Patent Application
20240400712
The content of the electronically submitted sequence listing (Name: 2149_0311_Sequence_Listing.txt, Size: 27,651 bytes, and Date of Creation: Dec. 5, 2023) is herein incorporated by reference in its entirety.
The present invention concerns a platform to obtain monoclonal antibodies, fragments and conjugates thereof that bind with high affinity processed, tumor-specific forms of proteins, for medical and industrial uses. More specifically, the invention concerns a method to obtain monoclonal antibodies, fragments and conjugates thereof that are able to bind with high affinity the Trop-2 protein in the processed form that is specific for tumors. The use of such method include the diagnostic, prognostic and therapeutic fields of Trop-2-expressing malignancies. The related tumors include, but are not limited to, cancers of the breast, head and neck, skin, colon-rectum, stomach, lung, ovary, thyroid, prostate, pancreas, endometrium, cervix, gallbladder, bile ducts, kidney, urinary bladder and chorocarcinomas.
Trop-2 (NCBI accession number NP_002344.2; SEQ ID NO: 1) is also known as tumor-associated calcium signal transducer 2 (TACSTD-2) (Fomaro, Dell'Arciprete et al. 1995, Calabrese, Crescenzi et al. 2001), GA733-1, EGP, MR23, MR54, RS-7 and T16 (Fradet, Cordon-Cardo et al. 1986, Stein, Chen et al. 1990, Alberti, Miotti et al. 1992). The authors of the present invention have shown that Trop-2 localizes along the cell membrane in epithelia (Alberti, Miotti et al. 1992), and functions as a signal transducer (Basu, Goldenberg et al. 1995, Ripani, Sacchetti et al. 1998), that induces an intracellular calcium signal after cross-linking with antibodies (Ripani, Sacchetti et al. 1998), and activates a growth-signaling network that converges on AKT (Guerra, Trerotola et al. 2016).
The extracellular domain of the Trop molecules (Trop-2 and the Trop-1/EpCAM paralog, with NCBI accession number NP_002345.2) contains a GA-733 (EGF-like) domain and a thyroglobulin repeat, that is host 12 conserved cysteine residues (Fomaro, Dell'Arciprete et al. 1995, El Sewedy, Fomaro et al. 1998, Calabrese, Crescenzi et al. 2001). This cysteine-rich region folds as a globular domain (aminoacids 31-145 of SEQ ID NO: 1) and is followed by a region devoid of cysteines (aminoacids 146-274 of SEQ ID NO: 1), that acts as a connecting “stem” to the transmembrane domain (
Trop-2 is overexpressed by most cancer cells in man (Trerotola, Cantanelli et al. 2013). Overexpression was found in breast cancer cells (Fradet, Cordon-Cardo et al. 1984, Govindan, Stein et al. 2004, Trerotola, Cantanelli et al. 2013, Ambrogi, Fomili et al. 2014), in bladder cancer (Fradet, Cordon-Cardo et al. 1984), ovarian serous papillary carcinomas (Santin, Zhan et al. 2004), and in numerous other tumors, e.g. non-small cell lung cancer (Stein, Chen et al. 1990), choriocarcinomas (Lipinski, Parks et al. 1981, Alberti, Miotti et al. 1992), colo-rectal, prostate and endometrial cancers (Ohmachi, Tanaka et al. 2006, Trerotola, Cantanelli et al. 2013). Transformation of keratinocytes with SV40 induced a 3- to 4-fold higher expression of Trop-2 compared to their normal counterparts (Schon and Orfanos 1995), consistent with a direct role in tumor progression. The authors of the present invention have shown that Trop-2 drives cancer development and progression through interaction and regulation of expression of several proteins involved in cell-cell and cell/matrix adhesion in epithelial tissues. Upregulation of TROP2 was shown to be both necessary and sufficient to stimulate tumor growth (Trerotola, Cantanelli et al. 2013), and Trop-2 was found to induce growth in direct proportion to its expression levels (Trerotola, Cantanelli et al. 2013). Further, expression of Trop-2 has been associated with poor prognosis of pancreatic (Fong, Moser et al. 2008), gastric (Muhlmann, Spizzo et al. 2009), lung (Kobayashi, Minami et al. 2010), oral (Fong, Spizzo et al. 2008), ovarian (Bignotti, Todeschini et al. 2010) and colo-rectal (Ohmachi, Tanaka et al. 2006) cancers. More specifically, membrane overexpression of Trop-2 (Lin, Huang et al. 2012) was associated with unfavourable prognosis of breast cancer (Ambrogi, Fomili et al. 2014). The authors of the present invention have also shown that Trop-2 can be revealed in intracellular compartments, in a heterogeneous manner across different tumor cases (Trerotola, Cantanelli et al. 2013) and that high intra-cellular Trop-2 expression levels are associated with improved outcome in breast cancer (Ambrogi, Fomili et al. 2014), thus indicating the usefulness of measuring Trop-2 sub-cellular localization for prognostic procedures.
Metastatic disease is the dominant cause of death in cancer patients (De Vita, Lawrence et al. 2008, Siegel, Miller et al. 2019), and is the greatest hurdle for cancer cure (Wan, Pantel et al. 2013), as metastatic cancer is largely resistant to therapy. The identification of proteins/genes linked to the metastatic phenotype (Nguyen, Bos et al. 2009, Polyak and Weinberg 2009, Hanahan and Weinberg 2011, Vanharanta and Massague 2013) is useful (Amit, Citri et al. 2007), as these markers can contribute to the identification of the aggressive cases at an early stage and provide new targets for novel therapies (De Vita, Hellman et al. 2001). This research was conducted by the authors of the present invention, who looked for genes that were concordantly dysregulated across independent cancer metastasis models. Following this approach, the authors of the present invention showed that TROP2 was the only gene consistently upregulated in metastatic cancers across different experimental settings, tumor types, and animal species. A large-scale analysis of Trop-2 expression in human primary cancers and their corresponding metastases revealed Trop-2 overexpression in the metastases from colon, stomach, breast, and ovary tumors. Trop-2 overexpression was then shown to drive metastasis and to affect the outcome of colon, stomach, breast, pancreas, lung, and ovary cancers. This showed that Trop-2 (Alberti, Miotti et al. 1992, Fomaro, Dell'Arciprete et al. 1995, El Sewedy, Fomaro et al. 1998, Ripani, Sacchetti et al. 1998, Trerotola, Cantanelli et al. 2013) is an inducer of tumor progression and metastatic diffusion (Guerra, Trerotola et al. 2008, Trerotola, Rathore et al. 2010, Stoyanova, Goldstein et al. 2012, Trerotola, Cantanelli et al. 2013).
The high expression levels of Trop-2 in many human tumors and in their metastases have made this molecule an attractive target for “adoptive” immunotherapy, i.e. based on the administration of experimentally-produced antibodies (Fradet, Cordon-Cardo et al. 1986, Stein, Chen et al. 1990, Alberti, Miotti et al. 1992). The Authors of the present invention have developed anti-Trop-2 monoclonal antibodies able to recognize and bind different regions of the Trop-2 molecule with high efficiency (WO2010089782 and WO2016087651) for diagnostic and therapeutic uses in cancer. Sacituzumab govitecan-hziy, a humanized anti-Trop-2 monoclonal antibody conjugated to the SN-38 topoisomerase I inhibitor, was shown to be effective in patients with metastatic triple-negative breast cancer (TNBC) (Bardia, Mayer et al. 2017, Bardia, Mayer et al. 2019) and was granted accelerated FDA approval as Trodelvy, for use in the clinics (www.fda.gov/drugs/drug-approvals-and-databases/drug-trial-snapshot-trodelvy). This indicated that Trop-2-targeted monoclonal antibodies can be useful to generate drugs to treat cancer, including advanced cases that have progressed to metastasis.
Trop-2 expression is not exclusively limited to tumors. Trop-2 is also expressed in normal human tissues, in particular at high levels in skin, oesophagus, tonsils, cornea, at lower levels in lung, exocrine pancreas, prostate, salivary glands, urothelium, bile ducts, breast, kidney, and sometimes in the stomach and in the endometrium (Alberti, Miotti et al. 1992, El Sewedy, Fomaro et al. 1998, Trerotola, Cantanelli et al. 2013). Unlike Trop-1, which is expressed by proliferating epithelial cells (Schon, Schon et al. 1994 1834), Trop-2 is expressed predominantly by epithelial cells at advanced stages of differentiation (Alberti, Miotti et al. 1992 109). In the epidermis, in particular, the expression of Trop-2 is detected in the keratinocytes of the suprabasal layer and increases towards the surface of the skin, with the highest expression levels in the stratum corneum (Alberti, Miotti et al. 1992 109).
Antigen expression in normal tissues poses serious problems of on-target toxicity that strongly limit the use of high-potency targeted therapeutics. A phase-1 dose-escalation study of bivatuzumab mertansine, which targets the tumor-associated v6-containing CD44 variant, also expressed in skin keratinocytes and other epithelia, showed serious skin toxicity and one fatal event due to the development of massive epidermal necrolysis after the second infusion at the highest dose (Tijink, Buter et al. 2006). BAY794620, an ADC targeting the CA9 antigen, also expressed within the gut, showed fatal gastrointestinal toxicity in two patients (Clinical Study Report No.: PH-37705/12671. Bayer Healthcare, 2014). Specifically, for Trop-targeted anti-cancer therapy, adverse effects were observed in patients that involved organs that normally express the molecule. The high-affinity humanized anti-Trop-1 monoclonal antibody ING-1 caused cases of acute pancreatitis in patients (de Bono, Tolcher et al. 2004). Moreover in a recent phase 1, dose-escalation study in patients with advanced or metastatic solid tumors the anti-Trop-2/Aur0101 antibody-drug conjugate PF-06664178 showed excess toxicity that manifested with neutropenia, skin rash and mucosal inflammation (King, Eaton et al. 2018). In both these cases is clinical development was terminated.
Obtaining tumor-specific monoclonal antibodies would allow the design of novel targeted drugs that combine high antitumor potency and low on-target toxicity for a highly improved therapeutic index. Classical oncogenes/tumor suppressors acquire transforming capabilities following mutations in coding or regulatory sequences. Recurrent, high-frequency oncogenic mutations that alter the amino acid sequence may offer potential for tumor-specific targeting. However, this is not the case for Trop-2: while germline mutations of the TROP2 gene have been described and cause the inherited corneal amyloidosis known as Gelatinuos Drop-Like Dystrophy (GDLD) (Tsujikawa, Kurahashi et al. 1999), Trop-2 is substantially wild-type in tumors (Trerotola, Cantanelli et al. 2013) (https://cancer.sanger.ac.uk/cosmic/search?g=TACSTD2).
In order to improve the effectiveness of targeted anticancer therapy with monoclonal antibodies, while at the same time preventing toxicity, it is important that such monoclonal antibodies recognize specific epitopes of the target molecule that are exposed in tumor cells (Johnson and Janne 2006). This makes also possible to bind higher numbers of tumor cells, when the epitopes of the individual antibodies are selectively expressed at different developmental stages of the tumor, while sparing normal cells.
The authors of the present invention have discovered here that Trop-2 post-translational processing by ADAM10 is a feature of malignancies. 3D modeling of Trop-2 structure predicts that such processing causes a spatial rearrangement of the extracellular portion of the molecule and the consequent exposure of domains that would be normally inaccessible. These domains provide novel tumor-specific targets. Therefore, it is object of the present invention a platform that uses immunization methods to obtain monoclonal antibodies, fragments and conjugates thereof with maximal epitope heterogeneity, and screening methods of such antibodies, fragments and conjugates thereof for differential recognition of the antigen in tumors versus normal tissues. This platform can be applied to proteins that undergo tumor-specific processing, to obtain monoclonal antibodies, fragments, and conjugates thereof that specifically bind to cancer tissues, thus providing means to reduce the toxicity of targeted therapies and to improve anticancer therapies.
Post-translational processing is a key activatory step of several tumor growth inducers and adhesion molecules. The present invention refers to a platform to obtain monoclonal antibodies, fragments, and conjugates thereof, that are specific for such processed forms
It is therefore an object of the present invention a method to obtain monoclonal antibodies, or fragments or conjugates thereof, which recognise and bind with high affinity processed proteins that are specifically expressed in local and metastatic tumors, said method characterized by immunization and screening procedures according to the present invention.
Protein lysates from tumor cells are typically used as immunogenic material in procedures aimed to obtain monoclonal antibodies targeting tumor antigens. However, this may result in establishing one main (immunodominant) epitope that will correspondingly skew antibody generation and produce one single antibody species even from independent immunization procedures. This was shown by the Authors of the present invention who found that the anti-Trop-2 162-46.2 obtained through immunization with the human chorocarcinoma BeWo cell line (Lipinski, Parks et al. 1981), T16, obtained through immunization with bladder cancer cells (Fradet, Cordon-Cardo et al. 1986), and RS7-3G11 monoclonal antibody obtained through immunization with tissue from lung squamous carcinoma (U.S. Pat. No. 7,238,785B2) all recognize the same epitope, which is also expressed in normal tissues (Fradet, Cordon-Cardo et al. 1986). Hence an object of the present invention are immunization procedures aimed to increase the probability to obtain monoclonal antibodies able to recognize diverse epitopes, wherein the immunogenic material consists of the target protein or fragments or conjugates thereof produced in different organisms, including tumor cells that naturally express the processed target protein and mammalian transformed and tumor cells, insect cells, yeast cells, that have been transfected with vectors that express the full-length target protein or fragments or conjugates thereof. Different domains of the target protein can be expressed as fusion proteins with tags that improve immunogenicity of small, non-immunogenic peptides, triggering the production of a wider variety of antibodies. These tags can also provide useful element for the visualization and purification of the corresponding fusion proteins.
Another object of the present invention consists of screening procedures especially designed to select monoclonal antibodies able to access and bind the target protein in its tumor-specific processed form. Therefore a specific object of the present invention is a method that comprises the steps of: a) contacting the target protein in its tumor-specific processed form with each one of the monoclonal antibodies or fragments or conjugates thereof under screening; b) measuring the binding between the target protein in its tumor-specific processed form and each one of the monoclonal antibodies or fragments or conjugates thereof under screening; c) contacting the target protein in its normal-tissue unprocessed form with each one of the monoclonal antibodies or fragments or conjugates thereof under screening; d) measuring the binding between the target protein in its normal-tissue unprocessed form and each one of the monoclonal antibodies or fragments or conjugates thereof under screening; e) contacting a negative control, comprising or consisting of protein or proteins different from the target protein and/or cells that do not express the target protein; f) measuring the binding between the negative control and the monoclonal antibody or fragments or conjugates thereof; g) selecting the monoclonal antibodies or fragments or conjugates thereof that show absence of binding to the negative control and binding to the target protein in its tumor-specific processed form that is at least 10 times higher than the binding to the target protein in its normal-tissue unprocessed form. These different steps can be performed sequentially or in parallel. This binding between antigen and antibody, fragments and conjugates thereof can be measured by flow cytometry and/or ELISA assay and/or cell-based ELISA assay and/or microscopy and/or bio-layer interferometry and/or isothermal titration calorimetry and/or microscale thermophoresis and/or surface plasmon resonance.
The target protein in its processed form can be expressed by tumors that express it endogenously or can be expressed by tumor cells that have been transfected with suitable vectors. The target protein in its unprocessed form can be expressed by normal tissues that express it endogenously or can be expressed by normal cells that have been transfected with suitable vectors. The negative control can be a protein or proteins different from the target protein, or cells that do not express the target protein, or cells that do not express the target protein and have been transfected with the empty vector. Expressing and non-expressing cells can be identified by means of target-specific antibodies that are already known in the art. The negative control can comprise or consist of negative cells that have been transfected with the empty vector. Using transfectants with expression vectors for the target protein and with the empty vector as negative control offers a stringent comparison as the target protein is the only variable between the two systems. The target protein can be in its wild-type form or can be engineered at the processing sites, to make them either constitutively fully activated or inactivated/processing-resistant.
In a preferred object of the present invention the processing of the target protein is post-translational and comprises at least one of the modifications selected from the group consisting of: peptide bond cleavage, amino acid modifications, including deamidation, addition of chemical groups, including phosphorylation, acetylation, hydroxylation, methylation, addition of complex organic molecules, including lipidation, AMPylation, ubiquitination, SUMOylation. More preferably the processing consists of the cleavage of at least one peptide bond of the target protein. Also, more preferably the target protein is Trop-2.
In a preferred object of the present invention the tumors where specific processing occurs include cancers of the breast, head and neck, skin, colon-rectum, stomach, lung, ovary, thyroid, prostate, pancreas, endometrium, cervix, gallbladder, bile ducts, kidney, urinary bladder, choriocarcinomas, and their metastases.
An object of the present invention is a platform to obtain monoclonal antibodies or fragments, or conjugates thereof directed against processed tumor-specific antigens for use as a medicament, preferably for use in the prevention and/or treatment of tumors and metastases, more preferably of the tumors and metastases that express Trop-2, even more preferably in combination with at least one therapeutic agent or treatment. In an object of the present invention, the therapeutic agents are cytotoxic substances, including radioactive isotopes, chemotherapeutic agents, and toxins of bacterial, fungal, plant or animal origin, and their fragments. A chemotherapeutic agent is a chemical compound useful in the treatment of cancer, including adriamycin, doxorubicin, 5-fluorouracil, cytosine-arabinoside (“Ara-C”), cyclophosphamide, thiotepa, busulfan, taxol, methotrexate, cisplatin, melphalan and other nitrogen mustards, vinblastine, bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide, aminopterin, dactinomycin, esperamycins. The therapeutic agent can be an intercellular mediator, for example a cytokine, including non-exclusively lymphokines, monokines, hormones, growth factors, interferon, interleukins, coming from natural sources or from recombinant cell cultures, as well as biologically active equivalents of native cytokines. Therapeutic treatments can be surgical removal of the tumor and related metastases, and/or radiotherapy.
Another object of the present invention is a platform to obtain monoclonal antibodies or fragments or conjugates thereof directed against processed tumor-specific antigens for use in the diagnosis and/or prognosis of tumors or metastases, in assessing the risk of developing a tumor or metastases, in monitoring the progression of a tumor or metastases, in monitoring the efficacy of an antitumor or antimetastasis therapeutic treatment, in the screening of antitumor or antimetastasis therapeutic treatments.
The present invention finds application in the field of monoclonal antibody generation for clinical use, where it teaches new immunization procedures and screening methods to obtain specificity towards processed tumor-specific forms of target proteins. Hybridomas that produce monoclonal antibodies can be obtained through techniques known in the art, by fusion of immunoglobulin-producing cells isolated from immunized animals, for example from the spleen of Balb-C mice, with cells from immortalized cell lines, for example murine myelomalines. Immunization can take place through injections of immunogenic material in compositions and according to methods and schedules of administration known in the art. In one embodiment of the present invention the immunogenic material contains the target protein that has been produced by various organisms, such as human tumor cells that naturally express the target protein and transformed or tumor mammalian cells, insect cells, yeast cells that have been transfected with vectors that express the whole target protein, its fragments and/or conjugates. Cancer cells include, but are not limited to, cells from cancers of the breast, head and neck, skin, colorectal, stomach, lung, ovary, thyroid, prostate, pancreas, endometrium, cervix, gallbladder, bile ducts, kidney, urinary bladder, and cells from choriocarcinomas. Transformed cells include, but are not limited to, 293 human embryonic kidney cells that have been transformed with adenoviral DNA.
A nucleic acid molecule that codes for the target protein or mutated forms or fragments or conjugates thereof according to the present invention can be generated according to technologies known in the art, for example PCR amplification of template molecules or gene synthesis. A fragment of the target protein can be, in the case of membrane proteins, the extracellular portion. A fragment of the target protein can also be a particular structural and/or functional domain, that is identified based on sequence homology, structure predictions, structure determinations or functional studies known in the art. The target protein can be conjugated by means of recombinant DNA technologies known in the art to fluorescent molecules, enzymes, or specific sequences (tags) to obtain fusion proteins for detection, purification, and to increase immunogenicity. Specific sequences can also be added to the N-terminus to guide secretion (leader sequences). Conjugation can also per performed with the inclusion of linker sequences that are positioned upstream of the tag and can be cut in vitro by enzymes known in the art to obtain the native target protein. Examples of tags to facilitate purification include the 6-histidine tail, glutathione S-transferase, maltose-binding protein, chitin-binding protein, FLAG sequence, myc-tag, hemagglutinin. Tags to increase immunogenicity are known in the art and include immunoglobulin domains and short peptide chains formed by 2 glycine residues followed by 5 isoleucine or 5 proline or 5 arginine residues (Rahman, Islam et al. 2020).
The nucleic acid molecule can be cloned into an expression vector by means of recombinant DNA technologies known in the art. Plasmid and viral expression vectors are known that are suitable for various eukaryotic organisms, such as mammals, insects, yeasts, and prokaryotes, such as bacteria. The vector for the expression of the target protein or mutated forms, fragments, or conjugates thereof can be inserted into the host cells through chemical transfection or by electroporation. Transfection methods are known in the art and transfection kits and electroporation equipment are available on the market. Viral expression vectors can be transfected into competent host cells together with vectors coding for viral structural proteins, to obtain viruses that are capable of infecting the host cells that will be used for the production of the recombinant protein. For example, viruses can be baculoviruses, lentiviruses, retroviruses, adenoviruses, adeno-associated viruses.
The cells that express the target protein, fragments or conjugates thereof can be grown in culture media and conditions known in the art. In one embodiment, the immunogen can contain the target protein that has been purified from cell lysates and/or from culture media in which its soluble forms are secreted. In another embodiment, the immunogen can derive from unfractionated cell lysates and/or culture media as above. The target protein, fragments or conjugates thereof can be purified by experts in the art using procedures known in the art, for example affinity chromatography or molecular exclusion chromatography. A fragment of the target protein can be, in the case of membrane proteins, the extracellular portion, which can thus be secreted into the culture medium.
The monoclonal antibody screening method according to the present invention measures and compares the binding of the antibody to the tumor-specific processed target protein, fragments, or conjugates thereof with the binding to the unprocessed target protein, fragments or conjugates thereof expressed by untransformed normal cells, and with the negative control as defined above. Methods for measuring antibody-antigen binding are known in the art. In one embodiment, the method is flow cytometry, in which the amount of antibody, fragments or conjugates thereof is measured that bind to each cell expressing the antigen, preferably a membrane antigen and preferably expressed by living cells, for example live tumor cell lines and cells isolated from primary tumors and metastases, or transfected cells. The expression of the target protein in its processed and unprocessed forms is detected by antibodies known in the art that are not tumor-specific. The processing is performed on the wild-type target protein by the specific molecular machinery of the tumor cell. Flow cytometry measurement is performed by measuring the fluorescence that is associated with the antibody, directly if the antibody is conjugated to a fluorescence molecule, or indirectly if the antibody is bound by a secondary antibody that is conjugated to a fluorescent molecule. Fluorescent molecules with specific emission and excitation spectra and corresponding flow cytometers are known in the art.
In one embodiment of the present invention antibody-antigen binding is measured by cell-based ELISA assays or by optical or fluorescence microscopy, using antibodies or secondary antibodies that are labelled by enzymatic or fluorescent tags. In another aspect of the present invention antibody-antigen binding is measured by classical ELISA assays on target proteins, fragments or conjugates thereof that have been purified from endogenously expressing or transfected tumor cells and from cells isolated from normal tissues. In another aspect of the present invention antibody-antigen binding is measured by bio-layer interferometry, isothermal titration calorimetry, microscale thermophoresis, surface plasmon resonance.
The present invention will be now described by way of illustration and example, according, but not limited, to, some of its preferred embodiments, with particular reference to the figures of the enclosed drawings.
Amino acid sequence of Trop-2 (SEQ ID NO: 1). The different regions of the molecule are indicated; aa: amino acids.
A. (top) Alignment between the amino acid sequence obtained by Edman degradation of the E1 immunoprecipitate (underlined; SEQ ID NO: 2) and the canonical Trop-2 sequence (aa 88-97 of SEQ ID NO: 1); (bottom) alignment between the proteolytic sites of Trop-2 and Trop-1 (Schön, Schön et al. 1993). Arrowhead: cleavage site.
B. (left) Western blotting (WB) of purified Trop-2, run in SDS-PAGE gradient gel under native (non-reducing) conditions; (right) Coomassie blue staining of purified Trop-2 run under native or reducing conditions, as indicated.
C. Competition studies between the E1 antibody and anti-Trop-2 monoclonal antibodies (mAbs) known in the art were performed by staining reconstituted mixtures of 70% parental L cells and 30% L/TROP2 transfectants (Alberti, Bucci et al. 1991) with the FITC-E1 mAb after preincubation with 100× excess of each of the indicate mAbs.
Competition between antibodies, which depends on recognition of a shared epitope, was revealed by the disappearance of the peak of FITC-E1 stained cells. Control mAb: mAb that does not recognize Trop-2. Black is line: FITC-E1 stained sample; gray line: unstained sample:
D. Immunofluorescence confocal microscopy image of L/TROP2 transfectants stained with the FITC-E1 mAb.
A. Alignment between the amino acid sequence of the protein under study (Trop-2) and that of the template protein (p41), to generate an input file for structure homology-modelling.
B. RODS-Raster3D stick representation of the 3D structure of Trop-2 thyroglobulin domain. Amino acid residues of different classes are indicated in different shades of gray. The three white arrows indicate the disulfide bridges. The Arg87 and Thr88 amino acids are indicated that flank the processing site.
C. Ramachandran plot of the Trop-2 thyroglobulin domain; the conformations that do not have any steric hindrance (core regions) are shown in dark gray.
D. 3D MOLMOL ribbon diagram of superimposed Trop-2 thyroglobulin domain (dark gray) and corresponding p41 domain (light gray). The cleavage site is indicated by the arrow.
E. 3D backbone of the first loop of the Trop-2 thyroglobulin domain. The two cysteines that are engaged in a disulfide bridge and the two amino acid residues flanking the cleavage site are indicated, with their side chains.
E. E con. Consensus sequences at the processing sites of ADAM10 substrates. The table is from the Merops database (merops.sanger.ac.uk) integrated with additional data of processing sites in native proteins. Uniprot codes of processed proteins are listed. Amino acids flanking the processing site are indicated (SEQ ID NOs: 3-44).Corresponding residues/sites in the Trop-2 sequence are highlighted in light gray.
Immunofluorescence analysis of ADAM10 and Trop-2 localization. The two proteins colocalize at the cell membrane of MTE 4-14 cells transfected with wt (left) or mutated (right) Trop-2. White arrowheads indicate areas of colocalization. Full colocalization was shown in 72.2% of the cells (n=54), partial colocalization in 22.2% and no colocalization in 5.5%. wt: wild-type Trop-2 (SEQ ID NO: 1); R87A-T88A: processing site mutant (SEQ ID NO: 45), where R87 and T88 are substituted with two alanines (A).
Trop-2 coimmunoprecipitated material was analysed by mass-spectrometry (MS) to identify Trop-2 binding partners. Four independent peptides (panel A, analysis output; SEQ ID NOs: 46-49) mapping on ADAM10 were identified (panel B, the MS peptides are highlighted in gray on the ADAM10 sequence: SEQ ID NO: 50), across multiple analytical procedures.
ADAM 10 activity was inhibited in the MTE 4-14/Trop-2 transfectants by treatment with chemical inhibitors or mRNA silencing, and the effect on Trop-2 processing and on Trop-2-dependent cell growth was evaluated.
A. WB analysis following treatment with the broad-spectrum metalloprotease inhibitor GM6001 or with the specific ADAM10 inhibitor G1254023X (Ludwig, Hundhausen et al. 2005) or ADAM10 siRNA silencing, as indicated. Chemical inhibition of ADAM10 activity led to a reduction in the 40kD processed Trop-2 band and a corresponding increase in the native form of Trop-2 (the bar graph shows the quantification by image analysis of the intensity of the WB bands corresponding to the native and processed forms of Trop-2 following treatment with G1254023X, compared with the control where the vehicle only was administered to the cells. ADAM10 inhibition upon specific siRNA treatment, as shown by the disappearance of the corresponding band (right, upper panel: ADAM10 WB) inhibited Trop-2 processing, as shown by the disappearance of the 40 and 10 kD bands (right, bottom panel: Trop-2 WB). T2-p: processed Trop-2; na: native form; pr processed form.
B. (left) ADAM10 mRNA levels measured by RT-PCR 48 hours after transfection with specific siRNA or control siRNA (human CD133) in MTE 4-14 cells transfected with the empty vector, wtTrop-2 or the processing-resistant RA87-TA88 Trop-2 mutant. (right) mRNA levels of the MMP3, MMP9, Presenilin-2 (PSEN2) and ADAM10 metalloproteases, upon treatment with specific siRNAs or irrelevant control siRNAs. Bars: standard deviation.
C. In vitro growth curves of MTE 4-14 cells transfected with the empty vector, wtTrop-2 or R87A-T88A Trop-2 upon ADAM10 (upper graphs, black,), MMP9 (lower graphs, black,) or control (human CD133, gray), siRNA-mediated inhibition. Bars: SEM. The p value for the only significant difference (ANOVA analysis) is indicated. Stars indicate post-hoc Bonferroni's t test p values (*≤0.05, ***≤0.001).
A. WB analysis of MTE 4-14/Trop-2 transfectants growing in culture at different densities (upper panel) or for different lengths of time (lower panel). Trop-2 processing increases when cells come into contact in high confluency conditions (reached either by high density seeding or by prolonged time in culture).
B. Partial sequence of the R87A-T88A mutant (SEQ ID NO: 45) versus wild-type Trop-2 (SEQ ID NO: 1) (upper panel) and flow cytometry analysis of the corresponding MTE 4-14 transfectants with the T16 anti-Trop-2 mAb known in the art (lower panel).
C. WB analysis of MTE4-14/Trop-2 transfectants using polyclonal antibodies that bind either the extracellular domain (extra) or the cytoplasmic tail (intra). Processed Trop-2 (T2-p) retains the cytoplasmic tail.
D. WB analysis of KM12SM cells transfected with wtTrop-2 or with the R87A-T88A mutant. The RT-to-AA mutagenesis makes Trop-2 resistant to processing.
E. In vitro growth curves of KM12SM and MTE 4-14 cells transfected with wtTrop-2, the R87A-T88A mutant or the empty vector as a control, as indicated.
A. Tumor growth curves in vivo of L (left) and 293 (right) cells transfected with wtTrop-2 or with the R87A-T88A mutant. Bars: standard error of the mean (SEM).
B. Boxplot analysis of liver metastasis volume from in vivo assays using metastatic KM12SM colon cancer cells transfected with wtTrop-2 or with the R87A-T88A mutant.
C. Distribution curves of the volumes of Individual metastasis from the in vivo assays described above. Distributions were analyzed using the Mann-Whitney non-parametric statistical test. This showed a significant (p value=0.0436) reduction of volumes in the R87-T88 metastases (black solid line) versus wtTrop-2 (dashed line).
A. WB analysis of Trop-2 in frozen samples from a consecutive breast cancer case series T2-p: processed Trop-2.
B. WB analysis of Trop-2 in frozen samples of normal breast tissues (8 individuals). T2-p marks the molecular weight corresponding to the processed Trop-2 band.
C. Image analysis distribution of the fraction of processed Trop-2 in the individual breast cancer samples from the WB shown in (A). Dashed lines: inflection points of the distribution curve.
A. WB analysis of Trop-2 expression in cells from normal epidermis and in skin tumors (SCC: squamous cell carcinoma; KA: keratoacanthoma; BCC: basal cell carcinoma).
B. WB analysis of Trop-2 expression in normal tissues from rhesus monkey (Macaca mulatta): (top) 1: tongue; 2: urinary bladder; 3: heart; 4: salivary gland; 5: mammary gland; 6: skin; 7: kidney; (mid) 1: parotid gland; 2: oesophagus; 3: pancreas; 4: stomach; 5: thymus; (bottom) 1: brain; 2: eye; 3: thyroid; 4: parotid gland; 5: oesophagus; 6: lung; 7: liver; 8: pancreas.
C. WB analysis of in-vitro and in-vivo Trop-2 expression in cancer cells endogenously expressing Trop-2 (HT-29, colon; MCF-7, breast; OVCA: OVCA-432, ovary) or transfected with Trop-2-expressing vectors (MTE4-14, transformed thymus cells; HCT116, colon cancer; NS-0, myeloma; L, fibrosarcoma; 293, transformed embryonic kidney cells) in culture (left) is or grown as tumors in nude mice (right). N: Untransfected cells; V: cells transfected with vector alone; T2: Trop-2-transfected cells; hiT2, loT2: Trop-2-transfected NS-0 cells selected to express TROP2 at high or low levels, respectively.
T2-p: processed Trop-2.
Murine fibrosarcoma L cells transfected with wtTrop-2 or with the empty vector (control) were incubated with mAbs generated according to the present invention. Antibody binding was detected by means of incubation with a goat-anti-mouse antiserum conjugated with the Alexa488 fluorophore followed by flow citometry analysis. Two of the anti-Trop-2 mAbs that were identified are shown in this example.
Human colon cancer KM12-SM cells transfected with wtTrop-2, or the processing-resistant R87A-T88A Trop-2 mutant (see
Human colon cancer KM12-SM cells transfected with wtTrop-2 were incubated with either (i) one of the anti-Trop-2 mAbs generated according to the present invention, which recognize the processed tumor-specific Trop-2 or (ii) with the T16 anti-Trop-2 mAb known in the art, conjugated with Alexa488, pre-mixed with a 10-times excess of the indicated unlabelled mAb. Antibody binding was revealed by flow cytometry analysis. Antibody competition for a shared epitope is revealed by the corresponding reduction in the fluorescence signal (arrows: 1A9 versus 1B4 and vice versa). Each mAb shows competition with itself, as expected.
Cells were transfected with purified DNA (Alberti and Fomaro 1990) in Lipofectamine 2000 or LTX (Invitrogen) according to the manufacturer instructions
Cell staining for flow cytometry was performed as described (Dell'Arciprete, Stella et al. 1996). Subtraction of cell autofluorescence and displacement of Alexa488- or FITC-stained cells in the red channel were applied (Alberti, Parks et al. 1987, Alberti, Bucci et al. 1991). Trop-2 transfectants were selected for expression levels comparable to those of endogenously expressing cancer cells (Alberti and Herzenberg 1988, Ripani, Sacchetti et al. 1998). Reconstituted mixtures of L cells and Trop-2 transfectants (Alberti, Bucci et al. 1991) were utilized for E1 mAb binding and competition studies of E1 with other anti-Trop-2 mAb, whereby cell mixtures were preincubated with 100× amounts of the indicated antibodies.
Cells grown on glass coverslips were fixed with 4% paraformaldehyde/PBS for 20 min. Staining was performed with the 162-46.2 (Alberti and Herzenberg 1988), T16 (Alberti, Miotti et al. 1992), E1 anti-Trop-2 (Alberti, Nutini et al. 1994), anti-ADAM10 and anti-CD9 antibodies, after permeabilization and blocking in 10% FBS, 0.1% saponin (Polishchuk, Polishchuk et al. 2000). Slides were viewed with an LSM-510 META (Zeiss) confocal microscope.
Live cells cultured on glass slides were analyzed in Leibovitz's F15 culture medium without phenol red and bicarbonate, supplemented with 10% FBS, 100 IU/ml penicillin, 100 μg/ml streptomycin (Euroclone) and 2 mM N-acetyl-cysteine (Sigma), to reduce free-radical damage. Cells were viewed with an LSM-510 META (Zeiss) confocal microscope. Images were captured at 1 min intervals. Excitation of EGFP was at 488 nm; excitation mRFP was at 546 nm.
Patients with breast cancer (N=453) with T1/T2 tumor diameter, without lymph node (Querzoli, Pedriali et al. 2006, Biganzoli, Pedriali et al. 2010) and distant metastases at the time of diagnosis were analyzed. Clinical and pathological status, tumor type, grading, expression of ERα, PgR, Ki-67-index were determined (Ambrogi, Biganzoli et al. 2006). Expression of Trop-2, uPA/PAI-1, MMP11, cathepsin D was assessed as indicated. The frozen material was lysed and processed as for Western blotting.
The human mammary MCF-7 cancer cell lines and the murine myeloma NS-0 cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum. The human 293 transformed kidney and murine L fibrosarcoma (Alberti and Herzenberg 1988) cell lines were maintained in DMEM supplemented with 10% fetal calf serum. Stable transfectants were propagated in complete medium supplemented with 100 μg/ml of G418.
Frozen samples from various organs of Rhesus monkey (Macaca mulatta) were analyzed by WB to reveal Trop-2 expression and processing level in primate normal tissues. A rabbit polyclonal anti-human wtTrop-2 was utilized; this was shown to be highly specific for the monkey Trop-2. Tissue samples included: tongue; urinary bladder; heart; salivary gland; mammary gland; skin; kidney, parotid gland; esophagus; pancreas; stomach; thymus, brain; eye; thyroid; lung; liver.
Cell transfectants were seeded at 1.5-3.0×103 cells/well in 96-well plates (five replica wells per data point). Cell numbers were quantified by staining with crystal violet (Orsulic, Li et al. 2002).
Cell numbers were normalized against a standard reference curve of two-fold serially diluted cell samples.
E1 mAb: Balb/c mice were immunized with Fe cells. Cell fusion and hybridoma cloning were carried out as previously described (Zardi, Camemolla et al. 1980). A screening for cell surface-reactive hybridomas was performed by immunohistochemistry on Fe cells (Camemolla, Ner et al. 1996) and by flow cytometry on Trop-2 transfectants. Monoclonal antibodies from the E1 hybridoma were purified by affinity chromatography on Protein-A Sepharose and conjugated to fluorescein-isothiocyanate (FITC) or NHS-Alexa Fluor 488 as described (Alberti, Nutini et al. 1994).
Rabbit polyclonal antibodies: Rabbit polyclonal anti Trop-2 antisera were generated by subcutaneous immunization with the recombinant extracellular domain of human Trop-2 synthesized in bacteria (El Sewedy, Fomaro et al. 1998), or with KLH-conjugated, N-ter biotinylated peptides corresponding to the cytoplasmic tail of human Trop-2. Anti Trop-2 polyclonal antibodies were purified by affinity-chromatography on recombinant Trop-2 conjugated to NHS-Sepharose (GE Healthcare) or biotinylated Trop-2 cytoplasmatic tails conjugated to Streptavidin-Agarose (Sigma-Aldrich). Purified antibodies were eluted with 0.2 M glycine pH 2.5.
Polyclonal antibodies known in the art: AF650 polyclonal goat anti-Trop-2 was purchased from R&D Systems (Minneapolis, MN, USA). Rabbit polyclonal anti-ADAM10 was purchased from Calbiochem (Merck Chemicals Ltd., Nottingham, UK); goat polyclonal anti-ADAM10 (sc-31853) and rat monoclonal anti-CD9 (so-18869) were obtained from Santa Cruz (Santa Cruz Biotechnology, CA). Secondary Alexa Fluor (488, 546, and 633) conjugated antibodies were provided by Invitrogen.
Cells were washed with cold wash buffer (10 mM HEPES, 150 mM NaCl, 1 mM CaCl2, 1 mM MgC2, pH 8) and lysed with 3 ml of lysis buffer (1% Triton, 0.1% SDS, 20 mM NaF, 1 mM NaVO4, 2 mM PMSF, 5 μM Pepstatin, 5 μM Leupeptin). After centrifugation at 15,000 rpm for 5 min at 4° C., protein concentration of the supernatant was quantified with a Bradford protein assay and the supernatant was used for immunoprecipitation (IP). Four mg of lysate were incubated with T16-NHS Sepharose on a rotating wheel at 4° C. for 3 h. After centrifugation at 1,000 rpm, the resin was washed 3 times with PBS. T16-bound Trop-2/protein complexes were eluted 3 times with 150 μl of 0.1 M glycine buffer pH 2.5. Eluted solutions were immediately neutralized with TRIS 1 M pH 11.5.
Trop-2 was purified by affinity chromatography over a Sepharose-E1 mAb column. Briefly, Fe cell monolayers were extensively washed in PBS and lysed in 20 mM TRIS-Cl pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 50 U/ml aprotinin, 1 mM AEBSF for 20 min at 4° C. The cell lysate was centrifuged at 1,500 g for 10 min; the supernatant was cleared by centrifugation at 12,000 g for 20 min and passed through a hydroxyapatite column (DNA-grade Bio-Gel HTP, Richmond, CA). The unbound fraction was loaded onto a Sepharose-E1 mAb column. Bound material was eluted with 0.1 M glycine pH 2.7, 0.1% Triton X-100. The eluate was immediately neutralized with 1 M TRIS pH 9, and eluted fractions were analyzed by SDS-PAGE on 4-18% gradient gels and Western blotting. Fractions containing the E1-immunoreactive material were pooled, concentrated and transferred to a PVDF membrane. N-terminal sequences of the blotted protein samples were obtained by Edman degradation. The N-terminus of unprocessed Trop-2 appeared blocked/resistant to sequencing procedures.
Immunoprecipitated material was incubated at 37° C. with sequencing-grade porcine trypsin (Promega). A 1 ml aliquot of each peptide mixture was analyzed by MALDI Mass Spectrometry (MS) (www.york.ac.uk/depts/biol/tf/proteomics/). Positive-ion mass spectra were obtained using a Bruker Ultraflex Ill in reflectron mode, equipped with a Nd:YAG smart-beam laser. Identifications were obtained by MS-Fit web-application of ProteinProspector v5.7.3 (prospector.ucsf.edulprospector/cgi-bin/msform.cgi?form=msfitstandard; UCSF). The peaks list by the mass spectrometer was used as input. We imposed as constant modification the carbamido-methylation, by reaction with iodoacetamide. Methionine oxidation, N-terminus acetylation, and transformation of peptide N-terminal Gln to pyroGlu were imposed as recognizable modifications. The mass tolerance was set at 100 ppm. Contaminant masses of the matrix a-cyano-4-hydroxycinnamic acid were excluded from analysis. Protein identification were ranked on MOWSE score. The MOWSE score is reported by MS-Fit is based on an updated scoring system (Pappin, Hojrup et al. 1993).
To identify proteolytic processing sites of Trop-2, PMF-mass spectrometer-peak lists was analyzed by MS-Fit as above. To identify processing in vivo, spectra were scanned for peptide sequences that did not fit trypsin cleavage consensus sites. The mass tolerance was set at 20 or 50 ppm (50 ppm data are shown). Contaminant masses of matrix α-cyano-4-hydroxycinnamic acid were excluded from analysis.
Western blotting was performed as described (El Sewedy, Fomaro et al. 1998). Tumors from experimental animals were rapidly frozen upon surgical removal and stored at −80° C. Frozen samples were quickly thawed and homogenized with a Dounce homogenizer. Cleared homogenates of tumors and cultured cell lysates were analyzed by SDS-PAGE and transferred to nitrocellulose filters. Filters were hybridized with anti-human Trop-2 (affinity-purified polyclonal rabbit antiserum) or anti-ADAM10 antibodies. Antibody binding was revealed by goat anti-rabbit or rabbit anti-goat peroxidase (Calbiochem, La Jolla, CA) via chemiluminescence (ECL; Amersham, Aylesbury, UK) (El Sewedy, Fomaro et al. 1998).
The pBJI-neo vector was provided by Dr. M. Davis. The Bluescript vector was obtained from Stratagene (La Jolla, CA). The pcDNA3 expression vector was obtained from Invitrogen (Graningen, The Netherlands). The pEYFP-N1 was obtained by Clontech (Palo Alto, CA).
A 970 bp full-length E1/TROP2 cDNA from Fe cells was isolated by RT-PCR, using the Titan System kit from Boehringer (Mannheim, Germany) and total Fe cell RNA as template. The amplified band was digested with Bam HI and Eco RI and subcloned in the corresponding sites of the pcDNA3 expression vector using the following primers:
A pEYFP-derived vector devoid of the coding sequence of the EYFP fluorescent protein (pΔEYFP vector) was used to express Trop-2 in mammalian cells. Cells transfected with pΔEYFP are indicated as “control”, or “vector-alone” cells. Wild-type TROP2 was obtained by PCR from the original full length genomic TROP2 clone (Fomaro, Dell'Arciprete et al. 1995), and inserted in the vector at the XhoI/KpnI sites, using the following primers:
R87A-T88A Trop-2 mutant was generated by site-specific mutagenesis of the Trop-2 processing site with the Quikchange® Site-directed mutagenesis kit (Stratagene) following the instruction of the manufacturer, using the following primers:
The mutagenized coding region was entirely sequenced, to verify the absence of any Taq polymerase-induced mutations.
Cells were treated with ADAM10 inhibitors for 24 hours at the minimum concentration found to be effective in inhibiting Trop-2 cleavage, then assayed as indicated. The GM6001 metalloprotease family inhibitor (Calbiochem) was dissolved in DMSO and used at 6.5-13 μM for 24 hours; up to 50 μM GM6001 were found effective. The G1254023X selective ADAM10 inhibitor, dissolved in DMSO, was kindly provided by Dr. A. Ludwig. Cells received two doses (10 μM) every 24 hours for 48 hours; up to 20 μM G1254023X were found effective. Control cells received vehicle alone.
ADAM10 siRNA
Complementary strategies were utilized for siRNA design. Briefly, Tuschl' criteria were applied, i.e. position in the mRNA, GC content, base composition and flanking sequences (Elbashir, Harborth et al. 2001). Invitrogen (maidesigner.invitrogen.com/maiexpress/) provides a proprietary algorithm that performs a statistical analysis of the target sequence, based on sequence composition, nucleotide content and thermodynamic properties, and compares candidates with validated siRNA sequences. The Whitehead Institute web site (jura.wi.mit.edu/bioc/siRNAext/) combines Tuschl' criteria with predictions of binding energies for both sense and antisense siRNAs and with BLAST filtering of cross-hybridizing sequences (Semizarov, Frost et al. 2003). The Sonnhammer procedure (sonnhammer.cgb.ki.se/siSearchl) performs data mining (siSearch) on validated siRNA data-banks, using motif rules and energy parameters (Chalk, Wahlestedt et al. 2004). siRNA were synthesized that were identified by more than one method or considered optimal by any of the procedures above. Annealed oligos were subcloned into the pSUPER vector, kindly provided by Dr. R. Bemard (Brummelkamp, Bemards et al. 2002), under the control of the RNA polymerase Ill H1 gene promoter. siRNA expression constructs were transiently transfected MTE 4-14 transfectants, and cell growth was measured. siRNA-targeted transcript levels were quantified by real-time PCR. Negative control siRNAs directed towards irrelevant targets were used; these were chosen after extensive testing for lack of off-target influence on cell growth.
siRNA targeting murine ADAM10 (NM_007399.3, nt 292-309) (Schramme, Abdel-Bakky et al. 2008)
siRNA targeting murine MMP9 (NM_013599, nt 1780-1798)
Negative control siRNAs for murine cells targeting human CD133 (NM_006017.1, nt 1061-1079)
Negative control siRNAs for murine cells targeting human CD316 (NM_052868.2, nt 801-819)
One μg of total RNA was reverse transcribed with the ImProm-II Reverse Transcriptase (Promega) according to standard protocols. Quantitative RT-PCR was performed using an ABI-PRISM 7900HT Sequence Detection System and Power SYBR® Green PCR Master Mix (PE Applied Biosystems, Foster City, CA) according to the manufacturer instructions (Giulietti, Overbergh et al. 2001), using the listed primers:
Each sample was assayed in triplicate. The 2-ΔΔCT method was used to calculate relative changes in gene expression (Livak and Schmittgen 2001). A more accurate base of 1.834 was used (Guerra, Trerotola et al. 2008), as 1.1 cycles are required to double the amplified material. The ß2-microglobulin (B2M) housekeeping gene was used as an internal control. For set-up curves, ΔCT (CT, target gene—CT, GAPDH) were calculated for each cDNA dilution. The data were fit using least-squares linear regression analysis. As relative amplification efficiency was invariant over the range of RNA amounts used (Zanna, Trerotola et al. 2007, Guerra, Trerotola et al. 2008), amplification curves were used to calculate cross-over point values for siRNA-treated samples. Each sample was routinely assessed for genomic DNA contamination by using is non-retrotranscribed RNA isolates as templates for PCR reactions.
Subcloned PCR bands were sequenced using the method of Sanger (Sambrook, Fritsch et al. 1989), to confirm the correctness of the construct and the absence of PCR-induced mutations. DNA and protein sequences were analyzed using GCG and EMBOSS (www.uk.embnet.org/Software/EMBOSS/) programs. Analysis of ADAM10 substrates was performed through the Merops database (merops.sanger.ac.uk/cgi-bin/protsearch.pi). The interrogation output was cross-checked with additional, updated published data on native-protein cleavage-sites.
The structure of the thyroglobulin domain of the p41 isoform of the invariant chain of MHC class II (PDB code 1ICF, chain 1) (Guncar, Pungercic et al. 1999) was used as a template for the homology-modeling of the Trop-2 thyroglobulin region. The Trop-2 and p41 thyroglobulin domains (aa 71-145 di SEQ ID NO: 1 and aa 211-271 di NP_001020330.1 respectively) were aligned using GAP and NEEDLE software. Alignments were manually refined in regions with lower conservation, using conserved cysteines and secondary-structures as anchor sites. A short α-helix around the first cysteine of the Trop-2 thyroglobulin domain was concordantly predicted by secondary-structure prediction programs (PHDsec at cubic.bioc.columbia.edu/predictprotein/; jpred2 at jura.ebi.ac.uk:8888/; 3D-pssm at www.bmm.icnet.uk/˜3dpssm/), in good correspondence to that of p41. A model of the tertiary structure of the Trop-2 thyroglobulin domain was built using the programs MODELLER-4 (Sali and Blundell 1993) and WHATIF (bioslave.uio.no/Programs/MVL/index.php3). Similar results were obtained with the two software and only the model generated by MODELLER was further analyzed. The stereochemical properties of the is resulting models were assessed with the program PROCHECK. The graphic representations of the MODELLER outputs were prepared with the MOLMOL, Swiss-PdbViewer (www.expasy.ch/swissmod/SWISS-MODEL.html), RasMol (www.umass.edu/microbio/rasmol/) and Raster3D (asdp.bnl.gov/asda/LSD/Modeling/Raster3D.html) (Merritt and Bacon 1997) programs. Modelling and model evaluation were performed on a Silicon Graphics Octane r12000 workstation. PDB files of ADAM10-cleaved proteins were retrieved from the PDB data-bank (www.rcsb.org/pdb/home/home.do), and analyzed as described above. Structure images were generated with PyMol (www.pymol.org/).
Transformed cell lines and TROP2-transfectants were injected subcutaneously (SC) in 8-week old female athymic Crl:CD1-Foxn1nu mice (Charles River Laboratories). SC tumor growth was quantified as described (Rossi, Di Lena et al. 2008). To assess metastatic diffusion, KM12SM colon cancer cell (Morikawa, Walker et al. 1988) transfectants were injected in the spleen of 8-week old female athymic Crl:CD1-Foxn1nu mice. After 4 weeks mice were euthanized; tumor growth and diffusion to the liver or other organs were determined. All autoptic samples underwent microscopy histopathology analysis to detect minimal tumors and metastatic burdens.
Procedures involving animals and their care were conducted in compliance with institutional guidelines, national laws and international protocols (D.L. No. 116, G.U., Suppl. 40, Feb. 18, 1992; No. 8, G.U., July, 1994; UKCCCR Guidelines for the Welfare of Animals in Experimental Neoplasia; EEC Council Directive 86/609, OJL358. 1, Dec. 12, 1987; Guide for the Care and Use of Laboratory Animals, United States National Research Council, 1996).
The E1 antibody generated as described above and selected for the recognition of native Trop-2 in tumor cells was compared to anti-Trop-2 mAbs known in the art by flow cytometry competition experiments (
Identification of Processed Forms of Trop-2 in Tumors Trop-2 was immunoprecipitated from ovarian carcinoma cells using the E1 antibody, purified by affinity chromatography on an E1-NHS-Sepharose mAb column as described (purity>95%, as indicated by WB analysis,
The authors of the present invention generated a 3D model of the thyroglobulin domain of Trop-2 (
It is known in the art that clusters of proteases act sequentially for the processing of surface molecules, thus activating signalling pathways that are involved in metastatic diffusion (Chen, Zhao et al. 2018 33993). Cancer-related protease families such as uPA/PAI-1, MMP11 and cathepsin D have been evaluated in a series of breast cancer patients, but none were found to be directly related to Trop-2 processing. Other is metalloproteases involved in cell adhesion mechanisms and expressed in cancer were then investigated. Among these, the ADAM10 metalloprotease has been shown to cleave target molecules within loops that have structural characteristics similar to those identified in Trop-2 (
In order to confirm that ADAM10 is indeed responsible for the processing of Trop-2 described above, transfected cells expressing Trop-2 were treated with an inhibitor of metalloprotease enzymatic activity (GM6001) or specific for ADAM10 (G1254023X), or subjected to ADAM10 gene expression silencing by specific siRNAs (
Furthermore, it has been shown that R87-T88-processed Trop-2 is recognized by polyclonal antibodies directed against the cytoplasmic tail (
In order to investigate the functional role of Trop-2 processing, a mutated version of Trop-2 was created in which the two amino acids flanking the processing site were replaced with two alanines (SEQ ID NO: 45). WB analysis showed that this R87A-T88A mutant is resistant to processing (
In vivo tumor growth and metastatic spread assays were also performed, in which colon cancer cells transfected with wtTrop-2 or with the R87A-T88A processing-resistant mutant were injected in nude mice either subcutaneously (tumor growth model) or in the spleen (liver metastatic spread model) (
Taken together these data show that processing by ADAM10 is necessary for Trop-2 to stimulate tumor growth and metastatic spread.
Trop-2 processing as described above has been detected in cell lines is that were either transformed or tumor-derived. To better define how widespread this processing is in tumors, a WB analysis of a series of breast cancer patients was performed (
The Trop-2 sequence in primates is very similar to that of human Trop-2. In particular, Rhesus monkey (Macaca mulatta) Trop-2 (SEQ ID NO: 73) has 98.1% homology with human Trop-2, and perfect conservation of the processing sequence. An extensive panel of normal Rhesus monkey tissues was subjected to WB analysis for Trop-2 (
Therefore, Trop-2 processing by ADAM10 occurs specifically in tumors.
Production and Screening Procedures to Obtain mAbs that Recognize the Tumor-Specific Processed Trop-2
Trop-2-targeted analytical and therapeutic approaches known in the art have relied on anti-Trop-2 mAbs that recognize a single is immunodominant epitope, poised between the globular and the stem regions, which is accessible and recognized in all Trop-2 expressing cells. In the present invention, novel procedures for the generation and selection of new mAbs against different Trop-2 epitopes have been developed and successfully applied, with the aim of obtaining anti-Trop-2 mAbs able to recognize and bind with high affinity processed forms of the target molecule with specific and differential expression in tumor cells.
To maximize the recognition heterogeneity of epitopes corresponding to regions of the target molecule with distinct structural and functional characteristics, an immunogen was used comprising both the entire extracellular portion (amino acids 31-274 of SEQ ID NO: 1) and single domains of the Trop-2 molecule (globular domain: amino acids 31-145 of SEQ ID NO: 1; “stem”: amino acids 146-274 of SEQ ID NO: 1). These were produced in their native folding in human 293 transformed kidney epithelial cells and MCF-7 breast adenocarcinoma (Taylor-Papadimitriou, Burchell et al. 1999), and murine L fibrosarcoma and NS-0 myeloma, in insect Sf9 and yeast cells. Expression vectors for production were generated using PCR amplification of Trop-2 coding sequences fused to tags for purification or immunogenicity enhancement. The PCR fragments were subcloned in the vectors described above and expressed in the corresponding hosts. The Trop-2 proteins were purified by affinity chromatography. BALB/c mice were subjected to multiple immunization cycles with the immunogen described above, following best procedures known in the art (Weir, Herzenberg et al. 1986). Splenocytes from immunized mice were fused to Sp2/0 or NS-0 myeloma cells and corresponding hybridomas were obtained, according to the methods known in the art (Weir, Herzenberg et al. 1986). The antibodies produced by the hybridomas thus obtained were screened for specific and differential reactivity towards the processed Trop-2 that is expressed by is tumor cells. In one phase of the screening, the antibody ability to specifically bind Trop-2 was measured. An example is shown in
Therefore the immunization and screening procedures described in the present invention made it possible to obtain new antibodies that recognize with high affinity the processed antigen expressed in cancer cells, and not the unprocessed antigen expressed in normal tissues. Similar procedures can be applied to obtain antibodies for a variety of antigens that undergo specific and differential processing in tumor cells compared to normal tissues.
| Number | Date | Country | Kind |
|---|---|---|---|
| 102020000031838 | Dec 2020 | IT | national |
This application is the U.S. national phase of International Application No. PCT/EP2021/086654 filed Dec. 17, 2021, which designated the U.S. and claims priority to IT 102020000031838 filed Dec. 22, 2020, the entire contents of each of which are hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/086654 | 12/17/2021 | WO |
