Anti-Trop2 antibodies in immunosuppressed subjects

Abstract

An object of the present invention is to provide a composition comprising a 4F6 anti-Trop-2 antibody and / or a conjugate thereof for use in the treatment of a Trop-2-expressing tumor in a subject wherein the subject is immunosuppressed by an anti-cancer treatment, such as chemotherapy or radiation therapy.

Description

Background Art

[0001] Trop-2 (AC: P09758), also known as EGP-1, T16, MR23, MR54, is a type I transmembrane protein encoded by the tumor-associated calcium signal transducer 2 (TACSTD2 / M1S1 / GA733-1) gene [Fornaro, 1995], which is a retrotransposon of the TROP1 / TACSTD1 / EPCAM gene [Linnenbach, 1993]. The extracellular domain (ECD, residues 27-274) of human Trop-2 contains a cysteine-rich N-terminal region containing a GA733 type 1 motif (residues 27-69) and a thyroglobulin type 1 domain (residues 70-148), followed by a cysteine-free C-terminal domain (residues 149-274) [Fornaro, 1995]. A single transmembrane helix connects the ECD to a 26-amino acid (AA) intracellular tail that contains a HIKE motif [Ciccarelli, 2000] and two PKC phosphorylation sites at Ser303 [Basu, 1995] and Ser322 [Mori, 2019], and drives downstream Ca 2+ , PKC and AKT signaling [Guerra, 2022; Guerra, 2016].

[0002] Trop-2 induces tumor and cancer stem cell growth [Stoyanova, 2012; Trerotola, 2013a, 2013b]. Upregulation of Trop-2 is associated with poor prognosis in pancreatic, gastric, ovarian, lung, and colorectal cancers [Relli, 2018; Guerra, 2021], suggesting that this molecule plays a key role in tumor progression [Trerotola, 2013a, 2013b].

[0003] We have demonstrated that Trop-2 is expressed in the majority of human tumors [Trerotola, 2013a]. DNA microarray, EST, SAGE, Northern blot, and RT-PCR analyses have shown that TROP2 mRNA is expressed in ovarian, NSCLC, prostate, breast, and colon cancer cell lines [Guerra, 2021], as well as in gastric, colon, breast, endometrial, kidney, lung, and ovarian human tumors [Trerotola, 2021; Guerra; 2021]. TROP2 mRNA expression has also been detected in normal human breast tissue, lung, uterus, pancreas, prostate, salivary glands, respiratory tract, thymus, kidney, and placenta [Trerotola, 2013a]. Immunohistochemical analysis of Trop-2 protein expression in more than 3,000 human tumors has shown that Trop-2 protein is highly expressed in the vast majority of malignant cancers (ranging between 64% and 90%) ( Figure 1 B).

[0004] Trop-2 in growth control

[0005] A direct role for Trop-2 in tumor development has been demonstrated both in vitro and in vivo. De novo Trop-2 expression in the mouse thymic immortalized cell line MTE 4-14 and the human ovarian tumor Igrov-1 or Trop-2 overexpression in the human colon cancer cell line KM12SM resulted in growth stimulation in both fully transformed and simply immortalized cells [Trerotola, 2013a], indicating that Trop-2 expression is necessary and sufficient to stimulate the growth of tumor cells.

[0006] Similar results were obtained in experimental tumors (nude mice injected subcutaneously with 293 or L tumor cells) [Trerotola, 2013a]. Trop-2 expression was shown to induce increased mitotic activity, nuclear pleomorphism, and the appearance of multinucleated giant cells. Tumor development was proportional to the expression level of Trop-2.

[0007] Deletion of the Trop-2 cytoplasmic tail abolished Trop-2-dependent growth stimulation, indicating that this region plays a key role in Trop-2 signaling [Guerra, 2013; Guerra, 2022]. The tail contains a serine residue (S303) that is phosphorylated by PKC [Basu, 1995]. Mutagenesis of S303 abolished growth stimulation, indicating that the stimulatory activity of Trop-2 is phosphorylation-dependent [Guerra, 2013; Guerra, 2022].

[0008] Proteomic and phosphoproteomic analysis of antibody arrays identified downstream molecules involved in Trop-2 signaling [Guerra, 2013; Guerra, 2022]. Transmembrane tyrosine kinase receptors (PDGFR, Met, Ret, VEGFR), soluble tyrosine kinases, serine / threonine kinases, phosphatases, cell cycle and apoptosis regulatory molecules showed Trop-2-induced expression modifications. Western blot analysis validated the microarray analysis of the reference set of molecules and phosphorylation sites. The results obtained showed Trop-2-dependent activation of PKCα, FAK and Raf-1, involvement of the PTEN-Akt-GSK3β-S6K pathway, regulation of ERK, JNK and p38 MAPK, induction of NF-B and regulation of apoptotic factors p53 and Rb. The regulation of key players of this model (e.g. ERK, cyclin D1, NFkB) was shown to depend on the presence of an intact Trop-2 cytoplasmic tail.

[0009] Role of Trop-2 in metastatic spread

[0010] Metastatic spread is the leading cause of death in many human cancers. In many cases, common tumors such as colon, breast, and lung cancers will produce metastases at the time of surgery. Metastatic tumors are often resistant to most currently available therapies [De Vita, 2001]. Therefore, a better understanding of the molecular mechanisms of tumor spread is essential for better treatment of advanced neoplastic disease. The identification of new markers of tumor invasiveness and metastatic potential could help identify the most aggressive cases at an early stage and provide new targets for new therapies.

[0011] TROP2 is the only gene overexpressed in metastatic cells in different experimental systems. Large-scale analysis of Trop-2 expression in human primary cancers and their corresponding metastases showed that Trop-2 is overexpressed in metastases of colon, gastric, breast, and ovarian tumors [Guerra, 2021; Trerotola, 2021]. This was confirmed by Northern and Western blots and immunohistochemical analysis.

[0012] The pathogenic role of Trop-2 in metastatic spread was demonstrated using HCT116 U.5.5 colon cancer cells and by transfecting the KM12SM colon cancer cell line with wild-type or mutated Trop-2. The transfected cells were then injected into the spleen of nude mice and the metastatic potential was assessed. The expression of Trop-2 was shown to induce increased metastatic spread to the liver (90% of cases). The expression of Trop-2 also altered the growth pattern, apoptosis induction, cell morphology, cell replication rate, and metastatic dimensions [Guerra, 2021; Trerotola, 2021].

[0013] The high frequency and high expression levels of Trop-2 in human tumors and corresponding metastases make this molecule an attractive target for adoptive immunotherapy. Most anti-Trop-2 monoclonal antibodies (mAbs), among which MOv16 [Alberti, 1992], cAR47A6.4.2 [Truong, 2007], 162-46.2 [Alberti, 1992], Pr1E11 [Ikeda, 2015], AbT16 [Trerotola, 2021] bind to one and the same immunodominant epitope in the D146-L274 region [Ikeda, 2015] [Alberti, 1992].

[0014] Current anti-Trop-2 targeted antibody therapies are severely hampered by limited efficacy and unmanageable toxicity. In this context, the main obstacle to next-generation therapeutic strategies remains the widespread expression of Trop-2 in normal tissues [Trerotola, 2010]. We found that activation of Trop-2 to drive tumor progression requires proteolytic activation of ADAM10 in cancer cells [Trerotola, 2021; Guerra, 2021]. Importantly, it was found that cleavage of Trop-2 by ADAM10 does not occur in normal tissues [Trerotola, 2021; Guerra, 2021].

[0015] This provides potential for a cancer Trop-2-only targeted therapeutic strategy. Structure / function-guided analysis was performed to identify the cleavage activation signaling trigger region of Trop-2. Then, accessibility-guided activation site identification was performed to target cancer-exposed sites to exploit this unique cancer vulnerability in patients [Kim, 2022; Alberti, 2022]. In fact, it is well known how important it is for anti-tumor monoclonal antibodies (i.e., targeting Her2 / neu) to target specific parts of the target molecule, which plays an important role in determining the efficacy of anti-tumor treatment [Johnson, 2006].

[0016] This also includes the possibility that antibody binding can produce direct functional inhibition of antigen-promoting growth and pro-metastatic signaling, leading to tumor growth inhibition independent of the recruitment of immune system cells (i.e., T and B lymphocytes). This is particularly important because most anticancer immunotherapy regimens are administered to patients in combination with proliferation-inhibiting chemotherapy, which includes paclitaxel or anthracyclines and cyclophosphamide. These can cause immunosuppression directly by killing anticancer T and B lymphocytes, or indirectly by inducing immunosuppressive cell types or soluble circulating factors [Cersosimo, 2003]. Tumors themselves can cause immunosuppression, which has led to the use of antibodies directed against immune checkpoints to counteract it [Brody, 2011]. Different chemotherapy regimens can be identified that can differentially modulate the response to immune checkpoint inhibitors [Voorwerk, 2019].

[0017] Damage caused by chemotherapy can induce metastasis. Tumor cell repair responses to chemotherapy are carried out through local invasion, intravasation, hematogenous dissemination and extravasation of tumor cells, and effective colonization at metastatic sites [Karagiannis, 2018].

[0018] Experimental evidence suggests that cancer treatments induce host-mediated local and systemic responses that can support tumor progression. Treatment with doxorubicin, but not cisplatin, induces the expression of complement factors in lung fibroblasts and modulates an immunosuppressive prometastatic microenvironment that supports lung metastasis [Monteran, 2022]. Other mechanisms include treatment-induced immunity, angiogenesis, and pro-metastatic effects [Shaked, 2019]. In response to chemotherapy and radiotherapy, cancer-associated fibroblasts and endothelial cells produce elevated levels of systemic proinflammatory molecules such as interleukin-6 (IL-6) and IL-8, respectively. These secreted factors regulate the activity of myeloid cells, including macrophages, which can act as inhibitors of antitumor T and B lymphocytes, thereby promoting tumor immune escape [Shaked, 2019]. Treatment of healthy animal models with paclitaxel, cisplatin, or gemcitabine chemotherapy results in increased lung metastasis after tumor cell inoculation, due to increased levels of circulating matrix metalloproteinase 9, SDF1, and granulocyte colony-stimulating factor (G-CSF). Circulating growth factors and cytokines induced by chemotherapy support tumor angiogenesis. Oxaliplatin induces upregulation of circulating VEGFA. In addition, paclitaxel leads to increased circulating levels of SDF1, G-CSF, and VEGFC, increasing angiogenesis and tumor growth. Radiation therapy has also been reported to induce a pro-angiogenic shift [Shaked, 2019].

[0019] In a parallel context, the function of T and B lymphocytes has been shown to require topoisomerase I activity, as lymphocytes proliferate and activate immune responses upon encountering target antigens. Therefore, chemotherapeutic drugs that inhibit topoisomerase activity may alter systemic innate immune cell function and lead to pro-tumor activity [Shaked, 2019]. In addition, in response to chemotherapy, B cells release extracellular vesicles systemically, leading to impaired CD8+ T cell function, thereby promoting immunosuppression [Shaked, 2019]. In response to oxaliplatin, doxorubicin, or cyclophosphamide chemotherapy, ATP released by tumor cells is converted to adenosine, leading to abnormal CD8+ T cell function [Shaked, 2019]. In response to 5-fluorouracil or gemcitabine chemotherapy, myeloid suppressor cells promote pro-inflammatory local and systemic responses, thereby aiding tumor progression [Shaked, 2019].

[0020] All of these immunosuppressive mechanisms severely affect cells that mediate antibody-dependent cell-mediated cytotoxicity (ADCC), such as NK cells and antitumor cytotoxic T lymphocytes, and may contribute to resistance to antibody-based anticancer therapies.

[0021] Therefore, there is an urgent need to generate new, homogeneous and high-affinity anti-Trop-2 monoclonal antibodies, especially those targeting specific functional regions of the molecule, which, on the basis of ADCC, can also directly inhibit the tumorigenic effects of Trop-2 for effective utilization in biomedical applications. Summary of the invention

[0022] New anti-Trop-2 monoclonal antibodies were obtained with the goal of optimizing their recognition of specific functional regions of the target molecule. To achieve this goal, a strategy for producing monoclonal antibodies by immunization with recombinant Trop-2 proteins produced in baculovirus and mammalian cells was developed, as they are expected to maintain the structure, folding, processing and glycosylation properties of the native molecule. This led to the selection of anti-Trop-2 hybridomas by ELISA testing. These are performed on recombinant Trop-2 proteins (full-length or partial proteins) to optimize reactivity with native Trop-2 proteins with correct folding and glycosylation. The second step of anti-Trop-2 hybridoma selection was performed by cytofluorimetry for the reactivity of L cells transfected with TROP2, using a vector-only control transfectant as a negative control.

[0023] We found that in cancer cells, Trop-2 is proteolytically activated by ADAM10 [Trerotola, 2021; Guerra, 2021]. This exposes a previously inaccessible protein groove flanked by two glycosylation sites. We designed a strategy to identify these targets to exploit selective cancer vulnerabilities in patients. Our strategy was designed based on structure-function analysis of the Trop-2 ECD [Trerotola, 2021; 2021]. This led to the realization that the N-terminal small subunit, which is cleaved from Trop-2 by ADAM10 cleavage at R87-T88 [Trerotola, 2021], masks the groove of the Trop-2 ECD, rendering it largely inaccessible in the wt / uncleaved conformation. Following ADAM10-mediated cleavage, the N-terminal small subunit is bound to the core Trop-2 structure only via the Cys73-Cys108 disulfide bond, and the Trop-2 structure undergoes a dramatic rearrangement to enable targeting of the groove thus exposed.

[0024] Through a selective immunization and screening strategy, we successfully generated a family of mAbs that recognize this region in an ADAM10 cleavage-dependent manner. The requirement for glycosylation of flanking sites supports efficient recognition of the target groove in Trop-2 exposed to cleavage ( Figure 2 ).

[0025] Specifically, it is necessary to find high-affinity anti-Trop-2 monoclonal antibodies targeting specific functional regions of the molecule, which can exert direct inhibition on the growth of cancer cells on the basis of ADCC. This new therapeutic strategy is also suitable for patients who are immunosuppressed by chemotherapy because it does not require interaction with functional immune cells (such as cytotoxic T lymphocytes, B lymphocytes and NK cells).

[0026] WO2010089782 describes the 4F6 monoclonal antibody, which specifically recognizes activated Trop-2 in cancer cells. Therefore, the direct inhibition of cancer cell growth by the 4F6 anti-Trop-2 monoclonal antibody was measured in vitro in the absence of immune system cells. To avoid complement-dependent cytotoxicity (CDC), we used heat-inactivated serum products in all growth media. Heat inactivation at 56°C for 30 minutes destroyed complement activity without affecting the growth-promoting ability of the serum. Surprisingly, we observed for the first time that the 4F6 antibody was able to inhibit cancer cell growth in vitro in the absence of ADCC and CDC ( Figure 4 , Figure 5 Growth inhibition was specific for cells expressing Trop-2, as control vector-only transfectants were unaffected ( Figure 4 , Figure 5).

[0027] These results suggest that 4F6 has the potential to be used for effective anticancer therapy in patients even in the presence of cancer- and chemotherapy-associated immunosuppressive mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : Expression of Trop-2 in human tumors. (A) Expression of Trop-2 protein in cancer. IHC analysis of Trop-2 protein expression in human breast cancer samples. (Left) Staining with anti-Trop-2 mouse monoclonal antibody. Reactivity to intracellular Trop-2 deposits is prominent [Ambrogi, 2014]. (Right) Staining with anti-Trop-2 goat polyclonal antibody. A general reactivity to the activated form of Trop-2 on the cancer cell membrane was observed [Ambrogi, 2014]. Bar = 30 μm. (B) Frequency of expression of Trop-2 in human tumors. The tumor categories analyzed are listed on the X-axis. The height of the band is proportional to the frequency of expression of Trop-2 (the total frequency is listed at the top of the band). The absolute number of cases analyzed is listed within each frequency bar.

[0030] Figure 2 : 3D structure of Trop-2. (A) Side and top (i.e., extracellular) views of the human Trop-2 dimer model [ 2021#36406], truncated in the middle of the transmembrane helix (horizontal black bar). The two views are related to each other by rotating 90° about the horizontal direction, as indicated by the arrow symbol. The black circle and black rectangle surround the groove between the glycans at N120 and N208, which becomes more accessible after ADAM10 cleavage and rearrangement of the N-terminal ADAM10 cleavage subunit. The Trop-2 molecule in dimer is shown as a ribbon diagram. Residues N120 and N208 are shown as spheres, and the N-glycans located at these sites are shown as sticks. Residue N168 is shown as a light gray sphere, and its N-glycans are shown as light gray sticks. The two N-terminal ADAM10 cleavage subunits (aa 27-87) are shown as dark gray spheres. The N33-linked glycans on this part of the molecule are shown as dark gray sticks. The C73-C108 disulfide bond that tethers the ADAM10 cleavage subunit to the rest of the protein is shown as a black sphere. (Top) Model of the Trop-2 dimer before cleavage. (Bottom) Model of the cleaved Trop-2 dimer, with the two N-terminal small subunits (aa 27-87) likely in a new orientation after ADAM-10 cleavage, retaining the C73-C108 disulfide bond. (B) Full-length Trop-2 ECD. The N-terminal subunit is located at the top of the structure (grey sphere in the center figure). The surface sphere model is shown with the ribbon diagram (right).

[0031] Figure 3: Inhibition of tumor growth (human cancer cells) after treatment with 4F6 anti-Trop-2 antibody. Non-metastatic HT29 colon cancer cells (top) or metastatic HCT116 U5.5 colon cancer cells (bottom) xenografts treated with 4F6 anti-Trop-2 mAb (solid black line) or PBS vehicle alone (dashed line) from the time of injection. Bars represent standard error of the mean of tumor volume measurements.

[0032] Figure 4 : Inhibition of ovarian cancer cell growth after treatment with 4F6 anti-Trop-2 antibody. (A) OVCAR-3 cancer cells expressing Trop-2 were treated with 4F6 antibody (solid black line) or PBS vehicle alone (dashed line). (B) Igrov-1 cancer cells transfected with Trop-2 (solid black line) or vector alone (control, dashed line) were treated with 4F6 anti-Trop-2 antibody. Bars represent standard error of the mean of cell number measurements.

[0033] Figure 5 : Inhibition of MTE4-14 transformed cell growth after 4F6 anti-Trop-2 antibody treatment. (A) MTE4-14 transfected with wtTrop-2 were treated with 4F6 antibody (solid black line) or PBS vehicle alone (dashed line) as indicated. (B) MTE4-14 control vector-only transfectants were treated with 4F6 antibody (solid black line) or PBS vehicle alone (dashed line) as indicated. Bars represent standard error of the mean of cell number measurements.

[0034] Figure 6 : Growth inhibition of the metastatic HCT116U5.5 colon cancer cell line treated with the 2G10 (comparative) or 4F6 (according to the present invention) anti-Trop-2 monoclonal antibodies. Control tumors were treated with an antibody with irrelevant specificity (anti-dansyl: dotted line). The cells were treated in a prophylactic manner, i.e. by co-injection of tumor cells and antibodies. The antibodies were administered intraperitoneally (ip) once a week for a total of 4 treatments. The bars represent the standard error of the mean value measured. The 4F6 anti-Trop-2 antibody taught in the present invention is much more effective than the comparative 2G10 anti-Trop-2 antibody when treating metastatic cell lines.

[0035] Figure 7 : Alignment of the VL sequences of 2G10 (SEQ ID NO: 5) and 4F6 (SEQ ID NO: 4) antibodies. DETAILED DESCRIPTION OF THE INVENTION

[0037] The invention presented herein is directed to the use of 4F6 anti-Trop-2 monoclonal antibodies and fully humanized or chimeric antibodies thereof (in which the mouse constant region is replaced by a human constant region [Oi, 1983 #11830]) or variants containing at least one complementary determining region (CDR) of the variable region of the corresponding light chain and / or heavy chain (these variants may be mutagenized to alter their affinity for the target) or antibody fragments, Fv, Fab, F(ab)2 fragments, single chain or multimeric 4F6 anti-Trop-2 antibodies in anti-cancer therapy. In one embodiment, the antibodies, fragments or antibody chimeras comprised in the invention described herein are from, or engineered in, IgM, IgD, IgG, IgA or IgE isotypes.

[0038] Another object of the invention proposed herein is the conjugation of the 4F6 antibody, wherein the antibody is conjugated to a biologically active partner, such as avidin or its derivatives, growth factors or toxins, cytokines, anti-tumor drugs and radioactive isotopes or other biologically active and / or biologically active partners that can be used to enhance the anti-tumor therapeutic effect. For example, the conjugate is selected from the group consisting of:

[0039] (a) Toxicity payload, optimized for anticancer efficacy;

[0040] (b) a linker optimized for conjugation of a toxic payload with high anticancer potency;

[0041] (c) emit beta particles of radioactive isotopes;

[0042] (d) emitting alpha particles of radioisotopes for use in radioimmunotherapy;

[0043] (e) Positron emitters for PET and PET-CT to image Trop-2 expressing tumors and metastases.

[0044] The present invention aims to use 4F6 anti-Trop-2 antibodies conjugated to sequences, single residues or synthetic molecules (tags) for affinity chromatography purification in experiments, diagnosis and therapy. These tags can be used as detection molecules (e.g., radioisotopes or fluorescent tags) or enzyme tags that can catalyze the modification of visible substrates, both for laboratory diagnostic purposes and for imaging.

[0045] The invention presented herein is intended for use in diagnostic applications including, but not limited to, optical, confocal, multiphoton and electron microscopy, ELISA, Western blotting, immunoprecipitation, radioimmunoassay and similar techniques.

[0046] In another embodiment, an object of the present invention is to provide a composition comprising 4F6 anti-Trop-2 antibody and / or its conjugate for use in treating a subject having a tumor expressing Trop-2, wherein the subject is immunosuppressed.

[0047] In another embodiment, an object of the invention is to provide a method of treating a subject affected by a tumor expressing Trop-2, wherein the subject is immunosuppressed, using the 4F6 antibody or its conjugate alone, or in combination with other therapeutic interventions (e.g., other antibody-drug conjugates or other engineered molecules) before or after tumor removal.

[0048] The invention presented herein is directed to the use in therapy, in particular the systemic or local (e.g., intraperitoneal, intrahepatic arterial or intratumoral) administration of the 4F6 anti-Trop-2 antibody and its conjugates. An indicative but non-exhaustive list of therapeutic targets based on anti-Trop-2 antibodies includes endometrial, breast, head and neck, colorectal, gastric, lung, ovarian, prostate, pancreatic, cervical and bladder (urothelial) tumors.

[0049] The objects of the invention presented herein also include therapeutic compositions containing the 4F6 antibody or its conjugate. These can be provided as a composition or carrier or macro / micro or nano container or nano structure containing the antibody or its conjugate and a pharmaceutically acceptable delivery agent or diluent. The therapeutic composition can be used to treat diseases in mammals such as humans. According to conventional practices in the art, the 4F6 anti-Trop-2 antibody will be included in a pharmaceutical composition or a diagnostic kit.

[0050] Another object of the invention proposed herein is to use the 4F6 anti-Trop-2 antibody conjugate in patient imaging by labeling the 4F6 anti-Trop-2 antibody with a radioisotope or a fluorescent tracer (e.g., quantum dots or organic chromophores or enzymes detectable by chemiluminescence) by techniques and procedures known in the art. The signal generated by the labeled anti-Trop-2 antibody can be detected by a scanner or tomography instrument according to the principles of currently used, e.g., TAC / PET equipment.

[0051] Another object of the present invention is the use of radioisotope-conjugated 4F6 anti-Trop-2 antibody in anti-cancer therapy.

[0052] The mouse-derived 4F6 anti-Trop-2 monoclonal antibody and its conjugates can be produced in reasonable quantities by expert researchers in the field.

[0053] The coding regions (open reading frames, ORFs) and amino acid sequences corresponding to the 4F6 monoclonal antibody VH and VL are as follows:

[0054] VH (SEQ ID NO: 1)

[0055] CAGCTGCAGCAGTCTGGAGCTGAGGTGGTGAAGCGTGGGGCTTCAGTGAAGCTGTCCTGCAAGACTTCTGGCTTCACCTTCAGCAGTAGCTATATAAGTTGGTTGAAGCAGAAGCCTCGACAGAGTCTTGAGTGGATTGCATGGATTATGCTGGAACTGGTGGTACTAGCTATAATCAGAAGTTCACAGGCAAGGCCCAACTGACTGTAGACACATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCCATCTATTACTGTGCAAGACATAACCCTTGTTACTATGCTATGGATTACTGGGGTCAAGG

[0056] VH amino acid sequence (SEQ ID NO: 2)

[0057] DVQLEQFGAELVRPGTSVKMSCKAAGYTFTNYWIGWVKQRPGHGLEWIGDIYPGGGYTNYNEKFKGKATLTADTSSSTAYMQLSSLTFEDFAIYYCARGTGGGDYWGQG

[0058] VL (SEQ ID NO: 3):

[0059] ATGTTCTCACTAGCTCTTCTCCTCAGTCTTCTTCTCCTCTGTGTCTCTGATTCTAGGGGCAGAAACAACTGTGACCCAGTCTCCAGCATCCCTGTCCATGGCTATAGGAGAAAAAGTCTCCATCAGATGCATAACCAGCACTGATATTGATGATGATATGAACTGGTACCAGCAGAAGCCAGGGGAACCTC CTAAGCTCCTTATTTCAGAAGGCAATACTCTTCGTCCTGGAGTCCCATCCCGATTCTCCAGCAGTGGCTATGGTACAGATTTTGTTTTTACAATTGAAAACATGCTCTCAGAAGATGTTGCAGATTACTACTGTTTGCAAAGTGATAACTTGCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[0060] VL amino acid sequence (SEQ ID NO: 4)

[0061] MFSLALLLSLLLLCVSDSRAETTVTQSPASLSMAIGEKVSIRCITSTDIDDDMNWYQQKPGEPPKLLISEGNTLRPGVPSRFSSSGYGTDFVFTIENMLSEDVADYYCLQSDNLPYTFGGGTKLEIK

[0062] Notably, the 4F6 VH and VL presented herein are highly related to the 2G10 VH and VL sequences described in WO2010 / 089782. Figure 7 An alignment of the VL sequences of 2G10 (SEQ ID NO: 5) and 4F6 (SEQ ID NO: 4) is shown. Surprisingly, this difference between the two antibodies was sufficient to increase the selective activity of 4F6 against metastatic cancer cells relative to the activity observed with 2G10.

[0063] Similar procedures are also applicable to antibodies and their fragments, such as Fv, Fab and F(ab)2 fragments and single-chain or multimeric anti-Trop-2 antibodies, including engineered versions thereof to obtain different isotypes (IgM, IgD, IgG, IgA, IgE) and fully or partially humanized mouse variable chains.

[0064] Nucleic acid molecules encoding anti-Trop-2 immunoglobulins can be generated by experts in the field using established techniques (e.g., oligonucleotide synthesis or PCR amplification). Once the nucleic acid molecules are synthesized, they can be cloned into expression vectors, as known in the art.

[0065] Another method of implementing the present invention proposed herein includes obtaining the cells expressing 4F6 included in the present invention, for example, by transfection. Transfection methods are known, and transfection kits can be purchased from commercial sources (e.g., Invitrogen, Carlsbad, Ca; Stratagene, La Jolla, Calif.).

[0066] Another method for implementing the present invention proposed herein is to detect or diagnose neoplastic, pre-neoplastic and non-neoplastic diseases expressing the target molecules of these antibodies by IHC or immunofluorescence, and these antibodies can be used for the identification and quantification of these diseases. Further diagnostic methods include Western blotting and its derivatives, in which proteins are fractionated by gel electrophoresis and then detected by specific antibodies. Example

[0067] The following are examples that illustrate methods of practicing the present invention. These examples should not be construed as limiting.

[0068] Example 1: In vivo inhibition of human cancer cell growth after treatment with 4F6 anti-Trop-2 antibody.

[0069] Colon cancer cell lines were injected subcutaneously (5-10x10 6 cells) into 8-week-old female athymic CD1-Foxn1 nu / nu mice. Figure 3 ) were treated with 4F6 anti-Trop-2 mAb or PBS vehicle only, with 800 μg / mouse of antibody administered intraperitoneally weekly in sterile PBS. Treatment was administered weekly for 4 weeks starting from injection. The longest / shortest diameter (D / d) of the tumor was measured every 5-7 days, and the tumor volume was calculated as an ellipsoid (Dxd 2 / 2) [Guerra, 2021]. Taking into account the standard error of the mean of tumor volume measurements, Sigma Stat (SPSS Science Software UK Ltd.) and GraphPadPrism (GraphPad Software Inc., La Jolla, Ca) were used to compare tumor growth curves using ANOVA [Rossi, 2008] and t-tests with post hoc Bonferroni correction.

[0070] Example 2: Efficacy of 4F6 treatment in inhibiting cancer cell growth.

[0071] OVCAR-3 and IGROV-1 ovarian cancer cells were cultured at 1.5-3×103 1 μg / well of purified 4F6 in sterile PBS or vehicle alone as a control was added to the cell culture. Cell number was quantified by crystal violet staining every 24 hours [Orsulic, 2002]. The standard error of the mean of the cell number measurements was determined ( Figure 4 ).

[0072] Example 3: Direct inhibition of human cancer growth following treatment with 4F6 due to specificity for cells expressing Trop-2 The growth of disease cells.

[0073] IGROV-1 and MTE4-14 cells expressing Trop-2 were transfected with Trop-2 at a rate of 1.5-3×10 3 Cells / well were seeded in 96-well plates (5 replicate wells per data point). Cells transfected with empty vector were used as controls. Purified 4F6 or vehicle alone (for MTE4-14 transfectants) at 1 μg / well in sterile PBS was added to the cell cultures and cell number was quantified by crystal violet staining every 24 hours [Orsulic, 2002]. The standard error of the mean (SD) of the cell number measurements was determined. Figure 4 B, Figure 5 ).

[0074] Example 4: Inhibition of metastatic human leukemia following treatment with 4F6 due to specificity for cells expressing Trop-2 Cancer cell growth.

[0075] The metastatic HCT116 U5.5 colon cancer cell line expressing Trop-2 was injected into the flanks of nude mice (i.e., immunosuppressed mice) at a rate of 5 × 10 cells per injection site. 6 cells. Mice were treated with 2G10 (comparative) or 4F6 (according to the present invention) anti-Trop-2 monoclonal antibodies. Control tumors were treated with an antibody with irrelevant specificity (anti-dansyl: dotted line). Cells were treated in a prophylactic manner, i.e. by co-injection of tumor cells and antibodies. Antibodies were administered intraperitoneally (ip) once a week for a total of 4 treatments. The bars represent the standard error of the mean measured. The 4F6 anti-Trop-2 antibody taught in the present invention was much more effective than the comparative 2G10 anti-Trop-2 antibody in treating metastatic cell lines.

[0076] Materials and methods

[0077] Flow cytometry. Binding of monoclonal antibodies to the surface of tumor cells and transfected cells was analyzed by flow cytometry on non-transformed and transformed cell lines. Staining and analysis were performed essentially as described [Alberti, 1994]. Unstained negative controls (i.e., untransfected or mock-transfected cells) were used and the amount of monoclonal antibody that stained positive cells was as close to saturation as possible. In most cases, 2 x 10 cells / mL were added to 200 μl. 50.3 μg MAb per cell proved to be sufficient. To improve the detection of expressing cells, subtraction of cellular autofluorescence and overcompensation in the red channel were performed [Alberti, 1991]. The latter approach allowed us to distinguish between truly expressing cells and cells that took up shed antigen. Forward scatter, side scatter, and propidium iodide gating were routinely used to eliminate dead cells and debris from the analysis.

[0078] Trop-2 structural analysis. A homology model of the ECD thyroglobulin-like repeats was constructed on the p41 splice variant of the major histocompatibility complex class II-related invariant chain (PDB ID 1ICF, chain I) [Trerotola, 2021; Guncar, 1999]. The mature portion of human Trop-2, residues 27–323 (Uniprot P09758 (TACD2_HUMAN) was subsequently modeled as a cis-dimer, similar to the dimer in human Trop-1 / EpCAM crystals (PDB ID 4MZV [Pavsic, 2014; Fornaro, 1995; Zanna, 2007]) and the dimer in the NMR-derived model of the transmembrane region of rat p75 protein (PDB ID 4MZV, residues 278–298 [Nadezhdin, 2016]). These protein models were used as templates. The cytoplasmic domain of human Trop-2 (portion 299–323) was inherited from the NMR structure of the C-terminus of Trop-2 (PDB ID 2MAE [Pavsic, 2015]).

[0079] The analysis of human Trop-2 ECD was verified by the crystal structure in PDB ID 7PEE [Pavsic, 2015]. The structure file was obtained from the Protein Data Bank (www.rcsb.org / ). Swiss-PdbViewer (www.expasy.ch / swissmod / SWISS-MODEL.html) and PyMol (pymol.org / 2 / ) were used for graphical rendering of the 3D structure. N-glycosylation was added to Asn residues N33, N120, N168, and N208 using the GlyProt server (www.glycosciences.de / modeling / glyprot / php / main.php). The list of residues at the interface between the N-terminal cleavage portion and the transmembrane portion of Trop-2, as well as the buried region in this interface, were calculated on the PISA server (www.ebi.ac.uk / pdbe / pisa / ; www.ebi.ac.uk / pdbe / prot_int / pistart.html).

[0080] DNA transfection. Cells were transfected with DNA in Lipofectamine 2000 or LTX (Invitrogen) according to the manufacturer's instructions [Alberti, 1994]. Stable transfectants were selected in medium containing G-418.

[0081] ELISA. ELISA assay plates were coated overnight at 4°C with 100 μl / well of 1 μg / ml recombinant human Trop-2-IgFc chimeric protein (rhTROP-2; R&D, catalog number 650-T2-100) in 0.2 M sodium carbonate buffer (pH 9.4). The well surface was blocked with 300 μl / well of blocking buffer (2% skim milk, 0.05% Tween-20 in PBS) for 30 minutes at room temperature. The plates were washed twice with washing buffer (PBS, 0.05% Tween-20). Purified antibodies or supernatants were added to the plates at 100 μl / well in serial 3-fold dilutions starting from 5-10 μg / ml. All dilutions were performed in blocking buffer. The antibody-containing plates were incubated at room temperature for 1 hour. Then, the plates were washed 3 times with washing buffer. Antibody binding was visualized with 100 μl / well of 1:2000 dilution of goat anti-human kappa HRP (Southern Biotech, catalog number 2060-05) in blocking buffer, incubated at room temperature for 30 minutes, and washed 4 times with wash buffer. HRP activity was visualized with 100 μl / well ABTS substrate (AMRESCO, Solon, OH), activated with 20 μl 30% H2O2 per 10 ml ABTS solution. The reaction was stopped with 100 μl 2% oxalic acid per well. Absorbance was read at 405 nm.

[0082] Immunohistochemistry (IHC). IHC was performed on normal and neoplastic human tissues as described previously [Ambrogi, 2014]. Briefly, specimens were fixed in phosphate-buffered formalin, pH 7.2, and embedded in paraffin. 5 μm sections were mounted on silanized slides, dewaxed, and rehydrated through graded alcohols to water. Endogenous peroxidase activity was eliminated by incubation with 3% H2O2 for 5 min. Antigen retrieval was performed by microwave treatment at 750 W for 10 min in 1 M urea buffer, pH 8.0.

[0083] The sections were incubated with 162-46.2 anti-Trop-2 mAb (ATCC clone HB187) at a dilution of 1:500. Figure 1 A, left) or AF650 anti-Trop-2 goat pAb (R&D Systems) ( Figure 1A, right) and incubated for 30 min. To control for nonspecific reactions, non-immune serum or isotype-matched immunoglobulins (DAKO) were used instead of specific primary antibodies. Anti-mouse (K4001, EnVision kit; DAKO) and anti-goat (K0679, LSAB kit; DAKO) secondary pAbs were used for signal amplification as needed. Slides were washed in Tris-buffered saline-Tween 20 and incubated in 3,3'-diaminobenzidine (DAKO) for 10 min. Counterstaining was performed with hematoxylin. Slides were mounted with Immunomount (Shandon).

[0084] Trop-2 expression was quantified as the percentage of stained cells and the intensity of staining. An IHC score (H score) was obtained (range: 0 to 12). The expression rate was divided into five categories: 0 (0% of positive cells), 1 (<10% of positive cells), 2 (10-50% of positive cells), 3 (50-80% of positive cells), 4 (>80% of positive cells). The intensity score divided the average intensity of positive cells into 1 (weak staining), 2 (moderate staining) or 3 (strong staining). Then, the positivity rate and the intensity score were multiplied to obtain the final H score to rank the overall Trop-2 expression of the tumor ( Figure 1 B).

[0085] In vitro cell growth assay. OVCAR-3 cells expressing Trop-2 or IGROV-1 and MTE 4-14 cells transfected with empty vector or Trop-2 were cultured at 1.5-3 × 10 3 Cells / well were seeded in 96-well plates (5 replicate wells per data point). 1 μg of purified 4F6 in sterile PBS or vehicle alone as a control was added to the cell cultures and the effect on cell growth was measured. Cell number was quantified by crystal violet staining [Orsulic, 2002 #4340].

[0086] Experimental tumors. Cancer cell lines expressing Trop-2 were injected subcutaneously (5-10x10 6 Cells) were injected into 8-week-old female athymic CD1-Foxn1 nu / nu mice (Charles River Laboratories, Calco, Lecco, Italy). The longest / shortest diameter (D / d) of the tumors was measured every 5-7 days. The tumor volume was calculated as an ellipsoid (Dxd 2 / 2) [Guerra, 2021]. Anti-Trop-2 mAb or vehicle treatment alone was administered intraperitoneally weekly for 4 weeks starting from the day of inoculation at 800 μg / mouse of the antibody in sterile PBS.

[0087] Statistical analysis. Student t test was used to compare the mean protein levels of control and TROP2 transfectants. The normality of the distribution of the measured values ​​was verified (www.graphpad.com). Two-tailed Fisher's exact test was used to compare protein expression levels in normal and tumor samples. 50 Values ​​are calculated from dose-response data fitted to a 4-parameter logistic nonlinear regression model. Spearman nonparametric correlation coefficients were calculated for protein expression levels in human cancer samples. Analysis of variance [Rossi, 2008#14010] and t-tests with post-hoc Bonferroni correction were used to compare and evaluate tumor growth curves. Data were analyzed using Sigma Stat (SPSS Science Software UK Ltd.) and GraphPad Prism (GraphPad Software Inc., La Jolla, Ca).

[0088] result

[0089] The therapeutic efficacy of 4F6 anti-Trop-2 mAb was tested in vivo using human cancer cells injected into nude mice. Two colon cancer models that endogenously express Trop-2 were used: HT29 colon cancer cells and the metastatic HCT116U5.5 clone, which was derived from the non-metastatic HCT116 colon cancer cell line and was selected in vivo for metastatic capacity after upregulation of Trop-2 expression [Guerra, 2021].

[0090] HT29( Figure 3 , above) and HCT116 U5.5 ( Figure 3 , lower figure) Colon cancer cell lines were injected subcutaneously into the flanks of nude mice, and tumor volume was measured every 5-7 days [Guerra, 2021]. Mice bearing xenografts were treated with 800 μg of endotoxin-free 4F6 mAb once a week for four weeks starting at the time of cancer cell injection. Mice in the control group received only PBS vehicle, which has been shown in previous experiments to be comparable to irrelevant isotype-matched mAbs. Tumor growth curves were evaluated using analysis of variance [Rossi, 2008] and t-tests with post hoc Bonferroni corrections, taking into account the standard error of the mean of tumor volume measurements 35 days after tumor cell injection ( Figure 3 ). These experiments showed that tumor growth was statistically significantly inhibited in mice injected with the 4F6 antibody, indicating the feasibility of 4F6 for anti-cancer therapeutic purposes.

[0091] Using the same method, the efficacy of 4F6 anti-Trop-2 antibody and 2G10 anti-Trop-2 antibody in inhibiting the growth of metastatic human cancer cells was compared. The metastatic HCT116 U5.5 colon cancer cell line expressing Trop-2 was injected into the flank of nude mice (i.e., immunosuppressed mice) at 5×10 cells / mL per injection site. 6 cells. Mice were treated with 2G10 (comparison) or 4F6 (according to the invention) anti-Trop-2 monoclonal antibodies. Control tumors were treated with an antibody with irrelevant specificity (anti-dansyl: dashed line). Cells were co-injected with the listed antibodies. 800 μg of endotoxin-free antibody / mouse was administered intraperitoneally once a week for a total of 4 treatments. Tumor growth curves were evaluated using analysis of variance [Rossi, 2008] with a t-test with a post hoc Bonferroni correction, taking into account the standard error of the mean of tumor volume measurements ( Figure 6 ). These experiments showed that the tumor growth of mice injected with 4F6 antibody was statistically significantly inhibited. These experiments also showed that the 4F6 anti-Trop-2 antibody taught in the present invention was much more effective than the comparative 2G10 anti-Trop-2 antibody in treating metastatic cell lines, indicating the feasibility of 4F6 for anti-cancer treatment purposes in immunosuppressed patients with advanced stage (i.e., tumor metastasis spread).

[0092] Chemotherapy-induced immunosuppression impedes ADCC cytotoxicity and may lead to resistance to antibody-based anticancer therapy. The function of T and B lymphocytes requires topoisomerase I activity, and chemotherapy drugs that inhibit topoisomerase activity alter immune cell function [Shaked, 2019]. The corresponding inhibitory signal is derived from ATP released by tumor cells in response to chemotherapy such as oxaliplatin, doxorubicin or cyclophosphamide [Shaked, 2019]. In response to 5-fluorouracil or gemcitabine chemotherapy, myeloid suppressor cells contribute to tumor progression [Shaked, 2019].

[0093] Therefore, there is an urgent need to generate new, homogeneous and high-affinity anti-Trop-2 monoclonal antibodies that target specific functional regions of the molecule and that can directly inhibit the tumorigenic effects of Trop-2 on the basis of ADCC so that they can be effectively utilized in biomedical applications. As described below, our results show that 4F6 possesses this important property.

[0094] Since 4F6 can directly inhibit the growth of cancer cells, it is important to show that 4F6 can exert a direct inhibitory effect on the tumorigenic effects of Trop-2. Therefore, a cell culture growth assay was established to show that the efficacy of 4F6 treatment is specific to the expressed Trop-2 target protein.

[0095] IGROV-1 and MTE4-14 transfectants expressing Trop-2 were cultured at 1.5-3×10 3 Cells / well were seeded on a 96-well plate. Cells transfected with an empty vector were used as controls. After attachment to the substrate, 1 μg of purified 4F6 in sterile PBS or only the loading material was added to the cell culture as a control for the MTE4-14 transfectant. At 72 hours after inoculation, the cell number was quantified by crystal violet staining [Orsulic, 2002#4340] to measure the effect of 4F6 treatment on cell growth. Specific inhibition of cell growth by treatment with 4F6 anti-Trop-2 antibodies was obtained in IGROV-1 ovarian cancer cells expressing Trop-2. On the other hand, no results were found for the empty vector mock transfectant ( Figure 4 B). Compared with empty vector mock transfection and vehicle-only treatment, MTE4-14 cells expressing Trop-2 also showed further specific inhibition of cell growth after treatment with 4F6 anti-Trop-2 antibody ( Figure 5 ).

[0096] Thus, the 4F6 anti-Trop-2 antibody produced significant direct inhibition of cancer cell growth, suggesting that 4F6 can inhibit tumor growth even in the absence of ADCC, such as in patients who are immunosuppressed by chemotherapy or radiation therapy.

[0097] Finally, the 4F6 antibody was tested on HCT116 U5.5 cells and compared with the 2G10 antibody. Colon cancer cells were co-injected subcutaneously into the flanks of nude mice with the indicated antibodies (4F6 according to the invention; 2G10 for comparison, or anti-dansyl for control), and tumor volume was measured every 5-7 days. The experiment showed that the tumor growth of mice co-injected with the 4F6 antibody was statistically significantly inhibited. The inhibitory effect was poor when the 2G10 antibody was injected ( Figure 6 ). Of note, different activities of the two antibodies were observed on the colon cancer metastatic clone HCT116U5.5 cell line. When tested on HT29 cells (non-metastatic tumor cells), the 4F6 antibody was not more effective. These data confirm the favorable selective effect of the 4F6 antibody on activated Trop-2, which induces metastasis, and indicate the feasibility of 4F6 for anticancer therapeutic purposes in advanced immunosuppressed patients and tumor metastatic spread.

[0098] It is noteworthy that the 4F6 VH and VL presented herein are highly related to the 2G10 VH and VL sequences described in WO2010 / 089782. Surprisingly, the differences in the VL sequences between the two antibodies ( Figure 7) is sufficient to increase the selective activity and recognition of 4F6 for Trop-2 in metastatic cancer cells.

[0099] References:

[0100] Alberti, S., C. Bucci, M. Fornaro, A. Robotti and M. Stella (1991). "Immunofluorescence analysis in flow cytometry: better selection of antibody-labeled cells after fluorescence overcompensation in the red channel." J. Histochem. Cytochem. 39(5): 701-706.

[0101] Alberti, S., S. Miotti, M. Stella, CE Klein, M. Fornaro, S. Ménard and MIColnaghi (1992). "Biochemical characterization of Trop-2, a cell surface molecule expressed by human carcinomas: formal proof that the monoclonal antibodies T16 and MOv-16 recognize Trop-2." Hybridoma 11:539-535.

[0102] Alberti, S., M. Trerotola, and E. Guerra (2022). "The Hu2G10 tumor-selectiveanti–Trop-2 monoclonal antibody targets the cleaved-activated Trop-2and shows therapeutic efficacy against multiple human cancers." Cancer Res 82(12_Supplement):340.

[0103] Alberti, S., M. Nutini, and L. A. Herzenberg (1994). "DNA methylation prevents the amplification of TROP1, a tumor associated cell surface antigen gene." Proc. Natl. Acad. Sci. USA 91:5833-5837.

[0104] Ambrogi, F., M. Fornili, P. Boracchi, M. Trerotola, V. Relli, P. Simeone, R. La Sorda, R. Lattanzio, P. Querzoli, M. Pedriali, M. Piantelli, E. Biganzoli, and S. Alberti (2014). "Trop-2 is a determinant of breast cancer survival." PLoS One 9(5):e96993.

[0105] Basu, A., D. M. Goldenberg, and R. Stein (1995). "The epithelial / carcinoma antigen EGP-1, recognized by monoclonal antibody RS7-3G11, is phosphorylated on serine 303." Int J Cancer 62(4):472-479.

[0106] Brody, J., H. Kohrt, A. Marabelle, and R. Levy (2011). "Active and passive immunotherapy for lymphoma: proving principles and improving results." J Clin Oncol 29(14):1864-1875.

[0107] Cersosimo, R. J. (2003). "Monoclonal antibodies in the treatment of cancer, Part 2." Am J Health Syst Pharm 60(16):1631-1641.

[0108] Ciccarelli, F., A. Acciarito and S. Alberti (2000). "Large and diverse numbers of human diseases with HIKE mutations." Hum. Mol. Genet. 9: 1001 - 1007.

[0109] De Vita, V. T., S. Hellman and S. A. Rosenberg (2001). Cancer - Principles and Practice of Oncology. Philadelphia, Lippincott J. B. Co.

[0110] Fornaro, M., R. Dell'Arciprete, M. Stella, C. Bucci, M. Nutini, M. G. Capri and S. Alberti (1995). "Cloning of the gene encoding TROP - 2, a cell - surface glycoprotein expressed by human carcinomas." Int. J. Cancer 62: 610 - 618.

[0111] Guerra, E., V. Relli, M. Ceci, R. Tripaldi, P. Simeone, A. L. Aloisi, L. Pantalone, R. La Sorda, R. Lattanzio, A. Sacchetti, K. Havas, S. Guarnieri, D. Vergara, I. Fournier, M. Salzet, N. Tinari, M. Piantelli, M. Trerotola and S. Alberti (2022). "Trop - 2, Na+ / K+ ATPase, CD9, PKCα, cofilin assemble a membrane signaling super - complex that drives colorectal cancer growth and invasion." Oncogene 41(12): 1795 - 1808.

[0112] Guerra, E., M. Trerotola, A. L. Aloisi, R. Tripaldi, G. Vacca, R. La Sorda, R. Lattanzio, M. Piantelli and S. Alberti (2013). "The Trop-2 signalling network in cancer growth." Oncogene 32:1594-1600.

[0113] Guerra, E., M. Trerotola, V. Relli, R. Lattanzio, R. Tripaldi, G. Vacca, M. Ceci, K. Boujnah, V. Garbo, A. Moschella, R. Zappacosta, P. Simeone, R. de Lange, U. H. Weidle, M. T. Rotelli, A. Picciariello, R. Depalo, P. Querzoli, M. Pedriali, E. Bianchini, D. Angelucci, G. Pizzicannella, C. Di Loreto, M. Piantelli, L. Antolini, X.-F. Sun, D. F. Altomare and S. Alberti (2021). "Trop-2 induces ADAM10-mediated cleavage of E-cadherin and drives EMT-less metastasis in colon cancer." Neoplasia 23(9):898-911.

[0114] Guerra, E., M. Trerotola, R. Tripaldi, A. L. Aloisi, P. Simeone, A. Sacchetti, V. Relli, D. A. A, R. La Sorda, R. Lattanzio, M. Piantelli and S. Alberti (2016). "Trop-2 induces tumor growth through Akt and determines sensitivity to Akt inhibitors." Clin Cancer Res 22(16):4197-4205.

[0115] Guncar, G., G. Pungercic, I. Klemencic, V. Turk and D. Turk (1999). "Crystal structure of MHC class II-associated p41 Ii fragment bound to cathepsin L reveals the structural basis for differentiation between cathepsins L and S." EMBO J. 18:793-803.

[0116] Ikeda, M., M. Yamaguchi, K. Kato, K. Nakamura, S. Shiina, T. Ichikawa-Ando, H. Misaka, K. Myojo, Y. Sugimoto and H. Hamada (2015). "Pr1E11, a novel anti-TROP-2 antibody isolated by adenovirus-based antibody screening, recognizes a unique epitope." Biochem Biophys Res Commun 458(4):877-882.

[0117] Johnson, B. E. and P. A. Janne (2006). "Rationale for a phase II trial of pertuzumab, a HER-2 dimerization inhibitor, in patients with non-small cell lung cancer." Clin Cancer Res 12(14 Pt 2):4436s-4440s.

[0118] Karagiannis GS, Condeelis JS, Oktay MH. Chemotherapy-induced metastasis: mechanisms and translational opportunities. Clin Exp Metastasis. 2018 Apr;35(4):269-284.

[0119] Kim H, Guerra E, Baek E, Jeong Y, You H, Yu B, Jang T, Saverio A, Chung C-W, Park C. (2022) LCB84, a TROP2-targeted ADC, for treatment of solid tumors that express TROP-2 using the hu2G10 tumor-selective anti-TROP2 monoclonal antibody, a proprietary site-directed conjugation technology and plasma-stable tumor-selective linker chemistry Cancer Res 82(12_Suppl): Abstract nr 328.

[0120] Linnenbach, A.J., B.A. Seng, S. Wu, S. Robbins, M. Scollon, J.J. Pyrc, T. Druck and K. Huebner (1993). "Retroposition in a family of carcinoma-associated antigen genes." Mol Cell Biol 13(3): 1507-1515.

[0121] Monteran L, Ershaid N, Doron H, Zait Y, Scharff Y, Ben-Yosef S, Avivi C, Barshack I, Sonnenblick A, Erez N. Chemotherapy-induced complement signaling modulates immunosuppression and metastatic relapse in breast cancer. Nat Commun. 2022 Oct 2;13(1):5797.

[0122] Mori, Y., K. Akita, K. Ojima, S. Iwamoto, T. Yamashita, E. Morii and H. Nakada (2019). "Trophoblast cell surface antigen 2 (Trop-2) phosphorylation by protein kinase C alpha / delta (PKC alpha / delta) enhances cell motility." J Biol Chem 294(30):11513-11524.

[0123] Nadezhdin, K.D., I. García-Carpio, S.A. Goncharuk, K.S. Mineev, A.S. Arseniev and M. Vilar (2016). "Structural Basis of p75 Transmembrane Domain Dimerization." J Biol Chem 291(23):12346-12357.

[0124] Orsulic, S., Y. Li, R.A. Soslow, L.A. Vitale-Crosss, J.S. Gutkind and H.E. Varmus (2002). "Induction of ovarian cancer by defined multiple genetic changes in a mouse model system." Cancer Cell 1(1):53-62.

[0125] M. (2021). "Trop2 Forms a Stable Dimer with Significant Structural Differences within the Membrane-Distal Region as Compared to EpCAM." International Journal of Molecular Sciences 22(19).

[0126] Pavsic, M., G. Guncar, K. Djinovic-Carugo, and B. Lenarcic (2014). "Crystal structure and its bearing towards an understanding of key biological functions of EpCAM." Nature Communications 5.

[0127] Relli, V., M. Trerotola, E. Guerra, and S. Alberti (2018). "Distinct lung cancer subtypes associate to distinct drivers of tumor progression." Oncotarget 9(85):35528 - 35540.

[0128] Rossi, C., A. Di Lena, R. La Sorda, R. Lattanzio, L. Antolini, C. Patassini, M. Piantelli, and S. Alberti (2008). "Intestinal tumour chemoprevention with the antioxidant lipoic acid stimulates the growth of breast cancer." Eur J Cancer 44:2696–2704.

[0129] Shaked Y. The pro-tumorigenic host response to cancer therapies. Nat Rev Cancer. 2019 Dec;19(12):667 - 685.

[0130] Stoyanova, T., A. S. Goldstein, H. Cai, J. M. Drake, J. Huang, and O. N. Witte (2012). "Regulated proteolysis of Trop2 drives epithelial hyperplasia and stem cell self-renewal via beta-catenin signaling." Genes Dev 26(20):2271 - 2285.

[0131] Trerotola, M., P. Cantanelli, E. Guerra, R. Tripaldi, A. L. Aloisi, V. Bonasera, R. Lattanzio, R. de Lange, U. H. Weidle, M. Piantelli and S. Alberti (2013a). "Up-regulation of Trop-2 quantitatively stimulates human cancer growth." Oncogene 32 222-233.

[0132] Trerotola, M., E. Guerra and S. Alberti (2010). "Letter to the editor: efficacy and safety of anti-Trop antibodies." Biochim Biophys Acta 1805(2):119-120.

[0133] Trerotola, M., E. Guerra, Z. Ali, A. L. Aloisi, M. Ceci, P. Simeone, A. Acciarito, P. Zanna, G. Vacca, A. D'Amore, K. Boujnah, V. Garbo, A. Moschella, R. Lattanzio and S. Alberti (2021). "Trop-2 cleavage by ADAM10 is an activator switch for cancer growth and metastasis." Neoplasia 23(4):415-428.

[0134] Trerotola, M., D. Jernigan, Q. Liu, J. Siddiqui, A. Fatatis and L. Languino (2013b). "Trop-2 promotes prostate cancer metastasis by modulating β1 integrin functions." Cancer Res. 73(10):3155-3167.

[0135] Truong, A.H.L., N. Feng, D. Sayegh, B.C. Mak, K. O'Reilly, S.W. Fung, N. Ceric, S.H. Hahn, D. Pereira and H. Findlay (2007). "AR47A6.4.2, a functional naked monoclonal antibody targeting Trop-2, demonstrates in vivo efficacy in human pancreatic, colon, breast and prostate cancer models." Molecular Cancer Therapeutics 6(12):3334s-3334s.

[0136] Voorwerk L, Slagter M, Horlings HM, Sikorska K, van de Vijver KK, de Maaker M, Nederlof I, Kluin RJC, Warren S, Ong S, Wiersma TG, Russell NS, Lalezari F, Schouten PC, Bakker NAM, Ketelaars SLC, Peters D, Lange CAH, van Werkhoven E, van Tinteren H, Mandjes IAM, Kemper I, Onderwater S, Chalabi M, Wilgenhof S, Haanen JBAG, Salgado R, de Visser KE, Sonke GS, Wessels LFA, Linn SC, Schumacher TN, Blank CU, Kok M. Immune induction strategies in metastatic triple-negative breast cancer to enhance the sensitivity to PD-1 blockade: the TONIC trial. Nat Med. 2019 Jun;25(6):920-928.

[0137] Zanna, P., M. Trerotola, G. Vacca, V. Bonasera, B. Palombo, E. Guerra, C. Rossi, R. Lattanzio, M. Piantelli and S. Alberti (2007). "Trop-1 Are Conserved Growth Stimulatory Molecules That Mark Early Stages of Tumor Progression." Cancer 110(2):452 - 464.

Claims

1. A composition comprising 4F6 anti-Trop-2 antibody and / or its conjugate for use in treating a Trop-2 expressing tumor in a subject, wherein the subject is immunosuppressed by anti-tumor therapy including chemotherapy and radiation therapy.

2. A composition for use according to claim 1, wherein the treatment is performed prior to tumor removal.

3. A composition for use according to claim 1, wherein the treatment is performed after tumor removal.

4. The composition for use according to claim 1, wherein the treatment is performed in combination with other therapeutic interventions.

5. The composition for use according to claim 4, wherein the additional therapeutic intervention is the administration of an antibody-drug conjugate.

6. The composition for use according to any one of claims 1 to 5, wherein the 4F6 anti-Trop-2 antibody conjugate is a radioisotope-bound conjugate, such as a beta particle emitter or an alpha particle emitter.

7. A composition for use according to any one of claims 1 to 6, wherein the tumor is selected from endometrial tumors, breast tumors, colorectal tumors, gastric tumors, lung tumors, ovarian tumors, prostate tumors, pancreatic tumors, head and neck tumors, kidney tumors, bladder tumors and cervical tumors. 8.4F6 anti-Trop-2 antibody conjugated to a positron emitter for imaging of metastatic immunosuppressed patients by positron emission tomography or conjugated to a fluorescent tracer for detection of Trop-2 expressing tumors and metastases during surgery.

Images