U.S. patent application number 09/954166 was filed with the patent office on 2002-09-12 for soluble divalent and multivalent heterodimeric analogs of proteins.
Invention is credited to O'Herrin, Sean, Schneck, Jonathan.
Application Number | 20020127231 09/954166 |
Document ID | / |
Family ID | 21765064 |
Filed Date | 2002-09-12 |
United States Patent
Application |
20020127231 |
Kind Code |
A1 |
Schneck, Jonathan ; et
al. |
September 12, 2002 |
Soluble divalent and multivalent heterodimeric analogs of
proteins
Abstract
Specificity in immune responses is in part controlled by the
selective interaction of T cell receptors with their cognate
ligands, peptide/MHC molecules. The discriminating nature of this
interaction makes these molecules, in soluble form, good candidates
for selectively regulating immune responses. Attempts to exploit
soluble analogs of these proteins has been hampered by the
intrinsic low avidity of these molecules for their ligands. To
increase the avidity of soluble analogs for their cognates to
biologically relevant levels, divalent peptide/MHC complexes or T
cell receptors (superdimers) were constructed. Using a recombinant
DNA strategy, DNA encoding either the MHC class II/peptide or TCR
heterodimers was ligated to DNA coding for murine Ig heavy and
light chains. These constructs were subsequently expressed in a
baculovirus expression system. Enzyme-linked immunosorbant assays
(ELISA) specific for the Ig and polymorphic determinants of either
the TCR or MHC fraction of the molecule indicated that infected
insect cells secreted approximately 1 .mu.g/ml of soluble,
conformnationally intact chimeric superdimers. SDS PAGE gel
analysis of purified protein showed that expected molecular weight
species. The results of flow cytometry demonstrated that the TCR
and class II chimeras bound specifically with high avidity to cells
bearing their cognate receptors. These superdimers will be useful
for studying TCR/MHC interactions, lymphocyte tracking, identifying
new antigens, and have possible uses as specific regulators of
immune responses.
Inventors: |
Schneck, Jonathan; (Silver
Spring, MD) ; O'Herrin, Sean; (Baltimore,
MD) |
Correspondence
Address: |
BANNER & WITCOFF
1001 G STREET N W
SUITE 1100
WASHINGTON
DC
20001
US
|
Family ID: |
21765064 |
Appl. No.: |
09/954166 |
Filed: |
September 18, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09954166 |
Sep 18, 2001 |
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09324782 |
Jun 3, 1999 |
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09324782 |
Jun 3, 1999 |
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08828712 |
Mar 28, 1997 |
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6015884 |
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60014367 |
Mar 28, 1996 |
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Current U.S.
Class: |
424/178.1 ;
514/1.2; 514/19.3 |
Current CPC
Class: |
A61K 39/00 20130101;
A61K 2039/605 20130101; A61K 38/00 20130101; A61P 37/00 20180101;
C07K 2319/00 20130101; C07K 14/7051 20130101; C07K 14/70539
20130101; A61K 2039/6056 20130101; C07K 2319/30 20130101; C12N
2799/026 20130101 |
Class at
Publication: |
424/178.1 ;
514/12 |
International
Class: |
A61K 039/395; A61K
038/17 |
Claims
We Claim:
1. A soluble recombinant divalent protein composition comprising
the extracellular domains of a heterodimeric protein operatively
linked to immunoglobulin heavy and light chain polypeptides.
2. The recombinant protein composition of claim 1, wherein said
immunoglobulin is selected from the group consisting of IgM, IgD,
IgG3, IgG1, IgG2b, IgG2a, IgE, and IgA.
3. The recombinant protein composition of claim 1, wherein said
extracellular domains of the heterodimeric protein minimally
contain a binding site involved in immune recognition.
4. The recombinant protein composition of claim 1 further
comprising a linker domain between said extracellular domains of
the heterodimeric protein and said immunoglobulin polypeptides.
5. The recombinant protein composition of claim 3, wherein said
heterodimeric protein comprises an a polypeptide chain and .beta.
polypeptide chain.
6. The recombinant protein composition of claim 3, wherein said
heterodimeric protein comprises a .gamma. polypeptide chain and
.delta. polypeptide chain.
7. The recombinant protein composition of claim 5 wherein said
heterodimeric protein is an MHC class II molecule.
8. The recombinant protein composition of claim 5 wherein said
heterodimeric protein is a TcR molecule.
9. The recombinant protein composition of claim 7 additionally
comprising an antigenic peptide in the peptide binding groove of
said MHC class II molecule.
10. A soluble recombinant multivalent protein composition
comprising the extracellular domains of a heterodimeric protein
operatively linked to immunoglobulin heavy and light chain
polypeptides.
11. The recombinant protein composition of claim 10, wherein said
immunoglobulin is selected from the group consisting of IgM, IgD,
IgG3, IgG1, IgG2b, IgG2a, IgE, and IgA.
12. The recombinant protein composition of claim 10, wherein said
extracellular domains of the heterodimeric protein minimally
contain a binding site involved in immune recognition.
13. The recombinant protein composition of claim 10 further
comprising a linker domain between said extracellular domains of a
divalent heterodimeric protein and said immunoglobulin
polypeptides.
14. The recombinant protein composition of claim 10, wherein said
heterodimeric protein comprises an .alpha. polypeptide chain and
.beta. polypeptide chain.
15. The recombinant protein composition of claim I0, wherein said
heterodimeric protein comprises a .gamma. polypeptide chain and
.delta. polypeptide chain.
16. The recombinant protein composition of claim 14 wherein said
heterodimeric protein is an MHC class II molecule.
17. The recombinant protein composition of claim 14 wherein said
heterodimeric protein is a TcR molecule.
18. The recombinant protein composition of claim 16 additionally
comprising an antigenic peptide in the peptide binding groove of
said MHC class II molecule.
19. A method for producing an expression vector encoding the
recombinant protein composition according to any one of claims 1-8
and 10-17, comprising modifying an expression vector for
immunoglobulin heavy and light chains by inserting DNA sequences
which code for the extracellular domains of a heterodimeric
integral membrane protein such that fusion proteins are produced
which comprise at the amino terminus one of said extracellular
domains operatively linked to said immunoglobulin heavy or light
chain.
20. The method of claim 19 wherein said heavy and light chain
fusion proteins are encoded on separate expression vectors.
21. An expression vector comprising DNA sequences encoding a
soluble heterodimeric protein each operatively linked to a DNA
sequence encoding either an immunoglobulin heavy or light chain
polypeptide, respectively.
22. An expression vector comprising a DNA sequence encoding a
soluble analog of one member of a heterodimeric protein operatively
linked to a DNA sequence encoding either an immunoglobulin heavy or
light chain polypeptide.
23. The expression vector of claim 21 further comprising linker
domains between said DNA sequences encoding said soluble
heterodimeric protein and said respective DNA sequences encoding
said immunoglobulin heavy and light chain polypeptides.
24. The expression vector of claim 22 further comprising a linker
domain between said DNA sequence encoding one member of a soluble
heterodimeric protein and said DNA sequence encoding said
immunoglobulin heavy or light chain polypeptide.
25. A host cell comprising any one of the vectors of claims
21-24.
26. A host cell comprising two vectors according to either claims
22 or 24, wherein one of said two vectors comprises a DNA sequence
encoding one member of said heterodimeric protein operatively
linked to a DNA sequence encoding an immunoglobulin heavy chain
polypeptide, and the other of said two vectors comprises a DNA
sequence encoding the other member of said heterodimeric protein
operatively linked to a DNA sequence encoding an immunoglobulin
light chain polypeptide.
27. A pharmaceutical composition comprising the soluble recombinant
divalent protein composition according to any one of claims 1-9 in
a pharmaceutically-acceptable carrier.
28. A pharmaceutical composition comprising a soluble recombinant
multivalent protein composition according to any one of claims
10-18 in a pharmaceutically-acceptable carrier.
29. A method for selectively inhibiting or decreasing an immune
response, comprising administering to a patient an effective amount
of a soluble recombinant divalent protein composition according to
any one of claims 1-9 in a pharmaceutically-acceptable carrier such
that said immune response is inhibited or decreased.
30. A method according to claim 29 wherein said immune response is
directed to a foreign transplantation antigen.
31. A method according to claim 29 wherein said immune response
results in an autoimmune disease.
32. A method for inhibiting or decreasing an immune response,
comprising administering to a patient an effective amount of a
soluble recombinant multivalent protein composition according to
any one of claims 10-18 in a pharmaceutically-acceptable carrier
such that said immune response is inhibited or decreased.
33. A method according to claim 32 wherein said immune response is
directed to a foreign transplantation antigen.
34. A method according to claim 32 wherein said immune response
results in an autoimmune disease.
35. A method for stimulating an antigen-specific T-cell response
comprising immobilizing a soluble recombinant divalent protein
composition according to claim 9 on a substrate and exposing said
immobilized protein composition to a population of T cells such
that an antigen-specific T cell response is stimulated.
36. A method for stimulating an antigen-specific T-cell response
comprising immobilizing a soluble recombinant multivalent protein
composition according to claim 18 on a substrate and exposing said
immobilized protein composition to a population of T cells such
that an antigen-specific T cell response is stimulated.
37. A method according to claim 35 or 36 wherein said method is
used for the identification and purification of specific T cell
subsets.
38. A method for identifying and purifying an unknown peptide/MHC
complex comprising immobilizing a soluble recombinant divalent
protein composition according to claim 8 on a substrate and
exposing said immobilized protein composition to a population of
peptide/MHC complexes such that a particular peptide/MHCcomplex is
identified.
39. A method for identifying and purifying an unknown peptide/MHC
complex comprising immobilizing a soluble recombinant multivalent
protein composition according to claim 17 on a substrate and
exposing said immobilized protein composition to a population of
peptide/MHC complexes such that a particular peptide/MHCcomplex is
identified.
40. A method according to claim 38 or 39 wherein said peptide is a
tumor or viral antigen.
41. A soluble recombinant multivalent protein composition
comprising the extracellular domains encoding a binding site
operatively linked to both immunoglobulin heavy and light chain
polypeptides.
42. The protein composition of claim 42 wherein said binding site
is encoded by a polypeptide, a carbohydrate or a glycoprotein.
Description
[0001] This application is a continuation-in-part of Provisional
Application Ser. No. 60/014,367 which was filed Mar. 28, 1996.
TECHNICAL FIELD
[0002] This invention is directed to compositions comprising
soluble divalent and multivalent heterodimeric analogs of proteins
that are involved in immune regulation and methods of making and
using the same. The high affinity that these complexes have for
their cognate ligands enables them to be effective competitors to T
cell receptors and MHC molecules normally involved in transplant
rejection and autoimmune disease. Molecules such as divalent T cell
receptors may also have an impact on the diagnosis and treatment of
cancer in that they may be used to augment antitumor responses, or
may be conjugated to toxins which may then be used to help
eliminate tumors. Use of such compositions will allow one to
accomplish selective immune modulation without compromising the
general performance of the immune system.
BACKGROUND OF THE INVENTION
[0003] The process of signal transduction often involves proteins
that have extracellular domains, transmembrane domains, and
intracellular domains. During ligand binding there is often
oligomerization of receptor molecules in order to transmit
effectively the signal to the intracellular component of the cell.
The immune system is an excellent example of a signal transduction
pathway that works by these methods (Rosen et al. J. Med. Chem.
38:48-55).
[0004] The immune system is a defense system found in most advanced
forms of higher vertebrates. A properly functioning lymphatic and
immune system distinguishes between self and nonself. A healthy
body protects against foreign invaders, such as bacteria, viruses,
fungi, and parasites. As the body encounters foreign material
(nonself), also known as an antigens, the immune system becomes
activated. An antigen is recognized by characteristic shapes or
epitopes on its surface. This defense mechanism provides a means of
rapid and highly specific responses that are used to protect an
organism against invasion by pathogenic microorganisms. It is the
myriad of pathogenic microorganisms that have principally caused
the evolution of the immune system to its current form. In addition
to protection against infectious agents, specific immune responses
are thought to be involved in surveillance against alterations in
self antigens as seen in tumor development. Immune responses are
also involved in the development of autoimmune disease, AIDS, as
well as rejection of transplanted tissues.
[0005] Lymphocytes
[0006] Within the immune system, lymphocytes play a central role.
Lymphocyte responses to foreign organisms orchestrate the effector
limbs of the immune system, and ultimately, determine the fate of
an infection. Lymphocytes can be divided into two main categories,
B and T cells. These two types of lymphocytes are specialized in
that they have different effector functions and play different
roles in the development of specific immune responses. Individual
lymphocytes are specialized in that they are committed to respond
to a limited set of structurally related antigens. Specificity is
conferred by an unique set of cell surface receptors expressed on
individual lymphocytes. These receptors interact with soluble
proteins, in the case of B cells, and with antigenic peptide/major
histocompatibility complex (MHC) molecules in the case of T
lymphocytes. The nature of the interaction with their ligands also
differs between B and T cells. The antigen receptors produced by B
cells, immunoglobulins (Igs), interact with their ligands with a
high affinity. In contrast, T cell receptors interact with their
ligands with low affinity. Thus, the T cell response is driven by
the interaction of many T cell receptors (TcR) on the surface of an
individual T cell interacting with multiple antigenic peptide/MHC
complexes on the surface of the antigen presenting cell. Thus,
these two diverse groups of cell-surface glycoproteins, the TcRs
and the MHC glycoproteins, form key components of specificity in
the T lymphocyte response to antigens.
[0007] T cells are a major regulatory cell of the immune system.
Their regulatory functions depend not only on expression of a
unique T cell receptor, but also on expression of a variety of
accessory molecules and effector functions associated with an
individual T cell response. Effector functions include responses
such as cytotoxic responses or other responses characterized by
secretion of effector molecules, i.e., lymphokines. It is this
regulatory function that often goes awry in the development of
autoimmune diseases. The different effector functions also play a
large role in tissue graft rejection, and can be important in tumor
rejection.
[0008] T cells respond to antigens in the context of either Class I
or Class II MHC molecules. Cytotoxic T cells respond mainly against
foreign antigens in the context of Class I glycoproteins, such as
viral-infected cells, tumor antigens and transplantation antigens.
In contrast, helper T cells respond mainly against foreign antigens
in the context of Class II molecules. Both types of MHC molecules
are structurally distinct, but fold into very similar shapes. Each
MHC molecule has a deep groove into which a short peptide, or
protein fragment, can bind. Because this peptide is not part of the
MHC molecule itself, it varies from one MHC molecule to the next.
It is the presence of foreign peptides displayed in the MHC groove
that engages clonotypic T cell receptors on individual T cells,
causing them to respond to foreign antigens.
[0009] Antigen-specific recognition by T cells is based on the
ability of clonotypic T cell receptor to discriminate between
various antigenic-peptides resident in MHC molecules. These
receptors have a dual specificity for both antigen and MHC
(Zinkernagel et al. Nature 248: 701-702 (1974)). Thus, T cells are
both antigen-specific and MHC-restricted. A simple molecular
interpretation of MHC-restricted recognition by T cells is that
TcRs recognize MHC residues as well as peptide residues in the
MHC-peptide complex. Independent of the exact mechanism of
recognition, the clonotypic T cell receptor is the molecule that is
both necessary and sufficient to discriminate between the multitude
of peptides resident in MHC.
[0010] T cells can be divided into two broad subsets; those
expressing .alpha./.beta. TcR and a second set that expresses
.gamma./.delta. TcR. Cells expressing .alpha./.beta. TcR have been
extensively studied and are known to comprise most of the
antigen-specific T cells that can recognize antigenic peptide/MHC
complexes encountered in viral infections, autoimmune responses,
allograft rejection and tumor-specific immune responses. Cells
expressing .alpha./.beta. TcRs can be further divided into cells
that express CD8 accessory molecules and cells that express CD4
accessory molecules. While there is no intrinsic difference between
the clonotypic .alpha./.beta. T cell receptors expressed either on
CD4 and CD8 positive cells, the accessory molecules largely
correlate with the ability of T cells to respond to different
classes of MHC molecules. Class I MHC molecules are recognized by
CD8+, or cytotoxic, T cells and class II MHC molecules by CD4+, or
helper, T cells.
[0011] .gamma./.delta. T cells make up another significant
population of T cells seen in circulation as well as in specific
tissues. These cells are not well understood; their antigen/MHC
specificity is poorly defined and in most cases their ligands are
completely unknown. These cells are present in high quantities in
certain tissues, including skin and gut epithelium, and are thought
to play a significant role in immune responses of those organs.
They have also been implicated in autoimmune responses and may be
involved in the recognition of heat shock proteins. A general
approach to the identification of antigenic complexes, as outlined
in the present invention, would greatly facilitate understanding of
how these cells influence the development of both normal and
abnormal immune responses. There is a large degree of homology
between both .alpha./.beta. and .gamma./.delta. TcR expressed in
rodents and humans. This extensive homology has, in general,
permitted one to develop murine experimental models from which
results and implications may be extrapolated to the relevant human
counterpart.
[0012] MHC Molecules in Health and Disease
[0013] Major histocompatibility antigens consist of a family of
antigens encoded by a complex of genes called the major
histocompatibility complex. In mice, MHC antigens are called H-2
antigens (Histocompatibility-2 antigens). In humans MHC antigens
are called HLA antigens (Human-Leukocyte-associated Antigens). The
loci that code for MHC glycoproteins are polymorphic. This means
that each species has several different alleles at each locus. For
example, although a large number of different Class I antigens may
be seen in a species as a whole, any individual inherits only a
single allele from each parent at each locus, and therefore
expresses at most two different forms of each Class I antigen.
[0014] In the murine system, the class II MHC molecules are encoded
by I-A and I-E loci, and in humans, class II molecules are encoded
by the DR, DP and DQ loci. Polymorphism of class II alleles is
attributed to the alpha and beta chains and specificities are
designated using the nomenclature set forth by the World Health
Organization (Immunogenetics (1992) 36:135).
[0015] The Role of MHC Molecules- Transplantation
[0016] MHC molecules play an essential role in determining the fate
of grafts. Various species display major immunological functional
properties associated with the MHC including, but not limited to,
vigorous rejection of tissue grafts, stimulation of antibody
production, stimulation of the mixed lymphocyte reaction (MLR),
graft-versus-host reactions (GVH), cell-mediated lympholysis (CML),
immune response genes, and restriction of immune responses.
Transplant rejection occurs when skin, organs (e.g., kidney, liver,
lung), or other tissues (e.g., blood, bone marrow) are transplanted
across an MHC incompatibility. A vigorous graft rejection occurs
when the immune system is activated by mismatched transplantation
antigens that are present in donor tissue but not in recipient.
Graft rejection may occur in the graft itself by exposure of
circulating immune cells to foreign antigens, or it may occur in
draining lymph nodes due to the accumulation of trapped
transplantation antigens or graft cells. Because of the extensive
diversity of MHC antigens, numerous specificities are possible
during physiological and pathophysiologic immune-related
activities, (e.g., transplantation, viral infections, and tumor
development). The recognized HLA specificities are depicted, for
example, in a review by Bodmer et al. (In: Dupont B. (Ed.)
Immunobiology of HLA (Volume I) New York: Springer-Verlag
(1989)).
[0017] The Role of MHC Molecules- Autoimmune Response
[0018] Susceptibility to many autoimmune disease shows a
significant genetic component and familial linkage. Most genetic
linkages of autoimmune diseases are with certain class II MHC
alleles (see Table 1 for Overview). The level of association
between a particular disease and an allele at one of the MHC loci
is defined by a term called "relative risk". This term reflects the
frequency of the disease in individuals who have the antigen
compared to the frequency of the disease among individuals who lack
the antigens. For example, there is a strong association with
DQ.beta. genotype in insulin-dependent diabetes mellitus; the
normal DQ.beta. sequence has an aspartic acid at position 57,
whereas in Caucasoid populations, patients with diabetes most often
have valine, serine or alanine at that position.
1TABLE 1 Associations of HLA genotype with susceptibility to
autoimmune disease Disease HLA allele Relative risk Goodpasture's
syndrome DR2 15.9 Multiple Sclerosis DR2 4.8 Graves' disease DR3
3.7 Myasthenia gravis DR3 2.5 Systemic lupus DR3 5.8 erythematosus
Insulin-dependent diabetes DR3 and DR4 3.2 mellisis Rheumatoid
arthritis DR4 4.2 Pemphigus vulgaris DR4 14.4 Addison's disease DR3
8.8 Dermatitis herpetiformis DR3 13.5 Celiac disease DR3 73.0
Hashimoto's thyroiditis DR5 3.2
[0019] Regulation of Immune Reponses
[0020] Interest in analyzing both normal and abnormal T
cell-mediated immune responses led to the development of a series
of novel soluble analogs of T cell receptors and MHC molecules to
probe and regulate specific T cell responses. The development of
these reagents was complicated by several facts. First, T cell
receptors interact with peptide/MHC complexes with relatively low
affinities (Matsui et al Science 254:1788-1891 (1991) Sykulev et al
Immunity 1:15-22 (1994) Corr et al Science 265:946-949 (1994)). In
order to specifically regulate immune responses, soluble molecules
with high affinities/avidities for either T cell receptors or
peptide/MHC complexes are needed. However, simply making soluble
monovalent analogs of either T cell receptors or peptide/MHC
complexes has not proven to be effective at regulating immune
responses with the required specificity and avidity.
[0021] To regulate immune responses selectively, investigators have
made soluble versions of proteins involved in immune responses.
Soluble divalent analogs of proteins involved in regulating immune
responses with single transmembrane domains have been generated by
several laboratories. Initially, CD4/Ig chimeras were generated
(Capon et al Nature 337:525-531 (1989); Bryn et al Nature
344:667-670 (1990)), as well as CR2/Ig chimeras (Hebell et al
Science 254:102-105 (1991)). Later it was demonstrated that immune
responses could be modified using specific CTLA-4/Ig chimeras
(Linsley et al Science 257:7920-795 (1992); U.S. Pat. No.
5,434,131; Lenschow et al Science 257:789-791 (1992)). In addition,
class I MHC/Ig chimeras were used to modify in vitro allogeneic
responses (Dal Porto, supra). However, these examples include only
soluble divalent analogs of single transmembrane polypeptide
molecules and not chimeric molecules of heterodimeric proteins in
which the heterodimer consists of .alpha. and .beta. polypeptides
that are both transmembrane polypeptides. The present invention
reports the generation of soluble divalent and multivalent
heterodimeric analogs of integral membrane protein complexes, which
consist of alpha and beta polymorphic integral membrane
polypeptides that properly fold to form a functional unit that has
potential use in immune modulation.
[0022] Previously, replacement of two transmembrane domains in the
generation of multivalent analogs has not been achieved. The
challenge of generating these molecules lies in achieving the
proper folding and expression of two polypeptides, both of which
ordinarily require transmembrane domains (FIG. 1). In addition,
soluble multivalent analogs of heterodimeric proteins generally
have increased affinity and, therefore, are preferred therapeutic
agents. These soluble protein complexes, which consist of .alpha.
and .beta. polymorphic integral membrane polypeptides that properly
fold to form a functional unit, have potential use as immune
modulating agents.
SUMMARY OF THE INVENTION
[0023] It is one objective of this invention to provide soluble
recombinant divalent and multivalent analogs of heterodimeric
proteins, which are capable of specifically binding target
molecules to regulate immune responses.
[0024] It is another object of this invention to provide soluble
recombinant divalent heterodimeric proteins that possess enhanced
affinity for their target molecules.
[0025] It is still another object of this invention to claim a
method for producing an expression vector encoding soluble divalent
analogs of heterodimeric integral membrane proteins. This comprises
modifying an expression vector for an immunoglobulin molecule by
inserting at least two DNA sequences, such as an .alpha.
polypeptide fused to an immunoglobulin heavy chain and a .beta.
polypeptide fused to an immunoglobulin light chain (FIG. 2). This
could also be done by inserting at least two DNA sequences, such as
a .beta. polypeptide fused to an immunoglobulin heavy chain and an
a polypeptide fused to an immunoglobulin light chain. The .alpha.
and .beta. polypeptides are ones that encode a binding or
recognition site. It is also possible for the fusion proteins of
the present invention to be encoded by two compatible expression
vectors.
[0026] A host cell containing the vector or vectors, which is
capable of expressing soluble divalent heterodimeric proteins
containing an .alpha. and .beta. polypeptide subunit is also an
object of this invention.
[0027] Also included in this invention is a method for inhibiting
or decreasing immune responses. Specifically, antigen-specific
interactions between T cells and cells presenting antigens may be
inhibited using the soluble divalent analogs of either the TcR or
class II MHC molecules. An example of this could be suppression of
an autoimmnune response as seen in Myasthenia gravis, multiple
sclerosis, arthritis, and allergic diseases. Adhesion of cells
mediated through the interactions of integrins can also be
inhibited using soluble divalent analogs of integrin molecules.
Inhibition of cytokine-mediated cell stimulation is also included,
in that soluble divalent versions of cytokine receptors could bind
to soluble cytokines, thereby inhibiting the ability of the
cytokines to mediate cellular proliferation.
[0028] Also included in this invention is a method for augmenting
immune responses. Specifically, antigen-specific interactions
between T cells and cells presenting antigens may be augmented
using the soluble divalent analogs of either the TcR or class II
MHC molecules immobilized on a substrate to stimulate
antigen-specific T cell responses. Such a system may also be used,
in the case of immobilized MHC/Ig molecules presenting antigenic
peptides, to identify and purify specific T cell subsets, i.e. for
the identification of the clonotypic TcR. Along the same lines,
immobilized TcR/Ig may be used to identify and purify unknown
peptide/MHC complexes which may be involved in cancer or infectious
diseases such as AIDS. Stimulation of cells via adhesion receptors
can also be accomplished using soluble divalent analogs of integrin
molecules that have been immobilized on a solid substrate, such as
a tissue culture plate or bead.
[0029] The invention further includes a method for treating
diseases by administering soluble recombinant divalent
heterodimeric analogs of proteins whereby the .alpha. and .beta.
polypeptides form a unit, and whereby the claimed constructs
selectively increase or decrease cellular activation,
proliferation, anergy, or deletion of specific T cell subsets. Such
diseases include autoimmune disorders, transplant rejection, cancer
and AIDS. Since divalent and multivalent complexes of the present
invention will have increased affinity for their respective
targets, administration of such compounds should selectively
suppress or block T cell recognition of specific transplantation
antigens and self antigens by binding to the designated target
molecule and inhibiting cell-to-cell interaction.
[0030] It is also possible using techniques known in the art to
conjugate toxin molecules, such as ricin and pseudomonas exotoxin,
to the compounds of the present invention. The invention also
includes methods of treating cancer and AIDS with such conjugate
molecules. For example, following the identification of virus- or
tumor-specific peptides displayed on the MHC molecules of
viral-infected or tumor cells, toxin-conjugated soluble
heterodimeric TCR molecules may be designed that bind to and
destroy cells harboring the HIV virus, or cancerous cells,
respectively. In addition, soluble divalent or multivalent MHC/IG
molecules displaying tumor- or AIDS-related peptides might have
potential use in immunization protocols.
[0031] Accordingly, also included in the invention are methods of
identifying unknown antigens or peptides derived using soluble
divalent TcR. A distinct advantage of soluble high affinity TCR/Ig
chimeras is that even in the absence of any a priori knowledge
about their ligands, they may be useful in defining the specific
peptide/MHC ligands recognized by uncharacterized tumor-specific T
cells and T cells involved in autoimmune responses. Not only are
soluble divalent TCR/IG molecules efficient probes for the
quantitative detection of specific peptide/MHC complexes, but due
to their strong affinity for the target molecule, they will
consequently play an important role in the purification of such
complexes and facilitate their characterization.
[0032] Soluble divalent heterodimeric analogs of integral membrane
proteins of this invention provide significant benefits because
these recombinant proteins possess enhanced binding affinities for
modulating immune responses. High affinity divalent ligands, such
as the divalent chimeric molecules of this invention, can be used
to selectively modulate specific T cell responses and to study
cell-cell interactions that are driven by multivalent
ligand-receptor interactions.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1A. A typical configuration of a heterodimeric double
transmembrane protein.
[0034] FIG. 1B. Heterodimeric transmembrane protein made divalent
and soluble by covalent linkage of outer-membrane region to
antibody.
[0035] FIG. 1C. Outer-membrane region of MHC class II covalently
linked to an antibody.
[0036] FIG. 1D. A schematic of the chimeric protein showing the TcR
.alpha. polypeptide (shaded) linked to IgG1 heavy chain and TcR
.beta. polypeptide (shaded) linked to Ig kappa light chain is
shown. The linkers between the chimeric chains consist of short
glycine/serine spacers. Presumptive binding sites of two monoclonal
antibodies (mAb), H57 (TcR specific) and 1B2 (2C TcR specific), on
the putative 2C TcR/Ig structure are also noted.
[0037] FIG. 2. Map of the expression vector, which encodes soluble
divalent heterodimeric proteins. Multi-step construction schematic
is shown to depict fusion of .alpha. and .beta. polypeptide subunit
linked to Ig heavy and light chains to form the chimeric
Immunoglobulin molecules.
[0038] FIG. 3. Detail of the DNA sequences introduced into the
plasmid construct.
[0039] FIG. 4. Schematic of TcR/MHC interactions.
[0040] FIGS. 5A-5C. Detection of soluble heterodimeric proteins by
ELISA assays.
[0041] FIG. 6. SDS-PAGE analysis of I-E.sup.k/Ig and 2C TcR/Ig
chimeric proteins.
[0042] FIG. 7. Graph showing that the affinity of soluble divalent
2C TcR/Ig for peptide/H-2 L.sup.d complexes is higher than that of
soluble monovalent 2C TcR is depicted in FIG. 11. RMA S-L.sup.d
cells were loaded with peptides (QL9; p2Ca; or pMCMV) and
subsequently incubated with a constant amount of FITC labeled
30.5.7 Fab and varying concentrations of either 2C TcR/Ig (solid
lines) or soluble monovalent 2C TcR, sm2C TcR (dashed line) Binding
of FITC-30.5.7 Fab was determined by flow cytometry. Plotted as the
% maximal (no 2C TcR Analog) 30.5.7 binding vs. the concentration
of 2C TcR analog. Apparent affinities were determined from a replot
of 1/(% maximal 30.5.7 binding) vs [TcR Analog] see text and Table
II for further discussion. Data shown are from one representative
experiment that has been repeated at least three times. Each data
point is the average of duplicates.
[0043] FIG. 8. Cells were then harvested as described above and
processed for flow cytometry analysis. Cells were stained with
either purified mAb, 30.5.7 (Panels A-D), or 2C TcR/Ig culture
supernatants (Panels E-H) diluted to 20-40 .mu.g/ml final
concentration. In each panel the histogram of treated cells (solid
line) is contrasted with that of cells not treated with any peptide
and cultured for one hour at 37.degree. C. (broken line).
Histograms shown are from one representative experiment that has
been repeated at least three times.
[0044] FIG. 9. A comparison of 2C TcR/Ig reactivity versus mAb
30.5.7 reactivity in peptide-stabilized H-2L.sup.d molecules. RMA-S
L.sup.d cells were incubated under various conditions. Following
overnight incubation of RMA-S L.sup.d cells at 27.degree. C., cells
were cultured in the presence or absence of various H-2 L.sup.d
binding peptides: no peptide cells maintained at 27.degree. C.,
(Panel A and E); tum, (Panel B and F); p2Ca, (Panel C and G); and
QL9, (Panel D and H), were added to cultures as described in
section.
[0045] FIG. 10. Graph demonstrating inhibition of in vitro 2C T
cell mediated lysis by soluble 2C TcR/Ig superdimers.
[0046] FIG. 11. Fluorescence data showing that soluble divalent 2C
TcR/Ig interacts with SIY/MHC complexes but not with dEV-8/MHC
complexes is depicted in FIG. 11. T2 cells transfected with either
H-2 K.sup.b, H-2 K.sup.bm3, or H-2 K.sup.bm11 were incubated
overnight at 27.degree. C. and loaded with peptides dEV-8 (-----),
SIY (QQ), or pVSV (aaaaa) as described below. Cells were stained
with purified 2C TcR/Ig (.about.50 mg/ml) and GAM-IgG-RPE as
described in Methods, and analyzed by FACS. Resultant histograms
are shown; Panel A, T2-Kb cells; B, T2-K.sup.bm3; C, T2-K.sup.bm11.
In the histograms presented 2C TcR/Ig reactivity with either dEV-8
(-----) or pVSV (aaaaa) was virtually identical leading to
difficulty in discriminating between these two histograms.
[0047] FIG. 12. 2C CTL mediated lysis on various peptide/MHC
targets is depicted in this figure. T2 cells transfected with
either H-2 L.sup.d (Panel A), H-2 K.sup.b (Panel B), or H-2
K.sup.bm3 (Panel C), were chromium labeled as described and then
loaded with peptides by incubating at 25 {C. for 1.5 hrs. in the
presence of variable amounts of peptides: p2Ca ( ) and pMCMV ( )
(Panel A); and dEV-8 ( ); SIY ( ); or pVSV ( ) (Panels B and C).
Peptide loaded target cells were then incubated at an effector to
target ratio of 10:1 and specific lysis calculated as described
below. Data shown are representative of three separate
experiments.
[0048] FIG. 13. Fluorescence data showing modulation of endogenous
2C specific peptide/H-2 L.sup.d complexes on the surface of RENCA
cells by .gamma.-IFN is depicted in FIG. 13. RENCA cells were
cultured for 48 hrs. with 0 (panels A & E), 5 (panels B &
F), 10 (panels C & G), or 50 (panels D & H), units/ml
.gamma.-IFN. As described in the results, .gamma.-IFN is known to
have a direct effect on class I expression, making it necessary to
establish background binding of 2C TcR/Ig to .gamma.-IFN treated
cells. This was accomplished by incubating RENCA cells with
saturating amounts of the H-2 L.sup.d binding peptide, MCMV. which
efficiently displaced the endogenous H-2 L.sup.d bound peptides,
including any 2C-reactive peptides. Cells were harvested, stained
with 2C TcR/Ig (75 mg/ml), panels A-D, or the mAb 30.5.7 (45
mg/ml), panels E-H, as described below. Cells were subsequently
stained with GAM-IgG-RPE and analyzed by FACS. Resultant histograms
are shown. Solid lines (Q) represent histograms of cultures with no
added peptide while dotted lines (aaa) represent histograms from
cultures incubated with pMCMV. All experiments were done in
duplicate and repeated at least three times. Note the differences
in the extents of fluorescence (see the scales on the histograms)
upon staining with 2C-TcR/Ig vs. staining with 30.5.7.
DETAILED DESCRIPTION OF THE INVENTION
[0049] Soluble recombinant divalent and multivalent analogs of
heterodimeric proteins were generated that specifically bind to
target molecules that regulate activities of the immune system. The
construction and expression of soluble recombinant divalent and
multivalent analogs of heterodimeric proteins involved linking
polypeptide sequences from the heterodimeric proteins to
immunoglobulin heavy and light chains. Specifically, soluble
recombinant divalent and multivalent analogs of heterodimeric
proteins link a polypeptide chain of a heterodimeric transmembrane
protein to an immunoglobulin heavy chain and a second polypeptide
chain of a heterodimeric transmembrane protein to an immunoglobulin
light chain. These soluble hybrid constructs contain two or more
binding sites for the same ligand. "Polypeptide" refers to any
polypeptide of a heterodimeric protein. "Polypeptide" may refer to
a protein in its entirety, or to a portion thereof. Selected
polypeptide sequences will minimally contain any binding site
involved in a specific immune response for regulation, including
regions of the protein required for proper folding and conformation
of the binding site or any other region necessary for the function
of the molecule. "Binding site" refers to the domain or sequence of
amino acids from the protein of interest that mediates interaction
or association with the ligand or target cell. The binding site may
be formed from a nonconsecutive sequence of amino acids that
associate in a tertiary conformation. A binding site may also be
found within the extracellular domains of a glycoprotein. A
glycoprotein is a protein that contains at least one carbohydrate
group.
[0050] Polypeptide sequences contain about 5 amino acid sequences
to about 1000 amino acid sequences. Preferably, the polypeptide
sequences contain 200 amino acid sequences or less. Mammalian
polypeptides are preferred, and more preferably, human polypeptides
from transmembrane proteins. DNA, RNA, and amino acid sequences
which have slight and non-consequential sequence variations from
the actual sequences containing more two or more binding sites for
the same ligand are within the scope of the present invention.
Conventional abbreviations for amino acids, peptides and their
derivatives are used as generally accepted in the peptide art and
as recommended by the IUPAC-IUB Commission on Biochemical
Nomenclature (European J. Biochem (1984) 138:9-37). "Slight and
non-consequential" sequence variations mean that the homologous
sequences will function in substantially the same manner to produce
substantially the same proteins and polypeptides of the present
invention. Functionally equivalent polypeptides are also
encompassed within this invention. Conservative substitutions may
be made in such amino acid sequences without losing biological or
chemical functionality.
[0051] As used herein the term "soluble" means that the composition
of interest is sufficiently soluble at 37.degree. C. or in bodily
fluids, plasma, etc. such that it may be used at the specified
range of concentrations required to enable the composition to serve
its intended function according to the present invention.
[0052] "Divalent" means that the naturally occurring or genetically
engineered chimeric protein or polypeptide of interest that has two
binding sites for the same ligand. This is in contrast to
bifunctional in which a chimeric protein has two binding sites for
different ligands on the same polypeptide. Thus all immunoglobulins
are both bifunctional and also minimally divalent. There are
bifunctional in that they all have at least one binding site for
antigen and a separate site for Fc-receptor binding.
Immunoglobulins are also minimally divalent in that they have at
least two identical but separate binding sites for antigen.
[0053] "Multivalent" means that the naturally occurring or
genetically engineered chimeric proteins or polypeptides of
interest have more than two binding sites for the same ligand. For
example, "multivalent" would encompass IgM and IgA chimeric
molecules according to the present invention, which are pentavalent
and tetravalent, respectively. In addition, "multivalent" might
indicate a composition having more than one chimeric antibody
molecule. Since each divalent heterodimeric IgG molecule has two
binding sites (divalent), a chimeric antibody complex containing
four IgG molecules would have eight antigen binding sites
(octavalent). Similar multivalent antibody complexes that are
non-chimeric have been constructed using methods known in the art.
For instance, Sano and Cantor disclose a method for making a
multivalent antibody U.S. Pat. No. 5,328,985 using
streptavidin-proteinA, which has four or more IgG binding sites per
molecule. The number of antibody molecules per conjugate molecule
is controlled by mixing the streptavidin-Protein A and antibody of
interest at an appropriate ratio. Other methods of conjugating
antibodies known in the art could also be used to form soluble
multivalent chimeric compositions according to the present
invention.
[0054] "Linker" refers to the linker region inserted between the
immunoglobulin molecules and the heterodimeric polypeptides. The
length of the linker sequence will vary depending upon the desired
flexibility to regulate the degree of antigen binding and
cross-linking. The "linker" should be considered to be an optional
feature. Constructs may be designed such that the heterodimeric
polypeptides are directly and covalently attached to the
immunoglobulin molecules without an additional linker region. If a
linker region is included, this region will preferably contain at
least 3 and not more than 30 amino acids. More preferably, the
linker is about 5 and not more than 20 amino acids and most
preferably, the linker is less than 10 amino acids. Generally, the
linker consists of short glycine/serine spacers, but any known
amino acid may be used.
[0055] "lmmunoglobulin(s) or Ig(s)" means a group of proteins that
are products of antibody secreting cells. Igs are constructed of
one, or several, units, each of which consists of two heavy (H)
polypeptide chains and two light (L) polypeptide chains. Each unit
possesses two combining sites for antigen. The H and L chains are
made up of a series of domains. The L chains, of which there are
two major types ( .kappa. and .lambda.), consists of two domains.
The H chains of Ig molecules are of several types, including .mu.,
.delta., and .gamma.(of which there are several subclasses),
.alpha. and .epsilon.. There are eight genetically and structurally
identified Ig classes and subclasses as defined by heavy chain
isotypes: IgM, IgD, IgG3, IgG1 , IgG2b, IgG2a, IgE, and IgA.
Further, for example, "IgG" means an immunoglobulin of the G class,
and that, "IgGI" refers to an IgG molecules of subclass 1 of the G
class. "Fab" and "F(ab').sub.2" are fragments of Ig molecules that
can be produced by proteolytic digestion of an intact Ig molecule.
Digestion of an IgG molecule with papain will produce two Fab
fragments and an Fc fragment and digestion with pepsin will produce
an F(ab').sub.2 fragment and subfragments of the Fc portion.
[0056] The "transplantation antigens" referred to in the present
invention are molecules responsible for graft recognition and
rejection. Since the immunological status of the recipient is a
critical factor affecting graft survival, diverse antigen systems
may be involved in the acceptance/rejection process. These not only
include the well recognized HLA system, such as class I and class
II MHC molecules, but also include other minor histocompatibility
antigens, such as the ABO blood group system, (including
carbohydrates, which includes but is not limited to, disaccharides,
trisaccharides, tetrasaccharides, pentasaccharides,
oligosaccharides, polysaccharides, and more preferably, the
carbohydrate .alpha.(1,3) Galactosyl epitope [.alpha.(1,3) Gal]),
autoantigens on T and B cells, and monocyte/endothelial cell
antigens. Since the present invention is primarily concerned with
divalent and multivalent heterodimeric compounds comprising two
subunit molecules, each generally known in the native state to
possess a transmembrane domain, transplantation antigens in the
context of the present invention include MHC class II antigens. In
clinical applications concerning treatment or therapy to inhibit or
reduce graft rejection, selective suppressing antigen soecific
responses are targeted. A transplantation antigen may be any class
I or class II MHC molecule, or more specifically for humans, any
MHC molecules including HLA specificities such as A (e.g. A1-A74),
B (e.g., B1-B77), C (e.g., C1-C11), D (e.g., D1-D26), DR (e.g.,
DR1-DR8), DQ (e.g., DQ1-DQ9) and DP (e.g. DP1-DP6). More
preferably, HLA specificities include A1, A2, A3, A11, A23, A24,
A28, A30, A33, B7, B8, B35, B44, B53, B 60, B62, DR1, DR2, DR3,
DR4, DR7, DR8, and DR11 (Zachary et al., Transplant. 62: 272-283).
In clinical applications concerning the therapy of autoimmune
disease, a transplantation antigen is any MHC class II molecule
associated or linked with the disease of interest. Such
transplantation antigens particularly include any D and DR allele,
but DQ and DP alleles that are shown to be associated with
autoimmune disease are also encompassed. Therapeutic applications
involve the specific suppression of transplantation antigens using
soluble proteins (also referred to as "specific antigen
suppressors") of the present invention. In particular, one
therapeutic application involves specific suppression of preformed
anti-carbohydrate antibody responses using specific antigen
suppressors.
[0057] "Heterodimeric" means that the protein of interest is
comprised of two separate polypeptide chains. In this description
we will consider only those polypeptide chains that have
transmembrane and intracellular domains. Different classes of
heterodimeric transmembrane proteins, which contain .alpha. and
.beta. polymorphic integral membrane polypeptides that bind each
other forming a functional unit involved in immune recognition,
include, but are not limited to, proteins such as T cell receptors,
and class II MHC molecules, integrins (e.g., including more than 20
cell surface heterodimers), and cytokine receptors (e.g., IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, erythropoietin (EPO), leukemia
inhibitor factor (LIF), G-CSF, Oncostatin M, ciliary neurotrophic
factor (CNTF), growth hormone, and prolactin).
[0058] It is also possible for the compositions of the present
invention to be prepared such that both the heavier and light
immunoglobulin chains are fused to the same extracellular domain
(i.e. the extracellular domain from a class I MHC molecule or
glycoprotein). Protein expression and folding would then result in
a chimeric homotetrameric composition comprising two light chains
and two heavy chains, all fused to the same polypeptide.
[0059] "Integrin" refers to a class of proteins defined as having
adhesive properties and known to be involved in mediating adhesion
between both like and different cells. These molecules are also
heterodimeric transmembrane proteins consisting of .alpha. and
.beta. polypeptides.
[0060] "Superdimer" refers to dimers of heterodimeric proteins.
This term has been coined to describe what may be the conformation
of MHC molecules on the surface of antigen presenting cell. In this
application this term will be used to describe only soluble
"superdimers", such as the soluble divalent or multivalent versions
of either class II MHC or TcR molecules.
[0061] "Cytokine" refers to proteins that affect the behavior of
other cells. Cytokines made by lymphocytes are often called
lymphokines or interleukins, but the generic term "cytokine" is
used most often in the literature. Cytokines act on specific
"cytokine receptors" on the cells they affect. Cytokine receptors
also belong to a family of molecules in which at least two
component polypeptides are transmembrane spanning proteins. This
system is central in the growth and regulation of many cell types
including cells of the immune system. Cytokines/cytokine receptors
include the following examples, but are not limited to this
listing: I) hematopoietin family (e.g., erythropoietin(Epo)/EpoR;
IL-2(T-cell growth factor)/CD25, CD122; IL-3 (multicolony
CSF)/CD123; IL-4 (BCGF-1, BSF-1)/CD124; IL-5 (BCGF-2)/CD125; IL-6
(INF-.beta..sub.2, BSF-2, BCDF)/CD126, Cdw130; IL-7/CDw127;
IL-9/IL-9R; IL-11/IL-11R, Cdwl130; IL-13 (P600)/IL-13R; IL-15
(T-cell growth factor)/IL-15R; GM-CSF (granulocyte macrophage
colony stimulating family)/CDw116; OSM (OM, oncostatin M)/OMR,
CDw130; LIF (leukemia inhibitory factor)/LIFR, Cdw130); II)
Interferon Family (e.g., IFN-.gamma./CD119; INF-.alpha./CD118;
INF-.beta./CD118); III) Immunoglobulin Superfamily (e.g., B7.1
(CD80)/CD28; CTLA-4; B7.2/CD28, CTLA-4); IV) TNF Family (e.g.,
TNF-.alpha.(cachectin)/p55, p75, CD120a, CD120b;
TNF-.beta.(lymphotoxin, LT, LT-.alpha.)/p55, p75, CD120a, CD120b),
LT-.beta.), CD40 ligand (CD40-L)/CD40; Fas ligand/CD95 (Fas); CD27
ligand/CD27; CD30 ligand/CD-30; 4-1BBL/4-1BB; V) Chemokine Family
(e.g., IL-8 (NAP-1)/CDw128; MP-1 (MCAP); MIP-1.alpha.; MIP-1.beta.;
RANTES); and VI) others (TFG-.beta.; IL-1.alpha.; IL-1.beta.; IL-10
(cytokine synthesis inhibitor F); IL-12 (natural killer cell
stimulatory factor); and MIF).
[0062] DNA constructs encoding the chimeric compounds of the
present invention generally comprise sequences coding for the
signal sequence and extracellular domain of one polypeptide of the
heterodimeric complex (i.e. TCR.alpha. or .beta., or MHC class II
.alpha. or .beta.) fused to the first amino acid of either the
heavy or light chain immunoglobulin variable region sequence. Such
a DNA construct results in the expression and secretion of a
protein comprising the extracellular portion of the polypeptide of
interest at the N terminus (transmembrane regions are not included)
spliced to the intact variable region of the immunoglobulin
molecule (see FIG. 1). Variations or truncations of this general
structure in which one or more amino acids are inserted or deleted
but which retain the ability to bind to the target ligand are
encompassed in the present invention.
[0063] Standard Cloning Methods:The techniques for cloning and
expressing DNA sequences encoding the amino acid sequences
corresponding to binding sites of divalent heterodimeric analogs of
integral membrane proteins, such as TcR and MUC molecules, soluble
fusion proteins and hybrid fusion proteins consisting of an .alpha.
and .beta. polypeptide subunit, e.g., synthesis of
oligonucleotides, PCR, transforming cells, constructing vectors,
expression systems, and the like are well-established in the art,
and skilled artisans are familiar with the standard resource
materials for specific conditions and procedures.
[0064] In general, various expression systems are well known in the
art. Prokaryotes are useful for cloning variant DNA sequences. For
example, E. coli strain SR101 (Messing et al Nucl Acids Res
9(2):309-321 (1981), E. coli K12 strain 294 (ATTC No. 31446), E.
Coli B, UM101, and E. coli .sub..chi.1776 (ATTC No. 31537) are
particularly useful. Constructs are inserted for expression into
vectors containing promoters and control sequences, which are
derived from species compatible with the intended host cell. The
vector ordinarily, but not necessarily, carries a replication site
as well as one or more marker sequences, which are capable of
providing phenotypic selection in transformed cells. For example,
E. coli is typically transformed using a derivative of pBR322,
which is a plasmid derived from an E. coli species (Bolivar et al
Gene 2:95 (1977). pBR322 contains genes for ampicillin and
tetracycline resistance and thus provides easy means for
identifying transformed cells. The pBR322 plasmid, or other
microbial plasmid must also contain or be modified to contain
promoters and other control elements commonly used in recombinant
DNA constructions.
[0065] Promoters suitable for use with prokaryotic hosts include,
but are not limited to, the beta-lactamase and lactose promoter
systems (Chang et al Nature 275:615 (1978); Goeddel et al Nature
281:544 (1979), alkaline phosphatase, the tryptophan (trp) promoter
system (Goeddel et al Nucl Acid Res 8:4057 (1980), and hybrid
promoters, such as the tac promoter (de Boer et al Proc Natl Acad
Sci USA 80:21-25 (1983). Other functional bacterial promoters are
suitable. Nucleotide sequences called linkers or adaptors are
generally known which enable the skilled artisan to operably ligate
DNA sequences of interest (Siebenlist et al Cell 20:269 (1980)).
Promoters for use in a bacterial system will also contain a
Shine-Dalgarno sequence.
[0066] In addition to prokaryotes, eukaryotic microbes, such as
yeast cultures, are useful as cloning or expression hosts. In
particular, Saccharomyces cerevisiae, or common baker's yeast, is
commonly used (although other strains are commonly available). For
expression in Sarraromyces, the plasmid YRp7, for example, is
commonly used (Stinchcomb et al Nature 282:39 (1979). This plasmid
already contains the trp1 gene, which provides a selection marker
for a mutant strain of yeast lacking the ability to grow in
tryptophan (ATTC No. 44076). The presence of the trp1 lesion as a
characertistic of the yeast host cell genome then provides an
effective means of selection by growth in the absence of
tryptophan. Suitable promoting sequences for use with yeast hosts
include the promoters for 3-phosphoglycerate kinase or other
glycolytic enzymes, such as enolase, hexokinase, pyruvate kinase,
and glucokinase. Other yeast promoters, which are inducible
promoters having the additional advantage of transcription
controlled by growth conditions, are the promoter regions for
alcohol dehydrogenase 2, acid phosphatase, metallothionenin, for
example. Suitable vectors and promoters for use in yeast expression
are further described in R. Hitzeman et al European Patent
Publication No. 73,657A.
[0067] Promoters for controlling transcription from vectors in
mammalian host cells may be obtained from various sources, for
example, the genomes of viruses, such as polyoma, Simian Virus 40
(SV40), adenovirus, retroviruses, hepatitis B virus, and most
preferably cytomegalovirus (CMV), or from heterologous mammalian
promoters, e.g., the beta actin promoter. The early and late
promoters of the SV40 virus are conveniently obtained as an SV40
restriction fragment, which also contains the SV40 viral origin of
replication (Fiers et al Nature 273:113 (1978). The immediate early
promoter of the human CMV is conveniently obtained as a HindIII E
restriction fragment (Greenaway et al Gene 18:355-360 (1982).
[0068] DNA transcription in higher eukaryotes is increased by
inserting an enhancer sequence into the vector. Enhancers are
cis-acting elements of DNA, usually from 10 to 300 bp, that act to
increase the transcription initiation capability of a promoter.
Enhancers are relatively orientation and position independent
having been found 5' and 3' to the transcription unit within an
intron as well as within the coding sequence itself. Many enhancer
sequences are now known from mammalian genes (globin, elastase,
albumin, and insulin, for example). Typically, however, one will
use an enhancer from a eukaryotic cell virus. Examples include the
SV40 enhancer on the late side of the replication origin (bp
100-270), the CMV early promoter enhancer, the polyoma enhancer on
the late side of the replication origin, and adenovirus
enhancers.
[0069] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human or nucleated cells) may also
contain sequences necessary for the termination of transcription,
which may affect mRNA expression These regions are transcribed as
polyadenylated segments in the untranslated portion of the mRNA
encoding the desired sequence. The clones containing DNA encoding
soluble constructs are transfected into suitable host cells for
expression. Depending upon the host cell used, transfection is
performed using standard techniques (transfection into mammalian
cells is accomplished using DEAE-dextran mediated transfection,
CaPO.sub.4 co-precipitation, lipofection, electroporation. or
protoplast fusion, and still other procedures known in the art
including, but not limited to, lysozyme fusion or direct uptake,
osmotic or sucrose shock, direct microinjection, indirect
microinjection, and/or subjecting cells to electric currents.
[0070] Peptides, proteins, or molecules of the present invention
may be conjugated to a reporter group, including, but not limited
to, a radiolabel (e.g., .sup.32P), a fluorescent label, an enzyme,
a substrate, a solid matrix, or a carrier (e.g., biotin or avidin)
for use in the detection of specific levels of molecules or the
specific binding activity of particular molecules of the present
invention. Hybrid constructs of the present invention may be
further modified to include toxins.
[0071] The divalent and multivalent heterodimeric compounds of the
present invention may be used as immune modulating agents in
methods of regulating the immune system. For example,
immunoregulatory actions that may be activated or inhibited include
the ability to stimulate, depress or abrogate the following immune
responses: erythroid progenitor production, T-cell proliferation,
hematopoiesis production, B-cell activation, class switching (e.g.,
IgE switch), Eosinophil growth and differentiation, T-and B-cell
growth and differentiation, acute phase reaction, growth of pre-B
cells and pre-T cells, mast cell activity, IL-3 and IL-4
involvement in hematopoiesis, cytokine activation or inhibition;
differentiation of myeolomonocytic lineage; cancer cell growth and
development; macrophage activation, MHC expression, anti-viral
activity, T-cell respones, inflammation, anti-inflammation,
endothelial activation, B-cell activation, apoptosis,
calcium-independent cytotoxicity; chemotactic activity of
neutrophils, T-cells, eosinophils, and macrphages, fever, cell
(macrophage, T-cell, B-cell, neutrophils, eosinophils, natural
killer cells) functions, antigen processing, cytotoxicity, and
receptor crosslinking. In essence, the hybrid constructs of the
present invention selectively enhances, decreases, or abrogates
cellular activation, proliferation, anergy (tolerance), or deletion
of specific T-cell subsets (Hewitt et al. J Exp. Med. 175:1493
(1992); Choi et al. Proc. Natl. Acad. Sci. 86:8941 (1989); Kappler
et al. Science 244:811 (1989); Minasi et al. J Exp. Med. 177:1451
(1993); Sundstedt et al. Immunology 82:117 (1994); and White et al.
Cell 56:27 (1989).
[0072] In addition, the compounds of the present invention may also
be used in the treatment of diseases related to immune dysfunction.
Conditions which might benefit from the activation or inhibition of
immune responses include, but are not limited to, the following
disorders and diseases: autoimmune diseases, such as idiopathic
thrombocytopenia purpura, systemic lupus erythematosus, myasthenia
gravis, arthritis, autoimmune hemolysis, glomerulonephritis,
multiple sclerosis, psoriasis, juvenile diabetes, primary
idiopathic myxedema, systemic lupus erythematosus, autoimmune
asthma, scleroderma, chronic hepatitis, Addison's disease,
hypogonadism, pernicious anemia, vitiligo, alopecia areata, Coeliac
disease, autoimmune enteropathy syndrome, idiopathic thrombocytic
purpura, acquired spenic atrophy, idiopathic diabetes insipidus,
infertility due to antispermatazoan antibodies, sudden hearing
loss, sensoneural hearing loss, polymyositis, autoimmune
demyelinating diseases, traverse myelitis, ataxic sclerosis,
progressive systemic sclerosis, dermatomyositis, polyarteritis
nodosa, hemolytic anemia, glomerular nephritis, idiopathic facial
paralysis, Pemphigus vulgaris, cryoglobulinemia, and AIDS, Epstein
Barr virus associated diseases, such as Sjorgren's Syndrome,
rheumatoid arthritis, Burkitt's lymphoma, Hodgkin's disease, virus
(AIDS or EBV) associated B cell lymphoma, chronic fatigue syndrome,
parasitic diseases, such as Lesihmania and immunosuppressed disease
states, such as viral infections following allograft
transplantation or AIDS, cancers, chronic active hepatitis
diabetes, toxic shock syndrome, food poisoning, and transplant
rejection.
[0073] Since the vector constructs of the present invention
incorporate a signal sequence for the secretion of each member of
the chimeric heterodimeric molecule, it is possible that the
therapeutic methods of the present invention may also be performed
with polynucleotides or vectors designed for gene therapy. The
polynucleotide may be DNA or RNA. When the polynucleotide is DNA,
it can also be a DNA sequence which is itself non-replicating, but
is inserted into a replicating plasmid vector. The polynucleotide
may be engineered such that it is not integrated into the host cell
genome. Alternatively, the polynucleotide may be engineered for
integration into the chromosome provided the expression of the
polypeptide may be controlled. Such regulatable gene expression
systems having in vivo applicability are known in the art, and may
be used in the present invention. For example, selective killing of
transfected cells may be mediated by including in the
polynucleotide or vector a gene sequence encoding a cytotoxic
peptide such as HSV thymidine kinase (Borrelli et al. Proc. Nat.
Acad. Sci. USA 85:7572, 1988). The thymidine kinase gene acts as a
suicide gene for transfected cells if the patient is exposed to
gancyclovir. Thus, if expression of the encoded peptides of the
invention is too high, gancyclovir may be administered to reduce
the percentage of cells expressing the peptides.
[0074] The compositions of the present invention, or more
specifically different classes of heterodimeric transmembrane
proteins or polynucleotides encoding the same, which contain
.alpha. and .beta. polymorphic integral membrane polypeptides that
bind each other forming a functional unit involved in immune
recognition, may be made into pharmaceutical compositions with
appropriate pharmaceutically acceptable carriers or diluents, such
as a macromolecule, which is soluble in the circulatory system and
which is physiologically acceptable where physiological acceptance
means that those skilled in the art would accept injection of said
carrier into a patient as part of a therapeutic regime. The carrier
preferably is relatively stable in the circulatory system with an
acceptable plasma half-life for clearance. Suitable carriers
include, but are not limited to, water, alcoholic/aqueous
solutions, emulsions, or suspensions, including saline and buffered
media, and proteins such as serum albumin, heparin, immunoglobulin,
polymers such as polyethylene glycol or polyoxyethylated polyols or
proteins modified to reduce antigenicity by, for example,
derivitizing with polyethylene glycol. Suitable carriers are well
known in the art and are described, for example, in U.S. Pat. Nos.
4,745,180, 4,766,106, and 4,847,325, and references cited therein.
If appropriate, pharmaceutical compositions may be formulated into
preparations including, but not limited to, solid, semi-solid,
liquid, or gaseous forms, such as tablets, capsules, powders,
granules, ointments, solutions, suppositories, injections,
inhalants, and aerosols, in the usual ways for their respective
route of administration. Methods known in the art can be utilized
to prevent release or absorption of the composition until it
reaches the target organ or to ensure time-release of the
composition. A pharmaceutically-acceptable form should be employed
which does not ineffectuate the compositions of the present
invention. In pharmaceutical dosage forms, the compositions may be
used alone or in appropriate association, as well as in combination
with, other pharmaceutically-active compounds. For example, in
applying the method of the present invention for delivery of a
soluble constructs of the invention, or more specifically different
classes of heterodimeric transmembrane proteins, which contain
.alpha. and (.alpha./.beta. integral membrane polypeptides that
bind each other forming a functional unit involved in immune
recognition, such delivery may be employed in conjunction with
other means of treatment of infectious diseases, autoimmunity,
cancers, for example. The compounds of the present invention may be
administered alone or in combination with other diagnostic,
therapeutic or additional agents. Therapeutic agents may include
cytokines or lymphokines, such as IL-2, .alpha.-interferon and
interferon-.gamma..
[0075] Accordingly, the pharmaceutical compositions of the present
invention can be delivered via various routes and to various sites
in an animal body to achieve a particular effect. Local or system
delivery can be accomplished by administration comprising
application or instillation of the formulation into body cavities,
inhalation, or insufflation of an aerosol, or by parenteral
introduction, comprising intramuscular, intravenous, peritoneal,
subcutaneous intradermal, as well as topical administration.
[0076] The composition of the present invention can be provided in
unit dosage form, wherein each dosage unit, e.g., a teaspoon,
tablet, solution, or suppository, contains a predetermined amount
of the composition, alone or in appropriate combination with other
pharmaceutically-active agents. The term "unit dosage form" refers
to physically discrete units suitable as unitary dosages for human
and animal subjects, each unit containing a predetermined quantity
of the composition of the present invention, alone or in
combination with other active agents, calculated in an amount
sufficient to produce the desired effect, in association with a
pharmaceutically-acceptable diluent, carrier (e.g., liquid carrier
such as a saline solution, a buffer solution, or other
physiological aqueous solution), or vehicle, where appropriate. The
specifications for the novel unit dosage forms of the present
invention depend on the particular effect to be achieved and the
particular pharmacodynamics associated with the pharmaceutical
composition in the particular host.
[0077] Additionally, the present invention specifically provides a
method of administering soluble constructs of the invention to a
host, which comprises administering the composition of the present
invention using any of the aforementioned routes of administration
or alternative routes known to those skilled in the art and
appropriate for the particular application. The "effective amount"
of the composition is such as to produce the desired effect in a
host which can be monitored using several end-points known to those
skilled in the art. For example, one desired effect might comprise
effective nucleic acid transfer to a host cell. Such transfer could
be monitored in terms of a therapeutic effect, e.g., alleviation of
some symptom associated with the disease being treated, or further
evidence of the transferred gene or expression of the gene within
the host, e.g., using PCR, Northern or Southern hybridization
techniques, or transcription assays to detect the nucleic acid in
host cells, or using immunoblot analysis, antibody-mediated
detection, or particularized assays, as described in the examples,
to detect protein or polypeptide encoded by the transferred nucleic
acid, or impacted level or function due to such transfer. These
methods described are by no means all-inclusive, and further
methods to suit the specific application will be apparent to the
ordinary skilled artisan.
[0078] The particular dosages of divalent and multivalent
heterodimeric compounds employed for a particular method of
treatment will vary according to the condition being treated, the
binding affinity of the particular reagent for its target, the
extent of disease progression, etc. However, the dosage will
generally fall in the range of 1 pg/kg to 100 mg/kg of body weight
per day. Where the active ingredient of the pharmaceutical
composition is a nucleic acid, dosage will generally range from 1
nM to 50 .mu.M per kg of body weight. The amounts of each active
agent included in the compositions employed in the examples
described herein provide general guidance of the range of each
component to be utilized by the practitioner upon optimizing the
method of the present invention for practice either in vitro or in
vivo. Moreover, such ranges by no means preclude use of a higher or
lower amount of a component, as might be warranted in a particular
application. For example, the actual dose and schedule may vary
depending on whether the compositions are administered in
combination with other pharmaceutical compositions, or depending on
individual differences in pharmacokinetics, drug disposition, and
metabolism. Similarly, amounts may vary in vitro applications
depending on the particular cell line utilized, e.g., the ability
of the plasmid employed to replicate in that cell line.
Furthermore, the amount of nucleic acid to be added per cell or
treatment will likely vary with the length and stability of the
nucleic acid, as well as the nature of the sequence, and is
particularly a parameter which needs to be determined empirically,
and may be altered due to factors not inherent to the method of the
present invention. e.g., the cost associated with synthesis, for
instance. One skilled in the art can easily make any necessary
adjustments in accordance with the necessities of the particular
situation.
[0079] The following examples merely illustrate the best mode now
contemplated for practicing the invention and should not be
construed to limit the invention.
EXAMPLES
[0080] Cells and Culture Conditions: RMA-S, RMA-S L.sup.d, T2, T2
K.sup.b, T2 Kbm3, T2 K.sup.bm11, and RENCA cells were maintained by
1:10 passage three times weekly in RPMI-1640 supplemented with 2 mM
glutamine, nonessential amino acids, 50 5g/ml of gentamicin,
5.times.10-5M 2-mercaptoethanol, and 10% fetal calf serum.
[0081] Expression of soluble 2C TcR analogs: The details of
construction, expression, purification and characterization of
soluble divalent 2C TcR/Ig were carried out as described elsewhere
(O'Herrin et al manuscript in preparation). Briefly, to generate
the soluble divalent 2C TcR, cDNAs encoding the 2C TcR .alpha. and
.beta. chains were genetically linked via a six amino acid
glycine/serine spacer to cDNAs encoding IgGI heavy chains and
.kappa. light chains, respectively (see FIG. 1 for protein
schematic). Soluble monovalent 2C TcR was made and purified as
previously described (Corr et al Science 265:946-949 1994).
[0082] Peptide Loading of Cells: RMA-S and T2 cell lines are
defective in antigen processing and express functionally empty
class I MHC on their cell surface (Spies et al Nature 355:644-646
(1992); Townsend et al Nature 340:443-448 (1989). These empty MHC
molecules may be loaded with peptides as described (Catipovic et al
Journal of Experimental Medicine 176:1611-1618 (1992); Townsend et
al (1989) supra. Briefly, cells (RMA-S, RMA-S L.sup.d, T2, T2
L.sup.d, T2 Kb, T2 Kbm3 or T2 K.sup.bm11) were cultured at
27.degree. C. overnight. Subsequently, cells were incubated in the
presence or absence of various antigenic peptides (100 5M final
concentration) for an additional 1.5 hours at 27.degree. C. and
then for one hour at 37.degree. C.
[0083] RENCA cells were loaded with peptides by incubation with
peptides (100 5M final concentration) for >2 hrs. at 37.degree.
C. Cells were then harvested and processed for FACS analysis as
described.
[0084] Measurement of the affinity of soluble 2C TcR for H-2
L.sup.d molecules: Affinities of soluble 2C TcR analogs for peptide
loaded cells were determined in a competition assay with
FITC-30.5.7 Fab similar to one previously described (Schlueter et
al Journal of Molecular Biology 256:859-869 (1996). 30.5.7 is a
monoclonal antibody that recognizes an epitope near the
peptide-exposed face of H-2 L.sup.d; thus 30.5.7 and 2C TcR compete
for binding to the peptide exposed face of H-2 L.sup.d. Kd of
30.5.7 Fab for peptide-loaded RMA-S L.sup.d cells were determined
as follows. Cells (0.3.times.10.sup.6/0 ml) were loaded with
peptide as described above. Subsequently, peptide-loaded or control
cells were incubated with varying concentrations of FITC-30.5.7 Fab
for 1 hr. at 4.degree. C., and then diluted 1:6 with FACS wash
buffer (PBS, 1% FCS, 0.02% NaN.sub.3) immediately prior to analysis
by flow cytometry. Kd were estimated from a plot of 1/(mean channel
fluorescence) vs. 1/[FITC-30.5.7 Fab].
[0085] Affinities of 2C TcR analogs were determined by competition
with constant concentrations of FITC-30.5.7 Fab. Cells were loaded
with peptide, and subsequently incubated with a constant
concentration of FITC-30.5.7 Fab and varying concentration of 2C
TcR analogs for 1 hour at 4.degree. C. Cells were diluted 1:6 with
FACS wash buffer immediately prior to analysis by flow cytometry.
Maximal inhibition of FITC-30.5.7 Fab binding was determined by
incubation in the presence of 30.5.7 mAb (75 mg). Kapp was
determined from a plot of 1/(% maximal inhibition) vs. [2C TCR
analog]. Kapp was corrected for the affinity of FITC-30.5.7 Fab for
peptide loaded cells according to the equation
Kd,TcR=Kapp/(1+([FITC 30.5.7 Fab]/Kd,30.5.7)) (Schlueter et al
(1996) supra.
[0086] Direct Flow Microfluorimetry: Approximately 3.times.10.sup.5
peptide-loaded or control cells were incubated for 60 min. at
4.degree. C. with either .about.50 mg/ml mAb 30.5.7 culture
supernatants in a 30-50 ml volume, 50 ml of 2C TcR/Ig culture
supernatants (10 5g/ml final concentration), or 25-50 mg/ml
purified 2C TcR/Ig in a 30 ml volume. Cells were washed once in
1.times.PBS, 1% FBS, 0.02% Na-azide (FACS wash Buffer) and then
incubated for an additional 60 min. at 4.degree. C. in 20 5I of a
{fraction (1/20)} dilution goat anti-mouse IgG-RPE (Southern
Biotechnology Associates, Inc.). Cells were subsequently washed
once with FACS wash buffer, resuspended in 250 ul FACS wash buffer
and analyzed on a Becton Dickinson FACScan flow cytometer.
[0087] CTL Assays (Generation of CTL) - Splenocytes from 2C TCR
transgenic mice (Sha et al Nature 336:73-76 (1988 ) were
resuspended at 1.25.times.10.sup.6 per ml and stimulated with
1.75.times.10.sup.6 BALB/c splenocytes that had been exposed to
3,000 cGy radiation. On day 7, the 2C T cell-enriched cultures were
restimulated at 5.times.10.sup.5 per ml with 2.5.times.10.sup.6 per
ml BALB/c splenocytes. Experiments were performed on this and
subsequent stimulationUs on day 4. All subsequent stimulation was
performed with 3.75.times.10.sup.5 per ml 2C splenocytes and
2.5.times.10.sup.6 per ml BALB/c cells in the presence of IL-2 (5
U/ml). Assays were performed in triplicate according to established
CTL protocols. Briefly, target cells (2-4.times.10.sup.6) were
incubated with 100 5Ci.sup.51 [Cr] at 37.degree. C. for 1 h. After
three washes, cells were added to V-bottom 96 well plates
(3.times.10.sup.3/100 51) and incubated (25.degree. C. for 1.5 h)
with peptides at the indicated concentrations. 2C T cells
(3.times.10.sup.4/10051) were added to targets and plates were
incubated at 37.degree. C. for 4.5 h. Maximum release was achieved
by incubating targets with 5% Triton .times.100. Percent specific
lysis was calculated from raw data using [(experimental
release-spontaneous release )/(maximum release-spontaneous
release)].times.100.
[0088] General Construction and Biochemical Characterization of
Chimeric Molecules
[0089] Using imnmunoglobulin as a backbone, a general system has
been designed for expression of soluble recombinant divalent
analogs of heterodimeric transmembrane proteins (FIGS. 1B-D and
FIG. 2). As shown in FIG. 2, site-directed mutagenesis was used to
insert restriction enzyme sites, such as KpnI and Hind III, into
the 5' region of the Ig heavy and light chains, respectively. The
enzyme sites were introduced immediately after the leader sequence
prior to the start of the mature protein encoding the intact
variable domains. This strategy leads to a generic system for
expression of chimeric polypeptides and serves as a foundation
molecule for construction of soluble divalent analogs of two
different classes of heterodimeric proteins. The different classes
of heterodimeric transmembrane proteins, which contain .alpha. and
.beta. polymorphic integral membrane polypeptides that bind each
other forming a functional unit involved in immune recognition,
include, but are not limited to, proteins such as T cell receptors,
and class II MHC molecules, integrins, and cytokine receptors.
[0090] A multi-step construction was used to genetically fuse
.alpha. and .beta. polypeptides to Ig heavy and light chains to
form the chimeric IgG molecules. As chimeric fusion partners
consisted of cDNA encoding a murine IgGI arsonate-specific heavy
chain, 93G7, and .kappa. light chain, 91A3 (Haseman et al Proc Natl
Acad Sci USA 87:3942-3946 (1990). Both of these Ig polypeptides
have been expressed and produce intact soluble intact IgGI
molecules in baculovirus infected cells. cDNA encoding the light
chain clone 91A3 was modified by introduction of 5' HindIII site
and linker immediately prior to position one amino acid residue,
Asp, at the start of the mature protein. A KpnI restriction enzyme
endonuclease site was introduced after the stop codon in the mature
.kappa. polypeptide. cDNA encoding the 93G7 clone was modified but
introduction of a KpnI restriction enzyme endonuclease site
immediately prior to 5' to amino acid residue position Glu located
at the start of the mature protein, and an SpHI restriction enzyme
endonuclease site 3' to the stop codon in the mature IgGI
protein.
EXAMPLE 1
Construction and Expression of Soluble Divalent Class II MHC
Molecules and T Cell Receptors
[0091] The difficulty experienced with the construction and
expression of soluble heterodimeric integral proteins, such as
soluble divalent class II MHC molecules and T cell receptors
(TcRs), was overcome by linking .alpha. and .beta. chain
polypeptides to immunoglobulin heavy and light chains (FIGS. 1 and
2). Using the soluble divalent TcRs, data are presented to show
that soluble proteins are high affinity ligands for peptide/MHC
complexes.
[0092] TcR Rationale and Construction:
[0093] The 2C TcR was selected to generate soluble divalent TcR
analogs. 2C is a well characterized alloreactive, peptide-specific
cytotoxic T lymphocyte (CTL) clone (Kranz et al Proc Natl Acad Sci
USA 81:573-577 (1984). This clone is specific for a naturally
processed endogenous peptide derived from alpha-ketoglutarate
dehydrogenase bound by the murine class I molecule H-2L.sup.d
(Udaka et al Cell 69:989-998 (1992). The original 2C reactive
peptide, called p2Ca, identified as an eight amino acids residue.
Both higher and lower affinity variants of p2Ca reactive with 2C
cells have been defined (Sykulev, Immunity, supra; Sykulev et al
Proc Natl Acad Sci USA 91:11487-11491 (1994) (see Table 3).
[0094] A clonotypic monoclonal antibody, 1B2, specific for the 2C
TcR has also been developed (Kranz Proc. Natl. Acad. Sci. USA
81:573-577 (1984). The TcR conferring 2C specificity has been
cloned and transgenic mice expressing 2C TcR have also been derived
(Sha et al Nature 336:73-76 (1988); Sha et al Nature 335:271-274
(1988). The above mentioned prior art makes 2C TcRs an excellent
model to study.
[0095] To generate the soluble divalent TcR, cDNA encoding the TcR
.alpha. and .beta. chains of TcR was genetically linked to cDNA
encoding IgGI heavy chains and .kappa. light chains, respectively.
Site-directed mutagenesis was used to introduce restriction
endonuclease enzyme sites into the 5' region prior to the leader
sequence and into the 3' region of the TcR .alpha. and .beta. genes
immediately preceding the regions encoding the transmembrane
domains (see FIGS. 1 B-D for schematic of proteins, FIG. 2 for
expression vector and FIG. 3 for oligonucleotides used to induce
mutations). The sites introduced in the 3' region in the TcR
.alpha. and .beta. cDNA were compatible with the sites introduced
into the immunoglobulin (Ig) heavy and light chain cDNA,
respectively. For expression, the constructs were cloned into a
modified version of baculovirus expression vector, pAcUW51, and
other baculovirus expression systems (Kozono et al Nature
369:151-154 (1994). This expression vector has two separate viral
promoters, polyhedron and P10, allowing expression of two
polypeptides in a single virally infected cell.
[0096] The 2C TcR .alpha.: chain was modified by introduction of a
linker and a KpnI restriction enzyme endonuclease site immediately
3' to the Gln residue at interface between the extracellular and
transmembrane domains of the .alpha. polypeptide. The 5'0 regions
of the genes already expressed the appropriate restriction enzyme
endonuclease sites and did not require any additional
modifications.
[0097] The 2C TcR .beta., chain was modified by introduction of a
Xho 1 site 5' to the start of the signal sequence for the .beta.
chain and a HindIII restriction enzyme endonuclease site
immediately 3' to the Ile residue at interface between the
extracellular ad transmembrane domains of the .beta.
polypeptide.
[0098] Class II MHC Rational and Construct:
[0099] To study class II MHC molecules, the well-characterized
murine I-E.sup.k molecule was chosen as a model antigen. Other
class II molecules that could have been chosen include murine I-A
molecules and human HLA-DR, DP, and DQ molecules. Murine I-E.sup.k
is a known restriction element for a model class II antigen moth
cytochrome C (MCC). Soluble monovalent versions of relevant TcR and
class II MHC/peptide complexes have been generated (Wettstein et al
J Exp Med 174:219-228 (1991); Lin et al Science 249:251 (1990). T
cell responses to this complex have been well characterized
(Jorgensen et al Nature 355:224 (1992) and the affinity of specific
T cell clones to MCC/I-E.sup.k complexes have been measured (Matsui
Proc. Natl. Acad. Sci. 91:12862-12866 (1994). A genetically
engineered soluble version of murine I-E.sup.k that was covalently
linked to MCC has also been shown to stimulate MCC-specific T cells
(Kozono, supra). Thus, this well characterized MHC system was used
as a model to study the influence of divalent class II MHC on T
cell reactivity.
[0100] For expression of soluble divalent class II MHC molecules,
cDNA encoding the I-E.sup.k.sub..beta. chain was genetically linked
to a cDNA encoding an IgG heavy chain. A cDNA encoding an
I-E.sub..alpha.. chain was linked to the one encoding the kappa
light chain. The 5' amino terminus of the .beta. chain was
previously genetically linked via a thrombin cleavage site to the
I-E.sup..kappa.-restricted antigenic-peptide derived from MCC
(81-101) (Kozono, supra). Site-directed mutagenesis was used to
introduce a KpnI restriction enzyme endonuclease enzyme site into
the 3' region of the I-E.sup..kappa..sub..beta. immediately
preceding the regions encoding the transmembrane domains. The cDNA
encoding the I-E.sub..alpha. chain was modified by introduction of
a HindIII restriction enzyme endonuclease immediately preceeding
the transmembrane domains. The 5'I-E.sub..alpha. and I-E.sub..beta.
regions of the genes did not require any additional
modifications.
[0101] General Linker Region Rational and Construction:
[0102] A linker of six amino acid residues was also added at the
junctions between the end of the TcR .alpha. and .beta. and I-E
.alpha. and .beta. polypeptides and the start of the mature IgG
polypeptides. For the junction with Ig.gamma.1 polypeptides the
linker consists of Gly-Gly-Gly-Thr-Ser-Gly. For the junction with
Ig.kappa. polypeptides the linker consists of
Gly-Ser-Leu-Gly-Gly-Ser. Oligonucleotides used to introduce all the
above mutations are described in FIG. 3.
[0103] The expression vector used to generate a soluble divalent T
cell receptor analog was derived from the baculovirus expression
vector pAcUW51 (Pharmingen, Calif.). This vector has two separate
viral promoters, polyhedron and P 10, allowing one to express both
chimeric polypeptide chains in the same cell. To facilitate cloning
of different genes into the vector, multiple cloning sites were
previously introduced after each of the promoters (Kozono,
supra).
EXAMPLE 2
Detection of Soluble Heterodimeric Proteins
[0104] Cells infected with baculovirus containing transfer vectors
encoding the soluble chimeric Ig constructs described above secrete
a soluble chimeric Ig-like molecule detected by specific ELISA
assays 4-5 days post infection. For 2C TcR/IgG, the assay was based
on a primary antibody specific for murine IgG1 Fc (plated at 10
.mu.g/ml) and a biotinylated secondary antibody, H57 (used at
1:5000 final dilution), specific for a conformational epitope
expressed on the .beta. chain of many TcR (FIG. 5, Panel A) or
biotinylated 1B2 or a monoclonal antibody specific for a clontoypic
epitope expressed on 2C TcR (FIG. 5, Panel B). For detection of
I-E/IgG chimeric molecules, the same primary antibody was used and
the biotinylated secondary antibody was 14.4.4, which is specific
for I-E.alpha. chain (FIG. 5, Panel C). Supernatants from infected
cells were incubated for 1 hour at room temperature. Plates were
washed extensively with phosphate buffer saline, incubated with the
biotinylated secondary antibody for 1 hour at room temperature. The
plates were then washed and incubated with HRP-conjugated
strepatvidin (100 .mu.l of a 1:10000 dilution) (Sigma, St. Louis,
Mo.) for 1 hour at room temperature, washed and developed with 3,
3', 5, 5'-Tetramethylbenzidine Dihydrochloride (TMB) substrate for
3-5 minutes. Supernatants from cells infected with baculovirus
containing the 2C TcR/Ig and I-E/Ig transfer vectors were compared
to control supernatant from cells infected with the wild type
baculovirus.
[0105] The chimeric proteins are conformationally intact as shown
in FIG. 5. The soluble divalent 2C TcR/Ig is reactive with H57, a
monoclonal antibody specific for a conformational epitope expressed
on most TcR .beta. chains as well as with 1B2, the anti-clonotypic
monoclonal antibody determinant specific for the 2C TcR as shown in
FIGS. 5A and 5B. Soluble divalent class II molecules are reactive
with the conformationally dependent monoclonal antibody specific
for a native alpha chain determinant only expressed on intact I-E
molecules, monoclonal antibody 14.4.4 as shown in FIG. 5C. The
immunoglobulin portion of the chimeric molecules is also
conformationally intact. It is reactive immunoglobulin specific
ELISA, as mentioned above, and can be used to purify the chimeric
molecules. Protein G or arsenate-sepharose affinity purification
column methods can also be used (data not shown).
[0106] The purified material has the expected molecular weights
when analyzed by SDA-PAGE as depicted in FIG. 6. The chimeric
TcR.beta./Ig.kappa. has an apparent molecular weight (MW) of 55,000
and the chimeric TcR.alpha./Ig.gamma.1 has an apparent MW of
approximately 89,000. The chimeric I-E.alpha./Ig.kappa. has an
approximate MW of 44,000 and the chimeric I-E.beta./Ig.gamma.1 has
an apparent MW of approximately 76,000 (FIG. 6).
EXAMPLE 3
Affinity measurements of soluble divalent TCR interaction with
peptide/MHC complexes.
[0107] A competitive inhibition assay was developed to measure the
affinity of soluble 2C TCR/Ig for peptide/MHC complexes. This
assay, similar to one previously used to determine the affinity of
soluble monovalent 2C TCR for peptide/MHC complexes (Schlueter et
al Journal of Molecular Biology 256:859-869 (1996), is based on mAb
30.5.7 binding to a region of the a2 helix of H-2 Ld that overlaps
with TCR receptor binding (Solheim et al Journal of Immunology
154:1188-1197 (1995); Solheim et al Journal of Immunology
150:800-811 (1993). Briefly, affinities of 30.5.7 Fab fragments for
RMA-S L.sup.d cells were determined by direct saturation analysis
of 30.5.7 Fab binding to cells analyzed by flow cytometry. Cells
were incubated with increasing amounts of FITC labeled 30.5.7 Fab,
and K.sub.d's were estimated from a plot of 1/MCF vs. 1/[30.5.7
Fab]. Affinities of 2C TCR analogs were determined by competition
of the 2C TCR analog with a constant amount of FITC labeled 30.5.7
Fab fragments for RMA-S L.sup.d cells as described in Methods.
K.sub.app was calculated from a plot of (% maximal 30.5.7 Fab
binding).sup.-1 vs. [2C TCR analog]. The K.sub.app was corrected
for the affinity of 30.5.7 Fab for RMA-S L.sup.d cells according to
the equation K.sub.d.TCR=K.sub.app'(1+{30.5.7 Fab]'K.sub.d 30 5 7)
(Schlueter et al., 1996). The values reported in the Table 2 are
from one representative experiment that has been repeated at least
three times. Each data point used in determination of the K.sub.d
is the average of duplicate points. Hence, the affinity of soluble
TCR analogs was measured in terms of their inhibition of 30.5.7
binding.
[0108] To determine the affinity of the soluble 2C TCR analogs, one
has to first determine the Kd of 30.5.7 Fab fragments for
peptide-loaded H-2 Ld molecules. This measurement was determined by
direct saturation analysis of 30.5.7-FITC Fab binding to H-2 Ld
molecules on the surface of RMA-S Ld cells. RMA-S cells were chosen
since these cells express empty MHC molecules that can be readily
loaded with specific peptides of interest (Catipovic et al Journal
of Experimental Medicine 176:1611-1618 (1992); Townsend et al
Nature 340:443-428 (1989). The affinity of 30.5.7 for H-2 Ld
molecules is dependent on the peptide loaded into H-2 L.sup.d
(Table 2). The affinity of the 30.5.7 for QL9 loaded H-2 L.sup.d
molecules is 12.2 nM while the affinities for p2Ca, pMCMV and SL9
loaded H-2 L.sup.d molecules range between 4.8-6.4 nM. These small,
peptide-dependent, differences in affinity are reproducible and
variations in affinity were accounted for in the competitive
binding assays. These values are in good agreement with the
previously measured affinities of 125I-30.5.7 Fab for the same
peptide/H-2 L.sup.d complexes (8.8 to 16 nM (Schlueter et al.,
1996)).
[0109] 2C TCR/Ig inhibited binding of 30.5.7 Fab to H-2 L.sup.d
molecules loaded with either QL9 or p2Ca peptides but did not
inhibit 30.5.7 Fab binding to pMCMV loaded H-2 L.sup.d molecules
(FIG. 7). The affinity of soluble divalent 2C TCR/Ig for QL9 loaded
molecules is 13.3 nM (FIG. 7 and Table 2). As expected, the
affinity of 2C TCR/Ig for p2Ca loaded molecules, 90 nM, is lower
than that for QL9 loaded H-2 L.sup.d. Although a small amount of
competitive inhibition was seen with SL9 loaded cells, the affinity
of the soluble divalent 2C TCR/Ig chimeras for SL9 loaded molecules
is too low to be accurately measured under the conditions tested
(data not shown).
[0110] In all cases analyzed, the affinity of the soluble divalent
2C TCR/Ig was significantly higher than the affinity of the soluble
monovalent 2C TCR for its cognate ligand (FIG. 7 and Table 2). The
affinity of soluble divalent 2C TCR/Ig was 50-fold higher for
QL9-loaded H-2 L.sup.d and at least 20-fold higher for p2Ca-loaded
H-2 L.sup.d molecules than that of soluble monovalent 2C TCR for
the same peptide/MHC complexes (Table 2). Thus, the divalent nature
of soluble 2C TCR/Ig chimeras significantly increased the affinity
of the TCR analog for its cognate ligands. The finding that the
chimeric molecules of the present invention demonstrate increased
affinity for their specific ligands over what is seen for
monovalent molecules was not an expected result. In fact, the
chimeric CD4-IgG molecules disclosed in Capon et al. do not
demonstrate improved target affinity, which is further evidence to
the value and novelty of the compositions of the present
invention.
2TABLE 2 Measured Affinities of TCR analogs for peptide loaded
RMA-SL.sup.d cells Peptide/MHC 30.5.7 Fab 2C TCR/Ig 2C-sm TCR
complex K.sub.d (nM) K.sub.app (nM) K.sub.d (nM) K.sub.app (nM)
K.sub.d (nM) QL9 12.2 18.3 13.3 953.4 613.6 p2Ca 5.8 107.7 90.5
>2000.sup.2 >2000.sup.2 pMCMV 4.8 ndc.sup.1 ndc.sup.1
--.sup.3 --.sup.3 .sup.1ndc - no detectable competition with 30.5.7
Fab fragments .sup.2Competition was detected at the highest
concentration of 2C-smTCR used, but an accurate measure of the
K.sub.d could not be determined. .sup.3-- not done.
EXAMPLE 4
Binding specificity of soluble divalent TCR chimeras to
peptide-loaded H-2 Ld molecules.
[0111] Based on the relatively high affinity of soluble divalent 2C
TCR/Ig for peptide/MHC complexes, we postulated that these
molecules might be useful in analysis of peptide/MHC complexes by
direct flow cytometry based assays. To study peptide specificity of
2C TcR/Ig, we compared reactivity of 2C TcR/Ig with that of H-2
L.sup.d reactive mAb, 30.5.7, in direct flow cytometry assays.
Specific peptides (see Table 3 for sequences) were loaded into H-2
Ld molecules on RMA-S L.sup.d cells. Peptides listed in Table 2 are
a collection of H-2 L.sup.d and H-2 K.sup.b binding peptides used
in analysis of the reactivity of the soluble divalent 2C TCR/Ig.
Lysis and affinity data are summarized from their primary
references (Corr et al., 1994; Huang et al., 1996; Solheim et al.,
1993; Sykulev et al., 1994a; Sykulev et al., 1994b; Tallquist et
al., 1996; Udaka et al., 1996; Van Bleek and Nathanson, 1990).
3TABLE 3 Peptides used in this study: Their reported effectiveness
in 2C CTL assays and affinities of 2C TCR for peptide/MHC
complexes. peptide peptide MHC 2C-mediated name sequence
restriction lysis K.sub.d(.mu.M) p2Ca LSPFPFDL H-2 L.sup.d +++
0.5-0.1 QL9 QLSPFPFDL H-2 L.sup.d ++++ 0.066 SL9 LSPFPFDLL H-2
L.sup.d +/- 71 turn TQNHRALDL H-2 L.sup.d na na pMCMV YPHFMPTNL
H-2L.sup.d -- nd gp 70 SPSYVYHQF H-2 L.sup.d na na dEV-8 EQYKFYSV
H-2 K.sup.b -- na dEV-8 H-2 K.sup.bm3 +++ unknown SIY SIYRYYGL H-2
K.sup.b +++ unknown SIY H-2 K.sup.bm3 unknown unknown pVSV
NP(52-59) RGYVYQGL H-2 K.sup.b -- nd na - not available. nd - none
detected. The affinity were below the sensitivity of the assay
used.
[0112] The temperature-dependent reactivity of RMA-S L.sup.d with
2C TCR/Ig was significantly different than the reactivity of RMA-S
L.sup.d with mAb 30.5.7. As expected (Solheim et al (1995) supra;
Solheim et al (1993) supra), RMA-S L.sup.d cells expressed more
serologically reactive H-2 L.sup.d molecules recognized by mAb
30.5.7 on cells cultured at 27.degree. C. than when cells were
cultured at 37.degree. C. (FIG. 8A); mean channel fluorescence
(MCF) increased approximately 5 fold. Thus the epitope on H-2
L.sup.d molecules recognized by mAb 30.5.7 can be stabilized by
incubating cells at low temperatures. In contrast, RMA-S L.sup.d
cells expressed very low amounts of H-2 L.sup.d molecules
recognized by 2C TcR/Ig on cells cultured at either 27.degree. C.
or at 37.degree. C. (FIG. 8, Panel E). This finding is consistent
with the expected peptide-dependent reactivity of 2C TcR/Ig which
should not recognize unloaded MHC even when conformationally
stabilized by decreased temperature.
[0113] 2C TcR/Ig reactivity also showed exquisite peptide
specificity. As expected, all H-2 L.sup.d-binding peptides
stabilized expression of the epitope recognized by mAb 30.5.7 (FIG.
8, Panels B-D and FIG. 9). Only H-2 L.sup.d molecules loaded with
2C reactive peptides, peptides p2Ca, QL9, and SL9 expressed
peptide/H-2L.sup.d epitopes that reacted with 2C TcR/Ig (FIG. 8,
Panels F-H and FIG. 9). MCF increased approximately 10-200 fold,
from a MCF of 10 for either unloaded cells or cells loaded with an
irrelevant H-2 L.sup.d binding peptide, to as high as 2200 for
RMA-S L.sup.d cells loaded with peptide QL9 (FIG. 9). The pattern
of reactivity mimicked the known affinities of monovalent 2C TcR
for peptide/H-2 L.sup.d complexes (see Table 3 for affinities).
RMA-S L.sup.d cells loaded with peptide QL9, p2Ca, or SL9 had MCF
values of 2200, 550, and 100, respectively, when stained with 2C
TcR/Ig. Thus, soluble divalent 2C TcR/Ig chimeras reacted strongly
with QL9/H-2 L.sup.d complexes, modestly with p2Ca/H-2 L.sup.d
complexes, and weakly with SL9/H-2 L.sup.d complexes. The fact that
2C TcR/Ig bound to SL9-loaded H-2 L.sup.d molecules indicates, that
even in a direct flow cytometry assay, soluble divalent 2C TcR/Ig
chimeras could be used to detect specific peptide/MHC complexes
that have affinities as weak as 71 mM for monovalent 2C TcR.
EXAMPLE 5
Inhibition of In Vitro 2C T cell Mediated Lysis by Soluble Divalent
2C TcR/Ig Molecules
[0114] Soluble divalent 2C TcR/Ig blocks 2C reactive T cell
responses. Since soluble divalent 2C TcR/Ig interacts with high
avidity with H-2 L.sup.d molecules loaded with the appropriate
peptides in the flow cytometry assay, it was explored whether the
reagent could effectively inhibit 2C T cells in vitro cytotoxicity
CTL assays. This was analyzed using a cell line derived from 2C
transgenic mice to lyse tumor target cells expressing H-2 L.sup.d.
CTL were tested in a routine 4 hour .sup.51 Cr cytotoxicity assay.
As targets for all the CTL assays, untransfected, MC57G, and
L.sup.d transfected, MC57G L.sup.d, cells were used as targets. The
percent specific lysis was determined as: .sup.51 Cr cpm
(experimental) -CPM (spontaneous)/cpm (maximum) -cpm (spontaneous).
Standard errors were routinely less than 5% and spontaneous release
was usually 10-15% of maximal release.
[0115] Using both untransfected MC57G and L.sup.d transfected,
MC57G L.sup.d, we were able to establish a window of H-2L.sup.d
specific lysis mediated by the 2C CTL line. To test the influence
of 2C TcR/Ig, target cells were pretreated with either 2C TcR/Ig,
or I-E.sup.k/Ig and analyzed for lysis by the CTL cell line derived
from 2C transgenic mice. Significant inhibition of lysis was seen
at each effector to target cell ratio analyzed when cells were
treated with 2C TcR/Ig (see FIG. 10). While some non-specific
inhibition was seen in the I-E.sup.k/Ig treated target cells,
significantly more inhibition was seen in the 2C TcR/Ig treated
target cells.
[0116] In this assay, the target cells were normal tumor cells that
load their cell surface MHC molecules with a variety of different
endogenous peptides. Using these target cells, one does not need to
specifically load H-2 L.sup.d molecules with the p2Ca peptide since
p2Ca or p2Ca-like peptides along with a large number of irrelevant
peptides are endogenously loaded onto cellular MHC molecules.
Inhibition of CTL-mediated lysis indicates that soluble divalent 2C
TcR/Ig can effectively interact with the relevant peptides even
within a milieu of a large number of irrelevant peptides. Thus,
this approach could be used to search the universe of peptide/MHC
complexes to identify only those complexes relevant to specific T
cell responses of interest. In particular, these high avidity
soluble analogs of heterodimeric proteins may specifically be
useful in identification of unknown tumor and autoimmune
antigens.
EXAMPLE 6
Binding of soluble divalent TCR chimeras to self restricted
peptide/MHC complexes.
[0117] In addition to recognizing peptide/H-2 L.sup.d ligands, two
peptides, SIY and dEV-8, that sensitize either H-2 K.sup.b or H-2
K.sup.bm3 targets for lysis by 2C CTL have also been defined (see
Table 3 for sequences). To analyze the ability of 2C TcR/Ig to bind
to these alternate 2C-reactive complexes, the binding of 2C TcR/Ig
to peptide loaded transfected T2 cells was studied. Since T2 cells
are derived from a human cell line, T2 cells do not naturally
express H-2 K.sup.b as do RMA-S cells. Thus to study the binding of
2C TcR/Ig to peptide-loaded H-2 K.sup.b or various H-2 K.sup.bm
mutant molecules, the T2 system was chosen since it is not
complicated by the expression of MHC molecules from the parental
cell line. Similar to RMA-S L.sup.d cells, T2 cells also express
empty MHC molecules that can be readily loaded with different
peptides. For these studies peptide-loaded T2 cells transfected
with: H-2 K.sup.b, T2 K.sup.b;. H-2 K.sup.bm3, T2 K.sup.bm3; and
H-2 K.sup.bm11, T2 K.sup.bm11 (Tallquist et al Journal of
Immunology 155:2419-2426 (1995); Tallquist et al Journal of
Experimental Medicine 184:1017-1026 (1996) were utilized.
[0118] Peptide SIY-loaded T2 K.sup.b or T2 K.sup.bm11 cells both
expressed epitopes recognized by 2C TcR/Ig (FIG. 11A,C). MCF of
cells incubated with 2C TcR/Ig increased approximately 20 fold from
14 for pVSV loaded- to 276 for SIY loaded-T2 K.sup.b and from 16
for pVSV loaded- to 250 for SIY loaded-T2 K.sup.bm11. SIY-loaded T2
K.sup.bm3 cells showed a much weaker but still significant
interaction with 2C TcR/Ig (FIG. 11B); compare SIY-loaded (solid
lines; MCF, 36) to pVSV-loaded (dotted lines; MCF, 12) T2 K.sup.bm3
cells. The 2C TcR/Ig binding data to SIY/MHC complexes was
consistent with 2C CTL mediated lysis on various SIY/MHC targets
(FIG. 12). 2C CTL mediated efficient lysis of SIY loaded T2 K.sup.b
and T2 K.sup.bm11 cells (FIG. 12B and data not shown, LD50.about.10
ng/ml for SIY/T2 K.sup.b). 2C CTL mediated lysis of SIY loaded T2
K.sup.bm3 cells was significantly less efficient (FIG. 12C,
LD50.about.100 ng/ml).
[0119] The binding of 2C TcR/Ig to dEV-8 loaded cells revealed a
striking difference between the affinity of 2C TcR/Ig for dEV-8/MHC
complexes and the ability of that same peptide/MHC complex to
mediate lysis by 2C CTL. As expected, dEV-8 loaded T2 K.sup.b cells
were neither lysed by 2C CTL (FIG. 12B), nor were they recognized
by 2C TcR/Ig in flow cytometry assays (FIG. 12A). Interestingly, no
significant binding of 2C TcR/Ig could be found to dEV-8 loaded T2
K.sup.bm3 cells (FIG. 12B). MCF of cells stained with 2C TcR/Ig was
similar whether cells were loaded with either dEV-8 or a control
H-2 Kb-binding peptide, pVSV (FIG. 12; compare dotted to dashed
lines). This is most surprising in that, consistent with previous
reports (Tallquist et al (1996) supra, dEV-8 loaded T2 K.sup.bm3
cells were efficiently lysed by 2C CTL (FIG. 12C). In fact, dEV-8
loaded T2 K.sup.bm3 cells were much better target cells
(LD50.about.0.5-1.0 ng/ml), than SIY loaded T2 K.sup.bm3 cells
(LD50.about.100 ng/ml), where a significant binding of 2C TcR/Ig
was seen (FIG. 12B). The efficiency of lysis by 2C CTL of dEV-8
loaded T2 K.sup.bm3 cells, was on the same order of magnitude as
that of p2Ca loaded T2 L.sup.d cells (FIG. 12A, LD50.about.0.5
ng/ml) which was also efficiently recognized in the 2C TcR/Ig
binding assay (FIG. 8). A similar, although significantly less
dramatic, lack of correlation between cytolysis and 2C TcR/Ig
binding was seen for dEV-8 loaded T2 K.sup.bm11 cells. dEV-8 loaded
T2 Kb.sup.m11 cells are relatively poor targets for 2C CTL
(Tallquist et al (1996) supra) (data not shown), but were also not
reactive with 2C TcR/Ig in flow cytometry assays (FIG. 11C).
EXAMPLE 7
Analysis of the effects of .gamma.-IFN on expression of endogenous
2C-specific peptide/MHC complexes.
[0120] The specificity and affinity of 2C TcR/Ig for peptide/MHC
complexes suggested that one might be able to use this reagent to
probe the influence of lymphokines on endogenous, cell surface,
peptide/MHC complexes. To analyze this possibility and follow the
expression of endogenous 2C-reactive peptide/H-2 L.sup.d complexes
within a heterogeneous peptide/MHC environment, the influence of
.gamma.-IFN on the H-2 L.sup.d expressing murine cell line, RENCA
was studied. RENCA cells were cultured in the presence of variable
amounts of .gamma.-IFN to induce up-regulation of naturally loaded
peptide/MHC complexes. 2C TcR/Ig binding to RENCA cells increased
as a function of .gamma.-IFN induction (FIG. 13A-D, solid lines).
The effect of .gamma.-IFN was dose dependent with a maximal 2-3
fold increase seen on cells treated with 10 units/ml of
.gamma.-IFN. Since .gamma.-IFN is known to have a direct effect on
class I expression (FIG. 13E-H) (Hengel et al Journal of Virology
68:289-297 (1994)), it is necessary to normalize for any
non-specific 2C TcR/Ig binding secondary to increased expression of
H-2 L.sup.d. This was accomplished by incubating RENCA cells with a
control irrelevant H-2 L.sup.d binding peptide, pMCMV. Since p2Ca
is known to have a weak affinity for H-2 L.sup.d (Sykulev et al
Immunity 1:15-22 (1994a) exchange with a higher affinity H-2 Ld
binding peptide like pMCMV (Sykulev et al (1994a) supra) should be
very efficient. Therefore background reactivity of 2C TcR/Ig could
be determined by the efficient displacement of endogenous p2Ca or
p2Ca-like peptides by incubating the cells with saturating amounts
of the control pMCMV peptide. In all cases, 2C TcR/Ig binding could
be blocked by prior incubation of cells with the control H-2
L.sup.d binding, pMCMV (FIG. 13A-D, dotted lines). Prior incubation
of RENCA cells with a 2C specific peptide, QL9, induced a dramatic
increase in 2C TcR/Ig binding (data not shown). The results of
these experiments indicate that 2C TcR/Ig could be used as a
sensitive probe to analyze cell surface expression of endogenous
2C-reactive peptide/MHC complexes.
[0121] The effect of .gamma.-IFN on 2C TcR/Ig reactivity was
distinct from its effects on 30.5.7 reactivity. At all
concentrations analyzed, 5-50 units/ml, .gamma.-IFN induced a 5-6
fold increase in serologically reactive H-2 L.sup.d, as recognized
by mAb 30.5.7 (FIG. 13E-H). MCF of unstimulated RENCA cells was
500, while the MCF of .gamma.-IFN stimulated cells was between 2666
and 3038. The maximal effect of .gamma.-IFN was seen at the lowest
dose used, in the experiment presented, 5U/ml, and in other
experiments was seen even at dose of .gamma.-IFN as low as 1
unit/ml (data not shown). Interestingly, the dose response curve of
.gamma.-IFN on 2C TcR/Ig reactivity was shifted. .gamma.-IFN at
5U/ml had a relatively small but significant effect on 2C TcR/Ig
reactivity. Maximal effects of .gamma.-IFN on 2C TcR/Ig reactivity
required .gamma.-IFN treatment at 10 units/ml, approximately ten
fold more than needed for maximal effects of .gamma.-IFN on 30.5.7
reactivity. These results indicate a differential effect of
.gamma.-IFN on MHC heavy chain expression than that of .gamma.-IFN
on specific peptide antigen/MHC complex expression.
[0122] These results show that this approach is a general one for
producing soluble divalent versions of heterodimeric proteins.
Soluble divalent analogs of heterodimeric proteins of this
invention are characterized as having high avidity for their
targets.
[0123] The same way that this was done for a single murine class II
MHC and .alpha./.beta. TcR, so to the same technology, generating
soluble divalent heterodimeric proteins, can be used to develop
other mammalian systems. These will include both rodent and human
class II HLA molecules and .alpha./.beta. and .gamma./.delta. T
cell receptors.
[0124] The present invention may be embodied in forms other than
those specifically disclosed above without departing from the
spirit or essential characteristics of the invention. The
particular embodiments of the invention described above, are
therefore, to be considered as illustrative and not restrictive.
All references and patent applications are herein incorporated by
reference to the same extent as if each individual publication or
patent application was specifically and individually indicated to
be incorporated by reference.
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Sequence CWU 1
1
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