Anti-activated Trop-2 chimeric antigen receptor constructs for cancer treatment

Abstract

The present invention relates to chimeric antigen receptors (Chimeric Antigen Receptors, CARs) that can be expressed by cells of the immune system, such as T cells or NK cells (CAR-T and CAR-NK). The CAR comprises an scFv antibody fragment directed against a specific antigen of tumor tissue (tumor activated Trop-2 antigen), wherein the activated Trop-2 is a Trop-2 molecule that is proteolytically cleaved between R87 and T88. Another object of the invention is the use of said CAR constructs in anti-tumor cell therapy.

Description

Background Art

[0001] The development of anticancer immunotherapies is one of the major challenges in oncology today. Chimeric antigen receptor (CAR)-T or NK cell therapy is a promising engineering practice for T / NK cell therapy. It is based on artificial CARs that target specific tumor-associated antigens, which can directly link antibody-antigen recognition to the cytotoxic activity of immune effector cells. Immune cells are isolated from the patient's peripheral blood, a process called leukapheresis, to be engineered in vitro to express a specific CAR, and once injected back into the patient, they can trigger an immune response against the corresponding target antigen and cells expressing it.

[0002] Gene modification of peripherally derived T cells using CARs has evolved from the concept of adoptive immunotherapy using tumor-infiltrating lymphocytes (TILs), whose T cell receptors (TCRs) recognize tumor-associated antigens. CAR-T recognizes target antigens through the immunoglobulin antigen-binding region, so MHC presentation is not required.

[0003] CARs typically comprise an antibody fragment, such as an scFv or Fab fragment, incorporated into a fusion protein that also comprises other components, such as a CD3-ζ or CD28 transmembrane domain and a selective T cell activating portion, including the endodomain of CD3-ζ, CD28, OX40, 4-IBB, Lek, and / or ICOS ( Figure 1 ).

[0004] CAR constructs have accordingly been used to direct the activity of natural killer (NK) cells, which can be easily isolated from donors by a simple blood draw. Cancer cells that do not cause inflammation are often viewed by the immune system as self cells and cannot effectively stimulate T cell responses. NK cells are more easily activated and produce a variety of cytokines, including tumor necrosis factor alpha, interferon gamma, and IL-10 (Jiang, Zhang, et al., 2014). NK cell activation leads to the gradual formation of immune effector cells (such as dendritic cells, macrophages, and neutrophils), thereby promoting antigen-specific T cell and B cell responses. NK cell-mediated tumor cell lysis involves different receptors, including NKp44, NKp46, NKG2D, NKp30, and DNAM. In addition to ULBP and MICA, malignant cells often express NKG2D (Romanski, Uherek, et al., 2016).

[0005] Like T cells, NK cells can also be transfected with CAR constructs for inducing anticancer immune responses. NK cells do not require HLA matching; therefore, they can be used as allogeneic effector cells (Hermanson and Kaufman 2015). Unlike T cells, NK cells are easily redirected by CAR due to the lack of endogenous antigen specificity for targeting cells to be killed. Cell-targeting scFv or Fab can be connected to one or more intracellular signaling domains via a transmembrane domain to induce lymphocyte activation. Signaling domains used with CAR-NK cells include CD3-ζ, CD28, 4-IBB, DAPIO, and OX40. NK cell lines with cytotoxic activity (e.g., NK-92, NKG, YT, NK-YS, HANK-I, YTS, NKL) have also been used as receptors for CAR constructs. A major advantage of these cell lines is their essentially unlimited availability as "off-the-shelf" reagents that can be injected into multiple unrelated patients.

[0006] The clinical efficacy of CAR-T or NK cells in hematological malignancies is rarely replicated in solid tumors. Hematological malignancies are often circulating / disseminated and therefore lack many of the physical immunosuppressive factors that prevent adoptively transferred cells from reaching solid tumors. In addition, the target antigens present on hematological cancers are often homogeneous and expressed on most, if not all, tumor cells. In contrast, target antigens on solid tumors are often heterogeneous, varying not only within one tumor but also between primary and metastatic tumors. Therefore, CAR-T cell therapy faces multiple obstacles for solid tumors.

[0007] Trop-2 is a cell surface signal transducer (Basu, Goldenberg et al., 1995, Ripani, Sacchetti et al., 1998) that can induce intracellular calcium spikes after cross-linking with antibodies (Ripani, Sacchetti et al., 1998). Trop-2 has been shown to drive cancer development and progression by interacting with and regulating the expression and activity of cyclin D1, ERK, NFkB (Guerra, Trerotola et al., 2008, Guerra, Trerotola et al., 2013, Trerotola, Cantanelli et al., 2013), FAK, Rac1 and integrins (Trerotola, Li et al., 2012, Trerotola, Jernigan et al., 2013).

[0008] Trop-2 greatly regulates the E-cadherin / β-catenin pathway during metastasis. Overexpression of Trop-2 leads to the release of E-cadherin from the cytoskeleton, resulting in loss of cell-cell adhesion and activation of β-catenin transcription and anti-apoptotic signaling, thereby inducing a malignant phenotype. This systemic pro-metastatic program driven by Trop-2 / E-cadherin / β-catenin has a significant impact on the survival of patients with breast, colon, ovarian, uterine, and gastric cancers (Guerra, Trerotola et al., 2021, Trerotola, Guerra et al., 2021).

[0009] Trop-2 is expressed at high levels in a variety of cancer types (Alberti, Miotti et al., 1992, Stepan, Trueblood et al., 2011, Trerotola, Cantanelli et al., 2013, Guerra, Trerotola et al., 2021, Trerotola, Guerra et al., 2021, Guerra, Relli et al., 2022), supporting the use of Trop-2 as a therapeutic target. However, Trop-2 has also been shown to be highly expressed in several normal tissues (Alberti, Miotti et al., 1992, Stepan, Trueblood et al., 2011, Trerotola, Cantanelli et al., 2013, Guerra, Trerotola et al., 2021, Trerotola, Guerra et al., 2021, Guerra, Relli et al., 2022), including epidermis, endometrium, esophagus, tonsil, lung, kidney, salivary gland and breast (Guerra, Relli et al., 2022). This would be expected to result in toxicity in patients treated with anti-Trop-2 agents due to on-target / off-tumor recognition.

[0010] The authors of the present invention found that ADAM10-mediated processing triggers Trop-2 activation (Guerra, Trerotola et al., 2021, Trerotola, Guerra et al., 2021). Trop-2 activation is associated with cleavage of the first thyroglobulin domain loop (between R87-T88) of the Trop-2 extracellular domain. Molecular modeling suggests that this induces a deep rearrangement of Trop-2 structure, consistent with a regulatory function. Using in vitro and in vivo systems (including colon cancer xenografts), the role of Trop-2 processing on metastatic spread was shown.

[0011] It is noteworthy that processed Trop-2 was not detected in normal human keratinocytes or other normal tissues of humans and primates (tongue, bladder, heart, salivary gland, breast, skin, kidney, parotid gland, esophagus, lung, liver and pancreas). On the other hand, proteolytic processing has been shown in breast cancer, ovarian cancer and colon cancer (Guerra, Trerotola et al., 2021, Trerotola, Guerra et al., 2021).

[0012] WO2010089782 describes anti-Trop-2 murine monoclonal antibodies (mAbs), including 2G10 mAb (hybridoma cell line, deposited at the Advanced Biotechnology Center - ICLC, Genoa, number PD 08020), which are active against the growth of various types of tumors, such as Colo-205, HCT-116 and HT29 colon cancer; SKOV ovarian cancer; SKBR3 and MDA MB468 breast cancer.

[0013] WO2016087651 describes anti-Trop-2 human mAbs designed to reduce the immune response to the injected mAbs in patients with target cancers.

[0014] There is a fundamental need for improved designs of CAR-T and CAR-NK constructs and CAR strategies to achieve better efficacy and reduce systemic toxicity.

[0015] Description of the invention

[0016] The authors of the present invention unexpectedly discovered a solution to the practical problem while producing a chimeric antigen receptor (CAR) comprising an activated Trop-2 binding domain, wherein the activated Trop-2 is a Trop-2 molecule that is proteolytically cleaved between R87 and T88.

[0017] Therefore, an object of the present invention is to provide a CAR construct for binding to the cleavage-activated Trop-2 target in cancer cells while protecting normal cells expressing wild-type, uncleaved Trop-2. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : (A) CAR construct, comprising CD3ζ as an activation domain, 4-1BB and CD28 as co-stimulatory domains, CD28 transmembrane domain (TM), tag F, and signal sequence S. (B) Schematic diagram of the expression construct, showing that the scFv is connected to the intracellular T cell signaling domain via the transmembrane domain, specifically CD3ζ, which is the main activation domain for TCR-mediated T cell activation.

[0019] Figure 2 : Flow cytometric analysis of Jurkat cells transduced with an anti-activated Trop-2 CAR, wherein the activated Trop-2 is a Trop-2 molecule proteolytically cleaved between R87 and T88. Horizontal axis: fluorescent staining. Vertical axis: cell forward scatter, related to cell size. Each point represents the reading of a single cell. Upper figure: CAR element order is "light chain-linker-heavy chain" (control); lower figure: order is "heavy chain-linker-light chain" (according to the present invention). (A) Control map of unstained cells. (B, C) Anti-FLAG-Alexa488 antibody binds to the synthetic marker Flag added to the CAR construct. The oval includes positive CAR expressing cells. DETAILED DESCRIPTION OF THE INVENTION

[0021] Unless specifically defined otherwise herein, all technical and scientific terms used have the same meanings as commonly understood by those skilled in the fields of gene therapy, biochemistry, genetics, and molecular biology.

[0022] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are fully explained in the literature. See, for example, Molecular Cloning: A Laboratory Manual, 3rd edition, (Sambrook et al., 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press).

[0023] The object of the present invention is to provide a Trop-2 specific chimeric antigen receptor (Trop-2-CAR), according to Figure 1 , which includes: an extracellular ligand binding domain, which contains VH and VL from a monoclonal anti-Trop-2 antibody, a transmembrane domain and a cytoplasmic domain.

[0024] In one embodiment, the anti-Trop-2 antibody is an antibody specific for activated Trop-2, wherein the activated Trop-2 is a Trop-2 molecule that is proteolytically cleaved between R87 and T88.

[0025] Antibodies against activated Trop-2 were generated using immunogens containing the entire extracellular portion of the Trop-2 molecule (SEQ ID NO: 1, corresponding to amino acids 31-274 of SEQ ID NO: 2) and individual domains (SEQ ID NO: 3, globular domain: amino acids 31-145 of SEQ ID NO: 1; SEQ ID NO: 4 "stem": amino acids 146-274 of SEQ ID NO: 2), wherein the activated Trop-2 is a Trop-2 molecule proteolytically cleaved between R87 and T88. These were produced in human 293 transformed renal epithelial cells and MCF-7 breast adenocarcinoma, mouse L fibrosarcoma and NS-0 myeloma, insect Sf9 and yeast cells in their native folded form. Expression vectors for production were generated using PCR amplification to fuse the Trop-2 coding sequence with tags for purification or to enhance immunogenicity. The PCR fragments were subcloned into the above vectors and expressed in the corresponding hosts. The Trop-2 protein was purified by affinity chromatography. According to the best procedures known in the art, BALB / c mice were subjected to multiple immunization cycles with the above-mentioned immunogens. According to methods known in the art, the spleen cells of the immunized mice were fused with Sp2 / 0 or NS-0 myeloma cells and the corresponding hybridomas were obtained. The antibodies produced by the hybridomas thus obtained were screened to determine their specific and differential responses to the processed Trop-2 expressed by tumor cells. mAbs 1A9 and 1B4 have been selected because they recognize and bind only to the tumor-specific processed form of Trop-2 with high affinity, rather than the unprocessed Trop-2 found in normal tissues. Flow cytometry cross-competition experiments between 1A9, 1B4 and T16 mAbs on KM12SM / wtTrop-2 and KM12SM / vector transfectants showed that 1A9 and 1B4 blocked each other's binding, thereby indicating that they recognized the same epitope. On the contrary, there was no competition between 1A9 and 1B4 and T16, which shows that the procedures described herein effectively obtain mAbs for epitopes different from the immunodominant epitope.

[0026] In one embodiment, the VH domain is the VH domain of the 1B4 anti-Trop-2 mAb, which is secreted by a hybridoma deposited at the following International Depository Authority (IDA): Interlab Cell Line Collection, IRCCSOspedale Policlinico San Martino, Genoa, Italy, and assigned the deposit number PD21005.

[0027] In one embodiment, the VH domain is the VH domain of the 1A9 anti-Trop-2 mAb, which is secreted by a hybridoma deposited at the following International Depository Authority (IDA): Interlab Cell Line Collection, IRCCSOspedale Policlinico San Martino, Genoa, Italy, and assigned the deposit number PD21006.

[0028] In one embodiment, the VL domain is the VL domain of the 1B4 anti-Trop-2 mAb, which is secreted by a hybridoma deposited at the following International Depository Authority (IDA): Interlab Cell Line Collection, IRCCSOspedale Policlinico San Martino, Genoa, Italy, and assigned the deposit number PD21005.

[0029] In one embodiment, the VL domain is the VL domain of the 1A9 anti-Trop-2 mAb, which is secreted by a hybridoma deposited at the following International Depository Authority (IDA): Interlab Cell Line Collection, IRCCSOspedale Policlinico San Martino, Genoa, Italy, and assigned the deposit number PD21006.

[0030] In one embodiment, the VH domain has a protein sequence as shown in SEQ ID NO:5, a DNA sequence as shown in SEQ ID NO:6, or a variant thereof having at least 80% sequence identity, wherein the variant retains the ability to bind to activated Trop-2, wherein the activated Trop-2 is a Trop-2 molecule that is proteolytically cleaved between R87 and T88.

[0031] SEQ ID NO:5

[0032] QVQLVQSGAEVKKPGASVKVSCKASGFTFSSSYISWLRQAPGQRLEWIAWIYAGTGGTSYNQKFTGKATLTVDTSASTAYMELSSLRSEDTAVYYCARHNPRYYAMDYWGQGTTVTVSS

[0033] SEQ ID NO:6

[0034] CAAGTGCAGCTCGTCCAGTCTGGAGCTGAAGTCAAAAAGCCTGGGGCTTCAGTGAAAGTCTCCTGCAAGGCTTCTGGCTTCACCTTCAGCAGTAGCTATATCAGTTGGTTGAGGCAGGCCCTGGACAGAGACTTGAGTGGATTGCATGGATTTATGCTGGAACTGGCGGAACTAGCT ATAATCAGAAGTTCACAGGCAAGGCCACACTGACTGTAGACACATCCGCCAGCACAGCCTACATGGAACTCAGCAGCCTGAGATCTGAGGACACTGCCGTCTATTACTGTGCAAGACATAACCCTCGTTACTATGCTATGGACTACTGGGGCCAAGGAACCACAGTCACCGTCTCCTCA

[0035] In one embodiment, the VL domain has a protein sequence as shown in SEQ ID NO:7, a DNA sequence as shown in SEQ ID NO:8, or a variant thereof having at least 80% sequence identity, wherein the variant retains the ability to bind to activated Trop-2, wherein the activated Trop-2 is a Trop-2 molecule that is proteolytically cleaved between R87 and T88.

[0036] SEQ ID NO:7

[0037] DTQMTQSPSSSLSASVGDRVTITCITSTDIDDDMNWYQQKPGKAPKLLISEGNTLRPGVPSRFSGSGYGTDFTFTISSLQPEDIATYYCLQSDNLPYTFGGGTKVEIKR

[0038] SEQ ID NO:8

[0039] GACACCCAGATGACCCAGTCTCCAAGCTCCCTGTCCGCCAGCGTGGGAGATAGAGTCACCATCACATGCATCACCAGCACTGATATTGATGATGATATGAACTGGTACCAGCAGAAGCCAGGGAAAGTCCTTAAGCTCCTGATTTCAGAAGGCAATACTCTG CGCCCTGGAGTCCCATCCCGATTCTCCGGCAGTGGCTATGGAACAGATTTTACCTTTACAATTAGCTCCCTGCAGCCAGAAGATATTGCAACCTACTACTGTTTGCAAAGTGATAACCTGCCCTACACCTTCGGAGGGGGGACCAAAGTCGAAATCAAACGG

[0040] In one embodiment, the antibodies taught in the present application are humanized antibodies or chimeric antibodies in which the mouse constant region is replaced by a human constant region, or are variants comprising at least one CDR of the corresponding light chain and / or heavy chain variable region, thereby maintaining the specificity of the antibody for the target, and can be subjected to mutagenesis to change their affinity for the target.

[0041] In one embodiment, the two domains VH and VL are connected by a spacer. Preferably, the spacer is GlyGlyGlyGlySer, more preferably, the motif is repeated 4 times.

[0042] In one embodiment, the domains are linked in this manner: VH-spacer-VL.

[0043] In one embodiment, the CAR includes activation and costimulatory domains, and in one embodiment, they are selected from CD28, 4-1BB or CD137, inducible T cell costimulators (ICOS) or CD278, OX40 or CD134 or CD3 ζ. Preferably, they are CD3 ζ, CD28 and 4-1BB.

[0044] In some of any such embodiments, the transmembrane portion of CD28 is human CD28.

[0045] In one embodiment, the CAR comprises a Flag epitope, which is recognized by an anti-Flag mAb to reveal the expression of CAR on the cell surface and to identify CAR transduced cells. In one embodiment, the Flag epitope has a sequence motif DYKDDDDK (SEQ ID NO: 9), which is a highly specific artificial antigen recognized by an anti-FLAG mAb known in the art.

[0046] In one embodiment, the CAR comprises a CD8 alpha signal sequence. In one embodiment, the CAR comprises a CD8 alpha hinge domain.

[0047] refer to Figure 1 Schematic diagram of A, in a preferred embodiment, the construct includes: a CD3ζ activation domain, two co-stimulatory domains (CD28 and 4-1BB), a tag F, a signal sequence S, and a variable region of an antibody against activated Trop-2, wherein the activated Trop-2 is a Trop-2 molecule that is proteolytically cleaved between R87 and T88.

[0048] In a second embodiment, a cell is claimed, which is a T cell or a NK cell, and which expresses the CAR described in the present specification.

[0049] refer to Figure 1 B, Once expressed by T cells, the construct according to the invention displays the extracellular scFv portion connected via the transmembrane domain to the intracellular signaling domains represented by CD28, the 4-1BB co-stimulatory domain and the CD3ζ activation domain.

[0050] In a third embodiment, a method for preparing a cell according to the present specification is claimed, the method comprising the step of introducing the nucleic acid sequence according to the present specification into the cell ex vivo. Preferably, the cell is a T cell or a NK cell.

[0051] In a fourth embodiment, a pharmaceutical composition is claimed, comprising a plurality of cells according to the present description.

[0052] In a fifth embodiment, the pharmaceutical composition is claimed for use in a method of treating a disease in a subject. In one embodiment, the disease is a tumor.

[0053] In another embodiment, described herein is a method of eliciting a regulatory immune response to a disease, the method comprising administering to a patient cells of the immune system, e.g., T cells or NK cells that have been transfected with a CAR according to the invention.

[0054] The authors of the present invention surprisingly demonstrated that CAR T or CAR NK cells expressing the anti-Trop-2 antibody V gene, once administered to a subject, are able to induce an immune response against target cells expressing Trop-2, because the binding of the CAR on the surface of the transduced T cells or NK cells to the antigen expressed by the target cells activates the transduced T cells or NK cells.

[0055] The following are examples that illustrate methods of practicing the present invention. These examples should not be construed as limiting. Example

[0056] Embodiment 1:

[0057] Jurkat cells were transduced with an anti-activated Trop-2 CAR, wherein the activated Trop-2 was a Trop-2 molecule proteolytically cleaved between R87 and T88, wherein the VH had a protein sequence as shown in SEQ ID NO:5, a DNA sequence as shown in SEQ ID NO:6, and the VL had a protein sequence as shown in SEQ ID NO:7, a DNA sequence as shown in SEQ ID NO:8, and the linker consisted of 4 repeated GlyGlyGlyGlySer motifs. In a first embodiment (control), the CAR element is VL-linker-VH. In a second embodiment, according to the present invention, the CAR element is VH-linker-VL. The cells were then analyzed by FACS after staining with anti-FLAG-Alexa488 antibodies ( Figure 2 BC). Unstained cells were also analyzed as a control. Only the VH-linker-VL CAR element showed fluorescent staining, i.e., CAR was expressed at the membrane level.

[0058] References

[0059] Alberti, S., S. Miotti, M. Stella, CE Klein, M. Fornaro, S. Ménard and MIColnaghi (1992). "Biochemical characterization of Trop-2, a cellsurfacemolecule expressed by human carcinomas: formal proof that themonoclonalantibodies T16 and MOv-16recognize Trop-2." Hybridoma 11:539-535.

[0060] Basu, A., DM Goldenberg and R. Stein (1995). "The epithelial / carcinomaantigen EGP-1, recognized by monoclonal antibody RS7-3G11, is phosphorylated onserine 303." Int J Cancer 62(4):472-479.

[0061] 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.

[0062] 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.

[0063] Guerra, E., M. Trerotola, R. Dell’Arciprete, V. Bonasera, B. Palombo, T. El-Sewedy, T. Ciccimarra, C. Crescenzi, F. Lorenzini, C. Rossi, G. Vacca, R. Lattanzio, M. Piantelli and S. Alberti (2008). "A bi-cistronic CYCLIN D1-TROP2 mRNA chimera demonstrates a novel oncogenic mechanism in human cancer." Cancer Res 68(19):8113-8121.

[0064] 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.

[0065] Hermanson, D. L. and D. S. Kaufman (2015). "Utilizing chimeric antigen receptors to direct natural killer cell activity." Front Immunol 6: 195.

[0066] Jiang, H., W. Zhang, P. Shang, H. Zhang, W. Fu, F. Ye, T. Zeng, H. Huang, X. Zhang, W. Sun, D. Man-Yuen Sze, Q. Yi and J. Hou (2014). "Transfection of chimeric anti-CD138 gene enhances natural killer cell activation and killing of multiple myeloma cells." Mol Oncol 8(2): 297-310.

[0067] Ripani, E., A. Sacchetti, D. Corda and S. Alberti (1998). "The human Trop-2 is a tumor-associated calcium signal transducer." Int. J. Cancer 76: 671-676.

[0068] Romanski, A., C. Uherek, G. Bug, E. Seifried, H. Klingemann, W. S. Wels, O. G. Ottmann and T. Tonn (2016). "CD19-CAR engineered NK-92 cells are sufficient to overcome NK cell resistance in B-cell malignancies." J Cell Mol Med 20(7): 1287-1294.

[0069] Stepan, L. P., E. S. Trueblood, K. Hale, J. Babcock, L. Borges and C. L. Sutherland (2011). "Expression of Trop2 Cell Surface Glycoprotein in Normal and Tumor Tissues: Potential Implications as a Cancer Therapeutic Target." J Histochem Cytochem.

[0070] 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 (2013). "Up-regulation of Trop-2 quantitatively stimulates human cancer growth." Oncogene 32 222-233.

[0071] 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.

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[0073] Trerotola, M., J. Li, S. Alberti and L. R. Languino (2012). "Trop-2 inhibits prostate cancer cell adhesion to fibronectin through the β1 integrin-RACK1 axis." J Cell Physiol 227(11): 3670-3677.

[0074]

[0075]

Claims

1. A Trop-2 specific chimeric antigen receptor (CAR), wherein the CAR has a structure comprising an extracellular ligand binding domain, wherein the extracellular ligand binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) from a monoclonal anti-Trop-2 antibody, a transmembrane domain, and a cytoplasmic domain.

2. The CAR of claim 1, wherein the antibody is a humanized 2G10 antibody.

3. The CAR according to claim 1, wherein the antibody specifically recognizes activated Trop-2, wherein the activated Trop-2 is a Trop-2 molecule that is proteolytically cleaved between R87 and T88.

4. The CAR according to claim 3, wherein the VH is selected from the group consisting of: the VH domain of 1B4 anti-Trop-2 mAb, which is secreted by a hybridoma deposited in the following international depository (IDA): Interlab Cell Line Collection, IRCCS Ospedale Policlinico San Martino, Genoa, Italy, and assigned the deposit number PD21005; and the VH domain of 1A9 anti-Trop-2 mAb, which is secreted by a hybridoma deposited in the following international depository (IDA): Interlab Cell Line Collection, IRCCS Ospedale Policlinico San Martino, Genoa, Italy, and assigned the deposit number PD21006.

5. The CAR according to claim 3, wherein the VL is selected from the group consisting of: the VL domain of 1B4 anti-Trop-2 mAb, which is secreted by a hybridoma deposited in the following international depository unit (IDA): Interlab Cell Line Collection, IRCCS Ospedale Policlinico San Martino, Genoa, Italy, and assigned the deposit number PD21005; and the VL domain of 1A9 anti-Trop-2 mAb, which is secreted by a hybridoma deposited in the following international depository unit (IDA): Interlab Cell Line Collection, IRCCS Ospedale Policlinico San Martino, Genoa, Italy, and assigned the deposit number PD21006.

6. The CAR according to claim 3, wherein the VH has a protein sequence as shown in SEQ ID NO:5, a DNA sequence as shown in SEQ ID NO:6, or a variant having at least 80% sequence identity thereto, wherein the variant retains the ability to bind to activated Trop-2, wherein the activated Trop-2 is a Trop-2 molecule that is proteolytically cleaved between R87 and T88.

7. The CAR according to claim 3, wherein the VL has a protein sequence as shown in SEQ ID NO:7, a DNA sequence as shown in SEQ ID NO:8, or a variant thereof having at least 80% sequence identity, and the variant retains the ability to bind to activated Trop-2, wherein the activated Trop-2 is a Trop-2 molecule that is proteolytically cleaved between R87 and T88.

8. The CAR according to claim 3, wherein the VL has a protein sequence as shown in SEQ ID NO: 5 and a DNA sequence as shown in SEQ ID NO:

6.

9. The CAR according to claim 3, wherein the VH has a protein sequence as shown in SEQ ID NO: 7 and a DNA sequence as shown in SEQ ID NO:

8.

10. The CAR according to claim 3, wherein the VL has a protein sequence as shown in SEQ ID NO: 5, a DNA sequence as shown in SEQ ID NO: 6, and the VH has a protein sequence as shown in SEQ ID NO: 7, a DNA sequence as shown in SEQ ID NO: 8, and the VH and VL are connected by a spacer.

11. The CAR according to claim 10, wherein the spacer is GlyGlyGlyGlySer.

12. The CAR of claim 11, wherein the motif is repeated 4 times.

13. The CAR according to any one of claims 10-12, wherein the domains are connected in this manner: VH-spacer-VL.

14. A T cell (CAR-T) or NK cell (CAR-NK), which is transduced with the CAR according to any one of claims 1-13.

15. A pharmaceutical composition comprising the CAR-T or CAR-NK cell according to claim 14.

16. The CAR according to any one of claims 1 to 13, for use in treating a disease.

17. The CAR for use according to claim 16, wherein the disease is a solid cancer expressing activated Trop-2, wherein the activated Trop-2 is a Trop-2 molecule that is proteolytically cleaved between R87 and T88.

18. A CAR for use according to any one of claims 16 or 17, wherein the CAR is used together with at least one additional activating agent selected from the group comprising: (i) interferon; (ii) checkpoint inhibitory antibodies; (iii) antibody-drug conjugates (ADC); (iv) anti-HLA-DR antibodies; (v) anti-CD74 antibodies.

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