Mabs for inhibiting human rage by blocking binding to heparan sulfate

Abstract

Provided are monoclonal antibodies (mAbs) that target the heparin sulfate (HS) binding site. The mAbs can be used for inhibiting Receptor for Advanced Glycation Endproducts (RAGE) signaling. The mAbs can also be used for prophylaxis or therapy for atherosclerosis, arthritis, congestive heart failure, Alzheimer's disease, diabetes, myocardial infraction, peripheral vascular disease, psoriasis, sepsis, cancer, osteoporosis, periodontitis, and drug-induced liver toxicity, and cancer.

Description

[0001] mAbs For Inhibiting Human RAGE by Blocking Binding to Heparan Sulfate

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 610,259, filed December 14, 2023, the entire disclosure of which is incorporated herein by reference.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under grant number HL094463 awarded by National Institutes of Health. The government has certain rights in the invention.

[0006] SEQUENCE LISTING

[0007] The instant application contains a Sequence Listing which has been submitted in .xml format and is hereby incorporated by reference in its entirety. Said .xml copy was created on December 13, 2024, is named “011520.01862. xml” and is 30,463 bytes in size.

[0008] BACKGROUND

[0009] Studies in the past three decades has shown that heparan sulfate (HS) plays important roles in regulating cell signaling, cell-cell interactions and cell-pathogen interactions (Bishop et al., 2007; Lindahl and Kjellen, 2013). The regulatory role of HS in various biological systems hinges upon its interactions with specific HS- binding proteins (HSBP) (Gomez Toledo et al., 2021 ; Xu and Esko, 2014). In situations where the normal functions of HSBPs are dependent upon their interactions with HS, manipulating the specific interactions between HS and HSBPs would be a way to control (either promote or inhibit) the biological functions of HSBPs (Li et al., 2023; Lindahl, 2007; Weiss et al., 2017). An example of this therapeutic approach is the use of heparin as an anticoagulant drug, in which heparin promotes the inhibitory activity of antithrombin towards thrombin (Li et al., 2004; Linhardt, 2003; Olson and Bjork, 1991 ). In previous work related to to targeting HS-HSBP interactions pharmacologically, an emphasis has been placed upon using HS oligosaccharides or HS mimetics to occupy the HS-binding site of HSBPs (Farrugia et al., 2018; Mulloy, 2019).

[0010] Due to its involvement in many pathological conditions, the Receptor for Advanced Glycation Endproducts (RAGE) has been proposed as a therapeutic target to treat diseases such as atherosclerosis, diabetes, Alzheimer’s, ischemia, arthritis and cancer (Deane et al., 2012; Hudson and Lippman, 2018; Kang et al., 2010; Taguchi et al., 2000; Yan et al., 2010). There is an ongoing and unmet need for improved compositions and methods for targeting HS-HSBP interactions, and conditions for which these interactions could provide prophylactic or therapeutic approaches. The present disclosure is pertinent to this need.

[0011] SUMMARY OF THE DISCLOSURE

[0012] The present disclosure provides compositions and methods for inhibition of RAGE activation and signaling. The disclosure provides isolated antibodies, including monoclonal and polyclonal antibodies and fragments and variants thereof that are specific for the heparan-binding site of RAGE. The disclosure provides compositions comprising the antibodies, nucleic acid molecules encoding the antibodies or portions thereof or variants thereof, vectors comprising the nucleic acid molecules, cells comprising the antibodies and / or nuclei acid molecules, kits comprising one or more antibodies or nucleic acid molecules, and methods of using the antibodies or nucleic acid molecules or cells comprising the antibodies or nucleic acid molecules to inhibit RAGE activation and signaling.

[0013] This disclosure also provides a description of a process used to identify mAbs that specifically target the HS-binding site of human RAGE from a pool of 94 anti- RAGE murine hybridoma clones. The disclosure reveals that the chance of obtaining mAbs that target the HS-binding site is relatively high (19%), and epitopes within the HS-binding site can display vastly different immunogenicity. A common feature of the anti-HS-binding site mAbs is that they all contain high abundance of acidic residues in their complementarity determining regions (CDRs), and the vast majority of these acidic residues are harbored in the gamma chain. The disclosure also provides mAbs that target different epitopes within the HS-binding site and impair HS-RAGE interaction to different degrees, which could be used to tune biological potency of anti-HS-binding site mAbs. By providing the described analysis of mAbs that target the HS-binding site of a HS-binding protein, the disclosure provides a method of targeting HS-HSBP interactions.

[0014] In one aspect, the disclosure provides an isolated antibody, which may be a polyclonal or a monoclonal antibody (mAb), which is specifically reactive against the HS binding site of RAGE. The antibody may be generated in response to administration of a RAGE or a peptide thereof or a modification thereof.

[0015] The antibodies of the present disclosure may be chimeric, human, or humanized antibodies. In a chimeric or humanized antibodies, some portions of the heavy and / or light chains may be identical or homologous to sequences from one species while other portions may be identical or homologous to sequences from a different species. For example, murine or rabbit monoclonal antibodies may be isolated or generated and then portions of these antibodies (or sequence information derived therefrom) used for generating chimeric or humanized antibodies. For example, rabbits may be immunized with RAGE or RAGE peptides and then ascites fluid samples can be collected. The samples can be screened and selected to develop a panel of monoclonal antibodies and corresponding hybridoma cell lines. Portions or sequences from the monoclonal antibodies can then be used to generate chimeric or humanized antibodies. An antibody of the present disclosure can also be an antibody fragment, a single chain, a bispecific or multispecific antibody.

[0016] The disclosure provides nucleic acid molecules comprising sequences encoding portions or all of the antibodies (including mAbs) sequences. The disclosure also provides cells comprising nucleic acid molecules.

[0017] This disclosure provides a method of treatment of a condition associated with RAGE or abnormal RAGE function. Conditions in which RAGE function is abnormal or altered include atherosclerosis, arthritis, congestive heart failure, Alzheimer’s disease, diabetes, myocardial infraction, peripheral vascular disease, psoriasis, sepsis, cancer, osteoporosis, periodontitis, tumors and drug-induced liver toxicity, and cancer. The method comprises administering to an individual afflicted with a condition in which RAGE function is altered a therapeutically effective amount of one or more antibodies that are specific for the heparin binding site of RAGE. BRIEF DESCRIPTION OF FIGURES

[0018] Figure 1. Project design, screening method and project outcome. (A) Cartoon representation of HS-induced RAGE hexamer (PDB:4IM8). The hexamer is organized as a trimer of dimers, with each dimer stabilized by one molecule of HS oligosaccharide (shown in spheres). (B) The HS-binding site of RAGE is located in a positively charged cleft between dimer-related subunits and includes 14 basic residues (7 from each monomer). The HS oligosaccharide is shown in spheres, and the HS-binding residues are shown in stick (C) Epitopes screened in this project. Five epitopes are chosen to screen for mAbs, which include three epitopes within the HS-binding site (HS-1 , -2 and -3); one epitope around residue R98 and K110 a surface that is involved in ligand binding; and the whole C2 domain. (D) Supernatant of hybridoma clones were screened by ELISA against immobilized wild-type (WT) sRAGE or mutant sRAGE. Mutant sRAGE used are R216A-R218A, K39A-K43A- K44A, R104A-K107A, R98A-K110A and C2 domain truncated sRAGE. Comparison between the binding signals from WT plate and mutant plate reveals the clones that depend on the mutated epitope for binding. (E) Number of mAbs identified for each epitope.

[0019] Figure 2. Confirmation of epitope specificity of identified mAbs. (A-F) Binding of murine mAbs 91 E12, 88A5, 99G6, 44C6, 84A6, 93B10 to immobilized wild-type sRAGE or mutant sRAGE were determined by ELISA. The corresponding epitope of each clone is indicated after the clone name in parenthesis.

[0020] Figure 3. Sequence alignment of kappa and gamma chains of identified mAbs. (A-B) The variable regions of kappa / lambda (A) and gamma (B) chains were aligned using Clustal Omega. Kappa / The homologous sequences are clustered together. Residues are color coded with acidic residues in Blue, basic residues in Purple, hydrophobic residues in Red and polar residues in Green. The CDRs are indicated by dashed boxes in black. The corresponding epitope of each clone is indicated after the clone name. (C-D) Total number of acidic residues found in the CDRs of the light chains (C) and the heavy chains (D). Kappa / lambda chains as described herein are also referred to as light chain. Gamma chains are also referred to herein as heavy chains. The kappa / lambda chain sequences are shown in Fig. 3A. The gamma chains sequences are shown in Fig. 3B. The sequences shown in Fig. 3 are: 84A6 Heavy chain

[0021] MKCSWIIFFLMAWTGVNSEVQLQQSGAELVKPGASVKLSCTASGFNIKDYYIHWVK QRTEQGLQWIGRIDPEDGETKYAPKFQDKATITADTSSNTAYLQLSSLTSEDTAVYY CARGGDGEGDYWGQGTTLTVSS (SEQ ID NO:1 )

[0022] 84A6 Light chain

[0023] MVSTPQFLVFLLFWIPASSGDILLIQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQ RTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDVADYYCQQSDSWP LTFGAGTKLELK (SEQ ID NO:5)

[0024] 44C6 Heavy chain

[0025] MKCSWVIFFLMAVVIGINSEVPLQQSGAELVRSGASVKLSCTASGFNIKDYYMQWV KQRPEQGLEWIGWIDPENGDTEYAPKFQGKATMTADTSSNTAYLHLSSLTSEDSAV YFCKGDYDRAYWGQGTLVTVSA (SEQ ID NO:9)

[0026] 44C6 Light chain

[0027] MMSPAQFLFLLVLWIRETYGDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGMTY LNWLFQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC WQGTHFPQTFGGGTKLEIK (SEQ ID NO:13)

[0028] 91 E12 Heavy chain

[0029] MGWSYIILFLVATATGVHSQVQLQQPGAELVKPGTSVKLSCKASGYNFTSYWINWV RLGPGQGLEWIGDIHPGSGGNNYNEKFKRKATLTVDTSSSTAYMQLSSLASEDSAL YYCASDSLYDHVEVPFAYWGQGTLVTVSA (SEQ ID NO:17)

[0030] 91 E12 Light chain

[0031] MDFQVQIFSFLLISASVKLSRGQIVLTQSPAIMSASPGEKVTMTCSARSSVSYMYWH QQKPGSSPRLLIYDTSNLASGVPVRFSGGGSGTSYSLTISRMEAEDAATYFCQQWS TYPPTFGGGTRLEIK (SEQ ID NO:21 )

[0032] 93B10 Heavy chain

[0033] MA WWTLLFLMAAAQSIQAQIQLVQSGPELKKPGETVKISCKASGYTFTDFSMHVW KQAPGKGLKWMGWIDTETGEPTYADDFKGRFAFSLETSASTVYLQINNLKDEDTAT YFCASDYYAMDYWGQGTSVTVSSAKTTPPS (SEQ ID NO:25) 93B10 Light chain

[0034] MAWISLILSLLALSSGAISQAWTQESALTTSPGETVTLTCRSSTGVVTTSNYANVWQ EKPDHLFTGLIGGTNNRISGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNH LVFGGGTKLTVL (SEQ ID NO:26)

[0035] 99G6 Heavy chain

[0036] MEWTWVFLFLLSVTAGVHSQVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEW VKQRPGHGLEWIGEILPGSDSTNYNEKFKGKATFTADTSSNTAYMQFTSLTSEDSAV YYCARGPNYPDYFDYWGQGTTLTVSSAKTTPPSV (SEQ ID NO:27)

[0037] 99G6 Light chain

[0038] MDFQVQIFSFLLISASVIMSRGESVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWY QQKSSTSPKLWIYDTSKLASGVPGRFSGSGSGNSYSLTISSMEAEDVAAYYCFQGN GYPYTFGGGTKLEIK (SEQ ID NO:28)

[0039] 53C2 Heavy chain

[0040] MGWSWIFLFLLSGTAGVLSEDQLQQSGPELVKPGASVKISCKASGYSFTGYYMHW VRQSHVKSLEWIGRIIPYNGAASYNQNFKDKASLTVDTSSSTAYMELHSLTSEDSAV YYCTRAETGPLAYWGQGTLVTVSA (SEQ ID NO:29)

[0041] 53C2 Light chain

[0042] MDSQAQVLMLLLLWVSGTCGDIVMSQSPSSLAVSVGEKVTLSCKSSQSLLYSSNQK NYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVY YCQQYYSYPWTFGGGTKLEIK (SEQ ID NQ:30)

[0043] 88A5 Heavy chain

[0044] MEWTVWFLFLLSVTAGVHSQVQLQQSGAELMKPGASVKLACKATGYTFTGHWIE VWNQRPGHGLEWIGEILPGSGSTNYNEKFKGKATFIADSSSNTAYLQLSSLTTEDSA IYYCAAGGAFDYWGQGTTLTVSS (SEQ ID N0:31 )

[0045] 88A5 Light chain

[0046] MGTFDSPYQVRRMRFSAQLLGLLVLWIPGSTADIVMTQAAFSNPVSLGTSASISCRS

[0047] SKSLLHSDGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISR VEAEDVGVYYCAQHLELPTFGGGTKLEIK (SEQ ID NO:32) Figure 4.SPR analysis of the binding kinetics between mAbs and human sRAGE. (A-F) SPR sensorgrams of mAbs binding to immobilized biotinylated human-sRAGE. The black lines represent fitted curve based on 1 :1 Langmuir binding model using TraceDrawer 1 .9.2. The corresponding epitope of each clone is indicated after the clone name in parenthesis.

[0048] Figure 5. Anti-HS-binding site mAbs inhibit sRAGE-heparin interaction.

[0049] (A-B) WT sRAGE were either directly loaded onto heparin Sepharose column, or loaded after pre-incubation with mAbs (91 E12 or 99G6) targeting the HS-1 eiptope (A), or mAbs (44C6 or 84A6) targeting the HS-2 epitope (B). The red line represents the salt gradient (in conductivity mS / cm, from 150 mM to 1.5 M). The elution salt concentration for each peak was indicated above the peak.

[0050] Figure 6. Anti-HS-binding site mAbs inhibit osteoclastogenesis in vitro.

[0051] Human bone marrow macrophages were cultured in complete aMEM medium supplemented with 20 ng / mL M-CSF and 100 ng / mL RANKL for 5 days to induce osteoclastogenesis. Cells were incubated with either control murine IgG 1 , or anti- RAGE mAbs at 20 ug / mL . (A) Representative images of TRAP staining of osteoclasts. Osteoclasts are multi-nucleated (>3 nuclei) TRAP positive cells (stained purple). The corresponding epitope of each clone is indicated after the clone name in parenthesis. (B-D) Osteoclasts were sub-grouped into three categories based on the number of nuclei and quantified. (E-G) Dose-dependent inhibition of osteoclastogenesis by 91 E12 and 84A6. mAbs were used at 2, 5 or 10 pg / ml . Osteoclast were sub-grouped into three categories based on the number of nuclei and quantified. n=14 fields taken in 10X magnification. * represents p<0.05, ** represents p<0.01 , *** represents p< 0.001.

[0052] Figure 7 shows that mAbs 91 E12, 44C6 and 84A6 specifically binds the HS- binding site of RAGE. The apparent Ko of the mAbs to RAGE was analyzed by ELISA. Briefly, WT-RAGE or mutant RAGEs (with mutated HS-binding residues: K39A-K43A-K44A or R216A-R218A) were coated on a 96-well plate. mAbs ranging from 3 ng / ml to 3 pg / ml and 30 ng / ml to 30 pg / ml was used for the binding to WT- RAGE or mutant RAGE, respectively. This assay maps the epitope of 84A6 and 44C6 to K39-K43-K44 region, and the epitope of 91 E12 to R216-R218 region. Results are shown in the graphs, as indicated. A ribbon diagram is provided, as annotated.

[0053] Figure 8 provides the variable regions of 91 E12, 44C6 and 84A6. The heavy chain sequences shown in the top panel. The light chain sequences are shown in the bottom panel. The CDRs are indicated. The sequences of the variable regions of the mAbs were obtained by Rapid amplification of cDNA ends. Briefly, the RNAs of the hybridoma cells were isolated and a reverse transcription assay was performed to amplify the cDNA coding for the variable region. The cDNA was then sequenced. The sequences shown in Fig. 8 with CDRs as indicated are:

[0054] 84A6 Heavy chain

[0055] MKCSWIIFFLMAWTGVNSEVQLQQSGAELVKPGASVKLSCTASGFNIKDYYIHWVK QRTEQGLQWIGRIDPEDGETKYAPKFQDKATITADTSSNTAYLQLSSLTSEDTAVYY CARGGDGEGDYWGQGTTLTVSS (SEQ ID NO:1)

[0056] 84A6 Heavy chain CDRs:

[0057] 84A6 Heavy chain CDR1 FNIKDYYIH (SEQ ID NO:2);

[0058] 84A6 Heavy chain CDR2 GRIDPEDGETKYAPKFQD (SEQ ID NO:3)

[0059] 84A6 Heavy chain CDR3 ARGGDGEGDY (SEQ ID NO:4)

[0060] 84A6 Light chain

[0061] MVSTPQFLVFLLFWIPASSGDILLIQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQ RTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDVADYYCQQSDSWP LTFGAGTKLELK (SEQ ID NO:5)

[0062] 84A6 Light chain CDRs:

[0063] 84A6 Light chain CDR1 RASQSIGTSIH (SEQ ID NO:6)

[0064] 84A6 Light chain CDR2 LLIKYASESIS (SEQ ID NO:7)

[0065] 84A6 Light chain CDR3 QQSDSWPLTF (SEQ ID NO:8)

[0066] 44C6 Heavy chain

[0067] MKCSWVIFFLMAWIGINSEVPLQQSGAELVRSGASVKLSCTASGFNIKDYYMQWV KQRPEQGLEWIGWIDPENGDTEYAPKFQGKATMTADTSSNTAYLHLSSLTSEDSAV YFCKGDYDRAYWGQGTLVTVSA (SEQ ID NO:9) 44C6 Heavy chain CDRs:

[0068] 44C6 Heavy chain CDR1 FNIKDYYMQ (SEQ ID NO:10)

[0069] 44C6 Heavy chain CDR2 GWIDPENGDTEYAPKFQG (SEQ ID N0:11)

[0070] 44C6 Heavy chain CDR3 KGDYDRAY (SEQ ID N0:12)

[0071] 44C6 Light chain

[0072] MMSPAQFLFLLVLWIRETYGDWMTQTPLTLSVTIGQPASISCKSSQSLLDSDGMTY LNWLFQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC WQGTHFPQTFGGGTKLEIK (SEQ ID NO: 13)

[0073] 44C6 Light chain CDRs:

[0074] 44C6 Light chain CDR1 KSSQSLLDSDGMTYLN (SEQ ID NO:14)

[0075] 44C6 Light chain CDR2 RLIYLVSKLDS (SEQ ID NO:15)

[0076] 44C6 Light chain CDR3 WQGTHFPQTF (SEQ ID NO:16)

[0077] 91 E12 Heavy chain

[0078] MGWSYIILFLVATATGVHSQVQLQQPGAELVKPGTSVKLSCKASGYNFTSYWINWV RLGPGQGLEWIGDIHPGSGGNNYNEKFKRKATLTVDTSSSTAYMQLSSLASEDSAL YYCASDSLYDHVEVPFAYWGQGTLVTVSA (SEQ ID NO:17)

[0079] 91 E12 Heavy chain CDRs:

[0080] 91 E12 Heavy chain CDR1 YNFTSYWIN (SEQ ID NO:18)

[0081] 91 E12 Heavy chain CDR2 GDIHPGSGGNNYNEKFKR (SEQ ID NO:19)

[0082] 91 E12 Heavy chain CDR3 ASDSLYDHVEVPFAY (SEQ ID NO:20)

[0083] 91 E12 Light chain

[0084] MDFQVQIFSFLLISASVKLSRGQIVLTQSPAIMSASPGEKVTMTCSARSSVSYMYWH QQKPGSSPRLLIYDTSNLASGVPVRFSGGGSGTSYSLTISRMEAEDAATYFCQQW STYPPTFGGGTRLEIK (SEQ ID NO:21)

[0085] 91 E12 Light chain CDRs:

[0086] 91 E12 Light chain CDR1 SARSSVSYMY (SEQ ID NO:22)

[0087] 91 E12 Light chain CDR2 LLIYDTSNLAS (SEQ ID NO:23) 91 E12 Light chain CDR3 QQWSTYPPTF (SEQ ID NO:24)

[0088] Figure 9 shows affinity determination by surface plasmon resonance (SPR). SPR analysis of the binding kinetics between mAbs and human sRAGE. SPR sensorgrams of mAbs binding to immobilized biotinylated human-sRAGE. The black lines represent fitted curve based on 1 :1 Langmuir binding model using TraceDrawer 1.9.2. A table summarizing data is provided.

[0089] Figure 10 shows the mAbs block RAGE-dependent osteoclastogenesis. The osteoclastogenesis assay was carried out using human monocytes isolated from whole bone marrow. A photographic representation is shown at the top. During the differentiation process, 20 pg / ml of mAbs, or control murine IgG, were added to the culture. After 5 days, cells were stained for the presence of TRAP, a specific marker for mature osteoclasts. Quantification was done based on the size of the osteoclast, n=3. *P<0.05, **P<0.01. Results are shown in the left bottom graph. The inhibitory potency of 91 E12 and 84A6 were further compared using lower concentration of mAbs at 5 and 10 pg / ml. Quantification was done based on the size of the osteoclast (number of nuclei per osteoclasts), n=3. *P<0.05, **P<0.01 , ***P<0.001 , ****p<0.0001 . The result indicate that the efficacy of 84A6 is superior to 91 E12 in this assay, which can be effective at concentration as low as 5 pg / ml. The results are shown in the bottom right two graphs.

[0090] Figure 11 shows 91 E12, 44C6 and 84A6 inhibit the binding of RAGE to Heparin column. To confirm the mechanism of action of the shown mAbs, we examined the inhibitory effect of the mAbs on binding of RAGE to a column immobilized with heparin as a special form of heparan sulfate. RAGE alone binds heparin column strongly and requires 60ms / cm salt concentration for elution. 91 ENRAGE complex showed a significant reduction of binding to heparin column, requires only 48ms / cm salt for elution. 44C6-RAGE and 84A6-RAGE complexes displayed a dramatic reduction of binding to heparin column, requiring only 25.3ms / cm salt for elution. Results are shown in the graphs, as indicated.

[0091] DETAILED DESCRIPTION

[0092] The present disclosure is related to antibodies that specifically target the HS binding of RAGE with high specificity, and methods of using the antibodies. Throughout this application, the use of the singular form encompasses the plural form and vice versa. For example, “a”, or “an” also includes a plurality of the referenced items, unless otherwise indicated.

[0093] Where a range of values is provided in this disclosure, it should be understood that each intervening value to the tenth decimal place of the lowest value, and all intervening ranges, between the upper and lower limit of that range is also included, unless clearly indicated otherwise. The upper and lower limits from within the broad range may independently be included in the smaller ranges encompassed within the disclosure.

[0094] The term “therapeutically effective amount” as used herein refers to an amount of an agent sufficient to achieve, in a single or multiple doses, the intended purpose of treatment. Treatment does not have to lead to complete cure, although it may. Treatment can mean alleviation of one or more of the symptoms or markers of the indication. The exact amount desired or required will vary depending on the particular compound or composition used, its mode of administration, patient specifics and the like. Appropriate effective amount can be determined by one of ordinary skill in the art informed by the instant disclosure using only routine experimentation. Within the meaning of the disclosure, “treatment” also includes prophylaxis and treatment of relapse, as well as the alleviation of acute or chronic signs, symptoms and / or malfunctions associated with the indication. Treatment can be orientated symptomatically, for example, to suppress symptoms. It can be effected over a short period, over a medium term, or can be a long-term treatment, such as, for example within the context of a maintenance therapy. Administrations may be intermittent, periodic, or continuous.

[0095] This disclosure provides isolated antibodies and fragments or variants thereof directed to HS binding site of RAGE, isolated nucleic acid molecules encoding antibodies or fragments or variants thereof, cells producing antibodies or fragments or variants thereof, vectors or cells comprising nucleic acids encoding antibodies or fragments or variants thereof, compositions comprising any of the foregoing, methods of making any of the foregoing, and methods of using the antibodies and fragments thereof, or nucleic acid molecules in the treatment of conditions associated with RAGE. Also provided are conjugates of the antibodies, fragments and variants thereof and drugs - generally referred to as antibody-drug conjugates or ADCs.

[0096] In particular, in the present disclosure, HS-RAGE specific antibodies are used to block HS-RAGE interaction that is considered to prevent RAGE oligomerization and activation. It is considered that HS-RAGE interaction and RAGE oligomerization are required for RAGE signaling. Pharmacologically, the present strategy is different from the conventional strategy in terms of the molecular mechanism of action. By targeting the surfaces that are not involved in ligand binding, this strategy is noncompetitive in nature. A significant advantage of a non-competitive inhibitor over a competitive inhibitor is that the IC50 is unaffected by the ligand concentration. This is advantageous because it helps maintain efficacy at low drug concentrations. Since HS-dependent RAGE oligomerization appears to be required for all RAGE ligands, blocking HS-RAGE interactions may be a universal way to block RAGE signaling.

[0097] In non-limiting examples, this disclosure provides three new murine mAbs that display high affinity to human RAGE and bind to different parts of the HS-binding site of RAGE. One of the mAbs, referred to herein as 91 E12, targets one part of the HS binding site of RAGE, which is the same epitope targeted by an antibody referred to as “B2” which is described in PCT publication WO 2021 / 087462. The other two mAbs, referred to herein as 44C6 and 88A6 target different parts of the HS-binding site of RAGE. 91 E12, 44C6 and 84A6, were found to specifically bind part of the heparan sulfate (HS)-binding site of RAGE and inhibits HS-RAGE interaction. Functional assay confirmed that these mAbs block differentiation of osteoclasts from human monocytes. All three mAbs show very high affinity to human RAGE, affinity ranging from 3 to 136 Pm. The mechanism of action of 91 E12, 44C6 and 84A6 involves blocking RAGE-HS interaction, which is required for RAGE activation. Mouse mAbs 91 E12, 44C6 and 84A6 specifically bind the HS-binding site of human RAGE. The variable regions of 91 E12, 44C6 and 84A6 were sequenced. The affinities of the mAbs to human RAGE were determined by SPR. Mouse mAbs 91 E12, 44C6 and 84A6 block RAGE-dependent osteoclastogenesis.91 E12, 44C6 and 84A6 block RAGE binding to heparin.

[0098] The gamma-heavy chain and kappa-light chain sequences for each of the described antibodies are provided in Fig. 8. The CDRs are indicated. Kappa / lambda chains as described herein are also referred to as light chains. Gamma chains are also referred to herein as heavy chains. The disclosure includes the following sequences:

[0099] 84A6 Heavy chain

[0100] MKCSWIIFFLMAWTGVNSEVQLQQSGAELVKPGASVKLSCTASGFNIKDYYIHWVK QRTEQGLQWIGRIDPEDGETKYAPKFQDKATITADTSSNTAYLQLSSLTSEDTAVYY CARGGDGEGDYWGQGTTLTVSS (SEQ ID NO:1)

[0101] 84A6 Heavy chain CDRs:

[0102] 84A6 Heavy chain CDR1 FNIKDYYIH (SEQ ID NO:2);

[0103] 84A6 Heavy chain CDR2 GRIDPEDGETKYAPKFQD (SEQ ID NO:3)

[0104] 84A6 Heavy chain CDR3 ARGGDGEGDY (SEQ ID NO:4)

[0105] 84A6 Light chain

[0106] MVSTPQFLVFLLFWIPASSGDILLIQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQ RTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDVADYYCQQSDSWP LTFGAGTKLELK (SEQ ID NO:5)

[0107] 84A6 Light chain CDRs:

[0108] 84A6 Light chain CDR1 RASQSIGTSIH (SEQ ID NO:6)

[0109] 84A6 Light chain CDR2 LLIKYASESIS (SEQ ID NO:7)

[0110] 84A6 Light chain CDR3 QQSDSWPLTF (SEQ ID NO:8)

[0111] 44C6 Heavy chain

[0112] MKCSWVIFFLMAWIGINSEVPLQQSGAELVRSGASVKLSCTASGFNIKDYYMQWV KQRPEQGLEWIGWIDPENGDTEYAPKFQGKATMTADTSSNTAYLHLSSLTSEDSAV YFCKGDYDRAYWGQGTLVTVSA (SEQ ID NO:9)

[0113] 44C6 Heavy chain CDRs:

[0114] 44C6 Heavy chain CDR1 FNIKDYYMQ (SEQ ID NO:10)

[0115] 44C6 Heavy chain CDR2 GWIDPENGDTEYAPKFQG (SEQ ID NO:11)

[0116] 44C6 Heavy chain CDR3 KGDYDRAY (SEQ ID NO:12)

[0117] 44C6 Light chain MMSPAQFLFLLVLWIRETYGDWMTQTPLTLSVTIGQPASISCKSSQSLLDSDGMTY LNWLFQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC WQGTHFPQTFGGGTKLEIK (SEQ ID NO: 13)

[0118] 44C6 Light chain CDRs:

[0119] 44C6 Light chain CDR1 KSSQSLLDSDGMTYLN (SEQ ID NO:14)

[0120] 44C6 Light chain CDR2 RLIYLVSKLDS (SEQ ID NO:15)

[0121] 44C6 Light chain CDR3 WQGTHFPQTF (SEQ ID NO:16)

[0122] 91 E12 Heavy chain

[0123] MGWSYIILFLVATATGVHSQVQLQQPGAELVKPGTSVKLSCKASGYNFTSYWINWV RLGPGQGLEWIGDIHPGSGGNNYNEKFKRKATLTVDTSSSTAYMQLSSLASEDSAL YYCASDSLYDHVEVPFAYWGQGTLVTVSA (SEQ ID NO:17)

[0124] 91 E12 Heavy chain CDRs:

[0125] 91 E12 Heavy chain CDR1 YNFTSYWIN (SEQ ID NO:18)

[0126] 91 E12 Heavy chain CDR2 GDIHPGSGGNNYNEKFKR (SEQ ID NO:19)

[0127] 91 E12 Heavy chain CDR3 ASDSLYDHVEVPFAY (SEQ ID NO:20)

[0128] 91 E12 Light chain

[0129] MDFQVQIFSFLLISASVKLSRGQIVLTQSPAIMSASPGEKVTMTCSARSSVSYMYWH QQKPGSSPRLLIYDTSNLASGVPVRFSGGGSGTSYSLTISRMEAEDAATYFCQQW STYPPTFGGGTRLEIK (SEQ ID NO:21)

[0130] 91 E12 Light chain CDRs:

[0131] 91 E12 Light chain CDR1 SARSSVSYMY (SEQ ID NO:22)

[0132] 91 E12 Light chain CDR2 LLIYDTSNLAS (SEQ ID NO:23)

[0133] 91 E12 Light chain CDR3 QQWSTYPPTF (SEQ ID NO:24)

[0134] The disclosure includes all sequences that are 80-100% identical to the figures shown in Fig. 8. The disclosure includes the heavy chain and light chain CDRs that are in the context of different framework sequences, such as for humanization. In this regard, the terms “antibody” as used herein can encompass whole antibody molecules, full-length immunoglobulin molecules, such as naturally occurring full-length immunoglobulin molecules or full-length immunoglobulin molecules formed by immunoglobulin gene fragment recombinatorial processes, as well as antibody fragments including scFvs. Antibody fragments can be fragments comprising at least one antibody-antigen binding site. Antibody fragments can, for example, exhibit specific binding to HS binding site of RAGE.

[0135] The term “antibody” can include e.g. monoclonal, polyclonal, multispecific (for example bispecific), recombinant, human, chimeric and humanized antibodies. The term “antibody” can also encompass recombinantly expressed antigen binding proteins and antigen binding synthetic peptides. Further, the term “antibody” as used herein encompasses minibodies, and diabodies, all of which preferably exhibit specific binding to HS binding site of RAGE, especially HD binding site of human RAGE. The term “antibody”, as used herein, can also encompass antibodies produced in vivo, as well as those produced in vitro, such as, for example, by a bacterial expression system or a mammalian hybridoma cell line.

[0136] An antibody of the present disclosure may be modified by, for example, acetylation, formylation, amidation, phosphorylation, or polyethylene glycolation (PEGylation), as well as glycosylation. The term “an antibody” as used herein is intended to cover all antibodies disclosed herein. For example, the term “an antibody” can refer to monoclonal, polyclonal, scFv, chimeric, human, or humanized antibodies, or antigen (i.e. , HS binding portion of RAGE) binding fragments thereof. The fragments or other derivatized molecules (such as scFvs and the like) may generally be referred to as “antibodies” in this disclosure.

[0137] In an example, the present antibodies are PEGylated to increase half-life and bioavailability. Various types of PEG molecules can be added to the antibodies, such as scFvs, including but not limited to PEG(5K), PEG(10K), PEG(20K), and PEG(40K). The antibodies or fragments, derivatives thereof, such as scFvs can be modified by the addition of a cysteine, lysine or serine amino acid to facilitate PEGylation. This modification can occur at, but is not limited to, the C-terminus of the antibodies. The antibodies of the disclosure may be whole immunoglobulin molecules such as polyclonal or monoclonal antibodies or may be antigen-binding fragments thereof, including but not limited to, Fab, F(ab'), F(ab')2, Fv, dAb, Fd, CDR fragments, single-chain variable fragment antibodies (scFv), bivalent single-chain antibodies, single-chain phage antibodies, diabodies, nanobodies, BiKes, BiTes and the like. The fragments of the antibodies may be produced synthetically or by enzymatic or chemical cleavage of intact immunoglobulins or may be genetically engineered by recombinant DNA techniques. These techniques are well known in the art.

[0138] In one example, this disclosure provides isolated antibodies. By the term “isolated” it is meant that the antibody or the fragment thereof, is separated and / or recovered from its natural environment. The isolation of the antibody from its natural environment can be such that the antibody can be used without interference from other active agents (such as other proteins) that normally are present in its natural environment.

[0139] In one example, this disclosure provides generating and isolating single domain antibodies or nanobodies produced by camelids in response to introducing RAGE or RAGE peptides into the camelids. The nanobodies are typically heavy chain antibodies and thus contain heavy chain homodimers and do not contain antibody light chains. These antibodies typically comprise a single variable domain and two constant domains (CH2 and CH3).

[0140] The antibodies of the present disclosure may be obtained from a human or a non-human animal. In many mammals, intact immunoglobulins have two heavy chains and two light chains. Each of the light chains is covalently linked to a heavy chain by a disulfide bond. The two heavy chains are linked to each other by additional disulfide bonds. The light chain typically has one variable domain (VL) and one constant domain (CL). The heavy chain can also have one variable domain (VH). The variable domains contain complementarity-determining regions (CDRs). The heavy chain can further have three or four constant domains (CHI, CH2, CH3 and CH4). The variability of the constant domains results is various isotypes such as IgA, IgD, IgE, IgG, and IgM. The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1 , CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 (referred to as VH-CDR3 or CDRH3) is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a VL CDR1 (referred to as VL-CDR1 or CDRL1 ) is the CDR1 from the variable domain of the light chain of the antibody in which it is found. An antibody that binds to the HS binding site of RAGE, for example, will have a specific VH region and the VL region sequence, and thus specific CDR sequences. Antibodies with different specificities (i.e. different combining sites for different antigens) have different CDRs.

[0141] The terms VH or VH as used herein refer to the variable region of an immunoglobulin heavy chain, including a heavy chain of an Fv, scFv, dsFv or Fab, and the terms VL or VL refer to the variable region of an immunoglobulin light chain, including a light chain of an Fv, scFv, dsFv or Fab.

[0142] The term “monoclonal antibody” refers to an antibody produced by a single clone of B-lymphocytes or by a cell into which the light and / or heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibodyforming cells from a fusion of myeloma cells with immune spleen cells. For example, mice, rabbits (or other suitable animals) may be immunized with RAGE or RAGE peptides and then ascites fluid samples can be collected. The samples can be screened and selected to develop a panel of monoclonal antibodies and corresponding hybridoma cell lines. Animal monoclonal antibodies may be isolated or generated and then humanized, if desired.

[0143] An antibody of the present disclosure can be an antibody of any class. For example, an antibody of the present invention can be an antibody isotype lgG1 , lgG2, lgG3, lgG-4, IgM, IgA, IgD or IgE. For example, the antibody can be lgG2b. The term “isotype”, as used herein, can in particular refer to the antibody class (such as e.g. IgG) that is encoded by heavy chain constant region genes. Sequences of human immunoglobulin constant regions are known in the art and are available in public databases such as National Center for Biotechnology Information (NCBI), U.S. National Library of Medicine.

[0144] The term “chimeric antibody” refers to an antibody which has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species, such as a murine antibody that specifically binds the HS binding site of RAGE. In a chimeric antibody, some portions of the heavy and / or light chains may be identical or homologous to sequences from a particular species while other portions may be identical or homologous to sequences from a different species. Chimeric antibodies generally exhibit decreased immunogenicity and increased stability. Techniques for cloning murine immunoglobulin variable domains known in the art - such as, for example, see Orlandi et al., Proc. Natl Acad. Sci. USA 86: 3833 (1989), and Leung et al., Hybridoma 13:469 (1994). As an example of a chimeric antibody, polynucleotides encoding the variable domains of the light chain or the heavy chain of an antibody derived from an animal (e.g., mouse, rat, or chicken) other than human can be linked to polynucleotides encoding the constant domains of the light chain or the heavy chain derived from a human antibody to produce a polynucleotide (such as DNA) encoding a chimeric antibody.

[0145] A “human” antibody (also called a “fully human” antibody) is an antibody that includes human framework regions and all of the CDRs from a single or different human immunoglobulins. Thus, frameworks from one human antibody can be engineered to include CDRs from a different human antibody. Methods for producing human antibodies are known in the art - such as, for example, see Mancini et al., 2004, New Microbiol. 27:315-28; Conrad and Scheller, 2005, Comb. Chem. High Throughput Screen. 8:117-26.

[0146] A “humanized antibody” is typically a human antibody that has one or more amino acid residues imported into it (i.e. , introduced into it) from a source that is nonhuman. For example, a humanized antibody is a recombinant protein in which the CDRs of an antibody from a species such as rodent, rabbit, dog, goat, or horse are imported into human heavy and light variable domains. The constant domains (also referred to as framework regions) of the antibody molecule are generally the same as those of a human antibody. The non-human immunoglobulin providing the CDRs can be termed as “donor” and the human immunoglobulin providing the framework can be termed as “acceptor”. For example, all the CDRs can be from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be always present, but if they are, they can be substantially identical to human immunoglobulin constant regions, i.e. , at least about 85-90%, such as about 95% or more identical. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. Humanized immunoglobulins can be constructed by means of genetic engineering (see for example, U.S. Pat. No. 5,585,089, and U.S. Publication No. 2010 / 0196266).

[0147] Antibody fragments can be produced by enzymatic digestion. For example, papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, and a “Fc” fragment. The Fab fragment contains an entire L chain and the variable region domain of the H chain (VH), and the first constant domain of one heavy chain. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment that roughly corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is capable of cross-linking antigen. “Fv” is the minimum antibody fragment that contains a complete antigen-recognition and -binding site and single-chain Fv also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. The term “diabodies” refers to small antibody fragments prepared by constructing sFv fragments with short linkers between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., fragment having two antigen-binding sites. A single domain antibody (sdAb) is an antibody fragment which has a single monomeric variable antibody domain. ScAbs can be made from heavychain antibodies found in camelids. An antibody fragment can be a single variable region or a peptide consisting of or comprising a single CDR. A single-chain antibody has a heavy chain variable domain and a light chain variable domain linearly linked to each other via a linker. A polynucleotide (such as DNA) encoding the single-chain antibody can be produced by binding a polynucleotide encoding the heavy chain variable domain, a polynucleotide encoding the linker (typically 10-20 nucleotides), and a polynucleotide encoding the light chain variable domain, with the heavy chain variable domain and the light chain variable domain being both derived from a human antibody.

[0148] The present disclosure also provides isolated nucleotide sequences encoding all or portions of the amino acid sequences disclosed herein. For example, the present disclosure provides an isolated nucleic acid molecule comprising the sequences of the CDRs, such as CDRL1 , CDRL2, CDRL3, CDRH1 , CDRH2, CDRH3 for the antibody as described in Figure 8. The heavy chain sequences are indicated in the top panel. The light chain sequences are indicated in the bottom panel.

[0149] In an example, the present disclosure also provides isolated nucleic acid molecules comprising or consisting of the sequence encoding one or more CDRs described herein. The disclosure also provides cells comprising an expression vector or other polynucleotide sequence encoding the antibodies provided herein (including but not necessarily limited to scFvs). Nucleotide sequences encoding the CDRs and the described heavy and light chains can be expressed using any suitable expression vector, many of which are known in the art and / or are commercially available. A vector generally includes nucleic acid sequences, such as origin or replication that enables it to replicate in a host cell. A vector can also include selectable marker genes. In an example, the disclosure provides a polynucleotide that is capable of hybridizing to a polynucleotide encoding a described heavy chain, a described light chain, or a combination thereof.

[0150] The isolated monoclonal antibodies or fragments thereof can be labeled, such as with enzymatic, fluorescent or radioactive tags or can be conjugated to effector molecules such as, for example, toxins.

[0151] The disclosure provides cells comprising an expression vector or other polynucleotide sequence encoding the antibodies provided herein (including mAbs) or fragments that bind to HS-binding site of RAGE. Nucleotide sequences encoding the mAbs or fragments thereof that bind to HS-binding site of RAGE can be expressed using any suitable expression vector, many of which are known in the art and / or are commercially available. A vector generally includes nucleic acid sequences, such as origin or replication that enables it to replicate in a host cell. A vector can also include selectable marker genes. Heavy and light chains can be expressed on a single expression vector, such as a plasmid or the heavy and light chains can be expressed on distinct plasmids in the same cell, after which the expressed heavy and light chains can form the conventional mAb architecture. The mAbs or RAGE binding fragments thereof can be isolated and / or purified using conventional techniques, given the benefit of the present disclosure.

[0152] This disclosure provides compositions comprising antibodies, antibody fragments or derivatives loaded onto liposomes. Standard approaches for ligand attachment to aqueous liposome nanoparticles make use of maleimides, succinimidyl esters and carbodiim ide-activated carboxylic acids. These can covalently react with amine and thiol groups of polypeptides. The use of maleimide- lipids has been explored extensively for antibody-conjugated liposomes. Conjugation yields may reach as high as 90% from an overnight reaction, but subsequent quenching of free maleimide groups and additional purification is required. Proteins may require a preparative step of thiolation and purification prior to conjugation. Antibody orientation is a major factor influencing the conjugated antibody target binding efficacy, but these approaches result in numerous antibody labeling sites and indiscriminate orientations. Biorthogonal synthetic strategies such as the click reaction have recently been applied to pre-formed liposomes, however these require the use exogenous catalysts and unconventional amino acids.

[0153] The liposomes in the present disclosure may be spherical or non-spherical. The size of the liposomes can be from 50 to 1000 nm or more. In one example, the liposomes have a size (e.g., a longest dimension such as, for example, a diameter) of 50 to 1000 nm, including all integer nm values and ranges therebetween. For example, the size may be from 50 to 200 nm or from 20 to 1000 nm. If the liposomes are not spherical, the longest dimension can be from 50 to 1000 nm. These dimensions can be achieved while preserving the nanostructure width of the monolayer or the bilayer. The liposomes can carry cargo in the aqueous compartment. The cargo, or part thereof, can also, or alternatively, be incorporated in the monolayer or the bilayer.

[0154] In an example the present antibodies, fragments or derivatives can be used in conjunction with cytotoxic drugs, such as in the form of antibody drug conjugates. For example the antibodies, fragments, or derivatives may be conjugated to campothecin, calicheamicin, maytansinoid, auristatin E (MMAE), pyrrolobenzodiazepine, or a derivative, analog or metabolite of any of the aforementioned drugs. The drugs may be anti-tumor drugs or drugs that inhibit the growth of cells.

[0155] In addition, the subjection invention provides an antibody or fragment thereof comprising a light chain variable region wherein one or more of residues 53, 117 or 121 is D and / or comprising a heavy chain variable region wherein residue 51 is D. Without being bound by any theory, these residues are believed to be the critical residues.

[0156] As used herein, antibody fragments include, but are not limited to, scFv, Fab, F(ab’)2, F(ab’), scFab, scFV-CH, scFV-Fc, scFv-zipper (2 scFvs connected by long linkers such as leucine zippers), diabodies (Db), scDb, triabodies, tetrabodies and minibodies.

[0157] Either I- or d-amino acids may be used in the invention. In certain examples, conservative substitutions of the amino acids are contemplated. The term, "conservative substitution," is used to reflect amino acid substitutions that do not substantially alter the activity (e.g., antifungal activity and / or specificity) of the molecule. Typically, conservative amino acid substitutions involve substitution of one amino acid for another amino acid with similar properties (e.g. charge, hydrophobicity and size). Conservative substitutions include, but are not limited to Gly / Ala; Arg / Lys; Ser / Tyr / Thr; Leu / lle / Val; Asp / Glu; Gln / Asn; and Phe / Trp / Tyr. Other examples of substitutions within the scope of the invention include: Gly / Ala / Pro; Tyr / His; Arg / Lys / His; Ser / Thr / Cys; and Leu / lle / Val / Met.

[0158] Substitution can also be in the form of analog substitutions where a standard amino acid is replaced by a non-standard amino acid such as a synthetic or rare amino acid differing minimally from the parent residue from which it is typically derived. Some examples of amino acid analogs are: beta-alanine (betaaminopropionic acid), 2-aminobutyric acid, 4-aminobutyric acid, piperidinic acid, 6- aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4 diaminobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, n-ethylglycine, n-ethylasparagine, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine, n-methylglycine, sarcosine, n-methylisoleucine, 6-n-methyllysine, n- methylvaline, norvaline, norleucine, and ornithine.

[0159] The antibodies and fragments retain their ability to bind the HS binding site of RAGE.

[0160] In an example, the antibody or fragment thereof is monoclonal. Optionally, the antibody or fragment thereof may be polyclonal.

[0161] In certain examples, the antibody or fragment thereof may be fully or partially humanized.

[0162] The present disclosure provides antibody-drug conjugates (ADCs). Any antibody, fragment, variant or derivative described here may be used. It may be conjugated, directly or via a linker, to a drug. Examples of drugs are those that can inhibit the growth of cells, such as cancer cells. Examples of drugs include campothecin, calicheamicin, maytansinoid, auristatin E (MMAE), pyrrolobenzodiazepine, or a derivative, analog or metabolite of any of the aforementioned drugs.

[0163] The present disclosure provides pharmaceutical compositions comprising the antibodies or fragments thereof, or ADCs and pharmaceutically suitable carrier. Suitable carriers include excipients, or stabilizers which are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as acetate, Tris, phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; tonicifiers such as trehalose and sodium chloride; sugars such as sucrose, mannitol, trehalose or sorbitol; surfactant such as polysorbate; salt-forming counter-ions such as sodium; and / or non-ionic surfactants such as Tween or polyethylene glycol (PEG). The pharmaceutical compositions may comprise other therapeutic agents.

[0164] Compositions of the present disclosure can comprise only one described antibody or more than one described antibody. A composition of the disclosure can have one or more of an antibody or fragment or variant thereof, A composition can comprise one or more subtypes of antibodies. For example, a composition can comprise a mixture of IgG or IgM or a mixture of one or more of IgG 1 , lgG2, and lgG2b. A composition of the present disclosure can comprise an antibody as the only active ingredient, wherein the antibody may be monoclonal, polyclonal, chimeric, human, humanized or combinations thereof. By “active ingredient” is meant that the ingredient can interfere with binding of HS and RAGE, such as by binding to the HS- binding site of RAGE.

[0165] In an example the disclosure provides a method of treating a subject afflicted with a disease or condition associated with altered RAGE signaling by administering to the subject a therapeutically effective amount of the antibodies or fragments thereof, or antibody-drug conjugates. A disease or condition is associated with RAGE or altered RAGE signaling when symptoms of the disease or condition are ameliorated by inhibition of RAGE. Non-limiting examples of diseases or conditions associated with RAGE include atherosclerosis, arthritis (including rheumatoid arthritis), Takayasu’s arthritis, congestive heart failure, Alzheimer’s disease, diabetes (including diabetic nephropathy, diabetic retinopathy and diabetic neuropathy), myocardial infraction, peripheral vascular disease, psoriasis, sepsis, cancer, osteoporosis, periodontitis, tumors and drug-induced liver toxicity. It is considered that RAGE plays a prominent role in the aforementioned pathophysiological conditions.

[0166] In an example the disclosure provides a method of treating a subject afflicted with a disease associated with altered RAGE function or signaling, such as, but not limited to, atherosclerosis, arthritis (including rheumatoid arthritis), Takayasu’s arthritis, congestive heart failure, Alzheimer’s disease, diabetes (including diabetic nephropathy, diabetic retinopathy and diabetic neuropathy), myocardial infraction, peripheral vascular disease, psoriasis, sepsis, cancer, osteoporosis, periodontitis, a tumor and / or drug-induced liver injury by administering to the subject a therapeutically effective amount of the antibodies or fragments thereof. In an example, an individual who is at risk of developing a condition, such as arthritis or osteoporosis may be administered the present compositions. In an example, an individual who has developed a pre-condition, such as osteopenia, pre-diabetes, prearthritis and the like may be administered the present compositions to halt or delay the progression of disease.

[0167] In an example, the present disclosure provides a method for treatment of bone disorder comprising administering to an individual who is in need of treatment a therapeutically effective amount of a present antibody, fragment or variant. The bone disorder may be osteoporosis or osteopenia, or bone loss may be caused by trauma or surgery. The present antibody may inhibit bone resorption or may enhance bone regeneration or both.

[0168] In examples, an individual in need of a described antibody may be a human, the described antibodies may be used for veterinary purposes.

[0169] Drug-induced liver injury (DILI) is associated with prescription medications, over the counter drugs (OTC) and herbal and dietary supplements. Specific drugs and classes associated with DILI include: allopurinol, amiodarone, amoxicillin- clavulanate, anabolic steroids, androgen-containing steroids, anti-TNF agents, azathioprine, carbamazepine, flavocoxid, fluoroquinotones, green tea extract, inhaled anesthetics, interferon-alfa, interferon-beta, isoniazid, lamotrigine, macrolides, methotrexate - oral, minocycline, nitrofurantoin, NSAIDs, phenytoin, proton pump inhibitors, pyrrolizidine alkaloids, sulfasalazine, TMP-SMX, and valproate. In the United States, antibiotics and antiepileptic drugs are the most common drug classes associated with DILI.

[0170] In an example, the present disclosure provides a method for reducing drug induced liver injury, wherein the method comprises administering to an individual who is also being administered a drug that is associated with DILI, an effective amount of a present antibody or an antibody, fragment or modification thereof. A described antibody may be administered prior to, concurrently, or sequentially with the drug associated with DILI, and may be administered over the same or different periods of time, via same or different routes. An effective amount as used in this disclosure refers to an amount that will result in the intended effect. For example an effective amount to reduce drug induced liver injury is the amount of the present antibodies that will result in reduced liver injury compared to if the drug is administered without the antibody regimen.

[0171] In an example, this disclosure provides a method of inhibiting the growth of cancer cells. The disclosure provides a method of treating an individual afflicted with a cancer, comprising administering to an individual in need of treatment a therapeutically effective amount of a described antibody, fragment or modification thereof, or an antibody-drug conjugate to thereby reduce cancer cells and / or tumor growth. Examples of cancers that may be treated with the present compositions include blood cancers as well as tumors. Examples of tumors include but are not limited to, prostate cancer, testicular cancer, pancreatic cancer, lung cancer, which may be non-small cell lung cancer (NSCLC), which may be squamous cell (or epidermoid) carcinoma, adenocarcinoma and, large cell (or undifferentiated) carcinoma, or any other type, melanoma of the skin, kidney cancer, bladder cancer, liver cancer, colon cancer, head and neck cancers, breast cancer, ovarian cancer, cervical cancer, Hodgkin lymphoma, urinary tract cancers, and other types of cancers. The cancer, such as lung cancer or breast cancer may be refractory to current treatments. The breast cancer may be metastatic triple-negative breast cancer, all stages, and may be refractory to current treatments. Individuals who may receive the present compositions may include those who have already undergone other types of therapies, including chemotherapy, surgical intervention, or hormonal therapy and the like.

[0172] A pharmaceutical composition of the disclosure can comprise one or more antibodies at a concentration range from 0.1 mg / ml to 100 mg / ml, 1 mg / ml to 10 mg / ml, 1 mg / ml to 50 mg / ml, 1 mg / ml to 100 mg / ml, 10 mg / ml to 100 mg / ml, or 50 mg / ml to 100 mg / ml of each of the antibodies or total antibodies. For example, a pharmaceutical composition of the disclosure can comprise at least or about 0.1 mg / ml, at least or about 1 mg / ml, at least or about 5 mg / ml, at least or about 10 mg / ml, at least or about 50 mg / ml, at least or about 100 mg / ml of an antibody.

[0173] The compositions of the present disclosure may be administered by routine methods known in the art. For example, the compositions comprising antibodies or fragments thereof may be administered via intravenous, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes, or by intracerebral or intra-spinal convection enhanced delivery or direct intratumoral injection. The antibodies may be administered parenterally directly at the target site (such as at or within a tumor). The compositions may be introduced as a single administration or as multiple administrations and may be introduced in a continuous manner over a period of time. In one example, the composition may be administered daily for a period of at least 2 days such as, for example, for a period of 2-30 days (and all periods therebetween). In one example, it is administered daily for 7-10 days. It may alternatively be administered at desired intervals (such as every 2, 3, 4, 5 days and the like).

[0174] It will be recognized by those of skill in the art that the form and character of the particular dosing regimen employed in the method of the invention will be dictated by the route of administration and other well-known variables, such as the size of the individual and the stage of the disease. Further, the compositions can be provided in the form of unit dosage forms for administration to an individual in need of treatment. Antibodies can be provided in a lyophilized form to be reconstituted prior to administration. The reconstitution medium can be sterile 0.9% saline solution or a suitable physiological buffer or water, or any other solution known in the art for reconstituting proteins prior to administration.

[0175] The disclosure also provides kits which can be used for administration to individuals in need of treatment. A kit, for example, can comprise one or more antibodies, which may be in a lyophilized form, optionally reconstitution media, and instructions for administration. A kit can comprise a single dose or multiple doses.

[0176] The following Examples are intended to illustrate but not limit the disclosure. Any characterization of a described composition, method and / or result is not intended to be constrained by any particular theory or interpretation. Development of mAbs that specifically targets the HS-binding site of RAGE.

[0177] HS can induce the extracellular domains of RAGE to form a stable hexamer (Fig 1A) and that RAGE-HS interaction is indispensable for RAGE signaling in multiple biological systems(Li et al., 2022; Xu et al., 2013). The HS-binding site is found at the interface between two monomers, and binding of HS is required for stabilizing the dimer (Fig. 1 B). Altogether, 7 residues are involved in HS-RAGE interaction, with 5 in the V domain (K39, K43, K44, R104 and K107) and 2 in the C1 domain (R216 and R218) (Fig. 1 C). These residues cover a large surface area of V and C1 domain. As described above, this disclosure provides multiple mAbs that target different regions within the HS-binding site. In the described screening process, we split the HS-binding site in to three clusters of residues. These are HS-1 epitope, including R216 and R218; HS-2 epitope, including K39, K43 and K44; and HS-3 epitope, including R104 and K107 (Fig. 1 C). For comparison purpose, we also screened for mAbs that specifically bind the primary ligand binding site (including residue R98 and K110), and mAbs that bind to the C2 domain (Fig. 1 C).

[0178] After Immunizing mice with the extracellular domain of human RAGE (soluble RAGE, or sRAGE) and hybridoma fusion, we obtained 94 positive hybridoma clones that bind immobilized sRAGE. These clones were then subjected to a secondary screening to determine the epitopes to which the antibodies bind. To identify mAb clones with binding to desired epitopes, we compared the binding signals between the plates immobilized with wild-type (WT) sRAGE versus sRAGE mutants (for example, HS-1 epitope mutant R216A-R218A). If some clones display 50% or more reduction in binding signal towards R216A-R218A mutant compared to WT sRAGE , it indicates that such clones recognize HS-1 epitope (Fig. 1 D). By this method we identified 18 clones that target the HS-binding site, with the HS-1 and HS-2 epitopes are targeted by 8 and 9 clones, respectively, while the HS-3 epitope is only targeted by 1 clone. For comparison we also identified 4 clones that target the ligand-binding site, and 4 clones that target the C2 domain. Based on various factors including subclone stability, affinity and expression level, we selected 3 clones targeting the HS-1 epitope, 2 clones targeting the HS-2 epitope, 1 clone targeting the ligandbinding epitope and 1 clone targeting the C2 domain for further characterization. To further confirm the epitopes to whcih these selected mAbs bind, we compared the binding curves of selected mAbs to WT-sRAGE versus mutant sRAGE. We found that all mAbs that recognize the HS-1 epitope, including 91 E12, 88A5 and 99G6, showed a dependence on residue R216 and R218 for binding, as their binding to R216A-R218A mutant was reduced as least 300-fold compared to WT-sRAGE (Fig. 2A-C). For mAbs that recognize the HS-2 epitope, we found that while 84A6 displays a complete dependence on K39, K43 and K44, binding of 44C6 to K39A-K43A-K44A mutant only reduced 10 fold (Fig. 2D-E). This finding indicates that epitopes in addition to HS-2 also makes significant contribution to the binding energy of 44C6-sRAGE interaction. We also observed a dependence of clone 93B10 on residue R98 and K100 for binding to sRAGE.

[0179] Sequence analysis of the mAbs that target the HS-binding site of RAGE.

[0180] Coding sequences of the variable regions of the selected mAbs were determined by rapid amplification of cDNA ends using total mRNA purified from hybridoma clones. Sequence alignment of the light and heavy chain sequences revealed certain features of the mAbs that target the HS-binding site. We first observed that while light chain (kappa and lambda) sequences display no clear homology among mAbs targeting the same epitopes (Fig.3A, with clones targeting the same epitope displayed in like colors), the heavy chain (gamma) sequences display homology among mAbs targeting the same epitopes (Fig. 3B). This finding indicates that the heavy chains likely play a main role in recognizing the HS-binding residues, while the light chains likely play a supporting role and / or recognizing other epitopes near the HS-binding site. Secondly, we found a high occurrence of acidic residues (Asp and Glu) within the CDRs of the mAbs targeting the HS-binding site epitopes. The mAbs targeting the HS-1 epitope has a total of 6-7 acidic residues, and mAbs targeting the HS-2 epitope has a total of 10-11 acidic residues (Fig. 3 C- D). In contrast, clone 53C2, which targets the C2 domain, has only 3 acidic residues in its CDR regions. We also observed an unbalanced distribution of acidic residues between light and heavy chains, with the majority of acidic residues found on the heavy chain (Fig. 3 C-D). This finding provides additional evidence indicating that the CDRs of heavy chains likely makes the most contribution to engaging the basic residues in the HS-binding site. Because the ligand-binding site of RAGE is also positively charged, the mAb that targets the ligand binding site (93B10) also contains a total of 8 acidic residues.

[0181] Surface plasmon resonance (SPR) analysis of the binding properties of antiRAG E mAbs.

[0182] The SPR analysis was performed by injecting mAbs onto a sensor chip immobilized with biotinylated sRAGE. The sensorgrams revealed that all tested mAbs display similar association rates, ranging from 2.92 to 4.56 x 105M’1S’1(Table 1 ). However, the dissociation rates vary tremendously among tested mAbs, ranging from 3.94 x1 O’4to 1 .17 x 10’6S’1(Table 1 ). We found that mAbs that target the HS-2 epitope (44C6 and 84A6) had much slower dissociation rate compared to mAbs that target the HS-1 epitope (88A5, 99G6 and 91 E12). Because of this difference in the dissociation rates, mAbs that target the HS-2 epitope have much higher binding affinity towards sRAGE compared to mAbs that target the HS-1 epitope (Fig. 4). The binding affinity of 93B10, which targets the ligand-binding site, has an affinity similar to mAbs that target the HS-1 epitope. mAbs inhibit RAGE-HS interaction in an epitope-dependent manner.

[0183] To demonstrate that the described anti-HS binding site mAbs can clearly inhibit RAGE-HS interaction, we performed a binding assay to determine how our mAbs might affect the binding of sRAGE to heparin Sepharose column. mAbs that target either HS-1 or HS-2 epitope were able to reduce the binding of sRAGE to heparin column (Fig 5). However, the extent of reduction depended on which epitope the mAb targets. Remarkably, the disclosure demonstrates that mAbs that target the same epitope display identical effect on RAGE-heparin interaction, with mAbs that target the HS-2 epitope (Fig. 5B) displaying a much greater effect compared to mAbs that target the HS-1 epitope (Fig. 5A). This result suggests that targeting different epitopes within the HS-binding site could have different outcomes with regard to the extent of disruption of RAGE-HS interaction.

[0184] Anti-RAGE mAbs inhibits osteoclastogenesis.

[0185] To determine the biological activity of the anti-RAGE mAbs, we performed an osteoclastogenesis assay using human bone marrow monocytes. RAGE signaling is known to be important for normal osteoclastogenesis, and inhibition of RAGE signaling impairs osteoclastogenesis of murine bone marrow macrophages (Li et al., 2022). We found that the majority of the tested mAbs display inhibitory effect on osteoclastogenesis, with the exception of 53C2, which targets the C2 domain (Fig. 6A). However, the inhibitory efficacy of these mAbs varies widely. 84A6, which targets the HS-2 epitope, was found to have the highest efficacy because it inhibits formation of osteoclasts of all sizes (Fig. 6B-D). 44C6 and 91 E12 were found to have similar potency and they suppress formation of both medium (Fig. 6C) and large (Fig. 6D) osteoclasts. The other three mAbs, including 88A5, 99G6 and 93B10, were effective only in suppressing large osteoclasts (Fig. 6L). As 84A6 and 91 E12 performed best among all mAbs when used at 20 pg / ml, we further examined the efficacy of these two mAbs at lower doses (Fig. 6E-G). We found 84A6 was more potent than 91 E12 in inhibiting osteoclastogenesis, which could significantly inhibit osteoclasts of all sizes at 5 pg / ml. In contrast, 91 E12 required 10 pg / ml to show inhibition of medium and large osteoclasts.

[0186] Discussion

[0187] This disclosure provides functional blocking mAbs that specifically target the HS-binding site of human RAGE. The antibodies were initially developed by immunizing mice with the extracellular domain of human RAGE, which includes immunoglobulin like V, C1 and C2 domains. We screened 5760 hybridoma clones and identified 94 anti-RAGE clones, which represent a positive hit rate of 1 .6%. To increase the diversity of the clones, we immunized both C57BL / 6 and BALB / c strains and used mice from both strains to generate hybridomas.

[0188] Structural studies of RAGE have identified 7 residues that are involved in HS- RAGE interaction. Spatially, these 7 residues are separated into three clusters, one (HS-1 epitope) in the C1 domain and two in the V domain (HS-2 and HS-3 epitope) (Fig. 1 C). This property was used to prepare three mutant RAGE variants to screen for mAbs that specifically targets these three separate regions within the HS-binding site, and together it is considered that they cover the whole structural space encompassed by the HS-binding site. After the initial screening, the disclosure includes verification that the hits truly inhibit HS-HSBP interaction, as demonstrated by heparin Sepharose chromatography. This assay allows direct assessment of the extent of inhibition by each mAbs (Fig. 5). Sequence analysis of the anti-HS-binding site mAbs revealed common structural features of this type of mAbs. A finding is that all anti-HS-binding site mAbs contains many acidic residues (Asp and Glu) in their CDRs. This finding suggests that electrostatic interactions play a role in mediating binding of mAbs to the HS- binding site of RAGE. The highest occurrence of acidic residues is found in CDR-2 and -3 of gamma chains, which suggest these two CDRs likely play a primary role in binding and blocking the HS-binding site.

[0189] The functional study of anti-HS-binding site mAbs revealed that there is no simple way to predict the inhibitory efficacy of mAbs based on the binding epitope or the binding affinity, thus supporting an inventive step in arriving at described antibodies. Based on biochemical analysis, mAbs that targets the epitope HS-2 (84A6 and 44C6) appear to inhibit HS-HSBP interaction much more effectively compared to mAbs that target the epitope HS-1 (91 E12 and 99G6) (Fig. 5). However, in osteoclastogenesis assay the efficacy of 91 E12 is second only to 84A6 but superior to 44C6. When directly comparing 84A6 and 44C6, we found that while 44C6 has 5-fold higher affinity compared to 84A6, its efficacy was inferior to 84A6. Thus, the present disclosure demonstrates new anti-RAGE mAbs that unexpectedly specifically target the HS-RAGE interaction.

[0190] Materials and methods

[0191] Expression and purification of the extracellular domains of human RAGE

[0192] Recombinant human sRAGE was expressed in Freestyle 293-F cells. The sequence encoding the extracellular domain of human sRAGE (aa1-342) was cloned into pUNO1 (Invivogen) for protein expression. 293F cells were transfected transiently with pUNOI-sRAGE using FectoPRO transfection reagent (Polyplus) as per manufacturer’s protocol. Cells were incubated at 31 °C on a shaker for 5 days to allow protein expression. After 5 days, the supernatant of the transfected 293F cells was purified with HiTrap SP cation exchange column (Cytiva) at pH7.8 (25mM HEPES, NaCI gradient from 50 mM to 1 M), followed by HiTrap Heparin Sepharose column (Cytiva) at pH 7.1 (25mM HEPES, NaCI gradient from 150 mM to 1 M). The purity of the recombinant sRAGE is higher than 98% as judged by silver staining. Development and screening of mouse mAbs

[0193] The mouse hybridomas were generated at Genscript Biotech. Briefly, five Balb / c mice and five C57 mice were immunized with conventional protocol over 8 weeks with one primary immunization with 50 pg human sRAGE, and three boosts each with 25 pg human sRAGE. Mice with the best serum titer (three C57 and two Balb / c mice) were used for hybridoma cell fusion. A total of 60 plates were used for initial screening (5760 clones). The initial screening resulted in 94 clones that show reactivity against immobilized wildtype human sRAGE. The supernatants of the positive clones were then screened for mAbs that specifically bind the HS-binding site, the ligand binding site, and the C2 domain of human sRAGE. In this screening, plates were immobilized with various sRAGE mutants. These include three HS- binding site mutants (K39A-K43A-K44A, R104A-K107A, R216A-R218A), one ligandbinding site mutant (R98A-K110A), and a C2 domain truncated sRAGE. By comparing the binding signals of individual hybridoma clone on plates immobilized with wildtype versus mutant sRAGE, one can easily identify clones that display reduced binding to certain sRAGE mutants. By this method, we were able to identify hybridoma clones that bind to specific RAGE epitopes.

[0194] Mouse mAbs expression

[0195] Hybridoma clones were cultured in DMEM (Gibco) supplemented with 10% FBS and 1 % penicillin / streptomycin. Cells were passed every 3 days in a 1 :3 ratio to maintain cell density between 2 x 105to 1 x 106and the conditioned medium were collected for mAb purification by protein G agarose (Genscript).

[0196] Direct ELISA binding

[0197] 200 ng of sRAGE or mutants (K39A-K43A-K44A, R104A-K107A, R216A- R218A and R98A-K110A) were immobilized onto a 96 well high-binding ELISA plate. Plates were blocked with 5% bovine serum albumin (BSA) in PBS and incubated with 0.01 -100 nM of mouse mAbs for 1 hour at room temperature. After washing with PBS for three times, the plates were incubated with anti-mouse-HRP (1 :5000) for 1 hour at room temperature, which is followed by addition of HRP substrate (ThermoFisher). The color was allowed to develop for 2 minutes and then stopped with 1 M H2SO4. The absorbance at 450nm was determined by Biotek plate reader. Determination of the coding sequence of mAbs

[0198] 3x106hybridoma cells were used for RNA extraction using the RNeasy Plus Mini kit (Qiagen, #74134) as per manufacturer’s protocol. The extracted RNA was reverse transcribed to cDNA, which was then used as template to amplify the coding sequences of both heavy and light chains by PCR with universal primers and specific primers. Both cDNA synthesis and PCR amplification were performed using the SMARTer RACE 573’ Kit (Takara, #634858). The PCR products were then purified and sequenced.

[0199] Surface plasmon resonance analysis

[0200] SPR was performed on a OpenSPR instrument (Nicoya). Biotinylation of human sRAGE was prepared as previously described (ref). Biotinylated human sRAGE was immobilized to a streptavidin sensor chip (Nicoya) based on the manufacturer’s protocol. In brief, 150ul of solution of the biotin-sRAGE conjugate (80 ug / ml) in HBS-running buffer (0.025M HEPES, pH 7.1 , 0.15M NaCI, 0.05% Tween- 20) was injected to channel 2 of the flow cell of the sensor chip at a flow rate of 20ul / min. The successful immobilization of biotin-sRAGE was confirmed by the observation of a 100-200 resonance unit increase in the sensor chip. The flow cell channel 1 without any immobilization was served as a background control. Different dilutions of mAb samples (concentrations from 1 .67 to 26.70 nM) in HBS-running buffer were injected at a flow rate of 20 pl / min. At the end of the sample injection, the same buffer was flowed over the sensor surface to facilitate dissociation. After a 5- min dissociation time, the sensor surface was regenerated by injecting with 150ul regeneration buffer (0.025M HEPES, pH 7.1 , 2M NaCI) at a flow rate of 150 pl / min to get a fully regenerated surface. The sensorgrams were fit with 1 :1 Langmuir binding model from TraceDrawer 1 .9.2.

[0201] Effect of mAbs on RAGE binding on heparin

[0202] 22 pg of human sRAGE was either directly loaded onto heparin Sepharose column or loaded after incubating with 102ug of mAbs (1 :1 molar ratio) for 30 min at room temperature. The bound sRAGE was eluted with a salt gradient from 200 mM to 2 M NaCI, pH7.1 in HEPES buffer. Osteoclastogenesis inhibition assay

[0203] Isolation of human bone marrow mononuclear cell. 30 ml of freshly isolated human bone marrow (Lonza) were diluted at a ratio of 7:1 with cold sterile PBS, 2mM EDTA. The diluted bone marrow was passed through a 100 pm cell strainer to remove bone fragments and cell clumps, which is then layered over 15ml of Polymorphprep () and centrifuge at 445 x g for 35 minutes at 20 °C without brake. The upper layer was aspirated leaving the mononuclear cell layer undisturbed at the interphase. The bone marrow mononuclear cell (BM-MNC) layer was transferred to a new tube, washed with 40 ml cold sterile PBS with 2mM EDTA, and then centrifuged at 300 x g for 10 minutes to pellet cells. BM-MNC was then resuspended with 50 ml of PBS, 2mM EDTA and centrifuge at 200 x g for 15 minutes at 20 °C to remove platelets. BM-MNC are frozen at 10 x 106per tube in alpha-MEM medium with 10% FBS and 10% DMSO.

[0204] Osteoclastogenesis assay. Thawed BM-MNCs were cultured in complete alpha-MEM medium (with 10% FBS and 1x penicillin / streptomycin) and DNase I (20 ll / rnl) for 6 days in a Teflon beaker with medium change every 3 days. Then human MNCs are cultured in untreated dish supplemented with human m-CSF at 20 ng / ml in complete alpha-MEM for 6 days with medium change every 3 days. Cells were then seeded in 96 well plate at 1 x 105per well in complete alpha-MEM medium supplemented with human m-CSF (20ng / ml) and murine RANKL (100 ng / ml) for 6 days with medium change every 3 days. Cells were stained with TRAP assay kit (Sigma-Aldrich, #387A) as per manufacturer’s protocol.

[0205] Table 1: SPR binding kinetics References. This reference listing is not an indication that any reference is material to patentability.

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[0211] Gomez Toledo, A., J.T. Sorrentino, D.R. Sandoval, J. Malmstrdm, N.E. Lewis, and J.D. Esko. 2021. A systems view of the heparan sulfate interactome. J. Histochem. Cytochem. 69:105-119.

[0212] Hudson, B.I., and M.E. Lippman. 2018. Targeting RAGE Signaling in Inflammatory Disease. Annu. Rev. Med. 69:349-364.

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[0214] Li, M., C.Y Ong, C.J. Langouet-Astrie, L. Tan, A. Verma, Y. Yang, X. Zhang, D.K. Shah, E.P. Schmidt, and D. Xu. 2022. Heparan sulfate-dependent RAGE oligomerization is indispensable for pathophysiological functions of RAGE. Elife. 11. Li, M., L.C. Pedersen, and D. Xu. 2023. Targeting heparan sulfate-protein interactions with oligosaccharides and monoclonal antibodies. Front Mol Biosci. 10:1194293.

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[0217] Lindahl, U. 2007. Heparan sulfate-protein interactions - A concept for drug design? Thromb. Haemost. 98:109-115.

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Claims

What is claimed is:1 . An antibody that binds to heparan sulfate binding site of Receptor for Advanced Glycation Endproducts (RAGE) on a cell and does not interfere with the binding of RAGE ligands to RAGE on the cell, wherein the antibodies are selected from the group of antibodies comprising heavy and light chain complement determining regions (CDRs) of the sequences of: i) an 84A6 Heavy chain CDR1 comprising the sequence FNIKDYYIH (SEQ ID NO:2); and84A6 Heavy chain CDR2 comprising the sequence GRIDPEDGETKYAPKFQD (SEQ ID NO:3); and84A6 Heavy chain CDR3 comprising the sequence ARGGDGEGDY (SEQ ID NO:4) ; and84A6 Light chain CDR1 comprising the sequence RASQSIGTSIH (SEQ ID NO:6); and84A6 Light chain CDR2 comprising the sequence LLIKYASESIS (SEQ ID NO:7); and84A6 Light chain CDR3 comprising Q the sequence QSDSWPLTF (SEQ ID NO:8); and wherein said heavy and light chain CDRs are optionally comprised by an 84A6 Heavy chain sequence comprising a sequence that is at least 90% identical to the sequenceMKCSWIIFFLMAWTGVNSEVQLQQSGAELVKPGASVKLSCTASGFNIKDYYIHWVK QRTEQGLQWIGRIDPEDGETKYAPKFQDKATITADTSSNTAYLQLSSLTSEDTAVYYC ARGGDGEGDYWGQGTTLTVSS (SEQ ID NO:1 ); and an 84A6 light chain sequence comprising a sequence that is at least 90% identical to the sequenceMVSTPQFLVFLLFWIPASSGDILLIQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQ RTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDVADYYCQQSDSWP LTFGAGTKLELK; or ii) a 44C6 Heavy chain CDR1 comprising the sequence FNIKDYYMQ (SEQ ID NQ:10); andan 44C6 Heavy chain CDR2 comprising the sequence GWIDPENGDTEYAPKFQG (SEQ ID NO: 11 ); and a 44C6 Heavy chain CDR3 comprising the sequence KGDYDRAY (SEQ ID NO:12); and a 44C6 Light chain CDR1 comprising the sequence KSSQSLLDSDGMTYLN (SEQ ID NO:14); and a 44C6 Light chain CDR2 comprising the sequence RLIYLVSKLDS (SEQ ID NO:15); and a 44C6 Light chain CDR3 comprising the sequence WQGTHFPQTF (SEQ ID NO:16); and wherein said heavy and light chain CDRS are optionally comprised by a 44C6 Heavy chain sequence comprising a sequence that is at least 90% identical to the sequenceMKCSWVIFFLMAVVIGINSEVPLQQSGAELVRSGASVKLSCTASGFNIKDYYMQWV KQRPEQGLEWIGWIDPENGDTEYAPKFQGKATMTADTSSNTAYLHLSSLTSEDSAV YFCKGDYDRAYWGQGTLVTVSA (SEQ ID NO:9); and a 44C6 Light chain sequence that is at least 90% identical to the sequenceMMSPAQFLFLLVLWIRETYGDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGMTY LNWLFQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC WQGTHFPQTFGGGTKLEIK (SEQ ID NO:13); or iii) an 91 E12 Heavy chain CDR1 comprising the sequence YNFTSYWIN (SEQ ID NO:18); and a 91 E12 heavy chain CDR2 comprising the sequence GDIHPGSGGNNYNEKFKR (SEQ ID NO:19); and a 91 E12 heavy chain CDR3 comprising the sequence ASDSLYDHVEVPFAY (SEQ ID NQ:20); and a 91 E12 Light chain CDR1 comprising the sequence SARSSVSYMY (SEQ ID NO:22); and a 91 E12 Light chain CDR2 comprising the sequence LLIYDTSNLAS (SEQ ID NO:23);and a 91 E12 Light chain CDR3 comprising the sequence QQWSTYPPTF (SEQ ID NO:24); and wherein optionally said heavy and light chain CDRs are comprised by a sequence that is at least 90% identical to 91 E12 heavy chain sequence:MGWSYIILFLVATATGVHSQVQLQQPGAELVKPGTSVKLSCKASGYNFTSYWINWV RLGPGQGLEWIGDIHPGSGGNNYNEKFKRKATLTVDTSSSTAYMQLSSLASEDSAL YYCASDSLYDHVEVPFAYWGQGTLVTVSA (SEQ ID NO:17); and a 91 E12 Light chain sequence comprising a sequence that is at least 90% identical to the sequence:MDFQVQIFSFLLISASVKLSRGQIVLTQSPAIMSASPGEKVTMTCSARSSVSYMYWH QQKPGSSPRLLIYDTSNLASGVPVRFSGGGSGTSYSLTISRMEAEDAATYFCQQWS TYPPTFGGGTRLEIK (SEQ ID NO:21 ).

2. The antibody of claim 1 comprising the 84A6 Heavy chain sequence comprising a sequence that is at least 90% identical to the sequenceMKCSWIIFFLMAWTGVNSEVQLQQSGAELVKPGASVKLSCTASGFNIKDYYIHWVK QRTEQGLQWIGRIDPEDGETKYAPKFQDKATITADTSSNTAYLQLSSLTSEDTAVYYC ARGGDGEGDYWGQGTTLTVSS (SEQ ID NO:1 ); and an 84A6 light chain sequence comprising a sequence that is at least 90% identical to the sequenceMVSTPQFLVFLLFWIPASSGDILLIQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQ RTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDVADYYCQQSDSWP LTFGAGTKLELK.

3. The antibody of claim 1 , comprising the 44C6 Heavy chain sequence comprising a sequence that is at least 90% identical to the sequenceMKCSWVIFFLMAVVIGINSEVPLQQSGAELVRSGASVKLSCTASGFNIKDYYMQVW KQRPEQGLEWIGWIDPENGDTEYAPKFQGKATMTADTSSNTAYLHLSSLTSEDSAV YFCKGDYDRAYWGQGTLVTVSA (SEQ ID NO:9); and a 44C6 Light chain sequence that is at least 90% identical to the sequenceMMSPAQFLFLLVLWIRETYGDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGMTY LNWLFQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC WQGTHFPQTFGGGTKLEIK (SEQ ID N0:13).

4. The antibody of claim 1 , comprising the 91 E12 Heavy chain sequence that is at least 90% identical to the sequence:MGWSYIILFLVATATGVHSQVQLQQPGAELVKPGTSVKLSCKASGYNFTSYWINWV RLGPGQGLEWIGDIHPGSGGNNYNEKFKRKATLTVDTSSSTAYMQLSSLASEDSAL YYCASDSLYDHVEVPFAYWGQGTLVTVSA (SEQ ID NO:17); and a 91 E12 Light chain sequence that is at least 90% identical to the sequenceMDFQVQIFSFLLISASVKLSRGQIVLTQSPAIMSASPGEKVTMTCSARSSVSYMYWH QQKPGSSPRLLIYDTSNLASGVPVRFSGGGSGTSYSLTISRMEAEDAATYFCQQWS TYPPTFGGGTRLEIK (SEQ ID NO:21 ).

5. The antibody of any one of claims 1-4, wherein the antibody is in the form of a chimeric antibody, a humanized antibody, a single chain antibody, a bispecific antibody or a multispecific antibody.

6. A method for inhibiting Receptor for Advanced Glycation Endproducts (RAGE) signaling in a cell comprising contacting the cell with an antibody of any one of claims 1-4, and wherein said RAGE signaling is inhibited in the cell after contacting the cell with said antibody.

7. The method of claim 6, wherein the cell is an endothelial cell, a neutrophil or an osteoclast.

8. A method for treatment of a bone disorder comprising administering to an individual in need of the treatment a composition comprising an antibody of any one of claims 1 -4, wherein the administration of the antibody results in inhibiting bone resorption or enhancing bone growth.

9. The method of claim 8, wherein the bone disorder is osteopenia or osteoporosis, or bone loss because of trauma or surgery.

10. A method for treatment of cancer comprising administering to an individual in need of the treatment a composition comprising an antibody any one of claims 1-4, and wherein the administration of the antibody results in inhibiting growth of cancer cells.11 . The method of claim 10, wherein the cancer is blood cancer or a solid tumor.

12. The method of claim 11 , wherein the antibody is conjugated to a drug that inhibits the growth of the cancer cells.

13. A pharmaceutical composition comprising an antibody of any one of claims 1 - 4, and a pharmaceutically acceptable carrier.

14. A polynucleotide encoding an antibody of any one of claims 1 -4.

15. The polynucleotide of claim 14, wherein the polynucleotide is present in an expression vector.

16. The polynucleotide of claim 15, wherein the polynucleotide is present within a cell.

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