U.S. patent application number 10/778394 was filed with the patent office on 2005-09-22 for agonist antibodies.
Invention is credited to Adams, Camellia W., Carter, Paul J., Fendly, Brian M., Gurney, Austin L..
Application Number | 20050208585 10/778394 |
Document ID | / |
Family ID | 32302025 |
Filed Date | 2005-09-22 |
United States Patent
Application |
20050208585 |
Kind Code |
A1 |
Adams, Camellia W. ; et
al. |
September 22, 2005 |
Agonist antibodies
Abstract
Various forms of c-mpl agonist antibodies are shown to influence
the replication, differentiation or maturation of blood cells,
especially megakaryocytes and megakaryocyte progenitor cells.
Accordingly, these compounds may be used for treatment of
thrombocytopenia.
Inventors: |
Adams, Camellia W.;
(Mountain View, CA) ; Carter, Paul J.; (San
Francisco, CA) ; Fendly, Brian M.; (Half Moon Bay,
CA) ; Gurney, Austin L.; (Belmont, CA) |
Correspondence
Address: |
Supervisor, Patent Prosecution Services
PIPER RUDNICK LLP
1200 Nineteenth Street, N.W.
Washington
DC
20036-2412
US
|
Family ID: |
32302025 |
Appl. No.: |
10/778394 |
Filed: |
February 17, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10778394 |
Feb 17, 2004 |
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09138091 |
Aug 21, 1998 |
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6737249 |
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60056736 |
Aug 22, 1997 |
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Current U.S.
Class: |
435/7.1 ;
424/144.1; 435/320.1; 435/334; 435/69.1; 506/14; 506/18;
530/388.22; 536/23.53 |
Current CPC
Class: |
C07K 2317/74 20130101;
C07K 2317/55 20130101; C07K 2317/21 20130101; C07K 2317/75
20130101; C07K 2317/54 20130101; C07K 2317/622 20130101; C07K 16/24
20130101; A61K 2039/505 20130101; C07K 2317/567 20130101; C07K
2317/92 20130101; C07K 16/40 20130101; C07K 16/2866 20130101 |
Class at
Publication: |
435/007.1 ;
435/069.1; 435/320.1; 435/334; 530/388.22; 536/023.53;
424/144.1 |
International
Class: |
G01N 033/53; C07H
021/04; C12P 021/06; A61K 039/395; C12N 015/09; C12N 005/06; C07K
016/28 |
Claims
1. An agonist antibody, fragment, or variant thereof which binds to
a thrombopoietin receptor.
2-19. (canceled)
20. A library of different single chain antibodies, comprising a
plurality of the antibody of claim 1.
21. The library of claim 20, wherein the single chain antibodies
are displayed on phage.
22. The library of claim 21, wherein the phage is M13 and the
antibodies are displayed as fusions of coat protein 3.
23. The library of claim 22, wherein less than 20% of the phage
display more than one fusion on the surface thereof.
24. A phage displaying on the surface thereof, the antibody of
claim 1.
25. The phage of claim 24, wherein the phage is M13 and the
antibodies are displayed as fusions of coat protein 3.
26. The phage of claim 25, wherein the phage displays only one
fusion on the surface thereof.
27. A fusion protein, comprising at least a portion of a phage coat
protein fused at the amino terminus thereof to the antibody of
claim 1.
28. The fusion protein of claim 27, wherein the phage coat protein
is M13 coat protein 3.
29. A method of stimulating proliferation, differentiation or
growth of megakaryocytes, comprising contacting megakaryocytes with
an effective amount of the antibody of claim 1.
30. The method of claim 29, comprising administering the antibody
of claim 1 to a patient in need thereof.
31. A method of increasing platelet production, comprising
contacting megakaryocytes with an effective amount of the antibody
of claim 1.
32. The method of claim 31, comprising administering the antibody
of claim 1 to a patient in need thereof.
33. Isolated nucleic acid encoding the antibody of claim 1.
34. A vector comprising the nucleic acid of claim 33.
35. A host cell comprising the vector of claim 34.
36. A method of producing an agonist antibody comprising culturing
the cell of claim 35 under conditions wherein the nucleic acid is
expressed.
37. An agonist antibody, fragment or variant thereof which binds to
a MuSK receptor.
38. A method of activating a receptor protein having two sub-units,
comprising contacting the receptor with a single chain antibody
which binds to the receptor.
39-42. (canceled)
Description
[0001] This application is a non-provisional application filed
under 37 CFR 1.53(b), claiming priority under 35 USC 119(e) to
provisional application No. 60/056,736, filed 22 Aug. 1997, the
contents of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to the recombinant synthesis and
purification of protein antibodies that influence survival,
proliferation, differentiation or maturation of hematopoietic
cells, especially platelet progenitor cells and to antibodies that
influence the growth and differentiation of cells expressing a
protein kinase receptor. This invention also relates to the cloning
and expression of nucleic acids encoding antibody ligands
(thrombopoietin receptor agonist antibodies) capable of binding to
and activating a thrombopoietin receptor such as c-mpl, a member of
the cytokine receptor superfamily. This invention further relates
to the use of these antibodies alone or in combination with other
cytokines to treat immune or hematopoietic disorders including
thrombocytopenia and to uses in assays.
BACKGROUND OF THE INVENTION
[0003] In 1994 several groups reported the isolation and cloning of
thrombopoietin (F. de Sauvage et al., Nature 369:533 (1994); S. Lok
et al., Nature 369:565 (1994); T D. Bartley et al., Cell 77:1117
(1994); Y. Sohma et al., FEBS Letters 353:57 (1994); D J. Kuter et
al., Proc. Natl. Acad. Sci. 91:11104 (1994)). This was the
culmination of more than 30 years of research initiated in the late
50's when Yamamoto (S. Yamamoto, Acta Haematol Jpn. 20:163-178.
(1957)) and Kelemen (E. Kelemen et al., Acda Haematol (Basel).
20:350-355 (1958)) proposed that physiological platelet production
is controlled by a humoral factor termed "thrombopoietin" (TPO).
Although routinely detected in urine, plasma and serum from
thrombocytopenic animals and patients, as well as kidney cell
conditioned media, purification of TPO proved to be a daunting task
(for a review see M S. Gordon et al., Blood 80:302 (1992); W.
Vainchenker et al., Critical Rev. Oncology/Hematology 20:165
(1995)). In the absence of purified TPO and the apparent fact that
numerous plieotrophic cytokines affected megakaryocytopoiesis (M S.
Gordon et al., Blood 80:302 (1992); W. Vainchenker et al., Critical
Rev. Oncology/Hematology 20:165 (1995)), the existence of a lineage
specific factor that regulated platelet production was doubted
until the discovery of the orphan cytokine receptor c-Mpl in 1990
(M. Souyri et al., Cell 63:1137 (1990); I. Vigon et al., Proc.
Nail. Acad. Sci. 89:5640 (1992)). The expression of c-Mpl was found
to be restricted to progenitor cells, megakaryocytes and platelets,
and c-Mpl antisense oligonucleotides selectively inhibited in vitro
megakaryocytopoiesis (M. Methia et al., Blood 82:1395 (1993)). From
this it was postulated that c-Mpl played a critical role in
regulating megakaryocytopoiesis and that its putative ligand may be
the long sought TPO (M. Methia et al., supra). Following this
discovery several groups utilizing c-Mpl ligand specific cell
proliferation assays and c-Mpl as a purification tool isolated and
cloned the ligand for c-Mpl (F. de Sauvage et al., supra; S. Lok et
al., supra; T D. Bartley et al., supra). In addition two other
groups independently reported the purification of the Mpl-ligand
using standard chromatography techniques and megakaryocyte assays
(Y. Sohma et al., supra; D J. Kuter et al., supra). In the years
since its reported discovery numerous studies clearly indicate that
the Mpl-ligand possess all the characteristics that have long been
attributed to the purported regulator of megakaryocytopoiesis and
thrombopoiesis and consequently, is now referred to as TPO. The Mpl
ligand is currently referred to as either TPO or as megakaryocyte
growth and differentiation factor (MGDF).
[0004] Human TPO consists of 332 amino acids that can be divided
into 2 domains; an amino terminal domain of 153 amino acids showing
23% identity (50% similarity) to erythropoietin (EPO) and a unique
181 amino acid C-terminal domain that is highly glycosylated ((F.
de Sauvage et al., supra; S. Lok et al., supra; T D. Bartley et
al., supra). The EPO-like domain of TPO contains 4 cysteines, 3 of
which are conserved with EPO The first and last and the two middle
cysteines form two disulfide bridges, respectively, which are both
required for activity (T. Kato et al., Blood 86 (suppl 1):365
(1995)). None of the Asn-linked glycosylation sites present in EPO
are conserved in the EPO-like domain of TPO, however, the EPO-like
domain of recombinant TPO (rTPO) contains 2-3 O-linked
glycosylations (M. Eng et al., Protein Science 5(suppl 1):105
(1996)). A recombinant truncated form of TPO (rTPO153), consisting
of only the EPO-like domain, is fully functional in vitro,
indicating that this domain contains all the required structural
elements to bind and activate Mpl (F. de Sauvage et al., supra; D
L. Eaton et al., Blood 84(suppl 1):241 (1994)). The carboxy
terminal domain of TPO contains 6 N-linked and 18 O-linked
glycosylated sites and is rich in proline, serine and threonine (M.
Eng et al., supra). The function of this domain remains to be
elucidated. However, because of its high degree of glycosylation
this region may act to stabilize and increase the half life of
circulating TPO. This is supported by the observation that rTPO153
has a half life of 1.5 hours compared to 18-24 hours for full
length glycosylated rTPO (G R. Thomas et al., Stem Cells 14(suppl
1) (1996). The two domains of TPO are separated by a potential
dibasic proteolytic cleavage site that is conserved among the
various species examined. Processing at this site could be
responsible for releasing the C-terminal region from the EPO domain
in vivo. The physiological relevance of this potential cleavage
site is unclear at this time. Whether TPO circulates as an intact
full length molecule or as a truncated form is also equivocal. When
aplastic porcine plasma was subjected to gel filtration
chromatography, TPO activity present in this plasma resolved with a
Mr. of .about.150,000 ((F. de Sauvage et al., supra). Purified full
length rTPO also resolves at this Mr., whereas the truncated forms
resolve with Mr. ranging from 18,000-30,000. Using TPO ELISAs that
selectively detect either full length or truncated TPO it has also
been shown that full length TPO is the predominant form in the
plasma of marrow transplant patients (Y G. Meng et al., Blood
86(suppl. 1):313 (1995)).
[0005] Prior to the discovery of c-Mpl and the isolation of TPO, it
was thought that megakaryocytopoiesis 30, was regulated at multiple
cellular levels (M S. Gordon et al., supra; W. Vainchenker et al.,
supra; Y G. Meng et al., supra) This hypothesis was based on the
observation that certain hematopoietic growth factors stimulated
proliferation of megakaryocyte progenitors while others primarily
affected maturation (M S. Gordon et al., supra; W Vainchenker et
al., supra; Y G. Meng et al., supra). Other data indicated that
plasma from thrombocytopenic animals contained distinct activities
that either affected proliferation (meg-CSF) or maturation (TPO) of
megakaryocytes (R J. Hill et al., Exp. Hematol 20:354 (1992)).
Wendling and her colleagues (F. Wendling et al., Nature 369:571
(1994)) initially dispelled this theory by demonstrating that all
the megakaryocyte colony-stimulating and thrombopoietic activities
in thrombocytopenic plasma could be neutralized by soluble Mpl.
This indicated that these activities are due to a single factor,
the Mpl-ligand. Numerous studies have now shown that recombinant
forms of TPO not only induce proliferation of progenitor
megakaryocytes but also their maturation (K. Kaushansky et al.,
Nature 369:568 (1994); F C. Zeigler et al., Blood 84:4045 (1994); V
C. Broudy et al., Blood 85:1719 (1995); J L. Nichol et al., J.
Clin. Invest. 95:2973 (1995); N. Banu et al., Blood 86:1331 (1995);
N. Debili et al., Blood 86:2516 (1995); P. Angchaisuksiri et al.,
Br. J. Haematol. 93:13 (1996); E S. Choi et al., Blood 85:402
(1995)). Human CD34+, CD34+CD41+cells (F C. Zeigler et al., supra;
V C. Broudy et al., supra; J L. Nichol et al., supra; N. Banu et
al., supra;) or purified murine stem cells (sca+, lin-, kit+) (K.
Kaushansky et al., supra; F C. Zeigler et al., supra) cultured with
rTPO selectively differentiate to megakaryocytes. rTPO induces the
differentiation and proliferation of megakaryocyte colonies in
semisolid cultures and single megakaryocytes in liquid suspension
cultures. This activity appears to be a direct effect of TPO as
limiting dilution experiments show a direct correlation between
progenitors seeded and megakaryocytes obtained (N. Debili et al.,
supra). In addition comparable results are obtained in serum free
or serum containing culture conditions (N. Banu et al., supra; N.
Debili et al., supra; P. Angchaisuksiri et al., supra,). These
observations indicate that neither accessory cells or serum
components are required for TPO to induce megakaryocyte growth and
differentiation in vitro.
[0006] The effect of rTPO on the megakaryocyte maturation process
is dramatic. rTPO induces highly purified murine or human
progenitor cells in liquid culture to differentiate into very large
mature polyploid megakaryocytes (F C. Zeigler et al., supra; V C.
Broudy et al., supra; J L. Nichol et al., supra; N. Debili et al.,
supra) Megakaryocytes from such cultures exhibit ploidy of 4N-16N
with ploidy classes of 64N and 128N also being detected in these
cultures (N. Debili et al., supra). In addition, megakaryocytes
produced from these cultures undergo a terminal maturation process
and appear to develop proplatelets and shed platelet like
structures into the medium (F C. Zeigler et al., supra; N. Debili
et al., supra; E S. Choi et al., supra). Significantly, the
platelets produced from such cultures have been shown to be
morphologically and functionally indistinct from plasma-derived
platelets (E S. Choi et al., supra).
[0007] Although, rTPO appears to act directly on hematopoietic
progenitors to induce megakaryocyte differentiation, it also acts
synergistically and additively with early and late acting
hematopoietic factors. In murine megakaryocytopoiesis assays IL-11,
kit ligand (KL) or EPO act synergistically and IL-3 and IL-6 act
additively with rTPO to stimulate proliferation of megakaryocyte
progenitors (V C. Broudy et al., supra). In human
megakaryocytopoiesis assays IL-3 and IL-6 effects are additive to
rTPO, while KL acts synergistically with rTPO (J L. Nichol et al.,
supra; N. Banu et al., supra; N. Debili et al., supra; P.
Angchaisuksiri et al., supra) None of the cytokines mentioned above
affect the megakaryocyte maturational activity of rTPO.
[0008] The initial studies with rTPO clearly indicate that TPO
predominantly affects the megakaryocytic lineage. However, like all
other hematopoietic regulators, TPO affects other hematopoietic
lineages as well. In the presence of EPO, rTPO has been shown to
enhance erythroid burst (BFU-E) formation in human CD34+ colony
assays (M. Kobayashi et al., Blood 86:2494 (1995); T.
Papayannopoulou et al., Blood 87:1833 (1996)). The burst promoting
activity of rTPO is comparable to GM-CSF and KL and increases both
the number and size of BFU-E colonies (M. Kobayashi et al., supra).
In addition rTPO also stimulates CFU-E development, indicating that
TPO acts on both early and late erythroid progenitors (M. Kobayashi
et al., supra; T. Papayannopoulou et al., supra). In the absence of
EPO, however, rTPO has no effect on erythropoiesis. An effect of
rTPO on myeloid colony growth in normal hematopoietic cultures has
not been demonstrated in vitro; however.
[0009] rTPO has a dramatic effect on platelet production when
administered to normal animals. Pharmacological doses of
recombinant forms of TPO cause as much as a 10 fold increase in
platelet levels in mice and non-human primates (E F. Winton et al.,
Exp. Hematol. 23:879 (1995); A M. Farese et al., Blood 86:54
(1995); K H. Sprugel et al., Blood 86(suppl 1):20 (1995); L A.
Harker et al., Blood 87:1833 (1996); K. Kaushansky et al., Exp.
Hematol 24:265 (1996); T R. Ulich et al., Blood 87:5006 (1996); K.
Ault et al., Blood 86(suppl 1): 367 (1995); N C. Daw et al., Blood
86 (suppl 1):5006 (1995)). This effect of rTPO is due to an
increase in the synthesis of new platelets as reticulated platelets
increase within 24 hours after rTPO administration (K. Ault et al.,
supra). Preceding this effect is a dramatic increase in CFU-MK in
both the marrow and spleen (A M. Farese et al., supra; K.
Kaushansky et al., supra; T R. Ulich et al., supra). Megakaryocytes
from rTPO treated animals exhibit a higher mean ploidy and are
larger in size than megakaryocytes from control animals. These
later two observations again demonstrate the proliferative and
maturational activities of TPO on the megakaryocytic lineage.
Because the effect of TPO on megakaryocytes precedes its effect on
platelet production it has been suggested that TPO primarily
affects megakaryocyte progenitors rather than inducing platelet
release from mature megakaryocytes (N C. Daw et al., supra). No
significant effect on red blood cell (RBC) or white blood cell
(WBC) production occurs in normal animals following rTPO
administration. However, rTPO treatment caused an expansion of
BFU-E and CFU-GM and a redistribution CFU-E in normal mice (K.
Kaushansky et al., supra) and expanded CFU-mixed in rhesus monkeys
(A M. Farese et al., supra).
[0010] Even though rTPO dramatically stimulates platelet
production, it only has a modest effect on platelet function. In
vitro studies show that rTPO has no effect on platelet aggregation
itself, but does enhance agonist induced aggregation (G.
Montrucchio et al., Blood 87:2762 (1996); A. Oda et al., Blood
87:4664 (1996); C F. Toombs et al., Thromb. Res. 80:23 (1995); C F.
Toombs et al., Blood 86(suppl 1):369 (1995)). rTPO appears to
sensitize platelets making them moderately more responsive to
aggregation agonist. This raises the possibility that rTPO may have
prothrombotic effects in vivo. However, an increase in thrombotic
episodes in animals treated with rTPO has never been observed, even
when platelet levels were 4-10 fold above normal. In vivo
thrombosis models also indicate that elevated platelet levels
following rTPO treatment is not associated with an increase in
platelet dependent thrombosis (L A. Harker et al., supra; C F.
Toombs et al., supra). These results indicate that stimulation of
platelet production by rTPO will unlikely be associated with an
increase in thrombo-occulsive events.
[0011] The involvement of c-Mpl and TPO in the control of platelet
production and its effect on other hematopoietic lineages is
further demonstrated by the phenotype of mice deficient in either
the c-mpl or the TPO genes (W S. Alexander et al., Blood 87:2162
(1996); F J. de Sauvage et al., J. Exp. Med 183:651 (1996); A L.
Gurney et al., Science 265:1445 (1994)). In both cases a dramatic
85 to 90% drop in platelet counts is observed with a similar
decrease of megakaryocytes in the spleen and bone marrow. In
addition, the megakaryocytes of the knockout mice are smaller and
exhibit a lower ploidy than those of control mice. The similarity
in phenotype observed for these knock-outs (KO) indicates that the
system is non-redundant and that there is probably only one
receptor for TPO and one ligand for c-Mpl. Although the platelet
number is reduced in the KO mice their platelets appear normal,
both structurally and functionally, and are sufficient to prevent
overt bleeding. The genes and factors involved in the production of
this basal level of platelets and megakaryocytes still remain to be
identified. However, treatment of either the TPO or c-mpl knockout
mice with other cytokines with megakaryopoietic activity (IL-6,
IL-11 and stem cell factor) results in a modest stimulation of
platelet production (A L. Gurney et al., supra). This suggest that
these cytokines do not require TPO or c-mpl to exert their
thrombopoietic activity and, therefore, may be involved in the
maintenance of a basal level of megakaryocytes and platelets.
[0012] Comparison of CFU-megakaryocyte (CFU-Meg) from TPO or c-mpl
deficient and normal mice shows that the number of megakaryocytes
progenitors is decreased in both knock-outs compared to control,
suggesting that TPO acts on very early megakaryocyte progenitors.
In addition, both erythroid and myeloid progenitors are also
reduced in the TPO and c-Mpl knockout mice (W S. Alexander et al.,
supra; K. Carver-Moore et al., 88:803 (1996)). This reduction in
progenitors from all lineages indicates that TPO probably acts on a
very early pluripotent progenitor cell. The involvement of TPO and
c-Mpl at an early stage of hematopoiesis correlates with the
detection of c-Mpl expression in AA4+Sca+ murine stem cell
population (F C. Zeigler et al., supra). The effect of TPO on this
most primitive stem cell population still remains to be
investigated, however, preliminary data indicate that TPO may
directly affect the proliferation of primitive murine hematopoietic
stem or progenitor cells (E. Stinicka et al., Blood 87:4998 (1996);
M. Kobayashi et al., Blood 88:429 (1996); H. Ku et al., Blood
87:4544 (1996)). This, in part, may explain the effect TPO has on
erythropoiesis and myelopoiesis in vitro and in vivo.
[0013] It has long been observed that an inverse correlation exists
between plasma megakaryopoietic and thrombopoietic activity and
platelet levels (reviewed in T P. McDonald, Am. J. Pediair.
Hematol/Oncol. 14:8 (1992)). TPO specific ELISAs and cell
proliferation assays have now confirmed that TPO levels increase
and decrease inversely with platelet mass (J L. Nichol et al.,
supra; E V B. Emmons et al., Blood 87:4068 (1996); H. Oh et al.,
Blood 87:4918 (1996); M. Chang et al., Blood 86(suppl 1):368
(1995)). Unlike erythropoietin, however, TPO does not appear to be
regulated at the transcriptional level, but rather by platelet
mass. This was initially proposed de Gabriele and Pennington (G. de
Gabriele et al., Br. J. Haematol. 13:202 (1967); G. de Gabriele et
al., Br. J. Haematol. 13:210 (1967)) and subsequently confirmed by
Kuter and Rosenberg (D J. Kuter et al., Blood 84:1464 (1994)) who
showed direct regulation of circulating TPO levels by exogenously
administering platelets to thrombocytopenic mice. More recently, it
was demonstrated that TPO mRNA levels in thrombocytopenic mice are
not increased even though TPO levels are elevated by at least 10
fold (P J. Fielder et al., Blood 87:2154 (1996); R. Stoffel et al.,
Blood 87:567 (1996)). In addition, the gene dosage effect observed
in TPO heterozygous knockout mice refute the regulation of TPO
production by platelet mass (F J. de Sauvage et al., supra). Taken
together, these results strongly support the hypothesis that TPO
expression is constitutive and it is the sequestering by platelets
that regulates TPO levels. Platelets bind TPO with high affinity
(Kd(100-400 pM) and internalize and degrade TPO (P J. Fielder et
al., supra). Platelets from c-Mpl knockout mice do not bind TPO and
the clearance of TPO by these mice is 5 fold slower than that
observed for wild type mice (P J. Fielder et al., supra). These
results indicate that TPO clearance is mediated by platelet binding
via c-Mpl. It is also likely that megakaryocyte mass plays a role
in regulating circulating TPO levels. This is supported by the
observation that both ITP patients and mice deficient in the NF-E2
transcription factor are highly thrombocytopenic, exhibit
megakaryocytosis, but have normal TPO levels (E V B. Emmons et al.,
supra; R A. Shivdasani et al., Cell 81:695 (1995)). In situ studies
with radiolabeled TPO show that marrow megakaryocytes of the NF-E2
mice bind significant amounts of labeled TPO (R A. Shivdasani et
al., Blood submitted (1996)). The phenotype of the ITP and NF-E2
knockout mice, therefore, suggest that binding of TPO to
megakaryocytes may also regulate TPO levels.
[0014] The dramatic effect of rTPO on platelet production in normal
mice and monkeys and subsequent clinical trials indicate that rTPO
is clinically useful in alleviating thrombocytopenia associated
with myelosuppressive and myeloablative therapies for cancer
patients. In several myelosuppressive and myeloablative murine and
monkey preclinical models recombinant forms of TPO have been shown
to significantly affect platelet recovery. In mice treated with
carboplatin and sublethal irradiation in combination (J P Leonard
et al., Blood 83:1499 (1994)), daily treatment with rTPO both
reduced the severity of the platelet nadir and accelerated platelet
recovery by 10-12 days when compared to excipient treated animals
(G R. Thomas et al, supra; K. Kaushansky et al., supra; M M. Hokom
et al., Blood 86:4486 (1995)). Similar results were obtained in a
murine sublethal irradiation model (G R. Thomas et al., supra). In
murine myeloablative transplantation models rTPO has been shown to
reduce the extent of the nadir and accelerate platelet recovery by
2-3 weeks (G R. Thomas et al., supra; K. Kabaya et al., Blood
86(suppl 1):114 (1995); G. Molineux et al., Blood 86(suppl 1):227
(1995)). Treatment of sublethally irradiated rhesus monkeys with
rTPO accelerated platelet recovery by 3 weeks and prevented
platelet nadirs below 40,000 (A M. Farese et al., J. Clin. Invest.
97:2145 (1996); K J. Neelis et al., Blood 86(suppl 1):256 (1995)).
Even more impressively, rTPO completely prevented post-chemotherapy
thrombocytopenia following the treatment of rhesus monkeys with
hepsulfam (A M. Farese et al., supra). In contrast to these
promising results, two groups have reported that rTPO had no effect
on the hematopoietic recovery of lethally irradiated mice or
monkeys rescued with a marrow transplant (K J. Neelis et al.,
supra; W E. Fibbe et al., Blood 86:3308 (1995)). The reason for
this discrepancy is unclear, however it is possible that lethal
radiation may destroy stromal cells or components essential for TPO
activity in vivo. In support of this, lethally irradiated mice
transplanted with marrow cells from rTPO treated donor mice show
accelerated recovery of platelets and RBCs, however,
post-transplant administration of rTPO had no further effect on
this accelerated recovery (W E. Fibbe et al., supra). This result
suggests that although the transplanted cell population was
enriched for megakaryocyte progenitors, TPO had no effect on these
progenitors in a lethally irradiated marrow.
[0015] Although rTPO only modestly affects erythroid and myeloid
lineages in normal mice it dramatically accelerates the recovery of
all progenitor classes in myelosuppressed mice and monkeys
resulting in a significant acceleration of RBC and WBC recovery (K.
Kaushansky et al., supra; A M. Farese et al., supra; K. Kalisntnsky
et al., J. Clin. Invest. 96:1683 (1995)). The effect of rTPO on
neutrophil recovery has been shown to be additive to that of G-CSF
(A M. Farese et al., supra). These results indicate that the
clinical utility of rTPO may be broader than originally
anticipated.
[0016] The difference between the effect of rTPO on hematopoiesis
in normal and myelosuppressed animals is likely due to the change
in the cytokine environment that occurs following myelosuppressive
therapy. It is likely that elevated levels of EPO, G-CSF or other
cytokines essential for erythropoiesis and myelopoiesis present
following myelosuppressive treatment interact with rTPO to have a
multilineage effect (K. Kaushansky et al., supra). In normal mice
the level of these cytokines are insufficient and the effects of
rTPO on erythroid and myeloid lineages are less significant. This
hypothesis is supported by the above mentioned synergistic
interaction of rTPO and EPO to stimulate in vitro erythropoiesis (E
S. Choi et al., supra). It has also been proposed that production
of hemopoietic factors from megakaryocytes themselves may also play
a role in the multilineage effect of rTPO (A M. Farese et al.,
supra).
[0017] In the above mentioned animal studies rTPO was administered
daily for 14-28 days, which was based on previous experience in
dosing other hematopoietic growth factors. However, it has recently
been shown that a single dose of rTPO following myelosuppressive
treatment of mice with carboplatin and sublethal irradiation is as
effective as multiple doses in reducing nadirs and accelerating
platelet and RBC recovery (G R. Thomas et al., supra). This effect
is likely due to the potency and long half life of rTPO.
[0018] (G R. Thomas et al., supra). This is supported by the fact
that single doses of unglycosylated rTPO153 are not effective in
this model. These observations indicate that the frequency of rTPO
dosing required to affect hematopoietic recovery following
myelosuppressive treatment may be significantly less than that for
other currently used cytokines.
[0019] Early results from human clinical trails show that rTPO also
stimulates platelet production in humans.
[0020] In phase I trials, a pegylated and truncated form of rTPO
(MGDF) administered daily for 10 days at 0.03-5.0 .mu.g/kg to
cancer patients prior to chemotherapy caused up to a four fold
increase in circulating platelet levels (R. Basser et al., Blood
86(suppl 1): 257 (1995); J E J. Rasko et al., Blood 86(suppl 1):497
(1995)). Similarly, patients given a single dose of rTPO had
platelet levels increase by four fold (S. Vaden-Raj et al.,
Stimulation of megakaryocyte and platelet production by a single
dose of recombinant human thrombopoietin in cancer patients.
Submitted. (1996)). In both studies platelet increases are observed
by day four and peak about 12-16 days later. No drug related
toxicity's were reported and, although platelet levels greater then
1.times.106/.mu.l were observed in some of the patients, no
thrombotic events were observed. This indicates that TPO will be
well tolerated in humans. In myelosuppressed patients, pegylated
rTPO153(MGDF) given post chemotherapy has been shown to reduce the
extent of the platelet nadir following chemotherapy (G. Begley et
al., Proceedings of ASCO 15:271 (1996); M. Fanucchi et al.,
Proceedings of ASCO 15:271 (1996)). As seen in the preclinical
animals studies, TPO also expanded marrow progenitors of
megakaryocyte, erythroid, myeloid and multipotential lineages (S.
Vaden-Raj et al., supra). This later observation suggests that rTPO
may be useful as a priming agent.
[0021] It is believed that the proliferation and maturation of
hematopoietic cells is tightly regulated by factors that positively
or negatively modulate pluripotential stem cell proliferation and
multilineage differentiation. These effects are mediated through
the high-affinity binding of extracellular protein factors
(ligands) to specific cell surface receptors. These cell surface
receptors share considerable homology and are generally classified
as members of the cytokine receptor superfamily. Members of the
superfamily include receptors for: IL-2 (b and g chains)
(Hatakeyama et al., Science, 244:551-556 (1989); Takeshita et al.,
Science, 257:379-382 (1991)), IL-3 (Itoh et al., Science,
247:324-328 (1990); Gorman et al., Proc. Natl. Acad. Sci. USA,
87:5459-5463 (1990); Kitamura et al., Cell, 66:1165-1174 (1991a);
Kitamura et al., Proc. Natl. Acad. Sci. USA, 88:5082-5086 (1991b)),
IL-4 (Mosley et al., Cell, 59:335-348 (1989), IL-5 (Takaki et al.,
EMBO J., 9:4367-4374 (1990); Tavernier et al., Cell, 66:1175-1184
(1991)), IL-6 (Yamasaki et al., Science, 241:825-828 (1988); Hibi
et al., Cell, 63:1149-1157 (1990)), IL-7 (Goodwin et al., Cell,
60:941-951 (1990)), IL-9 (Renault et al., Proc. Natl. Acad. Sci.
USA, 89:5690-5694 (1992)), granulocyte-macrophage
colony-stimulating factor (GM-CSF) (Gearing et al., EMBO J.,
8:3667-3676 (1991); Hayashida et al., Proc. Natl. Acad. Sci. USA,
244:9655-9659 (1990)), granulocyte colony-stimulating factor
(G-CSF) (Fukunaga et al., Cell, 61:341-350 (1990a); Fukunaga et
al., Proc. Natl. Acad. Sci. USA, 87:8702-8706 (1990b); Larsen et
al., J. Exp. Med, 172:1559-1570 (1990)), EPO (D'Andrea et al.,
Cell, 57:277-285 (1989); Jones et al., Blood, 76:31-35 (1990)),
Leukemia inhibitory factor (LIF) (Gearing et al., EMBO J.,
10:2839-2848 (1991)), oncostatin M (OSM) (Rose et al., Proc. Natl.
Acad. Sci. USA, 88:8641-8645 (1991)) and also receptors for
prolactin (Boutin et al., Proc. Natl. Acad. Sci. USA, 88:7744-7748
(1988); Edery et al., Proc. Natl. Acad. Sci. USA, 86:2112-2116
(1989)), growth hormone (GH) (Leung et al., Nature, 330:537-543
(1987)) and ciliary neurotrophic factor (CNTF) (Davis et al.,
Science, 253:59-63 (1991).
[0022] Members of the cytokine receptor superfamily may be grouped
into three functional categories (for review see Nicola et al.,
Cell, 67:1-4 (1991)). The first class comprises single chain
receptors, such as erythropoietin receptor (EPO-R) or granulocyte
colony stimulating factor receptor (G-CSF-R), which bind ligand
with high affinity via the extracellular domain and also generate
an intracellular signal. A second class of receptors, so called
a-subunits, includes interleukin-6 receptor (IL6-R),
granulocyte-macrophage colony stimulating factor receptor
(GM-CSF-R), interleukin-3 receptor (IL3-Ra) and other members of
the cytokine receptor superfamily. These a-subunits bind ligand
with low affinity but cannot transduce an intracellular signal. A
high affinity receptor capable of signaling is generated by a
heterodimer between an a-subunit and a member of a third class of
cytokine receptors, termed b-subunits, e.g., b.sub.c, the common
b-subunit for the three a-subunits of IL-3-R, IL-5-R and GM-CSF-R
(Nicola N. A. et. al. Cell 67:1-4 (1991)).
[0023] Evidence that mpl is a member of the cytokine receptor
superfamily comes from sequence homology. (Gearing, EMBO J.,
8:3667-3676 (1988); Bazan, Proc. Natl. Acad. Sci. USA, 87:6834-6938
(1990); Davis et al., Science, 253:59-63 (1991) and Vigon et al.,
Proc. Natl. Acad. Sci. USA, 89:5640-5644 (1992)) and its ability to
transduce proliferative signals.
[0024] Deduced protein sequence from molecular cloning of murine
c-mpl reveals this protein is homologous to other cytokine
receptors. The extracellular domain contains 465 amino acid
residues and is composed of two subdomains each with four highly
conserved cysteines and a particular motif in the N-terminal
subdomain and in the C-terminal subdomain. The ligand-binding
extracellular domains are predicted to have similar double b-barrel
fold structural geometries. This duplicated extracellular domain is
highly homologous to the signal transducing chain common to IL-3,
IL-5 and GM-CSF receptors as well as the low-affinity binding
domain of LIF (Vigon et al., Oncogene, 8:2607-2615 (1993)). Thus
mpl may belong to the low affinity ligand binding class of cytokine
receptors.
[0025] A comparison of murine mpl and mature human mpl P, reveals
these two proteins show 81% sequence identity. More specifically,
the N-terminus and C-terminus extracellular subdomains share 75%
and 80% sequence identity respectively. The most conserved mpl
region is the cytoplasmic domain showing 91% amino acid identity,
with a sequence of 37 residues near the transmembrane domain being
identical in both species. Accordingly, mpl is reported to be one
of the most conserved members of the cytokine receptor superfamily
(Vigon supra).
[0026] Activation of certain hematopoietic receptors is believed to
cause one or more effects including; stimulation of proliferation,
stimulation of differentiation, stimulation of growth and
inhibition of apoptosis (Libol et al Proc. Natl. Acad. Sci. 248:378
(1993). Activation of hematopoietic receptors upon ligand binding
may be due to dimerization of two or more copies of the receptor.
In addition to the naturally occurring ligand causing this
dimerization, agonist antibodies may also activate receptors by
crosslinking or otherwise causing dimerization of a receptor. Such
antibodies are useful for the same indications as the natural
ligand and may have advantageous properties such as a longer
half-life. An example of a monoclonal antibody to a cytokine
receptor that activates the erythropoietin receptor (EPO-R) is
described in WO 96/03438 (published 8 Feb. 1996). These agonist
antibodies to EPO-R are about 3-4 orders of magnitude weaker in
activity based on weight than the natural EPO ligand.
[0027] There is a current and continuing need to isolate and
identify molecules, especially antibodies, fragments and
derivatives thereof, capable of stimulating proliferation,
differentiation and maturation and/or modulation of apoptosis of
cells, for example hematopoietic cells, including megakaryocytes or
their predecessors for therapeutic use in the treatment of
hematopoietic disorders including thrombocytopenia.
SUMMARY OF THE INVENTION
[0028] Accordingly, It is an object of this invention to obtain a
pharmaceutically or essentially pure antibody or fragments or
derivatives thereof capable of stimulating proliferation,
differentiation and/or maturation of hematopoietic cells, including
megakaryocytes or their predecessors, or to modulate apoptosis of
hematopoietic cells:
[0029] It is a specific object of the present invention to isolate
antibody ligands capable of binding in vivo a hematopoietic growth
factor superfamily receptor and to activate the receptor, the
antibody having a biological activity equal to or not less than 2
orders of magnitude below that of the naturally occurring ligand on
a weight basis.
[0030] It is also an object of the present invention to isolate
antibody ligands capable of binding to and activating any of the
three functional categories of cytokine superfamily receptors (see
Nicola et al., Cell, 67:1-4 (1991)).
[0031] In one embodiment, the objects of the invention are achieved
by providing an antibody or fragment thereof that activates a
hematopoietic growth factor superfamily receptor having a
biological activity within 2 orders of magnitude (100), preferably
within one order of magnitude (10), of the natural ligand on a
weight basis Preferably, the antibody activates the thrombopoietin
(TPO) receptor. This antibody, referred to as an agonist antibody,
activates a thrombopoietin receptor which preferably comprises a
mammalian c-mpl, more preferably human c-mpl. Usually the antibody
will be a full length antibody such as an IgG antibody. Suitable
representative fragment agonist antibodies include Fv, ScFv, Fab,
F(ab').sub.2 fragments, as well as diabodies and linear antibodies.
These fragments may be fused to othersequences including, for
example, the F" or Fc region of an antibody, a "leucine zipper" or
other sequences including pegylated sequences or Fc mutants used to
improve or modulate half-life. Normally the antibody is a human
antibody and may be a non-naturally occurring antibody, including
affinity matured antibodies. Representative antibodies that
activate c-mpl are selected from the group 12E10, 12B5, 10F6 and
12D5, and affinity matured derivatives thereof. Other preferred
agonist antibodies to c-mpl are selected from the group consisting
of Ab1, Ab2, Ab3, Ab4, Ab5 and Ab6, wherein each Ab1-Ab6 contains a
VH and VL chain and each VH and VL chain contains complementarity
determining region (CDR) amino acid sequences designated CDR1, CDR2
and CDR3 separated by framework amino acid sequences, the amino
acid sequence of each CDR in each VH and VL chain fAb1-Ab6 is shown
in Table 1.
1TABLE 1 Ab1: VH.sup.CDR1 VH.sup.CDR2 VH.sup.CDR3 DNA (SEQ ID NO:
1) (SEQ ID NO: 3) (SEQ ID NO: 5) protein (SEQ ID NO: 2) (SEQ ID NO:
4) (SEQ ID NO: 6) VL.sup.CDR1 VL.sup.CDR2 VL.sup.CDR3 DNA (SEQ ID
NO: 7) (SEQ ID NO: 9) (SEQ ID NO: 11) protein (SEQ ID NO: 8) (SEQ
ID NO: 10) (SEQ ID NO: 12) Ab2: VH.sup.CDR1 VH.sup.CDR2 VH.sup.CDR3
DNA (SEQ ID NO: 13) (SEQ ID NO: 15) (SEQ ID NO: 17) protein (SEQ ID
NO: 14) (SEQ ID NO: 16) (SEQ ID NO: 18) VL.sup.CDR1 VL.sup.CDR2
VL.sup.CDR3 DNA (SEQ ID NO: 19) (SEQ ID NO: 21) (SEQ ID NO: 23)
protein (SEQ ID NO: 20) (SEQ ID NO: 22) (SEQ ID NO: 24) Ab3:
VH.sup.CDR1 VH.sup.CDR2 VH.sup.CDR3 DNA (SEQ ID NO: 25) (SEQ ID NO:
27) (SEQ ID NO: 29) protein (SEQ ID NO: 26) (SEQ ID NO: 28) (SEQ ID
NO: 30) VL.sup.CDR1 VL.sup.CDR2 VL.sup.CDR3 DNA (SEQ ID NO: 19)
(SEQ ID NO: 21) (SEQ ID NO: 23) protein (SEQ ID NO: 20) (SEQ ID NO:
22) (SEQ ID NO: 24) Ab4: VH.sup.CDR1 VH.sup.CDR2 VH.sup.CDR3 DNA
(SEQ ID NO: 25) (SEQ ID NO: 31) (SEQ ID NO: 33) protein (SEQ ID NO:
26) (SEQ ID NO: 32) (SEQ ID NO: 34) VL.sup.CDR1 VL.sup.CDR2
VL.sup.CDR3 DNA (SEQ ID NO: 35) (SEQ ID NO: 21) (SEQ ID NO: 23)
protein (SEQ ID NO: 20) (SEQ ID NO: 22) (SEQ ID NO: 24) Ab5:
VH.sup.CDR1 VH.sup.CDR2 VH.sup.CDR3 DNA (SEQ ID NO: 36) (SEQ ID NO:
38) (SEQ ID NO: 46) protein (SEQ ID NO: 37) (SEQ ID NO: 39) (SEQ ID
NO: 41) VL.sup.CDR1 VL.sup.CDR2 VL.sup.CDR3 DNA (SEQ ID NO: 19)
(SEQ ID NO: 21) (SEQ ID No: 23) protein (SEQ ID NO: 20) (SEQ ID NO:
22) (SEQ ID No: 24) Ab6: VH.sup.CDR1 VH.sup.CDR2 VH.sup.CDR3 DNA
(SEQ ID NO: 42) (SEQ ID NO: 44) (SEQ ID No: 46) protein (SEQ ID NO:
43) (SEQ ID NO: 45) (SEQ ID No: 47) VL.sup.CDR1 VL.sup.CDR2
VL.sup.CDR3 DNA (SEQ ID NO: 48) (SEQ ID NO: 50) (SEQ ID No: 52)
protein (SEQ ID NO: 49) (SEQ ID NO: 51) (SEQ ID No: 53)
[0032] Other preferred c-mpl agonist antibodies of this invention
include those that activate platelets in a manner similar to TPO or
in a manner similar to ADP, collagen and the like. Optionally the
c-mpl agonist antibodies of this invention do not activate
platelets. The c-mpl agonist antibodies of this invention are used
in a manner similar to TPO.
[0033] In another embodiment, substantially pure single chain
antibodies are provided which bind to and act as agonist or
antagonist antibodies to a cytokine receptor or to a kinase
receptor.
[0034] The invention also provides a method of obtaining these
antibodies, in particular a method of screening a library of phage
displayed antibodies, preferably human single chain antibodies.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 shows examples of single chain antibody (scFv)
fragments denominated 10F6, 5E5, 10D10, 12B5, 12D5 and 12E10 having
sequences for CDRs and framework regions.
[0036] FIG. 2 illustrates a method for the construction of a phage
library containing single-chain antibodies fused to a coat protein
of a phage.
[0037] FIG. 3 shows a single-chain antibody displayed as a fusion
protein on coat protein 3 of a filamentous phage.
[0038] FIG. 4 illustrates a method of selecting scFv in a phage
library by one or more binding selection cycles.
[0039] FIG. 5 illustrates a method of panning high affinity phage
using biotinylated antigen and streptavidin coated paramagnetic
beads.
[0040] FIG. 6 shows a process for identifying c-mpl binding phage
using a phage ELISA method.
[0041] FIG. 7 illustrates DNA fingerprinting of clones to determine
diversity by BstNI restriction enzyme analysis
[0042] FIG. 8A-C show a typical BstNI analysis on a 3% agarose gel;
see Example 2.
[0043] FIG. 9 shows the results of agonist antibodies relative to
TPO in the KIRA-ELISA assay.
[0044] FIG. 10A-F show the results of TPO-antibody competitive
binding assays for HU-03 cells. See Example 1.
[0045] FIG. 11 shows activity for MuSK agonist antibodies of
Example 5.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0046] I. Definitions
[0047] In general, the following words or phrases have the
indicated definition when used in the description, examples, and
claims.
[0048] The terms "agonist" and "agonistic" when used herein refer
to or describe a molecule which is capable of, directly or
indirectly, substantially inducing, promoting or enhancing cytokine
biological activity or cytokine receptor activation.
[0049] "Agonist antibodies" (aAb) are antibodies or fragments
thereof that possess the property of binding to a cytokine
superfamily receptor and causing the receptor to transduce a
survival, proliferation, maturation and/or differentiation signal.
Included within the definition of transducing a survival signal is
a signal which modulates cell survival or death by apoptosis. To be
therapeutically useful the agonist antibodies of this invention
will be capable of inducing or causing survival, proliferation,
maturation or differentiation at a concentration equal to or not
less than 2 orders of magnitude (100-fold) below that of the
natural in vivo ligand on a weight basis.
[0050] "Activate a receptor", as used herein, is used
interchangeably with transduce a growth, survival, proliferation,
maturation and/or differentiation signal.
[0051] "Activate platelets", as used herein, means to stimulate
platelets to make them more likely to aggregate by comparison to
unactivated platelets. For example, ADP and collagen are substances
known to activate platelets.
[0052] "Affinity matured antibodies" are antibodies that have had
their binding affinity and/or biological activity increased by
altering the type or location of one or more residues in the
variable region. An example of alteration is a mutation which may
be in either a CDR or a framework region. An affinity matured
antibody will typically have its binding affinity increased above
that of the isolated or natural antibody or fragment thereof by
from 2 to 500 fold. Preferred affinity matured antibodies will have
nanomolar or even picomolar affinities to the receptor antigen.
Affinity matured antibodies are produced by procedures known in the
art. Marks, J. D. et al., Bio/Technology 10:779-783 (1992)
describes affinity maturation by VH and VL domain shuffling Random
mutagenesis of CDR and/or framework residues is described by;
Barbas, C. F. et al. Proc Nat. Acad. Sci., USA 91:3809-3813 (1994),
Schier, R. et al. Gene 169:147-155 (1995), Yelton, D. E. et al J.
Immunol. 155:1994-2004 (1995), Jackson, J. R. et al. J. Immunol.
154(7):3310-9 (1995), and Hawkins, R. E. et al., J. Mol. Biol.
226:889-896 (1992).
[0053] "Cytokine" is a generic term for proteins released by one
cell population which act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines,
and traditional polypeptide hormones. Included among the cytokines
are growth hormone, insulin-like growth factors, human growth
hormone, N-methionyl human growth hormone, bovine growth hormone,
parathyroid hormone, thyroxine, insulin, proinsulin, relaxin,
prorelaxin, glycoprotein hormones such as follicle stimulating
hormone (FSH), thyroid stimulating hormone (TSH), and leutinizing
hormone (LH), hematopoietic growth factor, hepatic growth factor,
fibroblast growth factor, prolactin, placental lactogen, tumor
necrosis factor-a (TNF-a and TNF-b) mullerian-inhibiting substance,
mouse gonadotropin-associated peptide, inhibin, activin, vascular
endothelial growth factor, integrin, nerve growth factors such as
NGF-b, platelet-growth factor, transforming growth factors (TGFs)
such as TGF-a and TGF-b, insulin-like growth factor-I and -II,
erythropoietin (EPO), osteoinductive factors, interferons such as
interferon-a, -b, and -g, colony stimulating factors (CSFs) such as
macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and
granulocyte-CSF (G-CSF), thrombopoietin (TPO), interleukins (IL's)
such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,
IL-9, IL-11, IL-12 and other polypeptide factors including LIF,
SCF, and kit-ligand. As used herein the foregoing terms are meant
to include proteins from natural sources or from recombinant cell
culture. Similarly, the terms are intended to include biologically
active equivalents; e.g., differing in amino acid sequence by one
or more amino acids or in type or extent of glycosylation.
[0054] "Cytokine superfamily receptors" and "hematopoietic growth
factor superfamily receptors" are used interchangeably herein and
are a group of closely related glycoprotein cell surface receptors
that share considerable homology including frequently a WSXWS
domain and are generally classified as members of the cytokine
receptor superfamily (see e.g. Nicola et al., Cell, 67:1-4 (1991)
and Skoda, R. C. et al. EMBO J. 12:2645-2653 (1993)). Generally,
these receptors are interleukins (IL) or colony-stimulating factors
(CSF). Members of the superfamily include, but are not limited to,
receptors for: IL-2 (b and g chains) (Hatakeyama et al., Science,
244:551-556 (1989); Takeshita et al., Science, 257:379-382 (1991)),
IL-3 (Itoh et al., Science, 247:324-328 (1990); Gorman et al.,
Proc. Natl. Acad. Sci. USA, 87:5459-5463 (1990); Kitamura et al.,
Cell, 66:1165-1174 (1991a); Kitamura et al., Proc. Natl. Acad. Sci.
USA, 88:5082-5086 (1991b)), IL-4 (Mosley et al., Cell, 59:335-348
(1989), IL-5 (Takaki et al., EMBO J., 9:4367-4374 (1990); Tavernier
et al., Cell, 66:1175-1184 (1991)), IL-6 (Yamasaki et al., Science,
241:825-828 (1988); Hibi et al., Cell, 63:1149-1157 (1990)), IL-7
(Goodwin et al., Cell, 60:941-951 (1990)), IL-9 (Renault et al.,
Proc. Natl. Acad. Sci. USA, 89:5690-5694 (1992)),
granulocyte-macrophage colony-stimulating factor (GM-CSF) (Gearing
et al., EMBO J., 8:3667-3676 (1991); Hayashida et al., Proc. Natl.
Acad. Sci. USA, 244:9655-9659 (1990)), granulocyte
colony-stimulating factor (G-CSF) (Fukunaga et al., Cell,
61:341-350 (1990a); Fukunaga et al., Proc. Natl. Acad. Sci. USA,
87:8702-8706 (1990b); Larsen et al., J. Exp. Med., 172:1559-1570
(1990)), EPO (D'Andrea et al., Cell, 57:277-285 (1989); Jones et
al., Blood, 76:31-35 (1990)), Leukemia inhibitory factor (LIF)
(Gearing et al., EMBO J., 10:2839-2848 (1991)), oncostatin M (OSM)
(Rose et al., Proc. Natl. Acad. Sci. USA, 88:8641-8645 (1991)) and
also receptors for prolactin (Boutin et al., Proc. Natl. Acad. Sci.
USA, 88:7744-7748 (1988); Edery et al., Proc. Natl. Acad. Sci. USA,
86:2112-2116 (1989)), growth hormone (GH) (Leung et al., Nature,
330:537-543 (1987)), ciliary neurotrophic factor (CNTF) (Davis et
al., Science, 253:59-63 (1991) and c-Mpl (M. Souyri et al., Cell
63:1137 (1990); 1. Vigon et al., Proc. Natl. Acad. Sci. 89:5640
(1992)).
[0055] "Thrombocytopenia" in humans is defined as a platelet count
below 150.times.10.sup.9 per liter of blood.
[0056] "Thrombopoietic activity" is defined as biological activity
that consists of accelerating the proliferation, differentiation
and/or maturation of megakaryocytes or megakaryocyte precursors
into the platelet producing form of these cells. This activity may
be measured in various assays including an in vivo mouse platelet
rebound synthesis assay, induction of platelet cell surface antigen
assay as measured by an anti-platelet immunoassay
(anti-GPII.sub.bIII.sub.a) for a human leukemia megakaryoblastic
cell line (CMK), and induction of polyploidization in a
megakaryoblastic cell line (DAMI). A "thrombopoietin receptor" is a
mammalian polypeptide receptor which, when activated by a ligand
binding thereto, includes, causes or other vise gives rise to
"thrombopoietic activity" in a cell or mammal, including a
human.
[0057] "Control sequences" when referring to expression means DNA
sequences necessary for the expression of an operably linked coding
sequence in a particular host organism. The control sequences that
are suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, a ribosome binding site, and
possibly, other as yet poorly understood sequences. Eukaryotic
cells are known to utilize promoters, polyadenylation signals, and
enhancers.
[0058] "Operably linked" when referring to nucleic acids means that
the nucleic acids are placed in a functional relationship with
another nucleic acid sequence. For example, DNA for a presequence
or secretory leader is operably linked to DNA for a polypeptide if
it is expressed as a preprotein that participates in the secretion
of the polypeptide; a promoter or enhancer is operably linked to a
coding sequence if it affects the transcription of the sequence; or
a ribosome binding site is operably linked to a coding sequence if
it is positioned so as to facilitate translation. Generally,
"operably linked" means that the DNA sequences being linked are
contiguous and, in the case of a secretory leader, contiguous and
in reading phase. However, enhancers do not have to be contiguous.
Linking is accomplished by ligation at convenient restriction
sites. If such sites do not exist, the synthetic oligonucleotide
adapters or linkers are used in accord with conventional
practice.
[0059] "Exogenous" when referring to an element means a nucleic
acid sequence that is foreign to the cell, or homologous to the
cell but in a position within the host cell nucleic acid in which
the element is ordinarily not found.
[0060] "Cell," "cell line," and "cell culture" are used
interchangeably herein and such designations include all progeny of
a cell or cell line. Thus, for example, terms like "transformants"
and "transformed cells" include the primary subject cell and
cultures derived therefrom without regard for the number of
transfers. It is also understood that all progeny may not be
precisely identical in DNA content, due to deliberate or
inadvertent mutations. Mutant progeny that have the same function
or biological activity as screened for in the originally
transformed cell are included. Where distinct designations are
intended, it will be clear from the context.
[0061] "Plasmids" are autonomously replicating circular DNA
molecules possessing independent origins of replication and are
designated herein by a lower case "p" preceded and/or followed by
capital letters and/or numbers. The starting plasmids herein are
either commercially available, publicly available on an
unrestricted basis, or can be constructed from such available
plasmids in accordance with published procedures. In addition,
other equivalent plasmids are known in the art and will be apparent
to the ordinary artisan.
[0062] "Restriction enzyme digestion" when referring to DNA means
catalytic cleavage of internal phosphodiester bonds of DNA with an
enzyme that acts only at certain locations or sites in the DNA
sequence. Such enzymes are called "restriction endonucleases". Each
restriction endonuclease recognizes a specific DNA sequence called
a "restriction site" that exhibits two-fold symmetry. The various
restriction enzymes used herein are commercially available and
their reaction conditions, cofactors, and other requirements as
established by the enzyme suppliers are used. Restriction enzymes
commonly are designated by abbreviations composed of a capital
letter followed by other letters representing the microorganism
from which each restriction enzyme originally was obtained and then
a number designating the particular enzyme. In general, about 1
.mu.g of plasmid or DNA fragment is used with about 1-2 units of
enzyme in about 20 .mu.l of buffer solution. Appropriate buffers
and substrate amounts for particular restriction enzymes are
specified by the manufacturer. Incubation of about 1 hour at
37.degree. C. is ordinarily used, but may vary in accordance with
the supplier's instructions. After incubation, protein or
polypeptide is removed by extraction with phenol and chloroform,
and the digested nucleic acid is recovered from the aqueous
fraction by precipitation with ethanol. Digestion with a
restriction enzyme may be followed with bacterial alkaline
phosphatase hydrolysis of the terminal 5' phosphates to prevent the
two restriction-cleaved ends of a DNA fragment from "circularizing"
or forming a closed loop that would impede insertion of another DNA
fragment at the restriction site. Unless otherwise stated,
digestion of plasmids is not followed by 5' terminal
dephosphorylation. Procedures and reagents for dephosphorylation
are conventional as described in sections 1.56-1.61 of Sambrook et
al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring
Harbor Laboratory Press, 1989).
[0063] "Recovery" or "isolation" of a given fragment of DNA from a
restriction digest means separation of the digest on polyacrylamide
or agarose gel by electrophoresis, identification of the fragment
of interest by comparison of its mobility versus that of marker DNA
fragments of known molecular weight, removal of the gel section
containing the desired fragment, and separation of the gel from
DNA. This procedure is known generally. For example, see Lawn et
al., Nucleic Acids Res., 9:6103-6114 (1981), and Goeddel et al.,
Nucleic Acids Res., 8:4057(1980).
[0064] "Southern analysis" or "Southern blotting" is a method by
which the presence of DNA sequences in a restriction endonuclease
digest of DNA or DNA-containing composition is confirmed by
hybridization to a known, labeled oligonucleotide or DNA fragment.
Southern analysis typically involves electrophoretic separation of
DNA digests on agarose gels, denaturation of the DNA after
electrophoretic separation, and transfer of the DNA to
nitrocellulose, nylon, or another suitable membrane support for
analysis with a radiolabeled, biotinylated, or enzyme-labeled probe
as described in sections 9.37-9.52 of Sambrook et al., supra.
[0065] "Northern analysis" or "Northern blotting" is a method used
to identify RNA sequences that hybridize to a known probe such as
an oligonucleotide, DNA fragment, cDNA or fragment thereof, or RNA
fragment. The probe is labeled with a radioisotope such as
.sup.32P, or by biotinylation, or with an enzyme. The RNA to be
analyzed is usually electrophoretically separated on an agarose or
polyacrylamide gel, transferred to nitrocellulose, nylon, or other
suitable membrane, and hybridized with the probe, using standard
techniques well known in the art such as those described in
sections 7.39-7.52 of Sambrook et al., supra.
[0066] "Ligation" is the process of forming phosphodiester bonds
between two nucleic acid fragments. For ligation of the two
fragments, the ends of the fragments must be compatible with each
other. In some cases, the ends will be directly compatible after
endonuclease digestion. However, it may be necessary first to
convert the staggered ends commonly produced after endonuclease
digestion to blunt ends to make them compatible for ligation. For
blunting the ends, the DNA is treated in a suitable buffer for at
least 15 minutes at 15.degree. C. with about 10 units of the Klenow
fragment of DNA polymerase I or T4 DNA polymerase in the presence
of the four deoxyribonucleotide: triphosphates. The DNA is then
purified by phenol-chloroform extraction and ethanol precipitation.
The DNA fragments that are to be ligated together are put in
solution in about equimolar amounts. The solution will also contain
ATP, ligase buffer, and a ligase such as T4 DNA ligase at about 10
units per 0.5 .mu.g of DNA. If the DNA is to be ligated into a
vector, the vector is first linearized by digestion with the
appropriate restriction endonuclease(s). The linearized fragment is
then treated with bacterial alkaline phosphatase or calf intestinal
phosphatase to prevent self-ligation during the ligation step.
[0067] "Preparation" of DNA from cells means isolating the plasmid
DNA from a culture of the host cells.
[0068] Commonly used methods for DNA preparation are the large- and
small-scale plasmid preparations described in sections 1.25-1.33 of
Sambrook et al., supra. After preparation of the DNA, it can be
purified by methods well known in the art such as that described in
section 1.40 of Sambrook et al., supra.
[0069] "Oligonucleotides" are short-length, single- or
double-stranded polydeoxynucleotides that are chemically
synthesized by known methods (such as phosphotriester, phosphite,
or phosphoramidite chemistry, using solid-phase techniques such as
described in EP 266,032 published 4 May 1988, or via
deoxynucleoside H-phosphonate intermediates as described by
Froehler et al., Nucl. Acids Res., 14:5399-5407 (1986)). Further
methods include the polymerase chain reaction defined below and
other autoprimer methods and oligonucleotide syntheses on solid
supports. All of these methods are described in Engels et al.,
Agnew. Chem. Int. Ed. Engl., 28:716-734 (1989). These methods are
used if the entire nucleic acid sequence of the gene is known, or
the sequence of the nucleic acid complementary to the coding strand
is available. Alternatively, if the target amino acid sequence is
known, one may infer potential nucleic acid sequences using known
and preferred coding residues for each amino acid residue. The
oligonucleotides are then purified on polyacrylamide gels.
[0070] "Polymerase chain reaction" or "PCR" refers to a procedure
or technique in which minute amounts of a specific piece of nucleic
acid, RNA and/or DNA, are amplified as described in U.S. Pat. No.
4,683,195 issued 28 Jul. 1987. Generally, sequence information from
the ends of the region of interest or beyond needs to be available,
such that oligonucleotide primers can be designed; these primers
will be identical or similar in sequence to opposite strands of the
template to be amplified. The 5' terminal nucleotides of the two
primers may coincide with the ends of the amplified material. PCR
can be used to amplify specific RNA sequences, specific DNA
sequences from total genomic DNA, and cDNA transcribed from total
cellular RNA, bacteriophage or plasmid sequences, etc. See
generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol.,
51:263 (1987); Erlich, ed., PCR Technology, (Stockton Press, NY,
1989). As used herein, PCR is considered to be one, but not the
only, example of a nucleic acid polymerase reaction method for
amplifying a nucleic acid test sample comprising the use of a known
nucleic acid as a primer and a nucleic acid polymerase to amplify
or generate a specific piece of nucleic acid.
[0071] "Native antibodies and immunoglobulins" are usually
heterotetrameric glycoproteins of about 150,000 daltons, composed
of two identical light (L) chains and two identical heavy (H)
chains. Each light chain is linked to a heavy chain by one covalent
disulfide bond, while the number of disulfide linkages varies
between the heavy chains of different immunoglobulin isotypes. Each
heavy and light chain also has regularly spaced intrachain
disulfide bridges. Each heavy chain has at one end a variable
domain (V.sub.H) followed by a number of constant domains. Each
light chain has a variable domain at one and (V.sub.L) and a
constant domain at its other end; the constant domain of the light
chain is aligned with the first constant domain of the heavy chain,
and the light chain variable domain is aligned with the variable
domain of the heavy chain. Particular amino acid residues are
believed to form an interface between the light and heavy chain
variable domains (Clothia et al., J. Mol. Biot., 186:651-663
(1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA, 82:4592-4596
(1985)).
[0072] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed through the variable domains
of antibodies. It is concentrated in three segments called
complementarity determining regions (CDRs) or hypervariable regions
both in the light chain and the heavy chain variable domains. The
more highly conserved portions of variable domains are called the
framework (FR). The variable domains of native heavy and light
chains each comprise four FR regions, largely adopting a b-sheet
configuration, connected by three CDRs, which form loops
connecting, and in some cases forming part of, the b-sheet
structure. The CDRs in each chain are held together in close
proximity by the FR regions and, with the CDRs from the other
chain, contribute to the formation of the antigen binding site of
antibodies (see Kabat et al., Sequences of Proteins of
Immunological Interest, National Institute of Health, Bethesda, Md.
(1987)). The constant domains are not involved directly in binding
an antibody to an antigen, but exhibit various effector functions,
such as participation of the antibody in antibody-dependent
cellular toxicity.
[0073] Papain digestion of antibodies produces two identical
antigen binding fragments, called "Fab" fragments, each with a
single antigen binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab').sub.2 fragment that has two antigen combining
sites and is still capable of cross-linking antigen.
[0074] "Fv" is the minimum antibody fragment which contains a
complete antigen recognition and binding site. This region consists
of a dimer of one heavy and one light chain variable domain in
tight, non-covalent association. It is in this configuration that
the three CDRs of each variable domain interact to define an
antigen binding site on the surface of the V.sub.H-V.sub.L dimer.
Collectively, the six CDRs confer antigen binding specificity to
the antibody. However, even a single variable domain (or half of an
Fv comprising only three CDRs specific for an antigen) has the
ability to recognize and bind antigen, although at a lower affinity
than the entire binding site.
[0075] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CHI) of the heavy chain.
Fab" fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear a free thiol group.
F(ab').sub.2 antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Other,
chemical couplings of antibody fragments are also known.
[0076] The "light chains" of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct
types, called kappa and lambda (1), based on the amino acid
sequences of their constant domains.
[0077] Depending on the amino acid sequence of the constant domain
of their heavy chains, immunoglobulins can be assigned to different
classes. There are five major classes of immunoglobulins: IgA, IgD,
IgE, IgG and IgM, and several of these may be further divided into
subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1
and IgA-2. The heavy chain constant domains that correspond to the
different classes of immunoglobulins are called alpha, delta,
epsilon, gamma, and .mu., respectively. The subunit structures and
three-dimensional configurations of different classes of
immunoglobulins are well known.
[0078] The term "antibody" is used in the broadest sense and
specifically covers single monoclonal antibodies (including agonist
and antagonist antibodies), antibody compositions with polyepitopic
specificity, as well as antibody fragments (e.g., Fab,
F(ab').sub.2, scFv and Fv), so long as they exhibit the desired
biological activity.
[0079] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to conventional
(polyclonal) antibody preparations which typically include
different antibodies directed against different determinants
(epitopes), each monoclonal antibody is directed against a single
determninant on the antigen. In addition to their specificity, the
monoclonal antibodies are advantageous in that they are synthesized
by the hybridoma culture, uncontaminated by other immunoglobulins.
The modifier "monoclonal" indicates the character of the antibody
as being obtained from a substantially homogeneous population of
antibodies, and is not to be construed as requiring production of
the antibody by any particular method. For example, the monoclonal
antibodies to be used in accordance with the present invention may
be made by the hybridoma method first described by Kohler &
Milstein, Nature, 256:495 (1975), or may be made by recombinant DNA
methods (see, e.g., U.S. Pat. No. 4,816,567 (Cabilly et al.)).
[0080] The monoclonal antibodies herein specifically include
"chimeric" antibodies (immunoglobulins) in which a portion of the
heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass, as well
as fragments of such antibodies, so long as they exhibit the
desired biological activity, e.g. binding to and activating mpl
(U.S. Pat. No. 4,816,567 (Cabilly et al.); and Morrison et al.,
Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
[0081] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) which contain minimal
sequence derived from non-human immunoglobulin. For the most part,
humanized antibodies are human immunoglobulins (recipient antibody)
in which residues from a complementary determining region (CDR) of
the recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity and capacity. In some instances, Fv
framework residues of the human immunoglobulin are replaced by
corresponding non-human residues. Furthermore, humanized antibody
may comprise residues which are found neither in the recipient
antibody nor in the imported CDR or framework sequences. These
modifications are made to further refine and optimize antibody
performance. In general, the humanized antibody will comprise
substantially all of at least one, and typically two, variable
domains, in which all or substantially all of the CDR regions
correspond to those of a non-human immunoglobulin and all or
substantially all of the FR regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin. For further
details see: Jones et al., Nature, 321:522-525 (1986); Reichmann et
al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct.
Biol., 2:593-596 (1992)).
[0082] "Single-chain Fv" or "sFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain. Generally, the Fv
polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the sFv to form the
desired structure for antigen binding. For a review of sFv see
Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315
(1994).
[0083] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy chain
variable domain (V.sub.H) connected to a light chain variable
domain (V.sub.L) in the same polypeptide chain (V.sub.H and
V.sub.L). By using a linker that is too short to allow pairing
between the two domains on the same chain, the domains are forced
to pair with the complementary domains of another chain and create
two antigen-binding sites. Diabodies are described more fully in,
for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc.
Natl. Acad. Sci. USA 90:6444-6448 (1993).
[0084] The expression "linear antibodies" when used throughout this
application refers to the antibodies described in Zapata et al.
Protein Eng. 8(10):1057-1062 (1995). Briefly, these antibodies
comprise a pair of tandem Fd segments
(V.sub.H-C.sub.H1-V.sub.H-C.sub.H1) which form a pair of antigen
binding regions. Linear antibodies can be bispecific or
monospecific.
[0085] A "variant" antibody, refers herein to a molecule which
differs in amino acid sequence from a "parent" antibody amino acid
sequence by virtue of addition, deletion and/or substitution of one
or more amino acid residue(s) in the parent antibody sequence. In
the preferred embodiment, the variant comprises one or more amino
acid substitution(s) in one or more hypervariable region(s) of the
parent antibody. For example, the variant may comprise at least
one, e.g. from about one to about ten, and preferably from about
two to about five, substitutions in one or more hypervariable
regions of the parent antibody. Ordinarily, the variant will have
an amino acid sequence having at least 75% amino acid sequence
identity with the parent antibody heavy or light chain variable
domain sequences, more preferably at least 80%, more preferably at
least 85%, more preferably at least 90%, and most preferably at
least 95%. Identity or homology with respect to this sequence is
defined herein as the percentage of amino acid residues in the
candidate sequence that are identical with the parent antibody
residues, after aligning the sequences and introducing gaps, if
necessary, to achieve the maximum percent sequence identity. See
FIG. 1. None of N-terminal, C-terminal, or internal extensions,
deletions, or insertions into the antibody sequence shall be
construed as affecting sequence identity or homology. The variant
retains the ability to bind the receptor and preferably has
properties which are superior to those of the parent antibody. For
example, the variant may have a stronger binding affinity, enhanced
ability to activate the receptor, etc. To analyze such properties,
one should compare a Fab form of the variant to a Fab form of the
parent antibody or a full length form of the variant to a full
length form of the parent antibody, for example, since it has been
found that the format of the antibody impacts its activity in the
biological activity assays disclosed herein. The variant antibody
of particular interest herein is one which displays at least about
10 fold, preferably at least about 20 fold, and most preferably at
least about 50 fold, enhancement in biological activity when
compared to the parent antibody.
[0086] The "parent" antibody herein is one which is encoded by an
amino acid sequence used for the preparation of the variant.
Preferably, the parent antibody has a human framework region and
has human antibody constant region(s). For example, the parent
antibody may be a humanized or human antibody.
[0087] An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, the antibody will be purified (1) to greater than 95%
by weight of antibody as determined by the Lowry method, and most
preferably more than 99% by weight, (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain. Isolated antibody
includes the antibody in situ within recombinant cells since at
least one component of the antibody's natural environment will not
be present. Ordinarily, however, isolated antibody will be prepared
by at least one purification step.
[0088] The term "epitope tagged" when used herein refers to an
antibody fused to an "epitope tag". The epitope tag polypeptide has
enough residues to provide an epitope against which an antibody
thereagainst can be made, yet is short enough such that it does not
interfere with activity of the antibody. The epitope tag preferably
is sufficiently unique so that the antibody thereagainst does not
substantially cross-react with other epitopes. Suitable tag
polypeptides generally have at least 6 amino acid residues and
usually between about 8-50 amino acid residues (preferably between
about 9-30 residues). Examples include the flu HA tag polypeptide
and its antibody 12CA5 (Field et al. Mol. Cell. Biol. 8:2159-2165
(1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10
antibodies thereto (Evan et al., Mol. Cell. Biol. 5(12):3610-3616
(1985)); and the Herpes Simplex virus glycoprotein D (gD) tag and
its antibody (Paborsky et al., Protein Engineering 3(6):547-553
(1990)). In certain embodiments, the epitope tag is a "salvage
receptor binding epitope". As used herein, the term "salvage
receptor binding epitope" refers to an epitope of the Fc region of
an IgG molecule (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, or
IgG.sub.4) that is responsible for increasing the in vivo serum
half-life of the IgG molecule.
[0089] The terms "mpl ligand", mpl ligand polypeptide", "ML",
"thrombopoietin" or "TPO" are used interchangeably herein and
include any polypeptide that possesses the property of binding to
mpl, a member of the cytokine receptor superfamily, and having a
biological property of mpl ligand. An exemplary biological property
is the ability to stimulate the incorporation of labeled
nucleotides (e.g. .sup.3H-thymidine) into the DNA of IL-3 dependent
Ba/F3 cells transfected with human mpl. Another exemplary
biological property is the ability to stimulate the incorporation
of .sup.35S into circulating platelets in a mouse platelet rebound
assay. This definition encompasses a polypeptide isolated from a
mpl ligand source such as aplastic porcine plasma described herein
or from another source, such as another animal species, including
humans, or prepared by recombinant or synthetic methods. Examples
include TPO(332) and rhTPO.sub.332. Also included in this
definition is the thrombopoietic ligand described in WO 95/28907
having a molecular weight of about 31,000 daltons (31 kd) as
determined by SDS gel under reducing conditions and 28,000 daltons
(28 kd) under non-reducing conditions. The term "TPO" includes
variant forms, such as fragments, alleles, isoforms, analogues,
chimera thereof and mixtures of these forms. For convenience, all
of these ligands will be referred to below simply as "TPO"
recognizing that all individual ligands and ligand mixtures are
referred to by this term.
[0090] Preferably, the TPO is a compound having thrombopoietic
activity or being capable of increasing serum platelet counts in a
mammal. The TPO is preferably capable of increasing endogenous
platelet counts by at least 10%, more preferably by 50%, and most
preferably capable of elevating platelet counts in a human to
greater than about 150.times.10.sup.9 per liter of blood.
[0091] The TPO of this invention preferably has at least 70%
overall sequence identity with the amino acid sequence of the
highly purified substantially homogeneous porcine mpl ligand
polypeptide and at least 80% sequence identity with the
"EPO-domain" of the porcine mpl ligand polypeptide. Alternatively,
the TPO of this invention may be a mature human mpl ligand (hML),
or a variant or post-transcriptionally modified form thereof or a
protein having about 80% sequence identity with mature human mpl
ligand. Alternatively, the TPO may be a fragment, especially an
amino-terminus or "EPO-domain" fragment, of the mature human mpl
ligand. Preferably, the amino terminus fragment retains
substantially all of the human ML sequence between the first and
fourth cysteine residues but may contain substantial additions,
deletions or substitutions outside that region. According to this
embodiment, the fragment polypeptide may be represented by the
formula:
X-hTPO(7-151)-Y
[0092] Where hTPO(7-151) represents the human TPO (hML) amino acid
sequence from Cys.sup.7 through Cys.sup.151 inclusive; X represents
the amino group of Cys.sup.7 or one or more of the amino-terminus
amino acid residue(s) of the mature TPO or amino acid residue
extensions thereto such as Met, Lys, Tyr or amino acid
substitutions thereof such as arginine to lysine or leader
sequences containing, for example, proteolytic cleavage sites (e.g.
Factor Xa or thrombin); and Y represents the carboxy terminal group
of Cys.sup.151 or one or more carboxy-terminus amino acid
residue(s) of the mature TPO or extensions thereto.
[0093] A "TPO fragment" means a portion of a naturally occurring
mature full length mpl ligand or TPO sequence having one or more
amino acid residues or carbohydrate units deleted. The deleted
amino acid rcsidue(s) may occur anywhere in the peptide including
at either the N-terminal or C-terminal end or internally, so long
as the fragment shares at least one biological property in common
with mpl ligand. Mpl ligand ZS fragments typically will have a
consecutive sequence of at least 10, 15, 20, 25, 30 or 40 amino
acid residues that are identical to the sequences of the mpl ligand
isolated from a mammal including the ligand isolated from aplastic
porcine plasma or the human or murine ligand, especially the
EPO-domain thereof. Representative examples of N-terminal fragments
are TPO(153), hML.sub.153 or TPO(Met.sup.-1 1-153).
[0094] The terms "TPO isoform(s)" and "TPO sequence isoform(s)" or
the term "derivatives" in association with TPO, etc. as used herein
means a biologically active material as defined below having less
than 100% sequence identity with the TPO isolated from recombinant
cell culture, aplastic porcine plasma or the human mpl ligand.
Ordinarily, a biologically active mpl ligand or TPO isoform will
have an amino acid sequence having at least about 70% amino acid
sequence identity with the mpl ligand/TPO isolated from aplastic
porcine plasma or the mature murine, human mpl ligand or fragments
thereof, preferably at least about 75%, more preferably at least
about 80%, still more preferably at least about 85%, even more
preferably at least about 90%, and most preferably at least about
95%.
[0095] TPO "analogues" include covalent modification of TPO or mpl
ligand by linking the TPO polypeptide to one of a variety of
nonproteinaceous polymers, e.g. polyethylene glycol, polypropylene
glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat.
Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or
4,179,337. TPO polypeptides covalently linked to the forgoing
polymers are referred to herein as pegylated TPO.
[0096] Still other preferred TPO polypeptides of this invention
include mpl ligand sequence variants and chimeras. Ordinarily,
preferred mpl ligand sequence variants and chimeras are
biologically active mpl ligand variants that have an amino acid
sequence having at least 90% amino acid sequence identity with the
human mpl ligand and most preferably at least 95%. An exemplary
preferred mpl ligand variant is a N-terminal domain hML variant
(referred to as the "EPO-domain" because of its sequence homology
to erythropoietin). The preferred hML EPO-domain comprises about
the first 153 amino acid residues of mature hML and is referred to
as hML.sub.153. An optionally preferred hML sequence variant
comprises one in which one or more of the basic or dibasic amino
acid residue(s) in the C-terminal domain is substituted with a
non-basic amino acid residue(s) (e.g., hydrophobic, neutral,
acidic, aromatic, Gly, Pro and the like). A preferred hML
C-terminal domain sequence variant comprises one in which Arg
residues 153 and 154 are replaced with Ala residues. This variant
is referred to as hML.sub.332(R153A, R154A).
[0097] A preferred chimera is a fusion between mpl ligand or
fragment (defined below) thereof with a heterologous polypeptide or
fragment thereof. For example, hML.sub.153 may be fused to an IgG
fragment to improve serum half-life or to IL-3, G-CSF or EPO to
produce a molecule with enhanced thrombopoietic or chimeric
hematopoietic activity.
[0098] Other preferred mpl ligand fragments have a Met preceding
the amino terminus Ser (e.g. Met.sup.-1TPO.sub.153). This is
preferred when, for example, the protein is expressed directly in a
microorganism such as E. coli. Optionally, these mpl ligand
fragments may contain amino acid substitutions to facilitate
derivitization. For example, Arg 53 or other residues of the
carbohydrate domain may be substituted with Lys to create
additional sites to add polyethylene glycol. Preferred mpl ligand
fragments according to this option include Met.sup.-1TPO(1-X) where
X is about 153, 164, 191, 199, 205, 207, 217, 229, or 245 for the
sequence of residues I-X. Other preferred mpl ligand fragments
include those produced as a result of chemical or enzymatic
hydrolysis or digestion of the purified ligand.
[0099] "Essentially pure" protein means a composition purified to
remove contaminating proteins and other cellular components,
preferably comprising at least about 90% by weight of the protein,
based on total weight of the composition, more preferably at least
about 95% by weight. "Essentially homogeneous" protein means a
composition comprising at least about 99% by weight of protein,
based on total weight of the composition.
[0100] II. Preferred Embodiments of the Invention
[0101] In one embodiment, preferred antibodies of this invention
are substantially homogeneous antibodies and variants thereof,
referred to as agonist antibodies (aAb), that possess the property
of binding to c-mpl, a member of the hematopoietic growth factor
receptor superfamily, and transducing a survival, proliferation,
maturation and/or differentiation signal. Such signal transduction
may be determined by measuring stimulation of incorporation of
labeled nucleotides (3H-thymidine) into the DNA of IL-3 dependent
Ba/F3 cells transfected with human mpl P, or with a CMK Assay
measuring Induction of the platelet antigen GPII.sub.bIII.sub.a
expression. Signal transduction may also be determined by KIRA
ELISA by measuring phosphorylation of the c-mpl-Rse.gD chimeric
receptor, in a c-mpl/Mab HU-03 cell proliferation assay or in a
liquid suspension megakaryocytopoiesis assay.
[0102] Preferred c-mpl agonist antibodies of this invention are
also capable of inducing or causing survival, proliferation,
maturation or differentiation of CD34+ cells into the platelet
producing form at a concentration equal or not less than 2 orders
of magnitude (100-fold) below that of thrombopoietin on a weight
basis.
[0103] More preferred c-mpl aAb(s) are substantially purified
aAb(s) having hematopoietic, especially megakaryocytopoietic or
thrombocytopoietic activity--namely, being capable of stimulating
proliferation, maturation and/or differentiation of immature
megakaryocytes or their predecessors into the mature
platelet-producing form that demonstrate a biological activity
equal to or within 2 orders of magnitude of that of rhTPO or a
weight basis Most preferred aAb(s) of this invention are human
aAb(s) including full length antibodies having an intact human Fc
region and including fragments thereof having hematopoietic,
megakaryocytopoietic or thrombopoietic activity Exemplary fragments
having the above described biological activity include; Fv, scFv,
F(ab'), F(ab').sub.2.
[0104] Preferred scFv fragments denominated 10F6, 5E5, 10D10, 12B5,
12D5 and 12E10 having sequences for CDRs and Framework regions
provided in FIG. 1. Alternatively, the above enumerated scFvs are
affinity matured by mutating 1-3 amino acid residues in one or more
of the CDRs or in the framework regions between the CDRs.
[0105] The framework regions may be derived from a "consensus
sequence" (ie. the most common amino acids of a class, subclass or
subgroup of heavy or light chains of human immunoglobulins) or may
be derived from an individual human antibody framework region or
from a combination of different framework region sequence Many
human antibody framework region sequences are compiled in Kabat et
al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991), pages 647-669), for example.
[0106] A suitable method for purifying mpl antibodies comprises
contacting an antibody source containing the mpl antibody molecules
with an immobilized receptor polypeptide, specifically mpl or a mpl
fusion polypeptide, under conditions whereby the mpl antibody
molecules to be purified are selectively adsorbed onto the
immobilized receptor polypeptide, washing the immobilized support
to remove non-adsorbed material, and eluting the molecules to be
purified from the immobilized receptor polypeptide with an elution
buffer. The source containing the mpl antibody may be a library of
antibodies having different binding epitopes and the receptor may
be immobilized on a plate, tube, particle or other suitable surface
using known methods.
[0107] Alternatively, the source containing the antibody is
recombinant cell culture where the concentration of antibody in
either the culture medium or in cell lysates is generally higher
than in plasma or other natural sources. The preferred purification
method to provide substantially homogeneous antibody comprises:
removing particulate debris, either host cells or lysed fragments
by, for example, centrifugation or ultrafiltration; optionally,
protein may be concentrated with a commercially available protein
concentration further; followed by separating the antibody from
other impurities by one or more steps selected from;
immunoaffinity, ion-exchange (e.g., DEAE or matrices containing
carboxymethyl or sulfopropyl groups), Blue-SEPHAROSE, CM
Blue-SEPHAROSE, MONO-Q, MONO-S, lentil lectin-SEPHAROSE,
WGA-SEPHAROSE, Con A-SEPHAROSE, Ether TOYPEARL, Butyl TOYPEARL,
Phenyl TOYPEARL, protein A SEPHAROSE, SDS-PAGE, reverse phase HPLC
(e.g., silica gel with appended aliphatic groups) or SEPEADEX
molecular sieve or size exclusion chromatography, and ethanol or
ammonium sulfate precipitation. A protease inhibitor such as
methylsulfonylfluoride (PMSF) may be included in any of the
foregoing steps to inhibit proteolysis.
[0108] Preferably, the isolated antibody is monoclonal (Kohler and
Milstein, Nature, 256:495-497 (1975); Campbell, Laboratory
Techniques in Biochemistry and Molecular Biology, Burdon et al.,
Eds, Volume 13, Elsevier Science Publisrers, Amsterdam (1985); and
Huse et al., Science, 246:1275-1281 (1989)). A preferred mpl
antibody is one that binds to mpl receptor with an affinity of at
least about 10.sup.6 l/mole. More preferably the antibody binds
with an affinity of at least about 10.sup.7 l/mole or even at least
10.sup.9 l/mole. Most preferably, the antibody is raised against a
mpl receptor having one of the above described effector functions.
The isolated antibody capable of binding to the mpl receptor may
optionally be fused to a second polypeptide and the antibody or
fusion thereof may be used to isolate and purify mpl from a source
as described above for immobilized mpl polypeptide. In a further
preferred aspect of this embodiment, the invention provides a
method for detecting the mpl ligand in vitro or in vivo comprising
contacting the antibody with a sample, especially a serum sample,
suspected of containing the ligand and detecting if binding has
occurred.
[0109] The invention also provides an isolated nucleic acid
molecule encoding the mpl antibody or fragments thereof, which
nucleic acid molecule may be labeled or unlabeled with a detectable
moiety, and a nucleic acid molecule having a sequence that is
complementary to, or hybridizes under stringent or moderately
stringent conditions with, a nucleic acid molecule having a
sequence encoding a mpl antibody. A preferred mpl antibody nucleic
acid is RNA or DNA that encodes a biologically active human
antibody.
[0110] In a further preferred embodiment of this invention, the
nucleic acid molecule is cDNA encoding the mpl antibody and further
comprises a replicable vector in which the cDNA is operably linked
to control sequences recognized by a host transformed with the
vector. This aspect further includes host cells transformed with
the vector and a method of using the cDNA to effect production of
antibody, comprising expressing the cDNA encoding the antibody in a
culture of the transformed host cells and recovering the antibody
from the host cell culture. The antibody prepared in this manner is
preferably substantially homogeneous human ZS antibody. A preferred
host cell for producing the antibody is Chinese hamster ovary (CHO)
cells. An alternative preferred host cell is E. coli.
[0111] The invention further includes a preferred method for
treating a mammal having an immunological or hematopoietic
disorder, especially thrombocytopenia comprising administering a
therapeutically effective amount of a: mpl agonist or antagonist
antibody to the mammal. Optionally, the antibody is administered in
combination with a cytokine, especially a colony stimulating factor
or interleukin. Preferred colony stimulating factors or
interleukins include; kit-ligand, LIF, G-CSF, GM-CSF, M-CSF, EPO,
IL-1, IL-2, IL-3, IL-5, IL-6, IL-7, IL-8, IL-9 or IL-II.
Alternatively, the antibody is administered in combination with an
Insulin-like growth factor (e.g., IGF-1) or a tumor necrosis factor
(e.g., lymphotoxin (LT)).
[0112] III. Methods of Making
[0113] Nucleic acid encoding the agonist and/or antagonist
antibodies of the invention can be prepared from a library of
single chain antibodies displayed on a bacteriophage. The
preparation of such a library is well known to one of skill in this
art. Suitable libraries may be prepared by the methods described in
WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO
95/01438 and WO 95/15388. In a preferred embodiment, a library of
single chain antibodies (scFv) may be generated from a diverse
population of human B-cells from human donors. mRNA corresponding
to the VH and VL antibody chains is isolated and purified using
standard techniques and reverse transcribed to generate a
population of cDNA. After PCR amplification, DNA coding for single
chain antibodies is assembled using a linker, such as Gly.sub.4Ser,
and cloned into suitable expression vectors. A phage library is
then prepared in which the population of single chain antibodies is
displayed on the surface of the phage. Suitable methods for
preparing phage libraries have been reviewed and are described in
Winter et. al., Annu. Rev. Immunol., 1994, 12:433-55; Soderlind et.
al., Immunological Reviews, 1992, 130:109-123; Hoogenboom, Tibtech
February 1997, Vol. 15; Neri et. al., Cell Biophysics, 1995,
27:47-61, and the references described therein.
[0114] The antibodies of the invention having agonist or antagonist
properties may be selected by immobilizing a receptor and then
panning a library of human scFv prepared as described above using
the immobilized receptor to bind antibody. Griffiths et. al.,
EMBO-J, 1993, 12:725-734. The specificity and activity of specific
clones can be assessed using known assays. Griffiths et. al.;
Clarkson et. al., Nature, 1991, 352:642-648. After a first panning
step, one obtains a library of phage containing a plurality of
different single chain antibodies displayed on phage having
improved binding to the receptor. Subsequent panning steps provide
additional libraries with higher binding affinities. When avidity
effects are a problem, monovalent phage display libraries may be
used in which less than 20%, preferably less than 10%, and more
preferably less than 1% of the phage display more than one copy of
an antibody on the surface of the phage. Monovalent display can be
accomplished with the use of phagemid and helper phage as
described, for example, in Lowman et al. Methods: A Companion to
Methods in Enzymology, 1991, 3(3):205-216. A preferred phage is M13
and display is preferably as a fusion protein with coat protein 3
as described in Lowman et. al., supra. Other suitable phage include
fl and fd filamentous phage. Fusion protein display with other
virus coat proteins is also known and may be used in this
invention. See U.S. Pat. No. 5,223,409.
[0115] Amino acid sequence variants of the antibody are prepared by
introducing appropriate nucleotide changes into the antibody DNA,
or by peptide synthesis. Such variants include, for example,
deletions from, and/or insertions into and/or substitutions of,
residues within the amino acid sequences of the antibodies of the
examples herein. Any combination of deletion, insertion, and
substitution is made to arrive at the final construct, provided
that the final construct possesses the desired characteristics. The
amino acid changes also may alter post-translational processes of
the humanized or variant antibody, such as changing the number or
position of glycosylation sites. A useful method for identification
of certain residues or regions of the antibody that are preferred
locations for mutagenesis is called "alanine scanning mutagenesis,"
as described by Cunningham and Wells Science, 244:1081-1085 (1989).
Here, a residue or group of target residues are identified (e.g.,
charged residues such as arg, asp, his, lys, and glu) and replaced
by a neutral or negatively charged amino acid (most preferably
alanine or polyalanine) to affect the interaction of the amino
acids with the receptor. Those amino acid locations demonstrating
functional sensitivity to the substitutions then are refined by
introducing further or other variants at, or for, the sites of
substitution. Thus, while the site for introducing an amino acid
sequence variation is predetermined, the nature of the mutation per
se need not be predetermined. For example, to analyze the
performance of a mutation at a given site, ala scanning or random
mutagenesis is conducted at the target codon or region and the
expressed antibody variants are screened for the desired
activity.
[0116] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue or the antibody fused to an epitope
tag. Other insertional variants of the antibody molecule include
the fusion to the N- or C-terminus of the antibody of an enzyme or
a polypeptide which increases the serum half-life of the
antibody.
[0117] Another type of variant is an amino acid substitution
variant. These variants have at least one amino acid residue in the
antibody molecule removed and a different residue inserted in its
place. The sites of greatest interest for substitutional
mutagenesis include the hypervariable regions, but FR alterations
are also contemplated. Conservative substitutions are shown in
Table 2 under the heading of "preferred substitutions". If such
substitutions result in a change in biological activity, then more
substantial changes, denominated "exemplary substitutions" in Table
2, or as further described below in reference to amino acid
classes, may be introduced and the products screened.
2 TABLE 2 Exemplary Preferred Original Residue Substitutions
Substitutions Ala (A) val; leu; ile val Arg (R) lys; gln; asn lys
Asn (N) gln; his; asp, lys; arg gln Asp (D) glu; asn glu Cys (C)
ser; ala ser Gln (Q) asn; glu asn Glu (E) asp; gln asp Gly (G) ala
ala His (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe;
leu norleucine Leu (L) norleucine; ile; val; met; ile ala; phe Lys
(K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val;
ile; ala; tyr tyr Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser
Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile;
leu; met; phe; ala; leu norleucine
[0118] Substantial modifications in the biological properties of
the antibody are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Naturally occurring residues are
divided into groups based on common side-chain properties:
[0119] (1) hydrophobic: norleucine, met, ala, val, leu, ile;
[0120] (2) neutral hydrophilic: cys, ser, thr;
[0121] (3) acidic: asp, glu;
[0122] (4) basic: asn, gin, his, lys, arg;
[0123] (5) residues that influence chain orientation: gly, pro;
and
[0124] (6) aromatic: trp, tyr, phe.
[0125] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0126] Any cysteine residue not involved in maintaining the proper
conformation of the humanized or variant antibody also may be
substituted, generally with serine, to improve the oxidative
stability of the molecule and prevent aberrant crosslinking.
Conversely, cysteine bond(s) may be added to the antibody to
improve its stability (particularly where the antibody is an
antibody fragment such as an Fv fragment).
[0127] A particularly preferred type of substitutional variant
involves substituting one or more hypervariable region residues of
a parent antibody (e.g. a humanized or human antibody). Generally,
the resulting variant(s) selected for further development will have
improved biological properties relative to the parent antibody from
which they are generated. A convenient way for generating such
substitutional variants is affinity maturation using phage using
methods known in the art. Briefly, several hypervariable region
sites (e.g. 3-7 sites) are mutated to generate all possible amino
substitutions at each site. The antibody variants thus generated
are displayed in a monovalent fashion from filamentous phage
particles as fusions to the gene III product of M13 packaged within
each particle. The phage-displayed variants are then screened for
their biological activity (e.g. binding affinity) as herein
disclosed. In order to identify candidate hypervariable region
sites for modification, alanine scanning mutagenesis can be
performed to identified hypervariable region residues contributing
significantly to antigen binding. Alternatively, or in addition, it
may be beneficial to analyze a crystal structure of the
antigen-antibody complex to identify contact points between the
antibody and receptor. Such contact residues and neighboring
residues are candidates for substitution according to the
techniques elaborated herein. Once such variants are generated, the
panel of variants is subjected to screening as described herein and
antibodies with superior properties in one or more relevant assays
may be selected for further development.
[0128] Another type of amino acid variant of the antibody alters
the original glycosylation pattern of the antibody. By altering is
meant deleting one or more carbohydrate moieties found in the
antibody, and/or adding one or more glycosylation sites that are
not present in the antibody.
[0129] Glycosylation of antibodies is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences asparagine-X-serine and asparagine-X-threonine, where X
is any amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tripeptide
sequences in a polypeptide creates a potential glycosylation site.
O-linked glycosylation refers to the attachment of one of the
sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino
acid, most commonly serine orthreonine, although 5-hydroxyproline
or 5-hydroxylysine may also be used.
[0130] Addition of glycosylation sites to the antibody is
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tripeptide
sequences (for N-linked glycosylation sites) The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the sequence of the original
antibody (for O-linked glycosylation sites).
[0131] Nucleic acid molecules encoding amino acid sequence variants
of the antibody are prepared by a variety of methods known in the
art. These methods include, but are not limited to, isolation from
a natural source (in the case of naturally occurring amino acid
sequence variants) or preparation by oligonucleotide-mediated (or
site-directed) mutagenesis, PCR mutagenesis, and cassette
mutagenesis of an earlier prepared variant or a non-variant version
of the antibody.
[0132] Preferably, the antibodies are prepared by standard
recombinant procedures which involve production of the antibodies
by culturing cells transfected to express antibody nucleic acid
(typically by transforming the cells with an expression vector) and
recovering the antibody from the cells of cell culture.
[0133] The nucleic acid (e.g., cDNA or genomic DNA) encoding mpl
antibody selected as described above is inserted into a replicable
vector for further cloning (amplification of the DNA) or for
expression. Many vectors are available, and selection of the
appropriate vector will depend on (1) whether it is to be used for
DNA amplification or for DNA expression, (2) the size of the
nucleic acid to be inserted into the vector, and (3) the host cell
to be transformed with the vector. Each vector contains various
components depending on its function (amplification of DNA or
expression of DNA) and the host cell with which it is compatible.
The vector components generally include, but are not limited to,
one or more of the following: a signal sequence, an origin of
replication, one or more marker genes, an enhancer element, a
promoter, and a transcription termination sequence.
[0134] (i) Signal Sequence Component
[0135] The mpl antibody of this invention may be expressed not only
directly, but also as a fusion with a heterologous polypeptide,
preferably a signal sequence or other polypeptide having a specific
cleavage site at the N-terminus of the mature protein or
polypeptide. In general, the signal sequence may be a component of
the vector, or it may be a part of the mpl antibody DNA that is
inserted into the vector. The heterologous signal sequence selected
should be one that is recognized and processed (i.e., cleaved by a
signal peptidase) by the host cell For prokaryotic host cells a
prokaryotic signal sequence selected, for example, from the group
of the alkaline phosphatase, penicillinase, lpp, or heat-stable
enterotoxin II leaders. For yeast secretion the native signal
sequence may be substituted by, e.g., the yeast invertase, alpha
factor, or acid phosphatase leaders, the C. albicans glucoamylase
leader (EP 362,179 published 4 Apr. 1990), or the signal described
in WO 96/13646 published 15 Nov. 1990. In mammalian cell expression
the native signal sequence (i.e., the mpl ligand presequence that
normally directs secretion of mpl ligand from its native mammalian
cells in vivo) is satisfactory, although other mammalian signal
sequences may be suitable, such as signal sequences from other mpl
ligand polypeptides or from the same mpl ligand from a different
animal species, signal sequences from a mpl ligand, and signal
sequences from secreted polypeptides of the same or related
species, as well as viral secretory leaders, for example, the
herpes simplex gD signal.
[0136] (ii) Origin of Replication Component
[0137] Both expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Generally, in cloning vectors this sequence is
one that enables the vector to replicate independently of the host
chromosomal DNA, and includes origins of replication or
autonomously replicating sequences. Such sequences are well known
for a variety of bacteria, yeast, and viruses. The origin of
replication from the plasmid pBR322 is suitable for most
Gram-negative bacteria, the 2.mu. plasmid origin is suitable for
yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or
BPV) are useful for cloning vectors in mammalian cells. Generally,
the origin of replication component is not needed for mammalian
expression vectors (the SV40 origin may typically be used only
because it contains the early promoter).
[0138] Most expression vectors are "shuttle" vectors, i.e., they
are capable of replication in at least one class of organisms but
can be transfected into another organism for expression. For
example, a vector is cloned in E. coli and then the same vector is
transfected into yeast or mammalian cells for expression even
though it is not capable of replicating independently of the host
cell chromosome.
[0139] DNA may also be amplified by insertion into the host genome.
This is readily accomplished using Bacillus species as hosts, for
example, by including in the vector a DNA sequence that is
complementary to a sequence found in Bacillus genomic DNA.
Transfection of Bacillus with this vector results in homologous
recombination with the genome and insertion of antibody DNA.
However, the recovery of genomic DNA encoding antibody is more
complex than that of an exogenously replicated vector because
restriction enzyme digestion is required to excise the antibody
DNA.
[0140] (iii) Selection Gene Component
[0141] Expression and cloning vectors should contain a selection
gene, also termed a selectable marker. This gene encodes a protein
necessary for the survival or growth of transformed host cells
grown in a selective culture medium. Host cells not transformed
with the vector containing the selection gene will not survive in
the culture medium. Typical selection genes encode proteins that
(a) confer resistance to antibiotics or other toxins, e.g.,
ampicillin, neomycin, methotrexate, or tetracycline, (b) complement
auxotrophic deficiencies, or (c) supply critical nutrients not
available from complex media, e.g., the gene encoding D-alanine
racemase for Bacilli.
[0142] One example of a selection scheme utilizes a drug to arrest
growth of a host cell. Those cells that are successfully
transformed with a heterologous gene express a protein conferring
drug resistance and thus survive the selection regimen. Examples of
such dominant selection use the drugs neomycin (Southern et al., J.
Molec. Appl. Genet, 1:327 (1982)) mycophenolic acid (Mulligan et
al., Science, 209:1422 (1980)) or hygromycin Sugden et al., Mol.
Cell. Biol., 5:410-413 (1985)). The three examples given above
employ bacterial genes under eukaryotic control to convey
resistance to the appropriate drug G418 or neomycin (geneticin),
xgpt (mycophenolic acid), or hygromycin, respectively.
[0143] Examples of other suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the antibody nucleic acid, such as dihydrofolate
reductase (DHFR) or thymidine kinase. The mammalian cell
transformants are placed under selection pressure that only the
transformants are uniquely adapted to survive by virtue of having
taken up the marker. Selection pressure is imposed by culturing the
transformants under conditions in which the concentration of
selection agent in the medium is successively changed, thereby
leading to amplification of both the selection gene and the DNA
that encodes antibody. Amplification is the process by which genes
in greater demand for the production of a protein critical for
growth are reiterated in tandem within the chromosomes of
successive generations of recombinant cells. Increased quantities
of antibody are synthesized from the amplified DNA.
[0144] For example, cells transformed with the DHFR selection gene
are first identified by culturing all of the transformants in a
culture medium that contains methotrexate (Mtx), a competitive
antagonist of DHFR. An appropriate host cell when wild-type DHFR is
employed is the Chinese hamster ovary (CHO) cell line deficient in
DHFR activity, prepared and propagated as described by Urlaub and
Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980). The transformed
cells are then exposed to increased levels of Mtx. This leads to
the synthesis of multiple copies of the DHFR gene, and,
concomitantly, multiple copies of other DNA comprising the
expression vectors, such as the DNA encoding antibody. This
amplification technique can be used with any otherwise suitable
host, e.g., ATCC No. CCL61 CHO-K1, notwithstanding the presence of
endogenous DHFR if, for example, a mutant DHFR gene that is highly
resistant to Mtx is employed (EP 117,060). Alternatively, host
cells (particularly wild-type hosts that contain endogenous DHFR)
transformed or co-transformed with DNA sequences encoding antibody,
wild-type DHFR protein, and another selectable marker such as
aminoglycoside 3 phosphotransferase (APH) can be selected by cell
growth in medium containing a selection agent for the selectable
marker such as an aminoglycosidic antibiotic, e.g. kanamycin,
neomycin, or G418. See U.S. Pat. No. 4,965,199.
[0145] A suitable selection gene for use in yeast is the trp1 gene
present in the yeast plasmid YRp7 (Stinchcomb et al., Nature,
282:39 (1979); Kingsman et al., Gene, 7:141 (1979); or Tschemper et
al., Gene, 10:157 (1980)). The trp1 gene provides a selection
marker for a mutant strain of yeast lacking the ability to grow in
tryptophan, for example, ATCC No. 44076 or PEP4-1 (Jones, Genetics,
85:12 (1977)). The presence of the trp1 lesion in the yeast host
cell genome then provides an effective environment for detecting
transformation by growth in the absence of tryptophan. Similarly,
Leu2-deficient yeast strains (ATCC No. 20,622 or 38,626) are
complemented by known plasmids bearing the Leu2 gene.
[0146] (iv) Promoter Component
[0147] Expression and cloning vectors usually contain a promoter
that is recognized by the host organism and is operably linked to
the antibody nucleic acid. Promoters are untranslated sequences
located upstream (5') to the start codon of a structural gene
(generally within about 100 to 1000 bp) that control the
transcription and translation of particular nucleic acid sequence,
such as the antibody nucleic acid sequence, to which they are
operably linked. Such promoters typically fall into two classes,
inducible and constitutive. Inducible promoters are promoters that
initiate increased levels of transcription from DNA under their
control in response to some change in culture conditions, e.g., the
presence or absence of a nutrient or a change in temperature. At
this time a large number of promoters recognized by a variety of
potential host cells are well known. These promoters are operably
linked to antibody encoding DNA by removing the promoter from the
source DNA by restriction enzyme digestion and inserting the
isolated promoter sequence into the vector. Both the native
antibody promoter sequence and many heterologous promoters may be
used to direct amplification and/or expression of the antibody DNA.
However, heterologous promoters are preferred, as they generally
permit greater transcription and higher yields of expressed
antibody as compared to the native promoter.
[0148] Promoters suitable for use with prokaryotic hosts include
the .beta.-lactamase and lactose promoter systems (Chang et al.,
Nature, 275:615 (1978); and Goeddel et al., Nature, 281:544
(1979)), alkaline phosphatase, a tryptophan (trp) promoter system
(Goeddel, Nucleic Acids Res., 8:4057 (1980) and EP 36,776) and
hybrid promoters such as the tac promoter (deBoer et al., Proc.
Natl. Acad. Sci. USA, 80:21-25 (1983)). However, other known
bacterial promoters are suitable. Their nucleotide sequences have
been published, thereby enabling a skilled worker operably to
ligate them to DNA encoding antibody (Siebenlist et al., Cell,
20:269 (1980)) using linkers or adapters to supply any required
restriction sites. Promoters for use in bacterial systems also will
contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA
encoding antibody polypeptide.
[0149] Promoter sequences are known for eukaryotes. Virtually all
eukaryotic genes have an AT-rich region located approximately 25 to
30 bases upstream from the site where transcription is initiated.
Another sequence found 70 to 80 bases upstream from the start of
transcription of many genes is a CXCAAT region where X may be any
nucleotide. At the 3 end of most eukaryotic genes is an AATAAA
sequence that may be the signal for addition of the poly A tail to
the 3 end of the coding sequence. All of these sequences are
suitably inserted into eukaryotic expression vectors.
[0150] Examples of suitable promoting sequences for use with yeast
hosts include the promoters for 3-phosphoglycerate kinase (Hitzeman
et al., J. Biol. Chem., 255:2073 (1980)) or other glycolytic
enzymes (Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); and
Holland, Biochemistry, 17:4900 (1978)), such as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase.
[0151] Other yeast promoters, which are inducible promoters having
the additional advantage of transcription controlled by growth
conditions, are the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, degradative enzymes associated
with nitrogen metabolism, metallothionein,
glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible
for maltose and galactose utilization. Suitable vectors and
promoters for use in yeast expression are further described in
Hitzeman et al., EP 73,657A. Yeast enhancers also are
advantageously used with yeast promoters.
[0152] Antibody transcription from vectors in mammalian host cells
may be controlled, for example, by promoters obtained from the
genomes of viruses such as polyoma virus, fowlpox virus (UK
2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus
2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a
retrovirus, hepatitis-B virus and most preferably Simian Virus 40
(SV40), from heterologous mammalian promoters, e.g., the actin
promoter or an immunoglobulin promoter, from heat-shock promoters,
and from the promoter normally associated with the antibody
sequence, provided such promoters are compatible with the host cell
systems.
[0153] The early and late promoters of the SV40 virus are
conveniently obtained as an SV40 restriction fragment that also
contains the SV40 viral origin of replication. Fiers et al.,
Nature, 273:113 (1978); Mulligan and Berg, Science, 209:1422-1427
(1980); Pavlakis et al., Proc. Nail. Acad. Sci. USA, 78:7398-7402
(1981). The immediate early promoter of the human cytomegalovirus
is conveniently obtained as a HindIII E restriction fragment.
Greenaway et al., Gene, 18:355-360 (1982). A system for expressing
DNA in mammalian hosts using the bovine papilloma virus as a vector
is disclosed in U.S. Pat. No. 4,419,446. A modification of this
system is described in U.S. Pat. No. 4,601,978. See also Gray et
al., Nature, 295:503-508 (1982) on expressing cDNA encoding immune
interferon in monkey cells; Reyes et al., Nature, 297:598-601
(1982) on expression of human B-interferon cDNA in mouse cells
under the control of a thymidine kinase promoter from herpes
simplex virus; Canaani and Berg, Proc. Nail. Acad. Sci. USA,
79:5166-5170 (1982) on expression of the human interferon BI gene
in cultured mouse and rabbit cells; and Gorman et al., Proc. Natl.
Acad. Sci. USA, 79:6777-6781 (1982) on expression of bacterial CAT
sequences in CV-1 monkey kidney cells, chicken embryo fibroblasts,
Chinese hamster ovary cells, HeLa cells, and mouse NIH-3T3 cells
using the Rous sarcoma virus long terminal repeat as a
promoter.
[0154] (v) Enhancer Element Component
[0155] Transcription of a DNA encoding the antibody of this
invention by higher eukaryotes is often increased by inserting an
enhancer sequence into the vector. Enhancers are cis-acting
elements of DNA, usually about from 10 to 300 bp, that act on a
promoter to increase its transcription. Enhancers are relatively
orientation and position independent, having been found 5
(Laiminset al., Proc. Natl. Acad. Sci. USA, 78:993 (1981)) and 3
(Luskyet al., Mol. Cell Bio., 3:1108 (1983)) to the transcription
unit, within an intron (Banerji et al., Cell, 33:729 (1983)), as
well as within the coding sequence itself (Osborne et al., Mol.
Cell Bio., 4:1293 (1984)). Many enhancer sequences are now known
from mammalian genes (globin, elastase, albumin, a-fetoprotein, and
insulin). Typically, however, one will use an enhancer from a
eukaryotic cell virus. Examples include the SV40 enhancer on the
late side of the replication origin (bp 100-270), the
cytomegalovirus early promoter enhancer, the polyoma enhancer on
the late side of the replication origin, and adenovirus enhancers.
See also Yaniv, Nature, 297:17-18 (1982) on enhancing elements for
activation of eukaryotic promoters. The enhancer may be spliced
into the vector at a position 5 or 3 to the antibody encoding
sequence, but is preferably located at a site 5 from the
promoter.
[0156] (vi) Transcription Termination Component
[0157] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human, or nucleated cells from other
multicellular organisms) will also contain sequences necessary for
the termination of transcription and for stabilizing the mRNA. Such
sequences are commonly available from the 5 and, occasionally 3
untranslated regions of eukaryotic or viral DNAs or cDNAs. These
regions contain nucleotide segments transcribed as polyadenylated
fragments in the untranslated portion of the mRNA encoding
antibody.
[0158] (vii) Construction and Analysis of Vectors
[0159] Construction of suitable vectors containing one or more of
the above listed components employs standard ligation techniques.
Isolated plasmids or DNA fragments are cleaved, tailored, and
religated in the form desired to generate the plasmids
required.
[0160] For analysis to confirm correct sequences in plasmids
constructed, the ligation mixtures are used to transform E. coli
K12 strain 294 (ATCC No. 31,446) and successful transformants
selected by ampicillin or tetracycline resistance where
appropriate. Plasmids from the transformants are prepared, analyzed
by restriction endonuclease digestion, and/or sequenced by the
method of Messing et al., Nucleic Acids Res., 9:309 (1981) or by
the method of Maxam et al., Methods in Enzymology, 65:499
(1980).
[0161] (viii) Transient Expression Vectors
[0162] Particularly useful in the practice of this invention are
expression vectors that provide for the transient expression in
mammalian cells of DNA encoding the antibody polypeptide. In
general, transient expression involves the use of an expression
vector that is able to replicate efficiently in a host cell, such
that the host cell accumulates many copies of the expression vector
and, in turn, synthesizes high levels of a desired polypeptide
encoded by the expression vector. Sambrook et al., supra, pp.
16.17-16.22. Transient expression systems, comprising a suitable
expression vector and a host cell, allow for the convenient
positive identification of polypeptides encoded by cloned DNAs, as
well as for the rapid screening of such polypeptides for desired
biological or physiological properties. Thus, transient expression
systems are particularly useful in the invention for purposes of
identifying analogues and variants of antibody polypeptide that
have antibody polypeptide biological activity.
[0163] (ix) Suitable Exemplary Vertebrate Cell Vectors
[0164] Other methods, vectors, and host cells suitable for
adaptation to the synthesis of the antibody in recombinant
vertebrate cell culture are described in Gething et al., Nature,
293:620-625 (1981); Mantei et al., Nature, 281:40-46 (1979);
Levinson et al.; EP 117,060; and EP 117,058. A particularly useful
plasmid for mammalian cell culture expression is pRK5 (EP 307,247
U.S. Pat. No. 5,258,287) or pSV16B (PCT Publication No. WO
91/08291).
[0165] Suitable host cells for cloning or expressing the vectors
herein are the prokaryote, yeast, or higher eukaryotic cells
described above. Suitable prokaryotes include eubacteria, such as
Gram-negative or Gram-positive organisms, for example, E. coli,
Bacilli such as B. subtilis, Pseudomonas species such as P.
aeruginosa, Salmonella typhimurium, or Serratia marcescans. One
preferred E. coli cloning host is E. coli 294 (ATCC No. 31,446),
although other strains such as E. coli B, E. coliXI776 (ATCC No.
31,537), and E. coli W3110 (ATCC No. 27,325) are suitable. These
examples are illustrative rather than limiting. Preferably the host
cell should secret minimal amounts of proteolytic enzymes.
Alternatively, in vitro methods of cloning, e.g., PCR or other
nucleic acid polymerase reactions, are suitable.
[0166] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable hosts for antibody encoding
vectors. Saccharomyces cerevisiae, or common baker's yeast, is the
most commonly used among lower eukaryotic host microorganisms.
However, a number of other genera, species, and strains are
commonly available and useful herein, such as Schizosaccharomyces
pombe (Beach and Nurse, Nature, 290-140(1981); EP 139,383 published
2 May 1985), Kluyveromyces hosts (U.S. Pat. No. 4,943,529) such as,
e.g., K. lactis (Louvencourt et al., J. Bacteriol., 737 (1983)), K.
fragilis, K. bulgaricus, K. thermotolerans, and K. maxianus,
yarrowia (EP 402,226), Pichia pastoris (EP 183,070; Sreekrishna et
al, J. Basic Microbiol., 28:265-278 (1988)), Candida, Trichoderma
reesia (EP 244,234), Neurospora crassa (Case et al., Proc. Natl.
Acad. Sci. USA, 76:5259-5263 (1979)), and filamentous fungi such
as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357
published 10 Jan. 1991), and Aspergillus hosts such as A. nidulans
(Ballance et al., Biochem. Biophys. Res. Commun., 112:284-289
(1983); Tilburn et al., Gene, 26:205-221 (1983); Yelton et al.,
Proc. Natl. Acad. Sci. USA, 81:1470-1474 (1984)) and A. niger
(Kelly and Hynes, EMBO J., 4:475-479 (1985)).
[0167] Suitable host cells for the expression of glycosylated
antibody are derived from multicellular organisms. Such host cells
are capable of complex processing and glycosylation activities. In
principle, any higher eukaryotic cell culture is workable, whether
from vertebrate or invertebrate culture. Examples of invertebrate
cells include plant and insect cells. Numerous baculoviral strains
and variants and corresponding permissive insect host cells from
hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti
(mosquito), Aedes albopictus (mosquito), Drosophila nielanogaster
(fruitfly), and Bombyx mori have been identified. See, e.g., Luckow
et al., Bio/Technology, 6:47-55 (1988); Miller et al., Genetic
Engineering, Setlow et al., eds., Vol. 8 (Plenum Publishing, 1986),
pp. 277-279; and Maeda et al., Nature, 315:592-594 (1985). A
variety of viral stains for transfection are publicly available,
e.g., the L-1 variant of Autographa californica NPV and the Bm-5
strain of Bombyx mori NPV, and such viruses may be used as the
virus herein according to the present invention, particularly for
transfection of Spodoptera frugiperda cells.
[0168] Plant cell cultures of cotton, corn, potato, soybean,
petunia, tomato, and tobacco can be utilized as hosts. Typically,
plant cells are transfected by incubation with certain strains of
the bacterium Agrobacterium tumefaciens, which has been previously
manipulated to contain the antibody DNA. During incubation of the
plant cell culture with A. tumefaciens, the DNA encoding the
antibody is transferred to the plant cell host such that it is
transfected, and will, under appropriate conditions, express the
antibody DNA. In addition, regulatory and signal sequences
compatible with plant cells are available, such as the nopaline
synthase promoter and polyadenylation signal sequences. Depicker et
al., J. Mol. Appl. Gen., 1:561 (1982). In addition, DNA segments
isolated from the upstream region of the T-DNA 780 gene are capable
of activating or increasing transcription levels of
plant-expressible genes in recombinant DNA-containing plant tissue.
EP 321,196 published 21 Jun. 1989.
[0169] However, interest has been greatest in vertebrate cells, and
propagation of vertebrate cells in culture (tissue culture) has
become a routine procedure in recent years (Tissue Culture,
Academic Press, Kruse and Patterson, editors (1973)). Examples of
useful mammalian host cell lines are monkey kidney CV1 line
transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney
line (293 or 293 cells subcloned for growth in suspension culture,
Graham et al., J. Gen Virol., 36:59 (1977)); baby hamster kidney
cells (BHK, ATCC CCL 70); Chinese hamster ovary cells/-DHFR(CHO,
Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980));
mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251
(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green
monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical
carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC
CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human
lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB
8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells
(Mather et al., Annals N.Y. Acad. Sci., 383:44-68 (1982)); MRC 5
cells; FS4 cells; and a human hepatoma line (Hep G2).
[0170] Host cells are transfected and preferably transformed with
the above-described expression or cloning vectors of this invention
and cultured in conventional nutrient media modified as appropriate
for inducing promoters, selecting transformants, or amplifying the
genes encoding the desired sequences.
[0171] Transfection refers to the taking up of an expression vector
by a host cell whether or not any coding sequences are in fact
expressed. Numerous methods of transfection are known to the
ordinarily skilled artisan, for example, CaPO.sub.4 and
electroporation. Successful transfection is generally recognized
when any indication of the operation of this vector occurs within
the host cell.
[0172] Transformation means introducing DNA into an organism so
that the DNA is replicable, either as an extrachromosomal element
or by chromosomal integrant. Depending on the host cell used,
transformation is done using standard techniques appropriate to
such cells. The calcium treatment employing calcium chloride, as
described in section 1.82 of Sambrook et al., supra, is generally
used for prokaryotes or other cells that contain substantial
cell-wall barriers. Infection with Agrobacterium tumefaciens is
used for transformation of certain plant cells, as described by
Shaw et al., Gene, 23:315 (1983) and WO 89/05859 published 29 Jun.
1989. In addition, plants may be transfected using ultrasound
treatment as described in WO 91/00358 published 10 Jan. 1991. For
mammalian cells without such cell walls, the calcium phosphate
precipitation method of Graham and van der Eb, Virology, 52:456-457
(1978) is preferred. General aspects of mammalian cell host system
transformations have been described by Axel in U.S. Pat. No.
4,399,216 issued 16 Aug. 1983. Transformations into yeast are
typically carried out according to the method of Van Solingen et
al., J. Bact., 130:946 (1977) and Hsiao et al., Proc. Natl. Acad.
Sci. (USA), 76:3829 (1979). However, other methods for introducing
DNA into cells such as by nuclear injection, electroporation, or
protoplast fusion may also be used.
[0173] Prokaryotic cells used to produce the antibody polypeptide
of this invention are cultured in suitable media as described
generally in Sambrook et al., supra.
[0174] The mammalian host cells used to produce the antibody of
this invention may be cultured in a variety of media. Commercially
available media such as Ham's FIO (Sigma), Minimal Essential Medium
((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's
Medium ((DMEM), Sigma) are suitable for culturing the host cells.
In addition, any of the media described in Ham and Wallace, Meth.
Enz, 58:44 (1979), Barnes and, Sato, Anal. Biochem., 102:255
(1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; or
4,560,655; WO 90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985;
the disclosures of all of which are incorporated herein by
reference, may be used as culture media for the host cells. Any of
these media may be supplemented as necessary with hormones and/or
other growth factors (such as insulin, transferrin, or epidermal
growth factor), salts (such as sodium chloride, calcium, magnesium,
and phosphate), buffers (such as HEPES), nucleosides (such as
adenosine and thymidine), antibiotics (such as Gentamycin.TM.
drug), trace elements (defined as inorganic compounds usually
present at final concentrations in the micromolar range), and
glucose or an equivalent energy source. Any other necessary
supplements may also be included at appropriate concentrations that
would be known to those skilled in the art. The culture conditions,
such as temperature, pH, and the like, are those previously used
with the host cell selected for expression, and will be apparent to
the ordinarily skilled artisan.
[0175] The host cells referred to in this disclosure encompass
cells in in vitro culture as well as cells that are within a host
animal.
[0176] Gene amplification and/or expression may be measured in a
sample directly, for example, by conventional Southern blotting,
northern blotting to quantitate the transcription of mRNA (Thomas,
Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)), dot blotting (DNA
analysis), or in situ hybridization, using an appropriately labeled
probe, based on the sequences provided herein. Various labels may
be employed, most commonly radioisotopes, particularly .sup.32P.
However, other techniques may also be employed, such as using
biotin-modified nucleotides for introduction into a polynucleotide.
The biotin then serves as the site for binding to avidin or
antibodies, which may be labeled with a wide variety of labels,
such as radionuclides, fluorescers, enzymes, or the like.
Alternatively, antibodies may be employed that can recognize
specific duplexes, including DNA duplexes, RNA duplexes, and
DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in
turn may be labeled and the assay may be carried out where the
duplex is bound to a surface, so that upon the formation of duplex
on the surface, the presence of antibody bound to the duplex can be
detected.
[0177] Gene expression, alternatively, may be measured by
immunological methods, such as immunohistochemical staining of
tissue sections and assay of cell culture or body fluids, to
quantitate directly the expression of gene product. With
immunohistochemical staining techniques, a cell sample is prepared,
typically by dehydration and fixation, followed by reaction with
labeled antibodies specific for the gene product coupled, where the
labels are usually visually detectable, such as enzymatic labels,
fluorescent labels, luminescent labels, and the like. A
particularly sensitive staining technique suitable for use in the
present invention is described by Hsu et al., Am. J. Clin. Path.,
75:734-738 (1980).
[0178] Antibody preferably is recovered from the culture medium as
a secreted polypeptide, although it also may be recovered from host
cell lysates when directly expressed without a secretory
signal.
[0179] When the antibody is expressed in a recombinant cell other
than one of human origin, the antibody is completely free of
proteins or polypeptides of human origin. However, it is still
usually necessary to purify the antibody from other recombinant
cell proteins or polypeptides to obtain preparations that are
substantially homogeneous as to the mpl ligand per se. As a first
step, the culture medium or lysate is centrifuged to remove
particulate cell debris. The membrane and soluble protein fractions
are then separated. Alternatively, a commercially available protein
concentration filter (e.g., AMICON or Millipore PELLICON
ultrafiltration units) may be used The antibody may then be
purified from the soluble protein fraction. The antibody thereafter
is purified from contaminant soluble proteins and polypeptides by
salting out and exchange or chromatographic procedures employing
various gel matrices. These matrices include; acrylamide, agarose,
dextran, cellulose and others common to protein purification.
Exemplary chromatography procedures suitable for protein
purification include immunoaffinity, receptor affinity (e.g.,
mpl-IgG or protein A SEPHAROSE), hydrophobic interaction
chromatography (HIC) (e.g., ether, butyl, or phenyl Toyopearl),
lectin chromatography (apt, Con A-SEPHAROSE,
lentil-lectin-SEPHAROSE), size exclusion (e.g., SEPHADEX G-75),
cation- and anion-exchange columns (e.g., DEAE or carboxymethyl-
and sulfopropyl-cellulose), and reverse-phase high performance
liquid chromatography (RP-HPLC) (see e.g., Urdal et al., J.
Chromatog., 296:171 (1984) where two sequential RP-HPLC steps are
used to purify recombinant human IL-2). Other purification steps
optionally include; ethanol precipitation; ammonium sulfate
precipitation; chromatofocusing; preparative SDS-PAGE, and the
like.
[0180] Antibody variants in which residues have been deleted,
inserted, or substituted are recovered in the same-fashion, taking
account of any substantial changes in properties occasioned by the
variation. For example, preparation of a an antibody fusion with
another protein or polypeptide, e.g., a bacterial or viral antigen,
facilitates purification; an immunoaffinity column containing
antibody to the antigen can be used to adsorb the fusion
polypeptide. Immunoaffinity columns such as a rabbit polyclonal
anti-antibody column can be employed to absorb the antibody variant
by binding it to at least one remaining immune epitope.
Alternatively, the antibody may be purified by affinity
chromatography using a purified receptor-IgG coupled to a
(preferably): immobilized resin such as AFFI-Gel 10 (Bio-Rad,
Richmond, Calif.) or the like, by means well known in the art. A
protease inhibitor such as phenyl methyl sulfonyl fluoride (PMSF)
also may be useful to inhibit proteolytic degradation during
purification, and antibiotics may be included to prevent the growth
of adventitious contaminants. One skilled in the art will
appreciate that purification methods suitable for native the
antibody may require modification to account for changes in the
character of the antibody or its variants upon expression in
recombinant cell culture.
[0181] In a most preferred embodiment of the invention, the
antibodies are agonist antibodies (aAb). By `agonist antibody` is
meant an antibody which is able to bind to and to activate, a
particular hematopoietic growth factor receptor. For example, the
agonist may bind to the extracellular domain of the receptor and
thereby cause differentiation and proliferation of megakaryocyte
colonies in semisolid cultures and single megakaryocytes in liquid
suspension cultures and platelet production in vitro and/or in
vivo. The agonist antibodies are preferably against epitopes within
the extracellular domain of the receptor Accordingly, the antibody
preferably binds to substantially the same epitope as the 12E10,
12B5, 10F6, and 12D5 monoclonal antibodies specifically disclosed
herein. Most preferably, the antibody will also have substantially
the same or greater antigen binding affinity as the monoclonal
antibodies disclosed herein. To determine whether a monoclonal
antibody has the same specificity as an antibody specifically
disclosed, one can, for example, use a competitive ELISA binding
assay.
[0182] DNA encoding the monoclonal antibodies useful in the method
of the invention is readily isolated and sequenced using
conventional procedures (e.g., by using oligonucleotide probes that
are capable of binding specifically to genes encoding the heavy and
light chains of human antibodies). The phage of the invention serve
as a preferred source of such DNA. Once isolated, the DNA may be
placed, into expression vectors, which are then transfected into
host cells such as E. coli cells, simian COS cells, Chinese Hamster
Ovary (CHO) cells, or myeloma cells that do not otherwise produce
immunoglobulin protein, to obtain the synthesis of monoclonal
antibodies in the recombinant host cells.
[0183] IV. Utility
[0184] The antibodies disclosed herein are useful for in vitro
diagnostic assays for activating the receptor of interest This is
useful in order to study the role of the receptor in megakaryocyte
growth and/or differentiation and platelet production.
[0185] The biologically active c-mpl agonist antibody capable of
stimulating either proliferation, differentiation or maturation
and/or modulation (either stimulation or inhibition) of apoptosis
of hematopoietic cells may be used in a sterile pharmaceutical
preparation or formulation to stimulate megakaryocytopoietic or
thrombopoietic activity in patients suffering from thrombocytopenia
due to impaired production, sequestration, or increased destruction
of platelets. Thrombocytopenia-associated bone marrow hypoplasia
(e.g., aplastic anemia following chemotherapy or bone marrow
transplant) may be effectively treated with the aAb compounds of
this invention as well as disorders such as disseminated
intravascular coagulation (DIC), immune thrombocytopenia (including
HIV-induced ITP and non HIV-induced ITP), chronic idiopathic
thrombocytopenia, congenital thrombocytopenia, myelodysplasia, and
thrombotic thrombocytopenia.
[0186] Preferred uses of the megakaryocytopoietic or
thrombocytopoietic biologically active c-mpl agonist antibody of
this invention are in: myelotoxic chemotherapy for treatment of
leukemia or solid tumors, myeloablative chemotherapy for autologous
or allogeneic bone marrow transplant, myelodysplasia, idiopathic
aplastic anemia, congenital thrombocytopenia, and immune
thrombocytopenia.
[0187] The biologically active c-mpl agonist antibody of the
instant invention may be employed alone or in combination with
other cytokines, hematopoietins, interleukins, growth factors, or
antibodies in the treatment of the above-identified disorders and
conditions. Thus, the instant compounds may be employed in
combination with other protein or peptide having hematopoietic
activity including G-CSF, GM-CSF, LIF, M-CSF, IL-1, IL-3,
erythropoietin (EPO), kit ligand, IL-6, and IL-11.
[0188] The biologically active c-mpl agonist antibody of the
instant invention may be used in the same way and for the same
indications as thrombopoietin (TPO). Some forms of the aAb have a
longer half-life than native or pegylated TPO and thus are used in
indications where a longer half-life are indicated.
[0189] When used for in vivo administration, the antibody
formulation must be sterile. This is readily accomplished by
filtration through sterile filtration membranes, prior to or
following lyophilization and reconstitution. The antibody
ordinarily will be stored in lyophilized form or in solution.
[0190] Therapeutic antibody compositions generally are placed into
a container having a sterile access port, for example, an
intravenous solution bag or vial having a stopper pierceable by a
hypodermic injection needle.
[0191] The route of antibody administration is in accord with known
methods, e.g., injection or infusion by intravenous,
intraperitoneal, intracerebral, intramuscular, intraocular,
intraarterial, intrathecal, inhalation or intralesional routes, or
by sustained release systems as noted below. The antibody is
preferably administered continuously by infusion or by bolus
injection.
[0192] An effective amount of antibody to be employed
therapeutically will depend, for example, upon the therapeutic
objectives, the route of administration, and the condition of the
patient. Accordingly, it will be necessary for the therapist to
titer the dosage and modify the route of administration as required
to obtain the optimal therapeutic effect. Typically, the clinician
will administer antibody until a dosage is reached that achieves
the desired effect. The progress of this therapy is easily
monitored by conventional assays.
[0193] The antibodies of the invention may be prepared in a mixture
with a pharmaceutically acceptable carrier. This therapeutic
composition can be administered intravenously or through the nose
or lung, preferably as a liquid or powder aerosol (lyophilized).
The composition may also be administered parenterally or
subcutaneously as desired. When administered systematically, the
therapeutic composition should be sterile, pyrogen-free and in a
parenterally acceptable solution having due regard for pH,
isotonicity, and stability. These conditions are known to those
skilled in the art. Briefly, dosage formulations of the compounds
of the present invention are prepared for storage or administration
by mixing the compound having the desired degree of purity with
physiologically acceptable carriers, excipients, or stabilizers.
Such materials are non-toxic to the recipients at the dosages and
concentrations employed, and include buffers such as TRIS HCl,
phosphate, citrate, acetate and other organic acid salts;
antioxidants such as ascorbic acid; low molecular weight (less than
about ten residues) peptides such as polyarginine, proteins, such
as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers
such as polyvinylpyrrolidinone; amino acids such as glycine,
glutamic acid, aspartic acid, or arginine; monosaccharides,
disaccharides, and other carbohydrates including cellulose or its
derivatives, glucose, mannose, or dextrins; chelating agents such
as EDTA; sugar alcohols such as mannitol or sorbitol; counterions
such as sodium and/or nonionic surfactants such as TWEEN, PLURONICS
or polyethyleneglycol.
[0194] Sterile compositions for injection can be formulated
according to conventional pharmaceutical practice. For example,
dissolution or suspension of the active compound in a vehicle such
as water or naturally occurring vegetable oil like sesame, peanut,
or cottonseed oil or a synthetic fatty vehicle like ethyl oleate or
the like may be desired. Buffers, preservatives, antioxidants and
the like can be incorporated according to accepted pharmaceutical
practice.
[0195] Suitable examples of sustained-release preparations include
semipermeable matrices of solid hydrophobic polymers containing the
polypeptide, which matrices are in the form of shaped articles,
e.g., films, or microcapsules. Examples of sustained-release
matrices include polyesters, hydrogels (e.g.,
poly(2-hydroxyethyl-methacrylate) as described by Langer et al, J.
Biomed. Mater. Res., 15:167-277 (1981) and Langer, Chem. Tech.,
12:98-105 (1982) or poly(vinylalcohol)), polylactides (U.S. Pat.
No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma
ethyl-L-glutamate (Sidman et al., Biopolymers, 22:547-556 (1983)),
non-degradable ethylene-vinyl acetate (Langer et al., supra),
degradable lactic acid-glycolic acid copolymers such as the LUPRON
Depot.TM. (injectable microspheres composed of lactic acid-glycolic
acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid (EP 133,988).
[0196] While polymers such as ethylene-vinyl acetate and lactic
acid-glycolic acid enable release of molecules for over 100 days,
certain hydrogels release proteins for shorter time periods. When
encapsulated proteins remain in the body for a long time, they may
denature or aggregate as a result of exposure to moisture at
37.degree. C., resulting in a loss of biological activity and
possible changes in immunogenicity. Rational strategies can be
devised for protein stabilization depending on the mechanism
involved. For example, if the aggregation mechanism is discovered
to be intermolecular S--S bond formation through disulfide
interchange, stabilization may be achieved by modifying sulfhydryl
residues, lyophilizing from acidic solutions, controlling moisture
content, using appropriate additives, and developing specific
polymer matrix compositions. Sustained-release compositions also
include liposomally entrapped TPO. Liposomes containing TPO are,
prepared by methods known per se: DE 3,218,121; Epstein et al.,
Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); Hwang et al.,
Proc. Natl. Acad. Sci. USA, 77:4030-4034 (1980); EP 52,322; EP
36,676; EP 88;046; EP 143,949; EP 142,641; Japanese patent
application 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and
EP 102,324. Ordinarily the liposomes are of the small (about
200-800 Angstroms) unilamellar type in which the lipid content is
greater than about 30 mol. % cholesterol, the selected proportion
being adjusted for the optimal therapy.
[0197] The dosage of the antibody will be determined by the
attending physician taking into consideration various factors known
to modify the action of drugs including severity and type of
disease, body weight, sex, diet, time and route of administration,
other medications and other relevant clinical factors.
Therapeutically effective dosages may be determined by either in
vitro or in vivo methods.
[0198] An effective amount of the agonist antibody to be employed
therapeutically will depend, for example, upon the therapeutic
objectives, the route of administration, and the condition of the
patient. Accordingly, it will be necessary for the therapist to
titer the dosage and modify the route of administration as required
to obtain the optimal therapeutic effect. A typical daily dosage
might range from about 1 .mu.g/kg to up to 1000 mg/kg or more,
depending on the factors mentioned above. Typically, the clinician
will administer the molecule until a dosage is reached that
achieves the desired effect. The progress of this therapy is easily
monitored by conventional assays.
[0199] Depending on the type and severity of the disease, from
about 0.001 mg/kg to about 1000 mg/kg, more preferably about 0.01
mg to 100 mg/kg, more preferably about 0.010 to 20 mg/kg of the
agonist antibody might be an initial candidate dosage for
administration to the patient, whether, for example, by one or more
separate administrations, or by continuous infusion. For repeated
administrations over several days or longer, depending an the
condition, the treatment is repeated until a desired suppression of
disease symptoms occurs or the desired improvement in the patient's
condition is achieved. However, other dosage regimens may also be
useful.
EXAMPLES
[0200] Without further description, it is believed that one of
ordinary skill in the art can, using the preceding description and
illustrative examples, make and utilize the present invention to
the fullest extent. The following working examples therefore
specifically point out preferred embodiments of the present
invention, and are not to be construed as limiting in any way of
the remainder of the disclosure.
Example 1
[0201] Assays
[0202] The mpl agonist antibody assays were conducted essentially
as described in WO 95/18858.
[0203] (a) Ba/F3 Cell Proliferation Assay
[0204] The Ba/F3-mpl cell line was established (F. de Sauvage et
al., Nature, 369:533 (1994)) by introduction of the cDNA encoding
the entire mpl receptor into the IL-3 dependent murine
lymphoblastoid cell line Ba/F3. Simulation of proliferation of
Ba/F3-mpl cells in response to various concentrations of antibodies
or TPO was measured by the amount of incorporation of
.sup.3H-thymidine as previously described (F. de Sauvage et al.,
supra).
[0205] (b) CMK Assay for Induction of Platelet Antigen
GPII.sub.bIII.sub.a Expression
[0206] CMK cells are maintained in RMPI 1640 medium (Sigma)
supplemented with 10% fetal bovine serum and 10 mM glutamine. In
preparation for the assay, the cells are harvested, washed and
resuspended at 5.times.10.sup.5 cells/ml in serum-free GIF medium
supplemented with 5 mg/l bovine insulin, 10 mg/l apo-transferrin,
1.times. trace elements. In a 96-well flat-bottom plate, the TPO
standard or experimental agonist antibody samples are added to each
well at appropriate dilutions in 100 ml volumes. 100 ml of the CMK
cell suspension is added to each well and the plates are incubated
at 37.degree. C., in a 5% CO.sub.2 incubator for 48 hours. After
incubation, the plates are spun at 1000 rpm at 4.degree. C. for
five minutes. Supernatants are discarded and 100 ml of the
FITC-conjugated GPII.sub.bIII.sub.a monoclonal 2D2 antibody is
added to each well. Following incubation at 4.degree. C. for 1
hour, plates are spun again at 1000 rpm for five minutes. The
supernatants containing unbound antibody are discarded and 200 ml
of 0.1% BSA-PBS wash is added to each well. The 0.1% BSA-PBS wash
step is repeated three times. Cells are then analyzed on a FASCAN
using standard one parameter analysis measuring relative
fluorescence intensity.
[0207] (c) KIRA ELISA for Measuring Phosphorylation of the
mpl-Rse.gD Chimeric Receptor
[0208] The human mpl receptor has been disclosed by Vigon et al.,
PNAS, USA 89:5640-5644 (1992). A chimeric receptor comprising the
extracellular domain (ECD) of the mpl receptor and the
transmembrane and intracellular domain (ICD) of Rse (Mark et al.,
J. of Biol. Chem. 269(14):10720-10728 (1994)) with a
carboxyl-terminal flag polypeptide (i.e. Rse.gD) was made for use
in the KIRA ELISA described herein.
[0209] (i) Capture Agent Preparation
[0210] Monoclonal anti-gD (clone 5B6) was produced against a
peptide from Herpes simplex virus glycoprotein D (Paborsky et al.,
Protein Engineering 3(6):547-553 (1990)). The purified stock
preparation was adjusted to 3.0 mg/ml in phosphate buffered saline
(PBS), pH 7.4 and 1.0 ml aliquots were stored at -20.degree. C.
[0211] (ii) Anti-Phosphotyrosine Antibody Preparation
[0212] Monoclonal anti-phosphotyrosine, clone 4G10, was purchased
from UBI (Lake Placid, N.Y.) and biotinylated using long-arm
biotin-N-hydroxysuccinamide (Biotin-X-NHS, Research Organics,
Cleveland, Ohio).
[0213] (iii) Ligand
[0214] The mpl ligand was prepared by the recombinant techniques
described herein. The purified mpl ligand was stored at 4.degree.
C. as a stock solution.
[0215] KIRA ELISA results for agonist antibodies of the invention
are shown in FIG. 9. This assay indicates that the antibodies of
the invention activate the mpl receptor to a degree similar to the
cognate ligand TPO.
[0216] (d) TPO Receptor-Binding Inhibition Assay
[0217] NUNC 96-well immunoplates were coated with 50 .mu.l of
rabbit anti-human IgG Fc (Jackson Labs) at 2 .mu.g/ml in carbonate
buffer (pH9.6) overnight at 4.degree. C. After blocking with ELISA
buffer (PBS, 1% BSA, 0.2% TWEEN 20), the plates were incubated for
2 hr with conditioned media from mpl-Ig-transfected 293 cells.
Plates were washed, and 2.5 ng/ml biotinylated TPO was added in the
presence or absence of various concentrations of antibodies. After
incubation for 1 hr and washing, the amount of TPO bound was
detected by incubation with streptAvidin-HRP (Sigma) followed by
TMB peroxidase substrate (Kirkegaard & Perry). All dilutions
were performed in ELISA buffer, and all incubations were at room
temperature. Color development was quenched with H3PO.sub.4 and
absorbance was read at 450-650 nm.
[0218] (e) HU-03 Cell Proliferation Assay
[0219] The HU-01 cell line (D. Morgan, Hahnemann University) is
derived from a patient with acute megakaryoblastic leukemia and is
dependent on granulocyte-macrophage colony stimulating factor
(GM-CSF) for growth. The HU-03 cell line used here was derived from
HU-01 cells by adaptation to growth in rhTPO rather than
GM-CSF.
[0220] HU-03 cells were maintained in RPMI 1640 supplemented with
2% heat-inactivated human male serum and 5 ng/ml rhTPO. Before
assay, cells were starved by removing TPO, decreasing serum
concentration to 1%, and adjusting the concentration of cells to
2.5.times.10.sup.5 cells/ml, followed by incubation for 16 hr.
Cells were then washed and seeded into 96-well plates at a density
of 5.times.10.sup.4 cells per well in medium containing TPO or
antibodies at various concentrations. Quadruplicate assays were
performed. 1 .mu.Ci 3H-thymidine was added to each well before
incubation for 24 hr. Cells were collected with a Packard cell
harvester and incorporation of .sup.3H-thymidine was measured with
a Top Count Counter (Packard).
[0221] (f) Liquid Suspension Megakaryocytopoeisis Assay
[0222] The effect of Mpl agonist antibodies on human
megakaryocytopoiesis was determined using a modification of the
liquid suspension assay previously described (Grant et al, Blood
69:1334-1339 (1997)).
[0223] Buffy coats were collected from human umbilical cord blood
and cells washed in phosphate-buffered saline (PBS) by
centrifugation at 120 g for 15 min at room temperature to remove
platelet-rich plasma. Cell pellets were resuspended in Iscove's
modified Dulbecco's medium (IMDM, GIBCO) (supplemented with 100
units per ml penicillin and streptomycin), layered onto 60% percoll
(density=1.077 gm/ml, Pharmacia), and centrifuged at 800 g for 20
min at room temperature. The light-density mononuclear cells were
collected from the interface and washed twice with IMDM. Cells were
seeded at 1.times.10.sup.6 cells per ml in IMDM supplemented with
30% fetal bovine serum (FBS), 100 units per ml penicillin and
streptomycin, and 20 .mu.M 2-mercaptoethanol, into 24-well tissue
culture plates (COSTAR). Serial dilutions of thrombopoietin (TPO)
or the Fab'2 forms of antibody 12B5 or antibody 12D5 were added to
quadruplicate wells; control wells contained no additional
supplements. Final volumes were 1 ml per well. The cultures were
grown in a humidified incubator at 31.degree. C. in 5% CO.sub.2 for
14 days. Megakaryocytopoiesis was quantified using radiolabelled
murine monoclonal antibody HP1-1D (provided by W. L. Nichols, Mayo
Clinic) which has been shown to be specific for the human
megakaryocyte glycoprotein IIb/IIIa (Grant et al., supra). Cells
were harvested from the tissue culture plates, washed twice with
assay buffer (20% FBS, 0.002% EDTA in PBS), and resuspended in 100
.mu.l assay buffer containing 20 ng iodinated HP1-1D
(approximatedly 100,000 cpm). After incubation at room temperature
for 1 hr, the cells were washed twice with assay buffer and the
cell pellets counted with a gamma counter.
[0224] FBS used in this assay was treated with Dextran T40 at 1
mg/ml and charcoal at 10 mg/ml for 30 min, centrifuged, decanted,
filter sterilized and heat inactivated at 56.degree. C. for 30
min.
[0225] (g) TPO-Antibody Competitive Binding Assays for HU-03 Cells
and Human Platelets
[0226] HU-03 cells were cultured as described above. Platelet rich
plasma (PRP) was prepared by centrifugation of citrated whole blood
at 400 g's for 5 minutes. Binding studies were conducted within
three hours of collection. .sup.125I-TPO was prepared by indirect
iodination (Fielder, P. J., Hass, P., Nagel, M., Stefanich, E.,
Widmer, R., Bennett, G. L., Keller, G., de Savage, F. J., and
Eaton, D. 1997. Human platelets as a model for the binding and
degradation of thrombopoietin. Blood 89: 2782-2788) and yielded a
specific activity of 15-50 .mu.Ci/.mu.g protein.
[0227] In a volume of 110 microliters containing 100 pM iodinated
TPO, 2.times.10.sup.6 washed HU-03 cells in Hank's Balanced Salt
Solution, 5 mg/ml bovine serum albumin (HBSSB), or 4.times.10.sup.7
platelets in plasma, were incubated at 37.degree. C. for 30 minutes
with varying concentrations of antibody in triplicate. HU-03 cells
were agitated during the incubation period to keep them in
suspension. The reaction mixture was overlayed on 1 ml 20%
sucrose-HBSSB and microcentrifuged at 13,500 rpm for five minutes.
The supernatants were aspirated, tube bottoms containing the cell
pellets were cut off, and cell- or platelet-associated
radioactivity was measured with an Iso Data Model 120 gamma
counter.
[0228] Results for several agonist antibodies on the invention in
this assay are shown in FIG. 10A-F. Longer bars in the graphs
indicate greater amounts of bound radiolabeled TPO and less
competition by the agonist antibody at a particular
concentration.
[0229] (h) Affinity Determinations.
[0230] The receptor-binding affinities of several Fab fragments
were calculated (Lofas & Johnsson, 1990) from association and
dissociation rate constants measured using a BIACORE surface
plasmon resonance system (Pharmacia Biosensor). A biosensor chip
was activated for covalent coupling of gD-mpl receptor using
N-ethyl-N'"-(3-dimethylaminopropyl)-car- bodiimide hydrochloride
(EDC) and N-hydroxysuccinimide (NHS) according to the supplier's
(Pharmacia Biosensor) instructions. gD-mpl was buffer-exchanged
into 10 mM sodium acetate buffer (pH 4.5) and diluted to
approximately 30 .mu.g/mL. An aliquot (35 .mu.L) was injected at a
flow rate of 1 .mu.L/min to achieve approximately 6400 response
units (RU) of coupled protein. Finally, 1M ethanolamine was
injected as a blocking agent. For kinetics measurements, 1.5 serial
dilutions of Fab were injected in PBS/Tween buffer (0.05% Tween-20
in phosphate buffered saline) at 25.degree. C. using a flow rate of
20 .mu.L/min. Equilibrium dissociation constants, K.sub.d's, from
SPR measurements were calculated as k.sub.off/k.sub.on. Standard
deviations, s.sub.on for k.sub.on and s.sub.off for k.sub.off, were
obtained from measurements with >4 protein concentrations
(k.sub.on) or with >7 protein concentrations (k.sub.off).
Dissociation data were fit to a simple AB-->A+B model to obtain
koff+/-s.d. (standard deviation of measurements). Pseudo-first
order rate constant (ks) were calculated for each association
curve, and plotted as a function of protein concentration to obtain
kon+/-s.e. (standard error of fit). The resulting errors e[K] in
calculated K.sub.d's were estimated according to the following
formula for propagation of errors:
e[K]=[(k.sub.on).sup.-2(s.sub.off).sup.2+(k.sub.of-
f).sup.2(k.sub.on).sup.-4(s.sub.on).sup.2].sup.1/2 where s.sub.off
and s.sub.on, are the standard errors in k.sub.on and k.sub.off,
respectively.
Example 2
[0231] Isolation of Antibodies from the CAT Library
[0232] For construction of a library of antibodies displayed of a
phage see the following references: WO 92/01047, WO 92/20791, WO
93/06213, WO 93/11236, WO 93/19172, WO 95/01438 and WO 95/15388.
Briefly, FIGS. 2 and 3 presents a cartoon of the construction of a
phage library containing 6.times.10.sup.9 different clones
containing single-chain Fv (scFv) antibodies fused to gene 3 of a
phage. Binding selection against an antigen, in this case c-mpl,
can be carried out as shown in FIG. 4 and described in greater
detail below.
[0233] (a) The Antigen
[0234] Human c-mpl was cloned as described by F. de Sauvage et al.,
Nature 369:533 (1994).
[0235] (b) Phage Selection on Immunotubes
[0236] NUNC immunotubes were coated with 2 ml of a solution of 10
microg/ml of gD-c-mpl in PBS at 4.degree. C. overnight. After
rinsing with PBS, tubes were blocked with 3% dry milk in PBS (MPBS)
for 2 hr at room temperature. For the first round, 10 .mu.l of
C.A.T. antibody phage library containing .about.1.times.10.sup.12
c.f.u. were added to 1 ml MPBS for blocking for 1 hr at room
temperature. Blocked phage were added to coated tubes, and binding
of phage to antigen allowed to continue for 2 hr at 37.degree. C.
on a rotating wheel. Tubes were washed 6 times with PBS-TWEEN and 6
times with PBS, and phage were then eluted with 100 mM TEA for 10
min at room temperature, neutralized with 500 .mu.l of 1 M TRIS (pH
7.4), and stored on ice until needed. For subsequent rounds,
washing was increased to 20 times with PBS-TWEEN, and 20 times with
PBS.
[0237] Eluted phage were used to infect 5 ml of log phase E. coli
TG1 cells and plated on 2YT agar supplemented with 2% glucose and
100 .mu.g/ml carbenicillin. After overnight growth at 30.degree.
C., colonies were scraped into 10 ml 2YT. 50 .mu.l of this solution
was used to inoculate 25 ml of 2YT with carbenicillin and glucose
and incubated, shaking, for two hours at 37.degree. C. Helper phage
M13K07 (Pharmacia) were added at an m.o.i. of 10. After adsorption,
the cells were pelleted and resuspended in 25 ml of 2YT with
carbenicillin (100 g/ml) and kanamycin (50 .mu.g/ml) and growth
continued at 30.degree. C. for 4 hr. E. coli were removed from the
phage by centrifugation, and 1 ml of these phage (approx. 1012
c.f.u.) were used in subsequent rounds of selection.
[0238] (c) Antibody Phage Selection Using Streptavidin-Coated
Paramagnetic Beads
[0239] The library was also selected using soluble biotinylated
antigen and streptavidin-coated paramagnetic beads (see FIG. 5).
gD-c-mpl was biotinylated using IMMUNOPURE NHS-biotin
(biotiny-N-hydroxy-succinimide, Pierce) according to manufacturer's
recommendations.
[0240] For the first round of panning, 10 .mu.l of the phage
library were blocked with 1 ml of MPBST (3% dry milk powder,
1.times.PBS, 0.2% TWEEN 20) for 1 hour on a rotating wheel at room
temperature. Biotinylated gD-c-mpl was then added to a final
concentration of 100 nM, and phage were allow to bind antigen for 1
hour at 37.degree. C. on a rotator. Meanwhile, 300 .mu.l of
DYNABEADS M-280, coated with streptavidin (DYNAL) were washed 3
times with 1 ml MPBST (using a DYNAL Magnetic Particle
Concentrator) and then blocked for 2 hr at 37.degree. C. with 1 ml
fresh MPBST on a rotator. The beads and were collected with the
MPC, resuspended in 50 .mu.l of MPBST, and added to the
phage-plus-antigen solution. Mixing continued on a wheel at room
temperature for 15 min. The DYNABEADS and attached phage were then
washed a total of 7 times: 3 times with 1 ml PBS-TWEEN, once with
MPBS, followed by 3 times with PBS. Phage were eluted from the
beads by incubating 5 min at room temperature with 300 .mu.l of 100
mM triethylamine. The phage-containing supernatant was removed and
neutralized with 150 .mu.l of 1M TRIS-HCl (pH 7.4). Neutralized
phage were used to infect mid-log TG1 host cells as described
above. Plating, induction and harvesting of phage were also as for
selection on tubes.
[0241] For the second and subsequent rounds of selection on
biotinylated gD-c-mpl, 1 ml of harvested phage (approximately
10.sup.12 cfu) were blocked with 200 .mu.l 10% dry milk,
6.times.PBS, 0.3% TWEEN 20. Antigen concentration was decreased at
each round of selection. In one series the concentrations were:
first round, 100 nM; second round, 10 nM; third round, 1 nM. A
second panning was performed using: first round 100 nM; second
round 100 nM; third round, 50 nM; fourth round, 10 nM; and fifth
round, 1 nM. Washing stringency was increased to two cycles of 7
washes for rounds 2, and three cycles for rounds 3 and beyond.
[0242] (d) ELISA Screening of Selected Clones
[0243] After each round of selection, individual
carbenicillin-resistant colonies were screened by ELISA to identify
those producing c-mpl-binding phage. Only those clones which were
positive in two or more assay formats were further studied. FIG. 6
illustrates the phage ELISA process.
[0244] Individual clones were inoculated into 2TY with 2% glucose
and 100 .mu.g/ml carbenicillin in 96-well tissue culture plates and
grown until turbid. Cultures were then infected at an m.o.i. of 10
with M12KO7 helper phage, and infected cells were transferred to
2YT media containing carbenicillin (100 .mu.g/ml) and kanamycin (50
.mu.g/ml) for growth overnight at 30.degree. C. with gentle
shaking.
[0245] NUNC MAXISORP microtiter plates were coated with 50 .mu.l
per well of gD-c-mpl, BSA, or gD-gp120, at 2 .mu.g/ml in 50 mM
carbonate buffer (pH 9.6), at 4.degree. C. overnight. After
removing antigen, plates were blocked with 3% dry milk in PBS
(MPBS) for 2 hours at room temperature.
[0246] Phage cultures were centrifuged and 100 .mu.l of
phage-containing supernatants were blocked with 20 .mu.l of
6.times.PBS/18% dry milk for 1 hour at room temperature. Block was
removed from titer plates and blocked phage added and allowed to
bind for 1 hour at room temperature. After washing, phage were
detected with a 1:5000: dilution of horseradish
peroxidase-conjugated anti-M13 antibody (Pharmacia) in MPBS
followed by 3',3',5',5'-tetramethylbenzidine (TMB). Reactions were
stopped by the addition of H.sub.2SO.sub.4 and readings taken by
subtracting the A.sub.405nm from the A.sub.450nm.
[0247] (e) Soluble scFv ELISA
[0248] Soluble scFv was induced in the bacterial supernatants of
clones by growth in 2YT containing carbenicillin (100 .mu.g/ml) and
IPTG (1 mM) ON at 30.degree. C. ELISA plates were either coated
with gD-c-mpl or, for capture ELISA, with anti-c-myc Mab 9E10.
Plates were blocked with 1.times. ELISA diluent (PBS supplemented
with 0.5% BSA, 0.05% Tween 20, pH 7.4), and soluble scFv was
blocked by adding 20 .mu.l of 6.times. ELISA dil to 100 .mu.l of
supernatant. After binding to antigen coated plates, soluble scFv
was detected by adding 50 .mu.l of 1 .mu.g/ml Mab 9E10 per well,
followed by horseradish peroxidase-conjugated goat anti-murine Ig,
and then TMB as described above. For capture ELISA, soluble scFv
was detected by addition of biotinylated c-mpl, followed by
streptavidin-peroxidase conjugate and then TMB as above.
[0249] The number of clones screened by ELISA from each round, and
the number of clones positive by phage ELISA are shown in Table
3.
3TABLE 3 Anti-c-mpl scFv antibodies from CAT library Clones
screened: 1534 Clones positive by ELISA: 361 Clones different by
BstNI and sequencing: 24 Clones that express protein well 17 clones
that are agonists by KIRA: 9 clones that are agonists by BaF3
proliferation assay: 6 clones that are agonists by Hu3
proliferation assay: 4
[0250] (f) DNA Fingerprinting: of Clones
[0251] The diversity of c-mpl-binding clones was determined by PCR
amplifying the scFv insert using primers pUC19R (5 AGC GGA TAA CAA
TTT CAC ACA GG 3) (SEQ. ID. NO: 54) which anneals upstream of the
leader sequence and fdtetseq (5 GTC GTC TTT CCA GAC GGT AGT 3)
(SEQ. ID. NO: 55) which anneals in the 5 end of gene III, followed
by digestion with the frequent-cutting restriction enzyme BstNI
(see
[0252] Typical patterns seen after analysis on a 3% agarose gel are
shown in FIG. 8A-C.
[0253] DNA Fingerprinting: Protocol
4 Mix A: dH20 67 .mu.l 10 x ampliTaq buffer 10 25 mM MgCl2 10 DMSO,
50% 2 forward primer 1 Mix B: 2.5 mM dNTPs 8 .mu.l AMPLITAQ 0.5
reverse primer 1.0
[0254] Place 90 .mu.l of Mix A in reaction tube
[0255] Inoculate with very small portion of E. coli colony using a
yellow tip
[0256] Heat in PCR block to 98.degree. C., 3 min. Remove to
ice.
[0257] Add 10 .mu.l Mix B
[0258] Cycle: 95.degree. C., 30 sec, 55.degree. C. 30 sec,
72.degree. C. 1 min 20 sec, for 25 cycles, in Perkin Elmer 2400
[0259] Remove 10 .mu.l ro run on a 1% agarose gel, test for a 1 kB
band
[0260] Make remaining mix to 1.times.BstNI reaction buffer
[0261] Add 5 units BstNI
[0262] 60.degree. C., 2 hours
[0263] Electrophorese samples on 3% METAPHORE agarose gel
[0264] (P) Sequencing of Clones
[0265] The nucleotide sequence of representative clones of each
fingerprint were obtained. Colonies were inoculated into 50 ml of
LB medium supplemented with 2% glucose and 100 .mu.g/ml
carbenicillin, and grown overnight at 30.degree. C. DNA was
isolated using Qiagen Tip-100s and the manufacturer's protocol and
cycle sequenced with fluorescent dideoxy chain terminators (Applied
Biosystems). Samples were run on an Applied Biosystems 373A
Automated DNA Sequencer and sequences analyzed using the program
"Sequencher" (Gene Codes Corporation). The VH and VL genes were
assigned to a germline segment using the antibody database,
V-BASE.
[0266] DNA sequence was obtained for 39 clones and resulted in 24
different c-mpl-binding scFvs.
[0267] (h) Purification of scFvs with (his).sub.6
[0268] For protein purification of soluble antibody, E. coli strain
33D3 was transformed with phagemid DNA. Five ml of 2YT with
carbenicillin and glucose was used to grow overnight cultures at
30.degree. C. 0.2 ml of these cultures were diluted into 200 ml of
the same media and grown to an OD.sub.600 of approximately 0.9. The
cells were pelleted and resuspended in 250 ml of 2YT containing
IPTG (1 mM) and carbenicillin (100 .mu.g/ml) and to induce
expression and grown for a further 5 hours at 30.degree. C. Cell
pellets were harvested and frozen at -20.degree. C.
[0269] The antibodies were purified by immobilized metal chelate
affinity chromatography (IMAC). Frozen pellets were resuspended in
10 ml of ice-cold shockate buffer (25 mM TRIS-HCl, 1 mM EDTA, 200
mM NaCl, 20% sucrose, 1 mM PMSF) by shaking on ice for 1 hr.
Imidazole was added to 20 mM, and cell debris removed by
centrifugation. The supernatants were adjusted to 1 mM MgCl.sub.2
and 50 mM phosphate buffer pH 7.5. Ni-NTA agarose resin from Qiagen
was used according to the manufacturers instructions. The resin was
equilibrated with 50 mM sodium phosphate buffer pH 7.5, 500 mM
NaCl, 20 mM imidazole, and the shockate added Binding occurred in
either a batch mode or on a gravity flow column. The resin was then
washed twice with 10 bed volumes of equilibration buffer, and twice
with buffer containing imidazole increased to 50 mM. Elution of
proteins was with 50 mM phosphate buffer pH 7.5, 500 mM NaCl and
250 mM imidazole. Excess salt and imidazole was removed on a PD-10
column (Pharmacia), and proteins were concentrated using a
Centricom 10 to a volume of about 1 ml.
[0270] Concentration was estimated spectrophotometrically assuming
an A280 nm of 1.0=0.6 mg/ml.
[0271] Portions of these protein preparations were submitted for
KIRA assay, c-mpl-Ba/F3 cell proliferation assay, and Hu3 cell
proliferation assay.
[0272] Plasmid DNA for scFv clones 12B5, 12D5, 12E10, 10D10, 10F6
and 5E5 (named pMpl.12B5.scFv.his; pMpl.12D5.scFv.his;
pMpl.12E10.scFv.his; pMpl.10D10.scFv.his; pMpl.10F6.scFv.his; and
pMpl.5E5.scFv.his, respectively) has been deposited with ATCC,
Manassas, Va., USA.
[0273] (a) Reformatting of Antibodies to scFv with tD tag, Fab',
Fab'2, and Full Length Molecules.
[0274] For improved expression of scFv, and for Fab', and Fab'2
forms of antibodies, some of the anti-c-mpl clones were cloned into
derivatives of the expression vector pAK19 (Carter et al. METHODS:
A companion to Methods in Enzymology. 3:183-192 (1991). Expression
is under the transcriptional control of the E. coli alkaline
phosphatase (phoA) promoter (Chang, et al Gene 44:121-125 (1986)
which is inducible by phosphate starvation Each peptide chain is
preceded by the E. coli enterotoxin II (stII) signal sequence
(Picken, et al.) to direct secretion to the periplasmic space of E.
coli. This vector also contains the human k.sub.1 C.sub.L (Palm et
al. Infect Immun. 42:269-275 (1983)) and the human IgG1 C.sub.H1
(Ellison, et al, Nucleic Acids Res. 10: 4071-4079 (1982)) constant
domains. The C.sub.H1 gene is immediately followed by the
bacteriophage 1 to transcriptional terminator (Scholtissek and
Grosse Nucleic Acids Res. 15: 3185 (1987)).
[0275] (i) Fab' and Fab'2 Construction
[0276] Construction of the Fab' and Fab'2 variants was facilitated
by insertion into pAK19 of unique restriction sites at the
junctions of the stlI and V.sub.L domain (Sfi 1), the V.sub.1 and
Ck domains (Rsr II), the stIl and V.sub.H domain (MluI), and the
V.sub.H and C.sub.H1 domains (Apa 1), using oligonucleotide
directed mutagenesis. In order to insure expression of monovalent
Fab' molecules, the free cysteine at the 3' end of the CH1 domain
was mutated to a threonine, these Fab' molecules thus end in the
amino acid sequence thr-ala-ala-pro, rather than thr-cys-ala-ala as
in pAKI9. This vector for the expression of Fab' molecules is named
pXCA730.
[0277] Since some of the antibodies derived from the library had
light chains which were derived from lambda rather than kappa light
chain families, the human .lambda.C.sub.L was subcloned from pB11.2
(Carter, P, Garrard, L., Henner, D. 1991. Methods: A Companion to
Methods in Enzymology. 3:183-192) into a derivative of pXCA730 to
give vector pXCA970.
[0278] For expression of the antibodies as Fab'2 molecules, a
vector was constructed which adds the human IgG1 hinge region onto
the C.sub.H1 domain of pXCA730. This is followed by the yeast GCN4
leucine zipper domain (Hu, et al. Science 250:1400-1403 (1990)) for
stability. These DNA fragments were constructed using synthesized
oligonucleotides and encode the amino acid sequence:
cys-pro-pro-cys-ala-pro-glu-leu-leu-gly-gly-arge-
t-lys-gln-Ieu-glu-asp-lys-val-glu-glu-leu-leu-ser-lys-asn-tyr-his-leu-glu--
asn-glu-val-ala-arg-leu-lys-lys-leu-val-gly-glu-arg (SEQ. ID. NO:
56). The resultant plasmid is named pXCA740.
[0279] The variable domains of the scFvs were amplified and
restriction sites added for subcloning into the vectors described
above by the PCR technique. Specific oligonucleotides were designed
for each V.sub.L or V.sub.H region as shown below.
[0280] 12B5,12D5, and 10D10 Light chain variable domains:
5 5 primer (SEQ. ID. NO: 57) GCT TCT GCG GCC ACA CAG GCC TAC GCT
GAC ATC GTG ATG ACC C 3 primer (SEQ. ID. NO: 58) ATG ATG ATG TGC
CAC GGT CCG TTT GAT CTC CAG TTC GGT C
[0281] 12E10 Light chain variable domain:
6 5 primer (SEQ. ID. NO: 59) GCT TCT GCG GCC ACA CAG GCC TAC GCT
TCC TAT GTG CTG ACT C 3 primer (SEQ. ID. NO: 60) CCT TCT CTC TTT
AGG TTG GCC AAG GAC GGT CAG CTT GGT C
[0282] 10F6 Light chain variable domain
7 5 primer (SEQ. ID. NO: 61) GCT TCT GCG GCC ACA CAG GCC TAC GCT
CAG TCT GTG CTG ACT G 3 primer (SEQ. ID. NO: 60) CCT TCT CTC TTT
AGG TTG GCC AAG GAC GGT CAG CTT GGT C
[0283] 12B5 Heavy chain variable domain
8 5 primer (SEQ. ID. NO: 62) CAT TCT ACA AAC GCG TAC GCT CAG GTG
CAG CTG GTG CAG 3 primer (SEQ. ID. NO: GTA AAT GTA TGG GCC CTT GGT
GGA GGA GGC ACT CGA GAC GGT GAC
[0284] 12D5 Heavy chain variable domain
9 5 primer (SEQ. ID. NO: 64) CAT TCT ACA AAC GCG TAC GCT CAG GTG
CAG CTG GTG GAG 3 primer (SEQ. ID. NO: GTA AAT GTA TGG GCC CTT GGT
GGA GGA GGG ACT CGA GAC GGT GAC
[0285] 10D10 Heavy chain variable domain
10 5 primer (SEQ. ID. NO: 65) CAT TCT ACA AAG GCG TAG GCT GAC GTG
CAG CTG GTG CAG 3 primer (SEQ. ID. NO: 66) GTA AAT GTA TGG GCC CTT
GGT GGC GGC TGA GGA GAC GGT GAC
[0286] 12E10 Heavy chain variable domain
11 5 primer (SEQ. ID. NO: 67) CAT TCT ACA AAC GCG TAC GCT CAG GTG
CAG CTG CAG CAG 3 primer (SEQ. ID. NO: GTA AAT GTA TGG GCC CTT GGT
GGA GGA GGC ACT CGA GAG GGT GAG
[0287] 63).
[0288] 10F6 Heavy chain variable domain
12 5 primer (SEQ. ID. NO: 68) CAT TCT ACA AAC GCG TAC GCT CAG GTG
GAG CTG CAG GAG 3 primer (SEQ. ID. NO: 69) GTA AAT GTA TGG GCC CTT
GGT GGA GGC TGA AGA GAC GGT AAC
[0289] PCR reactions were carried out using 100 ng of plasmid DNA
containing the scFv, 0.5 .mu.M of the appropriate 5 and 3 primer,
200 .mu.M each dNTP, 10 mM KCl, 6 mM (NH.sub.4).sub.2SO.sub.4, 20
mM TRIS-HCl, pH 8.0, 2 mM MgCl.sub.2, 1% Triton X-100, 100 .mu.M
BSA and 2.5 units of Pfu DNA polymerase (Stratagene). Amplification
was for 30 cycles of: 30 sec at 95.degree. C., 30 sec at 55.degree.
C., 30 sec at 72.degree. C. After digestion with the appropriate
restriction enzymes, the reaction products were separated by
agarose gel electrophoresis and the approximately 350 bp band was
isolated using a Gene Clean II kit (BIO 101, Vista, Calif.). The
fragments for the light chain variable regions were ligated into
the vectors previously digested with Sfi I and Rsr II for the kappa
isotypes, or Sfi I and Msc I for the lambda isotypes, and
transformed into E. coli DH5a. Desired recombinants were identified
using restriction enzyme analysis and sequenced to confirm the
presence of the desired fragments. The heavy chain variable domains
were then cloned similarly into the plasmids containing the light
chains using the restriction enzymes Mlu I and Apa I, and the final
constructions were again checked by DNA sequencing.
[0290] (k) Construction of scFv with gD Taps.
[0291] For increased and regulated expression in high density
fermentation tanks, the Sfi I to Not I fragments J5 of the scFv
forms of p12B5, p12D5, p10F6, and p12E10 were subcloned into a
derivative of pAK19 containing the phoA promoter and stII signal
sequence rather than the lacZ promoter and hybrid signal sequence
of the original library. For ease of purification, a DNA fragment
coding for 12 amino acids
(met-ala-asp-pro-asn-arg-phe-arg-gly-lys-asp-leu) (SEQ. ID. NO: 70)
derived from herpes simplex virus type I glycoprotein D (Lasky and
Dowbenko DNA (N.Y.) 3:23-29 (1984.)) was synthesized and inserted
at the 3 end of the V.sub.L domain in place of the (his).sub.6 and
c-myc epitope originally present in the C.A.T. library clones.
[0292] (l) Expression in E. coli
[0293] Plasmids containing genes for scFv-gD, Fab' or Fab'2
molecules were expressed in E. coli strain 33B6 (W3110 DfhuA
phoADE15 deoC2 i/vG2096(val.sup.R) degP41 (DPstI-Kan.sup.R)
D(argF-/ac) 169 IN(rrnD-rrnE)1) grown for approximately 40 hr at
30.degree. C. in an aerated 10-liter fermentor as described
previously (Carter et al Bio/Technology 10:163-167(1992.)).
Example 3
Cloning and Expression of Full Length Human Antibody Derivatives of
12B5, 12D5, and 12E10
[0294] For expression of full length antibodies in mammalian cells,
the heavy chain variable domains were subcloned from the Fab
constructs into a derivative of expression vector pRK (Suva et al.,
Science 237:893-896 (1987)) which contains the human IgG1 CH1, CH2,
and CH3 domains and a human antibody signal sequence (Carter et
al., Proc. Natl. Acad. Sci. USA. 89:4285-4289 (1992)). The light
chain was cloned into a separate pRK plasmid. The light and heavy
chain expression vectors were cotransfected into
adenovirus-transformed human embryonic kidney cell line 293 by a
high-efficiency procedure (Gorman et al, DNA Protein Eng. Technol.
2:3-10 (1990)). Harvested conditioned media was shown to contain
anti-mpl antibody by ELISA.
[0295] For production of a more stable cell line and high-level
antibody production, the light and heavy chains were moved into the
SVI.DI expression vector previously described (Lucas et al.,
Nucleic Acids Res. 24: 1774-1779 (1996). This vector contains the
mouse DHFR cDNA in the intron of the expression vector pRK and
allows for amplification of expression by selection in
methotrexate. The light chain is cloned into the same plasmid with
expression driven by a second SV40 promoter/enhancer. The plasmid
was linearized and transfected into CHO cells using lipofectamine
(Gibco-BRL) following manufacturer's instructions. Seven to ten
days after transfer to selective medium, clones were isolated into
96 well plates for later study, or pooled and expanded for culture
in roller bottles.
[0296] Conditioned media for purification of the antibodies was
generated in roller bottles. Cells were seeded into the roller
bottles at an initial cell density of 2.times.10.sup.7 cells in 200
ml rich medium (DMEM: Ham's F12 (1:1) supplemented with 5% fetal
bovine serum. At approximately 80% confluency, the media was
replaced with serum-free PS-24 production medium supplemented with
insulin (10 .mu.g/ml), transferrin (10 .mu.g/ml), trace elements
and lipid alcohol. Conditioned media was harvested after 10
days.
Example 4
Purification Of Agonist Antibodies
[0297] (a) Purification of scFv with gD Tag
[0298] Frozen cell paste was resuspended at 1 gm/ml TE (25 mM TRIS,
1 mM EDTA, pH 7.4) and gently agitated 18 hr on ice. Cell debris
was removed by centrifugation at 10,000.times.g for 30 min. The
supernatant was loaded onto an affinity column (2.5.times.9.0 cm)
consisting of an anti-gD monoclonal antibody 5B6 (Paborsky, L. R.
et al., Protein Eng. 3: 547-553 (1990)) coupled to CNBr SEPHAROSE
which had been equilibrated with PBS. The column was washed 18 hr
with PBS, and then washed with PBS containing 1 M NaCl until the
absorbance of the column effluent was equivalent to baseline. All
steps were done at 4.degree. C. at a linear flow rate of 25 cm/hr.
Elution was performed with 0.1 M acetic acid, 0.5 M NaCl, pH 2.9.
Column fractions were monitored by absorbance at 280 nm and peak
fractions pooled, neutralized with 1.0 M TRIS, pH 8.0, dialyzed
against PBS, and sterile filtered. The resultant protein
preparations were analyzed by non-reducing SDS-PAGE
[0299] (b) Purification of Fab' Molecules
[0300] For purification of Fab' molecules, 5 g of frozen cell paste
was resuspended in 5 ml of TE (25 mM TRIS, 1 mM EDTA, pH 7.4) and
gently stirred 18 hr on ice. The pH of the shockate was adjusted to
5.6 with 2 M HCl and the precipitate and cell debris removed by
centrifugation at 10,000.times.g for 30 min. The supernatant was
loaded onto a 1 ml BAKERBOND ABx column (0.5.times.5.0 cm) (J. T.
Baker, Phillipsburg, N.J.) pre-equilibrated with 20 mM MES, pH 5.5.
After washing with 20 mM MES to baseline, the Fab' was eluted using
a 10 ml linear gradient from 0 to 100% of 20 mM NaOAc, 0.5 M
(NH.sub.4).sub.2SO.sub.4, pH 7.2, with a flow rate of 153 cm/hr.
Fractions containing Fab' were pooled, and buffer exchanged into
PBS.
[0301] (c) Purification of Fab'2 Molecules
[0302] Frozen cell paste (100 gm) was thawed into 10 volumes of 25
mM TRIS, 5 mM EDTA, 1 mM NaN3, pH 7.4 and disrupted by three
passages through a microfluidizer (TECH-MAR). PMSF was added to 1
mM and the cell debris removed by centrifugation at 10,000.times.g
for 30 min. The supernatant was filtered sequentially through a
0.45 .mu.m, and a 0.2 .mu.m SUPORCAP filter (Gelman), and loaded
onto a 50 ml SEPHAROSE-fast-flow Protein-G column (Pharmacia)
pre-equilibrated with PBS. After washing to baseline with PBS,
Fab'2 was eluted with 0.1% M glycine ethyl ester, pH 2.3, into
tubes with contained {fraction (1/10)} volume of 1 M TRIS, pH 8.0.
Fractions containing Fab'2 were pooled and concentrated by
Ultrasette with a 30 kilodalton molecular weight cut off, and
buffer exchanged into 20 mM NaOAc, 0.01% octylglucoside, pH 5.5.
This material was loaded onto a 30 ml S-SEPHAROSE column
(Pharmacia) pre-equilibrated with 20 mM NaOAc, washed to baseline
with 20 mM NaOAc, pH 5.5, and eluted with a linear gradient of 0-1
M NaCl in 25 mM NaOAc over 10 column volumes Fractions containing
Fab'2 were pooled and buffer exchanged to PBS.
[0303] (d) Purification of Full Length Antibodies from Transfected
CHO Cell Supernatants.
[0304] Conditioned medium harvested from roller bottles was loaded
onto a 5 ml Protein-A SEPHAROSE column (1.0.times.50 cm)
pre-equilibrated with PBS, washed with PBS, and then washed to
baseline with PBS containing I. M NaCl. Antibody was eluted with
0.1 M HOAc, 0.5 N NaCl, pH 2.9, neutralized with 1 M TRIS, and
buffer exchanged to PBS.
[0305] A summary of agonist antibody activities for several
antibodies and fragments thereof is shown in Table 4 below
13TABLE 4 Summary of Mpl Agonist Antibody Activities Hu3 Prolifer-
Hu3 Mpl/TPO MK Anti- ation KIRA Binding ELISA Platelets As- body
(ED50) (ED50) (IC50) (IC50) (IC50) say 12B5 scFv 20 pM 1 nM 10 nM
17 nM 100 nM ++ Fab >1 .mu.M 3 nM 900 nM none >1 .mu.M -
Fab'2 5 pM 1 nM 5 nM 1 nM 300 nM + IgG 30 pM 400 pM 10 nM 152 pM
300 nM - 12E10 scFv 5 pM 60 pM 5 nM 1.6 nM 5 nM Fab >1 .mu.M
>1 .mu.M 500 nM 180 nM >1 .mu.M Fab'2 >1 .mu.M 160 pM 10
nM 640 nM 500 nM IgG >1 .mu.M 480 pM 50 nM 450 pM 500 nM 12D5
scFv 1.2 nM 280 pM 10 nM 24 nM >1 .mu.M Fab >1 .mu.M 4 nM 500
nM 1 .mu.M >1 .mu.M Fab'2 4.8 pM 600 pM 4 nM 1 nM 100 nM + IgG
>1 .mu.M 3 nM 10 nM 450 pM 500 nM
Example 5
[0306] In another embodiment, the invention provides a method of
selecting an antibody which binds to and dimerizes a receptor
protein. In this method, a library of antibodies is panned using a
receptor protein having two protein subunits as the binding target.
The library is panned as described above for mpl agonist
antibodies. Preferably, the antibodies are human and more
preferably monoclonal. The library is conveniently a library of
single chain antibodies, preferably displayed on the surface of
phage. The display of proteins, including antibodies, on the
surface of phage is well known in the art as discussed above and
these known methods may be used in this invention. Antibody
libraries are also commercially available, for example, from
Cambridge Antibody Technologies (CAT), Cambridge, UK. Preferably,
the antibody selected by the method of the invention activates the
receptor by dimerizing the receptor and thereby achieves an
effector result similar to the effector result generated when the
natural endogenous ligand for the receptor binds the receptor.
[0307] The method of the invention can be used to find agonist
antibodies to any receptor having two components which is known
and/or can be cloned. It is not necessary to know the primary,
secondary or tertiary structure of the receptor protein, although
this information is useful for cloning, etc., since the method of
the invention allows selection of antibodies which will bind any
displayed receptor which is activated by dimerization. Many known
receptor proteins are activated by dimerization and any of these
known receptors may be used in the invention. Suitable receptors
include tyrosine kinase receptors and hematopoietic receptors that
lack kinase activity.
[0308] Activation of a receptor such as a tyrosine kinase receptor
by a scFv is an unexpected result. Current understanding of
receptor activation argues that for many classes of receptors,
including tyrosine kinase receptors and hematopoietic cytokine
receptors that lack intrinsic tyrosine kinase activity (but
associate with intracellular kinases), it is a dimerization event
mediated by a ligand that is the key event in receptor activation.
This view is supported by crystal structures of receptor ligand
complexes as well as the demonstrated agonist ability of certain
monoclonal antibodies (but not the Fab' fragments of these
antibodies). A single chain antibody would not, therefore, be
expected to be able to cause receptor dimerization and
activation.
[0309] MuSK is a recently identified tyrosine kinase localized to
the postsynaptic surface of the neuromuscular junction. (Valenzuela
et. al. 1995. Neuron 15 573-584.) Mice made deficient in MuSK fail
to form neuromuscular junctions (Dechiara et. al. 1996. Cell 85
501-512.), a phenotype highly similar to that observed in mice
lacking the nerve derived signaling molecule agrin (Gautam et. al.,
1996, Cell 85 525-535). The likely involvement of MuSK in agrin
signaling is strengthened by the observations that agrin induces
the rapid tyrosine phosphorylation of MuSK and that labeled agrin
can be chemically crosslinked to MuSK (Glass et. al., 1996, Cell 85
513-523.).
[0310] Formation of the neuromuscular junction is achieved through
a process that includes the differentiation of membrane on the
muscle fiber proximal to the neuron terminus and changes in gene
expression within the nuclei proximal to this junction (reviewed by
Bowe et. al., 1995, Annu-Rev-Neurosci. 18 443-462 and Kleiman et.
al., 1996, Cell 85 461-464.). A striking feature of this complex
process is the redistribution and concentration of AChRs within the
myotube membrane. Agrin is able to the induce this clustering of
AChRs as well as changes in the extracellular matrix and
cytoskeletal components of the synaptic apparatus (Bowe et. al.,
supra; Godfrey et. al., 1984, J. Cell Biol. 99 615-627; Nitkin et.
al., 1987, J. Cell Biol. 105 2471-2478). Agrin is a secreted
protein with a core molecular weight of 200 kDa that contains
several copies of EGF repeats, laminin-like globular domains and
sequences that resemble protease inhibitors. It is released by
motor neuron terminals and maintained within the basil lamina of
the synaptic cleft. While agrin apparently does not to bind MuSK
with high affinity (Glass et. al., supra), it has been reported to
interact with other molecules present at the neuromuscular
junction, most notably alpha-dystroglycan (O'Toole et. al., 1996,
Natl. Acad. Sci. USA 93 7369-7374) thereby complicating the
analysis of MuSK's role in the signaling events initiated by
agrin.
[0311] Antigen specific scFv, identified by panning a diverse
library of scFv expressed, for example, on M13 phage provide a
source of molecules capable of mediating specific therapeutic
activities, and offer a rapid new approach to study the function of
novel or recently identified molecules such as MuSK. scFv are
identified below that mediate receptor activation and that direct
MuSK activation induces changes in AChR distribution and tyrosine
phosphorylation similar to that observed with agrin.
[0312] The induction of AChR clustering and tyrosine
phosphorylation by scFv antibodies provides direct evidence to
support conclusions drawn from studies of knockout mice deficient
in MuSK indicating this recently discovered tyrosine kinase acts to
induce key events in the formation of the neuromuscular junction.
As a potential signal transducer of agrin, it is noteworthy that
MuSK does not display high affinity binding to agrin, leading to
speculation that there must be an additional agrin binding
component(s) involved in mediating the agrin signal. The molecular
nature of this component is unknown. It is interesting that it is
possible to induce the receptor clustering, the hallmark activity
of agrin, with an agent directed specifically to MuSK.
[0313] The marked upregulation of MuSK expression in muscle
following denervation or muscle immobilization as well as the
chromosomal localization of MuSK within a region associated with
fukiyama muscular dystrophy point to an important role for this
molecule in regulation of the neuromuscular junction (Valenzuela et
al., supra) and indicates the possibility that therapeutic benefit
is possible through the controlled regulation of MuSK activity. As
agrin is expressed not only at the neuromuscular junction, but in a
wide variety of peripheral and central neurons (Bowe et. al.,
supra; Rupp et al., 1991, Neuron 6 811-823; Tsim et. al., 1992,
Neuron 8 677-689) it may not be an optimal candidate molecule
through which to manipulate MuSK function as exogenously introduced
agrin derivatives might elicit consequences not restricted to the
neuromuscular junction. Thus, in comparison, the ability to obtain
direct activation of MuSK through scFv offers an attractive
alternative. Each of the scFv that were tested displayed affinity
for MuSK in the nM range demonstrating the utility of phage
displayed scFv libraries as a rich source of high affinity and
highly specific molecules.
[0314] The antibodies of the invention are, therefore, useful in
assaying the upregulation of MuSK receptors in sample tissues to
determine the degree of neuromuscular damage associated with this
upregulation. The antibodies are also useful for activating the
MuSK receptor and inducing AChR clustering at neuromuscular
junctions as a direct result of the agonist properties of these
antibodies. Administration of the antibodies to a person suffering
from denervation or muscle immobilization, e.g. muscular dystrophy,
provides a method of improving the function of the neuromuscular
junctions in these people.
[0315] To prepare scFv having agonist activity, antibodies were
selected which induce a proliferative response in a factor
dependent cell line through a chimeric MuSK-Mpl receptor comprised
of the extracellular domain of MuSK and the intracellular domain of
the hematopoietic cytokine receptor c-Mpl (the receptor for
thrombopoietin, TPO). Activation of c-Mpl is believed to require
homodimerization, as is the case for the growth hormone receptor,
the erythropoietin receptor and other related receptors of this
class (Carter et al., 1996, Annu-Rev-Physiol 58 187-207; Gurney et
al., 1995, Proc. Natl. Acad. Sci. USA 92 5292-5296). Ba/F3 cells
expressing MuSK-Mpl were starved of IL-3 and exposed to a range of
concentrations of each scFv expressed as soluble protein.
Surprisingly, 4 of the 21 scFv were able to induce a robust
proliferative response in the MuSK-Mpl expressing cells (FIG. 11).
This activity was observed at nM concentrations of scFv. The scFv
were without effect on the parental, untransfected Ba/F3. Agonist
activity was also present among those IgG that were derived from
agonist scFv but was not noted among IgG derived from non-agonist
scFv. Soluble agrin c-terminal domain (c-agrin) was without effect
supporting previous observations that agrin does not bind MuSK
directly. The c-terminal domain of agrin is known to contain the
AChR clustering activity of agrin and is essential for
neuromuscular junction formation (Ruegg et al., 1992, Neuron 8
691-699; Tsim et. al., supra). The EC.sub.50 for the ability to
induce proliferation was 5 nM for the most active agonist clone
when expressed as either scFv or IgG. The affinity of these scFv
and IgG for MuSK was determined using BIAcore.TM. analysis. The
agonist scFv and several non-agonist scFv each displayed affinity
for MuSK within the range of 5-25 nM. In contrast, the affinities
of the IgG for MuSK were 10-30 pM. See Table 5 below.
14TABLE 5 clone # Agonist k.sub.d k.sub.a Affinity musk #2-scFv +
3.34 .times. 10.sup.-3 8.78 .times. 10.sup.5 3.8 nM musk #3-scFv -
2.39 .times. 10.sup.-3 1.05 .times. 10.sup.5 23 nM musk #4-scFv +
1.57 .times. 10.sup.-3 1.84 .times. 10.sup.5 8.5 nM musk #5-scFv -
2.49 .times. 10.sup.-3 5.29 .times. 10.sup.5 4.7 nM musk #6-scFv -
4.95 .times. 10.sup.-3 1.05 .times. 10.sup.5 4.7 nM musk #13-scFv +
2.32 .times. 10.sup.-3 4.53 .times. 10.sup.5 5.1 nM musk #22-scFv +
6.09 .times. 10.sup.-3 1.27 .times. 10.sup.5 4.8 nM musk #13-IgG +
1.01 .times. 10.sup.-5 8.05 .times. 10.sup.5 12.5 pM musk #22-IgG +
4.86 .times. 10.sup.-5 1.65 .times. 10.sup.6 29.5 pM
[0316] To probe this agonist activity further, scFv were examined
for the ability to induce tyrosine phosphorylation of full length
MuSK tyrosine kinase. The murine myoblastic cell line C2C12 was
cultured under conditions that promote myotube differentiation and
subsequently exposed to scFv, IgG or c-agrin. In correspondence
with previous data (Glass et. al., supra), c-agrin was able to
induce MuSK tyrosine phosphorylation. The agonist scFv and IgG were
also found to rapidly induce tyrosine phosphorylation of MuSK as
determined by western blot analysis with anti-phosphotyrosine
antibody whereas other scFv and non-agonist anti-MuSK IgG were
without effect.
[0317] The ability of the scFv MuSK agonists to induce AChR
clustering in cultured C2C12 myotubes was examined. Following
stimulation, the cells were fixed and the distribution of cell
surface AChR was revealed with rhodamine labeled bungarotoxin. In
undifferentiated myoblasts, AChR were dispersed and unfocused in
the presence of c-agrin, scFv, or IgG. In contrast, upon myotube
differentiation, c-agrin and agonist scFv and IgG induced marked
aggregation of AChR into large and intensely stained clusters.
Non-agonist scFv and non-agonist IgG directed against MuSK or an
irrelevant antigen were without effect. An additional consequence
of agrin action, tyrosine phosphorylation of subunits of the AChR
was also examined utilizing an antisera that recognizes the and
chains of the receptor. Tyrosine phosphorylation levels of both the
and chains were markedly induced by c-agrin as well as the agonist
scFv and agonist IgG but were unaffected by control scFv and
IgG.
[0318] Variants of the MuSK agonist antibodies of the invention may
be prepared as described above for thrombopoietic antibodies.
[0319] Construction of expression vectors. Coding sequence for
murine MuSK was obtained by PCR amplification. MuSK-Fc was prepared
by fusion of the extracellular domain of MuSK (a.a. 1492) in frame
with the Fc region of human IgG1 in the eukaryotic expression
vector pRK5tkNEO. MuSK-Fc was transiently expressed in 293 cells
and purified over a protein G column. A chimeric receptor,
MuSK-Mpl, comprised of the extracellular domain of MuSK (amino
acids 1492) and the transmembrane and intracellular domain of the
human c-Mpl receptor (amino acids 491-635) was prepared by
sequential PCR and cloned into pRK5tkNEO. Stable cell lines
expressing the chimeric receptor were obtained by electroporation
(5 million cells, 250 volts, 960 .mu.F) of linearized vector (20
.mu.g) into Ba/F3 cells followed by selection for neomycin
resistance with 2 mg/ml G418. Full length MuSK in pRK5tkNEO was
transfected into 293 cells and stable transformants were obtained
following two weeks of G418 selection (400 .mu.g/ml). The sequence
of the DNA constructs were confirmed by DNA sequencing. Expression
of MuSK was assessed by flow cytometry analysis as described below.
Ba/F3 cells were maintained in RPMI 1640 media supplemented with
10% fetal calf serum and 5% conditioned media from WEHI-3B cells as
source of IL-3. C-agrin (amino acids 1137-1949 of the rat agrin (Ag
+8 active splice form (Ferns et al., 1993, Neuron 11 491-502.)) was
expressed by transient transfection from 293 cells in serum free
media with an expression vector, pRK-gD-c-Agrin, as a fusion
protein with the gD signal sequence and epitope tag and a genenase
cleavage site (MGGAAARLGAVILFVV
IVGLHGVRGKYALADASLKMADPNRFRGKDLPVLDQLLEGGAAHYALLPG) (SEQ ID NO. 71)
fused to the N-terminus.
[0320] Isolation of scFv and IgG MuSK-Fc immunoadhesin was coated
on Maxisorp tubes (Nunc) at 10 .mu.g/ml. A library of human scFv
(Cambridge Antibody Technology, England) was panned through two
rounds of enrichment essentially as described (Griffiths et al,
199, EMBO-J 12 725-734). The specificity of individual clones was
assessed first by elisa (Griffiths et al, supra) using MuSK-Fc and
a control immunoadhesin (CD4-Fc). Positive clones were screened by
PCR and "fingerprinted" by BstNI digestion (Clackson et al., 1991,
Nature 352 642-648.). Examples of clones with unique patterns were
sequenced and subjected to FACS analysis with cells expressing or
not expressing MuSK. For FACS analysis, cells (10.sup.5) were
incubated for 60 minutes at 4 C in 200 .mu.l 2% FBS/PBS (fetal
bovine serum/phosphate buffered saline) with 10.sup.10 phage that
were first blocked by incubation in 30 .mu.l 10% FBS/PBS. Cells
were then washed with 2% FBS/PBS, stained with anti-M13 antibody
(Pharmacia, Piscataway N.J.) and R-phycoerytherin-conjugated donkey
anti-sheep antibody (Jackson Immunoresearch, West Grove Pa.), and
analyzed by FACS analysis. ScFv were expressed in bacteria as
epitope tagged proteins containing a c-myc tag sequence recognized
by monoclonal antibody 9E10 (Griffiths et al, supra) and a
polyhistidine tail (his.sub.6) and were purified over Ni-NTA column
with imidazole elution as recommended by manufacturer (Qiagen). For
expression of clones as IgG the sequences encoding the V.sub.H and
V.sub.L regions of the scFv were introduced by PCR into mammalian
expression vector pIgG-kappa which was designed to enable the
expression of fully human light and heavy chains of kappa type IgG.
Expression vectors for the individual clones were transfected into
CHO cells and IgG were harvested from conditioned serum free media
and purified over a protein A column.
[0321] Proliferation assays. Cells were cultured in the absence of
IL-3 for twenty-two hours (in RPMI supplemented with 10% FBS).
Cells were then washed twice with RPMI and plated in 96 well dishes
at 50,000 cells per well in 0.2 ml of 7.5% FBS RPMI supplemented
with the indicated concentrations of scFv or IgG. Each
concentration was tested in duplicate. After an incubation of
sixteen hours, 1 .mu.Ci of [.sup.3H]-thymidine was added per well
and incubation was continued for an additional six hours.
Incorporation of [3H]-thymidine was measured with a Top Count
Counter (Packard Instruments, CA).
[0322] AChR clustering assay. C2C12 were maintained in 10% FBS in
high glucose DMEM at subfluency. For AChR clustering assays C2C12
were seeded on glass slides coated with fibronectin and poly-lysine
and myotube differentiation was induced by 48 hour incubation in 2%
horse serum high glucose DMEM. scFv or c-agrin were added to the
culture medium and incubated overnight (16 hours). Cells were then
washed with PBS and fixed in 4% paraformaldehyde. Rhodamine
conjugated bungarotoxin (Molecular Probes, Eugene Oreg.) was used
to reveal the localization of AChRs as described (Ferns et al,
supra).
[0323] Binding affinity analysis. Protein interaction analysis
using BIAcore.TM. instruments was performed as described (Mark et
al, 1996, J. Biol. Chem. 271 9785-9789). Briefly, research grade
CM5 sensor chips were activated by injection of 20 .mu.l of 1:1
N-Ethyl-N'-(3-dimethylaminoprop- yl)carboiimide hydrochloride and
N-hydroxysuccinimide at 5 .mu.l/min flow rate. 20 .mu.l of MuSK-Fc
at 20 .mu.g/min in 10 mM sodium acetate, pH 5.0 was injected over
the sensor chip, followed by 30 .mu.l of ethanolamine. scFv or IgG
were purified and concentrations determined by Pearce BCA kit.
Thirty .mu.l protein samples in PBS with 0.05% Tween 20 were
injected at a flow rate of 10 .mu.l min by the Kinject method.
Proteins were allowed to dissociate for 20 min in a flow of PBS
with 0.05% Tween 20. Sensorgrams were analyzed with BIAevaluation
2.1 software from Pharmacia Biosensor AB. Apparent dissociation
rate constants (k.sub.d) and association rate constants (k.sub.a)
were obtained by evaluating the sensorgram with A+B=AB type I
fitting. Equilibrium dissociation constant K.sub.d was calculated
as k.sub.d/k.sub.a.
[0324] Immunoprecipitation and Western Blot Analysis. C2C12 were
maintained in 10% FBS high glucose DMEM and induced to
differentiate by 72 hour incubation in 2% horse serum. Cells were
then stimulated by addition of c-agrin, scFv or IgG for the time
indicated in the figures. ScFv and IgG were used at 50 nM. The
c-agrin containing conditioned media was used at level that
provided maximal tyrosine phosphorylation. Cell extracts were
prepared as described (Gurney et al, supra). Extracts were
incubated for 60 minutes at 4.degree. C. with 30 .mu.l agarose
conjugated antiphosphotyrosine monoclonal antibody 4G10 (UBI inc.,
Lake Placid N.Y.) or 1 .mu.g of anti-MuSK IgG #13 followed by 30
.mu.l protein A sepharose beads. Western blot analysis with
antiphosphotyrosine antibody 4G10 or anti AChR antibody (Affinity
Bioreagents, Golden Colo.) was performed as recommended by the
manufacturer and revealed with HRP conjugated secondary antibody
and ECL (Amersham).
[0325] MuSK scFv are readily observed as dimers when resolved by
nondenaturing gel electrophoresis. Additionally, the abundance of
dimeric species may be significantly altered in the local context
of scFv bound to receptor on the cell surface. Alternatively,
screening an scFv phage library with a divalent antigen, in this
case MuSK-Fc, allows direct selection of scFv that bind to and
facilitate the formation of a receptor dimer.
[0326] While the invention has necessarily been described in
conjunction with preferred embodiments and specific working
examples, one of ordinary skill, after reading the foregoing
specification, will be able to effect various changes,
substitutions of equivalents, and alterations to the subject matter
set forth herein, without departing from the spirit and scope
thereof. Hence, the invention can be practiced in ways other than
those specifically described herein. It is therefore intended that
the protection granted by letters patent hereon be limited only by
the appended claims and equivalents thereof.
[0327] All references cited herein are hereby expressly
incorporated by reference.
[0328] Deposit of Material
[0329] The following materials have been deposited with the
American Type Culture Collection, 10801 University Boulecard,
Manassas, Va., USA (ATCC):
15 Material ATCC Dep. No. Deposit Date pMpl.12B5.scFv.his Aug. 18,
1998 pMpl.12D5.scFv.his Aug. 18, 1998 pMpl.12E10.scFv.his Aug. 18,
1998 pMpl.10D10.scFv.his Aug. 18, 1998 pMpl.10F6.scFv.his Aug. 18,
1998 pMpl.5E5.scFv.his Aug. 18, 1998
[0330] This deposit was made under the provisions of the Budapest
Treaty on the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure and the
Regulations thereunder (Budapest Treaty). This assures maintenance
of a viable culture of the deposit for 30 years from the date of
deposit. The deposit will be made available by ATCC under the terms
of the Budapest Treaty, and subject to an agreement between
Genentech, Inc. and ATCC, which assures permanent and unrestricted
availability of the progeny of the culture of the deposit to the
public upon issuance of the pertinent U.S. patent or upon laying
open to the public of any U.S. or foreign patent application,
whichever comes first, and assures availability of the progeny to
one determined by the U.S. Commissioner of Patents and Trademarks
to be entitled thereto according to 35 USC .sctn.122 and the
Commissioner's rules pursuant thereto (including 37 CFR .sctn.1.14
with particular reference to 8860G 638).
[0331] The assignee of the present application has agreed that if a
culture of the materials on deposit should die or be lost or
destroyed when cultivated under suitable conditions, the materials
will be promptly replaced on notification with another of the same.
Availability of the deposited material is not to be construed as a
license to practice the invention in contravention of the rights
granted under the authority of any government in accordance with
its patent laws.
[0332] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The present invention is not to be limited in scope by
the construct deposited, since the deposited embodiment is intended
as a single illustration of certain aspects of the invention and
any constructs that are functionally equivalent are within the
scope of this invention. The deposit of material herein does not
constitute an admission that the written description herein
contained is inadequate to enable the practice of any aspect of the
invention, including the best mode thereof, nor is it to be
construed as limiting the scope of the claims to the specific
illustrations that it represents. Indeed, various modifications of
the invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
Sequence CWU 1
1
77 1 15 DNA Homo sapiens 1 acc tct tgg atc ggc 15 Thr Ser Trp Ile
Gly 1 5 2 5 PRT Homo sapiens 2 Thr Ser Trp Ile Gly 1 5 3 66 DNA
Homo sapiens 3 atc atg tat cct ggg aac tct gat acc aga cac aac 36
Ile Met Tyr Pro Gly Asn Ser Asp Thr Arg His Asn 1 5 10 ccg tcc ttc
gaa gac cag gtc acc atg tca 66 Pro Ser Phe Glu Asp Gln Val Thr Met
Ser 15 20 22 4 22 PRT Homo sapiens 4 Ile Met Tyr Pro Gly Asn Ser
Asp Thr Arg His Asn Pro Ser Phe 1 5 10 15 Glu Asp Gln Val Thr Met
Ser 20 5 30 DNA Homo sapiens 5 gct ggg gtc gcg ggc ggt gct ttt gat
ctc 30 Ala Gly Val Ala Gly Gly Ala Phe Asp Leu 1 5 10 6 10 PRT Homo
sapiens 6 Ala Gly Val Ala Gly Gly Ala Phe Asp Leu 1 5 10 7 42 DNA
Homo sapiens 7 act gga acc agc agt ggc gtt ggt ggt tat aac tat 36
Thr Gly Thr Ser Ser Gly Val Gly Gly Tyr Asn Tyr 1 5 10 gtc tcc 42
Val Ser 14 8 14 PRT Homo sapiens 8 Thr Gly Thr Ser Ser Gly Val Gly
Gly Tyr Asn Tyr Val Ser 1 5 10 9 21 DNA Homo sapiens 9 ggt aac agc
aat cgg ccc tca 21 Gly Asn Ser Asn Arg Pro Ser 1 5 7 10 7 PRT Homo
sapiens 10 Gly Asn Ser Asn Arg Pro Ser 1 5 11 30 DNA Homo sapiens
11 agc aca tat gca ccc ccc ggt att att atg 30 Ser Thr Tyr Ala Pro
Pro Gly Ile Ile Met 1 5 10 12 10 PRT Homo sapiens 12 Ser Thr Tyr
Ala Pro Pro Gly Ile Ile Met 1 5 10 13 15 DNA Homo sapiens 13 gac
tac tac atg agc 15 Asp Tyr Tyr Met Ser 1 5 14 5 PRT Homo sapiens 14
Asp Tyr Tyr Met Ser 1 5 15 66 DNA Homo sapiens 15 tac att agt agt
agt ggt agt acc ata tac tac gca 36 Tyr Ile Ser Ser Ser Gly Ser Thr
Ile Tyr Tyr Ala 1 5 10 gac tct gtg aag ggc cga ttc acc atc tcc 66
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 15 20 22 16 22 PRT Homo
sapiens 16 Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser
Val 1 5 10 15 Lys Gly Arg Phe Thr Ile Ser 20 17 27 DNA Homo sapiens
17 tgg agt ggt gag gat gct ttt gat atc 27 Trp Ser Gly Glu Asp Ala
Phe Asp Ile 1 5 9 18 9 PRT Homo sapiens 18 Trp Ser Gly Glu Asp Ala
Phe Asp Ile 1 5 19 33 DNA Homo sapiens 19 cgg gcc agt gag ggt att
tat cac tgg ttg gcc 33 Arg Ala Ser Glu Gly Ile Tyr His Trp Leu Ala
1 5 10 20 11 PRT Homo sapiens 20 Arg Ala Ser Glu Gly Ile Tyr His
Trp Leu Ala 1 5 10 21 21 DNA Homo sapiens 21 aag gcc tct agt tta
gcc agt 21 Lys Ala Ser Ser Leu Ala Ser 1 5 22 7 PRT Homo sapiens 22
Lys Ala Ser Ser Leu Ala Ser 1 5 23 27 DNA Homo sapiens 23 caa caa
tat agt aat tat ccg ctc act 27 Gln Gln Tyr Ser Asn Tyr Pro Leu Thr
1 5 24 9 PRT Homo sapiens 24 Gln Gln Tyr Ser Asn Tyr Pro Leu Thr 1
5 25 15 DNA Homo sapiens 25 acc tac ggc atg cac 15 Thr Tyr Gly Met
His 1 5 26 5 PRT Homo sapiens 26 Thr Tyr Gly Met His 1 5 27 66 DNA
Homo sapiens 27 ggt ata tcc ttt gac gga aga agt gaa tac tat gca 36
Gly Ile Ser Phe Asp Gly Arg Ser Glu Tyr Tyr Ala 1 5 10 gac tcc gtg
aag ggc cga ttc acc atc tcc 66 Asp Ser Val Lys Gly Arg Phe Thr Ile
Ser 15 20 28 22 PRT Homo sapiens 28 Gly Ile Ser Phe Asp Gly Arg Ser
Glu Tyr Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly Arg Phe Thr Ile Ser
20 29 27 DNA Homo sapiens 29 gat agg ggg tcc tac ggt atg gac gtc 27
Asp Arg Gly Ser Tyr Gly Met Asp Val 1 5 30 9 PRT Homo sapiens 30
Asp Arg Gly Ser Tyr Gly Met Asp Val 1 5 31 66 DNA Homo sapiens 31
ggt ata tcc ttt gac gga aga agt gaa tac tat gca 36 Gly Ile Ser Phe
Asp Gly Arg Ser Glu Tyr Tyr Ala 1 5 10 gac tcc gtg cag ggc cga ttc
acc atc tcc 66 Asp Ser Val Gln Gly Arg Phe Thr Ile Ser 15 20 22 32
22 PRT Homo sapiens 32 Gly Ile Ser Phe Asp Gly Arg Ser Glu Tyr Tyr
Ala Asp Ser Val 1 5 10 15 Gln Gly Arg Phe Thr Ile Ser 20 33 24 DNA
Homo sapiens 33 gga gca cat tat ggt ttc gat atc 24 Gly Ala His Tyr
Gly Phe Asp Ile 1 5 34 8 PRT homo sapiens 34 Gly Ala His Tyr Gly
Phe Asp Ile 1 5 35 33 DNA Homo sapiens 35 cgg gcc agc gag ggt att
tat cac tgg ttg gcc 33 Arg Ala Ser Glu Gly Ile Tyr His Trp Leu Ala
1 5 10 36 15 DNA Homo sapiens 36 agc cat aac atg aac 15 Ser His Asn
Met Asn 1 5 37 5 PRT Homo sapiens 37 Ser His Asn Met Asn 1 5 38 66
DNA Homo sapiens 38 tcc att agt agt agt agt agt tac ata tac tac gca
36 Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala 1 5 10 gac tca
gtg aag ggc cga ttc acc atc tcc 66 Asp Ser Val Lys Gly Arg Phe Thr
Ile Ser 15 20 39 22 PRT Homo sapiens 39 Ser Ile Ser Ser Ser Ser Ser
Tyr Ile Tyr Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly Arg Phe Thr Ile
Ser 20 40 27 DNA Homo sapiens 40 gat cgc ggg agt acc ggt atg gac
gtc 27 Asp Arg Gly Ser Thr Gly Met Asp Val 1 5 41 9 PRT Homo
sapiens 41 Asp Arg Gly Ser Thr Gly Met Asp Val 1 5 42 15 DNA Homo
sapiens 42 agt tac tac tgg agc 15 Ser Tyr Tyr Trp Ser 1 5 43 5 PRT
Homo sapiens 43 Ser Tyr Tyr Trp Ser 1 5 44 63 DNA Homo sapiens 44
tat atc tat tac agt ggg agc acc aac tac aac ccc 36 Tyr Ile Tyr Tyr
Ser Gly Ser Thr Asn Tyr Asn Pro 1 5 10 tcc ctc aag agt cga gtc acc
ata tca 63 Ser Leu Lys Ser Arg Val Thr Ile Ser 15 20 45 21 PRT Homo
sapiens 45 Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu
Lys 1 5 10 15 Ser Arg Val Thr Ile Ser 20 46 18 DNA Homo sapiens 46
ggg agg tat ttt gac gtc 18 Gly Arg Tyr Phe Asp Val 1 5 47 6 PRT
Homo sapiens 47 Gly Arg Tyr Phe Asp Val 1 5 48 42 DNA Homo sapiens
48 act gga acc agc agt gac gtt ggt ggt tat aac tat 36 Thr Gly Thr
Ser Ser Asp Val Gly Gly Tyr Asn Tyr 1 5 10 gtc tcc 42 Val Ser 14 49
14 PRT Homo sapiens 49 Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn
Tyr Val Ser 1 5 10 50 21 DNA Homo sapiens 50 gag ggc agt aag cgg
ccc tca 21 Glu Gly Ser Lys Arg Pro Ser 1 5 51 7 PRT Homo sapiens 51
Glu Gly Ser Lys Arg Pro Ser 1 5 52 30 DNA Homo sapiens 52 agc tca
tat aca acc agg agc act cga gtt 30 Ser Ser Tyr Thr Thr Arg Ser Thr
Arg Val 1 5 10 53 10 PRT Homo sapiens 53 Ser Ser Tyr Thr Thr Arg
Ser Thr Arg Val 1 5 10 54 23 DNA Artificial Sequence PCR primer 54
agcggataac aatttcacac agg 23 55 21 DNA Artificial Sequence PCR
primer 55 gtcgtctttc cagacggtag t 21 56 44 PRT Artificial Sequence
Fab'2 antibody fragment 56 Cys Pro Pro Cys Ala Pro Glu Leu Leu Gly
Gly Arg Met Lys Gln 1 5 10 15 Leu Glu Asp Lys Val Glu Glu Leu Leu
Ser Lys Asn Tyr His Leu 20 25 30 Glu Asn Glu Val Ala Arg Leu Lys
Lys Leu Val Gly Glu Arg 35 40 57 43 DNA Artificial Sequence PCR
primer 57 gcttctgcgg ccacacaggc ctacgctgac atcgtgatga ccc 43 58 40
DNA Artificial Sequence PCR primer 58 atgatgatgt gccacggtcc
gtttgatctc cagttcggtc 40 59 43 DNA Artificial Sequence PCR primer
59 gcttctgcgg ccacacaggc ctacgcttcc tatgtgctga ctc 43 60 40 DNA
Artificial Sequence PCR primer 60 ccttctctct ttaggttggc caaggacggt
cagcttggtc 40 61 43 DNA Artificial Sequence PCR primer 61
gcttctgcgg ccacacaggc ctacgctcag tctgtgctga ctc 43 62 39 DNA
Artificial Sequence PCR primer 62 cattctacaa acgcgtacgc tcaggtgcag
ctggtgcag 39 63 45 DNA Artificial Sequence PCR primer 63 gtaaatgtat
gggcccttgg tggaggaggc actcgagacg gtgac 45 64 39 DNA Artificial
Sequence PCR primer 64 cattctacaa acgcgtacgc tcaggtgcag ctggtggag
39 65 39 DNA Artificial Sequence PCR primer 65 cattctacaa
acgcgtacgc tgacgtgcag ctggtgcag 39 66 42 DNA Artificial Sequence
PCR primer 66 gtaaatgtat gggcccttgg tggcggctga ggagacggtg ac 42 67
39 DNA Artificial Sequence PCR primer 67 cattctacaa acgcgtacgc
tcaggtgcag ctgcagcag 39 68 39 DNA Artificial Sequence PCR primer 68
cattctacaa acgcgtacgc tcaggtgcag ctgcaggag 39 69 42 DNA Artificial
Sequence PCR primer 69 gtaaatgtat gggcccttgg tggaggctga agagacggta
ac 42 70 12 PRT Artificial Sequence gD tag 70 Met Ala Asp Pro Asn
Arg Phe Arg Gly Lys Asp Leu 1 5 10 71 66 PRT Artificial Sequence
Partial fusion protein sequence 71 Met Gly Gly Ala Ala Ala Arg Leu
Gly Ala Val Ile Leu Phe Val 1 5 10 15 Val Ile Val Gly Leu His Gly
Val Arg Gly Lys Tyr Ala Leu Ala 20 25 30 Asp Ala Ser Leu Lys Met
Ala Asp Pro Asn Arg Phe Arg Gly Lys 35 40 45 Asp Leu Pro Val Leu
Asp Gln Leu Leu Glu Gly Gly Ala Ala His 50 55 60 Tyr Ala Leu Leu
Pro Gly 65 72 249 PRT Artificial Sequence single chain antibody
(scFv) fragments 72 Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Glu
Met Lys Lys Pro 1 5 10 15 Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly
Tyr Gly Tyr Ser Phe Ala 20 25 30 Thr Ser Trp Ile Gly Trp Val Arg
Gln Met Pro Gly Arg Gly Leu Glu 35 40 45 Trp Met Ala Ile Met Tyr
Pro Gly Asn Ser Asp Thr Arg His Asn Pro 50 55 60 Ser Phe Glu Asp
Gln Val Thr Met Ser Ala Asp Thr Ser Ile Asn Thr 65 70 75 80 Ala Tyr
Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95
Tyr Cys Ala Arg Ala Gly Val Ala Gly Gly Ala Phe Asp Leu Trp Gly 100
105 110 Lys Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr
Gln Pro Ala 130 135 140 Ser Val Ser Gly Ser Pro Gly Gln Ser Ile Thr
Ile Ser Cys Thr Gly 145 150 155 160 Thr Ser Ser Gly Val Gly Gly Tyr
Asn Tyr Val Ser Trp Tyr Gln Gln 165 170 175 His Pro Gly Lys Ala Pro
Lys Leu Leu Ile Tyr Gly Asn Ser Asn Arg 180 185 190 Pro Ser Gly Val
Pro Asp Arg Phe Ser Ala Ser Lys Ser Gly Asn Thr 195 200 205 Ala Ser
Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr 210 215 220
Phe Cys Ser Thr Tyr Ala Pro Pro Gly Ile Ile Met Phe Gly Gly Gly 225
230 235 240 Thr Lys Leu Thr Val Leu Gly Ala Ala 245 73 245 PRT
Artificial Sequence single chain antibody (scFv) fragments 73 Met
Ala Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro 1 5 10
15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
20 25 30 Asp Tyr Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly
Leu Glu 35 40 45 Trp Val Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile
Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Trp Ser
Gly Glu Asp Ala Phe Asp Ile Trp Gly Gln 100 105 110 Gly Thr Met Val
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser
Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser 130 135 140
Thr Leu Ser Ala Ser Val Gly Asp Arg Val Ala Ile Thr Cys Arg Ala 145
150 155 160 Ser Glu Gly Ile Tyr His Trp Leu Ala Trp Tyr Gln Gln Lys
Pro Gly 165 170 175 Lys Ala Pro Lys Leu Leu Ile Tyr Lys Ala Ser Ser
Leu Ala Ser Gly 180 185 190 Ala Pro Ser Arg Phe Ser Gly Ser Gly Ser
Gly Ala Asp Phe Thr Leu 195 200 205 Thr Ile Ser Ser Leu Gln Pro Asp
Asp Phe Ala Thr Tyr Tyr Cys Gln 210 215 220 Gln Tyr Ser Asn Tyr Pro
Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu 225 230 235 240 Val Lys Arg
Ala Ala 245 74 245 PRT Artificial Sequence single chain antibody
(scFv) fragments 74 Met Ala Glu Val Gln Leu Val Gln Ser Gly Gly Gly
Val Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Ser Leu Ser Cys Ala Val
Ser Gly Ile Thr Leu Arg 20 25 30 Thr Tyr Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Gly Ile Ser
Phe Asp Gly Arg Ser Glu Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95
Tyr Cys Ala Arg Asp Arg Gly Ser Tyr Gly Met Asp Val Trp Gly Arg 100
105 110 Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln
Ser Pro Ser 130 135 140 Thr Leu Ser Ala Ser Ile Gly Asp Arg Val Thr
Ile Thr Cys Arg Ala 145 150 155 160 Ser Glu Gly Ile Tyr His Trp Leu
Ala Trp Tyr Gln Gln Lys Pro Gly 165 170 175 Lys Ala Pro Lys Leu Leu
Ile Tyr Lys Ala Ser Ser Leu Ala Ser Gly 180 185 190 Ala Pro Ser Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 195 200 205 Thr Ile
Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln 210 215 220
Gln Tyr Ser Asn Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu 225
230 235 240 Ile Leu Arg Ala Ala 245 75 244 PRT Artificial Sequence
single chain antibody (scFv) fragments 75 Met Ala Gln Val Gln Leu
Val Gln Ser Gly Gly Gly Leu Val Arg Pro 1 5 10 15 Gly Gly Ser Leu
Ser Leu Ser Cys Ala Val Ser Gly Ile Thr Leu Arg 20 25 30 Thr Tyr
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45
Trp Val Ala Gly Ile Ser Phe Asp Gly Arg Ser Glu Tyr Tyr Ala Asp 50
55 60 Ser Val Gln Gly Arg Phe Thr Ile Ser Arg Asp Ser Ser Lys Asn
Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Gly Ala His Tyr Gly Phe Asp
Ile Trp Gly Gln Gly 100 105 110 Thr Met Val Thr Val Ser Ser Gly Gly
Gly Gly Thr Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp
Ile Gln Met Thr Gln Ser Pro Ser Thr 130 135 140 Leu Ser Ala Ser Ile
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 145 150 155
160 Glu Gly Ile Tyr His Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
165 170 175 Ala Pro Lys Leu Leu Ile Tyr Lys Ala Ser Ser Leu Ala Ser
Gly Ala 180 185 190 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr 195 200 205 Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln 210 215 220 Tyr Ser Asn Tyr Pro Leu Thr Phe
Gly Gly Gly Thr Glu Leu Glu Ile 225 230 235 240 Lys Arg Ala Ala 76
245 PRT Artificial Sequence single chain antibody (scFv) fragments
76 Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro
1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser 20 25 30 Ser His Asn Met Asn Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu 35 40 45 Trp Val Ser Ser Ile Ser Ser Ser Ser Ser
Tyr Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg
Asp Arg Gly Ser Thr Gly Met Asp Val Trp Gly Arg 100 105 110 Gly Thr
Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125
Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser 130
135 140 Thr Leu Ser Ala Ser Ile Gly Asp Arg Val Thr Ile Thr Cys Arg
Ala 145 150 155 160 Ser Glu Gly Ile Tyr His Trp Leu Ala Trp Tyr Gln
Gln Lys Pro Gly 165 170 175 Lys Ala Pro Lys Leu Leu Ile Tyr Lys Ala
Ser Ser Leu Ala Ser Gly 180 185 190 Ala Pro Ser Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Xaa 195 200 205 Thr Ile Ser Ser Leu Gln
Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln 210 215 220 Gln Tyr Ser Asn
Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu 225 230 235 240 Ile
Lys Arg Ala Ala 245 77 244 PRT Artificial Sequence single chain
antibody (scFv) fragments 77 Met Ala Gln Val Gln Leu Gln Gln Ser
Gly Pro Gly Leu Val Lys Pro 1 5 10 15 Ser Glu Thr Leu Ser Leu Thr
Cys Thr Val Ser Gly Asp Ser Ile Ser 20 25 30 Ser Tyr Tyr Trp Ser
Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly
Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser 50 55 60 Leu
Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Ser Gln Phe 65 70
75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
Tyr 85 90 95 Cys Ala Arg Gly Arg Tyr Phe Asp Val Trp Gly Arg Gly
Thr Met Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Ser Tyr Val Leu Thr Gln
Pro Pro Ser Val Ser Gly Ser 130 135 140 Pro Gly Gln Ser Ile Thr Ile
Ser Cys Thr Gly Thr Ser Ser Asp Val 145 150 155 160 Gly Gly Tyr Asn
Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala 165 170 175 Pro Lys
Leu Met Ile Tyr Glu Gly Ser Lys Arg Pro Ser Gly Val Ser 180 185 190
Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile 195
200 205 Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser
Tyr 210 215 220 Thr Thr Arg Ser Thr Arg Val Phe Gly Gly Gly Thr Lys
Leu Thr Val 225 230 235 240 Leu Gly Ala Ala
* * * * *