U.S. patent application number 11/313230 was filed with the patent office on 2007-05-24 for lymphotoxin-beta, lymphotoxin-beta complexes, pharmaceutical preparations and therapeutic uses thereof.
Invention is credited to Jeffrey L. Browning, Carl F. Ware.
Application Number | 20070116668 11/313230 |
Document ID | / |
Family ID | 27617796 |
Filed Date | 2007-05-24 |
United States Patent
Application |
20070116668 |
Kind Code |
A1 |
Browning; Jeffrey L. ; et
al. |
May 24, 2007 |
Lymphotoxin-beta, lymphotoxin-beta Complexes, pharmaceutical
preparations and therapeutic uses thereof
Abstract
This invention relates to lymphotoxin-.beta., a lymphocyte
membrane type protein. This protein is found on the surface of a
number of cells, including phorbol ester (PMA) stimulated T cell
hybridoma II-23.D7 cells. This invention also relates to complexes
formed between lymphotoxin-.beta. and other peptides such as
lymphotoxin-.alpha. and to complexes comprising multiple subunits
of lymphotoxin-.beta.. These proteins and complexes are useful in
holding LT-.alpha. formed within the cell on the cell surface where
the LT-.alpha./LT-.beta. complex may act as an inflammation
regulating agent, a tumor growth inhibiting agent, a T cell
inhibiting agent, a T cell activating agent, an autoimmune disease
regulating agent, or an HIV inhibiting agent. Furthermore, the
antitumor activity of the LT-.alpha./LT-.beta. complex may be
delivered to tumor cells by tumor infiltrating lymphocytes (TILs)
transfected with the gene for LT-.beta..
Inventors: |
Browning; Jeffrey L.;
(Brookline, MA) ; Ware; Carl F.; (Riverside,
CA) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP
ONE POST OFFICE SQUARE
BOSTON
MA
02109-2127
US
|
Family ID: |
27617796 |
Appl. No.: |
11/313230 |
Filed: |
December 20, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10040281 |
Nov 7, 2001 |
7030080 |
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11313230 |
Dec 20, 2005 |
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08466254 |
Jun 6, 1995 |
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10040281 |
Nov 7, 2001 |
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08222614 |
Apr 1, 1994 |
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08466254 |
Jun 6, 1995 |
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07990304 |
Dec 4, 1992 |
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08222614 |
Apr 1, 1994 |
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PCT/US91/04588 |
Jun 27, 1991 |
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07990304 |
Dec 4, 1992 |
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07544862 |
Jun 27, 1990 |
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07990304 |
Dec 4, 1992 |
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Current U.S.
Class: |
424/85.1 ;
435/320.1; 435/325; 435/69.5; 530/351; 536/23.5 |
Current CPC
Class: |
C07K 14/5255 20130101;
A61K 38/00 20130101; C07K 14/525 20130101; C07K 16/242 20130101;
C07K 2317/76 20130101; C07K 2317/73 20130101 |
Class at
Publication: |
424/085.1 ;
435/069.5; 435/320.1; 435/325; 530/351; 536/023.5 |
International
Class: |
C07K 14/52 20060101
C07K014/52; A61K 38/19 20060101 A61K038/19; C07H 21/04 20060101
C07H021/04; C12P 21/02 20060101 C12P021/02 |
Goverment Interests
[0002] The invention described herein was made in part during the
course of work under Grant No. CA 35638-07-10 from the National
Institutes of Health. The U.S. Government has certain rights in
this invention.
Claims
1-28. (canceled)
29. A composition comprising an antibody, or fragment thereof, that
specifically binds a lymphotoxin-.beta. polypeptide, and a
pharmaceutically-acceptable carrier.
30. The composition of claim 29, wherein the antibody specifically
binds a soluble lymphotoxin-.beta. polypeptide.
31. The composition of claim 29, wherein the antibody specifically
binds a polypeptide complex comprising a first polypeptide which is
lymphotoxin-.beta. or a fragment thereof and a second polypeptide
selected from the group consisting of lymphotoxin-.alpha., native
human or animal lymphotoxin, recombinant lymphotoxin, soluble
lymphotoxin, secreted lymphotoxin, or lymphotoxin or
lymphotoxin-active fragments of any of the above.
32. The composition of claim 31, wherein the second polypeptide is
lymphotoxin-.alpha..
33. The composition of claim 29, wherein the antibody specifically
binds surface lymphotoxin.
34. The composition of claim 29, wherein the antibody specifically
binds a polypeptide selected from the group consisting of: (a) an
amino acid sequence defined by SEQ ID NO:2; (b) an amino acid
sequence defined by SEQ ID NO:4; (c) an amino acid sequence defined
by SEQ ID NO:6; and (d) an amino acid sequence represented by the
following formula: X-SEQ ID NO:6, wherein X comprises one or more
of the amino acid residues starting from the carboxyl terminus
reading toward the amino terminus of SEQ ID NO:8.
35. The composition of claim 29, wherein the antibody specifically
binds a polypeptide comprising an amino acid sequence that is
encoded by a DNA molecule selected from the group consisting of:
(a) a DNA sequence defined by SEQ ID NO:1; (b) a DNA sequence
defined by SEQ ID NO:3; (c) a DNA sequence defined by SEQ ID NO:5;
and (d) a DNA sequence represented by the following formula: X-SEQ
ID NO:5, wherein X comprises one or more of the nucleoside triplets
starting from the 3' end reading toward the 5' of SEQ ID NO:7.
36. The composition of claim 29, wherein the antibody specifically
binds a polypeptide complex comprising a first polypeptide selected
from the group consisting of an amino acid sequence selected from
the group consisting of: (a) an amino acid sequence defined by SEQ
ID NO:2; (b) an amino acid sequence defined by SEQ ID NO:4; (c) an
amino acid sequence defined by SEQ ID NO:6; and (d) a soluble
lymphotoxin-beta peptide represented by the following formula:
X-SEQ ID NO:6, wherein X comprises one or more of the amino acid
residues starting from the carboxyl terminus reading to the amino
terminus of SEQ ID NO:8; and a second polypeptide selected from the
group consisting of lymphotoxin-alpha, native human or animal
lymphotoxin, recombinant lymphotoxin, soluble lymphotoxin, secreted
lymphotoxin, or lymphotoxin or lymphotoxin-active fragments of any
of the above.
37. The composition of any of claims 29-36, wherein the antibody is
a monoclonal antibody.
38. The composition of any of claims 29-36, wherein the antibody is
a neutralizing antibody.
39. The composition of claim 37, wherein the monoclonal antibody is
a neutralizing antibody.
40. The composition of any of claims 29-36, wherein the composition
is formulated for parenteral administration.
41. The composition of claim 40, wherein the parenteral
administration is selected from the group consisting of
subcutaneous and intravenous administration.
Description
[0001] The present application is a continuation-in-part of
co-pending applications PCT/US91/04588, filed Jun. 27, 1991 and
U.S. patent application Ser. No. 07/544,862, filed Jun. 27,
1990.
TECHNICAL FIELD OF THE INVENTION
[0003] This invention relates to lymphotoxin-.beta., a lymphocyte
membrane-type polypeptide. Lymphotoxin-.beta., also referred to as
p33, has been identified on the surface of T lymphocytes, T cell
lines, B cell lines and lymphokine-activated killer cells.
[0004] This invention also relates to complexes formed between
lymphotoxin-.alpha. (LT-.beta.) and other lymphotoxin-type peptides
such as lymphotoxin (which we refer to herein as
"lymphotoxin-.alpha. (LT-.alpha.)" to distinguish it from
LT-.beta.) and to complexes comprising multiple subunits of
LT-.beta.. The LT-.beta. polypeptide of this invention is expected
to be useful in holding LT-.alpha. formed within the cell on the
cell surface where either LT-.beta. or the LT-.alpha./LT-.beta.
complex may act as an inflammation regulating agent, a tumor growth
inhibiting agent, a T cell inhibiting agent, a T cell activating
agent, an immunomodulatory agent, an autoimmune disease regulating
agent or an HIV regulating agent. Furthermore, the antitumor
activity of the LT-.alpha./LT-.beta. complex may be delivered to
tumor cells by tumor infiltrating lymphocytes (TILs) transfected
with the gene for LT-.beta..
BACKGROUND OF THE INVENTION
[0005] The initiation of the immune response involves a complex
array of intercellular signals. These signals typically involve
soluble cytokines coupled with a number of cell-cell contact
dependent-sign signals. The contact dependent events, most notably
activation of the T-cell receptor, lend specificity to the response
whereas the soluble mediators are generally responsible for
maintenance of cell differentiation and proliferation. Tumor
Necrosis Factor (TNF) and LT-.alpha. are two polypeptides generally
recognized for involvement with the initiation of the immune
response.
[0006] TNF and LT-.alpha. are soluble proteins noted originally for
their ability to inhibit the growth of tumors. [L. Old, "Tumor
Necrosis Factor," Science, 230:630 (1985)]. Further research
demonstrated that both proteins exhibit a wide range of activities.
TNF is synthesized in response to various inflammatory insults by a
variety of cell types including both hematopoietic and
nonhematopoietic cells, while LT-.alpha., in contrast, is made
specifically by lymphocytes. The two known TNF receptors do not
appear to discriminate between LT-.alpha. and TNF. [T. Schall et
al., "Molecular Cloning and Expression of a Receptor for Human
Tumor Necrosis Factor," Cell, 61:361-370 (1990); C. Smith et al.,
"A Receptor for Tumor Necrosis Factor Defines an Unusual Family of
Cellular and Viral Proteins," Science 248:1019 (1990)]. In general,
LT-.alpha. and TNF display similar spectra of activities in in
vitro systems, although LT-.alpha. is often less potent. [J.
Browning et al., Studies Of The Differing Affects Of Tumor Necrosis
Factor And Lymphotoxin On The Growth Of Several Human Tumor Lines,"
J. Immunol., 143:1859 (1989)].
[0007] TNF appears to play a major role in specific aspects of
metabolic control, in the response to endotoxin shock, and in the
regulation of hematopoietic cell development. [B. Beutler et al.,
"The History, Properties, and Biological Effects of Cachectin,"
Biochemistry, 27, (1988); M. Akashi et al., "Lymphotoxin:
Stimulation And Regulation of Colony Stimulating Factors in
Fibroblasts," Blood, 74:2383 (1989); G. Roodman et al., "Tumor
Necrosis Factor-alpha and Hematopoietic Progenitors: Effects Of
Tumor Necrosis Factor On The Growth Of Erythroid Progenitors CFU-E
And BFU-E And The Hematopoietic Cell Lines k562, HL60, And HEL
Cells," Exp. Hematol., 15:928 (1987)].
[0008] Along with IL-1 and IL-6, TNF is also a major mediator of
the inflammatory response. [D. Cavender et al., "Endothelial Cell
Activation Induced By Tumor Necrosis Factor And Lymphotoxin," Amer.
Jour. Path., 134:551 (1989); R. Cotran et al., "Endothelial
Activation Its Role In Inflammatory And Immune Reactions," in
Endothelial Cell Biology, (Plenum Press, Simonescu & Simonescu,
eds., 1988) 335]. TNF also appears to be involved in T cell
activation under certain conditions. [M. Shalaby et al., "The
Involvement Of Human Tumor Necrosis Factors-.alpha. And -.beta. In
The Mixed Lymphocyte Reaction," J. Immunol., 141:499 (1988); N.
Damle et al., "Distinct Regulatory Effects of IL-4 and TNF-.alpha.
During CD3-Dependent and CD3-Independent Initiation Of Human T-Cell
Activation," Lymph. Res., 8:85 (1989); G. Ranges et al., "Tumor
Necrosis Factor-.alpha. As A Proliferative Signal For An
IL-2-Dependent T Cell Line: Strict Species Specificity of Action,"
Amer. Assoc. Immunol., 142:1203 (1989); G. Ranges et al., "Tumor
Necrosis Factor .alpha./Cachectin Is A Growth Factor For
Thymocytes," J. Exp. Med., 167:1472 (1988); P. Scheurich et al.,
"Immunoregulatory Activity Of Recombinant Human Tumor Necrosis
Factor (TNF)-.alpha.: Induction Of TNF Receptors On Human T Cells
And TNF-.alpha.-Mediated Enhancement Of T Cell Responses," J.
Immunol., 138:1786 (1987)].
[0009] TNF is produced by several types of cells, including
monocytes, fibroblasts, T cells and Natural Killer (NK) cells. [D.
Goeddel et al., "Tumor Necrosis Factors: Gene Structure And
Biological Activities," Cold Spring Harbor Symposium Quant. Biol.,
51, 597 (1986); D. Spriggs et al., "Tumor Necrosis Factor
Expression In Human Epithelial Tumor Cell Lines," J. Clin. Invest.,
81:455:(1988); M. Turner et al., "Human T cells From Autoimmune and
Normal Individuals Can Produce Tumor Necrosis Factor," Eur. J.
Immunol., 17:1807 (1987)]. Investigators have also detected murine
and human forms of TNF that are associated with the surface of
various cells either as a transmembrane protein or a receptor-bound
molecule. [B. Luettig et al., "Evidence For the Existence of Two
Forms of Membrane Tumor Necrosis Factor: An Integral Protein and a
Molecule Attached To Its Receptor," J. Immunol., 143:4034 (1989);
M. Kriegler et al., "A Novel Form of TNF/Cachectin Is a Cell
Surface Cytotoxic Transmembrane Protein: Ramifications For the
Complex Physiology of TNF," Cell, 53, pp. 45-53 (1988); and M.
Kinkhabwala et al. "A Novel Addition To the T Cell Repertory," J.
Exp. Med., 171:941-946 (1990)].
[0010] LT-.alpha. also has many activities, generally similar, but
not identical to those of TNF, including tumor necrosis, induction
of an antiviral state, activation of polymorphonuclear leukocytes,
induction of class I major histocompatibility complex antigens on
endothelial cells, induction of adhesion molecules on endothelium
and growth hormone stimulation. [N. Ruddle and R. Homer, "The Role
of Lymphotoxin in Inflammation," Prog. Allergy, 40:162-182 (1988)].
Both LT-.alpha. and TNF are ligands to members of the nerve growth
factor (NGF) receptor family. [S. Mallett and A. N. Barclay, "A New
Superfamily Of Cell Surface Proteins Related To The Nerve Growth
Factor Receptor," Immunology Today, 12:7:220-223 (1991).]
[0011] In contrast to TNF, LT-.alpha. secretion appears to be a
specific property of only activated T cells and certain
B-lymphoblastoid tumors. [N. Paul et al., "Lymphotoxin," Ann. Rev.
Immunol., 6:407 (1988)]. Some researchers have also indicated that
a membrane-associated form of LT-.alpha. may be expressed on the
surface of lymphocytes under certain circumstances [J. Hiserodt, et
al., "Identification of Membrane-Associated Lymphotoxin (LT) On
Mitogen-Activated Human Lymphocytes Using Heterologous Anti-LT
Antisera In Vitro," Cell. Immunol., 34:326-339 (1977); C. Ware et
al., "Mechanisms of Lymphocyte-Mediated Cytotoxicity," J. Immunol.,
126:1927-1933 (1981); U. Anderson et al. J. Immunol. Methods, 123,
233 (1989); Y. Abe et al., Jpn. J. Canc. Res., 82:23 (1991); Y. Abe
et al., "Studies of Membrane Associated and Soluble (Secreted)
Lymphotoxin In Human Lymphokine-Activated T-Killer Cells In Vitro,"
Lymphokine and Cytokine Research, 11, 2: 15-121 (1992)].
[0012] In recent years genes for both TNF and LT-.alpha. have been
isolated and cloned, leading to their complete characterization and
to the availability of recombinant forms of both proteins. [P. Gray
et al., "Cloning and Expression of cDNA For Human Lymphotoxin, A
Lymphokine With Tumor Necrosis Activity," Nature, 312:121-124
(1984); D. Pennica et al., "Human Tumor Necrosis Factor: Precusor
Structure, Expression And Homology To Lymphotoxin," Nature, 312:724
(1984)].
[0013] Other "cytokine-like" cell surface proteins including the
CD40 protein have recently been shown to share certain similarities
with TNF and LT-.alpha.. Like TNF and LT-.alpha., the CD40 protein
is a ligand to members of the TNF/nerve growth factor (NGF)
receptor family. [S. Mallett and N. Barclay, Immunology Today,
12:220-223 (1991)]. The CD40 protein is a 277-amino acid protein
expressed on the surface of B lymphocytes, epithelial cells, and
some carcinoma cell lines. [R. Armitage et al., Nature, 357:80-82
(1992); T. Farrah and C. Smith, "Emerging Cytokine Family," Nature,
358:26 (1992)].
[0014] We have now identified a novel surface protein,
lymphotoxin-.beta. (LT-.beta.) or p33. LT-.beta. has been
identified on the surface of several types of lymphocyte cells,
including OKT3-stimulated primary T cells, antigen specific IL-2
dependent CTL clones, and a PMA-stimulated human T cell hybridoma
II-23.D7. LT-.beta. targets LT-.alpha. produced in the cell to the
cell membrane where LT-.beta. and LT-.alpha. appear as a complex
(designated "LT-.alpha./LT-.beta." throughout this disclosure). The
LT-.alpha./LT-.beta. complex is believed to be a novel mechanism
for membrane expression of LT-.alpha. by activated T-cells.
[Androlewicz et al., "Lymphotoxin Is Expressed As a Heteromeric
complex With A Distinct 33 kDa Glycoprotein On The Surface Of An
Activated Human T Cell Hybridoma," Journal Of Biological Chemistry,
267:2542-2547 (1992)]. The LT-.alpha./LT-.beta. complex may exhibit
cytolytic and cell regulatory activity similar to the soluble
LT-.alpha., TNF and CD40 proteins. The membrane-associated
LT-.beta. complexed with LT-.alpha. may represent, as a complex, a
novel ligand for T cell interactions with other cells and may also
be useful in targeted cell lysis.
SUMMARY OF THE INVENTION
[0015] The novel protein of the present invention has been named
lymphotoxin-.beta. (LT-.beta.). This protein is found on the
surface of several types of lymphocyte cells, including
OKT3-stimulated primary T cells, antigen-specific IL-2 dependent
CTL clones, and a PMA-stimulated human T cell hybridoma, II-23.D7.
It forms a novel complex with LT-.alpha. and forms complexes with
other LT-.beta. subunits (e.g., (LT-.beta.).sub.2 LT-.alpha.
complexes).
[0016] LT-.beta. has a molecular weight of 31-35 kD as determined
by immunoprecipitation and SDS-PAGE. LT-.beta. exhibits N-linked
glycosylation. The amino acid sequence of lymphotoxin-.beta. is set
forth in SEQ ID NO:2, and the amino acid sequences of several
soluble lymphotoxin-.beta. peptides are set forth in SEQ ID NO:4,
SEQ ID NO:6 and SEQ ID NO:8. The DNA sequence coding for
lymphotoxin-.beta. is set forth in SEQ ID NO:1 and DNA sequences
coding for several soluble lymphotoxin-.beta. peptides are set
forth in SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7.
[0017] LT-.beta. as a cell membrane protein binds LT-.alpha. during
synthesis, thus "targeting" the LT-.alpha. to the cell membrane. In
the absence of LT-.beta., LT-.alpha. is secreted into the
extracellular medium. The LT-.alpha./LT-.beta. complex is
recognized by polyclonal antisera raised against recombinant
lymphotoxin-.alpha. (rLT-.alpha.) expressed in CHO cells or by
monoclonal antibodies (mAbs) raised against natural LT-.alpha..
Furthermore, antisera that recognize the LT-.alpha./LT-.beta.
complex and (LT-.alpha.).sub.3 block the mixed lymphocyte reaction
(MLR), a standard immunological assay of the expected proliferative
response of T lymphocytes to allogenic stimulation, i.e., the
introduction of T lymphocytes from another individual, which are
recognized as foreign (non-self) by the "responder" lymphocytes
[See, e.g., M. Shalaby et al., J. Immunol., 141:499 (1988)].
[0018] The LT-.beta. protein was purified by affinity
chromatography, partially sequenced and a specific oligonucleotide
probe was designed. The cDNA encoding the LT-.beta. was isolated by
probing a cDNA library from activated II-23.D7 cells, a human
T-cell hybridoma that displays large amounts of surface lymphotoxin
upon phorbol ester activation. The identified amino acid sequence
encodes a 240-244 amino acid sequence (a molecular mass of the
unmodified protein of about 25130-25390 kDa). See SEQ ID NO:2. The
amino acid sequence and the placement of the transmembrane region
are typical of a type II membrane protein.
[0019] This sequence comprises a short 14-18 amino acid N-terminal
"cytoplasmic" domain. Following this cytoplasmic domain there is an
extensive stretch of 30 hydrophobic amino acids which presumably
acts as a membrane anchoring domain. No identical sequences were
found within available databases. There is one cysteine residue in
the extracellular domain and two methionines within the last
C-terminal 17 amino acids. This is consistent with the very limited
cyanogen bromide cleavage pattern exhibited by this protein.
[0020] Comparison of the LT-.beta. sequence with other proteins
known to bind to members of the TNF/NGF receptor family reveals
considerable structural similarity. Four of the ligands to members
of the TNF/NGF receptor family (TNF, LT-.alpha., LT-.beta. and the
CD40 ligand) resemble type II membrane proteins and share at least
four regions of sequence conservation in the extracellular domain
as indicated in FIG. 14. The conserved TNF and LT-.alpha. domains
shared with LT-.beta. are likely to be involved in intersubunit
interactions and .beta. sheet organization. These regions of
conservation can account for the association between LT-.alpha. and
LT-.beta.. The existence of these homology regions may facilitate
engineering the polypeptide to form complexes with, for example,
TNF or the CD40 ligand. Such a molecule would have mixed functions
and could possibly be used as a custom designed drug. [See J. Fuh
et al., "Rational Design Of Potent Antagonists To The Human Growth
Hormone Receptor," Science, 256:1677 (1992)].
[0021] We believe that the polypeptide complexes of this invention
are important in T cell activation events and are useful in
compositions and methods for T cell activation or T cell
suppression and as therapeutic agents in the treatment of
inflammation or applications requiring cytolytic activities, such
as inhibition of tumor cell or neoplasia growth. We also believe
that the polypeptide complexes may be important in cellular
immunotherapies, including enhancing the tumoricidal properties of
tumor infiltrating lymphocytes in Tumor Infiltrating Lymphocyte
("TIL") therapy. TIL immunotherapy may be improved by gene transfer
techniques. For example, a gene may be added to tumor cells for the
purpose of inducing the body's immune system to mediate an
effective tumor-directed immune response. [See, e.g., W. F.
Anderson, "Human Gene Therapy." Science, 256:808-813 (1992)].
[0022] We also believe, based upon similarities between a molecule
identified as "Fas" and members of the TNF/NGF receptor family,
that the polypeptide complexes of this invention may be involved in
the internal cell process known as programmed cell death or
apoptosis, and may therefore be involved in mediating autoimmune
disease. [See, e.g., N. Itoh et al., "The Polypeptide Encoded By
The cDNA For Human Cell Surface Antigen Fas Can Mediate Apoptosis,"
Cell, 66:233-243 (1991); R. Watanabe-Fukunaga et al.,
"Lymphoproliferation Disorder In Mice Explained By Defects In Fas
Antigen That Mediates Apoptosis," Nature 356:314 (1992)].
[0023] Antibodies to LT-.beta., its related polypeptides, the
LT-.alpha./LT-.beta. complex or the other polypeptide complexes of
this invention may also disrupt critical LT interactions with
particular receptors, thus specifically affecting LT-mediated
events other than those mediated through the known TNF receptor
forms. Likewise, receptors for TNF, LT-.alpha. or
LT-.alpha./LT-.beta., or their derivatives (e.g., soluble receptors
and IgG/receptor fusion proteins) may be used to inhibit the
polypeptides and complexes of this invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 depicts flow cytofluorometric analysis of
OKT3-stimulated, IL-2 expanded peripheral blood lymphocytes (PBL)
showing reaction with 3 different rabbit anti-rLT-.alpha. antisera
and showing essentially no reaction with rabbit anti-rTNF
antisera.
[0025] FIG. 2 depicts flow cytofluorometric analysis of a human T
cell hybridoma, II-23.D7, showing the presence (following PMA
treatment) of a LT-.alpha.-related epitope on the cell surface.
[0026] FIG. 3 depicts the ability of PMA activated 11-23.D7 cells
to bind anti-rLT-.alpha.antibodies. Samples of anti-rLT-.alpha.
antisera were incubated with PMA-treated U937 (non-LT-producing)
cells, (--O--), PMA-activated 11-23.D7 hybridoma cells (10.sup.8, -
-; 10.sup.7, - -), and no cells (control, - -). Serial dilutions of
the cell-free antisera after incubation were added to rLT-.alpha.
and used in a cytotoxicity assay against L929
(LT-.alpha.-sensitive) cells. The plots indicate that rLT-.alpha.
neutralizing antibodies were removed from the antisera by the
activated II-23.D7 cells.
[0027] FIG. 4 shows two autoradiographs (A and B) depicting
immunoprecipitation of .sup.125I-labeled surface proteins from
PMA-activated UI-23.D7 cells. FIG. 4A shows immunoprecipitation of
an approximately 25 kD surface protein (LT-.alpha.) and an
approximately 33 kD surface protein (p33, or LT-.beta.) by
post-immune rabbit anti-rLT-.alpha.antiserum but not by pre-immune
rabbit serum. FIG. 4B shows a 1-dimensional CNBr cleavage map of
the 25 kD and 33 kD bands from FIG. 4A, compared against
recombinant TNF (rTNF) produced in E. coli, and recombinant
lymphotoxin-.alpha. (rLT-.alpha.) produced in CHO cells, [J.
Browning et al., J. Immunol., 143:1859 (1989)] both with (+) and
without (-) CNBr cleavage.
[0028] FIG. 5 presents autoradiographs showing immunoprecipitation
of TNF- and LT-.alpha.-related proteins from PMA-stimulated
II-23.D7 cells metabolically labeled with .sup.35S-methionine or
.sup.35S-cysteine. The figure shows recognition by anti-rLT-.alpha.
antisera (L), but not preimmune (P) or anti-rTNF antisera (T), of
an approximately 25 kD methionine-containing surface protein
(LT-.alpha.) and an approximately 33 kD methionine- and
cysteine-containing surface protein (p33, or LT-.beta.). The
autoradiographs also indicate that activated II-23.D7 cells also
produce a 26 kD form of TNF and secrete soluble
lymphotoxin-.alpha..
[0029] FIG. 6 depicts affinity purification 1-D CNBr peptide
mapping of those proteins from PMA-activated II-23.D7 cells
recognized by anti-rLT-.alpha. serum. FIG. 6A represents SDS PAGE
analysis of the proteins eluted from an anti-LT-.alpha. affinity
column prepared from either pre-immune (PRE) or post-immune (POST)
rabbit sera. FIG. 6A shows the .about.33 kD and .about.20 kD
protein bands did not bind to an affinity column prepared using
preimmune serum (PRE) but did bind to an affinity column prepared
using anti-rLT-.alpha. antiserum (POST). FIG. 6B shows partial CNBr
cleavage of the .sup.-33 kD and .sup.-20 kD proteins eluted from
the POST column; compared against rTNF and rLT-.alpha. run in
parallel. The gels were visualized by silver staining.
[0030] FIG. 7 presents autoradiographs of the .sup.-25 kD and
.sup.-33 kD .sup.125I-labeled proteins (designated LT-.alpha. and
LT-.beta., respectively) immunoprecipitated from activated II-23.D7
cells, treated with N-glycanase (N-gly), with a mixture of
neuraminidase and O-glycanase (O-gly), or with all three
enzymes.
[0031] FIG. 8 depicts the results of a reimmunoprecipitation of the
coprecipitated p33 (LT-.beta.) and p25 (LT-.alpha.) proteins to
further investigate whether they are immunogenically related.
[0032] FIG. 9 (comprising parts 9A and 9B) shows the results of
isoelectric focusing under denaturing conditions of the
immunoprecipitated p33 (LT-.beta.) and p25 (LT-.alpha.)
proteins.
[0033] FIG. 10 (comprising parts 10A and 10B) shows the results of
isoelectric focusing under native conditions of the
immunoprecipitated p33 (LT-.beta.) and p25 (LT-.alpha.) proteins.
Together FIGS. 9 and 10 indicate that LT-.beta. and LT-.alpha. form
a denaturable complex.
[0034] FIG. 11 depicts flow cytofluorometric analysis of surface
protein differentially expressed on T cells and monocytes after
stimulation with a mixture of LPS, IFN-.gamma. and OKT3. From a
stimulated PBL pool, separated T cells were observed to express a
surface protein recognized by anti-rLT-.alpha. antisera (LT),
whereas separated monocytes expressed a surface protein recognized
by anti-rTNF antisera (TNF).
[0035] FIG. 12 shows flow cytofluorometric analysis of surface LT
forms on leu-19.sup.- and leu-19.sup.+ (i.e., natural killer) cells
treated with IL-2. Analysis of IL-2 treated PBL with both labeled
leu-19 and anti-rLT-.alpha. confirms that lymphokine-activated
killer (LAK) cells express a surface LT form.
[0036] FIG. 13 depicts amino acid sequence of LT-.beta. fragments
obtained by direct N-terminal sequencing and in situ trypsin
digestion followed by reverse phase HPLC resolution of the digested
peptides.
[0037] FIG. 14 depicts an amino acid sequence comparison of four
members of the family of ligands binding to members of the
TNF/NGF-receptor family. Homology regions are shown in bold type
face with sequence identity indicated with a dot and conserved
sequences with an asterisk. Putative N-linked glycosylation sites
are boxed.
[0038] FIG. 15 depicts northern analysis of LT-.alpha. and
LT-.beta. expression. A) Northern blot of several cell lines
showing specific expression of both LT genes in only Hut-78 and
II-23.D7 cells. B) Time course of PMA induction of LT-.alpha. and
LT-.beta. mRNAs in II-23.D7 cells. C) Similar analysis of human
peripheral blood lymphocytes activated with either anti-CD3 or IL-2
alone.
[0039] FIG. 16 depicts expression of LT-.alpha. and LT-.beta. in
CHO cells. A) FACS analysis of CHO cells transiently transfected
with the LT-.beta. cDNA. Either parental dHFR-CHO or LT-.alpha.
expressing CHO cells were electroporated with either pCDM8
containing an irrelevant insert (clone 4) or the pCDM8/LT-.beta.
plasmid and stained with control IgG(- - -) or anti LT-.alpha.
monoclonal antibodies (______). B) Panel B depicts expression of
LT-.alpha. and LT-.beta. in COS cells. Cells were transfected with
control DNA or the pCDM8/LT.beta. plasmid, either with or without
pCDM8/LT.alpha. and stained as per panel A.
DETAILED DESCRIPTION OF THE INVENTION
[0040] In order that the invention herein described may be fully
understood, the following detailed description is set forth.
[0041] This invention relates to lymphotoxin-.alpha., a lymphocyte
membrane-type polypeptide. The amino acid sequence of
lymphotoxin-.beta. is set forth in SEQ ID NO:2.
[0042] This polypeptide, also referred to as p33, has a molecular
weight of 31 to 35 kD. The polypeptides of this invention may be
associated with a cell surface or not associated with such a
surface.
[0043] This invention also relates to soluble forms of
lymphotoxin-.beta.. Soluble lymphotoxin-.beta. peptides are defined
by the amino acid sequence of lymphotoxin-.beta. wherein the
sequence is cleaved at any point between the end of the
transmembrane region (i.e., at about amino acid #44) and the first
homology region at about amino acid #95. Amino acid sequences of
two soluble lymphotoxin-.beta. peptides are defined by SEQ ID NO:4
and SEQ ID NO:6. Several additional soluble lymphotoxin-.beta.
polypeptides comprise the amino acid sequence as defined by SEQ ID
NO:6 with additional amino acid residues at the 5' end. The
additional residues may comprise the 52 amino acid residues as
defined by SEQ ID NO:8. The soluble lymphotoxin may also comprise
an amino acid sequence defined by SEQ ID NO:6 plus a portion of SEQ
ID NO:8 comprising 1 to 51 of the amino acid residues beginning
from the 3' end. Such soluble peptides may include any number of
well known-leader sequences at the 5' end. Such a sequence would
allow the peptides to be expressed in a eukaryotic system. [See,
e.g., Ernst et al. U.S. Pat. No. 5,082,783 (1992)].
[0044] The polypeptide complexes of this invention comprise a first
polypeptide comprising an amino acid sequence selected from the
group of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and the sequence
defined by SEQ ID NO:6 plus the entirety or a portion of SEQ ID
NO:8 as described above, complexed with a second polypeptide
selected from the same group and/or with a second polypeptide such
as: lymphotoxin-.alpha., native human or animal
lymphotoxin-.alpha., recombinant lymphotoxin-.alpha., soluble
lymphotoxin-.alpha., secreted lymphotoxin-.alpha., or
lymphotoxin-.alpha.-active fragments of any of the above.
[0045] The novel LT-.beta. peptide forms a complex with LT-.alpha.
and forms complexes with other LT-.beta. subunits (e.g.
(LT-.beta.).sub.2 LT-.alpha. complexes). These complexes may be
cell associated or not associated with a cell, and may complex with
other type II membrane proteins sharing common homology regions as
described above.
[0046] The polypeptide complexes are recognized by polyspecific
antisera that recognize recombinant LT-.alpha., suggesting that the
complex exhibits LT-.alpha. epitopes. These antisera include a
commercial anti-LT-.alpha. monoclonal antibody (Boehringer
Mannheim), and polyspecific antisera raised against recombinant
lymphotoxin-.alpha. (rLT-.alpha.) expressed in transfected Chinese
Hamster Ovary (CHO) cells. Polyclonal antisera that recognize these
complexes also block the mixed lymphocyte reaction (MLR), but a
monoclonal anti-rLT-.alpha. antibody that recognizes soluble
LT-.alpha. do not block the MLR. The complexes of the present
invention thus appear to play an important role in T cell
activation. We also expect these complexes to have T cell
regulatory activities and cytotoxic activities similar to those of
soluble LT-.alpha. or TNF.
[0047] This invention also relates to DNA consisting essentially of
DNA sequences that code for the polypeptides comprising the amino
acid sequences described above, recombinant DNA molecules
characterized by that DNA, hosts selected from the group of
unicellular hosts or animal and human cells in culture transfected
with that DNA, and recombinant methods of using that DNA and those
recombinant DNA molecules and hosts to produce the polypeptides
coded thereby.
[0048] More specifically, this invention relates to an isolated DNA
sequence comprising the nucleotide sequence as defined by SEQ ID
NO:1.
[0049] This invention also relates to polypeptides encoded by that
sequence, DNA sequences that hybridize with that DNA sequence that
code for a polypeptide that is substantially homologous with
lymphotoxin-.beta., and degenerate DNA sequences comprising
nucleotide sequences that code for lymphotoxin-.beta..
[0050] This invention also relates to DNA sequences that code for
soluble lymphotoxin-.beta. peptides. These DNA sequences are
defined by SEQ ID NO:3 and SEQ ID NO:5. This invention also relates
to several additional soluble lymphotoxin-.beta. peptides that are
coded for by the DNA of SEQ ID NO:5 plus several additional
nucleotide triplets at the 5' end. The additional nucleotide
triplets may comprise the 52 triplets as defined by SEQ ID NO:7.
The soluble lymphotoxin peptide may also be encoded by SEQ ID NO:5
plus a portion of SEQ ID NO:7 comprising 1 to 51 nucleotide
triplets beginning from the 3' end.
[0051] This invention also relates to DNA sequences that hybridize
to any of the sequences identified above that code on expression
for lymphotoxin-.beta. or soluble lymphotoxin-.beta. peptides. This
invention also relates to degenerate nucleotide sequences that code
for lymphotoxin-.beta. or a soluble lymphotoxin-.beta. peptide, and
to DNA sequences that code for polypeptides that are substantially
homologous with a soluble lymphotoxin-.beta. peptide.
[0052] Lymphotoxin-.beta. was identified, isolated and
characterized using the techniques described below:
Flow Cytofluorometric Analysis
[0053] First we demonstrated the expression of LT-.alpha. epitopes
on the surface of T cells using flow cytofluorometric analysis. We
observed that human peripheral blood mononuclear cells activated
with OKT3 monoclonal antibody demonstrated expression of LT-.alpha.
epitopes by reacting with anti-rLT-.alpha. antisera. Only
anti-rLT-.alpha. antisera, not anti-rTNF antisera, bound to
OKT3-stimulated primary T cells.
[0054] We also observed that a human T cell hybridoma, II-23.D7 [C.
Ware et al., "Human T Cell Hybridomas Producing Cytotoxic
Lymphokines: Induction of Lymphotoxin Release And Killer Cell
Activity By Anti-CD3 Monoclonal Antibody Or Lectins And Phorbol
Ester," Lymph. Res., 5:313 (1986)], secreted LT-.alpha. upon PMA
stimulation and also expressed surface LT-.alpha.-related epitopes
upon PMA stimulation. We also demonstrated that
PMA-activated-II-23.D7 cells were able to remove LT-neutralizing
antibodies from the rabbit anti-rLT-.alpha. antisera, while control
U937 cells, which lack all surface LT-.alpha. forms, were not. We
further ruled out the possibility that the rabbit anti-rLT-.alpha.
antisera had bound (complexed) with rabbit LT-.alpha. (the
resulting complexes subsequently binding to cellular LT-.alpha./TNF
receptors on the II-23.D7 cells) by saturating the cellular
receptors with excess soluble TNF or LT-.alpha. and observing that
this had no effect on the staining. These assays demonstrate that
the LT-.alpha.-related epitopes on this hybridoma are genuinely
related to LT-.alpha..
[0055] We also observed that pretreatment of the antisera with
excess rLT-.alpha. blocked the ability of the antisera to stain
II-23.D7 cells, while pretreatment with rTNF had no effect. This
assay demonstrated the specificity of the antisera for
LT-.alpha.-related epitopes.
[0056] Trypsinization of the stimulated II-23.D7 cells prior to
staining led to loss of the signal, demonstrating that the epitopes
recognized by the antisera were proteins.
[0057] We also demonstrated that CHO-derived contaminants did not
contribute to the antisera recognition of induced proteins on the
surface of activated II-23.D7 cells by showing that CHO cells
stably transfected with the LT-.alpha. gene, which produce only
soluble LT-.alpha., were not stained by the anti-LT-.alpha.
antisera.
Immunoprecipitation
[0058] We further characterized these surface LT-.alpha.-related
proteins by either surface iodination (.sup.125 I-labelling) or
metabolic labelling (.sup.35S-Met or .sup.35S-Cys) of PMA-activated
H-23.D7 cells, followed by solubilization of the plasma membrane
with detergent and immunoprecipitation of the labeled
LT-.alpha.-related proteins.
[0059] Surface iodination coupled with immunoprecipitation revealed
two proteins recognized by the anti-LT-.alpha. antisera: a 25-26 kD
form subsequently referred to as LT-.alpha., and a 31-35 kD form
subsequently referred to as LT-.beta. or p33. We observed that
neither the preimmune serum from the same rabbit nor anti-rTNF
rabbit serum immunoprecipitated these bands from the iodinated,
PMA-activated II-23.D7 cells. One dimensional partial CNBr peptide
mapping of the iodinated, immunoprecipitated bands showed that the
25-26 kD form (LT-.alpha.) cleaves in a pattern identical to that
of iodinated recombinant LT-.alpha., reinforcing the correlation
between surface LT-.alpha. and soluble LT-.alpha.. The iodinated
31-35 kD form (LT-.beta., or p33) was not cleaved by CNBr,
indicating that it is distinct from the known LT-.alpha. gene
product.
[0060] We further characterized the LT-.alpha. and LT-.beta.
proteins by metabolic labelling of PMA-activated II-23.D7 cells
with .sup.35S-methionine or .sup.35S-cysteine, followed by
immunoprecipitation. The distribution of cysteine and methionine
provides a means of distinguishing between TNF and LT-.alpha. and
between forms of each with and without their signal sequences-[M.
Kriegler et al., Cell, 53:45 (1988)]. Secreted TNF contains
cysteine, but not methionine, while secreted LT-.alpha. contains
only methionine and no cysteine residues. LT-.alpha., however, has
one cysteine residue in its signal sequence, while TNF contains two
methionine residues in its signal sequence.
[0061] We labeled separate cultures of PMA-treated II-23.D7 cells
with either .sup.35S-methionine or .sup.35S-cysteine and
precipitated immunoreactive proteins from the culture media and the
cells. Subsequent SDS-PAGE analysis of the immunoprecipitates from
the culture media of cells labeled with .sup.35S-methionine
revealed a 25 kD form of LT-.alpha. while the immunoprecipitates
from the culture media of cells labeled with .sup.35S-cysteine did
not, a pattern expected for secreted LT-.alpha.. Analysis of the
washed cells showed both the 25-26 kD LT-.alpha. form and the 33 kD
LT-.beta. form. These results paralleled the membrane-associated
forms observed using surface iodination.
[0062] The 25-26 kD LT-.alpha. lacked cysteine, indicating
processing of the leader sequence. We also observed that the 33 kD
LT-.beta. incorporated both .sup.35S-methionine and
.sup.35S-cysteine, distinguishing itself as different from the 25
kD LT-.alpha. form. Typically, LT-.alpha. bound to its receptor can
be cross-linked to the receptor using a chemical linker such as
BOSCOES (i.e., (bis[2-[succinimidooxy-carbonyloxy]ethyl]sulfone;
Pierce, Rockford, Ill.). [J. S. Andrews et al., "Characterization
of the Receptor for Tumor-Necrosis Factor (TNF) and Lymphotoxin
(LT) on Human T Lymphocytes," J. Immunol., 144:2582 (1990)]. We
observed that when surface iodinated II-23.D7 cells were treated
with a cross-linking agent, there was no association of either the
25-26 kD) LT-.alpha. or the 33 kD LT-.beta. related form with an
additional membrane protein. This assay demonstrated that receptor
binding is not the mechanism by which LT-.alpha. and LT-.beta.
remain associated with the cell membrane. Sequence analysis of
LT-.beta. showed no relationship to either of the two TNF-receptor
forms [C. Smith et al., Science, 248:1019 (1990); T. J. Schall et
al., Cell, 61-61 (1990)].
Affinity Chromatography
[0063] Further characterization of the LT-.beta. and LT-.alpha.
proteins on the surface of II-23.D7 cells was obtained through
affinity chromatography. We observed that
LT-.alpha./LT-.beta.complex on the surface of PMA-treated II-23.D7
cells bound to lentil lectin, indicating a glycoprotein structure
for each form. Hence a lentil lectin chromatography step was used
as a purification step prior to antisera affinity chromatography.
We bound detergent-solubilized PMA-treated II-23.D7 proteins to
lentil lectin sepharose and eluted with .alpha.-methyl mannoside.
We prepared both control IgG and anti-LT-.alpha. IgG affinity
columns to accurately assess those proteins specifically recognized
by the anti-LT-.alpha. antiserum. We then applied the proteins that
bound to lentil lectin to these columns. We observed that low pH
elution of the columns led to the release of the LT-.beta. and
LT-.alpha. proteins from the anti-LT-.alpha. affinity column.
SDS-PAGE analysis of the eluate closely resembled the SDS-PAGE
analysis of immunoprecipitated proteins from surface iodinated
PMA-treated II-23.D7 cells. This comparison demonstrated that
similar proteins were purified by the two methods.
[0064] We observed that during affinity purification, the .sup.-25
kD LT-.alpha. form appeared to be cleaved to a 19-20 kD form, or a
"des 20" form. The original isolation of natural LT-.alpha.from the
RPMI 1788 tumor cell line [B. Aggarwal et al., "Primary Structure
of Human Lymphotoxin Derived From 1788 Lymphoblastoid Cell Line,"
J. Biol. Chem., 260:2334-2344 (1985)] also yielded an N-terminally
cleaved 20 kD LT-.alpha. form. One of the methionines is lost in
this "des-20" natural LT-.alpha. form, producing a different CNBr
cleavage pattern from the intact molecule. One-dimensional CNBr
digests of the affinity-purified LT-.alpha. protein demonstrated a
cleavage pattern that is consistent with the truncated natural
LT-.alpha. form, and we concluded that the affinity-purified
"des-20" LT-.alpha.form probably results from a similar cleavage as
observed with the natural LT-.alpha. "des-20" form.
[0065] We further observed that LT-.beta. generates a doublet upon
partial CNBr cleavage. The cleavage pattern generated by the
LT-.beta. protein demonstrated that methionine residue(s) were
present, and at least one methionine was within 5-20 residues from
either the C- or N-terminus (methionine residues within 1-5
residues of either end would not be detected when cleaved using
this mapping technique). This pattern suggested that LT-.beta. does
not contain the entire known LT-.alpha. sequence.
[0066] We observed that the LT-.beta. protein is also expressed by
antigen-activated primary cytotoxic T lymphocyte clones. Metabolic
labelling of these cells followed by immunoprecipitation with
anti-rLT-.alpha. revealed L-T-6 along with small amounts of
LT-.alpha.. These results demonstrated that LT-.beta. is made by
primary T cells as well as by the II-23.D7 hybridoma.
Initial Purification of the LT-.beta. and LT-.alpha. Proteins
[0067] We purified these LT-.alpha. and LT-.beta. proteins using
the following general steps. We first added phorbol myristic
acetate (PMA) to II-23.D7 cells. After 24 hours we harvested the
cells and washed them with cold serum-free RPMI medium. To the
chilled cell pellet we added ice-cold lysis buffer (HEPES, NP-40,
EDTA, NaCl, and sodium azide) to which benzamidine, phenyl methyl
sulfonyl chloride (PMSF), and N-ethyl maleimide (NEM), soybean
trypsin inhibitor, pepstatin and aprotinin had been freshly added.
We homogenized the cells gently in a Dounce homogenizer and
centrifuged the lysate. We centrifuged and collected the
supernatant. We loaded the supernatant onto a lentil-lectin
sepharose column equilibrated in lysis buffer to which we had added
CaCl.sub.2 and MnCl.sub.2. We washed the column with lysis buffer
with CaCl.sub.2 and MnCl.sub.2 and then eluted with lysis buffer
containing .alpha.-methyl mannoside. We pooled the eluate fractions
and loaded directly onto a rabbit nonspecific IgG sepharose
affinity column which was directly connected to a rabbit
anti-rLT-.alpha. sepharose affinity column. We washed both columns
with the same lysis buffer with EDTA followed by lysis buffer
wherein the NP-40 had been replaced with MEGA-8 (Octanoyl-N-methyl
glucamide, Boehringer-Mannheim). We eluted the washed columns
individually with a solution of 5 mM MEGA-8, 50 mM glycine, NaCl,
benzamidine, and EDTA as described in Browning et al., "Lymphdtoxin
And An Associated 33-kDa Glycoprotein are Expressed On The Surface
Of An Activated Human T-Cell Hybridoma," J. Immunol. 147:1230-1237
(1991). The first fractions following the pH shift were pooled,
lyophilized and resuspended in water with SDS, and dialyzed against
a solution of HEPES and SDS. We dried the dialyzed fractions on a
speed-vac and resuspended in water. We mixed aliquots with Laemmli
loading buffer and electrophoresed on SDS-PAGE. We visualized
proteins by silver staining.
[0068] We observed that LT-.alpha. epitope(s) are present on the
surface of the II-23.D7 T cell hybridoma only following cell
activation such as occurs with PMA treatment. In contrast, when
present on primary T-cells, PMA treatment leads to loss of the
surface antigen. Additionally, we found that rabbit polyclonal
antisera to either recombinant LT-.alpha. (produced in CHO cells)
or natural LT-.alpha. (e.g., Genzyme, Boston, Mass.) recognized the
LT-.alpha. epitope(s).
[0069] We have also observed that our antisera recognizing the
LT.alpha./LT-.beta. complex blocks the MLR, whereas a particular
monoclonal antibody recognizing a soluble LT-.alpha.does not [M.
Shalaby et al., J. Immunol., 141:499 (1988)]. The
LT-.alpha./LT-.beta. complex of the invention, therefore, may be a
mediator in T cell activation.
[0070] The presence of LT-.beta. with LT-.alpha. in
immunoprecipitates from cell lysates suggested that either
LT-.beta. is antigenically related to LT-.alpha. or that LT-.beta.
is bound to LT-.alpha.or both. To address this issue 25 kD and 33
kD bands from .sup.35S-methionine labeled cells were
immunoprecipitated with rabbit polyclonal anti-rLT.alpha. serum,
eluted from excised gel slices and subjected to
reimmunoprecipitation with either anti-rLT-.alpha. polyclonal serum
or anti-rLT-.alpha. mAb. LT-.alpha., but not LT-.beta., could be
immunoprecipitated with either of the anti-rLT-.alpha. antibodies,
suggesting that LT-.beta. is not antigenically related to
LT-.alpha.. These observations indicated that LT-.beta. is
physically associated with LT-.alpha..
[0071] To further investigate the hypothesis that surface
LT-.alpha. and LT-.beta. form a complex, we performed isoelectric
focusing (IEF) experiments under both denaturing and native
conditions, the rationale being that if LT-.alpha. and LT-.beta.
are physically associated, then they should focus as a complex
under native conditions but as separate entities under denaturing
conditions. The individual isoelectric points (pI's) for LT-.alpha.
and LT-.beta. were determined by two-dimensional gel analysis
(denaturing conditions) (FIG. 9A). LT-.alpha.possesses five charged
isomers ranging in pI from 6.5 to 7.3, whereas LT-.beta. possesses
four charged isomers ranging in pI from 5.5 to 6.0. When focusing
was performed under native conditions, however, LT-.alpha. and
LT-.beta. focused together as a broad band ranging in pI from 6.3
to 7.2 (FIG. 10A, lanes 6-8). Therefore, the migration of LT-.beta.
was significantly retarded under native conditions.
Further Purification and Identification of LT-.alpha. and
LT-.beta.
[0072] We later purified these LT-.alpha. and LT-.beta. proteins
using the following general steps. We grew II-23.D7 cells in RPMI
medium with fetal bovine serum and we harvested the cells from 501
of RPMI and resuspended them in medium and we added phorbol
myristoyl acetate (PMA). After activation for 6 hours, we harvested
the cells by centrifugation and washed them with Dulbecco's
phosphate buffered saline. We suspended the final cell pellet in
cold lysis buffer and passed the pellet once through a nitrogen
cavitator. We centrifuged the lysed cells and discarded the
supernatant. We extracted the pellet overnight in lysis buffer with
detergent and then centrifuged it again.
[0073] We added the supernatant containing the detergent
solubilized membranes to affinity resin composed of monoclonal
anti-LT-.alpha. coupled to Affi-gel (10) and rocked the suspension
overnight. We collected the resin into a small column and washed it
with HEPES with nonidet P40, and then with the same buffer with 1%
w/v MEGA-8, we eluted the bound proteins with MEGA-8 in glycine
buffer and neutralized the fractions immediately with Tris base. We
determined the presence of LT-.beta. and LT-.alpha. in the
fractions by SDS-PAGE analysis and silver staining. We pooled
fractions containing these proteins and added SDS, and we dialyzed
the pool against O.1.times. laemmli sample buffer (using multiple
changes to remove the MEGA-8 detergent). We lyophilized the
dialyzed solution to dryness and resuspended it in 1/10th the
original volume of water.
[0074] We ran the sample on an SDS-PAGE gel, blotted onto a ProBlot
membrane and stained with Coomassie blue dye. We excised the
LT-.beta. and LT-.alpha. bands and loaded them into a protein
sequencer. We obtained the N-terminal sequence by Edman degradation
We found the sequence of the membrane associated LT-.alpha. band to
exactly match that described for secreted LT-.alpha., i.e., Leu Pro
Gly Val Gly Leu Thr Pro Ser (amino acid No. 1 to 9.) [P. Gray et
al., Nature, 312:121-124 (1984)]. The Edman degradation analysis
revealed that the N-terminal portion of the associated LT-.beta.
protein included two possible amino acid sequences: Gly Leu Glu Gly
Arg Gly Gin Arg Leu Gin or Gly Leu Glu Gly Arg Leu Gin Arg Leu Gin.
Subsequent DNA analysis using more accurate cDNA techniques
confirmed that the correct sequence was Gly Leu Glu Gly Arg Gly Gly
Arg Leu Gin.
[0075] In each case where a surface LT-.alpha. form was detected,
we were also able to detect LT-.beta. (i.e., in PMA-activated
II-23.D7, activated CTL clones, and Hut-78 cells constitutively
expressing a surface LT-.alpha. form). Because LT-.alpha. is
secreted from transfected CHO cells in the absence of a surface
LT-.alpha. form, and because the presence of LT-.beta. is
associated with surface-bound LT-.alpha., we concluded that
LT-.beta. complexes with LT-.alpha. to target it to the cell
surface. Biochemically, LT-.beta. and LT-.alpha. co-migrate on a
non-denaturing isoelectric focusing gel, but when the complex is
dissociated with urea, the two proteins run separately. [See FIGS.
9A, 10A.] The observations have led us to conclude that LT-.alpha.
and LT-.beta. exist as a complex on the cell surface.
Identifying DNA Sequences That Code For Lymphotoxin-.beta. And
Soluble Lymphotoxin-.beta. Peptides
[0076] Lymphotoxin-6 was purified by immunoaffinity-chromatography
as described above. Direct N-terminal sequencing and in situ
trypsin digestion followed by reverse phase HPLC resolution of the
digested peptides was performed. [See, Abersold et al., "Internal
Amino Acid Sequence Analysis Of Proteins Separated By One Or Two
Dimensional Gel Electropherisis After In Situ Protease Digestion On
Nitrocellulose," PNAS, 84:6970-6974 (1987)]. The resulting
N-terminal and internal tryptic fragment peptides were then
sequenced using conventional methods. The sequencing of the
N-terminus and internal peptides, designated as T105, T87/88, T110
and T67 are shown in FIG. 13.
[0077] Two antisense 17-mer oligonucleotide probes
GTYTCNGGCTCYTCYTC [SEQ ID NO:9] and GTYTCNGGTTCYTCYTC [SEQ ID
NO:10], designated 1368 and 1369, respectively, were synthesized to
match a portion the sequence of peptide T-87/T-88 and radiolabelled
with .sup.32P. Northern analysis showed that the probe designated
1368 hybridized strongly to a 0.9-1.1 kb mRNA band that was
strongly induced in II-23.D7 cells that had been pretreated with
phorbol ester as previously described.
[0078] A cDNA library in the vector pCDM8 was constructed from poly
A+ mRNA isolated from H-23.D7 cells induced with PMA for 6 hours.
The library was screened with the labelled oligomer designated 1368
and positive clones were isolated following washing with 3 M
tetramethylammonium chloride at 50.degree. C. Several (>16)
clones containing 0.8-0.9 kb inserts were subjected to DNA sequence
analysis.
[0079] Clone pCDM8/LT-.beta.-12 contained the coding sequence of
lymphotoxin-.beta. as shown in SEQ ID NO:1. The other clones were
identical except for various truncations at the 5' end. The clone
12 cDNA codes for a functional lymphotoxin-.beta.. Using standard
primer extension methods, three additional codons encoding the
amino acid residues--MET GLY ALA--were identified. A termination
sequence AATAAA at position 862-867 was found just prior to the 3'
poly A tract indicating that the entire 3' end had been identified.
The protein coding sequence encodes for 240 amino acids with a
calculated unmodified molecular weight of approximately 25,390
kDa.
[0080] The 5' end of the LT-.beta. DNA sequence identified from the
clone 12 cDNA and primer extension, ATGGGGGCACTGGGGCTG [SEQ ID
NO:11] reveals three possible start codons (underlined). [See,
e.g., M. Kozak, "An Analysis Of Vertebrate mRNA Sequences:
Intimations Of Translational Control," J. Cell. Biol., 115,
4:887-903 (1991).] Engineered LT-.beta. polypeptides and DNA
sequences may be derived by cleaving all or part of this 5'
sequence and substituting a single start codon.
[0081] This amino acid sequence profile is typical of a type II
membrane protein. Following a short (maximum 17) amino acid
N-terminal "cytoplasmic" domain there is an extensive stretch of 30
hydrophobic amino acids which presumably acts as a membrane
anchoring domain. No identical sequences were found within the
available databases. There is one cysteine residue in the
extracellular domain and two methionines within the last C-terminal
17 amino acids. This is consistent with the very limited cyanogen
bromide cleavage pattern exhibited by this protein.
[0082] After the sequence of LT-.beta. was determined, subsequent
comparison revealed that LT-.beta. is a type-II membrane protein
with significant homology to TNF, LT-.alpha. and the CD40 protein.
These polypeptides share four regions of sequence conservation in
the extracellular domain. See FIG. 14. Such conservation regions
are likely to enable those polypeptides to form complexes with each
other. [See, e.g., M. Eck and S. Sprang, "The Structure Of Tumor
Necrosis Factor-.alpha. At 2.6 .ANG. Resolution, Implications For
Receptor Binding," J. Biological Chemistry, 264, 29:17595-17605
(1989); E. Jones et al., "Structure Of Tumor Necrosis Factors,"
Nature, 338:225-228 (1989); M. Eck et al., "The Structure of Human
Lymphotoxin (Tumor Necrosis Factor-.beta.) At 1.9-.ANG. Resolution,
J. Biological Chemistry, 267, 4:2119-2112 (1992); J. Tavernier et
al., "Conserved Residues Of Tumor Necrosis Factor And Lymphotoxin
Constitute the Framework Of The Trimeric Structure," Fed. Eur.
Biochem. Soc. Lett., 257:2 (1989)].
Expression of Cloned LT-.beta.
[0083] The pCDM8/LT-.beta. clone 12* or a control plasmid, Clone 4
(pCDM8 with a non-functional, LT-.beta. cDNA insert), were
introduced by electroporation into CHO dhfr.sup.- cells and CO
cells stably transfected with human LT-.alpha.. After three days,
cells were removed with Ca/Mg-free Hank's solution with 5 mM EDTA
and stained for FACS analysis as described above using either 10
.mu.g/ml control IgG1 or anti-LT.alpha. monoclonal antibody
(Boehringer-Mannheim) followed by labelling of bound immunoglobulin
with either a FITC or phycoerythrin labelled goat anti-mouse
preparation. *E. coli K12 bearing a plasmid pCDM8/LT-.beta. clone
12, designated BN1289 (MC1061/P3/P33-clone-12) was deposited with
ATCC on Nov. 13, 1992.
[0084] In other experiments, COS cells were electroporated with
either clone 4 or clone 12 LT-.beta. cDNA in pCDM8 in the presence
or absence of an equal amount of human LT-.alpha.cDNA also in the
pCDM8 vector and stained for FACS analysis after three days as
above. Only CHO cells expressing LT-.alpha. displayed surface
lymphotoxin upon transfection with a functional LT-.beta. DNA,
i.e., clone 12.
[0085] Clone 12 lacks an initiating ATG codon, but does possess
several CTG initiating codons and hence this expression experiment
shows that one or several of the 5' CTG codons must initiate
translation. CTG codons are known to serve as initiating sites for
translation in several eukaryotic proteins [M. Kozak, J. Cell.
Biol., 115, 4:887-903 (1991)]. Similar results were observed using
the dual transfection system in COS cells, such that only COS cells
receiving both LT-.alpha. and LT-.beta. DNA displayed substantial
surface LT-.alpha. in a FACS analysis.
Potential Uses of LT-.beta. and LT-.alpha. and the
LT-.alpha./LT-.beta. Complex
[0086] As noted above, there is considerable structural similarity
between LT-.beta., LT-.alpha., TNF and the CD40 ligand. LT-.beta.,
LT-.alpha., TNF and the CD40 ligand are type II membrane proteins
and share at least four regions of sequence conservation in the
extracellular domain.
[0087] In light of this structural similarity, it is of interest
that LT-.alpha. is found on the surface of activated lymphocytes in
a form identical to the secreted molecule but complexed with an
additional integral membrane protein presumably anchoring
LT-.alpha. to the surface. We believe that this unique complex, now
determined to be LT-.alpha./LT-.beta., represents a more relevant
form of LT-.alpha. and imparts specificity relative to TNF.
[0088] The existence of a heterometric complex of lymphokines,
while unique to the immune system is reminiscent of signalling
molecules in other areas, e.g, the PDGF and inhibin/activin
heteromeric complexes. Delineation of the LT-.alpha./LT-.beta.
complex poses the possibility of immunoregulatory activities unique
to the complex which cannot be mimicked by the LT-.alpha.
homotrimer. The complex may bind to a unique receptor or receptor
chain combinations leading to a high affinity interaction and
biologically relevant signalling. The hypothesis of an
LT-.alpha./LT-.beta. interaction with a unique receptor complex
could account for the relatively poor activity of the LT-.alpha.
homotrimer relative to TNF in many systems, an observation which
cannot be explained by studies on the two known TNF receptors. [T.
Schall et al., Cell, 61:361-370 (1990); C. Smith et al., Science,
248:019 (1990)].
[0089] The tethering of soluble LT-.alpha. to the cell surface via
complexation with LT-.beta. indicates that cell-ell contact
specific signalling through LT-.alpha./LT-.beta. may be an
important aspect of immune regulation. Because the TNF and related
LT-.alpha. forms are secreted we believe that LT-.beta. may also be
secreted. This may be verified in studies using anti-LT-.beta.
monoclonal antibodies. Such antibodies may also be used to
determine whether LT-.beta. homo-oligomers occur naturally.
[0090] In general, LT-.alpha. and TNF exhibit qualitatively the
same spectra of activities, and LT-.alpha. and TNF are believed to
interact with the same set of receptors (designated the 55 and 80
kD TNF receptors). [C. Smith et al., Science, 248:019 (1990); T.
Schall et al., Cell, 61:361 (1990)]. Nonetheless, the quantitative
patterns of biological potency exhibited by LT-.alpha. and TNF are
dramatically different, with LT-.alpha. often being much less
potent than TNF [see, e.g., Browning et al., J. Immunol., 143:1859
(1989)]. These observations are difficult to reconcile with the
existing receptor binding data. It is possible that the
LT-.alpha./LT-.beta. complex imparts unique properties on
LT-.alpha. such that it now interacts with other as yet undefined
receptors. In this case, a LT-.alpha./LT-.beta. complex and the
other complexes of this invention would have unique biological
properties distinguishing them from either LT-.alpha. or TNF. The
LT-.alpha./LT-.beta. complex may be used to identify and clone such
LT-.alpha./LT-.beta. or LT-.beta. specific receptors. Moreover,
further use of the complex may reveal novel biological
activities.
[0091] Also, while a number of T cell and macrophage cell lines are
known to be injectable by the HIV virus, in practice only a small
number of cell lines have been useful in propagating the virus in
tissue culture. For example, the H9 line, a derivative of Hut-78
originally exploited by Gallo et al. [M. Popovic et al., Science,
224:497-500 (1984)], and another human lymphocytic line, C8166,
have been valuable for HIV propagation [M. Somasundaran and H.
Robinson, Science, 242:1554-1557 (1988)]. It is possible that
surface LT-.alpha. expression or the capacity for expression of
surface LT-.alpha. makes a given cell a good target for HIV
proliferation.
[0092] A role for TNF has been proposed in enhancing HIV
proliferation [L. Osborn et al., Proc. Natl. Acad. Sci. USA,
86:2336 (1989); Z. Rosenberg and A. Fauci, Immunol. Today, 11: [76
(1990); C. Locardi et al., J. Virology, 64:5874 (1990); G. Oli et
al., Proc. Natl. Acad. Sci. USA, 87:782 (1990)]. We have found that
the 1]-23.D7 line is infectable with the HIV strain IIIB, but upon
PMA treatment the infection by the virus is dramatically increased.
The Hut-78 cell line was found to constitutively express a surface
LT form, and the C8166 line resembles II-23.D7 in that surface LT
appears following PMA treatment. [Ware et al., J. Immunol., article
in press (1992)].
[0093] Considering these results on the infectability of II-23.D7
by HIV and the relationship between infectable cell lines and
surface LT expression, we propose that those lines may be good
hosts for HIV infection and replication because the
LT-.alpha./LT-.beta. complex and the other polypeptides and
complexes of this invention serve a regulatory role. It has been
demonstrated that the LT-.alpha. gene is induced by expression of
the HIV transcriptional activator TAT [K. Sastry et al., J. Biol.
Chem., 265:20091 (1990)] and, moreover, HTLV-1 infection has also
been shown to induce LT-.alpha. expression [N. Paul, et al., J.
Virol., 64:5412 (1990); E. Tschachler et al., in Human
Retrovirology (Raven Press 1990), W. Blattner, eds., p. 105]. Thus,
induction of LT-.alpha. by HIV infection and consequent
LT-.alpha./LT-.beta. complex or other complex expression, or
induction of LT-.alpha./LT-.beta. or other complex expression by
PMA treatment in cell lines competent to make these proteins, may
serve to enhance viral replication. For this reason, antibodies or
specific binding proteins (e.g., soluble receptors) to the
LT-.alpha./LT-.beta. complex or the other polypeptide complexes of
this invention or to soluble forms of those complexes or to
LT-.beta. and the other polypeptides of this invention may inhibit
HIV proliferation or block HIV-induced T cell death.
[0094] Parallels may be drawn between LT-.alpha./LT-.beta. and the
CD40 receptor ligand pair where signalling from a T cell surface
CD40 ligand provides "help" to the B cell via the CD40 receptor.
One could therefore postulate that surface LT-.alpha./LT-.beta. may
be a component of T cell regulation of T cells or other cells of
hematopoietic lineage such as LAK or NK cells and B-cells.
Moreover, this interaction may be dysfunctional in some autoimmune
diseases. [See, e.g., R. Watanabe-Fukunaga, Nature, 356:314-317
(1992).]
[0095] Furthermore, a cell surface protein designated as the Fas
antigen has been shown to have considerable structural homology
with a number of cell-surface receptors including TNF, NGF and the
CD40 protein. The Fas antigen has been implicated in mediating
apoptosis, a process also referred to as programmed cell death [R.
Watanabe-Fukunaga, Nature, 356:314-317 (1992); N. Itoh et al.,
Cell, 66:233-243 (1991)]. A strain of mice that demonstrates
defects in the Fas antigen develop a systemic lupus
erythematosus-like autoimmune disease. This suggests that the
structurally similar LT-.beta. or LT-.alpha./LT-.beta. complexes
may also play a role in mediating systemic lupus erthyematosus and,
therefore, intervention in this pathway may be clinically useful in
treating various autoimmune diseases. Alternatively, LT-.beta. or
an LT-.alpha./LT-.beta. complex may be involved in inducing
programmed cell death through a cell-cell contact dependent
mechanism. The emergence of this family of TNF related ligands to
complement the already extensive family of TNF/NGF type receptors
suggests the existence of an additional array of important
regulatory elements within the immune system.
[0096] LT-.beta. or a LT-.beta./LT-.alpha. complex may similarly
play a role in suppressing the immune system and may be potentially
useful in treating allergy and inducing tolerance.
[0097] The location of the TNF/LT locus in the MHC region of the
genome has led workers to examine linkage to various autoimmune
disease, especially insulin-dependent diabetes melitis. [See, e.g.,
F. Pociot et al., "A Tumor Necrosis Factor Beta Gene Polymorphism
In Relation To Monokine Secretion And Insulin-Dependent Diabetes
Mellitus," Scand. J. Immunol., 33:3749 (1991); K. Badenhoop et al.,
"TNF-.alpha. Gene Polymorphisms In type 1 (Insulin-Dependent)
Diabetes Mellitus," Diabetologia, 32:445-448 (1989).] Because we
found that the LT-.beta. gene is located next to the TNF/LT locus,
it is possible that the LT-.beta. gene or its receptor may be
involved in this autoimmune condition. Hence, LT-.beta. or its
receptor or antibodies to LT-.beta. may comprise a replacement
therapy in this form of diabetes.
[0098] As discussed above, the LT-.beta. polypeptide, and the
polypeptide complexes of this invention, are expected to have a
number of potential uses including anti-tumor, T cell activating,
or T cell suppressing applications, application involving the
treatment of systemic lupus erythematosus, as well as uses in
anti-inflammatory compositions and methods. DNA sequences coding
for LT-.beta. polypeptides, recombinant DNA molecules including
such DNA sequences, and unicellular hosts and animal or human cells
in culture transfected with such recombinant DNA molecules may then
be employed to produce large amounts of the polypeptides of this
invention, substantially free from other human proteins, for use in
the compositions and therapies noted above.
[0099] Lymphocytes expressing on their surfaces the polypeptide
complexes of this invention, and preferably an LT-.alpha./LT-.beta.
complex, represent a subset of lymphocytes that may have enhanced
abilities to kill tumor cells. As such, this subset would be useful
in LAK (lymphokine-activated killer) cell or TIL (Tumor
Infiltrating Lymphocyte) cell therapies. [H. Thomas, K. Sikora,
"Biological Approaches to Cancer Therapy," Jour. Int. Med. Res.,
17:191 (1989)]. TIL immunotherapy may be improved by gene transfer
techniques. Recombinant genes for LT-.beta. and related
polypeptides based thereon will be useful therapeutically, for
example in TIL therapy, where a LT-.beta. gene, either with or
without an LT-.alpha. gene, is introduced into T cells isolated
from a tumor and introduced to the patient. More preferably, the
cells are taken from the patient, transfected with a DNA sequence
encoding on expression a polypeptide of this invention, before or
after that transfection incubated with a lymphokine, preferably
IL-2, and returned to the patient. The transfected T cells (now
expressing LT-.beta. and also consequently complexing LT-.alpha.)
home in on the tumors from which they were removed, where the
tumorcidal action of LT-.alpha. is delivered directly to the
tumors. Likewise, it is contemplated that a LT-.beta. gene
introduced into LAK cells would increase the number of surface
complexes on the cells and enhance their activity. Alternatively,
introduction of the LT-.beta. gene into a patient's tumor cells may
be useful in creating a tumor vaccine in which the LT-.beta.
modified tumor would trigger an enhanced immune response to the
tumor itself. [See e.g., W. F. Anderson, Science, 256:808-13
(1992)].
[0100] Antibodies or antibody derivatives to the polypeptides and
polypeptide complexes of this invention are also useful in
conventional immunological methods, e.g., panning or flow
cytofluorometric sorting, to enrich for this cell population. [(J.
Wysocki and V. L. Sato, "Panning for Lymphocytes: A method for Cell
Selection," PNAS 75:2844 (1978)].
[0101] It is also contemplated that the polypeptides and
polypeptide complexes of this invention, or fragments or
derivatives thereof, will be useful in cell regulatory or
therapeutic applications similar to those in which
lymphotoxin-.alpha. and tumor necrosis factors are used.
[0102] The compositions of this invention will be administered at
an effective dose to treat the particular clinical condition
addressed. Determination of the particular dose for a given
application is well within the skill of the art taking into
consideration, for example, the condition and weight of the
patient, the extent of desired treatment and the tolerance of the
patient for the treatment. Administration of the complexes and
polypeptides of this invention, or perhaps peptides derived or
synthesized therefrom or using their amino acid sequences,
including isolated and purified forms of the polypeptides, or their
salts or pharmaceutically acceptable derivatives thereof, may be
via any of the conventionally accepted modes of administration of
agents which exhibit anti-tumor, T cell-activating, T
cell-suppressing or anti-inflammatory activity.
[0103] The compositions used in these therapies may also be in a
variety of forms. These include, for example, solid, semi-solid and
liquid dosage forms, such as tablets, pills, powders, liquid
solutions or suspensions, suppositories, injectable and infusible
solutions, and genetic therapy. The preferred form depends on the
intended mode of administration and therapeutic application. Modes
of administration may include oral, parenteral, subcutaneous,
intravenous, intralesional or topical administration. The
compositions also will preferably include conventional
pharmaceutically acceptable carriers and may include other
medicinal agents, carriers, genetic carriers, adjuvants,
excipients, etc., e.g., human serum albumin or plasma preparations.
Preferably, the compositions are in the form of a unit dose and
will usually be administered one or more times a day.
[0104] The following are examples which illustrate the LT-.beta.
and the LT-.alpha./LT-.beta. complex of this invention and the
methods used to characterize them. These examples should not be
construed as limiting: the examples are included for purposes of
illustration and the present invention is limited only by the
claims.
EXAMPLES
[0105] We used the following experimental procedures in the
examples:
Antisera
[0106] Recombinant human LT-.alpha.(rLT-.alpha.) was expressed and
secreted by a stably transfected chinese hamster ovary (CHO) cell
line into serum-free conditioned media. We purified the secreted
rLT-.alpha. from the serum-free conditioned media by a series of
Sepharose S, lentil lectin-sepharose and FPLC Mono Q column
chromatography steps. The properties of the CHO cell-derived r
LT-.alpha. preparation have been described. [J. Browning et al., J.
Immunol., 143:1859 (1989)]. We immunized two rabbits (4 and 5) by a
lymph node procedure [M. Sigel et al., "Production Of Antibodies By
Inoculation Into Lymph Nodes," Met. Enz., 93:3 (1983)] with 25
.mu.g of native r LT-.alpha. in complete Freund's adjuvant. A third
rabbit (6) was immunized via the same route with 25 .mu.g of
denatured r LT-.alpha. in complete Freund's adjuvant. We prepared
denatured r LT-.alpha. by SDS-PAGE followed by electroelution into
0.1% SDS-carbonate buffer.
[0107] Using the above methods, three anti-rLT-.alpha. antisera
were generated, two directed against native rLT-.alpha. and a third
against SDS-denatured rLT-.alpha.. The antisera raised by
immunization with native protein (rabbits 4 and 5) could neutralize
a 50 unit/ml solution at a dilution of 1:2000-5000. The serum
raised against denatured rLT-.alpha. (rabbit 6) lacked neutralizing
titer, but was weakly reactive with rLT-.alpha. on a Western blot.
None of the antisera could neutralize r-human TNF nor could they
recognize r-human TNF bound to an ELISA plate except for a very
weak titer in the antiserum from rabbit 6. Only antiserum from
rabbit 6 was capable of recognizing rLT in a western analysis.
[0108] We immunized a fourth rabbit with recombinant human TNF. We
prepared the polyclonal anti-rTNF rabbit serum via a classical
immunization scheme using recombinant human TNF (E. coli derived
[D. Weir et al., Handbook Of Experimental Immunology In Four
Volumes, Chapter 8 "Immunization Of Experimental Animals"]) in
complete Freund's adjuvant followed by a boost in incomplete
Freund's adjuvant. The serum raised against rTNF by immunization
had a good neutralizing titer. A neutralizing monoclonal antibody
to TNF has been described [Liang et al., "Production And
Characterization Of Monoclonal Antibodies Against Recombinant Human
Tumor Necrosis Factor/Cachectin," Biochem. Biophys. Res. Comm.,
137:847 (1986)]. Pre-immunization serum was collected from all
animals for use as controls.
Cell Growth and T Cell Activation
[0109] All cells were obtained from the American Type Culture
Collection (ATCC), except for the LT-.alpha. transfected Chinese
hamster ovary (CHO) line that was described previously [Browning,
J. Immunol., 143, pp. 1859-1867 (1989)].
[0110] Cells were grown in RPMI 1640 supplemented with 1%
glutamine, 10 mM HEPES buffer, pH 7.5, penicillin/streptomycin and
10% fetal bovine serum (Hyclone-defined) (designated "complete
RPMI"), except for the transfected CHO cells which were grown in
Dulbecco's modified Eagle's medium supplemented as above. The human
T cell hybridoma, 11-23, was a result of a fusion of the human CEM
tumor line with activated peripheral T lymphocytes and was further
subcloned (II-23.D7) [C. Ware et al., "Human T Cell Hybridomas
Producing Cytotoxic Lymphokines: Induction of Lymphotoxin Release
And Killer Cell Activity By Anti-CD3 Monoclonal Antibody Or Lectins
And Phorbol Ester," Lymph. Res., 5, 313 (1986)]. Human peripheral
blood lymphocytes (PBL) were drawn into heparinized glass tubes,
isolated by Ficoll-Hypaque centrifugation, washed and resuspended
in complete RPMI medium. We treated PBL at 2.times.10.sup.6
cells/ml with a 1:500 dilution of OKT3-conditioned medium (-2 ng/ml
final) in the presence of 1 .mu.g/ml indomethacin and, in some
experiments, with 10 ng/ml rIL-2 (Biogen, Inc., Cambridge, Mass.)).
Human CTL-clones were generated as described [L. Green et al.,
"Cytotoxic Lymphokines Produced By Cloned Human Cytotoxic T
Lymphocytes," J. Immunol., 135:4034 (1985)] and activated either
with irradiated stimulator cells (antigen) or a combination of
anti-CD2 monoclonal antibodies (T11.sub.2+T11.sub.3) provided by E.
Reinherz.
Flow Cyometry
[0111] We resuspended cells in RPMI 1640 medium with 10% fetal
bovine serum (FBS), 0.1% sodium azide and 0.1 mg/ml human IgG at
0.degree. C. Following preincubation with the human IgG, we added
additional media containing the desired antisera. Typically the
cells were incubated with a final dilution of the anti-rLT-.alpha.
and anti-rTNF sera of 1:200 for 60-90 min. We washed cells twice
with Dulbecco's phosphate buffered saline (PBS) and then incubated
them with a 1:500 dilution of fluorescein-labeled goat anti-rabbit
IgG (Cappel Durham, N.C.) in the above medium for a minimum of 60
min. Cells were then washed once and either analyzed directly or,
in some cases, analyzed following fixation for 10 min. at 0.degree.
C. with 0.5% paraformaldehyde. We performed two color analyses as
above, except that we added phycoerythrin labeled leu-4, leu-2,
leu-M3 or leu-16 or leu-19 (Becton-Dickinson, Mountain View,
Calif.) at the second antibody stage. The comparison of
surface-bound LT-.alpha. with IL-2 receptor levels was done with
separate single color analyses with fluorescein-labeled anti-IL-2
receptor (CD25) antibody (Becton-Dickinson, Mountain View, Calif.).
Analyses were performed with a FACStar instrument
(Becton-Dickinson).
Adsorption of Neutralizing Anti-rLT-.alpha. Antibodies by Activated
II-23.D7 Cells
[0112] We stimulated 11-23.D7 and U937 premonocytic cells at
1.times.10.sup.6 cells/ml for 8 hours with 10 ng/ml of PMA in
complete RPMI medium. We washed the cells (1.times.10.sup.8) three
times in medium and aspirated the supernatant to obtain a dry
pellet. The cells were then resuspended in 1 ml of medium
containing a 1:1000 dilution of anti-rLT-.alpha. serum (from rabbit
4) and incubated on ice for 1.5 hours with mixing. Cells were
cleared from the antiserum by centrifugation. We mixed the absorbed
antiserum (both pre- and post-immune) with an equal volume (50
.mu.l) of medium containing 15 U/ml of rLT-.alpha. and incubated
for 20 minutes at room temperature. The mixtures were diluted
serially into medium and added to L929 cells (in 0.1 ml) and
incubated a further 24 hours. We assessed cell viability by the MTT
assay as described [L. Green et al., "Rapid Colorimetric Assay For
Cell Viability: Application To The Quantitation of Cytotoxic And
Growth Inhibitory Lymphokines" Jour. Immunol. Meth., 70:257-268
(1984)].
.sup.35S-Methionine or .sup.35S-Cysteine Metabolic Labelling of T
Cells
[0113] We transferred cells into either cysteine-free or
methionine-free RPMI 1640 supplemented with
penicillin/streptomycin, glutamine, 10 mM HEPES pH 7.5, 10% v/v
dialyzed PBS and 2% v/v conventional RPMI (cold carrier addition).
We adjusted the cell concentration to 2-3.times.10.sup.5 cells/ml
and added .sup.35S-methionine or .sup.35S-cysteine to the
appropriate medium to a level of 100-200 .mu.Ci/ml. In the case of
freshly activated PBL, the supernatants were gently removed, and
the cells were centrifuged, resuspended in labelling medium and
added back to the original adherent population. Following a 12-18
hour labelling period, we washed and lysed the cells as described
below. With the PBL, cells were removed by pipetting and the
adherent population partially removed by treatment with 5 mM EDTA
in PBS.
Immunoprecipitations
[0114] To 0.2-0.5 ml of labeled cell lysate we added 24 .mu.l of
rabbit serum. The sample was left for 1-2 hours at 4.degree. C. We
then added a 60 .mu.l aliquot of a 60-75% suspension of washed
Protein A sepharose (Pharmacia, Piscataway, N.J.) and rocked the
sample for 6-18 hours at 4.degree. C. We washed the Protein A
sepharose pellets 3 times with 1% NP-40 in calcium/magnesium free
PBS and resuspended them in 50 .mu.l of Laemmli SDS loading buffer.
Typically a single lysate sample was cycled through sequential
immunoprecipitations with preimmune anti-rLT-.alpha. serum,
anti-rTNF antiserum and finally post immune anti-rLT-.alpha.
antiserum. In one set of experiments, we added 5 mM CaCl.sub.2 and
MnCl.sub.2 to the lysate and the lysate was rocked overnight with
75 .mu.l of 75% suspension of washed lentil lectin-sepharose. The
sepharose was washed twice with NP-40/PBS and then eluted with 3
consecutive additions of 75 .mu.l of 1% NP-40/PBS with 0.25 M
.alpha.-methyl mannoside. We subjected the pooled washes to the
immunoprecipitation protocol.
Rabbit Anti-rLT Affinity Column
[0115] We purified the immunoglobulin fraction from the anti-rLT
serum (from rabbit 4) using Protein A sepharose with acid pH
elution. The eluted IgG-containing fractions were dialyzed against
PBS and concentrated by amicon filtration. The
anti-rLT-.alpha.-IgG-solution (15 ml of 6 mg/ml) was coupled to 8
ml of Affi-gel 10 resin (Biorad, Richmond, Calif.) as per
instructions. We prepared an identical affinity column using
nonspecific rabbit IgG (Cappel,. Durham, N.C.). Both columns were
washed with PBS, 1 M acetate pH 3.0 with 1% NP-40 and finally with
lysis buffer lacking protease inhibitors.
Initial Purification of LT-.beta. and LT-.alpha.
[0116] We grew II-23.D7 cells (15 l) to a density of
5.times.10.sup.5 cells/ml and added phorbol myristic acetate (PMA)
to give a final concentration of 25 ng/ml. After 24 hours, we
harvested the cells and washed them into cold serum-free RPMI
medium. To the chilled cell pellet containing 7.times.10.sup.9
cells we added 100 ml of ice-cold lysis buffer (50 mM HEPES pH 7.5,
1% v/v NP-40, 2 mM EDTA, 0.15 M NaCl and 0.1% sodium azide) to
which 5 mM benzamidine, 1 mM phenyl methyl sulfonyl chloride (PMSF)
and 0.25 mM N-ethyl maleimide (NEM), 10 g/ml soybean trypsin
inhibitor, 0.7 .mu.g/ml pepstatin and 10 .mu.g/ml aprotinin had
been freshly added. We gently homogenized the cells in a dounce
homogenizer and centrifuged the lysate at 10,000.times.g for 10
minutes. We centrifuged the supernatant at 60,000.times.g for 90
minutes and collected the supernatant.
[0117] To the supernatant from the high speed centrifugation
we-added 5 mM CaCl.sub.2 and 5 mM MnCl.sub.2. The supernatant was
then loaded onto a 20 ml lentil-lectin sepharose column (Pharmacia,
Piscataway, N.J.) equilibrated in lysis buffer plus CaCl.sub.2 and
MnCl.sub.2. We washed the column with lysis buffer (with CaCl.sub.2
and MnCl.sub.2) and then eluted the column with lysis buffer
containing 0.25 M .alpha.-methyl mannoside.
[0118] We pooled the lentil lectin eluate fractions to give a
volume of 50 ml and loaded them directly onto a 2 ml rabbit
nonspecific IgG sepharose affinity column. We connected this column
directly to a 2 ml rabbit anti-rLT-.alpha. sepharose affinity
column. We washed both columns with the same lysis buffer with
EDTA, followed by lysis buffer wherein the 1% NP-40 had been
replaced with 1% w/v MEGA-8 (Octanoyl-N-methyl glucamide,
Boehringer-Mannheim, Indianapolis, Ind.).
[0119] We eluted the washed columns individually with 1% MEGA-8, 50
mM glycine pH 2.5, 0.05 M NaCl, 5 mM benzamidine, and 2 mM EDTA. We
pooled the first 20 ml following the pH shift, lyophilized the pool
and resuspended it in 1 ml of water with 0.05% SDS, and dialyzed it
against 10 mM HEPES pH 7.5, 0.05% SDS and 0.1% MEGA-8. We dried the
dialyzed fractions on a speed-vac and resuspended them in 0.15 ml
of water. We mixed aliquots with Laemmli loading buffer and
electrophoresed them on SDS-PAGE. The LT-.beta.and LT-.alpha.
proteins were visualized by silver staining.
Iodination of II-23.D7 Cell Surface
[0120] Either control or PMA-induced II-23.D7 cells were washed
extensively in calcium/magnesium-free PBS, treated with 1 mM PMSF
and 0.25 mM NEM and then washed twice. To a 12.times.75 mm glass
tube that was coated with 50 .mu.g of iodogen (Pierce) we added 0.3
ml of cells (1.times.10.sup.7 total) and 1-2 mCi of .sup.125sodium
iodide. Cells were left with periodic swirling for 25 minutes at
room temperature, washed 3 times in PBS with 10% FBS and
resuspended in lysis buffer as described above. We then removed the
nuclei with a 2 minute centrifugation in an Eppendorf centrifuge.
We then centrifuged the supernatant an additional 15 minutes. The
cleared supernatant was subjected to the immunoprecipitation
protocol.
1-Dimensional CNBr Peptide Mapping
[0121] We electrophoresed samples on a 12% acrylamide SDS-PAGE
Laemmli system gel for a short distance and excised the appropriate
gel sections. We soaked the gel slices for 1 hour in 1.0 ml of 0.1
N HCl, 0.2% 2-mercaptoethanol with 15 .mu.l of 700 mg/ml fresh CNBr
in 90% formic acid. The slices were then removed and washed for 5
minutes with 0.1 M Tris-Cl pH 8.0, 5 min with 25 mM Tris-Cl pH 8.0
and finally 10 min with 1.times. Laemmli SDS-PAGE loading buffer.
We loaded the slices onto a 15% SDS-PAGE Laemmli gel with a 12%
acrylamide stacking gel. We visualized the peptide bands by silver
staining or autoradiography of the dried gel.
Reimmunoprecipitation
[0122] Reimmunoprecipitation of SDS-PAGE-separated antigens was
carried out by excising labeled bands from gels, rehydrating them
for 10 minutes in TBS, 0.2% SDS, and then dicing the gel slices
into small pieces. The proteins were eluted by incubation in 1 ml
TBS, 0.2% SDS for 8 hours at room temperature, with rotation. After
elution the gel pieces were removed by centrifugation, and NP-40
was added to the supernatant to a final concentration of 2%. The
eluted proteins were then immunoprecipitated as above, and
reanalyzed by SDS-PAGE.
Isoelectric Focusing (IEF)
[0123] Two-dimensional IEF was performed essentially as described
by P. H. O'Farrell [J. Biol. Chem., 250:4007-4021 (1975)].
.sup.125I-labeled antigens were immunoprecipitated from II-23.D7
cell extracts, and the immunoprecipitated proteins were eluted by
heating at 100.degree. C. for 5 minutes in 100 .mu.l O'Farrell
sample buffer containing 9.5 M urea. The eluted proteins were then
focused (first dimension) on 14 cm.times.3 mm tube gels possessing
a 2% final concentration of ampholines (range pH 3-10, Sigma) at
room temperature for 16 hours at a constant voltage (400 V). The
second dimension was 12% SDS-PAGE.
[0124] For IEF under native (non-denaturing) conditions,
.sup.125I-labeled cell extracts were focused directly on tube gels
identical to those above except for the presence of urea. The
labeled extract (200 .mu.l volume) was centrifuged at
100,000.times.g (30 psi, airfuge) for 10 minutes prior to loading
onto the tube get. The focusing was performed at 4.degree. C. under
the same conditions as described above. The tube gel was then
removed and sliced into 1 cm sections, and the proteins were,
eluted by incubating each slice in 1 ml TBS, 2% NP-40, 2 mM PMSF
for 8 hours at room temperature, with rotation. The supernatants
containing the eluted proteins were then immunoprecipitated and
analyzed by SDS-PAGE. The pH gradients for both the denatured and
native tube gels were determined by measuring the pH's of
individual slices from gels run in parallel.
T Cell Proliferation Assays
[0125] We isolated and resuspended PBL in complete RPMI as
described above except for the substitution of fetal bovine serum
with 10% human autologous serum, 1 .mu.g/ml indometacin and 50 U/ml
polymyxin B. In the MLR experiments, autologous serum was the
responder's serum. We irradiated stimulator cells from a different
donor with 3000 rads. We preheated rabbit sera for 1 hour at
56.degree. C., and diluted and sterile filtered the sera prior to
use in proliferation assays. Cells (1.times.10.sup.5 total) in 0.2
ml in a round bottom 96-well plate were treated with either 5
.mu.g/ml phytohemagglutinin, 1-2 ng/ml OKT3 or 1.5-2.times.10.sup.5
irradiated stimulator cells in the presence or absence of various
antisera or cytokines. After 3 days (PHA or OKT3 activation) or 5
days (MLR), cells were pulsed with .sup.3H-thymidine, harvested and
counted.
Further Purification of LT-.alpha. and LT-.beta.
[0126] We grew II-23.D7 cells in RPMI medium with 10% fetal bovine
serum and we harvested the cells from 50 1 RPMI medium and
resuspended them in medium at a concentration of 4.times.10.sup.6
cells/ml and we added 50 ng/ml phorbol myristoyl acetate (PMA).
After activation for 6 hours we harvested the cells by
centrifugation and washed them with Dulbecco's phosphate buffered
saline. We suspended the final cell pellet of 4.times.10.sup.10
cells in 200 r[1 of cold lysis buffer (50 mM HEPES buffer, pH 7.0;
0.1 M NaCl, 10 mM EDTA, 5 mM benzamidine, 10 .mu.g/ml each of
soybean trypsin inhibitor, aprotinin, chymostatin, leupeptin,
antipain, 1 .mu.g/ml pepstatin and 1 mM phenylmethyl sulfonyl
fluoride) and passed the pellet once through a nitrogen cavitator.
We centrifuged the lysed the cells at 40,000 rpm for 60 minutes in
a 50.2 Ti rotor and discarded the supernatant. We extracted the
pellet overnight in 120 ml of lysis buffer with 1% W/v Nonidet P40
detergent and then centrifuged it as above.
[0127] We added the supernatant containing the detergent
solubilized membrane proteins to 2 ml of affinity resin composed of
monoclonal anti-lymphotoxin (anti-tumor necrosis factor-.beta. from
Boehringer Mannheim) coupled to Affi-gel 10 (BioRad) and rocked the
suspension overnight. We collected the resin into a small column
and washed it with 50 mM HEPES, pH 7.0 with 1% Nonidet P40, and
then with the same buffer with 1% w/v MEGA-8 (Boehringer Mannheim).
We eluted the bound proteins with 1% MEGA-8 in 50 mM glycine buffer
pH 2.5 and the fractions immediately neutralized with Tris base. We
determined the presence of p33 and LT in the fractions by SDS-PAGE
analysis and silver staining. We pooled factions containing these
proteins and added SDS to a final concentration of 0.1% w/v and we
dialyzed the pool against 0.1.times. Laemmli sample buffer
(multiple changes to remove the MEGA-8 detergent). We lyophilized
the dialyzed solution to dryness and resuspended it in 1/10th the
original volume of water. We ran the sample on an SDS-PAGE gel,
blotted onto a ProBlot membrane (Applied Biosystems) and stained
with coomassie blue dye.
[0128] This scheme allows one to purify LT-.beta. to a band on a
blot. It should be possible for anyone skilled in the art to
separate the proteins eluted from the affinity resin by ion
exchange chromatography. For example, the complex can be
dissociated with urea and the LT-.alpha. and LT-.beta. proteins can
be separated by, e.g., MONO Q FPLC (Pharmacia) anion exchange
chromatography in Tris-Cl buffer pH 8.0 with 1% nonionic detergent
(e.g., MEGA-8, Boehringer-Mannheim) and urea, using a salt gradient
elution. This chromatographic technique separates on the basis of
differing charges on the proteins. The two proteins are separable
in an isoelectric focusing experiment (see, supra) on the basis of
charge differences, wherein urea is used to dissociate the
LT-.alpha./LT-.beta. complex. Such a combination of affinity
chromatography, dissociation in urea/nonionic detergent and ion
exchange chromatography allows purification of soluble LT-.beta. or
the LT-.alpha./LT-.beta.complex.
Peptide Sequencing Assays
[0129] We excised the LT-.beta. and LT-.alpha. bands from the
Problot and loaded them into a protein sequencer. We obtained
N-terminal sequence information by Edman degradation with a model
470A Applied Biosystems sequencer coupled to a 120A PTH amino acid
analyzer. LT-.beta. was purified by immunoaffinity-chromatography
as described above and tryptic fragment sequence was obtained. [See
Abersold et al., "Internal Amino Acid Sequence Analysis Of Proteins
Separated By One Or Two-Dimensional Gel Electrophoresis After In
Situ, Protease Digestion On Nitrocellulose," PNAS:84:6970-6974
(1987)]. That is, protein on the blot was digested with trypsin in
situ followed by reverse phase HPLC resolution of the digested
peptides. The resulting N-terminal and internal tryptic fragment
peptides were then sequenced by Edman degradation. The sequencing
of the N-terminal and internal peptides designated as T105, T87/88,
T100 and T67 are shown in FIG. 13.
Construction of Oligonucleotide Probes
[0130] From the sequence of T87/88 the following antisense probes
were designed: TABLE-US-00001 1368 GTYTCNGGCTCYTCYTC [SEQ ID NO: 9]
1369 GTYTCNGGTTCYTCYTC [SEQ ID NO: 10]
and synthesized by standard methods. [See, e.g., J. Sambrook et
al., Molecular Cloning, A Laboratory Manual, 2ed. (1989)].
Preparation Of An Induced II-23 cDNA Library
[0131] We prepared a cDNA sublibrary as follows:
[0132] We stimulated II-23.D7 cells for six hours with 50 ng/ml PMA
to ensure the presence of LT-.beta. mRNA. We isolated the mRNA from
these cells and reverse-transcribed it into cDNA using techniques
well known to the art. [B. Seed and A. Aruffo, "Molecular Cloning
Of The CD2 Antigen, The T-Cell Erythrocyte Receptor, By A Rapid
Immunoselection Procedure," PNAS, 84:3365-3369 (1987)]. Using
standard procedures, we ligated double stranded cDNA to a
NotI-BstXI linker/adaptor having the following sequence:
TABLE-US-00002 5' GCG GCC GCT TTA GAG CAC A 3' [SEQ ID NO: 12] 3'
CGC CGG CGA AAT CTC 5'
We then size-selected the cDNA on a 4.2 ml 5-20% potassium acetate
gradient, 2 mM EDTA, 1 .mu.g/ml ethidium bromide, in a Beckman.RTM.
SW60 Rotor for 3 hours at 50,000 rpm at 22.degree. C. according to
standard methods. We pooled the cDNA fragments of greater than 500
base pairs. Then we prepared the vector, pCDM8 (a gift from Brian
Seed (Massachusetts General Hospital). We digested this plasmid
with BstXI. To remove the 400 base pair stuffer fragment we
centrifuged the mixture on a potassium acetate gradient, as above,
and isolated the large fragment. We further purified this fragment
by agarose gel electrophoresis, and then ligated the cDNA to the
vector. In this way, we created recombinant DNA molecules
containing DNA sequences for mRNA expressed in induced II-23.D7
cells. We used these plasmids to transform E. coli MC1061 P3. The
result was a collection of over 1.times.10.sup.6 recombinant clones
comprising a cDNA library for PMA induced II-23.D7 mRNA. Screening
and DNA Sequencing of Clones
[0133] The pCDM8 II-23.D7 library was screened with .sup.32P
labelled oligomer 1368 and positive clones were isolated following
washing with 3M tetramethylammonium chloride at 50.degree. C. [J.
Sambrook et al., Molecular Cloning, A Laboratory Manual, (1989);
Jacobs et al., "The Thermal Stability Of Oligonucleotide Duplexes
In Sequence Independent In Tetraalkylammonium Salt Solutions:
Application To Identifying Recombinant DNA Clones", Nucleic Acids
Research, 16:10:4637-4649 (1988)]. Several clones containing 0.9 kb
inserts were subject to dideoxynucleotide DNA sequence analysis
[Id.].
Expression of LT-.beta. cDNA
[0134] The pCDM8/LT-.beta. clone 12 or a control plasmid, clone 4
(pCDM8 with an irrelevant cDNA insert), was introduced by
electroporation into CHO dhfr.sup.- and a CHO cell stably
transfected with human LT-.alpha.. After three days, cells were
removed with Ca/Mg-free Hank's solution with 5 mM EDTA and stained
for FACS analysis as described above using either 10 .mu.g/ml
control IgG.sub.1 or anti-LT monoclonal antibody
(Boehringer-Mannheim) followed by labelling of bound immunoglobulin
with either a FITC or phycoerythrin labelled goat anti-mouse
preparation. In other experiments, COS cells were electroporated
with either clone 4 or clone 12 LT-.beta. cDNA in pCDM8 in the
presence or absence of an equal amount of human LT-.alpha. cDNA
also in the pCDM8 vector and stained for FACS analysis after three
days as above.
Northern Analysis of LT-.beta. Expression
[0135] Poly A+ RNA was isolated from either 11-23.D7 cells or
peripheral blood mononuclear cells (PMBC) using the Fast-Track.TM.
system provided by Invitrogen. Northern blots were prepared using 2
.mu.g/lane of RNA and electrophoresis on a formamide gel
essentially as described in J. Sambrook et al., Molecular Cloning,
A Laboratory Manual, (1989), followed by transfer onto Gene Screen
nylon membrane and UV crosslinking. Blots were probed with random
primed BstEII/Xmn-I fragment of the LT-.beta. cDNA which had been
gel purified, or a fragment of human LT-.alpha. or actin. II-23.D7
cells were inducted with 50 ng/ml PMA for varying times and both
LT-.alpha. and LT-.beta. expression was found to be induced. PBMC
were either cultured in RPMI medium alone or in the presence of
1000 units/ml of IL-2 or with OKT3 to activate the T-cells.
Determination of the 5' End of LT-.beta.
[0136] The 5' mRNA sequence was determined by primer extension
analysis. [B. Wallner et al., Nature 320:77-81 (1986).] Primer
extension using an oligonucleotide primer (probe 360-121
5'GACAGTGATAGGCACCGCCAGCAACAA-3') [SEQ ID NO:13] yielded a roughly
128-130 bp product that upon sequencing using Maxam and Gilbert
methodology [A. Maxam and W. Gilbert, "Sequencing End-Labeled DNA
With Base-Specific Chemical Cleavages," Methods In Enzymology,
65:499 (1988)] showed the transcriptional start site to be 7-9 bp
upstream of the methionine ATG. The expression exhibited by clone
12 in transient experiments indicates that one or both of the Leu4
or Leu-6 start site is functional. To verify the 5' mRNA sequence,
a cosmid clone, 031A [Spies et al., Science 243:214 (1989)], was
digested with several restriction enzymes, electrophoresed, blotted
and probed with a BST E2/Xmm-1 fragment of the LT-.beta. cDNA. The
cosmid contained the LT-.beta. gene within a 6 kb EcoR1 fragment
which was subcloned into a pUC derivative called pNN 109 which
contained a kanamycin resistance gene. Dideoxynucleic acid
sequencing gave the entire genomic sequence.
Example 1
T cells Express LT-related Epitopes on their Surfaces
[0137] Under the conditions described above, we activated human
peripheral mononuclear cells (PMN) with OKT3 monoclonal antibody
and, after two days in culture, we analyzed them for expression of
LT-.alpha./LT-.beta. complex related forms using flow
cytofluorometric analysis. In one experiment, the results of which
are shown in FIG. 1, we cultured fresh PBL for 3 days with OKT3 and
IL-2 and stained them with a 1:200 dilution of antisera to native
rLT-.alpha. ("LT-4" and "LT-5" panels on FIG. 1, from rabbits 4 and
5 respectively), denatured rLT.alpha. ("LT-.beta." panels on FIG.
1, from rabbit 6) and native rTNF ("TNF" panel on FIG. 1, from
rabbit 7). We stained cells with postimmune serum (solid lines on
FIG. 1 panels) or with preimmune serum from each animal (dotted
lines on FIG. 1 panels) FIG. 1 shows that only anti-rLT-.alpha.
sera from rabbits 4 and 5 recognized epitopes on the activated
peripheral T cells.
[0138] In the experiment shown in FIG. 2, we treated II-23.D7 cells
with or without 10 ng/ml PMA for 15 hours and stained them as
described in the FIG. 1 experiment, with rabbit 4 anti-rLT-.alpha.
postimmune serum (solid line on, FIG. 2 panels) or with rabbit 4
preimmune serum (dotted line on FIG. 2 panels). As shown in FIG. 2,
we found that the T cell hybridoma II-23.D7, which synthesizes
LT-.alpha. upon phorbol ester (PMA) stimulation, expressed surface
LT-related epitopes upon PMA activation.
[0139] To establish that the LT-.alpha.-related epitopes on T cells
were related to LT and not to some contaminant in the CHO-cell
derived recombinant LT-.alpha. preparation, we treated a 1:1000
dilution of antiserum from rabbit 4 with PMA-activated, washed
11-23.D7 or U937 cells. In the experiment shown in FIG. 3, we
treated a 1 ml sample of anti-rLT-.alpha.sera (1:1000 anti-LT-4)
with either no cells (- -), 1.times.10.sup.8 U937 cells (--O--),
1.times.10.sup.8 (- -) PMA-activated II-23.D7 cells or
1.times.10.sup.7 (- -) PMA-activated II-23.D7 cells. We added
dilutions of absorbed antisera to a limiting amount of rLT-.alpha.
in a L929 cytotoxicity assay such that a 1:4000 final dilution was
present in the first well. This assay measures the ability of
LT-.alpha. to kill a mouse fibroblast cell line, L929, within a 24
hour period [L. Green, J. L. Reade, C. F. Wate, "Rapid Colorimetric
Assay for Cell Viability: Application to the Quantitation of
Cytotoxic and Growth Inhibitory Lymphokines," J. Immunol. Methods,
70:257 (1984)]. After 24 hours, we assessed cell viability using a
MIT readout. Plotted on FIG. 3 is optical density (which is
proportional to cell viability) vs. the dilution of absorbed
antisera. Data represent the average of duplicate wells and
duplicates generally were within the range defined by the symbol.
As shown in FIG. 3, analysis of the neutralizing titer of the
absorbed antisera in the standard L929 cytotoxicity assay
demonstrated that the activated 11-23.D7 cells removed the
LT-.alpha.neutralizing antibodies, whereas. U937 cells were
ineffective. These data indicate that the antigenic structures on
the membrane surface are actually related to LT-.alpha..
[0140] We subjected the hybridoma II-23.D7 to a number of further
treatments to examine a number of trivial explanations for the
apparent existence of LT-.alpha. related epitopes on T cell
surfaces. First we ruled out the possibility that
LT-.alpha.:antibody complexes in the antisera could bind to
TNF/LT-.alpha. receptors on the hybridoma. Both TNF and LT-.alpha.
have trimeric structures which could allow for the presence of
antibody binding epitopes within the complex. However, prior
saturation of the cellular TNF receptors with soluble TNF or
LT-.alpha. had no effect on the surface staining. Such saturation
should have prevented such an immune complex from binding to such a
receptor.
[0141] A pH 3 lactic acid treatment, which can release bound TNF
from its receptor, had no effect on the signal, suggesting that the
LT-.alpha. is not receptor bound. However, experiments utilizing
.sup.125]-LT-.alpha. binding to 11-23.D7 cells indicated that
receptor bound LT-.alpha. was more difficult to remove from its
receptor at acidic pH's than TNF.
[0142] Mild trypsinization of the cells prior to staining led to a
loss of the signal, indicating that the epitope is a protein. To
determine whether surface-associated LT-.alpha.was
phosphatidylinositol linked, the cells were treated with a
phosphatidylinositol specific phospholipase C. Under conditions
where a PI-linked antigen, LFA-3, could be released [A. Peterson et
al., "Monoclonal Antibody And Ligand Binding Sites Of The T Cell
Erythrocyte Receptor (CD2)," Nature, 329:842 (1987)], no effect was
observed on the LT-.alpha. epitope.
[0143] We could not stain CHO cells stably transfected with the
LT-.alpha. gene, either with or without prior PMA activation,
indicating that antibodies to CHO derived contaminants in the
original rLT-.alpha. used to immunize the rabbits were not present
in sufficient amounts to contribute to the staining of 11-23.D7
cells. Likewise, antibodies generated against any fetal bovine
serum proteins contaminating the LT-.alpha. preparation would be
ineffective in staining T cells since the staining was performed in
10% fetal calf serum.
[0144] Pretreatment of the anti-rLT-.alpha. serum with rLT-.alpha.
blocked the staining of LT-.alpha.forms on II-23.D7 cells whereas
pretreatment with rTNF did not.
Example 2
Immunoprecipitation of LT-.alpha.-related Proteins on the T cell
Hybridoma II-23.D7
[0145] We surface iodinated PMA activated II-23.D7 cells and lysed
and solubilized the cells in detergent. Immunoprecipitation and
SDS-PAGE analysis of the labeled membrane proteins showed that two
proteins were recognized by anti-rLT-.alpha. antisera. FIG. 4A
shows the results of SDS-PAGE analysis of the iodinated surface
proteins precipitated with either pre-immune (PRE) or post-immune
(POST) anti-rLT-.alpha. serum (from rabbit 4).
[0146] As shown in FIG. 4A, we observed a 25-26 kD molecular weight
form ("LT-.alpha.") that correlated with the expected size of
LT-.alpha., and we also saw an additional form of approximately 33
kD ("LT-.beta. or p33"). Neither the preimmune serum from the same
rabbit (FIG. 4A column PRE) nor anti-rTNF rabbit serum were able to
immunoprecipitate any bands from the iodinated, PMA-activated
II-23.D7 cells.
[0147] 1-D partial CNBr peptide mapping of the Iodinated bands
showed that the 25-26 kD form was cleaved in a pattern identical to
that of iodinated rLT-.alpha., thus identifying this band as
LT-.alpha.. In the experiment shown in FIG. 4B, the 25-26 kD and 33
kD bands from panel A were excised, subjected to limited CNBr
cleavage and electrophoresed on a SDS-PAGE system. For comparison,
cleavages of both rTNF and rLT-.alpha. performed in parallel are
shown in FIG. 4B. The gels were visualized by autoradiography. Lane
1 represents rTNF, lane 2 represents rLT, lane 3 represents LT, and
lane 4 represents LT-.beta.. The increased sizes of the CNBr
fragments reflect the increased amount of carbohydrate on natural
LT-.beta.. The iodinated 33 k[D form was not cleaved by CNBr (lane
4), indicating that it is different from the known LT-.alpha. gene
product. rTNF was not cleaved with CNBr (lane 1) due to the absence
of methionine in this protein.
[0148] We undertook metabolic labelling with .sup.35S-methionine
and .sup.35S-cysteine coupled with immunoprecipitation to
characterize further these LT-.alpha.-related surface forms. In the
case of the TNF/LT-.alpha. pair, the distribution of cysteine and
methionine allows one to distinguish both between TNF and
LT-.alpha. and between forms with and without their signal
sequences, as was exploited in studies on the membrane TNF form [M.
Kriegler et al., Cell, 53:45-53 (1988)]. In the case of the fully
processed cytokines, i.e., secreted forms, TNF contains cysteine
and not methionine, while LT-.alpha. contains only methionine and
not cysteine. LT-.alpha., however, has two cysteine residues in the
signal sequence domain and TNF contains a methionine residue in
this N-terminal region. Separate cultures of II-23.D7 hybridoma
cells were labeled with either .sup.35S-methionine or
.sup.35S-cysteine and immunoreactive proteins were precipitated. In
an experiment, the results of which are shown in FIG. 5, II-23-D7
cells were activated with 10 ng/ml PMA and simultaneously labeled
for 8 hours with either .sup.35S-methionine or .sup.35S-cysteine.
Both the medium and lysed cells were subjected to consecutive
immunoprecipitations with preimmune (rabbit 4) (P), anti-rTNF (T)
and anti-rLT-.alpha. (rabbit 4) (L) sera in that order. FIG. 5
shows an SDS-PAGE auto-radiographic analysis of the
immunoprecipitates from either supernatants containing secreted
proteins or the washed cells. "s-TNF" marks the .sup.35S-methionine
labeled anti-rTNF immunoprecipitated band from the cells that was
putatively assigned as the unprocessed 26 kD form of TNF. "Met" and
"Cys" refer to the .sup.35S-labeled amino acid employed. Those
lanes containing .sup.35S-cysteine were exposed for longer periods
of time than the lanes containing .sup.35S-methionine. In the
supernatants from these cells (lanes labeled "secreted"), a 25 kD
form of LT ("LT-.alpha.") was released following PMA treatments by
those cells that were labeled with .sup.35S-methionine, but not by
those labeled with .sup.35S-cysteine. This pattern is expected for
fully processed, secreted LT-.alpha.. Longer exposures showed trace
amounts of TNF in the supernatant, and the incorporation of label
was as expected for fully processed, secreted TNF. We observed the
expression of predominantly LT-.alpha. with low levels of TNF also
at the mRNA level (Shamansky and Ware, unpublished observation).
Analysis of the washed cells (lanes labeled "cellular") showed that
both the 25-26 kD LT-.alpha., along with the 33 kD LT-.beta., were
present. The relative amounts of the 25-26 kD and 33 kD forms
paralleled those observed using surface iodination. The 25-26 kD
surface LT-.alpha. form lacked cysteine, indicating processing of
the leader sequence. The 33 kD form incorporated both
.sup.35S-methionine and .sup.35S-cysteine. Longer exposures (not
shown) of the film shown in FIG. 5 revealed the presence of an
anti-TNF immunoprecipitated band from the cells at about 26-27 kD.
The band showed incorporation of both labeled cysteine and labeled
methionine. The labelling was stronger with cysteine. Since the
cys:met ratio is 4:1 in the 26 kD-TNF form, this labelling pattern
confirms the identity of this band.
[0149] The presence of LT-.beta. with LT-.alpha. in
immunoprecipitates from cell lysates suggested that either
LT-.beta. is antigenically related to LT-.alpha. or that LT-.beta.
is bound to LT-.alpha. or both. To address this issue 25 kD and 33
kD bands from .sup.35S-methionine labeled cells were
immunoprecipitated with rabbit polyclonal anti-rLT-.alpha. serum,
eluted from excised gel slices and subjected to
reimmunoprecipitation with either anti-rLT-.alpha. polyclonal serum
or mAb. LT-.alpha., but not LT-.beta., could be immunoprecipitated
with either anti-rLT-.alpha. antibodies suggesting that LT-.beta.
is not antigenically related to LT-.alpha.. These observations
indicated that LT-.beta. is physically associated with LT-.alpha..
We believe that the 33 kD protein is unrelated antigenically to
LT-.alpha. and simply co-precipitated with LT-.alpha..
[0150] With either surface iodination or metabolic labelling, we
were unable to detect either of the known 55 or 80 kD
TNF/LT-.alpha. receptor forms associated with LT-.alpha. or TNF.
Presumably, this is because the receptors are rapidly lost during
activation of T cells. [C. Ware et al., "Regulation Of The CTL
Lytic Pathway By Tumor Necrosis Factor,". Cellular Immunity And The
Immunotherapy Of Cancer, UCLA Symposia on Molecular and Cell
Biology M. T. Lotze and O. J. Finn, Eds. Vol. 135: 121-128
(Wiley-Liss, Inc. New York) 1990].
Example 3
Biochemical Characterization of Surface LT-forms on 11-23.D7
Cells
[0151] We purified the LT-related forms on the surface of
PMA-treated 1]-23.D7 cells using affinity chromatography. Using
immunoprecipitation techniques, we had noted that both of LT-.beta.
and LT-.alpha. bound to lentil lectin sepharose, indicating a
glycoprotein structure. We bound detergent solubilized PMA-treated
II-23.D7 proteins to lentil lectin sepharose and eluted with
a-methyl mannoside prior to affinity purification. We prepared both
control IgG and anti-IgG columns to more accurately assess those
proteins specifically recognized by the anti-rLT-.alpha. serum. Low
pH elution of the columns led to the release of about 100-200 ng of
the two LT forms from the anti-rLT-.alpha. column.
[0152] FIG. 6A reflects SDS PAGE analysis of the proteins eluted
from anti-rLT-.alpha.affinity column prepared from either
pre-immune (PRE) or post-immune (POST) rabbit sera. In FIG. 6B, the
33 kD and 20 kD bands from the gel in panel A were excised and
subjected to limiting CNBr cleavage and electrophoresed on a
SDS-PAGE system. For comparison, FIG. 6B showed CNBr cleavages of
rTNF and rLT-.alpha. (CHO-derived) performed in parallel. The gels
were visualized by silver staining. SDS-PAGE gels of the eluate
resembled closely gels of immunoprecipitated, surface iodinated
PMA-treated II-23.D7-cells, indicating that similar proteins had
been purified.
[0153] During the affinity purification, the 25 kD LT-.alpha. form
appeared to be cleaved to a 19-20 kD) form i.e., it now co-migrated
with the intact recombinant CHO cell-derived LT-.alpha.. The
original isolation of natural LT-.alpha. from the RPMI 1788 tumor
line also yielded an N-terminally cleaved "des-20" LT-.alpha. form.
One-dimensional CNBr digests of the affinity purified proteins
showed the cleaved 20 kD LT-.alpha. form to have a CNBr cleavage
pattern that presumably reflects the truncated nature of this
LT-.alpha. form. One of the methionines is lost in the "des-20".
LT-.alpha. forms and hence the cleavage-pattern would be different
from that of the intact LT-.alpha. form. The 33 kD protein
(LT-.beta.) generated a doublet upon CNBr cleavage, and from this
it was estimated that the single methionine must lie within 5-20
residues from either the C- or N-terminus. This cleavage pattern
shows that the 33 kD protein is significantly different from known
LT forms. CNBr cleavage analysis of the surface iodinated 33 kD
protein probably gave a similar result; however, the resolution
achievable with iodination was insufficient to visualize the
doublet. Staphylococcus V8 digestion of the iodinated rLT-.alpha.,
rTNF and II-23.D7 LT forms showed the rLT-.alpha. and 25-26 kD
II-23.D7 LT-.alpha. form to be resistant to digestion, confirming
the assignment of this protein as LT-.alpha.. The 33 kD protein was
cleaved into several smaller fragments with the pattern resembling
closely that of rTNF.
[0154] In FIG. 7, immunoprecipitated, surface iodinated proteins
were resolved on SDS-PAGE analysis and the surface-associated 25-26
kD protein ("sLT-.alpha.") and the 33 kD protein ("LT-.beta.")
bands were excised. Slices were digested with N-glycanase (N-Gly),
with a mixture of neuraminidase and O-glycanase (O-Gly), or with
all three enzymes. The digested slices were rerun on SDS-PAGE and
an autoradiogram of the dried gel is shown. As shown in FIG. 7,
immunoprecipitation of iodinated surface LT forms followed by
digestion with either or both N- and O-glycanases showed the 25-26
kD LT-.alpha. form to contain an N-linked oligosaccharide. The
25-26 kD LT-.alpha. form contains only one N-linked site which
would correlate well with the size change upon N-glycanase
digestion. Likewise, the 33 kD form (LT-.beta.) lost about 3 kD of
size upon treatment with N-glycanase, suggesting the presence of
one N-linked oligosaccharide. In contrast to the 25-26 kD
LT-.alpha. form, O-glycanase treatment did not affect the molecular
weight of LT-.beta.. The lack of cleavage by a glycanase, however,
is not definitive evidence for the lack of a carbohydrate.
Example 4
Reimmunoprecipitation of LT-.alpha.
[0155] The coprecipitation of LT-.beta. with LT-.alpha. suggested
that these proteins are either antigenically related or physically
associated. To address this issue we tested whether or not SDS-PAGE
separated LT-.alpha. (p25) and LT-.beta. proteins could be
immunoprecipitated. LT-.alpha. and LT-.beta. labeled with .sup.125I
or .sup.35S-Met were first partially purified by
immunoprecipitation and separated by SDS-PAGE. The labeled bands
were excised, rehydrated in buffer, and the proteins eluted. The
eluted proteins were then subjected to a second round of
immunoprecipitation using either polyclonal or monoclonal
anti-rLT-.alpha.antibodies (FIG. 10). Rabbit anti-rLT-.alpha.
reimmunoprecipitated LT-.alpha. ("p25", lane 2) but not
LT-.beta.(lane 3). The anti-rLT-.alpha. mAb precipitated LT-.alpha.
(lane 5) and a 21 kD protein ("p21", lane 4), which, as shown
below, is a precursor of LT-.alpha.; however, it did not
precipitate LT-.beta. (lane 6). The results indicate that after
LT-.alpha. and LT-.beta. are separated by SDS-PAGE, both polyclonal
and monoclonal anti-rLT-.alpha. antibodies are capable of reacting
with LT-.alpha. but not with LT-.beta.. This data provides evidence
that LT-.beta.is not antigenically related to LT-.alpha.. However,
we cannot rule out the possibility that putative LT-.beta.
cross-reactive epitopes are lost after denaturation, whereas the
LT-.alpha. epitopes remain intact.
[0156] FIG. 8 shows the results of reimmuno-precipitation of
.sup.125I-labeled and .sup.35S-Met-labeled p25 and p33 proteins
eluted from SDS-PAGE gels. LT-.alpha. and LT-.beta. species from
.sup.125I-labeled 11-23.D7 cells (lanes 2,3) and .sup.35S-labeled
cells (lanes 4,5,6) were eluted from gel slices as described above.
The elutes were immunoprecipitated with either the anti-rLT-.alpha.
serum (lanes 2,3) or the anti-rLT-.alpha. mAb (lanes 4,5,6), and
the reprecipitated proteins analyzed by SDS-PAGE and
autoradiography. Lane 1 is a control lane for the identification of
LT-.alpha. and LT-.beta..
Example 5
Isoelectric Focusing of LT-.alpha. and LT-.beta.
[0157] FIGS. 9 and 10 (each including an autoradiograph (9A, 10A)
and a calibration curve graphing migration distance vs. pH (9B,
10B)) depict isoelectric focusing analysis under denaturing (FIG.
9) and native (FIG. 10) conditions. Two-dimensional gel analysis
was carried out as described above on .sup.125I-labeled LT-.alpha.
and LT-.beta. that had been immuno-precipitated from II-23.D7 cell
extracts. The 2-D gel analysis was performed under denaturing
conditions in the presence of urea (FIG. 9A). In contrast, native
IEF was performed in 1% NP-40 without urea. .sup.125I-labeled
II-23.D7 cell extract was first focused on a tube gel at 4.degree..
After focusing, the tube gel was cut into 1 cm sections, the
focused proteins eluted from those sections, immunoprecipitated,
and analyzed by SDS-PAGE (FIG. 10A). Immunoprecipitated material
from gel lanes 1-12 correspond to tube gel slices 2-13. pH
gradients were generated for both the denatured and native tube
gels based on 1 cm gel increments. These are also shown below each
autoradiogram as 9B and 10B, respectively. Biochemically, LT-B and
LT-.alpha. comigrate on a non-denaturing isoelectric focusing gel,
but when the complex is dissociated with urea, the two proteins run
separately. [See FIGS. 9A, 10A] These observations led us to
conclude that LT-.alpha. and LT-.beta. exist as a complex on the
cell surface.
Example 6
Regulation of LT-.alpha. Expression
[0158] Table I set forth below, summarizes the results of a survey
using flow cytofluorometric analysis of various cell types for the
expression of a surface form of LT-.alpha.. TABLE-US-00003 TABLE I
Expression of LT-.alpha. and TNF Related Epitopes on the Surfaces
of Different Cells Suirface Expression of: Cell Treatment
LT-.alpha. TNF Peripheral Resting + - Mononuclear cells OKT3 ++ -
Leu-4.sup.+(CD3) Resting + - OKT3 ++ - PMA - nd. IL-2 ++ nd.
Leu-2.sup.+(CD8) OKT3 ++ nd. Leu-3.sup.+(CD4) OKT3 ++ nd. Leu-M3
Resting - + OKT3/LPS/IFN-.gamma. - ++ Leu-19.sup.+(NK) IL-2 (LAKs)
++ nd. Leu-16.sup.+(B's) Resting +/- - PWM + nd. CTL-clones Control
+/- - PMA nd. nd. Allogeneic Stim. ++ nd. Anti-T11 2 + 3 ++ - T
cell Hybridoma Control - - (II-23.D7) PMA ++ - PMA + A23187 ++ -
Hut-78 Control + - PMA + - C8166 Control - nd. PMA + nd. RPMI-1788
Control - - PMA - - rLT-producing Control - - CHO cell line PMA - -
Jurkat +/- nd. HL-60 - - U937 - nd. Raji + nd. K562 +/- nd.
[0159] The most striking observation from these studies was the
restriction of surface LT-.alpha. expression to T and B cells.
Leu-M3, a monocyte marker, and leu-4 (CD3) antibodies were used in
two-color flow cytofluorometric analysis to observe each cell
population separately. There was an excellent distinction between
surface TNF and surface LT-.alpha. in this analysis in that
monocytes expressed only surface TNF whereas T cells displayed only
surface LT-.alpha.. This result is shown in FIG. 11.
[0160] In the experiment depicted in FIG. 11, PBL were treated for
8 hours with a mixture of LPS (1 .mu.g/ml), Interferon-.gamma. (200
U/ml) and OKT3 (1 ng/ml) and then stained for LT-.alpha.
(anti-rLT-.alpha. serum from rabbit 5) or TNF (anti-rTNF serum from
rabbit 7) followed by FITC anti-rabbit labelling. Cells were
counterstained with either phycoerythrin-leu-4, a pan T cell
marker, or phycoerythrin leu-M3, a monocyte marker. "T cell panels"
were gated for leu-4.sup.+ cells while the "monocyte" panels were
gated for leu-M3.sup.+ cells. Cells were stained with preimmune
(dotted lines) or postimmune sera (solid lines). The monocytic
tumor lines HL-60 and U937 did not stain for LT-.alpha.. By using
two color flow cytofluorometric analysis, the T4 and T8 subclasses
of activated PBL were found to display similar levels of
surface-associated LT-.alpha.. In general, it appears that primary
T-cells capable of expressing LT-.alpha. are also capable of
displaying surface LT-.alpha. form(s).
[0161] Examination of three different human donors showed that a
surface LT-.alpha. form was present on freshly isolated, resting
peripheral T cells. In the case of PBL, OKT3 activation or simply
IL-2 treatment of the cells led to increased expression. By using
fluorescence channel numbers, we have attempted to quantitate both
surface LT-.alpha. and IL-2 receptor (CD25) expression during OKT3
activation. Maximal surface LT-.alpha. induction by OKT3 appeared
to precede the peak expression of IL-2 receptor (TAC expression) in
the bulk culture, thus the surface-bound LT form
(LT-.alpha./LT-.beta. complex) appears to be an early T-cell
activation antigen. It was found that both anti-T11 and allogeneic
antigen were capable of causing the appearance of LT-.alpha. on the
surface of cloned cytotoxic T cells. Likewise, PMA stimulation was
necessary to induce the appearance of LT-.alpha. on the surface of
the II-23.D7 hybridoma. It appears that T cell activation increases
surface LT-.alpha. form(s). Peripheral lymphocytes, in contrast to
the II-23.D7 hybridoma, down-regulate surface LT-.alpha. form(s)
very quickly following PMA treatment. Likewise, in a two-color
analysis of OKT3 activated PBL populations, Dr.sup.+ cells, which
should include T cells in advanced stages of activation, lacked
surface LT-.alpha.form(s).
[0162] Activation of fresh PBL with high levels of IL-2 generated
lymphokine-activated killer cells (LAK cells). As shown in FIG. 12,
two color flow cytofluorometric analysis using anti-rLT-.alpha. and
leu-19, a NK/LAK cell marker, showed LAK cell expression of surface
LT-.alpha. forms to resemble the T cell hybridoma, 11-23.D7. In the
experiment depicted in FIG. 12, PBL were cultured for 5 days with
20 ng/ml IL-2 and then stained for a two color analysis with
phycoerythrin labeled leu-19 and anti-rLT (rabbit 5) as described
above. FIG. 12 shows surface LT-.alpha. levels on leu-19.sup.+
cells that were stained with preimmune (dotted lines) or postimmune
sera (solid lines). Thus, LAK cells appeared to have the highest
levels of surface LT-.alpha. forms of any primary cell type.
Example 7
Functional Relevance of Total TNF or LT-.alpha. to T-cell
Activation
[0163] To examine the functional relevance of TNF and LT-.alpha. to
the T cell activation process, we included the rabbit
anti-rLT-.alpha. and anti-rTNF sera in mixed lymphocyte response
(MLR) and OKT3 activation assays. MLR is a standard immunological
assay which tests the ability of an individual's T cells to
recognize another person's T cells as foreign and respond to their
presence by proliferating. Table II, set forth below, presents data
from MLR experiments using various responder/stimulator
combinations. TABLE-US-00004 TABLE II Effects of LT and TNF
Antibodies on a 5-Day Mixed Lymphocyte Culture .sup.3H-Thy. (S.D.)
% Change.sup.a Cells.sup.b Addition cpm .times. 1000 Responder A
none 4.7 (0.8) -- Stimulator B* none 4.8 (0.5) -- A + B* none 20.3
(3.6) 0% A + B* r-TNF.sup.c 28.0 (1.4) +49% A + B* r-LT 32.8 (3.2)
+80% Responder C none 6.3 (0.8) -- Stimulator B* none 4.8 (0.7) --
C + B* none 30.0 (5.4) 0% C + B* r-TNF 36.9 (6.0) +28% C + B* r-LT
36.1 (5.8) +24% Responder A none 5.3 (0.6) -- Stimulator D* none
1.5 (0.5) -- A + D* none 22.2 (2.6) 0% Responder D none 7.8 (1.0)
-- Stimulator A none 1.6 (0.3) -- D + A* none 24.3 (7.0) 0%
Preimmune Postimmune A + B* Anti-LT-4.sup.d 26.8 (2.4) +41% 8.6
(0.2) -74% A + B* Anti-LT-5 27.4 (4.4) +45% 11.0 (1.2) -59% A + B*
Anti-LT-6 23.1 (1.0) +18% 25.7 (5.7) +35% A + B* Anti-TNF 26.0
(2.2) +36% 12.6 (2.7) -49% A + B* Anti-TNF mAb* 15.1 (2.2) -33% 4.7
(0.7) -99% C + B* Anti-LT-4.sup.d 41.1 (3.5) +44% 20.9 (5.8) -36% C
+ B* Anti-LT-5 35.5 (6.9) +22% 17.8 (2.6) -49% C + B* Anti-LT-6
39.4 (7.9) +38% 39.1 (5.3) +36% C + B* Anti-TNF 39.8 (4.3) +39%
24.4 (3.2) -22% C + B* Anti-TNF mAb.sup.e 37.8 (7.3) +31% 20.6
(1.8) -37% A + D* Anti-LT-5 28.6 (2.1) +37% 12.5 (2.4) -59% A + D*
Anti-LT-5 32.8 (6.4) +63% 14.5 (3.6) -47% D + A* Anti-LT-5 28.0
(1.2) +23% 20.8 (1.4) -21% D + A* Anti-LT-5.sup.f 28.1 (2.1) +24%
19.2 (0.7) -31% See notes below. .sup.aPercent change refers to the
increase or decrease in .sup.3H-thymidine incorporation after
correction for a background of responder cells alone.
.sup.bStimulator cells were irradiated with 3000 rads and are
denoted with a "*". Low level proliferation was still evident in
stimulator population. The ratio of responder to stimulator was
1/1.5. .sup.crTNF and rLT were added to a level of 10 ng/ml.
.sup.dAntisera were heat inactivated for 1 hour at 56.degree. C.,
filtered and used at a final dilution of 1:250. .sup.eThe
monoclonal anti-TNF was a purified mouse IgG.sub.2a antibody used
at 2 .mu.g/ml and the control in this case was pure mouse UPC 10
(IgG.sub.2a). .sup.fIn these cases, the immunoglobulin fraction was
purified from the serum and used at a final concentration of 50
.mu.g/ml.
[0164] As shown in Table 11, the neutralizing anti-rLT-.alpha. sera
(rabbits 4 and 5) inhibited the proliferative response as assessed
at five days whereas preimmune sera or the non-neutralizing
anti-rLT-.alpha. (rabbit 6) sera had mild stimulatory effects. As
previously reported [M. Shalaby et al., J. Immunol., 141:499
(1988)], polyclonal and a monoclonal anti-TNF preparations were
also inhibitory. These assays were carried out under excess
stimulator cell conditions and hence the inhibition may not be
optimized. The serum levels employed in Table II are rather high,
but in other experiments (data not shown), antibody dilutions up to
1:1000 were still inhibitory. PHA or OKT3 stimulated T cell
proliferation was also inhibited to a lesser extent (data not
shown). These data indicated that LT or LT-related epitopes on T
cell surfaces may be involved in T cell activation.
[0165] A previous study using the MLR assay and neutralizing
monoclonal antibodies implicated TNF but not LT-.alpha. in T cell
activation and subsequent proliferation in this system. [M. Shalaby
et al., J. Immunol., 141:499 (1988)]. In that study, monoclonal
anti-rLT-.alpha. antibodies had no effect on the MLR assay. Our
studies indicate that neutralizing polyclonal
anti-CHO-cell-derived-rLT-.alpha. sera were able to partially
inhibit the MLR, suggesting a role for some form of LT in this
system. The reasons for this discrepancy are not clear, although
there may be some differences in the nature of these antibody
preparations. The monoclonal antibodies were generated against
glutaraldehyde-cross linked natural LT-.alpha. (RPMI 1788
secreted), whereas polyclonal anti-rLT-.alpha. sera were prepared
using native r-LT-.alpha. (recombinant CHO-cell derived) with
Freund's adjuvant injected directly into the lymph nodes. The depot
effect in the success of the latter system was probably important
considering the difficulties reported in immunizing mice [T.
Bringman et al., "Monoclonal Antibodies to Human Tumor Necrosis
Factors Alpha and Beta: Application For Affinity Purification,
Immunoassays, And As Structural Probes," Hybridoma, 6:489 (1987)].
These data suggest that the blocking effect of our polyclonal
anti-rLT-.alpha. sera on the MLR is a result of recognition of the
surface LT form(s) by the sera rather than recognition of the
conventional soluble LT-.alpha. form.
Example 8
Purification And Initial Sequencing Of LT-.alpha. And LT-.beta.
[0166] We obtained N-terminal sequence information for LT-.alpha.
and LT-.beta. by Edman degradation as described above. We found the
sequence of the membrane associated LT-.alpha. band to be as
follows: Leu Pro Gly Val Gly Leu Thr Pro Ser. This sequence matches
with the known sequence of secreted LT-.alpha.. The Edman
degradation analysis revealed that the N-terminal portion of the
associated LT-.beta. protein included two possible amino acid
sequences: Gly Leu Glu Gly Arg Gly Gin Arg Leu Gin or Gly leu Glu
Gly Arg Leu Gln Arg Leu Gln. Subsequent DNA analysis confirmed the
former sequence except that glycine was incorrectly identified as
glutamine at cycle 7.
Example 9
Sequencing And Cloning LT-.beta.
[0167] As described above, we obtained sequences for several
peptides using the method of Abersold et al. Two antisense 17-mer
oligonucleotide probes GTYTCNGGCTCYTCYTC [SEQ ID NO:9] and
GTYTCNGGTTCYTCYTC [SEQ ID NO:10] were synthesized to match a
portion of the sequence of one of those peptides, T-87/T-88. Those
probes were radiolabelled with .sup.32P. Northern analysis (as
described in J. Sambrook et al., Molecular Cloning a Laboratory
Manual, 2d ed. (1989)) showed that probe 1368 (GTYTCNGGTCYTCYTC)
[SEQ ID NO:10] hybridized strongly to a 0.9-1.1 kb mRNA band that
was strongly induced in II-23.D7 cells which had been pretreated
with phorbol ester as previously described. A cDNA library in the
vector pCDM8 was constructed from poly A+ mRNA isolated from
Hl-23.D7 cells induced with PMA for 6 hours. [See, B. Seed and A.
Aruffo, PNAS, 84:3365-3369 (1987)]. The library was screened with
labelled oligomer 1368 and positive clones were isolated following
washing with 3 M tetramethylammonium chloride at 50.degree. C. [See
Jacobs et al., Nucleic Acids Research, 16, 10:4637-4649 (1988)].
Several clones containing 0.9 kb inserts were subjected to DNA
sequence analysis. Clone pCDM8/LT-.beta.-12 (clone 12) was found to
contain the coding sequence. The other clones were identical except
for various 1-30 bp truncations at the 5' end. One potential clone
(clone 4) contained a frameshift and was used as a control in
transfection experiments. A termination sequence, AATAAA at
position 862 was found just prior to a poly A tract indicating that
the entire 3' end had been identified. The identified protein
sequence encodes for at least 240 amino acids with a calculated
molecular weight of 25,390 and a domain structure typical of a type
II membrane protein. The present data suggest that the initiating
CTG leads to a processed N-terminus starting with gly 5 (met=1),
i.e., the CTG encoded leucine is either not translated or the
leucine-4 residue is further processed yielding the mature
N-terminus obtained by amino acid sequencing.
[0168] The 33 kDa size of LT-.beta. results from N-linked
glycosylation as previously defined and this result is corroborated
by the presence of one potential N-linked carbohydrate site in the
amino acid sequence at a position identical with a similar site
found in the CD40 ligand. A typical N-linked sugar-residue can add
approximately 3-4 kDa MW, hence, the final molecular weight is
close to the observed 30-33 kDa.
[0169] No identical sequences were found within the databases.
There is one cysteine residue in the extracellular domain and two
methionines within the last C-terminal 17 amino acids, in agreement
with the very limited cyanogen bromide cleavage pattern exhibited
by this protein.
Example 10
Expression Of LT-.beta.
[0170] The pCDM8/LT-.beta. clone 12 or a control plasmid, clone 4
(pCDM8 with a non-functional cDNA insert), were introduced by
electroporation into CHO dhfr- and a CHO cell stably transfected
with human LT-.alpha.. After three days, cells were removed with
Ca/Mg-free Hank's solution with 5 mM EDTA and stained for FACS
analysis as described above using either 10 .mu.g/ml control IgG1
or anti-LT monoclonal antibody (Boehringer-Mannheim) followed by
labelling of bound immunoglobulin with either a FITC or
phycoerythrin labelled goat anti-mouse preparation.
[0171] In a different experiment, COS cells were electroporated
with either clone 4 or clone 12 LT-.beta. cDNA in pCDM8 in the
presence or absence of an equal amount of human LT-.alpha. cDNA
also in the pCDM8 vector and stained for FACS analysis after three
days as above. Only COS cells expressing LT-.alpha. displayed
surface lymphotoxin upon transfection with a functional LT-.beta.
DNA, i.e., clone 12.
[0172] Clone 12 lacks an initiating ATG codon, but does possess
several CTG initiating codons and hence this expression experiment
shows that one or several of the 5'CTG codons can initiate
translation. CTG codons are known to serve as initiating sites for
translation in several eukaryotic proteins. [M. Kozak, J. Cell.
Biol., 115:4 (1991)]. Similar results were observed using the dual
transfection system such that only COS cells receiving both
LT-.alpha. and LT-.beta. DNA displayed substantial surface
LT-.alpha. in a FACS analysis.
[0173] Northern analysis of II-23.D7 cells showed hybridization of
the LT-.beta. cDNA to a 0.9-1.0 kb nRNA indicating that the cloned
cDNA represents essentially all of the transcribed gene. See FIG.
15. The LT-.beta.gene was expressed at low levels in untreated
II-23.D7 hybridoma cells; however, upon cell activation with
phorbol ester mRNA levels increased dramatically. See FIG. 16.
Example 11
Homology Between LT-.beta. and Other Members of the TNF Family Of
Lymphokines
[0174] Cloning of the cDNA encoding LT-.beta. revealed that
LT-.beta. is a type-II membrane protein with significant homology
to TNF, LT-.alpha. and the ligand for the CD40 receptor. These
proteins are known to bind to members of the TNF/NGF receptor
family. LT-.beta., TNF, LT-.alpha. and the ligand for the CD-40
receptor share four regions of sequence conservation in the
extracellular domain. See FIG. 13. These domains are located on the
face of the TNF and LT-.alpha. crystal structures and are likely to
be involved in intersubunit interactions.
Example 12
Determination of the 5'. End of LT-.beta. Reveals Several Possible
Start Sites
[0175] The 5' mRNA sequence was determined by primer extension
analysis. Primer extension analysis revealed that the
transcriptional start site for the LT-.beta. protein was
approximately 7-9 base pairs upstream of the methionine ATG. Thus,
the mRNA possesses at least 3 possible translation start sites, the
Met-1, Leu4 and Leu-6 codons. Transient experiments with clone 12
showed that one or both of the Leu4 or Leu-6 start sites is
functional. The 5' mRNA sequence was verified by determining the
LT-.beta.genomic sequence using a cosmid clone 031 A, described
above, and dideoxynucleic acid sequencing.
Sequence CWU 1
1
23 1 726 DNA Homo Sapien 1 ctggggctgg agggcagggg tgggaggctc
caggggaggg gttccctcct gctagctgtg 60 gcaggagcca cttctctggt
gaccttgttg ctggcggtgc ctatcactgt cctggctgtg 120 ctggccttag
tgccccagga tcagggagga ctggtaacgg agacggccga ccccggggca 180
caggcccagc aaggactggg gtttcagaag ctgccagagg aggagccaga aacagatctc
240 agccccgggc tcccagctgc ccacctcata ggcgctccgc tgaaggggca
ggggctaggc 300 tgggagacga cgaaggaaca ggcgtttctg acgagcggga
cgcagttctc ggacgccgag 360 gggctggcgc tcccgcagga cggcctctat
tacctctact gtctcgtcgg ctaccggggc 420 cgggcgcccc ctggcggcgg
ggacccccag ggccgctcgg tcacgctgcg cagctctctg 480 taccgggcgg
ggggcgccta cgggccgggc actcccgagc tgctgctcga gggcgccgag 540
acggtgactc cagtgctgga cccggccagg agacaagggt acgggcctct ctggtacacg
600 agcgtggggt tcggcggcct ggtgcagctc cggaggggcg agagggtgta
cgtcaacatc 660 agtcaccccg atatggtgga cttcgcgaga gggaagacct
tctttggggc cgtgatggtg 720 gggtga 726 2 241 PRT Homo Sapien 2 Leu
Gly Leu Glu Gly Arg Gly Gly Arg Leu Gln Gly Arg Gly Ser Leu 1 5 10
15 Leu Leu Ala Val Ala Gly Ala Thr Ser Leu Val Thr Leu Leu Leu Ala
20 25 30 Val Pro Ile Thr Val Leu Ala Val Leu Ala Leu Val Pro Gln
Asp Gln 35 40 45 Gly Gly Leu Val Thr Glu Thr Ala Asp Pro Gly Ala
Gln Ala Gln Gln 50 55 60 Gly Leu Gly Phe Gln Lys Leu Pro Glu Glu
Glu Pro Glu Thr Asp Leu 65 70 75 80 Ser Pro Gly Leu Pro Ala Ala His
Leu Ile Gly Ala Pro Leu Lys Gly 85 90 95 Gln Gly Leu Gly Trp Glu
Thr Thr Lys Glu Gln Ala Phe Leu Thr Ser 100 105 110 Gly Thr Gln Phe
Ser Asp Ala Glu Gly Leu Ala Leu Pro Gln Asp Gly 115 120 125 Leu Tyr
Tyr Leu Tyr Cys Leu Val Gly Tyr Arg Gly Arg Ala Pro Pro 130 135 140
Gly Gly Gly Asp Pro Gln Gly Arg Ser Val Thr Leu Arg Ser Ser Leu 145
150 155 160 Tyr Arg Ala Gly Gly Ala Tyr Gly Pro Gly Thr Pro Glu Leu
Leu Leu 165 170 175 Glu Gly Ala Glu Thr Val Thr Pro Val Leu Asp Pro
Ala Arg Arg Gln 180 185 190 Gly Tyr Gly Pro Leu Trp Tyr Thr Ser Val
Gly Phe Gly Gly Leu Val 195 200 205 Gln Leu Arg Arg Gly Glu Arg Val
Tyr Val Asn Ile Ser His Pro Asp 210 215 220 Met Val Asp Phe Ala Arg
Gly Lys Thr Phe Phe Gly Ala Val Met Val 225 230 235 240 Gly 3 606
DNA Homo Sapien 3 ctggccttag tgccccagga tcagggagga ctggtaacgg
agacggccga ccccggggca 60 caggcccagc aaggactggg gtttcagaag
ctgccagagg aggagccaga aacagatctc 120 agccccgggc tcccagctgc
ccacctcata ggcgctccgc tgaaggggca ggggctaggc 180 tgggagacga
cgaaggaaca ggcgtttctg acgagcggga cgcagttctc ggacgccgag 240
gggctggcgc tcccgcagga cggcctctat tacctctact gtctcgtcgg ctaccggggc
300 cgggcgcccc ctggcggcgg ggacccccag ggccgctcgg tcacgctgcg
cagctctctg 360 taccgggcgg ggggcgccta cgggccgggc actcccgagc
tgctgctcga gggcgccgag 420 acggtgactc cagtgctgga cccggccagg
agacaagggt acgggcctct ctggtacacg 480 agcgtggggt tcggcggcct
ggtgcagctc cggaggggcg agagggtgta cgtcaacatc 540 agtcaccccg
atatggtgga cttcgcgaga gggaagacct tctttggggc cgtgatggtg 600 gggtga
606 4 201 PRT Homo Sapien 4 Leu Ala Leu Val Pro Gln Asp Gln Gly Gly
Leu Val Thr Glu Thr Ala 1 5 10 15 Asp Pro Gly Ala Gln Ala Gln Gln
Gly Leu Gly Phe Gln Lys Leu Pro 20 25 30 Glu Glu Glu Pro Glu Thr
Asp Leu Ser Pro Gly Leu Pro Ala Ala His 35 40 45 Leu Ile Gly Ala
Pro Leu Lys Gly Gln Gly Leu Gly Trp Glu Thr Thr 50 55 60 Lys Glu
Gln Ala Phe Leu Thr Ser Gly Thr Gln Phe Ser Asp Ala Glu 65 70 75 80
Gly Leu Ala Leu Pro Gln Asp Gly Leu Tyr Tyr Leu Tyr Cys Leu Val 85
90 95 Gly Tyr Arg Gly Arg Ala Pro Pro Gly Gly Gly Asp Pro Gln Gly
Arg 100 105 110 Ser Val Thr Leu Arg Ser Ser Leu Tyr Arg Ala Gly Gly
Ala Tyr Gly 115 120 125 Pro Gly Thr Pro Glu Leu Leu Leu Glu Gly Ala
Glu THr Val Thr Pro 130 135 140 Val Leu Asp Pro Ala Arg Arg Gln Gly
Tyr Gly Pro Leu Trp Tyr Thr 145 150 155 160 Ser Val Gly Phe Gly Gly
Leu Val Gln Leu Arg Arg Gly Glu Arg Val 165 170 175 Tyr Val Asn Ile
Ser His Pro Asp Met Val Asp Phe Ala Arg Gly Lys 180 185 190 Thr Phe
Phe Gly Ala Val Met Val Gly 195 200 5 450 DNA Homo Sapien 5
ccgctgaagg ggcaggggct aggctgggag acgacgaagg aacaggcgtt tctgacgagc
60 gggacgcagt tctcggacgc cgaggggctg gcgctcccgc aggacggcct
ctattacctc 120 tactgtctcg tcggctaccg gggccgggcg ccccctggcg
gcggggaccc ccagggccgc 180 tcggtcacgc tgcgcagctc tctgtaccgg
gcggggggcg cctacgggcc gggcactccc 240 gagctgctgc tcgagggcgc
cgagacggtg actccagtgc tggacccggc caggagacaa 300 gggtacgggc
ctctctggta cacgagcgtg gggttcggcg gcctggtgca gctccggagg 360
ggcgagaggg tgtacgtcaa catcagtcac cccgatatgg tggacttcgc gagagggaag
420 accttctttg gggccgtgat ggtggggtga 450 6 149 PRT Homo Sapien 6
Pro Leu Lys Gly Gln Gly Leu Gly Trp Glu Thr Thr Lys Glu Gln Ala 1 5
10 15 Phe Leu Thr Ser Gly Thr Gln Phe Ser Asp Ala Glu Gly Leu Ala
Leu 20 25 30 Pro Gln Asp Gly Leu Tyr Tyr Leu Tyr Cys Leu Val Gly
Tyr Arg Gly 35 40 45 Arg Ala Pro Pro Gly Gly Gly Asp Pro Gln Gly
Arg Ser Val Thr Leu 50 55 60 Arg Ser Ser Leu Tyr Arg Ala Gly Gly
Ala Tyr Gly Pro Gly Thr Pro 65 70 75 80 Glu Leu Leu Leu Glu Gly Ala
Glu Thr Val Thr Pro Val Leu Asp Pro 85 90 95 Ala Arg Arg Gln Gly
Tyr Gly Pro Leu Trp Tyr Thr Ser Val Gly Phe 100 105 110 Gly Gly Leu
Val Gln Leu Arg Arg Gly Glu Arg Val Tyr Val Asn Ile 115 120 125 Ser
His Pro Asp Met Val Asp Phe Ala Arg Gly Lys Thr Phe Phe Gly 130 135
140 Ala Val Met Val Gly 145 7 156 DNA Homo Sapien 7 ctggccttag
tgccccagga tcagggagga ctggtaacgg agacggccga ccccggggca 60
caggcccagc aaggactggg gtttcagaag ctgccagagg aggagccaga aacagatctc
120 agccccgggc tcccagctgc ccacctcata ggcgct 156 8 52 PRT Homo
Sapien 8 Leu Ala Leu Val Pro Gln Asp Gln Gly Gly Leu Val Thr Glu
Thr Ala 1 5 10 15 Asp Pro Gly Ala Gln Ala Gln Gln Gly Leu Gly Phe
Gln Lys Leu Pro 20 25 30 Glu Glu Glu Pro Glu Thr Asp Leu Ser Pro
Gly Leu Pro Ala Ala His 35 40 45 Leu Ile Gly Ala 50 9 17 DNA Homo
Sapien misc_feature 6 n = A,T,C or G 9 gtytcnggct cytcytc 17 10 17
DNA Homo Sapien misc_feature 6 n = A,T,C or G 10 gtytcnggtt cytcytc
17 11 18 DNA Homo Sapien 11 atgggggcac tggggctg 18 12 19 DNA Homo
Sapien 12 gcggccgctt tagagcaca 19 13 27 DNA Homo Sapien 13
gacagtgata ggcaccgcca gcaacaa 27 14 34 PRT Homo Sapien Variant 7,
30, 31 Xaa = Any Amino Acid 14 Gly Leu Glu Gly Arg Gly Xaa Arg Leu
Gln Gly Arg Gly Ser Leu Leu 1 5 10 15 Leu Ala Val Ala Gly Ala Thr
Gly Leu Val Thr Leu Leu Xaa Xaa Val 20 25 30 Pro Ile 15 25 PRT Homo
Sapien 15 Leu Pro Glu Glu Glu Pro Glu Thr Asp Leu Ser Pro Gly Leu
Pro Ala 1 5 10 15 Ala His Leu Ile Gly Ala Pro Leu Lys 20 25 16 25
PRT Homo Sapien Variant 1 Xaa = Any Amino Acid 16 Xaa Gln Ala Phe
Leu Thr Ser Gly Thr Gln Phe Ser Asp Ala Glu Gly 1 5 10 15 Leu Ala
Leu Pro Gln Asp Gly Leu Tyr 20 25 17 8 PRT Homo Sapien Variant 1,
5, 6 Xaa = Any Amino Acid 17 Xaa Gln Gly Leu Xaa Xaa Glu Thr 1 5 18
5 PRT Homo Sapien 18 Ser Ser Leu Tyr Arg 1 5 19 26 PRT Homo Sapien
19 Ala Gly Gly Ala Tyr Gly Pro Gly Thr Pro Glu Leu Leu Leu Leu Glu
1 5 10 15 Gly Ala Glu Thr Val Thr Pro Val Leu Asp 20 25 20 233 PRT
Homo Sapien 20 Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala
Glu Glu Ala 1 5 10 15 Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser
Arg Arg Cys Leu Phe 20 25 30 Leu Ser Leu Phe Ser Phe Leu Ile Val
Ala Gly Ala Thr Thr Leu Phe 35 40 45 Cys Leu Leu His Phe Gly Val
Ile Gly Pro Gln Arg Glu Glu Phe Pro 50 55 60 Arg Asp Leu Ser Leu
Ile Ser Pro Leu Ala Gln Ala Val Arg Ser Ser 65 70 75 80 Ser Arg Thr
Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro 85 90 95 Gln
Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu 100 105
110 Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser
115 120 125 Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly
Gln Gly 130 135 140 Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile
Ser Arg Ile Ala 145 150 155 160 Val Ser Tyr Gln Thr Lys Val Asn Leu
Leu Ser Ala Ile Lys Ser Pro 165 170 175 Cys Gln Arg Glu Thr Pro Glu
Gly Ala Glu Ala Lys Pro Trp Tyr Glu 180 185 190 Pro Ile Tyr Leu Gly
Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu 195 200 205 Ser Ala Glu
Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly 210 215 220 Gln
Val Tyr Phe Gly Ile Ile Ala Leu 225 230 21 260 PRT Murinae gen. sp.
21 Met Ile Glu Thr Tyr Ser Gln Pro Ser Pro Arg Ser Val Ala Thr Gly
1 5 10 15 Leu Pro Ala Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val
Phe Leu 20 25 30 Ile Thr Gln Met Ile Gly Ser Val Leu Phe Ala Val
Tyr Leu His Arg 35 40 45 Arg Leu Asp Lys Val Glu Glu Glu Val Asn
Leu His Glu Asp Phe Val 50 55 60 Phe Ile Lys Lys Leu Lys Arg Cys
Asn Lys Gly Glu Gly Ser Leu Ser 65 70 75 80 Leu Leu Asn Cys Glu Glu
Met Arg Arg Gln Phe Glu Asp Leu Val Lys 85 90 95 Asp Ile Thr Leu
Asn Lys Glu Glu Lys Lys Glu Asn Ser Phe Glu Met 100 105 110 Gln Arg
Gly Asp Glu Asp Pro Gln Ile Ala Ala His Val Val Ser Glu 115 120 125
Ala Asn Ser Asn Ala Ala Ser Val Leu Gln Trp Ala Lys Lys Gly Tyr 130
135 140 Tyr Thr Met Lys Ser Asn Leu Val Met Leu Glu Asn Gly Lys Gln
Leu 145 150 155 160 Thr Val Lys Arg Glu Gly Leu Tyr Tyr Val Tyr Thr
Gln Val Thr Phe 165 170 175 Cys Ser Asn Arg Glu Pro Ser Ser Gln Arg
Pro Phe Ile Val Gly Leu 180 185 190 Trp Leu Lys Pro Ser Ile Gly Ser
Glu Arg Ile Leu Leu Lys Ala Ala 195 200 205 Asn Thr His Ser Ser Ser
Gln Leu Cys Glu Gln Gln Ser Val His Leu 210 215 220 Gly Gly Val Phe
Glu Leu Gln Ala Gly Ala Ser Val Phe Val Asn Val 225 230 235 240 Thr
Glu Ala Ser Gln Val Ile His Arg Val Gly Phe Ser Ser Phe Gly 245 250
255 Leu Leu Lys Leu 260 22 240 PRT Homo Sapien 22 Gly Leu Glu Gly
Arg Gly Gly Arg Leu Gln Gly Arg Gly Ser Leu Leu 1 5 10 15 Leu Ala
Val Ala Gly Ala Thr Ser Leu Val Thr Leu Leu Leu Ala Val 20 25 30
Pro Ile Thr Val Leu Ala Val Leu Ala Leu Val Pro Gln Asp Gln Gly 35
40 45 Gly Leu Val Thr Glu Thr Ala Asp Pro Gly Ala Gln Ala Gln Gln
Gly 50 55 60 Leu Gly Phe Gln Lys Leu Pro Glu Glu Glu Pro Glu Thr
Asp Leu Ser 65 70 75 80 Pro Gly Leu Pro Ala Ala His Leu Ile Gly Ala
Pro Leu Lys Gly Gln 85 90 95 Gly Leu Gly Trp Glu Thr Thr Lys Glu
Gln Ala Phe Leu Thr Ser Gly 100 105 110 Thr Gln Phe Ser Asp Ala Glu
Gly Leu Ala Leu Pro Gln Asp Gly Leu 115 120 125 Tyr Tyr Leu Tyr Cys
Leu Val Gly Tyr Arg Gly Arg Ala Pro Pro Gly 130 135 140 Gly Gly Asp
Pro Gln Gly Arg Ser Val Thr Leu Arg Ser Ser Leu Tyr 145 150 155 160
Arg Ala Gly Gly Ala Tyr Gly Pro Gly Thr Pro Glu Leu Leu Leu Glu 165
170 175 Gly Ala Glu Thr Val Thr Pro Val Leu Asp Pro Ala Arg Arg Gln
Gly 180 185 190 Tyr Gly Pro Leu Trp Tyr Thr Ser Val Gly Phe Gly Gly
Leu Val Gln 195 200 205 Leu Arg Arg Gly Glu Arg Val Tyr Val Asn Ile
Ser His Pro Asp Met 210 215 220 Val Asp Phe Ala Arg Gly Lys Thr Phe
Phe Gly Ala Val Met Val Gly 225 230 235 240 23 205 PRT Homo Sapien
23 Met Thr Pro Pro Glu Arg Leu Phe Leu Pro Arg Val Cys Gly Thr Thr
1 5 10 15 Leu His Leu Leu Leu Leu Gly Leu Leu Leu Val Leu Leu Pro
Gly Ala 20 25 30 Gln Gly Leu Pro Gly Val Gly Leu Thr Pro Ser Ala
Ala Gln Thr Ala 35 40 45 Arg Gln His Pro Lys Met His Leu Ala His
Ser Thr Leu Lys Pro Ala 50 55 60 Ala His Leu Ile Gly Asp Pro Ser
Lys Gln Asn Ser Leu Leu Trp Arg 65 70 75 80 Ala Asn Thr Asp Arg Ala
Phe Leu Gln Asp Gly Phe Ser Leu Ser Asn 85 90 95 Asn Ser Leu Leu
Val Pro Thr Ser Gly Ile Tyr Phe Val Tyr Ser Gln 100 105 110 Val Val
Phe Ser Gly Lys Ala Tyr Ser Pro Lys Ala Thr Ser Ser Pro 115 120 125
Leu Tyr Leu Ala His Glu Val Gln Leu Phe Ser Ser Gln Tyr Pro Phe 130
135 140 His Val Pro Leu Leu Ser Ser Gln Lys Met Val Tyr Pro Gly Leu
Gln 145 150 155 160 Glu Pro Trp Leu His Ser Met Tyr His Gly Ala Ala
Phe Gln Leu Thr 165 170 175 Gln Gly Asp Gln Leu Ser Thr His Thr Asp
Gly Ile Pro His Leu Val 180 185 190 Leu Ser Pro Ser Thr Val Phe Phe
Gly Ala Phe Ala Leu 195 200 205
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