Monoclonal antibodies to b0at1

Abstract

The present invention provides monoclonal antibodies or antigen-binding portions thereof against B0AT1 and their use in the treatment of disorders involving amino acid dysregulation. The invention also provides an in-vitro method for screening a monoclonal antibody or antigen-binding portion thereof that specifically binds to an extracellular epitope of the B0AT1 protein and an in-vitro method for detecting B0AT1 protein in a biological sample.

Description

[0001] 1 BW1384R

[0002] MONOCLONAL ANTIBODIES TO B°AT1

[0003] TECHNICAL FIELD OF THE INVENTION

[0004] The present invention provides monoclonal antibodies or antigen-binding portions thereof against B°AT1 and their use in the treatment of disorders involving amino acid dysregulation. The invention also provides an in-vitro method for screening monoclonal antibodies or antigen-binding portions thereof that specifically bind to an extracellular epitope of the B°AT1 protein and an in-vitro method for detecting B°AT1 protein in a biological sample.

[0005] STATE OF THE ART

[0006] The sodium-dependent neutral amino acid transporter B°AT1 (SLC6A19) is essential for amino acid homeostasis, with mutations causing Hartnup disease and dysregulation linked to metabolic disorders. It plays a central role in amino acid absorption and homeostasis in renal and intestinal epithelial cells by coupling amino acid transport to the electrochemical sodium gradient (Brber, S. Amino acid transport across mammalian intestinal and renal epithelia. Physiol Rev 88, 249-286 (2008)). In the kidney, B°AT1 requires collectrin (CLTRN) for proper trafficking and function (Danilczyk, U. et al. Essential role for collectrin in renal amino acid transport. Nature 444, 1088-1091 (2006)), whereas in the intestine angiotensin converting enzyme 2 (ACE2) serves as its key accessory protein (Kowal czuk, S. et al. A protein complex in the brush-border membrane explains a Hartnup disorder allele. FASEB J 22, 2880-2887 (2008)). These interactions highlight the tissue-specific complexity of B°AT1 regulation.

[0007] Loss-of-function mutations in SLC6A19 cause Hartnup disease, an autosomal recessive disorder characterised by defective neutral amino acid transport, resulting in hyperaminoaciduria, pellagra-like dermatitis, and cerebellar ataxia (Seow, H. F. et al. Hartnup disorder is caused by mutations in the gene encoding the neutral amino acid transporter SLC6A19. Nat Genetics 36, 1003-1007 (2004), Kleta, R. et al. Mutations in SLC6A19, encoding B°AT1, cause Hartnup disorder. Nat Genet 36, 999-1002 (2004)). Beyond this rare genetic disorder, dysregulated B°AT1 function has been linked to metabolic disorders, including insulin resistance and type 2 diabetes (T2D), in which amino acid imbalance contributes to impaired nutrient signalling (Brber, A.2 BW1384R et al. Impaired nutrient signaling and body weight control in a Na+ neutral amino acid cotransporter (Slc6al9)-deficient mouse. J Biol Chem 286, 26638-26651 (2011), Jiang, Y. et al. Mice lacking neutral amino acid transporter B(O)AT1 (Slc6al9) have elevated levels of FGF21 and GLP-1 and improved glycaemic control. Mol Metab 4, 406-417 (2015), Cheng, Q. et al. Identification of novel inhibitors of the amino acid transporter B°AT1 (SLC6A19), a potential target to induce protein restriction and to treat type 2 diabetes. British Journal of Pharmacology 174, 468-482 (2017)). Recent studies have demonstrated that modulation of amino acid transport can influence metabolic disease progression, suggesting therapeutic potential for B°AT1 -targeted interventions. B°AT1 plays a critical role in phenylalanine reabsorption in the kidney and intestine. Inhibition of SLC6A19 has been shown to reduce systemic and brain phenylalanine levels, offering a novel therapeutic approach for the treatment of phenylketonuria (PKU) (Belanger, A. M. et al. Inhibiting neutral amino acid transport for the treatment of phenylketonuria. JCI Insight 3, el21762 (2018)) suggesting broader implications for modulating B°AT1 function in metabolic disorders. Furthermore, recent fundings show a potential role of B°AT1 in tumoral proliferation, widening the range of clinical applications of this protein and the importance of having research tools useful for its study (Bogatikov E, Munoz C, Pakladok T, Alesutan I, Shojaiefard M, Seebohm G, Fbller M, Palmada M, Bbhmer C, Brber S, Lang F. Upregulation of amino acid transporter SLC6A19 activity and surface protein abundance by PKB / Akt and PIKfyve. Cell Physiol Biochem. 2012;30(6): 1538-46. doi: 10.1159 / 000343341. Epub 2012 Dec 10. PMID: 23234856; Xing Y, Li G, Li G, Xu J, Zhang T, Li M, Gao C, Fu M, Zheng P, Chu X. MT1H inhibits the growth of gastric cancer by regulating SLC6A19 / TTC39B / ADM2 and activating p53-dependent autophagy. Sci Rep. 2025 Mar 18; 15(1):9339. doi: 10.1038 / s41598-025-91319-y. PMID: 40102553; PMCID: PMC11920260). Moreover, has been recently explored the potential role of B°AT1 in insulin resistance and type 1 and 2 diabetes (T1D and T2D), suggesting that inhibition of this receptor could be a strategy in the treatment of cardiovascular diseases (Waters MF, Delghingaro-Augusto V, Shamoon M, Javed K, Burgio G, Dahlstrom JE, Brber S, Nolan CJ. Interaction of B0AT1 Deficiency and Diet on Metabolic Function and Diabetes Incidence in Male Nonobese Diabetic Mice. Endocrinology. 2025 Feb 5;166(3):bqafO16. doi: 10.1210 / endocr / bqaf016. PMID: 39844660; PMCID: PMC11815506).

[0008] Current approaches to studying B°AT1 function rely primarily on genetic manipulation and chemical inhibitors, which have significant limitations for therapeutic development. Despite its physiological importance, B°AT1 has remained3 BW1384R difficult to study due to its structural complexity as a multi-pass transmembrane protein, which has hindered the development of high-affinity monoclonal antibodies (mAbs) for functional characterisation and therapeutic targeting (Dodd, R., Schofield, D. J., Wilkinson, T. & Britton, Z. T. Generating therapeutic monoclonal antibodies to complex multi -spanning membrane targets: Overcoming the antigen challenge and enabling discovery strategies. Methods 180, 111-126 (2020), Carpenter, E. P., Beis, K., Cameron, A. D. & Iwata, S. Overcoming the challenges of membrane protein crystallography. Curr Opin Struct Biol 18, 581-586 (2008)). Conventional antibody discovery approaches often fail with membrane transporters due to poor antigen accessibility and epitope instability. Traditional immunization strategies typically generate antibodies against denatured or linear epitopes, which rarely translate to functional modulation of the native protein. As a result, there remains a critical gap in molecular tools to dissect B°AT1 function and develop transporter-targeted therapies and despite its therapeutic potential, developing functional antibodies against multipass transmembrane proteins like B°AT1 has remained challenging due to complex protein topology, conformational requirements and limited extracellular accessibility (Schlessinger, A., Zatorski, N., Hutchinson, K. & Colas, C. Targeting SLC transporters: small molecules as modulators and therapeutic opportunities. Trends Biochem Sci 48, 801-814 (2023); Thomas, B., Chockalingam, K. & Chen, Z. Methods for Engineering Binders to Multi-Pass Membrane Proteins. Bioengineering (Basel) 10, 1351 (2023); Stephens, A. D. & Wilkinson, T. Discovery of Therapeutic Antibodies Targeting Complex Multi-Spanning Membrane Proteins. BioDrugs 38, 769-794 (2024)).

[0009] SUMMARY OF THE INVENTION

[0010] The Authors have surprisingly developed the first monoclonal antibodies (mAb#51, mAb#A01 also referred to as A001, m Ab# A03 also referred to as A003, and mAb#D03 also referred to as D003) that selectively bind B°AT1 on the cell surface and inhibit its amino acid transport function. This advance not only enables the study of amino acid transport mechanisms but also provides a potential therapeutic strategy for disorders of amino acid homeostasis. Through strategic immunogen selection, the Authors have surprisingly developed functionally active chimeric monoclonal antibodies against B°AT1, overcoming the above-mentioned challenges and establishing a framework for extending antibody-based inhibition to other SLC transporters. These findings have broader implications beyond therapeutic applications, in fact recent studies have shown that B°AT1 and its accessory protein4 BW1384R ACE2 are critical for preimplantation embryo development, with Slc6al9- / - mice exhibiting reduced fertility and impaired embryonic development (Treleaven, T. et al. Stage-Specific L-Proline Uptake by Amino Acid Transporter Slc6al9 / B°AT1 Is Required for Optimal Preimplantation Embryo Development in Mice. Cells 12, 18 (2022)).

[0011] The monoclonal antibody mAb#51 specifically recognizes a conformational epitope uniquely accessible on native B°AT1 and potently inhibits its transport function (IC50 ~1 nM). Using a dual immunization strategy combining cell-based and recombinant approaches, the Authors of the present invention have demonstrated high specificity of mAb#51 across cell types, including cross-reactivity with the murine ortholog, revealing unexpected insights into B°AT1 trafficking. The monoclonal antibody mAb#A01 presents a distinct clonotype with respect to mAb#51, although maintaining some structural similarities. Experimental data indicates that mAb#A01 also exerts a potent inhibitory action on B°AT1 transport function, analogously to mAb#51. mAb#A03 and mAb#D03 have also been shown to bind B°AT1 on the cell surface. These findings establish mAb#51, mAb#A01, mAb#A03, and mAb#D03 as powerful research tools. Furthermore, the antibodies’ exceptional potency compared to existing inhibitors suggests therapeutic potential for disorders involving amino acid dysregulation, providing a framework for targeting complex membrane proteins, with implications for both basic research and therapeutic development.

[0012] The present application refers also to a targeted monoclonal antibody discovery strategy designed to overcome the above-mentioned challenges. By integrating cellbased expression of full-length B°AT1 with recombinant domain-based immunization, the authors have identified mAb#51, mAb#A01, mAb#A03, and mAb#D03, the first chimeric monoclonal antibodies that recognise a conformational epitope uniquely accessible in the native transporter. mAb#51, mAb#A01, mAb#A03, and mAb#D03 provide new opportunities for investigating amino acid transport mechanisms and developing targeted therapies for disorders involving amino acid dysregulation. Given their ability to modulate B°AT1 function, mAb#51, mAb#A01, mAb#A03, and mAb#D03 represent both powerful tools for investigating amino acid transport mechanisms and a prototype for antibody-based therapeutic strategies targeting amino acid transport disorders. Beyond advancing the understanding of B°AT1 biology, the invention establishes a versatile framework for generating functional monoclonal antibodies against structurally complex membrane transporters, with broad implications for molecular medicine and targeted therapies. The development of mAb#51, mAb#A01, mAb#A03, and mAb#D03 as functional inhibitors of B°AT15 BW1384R represents a significant advance in membrane protein targeting. This study provides a powerful tool for investigating amino acid transport mechanisms while establishing a framework for antibody-based therapeutic strategies in metabolic and genetic disorders. mAb#51, mAb#A01, mAb#A03, and mAb#D03 hold potential for clinical applications in PKU, Hartnup disease, other aminoacidopathies, insulin resistance, type 1 and type 2 diabetes (T1D and T2D) and cancer.

[0013] Therefore, objects of the present invention are:

[0014] Monoclonal antibodies or antigen-binding portions thereof that specifically bind to an extracellular epitope of the B°AT1 protein; preferably wherein said monoclonal antibodies or antigen-binding portions thereof are selected from a chimeric monoclonal antibody, or a bispecific antibody, or a diabody, or a nanobody, or any polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding to the polypeptide.

[0015] An in-vitro method for screening monoclonal antibodies or antigen-binding portions thereof that specifically bind to an extracellular epitope of the B°AT1 protein comprising the following steps:

[0016] a) isolating B°AT1 -specific plasma cells from bone marrow samples collected from two groups of animals wherein group (1) is immunized with a first antigen wherein said first antigen is cells co-expressing B°AT1 protein and collectrin protein (CLTRN) and / or group (2) is immunized with a second antigen wherein said second antigen is a recombinant protein comprising residues from 326 to 413 of loop 4 of B°AT1 protein; b) performing a screening to select the B°AT1 -specific antibody-secreting cells that bind the first antigen and / or the second antigen.

[0017] An in-vitro method for detecting B°AT1 protein in a biological sample comprising the following steps:

[0018] a) incubating the biological sample with an anti-B°ATl protein antibody of the invention,

[0019] b) detecting the bound antibody.

[0020] DETAILED DESCRIPTION OF THE FIGURES

[0021] Figure 1: A) mAb#51 specifically binds to B°AT1 on the cell surface, independent of collectrin expression. Surface binding of mAb#51 to B°AT1 was assessed by flow cytometry using CHO-B (B°AT1 without collectrin) and CHO-B+C (B°AT1 with collectrin) stable cell lines, with parental CHO cells serving as a negative control. Binding was detected with a DyLight™ 650-conjugated goat anti-human IgG Fc6 BW1384R secondary antibody. Analysis was restricted to EGFP-positive (EGFP+) cells to confirm B°AT1 expression in CHO-B and CHO-B+C lines, while EGFP-negative (EGFP ) parental CHO cells provided a gating control. mAb#51 exhibited specific binding to both CHO-B and CHO-B+C cells, validating its selectivity for B°AT1. B) Quantitative assessment of mAb#51 binding to B°AT1.

[0022] Flow cytometry analysis was performed to evaluate the specificity and affinity of monoclonal antibody mAb#51 for B°AT1. CHO-B+C cells co-expressing B°AT1 and collectrin (right panel), CHO-B cells expressing B°AT1 alone (middle panel), and parental CHO cells lacking B°AT1 (left panel) were analyzed. Serial 3-fold dilutions of mAb#51 (30 pg / mL to 1 ng / mL) were used to assess dose-dependent binding. Control antibodies included mAb#56, a non-conformational B°AT1 -specific monoclonal antibody, and an unrelated isotype control. Binding was quantified as mean fluorescence intensity (MFI), with data presented as mean ± s.d. from triplicate experiments. C) ACE2 co-expression does not alter mAb#51 binding to B°AT1.

[0023] Left: Validation of ACE2 and nuclear-localized blue fluorescent protein (BFP) coexpression in CHO cells transiently transfected with an ACE2-IRES-NLS-BFP construct (CHO wt+A). ACE2 expression was assessed via indirect immunofluorescence using a monoclonal anti-ACE2 antibody and Alexa Fluor™ 647-conjugated F(ab')2 goat anti-mouse IgG secondary antibody. Right: Flow cytometric analysis of mAb#51 binding to surface-localized B°AT1 in CHO-B cells (B°AT1-expressing CHO cells) co-transfected with the ACE2-IRES-NLS-BFP construct. Binding was detected with DyLight™ 650-conjugated goat anti-human IgG Fc secondary antibody. Cells were gated into BFP (ACE2 -negative) and BFP+(ACE2-positive) populations based on IRES-driven co-expression. Data represent fluorescence intensity from two independent experiments.

[0024] Figure 2: Validation of B° ATI -specific monoclonal antibodies. Western blot analysis demonstrating B°AT1 specificity of mAb#51 and mAb#56 in CHO cells. mAb#51 was compared with mAb#56 (generated against recombinant B°AT1 loop 4). Specific immunoreactive bands were detected at ~70 kDa (red arrows) in CHO cells stably expressing B°AT1 and collectrin (CHO-B+C, lane 2) but not in parental CHO cells (lane 1). M, molecular weight markers.

[0025] Figure 3: mAb#51 specifically binds B°AT1 in wild-type KYO-1 cells but not in B°AT1 -knockout KYO-1 ko#4 cells, confirming target specificity. Flow cytometry analysis of mAb#51 binding using DyLight™ 650-conjugated anti-human IgG Fc7 BW1384R secondary antibody, a Wild-type KYO-1 cells demonstrate positive surface staining, confirming B°AT1 recognition, b CRISPR / Cas9-generated B°AT1 knockout cells (KYO-1 KO#4) show no detectable signal, validating antibody specificity.

[0026] Figure 4: Cell surface recognition of mouse SLC6A19 by mAb#51. Flow cytometric analysis of CHO cells 48 h after transient transfection with mouse SLC6A19-IRES-EGFP construct. Cell surface binding was detected using DyLight™ 650-conjugated anti-human IgG Fc antibody. Top: Representative histograms showing control and mAb#51 staining in GFP-negative population. Bottom: Control and mAb#51 staining in GFP-positive population.

[0027] Figure 5 A) B° ATI-mediated transport activity assessed through membrane potential measurements. Membrane depolarization was monitored by fluorescence (kex / em = 545 / 625 nm) in response to isoleucine concentrations (1.5-300 mM) in CHO cells stably expressing B°AT1 with collectrin (CHO-B+C), B°AT1 alone (CHO-B), or neither (parental CHO). Measurements were recorded at 10-s intervals for 5 min. CHO-B+C cells exhibited dose-dependent membrane depolarization with maximal response at 15 mM isoleucine, while CHO-B and parental cells showed no significant changes, demonstrating collectrin-dependent B°AT1 transport activity.

[0028] B) Dose-dependent inhibition of B°AT1 transport by mAb#51. B°AT1 -mediated isoleucine (15 mM) transport was assessed in CHO-B+C cells using membrane potential measurements (kex / em = 545 / 625 nm) in the presence of serially diluted mAb#51 (6.7 pM-200 nM) or nimesulide (0.16-500 pM). Fluorescence was monitored at 10-s intervals for 5 min. mAb#51 demonstrated dose-dependent inhibition of B°AT1 activity, while an isotype-matched control antibody showed no effect, confirming specific B°AT1 antagonism by mAb#51.

[0029] Figure 6: Validation of the CHO-B+C stable cell line, a Western blot analysis of B°AT1 expression in CHO-B+C cells using an anti-SLC6A19 polyclonal antibody. A ~71 kDa band corresponding to B°AT1 was detected in CHO-B+C cells (lane 2) but was absent in parental CHO cells (lane 1). b Flow cytometry analysis of surface CLTRN expression in CHO-B+C cells (lower panels) using an anti-cMyc monoclonal antibody (right panels), detected with Alexa Fluor 647-conjugated anti-mouse IgG F(ab')2. GFP fluorescence marks B°AT1 expression. Quadrant analysis reveals a population of double-positive (B°AT1+ / CLTRN+) cells in the top-right quadrant. Parental CHO wild-type (wt) cells served as a negative control.8 BW1384R

[0030] Figure 7: Generation of B°AT1 -specific antibodies through a dual immunization strategy: immunization schedule and polyclonal antibody titer evaluation.

[0031] A) The immunization and blood sampling schedules are depicted for both immunization approaches. The left panel shows the timeline for mice immunized with B°AT1 loop4 recombinant fragment, while the right panel illustrates the schedule for mice immunized with CHO-B+C stable line cells. The timing of immunizations and blood collections are indicated chronologically.

[0032] B) ELISA analysis demonstrates antigen-specific antibody responses. Left panel shows antibody titers from mice immunized with B°AT1 loop4 recombinant fragment, while right panel displays titers from mice immunized with CHO-B+C stable line cells. All samples were tested against the B°AT1 loop4 recombinant fragment as the target antigen. Pre-immune sera were used as negative controls to establish baseline responses, with immune sera showing successful antibody generation in both immunization groups.

[0033] Figure 8: SLC6A19 mRNA expression in KYO-1 cells. Transcription of SLC6A19 in KYO-1 cells was assessed using three distinct amplicons (Amp#l, Amp#2, and Amp#3). The ACt values for each SLC6A19 amplicon were normalized to the threshold cycle (Ct) of the P-actin housekeeping gene.

[0034] Figure 9: mAbs mAb#A01(A001), mAb#A03 (A003) and mAb#D03 (D003) specifically bind cell-surface B°AT1. Flow cytometry analysis of parental CHO cells (CHO wt), CHO cells co-expressing B°AT1 and collectrin (CHO-B+C).

[0035] DETAILED DESCRIPTION OF THE INVENTION

[0036] The term "polypeptide" encompasses native or artificial proteins, protein fragments and polypeptide analogues of a protein sequence. A polypeptide may be monomeric or polymeric. The term "isolated protein", "isolated polypeptide" or "isolated antibody" is a protein, polypeptide or antibody that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is free of other proteins from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the9 BW1384R cell from which it naturally originates will be "isolated" from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art. The term "polypeptide fragment" as used herein refers to a polypeptide that has an amino-terminal and / or carboxy -terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the naturally occurring sequence. In some embodiments, fragments are at least 5, 6, 8 or 10 amino acids long. In other embodiments, the fragments are at least 14, at least 20, at least 50, or at least 70, 80, 90, 100, 150 or 200 amino acids long. The term "polypeptide analogue" as used herein refers to a polypeptide that comprises a segment that has substantial identity to a portion of an amino acid sequence and that has at least one of the following properties: (1) specific binding to B°AT1 protein under suitable binding conditions, (2) ability to inhibit the neutral amino acid transport mediated by the B°AT1 protein. Typically, polypeptide analogues comprise a conservative amino acid substitution (or insertion or deletion) with respect to the native sequence. Analogues typically are at least 20 or 25 amino acids long, preferably at least 50, 60, 70, 80, 90, 100, 150 or 200 amino acids long or longer, and can often be as long as a full-length polypeptide. Some embodiments of the invention include polypeptide fragments or polypeptide analogue antibodies with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 substitutions from the germline amino acid sequence. Analogues can include various muteins of a sequence other than the normally occurring peptide sequence. For example, single or multiple amino acid substitutions, preferably conservative amino acid substitutions, may be made in the normally occurring sequence, preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts. A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence.

[0037] Where an "antibody" is referred to herein with respect to the invention, it is normally understood that an antigen-binding portion thereof may also be used. An antigenbinding portion competes with the intact antibody for specific binding. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., second ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes). Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical10 BW1384R cleavage of intact antibodies. In some embodiments, antigen-binding portions include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies, nanobodies and any polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding to the polypeptide.

[0038] From N-terminus to C-terminus, both the mature light and heavy chain variable domains comprise the regions FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain herein is in accordance with the definitions of IMGT convention described in Lefranc et al. (2003), Developmental & Comparative Immunology 27.1 (2003): 55-77.

[0039] In some embodiments, the antibody is a single-chain antibody (scFv) in which a VL and VH domains are paired to form a monovalent molecule via a synthetic linker that enables them to be made as a single protein chain. (Bird et al, Science 242:423-426 (1988) and Huston et al, Proc. Natl Acad. ScL USA 85:5879-5883 (1988)). In some embodiments, the antibodies are diabodies, i.e., are bivalent antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites. (See e.g., Holliger P. et al, Proc. Natl. Acad. ScL USA 90:6444-6448 (1993), and Poljak R. J. et al, Structure 2:1121-1123 (1994)). In such embodiments, the CDR(s) may be incorporated as part of a larger polypeptide chain, may be covalently linked to another polypeptide chain, or may be incorporated noncovalently.

[0040] The term "polynucleotide" as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms.

[0041] The term "isolated polynucleotide" as used herein means a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the "isolated polynucleotide" (1) is not associated with all or a portion of a polynucleotides with which the "isolated polynucleotide" is found in nature, (2) is11 BW1384R operably linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.

[0042] The term "naturally occurring nucleotides" as used herein includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" as used herein includes nucleotides with modified or substituted sugar groups and the like. The term "oligonucleotide linkages" referred to herein includes oligonucleotides linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, phosphoroamidate, and the like. See e.g., LaPlanche et al., Nucl. Acids Res. 14:9081 (1986); Stec et al, J. Am. Chem. Soc. 106:6077 (1984); Stein et al., Nucl. Acids Res. 16:3209 (1988); Zon et al., Anti-Cancer Drug Design 6:539 (1991); Zon et al.. Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); U.S. Patent No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90:543 (1990), the disclosures of which are hereby incorporated by reference. An oligonucleotide can include a label for detection, if desired. "Operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. The term "expression control sequence" as used herein means polynucleotide sequences that are necessary to affect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term "control sequences" is intended to include, at a minimum, all components whose presence is essential for expression and processing and can also include additional components whose presence is advantageous, for example, leader sequences and12 BW1384R fusion partner sequences. The term "vector", as used herein, means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In some embodiments, the vector is a plasmid, i.e., a circular double stranded piece of DNA into which additional DNA segments may be ligated. In some embodiments, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. In some embodiments, the vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). In other embodiments, the vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors").

[0043] The term "recombinant host cell" (or simply "host cell"), as used herein, means a cell into which a recombinant expression vector has been introduced. It should be understood that "recombinant host cell" and "host cell" mean not only the particular subject cell but also the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0044] The term "percent sequence identity" in the context of nucleotide or aminoacidic sequences means the residues in two sequences that are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48 or more nucleotides. There are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs available, provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219 (2000); Pearson, Methods13 BW1384R Enzymol. 266:227-258 (1996); Pearson, J Mol. Biol 276:71-84 (1998); incorporated herein by reference). The term "substantial similarity" or "substantial sequence similarity," when referring to a nucleic acid or fragment thereof, or aminoacidic means that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 85%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above. As applied to polypeptides, the term "substantial identity" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights as supplied with the programs, share at least 70%, 75% or 80% sequence identity, preferably at least 90% or 95% sequence identity, and more preferably at least 97%, 98% or 99% sequence identity. In certain embodiments, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson, Methods Mol. Biol. 243:307-31 (1994). Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulphur-containing side chains: cysteine and methionine. Conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysinearginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the14 BW1384R PAM250 log-likelihood matrix disclosed in Gonnet et al, Science 256: 1443-45 (1992), incorporated herein by reference. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix. Sequence identity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as "Gap" and "Bestfit" which can be used with default parameters as specified by the programs to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. The present invention refers to monoclonal antibodies or antigen-binding portions thereof that specifically bind to an extracellular epitope of the B°AT1 protein. According to the present invention, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor or otherwise interacting with a molecule. Epitopes or antigenic determinants generally consist of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and generally have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope may be "linear" or "conformational." In a linear epitope, all of the points of interaction between the protein and the interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction occur across amino acid residues on the protein that are separated from one another. An “extracellular epitope” is an epitope localized in the extracellular environment and not in the transmembrane or intracellular environments.

[0045] The expression "specifically binds" refers to the ability of an antibody to selectively and tightly interact with a particular target molecule, an antigen wherein this binding is highly specific, meaning that the antibody recognizes and attaches to a unique epitope on the antigen's surface.

[0046] In one embodiment the B°AT1 protein is identified as the B°AT1 human protein (Uniprot #Q695T7) having SEQ ID NO: 1.

[0047] In one embodiment, said extracellular epitope comprises or consists of the residues15 BW1384R from 327 to 413 in the loop 4 of the B°AT1 protein, in particular wherein residues from 327 to 413 corresponding to residues number of SEQ ID NO: 1.

[0048] In one preferred embodiment, said extracellular epitope comprises or consists of at least 1 or 2 residues, preferably 3, 4, 5, 6, 7, 8 residues, more preferably 10, 11, 12 or more residues of the amino acid in the position 327 to 413 in the loop 4 of the B°AT1 protein. In one embodiment residues from 327 to 413 in the loop 4 of the B°AT1 protein have SEQ ID NO:2.

[0049] In one embodiment, said monoclonal antibodies or antigen-binding portions thereof are selected from a chimeric monoclonal antibody, or a bispecific antibody, or a diabody, or a nanobody, or any polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding to the polypeptide.

[0050] In one embodiment, said extracellular epitope does not comprise residues that interact partially or completely with the angiotensin converting enzyme 2 (ACE2) and / or with the collectrin protein (CLTRN).

[0051] In one embodiment, said antibodies or antigen-binding portions thereof inhibit the neutral amino acid transport mediated by the B°AT1 protein.

[0052] In one embodiment, said B°AT1 protein is the human B°AT1 protein and / or the murine B°AT1 protein.

[0053] In one embodiment, said monoclonal antibodies or antigen-binding portions thereof are human, murine or humanized.

[0054] In one embodiment, said antibodies or antigen-binding portions thereof shows a 50% inhibitory concentration (IC50) of less than 10 nM, preferably less than 1 nM, in particular when tested in a surface binding assay carried out through flow cytometry. In fact, as stated above and demonstrated in the experimental section of the specification, the monoclonal antibodies of the present invention have a surprising specificity for an extracellular epitope of the B°AT1 protein, having an inhibitory concentration (IC50) of 1 nM when measured in surface binding assays such as flow cytometry, unambiguously performable following the materials and methods provided in the experimental section of the present application.

[0055] In one preferred embodiment, a monoclonal antibody or antigen-binding portion thereof herein described comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein said VH and VL comprise the following complementarity-16 BW1384R determining regions (CDRs):

[0056] - CDR1 of VH having SEQ ID NO: 3,

[0057] -CDR2 of VH having SEQ ID NO: 4,

[0058] -CDR3 of VH having SEQ ID NO: 5,

[0059] -CDR1 of VL having SEQ ID NO: 6,

[0060] -CDR2 of VL having the sequence YAS (tyr-ala-ser) SEQ ID NO: 7,

[0061] -CDR3 of VL having SEQ ID NO: 8.

[0062] In one preferred embodiment, the monoclonal antibody or antigen-binding portion thereof herein described comprises a heavy chain variable domain (VH) and a light chain variable domain (VL),

[0063] wherein said VL and said VH is at least 85% identical in amino acid sequence, preferably at least 95% identical in amino acid sequence, more preferably at least 99% identical in amino acid sequence to said VH having SEQ ID NO: 9 and said VL having SEQ ID NO: 10, in particular wherein said VH has SEQ ID NO: 9 and said VL has SEQ ID NO: 10. In one preferred embodiment the heavy chain (HC) of said antibody has SEQ ID NO: 11 and the light chain (LC) of said antibody has SEQ ID NO: 12.

[0064] In one preferred embodiment, a monoclonal antibody or antigen-binding portion thereof herein described comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein said VH and VL comprise the following complementaritydetermining regions (CDRs):

[0065] - CDR1 of VH having SEQ ID NO: 3,

[0066] -CDR2 of VH having SEQ ID NO: 17,

[0067] -CDR3 of VH having SEQ ID NO: 18,

[0068] -CDR1 of VL having SEQ ID NO: 19,

[0069] -CDR2 of VL having the sequence YAS (tyr-ala-ser) SEQ ID NO: 7,

[0070] -CDR3 of VL having SEQ ID NO: 20.

[0071] In one preferred embodiment, the monoclonal antibody or antigen-binding portion thereof herein described comprises a heavy chain variable domain (VH) and a light chain variable domain (VL),

[0072] wherein said VL and said VH is at least 85% identical in amino acid sequence, preferably at least 95% identical in amino acid sequence, more preferably at least 99% identical in17 BW1384R amino acid sequence to said VH having SEQ ID NO: 21 and said VL having SEQ ID NO: 22, in particular wherein said VH has SEQ ID NO: 21 and said VL has SEQ ID NO: 22. In one preferred embodiment the heavy chain (HC) of said antibody has SEQ ID NO: 11 and the light chain (LC) of said antibody has SEQ ID NO: 12.

[0073] In one preferred embodiment, a monoclonal antibody or antigen-binding portion thereof herein described comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein said VH and VL comprise the following complementaritydetermining regions (CDRs):

[0074] - CDR1 of VH having SEQ ID NO: 23,

[0075] -CDR2 of VH having SEQ ID NO: 24,

[0076] -CDR3 of VH having SEQ ID NO: 25,

[0077] -CDR1 of VL having SEQ ID NO: 26,

[0078] -CDR2 of VL having the sequence RTS (arg-thr-ser) SEQ ID NO: 27,

[0079] -CDR3 of VL having SEQ ID NO: 28.

[0080] In one preferred embodiment, the monoclonal antibody or antigen-binding portion thereof herein described comprises a heavy chain variable domain (VH) and a light chain variable domain (VL),

[0081] wherein said VL and said VH is at least 85% identical in amino acid sequence, preferably at least 95% identical in amino acid sequence, more preferably at least 99% identical in amino acid sequence to said VH having SEQ ID NO: 29 and said VL having SEQ ID NO: 30, in particular wherein said VH has SEQ ID NO: 29 and said VL has SEQ ID NO: 30. In one preferred embodiment the heavy chain (HC) of said antibody has SEQ ID NO: 11 and the light chain (LC) of said antibody has SEQ ID NO: 12.

[0082] In one preferred embodiment, a monoclonal antibody or antigen-binding portion thereof herein described comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein said VH and VL comprise the following complementaritydetermining regions (CDRs):

[0083] - CDR1 of VH having SEQ ID NO: 31,

[0084] -CDR2 of VH having SEQ ID NO: 32,

[0085] -CDR3 of VH having SEQ ID NO: 33,18 BW1384R -CDR1 of VL having SEQ ID NO: 34,

[0086] -CDR2 of VL having the sequence SAS (ser-ala-ser) SEQ ID NO: 35,

[0087] -CDR3 of VL having SEQ ID NO: 36.

[0088] In one preferred embodiment, the monoclonal antibody or antigen-binding portion thereof herein described comprises a heavy chain variable domain (VH) and a light chain variable domain (VL),

[0089] wherein said VL and said VH is at least 85% identical in amino acid sequence, preferably at least 95% identical in amino acid sequence, more preferably at least 99% identical in amino acid sequence to said VH having SEQ ID NO: 37 and said VL having SEQ ID NO: 38, in particular wherein said VH has SEQ ID NO: 37 and said VL has SEQ ID NO: 38. In one preferred embodiment the heavy chain (HC) of said antibody has SEQ ID NO: 11 and the light chain (LC) of said antibody has SEQ ID NO: 12.

[0090] An antibody is made up of two heavy chains (HC) and two light chains (LC) joined by disulfide bonds so that each heavy chain is linked to a light chain and the two heavy chains are linked together. The amino-terminal variable or V domains of the heavy and light chains (VH and VL, respectively) together make up the V region of the antibody and confer on it the ability to bind specific antigens, while the constant domains (C domains) of the heavy and light chains (CH and CL, respectively) make up the C region.

[0091] In one embodiment the monoclonal antibodies or antigen-binding portions thereof described as far are for use as a medicament.

[0092] In one embodiment, the monoclonal antibodies or antigen-binding portions thereof described as far are for use in the treatment of disorders involving amino acid dysregulation, Hartnup disease, metabolic syndrome and cancer, preferably, wherein said amino acid dysregulation is selected from phenylketonuria (PKU), insulin resistance and type 1 and 2 diabetes (T1D and T2D), preferably wherein said monoclonal antibodies or antigenbinding portions thereof are administered by intravenous, subcutaneous, intraperitoneal or intramuscular route. The present invention also refers to a pharmaceutical composition comprising one or more of the monoclonal antibodies or antigen-binding portions thereof according to the present invention and a pharmaceutically acceptable carrier.

[0093] As used herein, "pharmaceutically acceptable carrier" means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption19 BW1384R delaying agents, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelflife or effectiveness of the antibody. The compositions of this invention may be in a variety of forms, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In one embodiment, the antibodies are administered by intravenous infusion or injection. In still another embodiment, the antibodies are administered by intramuscular or subcutaneous injection. Therapeutic compositions are typically sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration.

[0094] In one preferred embodiment, said pharmaceutical composition is for use in the treatment of disorders involving amino acid dysregulation, Hartnup disease, metabolic syndromes and cancer, preferably wherein said disorders involving amino acid dysregulation are phenylketonuria (PKU), insulin resistance and type 1 and type 2 diabetes (T1D and T2D).

[0095] The present invention also refers to an in-vitro method for screening monoclonal antibodies or antigen-binding portions thereof that specifically bind to an extracellular epitope of the B°AT1 protein comprising the following steps:

[0096] a) isolating B°AT1 -specific plasma cells from bone marrow samples collected from two groups of animals wherein group (1) is immunized with a first antigen wherein said first antigen is cells co-expressing B°AT1 protein and collectrin protein (CLTRN)20 BW1384R and / or group (2) is immunized with a second antigen wherein said second antigen is a recombinant protein comprising residues from 327 to 413 of loop 4 of B°AT1 protein; b) performing a screening to select the B°AT1 -specific antibody-secreting cells that bind the first antigen and / or the second antigen.

[0097] Example of biological samples to use in the methods herein disclosed are urine, stool, blood, saliva, biopsies, cerebrospinal fluid, nasopharyngeal and oropharyngeal wash, sputum, endotracheal aspirate, bronchoalveolar lavage or other biological samples obtainable from a human subject. The biological samples can be recombinant cells or non-recombinant cells, such as cultured cells.

[0098] In jurisdictions wherein methods comprising in-vivo steps on humans or animals are allowed, the present invention refers to a method for isolating monoclonal antibodies or antigen-binding portions thereof that specifically bind to an extracellular epitope of the B°AT1 protein comprising the following steps:

[0099] a) performing a first immunization of a first group of animals with cells co-expressing B°AT1 protein and collectrin protein (CLTRN) and / or

[0100] b) performing a second immunization of a second group of animals with a recombinant protein comprising residues of loop 4 of B°AT1 protein

[0101] c) collecting bone marrow samples from said first and second groups of animals d) isolating B°AT1 -specific plasma cells

[0102] e) performing a screening to select the B°AT1 -specific antibody-secreting cells that bind the first antigen and / or the second antigen.

[0103] In one embodiment, the present invention refers to a method for isolating monoclonal antibodies or antigen-binding portions thereof that specifically bind to an extracellular epitope of the B°AT1 protein comprising the following steps:

[0104] a) generating a stable cell line co-expressing B°AT1 and collectrin

[0105] b) synthesizing the extracellular loop of B°AT1, preferably loop 4 comprising the residues 327-413

[0106] c) performing a transient transfection in a cell line and harvesting the supernatant at least 5 days post-transfection, preferably 8 days post-transfection.

[0107] d) purifying the recombinant B°AT1 loop protein

[0108] e) collecting bone marrow of mice immunized with either the transfectants coexpressing B°AT1 DNA and collectrin DNA and with the recombinant B°AT1 loop21 BW1384R protein obtained at step b) at least 5 days after the last immunization, preferably 7 days after the last immunization

[0109] f) isolating B°AT1 -specific plasma cells

[0110] g) resuspending plasma cells in antibody expression medium and performing a screening to select the B°AT1 -specific antibody-secreting cells

[0111] h) extracting RNA and performing molecular RT-PCR and nested PCR to obtain VH and VL sequences

[0112] In one embodiment, the method described further comprises the following steps: i) using PCR products obtained at step h) to generate TAP minigenes encoding the human IgGl heavy chain and kappa light chain constant regions.

[0113] j) transiently transfecting said minigenes into cells

[0114] k) culturing and shaking the transfected cells

[0115] l) collecting the supernatants containing the expressed antibodies, at least 24 hours post-transfection, preferably 72 hours post-transfection.

[0116] In one embodiment, the method described further comprises a step of evaluating antibody binding by ELISA and flow cytometry.

[0117] In one embodiment, the expression of B°AT1 and CLTRN in said stable cell line is validated using Western blot and flow cytometry.

[0118] In one embodiment, said purification of the recombinant protein is performed by using Immobilized Metal Affinity Chromatography (IMAC).

[0119] In one embodiment, before transiently transfecting said minigenes into cells, antibody titers in plasma samples collected from said mice are determined using ELISA.

[0120] In one embodiment, said plasma cells are CD 138+ plasma cells

[0121] In one embodiment, said antibody expression medium comprises: RPMI1640 with 10% FBS, 1xMEM Non-Essential Amino Acids Solution, Recombinant mouse IL-6, 25% filtered supernatant from day 3 M210B4 cell culture, 10 pM 2-mercaptoethanol. In one embodiment, said transient transfecting at step o) is performed using 30% heavy chain-encoding minigenes and 70% light chain-encoding minigenes for each transfection.

[0122] In one embodiment, said shaking is performed at least 500 rpm, preferably at 1000 rpm.

[0123] The invention also provides an in vitro method for detecting B°AT1 protein in a22 BW1384R biological sample comprising the following steps:

[0124] a) incubating the biological sample with an anti-B°ATl protein antibody of the invention

[0125] b) detecting the bound antibody.

[0126] Incubating means the process of mixing the biological sample with the anti-X protein antibody under controlled conditions (such as temperature and time) to allow the antibody to bind specifically to the target antigen in the sample. This process allows the antibody to interact with the target molecule and form an antibody-antigen complex.

[0127] Detecting refers to the process of identifying and measuring the presence or amount of the antibody that has bound to the target antigen in the sample. This can be done using various methods such as enzyme-linked immunosorbent assay (ELISA), Western blotting, fluorescence microscopy, or other techniques that allow for the visualization or quantification of the antibody-antigen interaction. In one embodiment, the anti- B°AT1 protein antibody is directly labelled with a detectable label. In another embodiment, the anti- B°AT1 protein antibody (the first antibody) is unlabelled and a second antibody or other molecule that can bind the anti- B°AT1 protein antibody is labelled. As is well known to one of skill in the art, a second antibody is chosen that is able to specifically bind the particular species and class of the first antibody. For example, if the anti- B°AT1 protein antibody is a human IgG, then the secondary antibody could be an anti-human-IgG.

[0128] A biological sample for methods like Western blot or ELISA can be extracted from human, animal or recombinant sources and can come from tissues, cells, body fluids (e.g., serum, plasma, urine), or secretions. The sample should comprise proteins, which can be extracted from cells or tissues.

[0129] In one embodiment, the invention refers to monoclonal antibodies that compete for the binding to the B°AT1 protein with the monoclonal antibodies according to the present specification.

[0130] In certain aspects, the invention provides an isolated cell line that produces the antibodies or antigen-binding portions thereof according to any one of the embodiments herein disclosed.

[0131] In certain aspects, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes and / or translate an antibody or antigen-binding23 BW1384R portion thereof according to any one of the embodiments herein disclosed, such isolated nucleic acid molecule is DNA or mRNA. The nucleic acid molecules may be also used to produce chimeric antibodies, bispecific antibodies, nanobodies, single chain antibodies, immunoadhesins, diabodies, mutated antibodies and antibody derivatives.

[0132] In certain aspects, the invention provides a vector comprising the nucleic acid molecule encoding and / or translating an antibody or antigen-binding portion thereof according to any one of the embodiments herein disclosed, wherein the vector optionally comprises an expression control sequence operably linked to the nucleic acid molecule. In one embodiment, said vector is a selected from RNA virus vectors, DNA virus vectors, plasmid viral vectors, adenovirus vectors, adenovirus associated virus vectors, herpes virus vectors and retrovirus vectors.

[0133] In one embodiment, the invention provides a host cell comprising the vector or the nucleic acid molecule according to the present specification.

[0134] In certain aspects, the invention provides a non-human transgenic animal or transgenic plant comprising the nucleic acid according to any one of the preceding embodiments, wherein the non-human transgenic animal or transgenic plant expresses said nucleic acid. In certain embodiments, said non-human transgenic animal is a mammal.

[0135] In some embodiments such nucleic acid molecules and vectors are formulated in a nanoparticle, for example in lipid nanoparticle, cationic lipid nanoparticle.

[0136] The monoclonal antibodies or antigen-binding portions thereof herein disclosed may be used for the preparation of mimotopes, such as for example anti -idiotype antibodies, peptides, or artificial forms or others, endowed with the ability of evoking the antibodies herein disclosed. Among these, the anti-idiotype antibodies are preferred. The anti-idiotype antibodies are antibodies specifically directed against the idiotype of the neutralizing antibodies used for the manufacture thereof, and thus are able to mimic the key epitopes that they recognize. The manufacture of anti-idiotype antibodies is carried out by per se known methodologies that do not need further detailed explanations here. Thus, also mimotopes, preferably anti-idiotype antibodies, directed against an antibody of the invention fall within the scope of the invention. The monoclonal antibodies or antigen-binding portions thereof herein disclosed may be used for the manufacture of anti-idiotype antibodies according to methods per se known.

[0137] The monoclonal antibodies or antigen-binding portions thereof herein disclosed may be used as a carrier to specifically bind to the target molecule, wherein said antibodies or antigen-binding portions thereof are engineered to carry or deliver another24 BW1384R molecule, such as a drug, toxin, or imaging agent, to the targeted cells or tissues. The monoclonal antibodies or antigen-binding portions thereof herein disclosed may be used as a scaffold or base for the design of bispecific antibodies.

[0138] In one embodiment the monoclonal antibodies or antigen-binding portions thereof herein disclosed may be used in vitro as research tool for studying B°AT1 -mediated amino acid transport.

[0139] Unless otherwise defined herein, scientific, and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0140] In any point of the present specification or of the claims, the expression “comprising” or “comprise(s)” can be replaced by “consisting of’ or “consist(s) of’.

[0141] ***

[0142] In compliance with Art. 170bis, paragraph 2 of the IPC and in accordance with Art.

[0143] 21, paragraph 2 of the Implementing Regulation of the IPC, adopted by Ministerial Decree no. 33 of 13.1.2010, it is hereby declared that:

[0144] the material of animal origin at the basis of the invention covered by said application is from:

[0145] Flp-In™ CHO cells: (Invitrogen, #R75807)

[0146] KYO-1 cells: (DSMZ, #ACC601)

[0147] Expi293F™ cells: (Gibco, #A14527)

[0148] ExpiCHO-S™ cells: (Gibco, #A29127).

[0149] In compliance with Art. 170bis, paragraph 4 of the IPC, it is hereby declared that: With reference to biological material, containing microorganisms or genetically modified organisms, subject to or used in this application, the obligations deriving from national or EU regulations, and in particular, from the provisions referred to in paragraph 6 of Legislative Decree No. 206 of 12 April 2001 and No. 224 of 8 July 2003, concerning such amendments, have been complied with.

[0150] EXAMPLES

[0151] Results

[0152] Generation of a conformation-specific monoclonal antibody against B°AT1

[0153] To generate monoclonal antibodies against B°AT1, stable CHO-B and CHO-B+C cell lines were generated using a bicistronic construct encoding B°AT1 followed by an internal ribosome entry site (IRES) and GFP, allowing real-time monitoring of protein expression (Mancia, F. et al. Optimization of protein production in mammalian cells25 BW1384R with a coexpressed fluorescent marker. Structure 12, 1355-1360 (2004)). B°AT1 expression in CHO-B+C stable cells was confirmed by Western blot using a rabbit anti-SLC6A19 polyclonal antibody, detecting a band at ~71 kDa, with P-actin as a loading control. Flow cytometry showed that GFP fluorescence correlated with CLTRN expression, detected using an anti-c-Myc antibody against the c-Myc tag fused to the extracellular domain of CLTRN (Figure 6).

[0154] A dual immunisation strategy (Figure 7) was used to generate B°AT1 -specific mAbs. The first approach used a recombinant B°AT1 fragment corresponding to loop #4 (amino acids 327-413), the largest extracellular domain predicted to harbour immunogenic epitopes. The second strategy involved immunisation with intact CHO B+C cells, allowing the isolation of mAbs that recognise native conformational epitopes on the cell surface. Rather than conventional hybridoma technology, monoclonal antibodies were generated using a molecular approach. Single antigenspecific antibody-secreting cells (ASCs) were isolated, followed by VH and VL sequence retrieval via RT-PCR. Transcriptionally active PCR (TAP) minigenes were then recombinantly expressed through transient transfection in Expi293 cells, enabling the production of recombinant mAbs in cell culture supernatants (Strazza, V. et al. Rapid generation of human recombinant monoclonal antibodies from antibodysecreting cells using ferrofluid-based technology. Front Immunol 15, 1341389 (2024), Tiller, T., Busse, C. E. & Wardemann, H. Cloning and expression of murine Ig genes from single B cells. J Immunol Methods 350, 183-193 (2009)). This method allows fast and direct selection of high-affinity B cell clones from immunised animals while preserving native antibody pairing.

[0155] Following immunisation and antigen-specific plasma cell isolation, 19 mAbs were identified; 14 from recombinant protein immunisation and 5 from whole-cell immunisation (Table 1).

[0156] Table 1: FT . IS A reactivity of anti-BATl monoclonal antibodies

[0157] ELISA

[0158] mAb ID Immunogen

[0159] reactivity*

[0160] #02 Loop4 +

[0161] #05 Loop4 +

[0162] #11 Loop4 +++

[0163] #12 Loop4 +++

[0164] #21 Loop4 +++

[0165] #25 Loop4 +++

[0166] #26 Loop4 +++

[0167] #35 Loop4 +++

[0168]

[0169] #55 Loop4 +++26 BW1384R #56 Loop4 +++

[0170] #60 Loop4 +

[0171] #62 Loop4 +++

[0172] #69 Loop4 +

[0173] #72 Loop4 +++

[0174] #44 CHO-B+C +

[0175] #46 CHO-B+C +++

[0176] #47 CHO-B+C +

[0177] #51 CHO-B+C ++

[0178]

[0179] #54 CHO-B+C ++

[0180] * ELISA reactivity scoring: + (OD450 < 0.9); ++ (0.9 < OD450 < 1.9); +++ (OD450 > 1.9)

[0181] The ELISA screening identified 19 mAbs targeting the largest extracellular domain of B°AT1: 14 mAbs were generated through immunization with recombinant B°AT1 loop4 fragment, while 5 mAbs were obtained using CHO cells stably expressing B°AT1 and collectrin (CHO-B+C).

[0182] Among these, mAb#51 showed the highest specificity, binding strongly to CHO-B+C cells in a dose-dependent manner, while showing no reactivity with parental CHO cells (Fig. 1A, B). Unexpectedly, mAb#51 also bound CHO-B cells lacking CLTRN, suggesting that CLTRN is not strictly required for B°AT1 surface localisation. This finding challenges the prevailing assumption that CLTRN is indispensable for B°AT1 trafficking. The specificity of mAb#51 remained unchanged when tested in the presence of ACE2, demonstrating that the recognised epitope remains accessible in different physiological environments (Fig. 1C). These results establish mAb#51 as a highly specific antibody for B°AT1, providing a valuable tool for studying its membrane localisation and protein-protein interactions.

[0183] mAb#51 shows high specificity and cross-species reactivity

[0184] To validate mAb#51 specificity, it was implemented a comprehensive strategy combining biochemical and cellular approaches. Western blot analysis of CHO-B+C lysates revealed a single band at B°ATl's predicted molecular weight, without nonspecific signals. mAb#56, an independent monoclonal antibody generated in this study against a distinct linear B°AT1 epitope, showed identical band patterns, corroborating mAb#51 specificity (Fig. 2). It was next evaluated mAb#51 in human cells using KYO-1, a chronic myeloid leukemia line expressing SLC6A19 mRNA according to public datasets (E-MTAB-2770) (Ghandi, M. et al. Next-generation characterization of the Cancer Cell Line Encyclopedia. Nature 569, 503-508 (2019)) and the RT-qPCR validation (Figure 8). Flow cytometry demonstrated robust mAb#51 binding to KYO-1 cells, indicating plasma membrane-localized endogenous B°AT1. Target specificity27 BW1384R was further confirmed using CRISPR / Cas9-mediated B°AT1 knockout (KYO-1 KO#4) with an established sgRNA delivery system 16, which completely abolished mAb#51 binding (Fig. 3 A, B).

[0185] To assess cross-species reactivity, it was transiently expressed murine SLC6A19 in CHO cells using a bicistronic IRES-GFP vector, enabling concurrent monitoring of gene expression and protein detection. mAb#51 recognized murine B°AT1, particularly in high GFP-expressing cells (Fig. 4), establishing its utility for preclinical studies in mouse models.

[0186] mAb#51 potently inhibits B°AT1 -mediated amino acid transport

[0187] To assess the impact of mAb#51 on B°AT1 function, it was employed a fluorescencebased FLIPR membrane potential assay to measure sodium-dependent transporter activity (Danthi, S. J. et al. Identification and Characterization of Inhibitors of a Neutral Amino Acid Transporter, SLC6A19, Using Two Functional Cell-Based Assays. SLAS Discov 24, 111-120 (2019)). In a dose-response setup, CHO-B+C cells exhibited a strong fluorescence signal in response to 15 mM isoleucine, confirming B°AT1 -mediated amino acid transport (Fig. 5a). In contrast, collectrin-deficient CHO-B cells displayed minimal or no transport activity, reinforcing the essential role of CLTRN in amino acid transport rather than merely facilitating B°AT1 surface localization.

[0188] To evaluate the inhibitory potency of mAb#51, it was performed a dose-response analysis. Preincubation of CHO-B+C cells with mAb#51 resulted in a dose-dependent inhibition of B° ATI -mediated isoleucine uptake, with an IC50 of approximately 1 nM (Fig. 5b). This potency was nearly 10,000-fold greater than that of nimesulide, a smallmolecule B°AT1 inhibitor (Pochini, L. et al. Nimesulide binding site in the B°AT1 (SLC6A19) amino acid transporter. Mechanism of inhibition revealed by proteoliposome transport assay and molecular modelling. Biochem Pharmacol 89, 422-430 (2014)), which exhibited an IC50 of -100 pM under identical conditions. Notably, an isotype control antibody had no effect on transporter activity, ruling out non-specific inhibition.

[0189] These findings establish mAb#51 as a highly potent and selective inhibitor of B°AT1, providing a valuable tool for dissecting neutral amino acid transport mechanisms. Its exceptional inhibitory efficacy highlights its therapeutic potential, warranting further evaluation in preclinical models.

[0190] mAb#51 selectively inhibits B°AT1 with nanomolar potency by binding an extracellular epitope, as demonstrated by surface binding assays (Figures 1 and 3) and28 BW1384R inhibition of isoleucine transport (Figure 5). This mechanism represents a distinct approach from conventional small-molecule inhibitors, which typically act through binding sites located within the central transport pathway (Penmatsa, A. & Gouaux, E. How LeuT shapes our understanding of the mechanisms of sodium-coupled neurotransmitter transporters. J Physiol 592, 863-869 (2014)). Recent structural insights into the molecular basis of B°AT1 inhibition (Xu, J. et al. Molecular basis of inhibition of the amino acid transporter B°AT1 (SLC6A19). Nat Commun 15, 7224 (2024)) suggest that antibody -mediated inhibition may complement or synergize with existing small-molecule inhibitors.

[0191] Beyond its functional inhibition, mAb#51 provides novel insights into B°AT1 regulation. Contrary to previous models suggesting that collectrin is essential for B°AT1 surface expression (Fairweather, S. J. et al. Molecular basis for the interaction of the mammalian amino acid transporters B°AT1 and B0AT3 with their ancillary protein collectrin. Journal of Biological Chemistry 290, 24308-24325 (2015)), the results demonstrate that B°AT1 can reach the plasma membrane independently of auxiliary proteins such as CLTRN or ACE2. This suggests the existence of tissuespecific regulatory mechanisms governing B°AT1 trafficking, similar to those observed for other SLC family transporters (Nishimura, M. & Naito, S. Tissue-specific mRNA expression profiles of human solute carrier transporter superfamilies. Drug metabolism and pharmacokinetics 23, 22-44 (2008)) and raises the possibility that alternative chaperones or post-translational modifications may compensate for the absence of known accessory proteins in specific cell types. Notably, mAb#51 binds B°AT1 even when ACE2 is present, suggesting that its epitope remains accessible despite ACE2’s bulky extracellular domain. This implies that the binding sites for ACE2 and mAb#51 are distinct, or that the dynamic B°AT1-ACE2 complex intermittently exposes the epitope, allowing mAb#51 to potentially modulate the complex and influence ACE2-related functions. The ACE2-B°AT1 complex, critical for amino acid uptake in the intestine and known for mediating SARS-CoV-2 entry (Yan, R. et al. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science 367, 1444-1448 (2020)), suggests that mAb#51 might also impact viral infection dynamics.

[0192] From a translational perspective, mAb#51 offers a highly specific approach to modulating B°AT1 function, providing a potential therapeutic avenue for amino acid dysregulation disorders, including Hartnup disease and metabolic syndromes. Additionally, the antibody's specificity could enable development of diagnostic tools for prenatal screening, particularly given B°ATl's critical role in early embryonic29 BW1384R development. However, key challenges remain in advancing this approach towards clinical application. First, cross-species reactivity must be carefully assessed, as murine models may not fully recapitulate human B°AT1 function, necessitating the development of ortholog-specific antibody variants. Second, the potential for off-target effects must be evaluated given the high sequence conservation among SLC transporters (Leabman, M. K. et al. Natural variation in human membrane transporter genes reveals evolutionary and functional constraints. Proceedings of the National Academy of Sciences 100, 5896-5901 (2003)). Given B°ATTs role in embryonic development and fertility, therapeutic applications must carefully consider developmental timing and potential impacts on reproductive outcomes. Comparative studies with small molecule inhibitors will be critical to assess the advantages of each approach, particularly regarding pharmacokinetics, pharmacodynamics, and delivery. Given B°ATl's role in amino acid transport, its inhibition could significantly impact amino acid-sensitive signaling pathways, particularly mTOR, which is implicated in metabolic diseases, aging, and cancer (Goberdhan, D. C. I. et al. Amino acid sensing and mTOR regulation: inside or out? Biochem Soc Trans 37, 248-252 (2009); Poncet, N. & Taylor, P. M. The role of amino acid transporters in nutrition. Curr Opin Clin Nutr Metab Care 16, 57-65 (2013); Ragni, M., Fornelli, C., Nisoli, E. & Penna, F. Amino Acids in Cancer and Cachexia: An Integrated View. Cancers (Basel) 14, 5691 (2022)).

[0193] To further advance the therapeutic potential of mAb#51, structural characterization of its binding epitope will be critical for guiding ortholog-specific variants design and improving specificity (Yu, G. et al. Single-step antibody -based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies. J Struct Biol 187, 1-9 (2014); Ewert, S., Honegger, A. & Pliickthun, A. Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based framework engineering. Methods 34, 184-199 (2004)). Additionally, proteomic analysis and CRISPR-based screens may identify compensatory pathways that regulate B°AT1 trafficking and activity, further elucidating its regulatory mechanisms (Okanishi, H., Ohgaki, R., Okuda, S., Endou, H. & Kanai, Y. Proteomics and phosphoproteomics reveal key regulators associated with cytostatic effect of amino acid transporter LAT1 inhibitor. Cancer Sci 112, 871-883 (2021)).

[0194] This study enhances the understanding of B°AT1 biology and highlights the potential of antibody-based approaches to target multi-pass membrane proteins. The development of mAb#51 not only provides a complementary tool to small-molecule30 BW1384R inhibitors but also establishes a framework for antibody discovery against challenging membrane proteins. These findings lay the groundwork for future therapeutic exploration of B°AT1 in metabolic disorders and contribute to the broader field of functional antibody discovery.

[0195] Materials and Methods

[0196] Cell Maintenance and Culture Conditions

[0197] Expi293F™ (Gibco, #A14527) and ExpiCHO-S™ (Gibco, #A29127) cells were maintained as suspension cultures in Expi293™ (Gibco, #A1435101) and ExpiCHO™ Expression Medium (Gibco, #A14527) at 37°C in a humidified atmosphere of 8% CO2 with continuous shaking at 125 rpm in 250 mL vented Erlenmeyer flasks (Thermo Scientific, #4115). Flp-In™ CHO cells (Invitrogen#R75807) were maintained in Ham's F12 Nutrient Mix (Gibco, #11765054) supplemented with 10% (v / v) heat-inactivated fetal bovine serum (FBS; Gibco, #10082147), 2 mM GlutaMAX™ (Thermo Scientific, #350500), and 1% (v / v) penicillin-streptomycin (P / S; Gibco, #15140122) in vented 100-mm tissue culture plates (Corning) at 37°C with 5% CO2.

[0198] The human chronic myeloid leukaemia cell line KYO-1 (DSMZ, #ACC601) was cultured in RPMI 1640 medium (Gibco, #A1049101) supplemented with 20% (v / v) heat-inactivated FBS at 37°C with 5% CO2. Cell viability was routinely assessed using trypan blue exclusion (Thermo Scientific). All cell lines were routinely tested for mycoplasma contamination by PCR-based screening.

[0199] Generation of Stable CHO Cell Lines Expressing B°AT1 and CLTRN

[0200] To generate a stable CHO cell line co-expressing B°AT1 and collectrin (CLTRN) (CHO-B+C), a codon-optimised human B°AT1 DNA (amino acids 1-634, GenBank: NC_000005.10) was synthesised (GeneArt Gene Synthesis, Invitrogen) and cloned into the pcDNA5 / FRT™ vector (Invitrogen, #V601020) under the cytomegalovirus (CMV) promoter. An internal ribosome entry site (IRES)-EGFP cassette was inserted downstream of B°AT1 to allow real-time expression monitoring and facilitate selection. A codon-optimised human CLTRN DNA (amino acids 1-222, GenBank: NC_000023.11) with an N-terminal c-Myc epitope tag was synthesised (GeneArt Gene Synthesis, Invitrogen) and cloned downstream to the mouse IgH leader sequence into pcDNA3.4 TOPO™ (Invitrogen).

[0201] Flp-In™ CHO cells (2*105) were co-transfected with pcDNA5_B°ATl-IRES-EGFP (2 pg) and pcDNA3.4_cMyc-CLTRN (1 pg) together with pOG44 Flp recombinase expression vector (Invitrogen, #V600520, 2 pg) using Lipofectamine™ 200031 BW1384R (Invitrogen, #11668, 5 pL per well) in 6-well plates (Coming). Selection was initiated 48 hours post-transfection in Ham's F12 medium supplemented with 400 pg / mL Hygromycin B (Gibco, #10687010) and 600 pg / mL Geneticin™ (Gibco, #101310). Stable expression of B°AT1 was confirmed by EGFP fluorescence using live cell imaging (EVOS M5000, Invitrogen). Expanded clones were further validated for B°AT1 and CLTRN expression by Western blot and flow cytometry. The CHO-B cell line was generated omitting the co-transfection with the pcDNA3.4_cMyc-CLTRN plasmid and relative Geneticin™ selection.

[0202] Recombinant Proteins Expression and Purification

[0203] The largest extracellular loop of B°AT1 (loop 4, amino acids 327413) was synthesised (GeneArt Gene Synthesis, Invitrogen) and cloned downstream to the mouse IgH leader sequence into pcDNA™3.4 TOPO™ (Invitrogen) with a C-terminal 6*His tag. Transient transfection was performed in ExpiCHO-S™ cells (Gibco, #A29127) (107cells / mL, 30 mL cultures) using ExpiFectamine™ CHO Transfection Kit (Gibco, #A29129) according to the manufacturer's protocol. Supernatants were harvested 8 days post-transfection and recombinant protein was purified by immobilised metal affinity chromatography (IMAC) on a HisTrap™ FF 1 mL column (Cytiva, 11000458). Protein purity was assessed by SDS-PAGE and concentrations were determined using the Pierce™ BCA Protein Assay Kit (Thermo Scientific, #23225).

[0204] Ethical Statement

[0205] Animal experiments were carried out in accordance with experimental protocols reviewed and approved (AWB-072014; OPBA-P-052020 TLS PILERI) by the Animal Ethical Committee of Toscana Life Sciences (Siena, Italy) and by the Italian Ministry of Health (434 / 2015-PR; 1073 / 2020-PR).

[0206] Recombinant Antibody Generation and Isolation

[0207] BALB / c mice (Charles River, 4 weeks old) were housed under specific pathogen-free (SPF) conditions with ad libitum access to food and water. Mice were immunised intraperitoneally (i.p.) with either CHO-B+C cells (1 x 107cells per injection in 100 pL 1X DPBS, (Gibco, #14190) or purified B°ATlLoop4-6His protein (20 pg per injection) mixed with complete Freund's adjuvant (Invitrogen). Boosts with incomplete Freund's adjuvant were administered every 14 days, with three injections for the recombinant protein group and five injections for the cell-based group. Serum samples were32 BW1384R collected 7 days after the last immunisation and antibody titres were determined by ELISA.

[0208] Following completion of the immunisation schedule, bone marrow was collected to isolate B°AT1 -specific CD138+ plasma cells, which were enriched using the CD138+ plasma cell isolation kit (Miltenyi Biotec, #130092530) according to the manufacturer's protocol. B°AT1 -specific single antibody-secreting cells were identified as previously described by Strazza et al. 13. Briefly, enriched CD 138+ plasma cells were resuspended in antibody expression medium containing RPMI1640 with 10% FBS, lxMEM Non-Essential Amino Acids Solution (Gibco, #11140050), lOng / mL recombinant mouse IL-6 (Abeam, #ab 198572), 25% filtered supernatant from day 3 M210B4 cell culture, and lOpM 2 -mercaptoethanol (Gibco, #21985023). Cells were seeded at a density of 50 cells per well in 25pL final volume using Coming™ Low Volume 384-well TC-treated Microplates (Corning, #3542) and incubated overnight at 37°C in a humidified 5% CO2 atmosphere. The overnight cell culture supernatants were screened by ELISA using SpectraPlate-384 High Binding Plates (PerkinElmer, #6007500) coated with B°ATlLoop#4-6His (10 pg / mL). Cells from ELISA-positive wells were subsequently plated at limiting dilution (0.3 cells / well) and re-screened. Antibody VH and VL sequences were obtained by RT-PCR and nested PCR according to the method of Strazza, V. et al. Rapid generation of human recombinant monoclonal antibodies from antibody-secreting cells using ferrofluid-based technology. Front Immunol 15, 1341389 (2024) using primers adapted from Tiller, T., Busse, C. E. & Wardemann, H. Cloning and expression of murine Ig genes from single B cells. J Immunol Methods 350, 183-193 (2009).

[0209] The PCR products were used to generate transcriptionally active PCR (TAP) minigenes encoding human IgGl heavy chain and kappa light chain constant regions. These minigenes were transiently transfected into Expi293F™ cells using ExpiFectamine™ 293 (Gibco, #A14525). For each transfection, using 400ng of DNA (30% heavy chain-encoding minigenes and 70% light chain-encoding minigenes) was used to transfect 1x106 viable cells in a Deepwell Plate 96 / 2mL (Eppendorf, #0030501330). Cells were cultured at 37°C with 8% CO2 and shaken at l,000rpm using a MixMateTM Shaker (Eppendorf, #5353000510). Supernatants containing the expressed antibodies were collected 72 h post-transfection and evaluated for binding by ELISA and flow cytometry. This procedure generated chimeric antibodies comprising mouse-derived variable regions and human-derived constant regions.

[0210] ELISA33 BW1384R SpectraPlate-384 High Binding Plates (PerkinElmer, #6007500) were coated with 20 pL / well of purified B°ATl-Loop#4-6His fragment at lOpg / mL in lx DPBS per well and incubated overnight at 4°C. Plates were sealed with adhesive film during all incubation steps. The plates were washed three times with 100 pL / well of wash buffer (0.05% Tween 20 in 1 x DPBS) at room temperature. Blocking was performed with 30 pL / well of blocking buffer (lx DPBS containing 1% ([Fisher Scientific, #BP9702] and 1% FBS) for 1 h at 37°C. After washing as described above, 30 pL / well of serial 3-fold dilutions (1:300 to 1:72,900) of pre-immune and immune sera were applied in blocking buffer and incubated for 1 h at 37°C. Each sample was tested in technical triplicates. The plates were washed five times with wash buffer, followed by incubation with 30 pL / well of HRP-conjugated goat anti-mouse IgG (H+L) antibody (Bio-Rad #170-6516; 1:5,000 in blocking buffer) for 1 h at 37°C. After six washes, 30 pL / well of 1-Step™ Ultra TMB ELISA Substrate Solution (Thermo Scientific, #34028) was added and developed for 30 min at room temperature protected from light. The reaction was stopped with 30 pL / well of 0.5 M HC1. Absorbance was measured at 450 nm using a Spectramax M2 microplate reader (Molecular Devices). Each plate included blank wells (no primary antibody) and positive control sera. The threshold for positive sample identification was set at twice the mean blank optical density (OD)

[0211] Western Blot Analysis

[0212] Viable cells (1 x 106) were harvested and lysed in 600 pL RIPA lysis and extraction buffer (Thermo Scientific, #89900) supplemented with protease and phosphatase inhibitors (Roche). Total protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Scientific). Equal amounts of protein (20 pg per lane), together with a SeeBlue™ Plus2 pre-stained protein standard (Invitrogen, #LC5925) for molecular weight reference, were separated by SDS-PAGE on NuPAGE™ 4-12% Bis-Tris gels (Invitrogen, #NP0322) at 200 V for 45 min and transferred to PVDF membranes (Invitrogen, #IB4010) using the iBlot™ 2 Dry Blotting System (Invitrogen, #IB21001). Membranes were blocked in 5% non-fat milk in TBST (50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20, pH 7.6) for 1 h at room temperature and then incubated with primary antibodies at 4°C overnight. After washing, the membranes were incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. Protein detection was performed using Pierce™ ECL Western Blotting Substrate (Thermo Scientific, #32209) and chemiluminescence signals were captured using the ImageQuant LAS4000 system (Cytiva).34 BW1384R

[0213] Flow Cytometric Analysis (FACS)

[0214] Cells were harvested, washed with Dulbecco's phosphate buffered saline (DPBS; Gibco) supplemented with 5% FBS and resuspended in staining buffer (PBS + 2% FBS + 2 mM EDTA). Surface staining was performed by incubating 5 x 105cells per tube with primary antibodies (e.g. mAb#51, anti-cMyc or anti-ACE-2) at the recommended dilution for 30 min on ice. After washing, cells were incubated with fluorophore-conjugated secondary antibodies (Alexa Fluor™ 647, DyLight™ 650 or FITC-conjugated IgG) for 30 min on ice in the dark. After final washes, samples were resuspended in 200 pL staining buffer and acquired using a FACSCanto™ II flow cytometer (BD Biosciences) with FACSDiva™ software. Data were analysed using FlowJo vlO (BD Biosciences).

[0215] Production of Recombinant Antibodies

[0216] Antibody VH and VL sequences were obtained from single antigen-specific plasma cells by RT-PCR and nested PCR, following an adapted protocol from Tiller et al. (2009). PCR products were cloned into plasmids encoding human IgGl or kappa constant regions for mammalian expression using standard restriction enzyme digestion, ligation, and cloning techniques. Plasmids encoding the heavy (VH) and light (VL) chains were transiently transfected into Expi293F™ cells using the ExpiFectamine™ 293 Transfection Kit (Gibco, #A14525). Cultures were maintained in shaking suspension at 37°C in 8% CO2 for 72 hours, after which supernatants were harvested by centrifugation at 300 x g for 10 minutes. Recombinant antibodies were purified by Protein G affinity chromatography (HiTrap™ Protein G HP 1 mL, Cytiva, #17040401) followed by buffer exchange to DPBS using PD-10 desalting columns (Cytiva, # 17085101). Antibody purity was verified by SDS-PAGE and Coomassie staining, and concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Scientific, #23225).

[0217] CRISPR / Cas9-Mediated Knockout of SLC6A19 in KYO-1 Cells

[0218] A knockout (KO) KYO-1 cell line for the SLC6A19 (B°AT1) gene was generated using CRISPR / Cas9 ribonucleoprotein (RNP) transfection. Guide RNAs targeting exon 4 of SLC6A19 (chr5: 1212418-1212599) were designed using CRISPOR (www.crispor.tefor.net) 37 and selected based on specificity and predicted efficiency scores based on published algorithms 16,38. The sgRNA sequences were: sgRNAl (SEQ ID NO: 15): 5’-TCCCGGTGGTCTCGATGCCG-3’ (PAM: CGG) and35 BW1384R sgRNA2(SEQ ID NO: 16): 5’-CGGTACCAGAAGTAGTCCAC-3’ (PAM: AGG). RNP complexes were assembled by incubating 150 pmol total sgRNA with 75 pmol Alt-R™ S.p. Cas9 Nuclease V3 (IDT) and 150 pg polyglutamic acid (PGA), (Sigma-Aldrich) for 15 min at room temperature. KYO-1 cells (1 x 106) were washed twice with Opti-MEM™ I Reduced Serum Medium (Gibco) and resuspended in 100 pL electroporation buffer. RNP complexes were added to the cell suspension and electroporation was performed using a Super Electroporator NEPA21 (Nepa Gene) with a poring pulse of 175 V, 2.5 ms and transfer pulses of 20 V, 50 ms (5 pulses, 40% decay, bipolar polarity). Immediately after electroporation, cells were transferred to pre-warmed RPMI 1640 medium supplemented with 20% FBS and incubated at 37°C with 5% CO2. Gene editing efficiency was assessed 96 hours post-electroporation by flow cytometry using mAb#51, with wild-type KYO-1 cells as a positive control. Cells were stained with goat anti-human IgG Fc cross-adsorbed DyLight™ 650 conjugated secondary antibody (Invitrogen, 1:200 dilution) and data were acquired using a FACSCanto™ II flow cytometer (BD Biosciences).

[0219] FLIPR Membrane Potential Assay for B°AT1 Transport Activity

[0220] B°AT1 transport activity was assessed using the FLIPR Membrane Potential Assay Kit (Red, Explorer) (Molecular Devices). CHO-B+C or CHO-B cells were seeded at l*104cells per well in 384-well TC-treated microplates and incubated at 37°C, 5% CO2 for 24 hours. Cells were then washed with HBSS (Gibco) + 20 mM HEPES (Gibco) and incubated with serial dilutions of monoclonal antibodies (200 nM-6.7 pM) or nimesulide (500 pM-160 nM) for 1 hour. Control wells were treated with vehicle alone. Twenty-five pL of the fluorescent probe mixture (component A reconstituted in component B: 20 mM HEPES, lx HBSS, pH 7.4) was then added, followed by incubation at 37°C for 60 minutes. Baseline fluorescence was recorded using a SpectraMax™ M2 microplate reader (excitation: 545 nm, emission: 625 nm). Transport was initiated by the addition of 15 mM isoleucine (Sigma- Aldrich) and fluorescence changes were measured every 10 seconds for 5 minutes. Data were analysed using SoftMax Pro software, with transport activity quantified as the relative fluorescence change (AF / Fo). Info on the calculations should be added.

[0221] Statistical analysis

[0222] If not stated otherwise, all experiments were performed at least three times independently. Statistical analyses were performed using Prism 8 Software.36 BW1384R

[0223] Additional monoclonal antibodies against B°AT1.

[0224] Similarly, additional monoclonal antibodies specifically binding B°AT1 were generated: mAb#A01, mAb#A03, and mAb#D03. Specifically, also the additional monoclonal antibodies generated were capable of binding B°AT1 on the cell surface. mAb#A01 represents a different clonotype with respect to mAb#51, although maintaining some structural similarity. Experimental data indicated that also mAb#A01 exerts an inhibitory action on the transport of neutral amino acids, analogously to what was observed for mAb#51.

[0225] mAb#A01 was obtained from a different mouse by a separate immunization, which led to the development of a different clonotype, although with some affinities with the previous one.

[0226] mAb#A01 efficiently recognized the native form of the target on cell surface and showed high reactivity when tested by ELISA, even increased with respect to mAb#51. Thus, mAb#A01 showed similar results to those obtained with mAb#51 with respect to the inhibition of amino acid transport and cell surface binding. mAb#A03 and mAb#D03 also recognized the target both when tested by ELISA and on the cell surface. Figure 9 shows the binding of mAb#A01, mAb#A03, and mAb#D03 on the cell surface.37 BW1384R DETAILED DESCRIPTION OF THE SEQUENCES

[0227] > SEQ ID NO: 1 B°AT1 human protein (Uniprot #Q695T7) MVRLVLPNPGLDARIPSLAELETIEQEEASSRPKWDNKAQYMLTCLGFCVGLGNVWRFP YLCQSHGGGAFMIPFLILLVLEGIPLLYLEFAIGQRLRRGSLGVWSSIHPALKGLGLAS MLTSFMVGLYYNTI ISWIMWYLFNSFQEPLPWSDCPLNENQTGYVDECARSSPVDYFWY RETLNISTSISDSGSIQWWMLLCLACAWSVLYMCTIRGIETTGKAVYITSTLPYWLTI FLIRGLTLKGATNGIVFLFTPNVTELAQPDTWLDAGAQVFFSFSLAFGGLISFSSYNSV HNNCEKDSVIVS I INGFTSVYVAIWYSVIGFRATQRYDDCFSTNILTLINGFDLPEGN VTQENFVDMQQRCNASDPAAYAQLVFQTCDINAFLSEAVEGTGLAFIVFTEAITKMPLS PLWSVLFFIMLFCLGLSSMFGNMEGVWPLQDLRVIPPKWPKEVLTGLICLGTFLIGFI FTLNSGQYWLSLLDSYAGSIPLLIIAFCEMFSWYVYGVDRFNKDIEFMIGHKPNIFWQ VTWRWSPLLMLIIFLFFFWEVSQELTYSIWDPGYEEFPKSQKISYPNWVYWWIVA GVPSLTIPGYAIYKLIRNHCQKPGDHQGLVSTLSTASMNGDLKY

[0228] > SEQ ID NO: 2 Loop 4 of B°AT1 human protein FRATQRYDDCFSTNILTLINGFDLPEGNVTQENFVDMQQRCNASDPAAYAQLVFQTCDI NAFLSEAVEGTGLAFIVFTEAITKMPLS

[0229] > SEQ ID NO: 3 CDR 1 of variable domain of Heavy chain of mAb#51 GFTFNTYA

[0230] > SEQ ID NO: 4 CDR 2 of variable domain of Heavy chain of mAb#51 IRRKSDNSVT

[0231] > SEQ ID NO: 5 CDR 3 of variable domain of Heavy chain of mAb#51 VRQLRLHALDY

[0232] > SEQ ID NO: 6 CDR 1 of variable domain of Light chain of mAb#51 QSVSSSGYSY

[0233] > SEQ ID NO: 7 CDR 2 of variable domain of Light chain of mAb#51

[0234] YAS

[0235] > SEQ ID NO: 8 CDR 3 of variable domain of Light chain of mAb#51 QHSWEIPLT

[0236] > SEQ ID NO: 9 Heavy chain variable domain (VH) of mAb#51

[0237] EVQLQESGGRLVQPKGSMKLSCVASGFTFNTYAMNWVRQAPGKGLEWVARIRRKSDNSV38 BW1384R TNYADSVKDRFTISRDDSESMLYLQMNNMKTEDTAMYFCVRQLRLHALDYWGQGTSVTV SS

[0238] > SEQ ID NO: 10 Light chain variable domain (VL) of mAb#51 DIQMTQSPASLTVFLGQRATISCRASQSVSSSGYSYLHWYQQKPGQPPKLLIKYASNLE SGVPARFSGGGSGTDFTLNIHPVEEEDTATYYCQHSWEIPLTFGAGTKL

[0239] > SEQ ID NO: 11 Signal peptide VH of mAb#51, mAb#A01, mAb#A03, mAb#D03 MGWSCI ILFLVATATGVHS

[0240] > SEQ ID NO: 12 Signal peptide VL ofmAb#51, mAb#A01, mAb#A03, mAb#D03 MGWSCI ILFLVATATGVHS

[0241] > SEQ ID NO: 13 Human constant region IgGl HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0242] > SEQ ID NO: 14 Human constant region k LC RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0243] > SEQ ID NO: 15 sgRNAl

[0244] TCCCGGTGGTCTCGATGCCG

[0245] > SEQ ID NO: 16 sgRNA2

[0246] CGGTACCAGAAGTAGTCCAC

[0247] >SEQ ID NO: 3 CDR 1 of variable domain of Heavy chain of mAb#A01 GFTFNTYA

[0248] > SEQ ID NO: 17 CDR 2 of variable domain of Heavy chain of m Ab# API39 BW1384R ITRKSNNYAT

[0249] SEQ ID NO: 18 CDR 3 of variable domain of Heavy chain of m Ab# API VRHERSNYFAY

[0250] > SEQ ID NO: 19 CDR 1 of variable domain of Light chain of mAb#A01 QSVSTSSYNY

[0251] > SEQ ID NO: 7 CDR 2 of variable domain of Light chain of mAb#A01

[0252] YAS

[0253] > SEQ ID NO: 20 CDR 3 of variable domain of Light chain of mAb#A01 QHSWEIPYT

[0254] SEQ ID NO: 21 Heavy chain variable domain (VH) of mAb#A01

[0255]

[0256] > SEQ ID NO 22 Light chain variable domain (VL) of mAb#A01

[0257]

[0258] >SEQ ID NO: 23 CDR 1 of variable domain of Heavy chain of mAb#A03

[0259] SEQ ID NO: 24 CDR 2 of variable domain of Heavy chain of mAb#A03

[0260] SEQ ID NO: 25 CDR 3 of variable domain of Heavy chain of mAb#A0340 BW1384R > SEQ ID NO: 26 CDR 1 of variable domain of Light chain of mAb#A03

[0261] > SEQ ID NO: 27 CDR 2 of variable domain of Light chain of mAb#A03

[0262] > SEQ ID NO: 28 CDR 3 of variable domain of Light chain of mAb#A03

[0263] > SEQ ID NO: 29 Heavy chain variable domain (VH) of mAb#A03

[0264]

[0265] > SEQ ID NO: 30 Light chain variable domain (VL) of mAb#A03

[0266]

[0267] >SEQ ID NO: 31 CDR 1 of variable domain of Heavy chain of mAb#D03 GFTFSDYG

[0268] SEQ ID NO: 32 CDR 2 of variable domain of Heavy chain of mAb#D03 INSGSVTM

[0269] SEQ ID NO: 33 CDR 3 of variable domain of Heavy chain of mAb#D03 AISLVRYWYFDV

[0270] > SEQ ID NO: 34 CDR 1 of variable domain of Light chain of mAb#D03 QDVRTA

[0271] > SEQ ID NO: 35 CDR 2 of variable domain of Light chain of mAb#D03

[0272] SAS41 BW1384R

[0273] > SEQ ID NO: 36 CDR 3 of variable domain of Light chain of mAb#D03 QQHYSTPLT

[0274] > SEQ ID NO: 37 Heavy chain variable domain (VH) of mAb#D03

[0275]

[0276] > SEQ ID NO: 38 Light chain variable domain (VL) of mAb#D03

[0277]

Claims

42 BW1384R CLAIMS1. A monoclonal antibody or antigen-binding portion thereof that specifically binds to an extracellular epitope of the B°AT1 protein.

2. The monoclonal antibody or antigen-binding portion thereof according to claim 1, wherein said extracellular epitope comprises or consists of one or more amino acids in the positions 327 to 413 in the loop 4 of the B°AT1 protein having SEQ ID NO:1, preferably said amino acids in the positions 327 to 413 having SEQ ID NO:2.

3. The monoclonal antibody or an antigen-binding portion thereof according to any one of the claims from 1 to 2, wherein said monoclonal antibody or antigen-binding portion thereof is selected from a chimeric monoclonal antibody, bispecific antibody, diabody, nanobody, Fab(2), Fab, scFV, minibody, or any polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding to the target polypeptide.

4. The monoclonal antibody or antigen-binding portion thereof according to any one of claims from 1 to 3, wherein said antibody or antigen-binding portion thereof inhibits the neutral amino acid transport mediated by the B°AT1 protein.

5. The monoclonal antibody or an antigen-binding portion thereof according to any one of the claims from 1 to 4, wherein said B°AT1 protein is the human B°AT1 protein and / or the murine B°AT1 protein.

6. The monoclonal antibody or an antigen-binding portion thereof according to any one of the claims from 1 to 5, wherein said antibody or antigen-binding portion thereof shows a 50% inhibitory concentration (IC50) of less than 10 nM, preferably less than 1 nM, in particular when tested in a surface binding assay carried out through flow cytometry, in particular through a fluorescence-based FLIPR membrane potential assay.

7. The monoclonal antibody or antigen-binding portion thereof according to any one of the43 BW1384R claims from 1 to 6, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein said VH and VL comprise the following complementaritydetermining regions (CDRs):- CDR1 of VH having SEQ ID NO: 3,-CDR2 of VH having SEQ ID NO: 4,-CDR3 of VH having SEQ ID NO: 5,-CDR1 of VL having SEQ ID NO: 6,-CDR2 of VL having the sequence YAS (tyr-ala-ser),-CDR3 of VL having SEQ ID NO: 8.

8. The monoclonal antibody or antigen-binding portion thereof according to any one of the claims from 1 to 7, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein said VH has SEQ ID NO: 9 and said VL has SEQ ID NO: 10, or wherein said VL and said VH is at least 85%, preferably at least 95%, more preferably at least 99% identical in amino acid sequence to a VH having SEQ ID NO: 9 and a VL having SEQ ID NO: 10.

9. The monoclonal antibody or antigen-binding portion thereof according to any one of the claims from 1 to 6, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein said VH and VL comprise the following complementaritydetermining regions (CDRs):- CDR1 of VH having SEQ ID NO: 3,-CDR2 of VH having SEQ ID NO: 17,-CDR3 of VH having SEQ ID NO: 18,-CDR1 of VL having SEQ ID NO: 19,-CDR2 of VL having the sequence YAS (tyr-ala-ser),-CDR3 of VL having SEQ ID NO: 20.

10. The monoclonal antibody or antigen-binding portion thereof according to any one of the claims from 1 to 6, or 9, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein said VH has SEQ ID NO: 21 and said VL has SEQ ID NO: 22, or wherein said VL and said VH is at least 85%, preferably at least 95%, more preferably44 BW1384R at least 99% identical in amino acid sequence to a VH having SEQ ID NO: 21 and a VL having SEQ ID NO: 22.

11. The monoclonal antibody or antigen-binding portion thereof according to any one of the claims from 1 to 6, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein said VH and VL comprise the following complementarity - determining regions (CDRs):- CDR1 of VH having SEQ ID NO: 23,-CDR2 of VH having SEQ ID NO: 24,-CDR3 of VH having SEQ ID NO: 25,-CDR1 of VL having SEQ ID NO: 26,-CDR2 of VL having the sequence RTS (arg-thr-ser),-CDR3 of VL having SEQ ID NO: 28.

12. The monoclonal antibody or antigen-binding portion thereof according to any one of the claims from 1 to 6, or 11, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein said VH has SEQ ID NO: 29 and said VL has SEQ ID NO: 30, or wherein said VL and said VH is at least 85%, preferably at least 95%, more preferably at least 99% identical in amino acid sequence to a VH having SEQ ID NO: 29 and a VL having SEQ ID NO: 30.

13. The monoclonal antibody or antigen-binding portion thereof according to any one of the claims from 1 to 6, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein said VH and VL comprise the following complementarity - determining regions (CDRs):- CDR1 of VH having SEQ ID NO: 31,-CDR2 of VH having SEQ ID NO: 32,-CDR3 of VH having SEQ ID NO: 33,-CDR1 of VL having SEQ ID NO: 34,45 BW1384R 14. The monoclonal antibody or antigen-binding portion thereof according to any one of the claims from 1 to 6, or 13, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein said VH has SEQ ID NO: 37 and said VL has SEQ ID NO: 38, or wherein said VL and said VH is at least 85%, preferably at least 95%, more preferably at least 99% identical in amino acid sequence to a VH having SEQ ID NO: 37 and a VL having SEQ ID NO: 38.

15. The monoclonal antibody or antigen-binding portion thereof according to any one of the claims from 1 to 14, for use as a medicament.

16. The monoclonal antibody or antigen-binding portion thereof according to any one of the claims from 1 to 15, for use in the treatment of disorders involving amino acid dysregulation, Hartnup disease, metabolic syndrome and cancer, preferably, wherein said amino acid dysregulation is selected from phenylketonuria (PKU), insulin resistance and type 1 and type 2 diabetes (T1D and T2D).

17. A pharmaceutical composition comprising the monoclonal antibody or antigen-binding portion thereof according to any one of the claims from 1 to 16 and a pharmaceutically acceptable carrier.

18. An in-vitro method for screening a monoclonal antibody or antigen-binding portion thereof that specifically binds to an extracellular epitope of the B°AT1 protein comprising the following steps:a) isolating BOAT 1 -specific plasma cells from bone marrow samples collected from two groups of animals wherein group (1) is immunized with a first antigen wherein said first antigen is cells co-expressing B°AT1 protein and collectrin protein (CLTRN) and / or group (2) is immunized with a second antigen wherein said second antigen is a recombinant protein comprising residues from 327 to 413 of loop 4 of B°AT1 protein b) performing a screening to select the B°AT1 -specific antibody-secreting cells that bind the first antigen and / or the second antigen19. An in-vitro method for detecting B°AT1 protein in a biological sample comprising46 BW1384R the following steps:a) incubating the biological sample with an anti-B°ATl protein antibody of the inventionb) detecting the bound antibody.

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