as an agent
to stimulate an immune response to obtain effective rejection. This
suggests that CTLA-4 blockade could be used at the time of, or
shortly after, irradiation or chemotherapy.
EXAMPLE 11
CTLA-4 Blockade Enhances Immunogenicity of Tumor Fragments
[0186] B16 is a very poorly immunogenic melanoma which is resistant
to rejection induced by B7 expression. We have explored ways of
attacking it by CTLA-4 blockade. In the experiment shown in FIG.
13, mice received s.c. implants of unmodified tumor cells and the
indicated treatments at days 0, 3 and 6. CTLA-4 blockade by itself
(100 .mu.g 9H10/dose) had no effect, nor did immunization with
irradiated B 16 cells at a contralateral site. However, treatment
with both showed a small, but significant and reproducible
inhibition of tumor growth, although no cures were obtained.
[0187] This approach was also used in a protective immunization
setting. In the experiment shown in FIG. 14, mice were immunized
with irradiated B16 cells with and without CTLA-4 blockade (100
.mu.g 9H10/dose) and with and without cytokine-containing gelatin
microspheres (containing 50 ng .gamma. interferon and 50 ng
GM-CSF). The mice were rechallenged with live, unmodified tumor
cells two weeks later. Mice immunized with irradiated cells with
CTLA-4 blockade showed significantly impaired tumor growth compared
to mice receiving irradiated cells alone. The best protective
effect was obtained with cytokine-containing microspheres together
with CTLA-4 blockade.
[0188] Together, these data indicated that CTLA-4 blockade can
enhance immunization strategies employing active immunization with
modified tumor cells or tumor fragments, and that it can have a
synergistic effect with cytokines.
[0189] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0190] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
Sequence CWU 1
1
2 1 35 DNA Artificial Synthetic 1 ttactctact ccctgaggag ctcagcacat
ttgcc 35 2 35 DNA Artificial Synthetic 2 tatacttacc agaatccggg
catggttctg gatca 35
* * * * *