The term “antigen-binding portion” of an antibody (or simply “antibody portion”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a RANKL protein). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment which may comprise two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; (vi) an isolated complementarity determining region (CDR); and (viii) a nanobody, a heavy chain variable region containing a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

An “isolated antibody”, as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds a RANKL protein is substantially free of antibodies that specifically bind antigens other than RANKL proteins). An isolated antibody that specifically binds a human RANKL protein may, however, have cross-reactivity to other antigens, such as RANKL proteins from other species. Moreover, an isolated antibody can be substantially free of other cellular material and/or chemicals.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translation modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including, for example, the hybridoma method.

The term “mouse antibody”, as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from mouse germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from mouse germline immunoglobulin sequences. The mouse antibodies of the disclosure can include amino acid residues not encoded by mouse germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “mouse antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species have been grafted onto mouse framework sequences.

The term “chimeric antibody” refers to an antibody made by combining genetic material from a nonhuman source with genetic material from a human being. Or more generally, a chimeric antibody is an antibody having genetic material from a certain species with genetic material from another species.

The term “humanized antibody”, as used herein, refers to an antibody from non-human species whose protein sequences have been modified to increase similarity to antibody variants produced naturally in humans.

The term “isotype” refers to the antibody class (e.g., IgM or IgG1) that is encoded by the heavy chain constant region genes.

The phrases “an antibody recognizing an antigen” and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen.”

As used herein, an antibody that “specifically binds to human RANKL” is intended to refer to an antibody that binds to human RANKL protein (and possibly a RANKL protein from one or more non-human species) but does not substantially bind to non-RANKL proteins. Preferably, the antibody binds to human RANKL protein with “high affinity”, namely with a K_Dof 5.0×10⁻⁸M or less, more preferably 1.0×10⁻⁸M or less, and more preferably 3.0×10⁻⁹M or less.

The term “does not substantially bind” to a protein or cells, as used herein, means does not bind or does not bind with a high affinity to the protein or cells, i.e. binds to the protein or cells with a K_Dof 1.0×10⁻⁶M or more, more preferably 1.0×10⁻⁵M or more, more preferably 1.0×10⁻⁴M or more, more preferably 1.0×10⁻³M or more, even more preferably 1.0×10⁻²M or more.

The term “high affinity” for an IgG antibody refers to an antibody having a K_Dof 1.0×10⁻⁶M or less, more preferably 5.0×10⁻⁸M or less, even more preferably 1.0×10⁻⁸M or less, and even more preferably 3.0×10⁻⁹M or less and even more preferably 1.0×10⁻⁹M or less for a target antigen. However, “high affinity” binding can vary for other antibody isotypes. For example, “high affinity” binding for an IgM isotype refers to an antibody having a K_Dof 10⁻⁶M or less, more preferably 10⁻⁷M or less, even more preferably 10⁻⁸M or less.

The term “K_assoc” or “K_a”, as used herein, is intended to refer to the association rate of a particular antibody-antigen interaction, whereas the term “K_dis” or “K_d”, as used herein, is intended to refer to the dissociation rate of a particular antibody-antigen interaction. The term “K_D”, as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of K_dto K_a(i.e., K_d/K_a) and is expressed as a molar concentration (M). K_Dvalues for antibodies can be determined using methods well established in the art. A preferred method for determining the K_Dof an antibody is by using surface plasmon resonance, preferably using a biosensor system such as a Biacore™ system.

The term “EC₅₀”, also known as half maximal effective concentration, refers to the concentration of an antibody which induces a response halfway between the baseline and maximum after a specified exposure time.

The term “IC₅₀”, also known as half maximal inhibitory concentration, refers to the concentration of an antibody which inhibits a specific biological or biochemical function by 50% relative to the absence of the antibody.

The term “subject” includes any human or nonhuman animal. The term “nonhuman animal” includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals are preferred, such as non-human primates, sheep, dogs, cats, cows and horses.

The term “therapeutically effective amount” means an amount of the antibody of the present disclosure sufficient to prevent or ameliorate the symptoms associated with a disease or condition (such as a cancer) and/or lessen the severity of the disease or condition. A therapeutically effective amount is understood to be in context to the condition being treated, where the actual effective amount is readily discerned by those of skill in the art.

Various aspects of the disclosure are described in further detail in the following subsections.

Anti-RANKL Antibodies Having Increased Binding Affinity to Human RANKL and Better Blocking Activity on RANKL-RANK Interaction

The antibody, or the antigen-binding portion thereof, of the disclosure specifically binds to human RANKL with comparable, if not better, binding affinity/capacity as compared to previously described anti-RANKL antibodies, such as XGEVA® Denosumab.

The antibody, or antigen-binding portion thereof, of the present disclosure has comparable, if not higher, blocking capacity on RANK-RANKL interaction than prior art anti-RANKL antibodies such as XGEVA® Denosumab.

Preferred antibodies of the disclosure are humanized monoclonal antibodies. Additionally or alternatively, the antibodies can be, for example, chimeric monoclonal antibodies.

Monoclonal Anti-RANKL Antibody

The antibody or antigen-binding portion of the disclosure is structurally and chemically characterized below and in the following Examples. The amino acid sequence ID numbers of the heavy/light chain variable regions of the antibodies are summarized in Table 1 below, some antibodies sharing the same V_Hor V_L. The heavy chain constant region for the antibodies may be human IgG2 heavy chain constant region having an amino acid sequence set forth in, e.g., SEQ ID NO.:35 or human IgG4 heavy chain constant region having an amino acid sequence of SEQ ID NO.:36, and the light chain constant region for the antibodies may be human kappa constant region having an amino acid sequence set forth in, e.g., SEQ ID NO.:37. These antibodies may also contain mouse heavy/light chain constant regions as needed.

The heavy chain variable region CDRs and the light chain variable region CDRs in Table 1 have been defined by the Kabat numbering system. However, as is well known in the art, CDR regions can also be determined by other systems such as Chothia, and IMGT, AbM, or Contact numbering system/method, based on heavy chain/light chain variable region sequences.

The V_Hand V_Lsequences (or CDR sequences) of other anti-RANKL antibodies which bind to human RANKL can be “mixed and matched” with the V_Hand V_Lsequences (or CDR sequences) of the anti-RANKL antibody of the present disclosure. Preferably, when V_Hand V_Lchains (or the CDRs within such chains) are mixed and matched, a V_Hsequence from a particular V_H/V_Lpairing is replaced with a structurally similar V_Hsequence. Likewise, preferably a V_Lsequence from a particular V_H/V_Lpairing is replaced with a structurally similar V_Lsequence.

TABLE 1

Amino acid sequence ID numbers of heavy/light chain variable regions and CDRs

mAb ID
V_HCDR1
V_HCDR2
V_HCDR3
V_H

D1A1
SEQ ID NO.: 1
SEQ ID NO.: 5
SEQ ID NO.: 10
SEQ ID NO.: 25

huD1A1-V1
SEQ ID NO.: 1
SEQ ID NO.: 5
SEQ ID NO.: 10
SEQ ID NO.: 26, X1 = A,

X2 = R, X3 = T

huD1A1-V2
SEQ ID NO.: 1
SEQ ID NO.: 5
SEQ ID NO.: 10
SEQ ID NO.: 26, X1 = S,

X2 = K, X3 = K

huD1A1-V3
SEQ ID NO.: 1
SEQ ID NO.: 5
SEQ ID NO.: 10
SEQ ID NO.: 26, X1 = A,

X2 = K, X3 = K

huD1A1-V4
SEQ ID NO.: 1
SEQ ID NO.: 5
SEQ ID NO.: 10
SEQ ID NO.: 26, X1 = A,

X2 = R, X3 = T

huD1A1-V5
SEQ ID NO.: 1
SEQ ID NO.: 5
SEQ ID NO.: 10
SEQ ID NO.: 26, X1 = S,

X2 = K, X3 = K

huD1A1-V6
SEQ ID NO.: 1
SEQ ID NO.: 5
SEQ ID NO.: 10
SEQ ID NO.: 26, X1 = A,

X2 = K, X3 = K

B1A8
SEQ ID NO.: 2
SEQ ID NO.: 6
SEQ ID NO.: 11
SEQ ID NO.: 27, X1 = P,

X2 = Q

B3D8
SEQ ID NO.: 2
SEQ ID NO.: 7
SEQ ID NO.: 11
SEQ ID NO.: 27, X1 = S,

X2 = K

D1E3
SEQ ID NO.: 3
SEQ ID NO.: 8
SEQ ID NO.: 12
SEQ ID NO.: 28

B4H5
SEQ ID NO.: 4
SEQ ID NO.: 9
SEQ ID NO.: 13
SEQ ID NO.: 29

mAb ID
V_LCDR1
V_LCDR2
V_LCDR3
V_L

D1A1
SEQ ID NO.: 14
SEQ ID NO.: 18
SEQ ID NO.: 21
SEQ ID NO.: 30

huD1A1-V1
SEQ ID NO.: 14
SEQ ID NO.: 18
SEQ ID NO.: 21
SEQ ID NO.: 31, X1 = A,

X2 = I

huD1A1-V2
SEQ ID NO.: 14
SEQ ID NO.: 18
SEQ ID NO.: 21
SEQ ID NO.: 31, X1 = A,

X2 = I

huD1A1-V3
SEQ ID NO.: 14
SEQ ID NO.: 18
SEQ ID NO.: 21
SEQ ID NO.: 31, X1 = A,

X2 = I

huD1A1-V4
SEQ ID NO.: 14
SEQ ID NO.: 18
SEQ ID NO.: 21
SEQ ID NO.: 31, X1 = S,

X2 = L

huD1A1-V5
SEQ ID NO.: 14
SEQ ID NO.: 18
SEQ ID NO.: 21
SEQ ID NO.: 31, X1 = S,

X2 = L

huD1A1-V6
SEQ ID NO.: 14
SEQ ID NO.: 18
SEQ ID NO.: 21
SEQ ID NO.: 31, X1 = S,

X2 = L

B1A8
SEQ ID NO.: 15
SEQ ID NO.: 19
SEQ ID NO.: 22
SEQ ID NO.: 32

B3D8
SEQ ID NO.: 15
SEQ ID NO.: 19
SEQ ID NO.: 22
SEQ ID NO.: 32

D1E3
SEQ ID NO.: 16
SEQ ID NO.: 18
SEQ ID NO.: 23
SEQ ID NO.: 33

B4H5
SEQ ID NO.: 17
SEQ ID NO.: 20
SEQ ID NO.: 24
SEQ ID NO.: 34

Accordingly, in one embodiment, an antibody of the disclosure, or an antigen binding portion thereof, comprises:

- (a) a heavy chain variable region comprising an amino acid sequence listed above in Table 1; and
- (b) a light chain variable region comprising an amino acid sequence listed above in Table 1, or the V_Lof another anti-RANKL antibody, wherein the antibody specifically binds human RANKL.

In another embodiment, an antibody of the disclosure, or an antigen binding portion thereof, comprises:

- (a) the CDR1, CDR2, and CDR3 regions of the heavy chain variable region listed above in Table 1; and
- (b) the CDR1, CDR2, and CDR3 regions of the light chain variable region listed above in Table 1 or the CDRs of another anti-RANKL antibody, wherein the antibody specifically binds human RANKL.

In yet another embodiment, the antibody, or antigen binding portion thereof, includes the heavy chain variable CDR2 region of anti-RANKL antibody combined with CDRs of other antibodies which bind human RANKL, e.g., CDR1 and/or CDR3 from the heavy chain variable region, and/or CDR1, CDR2, and/or CDR3 from the light chain variable region of a different anti-RANKL antibody.

In addition, it is well known in the art that the CDR3 domain, independently from the CDR1 and/or CDR2 domain(s), alone can determine the binding specificity of an antibody for a cognate antigen and that multiple antibodies can predictably be generated having the same binding specificity based on a common CDR3 sequence. See, e.g., Klimka et al., British J. of Cancer 83(2):252-260 (2000); Beiboer et al., J. Mol. Biol. 296:833-849 (2000); Rader et al., Proc. Natl. Acad. Sci. U.S.A. 95:8910-8915 (1998); Barbas et al., J. Am. Chem. Soc. 116:2161-2162 (1994); Barbas et al., Proc. Natl. Acad. Sci. U.S.A. 92:2529-2533 (1995); Ditzel et al., J. Immunol. 157:739-749 (1996); Berezov et al., BIAjournal 8: Scientific Review 8 (2001); Igarashi et al., J. Biochem (Tokyo) 117:452-7 (1995); Bourgeois et al., J. Virol 72:807-10 (1998); Levi et al., Proc. Natl. Acad. Sci. U.S.A. 90:4374-8 (1993); Polymenis and Stoller, J. Immunol. 152:5218-5329 (1994) and Xu and Davis, Immunity 13:37-45 (2000). See also, U.S. Pat. Nos. 6,951,646; 6,914,128; 6,090,382; 6,818,216; 6,156,313; 6,827,925; 5,833,943; 5,762,905 and 5,760,185. Each of these references is hereby incorporated by reference in its entirety.

Accordingly, in another embodiment, antibodies of the disclosure comprise the CDR2 of the heavy chain variable region of the anti-RANKL antibody and at least the CDR3 of the heavy and/or light chain variable region of the anti-RANKL antibody, or the CDR3 of the heavy and/or light chain variable region of another anti-RANKL antibody, wherein the antibody is capable of specifically binding to human RANKL. These antibodies preferably (a) compete for binding with RANKL; (b) retain the functional characteristics; (c) bind to the same epitope; and/or (d) have a similar binding affinity as the anti-RANKL antibody of the present disclosure. In yet another embodiment, the antibodies further may comprise the CDR2 of the light chain variable region of the anti-RANKL antibody, or the CDR2 of the light chain variable region of another anti-RANKL antibody, wherein the antibody is capable of specifically binding to human RANKL. In another embodiment, the antibodies of the disclosure may include the CDR1 of the heavy and/or light chain variable region of the anti-RANKL antibody, or the CDR1 of the heavy and/or light chain variable region of another anti-RANKL antibody, wherein the antibody is capable of specifically binding to human RANKL.

Conservative Modifications

In another embodiment, an antibody of the disclosure comprises a heavy and/or light chain variable region sequences of CDR1, CDR2 and CDR3 sequences which differ from those of the anti-RANKL antibodies of the present disclosure by one or more conservative modifications. It is understood in the art that certain conservative sequence modification can be made which do not remove antigen binding. See, e.g., Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng. 10:835-41; Komissarov et al., (1997) J. Biol. Chem. 272:26864-26870; Hall et al., (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al., (1998) Int. Immunol. 10:341-6 and Beers et al., (2000) Clin. Can. Res. 6:2835-43.

Accordingly, in one embodiment, the antibody comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and/or a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein:

- (a) the heavy chain variable region CDR1 sequence comprises a sequence listed in Table 1 above, and/or conservative modifications thereof, and/or
- (b) the heavy chain variable region CDR2 sequence comprises a sequence listed in Table 1 above, and/or conservative modifications thereof, and/or
- (c) the heavy chain variable region CDR3 sequence comprises a sequence listed in Table 1 above, and conservative modifications thereof; and/or
- (d) the light chain variable region CDR1, and/or CDR2, and/or CDR3 sequences comprise the sequence(s) listed in Table 1 above; and/or conservative modifications thereof; and
- (e) the antibody specifically binds human RANKL.

The antibody of the present disclosure possesses one or more of the following functional properties described above, such as high affinity binding to human RANKL, and the ability to induce weak or no ADCC or CDC against RANKL-expressing cells.

In various embodiments, the antibody can be, for example, a mouse, human, humanized or chimeric antibody.

As used herein, the term “conservative sequence modifications” is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody of the disclosure can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function (i.e., the functions set forth above) using the functional assays described herein.

Engineered and Modified Antibodies

Antibodies of the disclosure can be prepared using an antibody having one or more of the V_H/V_Lsequences of the anti-RANKL antibody of the present disclosure as starting material to engineer a modified antibody. An antibody can be engineered by modifying one or more residues within one or both variable regions (i.e., V_Hand/or V_L), for example within one or more CDR regions and/or within one or more framework regions. Additionally or alternatively, an antibody can be engineered by modifying residues within the constant region(s), for example to alter the effector function(s) of the antibody.

In certain embodiments, CDR grafting can be used to engineer variable regions of antibodies. Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann et al., (1998) Nature 332:323-327; Jones et al., (1986) Nature 321:522-525; Queen et al., (1989) Proc. Natl. Acad. See also U.S.A. 86:10029-10033; U.S. Pat. Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

Accordingly, another embodiment of the disclosure pertains to an isolated monoclonal antibody, or antigen binding portion thereof, comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising the sequences of the present disclosure, as described above, and/or a light chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising the sequences of the present disclosure, as described above. While these antibodies contain the V_Hand V_LCDR sequences of the monoclonal antibody of the present disclosure, they can contain different framework sequences.

Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the “VBase” human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase), as well as in Kabat et al., (1991), cited supra; Tomlinson et al., (1992) J. Mol. Biol. 227:776-798; and Cox et al., (1994) Eur. J. Immunol. 24:827-836; the contents of each of which are expressly incorporated herein by reference. As another example, the germline DNA sequences for human heavy and light chain variable region genes can be found in the Genbank database. For example, the following heavy chain germline sequences found in the HCo7 HuMAb mouse are available in the accompanying Genbank Accession Nos.: 1-69 (NG-0010109, NT-024637 & BC070333), 3-33 (NG-0010109 & NT-024637) and 3-7 (NG-0010109 & NT-024637). As another example, the following heavy chain germline sequences found in the HCo12 HuMAb mouse are available in the accompanying Genbank Accession Nos.: 1-69 (NG-0010109, NT-024637 & BC070333), 5-51 (NG-0010109 & NT-024637), 4-34 (NG-0010109 & NT-024637), 3-30.3 (CAJ556644) & 3-23 (AJ406678).

Antibody protein sequences are compared against a compiled protein sequence database using one of the sequence similarity searching methods called the Gapped BLAST (Altschul et al., (1997), supra), which is well known to those skilled in the art.

Preferred framework sequences for use in the antibodies of the disclosure are those that are structurally similar to the framework sequences used by antibodies of the disclosure. The V_HCDR1, CDR2, and CDR3 sequences can be grafted onto framework regions that have the identical sequence as that found in the germline immunoglobulin gene from which the framework sequence derives, or the CDR sequences can be grafted onto framework regions that contain one or more mutations as compared to the germline sequences. For example, it has been found that in certain instances it is beneficial to mutate residues within the framework regions to maintain or enhance the antigen binding ability of the antibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

Another type of variable region modification is to mutate amino acid residues within the V_Hand/or V_LCDR1, CDR2 and/or CDR3 regions to thereby improve one or more binding properties (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s) and the effect on antibody binding, or other functional property of interest, can be evaluated in in vitro or in vivo assays as known in the art. Preferably conservative modifications (as known in the art) are introduced. The mutations can be amino acid substitutions, additions or deletions, but are preferably substitutions. Moreover, typically no more than one, two, three, four or five residues within a CDR region are altered.

Accordingly, in another embodiment, the disclosure provides isolated anti-RANKL monoclonal antibodies, or antigen binding portions thereof, comprising a heavy chain variable region comprising: (a) a V_HCDR1 region comprising the sequence of the present disclosure, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; (b) a V_HCDR2 region comprising the sequence of the present disclosure, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; (c) a V_HCDR3 region comprising the sequence of the present disclosure, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; (d) a V_LCDR1 region comprising the sequence of the present disclosure, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; (e) a V_LCDR2 region comprising the sequence of the present disclosure, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; and (f) a V_LCDR3 region comprising the sequence of the present disclosure, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions.

Engineered antibodies of the disclosure include those in which modifications have been made to framework residues within V_Hand/or V_L, e.g. to improve the properties of the antibody. Typically, such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to “backmutate” one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation can contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived.

Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as “deimmunization” and is described in further detail in U.S. Patent Publication No. 20030153043.

In addition, or as an alternative to modifications made within the framework or CDR regions, antibodies of the disclosure can be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. Furthermore, an antibody of the disclosure can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody.

In one embodiment, the hinge region of C_H1is modified in such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is described further in U.S. Pat. No. 5,677,425. The number of cysteine residues in the hinge region of C_H1is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

In another embodiment, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the C_H2-C_H3domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcal protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Pat. No. 6,165,745.

In still another embodiment, the glycosylation of an antibody is modified. For example, a glycosylated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such a glycosylation may increase the affinity of the antibody for antigen. See, e.g., U.S. Pat. Nos. 5,714,350 and 6,350,861.

Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the disclosure to thereby produce an antibody with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (α (1,6)-fucosyltransferase), such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8−/− cell lines were created by the targeted disruption of the FUT8 gene in CHO/DG44 cells using two replacement vectors (see U.S. Patent Publication No. 20040110704 and Yamane-Ohnuki et al., (2004) Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating the α-1,6 bond-related enzyme. EP 1,176,195 also describes cell lines which have a low enzyme activity for adding fucose to the N-acetylglucosamine that binds to the Fc region of the antibody or does not have the enzyme activity, for example the rat myeloma cell line YB2/0 (ATCC CRL 1662). PCT Publication WO 03/035835 describes a variant CHO cell line, Lec13 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). Antibodies with a modified glycosylation profile can also be produced in chicken eggs, as described in PCT Publication WO 06/089231. Alternatively, antibodies with a modified glycosylation profile can be produced in plant cells, such as Lemna. Methods for production of antibodies in a plant system are disclosed in the U.S. patent application corresponding to Alston & Bird LLP, filed on Aug. 11, 2006. PCT Publication WO 99/54342 describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al., (1999) Nat. Biotech. 17:176-180). Alternatively, the fucose residues of the antibody can be cleaved off using a fucosidase enzyme; e.g., the fucosidase α-L-fucosidase removes fucosyl residues from antibodies (Tarentino et al., (1975) Biochem. 14:5516-23).

Another modification of the antibodies herein that is contemplated by this disclosure is pegylation. An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term “polyethylene glycol” is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C₁-C₁₀) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the disclosure. See, e.g., EPO 154 316 and EP 0 401 384.

Antibody's Physical Properties

Antibodies of the disclosure can be characterized by their various physical properties, to detect and/or differentiate different classes thereof.

For example, antibodies can contain one or more glycosylation sites in either the light or heavy chain variable region. Such glycosylation sites may result in increased immunogenicity of the antibody or an alteration of the pK of the antibody due to altered antigen binding (Marshall et al (1972) Annu Rev Biochem 41:673-702; Gala and Morrison (2004) J Immunol 172:5489-94; Wallick et al (1988) J Exp Med 168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al (1985) Nature 316:452-7; Mimura et al., (2000) Mol Immunol 37:697-706). Glycosylation has been known to occur at motifs containing an N-X-S/T sequence. In some instances, it is preferred to have an anti-RANKL antibody that does not contain variable region glycosylation. This can be achieved either by selecting antibodies that do not contain the glycosylation motif in the variable region or by mutating residues within the glycosylation region.

In a preferred embodiment, the antibodies do not contain asparagine isomerism sites. The deamidation of asparagine may occur on N-G or D-G sequences and result in the creation of an isoaspartic acid residue that introduces a link into the polypeptide chain and decreases its stability (isoaspartic acid effect).

Each antibody will have a unique isoelectric point (pI), which generally falls in the pH range between 6 and 9.5. The pI for an IgG1 antibody typically falls within the pH range of 7-9.5 and the pI for an IgG4 antibody typically falls within the pH range of 6-8. There is speculation that antibodies with a pI outside the normal range may have some unfolding and instability under in vivo conditions. Thus, it is preferred to have an anti-RANKL antibody that contains a pI value that falls in the normal range. This can be achieved either by selecting antibodies with a pI in the normal range or by mutating charged surface residues.

Nucleic Acid Molecules Encoding Antibodies of the Disclosure

In another aspect, the disclosure provides nucleic acid molecules that encode heavy and/or light chain variable regions, or CDRs, of the antibodies of the disclosure. The nucleic acids can be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is “isolated” or “rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques. A nucleic acid of the disclosure can be, e.g., DNA or RNA and may or may not contain intronic sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

Nucleic acids of the disclosure can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described further below), cDNAs encoding the light and heavy chains of the antibody made by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), a nucleic acid encoding such antibodies can be recovered from the gene library.

Preferred nucleic acids molecules of the disclosure include those encoding the V_Hand V_Lsequences of the SLAMF7 monoclonal antibody or the CDRs. Once DNA fragments encoding V_Hand V_Lsegments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene. In these manipulations, a V_L- or V_H-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term “operatively linked”, as used in this context, is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the V_Hregion can be converted to a full-length heavy chain gene by operatively linking the V_H-encoding DNA to another DNA molecule encoding heavy chain constant regions (C_H1, C_H2and C_H3). The sequences of human heavy chain constant region genes are known in the art and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably is an IgG1 or IgG4 constant region. For a Fab fragment heavy chain gene, the V_H-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain C_H1constant region.

The isolated DNA encoding the V_Lregion can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the V_L-encoding DNA to another DNA molecule encoding the light chain constant region, C_L. The sequences of human light chain constant region genes are known in the art and DNA fragments encompassing these regions can be obtained by standard PCR amplification. In preferred embodiments, the light chain constant region can be a kappa or lambda constant region.

To create a scFv gene, the V_H- and V_L-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3, such that the V_Hand V_Lsequences can be expressed as a contiguous single-chain protein, with the V_Land V_Hregions joined by the flexible linker (see e.g., Bird et al., (1988) Science 242:423-426; Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

Production of Monoclonal Antibodies of the Disclosure

Monoclonal antibodies (mAbs) of the present disclosure can be produced using the well-known somatic cell hybridization (hybridoma) technique of Kohler and Milstein (1975) Nature 256: 495. Other embodiments for producing monoclonal antibodies include viral or oncogenic transformation of B lymphocytes and phage display techniques. Chimeric or humanized antibodies are also well known in the art. See e.g., U.S. Pat. Nos. 4,816,567; 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370, the contents of which are specifically incorporated herein by reference in their entirety.

Generation of Transfectomas Producing Monoclonal Antibodies of the Disclosure

Antibodies of the disclosure also can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art (e.g., Morrison, S. (1985) Science 229:1202). In one embodiment, DNA encoding partial or full-length light and heavy chains obtained by standard molecular biology techniques is inserted into one or more expression vectors such that the genes are operatively linked to transcriptional and translational regulatory sequences. In this context, the term “operatively linked” is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene.

The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody genes. Such regulatory sequences are described, e.g., in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, e.g., the adenovirus major late promoter (AdMLP) and polyoma. Alternatively, non-viral regulatory sequences can be used, such as the ubiquitin promoter or β-globin promoter. Still further, regulatory elements composed of sequences from different sources, such as the SRα promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1 (Takebe et al., (1988) Mol. Cell. Biol. 8:466-472). The expression vector and expression control sequences are chosen to be compatible with the expression host cell used.

The antibody light chain gene and the antibody heavy chain gene can be inserted into the same or separate expression vectors. In preferred embodiments, the variable regions are used to create full-length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype such that the V_Hsegment is operatively linked to the CH segment(s) within the vector and the V_Lsegment is operatively linked to the C_Lsegment within the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the disclosure can carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216; 4,634,665 and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).

For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains is transfected into a host cell by standard techniques. The various forms of the term “transfection” are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. Although it is theoretically possible to express the antibodies of the disclosure in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells, and most preferably mammalian host cells, is the most preferred because such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody.

Preferred mammalian host cells for expressing the recombinant antibodies of the disclosure include Chinese Hamster Ovary (CHO cells) (including dhfr− CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) J. Mol. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular for use with NSO myeloma cells, another preferred expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

Bispecific Molecules

In another aspect, the present disclosure features bispecific molecules comprising one or more antibodies of the disclosure linked to at least one other functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. Thus, as used herein, “bispecific molecule” includes molecules that have three or more specificities.

In an embodiment, a bispecific molecule has, in addition to an anti-RANKL binding specificity, a third specificity. The third specificity can be for another immune checkpoint molecules such as PD-1, PD-L1, CTLA-4 or LAG-3.

Bispecific molecules may be in many different formats and sizes. At one end of the size spectrum, a bispecific molecule retains the traditional antibody format, except that, instead of having two binding arms of identical specificity, it has two binding arms each having a different specificity. At the other extreme are bispecific molecules consisting of two single-chain antibody fragments (scFv's) linked by a peptide chain, a so-called Bs(scFv) 2 construct. Intermediate-sized bispecific molecules include two different F(ab) fragments linked by a peptidyl linker. Bispecific molecules of these and other formats can be prepared by genetic engineering, somatic hybridization, or chemical methods. See, e.g., Kufer et al, cited supra; Cao and Suresh, Bioconjugate Chemistry, 9 (6), 635-644 (1998); and van Spriel et al., Immunology Today, 21 (8), 391-397 (2000), and the references cited therein.

Pharmaceutical Compositions

In another aspect, the present disclosure provides a pharmaceutical composition comprising one or more antibodies (or antigen-binding portion thereof, the bispecific molecules, or nucleic acid molecules or expression vectors expressing the same) of the present disclosure formulated together with a pharmaceutically acceptable carrier. The antibodies (or antigen-binding portion thereof, the bispecific molecules, or nucleic acid molecules or expression vectors expressing the same) can be dosed sperapartely when the composition contains more than one antibody (or antigen-binding portion thereof, the bispecific molecule, or nucleic acid molecule or expression vector expressing the same). The composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or a drug, such as an anti-tumor drug.

The pharmaceutical composition can comprise any number of excipients. Excipients that can be used include carriers, surface active agents, thickening or emulsifying agents, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coatings, disintegrating agents, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof. The selection and use of suitable excipients are taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient can be coated in a material to protect it from the action of acids and other natural conditions that may inactivate it. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, an antibody of the disclosure can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, e.g., intranasally, orally, vaginally, rectally, sublingually or topically.

Pharmaceutical compositions can be in the form of sterile aqueous solutions or dispersions. They can also be formulated in a microemulsion, liposome, or other ordered structure suitable to high drug concentration.

The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration and will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01% to about ninety-nine percent of active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30% of active ingredient in combination with a pharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Alternatively, antibody can be administered as a sustained release formulation, in which case less frequent administration is required.

For administration of the composition, the dosage may range from about 0.0001 to 100 mg/kg. An exemplary treatment regime entails administration once per week. Exemplary dosage regimens include intravenous administration.

A “therapeutically effective dosage” of an anti-RANKL antibody, or antigen-binding portion thereof, the bispecific molecules, or nucleic acid molecules or expression vectors expressing the same of the disclosure preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. For example, for the treatment of tumor-bearing subjects, a “therapeutically effective dosage” preferably inhibits tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects. A therapeutically effective amount of a therapeutic antibody can decrease tumor size, or otherwise ameliorate symptoms in a subject, which is typically a human or can be another mammal. For the treatment of bone loss, a “therapeutically effective dosage” preferably inhibits bone loss by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects.

The pharmaceutical composition can be a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, poly-orthoesters, and polylactic acid. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Therapeutic compositions can be administered via medical devices such as (1) needleless hypodermic injection devices (e.g., U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and 4,596,556); (2) micro-infusion pumps (U.S. Pat. No. 4,487,603); (3) transdermal devices (U.S. Pat. No. 4,486,194); (4) infusion apparatuses (U.S. Pat. Nos. 4,447,233 and 4,447,224); and (5) osmotic devices (U.S. Pat. Nos. 4,439,196 and 4,475,196); the disclosures of which are incorporated herein by reference.

In certain embodiments, the monoclonal antibodies or antigen-binding portion thereof, the bispecific molecules, or nucleic acid molecules or expression vectors expressing the same of the disclosure can be formulated to ensure proper distribution in vivo. For example, to ensure that the therapeutic antibody of the disclosure cross the blood-brain barrier, they can be formulated in liposomes, which may additionally comprise targeting moieties to enhance selective transport to specific cells or organs. See, e.g. U.S. Pat. Nos. 4,522,811; 5,374,548; 5,416,016; and 5,399,331; V. V. Ranade (1989) J. Clin. Pharmacol. 29:685; Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038; Bloeman et al., (1995) FEBS Lett. 357:140; M. Owais et al., (1995) Antimicrob. Agents Chemother. 39:180; Briscoe et al., (1995) Am. J. Physiol. 1233:134; Schreier et al., (1994) J. Biol. Chem. 269:9090; Keinanen and Laukkanen (1994) FEBS Lett. 346:123; and Killion and Fidler (1994) Immunomethods 4:273.

Uses and Methods of the Disclosure

The composition comprising the antibodies or the antigen-binding portion thereof, the bispecific molecules, nucleic acid molecules or expression vectors expressing the same of the present disclosure have numerous in vitro and in vivo utilities involving, for example, treatment of RANKL associated diseases, such as bone loss and cancers.

Given the ability of anti-RANKL antibodies or antigen-binding portion thereof, the bispecific molecules, or nucleic acid molecules or expression vectors expressing the same of the disclosure to inhibit bone destruction, the disclosure provides methods for inhibiting bone loss in a subject comprising administering to the subject the pharmaceutical composition of the disclosure. The bone loss may be bone destruction in bone metastases, multiple myeloma or postmenopausal osteoporosis.

Given the anti-tumor activity of anti-RANKL antibodies or antigen-binding portion thereof, the bispecific molecules, or nucleic acid molecules or expression vectors expressing the same of the disclosure, the disclosure provides methods for inhibiting RANKL related cancers in a subject comprising administering to the subject the pharmaceutical composition of the disclosure such that growth of the tumor is inhibited in the subject. Non-limiting examples of tumors that can be treated by the antibodies or antigen-binding portion thereof, the bispecific molecules, or nucleic acid molecules or expression vectors expressing the same of the disclosure include, but not limited to, breast cancer, melanoma, prostate carcinoma, colon carcinoma, fibrosarcoma, lungcarcinoma, giant cell tumor of bone, multiple myeloma, and bone metastases.

More generally, the antibodies or antigen-binding portion thereof, the bispecific molecules, or nucleic acid molecules or expression vectors expressing the same of the disclosure can be used to enhance an immune response in a subject.

Combination Therapy

In another aspect, the disclosure provides methods of combination therapy in which the anti-RANKL antibodies or antigen-binding portion thereof, the bispecific molecules, or nucleic acid molecules or expression vectors expressing the same of the present disclosure are co-administered with one or more additional agents that are effective in inhibiting bone loss or tumor growth in a subject. The agent for preventing or inhibiting bone loss includes, but not limited to bisphosphonates, such as bisphosphonate zoledronic acid. In certain embodiments, the subject is human.

In one embodiment, the disclosure provides a method for inhibiting tumor growth in a subject comprising administering to the subject an anti-RANKL antibody (or antigen-binding portion thereof, the bispecific molecule, nucleic acid molecule or expression vector expressing the same) and one or more additional antibodies, such as an anti-VISTA antibody, an anti-LAG-3 antibody, an anti-PD-L1 antibody, and anti-PD-1 antibody and/or an anti-CTLA-4 antibody, especially the immune checkpoint inhibitors. In certain embodiments, the subject is human.

The RANKL signaling activation can also be further combined with standard cancer treatments. For example, RANKL signaling activations can be combined with CTLA-4 and/or LAG-3 and/or PD-1 blockade and also chemotherapeutic regimes. For example, a chemotherapeutic agent can be administered with the anti-RANKL antibodies, which may be a cytotoxic agent. For example, epirubicin, oxaliplatin, and 5-FU are administered to patients receiving anti-RANKL therapy.

Optionally, the combination of anti-RANKL and one or more additional antibodies (e.g., anti-CTLA-4 and/or anti-LAG-3 and/or anti-PD-1 antibodies) can be further combined with an immunogenic agent, such as cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), and cells transfected with genes encoding immune stimulating cytokines (He et al., (2004) J. Immunol. 173:4919-28). Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as peptides of gp100, MAGE antigens, Trp-2, MART1 and/or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF.

Other therapies that may be combined with anti-RANKL antibody includes, but not limited to, interleukin-2 (IL-2) administration, radiation, surgery, or hormone deprivation.

The combination of therapeutic agents discussed herein can be administered concurrently as a single composition in a pharmaceutically acceptable carrier, or concurrently as separate compositions with each agent in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic agents can be administered sequentially.

Furthermore, if more than one dose of the combination therapy is administered sequentially, the order of the sequential administration can be reversed or kept in the same order at each time point of administration, sequential administrations can be combined with concurrent administrations, or any combination thereof.

The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and all references, Genbank sequences, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

EXAMPLES
Example 1 Generation of Mouse Anti-RANKL Monoclonal Antibodies Using Hybridoma Technology

Immunization

Mice were immunized according to the method as described in E Harlow, D. Lane, Antibody: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998. In house made recombinant human RANKL protein (amino acid residue 136-317 of uniport #O14788) with human IgG1 Fc at the C-terminus was used as the immunogen, and also used for determining anti-sera titer and for screening hybridomas secreting antigen-specific antibodies.

Immunizing dosages contained 25 μg human RANKL-Fc protein/mouse/injection for both primary and boost immunizations. To increase immune response, the complete Freud's adjuvant and incomplete Freud's adjuvant (Sigma, St. Louis, Mo., USA) were used respectively for primary and boost immunizations. Briefly, adjuvant-antigen mixture was prepared as follows. First, the adjuvant was mixed in a vial using a vortex. The desired amount of adjuvant was transferred to an autoclaved 1.5 mL micro-centrifuge tube. The antigen was prepared in PBS or saline with concentration ranging from 0.5-1.0 mg/ml. The calculated amount of antigen was then added to the micro-centrifuge tube with the adjuvant, and the resulting mixture was mixed by gently vortexing for 2 minutes to generate water-in-oil emulsions. The adjuvant-antigen emulsion was then drawn into the proper syringe for animal injection. A total of 25 μg antigen was injected in a volume of 50-100 μl. Each animal was immunized, and then boosted for 3 to 4 times depending on the anti-sera titer. Animals with good titers were given a final boost by intraperitoneal injection before fusion.

Hybridoma Fusion and Screening

Cells of murine myeloma cell line (SP2/0-Ag14, ATCC #CRL-1581) were cultured to reach the log phase stage right before fusion. Spleen cells from immunized mice were prepared sterilely and fused with myeloma cells according to the method as described in Kohler G, and Milstein C, “Continuous cultures of fused cells secreting antibody of predefined specificity,” Nature, 256: 495-497(1975). Fused “hybrid cells” were subsequently dispensed into 96-well plates in DMEM/20% FCS/HAT medium. Surviving hybridoma colonies were observed under the microscope seven to ten days post fusion. After two weeks, the supernatant from each well was subjected to ELISA-based screening using in house made recombinant human RANKL-Fc protein. Positive hybridomas secreting antibodies that bound to human RANKL-Fc proteins were then selected and transferred to 24-well plates. These hybridomas were further tested for their activities for blocking human RANKL binding to RANK. Hybridoma clones producing antibodies that showed high specific human RANKL binding and RANKL-RANK blocking activities were subcloned by limiting dilution to ensure the clonality of the cell line, and then monoclonal antibodies were purified. Briefly, Protein A sepharose column (from bestchrom (Shanghai) Biosciences, Cat #AA0273) was washed using PBS buffer in 5 to 10 column volumes. Cell supernatants were passed through the columns, and then the columns were washed using PBS buffer until the absorbance for protein reached the baseline. The columns were eluted with elution buffer (0.1 M Glycine-HCl, pH 2.7), and immediately collected into 1.5 ml tubes with neutralizing buffer (1 M Tris-HCl, pH 9.0). Fractions containing immunoglobulins were pooled and dialyzed in PBS overnight at 4° C. Subsequently, the in vitro functional activities of purified monoclonal antibodies were characterized as follows.

Example 2 Affinity Determination of Mouse Anti-RANKL Monoclonal Antibodies Using Octet Bio-Layer Interferometry Technology

The purified anti-RANKL mouse monoclonal antibodies generated in Example 1 were characterized for affinities and binding kinetics by Octet system (ForteBio, Octet RED 96).

Briefly, AHC/AMC biosensors (anti-human/anti-mouse IgG Fc capture, ForteBio) were presoaked with 10 mM glycine (pH 1.5) for 3 seconds, and then dipped in a well with running buffer (0.5% w/v BSA in PBST) for 3 seconds, the soaking and dipping steps were repeated for three times. Then, the sensors were dipped in a well with anti-RANKL antibodies of the disclosure or a benchmark (denosumab, prepared by using heavy and light chain amino acid sequences of SEQ ID NOs.: 38 and 39, respectively) in HBS-EP⁺ at 5.0 μg/mL for 100 seconds, and then immersed in a well with in HBS-EP⁺ for 5 minutes. A new baseline was run for 180 seconds in another well with HBS-EP⁺. Then the sensors were dipped in a well with serially diluted human RANKL-his protein (prepared in-house with SEQ ID NO: 43, starting at 40 nM with a two-fold serial dilution) in HBS-EP⁺ for 100 seconds, and then immersed in a baseline well for 10 minutes. Finally, sensors were presoaked with 10 mM glycine (pH 1.5) for 3 seconds, and then dipped in a well with running buffer for 3 seconds, the soaking and dipping steps repeated for three times. The association and dissociation curves were fit to a 1:1 Langmuir binding model using Octet evaluation software. The K_a, K_dand K_Dvalues were determined and summarized in Table 2 below.

TABLE 2

Binding affinity of mouse anti-RANKL antibodies

Kinetics on Octet

Human RANKL

K_a
K_d
K_D

Mouse mAb ID#
(M⁻¹s⁻¹)
(s⁻¹)
(M)

D1E3
not tested
not tested
not tested

B3D8
6.13E+05
2.77E−04
4.52E−10

D1A1
1.10E+06
7.90E−05
7.18E−11

B1A8
6.11E+05
2.36E−04
3.86E−10

B4H5
not tested
not tested
not tested

Benchmark
3.20E+05
5.17E−05
1.62E−10

The mouse anti-RANKL antibodies of the present disclosure, including B3D8, D1A1 and B1A8, specifically bound to human RANKL with comparable binding affinities compared to the benchmark, with D1A1 showing the highest binding affinity.

Example 3 Binding Activity of Mouse Anti-RANKL Monoclonal Antibodies

The binding activities of mouse anti-RANKL antibodies were determined by Capture ELISA.

Briefly, 96-well micro plates were coated with 2.0 μg/ml Affinipure goat anti-mouse IgG F(ab′)₂fragment specific (Jackson Immuno Research, Cat #115-005-072, 100 μl/well) in PBS overnight at 4° C. Plates were washed once with wash buffer (PBS+0.05% v/v Tween-20, PBST) and then blocked with 200 μl/well blocking buffer (5% w/v non-fatty milk in PBST) for 2 hours at 37° C. Plates were washed again and incubated with 100 μl/well serially diluted anti-RANKL antibodies, the benchmark and negative control hIgG (human immunoglobulin (pH4) for intravenous injection, Hualan Biological Engineering Inc.) (5-fold dilution in 2.5% non-fatty milk in PBST, starting with 10000 ng/ml) for 40 minutes at 37° C., and then washed 4 times. Plates containing the captured anti-RANKL antibodies were incubated with biotin-labeled human RANKL-Fc proteins (prepared in house, SEQ ID NO.:40, 22 ng/mL in 2.5% non-fatty milk in PBST, 100 μl/well) for 40 minutes at 37° C., washed 4 times, and incubated with streptavidin conjugated HRP (1:10000 dilution in PBST, Jackson Immuno Research, Cat #016-030-084, 100 μl/well) for 40 minutes at 37° C. After a final wash, plates were incubated with 100 μl/well ELISA substrate TMB (Innoreagents, Cat #TMB-S-002) at room temperature. The reaction was stopped in 3-10 minutes with 50 μl/well 1M H₂SO₄, and the absorbance of each well was read on a microplate reader using dual wavelengths mode with 450 nm for TMB and 630 nm as the reference wavelength. The OD (450-630) values were plotted against antibody concentration. Data were analyzed using Graphpad Prism software and EC₅₀values were reported.

The results were shown in FIGS. 1A and 1B. The results indicated that the mouse anti-RANKL antibodies of the disclosure bound to human RANKL specifically. The maximum absorbance of D1E3/B3D8/D1A1/B1A8/B4H5 was similar to or a bit lower than that of the benchmark, with similar or slightly higher EC₅₀. Especially, the D1E3 and D1A1 antibodies showed lower EC₅₀values than the benchmark, suggesting that they more efficiently bound to more human RANKL proteins.

Example 4 Competitive ELISA Assays on Mouse Anti-RANKL Antibodies

4.1 Ligand Blocking ELISA

The abilities of anti-RANKL antibodies to block RANKL-RANK binding was measured using a competitive ELISA assay. Briefly, human RANK-Fc proteins (prepared in house, SEQ ID NO.: 41) were coated on 96-well micro plates at 2.0 μg/mL in PBS, 100 μl/well, and incubated overnight at 4° C. The next day, plates were washed with wash buffer (PBS+0.05% w/v Tween-20, PBST), and blocked with 5% w/v non-fatty milk in PBST, 200 μl/well, for 2 hours at 37° C.

The anti-RANKL antibodies of the present disclosure or controls were diluted with biotin labeled human RANKL-Fc protein (prepared in house, SEQ ID NO.: 40, 14.67 ng/mL in 2.5% w/v non-fatty milk in PBST), starting at 20 nM with a 5-fold serial dilution, and incubated at room temperature for 40 minutes. After plate washing, the antibody/RANKL-Fc mixtures were added to the RANK-Fc coated plates, 100 μl/well, and incubated for 40 minutes at 37° C. Plates were washed 4 times using wash buffer, and added and incubated with 100 μl/well of streptavidin conjugated HRP (1:10000 dilution in PBST buffer, Jackson ImmunoResearch Laboratories, Inc., Cat #016-030-084) for 40 minutes at 37° C. Plates were washed again using wash buffer. Finally, TMB was added and the reaction was stopped using 1M H₂SO₄, and the absorbance of each well was read on a microplate reader using dual wavelengths mode with 450 nm for TMB and 630 nm as the reference wavelength. The OD (450-630) values were plotted against antibody concentration. Data were analyzed using Graphpad Prism software and IC₅₀values were reported.

4.2 Benchmark Blocking ELISA

The abilities of the anti-RANKL antibodies to block Benchmark-human RANKL binding was measured using a competitive ELISA assay. Briefly, the benchmark denosumab was coated on 96-well micro plates at 2 μg/mL in PBS, 100 μl/well, and incubated overnight at 4° C. The next day, plates were washed with wash buffer, and blocked with 5% w/v non-fatty milk in PBST, 200 μl/well, for 2 hours at 37° C. While blocking, the anti-RANKL antibodies of the present disclosure or controls were diluted with biotin labeled human RANKL-Fc protein (SEQ ID NO.: 40, 17.6 ng/mL in 2.5% w/v non-fatty milk in PBST), starting at 80.0 nM with a 4-fold serial dilution, and incubated at room temperature for 40 minutes. After plate washing, the antibody/RANKL-Fc-biotin mixtures were added to the benchmark coated plates, 100 μl/well. After incubation at 37° C. for 40 minutes, plates were washed 4 times using wash buffer. Then the plates were added and incubated with 100 μl Peroxidase Streptavidin (1:5000 dilution in PBST buffer, Jackson Immunoresearch, Cat #016-030-084) for 40 minutes at 37° C. Plates were washed again using wash buffer. Finally, TMB was added and the reaction was stopped using 1M H₂SO₄. The absorbance of each well was read on a microplate reader using dual wavelengths mode with 450 nm for TMB and 630 nm as the reference wavelength, and the OD (450-630) values were plotted against antibody concentration. Data were analyzed using Graphpad Prism software and IC₅₀values were reported.

The results of the two competition ELISA assays were shown in FIGS. 2A-2B and 3A-3B.

It can be seen from FIGS. 2A-2B that the anti-RANKL antibodies of the disclosure were capable of blocking human RANK-human RANKL interaction. The D1E3 and D1A1 antibodies had lower IC₅₀values as compared to the benchmark, indicating their better blocking activities.

FIGS. 3A and 3B showed that some of the antibodies of the disclosure were able to block human RANKL-benchmark interaction, suggesting that they bound to the same or similar epitope as the benchmark did. The B4H5 antibody showed no blocking activity and may bind to a different epitope.

Example 5 Cell Based Functional Assay of Mouse Anti-RANKL Antibodies

RAW264.7 cells (mouse monocyte-macrophage leukemia cells) may be induced by RANKLs to differentiate into osteoclasts, and such cells may be used to test the anti-RANKL antibodies's capacities of inhibiting RANKL binding to cell surface RANK.

Briefly, RAW 264.7 cells (Cell Bank of Chinese Academy of Sciences, Cat #SCSP-5036) at the log phase were harvested from cell culture flasks, and resuspended in α-MEM medium (Gibco #12561056) supplemented with 10% FBS (Gibco #10091148). 2.0×10³of RAW 264.7 cells in 150 μl culture medium were plated into 96-well plates, and the plates were incubated in a CO₂incubator at 37° C. for 1 hour. Recombinant human RANKL-his proteins (R&D, Cat #6449-TEC-010) were diluted at 240 ng/ml in α-MEM medium supplemented with 10% FBS. Serially diluted antibodies (final concentration at 16.67 nM, 6.67 nM, 2.67 nM, 1.33 nM, 0.67 nM, 0.33 nM, 0.17 nM, 0.067 nM and 0.0267 nM) in the culture medium prepared above containing RANKL-his protein were added to the plates containing RAW 264.7 cells, 50 μl per well, and incubated in the CO₂incubator at 37° C. for 4 days. After the incubation, the supernatants were removed and the cultured cells in 96-well plates were fixed with 50 μl of ethanol-acetone (50:50, v/v) for 1 min. The plates were centrifuged at 300 g for 3 min and the supernatants were removed gently. Plates were dried at room temperature for 10 min, added with 100 μl pNPP chromogenic solution (Sigma, Cat #S0942-200TAB, for osteoclast staining), and incubated at 37° C. for 30 min. After incubation, the reaction was stopped with 50 μl of 0.5 N NaOH, and absorbance was read at 405 nm. Data were analyzed using Graphpad Prism software and IC₅₀values were reported.

The results of the functional assay were shown in FIGS. 4A-4D.

The anti-RANKL antibodies all blocked RANKL binding to cell surface RANK to some extent. FIGS. 4A and 4C showed that the B1A8 and D1E3 had very close blocking capacity to the benchmark, although with a bit higher EC₅₀value.

Example 6 Generation and Characterization of Chimeric Antibodies

The variable domains of the heavy and light chain of the anti-RANKL mouse mAbs were sequenced and the sequence ID numbers were summarized in Table 1.

The variable domains of the heavy and light chain of the anti-RANKL mouse mAbs were cloned in frame to human IgG2 heavy-chain (SEQ ID NO.: 35) and human kappa light-chain constant regions (SEQ ID NO.: 37), respectively, wherein the C terminus of variable region was linked to the N terminus of the respective constant region.

The vectors each containing a nucleotide encoding a heavy chain variable region linked to human IgG2 heavy-chain constant region, and the vectors each containing a nucleotide encoding a light chain variable region linked to human kappa light-chain constant region were transiently transfected into 50 ml of 293F suspension cell cultures in a ratio of 1.1:1 light to heavy chain construct, with 1 mg/mL PEI.

Cell supernatants were harvested after six days in shaking flasks, spun down to pellet cells, and then chimeric antibodies were purified from cell supernatants as described above. The purified antibodies were tested in the capture ELISA, competitive ELISA assay and Octet affinity test following the protocols in the foregoing Examples.

The results were shown in Table 3, FIGS. 5A-5D, 6 and 7.

TABLE 3

Binding affinities of Chimeric Antibodies

Clone ID#
Octet Affinity to human RANKL (KD, M)

Mouse D1E3
Not tested

Chimeric D1E3
6.12E−10

Mouse B3D8
4.52E−10

Chimeric B3D8
2.65E−09

Mouse D1A1
7.18E−11

Chimeric D1A1
1.97E−10

Mouse B1A8
3.86E−10

Chimeric B1A8
1.62E−09

Benchmark
1.62E−10

The data showed that the chimeric D1A1 antibody a similar binding affinity/capacity to human RANKL compared to its parental mouse antibody and the benchmark.

Example 7 Humanization of Anti-RANKL Mouse Monoclonal Antibody D1A1

Mouse anti-RANKL antibody D1A1 was selected for humanization and further investigations. Humanization of this mouse antibody was conducted using the well-established CDR-grafting method as described in detail below.

To select acceptor frameworks for humanization of mouse antibody D1A1, the light and heavy chain variable region sequences of mouse D1A1 were blasted against the human immunoglobulin gene database. The human germlines with the highest homology to mouse D1A1 were selected as the acceptor frameworks for humanization. The mouse antibody heavy/light chain variable region CDRs were inserted into the selected frameworks, and the residue(s) in the frameworks was/were further mutated to obtain more candidate heavy chain/light chain variable regions. A total of 6 exemplary humanized D1A1 antibodies (namely from huD1A1-V1 to huD1A1-V6) were obtained whose heavy/light chain variable region sequence number IDs were listed in Table 1.

The vectors each containing a nucleotide sequence encoding the humanized D1A1 heavy chain variable region linked to human IgG4 heavy-chain constant region (SEQ ID NO: 36), and the vectors each containing a nucleotide sequence encoding the humanized D1A1 light chain variable region linked to human kappa light-chain constant region (SEQ ID NO: 37) were transiently transfected into 50 ml of 293F suspension cell cultures in a ratio of 60% to 40% light to heavy chain construct, with 1 mg/ml PEI.

Example 8 Characterization of Humanized D1A1 Antibodies

Cell supernatants containing humanized antibodies were harvested after six days in shaking flasks and tested for binding affinities to human RANKL by Octet affinity test following the protocol described above. The chimeric antibody when tested was in HBS-EP⁺ at 5 μg/ml concentration. The K_a, K_dand K_Dvalues were determined and summarized in Table 4 below.

The results indicated that all humanized D1A1 antibodies had similar human RANKL binding affinities to the chimeric antibody.

TABLE 4

Binding affinities of Humanized D1A1 mAbs

Octet Kinetics of Humanized D1A1 mAbs Binding to Human RANKL

Kinetics on Octet

Human RANKL his

K_a
K_d
K_D

mAb
(M⁻¹s⁻¹)
(s⁻¹)
(M)

huD1A1-V1
1.24E+05
<1.0E−07
<1.0E−12

huD1A1-V2
1.12E+05
<1.0E−07
<1.0E−12

huD1A1-V3
1.06E+05
<1.0E−07
<1.0E−12

huD1A1-V4
1.01E+05
<1.0E−07
<1.0E−12

huD1A1-V5
1.20E+05
<1.0E−07
<1.0E−12

huD1A1-V6
1.15E+05
<1.0E−07
<1.0E−12

Chimeric D1A1
1.28E+05
<1.0E−07
<1.0E−12

The humanized antibodies huD1A1-V2 and huD1A1-V5 were purified as described above and tested for their binding affinities/capacities to human and cynomolgus RANKL and other functional activities by Biacore, binding capture ELISA, protein thermal shift assay, competitive ELISA and cell-based functional assay, following the protocols in the foregoing examples and described below.

The humanized antibodies huD1A1-V2 and huD1A1-V5 were characterized for affinities and binding kinetics by Biacore T200 system (GE healthcare, Pittsburgh, PA, USA) as follows. Briefly, goat anti-human IgG (GE healthcare, Cat #BR100839, Human Antibody Capture Kit) was covalently linked to a CM5 chip (carboxy methyl dextran coated chip from GE healthcare #BR10030) via primary amines, using a standard amine coupling kit provided by Biacore. Un-reacted moieties on the biosensor surface were blocked with ethanolamine. Then purified anti-RANKL antibodies at the concentration of 2.0 μg/mL were flown onto the chip at a flow rate of 10 μL/min. Then, recombinant human RANKL-his (prepared in-house with SEQ ID NO: 43) or cynomolgus monkey RANKL-his protein (prepared in-house with SEQ ID NO.:42), 2-fold dilution in HBS-EP⁺ buffer (provided by Biacore) starting at 80 nM, were flown onto the chip at a flow rate of 30 μL/min. The antigen-antibody association kinetics was followed for 2 minutes and the dissociation kinetics was followed for 10 minutes. The association and dissociation curves were fit to a 1:1 Langmuir binding model using BIAcore evaluation software. The K_Dvalues were determined and summarized in Table 6 below.

To determine the thermal stabilities of humanized antibodies, a protein thermal shift assay was used to determine melting temperature (Tm) using a GloMelt™ Thermal Shift Protein Stability Kit (Biotium, Cat #33022-T). Briefly, the GloMelt™ dye was allowed to thaw and reach room temperature. The vial containing the dye was vortexed and centrifuged. Then, 10× dye was prepared by adding 5 μL 200× dye to 95 μL PBS. 2 μL 10× dye and 10 μg humanized antibodies were added, and PBS was added to a total reaction volume of 20 μL. The tubes containing the dye and antibodies were briefly spun and placed in real-time PCR thermocycler (Roche, LightCycler 480 II) set up with a melt curve program having the parameters in Table 5.

TABLE 5

Parameters for Melt Curve Program

Profile step
Temperature
Ramp rate
Holding Time

Initial hold
25° C.
NA
30 s

Melt curve
25-99° C.
0.1° C./s
NA

For the capture ELISA, 96-well micro plates were coated with 2 μg/ml goat anti-human IgG (AffiniPure Goat Anti-Human IgG, F(ab′)₂fragment specific, Jackson Immunoresearch, Cat #109-005-097) instead of AffiniPure Goat Anti-Mouse IgG, Fcγ fragment specific, 100 μl/well.

The results were shown in Table 6, Table 10, FIGS. 8-10, FIGS. 11A-11C, and FIGS. 12A-12B.

TABLE 6

Binding affinities of Humanized mAbs huD1A1-V2 and huD1A1-V5

Binding assay

Human RANKL
Cynomolgus RANKL
TM (melting temperature), ° C.

mAbs
Biacore (KD, M)
Biacore (KD, M)
T1
T2

huD1A1-V2
1.34E−10
4.2E−10
66.0
78.5

huD1A1-V5
1.79E−10
4.14E−10
66.0
78.0

Chimeric D1A1
1.49E−10
4.2E−10
not tested
not tested

Mouse D1A1
not tested
not tested
not tested
not tested

Denosumab
1.2E−10
4.61E−10
not tested
not tested

The data showed that huD1A1-V2 and huD1A1-V5 showed comparable in vitro activities to chimeric D1A1 antibodies. In particular, chimeric and humanized D1A1 antibodies exhibited high and comparable binding affinities as compared with denosumab in Biacore test, and also showed comparable binding capacities to human RANKL in the ELISA. As shown in FIGS. 9, and 11A-11C, the huD1A1-V2 and huD1A1-V5 blocked binding of human RANKL to RANK at comparable activities to the benchmark.

While the disclosure has been described above in connection with one or more embodiments, it should be understood that the disclosure is not limited to those embodiments, and the description is intended to cover all alternatives, modifications, and equivalents, as may be included within the spirit and scope of the appended claims. All referenced cited herein are further incorporated by reference in their entirety.

Sequences in the present application are summarized below.

Description/

Sequence/SEQ ID NO.

VH CDR1 for mouse, chimeric and humanized D1A1

NYAMS (SEQ ID NO.: 1)

VH CDR2 for mouse, chimeric and humanized D1A1

GISSGGTYTYYPDTVKG (SEQ ID NO.: 5)

VH CDR3 for mouse, chimeric and humanized D1A1

RDYGDVYEWYFDV (SEQ ID NO.: 10)

VL CDR1 for mouse, chimeric and humanized D1A1

KASQDVRTSIA (SEQ ID NO.: 14)

VL CDR2 for mouse, chimeric and humanized D1A1

SASYRYT (SEQ ID NO.: 18)

VL CDR3 for mouse, chimeric and humanized D1A1

QQHYSSPWT (SEQ ID NO.: 21)

VH for mouse and chimeric D1A1

EVQLVESGGGLVKSGGSLKLSCAASGFTFRNYAMSWVRQTPEKRLEWVAGISSGGTYTYYPD

TVKGRFTISRDNARNTLFLQMSSLRSEDTAMYYCARRDYGDVYEWYFDVWGTGTTVTVSS

(SEQ ID NO.: 25)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGTCTGGAGGGTCCCTGAAACTC

TCCTGTGCAGCCTCTGGATTCACTTTCCGTAACTATGCCATGTCTTGGGTTCGCCAGACTCC

GGAGAAGAGGCTGGAGTGGGTCGCAGGAATTAGTAGTGGTGGTACTTACACCTACTATCC

AGACACTGTGAAGGGACGATTCACCATCTCCAGAGACAATGCCAGGAACACCCTGTTCCTG

CAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGAAGGGACTAT

GGTGATGTCTACGAATGGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCT

CA (SEQ ID NO.: 44)

VH for D1A1-V1 and huB1H2-V4

EVQLVESGGGLVKPGGSLRLSCAASGFTFRNYAMSWVRQAPGKGLEWVX1GISSGGTYTYYP

DTVKGRFTISRDNAX2NSLYLQMNSLRAEDTAVYYCARRDYGDVYEWYFDVWGX3GTTVTV

SS (SEQ ID NO.: 26) X1 = A, X2 = R, X3 = T

EVQLVESGGGLVKPGGSLRLSCAASGFTFRNYAMSWVRQAPGKGLEWVAGISSGGTYTYYPD

TVKGRFTISRDNARNSLYLQMNSLRAEDTAVYYCARRDYGDVYEWYFDVWGTGTTVTVSS

VH for hu D1A1-V2 and hu D1A1-V5

EVQLVESGGGLVKPGGSLRLSCAASGFTFRNYAMSWVRQAPGKGLEWVX1GISSGGTYTYYP

DTVKGRFTISRDNAX2NSLYLQMNSLRAEDTAVYYCARRDYGDVYEWYFDVWGX3GTTVTV

SS (SEQ ID NO.: 26) X1 = S, X2 = K, X3 = K

EVQLVESGGGLVKPGGSLRLSCAASGFTFRNYAMSWVRQAPGKGLEWVSGISSGGTYTYYPD

TVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRDYGDVYEWYFDVWGKGTTVTVSS

VH for hu D1A1-V3 and hu D1A1-V6

EVQLVESGGGLVKPGGSLRLSCAASGFTFRNYAMSWVRQAPGKGLEWVX1GISSGGTYTYYP

DTVKGRFTISRDNAX2NSLYLQMNSLRAEDTAVYYCARRDYGDVYEWYFDVWGX3GTTVTV

SS (SEQ ID NO.: 26) X1 = A, X2 = K, X3 = K

EVQLVESGGGLVKPGGSLRLSCAASGFTFRNYAMSWVRQAPGKGLEWVAGISSGGTYTYYPD

TVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRDYGDVYEWYFDVWGKGTTVTVSS

VL for mouse and chimeric D11

DIVMTQSHKFMSTSVGDRVSITCKASQDVRTSIAWYQQKPGQSPNLLIYSASYRYTGVPDRFTG

SGSGTDFTFTISSVQAEDLAVYYCQQHYSSPWTFGGGTKLEIK (SEQ ID NO.: 30)

GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCA

TCACCTGCAAGGCCAGTCAGGATGTGAGAACTTCTATTGCCTGGTATCAACAGAAACCAGG

ACAATCTCCTAACCTACTGATTTACTCGGCATCCTACCGGTACACTGGAGTCCCTGATCGCT

TCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGCAGGCTGAAGA

CCTGGCAGTTTATTACTGTCAACAACATTATAGTTCTCCGTGGACGTTCGGTGGAGGCACC

AAGCTGGAAATCAAA (SEQ ID NO.: 45)

VL for hu D1A1-V1-hu D1A1-V3

DIQMTQSPSSLSASVGDRVTITCKASQDVRTSIAWYQQKPGKX1PKLLIYSASYRYTGVPSRFSG

SGSGTDFTFTISSLQPEDX2ATYYCQQHYSSPWTFGGGTKVEIK (SEQ ID NO.: 31)

X1 = A, X2 = I

DIQMTQSPSSLSASVGDRVTITCKASQDVRTSIAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGS

GSGTDFTFTISSLQPEDIATYYCQQHYSSPWTFGGGTKVEIK

VL for hu D1A1-V4-hu D1A1-V6

DIQMTQSPSSLSASVGDRVTITCKASQDVRTSIAWYQQKPGKX1PKLLIYSASYRYTGVPSRFSG

SGSGTDFTFTISSLQPEDX2ATYYCQQHYSSPWTFGGGTKVEIK (SEQ ID NO.: 31)

X1 = S, X2 = L

DIQMTQSPSSLSASVGDRVTITCKASQDVRTSIAWYQQKPGKSPKLLIYSASYRYTGVPSRFSGS

GSGTDFTFTISSLQPEDLATYYCQQHYSSPWTFGGGTKVEIK

VH CDR1 for mouse and chimeric B1A8

RYGMS (SEQ ID NO.: 2)

VH CDR2 for mouse and chimeric B1A8

SISSGGTYTSYPDSVQG (SEQ ID NO.: 6)

VH CDR3 for mouse and chimeric B1A8

RNFITSATDCYFDV (SEQ ID NO.: 11)

VL CDR1 for mouse and chimeric B1A8

RASQSISNNLH (SEQ ID NO.: 15)

VL CDR2 for mouse and chimeric B1A8

ASQSIS (SEQ ID NO.: 19)

VL CDR3 for mouse and chimeric B1A8

QQSNSWPFT (SEQ ID NO.: 22)

VH for mouse and chimeric B1A8

EVQLVESGGDLVKX1GGSLKLSCAASGFTFSRYGMSWVRQTPDKRLEWVASISSGGTYTSYPD

SVX2GRFTITRDSAKNTLYLQMSSLKSEDTAMYYCARRNFITSATDCYFDVWGAGTTVTVSS

(SEQ ID NO.: 27) X1 = P, X2 = Q

EVQLVESGGDLVKPGGSLKLSCAASGFTFSRYGMSWVRQTPDKRLEWVASISSGGTYTSYPDS

VQGRFTITRDSAKNTLYLQMSSLKSEDTAMYYCARRNFITSATDCYFDVWGAGTTVTVSS

VL for mouse and chimeric B1A8

DTVLTQSPATLSVTPGASVSLSCRASQSISNNLHWYQQKSHESPRLLIIYASQSISGIPSRFSGSGS

GTDFTLSINSVETEDFGVYFCQQSNSWPFTFGGGTKLEIK (SEQ ID NO.: 32)

VH CDR1 for mouse and chimeric B3D8

RYGMS (SEQ ID NO.: 2)

VH CDR2 for mouse and chimeric B3D8

SISSGGTYTSYPDSVKG (SEQ ID NO.: 7)

VH CDR3 for mouse and chimeric B3D8

RNFITSATDCYFDV (SEQ ID NO.: 11)

VL CDR1 for mouse and chimeric B3D8

RASQSISNNLH (SEQ ID NO.: 15)

VL CDR2 for mouse and chimeric B3D8

ASQSIS (SEQ ID NO.: 19)

VL CDR3 for mouse and chimeric B3D8

QQSNSWPFT (SEQ ID NO.: 22)

VH for mouse and chimeric B3D8

EVQLVESGGDLVKX1GGSLKLSCAASGFTFSRYGMSWVRQTPDKRLEWVASISSGGTYTSYPD

SVX2GRFTITRDSAKNTLYLQMSSLKSEDTAMYYCARRNFITSATDCYFDVWGAGTTVTVSS

(SEQ ID NO.: 27) X1 = S, X2 = K

EVQLVESGGDLVKSGGSLKLSCAASGFTFSRYGMSWVRQTPDKRLEWVASISSGGTYTSYPDS

VKGRFTITRDSAKNTLYLQMSSLKSEDTAMYYCARRNFITSATDCYFDVWGAGTTVTVSS