Anti-LRRC15 antibodies and uses thereof

Abstract

The invention relates to novel anti-LRRC15 antibodies and uses thereof, the antibodies exhibiting specific binding and having higher internalization rates than the anti-LRRC15 antibodies samrotamab and TPP-17421. The invention further provides conjugates, nucleic acids and vectors comprising the antibodies.

Description

[0001] ANTI-LRRC15 ANTIBODIES AND USES THEREOF

[0002] FIELD OF THE INVENTION

[0003] The invention relates to novel anti-LRRC15 antibodies and uses thereof, the antibodies exhibiting specific binding and having higher internalization rates than the anti-LRRC15 antibodies samrotamab and TPP- 17421. The invention further provides conjugates, nucleic acids and vectors comprising the antibodies.

[0004] BACKGROUND OF DISCLOSURE

[0005] Human Leucine-rich repeat containing 15 (LRRC15), also known as LIB (LRR protein induced by P-amyloid treatment) or LFL2, is a 581 amino acid type I transmembrane protein with no obvious intracellular signaling domain (Uniprot number Q8TF66, SEQ ID NO: 1). LRRC15 belongs to the LRR superfamily, which is involved in cell-cell and cell-ECM interaction (Dolan et al. 2007). The LRRC15 gene is located on chromosome 3 at 3q29. Leucine-rich repeats (LRR) are short (19-29 amino acids) sequence motifs present in a large number of proteins of different structures, functions and localization, and the LRR domains generally organize themselves in a horseshoe structure (Ray et al. 2022). Proteins with LRR domains have been involved in a variety of functions including adhesion, receptor-ligand binding and target recognition (Kobe and Deisenhofer 1994; Buchanan and Gay 1996). Many proteins with LRR motifs have recognized functions in the innate immune pathway, such as toll-like receptors (TLR) and NOD-like receptors (NLR), sensing pathogen- associated molecular patterns (PAMPs) through their LRR domain (Ray et al. 2022).

[0006] LRRC15 is mostly not expressed in healthy tissues, except for localized regions within hair follicles, tonsil, stomach (pylorus and cardia regions), spleen (peritrabecular region), osteoblasts and sites of wound healing (Purcell et al. 2018; Ray et al. 2022). LRRC15 may also be expressed at the leading edge of migrating cytotrophoblast cells of the placenta (Satoh, Hata, and Yokota 2002) and in a subset of fibroblasts and lymphatic endothelial cells within the lung (Song et al. 2022). In line with other LRR proteins and given that normal expression of LRRC15 is localized to areas that make up innate immune barriers (placenta, skin, activated fibroblasts in wound and lymphoid tissues such as the spleen), it appears that LRRC15 may play a role in innate immunity (Ray et al. 2022). LRRC15 has also been found to be a novel SARS-CoV-2 spike-binding receptor that can help control viral load and regulate antiviral and antifibrotic transcriptional programs in the context of COVID-19 infection (Loo et al. 2023).

[0007] In cancer tissue, LRRC15 is expressed either only in the tumor stroma (cancer negative - stromal positive) or directly expressed in solid tumors of mesenchymal origin (cancer positive - stromal positive) (Purcell et al. 2018). When present in the tumor stroma, LRRC15 is expressed in cancer- associated fibroblast (CAFs). Cancer negative - stromal positive tumors for LRRC15 include pancreatic, lobular and ductal breast, squamous and adeno lung, head and neck, bladder, ovarian, testicular, hepatocellular, endometrial, renal, gastric and colorectal tumors. Stromal expression has also been observed in metastases from lymph nodes, bone and liver. Tumors of mesenchymal origin cancer positive - stromal positive for LRRC15 include glioblastomas, osteosarcomas, pleiomorphic undifferentiated sarcoma and melanoma.

[0008] Antibodies targeting LRRC15 or their use in the treatment of cancer have been previously published. W02005037999 relates to the treatment of cancer using antibodies binding LRRC15. W02005094348 discloses anti-LRRC15 antibodies including murine antibody M25. W02017095805 and W02017095808 relate to auristatin-based antibody-drug conjugates (ADCs) targeting LRRC15, wherein the antibodies are defined by sequence. Both applications disclose the huM25 (heavy chain SEQ ID NO: 53; light chain SEQ ID NO: 54), which is a humanized antibody version of the murine precursor M25 described in W02005094348. Antibody huM25 has been given the nonproprietary name samrotamab. Other anti-LRRC15 antibodies are disclosed in WO20 18227018 and WO2019084060. WO2021067673 discloses a pH-engineered anti-LRRC15 hul39.10 (hul39.10 was previously disclosed in W02017095805 and W02017095808). WO2021202642 discloses the anti-LRRC15 antibody DUNP19, further developed as a radiolabeled antibody with Cu64 and Lul77 isotopes. WO2022157094 discloses among others the anti-LRRC15 antibody TPP-17421 (heavy chain SEQ ID NO: 55; light chain SEQ ID NO: 56). Incidentally, WO2022157094 in Table 1, Table 2 and Table 4 also discloses that samrotamab shows off-target binding to EPHB6 and is thus a polyreactive antibody. WO2022216653 discloses pH-engineered anti-LRRC15 antibodies derived from antibodies samrotamab, hul39.10 and huAD208.12.1. HuAD208.12.1 was previously disclosed in W02017095805 and W02017095808. WO2024081729 discloses further pH-engineered antibodies 15G7, 24D9 and 29F1. WO2021102332 discloses 13 anti-LRRC15 antibodies (F36, J48, J66, K713, F32, F65, J24, K412, K55, K611, K72, K84, and 14K) conjugated to a TGFbR2 inhibitor. WO2024158047 discloses antibodies binding to various epitopes within amino-acids 21 and 315 of the LRRC15 sequence SEQ ID NO: 1. Each epitope has been defined as one of the first 12 leucine-rich repeats domains (LRR), LRR1 to 12. WO2024158047 further states that the disclosed antibodies do not bind to the LRR13 (amino acid residues 342 to 363 of SEQ ID NO: 1) and LRR14 domains (amino acid residues 366 to 387 of SEQ ID NO: 1). It also discloses antibodies defined by their CDR sequences, and pyrrolobenzodiazepine-conjugated antibody-drug conjugates (ADCs) based on the antibodies.

[0009] Samrotamab has been conjugated to the antimitotic drug monomethyl auristatin E (MMAE) through a protease-cleavable valine-citrulline linker and has been named as the ADC ABBV-085 (Demetri et al. 2021; Purcell et al. 2016). In preclinical experiments, ABBV-085 was able to kill LRRC 15 -positive cancer cells in in vitro systems and demonstrated in vivo efficacy in multiple solid tumor models representing different solid tumor indications expressing LRRC 15 (Purcell et al. 2018). ABBV-085 was tested in a phase I study (trial registration ID: NCT02565758) to assess safety and tolerability, evaluate the PK, and determine the maximum tolerated dose and recommended phase I expansion dose as monotherapy, and in combination with nivolumab or gemcitabine ± / / c / A-paclitaxel (Demetri et al. 2021). ABBV-085 dosing was associated with an acceptable safety profile, and preliminary antitumor activity was seen in patients with osteosarcoma and undifferentiated pleomorphic sarcoma (UPS), classes of tumor types where LRRC 15 can be expressed both on the cancer cell surface and in the stroma.

[0010] As already explained above, samrotamab shows off-target binding to EPHB6, potentially explaining why ABBV-085 was not further tested in clinical trial. Hence, there is the need to identify new anti-LRRC15 antibodies without off-target binding and with improved efficacy.

[0011] DEFINITIONS

[0012] "Antibodies" or “antibody”, also called "immunoglobulins" (Ig), generally comprise four polypeptide chains, two heavy (H) chains and two light (L) chains, and are therefore multimeric proteins, or comprise an equivalent Ig homologue thereof (e.g., a camelid antibody comprising only a heavy chain, single-domain antibodies (sdAb) or nanobody which can be either be derived from a heavy or light chain). The term “antibodies” includes antibody-based binding protein, modified antibody format retaining target binding capacity. The term “antibodies” also includes full length functional mutants, variants, or derivatives thereof (including, but not limited to, murine, chimeric, humanized and fully human antibodies) which retain the essential epitope binding features of an Ig molecule, and includes dual specific, bi specific, multi specific, and dual variable domain Igs. Ig molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) and allotype. Ig molecules may also be mutated e.g. to enhance or reduce affinity for Fey receptors or for the neonatal Fc receptor (FcRn). mAb stands for monoclonal antibody.

[0013] The term "monoclonal antibody", as used herein, refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of different antibodies having different amino acid sequences in their variable domains, particularly their CDRs, which are often specific for different epitopes. The term "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method.

[0014] An "antibody fragment", as used herein, relates to a molecule comprising at least one polypeptide chain derived from an antibody that is not full length and exhibits target binding. Antibody fragments are capable of binding to the same epitope or target as their corresponding full-length antibody. Antibody fragments include, but are not limited to (i) a Fab fragment, which is a monovalent fragment consisting of the variable light (VL), variable heavy (VH), constant light (CL) and constant heavy 1 (CHI) domains; (ii) a F(ab')2fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region (reduction of a F(ab')2fragment result in two Fab’ fragment with a free sulfhydryl group); (iii) a heavy chain portion of a Fab (Fa) fragment, which consists of the VH and CHI domains; (iv) a variable fragment (Fv) fragment, which consists of the VL and VH domains of a single arm of an antibody; (v) a domain antibody (dAb) fragment, which comprises a single variable domain; (vi) an isolated complementarity determining region (CDR); (vii) a single chain Fv fragment (scFv); (viii) a diabody, which is a bivalent, bispecific antibody 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 the complementarity domains of another chain and creating two antigen binding sites; (ix) a linear antibody, which comprises a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementarity light chain polypeptides, form a pair of antigen binding regions; (x) Dual-Variable Domain Immunoglobulin; (xi) other non-full length portions of immunoglobulin heavy and / or light chains, or mutants, variants, or derivatives thereof, alone or in any combination.

[0015] An "antibody-based binding protein", as used herein, may represent any protein that contains at least one antibody-derived VH, VL, or CH immunoglobulin domain in the context of other nonimmunoglobulin, or non-antibody derived components. Such antibody -based proteins include, but are not limited to (i) Fc-fusion proteins of binding proteins, including receptors or receptor components with all or parts of the immunoglobulin CH domains, (ii) binding proteins, in which VH and or VL domains are coupled to alternative molecular scaffolds, or (iii) molecules, in which immunoglobulin VH, and / or VL, and / or CH domains are combined and / or assembled in a fashion not normally found in naturally occurring antibodies or antibody fragments.

[0016] The term "modified antibody format", as used herein, encompasses antibody-drug-conjugates (ADCs), polyalkylene oxide-modified scFv, monobodies, diabodies, camelid antibodies, domain antibodies, bi- or trispecific antibodies, IgA, or two IgG structures joined by a J chain and a secretory component, shark antibodies, new world primate framework and non-new world primate CDR, IgG4 antibodies with hinge region removed, IgG with two additional binding sites engineered into the CH3 domains, antibodies with altered Fc region to enhance or reduce affinity for Fc gamma receptors, dimerized constructs comprising CH3, VL, and VH, and the like.

[0017] The terms "cancer", "cancerous", “tumor”, “neoplastic disease” or "malignant" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.

[0018] The term “epitope” refers to the region of an antigen to which an antibody binds preferentially and specifically. A monoclonal antibody binds preferentially to a single specific epitope of a molecule that can be molecularly defined.

[0019] The term “linker” refers to a molecule or group of molecules (e.g., peptide) connecting two polypeptides. The linker may be comprised of a single linking molecule or may comprise a linking molecule and a spacer molecule, intended to separate the linking molecule and a compound by a specific distance.

[0020] The term “spacer” as used herein refers to a peptide that joins the proteins comprising a fusion protein. Generally, a spacer has no specific biological activity other than to join the proteins or to preserve some minimum distance or other spatial relationship between them. However, the constituent amino acids of a spacer may be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity of the molecule.

[0021] The term “chimeric” as used herein, refers to both nucleic acid and protein which are the products of gene fusion by genetic manipulation. Gene manipulation methods are well known in the art (e.g., see Green and Sambrook (Green and Sambrook 2012)).

[0022] The terms “peptide,” “protein,” and “polypeptide” are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another.

[0023] The term "subject" includes any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, cynomolgus monkey and human). "Patient" or "subject" refers to any single subject for which therapy is desired or that is participating in a clinical trial, epidemiological study or used as a control, including humans and mammalian veterinary patients such as mouse, rat and cynomolgus monkey. As used herein, the term “patient” refers to a human or non-human animal. Typically, the terms “subject”, “individual”, and “patient” may be used interchangeably herein in reference to a subject. As such, a “patient” includes a human or non-human mammal that is being treated and / or diagnosed for / with a disease, such as cancer. In a preferred embodiment “subject” refers to a human patient being diagnosed for cancer.

[0024] The term “pharmaceutically effective amount” refers to an amount of a therapeutic agent effective to “treat” a cancer in a subject or mammal by achieving at least one positive therapeutic effect, such as for example, reduced number of cancer cells, reduced tumor size, reduced rate of cancer cell infiltration into peripheral organs, and reduced rate of tumor metastasis or tumor growth. Positive therapeutic effects in cancer can be measured in a number of ways, see Weber (2009).

[0025] Terms “treat” or “treating” means to administer a therapeutic agent, such as a composition containing any of the antibodies or antigen binding fragments of the present invention, internally or externally to a subject or patient having one or more disease symptoms, or being suspected of having a disease, for which the agent has therapeutic activity. Typically, the agent is administered in an amount effective to alleviate one or more disease symptoms in the treated subject or population, whether by inducing the regression of or inhibiting the progression of such symptom(s) by any clinically measurable degree. The amount of a therapeutic agent that is effective to alleviate any particular disease symptom (also referred to as the “therapeutically effective amount”) may vary according to factors such as the disease state, age, and weight of the patient, and the ability of the drug to elicit a desired response in the subject. Whether a disease symptom has been alleviated can be assessed by any clinical measurement typically used by physicians or other skilled healthcare providers to assess the severity or progression status of that symptom.

[0026] The term "variant" refers to an amino acid or peptide sequence having conservative amino acid substitutions, non-conservative amino acid substitutions (i.e., a degenerate variant), deletions or insertions, whereas conservative substitutions are preferred. Conservative amino acid substitution refer to a substation of an amino acid, where an aliphatic amino acid (i.e. Glycine, Alanine, Valine, Leucine, Isoleucine) is substituted by another aliphatic amino acid, a hydroxyl or sulfur / selenium- containing amino acid (i.e., Serine, Cysteine, Selenocysteine, Threonine, Methionine) is substituted by another hydroxyl or sulfur / selenium-containing amino acid, an aromatic amino acid (i.e.. Phenylalanine, Tyrosine, Tryptophan) is substituted by another aromatic amino acid, a basic amino acid (i.e. Histidine, Lysine, Arginine) is substituted by another basic amino acid, or an acidic amino acid or its amide (Aspartate, Glutamate, Asparagine, Glutamine) is replaced by another acidic amino acid or its amide. Variants substantially maintain the biological activity of the wildtype protein or domain of a protein, wherein substantially in this context shall mean at least 10% of the respective biological activity of such wildtype protein or domain of a wildtype protein, preferably at least 50%, more preferably 80%, especially 90%.

[0027] Where the term “comprising” is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term “consisting of’ is considered to be a preferred embodiment of the term “comprising of’. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group, which preferably consists only of these embodiments. Where an indefinite or definite article is used when referring to a singular noun, e.g. “a”, “an” or “the”, this includes a plural of that noun unless something else is specifically stated.

[0028] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, z.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, z.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0029] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, z.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (z.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either”, “one of’, “only one of’, or “exactly one of’. “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0030] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non- limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0031] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0032] Technical terms are used by their common sense. If a specific meaning is conveyed to certain terms, definitions of terms will be given in the following in the context of which the terms are used. All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents cited or incorporated by reference, and / or ordinary meanings of the defined terms.

[0033] FIGURES

[0034] FIG. 1 : Expression of LRRC15 at the cell membrane of the U-118, G-292 and 143B cell lines. Representative flow cytometry histograms for each cell line are shown, using the 2A6 VH4 VL3 m Ab to detect LRRC 15.

[0035] FIG. 2: Binding of mAbs measured by flow cytometry to cell lines with different levels of LRRC15 expression. mAbs 2A6 (named chim 2A6), 2A6 VH1 VL4, 2A6 VH4 VL3, TPP-17421, samrotamab (named hABA) and casivirimab (monoclonal antibody against spike protein of SARS-CoV-2, used here as isotype control) were incubated at different concentrations in the presence of: the U-118 cell line (high expression of LRRC 15) (2 A), the G-292 cell line (intermediate expression of LRRC 15) (2B) and the 143B cell line (limited to no expression of LRRC 15) (2C). After incubation of the mAbs with the cell line, a phycoerythrin (PE)-conjugated secondary antibody goat anti-human IgG Fc and DAPI were added to the cell suspension and binding was measured by flow cytometry. Binding is shown as percentage of PE-positive cells (upper panel) and as mean fluorescence intensity (MFI, lower panel) from live cells in dependence on the concentration of the respective mAbs.

[0036] FIG. 3: mAbs internalization by cell lines with different levels of LRRC15 expression. mAbs 2A6 (named chim 2A6), 2A6 VH1 VL4, 2A6 VH4 VL3, TPP-17421, samrotamab (named ABA) and casivirimab (isotype control), all labelled with the secondary anti-human pHrodo Red Fab fragment, where incubated at different concentrations in the presence of the U-118 cell line (high expression of LRRC15) (3A), the G-292 cell line (intermediate expression of LRRC15) (3B) and the 143B cell line (limited to no expression of LRRC15) (3C). Internalization was measured by flow cytometry after 8 h and 24 h of incubation of the mAbs with the cells. Internalization is shown as percentage of pHrodo Red-positive cells (upper panel) and as mean fluorescence intensity (MFI) from live cells (lower panel) for the respective antibodies.

[0037] FIG. 4: mAbs epitope mapping

[0038] Visual representation of the epitope bound by mAbs 2A6 VH1 VL4 and 2A6 VH4 VL3. The number on top shows the amino-acid position within the LRRC15 protein and the darkened / grayed area shows the part of the LRRC15 protected from hydrogen-deuterium exchange when the anti- LRRC15 mAb is bound. The bound region corresponds to the epitope of the antibody.

[0039] FIG. 5: Immunohistochemistry pictures of different cancer tissues showing the expression levels of LRRC15 in cancer positive - stroma negative tumors (A and B) and cancer negative - stroma positive tumors (C).

[0040] Representative immunohistochemistry pictures of tissue samples from: chondrosarcoma, fibrosarcoma, leiomyosarcoma (5A, top to bottom); osteosarcoma, soft tissue sarcoma and undifferentiated pleomorphic sarcoma (5B, top to bottom); and head & neck squamous cell sarcoma and non-small squamous cell lung cancer (5C, top to bottom). Tissues samples were stained with the anti-LRRC15 rabbit recombinant antibody - clone E4X8J (Cell Signaling Technology, catalog number 50546S).

[0041] FIG. 6: ADCC activity

[0042] The antibody-dependent cellular cytotoxicity (ADCC) of mAb 2A6 VH4 VL3 and of a control mAb, both with and without the L234A / L235A mutation (LALA), was tested in A549 cells expressing LRRC15 (top), the target of the control mAb (middle) or not expressing an mAb target (A549 WT cells, bottom). ADCC activity was measured using an LDH assay kit for cytotoxicity. ADCC activity is shown as percentage of dead cells over increased concentration of mAb.

[0043] DESCRIPTION OF THE INVENTION

[0044] The inventors have identified novel anti-LRRC15 antibodies as further described in the following embodiments, which exhibit specific binding to a peptide of the amino acid sequence SEQ ID NO: 48 and have higher internalization rates than the anti-LRRC15 antibodies samrotamab and TPP- 17421.

[0045] The invention provides an antibody or fragment thereof binding to a peptide of the amino acid of sequence SEQ ID NO: 48. The peptide of sequence SEQ ID NO: 48 is the epitope of the antibodies of the invention. The epitope of an antibody can be determined by multiple methods (Nilvebrant and Rockberg 2018). More specifically, the epitope can be determined by hydrogen-deuterium exchange mass spectrometry, as shown in Example 5 and FIG. 4. The invention relates to any antibody binding the epitope defined by a peptide of the amino acid sequence SEQ ID NO: 48 or competing with mAbs 2A6 (chimeric), 2A6 VH1 VL4 (humanized) and / or 2A6 VH4 VL3 (humanized) for the binding to LRRC 15. The three mAbs 2A6, 2A6 VH1 VL4 and 2A6 VH4 VL3 are jointly referred to 2A6 mAbs. Epitope binning may be used to determine whether given antibodies bind to the same epitope, as disclosed in Chan BM et al (2018).

[0046] In another embodiment, the invention provides an antibody or fragment thereof binding to an epitope that has at least a portion thereof within a region consisting of amino acid residues at positions 346 to 364 of SEQ ID NO: 1.

[0047] In yet another embodiment, the invention provides an antibody or fragment thereof binding to an epitope that has at least a portion thereof within a region consisting of amino acid residues at positions 342 to 364 of SEQ ID NO: 1, and comprising the LRR13 domain of LRRC15.

[0048] In another embodiment, the invention provides an antibody or fragment thereof binding to an epitope that overlaps with the epitope consisting of amino acid residues at positions 346 to 364 of SEQ ID NO: 1.

[0049] In yet another embodiment, the invention provides an antibody or fragment thereof binding to an epitope that overlaps with the epitope consisting of amino acid residues at positions 342 to 364 of SEQ ID NO: 1 and comprises the LRR13 domain of LRRC15 Importantly, Example 7 and Table 12 and Table 13 show that, unlike samrotamab also binding EPHB6, the 2A6 mAbs bind specifically to LRRC15, and do not bind specifically to any other antigen.

[0050] The invention also provides an antibody or fragment thereof binding to LRRC15, which comprises HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 10, SEQ ID NO: 3 and SEQ ID NO: 4, respectively and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 11, SEQ ID NO: 6 and SEQ ID NO: 7, respectively. The sequence of the CDRs is here defined by the Contact numbering scheme. The Contact numbering scheme is based on an analysis of the available complex crystal structures of known antibodies (http: / / www.bioinf.org.uk / abs / info.html).

[0051] In one embodiment, the invention provides an antibody or fragment thereof binding to LRRC15, which comprises HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 10, SEQ ID NO: 3 and SEQ ID NO: 4, respectively and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 11, SEQ ID NO: 6 and SEQ ID NO: 7, respectively, and wherein the antibody is binding to the peptide of the amino acid sequence of SEQ ID NO: 48.

[0052] The invention also provides an antibody or fragment thereof binding to LRRC15 comprising the heavy chain HCDR3 sequence of SEQ ID NO: 4 and the light chain LCDR3 sequence of SEQ ID NO: 7 or the respective HCDR3 and LCDR3 of Table 1 and Table 2, using the Kabat, AbM, Chothia or IMGT CDR numbering scheme.

[0053] In another embodiment, the invention provides for an antibody or fragment thereof, comprising: a. HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, respectively; or b. HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 8, SEQ ID NO: 3 and SEQ ID NO: 4, respectively and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 9, SEQ ID NO: 6 and SEQ ID NO: 7, respectively.

[0054] The invention further provides for an antibody or fragment thereof binding to LRRC15, which comprises the antibody heavy chain CDRs defined in Table 1 and the antibody light chain CDRs defined in Table 2, with CDRs defined using the Kabat CDR numbering scheme (Dondelinger et al. 2018).

[0055] The invention provides in another embodiment for an antibody or fragment thereof binding to LRRC15, which comprises the antibody heavy chain CDRs defined in Table 1 and the antibody light chain CDRs defined in Table 2, with CDRs defined using the AbM CDR numbering scheme (Dondelinger et al. 2018).

[0056] The invention further provides in another embodiment for an antibody or fragment thereof binding to LRRC15, which comprises the antibody heavy chain CDRs defined in Table 1 and the antibody light chain CDRs defined in Table 2, with CDRs defined using the Chothia CDR numbering scheme (Dondelinger et al. 2018).

[0057] In yet another embodiment, the invention provides in another embodiment for an antibody or fragment thereof binding to LRRC15, which comprises the antibody heavy chain CDRs defined in Table 1 and the antibody light chain CDRs defined in Table 2, with CDRs defined using the IMGT CDR numbering scheme (Dondelinger et al. 2018).

[0058] It is understood that any antibody of the invention having CDRs defined by any of the CDRs numbering schemes binds to LRRC15 at the identical epitope, the peptide of the amino acid sequence of SEQ ID NO: 48. In any embodiment of the invention, one or more of HC CDR1-3 and / or one or more of LC CDR1-3 respectively has up to 1 amino acid exchange. In any embodiment of the invention, one or more of HC CDR1-3 and / or one or more of LC CDR1-3 respectively has up to 2 amino acid exchanges. In any embodiment of the invention, one or more of HC CDR1-3 and / or one or more of LC CDR1-3 respectively has up to 3 amino acid exchanges.

[0059] Table 1: VH CDRs

[0060]

[0061] Table 2: VL CDRs

[0062] In another embodiment, the invention provides for an antibody or fragment thereof having the CDRs of Table 1 and Table 2 and further comprising: a. a VH sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 42; b. a VH sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 44; or c. a VH sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 46 and d. a VL sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 43; e. a VL sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 45; or f. a VL sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 47

[0063] In a preferred embodiment, the invention provides for an antibody comprising: a. a VH sequence of SEQ ID NO: 42; b. a VH sequence of SEQ ID NO: 44; or c. a VH sequence of SEQ ID NO: 46; and d. a VL sequence of SEQ ID NO: 43; e. a VL sequence of SEQ ID NO: 45; or f. a VL sequence of SEQ ID NO : 47.

[0064] In yet another embodiment, the invention provides for an antibody or fragment thereof comprising: a. a VH sequence of SEQ ID NO: 42 and a VL sequence of SEQ ID NO: 43; b. a VH sequence of SEQ ID NO: 44 and a VL sequence of SEQ ID NO: 45; or c. a VH sequence of SEQ ID NO: 46 and a VL sequence of SEQ ID NO: 47.

[0065] The invention further provides for an antibody or fragment thereof consisting of: a. a heavy chain sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 58 and a light chain sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 60; b. a heavy chain sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 61 or SEQ ID NO: 62 and a light chain sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 64; or c. a heavy chain sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 65 or SEQ ID NO: 66 and a light chain sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 68; preferably consisting of: d. the heavy chain sequence of SEQ ID NO: 57 or SEQ ID NO: 58 and light chain sequence of SEQ ID NO: 60; e. the heavy chain sequence of SEQ ID NO: 61 or SEQ ID NO: 62 and light chain sequence of SEQ ID NO: 64; or f. the heavy chain sequence of SEQ ID NO: 65 or SEQ ID NO: 66 and light chain sequence of SEQ ID NO: 68.

[0066] These antibodies may further be modified in the carboxyterminal end of the heavy and / or light chain, e.g., by the addition of protein tags which may be useful for conjugation of the antibody with an additional moiety. Such a tag may be a peptide of amino acid sequence of SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73 or SEQ ID NO: 74. Flexible amino acid linkers may also be added between the antibody and the tag. The conjugated moiety (additional moiety) may be a radioligand, a toxin, a fluorescent tag or other peptides such as biotin, streptavidin or any other peptide tag known in the art. Another potential modification of the carboxyterminal end of the heavy chains is the deletion of the terminal lysin.

[0067] In another embodiment, the invention provides an antibody or fragment thereof binding to LRRC15, the antibody being an IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, synthetic IgG, IgM, F(ab)2, Fv, scFv, IgGACH2, F(ab’)2, scFvCH3, Fab, VL, VH, scFv4, scFv3, scFv2, dsFv, scFv-Fc, (SCFV)2, a non-depleting IgG, a diabody, a bivalent antibody or Fc-engineered versions thereof. In a preferred embodiment, the antibody is an IgGl type of antibody. The Fc region of immunoglobulins interacts with multiple Fey receptors (FcyR) and complement proteins (e.g. Clq), and mediates immune effector functions, such as elimination of targeted cells via antibodydependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) or complement-dependent cytotoxicity (CDC). For therapeutic approaches, it may be beneficial to enhance or silence Fc related effector functions. The antibodies of the invention may have reduced FcyR and Clq binding via the L234A / L235A mutations in the Fc domain (see also Table 3), as disclosed in SEQ ID NO: 58, SEQ ID NO: 62 and SEQ ID NO: 66. The antibodies of the invention may have even further reduced FcyR and Clq binding via the L234A / L235A / P329G mutations in the Fc domain (see also Table 3), as disclosed in SEQ ID NO: 59, SEQ ID NO: 63 and SEQ ID NO: 67.

[0068] The type of immunoglobulin (IgA, IgD, IgE, IgG, IgM) may be selected according to the desired effector function of the antibody related to the Fc domain. One may also employ a synthetic immunoglobulin, such as an immunoglobulin with the IgG2 amino acids 118 to 260 and the IgG4 amino acids 261 to 447 or an IgG2 variant with point mutations from IgG4 (e.g. H268Q / V309L / A30S / P331S). Such synthetic immunoglobulins reduce effector functions of the antibody. Fc-engineered immunoglobulins may also be employed to modulate antibody effector function. Table 3 shows examples of such Fc engineering. Expression in production cell lines with altered fucosylation may also impact FcyR binding.

[0069] Table 3: Examples of modifications to modulate antibody effector function. Unless otherwise noted, the mutations are on the IgGl subclass (Wang, Mathieu, and Brezski 2018).

[0070] Half-life of antibodies may also be modulated. The Fc domain plays a central role in the stability and serum half-life of antibodies. For therapeutic approaches, antibody half-life may be reduced by using an antibody fragment missing the Fc domain or with a truncated Fc domain, such as F(ab)2, scFv, IgGACH2, F(ab’)2, scFvCH3, Fab, VL, VH, scFv4, scFv3, scFv2, dsFv, Fv, scFv-Fc or (SCFV)2.

[0071] The terminal lysine of the heavy chain is preferably removed to increase homogeneity of an antibody preparation.

[0072] The antibodies may also be in the form of diabodies or bivalent antibodies. Diabodies or bivalent antibodies may be used to increase the affinity to the target allowing lower dosage. Functional fragments missing the Fc domain or with truncated Fc domains may also be used in the development of other therapeutic approaches such as chimeric antigen receptor T cell (CAR T cells) or bispecific T cell engagers (BiTEs). In CAR constructs, one VH and one VL domain are typically connected by a short peptide linker to form a single-chain variable fragment (scFv), and the scFv fragment is further linked to a transmembrane domain and an intracytoplasmic T cell immunoreceptor tyrosine-based activation motif (from e.g. CD3Q and further domains of costimulatory molecules (from e.g. CD28, 4-1BB (CD127), or 0X40) (Chang and Chen 2017). The VH and VL domains used in the scFv fragment may be the ones of the 2A6 mAb of SEQ ID NO: 42 and SEQ ID NO: 43, respectively, or the 2A6 VH4 VL3 mAb of SEQ ID NO: 44 and SEQ ID NO: 45, respectively or of the 2A6 VH1 VL4 mAb of SEQ ID NO: 46 and SEQ ID NO: 47, respectively. Bispecific antibodies / bispecific T cell engagers typically consist of the fusion of two scFv of two different antibodies. One scFv domain may be of the isolated antibodies binding LRRC15 as mentioned above for the CAR constructs, while the other scFv domain may be from an antibody that binds, e.g., to CD3, CD16, NKG2D, NKp46, CD2, CD28 or CD25. Ample guidance on BiTE antibody formats and other bispecific antibody formats used for T-cell redirecting may be found in the review by Diego Ellerman (2019).

[0073] In yet another embodiment, the invention relates to an antibody that competes for binding with an antibody described herein. In one embodiment, the antibody competes for binding with an antibody comprising a heavy chain of sequence SEQ ID NO: 57 and a light chain of sequence SEQ ID NO: 60, a heavy chain sequence of SEQ ID NO: 61 and a light chain of sequence SEQ ID NO: 64 or heavy chain of sequence SEQ ID NO: 65 and a light chain of sequence SEQ ID NO: 68. Suitable methods to detect binding of antibodies to the same antigen include approaches to map the antigenantibody interactions. Such approaches have been described in Abbott et al. (2014) and in Example 5 below. Suitable methods to detect competition include competitive assays by epitope binning, as described in Abdiche et al. (2009). Suitable methods for detecting competitive inhibition include ELISA assays.

[0074] In another embodiment, the invention provides an antibody or fragment thereof binding to LRRC15, wherein the antibody or fragment thereof is humanized. Humanization of monoclonal antibodies is well-established. The Handbook of Therapeutic Antibodies, Second Edition, gives ample information on humanization of monoclonal antibodies (Saldanha 2014), bioinformatics tools for analysis of such antibodies (Martin and Allemn 2014) and development and manufacture of therapeutic antibodies (Jacobi et al. 2014). The examples below show that binding properties of the humanized antibodies 2A6 VH1 VL4 and 2A6 VH4 VL3 were not affected when compared to the chimeric antibody 2A6. Surprisingly, humanization improved the internalization of the 2A6 VH1 VL4 and 2A6 VH4 VL3 antibodies, compared to chimeric antibody 2A6, as shown in Example 3 and FIG. 3.

[0075] In another embodiment, the antibody or fragment thereof is an isolated antibody or isolated fragment binding to LRRC15. The isolated antibody may be produced based on its amino acid sequence in various expression systems. Ample guidance can be found in Jacobi et al. (2014).

[0076] In yet another embodiment, the invention provides an antibody or fragment thereof exhibiting a higher internalization rate in cells expressing LRRC15 as compared to antibody TPP- 17421 (heavy chain of sequence SEQ ID NO: 55 and light chain of sequence SEQ ID NO: 56) or antibody samrotamab (heavy chain of sequence SEQ ID NO: 53 and light chain of sequence SEQ ID NO: 54). The internalization of an antibody can be measured by flow cytometry, when the antibody is bound by a pH sensitive fluorophore such as a pHrodo-labeled secondary antibody recognizing the primary antibody. Internalization may also be measured by live-cell microscopy with fluorophore- labeled antibodies. Results of internalization measured by flow cytometry are shown in Example 3. Surprisingly, all three antibodies of the invention have a better internalization rate than samrotamab and TPP-17421. The internalization rate of all 2A6 mAbs is higher at measured time points (8 h and 24 h) than the internalization rate of TPP-17421 or samrotamab when the cell lines express LRRC15 (U-118 and G-292 cell lines) (see Table 9 in Example 3). No difference in internalization is seen when the cell line does not express LRRC15 (143B cell line), indicating that binding to LRRC15 of the mAbs is critical for internalization, and that internalization does not occur via pinocytosis or other forms of endocytosis not dependent on binding of an antibody to its target. When internalization is a critical component of the mAbs efficacy, the 2A6 mAbs may be superior as therapeutic antibodies to TPP-17421 and samrotamab.

[0077] In one embodiment, the invention relates to anti-LRRC15 antibodies or fragments thereof exhibiting a higher internalization rate in cells expressing LRRC15 as compared to antibody TPP- 17421 (heavy chain SEQ ID NO: 55 and light chain SEQ ID NO: 56) or antibody samrotamab (heavy chain SEQ ID NO: 53 and light chain SEQ ID NO: 54), where the invention the internalization rate is measured by flow cytometry on the U-118 glioblastoma cells line or G-292 osteosarcoma cell line expressing LRRC15. As shown in Example 1, the U-l 18 cell line has a high expression of LRRC15 and the G-292 cell line has an intermediate level expression of LRRC15. The inventors have now shown that the antibodies 2A6, 2A6 VH4 VL3 and 2A6 VH1 VL4 of the invention show better internalization after 8 h and 24 h of exposure to the antibodies than the anti- LRRC15 antibodies TPP- 17421 and samrotamab (see Example 3 below and FIG. 3). As seen in Table 9, when the internalization rate is normalized to the internalization of the benchmark samrotamab at 24 h in U-118 cells, all 2A6 mABs have a higher internalization rate than samrotamab. At all timepoints and in all cell lines expressing LRRC15, the best internalizing antibody is the humanized antibody 2A6 VH1 VL4, followed closely by the humanized antibody 2A6 VH4 VL3 and the chimeric antibody 2A6. All three 2A6 mAbs have also a better internalization rate than TPP- 17421. Furthermore, the internalization rate is proportional to the expression levels of LRRC15 (highest in U-118 cell with the highest expression of LRRC15, less high in the G-292 cell line with intermediate expressing of LRRC15 compared to the U-118 cell line, almost no internalization with the 143B cell line with low to no expression of LRRC15), showing that internalization is clearly depending on binding to LRRC15 of the 2A6 mAbs. The epitope on LRRC15 bound by the mAbs may provide the internalization advantage of the 2A6 mAbs over samrotamab or TPP-1741, especially since the binding affinity of the 2A6 mAbs and samrotamab or TPP-1741 to LRRC15 is within a similar range. Thus, the effect may be attributed to the epitope recognized by these antibodies.

[0078] In one embodiment, the invention relates to anti-LRRC15 antibodies or fragments thereof exhibiting a higher internalization rate in cells expressing LRRC15 as compared to antibody TPP- 17421 (heavy chain SEQ ID NO: 55 and light chain SEQ ID NO: 56) or antibody samrotamab (heavy chain SEQ ID NO: 53 and light chain SEQ ID NO: 54), wherein the measured internalization rate of the antibody or fragment thereof is at least 10% higher, at least 20% higher, at least 40% higher at least 50% higher or at least 70% higher than the internalization rate of antibody TPP- 17421 or is at least 10% higher, at least 20% higher, at least 40% higher at least 50% higher or at least 70% higher than the internalization rate of antibody samrotamab. Preferably, internalization is increased at most at 80%. In a preferred embodiment, the internalization rate is measured in the U-118 cell line.

[0079] In yet another embodiment, the invention provides for an antibody or fragment thereof binding to LRRC15, the antibody inducing target-specific killing of tumor cell via its ADCC (antibodydependent cellular cytotoxicity) and / or CDC (complement-dependent cytotoxicity) activity. ADCC and CDC activity may be measured as shown Bruckheimer et al (Bruckheimer et al. 2009) and Wang et al (Wang et al. 2020), respectively. In one embodiment, the antibodies of the invention have a higher ADCC / CDC activity than antibody TPP-17421 or antibody samrotamab. In another embodiment, the antibodies of the invention and antibodies TPP-17421 or antibody samrotamab have a similar ADCC / CDC activity. In yet another embodiment, antibody TPP-17421 or antibody samrotamab have a higher ADCC / CDC activity than the antibodies of the invention. With respect to modifying ADCC / CDC activity it is referred to Table 3.

[0080] In another embodiment, the invention provides for an antibody or fragment thereof binding to LRRC15, the antibody having attenuated ADCC and / or CDC activity. ADCC / CDC activity of an antibody may be attenuated by mutations in the Fc domain of the antibody such as L234A / L235A or L234A / L235A / P329G mutations when the antibody is in the IgGl format, or L235A or F234A / L235A when the antibody is in the IgG4 format, or H268Q / V309L / A330S / P331S or V234A / G237A / P238S / H268A / V309L / A330S / P331S when the antibody is in the IgG2 format. ADCC / CDC activity of an antibody may also be attenuated by producing an a-glycosylated antibody, by removing the glycan at N297. ADCC activity may be measured as disclosed in Bruckheimer et al. (2009).

[0081] In one embodiment, the ADCC / CDC activity of an antibody may be attenuated by mutations in the Fc domain of the antibody via the L234A / L235A mutation, preferably of an IgGl antibody. The CHi-Fc domain of the antibody may have the sequence of SEQ ID NO: 51.

[0082] In another embodiment, the ADCC / CDC activity of an antibody may be attenuated by mutations in the Fc domain of the antibody via the L234A / L235A / P329G mutation, preferably of an IgGl antibody. The CHi-Fc domain of the antibody may have the sequence of SEQ ID NO: 52.

[0083] In one embodiment, the invention provide a conjugate comprising one of the 2A6 antibodies or fragment thereof binding to LRRC15, further comprising by covalent linkage at least one of the following moi eties: i) a linker ii) a tag iii) an antigen-binding moiety iv) a toxin v) an immune-modifying moiety vi) a chelating agent suitable to complex a radioligand vii) a chromophore or fluorophore.

[0084] Means to obtain such conjugate have been previously described (Chis et al. 2024; Tsuchikama et al. 2024). Linkers allow to conjugate a toxin, chelating agent suitable to complex a radioligand, chromophore or fluorophore to the antigen-binding moiety. Linkers may be cleavable (enzymatically or chemically) or non-cleavable. Tags may be used to attach the linker to the antigen-binding moiety, such as the tags described above (SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73 or SEQ ID NO: 74). Toxins or payloads may be toxins inducing, once internalized and released in the target cancer cell, microtubule polymerization inhibitors such as maytansinoids or auri statins or DNA-affecting agents such as topoisomerase inhibitors or DNA intercalators. A chromophore or fluorophore may be used for imaging purposes or flow cytometry. The immune- modifying moiety may be a cytokine (Shi, Liu, and Lu 2024), a toll-like receptor agonists (Ackerman et al. 2021) or any compounds capable of modifying the immune system, including inducing immunogenic cell death (Kroemer et al. 2022). Ample guidance for selecting a chelating agent suitable to complex a radioligand may be found in Price and Orvig (Price and Orvig 2014). An antibody with two antigen-binding moieties forms a bi-specific antibody. The antigen-biding moiety may be binding to a different target than LRRC15, such as target expressed on T cells (Fenis et al. 2024) or target expressed on tumor cells (Klein et al. 2024).

[0085] In yet another embodiment, the invention provides nucleic acid sequences encoding that antibody of fragment thereof that bind LRRC15. The nucleic acid sequences may encode for the CDRs alone, for the VH and VL regions, or for the entire heavy and light chains of the antibodies. These nucleic acid sequences may be derived from the amino acid sequences of the antibodies. The nucleic acid sequence may also encode for F(ab)2, Fv, scFv, IgGACH2, F(ab’)2, scFvCH3, Fab, VL, VH, scFv4, scFv3, scFv2, dsFv, scFv-Fc, (scFv)2, a non-depleting IgG, a diabody, a bivalent antibody or Fc-engineered versions thereof. The encoded immunoglobin may be an IgAl, IgA2, IgD, IgE, IgGl, IdG2, IgG3, IgG4, synthetic IgG, IgM or mutated and Fc-engineered versions thereof.

[0086] In yet another embodiment, the nucleic acid sequence may also encode a CAR (chimeric antigen receptor) construct that binds to LRRC15. Ample guidance on construction of CAR T cells may be found in Chang and Chen (2017) or June and Sadelain (June and Sadelain 2018). Common designs of CARs are shown in Figure 1 and Box 1 of Chen (2017). In one embodiment, the invention provides a T cell that has been genetically engineered to produce an artificial T-cell receptor, e.g., a chimeric antigen receptor (CAR), wherein the artificial T-cell receptor comprises the antibody or functional fragment thereof of the present invention that binds to LRRC15.

[0087] In yet another embodiment, the invention provides a tumor-specific antibody -based binding protein that specifically binds to LRRC15. Such binding protein may contain at least an LRRC15 binding domain and at least one other protein domain not related to antibodies. The term antibody also includes modified antibody formats that bind to LRRC15.

[0088] The invention also provides an expression vector comprising a nucleic acid of the invention or a degenerate nucleic acid as a result of codon degeneracy. The expression vector may be an expression vector for protein expression in mammalian cells, bacteria, fungal or insect cells, and chosen for the type of host cell bearing the expression vector comprising the nucleic acid encoding the antibodies or functional fragments thereof. Ample guidance for the construction of such vectors may be found in Green and Sambrook (Green and Sambrook 2012).

[0089] In another embodiment, the invention provides a host cell comprising a nucleic acid or an expression vector of the present invention. The host cell may be a mammalian cell or cell line, a bacterial cell, a fungal cell or an insect cell.

[0090] In another embodiment, the invention relates to an antibody or fragment thereof binding to LRRC15, the nucleic acid encoding the antibody or fragment thereof, the vector comprising the nucleic acid or the host cells comprising the nucleic acid or the vector, for use as a medicament, preferably for use in the treatment of a subject that is suffering from a neoplastic disease. Preferably, the antibody or fragment thereof binding to LRRC15, the nucleic acid encoding the antibody or fragment thereof, the vector comprising the nucleic acid are formulated with a pharmaceutically acceptable buffer and at least one pharmaceutically acceptable excipient.

[0091] In another embodiment, the invention relates to an antibody or fragment thereof binding to LRRC15, the nucleic acid encoding the antibody or fragment thereof, the vector comprising the nucleic acid or the host cells comprising the nucleic acid or the vector, for use in the treatment of a subject that is at risk of developing a neoplastic disease, and / or for use in the treatment of a subject being diagnosed for a neoplastic disease. In one embodiment, the invention relates to an antibody or fragment thereof binding to LRRC15, the nucleic acid encoding the antibody or fragment thereof, the vector comprising the nucleic acid or the host cells comprising the nucleic acid or the vector for use in treatment.

[0092] In one embodiment, the invention relates to an antibody or fragment thereof binding to LRRC15, the nucleic acid encoding the antibody or fragment thereof, the vector comprising the nucleic acid or the host cells comprising the nucleic acid or the vector for use in the treatment of a neoplastic disease.

[0093] The disclosed antibodies or fragments thereof may be used as monotherapy. In a preferred embodiment, the disclosed antibodies or fragments thereof are used in combination with the established standard of care of the neoplastic disease.

[0094] The neoplastic disease may be at least one disease selected from the group consisting of glioblastomas, osteosarcomas, undifferentiated pleiomorphic sarcoma, melanoma, pancreatic cancer, lobular and ductal breast cancer, squamous and adeno lung cancer, head and neck cancer, bladder cancer, ovarian cancer, testicular cancer, hepatocellular cancer, endometrial cancer, renal cancer, gastric cancer and colorectal tumors cancer. It is understood that the cells relating to the neoplastic disease to be treated express LRRC15. These cells may either be the neoplastic / tumor cells, and / or the cancer-associated fibroblasts.

[0095] In one embodiment, the subject is a mammal. In a preferred embodiment, the subject is a human.

[0096] Another embodiment of the invention provides a method for treating a neoplastic disease, including glioblastomas, osteosarcomas, undifferentiated pleiomorphic sarcoma, melanoma, pancreatic cancer, lobular and ductal breast cancer, squamous and adeno lung cancer, head and neck cancer, bladder cancer, ovarian cancer, testicular cancer, hepatocellular cancer, endometrial cancer, renal cancer, gastric cancer and colorectal tumors cancer with an antibody or functional fragment thereof that binds to LRRC15, wherein the method comprises administering a pharmaceutically effective amount of the antibody or functional fragment thereof to a subject in need thereof, typically formulated with at least one pharmaceutically acceptable buffer and at least one pharmaceutically acceptable excipient. The treatment may be a monotherapy or preferably a combination therapy with the established standard of care of the neoplastic disease to be treated. Another embodiment of the invention provides a method for treating a neoplastic disease, including glioblastomas, osteosarcomas, undifferentiated pleiomorphic sarcoma, melanoma, pancreatic cancer, lobular and ductal breast cancer, squamous and adeno lung cancer, head and neck cancer, bladder cancer, ovarian cancer, testicular cancer, hepatocellular cancer, endometrial cancer, renal cancer, gastric cancer and colorectal tumors cancer with an antibody or functional fragment thereof that binds to LRRC15, wherein the method comprises administering a pharmaceutically effective amount of the antibody or functional fragment thereof to a subject in need thereof. The method of treatment may be monotherapy or preferably a combination therapy with the established standard of care of the neoplastic disease. Expression of LRRC15 on the cancer cell or in the tumor stroma of the patient to be treated may be confirmed by immunohistochemistry as shown in Example 6 and FIG. 5.

[0097] Yet another embodiment of the invention provides means for binding to the peptide of sequence SEQ ID NO: 48 of LRRC 15, comprising an anti-LRRC 15 antibody or fragment thereof for exerting ADCC and / or CDC activity for the treatment of a patient having a LRRC 15 -expressing tumor. A patient having a LRRC15-expressing tumor may be identified by performing immunohistochemistry staining on tumor tissue obtained from the patient as shown in Example 6 and FIG. 5.

[0098] In another embodiment, the invention provides means for binding to the peptide of sequence SEQ ID NO: 48 of LRRC15, comprising an anti-LRRC15 antibody or fragment thereof, for targeting a toxin or ligand to a LRRC 15 -expressing cell. Examples of toxin may be found in Wang Z et al. (2023). A ligand may be an immunomodulating molecule.

[0099] In one embodiment, the invention provides a method of treatment of a patient by administering an anti-LRRC 15 antibody or fragment thereof binding LRRC 15 comprising a means for binding to the peptide of sequence SEQ ID NO: 48 of LRRC15, and wherein the patient is selected for having an LRRC15-expressing tumor.

[0100] OTHER EMBODIMENTS

[0101] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. From the above description, one of skill in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

[0102] EQUIVALENTS

[0103] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0104] GENERAL TECHNIQUES

[0105] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I. Freshney, ed. 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. (1985); Transcription and Translation (B.D. Hames & S.J. Higgins, eds. 1984); Animal Cell Culture (R.I. Freshney, ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); and B. Perbal. A practical Guide To Molecular Cloning (F.M. Ausubel et al. eds. 1984).

[0106] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present disclosure to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

[0107] EXAMPLES

[0108] The following examples are intended only to illustrate methods and embodiments in accordance with the invention, and as such should not be construed as imposing limitations upon the claims. Example 1 - Cell lines and mAb clones

[0109] Cell lines used in binding and internalization assays are enlisted in Table 4. Cell lines were cultivated in media according to the provider’s recommendations in culture flasks at 37°C, 5% CO2. Table 4: List of cell lines mAb clones used in binding and internalization assays are summarized in Table 5.

[0110] Table 5: List of mAb clones

[0111] Expression of LRRC15 in the U-l 18 MG, G-292 clone A141B1 and 143B cell lines was measured by flow cytometry. In brief, Antibody Binding Capacity (ABC) values were determined using Quantum Simply Cellular (QSC) microspheres (catalog number 816, Bangs Laboratories, USA). Cells were transferred to a 96-well V-bottom plate (approximately 200,000 cells per well). The plate was centrifuged at 1200 rpm for 5 min at room temperature, and the supernatant was discarded. The cells were washed twice by resuspending the pellet in 50 pl of FACS buffer (PBS + 2% FBS) per well followed by centrifugation at 1200 rpm for 5 min at room temperature. Cells were stained with the 2A6 VH4 VL3 mAb clone (2 pg / ml), and the plate was incubated in the dark at room temperature for 30 min. After incubation, 50 pl of FACS buffer was added to each well. The plate was centrifuged at 1200 rpm for 5 min at room temperature, and the supernatant was discarded. Cells were resuspended in 50 pl of secondary antibody Goat anti-Human IgG Fc Secondary Antibody, PE (catalog number. 12-4998-82) at a 1 / 200 dilution and the plate was incubated on ice for 30 min. The cells were washed twice and the pellet resuspended with 1-3 pl of DAPI + 100 pl of FACS buffer (PBS + 2% FBS). The cell suspension was measured by flow cytometry on a flow cytometer LSR Fortessa (Becton Dickinson, US), using identical settings for both the cells and QSC microspheres. ABC values were calculated according to the QSC microspheres manufacturer’s instructions. FIG. 1 shows representative flow cytometry histograms for each cell line. Table 6 shows the ABC value obtained for each cell line. U-l 18MG cell line has a high expression of LRRC15 at the cell membrane and the G-292 clone A141B1 cell line has a medium expression of LRRC15. The expression LRRC15 in the 143B cell line can be considered as very low.

[0112] Table 6: expression of LRRC15 in tested cell lines

[0113] Example 2 - Binding of the 2A6 mAbs to LRRC15

[0114] Bio-layer interferometry kinetics

[0115] Bio-layer interferometry (BLI) measuring biomolecular interactions is an optical technique that monitors the interference pattern of white light reflected from an internal reference layer and a biomolecular layer (Sultana and Lee 2015). A bait is immobilized through various interactions onto the tip of a fiber-optic biosensor to form a biomolecular layer. The biosensor is dipped into a solution of the interacting partner (analyte). Upon binding of a biomolecule, the interference pattern of the reflected light is shifted due to a change in optical thickness at the biosensor tip. The bait may be an antibody binding to a specific target protein and the analyte may be the target protein.

[0116] More specifically, antibody at 2 pg / ml was immobilized to a protein A sensor. Concentration series between 158 and 5 nM of human or cynomolgus LRRC15 were used. The association rate (ka) was measured with soluble LRRC 15 protein and the dissociation rate (kd) with the assay blocking buffer (1% BSA in PBS with 0.05% Tween 20®). A global fitting 1 : 1 was used for the modelling of the association and dissociation curves. The BLI kinetics results are shown in Table 7. Samrotamab and the 2A6 antibodies used in these experiments have the L234A / L235A mutation in their Fc domain. The KD and kdof the 2A6 mAbs are higher than the KD and kdof samrotamab but are within the standard range for therapeutic antibodies. The higher KD or kdof the 2A6 mAbs may provide an advantage to the 2A6 mAbs over samrotamab when used as antibody-drug conjugates (ADCs), as it has been shown that ADCs with higher kdvalues can distribute more efficiently within tumor tissues (Tsumura et al. 2018).

[0117] Table 7: Bio-layer interferometry kinetics

[0118] Binding assay of mAbs to cell lines measured by flow cytometry

[0119] Binding assays of the antibodies to LRRC15 were performed on the U-l 18 MG glioblastoma cell line (ATCC catalogue number HTB-15™), the G-292 osteosarcoma cell line, clone A141B1 (ATCC catalogue number CRL-1423™) and the 143B osteosarcoma cell line (ATCC catalogue number CRL-8303™). Cells were transferred in 96-well V shaped plate (approximately 100 000- 300 000 cells / well). The plate was centrifuged at 2200 rpm for 2 min at 4°C and the supernatant was discarded. Cells were washed by resuspending pellets in 100 pl of flow cytometry buffer (PBS + 2% FBS) per well. Centrifugation and washing were repeated twice. mAbs listed in Table 5 were added to the cells at concentrations ranging between 66.6 nM to 0.000259 nM via serial dilutions and the plate was incubated on ice for 30 min. After incubation, 100 pl of flow cytometry buffer (PBS + 2% FBS) was added to cells, the plate was centrifuged at 2200 rpm for 2 min at 4°C and supernatant was discarded. The pellet was resuspended with 100 pl of flow cytometry buffer (PBS + 2% FBS). Centrifugation and washing were repeated twice. Supernatant was discarded. The secondary antibody goat anti-human IgG Fc conjugated to phycoerythrin (PE) (Thermo Fisher Scientific, catalog number 12-4998-82) was added to the cells at concentration of 0.5 pg / ml in flow cytometry buffer (PBS + 2% FBS). The plate was incubated on ice for 30 min. After incubation, cells were washed twice by resuspending pellets in 100 pl of flow cytometry buffer (PBS + 2% FBS) per well and centrifuging the plate at 2200 rpm for 2 min at 4°C followed by discarding the supernatant. The pellet was resuspended with 1-3 pl of DAPI + 100 pl of flow cytometry buffer (PBS + 2% FBS) and the cell suspension was measured by flow cytometry to determine the binding of the mAbs to LRRC15 expressed on the surface of the cells. FIG. 2 shows the binding curves of the mAbs listed in Table 5 against the cell lines listed in Table 4 expressing LRRC15. Table 8 shows the IC50values calculated from the curves for each antibody tested on the different cell lines. The IC50values were generated with the Prism-GraphPad ™ software from MFI data using the four-parameter non-linear dose response model.

[0120] FIG. 2 shows that the binding affinity of the 2A6 mAbs is within the same range of the affinity of TPP-1741 and samrotamab (hABA in FIG. 1), as all the binding curves overlap. Casivirimab (with the L234A / L235A mutation in the heavy chain) was used as antibody isotype control and does not show any binding to LRRC15. The difference in the MFI plateaus of the binding curves between the U-l 18 cell line (FIG. 2 A) and the G-292 cell line (FIG. 2B) reflects the difference in expression of LRRC15 between both cell lines, whereas the reached plateau is higher for the U-l 18 cell line as compared to the G-292 cell line. Likewise, Table 8 shows that the IC50of all tested antibodies is the highest for the U-l 18 cell line with highest expression of LRRC15. The IC50decreased when the mAb of the invention was humanized, but without significantly compromising the binding of the humanized mAbs to LRRC15. Overall, the 2A6 mAbs have a slightly lower IC50 than TPP- 17421 and samrotamab, but all measured IC50values are within the same nanomolar range, indicative of the overlapping binding curves shown in FIG. 2.

[0121] Table 8: IC50values (nM) of respective mAbs.

[0122] (-): IC50 not measurable

[0123] Example 3 - mAbs Internalization Measured by Flow Cytometry

[0124] Cells were seeded in 96-well flat plates. Seeding concentrations were selected according to titration for respective cell lines (U-l 18: 20.000 cells / well, G-292: 40.000 cells / well, 143B: 30.000 cells / well) and plates were incubated overnight at 37°C, 5% CO2. Secondary antibody Fab fragment Goat Anti -Human IgG Fey (Jackson ImmunoResearch, catalog number 109-007-008) was labeled using the pHrodo Red iFL Microscale Protein Labeling Kit (Thermo Fisher Scientific, catalog number P36014). mAb clones (Table 5) were diluted to a concentration of 4 pg / ml in cell culture media. A 50 pl aliquot of mAb was added to wells containing 100 pl of culture media, resulting in a final concentration of 0.2 pg mAb per 200 pl of cell culture media. The plates were incubated for 30 min at 37°C. During the incubation, a serial dilution of mAbs labeled with the pHrodo Red secondary antibody was prepared in the appropriate cell culture medium at a 1 :3 ratio (primary antibody: pHrodo). After the incubation, 50 pl of the diluted, labeled secondary antibody, at a concentration of 12 pg / ml, was added to the cells, resulting in a final concentration of 0.6 pg pHrodo Red per 200 pl of cell culture media. Plates with the treated cells were incubated for 8 and 24 h in a cell incubator set at 37°C and 5% CO2. After incubation, the medium was carefully removed from wells without disturbing the monolayer of the cells and cells were detached using a trypsin solution and the cell suspensions were transferred into a 96-well V-plate on ice. The plates were centrifuged at 2200 rpm for 2 min and the supernatant was discarded. The cells were washed with ice-cold flow cytometry buffer (PBS + 2% FBS). The pellet was resuspended in ice-cold flow cytometry buffer with DAPI (1 pl DAPI + 120 pl buffer) and measured by flow cytometry. FIG. 3 shows the internalization rates of the tested antibodies on the different cell lines.

[0125] Table 9: Internalization rates measured as percentage of pHrodo Red positive cells after 8 h and 24 h of incubation with the pHrodo Red-labelled antibody

[0126] Table 10: Internalization normalized to the highest internalization at timepoint 24 h of mAb

[0127] 2A6 VH1 VL4 measured in the U-118 cell line Table 11: Internalization normalized to the highest internalization at timepoint 24 h of the

[0128] ABA / samrotamab benchmark measured in the U-118 cell line

[0129] FIG. 3 and Table 9, 10 and 11 clearly show that the 2A6 mAbs have a higher internalization rate than the antibodies TPP-17421 and samrotamab. The internalization rate is highest in the U-118 cell line correlating with its highest expression of LRRC15, allowing to conclude that internalization of the antibodies depends on the binding of the antibody to its target, and does not occur nonspecifically for instance via pinocytosis. Internalization of the 2A6 mAbs is higher than internalization of antibodies TPP-17421 and samrotamab at any measured timepoint .

[0130] Example 4 - mAbs developability

[0131] 2A6, 2A6 VH1 VL4 and 2A6 VH4 VL3 mAbs satisfy the developability standards of therapeutic antibodies. The hydrophobicity of the 2A6 mAbs was within the range expected for a therapeutic antibody with a HIC-HPLC retention time shorter of about 3.5 min than pembrolizumab. The melting temperature Tmof the mAbs was between 72°C and 73°C, within the range expected for a therapeutic antibody. Binding of the mAbs to the neonatal fragment crystallizable receptor (FcRn) at pH 6 was comparable to the binding of the clinical benchmark trastuzumab (heavy chain variable region SEQ ID NO: 75, light chain variable region SEQ ID NO: 76) (2A6 VH1 VL4: KD= 164.6; 2A6 VH4 VL3: KD= 631.2; trastuzumab: KD=40831). The polyreactivity of the 2A6 mAbs, measured by ELISA assays (binding to LPS, DNA, lysozyme and cell lysate (“stickiness”)), showed also improved properties of the mAbs when compared to palivizumab (heavy chain variable region SEQ ID NO: 77, light chain variable region SEQ ID NO: 78). The binding of 2A6, 2A6 VH1 VL4 and 2A6 VH4 VL3 relative to palivizumab was of 0.5, 0.6 and 0.3, respectively. An accelerated stability study showed that the 2A6 mAbs were only minimally degraded after one month incubation at 40°C when compared to one month incubation at 5°C. Example 5 - mAbs epitope mapping by hydrogen-deuterium Exchange Mass Spectrometry (HDX-MS)

[0132] The epitope of the 2A6 mAbs was determined as described in Fojtik L. et al (2024). In brief, hydrogen-deuterium exchange (HDX) of mAbs, LRRC15 and the LRRC15-mAb complex was initiated by a 10-fold dilution in deuterated buffer (50 mM HEPES pH 7.5, 150 mM NaCl). The final protein concentration of LRRC15 was 1.7 pM. The molar ratio between LRRC15 and the mAbs in the complex was 1 : 1. Aliquots were collected after 20 s, 2 min, 20 min, 2 h and two time points (20 s and 20 min) were replicated (n = 3). The exchange was stopped by adding 0.5 M glycine-HCl buffer pH 2.3, 8 M Urea, 1 M TCEP in a 1 : 1 ratio. Samples were incubated for 5 min on ice for disulfide bond reduction and injected onto an LC system by a PAL DHR robot operating in the automatic mode, controlled by Chronos software (Axel Semrau). The LC system consisted of co-immobilized nepenthesin-2 / pepsin (bed volume 66 pl, Affipro) connected in series, a trapping column (SecurityGuard™ ULTRA Cartridge UHPLC Fully Porous Polar Cl 8, 2.1 mm ID, Phenomenex), and an analytical column (Luna Omega Polar Cl 8, 1.6 pm, 100 A, 1.0 x 100 mm, Phenomenex). To minimize back-exchange, the LC system, excluding the protease column, was cooled to 0 °C. Samples were digested and peptides were desalted with 0.4% formic acid in water driven by the 1260 Infinity II Quarternary pump at 200 pl / min. To elute and separate the desalted peptides, a water-acetonitrile gradient (10%-45%; solvent A: 0.1% FA in water, solvent B: 0.1% FA, 2% water in acetonitrile) was used. Gradient elution was done on an Agilent 1290 UP LC system at a flow rate of 40 pl / min. The LC system was coupled to the electrospray ionization source of timsTOF SCP (Bruker Daltonics) mass spectrometer operating in the MS mode with 1 Hz data acquisition and with the tims turned off.

[0133] The acquired data were peak picked in DataAnalysis, exported to text files and processed using DeutEx software. Data were visualized in MSTools. For peptide identification, the same LC setup was used, but with the mass spectrometer operating in MS / MS mode with PASEF active. The MASCOT (v 2.7, Matrix Science) search engine was used to search LC-MS / MS data against a custom-built database combining a common cRAP.fasta and the sequences of antibody, LRRC15, pepsin, nepethesin-2 and PNGaseF. Search parameters were set as follows: precursor tolerance 10 ppm, fragment ion tolerance 0.05 Da, variable modifications: Asn deamidation, Cys dehydration, protein N-term acetylation decoy search enabled, FDR <1%, lonScore >20, and peptide length >5. FIG. 4 shows the results of the epitope mapping for mAbs 2A6 VH1 VL4 and 2A6 VH4 VL3. Both mAbs bind to the same epitope, namely the peptide of the amino acid sequence of SEQ ID NO: 48 of LRRC15. This corresponds to the amino acid residues at positions 346 to 364 of the LRRC15 sequence SEQ ID NO: 1, which is approximately comprised within the LLR13 domain of LRRC15. The better internalization rate of the 2A6 mAbs over antibodies samrotamab and TPP- 17421 may be due to the epitope bound by the antibodies. The bound epitope may provide a unique advantage over the epitopes bound by samrotamab and TPP- 17421, allowing the higher internalization rate of the bound antibodies.

[0134] Example 6 - Immunohistochemistry

[0135] Paraffin-embedded cancer tissue slides were deparaffinized in xylene and ethanol and rehydrated in water. The tissue slides were then subjected to heat-induced epitope retrieval using a retrieval solution at pH 9 (1 :9 in dH2O, EDTA-based epitope retrieval solution, Leica catalog number RE7119-CE). After 30 min of incubation in the retrieval solution at 97°C, the slides were let to cool down and washed in TBS for 5 min. The blocking of non-specific antigen sides was subsequently performed: slides were incubated in 3% H2O2for 20 min at room temperature and blocked with the blocking solution (DAKO, catalog number S2002) for 15 min at room temperature. After blocking, the staining for LRRC15 was performed as follows: the slides were incubated with the primary antibody anti-LRRC15 rabbit recombinant antibody - clone E4X8J (Cell Signaling Technology, catalog number 50546S) diluted in antibody diluent (DAKO, catalog number S0809) to a concentration of 10 pg / ml for 60 min at room temperature. After washing in TBS with 0,04% Tween™-20, slides were incubated in the secondary antibody (HRP -labelled polymer - DAKO catalog number K4003) for 30 min at room temperature, washed in TBS and water, dried and counterstained for 3 min in hematoxylin solution (5 g / 1), washed again in water, dried and covered with a coverslip using EcoMount mounting medium (Biocare Medical, catalog number EM897L).

[0136] Representative images of IHC staining show that LRRC15 is expressed in the tumor cells of chondrosarcoma, fibrosarcoma, leiomyosarcoma (see FIG. 5A), and osteosarcoma, soft tissue sarcoma and undifferentiated pleomorphic sarcoma (see FIG. 5B). For these types of cancer, LRRC15 is exclusively expressed in cancer cells but not in stromal cells. LRRC15 may also be expressed in the tumor stroma and not on the cancer cell. For instance, for head and neck squamous cell carcinoma or non-small squamous cell lung cancer, expression of LRRC15 can be found in cancer associated fibroblast present in the stroma of the tumor but not on the cancer cell (see FIG. 5C). In FIG. 5, the stroma of the tumor showing strong staining for LRRC15 can be clearly delineated from the cancer cell showing no staining for LRRC15. The HRP staining obtained with the HRP -labelled secondary antibody recognizing the anti-LRRC15 rabbit recombinant antibody, visualized in FIG. 5, shows LRRC15 present at the cell membrane of the positive cells. The nuclei stained with hematoxylin marking individual cells can be faintly seen in FIG. 5, .

[0137] Example 7 - mAbs off-target screening

[0138] The 2A6 mAbs were generated to target LRRC15. However, it is critical to verify that the anti- LRRC15 mAbs do not interact with additional proteins (secondary targets or off-targets), and to identify those potential off-targets. Off-target interactions can have a significant impact on safety or toxicology and can unfavorably affect pharmaco-kinetics. Off-target screening of the 2A6 mAbs was performed as described in Freeth and Soden (2020). Briefly, antibodies 2A6, 2A6 VH1 VL4, 2A6 VH4 VL3 at a concentration of 2 pg / ml and samrotamab and rituximab at a concentration of 1 pg / ml, the 2A6 mAbs having the L234A / L235A mutations in their IgGl Fc domain, were screened for binding against fixed HEK293 cells expressing more than 6000 human plasma membrane proteins, secreted and cell surface tethered human secreted proteins and more than 400 human heterodimers arrayed across cell microarray slide sets. An AlexaFluor647 anti-hlgG Fc detection antibody was used to visualize binding of the antibodies to the fixed HEK293 cells. Signal to noise ratios (SNR) were calculated for each interactor. Interactions were categorized as specific, or non-specific (i.e., interaction also observed with the negative control). Significant interactions (Median SNR >1), specific to each test antibody, were identified. Results can be seen in Table 12 and Table 13. All 3 test antibodies and samrotamab showed specific interactions with LRRC15. However, samrotamab showed an additional significant specific interaction with EPHB6 primary target (see Table 12). Although the interaction of samrotamab is labelled as weak in Table 12, this interaction is specific as it was not seen with the PBS-only control. This finding also confirms the previous findings disclosed in WO2022157094 with regards to the polyreactivity of samrotamab and its binding to EPHB6 (see Table 1, Table 2 and Table 4 or WO2022157094). The non / very weak binding to common Fc gamma receptors such as FCGR3A+FCER1G or PAPPA of the 2A6 mAbs, compared to samrotamab or rituximab, is due to the L234A / L235A mutations in their Fc domain impairing such interactions.

[0139] Table 12: Library screen results for the 2A6 antibody and samrotamab. Strong: specific hits of weak / medium intensity or above (not seen with previous PBS only control); weak: specific hits of weak intensity (not seen with previous PBS only control); v. weak: non-specific hits; *: common

[0140] Fc gamma receptors; - : intensity lower than PBS only control.

[0141] Table 13: Library screen results for antibodies 2A6 VH1 VL4 and 2A6 VH4 VL3. Rituximab binding CD20 is used as positive control. Numbers represent the medium signal to noise ratio (medium NSR). Median SNR <1 : no significant interaction; median SNR >1 with negative control: non-specific interaction; median SNR >1 : significant interaction.

[0142] Example 7 - ADCC activity of antibody 2A6 VH4 VL3 with L234A / L235A mutations in the Fc domain

[0143] The antibody-dependent cellular cytotoxicity (ADCC) assay was performed using target cells expressing the antigen of interest and corresponding wild-type A549 control cells lacking target expression. The tested antibodies were antibody 2A6 VH4 VL3 without mutations in the Fc domain (2A6 VH4 VL3 WT mAb in FIG. 6) and 2A6 VH4 VL3 with the L234A / L235A (2A6 VH4 VL3 LALA mAb in FIG. 6). A control antibody with and without the LALA mutation with known ADCC activity was used. On Day 0, both antigen-positive and wild-type target cells were seeded into 96-well plates. On Day 1, serial dilutions of test antibodies were added, followed by the addition of NK92-CD16 effector cells at an effector-to-target (E:T) ratio of 10: 1. The co-cultures were incubated for 20 hours at 37 °C in a humidified 5 % CO2 atmosphere. After incubation, cytotoxicity was determined using an LDH release assay according to the manufacturer’s protocol (LDH Assay Kit (Cytotoxicity) ab65393, Abeam). Specific lysis was calculated relative to maximum- and minimum-lysis controls. This experimental setup enables assessment of FcyRIIIa- mediated, target-dependent killing while controlling for nonspecific background cytotoxicity using wild-type cells. FIG. 6 shows that, in A549 cells expressing LRRC15, the LALA in the Fc domain mutation surprisingly almost completely abolishes the ADCC activity of antibody 2A5 VH4 VL3 (FIG. 6 top) while the LALA mutated control antibody shows only attenuated ADCC activity (FIG. 6 middle) in A549 cells expressing the target of the antibody. No ADCC activity is detected when the cells do not express the antibody target (FIG. 6 bottom).

[0144] EMBODIMENTS

[0145] 1. An antibody or fragment thereof binding to a peptide of the amino acid sequence of SEQ ID NO: 48.

[0146] 2. An antibody or fragment thereof binding to LRRC15, which comprises HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 10, SEQ ID NO: 3 and SEQ ID NO: 4, respectively and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 11, SEQ ID NO: 6 and SEQ ID NO: 7, respectively.

[0147] 3. An antibody or fragment thereof of embodiment 2, comprising: a. HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, respectively; or b. HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 8, SEQ ID NO: 3 and SEQ ID NO: 4, respectively and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 9, SEQ ID NO: 6 and SEQ ID NO: 7, respectively.

[0148] 4. The antibody or fragment thereof of embodiment 2 and 3, comprising: a. a VH sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 42; b. a VH sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 44; or c. a VH sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 46 and d. a VL sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 43; e. a VL sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 45; or f. a VL sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 47 preferably comprising: g. a VH sequence of SEQ ID NO: 42; h. a VH sequence of SEQ ID NO: 44; or i. a VH sequence of SEQ ID NO: 46; and j . a VL sequence of SEQ ID NO : 43 ; k. a VL sequence of SEQ ID NO: 45; or l. a VL sequence of SEQ ID NO: 47. he antibody or fragment thereof of any of embodiments 2 to 4, comprising: a. a VH sequence of SEQ ID NO: 42 and a VL sequence of SEQ ID NO: 43; b. a VH sequence of SEQ ID NO: 44 and a VL sequence of SEQ ID NO: 45; or c. a VH sequence of SEQ ID NO: 46 and a VL sequence of SEQ ID NO: 47. he antibody or fragment thereof of any of embodiments 2 to 5, consisting of: a. a heavy chain sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 58 and a light chain sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 60; b. a heavy chain sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 61 or SEQ ID NO: 62 and a light chain sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 64; or c. a heavy chain sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 65 or SEQ ID NO: 66 and a light chain sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 68 preferably consisting of: d. the heavy chain sequence of SEQ ID NO: 57 or SEQ ID NO: 58 and light chain sequence of SEQ ID NO: 60; e. the heavy chain sequence of SEQ ID NO: 61 or SEQ ID NO: 62 and light chain sequence of SEQ ID NO: 64; or f. the heavy chain sequence of SEQ ID NO: 65 or SEQ ID NO: 66 and light chain sequence of SEQ ID NO: 68.

[0149] 7. An antibody or fragment thereof that competes for binding with an antibody or fragment thereof of any one of embodiments 2 to 6.

[0150] 8. The antibody or fragment thereof of any one of embodiments 1 to 7, wherein the format of the antibody or fragment thereof is selected from the group consisting of IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, synthetic IgG, IgM, F(ab)2, scFv, IgGACH2, F(ab’)2, scFvCH3, Fab, VL, VH, scFv4, scFv3, scFv2, dsFv, Fv, scFv-Fc, (scFv)2, a non-depleting IgG, a diabody, and a bivalent antibody, or Fc-engineered versions thereof.

[0151] 9. The antibody or fragment thereof of any of embodiments 1 to 7, wherein the antibody or fragment thereof a. is humanized or b. is isolated.

[0152] 10. The antibody or fragment thereof of any of embodiments 1 to 9, wherein the antibody or fragment thereof exhibits a higher internalization rate in cells expressing LRRC15 as compared to antibody TPP- 17421 (heavy chain SEQ ID NO: 55 and light chain SEQ ID NO: 56) or antibody Samrotamab (heavy chain SEQ ID NO: 53 and light chain SEQ ID NO: 54).

[0153] 11. The antibody or fragment thereof of embodiment 10 wherein internalization rate is measured by flow cytometry on the U-l 18 glioma cells line or G292 osteosarcoma cell line. 12. The antibody or fragment thereof of embodiment 10 wherein the measured internalization rate of the antibody or fragment thereof is at least 10% higher, at least 20% higher, at least 40% higher at least 50% higher or at least 70% higher than the internalization rate of antibody TPP- 17421 or antibody Samrotamab.

[0154] 13. The antibody or fragment thereof of embodiments 1 to 9, wherein the antibody or fragment thereof has ADCC and / or CDC activity.

[0155] 14. The antibody or fragment thereof of embodiments 1 to 9, wherein the ADCC and / or CDC activity is attenuated.

[0156] 15. A conjugate comprising the antibody or fragment thereof of embodiments 1 to 14, further comprising by covalent linkage at least one of the following moieties: i) a linker ii) a tag iii) an antigen-binding moiety iv) a toxin v) an immune-modifying moiety vi) a chelating agent suitable to complex a radioligand vii) a chromophore or fluorophore

[0157] 16. A nucleic acid encoding the antibody or fragment thereof of any of embodiments 1 to 15, or a chimeric antigen receptor comprising a fragment of the antibody of any of claims 1 to

[0158] 15.

[0159] 17. A vector comprising the nucleic acid of embodiment 16.

[0160] 18. A host cell comprising the nucleic acid of embodiment 16 or a vector of embodiment 17.

[0161] 19. The antibody or fragment thereof of embodiments 1 to 15, the nucleic acid of embodiment

[0162] 16, the vector of embodiment 17 or the host cell of embodiment 17 for use in the treatment of a subject a. suffering from, b. at risk of developing, and / or c. being diagnosed for a neoplastic disease.

[0163] 20. The antibody or fragment thereof for use of embodiment 19, wherein the neoplastic disease is selected from the group consisting of soft tissue sarcoma, glioblastomas, osteosarcomas, undifferentiated pleiomorphic sarcoma, melanoma, pancreatic cancer, lobular and ductal breast cancer, squamous and adeno lung cancer, head and neck cancer, bladder cancer, ovarian cancer, testicular cancer, hepatocellular cancer, endometrial cancer, renal cancer, gastric cancer and colorectal tumors cancer.

[0164] 21. Means for binding to a peptide of the amino acid sequence of SEQ ID NO: 48, comprising an anti-LRRC15 antibody or fragment thereof for exerting ADCC and / or CDC activity for the treatment of a patient having a LRRC 15 -expressing tumor.

[0165] 22. Means for binding to a peptide of the amino acid sequence of SEQ ID NO: 48, comprising an anti -LRRC 15 antibody or fragment thereof, for targeting a toxin or ligand to a LRRC 15- expressing cell.

[0166] 23. A method of treatment of a patient by administering an anti-LRRC15 antibody or fragment thereof binding LRRC 15 comprising a means for binding to a peptide of amino acid sequence of SEQ ID NO: 48, and wherein the patient is selected for having a LRRC 15- expressing tumor.

[0167] 24. The antibody or fragment thereof of any one of embodiments 1 to 7, wherein the format of the antibody or fragment thereof is selected from the group consisting of IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, synthetic IgG, IgM, F(ab)2, scFv, IgGACH2, F(ab’)2, scFvCH3, Fab, VL, VH, scFv4, scFv3, scFv2, dsFv, Fv, scFv-Fc, (scFv)2, a non-depleting IgG, a diabody, and a bivalent antibody, or Fc-engineered versions thereof. SEQUENCES:

[0168] SEQ ID NO : 1 - human LRRC 15

[0169] 001 MPLKHYLLLL VGCQAWGAGL AYHGCPSECT CSRASQVECT GARIVAVPTP 050

[0170] 051 LPWNAMSLQI LNTHITELNE SPFLNISALI ALRIEKNELS RITPGAFRNL 100

[0171] 101 GSLRYLSLAN NKLQVLPIGL FQGLDSLESL LLSSNQLLQI QPAHFSQCSN 150

[0172] 151 LKELQLHGNH LEYIPDGAFD HLVGLTKLNL GKNSLTHISP RVFQHLGNLQ 200

[0173] 251 VLRLYENRLT DIPMGTFDGL VNLQELALQQ NQIGLLSPGL FHNNHNLQRL 250

[0174] 251 YLSNNHISQL PPSVFMQLPQ LNRLTLFGNS LKELSPGI FG PMPNLRELWL 300

[0175] 301 YDNHISSLPD NVFSNLRQLQ VLILSRNQIS FISPGAFNGL TELRELSLHT 350

[0176] 351 NALQDLDGNV FRMLANLQNI SLQNNRLRQL PGNI FANVNG LMAIQLQNNQ 400

[0177] 401 LENLPLGI FD HLGKLCELRL YDNPWRCDSD ILPLRNWLLL NQPRLGTDTV 450

[0178] 451 PVCFSPANVR GQSLI I INVN VAVPSVHVPE VPSYPETPWY PDTPSYPDTT 500

[0179] 501 SVSSTTELTS PVEDYTDLTT IQVTDDRSVW GMTQAQSGLA IAAIVIGIVA 550

[0180] 551 LACSLAACVG CCCCKKRSQA VLMQMKAPNE C

[0181] SEQ ID NO: 2 - 2A6 HC CDR1 (Contact)

[0182] SDAWMH

[0183] SEQ ID NO: 3 - 2A6 / 2A6VH4VL3 / 2A6VH1 VL4 HC CDR2 (Contact)

[0184] WIAQVKAISHNFATY

[0185] SEQ ID NO: 4 - 2A6 / 2A6VH4VL3 / 2A6VH1 VL4 HC CDR3 (Contact)

[0186] CTSANRFNFGFA

[0187] SEQ ID NO: 5 - 2A6 LC CDR1 (Contact)

[0188] NTRMHWY

[0189] SEQ ID NO: 6 - 2A6 / 2A6VH4VL3 / 2A6VH1 VL4 LC CDR2 (Contact)

[0190] LLIYGTSNLE

[0191] SEQ ID NO: 7 - 2A6 / 2A6VH4VL3 / 2A6VH1 VL4 LC CDR3 (Contact)

[0192] QQSWNDPY

[0193] SEQ ID NO: 8 - 2A6VH4VL3 / 2A6VH1VL4 HC CDR1 (Contact)

[0194] SDAWLH SEQ ID NO: 9 - 2A6VH4VL3 / 2A6VH1VL4 LC CDR1 (Contact)

[0195] NTRLHWY

[0196] SEQ ID NO: 10 - 2A6 HC CDR1 consensus (Contact)

[0197] SDAWXiH (Xxis M or L )

[0198] SEQ ID NO: 11 - 2A6 LC CDR1 consensus (Contact)

[0199] NTRX2HWY (X2is M or L )

[0200] SEQ ID NO: 12 - 2A6 HC CDR1 (Kabat)

[0201] DAWMH

[0202] SEQ ID NO: 13 - 2A6VH4VL3 / 2A6VH1VL4 HC CDR1 (Kabat)

[0203] DAWLH

[0204] SEQ ID NO: 14 - 2A6 / 2A6VH4VL3 HC CDR2 (Kabat)

[0205] QVKAI SHNFATYYAES

[0206] SEQ ID NO: 15 - 2A6VH1VL4 HC CDR2 (Kabat)

[0207] QVKAI SHNFATYYAAS

[0208] SEQ ID NO: 16 - 2A6 HC CDR3 (Kabat / AbM)

[0209] IYYCTSAN

[0210] SEQ ID NO: 17 - 2A6VH4VL3 / 2A6VH1VL4 HC CDR3 (Kabat / AbM)

[0211] VYYCTSAN

[0212] SEQ ID NO: 18 - 2A6 HC CDR1 (AbM)

[0213] FT FS DAWMH

[0214] SEQ ID NO: 19 - 2A6VH4VL3 / 2A6VH1 VL4 HC CDR1 (AbM)

[0215] FT FS DAWLH

[0216] SEQ ID NO: 20 - 2A6 / 2A6VH4VL3 / 2A6VH1 VL4 HC CDR2 (AbM)

[0217] QVKAI SHNF

[0218] SEQ ID NO: 21 - 2A6 / 2A6VH4VL3 / 2A6VH1 VL4 HC CDR1 (Chothia)

[0219] FTFSDA

[0220] SEQ ID NO: 22 - 2A6 / 2A6VH4VL3 / 2A6VH1 VL4 HC CDR2 (Chothia)

[0221] KAISH

[0222] SEQ ID NO: 23 - 2A6 / 2A6VH4VL3 / 2A6VH1 VL4 HC CDR3 (Chothia) YYCTSA

[0223] SEQ ID NO: 24 - 2A6 / 2A6VH4VL3 / 2A6VH1 VL4 HC CDR1 (IMGT)

[0224] FTFSDAW

[0225] SEQ ID NO: 25 - 2A6 / 2A6VH4VL3 / 2A6VH1VL4 HC CDR2 (IMGT)

[0226] VKAISH

[0227] SEQ ID NO: 26 - 2A6HC CDR3 (IMGT)

[0228] TAIYYCTSAN

[0229] SEQ ID NO: 27 - 2A6VH4VL3 / 2A6VH1 VL4 HC CDR3 (IMGT)

[0230] TAVYYCTSAN

[0231] SEQ ID NO: 28 - 2A6 LC CDR1 (Kabat / AbM)

[0232] RASDSVNTRMH

[0233] SEQ ID NO: 29 - 2A6VH4VL3 / 2A6VH1 VL4 LC CDR1 (Kabat / AbM)

[0234] RASESVNTRLH

[0235] SEQ ID NO: 30 - 2A6 LC CDR2 (Kabat / AbM)

[0236] YGTSNLES

[0237] SEQ ID NO: 31 - 2A6VH4VL3 / 2A6VH1VL4 LC CDR2 (Kabat / AbM)

[0238] YGTSNLET

[0239] SEQ ID NO: 32 - 2A6 / 2A6VH4VL3 / 2A6VH1 VL4 LC CDR3 (Kabat / AbM / IMGT)

[0240] QQSWNDPYT

[0241] SEQ ID NO: 33 - 2A6 LC CDR1 (Chothia)

[0242] SDSVNTR

[0243] SEQ ID NO: 34 - 2A6VH4VL3 / 2A6VH1 VL4 LC CDR1 (Chothia)

[0244] SESVNTR

[0245] SEQ ID NO: 35 - 2A6 LC CDR2 (Chothia)

[0246] TSNLESG

[0247] SEQ ID NO: 36 - 2A6VH4VL3 / 2A6VH1 VL4 LC CDR2 (Chothia)

[0248] TSNLETG

[0249] SEQ ID NO: 37 - 2A6 / 2A6VH4VL3 / 2A6VH1 VL4 LC CDR3 (Chothia)

[0250] SWNDPY SEQ ID NO: 38 - 2A6 LC CDR1 (IMGT)

[0251] DSVNTR

[0252] SEQ ID NO: 39 - 2A6VH4VL3 / 2A6VH1 VL4 LC CDR1 (IMGT)

[0253] ESVNTR

[0254] SEQ ID NO: 40 - 2A6 LC CDR2 (IMGT)

[0255] TSNLES

[0256] SEQ ID NO: 41 - 2A6VH4VL3 / 2A6VH1 VL4 LC CDR2 (IMGT)

[0257] TSNLET

[0258] SEQ ID NO: 42 - 2A6 rat chimeric VH

[0259] 001 EVQLVETGGS LVRPGKSLKL TCATSGFTFS DAWMHWVRQS PEKQLEWIAQ 050

[0260] 051 VKAISHNFAT YYAESVKGRF TISRDDSKSS VYLQMNSLKE EDTAIYYCTS 100

[0261] 101 ANRFNFGFAY WGQGTLVTVS S

[0262] SEQ ID NO: 43 - 2A6 rat chimeric VL

[0263] 001 DTVLTQSPAL AVSPGERVTI SCRASDSVNT RMHWYQQKPG QQPKLLIYGT 050

[0264] 051 SNLESGVPAR FSGSGSGTDF TLTIDPVEAD DTATYFCQQS WNDPYTFGDG 100

[0265] 101 TKVELK

[0266] SEQ ID NO: 44 - 2A6 VH4 VL3 VH

[0267] 001 EVQLVESGGG LVQPGGSLRL SCATSGFTFS DAWLHWVRQA PGKQLEWIAQ 050

[0268] 051 VKAISHNFAT YYAESVKGRF TISRDDSKNS VYLQMNSLKT EDTAVYYCTS 100

[0269] 101 ANRFNFGFAY WGQGTLVTVS S

[0270] SEQ ID NO: 45 - 2A6 VH4 VL3 VL

[0271] 001 ETVLTQSPAT LALSPGERAT LSCRASESVN TRLHWYQQKP GQQPRLLIYG 050

[0272] 051 TSNLETGIPA RFSGSGSGTD FTLTIDPLEA EDTATYFCQQ SWNDPYTFGD 100

[0273] 101 GTKVEIK

[0274] SEQ ID NO: 46 - 2A6 VH1 VL4 VH

[0275] 001 EVQLVESGGG LVQPGGSLRL SCAASGFTFS DAWLHWVRQA PGKGLEWIAQ 050

[0276] 051 VKAISHNFAT YYAASVKGRF TISRDDSKNS LYLQMNSLKT EDTAVYYCTS 100

[0277] 101 ANRFNFGFAY WGQGTLVTVS S

[0278] SEQ ID NO: 47 - 2A6 VH1 VL4 VL 001 ETVLTQSPAT LSLSPGERAT LSCRASESVN TRLHWYQQKP GQAPRLLIYG 050

[0279] 051 TSNLETGVPA RFSGSGSGTD FTLTISSLEP EDFATYYCQQ SWNDPYTFGQ 100

[0280] 101 GTKVEIK

[0281] SEQ ID NO: 48 - 2A6VH1VL4 and 2A6VH4VL3 epitope

[0282] 001 LSLHTNALQD LDGNVFRML

[0283] SEQ ID NO: 49 - constant light chain - CL domain

[0284] 001 RTVAAPSVFI FPPSDEQLKS GTASWCLLN NFYPREAKVQ WKVDNALQSG 050

[0285] 051 NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK 100

[0286] 101 SFNRGEC

[0287] SEQ ID NO: 50 - constant heavy chain - CHI + Fc domain

[0288] 001 ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV 050

[0289] 051 HTFPAVLQSS GLYSLSSWT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP 100

[0290] 101 KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVWDVS 150

[0291] 151 HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRWSVLT VLHQDWLNGK 200

[0292] 201 EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC 250

[0293] 251 LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 300

[0294] 351 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK

[0295] SEQ ID NO: 51 - L234A / L235A mutation in constant heavy chain - CHI + Fc domain

[0296] 001 ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV 050

[0297] 051 HTFPAVLQSS GLYSLSSWT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP 100

[0298] 101 KSCDKTHTCP PCPAPEAAGG PSVFLFPPKP KDTLMISRTP EVTCVWDVS 150

[0299] 151 HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRWSVLT VLHQDWLNGK 200

[0300] 201 EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC 250

[0301] 251 LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 300

[0302] 301 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK

[0303] SEQ ID NO: 52 - L234A / L235A / P329G mutation in constant heavy chain - CHI + Fc domain

[0304] 001 ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV 050

[0305] 051 HTFPAVLQSS GLYSLSSWT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP 100

[0306] 101 KSCDKTHTCP PCPAPEAAGG PSVFLFPPKP KDTLMISRTP EVTCVWDVS 150

[0307] 151 HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRWSVLT VLHQDWLNGK 200 201 EYKCKVSNKA LGAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC 250

[0308] 251 LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 300

[0309] 301 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK

[0310] SEQ ID NO: 53 - huM25 (Samrotamab) heavy chain

[0311] 001 EVQLVQSGAE VKKPGASVKV SCKASGYKFS SYWIEWVKQA PGQGLEWIGE 050

[0312] 051 ILPGSDTTNY NEKFKDRATF TSDTS INTAY MELSRLRSDD TAVYYCARDR 100

[0313] 101 GNYRAWFGYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK 150

[0314] 151 DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTQT 200

[0315] 201 YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP 250

[0316] 251 KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN 300

[0317] 301 STYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ 350

[0318] 351 VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV 400

[0319] 401 LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 450

[0320] SEQ ID NO: 54 - huM25 (Samrotamab) light chain

[0321] 001 DIQMTQSPSS LSASVGDRVT ITCRASQDIS NYLNWYQQKP GGAVKFLIYY 050

[0322] 051 TSRLHSGVPS RFSGSGSGTD YTLTISSLQP EDFATYFCQQ GEALPWTFGG 100

[0323] 101 GTKVEIKRTV AAPSVFI FPP SDEQLKSGTA SWCLLNNFY PREAKVQWKV 150

[0324] 151 DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 200 201 LSSPVTKSFN RGEC

[0325] SEQ ID NO: 55 - TPP- 17421 heavy chain

[0326] 001 EVQLVQSGAE VKKPGASVKV SCKASGYKFS SYWIEWVKQA PGQGLEWIGE 050

[0327] 051 ILPGSDWTNY NEKFKDRATF TSDTS INTAY MELSRLRSDD TAVYYCARDR 100

[0328] 101 GNYRAWFQYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK 150

[0329] 151 DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTQT 200

[0330] 201 YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP 250

[0331] 251 KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN 300

[0332] 301 STYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ 351

[0333] 351 VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV 400

[0334] 401 LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG SEQ ID NO: 56 - TPP-17421 light chain

[0335] 001 DIQMTQSPSS LSASVGDRVT ITCRASQS IS SYLNWYQQKP GGAPKFLIYY 050

[0336] 051 ASSLQSGVPS RFSGSGSGTD YTLTISSLQP EDFATYYCQQ GLELPWTFGG 100

[0337] 101 GTKVEIKRTV AAPSVFI FPP SDEQLKSGTA SWCLLNNFY PREAKVQWKV 150

[0338] 151 DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 200

[0339] 251 LSSPVTKSFN RGEC

[0340] SEQ ID NO: 57 - 2A6 rat chimeric heavy chain

[0341] 001 EVQLVETGGS LVRPGKSLKL TCATSGFTFS DAWMHWVRQS PEKQLEWIAQ 050

[0342] 051 VKAISHNFAT YYAESVKGRF TISRDDSKSS VYLQMNSLKE EDTAIYYCTS 100

[0343] 101 ANRFNFGFAY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV 150

[0344] 151 KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSW TVPSSSLGTQ 200

[0345] 201 TYICNVNHKP SNTKVDKKVE PKSCDKTHTC PPCPAPELLG GPSVFLFPPK 250

[0346] 251 PKDTLMISRT PEVTCVWDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY 300

[0347] 301 NSTYRWSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP 350

[0348] 351 QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP 400

[0349] 401 VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG 450

[0350] 451 K

[0351] SEQ ID NO: 58 - 2A6 rat chimeric heavy chain L234A / L235A

[0352] 001 EVQLVETGGS LVRPGKSLKL TCATSGFTFS DAWMHWVRQS PEKQLEWIAQ 050

[0353] 051 VKAISHNFAT YYAESVKGRF TISRDDSKSS VYLQMNSLKE EDTAIYYCTS 100

[0354] 101 ANRFNFGFAY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV 150

[0355] 151 KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSW TVPSSSLGTQ 200

[0356] 201 TYICNVNHKP SNTKVDKKVE PKSCDKTHTC PPCPAPEAAG GPSVFLFPPK 250

[0357] 251 PKDTLMISRT PEVTCVWDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY 300

[0358] 301 NSTYRWSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP 350

[0359] 351 QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP 400

[0360] 401 VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG 450

[0361] 451 K SEQ ID NO: 59 - 2A6 rat chimeric heavy chain L234A / L235A / P329G

[0362] 001 EVQLVETGGS LVRPGKSLKL TCATSGFTFS DAWMHWVRQS PEKQLEWIAQ 050

[0363] 051 VKAISHNFAT YYAESVKGRF TISRDDSKSS VYLQMNSLKE EDTAIYYCTS 100

[0364] 101 ANRFNFGFAY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV 150

[0365] 151 KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSW TVPSSSLGTQ 200

[0366] 201 TYICNVNHKP SNTKVDKKVE PKSCDKTHTC PPCPAPEAAG GPSVFLFPPK 250

[0367] 251 PKDTLMISRT PEVTCVWDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY 300

[0368] 301 NSTYRWSVL TVLHQDWLNG KEYKCKVSNK ALGAPIEKTI SKAKGQPREP 350

[0369] 351 QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP 400

[0370] 401 VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG 450

[0371] 451 K

[0372] SEQ ID NO: 60 - 2A6 rat chimeric light chain

[0373] 001 DTVLTQSPAL AVSPGERVTI SCRASDSVNT RMHWYQQKPG QQPKLLIYGT 050

[0374] 051 SNLESGVPAR FSGSGSGTDF TLTIDPVEAD DTATYFCQQS WNDPYTFGDG 100

[0375] 101 TKVELKRTVA APSVFI FPPS DEQLKSGTAS WCLLNNFYP REAKVQWKVD 150

[0376] 151 NALQSGNSQE SVTEQDSKDS TYSLSSTLTL SKADYEKHKV YACEVTHQGL 200 251 SSPVTKSFNR GEC

[0377] SEQ ID NO: 61 - 2A6 VH4 VL3 heavy chain

[0378] 001 EVQLVESGGG LVQPGGSLRL SCATSGFTFS DAWLHWVRQA PGKQLEWIAQ 050

[0379] 051 VKAISHNFAT YYAESVKGRF TISRDDSKNS VYLQMNSLKT EDTAVYYCTS 100

[0380] 101 ANRFNFGFAY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV 150

[0381] 151 KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSW TVPSSSLGTQ 200

[0382] 201 TYICNVNHKP SNTKVDKKVE PKSCDKTHTC PPCPAPELLG GPSVFLFPPK 250

[0383] 251 PKDTLMISRT PEVTCVWDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY 300

[0384] 301 NSTYRWSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP 350

[0385] 351 QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP 400

[0386] 401 VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG 450

[0387] 451 K

[0388] SEQ ID NO: 62 - 2A6 VH4 VL3 VH heavy chain L234A / L235A 001 EVQLVESGGG LVQPGGSLRL SCATSGFTFS DAWLHWVRQA PGKQLEWIAQ 050

[0389] 051 VKAISHNFAT YYAESVKGRF TISRDDSKNS VYLQMNSLKT EDTAVYYCTS 100

[0390] 101 ANRFNFGFAY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV 150

[0391] 151 KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSW TVPSSSLGTQ 200

[0392] 201 TYICNVNHKP SNTKVDKKVE PKSCDKTHTC PPCPAPEAAG GPSVFLFPPK 250

[0393] 251 PKDTLMISRT PEVTCVWDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY 300

[0394] 301 NSTYRWSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP 350

[0395] 351 QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP 400

[0396] 401 VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG 450

[0397] 451 K

[0398] SEQ ID NO: 63 - 2A6 VH4 VL3 VH heavy chain L234A / L235A / P329G

[0399] 001 EVQLVESGGG LVQPGGSLRL SCATSGFTFS DAWLHWVRQA PGKQLEWIAQ 050

[0400] 051 VKAISHNFAT YYAESVKGRF TISRDDSKNS VYLQMNSLKT EDTAVYYCTS 100

[0401] 101 ANRFNFGFAY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV 150

[0402] 151 KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSW TVPSSSLGTQ 200

[0403] 201 TYICNVNHKP SNTKVDKKVE PKSCDKTHTC PPCPAPEAAG GPSVFLFPPK 250

[0404] 251 PKDTLMISRT PEVTCVWDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY 300

[0405] 301 NSTYRWSVL TVLHQDWLNG KEYKCKVSNK ALGAPIEKTI SKAKGQPREP 350

[0406] 351 QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP 400

[0407] 401 VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG 450

[0408] 451 K

[0409] SEQ ID NO: 64 - 2A6 VH4 VL3 light chain

[0410] 001 ETVLTQSPAT LALSPGERAT LSCRASESVN TRLHWYQQKP GQQPRLLIYG 050

[0411] 051 TSNLETGIPA RFSGSGSGTD FTLTIDPLEA EDTATYFCQQ SWNDPYTFGD 100

[0412] 101 GTKVEIKRTV AAPSVFI FPP SDEQLKSGTA SWCLLNNFY PREAKVQWKV 150

[0413] 151 DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 200 251 LSSPVTKSFN RGEC

[0414] SEQ ID NO: 65 - 2A6 VH1 VL4 VH heavy chain

[0415] 001 EVQLVESGGG LVQPGGSLRL SCAASGFTFS DAWLHWVRQA PGKGLEWIAQ 050

[0416] 051 VKAISHNFAT YYAASVKGRF TISRDDSKNS LYLQMNSLKT EDTAVYYCTS 100 101 ANRFNFGFAY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV 150

[0417] 151 KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSW TVPSSSLGTQ 200

[0418] 201 TYICNVNHKP SNTKVDKKVE PKSCDKTHTC PPCPAPELLG GPSVFLFPPK 250

[0419] 251 PKDTLMISRT PEVTCVWDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY 300

[0420] 301 NSTYRWSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP 350

[0421] 351 QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP 400

[0422] 401 VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG 450

[0423] 451 K

[0424] SEQ ID NO: 66 - 2A6 VH1 VL4 VH heavy chain L234A / L235A

[0425] 001 EVQLVESGGG LVQPGGSLRL SCAASGFTFS DAWLHWVRQA PGKGLEWIAQ 050

[0426] 051 VKAISHNFAT YYAASVKGRF TISRDDSKNS LYLQMNSLKT EDTAVYYCTS 100

[0427] 101 ANRFNFGFAY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV 150

[0428] 151 KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSW TVPSSSLGTQ 200

[0429] 201 TYICNVNHKP SNTKVDKKVE PKSCDKTHTC PPCPAPEAAG GPSVFLFPPK 250

[0430] 251 PKDTLMISRT PEVTCVWDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY 300

[0431] 301 NSTYRWSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP 350

[0432] 351 QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP 400

[0433] 401 VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG 450

[0434] 451 K

[0435] SEQ ID NO: 67 - 2A6 VH1 VL4 VH heavy chain L234A / L235A / P329G

[0436] 001 EVQLVESGGG LVQPGGSLRL SCAASGFTFS DAWLHWVRQA PGKGLEWIAQ 050

[0437] 051 VKAISHNFAT YYAASVKGRF TISRDDSKNS LYLQMNSLKT EDTAVYYCTS 100

[0438] 101 ANRFNFGFAY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV 150

[0439] 151 KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSW TVPSSSLGTQ 200

[0440] 201 TYICNVNHKP SNTKVDKKVE PKSCDKTHTC PPCPAPEAAG GPSVFLFPPK 250

[0441] 251 PKDTLMISRT PEVTCVWDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY 300

[0442] 301 NSTYRWSVL TVLHQDWLNG KEYKCKVSNK ALGAPIEKTI SKAKGQPREP 350

[0443] 351 QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP 400

[0444] 401 VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG 450

[0445] 451 K

[0446] SEQ ID NO: 68 - 2A6 VH1 VL4 light chain 001 ETVLTQSPAT LSLSPGERAT LSCRASESVN TRLHWYQQKP GQAPRLLIYG 050

[0447] 051 TSNLETGVPA RFSGSGSGTD FTLTISSLEP EDFATYYCQQ SWNDPYTFGQ 100

[0448] 101 GTKVEIKRTV AAPSVFI FPP SDEQLKSGTA SWCLLNNFY PREAKVQWKV 150

[0449] 151 DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 200

[0450] 251 LSSPVTKSFN RGEC

[0451] SEQ ID NO: 69 - casirivimab heavy chain L234A / L235A

[0452] 001 QVQLVESGGG LVKPGGSLRL SCAASGFTFS DYYMSWIRQA PGKGLEWVSY 050

[0453] 051 ITYSGSTIYY ADSVKGRFTI SRDNAKSSLY LQMNSLRAED TAVYYCARDR 100

[0454] 101 GTTMVPFDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK 150

[0455] 151 DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTQT 200

[0456] 201 YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPEAAGG PSVFLFPPKP 250

[0457] 251 KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN 300

[0458] 301 STYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ 350

[0459] 351 VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV 400

[0460] 401 LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 450

[0461] SEQ ID NO: 70 - casirivimab light chain

[0462] 001 DIQMTQSPSS LSASVGDRVT ITCQASQDIT NYLNWYQQKP GKAPKLLIYA 050

[0463] 051 ASNLETGVPS RFSGSGSGTD FTFTISGLQP EDIATYYCQQ YDNLPLTFGG 100

[0464] 101 GTKVEIKRTV AAPSVFI FPP SDEQLKSGTA SWCLLNNFY PREAKVQWKV 150

[0465] 151 DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 200

[0466] 251 LSSPVTKSFN RGEC

[0467] SEQ ID NO: 71 - tag

[0468] 001 GGGGGGGCVI M

[0469] SEQ ID NO: 72 - tag

[0470] 001 GGGGSLPQTG G

[0471] SEQ ID NO: 73 - tag

[0472] 001 LPQTG

[0473] SEQ ID NO: 74 - tag

[0474] 001 EGEE

[0475] SEQ ID NO: 75 - trastuzumab VH 001 EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR 050

[0476] 051 IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG 100 101 GDGFYAMDYW GQGTLVTVSS

[0477] SEQ ID NO: 76 - trastuzumab VL

[0478] 001 DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS 050

[0479] 051 ASFLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ 100

[0480] 101 GTKVEIK

[0481] SEQ ID NO: 77 - palivizumab VH

[0482] 001 QVTLRESGPA LVKPTQTLTL TCTFSGFSLS TSGMSVGWIR QPPGKALEWL 050

[0483] 051 ADIWWDDKKD YNPSLKSRLT ISKDTSKNQV VLKVTNMDPA DTATYYCARS 100

[0484] 101 MITNWYFDVW GAGTTVTVSS

[0485] SEQ ID NO: 78 - palivizumab VL

[0486] 001 DIQMTQSPST LSASVGDRVT ITCKCQLSVG YMHWYQQKPG KAPKLLIYDT 050

[0487] 051 SKLASGVPSR FSGSGSGTEF TLTISSLQPD DFATYYCFQG SGYPFTFGGG 100 101 TKLEIK

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[0497] WO2021102332

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Claims

1. CLAIMS1. An antibody or fragment thereof binding to a peptide of the amino acid sequence of SEQ ID NO: 48.

2. An antibody or fragment thereof binding to LRRC15, which comprises HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 10, SEQ ID NO: 3 and SEQ ID NO: 4, respectively and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 11, SEQ ID NO: 6 and SEQ ID NO: 7, respectively.

3. An antibody or fragment thereof of claim 2, comprising: a. HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, respectively; or b. HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 8, SEQ ID NO: 3 and SEQ ID NO: 4, respectively and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 9, SEQ ID NO: 6 and SEQ ID NO: 7, respectively.

4. The antibody or fragment thereof of claims 2 and 3, comprising: a. a VH sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 42; b. a VH sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 44; or c. a VH sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 46 and d. a VL sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 43; e. a VL sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 45; or f. a VL sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 47preferably comprising: g. a VH sequence of SEQ ID NO: 42; h. a VH sequence of SEQ ID NO: 44; or i. a VH sequence of SEQ ID NO: 46; and j . a VL sequence of SEQ ID NO : 43 ; k. a VL sequence of SEQ ID NO: 45; or l. a VL sequence of SEQ ID NO: 47.

5. The antibody or fragment thereof of any of claims 2 to 4, comprising: a. a VH sequence of SEQ ID NO: 42 and a VL sequence of SEQ ID NO: 43; b. a VH sequence of SEQ ID NO: 44 and a VL sequence of SEQ ID NO: 45; or c. a VH sequence of SEQ ID NO: 46 and a VL sequence of SEQ ID NO: 47.

6. The antibody or fragment thereof of any of claims 2 to 5, consisting of: a. a heavy chain sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 58 and a light chain sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 60; b. a heavy chain sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 61 or SEQ ID NO: 62 and a light chain sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 64; or c. a heavy chain sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 65 or SEQ ID NO: 66 and a light chain sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 68 preferably consisting of:d. the heavy chain sequence of SEQ ID NO: 57 or SEQ ID NO: 58 and light chain sequence of SEQ ID NO: 60; e. the heavy chain sequence of SEQ ID NO: 61 or SEQ ID NO: 62 and light chain sequence of SEQ ID NO: 64; or f. the heavy chain sequence of SEQ ID NO: 65 or SEQ ID NO: 66 and light chain sequence of SEQ ID NO: 68.

7. An antibody or fragment thereof that competes for binding with an antibody or fragment thereof of any one of claims 2 to 6.

8. The antibody or fragment thereof of any one of claims 1 to 7, wherein the format of the antibody or fragment thereof is selected from the group consisting of IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, synthetic IgG, IgM, F(ab)2, scFv, IgGACH2, F(ab’)2, scFvCH3, Fab, VL, VH, scFv4, scFv3, scFv2, dsFv, Fv, scFv-Fc, (scFv)2, a non-depleting IgG, a diabody, and a bivalent antibody, or Fc-engineered versions thereof, preferably wherein the antibody or fragment thereof a. is humanized or b. is isolated.

9. The antibody or fragment thereof of any of claims 1 to 8, wherein the antibody or fragment thereof exhibits a higher internalization rate in cells expressing LRRC15 as compared to antibody TPP-17421 (heavy chain SEQ ID NO: 55 and light chain SEQ ID NO: 56) or antibody Samrotamab (heavy chain SEQ ID NO: 53 and light chain SEQ ID NO: 54), preferably wherein internalization rate is measured by flow cytometry on the U-l 18 glioma cells line or G292 osteosarcoma cell line, and / or wherein the measured internalization rate of the antibody or fragment thereof is at least 10% higher, at least 20% higher, at least 40% higher at least 50% higher or at least 70% higher than the internalization rate of antibody TPP-17421 or antibody Samrotamab.

10. The antibody or fragment thereof of claims 1 to 9, wherein the ADCC and / or CDC activity is attenuated.

11. A conjugate comprising the antibody or fragment thereof of claims 1 to 10, further comprising by covalent linkage at least one of the following moieties: i) a linker ii) a tag iii) an antigen-binding moiety iv) a toxin v) an immune-modifying moiety vi) a chelating agent suitable to complex a radioligand vii) a chromophore or fluorophore12. A nucleic acid encoding the antibody or fragment thereof of any of claims 1 to 11, or a chimeric antigen receptor comprising a fragment of the antibody of any of claims 1 to 15.

13. A vector comprising the nucleic acid of claim 12.

14. A host cell comprising the nucleic acid of claim 12 or a vector of claim 13.

15. The antibody or fragment thereof of claims 1 to 11, the nucleic acid of claim 12, the vector of claim 13 or the host cell of claim 14 for use in the treatment of a subject a. suffering from, b. at risk of developing, and / or c. being diagnosed for a neoplastic disease, preferably, where the neoplastic disease is selected from the group consisting of soft tissue sarcoma, glioblastomas, osteosarcomas, undifferentiated pleiomorphic sarcoma, melanoma, pancreatic cancer, lobular and ductal breast cancer, squamous and adeno lung cancer, head and neck cancer, bladder cancer, ovarian cancer, testicular cancer, hepatocellular cancer, endometrial cancer, renal cancer, gastric cancer and colorectal tumors cancer.

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