Note: Descriptions are shown in the official language in which they were submitted.
CA 0222l234 l997-ll-l4
W O9~'3Ç358 PCT/u~ 01
COMPOSITIONS A~D ~ n~S
FOR INHIBITING XENOGRAFT REJECTION
I. BACKQROU~T~ OF T~F~ I NV1':N~1 lON
Organ transplantation is becoming an increasingly
effective medical therapy for the long-term treatment of
many otherwise fatal diseases. Prior to 1980, the major
limitation to routine transplantation as a surgical
procedure was that of organ rejection. In recent years,
however, the development of powerful new immunosuppressive
drugs has significantly improved organ graft survival. The
major factor currently acting to prevent those who need a
transplant from getting one is an acute shortage of human
donor organs. Those who do receive transplants are having
to wait longer to find a suitable match, compromising their
chances for optimum results, and sometimes, survival. This
shortage of donor tissue limits the application of
transplantation to a small proportion of patients who could
be expected to benefit from the operation and produces
higher health care costs because of the re~uirement for
continued intensive care of patients with chronic, terminal
diseases.
One practical solution to the shortage of human donors
is the use of organs from non-human donors. The ~ch~nge of
tissues between two different species ("xenogeneic
transplant"), however, results in a rejection reaction that
is more aggressive than that observed for the
transplantation of tissue between members of the same
species ("allogenic transplant~). In addition, this
reaction to xenografts has not responded well to the
traditional ;mmllnosuppressive treatment which is effective
to prevent loss of allograft, providing a clear suggestion
that the ;mmnnologic mechanism of rejection is different in
xenogeneic transplants than it is in allogenic transplants.
CA 02221234 1997-11-14
W 096136358 PCT~US~G/06r~1
A failure to completely understand the immune mechanisms
responsible for the rejection of xenografts has inhibited
the development of more effective treatment regimens.
A. The Rejectinn o:E ~e~og~n.~;c Tr:~n~l~nts T~3
~h~act~i7~ s~ ~ e~cl~te or Accelers~t~c~
Transplantation is by definition the placement of
foreign tissue ("donor") into a host recipient, thus an
immunological attack on the foreign tissue ("rejection") is
to be expected. The vigor with which this immunological
assault is mounted has been correlated with the genetic
disparity of the donor/recipient species. The greater the
genetic disparity, the swifter and more acute the
immunological rejection. Accordingly, the rejection of
xenografts is typically more vigorous than allografts.
Xenograft rejections have been classified into two general
categories on the basis of this genetic disparity of the
donor/recipient species and the rapidity of the graft
rejection as compared to allograft rejection untreated
recipients.
1. Hy~erAcl1te Reject;on ;s ~hAracter;st;c
of Tr~n.sDlAnts Retwee~ Discor~Ant
Species
In general, recipients of xenografts from genetically
distant ("discordant") donors react by mounting a rapid,
hyperacute rejection of the foreign graft. This form of
rejection is seen with pig-to-human xenogeneic transplants
and is associated with the loss of the xenograft within a
few minutes or hours. This type of xenograft rejection has
proven to be more difficult to control therapeutically than
the accelerated rejection characteristic of xenogeneic
transplants between concordant species, discussed below.
.
CA 02221234 1997-11-14
W 096/36358 PCTAUS96/06804
Therefore, these species combinations have been less
attractive as potential organ donors for hllmAn.~
Generally, the literature reports two hypothesis for
the pathogenesis of the hyperacute rejection. Some suggest
that anti-xenograft antibodies are produced by the recipient
prior to the xenogeneic transplant. These preformed
antibodies are believed to precipitate the violent,
hyperacute rejection of the xenograft as a result of the
immediate and diffuse deposition of these antibodies in the
graft and activation of the complement/coagulation systems.
The other commonly held hypothesis is that the alternative
complement pathway is directly activated by antigens
expressed by the xenograft in some donor/recipient species
combinations. Although there is some evidence in the
literature to support each of these hypothesis, none of the
evidence has proven sufficient to provide meaningful
prevention or intervention in the immune response which
results in hyperacute rejection of xenografts.
2. Accelerated Reject;on ;s ~hAr~cter;st;c
of X~no~rAfts ~etween Concor~Ant S~ec;es
In contrast, grafts that are ~xchAnged between more
closely-related ("concordant") species are rejected in an
accelerated, but not immediate, reaction. This form of
rejection is seen with baboon-to-human xenogeneic
transplants and is associated with the 1088 of the xenograft
within a few (2-4) days of transplant.
Accelerated reactions were originally considered to
represent an aggressive form of acute allograft rejection
directed at a different species. See, Calne R Y.,
T~An~pl~nt Proc. 2:550-553 (1970). However, it has
recently been reported that the accelerated rejection of
xenografts is due to an antibody-mediated rejection of the
donor graft that requires a period of a few days to result
CA 02221234 1997-11-14
W 096136358 PCTrUS~F'~6U01
in sufficient levels of antibody to cause the graft to fail.
See, Cosenza et al., J. He~rt Tnng Tr~n.s~l~nt, 13:489-497
(1994).
Humans, for example, do not exhibit high levels of
anti-xenograft antibodies to closely-related species, such
as baboons and other non-human primates, prior to a
xenogeneic transplant. Thus, these species have served as
the preferred donors of xenograft for human. The implicit
assumption is that the low level of preformed anti-xenograft
antibodies in a concordant reaction leads to the development
of a rejection reaction that may be more easily managed with
traditional forms of immunosuppressive therapy. Although
the rejection of the xenograft is mediated by the rapid
production of anti-xenograft antibodies post-transplant,
there is a small window of opportunity to intervene in the
immune response before the rejection of the xenograft is
complete.
3. Com~r~tive P~thogenes;s of the Hy~er~cllte
~n~ Acceler~te~ Reject;on of X~nog~ne;c
T;sslle
Rejection of both discordant and concordant xenografts
is associated with evidence of vascular damage, rather than
the extensive accumulation of inflammatory cells that is
characteristic of T cell-mediated reactions. In those cases
of xenogeneic transplants into hllm~n.q or experimental
animals when sequential ~m;n~tion has been conducted, the
pathological changes present in the xenografts are
consistent with antibody-mediated damage to the vessels of
the grafts. See, Rosengard, B.R., et al. J. Heart
Tr~n.q~l~nt, 5:263-266 (1986); Bailey, L. et al. J. Am. Me~.,
254:3321 (1985); Bogman, M.J.J.T., et al., Am.J.P~thol.,
100:727-735 (1980).; Linn, B.S., et al., Tr~ns~l~nt. Proc.,
3:527 (1971); Ertel, W. et al., Tr~nq~lant. Proc., 16:1259-
CA 02221234 1997-11-14
W 096/36358 PCTrUS~G~
1261 (1984). The primary pathological changes include
endothelial cell swelling, necrosis, interstitial edema,
platelet and fibrin thrombi, and hemorrhage. See,
t Xenotr~n.spl~ntat;on. The Tr~ns~l~ntation of Organs ~nd
5 T;~sue ~etween Spec;es, eds. Cooper, D.K.C., Kemp, E.,
Reemtsma, K., and White, D.J.G. Berlin:Springer-Verlag,
1991, p. 181-242.
Although the exact nature of the target antigens and
the humoral response that mediate accelerated and hyperacute
10 rejection reactions have largely been unknown, including
whether the anti-xenograft antibodies are polyclonal or
polyspecific, the changes are compatible with antibody
binding to antigens expressed on the endothelium of the
donor graft vessels and activation of the complement
15 cascade. In the case of hyperacute rejection of xenografts,
the antibodies are apparently immediately available to mount
an attack which results in rejection. While, in the case of
the accelerated rejection of xenografts, the antibodies
which mediate rejection must first be manufactured.
B. A Rel iabl e Mo~l ~or X~nngeneic Tr~ pl Ant
Rejec~-; on Is ~he ~m~ter-to-rat Tr~ nt Mo~l
~,n~tion of the rejection of heart xenografts
between different rodent species has demonstrated that the
accelerated rejection of hamster hearts by rat recipients is
a reliable model of the accelerated pattern of rejection
characteristic of baboon-to-human xenogeneic transplants.
See, Cramer, D.V. et al., Tr~ns~l~nt.Proc.,25:2864-2847
(1993); Makowka, L. and Cramer, D.V., "The use of xenografts
in experimental transplantation." In: ~ndbook of ~n;m~l
Mo~els jn Tr~nsp1~nt Resear~h, eds. Cramer, D.V. Podesta, L.
and Makowka, L. Boca Raton: CRC Press, Inc. 1993, p. 299-
310. Hamster heart xenografts are rejected by naive rats
within about four days due the rapid rise in anti-xenograft
CA 02221234 1997-11-14
W 096136358 PCT/U~G/U6~04
IgM antlbodies and the humoral destruction of the graft.
See, Wu G. D., et al., Tr~n.~ nt. Proc., 24:691-692 (1992).
In this rodent species combination, like the baboon-to-human
primate species combination, preformed anti-xenograft
antibodies appear to be present at low levels in the serum
of the recipient prior to transplantation, but are
apparently not in sufficient number to produce a hyperacute
response. Antibodies produced by the rat after transplant
with hamster tissue display a pattern of reaction with heart
membrane antigens in Western blots that is similar to that
seen with the preformed antibodies. See, Cramer D. V., et
al., Transpl~ntation 54:403-408 (1992).
The primary difference between the accelerated
rejection reaction occurring in this hamster-to-rat model
and the hyperacute rejection reaction observed in pig-to-
human xenogeneic transplants appears to be quantitative
rather than qualitative: the time period required for low
levels of preformed anti-xenograft antibodies in the rat
recipient to rise to sufficient levels to cause the loss of
the hamster xenograft. As discussed above, rejection of
both hyperacute and delayed xenografts is associated with
evidence of humoral vascular damage, rather than the
extensive accumulation of inflammatory cells that is
characteristic of T cell-mediated reactions.
Specie combinations that normally are characterized by
an accelerated rejection reactions can also be shown to
display hyperacute rejection following either sensitization
of the recipient with donor tissue immunogen prior to
transplant or by passive transfer of hyperimmune sera prior
to transplant. Immunizing recipient rats with hamster
lymphocytes or hamster cardiac grafts prior to a hamster-to-
rat xenogeneic transplant results in an antibody-mediated
hyperacute rejection of hamster xenografts. These hamster
tissue immunogens reportedly stimulate a rapid rise in the
CA 02221234 1997-11-14
W O 96/36358 PCTrUS9C/O~Y0~
normally low le~els of IgM antibody that react with hamster
lymphocytes and vascular endothelium and graft rejection
follows. Likewise, passive transfer of serum from a rat
that has rejected a hamster xenograft to a naive rat results
in hyperacute rejection of a hamster xenograft by the naive
rat. See e.g., Wu, G.D., et al., Tr~ns~lant Proc. 24:691
(1992).
There are, as described in a variety o~ current
reviews, apparently other important components of the
xenografts rejection reactions, including, for example,
direct activation of the alternative complement pathway,
modification of the rejection reaction by regulatory
complement proteins following antibody binding, and the
contribution of the cellular ;mml]n~ response. See, e.g.,
Auchincloss, H., Jr. Tr~ns~l~ntation 46:1-20 (1988);
X~notr~n~ ntat;on. The Tr~n~pl~ntat;on of Org~n~ ~n~
T;ssl]e Between Species, eds. Cooper, D.K.C., Kemp, E.,
Reemtsma, K., and White, D.J.G. Berlin:Springer-Verlag,
1991, p. 69-79; A~v~nces ;n Tr~nsplantation, eds. Hackel, B.
and AuBochon, J., Bethesda, Maryland: Amer. Assoc. Blood
Banks, 1993, p. 93-112; Johnston P. S., et al., Tr~n~l~nt.
Proc. 23:877-879 (1991). While each of these pathogenetic
mechanisms may be important in some or all xenograft
rejection reactions, it is abundantly clear that the
deleterious activity of preformed antibodies against the
xenograft must be controlled, preferably without
debilitating the entire immune system, if xenografts are to
provide a meaningful solution to the problems associated
with allograft transplants. Accordingly, there has existed
a need for methods and compositions to inhibit antibody-
mediated rejection of xenografts by recipient ~n;m~
.,
CA 0222l234 l997-ll-l4
W O9G'36358 PCT~US96/OG801
II. BRIEF DESCRIPTTON OF T~ INV~:N-llON
The present invention provides novel and powerful
immunological compositions and methods for inhibiting
antibody-mediated rejection of xenografts by a transplant
recipient. Through pre-transplant treatment of the
xenograft with these immunological compositions, preferably
supplemented by post-transplant therapeutic treatment of the
xenograft recipient with these immunological compositions
and, optionally, chemical immunosuppressive agents,
prolonged survival of the graft can be achieved, providing
the first, critical step to long-term xenograft survival.
This ability to intervene in the immune response to
xenogeneic tissue opens the door to the use of xenografts as
a meaningful alternative to the shortage of available
allogeneic organs.
Thus in accordance with the present invention there is
provided methods of inhibiting rejection of a donor
xenograft by a recipient animal, comprising modifying
antigen expressed by cells of the xenograft, without causing
lysis of the cells, to inhibit binding of recipient anti-
donor xenograft antibody to said antigen, wherein said
antigen present in unmodified form induces an antibody-
mediated immune response in the recipient animal. A
preferred method of modifying such antigen, particularly
those expressed by endothelial cells of the xenograft,
comprises contacting non-lytic, anti-donor xenograft
antibody material with said antigen for a time, at a
temperature, and at a pH suitable to bind the antibody
material to the antigen. Anti-donor xenograft antibody
material also provided by the present invention is
characterized as immunoreactive with antigen expressed by
endothelial cells of the xenograft and capable of inhibiting
~ CA 02221234 1997-11-14 - -I
~ ~9~ ~ ~ 9610~8~4
4 -, ~, " .' '_ '. : ' 'i'' ."-' 19g7
antibody-mediated rejection of the xenograft by a recipient
animal.
This invention also provided, for the first time,
monoclonal antibodies that immunoreact with antigen
expressed by endothelial cells of donor xenografts and which
are capable of inducing antibody-mediated rejection of the
xenograft. These monoclonal antibodies, as well as the
polypeptides from which they are formed and the
polynucleotides which encode them, will provide researchers
with powerful and reliable reagents greatly needed in the
search to better understand and control the xenogeneic
transplant rejection reaction.
The present invention also provides methods of using
the compositions of the present invention to isolate and
further characterize the antigen(s) responsible for
precipitating xenogeneic transplant rejection reaction.
III. R~I~F D~CRIP~ION OF T~ D~WING
Figure 1 (SEQ ID NO: 34) is the nucleotide sequence of
the variable heavy chain region of the rat anti-hamster
xenograft monoclonal antibody designated HAR-l. The sequence
of the SAX oligonucleotide has been artificially included
with the 5' end of the sequence. The 5' untranslated region
appears in lower case. The boundaries for the framework and
CDR regions are deduced from conventions estabilished by
Kabat and Wu for mouse and human immunoglobulin and are
indicated above the sequence. Primers which were used to
generate this and other sequences are labeled and designated
by underline. Figure 2 shows the cDNA nucleotide sequence
of the variable heavy chain segment of the rat anti-hamster
xenograft monoclonal antibody designated HAR-l (nucleic acid
residues 52 through 427 of SEQ ID NO: 34) aligned with its
germ-line counterpart VH1.1 (SEQ ID NO: 35) obtained by
genomic amplification of newborn LEW rat liver DNA. The two
AMENDED SH~Er
CA 02221234 1997-11-14
~f~ 9 6 l ~ 6 8-~
IP~ 'AN 1997
sequences are 99.2% (351/354 nucleotides) homologous. HAR-1
cDNA sequence differs at three nucleotides from the germ-
line sequence. The first difference leads to a replacement
Leu by Val in the leader sequence whereas the 2 others are
"silent". Intron 1 untranscribed region is presented in
lower case. "*" indicates identity.
Figure 3 shows the cDNA nucleotide sequences of the
variable heavy chain region of the rat anti-porcine
xenograft monoclonal antibodies designated HA75DBFl (SEQ ID
NO: 36) and IH21H7 (SEQ ID NO: 37) aligned to demonstrate
sequence homology. "*" indicates identity.
Figure 4 shows the cDNA nucleotide sequence of the
variable heavy chain segment of the rat anti-pig xenograft
monoclonal antibody designated HA75DBF1 (SEQ ID NO: 38)
aligned with its germ-line counterpart VHRAP.la (SEQ ID No:
39) obtained by genomic amplification of newborn LEW rat
liver DNA. The two sequences are 98.6% (286/290)
homologous. HA75DBF1 cDNA differs at three nucleotides from
the germ-line sequence. All three of these differences
occur in the framework regions and are indicated by a box.
"1" indicates identity.
IV. D~TAIr~n D~CRIPTTnN OF T~ ~Nv~r-~lON
The use of organ transplants for the treatment of end-
stage diseases has become an established and highly
effective therapeutic regimen. The success of organ
transplantation, however, has resulted in a shortage of
human donor organs creating a major limitation to the more
widespread use of this technology. One of the practical
solutions to the shortage of human donors is the use of
species other than humans for organ donation. Even if only
on a short-term basis, such organs could provide temporary
life-support until a more suitable organ became available.
However, the exchange of tissues between two different
- 10
AMEND~ SHE~T
..
CA 02221234 1997-11-14
W 096/363~8 PCT/US~6/OC804
species ("xenogeneic transplant~) results in a rejection
reaction that is more aggressive than that observed for the
transplantation of tissue between members of the same
species ("allogeneic transplant"). Although the rapidity of
rejection in a xenogeneic transplant can be somewhat
lessened (accelerated instead of hyperacute) by selecting
the donor from a concordant species, there still remains a
significant risk of transmitting potentially serious
pathogens to the recipient when xenogeneic transplant is
performed between concordant species. These xenograft
rejection reaction have not responded to traditional
immunosuppressive treatment and the lack of understanding of
the immune mechanisms responsible for the loss of the graft
has inhibited the development of more effective treatment
regimens.
The present invention provides novel and powerful
immunological compositions and methods for inhibiting the
antibody-mediated component of the rejection of xenografts.
Through pre-transplant treatment of the xenograft with these
immunological compositions, preferably supplemented by post-
transplant therapeutic treatment of the xenograft recipient
with these immunological compositions, prolonged survival of
the graft can be achieved, providing the first, critical
step to long-term xenograft survival. This ability to
intervene in the immune response opens the door to the use
of xenografts as a me~n; ngful alternative to the shortage of
available allogeneic organs.
This invention also provided, for the first time,
monoclonal antibodies that immunoreact with antigen
expressed by endothelial cells of donor xenografts and which
are capable of inducing antibody-mediated rejection of the
xenograft. These monoclonal antibodies, as well as the
polypeptides from which they are formed and the
polynucleotides which encode them, are valuable tools in the
CA 02221234 1997-11-14
W 0~6/36~58 PCT~US96/06YOq
hands of researches who seek to better understand the
xenogeneic rejection reaction and to devise new compositions
and methods to overcome it, but whose efforts have been
frustrated by the lack of available and reliable reagents
with which to conduct their work.
Another highlight of the present invention are methods
of using the compositions of the present invention to
isolate and further characterize the antigen(s) responsible
for precipitating xenogeneic transplant rejection.
A. ~nti-Do~or Xenograft ~nt;ho~ies
In accordance with the present invention there is
provided isolated and substantially purified anti-donor
xenograft antibody that is immunoreactive with antigen
expressed by endothelial cells of a xenograft from a donor
animal and that are capable of inducing antibody-mediated
rejection of the xenograft by a recipient animal.
As used herein the terms "isolated," "substantially
pure," or "recombinant" in their various grammatical forms
as a modifier of proteins including antibodies and antibody
materials, polypeptides, amino acid sequences,
polynucleotides, and nucleic acid sequences or molecules
means that the proteins, polypeptides, amino acid sequences,
polynucleotides, and nucleic acid sequences or molecules so
designated have been produced in such form by the hand of
man, and thus are separated from their native in vivo
cellular environment. As a result of this human
intervention, the isolated, pure and/or recombinant,
proteins, polypeptides, amino acid sequences,
polynucleotides, and nucleic acid sequences or molecules of
the invention can be produced in large quantities and are
useful in ways that the proteins, polypeptides, amino acid
CA 0222l234 l997-ll-l4
W 096/36358 PCT/US~6/OC~01
sequences, polynucleotides, and nucleic acid sequences or
molecules as they naturally occur are not.
The terms ~antibody~ and ~antibody molecule~ in their
various grammatical forms are used herein as collective
nouns to refer to a population of immunoglobulin molecules
which may be polyclonal or, more preferably, monoclonal in
origin and which may be of any isotype, preferably of the
IgM isotype.
The term "immunoreact" in its various grammatical forms
means specific binding between an antigenic determinant-
containing molecule, such as an antigen, and a molecule
containing an antibody combining site such as an antibody
molecule or antibody material.
As used herein the term ~xenograft~ re~ers to grafted
tissue or tissue intended for use in a transplant operation
between animals, including humans, that has been derived
from a donor animal that is a different species than that of
the recipient animal or intended recipient animal of the
tissue. Typically, the tissue is organized in the form of a
critical body organ. A variety of xenografts suitable for
use in the present invention are well-known in the art, such
as kidney, heart, liver, lung, pancreas, and the like.
The term "donor animal" as used herein is a collective
noun referring to the species of animal from which the
xenograft is taken for transplant and can include, for
example, domesticated animals such as pigs, non-human
primates such as baboons, rodents such as hamsters and
rabbits, and the like. Accordingly, the xenograft, being
tissue of the donor ~n;m~l, may be further designated herein
by the type (species) of donor ~n;m~l from which the
xenograft originates, e.g. hamster xenograft.
The term "recipient ~n;m~l " is used herein as a
collective noun referring to the species of ~n;m~l which
CA 0222l234 l997-ll-l4
W 096/36358 PCTrUS~6/0~01
receives the xenograft and includes, for example, humans,
domesticated animals, primates, rodents, and the like.
As used herein the phrase "antibody-mediated rejection
of the xenograft" refers to an immunological attack on the
xenograft which is driven by humoral, as opposed to cell
specific immunity, and which is thus mediated by antibody
molecules. Antibody-mediated rejection of a xenograft can
be accelerated or hyperacute. Preferably, anti-donor
xenograft antibody of the present invention is capable of
inducing hyperacute rejection of the xenograft.
Anti-donor xenograft antibodies of either monoclonal or
polyclonal form can be produced using techniques presently
known in the art. For example, polyclonal and monoclonal
antibodies can be produced as described, for example, in
Harlow and Lane, ~ntlho~;es: A T~hor~tory M~nl~l (Cold
Spring Harbor Laboratory 1988), which is incorporated herein
by reference. Altered antibodies, such as chimeric,
humanized, CDR-grafted or bifunctional antibodies can also
be produced by methods well known to those skilled in the
art. Such antibodies can also be produced by hybridoma,
chemical or recombinant methodology described, for example
in Ausubel et al., Curr~nt Protocols ;P Molec~ r R;ology
(Greene Publishing Associates, Inc. and John Wiley & Sons,
Inc. 1993), also incorporated herein by reference.
Exemplary methods of making and isolating monoclonal anti-
donor xenograft antibodies are provided in EXAMPLES below.
The phrase l'polyclonal antibody" in its various
grammatical forms refers to a population of antibody
molecules that contains more than one species of idiotope
capable of immunoreacting with epitopes on a particular
antigen. Polyclonal antibody of the present invention
specifically includes a mixture of more than one monoclonal
antibody that immunoreact with different epitopes on the
same antigen.
14
CA 0222l234 l997-ll-l4
W O~13~ PCTrUS~ 01
The phrase "monoclonal antibodyll in its various
grammatical forms refers to a population of antibody
molecules that contain only one species of idiotope capable
of immunoreacting with a particular epitope on an antigen.
A monoclonal antibody typically displays a single binding
affinity for an epitope with which it immunoreacts; however,
a monoclonal antibody may be a molecule having a plurality
of idiotopes, each immunospecific for a different epitope,
e.g., a bispeci~ic monoclonal antibody.
Monoclonal antibodies are typically composed of
antibodies produced by clones of a single cell called a
hybridoma that secretes (produces) but one kind of antibody
molecule. In accordance with the present invention
hybridomas capable of producing antibodies and antibody
materials having specific immunoreactivity with antigen
expressed by endothelial cells of a xenogra~t from a donor
animal is provided and described in greater detail below.
One of skill in the art will recognize that the hybridomas
disclosed herein can be used to produce other immortal cell
lines that produce antibody and antibody material of the
present invention.
A hybridoma cell is formed by fusing an antibody-
producing cell and a myeloma or other self-perpetuating cell
line. The preparation of such hybridomas was first
described by Kohler and Milstein, N~ture, 256:495-497
(1975), which description is incorporated by reference.
Polypeptide-induced hybridoma technology is also described
by Niman et al., Proc. N~tl. Sci. U.S.A., 80:4949-4953
(1983), which description is also incorporated herein by
reference.
To obtain an antibody-producing cell for fusion with an
immortalized cell, an animal can either be transplanted with
a xenogra~t from a donor animal or inoculated with a
xenogeneic immunogen. If the transplant technique is used,
CA 02221234 1997-11-14
W O9~/36358 PCTrUS~ C~0~
preferably, the animal receiving the transplant is of the
same species as the recipient animal.
The term "xenogeneic immunogen" in its various
grammatical forms is used herein to describe a composition
containing donor endothelial cell antigen as an active
ingredient used for the preparation of the antibodies
against antigen expressed by endothelial cells of a
xenograft from a donor animal. The amount of immunogen used
to inoculate the mammal should be sufficient to induce an
immune response to the immunizing antigen. This amount
depends, among other things, on the species of animal
inoculated, the body weight of the animal, the source and
form of the donor endothelium antigen in the immunogen, and
the chosen inoculation regimen as is well known in the art.
Antibody-producing cells, e.g. splenic lymphocytes,
- are harvested from the immunized animal, or the transplanted
animal after rejection of the xenograft, and can be fused
with myeloma cells using polyethylene glycol ("PEG"). Fused
hybrids are selected by their sensitivity to hypoxanthine,
aminopterin and thymidine selection medium ("HAT").
A monoclonal antibody of the present invention can also
be produced by initiating a monoclonal hybridoma culture
comprising a nutrient medium containing a hybridoma that
secretes anti-donor xenograft antibody molecules. The
culture is maintained under conditions and for a time period
sufficient for the hybridoma to secrete the antibody
molecules into the medium. The antibody-containing medium
is then collected. The antibody molecules can then be
further isolated by well known techniques.
Other methods of producing a monoclonal antibody, a
hybridoma cell, or a hybridoma cell culture are also well
known. See, for example, the method of isolating monoclonal
antibodies from an immunological repertoire as described by
Sastry et al., Proc. N~tl. Ac~. Scl., 86:5728-5732 (1989);
CA 0222l234 l997-ll-l4
W 096136358 PCT/U~ 6~01
Huse et al., Sc;ence, 246:1275-1281 (1981); and
International Patent Application No. PCT/US92/03091 all of
which are incorporated herein by reference.
Anti-donor xenograft antibody can be identified by
screening for the presence of antibody molecules that
immunoreact with antigen expressed by endothelial cells of
the donor animal and which are capable of inducing antibody-
mediated rejection of the xenograft. Screening methods for
such immunoreactivity can take any one of several commonly
used immunoassay formats, including for example,
radioimmunoassay (RIA), enzyme linked immunosorbent assay
(ELISA) or immunofluorescent assay. The source of antigen
in these immunoassays is endothelial cells or endothelial
cell-containing tissue sections from a donor ~n;m~l . More
preferably, the source of antigen for the immunoassay is
endothelial cells or endothelial cell-containing tissue
sections from the same type of tissue as the xenograft and,
most preferably, from the same individual animal that
donates the xenograft. An antibody is considered to
"immunoreact with antigen expressed by endothelial cells of
the donor animal" when binding by the test antibody exceeds
binding detected in the negative control by at least about
two times, preferably by at least about 2.5 times, and even
more preferably by about three times, particularly when flow
cytometric assay is employed. The negative control for such
assays is PBS or more preferably autologous serum. A
presently preferred method for screening for
immunoreactivity is described with greater detail in the
flow cytometry assay provided in the EXAMPLES below.
Screening methods to identify antibodies having the
ability to induce antibody-mediated rejection of the
xenograft include in vitro cytotoxicity assays such as or
example, flow cytometric cytotoxicity assay or MTT
cytotoxicity assay, and in vivo rejection experiments using
CA 02221234 1997-11-14
W Og~/36358 PCTrUS96~~0C~0~
animal models. In an in vitro format, antibody-mediated,
complement-dependent cytotoxicity indicates that antibody
tested has the ability to mediate rejection of the donor
xenograft by the recipient animal. The source of antigen
for the cytotoxicity assays can be as described above for
the immunoreactivity assays, or other whole cells from the
donor animal can be used. Preferably, the source of
complement is animal serum which produces low background,
e.g. Low Tox~ rabbit or mouse serum (Cedar Lane
Laboratories, Hornby Ontario, Canada). Preferably, as
measured in a flow cytometric cytotoxicity assay, antibody
is capable of inducing antibody-mediated rejection of a
xenograft by a recipient animal when more than about 20
cell death is detected. A presently preferred flow
cytometric cytotoxicity assay for detecting the ability of
antibody to induce antibody-mediated rejection of a
xenograft by a recipient animal is described with greater
detail in the EXAMPLES below. One of skill in the art will
appreciate that the assay described in Examples can easily
be adapted for specie combinations of interest.
Alternatively, in vivo rejection experiments can be
employed, where appropriate, to detect the capability of
antibody to induce antibody-mediated rejection of a
xenograft by a recipient An;mAl First, the median survival
times of a xenograft for the recipient animal must be
characterized, preferably, the mean rejection time of a
xenograft following pre-transplant passive transfer of
hyperimmune serum to a recipient animal is also determined.
A recipient animal then receives by passive transfer an
inoculation of the putative antibody. The quantity of
inoculum depends on the several factors such as the type and
weight of the recipient An;mAl, Transplant of the xenograft
is performed on the pre-treated recipient and xenograft
survival time is determined. Rejection is considered to
CA 02221234 1997-11-14
W 096/36358 PCT~US~G~'~C~
have occurred when the xenograft fails. An antibody is
capable of inducing antibody-mediated rejection of the
xenograft by the recipient if pre-transplant, passive
transfer of the antibody results in accelerated, or more
preferably hyperacute, rejection of a subsequently
transplanted xenograft. Histological ~m; n~tion of the
rejected xenograft can be performed to confirm that the
rejection was antibody-mediated.
Since the hyperacute rejection of hamster tissue by
sensitized rats and rats that have received passive transfer
of hyperimmune serum are regarded as reliable models of the
pattern of xenograft rejection in humans, these hamster-to-
rat transplant models were used to generate anti-donor
xenograft antibodies of the present invention and exemplify
screening techniques.
1. Xenograft Reject;on ;n the Rat Mo~el
As a point of reference and as reported in Table 1, a
series of experiments were conducted to quantify the
characteristic allograft rejection in naive LEW rats which
is generated by transplant between the same species ("ACI
rat --> LEW rat"), the characteristic accelerated
xenograft rejection in naive LEW rats which is generated by
transplant between concordant species ("Hamster --~ LEW
rat"), the characteristic hyperacute xenograft rejection
pattern in naive LEW rats which is generated by transplant
between discordant species ("Guinea pig --> LEW rat"), and
the characteristic hyperacute rejection pattern in LEW rats
which is generated by passive transfer of hYperimmune Lat
~erum followed by transplant between concordant species
("Hamster --> HRS+LEW rat"). The heterotopic cardiac
transplantation procedure was followed for all transplants.
19
CA 02221234 1997-11-14
W 096/36358 PCTrUS9~/OC80
Table 1. Cardiac Xenograft Survival in Rat Model
Group N Graft Survival
(Mean + 1 S.D.)
Allograft-type Rejection
ACI rat --~ LEW rat 5 7.0 + 0.5 days
Accelerated Rejection
Hamster --> LEW rat 5 3.9 + 0.2 days
Hyperacute Rejection
Guinea pig --> LEW rat 5 14.8 minutes
Hamster --> HRS + LEW rat 5 14.0 minutes
Naive LEW rats received passive transfer of hyperimmune rat
serum by intravenous injection of about 0.1 to 0.5 ml sera.
Rejection was considered to have occurred when the
xenogeneic heart stopped beating.
"Hyperimmune serum," as that term is used herein,
refers to sera from an animal that has rejected a xenograft
originating from the donor animal. Preferably, the
hyperimmune serum is from the same species of ~nl m~l as the
recipient ~n;m~l on which the in vivo rejection experiment
is to be performed. Hyperimmune serum may be further
designated herein by the ~nlm~l from which it was derived,
e.g., hyperimmune "rat" serum.
CA 02221234 1997-11-14
W O 961363~8 PCTrUS96/06804
2. The Ham.~ter-to-~t Mo~el M;m;cs the Humor~l
Component of x~nogra~t Reject;on ;n Humans
These transplant experiments also confirmed that the
- accelerated rejection by the rat of the hamster xenografts
is closely associated with the production of rat anti-
hamster IgM antibodies. Using an ELISA to detect the
immunoreactivity of serum antibodies it was demonstrated
that prior to transplantation, the serum of naive rats
contained a small amount of preformed IgM antibody
(detectable only at low dilutions, e.g., 1:2 to 1:4) that
bound to the endothelium of the normal hamster heart and
hamster lymphocytes. After transplantation, the total
amount of serum IgM (but not IgG) antibody rose rapidly
until rejection of the hamster heart xenograft at about Day
4 post-transplantation. Total IgM levels at about Day 4
post-transplant were ~ 2.4 mg/ml as quantified by
immunoprecipitation and compared to standards. This rise in
total IgM was paralleled by a rapid rise in the rat
recipient serum of IgM antibody that reacts exclusively with
the vascular endothelium of normal hamster hearts and normal
hamster splenic lymphocytes.
In addition, the Western blot binding patterns
conducted against hamster heart proteins using the pre- and
post-transplant sera in accordance with the EXAMPLES below
are strikingly similar. Serum from naive LEW rats detects
multiple (~8) protein bands by Western blot analysis of
hamster heart proteins. Rejection of the graft at 4 days
post-transplant is associated with serum antibodies that
recognize a similar pattern of Western blot protein bands,
most with greater intensity, reflecting a higher level of
antibody post-transplant, but with the same pattern of
reactivity. In addition, histopathological lesions seen in
the hamster-to-rat cardiac xenografts were consistent with
an acute, IgM antibody-mediated vascular rejection.
CA 02221234 1997-11-14
W096136358 PCTrUS36/OG804
3. Pre-tr~ns~l~nt Treatment with Anti-do~or
X~nograft Monoclonal ~ntibody Induces
Hyperacute ~nti~ody-me~;ated Reject;on
In accordance with a preferred embodiment of the
present invention, IgM producing B-cells from the spleen of
rats that had received hamster heart transplants were used
to generate antibodies of the present invention. Rat
hybridomas were produced by fusing rat myeloma cells (YB2/O)
with splenic lymphocytes from LEW rat recipients of hamster
cardiac xenografts. (See, EXAMPLES below.) The hybridomas
were screened for IgM antibody production in an ELISA format
as described in the EXAMPLES below. Antibodies from IgM-
producing hybridomas were screened, using the
immunofluorescent assay described in the EXAMPLES, for
immunoreactivity to hamster endothelial antigens. Such
antibodies were also screened for the ability to induce
antibody-mediate hyperacute rejection of hamster heart
xenografts in naive rats by in vivo rejection experiment.
As determined by the preliminary rejection experiments
described in Table 1 and used herein with regard to this
animal model, the term "hyperacute rejection" refers to the
rejection of a xenograft in less than about one hour.
Several hybridomas were created which produce anti-
donor xenograft antibodies, or more specifically, rat anti-
hamster xenograft monoclonal antibodies, including fourhybridoma cell lines stored in liquid nitrogen by Dr. Donald
Cramer in suite 250N of the Transplant Biology Research
Laboratory of Cedars-Sinai Medical Center, located at 150
North Robertson Blvd., Beverly Hills, California, 90211.
These three hybridoma cell lines are labeled and identified
by the following laboratory names: HAR-1, ID12BF3, ID12CF2,
and FC2EG11.
CA 02221234 1997-11-14
W 096/36358 PCT~US96/OC80q
These rat anti-hamster xenograft monoclonal antibodies
demonstrated binding to hamster endothelium derived from
various tissue sources including such vascularized organs as
the heart, kidney, gut, liver, lung, brain, and tongue, and
more specifically including arterial and capillary
endothelial cells, lymphatic endothelium, intestinal
epithelium, thymic epithelium and aortic endothelium in
tissue sections. The target antigen(s) are also associated
with the reticular cells and macrophages of the splenic red
pulp, the periarterial lymphoid sheath, and the thymic
medullary epithelium.
Rat anti-hamster xenograft monoclonal antibodies also
demonstrated a clear ability to induce antibody-mediated
rejection of the xenograft by naive ~EW rats. For example,
when a one milliliter aliquot of culture supernatant ~rom
HAR-l was passively transferred pre-transplant to naive LEW
rats, the mean rejection time of the subsequent hamster
CA 0222l234 l997-ll-l4
W 096/363S8 PCT~US96~0CY01
xenografts was 10 minutes (Table 2, Group "HAR-1"). Clearly,
the rejection induced by HAR-1 is hyperacute and is mediated
by binding to endothelial antigen, as was the rejections
Table 2. Passive Transfer of IgM Monoclonal Antibodies
Having Specificity for Hamster Endothelium
Induce Hyperacute Rejection Of Hamster
Xenografts in Naive Rats
Group NSurvival Median Survival
Time Time
HAR-1 58, 9, 10, 10, 10 10 minutes
Poly 510, 13, 15, 15, 18 15 minutes
9D6 54, 4, 4, 4, 4 4 days
induced by ID12BF3, ID12CF2 and FC2EG11 (median survival
time of 30 minutes each). Naive rats receiving hamster
cardiac xenografts and which are not subjected to pre-
transplant treatment with HAR-1 have a mean rejection time
of 3.9 days (Table 1, "Hamster --> LEW rat"). The mean
graft rejection time induced by pre-transplant treatment of
naive rats with culture supernatant from a hybridoma
producing IgM monoclonal antibody that lacked binding
specificity for endothelium (Table 2, "9D6") was four days,
the time to rejection normally seen in naive rats. The
hyperacute rejection precipitated by HAR-l was slightly more
CA 02221234 1997-11-14
W ~'3~3~ ~CT/U~ 66-~
severe, but comparable to, the hyperacute rejection
precipitated by pre-transpiant treatment of naive rats with
serum from a transplanted rat. (Table 2, Group "Poly").
HAR-l binding specificity was further characterized in
terms of its inability to bind tissue other than hamster
endothelium. Flow cytometric analysis and hemagglutination
assays of HAR-1 demonstrates limited binding of the antibody
to erythrocytes or splenic lymphocytes.
Through histopathologic ~ml n~tion of tissue using
immunohistochemistry to demonstrate antibody and complement
binding and thrombosis in vessels (See EXAMPLES below.), it
was determined that HAR-1 reacts specifically with vascular
endothelium, triggering complement activation and
intravascular thrombosis heart xenografts. The binding of
the rat anti-donor xenograft monoclonal antibodies and the
activation of complement with subsequent intravascular
thrombosis are the same lesions as those seen in models of
hyperacute rejection due to passive transfer of hyperimmune
serum.
Finally, it was determined it was determined through
Western blot analysis of hamster heart proteins that HAR-1
binds proteins producing 40 kDa and 80 kDa.
Thus, in accordance with more specific defined
embodiments of the present invention there is provided
isolated and substantially purified anti-donor xenograft
antibody ("anti-hamster xenograft antibody") characterized
as immunoreactive with antigen expressed by endothelial
cells of a hamster xenograft and capable of inducing
antibody-mediated rejection of the hamster xenograft by a
recipient animal. Preferably, the hamster xenograft is
heart tissue or the recipient animal is a rat. More
preferable the hamster xenograft is heart tissue and the
recipient ~n; m~l is a rat. Although such antibodies may be
of any isotype, preferably they are of the IgM isotype and
CA 0222l234 l997-ll-l4
W096/36358 PCTrUS~6/O~Y01
bind proteins producing 40 kDa and 80 kDa bands by Western
blot analysis.
In yet another embodiment of the present invention, the
isolated and substantially purified anti-hamster xenogra~t
antibody is further characterized as comprising at least one
polypeptide encoded by a nucleic acid sequence which
includes a sequence having at least about 90~ homology,
preferably at least about 95~ homology, more preferably at
least about 98~ homology, and most preferred about 100~
homology to the sequence defined by nucleic acid residues 58
through 351 of SEQ ID NO: 1 or nucleic acid residues 168
through 440 of SEQ ID NO: 9.
One of skill in the art will appreciate that having
provided the sequences of several anti-donor xenograft
antibodies, polypeptides, and antibody materials of the
~ present invention, additional embodiments of such
compositions can be generated which have amino acid residue
sequence substantially identical to a sequence specifically
shown herein merely by making conservative substitutions in
one or more residues of the sequence with a ~unctionally
similar residue and which displays the ability to mimic the
compositions as described herein. Examples of conservative
substitutions include the substitution of one non-polar
~hydrophobic) residue such as isoleucine, valine, leucine or
methionine for another, the substitution of one polar
(hydrophilic) residue for another such as between arginine
and lysine, between glutamine and asparagine, between
glycine and serine, the substitution of one basic residue
such as lysine, arginine or histidine for another, or the
substitution of one acidic residue, such as aspartic acid or
glutamic acid for another.
The phrase "conservative substitution" also includes
the use of a chemically derivatized residue in place of a
26
CA 02221234 1997-11-14
W 096/36358 PCTrUS~6/OC80
non-derivatized residue provided that such polypeptide
displays the requisite binding activity.
"Chemical derivative" refers to a subject polypeptide
having one or more residues chemically derivatized by
reaction o~ a ~unctional side group. Such derivatized
molecules include, for example, those molecules in which
free amino groups have been derivatized to form amine
hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy
groups, t-butyloxycarbonyl groups, chloroacetyl groups or
formyl groups. Free carboxyl groups may be derivatized to
form salts, methyl and ethyl esters or other types of esters
or hydrazides. Free hydroxyl groups may be derivatized to
form O-acyl or O-alkyl derivatives. The imidazole nitrogen
of histidine may be derivatized to form
N-im-benzylhistidine. Also included as chemical derivatives
are those peptides which contain one or more naturally
occurring amino acid derivatives of the twenty standard
amino acids. For examples: 4-hydroxyproline may be
substituted for proline; 5-hydroxylysine may be substituted
for lysine; 3-methylhistidine may be substituted for
histidine; homoserine may be substituted for serine; and
ornithine may be substituted for lysine. Polypeptides of
the present invention also include any polypeptide having
one or more additions and/or deletions of residues relative
to the sequence of a polypeptide whose sequence is shown
herein, so long as the requisite activity is maintained.
In an still another embodiment of the rat anti-hamster
xenograft antibody of the present invention, the antibody is
characterized as being immunoreactive with antigen
expressed by endothelial cells of a hamster xenograft,
capable of inducing antibody-mediated rejection o~ the
hamster xenograft by a recipient animal and comprising at
least one polypeptide encoded by a nucleic acid sequence
including the sequence defined by nucleic acid residues 58
CA 02221234 1997-11-14
W 096/363~8 PCTrUS~6/0~80~
through 420 o~ SEQ ID NO: 1 or by a nucleic acid sequence
including the sequence defined by nucleic acid residues 1
through 354 of SEQ ID NO: 3. Preferably, the anti-hamster
xenograft antibody further comprises at least one
S polypeptide encoded by a nucleic acid sequence including the
sequence defined by SEQ ID NO: 5.
4. Rat Anti-~orc;ne Xenogr~ft Ant;hodies
Demon.~trate Si~;lar Ant;gen B;n~;ng to Hl~m~n
Ant;-porc;ne Xenograft Ant;ho~ies
. 10 Having demonstrated through the hamster-to-rat
xenogeneic transplant model (1) that the accelerated
rejection reaction is mediated by preformed anti-xenograft
antibodies of the IgM isotype which are polyspecific for
antigens of the vascular endothelium of the transplant
tissue, and (2) that a hyperacute rejection reaction can be
induced by anti-donor xenograft antibodies, monoclonal
antibodies were then generated that exhibit binding
specificity representative of the human anti-porcine
xenograft antibodies which mediate the rejection of pig
tissue by humans.
Humans who have not undergone xenogeneic transplant or
immunization possess serum antibodies that are known to bind
a broad range of xenogeneic cell surface-associated
molecules including antigens present on erythrocytes,
vascular endothelium, platelets, and lymphocytes in a
variety of species, including pigs. Preformed human anti-
porcine xenograft antibodies bind at least six antigens
expressed by pig aortic endothelial cells ("PAEC") with
molecular weights of 44 kDa, 80 kDa, 115kDa, 125 kDa, 135
kda and 200 kDa by Western blot analysis. Three antigens of
similar molecular weights (115kD, 125kD, and 135kD) which
are expressed on pig platelet cells are also bound by these
28
CA 02221234 1997-11-14
W O9"3~3~ PCTrUS95'~
preformed human anti-porcine xenograft antibodies. In
addition, these preformed human anti-porcine antibodies bind
pig lymphocytes since absorption of human serum with pig
lymphocytes removes the binding of these preformed
antibodies to PAEC.
In general, the antibodies that appear to be most
closely associated with the xenograft reaction are of the
IgM isotype, although IgG and IgA antibodies have reported
to be involved in the rejection of pig tissue by humans.
However, the cytotoxic activity of human preformed
antibodies to pig endothelium is most clearly the result of
the binding of IgM and not IgG antibodies.
Similar to the rejection of pig xenografts by humans,
hyperacute rejection of pig xenografts by rats is initiated
by preformed IgM antibodies in the rat recipient's serum
that bind to antigens expressed by the pig xenograft.
Accordingly, a pig-to-rat model was employed to generate and
characterize anti-porcine xenograft antibodies. A panel of
83 rat monoclonal antibodies to PAEC ("rat anti-porcine
xenograft monoclonal antibodiesn) were generated using
traditional hybridoma techniques as described in the
EXAMPLES below and in Yokayama, W.M., Current Protocols ;n
I~mml~nology, Colifan, J, et al., (eds.). Greere Publishing
of Wiley - Interscience, 1991, 2.5.4, incorporated herein by
reference. Briefly, LEW rats were immllnized with PAEC and
splenic lymphocytes of the rat were harvested and fused with
rat YB2/O myeloma cells to produce hybridomas. Antibodies
secreted by these hybridomas were then subjected to several
assays including assays which identified antibodies which
are immunoreactive with antigen expressed by endothelial
cells of a xenograft from a donor animal and which are
capable of inducing antibody-mediated rejection of the
xenograft by a recipient animal.
29
CA 02221234 1997-11-14
W 096/36358 PCTrUS~'OC~03
of the approximately 250 hybridomas generated and
screened, 83 hybridomas were identified as secreting
antibody having immunoreactivity with antigen expressed by
PAEC. PAEC immunoreactivity was determined in an ELISA
format as described in the EXAMPLES below using PBS and pig
serum as negative controls.
Another selection criterion which is presently prefered
is the ability of the rat anti-porcine xenograft monoclonal
antibodies to immunoreact antigen expressed by pig platelets
as is characteristically observed with preformed human anti-
porcine xenograft antibodies. Using a flow cytometry assay,
the panel of 83 rat anti-porcine xenograft monoclonal
antibodies were tested for their ability to immunoreact with
pig platelets. Most of these monoclonal antibodies tested
positive. These results were confirmed in an ELISA assay
using pig platelets as targets and human serum as a positive
control.
Another selection criterion for anti-porcine xenograft
antibody which is presently prefered is the IgM isotype.
The isotypes of the monoclonal antibodies were determined in
an ELISA format as described in the EXAMPLES below. Twelve
of the 83 hybridomas having immunoreactivity for PAEC
secrete antibody of the IgG isotype, and the remainder are
IgM.
Anti-porcine xenograft monoclonal antibodies were
identified amongst the 83 monoclonal antibodies which
demostrated ;mml~noreactivity with antigen expressed by PAEC
by their ability to induce antibody-mediated rejection of
the xenograft by a recipient animal. Accordingly, these 83
monoclonal antibodies were their ability to kill PAEC in a
complement-mediated flow cytometric cytoxicity assay as
described in the EXAMPLES below. Eleven anti-porcine
xenograft monoclonal antibodies identified each being highly
cytotoxic (~80~) to PAEC in the complement-mediated
CA 02221234 1997-11-14
W 096/363~8 PCTrUS9~'OCYOl
cytotoxicity assays. The results were identical when
purified rabbit complement or rat serum was used as a source
of complement. Autologous complement, as expected, was not
cytotoxic for PAEC.
Hybridoma cell lines secreting these anti-porcine
~ xenograft antibodies are stored in liquid nitrogen by Dr.
Donald Cramer in suite 250N of the Transplant Biology
Research Laboratory of Cedars-Sinai Medical Center, located
at 150 North Robertson Blvd., Beverly Hills, California,
90211. These hybridoma cell lines are labeled and
identified by the following laboratory names: HA73C4;
HA71G4; HA73D7; HA75D8; HA72G3; HA71E3; HA73HB; IE31D8;
IG121H7; DA9lOE4; and IH21H7.
The present invention thus includes isolated and
substantially purified anti-donor xenograft antibody (~anti-
porcine xenograft antibody") characterized as being
immunoreactive with antigen expressed by endothelial cells
of a pig xenograft and capable of inducing antibody-mediated
rejection of the pig xenograft by a recipient animal. In a
particular embodiment of the invention, the pig xenograft is
heart tissue or liver tissue. In another embodiment of the
invention the recipient animal is a human.
Preferably, the anti-porcine xenograft antibodies of
the present invention are of the IgM isotype. Even more
preferably, the anti-porcine xenograft antibodies of the
present invention immunoreact with antigen expressed by pig
platelet cells.
In a related embodiment of anti-porcine xenograft
antibody of the present invention, the isolated and
substantially purified anti-porcine xenograft antibody is
further characterized as comprising at least one polypeptide
encoded by a nucleic acid sequence which includes a sequence
having at least about 90~ homology, preferably at least
about 95~ homology, more preferably at least about 98~
CA 0222l234 l997-ll-l4
W 096/36358 PCT~US9''0G801
homology, and most preferred about 100~ homology to the
sequence defined by nucleic acld residues 1 through 291 of
SEQ ID NO: 18 or nucleic acid residues 1 through 291 of SEQ
ID NO: 20.
In an still another embodiment of the anti-procine
xenograft antibody of the present invention, the antibody is
characterized as being immunoreactive with antigen expressed
by endothelial cells of a pig xenograft, capable of inducing
antibody-mediated rejection of the pig xenograft by a
recipient animal and comprising at least one polypeptide
encoded by a nucleic acid sequence including the sequence
defined by nucleic acid residues 1 through 345 of SEQ ID NO:
18 or defined by nucleic acid residues 1 through 357 of SEQ
ID NO: 20.
Another selection criterion for presently prefered
anti-porcine xenograft antibody is complement-mediated
cytotoxicity for pig spleen lymphocytes. Using the flow
cytotoxicity assay described herein and pig lymphocytes in
place of PAEC, the eleven anti-porcine xenograft monoclonal
antibodies were screened for cytotoxicity to pig splenic
lymphocytes. All eleven monoclonal antibodies also have the
capacity to bind and induce complement-mediated splenic
lymphocytes cell death; findings which are characteristic of
many preformed human anti-porcine xenograft antibodies.
Thus, anti-porcine xenograft antibodies of the present
invention preferably are also capable of inducing
complement-mediated death of pig spleenic lymphocytes.
Still another selection criterion for presently
prefered anti-porcine xenograft antibody is immunoreactivity
with proteins having molecular weights similar to those
which bind with preformed human anti-porcine xenograft
antibodies. Using immunoprecipitation of l25I labeled PAEC
surface antigens, it was demonstrated that several of the
monoclonal antibodies immunoprecipitate proteins of about
CA 02221234 1997-11-14
W 096136358 PCTrUS9~ r~~
the same molecular weight as antigens expressed by PAEC or
pig platelet cells and which are recognized by pre~ormed
hllm~n anti-porcine xenograft antibodies. Preformed human
r anti-porcine xenograft antibodies bind at least six antigens
5 expressed by pig aortic endothelial cells with molecular
weights of 44 kDa, 80 kDa, 115kDa, 12S kDa, 135 kda and 200
kDa by Western blot analysis. Three antigens of similar
molecular weights (115kD, 125kD, and 135kD) which are
expressed on pig platelet cells are also bound by these
10 preformed human anti-porcine xenogra~t antibodies.
Four of the eleven anti-porcine xenograft monoclonal
antibodies described above and secreted by hybridomas
HA73H8, HA72G3, IH21H7, or HA73D7, immunoreact with proteins
in the range of 38-44 kDa as demonstrated by
15 immunoprecipitation.
Thus, it is prefered that the anti-porcine xenograft
antibodies of the present invention be immunoreactive with
at least one antigen expressed by pig endothelial cells or
pig platelet cells which are also recognized by preformed
20 human anti-porcine xenograft antibodies. Even more
pre~erably, the anti-porcine xenograft antibodies of the
present invention are also capable of blocking preformed
human anti-porcine xenograft antibody from binding to PAEC
or LCPK1 pig kidney cells. (LCPK1 cells are included in
25 these screening assays because it has been reported that
they express the 115 kDa molecule recognized by preformed
hllm~n anti-porcine IgG antibodies and express ~Gal terminal
residues on surface antigens that are targets of preformed
hllm~n anti-porcine antibody binding and cytotoxicity. See,
30 Koren, E. et al., "Cytotoxic effects of human preformed
anti-gal IgG and complement on cultured pig cells." ~eCon~
Tnter~l CongreSS on ~notr~ns~l~ntat;on, England, Septe~mber
26-29, 1994; Neethling, F.A., et al., Tr~n.~ nt~t;on
57(6):959-963 1994.) The ability of anti-porcine xenograft
CA 02221234 1997-11-14
W 096/363S8 PCTrUS~G/06~C~
antibodies to block such binding can be screened for in the
ELISA format or the flow cytometric assay as described below
in the EXAMPLES.
The anti-porcine xenograft antibodies described above
were screened for their ability to block preformed human
anti-porcine xenograft antibody (as they occur in normal
human serum) from binding to PAEC or LCPK1 pig kidney cells.
These blocking studies were performed with groups of anti-
porcine xenograft monoclonal antibodies in order to screen
larger numbers of monoclonal antibodies more efficiently.
Each group, comprising four to five individual monoclonal
antibodies, was incubated with PAEC or LCPK1 cells. The
cells were then incubated with human serum as a source of
preformed human anti-porcine xenograft antibodies. Anti-
porcine xenograft monoclonal antibodies in Group 1 (HA75D8,IH21H7, HA72G3, HA73D7 and DElOlH2) were capable of
reproducibly blocking 50-65~ of preformed human IgM anti-
porcine xenograft antibody binding to LCPK1 cells as
indicated by a drop in mean channel shift from 242.5 with
human serum alone to 59.9 following preincubation with Group
1 monoclonal antibodies. Group 1 anti-porcine xenograft
antibodies were also capable of blocking binding of prefomed
human anti-porcine xenograft antibodies to PAEC, indicated
by a drop in mean channel shift from 73.6 with human serum
alone to 47.2 following preincubation with Group 1
monoclonal antibodies. The rat anti-porcine xenograft
monoclonal antibodies in Group 2 (HA73C4, IH27H6, HA71E3,
and DE103H6) did not significantly block preformed human IgM
anti-porcine xenograft antibody binding to pig cells (mean
channel shift of 219.2) in the same experiment but
demonstrated an equivalent level of binding to LCPK1 cells
as Group 1.
The individual monoclonal antibodies from Group 1 were
then each ~m; ned for their ability to block preformed
CA 02221234 1997-11-14
W O9f~58 PCTrUS9C~C~0
human IgM anti-porcine xenograft antibody binding to pig
cells. The results are reported in Table 3.
Table 3. Ability of Rat Anti-Porcine Xenograft Monoclonal
Antibodies To Block Binding of Preformed Human
Anti-Porcine Xenograft Antibodies to PAEC or LCPK-
1 Cells.
Mean Channel Shift (Fluorescence)
PAEC LCPK-1
Negative Control 3.3 8.7
lo Human Serum 53.1 87.7
HA75D8 + Human Serum 34.0 49.8
IH2IH7 + Human Serum 30.0 40.5
HA72G3 No blocking No blocking
HA73D7 No blocking No blocking
15 DElOlH2 No blocking No blocking
p<.0001 p~.0001
Incubation of HA75D8 monoclonal antibody with PAEC cells or
LCPK-1 cells prior to incubation with human serum resulted
in a 34.6% and a 37.9% decrease in binding of human serum to
PAEC cells and LCPK-1 cells, respectively. Incubation of
LCPK1 cells with monoclonal antibody IH2IH7 prior to
incubation with hl~m~n serum resulted in a 46~ decrease in
binding of human serum to LCPK1 cells. Dilution of
monoclonal antibody IH21H7 resulted in a corresponding
increase in preformed human IgM anti-porcine xenograft
antibody binding to LCPK1 cells, demonstrating specificity
of the blocking effect.
CA 02221234 1997-11-14
W 096/363~8 PCT/US96/06801
B. Anti-D~nnr XenoqFaft An~;hody Mater;~l
In accordance with another embodiment of the present
invention there is provided anti-donor xenograft antibody
material characterized as immunoreactive with antigen
expressed by endothelial cells of a donor xenograft and
capable of inhibiting antibody-mediated rejection of the
donor xenograft by a recipient animal.
The term "antibody material" in its various grammatical
forms is used herein as a collective noun that refers to a
popula~ion of immunologically active fragments of
immunoglobulin molecules, i.e., molecules that contain an
antibody combining site. Exemplary antibody materials of
the present invention include those portions of
immunoglobulin molecules known in the art as Fab, Fab',
F(ab' )2/ and F(v). Fab and F(ab' )2 portions of antibodies
are prepared by the proteolytic reaction of papain and
pepsin, respectively, on substantially intact antibodies by
methods that are well known. See, for example, U.S. Patent
No. 4,342,566 to Theofilopolous and Dixon. Fab' antibody
portions are also well known and are produced from F(ab') 2
portions followed by reduction of the disulfide bonds
linking the two heavy chain portions as with
mercaptoethanol, and followed by alkylation of the resulting
protein mercaptan with a reagent such as iodoacetamide. An
antibody combining site is that structural portion of the
antibody molecule comprised of a heavy and light chain
variable and hypervariable regions that specifically binds
(immunoreacts with) an antigen.
Thus, the term "anti-donor xenograft antibody material~
refers to antibody material that immunoreacts with antigen
expressed by endothelial cells of a xenograft and that
inhibits antibody-mediated rejection of the xenograft by the
recipient animal. In accordance with one embodiment of the
36
CA 0222l234 l997-ll-l4
W 096/363~8 PCTrUS~6/OC801
invention, anti-donor xenograft antibody material is
selected from the group consisting of Fab, Fab', F(ab' )21
F(v) and combinations of thereof. In yet another embodiment
of the present invention, anti-donor xenograft antibody
material is selected from the group consisting of F(ab~) 2
~ and Fab'. In a prefered embodiment of the invention, anti-
donor xenograft antibody material is comprises a combination
of F(ab' )2 and Fab'.
To produce the anti-donor xenograft antibody material
of the present invention, anti-donor xenograft antibody can
simply be reduced into fragments that retain their ability
to immunoreact with antigen expressed by endothelial cells
of the xenograft but that are incapable of inducing
complement-mediated death of xenograft cells.
Alternatively, more sophisticated methods, such as the phage
- display technique (descibed in greater detail below) can be
used to generate anti-donor xenograft antibody material of
the present invention.
Screening antibody material for immunoreactivity with
antigen expressed by endothelial cells of the xenograft is
similar to the method of screening antibodies for the same
attribute as described above and in the EXAMPLES, except
that an appropriate secondary antibody should be
substituted, such as for example enzyme-labeled anti-kappa
antibody. The same preference described above regarding the
sources of antigen for such screening as~ays would also
apply here.
Likewise, the skilled artisan will appreciate that with
the appropriate modifications the same in vivo rejection
experiments and in vitro complement-mediated cytotoxicity
assay formats described above for identify antibodies having
the ability to induce antibody-mediated rejection of the
donor xenograft by a recipient animal can be employed to
identify antibody material capable of inhibiting antibody-
CA 02221234 1997-11-14
W 096/363S8 PCT~US96'~6801
mediated rejection of the donor xenograft by a recipient
anlmal .
More specifically, in an in vitro format, the inability
to induce antibody-mediated, complement-dependent
cytotoxicity indicates that that antibody tested has the
ability to inhibit antibody-mediated rejection of the donor
xenograft by the recipient animal. The source of antigen
and complement for the cytotoxicity assays can be as
described above for the immunoreactivity assays, or other
whole cells from the donor animal can be used as a source of
antigen. Preferably, as measured in a flow cytometric
cytotoxicity assay, antibody is capable of inhibiting
antibody-mediated rejection of a xenograft by a recipient
animal when cell death is reduced by at least about 20~ as
compared to a control, preferably by at about 25~ as
compared to a control, more preferably by at least about 30
as compared to a control, even more preferably by at least
about 35~ and most preferably by at least about 40~. One of
skill in the art will appreciate that the flow cytometric
cytotoxicity assay described in EXAMPLES can easily be
adapted for specie combinations of interest.
Alternatively, and more preferably, the ability of
anti-donor xenograph antibody material to inhibit antibody-
mediated rejection of the xenograft is detected by the
ability of the antibody material to block binding of
preformed anti-donor xenograph antibodies in the recipient
animal serum to antigen expressed by endothelial cells of
the xenograft. The ability of anti-porcine xenograft
antibodies to block such binding can be screened for in the
ELISA format or the flow cytometric assay described in the
EXAMPLES below. The ability to inhibit antibody-mediated
rejection of the xenograft is detected by the ability of the
antibody material to block at least about 20~ of the binding
detected with normal recipient serum, more preferably at
CA 02221234 1997-11-14
W 096t3635~ PCTrUS9-'0680
least about 30~ of the binding detected with normal
recipient serum, even more preferably at least about 30~ of
the binding detected with normal recipient serum, and most
preferably at least about 50~ of the binding detected with
normal recipient serum.
The ability of the antibody material to inhibit
antibody-mediated rejection of a donor xenograft is
identified in the in vivo rejections experiments described
above when pre-transplant passive transfer of anti-donor
xenograft antibody material prolongs the median survival
time of the xenograft beyond the median survival time
characteristic of the hyperacute rejection of a xenograft by
the recipient animal, or more preferably beyond the median
survival time of the xenograft characteristic of the
accelerated rejection of a xenograft by the recipient
animal. When appropriate, post-transplant passive transfer
of hyperimmune sera or anti-donor xenograft antibody of the
present invention is administered to the transplant
recipient in such in vivo experiments to generate the
characteristic hyperacute rejection of the xenograft, such
as is exemplified in the following hamster-to-rat model.
1. Pre-tr~nspl~nt Tre~tment w;th Re~uced ~nt;-
~nogr~ft Monoclon~l ~nt;hody Mater;~
Tnh;h;ts ~y~er~cute Reject;o~
Having demonstrated that the rat anti-donor xenograft
monoclonal antibodies of the present invention have
specificity for vascular endothelium of hamsters and could
induce the hyperacute rejection reaction through activation
of the classic complement pathway, rat anti-hamster
xenograft antibodies were reduced to antibody material and
screened for their ability to inhibit antibody-mediated
rejection of the xenograft by rats.
CA 02221234 1997-11-14
W O9.~/36358 PCT~US~ C~1
An anti-hamster xenograft monoclonal antibody of the
present inventlon designated HAR-1 above, was reduced to its
Fab and F(ab') 2 form by protein reduction of the IgM
pentamers with 2-mercaptoethanol ("2-ME"). Naive LEW rats
were divided into five groups. In group 1, referred to as
the "Poly group," naive rats received pre-transplant
treatment of a single passive transfer of 0.5 ml rat
hyperimmune sera. In group 2, referred to as the "HAR-1
group," the naive rats received pre-transplant treatment of
1.O ml of HAR-1 (50-lO0 ~g/protein) in a single passive
transfer. In group 3, referred to as "HAR-lred/Poly group,"
the naive rats received pre-transplant treatment of 1.0 ml
HAR-l Fab and F(ab) 2 antibody material, followed by passive
transfer of 0.5 ml rat hyperimmune sera. Group 4, referred
to as "HAR-lr'd/HAR-l group" received pre-transplant
treatment of 1.O ml HAR-1 Fab and F(ab) 2 antibody material,
followed by passive transfer of 1.0 ml HAR-1. Group 5,
referred to as "9D6/Poly group," received pre-transplant
treatment of 1.0 ml 9D6 Fab and F(ab) 2 antibody material,
followed by passive transfer of 0.5 ml rat hyperimmune sera.
(9D6 is an IgM monoclonal antibody generated as described in
the EXAMPLES below which does not have binding specificity
for hamster endothelium and does not induce antibody-
mediated rejection of hamster xenografts by rats.) Each
group of rats were then transplanted with hamster cardiac
xenografts.
The results shown in Table 2 demonstrate that HAR-1 Fab
and HAR-1 F(ab) 2 antibody material significantly inhibit the
hyperacute rejection of hamster cardiac xenograft (p<0.01).
CA 02221234 1997-11-14
W 096136358 PCT~US~'068
Table 3. Protective Effect of Reduced HAR-1 Antibody
Material on Hyperacute Rejection of Hamster
Hearts Following Passive Antibody Transfer to
Naive Rats
Median
Group* N Survival TimeSurvival Time
(minutes) (minutes)
Poly 5 10, 13, 15, 15, 18 15
HAR-l 5 8, 9, 10, 10, 10 10
9D6/Poly 5 10, 20, 20, 25, 30. 20
HAR_1red/pOly 5 15, 50, 1440, 2580,
1440, 4200, 5400, 6120 2580**
HAR - lr~d/HAR - 1 3 1440, 4320, 4680 4320
**Significant at P<0.01 in Student's t test when compared
with the 9D6/Poly group.
The data indicates that (1) HAR-l has specificity for the
xenoantigen(s) which are primarily responsible for
generating antibody-mediated hyperacute rejection and that
are also recognized by the antibodies of transplanted rat
sera; and (2) the usage of anti-donor xenograft antibody
material (e.g., Fab, F(ab)2) can inhibit antibody-mediated
xenograft hyperacute rejection.
Thus, in accordance with the present invention there is
provided isolated and substantially purified anti-donor
xenograft antibody material ("anti-hamster xenograft
antibody material") which is characterized as ;mml~noreactive
with antigen expressed by endothelial cells of a hamster
CA 0222l234 l997-ll-l4
W 096/363S8 PCT~US9f'OG801
xenograft and capable of inhibiting antibody-mediated
rejection of the hamster xenograft by a recipient animal.
Preferably, the hamster xenograft i8 heart tissue or the
recipient animal is a rat. More preferable the hamster
xenograft is heart tissue and the recipient animal is a rat.
In a related embodiment of the anti-hamster xenograft
antibody material, the isolated and substantially purified
anti-hamster xenograft antibody material is further
characterized as comprising at least one polypeptide encoded
by a nucleic acid sequence which includes a sequence having
at least about 90~ homology, preferably at least about 95~
homology, more preferably at least about 98~ homology, and
most preferred about 100~ homology to the sequence defined
by nucleic acid residues 58 through 351 of SEQ ID NO: 1 or
by nucleic acid residues 168 through 440 of SEQ ID NO: 9.
In an still another embodiment of the anti-hamster
xenograft antibody material of the present invention, the
antibody material is characterized as being immunoreactive
with antigen expressed by endothelial cells of a hamster
xenograft, capable of inhibiting antibody-mediated rejection
of the hamster xenograft by a recipient animal and
comprising at least one polypeptide encoded by a nucleic
acid sequence including the sequence defined by nucleic acid
residues 58 through 420 of SEQ ID NO: 1 or by a nucleic acid
sequence including the sequence defined by nucleic acid
residues 1 through 354 of SEQ ID NO: 3. Preferably, the
anti-hamster xenograft antibody further comprises at least
one polypeptide encoded by a nucleic acid sequence including
the sequence defined by SEQ ID NO: 5.
In yet another embodiment of the present invention
there is provided isolated and substantially purified anti-
donor xenograft antibody material ("anti-porcine xenograft
antibody material") characterized as being immunoreactive
with antigen expressed by endothelial cells of a pig
CA 02221234 1997-11-14
W 096136358 PCT~US9-'~6e0
xenograft and capable of inhibiting antibody-mediated
rejection of the pig xenograft by a recipient animal. In a
particular embodiment of the invention, the pig xenograft i8
heart tissue or liver tissue. In another embodiment of the
invention the recipient animal is a human.
~ In a related embodiment of anti-porcine xenograft
antibody material of the present invention, the isolated and
substantially purified anti-porcine xenograft antibody
material is further characterized as comprising at least one
polypeptide encoded by a nucleic acid sequence which
includes a sequence having at least about 90~ homology,
preferably at least about 95~ homology, more preferably at
least about 98~ homology, and most preferred about 100~
homology to the sequence defined by nucleic acid residues 1
through 291 of SEQ ID NO: 18 or by nucleic acid residues 1
through 291 of SEQ ID NO: 20.
In an still another embodiment of the anti-porcine
xenograft antibody material of the present invention, the
antibody material is characterized as being immunoreactive
with antigen expressed by endothelial cells of a pig
xenograft, capable of inhibiting antibody-mediated rejection
of the pig xenograft by a recipient animal and comprising at
least one polypeptide encoded by a nucleic acid sequence
including the sequence defined by nucleic acid residues 58
through 345 of SEQ ID NO: 18 or defined by nucleic acid
residues 1 through 357 of SEQ ID NO: 20.
C. Ant; -~n~r }~nnS~rAft ~nn~cl ~n:~l An1-;ho~; es ~;h~w
Senl~n~e Homology to ~n~ Ann~h~ ~n~ to G~rmline
Se~ n~e~
A surprising and unexpected attribute of the anti-donor
xenograft antibody material of the present invention, as
demonstrated for example by HAR-1 antibody material, is that
anti-donor xenograft antibody material could inhibit
43
CA 02221234 1997-11-14
W 096/36358 PCTrUS~6/OCYO1
antibody-mediated rejection of a xenograft and that
polyclonal anti-donor xenograft antibody material was not
required. This result suggest that the preformed anti-donor
xenograft antibodies responsible for mediating xenograft
rejection are polyreactive. Without being bound by any
particular theory, it is presently believed that hyperacute
rejection of xenografts is mediated by preformed
polyreactive anti-donor xenograft antibodies having VH
germline configurations. This belief is substantiated by
the nucleic acid sequence homology in heavy chain variable
segments utilized in the anti-donor xenograft antibody
material sequenced to date and the homology of these
sequences to germline sequences of the animal from which the
antibody material originates.
Immunoglobulin heavy and light chain cDNA libraries
were constructed from mRNA of hybridomas producing rat anti-
xenograft monoclonal antibodies. As described in greater
detail in the EXAMPLES below, the PCR technique was employed
with primers for the proximal part of the constant region of
the heavy (~) or light (K and A) chain to synthesize a first
strand cDNA from mRNA isolated from the HAR-1-producing the
hybridoma and the ID12BF3-producing hybridoma. After the
synthesis of the second strand cDNA by standard techniques,
a double-stranded synthetic linker of known sequence was
ligated to the 5' end of the cDNA to facilitate the
amplification of all V, D, and J genes rearranged to the
constant region.
The cDNA was amplified by PCR using a second upstream
constant region primer and the sense strand of the linker.
The amplified product was cloned into the pCR~ vector of the
TA cloning kit~ (Invitrogen, San Diego, CA) to establish the
library. Recombinant clones were screened directly from
bacterial colonies by PCR and their nucleic acid sequences
characterized. This method was found to be more powerful
CA 02221234 1997-11-14
W 096/36358 PCT~US~'OC~O1
and practical to use than anchored PCR, which utilizes the
addition of a 3' tail to the first strand cDNA to clone
sequences with unknown 5' ends.
Due to the limited information available on rat
immunoglobulin variable region gene sequences, the
boundaries o~ individual V, D, and J segments, based on
consensus-conserved regions, were determined by computer-
assisted comparisons with mouse and rat sequences available
in the GenBank Database~ and by comparison with published
germ-line sequences. See, Kabat, E.A., et al. Sequences of
proteins of immunological interest. Department of Heal th
and Human Servi ces , Anonymous Washington, DC (1991),
incorporated herein by reference. The potential
introduction o~ mutations by PCR amplification was
compensated for by sequencing and aligning at least 3
individual clones and, whenever possible, by using another
DNA polymerase (Ultma DNA polymerase) that has high 3'-5'
exonuclease activity that leads to a "proof-reading"
activity and suppresses the small misincorporation rate seen
with the enzyme Taq DNA polymerase.
The HAR-1 cDNA sequence of the heavy chain variable
region is shown in SEQ ID NO. 1 and Figure 1. The ID12BF3
cDNA sequence of the heavy chain variable region is shown in
SEQ ID NO. 3. The VH segments of these two rat anti-hamster
xenograft monoclonal antibodies which were generated in
separate by fusions are nearly identical to one another.
When the sequence of this VH segment alone was compared to
sequences available on GeneBank~, three rat sequences
(Accession Nos. RRIGCD25H, RNIGHCAZ, and RNIGHNCS) were
identified as having about 91~ homology. The classification
of rat V~ segments into different families and subfamilies
is not yet available, however, using the criterion for
membership in human VH gene families, the four rat Vil gene
sequences would belong to the same VH i~amily. Six mouse
CA 02221234 1997-11-14
W Og-~63~8 PCTrUS~6/06801
sequences were also found to have about 87~ homology to the
rat anti-hamster xenograft monoclonal antibody VH segment.
HAR-1 and ID12BF3 monoclonal antibodies were found to
utilize different JH segments. The cDNA sequence of the
HAR-1 JH segment was found to be 98.2~ homoloyous to the rat
germline JH1 gene: identical except for a substitution C
for A in the first codon of germline JH1. The cDNA sequence
of the ID12BF3 JH segment was found to be 100~ homologous to
the rat germline JH2 gene.
Due to possible N region and P element variations
during the recombination process, it was not possible to
clearly set the boundaries for DH segment of HAR-1 and
ID12BF3, although it is evident that the D segments of these
antibodies differ from one another. Nevertheless, a search
was also conducted to identify any known sequences with
greater that 70~ homology to the totality of the cDNA
sequence between the end of the VH segment and the beginning
of the JH segment of HAR-1. No relevant match was found
with any of the sequences of the Genebank~ database.
The nucleotide and deduced amino acid sequences of the
variable region for HAR-1 kappa light chain, were also used
to search the GeneBank~ database for homologous sequences.
The nucleic acid sequence of the HAR-1 V~ segment has about
90~ sequence homology to three anti-DNA antibody VK segments
which are all members of the Vr8 family defined in mice. A
germline counterpart for the HAR-1 V~ segment has not yet
been identified, but a search of the Genebank~ database has
demonstrated that the J~ segment of HAR-1 matches the first
36 nucleotides of the rat germline Jr2 (Genebank~ Accession
No. RNIGKJCA) with 100~ identity.
To determine the level of homology between the VH
segment utilized in HAR-1, ID12BF3, and HA75D8F1 and their
germline counterpart, genomic liver DNA was amplified and
sequenced in accordance with the EXAMPLES below. See also,
46
, CA 02221234 1997-11-14 P ~ ~ 9 6 / 06 88 ~
- iPE~w l A~ A~ 19g7
Shirwan et al., J. Irmunol. 151:5228-5238 (1993) and Shirwan
et al., J. Immunol. 150:2295-2304 (1993), incorporated
herein by reference. The objective was to establish whether
the VH segments were in a germline configuration or if they
were displaying mutations suggesting an antigen-driven
affinity maturation. For this purpose, ~wo oligonucleotides
(SEQ ID NO. 7 and SEQ ID NO. 8, referred to as RVH1 and
RVH2, respectively in Figure 1) that allowed almost complete
recovery of the sequence information for the VH segment of
HAR-1 and ID12BF3 were used to amplify genomic liver DNA
extracted from a newborn LEW rat. Genomic sequence
information was obtained from 3 individual clones.
Likewise, two oligonucleotides (5' GGC ACA GAA GTA CAT GGC
CG 3' referred to herein as UH7RVH2" which anneals to the
framework 3 region of the variable heavy chain and 5' CGT
TTA GTT AAT TCA TTA TGC 3~ referred to herein as "HA7RVH3ll
which anneals upstream of the initiation codon of HA75D8F1)
that allow almost complete recovery of the sequence
information for the VH segment of HA75D8F1 were used to
amplify genomic liver DNA extracted from a newborn LEW rat
genomic sequence information was obtained from two
individual clones.
Alignment of germline DNA (SEQ ID NO. 35 and referred
to herein as VH1. 1) and cDNA sequences for the HAR-l VH
segment (nucleic acid residues 52 through 427 of SEQ ID NO:
34) is shown in Figure 2. Three differences were found over
a total of 376 nucleotides; the VH segment was 99%
homologous to the germline sequence. One transversion C for
G led to a replacement of leucine by valine in the eleventh
amino acid position of the leader sequence. The two other
differences, A for G at position 48 of framework region 1,
and T for C at position one of framework region 2, were
silent. Alignment of VH1.1 and the cDNA sequence for
ID12BF3 also showed about 99% homology to VH1.1, but as a
result of different nucleic acid substitutions.
AMEI\IGED S~l,-ET
, CA 02221234 1997-11-14 ~ 6 / ~ 8 e
IPEAi~v 1~ J.~N lgg7
Alignment of germline DNA (referred to herein as
VHRAP.la) (SEQ ID N0: 39) and the cDNA sequence for HA75DBF1
VH segment (SEQ ID N0: 38) is shown in Figure 4. Three
differences were found over a total of 290 nucleotides; the
5 VH segment was 98.6% homologous to the germline sequences.
All three of these differences occur in the framework
regions of the cDNA sequence of HA75D8F1.
The level of the sequence homology, the location of
nucleic acid substitutions, and failure of these
substitutions to significantly change the deduced amino acid
sequence of the VH segment, provide further evidence that
the hyperacute rejection of xenografts is mediated by
polyreactive anti-donor xenograft antibodies having VH
germline configurations.
For comparison, the genetic characteristics were also
determined for the 9D6 monoclonal antibody. The cDNA
sequence of the VH segment of 9D6 demonstrated a high
percentage of identity with three members of the VH4 family
as defined in mice. Comparison of heavy chain variable
regions of HAR-1 and 9D6 demonstrated an overall homology of
77~. The complementarity determining regions (CDRs)
exhibited homologies of 40%, 29~, and 33% for CDR1, CDR2,
and CDR3 regions, respectively. The low level of identity
in these antigen binding regions is consistent with
differences in the ability of these two antibodies to induce
hyperacute rejection of hamster hearts.
Finally, cDNA libraries of immunoglobulin VH genes were
constructed from splenic B lymphocytes of newborn rats,
naive adult rats, rats which received hamster-xenografts at
Day 4 post-transplant, and rats which received hamster-
xenografts at Day 21 post-transplant, to establish which
germline sequences was utilized for VH segments in vivo by
the recipient animals. The precursory frequency of the
VH1.1 germline sequence was established by dot hybridization
48
AMENDED SHE~T
CA 02221234 1997-11-14
W 096/36358 PCTrUS9GI'~6YO~
of immunoglobulin specific gene libraries with primers
specific for the VH1.1 gene segment. See, J. Sambrook, et
al., Molecular Cloning: A Laboratory Manual, 2nd Edition,
Cold Spring Harbor Laboratory Press, p. 9.52-9.55 (1989),
incorporated herein by reference. The frequency of VH1.1
gene expression was 1.2~ and 1.0~ in the newborn and naive
adult animals, respectively. The gene frequency was
increased to 16~ in the recipients of hamster xenografts at
Day 4 post-transplant and 10.3~ in the recipients at Day 21
post-transplant. Thus, the B cell subset(s) expressing the
specific IgM VH gene utilized in mediating the primary
humoral response of the rats to hamster xenografts exist- at
substantial levels in newborn and adult animals. These B
cells undergo rapid clonal expansion and express the VH gene
in a germline configuration after the challenge of rat
recipients with hamster xenografts. Sequence analysis of
clones from these libraries have demonstrated that the VH
genes used in response to the hamster heart xenografts as
well as the porcine xenografts are restricted to a small
number of closely-related genes.
The experiments demonstrating the use of Ig genes in a
germline configuration for controlling the immune response
to xenografts suggests that the B-la/B-lb B cell pathway is
important for the accelerated ("concordant") model of
xenograft rejection. This is an unexpected and potentially
important observation as this form of antibody production is
generally accepted to be responsible for the hyperacute
forms of xenograft rejection in more distantly related
species combinations. The data presented here suggest that
these two patterns of rejection (hamster-to-rat and pig-to-
hllm~n) share the same Ig gene usage for the control of the
humoral immune responses responsible for xenograft
rejection. The basic features of the xenograft rejection,
despite the involvement of widely divergent species,
49
CA 02221234 1997-11-14
W 096/36358 PCT~US9'!0C801
represent a rather stereotypical and relatively primitive
antibody response to endothelial antigens expressed by the
graft. The binding of these antibodies to similar antigens
expressed by many different species may be central theme in
the pathogenesis of the reaction and may provide the
opportunity to specifically target critical steps in the
rejection reaction. The ability of a single monoclonal
antibody to block the rejection of hamster heart xenografts
by rat recipients is clear support for the concept that
antibodies and/or their derived fragments will have a role
in preventing the xenograft reaction in many species,
including the pig-to-human combination.
D. Nucle;c Ac;~ An~ polypept;~s ~nro~;n~ An~ nn~r
Xenoaraft An t; ho~; es An~ An t~ ho~y M~ ter; Al
The present invention also encompasses isolated and
purified polynucleotide molecules encoding antibodies,
antibody material, and polypeptide of the present invention.
This invention also encompasses polynucleotide molecules
characterized by conservative changes in coding regions that
do not alter the phenotype of the polypeptide produced
therefrom when compared to the nucleic acid molecule
described herein.
This invention provides isolated and purified
polynucleotides comprising a nucleic acid sequence encoding
at least one of the following polypeptides: the polypeptide
defined by amino acid residues 1 through 354 of SEQ ID NO:
2; the polypeptide defined by amino acid residues 1 through
402 of SEQ ID NO: 5; the polypeptide defined by amino acid
residues 1 through 354 of SEQ ID NO: 3; the polypeptide
defined by amino acid residues 1 through 345 of SEQ ID NO:
18; the polypeptide defined by amino acid residues 106
through 151 of SEQ ID NO: 20. The present invention also
CA 02221234 1997-11-14
W 096/36358 PCTrUS96/OC80~
provides isolated and purified polypeptides defined by these
amino acid sequences.
As used herein, the term "polynucleotide" refers to a
contiguous sequence of DNA, RNA, or preferably cDNA. In
addition, as used herein, the term "polypeptide" encompasses
any naturally occurring allelic variant thereof as well as
man-made recombinant forms.
E. The Ph~ge Display Technique For Generating Anti-
Donor Xenogra~t ~nt;~ody Material
As discussed above, the present invention specifically
contemplates the use of the phage display technique in
combination with PCR amplification of immunoglobulin heavy
and light chain libraries as an alternative method of
producing and screening for anti-donor xenograft antibody
material of the present invention. The phage display
technique is particularly useful in producing and screening
human anti-donor xenograft antibody material. Although the
following discussion is provided in terms of a human as the
recipient animal and a pig as the donor animal, the skilled
artisan will appreciate that the same technique can be
modified for use with other animals and animal combinations.
Family specific primers are employed to generate
immunoglobulin heavy chain (consisting of VH region and part
of the C~,1) and light (kappa) chain (VK~ CK) CDNA libraries.
By combining these heavy and light chain fragments in a
random fashion and inserting them into a phagemid expression
vector, the heavy and light chain insert expression product
is targeted to the periplasm of E. coli for the assembly of
heterodimeric Fab molecules. In order to obtain expression
of antibody Fab libraries on a phage surface, the nucleotide
~ residue sequence encoding either the heavy or light ch~;n~
must be operatively linked to the nucleotide residue
sequence encoding a filamentous bacteriophage coat protein
CA 02221234 1997-11-14
W 096/36358 PCTrUS~G/0~0~
membrane anchor. A pre~erred coat protein for use in this
invention in providing a membrane anchor is cp III. In the
EXAMPLES described herein methods ~or operatively linking a
nucleotide residue sequence encoding a heavy chain to cp III
membrane anchor in a fusion protein of this invention are
described.
Preferably a phagemid vector is selected that contains
a single cistron consisting of an expression control
sequences operatively linked to a periplasmic secretion
signal (pelB leader) and a sequence encoding cpIII. The
presence of the pelB leader ~acilitates the secretion of the
heavy and light immunoglobulin chains from the bacterial
cytoplasm to the periplasmic space where it is cleaved off,
while cpIII provides a membrane anchor. The phagemid
expression vector suitable for production of the antibody
material of the present invention should also carry a
selectable resistance marker gene, a phage origin that
allows the vector to be replicated as a single stranded DNA
and subsequently packaged into phage particles and a
bacterial origin of replication that allows the vector to be
replicated in a suitable host as double-stranded DNA. A
presently prefered phagemid is the SurfZap~ Vector provided
in a kit by Statagene, La Jolla, California. Preferably,
the pelB leader sequence encodes a ~irst restriction site at
its 3' end and the cpIII sequence encodes a second
restriction site at its 5' end. These restriction site
allow nucleic acid sequences encoding immunoglobulin light
and heavy ch~; n~ to be inserted in the proper orientation as
one continuous strand.
Expression of the cistronic message encoding the pelB-
VK_VH_CP III fusion sequence leads to the formation of a
continuous amino acid seqence that is delivered to the
periplasmic space by the pelB leader sequence. The pelB
leader is subsequently cleaved and the VK - Vll chain is
CA 02221234 1997-11-14
W 09''36358 PCTrU~blOCYO~
anchored in the membrane by the cp III membrane anchor
domain. The heavy chain in the presence of the light chain
assembles to form Fab molecules.
With subsequent infection of E. coli with a helper
phage, as the assembly of the filamentous bacteriophage
progresses, the coat protein III is incorporated on the tail
of the bacteriophage thereby displaying the Fab on the
exterior of the phage particle. Phage particles diplaying
the Fab can then be screened for binding to antigen
expressed by endothelial cells o~ a xenograft by the panning
method described in the EXAMPLES. Positive phage can be
amplified and large quantities of pure Fab produced by co-
infection of phagmid and helper phage. The phagemid
immunoglobulin insert can also be sequenced either by
isolation of the insert or directly from the phagemid.
Thus, in accordance with the present invention there is
provided isolated and purified human anti-donor xenogra~t
antibody material that is immunoreactive with antigen
expressed by endothelial cells of the donor xenogra~t and is
capable of inhibiting antibody-mediated rejection of the
donor xenogra~t by a human. Pre~erably the donor xenogra~t
is donated by a pig, and more preferably, the pig xenograft
is a pig heart or pig liver.
In a related embodiment of the invention there is
provided the human anti-donor xenograft antibody material
described above, further comprising at least one polypeptide
encoded by a nucleic acid sequence including the sequence
defined by nucleic acid residues 1 through 345 of SEQ ID NO:
18 or residuesl through 357 of SEQ ID NO: 20, and at least
one human immunoglobulin light chain. Such antibody
materials can easily be generated by modification of the
[hage display technique described herein to use these heavy
chain sequences instead of the sequences obtained through
amiplification of the human heavy chain genome.
CA 02221234 1997-11-14
W 096/36358 PCTrUS9''~'U
In yet another embodiment of the present lnvention
there is provided polynucleotides and polypeptides encoding
the antibodies and antibody materials of the present
invention. Accordingly, this invention provides recombinant
anti-donor xenograft antibody material characterized as
immunoreactive with antigen expressed by endothelial cells
of a donor xenograft and capable of inhibiting antibody-
mediated rejection of the donor xenograft by a recipient
animal, wherein said antibody material comprises at least
one immunoglobulin light chain polypeptide and at least one
immunoglobulin heavy chain variable region polypeptide.
Preferably the immunoglobulin light chain polypeptide is
human or the immunoglobulin heavy chain variable region
polypeptide is human. Even more preferably, both
polypeptides are human.
In a related embodiment, nucleic acid encoding these
recombinant anti-donor xenograft antibody material is
provided. Such nucleic acids can be incorporated into
vectors, such as for example phagemid vectors including the
SurfZAP vector. As used herein the term "vector" refers to
a recombinant DNA molecule capable of autonomous
replication in a cell and to which a DNA segment, e.g., gene
or polynucleotide, can be operatively linked so as to bring
about replication of the attached segment. Vectors capable
of directing the expression of genes and coding for one or
more polypeptides are referred to herein as "expression
vectors."
This invention also provides a vector comprising an
isolated nucleic acid molecule such as DNA, cDNA or RNA
encoding anti-donor xenograft antibody material or the
peptide componetns thereof. Examples of additional vectors
useful herein are viruses, such as bacteriophages,
baculoviruses and retroviruses, cosmids, plasmids, and the
like. Nucleic acid molecules are inserted into vector
CA 02221234 1997-11-14
W 096/36358 PCTrUS9''~6~0
genomes by methods well known in the art. For example,
insert and vector DNA can both be exposed to a restriction
enzyme to create complementary ends on both molecules that
base pair with each other and which are then joined together
with a ligase. Alternatively, synthetic nucleic acid
linkers that correspond to a restriction site in the vector
DNA, can be ligated to the insert DNA which is then digested
with a restriction enzyme that recognizes a particular
nucleotide sequence. Additionally, an oligonucleotide
containing a termination codon and an appropriate
restriction site can be ligated for insertion into a vector
containing, for example, some or all of the following: a
selectable marker gene, such as neomycin gene for selection
of stable or transient transfectants in mAmmAlian cells;
enhancer/promoter sequences from the immediate early gene of
human CMV for high levels of transcription; transcription
termination and RNA processing signals from SV40 for mRNA
stability; SV40 polyoma origins of replication and ColE1 for
proper episomal replication; versatile multiple cloning
sites; and T7 and SP6 RNA promoters for in vitro
transcription of sense and antisense RNA. Other means are
available and can readily be accessed by those of skill in
the art.
Cells transformed with vectors of the present invention
are also provided, particularly including E. coli.
F. Methr~9~ of Tnh;h;1~ sr An~;hot~y-M~;~t~l X~n~r~ft
Rejec1-; on
The anti-donor xenograft antibody materials and anti-
donor antibodies of the present invention can readily be
used in the methods of the present invention.
- In accordance with the present invention there is
provided novel methods of inhibiting antibody-mediated
rejection of a xenograft from a donor An;mAl by a recipient
CA 0222l234 l997-ll-l4
W 096/363S8 PCTrUS9G/C~801
animal which comprises modifying antigen expressed by cells
of the xenograft, without causing lysis of the cells, so as
to inhibit specific binding of recipient anti-donor
xenograft antibody to said antigen. When in unmodified
form, these antigens expressed by cells of the xenograft are
capable of inducing an antibody-mediated immune response by
the recipient animal which, if untreated, results in the
rejection of the xenograft. Antigen targeted for
modification in accordance with the methods of the present
invention are preferably expressed by endothelial cells of
the donor xenograft.
One of skill in the art will appreciate that rejection
of a xenograft may involve several immunologic components
each having varying degrees of importance as rejection of
the xenograft proceeds to completion and is dependent upon
the combination of species selected for the xenograft
transplant. An objective of the present invention is to
disrupt the antibody-mediated (i.e., humoral as opposed to
cell-mediated) component of the rejection by directly or
indirectly modifying antigen presented by the xenograft in
such a manner that immunoreactivity (specific binding)
between antigen and recipient anti-donor xenograft antibody
is reduced or eliminated.
As used herein with regard to the methods of the
present invention, the term "recipient anti-donor xenograft
antibody" refers to antibody molecules produced by the
individual recipient of the xenograft that immunoreact with,
i.e., specifically bind, antigen expressed by the cells of
the xenograft and induce antibody-mediated rejection of the
donor xenograft. Accordingly, such antibodies may be
further designated herein by the species of the individual
recipient animal (e.g. human)~producing the antibodies
and/or the specific type of donor animal (e.g. porcine) from
which the xenograft originates. Thus, for example,
CA 02221234 1997-11-14
W 096/363S8 PCTAUS9~'0~80~
recipient anti-donor xenograft antibody produced by a human
and having specificity for a xenograft from a pig may be
refered to herein as human anti-porcine xenograft antibody.
Recipient anti-donor xenograft antibody include anti-donor
xenograft antibody that naturally occur in the recipient
prior to transplant of the xenograft (preforemd antibodies)
and anti-donor xenograft antibodies naturally produced by
the recipient after transplant of the xenogra~t.
One of skill in the art will appreciate that many
different strategies can be employed to directly or
indirectly modify antigen in accordance with the methods of
the present invention. In a presently prefered embodiment,
modifying antigen expressed by cells of the xenograft
comprises contacting non-lytic, anti-donor xenograft
antibody material with the antigen for a time, at a
temperature, and at a pH suitable to bind the antibody
material to the antigen, wherein said anti-donor xenograft
antibody material is characterized as immunoreactive with
antigen expressed by endothelial cells of the xenograft and
capable of inhibiting antibody-mediated rejection of the
xenograft by a recipient ~n;m~l . In a related embodiment,
the non-lytic, anti-donor xenograft antibody material is
derived from the same species of animal as the recipient
animal.
In accordance with the present methods, nonlytic, anti-
donor xenograft antibody material may be contacted with
antigen prior to transplant, after transplant or both.
Thus, antigen expressed by cells of the xenograft may be
modified by contacting the antibody material with the
antigen by ex vivo perfusion of the xenograft with a
solution comprising a preservative for the xenograft, such
as for example, Viaspir~ (DuPont-Merck Pharmaceuticals, Co.)
and the anti-donor xenograft antibody material for a time,
CA 0222l234 l997-ll-l4
W 096/36358 PCTrUS~6~ 01
at a temperature, and at a pH suitable to bind the antibody
material to the antigen.
The skilled artisan will appreciate that binding of
anti-donor xenograft antibody material with antigen
expressed by cells of the xenograft can be acheived and
optimized by adjusting such reaction parameters as time,
temperature and pH. The methods of the present invention
are typically performed at or below room temperature at
about physiological pH. Because the methods involve the use
of proteins, substantially higher temperatures acidity or
alkalinity which would substantially modify the tertiary and
quaternary structures of the proteins should be avoided.
Accordingly, conditions suitable for performing the methods
of the present invention generally range from about 1~C to
about 37~C, at about physiological pH. The time for
performing the methods, of course, will decrease in relation
to the increase in temperature at which the methods are
performed.
Alternatively or in addition to the ex vivo treatment
described above, anti-donor xenograft antibody material can
be administered to the recipient of the xenogeneic
transplant prior to and/or during the actual transplant
operation. A~lm;n; stration of the antibody material for this
purpose would be carried out along the lines and in amounts
generally known in this art. A therapeutically effective
amount would be predetermined and calculated to achieve the
desired effect, i.e., prolonging the survival time of the
xenograft. The required dosage will vary with the
particular treatment and with the duration of desired
treatment. Since the level of preformed anti-donor
xenograft antibody in the serum of a patient can readily be
determined by routine clinical analysis, dosages can be
taylored to the needs of the individual transplant
recipient. Thus, in yet another embodiment of the present
CA 0222l234 l997-ll-l4
W 096136358 PCTrUS96/OC801
invention, the continued administration of anti-donor
xenograft antibody material post-transplant is contemplated,
as needed.
In a related embodiment of the present method for
inhibiting inhibiting antibody-mediated rejection of a
~ xenograft from a donor animal by a recipient animal, in
addition to contacting the antigen with anti-donor xenograft
antibody material, said method further comprises
administering to said recipient animal a chemical
immunosuppressive agent.
When combination treatment (i.e., dual administration
of anti-donor xenograft antibody material and chemical
immunosuppressive agent) is employed, dosage levels for the
anti-donor xenograft antibody material are comparable to
levels presented above. Dosage levels for chemical
immunosuppressive agent typically fall in the range of about
1 to 1000 milligrams per kilogram of body weight.
Ordinarily, 5 to 750 and preferable 10-500 milligrams per
kilograms per dose is effective to obtain desired results.
Modes of administration as described above are suitable for
administration of chemical immunosuppressive agent.
Exemplary chemical immunosuppressive agents
contemplated for use in the practice of the present
invention are well-known in the art. Suitable
immunosuppressive agents include, for example, Cytoxan
(cyclophosphamide) azathioprine (AZA), corticosteroids (such
as prednisone), OKT3, FK506, mycophenolic acid or the
morpholinethylester thereof, 15-deoxyspergualin, rapamycin,
mizoribine, misoprostol, anti-interluekin-1 (Ih-2) receptor
antibodies, anti-lymphocyte globin (AhG), and the like.
co-administer post-transplant
In yet another embodiment of the present invention
there is provided a method of inhibiting antibody-mediated
rejection of a pig xenograft by a human, which comprises
59
CA 0222l234 l997-ll-l4
W 096/36358 PCTrUS9''0C~01
modifying antigen expressed by endothelial cells of the pig
xenograft, without causing lysis of the cells, to inhibit
binding of preformed human anti-porcine xenograft antibody
to said antigen, wherein said antigen present in unmodified
form induces an antibody-mediated immune response in the
human. In a related embodiment, antigen expresesed by
endothelial cells of the pig xenograft are modified by
contacting non-lytic, anti-porcine xenograft antibody
material with said antigen for a time, at a temperature, and
at a pH suitable to bind the antibody material to the
antigen, wherein said anti-porcine xenograft antibody
material is characterized as immunoreactive with antigen
expressed by endothelial cells of the xenograft and capable
of inhibiting antibody-mediated rejection of the xenograft
and is preferably characterized as a human antibody
- material. Anti-donor xenograft antibody material useful in
the methods of the present invention can be polyclonal or
more preferably monoclonol. Several such antibody materials
have been described above.
G. Me~h~A~ Of Tr~n~l~nt~n~ A ~n~gr~$t
In still another embodime~t of the present invention
there is provided methods for transplanting a xenograft in a
patient, said method comprising contacting said xenograft,
prior to transplantation,with anti-donor xenograft antibody
material for a time, at a temperature, and at a pH suitable
to allow said antibody material to immunoreact with antigen
expressed by said xenograft, and then transplanting said
xenograft. Such methods can further comprise administering
a therapeutically effective dose of said anti-donor antibody
material and optionally a chemical immunosuppressive agent
to said patient post-transplant
CA 02221234 1997-11-14
W 096136358 PCTrUS9f'0C80
H. Th~apeutic Compos;t; nn~
The present invention contemplates therapeutic
compositions useful for practicing the therapeutic methods
described herein. Therapeutic compositions of the present
~ 5 invention contain a physiologically tolerable carrier
together with anti-donor xenograft antibody material, as
described herein, dissolved or dispersed therein as an
active ingredient. In a preferred embodiment, the
therapeutic composition is not immunogenic when administered
to a mammal or human patient for therapeutic purposes.
As used herein, the terms "pharmaceutically
acceptable", "physiologically tolerable" and grammatical
variations thereof, as they refer to compositions, carriers,
diluents and reagents, are used interchangeably and
represent that the materials are capable of administration
to a mammal without the production of undesirable
physiological effects such as nausea, dizziness, gastric
upset and the like.
The preparation of a pharmacological composition that
contains active ingredients dissolved or dispersed therein
is well understood in the art. Typically such compositions
are prepared as injectables either as liquid solutions or
suspensions; however, solid forms suitable for solution, or
suspensions, in liquid prior to use can also be prepared.
The preparation can also be emulsified.
The active ingredient can be mixed with excipients
which are pharmaceutically acceptable and compatible with
the active ingredient and in amounts suitable for use in the
therapeutic methods described herein. Suitable excipients
are, for example, water, saline, dextrose, glycerol, ethanol
or the like and combinations thereof. In addition, if
desired, the composition can contain minor amounts of
auxiliary substances such as wetting or emulsifying agents,
61
CA 02221234 1997-11-14
W 096/363S8 PCT~US96/OC~01
pH buffering agents and the like which enhance the
effectiveness of the active ingredient.
The therapeutic composition of the present invention
can include pharmaceutically acceptable salts of the
components therein. Pharmaceutically acceptable nontoxic
salts include the acid addition salts (formed with the free
amino groups of the polypeptide) that are ~ormed with
inorganic acids such as, for example, hydrochloric or
phosphoric acids, or such organic acids as acetic, tartaric,
mandelic and the like. Also illustrative of such acid
addition salts are hydrobromide, sulphate, maleate, acetate,
citrate, benzoate, succinate, malate, ascorbate, and the
like. Salts formed with the free carboxyl groups can also
be derived from inorganic bases such as, for example,
sodium, potassium, ammonium, calcium, zinc, iron or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, 2-ethylamino ethanol, histidine, procaine
and the like.
Physiologically tolerable carriers are well known in
the art. Exemplary of liquid carriers are sterile aqueous
solutions that contain no materials in addition to the
active ingredients and water, or contain a buffer such as
sodium phosphate at physiological pH value, physiological
saline or both, such as phosphate-buffered saline. Still
further, aqueous carriers can contain more than one buffer
salt, as well as salts such as sodium and potassium
chlorides, dextrose, polyethylene glycol and other solutes.
Liquid compositions can also contain liquid phases in
addition to and to the exclusion of water. Exemplary of
such additional liquid phases are glycerin, vegetable oils
such as cottonseed oil, and water-oil emulsions.
62
CA 02221234 1997-11-14
W 096/36358 PCTrUS9G~ 1
I. Me~h~ Of T80l~;n~ Ant;g~n ~es~ed
l;!n~ t h~l; ;-1 C~l 18 Of the D~nr~r ~F~nr~r~t
The anti-donor xenograft antibodies and antibody
- materials of the present invention are particularly well
suited to isolate antigen expressed by endothelial cells of
a xenograft and which are characterized as inducing
antibody-mediated rejection of the xenograft by a recipient
animal. Accordingly, the present invention provides methods
of isolating such antigen comprising contacting anti-donor
xenograft antibody or anti-donor xenograft antibody material
with endothelial cell membrane lysate of the xenograft for a
time and at a temperature and pH suitable to form an
immunecomplex comprising said antibody, then seperating said
immune-complex from said non-complexed endothelial cell
membrane lysate, and seperating said anti-donor xenograft
antibody or antibody material from said antigen. Such
antigens include antigen which is characterized as inducing
antibody-mediated rejection of the xenograft by humans,
particularly those expressed by endothelial cells of porcine
xenografts-.
Such antigens can readily be isolated by
immunoprecipitation with the antibody or the antibody
material of the present invention. For example, PAEC can be
radiolabeled by growing the cells in the presence of
radioactive amino acids or radioactive amoino acid
precursors, as described for example in Harlow and Lane,
Ant;ho~;es: A T~horatory MAnnllAl,Cold Sprong Harbor
Laboratories, pp. 430-433 (1989), incorporated herein by
reference. Labeled cells can then be lysed as described by
Harlow and Lane, pp. 449, supra, incorporated herein by
reference, and preferably the membrane lysate seperated for
immunoprecitation. Antigen can then be ;mmllnoprecipitated
from the lysate as described by Harlow and Lane, pp. 465,
~upra, incorporated herein by reference. Immune-complex can
CA 0222l234 l997-ll-l4
W 096/36358 PCTrUS9f'0f801
then be seperated on two-dimensional SDS-PAGE gels,
extracted and seperated by high pressure liquid
chromatography.
The invention will now be described in greater detail
by reference to the following non-limiting examples.
V. Fi!~MPT.~;!.C:
The choice of the hamster-to-rat as the xenogeneic
transplant model was based on the consistently observed
patterns of rejection for cardiac xenografts among these
animals which are accepted as representing both accelerated
and hyperacute patterns of xenograft rejection. The pig-to-
rat model was also employed since pigs provide a preferred
choice for xenogeneic organ transplant to humans because
they can be bred easily, their organs are similar in size
and function to human organs, and there is a reduce risk of
disease transmission to man from the use of pig organs for
transplant as compared to non-human primate organs.
All rodent animals brought into the colony are
certified virus-free and the colony is monitored regularly
for accidental contamination with infectious diseases. The
animals are maintained in individual micro-isolator cages,
inspected twice daily and fed standard rodent pellet diet
and water ad libi tum. Any procedures that might have
produced pain or discomfort to these animals was conducted
under fluothane and/or pentobarbital anesthesia. The method
of euth~n~Ria is by exsanguination.
The animal facilities at Cedars-Sinai Medical Center
are accredited by the American Association for Accreditation
of Laboratory Animal Care and the ~n;m~l s included in these
studies were handled humanely in accordance with animal
64
CA 02221234 1997-11-14
W 096136358 PCTrUS3f'~C804
experimental protocols approved by the Institutional Animal
Care and Use Committee.
Adult LEW rats (6-8 weeks old) were purchased
commercially from Harlan Sprague-Dawley (Indianapolis, IN).
Donors for the appropriate xenografts and other lymphoid or
endothelial tissues included young Syrian Golden hamsters
(Harlan Sprague-Dawley), DBA/2 mice (Jackson Laboratories,
Bar Harbour, ME), newborn NZ rabbits (200-250 gm; Irish
Farms) and young (220-260 gm) Hartley guinea pigs (Charles
River Breeding Laboratories, Wilmington, MA).
Farm pigs used in the routine surgical training of
residents in the Department of Surgery at Cedars Sinai
Medical Center were used as a source of pig tissue for these
experiments.
Example 1 - Xenograft Transplant
Intra-abdominal heterotopic ACI rat, hamster, mouse,
rabbit and guinea pig cardiac xenograft transplants into
rats are performed in accordance with the techniques
described in Cramer, D.V. et al., Tr~n.~l~ntat;on, 53: 303-
308 (1992), incorporated herein by reference. The donoranimals are anesthetized with ketamine (100 mg/kg), xylazine
(10 mg/kg), and atropine (0.05 mg/kg) administered
intraperitoneally, and then maintained as necessary on
methoxyflurane via inhalation. The venae cavae and the
pulmonary veins are ligated with 5-0 silk, and the pulmonary
artery and aorta are transected 2-3 mm above their origins
in the heart. After perfusion of the ventricles and atria
with lactated Ringer's solution (containing 200 units/ml of
heparin), the heart is placed in a saline bath at 4~C.
Recipient animals are anesthetized as described above, a
midline incision is made, and the great abdominal vessels
are dissected free from the left renal vein to the
CA 02221234 1997-11-14
W 096/36358 PCTrUS9~'0C~04
bifurcation. The graft is implanted in the abdominal cavity
with end-to-side anastomoses of the donor to recipient
aortas and of the pulmonary artery to recipient vena cava in
a running continuous suture with 10-0 Novafil on a TE-70
needle. Operative times range from 30 to 45 min, with a
success rate of approximately 90~. The grafts are evaluated
for function by abdominal palpation and all grafts are
removed for ~m; n~tion at the termination of the
experiment. At removal the hearts are fixed in 10~ buffered
formalin for 24 hr and then stored for histological
processing.
Rejection is considered to have occurred when the
xenograft stops functioning, i.e., heart stops beating.
Hyperacute rejection is considered to have occurred when the
xenograft is rejected within one hour, or more preferably
within 10 minutes. Accelerated rejection is considered to
have occured when the xenograft has survived for for at
least about a
Example 2 - Histopathology and Antigen Distribution
Histologic and immunopathologic ~Am;n~tions of the
cardiac xenografts and the major organs from normal hamsters
to study rat anti-hamster xenograft monoclonal antibody
target antigen distribution are performed as described
previously in Cramer, D.V., et al. J. ~eart Jl~ng Tr~ns~1 ~nt,
11: 458-466 (1992), incorporated herein by reference.
Briefly, hamster heart samples are collected immediately
following rejection or at 48 hours in those instances in
which hyperacute rejection does not occur. After
harvesting, the cardiac xenografts are immediately washed
with a 0.9~ NaCl solution. One portion of the graft is
embedded in O.C.T. compound (Tissue-Tek, Miles Inc. Elkhart,
IN) and frozen by immersion in an iso-pentane solution
CA 02221234 1997-11-14
WO g~ 3Ç358 PCT/US9-'OC~Cq
prechilled in dry ice. The r~m~;n~er of the graft is fixed
in 10~ bu~fered ~ormalin and prepared for routine
histological ex~m;n~tion.
Five micron cryostat sections are cut from frozen
tissues and examined with immunofluorescent staining for the
deposition of rat immunoglobulins and complement. Tissue
sections are incubated with the antibody of interest,
followed by isotype specific antibody and an enzyme-labeled
secondary antibody, e.g., mouse monoclonal antibody against
rat IgM ~ chains (MARM-4, BPS, Inc., Indianapolis, Indiana),
goat-anti-rat complement C3 antibody (The Binding Site,
Birmingham, England) and FITC-conjugated goat-anti-mouse IgG
(Caltag Laboratory, Ontario, Canada) may be used.
To block nonspecific binding, normal hamster serum is
added to the secondary antibody in PBS (1:10; v/v). The
immunoperoxidase sections are chromonized with 3,3'-
Di~m;nohenzidine tetrahydrochloride (DAB, Sigma Chemical)
and counterstained with diluted hematoxylin.
Immunofluorescent slides are studied under a Nikon
fluorescent microscopy with absorbance at 490 nm.
Example 3 - Monoclonal Antibody Production
Rat anti-donor xenograft monoclonal antibodies (hamster
or pig donors) are prepared by transplantation of LEW rats
with hamster heart xenografts or immunization of LEW rats
with isolated PAEC in suspension. Although the following
example is discussed in terms of hamster/rat and pig/rat
species combinations, the skilled artisan will appreciate
that the following method can be modified to generate
monoclonal antibodies for other species combinations.
For the production of rat anti-hamster xenograft
monoclonal antibodies, LEW rats are transplanted with
hamster heart xenografts as described in EXAMPLE 1. The
CA 02221234 1997-11-14
W 096/36358 PCTrUS9~hEC1
xenograft is rejected at Day 4 post-transplant and the
recipient spleens harvested for cell fusion at rejection.
For the production of rat anti-porcine xenograft
monoclonal antibodies, LEW rats are initially immunized by
intraperitoneal injection with 105 whole PAEC in PBS
followed by a second immunization on day 14 with 107cells in
PBS. A third immuniztion of 5 x 106 cells is administered
at day 21. At day 24 the rat's spleen is harvested for cell
fusion.
The splenic lymphocytes (Io8 cells) are mixed with
YB2/0 rat myeloma cells (ATCC, Rockville, MD) in serum-free
DMEM at a 1:1 ratio, and centrifuged at 500 xg for 5 minutes
to pellet the cells. After removing the supernatant, 1 ml
of prewarmed (37~C) 50~ PEG is added to disrupt the cell
pellet. The suspension is swirled for 2 minutes at 37~C,
and the mixture diluted with 1 ml of DMEM for 1 minute,
followed by the addition of another 1 ml DMEM for one more
minute, then 7 ml of DMEM over a course of 2 to 3 minutes.
The cells are then centrifuged at 500 xg for 5 min. The
cell pellet is gently resuspended in 10 ml of 20~ complete
DMEM, then diluted to a concentration of 2.5 x 106 cells/ml.
Aliquots (0.2 ml) of the cell mixture are placed in
individual wells (96 well plates) and incubated at 37~C in
7~ COz.
Hybridoma cells are selected by incubation in HAT
medium for a minimum of 2 weeks, beginning on the day after
fusion. Supernatants from individual wells are screened for
production of IgG and IgM antibodies in an ELISA format as
described in EXAMPLE 5 and the ability of the antibodies to
immunoreact with antigen expressed by hamster endothelium as
described in EXAMPLE 6 or PAEC as described in EXAMPLES 7,
below. Clones derived from wells that are positive in the
screening process are cloned by limiting dilution in 20~
DMEM. Several weeks after the initial cloning, positive
68
CA 02221234 1997-11-14
W ~96136358 PCTrUS9G/OC801
clones are subcloned again by limiting dilution at 0.3
cells/well. The antibody secreting clones are then analyzed
by the assays described herein.
Example 4 - Cell Culture Technique~
Pi~ aortic endothel;al cells. Pig thoracic aortas are
removed using sterile conditions and placed in
RPMI/antibiotic medium (RPMI containing penicillin,
streptomycin and glutamine (lx antibiotic)). Three 50 ml
tubes are filled with RPMI/antibiotic medium, approximately
35 mls are needed to cover the length of the aorta.
Harvested aortas are each placed in a petri dish. Using the
forceps and scissors the connective tissue (CT) and RBCs are
completely removed ~rom the outside of the aorta. RBC
contamination will interfere with the culture. Once all the
CT is removed, the aorta is placed in the first 50 ml tube
containing media and shaken slightly to help remove any RBCs
inside the aorta. If the aorta looks clean, it is moved on
to tube 2 and then tube 3. Each well of a six-well tissue
culture plate (Baxter T4133-2, Corning) that has been coated
with 2 ml each of 2~ sterile gelatin in lx PBS (Sigma G-
1890) and brought to 37~C, washed with RPMI/antibiotic media
(approximately 3 mls) by pipetting media into well, allowed
to sit for approximately 15 seconds and discarded into
waste. RPMI/antibiotic media (0.5 ml) + 10~ FBS is added to
each well. The aorta is removed from the third tube and
placed in a clean petri dish. The aorta is turned dorsal
side up and the aorta cut open longways. A 1.5 cm section
of the aorta is gently scraped with surgical blade and the
blade is dipped into one well of the 6 well plate. Scraping
is repeated in 1.5 cm increments for all six wells. Each
well is mixed to help break up the cells. If sheets of
cells are seen under a microscope, resuspend more. If no
CA 02221234 1997-11-14
W 096/36358 PCTrUS9G/OC~O~
cells are seen, the aorta is scraped again. The plate is
placed in CO2 incubator.
Endothelial cells (PAEC) are passaged when they reach
confluence. Experiments are performed on cells between
passages 4 and 14, preferably using cells from the earlier
passage rather than the latter.
TCPKl cells. LCPKl cells (also referred to as minipig
kidney cells) can be obtained from the ATCC, Accession No.
CRL-1392. Once cells are thawed and rest overnight, the
cells are rinsed with serum free media (5 ml for T75 flask,
7 ml for T162) and the media is discarded. Three mililiters
lx Trypsin-Edta (Gibco) are added and the cells incubate at
37~C, 6~ CO2for 3-5 minutes. When 85-95~ are floating an
equal volume of growth media is added and the cells pelleted
at 1500 rpm at room temperature for 5 minutes. Supernatant
is discarded and the cells washed in 3-5 ml growth media and
pelleted again. Cells are resuspended in 1 ml growth media
per flask and incubated at 37~C, 6~ CO2. Cells are
maintained at 37~C in Ml99 growth media (Gibco, Grand
Island, NY) supplemented with 3~ fetal calf serum. (Irvine
Scientific) The cells are passaged when they reach 90~
confluence and are usually ready for use at passages 5 to
10 .
T~m~hocytes. Lymphocytes are isolated from the spleens
of miniature pigs (Charles River Breeding Laboratory,
Wilmington, MA). Under sterile conditions, 15-20 g of pig
spleen is passed through stainless steel sieves into 20-40
ml of RPMI containing antibiotics (300 ~/ml penicillin and
300 ~g/ml streptomycin), followed by passage through nylon
mesh (Tetko, Inc., Monterey Park, CA). The lymphocytes are
isolated by density centrifugation (Histopaque 1077; Sigma
Chemical Co., St. Louis, MO), washed 3 times with RPMI, and
CA 02221234 1997-11-14
W 096/3635S PCTAUS~'n~01
then pelleted by centrifugation at 1000 xg for absorption
studies.
Example 5 - T ~globulin Isotype E~ISA
The isotype of antibodies presents in the serum of
recipient ~n; m~ 1 S, secreted by hybridomas, and binding to
tissue or cells the donor, particularly endothelial cells
and lymphocytes were characterized and quantified in an
ELISA format.
After washing, the cells are incubated with monoclonal
antibodies (1:10) and antibody binding to the target cells
is detected using affinity-purified mouse anti-rat IgG or
IgM conjugated with alkaline phosphatase (Accurate Chemical
and Scientific Corporation, Westbury, NY). Absorbance at
405 nanometers is determined after the colorimetric reaction
is developed for one hour using 1 mg/ml of p-nitrophenyl
phosphate and 100 mM diethanolamine in 0.5 mM MgCl2 (Bio-Rad
Laboratories, Richmond, CA).
Hybridoma supernatants are screened for immunoglobulin
(IgM) production using ninety-six (96) well flat bottom
plates (Corning Costar Corporation, Cambridge, MA) coated
with goat ~rat IgM (Accurate) antibody (1:2500 of 1 mg/ml
stock) in lM carbonate-bicarbonate buffer (Sigma Chemical,
Saint Louis, MO) and incubated overnight at 4~C. The plates
are blocked with 5~ BSA, washed, and incubated at room
temperature for 1 hour with the undiluted (neat) hybridoma
supernatants. The plates are then washed and incubated with
goat ~rat IgM alkaline phosphatase conjugated antibody
(1:5000) (Accurate Chemical and Scientific, Westbury, NY) at
room temperature for 30 minutes. Absorbances at 405
nanometers are determined after the colorimetric reaction is
developed for 30 to 45 minutes using 1 mg/ml of p-
CA 02221234 1997-11-14
W 096/36358 PCT/U~,G~CC~0~
nitrophenyl phosphatase and 100 mM diethanolamine in 0.5 mM
MgCL2 (Bio-Rad Laboratories, Richmond, CA).
Example 6 - Antibody B;nA;ng to Hamster Cell
Since hamster endothelial cells can be difficult to
culture, hamster tissue sections is used as the source of
antigen to detect immunoreactivity of anti-hamster xenograft
antibodies, anti-rat xenograft antibody material and
preformed rat anti-hamster xenograft antibodies. Of course,
one of skill in the art will appreciate that tissue sections
for other donor animals can be substituted in the following
assay.
Frozen tissue ~ections are prepared as described above
in EXAMPLE 2 and incubated with the antibody, serum or
antibody material of interest, followed by isotype specific
antibody and an enzyme-labeled secondary antibody, e.g.,
mouse monoclonal antibody against rat IgM ,b chain or K
chain(MARM-4, BPS, Inc., Indianapolis, Indiana), goat-anti-
rat complement C3 antibody (The Binding Site, Birmingham,
England) and FITC-conjugated goat-anti-mouse IgG (Caltag
Laboratory, Ontario, Canada) may be used.
Example 7 -T~T.T.~z~ for T s~reactivity Antigen of Donor Cell~
The binding of preformed anti-donor xenograft
antibodies in serum, anti-donor xenograft monoclonal
antibodies, or anti-donor xenograft antibody material are
measured with this colorimetric assay. One of skill in the
art will appreciate that the following assay can be modified
to detect immunoreactivity with other types of cells than
the specific cells discussed below.
CA 0222l234 l997-ll-l4
W 096136358 PCT~U~9~ 01
Pig aortic endothelial cells (PAEC) are harvested as
described in EXAMPLE 4 and cultured in RPMI with 10~ fetal
calf serum in 96 well plates (Costar Corp., Cambridge, MA)
until confluence. The PAEC are then fixed with 0.1~
glutaraldehyde. Non-specific binding is blocked by a one
hour incubation in RPMI 1640. Blocking solution is removed
and monoclonal antibody supernatant, antibody material, or
human serum (1:10 dilution) is incubated for one hour at
room temperature. The wells are washed twice with RPMI 1640
and antibody binding is detected by adding affinity-purified
anti-donor isotype-specific secondary antibody conjugated
with an enzyme label, e.g., goat anti-rat IgG or IgM
antibody conjugated with alkaline phosphatase or goat anti-
hllm~n IgG or IgM antibody conjugated with alkaline
phosphatase (both available from Accurate Chemical and
Scientific Corporation, Westbury, New York). The secondary
antibody is allowed to incubate at room temperature for one
hour. Following three washes with RPMI 1640, the
colorimetric reaction is developed at room temperature for
one hour, using 1 mg/ml of p-nitrophenyl phosphate and lOOmM
diethanolamine in 0.5M MgCl (Bio-Rad Laboratories, Richmond,
CA) Absorbance is read on an automatic micro plate reader at
405nm. A reading of at least two times background or a
control, and more preferably at least 2.5 times background
or a control is considered positive.
Example 8 - Flow Cytometry
The results of the ELISAs to detect immunoreactivity of
antibody and antibody material can be compared to similar
experiments conducted with the same cells using flow
cytometric analysis. Flow cytometric analysis detects
surface expressed antigens on cells. The cells of interest
(lx106) are incubated with the antibody of interest
CA 0222l234 l997-ll-l4
W O9f~36358 PCTrUS9G/0~ 1
(monoclonal supernatant, serum, antibody material). The
cells are washed and incubated with 100 ~l of a 1/100
dilution (1 mg/ml initial concentration) of FITC-conjugated
affinity-purified anti-donor isotype-specific secondary
antibody, e.g., mouse anti-rat IgG or IgM (Serotec,
Cambridge, England). Followiny three washes with PBS at
room temperature, fluorescence is read on a FACScan (Becton
Dickinson).
Example 9 - Flow Cytometric Cytotoxicity AQRay
This assay measures antibody-mediated, complement-
dependent cytotoxicity and is used to monitor antibody or
antibody material cytotoxicity for antigen expression on
donor animal cells, such as for example, PAEC or hamster
lymphocytes. In order to perform this assay, 5 x 105 donor
cells (PAEC or hamster spleen lymphocytes) in RPMI are
incubated at room temperature or on ice for 30 minutes with
serial two-fold dilutions of the antibody or antibody
material of interest (e.g., rat anti-porcine xenograft
monoclonal antibody or the rat anti-hamster xenograft
monoclonal antibodies) in medium. The cells are washed and
rabbit serum (Low Tox, Cedarlane Laboratories) is used as a
source of complement. Sixty ~l rabbit complement is added
to the cells and allowed to incubate for another hour.
Propidium iodide is added to the samples, incubated for 5
minutes, and washed with PBS in accordance with Wetzsteon,
P.J., et al., A. ~llm. Immllnol . 35: 93-99 (1992),
incorporated herein by reference. The samples are then
ready to be read by the FACScan. The uptake of propidium
iodide is indicative of cell death. More than about 20~
cell death is considered to indicate that the antibody is
cytotoxic. Rat anti-hamster serum (1:20 dilution) or rat
74
CA 02221234 1997-11-14
W 096/36358 PCT/U~ o~r~
anti-pig serum (1:20 dilution) is employed as positive
controls and normal hamster or pig serum as negative
controls. The cytotoxic titre of the supernatants are
expressed as a reciprocal of the last dilution exhibiting
more than 20~ cytotoxicity.
Example 10 - We~tern Blots
Aortic endothelial cell or lymphocyte membranes are
extracted as described earlier by Tuso, P.J., et al.,
Tr~nsplantatio~ 56: 651-655 (1993), incorporated herein by
reference. Protein is extracted from the membranes with
0.5~ Triton X-100 in 0.15 M NaCl, 1 mM EDTA, 0.062 M Tris,
9.2 mM 6-aminocaproic acid, 1 mM N-ethylmaleimide (NEM), 1
mM PMSF at 4~C for 24 hours. The extract is precipitated
with ethanol at -70~C and the protein content determined by
dye-binding assay as described by Smith. P.K., et al., Proc.
N~tl. Ac~d. Sc;. USA 85: 4015 (1988), and incorporated
herein by re~erence.
Membrane proteins (10 ~g) are fractionated on a SDS-
PAGE gel using a discontinuous method using 10~ separating
and 3~ stacking gels. The proteins are then transferred to
nitrocellulose filter (8 hours at 200 mA). The
nitrocellulose filters are then incubated with saturating
concentrations of the antibody of interest for 1 hour,
followed by an alkaline phosphatase-conjugated mouse anti-
rat IgM or IgG (Accurate, dilution 1:2500). Thenitrocellulos filters are then developed using alkaline
phosphatase substrate kit (Bio-Rad Lab, Richmond, CA).
Example 11 - Radioi.. unoprecipitation
The molecular weight of the target antigens recognized
by rat anti-porcine xenograft monoclonal antibodies are
CA 0222l234 l997-ll-l4
W 096/363S8 PCT~U$9G/06801
identified by immunoprecipitation, and compared to the
molecular weights of the antigens recognized by preformed
anti-porcine xenograft antibodies in human serum that react
with PAEC.
Pig aortic endothelial cells (5x 106) cultured as
described above are harvested using trypsin-EDTA (Gibco-BRL,
Gaithersburg,Maryland), resuspended in fresh RMPI-1640
medium supplemented with 10~ FCS and incubated for 30
minutes at 37 C to minimize any proteolytic damage to the
relevant antigens. The cells are washed with supplemental
PBSxl, resuspended in PBS containing 0.5M sodium phosphate
buffer, pH 7.4, and labeled with l2sI by adding, over a
period of 15 minutes, 0.5 mCi NaIl2s, 200 ~1 of 20 ~M
lactoperoxidase, (Signman,St.Louis, MO) and 250 ~l 0.03
hydrogen peroxide, and incubating at 4~C for 15 minutes.
Radiolabeled cells are washed three times with 15mM NaI
in PBS and incubated with monoclonal antibody supernatants
for one hour at 4 C. The cells are then lysed with double
lysis buffer (1~ NP-40 and 0.1~ SDS) and antigens are
precipitated by incubation of the lysate with CNBR-activated
Sepharose 4B beads (Pharmacia, Piscataway, NJ) coupled to
mouse anti-rat IgG or IgM monoclonal antibodies
(Serotec,Oxford, England). The immunoprecipitated pellet is
washed extensively, then denatured in sample buffer (dd H2O
4 ml, Tris 1 ml, Glycerol 0.8 ml, 10~, 1.6 ml 2-ME and
0.4 ml 0.05~ BPB). The eluted material is subjected to
electrophoresis on a 10~ discontinuous polyacrylamide slab
gel and transferred to nitrocellulose membrane. Bands were
visualized by exposure of dried nitrocellulose blots to
Kodak X-Omat film.
CA 02221234 1997-11-14
W 09~363~8 PCTrU~/OG8C1
Example 12 - Construction of V~ and VL Specific
Rat Anti-Donor cDNA Libraries
a. RNA Isolation
Total RNA is extracted from hybridoma cells using a
method described by Chomczynsky, P. and Sacchi, N., ~n~lyt
~3;ochem 162:156 (1987), incorporated herein by reference.
Briefly, 106 hybridoma cells are washed twice in PBS and
resuspended in 1 ml of a solution containing; GuSCN, 4M;
NaCitrate pH 7.0, 1.5 mM; and Sarcosyl 0.5~. After
phenol/chloroform:isoamyl-OH extraction, total RNA is
precipitated twice in isopropanol at -70~C for one hour
periods. The final pellet is resuspended in 50 ~l DEPC
(Sigma Chemical, Saint-Louis, MO) treated deionized water
and the concentration of the RNA established in a
spectrophotometer.
b. Definition and synthesis of primers
All the oligonucleotides used for PCR amplification and
colony hybridization were defined using the PCGene~
software. The oliogonucleotides were synthesized using core
support facilities at the Cedars-Sinai Research Institute.
c. Construction of ~ and K cDNA libraries
A linker-mediated polymerase chain reaction (PCR)
procedure was used to construct the cDNA libraries. A
similar procedure for use with T cell receptors is described
at Shirwan, H., et al., J. Imml]nol . 150: 2295-2304 (1993),
and Shirwan, H., et al. J. Immllnol~ 151: 5228-5238 (1993),
incorporated herein by reference. This method employs a
double-stranded synthetic linker of known sequence ligated
to the 5' end of the double stranded DNA and allows the
77
CA 02221234 1997-11-14
W 096/36358 PCTrUSg~ O~
amplification and characterization of all unknown V, D~, and
J genes rearranged to the constant region.
~e~vy ch~;n ~ : The first strand of cDNA is transcribed
from 3 ~g of total RNA using a C~primer "RCM1" (SEQ ID NO:
13) and a cDNA synthesis kit according to the manufacturer's
instructions (Boerhinger-Mannheim, Indianapolis, IN). After
the synthesis of double-stranded cDNA duplex, a double
stranded synthetic linker "SAX" (SEQ ID NO: 14) is ligated
to the 5' end of the double-stranded cDNA. One ~l T4 DNA
ligase (Boehringer Mannheim) is added to 5 ~l cDNA sample, 1
~l SAX/XAS, 2 ~l dH2O, and 1 ~l T4 buffer and incubated at
16~C for 24 hours. The excess linker is removed by
microcentrifugation (Microcon-100, Amicon, Beverly, MA) and
the puri~ied cDNA used as a template for PCR amplification.
Amplification is carried out using SAX (SEQ ID NO: 14)
and the "RCM3" C~ primer (SEQ ID NO: 15) as forward and
inverse primers, respectively. RCM3 is located upstream of
RCM1 on the constant region. (See, Fig.1) Two microliters
of cDNA are used in a 25 ~l reaction that contains: RCM3 (50
ng/~l), 1 ~l; SAX (50 ng\~l), 1 ~l; Tris-HCl, 10mM; MgCl2,
1.5mM; KCl, 5OmM; dATP, dCTP, dGTP, dTTP, 0.2mM each; and
Taq DNA polymerase, 1 unit. The PCR conditions are:
denaturation at 94~C for 60 seconds, annealing at 60~C for 60
seconds, and extension a 72~C for 60 seconds; 35 cycles on a
DNA Thermal Cycler 480 (Perkin Elmer Cetus, Norwalk, CT).
Ten microliters of the reaction are run on a 1.4~ agarose
gel to check for amplification products and to confirm the
absence of contamination as demonstrated by the lack of a
signal when water alone is used as the negative control.
One microliter of PCR products is cloned at 12~C for 16
hours into the pCR~ vector of the TA cloning kit~
(Invitrogen, San Diego, CA)in accordance with the
manufacture's instructions. Two microliters of this
CA 0222l234 l997-ll-l4
W OgG,~36~58 PCT~US9~'0C~01
reaction (10 ng vector) are used to transform "One-Shot E.
Coli~ by thermic shock according to the manu~acturer~s
instructions (Invitrogen, San Diego, CA). Two hundred
microliters of the transformation reaction are spread on
~uria-Bertani petri dishes to which ampicillin, 100 ng/~l
(Sigma Chemical, Saint-Louis, MO) and X-Gal (50 ~1, 20
mg/ml) are added.
T,; ght ch~ n K: The synthesis of double stranded cDNA from
3 ~g of total RNA extracted from HAR-1 cells is similar to
the method described for the ~ chain. The synthesis of the
first strand, however, uses the oligonucleotide poly(dT) 15
from the cDNA synthesis kit. A ~irst round of ampli~ication
is carried out using SAX (SEQ ID NO 14) and a C~, primer
referred to as "RCK1" (SEQ ID NO 16) as ~orward and inverse
primers, respectively. A "proof-reading" DNA polymerase,
Ultma DNA polymerase (Perkin Elmer) is used to amplify 4 ~1
of cDNA in a 100~1 reaction that contained: 10X Ultma
Buffer, lX; MgCl2 25mM, 1.5mM; dATP, dCTP, dGTP, dTTP, 40~M
each; SAX (50 ng/~l), 4~1; RCK1 (50 ng/~l), 4~1; Ultma AND
polymerase, 0.5 unit. The PCR conditions are: denaturation
at 94~C for 45 seconds, annealing at 55~C for 1 second, and
extension at 72~C for 60 seconds; 30 cycles on a DNA Thermal
Cycler 480 (Perkin Elmer Cetus, Norwalk, CT). The PCR
products are fractionated on a 1.2~ low melting point
agarose gel (Gibco BRL, Gaithersburg, MD) and the cDNA
fragments of 492 to 1107 bp purified by phenol/chloroform
extraction-ethanol precipitation, resuspended in 15 ~1 Tris-
EDTA and subjected to another round of PCR amplification.
One microliter of this solution is reamplified using
SAX as forward primer and a C~ primer referred to a RCK3
(SEQ ID NO 17) as a reverse primer. RCK3 is located
upstream of RCKl on C~. This second "nested" amplification
uses the enzyme Taq as DNA polymerase so as to generate PCR
CA 02221234 1997-11-14
W 096/36358 PCT/US9~06~-1
products with unpaired deoxyadenosine on their 3' ends, a
condition necessary for efficient cloning into the pCR~
vector. The PCR conditions are as above for Taq, except
that the number of cycles is limited to 25. One microliter
of PCR products are cloned in the pCR~ vector, in accordance
with the manufacturer's direction, and E. coli bacteria
transformed as described above. Two hundred microliters of
the transformation reaction are spread on ~uria-Bertani
petri dishes to which ampicillin, 100 ng/~l (Sigma Chemical,
Saint-Louis, MO) and X-Gal (50 ~l, 20 mg/ml) are added.
Example 13 - Screening of ~ and K cDNA Librarie~
Recombinant colonies are first identified by color
screening based on the demonstration of the loss of ~-
complementation. White colonies are subsequently screenedby PCR using SAX/RCM3 and SAX/RCK3 as primers for the ~ and
K library, respectively. Briefly, each individual colony is
harvested with a sterile tooth-pick, resuspended in 100 ~l
sterile double distilled water, and incubated at room
temperature for 30 minutes. Each tube is vortexed briefly
and 4 ~l used as a template in a 10 ~l reaction that
includes: SAX (50 ng/~l), 0.8 ~l; anti-sense primer (50
ng/~l), 0.8 ~l; Tris-HCl, 10mM; MgCl2, 1.5mM; KCl, 50mM;
dATP, dCTP, dGTP, dTTP, 0.2mM each; and 1 unit of Taq DNA
polymerase. The PCR conditions are: denaturation at 94~C
for 20 seconds, annealing at 55~C for 60 seconds, and
extension at 72~C for 60 seconds; 28 cycles on a GeneAmp PCR
system 9600 (Perkin Elmer Cetus, Norwalk, CT). Colonies of
interest are identified by the demonstration of an insert of
the expected size (~ 600 bp for SAX/RCM3 and 460 bp for
SAX/RCK3). Within each library, a m; n; m~lm of 4 positive
colonies are used to obtain cDNA sequence information.
CA 02221234 1997-11-14
W 096/363S8 PCTAUS9~
Example 14 - Plasmid DNA Extraction and Sequencing
Each positive colony was incubated at 37~C ~or 18 hours
in culture medium containing ampicillin (100 ng/~l).
Plasmid DNA is extracted by alkaline lysis according to
Sambrook, J., et al., Molecul~r Clo~1ng. A Tl~horatory ~nual
Cold Spring Harbor Laboratory Press (1989) and sequenced on
both strands with the Sanger dideoxynucleotide chain
termination method as used in the Sequenase Plasmid
Sequencing kit (United States Biochemicals, Cleveland, OH).
Example 15 - Extraction and Ampli~ication
o$ Germline V~ DNA from Rat Liver
LEW liver is harvested and digested for 16 hours at
55~C in a solution o~ proteinase K, 10 mg/ml; Tris, 50 mM;-
EDTA, 100 mM; NaCl, 100mM and SDS 1~. After
phenol/chloroform extraction the DNA is precipitated with
ethanol. Ten nanograms of DNA are amplified with Ultma DNA
polymerase using the parameters already described ~or that
enzyme. Amplification primers were chosen based on the
sequence information for VH segment of HAR-l(Figure 1). The
sense primer referred to here as "RVH1" (SEQ ID NO 7)is
designed to anneal immediately before the initiation codon.
The anti-sense primer, referred to here as "RVH2" (SFQ ID
NO 8), is designed to anneal on framework region 3,
downstream o~ the first two complementarity determining
regions. The PCR conditions are as already described for
the Ultma enzyme.
The amplification products are treated with Taq DNA
polymerase so as to add unpaired deoxyadenosine to the 3'
end of the molecules and to allow for subsequent cloning in
the pCR~ vector. For that purpose, amplification products
generated with Ultma are fractionated by electrophoresis on
a 1.4~ LMP agarose gel, purified by phenol/chloroform,
81
CA 02221234 1997-11-14
W 096/36358 PCTrUSg~'.6~-1
precipitated in ethanol and resuspended in 20 ~1 Tris-EDTA.
One unit of Taq DNA polymerase and 4 ~1 lmM dATP are added
and the reaction carried out at 72~C for 10 minutes. The
excess of Taq is removed by phenol/chloroform, the reaction
products precipitated by ethanol, and the volume brought
back to 20 ~1. Two microliters of treated cDNA are cloned
in the pCR~ vector and E. col i transformed as already
described. Recombinant colonies are searched for using PCR
amplification with RVH1/RVH2. Four separate clones that
demonstrate an insert of the expected size (~460 bp) are
selected and both strands sequenced.
Example 16 - Anti-donor Xenograft Antibody Material
By Phage Di~play Technique
The following method of making anti-donor xenogen
antibody material is described in term of a the generation
and screening of a human IgM heavy chain, human IgK light
chain Fab library displayed through the use of filimentous
phage encoded by the SurfZAP~ Vector (Stratagene, La Jolla,
CA) and the SurfZAP~ Vector Kit, both available from
Stratagene (La Jolla, CA). Where additional detailed is
desired, reference should be made to the manufacturer's
directions provided with vector and kit, and incorporated
herein by reference. One of skill in the art will
appreciate that the method can be modified to accomodate the
production of other immunoglobulin Fab libraries of the
present invention and utilizing other vectors.
a. I~olation of Human Lymphocytes
Peripheral blood or perferably splenic lymphocytes are
isolated from a human transplant patient who has recieved a
porcine xenograft. For example, biopsy tissue is minced and
then incubated at 37~C for 30 minutes in sterile culture
CA 0222l234 l997-ll-l4
W 09~'35~58 PCTrUS96/06804
medium (RPMI 1640, 10~ FCS, and antibiotics) with 20 ~bg/ml
collagenase, 20 ~g/ml hyaluronidase, and 0.1~ DNase. Tissue
is then titrated through an 18 gauge needle until a cloudy
suspension is achieved. After washing, the resultant single
cell suspension is centrifuged on a Ficoll-Hypaque gradient
~ to obtain mononuclear cells. Peripheral blood mononuclear
cells ("PBMC") are isolated directly by Ficoll-Hypaque
fractionation.
b. Confirmation of IgM I~otype and PAEC B;~;~
Isolated lymphocytes are cultured at 37~C in a
humidified atmosphere of 5~ C02: 95~ air for 12 days at a
concentration of 2 x 106 cells/ml in RPMI 1640 (Irvine
Scientific, Santa Ana, CA) supplemented with 10~ fetal
bovine serum and antibiotics.
Supernatant is analyzed for concentrations of total
serum IgM by a standard ELISA method. Briefly, wells of
microtiter plates (Costar, Pleasanton, CA) are coated
overnight (4~C) with goat anti-human IgM (Accurate Chemical
and Scientific Corp., Westbury, NY) diluted in carbonate-
bicarbonate buffer, pH 9. 6 (Sigma, St. Louis, M0). The
plates were rinsed three times for 15 minutes with PBS +
0.5~ Tween-20 and incubated for 1 hour at 4~C with serial
dilutions for each serum sample, assayed in triplicate, then
stained for one hour at 4~C with goat anti-human IgM-
alkaline phosphatase. (Accurate Chemical and ScientificCorp., Westbury, NY) Absorbance of 405 nm was determined
after the colorimetric reaction was developed using 1 mg/ml
of p-nitrophenyl phosphate and 10 mM diethanolimine in 0.5
mM MgCl2 (Bio-Rad Laboratories, Richmond, CA) for 1 hour.
Supernatant are also analyzed for binding to PAEC cells
as described above.
CA 02221234 1997-11-14
W 096/36358 PCTrUS96/OC801
c. Library Construction
V~, ~n~ V3~ T.; hrary Generat;on for Phage Dls~lay Hl~m~3n ~nti
Porc;ne Xeno~r~ft ~ntlhody T; hrary. The nucleotide
sequences encoding the immunoglobulin protein CDRs are
highly variable. However, there are several regions of
conserved sequences in nucleotide sequences encoding human
immunoglobulins that flank the V region domains of either
the light or heavy chain, for instance and that contain
substantially conserved nucleotide sequences, i.e.,
sequences that will hybridize to the same primer sequence.
Polynucleotide synthesis (amplification) primers that
hybridize to the conserved sequences and incorporate
restriction sites into the DNA homolog produced that are
therefore suitable for operatively linking the synthesized
DNA fragments to a vector are employed. More specifically,
the primers are designed so that the resulting DNA homologs
produced can be inserted into an expression vector in
reading frame with the upstream translatable DNA sequence at
the region of the vector containing the first restriction
site. Alternatively, linkers containing the desired
restriction site (e.g., SEQ ID NO: 33) can be blunt end
ligated to the end of the Ig DNA fragment so that the
resulting DNA homologs produced can be inserted into an
expression vector in reading frame with the upstream
translatable DNA sequence at the region of the vector
containing the first restriction site. Amplification with
the primers described herein is performed on cDNA templates
produced from mRNA isolated from lymphocytes isolated as
described above.
V~ Pr,mers. For amplification of the VH region, primers are
designed to introduce cohesive termini compatible with
directional ligation into one of the two unique restriction
sites (NotI or SpeI) of the SurfZAP vector. In all cases,
84
CA 02221234 1997-11-14
W 096/363~8 PCTrUS9G~'~CYO~
the 5~ primers should be chosen to be complimentary to the
first strand cDNA in the conserved and N-terminus region
(anti-sense strand). Listed in SEQUENCE ID Nos: 26 through
31 are exemplary Ig ~amily speci~ic heavy chain Ig primers
which can be engineered to incorporate the desired
~ restriction site or used with restriction site encoding
linkers.
Additional VH amplification primers including the
unique 3' primer are designed to be complimentary to a
portion of the first constant region domain of the ~1 heavy
chain mRNA (SEQUENCE ID No: 32). These primers will
produce DNA homologs containing polynucleotides coding for
amino acids from the VH region and the first constant region
domains of the heavy chain. These DNA homologs can
therefore be used to produce Fab fragments rather than Fv.
Additional unique 3' primers designed to be hybridized
to similar regions of another class of immunoglobulin heavy
chain such as IgG, IgE and IgA are contemplated. Other 3'
primers that hybridize to a specific region of a specific
class of CHl constant region and are adapted for
transferring the VH region amplified using this primer to an
expression vector capable of expressing those VH region with
a different class of heavy or light chain constant regions
are also contemplated.
Either the VH or the Vc should contain a third
restriction, unique from either the first or second
restrictions site, to provide a means for directional
ligating heavy and light chain sequences together for
subesequent insert into the phagemeid vector.
Amplification is performed in six separate reactions,
éach cont~;n;ng one of the 5' family specific (VH1 - VH6)
primers and a 3' primer. The 5' primers preferably
incorporate a Not I site and the 3' primers preferably
incorporate a FSI 1 restriction site, for the insertion of
CA 02221234 1997-11-14
W 096/363~8 PCT~US96/~C~O1
the VH DNA homolog into the phagemid expression vector and
ligation to the Ig kappa light chain sequence, respectively.
VK Pr,mers. The nucleotide sec~uences encoding the VK CDRs
are highly variable. However, there are several regions of
conserved sequences that flank the VR CDR domains including
the JK~ VK framework regions and VK leader/promoter.
Therefore, amplification primers are constructed that
hybridize to the conserved sequences and incorporate
restriction sites that allow directional ligation of the
amplified fragment to the heavy chain fragment.
For amplification of the VK CDR domains, the 5' primers
are designed to be complimentary to the first strand cDNA in
the conserved N-terminus region. These primers pre~erably
introduce a FSI 1 restriction endonuclease site to allow the
VK DNA homolog to be ligated to the V~ homolog. The 3 ' VK
amplification primer is designed to hybridize to the
constant region of the kappa mRNA and to introduce the Spe I
restriction endonuclease site required to insert the VK DNA
homolog into the SurfZAP vector. These primers allow a DNA
homolog to be produced containing polynucleotide sequences
coding for constant region amino acids of the kappa chain.
These primers make it possible to produce a Fab fragment
rather than a Fv. Amplification with these primers is
performed in separate reactions, each containing one of the
family specfic 5' primers and onelof the 3 ' primers.
Amplification primers designed to amplify human light
chain variable regions of the lambda isotype are also
contemplated.
V~ ~n~ VK T~; hr~ry Conqtrllct;on. Total RNA is extracted from
1.15 x 107 lymphocytes using standard guanadinium
isothiocynate extraction protocols. See, for example,
86
CA 02221234 1997-11-14
W 096/36358 PCTrUS96/06804
Chomcynski, P. and Saochi, N., ~nAl. R;ochem. 162:156-159
(1987), incorporated herein by reference.
In preparation for PCR amplification, the mRNA,
prepared above, is used as a template for cDNA synthesis by
a primer extension reaction. Thus, 10 ~g RNA is reverse
~ transcribed to single-stranded cDNA using 1 ~g oligo-dT
primer with 10 mM dithiothreitol, RNasin~ (a protein RN~se
inhibitor of Promega Corporation, Madison, WI), 25 mM each
dATP, dCTP, dGTP, dTTP, lx reverse transcriptase buffer
(Bethesda Research Laboratories, Bethesda, MD), and 2~1 (two
hundred units) reverse transcriptase (Bethesda Research
Laboratories, TM Bethesda, MD) in 50 ~l volume ~or 10 minutes
at room temperature followed by 50 minutes at 42~C.
Following a 5 minute 90~C heat kill and 10 minutes on ice,
the reaction was treated with 1 ~l (one unit) RNase H
(Bethesda Research Laboratories) for 20 minutes at 37~C.
The cDNA generated above is amplified using the
polymerase chain reaction ("PCR") method. Family speci~ic
variable region and isotype specific constant region primers
as described above are used to create heavy chain IgM VH1_VH6
family-specific and light chain VK1_VK3 family-specific
libraries.
PCR amplification is performed in a 50 ~l reaction
containing the products of the reverse transcription
reaction (about 100 ~g of the cDNA/RNA hybrid), 50 ng of 5'
VH primer, 50 ng of the 3' primer, 500mM of the mixture of
dNTP's, 5 mM KCl, 100 mM Tris-HCl buffer at pH 8.3, and .25
units of Taq DNA polymerase (Boehringer M~nnh~im/
Indianopolis, Indiana). The reaction mixture is subjected
to 30 cycles of amplification using a DNA Thermal Cycler 480
(Perkin Elmer Cetus, Norwalk, CT) Each amplification cycle
included denaturing of cDNA at 94~C for 15 minutes, followed
by annealing of primers at 52~C for 50 minutes, and
CA 02221234 1997-11-14
W 096/36358 PCTrUS96/06804
amplification at 72~C for 90 minutes. This is followed by a
10 minute extension at 72~C.
After verifying by agarose gel electrophoresis that all
amplifications are successful and that similar yields are
achieved, heavy chain and light chain libraries are
separately pooled and gel purified on 0.8~ Seaplaque GTG
Agarose (FMC, Rockland, ME) according to the manufacturer's
directions.
Equal portions of the products from each light chain
primer extension reaction and each heavy chain primer
extension reaction is mixed to create randomization of
subsequent VH and V~ ligation. The mixed products are
digested with FPI 1 restriction endonuclease. (All
restriction enzymes are available from Boehringer-M~nnheim,
Indianapolis, IN.) Digested products are again gel purified
as described above, and the region of the gel containing DNA
fragments of appropraite size are excised, electroeluted
into a dialysis membrane and ethanol precipitated. The
resulting DNA fragments are again mixed to create
randomization and liogated to one another, and gel purified
as described above. Finally, ligated DNA is double-digested
with Not 1 and Spe 1 which will represent a repertoire of
polypeptide genes having cohesive termini adapted for
directional ligation into the to create an insert that can
be directionally ligated into the SurfZAP vector.
The SurfZAP vector is prepared in accordance with the
manufacturer's direction for inserting the V~_VK sequence.
Tr~n~forlr~t;on of Host w;th Ig T~; hr~ry Escherichia coli
provided in the SurfZAP Cloning Kit (Stratagene, La Jolla,
CA) are transformed with the SurfZAP vector containing the
Ig library in accordance with the manufacturer's directions.
Transformants are selected by antibioic resistance and
enriched by growth in liquid cultures.
CA 0222l234 l997-ll-l4
W O9C136358 PCTrUS~ 01
d. p~nn; n~
Panning is performed to select for phage displayed Fab
that bind PAEC. PAEC cells are grown in in RPMI 1640 growth
medium supplemented with 10~ FBS and antibiotics to
approximately 4 million cells, preferably at a low passage
(c2), in a T75 tissue culture flask. The cells are rinsed
5-10 times with PBS to remove all medium. Non-specific
binding is blocked with PBS supplemented with 3~ BSA for one
hour at 37~C. The cells are aspirated to remove all
blocking agent and about 1011 phage in SM broth are added to
the cells and gently rocked at 37~C for about 2-3 hours.
Cells are washed about 10 times with PBS supplemented with
.5~ Tween 20 to remove unbound phage.
Phage which bind the cells are eluted by incubating
cells with 2 ml of .lM HCl at pH 2.2 with BSA to a
concentration lmg/ml (w/v) for about 10 minutes at room
temperatire while gently rocking the flask. The reaction is
neutrilized with 2 M Tris base. The number of phage eluted
is monitored by CFU.
Eluted phage are amplified by reinfecting 2 ml E. coli
XL-Blue in growth medium supplemented with tetracycline with
50 ~l eluted phage. Ten ml SB with carbenicillin is added
to select for phagemids. Panning repeated until there was at
least 100 fold enrichment. For enrichment quantitation,
aliquots of the original library are re-panned in parallel
with each cycle of enrichment to control for daily
fluctuations in phage recovery. Enrichment is calculated by
ratio of phage on vs. off and compared to the unenriched
library run on the same day. Preferably, each round of
panning is conducted against PAEC from the same individual
and PAEC from another individual as a control.
. CA 0222l234 l997-ll-l4
~"~ 961û68C4
N Ig97
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Cedars-Sinai Medical Center
(B) STREET: 8700 Beverly Boulevard
(C) CITY: Los Angeles
(D) STATE: California
(E) COUNTRY: US
(F) POSTAL CODE (ZIP): 90048-1863
(G) TELEPHONE: (310) 855-5284
(H) TELEFAX: (310) 967-0101
(ii) TITLE OF lNV~NllON: COMPOSITIONS AND METHODS FOR INHIBITING
XENOGRAFT REJECTION
(iii) NUMBER OF SEQUENCES: 39
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPO)
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/440,621
(B) FILING DATE: 15-MAY-1995
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 420 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(B) STRAIN: LEW RAT
(F) TISSUE TYPE: Spleen, hyperimmunized
(G) CELL TYPE: Splenic lymphocyte
(vii) IMMEDIATE SOURCE:
(B) CLONE: HAR-1
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..420
(D) OTHER INFORMATION: /product= "Immunoglobulin Variable
~MENDED SHEET
, CA 0222l234 l997-ll-l4 ~ ............... ,~ -
s6/a6s~
IPE~ JAN 199~7
Region"
/standard_name= "Ig Heavy Chain Variable Region"
/label= VH-Region
/note= ~Variable Region o~ HAR-1 Heavy Chain~
(ix) FEA~lu~E:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 1..57
(D) OTHER INFORMATION: /standard_name= "Leader~
/label= Leader
(ix) FEAlu~E:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 58..351
(D) OTHER INFORMATION: /standard_name= "Ig Heavy Chain
Variable Segment"
/label= VH-Segment
/note= "Variable Segment of HAR-1 Heavy Chain
Variable Region~
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 352..366
(D) OTHER INFORMATION: /standard_name- "Ig Heavy Chain
Diversity Segment"
/label= D-Segment
/note= "Diversity Segment of HAR-l Heavy Chain
Variable Region~
(ix) FEA~lu~E:
(A) NAME/KEY: mi~c_RNA
(B) LOCATION: 367..420
(D) OTHER INFORMATION: /product= "Immunoglobulin Joining
Region"
/standard_name= "Ig Heavy Chain Joining Segment"
/label= JH-Segment
/note= "Joining Segment of HAR-1 Heavy Chain
Variable Region~
(ix) FEAluKE:
(A) NAME/KEY: misc_RNA
__ (B) LOCATION: 58.. 147
(D) OTHER INFORMATION: /standard_name= "FL~...e~.Jrk Region
1"
/label= FR-1
/note= "FLa~ JLh Region 1 o~ HAR-1 Heavy Chain
Variable Region~
(ix) FEAlu~E:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 148..162
(D) OTHER INFORMATION: /standard_name= "CDR-l"
/label= CDR-1
/note= "Compl;m~nt~rity Determining Region 1 of
HAR-l Heavy Chain Variable Region~
91
~MENDcD S~IEET
~ CA 0222l234 l997-ll-l4
~C7'1~ 'q6~ n~
J A \i 19~ 7
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 163..204
(D) OTHER INFORMATION: /standard_name= "Framework Region
2"
- /label= FR-2
/note= "Framework Region 2 of HAR-1 Heavy Chain
Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 205..255
(D) OTHER INFORMATION: /standard_name= "CDR-2"
/label= CDR-2
/note= "Complimentarity Determining Region 2 of
HAR-1 Heavy Chain Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 256..351
(D) OTHER INFORMATION: /standard_name= "Fla...ewJrk Region
3"
/label= FR-3
/note= "Framework Region 3 of HAR-1 Heavy Chain
Variable Region"
(ix) FEATURE:
(A) NAME/KEY: mi~c_RNA
(B) LOCATION: 352..387
(D) OTHER INFORMATION: /Rtandard_name= "CDR-3"
/label= CDR-3
/note= "Complimentarity Determining Region of
HAR-l Heavy Chain Variable Region"
(ix) FEATURE:
(A) NAME/KEY: miRc_RNA
(B) LOCATION: 388..420
(D) OTHER INFORMATION: /standard_name= "Framework Region
4"
/label= FR-4
/note= "Framework Region 4 of HAR-1 Variable Heavy
_ Chain Region"
(xi) ~U~N~ DESCRIPTION: SEQ ID NO:1:
ATG GAC ATC AGG CTC AGC TTG GCT TTC CTT CTC CTT TTC ATA AAA GGT 48
Met A~p Ile Arg Leu Ser Leu Ala Phe Leu Leu Leu Phe Ile Ly~ Gly
1 5 10 15
GTC CAG TGT GAG GTG CAG CTG GTG GAG TCT GGG GGA GGC TTA GTG CAG 96
Val Gln CYR Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
20 25 30
CCT GGA AGA TCC CTG A~A CTC TCC TGT GCA GCC TCA GGA TTC ACT TTC 144
AAI~ENDED St~EET
CA 0222l234 l997-ll-l4 ~Ti~S 96 1 0
3P~ 14 JAN 1~4
Pro Gly Arg Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
AGT AAC TAT GGC ATG GCT TGG GTC CGC CAG GCT CCA ACG AAG GGT CTG 192
Ser Asn Tyr Gly Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu
50 55 60
GAG TGG GTC GCA TCC ATT AGT ACT GGT GGT GGT AAC ACT TAC TAT CGA 240
Glu Trp Val Ala Ser Ile Ser Thr Gly Gly Gly Asn Thr Tyr Tyr Arg
65 70 75 80
GAC TCC GTG AAG GGC CGA TTC ACT ATC TCC AGA GAT AAT GCA AAA AAC 288
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
85 9O 95
ACC CTA TAC CTG CAA ATG GAC AGT CTG AGG TCT GAG GAC ACG GCC ACT 336
Thr Leu Tyr Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr
100 105 110
TAT TAC TGT GCA AGA CAT CGC GGG TAT AAC TCC TAC TGG TAC TTT GAC 384
Tyr Tyr Cys Ala Arg HiS Arg Gly Tyr Asn Ser Tyr Trp Tyr Phe Asp
115 120 125
TTC TGG GGC CCA GGA ACC ATG GTC ACC GTG TCC TCA 420
Phe Trp Gly Pro Gly Thr Met Val Thr Val Ser Ser
130 135 140
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 140 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(Xi) ~QU~N~ DESCRIPTION: SEQ ID NO:2:
Met Asp Ile Arg Leu Ser Leu Ala Phe Leu Leu Leu Phe Ile Lys Gly
1 5 10 15
~al Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Arg Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Asn Tyr Gly Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu
Glu Trp Val Ala Ser Ile Ser Thr Gly Gly Gly Asn Thr Tyr Tyr Arg
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
AMENDED SHEE~
, CA 0222l234 l997-ll-l4 C ~ 9 6 / &i ~ 8 ~ ~
d ~ 9g 7
Thr Leu Tyr Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr
100 105 110
Tyr Tyr Cys Ala Arg His Arg Gly Tyr Asn Ser Tyr Trp Tyr Phe Asp
115 120 125
Phe Trp Gly Pro Gly Thr Met Val Thr Val Ser Ser
130 135 140
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 354 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(B) STRAIN: LEW RAT
(F) TISSUE TYPE: Spleen, hyper;mm~ln;~ed
(G) CELL TYPE: Splenic lymphocyte
(vii) IMMEDIATE SOURCE:
(B) CLONE: ID12BF3
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..354
(D) OTHER INFORMATION: /product= "Immunoglobulin Variable
Region"
/standard_name= "Ig Heavy Chain Variable Region"
/label= VH-Region
/note= "Variable Region of ID12BF3 Heavy Chain"
(ix) FEATURE:
... (A) NAME/KEY: misc RNA
(B) LOCATION: 1..294
(D) OTHER INFORMATION: /standard_name= "Heavy Chain
Variable Segment"
/label= VH-Segment
/note= "Variable Segment o~ ID12BF3 Heavy Chain
Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 295..308
(D) OTHER INFORMATION: /standard_name= "Ig Heavy Chain
Diversity Segment"
/label= D-Segment
94
AMEND~D SttEEr
CA 0222l234 l997-ll-l4
~L~ 9~ 6 /
/note= "Diversity Segment of ID12BF3 Heavy Chain
Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 309..354
(D) OTHER INFORMATION: /product= "Immunoglobulin Joining
Region"
/standard_name= "Ig Heavy Chain Joining Segment"
/label= JH-Segment
/note= "Joining Segment of ID12BF3 Heavy Chain
Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 1..90
(D) OTHER INFORMATION: /standard_name= "Framework Region
1"
/label= FR-1
/note= "FLa~ .Jrk Region 1 of ID12BF3 Heavy Chain
Variable Region"
(ix) FEAlU~E:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 91..105
(D) OTHER INFORMATION: /standard_name= "CDR-1"
/label= CDR-1
/note= "Complimentarity Determining Region 1 of
ID12BF3 Heavy Chain Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 106..147
(D) OTHER INFORMATION: /standard_name= "Frd,..~Jrk Region
2"
/label= FR-2
/note= "Framework Region 2 of ID12BF3 Heavy Chain
Variable Region"
(ix) FEA~l~U~E:
(A) NAME/KEY: misc_RNA
_ (B) LOCATION: 148.. 198
(D) OTHER INFORMATION: /standard_name= "CDR-2"
/label= CDR-2
/note= "Compl;m~nt~rity Determining Region 2 of
ID12BF3 Heavy Chain Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 199..294
(D) OTHER INFORMATION: /standard_name= "Framework Region
3"
/label= FR-3
/note= "Framework Region 3 of ID12BF3 Heavy Chain
Variable Region"
AMENDE~ SHE~T
CA 0222l234 l997-ll-l4 PC~ 96 ~ ~6 8
,f ~ ~ r ~ 9 9 7
tix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 295..321
(D) OTHER INFORMATION: /standard_name= "CDR-3"
/label= CDR-3
/note= ~Complimentarity Determining Region 3 of
ID12BF3 Heavy Chain Variable Region~
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 322..354
(D) OTB R INFORMATION: /standard_name= "Framework Region
4"
/label= FR-4
/note= "Framework Region 4 of ID12BF3 Eeavy Chain
Variable Region~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GAG GTG CAG CTG GTG GAG TCT GGG GGA GGC TTA GTG CAG CCT GGA AGA 48
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
TCC CTG AAA CTC TCC TGT GCA GCC TCA GGA TTC ACT TTC AGT AAC TAT 96
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
GGC ATG GCT TGG GTC CGC CAG GCT CCA ACG AAG GGT CTG GAG TGG GTC 144
Gly Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val
35 40 45
GCA TCC ATT AGT ACT GGT GGT GGT AAC ACT TAC TAT CGA GAC TCC GTG 192
Ala Ser Ile Ser Thr Gly Gly Gly Asn Thr Tyr Tyr Arg Asp Ser Val
50 55 60
AAG GGC CGA TTC ACT ATC TCC AGA GAT AAT GCA AAA AAC ACC CTA TAC 240
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
CTG CAA ATG GAC AGT CTG AGG TCT GAG GAC ACG GCC ACT TAT TAC TGT 288
--Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
GCA AGA CCT TCC TAT AGC AGC TAC TTT GAT TAC TGG GGC CAA GGA GTC 336
I Ala Arg Pro Ser Tyr Ser Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Val
100 105 110
ATG GTC ACA GTC TCC TCA 354
Met Val Thr Val Ser Ser
115
(2) INFORMATION FOR SEQ ID NO:4:
96
N~ED St~EE~
. CA 0222l234 l997-ll-l4
PCTIUS 96/ 068~
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 118 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
Gly Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val
Ala Ser Ile Ser Thr Gly Gly Gly Asn Thr Tyr Tyr Arg Asp Ser Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr cy8
Ala Arg Pro Ser Tyr Ser Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Val
100 105 110
Met Val Thr Val Ser Ser
115
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 402 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(B) STRAIN: LEW RAT
(F) TISSUE TYPE: Spleen, hyper;mmlln;~ed
(G) CELL TYPE: Splenic lymphocyte
(vii) IMMEDIATE SOURCE:
(B) CLONE: HAR-1
(ix) FEATURE:
(A) NAME/KEY: CDS
AM~h~DED S~EE~
CA 02221234 1997-11-14 P C ~ ~ ~ 96 / ~!k 8
9g7
(B) LOCATION: 1..402
(D) OTHER INFORMATION: /product= "Immunoglobulin Variable
Region"
/standard_name= "Ig Kappa Chain Variable Region"
/label= VK-Region
/note= "Variable Region of HAR-1 Kappa Light
Chain"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 1..60
(D) OTHER INFORMATION: /standard_name= "Leader"
/label= Leader
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 61..129
(D) OTHER INFORMATION: /standard_name= "Framework Region
1"
/label= FR-1
/note= "Framework Region 1 o~ H~R-1 Kappa Light
Chain Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 130..180
(D) OTEER INFORMATION: /standard_name= "CDR-l"
/label= CDR-1
/note= "Complimentarity Determining Region 1 of
HAR-l Kappa Light Chain Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 181..22S
(D) OTHER INFORMATION: /standard_name= "Framework Region
2"
/label= FR-2
/note= "Framework Region 2 o~ HAR-l Kappa Light
Chain Variable Region"
(ix) FEATUKE:
- (A) NAME/KEY: misc_RNA
(B) LOCATION: 226..246
(D) OTHER INFORMATION: /standard_name= "CDR-2"
/label= CDR-2
/note= "Compl;m~nt~ity Determining Region 2 o~
HAR-1 Kappa Light Chain Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 247..342
(D) OTHER INFORMATION: /standard_name= "FLa,l.~ork Region
3"
/label= FR-3
/note= "FLd...ew~rk Region 3 o~ HAR-1 Kappa Light
98
P~MEN~En SHEE~
CA 0222l234 l997-ll-l4 P C ~U ~ 96 / n~ 8~, 4
~ J~ 1997
Chain Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 343..366
(D) OTHER INFORMATION: /standard_name= "CDR-3"
/label= CDR-3
/note= "Complimentarity Determining Region 3 of
HAR-1 Kappa Light Chain Variable Region~
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 367..402
(D) OTHER INFORMATION: /standard_name= "Framework Region
4"
/label= FR-4
/note= "F~ar"el~Jrk Region 4 of HAR-1 Kappa Light
Chain Variable Region"
(Xi) ~U~N~ DESCRIPTION: SEQ ID NO:5:
ATG GAA TCA CAG ACA CAG GTC CTC ATG TCC CTG CTG CTC TGG GTA TCT 48
Met Glu Ser Gln Thr Gln Val Leu Met Ser Leu Leu Leu Trp Val Ser
1 5 10 15
GGT ACC TGT GGG GAC ATT GTG ATG ACC CAG ACT CCA TCC TCC CAG GCT 96
Gly Thr Cys Gly ASp Ile Val Met Thr Gln Thr Pro Ser Ser Gln Ala
20 25 30
GTG TCA GCA GGG GAG AAG GTC ACT ATG AGC TGC AAG TCC AGT CAG AGT 144
Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser
35 40 45
CTT TTA TAC AAT GAA AAC AAA AAG AAC TAC TTG GCC TGG TAC CGG CAG 192
Leu Leu Tyr Asn Glu Asn Lys Lys Asn Tyr Leu Ala Trp Tyr Arg Gln
50 55 60
AAA CCA GGG CAG TCT CCT AAA CTG CTG ATC TAC TGG GCA TCC ACT AGG 240
Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg
65 70 75 80
GAA TCT GGG GTC CCT GAT CGC TTC ATA GGC AGT GGA TCT GGG ACA GAT 288
Glu Ser Gly Val Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp
85 90 95
TTC ACT CTG ACC ATC AGC AGT GTG CAG GCA GAA GAC CTG GCT GTT TAT 336
Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr
100 105 110
TAC TGC CAG CAG TAC TAT AAC TTG TAC ACG TTT GGA GCT GGG ACC AAG 384
Tyr Cys Gln Gln Tyr Tyr Asn Leu Tyr Thr Phe Gly Ala Gly Thr Lys
115 120 125
CTG GAA CTG AAA CGG GCT 402
~M~NDED SH~Er
CA 0222l234 l997-ll-l4 ~ ~ ~/U S 96 / ~ L
Leu Glu Leu Lys Arg Ala
130
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 134 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Met Glu Ser Gln Thr Gln Val Leu Met Ser Leu Leu Leu Trp Val Ser
1 5 10 15
~ly Thr Cys Gly Asp Ile Val Met Thr Gln Thr Pro Ser Ser Gln Ala
Val Ser Ala Gly Glu Lys Val Thr Met Ser Cy8 Lys Ser Ser Gln Ser
Leu Leu Tyr Asn Glu Asn Lys Lys Asn Tyr Leu Ala Trp Tyr Arg Gln
Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg
~lu Ser Gly Val Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp
~he Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr
100 105 110
~yr Cys Gln Gln Tyr Tyr Asn Leu Tyr Thr Phe Gly Ala Gly Thr Lys
115 120 125
Leu Glu Leu Lys Arg Ala
130
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
100
~M~NDED SI~EE~
. CA 02221234 1997-11-14
PCT~S 96~n~8~
~p~ r~ l l 1991
(B) LOCATION: 1..20
(D) OTHER INFORMATION: /label= RVHl
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
TACAGCACTG CACAGACTCC 20
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 8 ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_~eature
(B) LOCATION: 1..24
(D) OTHER INFORMATION: /label= RVH2
(xi) S~QU~N~ DESCRIPTION: SEQ ID NO:8:
GTGTCCTCAG ACCTCAGACT GTCC 24
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 440 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(B) STRAIN: LEW RAT
(F) TISSUE TYPE: Liver
(vii) IMMEDIATE SOURCE:
(B) CLONE: VHl.1
(ix) FEAluKE:
(A) NAME/KEY: CDS
(B) LOCATION: join(l..48, lS9..440)
(D) OTHER INFORMATION: /label= VH-Segment
/note= "Rat Ig Germline VH1.1 Heavy Chain Variable
Segment"
lûl
AMENDE~ S~EE~
CA 0222l234 l997-ll-l4 P~T~U~ 9 6 1 0 6 8 ~ 4
v ~ J r.i ~ ~g97
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: join(l..48, 159..167)
(D) OTHER INFORMATION: /standard_name= ~Leader~
/label= Leader
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 168..257
(D) OTHER INFORMATION: /standard_name= "Framework Region
1"
/label= FR-1
/note= "Framework Region 1 of Rat Ig Germline
VHl.l Eeavy Chain Variable Segment~
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 258..272
(D) OTHER INFORMATION: /standard_name= "CDR-l"
/label= CDR-1
/note= "Complim~ntArity Determining Region 1 of
Rat Ig Germline VHl.1 Heavy Chain Variable
Segment"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 273..315
(D) OTHER INFORMATION: /standard_name= "FLa".e~ork Region
2"
/label= FR-2
/note= "Framework Region 2 of Rat Ig Germline
VH1.1 Heavy Chain Variable Segment"
(ix) FEATURE:
(A) NAME/KEY: mi~c_RNA
(B) LOCATION: 316..365
(D) OTHER INFORMATION: /standard_name= "CDR-2"
/label= CDR-2
/note. "Complimentarity Determining Region 2 of
Rat Ig Germline VH1.1 Heavy Chain Variable
Segment~
(ix) FEALuKE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 366..440
(D) OTHER INFORMATION: /standard_name= ''FL~I.._~.Jrk Region
3"
/label= FR-3
/note= "Framework Region 3 of Rat Ig Germline
VH1.1 Heavy Chain Variable Segment"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
ATG GAC ATC AGG CTC AGC TTG GCT TTC CTT GTC CTT TTC ATA A~A GGT 48
lû2
A,~ ED S~EET
~ CA 0222l234 l997-ll-l4 j .;~,~J,~,~,~ 9 6 / 06 ~ ~4
~E~ I JAI'3 1937
Met Asp Ile Arg Leu Ser Leu Ala Phe Leu Val Leu Phe Ile Lys Gly
1 5 10 15
AATTGATAAA AGTGTGATCA T~l~l~ll~T GTGCACATGA GAATAAGAAA GTTTATTTTG 108
TTTTGTTGTG TTAGTGATGG 'l"l''l"l'~'l'AACC AGTATTCTCT GTTTGCAGGT GTC CAG 164
Val Gln
TGT GAG GTG CAG CTG GTG GAG TCT GGG GGA GGC TTA GTG CAG CCT GGA 212
Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
20 25 30
AGG TCC CTG AAA CTC TCC TGT GCA GCC TCA GGA TTC ACT TTC AGT AAC 260
Arg Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn
35 40 45 50
TAT GGC ATG GCC TGG GTC CGC CAG GCT CCA ACG AAG GGT CTG GAG TGG 308
Tyr Gly Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp
55 60 65
GTC GCA TCC ATT AGT ACT GGT GGT GGT AAC ACT TAC TAT CGA GAC TCC 356
Val Ala Ser Ile Ser Thr Gly Gly Gly Asn Thr Tyr Tyr Arg Asp Ser
70 75 80
GTG AAG GGC CGA TTC ACT ATC TCC AGA GAT AAT GCA AAA AAC ACC CTA 404
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu
85 90 95
TAC CTG CAA ATG GAC AGT CTG AGG TCT GAG GAC ACG 440
Tyr Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr
100 105 110
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 110 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Met Asp Ile Arg Leu Ser Leu Ala Phe Leu Val Leu Phe Ile Lys Gly
1 5 10 15
Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Arg Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Asn Tyr Gly ~et Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu
103
J v~ T
~ CA 0222l234 l997-ll-l4 ~ J q ~ f ~ 4
,Qi~3 199~7
Glu Trp Val Ala Ser Ile Ser Thr Gly Gly Gly Asn Thr Tyr Tyr Arg
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
85 90 95
Thr Leu Tyr Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr
100 105 110
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 348 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(B) STRAIN: LEW RAT
(F) TISSUE TYPE: Spleen, hyperimmunized
(G) CELL TYPE: Splenic lymphocyte
(vii) IMMEDIATE SOURCE:
(B) CLONE: 9D6
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..348
(D) OTHER INFORMATION: /product= "Immunoglobulin Variable
Region"
/standard_name= "Ig Heavy Chain Variable Region"
/label= VH-Region
/note= "Variable Region of 9D6 Heavy Chain"
(ix) FEATURE:
. (A) NAME/KEY: misc_RNA
(B) LOCATION: 1..294
(D) OTHER INFORMATION: /standard_name= "Ig Heavy Chain
Variable Segment"
/label= VH-Segment
/note= "Variable Segment of 9D6 Heavy Chain
Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 295..303
(D) OTHER INFORMATION: /standard_name= "Ig Heavy Chain
Diversity Segment"
/label= D-Segment
104
AMrl~lD~D SHEET
,CA 02221234 1997-11-14
P~ b l O 6 8 ~ 4
A~1397
/note= "Diversity Segment of 9D6 Heavy Chain
Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 304..348
(D) OTHER INFORMATION: /standard_name= "Ig Heavy Chain
Joining Segment"
/label= JX-Segment
/note= "Joining Segment of 9D6 Eeavy Chain
Variable Region"
(Xi) s~Qu~N~ DESCRIPTION: SEQ ID NO:ll:
GAG GTG AAA CTT GTC GAG TCT GGA GGT GGC CTG GTG CAA CCT GGA AGA 48
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
TCC TTG AAA CTC TCC TGT GCA GCC TCT GGA TTC AAT TTT AAT GAT TAC 96
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Asn Asp Tyr
20 25 30
TGG ATG GGC TGG GTC CGG CAG GCT CCA GGG AAG GGG CTA GAA TGG ATT 144
Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
GGA GAA ATT AAT AAG GAT AGC AGT ACA ATA AAC TAT ACT CCA TCC TTG 192
Gly Glu Ile Asn Lys Asp Ser Ser Thr Ile Asn Tyr Thr Pro Ser Leu
50 55 60
AAG GAT AAA TTC ACC ATC TCC AGA GAC AAT GCC CAA AAC ACT CTG TAC 240
Lys Asp Lys Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Thr Leu Tyr
65 70 75 80
CTG CAA ATG AGC AAA CTG GGA TCT GAG GAC ACG GCC ATT TAT TAC TGT 288
Leu Gln Met Ser Lys Leu Gly Ser Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
GCA AAA GCA ACT GGG AGC TTT GAT TAC TGG GGC CAA GGA GTC ATG GTC 336
Ala Lys Ala Thr Gly Ser Phe Asp Tyr Trp Gly Gln Gly Val Met Val
100 105 110
ACA GTC TCC TCA 348
Thr Val Ser Ser
115
(2) INFORMATION FOR SEQ ID NO:12:
(i) ~u~N~ CHARACTERISTICS:
(A) LENGTH: 116 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
105
.~ENDED SHEET
, CA 0222l234 l997-ll-l4
p~ 9b ~ ~ 8
J~,3 l~g~
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu ~ys Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Asn Asp Tyr
Trp Met Gly Trp Val Arg Gln Ala Pro Gly hys Gly Leu Glu Trp Ile
Gly Glu Ile Asn Lys Asp Ser Ser Thr Ile Asn Tyr Thr Pro Ser Leu
Lys Asp Lys Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Thr Leu Tyr
Leu Gln Met Ser Lys Leu Gly Ser Glu Asp Thr Ala Ile Tyr Tyr Cys
Ala Lys Ala Thr Gly Ser Phe Asp Tyr Trp Gly Gln Gly Val Met Val
100 105 110
Thr Val Ser Ser
115
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
. (B) LOCATION: 1.. 20
(D) OTHER INFORMATION: /label= RCMl
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
ACTGGCTCTG TGGTGAAGCC 20
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STR~Nn~nN~SS: double
106
A~ENDED SHEET
, CA 0222l234 l997-ll-l4
9 6 I Q 6 8 ~ 4
IP~ A~ g~7
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEA'l'URE:
~ (A) NAME/KEY: misc_feature
(B) LOCATION: 1..32
(D) OTHER INFORMATION: /label= SAX
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
CGAATTCGGG CCCTCGAGGC CTCTAGAATT CG 32
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pair~
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..23
(D) OTHER INFORMATION: /label_ RCM3
(xi) ~yu~N~ DESCRIPTION: SEQ ID NO:15:
GTTCTGGTAG TTCCAGGAGA AGG 23
(2) INFORMATION FOR SEQ ID NO:16:
(i) ~UU~N~ CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) sTR~Nn~nN~s single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEAlu~E:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..20
(D) OTHER INFORMATION: /label- RCK1
(xi) S~U~N~ DESCRIPTION: SEQ ID NO:16:
lû7
4MENi~ED SHEET
, CA 0222l234 l997-ll-l4
9 6 l Q 6 8 ~ bf
~PE~ i 1997
TTGAAGCTCT TGACGACGGG 20
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 ba~e pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..24
(D) OTHER INFORMATION: /label= RCK3
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
GCTAACTGTT CCGTGGATGG TGGG 24
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 345 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(B) STRAIN: LEW RAT
(F) TISSUE TYPE: Spleen, hyperimmunized
(G) CELL TYPE: Splenic lymphocyte
(vii) IMMEDIATE SOURCE:
_ (B) CLONE: HA75D8F1
(ix) FEAIu~E:
(A) NAME/KEY: CDS
(B) LOCATION: 1..345
(D) OTHER INFORMATION: /product= "Tmmlln~globulin Variable
Region"
/standard_name= "Ig Heavy Chain Variable Region"
/label= VH-Region
/note= "Variable Region of HA75D8F1 Heavy Chain"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 1..291
lû8
AMENtk~ SHEET
, CA 0222l234 l997-ll-l4
PG~ 96 ~ ~6 8~4'
~P~ J.~.~ 1997
(D) OTHER INFORMATION: /standard_name= "Ig Heavy Chain
Variable Segment"
/label= VH-Segment
/note= "Variable Segment of HA75D8Fl Heavy Chain
Variable Region"
( ix? FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 292..312
(D) OTHER INFORMATION: /standard_name= "Ig Heavy Chain
Diversity Segment"
/label= D-Segment
/note= "Diversity Segment of HA75D8Fl Heavy Chain
Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 313..345
(D) OTHER INFORMATION: /standard_name= "Ig Heavy Chain
Joining Segment"
/label= JH-Segment
/note= "Joining Segment of HA75D8Fl Heavy Chain
Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 1..90
(D) OTHER INFORMATION: /standard_name~ "Framework Region
1"
/label= FR-l
/note= "Framework Region 1 of HA75D8Fl Heavy Chain
Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) BOCATION: 91..105
(D) OTHER INFORMATION: /standard_name= "CDR-l"
/label= CDR-l
/note= "Complimentarity Determining Region 1 of
HA75D8Fl Heavy Chain Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 106..151
(D) OTHER INFORMATION: /standard_name= "F~d,~ .Jrk Region
2"
/label= FR-2
/note= "F~ ~work Region 2 of HA75D8Fl Heavy Chain
Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 152..198
(D) OTHER INFORMATION: /standard_name= "CDR-2"
/label= CDR-2
109
ENDE~ S~EE~
CA 0222l234 l997-ll-l4 P ~ 9 6 / 0 6 8
1397
/note= ~Complimentarity Determining Region 2 of
HA75D8F1 Heavy Chain Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 199..291
(D) OTHER INFORMATION: /standard_name= "Framework Region
3"
/label= FR-3
/note= "Framework Region 3 o~ HA75D8F1 Heavy Chain
Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 292..312
(D) OTHER INFORMATION: /standard_name= "CDR-3"
/label= CDR-3
/note= "Compl; -nt~rity Determining Region 3 of
HA75D8F1 Heavy Chain Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 313..345
(D) OTHER INFORMATION: /standard_name= "Fra~ .Jrk Region
4"
/label= FR-4
/note= "F ~...~.Jrk Region 4 o~ HA75D8F1-Heavy Chain
Variable Region"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
GAG GTG AAG CTG CAG GAG TCA GGA CCT GGT CTG GTA CAG CCC TCA CAG 48
Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
ACC CTG TCC CTC ACC TGC ACT GTC TCT GGG TTC TCA CTA AAC AAC TAT 96
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Asn Asn Tyr
20 25 30
GGT GTG ATC TGG GTT CGC CAG CCT CCA GGA AAG GGT CTG GAG TGG ATG 144
Gly Val Ile Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
GGA ATA ATT TGG AAT AAT GGA AAT ACA AAT TAT AAT TCA GCT CTC A~A 192
Gly Ile Ile Trp Asn Asn Gly Asn Thr Asn Tyr Asn Ser Ala Leu Lys
50 55 60
TCC CGA CTG AGC ATC AGC AGG GAC ACC TCC AAG AGC CAA GTT TTC TTA 240
Ser Arg Leu Ser Ile Ser Arg A8p Thr Ser Lys Ser Gln Val Phe Leu
65 70 75 80
A~A ATG AAC AAT CTG CAA ACT GAA GAC ACG GCC ATG TAC TTC TGT GCC 288
Lys Met Asn Asn Leu Gln Thr Glu Asp Thr Ala Met Tyr Phe Cys Ala
85 90 95
110
AMENDED SI~E~
, CA 02221234 1997-11-14
~T~ 96/~68~4
I~'t~3~ 99~7
AGA GGA GGA GTG GGG TTT GAT TTC TGG GGC CAA GGA GTC ATG GTC ACA 336
Arg Gly Gly Val Gly Phe Asp Phe Trp Gly Gln Gly Val Met Val Thr
100 105 110
GTC TCC TCA 345
Val Ser Ser
. 115
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 115 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Asn Asn Tyr
Gly Val Ile Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met
Gly Ile Ile Trp Asn Asn Gly Asn Thr Asn Tyr Asn Ser Ala Leu Lys
Ser Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Ser Gln Val Phe Leu
Lys Met Asn Asn Leu Gln Thr Glu Asp Thr Ala Met Tyr Phe Cys Ala
Arg Gly Gly Val Gly Phe Asp Phe Trp Gly Gln Gly Val Met Val Thr
100 105 110
Val Ser Ser
115
(2) INFORMATION FOR SEQ ID NO:20:
(i) S~OU~N~ CHARACTERISTICS:
(A) LENGTH: 357 base pairs
(B) TYPE: nucleic acid
(C) STE~N~ N~ s double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
111
AIIIEN~D SHEET
CA 0222l234 l997-ll-l4 ~ S 9b / 06 80
~P~AIlh 14 JAN ~
(vi) ORIGINAL SOURCE:
(B) STRAIN: LEW RAT
(F) TISSUE TYPE: Spleen, hyperimmunized
(G) CELL TYPE: Splenic lymphocyte
(vii) IMMEDIATE SOURCE:
(B) CLONE: IH2lH7
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..357
(D) OTHER INFORMATION: /product= "Immunoglobulin Variable
Region"
/standard_name= ~'Ig Heavy Chain Variable Region~
/label= VH-Region
/note= ~Variable Region o~ IH21H7 Heavy Chain~
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 1..291
(D) OTHER INFORMATION: /standard_name= "Ig Heavy Chain
Variable Segment"
/label= VH-Segment
/note= "Variable Segment of IH2lH7 Heavy Chain
Variable Region~
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 292..312
(D) OTHER INFORMATION: /standard_name= "Ig Heavy Chain
Diversity Segment"
/label= D-Segment
/note= "Diversity Segment o~ IH21H7 Heavy Chain
Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 313..357
(D) OTHER INFORMATION: /standard_name= "Ig Heavy Chain
Joining Segment"
/label= JH-Segment
/note= "Joining Segment o~ IH21H7 Heavy Chain
Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 1..90
(D) OTHER INFORMATION: /standard_name= "Framework Region
ln
/label= FR-1
/note= ~Framework Region 1 o~ IH21H7 Heavy Chain
Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
112
AM~ND~D ~HEET
~ CA 0222l234 l997-ll-l4
O ~ 8
~P~f~J~ 14 JAN ,397
(B) LOCATION: 91..105
(D) OTHER INFORMATION: /standard_name= "CDR-1"
/label= CDR-1
/note= "Complimentarity Determining Region 1 of
IH21H7 Heavy Chain Variable Region"
( ix? FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 106..151
(D) OTHER INFORMATION: /standard_name= "Framework Region
2"
/label= FR-2
/note= "Framework Region 2 of IH21H7 Heavy Chain
Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 152..198
(D) OTHER INFORMATION: /standard_name= "CDR-2"
/label= CDR-2
/note= "Compllm~nt~ry Determining Region 2 of
IH21H7 Heavy Chain Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 199..291
(D) OTHER INFORMATION: /standard_name= "Framework Region
3"
/label= FR-3
/note= "Framework Region 3 of IH21H7 Heavy Chain
Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 292..324
(D) OTHER INFORMATION: /standard_name= "CDR-3"
/label= CDR-3
/note= "Complimentarity Determining Region 3 of
IH21H7 Heavy Chain Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 325..357
(D) OTHER INFORMATION: /standard_name= "Framework Region
4"
/label5 FR-4
/note= "Fral.,ewJrk Region 4 of IH21H7 Heavy Chain
Variable Region"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
GAG GTC AAG CTG CAG CAG TCA GGA CCT GGC CTG GTG CAG CCC TCA CAG 48
Glu Val Lys Leu Gln Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 ' 5 10 15
113
~MF.ND~3 SHE~T
, CA 0222l234 l997-ll-l4
p~ S 96~6813
IPE~ JAN 1997
ACC CTG TCT CTC ACC TGC ACT GTC TCT GGG TTC TCA TTA ACC AAC TAT 96
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr
20 25 30
CAT GTG CAC TGG GTT CGA CAG CCT CCA GGA AAA GGT CTG GAG TGG ATG 144
His Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
GGA GTC ATG TGG GGT GAT GGA GAC ACA TCA TGT AAT TCA GCT CTC A~A 192
Gly Val Met Trp Gly Asp Gly Asp Thr Ser Cys Asn Ser Ala Leu Lys
50 55 60
TCC CGA CTG AGC ATC AGC AGG GAC ACC TCC AAG AGC CAA GTT TTC TTA 240
Ser Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Ser Gln Val Phe Leu
65 70 75 80
AAA TTG AGC AGT CTG CAA ACT GAA GAC ACA GCC ACT TAC TAC TGT GCC 288
Lys Leu Ser Ser Leu Gln Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala
85 90 95
AGA CTC CCT AGG GGG AAG GGA CCC CAC TTT GAT TAC TGG GGC CAA GGA 336
Arg Leu Pro Arg Gly Lys Gly Pro His Phe Asp Tyr Trp Gly Gln Gly
100 105 110
GTC ATG GTC ACA GTC TCC TCA 357
Val Met Val Thr Val Ser Ser
115
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 119 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Glu Val Lys Leu Gln Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr
His Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met
Gly Val Met Trp Gly Asp Gly Asp Thr Ser Cys Asn Ser Ala Leu Lys
Ser Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Ser Gln Val Phe Leu
114
~MEN~D SHEEr
CA 0222l234 l997-ll-l4 p ~ 9 6 / 0 6 8 ~ ~
~P~ 14 JAN ~9g7
Lys Leu Ser Ser Leu Gln Thr Glu Asp Thr Ala Thr Tyr Tyr Cy9 Ala
85 90 95
Arg Leu Pro Arg Gly Lys Gly Pro His Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Val Met Val Thr Val Ser Ser
115
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 354 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(B) STRAIN: LEW RAT
(F) TISSUE TYPE: Spleen, hyperimmunized
(G) CELL TYPE: Splenic lymphocyte
(vii) IMMEDIATE SOURCE:
(B) CLONE: ID12CF2
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..354
(D) OTHER INFORMATION: /product= ~ T ln~globulin Variable
Region"
/standard_name= "Ig Heavy Chain Variable Region"
/label= VH-Region
/note= "Variable Region of ID12CF2 Heavy Chain"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 1..90
_(D) OTHER INFORMATION: /standard_name= "Framework Region
/label= FR-1
/note= "F.d,.~ework Region l of ID12CF2 Heavy Chain
Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc RNA
(B) LOCATION: 91..105
(D) OTHER INFORMATION: /standard_name= "CDR-1"
/label= CDR-1
/note= "Complimentarity Determining Region 1 of
ID12CF2 Heavy Chain Variable Region"
115
D~D ~EET
CA 02221234 1997-11-14 ~ 9~ / 06 8
~PEAIU~ 14 JAN 1997
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 106..147
(D) OTHER INFORMATION: /standard_name= "Framework Region
2"
/label= FR-2
/note= "Framework Region 2 of ID12CF2 Heavy Chain
Variable Region"
(ix) FEAlu~E:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 148..198
(D) OTHER INFORMATION: /standard_name= "CDR-2"
/label= CDR-2
/note= "Complimentarity Determining Region 2 of
ID12CF2 Heavy Chain Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 199..294
(D) OTHER INFORMATION: /standard_name= "Framework Region
3"
/label= FR-3
/note= "Framework Region 3 of ID12CF2 Heavy Chain
Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 295..321
(D) OTHER INFORMATION: /~tandard_name= "CDR-3"
/label= CDR-3
/note= "Complimentarity Determining Region 3 of
ID12CF2 Heavy Chain Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 322..354
(D) OTHER INFORMATION: /standard_name= "Framework Region
4"
/label= FR-4
/note= "Framework Region 4 of ID12CF2 Heavy Chain
Variable Region"
(xi) ~u~N~ DESCRIPTION: SEQ ID NO:22:
GAG GTG CAG CTG GTG GAG TCT GGG GGA GGC TTA GTG CAG CCT GGA AGA 48
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
TCC CTG A~A CTC TCC TGT GCA GCC TCA GGA TTC ACT TTC AGT A~C TAT 96
Ser Leu Lys Leu Ser Cy5 Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
GGC ATG GCT TGG GTC CGC CAG GCT CC~ ACG AAG GGT CTG GAG TGG GTC 144
116
4MEND~D SHE~
CA 0222l234 l997-ll-l4
PC~US 96 ~ 06 8~4
~rEh~ JAN 1g97
Gly Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val
GCA TCC ATT AGT ACT GGT GGT GGT AAC ACT TAC TAT CGA GAC TCC GTG 192
Ala Ser Ile Ser Thr Gly Gly Gly Asn Thr Tyr Tyr Arg Asp Ser Val
50 55 60
AAG GGC CGA TTC ACT ATC TCC AGA GAT AAT GCA A~A AAC ACC CTA TAC 240
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
CTG CAA ATG GAC AGT CTG AGG TCT GAG GAC ACG GCC ACT TAT TAC TGT 288
Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
GCA AGA CCT TCC TAT AGC AGC TAC TTT GAT TAC TGG GGC CAA GGA GTC 336
Ala Arg Pro Ser Tyr Ser Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Val
100 105 110
ATG GTC ACA GTC TCC TCA 354
Met Val Thr Val Ser Ser
115
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 118 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(Xi) ~U~N~ DESCRIPTION: SEQ ID NO:23:
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
~er Leu Lys Leu Ser Cy5 Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
Gly Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val
Ala Ser Ile Ser Thr Gly Gly Gly Asn Thr Tyr Tyr Arg Asp Ser Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
~eu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
~la Arg Pro Ser Tyr Ser Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Val
100 105 110
117
AMENûED SHEET
CA 0222l234 l997-ll-l4 P~T/~ 9-6 ~ 06 8
~PE~ 14 lA~I 1997
Met Val Thr Val Ser Ser
115
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTE: 348 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(B) STRAIN: LEW RAT
(F) TISSUE TYPE: Spleen, hyper;mml-n;~ed
(G) CELL TYPE: Splenic lymphocyte
(vii) IMMEDIATE SOURCE:
(B) CLONE: FC2EG11
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..348
(D) OTHER INFORMATION: /product= "Tmmllnoglobulin Variable
Region"
/standard_name- "Ig Heavy Chain Variable Region"
/label= VH-Region
/notez "Variable Region of FC2EGll Heavy Chain"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 1..90
(D) OTHER INFORMATION: /standard_name= "Fr~ 7~rk Region
1"
/label= FR-1
/note= "FLa--,e~.Jrk Region 1 of FC2EGll Heavy Chain
Variable Region"
- (ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 91..105
(D) OTHER INFORMATION: /standard_name= "CDR-1"
/label= CDR-1
/note= "Complimentarity Determining Region 1 of
FC2EG11 Heavy Chain Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 106..147
(D) OTHER INFORMATION: /standard_name= "FLd..._~.Jrk Region
2"
/label= FR-2
118
~4~ENo~o SH~
CA 02221234 1997-11-14
~ f~ 9 ~ / n 6 8
1 4 JAN 1997
/note= "Framework Region 2 of FC2EGll Heavy Chain
Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 148..198
(D) OTHER INFORMATION: /standard_name= "CDR-2"
/label= CDR-2
/note= "Complimentarity Determining Region 2 of
FC2EGll Heavy Chain Variable Region"
(ix) FEAlu~E:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 199..294
(D) OTHER INFORMATION: /standard_name= "Framework Region
3"
/label= FR-3
/note= "Framework Region 3 of FC2EGll Heavy Chain
Variable Region"
(ix) FEAlU~E:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 295..315
(D) OTHER INFORMATION: /standard_name= "CDR-3"
/label= CDR-3
/note= "Compl;m~nt~rity Determining Region 3 of
FC2EGll Heavy Chain Variable Region"
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 316..348
(D) OTHER INFORMATION: /standard_name= "Framework Region
4"
/label= FR-4
/note= "Framework Region 4 of FC2EGll Heavy Chain
Variable Region"
(xi) ~Q~N~ DESCRIPTION: SEQ ID NO:24:
GAG GTG CAG CTG GTG GAG TCT GGG GGA GGC TTA GTG CAG CCT GGA AGA 48
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
TCC ATG A~A CTC TCC TGT GCA GCC TCA GGA TTC ACT TTC AGT AAC TAT 96
Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
TAC ATG GCC TGG GTC CGC CAG GCT CCA ACG AAG GGT CTG GAG TGG GTC 144
Tyr Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val
35 40 45
GCA TCC ATT AGT ACT GGT GGT GGT AAC ACT TAC TAT CGA GAC TCC GTG 192
Ala Ser Ile Ser Thr Gly Gly Gly Asn Thr Tyr Tyr Arg Asp Ser Val
50 55 60
119
AhlEN~D S~E~
. CA 0222l234 l997-ll-l4
PCTJUS 96J~68~4
f'~ 4 JAN 1997
AAG GGC CGA TTC ACT ATC TCC AGA GAT AAT GCA AAA AAC ACC CTA TAC 240
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
CTG CAA ATG GAC AGT CTG AGG TCT GAG GAC ACG GCC ACT TAT TAC TGT 288
Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
GCA AGA GGG GAG GCC TAC TTT GAT TAC TGG GGC CAA GGA GTC ATG GTC 336
Ala Arg Gly Glu Ala Tyr Phe Asp Tyr Trp Gly Gln Gly Val Met Val
100 105 110
ACA GTC TCC TCA 348
Thr Val Ser Ser
115
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 116 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
Tyr Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val
Ala Ser Ile Ser Thr Gly Gly Gly Asn Thr Tyr Tyr Arg Asp Ser Val
Lys Gly Arg Phe Thr Ile Ser Arg A~p Asn Ala Lys Asn Thr Leu Tyr
Leu Gln Met Asp Ser Leu Arg Ser Glu A~p Thr Ala Thr Tyr Tyr Cys
Ala Arg Gly Glu Ala Tyr Phe Asp Tyr Trp Gly Gln Gly Val Met Val
100 105 110
Thr Val Ser Ser
115
(2) INFORMATION FOR SEQ ID NO:26:
( i ) S~QU~N~ CHARACTERISTICS:
120
~n~ y~
. CA 0222l234 l997-ll-l4
~TIU~ 96/~68~4
4 JQN ~99~
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 1..18
(D) OTHER INFORMATION: /label= VH1
/note= "VH1 Family Specific Primer for Human Heavy
Chain Ig~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
CCATGGACTG GACCTGGA 18
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 ba~e pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc RNA
(B) LOCATION: 1..20
(D) OTHER INFORMATION: /label= VH2
/note= "VH2 Family Specific Primer for Human Heavy
Chain Ig"
(Xi) ~ U~N~ DESCRIPTION: SEQ ID NO:27:
ATGGACATAC ~lLl~LlC~AC 20
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomlc)
12
~ CA 0222l234 l997-ll-l4
9b / ~6 8
A~J 1997
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 1..20
(D) OTHER INFORMATION: /label= VH3
/note= "VH3 Family Specific Primer for Xuman Heavy
Chain Ig"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
CCATGGAGTT TGGGCTGAGC 20
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 1..20
(D) OTHFR INFORMATION: /label= VH4
/note= "VH4 Family Specific Primer for Human Heavy
Chain Ig"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
ATGAAACACC TGTGGTTCTT 20
(2) INFORMATION FOR SEQ ID NO:30:
(i) ~Qu~N~ CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: mi~c_RNA
(B) LOCATION: 1..20
(D) OTHER INFORMATION: /labels VH5
/note= "VH5 Family Specific Primer for Human Heavy
Chain Ig"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
122
D SI~EEr
CA 0222l234 l997-ll-l4 ~ J ~' 9 6 1 0 6 8 ~ 4
J~, t~ AN 1997
ATGGGGTCAA CCGCCATCCT 20
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 1..20
(D) OTHER INFORMATION: /label= VH6
/note= "VH6 Family Specific Primer for Human Heavy
Chain Ig"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
AL~l~L~lCT CCTTCCTCAT 20
(2) INFORMATION FOR SEQ ID NO-32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTE: 20 base pairs
(B) TYPE: nucleic acid
(C) sTRA~n~n~s: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 1..20
(D) OTHER INFORMATION: /label= HCM2
/note= "HCM2 Isotype Specific Primer for Human
Heavy Chain Ig"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
TCACAGGAGA CGAGGGGGAA 20
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 100 base pair~
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
123
~M',~ 3 ~
~ CA 0222l234 l997-ll-l4
P~96 f ~6 8 Gk
~p~ J ~ N 1997
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 1..100
(D) OTHER INFORMATION: /label= Notl-Linker
/note= "Oligonucleotide Linker Encoding Not 1
Restriction Site"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
AATTAACCCT CACTAAAGGG AACAAAAGCT GGAGCTTGAA TTCTTAACTA CTCGCCAAGG 60
AGACAGTCAT AATGAAATAC CTATTGCCTA CGGCGGCCGC 100
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 496 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(B) STRAIN: LEW RAT
(F) TISSUE TYPE: Spleen, hyperimmunized
(G) CELL TYPE: Splenic lymphocyte
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
CGAATTCGGG CCCTCGAGGC CTCTAGAATT CGCCCACTCA GTAATCAGTA CTACAGCACT 60
GCACAGACTC CTCACCATGG ACATCAGGCT CAGCTTGGCT TTCCTTCTCC TTTTCATAAA 120
AGGTGTCCAG TGTGAGGTGC AGCTGGTGGA GTCTGGGGGA GGCTTAGTGC AGCCTGGAAG 180
ATCCCTGAAA CTCTCCTGTG CAGCCTCAGG ATTCACTTTC AGTAACTATG GCATGGCTTG 240
GGTCCGCCAG GCTCCAACGA AGGGTCTGGA GTGGGTCGCA TCCATTAGTA CTGGTGGTGG 300
TAACACTTAC TATCGAGACT CCGTGAAGGG CCGATTCACT ATCTCCAGAG ATAATGCA~A 360
AAACACCCTA TACCTGCAAA TGGACAGTCT GAGGTCTGAG GACACGGCCA CTTATTACTG 420
TGCAAGACAT CGCGGGTATA ACTCCTACTG GTACTTTGAC TTCTGGGGCC CAGGAACCAT 480
GGTCACCGTG TCCTCA 496
(2) INFORMATION FOR SEQ ID NO:35:
124
AMEI\IC~D SI~E~
~ CA 0222l234 l997-ll-l4
PC~S 9~0~8~
v ~N 1997
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 464 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
tD) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(B) STRAIN: LEW RAT
(F) TISSUE TYPE: Liver
(Xi) ~Q~ DESCRIPTION: SEQ ID NO:35:
TACAGCACTG CACAGACTCC TCACCATGGA CATCAGGCTC AGCTTGGCTT TC~ll~,lCCT 60
TTTCATAAAA GGTAATTGAT AAAAGTGTGA TCAL~l~l~tl TGTGTGCACA TGAGAATAAG 120
AAAGTTTATT ~ll~llllGTT GTGTTAGTGA TG~llll~lA ACCAGTATTC T~L~,lllGCA 180
GGTGTCCAGT GTGAGGTGCA GCTGGTGGAG TCTGGGGGAG GCTTAGTGCA GCCTGGAAGG 240
TCCCTGAAAC TCTCCTGTGC AGCCTCAGGA TTCACTTTCA GTAACTATGG CATGGCCTGG 300
GTCCGCCAGG CTCCAACGAA GGGTCTGGAG TGGGTCGCAT CCATTAGTAC TGGTGGTGGT 360
AACACTTACT ATCGAGACTC CGTGAAGGGC CGATTCACTA TCTCCAGAGA TAATGCAAAA 420
AACACCCTAT ACCTGCAAAT GGACAGTCTG AGGTCTGAGG ACAC 464
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 346 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(B) STRAIN: LEW RAT
(F) TISSUE TYPE: Spleen, hyper;~lntzed
(G) CELL TYPE: Splenic lymphocyte
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
AGGTGAAGCT GCAGGAGTCA GGACCTGGTC TGGTACAGCC CTCACAGACC CTGTCCCTCA 60
125
~ CA 02221234 1997-11-14
rL~ b J ~
9 9 7
CCTGCACTGT CTCTGGGTTC TCACTAAACA ACTATGGTGT GATCTGGGTT CGCCAGCCTC 120
CAGGAAAGGG TCTGGAGTGG ATGGGAATAA TTTGGAATAA TGGAAATACA AATTATAATT 180
CAGCTCTCAA ATCCCGACTG AGCATCAGCA GGGACACCTC CAAGAGCCAA GTTTTCTTAA 240
AAATGAACAA TCTGCAAACT GAAGACACGG CCATGTACTT CTGTGCCAGA GGAGGAGTGG 300
GGTTTGATTT CTGGGGCCAA GGAGTCATGG TCACAGTCTC CTCAGA 346
(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 358 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(B) STRAIN: LEW RAT
(F) TISSUE TYPE: Spleen, hyperimmunized
(G) CELL TYPE: Splenic lymphocyte
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
AGGTCAAGCT GCAGCAGTCA GGACCTGGCC TGGTGCAGCC CTCACAGACC CTGTCTCTCA 60
CCTGCACTGT CTCTGGGTTC TCATTAACCA ACTATCATGT GCACTGGGTT CGACAGCCTC 120
CAGGAAAAGG TCTGGAGTGG ATGGGAGTCA TGTGGGGTGA TGGAGACACA TCATGTAATT 180
CAGCTCTCAA ATCCCGACTG AGCATCAGCA GGGACACCTC CAAGAGCCAA GTTTTCTTAA 240
AATTGAGCAG TCTGCAAACT GAAGACACAG CCACTTACTA CTGTGCCAGA CTCCCTAGGG 300
GGAAGGGACC CCACTTTGAT TACTGGGGCC AAGGAGTCAT GGTCACAGTC TCCTCAGA 358
(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 289 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(vi) ORIGINAL SOURCE:
(B) STRAIN: LEW RAT
126
A~NDED S~EFF
~ CA 0i22l234 l997-ll-l4
~T~L~J~ 9 6 / 0~ 8 ~ 4
1 1997
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
CAGGTGAAGC TGCAGGAGTC AGGACCTGGT CTGGTACAGC CCTCACAGAC CCTGTCCCTC 60
ACCTGCACTG TCTCTGGGTT CTCACTAAAC AACTATGGTG TGATCTGGGT TCGCCAGCCT 120
CCAGGAAAGG GTCTGGAGTG GATGGGAATA ATTTGGAATA ATGGAAATAC AAATTATAAT 180
TCAGCTCTCA AATCCCGACT GAGCATCAGC AGGGACACCT CCAAGAGCCA AGTTTTCTTA 240
AAAATGAACA ATCTGCAAAC TGAAGACACG GCCATGTACT TCTGTGCCA 289
(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 290 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
CAGGTGCAGC TGAAGGAGTC AGGACCTGGT CTGGTACAGC CCTCACAGAC CCTGTCCCTC 60
ACCTGCACTG l~l~lGGGTT CTCACTAAAC AACTATGGTG TGAl~lGG~l TCGCCAGCCT 120
CCAGGAAAGG GTCTGGAGTG GATGGGAATA ATTTGGAATA ATGGAAATAC AAATTATAAT 180
TCAGCTCTCA AATCCCGACT GAGCATCAGC AGGGACACCT CCAAGAGCCA A~1111~1 1A 240
AAAATGAACA AATTTGCAAA CTGAAGACAC GGCCATGTAC ll~l~lGCCA 290
127
AI~EN~EO S~
