An antigen binding molecule selective for cell FAP relative to soluble fibroblast activation protein (FAP)

Abstract

This invention relates to antigen-binding molecules that specifically bind to cell-bound fibroblast activation protein (cFAP). The antigen-binding molecules significantly distinguish cFAP from one or more soluble variants of fibroblast activation protein (FAP), including one or more soluble variants of FAP known to be present in bodily fluids such as blood, plasma, and serum. The antigen-binding molecules comprise one or more antibodies and / or one or more antibody-antigen-binding domains or fragments, and particularly include one or more camelid-specific antigen-binding domains, i.e., one or more V... H H sequence. The present invention further relates to a nucleic acid molecule encoding the antigen-binding molecule or a component domain thereof, and a vector comprising the nucleic acid molecule. The present invention also relates to a host cell comprising the vector of the present invention, and a method for generating the antigen-binding molecule of the present invention, the method comprising culturing the host cell of the present invention under suitable conditions and isolating the generated antigen-binding molecule. Furthermore, the present invention relates to an antigen-binding molecule obtainable by the method of the present invention, and to a composition comprising at least one of the antigen-binding molecule of the present invention, the nucleic acid molecule of the present invention, the vector of the present invention, the host cell of the present invention, or an antigen-binding molecule generated by the method of the present invention. The present invention also relates to the use of the antigen-binding molecule of the present invention for in vivo or in vitro detection and targeting of cFAP, particularly selectively targeting one or more soluble variants of FAP, as a therapeutic agent for treating diseases characterized by cFAP, or as a diagnostic agent.

Description

Technical Field

[0001] This invention relates to antigen-binding molecules that specifically bind to cell-bound fibroblast activation protein (cFAP). The antigen-binding molecules significantly distinguish cFAP from one or more soluble variants of fibroblast activation protein (FAP), including one or more soluble variants of FAP known to be present in bodily fluids such as blood, plasma, and serum. The antigen-binding molecules comprise one or more antibodies and / or one or more antibody-antigen-binding domains or fragments, and particularly include one or more camelid-specific antigen-binding domains, i.e., one or more V... H H sequence. The present invention further relates to a nucleic acid molecule encoding the antigen-binding molecule or a component domain thereof, and a vector comprising the nucleic acid molecule. The present invention also relates to a host cell comprising the vector of the present invention, and a method for generating the antigen-binding molecule of the present invention, the method comprising culturing the host cell of the present invention under suitable conditions and isolating the generated antigen-binding molecule. Furthermore, the present invention relates to an antigen-binding molecule obtainable by the method of the present invention, and to a composition comprising at least one of the antigen-binding molecule of the present invention, the nucleic acid molecule of the present invention, the vector of the present invention, the host cell of the present invention, or an antigen-binding molecule generated by the method of the present invention. The present invention also relates to the use of the antigen-binding molecule of the present invention for in vivo or in vitro detection and targeting of cFAP, particularly selectively targeting one or more soluble variants of FAP, as a therapeutic agent for treating diseases characterized by cFAP, or as a diagnostic agent. Background Technology

[0002] Fibroblast activating protein (FAP), also known as fibroblast activating protein-α (FAP-α), is a member of the prolyl-specific serine protease family, possessing both enzymatic and signal transduction activities. FAP exists in its full-length 760-amino acid (AA) cell-binding protein (cFAP) and in a soluble form in plasma, which can be many different variants with varying truncated sequences. Therefore, without further clarification, the term "FAP" can refer to cFAP as well as any of the various truncated extracellular, soluble, non-membrane-associated variants in human plasma (i.e., the soluble plasma variant of FAP (spFAP)). The cleavage initiation site of spFAP is thought to be approximately within the first 40 N-terminal residues of cFAP, but this has not been reproducibly determined. At least one spFAP sequence has been approximated by Lee et al., Blood 107 (2006), 1397-1404 (SEQ NO: 2).

[0003] FAP is typically expressed only in damaged tissue undergoing active repair. Therefore, it is considered a highly attractive therapeutic and diagnostic target. Besides being poorly expressed in normal tissues during active tissue repair, it is overexpressed in several disease states associated with extracellular remodeling, such as fibrosis, inflammatory diseases, arthritis, atherosclerosis, autoimmune diseases, metabolic diseases, and cancer; Fitzgerald and Weiner, *Cancer Metastasis Rev.* 39 (2020), 783-803. For example, its (over)expression has been identified in benign gynecological tumors such as endometriosis (WO2023 / 198677) and fibroids (Luo et al., *Translational Research* 163 (2014), 232-241).

[0004] However, antigen-binding molecules targeting FAP, such as the humanized monoclonal antibody sibrotuzumab (WO2002083171A2) and antibody 4B9 (Waldhauer et al., MAbs. 13(2021), 1913791), have not yet demonstrated clinically relevant therapeutic efficacy. The inventors have recognized that this lack of efficacy can be partly attributed to poor pharmacokinetics (PK) resulting from the absorption effect of spFAP (i.e., current anti-FAP antibodies bind to non-target spFAP, present in healthy individuals at an average concentration of 100 ng / mL). Therefore, binding molecules that selectively (e.g., preferentially or exclusively) bind to cFAP relative to spFAP have significant utility for therapeutic and diagnostic uses. In particular, such selective bindings are desirable because their use will avoid off-target tissue transport (causing unintended side effects), poor PK, and / or other side effects that may be related to the location of spFAP. In addition, the use of such selective conjugates could improve the potential therapies for current anti-FAP molecules because they do not have to be administered at higher levels to overcome absorption effects, thereby reducing dosage, cost and / or allowing for more attractive dosing regimens. Summary of the Invention

[0005] Despite the potentially significant overlap between cFAP and its soluble variants (e.g., present in plasma as multiple potential variants, “spFAP”), and the known problems in the art related to the development of antigen-binding molecules capable of distinguishing such highly similar molecules, the inventors have surprisingly developed an antigen-binding molecule characterized by selective binding to soluble forms of cFAP, but not FAP, particularly spFAP. The developed antigen-binding molecule is the first of its kind and is particularly characterized by its activity in distinguishing cFAP from one or more soluble variants (e.g., spFAP). The antigen-binding molecule exhibits remarkably high specificity for cFAP and also selectively does not bind to, for example, soluble variants (spFAP) present in human blood. Analysis of the identified antigen-binding molecule also allows for the development of common structures that can confer specific and selective binding to cFAP as defined herein. Therefore, the use of antigen-binding molecules and their use as targeting agents and / or diagnostic and prognostic tools for therapies (including diagnosis, treatment, or remission) of diseases characterized by cFAP expression is provided.

[0006] The antigen-binding molecules provided herein comprise or consist of one or more antibodies, antibody-antigen-binding domains, or antibody-antigen-binding fragments (e.g., single-domain antigen-binding fragments in a non-limiting example). The antigen-binding molecules of the present invention, as demonstrated by exemplary specific members of the described family, bind specifically to cFAP. As known in the art, FAP (also known as fibroblast protein α (FAP-α) and prolyl endopeptidase FAP) is a type II transmembrane glycoprotein having 760 amino acids, as represented by SEQ ID NO: 1. Therefore, the antigen-binding molecules of the present invention bind specifically to SEQ ID NO: 1 as expressed on the cell surface. Cellular expression of cFAP can be endogenous (cells express cFAP without recombinant manipulation), or cells can be recombinantly modified to express cFAP. Therefore, the antigen-binding molecules also bind specifically to cells expressing cFAP, whether endogenously or recombinantly. The cFAP-specific and selective antigen-binding molecules of the present invention may also be referred to interchangeably herein as anti-cFAP molecules, anti-cFAP antibodies, anti-cFAP antibody fragments, anti-cFAP antibody-derived domains, etc.

[0007] The antigen-binding molecule of the present invention specifically binds to the target antigen cFAP (e.g., as expressed on the surface of a cell membrane) and selectively binds to (or does not bind to) cFAP relative to soluble forms of FAP, for example, binding little or no to sFAP (spFAP, which may contain one or more variants of sFAP) present in human plasma, serum, or blood. In a non-limiting example, sFAP is represented by the amino acid sequence SEQ ID NO: 2. Thus, the antigen-binding molecule distinguishes cFAP as defined herein from soluble forms of FAP (e.g., sFAP). The antigen-binding molecule also additionally or selectively distinguishes cFAP as defined herein from soluble FAP variants (spFAP) present in plasma, serum, or blood (particularly human plasma, serum, or blood). Because it is understood that said plasma, serum, and blood contain spFAP, the selectivity of the antigen-binding molecule of the present invention can be assessed by comparing the binding to cFAP relative to the binding to plasma, serum, or blood known or expected to contain spFAP. Therefore, the antigen-binding molecules provided herein preferentially and selectively bind to the target antigen cFAP in the presence of soluble forms of FAP, such as sFAP and / or spFAP. The antigen-binding molecules provided herein also preferentially and selectively bind to the target antigen cFAP in the presence of plasma, serum, or blood (preferably human plasma, serum, or blood). Without limitation to specific interpretations, it is considered that the anti-cFAP antigen-binding molecules bind to cFAP epitopes not present in soluble variants of FAP. As understood in the art, "not present," as used in this context, can mean that the specific amino acid forming the recognized epitope is not present in the soluble variant (e.g., spFAP), or that the structure of the soluble variant has been altered relative to cFAP such that the three-dimensional epitope recognized by the antigen-binding molecules of the present invention is no longer present in the soluble variant, or is otherwise spatially inaccessible in the soluble variant. Therefore, if the target epitope is present, the anti-cFAP antigen-binding molecules and their antigen-binding fragments can also bind to the full-length FAP or a portion thereof when not expressed by cells.

[0008] As defined below, the terms “selective binding” and “distinguishing” are used interchangeably and indicate that the anti-cFAP antigen-binding molecules of the present invention have a higher activity and / or specificity in binding to specific antigen targets (i.e., FAPs that are endogenously or recombinantly expressed on the surface of cells (cFAPs)) than in binding to other antigens, particularly soluble forms of FAPs (sFAPs) as known in the art and / or soluble forms of FAPs present in human extracellular fluids such as serum or plasma (spFAPs). For example, as detailed below, the characteristic of selective binding or distinguishing a target antigen from / relative to a non-target antigen can be characterized by an anti-cFAP antigen-binding molecule (e.g., containing or consisting of at least one antibody-antigen-binding domain or region) that selectively binds to one or more soluble variants of FAP (as present in vivo, such as plasma, serum, blood, tissue, or body fluids, preferably human plasma, human serum, human blood, human tissue, or human body fluids).

[0009] As disclosed herein, specific and selective anti-cFAP antigen-binding molecules and antigen-binding fragments distinguish cFAP from one or more soluble variants. Therefore, it is most preferred that specific and selective anti-cFAP antigen-binding molecules and antigen-binding fragments substantially do not bind to SEQ ID NO: 2 or spFAP present in plasma, serum, or blood. As is known in the art, because plasma, serum, and blood, particularly human plasma, serum, and blood, are known to contain spFAP, it is not necessary to isolate spFAP or any specific soluble FAP variant from plasma, serum, or blood. Therefore, it is also most preferred that specific and selective anti-cFAP antigen-binding molecules and antigen-binding fragments do not exhibit significant binding activity to plasma, serum, or blood, particularly human plasma, serum, or blood.

[0010] Selective binding of cFAP to one or more soluble variants (e.g., SEQ ID NO: 2 and / or spFAP in blood) can be established by any assay or combination of assays known in the art or described herein, such as in which the binding of a specific and selective anti-cFAP antigen-binding molecule to cFAP is compared with its binding to one or more soluble variants of sFAP, and / or in which a competitive assay is performed by comparing the binding to cFAP in the presence and absence of one or more soluble FAP variants, or in the presence and absence of plasma, serum or blood known or believed to contain soluble FAP (e.g., spFAP).

[0011] As a non-limiting example, the binding EC50 of specific and selective anti-cFAP antigen-binding molecules to cFAP was measured. 50 And EC combined with one or more soluble variants50 Comparisons can be made to assess the specificity and selectivity of binding to cFAP relative to one or more soluble FAP variants (e.g., spFAP, known to be present in plasma, serum, blood, or tissue). For example, by comparing EC binding to cFAP and spFAP... 50 There are no limitations on the assays used to determine specific and selective binding, and any suitable assay known in the art or described herein may be used. Assays used for comparative studies can be calibrated by using controls (e.g., assay conditions adjusted to obtain standard results for control molecules such as the known anti-FAP molecules siroizumab and / or 4B9). For example, preferably, the assay used to determine the binding of EC to soluble FAP (e.g., spFAP) is... 50 The determination of EC with siroizumab or 4B9 bound to spFAP 50 The tests were conducted under conditions of less than 1 nM or less than 10 nM, respectively.

[0012] Used to determine EC in conjunction with cFAP 50 An exemplary assay is performed using flow cytometry. Preferably, the assay conditions are adjusted such that EC binds to cFAP. 50 The value is less than 5 nM. Further preferably, the measurement conditions are adjusted such that (a) EC binding to the cFAP is as evaluated under the same conditions. 50 Is it siroizumab or 4B9 EC? 50 (a) within at least 1 time; and / or (b) the maximum binding to the cFAP is within 1 time of the maximum binding to siroizumab or 4B9 as evaluated under the same conditions.

[0013] When the assays used for binding to cFAP and soluble FAP (e.g., spFAP) are calibrated as described above, for example adjusted to achieve preferred values ​​for or relative to a control molecule (such as siroizumab or 4B9), specific and selective binding can be achieved through EC50 of less than 5 nM with cFAP. 50 The value sum is EC combined with cFAP 50 The value of up to 1 / 20 of the EC with one or more soluble variants (e.g., spFAP) 50 The specific and selective binding of the antigen-binding molecules of the present invention to cFAP can also be identified by molecules exhibiting one or more of the following cFAP binding properties, as determined by flow cytometry: (a) an apparent Kd of less than 2 nM for cFAP binding; (b) an EC of less than 2 nM for cFAP binding. 50 Less than 2 nM; (c) EC combined with the cFAP 50 The EC of sirolimus or 4B9 was evaluated under the same conditions.50 (d) The maximum binding to the cFAP is within 1 time the maximum binding to siroizumab or 4B9 evaluated under the same conditions.

[0014] Alternatively, it can be determined by identifying the EC50 of (i) antigen-binding molecules against cFAP (such as those recombinantly expressed on the surface of HEK 293F cells). 50 and / or TOP (MFI), and (ii) ECs of the molecule bound to one or more soluble FAPs (e.g., spFAP). 50 To identify or establish specific and selective binding. In a non-limiting example, the anti-cFAP antigen-binding molecule of the present invention is specific and selective for cFAP (relative to sFAP), wherein (i) EC binds to FAP such as that expressed on the surface of cells (e.g., SEQ ID NO: 1 recombinantly expressed on the surface of HEK 293 cells). 50 TOP (MFI) is the EC5 of siroizumab when evaluated under the same conditions. 50 and at least 60% of TOP (MFI); and (ii) EC bound to undiluted human plasma containing at least 80 ng / mL of spFAP. 50 It is (a) weaker than 40 nM, (b) undetectable, or (c) when siroizumab or 4B9 binds to the undiluted plasma, respectively, under the same conditions having less than 1 nM or less than 10 nM, as in control ECs. 50 It is incalculable during the evaluation of the measurement.

[0015] In another non-limiting example, the specificity and selective binding activity of the anti-cFAP antigen-binding molecule of the present invention can be determined in a competitive binding assay, such as in the presence or absence of spFAP, for example in the presence or absence of plasma, serum, tissue, or blood, by detecting and comparing the binding activity of cFAP. For example, the antigen-binding molecule of the present invention is specific and selective for cFAP (compared to one or more soluble variants of FAP, such as spFAP), wherein the binding ratio of cFAP to the absence of said undiluted human plasma in the presence of known or believed to contain spFAP (e.g., at a concentration of at least 80 ng / mL) is at least 80% at a concentration of 0.8 nM antigen-binding molecule, at least 60% at a concentration of 0.16 nM antigen-binding molecule, or at least 50% at a concentration of 0.0032 nM antigen-binding molecule. Preferably, the comparative assays in this context, under the same conditions, show a binding rate of 4B9 or siroizumab to cFAP of less than 10% or less than 25% in the presence of the undiluted human plasma relative to the binding rate in the absence of the undiluted human plasma, wherein the concentration of 4B9 or siroizumab is less than or equal to 0.8 nM and greater than or equal to 0.0032 nM, for example, 0.8 nM, 0.016 nM, or 0.0032 nM.

[0016] Any cFAP known in the art can be used for comparative assays, but preferably, the cFAP has the amino acid sequence SEQ ID NO: 1. As disclosed herein, cFAP is also membrane-bound and therefore must be expressed on the surface of a cell or cell membrane to allow for the assessment of specific and selective binding. cFAP can be expressed in any suitable cell as known in the art or described herein. In a non-limiting example, cFAP having the amino acid sequence SEQ ID NO: 1 was recombinantly expressed on the surface of HEK293F cells. Similarly, any known soluble variant of FAP can be used. As explained herein, multiple variants of soluble FAP are believed to be present in plasma, serum, blood, and tissues and differ from cFAP by having different truncated amino acid sequences. Therefore, the use of any one or more soluble variants is contemplated. In a non-limiting example, the soluble FAP variant used for comparative evaluation is sFAP having the amino acid sequence SEQ ID NO: 2. Alternatively or additionally, the soluble variant can be one or more soluble FAP variants present in plasma, serum, blood, or tissues, i.e., spFAP. It is not expected that spFAP need to be isolated from plasma, serum, blood or tissue, but serum, plasma, blood or tissue can be used as (diluted or undiluted) spFAP.

[0017] Specific and selective anti-cFAP antigen-binding molecules comprise or consist of one or more antibodies, antibody-antigen-binding domains, and / or antibody-antigen-binding fragments, i.e., comprise or consist of one or more antibodies and / or domains and fragments derived therefrom that provide antigen-binding ability as known in the art. Therefore, the anti-cFAP antigen-binding molecules of the present invention may comprise or consist of one or more antibodies or antibody-antigen-binding domains or fragments, said antibody-antigen-binding domains or fragments including, but not limited to, Fv domains (i.e., paired heavy and light chain variable domains, such as Fab, Fab', F(ab')2, and Fv fragments, and recombinant constructs, such as single-chain Fv domains (scFv)) and antigen-binding fragments and domains comprising single, unpaired heavy or light chain variable domains as known in the art, which are reserved for target antigens as defined herein (including, but not limited to, single-domain antibodies (also referred to in the art as sdAb, dAb, and / or nanobodies) and V-type heavy chains based on camel family heavy chains). H The ability to specifically and selectively bind to H domains. Preferably, the anti-cFAP antigen-binding molecule of the present invention comprises one or more V domains. H The H-structure domain or is composed of it.

[0018] Antigen binding specificity is determined by the portion of the antibody antigen-binding domain and fragment that contacts the ligand. These are called complementarity-determining regions (CDRs). CDRs are the most variable parts of an antibody and contribute to antibody diversity. It is well known that there are three CDR regions, CDR1, CDR2, and CDR3, in each heavy and light chain variable domain, embedded between four frame regions (FWs) according to the general pattern FW1-CDR1-FW2-CDR2-FW3-CDR3-FW4. In the case of a single-domain antibody, antigen binding specificity is determined by a set of three CDRSs (e.g., the three CDRs of the heavy or light chain variable domain). For example, in V... H In H, antigen binding specificity is determined by V. H H CDR1, CDR2 and CDR3 are determined.

[0019] The boundaries and lengths of individual CDRs are subject to various classification and numbering systems known in the art, including but not limited to those referred to as the Kabat, Chothia, and IMGT systems, as described below: for example, Kabat et al., “Sequences of Proteins of Immunological Interest”, 5th ed., US Department of Health and Human Services, 1992; Chothia et al., J. Mol. Biol. 196 (1987), 901; and Lefranc et al., Dev Comp Immunol 27 (2003), 55-77. Unless otherwise stated, the CDR domains mentioned herein are defined according to IMGT.

[0020] As used herein, the term "comprising" means that in addition to the specifically listed sequences and / or components, it may include additional sequences / components. In antigen-binding molecules, one or more antibody-antigen binding domains or fragments (e.g., one or more V...) are included. H In those embodiments of the H antibody, additional amino acids and / or functional domains of the molecule of the present invention may be present at the N-terminus, C-terminus, or both of the antibody-antigen binding domain. Additional sequences may include, but are not limited to, sequences introduced for purification or detection, or sequences introduced as known in the art to confer additional activity (e.g., Fc region function / activity, such as Fc receptor binding). Furthermore, in cases where individual sequences "comprise" the aforementioned sequences, they may also include additional amino acids at the N-terminus, C-terminus, or both.

[0021] The specific and selective anti-cFAP antigen-binding molecule according to the present invention may include an Fc region. As used herein, the term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region capable of binding to one or more Fc receptors, preferably one or more FcγR receptors. The term includes native sequence Fc regions and variant Fc regions. As understood in the art, the term "Fc region" and similar phrases refer to a polypeptide containing a constant region of an antibody other than the first constant region immunoglobulin domain, and in some cases, is part of a hinge. Thus, "Fc region" and similar terms / phrases generally refer to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the N-terminus of the flexible hinge of these domains. For IgA and IgM, the Fc region may include a J chain. For IgG, the Fc region includes immunoglobulin domains Cγ2 and Cγ3 and a lower hinge region between Cγ1 and Cγ2. Although the boundaries of the Fc region may vary, the Fc region of the human IgG heavy chain is generally defined as including the C226 or P230 residue at the carboxyl terminus. It should be further understood that, depending on the production process, the sequence of the Fc region can be altered by post-translational modifications, particularly of the terminal residues. In a non-limiting instance, it should be understood that, depending on the production process, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Therefore, antigen-binding molecules as disclosed herein may contain variants of any of the aforementioned Fc regions. Unless otherwise stated herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system (also known as the EU index), e.g., as described in Kabat et al., *Protein Sequences of Immunological Significance*, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0022] The Fc region of the antigen-binding molecule of the present invention may be a modified Fc region relative to the natural (wild-type) Fc region sequence, for example exhibiting increased binding to one or more FcγR receptors and / or exhibiting increased ADCC or ADCP activity. Such modifications are well known in the art and include, but are not limited to, the EU-numbered mutation S239D / A303L / I332E (also known as the DEL modification) and the absence of fucosylation modification. Therefore, it is preferred that the Fc region exhibits increased activity relative to the natural (wild-type) sequence. "Natural (wild-type) sequence Fc region" comprises the same amino acid sequence as the Fc region found in nature. Because specific and selective anti-cFAP antigen-binding molecules are preferred for use in humans, the Fc region is expected to be a human Fc region or a variant / modified human Fc region. As described herein, the variant / modified Fc region may exhibit increased binding activity to one or more FcγR receptors and / or increased ADCC or ADCP activity relative to the natural or wild-type Fc region. Therefore, in cases where the specific and selective anti-cFAP antigen-binding molecule of the present invention contains a modified or variant Fc region, it is preferred that the reference antigen-binding molecule used to determine the increased Fc region activity, as described herein, contains the same antigen-binding region / domain / fragment as the native Fc region sequence of the test molecule and human IgG1 having the amino acid sequence SEQ ID NO: 22.

[0023] It is further anticipated that the Fc region of the disclosed antigen-binding molecule (where present) may include additional modifications known in the art to modulate the interaction of the antibody Fc with Fc ligands other than FcγR. Such ligands include FcRn and Fc receptor homologues, including but not limited to FcRH1, FcRH2, FcRH3, FcRH4, FcRH5, and FcRH6; see, for example, Davis et al., 2002, *Immunol. Reviews* 190:123-136.

[0024] As described in the examples, the inventors have developed antibodies specific and selective for FAP expressed on the surface of cells (i.e., cFAP), particularly against soluble variants of FAP, such as soluble recombinant FAP having the amino acid sequence of SEQ ID NO: 2 (sFAP) and / or soluble variants (spFAP) present in plasma, serum, tissue, or blood. Analysis of exemplary embodiments of the invention further allowed the inventors to identify V H Exemplary common sequences of H CDR and complete V HThe H sequence can confer the identified functional characteristics of the specific and selective anti-cFAP antigen-binding molecule of the present invention. The analysis was performed based on the understanding in the art that certain CDR / variable domain residues are primarily responsible for antibody binding activity, while the remaining residues have a smaller impact. Therefore, it is known that amino acid residues within the CDR and / or variable domain regions can be exchanged without necessarily leading to a (significant) loss of function. That is, it is known in the art that certain amino acid residues in the CDR and / or variable regions can be exchanged, and sequence variants that maintain the desired functional properties are readily identifiable.

[0025] Therefore, exemplary anti-cFAP antigen-binding molecules of the present invention comprise one or more V H H or composed of it, the one or more V H H includes

[0026] (i) CDR1, which has the sequence X1X2TX3X4X5YAX6G (SEQ ID NO: 3), wherein X1 is A, G or S; wherein X2 is P, R or G; wherein X3 is K, F, R or N; wherein X4 is S, R or G; wherein X5 is T, S or N; and wherein X6 is M, I or L;

[0027] (ii) CDR2, wherein CDR2 has the sequence X1IX2WX3X4X5X6TX7YX8DSVKG (SEQ ID NO: 4), wherein X1 is A or V; wherein X2 is N, W or S; wherein X3 is S or A; wherein X4 is G or N; wherein X5 is T or G; wherein X6 is I, L, S, T or V; wherein X7 is S, Q or N; and wherein X8 is S or T; and / or

[0028] (iii) CDR 3, which has the sequence AADX1DFRTVGSRPSY (SEQ ID NO: 5), wherein X1 is R, S or K.

[0029] The exemplary anti-cFAP antigen-binding molecules defined above include those containing one or more V H H or molecules composed thereof, the one or more V H H includes

[0030] (i) CDR1, CDR2 and CDR3, wherein CDR1 has a sequence of APTRSTYAMG (SEQ ID NO: 6) or GRTFSSYAIG (SEQ ID NO: 7), CDR2 has a sequence of VINWSGTLTSYADSVKG (SEQ ID NO: 8), and CDR3 has a sequence of AADRDFRTVGSRPSY (SEQ ID NO: 14);

[0031] (ii) CDR1, CDR2 and CDR3, wherein CDR1 has a sequence of APTRSTYAMG (SEQ ID NO: 6) or GRTFSSYAIG (SEQ ID NO: 7), CDR2 has a sequence of VINWAGTLTSYADSVKG (SEQ ID NO: 9), and CDR3 has a sequence of AADRDFRTVGSRPSY (SEQ ID NO: 14);

[0032] (iii) CDR1, CDR2 and CDR3, wherein CDR1 has a sequence of APTRSTYAMG (SEQ ID NO: 6) or GRTFSSYAIG (SEQ ID NO: 7), CDR2 has a sequence of VISWSGTLTSYADSVKG (SEQ ID NO: 10), and CDR3 has a sequence of AADRDFRTVGSRPSY (SEQ ID NO: 14);

[0033] (iv) CDR1, CDR2 and CDR3, wherein CDR1 has the sequence GRTFSSYAIG (SEQ ID NO: 7), CDR2 has the sequence AISWSGGTTQYTDSVKG (SEQ ID NO: 11), and CDR3 has the sequence AADRDFRTVGSRPSY (SEQ ID NO: 14);

[0034] (v) CDR1, CDR2, and CDR3, wherein CDR1 has the sequence GRTFSSYAIG (SEQ ID NO: 7), CDR2 has the sequence AISWSGGTTNYTDSVKG (SEQ ID NO: 12), and CDR3 has the sequence AADRDFRTVGSRPSY (SEQ ID NO: 14); or

[0035] (vi) CDR1, CDR2 and CDR3, wherein CDR1 has the sequence GRTFSSYAIG (SEQ ID NO: 7), CDR2 has the sequence AISWSGGTTNYADSVKG (SEQ ID NO: 13) and CDR3 has the sequence AADRDFRTVGSRPSY (SEQ ID NO: 14).

[0036] The anti-cFAP antigen-binding molecule of the present invention as defined above further includes molecules comprising one or more amino acid sequences selected from or composed of the following amino acid sequences:

[0037] (i)QVQLVESGGGLVQAGDSLSLSCVASAPTRSTYAMGWFRQAPGKEREFAAVINWSGTLTSYADSVKGRFTISRDNAKNTVFLQMNSLKPDDTAVYYCAADRDFRTVGSRPSYWGQGTPVTVSS (“ab1”, SEQ ID NO:15);

[0038] (ii)QVQLVESGGGLVEPGDSLRLSCAASGRTFSSYAIGWFRQAPGKEREFVAAISWSGGTTNYTDSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAADRDFRTVGSRPSYWSKGTRVTVSS (“ab3”, SEQ ID NO:16);

[0039] (iii)QVQLVESGGGLVQPGGSLRLSCSASAPTRSTYAMGWFRQAPGKEREFASVISWSGTLTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAADRDFRTVGSRPSYWGQGTTVTVSS (“ab1z12”, SEQ ID NO: 17);

[0040] (iv)QVQLVESGGGLVQPGGSLRLSCSASGRTFSSYAIGWFRQAPGKEREFVSAISWSGGTTQYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAADRDFRTVGSRPSYWGQGTTVTVSS (“ab3z7”, SEQ ID NO:18);

[0041] (v)QVQLVESGGGLVQPGGSLRLSCSASGRTFSSYAIGWFRQAPGKEREFVSAISWSGGTTNYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAADRDFRTVGSRPSYWGQGTTVTVSS (“ab3Z10”, SEQ ID NO:19).

[0042] The anti-cFAP antigen-binding molecule of the present invention may further comprise V HH CDR and / or one or more variants of the complete sequence as defined herein. As used herein, the terms variant CDR and / or variant sequence indicate a functional variant, i.e., an amino acid sequence that differs from the reference amino acid sequence, but which exhibits or maintains the same functional activity as the reference sequence. Specifically, as indicated throughout this disclosure, a variant V includes one or more variants. H H CDR and / or variant V H The exemplary antigen-binding molecules of the present invention with the H sequence exhibit specific and selective / discriminatory binding to cFAP as described herein.

[0043] The amino acid sequences of the variant CDRs and / or sequences described herein are defined by referring to amino acid sequences having amino acid substitutions at one or more positions. As used herein, the term "substitution" means that one amino acid is substituted by another amino acid. Thus, the total number of amino acids remains unchanged. The term "substitution" explicitly does not cover the deletion of an amino acid at one position or the introduction of one (or more) amino acids at different positions.

[0044] Regarding variant CDRs, modifications can occur within one or more CDRs, provided that the modifications substantially do not diminish or otherwise alter the desired functional properties of the reference antigen-binding molecule. Thus, in some embodiments, the CDRs provided herein may be unaltered, or may be variants with no more than one, two, or three amino acid substitutions relative to SEQ ID NO: 6 to 14, which exhibit specific selective binding to cFAP as defined herein. In other embodiments, the antigen-binding molecule of the present invention may comprise one or more V's having the amino acid sequence of SEQ ID NO: 15 to 19 or having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with it. H H domain / antibody, the molecule exhibiting specific and selective binding to cFAP relative to one or more soluble variants as defined herein (e.g., spFAP). In some embodiments, the antigen-binding molecule of the present invention comprises one or more of the amino acid sequences SEQ ID NO: 15 to 19.

[0045] Preferably, the amino acid substitution is a conserved residue substitution. The term "conserved amino acid substitution" is well known in the art and refers to the substitution of one amino acid with a different amino acid having similar biophysical properties. As used herein, a group of amino acids having similar biophysical properties is...

[0046] (a) Nonpolar hydrophobic amino acids composed of glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), and methionine (Met);

[0047] (b) Polar neutral amino acids composed of serine (Ser), threonine (Thr), asparagine (Asn), and glutamine (Gln);

[0048] (c) A positively charged basic amino acid composed of arginine (Arg), lysine (Lys), and histidine (His); and

[0049] (d) A negatively charged acidic amino acid composed of aspartic acid (Asp) and glutamic acid (Glu).

[0050] Therefore, a conservative substitution is the substitution of a residue with another residue from the same group, namely (i) replacing a nonpolar hydrophobic amino acid of group (a) with another amino acid of group (a); (ii) replacing a polar neutral amino acid of group (b) with another amino acid of group (b); replacing a positively charged basic amino acid of group (c) with another amino acid of group (c); and / or replacing a negatively charged acidic amino acid of group (d) with another amino acid of group (d). It should be understood that amino acids Cys and Pro are not included in the above groupings and / or lists of conservative substitutions because, as is well known in the art, these residues are not suitable as universal substituents. Where residues Cys or Pro are substituted, as used herein, the conservative substitution of Cys is with Ser or Ala, and the conservative substitution of Pro is with Ala.

[0051] As used herein, the terms “selectively binds to and / or distinguishes between two antigens” and similar terms relating to two antigens, such as the selective binding of an antigen-binding molecule or antigen-binding domain / fragment to cFAP or the distinguishing of cFAP from / relative to one or more soluble variants (e.g., spFAP present in plasma, serum, tissue, or blood), indicate that the antigen-binding molecule or antigen-binding domain / fragment specifically binds to the target antigen cFAP but not specifically to non-target antigens (i.e., their soluble variants, such as spFAP present in plasma, serum, tissue, blood, or body fluids in vivo). Therefore, the terms selective binding, distinguishing, and similar terms as used herein mean that an antibody or antigen-binding fragment “does not specifically bind to non-target antigens” / “does not significantly bind to non-target antigens” (which are used interchangeably). As is well known in the art, the terms “specifically binds” and “does not significantly bind” indicate the degree to which an antigen-binding molecule distinguishes between two antigens. This is because no antigen-binding molecule (especially one containing an antibody-derived antigen-binding domain or fragment) is known to have absolute specificity, meaning that it reacts with only one epitope regardless of conditions. In other words, in the presence of other (non-target) antigens, antigen-binding molecules, domains, or fragments can react to a certain extent with similar epitopes on these other (non-target) antigens. However, specific antigen-binding molecules, domains, or fragments have a significantly greater affinity for their target epitopes / antigens than for the related epitopes. This difference in affinity is used to establish assay conditions under which the antigen-binding molecule binds almost exclusively to the specific epitope. In this respect, the binding (or non-binding) of the antigen to the antigen by the antigen-binding molecule should not be understood as absolute. That is, the anti-cFAP antigen-binding molecule of the present invention may exhibit some (residual) binding activity to other (non-)targets, but its level is significantly reduced relative to the binding activity of FAP (cFAP) expressed on the surface of cells. The characteristics that distinguish the target antigen from / relative to the non-target antigen can be characterized by exemplary assays as known in the art or as described above. In some embodiments, the anti-cFAP antigen-binding molecule of the present invention may not exhibit detectable binding to non-target antigens.

[0052] As detailed herein, it is preferred to use the same experimental protocol and the same experimental conditions (e.g., the same binding assay, testing / measuring the concentration / density of antibody or antigen-binding fragments, antigen concentration / density / flow rate, etc.) to determine the comparison of distinguishing features, i.e., “specific binding”, “non-specific binding”, “selective binding” and / or “distinguishing” relative to the target antigen and non-target antigen.

[0053] The binding properties of anti-cFAP antigen-binding molecules can be established by any suitable method known in the art and / or as described herein, which allows for the quantification of binding parameters, and in particular, allows for their quantitative comparison. Methods for analyzing the binding specificity and binding parameters of antigen-binding molecules are described, for example, in Harlow and Lane (1988), *Antibodies: A Laboratory Manual*, Cold Spring Harbor Laboratory Press, and Harlow and Lane (1999), *Using Antibodies: A Laboratory Manual*, Cold Spring Harbor Laboratory Press. Non-limiting examples of suitable studies include binding studies and blocking / competition studies performed by methods such as IHC, IF, flow cytometry analysis, FACS analysis, flow cytometry titration analysis (FACS titration), surface plasmon resonance (SPR, e.g., using BIAcore®), isothermal titration calorimetry (ITC), fluorescence titration, or determination of binding by radiolabeled ligands. Other methods include, for example, Western blotting, ELISA (including competitive ELISA), RIA, ECL, IRMA assays, and physiological assays such as cytotoxicity assays.

[0054] The anti-cFAP antigen-binding molecule of the present invention can be produced by any technique described herein and / or as known in the art. For example, the molecule can be prepared, expressed, generated, or isolated by recombination, such as by expression using a recombinant expression vector transfected into a host cell, or by any other method involving splicing a coding gene sequence into another DNA sequence. Therefore, a polynucleotide encoding the anti-cFAP antigen-binding molecule of the present invention and / or encoding its component domains or fragments is provided, as well as a vector comprising such a polynucleotide. The vector need not necessarily be an expression vector, but can be a vector that allows for vector replication, and thus, for example, replication of the polynucleotide sequence of the present invention by culturing a host cell containing the vector. As known in the art, the vector can also be adapted to allow recombination manipulation of the polynucleotide sequence of the present invention. In a preferred embodiment, the vector is an expression vector comprising a polynucleotide encoding the anti-cFAP antigen-binding molecule as disclosed herein, which, when introduced into a suitable prokaryotic or eukaryotic cell according to standard methods known in the art, expresses the molecule of the present invention or its component fragments or domains. The host cells of this invention can be directly engineered cells, i.e., cells that have been directly transfected with a vector or polynucleotide as disclosed herein, or can be daughter cells or progeny of directly transfected cells. Therefore, a method is provided for generating anti-cFAP antigen-binding molecules by culturing host cells containing polynucleotides encoding said molecule or its component domains or fragments (e.g., contained in an expression vector). The method further comprises recovering and isolating the expressed molecule or component domains or fragments thereof from the culture (e.g., from cell fractions and / or culture media) using standard protein purification methods. Therefore, this invention also provides specific and selective anti-cFAP antigen-binding molecules obtainable by the methods disclosed herein.

[0055] Expression from host cells and / or their progeny is achieved by introducing one or more expression vectors encoding molecules of the present invention into host cells using standard techniques. The introduction of such expression vectors is known in the art and is referred to herein as transfection or transformation, encompassing a variety of standard techniques commonly used to introduce exogenous DNA into eukaryotic or prokaryotic host cells. Non-limiting examples of suitable transfection methods include chemical transfection (e.g., lipid transfection, calcium phosphate precipitation, and DEAE-glucan), physical transfection (e.g., microinjection, gene gun), viral vectors (e.g., viral vectors such as retroviruses, lentiviruses, adenoviruses), and non-viral vectors (plasmid vectors, transfection). Non-limiting examples of suitable transformation methods include chemical, physical, or phage-mediated methods.

[0056] Although the molecules of the present invention can be expressed in prokaryotic or eukaryotic host cells, expression in eukaryotic cells is preferred, as they contain antibody-derived domains and fragments (such as V).H The molecules of H antibodies are most preferably expressed in mammalian host cells because such eukaryotic cells (especially mammalian cells) are more likely than prokaryotic cells to assemble and secrete properly folded immune-active molecules. Non-limiting examples of mammalian host cells for expressing the molecules of the present invention include Chinese hamster ovary (CHO cells), NSO myeloma cells, COS cells, and SP2 cells.

[0057] Recombinant anti-cFAP antigen-binding molecules and / or their antigen-binding domains / fragments may have variable and constant regions (if present) derived from germline immunoglobulin sequences of the species from which the molecules and / or their antigen-binding domains / fragments were isolated following standard immunization and selection procedures known in the art, such as potentially including germline immunoglobulin sequences from alpacas. However, sequences can be mutagenized in vitro, particularly by combining CDR sequences with FW sequences from another species (e.g., known from humans during humanization). Therefore, the amino acid sequences of the molecules of the present invention disclosed herein may be sequences that, while derived from and associated with germline heavy or light chain sequences, may not be naturally present in any endogenous antibody germline library in vivo.

[0058] The anti-cFAP antigen-binding molecule of the present invention is contemplated as a therapeutic and / or diagnostic tool, for example, in therapies for treating diseases characterized by cFAP overexpression. As used herein, the term "therapeutic" does not mean the complete eradication of any disease or cause of disease, but also encompasses the permanent or temporary relief of any disease parameter or symptom. In non-limiting exemplary embodiments, the molecule of the present invention may be used as a targeting portion in the diagnostic and therapeutic uses disclosed herein. In this context, the molecule of the present invention may be conjugated with therapeutic or diagnostic portions as known in the art or described herein, such as cytotoxins, radioisotopes, or other active molecules as known in the art as therapeutic or diagnostic portions.

[0059] The molecules of this invention can be directly conjugated to diagnostic or therapeutic moieties, or indirectly conjugated, for example, by using a linker. The selection of a suitable linker can be based on criteria well known in the art. As recognized, a suitable linker can be selected for characteristics such as stability and biocompatibility. Linkers can also be designed and / or selected to maintain the structural integrity and binding affinity of the antibody while facilitating targeted delivery of the conjugated moieties, such as radiolabeled isotopes or therapeutic agents. Suitable linkers can include, but are not limited to, maleimide-thiol bonds, hydrazone bonds, or click chemistry-based constructs that allow site-specific conjugation and controlled release. In some embodiments, the linker can incorporate cleavable functional groups, such as pH-sensitive bonds or enzyme-cleavable groups, to ensure selective release of the radioligand in the tumor microenvironment or at the disease site.

[0060] In the context of in vivo diagnostics, and in non-limiting instances, the molecules of the present invention may be conjugated with radioisotopes, radiotracers, radionucleotides, luminescent probes, or fluorescent probes as known in the art (e.g., directly or indirectly via covalent linkers, DOTA, etc.). Known radioisotopes that can be used in the context of the diagnostic or therapeutic indications disclosed herein include, but are not limited to: 18 F, 18 F-FAC, 32 P, 33 P, 45 Ti、 47 Sc、 52 Fe、 59 Fe、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 75 Sc、 77 As、 86 Y、 89 Sr、 89 Zr、 94 Tc, 99 mTc, 99 Mo、 105 Pd, 105 Rh、 111 Ag、 111 In、 123 I, 124 I, 125 I, 131 I, 142 Pr、 143 Pr、 149 Pm, 149 Tb, 153 Sm、 154 – 158 Gd, 161 Tb, 166 Dy、 166 Ho、 169 Er、 175 Lu、 177 Lu、 186 Re、 188 Re、 189 Re、 194 Ir、 198 Au、 199 Au、 211 At、 211 Pb, 212 Bi、 212 Pb, 213 Bi、 223 Ra、227 Th、 225 Ac、 3 H, 11 C. 94 mTc, 203 Pb, 97 Ru、 88 Y、 121 Sn. Therefore, the molecules of the present invention are also conceived as diagnostic agents in assays that include the detection of cFAP, particularly for further distinguishing cFAP from / relative to other variants of FAP that may be present in a sample (particularly soluble variants present in plasma, serum, tissue, blood or body fluids).

[0061] Therefore, therapeutic and diagnostic compositions are provided comprising anti-cFAP antigen-binding molecules as disclosed herein, polynucleotides encoding such molecules (e.g., in the context of a vector), and host cells comprising the vector and / or a polynucleotide sequence encoding said molecule. The molecules of the present invention are suitable for any immunologically based diagnostic assays (including immunohistochemical assays) known in the art and / or described herein, particularly ex vivo and in vitro diagnostic assays and assay systems known in the art. For example, methods for immunohistochemical staining of biological samples (e.g., containing tissues or cells) obtained from a patient or for measuring the amount of cFAP in a specific tissue may be valuable.

[0062] The specific and selective anti-cFAP antigen-binding molecules and methods disclosed herein provide for the treatment and diagnosis of diseases characterized by cFAP expression. Therefore, specific and selective anti-cFAP antigen-binding molecules, their antigen-binding domains / fragments, polynucleotides encoding such molecules / domains or fragments, and cells expressing such molecules / domains or fragments are provided for use as medicaments. As understood, cFAP may or may not be expressed by the disease or pathogenic cells themselves. Diseases are also characterized by cFAP expression, wherein it is not expressed by the diseased cells themselves, but rather by cells residing within the disease parenchyma, and said cells are not diseased cells themselves. Such cells residing in the disease parenchyma that are not diseased cells but can express cFAP include, but are not limited to, CAFs, activated fibroblasts in fibrotic diseases, myofibroblasts in scar formation, and mesenchymal cells in inflammatory diseases.

[0063] Diseases characterized by cFAP expression and targeted by the antigen-binding molecules / domains / fragments of the present invention or cells expressing such molecules / domains / fragments of the present invention can be any disease known in the art, such as, but not limited to, cancer, fibrotic cancer, fibrotic diseases (including liver, kidney, and lung fibrosis), inflammatory diseases, scarring, heart diseases (including cardiac fibrosis and heart failure), arthritis, benign gynecological diseases (including endometriosis, fibroids, and Asherman's syndrome), and surgically induced fibrosis. Preferably, diseases characterized by cFAP expression and targeted by the antigen-binding molecules / domains / fragments of the present invention and their methods of use are endometriosis, fibroids, adenomyosis, atherosclerosis, cFAP+ tumors, cardiac fibrosis, idiopathic pulmonary fibrosis, or surgically induced fibrosis. Therefore, methods comprising administering single or multiple doses of the antigen-binding molecules / domains / fragments of the present invention or drug carriers such as VLP, LNP, or cells carrying such molecules / domains / fragments of the present invention are also provided.

[0064] The antigen-binding molecules / domains / fragments of the present invention and / or compositions comprising such antigen-binding molecules / domains / fragments of the present invention are also suitable for immunoassays, wherein they can be used in a liquid phase or bound to a solid-phase carrier. Examples of such immunoassays or immunohistochemical assays are direct or indirect forms of immunoassays or immunohistochemical assays and can be single-step or multi-step (e.g., heterologous) assays. Examples of such assays are enzyme-linked immunosorbent assays (ELISA), enzyme immunoassays (EIA), radioimmunoassays (RIA), Western blot assays, or immunoassays based on luminescence, fluorescence, chemiluminescence, or electrochemiluminescence detection. In some embodiments, the antigen-binding molecules / domains / fragments of the present invention or compositions comprising them can be used in methods for detecting cFAP. Alternatively or additionally, the antigen-binding molecules / domains / fragments of the present invention or compositions comprising them can be used in vivo or in vitro to distinguish cFAP from / relative to its soluble variants (including one or more soluble variants present in serum, plasma, blood, tissue, or body fluids, such as spFAP).

[0065] The immunoassays described herein (including immunohistochemical assays) can be performed on any suitable biological sample known in the art and / or described herein. Because the assays described herein are used for the specific and selective detection of cFAP, particularly to distinguish its soluble variants, the samples are biological samples from subjects who are expected to or have evidence of cFAP presence. Examples of biological samples suitable for the uses and methods disclosed herein include, but are not limited to, tissue samples and body fluid samples, such as, but not limited to, plasma, serum, and blood.

[0066] The term "composition," for example, when referring to therapeutic or diagnostic compositions used according to the invention, refers to a composition comprising at least one of the anti-cFAP antigen-binding molecules (or their antigen-binding domains or fragments) disclosed herein, a polynucleotide, a carrier, and / or a host cell. The composition may optionally further comprise other molecules capable of altering the properties of the compounds of the invention, thereby, for example, stabilizing, modulating, and / or enhancing the function of the compounds. The composition may be in solid or liquid form, and in particular may be in powder, tablet, or solution form.

[0067] The components of the composition can be packaged in one or more containers (e.g., sealed ampoules or vials) as an aqueous solution or as a lyophilized formulation for reconstitution. A working solution is prepared by reconstituted the lyophilized compound using, for example, water for injection for therapeutic purposes or another desired solvent (e.g., a buffer) for diagnostic purposes. Preservatives and other additives, such as antibacterial agents, antioxidants, chelating agents, and inert gases, may also be present. The various components of the composition can be packaged into a kit with instructions for use. Therefore, a kit comprising one or more compositions as disclosed herein is also provided. Attached Figure Description

[0068] Figure 1 Comparative ELISA assays of ab1 and ab3 variants fused to the human antibody Fc domain (hFc) of human donor plasma. For reference, the antibody siroizumab and an antibody construct containing the Fab domain of a 4B9 antibody fused to hFc (“4B9-hFc”) were used. The Fc domain used in 4B9-hFc is the same as that used in the ab1 and ab3 Fc fusions. Optical density (OD) measured in the ELISA assay was used to quantify the binding strength of the antibody / antibody construct to spFAP in human plasma.

[0069] Figure 2 The binding activity of antibody constructs to cell-bound FAP (cFAP) was determined by flow cytometry (FCM) analysis of HEK 293F cells transiently transfected with full-length human FAP (Uniport-id: Q12884). Mean fluorescence intensity (MFI) was measured and plotted against the difference between human FAP and isotype control human IgG (MFI). All antibody constructs tested showed cFAP binding activity.

[0070] Figure 3In HEK 293F cells transiently transfected with full-length human FAP (Uniport-id: Q12884), the binding activity of the antibody construct to cellular FAP (cFAP) was measured by flow cytometry (FCM) in the presence and absence of undiluted plasma. Mean fluorescence intensity (MFI) was measured and plotted against the percentage of cFAP binding in the presence and absence of spFAP. Controls were siroizumab, 4B9, and isotype control mouse IgG.

[0071] Figure 4 Cross-reactivity of antibody constructs with cynomolgus monkey and mouse cFAP. The binding affinity of antibody / antibody construct pairs on cells was measured by flow cytometry for (A) HEK239F transfected with cynomolgus monkey FAP accession number XP_005573377.1 and (B) HEK293F transfected with mouse FAP (accession number P97321). Higher affinity was reflected by a higher MFI.

[0072] Figure 5 The pharmacokinetic (PK) characteristics of the antibody constructs in mouse plasma were improved (n ≥ 3). (A) Mean plasma concentrations of 4B9-hFc (Pharma) and ab1z12-hFc (single injection of 10 mg / kg in BALB / c mice) over time were monitored using human IgG1 ELISA. (B) A summary of the PK parameters is presented in (B).

[0073] Figure 6 BALB / c mice were administered a specific and selective cFAP-mFc construct or a control molecule developed according to the method of the present invention, wherein the control molecule was formatted as a complete IgG containing a variable 4B9 domain. Both the specific and selective cFAP antigen-binding molecule and the 4B9-mFc construct used the mouse IgG2a domain as the Fc. The concentration of spFAP in mouse plasma was measured by a mouse FAP sandwich ELISA. The spFAP concentration was decreased in mice injected with 4B9-Fc, but not in mice injected with the specific and selective cFAP construct.

[0074] Figure 7The enzymatic activity of (A) recombinant human soluble FAP (Acro Biosytems, Switzerland; FAP-H5244) and (B) spFAP in human plasma was not affected by the antibody construct. FAP enzymatic activity was determined using FAP substrates and quenched dyes in the presence of different concentrations of antibody constructs ab1-hFc and ab3-hFc. Results are presented in comparison with controls, siroizumab, and 4B9-hFc. The measured fluorescence indicates the enzymatic activity of FAP. The enzymatic activity of recombinant FAP and spFAP in human plasma was not affected by the antibody / antibody construct tested.

[0075] Figure 8 ADCC reporter gene assay. Antibody constructs ab1-hFc and ab3-hFc exhibited dose-dependent ADCC activity in the following different fibroblast types: (A) endometrial / uterine (HUF), (B) cardiac (HCF), (C) skin fibroblasts (Detroit551), and (D) lung (HPF). Sirolizumab was used as a reference antibody. Relative luminescence was used to quantify ADCC activity / effect cell activation. Data points represent the mean of technical replicas. A dose-dependent increase in luminescence was observed, confirming the efficacy of the antibody constructs in mediating antibody-dependent cell-mediated cytotoxicity.

[0076] Figure 9 ADCP reporter gene assays were performed on HUF and HCF using ab1 and ab3 hFc constructs. The antibody constructs ab1-hFc and ab3-hFc exhibited low dose-dependent ADCP activity in the following different fibroblast types: (A) endometrial / uterine (HUF) and (B) cardiac fibroblasts (HCF). The antibody construct ab1-hFc (DEL) exhibited enhanced ADCP activity. Relative luminescence was used to quantify ADCP activity / effect cell activation. Data points represent the mean of technical replicas.

[0077] Figure 10 ADCP reporter gene assays were performed on HUF and HPF using the ab1-hFc construct containing either fucosylation-free or DEL-free Fc modification. The antibody construct ab1-hFc exhibited relatively low ADCP activity in (A) endometriosis / uterus (HUF) and (B) lung fibroblasts (HPF). ADCP activity was enhanced by increasing affinity for FcyR on effector cells through DEL modification of the Fc region or fucosylation-free Fc region (ahFc). No nonspecific ADCP activity was observed in the absence of target cells. Relative luminescence was used to quantify ADCP activity / effector cell activation. Data points represent the mean of technical replicas.

[0078] Figure 11 ADCC reporter gene assays were performed on HUF, HPF, HCF, and Detroit551 cells using the ab1-hFc construct containing either un-fucosylated (ahFc) or DEL-modified Fc amino acids. The antibody construct ab1-hFc exhibited ADCC activity in (A) endometrial / uterine (HUF), (B) lung (HPF), (C / D) heart (HCF), and (E) skin (Detroit551) fibroblasts. ADCC activity was enhanced by increasing effector function through DEL modification of the Fc region or un-fucosylation of the Fc region. No nonspecific ADCC activity was observed in the absence of target cells. Relative luminescence was used to quantify ADC activity / effector cell activation. Data points represent the average of technical replicas.

[0079] Figure 12 ADCP was measured using PBMC-derived macrophages. This assay demonstrated the superior phagocytic efficacy of ab3z7-ahFc compared to siroizumab in terms of EC50 and maximum phagocytosis.

[0080] Figure 13 The specific uptake of antibody fluorophore conjugates demonstrates the effectiveness of antibodies in targeting FAP-positive cells in complex biological processes such as cancer or other FAP+ diseases.

[0081] Figure 14 Detection of ab3-mFc binding to different tissues. FAP detection based on ab3-mFc staining was low or undetectable in all tested healthy adult tissues. Detailed Implementation

[0082] 5.1 Antigen-binding molecules that selectively and differentially bind to cFAP

[0083] This invention provides antigen-binding molecules comprising, or composed of, an antibody that specifically binds to cell-bound fibroblast activation protein (cFAP), an antibody-antigen-binding domain and / or an antibody-antigen-binding fragment, as well as a polynucleotide encoding such an antigen-binding molecule and a host cell expressing such an antigen-binding molecule. The antigen-binding molecules of this invention are particularly useful as therapeutic agents and reagents for the specific binding and targeting of cFAP, wherein the reagents also particularly distinguish the target antigen (i.e., cFAP) from its soluble variants (including one or more soluble FAP variants present in bodily fluids such as blood, serum, plasma, or other tissue fluids). Therefore, in some embodiments, the antibodies and antibody-binding fragments of this invention can also distinguish from non-target binding and selectively bind to cFAP in the presence of bodily fluids such as blood, serum, plasma, or other tissue fluids.

[0084] The antigen-binding molecules disclosed herein specifically and selectively bind to cFAP (i.e., cell membrane-bound FAP, known as a type II transmembrane glycoprotein having the amino acid sequence of SEQ ID NO: 1) and do not significantly bind to soluble versions of FAP (known as one or more variants of FAP having one or more truncated amino acid sequences relative to the amino acid sequence of cFAP). An exemplary soluble version of FAP has the amino acid sequence of SEQ ID NO: 2. Alternatively or additionally, the soluble version of FAP is one or more soluble forms known to be present in bodily fluids such as blood, plasma, serum, or other tissue fluids, i.e., spFAP. Additionally, in some embodiments, the antigen-binding molecules do not exhibit significant binding to bodily fluids (such as blood, plasma, or serum) that do not contain cFAP.

[0085] As demonstrated herein, the specific and selective antigen-binding molecules of the present invention distinguish cFAP from soluble versions of FAP (including spFAP). Without being bound by any particular interpretation, the antigen-binding molecules of the present invention recognize and specifically bind to target epitopes not present in or not presented in soluble FAP variants. As understood in the art, a target epitope that is absent or not presented can be an epitope comprising a linear contiguous segment of an amino acid missing in a soluble variant of FAP. Alternatively or additionally, a target epitope not present in or not presented by a soluble FAP variant can be a three-dimensional epitope formed by discontinuous residues in an amino acid sequence spatially close by the tertiary structure of cFAP, which is not formed in the soluble variant due to variations in amino acid sequence and / or folding. Target epitopes not present in or not presented by a soluble FAP variant may also be unavailable because steric hindrance is only relevant in soluble variants. Therefore, if the target epitope is present, the anti-cFAP antigen-binding molecule presented in this paper can also specifically bind to FAP variants other than cFAP.

[0086] Most preferably, the specific and selective anti-cFAP antigen-binding molecule specifically binds to cFAP having the amino acid sequence of SEQ ID NO: 1, as expressed on the surface of cells (e.g., HEK-293F cells), and is distinguished from / relative to (a) soluble FAP having the amino acid sequence of SEQ ID NO: 2; (b) blood, plasma, serum or other body fluids that do not contain cFAP; and / or (c) spFAP known to be present in blood, plasma, serum or other body fluids (whether or not spFAP has been isolated) (i.e., does not show specific binding to it).

[0087] The anti-cFAP antigen-binding molecules presented herein have been illustrated by several different working embodiments disclosed herein, including constructs containing the Fc region, which allow for the identification of common V H H CDR structure, which provides specific and selective binding to cFAP and provides differentiation from its soluble variants (such as one or more soluble variants of soluble FAP and / or spFAP having the amino acid sequence of SEQ ID NO: 2). However, it is well known in the art that some deviation from the common CDR sequence is possible while still retaining the specific and distinguishable binding function exhibited by exemplary antigen-binding molecules, for example, as demonstrated by at least standard humanization and affinity maturation protocols. That is, it is known that certain CDR / variable domain residues can be interchanged, and sequence variants that maintain the desired functional properties can be readily identified using only conventional knowledge in the art. Therefore, the present invention provides an antigen-binding molecule comprising or consisting of one or more antibodies, antibody-antigen-binding domains, or antibody-antigen-binding fragments that specifically and selectively / distinctly bind to cFAP, said antigen-binding molecule comprising one or more heavy chain variable domains (preferably one or more V... H H) or composed of thereof, wherein the one or more heavy-chain variable structural domains comprise:

[0088] (i) CDR1, which has the sequence X1X2TX3X4X5YAX6G (SEQ ID NO: 3), wherein X1 is A, G or S; wherein X2 is P, R or G; wherein X3 is K, F, R or N; wherein X4 is S, R or G; wherein X5 is T, S or N; and wherein X6 is M, I or L;

[0089] (ii) CDR2, wherein CDR2 has the sequence X1IX2WX3X4X5X6TX7YX8DSVKG (SEQ ID NO: 4), wherein X1 is A or V; wherein X2 is N, W or S; wherein X3 is S or A; wherein X4 is G or N; wherein X5 is T or G; wherein X6 is I, L, S, T or V; wherein X7 is S, Q or N; and wherein X8 is S or T; and / or

[0090] (iii) CDR 3, which has the sequence AADX1DFRTVGSRPSY (SEQ ID NO: 5), wherein X1 is R, S or K.

[0091] The aforementioned anti-cFAP antigen-binding molecules may contain one or more V H H or composed of it, the one or more V H H includes

[0092] (i) CDR1, CDR2 and CDR3, wherein CDR1 has a sequence of APTRSTYAMG (SEQ ID NO: 6) or GRTFSSYAIG (SEQ ID NO: 7), CDR2 has a sequence of VINWSGTLTSYADSVKG (SEQ ID NO: 8), and CDR3 has a sequence of AADRDFRTVGSRPSY (SEQ ID NO: 14);

[0093] (ii) CDR1, CDR2 and CDR3, wherein CDR1 has a sequence of APTRSTYAMG (SEQ ID NO: 6) or GRTFSSYAIG (SEQ ID NO: 7), CDR2 has a sequence of VINWAGTLTSYADSVKG (SEQ ID NO: 9), and CDR3 has a sequence of AADRDFRTVGSRPSY (SEQ ID NO: 14);

[0094] (iii) CDR1, CDR2 and CDR3, wherein CDR1 has a sequence of APTRSTYAMG (SEQ ID NO: 6) or GRTFSSYAIG (SEQ ID NO: 7), CDR2 has a sequence of VISWSGTLTSYADSVKG (SEQ ID NO: 10), and CDR3 has a sequence of AADRDFRTVGSRPSY (SEQ ID NO: 14);

[0095] (iv) CDR1, CDR2 and CDR3, wherein CDR1 has the sequence GRTFSSYAIG (SEQ ID NO: 7), CDR2 has the sequence AISWSGGTTQYTDSVKG (SEQ ID NO: 11), and CDR3 has the sequence AADRDFRTVGSRPSY (SEQ ID NO: 14);

[0096] (v) CDR1, CDR2, and CDR3, wherein CDR1 has the sequence GRTFSSYAIG (SEQ ID NO: 7), CDR2 has the sequence AISWSGGTTNYTDSVKG (SEQ ID NO: 12), and CDR3 has the sequence AADRDFRTVGSRPSY (SEQ ID NO: 14); or

[0097] (vi) CDR1, CDR2 and CDR3, wherein CDR1 has the sequence GRTFSSYAIG (SEQ ID NO: 7), CDR2 has the sequence AISWSGGTTNYADSVKG (SEQ ID NO: 13) and CDR3 has the sequence AADRDFRTVGSRPSY (SEQ ID NO: 14).

[0098] The above-mentioned anti-cFAP antigen-binding molecules may also contain or consist of one or more of the following amino acid sequences:

[0099] (i) QVQLVESGGGLVQAGDSLSLSCVASAPTRSTYAMGWFRQAPGKEREFAAVINWSGTLTSYADSVKGRFTISRDNAKNTVFLQMNSLKPDDTAVYYCAADRDFRTVGSRPSYWGQGTPVTVSS ("ab1", SEQ ID NO:15);

[0100] (ii) QVQLVESGGGLVEPGDSLRLSCAASGRTFSSYAIGWFRQAPGKEREFVAAISWSGGTTNYTDSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAADRDFRTVGSRPSYWSKGTRVTVSS (“ab3”, SEQ ID NO:16);

[0101] (iii) QVQLVESGGGLVQPGGSLRLSCSASAPTRSTYAMGWFRQAPGKEREFASVISWSGTLTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAADRDFRTVGSRPSYWGQGTTVTVSS ("ab1z12", SEQ IDNO: 17);

[0102] (iv) QVQLVESGGGLVQPGGSLRLSCSASGRTFSSYAIGWFRQAPGKEREFVSAISWSGGTTQYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAADRDFRTVGSRPSYWGQGTTVTVSS ("ab3z7", SEQ ID NO:18);

[0103] (v) QVQLVESGGGLVQPGGSLRLSCSASGRTFSSYAIGWFRQAPGKEREFVSAISWSGGTTNYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAADRDFRTVGSRPSYWGQGTTVTVSS ("ab3z10", SEQ ID NO: 19).

[0104] As disclosed above, CDR, variable structural domain and V H The H sequence may include the specific reference sequence described above, or may differ from it in one or more amino acid substitutions. It should be understood that the amino acid substitutions disclosed above may not be present in the CDR, variable domain, or V of the antigen-binding molecule of the present invention. H In an H sequence, it can also exist in one, more than one, or all CDRs, variable structural domains, or V sequences. H In the H sequence. As used herein, the term "substitution" refers to the replacement of one amino acid by another. The term "substitution" explicitly does not cover the deletion of an amino acid at a specific position or the introduction of one (or more) amino acids at different positions. As described above, the present invention specifically covers the CDR, variable domain, or V containing one or more conserved amino acid substitutions. H H sequence.

[0105] The above indicates the inclusion of one or more substituted CDRs, variable structural domains, and V. H The H sequence is referred to herein as a “variant” sequence. Clearly, a variant sequence is a functional variant, i.e., having an amino acid sequence that may differ from the reference amino acid sequence, but which exhibits or maintains the same functional activity as the reference sequence in the context of the described antigen-binding molecule, i.e., conferring or contributing to the specific and selective target antigen-binding activity of the antigen-binding molecule of the present invention. In this context, “exhibiting or maintaining the same functional activity” does not mean that any quantitative parameters of antigen binding (e.g., dissociation constant or equilibrium dissociation constant) are the same within the experimental error range. Rather, a variant sequence that “exhibits or maintains the same functional activity” means that, when tested in the same context and under the same conditions as the reference sequence, the variant sequence also contributes to or confers the specific and selective anti-cFAP binding of the antigen-binding molecule as defined herein. Therefore, if an antigen-binding molecule containing one or more variant sequences or composed thereof maintains the specific and selective anti-cFAP binding as defined herein, then the antigen-binding molecule and / or any component variant sequence of the antigen-binding molecule may exhibit improved, equivalent, or reduced quantitative binding parameters, respectively, compared to the non-variant molecule and / or one or more component sequences.

[0106] It is known in the art that antibody heavy or light chain variable domains (including antibody single-chain antigen-binding domains, such as V) H In addition to the three CDRs defined above, H) also includes four frame domains (“FW”). Therefore, as is well known in the art, the antigen-binding molecule of the present invention comprises one or more antibodies, antibody-antigen binding domains, or antibody-antigen binding fragments (including, for example, one or more CDRs, variable domains, or V) H The H sequence, in addition to containing the CDRs defined above, also contains the necessary framework domains. Those skilled in the art can select the necessary / surrounding FW sequences using standard methods conventionally practiced in the art. It should be understood that those skilled in the art will select appropriate sequences for the FW such that the resulting component of the antigen-binding molecule is one or more antibodies, antibody-antigen-binding domains, or antibody-antigen-binding fragments (containing at least one CDR, variable domain, or V as defined herein). H The H sequence confers or contributes to the specific binding and selective anti-cFAP binding activity of the molecule.

[0107] In certain embodiments, the anti-cFAP antigen-binding molecule of the present invention comprises one or more amino acid sequences having at least 85%, at least 90%, or at least 95% sequence identity with any of SEQ ID NO: 15, 16, 17, 18, and 19. As defined herein, an amino acid sequence having such sequence identity with any of SEQ ID NO: 15, 16, 17, 18, and 19 is a variant amino acid sequence, i.e., a functional variant as defined above. The total number of substitutions in the variant sequences of SEQ ID NO: 15, 16, 17, 18, and 19 may be up to 18, up to 17, up to 16, up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or the variant sequence may contain only one substitution relative to the reference sequence. One or more substitutions may be in one or more CDRs, one or more FWs, or at least one CDR and at least one FR.

[0108] As used herein, the term "sequence identity %" in relation to polypeptide / peptide and / or nucleic acid sequences or amino acid sequences of nucleic acid molecules describes the number of matches of the same amino acid or nucleic acid residues between two or more aligned sequences compared to the total length (or the total number of residues in the compared portion) of the sequence being compared. The percentage of identical residues can be determined using the alignment of two or more sequences or subsequences when the (sub)sequences are compared and aligned with maximum correspondence within a comparison window or a designated region measured using sequence comparison algorithms known in the art, or when manually aligned and visually inspected. Non-limiting examples of algorithms used to determine sequence identity include those based on, for example, the NCBI BLAST algorithm (Altschul et al., Nucleic Acids Research, 25(1997), 3389-3402), the CLUSTALW computer program (Thompson, Nucleic Acids Research, 2(1994), 4673-4680), or FASTA (Pearson and Lipman, Proceedings of the National Academy of Sciences, 85(1988), 2444). While the FASTA algorithm typically does not account for internal mismatches, deletions, or additions in the sequence—i.e., gaps—in its calculations, this can be manually corrected to avoid overestimating sequence identity percentages. However, CLUSTALW does account for sequence gaps in its identity calculations. BLAST and BLAST 2.0 algorithms are also provided (Altschul et al., Nucleic Acids Research, 25(1977), 3389).

[0109] The present invention also provides a second anti-cFAP antigen-binding molecule that binds to the same epitopes of cFAP as follows: (a) an amino acid sequence comprising or consisting of SEQ ID NO: 15, 16, 17, 18 or 19. H H; or (b) a first antigen-binding molecule comprising V having the amino acid sequence of SEQ ID NO: 15, 16, 17, 18 or 19. H H or composed of it. V, as defined above, can be identified by any suitable epitope mapping method known in the art. H The H struct may contain the V defined above. HThe antigen-binding molecule with the H domain binds to a specific epitope of cFAP or a portion thereof. Examples of such methods include, for example, mass spectrometry analysis of the antigen-binding molecules of the present invention binding to cFAP peptides of different lengths. In another example, NMR spectroscopy or X-ray crystallography can be used to identify the epitope bound by the antigen-binding molecule of the present invention. Once identified, the epitope fragment bound to the antigen-binding molecule of the present invention can be used as an immunogen to obtain additional antibodies that bind to the same epitope.

[0110] 5.2 Generation and Engineering of Antigen-Binding Molecules

[0111] As defined herein, the antigen-binding molecule of the present invention may comprise one or more antibodies, antibody-antigen binding domains, or antibody-antigen binding fragments (including, for example, one or more CDRs, variable domains, or V). H (H sequence), and may further include an antibody Fc region or an Fc-receptor binding domain. Unless otherwise stated, the terms "antibody," "antibodies," and similar terms refer to whole immunoglobulin molecules and encompass antibodies in naturally occurring forms (including, but not limited to, IgG, IgA, IgM, IgE) and recombinant antibody constructs, including, but not limited to, single-chain antibodies, chimeric antibodies, humanized antibodies, antibody fusion proteins, and multispecific antibodies; as well as all of the foregoing antigen-binding fragments and derivatives. As known in the art, antibodies comprise variable regions (referred to in the art as "Fv regions" and / or "Fv domains") formed by paired variable domains from the heavy and light chains, which interact with the antigen. The term Fv region does not include constant regions of the heavy and / or light chains.

[0112] As used herein, the terms “antibody,” “antibody,” and similar terms also refer to their antigen-binding fragments, which may be referred to herein as antibody-antigen-binding domains, antibody-antigen-binding fragments, and / or simply as antigen-binding fragments. These terms refer to one or more domains or fragments of an antibody that retain the ability to specifically bind to target antigens (e.g., cFAP) as known in the art, including, but not limited to, antigen-binding fragments containing an Fv domain, i.e., paired heavy and light chain variable domains such as Fab, Fab', F(ab')2, and Fv fragments, and recombinant constructs such as single-chain Fv domains, referred to in the art as scFv. The terms also include antibody-antigen-binding fragments containing a single unpaired heavy or light chain variable domain as known in the art, retaining the ability to specifically and selectively bind to antigens as defined herein, including, but not limited to, single-domain antibodies (also referred to in the art as sdAb, dAb, and / or nanobodies) and V-type heavy chains based on camel family antibodies. HH domain. Preferably, the antigen-binding molecule of the present invention comprises one or more antibodies, said antibody being V H H.

[0113] Antibody and antigen binding domains or fragments (including, for example, one or more CDRs, variable domains, or V) H The H sequence can be polyclonal or monoclonal, preferably monoclonal. As used herein, the terms “monoclonal,” “monoclonal composition,” and similar terms referring to antigen-binding molecules or component antibodies, antibody-antigen-binding domains, or antibody-antigen-binding fragments refer to a population of antibody peptides or fragments thereof derived from a single B cell clone, containing only one type of antigen-binding site capable of generating an immune response to a specific epitope of an antigen. This contrasts with “polyclonal” antibodies and compositions, which refer to a population of antibody peptides or antigen-binding fragments containing multiple types of antigen-binding sites. Modified forms (such as humanized or chimeric forms) and recombinant constructs (such as fusion proteins) of any antigen-binding molecule or component antibody, antibody-antigen-binding domain, or antibody-antigen-binding fragment are also included, wherein the antigen-binding molecule further comprises, for example, an Fc region or an Fc receptor-binding domain. Non-limiting examples of antigen-binding molecules of the present invention include antibody-antigen-binding domains or fragments recombinantly fused with an antibody Fc region or Fc receptor-binding domain (including those containing one or more CDRS, variable domains, or V... H (H sequence or composed thereof). Antigen-binding molecules may also contain additional structural domains, for example, for the isolation and / or preparation of recombinant-derived molecules as known in the art.

[0114] The antigen-binding molecules and / or components thereof of the present invention (including but not limited to antibodies, antibody-antigen binding domains or fragments, CDRs, variable domains, single-domain antigen conjugates (e.g., V)) HThe Fc region and Fc receptor-binding domain (H), Fc region, and Fc receptor-binding domain can be prepared using a variety of techniques conventionally used in the art. For example, a non-human animal is immunized with one or more antigens of interest (i.e., cFAP, which may be in one or more forms and / or derived from one or more species), followed by isolation of antigen-reactive B cells that produce antibodies. More preferably, positive clones are further subjected to negative selection to exclude clones that specifically or significantly react with soluble versions of FAP (including, but not limited to, FAP having the amino acid sequence of SEQ ID NO: 2 and / or spFAP). Alternatively or additionally, negative selection may exclude clones that significantly react with bodily fluids such as blood, serum, or plasma known or believed to contain spFAP (regardless of whether spFAP is detected in the specific bodily fluid used). Methods for isolating and / or selecting clones (positive or negative) that produce antibodies with the desired properties are well known in the art. As is known in the art, peptide immunogens of target antigens can be conjugated with adjuvant-carriers, such as keyhole hemocyanin (KLH), and / or administered with adjuvant compositions, such as Freund's complete or incomplete adjuvants, to enhance immunogenicity. Animals can be immunized according to a standard schedule, such as weekly, monthly, or a combination of weekly and monthly, depending on the animal, the antigen, and the resulting antibody titer. To determine the animal's response, antibody titers in serum can be tested according to standard procedures. Peripheral blood mononuclear cell (PBMC) fractions from positive animals can be isolated, and antigen-reactive B cells can be purified from serum using standard techniques, such as ELISA or column-based techniques, as described, for example, in Seeber et al., PLoS One, 9 (2014), e86184. As described above, such screening methods preferably include a negative selection step to identify and exclude clones exhibiting cross-reactivity with soluble versions of FAP, spFAP, and / or humors known or believed to contain spFAP. The selected clones can then be used for subsequent recombinant processing.

[0115] Another suitable method for generating or isolating the antibodies and antibody-antigen binding fragments of the present invention includes, but is not limited to, selecting recombinant antibodies or antibody-antigen binding domains or fragments (including single-domain conjugates, such as V) from peptide or protein libraries (e.g., but not limited to, bacteriophage, bacterial, mammalian cell, ribosome, oligonucleotide, RNA, cDNA, or yeast display libraries) using the binding activity of interest. HThe method of H). For example, as illustrated in the working examples, antigen-binding molecules or components thereof can be selected from such libraries by positive selection for specific binding to cFAP (e.g., as endogenously or recombinantly expressed on the surface of cells) and negative selection for binding to soluble FAP, spFAP, and / or bodily fluids such as blood, plasma, or serum. Demonstration libraries are well known in the art and are available, for example, from various commercial suppliers, including but not limited to Cambridge Antibody Technologies (Cambridgeshire, UK), MorphoSys (Martinsried / Planegg, Del., Germany), Biovation (Aberdeen, Scotland, UK), and Bioinvent (Lund, Sweden). Similarly, the selected clones can be processed according to conventional methods for subsequent recombinant processing into the antigen-binding molecules of the present invention.

[0116] Therefore, the present invention also provides encoding antigen-binding molecules and antigen-binding components thereof disclosed herein, preferably encoding specific and selective anti-cFAP V. H The nucleic acid molecule of H. As used herein, the terms “nucleic acid molecule,” “nucleic acid sequence,” “polynucleotide,” and similar terms include genomic DNA and cDNA, as well as RNA capable of driving the expression of the peptide or polypeptide antigen-binding molecules of the present invention. It should be understood that the term “RNA” as used herein includes all forms of RNA, including mRNA, tRNA, and rRNA, but also includes genomic RNA, as in the case of RNA viruses. Preferably, the detailed examples of “RNA” refer to mRNA. The nucleic acid molecule / nucleic acid sequence of the present invention can be natural, synthetic, or semi-synthetic. Thus, the nucleic acid molecule can be, for example, a nucleic acid molecule synthesized according to conventional organic chemistry schemes, a nucleic acid molecule synthesized according to recombination methods, or a nucleic acid molecule produced, for example, by combinatorial chemistry synthesis and semi-synthetic recombination methods. Those skilled in the art are familiar with the preparation and use of such nucleic acid molecules.

[0117] In particular, the present invention provides a polynucleotide encoding a polypeptide comprising any of SEQ ID NO: 3 to 19, preferably any of SEQ ID NO: 15, 16, 17, 18 or 19, or a variant thereof as defined above or composed thereof.

[0118] Vectors comprising nucleic acid molecules encoding the antigen-binding molecule of the present invention or one or more components thereof are also provided. As used herein, the term “vector” refers to a circular or linear nucleic acid molecule that can autonomously replicate in the host cell into which it is introduced. Non-limiting examples of vectors suitable for use in the present invention include entrapments, plasmids (e.g., naked or contained in liposomes), viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses), and bacteriophages. However, many suitable vectors are provided in the art, the choice of which depends on the desired function. The development and use of suitable vectors are well documented in the art; see, for example, Sambrook and Russell, “Molecular Cloning, A Laboratory Manual,” Cold Spring Harbor Laboratory, New York (2001), and Ausubel, “Current Protocols in Molecular Biology,” Green Publishing Associates and Wiley Interscience, New York (1989), (1994). Vectors used in conjunction with the present invention comprise nucleic acid sequences encoding anti-cFAP antigen-binding molecules as disclosed herein and / or one or more component domains / regions thereof. Thus, vectors used in conjunction with the present invention may encode any of SEQ ID NO: 3-19, preferably any of SEQ ID NO: 15-19, or peptides or polypeptides thereof.

[0119] With regard to the term "comprising a vector" as used herein, it should be understood in the art that the vector contains necessary and / or sufficient additional nucleic acid sequences for the desired vector activity in the host cell, such as those driving the replication of the vector (and the encoding nucleic acid sequence) and / or directing the host cell to express the antigen-binding molecule of the present invention or its component domains. Such additional nucleic acid sequences include, but are not limited to, sequences that control vector replication and / or expression of the desired sequence in a particular cellular system. For example, the vector may comprise a nucleic acid molecule encoding a desired peptide or polypeptide sequence, said peptide or polypeptide sequence being operatively linked to and / or under the control of a regulatory sequence. The term "regulatory sequence" refers to a DNA sequence necessary to achieve the expression of the encoding sequence operatively linked thereto. The term "control sequence" is intended to include at least all components that may be necessary for expression and may further include additional advantageous components, for example, to allow replication. As understood in the art, the nature of such regulatory and control sequences varies from host organism to host organism. For example, in prokaryotes, control sequences typically include promoters, ribosome binding sites, and terminators. In eukaryotes, control sequences typically include promoters, terminators, and in some cases, enhancers, transactivators, and / or transcription factors.

[0120] The vector used in this invention is preferably an expression vector. Expression vectors can direct the replication and expression of the nucleic acid molecules of this invention in host cells, thus providing expression of anti-cFAP antigen-binding molecules or their component domains as disclosed herein. Suitable expression vectors have been extensively described in the literature, and those skilled in the art can readily determine appropriate expression vectors for a particular cell system using conventional methods. Preferably, the vectors disclosed herein comprise a recombinant polynucleotide (i.e., a nucleic acid sequence encoding an anti-cFAP antigen-binding molecule or its component domains) and an operatively linked expression control sequence. Vectors provided herein may further comprise a promoter. Vectors described herein may also comprise selection marker genes and a replication origin ensuring replication in the host. Furthermore, vectors provided herein may also comprise a termination signal for transcription. Expression vectors known in the art can drive transient or constitutive expression in host cells.

[0121] The nucleic acid molecules and / or vectors of the present invention can be designed for transfection into prokaryotic or eukaryotic host cells by any means known in the art or described herein. Non-limiting examples of suitable methods include chemical-based methods (polyethyleneimine, calcium phosphate, liposomes, DEAE-glucan, nuclear transfection), non-chemical methods (electroporation, acoustic perforation, optical transfection, gene electrotransfer, hydrodynamic delivery, or transformation that occurs naturally after cells come into contact with the nucleic acid molecules of the present invention), particle-based methods (gene gun, magnetic transfection, puncture), phage vector-based methods, and viral methods. For example, expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpesvirus, Semliki Forest virus, or bovine papillomavirus can be used to transfect nucleic acid molecules into target cell populations. Additionally, baculovirus systems can also be used as vectors in eukaryotic expression systems for the nucleic acid molecules of the present invention.

[0122] The term "prokaryote" means all bacteria that can be transformed, transduced, or transfected with DNA or DNA or RNA molecules to express the proteins of the present invention. Prokaryotic hosts may include Gram-negative and Gram-positive bacteria, such as *Escherichia coli*, *Salmonella typhimurium*, *Serratia marcescens*, *Corynebacterium* (glutamate), *Pseudomonas* (fluorescent), *Lactobacillus*, *Streptomyces*, *Salmonella*, and *Bacillus subtilis*. The term "eukaryotic cell" means cells including yeast, higher plants, insects, and mammals. Non-limiting examples of mammalian host cells commonly used in the art include HeLa, HEK293 (including HEK-293F), H9, Per.C6, and Jurkat cells, mouse NIH3T3, NS / O, SP2 / O, and C127 cells, COS cells, such as COS 1 or COS 7, CV1, quail QC1-3 cells, mouse L cells, mouse sarcoma cells, Bowman's melanoma cells, and Chinese hamster ovary (CHO) cells.

[0123] When a recombinant expression vector encoding an antigen-binding molecule or a component domain thereof is introduced into a host cell, the molecule or component domain is generated by culturing the host cell for a period sufficient to allow expression of the molecule and / or domain in the host cell, or preferably sufficient to allow secretion of the molecule and / or domain into the culture medium in which the host cell is grown. The molecule of interest can be recovered from the culture medium using standard protein purification methods. The present invention further provides antigen-binding molecules obtainable by any of the methods disclosed herein.

[0124] It should be understood that variations of the above procedures are within the scope of this invention. For example, recombinant DNA technology can be used to remove or modify domains encoding antigen-binding molecules or their components disclosed herein (e.g., V as defined above). HDNA sequences containing H domains. For example, recombinant DNA technology can be used to remove portions of the coding sequence that are unnecessary for maintaining specific and selective binding to the antigen of interest. Molecules expressed from such truncated DNA molecules are also covered in this invention. Additionally, bifunctional antigen-binding molecules are provided, for example, comprising one or more antibodies, antibody-antigen-binding domains, or antibody-antigen-binding fragments (including, for example, one or more CDRs, variable domains, or V domains as defined herein) that provide specificity for cFAP. H H sequence) and antibodies or antibody-antigen binding domains or fragments specific to antigens other than cFAP.

[0125] As defined herein, an antigen-binding molecule or its component domains may comprise or consist of an antibody or a derivative thereof, such derivatives being produced, for example, by adding exogenous sequences to modify immunogenicity or reduce, enhance, or modify binding, affinity, association rate, dissociation rate, specificity, half-life, or any other suitable property. Typically, with respect to the antigen-binding domain, some or all of the non-human or human CDR sequence is maintained, while the non-human sequence in the FW and / or constant region is replaced by human or other amino acids.

[0126] Humanized versions of the antigen-binding molecules disclosed herein are also provided, namely those containing CDR and V as disclosed above. H H sequence. As is well known in the art, “humanization” (to produce a humanized version of the parent antibody) refers to the recombinant engineered antibody antigen-binding domain using a CDR derived from a non-human donor immunoglobulin in the context of a humanized FW and optionally a constant domain. During engineering, the FW and / or CDR residues can be modified to maintain binding affinity and activity, such as specific and selective binding to cFAP. Methods for humanizing antibodies and antibody domains are well known in the art, for example, as disclosed in Queen et al., Proceedings of the National Academy of Sciences 86 (1989), 10029-10032; Hodgson et al., Bio / Technology 9 (1991) 421.

[0127] 5.3 Characterization of Binding Activity

[0128] The antigen-binding molecules of the present invention exhibit specific binding to cFAP and are also distinguishable from soluble variants of FAP, such as soluble FAP having the amino acid sequence of SEQ ID NO: 2 and one or more soluble FAP variants (spFAP) present in blood, serum, or plasma. Therefore, the antigen-binding molecules of the present invention also do not exhibit non-specific binding to bodily fluids known or believed to contain spFAP (such as blood, serum, and plasma in non-limiting examples). The antigen-binding molecules of the present invention comprise one or more antibody-antigen binding domains or fragments as defined herein. Therefore, as used herein, in the context of an antigen-binding molecule reacting with cFAP, the phrase "specifically binds" indicates that cFAP binds to the antigen-binding molecule via an antigen-antibody reaction. In a particular embodiment, the reaction is V H H-antigen reaction. As defined above, the term “distinguishing with / relative to” indicates that the antigen-binding molecule binds to the target antigen (i.e., cFAP) but does not specifically bind to soluble variants of FAP, and / or does not exhibit significant binding or other reactions with body fluids (such as blood, serum, plasma or other tissue fluids) known or believed to contain one or more soluble FAP variants (spFAP).

[0129] The specific and selective binding of cFAP to one or more soluble FAP variants (including spFAP and / or body fluids known or believed to contain spFAP) can be assessed by any method known in the art or as described herein, particularly by comparing binding activity to cFAP with binding activity to one or more soluble variants of FAP. Because cFAP is a cell-bound FAP, it is preferred that the binding activity of the antigen-binding molecule of the present invention to cFAP be assessed by determining binding to cFAP, such as that expressed on the surface of cells. In this regard, it will be understood that the assay protocol for determining binding to cFAP differs from that for determining binding to soluble variants of FAP, for example, at least because ligand-soluble FAP can be used in solution or in assay formats unsuitable for cell or cell membrane binding of cFAP. However, those skilled in the art can readily modify assay parameters using standard methods in the art and / or as described herein to allow comparisons of binding activity to cFAP and one or more soluble FAP variants. As used herein, the term “distinguishing with / relative to” indicates that the binding activity to cFAP is at least 20-fold higher than that to one or more soluble variants (e.g., spFAP), which is at least an order of magnitude higher than that to one or more soluble variants of spFAP.

[0130] Preferably, the binding activity that determines the specific and selective binding to cFAP is the EC binding to cFAP.50 In particular, relative to one or more soluble variants of FAP. Further preferably, EC is used to determine the binding of one or more soluble variants of cFAP and / or FAP. 50 The determination of the value is calibrated by using a control FAP binding molecule, i.e., calibration to provide the determined results for the test control molecule (preferably siroizumab and / or 4B9).

[0131] Regarding the identification of EC binding to the soluble variant spFAP of FAP 50 It allows calibration / selection of assay conditions to enable ECMO of siroizumab or 4B9 with spFAP. 50 The amounts were less than 1 nM or less than 10 nM, respectively. As explained in this paper, spFAP is also known to be present in body fluids such as blood, plasma, and serum. Therefore, identifying EC that bind to spFAP is crucial. 50 The value does not necessarily need to be performed on spFAP or isolated soluble FAP variants, but can be evaluated on body fluids known to contain spFAP. When using body fluids, undiluted human plasma containing at least 80 ng / mL of spFAP is preferred. Therefore, in some embodiments, the EC value bound to soluble variants of FAP is determined. 50 EC is determined to bind to undiluted human plasma containing at least 80 ng / mL of spFAP. 50 The calibration / selection of assay conditions allows siroizumab or 4B9 to bind to undiluted human plasma EC. 50 Less than 1 nM or less than 10 nM respectively.

[0132] EC related to cFAP 50 The determination of EC is exemplified by flow cytometry. Preferably, the flow cytometry assay conditions are calibrated / set such that EC associated with cFAP... 50 The value is less than 5 nM. Further preferably, under calibrated measurement conditions, (a) EC bound to the cFAP as evaluated under the same conditions. 50 Is it siroizumab or 4B9 EC? 50The maximum binding of the cFAP is within 1 time the maximum binding of siroizumab or 4B9 as evaluated under the same conditions; and / or (b) the maximum binding of the cFAP is within 1 time the maximum binding of siroizumab or 4B9 as evaluated under the same conditions. The cFAP can be expressed by any suitable cell known in the art or described herein (e.g., as suitable for flow cytometry assay). In a particular embodiment, the cFAP has the amino acids of SEQ ID NO: 1, and the cells used to express the cFAP are HEK-293F cells. The specific and selective binding of the antigen-binding molecules of the present invention to cFAP can also be identified by antigen-binding molecules exhibiting one or both of the following: (a) an apparent Kd of less than 2 nM binding to cFAP or (b) an EC of less than 2 nM binding to cFAP. 50 Less than 2 nM, wherein the measurement conditions are calibrated such that (c) EC binds to the cFAP. 50 The EC of sirolimus or 4B9 was evaluated under the same conditions. 50 (d) The maximum binding to the cFAP is within 1 time the maximum binding to siroizumab or 4B9 as evaluated under the same conditions.

[0133] When the assay for binding with cFAP and one or more variants of soluble FAP (e.g., spFAP and / or body fluids known or believed to contain spFAP) is calibrated / set as described above, the EC binding with cFAP... 50 Values ​​less than 5 nM and EC values ​​of one or more soluble variants of FAP 50 The combination is EC with cFAP. 50 In cases where the value is at most 1 / 20, specificity and selectivity of binding are determined. Alternatively or additionally, assays calibrated / set as described above can also be used to determine and compare the TOP (MFI) of the antigen-binding molecule with cFAP relative to the TOP (MFI) of one or more soluble variants of FAP (such as those recombinantly expressed on the surface of HEK 293F cells) and (ii) the EC50 of the molecule with one or more soluble FAPs (e.g., spFAP). 50 To identify specific and selective binding. In a non-limiting example, the anti-cFAP antigen-binding molecule of the present invention is specific and selective for cFAP (relative to sFAP), wherein...

[0134] (i) EC in conjunction with cFAP 50 Or TOP (MFI) is the EC5 of siroizumab when evaluated under the same conditions. 50 Or at least 60% of TOP (MFI); and

[0135] (ii) ECs of the molecule bound to one or more soluble variants of FAP50

[0136] (a) Weaker than 40 nM,

[0137] (b) is undetectable, or

[0138] (c) Incalculable

[0139] When the EC is calibrated / conditions are set such that siroizumab or 4B9 binds to one or more soluble variants of the FAP. 50 When evaluating measurements below 1 nM or below 10 nM, respectively.

[0140] In certain embodiments, the anti-cFAP antigen-binding molecule of the present invention is specific and selective for cFAP (as opposed to sFAP), wherein

[0141] (i) ECs that bind to cFAP having the amino acid sequence of SEQ ID NO 1 and expressed on the surface of HEK-293F cells 50 Or TOP (MFI) is the ECG binding of siroizumab to the cFAP when evaluated under the same conditions. 50 Or at least 60% of TOP (MFI); and

[0142] (ii) ECs of the molecule bound to undiluted human plasma at a concentration of at least 80 ng / mL. 50

[0143] (a) Weaker than 40 nM,

[0144] (b) is undetectable, or

[0145] (c) Incalculable

[0146] When the EC is calibrated / its conditions are set to allow siroizumab or 4B9 to bind with the undiluted human plasma, 50 When evaluating measurements below 1 nM or below 10 nM, respectively.

[0147] In a non-restrictive instance, used to determine EC 50 Cells expressing cFAP (e.g., SEQ ID NO: 1) and / or MFI, with an average of approximately 30,000 copies of the cFAP molecule expressed per cell. Cells can be exposed to a series of dilutions of the antigen-binding molecule to be tested, and the bound molecule can be detected according to standard protocols known in the art or as described herein. S-shaped dose-response curves (e.g., a four-parameter logarithmic model) can be used to fit the data, and EC 50The concentration determined to be 50% of the maximum response achieved by the fitted curve is well known in the art. In a particular embodiment, the dilution series comprised 100,000 cFAP-expressing cells incubated with antigen-binding molecule concentrations of at least 25 nM, 5 nM, 1 nM, 0.2 nM, 0.04 nM, 0.008 nM, 0.0016 nM, and 0.00032 nM. The culture medium containing the antigen-binding molecule to be tested can be any suitable medium, such as, but not limited to, PBS supplemented with 1% BSA or undiluted human plasma, wherein the antigen-binding molecule is incubated in the medium at room temperature for 1 hour. To aid in detection or isolation, the antigen-binding molecule may optionally contain a known purification / isolation / detection tag or domain, such as, but not limited to, an antibody Fc domain, for example, mouse IgG2a (SEQ ID NO: 20). The antigen-binding molecule can be incubated with the cells at 4°C for 30 minutes and then detected by any suitable method following a standard washing protocol. For example, when the antigen-binding molecule contains an antibody Fc domain, a suitable secondary labeling agent is an anti-Fc antibody. When the antigen-binding molecule contains mouse IgG2a, a suitable secondary antibody could be anti-mIgG2a-PE. Quantification of binding can also be performed according to standard protocols, such as measuring mean fluorescence intensity (MFI) by flow cytometry. Appropriate controls may include unstained or isotype controls, for example, to allow correction for nonspecific signals or autofluorescence, and / or a fluorescence minus one (FMO) control for each fluorescent dye. MFI values ​​can be normalized by subtracting the isotype MFI from the treated sample.

[0148] In another non-limiting example, the specificity and selective binding activity of the anti-cFAP antigen-binding molecule of the present invention can be determined in competitive binding experiments, such as detecting and comparing the binding activity of cFAP in the presence or absence of one or more soluble variants of FAP (e.g., spFAP and / or body fluids known or believed to contain spFAP (e.g., but not limited to plasma or serum, preferably human plasma or human serum)). In a non-limiting example, the anti-cFAP antigen-binding molecule of the present invention is specific and selective for cFAP (relative to spFAP), wherein, in the absence of one or more soluble variants of FAP (e.g., in the absence of spFAP or in the absence of human plasma containing FAP), the MFI in a flow cytometry assay containing cFAP (e.g., having the amino acid sequence of SEQ ID NO: 1 and recombinantly expressed on the surface of HEK 293F cells) is reduced by less than 40%, less than 30%, less than 20%, less than 15%, or preferably less than 12% at a concentration of less than 1 nM compared to the MFI determined in the same assay under the same conditions but in the presence of one or more soluble variants of FAP (e.g., in the presence of spFAP, or preferably in the presence of human plasma containing FAP). Preferably, the assay is calibrated / its conditions are set such that the MFI of siroizumab or 4B9 determined in the same assay and under the same conditions in the presence of one or more soluble variants of FAP (e.g., in the presence of spFAP or in the presence of human plasma containing FAP) is reduced by at least 50%, at least 60%, at least 70%, at least 80%, or preferably at least 90% at a concentration below 1 nM compared to the MFI determined under the same conditions in the absence of one or more soluble variants of FAP (e.g., in the absence of spFAP, or preferably in the absence of human plasma containing FAP). The antigen-binding molecule of the present invention can also be specific and selective for cFAP (compared to one or more soluble variants of FAP, such as human plasma), wherein the binding ratio of cFAP to the absence of said undiluted human plasma in the presence of known or believed to contain spFAP (e.g., at a concentration of at least 80 ng / mL) is at least 80% at a concentration of 0.8 nM, at least 60% at a concentration of 0.16 nM, or at least 50% at a concentration of 0.0032 nM.Preferably, the comparative assays in this context, under the same conditions, show a binding rate of 4B9 or siroizumab to cFAP of less than 10% or less than 25% in the presence of the undiluted human plasma relative to the binding rate in the absence of the undiluted human plasma, wherein the concentration of 4B9 or siroizumab is less than or equal to 0.8 nM and greater than or equal to 0.0032 nM, for example, 0.8 nM, 0.016 nM, or 0.0032 nM.

[0149] Similar to the competitive binding assays outlined above, the specific and selective binding activity of the anti-cFAP antigen-binding molecules of the present invention can be determined by identifying and comparing any other activity of the antigen-binding molecules in the presence or absence of one or more soluble variants of FAP (e.g., spFAP and / or body fluids known or believed to contain spFAP, such as, but not limited to, plasma or serum, preferably human plasma or human serum)). Specific and selective activity can be determined without being affected by the presence of one or more soluble variants of FAP (within experimental error), and particularly without being affected by increasing the concentration of one or more variants of soluble FAP (e.g., concentrations of up to 100 ng / ml, 200 ng / ml, 500 ng / ml, or 1000 ng / ml). As a control, under the same conditions and in the same assay, a decrease in the activity of the known FAP-binding molecules siroizumab or 4B9 will be observed with increasing concentrations of soluble FAP. The cytotoxic activity of the antigen-binding molecule against cells expressing cFAP can be assessed in the presence and absence of one or more soluble variants of FAP, according to any method known in the art or described herein. In a non-limiting exemplary assay, cytotoxic activity can be assessed using the bispecific antigen-binding molecule of the present invention, for example, crosslinking cFAP with an antigen on an immune cell that mediates cytotoxicity (such as CD3 on T cells), and as known in the art, the assay further includes T cells. Resistance to soluble variants of FAP, as described herein, can be assessed by incubating the antigen-binding molecule of the present invention in the presence and absence of one or more soluble variants of FAP (e.g., spFAP) and / or in sequential dilutions (and / or equivalent increases in concentration of soluble FAP) prior to the assay and comparing changes in cytotoxic activity.

[0150] As described above, the present invention addresses many shortcomings of existing anti-FAP antigen-binding molecules in the art, particularly through their specific and selective binding to cFAP. Known anti-FAP binding molecules, such as siroizumab and 4B9, bind to soluble FAP and therefore cannot significantly distinguish cFAP from any soluble variants that may also be present in the target sample or tissue. Thus, soluble variants of FAP (often present in high concentrations in bodily fluids such as blood, serum, or plasma) can block therapeutic agents targeting cFAP or act as sinks for such agents, thereby preventing cFAP-expressing cells from recognizing and / or requiring prohibited doses of anti-cFAP agents to achieve their effects. In the present invention, it has been found that even in the presence of high concentrations of soluble variants of FAP, it is possible to generate antigen-binding molecules that specifically and selectively bind to cFAP. Because the antigen-binding molecules of the present invention do not bind significantly to soluble FAP, their activity is not significantly affected by the presence of soluble FAP (e.g., at levels of spFAP found in serum or plasma in vivo). In the context of cytotoxic antigen-binding molecules as described herein, the present invention therefore provides antigen-binding molecules having cytotoxic activity that is unaffected by or not resistant to the presence of soluble variants of FAP (e.g., spFAP), for example at levels greater than or equal to 40 ng / mL, 100 ng / mL, or physiologically relevant concentrations.

[0151] As provided herein, the present invention provides an anti-cFAP antigen-binding molecule that is particularly suitable for treating diseases characterized by the presence of cFAP. In some embodiments, the anti-cFAP antigen-binding molecule is suitable for treating subjects with a concentration of soluble FAP (which may comprise one or more soluble FAP variants) of at least 40 ng / mL.

[0152] Given the selectivity of the anti-cFAP antigen-binding molecules disclosed herein, the molecules and compositions of the present invention are considered to be unaffected by or resistant to one or more soluble FAP variants (e.g., spFAP). As used herein, the terms "resistant to soluble FAP," "resistant to one or more soluble FAP variants" (and related terms) mean that the activity (e.g., binding activity or cytotoxic activity) of the anti-cFAP antigen-binding molecules is unaffected by various physiological concentrations of spFAP.

[0153] 5.4 Diagnostic and Pharmaceutical Compositions

[0154] As described above, anti-cFAP antigen-binding molecules distinguish soluble forms / variants of FAP. Therefore, antigen-binding molecules are particularly well-suited for the specific targeting and detection of cFAP, unaffected by soluble variants of FAP known to exist in bodily fluids such as blood, serum, and plasma.

[0155] Therefore, the present invention further relates to compositions, such as diagnostic or pharmaceutical compositions, comprising at least one of the following: (i) an antigen-binding molecule of the present invention, (ii) a nucleic acid molecule of the present invention, (iii) a carrier of the present invention, (iv) a host cell of the present invention, and / or (v) an antigen-binding molecule produced or obtained by the method of the present invention.

[0156] 5.4.1 Diagnostic Compositions

[0157] As described above, the anti-cFAP antigen-binding molecule provided herein can be used for the detection of cFAP, particularly relative to and distinguishing it from soluble variants of FAP that may be present in a sample. In the most preferred embodiment, the anti-cFAP antigen-binding molecule of the present invention can be used to detect cFAP in the presence of one or more soluble variants of FAP that are known, suspected, or believed to contain it. Those skilled in the art will readily understand how to use the antigen-binding molecule of the present invention to determine whether a sample contains cFAP. Non-limiting examples of suitable methods include in vivo assays in which the antigen-binding molecule is conjugated to a suitable detectable reagent or portion such as a radionuclide or contrast agent (e.g., for MRI or CT); and in vitro assays such as immunohistochemical and immunocytochemical methods, Western blotting, ELISA, and immunoassays based on absorbance, luminescence, fluorescence, chemiluminescence, or electrochemiluminescence detection. Diagnostic methods may include the use of suitable controls to ensure that any positive or negative results are reliable. Those skilled in the art can design suitable positive and negative controls and include them in experimental apparatus using conventional methods and the teachings of this disclosure.

[0158] Biological samples for detecting cFAP according to the methods disclosed herein include subject-derived samples or preparations. Subject-derived samples can be any known, confirmed, or suspected sample containing cFAP, and include, but are not limited to, subject tissue samples, which may further contain or be contaminated with blood, plasma, serum, or other bodily fluids. The methods also cover the analysis of subject-derived samples where the presence of cFAP is unknown and / or where such presence is excluded.

[0159] As used herein, subject-derived formulations also include tissue formulations. In particular, the present invention provides methods and compositions for immunohistochemical analysis of such tissue articles, for tissue sections prepared according to standard methods known in the art. Preferably, the samples used for immunohistochemical analysis according to the method of the present invention are OCT-frozen.

[0160] 5.4.2 Pharmaceutical Composition

[0161] The anti-cFAP antigen-binding molecule of the present invention, as well as the methods for its production and use, are not only provided as diagnostic tools, but are also considered applicable to the treatment and relief of diseases and disease symptoms, and to model systems for researching disease therapies, specifically diseases characterized by cFAP expression. Therefore, the present invention provides pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers and (i) a specific and selective anti-cFAP antigen-binding molecule as disclosed herein; (ii) a polynucleotide encoding the antigen-binding molecule of (i); (iii) a carrier comprising the polynucleotide of (ii); or (iv) a host cell comprising the polynucleotide of (ii) and / or expressing the antigen-binding molecule of (iii).

[0162] The pharmaceutical compositions disclosed herein are formulated to be administered to human or animal subjects. In the manufacture of the pharmaceutical formulations, the antibody or antigen-binding fragment of the present invention is mixed with a pharmaceutically acceptable carrier, excipient, and / or diluent. Of course, the carrier, excipient, and / or diluent must be acceptable in the sense of compatibility with any other components of the formulation and must be harmless to the subject. Examples of suitable pharmaceutical carriers for use with antibody-based compositions are well known in the art and can be formulated using conventional methods.

[0163] The terms “treatment”, “treating”, etc., are generally used herein to mean achieving a desired pharmacological and / or physiological effect. Such effect may be preventative in relation to the complete or partial prevention of a disease or its symptoms, and / or therapeutic in relation to the partial or complete cure of a disease or symptom and / or side effects attributable to said disease or symptom. As used herein, the term “treatment” covers any treatment of a subject’s disease or symptom and includes: (a) preventing and / or alleviating a disease or symptom (or its symptoms) occurring in a subject, wherein the subject is known or believed to have said disease or symptom, or is susceptible to said disease or symptom; (b) inhibiting a disease or symptom, i.e., preventing its development, such as inhibiting disease progression; (c) alleviating a disease or symptom, even if the disease subsides; and / or (d) preventing, inhibiting, or alleviating any symptoms or side effects associated with the disease or symptom. Preferably, as used herein, the term “treatment” refers to a medical intervention for a condition that has already manifested, for example, treatment of a diagnosed disease or symptom characterized by cFAP expression. The term "treatment" also includes remission, such as improvement or reduction of one or more indicators, signs, or symptoms of a disease, condition, or symptom characterized by cFAP expression. Therefore, the term "treatment" can include delaying or mitigating the progression or severity of one or more indicators of a condition or disease characterized by cFAP expression. The progression or severity of the indicator can be determined by subjective or objective measurements known to those skilled in the art.

[0164] As used herein, the term “prevention” refers to delaying or stopping the onset, development, or progression of a disease, condition, or symptom characterized by cFAP expression for a period ranging from a few minutes to indefinitely.

[0165] Subjects to be treated using the methods and uses disclosed herein may be mammals, including but not limited to domesticated animals (e.g., cattle, sheep, cats, dogs, horses, and birds), rabbits, rodents (e.g., mice and rats), and primates (e.g., humans and non-human primates such as monkeys). Preferably, the subjects to be treated are humans. In particular, the subjects to be treated (e.g., human patients) are at risk of having, suspected of having, or have been identified as having a disease or symptom characterized by cFAP expression as described herein or as known in the art.

[0166] 5.5 Reagent Kit

[0167] The present invention also provides a kit comprising any article (e.g., packaging or container) containing at least one reagent of the present invention, namely one or more of the following: (i) the antigen-binding molecule of the present invention, (ii) the nucleic acid molecule of the present invention, (iii) the vector of the present invention, (iv) the host cell of the present invention, and / or (v) the antigen-binding molecule generated or obtained by the method of the present invention. The kit may be promoted, distributed, or sold as a unit for performing the method of the present invention.

[0168] The specific and selective anti-cFAP antigen-binding molecules disclosed herein can be used in cFAP detection kits. Such kits may contain a capture reagent, a detection reagent, and / or a solid phase. The anti-cFAP antigen-binding molecules of the present invention may or may not be conjugated to or otherwise linked to a solid phase (e.g., magnetic beads or plates). Furthermore, the anti-cFAP antigen-binding molecules of the present invention may or may not be detectably labeled. The anti-cFAP antigen-binding molecules of the present invention can be used as a capture reagent or a detection reagent. The detection reagent is preferably labeled. In such cases, the kit may additionally contain a substrate and / or reagent that allows for detection labeling.

[0169] The test kit may additionally contain a pretreatment solution (e.g., a solution containing cFAP), a washing solution, and / or a calibration standard for biological samples as positive or negative controls.

[0170] In the foregoing detailed description of the invention, numerous individual elements, characterizing features, techniques, and / or steps have been disclosed. It will be readily recognized that each of these is beneficial not only individually when considered or used, but also when considered and used in combination with one another. Therefore, to avoid excessive repetition and redundant paragraphs, this specification avoids repeating every possible combination and arrangement. However, whether explicitly stated or not, it should be understood that such combinations are entirely within the scope of the currently disclosed subject matter.

[0171] Unless otherwise defined, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques used herein are intended to refer to techniques commonly understood in the art, including variations of those techniques or substitutions for equivalent techniques that would be obvious to one of ordinary skill in the art.

[0172] All amino acid sequences presented herein begin with the N-terminal residue and end with the C-terminal residue (N→C), as is customary in the art, and the single-letter or three-letter code abbreviations used to identify amino acids in this invention correspond to abbreviations commonly used for amino acids.

[0173] Numerous references, including patent applications and manufacturer's manuals, are cited in this specification. Although the disclosures in these documents are not considered to be related to the patentability of this invention, they are incorporated herein by reference in their entirety. More specifically, all cited documents are incorporated by reference to the extent that each individual document is specifically and individually indicated as incorporated by reference.

[0174] In addition, the present invention relates to the following:

[0175] 1. An antigen-binding molecule that is specific to membrane-bound fibroblast activation protein (FAP) (cFAP) expressed on the surface of a cell, said antigen-binding molecule distinguishing said cFAP from extracellular soluble non-membrane-associated fibroblast activation protein (sFAP).

[0176] 2. The antigen-binding molecule according to Clause 1, wherein the cFAP has the amino acid sequence of SEQ ID NO: 1 and is expressed on the surface of HEK293 cells, and wherein the sFAP has the amino acid sequence of SEQ ID NO: 2 or is present in human plasma (spFAP).

[0177] 3. The antigen-binding molecule according to Clause 1 or 2, wherein the EC50 of the molecule binding to cFAP is less than 5 nM, and the EC50 of the molecule binding to sFAP is at most 1 / 20 of the EC50 of the molecule binding to cFAP.

[0178] 4. The antigen-binding molecule according to Clause 3, wherein the EC50 binding to sFAP is identified as the EC50 binding to human plasma known to or believed to contain spFAP.

[0179] 5. The antigen-binding molecule according to clause 3 or 4, wherein

[0180] (i) When measured in an assay, the EC of the molecule bound to spFAP 50 It is undetectable or incalculable in the assay, and when evaluated under the same conditions, the EC binding of siroizumab or 4B9 to the spFAP. 50 < 1 nM or < 10 nM respectively;

[0181] and

[0182] (ii) The antigen-binding molecule has one or more of the following cFAP binding properties as determined by flow cytometry:

[0183] (a) The apparent Kd binding to the cells expressing the cFAP is less than 2 nM;

[0184] (b) EC binding to cells expressing the cFAP 50 Less than 2 nM;

[0185] (c) EC binding to cells expressing the cFAP 50 Siroizumab or 4B9 EC were evaluated under the same conditions. 50 Within at least 1 time;

[0186] (d) The maximum binding to the cells expressing the cFAP was within 1 time the maximum binding of siroizumab or 4B9 as evaluated under the same conditions.

[0187] 6. The antigen-binding molecule according to any one of clauses 3 to 5, wherein the binding ratio of cFAP to undiluted human plasma having a concentration of at least 80 ng / mL in the presence of undiluted human plasma relative to the binding ratio in the absence of said undiluted human plasma is at least 80% at a concentration of 0.8 nM antigen-binding molecule, at least 60% at a concentration of 0.16 nM antigen-binding molecule, or at least 50% at a concentration of 0.0032 nM antigen-binding molecule; wherein, if evaluated in the same assay and under the same conditions,

[0188] (i) The binding ratio of 4B9 to cFAP in the presence of the undiluted human plasma to the binding ratio in the absence of the undiluted human plasma is less than 10% at a 4B9 concentration of less than 0.8 nM; or

[0189] (ii) The binding ratio of siroizumab to cFAP in the presence of the undiluted human plasma to the binding ratio in the absence of the undiluted human plasma is less than 25% at less than 0.8 nM.

[0190] 7. An antigen-binding molecule according to any one of clauses 1 to 6, wherein the molecule comprises or is composed of an antibody, an antibody-antigen binding domain, or an antibody-antigen binding fragment.

[0191] 8. The antigen-binding molecule according to Clause 7, wherein the molecule comprises more than one antibody, antibody-antigen binding domain, or antibody-antigen binding fragment.

[0192] 9. The antigen-binding molecule according to Clause 8, comprising more than one antibody-antigen-binding domain or more than one antibody-antigen-binding fragment having the same amino acid sequence.

[0193] 10. The antigen-binding molecule according to Clause 8, comprising more than one antibody-antigen-binding domain or more than one antibody-antigen-binding fragment, said more than one antibody-antigen-binding domain or fragment specifically binding to the same antigen and having different amino acid sequences.

[0194] 11. An antigen-binding molecule according to any one of clauses 7 to 10, comprising at least one antibody-antigen-binding domain or fragment, said at least one antibody-antigen-binding domain or fragment being Fab, Fab', F(ab')2, Fv, scFv or a single-domain binding fragment.

[0195] 12. The antigen-binding molecule according to Clause 11, wherein the single-domain binding fragment is an sdAb, dAb, nanobody, or V. H H-structure domain.

[0196] 13. The antigen-binding molecule according to any one of clauses 1 to 11, wherein the molecule comprises a human Fc domain.

[0197] 14. The antigen-binding molecule according to Clause 13, wherein the Fc domain (i) comprises the mutation S239D / A303L / I332E (DEL modification) according to EU number, (ii) is unfucosylated, or (iii) is modified to increase ADCC or ADCP function relative to the same antigen-binding molecule comprising the IgG1 Fc domain having the amino acid sequence of SEQ ID NO: 22.

[0198] 15. The antigen-binding molecule according to any one of Clauses 7 to 14, wherein the antibody, antibody-antigen-binding domain, or antibody-antigen-binding fragment is a human, chimeric, humanized, CDR-transplanted, and / or deimmunized antibody, antibody-antigen-binding domain, or antibody-antigen-binding fragment.

[0199] 16. The antigen-binding molecule according to any one of clauses 1 to 15, wherein said molecule comprises one or more V H The H-structure domain or is composed of it.

[0200] 17. The antigen-binding molecule according to Clause 16, wherein one or more of the V H The H domain contains

[0201] (i) CDR1, which has the sequence X1X2TX3X4X5YAX6G (SEQ ID NO: 3), wherein X1 is A, G or S; wherein X2 is P, R or G; wherein X3 is K, F, R or N; wherein X4 is S, R or G; wherein X5 is T, S or N; and wherein X6 is M, I or L;

[0202] (ii) CDR2, wherein CDR2 has the sequence X1IX2WX3X4X5X6TX7YX8DSVKG (SEQ ID NO: 4), wherein X1 is A or V; wherein X2 is N, W or S; wherein X3 is S or A; wherein X4 is G or N; wherein X5 is T or G; wherein X6 is I, L, S, T or V; wherein X7 is S, Q or N; and wherein X8 is S or T; and / or

[0203] (iii) CDR3, which has the sequence AADX1DFRTVGSRPSY (SEQ ID NO: 5), wherein X1 is R, S or K;

[0204] Furthermore, the CDR mentioned therein is defined based on the IMGT number.

[0205] 18. The antigen-binding molecule according to clause 16 or 17, wherein one or more of the V H The H domain contains

[0206] (i) CDR1, wherein the CDR1 has a sequence of APTRSTYAMG (SEQ ID NO: 6) or GRTFSSYAIG (SEQ ID NO: 7);

[0207] (ii) CDR2, said CDR2 having the sequence VINWSGTLTSYADSVKG (SEQ ID NO: 8), VINWAGTLTSYADSVKG (SEQ ID NO: 9), VISWSGTLTSYADSVKG (SEQ ID NO: 10), AISWSGGTTQYTDSVKG (SEQ ID NO: 11), AISWSGGTTNYTDSVKG (SEQ ID NO: 12) or AISWSGGTTNYADSVKG (SEQ ID NO: 13); and / or

[0208] (iii) CDR 3, which has the sequence AADRDFRTVGSRPSY (SEQ ID NO: 14),

[0209] Furthermore, the CDR mentioned therein is defined based on the IMGT number.

[0210] 19. The antigen-binding molecule according to any one of clauses 16 to 18, wherein the one or more V H The H domain contains

[0211] (i) CDR1, CDR2 and CDR3, wherein CDR1 has a sequence of APTRSTYAMG (SEQ ID NO: 6) or GRTFSSYAIG (SEQ ID NO: 7), CDR2 has a sequence of VINWSGTLTSYADSVKG (SEQ ID NO: 8), and CDR3 has a sequence of AADRDFRTVGSRPSY (SEQ ID NO: 14);

[0212] (ii) CDR1, CDR2 and CDR3, wherein CDR1 has a sequence of APTRSTYAMG (SEQ ID NO: 6) or GRTFSSYAIG (SEQ ID NO: 7), CDR2 has a sequence of VINWAGTLTSYADSVKG (SEQ ID NO: 9), and CDR3 has a sequence of AADRDFRTVGSRPSY (SEQ ID NO: 14);

[0213] (iii) CDR1, CDR2 and CDR3, wherein CDR1 has a sequence of APTRSTYAMG (SEQ ID NO: 6) or GRTFSSYAIG (SEQ ID NO: 7), CDR2 has a sequence of VISWSGTLTSYADSVKG (SEQ ID NO: 10), and CDR3 has a sequence of AADRDFRTVGSRPSY (SEQ ID NO: 14);

[0214] (iv) CDR1, CDR2 and CDR3, wherein CDR1 has the sequence GRTFSSYAIG (SEQ ID NO: 7), CDR2 has the sequence AISWSGGTTQYTDSVKG (SEQ ID NO: 11), and CDR3 has the sequence AADRDFRTVGSRPSY (SEQ ID NO: 14);

[0215] (v) CDR1, CDR2, and CDR3, wherein CDR1 has the sequence GRTFSSYAIG (SEQ ID NO: 7), CDR2 has the sequence AISWSGGTTNYTDSVKG (SEQ ID NO: 12), and CDR3 has the sequence AADRDFRTVGSRPSY (SEQ ID NO: 14); or

[0216] (vi) CDR1, CDR2 and CDR3, wherein CDR1 has the sequence GRTFSSYAIG (SEQ ID NO: 7), CDR2 has the sequence AISWSGGTTNYADSVKG (SEQ ID NO: 13) and CDR3 has the sequence AADRDFRTVGSRPSY (SEQ ID NO: 14).

[0217] 20. The antigen-binding molecule according to any one of clauses 16 to 19, wherein the one or more V H The H struct contains the following sequence:

[0218] (i) QVQLVESGGGLVQAGDSLSLSCVASAPTRSTYAMGWFRQAPGKEREFAAVINWSGTLTSYADSVKGRFTISRDNAKNTVFLQMNSLKPDDTAVYYCAADRDFRTVGSRPSYWGQGTPVTVSS (SEQ ID NO: 15);

[0219] (ii) QVQLVESGGGLVEPGDSLRLSCAASGRTFSSYAIGWFRQAPGKEREFVAAISWSGGTTNYTDSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAADRDFRTVGSRPSYWSKGTRVTVSS (SEQ ID NO: 16);

[0220] (iii) QVQLVESGGGLVQPGGSLRLSCSASAPTRSTYAMGWFRQAPGKEREFASVISWSGTLTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAADRDFRTVGSRPSYWGQGTTVTVSS (SEQ ID NO: 17);

[0221] (iv) QVQLVESGGGLVQPGGSLRLSCSASGRTFSSYAIGWFRQAPGKEREFVSAISWSGGTTQYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAADRDFRTVGSRPSYWGQGTTVTVSS (SEQ ID NO: 18);

[0222] (v) QVQLVESGGGLVQPGGSLRLSCSASGRTFSSYAIGWFRQAPGKEREFVSAISWSGGTTNYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAADRDFRTVGSRPSYWGQGTTVTVSS (SEQ ID NO: 19).

[0223] 21. The antigen-binding molecule according to any one of clauses 16 to 19, wherein the one or more V H The H domain contains a variant amino acid sequence having at least 85%, 90%, or 95% sequence identity with any of SEQ ID NO: 15 to 19, wherein the variant is a modification of any of SEQ ID NO: 15 to 19 by substitution of one or more conserved amino acids.

[0224] 22. The antigen-binding molecule according to Clause 21, wherein the conserved amino acid substitution is the substitution of one amino acid with another amino acid selected from the same group thereof, wherein the amino acid group is

[0225] a) Nonpolar hydrophobic amino acids composed of Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp and Met;

[0226] b) Polar neutral amino acids composed of Ser, Thr, Asn, and Gln;

[0227] c) Positively charged basic amino acids composed of Arg, Lys, and His, and

[0228] d) A negatively charged acidic amino acid composed of Asp and Glu.

[0229] Where Cys is conservatively replaced, it is replaced by Ser or Ala, and where Pro is conservatively replaced, it is replaced by Ala.

[0230] 23. A polynucleotide encoding an antigen-binding molecule according to any one of clauses 1 to 22.

[0231] 24. A carrier comprising the polynucleotide as described in Clause 23.

[0232] 25. A host cell comprising the polynucleotide described in claim 23 or the vector described in claim 24.

[0233] 26. A method for generating an antigen-binding molecule according to any one of clauses 1 to 22, the method comprising culturing a host cell according to clause 25 and isolating the antigen-binding molecule.

[0234] 27. A composition comprising (i) an antigen-binding molecule according to any one of Clauses 1 to 22 or obtainable by means of the method according to Clause 26, (ii) a polynucleotide according to Clause 23, (iii) a carrier according to Clause 24, or (iv) a host cell according to Clause 25.

[0235] 28. An antigen-binding molecule according to any one of Clauses 1 to 22, or obtainable by the method according to Clause 26, or a composition according to Clause 27, used as a medicine.

[0236] 29. A pharmaceutical composition comprising an antigen-binding molecule according to any one of Clauses 1 to 22, or obtainable by the method according to Clause 26, or the composition according to Clause 27.

[0237] 30. An antigen-binding molecule according to any one of Clauses 1 to 22, or obtainable by the method according to Clause 26, or a composition according to Clause 27, for the treatment of a disease characterized by cFAP overexpression.

[0238] 31. A method for treating a disease characterized by cFAP overexpression, the method comprising administering to a subject in need a therapeutically effective amount of an antigen-binding molecule according to any one of clauses 1 to 22, or obtainable by the method according to clause 26, or a composition according to clause 27.

[0239] 32. The antigen-binding molecule for use as described in Clause 30, wherein the treatment is for a subject with a blood spFAP concentration greater than 40 ng / mL.

[0240] 33. The method according to Clause 31, wherein the subject's blood spFAP concentration is greater than 40 ng / mL.

[0241] 34. An antigen-binding molecule according to any one of Clauses 1 to 22, or obtainable by the method according to Clause 26, or a composition according to Clause 27, which is a diagnostic agent for use in vitro or in vivo for the following purposes.

[0242] (i) Used for detecting cFAP; and / or

[0243] (ii) Used to distinguish between cFAP and spFAP in serum, plasma, tissue or body fluids.

[0244] 35. A kit comprising an antigen-binding molecule according to any one of claims 1 to 22 or obtainable by means of the method according to claim 26.

[0245] 6. Examples

[0246] 6.1 Materials and Methods

[0247] Unless otherwise stated, the following description of materials and methods generally applies to the embodiments described herein.

[0248] 6.1.1 Primary cells

[0249] Human uterine / endometrial fibroblasts (HUF), human lung fibroblasts (HPF), and human cardiac fibroblasts (HCF) were used as primary cells (Sigma-Aldrych / Merck / Promocell). Additionally, human skin fibroblasts (Detroit 551) and HEK293 were used. HUF and HPF were cultured in fibroblast growth medium 2 (with supplements) (PromoCell GmbH; Heidelberg, Germany). HCF was cultured in fibroblast growth medium 3 (with supplements) (Promocell). Detroit 551 was cultured in Eagle's minimal basal medium (catalog number 30-2003) supplemented with a final concentration of 10% fetal bovine serum.

[0250] 6.1.2 Human plasma samples

[0251] Human plasma was obtained from fresh EDTA-treated female blood and stored at 4°C for 24 hours until processing. The blood was centrifuged at 2000 rpm for 10 minutes to separate the plasma layer. The plasma was then filtered first through a 40 µm sieve and subsequently through a 0.45 µm filter. If not required for immediate use, the plasma was aliquoted into 5 ml volumes and stored at -80°C. All plasma processing was performed on ice under BSL2 conditions.

[0252] 6.1.3 FAP ELISA

[0253] The following FAP ELISA is used in the working examples detailed below.

[0254] a) Human FAP DuoSet ELISA (Biotechne, DY3715) Used to quantify human spFAP in plasma using a protocol recommended by the manufacturer.

[0255] b) Positive plasma ELISAThe affinity of the antigen-binding molecule for spFAP was used to quantify the affinity of the antigen-binding molecule for spFAP. A Human FAP DuoSet ELISA kit (Biotechne, DY3715) was used, in which the capture antibody was replaced with serially diluted concentrations of the antigen-binding molecule. The EC50 of the antigen-binding molecule for human spFAP was evaluated.

[0256] c) Reverse plasma ELISA This method utilizes an alternative orthogonal setup to quantify and validate the affinity of antigen-binding molecules for spFAP. The Human FAP DuoSet ELISA kit (Biotechne, DY3715) is used, in which the antigen-binding molecules of the present invention are used in place of the detection antibody at sequentially diluted concentrations. The EC50 of the antigen-binding molecules with human spFAP is evaluated. The antigen-binding molecules of the present invention are detected using biotinylated anti-human IgG1 Fc antibody (using 1:1000, B6775 Merck / Sigma-Aldrich) and streptavidin-HRP (e.g., DY998 Bio-Techne) or suitable alternatives.

[0257] c) Recombinant FAP ELISA. An internal protocol was established for measuring the EC50 of antigen-binding molecules and recombinant FAP (rFAP; SEQ NO: 2), in which plates were coated overnight at 4°C with 10 µg / ml or 5 µg / ml rFAP.

[0258] FAP in plasma and / or cell cultures was measured using the Human FAP DuoSet ELISA Kit (Biotechne, DY3715) and a high-binding 96-well plate (Corning, reference number 3590) according to the manufacturer’s guidance as detailed in (a).

[0259] To measure the binding of the antigen-binding molecules of the present invention (also referred to herein as "antibody constructs") to plasma (including antibody constructs ab1-hFc and ab3-hFc, as detailed below), forward and reverse ELISAs for FAP were established as described in (b) and (c). Forward and reverse ELISAs were used to exclude lower binding due to competition with other antibodies used in the same assay (e.g., coated antibodies whose binding overlaps with that of ab1-hFc and ab3-hFc used as detection antibodies).

[0260] A forward plasma FAP ELISA was performed using the antigen-binding molecule of the present invention, which acts as a capture antibody, and a plate coated with a detection antibody obtained from the DY3715 Human FAP ELISA Kit. The assay was performed in a high-binding 96-well EIA / RIA plate (Corning Electron) using coating buffer (1x PBS), wash buffer, freshly prepared and sterile filtered dilution buffer consisting of PBS containing 1% BSA, human plasma (see above), streptavidin-HRP (DY998 Bio-Techne, according to the manufacturer's dilution), HRP substrate (e.g., 1-Step UltraTMB ELISA Thermo Scientific Reference No. 34028), ELISA stop solution (Invitrogen SS04), and a plate sealer.

[0261] To coat the ELISA plate, a serially diluted buffer of the antigen-binding molecule of the present invention was prepared in PBS. A 96-well high-binding plate from Corning was then coated with 100 µL of the diluted buffer per well, immediately sealed with a plate sealer, and incubated overnight at 4°C. Next, each well was washed three times with 300 µL of wash buffer (e.g., Thermo Scientific catalog number: J63596.K2, diluted directly in dH2O before use), completely removing liquid after each step. The plate was then blocked by adding 200 µL of reagent diluent (e.g., PBS with 1% BSA and no protease (Biowest P6155-100 g or Sigma-Aldrich A3059-50 g)) to each well, followed by incubation at room temperature for 1 to 2 hours. After another round of washing, 100 µL of the prepared human plasma was added to each well, and the plate was covered and incubated at room temperature for 2 hours. Next, 100 µl of detection antibody diluted to 200 ng / ml was added to each well and incubated for 2 hours. Subsequently, 100 µl of working dilution of streptavidin-HRP was added to each well, covered, and incubated for 20 minutes, avoiding direct light exposure. After another washing step, 100 µl of HRP substrate was added to each well. The generated signal was monitored after an incubation period of up to 20 minutes. Finally, 100 µl of stop solution was added to each well and gently mixed by tapping. The optical density of each well was immediately measured at 450 nm using a microplate reader. If wavelength correction was available, the reading at 540 nm or 570 nm was subtracted from the initial 450 nm reading.

[0262] In the reverse plasma FAP ELISA, the standard procedure was modified by replacing the detection antibody from the human FAP DuoSet ELISA kit with the antigen-binding molecule of the present invention at different serial dilutions. Additionally, biotinylated anti-human IgG1 (B6775 from Sigma-Aldrich) was combined with streptavidin-HRP (DY998 Bio-Techne) for detection. The manufacturer-recommended FAP DuoSet ELISA kit protocol was used for the remaining steps of the procedure.

[0263] 6.1.4 Cell binding assay performed by flow cytometry

[0264] HEK 293 cells were transfected with DNA encoding the intact human fibroblast activator protein (FAP, SEQ ID NO: 1) using lipofectamine 2000 (Ingenieur Biotech) according to the manufacturer’s standard protocol.

[0265] 100,000 transfected cells were seeded into each well. The primary antibody was serially diluted in PBS and 1% bovine serum albumin (BSA). The primary antibody was then added to the corresponding wells and incubated at 4°C for one hour. The incubation period allowed the primary antibody to fully bind to the target antigen on the cells.

[0266] Subsequently, after washing, secondary antibody staining was performed. In short, a 1:100 dilution of anti-human Fc antibody conjugated with R-phycoerythrin (RPE) in 1% BSA was added to wells containing cells and primary antibodies, followed by incubation at 4°C for 30 minutes and two washing steps in PBS and 1% BSA. Flow cytometry was then performed using standard procedures.

[0267] 6.1.5 Cross-species responsiveness to FAP in mice and cynomolgus monkeys

[0268] According to a standard protocol, the cross-species reactivity of FAP antigen-binding molecules was measured against mouse and cynomolgus monkey FAP proteins with amino acid sequences (SEQ ID NO: 23 or 24, respectively) transiently expressed on HEK293 cells. Subsequent steps of the protocol are those outlined in Section 6.1.4.

[0269] 6.1.6 Competitive cell-binding fluidization cytometry in the presence of soluble FAP (plasma)

[0270] The antigen-binding construct containing the mouse IgG2a Fc region according to the present invention was incubated in human plasma at room temperature or 37°C for one hour. Next, human plasma containing the antigen-binding construct was added to transfected cells expressing complete human FAP protein (SEQ ID NO: 1) at a concentration ranging from 50,000 to 100,000 cells per well, and incubated at 4°C for 30 minutes. The cells were then washed and subsequently stained with fluorescently labeled anti-mouse IgG. Binding was quantified by measuring mean fluorescence intensity (MFI) using flow cytometry.

[0271] 6.1.7 FAP enzyme activity assay

[0272] FAP enzyme activity was measured in black 96-well plates in the presence of 50 μM Suc-Gly-Pro-Leu-Gly-Pro-AMC (#I-1350.0100, BACHEM, Bubendorf, Switzerland) in 20 mM Tris / HCl, 0.1 M NaCl, and 1 mM EDTA. Donor plasma in a total volume of 100 µl and soluble FAP (Bepsys) with an enzymatic activity of 0.4 ug / ml were used (alone). The reaction mixture was incubated at 37 °C for 60 min, and the fluorescence of FAP-induced cleavage-released AMCs was determined using a plate reader at an emission wavelength of 475 nm (excitation wavelength of 380 nm).

[0273] 6.1.8 ADCC (Antibody-dependent cell-mediated cytotoxicity) assay

[0274] The ADCC reporter gene bioassay core kit (Promega catalog numbers G7010 and G7018) is used to quantify the bioactivity of the antigen-binding molecule pair activating the pathway according to the present invention.

[0275] On Day 1, target cells were plated in white 96-well plates (TC-treated) in cell culture medium (20k cells / well). On Day 2, the antigen-binding molecule was diluted in assay medium (FBS-free). The medium was removed from the target cells in the 96-well plate, and 50 µl of serially diluted antigen-binding molecule was added to each well. The antigen-binding molecule was then incubated for 20 min, followed by the addition of 120k cells / well of effector cells to assay medium with 1% low IgG FBS. The cells were then incubated at 37°C for 18 h. On Day 3, the plate was equilibrated at RT for 10 min, and Bio-Glow was added to each well (75 µl) and incubated for 5 min for luminescence measurement according to the manufacturer's instructions.

[0276] ADCC in the presence of rFAP:

[0277] As described above, the assay is performed by serial dilution of the antigen-binding molecule in assay medium (without FBS) containing spFAP.

[0278] 6.1.9 ADCP (antibody-dependent cell-mediated phagocytosis) assay

[0279] ADCP reporter gene bioassays reflect the mechanism of action of biologics designed to bind to and activate FcγRIIa. The potency and stability of the antigen-binding molecule fused to human FC (Promega catalog number G9871) are measured using a bioluminescent cell-based assay, according to manufacturer guidance, which specifically binds to and activates FcγRIIa.

[0280] The assay utilizes a genetically engineered Jurkat T cell line expressing human FcγRIIa and a luciferase reporter gene driven by an NFAT response element (NFAT-RE). Briefly, on Day 1, target cells are plated in white 96-well plates (20k cells / well) in cell culture medium. On Day 2, the antigen-binding molecule is diluted in assay medium (FBS-free). Next, the medium is removed from the cells, and 50 µl of serially diluted antigen-binding molecule is added to each well and incubated for 20 min. Afterward, 120k effector cells / well are added to assay medium supplemented with 50 µl (total volume 100 µl) of 8% low-IgG FBS and incubated at 37°C for 18–20 h. On Day 3, the 96-well plates are equilibrated at room temperature for 10 min, then Bio-Glow is added to each well (75 µl) and incubated for 5 min for luminescence measurement according to the manufacturer's instructions.

[0281] 6.1.10 Assay for antigen-binding molecule internalization on cells expressing FAP

[0282] WI-38 cells were used to evaluate the internalization of the antigen-binding molecule according to the invention. Antibody was serially diluted 4-fold in assay buffer, starting at 200 nM. Ten thousand WI-38 cells were seeded per well (96-well plate) and grown overnight at 37°C. The antigen-binding molecule was added to the cells, and the cells were incubated at 4°C for 1 hour. Washing was then performed, followed by incubation at 4°C for 30 minutes with a pHrodo-labeled secondary antibody. The internalization process was activated by incubating the cells at 37°C for 4 hours and 24 hours after another washing step. At the end of the incubation period, the cells were stained with Hoechst and calcein AM at 37°C for 15 minutes. Readings were taken using the Operetta system, specifically counting the number of pHrodo spots per cell.

[0283] 6.1.11 Binding with human DPP4

[0284] Reactivity with FAP antigen-binding molecules was measured against human DPP4 (a near homolog of FAP). HEK 293F cells were transfected with DNA encoding complete human DPP4 (SEQ ID NO: 34) using a lipofectamine 2000 (Ingenieur Biosciences) according to a standard protocol. Subsequent steps of this protocol were those outlined in Section 6.1.4.

[0285] 6.1.12 In vivo imaging

[0286] 1 × 10 per mouse 6 MC38-hFAP cells were subcutaneously (SC) inoculated into 8-week-old female C57BL / 6 mice. At 28 days post-inoculation and 24 hours before sacrifice, mice were administered 7.5 mg / kg of Ab3z7p2-Scarlett (a fluorescently labeled Scarlett conjugate of Ab3) or PBS as a control via intraperitoneal (IP) administration. Fluorescence was measured at different time points post-inoculation (3 hours, 24 hours). Fluorescence imaging was performed using a provided imaging system to detect the Scarlett fluorophore. Imaging parameters included the excitation wavelength range of 570–604 nm and the emission wavelength range of 620–639 nm. The fluorescence data were visually analyzed to demonstrate uptake by specific tumors.

[0287] 6.1.13 ADCP assay using human macrophages

[0288] The ADCP assay utilizes monocyte-derived macrophages isolated from primary cells of healthy human blood donors. CD14+ monocytes are enriched from peripheral blood monocytes (PBMCs) and subsequently differentiated into macrophages through culture in the presence of recombinant human macrophage colony-stimulating factor (rhM-CSF). The assay measures the phagocytic activity of macrophages, particularly assessing the internalization of target cells by antibody opsonization. This quantification is achieved via fluorescence-based flow cytometry, which tracks the uptake of fluorescently labeled target cells by macrophages.

[0289] Effector cell preparation

[0290] CD14+ monocytes were isolated from PBMCs and differentiated into macrophages by culturing them for 6-8 days in RPMI-1640 medium supplemented with 50 ng / mL recombinant human macrophage colony-stimulating factor (rhM-CSF) at 37°C and 5% CO2.

[0291] Target cell preparation

[0292] HT1080-hFAP target cells were labeled with 1 nM CSFE (carboxyfluorescein succinimide) in the dark at 37°C for 20 minutes and then washed in PBS.

[0293] Antibody preparation

[0294] Antibodies of different concentrations were prepared, starting with 5 nM and serially diluted 8-fold in PBS.

[0295] Co-culture and phagocytosis assay

[0296] Preheat the target cells with the prepared antibody dilution at room temperature for 15-20 minutes. Then, co-culture the target cells with macrophages at a 1:1 effector-to-target (E:T) ratio. Maintain the co-culture at 37°C in a humidified environment with 5% CO2 for 4 hours.

[0297] Secondary antibody staining

[0298] After co-culturing, the cells were stained with CD14-APC antibody (1:150 dilution) in 1% bovine serum albumin (BSA) at a volume of 100 μL per well. Staining was performed on ice for 40 minutes.

[0299] Flow cytometry analysis

[0300] Samples were washed and resuspended in PBS before analysis by fluorescence-activated cell sorting (FACS). FACS analysis was used to measure phagocytosis by detecting CFSE-labeled target cells internalized by CD14+ macrophages.

[0301] 6.1.14 FDA tissue staining

[0302] Material

[0303] Tissue array: FDA standard frozen tissue array (catalog number T6234701-2, AMSBIO).

[0304] Primary antibody: ab3-mFc (mouse IgG2a).

[0305] Stock solution concentration: 1.72 mg / ml.

[0306] Dilution ratio: 1:8000.

[0307] Negative control: Mouse IgG2A isotype control, Biotechne (catalog number MAB0031).

[0308] Blocking: BloxAll blocking reagent and peroxide blocking agent.

[0309] The Human FDA Tissue Array (AMS-Bio) comprised fresh frozen tissue sections from major human organs of three donors of different ages and sexes. All sections were stained with ab3-mFc via IHC and scanned and quantified using a digital pathology AI algorithm (Qpath). In short, fresh frozen tissue sections were air-dried overnight at room temperature and then fixed in acetone at 4°C for 10 minutes. After fixation, sections were stained using the Leica Bond system according to the HRP Refine 60 Kryo + BloxAll protocol. For detection, the BOND Refine detection system (DS9800) was used. The antibody dilution was 1.72 mg / ml ab3-mFc 1:8000, and the control antibody was diluted accordingly.

[0310] 6.2 Example 1 - Generation of specific and selective antigen-binding molecules of cFAP

[0311] Alpacas were subcutaneously immunized four times with a mixture of different FAP species, the mixture comprising membrane truncated peptides of different lengths: full-length human FAP (SEQ ID NO: 1), a human FAP fragment spanning amino acids 20-45 of human FAP (SEQ ID NO: 25), a human FAP fragment fused with KLH spanning amino acids 24-50 of human FAP (SEQ ID NO: 26), recombinant cynomolgus monkey FAP (accession number XP_005573377.1, SEQ ID NO: 27), and recombinant mouse FAP (accession number P97321; SEQ ID NO: 28).

[0312] PBMCs from immunized alpacas were isolated, and phage display libraries were generated according to a standard protocol. Primary human cells overexpressing cellular FAP (cFAP), including HUF, HPF, HCF, and the dermal cell line Detroit551, were panned. A negative selection step was performed on human plasma to eliminate conjugates against soluble FAP (spFAP). Multiple rounds of positive and negative selection were performed using previously defined primary human cells and human plasma to enrich highly specific cFAP conjugates lacking binding to spFAP. The absence of specific cFAP conjugates in spFAP assays bound to human plasma was validated by an ELISA (positive FAP plasma ELISA).

[0313] The selected V, which binds specifically and selectively to spFAP and cFAP respectively. HThe H antibody was subsequently humanized according to: Sulea, *Methods in Molecular Biology* 2446(2022), 299-312. Additional mutations were introduced to improve the safety and scalability of commercial production by removing sites with potential post-translational modification responsibility. The obtained modified V antibodies were then classified according to IMGT numbers. H H antibodies were sequenced and aligned, and characterized by the following IMGT CDR sequence:

[0314] (i) CDR1, which has the sequence X1X2TX3X4X5YAX6G (SEQ ID NO: 3), wherein X1 is A, G or S; wherein X2 is P, R or G; wherein X3 is K, F, R or N; wherein X4 is S, R or G; wherein X5 is T, S or N; and wherein X6 is M, I or L;

[0315] (ii) CDR2, wherein CDR2 has the sequence X1IX2WX3X4X5X6TX7YX8DSVKG (SEQ ID NO: 4), wherein X1 is A or V; wherein X2 is N, W or S; wherein X3 is S or A; wherein X4 is G or N; wherein X5 is T or G; wherein X6 is I, L, S, T or V; wherein X7 is S, Q or N; and wherein X8 is S or T; and

[0316] (iii) CDR 3, which has the sequence AADX1DFRTVGSRPSY (SEQ ID NO: 5), wherein R, S or K is present.

[0317] Based on the exemplary antigen-binding molecules described above, including specific and selective anti-cFAP Camelidae V, H H antibodies ab1 and ab3, as well as humanized and optimized variant sequences ab1z12, ab3z7 and ab3z10, are shown in Table 1 below.

[0318] Table 1:

[0319]

[0320] 6.3 Example 2 – Selective Combination of spFAP and cFAP

[0321] According to Table 2, the humanized and optimized V1 in Example 1 was linked to the IgG1 Fc domain using a GS adapter. H The H sequence is formatted as a construct containing an antigen-binding Fc region (the “antigen-binding” construct is also referred to as the “antibody” construct in this document).

[0322] Table 2

[0323]

[0324] The selective binding of the construct to cFAP relative to spFAP (i.e., FAP as present in human plasma) was evaluated. Sandwich forward and reverse ELISA were performed on transiently transfected human plasma or HEK293 cell samples expressing cFAP (as described in the Materials and Methods section above). Commercially available anti-FAP antibodies 4B9 (WO 2020 / 198665A1) and siroizumab (WO2002083171A2), containing the same IgG1 Fc domain listed above, were also tested for comparison; an isotype control was used as a negative control.

[0325] The results of combining with spFAP Figure 1 The results are shown in Table 3.

[0326] Table 3: Binding affinity for soluble FAPs (spFAP) present in human plasma

[0327]

[0328] The tested humanized antibody constructs (including variants of ab1 and ab3) had EC50 values ​​for FAP (spFAP) in human plasma that were incalculable at most 100 nM. Therefore, these constructs did not bind to spFAP or only with very low affinity. In contrast, the control antibody 4B9 and siroizumab showed high binding affinity to spFAP, with EC50 values ​​of approximately 0.5 nM and 8.8 nM, respectively. Figure 1 ).

[0329] In the cell binding assay, HEK 293F cells transiently transfected with the intact FAP sequence (SEQ ID NO: 1) were used, and the binding of the construct to cFAP was also tested by flow cytometry. The EC50 of the tested humanized antibody construct to cFAP was comparable to that of the control antibody 4B9 and siroizumab (the EC50 of all tested antibody / antibody constructs was approximately 1 nM). Figure 2 And Table 4.

[0330] Table 4: Binding affinity for cellular FAP

[0331]

[0332] The results demonstrated that the antibody construct specifically binds to cFAP but not to sFAP, thus exhibiting selectivity for both antigens and the ability to distinguish between them. In contrast, the known control antibody siroizumab and the antibody construct containing the variable domain of the known antibody 4B9 bind to both sFAP and cFAP.

[0333] 6.4 Example 3 - Competitive flow cytometry demonstrating preferential binding to cellular FAP

[0334] Competitive flow cytometry assays were performed to further validate the selective binding of the construct from Example 2 to cFAP relative to spFAP. Specifically, the flow cytometry assays of Example 2 were repeated, wherein cells expressing cFAP were incubated with the antigen-binding construct in the presence or absence of human plasma. Human plasma is known to contain a concentration of 100 ng / ml of spFAP (median from multiple studies); see, for example, Busek et al., Frontiers in Bioscience 23 (2018), 1933–1968.

[0335] In competitive flow cytometry assays, siroizumab and 4B9 showed a significant decrease in MFI (measuring efficiency) for cells with cFAP in the presence of human plasma (spFAP) compared to assays in the absence of human plasma. In the presence of plasma (spFAP), siroizumab binding decreased by up to 83%, and 4B9 by up to 95%. This significant decrease in MFI highlights that soluble plasma FAP (spFAP) acts as a competitive trap, significantly impacting the potential efficacy of known anti-FAP antibodies and their derivatives.

[0336] In contrast, competitive flow cytometry assays showed that ab3z7-hFc preferentially binds to cellular FAP (cFAP) in the presence of plasma FAP (spFAP). The minimal decrease in ab3 binding capacity in the presence of plasma was similar to the minimal decrease in binding capacity of the isotype IgG used as a negative control. These results further confirm that ab3z7 selectively binds to cFAP and can distinguish cFAP from spFAP. Figure 3

[0337] 6.5 Example 4 - Flow cytometry demonstrates the cross-reactivity of cFAP-specific and selective antigen-binding molecules with cFAP from other mammalian species.

[0338] HEK293F cells transiently transfected with cynomolgus monkey or mouse cFAP were analyzed by flow cytometry (see Materials and Methods above). Antigen-binding molecule assays showed that ab1-hFc, ab1z12-hFc, ab3-hFc, ab3z7-hFc, and ab3z10-hFc exhibited binding affinity to cynomolgus monkey cFAP (…). Figure 4 A) or mouse cFAP ( Figure 4 The antigen-binding molecule provided and developed according to the present invention cross-reacts with at least one non-human mammalian cFAP, which is beneficial for preclinical development.

[0339] 6.6 Example 5 - PK Features

[0340] In a pharmacokinetic (PK) study, female BALB / c mice aged 6 to 8 weeks received a single intravenous injection of 10 mg / kg of either ab1-hFc or 4B9-hFc. Blood samples were systematically collected and preserved over a 2-week period following administration. At the end of the experiment, the samples were batch-analyzed using a human IgG1 ELISA to quantify the concentration of the compounds over time. Figure 5 A).

[0341] The obtained data were used to calculate PK parameters such as half-life, clearance rate, and area under the curve (AUC) to provide the pharmacokinetic characteristics of the construct. Figure 5 (B and Table 5). Each data point was obtained from at least 3 mice.

[0342] Molecular weight is related to clearance rate, with larger molecular weight peptides exhibiting longer half-lives and AUCs. Existing anti-FAP antibodies show poorer half-lives due to their binding to spFAP (see, for example, Kloft et al., *Invest New Drugs* 22(2004), 39-52; Waldhauer et al., *MAbs* 13(2021), 1913971). Figure 5 As shown in B and Table 5, ab1-hFc (molecular weight 80 kD) exhibited improved AUC / dose and AUC / dose / Mwt compared to control 4B9-hFc (formatted as intact IgG1, Mwt 148 kD). Parameters were calculated from the corresponding antibody concentrations derived from human IgG1 ELISA based on standard curves.

[0343] Table 5: PK Parameters

[0344]

[0345] These results indicate that antigen-binding molecules / constructs exhibit improved PK properties, not only due to their low molecular weight, but also due to their selective binding relative to spFAP and cFAP in plasma.

[0346] 6.7 Example 6 - Effects of specific and selective cFAP antigen-binding molecules on spFAP levels in an in vivo mouse model

[0347] The activity of specific and selective cFAP antigen-binding molecules at circulating spFAP levels was evaluated in an in vivo mouse model. Female Balb / C mice were administered intravenously three times weekly over a two-week period with either a mediator-only agent, a cFAP selective antigen-binding-Fc construct, or 4B9-Fc (both Fc formats using mouse IgG2a). On day 43, two weeks after the last administration of the antigen-binding construct, the animals were sacrificed and mouse plasma samples were collected.

[0348] The level of spFAP in mouse plasma samples (diluted 1:100 and 1:500 in dilution buffer) was assessed using a mouse sandwich ELISA (Mouse FAP DuoSet ELISA, Biotechne DY8647-05).

[0349] Compared to 4B9-Fc, the cFAP-Fc antigen-binding construct of the present invention exhibits higher affinity for cFAP and a lower molecular weight. Plasma spFAP was depleted in mice receiving 4B9-Fc, while sFAP was retained in mice receiving the specific and selective cFAP-Fc construct. Figure 6 This demonstrates that the cFAP antigen-binding construct does not interact with soluble FAP in plasma and maintains stable plasma FAP (spFAP) levels throughout and after treatment.

[0350] 6.8 Example 7 - FAP Enzymatic Activity

[0351] It should be recognized that FAP has important physiological functions, such as cleaving neuropeptides and extracellular matrix components. Therefore, it is important that selective cFAP antigen-binding molecules do not affect the enzymatic activity of FAP to avoid any unintended side effects. It has been demonstrated that cFAP-specific and selective antigen-binding molecules and constructs do not bind to spFAP in plasma and do not affect circulating spFAP plasma levels. To further confirm that the selective cFAP antigen-binding construct does not potentially negatively affect FAP function, FAP enzymatic activity was tested in the presence or absence of the selective cFAP construct of this invention or a known anti-FAP molecule as a control.

[0352] FAP enzyme activity assays (see General Materials and Methods above) demonstrate that, in the absence or presence of ab1-hFc and ab3-hFc, recombinant FAP (… Figure 7 A) and human plasma containing soluble FAP (spFAP; Figure 7 The enzymatic activity of B) was unaffected at all tested concentrations. Specific and selective cFAP constructs / antibodies, siroizumab, and 4B9-hFc did not affect the enzymatic activity of FAP. In contrast, it has been previously shown that anti-FAP antibodies can directly affect the enzymatic function of FAP (see, for example, WO2016 / 110598A1).

[0353] 6.9 Example 8 - Antibody-dependent cytotoxicity (ADCC) and antibody-dependent phagocytosis (ADCP) showed enhanced effector cell binding.

[0354] The cFAP antigen-binding construct binds specifically and selectively to cFAP. The construct, as reported in Example 2, is also designed to enhance Fc receptor binding through known modifications to the Fc domain, including, for example, the absence of fucosylation and / or modifications (e.g., DEL mutations) within the Fc region mentioned in Lazar et al., Proceedings of the National Academy of Sciences (PNAS) 103 (2006), 4005-4010. By containing the modified Fc domain, the cFAP antigen-binding construct can bind to Fc receptors on immune effector cells and mediate effector functions such as antibody-dependent cytotoxicity (ADCC) or antibody-dependent phagocytosis (ADCP).

[0355] Cytotoxic activity against primary human fibroblasts was demonstrated using the cFAP antigen-binding constructs ab1-hFc and ab3-hFc as target cells. Increased concentrations of ab1-hFc and ab3-hFc were incubated with fibroblasts and effector cells expressing FcγRIIIa. ADCC reporter gene assays showed that the tested constructs effectively induced ADCC in FAP-expressing target cells of the uterus (HUF), heart (HCF), skin (Detroit 551), and lung (HPF) fibroblasts (see [references]). Figure 8 (AD). Sirolizumab was included in assays targeting HCF and Detroit 551 cells for comparison.

[0356] The cytotoxic activity of the cFAP antigen-binding constructs was also tested in the ADCP assay. In short, the FcγRIIa ADCP bioassay (Promega) uses bioluminescent Jurkat T cells expressing FcγRIIa and target cells expressing cFAP, such as HPF, HCF, HUF, or Detroit 551. The specific and selective cFAP antigen-binding constructs tested were one or both of ab3-hFc, ab1-hFc, and ab1-hFc DEL (according to EU designations, containing DEL mutations Ser239Asp, Ala303Leu, and Ile332Glu with known enhanced effector functions) or fucosylated Fc regions (antibodies with the suffix -ahFc), as shown in Table 6. Siroizumab was also tested as a reference.

[0357] Table 6: Modified antibody constructs with enhanced effector functions

[0358]

[0359] The specific and selective cFAP construct was combined with ADCP effector cells and FAP-expressing target cells, HUF ( Figure 9 A and 10A), HCF ( Figure 9 B) or HPF ( Figure 10 B) Incubate together. Specific and selective cFAP constructs with enhanced FcγR binding activity were observed to exhibit ADCP activity.

[0360] Furthermore, modified variants containing DEL mutations or without fucosylated Fc regions were tested in the ADCC assay described above. Figure 11 As shown, modifications to enhance effector function enhanced the luminescence of each of the following target cell lines in the ADCC assay: HUF ( Figure 11 A), HPF ( Figure 11 B), HCF ( Figure 11 C / D) and Detroit 551 ( Figure 11 E). Thus, it is shown that ADCP activity can be endowed with the specific cFAP construct of the present invention, and ADCC activity can be enhanced by modification of the Fc region.

[0361] 6.10 Example 9 - Antigen-binding molecules with specificity and selectivity for cFAP are not internalized upon binding to cFAP.

[0362] When incubated with WI-38 cells expressing cFAP, the specific and selective anti-cFAP molecules developed according to the methods presented herein did not show internalization. At 4 and 24 hours, the internalization rate was comparable to that of the allotype control, 4B9-hfc, and siroizumab. Even using concentrations up to 200 nM (4-fold serial dilutions, 8 concentrations), the EC50 of all antibodies could not be calculated.

[0363] 6.11 Example 10 - Antigen-binding molecules with specificity and selectivity for cFAP do not bind to the cFAP homolog DPP4.

[0364] The specific and selective anti-cFAP molecules ab1z10-hFc, ab3z7-hFc, and ab3z10-hFc did not show significant binding to HEK293F cells expressing DPP4 at 100 nM. The binding of these antigen-binding molecules to DPP4 was similar to that of the allotype control used and siroizumab.

[0365] 6.12 Example 11 - Compared with siroizumab, ADCP of human PBMC-derived macrophages in ab3z7-hFc was improved.

[0366] Compared with siroizumab, the specific and selective anti-FAP molecule ab3z7-hFc showed superior phagocytic efficacy in terms of EC50 (10-fold increase) and maximum phagocytosis (44.86% increase). Figure 12 ).

[0367] Table 7: Results of ADCP Measurement

[0368]

[0369] 6.13 Example 12 - ab3 conjugated with a fluorescent probe showed specific uptake in mice with tumors positive for human FAP.

[0370] The specific and selective anti-FAP molecule ab3 was conjugated with a fluorescent probe and showed specific staining in mice with tumors positive for human FAP. Figure 13 ).

[0371] Table 7: Antibody constructs for in vivo imaging

[0372]

[0373] 6.14 Example 13-ab3-mFc has low binding to all healthy human tissues.

[0374] IHC staining using ab3-mFc showed low binding to tissues in all healthy male and female adults. Figure 14 ).Apart from Figure 14 Aside from the tissues in the brain, the following tissues did not show visually detectable staining for human FAP: bone marrow, cerebellum, brain, pituitary gland, fallopian tubes, kidneys, ovaries, prostate, spinal cord, thymus, thyroid gland, ureters, and cervix.

Claims

1. An antigen-binding molecule that is specific to membrane-associated fibroblast activation protein (FAP) (cFAP) expressed on the surface of a cell membrane, said antigen-binding molecule distinguishing said cFAP from extracellular soluble non-membrane-associated fibroblast activation protein (sFAP).

2. The antigen-binding molecule according to claim 1, wherein the cFAP has the amino acid sequence of SEQ ID NO: 1 and is expressed on the surface of HEK293 cells, and wherein the sFAP has the amino acid sequence of SEQ ID NO: 2 or is present in human plasma (spFAP).

3. The antigen-binding molecule according to claim 1 or 2, wherein the EC50 of the molecule binding to cFAP is less than 5 nM, and the EC50 of the molecule binding to sFAP is at most 1 / 20 of the EC50 of the molecule binding to cFAP.

4. The antigen-binding molecule of claim 3, wherein the EC50 binding to sFAP is identified as an EC50 binding to human plasma known to or believed to contain spFAP.

5. The antigen-binding molecule according to claim 3 or 4, wherein said molecule has the following property (A). (A) When evaluated in an assay, the EC of the molecule binding to spFAP 50 It is undetectable or incalculable in the assay, and when evaluated under the same conditions, the EC binding of sibrotuzumab or 4B9 to the spFAP. 50 < 1 nM or < 10 nM respectively; and The antigen-binding molecule has one or more of the following cFAP binding properties as determined by flow cytometry: (a) The apparent Kd binding to the cells expressing the cFAP is less than 2 nM; (b) EC binding to cells expressing the cFAP 50 Less than 2 nM; (c) EC binding to cells expressing the cFAP 50 Siroizumab or 4B9 EC were evaluated under the same conditions. 50 Within at least 1 time; (d) The maximum binding to the cells expressing the cFAP was within 1 time the maximum binding of siroizumab or 4B9 as evaluated under the same conditions; And / or has the following property (B) (B) The antigen-binding molecule according to any one of claims 3 to 5, wherein the binding ratio of cFAP to undiluted human plasma having a concentration of at least 80 ng / mL of spFAP in the presence of said undiluted human plasma is at least 80% at a concentration of 0.8 nM, at least 60% at a concentration of 0.16 nM, or at least 50% at a concentration of 0.0032 nM; wherein, as evaluated in the same assay and under the same conditions, (i) The binding ratio of 4B9 to cFAP in the presence of the undiluted human plasma to the binding ratio in the absence of the undiluted human plasma is less than 10% at a 4B9 concentration of less than 0.8 nM; or (ii) The binding ratio of siroizumab to cFAP in the presence of the undiluted human plasma to the binding ratio in the absence of the undiluted human plasma is less than 25% at less than 0.8 nM.

6. The antigen-binding molecule according to any one of claims 1 to 5, wherein the molecule comprises an antibody, an antibody-antigen binding domain, or an antibody-antigen binding fragment, or is composed of the same.

7. The antigen-binding molecule of claim 6, wherein the molecule comprises more than one antibody, antibody-antigen binding domain, or antibody-antigen binding fragment.

8. The antigen-binding molecule of claim 7, comprising more than one antibody-antigen-binding domain or more than one antibody-antigen-binding fragment, wherein the more than one antibody-antigen-binding domain or antibody-antigen-binding fragment has the same amino acid sequence, or the more than one antibody-antigen-binding domain or fragment specifically binds to the same antigen and has different amino acid sequences.

9. The antigen-binding molecule according to any one of claims 6 to 8, comprising at least one antibody-antigen binding domain or fragment, wherein the at least one domain or fragment is Fab, Fab', F(ab')2, Fv, scFv or a single-domain binding fragment.

10. The antigen-binding molecule according to claim 9, wherein the single-domain binding fragment is an sdAb, dAb, nanobody, or V. H H-structure domain.

11. The antigen-binding molecule according to any one of claims 1 to 10, wherein the molecule comprises a human Fc domain.

12. The antigen-binding molecule of claim 11, wherein the Fc domain (i) comprises the mutation S239D / A303L / I332E (DEL modification) according to EU number, (ii) is unfucosylated, or (iii) is modified to increase ADCC or ADCP function relative to the same antigen-binding molecule comprising the IgG1 Fc domain having the amino acid sequence of SEQ ID NO:

22.

13. The antigen-binding molecule according to any one of claims 7 to 12, wherein the antibody, antibody-antigen-binding domain, or antibody-antigen-binding fragment is a human, chimeric, humanized, CDR-transplanted, and / or deimmunized antibody, antibody-antigen-binding domain, or antibody-antigen-binding fragment.

14. The antigen-binding molecule according to any one of claims 1 to 13, wherein the molecule comprises one or more V H The H-structure domain or is composed of it.

15. The antigen-binding molecule of claim 14, wherein the one or more V H The H domain contains (i) CDR1, which has the sequence X1X2TX3X4X5YAX6G (SEQ ID NO: 3), wherein X1 is A, G or S; wherein X2 is P, R or G; wherein X3 is K, F, R or N; wherein X4 is S, R or G; wherein X5 is T, S or N; and wherein X6 is M, I or L; (ii) CDR2, wherein CDR2 has the sequence X1IX2WX3X4X5X6TX7YX8DSVKG (SEQ ID NO: 4), wherein X1 is A or V; wherein X2 is N, W or S; wherein X3 is S or A; wherein X4 is G or N; wherein X5 is T or G; wherein X6 is I, L, S, T or V; wherein X7 is S, Q or N; and wherein X8 is S or T; and / or (iii) CDR3, which has the sequence AADX1DFRTVGSRPSY (SEQ ID NO: 5), wherein X1 is R, S or K; Furthermore, the CDR mentioned therein is defined based on the IMGT number.

16. The antigen-binding molecule according to claim 14 or 15, wherein the one or more V H The H domain contains (i) CDR1, wherein CDR1 has a sequence of APTRSTYAMG (SEQ ID NO: 6) or GRTFSSYAIG (SEQ ID NO: 7); (ii) CDR2, said CDR2 having the sequence VINWSGTLTSYADSVKG (SEQ ID NO: 8), VINWAGTLTSYADSVKG (SEQ ID NO: 9), VISWSGTLTSYADSVKG (SEQ ID NO: 10), AISWSGGTTQYTDSVKG (SEQ ID NO: 11), AISWSGGTTNYTDSVKG (SEQ ID NO: 12) or AISWSGGTTNYADSVKG (SEQ ID NO: 13); and / or (iii) CDR 3, which has the sequence AADRDFRTVGSRPSY (SEQ ID NO: 14), Furthermore, the CDR mentioned therein is defined based on the IMGT number.

17. The antigen-binding molecule according to any one of claims 14 to 16, wherein the one or more V H The H domain contains (i) CDR1, CDR2 and CDR3, wherein CDR1 has a sequence of APTRSTYAMG (SEQ ID NO: 6) or GRTFSSYAIG (SEQ ID NO: 7), CDR2 has a sequence of VINWSGTLTSYADSVKG (SEQ ID NO: 8), and CDR3 has a sequence of AADRDFRTVGSRPSY (SEQ ID NO: 14); (ii) CDR1, CDR2 and CDR3, wherein CDR1 has a sequence of APTRSTYAMG (SEQ ID NO: 6) or GRTFSSYAIG (SEQ ID NO: 7), CDR2 has a sequence of VINWAGTLTSYADSVKG (SEQ ID NO: 9), and CDR3 has a sequence of AADRDFRTVGSRPSY (SEQ ID NO: 14); (iii) CDR1, CDR2 and CDR3, wherein CDR1 has a sequence of APTRSTYAMG (SEQ ID NO: 6) or GRTFSSYAIG (SEQ ID NO: 7), CDR2 has a sequence of VISWSGTLTSYADSVKG (SEQ ID NO: 10), and CDR3 has a sequence of AADRDFRTVGSRPSY (SEQ ID NO: 14); (iv) CDR1, CDR2 and CDR3, wherein CDR1 has the sequence GRTFSSYAIG (SEQ ID NO: 7), CDR2 has the sequence AISWSGGTTQYTDSVKG (SEQ ID NO: 11), and CDR3 has the sequence AADRDFRTVGSRPSY (SEQ ID NO: 14); (v) CDR1, CDR2, and CDR3, wherein CDR1 has the sequence GRTFSSYAIG (SEQ ID NO: 7), CDR2 has the sequence AISWSGGTTNYTDSVKG (SEQ ID NO: 12), and CDR3 has the sequence AADRDFRTVGSRPSY (SEQ ID NO: 14); or (vi) CDR1, CDR2 and CDR3, wherein CDR1 has the sequence GRTFSSYAIG (SEQ ID NO: 7), CDR2 has the sequence AISWSGGTTNYADSVKG (SEQ ID NO: 13) and CDR3 has the sequence AADRDFRTVGSRPSY (SEQ ID NO: 14).

18. The antigen-binding molecule according to any one of claims 14 to 17, wherein the one or more V H The H domain contains the following sequence: (i) QVQLVESGGGLVQAGDSLSLSCVASAPTRSTYAMGWFRQAPGKEREFAAVINWSGTLTSYADSVKGRFTISRDNAKNTVFLQMNSLKPDDTAVYYCAADRDFRTVGSRPSYWGQGTPVTVSS (SEQ ID NO: 15); (ii) QVQLVESGGGLVEPGDSLRLSCAASGRTFSSYAIGWFRQAPGKEREFVAAISWSGGTTNYTDSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAADRDFRTVGSRPSYWSKGTRVTVSS (SEQ ID NO: 16); (iii) QVQLVESGGGLVQPGGSLRLSCSASAPTRSTYAMGWFRQAPGKEREFASVISWSGTLTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAADRDFRTVGSRPSYWGQGTTVTVSS (SEQ ID NO: 17); (iv) QVQLVESGGGLVQPGGSLRLSCSASGRTFSSYAIGWFRQAPGKEREFVSAISWSGGTTQYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAADRDFRTVGSRPSYWGQGTTVTVSS (SEQ ID NO: 18); Or (vi) A variant amino acid sequence having at least 85%, 90% or 95% sequence identity with any of SEQ ID NO: 15 to 19, wherein the variant is a modification of any of SEQ ID NO: 15 to 19 by substitution of one or more conserved amino acids.

19. The antigen-binding molecule according to any one of claims 1 to 18, which is used as a drug.

20. The antigen-binding molecule according to any one of claims 1 to 18, for treating a disease characterized by cFAP overexpression, preferably wherein the treatment is performed on a subject with a blood spFAP concentration greater than 40 ng / ml.

21. The antigen-binding molecule according to any one of claims 1 to 18, wherein it is a diagnostic agent used in vitro or in vivo for the following purposes. (i) Used for detecting cFAP; and / or (ii) Used to distinguish between cFAP and spFAP in serum, plasma, tissue or body fluids.

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