U.S. patent application number 10/513539 was filed with the patent office on 2006-07-27 for therapeutic anti-tirc7 antibodies for use in immune related and other diseases.
Invention is credited to Nalan Utku.
Application Number | 20060165684 10/513539 |
Document ID | / |
Family ID | 8179669 |
Filed Date | 2006-07-27 |
United States Patent
Application |
20060165684 |
Kind Code |
A1 |
Utku; Nalan |
July 27, 2006 |
Therapeutic anti-tirc7 antibodies for use in immune related and
other diseases
Abstract
Provided are specific antibodies against T-cell immune response
cDNA7 (TIRC7) costimulatory molecule, which are capable of
inhibiting proliferation of peripheral blood mononuclear cells
(PBMCs). In particular, high affinity monoclonal and chimeric
anti-TIRC7 antibodies are described. Compositions comprising such
antibodies and their use for the treatment of immune diseases are
provided.
Inventors: |
Utku; Nalan; (Berlin,
DE) |
Correspondence
Address: |
COOPER & DUNHAM, LLP
1185 AVENUE OF THE AMERICAS
NEW YORK
NY
10036
US
|
Family ID: |
8179669 |
Appl. No.: |
10/513539 |
Filed: |
December 23, 2002 |
PCT Filed: |
December 23, 2002 |
PCT NO: |
PCT/EP02/14734 |
371 Date: |
December 19, 2005 |
Current U.S.
Class: |
424/143.1 ;
435/320.1; 435/336; 435/69.1; 514/44R; 530/388.22; 536/23.53 |
Current CPC
Class: |
A61P 29/00 20180101;
C07K 16/28 20130101; C07K 7/00 20130101; A61P 37/06 20180101; C07K
14/00 20130101; C07K 16/2803 20130101; A61K 45/06 20130101; A61K
39/39541 20130101; A61K 2300/00 20130101; A61K 39/39541 20130101;
C07K 2317/24 20130101; A61P 37/02 20180101; A61K 2039/505
20130101 |
Class at
Publication: |
424/143.1 ;
514/044; 530/388.22; 435/069.1; 435/336; 435/320.1; 536/023.53 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 48/00 20060101 A61K048/00; C12P 21/06 20060101
C12P021/06; C12N 5/06 20060101 C12N005/06; C07K 16/28 20060101
C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 21, 2001 |
EP |
01 130 730.3 |
Claims
1. A monoclonal antibody or antigen binding molecule which is
capable of binding to an antigen comprising or consisting of the
amino acid sequence of any one of SEQ ID NOs: 9 to 11.
2. The antibody or antigen binding molecule of claim 1, comprising
in its variable region at least one complementarity determining
region (CDR) of the V.sub.H and/or V.sub.L of the variable region
comprising (a) the amino acid sequence depicted in FIG. 4 (V.sub.H)
(SEQ ID NO: 2) and FIG. 5 (V.sub.L) (SEQ ID NO: 4); or (b) the
amino acid sequence depicted in FIG. 6 (V.sub.H) (SEQ ID NO: 6) and
FIG. 7 (V.sub.L) (SEQ ID NO: 8).
3. The antibody of claim 1, wherein said antibody is a chimeric or
humanized antibody.
4. The antibody of claim 1 comprising the amino acid sequence of
the V.sub.H and/or V.sub.L region as depicted in any one of FIGS. 4
to 7.
5. An antigen or an epitope thereof which is recognized by the
antibody of claim 1.
6. A polynucleotide encoding at least a variable region of an
immunoglobulin chain of the antibody of claim 1.
7. A vector comprising the polynucleotide of claim 6, optionally in
combination with a polynucleotide of claim 6 that encodes the
variable region of the other immunoglobulin chain of said
antibody.
8. A host cell comprising a polynucleotide of claim 6.
9. A method for preparing an antibody or a functional fragment or
immunoglobulin chain(s) thereof comprising (a) culturing the cell
of claim 8; and (b) isolating said antibody or functional fragment
or immunoglobulin chain(s) thereof from the culture.
10. An antibody, an immunoglobulin chain thereof or an antigen
binding fragment thereof encoded by a polynucleotide of claim
6.
11. A composition comprising the antibody of claim 1.
12. The composition of claim 11 which is a pharmaceutical
composition and further comprises a pharmaceutically acceptable
carrier.
13. The pharmaceutical composition of claim 12 further comprising
an immunosuppressive agent.
14. A diagnostic composition comprising the antibody of claim 8
claim 1; and optionally appropriate reagents conventionally used in
immuno or nucleic acid based diagnostic methods.
15. Use of the antibody of claim 1 for the preparation of a
pharmaceutical composition for inhibition of an immune
response.
16. The use of claim 15, wherein said pharmaceutical composition is
administered intravenious, intramuscular, subcutaneous,
intraperitoneal, or as an aerosol.
17. A method of modulating the immune response in a subject in need
thereof, comprising administering the antibody of claim 1.
18. Use of a ligand binding molecule comprising at least one CDR of
an antibody of claim 1 for diagnosing and/or treatment of a
disorder related to the aberrant expression or malfunction of
T-cell immune response cDNA 7 (TIRC7).
19. The use of claim 18, wherein said ligand binding molecule is an
antibody or an immunoglobulin chain thereof.
20. An oligonucleotide consisting essentially of the nucleotide
sequence of any one of SEQ ID NOs: 12 to 40.
21. Use of an oligonucleotide comprising a nucleotide sequence of
any one of SEQ ID NOs: 12 to 40 for the cloning of an anti-TIRC7
antibody or TIRC7 binding molecule.
22. A host cell comprising a vector of claim 7.
23. An antibody, an immunoglobulin chain thereof or an antigen
binding fragment thereof obtainable by the method of claim 9.
Description
[0001] The present invention relates to anti-T-cell immune response
cDNA 7 (TIRC7) antibodies and uses thereof. In particular, the
anti-TIRC7 antibodies of the invention are capable of suppressing
the proliferation of activated cells of the immune system.
Furthermore, the present invention relates to compositions
comprising said antibodies and to methods of modulating immune cell
proliferation, and treating immune response related diseases.
[0002] Several documents are cited throughout the text of this
specification. Each of the documents cited herein (including any
manufacturer's specifications, instructions, etc.) are hereby
incorporated herein by reference; however, there is no admission
that any document cited is indeed prior art as to the present
invention.
[0003] T-cell activation is a serial process involving multiple
signaling pathways and sequential changes in gene expression
resulting in differentiation of T-cells into distinct
subpopulations, i.e. Th1 and Th2, which are distinguishable by
their pattern of cytokine production and characterize the mode of
cellular immune response. The T-cell response is initiated by the
interaction of the antigen-specific T-cell receptor (TCR) with
peptide presented by major histocompatibility complex (MHC)
molecules on the surface of antigen presenting cells (APCs).
Additional signals are provided by a network of receptor-ligand
interactions mediated by a number of membrane proteins such as
CD28/CTLA4 and B7, CD40/CD40L, LFA-1 and ICAM-1 (Lenschow, Science
257 (1992), 789-792; Linsley, Annu. Rev. Immunol. 11 (1993),
191-212; Xu, Immunity 1 (1994), 423-431; Bachmann, Immunity 7
(1997), 549-557; Schwartz, Cell 71 (1992), 1065-1068) collectively
called costimulatory signals (Perez, Immunity 6 (1997), 411). These
membrane proteins can alter T-cell activation in distinct ways
(Bachmann, Immunity 7 (1997), 549-557) and regulate the immune
response by the integration of positive and negative signals
provided by these molecules (Bluestone, Immunity 2 (1995), 555-559;
Perez, Immunity 6 (1997), 411). Many of the agents which are
effective in modulating the cellular immune response either
interfere with the T-cell receptor (Cosimi, Transplantation 32
(1981), 535-539) block costimulatory signaling (Larsen, Nature 381
(1996), 434-438; Blazar J. Immuno. 157 (1996), 3250-3259; Kirk,
Proc. Natl. Acad. Sci. USA 94 (1997), 8789-8794; Linsley, Science
257 (1992), 792-95; Turka, Proc. Natl. Acad. Sci. USA 89 (1992),
11102-11105) or inhibit intracellular activation signals downstream
from these primary cell membrane triggers (Schreiber and Crabtree,
Immunology Today 13 (1992), 136-42). Therapeutic prevention of
T-cell activation in organ transplantation and autoimmune diseases
presently relies on panimmunosupressive drugs interfering with
downstream intracellular events. Specific modulation of the immune
response remains a longstanding goal in immunological research.
[0004] In view of the need of therapeutic means for the treatment
of diseases related to immune responses of the human body, the
technical problem of the present invention is to provide means and
methods for modulation of the immune response in a subject. The
solution to said technical problem is achieved by providing the
embodiments characterized in the claims, and described further
below.
[0005] Accordingly, the present invention generally relates to a
monoclonal antibody or antigen binding molecule which is capable of
binding to an antigen comprising or consisting of the amino acid
sequence of any one of SEQ ID NOs: 9 to 11. Those antibodies are
preferably capable of inhibiting proliferation of peripheral blood
mononuclear cells (PBMCs). In a particularly preferred embodiment
said antibody comprises in its variable region at least one
complementarity determining region (CDR) of the V.sub.H and/or
V.sub.L of the variable region comprising [0006] (a) the amino acid
sequence depicted in FIG. 4 (V.sub.H) (SEQ ID NO: 2) and FIG. 5
(V.sub.L) (SEQ ID NO: 4); or [0007] (b) the amino acid sequence
depicted in FIG. 6 (V.sub.H) (SEQ ID NO: 6) and FIG. 7 (V.sub.L)
(SEQ ID NO: 8).
[0008] The person skilled in the art knows that each variable
domain (the heavy chain V.sub.H and light chain V.sub.L) of an
antibody comprises three hypervariable regions, sometimes called
complementarity determining regions or "CDRs" flanked by four
relatively conserved framework regions or "FRs". The CDRs contained
in the variable regions of the antibody of the invention can be
determined, e.g., according to Kabat, Sequences of Proteins of
Immunological Interest (U.S. Department of Health and Human
Services, third edition, 1983, fourth edition, 1987, fifth edition
1990). The person skilled in the art will readily appreciate that
the variable domain of the antibody having the above-described
variable domain can be used for the construction of other
polypeptides or antibodies of desired specificity and biological
function. Thus, the present invention also encompasses polypeptides
and antibodies comprising at least one CDR of the above-described
variable domain and which advantageously have substantially the
same or similar binding properties as the antibody described in the
appended examples. The person skilled in the art will readily
appreciate that using the variable domains or CDRs described herein
antibodies can be constructed according to methods known in the
art, e.g., as described in EP-A1 0 451 216 and EP-A1 0 549 581.
Furthermore, the person skilled in the art knows that binding
affinity may be enhanced by making amino acid substitutions within
the CDRs or within the hypervariable loops (Chothia and Lesk, J.
Mol. Biol. 196 (1987), 901-917) which partially overlap with the
CDRs as defined by Kabat. Thus, the present invention also relates
to antibodies wherein one or more of the mentioned CDRs comprise
one or more, preferably not more than two amino acid substitutions.
Preferably, the antibody of the invention comprises in one or both
of its immunoglobulin chains two or all three CDRs of the above
mentioned variable regions shown in FIGS. 4-5 and FIGS. 6-7,
respectively.
[0009] As described in the examples, the antibody of the invention
recognizes a fragment of the amino acid sequence from T cell immune
response cDNA 7 (TIRC7) protein. The term "TIRC7" as used in
accordance with the present invention, denotes a protein which
initially has been described to be involved in the signal
transduction of T-cell activation and proliferation and that,
preferably in a soluble form is capable of inhibiting or
suppressing T-cell proliferation in response to alloactivation in a
mixed lymphocyte culture or in response to mitogens when
exogeneously added to the culture. In vitro translated TIRC7
protein has been shown to be able to efficiently suppress in a dose
dependent manner the proliferation of T-cells in response to
alloactivation in a mixed lymphocyte culture or in response to
mitogens. TIRC7 is known to the person skilled in the art and
described, inter alia, in WO99/11782, Utku, Immunity 9 (1998),
509-518 and Heinemann, Genomics 57 (1999), 398-406, which also
disclose the amino and nucleic acid sequences of TIRC7.
[0010] As it was shown by Utku et al. (Immunity, 1998), polyclonal
antibodies against TIRC7 suppressed the proliferation of activated
T-cells in MLR in a dose dependent manner. While these promising
results suggested the therapeutic use of such antibodies, there was
a need for antibodies that have high binding specificity and
affinity, and that efficiently suppress, for example, T cell
proliferation thereby allowing the use of such antibodies at low
doses in order to circumvent possible HAMA responses in a subject.
Furthermore, such antibodies may have different or differently
pronounced effects on, e.g., cytokine production which can be
important in the treatment of certain immune response related
diseases, for example graft rejection.
[0011] In order to find antibodies which supply the needs mentioned
above, mice were immunized with peptides from several domains of
TIRC7, which were thought to represent putatively appropriate
antigens; see FIG. 1 of WO99/11782. However, while most of these
peptides proofed to be good antigens for raising polyclonal
antibodies, several attempts failed to produce stable hybridomas
which secreted antibodies with the desired binding affinity and/or
biological activity. However, with three (see Table 1 and SEQ ID
NOs: 9 to 11, infra) of six peptides derived from the sequence of
several hypothetically extracellular domains of TIRC7, the
inventors eventually succeeded with generating stable hybridomas
producing the desired monoclonal antibodies. Thus, 192 stable
antibody producing hybridomas were received and 42 antibodies were
tested; see FIG. 1. From those antibodies 15 antibodies were
selected which inhibited cell proliferation (FIG. 2, proliferation
assay) as well as the secretion of IFN.gamma. and IL-2 (FIG. 2) of
PHA-stimulated human PBMC of healthy donors below 30% calculated in
relation to the positive control (100%). Finally three antibodies
were selected, #9 and #17, both descended from fusions performed
with spleen cells of mice that had been immunized with peptides
derived from the largest extracellular loop of TIRC7, and #18, in
this case the peptide used for immunization was derived from the
extracellular C-Terminus of TIRC7 (FIG. 3); see also Table 1. In
accordance with the present invention, it could then surprisingly
shown that chimeric recombinant antibodies comprising the V.sub.H-
and V.sub.L-variable regions of the murine monoclonal antibodies
and either the human gamma or kappa constant region exhibit
substantially the same specificity, binding affinity and biological
activity as the murine donor antibodies.
[0012] Accordingly, the antibodies of the present invention are
expected to be useful in the modulation of immune responses.
Modulating the immune response, as for example by activating or
inhibiting the proliferation and/or differentiation of T-cells,
B-cells, NK cells, LAK cells, dendritic cells, monocytes,
macrophages or other immune system cells, may be useful in treating
autoimmune diseases, allergic diseases, and in transplantation
therapies where graft vs. host or host vs. graft effects may be
undesirable. The antibodies could also be immune stimulants in
settings such as cancer, infectious disease, sepsis, wound healing,
or immunization. Alternatively, they could be immune suppressants.
They could also be used to detect inflammation, and preferably
modulate inflammation by activating or inhibiting activation of
immune or inflammatory cells. A preferred method involves detecting
(and preferably modulating) inflammation in tissues such as
inflamed vasculature or leukocytes. Furthermore, the antibodies of
the present invention can be used to induce or maintain immune
unresponsiveness.
[0013] The term "immune unresponsiveness" comprises
non-unresponsiveness of immune cell subsets like T-cell or B-cells,
NK-cells, monocytes and/or macrophages.
[0014] The terms "treatment", "treating" and the like are used
herein to generally mean obtaining a desired pharmacological and/or
physiological effect. The effect may be prophylactic in terms of
completely or partially preventing a disease or symptom thereof
and/or may be therapeutic in terms of partially or completely
curing a disease and/or adverse effect attributed to the disease.
The term "treatment" as used herein covers any treatment of a
disease in a mammal, particularly a human, and includes: (a)
preventing the disease from occurring in a subject which may be
predisposed to the disease but has not yet been diagnosed as having
it; (b) inhibiting the disease, i.e. arresting its development; or
(c) relieving the disease, i.e. causing regression of the
disease.
[0015] Furthermore, the term "subject" as employed herein relates
to animals in need of amelioration, treatment and/or prevention of
immunological diseases as disclosed herein. Most preferably said
subject is a human.
[0016] Hence, the antibodies described herein can be used for any
application described for anti-TIRC7 antibodies before, in
particular if therapeutic and in vivo diagnostic uses are
envisaged; see for example WO99/11782 and co-pending PCT
application no. PCT/EP02/13384, the disclosure content of which is
hereby incorporated by reference.
[0017] Without intending to be bound by theory, it is believed that
the described anti-TIRC7 antibodies are capable of modulating the
function (e.g., signaling or adhesive activities) of TIRC7, its
family members and/or their ligands, for example by interfering
with the interaction of TIRC7 with its ligand. However,
irrespective the theory behind the molecular mechanism of action,
the antibody of the invention can be characterized by (1) having
binding affinity to TIRC7 in the order of at least 10.sup.-7M,
preferably at least 10.sup.-8M, more preferably at least
0.5.times.10.sup.-8M, still more preferably at least 10.sup.-8M,
and most preferably at least 10.sup.-9M or 10.sup.-10M and (2)
being capable of inhibiting proliferation of mitogen-stimulated
PBMCs in an assay as described in Example 1. Preferably, the
antibody of the invention and any binding fragment derived thereof
is capable of inhibiting the proliferation as well as the secretion
of IFNg and IL-2 of PHA-stimulated human PBMC of healthy donors
below 30% calculated in relation to the positive control (100%).
Most preferably, the antibody or binding fragment is capable of
inhibiting the proliferation of PHA-stimulated human PBMC of
healthy donors below 25% or even below 20% or more calculated in
relation to the positive control (100%).
[0018] Thus the present antibodies are preferably capable of
modulating, preferably inhibiting proliferation of peripheral blood
mononuclear cells (PBMCs). Preferably, the antibodies of the
present invention modulate at least one of the following (which are
functions of TIRC7 proteins and/or ligands thereof): activation of
neutrophils; activation or inhibition of T-cells, B-cells, NK
cells, LAK cells, dendritic cells, or other immune system cells;
proliferation and/or differentiation of T-cells, B-cells, NK cells,
LAK cells, dendritic cells, or other immune system cells;
proliferation and/or differentiation of epithelial cells such as
breast or intestinal/colonic epithelium cells or keratinocytes. In
addition these antibodies preferably capable of altering homotypic
and/or heterotypic adhesion among TIRC7 proteins (i.e., TIRC7
family members) or adhesion of TIRC7 proteins to other TIRC7
ligands.
[0019] The antibody of the invention can be a monoclonal antibody,
a single chain antibody, chimeric antibody, humanized antibody,
xenogeneic antibody, or a fragment and/or a chemically modified
derivative of any one thereof that specifically binds TIRC7 antigen
also including bispecific antibody, synthetic antibody, antibody
fragment, such as Fab, Fv or scFv fragments etc., or a chemically
modified derivative of any of these. Antibodies or fragments
thereof can be obtained by using methods which are described, e.g.,
in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press,
Cold Spring Harbor, 1988. When derivatives of said antibodies are
obtained by the phage display technique, surface plasmon resonance
as employed in the BIAcore system can be used to increase the
efficiency of phage antibodies which bind to the same epitope as
that of any one of the antibodies described herein (Schier, Human
Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol.
Methods 183 (1995), 7-13). The production of chimeric antibodies is
described, for example, in WO89/09622. Methods for the production
of humanized antibodies are described in, e.g., EP-A1 0 239 400 and
WO90/07861. A further source of antibodies to be utilized in
accordance with the present invention are so-called xenogeneic
antibodies. The general principle for the production of xenogeneic
antibodies such as human antibodies in mice is described in, e.g.,
WO 91/10741, WO 94/02602, WO 96/34096 and WO 96/33735. As discussed
above, the antibody of the invention may exist in a variety of
forms besides complete antibodies; including, for example, Fv, Fab
and F(ab)2, as well as in single chains; see e.g. WO88/09344. In
case of bispecific antibodies where one specificity is directed to
TIRC7 and the other preferably to a T cell antigen such as CD3, it
is advantageous if the binding site recognizing TIRC7 has a high
affinity in order to capture the antigen target cells. On the other
hand, the binding affinity of the binding site recognizing, e.g., a
T cell stimulatory molecule should be in the order of those of the
natural T cell receptor/ligand interaction or of that usually found
for the interaction of the T-cell costimulatory molecules with
their receptor.
[0020] The antibodies of the present invention or their
corresponding immunoglobulin chain(s) can be further modified using
conventional techniques known in the art, for example, by using
amino acid deletion(s), insertion(s), substitution(s), addition(s),
and/or recombination(s) and/or any other modification(s) known in
the art either alone or in combination. Methods for introducing
such modifications in the DNA sequence underlying the amino acid
sequence of an immunoglobulin chain are well known to the person
skilled in the art; see, e.g., Sambrook, Molecular Cloning A
Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y.
Modifications of the antibody of the invention include chemical
and/or enzymatic derivatizations at one or more constituent amino
acid, including side chain modifications, backbone modifications,
and N-- and C-terminal modifications including acetylation,
hydroxylation, methylation, amidation, and the attachment of
carbohydrate or lipid moieties, cofactors, and the like. Likewise,
the present invention encompasses chimeric proteins which comprise
the described anti-TIRC7 antibody or some fragment thereof at the
amino terminus fused to heterologous molecule such as an
immunostimulatory ligand at the carboxyl terminus; see, e.g.,
WO00/30680 for corresponding technical details.
[0021] Hence, the present invention relates to any antibody and
similar binding molecules which recognize the same epitope and with
substantially the same affinity, or at least 1/10 of the affinity
as the antibodies of the invention exemplified herein. Such
antibodies and binding molecules can be tested for their binding
specificity and affinity by for example by using peptide 6 and/or
competitive assays with the an antibody described in the
examples.
[0022] In a preferred embodiment, the antibody of the invention is
a chimeric or a humanized antibody. Chimeric antibodies are
antibodies whose light and heavy chain genes have been constructed,
typically by genetic engineering, from immunoglobulin gene segments
belonging to different species. For example, the variable (V)
segments of the genes from the mouse TIRC7 monoclonal antibody may
be joined to human constant (C) segments, such as .gamma.1 and
.gamma.3. A typical therapeutic chimeric antibody is thus a hybrid
protein consisting of the V or antigen-binding domain from a mouse
antibody and the C or effector domain from a human antibody,
although other mammalian species may be used as well if for example
veterinary application is envisaged. Human constant region DNA
sequences can be isolated in accordance with well known procedures
from a variety of human cells, but preferably immortalized B-cells
(see, Kabat op. cit. and WO87/02671). For example, the human kappa
immunoglobulin constant and J region genes and sequences are
described in Heiter, Cell 22 (1980), 197-207 and the nucleotide
sequence of a human immunoglobulin C gene is described in Ellison,
Nucl. Acids Res. 10 (1982), 4071, both of which are incorporated
herein by reference. In a particularly preferred embodiment, the
antibody of the invention comprises the amino acid sequence of the
V.sub.H and/or V.sub.L region as depicted in FIGS. 4 and 5, and 6
and 7, respectively.
[0023] In a further embodiment, the present invention relates to an
antigen or an epitope thereof which is recognized by an antibody of
the invention. Said antigen or epitope may be glycosylated,
unglycosylated or partially deglycosylated. As discussed herein and
explained in the examples, the present invention features antigens
which are particularly suited for eliciting an immune response. For
the identification and isolation of antigen and epitopes of the
invention conventional epitope mapping can be used; see, e.g.,
Harlow and Lane, supra. Furthermore, e.g., cDNA libraries can be
screened by injecting various cDNAs into oocytes, allowing
sufficient time for expression of the cDNA gene products to occur,
and testing for the presence of the desired cDNA expression
product, for example, by using the antibody of the invention.
Alternatively, a cDNA expression library in E. coli can be screened
indirectly for peptides having at least one epitope of the
invention using antibodies of the invention (Chang and Gottlieb, J.
Neurosci., 8:2123, 1988). After having revealed the structure of
such antigens the rational design of binding partners and/or
domains may be possible. For example, folding simulations and
computer redesign of structural motifs can be performed using
appropriate computer programs (Olszewski, Proteins 25 (1996),
286-299; Hoffman, Comput. Appl. Biosci. 11 (1995), 675-679).
Furthermore, computers can be used for the conformational and
energetic analysis of detailed protein models (Monge, J. Mol. Biol.
247 (1995), 995-1012; Renouf, Adv. Exp. Med. Biol. 376 (1995),
37-45).
[0024] Preferably, the antigen of the invention does not comprise
more that 50, preferably not more than 40, and still more
preferably not more 30 consecutive amino acids from TIRC7 protein.
Preferably, the antigens of the present invention have about 12 to
30 amino acids derived from TIRC7. In a most preferred embodiment,
said antigen comprises or consists of the amino acid sequence of
peptide 6 (DLPDASVNGWSSDE, SEQ ID NO: 9), peptide 7c
(DLPDASVNGWSSDEEKAGGLDDEE, SEQ ID NO: 10) and/or peptide 4
(VEFQNKFYSGTGYKLSPFDFAATD, SEQ ID NO: 11). This includes peptides
that have been modified or derivatized, such as by glycosylation,
acetylation, phosphorylation, and the like.
[0025] In another embodiment the present invention relates to a
polynucleotide encoding at least a variable region of an
immunoglobulin chain of any of the before described antibodies of
the invention. One form of immunoglobulin constitutes the basic
structural unit of an antibody. This form is a tetramer and
consists of two identical pairs of immunoglobulin chains, each pair
having one light and one heavy chain. In each pair, the light and
heavy chain variable regions or domains are together responsible
for binding to an antigen, and the constant regions are responsible
for the antibody effector functions. In addition to antibodies,
immunoglobulins may exist in a variety of other forms (including
less than full-length that retain the desired activities),
including, for example, Fv, Fab, and F(ab').sub.2, as well as
single chain antibodies (e.g., Huston, Proc. Nat. Acad. Sci. USA 85
(1988), 5879-5883 and Bird, Science 242 (1988), 423-426); see also
supra. An immunoglobulin light or heavy chain variable domain
consists of a "framework" region interrupted by three hypervariable
regions, also called CDRs; see supra. The antibodies of the present
invention can be produced by expressing recombinant DNA segments
encoding the heavy and light immunoglobulin chain(s) of the
antibody invention either alone or in combination.
[0026] The polynucleotide of the invention encoding the above
described antibody may be, e.g., DNA, cDNA, RNA or synthetically
produced DNA or RNA or a recombinantly produced chimeric nucleic
acid molecule comprising any of those polynucleotides either alone
or in combination. Preferably said polynucleotide is part of a
vector. Such vectors may comprise further genes such as marker
genes which allow for the selection of said vector in a suitable
host cell and under suitable conditions. Preferably, the
polynucleotide of the invention is operatively linked to expression
control sequences allowing expression in prokaryotic or eukaryotic
cells. Expression of said polynucleotide comprises transcription of
the polynucleotide into a translatable mRNA. Regulatory elements
ensuring expression in eukaryotic cells, preferably mammalian
cells, are well known to those skilled in the art. They usually
comprise regulatory sequences ensuring initiation of transcription
and optionally poly-A signals ensuring termination of transcription
and stabilization of the transcript. Additional regulatory elements
may include transcriptional as well as translational enhancers,
and/or naturally-associated or heterologous promoter regions. In
this respect, the person skilled in the art will readily appreciate
that the polynucleotides encoding at least the variable domain of
the light and/or heavy chain may encode the variable domains of
both immunoglobulin chains or only one. Likewise, said
polynucleotides may be under the control of the same promoter or
may be separately controlled for expression. Possible regulatory
elements permitting expression in prokaryotic host cells comprise,
e.g., the P.sub.L, lac, trp or tac promoter in E. coli, and
examples for regulatory elements permitting expression in
eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the
CMV-, SV40-, RSV-promoter (Rous sarcoma virus), CMV-enhancer,
SV40-enhancer or a globin intron in mammalian and other animal
cells. Beside elements which are responsible for the initiation of
transcription such regulatory elements may also comprise
transcription termination signals, such as the SV40-poly-A site or
the tk-poly-A site, downstream of the polynucleotide. Furthermore,
depending on the expression system used leader sequences capable of
directing the polypeptide to a cellular compartment or secreting it
into the medium may be added to the coding sequence of the
polynucleotide of the invention and are well known in the art. The
leader sequence(s) is (are) assembled in appropriate phase with
translation, initiation and termination sequences, and preferably,
a leader sequence capable of directing secretion of translated
protein, or a portion thereof, into the periplasmic space or
extracellular medium. Optionally, the heterologous sequence can
encode a fusion protein including an C-- or N-terminal
identification peptide imparting desired characteristics, e.g.,
stabilization or simplified purification of expressed recombinant
product. In this context, suitable expression vectors are known in
the art such as Okayama-Berg cDNA expression vector pcDV1
(Pharmacia), pCDM8, pRc/CMV, pcDNA1, pcDNA3 (In-vitrogene), or
pSPORT1 (GIBCO BRL).
[0027] Preferably, the expression control sequences will be
eukaryotic promoter systems in vectors capable of transforming or
transfecting eukaryotic host cells, but control sequences for
prokaryotic hosts may also be used. Once the vector has been
incorporated into the appropriate host, the host is maintained
under conditions suitable for high level expression of the
nucleotide sequences, and, as desired, the collection and
purification of the immunoglobulin light chains, heavy chains,
light/heavy chain dimers or intact antibodies, binding fragments or
other immunoglobulin forms may follow; see, Beychok, Cells of
Immunoglobulin Synthesis, Academic Press, N.Y., (1979); see also,
e.g., the appended examples.
[0028] As described above, the polynucleotide of the invention can
be used alone or as part of a vector to express the (poly)peptide
of the invention in cells, for, e.g., gene therapy or diagnostics
of diseases related to immune diseases. The polynucleotides or
vectors of the invention are introduced into the cells which in
turn produce the antibody. Gene therapy, which is based on
introducing therapeutic genes into cells by ex-vivo or in-vivo
techniques is one of the most important applications of gene
transfer. Suitable vectors and methods for in-vitro or in-vivo gene
therapy are described in the literature and are known to the person
skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996),
534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science
256 (1992), 808-813; Isner, Lancet 348 (1996), 370-374; Muhlhauser,
Circ. Res. 77 (1995), 1077-1086; Wang, Nature Medicine 2 (1996),
714-716; WO94/29469; WO 97/00957 or Schaper, Current Opinion in
Biotechnology 7 (1996), 635-640, and references cited therein. The
polynucleotides and vectors of the invention may be designed for
direct introduction or for introduction via liposomes, or viral
vectors (e.g. adenoviral, retroviral) into the cell. Preferably,
said cell is a germ line cell, embryonic cell, or egg cell or
derived therefrom, most preferably said cell is a stem cell.
[0029] Furthermore, the present invention relates to vectors,
particularly plasmids, cosmids, viruses and bacteriophages used
conventionally in genetic engineering that comprise a
polynucleotide encoding a variable domain of an immunoglobulin
chain of an antibody of the invention; optionally in combination
with a polynucleotide of the invention that encodes the variable
domain of the other immunoglobulin chain of the antibody of the
invention. Preferably, said vector is an expression vector and/or a
gene transfer or targeting vector. Expression vectors derived from
viruses such as retroviruses, vaccinia virus, adeno-associated
virus, herpes viruses, or bovine papilloma virus, may be used for
delivery of the polynucleotides or vector of the invention into
targeted cell population. Methods which are well known to those
skilled in the art can be used to construct recombinant viral
vectors; see, for example, the techniques described in Sambrook,
Molecular Cloning A Laboratory Manual, Cold Spring Harbor
Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular
Biology, Green Publishing Associates and Wiley Interscience, N.Y.
(1994). Alternatively, the polynucleotides and vectors of the
invention can be reconstituted into liposomes for delivery to
target cells. The vectors containing the polynucleotides of the
invention (e.g., the heavy and/or light variable domain(s) of the
immunoglobulin chains encoding sequences and expression control
sequences) can be transferred into the host cell by well-known
methods, which vary depending on the type of cellular host. For
example, calcium chloride transfection is commonly utilized for
prokaryotic cells, whereas calcium phosphate treatment or
electroporation may be used for other cellular hosts; see Sambrook,
supra.
[0030] The present invention furthermore relates to host cells
transformed with a polynucleotide or vector of the invention. Said
host cell may be a prokaryotic or eukaryotic cell. The
polynucleotide or vector of the invention which is present in the
host cell may either be integrated into the genome of the host cell
or it may be maintained extrachromosomally. The host cell can be
any prokaryotic or eukaryotic cell, such as a bacterial, insect,
fungal, plant, animal or human cell. Preferred fungal cells are,
for example, those of the genus Saccharomyces, in particular those
of the species S. cerevisiae. The term "prokaryotic" is meant to
include all bacteria which can be transformed or transfected with a
DNA or RNA molecules for the expression of an antibody of the
invention or the corresponding immunoglobulin chains. Prokaryotic
hosts may include gram negative as well as gram positive bacteria
such as, for example, E. coli, S. typhimurium, Serratia marcescens
and Bacillus subtilis. The term "eukaryotic" is meant to include
yeast, higher plant, insect and preferably mammalian cells, most
preferably NSO and CHO cells. Depending upon the host employed in a
recombinant production procedure, the antibodies or immunoglobulin
chains encoded by the polynucleotide of the present invention may
be glycosylated or may be non-glycosylated. Antibodies of the
invention or the corresponding immunoglobulin chains may also
include an initial methionine amino acid residue. A polynucleotide
of the invention can be used to transform or transfect the host
using any of the techniques commonly known to those of ordinary
skill in the art. Furthermore, methods for preparing fused,
operably linked genes and expressing them in, e.g., mammalian cells
and bacteria are well-known in the art (Sambrook, Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y., 1989). The genetic constructs and methods
described therein can be utilized for expression of the antibody of
the invention or the corresponding immunoglobulin chains in
eukaryotic or prokaryotic hosts. In general, expression vectors
containing promoter sequences which facilitate the efficient
transcription of the inserted polynucleotide are used in connection
with the host. The expression vector typically contains an origin
of replication, a promoter, and a terminator, as well as specific
genes which are capable of providing phenotypic selection of the
transformed cells. Suitable source cells for the DNA sequences and
host cells for immunoglobulin expression and secretion can be
obtained from a number of sources, such as the American Type
Culture Collection ("Catalogue of Cell Lines and Hybridomas," Fifth
edition (1985) Rockville, Md., U.S.A., which is incorporated herein
by reference). Furthermore, transgenic animals, preferably mammals,
comprising cells of the invention may be used for the large scale
production of the antibody of the invention.
[0031] Thus, in a further embodiment, the present invention relates
to a method for the production of an antibody capable of inhibiting
proliferation of peripheral blood mononuclear cells (PBMCs) or a
functional fragment or immunglobulin chain(s) thereof comprising
[0032] (a) culturing the cell of the invention; and [0033] (b)
isolating said antibody or functional fragment or immunoglobulin
chain(s) thereof from the culture,
[0034] The transformed hosts can be grown in fermentors and
cultured according to techniques known in the art to achieve
optimal cell growth. Once expressed, the whole antibodies, their
dimers, individual light and heavy chains, or other immunoglobulin
forms of the present invention, can be purified according to
standard procedures of the art, including ammonium sulfate
precipitation, affinity columns, column chromatography, gel
electrophoresis and the like; see, Scopes, "Protein Purification",
Springer-Verlag, N.Y. (1982). The antibody or its corresponding
immunoglobulin chain(s) of the invention can then be isolated from
the growth medium, cellular lysates, or cellular membrane
fractions. The isolation and purification of the, e.g., microbially
expressed antibodies or immunoglobulin chains of the invention may
be by any conventional means such as, for example, preparative
chromatographic separations and immunological separations such as
those involving the use of monoclonal or polyclonal antibodies
directed, e.g., against the constant region of the antibody of the
invention. It will be apparent to those skilled in the art that the
antibodies of the invention can be further coupled to other
moieties for, e.g., drug targeting and imaging applications. Such
coupling may be conducted chemically after expression of the
antibody or antigen to site of attachment or the coupling product
may be engineered into the antibody or antigen of the invention at
the DNA level. The DNAs are then expressed in a suitable host
system, and the expressed proteins are collected and renatured, if
necessary.
[0035] Substantially pure immunoglobulins of at least about 90 to
95% homogeneity are preferred, and 98 to 99% or more homogeneity
most preferred, for pharmaceutical uses. Once purified, partially
or to homogeneity as desired, the antibodies may then be used
therapeutically (including extracorporeally) or in developing and
performing assay procedures.
[0036] The present invention also involves a method for producing
cells capable of expressing an antibody of the invention or its
corresponding immunoglobulin chain(s) comprising genetically
engineering cells with the polynucleotide or with the vector of the
invention. The cells obtainable by the method of the invention can
be used, for example, to test the interaction of the antibody of
the invention with its antigen.
[0037] Furthermore, the invention relates to an antibody, an
immunoglobulin chain thereof and to a binding fragment thereof
encoded by a polynucleotide according to the invention or
obtainable by the above-described methods or from cells produced by
the method described above. The antibodies of the present invention
will typically find use individually in treating substantially any
disease susceptible to monoclonal antibody-based therapy. In
particular, the immunoglobulins can be used as immunosuppressive
agents. For an antibody of the invention, typical disease states
suitable for treatment include inflammatory symptoms. The
antibodies can be used therapeutically in, e.g., patients suffering
an diseases related to immune response; see supra. Such therapy can
be accomplished by, for example, the administration of antibodies,
antigens or epitopes of the invention. Such administration can
utilize unlabeled as well as labeled antibodies or antigens.
Labeling agents can be coupled either directly or indirectly to the
antibodies or antigens of the invention. One example of indirect
coupling is by use of a spacer moiety. Furthermore, the antibodies
of the present invention can comprise a further domain, said domain
being linked by covalent or non-covalent bonds. The linkage can be
based on genetic fusion according to the methods known in the art
and described above or can be performed by, e.g., chemical
cross-linking as described in, e.g., WO 94/04686. The additional
domain present in the fusion protein comprising the antibody of the
invention may preferably be linked by a flexible linker,
advantageously a polypeptide linker, wherein said polypeptide
linker comprises plural, hydrophilic, peptide-bonded amino acids of
a length sufficient to span the distance between the C-terminal end
of said further domain and the N-terminal end of the antibody of
the invention or vice versa. The above described fusion protein may
further comprise a cleavable linker or cleavage site for
proteinases. These spacer moieties, in turn, can be either
insoluble or soluble (Diener et al., Science, 231:148, 1986) and
can be selected to enable drug release from the antigen at the
target site. Examples of therapeutic agents which can be coupled to
the antibodies, antigens and epitopes of the invention for
immunotherapy are drugs, radioisotopes, lectins, and toxins. The
drugs with which can be conjugated to the antibodies, antigens and
epitopes of the invention include compounds which are classically
referred to as drugs such as mitomycin C, daunorubicin, and
vinblastine. In using radioisotopically conjugated antibodies,
antigens or epitopes of the invention for, e.g., immunotherapy,
certain isotopes may be more preferable than others depending on
such factors as leukocyte distribution as well as stability and
emission. Depending on the autoimmune response, some emitters may
be preferable to others. In general, .alpha. and .beta.
particle-emitting radioisotopes are preferred in immunotherapy.
Preferred are short range, high energy a emitters such as
.sup.212Bi. Examples of radioisotopes which can be bound to the
antibodies, antigens or epitopes of the invention for therapeutic
purposes are .sup.125I, .sup.131I, .sup.90Y, .sup.67Cu, .sup.212Bi,
.sup.212At, .sup.211Pb, .sup.47Sc, .sup.109Pd and .sup.188Re. Other
therapeutic agents which can be coupled to the antibody, antigen or
epitope of the invention, as well as ex vivo and in vivo
therapeutic protocols, are known, or can be easily ascertained, by
those of ordinary skill in the art. Wherever appropriate the person
skilled in the art may use a polynucleotide of the invention
encoding any one of the above described antibodies, antigens or the
corresponding vectors instead of the proteinaeous material
itself.
[0038] Moreover, the present invention relates to compositions
comprising the aforementioned antibody, antigen or epitope of the
invention or chemical derivatives thereof, or the polynucleotide,
vector or cell of the invention. The composition of the present
invention may further comprise a pharmaceutically acceptable
carrier. The term "chemical derivative" describes a molecule that
contains additional chemical moieties that are not normally a part
of the base molecule. Such moieties may improve the solubility,
half-life, absorption, etc. of the base molecule. Alternatively the
moieties may attenuate undesirable side effects of the base
molecule or decrease the toxicity of the base molecule. Examples of
such moieties are described in a variety of texts, such as
Remington's Pharmaceutical Sciences. Examples of suitable
pharmaceutical carriers are well known in the art and include
phosphate buffered saline solutions, water, emulsions, such as
oil/water emulsions, various types of wetting agents, sterile
solutions etc. Compositions comprising such carriers can be
formulated by well known conventional methods. These pharmaceutical
compositions can be administered to the subject at a suitable dose.
Administration of the suitable compositions may be effected by
different ways, e.g., by intravenous, intraperitoneal,
subcutaneous, intramuscular, topical or intradermal administration.
Aerosol formulations such as nasal spray formulations include
purified aqueous or other solutions of the active agent with
preservative agents and isotonic agents. Such formulations are
preferably adjusted to a pH and isotonic state compatible with the
nasal mucous membranes. Formulations for rectal or vaginal
administration may be presented as a suppository with a suitable
carrier.
[0039] The dosage regimen will be determined by the attending
physician and clinical factors. As is well known in the medical
arts, dosages for any one patient depends upon many factors,
including the patient's size, body surface area, age, the
particular compound to be administered, sex, time and route of
administration, general health, and other drugs being administered
concurrently. A typical dose can be, for example, in the range of
0.001 to 1000 .mu.g (or of nucleic acid for expression or for
inhibition of expression in this range); however, doses below or
above this exemplary range are envisioned, especially considering
the aforementioned factors. Generally, the regimen as a regular
administration of the pharmaceutical composition should be in the
range of 1 .mu.g to 10 mg units per day. If the regimen is a
continuous infusion, it should also be in the range of 1 .mu.g to
10 mg units per kilogram of body weight per minute, respectively.
Progress can be monitored by periodic assessment. Dosages will vary
but a preferred dosage for intravenous administration of DNA is
from approximately 10.sup.6 to 10.sup.12 copies of the DNA
molecule. The compositions of the invention may be administered
locally or systemically. Administration will generally be
parenterally, e.g., intravenously; DNA may also be administered
directly to the target site, e.g., by biolistic delivery to an
internal or external target site or by catheter to a site in an
artery. Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's, or fixed oils. Intravenous vehicles include
fluid and nutrient replenishers, electrolyte replenishers (such as
those based on Ringer's dextrose), and the like. Preservatives and
other additives may also be present such as, for example,
antimicrobials, anti-oxidants, chelating agents, and inert gases
and the like. Furthermore, the pharmaceutical composition of the
invention may comprise further agents such as interleukins or
interferons depending on the intended use of the pharmaceutical
composition.
[0040] In a preferred embodiment, the pharmaceutical composition of
the present invention comprises at least one second agent,
preferably an agent which inhibits T-cell stimulation depending on
the intended use. Such agents include, for example, molecules that
are capable of blocking or mimicking receptor/ligand interaction or
the like which leads to T-cell suppression. Such agents comprise
those blocking the activity of, e.g., costimulatory molecules, such
as anti-TIRC7 antibodies, anti-TNF-.alpha. antibodies, integrins,
Ig-superfamily molecules, selectins as well as drugs blocking
chemokines and their respective receptor interactions, drugs
blocking IL2/IL2-receptor interaction and other conventional
immunosuppressive drugs such as IL-2R mAbs, IL-Toxins and
IL-Muteins. Examples for costimulatory molecules and their ligands
are described in the prior art, e.g., in Schwartz, Cell 71 (1992),
1065-1068. The interruption of the receptor/ligand interactions by
using mAbs or soluble CTLA41g for the interaction between CD28 to
the B7-2 and CTLA4 to B7-1 and B7-2 are described in Blazar, J.
Immunol. 157 (1996), 3250-3259; Bluestone, Immunity 2 (1995),
555-559; Linsley, Science 257 (1992), 792-95. Examples for blocking
the receptor/ligand interaction by using mAbs to CD40 or CD40L are
reported by Burden, Nature 381 (1996), 434-435; Kirk, Proc. Natl.
Acad. Sci. USA 94 (1997), 8789-8794. CD2 antigen and its ligand
LFA-3 are described in Bagogui Li et al., review in Adhesion
Molecules, Fusion proteins, Novel Peptides, and Monoclonal
Antibodies, Recent Developments in Transplantation Medicine, Vol.
II, 1995, Physicians&Scientists Publishing Co., Inc. and
blocking of their interaction by using of mAbs (anti-Leu-5b, OKT11,
T11) is reported in Bromberg, Transplantation 51 (1991) 219-225 or
CD2.1gG1 fusion protein. The use of monoclonal Abs agains CD4
molecule is described in Cosimi, Surgery 108 (1990), 406-414. CD47
blockade by mAbs is described by Rheinhold, J. Exp. Med. 185
(1997), 1-11. Integrins and Ig-superfamily molecules include LFA-1
with its ligand ICAM-1, -2, -3, Mac-1 with ist ligand ICAM-1, -3;
ICAM-1 with its ligand LFA-1, Mac-1, CD43; ICAM-2 with ist ligand
LFA-1; ICAM-3 with its ligand LFA-1, Mac-1; V.sub.LA4 and VCAM-1
see, e.g., David, Adams, review in Adhesion Molecules, Fusion
proteins, Novel Peptides, and Monoclonal Antibodies, Recent
Developments in Transplantation Medicine, Vol. II, 1995,
Physicians&Scientists Publishing Co., Inc.; Isobe, Science, 255
(1992), 1125-1127; Cosimi, J. Immunology 144 (1990), 4604-4612;
Hynes, Cell 69 (1992),11-25.
[0041] Furthermore selectively interfering agents with V.sub.LA-4
mAbs to the alpha4 integrin chain (CD49d) can be used, beta1
integrin chain (CD29), or an activation-induced neo-epitope of
V.sub.LA-4 as well as soluble V.sub.LA-4 ligands such as soluble
fibronectin or its relevant peptide (GPEILDVPST), or soluble VCAM-1
or its relevant peptide. More selectively blocking agents are
antisense oligonucleotides, designed to selectively hybridize with
cytoplasmic alpha4, beta1, or VCAM-1 mRNA; Fedoseyeva, J. Immunol.
57 (1994), 606-612. Another example is the drug pentoxifylline
(PTX) that is able to block expression of VCAM-1; Besler, J.
Leukoc. Biol. 40 (1986), 747-754. Furthermore, VCAM-1 mAb, M/K-2,
anti-murine, for example could prolong allograft survival, Orosz,
Transplantation, 56 (1993), 453-460. Blocking of members of the
integrin family and their ligands by using mAbs is decribed in
Kupiec-Weglinski, review in Adhesion Molecules, Fusion proteins,
Novel Peptides, and Monoclonal Antibodies, Recent Developments in
Transplantation Medicine, Vol. II, 1995, Physicians&Scientists
Publishing Co., Inc. Selectins, e.g., L-selectin (CD62L),
E-selectin (CD62E), P-selectin (CD62P) have been described in
Forrest and Paulson, Selectin family of adhesion molecules. In:
Granger and Schmid-Schonbein, eds. Physiology and Pathophysiology
of Leukocyte Adhesion. New York, Oxford Press, 1995, pp 68-146. The
combination of conventional immunosuppressive drugs, e.g., ATG,
ALG, OKT3, Azathioprine, Mycophenylate, Mofetyl, Cyclosporin A,
FK506, Sirolimus (Rapamune), Corticosteroids may be used as
decribed in Cosimi, Transplantation 32 (1981), 535-539; Shield,
Transplantation 38 (1984), 695-701, and Graft, June 2001, Vol 4
(4). The interruption of chemokines and interactions with their
respective receptor by using mAbs is reviewed in Luster,
Chemokines-chemotactic cytokines that mediate inflammation, New
Engl. J. Med. February (1998), 436-445. Thus, any agent as defined
above and referenced by way of example can be used in accordance
with the pharmaceutical composition of the invention or the methods
and uses described herein.
[0042] Furthermore, the pharmaceutical composition may also be
formulated as a vaccine, for example, if the pharmaceutical
composition of the invention comprises an antigen as described
above that is capable of eliciting an effective immune response
against TIRC7. Advantageously, the pharmaceutical composition of
the invention is intended for use in organ transplantation.
[0043] Therapeutic or diagnostic compositions of the invention are
administered to an individual in a therapeutically effective dose
sufficient to treat or diagnose disorders in which modulation of
TIRC7-related activity is indicated. The effective amount may vary
according to a variety of factors such as the individual's
condition, weight, sex and age. Other factors include the mode of
administration. The pharmaceutical compositions may be provided to
the individuals by a variety of routes such as by intracoronary,
intraperitoneal, subcutaneous, intravenous, transdermal,
intrasynovial, intramuscular or oral routes. In addition,
co-administration or sequential administration of other agents may
be desirable.
[0044] A therapeutically effective dose refers to that amount of
antibodies, antigens, polynucleotides and vectors of the invention
ameliorate the symptoms or condition. Therapeutic efficacy and
toxicity of such compounds can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals,
e.g., ED50 (the dose therapeutically effective in 50% of the
population) and LD50 (the dose lethal to 50% of the population).
The dose ratio between therapeutic and toxic effects is the
therapeutic index, and it can be expressed as the ratio,
LD50/ED50.
[0045] Thus, the present invention relates to the use of the
antibody and the antigen of the invention for the preparation of a
pharmaceutical composition for inhibition of an immune response,
preferably for the treatment of graft versus host disease,
autoimmune diseases, allergic diseases, infectious diseases,
sepsis, for the treatment of tumors, for the improvement of wound
healing or for inducing or maintaining immune unresponsiveness in a
subject; see also supra.
[0046] Accordingly, the present invention also relates to a method
of modulating the immune response in a subject in need thereof,
comprising administering the antibody or the antigen of the
invention. Compositions comprising the antibody or the antigen of
this invention can be added to cells in culture (in vitro) or used
to treat patients, such as mammals (in vivo). Where the antibody or
the antigen are used to treat a patient, the polypeptide is
preferably combined in a pharmaceutical composition with a
pharmaceutically acceptable carrier such as a larger molecule to
promote polypeptide stability or a pharmaceutically acceptable
buffer that serves as a carrier for the antibodies that has more
than one antibody coupled to a single entity. The methods of the
invention include administering to a patient, preferably a mammal,
and more preferably a human, the composition of the invention in an
amount effective to produce the desired effect. The antibody or the
antigen can be administered as a single dose or in multiple doses.
Useful dosages of the active agents can be determined by comparing
their in vitro activity and the in vivo activity in animal models.
Methods for extrapolation of effective dosages in mice, and other
animals, to humans are known in the art. The present invention also
provides a method of modulating (e.g., activating or inhibiting)
immune cell (e.g., T-cells, B-cells, NK cells, LAK cells, or
dendritic cells) activation, proliferation, and/or differentiation
that includes contacting an immune cell with a antibody or the
antigen described above.
[0047] From the foregoing, it is evident that the present invention
encompasses any use of a ligand binding molecule comprising at
least one CDR of the above described antibody, in particular for
diagnosing and/or treatment of a disorder related to the aberrant
expression or malfunction of T-cell immune response cDNA 7 (TIRC7).
Preferably, said ligand binding molecule is an antibody of the
present invention or an immunoglobulin chain thereof.
[0048] The biological activity of the antibodies identified here
suggests that they have sufficient affinity to make them potential
candidates for drug localization to cells expressing the
appropriate surface structures. This targeting and binding to cells
could be useful for the delivery of therapeutically active agents
(including targeting drugs, DNA sequences, RNA sequences, lipids,
proteins (e.g., human growth factors)) and gene therapy/gene
delivery. More preferably, the therapeutically active agent is an
anti-inflammatory agent.
[0049] Molecules/particles with an anti-TIRC7 antibody would bind
specifically to cells/tissues expressing TIRC7, and therefore could
have diagnostic and therapeutic use. Thus, the antibody or the
antigen of the present invention can be labeled (e.g., fluorescent,
radioactive, enzyme, nuclear magnetic) and used to detect specific
targets in vivo or in vitro including "immunochemistry" like assays
in vitro. In vivo they could be used in a manner similar to nuclear
medicine imaging techniques to detect tissues, cells, or other
material expressing TIRC7. Another method involves delivering a
therapeutically active agent to a patient. The method includes
administering at least one antibody or the antigen and the
therapeutically active agent to a patient. Preferably, the
therapeutically active agent is selected from drugs, DNA sequences,
RNA sequences, proteins, lipids, and combinations thereof. More
preferably, the therapeutically active agent is an antibacterial
agent, anti-inflammatory agent, or antineoplastic agent.
[0050] In another embodiment the present invention relates to a
diagnostic composition comprising any one of the above described
the antibodies, antigens, polynucleotides, vectors or cells of the
invention and optionally suitable means for detection. The antigens
and antibodies of the invention are, for example, suited for use in
immunoassays in which they can be utilized in liquid phase or bound
to a solid phase carrier. Examples of immunoassays which can
utilize the antigen of the invention are competitive and
non-competitive immunoassays in either a direct or indirect format.
Examples of such immunoassays are the radioimmunoassay (RIA), the
sandwich (immunometric assay) and the Western blot assay. The
antigens and antibodies of the invention can be bound to many
different carriers and used to isolate cells specifically bound to
said polypeptides. Examples of well-known carriers include glass,
polystyrene, polyvinyl chloride, polypropylene, polyethylene,
polycarbonate, dextran, nylon, amyloses, natural and modified
celluloses, polyacrylamides, agaroses, and magnetite. The nature of
the carrier can be either soluble or insoluble for the purposes of
the invention. There are many different labels and methods of
labeling known to those of ordinary skill in the art. Examples of
the types of labels which can be used in the present invention
include enzymes, radioisotopes, colloidal metals, fluorescent
compounds, chemiluminescent compounds, and bioluminescent
compounds; see also the embodiments discussed hereinabove.
[0051] By a further embodiment, the antibodies of the invention may
also be used in a method for the diagnosis of TIRC7 related
diseases in an individual by obtaining a body fluid sample from the
tested individual which may be a blood sample, a lymph sample or
any other body fluid sample and contacting the body fluid sample
with an antibody of the invention under conditions enabling the
formation of antibody-antigen complexes. The level of such
complexes is then determined by methods known in the art, a level
significantly higher than that formed in a control sample
indicating the disease in the tested individual. In the same
manner, the specific antigen bound by the antibodies of the
invention may also be used. Thus, the present invention relates to
an in vitro immunoassay comprising the antibody or the antigen of
the invention.
[0052] Furthermore, the present invention relates to an
oligonucleotide comprising a nucleotide sequence of any one of SEQ
ID NOs: 12 to 40 and their use for the cloning of an anti-TIRC7
antibody; see the appended examples.
[0053] These and other embodiments are disclosed and encompassed by
the description and Examples of the present invention. Further
literature concerning any one of the antibodies, methods, uses and
compounds to be employed in accordance with the present invention
may be retrieved from public libraries and databases, using for
example electronic devices. For example the public database
"Medline" may be utilized, which is hosted by the National Center
for Biotechnology Information and/or the National Library of
Medicine at the National Institutes of Health. Further databases
and web addresses, such as those of the European Bioinformatics
Institute (EBI), which is part of the European Molecular Biology
Laboratory (EMBL) are known to the person skilled in the art and
can also be obtained using internet search engines. An overview of
patent information in biotechnology and a survey of relevant
sources of patent information useful for retrospective searching
and for current awareness is given in Berks, TIBTECH 12 (1994),
352-364.
[0054] The above disclosure generally describes the present
invention. A more complete understanding can be obtained by
reference to the following specific examples which are provided
herein for purposes of illustration only and are not intended to
limit the scope of the invention.
[0055] The Figures show:
[0056] FIG. 1: Functional assays in the presence of TIRC7
antibodies; see Example 1.
[0057] FIG. 2: Functional assays in the presence of 15 selected
TIRC7 mAbs; see Example 1.
[0058] FIG. 3: Functional assays in the presence of three selected
therapeutic TIRC7 mAbs; see Example 1 and Table 1.
[0059] FIG. 4: V.sub.H sequence of clone 9 (Metiliximab) (CDRs are
underlined).
[0060] FIG. 5: V.sub.L sequence of clone 9 (Metiliximab) (CDRs are
underlined).
[0061] FIG. 6: V.sub.H sequence of clone 17-1 (Neliximab) (CDRs are
underlined).
[0062] FIG. 7: V.sub.L sequence of clone 17-1 (Neliximab) (CDRs are
underlined).
[0063] FIG. 8: Affinity measurement of monoclonal antibodies: 1.
Affinity measurement of murine and chimeric antibodies against Vol
6 had been done by the method of Friquet et al 1985. Thereby, the
antibody (in nM concentration) had been titrated with increasing
concentrations of Vol 6 until equilibrium had been reached. The
fraction of free antibody had been determined by ELISA. Symbols of
the Klotz-Blot representation: a=antigen concentration (peptide 6)
in nM, b=bound antibody. Bivalency of the antibodies had been
considered by blotting b.sup.1/2 against 1/a.
[0064] FIG. 9: Functional assays in the presence of chimerized
(termed humanized in FIG. 9 because of their human constant region,
but chimerized is meant) therapeutic TIRC7 mAb. Shown is the
functional capacity of inhibition of the chimerized anti-TIRC7 mAb
antibody on T cell proliferation (proliferation assay) as well as
IFN-gamma cytokine inhibition (cytokine ELISA) after 48 h
activation of human T cells in the presence of mitogen in a dose
dependent manner.
[0065] The examples illustrate the invention.
EXAMPLE 1
Generation and Selection of Monoclonal Antibodies Directed Against
TIRC7
[0066] Balb/c-mice were immunized in presence of Freunds adjuvans
with one of six peptides derived from the sequence of several
hypothetically extracellular domains of TIRC7. Priming of mice with
antigen was followed by several booster injections over a period of
3 months. Fusion of spleen cells with SP2/0-Ag14 myeloma cells was
carried out according to the PEG-fusion technique. All together 15
fusions were performed and pursued successfully. After 3 weeks of
selection in HAT-media, repeated separation of the cells according
to the limiting-dilution method and screening of the supernatants
using the ELISA technique 192 stable antibody producing
hybribodomas were received. Determination of the antibody isotype
revealed that 140 of 192 monoclonal antibodies were IgM antibodies
whereas 52 were IgG antibodies. All 52 IgG antibody producing
hybridomas were thawed, separated once more and tested regarding
their IgG-production. Hybridomas which produced less than 5 .mu.g
IgG per ml supernatant after cell death were excluded.
[0067] 42 antibodies were produced in small volumes of 150-200 ml
supernatant and purified using protein A or protein G on a HPLC
affinity chromatographic column. Purified antibodies were tested
regarding their capacity to inhibit immune response to mitogens
(FIG. 1, proliferation assay) as well as their effects on cytokine
expression in the supernatants of 48 h activated human cells (FIG.
1, IFNg and hIL 2 ELISA).
[0068] Radioactive Proliferation Assay--Incorporation of
.sup.3H-Thymidine:
[0069] PBMC of healthy donors were isolated according to the
Ficoll-Paque density centrifugation protocol. Samples of 50000
PBMC's/well were stimulated with PHA (1 .mu.g/ml) and incubated for
48 h at 5% CO.sub.2, 37.degree. C. in presence of TIRC7-antibodies
and IgG-control antibodies in a total volume of 100 .mu.l/well.
Samples were run in triplicates on 96 well-microtiter-plates
(MTPs). After 48 h 0.5 .mu.Ci .sup.3H-thymidine per well were added
and the cells were reincubated for additional 18 h. Cells were
harvested and lysed using a cell harvester and collected on
nitrocellulose-filter-MTP's. Plates were dried at room temperature
for 4 h. To enhance the radioactive signal produced by the samples
a scintillation fluid was added and counts per minute (cpm) were
measured with a beta counter.
[0070] Quantitation of Secreted Cytokines in PBMC-Supernatants:
[0071] PBMC of healthy donors were isolated according to the
Ficoll-Paque density centrifugation protocol. Samples of 50000
PBMCs/well were stimulated with PHA (1 .mu.g/ml) and incubated for
48 h at 5% CO.sub.2, 37.degree. C. in presence of TIRC7-antibodies
and IgG-control antibodies in a total volume of 100 .mu.l/well.
Samples were run in triplicates on 96 well-microtiter-plates
(MTPs). After 48 h MTPs were centrifuged at 300.times.g for 10 min
and supernatants collected from the wells. The quantitation of
cytokines in the supernatant was carried out on
anti-cytokine-antibody-coated microtiter strips provided with the
Cytoscreen.RTM. ELISA Kit, Biosource. The formerly collected
supernatants and diluted standards were incubated in presence of a
biotinylated secondary antibody recognizing the specific cytokine
for 1.5-3 h at room temperature on these strips depending on the
detemined cytokine. Afterwards excessive secondary antibody was
removed by washing 3 times with washing buffer. A
streptavidin-peroxidase conjugate was added and incubated for 45
min-1 h at room temperature. Excessive conjugate was removed by
washing. TMB-substrate-solution was added and the strips incubated
for additional 30 min in the dark followed by the addition of stop
solution. The colour development was measured at 450 nm and the
numbers were statistically analyzed.
[0072] The first functional screen led to 15 antibodies which
inhibited the proliferation (FIG. 2, proliferation assay) as well
as the secretion of IFN.gamma. and IL-2 (FIG. 2) of PHA-stimulated
human PBMC of healthy donors below 30% calculated in relation to
the positive control (100%).
[0073] The next selection process was performed based on production
stability of the hybridoma, stability of the antibody,
immunoprecipitating qualities and immunofluorescence staining.
Finally three antibodies were selected, #9 and #17, both descended
from fusions performed with spleen cells of mice that had been
immunized with peptides derived from the largest extracellular loop
of TIRC7, and #18, in this case the peptide used for immunization
was derived from the extracellular C-Terminus of TIRC7 (FIG. 3).
The following table shows the IgG-Isotype and peptide used for
immunization for the selected 3 antibodies: TABLE-US-00001 TABLE 1
Isotype determination antibody Isotype Peptide #9 IgG1, .kappa.
Peptide6 #17 IgG1, .kappa. Peptide7c #18 IgG2b, .kappa.
Peptide4
[0074] These antibodies have been further investigated.
Furthermore, in the antibody of Clone 9 has been designated
Metiliximab and that of Clone 17 has been designated Neliximab.
EXAMPLE 2
Development of Chimeric Antibodies
[0075] 1. Identification of the V.sub.H and V.sub.L Regions of the
Antibodies Metiliximab and Neliximab
[0076] 1.1. RNA isolation. As a RNA source hybridoma cells were
used expressing the antibodies described in Example 1, supra.
Isolation was done with the RNA isolation colums of QIAGEN (Mini)
according to the manufactors instructions.
[0077] 1.2. cDNA synthesis. cDNA-synthesis was done with total RNA:
3 .mu.g total RNA in 17 .mu.l volume was incubated with 2 .mu.l
cDNA-Primer mentioned in Table 2 and incubated for 10 min at
75.degree. C. TABLE-US-00002 TABLE 2 Primer sequences for
cDNA-synthesis and amplification of murine variable regions
(V.sub.H and V.sub.L) A: primer for cDNA-synthesis: of the V.sub.H
regions MOCG12Forcor: CAC AAT TTT CTT GTC CAC CTT GGT GC of the
V.sub.L regions MOCKFOR: CTC ATT CCT GTT GAA GCT CTT GAC AAT B:
primer for amplification of murine variable regions V.sub.H chain:
Back primer MHV.B1.NCoI GAA TAG GCC ATG GCG GAT GTG AAG CTG CAG GAG
TC MHV.B2.NCoI GAA TAG GCC ATG GCG CAG GTG CAG CTG AAG GAG TC
MHV.B3.NCoI GAA TAG GCC ATG GCG CAG GTG CAG CTG AAG CAG TC
MHV.B4.NCoI GAA TAG GCC ATG GCG CAG GTT ACT CTG AAA GAG TC
MHV.B5.NCoI GAA TAG GCC ATG GCG GAG GTC CAG CTG CAA CAA TCT
MHV.B6.NCoI GAA TAG GCC ATG GCG GAG GTC CAG CTG CAG CAG TC
MHV.B7.NCoI GAA TAG GCC ATG GCG CAG GTC CAA CTG CAG CAG CCT
MHV.B8.NCoI GAA TAG GCC ATG GCG GAG GTG AAC CTG GTG GAG TC
MHV.B9.NCoI GAA TAG GCC ATG GCG GAG GTG AAG CTG GTG GAA TC
MHV.B10.NCoI GAA TAG GCC ATG GCG GAT GTG AAC TTG GAA GTG TC
MHV.B11.NCoI GAA TAG GCC ATG GCG GAG GTC CAG CTG CAA CAG TC
MHV.B12.NCoI GAA TAG GCC ATG GCG GAG GTG CAG CTG GAG GAG TC Forward
primer MHC.F.HindIII GGC CAG TGG ATA AAC CTT TGG GGG TGT CGT TTT
GGC V.sub.L- chain: Back primer MKV.B1.MluI TAC AGG ATC CAC GCG TAG
ATG TTT TGA TGA CCC AAA CT MKV.B2.MluI TAC AGG ATC CAC GCG TAG ATA
TTG TGA TGA CGC AGG CT MKV.B3.MluI TAC AGG ATC CAC GCG TAG ATA TTG
TGA TAA CCC AG MKV.B4.MluI TAC AGG ATC CAC GCG TAG ACA TTG TGC TGA
CCC AAT CT MKV.B5.MluI TAC AGG ATC CAC GCG TAG ACA TTG TGA TGA CCC
AGT CT MKV.B6.MluI TAC AGG ATC CAC GCG TAG ATA TTG TGC TAA CTC AGT
CT MKV.B7.MluI TAC AGG ATC CAC GCG TAG ATA TCC AGA TGA CAC AGA CT
MKV.B8.MluI TAC AGG ATC CAC GCG TAG ACA TCC AGC TGA CTC AGT CT
MKV.B9.MluI TAC AGG ATC CAC GCG TAG AAA TTG TTC TCA CCC AGT CT
MKV.B10.MluI TAC AGG ATC CAC GCG TAG ACA TTG TGA TGA CCC AGT CT
Forward primer MKV.F.Not TGA CAA GCT TGC GGC CGC GGA TAC AGT TGG
TGC AGC ATC
[0078] A mix consisting of 8 .mu.l First-strand-buffer, 4 .mu.l
DTT, 4 .mu.l dNTP, 0.5 .mu.l RnaseInhibitor and 1 .mu.l Dnase was
added and further incubated for 30 min at 37.degree. C. Enzymes
were deactivated by incubation in 75.degree. C. for 5 minutes. 1
.mu.l reverse transcriptase and 1 .mu.l RnaseInhibitor was added
and cDNA was synthesized by incubation with 42.degree. C. for 45
minutes. Heat inactivation occured at 94.degree. C. for 5
minutes.
[0079] 1.3. PCR-amplification of the variable regions.
Amplification was done with the components of the CLONTECH
Advantage-high-fidelity Polymerase. The reaction occurred in 50
.mu.l volume with 1 .mu.l of the cDNA (200 pg), 5 .mu.l
reaction-buffer, 200 .mu.M of an equimolar mix of dNTP and 25 pmol
of the Forward Primer and 25 pmol Backprimer mentioned in Table 2.
Amplification was done with 36 Cycles, each with denaturation at
94.degree. C. for 15 seconds, annealing at 55.degree. C. to
65.degree. C. for 30 seconds and elongation for 30 second s at
72.degree. C. After the last amplification cycle, one additional
elongation for 5 min was added.
[0080] 1.4. Cloning of the PCR amplified V-regions into the
procaryotic expression vector pOPE-101 (Genbank# Y14585). PCR
products, which were amplified with the different annealing
temperatures were pooled and DNA was precipitated by the addition
of sodiumacetate pH 5.2 ( 1/10 volume), ethanol (2.5 volume) and 1
.mu.l glycogen (ROCHE) as a carrier. DNA was purified on an 1%
agarose gel, excised (QIAGEN Gel purification kit) and either
NotI/MluI (New England Biolabs) digested for the V.sub.L region or
NcoI/Hind III (New England Biolabs) for the V.sub.H region.
Digestion occurred in 50 .mu.l reaction volume with 45 .mu.l
purified DNA (about 2 .mu.g), 5 .mu.l recommended buffer and 5
units of enzyme for 3 hours at 37.degree. C.
[0081] Digested DNA was purified by running on a 1% agarose gel and
excised from the gel according to the manufactures instructions
(QIAGEN Gel purification kit). A 50 ng portion of the digested and
gel-purified V.sub.L region was ligated with 500 ng of the
appropriately digested and purified expression vector pOPE101 in a
final volume of 40 .mu.l with 1 .mu.l ligase (Boeringer Mannheim)
at 16.degree. C. overnight. DNA was precipitated, electroporated in
XL 1 blue (Epicurian coli; STRATAGENE), and bacteria were grown for
1 h in 1 ml SOC-medium to allow recuperation. Bacteria were plated
on SOB.sub.GAT plates (0.1 M glucose, 100 .mu.gml.sup.-1
ampicillin, 12.5 .mu.gml.sup.-1, tetracycline), and, after
overnight incubation, clones were scraped off and DNA was isolated
with a DNA purification column according to the manufactures
instructions (MACHEREY and NAGEL).
[0082] Vector DNA (containing the V.sub.L chain) was digested with
NcoI/HindIII, purified by running on a 1% agarose gel and excised
from the gel according to the manufactures instructions (QIAGEN Gel
purification kit).
[0083] Ligation of this purified and digested vector DNA with the
NcoI/HindIII digested V.sub.H-regions mentioned above was done as
described. After electroporation in E.coli independent clones were
picked and screened for the expression of functional scFv
(single-chains) with specificity against Peptide.
[0084] 1.5. Screening of the transfected bacteria for positive
binders. Bacterial expression was IPTG-induced and soluble scFv-myc
fusion protein was rescued from the periplasmatic compartment by
osmotic lysis of the bacteria. Supernatant containing the scFv-myc
fusion protein was blocked in 2% Milk PBS and incubated for 3 h in
wells of an ELISA-Plate previously coated with 100 ng peptide/well.
Detection of Peptid6-bound scFv was done by incubation with
anti-c-myc (mouse) and Horseradish-peroxidase conjugated anti-mouse
(rabbit). Vector DNA of positive clones were rescued and the
V.sub.H and V.sub.L regions nucleotide sequences were determined.
Sequences of the V.sub.H and V.sub.L regions are depicted in FIGS.
4 to 7.
[0085] 2. Construction of the Chimeric Antibodies
[0086] For the construction of the chimeric recombinant antibodies,
the V.sub.H and V.sub.L variable regions were either cloned into
the pConGamma1f-vector (for the V.sub.H region) or into the
pConKappa-vector (for the V.sub.L region) purchased by LONZA
Biologics, (Slough,UK). Thereby, upstream of the variable regions a
IgG-leader sequence and a Kozak-sequence was introduced for
secretion into the medium. The two vectors (pConGamm1f and
pConKappa) had been fused in order to facilitate transfection and
to achieve a balanced production of light and heavy chains.
[0087] 2.1. Introduction of the eukaryotic leader sequence by PCR:
components of the CLONTECH Advantage-high-fidelity Polymerase had
been used. The PCR reaction occurred in 50 .mu.l volume with 1
.mu.l (100 ng) of the pOPE vector containing either the V.sub.H or
the V.sub.L region as a template, 5 .mu.l reaction-buffer, 200
.mu.M of an equimolar mix of dNTP and 25 pmol of the Forward Primer
and 25 pmol Backprimer mentioned in Table 3. TABLE-US-00003 TABLE 3
Primer for the introduction of the leadersequence and cloning of
the V-regions: cloning of the V.sub.H chain in the pConGamma1f
Vector: 5'-primer: 5' #9Leader V.sub.H-HindIII: 5'- GCG CGC AAG CTT
GCC GCC ACC ATG GGA TGG AGC TGT ATC ATC CTC TTC TTG GTA GCA ACA GCT
ACA GGT GTC CAC TCC GAG GTG CAG CTG CAA CAG TC-3' 3'-primer: 3'
#9VH-ApaI: 5'- TTT ATA TGG GCC CTT GGT GGA GGC TGA GGA GAC GGT GAC
CGT GGT-3' cloning of the V.sub.H-chain in the pConKappa Vector:
5'-primer: 5' #9Leader V.sub.L-HindIII 5'- GCG CGC AAG CTT GCC GCC
ACC ATG GGA TGG AGC TGT ATC ATC CTC TTC TTG GTA GCA ACA GCT ACA GGT
GTC CAC TCC CAA ATT GTT CTC ACC CAG TCT -3' 3'-primer: 3'
#9V.sub.L-BsiWI: 5'- ATA TGG CGT ACG TTT GAT TTC CAA CTT GGT GCC
-3' auxilliary primer for the 5' primer: HindIII-Kozakbeg: 5'- GCG
CGC AAG CTT GCC GCC AC -3'
[0088] Amplification was done with 36 Cycles, each with
denaturation at 94.degree. C. for 15 seconds, annealing at
65.degree. C. for 30 seconds and elongation for 30 seconds at
72.degree. C. After the 10.sup.th cycle, 25 pmol of the primer
HindIII-Kozakbeg (see Table 3) had been added to the reaction mix.
After the last amplification cycle, one additional elongation
period for 5 min was added.
[0089] 2.2. Cloning into vectors containing the IgG-constant
region. The PCR product was purified by running on a 1% agarose
gel, digested with HindIII/ApaI (V.sub.H chain) or Hind III/BsiWI
(V.sub.L chain) and again gel purified. 50 ng of the digested
V.sub.H and V.sub.L regions were ligated into 200 ng of the
appropriately digested pConGamma1f and pConKappa vectors,
respectively. The V.sub.H expression cassette, containing the
promoter-region and the gene for the entire Heavy chain, was
rescued from the pConGamma1f-vector by digestion with NotI/SalI and
ligated in the appropriately digested and purified pConKappa
vector. The resulted double gene vector was linearized with Pvu I,
phenol/chloroform extracted and 1 .mu.g was used for the
transfection of either 1.times.10.sup.7 NSO or CHO cells.
[0090] Antibody was isolated and tested for binding and biological
activity. The binding and functional characteristics of the
chimeric antibodies as compared to the murine antibodies are shown
in FIGS. 8 and 9, and can be summarized as follows: TABLE-US-00004
ELISA: Specificity for the TIRC7 derived peptide Peptid6
WesternBlot: Same band pattern as the murine mAb T cell
proliferation assay: Inhibition of mitogen induced T cell
proliferation Affinity measurement: Affinity of chimeric Neliximab
against peptide6: Kd = 1 nM Affinity of murine Neliximab against
peptide6: Kd = 1 nM Affinity of murine Metiliximab against
peptide6: Kd = 0.5 nM Affinity of chimeric Neliximab against
peptide6: Kd = 1 nM
[0091] The complete disclosure of all patents, patent documents,
and publications cited herein are incorporated by reference. The
foregoing detailed description and examples have been given for
clarity of understanding only. No unnecessary limitations are to be
understood therefrom. The invention is not limited to the exact
details shown and described, for variations obvious to one skilled
in the art will be included within the invention claimed by the
claims.
Sequence CWU 1
1
40 1 396 DNA Mus musculus CDS (1)..(396) 1 gag gtg cag ctg caa cag
tct gga cct gag ctg gta aag cct ggg gct 48 Glu Val Gln Leu Gln Gln
Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 tca gtg aag atg
tcc tgc aag gct tct gga tac aca ttc act agc tat 96 Ser Val Lys Met
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 gtt ata
cac tgg gtg aaa cag aag cct ggg cag ggc ctt gag tgg att 144 Val Ile
His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45
gga tat att aat cct tac aac tat gat act aaa tac aat gag aag ttc 192
Gly Tyr Ile Asn Pro Tyr Asn Tyr Asp Thr Lys Tyr Asn Glu Lys Phe 50
55 60 aaa ggc gag gcc aca ctg act tca gac aaa tcc tcc aat aca gcc
tac 240 Lys Gly Glu Ala Thr Leu Thr Ser Asp Lys Ser Ser Asn Thr Ala
Tyr 65 70 75 80 atg gaa ctc agc agc ctg acc tct gag gac tct gcg gtc
tat tac tgt 288 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys 85 90 95 gcg gga ttt ttt act agg gca gta ggt ggg tcc
tac tgg tac ctc gat 336 Ala Gly Phe Phe Thr Arg Ala Val Gly Gly Ser
Tyr Trp Tyr Leu Asp 100 105 110 gtc tgg ggc gca ggg acc acg gtc acc
gtc tcc tca gcc aaa acg aca 384 Val Trp Gly Ala Gly Thr Thr Val Thr
Val Ser Ser Ala Lys Thr Thr 115 120 125 ccc cca aag ctt 396 Pro Pro
Lys Leu 130 2 132 PRT Mus musculus 2 Glu Val Gln Leu Gln Gln Ser
Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Val Ile His
Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly
Tyr Ile Asn Pro Tyr Asn Tyr Asp Thr Lys Tyr Asn Glu Lys Phe 50 55
60 Lys Gly Glu Ala Thr Leu Thr Ser Asp Lys Ser Ser Asn Thr Ala Tyr
65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr
Tyr Cys 85 90 95 Ala Gly Phe Phe Thr Arg Ala Val Gly Gly Ser Tyr
Trp Tyr Leu Asp 100 105 110 Val Trp Gly Ala Gly Thr Thr Val Thr Val
Ser Ser Ala Lys Thr Thr 115 120 125 Pro Pro Lys Leu 130 3 354 DNA
Mus musculus CDS (1)..(354) 3 caa att gtt ctc acc cag tct cca gca
atc atg tct gca tct cca ggg 48 Gln Ile Val Leu Thr Gln Ser Pro Ala
Ile Met Ser Ala Ser Pro Gly 1 5 10 15 gag aag gtc acc atg acc tgc
agt gcc agc tca agt ata agt tat ata 96 Glu Lys Val Thr Met Thr Cys
Ser Ala Ser Ser Ser Ile Ser Tyr Ile 20 25 30 cac tgg ttc cag cag
aag cca ggc acc tcc ccc aaa aga tgg att tat 144 His Trp Phe Gln Gln
Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 gac aca tcc
aaa ttg gtt tct gga gtc cct gct cgc ttc agt ggc agt 192 Asp Thr Ser
Lys Leu Val Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 ggg
tct ggg acc tct tat tct ctc aca atc agc aac atg gag gct gca 240 Gly
Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Asn Met Glu Ala Ala 65 70
75 80 gat gct gcc act tat tac tgc cat cag cgg agt gct tcc acg tgg
acg 288 Asp Ala Ala Thr Tyr Tyr Cys His Gln Arg Ser Ala Ser Thr Trp
Thr 85 90 95 ttc ggt gga ggc acc aag ttg gaa atc aaa cgg gct gat
gct gca cca 336 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp
Ala Ala Pro 100 105 110 act gta tcc gcg gcc gcc 354 Thr Val Ser Ala
Ala Ala 115 4 118 PRT Mus musculus 4 Gln Ile Val Leu Thr Gln Ser
Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met
Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Ile 20 25 30 His Trp Phe
Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp
Thr Ser Lys Leu Val Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55
60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Asn Met Glu Ala Ala
65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys His Gln Arg Ser Ala Ser Thr
Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala
Asp Ala Ala Pro 100 105 110 Thr Val Ser Ala Ala Ala 115 5 396 DNA
Mus musculus CDS (1)..(396) 5 gag gtc cag ctg cag cag tct gga ccg
gag ctg gta aag cct ggg gct 48 Glu Val Gln Leu Gln Gln Ser Gly Pro
Glu Leu Val Lys Pro Gly Ala 1 5 10 15 tca gtg aag atg tcc tgc aag
gct tct ggg tac act ttc act acc tat 96 Ser Val Lys Met Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 gtt atg cac tgg gtg
aag cag aag cct ggg cag ggc ctt gag tgg att 144 Val Met His Trp Val
Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga tat att
aat cct tac aat gat ggt act aac tac aat gag aag ttc 192 Gly Tyr Ile
Asn Pro Tyr Asn Asp Gly Thr Asn Tyr Asn Glu Lys Phe 50 55 60 aaa
ggc aag gcc aca ctg acc tca gac aaa tcc tcc agt aca gcc tac 240 Lys
Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70
75 80 atg gag ctc agc acc ctg acc tct gag gac tct gcg gtc tat tac
tgt 288 Met Glu Leu Ser Thr Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr
Cys 85 90 95 gcg gaa ttt att act aag aca gtc ggt ggg tcc aac tgg
tac ctc gat 336 Ala Glu Phe Ile Thr Lys Thr Val Gly Gly Ser Asn Trp
Tyr Leu Asp 100 105 110 gtc tgg ggc gca ggg acc acg gtc acc gtc tcc
tca gcc aaa acg aca 384 Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser
Ser Ala Lys Thr Thr 115 120 125 ccc cca aag ctt 396 Pro Pro Lys Leu
130 6 132 PRT Mus musculus 6 Glu Val Gln Leu Gln Gln Ser Gly Pro
Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Val Met His Trp Val
Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile
Asn Pro Tyr Asn Asp Gly Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys
Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70
75 80 Met Glu Leu Ser Thr Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr
Cys 85 90 95 Ala Glu Phe Ile Thr Lys Thr Val Gly Gly Ser Asn Trp
Tyr Leu Asp 100 105 110 Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser
Ser Ala Lys Thr Thr 115 120 125 Pro Pro Lys Leu 130 7 354 DNA Mus
musculus CDS (1)..(354) 7 caa att gtt ctc acc cag tct cca gca atc
atg tct gct tct cca ggg 48 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile
Met Ser Ala Ser Pro Gly 1 5 10 15 gag aag gtc acc atg acc tgc agt
gcc agc tca agt ata agt tac ata 96 Glu Lys Val Thr Met Thr Cys Ser
Ala Ser Ser Ser Ile Ser Tyr Ile 20 25 30 cac tgg ttc caa cag aag
cca ggc acc tcc ccc aaa aga tgg att tat 144 His Trp Phe Gln Gln Lys
Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 gac aca tcc aaa
ctg cct tct gga gtc cct gct cgc ttc agt ggc agt 192 Asp Thr Ser Lys
Leu Pro Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 ggg tct
ggg acc tct tat tct ctc aca atc agc agc atg gag gct gaa 240 Gly Ser
Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80
gat gct gcc act tat tac tgc cat cag cgg agt agt tac acg tgg acg 288
Asp Ala Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Tyr Thr Trp Thr 85
90 95 ttc ggt gga ggc acc aag ctg gaa atc aaa cgg gct gat gct gca
cca 336 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala
Pro 100 105 110 act gta tcc gcg gcc gcc 354 Thr Val Ser Ala Ala Ala
115 8 118 PRT Mus musculus 8 Gln Ile Val Leu Thr Gln Ser Pro Ala
Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys
Ser Ala Ser Ser Ser Ile Ser Tyr Ile 20 25 30 His Trp Phe Gln Gln
Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser
Lys Leu Pro Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly
Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70
75 80 Asp Ala Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Tyr Thr Trp
Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp
Ala Ala Pro 100 105 110 Thr Val Ser Ala Ala Ala 115 9 14 PRT
Artificial Sequence Oligopeptide 9 Asp Leu Pro Asp Ala Ser Val Asn
Gly Trp Ser Ser Asp Glu 1 5 10 10 24 PRT Artificial Sequence
Oligopeptide 10 Asp Leu Pro Asp Ala Ser Val Asn Gly Trp Ser Ser Asp
Glu Glu Lys 1 5 10 15 Ala Gly Gly Leu Asp Asp Glu Glu 20 11 24 PRT
Artificial Sequence Oligopeptide 11 Val Glu Phe Gln Asn Lys Phe Tyr
Ser Gly Thr Gly Tyr Lys Leu Ser 1 5 10 15 Pro Phe Asp Phe Ala Ala
Thr Asp 20 12 20 DNA Artificial Sequence Oligonucleotide 12
gcgcgcaagc ttgccgccac 20 13 35 DNA Artificial Sequence
Oligonucleotide 13 gaataggcca tggcgcaggt gcagctgaag gagtc 35 14 35
DNA Artificial Sequence Oligonucleotide 14 gaataggcca tggcgcaggt
gcagctgaag cagtc 35 15 35 DNA Artificial Sequence Oligonucleotide
15 gaataggcca tggcgcaggt tactctgaaa gagtc 35 16 36 DNA Artificial
Sequence Oligonucleotide 16 gaataggcca tggcggaggt ccagctgcaa caatct
36 17 35 DNA Artificial Sequence Oligonucleotide 17 gaataggcca
tggcggaggt ccagctgcag cagtc 35 18 36 DNA Artificial Sequence
Oligonucleotide 18 gaataggcca tggcgcaggt ccaactgcag cagcct 36 19 35
DNA Artificial Sequence Oligonucleotide 19 gaataggcca tggcggaggt
gaacctggtg gagtc 35 20 35 DNA Artificial Sequence Oligonucleotide
20 gaataggcca tggcggaggt gaagctggtg gaatc 35 21 35 DNA Artificial
Sequence Oligonucleotide 21 gaataggcca tggcggatgt gaacttggaa gtgtc
35 22 35 DNA Artificial Sequence Oligonucleotide 22 gaataggcca
tggcggaggt ccagctgcaa cagtc 35 23 35 DNA Artificial Sequence
Oligonucleotide 23 gaataggcca tggcggaggt gcagctggag gagtc 35 24 36
DNA Artificial Sequence Oligonucleotide 24 ggccagtgga taaacctttg
ggggtgtcgt tttggc 36 25 38 DNA Artificial Sequence Oligonucleotide
25 tacaggatcc acgcgtagat gttttgatga cccaaact 38 26 38 DNA
Artificial Sequence Oligonucleotide 26 tacaggatcc acgcgtagat
attgtgatga cgcaggct 38 27 35 DNA Artificial Sequence
Oligonucleotide 27 tacaggatcc acgcgtagat attgtgataa cccag 35 28 38
DNA Artificial Sequence Oligonucleotide 28 tacaggatcc acgcgtagac
attgtgctga cccaatct 38 29 38 DNA Artificial Sequence
Oligonucleotide 29 tacaggatcc acgcgtagac attgtgatga cccagtct 38 30
38 DNA Artificial Sequence Oligonucleotide 30 tacaggatcc acgcgtagat
attgtgctaa ctcagtct 38 31 38 DNA Artificial Sequence
Oligonucleotide 31 tacaggatcc acgcgtagat atccagatga cacagact 38 32
38 DNA Artificial Sequence Oligonucleotide 32 tacaggatcc acgcgtagac
atccagctga ctcagtct 38 33 38 DNA Artificial Sequence
Oligonucleotide 33 tacaggatcc acgcgtacaa attgttctca cccagtct 38 34
38 DNA Artificial Sequence Oligonucleotide 34 tacaggatcc acgcgtagac
attctgatga cccagtct 38 35 39 DNA Artificial Sequence
Oligonucleotide 35 tgacaagctt gcggccgcgg atacagttgg tgcagcatc 39 36
98 DNA Artificial Sequence Polynucleotide 36 gcgcgcaagc ttgccgccac
catgggatgg agctgtatca tcctcttctt ggtagcaaca 60 gctacaggtg
tccactccga ggtgcagctg caacagtc 98 37 45 DNA Artificial Sequence
Oligonucleotide 37 tttatatggg cccttggtgg aggctgagga gacggtgacc
gtggt 45 38 99 DNA Artificial Sequence Polynucleotide 38 gcgcgcaagc
ttgccgccac catgggatgg agctgtatca tcctcttctt ggtagcaaca 60
gctacaggtg tccactccca aattgttctc acccagtct 99 39 33 DNA Artificial
Sequence Oligonucleotide 39 atatggcgta cgtttgattt ccaacttggt gcc 33
40 35 DNA Artificial Sequence Oligonucleotide 40 gaataggcca
tggcggatgt gaagctgcag gagtc 35
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