U.S. patent application number 10/151564 was filed with the patent office on 2003-01-09 for lymphotactin uses.
This patent application is currently assigned to Schering Corporation, a New Jersey corporation. Invention is credited to Hedrick, Joseph A., Hudak, Susan A., Rennick, Donna M., Zlotnik, Albert.
Application Number | 20030007970 10/151564 |
Document ID | / |
Family ID | 27363782 |
Filed Date | 2003-01-09 |
United States Patent
Application |
20030007970 |
Kind Code |
A1 |
Hedrick, Joseph A. ; et
al. |
January 9, 2003 |
Lymphotactin uses
Abstract
This invention relates to the use of lymphotactin or
lymphotactin antagonists for administration to an animal. The
administration of these therapeutic entities will attract certain
cell types, or can be blocked to prevent such attraction. It also
provides means to protect stem cells.
Inventors: |
Hedrick, Joseph A.; (South
River, NJ) ; Hudak, Susan A.; (Redwood City, CA)
; Rennick, Donna M.; (Los Altos, CA) ; Zlotnik,
Albert; (Palo Alto, CA) |
Correspondence
Address: |
DNAX Research Institute
901 California Avenue
Palo Alto
CA
94304-1104
US
|
Assignee: |
Schering Corporation, a New Jersey
corporation
|
Family ID: |
27363782 |
Appl. No.: |
10/151564 |
Filed: |
May 20, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10151564 |
May 20, 2002 |
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09457501 |
Dec 8, 1999 |
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6395268 |
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09457501 |
Dec 8, 1999 |
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08956250 |
Oct 22, 1997 |
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6022534 |
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60031078 |
Oct 24, 1996 |
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Current U.S.
Class: |
424/144.1 |
Current CPC
Class: |
A61K 2039/505 20130101;
A61K 39/395 20130101; A61P 37/06 20180101; C07K 2317/76 20130101;
C07K 16/24 20130101 |
Class at
Publication: |
424/144.1 |
International
Class: |
A61K 039/395 |
Claims
What is claimed is:
1. A method of increasing the numbers of NK and/or CTL cells at a
location in an animal, said method comprising administering an
amount of lymphotactin effective to increase said numbers.
2. The method of claim 1, wherein said increasing is by recruitment
of cells to said location.
3. The method of claim 2, wherein said recruitment is to a tumor
cell.
4. The method of claim 3, wherein said tumor cell is from a solid
tumor.
5. The method of claim 1, wherein said increasing is by
proliferation of said cells.
6. The method of increasing of CTL cells of claim 1.
7. The method of claim 1, wherein said animal is a rodent.
8. The method of claim 1, wherein said effective amount is between
20 and 800 .mu.g.
9. The method of claim 1, wherein said administering is
parenteral.
10. A method of reducing allogeneic reaction from tissue transplant
in an animal, comprising a step of administering an effective
amount of an antagonist of lymphotactin to said animal.
11. The method of claim 10, wherein said antagonist comprises an
antigen binding site from an antibody which neutralizes mouse
lymphotactin.
12. The method of claim 11, wherein said antibody is administered:
a) at a dose of about 1-10 mg/kg body weight; or b) at about 10 to
about 100 .mu.g per milliliter of patient sera.
13. The method of claim 10, wherein said tissue is an organ.
14. The method of claim 10, wherein said antagonist reduces the
influx of NK or CTL cells to said tissue.
15. The method of claim 10, wherein said tissue is an organ
transplant, or bone marrow transplant.
16. A method of inducing cell cycle quiescence in a hematopoietic
stem cell, comprising a step of administering to said stem cell an
effective amount of lymphotactin.
17. The method of claim 16, wherein said lymphotactin is a primate
lympotactin.
18. The method of claim 16, wherein said quiescence imparts
insensitivity to a cell cycle dependent cytotoxic treatment.
19. The method of claim 18, wherein said treatment is a
chemotherapy or radiation therapy.
Description
[0001] This filing is a divisional of commonly assigned, co-pending
application U.S. Ser. No. 09/457,501, filed Dec. 8, 1999, now U.S.
Pat. No. 6,395,268, which is a continuation-in-part of U.S. Ser.
No. 08/956,250, filed Oct. 22, 1997, now U.S. Pat. No. 6,022,534,
which claims benefit of U.S. Provisional Patent Application No.
60/031,078, filed Oct. 24, 1996.
FIELD OF THE INVENTION
[0002] The present invention relates to the use of lymphotactin, a
recently discovered chemokine, for therapeutic administration to
animal or human patients. The administration of the chemokine will
attract cytotoxic T lymphocytes (CTL) and/or Natural Killer (NK)
cells. In addition, the biological effects of lymphotactin can
provide many of the effects of MIP-1, e.g., protecting
hematopoietic stem cells from the effects of cell cycling dependent
treatments, including chemotherapies and radiation therapies which
specifically target cycling cells. Lymphotactin also can attact
class I MHC expressing cells, e.g., which are the mediators of
tissue rejection.
BACKGROUND OF THE INVENTION
[0003] The circulating component of the mammalian circulatory
system comprises various cell types, including red and white blood
cells of the erythroid and myeloid cell lineages. See, e.g.,
Rapaport (1987) Introduction to Hematology (2d ed.) Lippincott,
Philadelphia, Pa; Jandl (1987) Blood: Textbook of Hematology,
Little, Brown and Co., Boston, Mass.; and Paul (ed.) (1993)
Fundamental Immunology (3d ed.) Raven Press, N.Y.
[0004] For some time, it has been known that the mammalian immune
response is based on a series of complex cellular interactions,
called the "immune network." Recent research has provided new
insights into the inner workings of this network. While it remains
clear that much of the response does, in fact, revolve around the
network-like interactions of lymphocytes, macrophages,
granulocytes, and other cells, immunologists now generally hold the
opinion that soluble proteins, known as lymphokines, cytokines, or
monokines, play a critical role in controlling these cellular
interactions. Thus, there is considerable interest in the
isolation, characterization, and mechanisms of action of cell
modulatory factors, an understanding of which should lead to
significant advancements in the diagnosis and therapy of numerous
medical abnormalities, e.g., immune system and other disorders.
[0005] Lymphokines apparently mediate cellular activities in a
variety of ways. They have been shown to support the proliferation,
growth, and differentiation of the pluripotential hematopoietic
stem cells into vast numbers of progenitors comprising diverse
cellular lineages making up a complex immune system. These
interactions between the cellular components are necessary for a
healthy immune response. These different cellular lineages often
respond in a different manner when lymphokines are administered in
conjunction with other agents.
[0006] The chemokines are a large and diverse superfamily of
proteins generally considered a subset of the cytokines. The
superfamily is subdivided into four branches, based upon whether
the first two cysteines in the classical chemokine motif are
adjacent (termed the "C--C" branch) or spaced by an intervening
residue ("C--X--C"), or a new branch which lacks two cysteines in
the corresponding motif, represented by the chemokines known as
lymphotactins. See, e.g., Schall and Bacon (1994) Current Opinion
in Immunology 6:865-873; and Bacon and Schall (1996) Int. Arch.
Allergy & Immunol. 109:97-109. A new fourth branch is
represented by a new chemokine designated CX3C chemokine.
[0007] Many factors have been identified which influence the
differentiation process of precursor cells, or regulate the
physiology or migration properties of specific cell types. These
observations indicate that other factors exist whose functions in
immune function were heretofore unrecognized. These factors provide
for biological activities whose spectra of effects may be distinct
from known differentiation or activation factors. The absence of
knowledge about the structural, biological, and physiological
properties of the regulatory factors which regulate cell physiology
in vivo prevents the modification of the effects of such factors.
Thus, medical conditions where regulation of the development or
physiology of relevant cells is required remains unmanageable.
SUMMARY OF THE INVENTION
[0008] This invention provides methods of increasing the numbers
various lymphocytes, e.g., NK cells and cytotoxic T lymphocytes
(CTL). The method comprises administering an amount of lymphotactin
where said amount is effective to either attract cytotoxic T cells
and/or NK cells, and/or to induce proliferation of resident cells.
A preferred lymphotactin is human lymphotactin, though rat or mouse
lymphotactin will function in their own, and biologically
cross-reacting species. The preferred single dosage of lymphotactin
is about 1 to 100 .mu.g/kg body weight. Alternatively the amount of
lymphotactin administered in a single dose is about 10-800 .mu.g,
or to reach a concentration of from pM to 1 .mu.M of patient
sera.
[0009] Alternatively, an antagonist will be effective in preventing
the recruitment of such cells. This may be important, e.g., in a
transplantation context, where NK and/or CTL function is
harmful.
[0010] This invention also provides methods of protecting
hematopoietic stem cells. The method comprises administering an
effective amount of lymphotactin where said amount is effective to
inhibit hematopoietic stem cell sensitivity to a cell cycle
dependent cytotoxic treatment, e.g., chemotherapy and/or radiation
therapy. In certain embodiments, the lymphotactin is administered
in combination with another active agent, e.g., another chemokine.
Such chemokines may include, e.g., MIP-1.alpha., MIP-1.beta.,
etc.
[0011] More particularly, the invention provides a method of
increasing the numbers of NK and/or CTL cells at a location in an
animal, comprising administering an amount of lymphotactin
effective to increase said numbers. In preferred embodiments, the
increasing is by recruitment of cells to that location; or the
recruitment is to a tumor cell. Other preferred embodiments include
where the tumor cell is from a solid tumor; where the increasing is
by proliferation of the cells; where the increasing is of CTL
cells; or where the animal is a rodent. Typically, the effective
amount is between 20 and 800 .mu.g; or the administering is
parenteral.
[0012] Also provided is a method of reducing allogeneic reaction
from tissue transplant in an animal, comprising a step of
administering an effective amount of an antagonist of lymphotactin
to the animal. Typically, the antagonist comprises an antigen
binding site from an antibody which neutralizes mouse lymphotactin;
the antagonist is administered at a dose of about 1-10 mg/kg body
weight; or at about 1 to about 100 .mu.g per milliliter of patient
sera; or the tissue is an organ. In preferred embodiments, the
antagonist reduces the influx of NK or CTL cells to the tissue; or
the tissue is an organ transplant, or bone marrow transplant.
[0013] The present invention further provides a method of inducing
cell cycle quiescence in a hematopoietic stem cell, comprising a
step of administering to the stem cell an effective amount of
lymphotactin. Preferably, the lymphotactin is a primate
lympotactin; the quiescence imparts insensitivity to a cell cycle
dependent cytotoxic treatment; or the treatment is a chemotherapy
or radiation therapy.
[0014] Conversely, an antagonist will be effective in preventing
the normal effect of natural lymphotactin, and may be useful in
inducing specific hematopoietic stem cells to start cell cycling,
and subsequent proliferation and/or development.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIGS. 1A and 1B show ELISA and Western blot analysis of
anti-mLptn. In FIG. 1A, the wells of a microtiter plate were coated
with 50 ng of either mLptn (squares) or BSA (circles) and then
reacted with serial dilutions of the 4D8 rat-anti-mLptn hybridoma
culture supernatant. Antibody binding was detected with
HRP-conjugated mouse anti-rat IgG (1:5000 dilution) and developed
with TMB peroxidase. Absorption was read at 450 nm. In FIG. 1B, for
Western blotting, 3 .mu.g of either mLptn of hLptn were
electrophoresed under reducing conditions on a 4-20% gradient
Tris-glycine gel. Protein was electroblotted onto Immobilon-IPVH
and detected with 4D8 hybridoma supernatant (1:20 dilution) and
HRP-conjugated goat anti-rat Ig. The blot was developed with ECL
chemiluminescence. The molecular weights of protein markers (in
kilodaltons) are indicated on the left hand side of the figure.
Data are representative of at least three separate experiments in
each case.
[0016] FIG. 2 shows an influx of cells into the peritoneal cavity
in response to Lptn. Mice were injected intraperitoneally with 200
.mu.l of PBS containing 10 pg of LPS, 200 .mu.l of PBS containing
10 .mu.g of mLptn, or 200 .mu.l of PBS containing 10 .mu.g of mLptn
and 500 .mu.g of purified 4D8 mAb. The mice were euthanized after
24 hours and peritoneal lavage collected and total cell numbers
calculated. The results shown are representative of seven separate
experiments (three using the 4D8 mAb) consisting of five
mice/condition/in each experiment and are expressed as a percent of
the PBS+LPS control (100%).
[0017] FIG. 3 shows flow cytometric analysis of cells migrating in
response to Lptn. Cells recovered from the peritoneal lavage of
PBS-injected mice (left column) or mLptn-injected mice (right
column) were analyzed by flow cytometry. Electronic gates were
placed around cells with a lymphocyte scatter profile and
3.times.10.sup.5 cells analyzed using FITC-conjugated CD19 (1:200
dilution), PE-conjugated NK1.1 (1:100 dilution), and
biotin-conjugated CD3 (1:1000) antibodies. Biotin-conjugated CD3
was detected with streptavidin-TriColor.TM.. The results shown are
representative of seven independent experiments, each consisting of
five animals/condition.
[0018] FIG. 4 shows changes in absolute numbers of peritoneal
lymphocyte populations (NK, T, and B cells) as a result of mLptn
injection. The average change in the absolute number of each
lymphocyte population was determined for each of seven independent
experiments and normalized to the average control values for that
particular experiment. The data were then combined to produce the
figure displayed. Results are expressed as percent increase or
decrease relative to the PBS control. Each independent experiment
contained five animals/condition.
[0019] FIG. 5 shows infiltration of T cells in response to
subcutaneous injection of Lptn. Frozen sections from the rear
footpads of mice that had been subcutaneously injected with either
PBS (top panel) or PBS containing 1 .mu.g of mLptn (bottom panel,
bottom panel inset) were analyzed by immunohistochemistry using the
CD3.sub..epsilon. mAb. Bound CD3.sub..epsilon. was detected with
biotin-conjugated goat anti-hamster IgG and the Vectastain Elite
ABC kit. Stained sections were developed with DAB. Results shown
from one out of two independent experiments. Bars are 5 .mu.m.
[0020] FIGS. 6A and 6B show in vitro chemotaxis of NK cells in
response to Lptn. Microchemotaxis assays were conducted for (FIG.
6A) murine NK cells and (FIG. 6B) human NK clones responding to
mLptn or hLptn, respectively. Migration is expressed as cell number
per five high power (400.times.) fields, with duplicate wells
counted for concentration of Lptn, versus molar concentration of
Lptn. Background migration in media alone: (FIG. 6A) freshly
isolated murine NK cells=21 .+-.3, IL-2 activated cells=30 .+-.4;
(FIG. 6B) human NK clone 576 A6-1=58.+-.5, 867 c20=56.+-.3, 867
d27=49.+-.4, and 867 c18=73.+-.7.
[0021] FIG. 7 shows expression of Lptn by murine NK cells.
CD3-activated A3.2 T cells (control, A & B) or IL-2-activated
NK cells (C & D) were fixed in formalin and stained
intracellularly with the 4D8 anti-mLptn mAb (A, B & D) or an
isotype control antibody (C). Bound antibody was detected with
biotin-conjugated goat anti-rat IgG and the Vectastain Elite ABC
kit. Slides were then developed with DAB. Bars are 5 .mu.m.
[0022] FIG. 8 shows expression of Lptn mRNA by human NK clones. 10
.mu.g of total RNA from each of four human NK clones was analyzed
by Northern blotting using human Lptn as a probe. The blot had been
previously probed with actin as a control for equal loading of RNA.
The mRNAs for actin and Lptn are indicated.
[0023] FIGS. 9A, 9B, and 9C show that human PBLs and murine
splenocytes fail to respond to CT-hLptn. Microchemotaxis assays
were conducted with either (FIG. 9A) human PBLs or (FIGS. 9B &
9C) murine splenocytes using (FIGS. 9A & 9B) full length hLptn
(squares) and CT-hLptn (circles) or (FIG. 9C) mLptn. Migration is
expressed as cell number per five high power (400.times.) fields,
with duplicate wells counted for concentration of Lptn. Background
migration in media alone: (FIG. 9A) human PBLs=41.+-.9, (FIG. 9B)
murine splenocytes=67.+-.7, (FIG. 9C) murine
splenocytes=65.+-.7.
[0024] FIGS. 10, 11, and 12 show the ability of lymphotactin, like
MIP-1.alpha., to delay the entry of hematopoietic stem cells into
cycle. These figures indicate the number of progenitor cells
remaining after an initial culture period of 7, 14, or 21 days. 400
purified mouse hematopoietic Sca1.sup.+, c-Kit.sup.+,
rhodamine.sup.lo, lin.sup.- stem cells per culture were stimulated
in the various growth factor combinations shown for 7, 14, and 21
days. At the end of the initial liquid culture period the cells
were harvested, washed, and replated in methylcellulose to detect
colony-forming cells (CFU-c). A combination of hematopoietic growth
factors, e.g., Stem Cell Factor, IL-3, IL-6, and erythropoietin,
was used in the secondary cultures to support the development of
all cell lineages. Colony numbers are expressed as the
mean.+-.standard deviation for triplicate cultures. The input value
indicates the number of progenitors contained in 400 cells at day
0.
DETAILED DESCRIPTION
[0025] This invention provides an effective means for increasing
the local number of cytotoxic T cells (CTL) and/or natural killer
(NK) cells. Lymphotactin, when administered, has been observed to
increase the abundance of these cell types. This increase may
result from attraction of said cell types, from proliferation of
exisitng cells, or a combination of both. Antagonists will also
block such effect on such cells in circumstances, e.g., in MHC
mismatched contexts, including tissue or organ rejection and graft
vs. host disease, where such responses are undesireable.
[0026] In addition, lymphotactin may have positive effects on
antiviral processes, whether directly by blocking a chemokine
receptor used as a viral coreceptor, or to attract the NK or CTL
cells attracted to sites of virally infected cells.
[0027] Lymphotactin also exhibits an activity of inducing cell
cycle quiescence in hematopoietic stem cells. As such, when various
drugs are administered which specifically act on proliferating
cells, these stem cells are largely unaffected. Such drugs include
chemotherapy reagents, e.g., nucleotide or nucleoside analogs, or
radiation therapy, which mutagenizes cells which have a slowed
repair process.
[0028] Mammalian lymphotactin (Itn) has been well described, e.g.,
in U.S. Ser. No. 08/329,704 and related cases, and Kelner, et al.
(1994) Science 266:1395-1399. The preferred mammalian lymphotactin
is a natural human lymphotactin.
[0029] Lymphotactin is produced in a variety of ways, both
conventional and less conventional. A general review of recombinant
protein production can be found, e.g., Sambrook, et al. (1989)
Molecular Cloning: A Laboratory Manual Cold Spring Harbor Press,
N.Y., or in Ausubel, et al. (Eds.; 1989 and Suppl.) Current
Protocols in Molecular Biology, Green/Wiley, NY, N.Y. Natural
sources of protein may be used as a source, and protein isolated
therefrom.
[0030] Another method for preparing protein or peptides includes
polypeptide synthesis methods. See, e.g., Merrifield (1988) Science
232:341-347; and Dawson, et al. (1994) Science 266:776-779.
[0031] In antagonist embodiments, the antagonist can be an antibody
or fragment specific for binding to lymphotactin, e.g., which
interferes with the chemokine binding to its receptor.
Anti-lymphotactin reagents are produced in a variety of ways, both
conventional and less conventional. A general review of antibody
production can be found, e.g., in Harlow and Lane (1988)
Antibodies: A Laboratory Manual Cold Spring Harbor Press, N.Y., or
in Colligan, et al. (Eds.; 1991 and Suppl.) Current Protocols in
Immunology, Green/Wiley, NY, N.Y. Antibodies can be polyclonal
mixture or monoclonal. Antibodies can be intact immunoglobulins or
fragments thereof, derived from natural sources or from recombinant
sources. Antibodies also include the immunoreactive portions of
intact immunoglobulins, e.g., antigen binding portions such as Fab,
Fv, etc.
[0032] In brief, methods to obtain anti-lymphotactin antibodies
involve administering an amount of antigen, e.g., a fragment,
sufficient to induce a humoral response in a mammal. The antibodies
are either collected from the mammal's sera or lymphocytes removed,
immortalized, and those cell clones secreting the desired
antibodies isolated and cultured for harvest of the desired
antibodies.
[0033] The quantities of reagents necessary for effective therapy
will depend upon many different factors, including means of
administration, target site, physiological state of the patient,
and other medicants administered. Thus, treatment dosages should be
titrated to optimize safety and efficacy. Typically, dosages used
in vitro may provide useful guidance in the amounts useful for in
situ administration of these reagents. Animal testing of effective
doses for treatment of particular disorders will provide further
predictive indication of human dosage. Various considerations are
described, e.g., in Gilman, et al. (eds.) (1990) Goodman and
Gilman's: The Pharmacological Bases of Therapeutics (8th ed.)
Pergamon Press; and (1990) Remington's Pharmaceutical Sciences
(17th ed.) Mack Publishing Co., Easton, Pa. Methods for
administration are discussed therein and below, e.g., for oral,
intravenous, intraperitoneal, or intramuscular administration,
transdermal diffusion, and others. Pharmaceutically acceptable
carriers will include water, saline, buffers, and other compounds
described, e.g., in the Merck Index, Merck & Co., Rahway, N.J.
Dosage ranges would ordinarily be expected to be in amounts lower
than 1 mM concentrations, typically less than about 10 .mu.M
concentrations, usually less than about 100 nM, preferably less
than about 10 .mu.M (picomolar), and most preferably less than
about 1 fM (femtomolar), with an appropriate carrier. Slow release
formulations, or a slow release apparatus will often be utilized
for continuous administration.
[0034] Thymokines, fragments thereof, and antibodies to it or its
fragments, antagonists, and agonists, may be administered directly
to the host to be treated or, depending on the size of the
compounds, it may be desirable to conjugate them to carrier
proteins such as ovalbumin or serum albumin prior to their
administration. Therapeutic formulations may be administered in any
conventional dosage formulation. While it is possible for the
active ingredient to be administered alone, it is preferable to
present it as a pharmaceutical formulation. Formulations typically
comprise at least one active ingredient, as defined above, together
with one or more acceptable carriers thereof. Each carrier should
be both pharmaceutically and physiologically acceptable in the
sense of being compatible with the other ingredients and not
injurious to the patient. Formulations include those suitable for
oral, rectal, nasal, or parenteral (including subcutaneous,
intramuscular, intravenous and intradermal) administration. The
formulations may conveniently be presented in unit dosage form and
may be prepared by any methods well known in the art of pharmacy.
See, e.g., Gilman, et al. (eds.) (1990) Goodman and Gilman's: The
Pharmacological Bases of Therapeutics (8th ed.) Pergamon Press; and
(1990) Remington's Pharmaceutical Sciences (17th ed.) Mack
Publishing Co., Easton, Pa.; Avis, et al. (eds.) (1993)
Pharmaceutical Dosage Forms: Parenteral Medications Dekker, N.Y.;
Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:
Tablets Dekker, N.Y.; and Lieberman, et al. (eds.) (1990)
Pharmaceutical Dosage Forms: Disperse Systems Dekker, N.Y. The
therapy of this invention may be combined with or used in
association with other chemotherapeutic or chemopreventive
agents.
[0035] These methods require adequate sources of lymphotactin as
antigens. The antigens can either be intact lymphotactin or
immunoreactive peptides. Recombinant expression of lymphotactin is
a convenient means for obtaining lymphotactin for use as antigens.
For a general review of the applicable recombinant technology see
Sambrook, et al. (1989) Molecular Cloning--A Laboratory Manual,
Cold Spring Harbor Press, CSH, NY Specific techniques for
expressing and purifying lymphotactin are known. Expression of
lymphotactin is described in PCT/US03554(WO/91/00349) and in
Malefyt, et al. (1992) Curr. Opin. Immunology 4:314-320.
Alternatively, peptide synthesis may be used to obtain intact or
immunoreactive portions of lymphotactin.
[0036] The antibodies for use in this invention are preferably
autologous for the patient thereby minimizing further immunological
problems. Immunodeficient individuals will tend to be less reactive
to non-self antibodies, and thus non-self antibodies derived from
cells of the same species are also useful. Antibodies of different
species are useful but means to control possible adverse
immunoreactions must be undertaken. For example, humanized rat
antibodies can minimize immune responses in human patients.
[0037] The antibodies for use in this invention are typically
neutralizing antibodies and will preferably have binding constants
which are greater than or approximates the affinity of lymphotactin
for its natural receptor. Antibodies having a binding constant
100-fold less than these cytokines for their corresponding
receptors are less preferred. Binding comparisons are carried out
using standard equilibrium methods. The basic technology is
described in Chpt 25 of Vol. 1: Immunochemistry, Ed. D. M. Weir,
4th Ed. 1986, Blackwell Scientific Publ. 25.1-25.30. Alternatively,
one can use an assay for determining the molar excess of antibody
which neutralizes a defined amount of IL-10 in a standard in vitro
bioassay. Examples of such assays are found in Mosmann and Fong
(1989) J. Immunol. Methods 116:151 (IL-4) and Fiorentino et al.,
1989, J. Exp. Med. 170:2081. A reasonable range are those
antibodies which neutralize a given amount of lymphotactin in a 10
to 1,000 fold excess.
[0038] The means of administration of the antagonists, e.g.,
.alpha.lymphotactin, are typically parenteral, preferably
intravenous. The antagonists are infused into the patient using
standard intravenous techniques. The antagonists are first
suspended into a sterile, physiologically-compatible media, such as
phosphate buffered saline. Pharmaceutically acceptable excipients
such as lecithin, glucose, dextrose, antibiotics may also be
included with the antagonists.
[0039] When the antagonists are antibodies, they are administered
in an amount which provides circulating levels of anti-lymphotactin
at about 1 to 150 .mu.g/ml and preferably 10 to 100 .mu.g/ml of
sera for each antibody. The antibodies typically will have a 2-7
day half-life and repeated administration is necessary when levels
of anti-lymphotactin are below these levels. Total amount of
anti-lymphotactin applied per administration are between 1 and 10
mg/kg of body weight for each antibody.
[0040] The broad scope of this invention is best understood with
reference to the following examples, which are not intended to
limit the invention to specific embodiments.
EXPERIMENTAL
EXAMPLE 1
[0041] In vivo Effects: Methodology
[0042] Abbreviations used herein: Lptn, lymphotactin; mLptn, murine
lymphotactin; hLptn, human lymphotactin; CT-hLptn, carboxy-terminal
truncated human lymphotactin; IPTG, isopropyl
.beta.-D-thiogalactopyranos- ide; HRP, horse radish peroxidase; AP,
alkaline phosphatase; RT, room temperature; min, minute.
[0043] Injection of lymphotactin (Lptn) into the peritoneum caused
an influx of lymphocytes at 24 hours. Phenotypic analysis of the
cellular influx showed that a large proportion of these cells were
T lymphocytes, however, a large number of natural killer (NK) cells
were also present. This effect of mLptn was specific since the
cellular influx was blocked with a mLptn-specific monoclonal
antibody (mAb). Similar results were observed when Lptn was
injected subcutaneously and the tissue analyzed by
immunohistochemistry using an anti-CD3.sub..epsilon. mAb.
Microchemotaxis assays confirmed that murine NK cells respond to
mLptn and also showed human NK clones to be similarly responsive to
recombinant human Lptn (hLptn). Immunohistochemical analysis of
IL-2 activated murine NK cells and Northern analysis of human NK
clones revealed that these cells also produce Lptn, suggesting that
a self-regulatory migration mechanism exists in NK cells. Together
these data confirm, in vivo, the lymphocyte-specificity of Lptn
previously observed in vitro and extend its chemotactic effects to
the NK cell lineage. The functional consequences of truncating the
carboxy terminus of hLptn (CT-hLptn) were also investigated. This
truncated molecule (which is missing the carboxy-terminal 22 amino
acids of hLptn) had no detectable activity on human peripheral
blood lymphocytes. In addition, while hLptn was found to attract
murine splenocytes in vitro, the carboxy-terminal truncated hLptn
was again inactive on murine splenocytes. This observation
indicates the presence of structural features in the carboxy
terminus of Lptn that are necessary for its biological
activity.
[0044] The chemokines are a large family of chemoattractant
cytokines whose members have been subdivided into three subfamilies
on the basis of the positions of four invariant cysteines (reviewed
in Baggiolini, et al. (1994) Adv. in Immun. 55:97-179; and Schall
(1994) pp. 419-460, in Thomson (ed.) The Cytokine Handbook Academic
Press, New York, N.Y.). The first two cysteines of the C--X--C or
.alpha. chemokine subfamily are separated by a single amino acid
residue, while the C--C or .beta. chemokine subfamily members all
have the first two cysteines immediately adjacent to one another.
We recently described the cloning of murine Lymphotactin (Lptn)
(Kelner, et al. (1994) Science 266:1395-1399) a third type of
chemokine. This protein, designated the C or .gamma. chemokine,
lacks two of the four invariant cysteines (C.sub.1 and C.sub.3)
normally found in chemokines (Kelner, et al. (1994); Kennedy, et
al. (1995) J. Immunol. 155:203-209; Muller, et al. (1995) Eur. J.
Immunol. 25:1744-1748; and Yoshida, et al. (1995) FEBS Lett.
360:155-159) and thus can form only one of the two intrachain
disulfide bonds found in the .alpha. and .beta. chemokines. A human
form of Lptn was subsequently described (Kennedy, et al. (1995) J.
Immunol. 155:203-209; Muller, et al. (1995) Eur. J. Immunol.
25:1744-1748; and Yoshida, et al. (1995) FEBS Lett. 360:155-159)
which also lacks two cysteine residues. Lptn also has an extended
carboxy terminus which is unusual among chemokines and further
distinguishes it structurally. The gene coding for Lptn has been
localized to chromosome 1 in both mouse and human, which is a
different location from either the C--C or C--X--C chemokines. Lptn
also differs functionally from other chemokines since it has no
activity on either macrophages or neutrophils, and appears instead
to be specific for lymphocytes. The in vitro chemotactic activity
of Lptn has been demonstrated on both CD4.sup.+ and CD8.sup.+ T
cells of mouse and human. Murine thymocytes, including the
CD4.sup.-CD8.sup.-, CD4.sup.+, and CD8.sup.+ populations, also
respond to Lptn. This finding is supported, e.g., by Dorner, et al.
(1997) J. Biol. Chem. 272:8817-8823. A recent report has suggested
that Lptn could be used in conjunction with IL-2 to enhance the
antitumor activity of T lymphocytes. See, e.g., Dilloo, et al.
(1996) Nature Med. 2:1090-1095. An exception are the
CD4.sup.+CD8.sup.+ thymocytes which do not respond to Lptn. Binding
of Lptn to either murine or human T cells has been shown to produce
an intracellular Ca.sup.++ flux similar to that observed in
response to other chemokines. Finally, Lptn is also unusual in that
it does not bind to the duffy antigen receptor for chemokines which
can bind many C--C and C--X--C chemokines (Szabo, et al. (1995) J.
Biol. Chem. 270:25348-25351.).
[0045] Expression of Lptn is essentially restricted to activated
murine CD4.sup.-CD8.sup.-CD3.sup.-CD25.sup.+CD44.sup.+ thymocytes
(pro-T cells), from which it was originally cloned and to
activated, class-I-MHC-restricted T cells, including CD8.sup.+ T
cells from both mouse and human and
TCR.alpha..beta..sup.+CD4.sup.-CD8.sup.- T cells in mouse. A low
level of expression is detectable in human CD4.sup.+ T cells though
it is likely that this signal arises from a human equivalent to the
murine NK1.1.sup.+CD4.sup.+ T cells, which also express Lptn.
[0046] Here are reported studies of the in vivo activity of Lptn.
The results of these studies confirm the previous in vitro studies
in which Lptn was found to be a potent chemoattractant for T
lymphocytes. In addition, it is demonstrated that Lptn attracts not
only T cells, but also natural killer cells. It is shown that both
murine NK cells and human NK clones respond to and produce Lptn in
vitro, and that the carboxy terminus of Lptn is necessary for its
activity. It is also shown that murine NK cells and human NK clones
produce Lptn. Finally, the production and characterization of a
neutralizing anti-mLptn mAb is described.
[0047] Cells and cell lines: Purified murine NK cells were obtained
from the spleens of Rag-1.sup.-/- mice by Fluorescence Activated
Cell Sorting (FACS) for NK1.1.sup.+Gr-1.sup.- cells utilizing a
FACstar flow cytometer (Becton Dickinson, Mountain View, Calif.).
The purity of NK cells obtained by this method was routinely
>97%. Human NK clones were the generous gift of Dr. J. Phillips
(DNAX). The A3.2 T cell hybridoma was produced by fusion of BW5147
with purified .alpha..beta.TCR.sup.+CD4.sup.- -CD8.sup.- thymocytes
using standard techniques. The resulting hybridomas were subcloned
and selected for high IL-4 production, a characteristic of
.alpha..beta.TCR.sup.+CD4.sup.-CD8.sup.- T cells. Vicari and
Zlotnik (1996) Immunol. Today 17:71-76.
[0048] Mice: Female CB6F1/Sim BR mice were obtained from Simonsen
Laboratories (Gilroy, Calif.). Rag-1.sup.-/- knockout mice were
obtained from The Jackson Laboratory (Bar Harbor, Me.) and have
subsequently been bred and housed in a specific pathogen-free
facility. Animals used were between six and ten weeks of age.
[0049] Recombinant murine lymphotactin (mLptn): The coding region
of murine Lptn was subcloned into the pET-3a expression vector
(Novagen, Madison, Wis.). Expression plasmids were then used to
transform the BL21 (DE3) strain of E. coli (Novagen) containing
pLys S. The transformed cells were grown in Luria broth until the
O.D. at 560 nm had reached 1.0, at which point they were induced
with 0.4M IPTG for an additional 4 h. The cells were then harvested
by centrifugation and resuspended in 50 mM Tris (pH 7.5), 5 mM
EDTA, 1 mM Pefa Bloc. Soluble protein was purified by
chromatography on Q sepharose (pH 8.5; Pharmacia), SP sepharose (pH
8.5; Pharmacia), and POROS R2/M. Fractions containing mLptn were
identified by migration of protein on SDS-PAGE. Fractions
containing highly pure mLptn were then pooled and quantitated. The
identity of the purified protein was confirmed by amino-terminal
amino acid sequencing. The recombinant mLptn produced in this
manner has an amino terminus sequence of MVGTEVLEQS-. The endotoxin
content was <6 EU/mg of purified protein. The FLAG-murine Lptn
(FLAG-mLptn) was produced as previously described in Kelner, et al.
(1994) Science 266:1395-1399.
[0050] Recombinant human lymphotactin: Full-length recombinant
hLptn was obtained from Genzyme Diagnostics (Cambridge, Ma.). The
carboxy terminal truncated version of hLptn (CT-hLptn) was produced
as follows: a synthetic gene encoding residues 1-71 of human Lptn
was constructed with codons optimized for expression in E. coli,
and ligated into Xba1 and Kpn1 sites of the T7-driven expression
vector pCC101. CT-hLptn was expressed in a BL21 (DE3) strain of E.
coli containing pLys S. Transformed cells were grown to an O.D., at
600 nm, of between 0.7 and 1.0, at which time expression was
induced with IPTG (0.5 mM). Cells were grown for an additional
three hours, harvested by centrifugation, and frozen at -80.degree.
C.
[0051] The cell pellets were sonicated in 50 ml of buffer (25 mM
sodium acetate, 1 mM EDTA, pH 5.0) and centrifuged (10,000 rpm, 15
min) to pellet the protein. The protein pellet was resuspended in a
minimal amount of 6 M guanidine HCl and diluted into 50 volumes of
sonication buffer containing 6 M urea. Soluble protein was then
loaded onto a 30 ml SP Sepharose Fast Flow column, washed with 2-3
bed volumes of the loading buffer, and eluted with a 0-0.4 M
gradient of NaCl. Protein-containing fractions were pooled and
dialyzed against the sonication buffer at 4.degree. C. to remove
the urea. Oxidation of the single disulfide bond in the protein was
accomplished by 1:1 dilution into a redox buffer (100 mM Tris, 1 mM
EDTA, 2 mM oxidized glutathione, 0.2 mM reduced glutathione pH=8.0)
with stirring overnight at 4.degree. C. Final purification was
achieved by reversed-phase HPLC on a Vydac C.sub.4 semi-prep
column. Mass spectral analysis confirmed oxidation of the cysteine
residues and retention of the amino-terminal methionine.
[0052] Preparation of monoclonal anti-Lptn antibody: A male Lewis
rat was immunized intraperitoneally with 25 .mu.g of FLAG-mLptn in
1.0 ml of complete Freund's adjuvant. The rat was immunized three
more times, at two week intervals, with 25 .mu.g of FLAG-mLptn in
incomplete Freund's adjuvant. Serum was collected after the third
and fourth immunizations and assayed for anti-FLAG-mLptn reactivity
by ELISA (described below). Four days after the last immunization
the animal was euthanized and its splenocytes fused with cells,
e.g., as described by Chrtien, et al. (1989) J. Immunol. Meth.
117:67-81. Hybridomas were initially screened against FLAG-mLptn by
ELISA followed by a secondary screening against mLptn (non-FLAG). A
third screening was performed to identify those hybridomas with
reactivity to mLptn, as judged by Western blotting. The 4D8
anti-mLptn hybridoma was selected for cloning by these criteria and
the mAb produced by this hybridoma was further characterized as
having an IgG2a isotype.
[0053] In vivo experiments: Mice were injected intraperitoneally
with 10 .mu.g of purified mLptn in 200 .mu.l of PBS (pH 7.4).
Alternatively, mice were injected with 200 .mu.l of PBS containing
10 .mu.g of mLptn that had been preincubated with 500 .mu.g of
purified anti-mLptn mAb 4D8 or 500 .mu.g of isotype control for 30
min on ice. Control animals were initially injected with either 200
.mu.l PBS or 200 .mu.l of PBS containing 10 pg of LPS (equivalent
to the amount of endotoxin present in the mLptn preparation) as an
endotoxin control. No differences were observed between the PBS and
PBS+LPS injected animals; therefore PBS+LPS was subsequently used
as a negative control. Animals were euthanized after 24 h or 72 h
and the peritoneal cavity was then washed with 5 ml of ice-cold
PBS. Cell counts were obtained microscopically and adjusted for the
amount of PBS recovered according to the formula: adjusted cell
number=total cell number.times.(vol. injected/vol. recovered). Cell
viability was consistently above 97%.
[0054] A group of mice was injected subcutaneously in the hind
footpads. Each animal was injected with 20 .mu.l of PBS containing
1 .mu.g mLptn in one footpad and with a PBS control containing 1 pg
of LPS (an amount equivalent to the endotoxin present in the mLptn
preparation) in the opposite footpad. The footpads and ankles of
the mice were examined and measured with Vernier calipers twice
daily for 3 days to monitor for inflammation and/or swelling. For
immunohistochemical analysis, some animals were euthanized after
20-24 h and the footpads were removed and frozen in O.C.T. compound
(Baxter Diagnostics Inc., McGraw Park, Ill.
[0055] Antibodies and flow cytometric analyses: Cells obtained from
peritoneal lavage were washed once in ice-cold PBS (2% FBS) and
then resuspended in the same buffer for staining with the
appropriate dilution of CD19-FITC, NK1.1-PE, and
CD3.epsilon.-biotin, followed by streptavidin-Tricolor.TM.. For
some experiments, varying combinations of CD4-FITC, CD8-PE,
Mac-1-PE, and Gr-1-biotin were also used. Flow cytometric analyses
were performed on a FACScan II (Becton Dickinson) and analyzed
using CellQuest software (Becton Dickinson). Antibodies used for
flow cytometry were obtained commercially (PharMingen, San Diego,
Calif.) as was the streptavidin-tricolor.TM. conjugate (Caltag, So.
San Francisco, Calif.).
[0056] ELISA and western blotting: For ELISA, 50 ng of either mLptn
or BSA, suspended in PBS (pH 7.2) was added to each well of a
96-well, PVC U-bottom plate (Dynatech, Chantilly, Va.) and the
plate was incubated for 2 h at 37.degree. C. The plate was then
washed 3.times. with PBS (pH 7.2)/0.005% Tween-20 in a Dynatech
plate washer and the supernatant from the 4D8 hybridoma was added
in serial dilution. RPMI+10% FCS was used as a medium control.
Following an incubation of one hour at room temperature (RT), the
plate was washed again and 50 .mu.l of horseradish peroxidase
(HRP)-conjugated mouse anti-rat IgG (1:5000 dilution in PBS (pH
7.2) containing 0.005% Tween-20 and 0.1% BSA; Jackson
ImmunoResearch, West Grove, Pa.) was added to each well. The plate
was incubated for one hour at RT, washed again, and the assay
developed using TMB-peroxidase (Kirkegaard and Perry, Gaithersburg,
Md.) as a substrate. Optical density was read at 450 nm.
[0057] For Western blotting, 3 .mu.g of either mLptn or hLptn
(Genzyme) were suspended in Laemmli's buffer (containing 2%
.beta.-mercaptoethanol)- , boiled 5 min, and loaded onto a
Tris-Glycine polyacrylamide gel with a 4-20% gradient (Novex, San
Diego, Calif.). The gel was electrophoresed and protein transferred
onto Immobilon-IPVH (Millipore, Bedford, Mass.) via
electroblotting. The resulting protein blot was blocked overnight
at 4.degree. C. in wash buffer (0.01 M Tris ,pH 7.4, 0.155 M NaCl,
0.02% Tween-20) containing 5% w/v nonfat dry milk. The blot was
subsequently incubated with the 4D8 rat-anti-mLptn mAb (1:500
dilution of hybridoma supernatant) for 1.5 h, at RT. The blot was
then washed 3.times. (5 min each wash) in wash buffer and incubated
with a 1:5000 dilution of HRP-conjugated goat anti-Rat Ig
(Amersham, Little Chalfont, UK) for one hour at room temp. The blot
was again washed (6.times., 5 min each wash) and developed with the
ECL chemiluminescence detection system (Amersham) according to the
manufacturer's instructions.
[0058] Immunohistochemistry: Tissue was sectioned into 5 .mu.m
slices which were thaw mounted onto organosilicone subbed slides
(American Histology Reagent Co., Stockton, Calif.) and fixed by
acetone immersion (5 min, -20.degree. C.). Sections were then
rinsed 3.times. in 0.01 M Hepes-buffered Hank's Balanced Salt
Solution (Hepes/HBSS) for 5 min, each wash. Sections were
subsequently blocked with 1% hydrogen peroxide, 0.2 M sodium azide
in Hepes/HBSS for 20 min at RT. An additional blocking step was
carried out using 10% normal goat serum in Hepes/HBSS (10 min,
RT).
[0059] Following fixation and blocking, sections were incubated
with hamster-anti-mouse CD3.sub..epsilon. (PharMingen) at a
concentration of 5 .mu.g/ml in Hepes/HBSS for 2 hours at RT. The
sections were washed as before and bound antibody detected with
biotin-conjugated, goat-anti-hamster IgG (2.0 .mu.g/ml in
Hepes/HBSS, 1 h, RT; Vector Laboratories, Burlingame, Calif.). The
sections were washed as before and final detection carried out
using the Vectastain Elite ABC kit (Vector Laboratories) according
to the manufacturer's instructions. Slides were then washed with
Hepes/HBSS (no saponin) and developed with 3,3'-diaminobenzidine
tetrahydrochloride (DAB, 0.5 mg/ml in 0.05 M Tris (pH 7.4)
containing 0.0075% H.sub.2O.sub.2; Sigma Chemical Co., St. Louis,
Mo.) as substrate. The reaction was quenched with distilled water
and the sections were dehydrated in graded ethanol, immersed in
xylene, and finally mounted with Permount (Fisher Scientific,
Springfield, N.J.) for examination and photography.
[0060] Intracellular detection of lymphotactin: Activated A3.2 T
cells were obtained by incubation of cells with RPMI 1640/10% FBS
in tissue culture flasks precoated with CD3.epsilon. (PharMingen;
10 .mu.g/ml in PBS) for 3 h. Purified murine NK cells were
activated by incubation with IL-2 (500 U/ml in RPMI 1640/10% FBS)
for 5 days. Following activation, cells were washed 2.times. in DME
(no serum) and 3.times. in Hepes/HBSS. Cell concentration was
adjusted to 1.times.10.sup.6/ml in Hepes/HBSS and 50 .mu.l of cell
suspension (5.times.10.sup.4 cells) pipetted into each of the wells
of an adhesion slide (Bio-Rad Laboratories, Hercules, Calif.).
Following adhesion (20 min, RT), cells were fixed in 3%
formalin-Hepes/HBSS (15 min, RT) and washed 2.times. in Hepes/HBSS.
The cells were then blocked sequentially with avidin D (2 drops/ml
of avidin blocking solution; Vector Laboratories) for 15 min,
followed by a biotin block (2 drops/ml of biotin blocking solution;
Vector Laboratories) for 15 min, a sodium azide-peroxide block (0.2
M NaN.sub.3, 0.1% H.sub.2O.sub.2) for 20 min, and finally normal
rabbit serum (10%) for 10 min. Blocking solutions were made in
permeabilization buffer (Hepes/HBSS/0.1% saponin). The 4D8
anti-mLptn mAb was then added (5 .mu.g/ml in permeabilization
buffer) and the slides allowed to incubate for one hour at RT. The
slides were washed in permeabilization buffer (3.times., 5 min each
wash) and incubated with a biotin-conjugated rabbit-anti-rat Ig
(1:300 dilution in permeabilization buffer; Vector Laboratories)
for 30 min at RT. Bound antibody was detected using the Vectastain
Elite ABC kit (Vector Laboratories) and the slides developed with
DAB as before.
[0061] In vitro microchemotaxis assay: The microchemotaxis assays
were carried out using a modified 48-well Boyden chamber migration
assay. See Bacon, et al. (1988) Br. J. Pharmacol. 95:966-974.
Serial dilutions of mLptn or hLptn were made into serum free RPMI.
Duplicate wells of the lower half of the microchemotaxis chamber
(Neuro Probe Inc., Cabin John, Md.) were filled with the
appropriate dilutions and the upper chambers of the assembly were
filled with 40 .mu.l, of the appropriate cell suspension
(1-2.times.10.sup.6 cells/ml). Data was obtained by counting five
non-overlapping high-power microscope fields
(400.times.magnificatio- n at eyepiece) from each of the wells.
Cells were considered to have chemotaxed if the chemotactic index
(Cl=# cells migrating in experimental well/# cells migrating in
media only) was greater than two.
[0062] Expression of Lptn message: Expression of Lptn mRNA by human
NK clones was determined by Northern blotting. Total RNA was
extracted from each clone using RNAsol (Tel-Test Inc. Friendswood,
Tex.) according to the manufacturer's instructions. Northern
blotting was performed by standard methods using 10 .mu.g of total
RNA from each clone.
EXAMPLE 2
[0063] Characterization of an Anti-mLptn mAb
[0064] Anti-Lptn mAbs were raised in Lewis rats by immunization
with purified recombinant FLAG-mLptn. Hybridomas produced from the
splenocytes of immunized animals were then screened by ELISA for
reactivity with FLAG-mLptn. The hybridomas which were positive in
this initial screen were then tested for ELISA reactivity against
non-FLAG mLptn in order to eliminate any hybridomas which were
reactive with the FLAG peptide. Those hybridomas with ELISA
reactivity to mLptn were then screened for their ability to
recognize mLptn in Western blotting. One hybridoma designated 4D8,
identified through this screening procedure, exhibited strong
reactivity with mLptn by ELISA but did not score with the Bovine
Serum Albumin protein control (FIG. 1A). Furthermore, 4D8 reacted
with purified recombinant mLptn by Western blotting but was
unreactive with full-length hLptn (FIG. 1B). Taken together, these
data indicate that the 4D8 mAb specifically recognizes mLptn.
EXAMPLE 3
[0065] Intraperitoneal Injection of Lptn Causes an Influx of
Cells
[0066] The in vitro chemotactic activity of mLptn on murine
thymocytes and mature murine T lymphocytes has been described. In
order to confirm this activity in vivo, mLptn was injected into the
peritoneal cavity of female CB6F1 mice. Initially, doses of 1 .mu.g
and 5 .mu.g of Lptn were used, however, no effect was observed
following injection of 1 .mu.g and the effects were marginal with 5
.mu.g. A strong influx of cells into the peritoneum was observed,
however, when 10 .mu.g of Lptn were injected. This response was
evident at 24 hours, but had largely disappeared by 72 hours
post-injection. This cellular influx into the peritoneum was not
observed in control animals injected with an endotoxin-matched (10
pg LPS) PBS control. Furthermore, this effect was abolished when
0.5 mg of the anti-mLptn mAb 4D8 was co-injected with 10 .mu.g
mLptn.
EXAMPLE 4
[0067] NK Cells and T Cells are Attracted by Lptn in Vivo
[0068] Cells recovered from the peritoneum of mice injected 24
hours previously with mLptn were analyzed by FACS to determine what
type of cells were attracted. The cells recovered from the
mLptn-injected mice showed a marked increase in the percentage of
cells matching the known scatter profile of lymphocytes. This
population was found to be significantly enriched for T cells
(CD3.sup.+NK1.1.sup.-), as was expected. Surprisingly this
population was also enriched for NK cells (NK1.1.sup.+CD3.sup.-).
Although the percentage of B cells (identified as
CD19.sup.+CD3.sup.- cells) in the peritoneal lymphocyte population
was reduced, the absolute number of B cells, based on the
percentages obtained by FACS and the total number of cells
recovered from the peritoneum, was not significantly affected. The
absolute numbers of NK cells in the peritoneum of Lptn-injected
mice increased about 5-fold while the absolute number of T cells
increased about 2-fold. In some experiments the CD4:CD8 phenotype
of the CD3.sup.+ cells was also examined, however, the relative
ratio of CD4:CD8 (approx. 1.2:1) observed in the Lptn-injected mice
as compared to control animals was not significantly different (see
Table 1). In contrast, the absolute number of macrophages
(Mac-1.sup.+ cells) present in the peritoneum did not show a
significant change in mLptn-injected animals.
[0069] This finding is confirmed, e.g., in Giancarlo, et al. (1996)
Eur. J. Immunol. 26:3238-3241; and Maghazaci, et al. (1997) FASEB
J. 11:765-774.
EXAMPLE 5
[0070] Subcutaneous Injection of mLptn Attracts T Lymphocytes
[0071] We sought to confirm the ability of Lptn to attract T
lymphocytes in vivo by utilizing a subcutaneous injection model. In
this model, mice were injected in one footpad with a PBS control,
while the other footpad received an injection of 1 .mu.g of mLptn.
The animals were sacrificed after 24 h, the footpads excised, and
analyzed by immunohistochemistry. Serial sections from the
PBS-injected footpads showed no significant staining with the
anti-CD3.sub..epsilon. mAb used while the footpads receiving mLptn
showed numerous cells with anti-CD3.sub..epsilon. reactivity.
Interestingly, at no time did any animal show signs of inflammation
in either the PBS- or mLptn-injected footpads.
EXAMPLE 6
[0072] Lymphotactin Attracts NK Cells in Vitro
[0073] In vitro microchemotaxis assays were performed on purified
murine NK cells in order to confirm their in vivo
Lptn-responsiveness that had been observed for these cells. Murine
NK cells (NK1.1.sup.+Gr-1.sup.-) were isolated from Rag-1.sup.-
deficient mice in order to insure that contamination of the NK
cells by other lymphocyte populations, particularly
NK1.1.sup.+CD3.sup.+ T cells, would not be a concern. The freshly
isolated NK cells were either tested immediately in the
microchemotaxis assay or were activated with IL-2 for 5 days and
then examined for chemotaxis to mLptn. The IL-2 activated NK cells
were responsive to Lptn in vitro with a peak response around
10.sup.-8 M. Freshly isolated NK cells, however, were found to be
unresponsive to mLptn in these assays. See also, e.g. Giancarlo, et
al. (1996) Eur. J. Immunol. 26:3238-3241; and Maghazaci, et al.
(1997) FASEB J. 11:765-774.
EXAMPLE 7
[0074] Chemotaxis of Human NK Cell Clones
[0075] Because murine NK cells were chemotactic in response to Lptn
it was useful to determine whether human NK cells would be
similarly responsive. To address this question, several human NK
clones were examined for their ability to respond to hLptn in the
microchemotaxis assay. Some NK clones were found to respond very
well while others showed little or no response. The clones which
did respond displayed a similar dose-response to that observed with
murine NK cells, with a peak response around 10.sup.-8 M.
Altogether, two of seven human NK clones examined showed
significant Lptn responsiveness (i.e., a chemotactic index of 2 or
more).
EXAMPLE 8
[0076] NK cells Produce Lymphotactin
[0077] Since NK cells were found to respond to Lptn, it was of
interest to determine whether or not these cells could also make
Lptn. In order to demonstrate that murine NK cells express Lptn,
the 4D8 anti-mLptn mAb was used for intracellular staining. While
no staining of IL-2 activated murine NK cells was detected with an
tso isotype control antibody, intracellular staining with the 4D8
mAb was easily detected. Since murine Lptn mRNA had previously been
found to be expressed in murine
.alpha..beta.TCR.sup.+CD4.sup.-CD8.sup.- thymocytes, the A3.2
.alpha..beta.TCR.sup.+CD4.sup.-CD8.sup.- thymocyte hybridoma was
examined as a control for expression of Lptn. A3.2 cells which had
been activated on solid phase anti-CD3.sub..epsilon. for 3 hours
showed a pattern of intracellular staining similar to that of the
IL-2 activated murine NK cells. The isotype control for these cells
was similarly negative. Neither freshly isolated NK cells nor
unactivated A3.2 cells showed staining for Lptn.
[0078] Human NK clones were also examined for expression of Lptn.
Since no antibody to hLptn is presently available, a panel of human
NK clones were analysed for expression of Lptn message. Total RNA
was extracted from a series of human NK clones and subjected to
Northern blotting with a probe corresponding to the coding sequence
of human Lptn. A signal of the appropriate size was observed in
each of the clones examined, although the intensity of the signal
varied from clone to clone. An actin probe, used as a control,
demonstrated equivalent mRNA loading.
EXAMPLE 9
[0079] Carboxyl-terminal Truncated Lymphotactin Lacks Chemotactic
Activity
[0080] As discussed above, Lptn has an unusually long
carboxy-terminal tail extending approximately 22 amino acids beyond
the last amino acid of most C--C chemokines. A truncated version of
human Lptn, lacking the carboxy-terminal 22 amino acids (CT-hLptn),
was produced as part of an effort to understand the structure of
lymphotactin. We took advantage of the existence of this truncated
molecule in order to investigate the role that the tail plays in
the activity of Lptn. The activity of the CT-hLptn protein on human
PBLs was compared with that of full length hLptn in the
microchemotaxis assay. The truncated protein was found to lack
detectable activity (FIG. 9A) when compared with full-length hLptn.
Human and mouse Lptn have considerable similarity at the amino acid
level, particularly in the carboxy-terminal 22 amino acids, so the
ability of hLptn to attract murine lymphocytes was also
investigated. For this series of microchemotaxis assays murine
splenocytes were utilized which have previously been shown to
respond to Lptn. Full length hLptn was found to attract murine
splenocytes although the dose response was shifted, with a peak at
10.sup.-7 M hLptn, as compared to the 10.sup.-8 M peak obtained
with mLptn. The CT-hLptn lacked chemotactic activity for murine
splenocytes, in accordance with the findings obtained using human
PBLs.
[0081] The ability of Lptn to attract lymphocytes in vitro has been
previously reported. It is, however, important to study the effects
of chemokines in vivo. Here, a novel activity for lymphotactin is
described, namely the ability to attract NK cells, and confirm in
vivo that Lptn is capable of recruiting T lymphocytes.
Intraperitoneal injection of Lptn causes a significant influx of NK
cells as well as T lymphocytes. This influx of T and NK cells was
specific as it was inhibited by co-injection of the anti-mLptn mAb,
4D8. Similarly, subcutaneous injection of Lptn causes an influx of
T cells, confirming that this protein has the T cell chemotactic
properties in vivo that have been observed in vitro.
[0082] The observation that Lptn chemoattracted NK cells in vivo
was an unexpected result. NK cell chemotaxis in vitro using
microchemotaxis assays was demonstrated. Interestingly, freshly
isolated NK cells fail to respond to mLptn in vitro, while
activated NK cells were found to respond very well. NK cells
recovered from the peritoneum of mLptn-injected mice were evaluated
for expression of Ly49 and CD69, two markers associated with NK
activation, but no significant expression of these molecules was
observed, suggesting that these cells were not activated. Other
investigators have obtained similar in vitro results with freshly
isolated versus IL-2 activated NK cells. See, e.g., Allavena, et
al. (1994) Eur. J. Immunol. 24:3233-3236; and Maghazachi, et al.
(1994) J. Immunol. 153:4969-4977. Taub et al. (1995) J. Immunol.
155:3877-3888, however, have reported that fibronectin coating of
the porous filters utilized in the microchemotaxis assays allows
migration of freshly isolated NK cells. Another possible
explanation for the increase in peritoneal NK cells is that Lptn is
expanding a pool of NK cells already present in the peritoneum. No
effect of Lptn on proliferation of NK, either in the presence or
absence of exogenous IL-2, was observed, suggesting that this is
unlikely. Taken together, these observations strongly suggest that
Lptn recruits NK cells in vivo.
[0083] It was also investigated whether human NK cells might be
similarly responsive to Lptn. Some, but not all, human NK clones
were found to respond to hLptn in microchemotaxis assays. This
further confirms the NK cell response to Lptn and demonstrates that
human Lptn exhibits similar properties to murine Lptn. The varying
ability of human NK clones to respond to lymphotactin may reflect
clone-specific differences in receptor expression or in the
relative mobility of particular clones. Alternatively, since these
clones are maintained in culture by constant restimulation, their
ability to respond to chemotactic stimuli may depend on how
recently they had been stimulated.
[0084] Although a number of chemokines have been reported to
attract NK cells in vitro, this may be the first report
demonstration that a chemokine is capable of attracting NK cells in
vivo.
[0085] In addition to the NK cell chemotactic response to
lymphotactin, it was also found that both IL-2 activated murine NK
cells and human NK cell clones express Lptn protein or Lptn
message, respectively. This raises the possibility that an
activated NK cell could recruit other NK cells or T lymphocytes to
the site of an infection or into a tumor mass. Indeed, Lptn message
has been reported to be rapidly up-regulated in activated T cells
and here it is observed that protein can be expressed by the A3.2
thymocyte hybridoma within three hours of activation, indicating
that Lptn protein is produced shortly after the appearance of its
mRNA.
[0086] As previously mentioned, one of the structural features
which distinguishes Lptn from other chemokines is the unusually
long carboxy-terminus of Lptn, that is highly conserved (68.2%
identity in the last 22 amino acids) between mouse and human
proteins. It was investigated whether this extended "tail" was
necessary for Lptn's function by examining the activity of a
carboxy-terminal truncated version of the human protein. This
truncation removes the carboxy-terminal 22 amino acids of Lptn and
results in a protein that is similar in size to other chemokines.
The ability of CT-hLptn to cause chemotaxis of human peripheral
blood lymphocytes was evaluated. In contrast to the full-length
hLptn, which showed clear chemotactic activity for human PBLs, the
CT-hLptn was completely inactive. Similarly, while the full-length
hLptn was active on murine splenocytes, the truncated version was
once again inactive. The results of these experiments demonstrate
that the carboxy terminus of Lptn is indeed critical to at least
these functions. This is in contrast to the data obtained with the
murine chemokine JE, which also possesses an extended C-terminus.
Truncation of the extended tail of that protein does not
significantly affect is biologic activity. One possible explanation
for these differences is that the C-terminus is directly or
indirectly involved in binding to the Lptn receptor. Another
possibility is that because Lptn lacks two of the four cysteine
residues normally found in chemokines (and must therefore also lack
one of the two disulfide linkages present in most C--C and C--X--C
chemokines) the extended carboxy-terminal tail is needed to
stabilize the protein's structure. Both the C--C and C--X--C
chemokines have been shown to possess a carboxy-terminal
.alpha.-helix. See, e.g., St. Charles, et al. (1989) J. Biol. Chem.
264:2092-2099; Clore, et al. (1990) Biochemistry 29:1689-1696; and
Baldwin, et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88:502-506. It
is reasonable to believe that the truncation of the
carboxy-terminal 22 amino acids disrupts the predicted
carboxy-terminal o-helix of lymphotactin and similarly destroys its
biologic activity.
[0087] Previous work has also demonstrated that amino-terminus of
the C--C and C--X--C chemokines is very sensitive to modification
and that even minor changes can result in the loss of their
chemotactic activity. See Zhang, et al. (1994) J. Biol. Chem.
268:15918-15924; and Gong and Clark-Lewis (1995) J. Exp. Med.
181:631-640. In contrast to this, the murine Lptn with a
amino-terminal FLAG sequence is active. This contrasts with the
C--C chemokine MCP-1, which loses biologic activity if an
amino-terminal FLAG is added to its sequence. In this report
another version of mLptn with an amino-terminal methionine residue
is active in vitro and in vivo. These data suggest that the
biologic activity of Lptn is retained even after alterations in its
amino-terminus. The structures of the full length and
carboxy-terminal truncated hLptn are currently being determined and
this information will certainly shed some light on the
structure-function relationship of lymphotactin.
[0088] Lymphotactin is an important chemokine, not only because of
its unique structure and chromosomal localization, but also because
it represents, with the possible exception of the monokine induced
by interferon-.gamma. (Mig) (Liao, et al. (1995) J. Exp. Med.
182:1301-1314) the only lymphocyte-specific chemokine. The present
invention shows that the lymphocyte specificity of Lptn is observed
in vivo. The characterization of Lptn in vivo has also uncovered a
novel activity for Lptn, namely chemoattraction of NK cells, which
in turn suggests the possibility for anti-viral and anti-tumor
effects. The data discussed herein shows that the NK activity for
Lptn observed in mice also applies to hLptn and human NK cells. The
development of a neutralizing anti-Lptn mAb is also described.
1TABLE 1 The CD4 and CD8 phenotype of CD3.sup.+ peritoneal
lymphocytes. The percent CD4.sup.+ or CD8.sup.+ cells was
determined by FACS analysis of peritoneal lymphocytes harvested
from PBS injected control mice or Lptn-injected mice and gated on
CD3+ cells. Cells were stained with FITC-conjugated CD4,
PE-conjugated CD8, and biotin-conjugated CD3. Bound biotin-CD3 was
detected with streptavidin-TriColor .TM.. CD4:CD8 PBS Lptn
Experiment 1: 58.2%:40.2% 48.3%:42.9% Experiment 2: 51.4%:45.7%
52.1%:44.3%
EXAMPLE 10
[0089] Lymphotactin and Tissue Rejection; Graft vs. Host
Disease
[0090] The expression pattern of Lptn is highly specific. The cells
that produce Lptn include: NK cells, dendritic epidermal
.gamma..delta. T cells, and class I restricted T cells. Dendritic
cells may produce Lptn as well. Of this list, by far the most
important producers, in terms of numbers, are the class I
restricted T cells. Furthermore, Lptn is a very early and important
product of these cells following activation. These cells play a
critical role in various immune responses, including immunity to
tumors as well as organ rejection.
[0091] The cells recruited by Lptn in vivo have been studied. These
include what are likely to be subsets of CD4 and CD8 T cells, but
the biggest change (as a percentage) is observed in the NK
compartment, where there is a 5-10 fold increase in the number of
NK cells. Taken together, these observations imply that Lptn likely
plays an important role in the modulation of these responses. As
such, it is likely that: (a) Lptn constitutes an early recruiting
signal for class I restricted T cells, as well as for NK cells.
Thus, Lptn would be necessary for the development of these immune
reponses, since failure to recruit these cells would abrogate them.
(b) Interference with this normal early signaling mechanism would
result in a significant reduction in the magnitude of these immune
responses. One important process where early activation of class
I-restricted T cells, as well as NK cells, is critical is in the
process of tissue or organ rejection, or in graft vs. host disease.
Thus, it is likely that neutralzation of Lptn, e;.g., using a
monoclonal antibody antagonist or a mutein antagonist, will result
in modulation of the fate of the transplant across MHC differences.
(c) The presence of Lptn in a tumor would result in the recruitment
into the tumor mass of cells capable of destroying the tumor cells,
including CD4 and CD8 T cells as well as NK cells.
EXAMPLE 11
[0092] Hematopoietic Effects of Lymphotactin
[0093] Lin.sup.- Sca-1.sup.+ rhodamine.sup.lo c-kit.sup.+ stem
cells were isolated. These cells were incubated for at 37.degree.
C. and 5% CO.sub.2 in 1.5 ml eppendorf tubes at 400 cells per 400
.mu.l. Growth factors, alone or in appropriate combinations, were
added to the Iscove's Modified Dulbecco's Medium (IMDM)+15% fetal
bovine serum (FBS)+penn-strep+HEPES at a final concentration of
50-100 ng/ml, in the 400 .mu.l/tube.
[0094] At day 7, an aliquot of each culture which is 1/4 of the
input volume is withdrawn and the culture is refed with 200 .mu.l
of growth factors in IMDM+15% FBS. 5 .mu.l is withdrawn from the
sample and counted.
[0095] The remaining cells were diluted with IMDM+15% FBS and
underlayed with FBS. The culture was spun at 1000 RPM for 5 min.
The supernate is aspirated and the cells resuspended in 100 .mu.l
IMDM+15% FBS.
[0096] CFU-c assays were set-up to contain IL-3+IL-6+SCF+epo. The
cells from each delta culture were added to a single set-up along
with methylcellulose (final conc 0.8%). The contents of each set-up
were mixed with a 3 ml syringe and 16 Ga needle and plated into
three 35 mm pertri dishes. Cultures were incubated at 37.degree. C.
and 5% CO.sub.2 for 7 days and colonies were enumerated.
[0097] Harvest of 1/4 of the culture is repeated on day 14, day 21,
and day 28.
[0098] The effects of lymphotactin, alone or in combination with
MIP-1.epsilon., Flt3 ligand, Stem Cell Factor, IL-6, and/or IL-3
are shown in FIGS. 10 (day 7), 11 (day 14), and 12 (day 21). These
data show that lymphotactin, in combination with other factors,
will have the effect of preventing hematopoietic stem cell from
entering cycle and producing colonies at early time points, e.g.,
day 7, but will allow later colony formation, e.g., day 21. This
data indicates the ability of lymphotactin to protect hematopoietic
stem cells from damage by chemotherapeutic reagents by preventing
their entry in cycle and the resultant cellular proliferation and
differentiation for up to 21 days. The colony formation seen at day
21 indicates that the progenitors remained quiescent rather than
dying during the initial culture period.
[0099] All references cited herein are incorporated herein by
reference to the same extent as if each individual publication or
patent application was specifically and individually indicated to
be incorporated by reference.
[0100] Many modification an variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only, and the
invention is to be limited only by the terms of the appended
claims, along with the full scope of the equivalents to which such
claims are entitled.
[0101] Although the present invention has been described in some
detail by way of illustration and example for purposes of clarity
and understanding, it will be recognized that certain changes and
modifications may be practiced within the scope of the claims.
* * * * *