Targeted-elimination compounds, methods and uses thereof in targeted degradation and / or clearance of target macromolecules

Abstract

The present disclosure relates to a targeted elimination and / or clearance of target macromolecules. More specifically, the present disclosure provides bifunctional targeted elimination compounds designed to specifically recognize, bind, optionally neutralize, and direct target macromolecules toward degradation and / or clearance organs. The targeted elimination compound comprises at least one target-recognition component, which selectively binds to and optionally neutralizes a target macromolecule, and at least one elimination and / or clearance-targeting component, which directs the bound macromolecule to a degradation and / or clearance organ in a eukaryotic organism. These compounds, as well as associated methods and therapeutic applications, are disclosed herein.

Description

[0001] TARGETED-ELIMINATION COMPOUNDS, METHODS AND USES THEREOF IN TARGETED DEGRADATION AND / OR CLEARANCE OF TARGET MACROMOLECULES

[0002] TECHNOLOGICAL FIELD

[0003] The present disclosure relates to targeted elimination and / or clearance of target macromolecules. More specifically, the present disclosure provides bifunctional compounds that specifically recognize, bind, optionally neutralize, and target a target macromolecule to degradation and / or clearance organs, methods and uses thereof in therapeutic applications.

[0004] BACKGROUND ART

[0005] References considered to be relevant as background to the presently disclosed subject matter are listed below:

[0006] 1. Busse WW. Role of antihistamines in allergic disease. Ann Allergy. 1994;72(4):371-375.

[0007] 2. Barnes PJ. Corticosteroids, IgE, and atopy. The Journal of Clinical Investigation. 2001;107(3):265-266.

[0008] 3. Incorvaia C, Mauro M, Russello M, Formigoni C, Riario-Sforza GG, Ridolo E. Omalizumab, an anti-immunoglobulin E antibody: state of the art. Drug design, development and therapy. 2014;8: 197-207.

[0009] 4. Godse K, Mehta A, Patil S, Gautam M, Nadkarni N. Omalizumab-A Review. Indian journal of dermatology. 2015;60(4):381-384.

[0010] 5. Limb SL, Starke PR, Lee CE, Chowdhury BA. Delayed onset and protracted progression of anaphylaxis after omalizumab administration in patients with asthma. J Allergy Clin Immunol. 2007;120(6):1378-1381.

[0011] 6. Lieberman PL, Umetsu DT, Carrigan GJ, Rahmaoui A. Anaphylactic reactions associated with omalizumab administration: Analysis of a case-control study. Journal of Allergy and Clinical Immunology. 2016;138(3):913-915.e912.

[0012] 7. Song CH, Stern S, Giruparajah M, Berlin N, Sussman GL. Long-term efficacy of fixed- dose omalizumab for patients with severe chronic spontaneous urticaria. Annals of allergy, asthma & immunology : official publication of the American College of Allergy, Asthma, & Immunology. 2013;l 10(2): 113-117. 8. Fox JA, Hotaling TE, Struble C, Ruppel J, Bates DJ, Schoenhoff MB. Tissue distribution and complex formation with IgE of an anti-IgE antibody after intravenous administration in cynomolgus monkeys. Journal of Pharmacology and Experimental Therapeutics. 1996;279(2): 1000- 1008.

[0013] 9. Zhu R, Owen R, Wilkins J, et al. Pharmacokinetics and exposure-efficacy relationships of omalizumab in patients with nasal polyps. Pulmonary Pharmacology & Therapeutics. 2021 ;71:102080.

[0014] 10. Ra C, Jouvin MH, Kinet JP. Complete structure of the mouse mast cell receptor for IgE (Fc epsilon RI) and surface expression of chimeric receptors (rat-mouse-human) on transfected cells. The Journal of biological chemistry. 1989;264(26):15323-15327.

[0015] 11. Hakimi J, Seals C, Kondas JA, Pettine L, Danho W, Kochan J. The alpha subunit of the human IgE receptor (FcERI) is sufficient for high affinity IgE binding. The Journal of biological chemistry. 1990;265(36):22079-22081.

[0016] Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.

[0017] BACKGROUND

[0018] Allergies are most common in the developed world. The World Allergy Organization (WAO) estimate of allergy prevalence of the whole population by country reaches up to 40% [ Prescott SL, Pawankar R, Allen KJ, et al. The World Allergy Organization journal. 2013 ;6( 1):21 -21 ] . Common allergens include pollen, certain foods, metals and other substances. Among these, food, insect bites and medications are major causes of severe reactions. The mechanism by which allergy develops is not completely understood but both genetic and environmental factors are involved [Galli SJ, et al. Nature. 2008;454(7203):445-454].

[0019] IgE antibodies are key mediators of allergic diseases [Gould HJ, Sutton BJ. Nature Rev Immunol. 2008;8(3):205-217; Galli SJ, Tsai M. Nat Med. 2012; 18(5):693-704] . IgE is bound to the high- affinity receptor FcaRI on the surface of mast cells and basophils. Thus, upon exposure to an allergen in allergic patients, this allergen is recognized by the bound IgE, leading to mast cell immediate activation and the release of inflammatory mediators such as histamine, which are responsible for allergic symptoms [Galli SJ, Tsai M. Nat Med. 2012; 18(5):693-704] . Allergy may result in a local hypersensitivity response but often develops into a severe systemic reaction that may lead to anaphylaxis and even death. The symptoms of many allergies can be treated with medications, most common are antihistamines and steroids [Ref. 1, 2]. However, these treatments provide only short-term relief and are often not effective in severe allergies. Antigen-specific immunotherapy (desensitization treatment) helps to abolish sensitivity to specific allergens, but this treatment is not always successful [Larsen JN, Broge L, Jacobi H. Drug Discovery Today. 2016;21 (l):26-37] . Another approach to treat allergies is to target plasma IgE using humanized monoclonal anti-IgE specific antibodies (Omalizumab) [Ref. 3]. This treatment reduces availability of free IgE to bind FcaRI on mast cells and basophils and may even lead to downregulation of FcaRI expression by these cells [Ref. 4]. However, while this treatment proved to be effective in preventing the onset of allergies, treatment with omalizumab is associated with many adverse reactions, ranging from skin inflammation at the injection site to anaphylaxis [Ref. 5, 6]. Moreover, omalizumab has no effect in about 25-30% of the patients, and about 20% of omalizumab-treated patients develop resistance to the drug [Ref. 4, 7]. Lastly, complexes of the omalizumab-IgE are slowly cleared from the circulation, resulting in the overall increase of total IgE and the possible release of the IgE antibody, thus requiring repeated treatments in relatively high frequency [Ref. 8, 9]. Thus, although targeting IgE appears to be most effective treatment for severe allergies, the only available drug, omalizumab, is insufficient and often associated with adverse effects. This is particularly important considering that overall medical costs resulting only from food allergies in US children alone reaches 28.8 billion / year [Gupta R, et al. JAMA pediatrics. 2013; 167(11): 1026-1031].

[0020] The FcsRl is expressed on the membrane of mast cells and basophils as a complex of three different protein subunits: a chain, chain and two y chains, where the a chain is the IgE binding subunit, and the other subunits mediate signaling and cell activation [Ref. 10]. It has been shown that the a subunit is sufficient for high affinity IgE binding [Ref. 11]. However, this by itself should not be sufficient to effectively and specifically eliminate the IgE in the plasma.

[0021] SUMMARY OF THE PRESENT DISCLOSURE

[0022] There is a need in the art for integrated approach that allows specific binding and removal of undesired macromolecule (such as IgE) via degradation and / or clearance organs that ensure a longterm effect.

[0023] Similarly, there is need for effective targeted elimination of additional circulating macromolecules that are associated with pathologic conditions. For example, inflammatory cytokines, such as TNF, IL1 and IL6, as well as growth factors and survival factors that promote cancer cells such as VEGF, growth hormone (in case of excess), IL-5 (for eosinophils), etc. Currently, most of these targets are targeted with antibodies that are introduced to the body. Yet, these antibodies form complexes, which neutralize the factor but not eliminating it or only slowly removing it. This slow elimination may allow dissociation of the complex and release of the factor, thus resulting in the requirement of repeated treatment. Taken together, the use of antibodies specific for undesired target macromolecules may provide a partial and temporally limited approach that cannot provide total elimination.

[0024] The need of an integrated binding and clearance approach is met by the present disclosure.

[0025] The present disclosure provides novel bifunctional compounds that are capable of binding, optionally, neutralizing and eliminating an undesired target macromolecule from body fluids, by specific targeting of these macromolecules to degradation and / or clearance organs in the body.

[0026] As a proof of concept, the present disclosure demonstrates targeted elimination of plasma IgE, by facilitating its degradation and elimination from the body by the liver, thus providing an effective long-term therapy for desensitization of allergic responses.

[0027] The compounds disclosed herein are applicable in the treatment of IgE-mediated pathologies. In some embodiments, these compounds are prepared from the extracellular portion of the Fc epsilon receptor I alpha (FcεRIα) (for IgE binding and neutralizing), this FcaRIa portion, which is associated with a degradation-targeting component to facilitate its clearance and / or degradation (removal).

[0028] This “bind and remove” concept can be applied for any unwanted target macromolecule in the body and thus can be used as a new therapeutic strategy in general.

[0029] Thus, a first aspect of the present disclosure provides a targeted elimination compound comprising the following components: In one component (a), at least one target-recognition component. The target-recognition component specifically binds either directly or indirectly, at least one target macromolecule. In some optional embodiments, the target recognition component may further neutralize the target macromolecule. The other component of the disclosed compound (b) comprises at least one elimination and / or removal-targeting component. More specifically, the elimination and / or removal-targeting component targets the at least one target macromolecule to the liver in a eukaryotic organism. The elimination and / or removal-targeting component is derived from at least one of: a microorganism, a natural and / or synthetic molecule and / or particle displaying liver tropism.

[0030] In some respects thereof, the present disclosure provides a targeted elimination compound comprising a fusion protein comprising the Fragment crystallizable epsilon receptor I alpha chain (FcεRlα) and a peptide of the circumsporozoite protein (CSP). The fusion protein of the disclosed compound comprises the amino acid sequence as denoted by any one of: SEQ ID NOs: 4, 6, and 7, or any variants or derivatives thereof.

[0031] Another aspect disclosed herein relates to a targeted elimination compound comprising a fusion protein comprising the Fragment crystallizable epsilon receptor I alpha chain (FcεRlα) and the Apolipoprotein A-I (Apo Al) protein. The fusion protein of the disclosed compound comprises the amino acid sequence as denoted by any one of: SEQ ID NOs: 5 and 23, or any variants or derivatives thereof.

[0032] In yet another aspect, the present disclosure provides a targeted elimination compound comprising a liposome or a lipid nanoparticle (LNP) conjugated to the Fragment crystallizable epsilon receptor I alpha chain (FcεRlα), or to an anti-IgE antibody. It should be understood that in some embodiments (i), the FcεRlα comprises the amino acid sequence as denoted by any one of: SEQ ID NOs: 1 and 2, or any variants or derivatives thereof. In yet another additional or alternative embodiment (ii), the liposome comprises (l,2-dioleoyl-sn-glycero-3-[(N-(5-amino-l- carboxypentyljiminodiacetic acidjsuccinyl] (Cobalt salt) (DGS / NTA). In yet another alternative and / or additional embodiment, (iii) the LNP Butanoic acid, 4-(dimethylamino)-, (10Z,13Z)-l- (9Z, 12Z)-9, 12-octadecadien- 1-yl- 10, 13-nonadecadien- 1 -yl ester (D-Lin-MC3-DMA), Cholesterol, 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), polyethylene glycol (PEG)- 1 ,2-dimyristoyl-rac-glycerol (DMG); and l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide(polyethylene glycol)-200 (DSPE-PEG-mal).

[0033] Another aspect of the present disclosure is related to a pharmaceutical composition comprising at least one targeted elimination compound, or any preparations thereof or any vehicle, matrix, nano- or micro-particle comprising the same, and / or at least one of pharmaceutically acceptable carrier / s, excipient / s, auxiliaries, and / or diluent / s. More specifically, the targeted elimination compound comprises: in one component (a), at least one target-recognition component. The target-recognition component specifically binds either directly or indirectly, at least one target macromolecule. In some optional embodiments, the target recognition component may further neutralize the target macromolecule. The other component (b) comprises at least one elimination and / or removaltargeting component. More specifically, the elimination and / or removal-targeting component targets the at least one target macromolecule to the liver in a eukaryotic organism. The elimination and / or removal-targeting component is derived from at least one of: a microorganism, a natural and / or synthetic molecule and / or particle displaying liver tropism.

[0034] Another aspect of the present disclosure is related to a method for targeted elimination and / or removal and / or clearance of at least one target macromolecule in a subject in need. The method comprising the step of administering to the subject an effective amount of at least one targeted elimination compound or any preparations thereof, or any vehicle, matrix, nano- or micro-particle comprising the same, and / or any composition thereof. The targeted elimination compound comprises in one component (a), at least one target-recognition component. The target-recognition component specifically binds either directly or indirectly, at least one target macromolecule. In some optional embodiments, the target recognition component may further neutralize the target macromolecule. The additional component (b) comprises at least one elimination and / or removaltargeting component. More specifically, the elimination and / or removal-targeting component targets the at least one target macromolecule to the liver in a eukaryotic organism. The elimination and / or removal-targeting component is derived from at least one of: a microorganism, a natural and / or synthetic molecule and / or particle displaying liver tropism.

[0035] Still further, another aspect of the present disclosure is related to a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one pathological disorder associated directly or indirectly with at least one target macromolecule in a subject. The method comprising the steps of administering to the subject a therapeutically effective amount of at least one targeted elimination compound, or any preparations thereof, or any vehicle, matrix, nano- or micro-particle comprising the same, and / or any composition thereof. The targeted elimination compound comprises:

[0036] In one component (a), at least one target-recognition component. The target -recognition component specifically binds either directly or indirectly, at least one target macromolecule. In some optional embodiments, the target recognition component may further neutralize the target macromolecule. The other component (b) comprises at least one elimination and / or removaltargeting component. More specifically, the elimination and / or removal-targeting component targets the at least one target macromolecule to the liver in a eukaryotic organism. The elimination and / or removal-targeting component is derived from at least one of: a microorganism, a natural and / or synthetic molecule and / or particle displaying liver tropism.

[0037] Another aspect of the present disclosure is related to a therapeutically effective amount of at least one targeted elimination compound, or any preparations thereof, or any vehicle, matrix, nano- or micro-particle comprising the same, and / or any composition thereof. The therapeutically effective amount of at least one targeted elimination compound is for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one pathological disorder associated directly or indirectly with at least one target macromolecule in a subject. The targeted elimination compound comprises: in one component (a), at least one target- recognition component. The target-recognition component specifically binds either directly or indirectly, at least one target macromolecule. In some optional embodiments, the target recognition component may further neutralize the target macromolecule. The other component (b) comprises at least one elimination and / or removal-targeting component. More specifically, the elimination and / or removal-targeting component targets the at least one target macromolecule to the liver in a eukaryotic organism. The elimination and / or removal-targeting component is derived from at least one of: a microorganism, a natural and / or synthetic molecule and / or particle displaying liver tropism.

[0038] These and other aspects of the present disclosure will become apparent by the hand of the following drawings.

[0039] BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0041] FIGURE 1. Cloning, synthesis, and purification of human soluble FcεRlα (sFcεRlα )

[0042] The extracellular fragment of the human FcεRlα (SEQ ID NO: 1), which was derived from cDNA prepared from human peripheral blood lymphocytes (PBL), was fused with myc and His tags (SEQ ID NO: 2) and cloned into an expression vector. Upon sequence verification, CHO cells were transfected, and the supernatant was collected. Protein (sFcεRlα) was purified on nickel column and run on SDS gel.

[0043] FIGURE 2A-2B. Efficiency of IgE binding in vitro by purified sFcεRlα

[0044] Fig. 2A. Direct ELISA. Plates were coated with the purified sFcεRlα , followed by incubation with IgE, IgA or IgM, and followed by secondary isotype-specific detecting Abs.

[0045] Fig. 2B. Flow cytometry analysis. IgE was pre-incubated for 5-10 min with different concentration of sFcεRlα and then placed over recombinant HEK cells expressing membrane FcεRlα . Subsequently, the cells were stained with a secondary fluorescently labeled anti-IgE and analyzed by FACS.

[0046] FIGURE 3A-3B. In vivo binding of IgE by sFcεRlα in the circulation - ELISA

[0047] Fig. 3A. Experimental design. BALB / c mice were intravenously injected with sFcεRlα (140 μg / mouse) and blood was collected at 3h, 24h, 48h and 96h post injection. Fig. 3B. ELISA analysis of mice serum samples. An ELISA plate was coated with anti-IgE as a primary antibody. Mouse serum was then placed on the ELISA wells, and HRP anti- sFcεRlα antibody was used to bind the injected sFcεRlα , thus revealing pairs of IgE-sFcεRlα . Triangles: control un-injected mouse, Squares: a mouse injected with the sFcεRlα. Circles: a second mouse injected with the sFcεRlα.

[0048] FIGURE 4A-4B. In vivo binding of IgE by sFcεRlα in the circulation - Flow cytometry

[0049] Recombinant HEK cells expressing the FcεRlα on their membrane were cultured in the presence of the mouse serum (Figure 3A) to allow binding of serum IgE to the recombinant HEK cells. Subsequently, fluorescently labeled anti-IgE antibodies were used to stain the bound IgE.

[0050] Fig. 4A. Representative results from injected mice.

[0051] Fig. 4B. Representative results from control (un-injected) mice.

[0052] Sera collected from mice injected with sFcεRlα at time 0, 3h, 24h, 48h and 96h after injection, as indicated by the arrows.

[0053] FIGURE 5A-5B. Neutralization efficiency of allergic response by sFcεRlα in vitro

[0054] Free IgE was pre-incubated with different concentrations of the purified sFcεRlα (400-12800 ng / ml) and then placed over Rat Basophilic Leukemia cells (RBL) cells. Cognate antigen / allergen was added to the culture media and secreted β-hexosaminidase was quantified (Fig. 5A). After activation and degranulation, the RBL cells were collected, washed and lysed to determine also intracellular β-hexosaminidase (Fig. SB).

[0055] FIGURE 6. A liposome-based targeted elimination compound

[0056] The figure shows a schematic illustration of a liposome coated with the sFcεRlα, specifically the model binding of a His-Tagged FcεRlα to an NT A (Nitrilotriacetic acid)-coated liposome.

[0057] FIGURE 7. A targeted elimination compound based on microorganisms displaying livertropism

[0058] The figure shows a schematic illustration of sFcεRlα fused to a small liver targeting peptide (e.g. derived from the surface antigen circumsporozoite protein (CSP)).

[0059] FIGURE 8. A targeted elimination compound based on molecules displaying liver-tropism The figure shows a schematic illustration of sFcεRlα fused to Apolipoprotein A-I.

[0060] FIGURE 9A-9C. Liposomes coated with sFcεRlα

[0061] Fig. 9A. DGS / NTA / Ni liposomes were incubated with His-Tag-sFcεRlα and washed 3 times. Along the preparation, the following samples were collected: total mix liposomes+sFcεRlα (marked T), supernatant after centrifugation of the liposomes (marked S) and washed liposome pellet (marked P). For control, liposomes without DGS / NTA / Ni were incubated with sFcεRlα and washed as above and the same samples (marked T, S, P) were collected. Samples were analyzed by western blotting using anti-His antibody to detect sFcεRlα.

[0062] Fig. 9B. Samples from total mix (T), supernatant after centrifugation (S) and the washed liposome pellet (P) were collected and analyzed by western blotting for bound IgE using goat anti kappa light chain.

[0063] Fig. 9C. A flow cytometry of the DGS / NTA / Ni- sFcεRlα liposomes were incubated with IgE, followed by staining with fluorescently-labeled anti-IgE PE antibody, and DiD-APC reagent to label membrane lipids.

[0064] FIGURE 10. DGS / NTA / Ni- sFcεRlα liposomes compete and neutralize free IgE

[0065] FACS analysis of the indicated various dilutions of DGS / NTA / Ni- sFcεRlα liposomes (from 1:50 to 1:800) mixed with IgE (0.25 μg / ml) and placed over RBL cells. RBL cells cultured with liposomes only (without pre incubation with IgE) were used as a negative control and RBL cells cultured without liposomes were used as a positive control.

[0066] FIGURE 11. DGS / NTA / Ni- sFcεRlα liposomes neutralize allergic response in vitro

[0067] Quantification of secreted β-hexosaminidase from RBL cells treated with various dilutions of DGS / NTA / Ni- sFcεRlα liposomes (from 1:0 to 1:1280) mixed with IgE (0.2 μg / ml) followed by addition of cognate antigen / allergen.

[0068] FIGURE 12. anti-IgE antibody bound to the LNPs

[0069] LNPs coated with monoclonal rat anti mouse IgE (anti mlgE; rat IgG clone 23G3 from SB A) were subjected to western blot analysis and the bound rat anti mouse IgE antibody was detected by mouse anti rat IgG (from Jackson) and followed by HRP goat anti mouse IgG.

[0070] FIGURE 13. Binding of serum IgE in vitro by LNPs coated with anti-IgE

[0071] Serum containing IgE antibodies was mixed with the anti-IgE coated LNPs at different ratios as indicated in the figure. The presence of remaining unbound IgE antibodies was detected by ELISA. ELISA plates were coated with rat anti mouse IgE (clone 23G3) as a primary antibody. The mixed samples were then placed on the ELISA wells and free IgE were allowed to bind to the primary antibody. Subsequently, HRP anti-mouse IgE was used to detect the bound IgE, thus enabling to quantify the amount of IgE in the serum not neutralized by the LNPs. The figure shows the percent of serum IgE bound to LNPs calculated accordingly. FIGURE 14. Clearance of IgE in vivo by LNPs coated with anti-IgE

[0072] Mice were injected intravenously with 50pl of LNPs coated with anti-IgE. Blood was collected before, and up to 110 days after the coated-LNPs administration. The IgE concentration in the collected blood at the different timepoints was measured by ELISA. ELISA plates were coated with rat anti mouse IgE (clone 23G3) as a primary antibody. Mouse serum was then placed on the ELISA wells and HRP anti-mouse IgE was used to detect the bound IgE.

[0073] FIGURE 15A-15C. sFcεRlα -CSP compounds

[0074] Fig. ISA. shows a schematic illustration of the three CSP-based compounds, CSP#1 in the N- terminal end (SEQ ID NO: 4), CSP#2 (SEQ ID NO: 6) between the sFcεRlα and the His-tag, and CSP#3 (SEQ ID NO: 7) at the C-terminal end of the construct.

[0075] Fig. 15B. CSP#3 protein was produced, purified and detected by western blot using anti-His antibodies.

[0076] Fig. 15C. ELISA assay of plates coated with IgE, incubated with the different supernatants of sFcεRlα-CSP compounds and followed by incubation with anti-His detecting antibody.

[0077] FIGURE 16. sFcεRlα -CSP compounds compete and neutralize free IgE

[0078] FACS analysis of supernatants collected for CSP#1, CSP#2 and CSP#3 compounds mixed with IgE (0.25μg / ml) and placed over RBL cells. HEK sup was used for negative control. After wash, the cells were stained with fluorescently labeled anti-IgE PE and analyzed by FACS. RBL cells cultured with anti-IgE only were used as negative control and RBL cells cultured with IgE were used as positive control.

[0079] FIGURE 17A-17B. sFcεRlα -ApoAl fusion protein

[0080] Fig. 17A. Schematic structure of the sFcεRlα-ApoAl fusion construct.

[0081] Fig. 17B. Western blot of the purified sFcεRlα-ApoAl fusion protein (right lane) relative to a purchased recombinant human Apo Al protein (left lane).

[0082] FIGURE 18. The sFcεRlα -ApoAl fusion protein binds and neutralize free IgE in vitro sFcεRlα-ApoAl fusion protein at different concentrations (125-500 ng / ml) were mixed with IgE (0.25 μg / ml) and were then placed with RBL cells. RBL cells cultured with media alone were use as negative control (dashed gray line). After wash, the cells were stained with fluorescently labeled anti-IgE PE and analyzed by FACS.

[0083] FIGURE 19A-19B. Dual binding of the sFcεRlα -ApoAl fusion protein

[0084] Plates were coated with monoclonal mouse anti-human ApoAl, followed by incubation with the sFcεRlα-ApoAl fusion protein or with recombinant ApoAl. Free IgE was then added and followed by detecting anti-IgE HRP antibody. The plates are read in a plate reader and results are presented in OD 450nm values.

[0085] Fig. 19A. A schematic presentation of the ELISA in which the sFcεRlα-ApoAl fusion protein is bound from one side with mouse anti-human ApoAl, and from the other side binds free IgE.

[0086] Fig. 19B. Binding of IgE (presented in OD 450nm values).

[0087] FIGURE 20. The “bind and remove” approach

[0088] The figure is a schematic illustration of the “bind and remove” approach using either antibodies or receptors as the target target-recognition component.

[0089] DETAILED DESCRIPTION OF EMBODIMENTS

[0090] Abolishing circulated IgE is the most effective approach to treat severe allergies. The only available drug capable doing so, omalizumab, is effective in only 70% of the patients and many develop resistance. This is probably due to genetic variations between individuals and the nature of the immune system to constantly mutate the produced antibodies, including the targeted IgE. Moreover, antibodies are relatively stable antibodies that are not easily cleared. This is true for pathogenic antibodies such as autoantibodies and allergy-mediating antibodies such as IgE. In general, when targeting any unwanted protein in the body, for example by using specific antibodies, complexes are formed. These complexes may be cleared slowly via different phagocytic pathways or not cleared at all. This limitation comprises a significant disadvantage as these complexes may dissociate, thus releasing the target unwanted protein. To overcome these limitations the antibodies are administered repeatedly at relatively high frequently.

[0091] A novel therapeutic strategy is firstly disclosed by the present disclosure, to eliminate any undesired target macromolecule, e.g., antibodies and / or any other stable protein in the body. This approach, designated herein as “bind and remove”, is based on the construction of novel compounds that are composed of two components: one component is a target-recognition component that specifically binds and neutralizes a target macromolecule (e.g. protein). The other component is an elimination and / or removal-targeting component that directs the undesired target macromolecule to degradation and / or clearance in a degradation and / or clearance organ such as the liver. This novel therapeutic approach opens the path for developing new innovating treatments for diseases in which unwanted proteins that are produced in the body and are associated with any clinical manifestations, need to be cleared.

[0092] For the purpose of the present disclosure and as a non-limiting proof of concept, the inventors designed this “bind and remove” approach to clear IgE antibodies from the circulation for treatment of disorders associated with activation of at least one white blood cell, specifically, allergies. The novel compounds disclosed herein are based on a target-recognition component that comprises recombinant FcεRlα (for IgE binding) fused to elimination and / or removal-targeting component that targets the undesired target macromolecule to the liver. The present disclosure provides and demonstrates three different successful means for liver targeting. This approach fundamentally differs from the existing drug Xolair (omalizumab), based on a monoclonal antibody directed against IgE, that results in non-effectiveness and drug resistance. Hence, the proposed products could overcome limitations raised with the standard of care treatment.

[0093] These compounds and the inventor's approach have a significant advantage over antibody-based therapy, e.g., omalizumab, as they bind to the Fc portion of IgE antibody, a conserved region in the population that is not subjected to mutations. Soluble receptor drugs also possess an advantage over antibodies in that they are highly specific, bind their targets with high affinity, and are less likely to induce an immune response that might attenuate their actions. The inventor's compound molecules allow more effective sequestering of serum IgE and desensitization of allergies in most of the populations, without development of resistance and / or other adverse effects.

[0094] Moreover, as the inventor's approach includes not only binding (and optionally neutralization), but also rapid clearance, this extends the period of time between treatments, that is determined by the rate of the production of the protein, rather than by stability of the antibody-protein complexes. Another advantage for the rapid removal of the complexes, is to avoid development of immune complexes-mediated pathologies, which are often developing when applying antibody-mediated therapy.

[0095] The products provided herein, offer a novel mechanism of action that would create a new niche of novel treatments for patients with allergies and other immune-related disorders associated with pathologies caused by activation of leukocytes.

[0096] Thus, in a first aspect, the present disclosure provides a targeted elimination compound comprising the following components: in one component (a), at least one target-recognition component. The target-recognition component specifically binds either directly or indirectly, at least one target macromolecule. In some optional embodiments, the target recognition component may further neutralize the target macromolecule. The other component (b) comprises at least one elimination and / or removal-targeting component. More specifically, the elimination and / or removal-targeting component targets the at least one target macromolecule to the liver in a eukaryotic organism. The elimination and / or removal-targeting component is derived from at least one of: a microorganism, a natural and / or synthetic molecule and / or particle displaying liver tropism.

[0097] The present disclosure provides a compound, specifically a dual- or bifunctional compound that is functionally characterized by leading to specific elimination of target macromolecules. The term "targeted elimination compound” as used herein, refers to a compound or moiety that is capable of specifically transporting other moieties associated to it, particularly, the target macromolecule, selectively into a target clearance and / or degradation organ (e.g., liver or kidney), in a subject, without significant transport of the target macromolecule, to other organs in the subject. More specifically, a targeted elimination compound is a compound capable of specifically recognizing, binding and transporting the target macromolecule to the target organ of an organism, thereby increasing the amount or concentration of the target molecule in the target organ. In some specific embodiments, the target organ eliminates, degrade and removes the target macromolecule. Thus, following administration of the organism with the disclosed one or more targeted-elimination compound, the concentration or amount of the compound bound to the associated or targeted target macromolecule, is increased in the target organ (e.g., liver), as compared to the concentration of the compound with the associated target macromolecule in other organs of the organism. An "increase", "elevation," "enhancement," "augmentation," "amplification," "expansion," "escalation," "growth," "intensification," "magnification," "maximization," "promotion," in the amount, the level and / or the concentration of the target macromolecule, specifically, the target molecule bound to the compound of the present disclosure in the target organ, is meant any increase of between about 5% to about 100%, specifically, 10% to 90%, 20% to 80%, 25% to 75%, 30% to 70%, 40% to 60%, 45% to 55%, 50% to 100%, 60% to 90%, 70% to 100%, specifically, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, as compared with the amount and / or the concentration of the target molecule in the target organ prior to the administration of the disclosed compound to the subject, and / or, as compared with the levels and / or the concentration of the target macromolecule in other organs of the subject (eukaryotic organism).

[0098] The target elimination compound comprises at least one target-recognition component and at least one elimination and / or removal-targeting component.

[0099] The term "target-recognition component" as used herein refers to the component of the target elimination compound, specifically, a structural or functional element, which specifically recognizes, identifies, detects, and / or selectively binds the target macromolecule. In some optional embodiments, the target recognition component may further neutralize the target macromolecule. "Specifically binds" as used herein refers to a moiety which interacts with a specific target molecule, while avoiding interactions with other molecules. Examples of moieties with specific binding capabilities include for example receptors, antibodies, enzymes, aptamers or fragments thereof as further detailed herein after (e.g. scFv, Fab, soluble receptor, etc.). The target recognition component specifically binds the target molecule in any stoichiometric ratio. In some embodiments, one target recognition component binds at least one, at least two, at least three, at least four, at least five, at least ten, at least hundred, at least thousand and even more, target molecules. In some embodiments, the target-recognition component comprises more than one moiety, wherein the plurality of moieties may be the same and / or may be different (e.g. different receptors, different antibodies, a receptor and an antibody, an antibody and an enzyme, etc.), all targeted to the same target macromolecule, although can recognize and bind different target sequences or structures (e.g., epitopes, domains) within the target macromolecule. In some embodiments, in addition to the specific binding and association, the disclosed targeting moiety of the compound of the present disclosure may also neutralize the target macromolecule. "Neutralizes" as used herein refers to a component which upon binding to the target molecule counteracts the activity or effect of the target molecule, for example by masking a specific functional moiety of the target molecule, by changing the conformation of the target macromolecule or any other structural or physical properties of the macromolecule (e.g., affinity, stability, activity).

[0100] In some embodiments, upon specific binding to the target macromolecule, the targeted elimination compound of the present disclosure, that displays at least dual properties, further transports the target molecule to the liver, via its elimination and / or removal-targeting component. The term "elimination and / or removal-targeting component" as used herein refers to the component of the targeted elimination compound which possesses tropism to a degradation and / or clearance and / or elimination and / or removal organ (for example, the liver), and thus functions as the component which directs and transports the undesired target macromolecule to degradation and / or clearance in a degradation and / or clearance organ (e.g. liver and / or kidney). Tropism to an organ or tissue, refers to the tendency of a given molecule to be attracted to a specific organ or sets of organs. Specifically, preferential targeting, accumulation, or activity of the targeting component to the target organ, specifically, the liver. Accordingly, a component which tends to be attracted to the liver displays a "liver tropism”.

[0101] It should be understood that a "degradation and / or clearance and / or elimination and / or removal organ” refers to a physiological organ or system of a eukaryotic organism responsible for the breakdown, metabolism, catabolism, excretion, elimination, or removal of substances, biomolecules, or other agents from the body, via enzymatic and / or chemical means. Such organs include for example the liver, which is the primary site for metabolic degradation via enzymatic pathways (e.g., cytochrome P450-mediated oxidation, glucuronidation, or sulfation), and the kidneys, which facilitate the excretion of water-soluble metabolites through glomerular filtration, tubular secretion, and active transport. Additionally, the spleen and mononuclear phagocyte system (MPS) contribute to the clearance of particulate matter, including nanoparticles and immune complexes, through macrophage-mediated phagocytosis and lysosomal degradation. The lungs may also function in the clearance of volatile compounds or inhaled particles, while the gastrointestinal (GI) tract can be involved in biliary excretion and fecal elimination of nonabsorbed substances. In some specific embodiments, the degradation and / or clearance organ is the liver, and the "elimination and / or removal-targeting component" of the disclosed compound transports the target macromolecule to the liver, thereby facilitating degradation, elimination and clearance thereof from the body. Targeted elimination as used herein is exemplified by the significant reduction in the levels of the target molecule in the circulation (e.g., blood, serum) or other organs of the eukaryotic organism, caused by directing the target macromolecule to the liver. It should be understood that "elimination and / or removal” of the target molecule in some embodiments, relates to removal and clearance of the target macromolecule from the body of a subject in need. In some embodiments, it is meant that the removal, clearance and / or elimination of the target macromolecule leads to a decrease, elimination, removal of the target macromolecule from the circulation of the eukaryotic organism, and / or any body cavity, and / or any organ and / or tissue of the eukaryotic organism. More specifically, "elimination and / or removal" is meant in some embodiments any specific extraction, expulsion, ejection, deletion, subtraction, reduction of the target macromolecule, thereby resulting in a decreased amount of the target macromolecule by about 5% to 100%, specifically, about 1% to 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%, as compared with the amount, level or concentration of the target macromolecule in the circulation, body fluids, body cavities, tissues or organs of the eukaryotic organism, prior to administration of the compound of the present disclosure. Still further, in some embodiments "circulation of the eukaryotic organism" as used herein encompasses blood or lymph of the eukaryotic organism. The "target macromolecule" as used herein, refers to any molecule in a eukaryotic organism which is intended to be cleared from the body of said eukaryotic organism. More specifically, a macromolecule is a large molecule composed of subunits, typically with a high molecular weight (e.g., being larger than 1,000 daltons (Da)). These molecules are often complex and have substantial size and structural complexity. In accordance with the present disclosure, macromolecules can include biological polymers such as proteins, nucleic acids (DNA and RNA), and polysaccharides. More specifically, the present disclosure refers to a target molecule which is intended to be cleared to the liver.

[0102] In some embodiments, the elimination and / or removal-targeting component is derived from at least one of the following: a microorganism, a natural and / or synthetic molecule and / or particle displaying liver tropism. The term "derived from " as used herein refers to a substance, material, or biological entity that originates from a specified source (e.g. a microorganism, a natural molecule and / or synthetic molecule and / or particle displaying liver tropism) through either extraction, modification, transformation, or synthesis.

[0103] Still further, in some embodiments of the compound of the present disclosure, the at least one target-recognition component of (a), is associated, linked, fused and / or conjugated to at least one elimination and / or removal-targeting component of (b). As used herein, the term "associated” or "association" refers herein to the chemical or physical force which holds two entities together. In some embodiments, the association is via at least one "linking moiety" or chemical bond, namely the target recognition component and the elimination and / or removal targeting component may be held together via at least one chemical bonding. Non-limiting examples of such association interactions involving chemical bonding are ionic bonding, electrostatic bonding, covalent bonding, coordination bonding, complexation, hydrogen bonding, van der Waals bonding, hydrophobicity-hydrophilicity interactions, dipole-dipole interactions, London dispersion force etc. In some embodiments, the association is via covalent bonding. It should be understood to a person skilled in the art that in some cases the associative interactions between two atoms or two chemical entities may involve more than one type of chemical and / or physical interactions. In some further embodiments, the at least one target recognition component is associated with at least one elimination and / or removal targeting component to form a complex or a conjugate or even a fusion protein. A "complex" refers herein to a group of two or more associated components, e.g., polypeptide chains linked by non-covalent protein-protein interactions. A "conjugate” pertains to a substance made up of two or more other compounds that have been chemically joined together. The term "conjugated” refers to the deliberate covalent attachment of two or more components to form a unified entity with enhanced or modified properties. Conjugation typically involves well-defined chemical reactions, such as maleimide-thiol coupling click chemistry, carbodiimide-mediated amide formation and / or PEGylation. A "fusion protein" is a protein created through the joining of two or more encoding nucleic acid sequences that originally coded for separate proteins. Translation of this fused nucleic acid sequence results in a single or multiple polypeptides with functional properties derived from each of the original proteins.

[0104] The association between the at least one target recognition component of (a), and the at least one elimination and / or removal targeting component of (b), may be direct. In the context of the present disclosure, the association of the two components or moieties enables delivery or translocation into the target organ (degradation and / or clearance organ, specifically, liver or kidney) together as a single entity. The association may be either direct or via a linker or spacer of any length or nature, forming a single molecule, aggregate, conjugate or complex formed by electrostatic bonds or hydrogen bonds. The association may use structure / s such as polymeric particle, matrix, micelle, or liposomes wherein the particle used herein as the elimination and / or removal targeting component and features on its surface used herein as the target recognition component. The association may be of the type that is dissociated inside the target organ (degradation and / or clearance organ, specifically, liver or kidney) or of the type that maintains its integrity inside the target organ.

[0105] Further, and in accordance with the present disclosure, the target recognition component is associated with the elimination and / or removal targeting component in any stoichiometric ratio. In some embodiments, the association involves a 1:1 ratio. In some embodiments, one target recognition component is associated with at least one, at least two, at least three, at least four, at least five, at least ten, at least hundred, at least thousand and even more, elimination and / or removal targeting component. In some other embodiments, at least one, at least two, at least three, at least four, at least five, at least ten, at least hundred, at least thousand and even more, target recognition component is associated with one elimination and / or removal targeting component. In some further embodiments, the association may involve at least one, at least two, at least three, at least four, at least five, at least ten, at least hundred, at least thousand and even more, target recognition components that may be associated with at least one, at least two, at least three, at least four, at least five, at least ten, at least hundred, at least thousand and even more, elimination and / or removal targeting component.

[0106] As indicated above, the at least two components of the disclosed compound may be either directly or indirectly linked. In some embodiments, the at least two components, specifically, the at least one target recognition component is associated with the at least one elimination and / or removal targeting component, indirectly, via at least one "linking moiety" , that may be a linker or spacer. In some embodiments, the linker can be composed of any assembly of atoms, including oligomeric and polymeric chains, which functions to connect one or more of target recognition component described above to one or more elimination and / or removal targeting components. In some cases, the linker may be an oligomeric and polymeric chain, such as an oligo- or polyethylene glycol chain, or an oligo- or poly(amino acid) chain. In certain embodiments, the linker may be hydrophilic to facilitate passage of the target elimination compound across biological membranes. In many cases, the linker is a linear chain; however, in some embodiments, the linker / s may contain one or more branch points. In the case of branched linker, the terminus of each branch point can be functionalized with a elimination and / or removal targeting component. In some embodiments, a dendritic linker is used, with the elimination and / or removal targeting component being bound to the focal point of the dendrimer, and multiple target binding components being bound to the ends of the dendritic branches.

[0107] In some embodiments, the linker includes one or more cleavable subunits, such as a disulfide group, a hydrazone group, or a peptide group which can be cleaved by proteolytic enzymes within a cell. In alternative embodiments, the linker contains one or more hydrolysable subunits, such as an ester group. The linker can also contain one or more covalent or non- covalent functional groups to facilitate the assembly and / or separation of the target recognition component from the attached degradation targeting component, including, but not limited to one or more metal complexes, such as polyhistidine-nickel chelate complexes, one or more heteroaromatic rings (such as triazole rings formed by the cycloaddition of an alkyne and an azide), one or more hydrogen bond donor-acceptor pairs, one or more biomolecule / bioconjugate pairs (such as biotin-avidin or biotin-streptavidin pair), as well as combinations thereof. It should be understood that in some embodiments, the linker may be served herein as an adaptor for a universal elimination and / or removal component. More specifically, the adaptor-linker, attached to the elimination and / or removal targeting component, may be adapted for various target recognition components adapted for any desired target macromolecule. In other words, the present disclosure further encompasses the use of an targeted elimination compound composed of at least one elimination and / or removal targeting component and at least one adaptor (e.g. of a bioconjugate pair such as avidin / biotin), that can attach any desired target recognition component, specific for any desired target macromolecule.

[0108] Still further, one or more ends of the linking moiety may include a functional group used to facilitate attachment of a target recognition component and an elimination and / or removal targeting component. The functional group may be an atom or group of atoms that contains at least one atom that is neither carbon nor hydrogen. In some embodiments, the functional group may be a halo functional group, such as a fluoro, chloro, bromo, or iodo group; an oxygen-containing functional group, such as a hydroxyl, carbonyl, aldehyde, acetal, hemiacetal, hemiketal, ester, orthoester, carboxylic acid, carboxylate, or ether group; a nitrogen- containing functional group, such as an amide, amine, imine, azide, cyanate, nitrate, nitrile, nitrite, or pyridyl group; phosphorus containing functional groups, such as a phosphate or phosphono group; or a sulfur-containing functional group, such as a sulfide, sulfonyl, sulfonamido, sulfino, sulfo, sulfmyl, sulfhydryl, carbonothioyl, or disulfide group.

[0109] In some embodiments, the targeted elimination compound of the present disclosure targets a target macromolecule to the liver of a eukaryotic organism, specifically, a mammalian subject. A "eukaryotic organism" refers herein to an organism characterized by cells containing membranebound organelles, including a nucleus enclosed within a nuclear envelope and containing linear chromosomal DNA. Eukaryotic cells of eukaryotic organisms exhibit compartmentalization, allowing for specialized functions such as transcription within the nucleus and translation in the cytoplasm. Eukaryotic organisms include for example animals, plants, fungi, and protists, each with distinct genetic and metabolic processes. As indicated above, in some embodiments, the disclosed organisms are mammalian subjects.

[0110] In some embodiments, the elimination and / or removal-targeting component is derived from a microorganism displaying liver tropism. In some embodiments, the microorganism is at least one of a parasite, a virus and / or bacteria. "Microorganism displaying liver tropism", refers herein to microorganism or proteins-derived from microorganism or peptides-derived from microorganism which tends to be attracted to the liver. "Microorganism" is an organism of microscopic size, which may exist in its single-celled form or as a colony of cells. Microorganisms include most unicellular organisms from all three domains of life they can be extremely diverse. Two of the three domains, Archaea and Bacteria, only contain microorganisms. The third domain Eukaryota includes all multicellular organisms as well as many unicellular protists and protozoans that are microbes.

[0111] In some embodiments, the elimination and / or removal-targeting component of (b) is derived from a parasite. More specifically, a "parasite" is an organism which lives on or inside another organism, the host, causing it some harm, and is adapted structurally to this way of life. "Parasites” include single-celled protozoans such as the agents of malaria, sleeping sickness, and amoebic dysentery. Still further, in some embodiments, the elimination and / or removal-targeting component is derived from a virus. As used herein, the term "virus" refers to a submicroscopic infectious agent that replicates only inside the living cells of an organism. In some specific non-limiting examples of viruses which infect liver cells and thus display liver tropism, is hepatitis viruses, including hepatitis A virus (HAV), hepatitis E virus (HEV), hepatitis B virus (HBV), hepatitis C virus (HCV) and hepatitis D virus (HDV).

[0112] In some embodiments, the elimination and / or removal-targeting component is derived from bacteria. It should be noted that the term "bacteria" as used herein refers to any of the prokaryotic microorganisms that exist as a single cell or in a cluster or aggregate of single cells. In more specific embodiments, the term "bacteria" specifically refers to Gram positive, Gram negative or Acid fast organisms. The Gram-positive bacteria can be recognized as retaining the crystal violet stain used in the Gram staining method of bacterial differentiation and therefore appear to be purple-colored under a microscope. The Gram-negative bacteria do not retain the crystal violet, making positive identification possible. In other words, the term 'bacteria' applies herein to bacteria with a thicker peptidoglycan layer in the cell wall outside the cell membrane (Grampositive), and to bacteria with a thin peptidoglycan layer of their cell wall that is sandwiched between an inner cytoplasmic cell membrane and a bacterial outer membrane (Gram-negative). This term further applies to some bacteria, such as Deinococcus, which stain Gram-positive due to the presence of a thick peptidoglycan layer, but also possess an outer cell membrane, and thus suggested as intermediates in the transition between monoderm (Gram-positive) and diderm (Gram-negative) bacteria. Acid fast organisms like Mycobacterium contain large amounts of lipid substances within their cell walls called mycolic acids that resist staining by conventional methods such as a Gram stain. The microorganism displaying liver tropism of interest, may be any commensal, mutual or infectious bacteria. Commensalism is a long-term biological interaction (symbiosis) in which members of one species gain benefits while those of the other species neither benefit nor are harmed. This is in contrast with mutualism, in which both organisms benefit from each other; An infection is the invasion of tissues by pathogens, their multiplication, and the reaction of host tissues to the infectious agent and the toxins they produce. Common bacteria with tropism to the liver include for example Klebsiella, Brucella and others (https: / / link.springer.com / chapter / 10.1007 / 978-3-7643-8558-3_8).

[0113] Yet, in some embodiments, other components may also exist that may direct the target macromolecule / s for elimination and / or removal and / or clearance in the liver and / or in the kidney and / or in other tissue, such as viral-like particles (VLPs). Virus-like particles (VLPs) are molecules that closely resemble viruses, but are non-infectious because they contain no viral genetic material. They can be naturally occurring or synthesized through the individual expression of viral structural proteins, which can then self-assemble into the virus-like structure. Combinations of structural capsid proteins from different viruses can be used to create recombinant VLPs. Both in-vivo assembly (i.e., assembly inside E. coli bacteria via recombinant co-expression of multiple proteins) and in-vitro assembly (i.e., protein self-assembly in a reaction vessel using stoichiometric quantities of previously purified proteins) have been successfully shown to form virus-like particles. VLPs have been produced from components of a wide variety of virus families including Parvoviridae (e.g. adeno-associated virus), Retroviridae (e.g. HIV), Flaviviridae (e.g. Hepatitis C virus), Paramyxoviridae (e.g. Nipah) and bacteriophages. VLPs can be produced in multiple cell culture systems including bacteria, mammalian cell lines, insect cell lines, yeast and plant cells. VLPs can also refer to structures produced by some LTR retrotransposons (under Ortervirales) in nature. These are defective, immature virions, sometimes containing genetic material, that are generally non-infective due to the lack of a functional viral envelope.

[0114] In yet some specific embodiments, the elimination and / or removal-targeting component is derived from the Plasmodium sporozoites, specifically, the Plasmodium sporozoites surface antigen, circumsporozoite protein (CSP). "Circumsporozoite protein ( CSP)” is the major surface protein of sporozoites of the malaria parasite. "Plasmodium sporozoites” are the infective stage of malaria parasites in the mosquitoes. The amino-acid sequence of CSP consists of an immunodominant central repeat region flanked by conserved motifs at the N- and C- termini that are implicated in protein processing as the parasite travels from the mosquito to the mammalian vector. The structure and function of CSP is highly conserved across the various strains of malaria that infect humans, non-human primates and rodents. CSP is involved in hepatocyte binding in the mammalian host. Here, the N-terminus and central repeat region initially facilitate parasite binding. On the hepatocyte surface proteolytic cleavage at region 1 of the N-terminus exposes the adhesive domain of the C-terminus, thereby priming the parasites for invasion of the liver. At least one CSP-derived peptide may thus serve as the elimination and / or removal-targeting component which directs a target molecule bound to the target recognition component to the liver.

[0115] In some embodiments, the CSP-derived peptide serving as the elimination and / or removaltargeting component of the targeted elimination compound of the present disclosure comprises the amino acid sequence as denoted by SEQ ID NO: 3, or any variants or derivatives thereof. In yet some further embodiments, the elimination and / or removal-targeting component is derived from a synthetic molecule or particle displaying liver tropism. In some embodiments, synthetic particle may comprise at least one of at least one nano- or micro-particle, micellar formulation, or liposomal formulation.

[0116] In some specific embodiments, the elimination and / or removal-targeting component comprises at least one liposome. A "synthetic molecule" as used herein refers to a molecule that is manufactured artificially using various chemical processes. The term "nanoparticle" or "microparticle” is used herein to denote any microscopic particle smaller than about 1000 nm in diameter. In some other embodiments, the carrier is an organized collection of lipids. When referring to the structure forming lipids, specifically, " micellar formulations" or "liposomal formulation" or "liposomes", or lipid nanoparticles (LNPs), it is to be understood to mean any biocompatible lipid that can assemble into an organized collection of lipids (organized structure). In some embodiments, the lipid may be natural, semi-synthetic or fully synthetic lipid, as well as electrically neutral, negatively or positively charged lipid. In some embodiments, the lipid may be a naturally occurring phospholipid. Examples of lipids forming glycerophospholipids include, without being limited thereto, glycerophospholipid. phosphatidylglycerols (PG) including dimyristoyl phosphatidylglycerol (DMPG); phosphatidylcholine (PC), including egg yolk phosphatidylcholine, dimyristoyl phosphatidylcholine (DMPC), l-palmitoyl-2- oleoylphosphatidyl choline (POPC), hydrogenated soy phosphatidylcholine (HSPC), distearoylphosphatidylcholine (DSPC); phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylserine (PS). Examples of cationic lipids may include, for example, l,2-dimyristoyl-3- trimethylammonium propane (DMTAP) l,2-dioleyloxy-3-(trimethylamino) propane (DOTAP); N-[l-(2,3,- ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE); N-[l-(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxy ethyl- ammonium bromide (DORIE); N-[l-(2,3-dioleyloxy) propyl]-N,N,N- trimethylammonium chloride (DOTMA); 3p[N- (N',N'- dimethylaminoethane) carbamoly] cholesterol (DC-Chol); and dimethyl-dioctadecylammonium (DD AB ), N- [2- [ [2, 5 -bis [3 - aminopropyl) amino] - 1 - oxopentyl]amino]ethyl]-N,N-dimethyl-2,3-bis[(l-oxo-9-octadecenyl)oxy]-l propanaminium (DOSPA), and ceramide carbamoyl spermine (CCS), or the neutral lipid dioleoylphosphatidyl ethanolamine (DOPE) derivatized with polylysine to form a cationic lipopolymer.

[0117] The lipids may be combined with other lipid compatible substances, such as, sterols, lipopolymers etc. A lipopolymer may be a lipid modified by inclusion in its polar headgroup a hydrophilic polymer. The polymer headgroup of a lipopolymer may be preferably water-soluble. In some embodiments, the hydrophilic polymer has a molecular weight equal or above 750Da. There are numerous polymers which may be attached to lipids to form such lipopolymers, such as, without being limited thereto, polyethylene glycol (PEG), polysialic acid, polylactic (also termed polylactide), polygly colic acid (also termed poly glycolide), apolylactic-polygly colic acid, polyvinyl alcohol, polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxyethyloxazoline, polyhydroxypropyloxazoline, polyaspartamide, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, polyvinylmethylether, polyhydroxyethyl acrylate, derivatized celluloses such as hydroxymethylcellulose or hydroxy ethylcellulose. The polymers may be employed as homopolymers or as block or random copolymers. The lipids derivatized into lipopolymers may be neutral, negatively charged, as well as positively charged. The most commonly used and commercially available lipids derivatized into lipopolymers are those based on phosphatidyl ethanolamine (PE), usually, distearoylphosphatidylethanolamine (DSPE).

[0118] In some embodiments, the structure forming lipids may be combined with other lipids, such as a sterol. Sterols and in particular cholesterol are known to have an effect on the properties of the lipid's organized structure (lipid assembly), and may be used for stabilization, for affecting surface charge, membrane fluidity. In some embodiments, a sterol, e.g. cholesterol is employed in order to control fluidity of the lipid structure. The greater the ratio steroldipids (the structure forming lipids), the more rigid the lipid structure is. Liposomes are often distinguished according to their number of lamellae and size. The liposomes employed in the context of the present disclosure may be multilamellar vesicles (MLVs), multivesicular vesicles (MVVs), small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs) or large multivesicular vesicles (LMVV). It should be appreciated that the target-recognition component of the compound of the present disclosure may be associated with any of the nanostructures described above, acting as the elimination and / or removal targeting component, specifically, any of the micellar formulations, liposomes, polymers, dendrimers, silicon or carbon materials, polymeric nano or micro-particles and nano or microparticles disclosed herein above.

[0119] Still further, in some specific and non-limiting embodiments, the liposome used herein as the elimination and / or removal-targeting component (b), is composed of hydrogenated soy phosphatidylcholine (HSPC). In some other embodiments, the liposome is composed of at least one of (l,2-dioleoyl-sn-glycero-3-[(N-(5-amino-l-carboxypentyl)iminodiacetic acid)succinyl] (Cobalt salt) DGS / NTA / NI liposome. In more specific embodiments, the liposome is composed of HSPC, Cholesterol, PEG 2000 and DGS-NTA (Ni). In further specific embodiment, the liposome s composed of HSPC, Cholesterol, PEG 2000 and DGS-NTA (Ni) at mole percent ratio of 55,37.5,5 and 2.5, respectively. In other specific embodiments, the liposome is composed of HSPC, Cholesterol and PEG 2000 at a mole percent ratio of 55, 40 and 5, respectively. In other embodiments, the liposome is composed of DLin-MC3-DMA, Cholesterol, DSPC, PEG-DMG and DSPE-PEG-mal in the following ratio: 50:38:10:1.9:0.1.

[0120] In some further embodiments, the elimination and / or removal-targeting component is derived from a natural molecule displaying liver tropism. The natural molecule is a protein produced by and / or a protein functioning in a liver tissue and / or protein that has a tropism to the liver. The term "natural molecule" as used herein refers to a natural compound or substance produced by a living organism that is found in nature. Natural molecules can also be prepared by chemical synthesis, both semi-synthesis and total synthesis. Semi-synthesis is used to create defined analogues of proteins by the chemical manipulation of peptide fragments largely derived from the natural protein and the subsequent reassembly of those fragments into a near-native conformation. In some embodiments of the present disclosure, the natural molecule as used herein is a protein produced by and / or a protein functioning in a liver tissue and / or a molecule that has a tropism to the liver. The term "a protein produced by a liver tissue " refers to a naturally occurring polypeptide that is synthesized, expressed, or secreted by hepatocytes or other liver-associated cell types, including Kupffer cells, hepatic stellate cells, and liver sinusoidal endothelial cells. Such proteins may be encoded by genes that are transcriptionally and translationally active in liver tissue under physiological or pathological conditions. The term "a protein functioning in a liver tissue" refers to a naturally occurring polypeptide that exerts its biological activity within the structural or cellular environment of the liver, regardless of its site of origin or synthesis. Non limiting examples include apolipoproteins, which regulate lipid transport and metabolism, glucokinase, an enzyme essential for glucose metabolism in hepatocytes and transferrin, which is involved in iron homeostasis. In some cases, proteins originating from extrahepatic tissues, such as insulin, exert crucial regulatory functions within liver cells by modulating glucose uptake and storage. The term "a molecule that has a tropism to the liver" as used in this context refers to a naturally occurring biochemical entity that preferentially accumulates, is selectively transported to, or primarily exerts its function within hepatic tissue. Liver tropism can arise from intrinsic physicochemical properties of the molecule, such as its affinity for hepatic transporters or receptors, or from physiological processes that direct its circulation to the liver. Examples include bile acids (e.g., cholic acid, chenodeoxy cholic acid), which are actively taken up by hepatocytes via sodium-dependent bile acid transporters; lipoproteins (e.g., low-density lipoproteins, high-density lipoproteins), which interact with hepatic receptors for lipid metabolism; and asialoglycoproteins, which are recognized by the asialoglycoprotein receptor (ASGPR) for hepatic clearance. Liver tropism is also observed in certain hormones and signaling molecules, such as hepcidin, which regulates systemic iron homeostasis. These molecules naturally accumulate in, or interact with, liver cells due to their role in hepatic metabolism, detoxification, or systemic regulation.

[0121] In some specific embodiments, the elimination and / or removaltargeting component is derived from at least one Apolipoprotein, or any functional fragments or peptides thereof. In some embodiments, "functional fragments" or functional peptides of the Apolipoprotein that directly mediate the association and tropism of the Apolipoprotein to the liver, or any tissue or cells thereof.

[0122] "Apolipoproteins" are proteins that bind lipids (oil-soluble substances such as fats, cholesterol and fat soluble vitamins). Apolipoproteins interact with lipid transport proteins and lipoprotein receptors, thereby participating in lipoprotein uptake and clearance. As lipids-binding proteins, apolipoproteins also stabilize lipoprotein structure and solubilize the lipid component. They transport lipids in blood, cerebrospinal fluid and lymph. The lipid components of lipoproteins are insoluble in water. However, because of their detergent-like (amphipathic) properties, apolipoproteins and other amphipathic molecules (such as phospholipids) can surround the lipids, creating a lipoprotein particle that is itself water-soluble, and can thus be carried through body fluids (i.e., blood, lymph). Apolipoproteins function as lipid transport proteins, ligands for cellsurface receptors and structural components of lipoprotein particles. There are different classes of apolipoproteins. Exchangeable apolipoproteins (apoA, apoC, and apoE) have the same genomic structure and are members of a multi-gene family that probably evolved from a common ancestral gene. Apo- Al and ApoA4 are part of the APOA1 / C3 / A4 / A5 gene cluster on chromosome 11, all are encompassed by the present disclosure.

[0123] In some embodiments, the elimination and / or removal-targeting component is Apolipoprotein A- I (Apo Al).

[0124] In some further specific embodiments, the at least one Apolipoprotein is Apolipoprotein A-I (Apo Al). In some optional specific embodiments, the elimination and / or removal-targeting component may comprise the amino acid sequence as denoted by SEQ ID NO: 17, or any variants or derivatives thereof.

[0125] "Apolipoprotein A-I (apoAl)" is the major structural protein component of high-density lipoproteins (HDL), although it is present in other lipoproteins in smaller amounts. As a component of HDL particles, it enables efflux of fat molecules by accepting fats from within cells (including macrophages within the walls of arteries which have become overloaded with ingested fats from oxidized LDL particles) for transport (in the water outside cells) elsewhere, including back to LDL particles or to the liver for excretion.

[0126] Apolipoprotein A-IV (apoA4) is present in chylomicrons, very-low-density lipoproteins (VLDL), and HDL. It is thought to act primarily in reverse cholesterol transport and intestinal lipid absorption via chylomicron assembly and secretion.

[0127] It should be understood that the elimination and / or removal-targeting component may comprise other Apolipoproteins. Some other Apolipoproteins are detailed herein:

[0128] Apolipoprotein B plays a particularly important role in lipoprotein transport being the primary organizing protein of many lipoproteins.

[0129] Apolipoprotein C-III (apoC3) plays an important role in lipid metabolism specific in regulating the metabolism of triglyceride-rich lipoproteins (TRLs).

[0130] Apolipoprotein D (apoD) is a soluble carrier protein of lipophilic molecules in neurons and glial cells within the central and peripheral nervous system and apoD can also modulate the stability and oxidation status of these molecules.

[0131] Apolipoprotein E (apoE) plays an important role in the transport and uptake of cholesterol by way of its high affinity interaction with lipoprotein receptors, including the low-density lipoprotein (LDL) receptor. ApoE is the major lipoprotein in the central nervous system. Basic amino acids important for LDL receptor binding are clustered into a surface patch on one long helix.

[0132] Apolipoprotein F (apoF) is one of the minor apolipoprotein in blood plasma and it is a lipid transfer inhibit protein to inhibit protein-mediated transfers of cholesteryl esters and triglycerides.

[0133] Apolipoprotein M (apoM) participates in the lipid metabolism and exhibit anti-atherosclerotic functions and it is presented in high-density lipoprotein (HDL), low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL).

[0134] Still further, in some embodiments, Apolipoprotein peptides that may be used in the targeted elimination compound of the present disclosure as the elimination and / or removal-targeting component (b), may be any peptides derived from ApoA, ApoB, ApoC and / or ApoE. In some specific embodiments, Apolipoprotein peptides used in the compounds of the present disclosure as the elimination and / or removal-targeting component of (b), may comprise the amino acid sequence HLRKLRKRLLRDADDLQKR (ApoE liver targeting sequence, as denoted by SEQ ID NO: 20, residues 140 to 158 of the human ApoE) or the amino acid sequence LSVKAQYKKNKHRHSI (ApoB liver targeting sequence, as denoted by SEQ ID NO: 21, residues 3144 to 3159of the human ApoE), or any derivatives or variants thereof. As mentioned above, the "target-recognition component" is the component of the target elimination compound of the present disclosure, which specifically binds and optionally neutralizes the target macromolecule. In some embodiments, the target-recognition component comprises an affinity molecule that comprises at least one of: an amino acid-based, a nucleic acidbased, a small molecule-based, a carbohydrate-based, a lipid-based affinity molecule. More specifically, an affinity molecule that may be mainly composed of amino acid molecule, nucleic acid molecule, a small molecule, a carbohydrate, a lipid, or any mixture or combinations thereof. In some embodiments, the affinity molecule may be a part of an affinity pair. For example, affinity pair of at least one of: a receptor-ligand, antibody-antigen, enzyme-substrate, aptamer-target affinity pair, or any combination thereof. Thus, in some embodiments, the affinity molecule may be a receptor that recognizes its specific ligand. In another embodiments, the affinity molecule may be a ligand that specifically recognizes its receptor molecule. Still further, affinity molecule may be derived from an antibody-antigen affinity pair. Accordingly, in some embodiments, the affinity molecule may be an antibody molecule that is specific for a specific antigen. In yet some other embodiment, the affinity molecule may be an antigen molecule that is specific for a certain antibody. Similarly, the affinity molecule may be derived from an enzyme-substrate pair, and thus being in some embodiments, the enzyme, and in other embodiments, the substrate. Still further the affinity molecule may be derived from aptamer-target pair, and thus, may be compose of an aptamer, or alternatively, from its target.

[0135] Still further, in some other embodiments, the target-recognition component comprises, or is derived from at least one of: at least one receptor molecule, at least one antibody and / or any fragments thereof and / or at least one aptamer, and / or any combinations thereof.

[0136] The affinity molecule may be amino acid-based, a nucleic acid-based, a small molecule-based, a carbohydrate-based and / or a lipid-based. The term "-based" as used herein refers to an affinity molecule which is composed of the building block specified prior to "-based". For example, amino acid-based molecules are affinity molecules which are composed of amino acids, such as proteins, peptides, glycoproteins, lipoproteins, etc. Still further, nucleic acid-based molecules are affinity molecules which are composed of nucleic acids such as polynucleotides, aptamers, etc. Small molecule-based molecules are affinity molecules which are composed of small molecules, which are low molecular weight organic compound, having a molecular weight lower than 900 Daltons. Carbohydrate-based molecules are affinity molecules which are composed of carbohydrates such as monosaccharides, disaccharides, oligosaccharides, and polysaccharide as well as proteoglycans, glycoproteins, etc. lipid-based molecules are affinity molecules which are composed of lipids such as fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, proteolipids, and others.

[0137] The term "affinity molecule" or "affinity pair” as used herein refers to a corresponding binding couple biomolecule partners. The term "Affinity" or "binding affinity" is the strength of the binding interaction between a binding biomolecule (e.g. a drug or inhibitor) to its binding partner or target (e.g., a protein or DNA). Binding affinity is typically measured and reported by the equilibrium dissociation constant (KD), which is used to evaluate and rank order strengths of bimolecular interactions. The smaller the KD value, the greater the binding affinity of the binding molecule for its target. The larger the KD value, the weaker the target molecule and affinity molecule are attracted to and bind to one another. Binding affinity is influenced by non-covalent intermolecular interactions such as hydrogen bonding, electrostatic interactions, and hydrophobic and van der Waals forces between the two molecules. In addition, binding affinity between a binding molecule and its target molecule may be affected by the presence of other molecules. In some embodiments, the affinity pair is at least one of a receptor-ligand, antibody-antigen, enzyme-substrate, aptamertarget affinity pair, or any combination thereof. It should be understood that the target recognition component may comprise either the receptor or any fragment thereof and the target molecule may comprise the ligand or any fragment thereof or vice versa. Similarly, the target recognition component may comprise either the antibody or any fragment thereof and the target molecule may comprise the ligand or any fragment thereof or vice versa. And so on for the other affinity pairs. Stil further, the target recognition component and the target molecule may comprise any combination of affinity pairs.

[0138] As mentioned, one type of affinity pair is receptor-ligand. "Receptors” are chemical structures, composed of protein, that receive and transduce signals that may be integrated into biological systems. These signals are typically chemical messengers which bind to a receptor and produce physiological responses such as change in the electrical activity of a cell. Receptor proteins can be classified by their location. Cell surface receptors also known as transmembrane receptors, include ligand-gated ion channels, G protein-coupled receptors, and enzyme- linked hormone receptors. Intracellular receptors are those found inside the cell, and include cytoplasmic receptors and nuclear receptors. A molecule that binds to a receptor is called a "ligand" and can be a protein, peptide (short protein), or another small molecule, such as a neurotransmitter, hormone, pharmaceutical drug, toxin, calcium ion or parts of the outside of a virus or microbe. An endogenously produced substance that binds to a particular receptor is referred to as its endogenous ligand. Receptors of a particular type are linked to specific cellular biochemical pathways that correspond to the signal. While numerous receptors are found in most cells, each receptor will only bind with ligands of a particular structure. This has been analogously compared to how locks will only accept specifically shaped keys. When a ligand binds to a corresponding receptor, it activates or inhibits the receptor's associated biochemical pathway, which may also be highly specialized. Still further, the target-recognition component can be a whole receptor, a receptor fragment, or a soluble receptor. In some embodiments of the present disclosure, the target-recognition component may be for example an immunoglobulin-binding receptor, more specifically, the receptor may be at least one of FcaRl and FcaR2 immunoglobulin E (IgE) binding receptor and any fragments and / or combination thereof, such as the soluble form of Fragment crystallizable epsilon receptor I alpha chain (FcεRlα) as exemplified in this disclosure and as detailed below.

[0139] Another well-known affinity pair is Antibody-antigen. An "antibody (Ab)" , also known as an "immunoglobulin (Ig)", is a large, Y-shaped glycoprotein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule called an "antigen" . Each tip of the "Y" of an antibody contains a paratope (analogous to a lock) that is specific for one particular epitope (analogous to a key) on an antigen, allowing these two structures to bind together with precision. Using this binding mechanism, an antibody can tag a microbe or an infected cell for attack by other parts of the immune system, or can neutralize it directly (for example, by blocking a part of a virus that is essential for its invasion).

[0140] To allow the immune system to recognize millions of different antigens, the antigen-binding sites at both tips of the antibody come in an equally wide variety. In contrast, the remainder of the antibody is relatively constant. In mammals, antibodies occur in a few variants, which define the antibody's class or isotype: IgA, IgD, IgE, IgG, and IgM. The constant region at the trunk of the antibody includes sites involved in interactions with other components of the immune system. The class hence determines the function triggered by an antibody after binding to an antigen, in addition to some structural features. Antibodies from different classes also differ in where they are released in the body and at what stage of an immune response.

[0141] In humans and most other mammals, an antibody unit consists of four polypeptide chains; two identical heavy chains and two identical light chains connected by disulfide bonds. Each chain is a series of domains: somewhat similar sequences of about 110 amino acids each. These domains are usually represented in simplified schematics as rectangles. Light chains consist of one variable domain VL and one constant domain CL, while heavy chains contain one variable domain VH and three to four constant domains CHI, CH2, CH3, CH4.

[0142] Structurally an antibody is also partitioned into two antigen-binding fragments (Fab), containing one VL, VH, CL, and CHI domain each, as well as the crystallizable fragment (Fc), forming the trunk of the Y shape. In between them is a hinge region of the heavy chains, whose flexibility allows antibodies to bind to pairs of epitopes at various distances, to form complexes (dimers, trimers, etc.), and to bind effector molecules more easily.

[0143] The variable domains can also be referred to as the FV region. It is the subregion of Fab that binds to an antigen. More specifically, each variable domain contains three hypervariable regions - the amino acids seen there vary the most from antibody to antibody. When the protein folds, these regions give rise to three loops of P-strands, localized near one another on the surface of the antibody. These loops are referred to as the complementarity-determining regions (CDRs), since their shape complements that of an antigen. Three CDRs from each of the heavy and light chains together form an antibody-binding site whose shape can be anything from a pocket to which a smaller antigen binds, to a larger surface, to a protrusion that sticks out into a groove in an antigen. Typically, however, only a few residues contribute to most of the binding energy.

[0144] The existence of two identical antibody-binding sites allows antibody molecules to bind strongly to multivalent antigen (repeating sites such as polysaccharides in bacterial cell walls, or other sites at some distance apart), as well as to form antibody complexes and larger antigen-antibody complexes.

[0145] The Fc region (the trunk of the Y shape) is composed of constant domains from the heavy chains. Its role is in modulating immune cell activity: it is where effector molecules bind to, triggering various effects after the antibody Fab region binds to an antigen. Effector cells (such as macrophages or natural killer cells) bind via their Fc receptors (FcR) to the Fc region of an antibody, while the complement system is activated by binding the Clq protein complex. IgG or IgM can bind to Clq, but IgA cannot, therefore IgA does not activate the classical complement pathway.

[0146] Another role of the Fc region is to selectively distribute different antibody classes across the body. Antibodies are glycoproteins, that is, they have carbohydrates (glycans) added to conserved amino acid residues. These conserved glycosylation sites occur in the Fc region and influence interactions with effector molecules. The target-recognition component can be a whole antibody and / or any fragments thereof, such as antigen-binding fragments (Fab) and single chain variable fragments (scFv). The target-recognition component can be a molecule having two or more specificities (e.g., bispecific, Tri specific), which possesses two or more different antigen-binding domains, each with different target specificity. Still further, in some embodiments, the target-recognition component can be a bi-specific antibody (such as Bi-specific T-cell engagers-BiTEs) or a tri-specific antibody. The antibody suitable for the present disclosure may also be a variable new antigen receptor antibody (V-NAR). VNARs are a class of small, immunoglobulin-like molecules from the shark immune system. Humanized versions of VNARs could be used to bind protein epitopes that are difficult to access using traditional antibodies. Still further, the target-recognition component can be a VHH antibody, that are derived from camelids (such as alpacas, llamas, and camels) heavy chain only antibodies (HCAbs, aka. single-domain antibodies).

[0147] As mentioned above, in some embodiments, the affinity pair is enzyme-substrate. "Enzymes" are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called "substrates" , and the enzyme converts the substrates into different molecules known as products. Other biocatalysts that may be useful in the present disclosure as binding pairs are catalytic RNA molecules, called ribozymes.

[0148] Enzymes differ from most other catalysts by being much more specific. An enzyme's specificity comes from its unique three-dimensional structure. Enzymes are usually much larger than their substrates. Specificity is achieved by binding pockets with complementary shape, charge and hydrophilic / hydrophobic characteristics to the substrates. Enzymes can therefore distinguish between very similar substrate molecules to be chemo selective, regioselective and stereospecific. The target-recognition component can be a whole enzyme, and / or any fragments thereof, such as the enzyme's active site or the enzyme's binding site as long as it's binding capability is maintained.

[0149] Still further, in some embodiments, the affinity pair may be aptamer-target affinity pair. "Aptamers" are short sequences of artificial DNA, RNA, XNA, or peptide that bind a specific target molecule, or family of target molecules. They exhibit a range of affinities (KD in the pM to pM range), and are sometimes classified as "chemical antibodies" or "antibody mimics". The nucleic acid-based structure of aptamers, which are mostly oligonucleotides, is very different from the amino acid-based structure of antibodies, which are proteins. Aptamers are usually obtained by selection from a large random sequence library, using methods well known in the art, such as SELEX and / or Molinex. In SELEX, the best aptamers from a starting DNA library made of about a quadrillion different randomly generated pieces of DNA or RNA are repeatedly selects. After SELEX, the chemistry of the aptamers might be mutated or changed and another selection can be done, or a rational design processes might be used to engineer improvements. Aptamers are optimized to achieve a variety of beneficial features. The most important feature is specific and sensitive binding to the chosen target. Some aptamers are engineered to fit into a biosensor or in a test of a biological sample. In various embodiments, aptamers may include single-stranded, partially single-stranded, partially double-stranded or double-stranded nucleic acid sequences; sequences comprising nucleotides, ribonucleotides, deoxyribonucleotides, nucleotide analogs, modified nucleotides and nucleotides comprising backbone modifications, branch points and non-nucleotide residues, groups or bridges; synthetic RNA, DNA and chimeric nucleotides, hybrids, duplexes, heteroduplexes; and any ribonucleotide, deoxyribonucleotide or chimeric counterpart thereof and / or corresponding complementary sequence. In certain specific embodiments, aptamers used by the invention are composed of deoxyribonucleotides.

[0150] In some embodiments, the aptamer that may be applicable herein may optionally comprise a spacer between the nucleic acid sequence and the reactive group. The spacer may be an alkyl chain such as (CH2)6 / 12, namely comprising six to twelve carbon atoms.

[0151] In some other embodiments, the target-recognition component comprises, or is derived from at least one receptor molecule.

[0152] In some embodiments of the present disclosure, the target macromolecule is at least one of: an amino acid-based, a nucleic acid-based, a small molecule-based, a carbohydrate-based, a lipid- based macromolecule molecule or any combination or complex thereof. It should be understood that the amino acid-based, a nucleic acid-based, a small molecule-based, a carbohydrate-based, a lipid-based macromolecule molecules defined herein above in connection with the affinity molecule, are also applicable for the target macromolecule as well.

[0153] In other embodiments, the macromolecule is an amino acid-based molecule. In yet some further embodiments, the macromolecule is a circulating protein of at least one of: an immunoglobulin molecule, a cytokine, a chemokine, a growth and / or survival factor, a hormone, a ligand and / or, enzyme, a soluble receptor.

[0154] "Circulating proteins" refers herein to proteins which circulate in the blood circulation system and / or lymphatic system. The blood circulatory system, which includes the cardiovascular system and pulmonary circulation, is a system of organs that includes the heart, blood vessels, and blood which is circulated throughout the entire body of a human or other vertebrate. The lymphatic system is complementary to the circulatory system, which carries excess plasma (filtered from the circulatory system capillaries as interstitial fluid between cells) away from the body tissues via accessory routes that return excess fluid back to blood circulation as lymph. The lymphatic system also works with the immune system. In some embodiments, the circulating proteins may be a cytokine, a chemokine, a growth and / or survival factor, a hormone.

[0155] "Cytokines" are small proteins (~5-25 kDa) important in cell signaling. Due to their size, cytokines cannot cross the lipid bilayer of cells to enter the cytoplasm and therefore typically exert their functions by interacting with specific cytokine receptors on the target cell surface. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors, but generally not hormones or growth factors (despite some overlap in the terminology). Cytokines are produced by a broad range of cells, including immune cells like macrophages, B lymphocytes, T lymphocytes and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells; a given cytokine may be produced by more than one type of cell. Cytokines have been shown to be involved in autocrine, paracrine and endocrine signaling as immunomodulating agents.

[0156] Cytokines modulate the balance between humoral and cell-based immune responses. Non limiting examples of cytokines include the IL-2 subfamily (which include IL-2, IL-4, IL-7, IL-9, IL- 15 and IL-21), the interferon (IFN) subfamily (which includes for example IFN-a, IFN- , IFN- 8, IFN-K and IFN-co, IFN-y), the IL-10 subfamily (including for example IL-19, IL-20, IL-22, IL- 24 (Mda-7), IL-26), the IL- 1 family, which primarily includes IL-1 and IL-18, the cysteine knot cytokines (IPR029034) include members of the transforming growth factor beta superfamily, including TGF- i, TGF- 2 and TGF- 3 and the IL- 17 family.

[0157] In some embodiments, the target molecule is a chemokine. The term "chemokines" or "chemotactic cytokines" refers to a family of small cytokines or signaling proteins secreted by cells that induce directional movement of cells. Cytokine proteins are classified as chemokines according to behavior and structural characteristics. In addition to being known for mediating chemotaxis, chemokines are all approximately 8-10 kilodaltons in mass and have four cysteine residues in conserved locations that are key to forming their 3-dimensional shape. These proteins have historically been known under several other names including the SIS family of cytokines, SIG family of cytokines, SCY family of cytokines, Platelet factor-4 superfamily or intercrines. The major role of chemokines is to act as a chemoattractant to guide the migration of cells. Cells that are attracted by chemokines follow a signal of increasing chemokine concentration towards the source of the chemokine. Some chemokines control cells of the immune system during processes of immune surveillance, such as directing lymphocytes to the lymph nodes so they can screen for invasion of pathogens by interacting with antigen-presenting cells residing in these tissues. These are known as homeostatic chemokines and are produced and secreted without any need to stimulate their source cells. Some chemokines have roles in development; they promote angiogenesis (the growth of new blood vessels), or guide cells to tissues that provide specific signals critical for cellular maturation. Other chemokines are inflammatory and are released from a wide variety of cells in response to bacterial infection, viruses and agents that cause physical damage such as silica or the urate crystals that occur in gout. Chemokines are functionally divided into two groups: Homeostatic: are constitutively produced in certain tissues and are typically responsible for basal leukocyte migration. These include: CCL14, CCL19, CCL20, CCL21, CCL25, CCL27, CXCL12 and CXCL13. Inflammatory: that are formed under pathological conditions (on pro-inflammatory stimuli, such as IL-1, TNF-alpha, LPS, or viruses) and actively participate in the inflammatory response attracting immune cells to the site of inflammation. Examples are: CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, CXCL10. All of these proteins exert their biological effects by interacting with G protein-linked transmembrane receptors called chemokine receptors, that are selectively found on the surfaces of their target cells. It should be understood that the target molecule may be a growth factor. As used herein, "growth factors" refers to naturally occurring substances capable of stimulating cell proliferation, wound healing, and often promote cell differentiation and maturation. Growth factors typically act as signaling molecules between cells. Examples are some cytokines and hormones that bind to specific receptors on the surface of their target cells. While growth factor implies a positive effect on cell proliferation, cytokine is a neutral term with respect to whether a molecule affects proliferation. While some cytokines can be growth factors, such as G-CSF and GM-CSF, others have an inhibitory effect on cell growth or cell proliferation.

[0158] Individual growth factor proteins tend to occur as members of larger families of structurally and evolutionarily related proteins. There are many families, some of which are listed herein: Adrenomedullin (AM), Angiopoietin (Ang), Autocrine motility factor, Bone morphogenetic proteins (BMPs), Ciliary neurotrophic factor family (such as Ciliary neurotrophic factor (CNTF), Leukemia inhibitory factor (LIF), Interleukin-6 (IL-6)), Colony-stimulating factors (such as Macrophage colony-stimulating factor (M-CSF), Granulocyte colony-stimulating factor (G-CSF), Granulocyte macrophage colony-stimulating factor (GM-CSF)), Epidermal growth factor (EGF), Ephrins, Erythropoietin (EPO), Fibroblast growth factor (FGF), Foetal Bovine Somatotrophin (FBS), GDNF family of ligands (such as Glial cell line-derived neurotrophic factor (GDNF), Neurturin, Persephin, Artemin), Growth differentiation factor-9 (GDF9), Hepatocyte growth factor (HGF), Hepatoma-derived growth factor (HDGF), Insulin, Insulin-like growth factors, Interleukins, Keratinocyte growth factor (KGF), Migration-stimulating factor (MSF), Macrophage-stimulating protein (MSP), also known as hepatocyte growth factorlike protein (HGFLP), Myostatin (GDF-8), Neuregulins, Neurotrophins, Placental growth factor (PGF), Platelet-derived growth factor (PDGF), Renalase (RNLS) - Anti-apoptotic survival factor, T-cell growth factor (TCGF), Thrombopoietin (TPO), Transforming growth factors (such as TGF-a and TGF-P), Tumor necrosis factor-alpha (TNF-a), Vascular endothelial growth factor (VEGF), Wnt Signaling Pathway.

[0159] In some embodiments, the target molecule is a survival factor. A "survival factor" as used herein refers to a biomolecule, such as a protein, peptide, or small molecule, that promotes cell viability by preventing apoptosis or other forms of programmed cell death. These factors function by activating intracellular signaling pathways that regulate gene expression, inhibit pro-apoptotic proteins, or enhance metabolic stability. Survival factors include growth factors, cytokines, and neurotrophic factors, which are essential for cellular homeostasis, tissue regeneration, and immune response.

[0160] Still further, in some embodiments the target molecule is a hormone. The term "hormone" is a class of signaling molecules in multicellular organisms that are sent to distant organs by complex biological processes to regulate physiology and behavior. Hormones are required for the correct development of animals, plants and fungi. Due to the functional definition of a hormone (as a signaling molecule that exerts its effects far from its site of production), numerous kinds of molecules can be classified as hormones. Among the substances that can be considered hormones are eicosanoids (e.g. prostaglandins and thromboxanes), steroids (e.g. oestrogen and brassinosteroid), amino acid derivatives (e.g. epinephrine and auxin), protein or peptides (e.g. insulin and CLE peptides), and gases (e.g. ethylene and nitric oxide). Hormones are used to communicate between organs and tissues.

[0161] In some other embodiments, the target molecule may be a ligand. As used herein, the term "ligand” refers to a molecule that binds specifically to a target biomolecule, typically a receptor, enzyme, or ion channel, to induce a biochemical or physiological response. Ligands can be endogenous or exogenous and include proteins, peptides, small molecules, hormones, neurotransmitters, and synthetic compounds. The interaction between a ligand and its target is governed by molecular recognition, affinity, and specificity, often triggering conformational changes or signaling cascades.

[0162] Still further, in some embodiments, the target molecule may be a soluble receptor. A "soluble receptor" as used herein refers to a truncated or secreted form of a membrane-bound receptor that retains the ability to bind its ligand in the extracellular environment. These receptors, which may be naturally occurring or engineered, function as decoys or modulators by sequestering ligands, thereby regulating downstream signaling events. Soluble receptors are commonly used to neutralize cytokines, inhibit growth factor signaling, or modulate immune responses.

[0163] In some specific embodiments, the target macromolecule targeted by the target recognition component of the targeted-elimination compound of the present disclosure, is immunoglobulin E (IgE).

[0164] "Immunoglobulin E (IgE)" as used herein is a type of antibody (or immunoglobulin (Ig) "isotype"), that is synthesized by plasma cells. Monomers of IgE consist of two heavy chains (a chain) and two light chains, with the a chain containing four Ig-like constant domains (Cel-Ce4). IgE has an essential role in type I hypersensitivity, which manifests in various allergic diseases, such as allergic asthma, most types of sinusitis, allergic rhinitis, food allergies, and specific types of chronic urticaria and atopic dermatitis. IgE also plays a pivotal role in responses to allergens, such as: anaphylactic reactions to drugs, bee stings, and antigen preparations used in desensitization immunotherapy. IgE is thought to be an important part of the immune response against infection by certain parasitic worms, including Schistosoma mansoni, Trichinella spiralis, and Fasciola hepatica. IgE is also utilized during immune defense against certain protozoan parasites such as Plasmodium falciparum. IgE may have evolved as a defense to protect against venoms.

[0165] Although IgE is typically the least abundant isotype (blood serum IgE levels in a normal ("nonatopic") individual are only 0.05% of the Ig concentration, compared to 75% for the IgGs at 10 mg / ml) and is the isotype responsible for most of the classical adaptive immune response, it is capable of triggering anaphylaxis, one of the most rapid and severe immunological reactions. Other non-limiting examples of potential target molecules include the following:

[0166] Proprotein convertase subtilisin / kexin type 9 (PCSK9): this enzyme is important for clearance of cholesterol via the LDL receptor on liver cells. Enhanced expression and / or function cause elevated blood LDL, cholesterol and atherosclerosis. The PCSK9 is now a biological target for drug discovery and current drugs are based on small molecule inhibitors, gene silencing, monoclonal Abs and so. The inventors propose to use an anti-PCSK9 antibody (or single chain) that will be linked to the removal segment to generate a “bind and remove” compound specific for PCSK9 that can be used for treatment of hyperlipoproteinemia (e.g. Example 22).

[0167] Another potential target for the targeted elimination compound of the present disclosure is Transthyretin Amyloidosis (ATTR). This is a protein disorder disease in which the liver produces faulty transthyretin (TTR) proteins, which clump and deposit in various tissues including the heart where it causes severe cardiomyopathy. Current treatments are based on gene silencing and other mediations that stabilize the TTR protein. The inventors propose to use an anti-TTR antibody (or single chain) that will be linked to the removal segment to generate a “bind and remove” compound specific for TTR that can be used for treatment of ATTR-mediated cardiomyopathy.

[0168] Another potential target for the targeted elimination compound of the present disclosure is amyloid beta (AP) which accumulate and deposit in the brain and is a hallmark of Alzheimer’s disease (AD). Removing A plaques is most desired for treatment of AD but till now this goal has not been reached. Current treatments are based on antibody therapy (which is associated with severe adverse effects in AD patients). Though, the use of extracorporeal blood removal of Ap showed beneficial effect. The inventors propose to use an anti-Ap antibody (or single chain) that will be linked to the removal segment to generate a “bind and remove” compound specific for Ap that can be used for treatment of AD by removing the pathogenic Ap.

[0169] In some embodiments of the present disclosure, the at least one receptor molecule used herein as a target recognition component, may comprise an immunoglobulin-binding receptor.

[0170] In other embodiments, the immunoglobulin-binding receptor is an immunoglobulin E (IgE) binding receptor or any fragment / s thereof. In some specific embodiments, the immunoglobulin E binding receptor may be at least one of FcaRl and FcaR2 and any fragments and / or combination thereof.

[0171] Still further, in some embodiments said at least one immunoglobulin E binding receptor is the FcεRlα. Soluble form of FcεRlα extracellular fraction shall enable binding to the IgE in the circulation.

[0172] In some embodiments of the present disclosure, the target-recognition component is derived from the FcεRlα, and comprises the amino acid sequence:

[0173] MAPAMESPTLLCVALLFFAPDGVLAVPQKPKVSLNPPWNRIFKGENVTLTCNGNNFFEV SSTKWFHNGSLSEETNSSLNIVNAKFEDSGEYKCQHQQVNESEPVYLEVFSDWLLLQAS AEVVMEGQPLFLRCHGWRNWDVYKVIYYKDGEALKYWYENHNISITNATVEDSGTYY CTGKVWQLDYESEPLNITVIKAPREKYWLQ, as denoted by SEQ ID NO: 1, or any variant or derivatives thereof. For example, the His Tagged sequence according to SEQ ID No; 2.

[0174] As used herein, the "immunoglobulin-binding receptor" refers to Fc receptor (FcR). The Fc receptor is a protein found on the surface of certain cells, including, among others, B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, human platelets, and mast cells, that contribute to the protective functions of the immune system. Its name is derived from its binding specificity for a part of an antibody known as the Fc (fragment crystallizable) region. Fc receptors bind to antibodies that are attached to infected cells or invading pathogens. Their activity stimulates phagocytic or cytotoxic cells to destroy microbes, or infected cells by antibody- mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity. There are several different types FcR, which are classified based on the type of antibody that they recognize. For example, those that bind the most common class of antibody, IgG, are called Fc-gamma receptors (FcyR), those that bind IgA are called Fc-alpha receptors (FcaR) and those that bind IgE are called Fc-epsilon receptors (FcaR). The classes of FcR's are also distinguished by the cells that express them (macrophages, granulocytes, natural killer cells, T and B cells) and the signaling properties of each receptor.

[0175] In some embodiments, the IgE binding receptor refers to at least one Fc-epsilon receptors (FcaR) and any fragments and / or combination thereof. The FcaR refers to at least one of FceRI and FceRII. FceRI (type I Fee receptor) is a high-affinity IgE receptor and FceRII (type II Fee receptor), also known as CD23 is a low-affinity IgE receptor. IgE can upregulate the expression of both types of Fee receptors. FceRI is expressed on mast cells, basophils, and the antigen-presenting dendritic cells in both mice and humans. Binding of antigens to IgE already bound by the FceRI on mast cells causes cross-linking of the bound IgE and the aggregation of the underlying FceRI, leading to degranulation (the release of mediators) and the secretion of several types of type 2 cytokines like interleukin (IL)-3 and stem cell factor (SCF), which both help the mast cells survive and accumulate in tissue, and IL-4, IL-5, IL-13, and IL-33, which in turn activate group 2- innate lymphoid cells (ILC2 or natural helper cells). Basophils share a common haemopoietic progenitor with mast cells; upon the cross-linking of their surface bound IgE by antigens, also release type 2 cytokines, including IL-4 and IL-13, and other inflammatory mediators. The low- affinity receptor (FceRII) is always expressed on B cells; but IL-4 can induce its expression on the surfaces of macrophages, eosinophils, platelets, and some T cells.

[0176] FceRla is a subunit of the high-affinity receptor for IgE to which IgE directly binds. FceRI is a tetrameric complex consisting of one a, one , and two y subunits. The latter two subunits are required for signal transduction activity. The FceRI complex plays an important role in triggering allergic responses. The soluble form of FceRla extracellular fraction shall enable binding to the IgE in the circulation.

[0177] It should be noted that in some embodiments, the target-recognition component comprises, or is derived from at least one antibody and / or any fragment thereof. In other embodiments, the targetrecognition component comprises, or is derived from at least one antibody and / or any fragment thereof that binds at least one immunoglobulin molecule. In a more specific embodiments, the target-recognition component comprises, or is derived from at least one antibody and / or any fragment thereof that binds immunoglobulin E (IgE). This specific embodiments was exemplified in Examples 9-11.

[0178] It should be understood that in some embodiments, the disclosed targeted elimination compound of the present disclosure comprises, at least in part a proteinaceous compound, for example, any of the fusion proteins as disclosed in SEQ ID NO; 4, 5, 6, 7, 8, 23 or any derivatives or variants thereof, as well as the soluble FcεRlα conjugated to liposome or the lipid nanoparticles, as denoted by SEQ I NO; 1 or 2, or any derivatives or variants thereof. The term "derivative” is used to define amino acid sequences (polypeptide), with any insertions, deletions, substitutions and modifications to the amino acid sequences (polypeptide) that do not alter the activity of the original polypeptides, namely, the function of targeted elimination of a target macromolecule. By the term “derivative” it is also referred to homologues, variants and analogues thereof. Proteins orthologs or homologues having a sequence homology or identity to the proteins of the present disclosure, may share at least 50%, at least 60% and specifically 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher, specifically as compared to the entire sequence of the proteins (e.g., fusion proteins used as the targeted elimination compound of the present disclosure), specifically, any one of SEQ ID NO; 4, 5, 6, 7, 8, 23 or any derivatives thereof, as well as the soluble FcεRlα conjugated to liposome or the lipid nanoparticles, as denoted by SEQ I NO; 1 or 2, or any derivatives or variants thereof.

[0179] In some embodiments, derivatives refer to polypeptides, which differ from the polypeptides specifically defined in the present invention by insertions, deletions or substitutions of amino acid residues. It should be appreciated that by the terms "insertion / s", "deletion / s" or "substitution / s", as used herein it is meant any addition, deletion or replacement, respectively, of amino acid residues to the polypeptides disclosed by the invention as indicated above, of between 1 to 50 amino acid residues, between 20 to 1 amino acid residues, and specifically, between 1 to 10 amino acid residues. More particularly, insertion / s, deletion / s or substitution / s may be of any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. It should be noted that the insertion / s, deletion / s or substitution / s encompassed by the invention may occur in any position of the modified peptide, as well as in any of the N' or C termini thereof. With respect to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologues, and alleles of the invention. For example, substitutions may be made wherein an aliphatic amino acid (G, A, I, L, or V) is substituted with another member of the group, or substitution such as the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine. Each of the following eight groups contains other exemplary amino acids that are conservative substitutions for one another:

[0180] 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M).

[0181] More specifically, amino acid “substitutions” are the result of replacing one amino acid with another amino acid having similar structural and / or chemical properties, i.e., conservative amino acid replacements. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved. For example, nonpolar “hydrophobic” amino acids are selected from the group consisting of Valine (V), Isoleucine (I), Leucine (L), Methionine (M), Phenylalanine (F), Tryptophan (W), Cysteine (C), Alanine (A), Tyrosine (Y), Histidine (H), Threonine (T), Serine (S), Proline (P), Glycine (G), Arginine (R) and Lysine (K); “polar” amino acids are selected from the group consisting of Arginine (R), Lysine (K), Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q); “positively charged” amino acids are selected form the group consisting of Arginine (R), Lysine (K) and Histidine (H) and wherein “acidic” amino acids are selected from the group consisting of Aspartic acid (D), Asparagine (N), Glutamic acid (E) and Glutamine (Q). Variants of the polypeptides of the invention may have at least 80% sequence similarity or identity, often at least 85% sequence similarity or identity, 90% sequence similarity or identity, or at least 95%, 96%, 97%, 98%, or 99% sequence similarity or identity at the amino acid level, with the protein of interest, such as the various polypeptides of the invention. It should be understood that the percentage of similarity or identity refer to the similarity or identity to the entire sequences as denoted by any one of SEQ ID NO; 4, 5, 6, 7, 8, 23, as well as the soluble FcεRlα conjugated to liposome or the lipid nanoparticles, as denoted by SEQ ID NO: 1 or 2, or any derivatives or variants thereof. It should be appreciated that in some embodiments, the disclosed targeted elimination compound may be any of the targeted elimination compounds disclosed by, and encompassed by the present disclosure, with the proviso that the elimination and / or removal targeting component thereof is not a liposome or a lipid nanoparticle.

[0182] In some respects thereof, the present disclosure provides a targeted elimination compound comprising a fusion protein comprising the Fragment crystallizable epsilon receptor I alpha chain (FcεRlα) and a peptide of the circumsporozoite protein (CSP). The fusion protein of the disclosed compound comprises the amino acid sequence as denoted by any one of: SEQ ID NOs: 4, 6, and 7, or any variants or derivatives thereof. More specifically, in one embodiment, the sFcεRlα-CSP FUSION #1 that comprises the amino acid sequence:

[0183] MAPAMESPTLLCVALLFFAPDGVLADNEKLRKPKHKKLKQPADGGGGGSVPQKPKVSL NPPWNRIFKGENVTLTCNGNNFFEVSSTKWFHNGSLSEETNSSLNIVNAKFEDSGEYKC QHQQVNESEPVYLEVFSDWLLLQASAEVVMEGQPLFLRCHGWRNWDVYKVIYYKDGE ALKYWYENHNISITNATVEDSGTYYCTGKVWQLDYESEPLNITVIKAPREKYWLQEQKL ISEEDLNSAVDHHHHHH, as denoted by SEQ ID NO: 4.

[0184] In another in one embodiment, the sFcεRlα-CSP FUSION #2 that comprises the amino acid sequence:

[0185] MAPAMESPTLLCVALLFFAPDGVLAVPQKPKVSLNPPWNRIFKGENVTLTCNGNNFFEV SSTKWFHNGSLSEETNSSLNIVNAKFEDSGEYKCQHQQVNESEPVYLEVFSDWLLLQAS AEVVMEGQPLFLRCHGWRNWDVYKVIYYKDGEALKYWYENHNISITNATVEDSGTYY CTGKVWQLDYESEPLNITVIKAPREKYWLQEQKLISEEDLDNEKLRKPKHKKLKQPADG NSAVDHHHHHH, as denoted by SEQ ID NO: 6.

[0186] In another in one embodiment, the sFcεRlα-CSP FUSION #3 that comprises the amino acid sequence:

[0187] MAPAMESPTLLCVALLFFAPDGVLAVPQKPKVSLNPPWNRIFKGENVTLTCNGNNFFEV SSTKWFHNGSLSEETNSSLNIVNAKFEDSGEYKCQHQQVNESEPVYLEVFSDWLLLQAS AEVVMEGQPLFLRCHGWRNWDVYKVIYYKDGEALKYWYENHNISITNATVEDSGTYY CTGKVWQLDYESEPLNITVIKAPREKYWLQEQKLISEEDLNSAVDHHHHHHDNEKLRK PKHKKLKQPADG, as denoted by SEQ ID NO: 7.

[0188] Another aspect disclosed herein relates to a targeted elimination compound comprising a fusion protein comprising the Fragment crystallizable epsilon receptor I alpha chain (FcεRlα) and the Apolipoprotein A-I (Apo Al) protein. The fusion protein of the disclosed compound comprises the amino acid sequence as denoted by any one of: SEQ ID NOs: 5 and 23, or any variants or derivatives thereof.

[0189] In some in one embodiment, the sFcεRl - Apolipoprotein A-I fusion that comprises the amino acid sequence:

[0190] MAPAMESPTLLCVALLFFAPDGVLAVPQKPKVSENPPWNRIFKGENVTLTCNGNNFFEV SSTKWFHNGSESEETNSSENIVNAKFEDSGEYKCQHQQVNESEPVYLEVFSDWELLQAS AEVVMEGQPEFLRCHGWRNWDVYKVIYYKDGEALKYWYENHNISITNATVEDSGTYY CTGKVWQLDYESEPLNITVIKAPREKYWLQGPADEPPQSPWDRVKDLATVYVDVLKDS GRDYVSQFEGSAEGKQENEKEEDNWDSVTSTFSKEREQEGPVTQEFWDNEEKETEGER QEMSKDEEEVKAKVQPYEDDFQKKWQEEMEEYRQKVEPERAEEQEGARQKEHEEQEK ESPEGEEMRDRARAHVDAERTHEAPYSDEERQREAAREEAEKENGGAREAEYHAKATE HESTESEKAKPAEEDERQGEEPVEESFKVSFESAEEEYTKKENTQ, as denoted by SEQ ID NO: 5.

[0191] In another in one embodiment, the sFcεRl - Apolipoprotein A-I fusion that comprises the amino acid sequence:

[0192] MAPAMESPTLLCVALLFFAPDGVLAVPQKPKVSENPPWNRIFKGENVTLTCNGNNFFEV SSTKWFHNGSESEETNSSENIVNAKFEDSGEYKCQHQQVNESEPVYLEVFSDWELLQAS AEVVMEGQPEFLRCHGWRNWDVYKVIYYKDGEALKYWYENHNISITNATVEDSGTYY CTGKVWQLDYESEPLNITVIKAPREKYWLQGPADEPPQSPWDRVKDLATVYVDVLKDS GRDYVSQFEGSAEGKQENEKEEDNWDSVTSTFSKEREQEGPVTQEFWDNEEKETEGER QEMSKDEEEVKAKVQPYEDDFQKKWQEEMEEYRQKVEPERAEEQEGARQKEHEEQEK ESPEGEEMRDRARAHVDAERTHEAPYSDEERQREAAREEAEKENGGAREAEYHAKATE HESTESEKAKPAEEDERQGEEPVEESFKVSFESAEEEYTKKENTQGPEQKEISEEDENSA VDHHHHHH, as denoted by SEQ ID NO: 23.

[0193] In yet another aspect, the present disclosure provides a targeted elimination compound comprising a liposome or a lipid nanoparticle (ENP) conjugated to the Fragment crystallizable epsilon receptor I alpha chain (FcεRlα), or to an anti-IgE antibody. It should be understood that in some embodiments (i), the FcεRlα comprises the amino acid sequence as denoted by any one of: SEQ ID NOs: 1 and 2, or any variants or derivatives thereof.

[0194] In yet another additional or alternative embodiment (ii), the liposome comprises (1,2-dioleoyl-sn- glycero-3-[(N-(5-amino-l-carboxypentyl)iminodiacetic acidjsuccinyl] (Cobalt salt) (DGS / NTA). In yet another alternative and / or additional embodiment, (iii) the LNP Butanoic acid, 4- (dimethylamino)-, ( 1 OZ, 13Z)- 1 -(9Z, 12Z)-9, 12-octadecadien- 1 -yl- 10, 13-nonadecadien- 1 -yl ester (D-Lin-MC3-DMA), Cholesterol, 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), polyethylene glycol (PEG)-l,2-dimyristoyl-rac-glycerol (DMG); and 1 ,2-distearoyl-sn-glycero- 3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-200 (DSPE-PEG-mal).

[0195] More specifically, it should be understand that the content of the lipid nanoparticles used herein in the attached nanoparticles can be in any ratio. In some specific and non limiting embodiments, the LPNs are in the ratio of 50:38:10:1.9:0.1. Still further, the final concentration of 9.6mM LNPs were prepared by using microfluidic micro mixture (Precision NanoSystems, Vancouver, BC). Still further, in some embodiments, the disclosed nanoparticles, referred to herein as Lipid mixture C contains Butanoic acid, 4-(dimethylamino)-, (10Z,13Z)-l-(9Z,12Z)-9,12-octadecadien-l-yl- 10,13-nonadecadien-l-yl ester (D-Lin-MC3-DMA), Cholesterol, 1,2- distearoyl-sn-glycero-3- phosphocholine (DSPC), polyethylene glycol (PEG)-l,2-dimyristoyl-rac-glycerol (DMG) and l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-200 (DSPE-PEG-mal) in the following ratio: 50:38:10:1.9:0.1 (final concentration of 9.6mM LNPs were prepared by using microfluidic micro mixture (Precision NanoSystems, Vancouver, BC).

[0196] In the lipid mixture C, DSPE-PEG-mal was used to chemically conjugate protein to LNPs based on the cysteine bioconjugation strategy. It should be understood however that other conjugation options are available, for example, conjugating chemistry using the N coupling reaction. For this, the DSPE-PEG-mal is replaced with another lipids such as l,2-dioleoyl-sn-glycero-3-succinate (DGS 18:1) or 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [carboxy(polyethylene glycol)-2000, NHS ester] (sodium salt) (DSPE-PEG(2000) Carboxy NHS) or 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt) (DSPE- PEG(2000) Amine), that may also be considered to covalently conjugate protein to liposome based on N coupling reaction. Still further embodiment for conjugating the LNP to the target recognition molecule (e.g. the FcεRlα, or the anti-IgE antibody), is using the aldehyde reaction. . For this, the DSPE-PEG-mal is replaced with another lipid such as DSPE-PEG-Hydrazide.

[0197] Still further, it should be understood that all definitions of terms provided in the present disclosure, as set forth herein concerning the present aspect, apply equally to each and every aspect of the disclosure.

[0198] Another aspect of the present disclosure involves a pharmaceutical composition comprising at least one targeted elimination compound or any preparations thereof, or any vehicle, matrix, nano- or micro-particle comprising the same, and / or at least one of pharmaceutically acceptable carrier / s, excipient / s, auxiliaries, and / or diluent / s. The targeted elimination compound comprises the following components: In one component (a), at least one target-recognition component. The target-recognition component specifically binds either directly or indirectly, at least one target macromolecule. In some optional embodiments, the target recognition component may further neutralize the target macromolecule. The other component (b) comprises at least one elimination and / or removal-targeting component. More specifically, the elimination and / or removal-targeting component targets the at least one target macromolecule to the liver in a eukaryotic organism. The elimination and / or removal-targeting component is derived from at least one of: a microorganism, a natural and / or synthetic molecule and / or particle displaying liver tropism.

[0199] In some embodiments, the compounds of the compositions of the present disclosure, may be any of the compounds disclosed by the present disclosure, specifically, any of the compounds as defined above, in connection with other aspects of the present disclosure.

[0200] The term “pharmaceutical composition” in the context of the invention means that the composition is of a grade and purity suitable for therapeutic administration to human subjects. As mentioned above, the pharmaceutical composition comprising at least one targeted elimination compound or any preparations thereof, or any vehicle, matrix, nano- or micro-particle comprising the same. The phrase "any preparations thereof as used herein refers to any formulated, processed, or modified forms of the given compound, including but not limited to solutions, suspensions, emulsions, lyophilized powders, encapsulated forms, or chemically stabilized derivatives. These preparations may be designed to enhance stability, bioavailability, controlled release, or targeted delivery. The term "vehicle" as used in this context refers to a carrier or delivery medium that facilitates the administration, transport, or stabilization of the given compound. Vehicles can be aqueous or nonaqueous solutions, lipid-based formulations, polymeric carriers, or other biocompatible systems that enhance solubility, bioavailability, or targeted delivery. The term "matrix" refers to a structured or semi-structured material that serves as a support or scaffold for incorporating, encapsulating, or immobilizing the given compound. Matrices may be composed of biopolymers, hydrogels, ceramics, or synthetic polymers and can be used for controlled release, tissue engineering, or bioreactor applications. A "nano- or micro-particle" as further detailed below refers to a particulate system in the nanometer (1-1000 nm) or micrometer (1-1000 pm) size range that encapsulates, adsorbs, or conjugates the specified compound. These particles can be composed of lipids, polymers, metals, or other biocompatible substances and are used for drug delivery or controlled release applications.

[0201] More specifically, the pharmaceutical composition is present together with at least one of carrier / s, excipient / s, auxiliaries, and / or diluent / s that are pharmaceutically acceptable. The term "carrier" denotes to a diluent, adjuvant, excipient with which the therapeutic is administered. Example of such pharmaceutical carriers are sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Suitable pharmaceutical excipients may include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. A composition can further contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Examples of suitable pharmaceutical carriers are described for example in "Remington's Pharmaceutical Sciences" by E. W. Martin. An "excipient" as used herein refers to an inactive substance formulated alongside an active pharmaceutical ingredient (API) to aid in manufacturing, stability, bioavailability, or administration. Excipients include binders, fillers, disintegrants, lubricants, preservatives, and stabilizers, among others. They play a crucial role in controlling drug release, enhancing solubility, and improving patient compliance. "Auxiliaries " refers to additional substances incorporated into a pharmaceutical or biotechnological formulation to facilitate processing, enhance therapeutic effects, or improve the stability of an active ingredient. These may include solubilizers, buffering agents, emulsifiers, preservatives, or stabilizers. A "diluent" refers to an inert substance added to a formulation to increase volume, facilitate dosing, or modify the concentration of an active ingredient without affecting its therapeutic properties. Diluents can be solid (e.g., lactose, starch) or liquid (e.g., water, saline, ethanol) and are commonly used in tablet, capsule, and injectable formulations.

[0202] The pharmaceutical composition may be suitable for any mode of administration whether oral or parenteral, by injection or by topical administration by inhalation, intranasal spray or intraocular drops.

[0203] Thus, some embodiments consider the composition / s according to the present disclosure, particularly for treating allergic conditions, for example, respiratory diseases such as asthma. According to one embodiment, such disclosed targeted elimination compound may be particularly adapted for pulmonary delivery by oral or nasal inhalation. More specifically, pulmonary delivery may require the use of liquid nebulizers, aerosol-based metered dose inhalers (MDI's), or dry powder dispersion devices. Still further, it should not be overlooked that the degradation targeting compound of the present disclosure, particularly when used for treating allergic-skin disorders such as eczema, dermatitis, or any disorders disclosed in the present disclosure, may be an acceptable topically applied composition as will be described in more detail herein after. Alternatively, the administration may be systemic such as by sublingual, rectal, vaginal, buccal, parenteral, intravenous, intramuscular, subcutaneous modes transdermal, intraperitoneal or intranasal modes of administration. However, oral, transmucosal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as rectal, intrathecal, direct intraventricular, intravenous, intraocular injections or any other medically acceptable methods of administration can be considered as well.

[0204] The targeted elimination compounds and the compositions of the present disclosure may comprise carriers suitable for pulmonary delivery that may involve nasal and / or oral administration. In specific embodiments, such carrier may be any one of spray, mist, patch, foam, alcoholic foam, oily foam, aqueous foam, bandage, membrane, gel, cream, emulsion, oily solution, aqueous solution, hydroethanolic solution, hydro-alcoholic-glycolic solution, mixture of alcohol and glycols, microemulsion, double emulsions, nanoemulsion, nanoparticles, microparticles, microcapsules, lipid particles, lipospheres, liposomes, lipid vesicles, solid lipid nanoparticles, liquid crystals, eutectic mixtures, eutectic crystsls, cubosomes, hexazomes, micelosomes, liposomal systems, vesicular systems, nanocubes, ethosomes, hydroethanolic systems, mixtures of alcohols and glycols, aqueous mixtures of alcohols and glycols, buffer solutions, polymer based delivery systems, hydrophilic or lipophilic suppository bases, chitosan and derivatives bases. For nasal administration, suitable carriers are preferably water-soluble and include water, propylene glycol and other pharmaceutically acceptable alcohols, xanthan gum, locust bean gum, galactose, other saccharides, oligosaccharides and / or polysaccharides, starch, starch fragments, dextrins, British gum and mixtures thereof. For buccal aministration, suitable carriers are water-soluble carrier materials, for example (poly) saccharides like hydrolysed dextran, dextrin, mannitol, and alginates, or mixtures thereof, or mixtures thereof with other carrier materials like polyvinylalcohol, polyvinylpyrrolidine and water-soluble cellulose derivatives, like hydroxypropyl cellulose. In specific embodiments, the buccal carrier material may be gelatin, especially partially hydrolysed gelatin.

[0205] Pharmaceutical formulations adapted for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient. Suitable formulations wherein a semisolid carriers such as gel, cream or ointment may be also used for nasal administration according to the present disclosure.

[0206] As indicated herein above, targeted elimination compound of the present disclosure can be administered via an administration device suitable for nasal administration. As used herein, an administration device is any pharmaceutically acceptable device adapted to deliver a composition of the invention to a subject's nose. A nasal administration device can be a metered administration device (metered volume, metered dose, or metered-weight) or a continuous (or substantially continuous) aerosol-producing device. Suitable nasal administration devices also include devices that can be adapted or modified for nasal administration. In some embodiments, the nasally administered dose can be absorbed into the bloodstream of a subject.

[0207] A metered nasal administration device delivers a fixed (metered) volume or amount (dose) of a nasal composition upon each actuation. Exemplary metered dose devices for nasal administration include, by way of example and without limitation, an atomizer, sprayer, dropper, squeeze tube, squeeze-type spray bottle, pipette, ampule, nasal cannula, metered dose device, nasal spray inhaler, breath actuated bi-directional delivery device, pump spray, pre-compression metered dose spray pump, monospray pump, bispray pump, and pressurized metered dose device. The administration device can be a single-dose disposable device, single-dose reusable device, multi-dose disposable device or multi-dose reusable device.

[0208] Another aspect of the present disclosure is related to a method for targeted elimination and / or removal and / or clearance of at least one target macromolecule in a subject in need. The method comprising the step of administering to the subject an effective amount of at least one targeted elimination compound or any preparations thereof, or any vehicle, matrix, nano- or micro-particle comprising the same, and / or any composition thereof. More specifically, in some embodiments, the targeted elimination compound used by the disclosed methods, comprises the following components: In one component (a), at least one target-recognition component. The targetrecognition component specifically binds either directly or indirectly, at least one target macromolecule. In some optional embodiments, the target recognition component may further neutralize the target macromolecule. Another component (b) comprises at least one elimination and / or removal-targeting component. More specifically, the elimination and / or removal-targeting component targets the at least one target macromolecule to the liver in a eukaryotic organism. The elimination and / or removal-targeting component is derived from at least one of: a microorganism, a natural and / or synthetic molecule and / or particle displaying liver tropism. In some embodiments, the compound used in the methods of the present disclosure is any of the compounds disclosed herein, specifically, any of the compounds as defined above.

[0209] The method of the present disclosure relates to a targeted elimination and / or removal and / or clearance of at least one target macromolecule in a subject in need. "Targeted elimination” as used herein refers to the selective eradication of a specific substance or biomolecule from a biological or environmental system. In some embodiments, the term "targeted removal" as used in this context involves the selective displacement of a specific substance or biomolecule from a biological or environmental system. In some embodiments, the term " clearance" as used herein refers to the accelerated degradation, metabolism, and / or excretion of a specific substance or biomolecule from a biological or environmental system, specifically, from the body of the subject in need. In some embodiments, "elimination and / or removal" of the target macromolecule refers to removal and / or clearance of the target macromolecule from the body of a subject in need leading to a decrease, elimination, removal of the target macromolecule from the circulation, and / or any body cavity, and / or any organ and / or tissue of the subject. More specifically, "removal" is meant in some embodiments any specific extraction, expulsion, ejection, deletion, subtraction, reduction of the target macromolecule, thereby resulting in a decreased amount, level or concentration of the target macromolecule in the circulation, body fluids, body cavities, tissues or organs of the subject by about 5% to 100%, specifically, about 1% to 5%, about 5% to 10%, about 10% to 15%, about

[0210] 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about

[0211] 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about

[0212] 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about

[0213] 95% to 99%, or about 99% to 99.9%, as compared with its amount, level or concentration prior to administration of the compound of the present disclosure. In some embodiments of the present invention, the disclosed methods are specifically applicable for subjects suffering from a pathological disorder associated directly or indirectly with the at least one target macromolecule. A pathological disorder associated directly or indirectly with the at least one target macromolecule refers to a disease, condition, or physiological dysfunction that results from or is influenced by the presence, malfunction, or abnormal regulation of the at least one target macromolecule. Direct association implies that the target macromolecule itself is the primary cause of the disorder due to genetic mutations, structural defects, dysregulation, or pathological modifications. Indirect association refers to cases where the target macromolecule contributes to the pathological condition through intermediate biological pathways, interactions with other biomolecules, or secondary effects, such as inflammatory cascades, immune dysregulation, or altered signaling networks.

[0214] In some further embodiments, the pathological disorder is at least one immune-related disorder.

[0215] In some other embodiments, the methods of the present disclosure may be applicable for at least one immune-related disorder, specifically, as defined herein after. In yet some further embodiments, such immune-related disorder may be a medical condition associated with or induced by activation of at least one white blood cell, for example, leukocytes.

[0216] The term "white blood cells" or "leukocyte" refers to any of the three main subtypes of immune cells: granulocytes, lymphocytes and monocytes. All white blood cells have nuclei, which distinguishes them from the other blood cells, the non-nucleated red blood cells (RBCs) and platelets. The different white blood cells are usually classified by cell lineage (myeloid cells or lymphoid cells). White blood cells are part of the body's immune system, they help the body fight infection and other diseases. Types of white blood cells are granulocytes (neutrophils, eosinophils, and basophils), and agranulocytes (monocytes, and lymphocytes (T cells and B cells)). Myeloid cells (myelocytes) include neutrophils, eosinophils, mast cells, basophils, and monocytes. Monocytes are further subdivided into dendritic cells and macrophages. Monocytes, macrophages, and neutrophils are phagocytic. Lymphoid cells (lymphocytes) include T cells (subdivided into helper T cells, memory T cells, cytotoxic T cells), B cells (subdivided into plasma cells and memory B cells), and natural killer cells. Produced in the bone marrow, white blood cells defend the body against infections and disease. An excess of white blood cells is usually due to infection or inflammation. Less commonly, a high white blood cell count could indicate certain blood cancers or bone marrow disorders.

[0217] In some embodiments, the methods of the present disclosure may be applicable for subjects suffering of a medical condition involving activation of white blood cells (leukocyte). In some embodiments, such cell / s may be at least one of: mast cell, basophile and / or eosinophil. Thus, in some embodiments, the medical condition is associated with or induced by activation of at least one of the mast cells, basophils and / or eosinophils.

[0218] "Mast cells" (referred to herein as "MS”), also known as a "mastocytes” or a "labrocytes" are derived from the myeloid stem cells and contain many granules rich in histamine and heparin, very similar to basophil granulocytes. Mast cells comprise a normal component of the connective tissue that plays an important role in immediate (type I) hypersensitivity and inflammatory reactions by secreting a large variety of chemical mediators from storage sites in their granules upon stimulation. Mast cells, and their circulating counterparts the basophiles, possess surface receptors known as FcaRI that are specific for IgE heavy chains.

[0219] Still further, "Basophils" are a type of white blood cell. Basophils are the least common type of granulocyte, representing about 0.5% to 1% of circulating white blood cells. They are responsible for inflammatory reactions during immune response, as well as in the formation of acute and chronic allergic diseases, including anaphylaxis, asthma, atopic dermatitis and hay fever. They also produce compounds that coordinate immune responses, including histamine and serotonin that induce inflammation, and heparin that prevents blood clotting, although there are less than that found in mast cell granules. Mast cells were once thought to be basophils that migrated from the blood into their resident tissues (connective tissue), but they are now known to be different types of cells.

[0220] "Eosinophils" , also known as eosinophiles or, less commonly, acidophils, are a variety of white blood cells, which differentiate from myeloid precursor in the bone marrow, and produce and store many secondary granule proteins prior to their exit from the bone marrow. Eosinophils are one of the immune system components responsible for combating multicellular parasites and certain infections in vertebrates. Along with mast cells and basophils, they also control mechanisms associated with allergy and asthma. When eosinophils are activated, they undergo cytolysis, where the breaking of the cell releases eosinophilic granules found in extracellular DNA traps. High concentrations of these DNA traps are known to cause cellular damage, as the granules they contain are responsible for the ligand-induced secretion of eosinophilic toxins which cause structural damage. Following activation, eosinophils effector functions include production of mediators such as: cationic granule proteins and their release by degranulation, reactive oxygen species such as hypobromite, superoxide, and peroxide (hypobromous acid, which is preferentially produced by eosinophil peroxidase), lipid mediators like the eicosanoids from the leukotriene (e.g., LTC4, LTD4, LTE4) and prostaglandin (e.g., PGE2) families, enzymes, such as elastase, growth factors such as TGF beta, VEGF, and PDGF and cytokines such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, and TNF alpha.

[0221] The event that initiates immediate hypersensitivity is the binding of antigen to the antigen-binding site in IgE on the mast cell or basophil surface. Both cell types are activated by cross-linking of FcaRI molecules, which is thought to occur by binding multivalent antigens to the attached IgE molecules.

[0222] Therefore, mast cells and / or basophiles activation (or degranulation), as used herein, involve release of preformed granules and the secretion of eicosanoids, cytokines and chemokines. Mast cells may also be activated by mechanisms other than cross linking FcaRI, such as in response to mononuclear phagocyte-derived chemocytokines, to T cell-derived cytokines and to complement- derived anaphylatoxins.

[0223] In certain embodiments, the present disclosure relates to mast cell and / or basophil activation caused by cross-linking of FcaRI. Moreover, the cross-linking caused by and / or facilitated via IgE in the circulation.

[0224] More specifically, when antigen binds to IgE molecules attached to the surface of mast cells, a variety of mediators are released which give rise to increased vascular permeation, vasodilatation, bronchial, visceral smooth muscle contraction, and local inflammation.

[0225] Mediators released from mast cells upon their activation, may be divided into two broad classes, pre-formed or secretory granule associated mediators and non-preformed or newly synthesized mediators. The pre-formed mediators include biogenic amines, most notably histamine. The preformed mediators also comprise granule macromolecules such as proteoglycans, most notably heparin and chondroitin sulfate E; chemotactic factors such as eosinophil and neutrophil chemotactic factors of anaphylaxis; and enzymes such as proteases, tryptase, chymase, cathepsin G-like enzyme, elastase, carboxypeptidase A and acid hydrolases. The nonpreformed mediators include products of arachidonic acid, prostaglandin D2, leukotrienes C4 and B4 and platelet activating factor. Another class of mediators, the cytokines are produced by mast cells upon IgE- mediated activation, or by other cells, including recruited Th2 lymphocytes. The cytokines are predominantly responsible for the late phase reaction which begins two to four hours after elicitation of many immediate hypersensitivity reactions.

[0226] As also demonstrated by the attached Examples, it should be noted that the targeted elimination compounds of the present disclosure and any compositions thereof, lead to inhibition or decrease in mast cell activation or degranulation. Thus, the term “decrease in mast cell activation” refers to decrease, attenuation, reduction in cell degranulation and mediator secretion and release. In some particular embodiments, the degradation targeting compounds of the invention may lead to decrease of secretion or release of one or more of the following: serine proteases such as tryptase, histamine (2-5 μg / cell), serotonin, proteoglycans, mainly heparin (active as anticoagulant), Granzyme B, VEGF; newly formed lipid mediators (eicosanoids), for example, thromboxane, prostaglandin D2, leukotriene C4, platelet-activating factor, cytokines, Eosinophil chemotactic factor, IL-6, IL-4, IL-8 and TNF-a.

[0227] In some specific embodiments, the medical condition associated with or induced by activation of at least one of mast cells, basophiles and / or eosinophils is at least one of allergic reaction, a chronic or acute inflammatory condition involving airways, skin, and / or mucosal tissue, mastocytosis and mast cell tumors. According to some embodiments, the disclosed methods may be applicable for subjects suffering from any of the disclosed disorders.

[0228] An "allergic reaction” refers to an immune-mediated hypersensitivity response triggered by exposure to an allergen, resulting in the activation of mast cells, basophils, and other immune components. This reaction is primarily driven by immunoglobulin E (IgE) binding to FcaRI receptors on mast cells, leading to the release of histamine, cytokines, and other inflammatory mediators. The clinical manifestations can range from mild symptoms such as itching and localized swelling to severe, life-threatening anaphylaxis, affecting multiple organ systems.

[0229] A "chronic or acute inflammatory condition involving airways, skin, and / or mucosal tissue " refers to a pathological state characterized by sustained or transient immune activation and tissue inflammation affecting the respiratory tract, epidermis, or mucosal linings. Acute forms typically result from infections, allergic reactions, or environmental irritants, leading to symptoms such as swelling, erythema, and increased mucus production. Chronic forms, such as asthma, atopic dermatitis, or inflammatory bowel disease, involve prolonged immune dysregulation, tissue remodeling, and recurrent inflammatory episodes driven by immune cells, cytokines, and other inflammatory mediators.

[0230] "Mastocytosis" refers to a disorder characterized by the abnormal proliferation and accumulation of mast cells in various tissues, including the skin, bone marrow, gastrointestinal tract, and internal organs. It can present as cutaneous mastocytosis, which is limited to the skin, or systemic mastocytosis, which involves multiple organ systems and may be associated with activating mutations in the KIT proto-oncogene. Symptoms arise due to excessive mast cell degranulation, leading to histamine release and manifestations such as flushing, anaphylaxis, gastrointestinal distress, and bone pain.

[0231] "Mast cell tumors (MCTs)” refer to neoplastic proliferations of mast cells that can occur in various tissues, most commonly in the skin of dogs, cats, and other animals, and less frequently in humans. These tumors can range from benign to highly aggressive forms, with their pathogenesis often linked to dysregulated KIT receptor signaling and excessive mast cell activation. Clinical manifestations vary depending on tumor location and degranulation activity, leading to local inflammation, systemic hypersensitivity reactions, and, in severe cases, metastatic disease.

[0232] Another aspect of the present disclosure is related to a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one pathological disorder associated directly or indirectly with at least one target macromolecule in a subject, the method comprising the steps of administering to the subject a therapeutically effective amount of at least one targeted elimination compound, or any preparations thereof, or any vehicle, matrix, nano- or micro-particle comprising the same, and / or any composition thereof. More specifically, the targeted elimination compound comprises the following components: In one component (a), at least one target-recognition component. The target-recognition component specifically binds either directly or indirectly, at least one target macromolecule. In some optional embodiments, the target recognition component may further neutralize the target macromolecule. The additional component (b) comprises at least one elimination and / or removal-targeting component. More specifically, the elimination and / or removal-targeting component targets the at least one target macromolecule to the liver in a eukaryotic organism. The elimination and / or removal-targeting component is derived from at least one of: a microorganism, a natural and / or synthetic molecule and / or particle displaying liver tropism.

[0233] In some embodiments of the therapeutic methods of the present disclosure, the at least one targetrecognition component of (a) and the at least one elimination and / or removal-targeting component of (b), are fused, linked, associated and / or conjugated directly or indirectly via at least one linking moiety. In some further embodiments, the subject treated by the disclosed methods, may be a mammalian subject. In some other embodiments, the elimination and / or removal-targeting component (b) of the targeted elimination compound used by the therapeutic methods of the present disclosure, is derived form a microorganism displaying liver tropism. In some embodiments, such microorganism is at least one of a parasite, a virus and / or bacteria.

[0234] In some further embodiments, the elimination and / or removal-targeting component is derived from the Plasmodium sporozoites, specifically, the Plasmodium sporozoites surface antigen, CSP.

[0235] In some specific embodiments, the elimination and / or removal-targeting component of the targeted elimination compound used by the therapeutic methods of the present disclosure comprises at least one CSP-derived peptide. In some embodiments, such peptide may comprise the amino acid sequence as denoted by SEQ ID NO: 3, or any variants or derivatives thereof.

[0236] In some alternative embodiments of the therapeutic methods of the present disclosure, the elimination and / or removal-targeting component of the targeted elimination compound used, is derived form a synthetic molecule or particle displaying liver tropism. In some embodiments, the synthetic particle may be at least one of at least one nano- or micro-particle, micellar formulation, or liposomal formulation. In some further embodiments, the elimination and / or removal-targeting component of the targeted elimination compound used by the therapeutic methods of the present disclosure, may comprise at least one liposome.

[0237] In some alternative and / or additional embodiments, the elimination and / or removal-targeting component of the targeted elimination compound used by the therapeutic methods of the present disclosure, is derived form a natural molecule displaying liver tropism. In some embodiments, such molecule is a protein produced by and / or functions on the liver tissue.

[0238] In some embodiments, the at least one elimination and / or removal-targeting component of the targeted elimination compound used by the therapeutic methods of the present disclosure, is derived from at least one Apolipoprotein, or any functional peptides thereof. Specifically, Apo A, Apo B, Apo C, Apo D, Apo E or functional peptides thereof, specifically, peptides or derivatives that retain the function of liver tropism or targeting to liver cells or tissue.

[0239] In some specific embodiments, the at least one Apolipoprotein is Apolipoprotein A-I (Apo Al), or any functional peptides thereof. Optionally, the elimination and / or removal-targeting component comprises the amino acid sequence as denoted by SEQ ID NO: 17, or any variants or derivatives thereof, or any functional peptides thereof. Still further, in some embodiments, Apolipoprotein peptides that may be used in the targeted elimination compound of the present disclosure as the elimination and / or removal-targeting component (b), may be any peptides derived from ApoA, ApoB, ApoC and / or ApoE. In some embodiments, the Apolipoprotein functional peptide that may be used in the targeted elimination compound of the present disclosure as the elimination and / or removal-targeting component (b) comprises at least one peptide derived from ApoE. In some embodiments, a peptide derived from ApoE may comprise the amino acid sequence of residues 140 to 158 of the human ApoE. In some embodiments, such peptide may comprise the amino acid sequence as denoted by SEQ ID NO: 20 (ApoE liver targeting fragment, residues 140-158). In some other embodiments, the Apolipoprotein functional peptide that may be used in the targeted elimination compound of the present disclosure as the elimination and / or removal-targeting component (b) comprises at least one peptide derived from ApoB. In some embodiments, a peptide derived from ApoB may comprise the amino acid sequence of residues 3144 to 3159of the human ApoE. In some other embodiments, such peptide may comprise the amino acid sequence as denoted by SEQ ID NO: 21 (ApoB liver targeting fragment, residues 3144-3159).

[0240] In some embodiments, the target-recognition component of the targeted elimination compound used by the therapeutic methods of the present disclosure, is an affinity molecule comprising at least one of: an amino acid-based, a nucleic acid-based, a small molecule-based, a carbohydrate- based, a lipid-based molecule. In some embodiments, the affinity molecule may be a part of an affinity pair, or example, of a receptor-ligand, antibody-antigen, enzyme-substrate, aptamer-target affinity pair, or any combination thereof.

[0241] In some further embodiments, the target-recognition component of the targeted elimination compound used by the therapeutic methods of the present disclosure, is, or derived from at least one of: at least one receptor molecule, at least one antibody and / or any fragments thereof and / or at least one aptamer, and / or any combinations thereof.

[0242] In some additional embodiments, the target-recognition component of the targeted elimination compound used by the therapeutic methods of the present disclosure, comprises, or is derived from at least one receptor molecule.

[0243] In some additional embodiments, the target macromolecule targeted by targeted elimination compound used by the therapeutic methods of the present disclosure, is at least one of: an amino acid-based, a nucleic acid-based, a small molecule-based, a carbohydrate-based, a lipid-based molecule or any combination or complex thereof.

[0244] In some embodiments of the disclosure, the macromolecule is an amino acid-based molecule. In some embodiments, the targeted macromolecule may be a circulating protein of at least one of: an immunoglobulin molecule, a cytokine, a chemokine, a growth and / or survival factor, a hormone, a ligand and / or a soluble receptor.

[0245] In some other embodiments, the target macromolecule is an immunoglobulin molecule. In some specific embodiments, the immunoglobulin is immunoglobulin E (IgE).

[0246] In some other embodiments, the target recognition component of the targeted elimination compound used by the therapeutic methods of the present disclosure, may comprise at least one receptor molecule, specifically, an immunoglobulin-binding receptor.

[0247] In more specific embodiments, the immunoglobulin-binding receptor is at least one of IgE binding receptor or any fragment / s thereof. In some embodiments, the immunoglobulin E binding receptor may be at least one of FcaRl and FcaR2 and any fragments and / or combination thereof.

[0248] In some specific embodiments, the at least one immunoglobulin E binding receptor is the FcεRlα. It should be noted that soluble form of FcεRlα extracellular fraction shall enable binding to the IgE in the circulation.

[0249] In some further specific embodiments, the target-recognition component of the targeted elimination compound used by the therapeutic methods of the present disclosure, is derived from the FcεRlα. In more specific embodiments, such target recognition component may comprise the amino acid sequence as denoted by SEQ ID NO: 1, or any variants or derivatives thereof. It should be noted that in some embodiments, the target-recognition component comprises, or is derived from at least one antibody and / or any fragment thereof. In other embodiments, the targetrecognition component comprises, or is derived from, at least one antibody and / or any fragment thereof that binds at least one immunoglobulin molecule. In a more specific embodiments, the target-recognition component comprises, or is derived from at least one antibody and / or any fragment thereof that binds immunoglobulin E (IgE).

[0250] In some embodiments, the therapeutic methods of the present disclosure may be applicable for subject / s suffering from at least one immune-related disorder.

[0251] An "Immune-related disorder" or " Immune-mediated disorder" , as used herein encompasses any condition that is associated with the immune system of a subject, more specifically through inhibition of the immune system, or that can be treated, prevented, or ameliorated by reducing degradation of a certain component of the immune response in a subject, such as the adaptive or innate immune response. An immune-related disorder may include infectious condition (e.g., by a pathogen, specifically, viral, bacterial, or fungal infections), inflammatory disease, autoimmune disorders, immunodeficiency (e.g., primary or a secondary) metabolic disorders and proliferative disorders.

[0252] In some further embodiments, the at least one immune-related disorder is a medical condition associated with or induced by, activation of at least one white blood cell, specifically, leukocyte / s. In some additional embodiments, the medical condition treatable by the therapeutic methods disclosed herein, may be a disorder associated with activation of at least one of: mast cell, basophile and / or eosinophil. Thus, in some specific embodiments, the medical condition associated with or induced by activation of the mast cells, basophils and / or eosinophils may be any one of allergic reaction, a chronic or acute inflammatory condition involving airways, skin, and / or mucosal tissue, mastocytosis and mast cell tumors. More specific embodiments relate to disorders such as asthma, eczema, dermatitis, allergic rhinitis, allergic conjunctivitis, anaphylaxis, and the like.

[0253] Another aspect of the present disclosure is related to a therapeutically effective amount of at least one targeted elimination compound, or any preparations thereof, or any vehicle, matrix, nano- or micro-particle comprising the same, and / or any composition thereof, for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one pathological disorder associated directly or indirectly with at least one target macromolecule in a subject. The targeted elimination compound used herein, comprises the following components: In one component (a), at least one target-recognition component. The target -recognition component specifically binds either directly or indirectly, at least one target macromolecule. In some optional embodiments, the target recognition component may further neutralize the target macromolecule. The additional component (b) comprises at least one elimination and / or removaltargeting component. More specifically, the elimination and / or removal-targeting component targets the at least one target macromolecule to the liver in a eukaryotic organism. The elimination and / or removal-targeting component is derived from at least one of: a microorganism, a natural and / or synthetic molecule and / or particle displaying liver tropism.

[0254] In some embodiments, the therapeutically effective amount of at least one targeted elimination compound for use, in accordance with the present disclosure, the compound used herein, is any of the compounds of the present disclosure, specifically, as defined above. In yet some further embodiments, the disclosed use may be specifically applicable for a pathological disorder, specifically, at least one immune-related disorder. In yet some further embodiments, the at least one immune-related disorder may be a medical condition associated with, or induced by, activation of at least one leukocyte. In some further embodiments, the use herein may be applicable for a medical condition associated with, or induced by, at least one of mast cell basophile and / or eosinophil / s. In more specific embodiments, the medical condition associated with or induced by mast cell activation is any one of allergic reaction, a chronic or acute inflammatory condition involving airways, skin, and / or mucosal tissue, mastocytosis and mast cell tumors. Still further, in some embodiments, the use disclosed herein may be applicable for conditions such as asthma, eczema, dermatitis, allergic rhinitis, allergic conjunctivitis, and / or anaphylaxis.

[0255] In certain embodiments, the methods of the invention are particularly applicable for treating a disorder characterized by undesirable release of mediator from immunologically stimulated cells, particularly mast cells, basophils and / or eosinophils.

[0256] Mediators released from human mast cells and / or basophils are central to the pathophysiology of allergy, asthma and anaphylaxis. In particular, mast cells and their release of histamine and other mediators play an important role in the symptomatology of asthma and other human diseases. During the early phase of human lung hypersensitivity reactions upon exposure to antigen (i.e., pollens, cats, etc.), mast cells release and are the major source of histamine, and newly synthesized lipid products of arachidonic acid metabolism: prostaglandin D2 and leukotriene C4. These mediators produce immediate breathlessness, which subsides in one hour but returns within 2-4 hours (the "late phase" response).

[0257] More specifically, as used herein, the phrase "a medical condition associated with and / or induced by activation of mast cells (MCs), basophils and / or eosinophils" refers to a condition in which onset or progression of pathology is attributed at least in part to increase in activation of MCs, basophils and / or eosinophils. Non-limiting examples of diseases induced by activation of MCs and / or basophils include allergy, asthma, allergic rhinitis, allergic conjunctivitis, atopic dermatitis and atopic eczema, allergic disorders and responses to various allergens, systemic anaphylaxis, anaphylactic response to contrast media, anaphylactic response to muscle relaxants, systemic mastocytosis, mast cell activation syndrome (MAS), morphea / urticaria pigmentosa, mast cell leukemia, atherosclerosis, graft rejection, multiple sclerosis, fibrotic lung diseases, neurofibromatosis, keloids, scleroderma, acute gout, ocular cicatricial pemphigoid, inflammatory disease of the gut such as inflammatory bowel disease, colitis, interstitial cystitis, irritable bowel syndrome, and celiac disease and Crohn's disease, peritoneal adhesions, chronic graft versus host disease (GVHD), extrinsic bronchial asthma, nasal polyposis, Wegener's granulomatosis, intrinsic bronchial asthma, interstitial and other pulmonary diseases, hypersensitivity pneumonitis, allergic bronchopulmonary aspergillosis, sarcoidosis, idiopathic pulmonary fibrosis, toxocariasis, filariasis, schistosomiasis, trichinosis, neoplastic and myeloproliferative diseases, T cell lymphomas, Hodgkin's disease, psoriasis, diabetes mellitus, systemic lupus erythematosus (SLE), Sjogren's syndrome, rheumatoid arthritis, rheumatoid osteoarthritis, polymyositis, autoimmune thyroid disease, autoimmune gastritis and pernicious anemia, and autoimmune hepatitis.

[0258] Non-limiting examples of diseases induced by activation of eosinophils include asthma, chronic obstructive pulmonary diseases, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), acute eosinophilic pneumonia (AEP), chronic eosinophilic pneumonia (CEP), tumor- associated tissue eosinophilia (TATE), mainly in colon tumors, esophageal squamous cell carcinoma, nasopharyngeal cancer, penile cancer, laryngeal cancer, lung adenocarcinoma, bladder cancer, and prostate cancer.

[0259] In certain embodiments, the methods of the invention may be applicable for medical condition associated with or induced by mast cell and / or basophil activation. Such condition may be for example, any one of allergic reaction, asthma, eczema, dermatitis, allergic rhinitis allergic conjunctivitis, anaphylaxis, mastocytosis and mast cell tumors or basophil-associated cancer such as chronic myeloid leukemia, lung adenocarcinoma and pancreatic cancer.

[0260] In some embodiments, the invention provides methods for treating, preventing, ameliorating and inhibiting asthma. Asthma is a common chronic inflammatory disease of the airways characterized by variable and recurring symptoms, airflow obstruction, and bronchospasm. Symptoms include wheezing, coughing, chest tightness, and shortness of breath. Clinically, asthma is recognized by airway hyperactivity and reversible airways obstruction. Pathological derangements at the tissue level include constriction of airway smooth muscle, increased vascular permeability resulting in edema of airways, outpouring of mucus from goblet cells and mucus glands, parasympathetic nervous system activation, denudation of airway epithelial lining cells, and influx of inflammatory cells. Underlying these tissue effects are direct effects of potent mediators secreted following physical, inflammatory, or immunological activation and degranulation. The early phase of the asthmatic reaction is mediated by histamine and other mast cell mediators that induce rapid effects on target organs, particularly smooth muscle. The pathophysiologic sequence of asthma may be initiated by mast cell activation in response to allergen binding to IgE.

[0261] Asthma is clinically classified according to the frequency of symptoms, forced expiratory volume in one second (FEV 1), and peak expiratory flow rate. Asthma may also be classified as atopic (extrinsic) or non-atopic (intrinsic), based on whether symptoms are precipitated by allergens (atopic) or not (nonatopic).

[0262] Asthma is controlled by environmental and genetic factors. These factors influence how severe asthma is and how well it responds to medication. The interaction is complex and not fully understood.

[0263] Prevention of the development of asthma is different from prevention of asthma episodes. Aggressive treatment of mild allergy with immunotherapy has been shown to reduce the likelihood of asthma development. In controlling symptoms, the first step is establishing a plan of action to prevent episodes of asthma by avoiding triggers and allergens, regularly testing for lung function, and using preventive medications.

[0264] It should be appreciated that the compositions and methods of the invention may be applicable for treating asthma as quick-relief medications used to treat acute symptoms and as long-term control medications used to prevent further exacerbation.

[0265] It should be noted that medications for asthma and other reparatory-associated disorders are typically provided as metered-dose inhalers (MDIs) in combination with an asthma spacer or as a dry powder inhaler. The spacer is a plastic cylinder that mixes the medication with air, making it easier to receive a full dose of the drug. A nebulizer may also be used. Appropriate inhalers applicable for the methods of the present invention are described in detail herein before.

[0266] In certain embodiments, the methods of the present disclosure may be applicable for treating allergic rhinitis. Allergic rhinitis is a condition which results from the activation of mucosal mast cells beneath the nasal epithelium by allergens such as pollens. Allergic rhinitis is characterized by intense itching and sneezing, local oedema, nasal discharge and irritation of the nose as a result of histamine release. When caused by pollens of any plants, it is called pollinosis, and, if specifically caused by grass pollens, it is known as hayfever. In still further embodiments, the method / s and composition / s provided by the present disclosure may be used for treating allergic conjunctivitis. Allergic conjunctivitis is inflammation of the conjunctiva (the membrane covering the white part of the eye) due to allergy. Although allergens differ among patients, the most common cause is hay fever. Symptoms consist of redness (mainly due to vasodilation of the peripheral small blood vessels), oedema (swelling) of the conjunctiva, itching, and increased lacrimation (production of tears). If this is combined with rhinitis, the condition is termed allergic rhinoconjunctivitis. The symptoms are due to release of histamine and other active substances by mast cells, which stimulate dilation of blood vessels, irritate nerve endings, and increase secretion of tears.

[0267] As indicated above, the present invention contemplates methods for the treatment of different allergy- related respiratory diseases. In addition to asthma, such respiratory diseases may include any other acute allergy manifestations in airways, chronic rhinosinusitis (CRS), allergic rhinitis, COPD, nasal polyposis (NP), vasomotor rhinitis, airways hyper-responsiveness, or allergic sinusitis. The invention therefore provides methods and compositions for preventing, treating, ameliorating or inhibiting any of the respiratory diseases described above.

[0268] Thus, in certain embodiments, the invention provides methods and compositions for treating sinusitis. Sinusitis is inflammation of the paranasal sinuses, which may be due to infection, allergy or autoimmune issues.

[0269] Chronic sinusitis, by definition, lasts longer than three months and can be caused by many different diseases that share chronic inflammation of the sinuses as a common symptom. Chronic sinusitis cases are subdivided into cases with polyps and cases without polyps. When polyps are present, the condition is called chronic hyperplastic sinusitis; however, the causes are poorly understood and may include allergy, environmental factors such as dust or pollution, bacterial infection, or fungus (either allergic, infective, or reactive). Non-allergic factors, such as vasomotor rhinitis, can also cause chronic sinus problems.

[0270] Still further, the method / s and composition / s of the invention may be used for treating Nasal polyps. Nasal polyps are polypoidal masses arising mainly from the mucous membranes of the nose and paranasal sinuses. They are overgrowths of the mucosa that frequently accompany allergic rhinitis. They are freely moveable and non-tender. Nasal polyps are usually classified into antrochoanal polyps and ethmoidal polyps. Antrochoanal polyps arise from the maxillary sinuses and are the much less common, ethmoidal polyps arise from the ethmoidal sinuses. Antrochoanal polyps are usually single and unilateral whereas ethmoidal polyps are multiple and bilateral. Airway hyperresponsiveness (or other combinations with bronchial or hyperreactivity) is a state characterized by easily triggered bronchospasm (contraction of the bronchioles or small airways), and can be assessed with a bronchial challenge test. This most often uses products like metacholine or histamine. These chemicals trigger bronchospasm in normal individuals as well, but people with bronchial hyperresponsiveness have a lower threshold. Bronchial hyperresponsiveness is a hallmark of asthma but also occurs frequently in people suffering from chronic obstructive pulmonary disease (COPD). Still further embodiments of the invention provide methods for treating hypersensitivity. Hypersensitivity reactions are the result of immune responses acting inappropriately and can be provoked by many antigens. They are produced by a combination of inflammatory mediators released by several cell types, resulting in acute inflammatory reaction with symptoms such as asthma or rhinitis. Airway inflammation is central to the pathogenesis of asthma and involves the recruitment and activation of mast cells, eosinophils, neutrophils, and lymphocytes into lung tissue and bronchoalveolar space.

[0271] Still further, the methods of the present disclosure may be suitable for allergic hypersensitivity. The inflammatory response characteristic of allergic or hypersensitivity reactions can be elicited by extrinsic antigens such as pollen, dust, food, dust-mites and chemicals in the environment. There are four main classes of hypersensitivity reactions, which are distinguished by the type of immune cells and antibodies involved and the pathologies produced. In the most common IgE dependent allergic reactions, the inflammatory response involves mast cell degranulation, or emptying of the granules, triggered by allergen interaction with IgE molecules on the mast cell surface.

[0272] Inhaled allergens initiate respiratory allergies such as allergic rhinitis, hay fever and asthma, while ingested allergens may cause food allergies. Injected allergens, such as antibiotics and insect venoms, may cause life-threatening anaphylactic reactions.

[0273] Still further, it should be noted that the methods of the invention may be applicable for the treatment of anaphylaxis. Anaphylaxis is a serious allergic reaction that is rapid in onset and may cause death. It typically causes a number of symptoms including an itchy rash, throat swelling, and low blood pressure. Common causes include insect bites and stings, foods, and medications. More specifically, in anaphylaxis, body-wide degranulation of mast cells leads to vasodilation and, if severe, symptoms of life-threatening shock.

[0274] It should be also noted that the compositions and methods of the invention may be particularly useful in treating the underlying pathological changes in the airways associated with any of the diseases caused by MC activation, for example, basement membrane thickening, cell hypertrophy and hyperplasia, inflammatory cell influx, and other tissue remodeling.

[0275] Still further, certain embodiments of the invention relate to methods of treating dermatitis. The term "dermatitis" refers to inflammation of the skin, in general. The different kinds usually have in common an allergic reaction to specific allergens. The term may be used to refer to eczema, which is also known as dermatitis eczema or eczematous dermatitis. A diagnosis of eczema often implies atopic dermatitis (childhood eczema), but without proper context, it means nothing more than a "rash", i.e. a transient skin inflammation. In some embodiments, "dermatitis" and eczema are synonyms, while in other embodiments "dermatitis" implies an acute condition and "eczema" a chronic one, however, it should be noted that the two conditions are often classified together.

[0276] More specifically, dermatitis is characterized by itchy, erythematous, vesicular, weeping, and crusting patches. As noted above, the term eczema is also commonly used to describe atopic dermatitis or atopic eczema. The term eczema is broadly applied to a range of persistent skin conditions. These include dryness and recurring skin rashes that are characterized by one or more of these symptoms: redness, skin swelling, itching and dryness, crusting, flaking, blistering, cracking, oozing, or bleeding. Areas of temporary skin discoloration may appear and are sometimes due to healed injuries. Scratching open a healing lesion may result in scarring and may enlarge the rash.

[0277] Other embodiments of the invention relate to methods of treating urticaria. " Urticaria”, also known as Hives, is a kind of skin rash with red, raised, itchy bumps. Urticaria may burn or sting. The patches of rash may appear on different body parts, with variable duration from minutes to days, and does not leave any long-lasting skin change. Fewer than 5% of cases last for more than six weeks (a condition known as chronic urticaria). The condition frequently recurs. Urticaria frequently occur following an infection or as a result of an allergic reaction such as to medication, insect bites, or food. Psychological stress, cold temperature, or vibration may also be a trigger. In half of cases the cause remains unknown. Risk factors include having conditions such as hay fever or asthma. Whether the trigger is allergic or not, a complex release of inflammatory mediators, including histamine from cutaneous mast cells, results in fluid leakage from superficial blood vessels.

[0278] Allergic-related skin disorder or allergic-skin conditions that may be treated by the compositions and methods of the invention may include also insect bites and stings. Insect bites occur when an insect is agitated and seeks to defend itself through its natural defense mechanisms, or when an insect seeks to feed off the bitten person. Insects inject formic acid, which can cause an immediate skin reaction often resulting in redness and swelling in the injured area. The sting from fire ants, bees, wasps and hornets are usually painful, and may stimulate a dangerous allergic reaction called anaphylaxis for at-risk patients, and some wasps can also have a powerful bite along with a sting. Bites from mosquitoes, fleas, and mites are more likely to cause itching than pain. The skin reaction to insect bites and stings usually lasts for up to a few days. However, in some cases the local reaction can last for up to two years. The reaction to a sting is of three types. The normal reaction involves the area around the bite with redness, itchiness, and pain. A large local reaction occurs when the area of swelling is greater than five cm. Systemic reactions are when symptoms occur in areas besides that of the bites.

[0279] It should be further appreciated that the degradation targeting compounds of the present disclosure and any uses, methods and compositions thereof, may be applicable for treating any of the allergic disorders disclosed above, for example asthma or dermatitis, as well as any associated conditions. It is understood that the interchangeably used terms "associated" and "related", when referring to pathologies herein, mean diseases, disorders, conditions, or any pathologies which at least one of: share causalities, coexist at a higher than coincidental frequency, or where at least one disease, disorder condition or pathology causes the second disease, disorder, condition or pathology.

[0280] As noted above, the invention provides a method of treating, inhibiting, preventing or ameliorating MC activation related conditions.

[0281] The term “treatment” in accordance with disorders associated with activation of MC, specifically, allergic conditions may refer to one or more of the following: elimination of allergic reactions, reducing or decreasing the intensity or frequency of disorders associated with activation of MC, specifically, allergic conditions incidence. The treatment may be undertaken when a disorder associated with activation of MC, specifically, allergic conditions incidence is beginning or may be a continuous administration, for example by administration every 1 to 14 days, to prevent or decrease occurrence of allergic reaction in an individual prone to allergic reactions. Thus, the term “treatment” is also meant as prophylactic or ameliorating treatment.

[0282] The term "reduction" or "reduce", as referred to herein, relates to a decrease in value, amount, or rate of the intensity or frequency of allergic reactions in the treated subject. A decrease or a reduction as referred to herein, relate to a reduction or lessening of a quantity or intensity of allergic reactions by any one of about 1% to 99.9%. More specifically, about 1% to 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%. The term "inhibition" as referred to herein, relates to the retardation, retraining or reduction of the intensity or frequency of disorders associated with activation of MC, specifically, allergic conditions by about 1% to 99.9%. More specifically, by any one of about 1% to 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to

[0283] 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to

[0284] 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to

[0285] 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%.

[0286] The term "prophylaxis" refers to prevention or reduction the risk of occurrence of the biological or medical event, specifically, the occurrence or re occurrence of disorders associated with activation of MC, specifically, allergic conditions, that is sought to be prevented in a tissue, a system, animal or human by a researcher, veterinarian, medical doctor or other clinician, and the term “prophylactically effective amount” is intended to mean that amount of a pharmaceutical composition that will achieve this goal. Thus, in particular embodiments, the methods of the invention are particularly effective in the prophylaxis, i.e., prevention of conditions associated with activation of MC. Thus, subjects administered with said compositions are less likely to experience skin temperature rises or elevation in blood levels of mediators such as histamine, associated with allergic reactions, and said skin temperature rises or elevation in blood levels of mediators such as histamine are also less likely to re-occur in a subject who has already experienced them in the past. The term "amelioration" as referred to herein, relates to a decrease in the deviation of the body temperature or reduced elevation in blood levels of mediators such as histamine of a subject suffering from allergic condition, from the normal physiological temperature range for that subject or normal level of said mediators. More specifically, "amelioration" relates to an improvement in a subject's allergic reaction intensity. For instance, as a non-limiting example, the symptoms of a subject suffering from disorders associated with activation of MC, specifically allergic conditions, are ameliorated by the blood histamine levels or body-temperature reducing treatment with the composition of the invention or according to the method of the invention.

[0287] As used herein, “disease”, “disorder”, “condition” and the like, as they relate to a subject's health, are used interchangeably and have meanings ascribed to each and all of such terms.

[0288] The present invention relates to the treatment of subjects, or patients, in need thereof. By “patient” or “subject in need” it is meant any organism who may be affected by the above-mentioned conditions, and to whom the treatment and diagnosis methods herein described is desired, including humans, domestic and non-domestic mammals such as canine and feline subjects, bovine, simian, equine and murine subjects, rodents, domestic birds, aquaculture, fish and exotic aquarium fish. It should be appreciated that the treated subject may be also any reptile or zoo animal. More specifically, the composition / s and method / s of the invention are intended for mammals. By “mammalian subject” is meant any mammal for which the proposed therapy is desired, including human, equine, canine, and feline subjects, most specifically humans. It should be noted that specifically in cases of non-human subjects, the method of the invention may be performed using administration via injection, drinking water, feed, spraying, oral gavage and directly into the digestive tract of subjects in need thereof.

[0289] The terms "inhibition", "decrease", "moderation" or "attenuation" as referred to herein, relate to the retardation, restraining or reduction of a process, specifically the activation of mast cells, and / or release of mediator / s, by any one of about 1% to 99.9%, specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%.

[0290] With regards to the above, it is to be understood that, where provided, percentage values such as, for example, 10%, 50%, 120%, 500%, etc., are interchangeable with "fold change" values, i.e., 0.1, 0.5, 1.2, 5, etc., respectively.

[0291] It should be appreciated that the definition of the term "inhibition" as disclosed herein is applicable for any embodiment or aspect of the present disclosure.

[0292] It should be emphasized that the decrease in mast cell activation should be via a mechanism that does not involve mast cell death due to necrosis or apoptosis.

[0293] Another aspect of the present disclosure is related to a kit comprising: at least one targeted elimination compound comprising the following components: In one component (a), at least one target-recognition component. The target-recognition component specifically binds either directly or indirectly, at least one target macromolecule. In some optional embodiments, the target recognition component may further neutralize the target macromolecule. Additional component (b) comprises at least one elimination and / or removal-targeting component. More specifically, the elimination and / or removal-targeting component targets the at least one target macromolecule to the liver in a eukaryotic organism. The elimination and / or removaltargeting component is derived from at least one of: a microorganism, a natural and / or synthetic molecule and / or particle displaying liver tropism. The disclosed kit may further comprise (ii), at least one additional therapeutic agent. It should be understood that all definitions disclosed herein for each term appearing in the claims are applicable to every aspect of the present disclosure. Specifically, unless otherwise indicated, the definition of a term in connection with one particular aspect applies equally to all other aspects of the present disclosure.

[0294] Furthermore, as used herein in connection with all embodiments and aspects of the present disclosure, the phrase "at least one" refers to any number that is 1 or greater. More specifically, this includes, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more, such as several hundred, several thousand, or even higher numbers.

[0295] Still further, when referring to the terms "increase" or alternatively, "decrease", for example, in connection with specific embodiments as specified herein, relates to "inhibition", "moderation", “reduction” or "attenuation" of the specified parameter, as referred to herein, relate to the retardation, restraining or reduction of the specific parameter in the specified range. Alternatively, by any one of "increase," "elevation”, "rise", "elevation" "growth," "boost", "expansion”, "escalation”, of any parameter in connection with specific embodiments as specified herein, relate to the elevation, or enhancement of the specific parameter in the specified range. More specifically, for "increase" or alternatively, "decrease" the following rates are applicable: about 1% to 99.9%, specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%. It should be appreciated that 10%, 50%, 120%, 500%, etc. , are interchangeable with "fold change" values, i.e. , 0.1 , 0.5, 1.2, 5, etc., respectively. 10%, 50%, 120%, 500%, etc., are interchangeable with "fold change" values, i.e., 0.1, 0.5, 1.2, 5, etc., respectively. Therefore, the term inhibit or decrease or alternatively, increase or enhance refers to a change of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 folds or more, as compared with the previous level of the discussed parameter.

[0296] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0297] The term "about" as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range. In some embodiments, the term "about" refers to ± 10 %.

[0298] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise.

[0299] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0300] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of’ “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0301] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0302] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0303] Throughout this specification and the Examples and claims which follow, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Specifically, it should be understood as implying the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures. More specifically, the terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to". The term “consisting of means “including and limited to”. The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0304] It should be noted that various embodiments of this disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging / ranges between" a first indicate number and a second indicate number and "ranging / ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.

[0305] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0306] It is appreciated that certain features of the present disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub combination or as suitable in any other described embodiment of the present disclosure. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0307] Various embodiments and aspects of the present disclosure as delineated herein above and as claimed in the claims section below find experimental support in the following examples.

[0308] Disclosed and described, it is to be understood that this disclosure is not limited to the particular examples, methods steps, and compositions disclosed herein as such methods steps and compositions may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present disclosure will be limited only by the appended claims and equivalents thereof.

[0309] The following examples are representative of techniques employed by the inventors in carrying out aspects of the present disclosure. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the present disclosure, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the present disclosure.

[0310] It is to be understood that when specific values are given herein, they are meant to include a range of values acceptable within practical tolerances known in the pertinent field. Furthermore, for the sake of clarity, the term "substantially" or "approximately" is used herein to imply the possibility of variations in the specified values. For example, the term "pixels or pixel located substantially at the center of object", implies that the pixels may be located at the center but may also deviate from the exact center to some extent.

[0311] EXAMPLES

[0312] Experimental procedures

[0313] Mice

[0314] 6-8 weeks old Female BALB / c inbred mice were purchased from Envigo (Israel). Mice were maintained at the Animal Care-certified facility at the Faculty of Medicine, Technion (Israel) and all studies were approved by the institute’s committee for the supervision of animal experiments.

[0315] Cells and transfections

[0316] Baby hamster kidney cells (BHK-21; American Type Culture Collection [ATCC], Manassas, VA) were grown in Dulbecco's modified Eagle's medium (DMEM; Sigma, Israel), with 10% fetal Bovine serum (FBS). Cells were infected with a viral vector to express human FcεRlα on the membrane. This vector contained a construct encoding the extracellular domain of the human FcεRlα fused to the transmembrane and intracellular region of the human CD8 protein (SEQ ID NO. 24). Stable transfectants were selected and used for the Flow cytometry experiments to test IgE binding and neutralization.

[0317] Rat basophilic leukemia cell line (RBL; American Type Culture Collection [ATCC, CRL-2256], Manassas, VA)) were grown in low-glucose DMEM (Biological Industries, Beit-Haemek, Israel) supplemented with 10% FBS, 2 mM L-Glutamine, 100 μg / mL streptomycin and 100 U / mL penicillin.

[0318] Human Embryonic Kidney HEK293T cells are a popular derivative of the original HEK293 parent cell line. The HEK293T genome contains the SV40 large T antigen, which enables them to produce recombinant proteins within plasmid vectors containing the SV40 promoter. Cells were grown in high glucose DMEM (Biological Industries, Beit-Haemek, Israel) supplemented with 10% FBS, 2 mM L-Glutamine, 100 μg / mL streptomycin and 100 U / mL penicillin, ImM Sodium Pyruvate and 70pM [3ME. HEK 293T cells were transfected using PolyJet transfection reagent SignaGen Cat # SL100688 according to the standard protocol provided by the company.

[0319] Expi293F Cells (Gibco, Cat# A14527) are human cells derived from the 293F cell line. They are maintained in suspension culture and optimized to grow to high density in Expi293™ Expression Medium (Gibco, Cat# A1435101). Expi293F Cells were transfected using ExpiFectamine™ 293 Reagent (Gibco, Cat# A 14524) according to the user guide provided by the company. Lipid nanoparticles (LNPs) preparation

[0320] All lipids were weighted and dissolved in chloroform for making a stock solution in a sterile glass container. Lipid mixture A contains HSPC; Cholesterol; PEG 2000; DGS-NTA (Ni) at mole percent ratio of 55;37.5;5;2.5 (final concentration of lOrnM) and Lipid mixture B contains HSPC; Cholesterol; PEG 2000 at a mole percent ratio of 55;40;5 (final concentration of lOrnM). Each mixture was transferred with a Pasteur pipette to a clean corex tube. The evaporation and lipid film formation were made using an N2 environment, then films were desiccated overnight. Lipid films were resuspended under N2 environment with PBS to a concentration of 10 mM. Both mixtures were frozen in liquid nitrogen, thawed in RT water twice, sonicated and extruded through a 0.4pm polycarbonate membrane for at least 13 times. 200pl of aliquots were stored at -80°C.

[0321] Lipid mixture C contains DLin-MC3-DMA, Cholesterol, DSPC, PEG-DMG and DSPE-PEG- mal in the following ratio: 50:38:10:1.9:0.1 (final concentration of 9.6mM LNPs were prepared by using microfluidic micro mixture (Precision NanoSystems, Vancouver, BC). Briefly, one volume of lipid mixtures in ethanol and three volumes acetate buffer solutions were mixed through the micromixer at a combined flow rate of 12 mL min-1. The resultant mixture was dialyzed against phosphate-buffered saline (PBS) (pH 7.4) for 16 h to remove ethanol. hsFCeRa binding to liposomes

[0322] Liposomes from lipid mixture A and B were diluted to 2mM and subjected to 3 freeze / thaw cycles in liquid nitrogen. After extrusion in 0.4 pm membrane, liposomes (InM) were incubated with the sFCeRla (also denoted by SEQ ID NO: 1, or SEQ ID NO: 2) (125μg / ml final concentration) for 1 hour with titillation at 4°C. Liposomes were washed and resuspended in PBS. These are referred to as DGS / NTA / Ni- sFcεRlα liposomes.

[0323] Anti IgE Antibody binding to liposomes

[0324] Liposomes from lipid mixture C were coated with monoclonal rat anti mouse IgE (rat IgG clone 23G3 from SouthernBiotech Cat#l 130-01). The antibodies were conjugated to the LNPs using maleimide-thiol chemistry [Tarab-Ravski D, et al. Adv Sci (Weinh). 2023;10(21):e2301377]. Briefly, the anti mouse IgE was reduced with lx 103M dithiothreitol (Sigma-Aldrich) and 5 x 103M ED'TA (Sigma- Aldrich) for 1 h at room temperature. Dithiothreitol was later removed by using 7K Zeba spin desalting column (ThermoFischer Scientific) according to manufacturer protocol and the reduced antibody was immediately added to the LNPs at a ratio of 1 :40.7 antibody to LNPs (mg / mg) and incubated for 2 h at room temperature with gentle shaking and overnight at 4 C. To remove free unconjugated antibodies, LNPs were loaded on CL4B Sepharose beads (Sigma- Aldrich) and purified by gravity fed gel filtration chromatography column (BioRad Laboratories) using PBS as a mobile phase. The fractions were collected with a FC-203B fraction collector (Gilson).

[0325] Cloning

[0326] The gblock gene fragment encoding the signal peptide and the extracellular domain of the a chain from the human FCeRa (hFCeRa, Uniprot Accession: P12319) fused to CD8 transmembrane segment was obtained from Integrated DNA Technologies (IDT), (gBlock Flag tagged hFcεRlα- hCD8 FUSION, as denoted by SEQ ID NO: 18. The gblock fragment was amplified by PCR using Primer 1 ( A AT AGCT AGCGCC ACC ATGGCT : SEQ ID NO: 9) and Primer 2 (TAGAAGGGCCCTTGTAGCCAGTACTT: SEQ ID NO: 10) containing respectively Nhel and Apal restriction enzyme sites (underlined in the primer sequences). PCR fragment was digested using the same restriction enzymes and inserted into the mammalian expression vector pSecTag (Thermo) containing the Myc and His tag at the C- terminal extremity.

[0327] Circumsporozoite protein (CSP) sequence (SEQ ID NO: 3) was inserted in the pSecTag in the N- terminal end (CSP #1, as denoted by SEQ ID NO: 4) with primer 3 (TTA AAG CAA CCA GCG GAT GGT GGT GGT GGC GGT TCA GTCCCTCAGAAACCTAAGGTCT; as denoted by SEQ ID NO: 11) and primer 4 (TTT TTT ATG TTT TGG TTT CCT TAA TTT CTC GTT GTC TGCTAACACGCCATCTGGAG; as denoted by SEQ ID NO: 12), or between the hFCeRa and the His-tag (CSP #2, as denoted by SEQ ID NO: 6) with primer 5 (ATAAAAAATTAAAGCAACCAGCGGATGGTAATAGCGCCGTCGACCATCAT; as denoted by SEQ ID NO: 13) and primer 6

[0328] (GTTTTGGTTTCCTTAATTTCTCGTTGTCCAGATCCTCTTCTGAGATGAG; as denoted by SEQ ID NO: 14), or at the C-terminal end of the construct (CSP #3, as denoted by SEQ ID NO: 7) with Primer 7 (ATA AAA AAT TAA AGC AAC CAG CGG ATG GTT GAG TTT AAA CCC GC; SEQ ID NO: 15) and Primer 8 (GTT TTG GTT TCC TTA ATT TCT CGT TGT CAT GAT GAT GAT GAT G; SEQ ID NO: 16). Primers were designed with half of their sequence encoding for the inserted site in the plasmid and half sequence containing the CSP sequence. Then Pfu Turbo polymerase (Agilent) were used to amplify linearly the whole plasmid sequence using the respective primers. After Dpnl digestion, ligation was performed on the PCR product using the T4 ligase (NEB). To express stable membrane form of the hFcεRlα, a gblock gene fragment was synthetized from IDT containing the extracellular domain of the hFcεRlα fused with the transmembrane domain and the intracellular domain of human CD8 alpha subunit (Uniprot Accession: P01732-1) (SEQ ID NO: 8). BsiWI and BsrGI restriction enzymes sites were inserted respectively at the 5' and 3' extremities of the gblock to enable its cloning in the lenti viral vector MPH V2.

[0329] To express stable soluble form of the sFcεRlα-ApoAl, a gblock gene fragment was synthetized from IDT containing ApoAI sequence fused to myc and his tags (gblock myc his tagged ApoAI , as denoted by SEQ ID NO: 19). The gblock fragment was digested using Apal and Pmel restriction enzymes and inserted into the mammalian expression vector pSecTag (Thermo) containing the human FCeRa (as described previously) digested by the same restriction enzymes.

[0330] Protein production

[0331] For protein production, Expi293F cells were grown with Expi293 Expression chemically defined serum-free Medium (Thermo) in sterile Erlenmeyer flasks at 37°C and 8% CO2 on a shaker rotating at 125 rpm. Cells were grown at densities between 3-5xl06cells per mL at least 3 passages after thawing. The day before transfection, cells were split to a density of 2.5-3 xlO6cells and were transfected with the expression plasmid encoding the soluble extracellular domain of the a chain from the human FcεRlα (hsFCeRa) using the ExpiFectamine™ 293 transfection kit (Gibco™ A14524 according to manufacturer instruction. Five days post transfection the culture medium was harvested for purification of the secreted protein. hsFcεRlα CSP chimeras and sFcεRlα-ApoAl were produced in the same conditions.

[0332] Protein Purification

[0333] His tagged-hsFcεRlα protein was purified using the Ni-NTA purification system (Qiagen Cat# 20-30210). Briefly Ni-NTA agarose was equilibrate in a purification column with 5-10 volumes of Tris 50mM NaCl 300mM imidazole lOrnM pH 8. Ni-NTA agarose was allowed to settle by gravity and transfected Expi293F supernatant was loaded on the same column. After washing with 10 volumes of Tris 50mM NaCl 300mM imidazole lOmM pH 8, elution step was proceed using PBS imidazole 300mM and 0.5ml fractions were collected. Fractions containing high concentration of protein were dialyzed over-night in 1000-fold volume of PBS. hsFcεRlα CSP chimeras and sFcεRlα-ApoAl were purified in the same conditions.

[0334] Western Blot

[0335] Proteins were boiled for 5 min at 95 °C in sample buffer and separated on 10% Acrylamide SDS- PAGE. Following electrophoresis, gel was transferred in a PVDF membrane using a semi-dry transfer device. After blocking, membrane was incubated with primary antibody diluted in TBST at 4°C overnight. After washing, membrane was incubated with the appropriate horseradish peroxidase (HRP) conjugated secondary Ab. Development was conducted using an enhanced chemiluminescence (ECE) reagent.

[0336] For liposomes, liposome aliquots were first prepared for binding with hsFCeRa as described except that before washing, total preparate (liposome in hsFCeRa solution) and supernatants after the first ultracentrifugation were kept and prepared in sample buffer. After washing liposomes were resuspended at equal volume and prepared in sample buffer. Western blot protocol was conducted. For checking the binding of hsFCeRa to the Liposomes, HRP conjugated mouse anti His-tag antibody diluted in blocking buffer was used. When checking the binding of IgE to hsFCeRa liposomes, DGS / NTA / Ni- sFcεRlα liposomes (Mixture A) or liposomes from the mixture B were incubated with IgE (0.5μg / ml). After washing, liposomes were resuspended at equal volume and prepared in sample buffer. Biotinylated Goat anti mouse Kappa was used as secondary and HRP conjugated anti-Goat IgG antibody was used as secondary antibody.

[0337] To check IgE conjugation to the LNPs, a Western blot protocol was conducted. Denaturated and reduced IgE conjugated LNPs were separated by SDS-PAGE and transferred to polyvinylidene fluoride membrane (Millipore, Bedford, MA, United States). Blots were probed with mouse anti rat IgG antibody (Jackson Immunoresearch, Cat# 212-065-168) followed by HRP conjugated goat anti mouse IgG antibody (Jackson Immunoresearch, Cat#l 15-035-008).

[0338] To check sFcεRlα-ApoAl, aWestern blot protocol was conducted as described above. Blots were probed with mouse anti ApoAI antibody (Cell signalling, Cat# 3350S) followed by HRP conjugated goat anti mouse IgG antibody (Jackson Immunoresearch, Cat#l 15-035-008).

[0339] Neutralization of free IgE in vivo

[0340] BALB / c mice 8-12 weeks old were injected intravenously with 90-143.6 pg hsFCeRa in PBS. The mice were bled before the injection, and 24-96 hours after the injection. Sera was extracted by centrifugation and kept frozen until analyzed by ELISA.

[0341] C57B1 / 6 mice 8-12 weeks old were injected intravenously with 50, uL anti IgE conjugated LNPs. The mice were bled before the injection, and after the injection at different time points. Sera was extracted by centrifugation and kept frozen until analyzed by ELISA.

[0342] ELISA

[0343] 96 well plate was coated with hsFceRla (1.67μg / ml) in a carbonate-bicarbonate buffer and incubated overnight at 4°C. After washing and blocking steps, plates were loaded with the corresponding standard antibodies (125ng / ml) in blocking buffer and incubated for 1 hour at RT. After incubation with respective secondary biotinylated antibodies, wells were incubated with peroxidase conjugated streptavidin for 30 minutes at RT. Colorimetric reaction occurred using the TMB substrate and stop solution was added to stop the reaction. Absorbance was measured at 450nm and 620nm wavelength.

[0344] For hsFcεRlα CSP chimeras, 96 well plate was coated with purified IgE antibody (1.67μg / ml) in a carbonate-bicarbonate buffer and incubated overnight at 4°C. After washing and blocking steps, plates were loaded with the corresponding sFcεRlα CSP chimeras supernatant of transfected HEK cells and incubated for 1 hour at RT. Plate were incubated with HRP conjugated mouse anti His- tag antibody diluted in blocking buffer for 60 minutes at RT. Colorimetric reaction occurred using the TMB substrate and stop solution was added to stop the reaction. Absorbance was measured at 450nm and 620nm wavelength.

[0345] For IgE conjugated LNPs, 96 well plate was coated with the same rat anti IgE antibody used for LNPs conjugation (rat IgG clone 23G3 from Sou thernB iotech Cat#l 130-01) at 1.67μg / ml in a carbonate-bicarbonate buffer and incubated overnight at 4°C. After washing and blocking steps, plates were loaded with mouse serum or mouse serum previously incubated with IgE conjugated LNPs. The plate was incubated for 1.5 hour at RT. After incubation with Biotinilated goat anti mouse IgE (Southern Biotech, Cat#l 110-08) for one hour, wells were incubated with peroxidase conjugated streptavidin (Jackson Immunoresearch, Cat# 016-030-064) for 30 minutes at RT. Colorimetric reaction occurred using the TMB substrate and stop solution was added to stop the reaction. Absorbance was measured at 450nm and 620nm wavelength.

[0346] For sFcεRlα-ApoAl, 96 well plate was coated with mouse anti human ApoAI (Cell signalling, Cat# 3350S) at 1.67μg / ml in a carbonate-bicarbonate buffer and incubated overnight at 4°C. After washing and blocking steps, plates were loaded with purified sFcεRlα-ApoAl or recombinant ApoA (Iμg / ml). The plate was incubated for 1.5 hour at RT. After washes, plate was incubated with standard IgE (Iμg / ml) (Southern Biotech, Cat#0114-01) for 1 hour at RT. After washes and incubation with Biotinilated goat anti mouse IgE (Southern Biotech, Cat#l 110-08) for one hour, wells were incubated with peroxidase conjugated streptavidin (Jackson Immunoresearch, Cat# 016-030-064) for 30 minutes at RT. Colorimetric reaction occurred using the TMB substrate and stop solution was added to stop the reaction. Absorbance was measured at 450nm and 620nm wavelength. Flow cytometry hFCeRa transfected BHK cells (2,5.105 / sample) were incubated with anti-mouse CD16 / 32 (Fc- Blocker) (Biolegend, Cat#156603) for 15 minutes at RT. Then, diluted sera (1 / 3 in FACS Buffer) were added to the cells and incubated for 30 minutes at RT. After washing, cells were stained with FITC conjugated anti mouse IgE (Biolegend, Cat#406906) or with PE (phycoerythrin) conjugated anti mouse IgE (Biolegend, Cat#406908) and incubated for 20 minutes at RT in the dark. After washing, cells were acquired on LSRFortessaTM (BD Pharmingen) and analyzed using FlowJo software. For Liposomes, DGS / NTA / Ni-sFcεRlα liposomes (lipid mixture A, InM) were incubated with IgE (25μg / ml) (Southern Biotech, Cat#l 110-08), washed and stained with fluorescently-labeled anti-IgE PE antibody (25μg / ml) (Biolegend, Cat#406908), and DiD-APC (Invitrogen, Cat#V-22887 reagent to label membrane lipids (according to the manufacturer instruction).

[0347] For sFcεRlα-ApoAl, RBL cells (2,5.105 / sample) were incubated with mouse standard IgE (2.5μg / ml) (Southern Biotech, Cat#0114-01) previously mixed with decreasing concentration of sFcεRlα-ApoAl. After two washes, cells were stained with stained with fluorescently-labeled anti-IgE PE antibody (25μg / ml) (Biolegend, Cat#406908). After washing, cells were acquired on LSRFortessaTM (BD Pharmingen) and analyzed using FlowJo software.

[0348] Competition assay hFCeR transfected HEK cells (2,5.105 / sample) were incubated with anti-mouse CD16 / 32(Fc- Blocker) for 15 minutes at RT. Meanwhile, (2.5μg / ml) mouse IgE were pre-incubated with hsFCeRa at different concentrations for 15 minutes at RT and then added to the cells for 30 minutes at RT. After washing twice, cells were stained with FITC conjugated anti mouse IgE (Biolegend) and incubated for 20 minutes at RT in the dark. After washing, cells were acquired on LSRFortessaTM (BD Pharmingen) and analyzed using FlowJo software.

[0349] Neutralization of RBL activation assay

[0350] RBL cells were seeded onto 24-well plate at 4.105cells / well. Cells were sensitized overnight with 0.25 μg / mL mouse anti-DNP-specific monoclonal IgE with or without pre-incubation of different concentrations of hsFCeRla from 12.8μg / ml to 0.1 μg / ml. After 3 washes in Tyrode buffer, cells were stimulated at 37°C for 30 minutes with 50 ng / mL DNP-HAS (Human Serum Albumin) (antigen) in Tyrode buffer for measuring β-hexosaminidase secretion. Supernatants collect to 1.5 ml tube. Cells were lysed with 0.5% Triton X-100 and collected to 1.5 ml tube to determine the total β-hexosaminidase. 20pl- Aliquots of both Supernatants and lysates samples were incubated in 96-well plate for 90 minutes at 37°C with 50 pl substrate solution. Reaction was stopped by adding 180pl of 0.2 M glycine (PH 10.7). OD was measured at 405 nm 620nm using plate reader. Data is represented as percent activation by diving the β-hexosaminidase release in sample with total P- hexosaminidase content in the unstimulated cells.

[0351] For neutralization of RBL activation by DGS / NTA / Ni- sFcεRlα liposomes, all experiments were conducted in regular DMEM medium in the absence of serum. Cells were sensitized overnight with 0.2 μg / mL mouse anti-DNP-specific monoclonal IgE with or without pre-incubation with DGS / NTA / Ni- sFcεRlα liposomes diluted from 1:10 to 1:1280 (from ImM liposome stock solution). After 3 washes in Tyrode buffer, cells were stimulated at 37°C for 30 minutes with 50 ng / mL DNP-HAS in Tyrode buffer and β-hexosaminidase secretion was determined as detailed above.

[0352] Clearance of IgE in vivo by LNPs coated with anti-IgE

[0353] C57B1 / 6 mice 8-12 weeks old were injected intravenously with 50pL anti IgE conjugated LNPs. The mice were bled before the injection, and after the injection at different time points. Sera was extracted by centrifugation and kept frozen until analyzed by ELISA.

[0354] EXAMPLE 1

[0355] Cloning and synthesis of human soluble FcεRlα (sFcεRlα )

[0356] The FcsRl is expressed on the membrane of mast cells and basophils as a complex of three different protein subunits: a chain, p chain and two y chains, where the a chain is the IgE binding subunit, and the other subunits mediate signaling and cell activation [Ref. 10]. It has been shown that the a subunit is sufficient for high affinity IgE binding [Ref. 11]. The administration of the soluble form of FcεRlα extracellular fraction enables binding to the soluble IgE in the circulation. However, this by itself is not sufficient to eliminate the IgE in the plasma. As the liver is known to be a target of protein degradation, novel compounds were developed herein, in which the FcεRlα, acting as a target recognition component, was fused to different components that are capable to target and translocate the bound IgE for degradation in the liver.

[0357] As a first step, the inventors used synthetized gblock DNA fragment containing extracellular fragment of the human FcεRlα (that comprises the amino acid sequence as denoted by SEQ ID NO: 1 based on published sequence (UNIPROT P12319) (gBlock Flag tagged hFcεRlα -hCD8 FUSION, as denoted by SEQ ID NO: 18) to clone the extracellular fragment of the human FcsRl into an expression vector containing myc and his tags . Upon sequence verification of the myc and His tag FcεRlα fragment (as denoted by the amino acid sequence of SEQ ID NO: 2), HEK 293T cells were transfected, and the supernatant was collected. Protein was purified on nickel column and run on SDS gel. The purified protein is shown in Figure 1.

[0358] EXAMPLE 2

[0359] The purified sFcεRlα binds free IgE in vitro in 100% efficiency

[0360] To test for specific binding of IgE by the purified sFcεRlα , that comprises the amino acid sequence as denoted by SEQ ID NO: 1, the inventors used two approaches: direct ELISA, and flow cytometry. For direct ELISA, plates were coated with the purified sFcεRlα , followed by incubation with IgE, as well as IgA or IgM (as negative controls), and followed by secondary isotype-specific detecting Abs. As shown in Figure 2A only IgE was captured by the sFcεRlα with high affinity. For flow cytometry analysis, the inventors generated recombinant HEK cells expressing the FcεRlα on their membrane, thus capable to bind free IgE, which is then revealed by a secondary fluorescent-labeled anti-IgE antibody (Figure 2B). To test whether the purified sFcεRlα binds the free IgE, IgE was pre-incubated for 5-10 min with different concentration of sFcεRlα and then placed over the recombinant HEK cells expressing membrane FcεRlα . Subsequently, the cells were stained with the secondary fluorescently-labeled anti-IgE and analyzed by fluorescence-activated cell sorting (FACS). As shown in Figure 2B, pre-incubation with the sFcεRlα neutralized the free IgE in a dose dependent manner, reaching to 100% efficiency of binding (black graph).

[0361] EXAMPLE 3

[0362] The sFcεRlα binds 100% of IgE in circulation in vivo

[0363] To test whether the purified sFcεRlα is functional in vivo, the inventors intravenously injected sFcεRlα to mice (140μg / mouse) and collected blood 3h, 24h, 48h and 96h after injection (as illustrated in the experimental design scheme of Figure 3A). To detect the bound IgE, two approaches were taken. In the first approach, the inventors set up an ELISA in which the primary antibody is anti-IgE, and after placing the mouse serum the inventors used anti-tag HRP that binds to the injected sFcεRlα , thus revealing pairs of IgE- sFcεRlα in serum. As shown in Figure 3B, such pairs were not detected in serum of control un-injected mouse. In contrast, in the two mice injected with the sFcεRlα significant amounts of pairs of IgE- sFcεRlα were observed after 24h, which decreased with time, reaching to low level 96h after injection.

[0364] In the second approach, the inventors used a flow cytometry technology, which can also quantify the amount of the bound IgE in the serum. For this assay the inventors used recombinant HEK cells expressing the FcεRlα on their membrane (as also used in Figure 2B). In this assay, the recombinant HEK cells were cultured in the presence of the mouse serum to allow binding of serum IgE to the recombinant HEK cells. Subsequently, fluorescently-labeled anti-IgE antibodies were used to stain for the bound IgE. Figure 4 shows representative results from injected (Fig. 4A) and from control (Fig. 4B) mice. The inventors show that on day 0 (before injection) free IgE in sera bound the FcεRlα on the recombinant HEK cells, as revealed by significant positive staining for both mice (Figure 4). However, in sera collected from the injected mice 3h and 24h after injection there was no free IgE, as no staining was detected over the background (3h and 24h graphs, Figure 4A). This indicates that 100% of the IgE in the circulation was bound in complex with the injected sFcεRlα . An intermediate staining was observed for sera collected 48h after injection suggesting that some of these complexes dissociated and the IgE was released (Figure 4A). On 96h after injection, staining was similar to that of day 0, suggesting that all complexes were dissociated and all serum IgE was released (Figure 4A). In contrast, when sera collected from the control (un-injected mice) were used for the staining a significant fluorescence was observed, which was identical for all bleeding time points (Figure 4B). These results show that the purified soluble FcεRlα (sFcεRlα) effectively binds 100% of the IgE in the serum in vivo for at least 24h.

[0365] EXAMPLE 4

[0366] The sFcεRlα neutralizes allergic response in vitro by 100% efficiency

[0367] To look for functional activity of the sFcεRlα , the inventors used RBL cell line, which is a mast cell line that binds free IgE. Upon cross-linking of the bound IgE by cognate antigen / allergen, the cells get activated to de-granulate and secrete β-hexosaminidase, which can be quantified as an indication for allergic response [Wang W, et al. Pharmacol Rep. 2012;64(5): 1216-1222]. The inventors pre-incubated the free IgE with different concentrations of the purified sFcεRlα (400- 12800ng / ml), and then placed over the RBL cells. Subsequently, the cognate antigen / allergen was added to the culture media and the secreted β-hexosaminidase was quantified. As can be revealed in Figure 5A, the RBL activation was repressed upon treatment with the sFcεRlα in a dose dependent manner, reaching to 100% efficiency of inhibition at a concentration of ~2000ng / ml. To address this better, the inventors repeated this experiment but quantified also the intracellular β-hexosaminidase that remained in the RBL cells after activation and degranulation, in addition to that secreted to the media. To do so, after activation and degranulation, the RBL cells were collected, washed and lysed to determine intracellular β-hexosaminidase. The results in Figure 5B reveal that inhibition of degranulation by the sFcεRlα was also reflected by the increased amount of retained intracellular β-hexosaminidase. Thus, at a concentration of ~2000ng / ml, degranulation was completely abolished by 100% (bars), and the level of intracellular β-hexosaminidase was similar to that of unstimulated cells (red bars). This suggests that the soluble sFcεRlα can block allergic response in vitro in 100% efficiency.

[0368] Collectively, the results so far show 100% efficiency in binding and neutralizing of IgE antibodies by the soluble sFcεRlα in-vitro and in-vivo.

[0369] EXAMPLE 5

[0370] Designing the targeted elimination compounds

[0371] For effective elimination and clearance of the undesired target macromolecule, specifically, the IgE, three major approaches were designed, herein for targeting the target molecule, to the liver. These compounds are composed of the target recognition component, e.g., the soluble sFcεRlα , associated with various degradation-targeting components based on synthetic (e.g., liposomes), or natural molecules that display liver tropism.

[0372] Novel liposome coated with sFcεRlα

[0373] Another approach to facilitate clearance and degradation of the IgE- sFcεRlα complexes in the liver is by constructing nanoparticle composed of liposomes that are coated with the recombinant sFcεRlα chain. As liposomes are cleared by the liver [Kularatne RN, et al. Pharmaceuticals. 2022;15(7):897], they should function as efficient vehicles to bind and target the free IgE in the circulation to the liver.

[0374] There are many approaches to link proteins to liposomes. The inventors apply the following: 1) covalent linking to a lipid moiety using primary amine group-based coupling, 2) Using lipid-NTA (Nitrilotriacetic acid) to attach to the surface of the liposomes via the His-tag of the recombinant protein, 3) inserting a cysteine residue at the N-terminus of the recombinant FcεRlα and employing a thiol-based chemistry in conjunction with MeO-PEG-SH (Thiol PEG). A schematic illustration of the one of the proposed liposome-based compounds is shown in Figure 6. This figure illustrates only the model binding of a His-Tagged FcεRlα to an NTA-coated liposome.

[0375] As detailed below, LNPs coated with anti-IgE antibody (used herein as the target recognition component) are also presented. sFcεRlα fused to a small liver-targeting peptide

[0376] The inventors showed that their recombinant sFcεRlα is 100% efficient in binding and neutralizing serum IgE in-vivo and in-vitro. Upon administration in-vivo, the complexes formed between the sFcεRlα and the IgE dissociate within 48-96 hours and the IgE was released. The inventors are thus redesigning the recombinant molecule to facilitate clearance and degradation of the IgE-sFcεRlα complexes in the liver.

[0377] One of the approaches to achieve this is as follows:

[0378] Fusing the sFcεRlα to the small liver targeting peptide circumsporozoite protein (CSP), which is the predominant surface antigen of Plasmodium sporozoites, a microorganism that displays highly specific and highly efficient targeting to the liver and is required for sporozoite development and invasion of hepatocytes. The amino sequence of this small CSP fragment comprises the amino acid sequence DNEKLRKPKHKKLKQPADG, as denoted by SEQ ID NO: 3 [ Ma Y, et al. Applied Microbiology and Biotechnology. 2014;98(18):7923-7933]. This is achieved by standard PCR approach using appropriately designed primers and cloning into an expression vector and transfected into CHO cells. The recombinant protein is purified on nickel column using the HIS- tag quantified and tested. A schematic illustration of the proposed compound is shown in Figure 7. In some embodiments, the sFcεRlα-CSP fusion comprises the amino acid sequence as denoted by SEQ ID NO: 4 (CSP-fusion#l). Two additional CSP-sFcεRlα fusion proteins were also prepared (CSP-fusion#2, and CSP-fusion#3, as denoted by SEQ ID NO: 6, and 7, respectively).

[0379] Fusing the sFcεRlα to a small liver targeting peptide that is derived from human Apolipoprotein E, which is mediating ApoE binding to hepatic heparan sulfate proteoglycans (HSPG) in the absence of lipoproteins on hepathocytes. The amino sequence of this small fragment comprises the amino acid sequence HLRKLRKRLLRDADDLQKR (residues 140-158) as denoted by SEQ ID NO 20 (James J. Ludtke, et al. Drug Delivery 14:357-369, 2007 ) This is achieved by standard PCR approach using appropriately designed primers and cloning into an expression vector and transfected into CHO cells. The recombinant protein is purified on nickel column using the HIS- tag quantified and tested. A schematic illustration of the proposed compound is shown in Figure 7. The ApoE-targeting sequence is cloned at the N-terminal or at the C-terminal of the sFcεRlα , termed ApoE-target-sFcεRlα fusion#l and ApoE-target-sFcεRlα fusion#2, respectively.

[0380] Fusing the sFcεRlα to a small liver targeting peptide that is derived from human Apolipoprotein B, which reacts with hepatic heparan sulfate proteoglycans (HSPG) and the EDE receptor on hepathocytes. The amino sequence of this small fragment comprises the amino acid sequence LSVKAQYKKNKHRHSI (residues 3144-3159) as denoted by SEQ ID NO 21 (James J. Ludtke, et al. Drug Delivery 14:357-369, 2007) This is achieved by standard PCR approach using appropriately designed primers and cloning into an expression vector and transfected into CHO cells. The recombinant protein is purified on nickel column using the HIS-tag quantified and tested. A schematic illustration of the proposed compound is shown in Figure 7. The ApoB -targeting sequence is cloned at the N-terminal or at the C-terminal of the sFcεRlα , termed ApoB-target- sFcεRlα fusion#l and ApoB-target-sFcεRlα fusion#2, respectively.

[0381] Novel chimeric protein composed of the extracellular domain of the FcεRlα chain ligated to Apolipoprotein A-I

[0382] A third approach used by the inventors to facilitate clearance and degradation of the IgE- sFcεRlα complexes in the liver is by constructing a novel chimeric protein composed of the extracellular domain of the FcεRlα chain ligated to the Apolipoprotein A-I (SEQ ID NO: 17). A schematic illustration of this compound is provided by Figure 8. The sFcεRlα-ApoAl fusion protein comprises the amino acid sequence as denoted by SEQ ID NO: 5.

[0383] Apo A-I is a natural lipoprotein produced by the liver and secreted to the circulation where it binds to the HDL to function for clearing fats, including cholesterol, to reduce the risk for coronary artery disease. Apolipoprotein A-I as a soluble lipoprotein or after binding to the HDL has a natural tropism for the liver [ Ardaiz N, et al. Front Pharmacol. 2020;l 1:591293].

[0384] The liver is known to be a target for protein degradation and clearance. It should be noted that all segments detailed here are natural product of the body, thus minimizing any risk for side effects and / or lack of response. Hence, the inventors next evaluated the “bind and remove” approach that provides specific binding and elimination of plasma IgE by each of these compounds. Once achieved, the inventors apply this in a mouse model of food allergy. In this model, mice are presynthesized to a Ovalbumin (OVA) by intraperitoneal immunization with OVA mixed with aluminum hydroxide. Then, upon OVA gastric intubation the sensitized mice undergo mast cell- induced anaphylaxis [Johanna M. Smeekens, Front Allergy. 2021: 2: 810067]. Subsequently the inventors test this in humans (the cloned sFcεRlα is human protein that also functions to bind mouse IgE).

[0385] EXAMPLE 6

[0386] Generation of liposomes coated with sFcεRlα

[0387] Liposomes coated with sFcεRlα were generated using Ni-His interaction

[0388] A standard method was used to prepare liposomes (using HSPC, cholesterol, PEG and with or without DGS / NTA / Ni) [Luke van der Koog et al. 2022 Adv. Healthcare Mater.]. The DGS / NTA / Ni liposomes were then incubated with His-Tag-sFcεRlα and washed 3 times. Along the preparation, the following samples were collected: total mix liposomes+sFcεRlα (marked T), supernatant after centrifugation of the liposomes (marked S) and washed liposome pellet (marked P). For control, liposomes without DGS / NTA / Ni were incubated with sFcεRlα and washed as above and the same samples (marked T, S, P) were collected. Samples were analyzed by western blotting using anti-His antibody to detect sFcεRlα (Figure 9A). It is shown that in the liposome pellet of the DGS / NTA / NI liposome, a low but a significant amount of sFcεRlα was detected (circle, Figure 9A), which was not seen in the control liposome preparation (without DGS / NTA / Ni). Further, the DGS / NTA / Ni- sFcεRlα were incubated with IgE antibodies and washed. Samples from total mix (T), supernatant after centrifugation (S) and the washed liposome pellet (P) were collected and analyzed by western blotting for bound IgE using goat anti kappa light chain (Figure 9B). Liposomes without DGS / NTA / Ni were used for control. It is clearly shown that DGS / NTA / Ni- sFcεRlα liposomes bound IgE as revealed by the anti-kappa light chain antibody (arrow Figure 9B), which was not detected in the control liposomes. To further confirm this, a flow cytometry technique was used. The DGS / NTA / Ni- sFcεRlα liposomes were incubated with IgE, followed by staining with fluorescently-labeled anti-IgE PE antibody, and DiD-APC reagent to label membrane lipids. The results in Figure 9C clearly show that DGS / NTA / Ni- sFcεRlα liposomes were all positive for IgE binding (positive for both DiD and anti-IgE), whereas the control liposomes did not bind IgE (were all positive only for DiD).

[0389] Collectively, Figure 9 demonstrates successful generation of functional sFcεR lα-coated liposomes using the Ni-His interaction, that are able to bind soluble IgE in high efficiently. EXAMPLE 7

[0390] The DGS / NTA / Ni- sFcεRlα liposomes compete and neutralize free IgE on RBL cells

[0391] To find whether the DGS / NTA / Ni- sFcεRlα liposomes can compete and neutralize the free IgE in FcsRl -expressing RBL cells, different dilutions of DGS / NTA / Ni- sFcεRlα liposomes (from 1:50 to 1:800) were mixed with IgE (0.25μg / ml) and were then placed over RBL cells. RBL cells cultured with liposomes only (without pre incubation with IgE) were used as a negative control and RBL cells cultured without liposomes were used as a positive control. After wash, the cells were stained with fluorescently labeled anti- IgE PE and analyzed by FACS.

[0392] As shown in Figure 10, the DGS / NTA / Ni- sFcεRlα liposomes effectively bind and neutralize the soluble IgE in a dose dependent manner reaching 100% efficiency at a 1:100 dilution.

[0393] EXAMPLE 8

[0394] The DGS / NTA / Ni- sFcεRlα liposomes neutralize allergic response in vitro

[0395] To further find whether the DGS / NTA / Ni- sFcεRlα liposomes can bind the free IgE and neutralize the RBL-mediated allergic response, DGS / NTA / Ni- sFcεRlα liposomes at different dilutions (from 1:0 to 1:1280) were mixed with IgE (0.2μg / ml) and the mixture was then placed over RBL cells. Subsequently, the cognate antigen / allergen was added to the culture media and the secreted β-hexosaminidase was quantified. As can be revealed from Figure 11, RBL activation was repressed by the DGS / NTA / Ni- sFcεRlα liposomes in a dose dependent manner, reaching to 100% efficiency in dilutions of up to 1:40.

[0396] EXAMPLE 9

[0397] Generation of LNPs coupled to monoclonal antibody rat anti mouse IgE

[0398] Another compound was synthesized in which the liver-targeting components are lipid nanoparticles (LNPs), known for their liver clearance properties [Kularatne RN, Crist RM, Stern ST. Pharmaceuticals. 2022;15(7):897]. The LNPs were coated with monoclonal rat anti-mouse IgE antibodies (rat IgG, clone 23G3, sourced from SBA).

[0399] The LNPs were formulated using DLin-MC3-DMA, cholesterol, DSPC, PEG-DMG, and DSPE- PEG-mal in a ratio of 50:38:10:1.9:0.1. Antibody conjugation was achieved via maleimide-thiol chemistry [Tarab-Ravski D, et al. Adv Sci (Weinh). 2023;10(21):e2301377].

[0400] Western blot analysis confirmed the anti- IgE antibody conjugation, as detected using a mouse antirat IgG secondary antibody (Figure 12). EXAMPLE 10

[0401] Binding of serum IgE in vitro by LNPs coated with anti-IgE

[0402] The inventors next tested whether the LNPs coated with anti-IgE antibodies can bind serum IgE in vitro. Serum from mice, previously tested to the presence of IgE, was collected and mixed with the LNPs at different ratios as detailed in Figure 13. An ELISA was then performed to detect the presence of unbound IgE antibodies and the percent of LNPs binding to IgE was calculated. As shown in Figure 13, in the absence of LNPs no binding was detected (left bar) whereas in the presence of LNPs a significant binding was found which was dependent on the amount of LNPs: when 5 pl of LNPs were applied almost 90% binding was observed, which were reduced to about 10% when only 0.1 pl LNPs were applied. Figure 13 shows that LNPs coated with anti-IgE antibodies effectively bind free IgE in mouse serum.

[0403] EXAMPLE 11

[0404] Clearance of IgE in vivo by LNPs coated with anti-IgE

[0405] To test whether LNPs coated with anti-IgE can clear free IgE from circulation in vivo, the inventors injected 50microliter of LNPs intravenously to each mouse. Blood was collected from the mice before, and up to 110 days after LNPs administration, and IgE concentration in the blood was quantified by ELISA. The results in Figure 14 show that IgE concentration in blood dropped from an amount of 65 ng / ml to about 20 ng / ml (about 70%) within 3 days and continued to drop to below 10 ng / ml (about 85%) on day 14 and stayed very low until day 110. The results in Figure 14 clearly show that administration of LNPs coated with anti-IgE is effective for clearance of free IgE from the circulation to result in long-term elimination of IgE in vivo.

[0406] EXAMPLE 12

[0407] Construction and production of sFcεRlα fused to liver targeting sequence (CSP) - sFcεRlα - CSP compounds

[0408] The inventors have generated 3 sFcεRlα-CSP fusion compounds. The structure of the three compounds is illustrated in Figure I5A. The three fusion compounds were designed with different location of the CSP (which is composed of 19 amino acids): CSP#1 in the N-terminal end (SEQ ID NO: 4), CSP#2 (SEQ ID NO: 6) between the sFcεRlα and the His-tag, CSP#3 (SEQ ID NO: 7) at the C-terminal end of the construct. HEK cells were transfected with each of these constructs and the supernatants was collected to determine the presence of the fusion protein and its function (binding to IgE). It is important to note that all these experiments were conducted using the collected supernatants and not the purified proteins. Yet, for CSP#3 the inventors were able to purify some of the protein, which was clearly detected by western blot using anti-His antibodies (Figure 15B). The successful expression and function of the compounds was further tested by an ELISA. In this assay, plates were coated with IgE, followed by incubation with the different supernatants of sFcεRlα-CSP compounds and followed by anti-His detecting antibody. As shown in Figure 15C, all three compounds were clearly detected, and thus are functional in recognition of the IgE. It should be noted that the difference in the OD may account for the relative concentration of the protein rather than to binding function, but this needs to be further elucidated.

[0409] EXAMPLE 13

[0410] The sFcεRlα -CSP compounds compete and neutralize free IgE on RBL cells

[0411] To find whether the sFcεRlα-CSP compounds compete and neutralize free IgE from binding, RBL cells were used as described in EXAMPLE 7. The supernatants collected for CSP#1, CSP#2 and CSP#3 compounds were mixed with IgE (0.25μg / ml) and were then placed over RBL cells. HEK sup was used for negative control. After wash, the cells were stained with fluorescently labeled anti-IgE PE and analyzed by FACS. RBL cells cultured with anti-IgE only were used as negative control and RBL cells cultured with IgE were used as positive control.

[0412] As shown in Figure 16, sFcεRlα-CSP#2 and sFcεRlα-CSP#3 effectively bind and neutralize the soluble IgE in a dose dependent manner reaching 100% efficiency. The sFcεRlα-CSP#l did not show any effect; however this should be further elucidated as the ELISA results clearly show its ability to bind (Figure 15C).

[0413] EXAMPLE 14

[0414] Construct structure and generation of sFcεRlα -ApoAl fusion protein

[0415] Here, the inventors used the ApoAl, which is a key scaffold lipoprotein of HDL particles, as a live- targeting fragment of their compound (instead of the lipid nanoparticle or the CSP livertargeting moiety). ApoAl has a natural tropism to the liver, where it acts as ligand to its receptor on hepatocytes, facilitating the uptake of HDL and elimination of excess cholesterol [Brill A, et al. Arterioscler Thromb Vase Biol. 2012;32(8): 1841- 1847]. In these experiments, human ApoAl was cloned and a fusion protein was constructed with sFcεRlα , where the sFcεRlα functions as the binding moiety and the ApoAl functions as a liver targeting moiety in the compound. Figure 17A shows the schematic structure of the fusion construct (the nucleic acid sequence encoding this fusion construct is denoted by SEQ ID NO: 22; the amino acid sequence of this fusion protein is denoted by SEQ ID NO: 23) and Figure 8 shows a schematic structure of the compound protein. This plasmid was used to transfect Expi293F cells to produce the protein. The His-tagged sFcεRlα-ApoAl fusion was purified from the media using the Ni-NTA purification system. Figure 17B shows in western blot the purified protein (right lane) relative to a recombinant human Apo Al protein that was purchased (left lane). The detection of Apo Al was done using monoclonal mouse anti human ApoAl (product number 3350 from Cell Signaling Technology). The results show the formation of the sFcεRlα-ApoAl fusion.

[0416] EXAMPLE 15

[0417] The sFcεRlα -ApoAl fusion protein binds and neutralize free IgE in vitro

[0418] RBL cells express sFcεRl and bind free IgE. To find whether the sFcεRlα-ApoAl fusion protein can compete and neutralize the free IgE, sFcεRlα-ApoAl fusion protein at different concentrations (125-500 ng / ml) were mixed with IgE (0.25 μg / ml) and were then placed with RBL cells. RBL cells cultured with media alone were used as negative control (dashed gray line in Figure 18). After wash, the cells were stained with fluorescently labeled anti-IgE PE and analyzed by FACS. As shown in Figure 18, the sFcεRlα-ApoAl fusion protein effectively binds and neutralizes the soluble IgE in a dose dependent manner reaching 100% efficiency at 250-500 μg / ml ( dark gray and vertical strips). This experiment shows that the sFcεRlα-ApoAl fusion protein binds and neutralizes free IgE in 100% efficiency.

[0419] EXAMPLE 16

[0420] Dual binding of the sFcεRlα -ApoAl fusion protein

[0421] To determine whether the two components of the sFcεRlα-ApoAl fusion protein are biologically intact, a novel dual binding ELISA assay was designed in which the identity and / or function of each component of the fusion protein was confirmed. In this ELISA, plates were coated with monoclonal mouse anti-human ApoAl, followed by incubation with the sFcεRlα-ApoAl fusion protein or with recombinant ApoAl. Free IgE was then added and followed by detecting anti-IgE HRP antibody. The plates were read in a plate reader and results are presented in OD values. Figure 19A shows a schematic presentation of the ELISA in which the sFcεRlα-ApoAl fusion protein is bound from one side with mouse anti -human ApoAl, and from the other side binds free IgE. As shown in Figure 19B, an efficient binding of IgE was detected only in wells where sFcεRlα-ApoAl fusion protein was cultured, but not where recombinant ApoAl was cultured. These results clearly show that the sFcεRlα-ApoAl fusion protein has a dual recognition function encoded by the biological structure of each component.

[0422] EXAMPLE 17

[0423] Evaluating clearance of plasma IgE In vivo

[0424] Mice are intravenously introduced with the various targeted elimination compounds prepared herein, specifically, the purified sFcεRlα-ApoAl compound or with each of the sFcεRlα-CSP compounds (in groups of 10 mice each). Control mice (10) are injected with recombinant ApoAl, with each of the non-conjugated CSPs, or with the target recognition component sFcεRlα , alone. Each mouse is subjected to bleeding before administration of the compound, and at different time intervals up to 35 (or more) days after the administration. Levels of free IgE in the collected plasma samples are determined by ELISA.

[0425] EXAMPLE 18

[0426] Translocation of the IgE target molecule to the liver

[0427] Determination of the liver as the targeting organ for the LNPs- IgE conjugates

[0428] To determine whether the LNPs-IgE conjugates that are formed in the circulation in vivo are targeted for elimination to the liver, the inventors are loading the LNPs with non-sense RNA bound to allophycocyanin fluorochrome (APC). The APC fluorochrome can be traced in vivo using an in vivo live imaging system (IVIS), thus allowing to get live visualization of the initial spreading of the LNPs upon administration and the organ where they accumulate with time. To do so, LNPs decorated with anti-IgE antibodies, are loaded with RNA-APC and are injected intravenously to 10 anesthetized mice. The mice are then scanned all-body by the IVIS at different time intervals and the accumulation of APC is visualized and determined anatomically. Mice are then sacrificed and the identified organs are inspected for the presence of IgE antibodies by immunohistochemistry.

[0429] Determination of the liver as the targeting organ for the sFcsRla-ApoAl-IgE

[0430] To determine whether the sFcεRlα-ApoAl-IgE complexes that are formed in the circulation in vivo are targeted for elimination to the liver, sFcεRlα-ApoAl is injected intravenously to 10 mice. Mice are sacrificed 4-48 hours after administration of sFcεRlα-ApoAl (or the other conjugates) and liver is inspected for the presence of IgE antibodies by immunohistochemistry. Similarly, the liver translocation of IgE complexes with Liposomes coated with sFcεRlα, LNPs coated with anti-IgE antibodies, or the CSP-based compounds, is determined as specified above. Mice are injected with Liposomes coated with sFcεRlα , LNPs coated with anti-IgE antibodies, or the CSP-based compounds. Mice are sacrificed 4-48 hours after administration and liver is inspected for the presence of IgE antibodies by immunohistochemistry.

[0431] EXMPLE 19

[0432] Study with normal mice immunized with ovalbumin (OVA)

[0433] The elimination of IgE, but not other isotype of antibodies, is confirmed using an antigen-specific model. Since IgE is a TH2-type response, Balb / c mice (which have an enhanced TH2 response) are immunized with OVA in aluminum hydroxide (known as an adjuvant with a TH2-biassed response). The production of anti-OVA specific IgE and IgG antibodies is confirmed by ELISA. Liposomes coated with sFcεRlα or LNPs coated with anti-IgE or the CSP-based compounds or the sFcεRlα-ApoAl are then introduced intravenously, each group of 10 immunized mice, and the level of OVA-specific IgE and IgG antibodies is determined in serum at different time points following the injection with the above mentioned compounds, relative to the 10 control mice not injected with the above mentioned compounds or injected with a corresponding uncoated liposomes or unconjugated CSPs or a recombinant ApoAl. A reduction in the levels of OVA- specific IgE antibodies indicates that administration of sFcεRlα-based compounds enhance clearance of the specific IgE by liver targeting, whereas no change is expected in levels of OVA- specific IgG. To monitor liver targeting, mice are sacrificed in various time points, 24-96h after administration of the sFcεR lα-compounds (coated liposomes, CSP-fusions, and Apo-fusions) and the amount of the IgE- sFcεR lα-liposome complexes in the liver tissue is determined by immunohistochemistry.

[0434] EXAMPLE 20

[0435] Study with a mouse model of IgE-mediated allergy

[0436] There are several mouse models for IgE-mediated allergy. The inventors have set in the lab a Balb / c mouse model for food allergy (OVA) that is mediated by IgE. Another model to use is skin and / or bronchial sensitization. In all models, upon exposure of the sensitized mice to the allergen an anaphylactic response is developed that can be measured by mouse behavior, body temperature, general physical condition, body weight. Using these mouse models, the sFcεRlα-liposomes, the LNPs coated with anti-IgE or the CSP- based compounds or the sFcεRlα-ApoAl are injected to mice and the levels of IgE antibodies are measured in the serum of the injected mice. The mice are then exposed to the allergen (e.g. OVA) and the anaphylactic response in the sensitized mice treated with sFcεRlα-liposomes or the LNPs coated with anti-IgE or the CSP-based compounds or the sFcεRlα-ApoAl is monitored relative to the untreated mice or mice injected with uncoated / unconjugated / recombinant compounds. The anaphylactic response is monitored by movement tracking, behavior and body temperature.

[0437] EXAMPLE 21

[0438] Applying the “bind and remove” approach for novel therapy of other clinical conditions

[0439] To fit the compound to eliminate other “unwanted targets”, only the binding segment of the compound is changed. This can be achieved for example by changing the (1) specific antibody (or just as a single chain version) of this “unwanted targets”, (2) natural soluble receptor for this “unwanted targets” (if exist), or (3) ectodomain of a membrane receptor specific for this "unwanted target". This is illustrated in Figure 20.

[0440] EXAMPLE 22

[0441] LNPs coated with anti-PCSK9 antibodies to clear PCSK9 from plasma

[0442] The proprotein convertase subtilisin / kexin type 9 (PCSK9) is an enzyme primarily expressed in the liver and acts as a negative regulator of the low-density lipoprotein receptor (LDLR), through binding to and degrading the LDLR [Park SW, et al. J Biol Chem. 2004;279(48):50630-50638]. In familial hypercholesterolemia resulting from heterozygous loss-of-function mutations in LDLR or gain-of-function mutations in PCSK9, high levels of blood low-density lipoprotein (LDL) are developing, which are associated with hypercholesterolemia, atherosclerosis and early cardiovascular disease [Stein EA, et al. The Lancet. 2012;380(9836):29-36] . This places PCSK9 as a major biological target for drug discovery, as repressing PCSK9 levels results in enhanced clearance of low-density lipoprotein (LDL), and several therapeutics based on small molecule inhibitors, gene silencing, monoclonal Abs and so are tested [Rothgangl T, et al. Nature Biotechnology. 2021;39(8):949-957]. Using LNPs coated with anti-PCSK9 antibodies, the inventors are eliminating the PCSK9 from the circulation to reduce blood LDL. The LNPs are coated with monoclonal anti-PCSK9 antibodies (mouse IgG, clone 2F1, sourced from ThermoFisher Scientific). The LNPs are formulated using DLin-MC3-DMA, cholesterol, DSPC, PEG-DMG, and DSPE-PEG-mal in a ratio of 50:38:10:1.9:0.1. Antibody conjugation is achieved via maleimide-thiol chemistry [Tarab-Ravski D, et al. Adv Sci (Weinh). 2023;10(21):e2301377]. Western blot analysis using HRP anti-mouse IgG confirms the antibody conjugation.The coated LNPs or control uncoated LNPs are then injected intravenously to mice fed high-fat diet and exhibit high level of LDL cholesterol (10 mice in each group). The mice are bled at different time intervals up to 45 days after administration of the LNPs and levels of plasma PCSK9 are quantified by specific ELISA kit and levels of plasma LDL are quantified using a kit assay. A significant reduction in the amount of plasma PCSK9 indicates that liver-targeting of PCSK9 for degradation is successful and a significant reduction in LDL indicates that the therapeutic approach is successful.

Claims

CLAIMS:

1. A targeted elimination compound comprising:(a) at least one target-recognition component, wherein said component specifically binds and optionally, neutralizes at least one target macromolecule; and(b) at least one elimination and / or removal-targeting component, wherein said component targets said at least one target macromolecule to the liver in a eukaryotic organism, said component is derived from at least one of: a microorganism, a natural and / or synthetic molecule and / or particle displaying liver tropism.

2. The compound of claim 1 , wherein said at least one target-recognition component of (a) and said at least one elimination and / or removal-targeting component of (b) are fused, linked, associated and / or conjugated directly or indirectly via at least one linking moiety.

3. The compound of any one of claims 1 or 2, wherein said eukaryotic organism is a mammalian subject.

4. The compound of any one of claims 1 to 3, wherein said elimination and / or removaltargeting component is derived from a microorganism displaying liver tropism, said microorganism is at least one of a parasite, a virus and / or bacteria.

5. The compound of claim 4, wherein said elimination and / or removal-targeting component is derived from the Plasmodium sporozoites, specifically, the Plasmodium sporozoites surface antigen, circumsporozoite protein (CSP).

6. The compound of any one of claims 1 to 5, wherein said elimination and / or removaltargeting component comprises at least one CSP-derived peptide, and wherein at least one of said peptide comprises the amino acid sequence as denoted by SEQ ID NO: 3, or any variants or derivatives thereof.

7. The compound of any one of claims 1 to 3, wherein said elimination and / or removaltargeting component is derived from a synthetic molecule or particle displaying liver tropism, and wherein said synthetic particle is at least one of at least one nano- or micro-particle, micellar formulation, or liposomal formulation.

8. The compound of claim 7, wherein said elimination and / or removal-targeting component is at least one liposome or at least one lipid nanoparticles (LNPs).

9. The compound of any one of claims 1 to 3, wherein said elimination and / or removaltargeting component is derived from a natural molecule displaying liver tropism, said molecule is a protein produced by, and / or a protein functioning in a liver tissue and / or a protein that has a tropism to the liver.

10. The compound of claim 9, wherein said at least one elimination and / or removal-targeting component is derived from at least one Apolipoprotein or any functional peptides thereof.

11. The compound of claim 10, wherein at least one of:(a) said at least one Apolipoprotein is Apolipoprotein A-I (Apo Al);(b) said elimination and / or removal-targeting component comprises the amino acid sequence as denoted by SEQ ID NO: 17, or any variants or derivatives thereof.

12. The compound of any one of claims 1 to 11, wherein said target-recognition component comprises at least one of: an amino acid-based, a nucleic acid-based, a small molecule-based, a carbohydrate-based, a lipid-based affinity molecule, said affinity molecule is of at least one of: a receptor-ligand, antibody-antigen, enzyme-substrate, aptamer-target affinity pair, or any combination thereof.

13. The compound of claim 12, wherein said target-recognition component comprises, or is derived from at least one of: at least one receptor molecule, at least one antibody and / or any fragments thereof and / or at least one aptamer, and / or any combinations thereof.

14. The compound of any one of claims 1 to 13, wherein said target-recognition component comprises, or is derived from at least one receptor molecule, or any functional fragments thereof.

15. The compound of any one of claims 1 to 14, wherein said target macromolecule is at least one of: an amino acid-based, a nucleic acid-based, a small molecule-based, a carbohydrate-based, a lipid-based, macromolecule or any combination or complex thereof.

16. The compound of claim 15, wherein said macromolecule is an amino acid-based molecule, said macromolecule is a circulating protein of at least one of: an immunoglobulin molecule, a cytokine, a chemokine, a growth, a survival factor, a hormone, a ligand, an enzyme, and / or a soluble receptor.

17. The compound of claim 16, wherein said macromolecule is an immunoglobulin molecule, and wherein said immunoglobulin is immunoglobulin E (IgE).

18. The compound of any one of claims 14 to 17, wherein said at least one receptor molecule is an immunoglobulin-binding receptor.

19. The compound of claim 18, wherein said immunoglobulin-binding receptor is an immunoglobulin E (IgE) binding receptor or any fragment / s thereof.

20. The compound of claim 19, wherein said at least one immunoglobulin E binding receptor is Fragment crystallizable epsilon receptor I alpha chain (FcεRlα).

21. The compound of any one of claims 1 to 20, wherein said target-recognition component is derived from FcεRlα, and comprises the amino acid sequence as denoted by SEQ ID NO: 1, or any variant or derivatives thereof.

22. The compound of any one of claims 1 to 13, wherein said target-recognition component comprises, or is derived from at least one antibody and / or any fragment thereof.

23. The compound of claim 22, wherein said at least one antibody and / or any fragment thereof binds at least one immunoglobulin molecule.

24. The compound of claim 23, wherein said at least one antibody and / or any fragment thereof binds at least one immunoglobulin E (IgE) molecule.

25. A targeted elimination compound comprising a fusion protein comprising the Fragment crystallizable epsilon receptor I alpha chain (FcεRlα) and a peptide of the circumsporozoite protein (CSP), wherein said fusion protein comprises the amino acid sequence as denoted by any one of: SEQ ID NOs: 4, 6, and 7, or any variants or derivatives thereof.

26. A targeted elimination compound comprising a fusion protein comprising the Fragment crystallizable epsilon receptor I alpha chain (FcεRlα) and the Apolipoprotein A-I (Apo Al) protein, wherein said fusion protein comprises the amino acid sequence as denoted by any one of: SEQ ID NOs: 5 and 23, or any variants or derivatives thereof.

27. A targeted elimination compound comprising a liposome or a lipid nanoparticle (LNP) conjugated to the Fragment crystallizable epsilon receptor I alpha chain (FcεRlα), or to an anti- IgE antibody, wherein at least one of:(i) said FcεRlα comprises the amino acid sequence as denoted by any one of: SEQ ID NOs: 1 and 2, or any variants or derivatives thereof;(ii) said liposome comprises (l,2-dioleoyl-sn-glycero-3-[(N-(5-amino-l- carboxypentyljiminodiacetic acidjsuccinyl] (Cobalt salt) (DGS / NTA); and(iii) said LNP comprises Butanoic acid, 4-(dimethylamino)-, (10Z,13Z)-l-(9Z,12Z)-9,12- octadecadien-l-yl-10,13-nonadecadien-l-yl ester (D-Lin-MC3-DMA), Cholesterol, 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), polyethylene glycol (PEG)- 1,2- dimyristoyl-rac-glycerol (DMG); and l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N- [maleimide(polyethylene glycol)-200 (DSPE-PEG-mal).

28. A pharmaceutical composition comprising at least one targeted elimination compound or any preparations thereof, or any vehicle, matrix, nano- or micro-particle comprising the same, and at least one of pharmaceutically acceptable carrier / s, excipient / s, auxiliaries, and / or diluent / s, wherein said targeted elimination compound comprising:(a) at least one target-recognition component, wherein said component specifically binds and optionally, neutralizes at least one target macromolecule; and(b) at least one elimination and / or removal-targeting component, wherein said component targets said at least one target macromolecule to the liver in a eukaryotic organism, and wherein said component is derived form at least one of: a microorganism, a natural and / or synthetic molecule and / or particle displaying liver tropism.

29. The composition according to claim 28, wherein said compound is as defined by any one of claims 1 to 27.

30. A method for targeted elimination and / or removal and / or clearance of at least one target macromolecule in a subject in need, the method comprising the step of administering to said subject an effective amount of at least one targeted elimination compound or any preparations thereof, or any vehicle, matrix, nano- or micro-particle comprising the same, and / or any composition thereof, wherein said targeted elimination compound comprises:(a) at least one target-recognition component, wherein said component specifically binds and optionally, neutralizes at least one target macromolecule; and(b) at least one elimination and / or removal-targeting component, wherein said component targets said at least one target macromolecule to the liver in said subject, and wherein said component is derived form at least one of: a microorganism, a natural and / or synthetic molecule and / or particle displaying liver tropism.

31. The method of claim 30, wherein the compound is as defined in any one of claims 1 to 27.

32. The method of any one of claims 30 and 31, wherein said subject is suffering from a pathological disorder associated directly or indirectly with said at least one target macromolecule; optionally, wherein at least one of:(i) said pathological disorder is at least one immune -related disorder;(ii) said at least one immune-related disorder is a medical condition associated with or induced by activation of at least one white blood cell (leukocyte); and(iii) said leukocyte cell is at least one of: a mast cell, a basophil and / or an eosinophil, and wherein said medical condition is associated with, or induced by, activation of at least one of said mast cells, basophils and / or eosinophils.

33. The method of claim 32, wherein said medical condition associated with or induced by activation of at least one of mast cells, basophils and / or eosinophils is at least one of allergic reaction, a chronic or acute inflammatory condition involving airways, skin, and / or mucosal tissue, mastocytosis and mast cell tumors.

34. A method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one pathological disorder associated directly or indirectly with at least one target macromolecule in a subject, the method comprising the steps of administering to said subject a therapeutically effective amount of at least one targeted elimination compound, or any preparations thereof, or any vehicle, matrix, nano- or micro-particle comprising the same, and / or any composition thereof, wherein said targeted elimination compound comprises:(a) at least one target-recognition component, wherein said component specifically binds and optionally, neutralizes said at least one target macromolecule; and(b) at least one elimination and / or removal-targeting component, wherein said component targets said at least one target macromolecule to the liver in said subject, and wherein said component is derived form at least one of: a microorganism, a natural and / or synthetic molecule and / or particle displaying liver tropism.

35. The method of claim 34, wherein said at least one target-recognition component of (a), and said at least one elimination and / or removal-targeting component of (b), are fused, linked, associated and / or conjugated directly or indirectly via at least one linking moiety.

36. The method of any one of claims 34 and 35, wherein said subject is a mammalian subject.

37. The method of any one of claims 34 to 36, wherein said elimination and / or removaltargeting component is derived from a microorganism displaying liver tropism, said microorganism is at least one of a parasite, a virus and / or bacteria; optionally, at least one of:(i) wherein said elimination and / or removal-targeting component is derived form the Plasmodium sporozoites, specifically, the Plasmodium sporozoites surface antigen, CSP; and(ii) wherein said elimination and / or removal-targeting component comprises at least one CSP-derived peptide, and wherein said peptide comprises the amino acid sequence as denoted by SEQ ID NO: 3, or any variants or derivatives thereof.

38. The method of any one of claims 34 to 36, wherein said elimination and / or removaltargeting component is derived from a synthetic molecule or particle displaying liver tropism, wherein said synthetic particle is at least one of: at least one nano- or micro-particle, micellar formulation, or liposomal formulation; optionally, said elimination and / or removal-targeting component is at least one liposome and / or at least one LNP.

39. The method of any one of claims 34 to 36, wherein said elimination and / or removaltargeting component is derived from a natural molecule displaying liver tropism, said molecule is a protein produced by and / or functions on the liver tissue; optionally, wherein at least one of:(i) said at least one elimination and / or removal-targeting component is derived from at least one Apolipoprotein or any functional peptides thereof;(ii) wherein at least one of:(a) said at least one Apolipoprotein is Apolipoprotein A-I (Apo Al); and(b) said elimination and / or removal-targeting component comprises the amino acid sequence as denoted by SEQ ID NO: 17, or any variants or derivatives thereof;40. The method of any one of claims 34 to 39, wherein said target-recognition component is at least one of: an amino acid-based, a nucleic acid-based, a small molecule-based, a carbohydrate- based, a lipid-based affinity molecule of: a receptor-ligand, antibody-antigen, enzyme-substrate, aptamer-target, affinity pair, or any combination thereof; optionally, at least one of:(i) wherein said target-recognition component is, or derived from at least one of: at least one receptor molecule, at least one antibody and / or any fragments thereof and / or at least one aptamer, and / or any combinations thereof; and(ii) wherein said target-recognition component comprises, or is derived from at least one receptor molecule.

41. The method of any one of claims 34 to 40, wherein at least one of:(a) said target macromolecule is at least one of: an amino acid-based, a nucleic acid-based, a small molecule-based, a carbohydrate-based, a lipid-based, macromolecule or any combination or complex thereof; optionally, at least one of:(i) said macromolecule is an amino acid-based molecule, said macromolecule is a circulating protein of at least one of: an immunoglobulin molecule, a cytokine, a chemokine, a growth and / or survival factor, a hormone, a ligand and / or a soluble receptor; and(ii) wherein said macromolecule is an immunoglobulin molecule, and wherein said immunoglobulin is immunoglobulin E (IgE);(b) wherein said at least one receptor molecule is an immunoglobulin-binding receptor; optionally, wherein at least one of:(i) said immunoglobulin-binding receptor is at least one of IgE binding receptor or any fragment / s thereof; and(ii) said at least one immunoglobulin E binding receptor is FcεRlα, or any functional fragments thereof;(c) wherein said target-recognition component is derived from FcεRlα, said component comprising the amino acid sequence as denoted by SEQ ID NO: 1, or any variants or derivatives thereof; and(d) wherein said target-recognition component comprises, or is derived from at least one antibody and / or any fragment thereof; optionally, at least one of:(i) said at least one antibody and / or any fragment thereof binds at least one immunoglobulin molecule; and(ii) said at least one antibody and / or any fragment thereof binds at least one immunoglobulin E (IgE) molecule.

42. The method of any one of claims 34 to 41, wherein said targeted elimination compound comprises a fusion protein comprising the FcεRlα and a peptide of CSP, wherein said fusion protein comprises the amino acid sequence as denoted by any one of: SEQ ID NOs: 4, 6, and 7, or any variants or derivatives thereof.

43. The method of any one of claims 34 to 41, wherein said targeted elimination compound comprises a fusion protein comprising the Fragment crystallizable epsilon receptor I alpha chain (FcεRlα) and the Apolipoprotein A-I (Apo Al) protein, wherein said fusion protein comprises the amino acid sequence as denoted by any one of: SEQ ID NOs: 5 and 23, or any variants or derivatives thereof.

44. The method of any one of claims 34 to 41, wherein said targeted elimination compound comprises a liposome or a lipid nanoparticle (LNP) conjugated to the Fragment crystallizable epsilon receptor I alpha chain (FcεRlα), or to an anti-IgE antibody, wherein at least one of:(i) said FcεRlα comprises the amino acid sequence as denoted by any one of: SEQ ID NOs: 1 and 2, or any variants or derivatives thereof;(ii) said liposome comprises DGS / NTA; and(iii) said LNP comprises DSPE-PEG-mal.

45. The method of any one of claims 34 to 44, wherein said targeted elimination compound is as defined by any one of claims 1 to 27, and wherein the composition is as defined in any one of claims 28 to 29.

46. The method of any one of claims 34 to 45, wherein said subject is suffering from at least one immune-related disorder.

47. The method of claim 46, wherein said at least one immune-related disorder is a medical condition associated with, or induced by, activation of at least one white blood cell (leukocyte).

48. The method of claim 47, wherein said medical condition is associated with activation of at least one of: mast cell, basophil and / or eosinophil, and wherein said medical condition associated with, or induced by, activation of said mast cells, basophils and / or eosinophils is any one of allergic reaction, a chronic or acute inflammatory condition involving airways, skin, and / or mucosal tissue, mastocytosis and mast cell tumors.

49. A therapeutically effective amount of at least one targeted elimination compound, or any preparations thereof, or any vehicle, matrix, nano- or micro-particle comprising the same, and / or any composition thereof, for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one pathological disorder associated directly or indirectly with at least one target macromolecule in a subject wherein said targeted elimination compound comprises:(a) at least one target-recognition component, wherein said component specifically binds, and optionally, neutralizes said at least one target macromolecule; and(b) at least one elimination and / or removal-targeting component, wherein said component targets said at least one target macromolecule to the liver in said subject, and wherein said component is derived form at least one of: a microorganism, a natural and / or synthetic molecule and / or particle displaying liver tropism.

50. The therapeutically effective amount of at least one targeted elimination compound for use according to claim 49, wherein said targeted elimination compound is as defined by any one of claims 1 to 27, and wherein the composition is as defined in any one of claims 28 to 29.

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