FAP-targeted antibody-drug conjugate

Abstract

The present invention relates to an antibody-drug conjugate (ADC) targeting antifibroblast activation protein α (FAP) for use in a method of treating FAP+ sarcoma in mammalian subjects, in particular, an anti-FAP antibody-cytolysin conjugate having the formula A-(L-D)p (wherein A is an anti-FAP antibody that selectively binds to FAP, L is a linker, D is a drug comprising a cytolysin, and p is from 1 to 10), or a pharmaceutically acceptable salt or solvate thereof. Also disclosed is an in vitro method for selecting sarcoma patients for treatment with an FAP-targeted ADC.

Description

Technical Field

[0001] This application claims priority from EP22382713.0, filed on Jul. 26, 2022, the content and elements of which are incorporated herein by reference for all purposes.

[0002] Field of the Invention The present invention relates to antibody-drug conjugates (ADCs) and immunotoxins targeting fibroblast activation protein alpha (FAP), and their use in the treatment of sarcoma. The present invention also relates to the use of FAP as a biomarker for selecting sarcoma patients to be treated with FAP-targeted ADCs.

Background Art

[0003] Background Sarcoma is a type of cancer that occurs in tissues such as bone or muscle. Osteosarcoma and soft tissue sarcoma are composed of a heterogeneous group of rare solid tumors of mesenchymal origin Sarcoma tumors mainly arise from the embryonic mesoderm and can affect patients of any age at any site in the body. Although the treatment of these tumors has advanced, metastasis and death remain major problems for high-risk soft tissue sarcoma patients (Cormier et al. (2004), Guedes et al (2021), Niederhuber et al (2014)).

[0004] These solid tumors often exhibit a prominent stromal reaction, such as the so-called "fibroblastic stroma" or "reactive stroma", which occupies 20 - 60% of the entire tumor mass and is characterized by the presence of a large number of stromal cells and a high-density extracellular matrix (ECM). Recent studies have shown the tumor-promoting role of stromal cells represented by vascular cells, immune cells, fibroblasts, myofibroblasts, adipocytes, and bone marrow-derived progenitor cells (Weinberg, et al. (2007), Nieman, et al. (2011), Joyce, et al. (2009), Hanahan, et al. (2012), Gupta, et al (2006), Valastyan, et al (2011)). In particular, a significant number of cancer-associated fibroblasts (CAFs) are frequently observed in the tumor-associated stroma of various human cancers, such as breast cancer, lung cancer, colorectal cancer, and pancreatic cancer tumors (Kalluri, et al. (2006), Pietras, et al. (2010)). CAFs, which interact cooperatively with various components of the stroma, have the ability to promote angiogenesis and tumor growth. CAFs have also been shown to be extremely important in the development of aggressive tumors and tumor invasiveness during the progression of cancer (Orimo, et al. (2005), Erez, et al. (2010), Olumi, et al. (1999), Yang, et al. (2006), Hwang, et al. (2008), Hu, et al. (2009), Medema, et al. (2011), Malanchi, et al. (2012), Strell, et al. (2012), Horimoto, et al. (2012)). CAFs promote the spread and invasion of tumor cells to distant organs and contribute to the formation of metastases. Importantly, it has also been shown that stromal cells are involved in the failure of systemic drug delivery to tumors and the development of drug resistance (Meads, et al. (2009), Olive, et al. (2009), Acharyya, et al. (2012), Crawford, et al. (2009), Straussman, et al (2012)).

[0005] Leiomyosarcoma (LMS) is a malignant mesenchymal tumor derived from smooth muscle. It is one of the most common subtypes of sarcoma, accounting for up to 25% of all soft tissue sarcomas (Kannan (2022)). LMS has a tendency to metastasize via hematogenous spread. Since smooth muscle is present in many areas of the body, leiomyosarcoma can occur in a wide range of tissues such as the uterus (30% of all LMS), stomach, small intestine, and retroperitoneum (35% of all LMS). Some studies have pointed out that uterine LMS and extrauterine LMS exhibit different disease biologies.

[0006] Rhabdomyosarcoma is another malignant mesenchymal tumor. It is the most common among pediatric soft tissue sarcomas, accounting for up to 50% of all pediatric soft tissue sarcomas and 3% of all pediatric tumors (Amer (2019)). It is divided into six histological groups: fetal, alveolar, spindle cell, mixed, pleomorphic, and rhabdomyosarcoma with ganglionic differentiation.

[0007] Undifferentiated pleomorphic sarcoma (UPS) is a term referring to sarcomas that are not classified as differentiated. This subtype is composed of undifferentiated tumor cells that can appear as spindle cells, histiocytes, and giant cells.

[0008] Monoclonal antibody (MAb)-based drugs hold great promise in the fight against cancer. This is because they can precisely and specifically target the treatment at the molecular level. These advantages, along with commercial attractiveness (short development period, limited capabilities, easily exportable to other cancer types after approval), encourage many pharmaceutical companies to invest heavily in the development of new antibody-based molecules and the introduction of new molecules or technologies from biotechnology companies.

[0009] However, despite the clinical success of therapeutic antibodies, naked MAbs targeting cell surface tumor antigens rarely exhibit sufficient efficacy on their own. To enhance the low activity of MAbs, new strategies have focused on conjugating them to toxic molecules. Plant and bacterial toxins, as well as chemotherapeutic small molecules, can be good candidates because they are very potent and active at very low doses.

[0010] In the fields of immunotoxins (ITs) and antibody-drug conjugates (ADCs) for cancer treatment, recent technological advancements have led to increased development activities by pharmaceutical companies, aiming to address the initial issues regarding immunogenicity, unwanted toxicity, production, half-life, and resistance.

[0011] Immunoconjugates are human antibodies, humanized antibodies, or chimeric recombinant antibodies covalently linked to cytotoxic drugs. The main purpose of such constructs is to combine the potency of small cytotoxic agents (300 to 1000 Da) with the high specificity of MAbs targeting tumor-associated antigens (TAAs).

[0012] The Ab must be highly selective in reaching the antigen, and its expression should be limited to cancer cells. Additionally, the Ab must be efficiently internalized by cancer cells.

[0013] The cytotoxic agent selected as the effector moiety must kill cells only after internalizing into the cytoplasm and being released. The most commonly used payloads in ADCs are DNA-damaging agents such as calicheamicin and duocarmycin, or microtubule-targeting compounds such as auristatin and maytansinoid.

[0014] The Ab-cytotoxic linker is designed to be stable systemically and release the cytotoxic agent inside the target cell.

[0015] TAA is often a cell membrane protein that is overexpressed in diseased tissues or shows at least sufficient expression to promote internal translocation-activated cytotoxicity. Ideally, the antigen is expressed limitedly in normal tissues and shows low or no expression in vital organs. In addition to this, the tumor antigen must be recognized selectively and with high affinity by Ab.

[0016] In many types of human cancers, the fibroblast reaction is characterized by the induction of fibroblast activation protein α (FAPα), a cell surface protein that is a 95 kDa serine protease and whose expression is highly limited to the developing organs, wound healing, and tissue remodeling. FAPα is a well-studied type II membrane glycoprotein serine protease that is upregulated not only in cancer-associated fibroblasts (CAFs) but also in wound healing fibroblasts (Ramirez-Montagut et al. (2004)). In some tumor tissues, FAP expression is associated with tumor growth, invasiveness, angiogenesis, epithelial-mesenchymal transition (EMT), immunosuppression, and drug resistance (Xin et al. (2021)). FAP presents the following characteristics: · A type II membrane glycoprotein with SER protease activity (collagenase + DPP) · 89% human-mouse protein homology · Expressed in the tumor stroma in more than 90% of carcinomas (breast, pancreas, lung, bladder, and colon) · Temporally and highly limitedly expressed in normal adult tissues during wound healing and organ development. · FAP is expressed in tumor cells of various sarcomas such as leiomyosarcoma and rhabdomyosarcoma. · FAP(+) fibroblasts are located near the tumor vasculature · Very local expression · Internal translocation · Involved in extracellular matrix remodeling, tumor growth, and metastasis.

[0017] The expression of FAP has been found not only in pancreatic tumor cells but also in tumor-associated stromal fibroblasts. The expression of FAP correlates with shortened patient survival and worse prognosis, suggesting the possibility of the existence of an FAP-based autocrine / paracrine loop in this type of tumor (Shi, et al. (2012)).

[0018] In previous studies by Kontermann and Pfizenmaier (IZI, University of Stuttgart, Germany), anti-FAP MAb derivatives against both human and mouse proteins were developed (Mersmann, et al (2001), Brocks, et al. (2001)). They demonstrated in vitro that anti-FAP scFv immunoliposomes specifically bind to and internalize into FAP+ cells (Schmidt, et al. (2001)). They demonstrated the antitumor effect of nanoparticles coated with lipids and anti-FAP scFv and loaded with TNFα (Messerschmidt, et al. (2009)).

[0019] Treatment with the mouse MAb FAP5-DM1 immunotoxin induced long-term inhibition of tumor growth and complete regression without intolerance-related effects in pancreatic and lung cancer xenograft models (Ostermann, et al. (2008)).

[0020] The FAP protein has been widely validated as an interesting target for anticancer therapy in solid tumors and has thus been the subject of various CAF-targeting approaches (Kakarla et al (2012), Simkova et al.(2020)). In this context, OMTX705, a novel FAP(+)CAF-targeting antibody-drug conjugate, was developed as an alternative stromal-targeting therapeutic compound with potent antitumor effects. Preclinical studies in patient-derived xenograft mouse models of pancreatic cancer, NSCLC, triple-negative breast cancer, and diffuse gastric cancer have shown that FAP expression in these models is associated with the antitumor effect of OMTX705 treatment (Fabre et al. (2020)).

[0021] FAP, initially isolated from the reactive stroma of human sarcomas (Rettig et al. (1994), Rettig et al. (1988)), was found to be expressed also in tumor cells of osteosarcoma and subsets of soft tissue sarcomas in relation to mesenchymal origin (Rettig et al. (1993)). In recent studies on FAP protein as a cancer biomarker for sarcoma, it has been demonstrated that there is some correlation between FAP and poor prognosis of osteosarcoma (Zhang et al. (2019)). Furthermore, some 68 With the development of low molecular weight Ga-radiolabeled FAP inhibitors, studies have emerged to evaluate their clinical role in cancer imaging (Kuyumcu et al. (2021)). In various types of primary and metastatic tumors 68 Quantification studies of tumor uptake by Ga-FAPI PET / CT in sarcoma patients have shown that 68 sarcoma patients are included in the group with the highest Ga-FAPI ligand uptake. These high ratios have resulted in high image contrast and excellent tumor delineation in osteosarcoma and soft tissue sarcoma patients (Kratochwil et al. (2019), Koerber et al. (2021), Kessler et al. (2022)). Furthermore, FAP-targeted peptides are 17 effective for radionuclide therapy and imaging for various cancers such as Lu-FAP-2286 ( 68 Ga-FAP-2286, etc.) (Kwan et al. (2021)).

[0022] Despite such progress, there remain unmet needs for additional treatment strategies for sarcoma treatment such as leiomyosarcoma, rhabdomyosarcoma and UPS, and for the components used in such treatment strategies. The present invention addresses these and other needs. SUMMARY OF THE INVENTION

[0023] Generally, the present invention relates to anti-FAP antibodies, conjugates thereof, and optimized payloads for use in antibody conjugate strategies. In particular, the inventors have discovered that the anti-FAP antibodies described herein exhibit highly specific binding and rapid and efficient internalization. The present invention provides anti-FAP antibody-cytolysin conjugates of the type disclosed in WO 2015 / 118030 (which is hereby incorporated by reference in its entirety) for use in a method of treating sarcoma in a mammalian subject.

[0024] The cytolysin derivatives described herein are advantageously conjugated to anti-FAP antibodies for use in the treatment of sarcoma, which is characterized by FAP expression in tumors. Thus, the use of FAP-targeted ADCs is extended beyond tumors that express FAP in the stroma.

[0025] Accordingly, in a first aspect, the present invention provides a formula I for use in a method of treating a sarcoma (the sarcoma being characterized by FAP expression in a tumor) in a mammalian subject: A-(L-D) p (I) (wherein A is an anti-FAP antibody that selectively binds to FAP, L is a linker, D is a drug comprising a cytolysin, and p is from 1 to 10), an anti-fibroblast activation protein α (FAP) antibody-cytolysin conjugate or a pharmaceutically acceptable salt or solvate thereof.

[0026] In some cases, according to this and other aspects of the present invention, the anti-FAP antibody is a monoclonal antibody or a binding fragment thereof that selectively binds to the extracellular region of human FAP and / or mouse FAP. In some cases, the anti-FAP antibody may cross-react with both human FAP and mouse FAP.

[0027] In a particular case, the anti-FAP antibody has the following amino acid sequences: (i) CDRH1: SEQ ID NO: 7; (ii) CDRH2: SEQ ID NO: 8; (iii) CDRH3: SEQ ID NO: 9; (iv) CDRL1: SEQ ID NO: 10; (v) CDRL2: SEQ ID NO: 11; and (vi) CDRL3: SEQ ID NO: 12 may include heavy chain complementarity determining regions 1-3 (CDRH1-3) and light chain complementarity determining regions 1-3 (CDRL1-3) having

[0028] In some embodiments, the CDRH1 region may include a variant of SEQ ID NO: 7 having 1 or 2 amino acid substitutions compared to the sequence of SEQ ID NO: 7. In some embodiments, the CDRH2 region may include a variant of SEQ ID NO: 8 having 1 or 2 amino acid substitutions compared to the sequence of SEQ ID NO: 8. In some embodiments, the CDRH3 region may include a variant of SEQ ID NO: 9 having 1 or 2 amino acid substitutions compared to the sequence of SEQ ID NO: 9. In some embodiments, the CDRL1 region may include a variant of SEQ ID NO: 10 having 1 or 2 amino acid substitutions compared to the sequence of SEQ ID NO: 10. In some embodiments, the CDRL2 region may include a variant of SEQ ID NO: 11 having 1 or 2 amino acid substitutions compared to the sequence of SEQ ID NO: 11. In some embodiments, the CDRL3 region may include a variant of SEQ ID NO: 12 having 1 or 2 amino acid substitutions compared to the sequence of SEQ ID NO: 12.

[0029] In certain cases, CDRH1-3 each include the amino acid sequences of SEQ ID NOs: 7-9, and CDRL1-3 each include the amino acid sequences of SEQ ID NOs: 10-12.

[0030] In certain cases, the anti-FAP antibody includes a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 5 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the heavy chain variable region (VH) has at least 90%, 95% or 99% sequence identity with the full-length sequence of SEQ ID NO: 5. In some cases, the light chain variable region (VL) has at least 90%, 95% or 99% sequence identity with the full-length sequence of SEQ ID NO: 6.

[0031] In certain cases, the anti-FAP antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2. In some cases, the heavy chain has at least 90%, 95%, or 99% sequence identity with the full-length sequence of SEQ ID NO: 1. In some cases, the light chain has at least 90%, 95%, or 99% sequence identity with the full-length sequence of SEQ ID NO: 2.

[0032] In certain cases, the anti-FAP antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO: 4. In some cases, the heavy chain has at least 90%, 95%, or 99% sequence identity with the full-length sequence of SEQ ID NO: 3. In some cases, the light chain has at least 90%, 95%, or 99% sequence identity with the full-length sequence of SEQ ID NO: 4.

[0033] In certain cases, A can be a competing anti-FAP antibody that is structurally different from the anti-FAP antibody molecules exemplified herein. For example, A can be an anti-FAP antibody molecule that competes with the anti-FAP IgG1 antibody identified herein as "hu36" with respect to binding to immobilized recombinant human FAP. hu36 has the heavy chain amino acid sequence of SEQ ID NO: 3 and the light chain amino acid sequence of SEQ ID NO: 4. The anti-FAP antibody can, in some cases, bind to the same epitope as hu36. Methods for determining antibody binding competition and methods of epitope mapping are well known in the art. See, for example, "Epitope Mapping by Competition Assay" Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi:10.1101 / pdb.prot4277.

[0034] According to this and other aspects of the present invention, D can be a cytolysin. The cytolysin can, in some cases, be a compound disclosed in WO 2008 / 138561A1, the entire content of this publication being hereby expressly incorporated by reference herein (the compounds disclosed in the publication are also referred to as tubulysin derivatives). The cytolysin may be synthesized as described in WO 2008 / 138561. In certain cases, the cytolysin can be as defined by formula I or formula IV of WO 2008 / 138561A1. In certain cases, the cytolysin is of formula IV:

[0035]

Chemical formula

[0036] (wherein: R 2 is H or C1-C4 alkyl; R 6 is C1-C6 alkyl; R 7 is C1-C6 alkyl, CH2OR 19 or CH2OCOR 20 (wherein R 19 is alkyl, R 20 is C2-C6-alkenyl, phenyl or CH2-phenyl); R 9 is C1-C6 alkyl; R 10 is H, OH, O-alkyl or O-acetyl; f is 1 or 2; R 11 has the following structure:

[0037]

Chemical formula

[0038] (wherein, R 21is H, OH, halogen, NH2, alkyloxy, phenyl, alkylamino or dialkylamino; R 16 is H or a C1-C6-alkyl group; R 17 is connected directly or indirectly to the linker L; and q is 0, 1, 2 or 3; Also, the term "optionally substituted" relates to a group in which one or several H atoms may be replaced by F, Cl, Br or I or OH, SH, NH2 or NO2; also, the term "optionally substituted" relates to a group which may be exclusively or additionally substituted by an unsubstituted C1-C6 alkyl, C 2- C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C 10 cycloalkyl, C2-C9 heterocycloalkyl, C6-C 10 aryl, C1-C9 heteroaryl, C7-C 12 aralkyl or C2-C 11 heteroaralkyl group).

[0039] In some cases, R 2 is a bond to the linker L.

[0040] In some cases, R 17 is C(O)X, CONHNHX, OX, NHX or SX, where X is a bond to the linker L).

[0041] In some embodiments, the linker L comprises a spacer. In some cases, the spacer has a chain length of from 2 to 30 atoms. In some cases, the spacer comprises or consists of an alkylene (i.e. divalent alkyl) or heteroalkylene (i.e. divalent heteroalkyl) group. In some cases, the spacer comprises or consists of an alkylene or oxyalkylene group.

[0042] In some embodiments, the spacer is -(OCH2CH2)n -(wherein n is from 2 to 5). In some cases, the spacer is a group -(CH2) n - or -(OCH2CH2) n -(wherein n ≥ 1) or consists of them. In some cases, the spacer is the group -(OCH2CH2) n -(wherein n ≥ 1) or consists of them. In particular, n can be from 1 to 15, from 1 to 10, from 1 to 6, or from 2 to 5. For example, n can be 3 or 4. In some cases, the spacer contains between 1 and 6 ethylene glycol units, for example, triethylene glycol. In some cases, the spacer is directly connected to the group R 17 or may be connected to the group R via a crosslinking group 17 In some cases, the spacer is -C(O)X (where X is the bond with R 17 ) and is connected to the group R via a crosslinking group 17 In some cases, R 17 is CONHNHX, the spacer is connected to the group R via a -C(O)X crosslinking group 17 and X represents the bond between the spacer and R 17 In some cases, R 17 is CONHNHX, the spacer is -(OCH2CH2) connected to R via a -C(O)X crosslinking group 17 -(wherein n = 2, 3 or 4). n -(wherein n = 2, 3 or 4).

[0043] In some embodiments, L contains a linking group for connection to A.

[0044] In some embodiments, L contains a protease-cleavable moiety that includes a valine-citrulline unit. For example, L can include maleimidocaproyl-valine-citrulline-p-aminobenzylcarbamate.

[0045] In some cases, the cytolysin has the following structure:

[0046] [Chemical formula]

[0047] It has (wherein, * indicates the connection site to L).

[0048] In some cases, D is the following structure:

[0049] [Chemical formula]

[0050] It contains a cytolysin having the following structure:

[0051] In some cases, the double bond of maleimide reacts with the thiol group of the cysteine residue of antibody A to form a sulfur-carbon bond in order to link linker L to antibody A.

[0052] In some embodiments, -L-D has the structure:

[0053] [Chemical formula]

[0054] It has the following structure:

[0055] In other cases, -L-D is:

[0056] [Chemical formula]

[0057] [Chemical formula]

[0058] [Chemical formula]

[0059] [Chemical formula]

[0060] and

[0061] [Chemical formula]

[0062] has a structure selected from the group consisting of.

[0063] In certain cases, -L-D has the following structure:

[0064] [Chemical formula]

[0065] may have.

[0066] In certain cases, -L-D has the following structure:

[0067] [Chemical formula]

[0068] may have.

[0069] According to this and other aspects of the present invention, p can, in some cases, be in the range of 1 to 5, for example, 1 to 4, or 1 to 3. In special cases, p can be 1 or 2. In special cases, p can be 3 or 4.

[0070] In some embodiments, A has a heavy chain having the amino acid sequence of SEQ ID NO: 3 and a light chain having the amino acid sequence of SEQ ID NO: 4; L-D has the structure:

[0071] [Chemical formula]

[0072] (wherein, * indicates the connection point to A).

[0073] In some embodiments, the complex is the complex described herein as OMTX705. OMTX705 comprises a humanized anti-FAP mAb (OMTX005) conjugated to cytolysin TAM470 via a protease-cleavable vcPABA-(EG)3 optimized linker. OMTX705 is described in Fabre et al. (2020), and FIG. 7 is an exemplary depiction of OMTX705.

[0074] In some embodiments, the sarcoma is a FAP-expressing (FAP+) leiomyosarcoma. In certain cases, the complex is a complex for use in the treatment of leiomyosarcoma selected from the group consisting of uterine leiomyosarcoma, cutaneous leiomyosarcoma, gastrointestinal leiomyosarcoma, gastric leiomyosarcoma, small intestinal leiomyosarcoma, retroperitoneal leiomyosarcoma, or peritoneal leiomyosarcoma.

[0075] In some embodiments, the sarcoma is a FAP+ rhabdomyosarcoma. In some embodiments, the complex is a complex for use in the treatment of rhabdomyosarcoma selected from the group consisting of embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, spindle cell rhabdomyosarcoma, mixed rhabdomyosarcoma, pleomorphic rhabdomyosarcoma, and rhabdomyosarcoma with ganglionic differentiation.

[0076] In some embodiments, the sarcoma is FAP+ undifferentiated pleomorphic sarcoma (UPS).

[0077] In some embodiments, the complex is administered intravenously.

[0078] In some embodiments, the complex is administered weekly. In some embodiments, the complex is administered every two weeks. In some embodiments, the complex is administered monthly.

[0079] In some embodiments, the conjugate is administered in a dosage range of 0.1 to 30 mg / kg. In some embodiments, the conjugate is administered in a dosage range of 10 to 30 mg / kg. In some embodiments, the conjugate is administered at 10 mg / kg. In some embodiments, the conjugate is administered at 20 mg / kg. The conjugate is administered at 30 mg / kg. [[ID=,2]]

[0080] In some embodiments, the conjugate is administered in 4 divided doses.

[0081] In some cases, the conjugate for use in a method of treating sarcoma is for co - administration, sequential administration or separate administration with one or more other anti - tumor agents. The one or more other anti - tumor agents include cytotoxic chemotherapeutic agents, anti - angiogenic agents or immunotherapeutic agents. In some cases, the one or more other anti - tumor agents include gemcitabine, abraxane, bevacizumab, itraconazole, carboxyamidotriazole, anti - PD - 1 molecules or anti - PD - L1 molecules (e.g., nivolumab or pembrolizumab).

[0082] In a second aspect, the present invention provides an in vitro method of selecting a subject determined to have sarcoma for treatment with an FAP - targeted ADC, the method comprising measuring the FAP expression level in a biological sample taken from the subject; determining that the FAP expression level exceeds a threshold level, and selecting the subject for treatment with an FAP - targeted ADC. In some embodiments, the sarcoma is leiomyosarcoma. In some embodiments, the sarcoma is rhabdomyosarcoma. In some embodiments, the sarcoma is UPS. In some embodiments, the anti - FAP ADC has the formula A-(L - D)p, where A is an anti - FAP antibody having a heavy chain with the amino acid sequence of SEQ ID NO: 3 and a light chain with the amino acid sequence of SEQ ID NO: 4; and L - D has the structure:

[0083]

Chemical formula

[0084] (where * indicates the connection point to A) and p is from 1 to 10).

[0085] In some embodiments, the FAP-targeted ADC is OMTX705. In some embodiments, the subject is a mammalian subject. In some embodiments, the subject is a human. In some embodiments, the biological sample is taken from a tumor site. In some embodiments, the biological sample is a nucleic acid-containing cell-free sample, such as a plasma sample.

[0086] In a third aspect, the invention provides a method of treating a FAP+ sarcoma in a mammalian subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a conjugate as defined according to the first aspect of the invention. In some embodiments, the method is for treating FAP+ leiomyosarcoma. In some cases, the method may be for treating uterine leiomyosarcoma, cutaneous leiomyosarcoma, gastrointestinal leiomyosarcoma, gastric leiomyosarcoma, small intestine leiomyosarcoma, retroperitoneal leiomyosarcoma or peritoneal leiomyosarcoma. In some embodiments, the method is for treating FAP+ rhabdomyosarcoma. In some embodiments, the method may be for treating a rhabdomyosarcoma selected from the group consisting of: fetal rhabdomyosarcoma, alveolar rhabdomyosarcoma, spindle cell rhabdomyosarcoma, mixed rhabdomyosarcoma, pleomorphic rhabdomyosarcoma and rhabdomyosarcoma with ganglionic differentiation. In some embodiments, the method is for treating FAP+ UPS.

[0087] In a fourth aspect, the present invention provides the use of an anti-fibroblast activation protein α (FAP) antibody-cytolysin conjugate, or a pharmaceutically acceptable salt or solvate thereof, in the preparation of a medicament for the treatment of FAP+ sarcoma, wherein the antibody-cytolysin conjugate has the formula A-(L-D)p, where A is an anti-FAP antibody that selectively binds to FAP, L is a linker, D is a drug comprising a cytolysin, and p is from 1 to 10. In some embodiments, the sarcoma is FAP+ leiomyosarcoma. In some embodiments, the sarcoma is FAP+ rhabdomyosarcoma. In some embodiments, the sarcoma is FAP+ UPS.

[0088] Preferred features of the antibody-cytolysin conjugate or sarcoma as described in the first and second aspects are equally applicable to the third and fourth aspects.

[0089] The present invention includes combinations of the described aspects and preferred features, unless such combinations are clearly not permitted or are explicitly avoided. These and further aspects and embodiments of the present invention are described in more detail below with reference to the accompanying examples and drawings.

[0090] Sequence In the following sequences, the VH and VL domains are underlined, and the CDRH / CDRL regions are in bold. Mutations leading to ADCC and CDC deficiencies are shown in bold italics. The signal sequence (where applicable) is boxed. hu36 IgG1-HC with signal sequence

[0091]

Number

[0092] hu36-IgG1-LC with signal sequence:

[0093]

Number

[0094] hu36-IgG1-HC without signal sequence:

[0095]

Number

[0096] hu36-IgG1-LC without signal sequence:

[0097]

Number

[0098] hu36-VH:

[0099]

Number

[0100] hu36-VL:

[0101]

Number

[0102] hu36-CDRH1: ENIIH (Sequence Number 7)

[0103] hu36-CDRH2: WFHPGSGSIKYNEKFKD (Sequence Number 8)

[0104] hu36-CDRH3: HGGTGRGAMDY (Sequence Number 9)

[0105] hu36-CDRL1: RASKSVSTSAYSYMH (Sequence Number 10)

[0106] hu36-CDRL2: LASNLES (Sequence Number 11)

[0107] hu36-CDRL3: QHSRELPYT (SEQ ID NO: 12)

[0108] Human FAP It is also known as separase, 170 kDa melanoma membrane-bound gelatinase, fibroblast activation protein alpha or transmembrane serine protease. The amino acid sequence is disclosed by UniProt accession number Q12884 (Version 140, dated 11 December 2013): MKTWVKIVFGVATSAVLALLVMCIVLRPSRVHNSEENTMRALTLKDILNGTFSYKTFFPNWISGQEYLHQSADNNIVLYNIETGQSYTILSNRTMKSVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLATKYALWWSPNGKFLAYAEFNDTDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPVVRIFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAINIFRVTQDSLFYSSNEFEEYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSD (SEQ ID NO: 13)

[0109] Mouse FAP It is also known as fibroblast activation protein alpha or transmembrane serine protease. The amino acid sequence is disclosed in UniProt accession number P97321 (Version 117, dated 11 December 2013): MKTWLKTVFGVTTLAALALVVICIVLRPSRVYKPEGNTKRALTLKDILNGTFSYKTYFPNWISEQEYLHQSEDDNIVFYNIETRESYIILSNSTMKSVNATDYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLQNGEFVRGYELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITYTGRENRIFNGIPDWVYEEEMLATKYALWWSPDGKFLAYVEFNDSDIPIIAYSYYGDGQYPRTINIPYPKAGAKNPVVRVFIVDTTYPHHVGPMEVPVPEMIASSDYYFSWLTWVSSERVCLQWLKRVQNVSVLSICDFREDWHAWECPKNQEHVEESRTGWAGGFFVSTPAFSQDATSYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAIYIFRVTQDSLFYSSNEFEGYPGRRNIYRISIGNSPPSKKCVTCHLRKERCQYYTASFSYKAKYYALVCYGPGLPISTLHDGRTDQEIQVLEENKELENSLRNIQLPKVEIKKLKDGGLTFWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVKSVFAVNWITYLASKEGIVIALVDGRGTAFQGDKFLHAVYRKLGVYEVEDQLTAVRKFIEMGFIDEERIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASIYSERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGISSGRSQNHLYTHMTHFLKQCFSLSD (SEQ ID NO: 14)

[0110] Summary of the Drawings Next, embodiments and experiments demonstrating the principles of the present invention will be described with reference to the accompanying drawings:

Brief Description of the Drawings

[0111]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

[0112] Detailed Description of the Invention Next, aspects and embodiments of the present invention will be described with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in the text are hereby incorporated by reference into this specification.

[0113] In describing the present invention, the following terms are employed and are intended to be defined as set forth below.

[0114] FAP As used herein, the terms "Fibroblast activation protein", "fibroblast activating protein", "FAP" and "FAPα" are used interchangeably. FAP can be FAP from any mammalian species. In some cases, FAP is human FAP (also known as seprase, a 170 kDa melanoma membrane-bound gelatinase, fibroblast activation protein alpha or transmembrane serine protease), the amino acid sequence of which is disclosed by UniProt accession number Q12884 (Version 140, dated 11 December 2013) (SEQ ID NO: 13). In some cases, a molecule that binds to FAP (e.g., an antibody molecule or a complex thereof) can bind to the extracellular domain region of FAP. The extracellular domain of human FAP includes residues 26-760 of the full-length human FAP protein. In some cases, FAP is mouse FAP (also known as fibroblast activation protein alpha or transmembrane serine protease), the amino acid sequence of which is disclosed by UniProt accession number P97321 (Version 117, dated 11 December 2013) (SEQ ID NO: 14). The extracellular domain of mouse FAP includes residues 26-761 of the full-length mouse FAP protein.

[0115] Complex As used herein, "complex" includes the resultant structure formed by a linking molecule, specifically including antibody-drug conjugates (ADCs) and immunotoxins (ITs).

[0116] Bind selectively The terms selectively bind and selective binding refer to the binding of an antibody or a binding fragment thereof to a given molecule (e.g., an antigen) in a particular manner. For example, an antibody or a binding fragment thereof binds to at least about 1×10 7 M -1With an affinity that can bind to a FAP, for example, its extracellular portion, and can bind to a predetermined molecule with an affinity that is at least 2-fold higher (e.g., 5-fold or 10-fold higher) than the affinity for binding to a molecule other than the predetermined molecule.

[0117] Antibody molecule As used herein in connection with all aspects of the present invention, the terms "antibody" or "antibody molecule" include any immunoglobulin produced naturally or by partial or complete synthesis. The terms "antibody" or "antibody molecule" include monoclonal antibodies (mAbs) and polyclonal antibodies (including polyclonal antiserum). An antibody can be intact or a fragment derived from a complete antibody (see below). An antibody can be a human antibody, a humanized antibody or an antibody of non-human origin. A "monoclonal antibody" is a homogeneous, highly specific population of antibodies directed against a single antigenic site or "epitope" of a target molecule. A "polyclonal antibody" includes a heterogeneous population of antibodies directed against different antigenic epitopes of a target molecule. The terms "antiserum" or "antisera" refer to serum containing antibodies obtained from immunized animals.

[0118] Fragments of whole antibodies have been shown to be able to perform the function of binding to an antigen. Thus, references herein to antibodies, with reference to the methods, arrays and kits of the present invention, include intact antibodies and any polypeptide or protein that includes an antibody-binding fragment. Examples of binding fragments include: (i) a Fab fragment consisting of a V L , V H , C L and C H domain; (ii) an Fd fragment consisting of a V H and C H domain; (iii) an Fv fragment consisting of the V L and V H domains of a single antibody; (iv) a V HdAb fragments consisting of domains; (v) isolated CDR regions; (vi) F(ab’)2 fragments which are bivalent fragments comprising two linked Fab fragments; (vii) V H domains and V L domains are linked by a peptide linker that enables the two domains to associate to form an antigen-binding site, a single-chain Fv molecule (scFv); (viii) bispecific single-chain Fv dimers (WO 93 / 11161) and (ix) “diabodies”, multivalent or multispecific fragments constructed by gene fusion (WO94 / 13804; 58). Fv, scFv or diabody molecules can be stabilized by incorporation of disulfide bridges that link the VH and VL domains. Mini-bodies containing scFv linked to a CH3 domain can also be prepared.

[0119] In the context of antibody molecules, the term “selectively binds” can be used herein to refer to a situation where one member of a specific binding pair does not exhibit any significant binding to molecules other than its specific binding partner. This term is applicable, for example, when an antigen-binding site is specific for a particular epitope carried by several antigens, in which case the specific binding member carrying the antigen-binding site can bind to the various antigens carrying the epitope.

[0120] In some cases according to the present invention, the antibody can be a fully human antibody.

[0121] Cytotoxic chemotherapeutic agent In some cases according to any aspect of the present invention, the conjugate of the present invention can be administered with, or for administration (simultaneously, sequentially or separately) with, one or more other anti-tumor agents such as, but not limited to, cytotoxic chemotherapeutic agents or anti-angiogenic agents or immunotherapeutic agents.

[0122] Cytotoxic chemotherapeutic agents are well known in the art and include the following anti-cancer agents: Examples of alkylating agents include nitrogen mustards such as mechlorethamine (HN2), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin), and chlorambucil; ten ethyleneimines and methylmelamines such as hexamethylmelamine and thiotepa; alkyl sulfonates such as busulfan; nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), and streptozocin (streptozotocin); and triazenes such as dacarbazine (DTIC; dimethyltriazenoimidazole carboxamide). As metabolic antagonists, there are folic acid analogs such as methotrexate (amethopterin); pyrimidine analogs such as fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR) and cytarabine (cytosine arabinoside); as well as purine analogs and related inhibitors such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG) and pentostatin (2'-deoxycoformycin). As natural products, there are vinca alkaloids such as vinblastine (VLB) and vincristine; epipodophyllotoxins such as etoposide and teniposide; antibiotics such as dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin) and mitomycin (mitomycin Q); enzymes such as L-asparaginase; and biological response modifiers such as interferon alphenome. As various drugs, there are platinum coordination complexes such as cisplatin (cis-DDP) and carboplatin; anthracenediones such as mitoxantrone and anthracycline; substituted ureas such as hydroxyurea; methylhydrazine derivatives such as procarbazine (N-methylhydrazine, MIH); as well as adrenal cortex inhibitors such as mitotane (o,p'-DDD) and aminoglutethimide; taxol and analogs / derivatives; and hormone agonists / antagonists such as flutamide and tamoxifen. More preferred cytotoxic agents are gemcitabine (Gemzar®). More preferred cytotoxic agent is paclitaxel bound to human serum albumin (Abraxane®).

[0123] Angiogenesis inhibitors are well-known in the art and include anticancer agents such as bevacizumab, itraconazole, and carboxyamidotriazole.

[0124] Immunotherapeutic agents are known in the art and include, for example, anti-programmed cell death protein 1 (PD-1) antibodies and anti-programmed death ligand 1 (PD-L1) antibodies such as nivolumab (MDX1106) and pembrolizumab (MK-3475).

[0125] Pharmaceutical composition The complex of the present invention may be included in a pharmaceutical composition together with a pharmaceutically acceptable excipient.

[0126] Pharmaceutically acceptable excipients are compounds or combinations of compounds that do not induce side reactions and, for example, enable the promotion of the administration of the complex, the increase of its duration of survival and / or effectiveness in the body, or the increase of its solubility in solution. These pharmaceutically acceptable vehicles are well known and are adapted by those skilled in the art according to the mode of administration of the complex.

[0127] In some embodiments, the complex of the present invention may be provided in lyophilized form for reconstitution prior to administration. For example, the lyophilized complex may be reconstituted with sterile water and mixed with physiological saline prior to administration to an individual.

[0128] The complex of the present invention is usually administered in the form of a pharmaceutical composition that may contain at least one component in addition to the complex. Thus, the pharmaceutical composition may contain, in addition to the complex, pharmaceutically acceptable excipients, carriers, buffers, stabilizers or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the effectiveness of the complex. The exact nature of the carrier or other material depends on the route of administration, which can be, as described below, bolus, infusion, injection or any other suitable route.

[0129] For example, in the case of intravenous administration by injection, the pharmaceutical composition containing the complex can be in the form of a parenterally acceptable aqueous solution having pyrogen - free, appropriate pH, isotonicity, and stability. Those skilled in the art can adequately prepare an appropriate solution using an isotonic vehicle such as, for example, sodium chloride injection solution, Ringer's injection solution, lactated Ringer's injection solution, etc. If necessary, buffers such as phosphates, citrates, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3'-pentanol; and m - cresol, etc.); low - molecular - weight polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; saccharides such as sucrose, mannitol, trehalose or sorbitol; salt - forming counterions such as sodium; metal complexes (e.g., zinc - protein complexes); and / or preservatives, stabilizers, buffers, antioxidants and / or other additives including non - ionic surfactants such as TWEEN™, Pluronic™ or polyethylene glycol (PEG) may be used.

[0130] Subject The subject can be a human, a companion animal (e.g., a dog or a cat), a laboratory animal (e.g., a mouse, a rat, a rabbit, a pig or a non-human primate), a livestock or a farm animal (e.g., a pig, a cow, a horse or a sheep). Preferably, the subject is a human. In some cases, the subject can be a human diagnosed with cancer, e.g., an epithelial tumor, or classified as having a risk of developing it. In certain cases, the subject can be a laboratory animal, e.g., a mouse model of cancer. In certain cases, the subject can be a mammal (e.g., a human) diagnosed with an inflammatory condition such as rheumatoid arthritis (RA) or classified as having a risk of developing it. In particular, the subject can be a human having RA.

[0131] Cancer The anti-FAP complex described herein is found to be useful in the treatment of tumors in mammalian subjects. The tumor can be a solid tumor. The tumor can be a FAP+ sarcoma. The FAP+ sarcoma can be a leiomyosarcoma. In particular, the tumor can be a uterine leiomyosarcoma, a cutaneous leiomyosarcoma, a gastrointestinal leiomyosarcoma, a gastric leiomyosarcoma, a small intestinal leiomyosarcoma, a retroperitoneal leiomyosarcoma or an abdominal leiomyosarcoma. The FAP+ sarcoma can be a rhabdomyosarcoma. In particular, the rhabdomyosarcoma can be a fetal rhabdomyosarcoma, an alveolar rhabdomyosarcoma, a spindle cell rhabdomyosarcoma, a mixed rhabdomyosarcoma, a pleomorphic rhabdomyosarcoma and a rhabdomyosarcoma with ganglionic differentiation. The FAP+ sarcoma can be an undifferentiated pleomorphic sarcoma (UPS).

[0132] The features disclosed in the foregoing specification, or in the following claims, or in the accompanying drawings can be expressed, as appropriate, in their specific forms, or in terms of means for performing the disclosed functions, or in terms of methods or processes for obtaining the disclosed results, separately or in any combination of such features, and can be utilized to implement the present invention in various forms.

[0133] The present invention has been described in conjunction with the above exemplary embodiments, but upon reading this disclosure, many equivalent modifications and variations will be apparent to those skilled in the art. Accordingly, the above exemplary embodiments of the present invention are considered to be illustrative and not restrictive. Various changes can be made to the described embodiments without departing from the spirit and scope of the present invention.

[0134] To avoid doubt, the theoretical explanations provided herein are for the purpose of enhancing the reader's understanding. The inventors do not wish to be bound by these theoretical explanations.

[0135] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter being described.

[0136] Throughout this specification, including the following claims, unless the context requires otherwise, the words "comprise", "include", and variations such as "comprises", "comprising", "including" are to be understood to mean that the stated integer or step, or group of integers or steps, is included, but not to mean that other integers or steps, or group of integers or steps, are excluded.

[0137] It should be noted that the singular forms "a", "an", and "the" as used in this specification and the appended claims include plural referents unless the context clearly dictates otherwise. In this specification, ranges may be expressed as "about" one particular value and / or "about" another particular value. When such a range is expressed, another embodiment includes from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation using the antecedent "about", the particular value is understood to form another embodiment. The term "about" with respect to a numerical value is optional and means, for example, + / - 10%.

[0138] The following are presented as examples and should not be construed as limiting the claims.

Example

[0139] Materials and methods Clinical samples To construct a commercially available soft tissue sarcoma microarray (TMA), patients with various types of osteosarcoma and soft tissue sarcoma were selected. A full description including the clinicopathological characteristics of the patients can be found on the websites of US Biomax, Inc. (https: / / www.biomax.us / tissue-arrays / Soft_Tissue / SO809c, https: / / www.biomax.us / tissue-arrays / Bone_Cartilage / OS802c).

[0140] Immunohistochemistry Immunohistochemical (IHC) detection of FAP was performed on a DISCOVERY ULTRA instrument (F. Hoffman-La Roche, "Roche") using a rabbit monoclonal antibody against human FAP (diluted 1:25, Abcam-Ref. ab207178) diluted in an antibody diluent (Antibody diluent, Ventana - Ref. 760-108). The staining protocol included a standard antigen retrieval step with Tris-EDTA buffer pH8, incubation with the primary antibody at 37°C for 1 h, and incubation with the secondary antibody (Roche Omni Map Anti-rabbit, Ventana-Ref.760-4311) at 37°C for 20 min. The secondary antibody was detected by DAB (ChrommoMap DAB kit, Ventana-Ref.760-159).

[0141] Immunohistochemistry scoring and automated H-scoring The intensity of FAP staining in the tumor stromal compartment and tumor epithelium was analyzed. The FAP intensity was scored on an optical three-point intensity scale (0 to 2) by independent pathologists. The mean intensity value was calculated from both compartment scores, and case values were obtained. Subsequently, these three groups were reduced to two groups ("negative" and "positive").

[0142] The FAP-stained TMAs were scanned at a resolution of 2 pixels per μm using an Olympus dotSlide version 2.1 scanner. The digital image analysis procedure has been described in detail previously (Mezheyeuski et al. (2018)). Briefly, the FAP intensity was evaluated based on the marker expression level and quantified as the number of positive cells per unit area. The analysis was performed in the stromal compartment, and the samples were classified using the H-score considering a range from 0 to 300. The H-score combines the intensity and proportion of stained cells (Id et al. (2021)). The mean value of the H-score was calculated, and the samples were divided into two groups ("low H-score" and "high score").

[0143] For patients with clinicopathological characteristics, the binary FAP positive / negative and H-score low / high classifications of the cases were used.

[0144] Example 1 - Patients with leiomyosarcoma express high levels of FAP As shown in Table 1A, this cohort had a wide range of diagnoses from 17 to 78 years old, with 46.4% females and 57.1% males. The FAP status showed a significant association with various bone and soft tissue tumor types (p > 0.001), but no association with age or gender (p = 0.662 and p = 0.662). All fibrosarcoma samples were unexpectedly classified as FAP negative (100%), while the leiomyosarcoma group showed a high FAP positive rate (75%).

[0145] Based on the three-point intensity scale, further analysis was performed to investigate the FAP positivity rate and pathological diagnosis (Table 1B). Interestingly, a significant correlation was shown between higher FAP intensity and the histology of leiomyosarcoma: in fact, 83.3% of the FAP-positive samples diagnosed as leiomyosarcoma were scored as high-expression levels, and a significant association was found between high FAP intensity and the histological subgroup of leiomyosarcoma (p = 0.015). Similarly, in the digital H-score classification, high FAP expression was identified in most of the leiomyosarcoma samples investigated (Table 1C), and significant results were obtained (p = 0.007).

[0146] Figure 1 shows representative cases of FAP-negative and FAP-positive staining, such as a fibrosarcoma FAP-negative core (Figure 1A) and a high FAP intensity of a leiomyosarcoma core (Figure 1B).

[0147]

Table 1A

[0148] Table 1A: FAP positivity and clinicopathological variables in patients with fibrosarcoma and leiomyosarcoma (Chi-square exact test). Samples were classified as negative or positive by visual scoring by a pathologist.

[0149]

Table 1B

[0150] Table 1B - Frequency of FAP intensity in histological subtypes of fibrosarcoma and leiomyosarcoma (Chi-square exact test).

[0151]

Table 1C

[0152] Table 1C - Frequency of FAP H-score in histological subtypes of fibrosarcoma and leiomyosarcoma (Chi-square exact test).

[0153] For the entire cohort of bone and soft tissue tumor samples, leiomyosarcoma had the highest frequency (50%) of high FAP intensity compared to osteosarcoma (the largest histological group in this study), and only 7.69% of osteosarcoma samples showed high FAP intensity (Table 2). Digital H-scoring also identified leiomyosarcoma as the largest subset within the high FAP expression group (66.67%) (Table 3), and a significant association was shown between high FAP intensity and the subhistological groups of various bone and soft tissue tumors (p < 0.001). Among the top 15 groups with the highest FAP H-scores, leiomyosarcoma patients accounted for the largest proportion (60%) (Table 4).

[0154] The data showed that rhabdomyosarcoma exhibited considerable FAP expression, and 50% of rhabdomyosarcoma samples also showed moderate or high FAP intensity (Table 2). This trend was reflected in the digital H-scoring, and 60% of rhabdomyosarcoma samples were identified as having moderate or high FAP intensity (Table 3).

[0155]

Table 2

[0156] Table 2: Correlation between FAP intensity and subtypes of osteosarcoma and soft sarcoma (Chi-square exact test).

[0157]

Table 3

[0158] Table 3: Correlation between digital H-score of FAP and subtypes of osteosarcoma and soft sarcoma (Chi-square exact test).

[0159]

Table 4

[0160] Table 4: Pathological diagnosis of the top 15 samples with the highest H-scores.

[0161] Example 2 - Antitumor effect of OMTX705 in a leiomyosarcoma PDX mouse model Leiomyosarcoma has the highest incidence among all sarcoma subtypes, accounting for up to 20% of all sarcomas. Since there is no effective treatment method (Kannan et al. (2022), Kasper et al. (2022)), the anti-tumor effect of OMTX705 in this subtype was further investigated using a xenograft (PDX) model derived from FAP-positive patients.

[0162] The PDX mouse model was selected from a preliminary study using IHC FAP staining of FFPE sections of 12 different leiomyosarcoma tumor types. The PDX model SA4033 was selected due to its high FAP expression (Figure 2).

[0163] Female NOD / SCID mice aged 6 - 9 weeks were subcutaneously implanted with fragments of primary human tumor xenograft tumors (approximately 2 - 3 mm in diameter) for tumor formation (model SA4033). After tumor inoculation, the morbidity and mortality of the animals were examined daily. When the tumor volume reached approximately 150 mm 3 ³, the mice were randomly divided into groups (n = 10 / group) and treated intravenously with two different doses of OMTX705 (10 and 30 mg / kg) once a week for 4 weeks (Table 5).

[0164]

Table 5

[0165] Table 5: Treatment plan for the SA4033 model study. ROA: Route of administration, i.v.: Intravenous, BIW: Twice a week, QW: Once a week.

[0166] During regular monitoring, the effects of tumor growth and treatment on behaviors such as locomotor ability, food and water intake, weight gain / loss (weight was measured twice a week after randomization), gloss of the eyes / hair, and other abnormalities were examined in the animals. The mortality of individual animals and the observed clinical signs were recorded in detail.

[0167] After randomizing the tumor volume, it was measured two - dimensionally twice a week using calipers, and the volume was expressed in mm using the formula: V=(LxWxW) / 2. 3 Here, V is the tumor volume, L is the tumor length (the longest dimension of the tumor), and W is the tumor width (the longest tumor dimension perpendicular to L). Dose as well as tumor and body weight measurements were performed in a laminar flow cabinet. After the end of treatment on day 21, the post - treatment observation period for the group of mice treated with 10 mg / kg of OMTX705 was extended by 6 weeks.

[0168] In the SA4033PDX model of leiomyosarcoma, the efficacy of OMTX705 was measured in vivo by single - agent i.v. administration 4 times once a week at doses of 10 and 30 mg / kg. Tumor growth inhibition was found to be 92.04% and 93.11% respectively (Figure 3A). Interestingly, in this leiomyosarcoma model, when OMTX705 was administered as a single agent at 30 mg / kg, it had a similar anti - tumor effect compared to the low dose (10 mg / kg), and no significant weight loss was observed. Mice treated with OMTX705 at doses of 10 mg / kg and 30 mg / kg showed less than 4% change in body weight per group during the study period (Figure 3B). The percentage of tumor - inhibitory volume (TIV) of OMTX705 as a single agent in this model is shown in Figure 3C.

[0169] In the observation on day 42 after treatment, continuous tumor suppression and even tumor regression were observed in the group treated with 10 mg / kg of OMTX705, and the average tumor volume was 29.4 mm 3 which was much lower than the average of the tumor volumes recorded on day 0 (148.09 mm 3 ). By day 42, 4 out of 10 animals had no tumors. By the end of the post - treatment observation period, 100% of the mice in this group had no tumors. Therefore, these data demonstrate that the effect of weekly administration of 10 mg / kg of OMTX705 persists long - term even 6 weeks after the end of treatment.

[0170] Example 3 - Antitumor effect of OMTX705 in leiomyosarcoma PDX mouse models with different FAP expression levels. Following the promising results of Example 2 above, similar experiments were conducted to compare the antitumor effects of OMTX705 against sarcoma in patient-derived xenograft (PDX) mouse models with high to moderate FAP expression positive (SA4033 and SA3839) and low / zero FAP expression (SA10202).

[0171] As described above, the PDX mouse models were selected from a preliminary study using IHC FAP staining of FFPE sections of 12 different sarcoma tumor types. PDX models SA4033 and SA3839 were selected for high to moderate FAP expression (Figures 4A and 4B), and SA10202 was selected for low to zero FAP expression (Figure 4C).

[0172] In each model, female NOD / SCID mice at 6 - 9 weeks of age were subcutaneously implanted with primary human tumor xenograft tumor fragments (approximately 2 - 3 mm in diameter). After tumor inoculation, the morbidity and mortality of the animals were examined daily. When the tumor volume reached approximately 150 mm 3 ³, the mice were randomly grouped (n = 10 / group in the SA4033 model, n = 8 / group in the SA3839 model, n = 6 / group in the SA10202 model). In the SA4033 model, two different doses of OMTX705 (10 and 30 mg / kg) were administered intravenously, and in the SA3839 and SA10202 models, a single dose of 10 mg / kg was administered once a week for 4 weeks (Table 6).

[0173]

Table 6

[0174] Table 6: Treatment plan for SA4033, SA3839, SA10202 model studies. ROA: Route of administration, i.v.: Intravenous, BIW: Twice a week, QW: Once a week.

[0175] As in Example 2, during regular monitoring, the effects of tumor growth and treatment on behaviors such as motor ability, food and water intake, weight gain / loss (weight was measured twice a week after randomization), eye / coat shine, and other abnormalities were examined in animals. The mortality of individual animals and the observed clinical signs were recorded in detail.

[0176] After randomization, the tumor volume was measured two - dimensionally twice a week using calipers, and the volume was expressed in mm using the formula: V=(LxWxW) / 2. 3 Here, V is the tumor volume, L is the tumor length (the longest dimension of the tumor), and W is the tumor width (the longest tumor dimension perpendicular to L). Dosage and tumor and weight measurements were performed in a laminar flow cabinet. After the end of treatment on day 21, the post - treatment observation period for the group of mice treated with 10 mg / kg of OMTX705 was extended by 6 weeks.

[0177] In the SA4033PDX model of leiomyosarcoma, the efficacy of OMTX705 measured at doses of 10 and 30 mg / kg as a single agent was higher compared to the SA3839 and SA10202 PDX models (Figure 5A). In the observation on day 42 after treatment in SA4033 mice, continuous tumor suppression and even tumor regression were observed in the group treated with 10 mg / kg of OMTX705, and the average tumor volume was 29.4 mm 3 which was the average of the tumor volumes recorded on day 0 (148.09 mm 3) was much lower. On day 42, 4 out of 10 animals had no tumors. By the end of the post-treatment observation period, 100% of the SA4033 mice in this group had no tumors. This effect of weekly administration of 10 mg / kg of OMTX705 persisted long in SA4033 animals, and no tumor regrowth was observed even 6 weeks after the end of treatment (Figure 5A, upper). Interestingly, in the SA4033 model of leiomyosarcoma, when OMTX705 was administered as a single agent at 30 mg / kg, it had a similar anti-tumor effect compared to the low dose (10 mg / kg), and no significant weight loss was observed. Mice administered OMTX705 at doses of 10 mg / kg and 30 mg / kg showed a weight change of less than 4% per group during the SA4033 study period (Figure 5B, upper) (the data shown in the mouse model SA4033 in the upper parts of Figures 5A and 5B are the same as those described in Example 2, Figures 3A and 3B). In the SA3839 model, mice treated with weekly administration of 10 mg / kg of OMTX705 had a weight change of less than 9% per group during the study period, and OMTX705 could induce tumor growth inhibition and stasis but could not induce complete regression (Figures 5A and 5B, middle). In the low / zero FAP expression (SA10202) patient-derived xenograft (PDX) mouse model, mice treated at 10 mg / kg showed a weight change of less than 5% per group during the study period, and OMTX705 could not induce tumor regression (Figures 5A and 5B, lower).

[0178] As shown in Figure 5C, the percentages of tumor volume inhibition (TIV) of OMTX705 as a single agent in SA4033 (90.2%), SA3839 (74.86%), and SA10202 (42.6%) on day 24 (3 days after the last administration) demonstrate the different efficacies of OMTX705 among these models and support the correlation between the FAP expression level in leiomyosarcoma and the anti-tumor efficacy of OMTX705.

[0179] Further immunohistochemical studies of the SA4033 PDX model using different marker proteins indicated that the antitumor effect of OMTX705 in leiomyosarcoma is brought about by a different mechanism of action compared to carcinomas. On day 28, FFPE tissue samples of tumors treated with OMTX705 had very low cellularity (Figure 6B), very few Ki67, active caspase 3, and FAP-positive cells (Figure 6D, 6F, 6H) compared to tumors treated with vehicle (Figure 6A, 6C, 6E, 6G), indicating that FAP-positive tumor cells were directly and efficiently killed. Anti-TAM558 IHC revealed that the OMTX705 payload was still present in tumor samples even at least 1 week after the last treatment dose (Figure 6J). This may explain the long-lasting effect of the compound in preventing tumor recurrence even after treatment has ended.

[0180] Overall, these results support a direct and FAP-dependent effect on tumor cells, leading to a much more potent antitumor effect of OMTX705 in leiomyosarcoma at low doses compared to carcinomas where the compound acts via FAP-expressing CAFs.

[0181] Abstract Previous results obtained in the first-in-human and Phase I trial of ABBV-085, a novel ADC targeting the LRRC15 stromal protein for the treatment of sarcoma, showed high response rates across many solid tumors due to overall LRRC15 expression, regardless of sarcoma subtype (Demetri et al. (2021), Purcell et al. (2022)). Further experiments using PDX models of soft tissue sarcoma to study LRRC15 expression and ABBV-085 activity demonstrated significant antitumor activity in undifferentiated sarcoma (UPS), dedifferentiated liposarcoma, and leiomyosarcoma (Ben-Ami et al(2020)).

[0182] In fact, unexpectedly, our study found a high level of FAP(+) in leiomyosarcoma among other soft tissue sarcomas (i.e., liposarcoma or fibrosarcoma with very low or no FAP expression), thus identifying a subtype of leiomyosarcoma as a new highly specific indication for OMTX705 anti-tumor treatment among sarcomas.

[0183] The surprising finding that leiomyosarcoma tumor cells express FAP at high levels highlights the possibility of treating leiomyosarcoma by directly targeting cells with anti-FAP-cytolysin complexes. The in vivo data shown in Figure 3 demonstrate excellent tumor suppression rates of 92.04% and 93.11% at 10 and 30 mg / kg i.v. administration, respectively, and Figure 5 shows an excellent tumor suppression rate of 90.2% on day 24 at 10 mg / kg administration in a model with high FAP expression level (SA4033) (Figure 4). Furthermore, OMTX705 treatment actually resulted in complete regression of tumors at a later time point, with 100% of the mice reaching a tumor-free state and no tumor recurrence up to 6 weeks after the end of treatment. Interestingly, models with moderate (SA3839) or low / zero (SA10202) FAP expression levels showed a lower response to OMTX705, as indicated by %TVI of 74.86 and 42.6, respectively (Figure 5C). Therefore, the data provide a new mechanism of action for OMTX705, in addition to targeting FAP(+) CAFs that correlate with FAP expression levels, as first shown in the context of pancreatic cancer (Fabre et al. (2020)).

[0184] Both scoring systems (visual and digital) reached the same conclusion: the sarcoma subtype showing pan-total and strong FAP staining was leiomyosarcoma, followed by osteosarcoma with a large lag.

[0185] The data also revealed that rhabdomyosarcoma shows significant FAP expression, identifying an additional target for anti-FAP-cytolysin complexes.

[0186] This experimental data is the first preclinical evidence that FAP targeting by antibody-drug conjugates is a promising strategy for FAP-positive sarcoma patients.

[0187] Therefore, the findings presented in this study support a new strategy and new potential indication for the OMTX705 therapeutic compound in the treatment of FAP+ sarcoma patients, such as leiomyosarcoma patients, with a very promising antitumor effect compared to antibody-drug conjugates tested so far.

[0188] Furthermore, these results further support the use of FAP as a biomarker for selecting FAP+ sarcoma patients, such as leiomyosarcoma soft tissue tumor subtypes, who are likely to respond to the newly developed antibody-drug conjugate drug OMTX705.

[0189] References To more fully describe and disclose the present invention and the state of the art to which the present invention pertains, many publications have been cited above. The complete citations of these documents are shown below. The entire contents of each of these documents are incorporated herein by reference.

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Abstract LBA032: Pan-cancer analysis of fibroblast activation protein alpha (FAP) expression to guide tumor selection for the peptide-targeted radionuclide therapy FAP-2286. Mol. Cancer Ther. 20, LBA032-LBA032 (2021). Malanchi, I., et al., Nature, 2012, 481: 85-89 Meads, M.B, et al., Nat. Rev. Cancer, 2009, 9: 665-674 Medema, J.P., et al., Nature, 2011, 474: 318-326 Mersmann M., et al., Int. J. Cancer, 2001, 92: 240-248 Messerschmidt, S.K., et al., J Control Release, 2009, 137: 69-77 Mezheyeuski, A. et al. Multispectral imaging for quantitative and compartment-specific immune infiltrates reveals distinct immune profiles that classify lung cancer patients. J. Pathol. 244, 421-431 (2018). Niederhuber, J., Armitage, J., Doroshow, J., Kastan, M. & Tepper, J. Abeloff’s Clinical Oncology (5th ed.). Fac. Bookshelf (2014). Nieman, K.M., et al., Nat. 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Claims

1. For use in the treatment of sarcoma in mammalian subjects, formula A-(L-D) ｐ (wherein A is an anti-FAP antibody that selectively binds to FAP, L is a linker, D is a drug containing a cytolysin, and p is from 1 to 10), an anti-fibroblast activation protein α (FAP) antibody-cytolysin conjugate having the formula, or a pharmaceutically acceptable salt or solvate thereof, wherein the sarcoma is characterized by FAP expression in the tumor.

2. A has a heavy chain having the amino acid sequence of SEQ ID NO: 3 and a light chain having the amino acid sequence of SEQ ID NO: 4; L-D has the structure: 【Chemical 1】 (wherein * indicates the point of attachment to A), a conjugate for use according to claim 1.

3. The sarcoma is: i) a sarcoma selected from the group consisting of FAP-expressing (FAP+) leiomyosarcoma, optionally uterine leiomyosarcoma, cutaneous leiomyosarcoma, gastrointestinal leiomyosarcoma, gastric leiomyosarcoma, small intestinal leiomyosarcoma, retroperitoneal leiomyosarcoma, and / or abdominal wall leiomyosarcoma; ii) FAP+ rhabdomyosarcoma, optionally fetal rhabdomyosarcoma, alveolar rhabdomyosarcoma, spindle cell rhabdomyosarcoma, mixed rhabdomyosarcoma, pleomorphic rhabdomyosarcoma, and rhabdomyosarcoma with ganglionic differentiation; or iii) FAP+ undifferentiated pleomorphic sarcoma (UPS) A conjugate for use according to claim 1 or 2.

4. Intravenous administration of the conjugate; optionally, the conjugate is administered weekly, every two weeks, or monthly; optionally, the conjugate is administered within a dose range of 0.1 to 30 mg / kg, a conjugate for use according to any of the preceding claims.

5. An in vitro method of selecting a subject determined to have a sarcoma for treatment with an anti-FAP ADC, comprising: a) measuring the FAP expression level in a biological sample taken from the subject; b) determining that the FAP expression level exceeds a threshold level; and c) selecting the subject for treatment with the FAP-targeted ADC A method comprising.

6. The in vitro method of claim 5, wherein the sarcoma is selected from the group consisting of leiomyosarcoma, rhabdomyosarcoma, and undifferentiated pleomorphic sarcoma (UPS).

7. The anti-FAP ADC has the formula A-(L-D) ｐ (wherein A is an anti-FAP antibody having a heavy chain with the amino acid sequence of SEQ ID NO: 3 and a light chain with the amino acid sequence of SEQ ID NO: 4; L-D has the structure: [[Chemical Formula 2]] (wherein * indicates the point of attachment to A) and p is from 1 to 10), an in vitro method according to claim 5 or 6.

8. The in vitro method according to any of claims 5 to 7, wherein the subject is a mammalian subject, optionally, the mammalian subject is a human.

9. The anti-FAP antibody is a monoclonal antibody or a binding fragment thereof that selectively binds to the extracellular region of human FAP and / or mouse FAP; Optionally, the anti-FAP antibody has the following amino acid sequences: CDRH1: SEQ ID NO: 7; CDRH2: SEQ ID NO: 8; CDRH3: SEQ ID NO: 9; CDRL1: SEQ ID NO: 10; CDRL2: SEQ ID NO: 11; and CDRL3: SEQ ID NO: 12 A complex for use according to any one of claims 1, 3 or 4, comprising heavy chain complementarity determining regions 1 to 3 (CDRH1 to 3) and light chain complementarity determining regions 1 to 3 (CDRL1 to 3).

10. The complex for use according to claim 9, wherein the anti-FAP antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 5 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 6; or the anti-FAP antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO:

4.

11. The cytolysin has formula IV: [Chemical Formula 3] (wherein: R ２ is H or C １ -C ４ alkyl; R ６ is C １ -C ６ alkyl; R ７ is C １ -C ６ alkyl, CH ２ OR １９ or CH ２ O COR ２０ (wherein, R １９ is alkyl, R ２０ is C ２ -C ６ -alkenyl, phenyl or CH ２ -phenyl); R ９ is C １ -C ６ alkyl; R １０ is H, OH, O-alkyl or O-acetyl; f is 1 or 2; R １１ has the following structure: 【Chemical Formula 4】 (wherein, R ２１ is H, OH, halogen, NH ２ , alkyloxy, phenyl, alkylamino or dialkylamino; R １６ is H or C １ -C ６ -alkyl group; R １７ is directly or indirectly connected to the linker L; and q is 0, 1, 2 or 3; and the term "optionally substituted" relates to a group in which one or several H atoms can be replaced by F, Cl, Br or I or OH, SH, NH ２ or NO ２ and also the term "optionally substituted" relates to a group which can be exclusively or additionally substituted by an unsubstituted C １ -C ６ alkyl, C ２ -C ６ alkenyl, C ２ -C ６ alkynyl, C １ -C ６ heteroalkyl, C ３ -C １０ cycloalkyl, C ２ -C ９ heterocycloalkyl, C ６ -C １０ aryl, C １ -C ９ heteroaryl, C ７ -C １２ aralkyl or C ２ -C １１ heteroaralkyl group). A complex for use according to any one of claims 1, 3, 4 or 9 to 10.

12. L includes a spacer, optionally, the spacer being - (OCH ２ CH ２ ), ｎ - (where n is from 2 to 5), a complex for use according to any of claims 1, 3, 4 or 9 to 11.

13. The complex for use according to any one of claims 1, 3, 4, or 9 to 12, wherein L comprises a linking group for connecting to A and optionally L comprises a protease-cleavable moiety comprising a valine-citrulline unit.

14. The cytolysin has the formula: 【Chemical Formula 5】 (wherein * indicates the site of attachment to L), a complex for use according to any one of claims 11 to 13.

15. -L-D has the structure: 【Chemical Formula 6】 (wherein * indicates the point of attachment to A), a complex for use according to any one of claims 1, 3, 4 or 9 to 14.

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