DESIGNER OSTEOGEN IC PROTEINS
FIELD OF THE INVENTION
This application relates to the field of osteogenic proteins, methods of making improved
osteogenic proteins, and methods of treating patients with osteogenic proteins.
BACKGROUND OF THE INVENTION
The cystine knot cytokine superfamily is divided into subfamilies, which include, the transforming
growth factor b (TGFP) proteins, the glycoprotein hormones, the platelet-derived growth factor-like
(PDGF-like) proteins, nerve growth factors (NGF), and the differential screening-selected gene aberrative
in neuroblastoma (DAN) family (e.g. , cerberus). In turn, the TGFp superfamily comprises approximately
43 members, subdivided into three subfamilies: the TGFps, the activins and the bone
morphogenetic/growth differentiation factor proteins (BMP/GDF).
The TGF- b superfamily members contain the canonical cystine knot topology. That is, cystine
knots are the result of an unusual arrangement of six cysteine residues. The knot consists of bonds
between cysteines 1-4, cysteines 2-5, and the intervening sequence forming a ring, through which the
disulfide bond between cysteines 3-6 passes. The active forms of these proteins are homodimers or
heterodimers. In each case the monomer topology is stabilized by the cysteine knot and additional
cysteines contribute to additional intrachain bonds and/or mediate dimerization with another protein unit.
See Kingsley, 1994, Genes Dev. 8:1 33-1 46; Lander et al, 2001 , Nature 409:860-921 .
BMP/GDFs are the most numerous members of the TGF- b protein superfamily. The BMP/GDF
subfamily includes, but is not limited to, BMP2, BMP3 (osteogenin), BMP3b (GDF-1 0), BMP4 (BMP2b),
BMP5, BMP6, BMP7 (osteogenic protein-1 or OP1 ) , BMP8 (OP2), BMP8B (OP3), BMP9 (GDF2),
BMP1 0 , BMP1 1 (GDF1 1) , BMP1 2 (GDF7), BMP1 3 (GDF6, CDMP2), BMP1 5 (GDF9), BMP1 6, GDF1 ,
GDF3, GDF5 (CDMP1 ; MP52), and GDF8 (myostatin). BMPs are sometimes referred to as Osteogenic
Protein (OPs), Growth Differentiation Factors (GDFs), or Cartilage-Derived Morphogenetic Proteins
(CDMPs). BMPs are also present in other animal species. Furthermore, there is some allelic variation in
BMP sequences among different members of the human population.
BMPs are naturally expressed as pro-proteins comprising a long pro-domain, one or more
cleavage sites, and a mature domain. This pro-protein is then processed by the cellular machinery to
yield a dimeric mature BMP molecule. The pro-domain is believed to aid in the correct folding and
processing of BMPs. Furthermore, in some but not all BMPs, the pro-domain may noncovalently bind the
mature domain and may act as a chaperone, as well as an inhibitor (e.g., Thies et al., Growth Factors
18:251 -9 (2001 )).
BMP signal transduction is initiated when a BMP dimer binds two type I and two type I I
serine/threonine kinase receptors. Type I receptors include, but are not limited to, ALK-1 (Activin
receptor-Like Kinase 1) , ALK-2 (also called ActRIa or ActRI), ALK-3 (also called BMPRIa), and ALK-6
(also called BMPRIb). Type I I receptors include, but are not limited to, ActRlla (also called ActRII),
ActRllb, and BMPRII. The human genome contains 12 members of the receptor serine/threonine kinase
family, including 7 type I and 5 type I I receptors, all of which are involved in TGF- b signaling (Manning et
al., Science 298: 19 12-34 (2002)), the disclosures of which are hereby incorporated by reference). Thus,
there are 12 receptors and 43 superfamily members, suggesting that at least some TGF- b superfamily
members bind the same receptor(s). Following BMP binding, the type I I receptors phosphorylate the type
I receptors, the type I receptors phosphorylate members of the Smad family of transcription factors, and
the Smads translocate to the nucleus and activate the expression of a number of genes.
BMPs are among the most numerous members of TGF- b superfamily, and control a diverse set
of cellular and developmental processes, such as embryonic pattern formation and tissue specification as
well as promoting wound healing and repair processes in adult tissues. BMPs were initially isolated by
their ability to induce bone and cartilage formation. BMP signaling is inducible upon bone fracture and
related tissue injury, leading to bone regeneration and repair. BMP molecules which have altered affinity
for their receptors would have improved biological activity relative to the native proteins. Such BMPs
include proteins with increased in vivo activity and may provide potential improved therapeutics for,
among other things, tissue regeneration, repair, and the like, by providing greater or altered activity at
lower protein levels thereby providing improved protein therapeutics.
SUMMARY OF THE INVENTION
The invention includes a designer BMP protein comprising at least one mutation in at least one
type I or type I I receptor binding domain, wherein the mutation confers altered binding to the type I or type
I I BMP receptor compared with the binding to the type I or type I I receptor by a corresponding wild type
BMP.
In one aspect, the protein is selected from the group consisting of BMP2, BMP4, BMP5, BMP6,
BMP7, BMP8 and BMP9.
In another aspect, the protein comprises at least one mutation within: the type I I binding domain
A; the type I I binding domain B; the type I binding domain; and any combination thereof.
The invention also includes a designer osteogenic protein comprising an amino acid sequence
comprising at least one mutation in at least one type I or type I I receptor binding domain, wherein the
mutation confers altered binding to the type I or type I I BMP receptor compared with the binding to the
type I or type I I receptor by wild type BMP2.
In one aspect, the mutation is a mutation within the type I I binding domain A wherein said
mutation is at least one mutation selected from the group consisting of a mutation at V33, P36, H39, and
F41 with respect to the sequence of SEQ ID NO:1 .
In another aspect, the is a mutation within the type I I binding domain A wherein said mutation is
at least one mutation selected from the group consisting of V33I, P36K, P36R, H39A, and F41 N with
respect to SEQ ID NO:1 .
In yet another aspect, the mutation is a mutation within the type I I binding domain B wherein said
mutation is at least one mutation selected from the group consisting of a mutation at E83, S85, M89, L92,
E94, E96, K97, and V99 with respect to the sequence of SEQ ID NO: 1.
In a further aspect, the mutation is a mutation within the type I I binding domain B wherein said
mutation is at least one mutation selected from the group consisting of E83K, S85N, M89V, L92F, E94D,
E96S, K97N , and V99I with respect to of SEQ ID NO: 1.
In another aspect, the mutation is a mutation within the type I binding domain wherein said
mutation is at least one mutation selected from the group consisting of a mutation at H44, P48, A52, D53,
L55, S57, N68, S69, V70, an insertion of a single amino acid after N71 , S72, K73, I74, A77, and V80 with
respect to the sequence of SEQ ID NO:1 .
In yet another aspect, the mutation is a mutation within the type I binding domain wherein said
mutation is at least one mutation selected from the group consisting of H44D, P48S, A52N, D53A, L55M,
S57A, N68H, S69L, V70M, insertion of P after N71 , S72E, K73Y, I74V, A77P, and V80A with respect to
the sequence of SEQ ID NO:1 .
In a further aspect, the protein comprises a mutation at each of amino acids H44, P48, A52, D53,
L55, S57, N68, S69, V70, insertion of a single amino acid after N71 , S72, K73, I74, A77, and V80 with
respect to the sequence of SEQ ID NO:1 .
In another aspect, the protein comprises a mutation at each of amino acids H44, P48, A52, D53,
L55, S57, N68, S69, V70, insertion of a single amino acid after N71 , S72, K73, I74, A77, and V80 with
respect to the sequence of SEQ ID NO:1 wherein the mutations are H44D, P48S, A52N, D53A, L55M,
S57A, N68H, S69L, V70M, insertion of a P after N71 , S72E, K73Y, I74V, A77P, and V80A.
In yet another aspect, the protein comprises a mutation at each of amino acids V33, P36, H39,
S85, M89, L92, E94, E96, K97, and V99 with respect to the sequence of SEQ ID NO:1 .
In another aspect, the protein comprises a mutation at each of amino acids V33, P36, H39, S85,
M89, L92, E94, E96, K97, and V99 with respect to the sequence of SEQ ID NO:1 , wherein the mutations
are V33I, P36K, H39A, S85N, M89, L92F, E94D, E96S, K97N, and V99I.
In a further aspect, the protein comprises a mutation at each of amino acids V33, P36, H39, H44,
P48, A52, D53, L55, S57, N68, S69, V70, insertion of a single amino acid after N71 , S72, K73, I74, A77,
and V80, S85, M89, L92, E94, E96, K97, and V99 with respect to the sequence of SEQ ID NO:1 .
In yet another aspect, the protein comprises a mutation at each of amino acids V33, P36, H39,
H44, P48, A52, D53, L55, S57, N68, S69, V70, insertion of a single amino acid after N71 , S72, K73, I74,
A77, and V80, S85, M89, L92, E94, E96, K97, and V99 with respect to the sequence of SEQ ID NO:1
wherein the mutations are V33I , P36K, H39A, H44D, P48S, A52N, D53A, L55M, S57A, N68H, S69L,
V70M, insertion of a P after N71 , S72E, K73Y, I74V, A77P, and V80A, S85N, M89, L92F, E94D, E96S,
K97N , and V99I .
In yet another aspect, the protein comprises a mutation at each of amino acids V33, P36, H39,
H44, P48, A52, D53, L55, S57, N68, S69, V70, insertion of a single amino acid after N71 , S72, K73, I74,
A77, and V80, S85, M89, L92, E94, E96, K97, and V99 with respect to the sequence of SEQ ID NO:1
wherein the mutations are V33I , P36R, H39A, H44D, P48S, A52N, D53A, L55M, S57A, N68H, S69L,
V70M, insertion of a P after N71 , S72E, K73Y, I74V, A77P, and V80A, S85N, M89, L92F, E94D, E96S,
K97N , and V99I .
In another aspect, the protein binds: the ALK2 receptor with a K not greater than about 2 nM; the
ALK3 receptor with a K not greater than about 2 nM; the ALK6 receptor with a K not greater than about
1 nM; the ActRI IA receptor with a K not greater than about 2 nM; the ActRIIB receptor with a K not
greater than about 0.5 nM; and the BMPRIIA receptor with a K not greater than about 3.5 nM.
In one aspect, the protein further comprises 1, 2 , 3, 4 , 5 , 6 , 7, 8 , 9 , or 10 amino acid mutations
not located within the type I or the type I I binding regions.
The invention includes a designer osteogenic protein comprising the amino acid sequence of any
one of SEQ ID NOs:8-73.
The invention includes a designer osteogenic protein comprising the amino acid sequence of
SEQ ID NO:1 2 .
The invention includes a designer osteogenic protein comprising the amino acid sequence of
SEQ ID NO:1 4 .
The invention includes a designer osteogenic protein comprising the amino acid sequence of
SEQ ID NO:36.
The invention includes a designer osteogenic protein comprising the amino acid sequence of
SEQ ID NO:37.
The invention includes method of producing a designer BMP protein comprising at least one
mutation in at least one type I or type I I receptor binding domain, wherein the mutation confers altered
binding to the type I or type I I BMP receptor compared with the binding to the type I or type I I receptor by
a corresponding wild type BMP. The method comprises introducing a nucleic acid encoding the protein
into a host cell, culturing the cell under conditions where the protein is produced, and purifying the
protein.
In one aspect, the nucleic acid comprises a sequence selected from the nucleic acid sequence of
any one of SEQ ID NOs:74-1 39.
The invention includes a designer BMP6 protein comprising an amino acid sequence comprising
at least one mutation in at least one type I or type I I receptor binding domain, wherein the mutation
confers altered binding to the type I or type I I BMP receptor compared with the binding to the type I or
type I I receptor by wild type BMP6.
In one aspect, the mutation is a mutation within the type I I binding domain A wherein said
mutation is at least one mutation selected from the group consisting of a mutation at I57, K60, G61 , A63,
N65, Y66, and D68 with respect to the sequence of SEQ ID NO:4.
In another aspect, the mutation is a mutation within the type I I binding domain B wherein said
mutation is at least one mutation selected from the group consisting of K 108, N110 , A 111, V 114 , F 117 ,
D 119 , N 120, S 12 1, N122, V 123, and 1124 with respect to the sequence of SEQ ID NO:4.
In yet another aspect, the mutation is a mutation within the type I binding domain wherein said
mutation is at least one mutation selected from the group consisting of a mutation at S72, N76, A77, H78,
M79, N80, A81 , N83, V87, T89, H92, L93, M94, N95, P96, E97, Y98, V99, and P 100 with respect to the
sequence of SEQ ID NO:4.
In another aspect, the mutation is a mutation at each of amino acid residues I57, K60, G61 , A63,
N65, Y66, and D68 with respect to the sequence of SEQ ID NO:4.
In a further aspect, the mutation is a mutation at each of amino acid residues K 108, N 110 , A 111,
V 114 , F 117 , D 119 , N 120, S 12 1, N 122, V 123, and 1124 with respect to the amino acid sequence of SEQ
ID NO:4.
In yet another aspect, the mutation is a mutation at each of amino acid residues S72, N76, A77,
H78, M79, N80, A81 , N83, V87, T89, H92, L93, M94, N95, P96, E97, Y98, V99, or P 100 with respect to
the amino acid sequence of SEQ ID NO:4.
In another aspect, the designer BMP6 protein comprising an amino acid sequence comprising at
least one mutation in at least one type I or type I I receptor binding domain, wherein the mutation confers
altered binding to the type I or type I I BMP receptor compared with the binding to the type I or type I I
receptor by wild type BMP6 further comprises 1, 2 , 3 , 4 , 5 , 6, 7, 8 , 9 , or 10 amino acid mutations not
located within the type I or the type I I binding domains.
The invention includes an isolated nucleic acid molecule comprising a nucleotide sequence
encoding an amino acid sequence selected from the group consisting of the sequence of SEQ ID NOs:8
to 73.
In one aspect, the nucleic acid encodes a protein comprising an amino acid sequence selected
from the group consisting of the sequence of SEQ ID NO:1 2 , SEQ ID NO:1 4 , SEQ ID NO:36 and SEQ ID
NO:37.$$
The invention includes an isolated nucleic acid molecule comprising a nucleotide sequence
selected from the group consisting of SEQ ID NOs:74 to 139.
In one aspect, the nucleic acid comprises a nucleotide sequence selected from the group
consisting of the sequence of SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:1 02, and SEQ ID NO: 103.
The invention includes a method of producing the designer BMP6 protein comprising an amino
acid sequence comprising at least one mutation in at least one type I or type I I receptor binding domain,
wherein the mutation confers altered binding to the type I or type I I BMP receptor compared with the
binding to the type I or type I I receptor by wild type BMP6. The method comprises introducing a nucleic
acid encoding said protein into a host cell, culturing said cell under conditions where said protein is
produced, and purifying said protein.
The invention includes a method of treating a bone disease associated with bone loss in a patient
in need thereof. The method comprises administering a therapeutically effective amount of a designer
BMP protein comprising at least one mutation in at least one type I or type I I receptor binding domain,
wherein the mutation confers altered binding to the type I or type I I BMP receptor compared with the
binding to the type I or type I I receptor by a corresponding wild type BMP protein to the patient, thereby
treating bone disease in the patient.
The invention includes a method of treating fibrosis in a patient in need thereof. The method
comprises administering a therapeutically effective amount of a designer BMP protein comprising at least
one mutation in at least one type I or type I I receptor binding domain, wherein the mutation confers
altered binding to the type I or type I I BMP receptor compared with the binding to the type I or type I I
receptor by a corresponding wild type BMP to the patient, thereby treating fibrosis.
The invention includes a method of inducing bone formation in a tissue. The method comprises
contacting the tissue with a designer BMP protein comprising at least one mutation in at least one type I
or type I I receptor binding domain, wherein the mutation confers altered binding to the type I or type I I
BMP receptor compared with the binding to the type I or type II receptor by a corresponding wild type
BMP, thereby inducing bone formation in said tissue.
BRIEF DESCRIPTION OF THE DRAWINGS
For the purpose of illustrating the invention, there are depicted in the drawings certain
embodiments of the invention. However, the invention is not limited to the precise arrangements and
instrumentalities of the embodiments depicted in the drawings.
Figure 1, comprising panels A-C, is a diagram showing the alignment of various wild type and
designer BMP amino acid sequences and indicating (by being within a box) the regions of these proteins
potentially involved in type I and type I I receptor interactions. Figure 1A shows the amino acid sequence
alignment of wild type BMP2, BMP4, BMP5, BMP6, BMP7, BMP8 and BMP9. Figure 1B shows the
amino acid sequence alignment of various designer BMPs where the corresponding wild type BMP is
BMP2. Figure 1C shows the amino acid sequence alignment of various designer BMP6 molecules where
the corresponding wild type BMP is BMP6.
Figure 2 is an illustration of a structural model showing a wild type BMP2 homodimer binding to
two type I and two type I I BMP receptors.
Figure 3, comprising panels A and B, is an diagram of a structural model showing the position of
the histidine doorstop (H54) in human BMP2 produced in Chinese Hamster Ovary (CHO) (Figure 3A) and
E. coli cells (Figure 3B).
Figure 4 , comprising panels A and B, is a diagram illustrating the location of the glycan tether and
potential histidine (His) doorstop. Figure 4A shows the glycan tether (N-linked glycan at N56) and
histidine 54, in the non-doorstop orientation, as well as the interaction of the glycan tether with R 16 all in
CHO-produced BMP2. Figure 4B shows the glycan tether (N-linked glycan at N80) and the histidine in
the non-doorstop configuration at H78 in BMP6, as well as the R39 corresponding to R 16 in BMP2. The
sequence alignment of BMP2 ( 11-KSSCKRHP) and BMP6 (35-KTACRKHE) showing the corresponding
amino acids between BMP2 and BMP6 is shown along the top of the figure.
Figure 5 , comprising panels A-D, shows various steps in the process for purification of BMPs and
designer BMPs. Figure 5A shows a chromatogram showing gradient elution of BMPs using a cellufine
sulfate column. Figure 5B is an image of a Coomassie stained SDS-PAGE (non-reduced on the left and
reduced on the right side) gel containing samples of fractions from the cellufine sulfate column step.
Figure 5C shows a chromatogram showing the profile from preparative reversed phase purification step.
Figure 5D is an image of a Coomassie stained SDS-PAGE (non-reduced on the left and reduced on the
right) gel of BMP containing samples of the fractions obtained by the preparative reversed phase
purification step.
Figure 6 , comprising panels A-D, show images of Coomassie-stained SDS-PAGE protein gels
showing purified BMP2 wild type and various mutants as indicated along the top of each gel image. The
gels were run under either non-reducing (Figures 6A and 6B) and reducing (Figures 6C and 6D)
conditions.
Figure 7 shows alkaline phosphatase assay results in C2C1 2 pre-myoblasts comparing the
osteogenic activity of wild type BMP2 and BMP2/6 heterodimer to the various designer BMPs as
indicated in the graph legend.
Figure 8 shows the results of a C2C1 2 BMP-Response Element luciferase (BRE-luciferase)
assay indicative of Smad activity showing stronger signaling by BMPE compared to BMP2 and equivalent
signaling to BMP2/6.
Figure 9 , comprising panels A and B, shows the ectopic bone formation mediated by various
BMPs. Figure 9A is a graph showing the amount of ectopic bone (calculated as milligrams of
hydroxyapatite; mg HA) as determined by mOT analysis for each limb which was implanted with the
indicated BMP (BMP2, BMPE, and BMP2/6) at the dose indicated (0. 1 or 0.5 . Figure 9B is a graph
showing the amount of ectopic bone (calculated as milligrams of hydroxyapatite) as determined by mOT
analysis for each limb which was implanted with the indicated BMP (BMP2, BMPG, BMPA, and BMPF) at
the dose indicated (0. 1 or 0.5 mg). The data presented are from 2 separate experiments.
Figure 10 , comprising panels A-D, shows images of radiographs showing the results of a nonhuman
primate (NHP) fibula osteotomy model at 4 and 8 weeks. Radiographs are shown of the fibulas of
7 representative NHPs that received BMPE and BMPG, respectively, at 0.5 mg/ml (250 m total BMP
delivered/limb). Each NHP received WT BMP2 at the same dose in the contralateral limb. Figures 10A
and 10B show the radiographs for the NHPs indicated at the top of each diagram showing the effects of
BMPE compared with BMP2 wild type at 4 weeks and 8 weeks, respectively. Figures 10C and 10D show
the radiographs for the NHPs indicated at the top of each diagram showing the effects of BMPG
compared with BMP2 wild type at 4 weeks and 8 weeks, respectively.
Figure 11 is a graph showing the bone volume of the limbs treated with BMP-E versus
contralateral limbs treated with BMP-2.
Figure 12 is a graph showing results of an alkaline phosphatase assay in C2C1 2 pre-myoblasts
comparing the osteogenic activity of wild type BMP2 and BMP-GER, BMP-GEP, and BMP2/6
heterodimer.
Figure 13 is a graph showing the amount of ectopic bone (calculated as milligrams of
hydroxyapatite) as determined by mOT analysis for each limb which was implanted with the indicated
BMP (BMP-2, BMP-2/6, BMP-E, BMP-GER, and BMP-6) at the dose indicated (0.05 or 0.25Mg).
Figure 14 is a graph showing the amount of ectopic bone (calculated as milligrams of
hydroxyapatite) as determined by mOT analysis for each limb which was implanted with the indicated
BMP (BMP-2, BMP-2/6, BMP-E, BMP-GER, and BMP-6) at the dose indicated (0.05 or 0.25Mg). These
are the results from an experiment separate from that shown in Figure 13 .
Figure 15, comprising panels A and B, shows images of radiographs and mOT images showing
the results of a non-human primate (NHP) fibula wedge osteotomy model at 5 and 10 weeks. Figure 15A
shows images of 5-week radiographs obtained in a NHP fibula wedge osteotomy model. Figure 15A
shows images of the fibulas of 4 representative NHPs which received BMP-GER in one limb and WT
BMP-2 in the contralateral limb at 0.5 mg/ml (250 total BMP delivered/limb) at 5 weeks. Figure 15B
shows uCT images of the same limbs at 10 weeks showing the large calluses of the BMP-GER treated
limbs compared with the BMP2-treated contralateral limbs for each animal.
Figure 16, comprising panels A-C, shows graphs illustrating the strength (Figure 16A), stiffness
(Figure 16B), and callus bone volume (Figure 16C) of the BMP-GER treated limbs versus the BMP-2
treated contralateral limbs.
Figure 17, comprising panels A-C, shows radiographic images of the healing over time of 3 nonhuman
primate's (NHP) fibulas treated with BMP-GER at 0.5 mg/ml and BMP-2 in the contra lateral limb
at 1.5mg/ml using a calcium phosphate based cement as a carrier following the wedge defect model.
Figure 17A, upper panel, shows results for NHP number 1 left arm treated with 0.5 mg/ml GER as follows:
panels 1 and 2 show LAT (lateral) and AP (anterior-posterior) images, respectively, at the initial time
point; panels 3 and 4 show LAT and AP images, respectively, at 2 weeks; panels 5 and 6 show LAT and
AP images, respectively, at 4 weeks; panels 7 and 8 show LAT and AP images, respectively, at 6 weeks;
panels 9 and 10 show LAT and AP images, respectively, at 7 weeks; panels 11 and 12 show LAT and AP
images, respectively, at 8 weeks; Figure 17A, lower panel, shows results for NHP number 1 right arm
treated with 1.5 mg/ml BMP-2 as follows: panels 1 and 2 show LAT (lateral) and AP (anterior-posterior)
images, respectively, at the initial time point; panels 3 and 4 show LAT and AP images, respectively, at 2
weeks; panels 5 and 6 show LAT and AP images, respectively, at 4 weeks; panels 7 and 8 show LAT and
AP images, respectively, at 6 weeks; panels 9 and 10 show LAT and AP images, respectively, at 7
weeks; panels 11 and 12 show LAT and AP images, respectively, at 8 weeks; Figure 17B shows the
radiographic the results for NHP number 2 as described for NHP # 1 in Figure 17A; and Figure 17C sets
out the results for NHP number 3 as described for NHP # 1 in Figure 17A.
Figure 18 is a diagram of a structural model showing representations and comparison of the
crystal structures BMP-E and BMP-6 WT. The differences in the length of the glycan resolved is
highlighted showing that the glycan for BMPE that is resolved is much longer than that for BMP6. This
indicates that the BMPE glycan is more conformationally constrained than that of BMP6 such that more of
the glycan can be rendered in this model. The histidine doorstop residues for both BMPE and BMP6 are
shown in similar non-doorstop configurations. Also, the arginine glycan "tether" stabilizing the BMPE
glycan is shown by dotted lines representing the interactions of the arginine with the glycan.
Figure 19 is a closer view of the histidine doorstop and arginine tether of the BMPE and BMP6
comparison shown in Figure 18 . This image shows the similar conformation of the H54 histidine residue
of BMPE and the equivalent histidine of BMP6 both in the non-doorstop position. The image also shows
the R 16 tethering (via interactions of the BMPE glycan such that the glycan is more rigid and therefore
more is rendered by the model compared to the more "floppy" and less constrained glycan of BMP6 such
that less of the BMP6 glycan is visualized in this model. The diagram of this model also shows the similar
positioning of asparagine N56 of BMPE showing N-linked attachment of the glycan and the equivalent
and similarly positioned asparagine of BMP6. The diagram also illustrates the potential additional glycan
tethering interaction of BMPE E 110 shown by dotted lines between the amino acid residue and the distal
end of the glycan. The differences in the length of the glycan resolved is highlighted showing that less of
the darker BMP6 glycan can be resolved compared with the lighter shaded longer glycan rendered for
BMPE indicating that the BMPE glycan is more conformationally constrained and thus more is rendered
upon structural analysis.
Figure 20 is a graph showing the results of an alkaline phoshatase assay using C2C1 2 premyoblasts
comparing the osteogenic activity of BMP-2, BMPE and BMP-6 with their Endo-H treated
deglycosylated (Degly.) counterparts.
Figure 2 1 is a diagram illustrating the structural model of BMPE showing the location of the
glycan tether at R 16 and illustrating the stabilizing interactions between the arginine (R1 6) and glutamic
acid (E1 10 corresponding to E 109 of BMP2) residues. The diagram shows that R 16 and E 110 both form
multiple hydrogen bonds with the third (b-mannose) and fourth (a-mannose) glycan moieties. The
diagram also shows the position of H54 potential "doorstop" and asparagine 56 (N56) which provides the
N-linked attachment site of the glycan.
Figure 22 is a graph showing the results of an alkaline phoshatase assay using C2C1 2 premyoblasts
comparing the osteogenic activity of BMP-E, with BMP-E-NR, BMP-GER and BMP-GER-NR in
the presence of increasing doses of Noggin - a natural inhibitor of BMP-2. The data demonstrate that
BMP-GER-NR comprising sequences derived from activin was not inhibited by Noggin even at high
concentrations but that BMP-GER was sensitive to Noggin inhibition. Thus, addition of sequences
derived from activin caused BMP-GER to become Noggin resistant (NR). These results demonstrate that
at least in this in vitro assay, BMP-GER and BMPE, which are Noggin sensitive, become Noggin resistant
(NR) upon replacement of the C-terminal region of the protein with sequences derived from activin.
Figures 23 is a graph showing the bone score as determined by immunohistochemistry (IHC) for
rat ectopic implants treated with the indicated BMP at the specified dose. The data show that the bone
forming activity of BMP-GER was greatly decreased when the C-terminal sequence of the molecule was
replaced with a sequence derived from activin (NR). Thus, the data demonstrate that BMP-GER-NR was
much less active than BMP-GER in vivo.
Figures 24 is a graph showing the bone score as determined by immunohistochemistry (IHC) for
rat ectopic implants treated with the indicated BMP at the specified dose. The data show that the bone
forming activity of BMP-E was greatly decreased, indeed, it was completely abrogated , when the Cterminal
sequence of the molecule was replaced with a sequence derived from activin (NR).
DETAILED DESCRIPTION OF THE INVENTION
This invention relates a "designer" bone morphogenetic protein, referred to herein as "designer
BMP," "designer osteogenic protein" and "designer protein." The designer BMPs of the invention may
correspond to the amino acid sequences of wild type unmodified BMP, such as, but not limited to, BMP2,
BMP4, BMP5, BMP6, BMP7, BMP8, and BMP9. In particular embodiments, the designer BMPs show
altered binding to a type I and/or type I I BMP receptor when compared to its corresponding wild type
BMP. In further embodiments, the designer BMP may be modified to have altered half-life,
immunogenicity, or any pharmacokinetic/pharmacodynamic (PK/PD) parameter when compared to its
corresponding BMP.
Definitions
Unless otherwise defined herein, scientific and technical terms used in connection with the
present invention shall have the meanings that are commonly understood by those of ordinary skill in the
art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms
shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and
tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid
chemistry and hybridization described herein are those well known and commonly used in the art.
The methods and techniques of the present invention are generally performed according to
methods well known in the art and as described in various general and more specific references that are
cited and discussed throughout the present specification unless otherwise indicated. Such references
include, e.g. , Sambrook and Russell, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor
Press, Cold Spring Harbor, NY (2001 ) , Ausubel et al., Current Protocols in Molecular Biology, John Wiley
& Sons, NY (2002), and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY ( 1990), which are incorporated herein by reference. Enzymatic
reactions and purification techniques are performed according to manufacturer's specifications, as
commonly accomplished in the art or as described herein. The nomenclatures used in connection with,
and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and
medicinal and pharmaceutical chemistry described herein are those well known and commonly used in
the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical
preparation, formulation, and delivery, and treatment of patients.
As used herein, each of the following terms has the meaning associated with it in this section.
The articles "a" and "an" are used herein to refer to one or to more than one (i.e. , to at least one)
of the grammatical object of the article. By way of example, "an element" means one element or more
than one element.
In this application, the use of "or" means "and/or" unless stated otherwise.
Conventional notation is used herein to portray polypeptide sequences: the left-hand end of a
polypeptide sequence is the amino-terminus; the right-hand end of a polypeptide sequence is the
carboxyl-terminus. As used herein, the twenty conventional amino acids and their abbreviations follow
conventional usage. See Immunology-A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds.,
Sinauer Associates, Sunderland, Mass. ( 1991 )), which is incorporated herein by reference. As used
herein, amino acids are represented by the full name thereof, by the three letter code corresponding
thereto, or by the one-letter code corresponding thereto, as indicated as follows:
Full Name Three-Letter Code One-Letter Code
Aspartic Acid Asp D
Glutamic Acid Glu E
Lysine Lys K
Arginine Arg R
Histidine His H
Tyrosine Tyr Y
Cysteine Cys C
Asparagine Asn N
Glutamine Gin Q
Serine Ser S
Threonine Thr T
Glycine Gly G
Alanine Ala A
Valine Val V
Leucine Leu L
Isoleucine lie I
Methionine Met M
Proline Pro P
Phenylalanine Phe F
Tryptophan Trp W
A "conservative amino acid substitution" is one in which an amino acid residue is substituted by
another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or
hydrophobicity). In general, a conservative amino acid substitution will not substantially change the
functional properties of a protein. In cases where two or more amino acid sequences differ from each
other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted
upwards to correct for the conservative nature of the substitution. Means for making this adjustment are
well-known to those of skill in the art. See, e.g., Pearson, Methods Mol. Biol. 243:307-31 ( 1994).
Examples of groups of amino acids that have side chains with similar chemical properties include
1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains:
serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side
chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6)
acidic side chains: aspartic acid and glutamic acid ; and 7) sulfur-containing side chains: cysteine and
methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.
Alternatively, a conservative replacement is any change having a positive value in the PAM250
log-likelihood matrix disclosed in Gonnet et al., Science 256:1 443-1 445 ( 1992), herein incorporated by
reference. A "moderately conservative" replacement is any change having a nonnegative value in the
PAM250 log-likelihood matrix.
Preferred amino acid substitutions are those which: ( 1 ) reduce susceptibility to proteolysis, (2)
reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, and (4) confer or
modify other physicochemical or functional properties of such analogs. Analogs comprising substitutions,
deletions, and/or insertions can include various muteins of a sequence other than the specified peptide
sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid
substitutions) may be made in the specified sequence (preferably in the portion of the polypeptide outside
the domain(s) forming intermolecular contacts, e.g. , outside of the CDRs or the type I or type I I receptor
binding sites). A conservative amino acid substitution should not substantially change the structural
characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix
that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the
parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are
described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company,
New York ( 1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing,
New York, N.Y. ( 1991 )); and Thornton et al., Nature 354:1 05 ( 1991 ) , which are each incorporated herein
by reference.
The terms "polynucleotide", "nucleotide sequence", "nucleic acid", "nucleic acid molecule",
"nucleic acid sequence", and "oligonucleotide" refer to a series of nucleotide bases (also called
"nucleotides") in DNA and RNA, and mean any chain of two or more nucleotides. The polynucleotides can
be chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded . The
oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example,
to improve stability of the molecule, its hybridization parameters, etc. A nucleotide sequence typically
carries genetic information, including the information used by cellular machinery to make proteins and
enzymes. These terms include double- or single-stranded genomic and cDNA, RNA, any synthetic and
genetically manipulated polynucleotide, and both sense and antisense polynucleotides. This also
includes nucleic acids containing modified bases, for example, thio-uracil, thio-guanine, and fluoro-uracil,
or containing carbohydrate, or lipids.
In the context of a nucleotide sequence, the term "substantially identical" is used herein to refer to
a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are
identical to aligned nucleotides in a second nucleic acid sequence such that the first and second
nucleotide sequences encode a polypeptide having common functional activity, or encode a common
structural polypeptide domain or a common functional polypeptide activity. For example, nucleotide
sequences having at least about 85%, 90%, 9 1%, 92%, 93%, 94%, 95% , 96%, 97%, 98% or 99% identity
to a reference sequence.
By "designer BMP nucleic acids," and grammatical equivalents herein is meant nucleic acids that
encode designer BMPs.
The terms "protein" and "polypeptide" are used interchangeably herein. These terms refer to a
sequential chain of amino acids linked together via peptide bonds. The terms include one or more
proteins that function as a discrete unit. If a single polypeptide is the discrete functioning unit and does
not require permanent or temporary physical association with other polypeptides in order to form the
discrete functioning unit, the terms "polypeptide" and "protein" may be used interchangeably. If the
discrete functional unit is comprised of multiple polypeptides that physically associate with one another,
the term "protein" as used herein refers to the multiple polypeptides that are physically coupled and
function together as the discrete unit. A protein to be expressed according to the present invention can be
a protein therapeutic. A protein therapeutic is a protein that has a biological effect on a region in the body
on which it acts or on a region of the body on which it remotely acts via intermediates. Examples of
protein therapeutics are discussed in more detail below.
"Designer BMP," as the term is used herein, relates to a BMP protein comprising at least one
amino acid mutation compared to a corresponding wild type BMP without the mutation, wherein the
designer BMP has detectably altered binding for at least a type I receptor and/or at least one type II
receptor compared with the binding of the corresponding wild type BMP for the type I and/or type I I
receptor.
By "corresponding wild type protein" it is meant the wild type version of the designer BMP prior to
the introduction of any mutations. For example, if the designer BMP is a designer BMP2, the
corresponding wild-type BMP is wild-type BMP2. Thus, in one embodiment, design of a designer BMP
can, but need not, begin with a wild type BMP sequence wherein mutations (e.g., amino acid
substitutions, deletions and/or insertion) are introduced into the wild type sequence. Therefore, the
designer BMP can correspond with a wild type BMP, and the locations of the mutations can be said , for
instance, to correspond with, be relative to and/or be respective with the amino acid sequence of the wild
type corresponding or "reference" BMP sequence.
The proteins of the present invention include fragments, derivatives, analogs, or variants of the
polypeptides described herein, and any combination thereof. The terms "fragment," "variant," "derivative"
and "analog" when referring to proteins of the present invention include any proteins which retain at least
some of the functional properties of the protein from which it was derived.
By the term "fragment" as used herein refers to a polypeptide and is defined as any discrete
portion of a given polypeptide that is unique to or characteristic of that polypeptide. The term as used
herein also refers to any discrete portion of a given polypeptide that retains at least a fraction of the
activity of the full-length polypeptide. In certain embodiments, the fraction of activity retained is at least
10% of the activity of the full-length polypeptide. In certain embodiments, the fraction of activity retained is
at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the activity of the full-length polypeptide. In
certain embodiments, the fraction of activity retained is at least 95%, 96%, 97%, 98% or 99% of the
activity of the full-length polypeptide. In certain embodiments, the fraction of activity retained is 100% or
more of the activity of the full-length polypeptide. Alternatively or additionally, the term as used herein
also refers to any portion of a given polypeptide that includes at least an established sequence element
found in the full-length polypeptide. In some embodiments, the sequence element spans at least about 4-
5, 10 , 15 , 20, 25, 30, 35, 40, 45, 50 or more amino acids of the full-length polypeptide. Fragments of
proteins of the present invention include proteolytic fragments, as well as deletion fragments.
Variants of the proteins of the present invention include fragments as described above, and also
polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions.
Variants may occur naturally or be non-naturally occurring. Non-naturally occurring variants may be
produced using art-known mutagenesis techniques. Variant proteins may comprise conservative or nonconservative
amino acid substitutions, deletions or additions.
The proteins of the invention include proteins having one or more residues chemically derivatized
by reaction of a functional side group. Also included as proteins of the invention are polypeptides that
contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For
example, 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine;
3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and
ornithine may be substituted for lysine.
"Recombinantly expressed polypeptide" and "recombinant polypeptide" as used herein refer to a
polypeptide expressed from a host cell that has been manipulated to express that polypeptide. In certain
embodiments, the host cell is a mammalian cell. In certain embodiments, this manipulation may comprise
one or more genetic modifications. For example, the host cells may be genetically modified by the
introduction of one or more heterologous genes encoding the polypeptide to be expressed. The
heterologous recombinantly expressed polypeptide can be identical or similar to polypeptides that are
normally expressed in the host cell. The heterologous recombinantly expressed polypeptide can also be
foreign to the host cell , e.g. heterologous to polypeptides normally expressed in the host cell. In certain
embodiments, the heterologous recombinantly expressed polypeptide is chimeric. For example, portions
of a polypeptide may contain amino acid sequences that are identical or similar to polypeptides normally
expressed in the host cell, while other portions contain amino acid sequences that are foreign to the host
cell. Additionally or alternatively, a polypeptide may contain amino acid sequences from two or more
different polypeptides that are both normally expressed in the host cell. Furthermore, a polypeptide may
contain amino acid sequences from two or more polypeptides that are both foreign to the host cell. In
some embodiments, the host cell is genetically modified by the activation or upregulation of one or more
endogenous genes.
Calculations of homology or sequence identity between sequences (the terms are used
interchangeably herein) are performed as follows. To determine the percent identity of two amino acid
sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison
purposes (e.g. , gaps can be introduced in one or both of a first and a second amino acid or nucleic acid
sequence for optimal alignment and non-homologous sequences can be disregarded for comparison
purposes). In a typical embodiment, the length of a reference sequence aligned for comparison purposes
is at least 30%, at least 40%, at least 50% or 60%, or at least 70%, 80%, 90%, or 100% of the length of
the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the first sequence is occupied by the
same amino acid residue or nucleotide as the corresponding position in the second sequence, then the
molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent
to amino acid or nucleic acid "homology").
To determine the percent identity of two amino acid sequences or of two nucleic acids, the
sequences are aligned for optimal comparison purposes (e.g. , gaps can be introduced in the sequence of
a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid
sequence). The percent identity between the two sequences is a function of the number of identical
positions shared by the sequences (i.e. , % homology=# of identical positions/total # of positions X 100).
The determination of percent homology between two sequences can be accomplished using a
mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the
comparison of two sequences is the algorithm of Karlin et al., Proc Natl Acad Sci U S A 87:2264-8 ( 1990),
modified as in Karlin et al., Proc Natl Acad Sci U S A 90:5873-7 ( 1993). Such an algorithm is incorporated
into the NBLAST and XBLAST programs of Altschul et al. , J Mol Biol 2 15:403-1 0 ( 1990). BLAST
nucleotide searches can be performed with the NBLAST program , score=1 00, wordlength=1 2 .
BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3.
To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in
Altschul et al., Nucleic Acids Res 25:3389-402 ( 1997). When utilizing BLAST and Gapped BLAST
programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
The percent identity between the two sequences is a function of the number of identical positions
shared by the sequences, taking into account the number of gaps, and the length of each gap, which
need to be introduced for optimal alignment of the two sequences.
The comparison of sequences and determination of percent identity between two sequences can
be accomplished using a mathematical algorithm . In one embodiment, the percent identity between two
amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman et al., J Mol
Biol 48:443-53 ( 1970)) which has been incorporated into the GAP program in the GCG software package
(available on at gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16,
14 , 12 , 10 , 8 , 6 , or 4 and a length weight of 1, 2 , 3 , 4 , 5, or 6 . In yet another embodiment, the percent
identity between two nucleotide sequences is determined using the GAP program in the GCG software
package (available on the internet at gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40,
50, 60, 70, or 80 and a length weight of 1, 2 , 3 , 4 , 5, or 6. One typical set of parameters (and the one that
should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12 , a
gap extend penalty of 4 , and a frameshift gap penalty of 5.
The percent identity between two amino acid or nucleotide sequences can be determined using
the algorithm of E. Myers and W. Miller (Myers et al., Comput Appl Biosci 4:1 1-7 ( 1988)) which has been
incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length
penalty of 12 and a gap penalty of 4 .
"Instructional material," as that term is used herein, includes a publication, a recording, a
diagram , or any other medium of expression which can be used to communicate the usefulness of the
compound, combination, and/or composition of the invention in the kit for affecting, alleviating or treating
the various diseases or disorders recited herein. Optionally, or alternately, the instructional material can
describe one or more methods of alleviating the diseases or disorders in a cell, a tissue, or a mammal,
including as disclosed elsewhere herein.
The instructional material of the kit may, for example, be affixed to a container that contains the
compound and/or composition of the invention or be shipped together with a container which contains the
compound and/or composition. Alternatively, the instructional material may be shipped separately from
the container with the intention that the recipient uses the instructional material and the compound
cooperatively.
Except when noted , the terms "patient" or "subject" are used interchangeably and refer to
mammals such as human patients and non-human primates, as well as veterinary subjects such as
rabbits, rats, and mice, and other animals. Preferably, patient refers to a human.
"Effective amount", or "therapeutically effective amount," as the terms are used interchangeably
herein, is an amount that when administered to a tissue or a mammal, preferably a human, mediates a
detectable therapeutic response compared to the response detected in the absence of the compound. A
therapeutic response, such as, but not limited to, inhibition of and/or decreased fibrosis, increased bone
mass or bone density, and the like, can be readily assessed by a plethora of art-recognized methods,
including, e.g. , such methods as disclosed herein.
The skilled artisan would understand that the effective amount of the compound or composition
administered herein varies and can be readily determined based on a number of factors such as the
disease or condition being treated , the stage of the disease, the age and health and physical condition of
the mammal being treated , the severity of the disease, the particular compound being administered , and
the like
As used herein, to "treat" means reducing the frequency with which symptoms of a disease (e.g. ,
decreased bone density, fracture, fibrosis, and the like) are experienced by a patient. The term includes
the administration of the compounds or agents of the present invention to prevent or delay the onset of
the symptoms, complications, or biochemical indicia of a disease, alleviating the symptoms or arresting or
inhibiting further development of the disease, condition, or disorder. Treatment may be prophylactic (to
prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical
symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the
disease.
By the phrase "specifically binds," as used herein, is meant a compound, e.g., a protein, a nucleic
acid , an antibody, and the like, which recognizes and binds a specific molecule, but does not substantially
recognize or bind other molecules in a sample. For instance, an BMP protein, an antibody or a peptide
inhibitor which recognizes and binds a cognate receptor (e.g. , a BMP type I or type I I receptor, an
antibody that binds with its cognate antigen, and the like) in a sample, but does not substantially
recognize or bind other molecules in the sample. Thus, under designated assay conditions, the specified
binding moiety (e.g., a BMP or a receptor binding fragment thereof) binds preferentially to a particular
target molecule and does not bind in a significant amount to other components present in a test sample.
A variety of assay formats may be used to select an antibody that specifically binds a molecule of interest.
For example, solid-phase ELISA immunoassay, immunoprecipitation, BIAcore, FACS, Octet, and Western
blot analysis are among many assays that may be used to identify a BMP that specifically reacts with a
BMP receptor. Typically, a specific or selective reaction will be at least twice background signal or noise,
more preferably, at least five-fold greater than background signal or noise, and more typically, more than
10 times background, even more specifically, a BMP is said to "specifically bind" a BMP receptor when
the equilibrium dissociation constant (K ) is < 100 mM, more preferably < 10 mM, even more preferably < 1
mM, yet more preferably < 100 nM and most preferably < 10 nM.
The term "K " refers to the equilibrium dissociation constant of a particular ligand-receptor
interaction.
"Binding affinity" generally refers to the strength of the sum total of noncovalent interactions
between a binding site of a molecule (e.g. , a BMP ligand) and its binding partner (e.g., a BMP type I or
type I I receptor). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding
affinity which reflects a 1:1 interaction between members of a binding pair (e.g., BMP and its cognate
receptor). The affinity of a molecule X for its partner Y can generally be represented by the dissociation
constant (Kd).
Affinity can be measured by common methods known in the art, including those described herein.
Low-affinity BMPs generally bind a receptor slowly and tend to dissociate readily, whereas high-affinity
BMPs generally bind a receptor faster and tend to remain bound longer. A variety of methods of
measuring binding affinity are known in the art, any of which can be used for purposes of the present
invention. Specific illustrative embodiments are described elsewhere herein.
The term "ko " , as used herein is intended to refer to the association or on rate constant, or
specific reaction rate, of the forward, or complex-forming, reaction, measured in units: M~ sec 1 .
The term "koff" , as used herein, is intended to refer to the dissociation or off rate constant, or
specific reaction rate, for dissociation of an antibody from the antibody/antigen complex, measured in
units: sec 1 .
The term "Kd" , as used herein, is intended to refer to the dissociation constant of a particular
antibody-antigen interaction. It is calculated by the formula:
The term "altered binding" as used herein means the designer BMP comprises a different binding
specificity for at least a type I receptor and/or a type I I receptor when compared with the binding of a
corresponding wild type BMP to the same type I and/or type I I receptor. The designer BMP may bind
with greater or lesser affinity with the receptor compared to the binding of the wild type BMP to that
receptor. For instance, if the wild type BMP bound a certain type I receptor with a certain binding affinity,
the corresponding designer BMP binds that receptor with greater or lesser affinity compared with the wild
type BMP. It may even be that the designer BMP will specifically bind a receptor that the wild type BMP
did not detectably bind and vice-a-versa where the designer BMP will no longer detectably bind a
receptor that the wild type BMP binds. Thus, altered binding encompasses any detectable change in
binding by a designer BMP to a type I or type I I receptor compared with the binding of that receptor by the
corresponding wild type BMP. It may be that the designer BMP has a greater or lesser ko value
compared with the ko value for a corresponding wild type BMP and/or the designer BMP has a greater or
lesser koff value compared with the koff value of the corresponding wild type BMP such that the Kd of the
designer BMP is greater or lesser than the Kd of a corresponding wild type BMP for the same BMP
receptor. Thus, any difference in a binding characteristic and/or affinity value between a designer BMP
and a corresponding wild type BMP are encompassed by the term "altered binding" as used herein.
The term "surface plasmon resonance", as used herein, refers to an optical phenomenon that
allows for the analysis of real-time biospecific interactions by detection of alterations in protein
concentrations within a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor
AB, Uppsala, Sweden, and Piscataway, N.J .). For further descriptions, see, e.g., Johnsson, et al., Ann.
Biol. Clin. 5 1: 19-26 ( 1993); Johnsson, et al., Biotechniques 11: 620-627 ( 1991 ) ; Johnsson, et al. , J. Mol.
Recognit. 8 : 125-1 3 1 ( 1995); and Johnnson, et al., Anal. Biochem. 198: 268-277 ( 1991 ) .
As used herein, "substantially pure" means an object species is the predominant species present
(i.e. , on a molar basis it is more abundant than any other individual species in the composition), and
preferably a substantially purified fraction is a composition wherein the object species (e.g., a designer
BMP) comprises at least about 50 percent (on a molar basis) of all macromolecular species present.
Generally, a substantially pure composition will comprise more than about 80 percent of all
macromolecular species present in the composition, more preferably more than about 85%, 90%, 95%,
and 99%. Most preferably, the object species is purified to essential homogeneity (contaminant species
cannot be detected in the composition by conventional detection methods) wherein the composition
consists essentially of a single macromolecular species.
Description
Bone Morphoqenetic Proteins (BMPs)
As stated previously elsewhere herein, BMPs are members of the TGF- b protein superfamily all
of which are characterized by six-conserved cysteine residues (Lander et al, (2001 ) Nature, 409:860-921 .
The BMP/GDF subfamily includes, but is not limited to, BMP2 , BMP3 (osteogenin) (see, e.g., US Patent
No. 6,177,406), BMP3b (GDF-1 0) (see, e.g., US Patent No. 6,204,047), BMP4 (BMP2b) (see, e.g., US
Patent No. 6,245,889), BMP5 (see, e.g., US Patent No. 5,543,394), BMP6 (see, e.g., US Patent No.
6,61 3,744), BMP7 (osteogenic protein-1 or OP1 ) (see, e.g., US Patent No. 5,14 1,905), BMP8 (OP2) (see,
e.g., US Patent No. 5,688,678), BMP8B (OP3) (see, e.g., US Patent No. 5,854,071 ) , BMP9 (GDF2) (see,
e.g., US Patent No. 6,287,81 6), BMP1 0 (see, e.g., US Patent No. 5,703,043), BMP1 1 (GDF1 1) (see, e.g.,
US Patent No. 6,437,1 11) , BMP1 2 (GDF7) (see, e.g., US Patent No. 6,027,91 9), BMP1 3 (GDF6,
CDMP2) (see, e.g., US Patent No. 6,027,91 9), BMP1 5 (GDF9) (see, e.g., US Patent No. 6,034,229),
BMP1 6 (see, e.g., US Patent No. 6,331 ,61 2), GDF1 (see, e.g., US Application No. 2004/00391 62), GDF3
(see, e.g., US Patent No. 6,025,475), GDF5 (CDMP1 ; MP52) (see, e.g., US Patent No. 5,994,094), and
GDF8 (myostatin) (see, e.g., US Patent No. 5,827,733).
BMPs specifically bind their cognate receptors, which include Type I receptors: ALK-I, ALK-2
(also called ActRIa or ActRI), ALK-3 (also called BMPRIa), and ALK-6 (also called BMPRIb); and Type I I
receptors: ActRMa (also called ActRII), ActRllb, and BMPRI I . The BMP-receptor binding interactions have
been studied extensively, and the binding specificities of each wild type BMP for each type I and/or type I I
receptor is generally known in the art and are shown in Table 1. See, e.g. , Nickel et al., Cytokine Growth
Factor Rev 20:367-77 (2009); Heinecke et al., BMC Biol 7:59 (2009).
TABLE 1
ALK 1 ALK 2 ALK 3 ALK 6 ACTI IA ACTIIB BMPRII
BMP-7 No Binding No Binding ++ ++ ++++ ++++ ++++
BMP-9 +++++ No Binding No Binding No Binding ++ +++ ++++
Designer Bone Morphoqenetic Proteins with Improved Osteogenic Activity
This application is based , in part on the understanding that each BMP dimer binds to four BMP
receptors two type I receptors and two type I I receptors. The specificities of each BMP for each receptor
are known in the art as shown above in Table 1. Also, the receptor binding regions of various BMPs that
mediate binding of the BMP for each receptor have been mapped and are shown in Table 2 . For
instance, it is well established that wild type BMP2 and BMP4 bind type I BMP receptors Alk-3 and ALK-6
with high affinity and bind type I I BMP receptors with lower affinity. On the other hand , wild type BMP6
and BMP7 are known to have bind type I I receptors Actrl lA, Actrl lB, and BMPRI I with high affinity but bind
type I receptors with lower affinity than they do to type II. It is believed that the differing cellular responses
from the approximately forty-three TGFp superfamily members signaling through interaction with
approximately twelve receptors is believed to be due to each ligand utilizing a specific repertoire of
receptors with which it binds with differing affinities. The type I and II binding domains are described in
Table 2 .
TABLE 2
Rational amino acid substitution to alter receptor binding of designer BMPs
In one embodiment, the invention comprises introducing an amino acid mutation in at least one
receptor binding site thereby providing altered binding to type I and type I I BMP receptors by designer
BMPs compared to the binding of the corresponding wild type BMP to those receptors. That is, it is well
known in the art that wild type BMP2 shows a relatively high affinity for type I receptors, while wild type
BMP6 shows a high affinity for type I I receptors. It is further known in the art that heterodimers of wild
type BMP2 and BMP6 bind to both type I and type II receptors with relatively high affinity each BMP
apparently providing the higher affinity binding site for each receptor. See Table 3, below. The BMP2/6
heterodimers are known to be more active that BMP2 or BMP6 alone or as homodimers, in both in vitro
and in vivo bone formation assays. Table 3 shows an example of BMP2 and BMP6 binding affinities to
type I and I I receptors.
TABLE 3
Accordingly, it is an object of the invention to provide designer BMPs with improved binding to
type I and/or type I I receptors. As shown in Figure 1A and Table 2 , each BMP comprises three binding
sites that contribute to receptor binding. From N- to C-terminus, each BMP comprises a type I I receptor
binding site A, a type I receptor binding site, and a second type I I receptor binding site B. Although an
exemplary alignment of wild type BMP2, BMP4, BMP5, BMP6, BMP7, BMP8, and BMP9 is illustrated in
Figure 1, the skilled artisan will appreciate that there are well-known alignments providing the relative
positioning of various amino acids among the members of the TQBb superfamily. Such alignments are
provided , among others, in International Publication Nos. WO 2009/0861 3 1 (e.g. , Figures 15-1 7, Figure
31A), WO 2008/051 526 (Figures 9-1 2), WO 2005/1 18636 (Figure 6), WO 2005/1 18635, WO
2005/1 13585 (Figure 3), WO 2001 /92298 (Figure 6A-6C), Kirsch et al. , EMBO J. 19 :331 4-3324 (2000)
(Figure 1) , US Patent Application Publication No., 2007/0293425 (Figure 6), Groppe et al., Nature
420 :636-642 (2002), Nickel et al., J. Bone Joint Surg. Am. 83:7-1 4 (2001 ) , and Weber et al. , BMC
Structural Biol. 7 :6 (2007). Thus, using protein sequence alignment algorithms and tools well-known in
the art, including the alignments of the amino acid sequences of the various TGFp superfamily members,
as well as the disclosure provided herein, the corresponding amino acid in one BMP/GDF protein relative
to the amino acid at any position in another BMP/GDF protein can be determined. In one embodiment,
the corresponding amino acid residues in BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8 and BMP-9 are
shown (see, e.g. , Figure 1A).
In some embodiments of the invention, the designer BMP comprises mutations in a type I binding
domain or a type I I binding domain, wherein the mutations confer altered binding to a type I or type I I
BMP receptor. In some embodiments, the designer BMP comprises one or more mutations in both a type
I binding domain and a first (binding domain A) or second (binding domain B) type I I binding domain. In
other embodiments, the designer BMP comprises one or more mutations in both type I I binding domains.
In other embodiments, the designer BMP comprises one or more mutations in the first type I I binding
domain, in the second type I I binding domain, and in the type I binding domain. In some embodiments,
the designer BMP comprises one or more mutations in the type I binding domain.
In some embodiments, the mutations improve binding to a type I receptor. In other embodiments,
the mutations improve binding to a type I I receptor. In other embodiments, the mutations decrease
binding to a type I or type I I receptors. In some embodiments, the mutations create or destroy a glycan
tether as more fully set forth below. In some embodiments, the mutations create or destroy a His
doorstop as more fully set forth below.
Because BMPs are so well characterized and understood in the art, it would be understood , once
provided with the disclosure provided herein, the location of possible mutations that can be made that do
not further affect the activity of the designer BMPs would be understood . Accordingly, the designer BMPs
of the invention encompass variant BMPs which differ from a corresponding wild type or designer BMP in
that it contains additional insertions, deletions, or substitutions which do not affect the receptor binding
affinity of the variant BMPs. In some non-limiting embodiments, those of skill in the art would understand
that the cysteines involved in cysteine knot formation and amino acids involved in receptor interactions
should not be mutated or should be changed with conservative substitutions, while other amino acids may
be more freely substituted , inserted, or deleted without adversely affecting biological activity of the
designer BMP.
It should be noted that unless otherwise stated, all positional numbering of designed or modified
BMPs is based on the sequences of the mature native BMPs. Designer BMPs are characterized by the
predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or
interspecies variation of the BMP sequence. Variants of designer BMPs must retain at least 50% of the
activity of the corresponding wild type or designer BMP activity in one or more cell types, as determined
using an appropriate assay described below. Variants that retain at least 75%, 80%, 85% , 90% or 95% of
wild type activity are more preferred, and variants that are more active than wild type are especially
preferred. A designer BMP may contain insertions, deletions, and/or substitutions at the N- terminus, Cterminus,
or internally. In a preferred embodiment, designed or modified BMPs have at least 1 residue
that differs from the most similar human BMP sequence, with at least 2 , 3 , 4 , 5, 6, 7, 8 , 9 , 10 or more
different residues being more preferred .
Designer BMPs of the invention maintain at least 80% , at least 8 1%, at least 82%, at least 83%,
at least 84% , at least 85%, at least 86%, at least 87% , at least 88%, at least 89%, at least 90%, at least
9 1%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or
at least 99% identity with the corresponding wild-type BMP protein sequence.
Designer BMPs of the invention may maintain at least 80%, at least 8 1%, at least 82%, at least
83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at
least 9 1%, at least 92%, at least 93%, at least 94% , at least 95%, at least 96%, at least 97%, at least
98%, or at least 99% identity with the conserved cysteine domain of the C-terminal region of the
corresponding wild-type BMP protein sequence.
Designer BMPs may contain further modifications, for instance mutations that alter additional
protein properties such as stability or immunogenicity or which enable or prevent posttranslational
modifications such as PEGylation or glycosylation. Designer BMPs may be subjected to co- or post¬
translational modifications, including but not limited to synthetic derivatization of one or more side chains
or termini, glycosylation, PEGylation, circular permutation, cyclization, fusion to proteins or protein
domains, and addition of peptide tags or labels.
Due to the degeneracy of the genetic code, an extremely large number of nucleic acids may be
made, all of which encode the designer BMPs of the present invention, by simply modifying the sequence
of one or more codons in a way that does not change the amino acid sequence of the designer BMP. The
designer BMPs of the invention do not comprise these sequences set forth in WO2008/051 526 or
WO2009/0861 3 1.
As described above, BMPs are naturally expressed as pro-proteins comprising a long prodomain,
one or more cleavage sites, and a mature domain. This pro-protein is then processed by the
cellular machinery to yield a dimeric mature BMP molecule. In a preferred embodiment, the designer
BMPs of the invention are produced in a similar manner. The pro-domain is believed to aid in the correct
folding and processing of BMPs. Furthermore, in some but not all BMPs, the pro-domain may
noncovalently bind the mature domain and may act as a chaperone, as well as an inhibitor (e.g., Thies et
al. (2001 ) Growth Factors, 18:251 -259). Preferably, the modified BMPs of the invention are produced
and/or administered therapeutically in this form. Alternatively, BMPs may be produced in other forms,
including, but not limited to, where the mature domain is produced directly or refolded from inclusion
bodies, or comprises full-length intact pro protein. The designer BMPs of the invention will be useful in
these and other forms.
In particular embodiments, the designer BMP of the invention comprises a backbone BMP, i.e. ,
the wild type BMP, to which the designer BMP corresponds. In particular embodiments, this backbone
BMP may be a wild type BMP2, BMP4, BMP5, BMP6, BMP7, BMP8, or BMP9 backbone.
In some embodiments of the invention, the designer BMP comprises at least one mutation in a
type I binding domain and/or a type I I binding domain, wherein the mutation confers altered binding to a
type I or type I I BMP receptor compared with the binding of a corresponding wild type BMP not
comprising the mutation. In some embodiments, the designer BMP comprises at least one mutation in
both a type I binding domain and at least one mutation in a type II binding domain. In other
embodiments, the designer BMP comprises at least one mutation within the type I I binding domain A and
the type I I binding domain B. In other embodiments, the designer BMP comprises at least one mutation
in type I I binding domain A, type I I binding domain B, and a type I binding domain.
In certain embodiments, the mutation may comprise an amino or nucleic acid substitution,
deletion and/or insertion. In a preferred embodiment, the mutation comprises an amino acid substitution.
In some embodiments, the backbone BMP is a wild type BMP and the mutations are one or more
of the mutations listed in Tables 4 to 6 . The designer BMP may contain any combination and any number
of mutations listed in these tables.
In some embodiments, the backbone BMP is a wild type BMP and the mutations are one or more
of the mutations listed in Tables 4 to 6. The designer BMP may contain a permutation and any and all of
the mutations listed in these tables or disclosed elsewhere herein.
TABLE 4
TABLE 5
P36 P38 E59 K60 E60 Q60 K30 K, R, P, E, Q
G37 G39 G60 G61 G61 G61 E31 G, E
H39 Q41 A62 A63 A63 S63 E33 A, E, S, Q
F41 F43 F64 N65 Y65 Y65 Y35 N, Y, F
Y42 Y44 Y65 Y66 Y66 Y66 E36 Y, E
H44 H46 D67 D68 E68 E68 K38 H, D, K, R, E
TABLE 6
Type I I Binding Domain B Mutations
BMP2 BMP4 BMP5 BMP6 BMP7 BMP8 BMP9 Possible mutations
E83 E85 K 107 K 108 Q 108 K 108 K78 Q, K, E
S85 S87 N 109 N 110 N 110 S 110 S80 N, S
A86 A88 A 110 A 111 A 111 A 111 P81 P, A
M89 M91 V 113 V 114 V 114 V 114 V84 M, V
L92 L94 F 116 F 117 F 117 Y 117 K87 F, K, L, Y
E94 E96 D 118 D 119 D 119 D 118 D89 D, E
N95 Y97 S 119 N 120 S 120 S 119 M90 M, N, S
E96 D98 S 120 S 12 1 S 12 1 S 120 G91 S, G, D
K97 K99 N 12 1 N 122 N 122 N 12 1, V92 N, V, K
N 122
V98 V 100 V 122 V 123 V 123 V 123 P93 P, V
V99 V 10 1 11 23 11 24 11 24 11 24 T94 T, I , V
In some embodiments, the mutations improve binding to a type I receptor. In other embodiments
improve binding to a type II receptor. In other embodiments, the mutations decrease binding to a type I or
type I I receptors.
Tables 4-6 above provide a non-limiting compilation of example mutations of the present
invention where the position of the mutation is provided relative to the corresponding wild type BMP
amino acid sequence. Thus, in some embodiments, the designer BMP comprises the following preferred
combinations of mutations.
In certain embodiments, the corresponding wild type BMP to the designer BMP is BMP2.
Further, the at least one mutation within the type I I receptor binding domain A is a mutation selected from
the group consisting of V33, P36, G37, H39, F41 , Y42 AND H44.
In other embodiments, the designer BMP comprises at least one mutation within the type I I
receptor binding domain A and further comprises at least one additional mutation within a type I receptor
binding domain. The mutation within the type I receptor binding domain is at least one mutation at P48,
F49, A52, D53, H54, L55, N56, S57, N59, V63, T65, N68, S69, V70, N71 , S72, K73, I74, and P75 with
respect to the sequence of SEQ ID NO:1 .
In yet further embodiments, the designer BMP comprises at least one mutation within a type I I
receptor binding domain A, at least one mutation within the type I receptor binding domain, and further
comprises at least one additional mutation within a type MB receptor binding domain. The mutation within
the type II receptor binding domain B is at least one mutation at E83, S85, A86, M89, L92, E94, N95,
E96, K97, V98, and V99 with respect to the sequence of SEQ ID NO: 1.
In some embodiments, the designer BMP comprises mutations at each of amino acids H44, P48,
A52, D53, L55, S57, N68, S69, V70, insertion of P after N71 , S72, K73, I74, A77, and V80 with respect to
the sequence of SEQ ID NO:1 .
In one embodiment, the designer BMP comprises the following mutations: H44D, P48S, A52N,
D53A, L55M, S57A, N68H, S69L, V70M, insertion of a P after N71 , S72E, K73Y, I74V, A77P, and V80A
with respect to the sequence of SEQ ID NO:1 .
In some embodiments the designer BMP comprises mutations at each of amino acids V33, P36,
H39, S85, M89, L92, E94, E96, K97, and V99 with respect to the sequence of SEQ ID NO:1 .
In some embodiments, the designer BMP comprises mutations at each of amino acids V33I,
P36K, H39A, S85N, M89, L92F, E94D, E96S, K97N, and V99I with respect to the sequence of SEQ ID
NO: 1.
In other embodiments, the designer BMP comprises the following mutations: V33I, P36K, H39A,
H44D, P48S, A52N, L54M, S56M, N68H, V70M, S72E, K73E, insertion of a Y after K73, I74V, 77AP,
S85N , M89V, L92F, E94D, E96S, K97N , and V99I with respect to the sequence of SEQ ID NO:1 .
In yet other embodiments, the designer BMP comprises the following mutations: V33I, P36R,
H39A, H44D, P48S, A52N , L54M, S56M, N68H, V70M, S72E, K73E, insertion of a Y after K73, I74V,
77AP, S85N, M89V, L92F, E94D, E96S, K97N, and V99I with respect to the sequence of SEQ ID NO: 1.
In certain embodiments, the corresponding wild type BMP to the designer BMP is BMP4. In
certain embodiments, the at least one mutation within the type II receptor binding domain A is at V35,
P38, G39, Q41 , F43, Y44, and H46 of SEQ ID NO:2.
In other embodiments, the designer BMP4 comprises at least one mutation within the type I I
receptor binding domain A and further comprises at least one additional mutation within a type I receptor
binding domain. The mutation within the type I receptor binding domain is at least one mutation at P50,
A54, D55, H56, L57, N58, S59, N61 , V65, T67, N70, S71 , V72, N73, S74, S75, I76, and P77 of SEQ ID
NO:2.
In yet further embodiments, the designer BMP4 comprises at least one mutation within a type I I
receptor binding domain A, at least one mutation within the type I receptor binding domain, and further
comprises at least one additional mutation within a type MB receptor binding domain. The mutation within
the type I I receptor binding domain B is at least one mutation at E85, S87, A88, M91 , L94, E96, K97, V98
and V99 of SEQ ID NO:2.
In certain embodiments, the corresponding wild type BMP to the designer BMP is BMP5. In
certain embodiments, the mutation within the type I I receptor binding domain A is at least one mutation at
156, E59, G60, A62, F64, Y65, or D67 of SEQ ID NO:3.
In other embodiments, the designer BMP comprises at least one mutation within the type I I
receptor binding domain A and further comprises at least one additional mutation within a type I receptor
binding domain. The mutation within the type I receptor binding domain is at least one mutation at S71 ,
F72, N75, A76, H77, M78, N79, A80, N82, V86, T88, H91 , L92, M93, F94, P95, D96, H97, V98, or P99 of
SEQ ID NO:3.
In yet further embodiments, the designer BMP comprises at least one mutation within a type I I
receptor binding domain A , at least one mutation within the type I receptor binding domain, and further
comprises at least one additional mutation within a type MB receptor binding domain. The mutation within
the type I I receptor binding domain B is at least one mutation at K 107, N 109, A 110 , V 113 , F 116, D 118 ,
S 119 , S 120, N 12 1, V 122, or 11 23 of SEQ ID NO:3.
In certain embodiments, the corresponding wild type BMP to the designer BMP is BMP6. In
certain embodiments, the mutation within the type I I receptor binding domain A is at least one mutation at
157, K60, G61 , A63, N65, Y66, or D68 of SEQ ID NO:4.
In other embodiments, the designer BMP6 comprises at least one mutation within the type I I
receptor binding domain A and further comprises at least one additional mutation within a type I receptor
binding domain. The mutation within the type I receptor binding domain is at least one mutation at S72,
N76, A77, H78, M79, N80, A81 , N83, V87, T89, H92, L93, M94, N95, P96, E97, Y98, V99, or P 100 of
SEQ ID NO:4.
In yet further embodiments, the designer BMP6 comprises at least one mutation within a type I I
receptor binding domain A , at least one mutation within the type I receptor binding domain, and further
comprises at least one additional mutation within a type MB receptor binding domain. The mutation within
the type I I receptor binding domain B is at least one mutation at K 108, N 110 , A 111, V 114 , F 117, D 119 ,
N 120, S 12 1, N 122, V 123, or 11 24 of SEQ ID NO:4.
In certain embodiments, the corresponding wild type BMP to the designer BMP is BMP7. In
certain embodiments, the mutation within the type I I receptor binding domain A is at least one mutation at
I57, E60, G61 , A63, Y65, Y66, or E68 of SEQ ID NO:5.
In other embodiments, the designer BMP7 comprises at least one mutation within the type I I
receptor binding domain A and further comprises at least one additional mutation within a type I receptor
binding domain. The mutation within the type I receptor binding domain is at least one mutation at A72,
F73, N76, S77, Y78, M79, N80, A81 , N83, V87, T89, H92, F93, I94, N95, P96, E97, T98, V99, or P 100 of
SEQ ID NO:5.
In yet further embodiments, the designer BMP7 comprises at least one mutation within a type I I
receptor binding domain A , at least one mutation within the type I receptor binding domain, and further
comprises at least one additional mutation within a type MB receptor binding domain. The mutation within
the type I I receptor binding domain B is at least one mutation at Q 108, N 110 , A 111, V 114 , F 117, D 119 ,
S 120, S 12 1, N 122, V 123, or 11 24 of SEQ ID NO:5.
In certain embodiments, the corresponding wild type BMP to the designer BMP is BMP8. In
certain embodiments, the mutation within the type I I receptor binding domain A is at least one mutation at
I57, Q60, G61 , S63, Y65, Y66, or E68 of SEQ ID NO:6.
In other embodiments, the designer BMP8 comprises at least one mutation within the type I I
receptor binding domain A and further comprises at least one additional mutation within a type I receptor
binding domain. The mutation within the type I receptor binding domain is at least one mutation at S72,
F73, D76, S77, C78, M79, N80, A82, N83, L87, S89, H92, L93, M94, M95, P96, D97, A98, V99, or P 100
of SEQ ID NO:6.
In yet further embodiments, the designer BMP8 comprises at least one mutation within a type I I
receptor binding domain A, at least one mutation within the type I receptor binding domain, and further
comprises at least one additional mutation within a type MB receptor binding domain. The mutation within
the type I I receptor binding domain B is at least one mutation at K 108, S 110 , A 111, V 114 , Y 117, D 118 ,
S 119 , S 120, N 12 1, N 122, V 123, or 11 24 or SEQ ID NO:6
In certain embodiments, the mutation within the type II receptor binding domain A is at least one
mutation at I27, K30, E31 , E33, Y35, or E36 of SEQ ID NO:7.
In other embodiments, the designer BMP9 comprises at least one mutation within the type I I
receptor binding domain A and further comprises at least one additional mutation within a type I receptor
binding domain. The mutation within the type I receptor binding domain is at least one mutation at F42,
F43, A46, D47, D48, V49, T50, P51 , K53, V57, T59, H62, L63, K64, F65, P66, T67, K68, V69, or G70 of
SEQ ID NO:7.
In yet further embodiments, the designer BMP9 comprises at least one mutation within a type I I
receptor binding domain A, at least one mutation within the type I receptor binding domain, and further
comprises at least one additional mutation within a type MB receptor binding domain. The mutation within
the type I I receptor binding domain B is at least one mutation at K78, S80, P81 , V84, K87, D89, M90,
G91 , V92, P93, or T94 of SEQ ID NO:7.
Exemplary amino acid sequences of designer BMPs are set forth in Table 7, below. Table 7
shows the name and sequence of the designed molecules.
TABLE 7
NAME SEQUENCE SEQ ID
NO
BMP-E QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCDGECSFPLNAHMNATNHAIVQTLVH 12
LMNPEYVPKPCCAPTELSAISMLYLDENEKWLKNYQDMWEGCGCR
BMP-F QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWI IAPKGYAAFYCHGECPFPLADHLNSTNHAIVQTLVN 13
SVNSKIPKACCVPTELSAISMLYLDENEKWLKNYQDMWEGCGCR
BMP-G QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWI IAPKGYAAFYCHGECPFPLADHLNSTNHAIVQTLVN 14
SVNSKIPKACCVPTELNAISVLYFDDNSNVILKNYQDMWEGCGCR
BMP-H QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWI IAPKGYHAFYCHGECPFPLADHLNSTNHAIVQTLVN 15
SVNSKIPKACCVPTELNAISVLYFDENSNWLKKYQDMWRGCGCR
BMP-I QAKHKQRKRLKSSCKRHELYVSFQDLGWQDWI IAPKGYAANYCHGECPFPLADHLNSTNHAIVQTLVN 16
SVNSKIPKACCVPTELNAISVLYFDDNSNVILKKYRNMWRACGCR
BMP-J QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWI IAPKGYHAFYCDGECSFPLNAHMNATNHAIVQTLVH 17
LMNPEYVPKPCCAPTELNAISVLYFDENSNWLKKYQDMWRGCGCR
BMP-K QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTKHAIVQTLVN 18
SVNSKIPKACCVPTELSAISMLYLDENEKWLKNYQDMWEGCGCR
BMP-T QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTTHAIVQTLVN 19
SVNSKIPKACCVPTELSAISMLYLDENEKWLKNYQDMWEGCGCR
BMP-AP QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWI IAPPGYAANYCHGECPFPLADHLNSTNHAIVQTLVN 20
SVNSKIPKACCVPTELSAISMLYLDENEKWLKNYQDMWEGCGCR
BMP-AR QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWI IAPRGYAANYCHGECPFPLADHLNSTNHAIVQTLVN 2 1
SVNSKIPKACCVPTELSAISMLYLDENEKWLKNYQDMWEGCGCR
BMP-AK QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWI IAPKGYAANYCHGECPFPLADHLNSTKHAIVQTLVN 22
SVNSKIPKACCVPTELSAISMLYLDENEKWLKNYQDMWEGCGCR
BMP-AT QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWI IAPKGYAANYCHGECPFPLADHLNSTTHAIVQTLVN 23
SVNSKIPKACCVPTELSAISMLYLDENEKWLKNYQDMWEGCGCR
BMP-DP QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLNAHMNATNHAIVQTLVH 24
LMNPSKIPKACCVPTELSAISMLYLDENEKWLKNYQDMWEGCGCR
BMP-E9 QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCKGGCFFPLADDVTPTKHAIVQTLVH 25
LKFPTKVGKACCVPTELSAISMLYLDENEKWLKNYQDMWEGCGCR
BMP- QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCRGVCNYPLAEHLTPTKHAI IQALVH 26
E 10 LKNSQKASKACCVPTELSAISMLYLDENEKWLKNYQDMWEGCGCR
BMP-EK QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCDGECSFPLNAHMNATKHAIVQTLVH 27
LMNPEYVPKPCCAPTELSAISMLYLDENEKWLKNYQDMWEGCGCR
BMP-ET QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCDGECSFPLNAHMNATTHAIVQTLVH 28
LMNPEYVPKPCCAPTELSAISMLYLDENEKWLKNYQDMWEGCGCR
BMP-R QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPRGYHAFYCHGECPFPLADHLNSTNHAIVQTLVN 29
SVNSKIPKACCVPTELSAISMLYLDENEKWLKNYQDMWEGCGCR
BMP-G5 QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCEGLCEFPLRSHLEPTNHAVIQTLMN 30
SMDPESTPPTCCVPTELSAISMLYLDENEKWLKNYQDMWEGCGCR
BMP-ER QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPRGYHAFYCHGECPFPLADHLNSTNHAIVQTLVN 3 1
SVNSKIPKACCVPTELSAISMLYLDENEKWLKNYQDMWEGCGCR
BMP-GP QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWI IAPPGYAAFYCHGECPFPLADHLNSTNHAIVQTLVN 32
SVNSKIPKACCVPTELNAISVLYFDDNSNVILKNYQDMWEGCGCR
BMP- QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWI IAPRGYAAFYCHGECPFPLADHLNSTNHAIVQTLVN 33
GR SVNSKIPKACCVPTELNAISVLYFDDNSNVILKNYQDMWEGCGCR
BMP-GK QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWI IAPKGYAAFYCHGECPFPLADHLNSTKHAIVQTLVN 34
SVNSKIPKACCVPTELNAISVLYFDDNSNVILKNYQDMWEGCGCR
BMP-GT QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWI IAPKGYAAFYCHGECPFPLADHLNSTTHAIVQTLVN 35
SVNSKIPKACCVPTELNAISVLYFDDNSNVILKNYQDMWEGCGCR
BMP-GE QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWI IAPKGYAAFYCDGECSFPLNAHMNATNHAIVQTLVH 36
LMNPEYVPKPCCAPTELNAISVLYFDDNSNVILKNYQDMWEGCGCR
BMP-GE QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWI IAPRGYAAFYCDGECSFPLNAHMNATNHAIVQTLVH 37
R LMNPEYVPKPCCAPTELNAISVLYFDDNSNVILKNYQDMWEGCGCR
BMP-JP QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWI IAPPGYHAFYCDGECSFPLNAHMNATNHAIVQTLVH 38
LMNPEYVPKPCCAPTELNAISVLYFDENSNWLKKYQDMWRGCGCR
BMP-JR QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWI IAPRGYHAFYCDGECSFPLNAHMNATNHAIVQTLVH 39
LMNPEYVPKPCCAPTELNAISVLYFDENSNWLKKYQDMWRGCGCR
BMP-JK QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWI IAPKGYHAFYCDGECSFPLNAHMNATKHAIVQTLVH 40
LMNPEYVPKPCCAPTELNAISVLYFDENSNWLKKYQDMWRGCGCR
BMP-JT QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWI IAPKGYHAFYCDGECSFPLNAHMNATTHAIVQTLVH 4 1
LMNPEYVPKPCCAPTELNAISVLYFDENSNWLKKYQDMWRGCGCR
BMP-A9 QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWI IAPKEYEAYECHGECPFPLADHLNSTNHAIVQTLVN 42
SVNSKIPKACCVPTELSAISMLYLDENEKWLKNYQDMWEGCGCR

NAME SEQUENCE SEQ ID
NO
CH
BMP9E2 REKRSAGAGSHCQKTSLRVNFEDIGWDSWI IAPKEYEAYECHGECPFPLADHLNSTNHAIVQTLVNSV 65
NSKI PKACCVPTKLSPISVLYKDDMGVPTLKYHYEGMSVAECGCR
BMP9E6 SAGAGSHCQKTSLRVNFEDIGWDSWI IAPKEYEAYECDGECSFPLNAHMNATNHAIVQTLVHLMNPEY 66
VPKPCCAPTKLS PISVLYKDDMGVPTLKYHYEGMSVAECGCR
BMP6- VSSASDYNSSELKTACRKHELYVSFQDLGWQDWI IAPKGYAANYCDGECSFPLNAHMNATNHAIVQTL 67
Short VHLMNPEYVPKPCCAPTKLNAISVLYFDDNSNVILKKYRNMWRACGCH
BMP6- VSSASDYNSSELKTACRKHELYVSFQDLGWQDWI IAPKGYAANYCDGECSFPLNAAMNATNHAIVQTL 68
SA VHLMNPEYVPKPCCAPTKLNAISVLYFDDNSNVILKKYRNMWRACGCH
BMP6- VSSASDYNSSELKTACRKHELYVSFQDLGWQDWI IAPKGYAANYCDGECSFPLNAHLNATNHAIVQTL 69
SL VHLMNPEYVPKPCCAPTKLNAISVLYFDDNSNVILKKYRNMWRACGCH
BMP-E- QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCDGECSFPLNAHMNATNHAIVQTLVH 70
NR LMNPEYVPKPCCAPTELSAISMLYLDENEKWLKNYQDMWEGCGCR
BMP- QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWI IAPRGYAAFYCDGECSFPLNAHMNATNHAIVQTLVH 7 1
GER-NR LMNPEYVPKPCCAPTKLRPMSMLYYDDGQNI IKKDIQNMIVEECGCS
BMP-E- QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCDGECSFPLNAHMNATNHAIVQTLVH 72
NR-6 LMNPEYVPKPCCAPTKLNAISVLYFDDNSNVILKKYRNMWRACGCH
BMP- QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWI IAPRGYAAFYCDGECSFPLNAHMNATNHAIVQTLVH 73
GER- LMNPEYVPKPCCAPTKLNAISVLYFDDNSNVILKKYRNMWRACGCH
NR-6
Although the above listed designer BMPs comprise embodiments of the invention, the invention is
not limited in any way to any specific molecules. Instead, the invention encompasses any designer BMP
comprising altered receptor binding where the designer BMP comprises at least one mutation within a
type I I receptor binding domain A, even more preferably, the designer BMP comprises at least one further
mutation within a type I receptor binding domain, most preferably, the designer BMP comprises yet
another at least one further mutation within a type II receptor binding domain B.
In other embodiments, the designer BMP of the present invention comprises an amino acid
sequence at least about 70%, 75% , 80%, 85%, 87% , 90%, 92%, 95%, 96%, 97%, 98%, 99% or identical
to one of the sequences described above. In another embodiment, the designer BMP comprises an
amino acid sequence at least about 70%, 75%, 80%, 85%, 87%, 90%, 92%, 95%, 96% , 97%, 98%, 99%
or identical to the sequence of SEQ ID NOs:8-73.
In yet another embodiment, the designer BMP comprises an amino acid sequence as set forth in
any one of SEQ ID NOs:8-73. In another embodiment, the amino acid sequence of the designer BMP
consists of one of the sequences of SEQ ID NOs:8-73.
Further, in one embodiment, the designer BMP comprises an amino acid sequence at least about
70%, 75%, 80%, 85%, 87%, 90%, 92% , 95%, 96%, 97%, 98%, 99% or identical to the sequence of SEQ
ID NO:1 2 . In another embodiment, the amino acid sequence is the sequence of SEQ ID NO: 12 . In yet
another embodiment, the designer BMP is BMPE.
In an additional embodiment, the designer BMP comprises an amino acid sequence at least
about 70%, 75%, 80%, 85%, 87% , 90%, 92%, 95%, 96%, 97%, 98%, 99% or identical to the sequence of
SEQ ID NO: 14 . In another embodiment, the amino acid sequence is the sequence of SEQ ID NO:1 4 . In
yet another embodiment, the designer BMP is BMPG.
In another embodiment, the designer BMP comprises an amino acid sequence at least about
70%, 75%, 80%, 85%, 87%, 90%, 92% , 95%, 96%, 97%, 98%, 99% or identical to the sequence of SEQ
ID NO:36. In another embodiment, the amino acid sequence is the sequence of SEQ ID NO:36. In yet
another embodiment, the designer BMP is BMPGE.
In another embodiment, the designer BMP comprises an amino acid sequence at least about
70%, 75%, 80%, 85%, 87%, 90%, 92% , 95%, 96%, 97%, 98%, 99% or identical to the sequence of SEQ
ID NO:37. In another embodiment, the amino acid sequence is the sequence of SEQ ID NO:37. In yet
another embodiment, the designer BMP is BMPGER.
A designer BMP of the invention may comprise a fragment of any one of the sequences
described above. In an embodiment, a designer BMP fragment may comprise a fragment of at least an
uninterrupted 20, 22, 24, 25, 26, 27, 28, 30, 32, 33, 34, 35, 36, 37, 38, 40, 4 1, 43, 44, 45, 47, 50, 53, 54,
56, 58, 60, 62, 66, 68, 70, 7 1 , 74, 77, 80, 83, 85, 88, 90, 9 1, 93, 95, 97, 99, 100, 102, 105, 108, 110 , 112 ,
115 , 117, 119 , 120, 12 1, 122, or 125 amino acid sequence from the sequence of any one of the
sequences of SEQ ID NOs:8-73.
It is well known in the art that BMPs are often heterogeneous with respect to the amino and/or
carboxyl termini of the protein. That is, the present invention comprises a designer BMP comprising an
amino acid deletion/truncation at the amino and/or carboxyl terminus comprising a deletion of at least 10
amino acid residues, preferably, 9 amino acid residues, even more preferably, 8 amino acid residues, yet
more preferably, 7 amino acid residues, preferably 6 amino acid residues, even more preferably, 5 amino
acid residues, preferably 4 amino acid residues, more preferably 3 amino acid residues, even more
preferably 2 amino acid residues, and most preferably 1 amino acid reside from the C and or N terminus
of the designer BMP.
In another embodiment, the invention comprises a designer BMP protein comprising an amino
acid sequence of any one of the sequences of SEQ ID NO:8-73 and further comprising a
deletion/truncation from the amino and/or carboxyl termini of the protein. In another embodiment, the
invention comprises a designer BMP protein derived from a BMP protein comprising an amino acid
sequence of any of the sequences of SEQ ID NOs:8-73, wherein the protein comprises an amino acid
deletion/truncation at the amino and/or carboxyl terminus comprising a deletion of at least 10 amino acid
residues, preferably, 9 amino acid residues, even more preferably, 8 amino acid residues, yet more
preferably, 7 amino acid residues, preferably 6 amino acid residues, even more preferably, 5 amino acid
residues, preferably 4 amino acid residues, more preferably 3 amino acid residues, even more preferably
2 amino acid residues, and most preferably 1 amino acid reside from the C and or N terminus of the
designer BMP protein amino acid sequence.
Structural design of BMPs with altered receptor affinity mediated by qlycosylation
The data disclosed herein demonstrate that BMP2 homodimers produced in E. coli (referred to
herein as "E. coli BMP2"), which are not glycosylated, are less active than glycosylated BMP2 produced
in mammalian cells, such as CHO cells (referred to herein as "CHO BMP2"). In addition, data disclosed
herein further demonstrate that E. coli produced BMP6 homodimers are essentially non-functional
compared with BMP6 homodimers produced in mammalian cell culture.
The data disclosed herein demonstrate that there are significant variations in the crystal structure
of E. coli BMP2 compared with CHO BMP2 in the type I receptor binding region.
In one embodiment, the designer BMP comprises an altered conformation mediated by
glycosylation thereby affecting a binding motif that, in turn, mediates altered binding to a type I receptor.
This is based on the present discovery that in mammalian (e.g., CHO) cell produced wild type BMP2, D53
points towards the receptor interface while the H54 points away from the receptor. This is in contrast to
E. coli-produced BMP2 where the D53 residue points away from the receptor interface and the H54
residue lines up toward the receptor, stacking against a proline reside as illustrated in Figure 3 ,
apparently acting as a "doorstop." In addition, the data disclosed herein demonstrate for the first time that
CHO-produced BMP6, which is fully glycosylated and active, also comprises a histidine residue pointing
toward the incoming receptor, i.e. , a histidine "doorstop."
Without wishing to be bound by any particular theory, the data disclosed herein suggest, for the
first time, that moving a "doorstop" residue away from the receptor interface, can mediate increased
binding between the BMP ligand and its receptor. The data further demonstrate that the doorstop residue
may be either mutated itself to remove the doorstop or other residues may be mutated to shift the position
of the doorstop residue. Further, the data disclosed herein further demonstrate that other residues may
be mutated to provide a "glycan tether" which then, in turn, can orient a glycan such that the tethering of
the glycan will reorient the doorstop residue.
Therefore, in some embodiments, a designer BMP can be produced by incorporating at least one
amino acid mutation that affects the glycan tether and/or removes a histidine doorstop structure thereby
providing a designer BMP with altered receptor binding.
In summary, in some embodiments, the designer BMPs of the invention may comprise at least
one mutation in the type I and/or type II binding domains of BMPs that confer altered type I and/or type I I
receptor binding. In one embodiment, the BMP sequence is engineered to alter the receptor affinity of
BMPs in order to alter and improve the receptor binding and/or osteogenic activity of the engineered or
"designer" BMP. In one embodiment, this engineering involves identifying the residues involved in type I
and type I I receptor binding and replacing them to create designer BMP molecules that show, among
other things, higher affinity to both type I and type I I receptors than the parental BMP from which the
designer is derived.
In other embodiments, the designer BMPs of the invention comprise mutations that create a new
arginine "glycan tether" or destroy an existing one to reshape the type I receptor binding domain. That is,
the mutation to an arginine in the position two residues C-terminal from the first cysteine, equivalent to
R 16 of BMP2, appears to cause the glycan chain to be "tethered" onto the BMP surface and
consequently alter the conformation of the pre-helical loop region compared with the wild type BMP that
lacks the mutation. In other embodiments, the designer BMP of the invention may comprise at least one
mutation that alters, creates or destroys (abolishes) the "doorstop" residue that blocks type I receptor from
further engagement with BMP. That is, the mutation of H54 in the designer BMP, or a corresponding
equivalent residue thereof, that is oriented in such a way that it impedes or increases interaction of the
designer BMP with a type I receptor.
In some embodiments, the amino acid mutation affects the conformation of the designer BMP
such that the mutation mediates the creation and or abolishment of an arginine "glycan tether" otherwise
present in the corresponding wild type BMP. In some embodiments, the mutation mediates an altered
conformation which creates or removes/abolishes a histidine doorstop conformation in the designer BMP
where such doorstop conformation is either not present or active, respectively, in the corresponding wild
type BMP.
Therefore, the skilled artisan, once armed with the teachings provided herein, would appreciate
that the presence or absence of an arginine "glycan tether" and/or a histidine "doorstop" in a TGFp
superfamily member may be assessed using any method known in the art for the structural analysis of
proteins, including, but not limited to, the methods exemplified herein. Once the presence of a "doorstop"
residue has been identified, then at least one mutation can be introduced into the molecule to reorient the
histidine away from the receptor binding interface. Alternatively, a mutation can be introduced that will
create or enhance a "glycan tether" such that the inhibitory effect of the histidine "doorstop", if present, is
decreased or, more preferably, eliminated.
In one embodiment, where the TGFp superfamily member is BMP2, the mutation that removes
the histidine doorstop is substitution of another amino acid for H54. In some embodiments, the H54 is
replaced with alanine, glycine, serine, or threonine.
Although the present invention discloses such "doorstop' -removing mutations for BMP2, the
skilled artisan would understand, based on the knowledge in the art, how to identify corresponding
mutations for other TGFp superfamily members and readily produce mutants lacking a "doorstop," i.e.,
removing or reorienting a residue that would otherwise interfere with receptor binding by facing or
projecting into the binding interface. The effects of the mutation on protein conformation can be
determined using any art-recognized method for the structural analysis of proteins such as, but not limited
to, those disclosed herein. Alternatively, mutations that can remove the doorstop and increase ligand
binding to the type I receptor can be identified in silico using computer modeling methods available in the
art. Therefore, the present invention encompasses the design of TGFp superfamily members having
improved binding with the type I receptor in that they lack a histidine "doorstop" residue that would
otherwise be present in the receptor interface.
The present invention further provides the skilled artisan with the understanding of how to identify
mutations for other TGFp family members that would generate or destroy the arginine glycan tether.
Mutations that add the arginine glycan tether to a protein lacking the tether are contemplated by the
instant invention. Therefore, the present invention encompasses the design of TGFp superfamily
members having improved binding with the type I receptor in that they contain an arginine glycan tether
that alters the conformation of the type I receptor binding domain.
In some embodiments, the removal of the histidine doorstop thereby removing the requirement of
a glycan tether, provides a designer BMP that can be produced without glycosylation while maintaining
biological activity. For example, designer BMPs may be produced in cells with glycosylation activity that
differs from mammalian cells or is not present, such as bacterial cells, yeast cells, insect cells, or slime
mold cells. In particular embodiments, the designer BMPs may be produced in E. coli and maintain
biological activity.
Thus, in some embodiments, the invention provides methods for designing and producing BMPs
that can be produced in cells either lacking glycosylation or comprising altered glycosylation such that an
altered glycan is produced which differs from that produced by a mammalian cell. That is, the present
invention encompasses methods for introducing a mutation that removes a doorstop residue that would
otherwise impair or inhibit receptor binding. The skilled artisan would understand once provided with the
teachings of the invention that a doorstop residue that impinges upon the receptor-ligand interface may
be mutated to entirely remove the residue or other mutations can be introduced such that the residue is
oriented away from the interface. Such other mutations include, but are not limited to, providing a glycan
tether that will alter the conformation of a glycan and thereby alter the conformation of the ligand such
that the doorstop residue is orientated away from the binding interface.
Nucleic Acids Encoding Designer BMPs
The invention also includes nucleic acids encoding designer the BMPs described herein. Nucleic
acids encoding the designer BMPs described herein can be prepared according to a wide plethora of
methods known in the art.
In one, nucleic acids encoding designer BMPs are prepared by total gene synthesis, or by sitedirected
mutagenesis of a nucleic acid encoding wild type or modified BMPs. Methods including templatedirected
ligation, recursive PCR, cassette mutagenesis, site-directed mutagenesis or other techniques
that are well known in the art may be utilized (see for example Strizhov et al., Proc. Natl. Acad. Sci. USA
93: 1501 2-1 501 7 ( 1996); Prodromou and Perl, Prot. Eng. 5 : 827-829 ( 1992); Jayaraman and Puccini,
Biotechniques 12 : 392-398 ( 1992); and Chalmers et al. , Biotechniques 30: 249-252 (2001 )).
Thus, embodiments of the present invention can comprise nucleic acid molecules that encode the
designer BMPs of the present invention. In certain embodiments, the invention provides a nucleic acid
molecule that encodes for one of the amino acid sequences of SEQ ID NOs:8 to 66.
In other embodiments, the nucleic acid molecule encodes a designer BMP protein that comprises
an amino acid sequence at least 70%, 75% , 80%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% identical to the amino acid sequence of SEQ ID NO: 12 . In some embodiments, the nucleic
acid molecule encodes a designer BMP protein that comprises the amino acid sequence of SEQ ID
NO:1 2 . In another embodiment, the nucleic acid molecule encodes the amino acid sequence of BMPE as
set forth in Table 8 .
In other embodiments, the nucleic acid molecule encodes a designer BMP protein that comprises
an amino acid sequence at least 70%, 75% , 80%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% identical to the amino acid sequence of SEQ ID NO: 14 . In some embodiments, the nucleic
acid molecule encodes a designer BMP protein that comprises the amino acid sequence of SEQ ID
NO:1 4 . In another embodiment, the nucleic acid molecule encodes the amino acid sequence of BMPG
as set forth in Table 8 .
In other embodiments, the nucleic acid molecule encodes a designer BMP protein that comprises
an amino acid sequence at least 70%, 75% , 80%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% identical to the amino acid sequence of SEQ ID NO:36. In some embodiments, the nucleic
acid molecule encodes a designer BMP protein that comprises the amino acid sequence of SEQ ID
NO:36. In another embodiment, the nucleic acid molecule encodes the amino acid sequence of BMPGE
as set forth in Table 8 .
In other embodiments, the nucleic acid molecule encodes a designer BMP protein that comprises
an amino acid sequence at least 70%, 75% , 80%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% identical to the amino acid sequence of SEQ ID NO:37. In some embodiments, the nucleic
acid molecule encodes a designer BMP protein that comprises the amino acid sequence of SEQ ID
NO:37. In another embodiment, the nucleic acid molecule encodes the amino acid sequence of
BMPGER as set forth in Table 8 .
Exemplary nucleotide sequences encoding designer BMPs are set forth in Table 8 , below. Table
8 shows the name of the protein encoded and the nucleotide sequence encoding that protein. In general,
the mature protein coding sequence begins at nucleotide 847 of the sequences listed below.
TABLE 8
NAME SEQUENCE SEQ ID
NO
BMP-B ATGGTGGCCGGGACCCGCTGTCTTCTAGCGTTGCTGCTTCCCCAGGTCCTCCTGGGCGGCGCGGCTGG 75
CCTCGTTCCGGAGCTGGGCCGCAGGAAGTTCGCGGCGGCGTCGTCGGGCCGCCCCTCATCCCAGCCCT
CTGACGAGGTCCTGAGCGAGTTCGAGTTGCGGCTGCTCAGCATGTTCGGCCTGAAACAGAGACCCACC
CCCAGCAGGGACGCCGTGGTGCCCCCCTACATGCTAGACCTGTATCGCAGGCACTCAGGTCAGCCGGG
CTCACCCGCCCCAGACCACCGGTTGGAGAGGGCAGCCAGCCGAGCCAACACTGTGCGCAGCTTCCACC
ATGAAGAATCTTTGGAAGAACTACCAGAAACGAGTGGGAAAACAACCCGGAGATTCTTCTTTAATTTA
AGTTCTATCCCCACGGAGGAGTTTATCACCTCAGCAGAGCTTCAGGTTTTCCGAGAACAGATGCAAGA
TGCTTTAGGAAACAATAGCAGTTTCCATCACCGAATTAATATTTATGAAATCATAAAACCTGCAACAG
CCAACTCGAAATTCCCCGTGACCAGACTTTTGGACACCAGGTTGGTGAATCAGAATGCAAGCAGGTGG
GAAAGTTTTGATGTCACCCCCGCTGTGATGCGGTGGACTGCACAGGGACACGCCAACCATGGATTCGT
GGTGGAAGTGGCCCACTTGGAGGAGAAACAAGGTGTCTCCAAGAGACATGTTAGGATAAGCAGGTCTT
TGCACCAAGATGAACACAGCTGGTCACAGATAAGGCCATTGCTAGTAACTTTTGGCCATGATGGAAAA
GGGCATCCTCTCCACAAAAGAGAAAAACGTCAAGCCAAACACAAACAGCGGAAACGCCTTAAGTCCAG
CTGTAAGAGACACCCTTTGTACGTGGACTTCAGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCCC
CGGGGTATCACGCCTTTTACTGCCACGGAGAATGCCCTTTTCCTCTGGCTGATCATCTGAACTCCACT
AATCATGCCATTGTTCAGACGTTGGTCAACTCTGTTAACTCTAAGATTCCTAAGGCATGCTGTGTCCC
GACAAAGCTAAATGCCATCTCGGTTCTTTACTTTGATGACAACTCCAATGTCATTTTAAAGAACTATC
AGGACATGGTTGTGGAGGGTTGTGGGTGTCGCTGA
BMP-C ATGGTGGCCGGGACCCGCTGTCTTCTAGCGTTGCTGCTTCCCCAGGTCCTCCTGGGCGGCGCGGCTGG 76
CCTCGTTCCGGAGCTGGGCCGCAGGAAGTTCGCGGCGGCGTCGTCGGGCCGCCCCTCATCCCAGCCCT
CTGACGAGGTCCTGAGCGAGTTCGAGTTGCGGCTGCTCAGCATGTTCGGCCTGAAACAGAGACCCACC
CCCAGCAGGGACGCCGTGGTGCCCCCCTACATGCTAGACCTGTATCGCAGGCACTCAGGTCAGCCGGG
CTCACCCGCCCCAGACCACCGGTTGGAGAGGGCAGCCAGCCGAGCCAACACTGTGCGCAGCTTCCACC
ATGAAGAATCTTTGGAAGAACTACCAGAAACGAGTGGGAAAACAACCCGGAGATTCTTCTTTAATTTA
AGTTCTATCCCCACGGAGGAGTTTATCACCTCAGCAGAGCTTCAGGTTTTCCGAGAACAGATGCAAGA
TGCTTTAGGAAACAATAGCAGTTTCCATCACCGAATTAATATTTATGAAATCATAAAACCTGCAACAG
CCAACTCGAAATTCCCCGTGACCAGACTTTTGGACACCAGGTTGGTGAATCAGAATGCAAGCAGGTGG
GAAAGTTTTGATGTCACCCCCGCTGTGATGCGGTGGACTGCACAGGGACACGCCAACCATGGATTCGT
GGTGGAAGTGGCCCACTTGGAGGAGAAACAAGGTGTCTCCAAGAGACATGTTAGGATAAGCAGGTCTT
TGCACCAAGATGAACACAGCTGGTCACAGATAAGGCCATTGCTAGTAACTTTTGGCCATGATGGAAAA
GGGCATCCTCTCCACAAAAGAGAAAAACGTCAAGCCAAACACAAACAGCGGAAACGCCTTAAGTCCAG
CTGTAAGAGACACCCTTTGTACGTGGACTTCAGTGACGTGGGGTGGAATGACTGGATTATTGCACCCA
AGGGCTATGCTGCCAATTACTGCCACGGAGAATGCCCTTTTCCTCTGGCTGATCATCTGAACTCCACT
AATCATGCCATTGTTCAGACGTTGGTCAACTCTGTTAACTCTAAGATTCCTAAGGCATGCTGTGTCCC
GACAAAGCTAAATGCCATCTCGGTTCTTTACTTTGATGACAACTCCAATGTCATTTTAAAGAACTATC
AGGACATGGTTGTGGAGGGTTGTGGGTGTCGCTGA
BMP-D ATGGTGGCCGGGACCCGCTGTCTTCTAGCGTTGCTGCTTCCCCAGGTCCTCCTGGGCGGCGCGGCTGG 77
CCTCGTTCCGGAGCTGGGCCGCAGGAAGTTCGCGGCGGCGTCGTCGGGCCGCCCCTCATCCCAGCCCT
CTGACGAGGTCCTGAGCGAGTTCGAGTTGCGGCTGCTCAGCATGTTCGGCCTGAAACAGAGACCCACC
CCCAGCAGGGACGCCGTGGTGCCCCCCTACATGCTAGACCTGTATCGCAGGCACTCAGGTCAGCCGGG
CTCACCCGCCCCAGACCACCGGTTGGAGAGGGCAGCCAGCCGAGCCAACACTGTGCGCAGCTTCCACC
ATGAAGAATCTTTGGAAGAACTACCAGAAACGAGTGGGAAAACAACCCGGAGATTCTTCTTTAATTTA
AGTTCTATCCCCACGGAGGAGTTTATCACCTCAGCAGAGCTTCAGGTTTTCCGAGAACAGATGCAAGA
TGCTTTAGGAAACAATAGCAGTTTCCATCACCGAATTAATATTTATGAAATCATAAAACCTGCAACAG
CCAACTCGAAATTCCCCGTGACCAGACTTTTGGACACCAGGTTGGTGAATCAGAATGCAAGCAGGTGG
GAAAGTTTTGATGTCACCCCCGCTGTGATGCGGTGGACTGCACAGGGACACGCCAACCATGGATTCGT
GGTGGAAGTGGCCCACTTGGAGGAGAAACAAGGTGTCTCCAAGAGACATGTTAGGATAAGCAGGTCTT
TGCACCAAGATGAACACAGCTGGTCACAGATAAGGCCATTGCTAGTAACTTTTGGCCATGATGGAAAA
GGGCATCCTCTCCACAAAAGAGAAAAACGTCAAGCCAAACACAAACAGCGGAAACGCCTTAAGTCCAG
CTGTAAGAGACACCCTTTGTACGTGGACTTCAGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCCC
CGGGGTATCACGCCTTTTACTGCCACGGAGAATGCCCTTTTCCACTCAACGCACACATGAATGCAACC
AACCACGCGATTGTGCAGACCTTGGTTCACCTTATGAACTCTAAGATTCCTAAGGCATGCTGTGTCCC
GACAGAACTCAGTGCTATCTCGATGCTGTACCTTGACGAGAATGAAAAGGTTGTATTAAAGAACTATC
AGGACATGGTTGTGGAGGGTTGTGGGTGTCGCTGA
BMP-E ATGGTGGCCGGGACCCGCTGTCTTCTAGCGTTGCTGCTTCCCCAGGTCCTCCTGGGCGGCGCGGCTGG 78
CCTCGTTCCGGAGCTGGGCCGCAGGAAGTTCGCGGCGGCGTCGTCGGGCCGCCCCTCATCCCAGCCCT
CTGACGAGGTCCTGAGCGAGTTCGAGTTGCGGCTGCTCAGCATGTTCGGCCTGAAACAGAGACCCACC
CCCAGCAGGGACGCCGTGGTGCCCCCCTACATGCTAGACCTGTATCGCAGGCACTCAGGTCAGCCGGG
CTCACCCGCCCCAGACCACCGGTTGGAGAGGGCAGCCAGCCGAGCCAACACTGTGCGCAGCTTCCACC
ATGAAGAATCTTTGGAAGAACTACCAGAAACGAGTGGGAAAACAACCCGGAGATTCTTCTTTAATTTA
AGTTCTATCCCCACGGAGGAGTTTATCACCTCAGCAGAGCTTCAGGTTTTCCGAGAACAGATGCAAGA
TGCTTTAGGAAACAATAGCAGTTTCCATCACCGAATTAATATTTATGAAATCATAAAACCTGCAACAG
CCAACTCGAAATTCCCCGTGACCAGACTTTTGGACACCAGGTTGGTGAATCAGAATGCAAGCAGGTGG
GAAAGTTTTGATGTCACCCCCGCTGTGATGCGGTGGACTGCACAGGGACACGCCAACCATGGATTCGT
GGTGGAAGTGGCCCACTTGGAGGAGAAACAAGGTGTCTCCAAGAGACATGTTAGGATAAGCAGGTCTT
TGCACCAAGATGAACACAGCTGGTCACAGATAAGGCCATTGCTAGTAACTTTTGGCCATGATGGAAAA
GGGCATCCTCTCCACAAAAGAGAAAAACGTCAAGCCAAACACAAACAGCGGAAACGCCTTAAGTCCAG
CTGTAAGAGACACCCTTTGTACGTGGACTTCAGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCCC
NAME SEQUENCE SEQ ID
NO
CGGGGTATCACGCCTTTTACTGCGATGGAGAATGCTCCTTCCCACTCAACGCACACATGAATGCAACC
AACCACGCGATTGTGCAGACCTTGGTTCACCTTATGAACCCCGAGTATGTCCCCAAACCGTGCTGTGC
GCCGACAGAACTCAGTGCTATCTCGATGCTGTACCTTGACGAGAATGAAAAGGTTGTATTAAAGAACT
ATCAGGACATGGTTGTGGAGGGTTGTGGGTGTCGCTGA
BMP-F ATGGTGGCCGGGACCCGCTGTCTTCTAGCGTTGCTGCTTCCCCAGGTCCTCCTGGGCGGCGCGGCTGG 79

CLAIMS
1. A designer BMP protein comprising at least one mutation in at least one type I or type I I
receptor binding domain, wherein the mutation confers altered binding to the type I or type I I
BMP receptor compared with the binding to the type I or type I I receptor by a corresponding
wild type BMP.
2 . The protein of claim 1, wherein the protein is selected from the group consisting of BMP2,
BMP4, BMP5, BMP6, BMP7, BMP8 and BMP9.
3 . The protein of claim 1, comprising at least one mutation within:
a . the type I I binding domain A;
b. the type II binding domain B;
c . the type I binding domain; and
d . any combination of the above.
4 . A designer osteogenic protein comprising an amino acid sequence comprising at least
one mutation in at least one type I or type I I receptor binding domain, wherein the mutation confers
altered binding to the type I or type I I BMP receptor compared with the binding to the type I or type I I
receptor by wild type BMP2.
5 . The protein of claim 4 , wherein the mutation is selected from the group consisting of:
a . a mutation within the type I I binding domain A wherein said mutation is at least one
mutation selected from the group consisting of a mutation at V33, P36, H39, and
F41 with respect to the sequence of SEQ ID NO:1 ;
b. a mutation within the type I I binding domain A wherein said mutation is at least one
mutation selected from the group consisting of V33I, P36K, P36R, H39A, and F41 N
with respect to SEQ ID NO:1 ;
c . a mutation within the type I I binding domain B wherein said mutation is at least one
mutation selected from the group consisting of a mutation at E83, S85, M89, L92,
E94, E96, K97, and V99 with respect to the sequence of SEQ ID NO: 1;
d . a mutation within the type I I binding domain B wherein said mutation is at least one
mutation selected from the group consisting of E83K, S85N, M89V, L92F, E94D,
E96S, K97N, and V99I with respect to of SEQ ID NO:1 ;
e. a mutation within the type I binding domain wherein said mutation is at least one
mutation selected from the group consisting of a mutation at H44, P48, A52, D53,
L55, S57, N68, S69, V70, an insertion of a single amino acid after N71 , S72, K73,
I74, A77, and V80 with respect to the sequence of SEQ ID NO:1 ; and
f . a mutation within the type I binding domain wherein said mutation is at least one
mutation selected from the group consisting of H44D, P48S, A52N, D53A, L55M,
S57A, N68H, S69L, V70M, insertion of P after N71 , S72E, K73Y, I74V, A77P, and
V80A with respect to the sequence of SEQ ID NO:1 .
6. The protein of claim 4 , wherein said protein comprises mutations selected from the group
consisting of:
a . a mutation at each of amino acids H44, P48, A52, D53, L55, S57, N68, S69, V70,
insertion of a single amino acid after N71 , S72, K73, I74, A77, and V80 with respect
to the sequence of SEQ ID NO:1 ;
b. the protein of (a) wherein the mutations are H44D, P48S, A52N, D53A, L55M,
S57A, N68H, S69L, V70M, insertion of a P after N71 , S72E, K73Y, I74V, A77P, and
V80A;
c . a mutation at each of amino acids V33, P36, H39, S85, M89, L92, E94, E96, K97,
and V99 with respect to the sequence of SEQ ID NO:1 ;
d . the protein of (c) wherein the mutations are V33I, P36K, H39A, S85N, M89, L92F,
E94D, E96S, K97N , and V99I;
e. a mutation at each of amino acids V33, P36, H39, H44, P48, A52, D53, L55, S57,
N68, S69, V70, insertion of a single amino acid after N71 , S72, K73, I74, A77, and
V80, S85, M89, L92, E94, E96, K97, and V99 with respect to the sequence of SEQ
ID NO:1 ; and
f . the protein of (e) wherein the mutations are V33I, P36K, H39A, H44D, P48S, A52N,
D53A, L55M, S57A, N68H , S69L, V70M, insertion of a P after N71 , S72E, K73Y,
I74V, A77P, and V80A, S85N, M89, L92F, E94D, E96S, K97N , and V99I; and
g . the protein of (e) wherein the mutations are V33I , P36R, H39A, H44D, P48S, A52N,
D53A, L55M, S57A, N68H , S69L, V70M, insertion of a P after N71 , S72E, K73Y,
I74V, A77P, and V80A, S85N, M89, L92F, E94D, E96S, K97N , and V99I.
7. The protein of claim 1, wherein the protein binds the ALK2 receptor with a K not greater
than about 2 nM, the protein binds the ALK3 receptor with a K not greater than about 2 nM,
the protein binds the ALK6 receptor with a K not greater than about 1 nM, the protein binds the
ActRMA receptor with a K not greater than about 2 nM, the protein binds the ActRMB receptor
with a K not greater than about 0.5 nM, and protein binds the BMPRMA receptor with a K not
greater than about 3.5 nM.
8 . The protein of claim 1, further comprising 1, 2 , 3 , 4 , 5, 6, 7, 8 , 9 , or 10 amino acid
mutations not located within the type I or the type I I binding regions.
9 . A designer osteogenic protein selected from a protein comprising the amino acid
sequence of any one of SEQ ID NOs:8-73.
10 . A designer osteogenic protein comprising the amino acid sequence of SEQ ID NO:1 2 .
11. A designer osteogenic protein comprising the amino acid sequence of SEQ ID NO:1 4 .
12 . A designer osteogenic protein comprising the amino acid sequence of SEQ ID NO:36.
13. A designer osteogenic protein comprising the amino acid sequence of SEQ ID NO:37.
14 . A method of producing the protein of claim 1, the method comprising introducing a
nucleic acid encoding the protein into a host cell, culturing the cell under conditions where the protein is
produced, and purifying the protein.
15. The method of claim 14 , wherein said nucleic acid comprises a sequence selected from
the group consisting of the nucleic acid sequence of any one of SEQ ID NOs:74-1 39.
16. A designer BMP6 protein comprising an amino acid sequence comprising at least one
mutation in at least one type I or type I I receptor binding domain, wherein the mutation confers altered
binding to the type I or type I I BMP receptor compared with the binding to the type I or type I I receptor by
wild type BMP6.
17. The protein of claim 16, wherein the mutation is selected from the group consisting of:
a . a mutation within the type I I binding domain A wherein said mutation is at least one
mutation selected from the group consisting of a mutation at I57, K60, G61 , A63, N65,
Y66, and D68 with respect to the sequence of SEQ ID NO:4;
b. a mutation within the type I I binding domain B wherein said mutation is at least one
mutation selected from the group consisting of K 108, N 110 , A 111, V 114 , F 117, D 119 ,
N 120, S 12 1, N 122, V 123, and 11 24 with respect to the sequence of SEQ ID NO:4;
c . a mutation within the type I binding domain wherein said mutation is at least one mutation
selected from the group consisting of a mutation at S72, N76, A77, H78, M79, N80, A81 ,
N83, V87, T89, H92, L93, M94, N95, P96, E97, Y98, V99, and P 100 with respect to the
sequence of SEQ ID NO:4;
d. a mutation at each of amino acid residues I57, K60, G61 , A63, N65, Y66, and D68 with
respect to the sequence of SEQ ID NO:4;
e. a mutation at each of amino acid residues K 108, N 110 , A 111, V 114 , F 1 17, D 119 , N 120,
S 12 1, N122, V 123, and 1124 with respect to the amino acid sequence of SEQ ID NO:4;
and
f . a mutation at each of amino acid residues S72, N76, A77, H78, M79, N80, A81 , N83,
V87, T89, H92, L93, M94, N95, P96, E97, Y98, V99, or P 100 with respect to the amino
acid sequence of SEQ ID NO:4.
18 . The protein of claim 16, further comprising 1, 2 , 3, 4 , 5, 6, 7 , 8 , 9 , or 10 amino acid
mutations not located within the type I or the type I I binding domains.
19 . An isolated nucleic acid molecule comprising a nucleotide sequence encoding an amino
acid sequence selected from the group consisting of SEQ ID NOs:8 to 73.
20. The nucleic acid of claim 19 , said nucleic acid encoding a protein comprising an amino
acid sequence selected from the group consisting of the sequence of SEQ ID NO:1 2 , SEQ ID NO:1 4 ,
SEQ ID NO:36 and SEQ ID NO:37.
2 1. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the
group consisting of SEQ ID NOs:74 to 139.
22. The nucleic acid of claim 2 1, said nucleic acid comprising a nucleotide sequence
selected from the group consisting of the sequence of SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:1 02,
and SEQ ID NO: 103.
23. A method of producing the protein of claim 16 , the method comprising introducing a
nucleic acid encoding said protein into a host cell, culturing said cell under conditions where said protein
is produced , and purifying said protein.
24. A method of treating a bone disease associated with bone loss in a patient in need
thereof, the method comprising administering an effective amount of a designer osteogenic protein of
claim 1 to said patient, thereby treating bone disease in said patient.
25. A method of treating fibrosis in a patient in need thereof, the method comprising
administering an effective amount of a designer osteogenic protein of claim 1 to said patient, thereby
treating fibrosis.
26. A method of inducing bone formation in a tissue, said method comprising contacting said
tissue with a designer osteogenic protein of claim 1, thereby inducing bone formation in said tissue.