WO2025062271A1 - Antibodies against endometriosis - Google Patents

Abstract

The present invention relates to monoclonal antibodies or antigen-binding portion thereof that specifically binds the hNTRK2 receptor. The invention relates also to the use of such monoclonal antibodies or antigen-binding portion thereof in therapy and prophylaxis of endometriosis or related conditions.

Description

ANTIBODIES AGAINST ENDOMETRIOSIS DESCRIPTION

Technical field of the invention

The present invention relates to monoclonal antibodies or antigen-binding portion thereof that specifically binds the human NTRK2 receptor. The invention relates also to the use of such monoclonal antibodies or antigen-binding portion thereof in therapy, and prophylaxis of endometriosis or related conditions.

State of the art

Human monoclonal antibodies (mAbs) are an industrially mature technology with more than 50 products already approved in the field of cancer, inflammation and autoimmunity. The well-established safety profile and the large experience for their development make mAbs ideal candidates for rapid development especially in epidemic and pandemic settings. So far mAbs have rarely been used in the field of infectious diseases, mostly because the large quantities needed for therapy made them not cost effective. However, in recent years, the incredible technological progress in isolating and screening memory B cells allowed identification of highly potent neutralizing mAbs and further improvement of their potency by several orders of magnitude through established engineering procedures. This possibility resulted in a decreased quantity of antibodies necessary for therapy thus making non- intravenous delivery of potent neutralizing mAbs possible.

Among the many therapeutic options available, mAbs offer a series of advantages. First, they are the ones that can be developed in the shortest period of time. In fact, the extensive clinical experience with the safety of more than 50 commercial mAbs approved to treat cancer, inflammation and autoimmunity provides high confidence on their safety, support the possibility of having an accelerated regulatory pathway. In addition, the long industrial experience in developing and manufacturing mAbs decreases the risks usually associated with technical development of investigational products. Finally, the incredible technical progress in the field allows to shorten the conventional timelines and go from discovery to proof of concept trials in 5-6 months.

Endometriosis, a prevalent and debilitating gynecological disorder, is characterized by the presence of endometrial -like tissue outside the uterine cavity. This ectopic tissue responds to hormonal fluctuations, leading to inflammation, pain, and fertility challenges. Despite extensive research, the underlying mechanisms driving endometriosis pathophysiology remain incompletely understood, hampering the development of effective diagnostic tools and treatments.

Existing literature has revealed the significant regulatory influence of estrogen on BDNF expression within the uterus. Estradiol, a key estrogen hormone, has been shown to modulate BDNF levels across the menstrual cycle and in response to hormone replacement therapies. Studies have demonstrated that estrogen exposure leads to the upregulation of BDNF and its receptors, particularly the low affinity receptor NGFR. Furthermore, a direct correlation between circulating estrogen levels and BDNF concentrations has been observed, shedding light on the intricate communication between hormonal fluctuations and neurotrophin signaling. The interplay between BDNF and estrogen appears to play a pivotal role in uterine physiology, with the potential to influence processes such as cellular proliferation, angiogenesis, and apoptosis.

Of particular significance is the compelling association between BDNF dysregulation and endometriosis. Recent proteomics studies have highlighted elevated BDNF expression in the eutopic endometrium of women with endometriosis, indicating a potential role of BDNF in the pathogenesis of the disorder. Additionally, altered plasma BDNF levels in women with endometriosis suggest the feasibility of BDNF as a non-invasive biomarker for disease detection and therapeutic response assessment.

Beyond its role in endometrial tissue, BDNF appears to exert influence on nerve fibers associated with endometriotic lesions, which are implicated in the excruciating pain experienced by affected individuals. The proposed mechanism involves the interaction between BDNF and its receptor NTRK2, which enhances receptor affinity for BDNF. This interaction is suggested to contribute to sensory innervation of endometriotic lesions, exacerbating the pain associated with the disorder.

NTRK2, also known as neurotrophic receptor tyrosine kinase 2 or TrkB, is a receptor protein that plays a crucial role in the nervous system by binding to the brain-derived neurotrophic factor (BDNF) and other neurotrophins. Neurotrophins are a family of proteins that support the growth, survival, and function of nerve cells (neurons). The NTRK2 receptor is a key mediator of the effects of BDNF and other neurotrophins on neurons.

NTRK2 is a member of the tyrosine kinase receptor family, which means that it activates intracellular signaling pathways by adding phosphate groups to specific tyrosine residues on proteins. When BDNF or other neurotrophins bind to the NTRK2 receptor, it triggers a signaling cascade that can have various effects on neuronal development, function, and plasticity.

The NTRK2 receptor has been extensively studied in the context of the central nervous system, where it is involved in processes such as neuronal survival, differentiation, synaptic plasticity, and learning and memory. It has also been implicated in various neurological and psychiatric disorders.

In the context of endometriosis, recent research has suggested that NTRK2 might play a role in the pathophysiology of the disorder, potentially influencing cellular processes such as adhesion, proliferation, apoptosis resistance, angiogenesis, and pain perception within endometriotic lesions.

Nevertheless, it is important to produce specifically binding mAbs or antigen-binding portion thereof in order to inhibit the activity of the neurotrophic receptor tyrosine kinase 2 (NTRK2). Accordingly, there is a need for potent, broad spectrum antibody therapeutics specifically binding the NTRK2 receptor, for use in therapy and prophylaxis of endometriosis or related conditions.

Summary of the Invention

In certain aspects, the invention provides a monoclonal antibody or antigen-binding portion thereof capable of specifically binding the human NTRK2 receptor (hNTRK2).

In particular, such monoclonal antibody or antigen-binding portion thereof binds to the extracellular domain of the hNTRK2 receptor having SEQ ID NO. 51, and shows an equilibrium dissociation constant KD of less than 1 * 10'⁸ M, preferably of less than 8* 10'⁹ M, when tested in a BIACORE™ test against the hNTRK2 receptor.

In certain aspects, the invention provides a monoclonal antibody or antigen-binding portion thereof that specifically comprises the VL and VH domains that are at least 85%, 90%, 95%, 97%, 98% or 99% identical in amino acid sequence to the VL and VH domains, respectively, of a monoclonal antibody selected from the group consisting of R3P1-F1, R3P3-A11, R3P3- H4, R3P3-E1, R3P3-G5, R3P1-A1, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1- A6 and R4Pl-Fl. According to the present invention, each antibody corresponds to the following combination of VH and VL domains:

R3P1-F1 : VH having SED ID NO: 1 and VL having SED ID NO:22;

R3P3-A11 : VH having SED ID NO:2 and VL having SED ID NO:23;

R3P3-H4: VH having SED ID NO:3 and VL having SED ID NO:24;

R3P3-E1 : VH having SED ID NO:4 and VL having SED ID NO:25;

R3P3-G5: VH having SED ID NO:5 and VL having SED ID NO:26;

R3P1-A1 : VH having SED ID NO:6 and VL having SED ID NO:27;

R3P1-A4: VH having SED ID NO:7 and VL having SED ID NO:28;

R3P1-A10: VH having SED ID NO:8 and VL having SED ID NO:29;

R3P1-G12: VH having SED ID NO: 9 and VL having SED ID NO: 30;

R3P2-A1 : VH having SED ID NO: 10 and VL having SED ID NO:31;

R3P2-B 12: VH having SED ID NO: 11 and VL having SED ID NO:32;

R3P2-C3: VH having SED ID NO: 12 and VL having SED ID NO:33;

R3P3-E2: VH having SED ID NO: 13 and VL having SED ID NO:34;

R3P3-H3: VH having SED ID NO: 14 and VL having SED ID NO:35;

R3P1-A11 : VH having SED ID NO: 15 and VL having SED ID NO:36;

R3P1-G8: VH having SED ID NO: 16 and VL having SED ID NO: 37;

R3P1-H8: VH having SED ID NO: 17 and VL having SED ID NO:38;

R3P2-A5: VH having SED ID NO: 18 and VL having SED ID NO: 39;

R3P2-H6: VH having SED ID NO: 19 and VL having SED ID NO:40;

R4P1-A6: VH having SED ID NO:20 and VL having SED ID NO:41;

R4P1-F1 : VH having SED ID NO:21 and VL having SED ID NO:42

In certain aspects, the invention provides monoclonal antibody or an antigen-binding portion according to any embodiments herein disclosed, for use in a prophylactic or therapeutic treatment of endometriosis or related conditions.

The invention further provides monoclonal antibody or an antigen-binding portion according to any embodiments herein disclosed for use in the prophylaxis and/or treatment of a subject having, or at risk of developing, endometriosis or related conditions.

In certain aspects, the invention provides a pharmaceutical composition comprising at least one or more monoclonal antibodies or antigen-binding portions thereof according to any one of the embodiments herein disclosed and a pharmaceutically acceptable carrier and its use in the prevention and/or treatment of endometriosis or related conditions.

In certain aspects, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes the antibody or antigen-binding portion thereof according to any one of the embodiments herein disclosed.

In certain aspects, the invention provides a vector comprising the nucleic acid molecule encoding the 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 certain aspects, the invention provides a host cell modified to over-express the antibody or antigen-binding portion thereof embodiments according to any one of the embodiments herein disclosed.

In certain aspects, the invention provides an in vitro method for revealing the presence of hNTRK2 receptor comprising the following steps: i) Contacting the antibody or an antigen-binding portion thereof according to any one of the embodiments herein disclosed; ii) Detecting the binding of said antibody or an antigen-binding portion thereof to the hNTRK2 receptor.

The invention contemplates combinations of any of the foregoing aspects and embodiments of the invention.

Brief description of the drawings

Fig. 1 Purification profiles of the recombinant antibodies. Non-reduced PAGE with Coomassie blue staining. MW. Molecular weight marker (kDa). IN. Input. FT. Flowthrough.

E. Eluted fractions.

Fig. 2 Final sample QC. Coomassie blue staining. Reduced and Non-Reduced PAGE analysis. MW. Molecular weight marker (kDa). 2pg loaded per lane.

Fig. 3 Recombinant antibody titer against Human NTRK2 & BSA negative control.

5pg/mL of antigen & BSA was coated. BSA used as blocking buffer used.

Fig. 4 Schematic of a typical sensorgram. Bars below the curve indicate the solutions that pass over the sensor surface in one concentration test

Fig. 5 *hNTRK2 & R3P1-F1 Kinetics analysis of antibody/antigen interaction.

Fig. 6 *hNTRK2 & R3P3-A11 Kinetics analysis of antibody/antigen interaction. Fig. 7 *hNTRK2 & R3P3-H4 Kinetics analysis of antibody/antigen interaction.

Fig. 8 *hNTRK2 & R3P3-E1 Kinetics analysis of antibody/antigen interaction. Fig. 9 *hNTRK2 & R3P3-G5 Kinetics analysis of antibody/antigen interaction.

Fig. 10 Final SDS-PAGE QC of hNTRK2. Reduced SDS-PAGE analysis of pooled elution fractions. 2pg of sample loaded per lane. MW. Molecular weight marker.

Figure 11 Final SDS-PAGE QC of hBDNF. Reduced SDS-PAGE analysis of pooled elution fractions. 2pg of sample loaded per lane. MW. Molecular weight marker.

Figure 12 Epitope mapping in the NTRK2 structure.

Detailed description of the invention

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

The following terms, unless otherwise indicated, shall be understood to have the following meanings:

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 the 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. A protein or polypeptide is "substantially pure", "substantially homogeneous", or "substantially purified" when at least about 60 to 75% of a sample exhibits a single polypeptide. The polypeptide or protein may be monomeric or multimeric. A substantially pure polypeptide or protein will typically comprise about 50%, 60%, 70%, 80% or 90% W/W of a protein sample, more usually about 95%, and preferably will be over 99% pure. Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel with a stain well known in the art. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art for purification. 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 hNTRK2 receptor under suitable binding conditions, (2) ability to inhibit said at hNTRK2 receptor. 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. In certain embodiments, amino acid substitutions to an anti-hNTRK2 antibody or antigen-binding portion thereof are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity to form protein complexes, and (4) confer or modify other physicochemical or functional properties of such analogues, but still retain specific binding to at hNTRK2 receptor. 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; e.g., a replacement amino acid should not alter the anti-parallel [beta]-sheet that makes up the immunoglobulin binding domain that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence. In general, glycine and proline would not be used in an anti-parallel [beta]-sheet. Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et ai, Nature 354: 105 (1991), incorporated herein by reference.

Where an "antibody" is referred to herein with respect to the invention, it is normally understood that an anti gen -binding portion thereof may also be used. An antigen -binding 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 chemical 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. 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.

As used herein, a Fd fragment means an antibody fragment that consists of the VH and CH 1 domains; an Fv fragment consists of the VL and VH domains of a single arm of an antibody; and a dAb fragment (Ward et al, Nature 341 :544-546 (1989)) consists of a VH domain. 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. In embodiments having one or more binding sites, the binding sites may be identical to one another or may be different.

In certain preferred aspects of the invention, a monoclonal antibody according to any of the embodiments disclosed in the present specification and in the claims is a human antibody, i.e., a human monoclonal antibody, or an antigen -binding portion thereof.

As used herein, the term "human antibody" means any antibody in which the variable and constant domain sequences are human sequences or any of the CDRs of the variable domain sequences are human sequences. The term encompasses antibodies with sequences derived from human genes, but which have been changed, e.g. to decrease possible immunogenicity, increase affinity, eliminate cysteines that might cause undesirable folding, etc. The term encompasses such antibodies produced recombinantly in non-human cells, which might impart glycosylation not typical of human cells. The term "chimeric antibody" as used herein means an antibody that comprises regions from two or more different antibodies.

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 antibody is said to specifically bind an antigen when the dissociation constant is for example < 1 mM, preferably < 100 nM and most preferably < 10 nM. The dissociation constant may be measured by any of the methods available in the state of the art as for example using enzyme-linked immunoabsorbent assay (ELISAs), radioimmunoassays (RIAs), flow cytometry, surface plasmon resonance, preferably in BIACORE(™). For example, the expression “specifically binds to the hNTRK2 receptof’ as herein means an antibody or antigen-binding portion thereof showing an equilibrium dissociation constant KD of less than l*10'⁸ M when tested in a BIACORE™ test against hNTRK2 receptor, performed for example as disclosed herein in the examples.

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.

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) is 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.

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 and 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").

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.

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, Methods 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, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al, Science 256: 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.

As used herein, the terms "label" or "labelled" refers to incorporation of another molecule in the antibody. In one embodiment, the label is a detectable marker, e.g., incorporation of a radiolabelled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). In another embodiment, the label or marker can be therapeutic, e.g., a drug conjugate or toxin. Various methods of labelling polypeptides and glycoproteins are known in the art and may be used.

Human NTRK2 receptor (hNTRK2) hNTRK2, entry QI 6620 in UniProt, also known as neurotrophic receptor tyrosine kinase 2 or TrkB, is a receptor protein that plays a crucial role in the nervous system by binding to the brain-derived neurotrophic factor (BDNF) and other neurotrophins. The hNTRK2 receptor is a key mediator of the effects of BDNF and other neurotrophins on neurons and is a member of the tyrosine kinase receptor family, which means that it activates intracellular signaling pathways by adding phosphate groups to specific tyrosine residues on proteins. When BDNF or other neurotrophins bind to the hNTRK2 receptor, it triggers a signaling cascade that can have various effects on neuronal development, function, and plasticity.

The human BDNF (hBDNF) according to the present disclosure is the hBDNF, entry P23560 in UniProt, having SEQ ID NO. 50.

Anti-hNTRK2 Antibodies and Characterization Thereof

In one embodiment, the invention provides a monoclonal antibody or antigen-binding portion thereof capable of specifically binding the hNTRK2 receptor.

In particular, the monoclonal antibody or antigen-binding portion thereof binds to the extracellular domain of the hNTRK2 receptor having SEQ ID NO. 51.

In an embodiment, the antibody or antigen-binding portion thereof comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) of a monoclonal antibody selected from the group consisting of R3P1-F1, R3P3-A11, R3P3-H4, R3P3-E1, R3P3-G5, R3P1- Al, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1-A6 and R4Pl-Fl.

The amino acid sequences of the variable domain-comprising portions of heavy and light chains (VH and VL) of the antibodies herein noted as R3P1-F1, R3P3-A11, R3P3-H4, R3P3- El, R3P3-G5, R3P1-A1, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1-A6 and R4P1-F1 can be found in the sequence listing herein enclosed in the description.

In certain aspects, the invention provides a monoclonal antibody or antigen-binding portion thereof that is capable of specifically binding the hNTRK2 receptor: (a) a heavy chain variable domain amino acid sequence that comprises the amino acid sequence of the heavy chain variable domain of an antibody selected from: R3P1-F1, R3P3-A11, R3P3-H4, R3P3- El, R3P3-G5, R3P1-A1, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1-A6 and R4P1-F1; (b) a light chain variable domain amino acid sequence that comprises the amino acid sequence of the light chain variable domain of an antibody selected from: R3P1-F1, R3P3-A11, R3P3-H4, R3P3-E1, R3P3-G5, R3P1-A1, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1-A6 and R4P1-F1; (c) a heavy chain variable domain of (a) and a light chain variable domain of (b); or (d) heavy chain and light chain variable domain amino acid sequences comprising the heavy chain and light chain variable domain amino acid sequences, respectively, from the same antibody selected from: R3P1-F1, R3P3-A11, R3P3- H4, R3P3-E1, R3P3-G5, R3P1-A1, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1- A6 and R4Pl-Fl.

In certain aspects, the invention provides a monoclonal antibody or an antigen -binding portion thereof that is capable of specifically binding the hNTRK2 receptor, comprising: (a) a heavy chain variable domain amino acid sequence that comprises the heavy chain CDR1, CDR2 and CDR3 amino acid sequences of an antibody selected from: R3P1-F1, R3P3-A11, R3P3-H4, R3P3-E1, R3P3-G5, R3P1-A1, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1-A6 and R4P1-F1; (b) a light chain variable domain amino acid sequence that comprises the light chain CDR1, CDR2 and CDR3 amino acid sequences of an antibody selected from: R3P1-F1, R3P3-A11, R3P3-H4, R3P3-E1, R3P3-G5, R3P1-A1, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1-A6 and R4P1-F1 : (c) a heavy chain variable domain of (a) and a light chain variable domain of (b); or (d) the heavy chain variable domain and light chain variable domain of (c), comprising heavy chain and light chain CDR amino acid sequences from the same antibody selected from: R3P1-F1, R3P3- A11, R3P3-H4, R3P3-E1, R3P3-G5, R3P1-A1, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1-A6 and R4P1-F1.

In certain aspects, the invention provides a monoclonal antibody or an antigen -binding portion thereof that specifically binds to the hNTRK2 receptor, wherein the antibody comprises VH and VL amino acid sequences from an antibody selected from: R3P1-F1, R3P3-A11, R3P3-H4, R3P3-E1, R3P3-G5, R3P1-A1, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1-A6 and R4P1-F1.

In certain aspects, the invention provides a monoclonal antibody that is capable of specifically binding the hNTRK2 receptor, wherein said antibody comprises a heavy chain of an antibody selected from the group consisting of: R3P1-F1, R3P3-A11, R3P3-H4, R3P3- El, R3P3-G5, R3P1-A1, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1-A6 and R4P1-F1. In certain aspects, the invention provides a monoclonal antibody that is capable of specifically binding the hNTRK2 receptor, wherein said antibody comprises a light chain of an antibody selected from the group consisting of R3P1-F1, R3P3-A11, R3P3-H4, R3P3- El, R3P3-G5, R3P1-A1, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1-A6 and R4P1-F1. In certain aspects, the invention provides a monoclonal antibody that is capable of specifically binding the hNTRK2 receptor, wherein said antibody comprises a heavy chain and a light chain of the same antibody which is selected from the group consisting of R3P1- Fl, R3P3-A11, R3P3-H4, R3P3-E1, R3P3-G5, R3P1-A1, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1-A6 and R4P1-F1.

In certain aspects, the invention provides a monoclonal antibody or antigen-binding portion thereof that is capable of specifically binding the hNTRK2 receptor comprising VL and VH domains that are at least 85%, 90%, 95%, 97%, 98% or 99% identical in amino acid sequence to the VL and VH domains, respectively, of a monoclonal antibody selected from the group consisting of R3P1-F1, R3P3-A11, R3P3-H4, R3P3-E1, R3P3-G5, R3P1-A1, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1-A6 and R4P1-F1.

In certain aspects, the invention provides a monoclonal antibody or antigen-binding portion thereof that is capable of specifically binding thehNTRK2 receptor comprising the light chain and the heavy chain that are at least 85%, 90%, 95%, 97%, 98% or 99% identical in amino acid sequence to the light chain and the heavy chain, respectively, of a monoclonal antibody selected from the group consisting of R3P1-F1, R3P3-A11, R3P3-H4, R3P3-E1, R3P3-G5, R3P1-A1, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1-A6 and R4P1-F1.

In certain aspects, the invention provides a monoclonal antibody or antigen-binding portion thereof that is capable of specifically binding the hNTRK2 receptor comprising the VL and the VH of a monoclonal antibody selected from the group consisting of R3P1 -F1, R3P3- A11, R3P3-H4, R3P3-E1, R3P3-G5, R3P1-A1, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1-A6 and R4P1-F1, and a constant heavy chain region of SEQ ID NO. 45, and/or a kappa constant light chain region of SEQ ID NO. 46, and/or a lambda constant light chain region of SEQ ID NO. 47 or SEQ ID NO. 48.

The inventors utilized advanced computational epitope and paratope mapping techniques to define the critical binding interactions between the antibodies herein disclosed and the NTRK2 receptor, confirming that these interactions disrupt the binding of the natural ligand, BDNF. Using Al-driven molecular docking and 3D-modelling, key amino acid residues involved in the epitope-paratope interaction were identified and categorized according to their probability of involvement in the binding process. This analysis provided insight into the structural basis of the antibodies' efficacy in targeting NTRK2 in endometriotic stromal cells. The inventors identified the epitopes of the monoclonal antibodies herein disclosed with the higher affinity with the hNTRK2 receptor.

Therefore, in certain aspects, the invention provides a monoclonal antibody or an antigenbinding portion thereof that is characterized as binding an epitope comprising the following amino acid residues of hNTRK2 receptor (SEQ ID NO: 51):

(I) residues 288, 289, 291-296, 298-299 and/or 321-324 of SEQ ID NO. 51, or residues 297, 325, 328, 346 of SEQ ID NO. 51, or residues 290, 318-320, 327 and/or 344 of SEQ ID NO. 51, or residues 287, 300, 315, 326. 345 and/or 347-349 of SEQ ID NO. 51, or

(II) residues 288-291 and/or 321-327 of SEQ ID NO. 51, or residues 320 and/or 344-346 of SEQ ID NO. 51, or residues 292-294, 298, 319, 342, 343 and/or 347-349 of SEQ ID NO. 51, or residues 263, 287, 295-297, 299, 318 and/or 328 of SEQ ID NO. 51, or

(III) residues 288-289, 322-327 and/or 344-348 of SEQ ID NO. 51, or residues 263, 290, 321, 342, 343 and/or 349 of SEQ ID NO. 51, or residues 262, 291, 320, 328 and/or 350 of SEQ ID NO. 51, or residues 261, 264-266, 269, 292, 294, 296-299, 318, 319, 330 and/or 331 of SEQ ID NO. 51.

In one embodiment, the antibody or its portion, such for example R3P1-F1, binds specifically to an epitope comprising residues 288, 289, 291-296, 298-299 and 321-324 of SEQ ID NO. 51, or residues 297, 325, 328, 346 of SEQ ID NO. 51, or residues 290, 318-320, 327 and 344 of SEQ ID NO. 51, or residues 287, 300, 315, 326. 345 and 347-349 of SEQ ID NO. 51;

In one embodiment, the antibody or its portion, such for example the antibody R3P3-E1, binds specifically to an epitope comprising residues 288-291 and 321-327 of SEQ ID NO. 51, or residues 320 and 344-346 of SEQ ID NO. 51, or residues 292-294, 298, 319, 342, 343 and 347-349 of SEQ ID NO. 51, or residues 263, 287, 295-297, 299, 318 and 328 of SEQ ID NO. 51;

In one embodiment, the antibody or its portion, such for example antibody R3P3-H4, binds specifically to an epitope comprising residues 288-289, 322-327 and 344-348 of SEQ ID NO. 51, or residues 263, 290, 321, 342, 343 and 349 of SEQ ID NO. 51, or residues 262, 291, 320, 328 and 350 of SEQ ID NO. 51, or residues 261, 264-266, 269, 292, 294, 296-299, 318, 319, 330 and 331 of SEQ ID NO. 51. The number of the residue indicates the position of the residue in the specific sequence.

Moreover, the inventors unexpected found that the three more efficient antibodies specifically bind residues in a specific region of the extracellular domain of human NTRK2, in particular residues between position 288 and 348. In one preferred embodiment the invention provides a monoclonal antibody or an antigen-binding portion thereof that specifically binding an epitope between position 288 and 348 of the extracellular domain of human NTRK2.

Preferably, a monoclonal antibody according to any of the embodiments disclosed in the present specification and in the claims is a human antibody or a human monoclonal antibody. According to a preferred aspect, a monoclonal antibody or antigen binding portion thereof according to any of the embodiments disclosed in the present specification and in the claims is capable of specifically binding the hNTRK2 receptor.

One type of amino acid substitution that may be made is to change one or more cysteines in the antibody, which may be chemically reactive, to another residue, such as, without limitation, alanine, or serine. In one embodiment, there is a substitution of a non-canonical cysteine. The substitution can be made in a CDR or framework region of a variable domain or in the constant domain of an antibody. In some embodiments, the cysteine is canonical. Another type of amino acid substitution that may be made is to change any potential proteolytic sites in the antibody. Such sites may occur in a CDR or framework region of a variable domain or in the constant domain of an antibody. Substitution of cysteine residues and removal of proteolytic sites may decrease the risk of any heterogeneity in the antibody product and thus increase its homogeneity. Another type of amino acid substitution is to eliminate asparagine-glycine pairs, which form potential deamidation sites, by altering one or both of the residues. In some embodiments, the C-terminal lysine of the heavy chain of the anti-hNTRK2 protein antibody of the invention is cleaved. In various embodiments of the invention, the heavy and light chains of the anti-hNTRK2 receptor antibodies may optionally include a signal sequence.

The class and subclass of the antibodies according to the present invention may be determined by any method known in the art. In general, the class and subclass of an antibody may be determined using antibodies that are specific for a particular class and subclass of antibody. Such antibodies are commercially available. The class and subclass can be determined by ELISA, or Western blot (immunoblot) as well as other techniques. Alternatively, the class and subclass may be determined by sequencing all or a portion of the constant domains of the heavy and/or light chains of the antibodies, comparing their amino acid sequences to the known amino acid sequences of various class and subclasses of immunoglobulins, and determining the class and subclass of the antibodies.

In some embodiments, the antibody according to any of the embodiments disclosed herein is an IgG, an IgM, an IgE, an IgA, or an IgD molecule. In one embodiment, the antibody is an IgG and is an IgGl, IgG2, IgG3, IgG4 subclass. In still another embodiment, the human antibody subclass is IgGl. In some embodiments of the invention, the antibodies according to any of the embodiments disclosed herein bind to said hNTRK2 receptor, with high affinity. The binding affinity and dissociation rate of an antibody to the hNTRK2 receptor can be determined by methods known in the art. The binding affinity can be measured by ELISAs, RIAs, flow cytometry, surface plasmon resonance, such as BIACORE(TM). The dissociate rate can be measured by surface plasmon resonance. Preferably, the binding affinity and dissociation rate is measured by surface plasmon resonance. More preferably, the binding affinity and dissociation rate are measured using BIACORE(TM). One can determine whether an antibody has substantially the same KD as an anti-hNTRK2 antibody by using methods known in the art.

According to the present invention, a paratope is the specific region on an antibody molecule that binds to an antigen. It is formed by the variable regions of the antibody’s heavy and light chains, primarily consisting of the complementarity-determining regions (CDRs). The paratope interacts with a corresponding epitope on the antigen via non-covalent interactions, facilitating the immune recognition and subsequent neutralization of the antigen.

In one embodiment of the invention, the antibodies according to any of the embodiments disclosed herein have a paratope VH part of SEQ ID NO. 68-70 or 73-78.

In one embodiment of the invention, the antibodies according to any of the embodiments disclosed herein have a paratope VL part of SEQ ID NO. 71, 72 or 79.

Nucleic Acids, Vectors, Host Cells, and Recombinant Methods of Making Antibodies Nucleic Acids

The present invention also encompasses nucleic acid molecules encoding antibodies or antigen-binding portions thereof according to any of the embodiments disclosed herein. In some embodiments, different nucleic acid molecules encode a heavy chain and a light chain of an antibody according to the present invention. In other embodiments, the same nucleic acid molecule encodes a heavy chain and a light chain of an antibody according to the present invention. In one embodiment, the nucleic acid encodes an antibody, or antigen-binding portion thereof, of the invention. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a VL amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions and/or 1, 2, or 3 non- conservative substitutions compared to germline. Substitutions may be in the CDR regions, the framework regions, or in the constant domain. In some embodiments, the nucleic acid molecule encodes a VL amino acid sequence comprising one or more variants compared to germline sequence that are identical to the variations found in the VL of one of the antibodies selected from R3P1-F1, R3P3-A11, R3P3-H4, R3P3-E1, R3P3-G5, R3P1-A1, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1-A6 and R4P1-F1.

In some embodiments, the nucleic acid molecule encodes at least three amino acid substitutions compared to the germline sequence found in the VL of one of the antibodies selected from R3P1-F1, R3P3-A11, R3P3-H4, R3P3-E1, R3P3-G5, R3P1-A1, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1-A6 and R4P1-F1.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes the VL amino acid sequence of R3P1-F1, R3P3-A11, R3P3-H4, R3P3-E1, R3P3- G5, R3P1-A1, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1-A6 and R4Pl-Fl or a variant or portion thereof. In some embodiments, the nucleic acid encodes an amino acid sequence comprising the light chain CDRs of one of said above-listed antibodies. In some embodiments, said portion is a contiguous portion comprising CDR1-CDR3. In some embodiments, the nucleic acid encodes the amino acid sequence of the light chain CDRs of said antibody. In some embodiments, said portion encodes a contiguous region from CDR1- CDR3 of the light chain of an antibody according to the present invention.

In some embodiments, the nucleic acid molecule encodes a VL amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to a VL amino acid sequence of a VL region of any one of antibodies R3P1-F1, R3P3-A11, R3P3-H4, R3P3- El, R3P3-G5, R3P1-A1, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1-A6 and R4P1 -FL Nucleic acid molecules of the invention include nucleic acids that hybridize under highly stringent conditions, such as those described above, to a nucleotide sequence encoding the amino acid sequence of a VL region.

In another embodiment, the nucleic acid encodes a full-length light chain of an antibody selected R3P1-F1, R3P3-A11, R3P3-H4, R3P3-E1, R3P3-G5, R3P1-A1, R3P1-A4, R3P1- A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1- G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1-A6 and R4P1-F1, or a light chain comprising a mutation, such as one disclosed herein.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes at least a portion of the VH amino acid sequence of a monoclonal antibody selected from monoclonal antibodies R3P1-F1, R3P3-A11, R3P3-H4, R3P3-E1, R3P3-G5, R3P1- Al, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1-A6 and R4P1-F1, all three CDR regions, a contiguous portion including CDR1-CDR3, or the entire VH region, with or without a signal sequence. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes the amino acid sequence of one of R3P1-F1, R3P3-A11, R3P3-H4, R3P3-E1, R3P3-G5, R3P1-A1, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1-A6 and R4P1-F1, or said sequence lacking the signal sequence. In some preferred embodiments, the nucleic acid molecule comprises at least a portion of the nucleotide sequence of R3P1-F1, R3P3-A11, R3P3-H4, R3P3-E1, R3P3-G5, R3P1-A1, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1-A6 and R4P1-F1, or said sequence lacking the signal sequence. In some embodiments, said portion encodes the VH region (with or without a signal sequence), a CDR3 region, all three CDR regions, or a contiguous region including CDR1-CDR3.

In some embodiments, the nucleic acid molecule encodes a VH amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the VH amino acid sequences of any one of R3P1-F1, R3P3-A11, R3P3-H4, R3P3-E1, R3P3-G5, R3P1-A1, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1-A6 and R4Pl-Fl.

Nucleic acid molecules of the invention include nucleic acids that hybridize under highly stringent conditions, such as those described above, to a nucleotide sequence encoding the amino acid sequence of R3P1-F1, R3P3-A11, R3P3-H4, R3P3-E1, R3P3-G5, R3P1-A1, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1-A6 and R4P1-F1 or that encodes a VH region thereof. In another embodiment, the nucleic acid encodes a full-length heavy chain of an antibody selected from R3P1-F1, R3P3-A11, R3P3-H4, R3P3-E1, R3P3-G5, R3P1-A1, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1-A6 and R4P1-F1 or a heavy chain having the amino acid sequence of R3P1-F1, R3P3-A11, R3P3-H4, R3P3-E1, R3P3-G5, R3P1-A1, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1-A6 and R4P1-F1 with or without a signal sequence, or a heavy chain comprising a mutation, such as one of the variants discussed herein. Further, the nucleic acid may comprise the nucleotide sequence of R3P1-F1, R3P3-A11, R3P3-H4, R3P3-E1, R3P3-G5, R3P1-A1, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1-A6 and R4P1-F1, with or without a signal sequence, or a nucleic acid molecule encoding a heavy chain comprising a mutation, such as one of the variants discussed herein.

Vectors

The invention provides vectors comprising nucleic acid molecules that encode the heavy chain of an antibody of the invention or an antigen-binding portion thereof. The invention also provides vectors comprising nucleic acid molecules that encode the light chain of such antibodies or antigen-binding portion thereof. The invention further provides vectors comprising nucleic acid molecules encoding fusion proteins, modified antibodies, antibody fragments, and probes thereof. In some embodiments, the antibodies or antigen-binding portions of the invention are expressed by inserting DNAs encoding partial or full-length light and heavy chains, obtained as described above, into expression vectors such that the genes are operatively linked to necessary expression control sequences such as transcriptional and translational control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV derived episomes, and the like. The antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors. In one embodiment, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). A convenient vector is one that encodes a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that any VH or VL sequence can easily be inserted and expressed, as described above. In such vectors, splicing usually occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C domain, and also at the splice regions that occur within the human CH exons. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding regions. The recombinant expression vector also can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the immunoglobulin chain. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e. a signal peptide from a non -immunoglobulin protein). In addition to the antibody chain genes, the recombinant expression vectors of the invention carry regulatory sequences that control the expression of the antibody chain genes in a host cell. It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from retroviral LTRs, cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g. the adenovirus major late promoter (AdMLP)), polyoma and strong mammalian promoters such as native immunoglobulin and actin promoters. For further description of viral regulatory elements, and sequences thereof, see e.g., U.S. Patent No. 5,168,062, U.S. Patent No. 4,510,245 and U.S. Patent No. 4,968,615. Methods for expressing antibodies in plants, including a description of promoters and vectors, as well as transformation of plants is known in the art. See, e.g., United States Patent 6,517,529, incorporated herein by reference. Methods of expressing polypeptides in bacterial cells or fungal cells, e.g., yeast cells, are also well known in the art. In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes.

Non-Hybridoma Host Cells and Methods of Recombinantly Producing Protein

Nucleic acid molecules encoding antibodies and vectors according to the present invention comprising these nucleic acid molecules can be used for transfection or transformation of a suitable mammalian, plant, bacterial or yeast host cell. Transfection or transformation can be by any known method for introducing polynucleotides into a host cell. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene- mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei. In addition, nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art (see, e.g., U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455, incorporated herein by reference). Methods for transforming plant cells are well known in the art, including, e.g., Agrobacterium - mediated transformation, biolistic transformation, direct injection, electroporation and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art. Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, N50 cells, SP2 cells, HEK-293T cells, NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and a number of other cell lines. Cell lines of particular preference are selected through determining which cell lines have high expression levels. Other cell lines that may be used are insect cell lines, such as Sf9 or Sf21 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods. Plant host cells include, e.g., Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc. Bacterial host cells include E. coli and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae and Pichia pastoris. Further, expression of antibodies of the invention from production cell lines can be enhanced using a number of known techniques.

Modified Antibodies

In another embodiment, a fusion antibody or immunoadhesin may be made that comprises all or a portion of an antibody of the invention linked to another polypeptide. In one embodiment, only the variable domains of the antibody according to any of the embodiments disclosed herein are linked to the polypeptide. In still another embodiment, the VH domain of an antibody of the invention is linked to a first polypeptide, while the VL domain of an antibody of the invention is linked to a second polypeptide that associates with the first polypeptide in a manner such that the VH and VL domains can interact with one another to form an antigen binding site. In still another embodiment, the VH domain is separated from the VL domain by a linker such that the VH and VL domains can interact with one another (see below under Single Chain Antibodies). The VH-linker- VL antibody is then linked to the polypeptide of interest. The fusion antibody is useful for directing a polypeptide to a hNTRK2 receptor-expressing cell or tissue, such as a cell or tissue expressing the hNTRK2 receptor. The polypeptide may be a therapeutic agent, such as a toxin, chemokine or other regulatory protein. In addition, fusion antibodies can be created in which two (or more) single-chain antibodies are linked to one another. This is useful if one wants to create a divalent or polyvalent antibody on a single polypeptide chain, or if one wants to create a bispecific antibody or nanobody. To create a single chain antibody, (scFv) the VH- and VL -encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (GIy4 -Ser)3, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH domains joined by the flexible linker. See, e.g., Bird et al, Science 242:423-426 (1988); Huston et al, Proc. Natl. Acad. ScL USA 85:5879-5883 (1988); McCafferty et al., Nature 348:552-554 (1990). The single chain antibody may be monovalent, if only a single VH and VL are used, bivalent, if two VH and VL are used, or polyvalent, if more than two VH and VL are used. Bispecific or polyvalent antibodies may be generated that bind specifically to the hNTRK2 receptor. Bispecific antibodies or antigen-binding fragments can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79: 315-321 (1990), Kostelny et al, J. Immunol. 148: 1547-1553 (1992). In addition, bispecific antibodies may be formed as "diabodies" or "Janusins". In some embodiments, the bispecific antibody has a first heavy chain and a first light chain from monoclonal antibody R3P1-F1, R3P3-A11, R3P3-H4, R3P3-E1, R3P3-G5, R3P1-A1, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1- A6 and R4P1-F1 and an additional antibody heavy chain and light chain. In some embodiments, the additional light chain and heavy chain also are from one of the aboveidentified monoclonal antibodies but are different from the first heavy and light chains. In some embodiments, the modified antibodies described above are prepared using one or more of the variable domains or CDR regions from a monoclonal antibody provided herein.

Derivatized and Labelled Antibodies

An antibody or antigen-binding portion of the invention can be derivatized or linked to another molecule (e.g., another peptide or protein). In general, the antibodies or portion thereof are derivatized such that the specifically binding tosaid at hNTRK2 receptor is not affected adversely by the derivatization or labelling. Accordingly, the antibodies and antibody portions of the invention are intended to include both intact and modified forms of the antibodies described herein. For example, an antibody or antibody portion of the invention can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detection agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag). One type of derivatized antibody is produced by crosslinking two or more antibodies (of the same type or of different types, e.g., to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N- hydroxysuccinimide ester) or homobifunctional {e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, II. [0179], Another type of derivatized antibody is a labelled antibody. Useful detection agents with which an antibody or antigen-binding portion of the invention may be derivatized include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, phycoerythrin, 5- dimethylamine-l-napthalenesulfonyl chloride, lanthanide phosphors and the like. An antibody can also be labelled with enzymes that are useful for detection, such as horseradish peroxidase, [beta]-galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like. When an antibody is labelled with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a reaction product that can be discerned. For example, when the agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a coloured reaction product, which is detectable. An antibody can also be labelled with biotin, and detected through indirect measurement of avidin or streptavidin binding. An antibody can also be labelled with a predetermined polypeptide epitope recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance. Further, the radiolabel can be used therapeutically as a toxin for cancerous cells or tumours. In some embodiments, the antibody can be labelled with a paramagnetic, radioactive or florigenic ion that is detectable upon imaging. In some embodiments, the paramagnetic ion is chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) or erbium (III). In other embodiments, the radioactive ion is iodine 123, technetium 99, indium 111, rhenium 188, rhenium 186, copper 67, iodine 131, yttrium90, iodine 125, astatine 211, and gallium 67. In other embodiments, the antibody of the invention is labelled with an X-ray imaging agent such as lanthanum (III), gold (III) lead (II) and bismuth (III).

Compositions and Kits

The invention relates to compositions comprising any of the antibodies or antigen binding portions thereof of the invention and one or more pharmaceutical acceptable excipients and/or carriers.

In certain embodiments, the composition may comprise antibodies or a binding portion thereof of any of the preceding embodiments. In some embodiments, the subject of treatment is a human. In other embodiments, the subject is a veterinary subject. In some embodiments, an antagonist anti-hNTRK2 antibody that binds to the domain and one that binds to domain or antigen-binding portions of either or both, are both administered to a subject, either together or separately. In certain embodiments the antibodies are in a composition comprising a pharmaceutically acceptable carrier. In another embodiment, one or more of the antagonist antibodies of the invention are administered in combination with one or more additional antagonistic antibodies that bind different epitope on hNTRK2 receptor. As used herein, "pharmaceutically acceptable carrier" means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption 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 shelf life 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 antibody is administered by intravenous infusion or injection. In still another embodiment, the antibody is 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. Sterile injectable solutions can be prepared by incorporating the antibody according to any of the embodiments of the invention in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. The antibodies of the present invention can be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route/mode of administration is subcutaneous, intramuscular, or intravenous infusion. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Other modes of administration include intraperitoneal, intrabronchial, transmucosal, intraspinal, intrasynovial, intraaortic, intranasal, ocular, otic, topical and buccal. In certain embodiments, the active compound of the antibody compositions may be prepared with a carrier that will protect the antibody against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, poly anhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems (J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978). The invention also provides compositions suitable for administration by inhalation, which comprise one or more of any of the antibodies described herein. Any of the antibodies of the invention may be conveniently delivered to a subject in the form of an aerosol spray presentation from pressurized packs or from a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. Dellamary et al. (2004) J Control Release. ;95(3): 489-500 describes formulations for the pulmonary delivery of antibodies. The invention also provides compositions, suitable for administration through the oral mucosa, which comprise one or more of any of the antibodies described herein. Oral transmucosal delivery refers to the delivery of a delivery vehicle across a mucous membrane in the oral cavity, pharyngeal cavity, or esophagus, and may be contrasted, for example, with traditional oral delivery, in which absorption of a drug occurs in the intestine. Accordingly, routes of administration in which the antibodies are absorbed through the buccal, sublingual, gingival, pharyngeal, and/or esophageal mucosa are all encompassed within "oral transmucosal delivery," as that term is used herein. For administration through the transmucosal mucosa, any of the antibody of the invention may be formulated, for example, into chewing gums (see U.S. Pat No. 5,711,961) or buccal patches (see e.g. U.S. Patent No. 5,298,256). The invention also provides compositions suitable for administration through the vaginal mucosa, which comprise one or more of any of the antibodies described herein. The antibodies of the invention may be formulated into a vaginal suppository, foam, cream, tablet, capsule, ointment, or gel. In certain embodiments, the compositions comprising the antibodies are formulated with permeants appropriate to the transmucosal barrier to be permeated. Such penetrants are generally known in the art, and include, for example, for trans mucosal administration bile salts and fusidic acid derivatives. In certain embodiments, an antibody of the invention can be orally administered, for example, with an inert diluent or an assailable edible carrier. The compound (and other ingredients, if desired) can also be enclosed in a hard- or soft-shell gelatine capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the antibodies can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound of the invention by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. Additional active compounds also can be incorporated into the compositions. In certain embodiments, a specifically binding antibody of the invention is co-formulated with and/or co-administered with one or more additional therapeutic agents, particularly anti-viral agents. These therapeutic agents include, without limitation, antibodies that bind other targets, photosensitizers, androgen, oestrogen, nonsteroidal anti-inflammatory agents, antihypertensive agents, analgesic agents, antidepressants, antibiotics, anticancer agents, anaesthetics, antiemetics, anti-infectants, contraceptives, antidiabetic agents, steroids, anti- allergy agents, chemotherapeutic agents, anti-migraine agents, agents for smoking cessation, anti-viral agents, immunosuppressants, thrombolytic agent, cholesterol-lowering agents and anti-obesity agents. Therapeutic agents also include peptide analogues that inhibit hNTRK2 receptor. In one embodiment, the additional agents that inhibit hNTRK2 receptor activity comprise an antisense nucleic acid capable of hybridizing to hNTRK2 receptor mRNA, such as a hairpin RNA or siRNA, locked nucleic acids (LNA) or ribozymes. Sequence -specific nucleic acids capable of inhibiting gene function by RNA interference are well-known in the art. Such combination therapies may require lower dosages of the specifically binding antibody as well as the co-administered agents, thus avoiding possible toxicities or complications associated with the various monotherapies. In certain specific embodiments, the therapeutic agent(s) that is co-formulated with and/or co-administered with a specifically binding antibody of the invention is an antimicrobial agent. Antimicrobial agents include antibiotics (e.g. antibacterial), antiviral agents, antifungal agents, and anti -protozoan agents. Non-limiting examples of antimicrobial agents are sulfonamides, trimethoprimsulfamethoxazole, quinolones, penicillins, and cephalosporins. The compositions of the invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antigen-binding portion of the invention. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the antibody or antibody portion may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount may be less than the therapeutically effective amount. Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus can be administered, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the antibody or portion thereof and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an antibody for the treatment of sensitivity in individuals. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

In one embodiment the antibodies or binding portion thereof or composition comprising such antibodies according to any one of the embodiments herein disclosed are for use in the prevention or the treatment of patients affected with endometriosis or related conditions. The use of such antibodies and compositions of said Abs include, but are not limited to passive immunization in persons at risk of contracting the infection (e.g. professionally exposed personnel, people living in endemic areas) and therapy of acute cases, either hospitalized or not. The invention also relates to compositions for inhibiting viral infection, and in particular endometriosis or related conditions in a mammal comprising an amount of an antibody of the invention in combination with an amount of a further therapeutical agent, wherein the amounts of the antibody and of therapeutical agent are together effective in inhibiting the activity of the hNTRK2 receptor.

Therapeutic Methods of Use

In another embodiment, the invention provides a method for specifically binding the NTKR2 receptor, by administering an antibody according to any of the embodiments disclosed herein to a patient in need thereof. Any of the types of antibodies described herein may be used therapeutically. In various embodiments, said antibody is a human antibody. In some embodiments, the antibody, or antigen-binding portion thereof, binds to domain of said NTKR2 receptor. In some embodiments, the patient is a human patient. Alternatively, the patient may be a mammal affected by endometriosis or related conditions. In one embodiment, the invention provides methods of treating, aiding in the treatment, preventing or aiding in the prevention of endometriosis or related conditions, in a subject by administering to the subject a therapeutically-effective or prophylactically effective amount of an antibody of the invention. Antibodies and antigen-binding fragments thereof which are antagonists of said hNTRK2 receptor can be used as therapeutics for such infection. The antibody may be administered locally or systemically. The therapeutic compositions comprising one or more antibodies according to any of the embodiments disclosed herein may be administered to the subject, for example, orally, nasally, vaginally, buccally, rectally, via the eye, or via the pulmonary route, in a variety of pharmaceutically acceptable dosing forms, which will be familiar to those skilled in the art. For example, the antibodies may be administered via the nasal route using a nasal insufflator device. The antibodies can also be administered to the eye in a gel formulation. For example, before administration, a formulation containing one or more antibodies according to any of the embodiments disclosed herein may be conveniently contained in a two-compartment unit dose container, one compartment containing a freeze-dried antibody preparation and the other compartment containing normal saline. The dosage of antibody will generally be in the range of 0.1 -100 mg/kg, more preferably 0.5-50 mg/kg, more preferably 1-20 mg/kg, and even more preferably 1-10 mg/kg. The serum concentration of the antibody may be measured by any method known in the art.

In another embodiment, the antibodies of the present invention are administered to the subject in combination with other therapeutic agents. In one embodiment, the additional therapeutic agents may be treating the symptoms of the endometriosis or related conditions on their own, and may optionally synergize with the effects of the antibodies. The additional agent that is administered may be selected by one skilled in the art for treating the infection. Co-administration of the antibody with an additional therapeutic agent (combination therapy) encompasses administering a composition comprising the antibody and the additional therapeutic agent as well as administering two or more separate compositions, one comprising the antibody and the other(s) comprising the additional therapeutic agent(s). Further, although co-administration or combination therapy generally means that the antibody and additional therapeutic agents are administered at the same time as one another, it also encompasses instances in which the antibody and additional therapeutic agents are administered at different times. For instance, the antibody may be administered once every three days, while the additional therapeutic agent is administered once daily. Alternatively, the antibody may be administered prior to or subsequent to treatment with the additional therapeutic agent, for example after a patient has failed therapy with the additional agent. Similarly, administration of any of the antibodies of the invention may be administered prior to or subsequent to other therapy.

Said antibody and one or more additional therapeutic agents (the combination therapy) may be administered once, twice or at least the period of time until the condition is treated, palliated or cured. Preferably, the combination therapy is administered multiple times. The combination therapy may be administered from three times daily to once every six months. The administering may be on a schedule such as three times daily, twice daily, once daily, once every two days, once every three days, once weekly, once every two weeks, once every month, once every two months, once every three months and once every six months, or may be administered continuously via a minipump. The combination therapy may be administered via an oral, mucosal, buccal, intranasal, inhalable, intravenous, subcutaneous, intramuscular, or parenteral. In certain aspects, the invention provides a method for treating, preventing or alleviating the symptoms of at least one endometriosis or related conditions, in particular a disorder mediated by hNTRK2 receptor activity, in a subject in need thereof, comprising the step of administering to said subject an antibody or antigen-binding portion according to any one of the preceding embodiments, further comprising at least one additional therapeutic agent selected from the group consisting of: (a) one or more antibodies from the group consisting of: R3P1-F1, R3P3-A11, R3P3-H4, R3P3-E1, R3P3-G5, R3P1- Al, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1-A6 and R4P1-F1 : and

(b) one or more antibodies that specifically bind said hNTRK2 receptor; and/or

(c) one or more specifically binding antibodies that do not bind said hNTRK2 receptor; and/or

(d) one or more agents that bind hNTRK2 receptor; and/or

(e) one or more therapetuic agents. In certain aspects, the invention provides a kit for treating, preventing or alleviating the symptoms of endometriosis or related conditions in a subject in need thereof, in particular a disorder mediated by mediated by hNTRK2 receptor activity, comprising a) one or more antibodies from the group consisting of: R3P1-F1, R3P3-A11, R3P3-H4, R3P3-E1, R3P3- G5, R3P1-A1, R3P1-A4, R3P1-A10, R3P1-G12, R3P2-A1, R3P2-B12, R3P2-C3, R3P3-E2, R3P3-H3, R3P1-A11, R3P1-G8, R3P1-H8, R3P2-A5, R3P2-H6, R4P1-A6 and R4P1-F1; and

(b) one or more antibodies that specifically bind the hNTRK2 receptor; and/or

(c) one or more specifically binding antibodies that do not bind said hNTRK2 receptor; and/or

(d) one or more agents that bind hNTRK2 receptor; and/or

(e) one or more therapeutic agent.

The human monoclonal antibody or antigen-binding portion thereof herein disclosed may also be used advantageously for the design of a vaccine against endometriosis. As disclosed in Rappuoli, Rino et al. “ Reverse vaccinology 2.0: Human immunology instructs vaccine antigen design. ” The Journal of experimental medicine vol. 213,4 (2016): 469-81. doi: 10.1084/jem.20151960”, human mAb may be used to identify protective antigens/epitopes. Structural characterization of the Ab-antigen complex may be used to instruct antigen design. Thus, also a method or the use of the human monoclonal antibody or antigen-binding portion thereof herein disclosed for the design of a vaccine against endometriosis is within the scope of the invention.

In one embodiment, the present invention relates to a method of treating or adjuvating the treatment of a patient suffering of endometriosis or related conditions, comprising a step of administering an effective dose of the monoclonal antibody according to the present invention.

The human monoclonal antibody or antigen-binding portion thereof herein disclosed may be used for the preparation of mimotopes, such as for example anti-idiotype antibodies, peptides, truncated proteins 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 specifically binding 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 human monoclonal antibody or antigenbinding portion thereof herein disclosed may be used for the manufacture of anti -idiotype antibodies according to methods per se known. Anti-idiotype antibodies are antibodies specifically directed towards the idiotype of the broad-range specifically binding antibodies used to prepare them, and as such are able to mimic the key epitopes they recognize. Therefore, anti-idiotype antibodies directed against a monoclonal antibody of the invention are also included in the scope of the invention.

The following experimental section is provided solely by way of illustration and not limitation and does not intend to restrict the scope of the invention as defined in the appended claims. The claims are an integral part of the description.

EXAMPLES

Antigen Expression

1. Project information

Service type: recombinant protein antigen expression

Antigen:

- Extracellular domain of human NTRK2 (residues 32-430) with a twin-strep tag (Uniprot #Q 16620)

- Human BDNF (residues 129-247) with a polyhistidine tag (Uniprot #P23560)

Antigen alias for the project:

- hNTRK2

- hBDNF

2. Gene synthesis & sub-cloning in expression vector

2.1. E. coli cDNA of hBDNF [129-247] with a 6His-tag at C-ter was chemically synthesized with codon optimization for bacterial systems, then sub-cloned in ProteoGenix’s proprietary optimized expression vector. The protein sequence is as follows:

> hBDNF[129-247]-6his - 14.48 kDa* MGWSCIILFLVATATGVHSHSDPARRGELSVCDSISEWVTAADKKTAVDMSGGT VTVLEKVPVSKGQLKQYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRA LTMDSKKRIGWRFIRIDTSCVCTLTIKRGRGSHHHHHH

Features:

Signal peptide: [1-19] hBDNF sequence: [20-138]

6xHis-tag with linker: [139-146]

*Theoretical molecular weight based on amino acid sequence after cleavage of the signal peptide

2.2. Mammalian cells - XtenCHO™ cDNA of hNTRK2 with a twin-streptavidin tag at C-ter was chemically synthesized with codon optimization for mammalian cell expression, then sub-cloned in ProteoGenix’s proprietary optimized expression vector. The protein sequence is as follows:

> hNTRK2[32-430]-twin-strep - 47.24 kDa*

MGWSCIILFLVATATGVHSCPTSCKCSASRIWCSDPSPGIVAFPRLEPNSVDPENIT EIFIANQKRLEIINEDDVEAYVGLRNLTIVDSGLKFVAHKAFLKNSNLQHINFTRNK LTSLSRKHFRHLDLSELILVGNPFTCSCDIMWIKTLQEAKSSPDTQDLYCLNESSKN

IPLANLQIPNCGLPSANLAAPNLTVEEGKSITLSCSVAGDPVPNMYWDVGNLVSKH MNETSHTQGSLRITNISSDDSGKQISCVAENLVGEDQDSVNLTVHFAPTITFLESPTS DHHWCIPFTVKGNPKPALQWFYNGAILNESKYICTKIHVTNHTEYHGCLQLDNPT

HMNNGDYTLIAKNEYGKDEKQISAHFMGWPGIDDGANPNYPDVIYEDYGTAAND IGDTTNRSNEIPSTDVTDKTGREHSAWSHPQFEKGGGSGGGSGGSAWSHPQFEK Features:

Signal peptide: [1-19] hBDNF sequence: [20-418]

Twin-strep-tag with linker: [419-448]

*Theoretical molecular weight based on amino acid sequence after cleavage of the signal peptide

3. hNTRK2 3.1. Expression & purification tests

The test of 3 purifications i.e. medium, native protein extracts (NPE) obtained by cell lysis in PBS pH7.5, and denatured protein extracts (DPE) obtained by solubilization in urea 8M of the pellet obtained after native lysis in XtenCHO™ cells. hNTRK2 expression was mainly observed in medium (figure 1A). The protein migrated at slightly higher molecular weight. This is most likely due to the presence of glycosylation sites & their glycosylation.

The culture medium was purified by affinity with StrepTactin column. The purification of the native medium fraction was successful with a good solubility & purity. The quantity was insufficient for Phage Display thus the large-scale expression was performed.

Table 1. Production and purification yield from purification test

*Obtained for the 80ml-culture test. **Estimate based on 80mL-culture test. ***Based on the 2pg loading in Figure 1. Final buffer PBS, pH7.5

3.2. Scale-up

In order to obtain enough protein to perform the Phage Display project, a three-liter scale- up was performed & purified, following the conditions identified in the previous step. hNTRK2 was obtained from mammalian cells as follows:

Quantity: 5.57mg

Purity: > 90% (based on SDS-PAGE below)

Buffer: PBS pH7.5

Concentration: 0.62mg/mL

Quality validation for use as antigen? YES

4. hBDNF

The expression & purification conditions of hBDNF in E.coli had been identified in a previous project where multiple fractions were analyzed. Briefly, the protein expressed in the DPE & it was purified by affinity with a Nickel resin. The same conditions were used to obtain the protein. In order to obtain enough protein to perform the proj ect, a two-liter culture was performed. hBDNF was obtained from E.coli as followed:

Quantity: 1.49mg

Purity: > 80% (based on SDS-PAGE below)

Buffer: PBS pH7.5 + 0.02% NLS

Concentration: 0.22mg/mL

Quality validation for use as antigen? YES

Gene Synthesis & Sub-cloning

The cDNAs of the variable heavy (VH) and the variable light (VL) sequences previously obtained by screening ProteoGenix proprietary naive human Phage Display LiAb-SFMAX library were chemically synthesized with optimization for mammalian expression in CHO cells. They were then sub-cloned into ProteoGenix’ s proprietary mammalian cell expression vector pTXsl in order to obtain the full-length sequences of the heavy (HC) and light (LC) chains of human IgGl of Kappa or Lambda type. Expected corresponding proteins produced are illustrated below.

> 20236-R3P1-F1-VH

MKHLWFFLLLVAAPRWVLSOVOLQOSGPGLVKPSOTLSLTCAISGDSVSSNSAA WNWIROSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNOFSLQLNSVT PEDTAVYYCARGKNYAMGVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK

Features:

Signal peptide: [1:19]

Variable heavy chain region: 120: 1391

Human IgGl constant region: [140:469] > 20236-R3P3-A11-VH

MKHLWFFLLLVAAPRWVLSOVOLVQSGAEVKKPGESLKISCKGSGYSFSNSWIA

WVROMPGKGLEWMGIIYPGDSDTRYSPSFOGOVTISVDKSVNTAYLQWSSLKAS DTAFYYCATTGGYSPFDHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYIC

NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK

Features:

Signal peptide: [1:19]

Variable heavy chain region: [20:1371

Human IgGl constant region: [138:467]

> 20236-R3P3-H4-VH

MKHLWFFLLLVAAPRWVLSOVOLVQSGGGVVRPGGSLRLSCAASGFTFDDYG

MSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSL RAEDTAMYYCARGREVAALGKTYYHGLDVWGQGTTVTVSSASTKGPSVFPLAPS

SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Features:

Signal peptide: [1:19]

Variable heavy chain region: [20:1451

Human IgGl constant region: [146:475]

> 20236-R3P3-E 1-VH MKHLWFFLLLVAAPRWVLSOVOLVQSGAEVKKPGESLKISCKGSGYSFTSYWIG

WVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASD

TAMYYCARPLLGDGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG

CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYIC

NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT

PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH

QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL

TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG

NVFSCSVMHEALHNHYTQKSLSLSPGK

Features:

Signal peptide: [1:19]

Variable heavy chain region: [20:137]

Human IgGl constant region: [138:467]

> 20236-R3P3-G5-VH

MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGESLKISCKASGYDFTSYWI

GWVROMPGKGLEWMGVIYPGDSDARYSPSFOGOVTISADKSISTAYLQWSSLKAS

DTAIYYCARSTASDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK

DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN

HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT

CVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDW

LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV

KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS

CSVMHE ALHNHYTQKSLSLSPGK

Features:

Signal peptide: [1:19]

Variable heavy chain region: [20:134]

Human IgGl constant region: [135:464]

> 20236-R3P3-H4-VL

MVLOTOVFISLLLWISGAYGDIVMTQTPLSLPVTPGEPASISCRSSOSLLHSNGYN

YLDWYLQKPGOSPOLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CMOALOTFTFGPGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK

VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ

GLSSPVTKSFNRGEC

Features:

Signal peptide: [1:20]

Variable light chain region: [21:1311

Human kappa constant region: [132:238]

> 20236-R3P1-F 1-VL

MVLOTOVFISLLLWISGAYGOSVLTQPPSASGSPGOSVTISCTGTSSDVGLYNYV

SWYQORPGKAPKLMIYDVTKRPSGVPDRFSGSKSGNTASLTVSGLQTDDEADYYC

SSRTLNHIVLFGGGTQLTVLGOPKANPTVTLFPPSSEELQANKATLVCLISDFYPGA

VTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTH

EGSTVEKTVAPTECS

Features:

Signal peptide: [1:20]

Variable light chain region: [21:1301

Human lambda constant region: [131 :236]

> 20236-R3P3-A11-VL

MVLOTOVFISLLLWISGAYGOPVLTOPPSVSEAPRQRVTISCSGSSSNIENNAVN

WYQOLPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSASLAISALQSEDEGDYYCGV

WDDSLNGYVFGTGTKVTVLGOPKANPTVTLFPPSSEELQANKATLVCLISDFYPG

AVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVT

HEGSTVEKTVAPTECS

Features:

Signal peptide: [1:20]

Variable light chain region: [21:1301

Human lambda constant region: [131 :236]

> 20236-R3P3-E 1-VL MVLOTOVFISLLLWISGAYGSYELTOPPSVSVAPGETAQIPCGRPSIGRESVHWY QQTPGQAPVLVMYDDNDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVW DGVSGPYVFGPGTKLTVLGOPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAV TVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE GSTVEKTVAPTECS

Features:

Signal peptide: [1:20]

Variable light chain region: [21 : 1281

Human lambda constant region: [129:234]

> 20236-R3P3-G5-VL

MVLOTQVFISLLLWISGAYGQPVLTQPPSLSVSPGQAANIICSGHKLGEKFVSWY QOKPGOSPVLVVYODSKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCOTW DNGVVFGGGTOLTVLGOPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVA WKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGST VEKTVAPTECS

Features:

Signal peptide: [1:20]

Variable light chain region: [21 : 126]

Human lambda constant region: [127:231]

Production and purification of the recombinant antibodies

Description of the protocol

An endotoxin-free DNA preparation was done for the constructions obtained as described in the previous paragraph. The vectors were transfected with XtenCHO cells by Xten transfection protocol. Culture medium was collected when viability dropped under 50% (14 days after transfection) and purified by protein A purification. The fractions were analyzed by SDS-PAGE (figure 1). The elution fractions were pooled & underwent dialysis to exchange buffers. The final samples were filtered by 0.22pM Milipore flilter and assayed for endotoxin levels using a chromogenic LAL endotoxin assay.

Table 2: Summary of the antibody names & constructions

Short purification protocol description

Purification by Protein A:

-Clarification by 0.22pm filtration

-Equilibration, binding, and wash with PBS pH7.5

-Elution by pH shift with citric acid

-Neutralization with 1 M Tris-HCl pH 9.0

-Analysis by PAGE and pool of the fractions of interest. -Final QC by PAGE: qualitative and quantitative by SDS-PAGE

The purification test results and QC were illustrated in Figure 1 and Figure 2.

Table 3. Yield and purity obtained for each recombinant antibody after expression & purification.

*Obtained after purification for the 30ml -culture test. **Based on full length antibody observed on non-reduced PAGE analysis in Figure 2.

Final buffer: PBS, pH 7.5. To be stored at -20°C to -80°C. Avoid repeated freeze-thaw: aliquot according to your needs.

Conclusion: All antibodies could be purified with high yields & high purity, save for R3P3- G5 where the yield is below lmg/30mL.

Preliminary binding QC

The obtained recombinant antibodies were tested against their respective antigens. As a negative control, they were also tested against coated BSA.

Conclusion: In the tested experimental conditions, specific binding of all the recombinant antibodies to hNTRK2 could be observed. Binding could be observed at low concentrations, down to 15.625ng/mL. This could indicate the antibodies have high affinity.

General conclusion

The chosen phage-binder sequences could be expressed & purified as recombinant human IgGl antibodies. The activity of these antibodies against their respective antigen could be validated in ELISA. Yet, the recombinant antibodies should be validated in the appropriate functional tests in order to identify the best candidate(s) for the customer’s final use(s).

Determination of KD of antibody/antigen interaction via Surface Plasmon Resonance

(SPR) technology

Information of the samples:

- Origin of the antibodies: developed and produced by ProteoGenix

- Antigen name: hNTRK2 - Origin of the antigen: developed and produced by ProteoGenix

- General concept:

* hNTRK2 immobilized on sensor chip

*A solution containing each antibody at various concentrations is flown over hNTRK2

*Real time measurement of kinetics parameters: on/off rates (ka & kd)

*Binding affinity constant (KD) determination

Experimental conditions

• Specific buffers used:

SPR equipment: Biacore™ T200

• Sensor chips

In order to immobilize the antigen (hNTRK2), a CM5 sensor chip was used, which is a glass slide coated with a thin layer of gold, to which a matrix of carboxymethylated dextran is covalently attached. The gold is required for generation of the SPR response, while the dextran matrix allows covalent immobilization of biomolecules using mal eimide chemistry.

• Method

1) For each sensor chip, one channel was used as negative control: no coupling was performed.

2) Immobilization of hNTRK2 protein antigen on CM5 sensor chip using maleimide EDC/NHS coupling until response reaches defined resonance units (RU). The response measures changes in refractive index and is related to variations in mass close to the sensor surface. Therefore, the response is proportional to the number of antibody molecules interacting with antigen.

3) Each antibody at a defined concentration is flown over CM5 chip and response is captured over time, showing the progress of the interaction and association/dissociation cycle (see sensorgram example below).

4) Regeneration is performed to remove all remaining bound antibody analyte from the chip. 5) A new concentration is tested as in 3).

6) After different concentrations are successively tested, the kinetics parameters and affinity are calculated using BIAevaluation software.

Table 4. Kinetics parameters and affinity of antibody/antigen interaction

Note: KD is the equilibrium dissociation constant, a ratio of kd/ka between the antibody and its antigen.

KD and affinity are inversely related: the lower the KD value the higher the affinity of the antibody.

Discussion & Conclusion

KD is the equilibrium dissociation constant, a ratio of kd/ka between the antibody and its antigen. KD and affinity are inversely related: the lower the KD value the higher the affinity of the antibody:

=> Weak antigen/antibody interaction: 1 O'⁶- 1 O'⁷ M

=> Strong antigen/antibody interaction: 10'⁸-l O'¹⁰ M

=> Most antibodies have KD values in the low micromolar ( 1 O'⁶) to nanomolar ( 1 O'⁸- 1 O'⁹) range.

High affinity antibodies are generally considered to be in the low nanomolar range (1 O'⁹) and very high affinity antibodies in the picomolar ( 1 O'¹²) range.

Conclusion: Four over five tested antibodies show a strong binding affinity with hNTRK2 antigen (R3P1F1, R3P3-H4, R3P3-E1 and R3P3-A11) with determined KD in nanomolar range.

Affinity of R3P3-G5 antibody for hNTRK2 cannot be determined (probably due to epitope denaturation during the antigen coating).

These results are consistent with those previously got in ELISA for this project. Test on fresh cells of the antibodies

The inventors tested the antibodies R3P3-F1, R3P3-E1 and R3P3-H4 on fresh cells (human stromal and endometriosis cells) as endometriosis modelconfirming that the selective binding of extracellular domain of hNTRK2 with the monoclonal antibodies is efficient in the treatment of endometriosis, in particular the treatment of human stromal and endometriosis cells with the monoclonal of the present invention (R3P3-F1, R3P3-E1 and R3P3-H4) drastically increased cellular apoptosis of said cells. This result is consistent with the findings from a mouse model, as reported by Lee et al. (2021) (‘Inhibiting NTRK2 signalling causes endometriotic lesion regression,’ Reproduction, 161(1), 11-19). In this study, it was demonstrated that the NTRK2 antagonist ANA-12 inhibits the aberrant expression of NTRK2 in endometriotic stromal cells, which arises due to hypoxic stress and contributes to resistance to apoptosis.

NTRK2, a receptor tyrosine kinase, specifically binds brain-derived neurotrophic factor (BDNF), a key molecule overexpressed in endometriotic tissues. The interaction between BDNF and NTRK2 activates intracellular signalling pathways that promote cell survival and inflammation, which are central to the pathophysiology of endometriosis. Increased levels of BDNF have been correlated with more severe pain in endometriosis patients, and its expression is significantly higher in affected tissues compared to controls. Additionally, IL- ip, a cytokine known to be prevalent in endometriosis, enhances BDNF production via the NF-KB and JNK signalling pathways, further exacerbating inflammation and pain by promoting nerve fiber growth into endometriotic lesions.

Accordingly, the inventors’ studies with fresh cells confirms the critical role of the BDNF- NTRK2 axis in endometriosis and supports the therapeutic use the monoclonal antibodies herein disclosed to mitigate lesion growth and associated pain. Ongoing experiments on immortalized cells are expected to further validate these findings.

Sequence Listing in the description

Phage-binder sequences from Phage Display

Variable heavy chain

SEQ ID NO.l: R3P1-F1 VH

QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYY RSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARGKNYAMGV WGQGTMVTVSS

SEQ ID NO.2: R3P3-A11 VH

QVQLVQSGAEVKKPGESLKISCKGSGYSFSNSWIAWVRQMPGKGLEWMGIIYPGD SDTRYSPSFQGQVTISVDKSVNTAYLQWSSLKASDTAFYYCATTGGYSPFDHWGQ GTLVTVSS

SEQ ID NO.3: R3P3-H4 VH

QVQLVQSGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINW NGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAMYYCARGREVAALGK T YYHGLD VWGQGTT VT VS S

SEQ ID NO.4: R3P3-E1 VH

QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGD SDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARPLLGDGMDVWG QGTTVTVSS

SEQ ID NO.5: R3P3-G5 VH

QVQLVQSGAEVKKPGESLKISCKASGYDFTSYWIGWVRQMPGKGLEWMGVIYPG DSDARYSPSFQGQVTISADKSISTAYLQWSSLKASDTAIYYCARSTASDYWGQGTL VTVSS

SEQ ID NO. 6: R3P1-A1 VH

QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGD SDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLGYGMDVWGQG TMVTVSS

SEQ ID NO. 7: R3P1-A4 VH

QVQLVQSGAEVKKPGESLKISCKGSGYSFTTSWIGWVRQMPGKGLEWMGIIYPSD SDTRYSPSFQGQVTISVDKSISTAYLQWSSLKASDTAMYYCARGDGRYWGQGTLV TVSS SEQ ID NO.8: R3P1-A10 VH

QVQLVQSGAEVKKPGESLKISCKGSGYSFTDYWIGWVRQMPGKGLEWMGIIFPGD SDTRYSPSFQGQVTISVDKSISTAYLQWSSLKASDTAMYYCARTPDYWGQGTLVT vss

SEQ ID NO.9: R3P1-G12 VH

QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGD SDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARPDGYWGQGTLVT VSS

SEQ ID NO.IO: R3P2-A1 VH

QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGD SDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLLGFGESEQYWG QGTLVTVSS

SEQ ID NO.ll R3P2-B12 VH

QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMGIIYPG DSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLGSGSDSDYWG QGTLVTVSS

SEQ ID NO.12 R3P2-C3 VH

QVQLVQSGAEVKKPGESLKISCKGSGYSFSTYWIGWVRQMPGKGLEWMGIIYPGD SDTRYSPSFQGQVTISADKSISTAYLQWNSLKASDTAMYYCARQTYADYWGQGT LVTVSS

SEQ ID NO.13: R3P3-E2 VH

QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGD SDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARPLLGDGMDVWG QGTTVTVSS

SEQ ID NO.14: R3P3-H3 VH QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSVAWNWIRQSPSRGLEWLGRTYY RSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARGYNYAFDY WGQGTLVTVSS

SEQ ID NO.15: R3P1-A11 VH

QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGD SDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAFYYCATTGGYSPFDHWGQG TLVTVSS

SEQ ID NO.16: R3P1-G8 VH

QVQLVQSGAEVKKPGESLKISCKASGYDFTSYWIGWVRQMPGKGLEWMGVIYPG DSDARYSPSFQGQVTISADKSISTAYLQWSSLKASDTAIYYCARSTASDYWGQGTL VTVSS

SEQ ID NO.17: R3P1-H8 VH

QVQLVQSGAEVKKPGESLKISCKASGYSFTSYWIAWVRQMPGKGLEWMGIIYPGD SDTRYSPSFEGQVSISVDKSISTAYLQWSSLKASDTAMYYCARQSYYDYWGQGTL VTVSS

SEQ ID NO.18: R3P2-A5 VH

QVQLVQSGAEVKKPGESLKISCKGSGYKFANYWIAWVRQMPGKGLEWMGIIYPG DSDTRYSPSFQGQVIISADKSISTAYLQWSSLKASDTGMYYCARLAGDSTGYDNGL EIWGHGTMVTVSS

SEQ ID NO.19: R3P2-H6 VH

EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGD SDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCSYGMDVWGQGTMV TVSS

SEQ ID NQ.20: R4P1-A6 VH

QVQLVQSGTEVKKPGEPLKISCKGSGYSFSTHWIGWVRQMPGKGLEWMGIIYPGD SDTRYSPSFQGQVTISADKSINTAYLQWSSLKASDTAIYYCASFQALGDYGMDVW GQGTTVTVSS

SEQ ID NO.21: R4P1-F1 VH

QVQLVQSGAEVKKPGESLKISCKGSGYSFATHWIGWVRQMPGKGLEWMGIIYPG DSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGGDVWGQGT MVTVSS

Variable light chain

SEQ ID NO.22: R3P1-F1 VL

QSVLTQPPSASGSPGQSVTISCTGTSSDVGLYNYVSWYQQRPGKAPKLMIYDVTK

RPSGVPDRFSGSKSGNTASLTVSGLQTDDEADYYCSSRTLNHIVLFGGGTQLTVL

SEQ ID NO.23: R3P3-A11 VL

QPVLTQPPSVSEAPRQRVTISCSGSSSNIENNAVNWYQQLPGKAPKLLIYYDDLLPS

GVSDRFSGSKSGTSASLAISALQSEDEGDYYCGVWDDSLNGYVFGTGTKVTVL

SEQ ID NO.24: R3P3-H4 VL

DIVMTQTPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGS

NRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTFTFGPGTKLEIK

SEQ ID NO.25: R3P3-E1-VL

SYELTQPPSVSVAPGETAQIPCGRPSIGRESVHWYQQTPGQAPVLVMYDDNDRPSG IPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGVSGPYVFGPGTKLTVL R3P3-G5-VL

SEQ ID NO.26: R3P3-G5-VL

QPVLTQPPSLSVSPGQAANIICSGHKLGEKFVSWYQQKPGQSPVLVVYQDSKRPSG IPERFSGSNSGNTATLTISGTQAMDEADYYCQTWDNGWFGGGTQLTVLGQPKAN PTVTL

SEQ ID NO.27: R3P1-A1 VL

Q AVLTQPP S VSGAPGQRVTISCTGS S SNIGAGYD VHW YQQLPGTAPKLLIYGNSNR PSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSAFYVFGTGTKVTVL

SEQ ID NO.28: R3P1-A4 VL

QPVLTQPPSVSVSPGQTASITCSGDKLGDKFATWYQQKPGQSPVLVIYQDTKRPSG

IPERFSGSNSGSTATLTISGTQPMDEADYYCQAWDSGHYVFGTGTKVTVL

SEQ ID NO.29: R3P1-A10 VL

QPVLTQPPSVSVSPGQTASITCSGDKLGDKFATWYQQKPGQSPVLVIYQDTKRPSG

IPERFSGSNSGSTATLTISGTQPMDEADYYCQAWDSGHYVFGTGTKVTVL

SEQ ID NQ.30: R3P1-G12 VL

QAGLTQPPSVSVSPGQTASITCSGDKLGDKYASWYQQKPGQSPVLVIYQDSKRPSG

IPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSSTYVFGTGTKLTVL

SEQ ID NO.31: R3P2-A1 VL

QSVLTQPPSVSVSPGQTATITCSGDKLGDRFTWWYQQKAGQSPVLVIYEDNKRPS

GIPERFSGSNSGNAATLTISGTQAMDEADYYCQAWDTNTDSYVFGTGTKVTVL

SEQ ID NO.32: R3P2-B12 VL

QPVLTQPPSVSVSPGQTASITCSGHKLGDKYVSWYRHNPGQSPVLVIYQDTKRPSG

IPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSSNYVFGTGTKVTVL

SEQ ID NO.33: R3P2-C3 VL

QPVLTQPPSVSVSPGQTASITCSGDKLGDKYASWYQQKPGQSPVLVIYQDTKRPSG

IPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSRVVFGGGTKLTVL

SEQ ID NO.34: R3P3-E2 VL

SYELTQPPSVSVAPGETAQIPCGRPSIGRESVHWYQQTPGQAPVLVMYDDNDRPSG

IPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGVSGPYVFGPGTKLTVL

SEQ ID NO.35: R3P3-H3 VL

QSALTQPPSASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSK RPSGVSNRF SGSKSGNT ASLTISGLQ AEDEADYYC S S YTS S STRGVVFGGGTKLT V L

SEQ ID NO.36: R3P1-A11 VL

QPVLTQPPSVSVAPGQTAILTCGGNNIGGKSVHWLQQRPGQAPVPVIYNDRVRPSG

IPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDNSGYQYVFGAGTKLTVL

SEQ ID NO.37: R3P1-G8 VL

QPVLTQPPSLSVSPGQAANIICSGHKLGEKFVSWYQQKPGQSPVLVVYQDSKRPSG

IPERFSGSNSGNTATLTISGTQAMDEADYYCQTWDNGWFGGGTQLTVL

SEQ ID NO.38: R3P1-H8 VL

QPVLTQPPSVSVSPGQTASITCSGERLGDKFVGWYQQKPGQSPVLVIYQDAKRPAG

IPERFTGSNSGDSATLTISGTQAMDEADYYCQAWDSSANVFGTGTKVTVL

SEQ ID NO.39: R3P2-A5 VL

QAVLTQPPSVSVSPGQTATITCSGEKMGDKFVSWYQHKPGQSPVLVIYQDVKRPA

GIPERFSGSNSGNTATLTISGTQPMDEADYYCQAWDTSHYVFGTGTKVTVL

SEQ ID NQ.40: R3P2-H6 VL

QAGLTQPPSVSVSPGQTASITCSGDKLGDKFASWYQQKPGQSPVLVIYQDNERPSG IPERF SGSNS VNT ATLTISGTQAMDEAEYYCQ AWDNS AS S YVFGTGTKVTVL

SEQ ID NO.41: R4P1-A6 VL

QPVLTQPPSVSVSPGQTASITCSGDKLGDKFVSWYQQKPGQSPVLVIYQDSKRPSG

IPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSSHYVFGTGTKVTVL

SEQ ID NO.42: R4P1-F1 VL

NFMLTQPPSVSVSPGQTASITCSRDKLGDKYVSWYQQKPGQSPVLLIYQDKKRPS

GIPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSNTYVFGTGTKVTVL

SEQ ID NO.43: Signal peptide VH MKHLWFFLLLVAAPRWVLS

SEQ ID NO. 44: Signal peptide VL

MVLQTQVFISLLLWISGAYG

SEQ ID NO. 45: Human IgGl constant region

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV

LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC

PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV

HNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<ALPAPIEI<TISI<A

KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT

PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO. 46: Human kappa constant region

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES

VTEQDSKD ST YSLS STLTLSKAD YEKHKVYACEVTHQGLS SPVTKSFNRGEC

SEQ ID NO. 47: Human lambda constant region

GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVET

TKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

SEQ ID NO. 48: Human lambda constant region R3P3-G5-VL

FPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKY

AASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

SEQ ID NO. 49: Human NTRK2 receptor

MSSWIRWHGPAMARLWGFCWLVVGFWRAAFACPTSCKCSASRIWCSDPSPGIVA

FPRLEPNSVDPENITEIFIANQKRLEIINEDDVEAYVGLRNLTIVDSGLKFVAHKAFL

KNSNLQHINFTRNKLTSLSRKHFRHLDLSELILVGNPFTCSCDIMWIKTLQEAKSSP

DTQDLYCLNESSKNIPLANLQIPNCGLPSANLAAPNLTVEEGKSITLSCSVAGDPVP

NMYWDVGNLVSKHMNETSHTQGSLRITNISSDDSGKQISCVAENLVGEDQDSVNL

TVHFAPTITFLESPTSDHHWCIPFTVKGNPKPALQWFYNGAILNESKYICTKIHVTN HTEYHGCLQLDNPTHMNNGDYTLIAKNEYGKDEKQISAHFMGWPGIDDGANPNY PDVIYEDYGTAANDIGDTTNRSNEIPSTDVTDKTGREHLSVYAWVIASVVGFCLL VMLFLLKLARHSKFGMKGPASVISNDDDSASPLHHISNGSNTPSSSEGGPDAVIIG

MTKIPVIENPQYFGITNSQLKPDTFVQHIKRHNIVLKRELGEGAFGKVFLAECYNLC

PEQDKILVAVKTLKDASDNARKDFHREAELLTNLQHEHIVKFYGVCVEGDPLIMV

FEYMKHGDLNKFLRAHGPDAVLMAEGNPPTELTQSQMLHIAQQIAAGMVYLASQ HFVHRDLATRNCLVGENLLVKIGDFGMSRDVYSTDYYRVGGHTMLPIRWMPPESI MYRKFTTESDVWSLGVVLWEIFTYGKQPWYQLSNNEVIECITQGRVLQRPRTCPQ

EVYELMLGCWQREPHMRKNIKGIHTLLQNLAKASPVYLDILG

SEQ ID NO. 50: Human BDNF (residues 129-247)

HSDPARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFY ETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSC VCTLTIKRGR

SEQ ID NO. 51: Extracellular domain of human NTRK2 (residues 32-430)

CPTSCKCSASRIWCSDPSPGIVAFPRLEPNSVDPENITEIFIANQKRLEIINEDDVEAY VGLRNLTIVDSGLKFVAHKAFLKNSNLQHINFTRNKLTSLSRKHFRHLDLSELILVG NPFTCSCDIMWIKTLQEAKSSPDTQDLYCLNESSKNIPLANLQIPNCGLPSANLAAP

NLTVEEGKSITLSCSVAGDPVPNMYWDVGNLVSKHMNETSHTQGSLRITNISSDDS GKQISCVAENLVGEDQDSVNLTVHFAPTITFLESPTSDHHWCIPFTVKGNPKPALQ WFYNGAILNESKYICTKIHVTNHTEYHGCLQLDNPTHMNNGDYTLIAKNEYGKDE

KQISAHFMGWPGIDDGANPNYPDVIYEDYGTAANDIGDTTNRSNEIPSTDVTDKTG REH

SEQ ID NO. 52: Signal peptide hBDNF

MGWSCIILFLVATATGVHS

SEQ ID NO. 53: 6xHis-tag with linker hBDNF

GSHHHHHH SEQ ID NO. 54: Signal peptide hNTRK2

MGWSCIILFLVATATGVHS

SEQ ID NO. 55: Twin-strep-tag with linker hNTRK2

SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK

SEQ ID NO. 56: hBDNF[129-247]-6his - 14.48 kDa

MGWSCIILFLVATATGVHSHSDPARRGELSVCDSISEWVTAADKKTAVDMSGGTV

TVLEKVPVSKGQLKQYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRAL TMDSKKRIGWRFIRIDTSCVCTLTIKRGRGSHHHHHH

SEQ ID NO. 57: hNTRK2[32-430]-twin-strep - 47.24 kDa

MGWSCIILFLVATATGVHSCPTSCKCSASRIWCSDPSPGIVAFPRLEPNSVDPENITE

IFIANQKRLEIINEDDVEAYVGLRNLTIVDSGLKFVAHKAFLKNSNLQHINFTRNKL TSLSRKHFRHLDLSELILVGNPFTCSCDIMWIKTLQEAKSSPDTQDLYCLNESSKNIP LANLQIPNCGLPSANLAAPNLTVEEGKSITLSCSVAGDPVPNMYWDVGNLVSKHM NETSHTQGSLRITNISSDDSGKQISCVAENLVGEDQDSVNLTVHFAPTITFLESPTSD HHWCIPFTVKGNPKPALQWFYNGAILNESKYICTKIHVTNHTEYHGCLQLDNPTH

MNNGDYTLIAKNEYGKDEKQISAHFMGWPGIDDGANPNYPDVIYEDYGTAANDI GDTTNRSNEIPSTDVTDKTGREHSAWSHPQFEKGGGSGGGSGGSAWSHPQFEK

SEQ ID NO. 58: 20236-R3P1-F1-VH

MKHLWFFLLLVAAPRWVLSQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAW

NWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPE DTAVYYCARGKNYAMGVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT

PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 59: 20236-R3P3-A11-VH

MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGESLKISCKGSGYSFSNSWIA WVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISVDKSVNTAYLQWSSLKAS DTAFYYCATTGGYSPFDHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT

PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO. 60: 20236-R3P3-H4-VH

MKHLWFFLLLVAAPRWVLSQVQLVQSGGGVVRPGGSLRLSCAASGFTFDDYGMS WVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAE DTAMYYCARGREVAALGKTYYHGLDVWGQGTTVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR

VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO. 61: 20236-R3P3-E1-VH

MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIG WVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASD TAMYYCARPLLGDGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT

PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 62: 20236-R3P3-G5-VH

MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGESLKISCKASGYDFTSYWIG WVRQMPGKGLEWMGVIYPGDSDARYSPSFQGQVTISADKSISTAYLQWSSLKASD TAIYYCARSTASDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH

KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC

VVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK

SEQ ID NO. 63: 20236-R3P3-H4-VL

MVLQTQVFISLLLWISGAYGDIVMTQTPLSLPVTPGEPASISCRSSQSLLHSNGYNY LDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQALQTFTFGPGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG

LSSPVTKSFNRGEC

SEQ ID NO. 64: 20236-R3P1-F1-VL

MVLQTQVFISLLLWISGAYGQSVLTQPPSASGSPGQSVTISCTGTSSDVGLYNYVS

WYQQRPGKAPKLMIYDVTKRPSGVPDRFSGSKSGNTASLTVSGLQTDDEADYYCS SRTLNHIVLFGGGTQLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAV TVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE GSTVEKTVAPTECS

SEQ ID NO. 65: 20236-R3P3-A11-VL

MVLQTQVFISLLLWISGAYGQPVLTQPPSVSEAPRQRVTISCSGSSSNIENNAVNWY QQLPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSASLAISALQSEDEGDYYCGVWD DSLNGYVFGTGTKVTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVT VAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEG

STVEKTVAPTECS SEQ ID NO. 66: 20236-R3P3-E1-VL

MVLQTQVFISLLLWISGAYGSYELTQPPSVSVAPGETAQIPCGRPSIGRESVHWYQ QTPGQAPVLVMYDDNDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWD GVSGPYVFGPGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVT VAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEG STVEKTVAPTECS

SEQ ID NO. 67: 20236-R3P3-G5-VL

MVLQTQVFISLLLWISGAYGQPVLTQPPSLSVSPGQAANIICSGHKLGEKFVSWYQ QKPGQSPVLVVYQDSKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQTWD NGVVFGGGTQLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVA WKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGST VEKTVAPTECS

SEQ ID NO. 68: R3P3-F1 antibody paratope VH part#01 (CDR1):

SSNSA

SEQ ID NO. 69: R3P3-F1 antibody paratope VH part#02 (CDR2):

YYRSKWY

SEQ ID NO. 70: R3P3-F1 antibody paratope VH part#03 (CDR3):

KN

SEQ ID NO. 71: R3P3-F1 antibody paratope VL part#01 (CDR1): residue Y32 and Y34

SEQ ID NO. 72: R3P3-F1 antibody paratope VL part#02 (CDR3):

RTLNH

SEQ ID NO. 73: R3P3-E1 antibody paratope VH part#01 (CDR1):

SFTSYW SEQ ID NO. 74: R3P3-E1 antibody paratope VH part#02 (CDR2):

YPGDSD SEQ ID NO. 75: R3P3-E1 antibody paratope VH part#03 (CDR3): residue K74

SEQ ID NO. 76: R3P3-E1 antibody paratope VH part#04 (CDR4): PLLG

SEQ ID NO. 77: R3P3-H4 antibody paratope VH part#01 (CDR1): NWNGGSTGYAD SVKGRFTIS

SEQ ID NO. 78: R3P3-H4 antibody paratope VH part#02 (CDR3): LGKTY SEQ ID NO. 79: R3P3-H4 antibody paratope VL part#01 (CDR3): LQT

Claims

1. A monoclonal antibody or antigen-binding portion thereof that specifically binds the human NTRK2 (hNTRK2) receptor, in particular the extracellular domain of hNTRK2.

2. The monoclonal antibody or an antigen-binding portion thereof according to claim 1, wherein said antibody or antigen-binding portion thereof shows an equilibrium dissociation constant KD of less than 1 * 10'⁸ M when tested in a BIACORE™ test against the hNTRK2 receptor, preferably of less than 8* 10'⁹ M.

3. The monoclonal antibody or antigen-binding portion thereof according to anyone of claims 1 or 2, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein: said VH has amino acid sequence set forth in SED ID NO: 1 and said VL has amino acid sequence SED ID NO:22; or said VH has amino acid sequence set forth in SED ID NO:2 and said VL has amino acid sequence SED ID NO:23; or said VH has amino acid sequence set forth in SED ID NO:3 and said VL has amino acid sequence SED ID NO:24; or said VH has amino acid sequence set forth in SED ID NO:4 and said VL has amino acid sequence SED ID NO:25; or said VH has amino acid sequence set forth in SED ID NO:5 and said VL has amino acid sequence SED ID NO:26 said VH has amino acid sequence set forth in SED ID NO:6 and said VL has amino acid sequence SED ID NO:27; or said VH has amino acid sequence set forth in SED ID NO:7 and said VL has amino acid sequence SED ID NO:28; or said VH has amino acid sequence set forth in SED ID NO:8 and said VL has amino acid sequence SED ID NO:29; or said VH has amino acid sequence set forth in SED ID NO:9 and said VL has amino acid sequence SED ID NO:30; or said VH has amino acid sequence set forth in SED ID NO: 10 and said VL has amino acid sequence SED ID NO:31; or said VH has amino acid sequence set forth in SED ID NO: 11 and said VL has amino acid sequence SED ID NO:32; or said VH has amino acid sequence set forth in SED ID NO: 12 and said VL has amino acid sequence SED ID NO:33; or said VH has amino acid sequence set forth in SED ID NO: 13 and said VL has amino acid sequence SED ID NO:34; or said VH has amino acid sequence set forth in SED ID NO: 14 and said VL has amino acid sequence SED ID NO:35; or said VH has amino acid sequence set forth in SED ID NO: 15 and said VL has amino acid sequence SED ID NO:36; or said VH has amino acid sequence set forth in SED ID NO: 16 and said VL has amino acid sequence SED ID NO:37; or said VH has amino acid sequence set forth in SED ID NO: 17 and said VL has amino acid sequence SED ID NO:38; or said VH has amino acid sequence set forth in SED ID NO: 18 and said VL has amino acid sequence SED ID NO:39; or said VH has amino acid sequence set forth in SED ID NO: 19 and said VL has amino acid sequence SED ID NONO; or said VH has amino acid sequence set forth in SED ID NO:20 and said VL has amino acid sequence SED ID NON 1; or said VH has amino acid sequence set forth in SED ID NO:21 and said VL has amino acid sequence SED ID NON2.

4. The monoclonal antibody or antigen-binding portion thereof according to anyone of claims 1 to 3, wherein said VL is at least 85% identical in amino acid sequence, preferably at least 95% identical in amino acid sequence, more preferably at least 98% or at least 99% identical in amino acid sequence of:

VH having SED ID NO: 1 and VL having SED ID NO:22; or

VH having SED ID NO:2 and VL having SED ID NO:23; or

VH having SED ID NO:3 and VL having SED ID NO:24; or

VH having SED ID NON and VL having SED ID NO:25; or

VH having SED ID NO: 5 and VL having SED ID NO:26; or VH having SED ID N0:6 and VL having SED ID NO:27; or

VH having SED ID NO:7 and VL having SED ID NO:28; or

VH having SED ID NO: 8 and VL having SED ID NO:29; or

VH having SED ID NO:9 and VL having SED ID NO:30; or

VH having SED ID NO: 10 and VL having SED ID NO:31; or

VH having SED ID NO: 11 and VL having SED ID NO:32; or

VH having SED ID NO: 12 and VL having SED ID NO:33; or

VH having SED ID NO: 13 and VL having SED ID NO:34; or

VH having SED ID NO: 14 and VL having SED ID NO:35; or

VH having SED ID NO: 15 and VL having SED ID NO:36; or

VH having SED ID NO: 16 and VL having SED ID NO:37; or

VH having SED ID NO: 17 and VL having SED ID NO:38; or

VH having SED ID NO: 18 and VL having SED ID NO:39; or

VH having SED ID NO: 19 and VL having SED ID NO:40; or

VH having SED ID NO:20 and VL having SED ID NO:41; or

VH having SED ID NO:21 and VL having SED ID NO:42.

5. The monoclonal antibody or antigen-binding portion thereof according to anyone of claims 1 to 4, wherein said antibody or antigen-binding portion thereof specifically binds an epitope comprising:

(I) residues 288, 289, 291-296, 298-299 and/or 321-324 of SEQ ID NO. 51, or residues 297, 325, 328, 346 of SEQ ID NO. 51, or residues 290, 318-320, 327 and 344 of SEQ ID NO. 51, or residues 287, 300, 315, 326. 345 and/or 347-349 of SEQ ID NO. 51, or

(II) residues 288-291 and/or 321-327 of SEQ ID NO. 51, or residues 320 and 344-346 of SEQ ID NO. 51, or residues 292-294, 298, 319, 342, 343 and/or 347-349 of SEQ ID NO. 51, or residues 263, 287, 295-297, 299, 318 and/or 328 of SEQ ID NO. 51, or

(III) residues 288-289, 322-327 and/or 344-348 of SEQ ID NO. 51, or residues 263, 290, 321, 342, 343 and/or 349 of SEQ ID NO. 51, or residues 262, 291, 320, 328 and/or 350 of SEQ ID NO. 51, or residues 261, 264-266, 269, 292, 294, 296-299, 318, 319, 330 and/or 331 of SEQ ID NO.

51.

(IV) said epitope is between position 288 and 348 of the extracellular domain of human NTRK2 (SEQ ID NO:51).

6. The monoclonal antibody or an antigen-binding portion thereof according to any one of the claims from 1 to 5, for use in a prophylactic or therapeutic treatment of endometriosis or related conditions.

7. The monoclonal antibody or an antigen-binding portion according to any one of the claims from 1 to 6, for use in a prophylactic or therapeutic treatment of conditions mediated to the activity of the hNTRK2 receptor.

8. A pharmaceutical composition comprising one or more monoclonal antibody or antigen-binding portion thereof according to any one of the claims from 1 to 7 and a pharmaceutically acceptable carrier.

9. The composition according to claim 8, for use in a prophylactic or therapeutic treatment of endometriosis or related conditions or in a prophylactic or therapeutic treatment of conditions mediated to the activity of the hNTRK2 receptor.

10. An in vitro method for revealing the presence of the hNTRK2 receptor in a sample comprising the following steps: i) Contacting the antibody or an antigen-binding portion thereof according to any one of the claims from 1 to 7 with said sample; ii) Detecting the binding of said antibody or an antigen-binding portion thereof with the hNTRK2 receptor.

11. An isolated nucleic acid molecule comprising a nucleic acid sequence that encodes the antibody or antigen-binding portion thereof according to any one of claims 1 to 7, or an expression vector comprising said nucleic acid molecule encoding the antibody or antigenbinding portion thereof embodiments according to any one of claims 1 to 7, or a host cell transfected or transformed with said expression vector comprising said nucleic acid molecule encoding the antibody or antigen-binding portion thereof embodiments according to any one of claims 1 to 7. 12. A method of treating or adjuvating the treatment of a patient suffering of endometriosis or related conditions, comprising a step of administering an effective dose of the monoclonal antibody according to anyone of claims 1 to 7.