AU2003201322B2 - Agonist antibodies to the thrombopoietin receptor, and their therapeutic uses - Google Patents

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AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant: GENENTECH, INC.

Invention Title: AGONIST ANTIBODIES TO THE THROMBOPOIETIN

RECEPTOR,

AND THEIR THERAPEUTIC USES The following statement is a full description of this invention, including the best method of performing it known to me/us: AGONIST ANTIBODIES TO THE THROMBOPOIETiN RECEPTOR. AND THEIR THERAPEUTIC USES Field of the Invention This invention relates to the recombinant synthesis and purification of protein antibodies that influence survival, proliferation, differentiation or maturation of hematopoictic 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 (thrombopoictin 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 ofthe Invention In 1994 several groups reported the isolation and cloning of thrombopoietin de Sauvage er al., Nature 369:533 (1994): S. Lok et Nature 369:565 (1994); TD. Bartley et at., Cell 77:1117 (1994); Y.

Sohma et al., FEBS Letters 353:57 (1994); DJL Kuter et al., Proc. Nail. Acad Sci. 91:11104 (1994)). This was the culmination of more than 30 years of research initiated in the late 50's when Yamamoto Yamamoto.

Acta HaematolJpn. 20:163-178. (1957)) and Kelemen E. Kelemen et al., Acta Haematol (Basel). 20:350-355 (1958)) proposed that physiological platelet production is controlled by a humoral factor termed "thrombopoictin"(TPO). Although routinely detected in urine, plasma and serum from thrombocytopenic animals and patients, as well as kidney cell conditioned media, purification ofTPO proved to be a daunting task (for a review see MS. 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 plicotrophic cytokines affected megakaryocytopoiesis (MS. 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 Souyri et Cell 63:1137 (1990); I. Vigon et al., Proc. Natl. Acad Sci. 89:5640 (1992)). The expression of c-Mpl was found to be restricted to progenitor cells, megakaryocytes and platelets, and c-Mpl antiscnse oligonucleotides selectively inhibited in vitro megakaryocytopoicsis Methia etal., Blood 82:1395 (1993)). From this it was postulated that c-Mpl played a critical role in regulating megakaryocytopoicsis and that its putative ligand may be the long sought TPO 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 de Sauvage et al., .upra- S. Lok et supra; TD. Bartley et al., supra). In addition two other groups independently reported the purification of the Mpl-ligand using standard chromatography techniques and megakaryocyte assays Sohma et al., supra; DJ. Kuteret 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 mcgakaryocytopoiesis and thrombopoicsis 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).

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 crythropoietin (EPO) and a unique 181 amino acid C-terminal domain that is highly glycsylatcd de Sauvage el al., supra; S. Lok et al., supra: TD. Bartlcy 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 Kato el 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 0-linked glycosylations Eng et al., Protein Science 5(suppl 105 (1996)). A recombinant truncated form of TPO (rTP0153), 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 de Sauvage et al., supra, DL. 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 thrconine 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.

IS 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 (GR. 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-tcrminal 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 -150,000 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 (YG. Meng e: al., Blood 86(suppl. 1):313 (1995)).

Prior to the discovery of c-Mpl and the isolation of TPO, it was thought that megakaryocytopoiesis was regulated at multiple cellular levels (MS. Gordon el at, supra; W. Vainchenker et supra YG. 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 (MS. Gordon et at., supra, W. Vainchenker et al., supra; YG. Meng et al., supra). Other data indicated that plasma from thrombocytopenic animals contained distinct activities that either affected proliferation (mcg-CSF) or maturation (TPO) of megakaryocytes (RJ. Hil el al.. Exp.Hematol 20:354 (1992)). Wcndling and her colleagues Wendling et al., Nature 369:571 (1994)) initially dispelled this theory by demonstrating that all the megakaryocyte colony-stimulating and thrombopoictic 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 mcgakaryocytes but also their maturation K. Kaushansky et al., Nature 369:568 (1994); FC. Zeiglcr et al., Blood 84:4045 (1994); VC. Broudy et al.. Blood 85:1719 (1995); JL. Nichol et at., J. Clin. Invest. 95:2973 (1995); N. Banu et Blood 86:1331 (1995); N..Cebili et al., Blood 86:2516 (1995); P. Angchaisuksiri et al., Br. J. Haematol. 93.13 (1996); ES. Choi et al., Blood 85:402 (1995)). Human CD34+, CD34+CD4 I+ cells (FC.

Zeigler et al., supra: VC. Broudy e al., supra; 'JL. Nichol et al., supra; N. Banu el al., supra;) or purified murine stem cells (sca+,lin-, kit+) Kaushansky et al., supra; FC. 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 Debili et al., supra). In addition comparable results are obtained in serum free or serum containing culture conditions Banu et al. supra; N. Debili et al., supra: P.

Angchaisuksiri el al., supra;). These observations indicate that neither accessory cells or serum components are required for TPO to induce megakaryocyte growth and differentiation in vitro.

The effect of rTPO on the megakaryocytc maturation process is dramatic. rTPO induces highly purified murine or human progenitor cells in liquid culture to differentiate into very large mature polyploid megakaryocytes (FC. Zeigler et at, supra; VC. Broudy et al.. supra; JL. Nichol et al., supra: N. Debili et al.

supra). Megakaryocycs from such cultures exhibit ploidy of 4N-16N with ploidy classes of 64N andl28N also being detected in these cultures 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 (FC. Zeigler et al., supra; N. Debili et al., supra; ES. Choi el at, supra).

Significantly, the platelets produced from such cultures have been shown to be morphologically and functionally indistinct from plasma-derived platelets (ES. Choi ei at, supra).

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 mcgakaryocytopoiesis 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 (VC. Broudy et al., supra). In human megakaryocytopoicsis assays IL-3 and IL-6 effects are additive to rTPO, while KL acts syncrgistically with rTPO (JL. Nichol et al., supra; N. Banu t 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.

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 at, 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 Kobayashi el 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 mycloid colony growth in normal hematopoietic cultures has not been demonstrated in vitro, however.

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 (EF. Winton et,al., Erp. Hemalol. 23:879 (1995); AM. Farese et al., Blood 86:54 (1995); KH. Sprugel et al., Blood 86(suppl 1):20 (1995); LA. Harker er al., Blood 87:1833 (1996); K.

Kaushansky et al, Exp. Hematol. 24:265 (1996);'TR. Ulich er al., Blood 87:5006 (1996); K. Ault ei al., Blood 86(suppl 367 (1995); NC. 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 Ault et al., supra). Preceding this effect is a dramatic increase in CFU-MK in both the marrow and spleen AM. Farese et al., supra K. Kaushansky er al., supra; TR. 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 (NC. 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 Kaushansky et al., supra) and expanded CFU-mixed in rhesus monkeys (AM. Farese et al., supra).

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 Montrucchio et al., Blood 87:2762 (1996); A. Oda et al., Blood 87:4664 (1996); CF.

Toombs et al, Thromb. Res. 80:23 (1995); CF. 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 (LA. Harker et al., supra, CF. 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.

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-mpt or the TPO genes (WS. Alexander er al., Blood 87:2162 (1996); FJ. de Sauvage et aL, J.Exp.Med 183:651 (1996); AL. 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 megakaryocytcs 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- I and stem cell factor) results in a modest -4stimulation of platelet production (AL. Gurney et 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 megakaryocytcs and platelets.

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 (WS. Alexander et al., supra; K. Carver-Moore et at., 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 hematopoicsis correlates with the detection of c-Mpl expression in AA4+ Sca+ murine stem ceil population (FC. 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 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.

It has long been observed that an inverse correlation exists between plasma megakaryopoictic and thrombopoictic activity and platelet levels (reviewed in TP. McDonald, Am. J. Pediatr. 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 (JL. Nichol et al, supra, EVB. Emmons et Blood 87:4068 (1996); H.

Oh et al., Blood 87:4918 (1996); M. Chang er al., Blood 86(suppl 1):368 (1995)). Unlike crythropoietin, 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 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 (DJ.

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 (PJ.

Fielder r at., Blood 87:2154 (1996); R. Stoffel et al., Blood 87:567 (1996)). In addition, the gene dosage effect observed in TPO hetcrozygous knockout mice refute the regulation of TPO production by platelet mass (FJ. de Sauvage etat., 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(100-400pM) and internalize and degrade TPO (PJ. 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 (PJ. Fielder et at., supra). These results indicate that TPO clearance is mediated by platelet binding via c-Mpl. It is also likely that mcgakaryocyte 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 (EVB. Emmons et al.

supra; RA. Shivdasani et 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 (RA. 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.

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 mycloablative 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 (JP Leonard e 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 (GR. Thomas e al., supra; K. Kaushansky et al., supra, MM. Hokom et al., Blood 86:4486 (1995)). Similar results were obtained in a murine sublethal irradiation model (GR. 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 (GR. Thomas et supra' KI Kabaya et al., Blood 86(suppl 14 (1995); G. MolineuK el al., Blood 86(suppl 1):227 (1995)). Treatment of sublcthally irradiated rhesus monkeys with rTPO accelerated platelet recovery by 3 weeks and prevented platelet nadirs below 40,000 (AM. Faresc et al, J. Clin. Invest.

97:2145 (1996); KJ. Neelis et al., Blood86(suppl 1):256 (1995)). Even more impressively, rTPO completely prevented post-chemotherapy thrombocytopenia following the treatment of rhesus monkeys with hepsulfam (AM. Farese e al., supra). In contrast to these promising results, two groups have reported that rTPO had no effect on the hematopoictic recovery of lethally irradiated mice or monkeys rescued with a marrow transplant (KJ. Nelis et al., supra; WE. Fibbe et at, 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 (WE. Fibbe et at, 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.

Although rTPO only modestly affects crythroid and myeloid lineages in normal mice it dramatically accelerates the recovery of all progenitor classes in myelosupprcsscd mice and monkeys resulting in a significant acceleration of RBC and WBC recovery Kaushansky et al., supra; AM. Farese et al., supra; K.

Kaushansky eti J. Clin. Invest. 96:1683 (1995)). The effect of rTPO on neutrophil recovery has been shown to be additive to that of G-CSF (AM. Farese et al., supra). These results indicate that the clinical utility of rTPO may be broader than originally anticipated.

The difference between the effect of rTPO on hematopoiesis in normal and myclosuppressed 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 erythropoicsis and myclopoiesis present following myclosuppressive treatment interact with rTPO to have a multilineage effect Kaushansky et aL, supra). In normal mice the level of these cytokincs are insufficient and the effects of rTPO on erythroid and mycloid lineages are less significant. This hypothesis is supported by the above mentioned synergistic interaction of rTPO and EPO to stimulate in vitro erythropoiesis (ES. Choi et al.. supra). It has also been q 1' proposed that production of hemopoietic factors frptn megakaryocytcs themselves may also play a role in the multilineage effect ofrTPO (AM. Farese et supra).

In the above mentioned animal studies rTPO was administered daily for 14-28 days, which was based on previous experience in dosing other hematopoictic 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 (GR. Thomas et al., supra). This effect is likely due to the potency and long half life of rTPO (GR. 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 ofrTPO dosing required to affect hematopoictic recovery following myclosuppressive treatment may be significantly less than that for other currently used cytakines Early results from human clinical trails show that rTPO also stimulates platelet production in humans.

In phase I trials, a pegylated and truncated form of rTPO (MGD administered daily for 10 days at 0.03 pg/kg to cancer patients prior to chemotherapy caused up to a four fold increase in circulating platelet levels (R.

Basser el al., Blood 86(suppl 257 (1995); JEJ. Rasko et al., Blood 86(suppl I):497 (1995)). Similarly, patients given a single dose of rTPO had platelet levels increase by four fold Vaden-Raj et al., Stimulation of megakaryocyte and platelet production by a single dose of recombinant human thrombopoictin 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 IXl06/pl were observed in some of the patients, no thrombotic events were observed. This indicates that TPO will be well tolerated in humans. In myclosuppressed patients, pcgylated rTPO153(MGDF) given post chemotherapy has been shown to reduce the extent of the platelet nadir following chemotherapy Begley et al., Proceedings of ASCO 15271 (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, crythroid, myeloid and multipotential lineages Vaden-Raj et al, suprq). This later observation suggests that rTPO may be useful as a priming agent 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 etal., Proc. Nail Acad- Sci. USA, 87:5459-5463 (1990); Kitamura et al., Cell, 66:1165-1174 (1991a); Kitamura et 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 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 e al., Proc. Nail.

Acad Sci USA. 89:5690-5694 (1992)), granulocyte-macrophagc colony-stimulating factor (GM-CSF) (Gearing e al.. EMBO 8:3667-3676 (1991); Hayashida et al.. Proc. Natl. Acad Sci. USA, 244:9655-9659 (1990)).

C

granulocyte colony-stimulating factor (G-CSF) (Fukunaga ei al., Cell, 61:341-350 (1990a); Fukunaga et at..

Proc. Nat. Acad. Sci. USA, 87:8702-8706 (1990b); Larsen et al., J. Ep. Med.. 172:1559-1570 (1990)), EPO (D'Andrea el at. Cell, 57:277-285 (1989); Jones e al.. Blood, 76:31-35 (1990)), Leukemia inhibitory factor (LIF) (Gearing et al. EMBOJ., 10:2839-2848 (1991)), oncostatin M (OSM) (Rose et al.. Proc. Nal. Acad. Sci USA, 88:8641-8645 (1991)) and also receptors for prolactin (Boutin er Proc. atl. Acad. Sci. USA, 88:7744- 7748 (1988); Edery e al., Proc. Nail. Acad. Sci. USA, 86:2112-2116 (1989)), growth hormone (GH) (Leung e Nature, 330:537-543 (1987)) and ciliary neurotrophic factor (CNTF) (Davis et Science, 253:59-63 (1991).

Members of the cytokine receptor superfamily may be grouped into three functional categories (for review sec Nicola et al., Cell. 67:1-4 (1991)). The first class comprises single chain receptors, such as crythropoictin 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), granulocytc-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 ofa third class of cytokine receptors, termed b-subunits, be, the common b-subunit for the three asubunits of IL-3-R, IL-5-R and GM-CSF-R (Nicola N. A. et. al. Cell 67:1-4 (1991)) Evidence that mpl is a member of the cytokine receptor superfamily comes from sequence homology (Gearing, EMBO J. 8:3667-3676 (1988); Bazan, Proc. Nail. Acad. Sci. USA, 87:6834-6938 (1990); Davis et al.. Science, 253:59-63 (1991) and Vigon et al., Proc. Nal. Acad S. USA. 89:5640-5644 (1992)) and its ability to transduce proliferative signals.

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 cysteincs 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 bbarrel 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, 82607-2615 (1993)). Thus mpt may belong to the low affinity ligand binding class of cytokine receptors.

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 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).

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 aI Proc. NatL Acad. Sci. 248:378 (1993). Activation of hematopoictic receptors upon ligand binding Va 0 0 i 0 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 5 receptor that activates the erythropoictin receptor (EPO-R) is described in WO 96/03438 (published 8 February 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.

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 treamuent of hematopoictic disorders including thrombocytopenia.

Summary of the Invention In one aspect the present invention provides an antagonist antibody, fragment, or variant thereof which binds to a human c-mp[ which is selected from the group consisting of Abi, Ab2, Ab3, Ab4, Ab5 and Ab6, wherein each Abl-Ab6 comprises a VH and VL chain, each VH and VL chain comprising CDR amino acid sequences designated CDR I, CDR2 and CDR3 separated by framework amino acid sequences. the amino acid sequence of each CDR in each VH and VL chain ofAbl-Ab6 selected according to the following table: Abl: VHCDR1 VHCDR2 VHCDR3 (SEQ ID NO: 1) (SEQ ID NO: 2) VLCDR1 (SEQ ID NO: 7) (SEQ ID NO: 8) VHCDR1 (SEQ ID NO: 13) (SEQ ID NO: 14) VLCDR1 (SEQ ID NO: 19) (SEQ ID NO: 20) (SEQ ID NO: 3) (SEQ ID NO: 4) VLCDR2 (SEQ ID NO: 9) (SEQ ID NO: 10) VHCDR2 (SEQ ID NO: 15) (SEQ ID NO: 16) VLCDR2 (SEQ ID NO: 21) (SEQ ID NO: 22) (SEQ ID NO: (SEQ ID NO: 6) VLCDR3 (SEQ ID NO: 11) (SEQ ID NO: 12) VHCDR3 (SEQ ID NO: 17) (SEQ ID NO: 18) VLCDR3 (SEQ ID NO: 23) (SEQ ID NO: 24) Ab2: Ab3:

O

Va

O

0 0

O

i 0 Ab4: VHCDR1 (SEQ ID NO: 25) (SEQ ID NO: 26) VLCDR1 (SEQ ID NO: 19) (SEQ ID NO: 20) VHCDR1 (SEQ ID NO: 25) (SEQ ID NO: 26) VLCDR1 (SEQ ID NO: 35) (SEQ ID NO: 20) VHCDR1 (SEQ ID NO: 36) (SEQ ID NO: 37) VLCDR1 (SEQ ID NO: 19) (SEQ ID NO: 20)

VHCDRI

(SEQ ID NO: 42) (SEQ ID NO: 43) VLCDR1 (SEQ ID NO: 48) (SEQ ID NO: 49) VHCDR2 (SEQ ID NO: 27) (SEQ ID NO: 28) VLCDR2 (SEQ ID NO: 21) (SEQ ID NO: 22) VHCDR2 (SEQ ID NO: 31) (SEQ ID NO: 32) VLCDR2 (SEQ ID NO: 21) (SEQ ID NO: 22) VHCDR2 (SEQ ID NO: 38) (SEQ ID NO: 39) VLCDR2 (SEQ ID NO: 21) (SEQ ID NO: 22) VHCDR2 (SEQ ID NO: 44) (SEQ ID NO: 45) VLCDR2 (SEQ ID NO: 50) (SEQ ID NO: 51) VHCDR3 (SEQ ID NO: 29) (SEQ ID NO: VLCDR3 (SEQ ID NO: 23) (SEQ ID NO: 24) VHCDR3 (SEQ ID NO: 33) (SEQ ID NO: 34) VLCDR3 (SEQ ID NO: 23) (SEQ ID NO: 24) VHCDR3 (SEQ ID NO: (SEQ ID NO: 41) VLCDR3 (SEQ ID NO: 23) (SEQ ID NO: 24) VHCDR3 (SEQ ID NO: 46) (SEQ ID NO: 47) VLCDR3 (SEQ ID NO: 52) (SEQ ID NO: 53).

Ab6: 10 h:\MaraR\Keep\Speci\F493'. 3-4.L.06

ND

O In a further aspect the present invention provides an agonist antibody, fragment, or l variant thereof which binds to human c-mpl, comprising an amino acid sequence selected from the group consisting of 12E10, 12B5, 10F6 and 12D5.

5 Brief Description Of The Drawings 0 Fig. I shows examples of single chain antibody (scFv) fragments denominated 10F6, 5E5, 12B5, 12D5 and 12E10 having sequences for CDRs and framework regions.

l Fig. 2 illustrates a method for the constriction of a phage library containing single-chain antibodies v fused to a coat protein of a phage.

O 10 Fig. 3 shows a single-chain antibody displayed as. a fusion protein on coat protein 3 of a filamentous phage.

SFig. 4 illustrates a method of selecting scFv in a phage library by one or more binding selection cycles.

Fig. 5 illustrates a method of panning high affinity phage using biotinylated antigen and streptavidin coated paramagnetic beads.

Fig. 6 shows a process for identifying c-mpl binding phage using a phage ELISA method.

Fig. 7 illustrates DNA fingerprinting of clones to determine diversity by BstNI restriction enzyme analysis.

Fig. 8A-C show a typical BstNI analysis on a 3% agarosc gel; see Example 2.

Fig. 9 shows the results ofagonist antibodies relative to TPO in the KIRA-ELISA assay.

Fig. IOA-F show the results of TPO-antibody competitive binding assays for HU-03 cells. See Example I.

h:\H r.F\KepSpci \?49S3-. !,.Vi Fig. I shows activity for MuSK agonist antibodies of Example Detailed Description Of The Preferred Embodiments I. Definitions In general, the following words or phrases have the indicated definition when used in the description, examples, and claims.

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.

"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.

"Activate a receptor", as used herein, is used interchangeably with transduce a growth, survival.

proliferation, maturation and/or differentiation signal.

"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 arc substances known to activate platelets.

"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 at 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. el at 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. JR. et at Immunol 154(7):3310-9 (1995), and Hawkins, R.E. et al, J. Mo. BioL 226:889-896 (1992).

"Cylokine" is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokincs. 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, prordaxin, glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and leutinizing hormone hematopoictic growth factor, hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-a (TNF-a and TNFb) 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, and 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-I. IL-la, IL-2, IL-3. IL-4, IL-5, IL-6. IL-7. IL-8, IL-9, IL- S1, IL-12 and other polypcptide 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; differing in amino acid sequence by one or more amino acids or in type or extent of glycosylation.

"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 Cell. 67:1-4 (1991) and Skoda, R.C. er ol. EMBO J.

12:2645-2653 (1993)). Generally, these receptors arc interlcukins (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, 247324-328 (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 a, Cell, 59:335-348 (1989), IL-5 (Takaki et al., EMBOJ., 9:4367-4374 (1990); Tavrer er al.. Cell, 66:1175- 1184 (1991)), IL-6 (Yamasaki et al. Science, 241:825-828 (1988); Hibi etal., Cell, 63:1149-1157 (1990)), IL-7 (Goodwin et at. Cell, 60:941-951 (1990)). IL-9 (Renault et at, Proc. Nad. Acad Sci. USA, 89:5690-5694 (1992)). granulocyte-macrophage colony-stimulating factor (GM-CSF) (Gearing et at.. EMBO 8:3667-3676 (1991); Hayashida et al., Proc. Natl. Acad ScL OSA, 244:9655-9659 (1990)), granulocyte colony-stimulating factor (G-CSF) (Fukunaga et al.. Cell, 61:341-350 (1990a); Fukunaga et at., Proc. al. Acad Sci. USA, 87:8702-8706 (1990b); Larsen et al. J. Exp. Med, 172:1559-1570 (1990)). EPO (D'Andrea et Cell, 57:277-285 (1989); Jones et al.. Blood, 76:31-35 (1990)). Leukemia inhibitory factor (LIF) (Gearing et atl.

EMBOJ., 102839-2848 (1991)), oncostatin M (OSM) (Rose t al., Proc. lal. 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 Proc. Naol. Acad Sci. USA, 86:2112-2116 (1989)), growth hormone (GH) (Lcung et al., Nature, 330:537-543 (1987)), ciliary neurotrophic factor (CNTF) (Davis et al., Science, 253:59-63 (1991) and c-Mpl Souyri et al., Cell 63:1137 (1990); I. Vigon el at.. Proc. Nail. Acad. Sci 89:5640 (1992)).

"Thrombocytopenia" in humans is defined as a platelet count below ISO X 109 per liter of blood.

"Thrombopoictic activity" is defined as biological activity that consists of accelerating the proliferation, differentiation and/or maturation of megakaryocyes 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-GPllbIlla) for a human leukemia megakaryoblastic cell line (CMK), and induction of polyploidization in a mcgakaryoblastic cell line (DAMI). A "thrombopoietin receptor" is a mammalian polypeptide receptor which, when activated bya ligand binding thereto, includes, causes or otherwise gives rise to "thrombopoictic activity" in a cell or mammal, including a human.

"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.

"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 oligonuclcotide adapters or linkers are used in accord with conventional practice.

"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.

"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.

"Plasmids" are autonomously replicating circular DNA molecules possessing independent origins of replication and are designated herein by a lower case 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 an and will be apparent to the ordinary artisan.

"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 I Vg of plasmid or DNA fragment is used with about 1-2 units of enzyme in about 20 pl of buffer solution. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation of about I hour at 37*C is ordinarily used, but may vary in hccordance with the supplier's instructions. After incubation, protein or polypcptide 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).

"Recovery" or "isolation" of a given fragment of DNA from a restriction digest means separation of the digest on polyacrylamide or agarose gel by clectrophoresis, 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 at, Nucleic Acids Res.. 9:6103-6114 (1981), and Goeddel el al.. Nucleic Acids Res., 8:4057 (1980).

"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 radiolabcled, biotinylated, or enzyme-labeled probe as described in sections 9.37-9.52 of Sambrook el a, supra.

"Northern analysis" or "Northern blotting" is a method used to identify RNA sequences that hybridize to a known probe such as an oligonuclcotide, DNA fragment, cDNA or fragment thereof, or RNA fragment.

The probe is labeled with a radioisotope such as 3 2 P, 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.

"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 endonuclcase digestion. However, it may be necessary first to convert the staggered ends commonly produced after cndonuclease 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 0 C with about 10 units of the Klenow fragment of DNA polymerase I or T4 DNA polymcrase 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 units per 0,5 jg of DNA. If the DNA is to beligated 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.

"Preparation" of DNA from cells means isolating the plasmid DNA from a culture of the host cells.

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 at, supra.

"Oligonucleotides" are short-length, single- or double-stranded polydeoxynucleotides that are chemically synthesized by known methods (such as phosphotricster, phosphite, or phosphoramidite chemistry, using solid-phase techniques such as described in EP 266,032 published 4 May 1988, or via dcoxynucleosidc H-phosphanate intermediates as described by Froehler et al., Nucl. Acids Res., 14:5399-5407 (1986)). Further methods include the polymerasc 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 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.

"Polymerase chain reaction" or "PCR" refers to a procedure or technique in which minute amounts of a specific piece of nucleic acid, RNA andfor DNA, are amplified as described in U.S. Patent No. 4,683,195 issued 28 July 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 nucleotidcs 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.

51263 (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 acia 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 polymcrase to amplify or generate a specific piece of nucleic acid.

"Native antibodies and immunoglobulins" are usually heterotctrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy 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 (VH) followed by a number of constant domains. Each light chain has a variable domain at one and (VL) 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 tle light and heavy chain variable domains (Clothia e al., J.

Mot Biol.. 186:651-663 (1985); Novotny and Haber, Proc. Natl. Acad Sci. USA, 82:4592-4596 (1985)).

The tenm "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 tight chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework 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 ar 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.

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') 2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.

"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 VH-VL 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.

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab" fragments differ froii Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI 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') 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.

The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda based on the amino acid sequences of their constant domains.

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 (isorypes), IgG-1, igG-2, IgG-3, and IgG- 4; IgA-l and lgA-2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilpngainma, and p, respectively. The subunit structures and threedimensional configurations of different classes of immunoglobulins are well known.

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 Fab, F(ab') 2 scFv and Fv), so long as they exhibit the desired biological activity.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minoramounts. 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 determinant 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 (sec. U.S. Patent No. 4,816,567 (Cabilly et at)).

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 Patent No.

4,816,567 (Cabilly t at); and Morrison t Proc. Nat. Acad Sc. USA, 81:6851-6855 (1984)).

"Humanized" forms of non-human murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof'(such as Fv, Fab, Fab', F(ab') 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 nonhuman 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 typically that of a human immunoglobulin. For further details see: Jones e al., Nature, 321:522-525 (1986); Reichmann et al, Nature. 332:323-329 (1988); and Presta, Curr. Op. Struc. Biol., 2:593-596 (1992)).

"Single-chain Fv" or "sFv" antibody fragmhents comprise the V, and V L domains of antibody, wherein these domains are present in a single polypcptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review ofsFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Vcrlag, New York, pp. 269-315 (1994).

The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VF) connected to a light chain variable domain (VL) in the same polypeptide chain (VH and 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. Nall. Acaed Sci. USA 90:6444-6448 (1993).

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 (Vr-CHI-VH-CHI) which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

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 more preferably at least 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. I. 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 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.

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.

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 arc materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaccous or nonproteinaccous solutes. In preferred embodiments, the antibody will be purified to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, 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 to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassic 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.

The term "cpitope tagged" when used herein refers to an antibody fused to an "epitope tag". The cpitope 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 cpitope tag preferably is sufficiently unique so that the antibody thercagainst does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least 6 amino acid residues and usually between about 8amino 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. 82159-2165 (1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al., Mol. Cell. Bio 5(12):3610-3616 (1985)); and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et Protein Engineering 3(6):547- 553 (1990)). In certain embodiments, the cpitope tag is a "salvage receptor binding epitope". As used herein, the term "salvage receptor binding epitope" refers to an epitope ofthe Fe region of an IgG molecule IgG 1 IgG2, IgG 3 or [gG 4 that is responsible for increasing the in vivo serum half-life of the IgG molecule.

The terms "mpl ligand", mpl ligand, polypeptide". "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 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 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 3 32 Also included in this definition is the thrombopoietic ligand described in WO 95/28907 having a molecular weight of about 31,000 daltons (3 kd) as determined by SDS gel under reducing conditions and 28,000 daltons (28kd) 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 arc referred to by this term.

Preferably, the TPO is a compound havihg 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 X 109 per liter of blood.

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 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(7-15 1)-Y Where hTPO(7-151) represents the human TPO (hML) amino acid sequence from Cys through Cys t 5 1 inclusive; X represents the amino group of Cys 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 151 sites Factor Xa or thrombin); and Y represents the carboxy terminal group of Cys or one or more carboxy-terminus amino acid residue(s) of the mature TPO or extensions thereto.

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 residue(s) may occur anywhere in the peptide including at either the N-tenninal or C-terminal end or internally, so long as the fragment shares at least one biological property in common with mpl ligand. Mpl ligand 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 ofN-terminal fragments arc TPO(153). hML 3 5 or TPO(Met 1-153).

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 and most preferably at least about 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 polyoxyalkylcncs, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301.144; 4,670,417; -21- 4,791,192 or 4,179,337. TPO polypeptides covalcntly linked to the forgoing polymers are referred to herein as pegylated TPO.

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 hMLI53. 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) 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 33 2(RI53A, R154A).

A preferred chimera is a fusion between mpl ligand or fragment (defined below) thereof with a heterologous polypeptide or fragment thereof. For example, hMLi 5 3 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 thrombopoictic or chimeric hematopoictic activity.

Other preferred mpl ligand fragments have a Met preceding the amino terminus Ser (e.g.

Met-ITPOi53). This is preferred when, for example, the protein is expressed directly in a microorganism such as E coll Optionally, these mp! ligand fragments may contain amino acid substitutions to facilitate derivitization. For example, Arg153 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 Mct-lTPO(I-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.

"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.

II Preferred Embodiments of the Invention 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-mpt, 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 [L-3 dependent Ba/F3 cells transfected with human mpl P. or with a CMK Assay measuring Induction of the platelet antigen GPlbllla 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 cel proliferation assay or in a liquid suspension megakaryocytopoicsis assay 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 to or not less than 2 orders of magnitude (1 6 l0-fold) below that of thrombopoietin on a weight basis.

More preferred c-mpl aAb(s) are substantially purified aAb(s) having hematopoictic, especially megakaryocytopoictic or thrombocytopoictic activity namely, being capable of stimulating proliferation, maturation and/or differentiation of immature megakaryocytcs or their predecessors into the mature plateletproducing form that demonstrate a biological activity equal to or within 2 orders of magnitude of that of rhTPO on 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 hcmatopoietic, megakaryocytopoictic or thrombopoictic activity. Exemplary fragments having the above described biological activity include; Fv, scFv, F(ab')2.

Preferred scFv fragments denominated 10F6, 5E5, t1D10, 12B5, 12D5 and 12E10 having sequences for CDRs and Framework regions provided in Figure 1. Altematively, 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.

The framework regions may be derived from a "consensus sequence" 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 sequences. Many human antibody framework region sequences are compiled in Kabat et oa, Sequences of Proteins of mmunological Interest. 5th Ed. Public Health Service, National Institutes of Health. Bethesda, MD.

(1991), pages 647-669), for example.

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 polypcptide, 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 mp! 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.

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 filter; followed by separating the antibody from other impurities by one or more steps selected from; immunoaffinity, ion-exchange 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 silica gel with appended aliphatic groups) or SEPHADEX molecular sieve or size exclusion chromatography, and ethanol or ammonium sulfate precipitation. A protease inhibitor such as methy4 ulfonylfluoride (PMSF) may be included in any of the foregoing steps to inhibit proteolysis.

Preferably, the isolated antibody is monoclonal (Kohler and Milstein, Nature. 256:495-497 (1975); Campbell, Laboratory Techniques in Biochemistry and Molecular Biology, Burdon te al., Eds, Volume 13, Elsevier Science Publisrers, Amsterdam (1985); and Huse e al., Science. 246:1275-1281 (1989)). A preferred mpl antibody is one that binds to mpl receptor with an affinity of at least about 106 I/mole More preferably the antibody binds with an affinity ofat least about 107 I/mole or even at least 10 9 I/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 polypcptide 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.

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 mpt antibody. A preferred mpl antibody nucleic acid is RNA or DNA that encodes a biologically active human antibody.

In a further preferred embodiment of this invention, the nucleic acid molecule is cDNA encoding the mpt 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 antibody. A preferred host cell for producing .the antibody is Chinese hamster ovary (CHO) cells. An alternative preferred host cell is E coil.

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 cytokinc, 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 IGF- or a tumor necrosis factor lymphotoxin III. Methods of Making 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 93111236, 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 io 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 4 Scr, 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. at, Annu. Rev. Immunol., 1994, 12:433-55; Soderlind et. at., Immunological Reviews, 1992, 130:109-123; Hoogenboom, Tibtech February 1997, Vol. 15; Neri et. at, Cell Biophysics, 1995, 27:47-61, and the references described therein.

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 el. at: 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. 5,223,409.

Amino acid sequence variants of the antibody are prepared by introducing appropriate nuclcotide 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 ofglycosylation 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 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.

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 inscrtional 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.

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.

Table 2 rUngmal Kesidue Exemplary Preerred Substitutions Substitutions Ala val; lCu; Ile val Arg lys; gin; asn ys Asn gin; his; asp, lys; arg gin Asp glu; asn glu Cys scr; ala ser Lila asn; glu asn lu asp; gin asp ly ala ala His as; gin; lys; arg arg lie teu; val; met; ala; phe; noricucine l eu L- u norleucme; ile; val; met; ali phe ile Lys arg; gin; asn arg Met lcu; phe; ile leu Phe leu; val; ilc; ala; tyr tyr Pro ala ala Ser(S) thr: thr Thr(T) scr ser Trp tyr phe tyr Tyr trp; phe; thr; scr phe Val Lie; Icu; met; phe; ala; norleucine leu Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation. the charge or hydrophobicity of the molecule at the target site, or the bulk of tle side chain. Naturally occurring residues are divided into groups based on common side-chain properties: hydrophobic: norleucine, met, ala, val; ecu, ile; neutral hydrophilic: cys, ser, thr; acidic: asp, glu; basic: asn, gin, his, lys, arg; residues that influence chain orientation: gly, pro; and aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

Any cysteine residue not involved in maintaining the proper conformation of the humanized or variant antibody also may be substituted, generally with scrinc, 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).

A particularly preferred type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody 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 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 Ill product of MI3 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.

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.

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-Xscrine 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 or thrconine, although 5-hydroxyproline or 5-hydroxylysinc may also be used.

Addition of glycosylation sites to the antibgdy 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).

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 oligonucleotidcmediated (or site-directed) mutagenesis. PCR mutagencsis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the antibody.

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.

The nucleic acid 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 whether it is to be used for DNA amplification or for DNA expression, the size of the nucleic acid to be inserted into the vector, and 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.

Signal Sequence Component 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 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, Ipp, or heat-stable enterotoxin II leaders. For yeast secretion the native signal sequence may be substituted by, the yeast invertase, alpha factor, or acid phosphatase leaders, the C.

albicans glucoamylase leader (EP 362.179 published 4 April 1990), or the signal described in WO 90/13646 published 15 November 1990. In mammalian cell expression the native signal sequence the mpl ligand prescquence that normally directs secretion of mpt 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.

(ii) Origin of Replication Component

I

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 p plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adcnovirus, 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).

Most expression vectors are "shuttle" vectors, ie., 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.

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 gcnomic DNA. Transfcction 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.

(iii) Selection Gene Component 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 confer resistance to antibiotics or other toxins, eg., ampicillin, neomycin, methotrexate, or tetracycline, complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, the gene encoding D-alanine racemase for Bacilli.

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 e al., J. Molec.

Appl. Gene., 1:327 (1982)) mycophenolic acid (Mulligan ei 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.

Examples of other suitable selectable markers for mammalian cells arc 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 arc reiterated in tandem within the chromosomes of successive generations of recombinant cells. Increased quantities of antibody are synthesized from the amplified DNA.

For example, cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains mcthotrexate (Mtx), a competitive antagonist of DHFR. An appropriate host ceil 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. Nail. 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, ATCC No. CCL61 CHO-KI, 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, kanamycin, neomycin, or G418. See U.S. Patent No. 4,965,199.

A suitable selection gene for use in yeast is the trp gene present in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et at, Gene. 7:141 (1979); or Tschemper el aL, Gene.

10:157 (1930)). The trpl 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 trpl 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.

(iv) Promoter Component 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 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, 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.

Promoters suitable for use with prokaryotic hosts include the f-lactamase and lactose promoter systems (Chang et Nature, 275:615 (1978); and Goeddel el 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 at, 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 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 sequence operably linked to the DNA encoding antibody polypeptide.

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 nuclcotide. 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.

Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3phosphoglycerate kinase (Hitzcman et al., J. Bio. Chem., 2552073 (1980)) or other glycolytic enzymes (Hess t al., J. Adv. Enyme Reg., 7:149 (1968); and Holland, Biochemisry. 17:4900 (1978)). such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylasc, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinasc, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

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, metallothioncin. glyceraldehyde-3phosphate dchydrogenase, and enzymes responsible for maltose and galacose 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.

Antibody transcription from vectors in mammialian 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 July 1989), adenovirus (such as Adenovirus bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40), from heterologous mammalian promoters, 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.

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. Ficrs et at., Nature. 273:113 (1978); Mulligan and Berg, Science. 209:1422-1427 (1980); Pavlakis er al., Proc. Nad. Acad Sci USA. 78:7398-7402 (1981).

The immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindlll E restriction fragment. Grccnaway el at, Gene. 18:355-360 (1982). A system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is discosed in U.S. Patent No. 4,419,446. A modification of this system is described in U.S. Patent No. 4,601,978. See also Gray et Nature, 295:503-508 (1982) on expressing cDNA encoding immune interferon in'monkey cells; Reyes et Nature, 297:598-601 (1982) on expression of human 0-interferon cDNA in mouse cells under the control of a thymidine kinase promoter from herpes simplex virus; Canaani and Berg, Proc. Nad. Acad Sci. USA, 79:5166-5170 (1982) on expression of the human interferon 01 gene in cultured mouse and rabbit cells; and German 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.

Enhancer Element Component 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, afetoprotein, and insulin). Typically, however, one will use an enhancer from a cukaryotic 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.

(vi) Transcription Termination Component 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 cukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylatcd fragments in the untranslated portion of the IaRNA encoding antibody.

(vii) Construction and Analysis of Vectors 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.

For analysis to confirm correct sequences in plasmids constructed, the ligation mixtures arc 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 er aL. Nucleic Acids Res., 9:309 (1981) or by the method of Maxam et al., Methods in Enzymology, 65:499 (1980).

(viii) Transient Expression Vectors 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 polypeptidc. 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 e 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 polypcptides 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.

(ix) Suitable Exemplary Vertebrate Cell Vectors 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. patent no. 5,258,287) or pSVI6B (PCT Publication No. WO 91/08291).

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 Grampositive 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 coli B, E coli X1776 (ATCC No. 31,537), and E coli W3 10 (ATCC No. 27,325) are suitable. These examples are illustrative rather than limiting. Preferably the host cell should secrete minimal amounts of proteolytic enzymes. Alternatively, In vitro methods of cloning, eg., PCR or other nucleic acid polymerase reactions, are suitable.

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 Schirasaccharomyces pombe (Beach and Nurse, Nature, 290:140 (1981); EP 139.383 published 2 May 1985), Kluyveromyces hosts Patent No. 4,943,529) such as, K. lactis (Louvencourt et J. BacterioL.. 737 (1983)), K fragilis, K bulgaricus. K. thermroolerans, and K. marxianus. yarrowia (EP 402,226), Pichia pastoris (EP 183,070; Sreckrishna el al., J. Basic Microbiol..

28:265-278 (1988)), Candida, Trichoderma reesia (EP 244,234), Neuraspora crassa (Case et at., Proc. Nal.

Acad. Sci. USA, 76:5259-5263 (1979)), and filamentous fungi such as, eg., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10 January 1991), and Aspergillus hosts such as A. nidulans (Ballance at al., Biochem. Biophys. Res. Commun., 112:284-289 (1983); Tilbum et al., Gene. 26:205-221 (1983); Yelton et al., Proc. Nail. Acad. Sci. USA. 81:1470-1474 (1984)) and A. niger (Kclly and Hynes, EMBO J. 4:475-479 (1985)).

Suitable host cells for the expression of glycosylated antibody are derived from multicellular organisms. Such host cells arc capable of complex processing and glycosylation activities. In principle, any higher cukaryotic 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 Spodopterafrugiperdo (caterpillar), Aedes aegypli (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified. See, Luckow ea al., Bio/Technology, 6:47-55 (1988); Miller er aL, Genetic Engineering, Setlow e al., eds., Vol.

8 (Plenum Publishing, 1986), pp. 277-279; and Macda et al., Nature, 315:592-594 (1985). A variety of viral strains for transfection are publicly available, the L-I variant of Autographa californica NPV and the strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoprerafrugiperda cells.

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 rumefaciens, 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 polyadcnylation 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 June 1989.

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 CVI 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 at.. J. Gen irol., 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. NaIL Acad Sci USA, 77:4216 (1980)); mouse scrtoli cells (TM4, Mather, BioL Reprod, 23:243-251 (1980)): monkey kidney cells (CVI ATCC CCL 70); African green monkey kidney'cells (VERO-76, ATCC CRL-158); human cervical carcinoma cells (HELA, ATCC CCL canine kidney tells (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 CCLS1); TRI 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).

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.

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 4 and electroporation. Successful transfection is generally recognized when any indication of the operation of this vector occurs within the host cell.

Transformation means introducing DNA- into an organism so that the DNA is replicable, either as an extnchromosomal 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 a aL, supra, is generally used for prokaryotes or other cells that contain substantial cell-wall barriers. Infection with Agrobacterium tumcfaciens is used for tansformation of certain plant cells, as described by Shaw t at., Gene, 23:315 (1983) and WO 89105859 published 29 June 1989.

In addition, plants may be transfected using ultrasound treatment as described in WO 91/00355 published January 1991. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52:456457 (1978) is preferred. General aspects of mammalian cell host system transformations have been described by Axel in U.S. Patent No. 4,399,216 issued 16 August 1983.

Transformatiods into yeast are typically carried out according to the method of Van Solingen er at., J. Boer., 130:946 (1977) and Hsiao et al, Proc. Nal. ,cad Sc. (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.

Prokaryotic cells used to produce the antibody polypeptide of this invention are cultured in suitable media as described generally in Sambrook cl aL. supra.

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 ((MFM), 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, Meii. Er, 58:44 (1979), Barnes and Sato, Anal. Biochena. 102:255 (1980), U.S. Patent No. 4,767,704; 4,657,866; 4,927,762; or 4,560,655; WO 90103430; WO 87100195; or U.S. Patent 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 GentamycinT" 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 necessar y 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.

The host cells referred to in this disclosure encompass cells in in vitro culture as well as cells that are within a host animal.

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 RnRNA (Thomas, Proc. Nad.

Acadl 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 3 2 p. 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, fluorcsccrs, 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.

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 t at, Am. Clin Path., 75:734-738 (1980).

Antibody preferably is recovered from the culture medium as a secreted polypeptidc, although it also may be recovered from host cell lysates when directly expressed without a secretory signal.

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 perse. 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 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 mpl-IgG or protein A SEPHAROSE), hydrophobic interaction chromatography ether, butyl, or phenyl Toyopearl), lectin chromatography Con A-SEPHAROSE, lentil-lectin-SEPHAROSE), size exclusion SEPHADEX G-75), cation- and anion-exchange columns DEAE or carboxymethyl- and sulfopropyl-cellulose), and reverse-phase high performance liquid chromatography (RP-HPLC) (see eg., Urdal et at, J. Chromatog., 296:171 (1984) where two sequential RP-HPLC steps are used to purify recombinant human Other purification steps optionally include; ethanol precipitation; ammonium sulfate precipitation; chromatofocusing; preparative SDS-PAGE, and the like.

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 polypcptide, a bacterial or viral antigen, facilitates purification; an immunoaffinity column containing antibody to the antigen can be used to adsorb the fusion polypcptide. 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, CA) 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.

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 mcgakaryocyte 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.

DNA encoding the monoclonal antibodies useful in the method of the invention is readily isolated and sequenced using conventional procedures 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 ransfected 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.

IV. Utility 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.

The biologically active c-mpt agonist antibody capable of stimulating either proliferation, differentiation or maturation and/or modulation (either stimulation or inhibition) of apoptosis of hcmatopoietic cells may be used in a sterile pharmaceutical preparation or formulation to stimulate mcgakaryocytopoictic or thrombopoietic activity in patients suffering from thrombocytopenia due to impaired production, sequestration, or increased destruction of platelets. Thrombocytopenia-associated bone marrow hypoplasia 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 thrombocytopcnia, congenital thrombocytopenia, myelodysplasia, and thrombotic thrombocytopenia.

Preferred uses of the megakaryocytopoietic or thrombocytopoictic biologically active c-mpl agonist antibody of this invention are in: myelotoxic chemotherapy for treatment of leukemia or solid tumors, -37mycloablative chemotherapy for autologous or aUlgeneic bone marrow transplant, myclodysplasia, idiopathic aplastic anemia, congenital thrombocytopenia, and immune thrombocytopenia.

The biologically active c-mpl agonist antibody of the instant invention may be employed alone or in combination with other cytokines, hcmatopoictins, 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-I, IL- 3, erythropoictin (EPO), kit ligand, IL-6, and IL- 1.

The biologically active c-mpl agonist antibody of the instant invention may be used in the same way and for the same indications as thrombopoictin (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.

When used for in viva 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 lyophilizcd form or in solution.

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.

The route of antibody administration is in accord with known methods, injection or infusion by intravenous, intraperitoncal, intraccrebral, intramuscular, intraocular, intraarterial. intrathecal, inhalation or intralcsional routes, or by sustained release systems as noted below. The antibody is preferably administered continuously by infusion or by bolus injection.

An effective amount of antibody to be employed therapeutically will depend, for example, upon the therapeutic objectives, the mute 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.

The antibodies of the invention may be prepared in a mixture with a pharmaccutically 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 systemratically, 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 HCI, 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 polycthylneglycol.

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.

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, films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels poly(2hydroxyethyl-methacrylate) as described by Langer et at., Biomed Mater. Res., 15:167-277 (1981) and Langer, Chem. Tech.. 12:98-105 (1982) or poly(vinylalcohol)), polylactidcs Patent No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman el 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 DepotT (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid (EP 133,988).

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°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 e at., Proc. Nail. Acad SLe USA. 82:3688- 3692 (1985); Hwang el al., Proc. NatL Acad Sci. USA, 77:40304034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patent application 83-118008; U.S. Patent 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.

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.

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 titcr the dosage and modify the route of administration as required to obtain the optimal thcrapeutic effect. A typical daily dosage might range from about I pg/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.

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 on 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 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 I Assays The mpt agonist antibody assays were conducted essentially as described in WO 95/18858.

BaIF3 cell proliferation assay The Ba/F3-mpl cell line was established de Sauvage ei 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.

Stimulation of proliferation of BaF3-mpI cells in response to various concentrations of antibodies or TPO was measured by the amount of incorporation of 3 H-thymidine as previously described de Sauvage et al..

supra).

CMK Assay for Induction of Platelet Antigen GPllhIll Expression CMK cells are maintained in RMPI 1640.medium (Sigma) supplemented with 10% fetal bovine serum and 10mM glutamine. In preparation for the assay, the cells are harvested, washed and resuspended at 5x105 cells/ml in serum-free GIF medium supplemented with.Smg/1 bovine insulin, 10mg/1 apo-transferrin. I X trace elements. In a 96-well flat-botom 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*C, in a 5% CO 2 incubator for 48 hours. After incubation, the plates are spun at 1000rpm at 41C for five minutes. Supcmatants are discarded and 100 ml of the FITC-conjugated GPllblIa monoclonal 2D2 antibody is added to each well. Following incubation at 40C for I hour, plates are spun again at 1000rpm 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.

KIRA ELISA for Measurine Phosohorlation of the mpl-Rse.gD Chimeric Receptor The human mpl receptor has been disclosed by Vigon et al.. PNAS, USA 89:5640-5644 (1992). A chimeric receptor comprising the extraccltular domain (ECD) of the mpl receptor and the transmembrane and intraccllular domain (ICD) of Rse (Mark et al., J. ofiol. Chem. 269(14):10720-10728 (1994)) with a carboxylterminal flag polypeptide Rse.gD) was made for use in the KIRA ELISA described herein.

Capture agent preparation Monoclonal anti-gD (clone 586) was produced against a peptide from Herpes simplex virus glycoprotein D (Paborsky et at.. Protein Engineering 3(6):547-553 (1990)). The purified stock preparation was adjusted to 3.0mg/ml in phosphate buffered saline (PBS), pH 7.4 and 1.0ml aliquots were stored at -20° C.

(ii) Anti-phosphotyrosine antibody preparation Monoclonal anti-phosphotyrosinc, clone 4010, was purchased from UBI (Lake Placid. NY) and biotinylated using long-arm biotin-N-hydroxysuccinamide (Biotin-X-NHS, Research Organics, Cleveland,

OH).

(iii) Ligand The mpl ligand was prepared by the recombinant techniques described herein. The purified mpl ligand was stored at 4 OC. as a stock solution.

KIRA ELISA results for agonist antibodies or 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.

TPO receptor-biinding inhibition assay NUNC 96-well immunoplates were coated with 50 pl of rabbit anti-human IgG Fe (Jackson Labs) at 2 pg/ml in carbonate buffer (pH9.

6 overnight at 40C. After blocking with ELISA buffer (PBS, I BSA, 0.2 TWEEN 20), the plates were incubated for 2 hr with conditioned media from mpl-lg-transfccted 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 I 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 H3P04 and absorbance was read at 450-650 nm.

HU-03 cell proliferation assay The HU-01 cell line 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.

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 concentrration to I and adjusting the concentration of cells to 2.5 x 105 cellsrnl, followed by incubation for 16 hr. Cells were then washed and seeded into 96-well plates at a density of 5 x 104 cells per well in medium containing TPO or antibodies at various concentrations. Quadruplicate assays were performed. 1 iCi 3Hthymidine was added to each well before incubation for 24 hr. Cells were collected with a Packard cell harvester and incorporation of 3 H-thymidine was measured with a Top Count Counter (Packard).

Liquid suspension megakarocvtopoeisis assay 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)).

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'; medium (IMDM, G[BCO) (supplemented with 100 units per mi 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 mononuclcar cells were collected from the interface and washed twice with IMDM. Cells were seeded at I x 106 cells per ml in IMDM supplemented with 30%/ fetal bovine serum (FBS), 100 units per mt penicillin and streptomycin, and 20 pM 2mercaptoethanol, 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 I ml per well. The cultures were grown in a humidified incubator at 37 °C in 5% C02 for 14 days. Megakaryocytopoiesis was quantified using radiolabelled murine monoclonal antibody HP-ID (provided by W. L. Nichols, Mayo Clinic) which has been shown to be specific for the human megakaryocyte glycoprotein IIb/lla (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 l assay buffer containing 20 ng iodinated HPI-ID (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.

FBS used in this assay was treated with Dextran T40 at I mg/mi and charcoal at 10 mg/mi for 30 min, centrifuged, decanted, filter sterilized and heat inactivated at 56 °C for 30 min.

TPO-antibodv comptitive binding assays for HU-03 cells and human platelets HU-03 cells were cultured as described above. Platelet rich plasma (PRP) was prepared by ccntrifugation of citrated whole blood at 400 g's for 5 minutes. Binding studies were conducted within three hours of collection. 125 1-TPO was prepared by indirect iodination (Fielder, P. Hass, Nagel, M., Stefanich, Widmer, Bennett, G. Keller, de Savage, F. and Eaton, D. 1997. Human platelets as a model for the binding and degradation of thrombopoictin. Blood 89: 2782-2788) and yielded a specific activity of 15-50 pCi/lg protein.

In a volume of 110 microliters containing 100pM iodinated TPO, 2 x 106 washed HU-03 cells in Hank's Balanced Salt Solution,5 mg/ml bovine serum albumin (HBSSB), or 4 x 107 platelets in plasma, were incubated at 37*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 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.

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.

Affinity determinations.

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 actjvated for covalent coupling of gD-mpl receptor using Nethyl-N'"-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimidc (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 pg/mL. An aliquot (35 pL) was injected at a flow rate of 1 UL/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 0 C using a flow rate of UL/min. Equilibrium dissociation constants, Kd's, from SPR measurements were calculated as koflikon.

Standard deviations. son for kon and soff for koff were obtained from measurements with >4 protein concentrations (kon) or with >7 protein concentrations (koff). 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.c.

(standard error of fit). The resulting errors e[K] in calculated Kd's were estimated according to the following formula for propagation of errors: e[K] [(kon2(sof) 2 (off) (kon) 4(son)2 2 w h e r e off and son are the standard errors in kon and koff, respectively.

Example 2 Isolation of Antibodies from the CAT library 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, Figures 2 and 3 presents a cartoon of the construction of a phage library containing 6x10 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.

The Antigen Human c-mpl was cloned as described by F. de Sauvage et al., Nature 369:533 (1994).

Phage Selection on immunotubes NUNC immunotubes were coated with 2 ml of a solution of 10 microg/ml of gD-c-mpl in PBS at 4C overnight After rinsing with PBS, tubes were blocked with 3% dry milk in PBS (MPBS) for 2 hr at room temperature. Forthe first round, 10 pl of CA.T. antibody phage library containing -1 x 1012 c.f.u. were added to 1 ml MPBS for blocking for I 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*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 pl of I M TRIS (pH and stored on ice until needed. For subsequent rounds, washing was increased to 20 times with PBS-TWEEN, and 20 times with PBS.

Eluted phage were used to infect 5 ml of log phase E. coli TGI cells and plated on 2YT agar supplemented with 2% glucose and 100 pg/ml carbenicilin. After overnight growth at 30 0 C, colonies were scraped into 10 ml 2YT. 50 pl of this solution was used to inoculate 25 ml of 2YT with carbenicillin and glucose and incubated, shaking, for two hours at 37°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 (100Ig/ml) and kanamycin (50pg/ml) and growth continued at 301C for 4 hr- E. coli were removed from the phage by centrifugation, and 1 ml of these phagc'(approx. 10" were used in subsequent rounds of selection.

fc) Antibody Phage Selection using streptavidin-coated paramagnetic beads The library was also selected using soluble biotinylated antigen and streptavidin-coated paramagnetic beads (see Fig. gD-c-mpl was biotinylated using IMMUNOPURE NHS-biotin (biotiny-N-hydroxysuccinimide, Pierce) according to manufacturer's recommendations.

For the first round of panning, 10 pl of the phage library were blocked with 1 ml of MPBST dry milk powder, I x PBS, 0.2% TWEEN 20) for I hour on a rotating wheel at room temperature. Biotinylated gDc-mpl was then added to a final concentration of 100 nM, and phage were allow to bind antigen for I hour at 37 0 C on a rotator. Meanwhile, 300 pl of DYNABEADS M-280, coated with strcptavidin (DYNAL) were washed 3 times with I ml MPBST (using a DYNAL Magnetic Particle Concentrator) and then blocked for 2 hr at 37°C with I ml fresh MPBST on a rotator. The beads and were collected with the MPC, resuspended in pl 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 I ml PBS-TWEEN, once with MPBS, followed by 3 times with PBS. Phage were cluted from the beads by incubating 5 min at room temperature with 300 pl of 100 mM triethylamine. The phage-containing supernatant was removed and neutralized with 150 pl of IM TRIS-HCI (pH Neutralized phage were used to infect mid-log TGI host cells as described above. Plating, induction and harvesting of phage were also as for selection on tubes.

For the second and subsequent rounds of selection on biotinylatcd gD-c-mpl, I ml of harvested phage (approximately 1012 cfu) were blocked with 200 pl 10% dry milk. 6 X 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, I 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, I nM. Washing stringency was increased to two cycles of 7 washes for rounds 2. and three cycles for rounds 3 and beyond.

ELISA screenine of selected clones After each round of selection, individual carbnicillin-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.

Individual clones were inoculated into 2TY with 2% glucose and 100 pg/ml carbcnicillin in 96-well tissue culture plates and grown until turbid. Cultures were then infected at an m.o.i. of 10 with M12K07 helper phage, and infected cells were transferred to 2YT media containing carbenicillin (100 pg/ml) and kanamycin pg/ml) for growth overnight at 30°C with gentle shaking.

NUNC MAXISORP microtiter plates were coated with 50 pl per well of gD-c-mpi, BSA, or gDgpl20, at 2 pg/ml in 50 mM carbonate buffer (pH at 4°C overnight. After removing antigen, plates were blocked with 3% dry milk in PBS (MPBS) for 2 hours at room temperature.

Phage cultures were centrifuged and 100 pt of phage-containing supematants were blocked with pl of 6 x PBS 18 dry milk for I hour at room temperature. Block was removed from titer plates and blocked phage added and allowed to bind for I hour at room temperature. After washing, phage were detected with a -44- 1:5000 dilution of horseradish peroxidase-conjugated anti-M13 antibody (Phamnacia) in MPBS followed by 3',3'.5'.5'-tetramethylbcnzidinc (TMB). Reactions were stopped by the addition of H 2

SO

4 and readings taken by subtracting the A40n m from the A450n.

Soluble scFv ELISA Soluble scFv was induced in the bacterial supematants of clones by growth in 2YT containing carbenicillin (100 .g/mi) and IPTG (tmM) ON at 30*C. ELISA plates were either coated with gD-c-mpl or, for capture ELISA, with anti-c-myc Mab 9EIO. Plates were blocked with I x ELISA diluent (PBS supplemented with 0.5% BSA, 0.05% Tween 20, pH and soluble scFv was blocked by adding 20 pl of 6 x ELISA dil to 100 pl of supernatant. After binding to antigen coated plates, soluble scFv was detected by adding 50 pl of 1 pg/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.

The number of clones screened by ELISA from each round, and the number of clones positive by phage ELISA are shown in Table 3.

Table 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 proliferatioh assay: 4 DNA fingerprintine ofclones The diversity of c-mpl-binding clones was determined by PCR amplifying the scFv insert using primers pUCI9R (5 AGC GGA TAA CAA TIT CAC ACA GO 3 (SEQ. ID. NO: 54) which anneals upstream of the leader sequence and fdtetseq (5 GTC GTC TT CCA GAC GGT AGT 3 (SEQ. ID. NO: 55) which anneals in the 5 end of gene [II, followed by digestion with the frequent-cutting restriction enzyme BstNI (see Fig. 7).

Typical patterns seen after analysis on a 3% agarose gel are shown in Fig. 8A-C.

DNA Fingerprinting: Protocol Mix A: dH20 67 pl x ampliTaq buffer mM MgC12 DMSO, 50% 2 forward primer I Mix B: 2.5 mM dNTPs 8 pl AMPLITAQ reverse primer Place 90 pl of Mix A in reaction tube Inoculate with very small portion of E.coli colony using a yellow tip Heat in PCR block to 98*C, 3 min. Reniove to ice.

Add 10 pl Mix B Cycle: 95° C, 30 sec, 55°C 30 sec, 72*C Imin 20 sec, for 25 cycles, in Pcrkin Elmer 2400 Remove 10 pl ro run on a I agarose gel, test for a 1 kB band Make remaining mix to I x BstNl reaction buffer Add 5 units BstNI 60°C,2 hours Electrophorese samples on 3 METAPHORE agarose gel Sequencing of clones 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 pg/ml carbenicillin, and grown overnight at 30*C. DNA was isolated using Qiagen Tip-100s and the manufacturer's protocol and cycle sequenced with fluorescent dideoxy chain terminators (Applied Biosystcms). 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.

DNA sequence was obtained for 39 clones and resulted in 24 different c-mpl-binding scFvs.

Purification ofscFvs with (his) 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 30C. 0.2 ml of these cultures were diluted into 200 ml of the same media and grown to an OD600 of approximately 0.9. The cells were pelleted and resuspended in 250 ml of 2YT containing IPTG (1 mM) and carbenicillin (100 pg/ml) and to induce expression and grown fora further 5 hours at 30"C. Cell pellets were harvested and frozen at 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-HCI, I mM EDTA, 200 mM NaCI, sucrose, 1 mM PMSF) by shaking on ice for 1 hr. Imidazole was added to 20 rM. and cell debris removed by centrifugation. The supematants were adjusted to ImM MgCI 2 and 50 mM phosphate buffer pH Ni-NTA agarose resin from Qiagen was used according to the manufacturers instructions. The resin was equilibrated with 50 mM sodium phosphate buffer pH 500 mM NaCI, 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 Elution of proteins was with 50 mM phosphate buffer pH 7.5, 500 mM NaCI and 250 mM imidazolc. 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 I ml.

Concentration was estimated spectrophotomctrically assuming an A280 nm of 1.0 0.6 mg/ml.

Portions of these protein preparations wrq'submitted for KIRA assay, c-mpl-Ba/F3 cell proliferation assay, and Hu3 cell proliferation assay.

Plasmid DNA for scFv clones 1285, 12D5, 12E10, 10DIO, 10F6 and 5E5 (named pMpl.12BS.scFv.his; pMpl. 2D5.scFv.his; pMpl. 2E10.scFv.his; pMpl. 10Dl.scFv.his; pMpl.1OF6.scFv.his; and pMpl.5E5.scFv.his, respectively) has been deposited with ATCC, Manassas, Virginia, USA.

Reformatting of antibodies to scFv with RD tlag Fab'. Fab'2, and full length molecules.

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 a. METHODS: A companion to Methods in Enzymology. 3:183-192 (1991). Expression is under the transcriptional control of the E. co/i 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 (stil) signal sequence (Picken, et al.) to direct secretion to the periplasmic space of E coli.. This vector also contains the human k! CL (Palm et al.

Infect. Immun. 42:269-275 (1983)) and the human IgG1 CHI (Ellison, e al. Nucleic Acids Res. 10: 4071-4079 (1982)) constant domains. The ClH gene is immediately followed by the bacteriophage t t ranscriptional terminator (Scholtissek and Grosse Nucleic Acids Res. 15: 3185 (1987)).

Fab' and Fab'2 construction Construction of the Fab' and Fab'2 variants was facilitated by insertion into pAK19 of unique restriction sites at the junctions of the stil and VL domain (Sfi the VI and Ck domains (Rsr II), the stll and V

H

domain (MluI), and the VH and ClI domains (Apa using oligonuclcotide directed mutagenesis. In order to insure expression ofmonovalent Fab' molecules, the free cysteine at the 3' end of the CHI domain was mutated to a threonine, these Fab' molecules thus end in the amino acid sequence thr-ala-ala-pro, rather than thr-cysala-ala as in pAKI9. This vector for the expression of Fab' molecules is named pXCA730.

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 A CL was subcloned from pB 1.2 (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.

For expression of the antibodies as Fab'2 molecules, a vector was constructed which adds the human IgGI hinge region onto the CHt domain of pXCA730. .This is followed by the yeast GCN4 Icucine 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-glyarg-mct-lys-gin-leu-glu-asp-lys-val-glu-glu-cu-iu-ser-lys-asn-tyr-his-leu-glu-asn-glu-val-ala-arg-leu-lys-tysleu-val-gly-glu-arg (SEQ. ID. NO: 56). The resultant plasmid is named pXCA740.

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 1 or V, region as shown below.

12B5, 12D5, and 10DI0 Light chain variable domains: primer GCT TCT GCG GCC ACA CAG GCC TAC GCT GAC ATC GTG ATG ACC C (SEQ. ID. NO: 57) 3 primer ATG ATG ATG TGC CAC GGT CCG TrT GAT CTC CAG TTC GGT C (SEQ. ID. NO: 58) 12E10 Light chain variable domain: primer GCT TCT GCG GCC ACA CAG GCC TAC GCT TCC TAT OTG CTG ACT C (SEQ. NO: 59) 3 primer CCTTCT CTC TIT AGG 0TG GCC AAG GAC GGT CAG CTT GGT C (SEQ. ID. NO: 1 0F6 Light chain variable domain primer GCT TCT GCG GCC ACA CAG GCC TAC GCT CAG TCT GTG CTG ACT C (SEQ. ID. NO: 61) 3 primer CCT TCT CTC TIT AGG TG GCC AAG GAC GGT CAG CTT GGT C (SEQ. ID. NO: 1285 Heavy chain variable domain primer CAT TCT ACA AAC GCG TAC GCT CAG GTG CAG CT GTG CAG (SEQ. ID. NO: 62) IS 3 primer GTA AAT GTA TGG GCC CTT GGT GGA GGA GGC ACT CGA GAC GOT GAC (SEQ. ID. NO: 63) 12DS Heavy chain variable domain primer CAT TCT ACA AAC GCG TAC GCT CAG GTG CAG CTG GTG GAG (SEQ. ID. NO: 64) 3 primer GTA AAT GTA TGG GCC CTT GGOT GA GGA GGC ACT CGA GAC GGT GAC (SEQ. ID. NO: 63) Heavy chain variable domain 5 primer CAT TCT ACA AAC GCG TAC GCT GAC GTG CAO CTG GTG CAG (SEQ. ID. NO: 3 primer GTA AAT GTA TGO GCC CTT GGT GGC GGC TGA GGA GAC GGT GAC(SEQ. ID. NO: 66) 12E10 Heavy chain variable domain 5 primer CAT TCT ACA AAC GCG TAC GCT CAG GTG CAC CTG CAG CAG (SEQ. ID. NO: 67) 3 primer GTA AAT GTA TGG GCC CT T GT GGA GGA GGC ACT CGA GAC GGT GAC (SEQ. ID. NO: 63) I0F6 Heavy chain variable domain primer CAT TCT ACA AAC GCG TAC GCT CAG GTO CAG CTC CAG GAG (SEQ. ID. NO: 68) 3 primer OTA AAT GTA TGG GCC CTT GGT GGA GGC TGA AGA GAC GGT AAC (SEQ. ID. NO: 69) -48- PCR reactions were carried out using 106 ng of plasmid DNA containing the scFv, 0.5 pM of the appropriate 5 and 3 primer, 200 gM each dNTP, 10 mM KCI, 6 mM(NH 4 2

SO

4 20 mM TRIS-HCI, pH B.0O 2 mM MgCI 2 1% Triton X-100, 100 M BSA and2.5 units of Pfu DNA polymerase (Stratagene). Amplification was for 30 cycles of: 30 sec at 95 30 sec at 55*C, 30 sec at 72°C. After digestion with the appropriate restriction enzymes, the reaction products were separated by agarose gel clectrophoresis and the approximately 350 bp band was isolated using a Gene Clean II kit (BIO 101, Vista, CA). 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 colt 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.

Construction of scFv with eD tags.

For increased and regulated expression in high density fermentation tanks, the Sfi I to Not I fragments of the scFv forms ofpl 2B5, pl2DS, plOF6, and pI2E10 were subcloned into a derivative of pAKI9 containing the phoA promoter and stil signal sequence rather than the lacZ promoter and hybrid signal sequence of the original library. For case of purification, a DNA fragment coding for 12 amino acids (met-ala-asp-pro-asn-argphe-arg-gly-lys-asp-leu) (SEQ. ID. NO: 70) derived from herpes simplex virus type I glycoprotein D (Lasky and Dowbenko DNA 3:23-29 (1984.)) was synthesized and inserted at the 3 end of the V L domain in place of the (his) 6 and c-myc epitope originally present in the C.A.T. library clones.

Expression in E coli Plasmids containing genes for scFv-gD, Fab' or Fab'2 molecules were expressed in E. coi strain 33B6 (W3110 D/huA phoADEIS deoC2 l/vG2096(val) degP41(DPst-Kan) D(argF-lac)169 IN(rrnD-rrnE)l) grown for approximately 40 hr at 30*C in an aerated 10-liter fermentor as described previously (Carter et oa BiolTechnology 10:163-167(1992.)).

Example Clonin and expression of full length human antibody derivatives of 12B5. 12D5. and 12E10.

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 IgGI CHI, CH2, and CH3 domains and a human antibody signal sequence (Carter et al., Proc. Nail. 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 adenovirustransformed 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.

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 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

A

plasmid with expression driven by a second SV40 promoter/ enhancer. The plasmid was linearized and transfccted 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.

Conditioned media for purification of the antibodies was generated in roller bottles. Cells were seeded into the roller bottles at an initial cell density of2 x 107 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 pg/ml), transferrin (10 pg/rm), trace elements and lipid alcohol. Conditioned media was harvested after 10 days.

Example 4 Purification Of Agonist Antibodies Purification of scFv with gD tag Frozen cell paste was resuspended at I gm/ml TE (25 mM TRIS, I mM EDTA, pH 7.4) and gently agitated I1 hr on ice. Cell debris was removed by centrifugation at 10,000 x g for 30 min. The supematant was loaded onto an affinity column (2.5 x 9.0 cm) consisting of an anti-gD monoclonal antibody 5B6 (Paborsky, L.

RI e 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 NaCI until the absorbance of the column effluent was equivalent to baseline. All steps were done at 4°C at a linear flow rate of cm/hr. Elution was performed with 0.1 M acetic acid, 0.5 M NaCI, 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.

Purification of Fab' molecules For purification of Fab' molecules, 5 g of frozen cell paste was resuspended in 5 mI of TE (25 mM TRIS, 1 anM EDTA, pH 7.4) and gently stirred 1.8 hr on ice. The pH of the shockate was adjusted to 5.6 with 2 M HCI and the precipitate and cell debris removed by centrifugation at 10,000 x g for 30 min. The supernatant was loaded onto a I ml BAKERBOND ABx column (0.5 x 5.0 cm) T. Baker, Phillipsburg, NJ) preequilibrated with 20 mM MES, pH 5.S. 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 4 2 SO4, pH 7.2, with a flow rate of 153 cm/hr. Fractions containing Fab' were pooled, and buffer exchanged into PBS.

Purification of Fab'2 molecules Frozen cell paste (100 gm) was thawed into 10 volumes of 25 mM TRIS, 5 mM EDTA, I m M 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 x g for 30 min. The supernatant was filtered sequentially through a 0.

4 5pm, and a 0.2 pm SUPORCAP filter (Geman), 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 1/10 volume of 1 M TRIS, pH 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 prc-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 25mM NaOAc over 10 column volumes. Fractions containing Fab'2 were pooled and buffer exchanged to PBS.

Purification of full length antibodies from transfected CHO cell supernatants.

Conditioned medium harvested from roller bottles was loaded onto a 5 ml Protein-A SEPHAROSE column (1.0 x 5.0 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 NaCt, pH 2.9, neutralized with I M TRIS, and buffer exchanged to PBS.

A summary of agonist antibody activities for several antibodies and fragments thereof is shown in Table 4 below.

Table 4 Summary ofMpl Agonist Antibody Activities Antibody Hu3 Proliteration KIKA Hu3 Binding Mpl IPU tLbA Platelets MK. Assay (ED50) (ICSO) (IC50) (ICSO) scFv 20 pM I nM 10 nM 17nM 100 nM Fab >1gM 3nM 900 nM none >iM Fab'2 5 pM 1 M 5 a M 1nM 300 nM IgG 30 pM 400 pM 10 nM 152 pM 300 nM 12EIU scFv 5 pM 60 pM 5 nM 1.6 nM 5 nM Fab >lM M>M 500nM 180 nM >IM Fab'2 >1RM 160 pM 10 nM 640 nM 500 nM IgG >IgM 480pM 0O nM 450 pM 500 nM scFv 12 nM 280 pM 0nM 24 nM >IpM Fab >1IM 4nM 500 nM lpM Fab'2 4.8 pM 600 pM 4 nM I nM 100 nM IgG >IPM 3 nM 10 nM 450 pM 500 nM Example In another embodiment, the invention provide ai 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.

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 -51tertiary 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 hematopoictic receptors that lack kinase activity.

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 hcmatopoietic cytokine receptors that lack intrinsic tyrosine kinase activity (but associate with intracellular kinases), it is d 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.

MuSK is a recently identified tyrosine kinase localized to the postsynaptic surface of the neuromuscular junction. (Valenzuela e. at. 1995. Neuron 15 573-584.) Mice made deficient in MuSK fail to form neuromuscular junctions (Dechiara e at.. 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. at. 1996, Cell 85 513-523.).

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-ANeuroscL I8 443-462 and Kleiman et. at., 1996, Cell 85 461-464.). A striking feature of this complex process is the redistribution and concentration ofAChRs within the myotube membrane. Agrin is able to the induce this clustering of AChRs as well as changes in the extracellular matrix and cytoskelctal components of the synaptic apparatus (Bowe e. al..

supra, Godfrey et. al.,1984, J. Cell Biol. 99 615-627; Nitkin et. 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 (OToole et. al.. 1996, Nal. Acad Sci.

USA 93 7369-7374) thereby complicating the analysis of MuSK's role in the signaling events initiated by agrin.

Antigen specific scFv, identified by panning a diverse library ofscFv expressed, for example, on M1 3 phage provide a source ofmolecules 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.

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 ofagrin, with an agent directed specifically to MuSK.

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 e.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. 1991, Neuron 6 811-823; Tsim et.

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.

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 neuromuscularjunctions in these people.

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 intraccllular domain of the hematopoietic cytokine receptor c-Mpl (the receptor for thrombopoictin, 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 .Nal. 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. II1). This activity was observed at nM concentrations ofscFv. 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 ofagrin 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 e. al., supra).

The EC 50 for the ability to induce proliferation was 5 nM for the most active agonist clone when expressed as either seFv or IgG. The affinity of these scFv and IgG for MuSK was determined using BIAcore T M analysis.

The agonist scFv and several non-agonist scFveachidisplayed 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.

Table clone# Agonist k d ka Attmty musk #2-sc-v 334 x 10-- 8.78 x 105 3.g nM musk #3-scFv 2.39 x 10- 3 1.05 x 10 5 23 nM musk #4-scFv 1.57 x 10- 3 .84 x 105 8.5 nM musk #5-scFv -2.49 x 10- 3 5.29 x 105 4.7 nM musk #6-scFv 4.95 x 10- 3 1.05 x 105 4.7 nM musk #13-scFv 2.32 x 10- 3 4.53 x 105 5.1 nM musk #22-scFv 6.09 x 10-3 127 x105 4.8 nM musk #13-lgG 1.01 x 10- 5 .05 x 105 12.5 pM musk #22-lgG 4.86 x 10- 5 1.65 x 10 6 29.5 pM 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 nonagonist anti-MuSK IgG were without effect.

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 IgO. 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 nonagonist 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. Tyrosinc 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.

Variants of the MuSK agonist antibodies of the invention may be prepared as described above for thrombopoietic antibodies.

Construction of expression vctors. Coding sequence for murine MuSK was obtained by PCR amplification. MuSK-Fc was prepared by fusion, of the extracellular domain of MuSK 1-492) in frame with the Fc region of human IgGI in the cukaryotic 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 1-492) aid the transmembrane and intracellular domain of the human c-Mpl receptor (amino acids 491-635) was prepared by sequential PCR and cloned into Stable cell lines expressing the chimeric receptor were obtained by clcctroporation (5 million cells, 250 volts, 960 pF) of linearized vector (20 pg) into Ba/F3 cells followed by selection for neomycin resistance with 2 mg/ml G418. Full length MuSK in pRK5tkNECIO as transfected into 293 cells and stable transformants were obtained following two weeks of G418 selection (400 pg/ml). The sequence of the DNA constructs were confirmed by DNA sequencing. Expression of MuSK was assessed by flow cytomctry analysis as described below. Ba/F3 cells were maintained in RPMI 1640 media supplemented with 10% fetal calf serum and 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 theN-terminus.

Isolation of scFv and lgG MuSK-Fc immunoadhesin was coated on Maxisorp tubes (Nunc) at pig/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 BstNl 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 (105) were incubated for minutes at 4 C in 200 pl 2% FBS/PBS (fetal bovine serum/phosphate buffered saline) with 1010 phage that were first blocked by incubation in 30 pl 10%/ FBS/PBS. Cells were then washed with 2% FBS/PBS. stained with anti-M13 antibody (Pharmacia, Piscataway NJ) 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 (his6) and were purified over Ni-NTA column with imidazole clution as recommended by manufacturer (Qiagen). For expression of clones as IgG the sequences encoding the VH and VL 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.

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, I pCi of[ 3 H-thymidine was added per well and incubation was continued for an additional six hours. Incorporation of [3Hj-thymidine was measured with a Top Count Counter (Packard Instruments, CA).

AChR clustering assay. C2CI2 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 cagrin were added to the culture medium and incubated overnight (16 hours). Cells were then washed with PBS and fixed in 4% paraformaldehyde. Rhodamine conjugated bungartoxin (Molecular Probes, Eugene OR) was used to reveal the localization of AChRs as described (Ferns e al, supra).

Binding affinity analysis. Protein interaction analysis using BIAcorcTM instruments was performed as described (Mark e al, 1996, J. Biol. Chem. 271 9785-979). Briefly, research grade CM5 sensor chips were activated by injection of 20 pl of 1:1 N-Ethyl-N'-(3-dimethylaminopropyl)carboiimide hydrochloride and Nhydroxysuccinimidc at 5 pl /min flow rate. 20 pi ofMuSK-Fe at 20 pg/min in 10 mM sodium acetate, pH was injected over the sensor chip, followed by 30 pi of ethanolamine. scFv or IgG were purified and concentrations determined by Pearce BCA kit. Thirty 4 i protein samples in PBS with 0.05% Tween 20 were injected at a flow rate of 10 pl 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 BLAcvaluation 2.1 software from Pharmacia Biosensor AB. Apparent dissociation rate constants (kd) and association rate constants (ka) were obtained by evaluating the scnsorgram with A B- AB type I fitting. Equilibrium dissociation constant Kd was calculated as kdka.

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 cagrin containing conditioned media was used at level that provided maximal tyrosinc phosphorylation. Cell extracts were prepared as described (Gurney etat. supra). Extracts were incubated for 60 minutes at 4*C with pi agarose conjugated antiphosphotyrosine monoclonal antibody 4G10 CUBI inc., Lake Placid NY) or I pg of anti-MuSK IgG #13 followed by 30 pi protein A scpharose beads. Western blot analysis with antiphosphotyrosine antibody 4GIO or anti AChR antibody (Affinity Bioragents, Golden CO) was performed as recommended by the manufacturer and revecled with HRP conjugated secondary antibody and ECL (Amersham).

MuSK scFv are readily observed as dimers when resolved by nordenaturing gel electrophoresis.

Additionally, the abundance ofdimeric species may be significantly altered in the local context of scFv bound to receptor on the cell surface. Alternatively, screning an scFv phage library with a divalent antigen, in this case MuSK-Fc, allows direct selection ofscFv that bind tb and facilitate the formation of a receptor dimer.

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.

All references cited herein are hereby expressly incorporated by reference.

Deposit of Material The following materials have been deposited with the American Type Culture Collection.

10801 University Boulevard. Manassas, Virginia, 20110-2209 USA (ATCC): Material ATCC Dep. No. Deposit Date pMpl.12BS.scFv.his 203125 August 18. 1998 -56- 203121 August 18, 1998 pMpl.12E10.scFv.his 203120 August 18, 1998 pMpl.ODlO.scFv.his 203124 August 18. 1998 pMpl.lOF6.scFv.his 203122 August 1, 1998 pMpL5ES.scFv.his 203125 August IS, 1998 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 US. 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 US. Commissioner of Patents and Trademarks to be entitled thereto according to USC 1122 and the Commissioners rules pursuant thereto (including 37 CFR §1.14 with particular reference to 886 OG 638).

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 consrued as a license to practice the invention in contravention of thi rights granted under the authority of any government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The presnct invention is not *o 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 dtereof nor is it to be construed as limiting the scope of the claims to the specific illustrations that it represents. lndcld, 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.

Throughout this specification and the claims, the words "comprise", "comprises" and "comprising" are used in a non-exclusive sense, except where the context requires otherwise.

It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

Claims (20)

1. An agonist antibody, fragment, or variant thereof which binds to human c- mpl, which is selected from the group consisting ofAbl, Ab2, Ab3, Ab4, Ab5 and Ab6, wherein each Abl-Ab6 comprises a VH and VL chain, each VH and VL chain comprising CDR amino acid sequences designated CDRI, CDR2 and CDR3 separated by framework amino acid sequences, the amino acid sequence of each CDR in each VH and VL chain of Abl-Ab6 is selected according to the following table: Abl: VHCDR1 VHCDR2 VHCDR3 (SEQ ID NO: 1) (SEQ ID NO: 3) (SEQ ID NO: (SEQ ID NO: 2) (SEQ ID NO: 4) (SEQ ID NO: 6) VLCDR1 VLCDR2 VLCDR3 (SEQ ID NO: 7) (SEQ ID NO: 9) (SEQ ID NO: 11) (SEQ ID NO: 8) (SEQ ID NO: 10) (SEQ ID NO: 12) Ab2: VHCDR1 (SEQ ID NO: 13) (SEQ ID NO: 14) VLCDRI (SEQ ID NO: 19) (SEQ ID NO: 20) VHCDRI (SEQ ID NO: 25) (SEQ ID NO: 26) VHCDR2 (SEQ ID NO: 15) (SEQ ID NO: 16) VLCDR2 (SEQ ID NO: 21) (SEQ ID NO: 22) VHCDR3 (SEQ ID NO: 17) (SEQ ID NO: 18) VLCDR3 (SEQ ID NO: 23) (SEQ ID NO: 24) VHCDR3 (SEQ ID NO: 29) (SEQ ID NO: Ab3: VHCDR2 (SEQ ID NO: 27) (SEQ ID NO: 28) 71 O Va 0 O 0 i 0 Ab4: Ab6: VLCDR1 (SEQ ID NO: 19) (SEQ ID NO: 20) VHCDR1 (SEQ ID NO: 25) (SEQ ID NO: 26) VLCDR1 (SEQ ID NO: 35) (SEQ ID NO: 20) VHCDR1 (SEQ ID NO: 36) (SEQ ID NO: 37) VLCDR1 (SEQ ID NO: 19) (SEQ ID NO: 20) VHCDRI (SEQ ID NO: 42) (SEQ ID NO: 43) VLCDRI (SEQ ID NO: 48) (SEQ ID NO: 49) VLCDR2 (SEQ ID NO: 21) (SEQ ID NO: 22) VHCDR2 (SEQ ID NO: 31) (SEQ ID NO: 32) VLCDR2 (SEQ ID NO: 21) (SEQ ID NO: 22) VHCDR2 (SEQ ID NO: 38) (SEQ ID NO: 39) VLCDR2 (SEQ ID NO: 21) (SEQ ID NO: 22) VHCDR2 (SEQ ID NO: 44) (SEQ ID NO: 45) VLCDR2 (SEQ ID NO: 50) (SEQ ID NO: 51) VLCDR3 (SEQ ID NO: 23) (SEQ ID NO: 24) VHCDR3 (SEQ ID NO: 33) (SEQ ID NO: 34) VLCDR3 (SEQ ID NO: 23) (SEQ ID NO: 24) VHCDR3 (SEQ ID NO: (SEQ ID NO: 41) VLCDR3 (SEQ ID NO: 23) (SEQ ID NO: 24) VHCDR3 (SEQ ID NO: 46) (SEQ ID NO: 47) VLCDR3 (SEQ ID NO: 52) (SEQ ID NO: 53). 72 D 2. The antibody of Claim 1, wherein the antibody stimulates proliferation, 0 o differentiation or growth of megakaryocytes.

3. The antibody of Claim 1, wherein the antibody stimulates megakaryocytes to produce platelets. 0 4. The antibody of Claim 1, which is selected from the group consisting of ScFv, Fab, F(ab')2 and IgG. enl 5. The antibody of Claim 1, which is a human antibody. C6. The antibody of Claim 1, which is a non-naturally occurring antibody. o7. The antibody of Claim 1, having a detectable label.

8. The antibody of Claim 1, which is a monoclonal antibody.

9. The antibody of Claim 1, which is a single chain antibody. An antibody immobilized on an insoluble matrix, wherein the antibody is the antibody of Claim 1.

11. A composition, comprising the agonist antibody, fragment, or variant thereof of Claim 1 and a pharmaceutically acceptable carrier.

12. The composition of Claim 11 I, which is sterile.

13. The composition of Claim 11, which is lyophilized.

14. A library of different single chain antibodies, comprising a plurality of the antibody of Claim 9. The library of Claim 14, wherein the single chain antibodies are displayed on phage.

16. The library of Claim 15, wherein the phage is M 13 and the antibodies are displayed as fusions of coat protein 3. 73

17. The library of Claim 16, wherein less than 20% of the phage display more O than one fusion on the surface thereof.

18. A phage displaying on the surface thereof the antibody of Claim 9.

19. The phage of Claim 18, wherein the phage is MI3 and the antibodies are 0 displayed as fusions of coat protein 3. C 20. The phage of Claim 19, wherein the phage displays only one fusion on the C surface thereof. C 21. 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.

22. The fusion protein of Claim 21, wherein the phage coat protein is M13 coat protein 3.

23. A method of stimulating proliferation, differentiation or growth of megakaryocytes, comprising contacting megakaryocytes with an effective amount of the antibody of Claim 1.

24. The method of Claim 23, comprising administering the antibody of Claim 1 to a patient in need thereof. A method of increasing platelet production, comprising contacting megakaryocytes with an effective amount of the antibody of Claim 1.

26. The method of Claim 25, comprising administering the antibody of Claim 1 to a patient in need thereof.

27. An isolated nucleic acid encoding the antibody of any one of Claims 1 to 9.

28. A vector comprising the nucleic acid of Claim 27.

29. A host cell comprising the vector of Claim 28.

74- I O 30. A method of producing an agonist antibody comprising culturing the cell of 0 o Claim 29 under conditions wherein the nucleic acid is expressed. 31. An agonist antibody, fragment, or variant thereof which binds to human c- mpl, comprising an amino acid sequence selected from the group consisting of 12E10, 12B5, 10F6 and 12D5. Ci 32. An isolated nucleic acid encoding the antibody of Claim 31. eln 33. A vector comprising the nucleic acid of Claim 32. l 34. A host cell comprising the vector of Claim 33. 0 o 35. A method of producing an agonist antibody comprising culturing the cell of Claim 34 under conditions wherein the nucleic acid is expressed. Dated this 5 t h day of May 2006 GENENTEC H INC. By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia 75