Antibody-drug conjugates targeting FAP

Abstract

The present invention relates to anti-fibroblast activating protein alpha (FAP) targeted antibody drug conjugates (ADCs), in particular anti-FAP antibody-cytolysin conjugates having the formula A-(L-D) p, or a pharmaceutically acceptable salt or solvate thereof, for use in a method of treatment of FAP + sarcoma in a mammalian subject, where A is an anti-FAP antibody that selectively binds to FAP, L is a linker, and D is a hydrogen atom or a hydrogen atom, or a pharmaceutically acceptable salt or solvate thereof, for use in a method of treatment of FAP + sarcoma in a mammalian subject. D is a drug comprising cytolysin and p is 1 to 10. Also disclosed are in vitro methods of selecting sarcoma patients for ADC treatment with targeted FAP.

Description

[0001] This application claims priority to EP22382713.0 filed on July 26, 2022, the contents and elements of which are incorporated herein by reference for all purposes. Technical Field

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

[0003] Sarcomas are types of cancer that begin in tissues such as bone or muscle. Bone and soft tissue sarcomas consist of a heterogeneous group of rare solid tumors of mesenchymal origin. Sarcoma tumors originate primarily from the embryonic mesoderm (which can affect any part of the body in patients of any age). Despite the progress of therapy for these tumors, metastasis and death remain significant problems in patients with high-risk soft tissue sarcomas (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 "desmoplastic stroma" or "reactive stroma", which accounts for 20-60% of the total tumor mass and is characterized by the presence of abundant stromal cells and a dense extracellular matrix (ECM). Recent studies have indicated a pro-tumor role for stromal cells, as exemplified 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, cancer-associated fibroblasts (CAFs) are frequently observed in significant numbers within the tumor-associated stroma of various human cancers, including breast, lung, colon, and pancreatic cancers (Kalluri et al. (2006), Pietras et al. (2010)). Interacting in concert with different components of the stroma, CAFs have the ability to promote angiogenesis and tumor growth. CAFs have also been shown to be essential for the development of aggressive tumors and tumor invasiveness during cancer progression (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 infiltration of tumor cells in distant organs, thus contributing to the formation of metastases. Importantly, stromal cells have been shown to be associated with the failure of systemic drug delivery to tumors and with 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 that is 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. Because smooth muscle is found in many areas of the body, leiomyosarcoma can develop 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 indicated that uterine LMS and non-uterine LMS exhibit unique disease biology.

[0006] Rhabdomyosarcoma is another malignant mesenchymal tumor. It is the most common soft tissue sarcoma in children, 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: embryonal, alveolar, spindle cell, mixed, pleomorphic, and rhabdomyosarcoma with ganglionic differentiation.

[0007] Undifferentiated pleomorphic sarcoma (UPS) is a term for undifferentiated and unclassified sarcomas. This subtype consists of poorly differentiated tumor cells that may present as spindle-shaped cells, histiocytes, and giant cells.

[0008] Drugs based on monoclonal antibodies (MAbs) show great promise in the fight against cancer. This is because they allow for treatment aimed at a molecular level in an accurate and specific manner. These advantages, together with their commercial appeal (short development time, limited capacity, and their ability to be easily exported to other cancer types once approved), have prompted many pharmaceutical companies to invest heavily in developing new antibody-based molecules, as well as obtaining new molecules or technology licenses from biotechnology companies.

[0009] However, despite the clinical success of therapeutic antibodies, the naked MAb of the targeting cell surface tumor antigen rarely presents enough efficacy itself. In order to improve the low activity of MAb, new strategies focus on combining them to toxic molecules. Because plant and bacterial toxins and small chemotherapy molecules are very effective and have activity with very small amounts, they can be good candidates.

[0010] Due to the technological advances achieved in the past few years, the field of immunotoxins (ITs) and antibody-drug conjugates (ADCs) for the treatment of cancer has recently experienced increasing development by pharmaceutical companies aiming to solve their initial problems regarding immunogenicity, undesirable toxicity, yield, half-life and resistance.

[0011] Immunoconjugates are made by covalently linking a human, humanized or chimeric recombinant antibody to a cytotoxic drug. The main purpose of this type of structure is to combine the potency of a small cytotoxic agent (300 to 1000 Da) with the high specificity of a MAb targeting a tumor-associated antigen (TAA).

[0012] The Ab must be very selective to reach the antigen, the expression of which must be restricted to normal cells. The Ab must also be efficiently internalized into cancer cells.

[0013] The cytotoxic agent selected as the effector moiety must kill the cell only after internalization and release into the cytoplasm. The most commonly used payloads in ADCs are DNA damaging drugs such as calicheamicins, duocarmicins, or microtubule-targeted compounds such as auristatins and maytansinoids.

[0014] Ab-cytotoxic linkers are designed to be systemically stable and release the cytotoxic agent inside the target cell.

[0015] TAAs are often cell membrane proteins that are overexpressed in diseased tissues or at least express enough cytotoxins to promote internalization activation. Ideally, the antigens present restricted expression in normal tissues, with low or no expression in important organs. In addition, tumor antigens must be selectively and with high affinity recognized by Abs.

[0016] In many types of human cancer, the fibroblast response is characterized by the induction of the cell surface protein, fibroblast activation protein alpha (FAPα), a 95 kDa serine protease whose expression is highly restricted to developing organs, wound healing, and tissue remodeling. FAPα is a well-studied type II membrane glycoprotein serine protease that is upregulated in cancer-associated fibroblasts (CAFs) as well as wound healing fibroblasts (Ramirez-Montagut et al. (2004)). In several tumor tissues, FAP expression is associated with tumor proliferation, invasion, angiogenesis, epithelial-to-mesenchymal transition (EMT), immunosuppression, and drug resistance (Xin et al. (2021)). FAP exhibits the following characteristics:

[0017] Type II membrane glycoprotein with SER protease activity (collagenase + DPP)

[0018] 89% human-mouse protein homology

[0019] Tumor stroma expressed in >90% of cancers (breast, pancreatic, lung, bladder, and colon)

[0020] Transient and highly restricted expression in normal adult tissues during wound healing and organ development.

[0021] FAP is expressed in tumor cells of various sarcomas, including leiomyosarcoma and rhabdomyosarcoma.

[0022] FAP(+) fibroblasts are located near tumor blood vessels

[0023] Very focused expression

[0024] Internalization

[0025] • Involvement in extracellular matrix remodeling, tumor growth and metastasis.

[0026] FAP expression has been found in pancreatic tumor cells as well as tumor-associated stromal fibroblasts. FAP expression is associated with shorter patient survival and poor prognosis, suggesting the possibility of a FAP-based autocrine / paracrine loop in this type of tumor (Shi et al. (2012)).

[0027] Anti-FAP MAb derivatives against both human and murine proteins were developed by previous work by Kontermann and Pfizenmaier (IZI, University of Stuttgart, Germany) (Mersmann et al. (2001), Brocks et al. (2001)). They have shown in vitro that anti-FAP scFv immunoliposomes specifically bind to FAP+ cells and are internalized (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)).

[0028] Treatment with the murine MAb FAP5-DM1 immunotoxin induced durable inhibition of tumor growth and complete regression in pancreatic and lung cancer xenograft models without any intolerance-related effects (Ostermann et al. (2008)).

[0029] FAP protein has been widely validated as an interesting target for anticancer therapy in solid tumors and has therefore been subjected to various CAF targeting approaches (Kakarla et al. (2012), et al. (2020)). In this context, OMTX705, a novel antibody-drug conjugate targeting FAP(+)CAFs, was developed as an alternative stromal-targeted therapeutic compound with potent antitumor efficacy. 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 correlates with the antitumor efficacy of OMTX705 treatment in these models (Fabre et al. (2020)).

[0030] FAP was first isolated from reactive stroma of human sarcomas (Rettig et al. (1994), Rettig et al. (1988)) and has also been found to be expressed in tumor cells of a subset of bone and tissue sarcomas (Rettig et al. (1993)), correlating with their mesenchymal origin. Recent studies on FAP protein as a cancer biomarker in sarcomas have demonstrated some correlation between FAP and poor prognosis in osteosarcoma (Zhang et al. (2019)). In addition, several 68The development of Ga radiolabeled FAP inhibitor small molecules has led to studies evaluating its clinical role in cancer imaging (Kuyumcu et al. (2021)). 68 Quantitative study of tumor uptake on Ga-FAP IPET / CT reveals that sarcoma patients are included in the highest 68 These high ratios result in high image contrast and excellent tumor delineation in patients with bone and soft tissue sarcomas (Kratochwil et al. (2019), Koerber et al. (2021), Kessler et al. (2022)). In addition, peptides targeting FAP have been used in radionuclide therapies for various cancers (such as 17 Lu-FAP-2286) and imaging (such as 68 Ga-FAP-2286) (Kwan et al. (2021)).

[0031] Despite these advances, there remains an unmet need for further therapeutic strategies for treating sarcomas, including leiomyosarcoma, rhabdomyosarcoma, and UPS, as well as for components for such therapeutic strategies. The present invention addresses these and other needs. Summary of the invention

[0032] In a broad sense, the present invention relates to anti-FAP antibodies, conjugates thereof and optimized payloads for use in antibody conjugation strategies. In particular, the inventors have found that the anti-FAP antibodies as described herein exhibit high specific binding, as well as rapid and efficient internalization. The present invention provides anti-FAP antibody-cytolysin conjugates (of the type disclosed in WO 2015 / 118030 (which is incorporated herein by reference in its entirety)) for use in a method of treating a sarcoma in a mammalian subject.

[0033] The lysin derivatives described herein are advantageously coupled to anti-FAP antibodies for use in the treatment of sarcomas, wherein the sarcomas are characterized by the expression of FAP in the tumor.

[0034] Therefore, the utility of ADCs targeting FAP extends beyond tumors expressing FAP in the stroma.

[0035] Thus, in a first aspect, the present invention provides an anti-fibroblast activation protein alpha (FAP) antibody-cytolysin conjugate having formula I.

[0036] A-(LD) p (I)

[0037] or a pharmaceutically acceptable salt or solvate thereof, for use in a method of treating a sarcoma in a mammalian subject, wherein A is an anti-FAP antibody that selectively binds to FAP, L is a linker, D is a drug comprising a lysin and p is 1 to 10, wherein the sarcoma is characterized by expression of FAP in the tumor.

[0038] In some cases, according to this and other aspects of the 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 murine FAP. In some cases, the anti-FAP antibody can cross-react with both human and murine FAP.

[0039] In a specific case, the anti-FAP antibody may include a heavy chain complementary determining region 1-3 (CDRH1-3) and a light chain complementary determining region 1-3 (CDRL1-3) having the following amino acid sequence:

[0040] (i) CDRH1: SEQ ID NO: 7;

[0041] (ii) CDRH2: SEQ ID NO: 8;

[0042] (iii) CDRH3: SEQ ID NO: 9;

[0043] (iv) CDRL1: SEQ ID NO: 10;

[0044] (v) CDRL2: SEQ ID NO: 11; and

[0045] (vi) CDRL3: SEQ ID NO:12.

[0046] In some embodiments, the CDRH1 region may include a variant of SEQ ID NO:7 having up to 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 up to 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 up to 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 up to 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 up to 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 up to 1 or 2 amino acid substitutions compared to the sequence of SEQ ID NO:12.

[0047] In certain instances, CDRH1-3 comprise the amino acid sequences of SEQ ID NOs: 7-9, respectively, and CDRL1-3 comprise the amino acid sequences of SEQ ID NOs: 10-12, respectively.

[0048] In some cases, 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. In some embodiments, the heavy chain variable region (VH) has at least 90%, 95%, or 99% sequence identity to 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 to the full-length sequence of SEQ ID NO: 6.

[0049] In some 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 to the full-length sequence of SEQ ID NO: 1. In some cases, the light chain has at least 90%, 95%, or 99% sequence identity to the full-length sequence of SEQ ID NO: 2.

[0050] In some 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 to the full-length sequence of SEQ ID NO: 3. In some cases, the light chain has at least 90%, 95%, or 99% sequence identity to the full-length sequence of SEQ ID NO: 4.

[0051] In some cases, A can be a competitive binding 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" for binding to immobilized recombinant human FAP. hu36 has a heavy chain amino acid sequence of SEQ ID NO:3 and a light chain amino acid sequence of SEQ ID NO:4. In some cases, the anti-FAP antibody can bind to the same epitope as hu36. Methods for determining antibody binding competition and for epitope mapping are well known in the art, see, for example, "Epitope Mapping by Competition Assay" Harlow and David Lane, eds., Cold Spring Harb Protoc; 2006; doi: 10.1101 / pdb.prot4277.

[0052] According to this and other aspects of the invention, D may be a cytolysin. In some cases, the cytolysin may be a compound disclosed in WO 2008 / 138561A1 (the entire contents of which are expressly incorporated herein by reference) (wherein the disclosed compounds are also referred to as Tubulysine derivatives). The cytolysin may be synthesized as described in WO 2008 / 138561. In some cases, the cytolysin may be defined as Formula I or Formula IV in WO 2008 / 138561 A1. In some cases, the cytolysin may be Formula IV:

[0053]

[0054] in:

[0055] R 2 is H or C1-C4 alkyl;

[0056] R 6 is a C1-C6 alkyl group;

[0057] R 7 It is C1-C6 alkyl, CH2OR 19 or CH2OCOR 20 , where R 19 is an alkyl group, R20 is C2-C6-alkenyl, phenyl or

[0058] CH2-phenyl;

[0059] R 9 is a C1-C6 alkyl group;

[0060] R 10 is H, OH, O-alkyl or O-acetyl;

[0061] f is 1 or 2;

[0062] R 11 Has the following structure:

[0063]

[0064] in:

[0065] R 21 is H, OH, halogen, NH2, alkoxy, phenyl, alkylamino or dialkylamino;

[0066] R 16 is H or a C1-C6-alkyl group;

[0067] R 17 directly or indirectly attached to the connector L, and;

[0068] q is 0, 1, 2, or 3;

[0069] and wherein 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; the term "optionally substituted" further relates to a group which may be replaced solely or additionally by unsubstituted C1-C6 alkyl, C2-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 Arylalkyl or C2-C 11 Substitution with heteroarylalkyl groups.

[0070] In some cases, R 2 is the bond to the linker L.

[0071] In some cases, R 17 is C(O)X, CONHNHX, OX, NHX or SX, wherein X is a bond to the linker L.

[0072] In some embodiments, the linker L comprises a spacer. In some cases, the spacer has a chain length of 2 to 30 atoms. In some cases, the spacer comprises or is composed of an alkylene (i.e., a divalent alkyl) or heteroalkylene (i.e., a divalent heteroalkyl) group. In some cases, the spacer comprises or is composed of an alkylene or oxyalkylene group.

[0073] In some embodiments, the spacer comprises –(OCH2CH2) n -, wherein n is 2 to 5. In some cases, the spacer includes a group -(CH2) n -or-(OCH2CH2) n - or by the group -(CH2) n -or-(OCH2CH2) n -composition, wherein n≥1. In some cases, the spacer comprises a group -(OCH2CH2) n - or by the group -(OCH2CH2) n -composition, wherein n ≥ 1. In particular, n can be 1 to 15, 1 to 10, 1 to 6 or 2 to 5. For example, n can be 3 or 4. In some cases, the spacer includes between one and six ethylene glycol units, such as triethylene glycol. In some cases, the spacer can be directly attached to the group R 17 , or may be attached to the group R via a bridging group 17 In some cases, the spacer is attached to the group R via a -C(O)X bridging group. 17 , where X is to R 17 In some cases, R 17 is CONHNHX, and the spacer is attached to the group R via a -C(O)X bridging group 17 , where X represents the spacer and R 17 In some cases, R 17 is CONHNHX, and the spacer is attached to R via a -C(O)X bridging group 17 -(OCH2CH2) n -, where n=2, 3 or 4.

[0074] In some embodiments, L includes an attachment group for attachment to A.

[0075] In some embodiments, L comprises a protease cleavable portion comprising a valine-citrulline unit. For example, L may comprise maleimidocaproyl-valine-citrulline-p-aminobenzylcarbamate.

[0076] In some cases, a lysin has the following structure:

[0077]

[0078] where * indicates the site of attachment to L.

[0079] In some cases, D comprises a lysin having the structure:

[0080]

[0081] In some cases, the double bond of the maleimide reacts with the thiol group of the cysteine ​​residue of antibody A to form a sulfur-carbon bond to achieve the bonding effect of linker L to antibody A.

[0082] In some embodiments, -LD has the structure:

[0083]

[0084] In other cases, -LD has a structure selected from the group consisting of:

[0085]

[0086]

[0087] In some cases, –LD can have the following structures:

[0088]

[0089] In some cases, –LD can have the following structures:

[0090]

[0091] According to this and other aspects of the invention, in some cases, p may be in the range of 1 to 5, such as 1 to 4 or 1 to 3. In specific cases, p may be 1 or 2. In specific cases, p may be 3 or 4.

[0092] In some embodiments, A has a heavy chain with an amino acid sequence of SEQ ID NO: 3 and a light chain with an amino acid sequence of SEQ ID NO: 4; and LD has the structure:

[0093]

[0094] Where * indicates the point of attachment to A.

[0095] In some embodiments, the conjugate is a conjugate described herein as OMTX705. OMTX705 comprises a humanized anti-FAP mAb (OMTX005) conjugated to the cytolysin TAM470 via a protease-cleavable vcPABA-(EG)3 optimized linker. OMTX705 is described in Fabre et al. (2020), and Figure 7 is an exemplary depiction of OMTX705.

[0096] In some embodiments, the sarcoma is a leiomyosarcoma expressing FAP (FAP+). In some cases, the conjugate is used in the treatment of a leiomyosarcoma selected from the group consisting of uterine leiomyosarcoma, cutaneous leiomyosarcoma, gastrointestinal leiomyosarcoma, gastric leiomyosarcoma, small intestinal leiomyosarcoma, retroperitoneal leiomyosarcoma, or abdominal leiomyosarcoma.

[0097] In some embodiments, the sarcoma is FAP+ rhabdomyosarcoma. In some embodiments, the conjugate is for use in the treatment of a rhabdomyosarcoma selected from the group consisting of embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, spindle cell rhabdomyosarcoma, mixed rhabdomyosarcoma, pleomorphic rhabdomyosarcoma, and rhabdomyosarcoma with ganglionic differentiation.

[0098] In some embodiments, the sarcoma is FAP+ undifferentiated pleomorphic sarcoma (UPS).

[0099] In some embodiments, the conjugate is administered intravenously.

[0100] In some embodiments, the conjugate is administered weekly. In some embodiments, the conjugate is administered every 2 weeks. In some embodiments, the conjugate is administered monthly.

[0101] In some embodiments, the conjugate is administered in a dosage range of 0.1-30 mg / kg. In some embodiments, the conjugate is administered in a dosage range of 10-30 mg / kg. In some embodiments, the conjugate is administered at 10 mg / kg. In some embodiments, the conjugate is administered at 20 mg / kg. In some embodiments, the conjugate is administered at 30 mg / kg.

[0102] In some embodiments, the conjugate is administered in 4 doses.

[0103] In some cases, the conjugate used in the treatment method of sarcoma is administered simultaneously, sequentially or separately with one or more other anti-tumor drugs. The one or more other anti-tumor drugs include cytotoxic chemotherapeutic agents or anti-angiogenic agents or immunotherapeutic agents. In some cases, the one or more other anti-tumor drugs include gemcitabine, paclitaxel, bevacizumab, itraconazole, carboxyamidotriazole, anti-PD-1 molecules or anti-PD-L1 molecules (e.g., nivolumab or pembrolizumab).

[0104] In a second aspect, the present invention provides an in vitro method for selecting a subject determined to have a sarcoma for treatment with an ADC targeting FAP, the method comprising measuring the level of FAP expression in a biological sample obtained from the subject; determining that the level of FAP expression is above a threshold level, and selecting the subject for treatment with an ADC targeting FAP. In some embodiments, the sarcoma is a leiomyosarcoma. In some embodiments, the sarcoma is a rhabdomyosarcoma. In some embodiments, the sarcoma is a UPS. In some embodiments, the anti-FAP ADC has the formula A-(LD)p, wherein A is an anti-FAP antibody and has a heavy chain of the amino acid sequence SEQ ID NO:3 and a light chain of the amino acid sequence SEQ ID NO:4; LD has the structure:

[0105]

[0106] Where * represents the point of attachment to A and p is 1 to 10.

[0107] In some embodiments, the ADC targeting FAP 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 collected from the site of the tumor. In some embodiments, the biological sample is a cell-free sample containing nucleic acid, such as a plasma sample.

[0108] In a third aspect, the invention provides a method for 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 used to treat FAP+ leiomyosarcoma. In some cases, the method can be used to treat uterine leiomyosarcoma, cutaneous leiomyosarcoma, gastrointestinal leiomyosarcoma, gastric leiomyosarcoma, small intestinal leiomyosarcoma, retroperitoneal leiomyosarcoma, or abdominal leiomyosarcoma. In some embodiments, the method is used to treat FAP+ rhabdomyosarcoma. In some embodiments, the method can be used to treat a rhabdomyosarcoma selected from the group consisting of: embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, spindle cell rhabdomyosarcoma, mixed rhabdomyosarcoma, pleomorphic rhabdomyosarcoma, and rhabdomyosarcoma with ganglionic differentiation. In some embodiments, the method is used to treat FAP+UPS.

[0109] In a fourth aspect, the present invention provides the use of an anti-fibroblast activation protein alpha (FAP) antibody-lysin conjugate or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for treating FAP+ sarcoma, wherein the antibody-lysin conjugate has the formula A-(LD)p, wherein A is an anti-FAP antibody that selectively binds to FAP, L is a linker, D is a drug comprising a lysin and p is 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.

[0110] Any 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.

[0111] The invention includes combinations of the described aspects and preferred features unless such combinations are clearly impermissible or stated to be explicitly avoided.These and further aspects and embodiments of the invention are described in further detail below and with reference to the accompanying examples and drawings.

[0112] sequence

[0113] In the following sequences, the VH and VL domains are underlined and the CDRH / CDRL region is in bold. Mutations that result in ADCC and CDC deficiencies are shown in bold italics. Signal sequences (where applicable) are shown in boxes.

[0114] hu36IgG1-HC – with signal sequence:

[0115]

[0116] hu36-IgG1-LC–with signal sequence:

[0117]

[0118]

[0119] hu36-IgG1-HC – no signal sequence:

[0120]

[0121] hu36-IgG1-LC–no signal sequence:

[0122]

[0123] hu36-VH:

[0124]

[0125] hu36-VL:

[0126]

[0127] hu36-CDRH1:

[0128] ENIIH(SEQ ID NO:7)

[0129] hu36-CDRH2:

[0130] WFHPGSGSIKYNEKFKD(SEQ ID NO:8)

[0131] hu36-CDRH3:

[0132] HGGTGRGAMDY(SEQ ID NO:9)

[0133] hu36-CDRL1:

[0134] RASKSVSTSAYSYMH(SEQ ID NO:10)

[0135] hu36-CDRL2:

[0136] LASNLES(SEQ ID NO:11)

[0137] hu36-CDRL3:

[0138] QHSRELPYT(SEQ ID NO:12)

[0139] Human FAP

[0140] Also known as Seprase, 170 kDa melanoma membrane-bound gelatinase, fibroblast activation protein alpha, or integral membrane serine protease. The amino acid sequence is published under UniProt accession number Q12884 (version 140, dated December 11, 2013).

[0141] (SEQ ID NO:13)

[0142] Mouse FAP

[0143] Also known as fibroblast activation protein alpha or integral membrane serine protease. The amino acid sequence is published under UniProt accession number P97321 (version 117, dated December 11, 2013).

[0144] (SEQ ID NO:14)

[0145] BRIEF DESCRIPTION OF THE DRAWINGS

[0146] Now, embodiments and experiments illustrating the principles of the present invention will be discussed with reference to the accompanying drawings, in which:

[0147] Figure 1.FAP staining of fibrosarcoma and leiomyosarcoma histological subtypes. (A) Fibrosarcoma with negative FAP staining. (B) Leiomyosarcoma with high-level FAP staining in tumor cells. FAP was detected with DAB (brown) on hematoxylin-stained slides. Scale bar 500 μm.

[0148] Figure 2 . FAP staining of the PDX leiomyosarcoma model SA4033. FAP positive immunohistochemical staining in (A) negative control and (B) PDX tumor sections from the leiomyosarcoma model SA4033. Scale bar 2 mm.

[0149] Figure 3 . Antitumor effect of OMTX705 after 4 weeks of treatment in a humanized PDX leiomyosarcoma cancer model. (A) Mice bearing SA4033 PDX tumors were treated with: vehicle, OMTX705 monotherapy group 1 with 10 mg / kg, and OMTX705 monotherapy group 2 with 30 mg / kg, intravenously (iv), QW x 4 weeks (days 1, 8, 15, 22). (B) Evolution of body weight change (%). (C) Percentage of tumor volume inhibition in different treatment groups.

[0150] Figure 4 . Immunohistochemical staining of FAP in tumor samples from leiomyosarcoma PDX mouse models showing different expression levels. (A) High FAP intensity expression pattern in tumors from the SA4033 model. (B) Moderate FAP intensity expression pattern in tumors from the SA3839 model. (C) Low-null FAP intensity expression pattern in tumors from the SA10202 model. FAP was detected with DAB (brown) on hematoxylin-stained slides. Scale bar 1 mm.

[0151] Figure 5 . Antitumor effect of OMTX705 in SA4033, SA3839 and SA10202 leiomyosarcoma PDX cancer models after 4 weeks of treatment (weekly iv doses). (A) Evolution of tumor volume in mice bearing each PDX tumor treated with vehicle, OMTX705 monotherapy group 1 at 10 mg / kg, and in SA4033, OMTX705 monotherapy group 2 at 30 mg / kg (days 1, 8, 15, 22). Monitoring was maintained after the end of treatment until day 63 (SA4033) or day 49 (SA3839) to check tumor regression. (B) Evolution of body weight change (%). (C) Percentage of tumor suppression volume in different PDX models at day 24.

[0152] Figure 6. Immunohistochemical staining on FFPE sections of vehicle (left bar) versus OMTX 705 treated (right bar) tumor samples from leiomyosarcoma SA4033 PDX mice extracted on day 28 of treatment. (A,B) Hematoxylin and eosin. (C,D) Ki 67. (E,F) Active caspase 3. (G,H) FAP. (I,J) TAM558 (OMTX705 loading). (K,L) Human IgG. (Scale bar 200 μm).

[0153] Figure 7 . Illustrative description of the conjugate OMTX705, as described in Fabre et al. (2020). As shown, OMTX705 comprises a humanized anti-FAP mAb (OMTX005) conjugated to TAM558, which is formed by coupling the cytolysin TAM470 with a protease-cleavable vcPABA-(EG)3 optimized linker. DETAILED DESCRIPTION

[0154] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated herein by reference.

[0155] In describing the present invention, the following terminology will be used and is intended to be defined as indicated below.

[0156] FAP

[0157] As used herein, "Fibroblast activation protein", "fibroblast activating protein", "FAP" and "FAPα" are used interchangeably. FAP can be a FAP of any mammalian species. In some cases, FAP is human FAP (also known as Seprase, 170 kDa melanoma membrane-bound gelatinase, fibroblast activation protein α or integral membrane serine protease), whose amino acid sequence is disclosed as UniProt Accession No. Q12884 (version 140, dated December 11, 2013) (SEQ ID NO: 13). In some cases, a molecule that binds to FAP (such as an antibody molecule or a conjugate thereof) can bind to a region of the extracellular domain of FAP. The extracellular domain of human FAP includes residues 26-760 of the full-length human FAP protein. In some cases, the FAP is murine FAP (also known as fibroblast activation protein alpha or integral membrane serine protease), the amino acid sequence of which is disclosed as UniProt Accession No. P97321 (version 117, dated December 11, 2013) (SEQ ID NO: 14). The extracellular domain of murine FAP includes residues 26-761 of the full-length murine FAP protein.

[0158] Conjugate

[0159] As used herein, "conjugate" includes the resultant structure formed by linking molecules, and specifically includes antibody-drug conjugates (ADCs) and immunotoxins (ITs).

[0160] Selective Binding

[0161] The terms "selectively bind" and "selective binding" refer to the binding of an antibody or its binding fragment to a predetermined molecule (such as an antigen) in a specific manner. For example, an antibody or its binding fragment can bind to a predetermined molecule (such as an antigen) in a specific manner. 7 M -1 The antibody can bind to FAP (such as an extracellular portion thereof) with an affinity greater than 10 times greater than that with which it binds to a molecule other than the predetermined molecule, and can bind to the predetermined molecule with an affinity that is at least two times greater (such as five times or ten times greater) than that with which it binds to a molecule other than the predetermined molecule.

[0162] Antibody molecules

[0163] As used herein, with reference to all aspects of the invention, the term "antibody" or "antibody molecule" includes any immunoglobulin, whether natural or partially or completely synthetically produced. The term "antibody" or "antibody molecule" includes monoclonal antibodies (mAb) and polyclonal antibodies (including polyclonal antisera). Antibodies can be complete or fragments derived from complete antibodies (see below). Antibodies can be human antibodies, humanized antibodies, or antibodies of non-human origin. "Monoclonal antibodies" are homogeneous, highly specific antibody groups directed against a single antigenic site or "determinant" of a target molecule. "Polyclonal antibodies" include heterogeneous antibody groups directed against different antigenic determinants of a target molecule. The term "antiserum" or "antisera" refers to serum containing antibodies obtained from an immunized animal.

[0164] It has been shown that fragments of whole antibodies can function to bind antigens. Thus, references herein to antibodies (and to the methods, arrays and kits of the invention) encompass whole antibodies, and also encompass any polypeptide or protein comprising an antibody binding fragment. Examples of binding fragments are (i) L 、V H , C L and C H 1 domain; (ii) a Fab fragment consisting of V H and C H 1 domain; (iii) a Fd fragment consisting of a single antibody V L and V H Fv fragment composed of V H (v) isolated CDR regions; (vi) F(ab')2 fragments, i.e., bivalent fragments comprising two linked Fab fragments; (vii) single-chain Fv molecules (scFv), in which V H Domain and V L The domains are connected by a peptide linker, which allows the two domains to associate to form an antigen binding site; (viii) bispecific single-chain Fv dimers (WO 93 / 11161) and (ix) "diabodies", i.e. multivalent or multispecific fragments constructed by gene fusion (WO 94 / 13804; 58). Fv, scFv or diabody molecules can be connected by incorporating a linker V H and V L The CH3 domains are stabilized by disulfide bonds. Minibodies comprising scFvs linked to the CH3 domains can also be made.

[0165] With respect to antibody molecules, the term "selectively binds" may be used herein to refer to situations in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partner. The term also applies, for example, to situations in which an antigen binding site is specific for a particular epitope carried by many antigens, in which case a specific binding member carrying the antigen binding site will be able to bind to a variety of antigens carrying the epitope.

[0166] In some cases, according to the present invention, an antibody may be a fully human antibody.

[0167] Cytotoxic chemotherapeutic agents

[0168] In some cases, according to any aspect of the invention, the conjugate of the invention may be administered together with one or more other anti-tumor drugs (including but not limited to cytotoxic chemotherapeutic agents or anti-angiogenic agents or immunotherapeutic agents), or for administration together with one or more other anti-tumor drugs (including but not limited to cytotoxic chemotherapeutic agents or anti-angiogenic agents or immunotherapeutic agents) (whether simultaneously, sequentially or separately).

[0169] Cytotoxic chemotherapeutic agents are well known in the art and include anticancer agents such as:

[0170] Alkylating agents, including nitrogen mustards, such as mechlorethamine HN2, cyclophosphamide, ifosfamide, melphalan (L-sarcolysin), and chlorambucil; 10-ethyleneimines and methylmelamines, such as hexamethylmelamine, thiotepa; alkyl sulfonates, such as busulfan; nitrosoureas, such as carmustine (BCNU), lomustine (CCNLJ), semustine (methyl-CCN-U), and streptozoein (streptozotocin); and triazenes, such as dacarbazine (DTIC; dimethyltriazene imidazolecarboxamide);

[0171] Antimetabolites include folic acid analogs such as methotrexate (methotrexate); pyrimidine analogs such as fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR), and cytarabine (cytosine arabinoside); and purine analogs and related inhibitors such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG), and pentostatin (2'-deoxycofonnycin). Natural products include vinca alkaloids, such as vinblastine (VLB) and vincristine; epipodophyllotoxins, such as etoposide and teniposide; antibiotics, such as dactinomycin (actinomycin D), daunorabicin (daunomycin; daunomycin), doxorubicin, bleomycin, plicamycin (mithramycin), and mitomycin (mithramycin Q; enzymes, such as L-asparaginase; and Drug response modifiers, such as interferon α. ​​Miscellaneous agents, including platinum complexes, such as cis-DDP and carboplatin; anthraquinones, such as mitoxantrone and anthracyclines; substituted ureas, such as hydroxyurea; methylhydrazine derivatives, such as procarbazine (N-methylhydrazine, MIH); and adrenocortical hormone inhibitors, such as mitotane (o, p'-DDD) and aminoglutethimide; taxol and its analogs / derivatives; and hormone agonists / antagonists, such as flutamide and tamoxifen. Further preferably, the cytotoxic agent is gemcitabine Further preferably, the cytotoxic agent is paclitaxel bound to human serum albumin

[0172] Anti-angiogenic agents are well known in the art and include anti-cancer agents such as bevacizumab, itraconazole and carboxamidotriazole.

[0173] 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, including nivolumab (MDX1106) and pembrolizumab (MK-3475).

[0174] Pharmaceutical composition

[0175] The conjugate of the present invention may be included in a pharmaceutical composition together with a pharmaceutically acceptable excipient.

[0176] A pharmaceutically acceptable excipient may be a compound or combination of compounds that enters the pharmaceutical composition, which does not cause secondary reactions and which allows, for example, to facilitate the administration of the conjugate, increase its lifespan and / or its efficacy in the body or increase its solubility in solution. These pharmaceutically acceptable vehicles are well known and will be adjusted by those skilled in the art as a function of the mode of administration of the conjugate.

[0177] In some embodiments, the conjugates of the invention can be provided in a lyophilized form for reconstitution prior to administration. For example, the lyophilized conjugate can be reconstituted in sterile water and mixed with saline prior to administration to an individual.

[0178] The conjugate of the present invention is usually administered in the form of a pharmaceutical composition, which may include at least one component in addition to the conjugate. Therefore, the pharmaceutical composition may include pharmaceutically acceptable excipients, carriers, buffers, stabilizers or other materials well known to those skilled in the art in addition to the conjugate. Such materials should be nontoxic and should not interfere with the efficacy of the conjugate. The precise nature of the carrier or other materials will depend on the route of administration, which may be by bolus, infusion, injection or any other suitable route, as discussed below.

[0179] For intravenous administration, for example by injection, the pharmaceutical composition comprising the conjugate can be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has a suitable pH, isotonicity and stability. One skilled in the art will be able to use, for example, isotonic vehicles (such as sodium chloride infusion, Ringer's infusion, lactated Ringer's infusion) to prepare suitable solutions. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be used as needed, including buffers such as phosphates, citrates and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3'-pentanol; and m-cresol); 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; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN TM 、PLURONICS TMor polyethylene glycol (PEG).

[0180] Subjects

[0181] The subject can be a human, a companion animal (such as a dog or cat), an experimental animal (such as a mouse, a rat, a rabbit, a pig or a non-human primate), a domestic animal or a farm animal (such as 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 (such as an epithelial tumor) or classified as being at risk of developing cancer (such as an epithelial tumor). In some cases, the subject can be an experimental animal, such as a mouse model of cancer. In some cases, the subject can be a mammal (e.g., a human) diagnosed with an inflammatory condition (such as rheumatoid arthritis (RA)) or classified as being at risk of developing an inflammatory condition (such as rheumatoid arthritis (RA)). In particular, the subject can be a human suffering from RA.

[0182] cancer

[0183] The anti-FAP conjugates described herein can be used to treat a tumor in a mammalian subject. 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 an embryonal 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).

[0184] As the case may be, the features disclosed in the foregoing description or in the following claims or in the accompanying drawings (expressed in their specific form or from the perspective of means for performing the disclosed function or method or process for obtaining the disclosed result) may be utilized separately or in any combination of such features for implementing the invention in its various forms.

[0185] Although the present invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when this disclosure is given. Therefore, the exemplary embodiments of the present invention set forth above are considered to be illustrative and non-restrictive. Various changes may be made to the described embodiments without departing from the spirit and scope of the present invention.

[0186] To avoid any doubt, any theoretical explanations provided herein are for the purpose of improving the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations.

[0187] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0188] Throughout this specification, including the claims above, unless the context requires otherwise, the words "comprise" and "include" and variations such as "comprises", "comprising" and "including", will be understood to imply the inclusion of stated integers or steps or groups of integers or steps but not the exclusion of any other integers or steps or groups of integers or steps.

[0189] It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when a value is expressed as an approximation by using the preposition "about", it is understood that the particular value forms another embodiment. The term "about" in relation to a numerical value is optional and means, for example, + / - 10%.

[0190] The following is presented by way of example and should not be construed as limiting the scope of the claims.

[0191] Example

[0192] Materials and methods

[0193] Clinical samples

[0194] Patients affected by different types of bone and soft tissue sarcomas were selected to construct a commercial soft tissue sarcoma microarray (TMA). A full description can be found on the website of Biomax, Inc. (https: / / www.biomax.us / tissue-arrays / Soft_Tissue / SO809c and https: / / www.biomax.us / tissue-arrays / Bone_Cartilage / OS802c), including the clinicopathological characteristics of the patients.

[0195] Immunohistochemistry

[0196] Immunohistochemistry (IHC) detection of FAP was performed on a DISCOVERY ULTRA research instrument (F. Hoffman-La Roche, "Roche") using a rabbit monoclonal antibody against human FAP (1:25 dilution, Abcam-Cat. No. ab207178) diluted in antibody dilution buffer (Antibody Diluent, Ventana-Cat. No. 760-108). The staining protocol included a standard antigen retrieval step with Tris-EDTA buffer at pH 8, incubation with the primary antibody for 1 h at 37°C, and incubation with the secondary antibody (Roche Omni Map anti-rabbit, Ventana-Cat. No. 760-4311) for 20 min at 37°C. The secondary antibody was detected by DAB (Chrommo Map DAB Kit, Ventana-Cat. No. 760-159).

[0197] Immunohistochemistry scoring and automated H-score

[0198] The intensity of FAP staining was analyzed in the tumor stroma compartment and tumor epithelium. FAP intensity was scored by an independent pathologist on an optical 3-point intensity scale (0 to 2). Intensity mean values ​​were calculated from the two compartment scores to obtain case values. These three groups were subsequently reduced to two groups ("negative" and "positive").

[0199] The FAP-stained TMA was scanned with a scanner Olympus dotSlide version 2.1 at a resolution of 2 pixels per 1 μm. The digital image analysis procedure has been described in detail previously (Mezheyeuski et al. (2018)). In short, FAP intensity was evaluated based on marker expression levels and quantified as the number of positive cells per area unit. The analysis was performed in the matrix compartment and the H-score was used to classify the samples, considering a range from 0 to 300. The H-score combines the intensity and percentage of stained cells (Id et al. (2021)). The H-score average was calculated to divide the samples into two groups ("low H-score" and "high score").

[0200] Binarized FAP positive / negative and H-score low / high classification of cases was used in patients with clinicopathological features.

[0201] Example 1 - Leiomyosarcoma patients express high levels of FAP

[0202] As shown in Table 1A, the population showed a cohort with a wide range of diagnoses from 17 to 78 years old, 46.4% females and 57.1% males. FAP status showed significant correlation with different bone and soft tissue tumor types (p>0.001), but no correlation with age or gender (p=0.662 and p=0.662). All fibrosarcoma samples were unexpectedly classified as FAP negative (100%), while a high percentage of FAP positivity (75%) was shown by the leiomyosarcoma group.

[0203] Further analysis explored FAP positivity and pathological diagnosis based on the 3-point intensity scale (Table 1B). Interestingly, higher FAP intensity showed a significant correlation with leiomyosarcoma histology: in fact, 83.3% of FAP-positive samples diagnosed as leiomyosarcoma scored as high expression levels, and demonstrated a significant association between high FAP intensity and leiomyosarcoma histological subgroups (p = 0.015). Numerical H-score classification also identified high FAP expression in most leiomyosarcoma samples studied with significant results (p = 0.007) (Table 1C).

[0204] Figure 1 Representative cases of FAP-negative and FAP-positive staining are shown in Figure 2, including a fibrosarcoma with FAP-negative nuclei ( Figure 1 A) and high FAP intensity in leiomyosarcoma nuclei ( Figure 1 B).

[0205]

[0206]

[0207] Table 1A: FAP positivity and clinicopathological variables in fibrosarcoma and leiomyosarcoma patients (Chi-square exact test).

[0208] Samples were classified as negative or positive using visual scoring by a pathologist.

[0209]

[0210] Table 1B - Frequency of FAP intensity in fibrosarcoma and leiomyosarcoma histological subtypes (Chi-square exact test).

[0211]

[0212] Table 1C - Frequency of FAP H-scores in fibrosarcoma and leiomyosarcoma histological subtypes (Chi-square exact test).

[0213] Regarding the entire cohort of bone and soft tumor tissue samples, leiomyosarcoma showed the highest frequency of high FAP intensity (50%) compared to osteosarcoma (the largest histological group in the study): only 7.69% of osteosarcoma samples presented high FAP intensity (Table 2). The numerical H-score also identified leiomyosarcoma as the largest subset within the high FAP expression group (66.67%) (Table 3), which showed a significant correlation between high FAP intensity and different bone and soft tissue tumor subhistological groups (p<0.001). Among the top 15 groups with the highest values ​​of the FAP H-score, leiomyosarcoma patients were the most presenting (60%) (Table 4).

[0214] The data also highlighted that rhabdomyosarcoma had abundant FAP expression, with 50% of rhabdomyosarcoma samples having moderate or high FAP intensity (Table 2). This trend was reflected in the numerical H-score, which identified 60% of rhabdomyosarcoma samples as having moderate or high FAP intensity (Table 3).

[0215]

[0216]

[0217] Table 2: Correlation between FAP intensity within bone and chondrosarcoma subtypes (Chi-square exact test).

[0218]

[0219] Table 3: Correlation between numerical H-scores of FAP for bone and chondrosarcoma subtypes (Chi-square exact test).

[0220]

[0221]

[0222] Table 4: Pathological diagnosis of the top 15 samples with the highest H-scores.

[0223] Example 2 - Anti-tumor efficacy of OMTX705 in a leiomyosarcoma PDX mouse model

[0224] Since leiomyosarcoma has the highest incidence of all sarcoma subtypes, comprising up to 20% of all sarcomas, and lacks effective treatment (Kannan et al. (2022), Kasper et al. (2022)), we further investigated the antitumor efficacy of OMTX705 in this subtype using FAP-positive patient-derived xenograft (PDX) models.

[0225] PDX mouse models were selected from the pilot study using IHC FAP staining on FFPE sections from 12 different leiomyosarcoma tumor types. The PDX model SA4033 was selected for its high FAP expression ( Figure 2 ).

[0226] Primary human tumor xenograft tumor fragments (approximately 2-3 mm in diameter) were subcutaneously implanted into 6-9 week old female NOD / SCID mice for tumor development (model SA4033). Animals were examined daily for morbidity and mortality after tumor inoculation. When tumors reached approximately 150 mm 3 When the volume of OMTX705 reached 100 mg / kg, mice were randomly assigned to 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).

[0227]

[0228] Table 5: Treatment schedule for SA4033 model study. ROA: route of administration; iv: intravenous; BIW: twice weekly; QW: once weekly.

[0229] During routine monitoring, animals were examined for tumor growth and any effects of treatment on behavior, such as activity, food and water consumption, weight gain / loss (weight was measured twice a week after randomization), eye / hair matting, and any other abnormalities. Mortality and observed clinical signs of individual animals were recorded in detail.

[0230] After randomization, tumor volume was measured twice weekly in two dimensions using calipers and expressed in mm using the formula: V = (L x W x W) / 2 (where V is tumor volume, L is tumor length (longest tumor dimension), and W is tumor width (longest tumor dimension perpendicular to L)). 3 = represents volume. Dosing and 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.

[0231] OMTX705 efficacy was measured in vivo in the SA4033 PDX model of leiomyosarcoma, where OMTX705 was administered as a single agent at 10 and 30 mg / kg iv once weekly for four doses. Tumor growth inhibition was found to be 92.04% and 93.11%, respectively ( Figure 3 A). Interestingly, in this model for leiomyosarcoma, OMTX705 at a dose of 30 mg / kg as a single agent had similar antitumor effects compared to a lower dose (10 mg / kg), with no significant weight loss observed. Mice treated with 10 and 30 mg / kg doses of OMTX705 presented less than 4% body weight change per group during the study ( Figure 3 B). Figure 3C shows the percentage of tumor inhibition volume (TIV) for OMTX705 as a single agent in this model.

[0232] In the post-treatment observation on day 42, sustained tumor suppression and even tumor regression were observed in the group treated with 10 mg / kg OMTX705, where the mean tumor volume was 29.4 mm 3 , which is much lower than the mean tumor volume registered on day 0 (148.09 mm 3 ). On day 42, 4 of the 10 animals were tumor-free. By the end of the post-treatment observation period, 100% of the mice in this group were tumor-free. Therefore, these data demonstrate a lasting effect of OMTX705 at weekly doses of 10 mg / kg, even 6 weeks after the end of treatment.

[0233] Example 3 - Antitumor efficacy of OMTX705 in leiomyosarcoma PDX mouse models with different FAP expression levels effect.

[0234] Following the promising results of Example 2 above, similar experiments were performed to compare the anti-tumor efficacy of OMTX705 against leiomyosarcoma in high to moderate FAP-positive (SA4033 and SA3839) versus low / null FAP-positive (SA10202) patient-derived xenograft (PDX) mouse models.

[0235] As above, PDX mouse models were selected from the pilot study using IHC FAP staining on FFPE sections from 12 different leiomyosarcoma tumor types. PDX models SA4033 and SA3839 were selected for their high to moderate FAP expression ( Figure 4 A and 4B), and SA10202 as FAP low-null expression ( Figure 4 C).

[0236] For each model, 6-9 week old female NOD / SCID mice were subcutaneously implanted with primary human tumor xenograft fragments (approximately 2-3 mm in diameter). Following tumor inoculation, animals were examined daily for morbidity and mortality. When tumors reached approximately 150 mm 3 The mice were randomly assigned to groups (n=10 / group in SA4033 model, n=8 / group in SA3839 model, and n=6 / group in SA10202 model) at a volume of 1.25 μg / kg. Two different doses of OMTX705 (10 and 30 mg / kg) were administered intravenously in the SA4033 model, and one dose of 10 mg / kg was administered intravenously in the SA3839 and SA10202 models, once a week for 4 weeks (Table 6).

[0237]

[0238]

[0239] Table 6: Treatment schedule for SA4033, SA3839 and SA10202 model studies. ROA: route of administration; iv: intravenous; BIW: twice weekly; QW: once weekly.

[0240] As in Example 2, during routine monitoring, animals were examined for tumor growth and any effects of treatment on behavior, such as activity, food and water consumption, weight gain / loss (weight was measured twice a week after randomization), eye / hair matting, and any other abnormalities. Mortality and observed clinical signs of individual animals were recorded in detail.

[0241] After randomization, tumor volume was measured twice weekly in two dimensions using calipers and expressed in mm using the formula: V = (L x W x W) / 2 (where V is tumor volume, L is tumor length (longest tumor dimension), and W is tumor width (longest tumor dimension perpendicular to L)). 3 = represents volume. Dosing and 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.

[0242] In the SA4033 PDX model of leiomyosarcoma, the efficacy of OMTX705 measured as a single agent at 10 and 30 mg / kg was higher compared to the SA3839 and SA10202 PDX models ( Figure 5 A). In post-treatment observations on day 42 in SA4033 mice, sustained tumor suppression and even tumor regression were observed in the group treated with 10 mg / kg of OMTX705, with an average tumor volume of 29.4 mm 3 , which was much lower than the mean tumor volume registered on day 0 (148.09 mm 3 ). On day 42, 4 of 10 animals were tumor-free. By the end of the post-treatment observation period, 100% of the SA4033 mice in this group were tumor-free. This effect of weekly 10 mg / kg doses of OMTX705 in SA4033 animals was long-lasting: even 6 weeks after the end of treatment, no tumor regrowth was observed ( Figure 5 A, top). Interestingly, in the SA4033 model for leiomyosarcoma, a 30 mg / kg dose of OMTX705 as a single agent had similar antitumor effects compared to a lower dose (10 mg / kg), with no significant weight loss observed. During the SA4033 study, mice treated with 10 and 30 mg / kg doses of OMTX705 presented less than 4% body weight change per group ( Figure 5 B, top). (In Figure 5 The data shown in A and 5B (top) in the mouse model SA4033 are consistent with those in Examples 2, Figure 3 A and 3B). In the SA3839 model, mice treated with 10 mg / kg weekly doses in each group presented less than 9% body weight change during the study period, and OMTX705 was able to induce tumor growth inhibition and stasis, but not complete regression ( Figure 5 In a low / null FAP expression (SA10202) patient-derived xenograft (PDX) mouse model, mice treated with 10 mg / kg showed less than 5% body weight change per group during the study, and OMTX705 failed to induce tumor regression ( Figure 5 A and 5B, bottom).

[0243] exist Figure 5 The percentage of tumor inhibition volume (TIV) of OMTX705 as a single agent in SA4033 (90.2%), SA3839 (74.86%), and SA10202 (42.6%) at day 24 (3 days after the last dose), shown in C, demonstrates the differential efficacy of OMTX705 in these models and supports the correlation between FAP expression levels and the antitumor efficacy of OMTX705 in leiomyosarcoma.

[0244] Further immunohistochemistry studies of the SA4033 PDX model using different marker proteins suggest that the anti-tumor effects of OMTX705 in leiomyosarcoma are driven by different mechanisms of action compared to carcinoma. Figure 6 FFPE tissue samples from OMTX705-treated tumors showed very low cellularity ( Figure 6 B) and very few Ki67, active caspase 3, and FAP positive cells ( Figure 6 D, 6F, 6H), indicating direct and effective killing of FAP-positive tumor cells. Anti-TAM558 IHC revealed that OMTX705 loading was still present in tumor samples at least one week after the last dose ( Figure 6 J), which could explain the compound's persistent effect in preventing tumor recurrence after treatment has ended.

[0245] Together, these results support a direct and FAP-dependent effect on tumor cells, resulting in a more potent antitumor efficacy of OMTX705 at lower doses in leiomyosarcoma compared to carcinoma, where the compound acts through FAP-expressing CAFs.

[0246] summary

[0247] Previous results from a first-in-human phase I study of ABBV-085, a new ADC targeting the LRRC15 matrix protein, for the treatment of sarcomas showed high response rates independent of sarcoma subtype due to overall LRRC15 expression in many solid tumors (Demetri et al. (2021), Purcell et al. (2022)). Further experiments using PDX models of soft tissue sarcomas to study LRRC15 expression and ABBV-085 activity demonstrated significant antitumor activity in undifferentiated sarcomas (UPS), dedifferentiated liposarcoma, and leiomyosarcoma (Ben-Ami et al. (2020)).

[0248] Indeed, unexpectedly, our studies found highly FAP(+) leiomyosarcomas among other soft tissue sarcomas (i.e., liposarcoma or fibrosarcoma with very low or no FAP expression), thus identifying the leiomyosarcoma subtype as a new and highly specific indication for sarcoma for antitumor therapy with OMTX705.

[0249] The surprising finding that leiomyosarcoma tumor cells express FAP at high levels highlights the opportunity to treat leiomyosarcoma by directly targeting the cells with anti-FAP-cytolysin conjugates. Figure 3 The in vivo data presented in showed excellent tumor inhibition of 92.04% and 93.11% at 10 and 30 mg / kg intravenous doses, respectively, and Figure 5 Excellent tumor inhibition of 90.2% at 10 mg / kg on day 24 was demonstrated in a model with high FAP expression levels (SA4033) Figure 4 ). Moreover, OMTX705 treatment resulted in virtually complete tumor regression at later time points, with 100% of mice achieving a tumor-free state and no tumor recurrence up to 6 weeks after the end of treatment. Interestingly, models with moderate (SA3839) or low / null (SA10202) FAP expression levels showed a lower response to OMTX705, as indicated by %TVI of 74.86 and 42.6, respectively ( Figure 5 C). Thus, in addition to targeting FAP(+) CAFs, the data provide a novel mechanism of action for OMTX705, as originally shown in the context of pancreatic cancer (Fabre et al. (2020)), which would be related to FAP expression levels.

[0250] Both scoring systems (visual and numerical) reached the same conclusion: the sarcoma subtype with half-full and intense FAP staining was leiomyosarcoma, and far behind was osteosarcoma.

[0251] The data also revealed that rhabdomyosarcoma has significant FAP expression, thus identifying further targets for anti-FAP-cytolysin conjugates.

[0252] These data are the first preclinical evidence that targeting FAP with an antibody-drug conjugate is a promising strategy in patients with FAP-positive sarcomas.

[0253] Therefore, the findings presented in this study support a novel strategy and new potential indication for the OMTX705 therapeutic compound to treat patients with FAP+ sarcomas, such as leiomyosarcoma, with highly promising anti-tumor efficacy compared to previously tested antibody-drug conjugates.

[0254] Furthermore, these results further support the use of FAP as a biomarker for selecting patients with FAP+ sarcomas (such as the leiomyosarcoma soft tissue tumor subtype) with a higher potential response to the newly developed antibody-drug conjugate drug OMTX705.

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Claims

1. A compound having the formula A-(LD) p An anti-fibroblast activation protein alpha (FAP) antibody-lysin conjugate or a pharmaceutically acceptable salt or solvate thereof for use in a method for treating a sarcoma in a mammalian subject, wherein A is an anti-FAP antibody that selectively binds to FAP, L is a linker, D is a drug comprising a lysin and p is 1 to 10, wherein the sarcoma is characterized by FAP expression in the tumor.

2. The conjugate for use according to claim 1, wherein A has a heavy chain of the amino acid sequence SEQ ID NO: 3 and a light chain of the amino acid sequence SEQ ID NO: 4; and LD has the structure: Where * indicates the point of attachment to A.

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

4. A conjugate for use according to any preceding claim, wherein the conjugate is administered intravenously; optionally wherein the conjugate is administered weekly, biweekly or monthly; optionally wherein the conjugate is administered in a dose range of 0.1-30 mg / kg.

5. An in vitro method of selecting a subject identified as having a sarcoma for treatment with an anti-FAP ADC, the method comprising: a) measuring the level of FAP expression in a biological sample obtained from a subject; b) determining that the level of FAP expression is above a threshold level, and c) Selecting subjects for treatment with an ADC targeting FAP.

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

7. The in vitro method according to claim 5 or 6, wherein the anti-FAP ADC has the formula A-(LD) p , wherein A is an anti-FAP antibody and has a heavy chain with an amino acid sequence of SEQ ID NO: 3 and a light chain with an amino acid sequence of SEQ ID NO: 4; LD has the structure: Where * represents the point of attachment to A and p is 1 to 10.

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

9. The conjugate for use according to any one of claims 1, 3 or 4, wherein 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 wherein the anti-FAP antibody comprises a heavy chain complementary determining region 1-3 (CDRH1-3) and a light chain complementary determining region 1-3 (CDRL1-3) having the following amino acid sequence: 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.

10. The conjugate 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; Alternatively, 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 conjugate for use according to any one of claims 1, 3, 4 or 9 to 10, wherein the cytolysin comprises formula IV: in: R 2 is H or C1-C4 alkyl; R 6 is a C1-C6 alkyl group; R 7 It is C1-C6 alkyl, CH2OR 19 or CH2OCOR 20 , where R 19 is an alkyl group, R 20 is C2-C6-alkenyl, phenyl or CH2-phenyl; R 9 is a C1-C6 alkyl group; R 10 is H, OH, O-alkyl or O-acetyl; f is 1 or 2; R 11 Has the following structure: in: R 21 is H, OH, halogen, NH2, alkoxy, phenyl, alkylamino or dialkylamino; R 16 is H or a C1-C6-alkyl group; R 17 directly or indirectly attached to the connector L, and; q is 0, 1, 2, or 3; and wherein the term "optionally substituted" relates to groups in which one or several H atoms may be replaced by F, Cl, Br or I or OH, SH, NH2 or NO2; the term "optionally substituted" further relates to groups which may be replaced solely or additionally by unsubstituted C1-C6 alkyl, C2-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 The alkyl group is substituted with an aralkyl or C2-C11 heteroaralkyl group.

12. A conjugate for use according to any one of claims 1, 3, 4 or 9 to 11, wherein L comprises a spacer, optionally wherein the spacer comprises -(OCH2CH2) n –, where n is 2 to 5.

13. A conjugate for use according to any one of claims 1, 3, 4 or 9 to 12, wherein L comprises an attachment group for attachment to A; optionally wherein L comprises a protease cleavable portion comprising a valine-citrulline unit.

14. The conjugate for use according to any one of claims 11 to 13, wherein the lysin has the formula: where * indicates the site of attachment to L.

15. The conjugate for use according to any one of claims 1, 3, 4 or 9 to 14, wherein -LD has the structure: Where * indicates the point of attachment to A.

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