Bmp-alk3 antagonists and uses for promoting bone growth

ES 2 575 695 T3

DESCRIPTION

BMP-ALK3 antagonists and their uses to stimulate bone growth

Background of the invention

Bone-associated disorders, ranging from osteoporosis to fractures, represent a set of disease states for which there are few effective pharmaceutical agents. Instead, treatments focus on physical and behavioral interventions, such as immobilization, exercise, and dietary changes. It would be beneficial to have therapeutic agents that promote bone growth and increase bone density in order to treat a variety of bone disorders.

Bone growth and mineralization depend on the activities of the two types of cells, osteoclasts and osteoblasts, although chondrocytes and cells of the vasculature also participate in critical aspects of these processes. During development, bone formation occurs through two mechanisms, endochondral ossification and intramembranous ossification, the first being responsible for the formation of longitudinal bone and the second responsible for the formation of topologically flat bones, such as the bones of the skull. Endochondral ossification requires the sequential formation and degradation of cartilaginous structures in the growth plates that serve as templates for the formation of osteoblasts, osteoclasts, vasculature, and subsequent mineralization. During intramembranous ossification, bone is formed directly in connective tissues. Both processes require infiltration of the osteoblasts and subsequent deposition of the matrix.

Fractures and other structural disturbances of bone heal through a process that, superficially at least, resembles the sequence of developmental events of osteogenesis, including the formation of cartilage tissue and subsequent mineralization. The fracture healing process can occur in two ways. Direct or primary bone healing occurs without callus formation. Indirect or secondary bone healing occurs with a precursor stage of the callus. Primary fracture healing involves the reform of mechanical continuity through a tightly established break. Under suitable conditions, the bone resorption cells surrounding the rupture show a tunnel resorption response and establish pathways for blood vessel penetration and subsequent healing. Secondary bone healing follows a process of inflammation, soft callus formation, callus mineralization, and callus remodeling. In the inflammation stage, bruising and bleeding results in disruption of the periosteal and endosteal blood vessels at the site of injury. Inflammatory cells invade the area. In the soft callus stage, cells produce new vessels, fibroblasts, intracellular material, and support cells, forming granulation tissue in the space between the fracture fragments. The clinical attachment through the tear is established by fibrous or cartilaginous tissue (soft callus). Osteoblasts form and mediate the mineralization of the soft callus, which is then replaced by lamellar bone and undergoes normal remodeling processes.

In addition to fractures and other physical disturbances of bone structure, loss of bone mineral content and bone mass can be caused by a wide variety of conditions and can result in significant medical problems. Changes in bone mass occur in a relatively predictable way during an individual's life. Until about age 30, men's and women's bones grow to maximum mass through linear growth of the endochondral growth plates and radial growth. After approximately 30 years of age (for the trabecular bone, for example, flat bones such as the vertebrae and pelvis) and at 40 years of age (for the cortical bone, for example, long bones found in the extremities ), slow bone loss occurs in both men and women. In women, a final phase of substantial bone loss also occurs, probably due to post-menopausal estrogen deficiencies. During this phase, women may lose an additional 10% of bone mass from the cortical bone and 25% from the trabecular compartment. Whether progressive bone loss results in a pathological condition, such as osteoporosis, it depends largely on the person's initial bone mass and whether there are conditions that aggravate it.

Bone loss is sometimes characterized as an imbalance in the normal bone remodeling process. Healthy bone is constantly being remodeled. The remodeling begins with the resorption of bone by the osteoclasts. The resorbed bone is then replaced by new bone tissue, which is characterized by collagen formation by osteoblasts and subsequent calcification. In healthy people, the rates of resorption and formation are balanced. Osteoporosis is a progressive and chronic condition, marked by a shift towards resorption, resulting in an overall decrease in bone mass and bone mineralization. Osteoporosis in humans is preceded by clinical osteopenia (a bone mineral density that is greater than one standard deviation, but less than 2.5 standard deviations below the mean value for young adult bone). Worldwide, approximately 75 million people are at risk for osteoporosis.

Therefore, methods to control the balance between osteoclasts and osteoblast activity may be useful in promoting the healing of fractures and other bone damage, as well as treating disorders, such as osteoporosis, associated with loss. bone mass and bone mineralization.

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With regard to osteoporosis, as therapeutic interventions, all are used, estrogen, calcitonin, osteocalcin with vitamin K or high doses of calcium in the diet. Other therapeutic approaches for osteoporosis include bisphosphonates, parathyroid hormone, calcimimetics, statins, anabolic steroids, lanthanum and strontium salts, and sodium fluoride. Such therapeutic agents, however, are often associated with undesirable side effects.

Bone loss is also a major complication of many types of cancer and can be caused by tumor metastasis in the bone, activation of osteoclasts, or the effects of chemotherapy treatment. In particular, anti-estrogen therapies that are widely used in the treatment of breast cancer can cause significant bone loss.

Other bone disorders, such as osteogenesis imperfecta, can result as a consequence of genetics, development, nutrition, other pathologies and deficiencies.

Therefore, an object of the present disclosure is to provide compositions and methods for promoting bone growth and mineralization.

Summary of the invention

In a first aspect, the present invention provides a polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 7.

The polypeptide can comprise the amino acid sequence of SEQ ID NO: 7 and / or can be at least 95% pure, with respect to protein contaminants, as determined by size exclusion chromatography. The polypeptide can exhibit a dissociation constant for BMP2 or BMP4 of no more than 10^-8 M.

In a second aspect, the present invention provides an isolated polynucleotide comprising a coding sequence for the polypeptide of the first aspect. The isolated polynucleotide may comprise the nucleotide sequence of SeQ ID No: 12.

In a third aspect, the present invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of the second aspect.

In a fourth aspect, the present invention provides a BMP or ALK3 antagonist for use in a method of promoting bone growth, increasing bone density or increasing bone strength, or for use in a method of treating or preventing a bone disorder, wherein the BMP or ALK3 antagonist is selected from the group consisting of:

a) a soluble polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 3;

b) an antibody that binds to BMP2 and inhibits the interaction between BMP2 and ALK3;

c) an antibody that binds to BMP4 and inhibits the interaction between BMP4 and ALK3;

d) an antibody that binds to ALK3 and inhibits the interaction between ALK3 and one or more ALK3 ligands; Y

e) a polypeptide comprising an amino acid sequence that is at least 95% identical to amino acids 8-

117 of SEQ ID NO: 3 or 100% identical to amino acids 8-117 of SEQ ID NO: 3.

The polypeptide of (a) or (b) may have one or more of the following characteristics: i) binds to an ALK3 ligand with a Kd of at least 10^-7 M; and ii) inhibits ALK3 signaling in a cell, can include one or more modified amino acid residues selected from: a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid residue and an amino acid conjugated to an organic derivatizing agent and / or may not bind to human BMP6 or human BMP7.

The antagonist may be a fusion protein that includes, in addition to an ALK3 polypeptide domain, a portion of the polypeptide selected from the group consisting of: an immunoglobulin Fc domain and a serum albumin.

The bone disease that is treated or prevented can be selected from the group consisting of: primary osteoporosis, secondary osteoporosis, post-menopausal osteoporosis, hypogonadal bone loss, tumor-induced bone loss, bone loss induced by cancer therapy, bone metastases, multiple myeloma and Paget's disease.

In the antagonist for use of the fourth aspect, the method may further comprise administering a second bone active agent, which may be selected from the group consisting of: a bisphosphonate, an estrogen, a

ES 2 575 695 T3 selective estrogen receptor modulator, a parathyroid hormone, a calcitonin, a calcium supplement and a vitamin D supplement.

In a fifth aspect, the present invention provides a BMP or ALK3 polypeptide antagonist comprising a first amino acid sequence from the extracellular domain of human aLK3 and a heterologous amino acid sequence, wherein the first amino acid sequence consists of the sequence starting at any of positions 25 to 31 of SEQ ID NO: 1 and ending at any of positions 140 to 152 of SEQ ID NO: 1.

The heterologous amino acid sequence may comprise a constant domain from an antibody or an Fc domain from an IgG, preferably human IgG1.

In a sixth aspect, the present invention provides a polypeptide comprising or consisting of an amino acid sequence that is at least 95%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of : SEQ ID NOs: 11, 14, 20, 22, 23, 25, 26, 28, 29, 30, 31, 33, 34, 35, 36, 38, 39, 40 and 41.

The polypeptide of the sixth aspect may comprise or consist of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 11, 14, 20, 22, 23, 25, 26, 28, 29, 30, 31, 33 , 34, 35, 36, 38, 39, 40 and 41.

The polypeptide of the first and sixth aspects can be glycosylated, can stimulate bone formation or increase bone mineral density in an animal, or can be produced by expression in a CHO cell. The polypeptide of the sixth aspect can be encoded by an isolated polynucleotide comprising a nucleic acid sequence that is at least 95%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from: SEQ ID NOs: 12, 15, 21, 24, 27, 32 and 37. The polypeptide can comprise a nucleic acid sequence selected from: SEQ ID NOs: 12, 15, 21, 24, 27, 32 and 37.

The invention also provides a homodimer comprising two polypeptides of the first aspect or two polypeptides of the second aspect, as well as a pharmaceutical preparation comprising the polypeptide of the first or sixth aspect or said homodimer and a pharmaceutically acceptable excipient. The pharmaceutical preparation can be pyrogen-free.

Also provided by the invention is a cell comprising the polynucleotide of the second aspect or a polynucleotide comprising a nucleic acid sequence that is at least 95%, 97%, 98% or 99% identical to a nucleic acid sequence selected from from: SEQ ID NOs: 12, 15, 21, 24, 27, 32 and 37. The polypeptide may comprise a nucleic acid sequence selected from: SEQ ID NOs: 12, 15, 21, 24, 27, 32 and 37. The cell can be a mammalian cell, such as a CHO cell or a human cell.

In part, the disclosure demonstrates that molecules having ALK3 or BMP antagonist activity (ALK3 antagonists and BMP antagonists) can be used to increase bone density, promote bone growth, and / or increase bone strength. This observation is particularly surprising given the large body of literature and clinical experience indicating that many BMPs, and particularly BMP2, BMP4, and BMP7, are potent stimulators of bone formation. The disclosure demonstrates that a soluble form of ALK3 acts as an inhibitor of BMP-ALK3 signaling and promotes increased bone density, bone growth, and bone strength in vivo. Although it is not desired to be linked to any particular mechanism, it appears that the soluble form of ALK3 achieves this effect by inhibiting BMP2 and / or BMP4, and perhaps other ligands that signal through ALK3. Therefore, the disclosure states that antagonists of the BMP-ALK3 signaling pathway can be used to increase bone density and promote bone growth. While soluble ALK3 may affect bone through a mechanism other than, or in addition to, BMP antagonism, the disclosure, however, demonstrates that desirable therapeutic agents can be selected on the basis of BMP-ALK3 antagonist activity. Therefore, BMP-ALK3 antagonists can be used, including, for example, ALK3 polypeptides that bind to BMP, anti-BMP antibodies, anti-ALK3 antibodies, small molecule antibodies directed against BMP or ALK3, and ALK3 aptamers. and nucleic acids that decrease the expression of BMP and ALK3, to treat disorders associated with low bone density or low bone resistance, such as osteoporosis, or to promote bone growth in patients in need thereof, such as in patients who have bone fractures. In certain embodiments, the invention provides truncated forms of ALK3 polypeptides (eg, ALK3-Fc polypeptides) that have advantageous properties and that retain the appropriate binding of BMP2 or BMP4.

Described herein are polypeptides that comprise a soluble ALK3 polypeptide that binds to BMP2 and / or BMP4. The soluble ALK3 polypeptide can bind additional ligands as well. ALK3 polypeptides can be formulated as a pharmaceutical preparation comprising ALK3 polypeptide that binds to BMP and a pharmaceutically acceptable carrier. Preferably, the BMP-binding ALK3 polypeptide binds bMP2 and / or BMP4 with a Kd of less than 1 micromolar or less than 100, 10, or 1 nanomolar. Preferably, the composition is at least 95% pure, relative to other polypeptide components, as evaluated by size exclusion chromatography, and more preferably, the composition is at least 98% pure. An ALK3 polypeptide

ES 2 575 695 T3 that binds to BMP for use in a preparation can be any of those described herein, such as a polypeptide having an amino acid sequence selected from SEQ ID NOs: 3, 7, 11, 14, 20, 22, 23, 25, 26, 28, 29, 30, 31, 33, 34, 35, 36, 38, 39, 40, or 41, or has an amino acid sequence that is at least 80%, 85% , 90%, 95%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NOs: 3, 7, 11, 14, 20, 22, 23, 25, 26, 28, 29, 30, 31, 33, 34, 35, 36, 38, 39, 40 or 41, including N- and / or C terminal truncations of no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids of SEQ ID NO: 3, and optionally fused to an Fc fusion protein, with or without a linker. In particular, the disclosure provides ALK3 polypeptides with a truncation of 0 to 7 amino acids at the N-terminus of the ECD portion of ALK3 and 0 to 12 amino acids at the C-terminus of the ECD portion of ALK3, describing as thus a portion of the function corresponding to amino acids 8 to 117 of SEQ ID NO: 3 and polypeptides comprising a protein that is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence 8 to 117 of SEQ ID NO: 3. Notably, human ALK3 and murine ALK3 have 97 to 98% identity at the amino acid sequence level in the extracellular domain, and the proteins comprising such human or mouse protein domains are shown herein to exhibit similar activity in vitro and in vivo. An ALK3 polypeptide that binds to BMP can include a functional fragment of a wild-type ALK3 polypeptide, such as one comprising at least 10, 20, or 30 amino acids of a sequence selected from SEQ ID NOs: 1 or 3. Surprisingly, as demonstrated herein, ALK3 proteins that include an amino acid deletion in the C-terminal region of the extracellular domain of ALK3 retain activity against BMP2 and BMP4 while decreasing activity against other ligands (e.g., BMP6 and BMP7) thus providing an improvement in the selectivity of the ligand, that it is generally desirable to decrease unanticipated target-off effects in clinical development or commercialization. Such variations can include a deletion of no more than 6 or 7, no more than 12 or no more than 24 amino acids from the C-terminus of SEQ ID NO: 3. Optionally, a truncated form at the C-terminus may also be truncated. by no more than 1, 2, 3, 4, 5, 6 or 7 amino acids at the N-terminus. The aforementioned variations of ALK3 proteins can be included in an ALK3-Fc fusion protein, which can comprise any linker described herein (or no linker at all), including a linker having the sequence GGG or TGGG or SGGG, and a Fc portion derived from a human IgG1, IgG2, IgG3 or IgG4 or other mammalian immunoglobulin.

A soluble BMP-binding ALK3 polypeptide can include one, two, five, or more amino acid sequence alterations (eg, in the ligand-binding domain) relative to a naturally-occurring ALK3 polypeptide. The alteration in the amino acid sequence can be, for example, altering the glycosylation of the polypeptide when produced in a mammalian, insect or other eukaryotic cell or altering the proteolytic cleavage of the polypeptide relative to the naturally occurring ALK3 polypeptide.

An ALK3 polypeptide that binds to BMP can be a fusion protein having, as a domain, an ALK3 polypeptide (eg, a ligand-binding portion of ALK3) and one or more additional domains that provide a desirable property. such as improved pharmacokinetics, easier purification, targeting of particular tissues, etc. For example, a domain of a fusion protein can improve one or more of in vivo stability, in vivo half-life, absorption / delivery, localization or tissue distribution, protein complex formation, multimerization of the fusion protein and / or purification. An ALK3 fusion protein that binds to BMP may include an Fc immunoglobulin domain (wild-type or mutant) or a serum albumin or other polypeptide portion that provides desirable properties, such as improved pharmacokinetics, improved solubility or better stability. In a preferred embodiment, an ALK3Fc fusion comprises a relatively unstructured linker located between the Fc domain and the extracellular domain of ALK3. This unstructured linker can correspond to the C-terminal end of the extracellular domain of ALK3, or it can be an artificial sequence of 1, 2, 3, 4 or 5 amino acids or a length between 5 and 15, 20, 30, 50 or more. amino acids that are relatively free of secondary structure, or a mixture of both. A linker may be rich in glycine and proline residues and may, for example, contain a single threonine / serine and glycine sequence or threonine / serine and / or glycine repeat sequences (eg, singlets or repeats of GGG, GGGG , TG4, SG4, TG3 or SG3). A fusion protein can include a purification subsequence, such as an epitope tag, a FLAG tag, a polyhistidine sequence, and a GST fusion. Optionally, a soluble ALK3 polypeptide includes one or more modified amino acid residues selected from: a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid residue, and an amino acid. conjugated with an organic derivatizing agent. A pharmaceutical preparation can also include one or more additional compounds, such as a compound that is used to treat a bone disorder. Preferably, the pharmaceutical preparation is substantially pyrogen-free. In general, it is preferable that the ALK3 protein is expressed in a mammalian cell line that mediates natural adequate glycosylation of the ALK3 protein in order to decrease the likelihood of an unfavorable immune response in a patient. Human and CHO cell lines have been used with success and it is expected that other common mammalian expression systems will be useful.

The disclosure also provides nucleic acids that encode an ALK3 polypeptide that binds soluble BMP. An isolated polynucleotide may comprise a coding sequence for an ALK3 polypeptide that binds soluble BMP, as described above. For example, an isolated nucleic acid can include a sequence that encodes an extracellular domain (eg, a ligand-binding domain) of ALK3 and a

ES 2 575 695 T3 sequence encoding a part or all of the transmembrane domain and / or the cytoplasmic domain of ALK3, but for a stop codon located within the transmembrane domain or the cytoplasmic domain, or positioned between the extracellular domain and the transmembrane domain or the cytoplasmic domain. For example, an isolated polynucleotide may comprise a full-length ALK3 polynucleotide sequence such as sEq ID NO: two or 4, or a partially truncated version, said isolated polynucleotide further comprising a transcription termination codon of at least six hundred nucleotides before the 3 'end or otherwise positioned such that translation of the polynucleotide results in a fused extracellular domain optionally to a truncated part of a full-length ALK3. Preferred nucleic acid sequences are SEQ ID NO: 12, 13, 15, 16, 19, 21, 24, 27, 32, or 37 and nucleic acids that hybridize to such nucleic acids or complements thereof under conditions of stringent hybridization. The nucleic acids described herein can be operably linked to a promoter for expression and the description provides cells transformed with such recombinant polynucleotides. Preferably the cell is a mammalian cell such as a CHO cell.

The description provides methods for preparing an ALK3 polypeptide that binds soluble BMP. Such a method may include expressing any of the nucleic acids (eg, SEQ ID NO: 2, 4, 12, 13, 15, 16, 19, 21, 24, 27, 32, or 37) described herein in a suitable cell. , such as a Chinese Hamster Ovary (CHO) cell. Said method may comprise: a) cultivating a cell under conditions suitable for the expression of soluble ALK3 polypeptide, wherein said cell is transformed with a soluble ALK3 expression construct; and b) recovering the soluble aLK3 polypeptide thus expressed. Soluble aLK3 polypeptides can be recovered as crude, partially purified, or highly purified fractions. Purification can be achieved by a series of purification steps, including, for example, one, two, or three or more of the following, in any order: protein A chromatography, anion exchange chromatography (eg, Q Sepharose), hydrophobic interaction chromatography (eg, phenylsepharose), size exclusion chromatography, and cation exchange chromatography.

A BMP-ALK3 antagonist described herein, such as an ALK3 polypeptide that binds soluble BMP, can be used in a method of promoting bone growth or increasing bone density in a subject. The description provides methods for treating a disorder associated with low bone density or for promoting bone growth in patients in need thereof. One method may comprise administering to a subject in need thereof an effective amount of the BMP-ALK3 antagonist. The description provides uses of the BMP-ALK3 antagonist for the manufacture of a medicament for the treatment of a disorder or condition as described herein.

The description provides a method of identifying an agent that stimulates growth or increased mineralization of bone. The method comprises: a) identifying a test agent that binds to BMP or a ligand-binding domain of an ALK3 polypeptide; and b) evaluating the effect of the agent on bone growth or mineralization.

Brief description of the drawings

Figure 1 shows the native amino acid sequence of the human ALK3 precursor (SEQ ID NO: 1). The extracellular domain of ALK3 (residues 24-152) is underlined.

Figure 2 shows the native nucleotide sequence encoding the human ALK3 precursor (SEQ ID NO: 2). The sequence encoding the extracellular domain of ALK3 (nucleotides 70 to 456) is underlined.

Figure 3 shows the native amino acid sequence of the extracellular domain of human ALK3 (SEQ ID NO: 3).

Figure 4 shows the native nucleotide sequence encoding the extracellular domain of human ALK3 (SEQ ID NO: 4).

Figure 5 shows the native amino acid sequence of the human IgG1 Fc domain (SEQ ID NO: 5).

Figure 6 shows the native nucleotide sequence encoding the human IgG1 Fc domain (SEQ ID NO: 6).

Figure 7 shows the amino acid sequence hALK3 (24-152) -hFc without leader (SEQ ID NO: 7). The extracellular domain of human ALK3 (SEQ ID NO: 3) is underlined, and the TGGG linker sequence is in bold.

Figure 8 shows the complete amino acid sequence of hALK3 (24-152) -hFc with the TPA leader (SEQ ID NO: 11). The extracellular domain of human ALK3 (SEQ ID NO: 3) is underlined, and the TGGG linker sequence is in bold.

Figure 9 shows a nucleotide sequence encoding hALK3 (24-152) -hFc with the TPA leader. SEQ ID NO: 12 corresponds to the coding strand, and SEQ ID NO: 13 corresponds to the anti-coding strand. The sequence encoding the extracellular domain of human ALK3 (SEQ ID NO: 4) is underlined.

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Figure 10 shows the complete amino acid sequence of hALK3 (24-152) -mFc with the TPA leader (SEQ ID NO: 14). The extracellular domain of human ALK3 (SEQ ID NO: 3) is underlined and the TGGG linker sequence is in bold.

Figure 11 shows a nucleotide sequence encoding hALK3 (24-152) -mFc with the TPA leader. SEQ ID NO: 15 corresponds to the coding strand, and SEQ ID NO: 16 corresponds to the anti-coding strand. The sequence encoding the extracellular domain of human ALK3 (SEQ ID NO: 4) is underlined.

Figure 12 shows the effect of whole body hALK3 (24-152) -mFc treatment on bone mineral density in female mice. Measurements were made by dual energy X-ray absorptiometry (DEXA). Data are means (n ​​= 8 per group) + SEM. *, P <0.05 vs. vehicle using the unpaired t-test. HALK3 (24-152) -mFc increased whole body bone density significantly after 31 and 42 days of treatment.

Figure 13 shows the effect of hALK3 (24-152) -mFc treatment on vertebral bone mineral density in female mice. Measurements of a region containing the fourth and fifth lumbar vertebrae (L4, L5) were made by DEXA. Data are means (n ​​= 8 per group) + SEM. **, P <0.005 vs vehicle by unpaired t-test. HALK3 (24-152) -mFc significantly increased vertebral bone density after 31 and 42 days of treatment.

Figure 14 shows the effect of hALK3 (24-152) -mFc treatment on cortical bone thickness in female mice. Measurements of the right proximal tibia were made by microcomputed tomography (micro-CT). Data are means (n ​​= 8 per group), and error bars represent + twice SEM. **, P <0.005 vs vehicle by unpaired t-test. HALK3 (24-152) -mFc increased cortical bone thickness significantly after 6 weeks of treatment.

Figure 15 shows the effect of hALK3 (24-152) -mFc treatment on trabecular bone volume in female mice. Measurements of the right proximal tibia were made by micro-CT. Data are means (n ​​= 8 per group), and error bars represent + twice SEM. *** P <0.001 vs. pretreatment or vehicle baseline by unpaired t-test. HALK3 (24-152) -mFc more than doubled the proportion of trabecular bone after 4 weeks of treatment.

Figure 16 shows the effect of hALK3 (24-152) -mFc treatment on mean trabecular thickness in female mice. Measurements of the right proximal tibia were made by micro-CT. Data are group means (n ​​= 8 per group), and error bars represent + twice SEM. *** P <0.001 versus pretreatment or vehicle baseline by unpaired t-test. HALK3 (24-152) -mFc significantly increased trabecular thickness after 4 weeks of treatment.

Figure 17 shows the effect of treatment with hALK3 (24-152) -mFc treatment for 4 weeks on trabecular bone microarchitecture in female mice. Representative three-dimensional images of the proximal tibia trabecular bone were generated by micro-CT. Scale bars = 300 pm.

Figure 18 shows examples of the three approaches described herein to interfere with signaling along the BMP-ALK3 signaling axis for the purpose of stimulating bone formation. A: ALK3-Fc. B: antibody against the selected BMP ligand (s). C. Antibody against the ligand-binding region of the extracellular domain of ALK3. BMP2 is used to illustrate that BMP can be BMP2, BMP4, or another ALK3 high affinity ligand.

Figure 19 shows the effect of hALK3 (24-152) -mFc treatment for 6 weeks at maximum bone load in female mice. Unilateral analysis of the femur was performed ex vivo with an Instron mechanical tester. Data in newtons (N) are means (n ​​= 8 per group) + SEM. **, P <0.01 vs. vehicle. HALK3 (24-152) mFc increased maximum bone load by 30%.

Figure 20 shows the effect of hALK3 (24-152) -mFc treatment for 6 weeks on bone stiffness in female mice. Unilateral analysis of the femur was performed ex vivo with an Instron mechanical tester. Data in newtons (N) per mm are means (n ​​= 8 per group) + SEM. *, P <0.05 vs. vehicle. HALK3 (24-152) mFc increased bone stiffness by 14%.

Figure 21 shows the effect of hALK3 (24-152) -mFc treatment for 6 weeks on energy for marrow failure in female mice. Unilateral analysis of the femur was performed ex vivo with an Instron mechanical tester. The data in millijoules (mJ) are means (n ​​= 8 per group) + SEM. *, P <0.05 vs. vehicle. HALK3 (24-152) -mFc increases energy to failure by 32%.

Figure 22 shows the effect of mALK3 (24-152) -mFc treatment on trabecular bone volume in an OVX mouse model of established osteopenia. Measurements of the proximal tibia were made by micro-CT. Data are means (n ​​= 7-8 per group) and error bars represent + 2 SEM. *, P <0.05 vs. oVx + vehicle. Before administration, oVx mice had reduced trabecular bone volume compared to

ES 2 575 695 T3 sham operation mice. Compared to OVX controls, mALK3 (24-152) -mFc increased bone volume significantly at 28 and 56 days of treatment.

Figure 23 shows the effect of mALK3 (24-152) -mFc treatment on cortical bone thickness in an OVX mouse model of osteopenia. Cortical bone measurements were made by micro-CT. Data are means (n ​​= 7-8 per group) and error bars represent + 2 SEM. *, P <0.05 vs. OVX + vehicle. Compared to OVX controls, mALK3 (24-152) -mFc increased cortical thickness significantly at 56 days of treatment.

Figure 24 shows the effect of mALK3 (24-152) -mFc treatment on endosseous circumference in an OVX mouse model of osteopenia. Measurements of the tibial shaft were made by micro-CT. Data are means (n ​​= 7-8 per group), and error bars represent + 2 SEM. *, P <0.05 vs. OVX + vehicle. Compared with OVX controls, mALK3 (24-152) -niFc significantly reduced endosseous circumference at 56 days of treatment, thus providing additional evidence of cortical bone growth.

Figure 25 shows the effect of whole body mALK3 (24-152) -mFc treatment on bone mineral density in an OVX mouse model of osteopenia as determined by DEXA. Data are means (n ​​= 7-8 per group) + SEM. *, P <0.05 vs. OVX + vehicle. Compared to OVX controls, mALK3 (24-152) -mFc increased whole-body bone density significantly at 14, 28, 42, and 56 days of treatment.

Figure 26 shows the effect of mALK3 (24-152) -mFc treatment on vertebral bone mineral density in an OVX mouse model of osteopenia. Analysis of the lumbar spine (L1-L6 vertebrae) was carried out by DEXA. Data are means (n ​​= 7-8 per group) + SEM. *, P <0.05 vs. OVX + vehicle. Compared to OVX controls, mALK3 (24-152) -mFc significantly increased vertebral bone density at 14, 28, 42, and 56 days of treatment.

Figure 27 shows the effect of mALK3-mFc treatment on femur-tibia bone mineral density in an OVX mouse model of osteopenia as determined by DEXA. Analysis of the entire proximal tibia and femur was carried out by DEXA. Data are means (n ​​= 7-8 per group) + SEM. *, P <0.05 vs. OVX + vehicle. Compared to OVX controls, mALK3 (24-152) -mFc increased femorotibial bone density significantly at 28, 42, and 56 days of treatment.

Figure 28 shows the effect of mALK3 (24-152) -mFc for 56 days on vertebral bone microarchitecture in an OVX mouse model of osteopenia. Representative three-dimensional images of the trabecular bone in the lumbar vertebra (L5) were generated ex vivo by micro-CT. Scale bar = 300 microns.

Figure 29 shows the effect of mALK3 (24-152) -mFc on bone volume in female mice as assessed in the distal femur by histomorphometry. Data are means + SEM; n = 6 per group per time point. **, P <0.01 against the vehicle at the corresponding time points. Compared to vehicle, mALK3 (24152) -mFc significantly increased bone volume at all time points.

Figure 30 shows the effect of mALK3 (24-152) -mFc on the rate of bone formation in female mice as evaluated in the distal femur by histomorphometry. Data are means + SEM; n = 6 per group per time point. ***, P <0.001 versus vehicle at the corresponding time point. Compared to vehicle, mALK3 (24-152) -mFc increased the rate of bone formation significantly at 28 days of treatment, thus providing evidence of anabolic bone formation.

Figure 31 shows the effect of mALK3 (24-152) -mFc on the surface of bone mineralization in female mice as evaluated in the distal femur by histomorphometry. Data are means + SEM; n = 6 per group per time point. **, P <0.01; *, P <0.05 versus vehicle at the corresponding time points. Compared to vehicle, mALK3 (24-152) -mFc increased surface mineralization significantly at 14 and 28 days of treatment, providing additional evidence for anabolic bone formation.

Figure 32 shows the effect of mALK3 (24-152) -mFc on the surface of osteoclasts in female mice as evaluated in the distal femur by histomorphometry. Data are means + SEM; n = 6 per group per time point. **, P <0.01 versus the vehicle at the corresponding time point. Compared to vehicle, mALK3 (24-152) -mFc reduces osteoclast surface significantly at 28 days of treatment, thus providing evidence of antiresorptive bone formation.

Figure 33 shows the effect of mALK3 (24-152) -mFc on serum levels of RANKL (receptor activator for nuclear factor ligand kB) in female mice as determined by the Luminex xMAP® assay. Data are means + SEM; n = 6 per group per time point. **, P <0.01; *, P <0.05 versus vehicle at the corresponding time points. Compared to vehicle, mALK3 (24-152) -mFc reduces circulating RANKL levels significantly at all time points.

Figure 34 shows the effect of mALK3 (24-152) -mFc on serum levels of osteoprotegerin (OPG) in female mice as determined by the Luminex xMAP® assay. Data are means + SEM; n = 6 per group per time point. **, P <0.01; *, P <0.05 versus vehicle at the corresponding time points. On

ES 2 575 695 T3 compared to vehicle, mALK3 (24-152) -mFc increased circulating levels of OPG significantly at 28 and 42 days of treatment.

Figure 35 shows the effect of mALK3 (24-152) -mFc on sclerostin mRNA levels in femur and tibia of female mice as assessed by real-time polymerase chain reaction (RT-PCR). . Data are means + SEM. ***, P <0.001; *, P <0.05 versus vehicle at the corresponding time points. Compared to vehicle, mALK3 (24-152) -mFc reduced sclerostin mRNA levels significantly at 2, 7, and 28 days of treatment.

Figure 36 shows the effect of hALK3 (24-152) -hFc on bone volume in female mice. Bone volume was assessed in the proximal tibia by micro-CT on day 0 (baseline) and again on day 42 (ex vivo). Data are means + SEM; n = 6 per group. ***, P <0.001 vs vehicle. Over the course of the experiment, bone volume decreased by nearly 20% in vehicle-treated controls, but increased by more than 80% with the hALK3 (24-152) -hFc treatment.

Detailed description of the invention

1. Overview

In part, the present disclosure demonstrates the surprising result that inhibitors of the BMP-ALK3 signaling pathway, such as the ALK3-Fc protein, promote bone formation in animals. ALK3 is a receptor for members of the transforming growth factor beta (TGFbeta) / bone morphogenetic protein (BMP) superfamily. The TGF-beta / BMP superfamily contains a variety of growth factors that share common sequence elements and structural motifs. These proteins are known to exert biological effects on a wide variety of cell types in both vertebrates and invertebrates. Members of the superfamily perform important roles during embryonic development in pattern formation and tissue specification and can influence a variety of differentiation processes, including adipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis, neurogenesis, and differentiation of epithelial cells. By manipulating the activity of a member of the TGFbeta family, it is often possible to induce significant physiological changes in an organism. For example, Piedmontese and Belgian Blue cattle breeds carry a loss-of-function mutation in the GDF8 gene (also called myostatin) that causes a marked increase in muscle mass. Grobet et al., Nat Genet. 1997, 17 (1): 71-4. On the other hand, in humans, inactive GDF8 alleles are associated with increased muscle mass and, according to publications, exceptional endurance. Schuelke et al, N Engl J Med 2004, 350: 2682-8.

TGF-β signals are mediated by heteromeric complexes of type I and type II serine receptors / threonine kinase, which phosphorylate and activate subsequent Smad proteins after ligand stimulation (Massagué, 2000, Nat. Rev. Mol. Cell Biol. 1: 169-178). These type I and type II receptors are transmembrane proteins, composed of an extracellular ligand-binding domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with the predicted serine / threonine specificity. Type I receptors are essential for signaling; and type II receptors are required to bind ligands and for the expression of type I receptors. Type I and II activin receptors form a stable complex after ligand binding, resulting in phosphorylation of type I receptors by type II receptors.

Activin receptor kinase-3 (ALK3) is a type I receptor that mediates the effects of multiple ligands in the BMP family and is also known as the bone morphogenetic protein receptor, type IA (BMPR1A), or activin receptor. A, type II kinase (ACVRLK). Unlike several type I receptors with ubiquitous tissue expression, ALK3 shows a restricted expression pattern consistent with more specialized functionality (Ten Dijke 1993, Oncogene 8: 2879-2887). ALK3 is generally recognized as a high affinity receptor for BMP2, BMP4, BMP7, and other members of the BMP family. BMP2 and BMP-7 are potent stimulators of osteoblast differentiation and are now used clinically to induce bone formation in spinal fusions and certain unconsolidated fractures. ALK3 is considered a key receptor in mediating BMP2 and BMP4 signaling in osteoblasts (Lavery et al., 2008, J. Biol. Chem. 283: 20948-20958). An ALK3 mouse with homozygous knockout genes dies in early embryogenesis (day 9.5), however, it has been recently published that adult mice carrying a conditional alteration of ALK3 in osteoblasts exhibit increased bone mass, although the Newly formed bone showed evidence of disorganization (Kamiya, 2008, J. Bone Miner. Res. 23: 2007-2017; Kamiya, 2008 Development 135: 3801-3811). This finding is in striking contrast to the efficacy of BMP2 and BMP7 (ligands for ALK3) as bone-building agents in clinical use.

As demonstrated herein, a soluble ALK3 polypeptide (ALK3-Fc), which shows substantial preference in binding to BMP2 and BMP4, is effective in promoting bone growth and increasing bone density in vivo. Although not wishing to be bound to any particular mechanism, it is expected that the effect of ALK3 is primarily caused by an antagonistic effect of BMP, given the very strong binding of BMP2 and BMP4 (picomolar dissociation constant) exhibited by the particular soluble ALK3 construct. used in these studies. Regardless of the mechanism, it is clear from the data presented in this document that

ES 2 575 695 T3 BMP-ALK3 antagonists increase bone density in normal mice. Surprisingly, the bone generated by ALK3-Fc treatment shows no evidence of the type of disruption seen in mice with conditional knockout ALK3 genes. It should be noted that bone is a dynamic tissue, with growth or contraction and a density that increases or decreases depending on a balance of the factors that produce bone and stimulate mineralization (mainly osteoblasts) and the factors that destroy and demineralize. bone (mainly osteoclasts). Bone growth and mineralization can be increased by increasing the productive factors, by decreasing the destructive factors, or both. The terms promoting bone growth and increasing bone mineralization refer to observable physical changes in the bones and are intended to be neutral as to the mechanism by which changes in the bone occur.

The mouse model for bone growth / density that was used in the studies described herein is considered to be highly predictive of efficacy in humans and therefore ALK3 polypeptides and other BMP-ALK3 antagonists They can be used to promote bone growth and increase bone density in humans. According to a fourth aspect of the invention, BMPALK3 antagonists for such use include certain soluble ALK3 polypeptides that bind to BMP, antibodies that bind to BMP and disrupt binding to ALK3, and antibodies that bind to ALK3 and disrupt binding to BMP. Also described for such use are non-antibody proteins selected for BMP or ALK3 binding (see, for example, WO / 2002/088171, WO / 2006/055689, WO / 2002/032925, WO / 2005/037989, US 2003/0133939 and 2005/0238646 for examples of such proteins and methods for the design and selection thereof), randomized peptides selected for BMP or ALK3 binding, often attached to an Fc domain . Two different proteins (or other residues) with BMP or ALK3 binding activity, especially BMP binders that block type I (eg, a soluble type I activin receptor) and type II (eg, a soluble type II activin receptor), respectively, can be linked together to create a bifunctional binding molecule. Nucleic acid aptamers, small molecules, and other agents that inhibit the BMP-ALK3 signaling axis are also contemplated. Additionally, nucleic acids, such as antisense molecules, siRNAs or ribozymes that inhibit BMP or, particularly, the expression of ALK3, can be used as antagonists of BMP-ALK3.

The terms and expressions used in this specification generally have their ordinary meanings in the art, within the context of this invention and in the specific context where each term is used. Certain terms are discussed below or elsewhere in the specification to provide additional guidance to the practitioner in describing the compositions of the invention and how to prepare and use them. The scope or meaning of any use of a term will be apparent from the specific context in which the term is used.

About generally means an acceptable degree of error for the measured quantity given the nature or precision of the measurements. Typically, illustrative degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values.

Alternatively, and particularly in biological systems, the term "about" can mean values ​​that are within an order of magnitude, preferably within 5 times and more preferably within 2 times of a given value. The numerical amounts given in this document are approximate, unless otherwise indicated, which means that the term approximately can be inferred when not expressly indicated.

The sequences can be compared to each other, including the wild-type sequence for one or more mutants (sequence variants). These comparisons typically comprise polymer sequence alignments, eg, using sequence alignment algorithms and / or programs that are well known in the art (eg, BLAST, FASTA, and MEGALIGN, to name a few). One of skill in the art can readily appreciate that, in such alignments, where a mutation contains an insertion or deletion of residues, the sequence alignment will introduce a gap (usually represented by a dash, or A) in the polymer sequence that does not contain the remainder inserted or removed.

Homologous, in all its grammatical forms and spelling variations, refers to the relationship between two proteins that have a common evolutionary origin, including proteins from superfamilies in the same species of organism, as well as homologous proteins from different species of organism. Such proteins (and their encoding nucleic acids) have sequence homology, as reflected by their sequence similarity, either in terms of percent identity or by the presence of specific residues or motifs and conserved positions.

The term "sequence similarity", in all its grammatical forms, refers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin.

However, in common usage and in the present application, the term homologous, when modified with an adverb such as highly, may refer to sequence similarity and may or may not be related to a common evolutionary origin.

ES 2 575 695 T3

two. ALK3 polypeptides

ALK3 polypeptides are described herein. As used herein, the term ALK3 refers to a family of activin receptor-type kinase-3 (ALK3) proteins [also known as bone morphogenetic protein receptor, type IA (BMPR1A), or activin receptor A, type II kinase (ACVRLK)] of all species and variants derived from said ALK3 proteins by mutagenesis or other modification. Reference to ALK3 herein is understood to be a reference to any one of the currently identified forms. Members of the ALK3 family are generally transmembrane proteins, composed of an extracellular ligand-binding domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine / threonine kinase activity.

The term "ALK3 polypeptide" includes polypeptides that comprise any naturally occurring polypeptide of a member of the ALK3 family, as well as variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain useful activity. For example, ALK3 polypeptides include polypeptides derived from the sequence of any known ALK3 that have a sequence at least about 80% identical to the sequence of an ALK3 polypeptide and preferably at least 85%, 90%, 95%. , 97%, 99% or greater identity. For example, an ALK3 polypeptide can bind and inhibit the function of an ALK3 protein and / or bMp. Preferably, an ALK3 polypeptide promotes bone growth and bone mineralization. Examples of ALK3 polypeptides include human ALK3 precursor polypeptide (SEQ ID NO: 1) and soluble human ALK3 polypeptides (e.g., SEQ ID NOs: 3, 7, 11, 14, 20, 22, 23, 25, 26, 28, 29, 30, 31, 33, 34, 35, 36, 38, 39, 40 or 41).

The sequence of the human ALK3 precursor protein (SEQ ID NO: 1) is shown in Figure 1, and the nucleic acid sequence encoding the human ALK3 precursor protein (SEQ ID NO: 2; GenBank entry nucleotides 549-2144 NM_004329) is shown in Figure 2. The soluble human ALK3 (extracellular), processed polypeptide sequence (SEQ ID NO: 3) is shown in Figure 3, and the nucleic acid sequence encoding the extracellular domain of human ALK3 (SEQ ID NO: 4; nucleotides 618 -1004 from Genbank entry NM_004329) is shown in Figure 4.

Soluble ALK3 polypeptides are described herein. As described herein, the term "soluble ALK3 polypeptide" generally refers to polypeptides that comprise an extracellular domain of an ALK3 protein. The term "soluble ALK3 polypeptide," as used herein, includes any naturally occurring extracellular domain of an ALK3 protein, as well as any variants thereof (including mutants, fragments, and peptidomimetic forms). An ALK3 polypeptide that binds to BMP is one that retains the ability to bind to BMPs, especially BMP2 and BMP4. Preferably, an ALK3 polypeptide that binds to BMP will bind to BMP with a dissociation constant of 1 nM or less. The amino acid sequence of the human ALK3 precursor protein is provided in Figure 1. The extracellular domain of an ALK3 protein binds to BMP and is generally soluble and can therefore be termed a soluble ALK3 polypeptide that binds to BMP. . Examples of soluble ALK3 polypeptides that bind to BMP include the soluble polypeptide illustrated in SEQ ID NOs: 3, 7, 11, 14, 20, 22, 23, 25, 26, 28, 29, 30, 31, 33, 34, 35, 36, 38, 39, 40 or 41. SEQ ID NO: 7 is in accordance with the first aspect of the invention, is referred to as ALK3 (24-152) hFc, and is further described in the Examples. Other examples of soluble ALK3 polypeptides that bind to BMP comprise a signal sequence, in addition to the extracellular domain of an ALK3 protein, for example, the native ALK3 leader sequence (SEQ ID NO: 8), the tissue plasminogen activator leader ( TPA) (SeQ ID NO: 9) or the honey melittin leader (SEQ ID NO: 10). The ALK3-hFc polypeptide illustrated in SEQ ID NO: 11 uses a TPA leader.

Functionally active fragments of ALK3 polypeptides can be obtained by screening recombinantly produced polypeptides from the corresponding fragment of nucleic acid encoding an ALK3 polypeptide. In addition, fragments can be chemically synthesized using techniques known in the art such as conventional solid phase Merrifield chemistry f-Moc or t-Boc. The fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments that can function as antagonists (inhibitors) of the ALK3 protein or of BMP-mediated signaling.

Functionally active variants of ALK3 polypeptides can be obtained by screening libraries of modified polypeptides produced recombinantly from the corresponding mutagenized nucleic acids encoding an ALK3 polypeptide. Variants can be produced and tested to identify those that can function as antagonists (inhibitors) of ALK3 protein or BMP-mediated signaling. According to the sixth aspect of the invention, the functional variant comprises or consists of an amino acid sequence at least 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from SEQ ID NO: 11 , 14, 20, 22, 23, 25, 26, 28, 29, 30, 31, 33, 34, 35, 36, 38, 39, 40 or 41.

Functional variants can be generated by modifying the structure of an ALK3 polypeptide for purposes such as improving therapeutic efficacy or stability (eg, ex vivo half-life and resistance to proteolytic degradation in vivo). Such modified ALK3 polypeptides, when selected to retain BMP binding, are considered functional equivalents of naturally occurring ALK3 polypeptides.

ES 2 575 695 T3

Modified ALK3 polypeptides can also be produced, for example, by amino acid substitution, deletion or addition. For example, it is reasonable to expect that an isolated substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (eg, conservative mutations) it will have no major effect on the biological activity of the resulting molecule. Conservative substitutions are those that take place within a family of amino acids that are related in their side chains. When a change in the amino acid sequence of an ALK3 polypeptide results in a functional homologue, it can be readily determined by assessing the ability of the variant ALK3 polypeptide to elicit a response in cells in a manner similar to the wild-type ALK3 polypeptide. .

In certain embodiments, the present invention contemplates specific mutations of ALK3 polypeptides to alter glycosylation of the polypeptide. Such mutations can be selected to introduce or remove one or more glycosylation sites, such as O- or N-linked glycosylation sites. Asparagine-linked glycosylation recognition sites generally comprise a tripeptide sequence, asparagine-X-threonine (or asparagine-X-serine) (where X is any amino acid) that is specifically recognized by appropriate cellular glycosylation enzymes. Alteration can also be done by adding or substituting one or more serine or threonine residues to the wild-type ALK3 polypeptide sequence (for O-linked glycosylation sites). A variety of amino acid substitutions or deletions at one or both of the first or third amino acid positions of a glycosylation recognition site (and / or amino acid deletion at the second position) results in non-glycosylation in the tripeptide sequence modified. Another means of increasing the number of carbohydrate groups in an ALK3 polypeptide is by chemical or enzymatic coupling of glycosides to the ALK3 polypeptide. Depending on the mode of coupling used, the sugar (s) may be linked to (a) arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryl groups such as cysteine; (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline; (e) aromatic moieties such as phenylalanine, tyrosine, or tryptophan; or (f) the amide group of glutamine. These methods are described in WO 87/05330 published September 11, 1987, and in Aplin and Wriston (1981) CRC Crit. Rev. Biochem., Pp. 259306. Removal of one or more carbohydrate moieties present in an ALK3 polypeptide could be accomplished chemically and / or enzymatically. Chemical deglycosylation can involve, for example, exposure of the ALK3 polypeptide to the trifluoromethanesulfonic acid compound, or an equivalent compound. This treatment results in the cutting of most or all sugars except the binding sugar (N-acetylglucosamine or N-acetylgalactosamine), leaving the amino acid sequence intact. Chemical deglycosylation is further described in Hakimuddin et al. (1987) Arch. Biochem. Biophys. 259: 52 and in Edge et al. (1981) Anal. Biochem. 118: 131. Enzymatic cleavage of carbohydrate groups in ALK3 polypeptides can be achieved through the use of a variety of endo- and exo-glycosidases as described in Thotakura et al. (1987) Meth. Enzymol. 138: 350. The sequence of an ALK3 polypeptide can be adjusted, as appropriate, depending on the type of expression system used, such as mammalian, yeast, insect and plant cells, which can all introduce different glycosylation patterns that can be affected. by the amino acid sequence of the peptide. In general, ALK3 proteins for use in humans are expressed in a mammalian cell line that provides adequate glycosylation, such as the HEK293 or CHO cell lines, although other mammalian expression cell lines are expected, yeast cell lines with modified glycosylation enzymes and insect cells are useful as well.

A method of generating mutants is described herein, in particular combinatorial mutant pools of an ALK3 polypeptide, as well as truncation mutants; Combinatorial mutant pools are especially useful for identifying functional variant sequences. The purpose of the detection of such combinatorial libraries may be to generate, for example, ALK3 polypeptide variants that can act as either agonists or antagonists, or alternatively, that possess entirely novel activities. A variety of screening assays are provided below, and such assays can be used to evaluate variants. For example, an ALK3 polypeptide variant can be selected for its ability to bind to an ALK3 ligand, to prevent the binding of an ALK3 ligand to an ALK3 polypeptide, or to interfere with signaling elicited by an ALK3 ligand.

The activity of an ALK3 polypeptide or its variants can also be tested in an in vivo or cell-based assay. For example, the effect of an ALK3 polypeptide variant on the expression of genes involved in bone production or bone destruction can be evaluated. This can, as necessary, be carried out in the presence of one or more recombinant ALK3 ligand proteins (eg, BMP2 or BMP4), and the cells can be transfected in order to produce an ALK3 polypeptide and / or variants. thereof and, optionally, an ALK3 ligand. Similarly, an ALK3 polypeptide can be administered to a mouse or other animal, and one or more bone properties, such as density or volume, can be evaluated. The healing rate of bone fractures can also be evaluated. Dual X-ray absorptiometry (DEXA) is a well-established non-invasive quantitative technique for assessing bone density in an animal. In humans, central DEXA systems can be used to assess bone density in the spine and pelvis. These are the best predictors of overall bone density. Peripheral DEXA systems can be used to assess bone density in peripheral bones, including, for example, the bones of the hand, wrist, ankle, and foot. Traditional x-ray imaging systems can be used, including

ES 2 575 695 T3 CAT scans, to assess bone growth and fracture healing. The mechanical strength of the bone can also be evaluated.

Combinatorially derived variants can be generated that have higher overall or selective potency relative to a naturally occurring ALK3 polypeptide. Similarly, mutagenesis can result in variants that have drastically different intracellular half-lives than a wild-type ALK3 polypeptide. For example, the altered protein can be generated either more stable or less stable with respect to proteolytic degradation or other cellular processes that result in the destruction or inactivation of a native ALK3 polypeptide. Such variants, and the genes that encode them, can be used to alter ALK3 polypeptide levels by modulating the half-life of ALK3 polypeptides. For example, a short half-life can lead to more transient biological effects and can allow tighter control of recombinant ALK3 polypeptide levels within the patient. In an Fc fusion protein, mutations can be made in the linker (if any) and / or the Fc portion to alter the half-life of the protein.

A combinatorial library can be produced by means of a degenerate library of genes encoding a polypeptide library that each includes at least a portion of the possible ALK3 polypeptide sequences. For example, a mixture of synthetic oligonucleotides can be enzymatically linked into gene sequences such that the degenerate set of possible nucleotide sequences of the ALK3 polypeptide is expressible as individual polypeptides or, alternatively, as a set of larger fusion proteins (e.g. , for phage display).

There are many ways in which the library of potential homologues can be generated from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be carried out on an automated DNA synthesizer, and the synthetic genes can then be ligated into an appropriate vector for expression. The synthesis of degenerate oligonucleotides is well known in the art (see, for example, Narang, SA (1983) Tetrahedron 39: 3; Itakura et al, (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton , Amsterdam: Elsevier pp. 273-289; Itakura et al., (1984) Annu. Rev. Biochem. 53: 323; Itakura et al. (1984), Science 198: 1056; Ike et al., (1983) Nucleic Acid Res. 11: 477). Such techniques have been employed in the directed evolution of other proteins (see, for example, Scott et al, (1990) Science 249: 386-390; Roberts et al, (1992) PNAS USA 89: 2429-2433; Devlin et al , (1990) Science 249: 404-406; Cwirla et al, (1990) PNAS USA 87: 6378-6382; as well as US Patent Nos: 5,223,409, 5,198,346, and 5,096,815) .

Alternatively, other forms of mutagenesis can be used to generate a combinatorial library. For example, ALK3 polypeptide variants can be generated and isolated from a library by selection using, for example, alanine scanning mutagenesis and the like (Ruf et al, (1994) Biochemistry 33: 1565-1572; Wang et al, ( 1994) J. Biol. Chem. 269: 3095-3099; Balint et al, (1993) Gene 137: 109-118; Grodberg et al., (1993) Eur. J. Biochem. 218: 597-601; Nagashima et al., (1993) J. Biol. Chem. 268: 2888-2892; Lowman et al, (1991) Biochemistry 30: 10832-10838; and Cunningham et al, (1989) Science 244: 1081-1085), by linker scanning mutagenesis (Gustin et al, (1993.) Virology 193: 653-660; Brown et al, (1992) Mol. Cell Biol. 12: 2644-2652; McKnight et al, (1982) Science 232: 316); by saturation mutagenesis (Meyers et al, (1986) Science 232: 613); by PCR mutagenesis (Leung et al, (1989) Method Cell Mol Biol 1: 11-19); or by random mutagenesis, including chemical mutagenesis, etc. (Miller et al., (1992), A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor, New York, and Greener et al., (1994) Strategies in Mol Biol 7: 32-34). Linker scanning mutagenesis, particularly in a combinatorial environment, is an attractive method for the identification of truncated (bioactive) forms of ALK3 polypeptides.

A wide range of techniques are known in the art for selecting gene products from combinatorial libraries made by point mutations and truncations and, for that matter, for screening cDNA libraries for gene products that have a certain property. Such techniques will generally be adaptable for rapid screening of gene libraries generated by combinatorial mutagenesis of ALK3 polypeptides. The most widely used techniques for the selection of large gene libraries typically comprise cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting vector library, and expressing the combinatorial genes under conditions in which the detection of a desired activity facilitates the relatively easy isolation of the vector that encodes the gene whose product was detected. Preferred assays include BMP binding assays and BMP-mediated cell signaling assays.

In certain embodiments, the ALK3 polypeptides of the invention may further comprise post-translational modifications in addition to any that are naturally present in the ALK3 polypeptides. Such modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. As a result, modified ALK3 polypeptides can contain elements that are not amino acids, such as polyethylene glycols, lipids, poly- or mono-saccharides, and phosphates. The effects of such non-amino acid elements on the functionality of an ALK3 polypeptide can be tested as described herein for other ALK3 polypeptide variants. When an ALK3 polypeptide is produced in cells by cleavage of a nascent form of the ALK3 polypeptide, post-translational processing may also be important for proper folding and / or function of the protein. Different cells (such as CHO, HeLa, MDCK, 293, WI38, NIH-3T3, or HEK293) have specific cellular machinery and

ES 2 575 695 T3 characteristic mechanisms for such post-translational activities and can be chosen to ensure correct modification and processing of ALK3 polypeptides.

In certain aspects, functional variants or modified forms of ALK3 polypeptides include fusion proteins that have at least a portion of the ALK3 polypeptides and one or more fusion domains. Well known examples of such fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy chain constant region (Fc), protein binding to maltose (MBP) or human serum albumin. A fusion domain can be selected in order to confer a desired property. For example, some fusion domains are particularly useful for the isolation of fusion proteins by affinity chromatography. For the purpose of affinity purification, matrices relevant to affinity chromatography are used, such as glutathione, amylase, and nickel or cobalt conjugated resins. Many such matrices are available in kit form, such as the Pharmacia GST purification system and the useful QIAexpress ™ system (Qiagen) with fusion partners (HIS6). As another example, a fusion domain can be selected to facilitate detection of ALK3 polypeptides. Examples of such detection domains include the various fluorescent proteins (eg, GFP), as well as epitope markers, which are generally short peptide sequences for which a specific antibody is available. Well-known epitope markers for which specific monoclonal antibodies are readily available include FLAg, influenza virus hemagglutinin (HA), and cmyc markers. In some cases, the fusion domains have a protease cleavage site, such as for Factor Xa or thrombin, which allows the relevant protease to partially digest the fusion proteins and thereby release the recombinant proteins from them. . Released proteins can then be isolated from the fusion domain in subsequent chromatographic separation. In certain preferred embodiments, an ALK3 polypeptide is fused with a domain that stabilizes the ALK3 polypeptide in vivo (a stabilizer domain). By "stabilize" is meant anything that increases the serum half-life, regardless of whether this is due to less destruction, decreased clearance from the kidney, or another pharmacokinetic effect. Fusions with the Fc portion of an immunoglobulin are known to confer desirable pharmacokinetic properties on a wide range of proteins. Similarly, fusions to human serum albumin can confer desirable properties. Other types of fusion domains that can be selected include domain multimerization (eg, dimerization, tetramerization) and functional domains (conferring additional biological function, such as further stimulation of bone growth or muscle growth, as desired).

As a specific example, a fusion protein comprising a soluble extracellular domain of ALK3 fused to an Fc domain (eg, SEQ ID NO: 5 in Figure 5). Examples of Fc domains are shown below:

THTCPPCPAPELLGGPSVFLFPPKPKDTLMTSRTPEVTCVWD (A) VSHEDPEVKFNWYVDG

VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK (A) VSNKALPVPIEKTISKAK

GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG

PFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN (A) HYTQKSLSLSPGK *

Optionally, the Fc domain has one or more mutations at the residues, such as Asp-265, Lysine 322, and Asn-434. In certain cases, the mutant Fc domain having one or more of these mutations (eg, the Asp265 mutation) has reduced Fcy receptor binding ability relative to a wild-type Fc domain. In other cases, the mutant Fc domain that has one or more of these mutations (for example, the Asn-434 mutation) has increased ability to bind the MHC class I-related Fc receptor (FcRn) to an Fc domain of wild type.

It is understood that the different elements of the fusion proteins can be arranged in any way that is consistent with the desired functionality. For example, an ALK3 polypeptide can be C-terminal to a heterologous domain, or, alternatively, a heterologous domain can be C-terminal to an ALK3 polypeptide. The ALK3 polypeptide domain and the heterologous domain do not have to be adjacent in a fusion protein and C- or N-termini can be included to additional domains or amino acid sequences to either domain or between domains.

The ALK3 polypeptides of the present invention may contain one or more modifications that are capable of stabilizing the ALK3 polypeptides. For example, such modifications increase the in vitro half-life of ALK3 polypeptides, improve the circulatory half-life of ALK3 polypeptides, or reduce the proteolytic degradation of ALK3 polypeptides. Such stabilizing modifications include, but are not limited to, fusion proteins (including, for example, fusion proteins comprising an ALK3 polypeptide and a stabilizing domain), modifications of a glycosylation site (including, for example, the addition from a glycosylation site to an ALK3 polypeptide) and carbohydrate moiety modifications (including, for example, removal of carbohydrate moieties from an ALK3 polypeptide). In the case of fusion proteins, an ALK3 polypeptide is fused to a stabilizer domain such as an IgG molecule (e.g., a

ES 2 575 695 T3 domain Fc). As used herein, the term "stabilizing domain" not only refers to a fusion domain (eg, Fc) as in the case of fusion proteins, but also includes non-protein modifications such as a carbohydrate moiety. , or non-protein polymer, such as polyethylene glycol.

In certain embodiments, the present invention makes available isolated and / or purified forms of ALK3 polypeptides, which are isolated from, or otherwise, substantially free of other proteins. ALK3 polypeptides will generally be produced by the expression of recombinant nucleic acids.

3. Nucleic acids encoding ALK3 polypeptides

Described herein are isolated and / or recombinant nucleic acids that encode any of the ALK3 polypeptides (eg, soluble ALK3 polypeptides), including fragments, functional variants, and fusion proteins described herein. For example, SEQ ID NO: 2 encodes the naturally occurring human ALK3 precursor polypeptide, while SEQ ID NO: 4 encodes the processed extracellular domain of ALK3. The nucleic acids in question can be single-stranded or double-stranded. Said nucleic acids can be DNA or RNA molecules. These nucleic acids can be used, for example, in methods for the manufacture of ALK3 polypeptides or as direct therapeutic agents (eg, in a gene therapy approach).

The nucleic acids in question that encode ALK3 polypeptides are further understood to include nucleic acids that are variants of SEQ ID NO: 2 or 4. Variant nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions, or deletions. , such as allelic variants.

The second aspect of the invention provides an isolated polynucleotide comprising a coding sequence for the polypeptide of the first aspect. This may comprise the nucleic acid of SEQ. ID NO: 12. The polypeptide of the sixth aspect may be encoded by an isolated or recombinant polynucleotide comprising a nucleic acid sequence that is at least 95%, 97%, 98%, 99% or 100% identical to SeQ ID NO: 12, 15, 21, 24, 27, 32 or 37. Anyone of ordinary skill in the art will appreciate that nucleic acid sequences complementary to SEQ ID NO: 12, 15, 21, 24, 27, 32 or 37 and variants of SEQ ID NO: 12, 15, 21, 24, 27 , 32 or 37 are also within the scope of this invention. In additional embodiments, the nucleic acid sequences of the invention can be isolated, recombinant, and / or fused to a heterologous nucleotide sequence, or in a DNA library.

In other embodiments, the nucleic acids of the invention also include nucleotide sequences that hybridize under very stringent conditions to the nucleotide sequence designated in SEQ ID NO: 12, 15, 21, 24, 27, 32 or 37, complementary sequence of SEQ ID NO: 12, 15, 21, 24, 27, 32 or 37, or fragments thereof. As discussed above, one of ordinary skill in the art will readily understand that appropriate stringency conditions that promote DNA hybridization can be varied. One of ordinary skill in the art will readily understand that appropriate stringency conditions that promote DNA hybridization can be varied. For example, hybridization could be carried out in 6.0 x sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by a 2.0 x SSC wash at 50 ° C. For example, the concentration of the salt in the wash step can be selected from a low stringency of about 2.0 x SSC at 50 ° C to a high stringency of about 0.2 x SSC at 50 ° C. Furthermore, the temperature in the washing step can be increased from low stringency conditions at room temperature, about 22 ° C, to high stringency conditions at about 65 ° C. Both the temperature and the salt can be varied, or the temperature or concentration of the salt can be kept constant while the other variable is changed. In one embodiment, the invention provides nucleic acids that hybridize under low stringency conditions of 6 x SSC at room temperature followed by a 2 x SSC wash at room temperature.

Isolated nucleic acids that differ from nucleic acids set forth in SEQ ID NO: 12, 15, 21, 24, 27, 32, or 37, due to degeneracy in the genetic code, are also within the scope of the invention. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (eg, CAU and CAC are synonymous for histidine) can lead to silent mutations that do not affect the amino acid sequence of the protein. However, DNA sequence polymorphisms leading to changes in the amino acid sequences of the proteins in question are expected to exist among mammalian cells. Anyone skilled in the art will appreciate that these variations can exist in one or more nucleotides (up to about 3-5% of nucleotides) of the nucleic acids encoding a particular protein among individuals of a given species due to natural allelic variation. . Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this invention.

In certain embodiments, the recombinant nucleic acids of the invention can be operably linked to one or more regulatory nucleotide sequences in an expression construct. Regulatory nucleotide sequences will generally be appropriate for the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells. Typically, such one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites,

ES 2 575 695 T3 transcription initiation and termination, translation initiation and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters as known in the art are contemplated by the invention. Promoters can be either naturally-occurring promoters, or hybrid promoters that combine elements from more than one promoter. An expression construct can be present in a cell in an episome, such as a plasmid, or the expression construct can be inserted into a chromosome. In a preferred embodiment, the expression vector contains a selectable marker gene that allows for the selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used.

In certain aspects of the invention, the subject nucleic acid is provided in an expression vector comprising a nucleotide sequence encoding an ALK3 polypeptide and operably linked to at least one regulatory sequence. Regulatory sequences are recognized in the art and are selected to direct expression of the ALK3 polypeptide. Accordingly, the term "regulatory sequence" includes promoters, enhancers, and other elements of expression control. Illustrative regulatory sequences are described in Goeddel; Gene Expression Technology. Methods in Enzymology, Academic Press, San Diego, CA (1990). For example, any of a wide variety of expression control sequences that control the expression of a DNA sequence when operably linked to it can be used in these vectors to express DNA sequences that encode an ALK3 polypeptide. Such useful expression control sequences include, for example, the SV40 early and late promoters, the tet promoter, the adenovirus or cytomegalovirus immediate early promoter, the RSV promoters, the lac system, the trp system, the TAC or TRC, the T7 promoter, whose expression is driven by T7 RNA polymerase, the main operator and promoter regions of phage lambda, the control regions for the fd coat protein, promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, promoters for acid phosphatase, eg Pho5, promoters for yeast α-pairing factors, polyhedral promoter for the baculovirus system, and other sequences known to control expression of genes from prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. It should be understood that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed and / or the type of protein to be expressed. Furthermore, the vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, must also be considered.

A recombinant nucleic acid of the invention can be produced by ligation of the cloned gene, or a portion thereof, into a vector suitable for expression in prokaryotic cells, eukaryotic cells (yeast, avian, insect, or mammal), or both. Expression vehicles for the production of a recombinant ALK3 polypeptide include plasmids and other vectors. For example, suitable vectors include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences to facilitate propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. Vectors derived from pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with bacterial plasmid sequences, such as pBR322, to facilitate replication and selection for drug resistance in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as bovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derivative and p205) can be used for transient expression of proteins in eukaryotic cells. Examples of other viral (including retroviral) expression systems can be found below in the description of gene therapy delivery systems. The various methods employed in the preparation of plasmids and in the transformation of host organisms are well known in the art. For other expression systems suitable for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning A Laboratory Manual, 3^to Ed., Ed. By Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 2001). In some cases, it may be desirable to express the recombinant polypeptides through the use of a baculovirus expression system. Examples of such baculovirus expression systems include vectors derived from pVL (such as pVL1392, pVL1393, and pVL941), vectors derived from pAcUW (such as pAcUWl), and vectors derived from pBlueBac (such as pBlueBac III containing β-gal).

In a preferred embodiment, a vector was designed for the production of the target ALK3 polypeptides in CHO cells, such as a Pcmv-Script vector (Stratagene, La Jolla, Calif.), PcDNA4 vectors (Invitrogen, Carlsbad, Calif.) and pCI-Neo vectors (Promega, Madison, Wise). As will be apparent, the target gene constructs can be used to cause expression of the ALK3 target polypeptides in cells propagated in culture, eg, to produce proteins, including fusion proteins or variant proteins, for purification.

This invention also relates to a host cell transfected with a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 12, 15, 21, 24, 27, 32 or 37). The host cell can be any prokaryotic or eukaryotic cell. For example, an ALK3 polypeptide of the invention can be expressed in bacterial cells such as E. coli, insect cells (eg, using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those of skill in the art.

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Accordingly, methods of production of the subject ALK3 polypeptides are described herein. For example, a host cell transfected with an expression vector encoding an ALK3 polypeptide can be cultured under appropriate conditions to allow expression of the ALK3 polypeptide to occur. The ALK3 polypeptide can be secreted and isolated from a mixture of cells and medium containing the ALK3 polypeptide. Alternatively, the ALK3 polypeptide can be retained cytoplasmically or in a membrane fraction and the cells harvested, lysed and isolated from the protein. A cell culture includes host cells, media, and other by-products. Suitable media for cell culture are well known in the art. The target ALK3 polypeptides can be isolated from cell culture medium, host cells, or both, using techniques known in the art for purifying proteins, including ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, immunoaffinity purification with antibodies specific for particular epitopes of ALK3 polypeptides and affinity purification with an agent that binds to a domain fused to the ALK3 polypeptide (for example, a protein A column can be used to purify an ALK3 fusion -Fc). In a preferred embodiment, the ALK3 polypeptide is a fusion protein that contains a domain that facilitates its purification. Purification can be achieved by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q-sepharose chromatography, phenyl-sepharose chromatography, size exclusion chromatography. and cation exchange chromatography. Purification could be completed with virus filtration and buffer exchange. As demonstrated herein, the ALK3-hFc protein was purified to a purity of> 98%, determined by size exclusion chromatography and> 95% as determined by sDs PAGE. This level of purity was sufficient to achieve desirable effects on bone in mice and an acceptable safety profile in mice, rats, and non-human primates.

A fusion gene encoding a purification leader sequence, such as a poly (His) / enterokinase cleavage site sequence at the N-terminus of the desired portion of the recombinant ALK3 polypeptide, may allow purification of the protein. melt expressed by affinity chromatography using a Ni metal resin²⁺. The purification leader sequence can then be subsequently removed by enterokinase treatment to provide the purified ALK3 polypeptide (for example, see Hochuli et al., (1987) J. Chromatography 411: 177; and Janknecht et al, PNAS USA 88: 8972 ).

Techniques for preparing fusion genes are well known. Essentially, the joining of various DNA fragments encoding different polypeptide sequences is carried out according to conventional techniques, employing blunt or staggered ends of composition for ligation, digestion with restriction enzymes to provide appropriate ends, end filling cohesive as appropriate, alkaline phosphatase treatment to avoid undesirable binding and enzymatic ligation. The fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can then be hybridized to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology , eds. Ausubel et al, John Wiley & Sons: 1992).

Four. Alternative BMP and ALK3 antagonists

The data presented herein demonstrate that antagonists of BMP-ALK3 signaling can be used to promote bone growth and bone mineralization. Although soluble ALK3 polypeptides, and in particular ALK3-Fc, are preferred antagonists, and although such antagonists may affect bone through a mechanism other than BMP antagonism (e.g., inhibition of BMP may be an indicator of tendency of an agent to inhibit the activities of a spectrum of molecules, including, perhaps, other members of the TGF-beta superfamily, and such collective inhibition may lead to the desired effect on bone), Other types of BMP-ALK3 antagonists are expected to be useful, including anti-BMP antibodies (e.g., BMP2 or bMP4), anti-ALK3 antibodies, antisense, RNAi, or ribozyme nucleic acids that inhibit the production of ALK3, BMP2, or BMP4 and other BMP or ALK3 inhibitors, particularly those that disrupt the binding of BMP-ALK3.

An antibody that is specifically reactive with an ALK3 polypeptide (eg, a soluble ALK3 polypeptide), and that either competitively binds ligand with the ALK3 polypeptide or otherwise inhibits ALK3-mediated signaling, can be used. as an antagonist of ALK3 polypeptide activities. Similarly, an antibody that is specifically reactive with a BMP polypeptide and disrupts the binding of the ALK3 polypeptide can be used as an antagonist.

By using immunogens derived from an ALK3 polypeptide or a BMP polypeptide, anti-protein / anti-peptide antisera or monoclonal antibodies can be prepared by standard protocols (see, for example, Antibodies: A Laboratory Manual, ed. By Harlow and Lane ( Cold Spring Harbor Press: 1988)). A mammal, such as a mouse, hamster, or rabbit can be immunized with an immunogenic form of the ALK3 polypeptide, an antigenic fragment that is capable of eliciting an antibody response, or a fusion protein. Techniques for conferring immunogenicity to a protein or peptide include conjugation with carriers or other techniques well known in the art. An immunogenic portion of a BMP or ALK3 polypeptide can be administered in the presence of adjuvant. Immunization progress can be monitored by titre detection

ES 2 575 695 T3 of antibodies in plasma or serum. The standard ELISA or other immunoassays can be used with the immunogen as antigen to assess antibody levels.

After immunization of an animal with an antigenic preparation of an ALK3 polypeptide, antisera can be obtained and, if desired, polyclonal antibodies can be isolated from serum. To produce monoclonal antibodies, antibody-producing cells (the lymphocytes) can be grown from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to produce hybridoma cells. Such techniques are well known in the art, and include, for example, the hybridoma technique (originally developed by Kohler and Milstein, (1975) Nature, 256: 495-497), the human B-cell hybridoma technique (Kozbar et al, (1983) Immunology Today, 4:72), and the EBV hybridoma technique to produce human monoclonal antibodies (Cole et al, (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96 ). The hybridoma cells can be immunochemically selected for the production of antibodies specifically reactive with an ALK3 polypeptide and the monoclonal antibodies isolated from a culture comprising such hybridoma cells.

The term antibody, as used herein, is intended to include fragments thereof that are also specifically reactive with a subject polypeptide. Antibodies can be fragmented using standard techniques and fragments selected for utility in the same manner as described above for whole antibodies. For example, F (ab) 2 fragment can be generated by treating the antibody with pepsin. The resulting F (ab) 2 fragment can be treated to reduce disulfide bridges to produce Fab fragments. The antibody useful in the present invention is intended to further include chimeric single chain bispecific and humanized fully human molecules having affinity for a bMp or ALK3 polypeptide conferred by at least one CDR region of the antibody. An antibody may further comprise a marker attached thereto and capable of being detected (eg, the marker may be a radioisotope, fluorescent compound, enzyme, or enzyme co-factor).

The antibody may be a recombinant antibody, which term encompasses any antibody generated in part by molecular biology techniques, including CDR-grafted or chimeric antibodies, human or other antibodies assembled from antibody domains selected from the library, antibodies from single chain and single domain antibodies (eg human VH proteins or camelid Vhh proteins). In certain embodiments, a useful antibody of the invention is a monoclonal antibody. Methods for the generation of new antibodies are described herein. For example, a method of generating a monoclonal antibody that specifically binds to an ALK3 polypeptide or BMP polypeptide may comprise administering to a mouse an amount of an immunogenic composition comprising the antigen polypeptide effective to stimulate a detectable immune response, obtaining antibody-producing cells (for example, spleen cells) of the mouse and fusing the antibody-producing cells with myeloma cells to obtain antibody-producing hybridomas, and testing the antibody-producing hybridomas to identify a hybridoma that produces a monocolonal antibody that specifically binds to the antigen. Once obtained, a hybridoma can be propagated in cell culture, optionally under culture conditions where the hybridoma-derived cells produce the monoclonal antibody that specifically binds to the antigen. The monoclonal antibody can be purified from cell culture.

The adjective specifically reactive with, as used in reference to an antibody, is understood to mean, as is generally understood in the art, that the antibody is sufficiently selective between the antigen of interest (eg, an ALK3 polypeptide) and other antigens that are not of interest that the antibody is useful for, at a minimum, detecting the presence of the antigen of interest in a particular type of biological sample. In certain methods employing the antibody, such as therapeutic applications, a higher degree of specificity in binding may be desirable. Monoclonal antibodies generally have a greater tendency (compared to polyclonal antibodies) to efficiently discriminate between desired antigens and cross-reactive polypeptides. One characteristic that influences the specificity of an antibody: antigen interaction is the affinity of the antibody for the antigen. Although the desired specificity can be achieved with a range of different affinities, generally preferred antibodies will have an affinity (a dissociation constant) of about 10^-6, 10^-7, 10^-8, 10^-9 or less. Considering the extraordinarily tight binding between BMP and ALK3, it is expected that a neutralization of the anti-BMP or anti-ALK3 antibody would generally have a dissociation constant of 10^-9 or less.

Furthermore, the techniques used to detect antibodies in order to identify a desirable antibody can influence the properties of the obtained antibody. For example, if an antibody is to be used for the binding of an antigen in solution, it may be desirable to analyze the binding solution. A variety of different techniques are available for testing the interaction between antibodies and antigens to identify particularly desirable antibodies. Such techniques include ELISA, surface plasmon resonance binding assays (for example, the Biacore ™ binding assay, Biacore AB, Uppsala, Sweden), sandwich assays (for example, the paramagnetic bead system from IGEN International, Inc. ., Gaithersburg, Maryland), Western blots, immunoprecipitation assays, and immunohistochemistry.

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Examples of categories of nucleic acid compounds that are antagonists of BMP or ALK3 include antisense nucleic acids, RNAi constructs, and catalytic nucleic acid constructs. A nucleic acid compound can be single-stranded or double-stranded. A double stranded compound can also include cantilevered or non-complementary regions, where one or the other of the strands is single stranded. A single-stranded compound can include regions of self-complementarity, meaning that the compound forms a so-called hairpin or stem-loop structure, with a region of double-helix structure. A nucleic acid compound can comprise a nucleotide sequence that is complementary to a region consisting of no more than 1000, no more than 500, no more than 250, no more than 100, or no more than 50, 35, 30, 25 , 22, 20, or 18 nucleotides of the full-length ALK3 nucleic acid sequence or the BMP nucleic acid sequence. The region of complementarity will preferably be at least 8 nucleotides, and optionally at least 10 or at least 15 nucleotides, and optionally between 15 and 25 nucleotides. A region of complementarity may fall within an intron, coding sequence, or non-coding sequence of the target's transcription, such as the coding sequence portion. Generally, a nucleic acid compound will be from about 8 to about 500 nucleotides or base pairs in length, and optionally, the length will be from about 14 to about 50 nucleotides. A nucleic acid can be DNA (particularly for use as an antisense), RNA, or RNA: DNA hybrid. Any strand can include a mixture of DNA and RNA, as well as modified forms that cannot easily be classified as DNA or RNA. Similarly, a double-stranded compound can be DNA: DNA, DNA: RNA, or RNA: RNA, and any of the strands can also include a mixture of DNA and RNA, as well as modified forms that cannot be easily classified as DNA. or RNA. A nucleic acid compound can include any of a variety of modifications, including one or more modifications to the backbone (the sugar-phosphate portion in a natural nucleic acid, including internucleotide linkages) or the base portion (the purine portion or pyrimidine from a natural nucleic acid). An antisense nucleic acid compound will preferably be about 15 to about 30 nucleotides in length and often contains one or more modifications to enhance characteristics, such as stability in serum, in a cell, or in a location where it is likely to be. delivered the compound, such as the stomach in the case of orally delivered compounds and the lung for inhaled compounds. In the case of an RNAi construct, the strand complementary to the transcription of the target will generally be RNA or modifications thereof. The other strand can be RNA, DNA, or any other variation. The duplex portion of the double-stranded or single-stranded hairpin RNAi construct will preferably be 18 to 40 nucleotides in length and optionally about 21 to 23 nucleotides in length, as long as it serves as the Dicer substrate. The catalytic or enzymatic nucleic acids can be ribozymes or DNA enzymes and can also contain modified forms. Nucleic acid compounds can inhibit target expression by about 50%, 75%, 90% or more when it comes into contact with cells under physiological conditions and at a concentration where a nonsense or sense control has little or no effect. Preferred concentrations for testing the effect of nucleic acid compounds are 1, 5, and 10 micromolar. Nucleic acid compounds can also be tested for their effects on, for example, bone growth and mineralization.

5. Screening tests

ALK3 polypeptides (eg, soluble ALK3 polypeptides) and BMP polypeptides can be used to identify compounds (agents) that are agonists or antagonists of the BMP-ALK3 signaling pathway. Compounds identified through this screen can be tested for their ability to modulate bone growth or mineralization in vitro. Optionally, these compounds can be tested in animal models to assess their ability to modulate tissue growth in vivo.

There are numerous methods for the detection of therapeutic agents to modulate tissue growth by the recognition of BMP and ALK3 polypeptides. High-throughput screening of compounds can be carried out to identify agents that disrupt BMP- or ALK3-mediated effects on bone. The assay is carried out to detect and identify compounds that specifically inhibit or reduce the binding of an ALK3 polypeptide to BMPs. Alternatively, the assay can be used to identify compounds that enhance the binding of an ALK3 polypeptide to BMPs. Compounds can be identified by their ability to interact with a BMP or ALK3 polypeptide.

A variety of assay formats will suffice and, in light of the present disclosure, those not expressly described herein will, however, be understood by one of ordinary skill in the art. As described herein, the test compounds (agents) of the invention can be created by any combinatorial chemical method. Alternatively, the compounds in question can be naturally occurring biomolecules synthesized in vivo or in vitro. Compounds (agents) to be tested for their ability to act as tissue growth modulators can be produced, for example, by bacteria, yeast, plants or other organisms (e.g. natural products), they can be produced chemically (e.g. , small molecules, including peptidomimetics) or can be produced recombinantly. Test compounds include non-peptide organic molecules, peptides, polypeptides, peptidomimetics, sugars, hormones, and nucleic acid molecules. The test agent is a small organic molecule that has a molecular weight of less than about 2000 daltons.

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Test compounds of the invention can be provided as discrete, individual entities, or provided in more complex libraries, such as those made by combinatorial chemistry. These libraries can comprise, for example, alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers, and other classes of organic compounds. Presentation of the test compounds to the test system can be either in isolation or as mixtures of compounds, especially in the initial detection steps. Optionally, the compounds can be optionally derivatized with other compounds and have derivatizing groups that facilitate the isolation of the compounds. Non-limiting examples of derivatizing groups include biotin, fluorescein, digoxigenin, green fluorescent protein, isotopes, polyhistidine, magnetic beads, glutathione S transferase (GST), photoactivatable crosslinking agents, or any combination thereof.

In many drug screening programs that screen libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds studied in a given period of time. Assays that are performed in cell-free systems, such as that which can be derived with purified or semi-purified proteins, are often preferred as primary screens because they can be generated to allow rapid development and relatively easy detection of an alteration. on a molecular target that is mediated by a test compound. On the other hand, the effects of cellular toxicity or bioavailability of the test compound can be generally ignored in the in vitro system, instead of being focused mainly on the effect of the drug on the molecular target as it can be manifested in an alteration of the binding affinity between an ALK3 polypeptide and BMPs.

Merely to illustrate, in an exemplary screening assay, the compound of interest is contacted with an isolated and purified ALK3 polypeptide that is normally capable of binding to BMPs. To the mixture of the compound and the ALK3 polypeptide, a composition containing an ALK3 ligand is then added. Detection and quantification of ALK3 / BMP complexes provides a means of determining the compound's efficacy in inhibiting (or enhancing) complex formation between ALK3 polypeptide and BMPs. The efficacy of the compound can be evaluated by generating dose response curves from data obtained using various concentrations of the test compound. In addition, a control assay can also be performed to provide a baseline for comparison. For example, in a control assay, an isolated and purified BMP is added to a composition containing the ALK3 polypeptide, and the formation of the ALK3 / bMp complex is quantitated in the absence of the test compound. It will be understood that, in general, the order in which the reagents can be mixed can be varied, and they can be mixed simultaneously. On the other hand, instead of purified proteins, cell extracts and lysates can be used to set up a suitable cell-free assay system.

Complex formation between ALK3 polypeptide and BMPs can be detected by a variety of techniques. For example, modulation of complex formation can be quantified using, for example, detectably labeled proteins such as radiolabeled ALK3 polypeptide (for example,³²P, ³⁵Yes, ¹⁴C or ³H), fluorescently labeled (eg FITC) or enzymatically labeled ALK3 polypeptide or BMPs, by immunoassay or chromatographic detection.

Fluorescence polarization assays and fluorescence resonance energy transfer (FRET) assays can be used in measuring, either directly or indirectly, the degree of interaction between an ALK3 polypeptide and its binding protein. In addition, other detection modes can be used, such as those based on optical waveguides (PCT publication WO 96/26432 and US Patent No. 5,677,196), surface plasmon resonance (SPR), surface charge sensors, and surface resistance sensors.

In addition, an interaction trap assay, also known as the two-hybrid assay, can be used to identify agents that disrupt or enhance the interaction between an ALK3 polypeptide and its binding protein. See, for example, US Patent No. 5,283,317; Zervos et al. (1993) Cell 72: 223-232; Madura et al. (1993) J Biol Chem 268: 12046-12054; Bartel et al. (1993) Biotechniques 14: 920-924; and Iwabuchi et al. (1993) Oncogene 8: 1693-1696). Inverse two-hybrid systems can be used to identify compounds (eg, small molecules or peptides) that dissociate interactions between an ALK3 polypeptide and its binding protein. See, for example, Vidal and Legrain, (1999) Nucleic Acids Res 27: 919-29; Vidal and Legrain, (1999) Trends Biotechnol 17: 374-81; and US Patent Nos. 5,525,490; 5,955,280; and 5,965,368.

The present compounds can be identified by their ability to interact with an ALK3 or BMP polypeptide of the invention. The interaction between the compound and the ALK3 or BMP polypeptide can be covalent or non-covalent. For example, such an interaction can be identified at the protein level using in vitro biochemical methods, including photo-crosslinking, radiolabeled ligand binding, and affinity chromatography (Jakoby WB et al, 1974, Methods in Enzymology 46: 1). In certain cases, compounds can be screened in a mechanism-based assay, such as an assay to detect compounds that bind to a BMP or ALK3 polypeptide. This can include a solid phase or fluid phase binding event. Alternatively, the gene encoding a BMP or ALK3 polypeptide can be transfected with a reporter system (eg, β-galactosidase, luciferase, or green fluorescent protein) into a cell and screened against the library of choice by screening for high performance or with individual members of the library. Other mechanism-based binding assays can be used, for example, binding assays that detect changes in free energy. The trials of

ES 2 575 695 T3 binding can be performed with the target attached to a well, bead or chip or captured by an immobilized antibody or resolved by capillary electrophoresis. Bound compounds can generally be detected by colorimetric methods or fluorescence or surface plasmon resonance.

Described herein are methods and agents for modulating (stimulating or inhibiting) bone formation and increasing bone mass. Therefore, any identified compound can be tested in whole cells or tissues, in vitro or in vivo, to confirm its ability to modulate bone growth or mineralization. Various methods known in the art can be used for this purpose.

For example, the effect of ALK3 or BMP polypeptides or test compounds on bone or cartilage growth can be determined by measuring Msx2 induction or differentiation of osteoprogenitor cells into osteoblasts in cell-based assays ( see, for example, Daluiski et al., Nat Genet., 2001, 27 (1): 84-8; Hino et al, Front Biosci. 2004, 9: 1520-9). Another example of cell-based assays includes the analysis of the osteogenic activity of the subject ALK3 or BMP polypeptides and test compounds in mesenchymal and osteoblast progenitor cells. To illustrate, recombinant adenoviruses expressing a BMP or ALK3 polypeptide can be constructed to infect C3H10T1 / 2 pluripotent mesenchymal progenitor cells, preosteoblast C2C12 cells, and TE-85 osteoblast cells. Osteogenic activity is then determined by measuring the induction of alkaline phosphatase, osteocalcin, and matrix mineralization (see, for example, Cheng et al, J Bone Joint Surg Am 2003, 85-A (8): 1544-1552).

In vivo assays can be used to measure bone or cartilage growth. For example, Namkung-Matthai et al, Bone, 28: 80-86 (2001) describe an osteoporotic rat model in which bone repair is studied during the initial period after fracture. Kubo et al, Steroid Biochemistry & Molecular Biology, 68: 197-202 (1999) also describe an osteoporotic rat model in which bone repair is studied during the last period after fracture. Andersson et al., J. Endocrinol. 170: 529-537 describe a mouse model of osteoporosis in which mice are ovarioectomized, causing the mice to lose substantial bone mineral content and bone mineral density, with trabecular bone losing roughly 50% of the bone mineral density. Bone mineral density. Bone density could be increased in ovarioectomized mice by administering factors such as parathyroid hormone. Fracture healing tests that are known in the art can be used. These tests include fracture technique, histological analysis, and biomechanical analysis, which are described, for example, in US Patent No. 6,521,750, which describes experimental protocols for causing fractures as well as measurement of the extent of fractures and their repair process.

6. Exemplary therapeutic uses

In the fourth aspect of the invention, certain BMP-ALK3 antagonists (eg, ALK3 polypeptides) can be used to treat or prevent a disease or condition that is associated with bone damage, either, for example, through breakage, loss or demineralization. Damage to bone can be treated or prevented in an individual in need thereof by administering to the individual a therapeutically effective amount of a BMP-ALK3 antagonist, particularly an ALK3 polypeptide. Given the potential for a dual effect on bone resorption and formation, such compounds may be useful in a wide range of diseases that are currently treated with anabolics (eg, parathyroid hormone and derivatives thereof) or anti-resorption agents. (eg bisphosphonates). Described herein are methods of promoting bone growth or mineralization in an individual in need thereof through the administration to the individual of a therapeutically effective amount of a BMP-ALK3 antagonist, particularly an ALK3 polypeptide. These methods are optionally directed to therapeutic and prophylactic treatments of animals and, more preferably, of humans. The disclosure provides the use of BMP-ALK3 antagonists (particularly soluble ALK3 polypeptides and neutralizing antibodies directed to BMPs or ALK3s) for the treatment of disorders associated with low bone density or decreased bone strength.

As used herein, a "therapeutic agent that prevents a disorder or condition" refers to a compound that, in a statistical sample, reduces the occurrence of the disease or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample. The term "treat", as used herein, includes prophylaxis of the named condition or amelioration or elimination once it has been established. In either case, prevention or treatment can be distinguished in the diagnosis provided by a physician and the desired result of the administration of the therapeutic agent.

The description provides methods of inducing bone and / or cartilage formation, avoiding bone loss, increasing bone mineralization, or preventing demineralization of bone. For example, the subject BMP-ALK3 antagonists have application in the treatment of bone loss disorders, such as osteoporosis and the healing of bone fractures and cartilage defects or other bone defects, injuries and disorders in humans and others. animals. ALK3 or BMP polypeptides may be useful in patients diagnosed with subclinical low bone density, as a protective measure against the development of osteoporosis.

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The compositions of the present invention may find medical utility in the healing of bone fractures and cartilage defects in humans and other animals. The methods and compositions can also have prophylactic use in closed, as well as the reduction of open fractures and also in the better fixation of artificial joints. Osteogenic agent-induced de novo bone formation contributes to the repair of congenital, trauma-induced, or oncologic resection-induced craniofacial defects and is also useful in cosmetic plastic surgery. In certain instances, the subject BMP-ALK3 antagonists can provide an environment for attracting bone-forming cells, stimulating growth of bone-forming cells, or inducing differentiation of bone-forming cell progenitors. The BMPALK3 antagonists of the invention may also be useful in the treatment of osteoporosis.

Rosen et al. (ed) Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, 7^to ed. American Society for Bone and Mineral Research, Washington DC offers an extensive discussion of bone disorders that can be treated with BMP-ALK3 antagonists. A partial list is provided in this document. The compositions of the invention can be applied to conditions characterized or causing loss of bone mass, such as osteoporosis (including secondary osteoporosis), hyperparathyroidism, mineral and bone disorder associated with kidney disease, deprivation of sex hormones or ablation (for example , androgens and / or estrogens), glucocorticoid treatment, rheumatoid arthritis, severe burns, hyperparathyroidism, hypercalcaemia, hypocalcaemia, hypophosphatemia, osteomalacia (including tumor-induced osteomalacia), hyperphosphatemia, vitamin D deficiency, hyperparathyroidism (including familial hyperparathyroidism) and pseudohypoparathyroidism, tumor metastases to bone, bone loss as a result of tumor or chemotherapy, bone and bone tumors bone marrow (eg multiple myeloma), ischemic bone disorders, periodontal disease and oral bone loss, Cushing's disease, Paget's disease, thyrotoxicosis, chronic diarrhea or malabsorption state, renal tubular acidosis or anorexia nervosa. The compositions of the invention can also be applied to conditions characterized by failure of bone formation or healing, including unconsolidated fractures, fractures that are otherwise slow to heal, fetal and neonatal bone dysplasias (eg, hypocalcemia, hypercalcemia , calcium receptor defects and vitamin D deficiency), osteonecrosis (including osteonecrosis of the jaw) and osteogenesis imperfecta. Furthermore, the anabolic effects will cause such antagonists to decrease bone pain associated with bone damage or erosion. As a consequence of the anti-resorption effects, such antagonists may be useful to treat disorders of abnormal bone formation, such as osteoblastic tumor metastases (for example, associated with primary prostate or breast cancer), osteogenic osteosarcoma, osteopetrosis, dysplasia. progressive diaphysealis, endosseous hyperostosis, osteopoikilia and melorheostosis. Other conditions that can be treated include fibrous dysplasia and chondrodysplasias.

In addition to the above discussion, people with any of the following profiles may be candidates for treatment with an ALK3 antagonist: a woman who is post-menopausal and not taking estrogen or other hormone replacement therapy; a person with a personal or maternal history of hip fracture or a smoker; a post-menopausal woman who is tall (more than 5 feet 7 inches (170.18 centimeters)) or thin (less than 125 pounds (56.70 kg)); a man with clinical diseases associated with loss of bone mass; a person using medications known to cause bone loss, including corticosteroids, such as Prednisone ™, various anti-seizure medications such as Dilantin ™ and certain barbiturates, or high-dose thyroid replacement drugs; a person who has type 1 diabetes, liver disease, kidney disease, a family history of osteoporosis; a person who has high bone turnover (for example, excess collagen in urine samples); a person with a thyroid disease, such as hyperthyroidism; a person who has sustained a fracture after a single minor trauma; a person who has had x-ray evidence of a vertebral fracture or other signs of osteoporosis.

Osteoporosis (which means, in general terms, a state of low bone density or resistance) can be caused or is associated with several factors. Being a woman, especially a postmenopausal woman, who has a low body weight and a sedentary lifestyle are risk factors for osteoporosis (loss of bone mineral density, leading to risk of fracture).

Osteoporosis can also result as a condition associated with another disorder or from the use of certain medications. Osteoporosis resulting from drugs or some other medical condition is known as secondary osteoporosis. In a condition known as Cushing's disease, the excess amount of cortisol produced by the body results in osteoporosis and fractures. The most common medications associated with secondary osteoporosis are corticosteroids, a class of drugs that act like cortisol, a hormone produced naturally by the adrenal glands. Although adequate levels of thyroid hormones (which are produced by the thyroid gland) are needed for skeletal development, excess thyroid hormone can decrease bone mass over time. Aluminum-containing antacids can lead to bone loss when taken in high doses by people with kidney problems, particularly those on dialysis. Other medications that can cause secondary osteoporosis include phenytoin (Dilantin) and barbiturates that are used to prevent seizures; methotrexate (Rheumatrex, Immunex, folex PFS), a medicine for some forms of arthritis, cancer, and immune system disorders; cyclosporine (Sandimmune, Neoral), a drug used to treat some autoimmune diseases and to suppress the immune system in organ transplant patients; luteinizing hormone-releasing hormone agonists (Lupron, Zoladex), which is used to treat prostate cancer and endometriosis; heparin (Calciparin, Liquaemin), a medicine

ES 2 575 695 T3 anticoagulant; and cholestyramine (Questran) and colestipol (Colestid), which is used to treat high cholesterol. Bone loss resulting from cancer therapy is widely recognized and is called cancer therapy-induced bone loss (CTIBL). Bone metastases can create cavities in the bone that can be corrected by treatment with BMP-ALK3 antagonists.

Optionally, BMP-ALK3 antagonists, particularly a soluble ALK3, described herein, can be used in cancer patients. Patients who have certain tumors (eg, prostate, breast, multiple myeloma, or any tumor that causes hyperparathyroidism) are at high risk for bone loss due to tumor-induced bone loss, as well as bone metastases and therapeutic agents . These patients can be treated with BMP-ALK3 antagonists even in the absence of evidence of bone loss or bone metastases. Patients can also be monitored for evidence of bone loss or bone metastases, and can be treated with BMP-ALK3 antagonists if indicators suggest an increased risk. In general, DEXA scans are used to assess changes in bone density, while indicators of bone remodeling can be used to assess the likelihood of bone metastases. Serum markers can be monitored. Bone-specific alkaline phosphatase (BSAP) is an enzyme that is present in osteoblasts. Blood levels of BSAP are increased in patients with bone metastases and other conditions that result in increased bone remodeling. Osteocalcin and procollagen peptides are also associated with bone formation and bone metastases. Increases in BSAP have been detected in patients with bone metastases caused by prostate cancer and, to a lesser extent, in bone metastases from breast cancer. Bone morphogenetic protein-7 (BMP7) levels are elevated in prostate cancer that has metastasized to bone, but not in bone metastases due to bladder, skin, liver, or lung cancer. Type I carboxy-terminal telopeptide (CIFT) is a cross-linking agent found in collagen that is formed during bone resorption. Since bone is constantly being broken down and reshaped, ICTP is found throughout the body. However, at the site of bone metastasis, the level will be significantly higher than in an area of ​​normal bone. ICTP has been found at high levels in bone metastasis due to prostate, lung, and breast cancer. Another collagen crosslinking agent, Type I N-terminal telopeptide (NTx), is produced in conjunction with ICTP during bone turnover. The amount of NTx is increased in bone metastasis caused by many different types of cancer, including lung, prostate, and breast cancer. Furthermore, NTx levels increase with the progression of bone metastasis. Therefore, this marker can be used to detect both metastasis and to measure the extent of the disease. Other resorption markers include pyridinoline and deoxypyridoline. Any increase in bone resorption markers or metastasis markers indicates the need for BMP-ALK3 antagonist therapy in a patient.

BMP-ALK3 antagonists can be used in patients with a mineral and bone disorder associated with kidney disease (CKD-MBD), a broad syndrome of interrelated skeletal, cardiovascular and mineral metabolic disorders derived from kidney disease. CKD-MBD encompasses various skeletal pathologies often and is referred to as renal osteodystrophy (ROD), which is preferred for treatment with BMP-ALK3 antagonists. Depending on the relative contribution of the different pathogenic factors, ROD manifests itself as various pathological patterns of bone remodeling (Hruska et al., 2008, Chronic kidney disease mineral bone disorder (CKD-MBD); in Rosen et al (ed) Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, 7^to ed. American Society for Bone and Mineral Research, Washington DC, pp. 343-349). At one end of the spectrum is ROD with uremic osteodystrophy and low bone turnover, characterized by a low number of active remodeling sites, deeply suppressed bone formation, and low bone resorption. At the other extreme is ROD with hyperparathyroidism, high bone turnover, and osteitis fibrosa. Since BMP-ALK3 antagonists exert both anabolic and antiresorptive effects, these agents may be useful in patients across the spectrum of ROD pathology.

BMP-ALK3 antagonists can be administered in conjunction with other pharmaceutical agents. Co-administration can be carried out by administration of a single co-formulation, by simultaneous administration or by administration at separate times. BMP-ALK3 antagonists can be particularly advantageous when administered with other bone active agents. A patient may benefit from co-receiving the BMP-ALK3 antagonist and taking calcium supplements, vitamin D, appropriate exercise, and / or, in some cases, other medication. Examples of other medications include bisphosphonates (alendronate, ibandronate, and risedronate), calcitonin, estrogens, parathyroid hormone, and raloxifene. Bisphosphonates (alendronate, ibandronate, and risedronate), calcitonin, estrogens, and raloxifene affect the bone remodeling cycle and are classified as anti-resorption drugs. Bone remodeling consists of two distinct stages: bone resorption and bone formation. Antiresorptive medications slow or stop the bone resorption portion of the bone remodeling cycle but do not slow down the bone-building portion of the cycle. As a result, new formation continues at a faster rate than bone resorption, and bone density can increase over time. Teriparatide, a form of parathyroid hormone, increases the rate of bone formation in the bone remodeling cycle. Alendronate is approved for both the prevention (5 mg per day or 35 mg once a week) and the treatment (10 mg per day or 70 mg once a week) of postmenopausal osteoporosis. Alendronate reduces bone loss, increases bone density, and reduces the risk of spinal, wrist, and hip fractures. Alendronate is also approved for the treatment of glucocorticoid-induced osteoporosis in men and women, as a result of long-term use of glucocorticoids.

ES 2 575 695 T3 medicines (i.e. prednisone and cortisone) and for the treatment of osteoporosis in men. Alendronate and vitamin D are approved for the treatment of osteoporosis in postmenopausal women (70 mg once a week plus vitamin D), and for the treatment to improve bone mass in men with osteoporosis. Ibandronate is approved for the prevention and treatment of postmenopausal osteoporosis. Taken as a pill once a month (150 mg), ibandronate should be taken on the same day each month. Ibandronate reduces bone loss, increases bone density and reduces the risk of spinal fractures. Risedronate is approved for the prevention and treatment of postmenopausal osteoporosis. Taken daily (5 mg dose) or weekly (35 mg dose or 35 mg dose with calcium), risedronate reduces bone loss, increases bone density and reduces the risk of spinal and non-vertebral fractures. Risedronate is also approved for use by men and women to prevent and / or treat glucocorticoid-induced osteoporosis that results from long-term use of these medications (i.e., prednisone or cortisone). Calcitonin is a natural hormone involved in the regulation of calcium and bone metabolism. In women who are more than 5 years after menopause, calcitonin reduces bone loss, increases bone density in the spine, and can relieve pain associated with bone fractures. Calcitonin reduces the risk of spinal fractures. Calcitonin is available as an injection (50-100 IU per day) or nasal spray (200 IU per day). Estrogen therapy (ET) / hormone therapy (HT) is approved for the prevention of osteoporosis. ET has been shown to reduce bone loss, increase bone density in the spine and hip, and reduce the risk of hip and vertebral fractures in postmenopausal women. TE is most commonly administered in the form of a pill or skin patch that provides a low dose of approximately 0.3 mg per day or a standard dose of approximately 0.625 mg per day and is effective even when started after age 70 year old. When estrogens are taken alone, the risk in women of developing cancer of the uterine lining (endometrial cancer) may be increased. To eliminate this risk, medical professionals prescribe the hormone progestin in combination with estrogens (hormone replacement therapy or HT) for those women who have an intact uterus. TE / HT relieves symptoms of menopause and has been shown to have a beneficial effect on bone health. Side effects can include vaginal bleeding, breast tenderness, mood swings, and gallbladder disease. Raloxifene, 60 mg daily, is approved for the prevention and treatment of postmenopausal osteoporosis. It is from a class of drugs called selective estrogen receptor modulators (SERMs) that have been developed to provide beneficial effects of estrogens without their potential downsides. Raloxifene increases bone mass and reduces the risk of spinal fractures. No data are yet available to show that raloxifene can reduce the risk of hip and other non-vertebral fractures. Teriparatide, a form of parathyroid hormone, is approved for the treatment of osteoporosis in postmenopausal women and men who are at high risk for a fracture. This medicine stimulates the formation of new bone and significantly increases bone mineral density. In postmenopausal women, reduced fractures were observed in the spine, hip, feet, ribs, and wrist. In men, reduction of fractures was seen in the spine, but there were insufficient data to assess reduction of fractures at other sites. Teriparatide is administered as a daily self-injection for up to 24 months.

7. Pharmaceutical compositions

BMP-ALK3 antagonists (eg, ALK3 polypeptides) can be formulated with a pharmaceutically acceptable carrier. For example, an ALK3 polypeptide can be administered alone or as a component of a pharmaceutical formulation (therapeutic composition). The subject compounds can be formulated for administration in any manner convenient for use in human or veterinary medicine.

The composition can be administered systemically or locally, as an implant or device. When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free and physiologically acceptable form. Therapeutically useful agents other than ALK3 antagonists which may also optionally be included in the composition as described above, can be administered simultaneously or sequentially with the subject compounds (eg, ALK3 polypeptides) in the methods of the invention.

Typically, ALK3 antagonists will be administered parenterally. Pharmaceutical compositions suitable for parenteral administration may comprise one or more ALK3 polypeptides in combination with one or more pharmaceutically acceptable isotonic and sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into injectable solutions or dispersions. sterile just before use, which may contain antioxidants, buffers, bacteriostatic agents, solutes that make the formulation isotonic with the blood of the desired recipient or suspending or thickening agents. Examples of suitable aqueous and non-aqueous carriers that can be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as oil of olive, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by maintaining the required particle size in the case of dispersions and by the use of surfactants.

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In addition, the composition can be encapsulated or injected into a form for delivery to a target tissue site (eg, bone). In certain embodiments, the compositions of the present invention may include a matrix capable of delivering one or more therapeutic compounds (eg, ALK3 polypeptides) to a target tissue site (eg, bone), providing a structure for the tissue development and optimally capable of being reabsorbed into the body. For example, the matrix can provide for the slow release of ALK3 polypeptides. Such matrices can be formed from materials currently in use for other implanted medical applications.

The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic aspects, and interface properties. The particular application of the compositions in question will define the appropriate formulation. Potential matrices for the compositions can be biodegradable and chemically defined by calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, and polyanhydrides. Other potential materials are biodegradable and biologically well defined, such as bone or dermal collagen. Other matrices are composed of pure proteins or extracellular matrix components. Other potential matrices are non-biodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics. The matrices can be composed of combinations of any of the aforementioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. Bioceramics can be altered in composition, such as calcium aluminate phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.

In certain embodiments, the compositions of the invention can be administered orally, for example, in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as tablets (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as mouthwashes and the like, each containing a predetermined amount of an agent as the active ingredient. The agent can also be administered as a bolus, electuary, or paste.

In solid dosage forms for oral administration (capsules, tablets, pills, lozenges, powders, granules, and the like), one or more therapeutic compounds of the present invention can be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate. or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution of retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions can also comprise buffering agents. Solid compositions of a similar type can also be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol. , benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, peanut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and sorbitan fatty acid esters and mixtures thereof. In addition to inert diluents, oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming, and preserving agents.

Suspensions, in addition to active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

The compositions of the invention may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like in the compositions. Furthermore, prolonged absorption of the injectable pharmaceutical form can be caused by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

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It is understood that the dosage regimen will be determined by the attending physician considering various factors that modify the action of the compounds object of the invention (for example, ALK3 polypeptides). The various factors include, but are not limited to, the amount of bone weight to be formed, the degree of bone density loss, the site of bone damage, the condition of the damaged bone, the age of the patient, the sex and diet, severity of any disease that may be contributing to bone loss, timing of administration, and other clinical factors. Optionally, the dosage can vary with the type of matrix used in reconstitution and the types of compounds in the composition. The addition of other known growth factors to the final composition can also affect the dosage. Progress can be monitored by periodic evaluation of bone growth and / or repair, eg, X-rays (including DEXA), histomorphometric determinations, and tetracycline labeling.

Gene therapy can be used for the in vivo production of ALK3 polypeptides. Such therapy would achieve its therapeutic effect by introducing ALK3 polynucleotide sequences into cells or tissues having the disorders listed above. Administration of the ALK3 polynucleotide sequences can be accomplished using a recombinant expression vector, such as a chimeric virus or a colloidal dispersion system. The use of targeted liposomes is preferred for therapeutic delivery of ALK3 polynucleotide sequences.

Various viral vectors that can be used for gene therapy as taught herein include adenovirus, herpes virus, vaccinia, or, preferably, an RNA virus such as a retrovirus. Preferably, the retroviral vector is a derivative of a murine or avian retrovirus. Examples of retroviral vectors into which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus ( MuMTV) and Rous sarcoma virus (RSV). A series of additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that the transduced cells can be identified and generated. Retroviral vectors can be made specifically targeted by binding, for example, a sugar, a glycolipid, or a protein. The preferred target is achieved using an antibody. Those skilled in the art will recognize that specific polynucleotide sequences can be inserted into the retroviral genome or attached to a viral envelope to allow target-specific delivery of the retroviral vector containing the ALK3 polynucleotide. The vector is directed to bone or cartilage.

Alternatively, tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol and env, by conventional calcium phosphate transfection. These cells were then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium.

Another targeted delivery system for ALK3 polynucleotides is a colloidal dispersion system. Colloidal dispersion systems include complexes of macromolecules, nanocapsules, microspheres, beads, and lipid-based systems that include oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome. Liposomes are artificial membrane vesicles that are useful as in vitro and in vivo delivery vehicles. Intact RNA, DNA, and virions can be encapsulated within the aqueous interior and delivered to cells in a biologically active form (see, eg, Fraley, et al., Trends Biochem. Sci., 6:77, 1981). Methods for efficient gene transfer using a liposome carrier are known in the art, see, for example, Mannino, et al, Biotechniques, 6: 682, 1988. The composition of the liposome is usually a combination of phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids can also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

Examples of useful lipids in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine. Liposome recognition is also possible based on, for example, organ specificity, cell specificity, and organelle specificity and is known in the art.

Exemplification

The invention is now described generally; it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain embodiments of the present invention and are not intended to limit the invention.

Example 1. Generation of ALK3-Fc fusion proteins

The amino acid sequence and corresponding nucleotide sequence for native human ALK3 are shown in Figures 1 and 2. Applicants designed an ALK3-hFc fusion protein in which the extracellular domain (residues 24-152) of human ALK3 (Figures 3 and 4) are fused through the C-terminus with a human Fc domain (Figures 5 and 6) through a minimal linker (composed of the amino acid residues TGGG) to produce the protein shown in Figure 7. The following three leader sequences were considered:

ES 2 575 695 T3 (i) Native: MPQLYIYIRLLGAYLFIISRVQG (SEQ ID NO: 8) (ii) Tissue plasminogen activator (TPA): MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 9) (iii) Honey melitin MKFYFLYVYML: (SEQ ID NO: 10)

The selected form of hALK3 (24-152) -hFc (SEQ ID NO: 11) employs the TPA leader and has the raw amino acid sequence shown in Figure 8. A sense nucleotide sequence encoding this protein of fusion and the corresponding antisense sequence are indicated in Figure 9. An alternate sense nucleotide sequence encoding hALK3 (24-152) -hFc is shown below, incorporating a CT substitution at position 1137 (underlined) that does not alter the amino acid sequence.

<td> 1</td><td>ATGGATGCAA</td><td>TGAAGAGAGG</td><td>GCTCTGCTGT</td><td>GTGCTGCTGC</td>

<td> 41</td><td>TGTGTGGAGC</td><td>AGTCTTCGTT</td><td>TCGCCCGGCG</td><td>CCCAGAATCT</td>

<td> 81</td><td>GGATAGTATG</td><td>CTTCATGGCA</td><td>CTGGGATGAA</td><td>ATCAGACTCC</td>

<td> 121</td><td>GACCAGAAAA</td><td>AGTCAGAAAA</td><td>TGGAGTAACC</td><td>TTAGCACCAG</td>

<td> 161</td><td>AGGATACCTT</td><td>GCCTTTTTTA</td><td>AAGTGCTATT</td><td>GCTCAGGGCA</td>

<td> 201</td><td>CTGTCCAGAT</td><td>GATGCTATTA</td><td>ATAACACATG</td><td>CATAACTAAT</td>

<td> 241</td><td>GGACATTGCT</td><td>TTGCCATCAT</td><td>AGAAGAAGAT</td><td>GACCAGGGAG</td>

<td> 281</td><td>AAACCACATT</td><td>AGCTTCAGGG</td><td>TGTATGAAAT</td><td>ATGAAGGATC</td>

<td> 321</td><td>TGATTTTCAG</td><td>TGCAAAGATT</td><td>CTCCAAAAGC</td><td>CCAGCTACGC</td>

<td> 361</td><td>CGGACAATAG</td><td>AATGTTGTCG</td><td>GACCAATTTA</td><td>TGTAACCAGT</td>

<td> 401</td><td>ATTTGCAACC</td><td>CACACTGCCC</td><td>CCTGTTGTCA</td><td>TAGGTCCGTT</td>

<td> 441</td><td>TTTTGATGGC</td><td>AGCATTCGAA</td><td>CCGGTGGTGG</td><td>AACTCACACA</td>

<td> 481</td><td>TGCCCACCGT</td><td>GCCCAGCACC</td><td>TGAACTCCTG</td><td>GGGGGACCGT</td>

<td> 521</td><td>CAGTCTTCCT</td><td>CTTCCCCCCA</td><td>AAACCCAAGG</td><td>ACACCCTCAT</td>

<td> 561</td><td>GATCTCCCGG</td><td>ACCCCTGAGG</td><td>TCACATGCGT</td><td>GGTGGTGGAC</td>

<td> 601</td><td>GTGAGCCACG</td><td>AAGACCCTGA</td><td>GGTCAAGTTC</td><td>AACTGGTACG</td>

<td> 641</td><td>TGGACGGCGT</td><td>GGAGGTGCAT</td><td>AATGCCAAGA</td><td>CAAAGCCGCG</td>

<td> 681</td><td>GGAGGAGCAG</td><td>TACAACAGCA</td><td>CGTACCGTGT</td><td>GGTCAGCGTC</td>

<td> 721</td><td>CTCACCGTCC</td><td>TGCACCAGGA</td><td>CTGGCTGAAT</td><td>GGCAAGGAGT</td>

<td> 761</td><td>ACAAGTGCAA</td><td>GGTCTCCAAC</td><td>AAAGCCCTCC</td><td>CAGCCCCCAT</td>

<td> 801</td><td>CGAGAAAACC</td><td>ATCTCCAAAG</td><td>CCAAAGGGCA</td><td>GCCCCGAGAA</td>

<td> 841</td><td>CCACAGGTGT</td><td>ACACCCTGCC</td><td>CCCATCCCGG</td><td>GAGGAGATGA</td>

<td> 881</td><td>CCAAGAACCA</td><td>GGTCAGCCTG</td><td>ACCTGCCTGG</td><td>TCAAAGGCTT</td>

<td> 921</td><td>CTATCCCAGC</td><td>GACATCGCCG</td><td>TGGAGTGGGA</td><td>GAGCAATGGG</td>

<td> 961</td><td>CAGCCGGAGA</td><td>ACAACTACAA</td><td>GACCACGCCT</td><td>CCCGTGCTGG</td>

<td> 1001</td><td>ACTCCGACGG</td><td>CTCCTTCTTC</td><td>CTCTATAGCA</td><td>AGCTCACCGT</td>

<td> 1041</td><td>GGACAAGAGC</td><td>AGGTGGCAGC</td><td>AGGGGAACGT</td><td>CTTCTCATGC</td>

<td> 1081</td><td>TCCGTGATGC</td><td>ATGAGGCTCT</td><td>GCACAACCAC</td><td>TACACGCAGA</td>

<td> 1121</td><td>AGAGCCTCTC</td><td>CCTGTCTCCG</td><td>GGTAAATGA</td><td></td>

A hALK3 (24-152) -Fc variant with the TPA leader and with murine Fc substituted for human Fc is shown in Figure 10. A sense nucleotide sequence encoding this variant and its corresponding antisense sequence are indicated in Figure 11 Applicants constructed a form of hALK3 (24-152) -mFc having an asparagine at position 71 (position 70 in the native ALK3 ECD sequence). The protein was expressed in 15 CHO cell lines and the N-terminal sequence revealed a primary species with an N-terminal block, indicating an onset in the native glutamine (Q) remainder, consistent with the protein of SEQ ID NO: 7, and only one

ES 2 575 695 T3 minor sequence of GAQNLDSMLHGTGMK (SEQ ID NO: 17). Applicants further constructed a hALK3 (24-152) -hFc protein having the native ALK3 sequence. Another ALK3-Fc variant comprising the murine ALK3 extracellular domain (residues 24-152 native to the murine precursor) and the murine Fc domain was generated by similar methods. The amino acid sequence of this variant, mALK3 (24-152) -mFc, is shown below with the ALK3 domain underlined:

<td> 1</td><td>MDAMKRGLCC</td><td>VLLLCGAVFV</td><td>SPGAQNLDSM</td><td>LHGTGMKSDL</td><td>DQKKPENGVT</td>

<td> 51</td><td>LAPEDTLPFL</td><td>KCYCSGHCPD</td><td>DAINNTCITN</td><td>GHCFAIIEED</td><td>DQGETTLTSG</td>

<td> 101</td><td>CMKYEGSDFQ-</td><td>CKDSPKAQLR</td><td>RTIECCRTNL</td><td>CNQYLQPTLP</td><td>PVVIGPFFDG</td>

<td> 151</td><td>SIRTGGGEPR</td><td>VPITQNPCPP</td><td>LKECPPCAAP</td><td>DLLGGPSVFI</td><td>FPPKIKDVLM</td>

<td> 201</td><td>ISLSPMVTCV</td><td>WDVSEDDPD</td><td>VQISWFVNNV</td><td>EVHTAQTQTH</td><td>REDYNSTLRV</td>

<td> 251</td><td>VSALPIQHQD</td><td>WMSGKEFKCK</td><td>VNNRALPSPI</td><td>EKTISKPRGP</td><td>VRAPQVYVLP</td>

<td> 301</td><td>PPAEEMTKKE</td><td>FSLTCMITGF</td><td>lpaeiavdwt</td><td>SNGRTEQNYK</td><td>NTATVLDSDG</td>

<td> 351</td><td>SYFMYSKLRV</td><td>QKSIWERGSL</td><td>FACSVVHEGL</td><td>HNHLTTKTIS</td><td>RSLGK</td>

<td>(I KNOW THAT</td><td>ID NO: 18)</td><td></td><td></td><td></td><td></td>

Example 2. Ligand binding to ALK3-Fc

The Biacore ™ methodology was used to determine the binding affinity of Fc-ALK3 fusion proteins for more than 15 members of the BMP / GDF family. The mALK3-mFc derived from HEK 293 cells show high affinity binding to hBMP2 and hBMP4 (Kd = 2.43 x 10 '⁹ and 9.47 x 10 '¹⁰, respectively), as well as moderate affinity binding to various other ligands, including hBMP6 and hBMP7. HALK3 (24-152) -hFc shows a similar binding profile. Specifically, hALK3 (24-152) -hFc derived from HEK 293 cells bound to hBMP2 and hBMP4 with Kd of 6.53 x 10 ™ and 1.02 x 10⁹, respectively, while CHO cell-derived hALK3 (24-152) -hFc bound hBMP2 and hBMP4 with Kd of 4.53 x 10 ™ and 7.03 x 10 ™, respectively. Like mALK3 (24-152) -mFc, hALK3 (24-152) -hFc derived from both cell types showed moderate binding affinity to hBMP6 and hBMP7, among other ligands.

The overall selectivity of ALK3-Fc for BMP2 and BMP4 is remarkable. Although not wishing to be linked to any particular mechanism, applicants hypothesize, based on these results, that ALK3-Fc exerts its effects in vivo primarily by binding BMP2 and BMP4 and thus inhibiting the signaling by these ligands. Consequently, antibodies against BMP2 and / or BMP4 are also expected to stimulate bone formation. Alternatively, an antibody against the ligand binding domain of ALK3 is expected to inhibit ALK3-mediated signaling more broadly. Figure 18 schematically shows examples of three approaches proposed herein that interfere with BMP2 and BMP4 signaling, and potentially additional ligands in order to promote bone formation.

A series of ALK3-Fc proteins incorporating truncated variants of the human ALK3 extracellular domain (ECD) were generated and compared to hALK3 (24-152) -hFc for their ligand binding affinities. ALK3 ECD variants with N-terminal deletions of 6, 12, 27 or 31 amino acids, C-terminal deletions of 6 or 12 amino acids, and a double truncation were expressed in HEK 293 cells and purified by Mab chromatography (column Protein A). The Biacore ™ methodology was used to select members of the BMP / GDF / TGFp ligand superfamily for binding to these variants.

<td colspan="6">Binding Affinity (Kd, in pM) of Selected Human Ligands for Human ALK3 ECD Variants</td>

<td colspan="2">Construct Expressed in Cells 293</td><td colspan="4">Binding</td>

<td></td><td></td><td>hBMP2</td><td>hBMP4</td><td>hBMP6</td><td>hBMP7</td>

<td>Complete Chain</td><td>hALK3 (24-152) -hFc</td><td> 653</td><td> 1020</td><td> 17300</td><td> 5990</td>

<td>ALK3NA6</td><td>hALK3 (30-152) -hFc</td><td> 869</td><td> 1610</td><td> 12800</td><td> -</td>

<td>ALK3NA12</td><td>hALK3 (36-152) -hFc</td><td> 1040</td><td> -</td><td> 5280</td><td> -</td>

<td>ALK3NA27</td><td>hALK3 (51-152) -hFc</td><td> 1570</td><td> —</td><td> 8040</td><td> 4290</td>

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<td colspan="6">Binding Affinity (Kd, in pM) of Selected Human Ligands for Human ALK3 ECD Variants</td>

<td colspan="2">Construct Expressed in Cells 293</td><td colspan="4">Binding</td>

<td></td><td></td><td>hBMP2</td><td>hBMP4</td><td>hBMP6</td><td>hBMP7</td>

<td>ALK3NA31</td><td>hALK3 (55-152) -hFc</td><td> 663</td><td> -</td><td> 17000</td><td> 3670</td>

<td>ALK3CA6</td><td>hALK3 (24-146) -hFc</td><td> 532</td><td> 396</td><td> -</td><td> -</td>

<td>ALK3CA12</td><td>hALK3 (24-140) -hFc</td><td> 769</td><td> 446</td><td> -</td><td> 5900</td>

<td>ALK3NA6CA6</td><td>hALK3 (30-146) -hFc</td><td> 437</td><td> 329</td><td> -</td><td> -</td>

<td colspan="6">- binding not detectable</td>

As can be seen, the C-terminal truncations that were evaluated show similar or higher binding affinity for BMP2 / BMP4 compared to full-chain ALK3 ECD, with an overall reduction in binding to BMP6 / BMP-7, although ALK3CA12 retains binding to BMP-7 at an affinity similar to full-chain ALK3 ECDs. In contrast, N-terminal truncations tend to reduce BMP2 binding, abolish BMP4 binding, and show varying effects on BMP6 / BMP7 binding. Interestingly, doubly truncated ALK3NA6CA6 variants show higher affinity for BMP2 / BMP4 compared to full-length ALK3 ECDs, in combination with undetectable binding to BMP6 / BMP7. Molecules with higher selectivity for the desired targets, BMP2 and BMP4, are useful because they will have fewer off-target effects in patients. N-terminal sequencing showed that nucleic acid encoding a six amino acid truncation at the N-terminal end, when expressed in cell culture, resulted in a population of polypeptides having the six amino acid truncation and a population of polypeptides having a seven amino acid truncation. Taken together, these demonstrate that hALK3-HFC polypeptides containing up to a seven amino acid truncation at the N-terminus and up to a twelve amino acid truncation at the C-terminus retain useful activity and demonstrate the desirable reduction and surprising in binding to off-target ligands. Therefore, an ALK3 polypeptide comprising at least amino acids 8 to 117 of SEQ ID NO: 3 can be used for the purposes described herein.

Ligand binding properties were used to compare the quality of the hALK3 (24-152) -hFc protein derived from CHO cells with those derived from HEK 293 cells. As determined by the Biacore ™ methodology, the affinity (Kd) of BMP2 for hALK3 (24-152) -hFc did not differ depending on the source of the fusion protein; however, the percentage of active protein generated by the CHO cells was higher than that of the HEK 293 cells as a function of their respective Rmax values. Rmax is a measure of the quality of the protein equal to (PMa / PMl) X Rl X Sm, where PMa is the molecular weight of the analyte, PMl is the molecular weight of the ligand, Rl at the level of immobilization in response units, and Sm is the molar stoichiometry. Corresponding analysis of BMP4 binding revealed that the CHO cell-derived protein exhibited a higher affinity for BMP4 than that of HEK 293 cells (Kd of 314 pM vs. 1020 pM, respectively) and the Rmax value for the protein generated by CHO cells was three times that of the protein from HEK 293 cells, again indicating a higher percentage of active protein. Thus, the unexpected benefits of CHO cells as the source of the hALK3 (24-152) -hFc protein include higher binding affinity of the protein to BMP4 and higher predicted bioavailability to result in higher quality of protein (Rmax value).

Example 3. hALK3-mFC improves bone status in mice

The applicants investigated the ability of a version of ALK3-MFC to improve bone condition in mice. Twelve week old female C57BL / 6 mice (n = 8 per group) were treated with hALK3 (24-152) mFc, 10 mg / kg, or vehicle (Tris buffered saline) by intraperitoneal injection twice weekly for a total of six weeks. Compared to vehicle, hALK3 (24-152) -mFc significantly increased whole-body bone density, as determined by dual-energy X-ray absorptiometry (DEXA), on day 31 and this effect was maintained through completion of the study on day 42 (Figure 12). A similar effect of hALK3 (24-152) -mFc treatment on bone density was observed for localized analysis of lumbar vertebrae by DEXA at ​​these same time points (Figure 13). In addition, high-resolution measurements of the tibial shaft and proximal tibia were carried out using micro-computed tomography (micro-CT) to determine the effect of hALK3 (24-152) -mFc on cortical bone and trabecular bone. , respectively. Compared with the vehicle, the hALK3 (24-152) -mFc treatment significantly increased: i) the thickness of the

ES 2 575 695 T3 cortical bone at week 6 (Figure 14), ii) trabecular bone volume at Week 4 (Figure 15) and iii) mean trabecular thickness at week 4 (Figure 16). Representative three-dimensional reconstructions of micro-CT generated sections through the proximal tibia (Figure 17) highlight the robust stimulatory effect of hALK (24-152) -mFc treatment (4 weeks) on trabecular bone microarchitecture. . Importantly, hALK (24-152) -mFc treatment did not cause significant changes in lean tissue mass, fat mass, or red blood cell mass over the course of the study.

Taken together, the above data demonstrate that hALK3 (24-152) -mFc can be used in vivo to selectively improve bone status through increasing bone mineral density and increasing net formation of both cortical bone and bone. the trabecular.

Example 4. hALK3-mFc increases bone strength in mice

In the experiment described in Example 3, applicants also investigated the ability of hALK3 (24-152) mFc to increase bone strength. After 6 weeks of treatment, the femurs were harvested and stored frozen at -20 ° C. The bones were then thawed at room temperature and destructive four-point bending tests were performed on the shaft of the left femur with an Instron mechanical testing instrument (Instron 4465 retrofitted to 5500). The separation between the fixed supports was 7 mm and the separation between the two points of application of the load was 2.5 mm. The load was applied at a constant displacement speed of 3 mm / min until bone breakage and the maximum load, stiffness and energy absorption data were calculated with the Bluehill v 2.5 software. Compared to vehicle, hALK3 (24-152) -mFc significantly increased peak bone load by 30% (Figure 19), bone stiffness by 14% (Figure 20), and energy at bone failure by a 32% (Figure 21). These findings demonstrate that the increase in bone strength accompanies the improvement in bone composition observed with hALK3 (24-152) -mFc treatment (Example 3).

Example 5. Effects of mALK3-mFC on bone in an OVX mouse model of osteopenia

Estrogen deficiency in postmenopausal women promotes bone loss, particularly loss of trabecular bone. Applicants therefore investigated the ability of mALK3 (24-152) -mFc to improve bone status in an ovarioectomized (OVX) mouse model of osteopenia with established bone loss. Eight week old female C57BL / 6 mice underwent bilateral OVX or sham surgery, then remained untreated for an interval of eight weeks. At the end of eight weeks, baseline micro-CT and DEXA measurements confirmed significant bone loss in OVX mice compared to sham. Most notable was a 43% reduction in trabecular bone volume (Figure 22, time point day 0), as determined in the proximal tibia by micro-CT. Mice were then treated with mALK3 (24-152) -mFc, 10 mg / kg, or vehicle (Tris buffered saline), by ip injection twice weekly for 8 weeks.

Treatment with mALK3 (24-152) -mFc led to improvement in both trabecular and cortical bone despite continuing estrogen deficiency. At study completion on day 56, the volume of trabecular bone in the proximal tibia of OVX mice treated with mALK3 (24-152) -mFc was increased by almost 250% compared to OVX controls and by more than 80%. compared to sham controls (Figure 22). Treatment with mALK3 (24-152) -mFc also caused cortical bone growth, as indicated by increased cortical thickness (Figure 23) and reduced endosseous circumference (Figure 24) in the tibial axis in comparison. with OVX controls. These improvements were accompanied by an increase in bone mineral density. Compared to OVX controls, treatment with mALK3 (24-152) -mFc significantly increased whole body bone mineral density (as determined by DEXA) on day 14 and this improvement was maintained until study completion ( Figure 25). Similar effects were observed with mALK3 (24-152) -mFc treatment on mineral density in the lumbar spine (Figure 26) and femur-tibia (Figure 27). Three-dimensional images of vertebral trabecular bone derived from micro-CT analysis (Figure 28) highlight the robust improvement in bone status associated with mALK3 (24-152) -mFc treatment, despite deficiency of ongoing estrogens. These findings demonstrate that mALK3 (24-152) -mFc can reverse the deterioration of bone, including trabecular bone, associated with estrogen withdrawal in a mouse model of osteopenia. The ability of mALK3 (24-152) -mFc to transform bone from an osteopenic condition to one that exceeds the quantity (Figures 22-24, 28) and matches the quality (Figures 25-27) of the bones in intact gonad controls is evidence that this agent exerts effects that are not only antiresorptive but also anabolic.

Example 6. Effects of mALK3-mFC on Bone Histomorphometry and Serum Biomarkers in Mice

In a separate study, the applicants investigated the ability of mALK3-MFC to improve bone status in mice as assessed by histomorphometry and serum biomarkers. Twelve week old female C57BL / 6 mice were treated with mALK3 (24-152) -mFc, 10 mg / kg, or vehicle (Tris buffered saline) by intraperitoneal injection twice weekly. The mouse cohorts are autopsied after 14, 28 and 42 days of treatment to allow bone and serum collection. Fluorescent calcein (20 mg / kg) and demeclocycline (20 mg / kg) compounds were administered intraperitoneally to mice 9 days and 2 days before necropsy, respectively, for dynamic histomorphometric analysis.

ES 2 575 695 T3

The bone was prepared for histomorphometry as follows. At necropsy, the right femur was detached, and the distal quarter of the femur underwent a histological preparation consisting of dehydration, methyl methacrylate infiltration, and methyl methacrylate embedding. A rotary microtome was used to obtain sets of frontal sections in thicknesses of 4 and 8 microns. The thinner sections were stained with Goldner's trichrome and used for the analysis of static parameters, while the thicker sections were mounted without staining and were used for the analysis of the dynamic parameters. Histomorphometry was performed blind with a Nikon Eclipse E4000 light / epifluorescence microscope connected to a standard video subsystem with OsteoMeasure image analysis software.

Histomorphometric analysis of the distal femur revealed both anabolic and antiresorptive effects of ALK3-Fc. Compared to vehicle, mALK3 (24-152) -mFc significantly increased bone volume at all three time points by up to 90% (Figure 29). Importantly, mALK3 (24-152) -mFc increased bone formation rate by as much as 120% (Figure 30) and bone mineralization surface by as much as 115% (Figure 31). These latter parameters are considered indicative of anabolic bone growth, although additional markers of anabolic effects - osteoblast surface and osteoid surface showed more modest or insignificant increases. Histomorphometric analysis also provided evidence for temporary antiresorptive effects, as mALK3 (24-152) -mFc reduced the osteoclast surface significantly on day 28 only (Figure 32) and a similar effect was observed on the eroded surface.

The effects of mALK3 (24-152) -mFc treatment on serum biomarkers of bone status were also investigated. RANKL (nuclear factor-κΒ ligand receptor activator) is produced by osteoblasts and is a key activator of osteoclast differentiation, whereas osteoprotegerin (OPG) is an endogenous inhibitor of RANKL signaling. Therefore, the RANKL / OPG ratio is an important determinant of osteoclastic activity, bone mass, and bone quality (Boyce et al, 2008, Arch Biochem Biophys 473: 139-146). In the present experiment, serum levels of RANKL and OPG were measured with Millipore products (mBn2A-41k and 41K-MBN-1OPG) incorporating Luminex xMAP® technology. Treatment with mALK3 (24-152) -mFc significantly reduced serum RANKL levels at all three time points (Figure 33) and significantly increased serum OPG levels at 28 and 42 days (Figure 34) in comparison. with the vehicle. These results indicate that mALK3 (24-152) -mFc treatment stimulates bone formation, in part, through an antiresorptive action.

Example 7. Effects of mALK3-mFC on Sclerostin Gene Expression in Mice

Sclerostin protein is a key negative regulator in bone formation and interference with sclerostin signaling has been reported to exert anabolic effects on bone in vivo (Li et al, 2009, J Bone Miner Res. 24: 578-588 ). Therefore, the applicants investigated whether treatment with mALK3 (24-152) -mFc in vivo altered the expression of sclerostin genes in bone, and thus whether a reduction in sclerostin levels could potentially mediate some of the bone reconstructive effects of ALK3-Fc. Twelve week old female C57BL / 6 mice were treated with mALK3 (24-152) -mFc or vehicle (PBS) by intraperitoneal injection twice weekly. The mouse cohorts were autopsied after 2, 7, 14, and 28 days of treatment, which allowed for bilateral harvesting of the femurs and tibiae, which were separated and cleaned of any residual muscle tissue or connective.

Sclerostin gene expression was analyzed as follows. The bones were trimmed to expose the inner marrow axis, and the marrow cells were washed with sterile saline using a 21 gauge needle attached to a 3 ml syringe. The femurs and tibiae of each mouse were sprayed together, and RNA was extracted from the resulting powder with a RiboPure kit (Ambion) according to the manufacturer's instructions. The integrity of the RNA in the bone samples was confirmed with RNA nanochips (Agilent Technologies) run on an Agilent Technologies Bioanalyzer 2100 according to the manufacturer's instructions. RNA was reverse transcribed using TaqMan RT reagents (Applied Biosystems) and real-time polymerase chain reaction (PCR) was performed with sclerostin probe / primers and eukaryotic 18S rRNA endogenous control (both from Applied Biosystems). Amplifications were performed with an Applied Biosystems 7300 system, and the results were analyzed using method 2 '^ΔΔα

Compared to vehicle, mALK3 (24-152) -mFc treatment reduced bone sclerostin mRNA levels significantly at three of the four time points investigated (Figure 35). This finding indicates that reduced sclerostin expression may contribute to the anabolic and / or antiresorptive effects of mALK3 (24-152) -mFc on bone.

Example 8. Effect of hALK3-hFC on bone status in mice

Applicants investigated the effects of the human hALK3 (24-152) -hFc construct on bone status in mice. Twelve week old female C57BL / 6 mice (n = 6 per group) were treated with hALK3 (24-152) hFc, 10 mg / kg, or vehicle (Tris buffered saline) by intraperitoneal injection twice weekly for a total of 6 weeks. Over the course of the experiment, trabecular bone volume decreased by nearly 20% in vehicle-treated controls, but increased by more than 80% with hALK3 treatment (24-152)

ES 2 575 695 T3 hFc, as determined by micro-CT analysis of the proximal tibia (Figure 36). Significant increases from baseline in trabecular number (34%) and trabecular thickness (20%) were also observed with hALK3 (24-152) -hFc, but not with vehicle, per study conclusion. Compared to vehicle, hALK3 (24152) -hFc significantly increased whole-body bone mineral density, as determined by DEXA, at the conclusion of the study. Localized analysis of lumbar vertebrae (L1-L6) by DEXA also revealed a significant effect of stimulation (21% increase) of hALK3 (24-152) -hFc on bone mineral density at study conclusion compared to the vehicle.

These results show that the human construction of hALK3 (24-152) -hFc can improve bone status in mice, although it is expected that the magnitude of its effects in rodents would be decreased by the immune response. Taken together, the above results demonstrate that ALK3-Fc constructs 1) promote bone formation, both in the axial skeleton and in the appendicular skeleton through both antiresorptive and anabolic actions, 2) improve bone mechanical strength, and 3) reverses estrogen deficiency-induced bone loss in a mouse model of established osteopenia.

Example 9. Illustrative hALK3 HFC Nucleic Acids and Proteins

This example summarizes nucleic acid constructs used to express the ALK3 constructs in CHO cells, according to the methods provided herein, and provides the mature proteins isolated from cell culture.

A. The nucleic acid of SEQ ID NO: 19 was expressed in CHO cells and the following ALK3-Fc species were isolated:

(1) The hALK3 (24-152) -hFc sequence shown in SEQ ID NO: 7, starting with a glutamine (which tends to be blocked for N-terminal sequencing by Edman degradation).

(2) The sequence of hALK3 (GA, 24-152) -hFc shown below (SEQ ID NO: 20), which retains an initial alanineglycine from the leader sequence.

GAQNLDSM LHGTGMKSDS

DQKKSENGVT GHCFAIIEED RTIECCRTNL CPPCPAPELL VSHEDPEVKF LTVLHQDWLN PQVYTLPPSR QPENNYKTTP SVMHEALHNH

LAPEDTLPFL

DQGETTLASG

CNQYLQPTLP

GGPSVFLFPP

NWYVDGVEVH

GKEYKCKVSN

EEMTKNQVSL

PVLDSDGSFF

YTQKSLSLSP

KCYCSGHCPD CMKYEGSDFQ PVVIGPFFDG KPKDTLMISR NAKTKPREEQ KALPAPIEKT TCLVKGFYPS LYSKLTVDKS GK *

DAINNTCITN CKDSPKAQLR SIRTGGGTHT TPEVTCVVVD YNSTYRVVSV ISKAKGQPRE DIAVEWESNG RWQQGNVFSC (SEQ ID NO: 20)

B. A nucleic acid, encoding hALK3 (24-146) -hFc, is shown below (SEQ ID NO: 21); was expressed in CHO cells:

ΆΤ

GGATGCAATG

AAGAGAGGGC

TCTGCTGTGT

GCTGCTGCTG

TGTGGAGCAG

TCTTCGTTTC

GCCCGGCGCC

CAGAATCTGG

ATAGTATGCT

TCATGGCACT

GGGATGAAAT

CAGACTCCGA

CCAGAAAAAG

TCAGAAAATG

GAGTAACCTT

AGCACCAGAG

GATACCTTGC _CTTTTTTAAA

GTGCTATTGC

TCAGGGCACT

GTCCAGATGA

TGCTATTAAT

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AACACATGCA TAACTAATGG ACATTGCTTT GCCATCATAG

AAGAAGATGA CCAGGGAGAA ACCACATTAG CTTCAGGGTG

TAIGAAATAT GAAGGATCTG ATTTTCAGTG CAAAGATTCT

CCAAAAGCCC AGCTACGCCG GACAATAGAA TGTTGTCGGA

CCAATTTATG TAACCAGTAT TTGCAACCCA CACTGCCCCC

TGTTGTCATA GGTCCGTTTA CCGGTGGTGG AACTCACACA

TGCCCACCGT GCCCAGCACC TGAACTCCTG GGGGGACCGT

CAGTCTTCCT CTTCCCCCCA AAACCCAAGG ACACCCTCAT

GATCTCCCGG ACCCCTGAGG TCACATGCGT GGTGGTGGAC

GTGAGCCACG AAGACCCTGA GGTCAAGTTC AACTGGTACG

TGGACGGCGT GGAGGTGCAT AATGCCAAGA CAAAGCCGCG

GGAGGAGCAG TACAACAGCA CGTACCGTGT GGTCAGCGTC

CTCACCGTCC TGCACCAGGA CTGGCTGAAT GGCAAGGAGT

ACAAGTGCAA GGTCTCCAAC AAAGCCCTCC CAGCCCCCAT

CGAGAAAACC ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA

CCACAGGTGT ACACCCTGCC CCCATCCCGG GAGGAGATGA

CCAAGAACCA GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT

CTATCCCAGC GACATCGCCG TGGAGTGGGA GAGCAATGGG

CAGCCGGAGA ACAACTACAA GACCACGCCT CCCGTGCTGG

ACTCCGACGG CTCCTTCTTC CTCTATAGCA AGCTCACCGT

GGACAAGAGC AGGTGGCAGC AGGGGAACGT CTTCTCATGC

TCCGTGATGC ATGAGGCTCT GCACAACCAC TACACGCAGA

AGAGCCTCTC CCTGTCTCCG GGTAAATGA (SEQ ID NO: 21)

The following protein species were isolated:

(1) The hALK3 (24-146) -hFc is shown below (SEQ ID NO: 22), starting with a glutamine (which tends to be blocked for N-terminal sequencing by Edman degradation).

QNLDSMLHGT

GMKSDSDQKK SENGVTLAPE DTLPFLKCYC SGHCPDDAIN

NTCITNGHCF AIIEEDDQGE TTLASGCMKY EGSDFQCKDS

PKAQLRRTIE CCRTNLCNQY LQPTLPPWI GPFTGGGTHT

CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD

VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRWSV

LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE

PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG

QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC

SVMHEALHNH YTQKSLSLSP GK * (SEQ ID NO: 22) (2) The hALK3 (GA, 24-146) -hFc sequence shown below (SEQ ID NO: 23), which retains an initial glycinealanine from the leader sequence.

ES 2 575 695 T3

GA QNLDSMLHGT

GMKSDSDQKK SENGVTLAPE DTLPFLKCYC SGHCPDDAIN

NTCITNGHCF AIIEEDDQGE TTLASGCMKY EGSDFQCKDS

PKAQLRRTIE CCRTNLCNQY LQPTLPPVVI GPFTGGGTHT

CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVWD

VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV

LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE

PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG

QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC

SVMHEALHNH YTQKSLSLSP GK * (SEQ ID NO: 23)

C. A nucleic acid, encoding hALK3 (24-140) -hFc, is shown below (SEQ ID NO: 24); was expressed in CHO cells:

ATGG

ATGCAATGAA GAGAGGGCTC TGCTGTGTGC TGCTGCTGTG

TGGAGCAGTC TICGTTTCGC CCGGCGCCCA GAATCTGGAT

AGTATGCTTC ATGGCACTGG GATGAAATCA GACTCCGACC

AGAAAAAGTC AGAAAATGGA GTAACCTTAG CACCAGAGGA

TACCTTGCCT TTTTTAAAGT GCTATTGCTC AGGGCACTGT

CCAGATGATG CTATTAATAA CACATGCATA ACTAATGGAC

ATTGCTTTGC CATCATAGAA GAAGATGACC AGGGAGAAAC

CACATTAGCT TCAGGGTGTA TGAAATATGA AGGATCTGAT

TTTCAGTGCA AAGATTCTCC AAAAGCCCAG CTACGCCGGA

CAATAGAATG TTGTCGGACC AATTTATGTA ACCAGTATTT

GCAACCCACA CTGCCCCCTA CCGGTGGTGG AACTCACACA

TGCCCACCGT GCCCAGCACC TGAACTCCTG GGGGGACCGT

CAGTCTTCCT CTTCCCCCCA AAACCCAAGG ACACCCTCAT

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GATCTCCCGG ACCCCTGAGG TCACATGCGT GGTGGTGGAC gtgagccacg aagaccctga GGTCAAGTTC aactggtacg TGGACGGCGT GGAGGTGCAT AATGCCAAGA CAAAGCCGCG ggaggagcag tacaacagca cgtaccgtgt ggtcagcgtc CTCACCGTCC tgcaccagga ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc atctccaaag gccccgagaa ccaaagggca CCACAGGTGT ACACCCTGCC CCCATCCCGG GAGGAGATGA CCAAGAACCA GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT CTATCCCAGC GACATCGCCG TGGAGTGGGA GAGCAATGGG CAGCCGGAGA ACAACTACAA GACCACGCCT CCCGTGCTGG ACTCCGACGG CTCCTTCTTC CTCTATAGCA AGCTCACCGT GGACAAGAGC AGGTGGCAGC AGGGGAACGT CTTCTCATGC TCCGTGATGC ATGAGGCTCT GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG GGTAA NOATGA: 24)

The following protein species were isolated:

(1) The hALK3 (24-140) -hFc is shown below (SEQ ID NO: 25), starting with a glutamine (which tends to be blocked for N-terminal sequencing by Edman degradation.

QNLD

SMLHGTGMKS DSDQKKSENG VTLAPEDTLP FLKCYCSGHC

PDDAINNTCI TNGHCFAIIE EDDQGETTLA SGCMKYEGSD

FQCKDSPKAQ.LRRTIECCRT NLCNQYLQPT LPPTGGGTHT

CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD

VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV

LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE

PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG

QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC

SVMHEALHNH YTQKSLSLSP GK * (SEQ ID NO: 25) (2) The hALK3 (GA, 24-140) -hFc sequence shown below (SEQ ID NO: 26), which retains an initial glycinealanine from the leader sequence.

GAQNLD

SMLHGTGMKS DSDQKKSENG VTLAPEDTLP FLKCYCSGHC

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PDDAINNTCI TNGRCFAIIE EDDQGETTLA SGCMKYEGSD

FQCKDSPKAQ LRRTIECCRT NLCNQYLQPT LPPTGGGTHT

CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCWVD

VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRWSV

LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE

PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG

QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC

SVMHEALHNH YTQKSLSLSP GK * (SEQ ID NO: 26)

D. A nucleic acid encoding hALK3 (30-152) -hFc, shown below (SEQ ID NO: 27) was expressed in CHO cells:

AT GGATGCAATG AAGAGAGGGC

TCTGCTGTGT GCTGCTGCTG TGTGGAGCAG TCTTCGTTTC

GCCCGGCGCC CTTCATGGCA CTGGGATGAA ATCAGACTCC

GACCAGAAAA AGTCAGAAAA TGGAGTAACC TTAGCACCAG

AGGATACCTT GCCTTTTTTA AAGTGCTATT GCTCAGGGCA

CTGTCCAGAT GATGCTATTA ATAACACATG CATAACTAAT

GGACATTGCT TTGCCATCAT AGAAGAAGAT GACCAGGGAG

AAACCACATT AGCTTCAGGG TGTATGAAAT ATGAAGGATC

TGATTTTCAG TGCAAAGATT CTCCAAAAGC CCAGCTACGC

CGGACAATAG AATGTTGTCG C-ACCAATTTA TGTAACCAGT

ATTTGCAACC CACACTGCCC CCTGTTGTCA TAGGTCCGTT

TTTTGATGGC AGCATTCGAA CCGGTGGTGG AACTCACACA

TGCCCACCGT GCCCAGCACC TGAACTCCTG GGGGGACCGT

CAGTCTTCCT CTTCCCCCCA AAACCCAAGG ACACCCTCAT

GATCTCCCGG ACCCCTGAGG TCACATGCGT GGTGGTGGAC

GTGAGCCACG AAGACCCTGA GGTCAAGTTC AACTGGTACG

TGGACGGCGT GGAGGTGCAT AATGCCAAGA CAAAGCCGCG

GGAGGAGCAG TACAACAGCA CGTACCGTGT GGTCAGCGTC

CTCACCGTCC TGCACCAGGA CTGGCTGAAT GGCAAGGAGT

ACAAGTGCAA GGTCTCCAAC AAAGCCCTCC CAGCCCCCAT

CGAGAAAACC ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA

CCACAGGTGT ACACCCTGCC CCCATCCCGG GAGGAGATGA

CCAAGAACCA GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT

CTATCCCAGC GACATCGCCG TGGAGTGGGA GAGCAATGGG

CAGCCGGAGA ACAACTACAA GACCACGCCT CCCGTGCTGG

ACTCCGACGG CTCCTTCTTC CTCTATAGCA AGCTCACCGT

GGACAAGAGC AGGTGGCAGC AGGGGAACGT CTTCTCATGC

TCCGTGATGC ATGAGGCTCT GCACAACCAC TACACGCAGA

AGAGCCTCTC CCTGTCTCCG GGTAAATGA (SEQ ID NO: 27)

ES 2 575 695 T3

The following protein species were isolated:

(1) hALK3 (GA, 30-152) -hFc, shown below (SEQ ID NO: 28), retaining an initial glycine-alanine from the leader sequence.

GA LHGTGMKSDS

DQKKSENGVT LAPEDTLPFL KCYCSGHCPD DAINNTCITN

GHCFAIIEED DQGETTLASG CMKYEGSDFQ CKDSPKAQLR

RTIECCRTNL CNQYLQPTLP PWIGPFFDG SIRTGGGTHT

CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD

VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRWSV

LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE

PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG

QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC

SVMHEALHNH YTQKSLSLSP GK * (SEQ ID NO: 28) (2) The hALK3 (A, 30-152) -hFc, shown below (SEQ ID NO: 29), which retains an initial alanine from the leader sequence.

TO LHGTGMKSDS

DQKKSENGVT LAPEDTLPFL KCYCSGHCPD DAINNTCITN

GHCFAIIEED DQGETTLASG CMKYEGSDFQ CKDSPKAQLR

RTIECCRTNL CNQYLQPTLP PWIGPFFDG SIRTGGGTHT

CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD

VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRWSV

LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE

PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG

QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC

SVMHEALHNH YTQKSLSLSP GK * (SEQ ID NO: 29) (3) The sequence of hALK3 (31-152) -hFc, shown below (SEQ ID NO: 30), in which the leader and the initial 10 leucine are extracted, leaving an initial histidine (effectively NA7).

HGTGMKSDS

DQKKSENGVT LAPEDTLPFL KCYCSGHCPD DAINNTCITN

GHCFAIIEED DQGETTLASG CMKYEGSDFQ CKDSPKAQLR

RTIECCRTNL CNQYLQPTLP PWIGPFFDG SIRTGGGTHT

CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD

VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRWSV

LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE

PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG

QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC

SVMHEALHNH YTQKSLSLSP GK * (SEQ ID NO: 30) (4) An additional species, hALK3 (30-152) -hFc, shown below (SEQ ID NO: 31), was expected, but was not identified by N-terminal sequencing .

ES 2 575 695 T3

LHGTGMKSDS

DQKKSENGVT LAPEDTLPFL KCYCSGHCPD DAINNTCITN ghcfaiieed dqgettlasg cmkyegsdfq ckdspkaqlr

RTIECCRTNL CNQYLQPTLP PVVIGPFFDG 3IRTGGGTHT

CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCWVD

VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV

LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE

PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG

QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC

SVMHEALHNH YTQKSLSLSP GK * (SEQ ID NO: 31)

E. A nucleic acid encoding hALK3 (30-146) -hFc, shown below (SEQ ID NO: 32), was expressed in CHO cells:

ATGG

ATGCAATGAA GAGAGGGCTC TGCTGTGTGC TGCTGCTGTG

TGGAGCAGTC TTCGTTTCGC CCGGCGCCCT TCATGGCACT

GGGATGAAAT CAGACTCCGA CCAGAAAAAG TCAGAAAATG

ES 2 575 695 T3

GAGTAACCTT AGCACCAGAG GATACCTTGC CTTTTTTAAA

GTGCTATTGC TCAGGGCACT GTCCAGATGA TGCTATTAAT

AACACATGCA TAACTAATGG ACATTGCTTT GCCATCATAG

AAGAAGATGA CCAGGGAGAA ACCACATTAG CTTCAGGGTG

TATGAAATAT GAAGGATCTG ATTTTCAGTG CAAAGATTCT

CCAAAAGCCC AGCTACGCCG GACAATAGAA TGTTGTCGGA

CCAATTTATG TAACCAGTAT TTGCAACCCA CACTGCCCCC

TGTTGTCATA GGTCCGTTTA CCGGTGGTGG AACTCACACA

TGCCCACCGT GCCCAGCACC TGAACTCCTG GGGGGACCGT

CAGTCTTCCT CTTCCCCCCA AAACCCAAGG ACACCCTCAT

GATCTCCCGG ACCCCTGAGG TCACATGCGT GGTGGTGGAC

GTGAGCCACG AAGACCCTGA GGTCAAGTTC AACTGGTACG

TGGACGGCGT GGAGGTGCAT AATGCCAAGA CAAAGCCGCG

GGAGGAGCAG TACAACAGCA CGTACCGTGT GGTCAGCGTC

CTCACCGTCC TGCACCAGGA CTGGCTGAAT GGCAAGGAGT

ACAAGTGCAA GGTCTCCAAC AAAGCCCTCC CAGCCCCCAT

CGAGAAAACC ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA

CCACAGGTGT ACACCCTGCC CCCATCCCGG GAGGAGATGA

CCAAGAACCA GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT

CTATCCCAGC GACATCGCCG TGGAGTGGGA GAGCAATGGG

CAGCCGGAGA ACAACTACAA GACCACGCCT CCCGTGCTGG

ACTCCGACGG CTCCTTCTTC CTCTATAGCA AGCTCACCGT

GGACAAGAGC AGGTGGCAGC AGGGGAACGT CTTCTCATGC

TCCGTGATGC ATGAGGCTCT GCACAACCAC TACACGCAGA

AGAGCCTCTC CCTGTCTCCG GGTAAATGA (SEQ ID NO: 32)

The following protein species were isolated:

(1) hALK3 (GA, 30-146) -hFc, shown below (SEQ ID NO: 33), retaining an initial glycine-alanine from the leader sequence.

GALHGT

GMKSDSDQKK SENGVTLAPE DILPFLKCYC SGHCPDDAIN

NTCITNGHCF AIIEEDDQGE TTLASGCMKY EGSDFQCKDS ₅ PKAQLRRTIE CCRTNLCNQY LQPTLPPWI GPFTGGGTHT

CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVWD

VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV

LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE

PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG

QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC

SVMHEALHNH YTQKSLSLSP GK * (SEQ ID NO: 33)

ES 2 575 695 T3 (2) hALK3 (A, 30-146) -hFc, shown below (SEQ ID NO: 34), which retains an initial alanine from the leader sequence.

ALHGT

GMKSDSDQKK SENGVTLAPE DTLPFLKCYC SGHCPDDAIN

NTCITNGHCF AIIEEDDQGE TTLASGCMKY EGSDFQCKDS

PKAQLRRTIE CCRTNLCNQY LQPTLPPVVI GPFTGGGTHT

CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCWVD

VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV

LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE

PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG

QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC

SVMHEALHNH YTQKSLSLSP GK * (SEQ ID NO: 34) (3) The sequence of hALK3 (31-146) -hFc, shown below (SEQ ID NO: 35), in which the leader and the initial 5 leucine are extracted, leaving an initial histidine (effectively NA7CA6).

HGT

GMKSDSDQKK SENGVTLAPE DTLPFLKCYC SGHCPDDAIN

NTCITNGHCF AIIEEDDQGE TTLASGCMKY EGSDFQCKDS

PKAQLRRTIE CCRTNLCNQY LQPTLPPVVI GPFTGGGTHT

CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCWVD

VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV

LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE

PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG

QPENNYKTTP PVLDSDGSFF'LYSKLTVDKS RWQQGNVFSC

SVMHEALHNH YTQKSLSLSP GK * (SEQ ID NO: 35) (4) An additional species, hALK3 (30-146) -hFc, shown below (SEQ ID NO: 36), was expected, but was not identified by N-terminal sequencing .

LHGT

GMKSDSDQKK SENGVTLAPE DTLPFLKCYC SGHCPDDAIN

NTCITNGHCF AIIEEDDQGE TTLASGCMKY EGSDFQCKDS

PKAQLRRTIE CCRTNLCNQY LQPTLPPVVI GPFTGGGTHT

CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCWVD

VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV

LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE

PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG

QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC ₁₀ SVMHEALHNH YTQKSLSLSP GK * (SEQ ID NO: 36)

F. A nucleic acid encoding hALK3 (30-140) -hFc, shown below (SEQ ID NO: 37), can be expressed in CHO cells:

ES 2 575 695 T3

ATGGAT GCAATGAAGA GAGGGCTCTG

CTGTGTGCTG CTGCTGTGTG GAGCAGTCTT CGTTTCGCCC

GGCGCCCTTC ATGGCACTGG GATGAAATCA GACTCCGACC

AGAAAAAGTC AGAAAATGGA GTAACCTTAG CACCAGAGGA

TACCTTGCCT TTTTTAAAGT GCTATTGCTC AGGGCACTGT

CCAGATGATG CTATTAATAA CACATGCATA ACTAATGGAC

ATTGCTTTGC CATCATAGAA GAAGATGACC AGGGAGAAAC

CACATTAGCT TCAGGGTGTA TGAAATATGA AGGATCTGAT

TTTCAGTGCA AAGATTCTCC AAAAGCCCAG CTACGCCGGA

CAATAGAATG TTGTCGGACC AATTTATGTA ACCAGTATTT

GCAACCCACA CTGCCCCCTA CCGGTGGTGG AACTCACACA

TGCCCACCGT GCCCAGCACC TGAACTCCTG GGGGGACCGT

CAGTCTTCCT CTTCCCCCCA AAACCCAAGG ACACCCTCAT

GATCTCCCGG ACCCCTGAGG TCACATGCGT GGTGGTGGAC

GTGAGCCACG AAGACCCTGA GGTCAAGTTC AACTGGTACG

TGGACGGCGT GGAGGTGCAT AATGCCAAGA CAAAGCCGCG

GGAGGAGCAG TACAACAGCA CGTACCGTGT GGTCAGCGTC

CTCACCGTCC TGCACCAGGA CTGGCTGAAT GGCAAGGAGT

ACAAGTGCAA GGTCTCCAAC AAAGCCCTCC CAGCCCCCAT

CGAGAAAACC ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA

CCACAGGTGT ACACCCTGCC CCCATCCCGG GAGGAGATGA

CCAAGAACCA GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT

CTATCCCAGC GACATCGCCG TGGAGTGGGA GAGCAATGGG

CAGCCGGAGA ACAACTACAA GACCACGCCT CCCGTGCTGG

ACTCCGACGG CTCCTTCTTC CTCTATAGCA AGCTCACCGT

GGACAAGAGC AGGTGGCAGC AGGGGAACGT CTTCTCATGC

TCCGTGATGC ATGAGGCTCT GCACAACCAC TACACGCAGA

AGAGCCTCTC CCTGTCTCCG GGTAAATGA (SEQ ID NO: 37)

The following protein species can be isolated:

(1) hALK3 (GA, 30-140) -hFc, shown below (SEQ ID NO: 38), retaining an initial glycine-alanine of the leader sequence.

ES 2 575 695 T3

GALHGTGMKS

DSDQKKSENG

VTLAPEDTLP

FLKCYCSGHC

PDDAINNTCI

TNGHCFAIIE

EDDQGETTLA

SGCMKYEGSD

FQCKDSPKAQ

LRRTIECCRT

NLCNQYLQPT

LPPTGGGTHT

CPPCPAPELL

GGPSVFLFPP

KPKDTLMISR

TPEVTCVWD

VSHEDPEVKF

NWYVDGVEVH

NAKTKPREEQ

YNSTYRVVSV

LTVLHQDWLN

GKEYKCKVSN

KALPAPIEKT

ISKAKGQPRE

PQVYTLPPSR

EEMTKNQVSL tclvkgfyps

DIAVEWESNG

QPENNYKTTP

PVLDSDGSFF

LYSKLTVDKS

RWQQGNVFSC

SVMHEALHNH

YTQKSLSLSP

GK * (SEQ

ID NO: 38) (2) hALK3 (A, 30-140) -hFc, shown below (SEQ ID NO: 39), which retains an initial alanine from the leader sequence.

ALHGTGMKS DSDQKKSENG VTLAPEDTLP FLKCYCSGHC

PDDAINNTCI TNGHCFAIIE EDDQGETTLA SGCMKYEGSD

FQCKDSPKAQ LRRTIECCRT NLCNQYLQPT LPPTGGGTHT

CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVWD

VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRWSV

LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE

PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG

QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC

SVMHEALHNH YTQKSLSLSP GK * (SEQ ID NO: 39) (3) The sequence hALK3 (31-140) -hFc, shown below (SEQ ID NO: 40), in which the leader and initial leucine are extracted, leaving a initial histidine (effectively NA7CA12).

HGTGMKS DSDQKKSENG VTLAPEDTLP FLKCYCSGHC

PDDAINNTCI TNGHCFAIIE EDDQGETTLA SGCMKYEGSD

FQCKDSPKAQ LRRTIECCRT NLCNQYLQPT LPPTGGGTHT

CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVWD

VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV

LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE

PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG

QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC

SVMHEALHNH YTQKSLSLSP GK * (SEQ ID NO: 40) (4) An additional species, hALK3 (30-140) -hFc, is shown below (SEQ ID NO: 41).

ES 2 575 695 T3

LHGTGMKS DSDQKKSENG VTLAPEDTLP FLKCYCSGHC

PDDAINNTCI TNGHCFAIIE EDDQGETTLA SGCMKYEGSD

FQCKDSPKAQ LRRTIECCRT NLCNQYLQPT LPPTGGGTHT

CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVWD

VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV

LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE

PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG

QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC

SVMHEALHNH YTQKSLSLSP GK * (SEQ ID NO: 41)

Although specific embodiments of subject matter have been discussed, the foregoing specification is illustrative and not restrictive. Many variations will be apparent to those skilled in the art upon review of this specification and the claims that follow. The full scope of the invention is to be determined by reference to the claims and the specification.