Membrane alpha-enolase (ENOL)-binding chimeric antigen receptor (CAR) and therapeutic use thereof

Abstract

The invention relates to an isolated chimeric antigen receptor (CAR), comprising an extracellular domain capable of binding the membrane alpha-enolase (ENO1) antigen, a transmembrane domain, an intracellular signaling domain, and optionally one or more costimulatory domains. The invention also relates to an engineered immune cell expressing on its surface said CAR receptor and a pharmaceutical composition comprising said engineered immune cell, as well as their therapeutic use for the targeted treatment of a pancreatic tumor disease, in particular pancreatic ductal adenocarcinoma.

Description

[0001] Membrane alpha-enolase (ENOl)-binding chimeric antigen receptor (CAR) and therapeutic use thereof

[0002] Field of the invention

[0003] The present invention falls within the field of immunotherapy as a therapeutic approach for the treatment of tumor diseases. More specifically, the invention relates to a chimeric antigen receptor (CAR) and an engineered immune cell expressing said receptor for the treatment of pancreatic ductal adenocarcinoma, also referred to as “PDAC”.

[0004] State of the art

[0005] Pancreatic ductal adenocarcinoma (PDAC) is the most common of pancreatic tumors and the fourth leading cause of death in the United States and Europe but is expected to become the second leading cause of death by 2030, after lung cancer. PDAC is characterized by a poor prognosis with a 5-year survival rate from diagnosis of 13%. To date, the only curative treatment is surgery, but surgery is only applicable to 10-20% of patients. The overall 5-year survival after pancreaticoduodenectomy is approximately 25-30% in node-negative tumors and 10% in node-positive cases, as locoregional or distant recurrence often occurs in the rest of the patients. On the other hand, chemotherapy and radiotherapy treatments are only marginally successful in achieving an increase in patient survival.

[0006] In recent decades, immunotherapy has played an important role by identifying several tumor antigens, which, being expressed aberrantly in tumor cells, are an ideal target against which to induce a specific immune response leading to the death of the tumor cells themselves.

[0007] One of the most promising approaches is a new cell therapy based on the use of engineered T lymphocytes. More specifically, this approach involves the use of cells from the patient’s immune system, i.e., T lymphocytes, which are genetically modified to enhance their ability to recognize and destroy tumor cells. Therapeutic use of engineered T lymphocytes was found to be particularly effective in the treatment of certain hematological tumors, such as acute lymphoblastic leukemia, non-Hodgkin lymphoma and multiple myeloma.In the laboratory, T lymphocytes taken from the patient are engineered to express a chimeric antigen receptor, called “CAR,” on their surface. Typically, a CAR receptor consists of three distinct domains: (i) an extracellular domain (ectodomain) designed to recognize a specific antigen on the surface of target tumor cells, (ii) a transmembrane domain usually consisting of a hydrophobic a-helix extending across the plasma membrane and derived from membrane receptor proteins native to T lymphocytes, such as, for example, CD8a, and (iii) an intracellular domain (endodomain) responsible for the transduction of the lymphocyte activation signal, which typically consists of a dimer formed by a CD3 chain and a (^(CD3z) chain containing IT AMs (Immunoreceptor Tyrosine-based Activation Motif) in the terminal portion, which are capable of signalling within the cell the binding of the antigen recognition domain to its ligand.

[0008] CAR receptors having the structure illustrated above are defined as first generation receptors. Despite significant antigen recognition capacity, first-generation CARs have shown limited antitumor activity. For this reason, further generations of CAR receptors have been developed that carry further modifications, primarily at the intracellular domain level. In particular, second-generation CAR receptors are characterized by the insertion of a costimulatory domain, e.g., a CD28 or 41BB domain, within the intracellular portion of the receptor. Third-generation CAR receptors, on the other hand, were constructed by combining multiple costimulatory domains within the endodomain, for example, both CD28 or 4 IBB domains. Currently, CAR receptors approved for clinical applications belong to the second generation.

[0009] Patient's T cells, engineered in the laboratory and expressing a CAR receptor on their surface (CAR-T cells), after their expansion, are re-infused into the donor patient where they operate by specifically recognizing tumor cells and inducing the elimination of such cells, through the release of perforins and granzymes. Prior to infusion, the patient undergoes lymphodepletive chemotherapy to promote subsequent proliferation of the CAR-T cells. Once the CAR-T cells are re-infused, the patient is monitored for any treatment-related toxicities, such as cytokine release syndrome, neurotoxicity, macrophage activation syndrome, and B-cell aplasia.Clinical use of CAR-T cells has shown excellent results in patients unresponsive to conventional treatments due to their high specificity of action, thus reducing damage to healthy tissues, and their ability to persist for a long time, thereby maintaining an immunological memory that allows them to recognize tumor cells in the event of recurrence.

[0010] Among the tumor antigens associated with pancreatic ductal adenocarcinoma (PDAC) in humans, CA19.9 Lewis blood-type sialylated antigen is currently considered the most important diagnostic and prognostic serological marker, despite the significant amount of evidence indicating its low specificity. In order to identify markers that are more reliable for PDAC, in recent years studies have been performed on blood and tissues of PDAC patients with which, using techniques analysing large-scale RNA or protein expression, the expression levels of a large number of human proteins could be monitored in relation to the onset and progression of PDAC and its prognostic pattern. The research described in Tomaino B, Cappello P, Capello M, et al. Circulating autoantibodies to phosphorylated a-enolase are a hallmark of pancreatic cancer. J Proteome Res. 201 l;10(l): 105-112 on the serum-proteome profiles of a large cohort of PDAC patients and their controls revealed a specific association between this tumor and the increase in pancreatic levels of the glycolitic enzyme alpha-enolase (“ENO1” or “ENOA”) and, in particular, of its isoforms phosphorylated on serine in position 419. The ENO1 protein is present both on the cell surface and in the cytoplasm. In addition, circulating autoantibodies against the phosphorylated epitopes of the ENOA1-2 isoforms were found in 62% of patient sera, unlike what was found in the control group in which the aforementioned immunoreactivity was only present in 4% of samples. To further support the clinical value of these findings, the authors showed that the antibody response to phosphorylated alpha-enolase isoforms correlates in most cases with a more favourable disease prognosis and a significant increase in survival estimate. Furthermore, two parallel studies demonstrated the presence in patients of T lymphocytes capable of specifically recognizing the ENO1 protein and to become activated. These cells were isolated both from PDAC patients’ blood, where they correlate with the presence of circulating antibodies against ENO1 itself (Tomaino B et al. J. Proteome Res. 2007, 6, 10, 4025-4031 Publication Date: September 8, 2007), and from tumor tissue biopsies (Amedei A, et al. Ex vivo analysis of pancreatic cancer-infiltrating T lymphocytesreveals that ENO-specific Tregs accumulate in tumor tissue and inhibit Thl / Thl7 effector cell functions. Cancer Immunol Immunother. 2013;62(7): 1249-1260).

[0011] The specific linkage of the alpha-enolase antigen with PDAC has made this protein an ideal candidate for the development of a prognostic marker for this neoplasm. Italian Patent Application No. T02009000697 describes the use of the human alpha-enolase phosphorylated isoform as a biomarker for the diagnosis of PDAC, together with peptides derived therefrom and containing the phosphorylation site and with antibodies capable of specifically binding the phosphorylated epitope.

[0012] In addition to the immunodiagnostic application, research has also been conducted to evaluate the use of the ENO1 antigen in the therapeutic field, more specifically in the immunotherapy field, in order to develop approaches that, either alternatively or in combination with conventional strategies, enable effective action against pancreatic tumor cells, thereby slowing down the progression of the neoplastic disease.

[0013] Italian Patent Application T02009000697 suggests the therapeutic use of antibodies targeting an alpha-enolase phosphorylated epitope but does not provide any experimental support in this regard.

[0014] Italian Patent Application T02010A000966 describes a DNA vaccine for the therapeutic or prophylactic treatment of PDAC, which includes an expression vector containing a recombinant nucleotide sequence encoding the full-length alpha-enolase protein. A DNA vaccine based on a nucleotide sequence encoding for full-length human alpha-enolase (ENO1) is also described in Cappello P, Rolla S, Chiarle R, et al. Vaccination with ENO1 DNA prolongs survival of genetically engineered mice with pancreatic cancer. Gastroenterology. 2013;144(5): 1098-1106.

[0015] The papers by Moscato S. et al, Eur. J. Immunol. 2000, 30: 3575-3584, and Principe M. et al, Oncotarget. 2015 May 10;6(13): 11098-113, mention monoclonal antibodies to the enzyme alpha-enolase. However, the description of said antibodies does not provide any indication on how to obtain or identify them.In this context, therefore, there is a dramatic need for the development of therapeutic strategies which are capable of countering in a targeted manner the onset and progression of aggressive tumor diseases such as pancreatic ductal adenocarcinoma, thereby allowing for a significant improvement in patient survival rates while hampering the appearance of possible secondary adverse effects.

[0016] This and other needs are met by the isolated chimeric antigen receptor (CAR) of the invention as defined in the appended claim 1.

[0017] The invention also relates to a nucleic acid encoding for the CAR receptor of the invention, an engineered immune cell expressing said CAR receptor, a method for the preparation of said engineered immune cell, and their therapeutic use.

[0018] Further features and advantages of the invention are identified in the appended claims and illustrated in detail in the following description.

[0019] The appended independent and dependent claims form an integral part of the present specification.

[0020] As will be apparent from the following detailed description, the present invention provides an isolated chimeric antigen receptor (CAR) targeting the alpha-enolase antigen (ENO1), which is defined by a combination of characteristics capable of giving said receptor high specificity as well as the ability to effectively promote the activation of immune cells, in particular T lymphocytes.

[0021] In the context of the present description, the term “chimeric antigen receptor”, also referred to herein as “CAR”, refers to an engineered cell receptor capable of giving the cells in which it is expressed, e.g., naive T lymphocytes, central memory T lymphocytes, effector T lymphocytes, or combinations thereof, a particular antigen specificity.

[0022] The CAR receptor according to the invention comprises an extracellular domain capable ofbinding the alpha-enolase antigen (ENO1), which consists of a single-chain antibody fragment (scFv) comprising, from the N-terminal end to the C-terminal end, a light-chain variable region and a heavy-chain variable region.

[0023] The term “single-chain antibody fragment (scFv)”, as used herein, refers to a fusion protein comprising at least one variable region of an antibody light chain and at least one variable region of an antibody heavy chain linked together directly or via a linker peptide sequence. As is well known, an scFv fragment generally retains the antigen specificity of the parent antibody from which it is derived.

[0024] According to the present invention, the extracellular domain capable of binding the ENO1 antigen comprises a light chain variable region comprising or consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO. 1, and a heavy chain variable region comprising or consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO. 2.

[0025] The nucleotide sequences of SEQ ID NO. 1 and 2 are shown below:

[0026] SEQ ID NO. 1 5’gacattgtgatgacccaggctgcaccctctatacctgtcactcctggagagtcagtctccatctcctgcaggtctagtaagagtct cctgcatagtaatggcaacacttacttgtattggttcctgcagaggccaggccagtctcctcagctcctgatatatcggatgtccaacc ttgcctcaggagtcccagacaggttcagtggcagtgggtcaggaactgctttcgcactgagaatcagtagagtggaggctgagga tgtgggtgtttattactgtatgcaacatctagaatatcctttcacgttcggtgctgggaccaagctggagctgaaa 3 ’ ;

[0027] SEQ ID NO. 2 5’caggcctatctgcagcagtcaggggctgaactggtgaagccgggggcctcagtgaaggtgtcctgcaaggcttctgactaca gatttaccagttacaatttgcactgggtcaaacagacacctggtcagggcctggaatggattggagctatttggcctagaaatggtg atacctcctacaatcagaagttcaaaggcaaggccacattgactgcagacaaatcctccagaacagcctacatgcagctcgacagt ttgacatctgaggactctgcggtctattactgtgcaagatggggacttgatggtggtgcctggtttgcttactggggccaagggactc tggtcactgtctctaccacgacgccagcgccgcgacca 3 ’ .

[0028] In one embodiment, the light chain variable region comprises or consists of the amino acidsequence DIVMTQAAPSIPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMS NLASGVPDRFSGSGSGTAFALRISRVEAEDVGVYYCMQHLEYPFTFGAGTKLELK

[0029] (SEQ ID NO. 3), and the heavy chain variable region comprises or consists of the amino acid sequence QAYLQQSGAELVKPGASVKVSCKASDYRFTSYNLHWVKQTPGQGLEWIGAIWPR NGDTSYNQKFKGKATLTADKSSRTAYMQLDSLTSEDSAVYYCARWGLDGGAWF AYWGQGTLVTVSTTTPAPRP (SEQ ID NO. 4).

[0030] Preferably, in the CAR receptor according to the invention, the above-mentioned light chain and heavy chain variable regions are linked together via a peptide linker, more preferably a peptide linker comprising 5 to 25 amino acids, even more preferably a peptide linker comprising 10 to 20 amino acids, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.

[0031] In one embodiment, the peptide linker is a flexible peptide linker, such as a linker consisting of repeating units of glycine and serine residues. Preferably, the peptide linker is encoded by the nucleotide sequence 5’ ggcagcactagtggtagcggcaaaccaggttccggcgaaggctcgagcaaaggt 3’ (SEQ ID NO. 5). The amino acid sequence encoded by SEQ ID NO. 5 is GSTSGSGKPGSGEGSSKG (SEQ ID NO. 16).

[0032] The CAR receptor according to the invention is further characterized by comprising a transmembrane domain. Said transmembrane domain may include the transmembrane region of any membrane protein, including type I, II or III membrane proteins. Alternatively, the transmembrane domain of the CAR receptor of the invention may comprise an artificial hydrophobic amino acid sequence.

[0033] Non-limiting examples of transmembrane domains suitable for use in the CAR receptor according to the invention include the transmembrane domains of proteins selected from the group consisting of CD4, CD8, CD8a, CD3, CD3-zeta, CD28, 0X40 (CD134), and 4-1BB (CD137).Particularly preferred according to the invention is a transmembrane domain of the CD8a protein, more preferably a transmembrane domain of CD8a comprising or consisting of the amino acid sequence encoded by the nucleotide sequence SEQ ID NO. 6: 5’ ccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtg cacacgagggggctggacttcgcctgtgat 3 ’ .

[0034] The corresponding amino acid sequence encoded by SEQ ID NO. 6 is defined by the sequence SEQ ID NO.7: PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD.

[0035] According to one embodiment, the transmembrane domain of the CAR receptor is connected to the extracellular antigen-binding domain via a spacer element, e.g., an element consisting of a “hinge” region of an antibody, preferably a human antibody.

[0036] As is well known, the term “hinge region” of an antibody refers to a short sequence of the heavy chain (H) that connects the antigen-binding domain (Fab) to the Fc domain of said chain.

[0037] For example, in some embodiments, the spacer element may be a hinge region of a CD8a protein.

[0038] The CAR receptor of the present invention also includes an intracellular signaling domain.

[0039] The term “intracellular signaling domain”, as used herein, refers to the portion of a cell receptor designated for the intracellular transmission of the signal generated by the binding of the receptor to its ligand.

[0040] In the CAR receptor according to the invention, the intracellular signaling domain may comprise a cytoplasmic domain of a naturally occurring receptor, for example a cytoplasmic domain of a receptor of an immune cell such as a T lymphocyte, as well as any derivative or variant of said domain or any synthetic amino acid sequence having the same functional capacity. The intracellular signaling domain may comprise, for example, one or more signal transduction motifs known as immunoreceptor tyrosine-based activation motifs, commonlyreferred to by the acronym IT AM.

[0041] Preferably, the intracellular signaling domain of the CAR receptor of the invention comprises or consists of a signal transduction domain of a protein selected from the group consisting of CD3zeta, FcRgamma, FcRbeta, FcRepsilon, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

[0042] In one embodiment of the invention, the intracellular signaling domain comprises or consists of a signal transduction domain of the CD3zeta protein (also known as CD247), preferably a CD3zeta domain comprising or consisting of the amino acid sequence encoded by the nucleotide sequence SEQ ID NO.8: 5’ agagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacg aagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcag gaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggg gcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgcccc ctcgc 3’.

[0043] The corresponding amino acid sequence encoded by SEQ ID NO. 8 is illustrated by the sequence SEQ ID NO.9: RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPR.

[0044] According to the present invention, the CAR receptor may also comprise one or more costimulatory domains.

[0045] As is well known, the immune response of T lymphocytes, in addition to the specific interaction of the TCR receptor with the antigen, largely depends on the transmission of the signal by a series of costimulatory molecules expressed on the cell surface. The action of these molecules leads to an increase in lymphocyte proliferation and survival, as well as an enhancement of the cytotoxic function of these cells.The term “co stimulatory domain”, as used herein, therefore refers to the intracellular portion of a costimulatory molecule, which exhibits the functional activity as described above.

[0046] Preferably, the one or more costimulatory domains comprise or consist of a costimulatory domain of a protein selected from CD28, CD27, 4-1BB (CD137), 0X40 (CD134), ICOS (CD278), CD30, CD40, PD-1 (CD279), CD2, CD7, NKG2C (CD94), B7-H3 (CD276), and any combination thereof.

[0047] In a preferred embodiment, the CAR receptor of the invention comprises at least one costimulatory domain of a protein selected from CD28 and 4-1BB (CD137), even more preferably the CAR receptor comprises both the costimulatory domains of CD28 and 4- IBB (CD137).

[0048] In the embodiment described above, the CD28 costimulatory domain may comprise or consist of the amino acid sequence encoded by the nucleotide sequence SEQ ID NO. 10, and / or the 4-1BB (CD137) costimulatory domain may comprise or consist of the amino acid sequence encoded by the nucleotide sequence SEQ ID NO. 11.

[0049] The nucleotide sequences of SEQ ID NO. 10 and 11 are shown below:

[0050] SEQ ID NO. 10 5’ttctgggtgctggtcgttgtgggcggcgtgctggcctgctacagcctgctggtgacagtggccttcatcatcttttgggtgaggag caagcggagcagactgctgcacagcgactacatgaacatgaccccccggaggcctggccccacccggaagcactaccagccc tacgcccctcccagggatttcgccgcctaccggagc 3’

[0051] SEQ ID NO. 11 5’aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgt agctgccgatttccagaagaagaagaaggaggatgtgaactg 3 ’ .

[0052] In the present description, the amino acid sequences encoded by SEQ ID NO. 10 and 11 are designated as SEQ ID NO. 12 and 13, respectively, and shown below:SEP ID NO. 12 FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHY QPYAPPRDFAA YRS ;

[0053] SEO ID NO. 13

[0054] KRGRKKEEYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEE.

[0055] Optionally, the CAR receptor of the invention may also comprise a signal peptide capable of directing the nascent protein towards the secretory pathway. Preferably, the signal peptide precedes the alpha-enolase antigen-binding domain.

[0056] A preferred configuration of the CAR receptor according to the invention comprises, from the N-terminal end to the C-terminal end, an extracellular domain capable of binding the alpha-enolase antigen (ENO1), a transmembrane domain, one or more intracellular costimulatory domains, and an intracellular signaling domain, as defined above.

[0057] In this configuration, the CAR receptor preferably comprises or consists of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO. 14, wherein

[0058] - the sequence from nucleotide 1 to nucleotide 336 encodes the light chain variable region and corresponds to the sequence SEQ ID NO. 1;

[0059] - the sequence from nucleotide 337 to nucleotide 390 encodes the peptide linker and corresponds to the sequence SEQ ID NO. 5;

[0060] - the sequence from nucleotide 391 to nucleotide 771 encodes the heavy chain variable region and corresponds to the sequence SEQ ID NO. 2;

[0061] - the sequence from nucleotide 772 to nucleotide 882 encodes a transmembrane domain of CD8a and corresponds to the sequence SEQ ID NO. 6;

[0062] - the sequence from nucleotide 883 to nucleotide 1086 encodes a costimulatory domain of CD28 and corresponds to the sequence SEQ ID NO. 10;

[0063] - the sequence from nucleotide 1087 to nucleotide 1212 encodes a costimulatory domain of 4-1BB (CD137) and corresponds to the sequence SEQ ID NO. 11;

[0064] - the sequence from nucleotide 1213 to nucleotide 1548 encodes a signaling domain of CD3zeta and corresponds to the sequence SEQ ID NO. 8.The nucleotide sequence designated as SEQ ID NO. 14 is shown below: 5’gacattgtgatgacccaggctgcaccctctatacctgtcactcctggagagtcagtctccatctcctgcaggtctagtaagagtct cctgcatagtaatggcaacacttacttgtattggttcctgcagaggccaggccagtctcctcagctcctgatatatcggatgtccaacc ttgcctcaggagtcccagacaggttcagtggcagtgggtcaggaactgctttcgcactgagaatcagtagagtggaggctgagga tgtgggtgtttattactgtatgcaacatctagaatatcctttcacgttcggtgctgggaccaagctggagctgaaaggcagcactagtg gtagcggcaaaccaggttccggcgaaggctcgagcaaaggtcaggcctatctgcagcagtcaggggctgaactggtgaagccg ggggcctcagtgaaggtgtcctgcaaggcttctgactacagatttaccagttacaatttgcactgggtcaaacagacacctggtcag ggcctggaatggattggagctatttggcctagaaatggtgatacctcctacaatcagaagttcaaaggcaaggccacattgactgca gacaaatcctccagaacagcctacatgcagctcgacagtttgacatctgaggactctgcggtctattactgtgcaagatggggactt gatggtggtgcctggtttgcttactggggccaagggactctggtcactgtctctaccacgacgccagcgccgcgaccaccaacac cggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacga gggggctggacttcgcctgtgatttctgggtgctggtcgttgtgggcggcgtgctggcctgctacagcctgctggtgacagtggcc ttcatcatcttttgggtgaggagcaagcggagcagactgctgcacagcgactacatgaacatgaccccccggaggcctggcccca cccggaagcactaccagccctacgcccctcccagggatttcgccgcctaccggagcaaacggggcagaaagaaactcctgtata tattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaagga ggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagct caatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaagg aagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcga gcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgc aggccctgccccctcgc 3’.

[0065] The amino acid sequence encoded by SEQ ID NO. 14, designated as SEQ ID NO. 15, is shown below:

[0066] DIVMTQAAPSIPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQ LLIYRMSNLASGVPDRFSGSGSGTAFALRISRVEAEDVGVYYCMQHLEYP FTFGAGTKLELKGSTSGSGKPGSGEGSSKGQAYLQQSGAELVKPGASVKV SCKASDYRFTSYNLHWVKQTPGQGLEWIGAIWPRNGDTSYNQKFKGKATL TADKSSRTAYMQLDSLTSEDSAVYYCARWGLDGGAWFAYWGQGTLVTVST TTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVV VGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQP YAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG GKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPR.

[0067] It is understood, however, that the present invention is not limited to the CAR receptor configuration described above and that any possible CAR receptor configuration as defined above is encompassed by the present invention.

[0068] An isolated nucleic acid encoding for a CAR receptor as defined above and an expression vector comprising said nucleic acid also fall within the scope of the present invention.

[0069] In one embodiment, the nucleic acid according to the invention comprises both the nucleotide sequences SEQ ID NO. 1 and SEQ ID NO. 2.

[0070] In another embodiment, the nucleic acid according to the invention comprises or consists of the nucleotide sequence SEQ ID NO. 14.

[0071] Encoding nucleic acids according to the invention can be obtained by gene recombination techniques known and described in the state of the art. Alternatively, said nucleic acids can be produced synthetically.

[0072] The term “vector”, as used herein, refers to a molecule capable of artificially delivering foreign genetic material into a cell, where it can replicate or be expressed.

[0073] According to one embodiment, the expression vector according to the invention is a viral vector, e.g., a lentiviral, adenoviral, adeno-associated, or retroviral vector.

[0074] In an alternative embodiment, the expression vector of the invention is a plasmid, a linear expression vector, or an episome. In addition, the vector may include specific sequences capable of promoting the expression of the CAR receptor of the invention, such as, for example, promoter and / or enhancer sequences, and polyadenylation signal sequences.In a preferred embodiment, the sequence encoding for the CAR receptor in the expression vector is flanked by transposon sequences, such that the possible presence of a transposase allows the integration of said encoding sequence into the genome of a transfected cell.

[0075] The present invention is based on the results obtained by the inventors in the experimentation and research activities described in the following experimental section. In short, in vitro studies conducted by the present inventors revealed that engineered T lymphocytes expressing on their surface the CAR receptor of the invention (hereinafter also referred to as ENO1-CAR-T cells) are capable of specifically recognizing pancreatic tumor cells bearing the alpha-enolase antigen and of becoming activated, thus secreting cytokines with antitumor effector action. Moreover, using a cytotoxicity assay, the present inventors demonstrated that the ENO1-CAR-T cells activated as described above are capable of promoting the lysis of said tumor cells, and that this action is highly selective. Indeed, the effects mentioned above are no longer detectable when the expression of the alpha-enolase antigen is inhibited in the pancreatic tumor cells.

[0076] The above shows therefore that the CAR receptor of the invention advantageously gives the immune cell in which it is expressed high specificity for a target tumor cell, in particular for a tumor cell bearing the alpha-enolase antigen, while activating the effector properties of that cell and enhancing its anti-tumor response.

[0077] Therefore, a further object of the invention is an engineered immune cell comprising a nucleic acid and / or expression vector as defined above, preferably an engineered immune cell expressing a CAR receptor according to the invention on its surface.

[0078] The term “immune cell”, as used herein, refers to a cell of hematopoietic origin that is functionally involved in modulating the innate and / or adaptive immune response, for example in modulating the onset and / or execution of the mechanisms underlying such responses.

[0079] The term “engineered cell”, as used herein, refers to a cell genetically modified to express the CAR receptor of the invention.Immune cells suitable for use according to the invention include, but are not limited to, T lymphocytes, NK cells, NKT cells, monocytes, macrophages, dendritic cells, and any combination thereof.

[0080] Within the scope of the present description, the terms “T lymphocyte” and “T cell” are used interchangeably and include cytotoxic T lymphocytes (CTL, CD8+ T cells) and helper T lymphocytes (CD4+ T cells).

[0081] As is well known, macrophages are cells of the immune system, derived from the differentiation of monocytes present in peripheral blood. They act by phagocytising foreign particles. Another phagocyte subtype is dendritic cells (DC) also belonging to the class of antigen-presenting cells. Natural Killer (NK) lymphocytes, on the other hand, are a population of the immune system capable of eliminating cells recognized as foreign (nonself) without the need for antigen presentation.

[0082] Preferably, the engineered immune cell of the invention is an immune cell of human origin.

[0083] The scope of the present invention is intended to include engineered immune cells expressing the CAR receptor as defined herein either transiently or in a stable and permanent manner, depending on whether the nucleic acid encoding said receptor has integrated into the immune cell's genome or not.

[0084] As described above, the binding of the CAR receptor according to the invention, expressed on the surface of an engineered immune cell, with an antigen present on a tumor cell, preferably the alpha-enolase antigen of a PDAC cell, advantageously triggers the cytotoxic activity of the immune cell, thereby performing a striking antitumor activity with high antigen specificity.

[0085] Indeed, the antigen recognition domain of a CAR is known to be a critical determinant of its biological efficacy. The conversion of a monoclonal antibody into an scFv integrated in the CAR construct results in structural changes that can affect the antigen binding topology, theformation of immunological synapses, and the activation of engineered T lymphocytes. Several literature studies have shown that specific structural parameters, such as (i) the location of the epitope on the target, (ii) the distance of the antigen from the target cell membrane, and (iii) the length and composition of the hinge or spacer domain, significantly modulate the functional activity of the CAR, in some cases leading to a drastic reduction thereof due to steric hindrance or geometric incompatibility in the T-cell-target synapse (Hanssens, Heleen et al. “The antigen-binding moiety in the driver's seat of CARs.” Medicinal research reviews vol. 42,1 (2022): 306-342. doi:10.1002 / med.21818; Sterner, Robert C, and Rosalie M Sterner. “CAR-T cell therapy: current limitations and potential strategies.” Blood cancer journal vol. 11,4 69. 6 Apr. 2021, doi:10.1038 / s41408-021-00459-7; Mazinani, Marzieh, and Fatemeh Rahbarizadeh, “CAR-T cell potency: from structural elements to vector backbone components.” Biomarker research vol. 10,1 70. 19 Sep. 2022, doi:10.1186 / s40364-022-00417-w).

[0086] In light of the above, it is clear that the conversion of an effective monoclonal antibody in vitro does not ensure the maintenance of the same biological activity when inserted into a CAR construct. The present invention addresses this issue by designing and manufacturing a CAR construct derived from a monoclonal antibody specific for the ENO1 target antigen in which the structure ensures efficient recognition and robust signal transduction. The invention, in fact, shows that the parent monoclonal antibody, while showing binding activity and cytotoxicity in vitro, maintains full functional efficacy even in the CAR format, overcoming the structural limitations typically associated with the antibody-CAR transition.

[0087] It follows that the isolated CAR receptor, the isolated nucleic acid, the expression vector and / or the engineered immune cell as defined above are particularly suitable for use in the therapeutic treatment of a pancreatic tumor disease, in particular pancreatic ductal adenocarcinoma.

[0088] A further object of the present invention is a method for manufacturing an engineered immune cell according to the invention, which comprises introducing a nucleic acid or an expression vector as defined above into an immune cell and culturing said engineered immune cell under suitable conditions and for a time sufficient for the expression of the CARreceptor.

[0089] In the context of the present description, the terms “transduction” and “transfection” are used interchangeably and refer to a process by which an exogenous nucleic acid is introduced into a eukaryotic host cell, preferably a mammalian cell, preferably a human cell.

[0090] Transfection or transduction can be transient or stable, depending on whether the DNA introduced into the cell is maintained in the cytoplasm for a limited period of time or integrated into the cell genome.

[0091] Methods suitable for introducing a nucleic acid into an immune cell, preferably an immune cell of human origin, are known and described in the state of the art.

[0092] For example, a transfection or transduction method using non-viral vectors, such as plasmid vectors, may be used in the method according to the present invention. Typically, transfection / transduction methods using non-viral vectors are based on chemical-physical approaches designed to facilitate the entry of nucleic acid into the cell, including electroporation, ballistic methods such as the gene-gun, the use of liposomes and / or cationic polymers.

[0093] In one embodiment, the transfection / transduction system used in the method of the present invention is a transposon / transposase integration system, such as, for example, the Sleeping Beauty system (Aronovich EL et al, Hum. Mol. Genet. Volume 20, Issue Rl(201 l):R14-20). This system is based on the use of a transposon, i.e., a "mobile" DNA or RNA sequence containing within it the coding sequence to be introduced into the cell, for example the sequence coding for the CAR receptor of the invention, in combination with a transposase enzyme capable of transposing said transposon from a carrier plasmid (or other donor DNA) to the DNA of the immune cell.

[0094] Alternatively, in the method according to the invention, the introduction of the nucleic acid coding the CAR receptor or of the vector comprising said nucleic acid into an immune cell can occur through the use of a viral transduction system, preferably a viral vector selectedfrom retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses.

[0095] The selection of the most suitable transfection / transduction method for use within the scope of the present invention falls well within the skills of those of ordinary skill in the art.

[0096] An immune cell as defined above is preferably used in the method of the invention.

[0097] In one embodiment, the immune cell is from a patient suffering from pancreatic ductal adenocarcinoma. Advantageously, this embodiment allows the manufacture of an engineered immune cell suitable for use in an ex-vivo therapeutic approach in which the parent cell, e.g., a T lymphocyte, after being taken from the patient, is genetically modified ex vivo, for example by transfection, to express the CAR receptor of the invention, and subsequently reintroduced into the cancer patient, preferably by intravenous re-infusion.

[0098] Before being reintroduced into the patient, the engineered immune cell of the invention may be propagated in culture in order to generate a number of cells which is appropriate for therapeutic purposes, in particular for the treatment of pancreatic ductal adenocarcinoma.

[0099] According to this embodiment, the method of the invention can provide the use of autologous immune cells, i.e., immune cells originated from the same PDAC patient into which they are then reintroduced after their engineering. This advantageously prevents the onset of rejection phenomena by the immune system of the treated patient. Optionally, the immune cells can be isolated from the peripheral blood of the donor patient by leukapheresis - a process that allows selective separation of leukocytes from the collected blood using a cell separator.

[0100] Alternatively, the method of the invention may employ allogeneic immune cells that are characterized by being isolated from a donor other than the recipient patient. Preferably, the allogeneic immune cells are subjected to human leukocyte antigen (HLA) typing to determine an adequate level of compatibility with the recipient.

[0101] A pharmaceutical composition comprising an engineered immune cell as defined above, in combination with at least one pharmaceutically acceptable vehicle, excipient and / or diluent,is also within the scope of the invention.

[0102] According to the invention, the pharmaceutical composition is suitable for use in the above therapeutic medical applications relating to the engineered immune cell, in particular for the therapeutic treatment of pancreatic ductal adenocarcinoma.

[0103] The pharmaceutical composition of the present invention can be formulated into any suitable dosage form, for example, for subcutaneous, intravenous, intra-arterial, intraperitoneal, or intramuscular administration.

[0104] In an alternative embodiment, the pharmaceutical composition according to the invention can be formulated into a dosage form suitable for local intra-tumor administration, for example by injection under computed tomography guidance.

[0105] Of course, the selection of suitable vehicles, excipients and / or diluents is carried out depending on the desired form of administration and this selection is within the skills of those of ordinary skill in the art. The selection of the active principle dose and dosage regimen also falls within the skills of those of ordinary skill in the art, and the selection thereof depends on several factors, such as for example the age and weight of the patient, the degree of progression of the disease, as well as the size of the tumor mass to be treated.

[0106] In a preferred embodiment, the pharmaceutical composition according to the invention can be administered to a cancer patient, in particular a patient suffering from PDAC, at a dosage of between 104and 109ENO1-CAR-T cells / kg body weight, preferably at a dosage of between 105and 107ENO1-CAR-T cells / kg body weight.

[0107] The invention is further described in the examples below, with reference to the accompanying drawings, wherein:

[0108] Figure 1 shows a schematic representation of the CAR receptor of the invention;

[0109] Figure 2 shows the results of the T-Cell Receptor (TCR) activation analysis. ENO1-CAR-T cells and non-transduced T cells (Jurkat) were stimulated with recombinant ENO1 antigen (10 pg / mL) for 5, 30, 60, 90 minutes. Following this stimulation, lower phosphorylation ofLek tyrosine kinase is observed in the ENO1-CAR-T cells, together with higher phosphorylation of Zap70 and CD3z proteins (left panel) compared to non-transduced T cells (right panel), indicating increased TCR activation;

[0110] Figure 3 shows representative diagrams of IFNg (left) and IL- 10 (right) cytokine release after a 24-hour co-culture of PDAC tumor cells expressing ENO1 antigen (CFPAC) or lacking this antigen (CFPACshENO) with ENO1-CAR-T cells at different target: effector cell ratios (5:1; 2:1; 1:1). In the presence of PDAC tumor cells expressing ENO1 antigen, ENO1 CAR-T cells release a greater amount of IFNg, an anti-tumor cytokine, while the secretion of IL- 10, a pro-tumor cytokine, is reduced.

[0111] Declaration pursuant to Art. 170 bis, paragraphs 2, 3 and 4 of the Italian Industrial Property Code

[0112] The present invention has been made in accordance with the provisions of Article 170-bis, paragraphs 2, 3 and 4 of the Italian Industrial Property Code.

[0113] EXAMPLES

[0114] 1. Preparation of the construct encoding the CAR receptor of the invention and T lymphocyte transfection

[0115] The manufacture of a CAR receptor according to the invention falls well within the skills of those of ordinary skill in the art.

[0116] In order to generate the CAR receptor of the invention, a monoclonal antibody against ENO1 was sequenced, and from this, sequences corresponding to the variable portion of the light chain (SEQ ID NO. 1) and the variable portion of the heavy chain (SEQ ID NO. 2) as described above, respectively, were isolated.

[0117] For the manufacture of said monoclonal antibody, the inventors followed a procedure that involved immunizing a mouse model (BALB / c) with the ENO1 antigen and subsequently selecting antibody-producing splenocytes and obtaining the hybridoma through the fusionof the selected splenocytes with immortalized myeloma cells. The antigen used for the immunization was obtained by cloning and expression in Escherichia coli of a fragment of the ENO1 cDNA corresponding to amino acids 10-434 of the protein (Giallongo, A., et al, Molecular cloning and nucleotide sequence of a full-length cDNA for human a-enolase. Proc. Natl. Acad. Sci. USA 1986. 83:6741-6745.).

[0118] Hybridomas producing IgG immunoglobulins were selected through an immunological screening process by ELISA (Enzyme-Linked Immunosorbent Assay). This method included the step of incubating the supernatant from hybridoma cultures on polystyrene plates previously functionalized with the ENO1 antigen of interest, followed by a step to block non-specific binding sites to reduce spurious interactions.

[0119] Subsequently, culture fluids containing any antibodies secreted by the hybridomas were added to the plates, allowing for specific interaction between the immobilized antigen and the antibodies produced. Specific antigen- antibody binding was then detected by adding an anti-IgG secondary antibody conjugated to a reporter enzyme (alkaline phosphatase or horseradish peroxidase (HRP)). Enzymatic action on the appropriate chromogenic substrate resulted in the formation of a colorimetric signal, the intensity of which was found to be proportional to the amount of antibody bound, allowing the identification of hybridomas producing antibodies specific to the ENO1 antigen.

[0120] The positive hybridomas were subsequently selected and subjected to cloning procedures using the limiting dilution method, in order to ensure that stable and genetically homogeneous monoclonal cell lines capable of maintaining a consistent production of specific immunoglobulins were obtained.

[0121] In an alternative or complementary embodiment, the selection of the fused cells was confirmed by culture on a selective HAT medium (containing hypoxanthine, aminopterin, and thymidine). This selective system enables exclusive survival of functional hybrid cells, eliminating non-fused myeloma cells deficient in the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT), as well as non-transformed B cells, which are unable to proliferate indefinitely.In this way, the dual selective process based on the combined use of HAT medium and ELISA ensured that stable, highly productive hybridoma lines capable of secreting highly specific IgG monoclonal antibodies with high affinity towards the target antigen ENO1 were obtained.

[0122] Sequences encoding proteins constituting a CAR receptor of the invention, e.g., a CAR receptor consisting of the amino acid sequence SEQ ID NO. 15 as shown above, were then sequentially cloned in a transposon plasmid with a bidirectional promoter using molecular methods widely known in the state of the art (Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N. Y). The correct insertion of the above sequences into the transposon plasmid was confirmed by sequencing.

[0123] For the purpose of introducing into T cells the nucleic acid thus obtained encoding the CAR receptor of the invention, e.g., a nucleic acid comprising or consisting of the nucleotide sequence designated herein as SEQ ID NO. 14, the present inventors employed the non-viral “Sleeping Beauty” system based on the transposon-transposase copy-and-paste mechanism. In particular, Jurkat cells (ATCC, Virginia USA) were used, i.e., immortalized acute lymphocytic leukemia T-lymphocytes. The “Sleeping Beauty” system is based on the use of a transposon - a “mobile” DNA or RNA sequence - which contains the sequence coding for the CAR receptor, and a transposase - an enzyme that regulates its transposition into the lymphocyte's DNA (Aronovich EL et al, Hum. Mol. Genet. Volume 20, Issue Rl(2011):R14-20). Typically, this system is used in an attempt to overcome some limitations of viral transduction such as immunogenicity, production costs, and limitations on the size of the gene to be transduced.

[0124] CAR receptor transfection into Jurkat cells using the Sleeping Beauty system involved the following detailed procedure. Two plasmids were prepared: a first plasmid containing the CAR gene within the Sleeping Beauty transposon and a second plasmid expressing the Sleeping Beauty transposase. Jurkat cells were cultured in RPMI 1640 medium with 10% fetal bovine serum and appropriate antibiotics, ensuring that they were in the logarithmic growth phase. Electroporation or nucleofection was used for transfection, as Jurkat cells donot respond well to lipid reagents; the cells were mixed in the presence of both the first and second plasmids in a suitable electroporation buffer, and the electrical pulse was applied with a nucleofection device optimized for these cells. After transfection, the cells were transferred to an antibiotic-free complete medium and incubated at 37°C with 5% CO2 for recovery. Since the CAR construct of the invention incorporates a selectable marker, specifically a gene conferring antibiotic resistance, selection was initiated 24-48 hours posttransfection. Selection-positive cells were subsequently expanded in vitro.

[0125] Post-transfection CAR expression in the cells was confirmed by flow cytometry using specific antibodies, while successful integration of the CAR gene into the cell genome was verified by PCR or other molecular assays. The methodology illustrated herein enabled stable integration of the CAR gene into the genome of Jurkat cells via the Sleeping Beauty transposon system, ensuring long-term expression of the transgene.

[0126] 2. Activation of ENO1-CAR-T cells by the ENO1 antigen

[0127] In a particularly advantageous embodiment of the invention, the screening for antibodies produced by the hybridomas also included assessing the antibody's ability to selectively recognize and bind the target antigen of interest, i.e., the ENO1 antigen, as well as verifying that such specificity and binding affinity are maintained when the sequence of the antibody variable regions is used to generate CAR receptors. In this respect, antibody selection is crucial not only to ensure the effectiveness of the native antigen- antibody binding, but also to ensure that the derived recognition domain (scFv) retains its antigenic functionality and specificity when expressed in the context of a modified effector cell, such as an engineered T cell or NK cell.

[0128] In order to demonstrate that the recognition of the ENO1 antigen leads to activation of the ENO1-CAR-T cells, a pure population of ENO1-CAR-T cells (> 80%) was stimulated with the recombinant ENO1 protein at different time intervals, and the phosphorylation of Lek and Zap70 tyrosine kinase proteins and of the CD3 co-receptor was subsequently assessed.

[0129] T-cell receptor (TCR) activation occurs following recognition of a peptide-MHC complexon the antigen-presenting cell. This event induces a conformational rearrangement of the TCR-CD3 complex chains, exposing the ITAMs (Immunoreceptor Tyrosine-based Activation Motifs) present on the CD3(^, CD3s, CD35, and CD3y subunits.

[0130] The tyrosines contained in the ITAMs are phosphorylated by Lek tyrosine kinase associated with the CD4 or CD8 co-receptors. The phosphorylated tyrosines serve as docking sites for ZAP-70 kinase, which once recruited and activated by Lek, in turn phosphorylates cytoplasmic adaptors (LAT, SLP-76). This triggers a cascade of downstream signals involving the MAPK, PLCyl and NF-KB pathways, culminating in T lymphocyte transcriptional activation, proliferation and effector function.

[0131] In particular, the activity of the Lek protein is regulated by two major phosphorylation sites, Tyr394 and Tyr505. This protein, when dephosphorylated, leads to signal activation (Acuto O, et al, “T-cell receptor signals by proximal negative feedback mechanisms”. Nat Rev Immunol. 2008 Sep;8(9):699-712. doi: 10.1038 / nri2397. PMID: 18728635). In contrast, phosphorylations of the ITAM motifs present in the CD3 chains and of ZAP-70 tyrosine kinase represent events activating the TCR signalling cascade, as their phosphorylation allows the recruitment and sequential activation of the intracellular adaptor complexes responsible for signal transduction (Love P.E. & Hayes S.M., “IT AM-mediated signaling by the T-cell antigen receptor”, Nat Rev Immunol. 2010 May;10(5):337-347. doi:10.1038 / nri2741; Au- Yeung B.B. etal., “The structure, regulation, and function of ZAP-70”, Immunol Rev. 2018 Mar;285(l):45-55. doi: 10.1111 / imr.12690).

[0132] Briefly, to demonstrate the specificity of the CAR receptor of the present invention in binding the ENO1 antigen and leading to TCR activation, ENO1 CAR-T cells were stimulated in vitro with the ENO1 antigen for 5 minutes (5’), 30 minutes (30’), 60 minutes (60’), and 90 minutes (90’). Each stimulation was subsequently stopped by washing at 4°C to obtain a pellet of stimulated cells. The cells were then lysed to obtain a cell lysate, and the phosphorylation of Lek, CD3(^, and Zap70 was analysed by the Western Blot method.

[0133] As shown in Figure 2, stimulation with recombinant ENO1 antigen results in a lower degree of phosphorylation of the Lek protein (Tyr505) in ENO1-CAR-T cells and an increase in thephosphorylation of this protein in untransduced Jurkat cells. In contrast, there is an increase in the phosphorylation of the CD3(^ and Zap70 proteins in EN01-CAR-T cells and a lower phosphorylation of these proteins in untransduced Jurkat cells. These results demonstrate that recognition of ENO 1 by the CAR receptor leads to the activation of the T cell expressing said receptor.

[0134] 3. Activation of ENO1-CAR-T cells by PDAC tumor cells

[0135] The inventors conducted in vitro assays to demonstrate the responsiveness of ENO1-CAR-T cells in the presence of immortalized PDAC tumor cells expressing the ENO1 antigen of interest (CFPAC cells from ATCC, Virginia USA). In detail, CFPAC cells were co-cultured with increasing concentrations of ENO1-CAR-T cells to assess the production of anti-tumor effector cytokines, such as IFNg and TNFa, and suppressive pro-tumor cytokines, such as IL10. The extent of secretion of these cytokines by ENO1-CAR-T cells was assessed using the ELISA technique (Biolegend, California USA) and the actual amount of cytokines produced was assessed by subtracting the values measured in the medium (not stimulated with ENO1) and expressing the cytokine levels thus detected in pg / mL.

[0136] The diagrams in Figure 3 show that after 24 hours or 48 hours of co-culture with CFPAC cells, the production of IFNg and TNFa by ENO1-CAR-T cells is significantly higher than that measured in co-cultures of these cells with CFPACshENO cells in which the expression of the ENO1 antigen was silenced by using a short hairpin RNA (shRNA). In addition, the diagrams in Figure 3 show a significant decrease in IL10 production by ENO1-CAR-T cells after 24 hours or 48 hours of co-culture with CFPAC cells, compared to the levels of this cytokine measured when ENO1-CAR-T cells are cultured in the presence of CFPACshENO cells.

[0137] The data illustrated above provide clear evidence of the specific recognition of the ENO1 antigen present on the surface of PDAC tumor cells by ENO1-CAR-T cells and the subsequent activation of said cells with significant production of cytokines with anti-tumor effector action.4. Cytotoxicity assay

[0138] The inventors also demonstrated that the specific recognition and binding of the EN01 antigen present on the surface of CFPACs by EN01-CAR-T cells leads to the lysis of these tumor cells.

[0139] To this end, a cytotoxicity assay was carried out using an instrument that integrates flow cytometry with the ability to take pictures (Attune CytPix, ThermoFisher, Massachussetts, USA). Briefly, tumor cells expressing the EN01 antigen (CFPAC) or those silenced for this antigen (CFPACshENO) were stained with the CSFE marker (Merck, Germany) and cocultured with EN01 CAR-T cells stained with an anti-Fab-APC antibody (Jackson ImmunoResearch, UK). The cell samples were then analysed by acquiring fluorescence values with the Attune CytPix. As shown by the data in Table 1 below, using the aforementioned markers, after 30 minutes of incubation, the inventors detected a 24.01 percentage of double-positive cells, thus representing a mixed population of ENO1-CAR-T cells and CFPAC tumor cells. This double positivity percentage decreases significantly, up to 1.14%, when the ENO 1 -CAR-T cells are co-cultured with the CFPAC shENOl tumor cells which, as explained above, do not express the ENO1 antigen. This demonstrates the existence of a highly specific binding between the ENO 1 -CAR-T cells and the ENO1 antigen present on the surface of the tumor cells.

[0140] Table 1

[0141] Antigen recognition assay (30 minutes)

[0142] CFPAC + ENO 1- CFPAC ENO 1 -CAR-T

[0143] CAR-T

[0144] % cells 15.59 56.74 24.01

[0145] CFPACshENO 1 CFPACshENO 1 ENO 1 -CAR-T

[0146] + ENO 1 -CAR-T

[0147] % cells 26.9 65.71 1.14

[0148]

[0149] In addition, for the cytotoxicity assay, the target tumor cells (CFPAC and CFPACshENO cells) were stained with the CSFE marker and co-cultured with the effector cells (ENO1-CAR-T) at different effector: target ratios (1:1; 1:5; 1:10), for 4 or 8 hours. The cell lysis values in Table 2 below, expressed as the percentage of dead cells to total live cells, clearly indicate that the ENO1-CAR-T cells exert a highly specific cytotoxic action, since lysis of target tumor cells is only observed in the presence of ENO1 antigen expression.

[0150] Table 2

[0151] Cytotoxicity assay

[0152] CFPACshENO

[0153] 4 hours CFPAC (% lysis)

[0154] (% lysis)

[0155] T:E = 1:1 7.8 1.8

[0156] T:E = 1:5 37.6 7.3

[0157] T:E = 1:10 45.8 18.4

[0158] CFPACshENO

[0159] 8 hours CFPAC (% lysis)

[0160] (% lysis)

[0161] T:E = 1:1 2.6 2.3

[0162] T:E = 1:5 20.2 9.0

[0163] T:E = 1:10 56.3 13.3

[0164]

[0165] The cytotoxicity data illustrated above are also confirmed by the results of the “double positive” cell population imaging in Table 1 above, which show that the ENO 1 -CAR-T cells are linked to the tumor cells and that the latter are undergoing apoptotic death.

Claims

CLAIMS1. An isolated chimeric antigen receptor (CAR), comprising:an extracellular domain capable of binding the alpha-enolase antigen (ENO1), a transmembrane domain, andan intracellular signaling domain,wherein the extracellular domain capable of binding the ENO1 antigen is a single-chain antibody fragment (scFv) comprising, from the N-terminal end to the C-terminal end, a light chain variable region and a heavy chain variable region, the light chain variable region comprising or consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO. 1, and the heavy chain variable region comprising or consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO. 2.

2. The isolated CAR receptor according to claim 1, wherein the light chain variable region is linked to the heavy chain variable region through a peptide linker.

3. The isolated CAR receptor according to claim 2, wherein the peptide linker comprises or consists of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO. 5.

4. The isolated CAR receptor according to any of claims 1 to 3, wherein the transmembrane domain comprises or consists of a transmembrane domain of a protein selected from the group consisting of CD4, CD8, CD8a, CD3, CD3-zeta, CD28, 0X40 (CD134), and 4-1BB (CD137).

5. The isolated CAR receptor according to any of claims 1 to 4, wherein the intracellular signaling domain comprises or consists of a signaling domain of a protein selected from the group consisting of CD3zeta, FcRgamma, FcRbeta, FcRepsilon, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

6. The isolated CAR receptor according to any of claims 1 to 5, further comprising one or more costimulatory domains.

7. The isolated CAR receptor according to claim 6, wherein the one or more costimulatory domains comprise or consist of a costimulatory domain of a protein selected from the group consisting of CD28, CD27, 4-1BB (CD137), 0X40 (CD134), ICOS (CD278), CD30, CD40, PD-1 (CD279), CD2, CD7, NKG2C (CD94), B7-H3 (CD276), and any combination thereof.

8. The isolated CAR receptor according to any of claims 1 to 7, which comprises or consists of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO. 14.

9. An isolated nucleic acid encoding a CAR receptor according to any of claims 1 to 8.

10. The isolated nucleic acid according to claim 9, comprising the nucleotide sequence of SEQ ID NO. 1 and the nucleotide sequence of SEQ ID NO. 2.

11. The isolated nucleic acid according to claim 10, which comprises or consists of the nucleotide sequence of SEQ ID NO. 14.

12. An expression vector comprising a nucleic acid according to any of claims 9 to 11.

13. An engineered immune cell comprising a nucleic acid according to any of claims 9 to 11, and / or an expression vector according to claim 12.

14. The engineered immune cell according to claim 13, expressing on its surface a CAR receptor according to any of claims 1 to 8.

15. The engineered immune cell according to claim 13 or 14, which is selected from the group consisting of T lymphocytes, NK cells, NKT cells, monocytes, macrophages, dendritic cells, and any combination thereof.

16. A pharmaceutical composition comprising an engineered immune cell according to any of claims 13 to 15, and at least one pharmaceutically acceptable vehicle, excipient and / ordiluent.

17. The pharmaceutical composition according to claim 16, which is in a pharmaceutical form suitable for administration via subcutaneous, intravascular intravenous, intravascular intraarterial, intraperitoneal, or intramuscular route.

18. The pharmaceutical composition according to claim 16, which is in a pharmaceutical form suitable for intratumor administration.

19. An isolated CAR receptor according to any of claims 1 to 8, an isolated nucleic acid according to any of claims 9 to 11, an expression vector according to claim 12, an engineered immune cell according to any of claims 13 to 15, and / or a pharmaceutical composition according to any of claims 16 to 18, for use in the therapeutic treatment of pancreatic ductal adenocarcinoma.

20. The isolated CAR receptor, isolated nucleic acid, expression vector, engineered immune cell and / or pharmaceutical composition for use according to claim 19, wherein the treatment is a therapy that promotes the lysis of pancreatic tumor cells bearing the membrane alpha-enolase (ENO1) antigen in a subject suffering from pancreatic ductal adenocarcinoma.

21. An in vitro method of preparing an engineered immune cell according to any of claims 13 to 15, the method comprising introducing into an immune cell an isolated nucleic acid according to any of claims 9 to 11 or an expression vector according to claim 12, and culturing said engineered immune cell under suitable conditions and for a time sufficient for the expression of the CAR receptor.

22. The in vitro method according to claim 21, wherein the immune cell is from a patient suffering from pancreatic ductal adenocarcinoma.

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