Chimeric antigen receptor binding the globo series antigens

Abstract

A chimeric antigen receptor (CAR) is described, said chimeric antigen receptor comprising: a first endodomain CD3, and a single-chain antibody fragment (scFv). that recognizes the Globo series antigens, wherein the CAR comprises an amino acid sequence having a sequence identity between 80% and 100%, preferably between 85% and 95%, with respect to SEQ ID No: 9, 10, 11, or 12.

Description

[0001] Title: Chimeric antigen receptor binding the Globo series antigens

[0002] DESCRIPTION

[0003] Field of application

[0004] In its most general aspect, the present invention relates to chimeric antigen receptors (CARs) binding the Globo series antigens.

[0005] In particular, the present invention relates to a chimeric antigen receptor (CAR) including a single-chain antibody fragment (scFv) comprising VH and VL variable regions joined by a non-immunogenic linker.

[0006] The invention also relates to a method for producing a cell population comprising the chimeric antigen receptor.

[0007] Moreover, the invention also relates to said CAR for use in the treatment of solid tumors, in particular with the effect of inhibiting the growth of tumor cells.

[0008] Prior art

[0009] In recent years, the advent of immunotherapy, and especially of monoclonal antibodies inhibiting the immunological check-point, has improved the life expectancy of tumor-affected patients. These drugs, by enhancing the patient’s immune system, enable recognition and elimination of the tumor cells that are in the organism. Despite their important therapeutic activity toward some hematologic and non- hematologic neoplasms, different factors may limit their efficacy. The first factor is the activation of the immune system against the “self’, which is a possible cause of severe autoimmune reactions that can complicate the use of this type of therapies. Moreover, both the presence of an immunosuppressive tumor microenvironment and the extreme heterogeneity of tumor antigens, in particular in solid tumors, play an important role. Adoptive cell therapy (ACT) plays a role of primary importance among the immunotherapeutic approaches. A very important peculiarity among those that make ACT very attractive is its feature of being a tailored treatment requiring isolation of the cells of the patient’s immune system, successive engineering, ex- vivo expansion and reinfusion thereof1. Currently, ACT uses mainly three cell types: tumor-infiltrating lymphocytes (TILs), lymphocytes provided with engineered T-cell receptor (TCRs) and T cells engineered with sequences encoding the chimeric antigen receptor (CAR-T).

[0010] It is known that CAR-T cells are genetically engineered lymphocytes capable of specifically recognizing antigens expressed on the membrane of tumor- tissue cells2. Said cells are engineered so as to express an extracellular domain, namely a single-chain antibody fragment (scFv), designated to recognize tumor-associated surface antigens. The scFv within the CAR-coding sequences consist of both the variable heavy chain and the variable light chain of the antibody. Said scFv are capable of specifically recognizing the antigen, thereby enabling the activation of T- cell-specific signaling independently of the human-leukocyte-antigen (HLA) system, and thus, without the antigen being presented and processed3. Finally, the presence of cassettes for co-stimulatory domains, both in the case of second-generation constructs and in the case of third- generation constructs, is capable of influencing the effector function of the CAR-T cell. The first CAR-T cells were directed toward CD 19 so as to target cells of acute lymphoblastic leukemia (ALL) and B-cell lymphomas. Currently, the efforts are focused on the development of CAR-T cells directed toward antigens expressed on solid tumors. However, this objective is not easy to achieve due to some peculiarities characterizing this type of pathologies. The specificity to only one single extracellular target could be a limitation when its expression on the surface of the tumor cell is downregulated as a consequence of the resistance mechanisms carried out by the tumor. For this reason, the choice of a target having high specificity and expression on the surface of tumor cells is still a critical issue for the development of this type of technology. CA3204922 discloses CAR constructs capable of binding to Globo series antigens, including Globo-H, the constructs including an antigen binding fragment (Fab) or a single-chain variable fragment (scFv), and a method for treating a subject with cancer with such constructs.

[0011] US20 17 / 283489 discloses T cells expressing a CAR against stage-specific embryonic antigen 4 (SSEA-4).

[0012] The technical problem underlying the present invention is providing a new system for engineering and re-directing cell populations, in particular of T cells, expressing CAR that is highly specific against extracellular targets expressed on tumor cells, in particular of solid tumors, so as to solve the above-mentioned problems of the prior art.

[0013] Summary of the invention

[0014] Said problem was solved, according to the invention, by a chimeric antigen receptor (CAR) comprising: a first endodomain CD3, and a single-chain antibody fragment (scFv) that recognizes Globo-H antigen, wherein the CAR comprises an amino acid sequence having a sequence identity between 80% and 100%, preferably between 85% and 95%, with respect to SEQ ID No: 9, 10, 11, or 12.

[0015] The above-mentioned technical problem was also solved by a process for obtaining a cell population of the chimeric antigen receptor expression according to the invention comprising the following steps: a) providing a cell population; b) providing a vector that includes a nucleic acid sequence encoding a chimeric antigen receptor CAR according to the present invention; c) generating a cell population of chimeric antigen receptor expression by transducing the cell population with the aforementioned vector, thus obtaining a cell population expressing the chimeric antigen receptor (CAR).

[0016] The present invention also relates to a cell population expressing a chimeric antigen receptor (CAR) according to the present invention, in particular for use in the treatment of solid tumors.

[0017] The present invention also relates to a pharmaceutical composition for the treatment of solid tumors comprising a cell population expressing the chimeric antigen receptor according to the present invention.

[0018] The advantages and the features of the chimeric antigen receptor (CAR) according to the present invention will become clearer from the detailed description and from the following examples, which are given by way of a non-limiting example with reference to the accompanying figures.

[0019] Brief description of the drawings

[0020] Figure 1 shows the values of mean fluorescence intensity (MFI) detected through cytofluorimetry of Globo H in different tumor cell lines of different tumors, in particular ovarian tumor (Fig. la), breast tumor (Fig. lb), digestive system tumor (Fig. lc), and other tumor types (Fig. Id).

[0021] Figure 2 shows the Globo-H-expression profile in different tumor cell lines and tissues. In particular, Figure 2a shows tumor cell lines that are positive for a Globo-H-specific antibody, namely Globo-H-expressing tumor cells. Figure 2b shows tumor cell lines that are negative for a Globo-H-specific antibody, namely cells that do not express Globo H.

[0022] Figures 3a, 3b, 3c and 3d show Globo-H-expression profiles in different tissues, respectively, of breast tumor, of cholangiocarcinoma, of colorectal tumor, luminal-subtype breast tumor, detected through immunohistochemistry.

[0023] Figure 4a shows an outline of the scFv-sequence cloning into the second- generation retroviral vectors. Figure 4b shows the electrophoretic analysis identifying the second-generation SFG retroviral vectors containing the intracellular co-stimulatory domains CD28 or 4 IBB and the intracellular signaling domain CD3, and the scFvs that are not yet ligated. Figure 4c shows an outline of the different CARs according to the invention, in which scFv, the first endodomain, the second endodomain, the transmembrane region, and the hinge region are observed. Figure 4d shows the electrophoretic analysis identifying the constructs.

[0024] Figure 5 shows the percentage of HER 293-T-cell transfection for the production of retroviral supernatants expressing the CARs according to the invention. In particular, Figure 5a represents non-transfected HER 293-T cells. Figures 5b and 5c show the transfection percentage for the CAR constructs having 4 IBB as second endodomain, namely scFv in conformation VH_VL_41BB and VL_VH_41BB, respectively. Figure 5d shows the transfection percentage for the CAR construct having CD28 as a second endodomain, namely scFv in conformation VH_VL_CD28.

[0025] Figure 6 shows the transduction of the T lymphocytes from patients with the constructs according to the invention. In particular, Figure 6a shows the cytofluorimetry plot regarding non-transduced T lymphocytes from patients. Figure 6b shows the cytofluorimetry plot regarding T lymphocytes transduced with the CAR construct VH_VL_41BB, indicating the transduction percentage. Figure 6c shows the cytofluorimetry plot regarding T lymphocytes transduced with the CAR construct VL_VH_41BB, indicating the frequency of T lymphocytes expressing said CAR.

[0026] Figure 7 shows the percentage of expression of the CAR constructs in T lymphocytes. In particular, Figures 7a and 7c indicate the percentages of expression of the CAR constructs according to the invention. Said percentages were respectively obtained through the use of the FLAG APC antibody (Fig. 7a) for staining by detecting the TAG inserted in the sequence or through the use of the biotinylated Globo H (Fig. 7c). This results can be compared with NT samples in Figures 7b and 7d, namely non-transduced T cells used as a control in both experiments.

[0027] Figure 8 shows the analysis of the activation profile of T cells infected with the CAR construct following the activation through the specific antigen Globo H. In particular, Figure 8a shows the activation profile of CAR-T cells according to the invention in the cell line N87 expressing the target. Figure 8b shows the activation profile of CAR-T cells according to the invention following stimulation with the cell line A431 which does not express the target. NT: non-transduced T cells used as a control in both the experiments. Figure 8c shows the gating approach used to assess the frequency of activated T cells (CD69+CD25+) activated through the specific antigen Globo H.

[0028] Figure 9 shows the in-vitro tests to verify the recognition ability and the specificity of the CAR construct according to the invention transduced into the T lymphocytes by means of tests of inhibition of the tumor cell viability. Figures 9a, 9b, 9c and 9d show the proliferation of the tumor cells, expressing Globo H, in the presence of CAR-T cells, in the tumor cell lines N87 of gastric tumor, MCF7 of breast tumor, EGI 1 and HUCCT1 of cholangiocarcinoma, respectively. As a control, non-transduced (NT) T cells and tumor cells as such were used. Figure 9e shows the proliferation of tumor cells A431 from vulvar tumor, which do not express Globo H. As a control, non-transduced (NT) T cells and tumor cells as such were used.

[0029] Figures 10a and 10b show the cytotoxicity assays of the CAR construct according to the invention, transduced into T lymphocytes. The CAR-T constructs, VL_VH_41BB (Car A in Figure 10a) and VH_VL_CD28 (CAR B in Figure 10b), were cultured with the N87 and EGI- 1 cell lines, comparing CAR-T cells expanded for 7 days with those expanded for 14 days using the traditional method. Figures 11a and l ib show the phenotypic characterization of the transduced T cells, respectively of the subpopulations CD4 and CD8. In each plot, the differentiation degree of the populations within the CAR-T cells, in particular of the populations of T cell subtypes such as naive cells (Tn), central memory T cells (Tern), effector memory T cells (Tern), terminally differentiated T cells (Temra), for the first week, the second week and the third week is shown.

[0030] Detailed description

[0031] The expression “cell population” means cells coming and isolated from the fraction of peripheral blood mononuclear cells (PBMC).

[0032] According to the present invention, the expression “CAR-T cell” identifies a cell deriving from the T lymphocyte and transduced with the CAR construct according to the present invention.

[0033] The expression “T cells” and “T lymphocytes” are synonyms and are used interchangeably.

[0034] The expression “single-chain antibody fragment” or the acronym “scFv” comprises a variable fragment of a single-chain antibody (Fv) that recognizes a known protein of a tumor cell. The fragment Fv is engineered from monoclonal antibodies by using only the two N-terminal variable domains of the antibody and connecting them with each other with a linker.

[0035] The term “patient(s)” means a person affected by a disease, in this case a solid tumor.

[0036] The term “endodomain” encompasses all the intracellular domains. In particular, the first endodomains are signaling domains responsible for the activation, proliferation, and survival of the cell populations, in particular of T cells. The second endodomains are stimulation domains, i.e., they cause the signal transmission following antigen-TCR binding to occur in an optimal way.

[0037] The CAR binds the Globo series antigen, which may be selected from the stage-specific embryonic antigen 3 (SSEA-3), the stage-specific embryonic antigen 4 (SSEA-4) and the antigen Globo H.

[0038] According to the invention, said CAR specifically binds Globo H. Preferably, said scFv comprises an amino acid sequence having a sequence identity between 80% and 100%, preferably between 85% and 95%, with respect to SEQ ID No: 3 or 4.

[0039] In particular, said scFv has a variable light chain domain (VL) and a variable heavy chain domain (VH), which are assembled through a linker in the VH-linker-VL and VL-linker-VH conformations. The VH-linker-VL scFv comprises an amino acid sequence having a sequence identity between 80% and 100% with respect to SEQ ID No: 3.

[0040] The VL-linker-VH scFv comprises an amino acid sequence having a sequence identity between 80% and 100% with respect to SEQ ID No: 4.

[0041] In an embodiment, the chimeric antigen receptor may contain an antigenbinding fragment FAb, which comprises a variable heavy chain region (VH) having an amino acid sequence that has a sequence identity between 80% and 100%, preferably between 85% and 95%, with respect to SEQ ID No: 1 and comprises a variable light chain region (VL) having an amino acid sequence that has a sequence identity between 80% and 100%, preferably between 85% and 95%, with respect to SEQ ID No: 2.

[0042] In an embodiment, the first endodomain may be selected from CD3-zeta and CD3 gamma.

[0043] Preferably, the chimeric antigen receptor comprises a first endodomain CD3-zeta having an amino acid sequence that has a sequence identity between 80% and 100%, preferably between 85% and 95%, with respect to SEQ ID No: 8.

[0044] Preferably, the chimeric antigen receptor (CAR) comprises a hinge region and a transmembrane domain that is CD8 having an amino acid sequence that has a sequence identity between 80% and 100%, preferably between 85% and 95%, with respect to SEQ ID No: 5.

[0045] Preferably, said CAR further comprises a second endodomain selected from CD28, 4 IBB, 0X40, ICOS, CD27, CD40, CD40L or TLR, wherein scFv is fused to the second endodomain and the second endodomain is fused to the first endodomain.

[0046] The expression “second endodomain” may also be identified as intracellular domain, in particular co-stimulatory domain, or costimulation domain. In the present disclosure, said terms and said expressions are used interchangeably.

[0047] Preferably, the chimeric antigen receptor (CAR) comprises a second endodomain CD28 having an amino acid sequence that has a sequence identity between 80% and 100%, preferably between 85% and 95%, with respect to SEQ ID No: 6.

[0048] Preferably, the chimeric antigen receptor (CAR) comprises a second endodomain 4 IBB having an amino acid sequence that has a sequence identity between 80% and 100%, preferably between 85% and 95%, with respect to SEQ ID No: 7.

[0049] Preferably, the aforementioned process further comprises the step of expanding said cell population.

[0050] Preferably, in step b) the vector is selected from lentiviruses, retroviruses, or adeno-associated viruses.

[0051] In an embodiment, the vector is a retrovirus.

[0052] Preferably, the step b) comprises:

[0053] - assembly of the VL and VH variable regions in VH-linker-VL and / or VL- linker-VH conformation; and

[0054] - cloning within retroviral vectors in order to obtain said CAR constructs according to the invention.

[0055] Preferably, the cell population may be selected from T lymphocytes (T cells), gamma delta T lymphocytes (Ty5), Natural killer cells (NK) or Natural killer T lymphocytes (NKT). The cell population may also comprise macrophages generated by monocytes isolated from PBMC.

[0056] More preferably, the T lymphocytes may be selected from stem cell-like memory T cell (TSCM), central memory T lymphocytes (TCM), effector memory T lymphocytes (TEM) .

[0057] Preferably, said cell population expressing a chimeric antigen receptor is a T cell expressing a chimeric antigen receptor (CAR-T) .

[0058] Advantageously, the possibility of expressing said CAR construct in different cell populations allows to choose the cell population that is best for both the patient and the necessary anti-tumor therapy, thereby increasing the specificity of action. In fact, said cells may be engineered with a CAR and the choice of which cells to engineer depends on the tumor to be treated.

[0059] Preferably, the solid tumors comprise solid tumors expressing Globo series antigens.

[0060] More preferably, said solid tumors expressing Globo series antigens are selected from sarcoma, skin tumor, brain tumor, glioblastoma, lung tumor, breast tumor, oral tumor, head-and-neck tumor, nasopharyngeal tumor, bladder tumor, pancreas tumor, intestinal tumor, colorectal tumor, renal tumor, cervical tumor, endometrial tumor, ovarian tumor, mouth tumor, oropharyngeal tumor, laryngeal tumor, esophageal tumor, rectal tumor, bile-duct tumor, in particular cholangiocarcinoma, testis tumor, neuroendocrine tumor, adrenal tumor, thyroid tumor, bone tumor, basal tumor squamous-cell carcinoma, melanoma and prostate tumor.

[0061] Preferably, said pharmaceutical composition may be a pharmaceutical composition for parenteral administration, more preferably a pharmaceutical composition for subcutaneous administration, a pharmaceutical composition for intradermal administration, a pharmaceutical composition for intramuscular administration, and a pharmaceutical composition for intravenous administration.

[0062] In particular, when the pharmaceutical composition is administered intravenously, it is to be understood as an intravenous administration that may occur through a bolus or an infusion.

[0063] Advantageously, the CAR construct according to the invention expressed in cell populations such as CAR-Ts achieves the desired cytotoxic effect in significantly shorter time compared to traditional CAR-Ts. In fact, the CAR construct according to the invention achieves a cytotoxic effect such as to substantially totally inhibit the growth of tumor cells after just 7 days of expansion. On the contrary, the complex preparation system of traditional CAR-Ts requires 9 to 14 days to obtain the same cytotoxic effect. Therefore, advantageously, the CAR-Ts according to the invention can be prepared and used for the treatment of solid tumors in shorter time than traditional CAR-Ts. This results in shorter waiting times for therapy and, therefore, provides the possibility of more immediate action. The production costs of the CAR construct according to the present invention expressed in cell populations, such as CAR-Ts, are also lower because only 7 days of expansion are required for preparation compared to the 9- 14 days required for the preparation of traditional CAR-Ts. Hereafter, an example is described that explains the steps for obtaining the CAR construct and the cell population expressing the CAR construct, in particular a T cell expressing the CAR construct (CAR-T), according to the present invention and the functionality of said construct in terms of inhibition of the growth of Globo-H-overexpressing cells.

[0064] In general, the nomenclatures used in connection with and the described techniques of cell and tissue culture, molecular and chemical biology, and hybridization of proteins and oligo- or polynucleotides are the ones well-known and commonly used in the art.

[0065] EXAMPLE 1 : Production of the CAR-T cells

[0066] 1.1 Globo-H-expression profile in different tumor lines through cytofluorimetry

[0067] As can be observed in the plot of Figure 1, different cell lines were analyzed through cytofluorimetry, said cell lines being purchased from ATCC (indicated in Figure 1) and derived from different tumors, such as breast, colon or ovarian tumors, to identify in which tumor cell line the antigen Globo H is expressed. For each analyzed line, 5xl05cells in suspension in their culture medium were incubated for 30 minutes in ice with a Globo-H-specific antibody (mouse IgM) in a concentration of 10 gg / ml, wherein the culture medium consists of a formulation that is supplemented with nutrients necessary for cell proliferation and to which antibiotics necessary to avoid bacterial contaminations are added. Since the scFv expressed on the CAR surface according to the present invention must bind Globo H, it is important to know which cell lines expressing said antigen.

[0068] After incubation with the Globo-H-specific antibody, the cells were washed again with PBS and incubated for 30 minutes in ice with an antimouse IgM-Alexa488, a fluorescent marker capable of detecting the antibody bound to the antigen Globo H.

[0069] Reading of the results was carried out through a cytofluorimeter.

[0070] As can be observed in the plots of Figure 2, not all the considered cell lines express Globo H or, in any case, it is not in a sufficient amount for the recognition by the specific antibody.

[0071] 1.2 Amplifications of the variable VH and VL from the hybridoma, assembly of the scFv and cloning within a CAR vector

[0072] The hybridoma secreting the anti-Globo-H antibody (a murine antibody) was produced using the standard protocol of somatic hybridization.

[0073] The messenger RNA was extracted from the hybridoma cells using the Quiagen kit and subsequently reverse transcribed into cDNA using an Applied Biosystem kit. The variable, heavy and light, regions of the antibody were then amplified from the hybridoma cDNA through PCR and assembled using the linker (Gly-Gly-Gly-Ser)4, in VH-linker-VL or VL-linker-VH conformation, and inserted into the CAR cassette.

[0074] 1.3 Expression profile in different tissues using IHC

[0075] Immunohistochemistry (IHC) was carried out on tumor-tissue sections from different tumors, such as breast tumor, colorectal tumor, cholangiocarcinoma, and a luminal-subtype breast tumor. Said sections were fixed in formalin and embedded in paraffin (FFPE) of 3 pm thickness.

[0076] IHC was carried out on a BenchMark Ultra platform (Ventana Medical Systems, Tucson, AZ). The tumor tissue sections underwent antigen unmasking with a cell conditioning solution CC1 (at 100°C for 48 minutes) and were incubated with the mouse monoclonal anti-GloboH antibody (diluted 1:25; incubation at 37°C for 1 hour). The specific signal was detected using the OptiviewDAB kit with amplification (Ventana Medical Systems).

[0077] As can be observed in Figures 3a, 3b and 3c, the tissues isolated from breast tumor, cholangiocarcinoma and colorectal tumor are positive, whereas the luminal subtype is negative (Figure 3d).

[0078] 1.4 Sequencing of the single-chain variable fragments for the design of the CAR construct

[0079] The sequences encoding the 2 scFvs with different orientation of the variable chains, inserted in the phagemid vector pIT2, were sequenced using the Sanger method. The sequences are SEQ ID No: 3 and 4.

[0080] 1.5 Strategy for cloning the CAR constructs

[0081] The cleavage sites for the restriction enzymes Ncol (CCATGG) and Mlul (ACGCGT) were added at the two 5’ and 3’ ends of both the scFvs obtained in the two orientations (VH_VL e VL_VH) at step 1.2 to enable the successive cloning of the scFv sequences in retroviral vectors (see outline in Figure 4a).

[0082] Before adding the cleavage sites, a short nucleotide sequence encoding an 8-amino acid flag was also inserted at the 3’ end of both sequences, said flag being useful to detect the scFv once it is expressed by the T cells (DYKDDDDK).

[0083] The desired sequences, obtained by cutting with restriction enzymes at the cleavage sites, were ligated (16°C, overnight incubation) with traditional methods (DNA-ligase type) into previously validated second- generation SFG retroviral vectors (Fig. 4a and 4b) containing the intracellular co-stimulatory domains CD28 or 4 IBB in addition to the intracellular signaling domain CD3-zeta. In particular, the nucleotide sequence of the scFv to be cloned may be ligated to the retroviral vector using the DNA ligases. The recombinant vector is then introduced into a bacterial cell (AH5a E. coli) to amplify the construct. In fact, the bacterial colonies with the recombinant vector were cultured in the suitable culture broth supplemented with ampicillin, to prevent the proliferation of bacteria not containing the construct, and the vector was extracted through miniPrep (QIAprep Spin Miniprep kit). The vector obtained was then validated through sequencing and amplified through maxiPrep (GenElute HP Endotoxin Free Plasmid Maxiprep kit, Sigma- Aldrich) , before the use.

[0084] Thus, 4 constructs were obtained (Fig. 4c), whose identity was validated through both restriction analysis of the miniPrep (Fig. 4d) and Sanger sequencing.

[0085] 1.6 Packaging 293T cells

[0086] The human embryonic kidney cell line 293T was purchased from ATCC and kept in 1MDM (fscove’s Modified Dulbecco’s Medium, Lonza) supplemented with 10% fetal bovine serum (FBS) inactivated at 56 °C according to the indications of the manufacturer (Gibco), 100 lU / mL penicillin (Merck), 100 mg / mL streptomycin (Merck) and 2 mmol / L L- glutamine (Merck).

[0087] 1.7 Transfection of 293T cells with CAR sequences for the production of retroviral supernatants

[0088] The retroviral supernatants were produced through co-transfection of the 293T cells with 3 plasmids: PegPam-e (encoding MoMLV gag-pol), RDF (encoding RD 114 env), namely plasmids encoding the proteins required for the formation and the replication of the viral particles, and the desired CAR constructs CD28 VH_VL, 4 IBB VH_VL and 4 IBB VL_VH, respectively. An empty vector encoding the green fluorescent protein (GFP) was also used as a positive control to measure the cell-transfection efficiency through FACS analysis. The 293T cells were seeded 24 hours prior to transfection, in order to make cell density reach 80% confluency at the time of transfection. The cells were then transfected using the transfection reagent Gene Juice (Millipore) and a total DNA amount equal to 10 gg according to the following ratios:

[0089] PegPam3: RDF: CAR=3:2:3.

[0090] The retroviral supernatants were then harvested at 48 and 72 hours after transfection, filtered through 0.45-gm filters and frozen in liquid nitrogen to be subsequently used in the experiments of T lymphocytes transduction. As can be observed in Figure 5b, 5c and 5d, the transfection percentage is higher than 90%.

[0091] 1.8 Isolation ofT lymphocytes from patients’ peripheral blood

[0092] The mononuclear component (PBMC), namely the mononuclear cell population from peripheral blood, said population comprising all the mononuclear leukocytes, including also T lymphocytes, was isolated from 30 ml of peripheral blood taken from patients of the Fondazione IRCCS Istituto Nazionale dei Tumori of Milan after informed consent (INT 151 / 15) using the reagent Ficoll Paque Plus (Cytiva) following the manufacturer’s indications. Afterward, the PBMCs were counted and frozen in human serum and DMSO in a ratio 9: 1 or immediately used in the experiments of transduction with retroviral supernatants.

[0093] 1.9 Transduction ofT lymphocytes activated with retroviral supernatants

[0094] Before transduction, the PBMCs as previously isolated from the blood samples were activated with the CD3 antibody (Miltenyi Biotec, 100 gg / ml) and CD28 antibody (Pharmingen, 1 mg / ml). 48 hours after activation, the activated T lymphocytes were selected and expanded, and were then transduced using the retroviral supernatants obtained from the packaging 293T cells, through centrifugation in plates previously treated with retronectin (Takara) on which retroviral particles were adhered, also through centrifugation (2000g for 90 minutes). At the end of transduction, the transduced T lymphocytes are kept in RPMI medium supplemented with interleukin 15 (IL- 15) at a final concentration of 5 ng / ml and interleukin 7 (IL-7) at a final concentration of 10 ng / mL 72 hours after transduction, an aliquot of transduced T lymphocytes is used for cytofluorimetric assessment of transduction efficiency. As shown in Figure 6, the transduction percentages are higher than 80% in the lymphocytes isolated from the patient (Figg. 6b and 6c). The constructs used, in the above-mentioned experiment, are the constructs with the costimulation domain 4 IBB with the chains in the two orientations. These experiments show that the constructs used are capable of inducing expression of the CAR of interest, its correct conformation and localization on the cell membrane of the T cells.

[0095] 1.10 Expansion of transduced T cells

[0096] The CAR-T cells were expanded for the 21 days following the transduction under the same conditions described in 1.9 and the scFv expression by the CAR-T cells compared with non-transduced control T cells (NT) was assessed every 7 days through cytofluorimetry. As can be appreciated in Figure 7, 14 days after transduction the frequency of CAR-T cells in culture is higher than 80% (Fig. 7a). 1.11 FACS Staining with antiFlag and GloboH-protocol

[0097] To identify the surface expression of the CAR construct according to the invention, the transduced T cells were labeled with the APC-conjugated monoclonal Fc-specific antibody (APC anti-DYKDDDDK Tag Antibody, Biolegend) that specifically recognizes the flag-TAG epitope inserted in the construct of the anti-GloboH CAR-T according to the invention. The transduction of T lymphocytes with the CAR construct was also confirmed by incubating the T cells with biotinylated Globo H followed by incubation with PE-conjugated streptavidin.

[0098] The samples were acquired through cytofluorimetry (FACS Calibur-BD) and analyzed with the software FlowJo (v. 10.9.0, BD).

[0099] 1.12 Staining protocol for the characterization of the activation profile (CD69; CD25f.

[0100] For the characterization of the activation profile, the CAR-T cells and the non-transduced T cells (NT), namely T cells prepared as a control, were stimulated for 24h with the tumor line N87 (which is Globo-H-positive) and with the tumor line A431 (which is Globo-H-negative). After incubation, the supernatant containing the CAR-T cells and NT cells was harvested and the lymphocytes were labeled with anti-CD4 FITC, anti- CD8 APC, anti-CD25 PE and anti-CD69 PerCP-Cy5.5 monoclonal antibodies. The samples were acquired through FACS Canto (BD) and analyzed with the software FlowJo (v. 10.9.0). Specifically, living lymphocytes were selected and debris were discarded (plot of SSC-A vs FSC-A) and the selection of activated lymphocytes (CD25+CD69+) was made on the CD8+ population.

[0101] As can be observed in Figure 8, the CAR-T cells directed against the antigen Globo H when activated by the line N87 show a higher frequency of the markers CD69 and CD25. On the contrary, the stimulation with A431 does not cause any increase in the frequency of the considered markers. 1.13 Co-culture of CAR-T cells with Globo-H-positive and negative tumor cells for the validation of functional activity (Cell Titer Gio)

[0102] 1.13.1 Protocol of CAR-T co-culture test

[0103] With the purpose of verifying the anti-tumor activity and the specificity of the CAR-T cells produced, a test was carried out. Said test was based on the co-culture of CAR-T cells with different Globo-H-positive tumor lines such as N87 (gastric tumor), MCF7 (breast tumor), EGI1 and HUCCT1 (cholangiocarcinoma) and Globo-H-negative tumor lines such as A431 (vulvar tumor) (as already mentioned in Fig. 2).

[0104] The CAR-T cells and their corresponding NT cells were then cultured together with the previously described tumor cells, according to the effector- vs-tumor (E:T) ratios ranging from 5: 1 to 0.5: 1 (Fig.9).

[0105] At day 0, the different cell lines are seeded in 96-well plates at 5000 / 10000 cells / well in 100 pl.

[0106] At day 1, CAR-T or NT were added at different ratios, E:T from 10: 1 to 0,5: 1, in 100 pl medium. The CAR-T’s functional activity was assayed through the Cell Titer Gio test.

[0107] The test assesses the number of viable cells in culture by quantifying ATP (by means of luciferase), which indicates the presence of metabolically active cells.

[0108] Luminescence reading is directly proportional to the number of viable cells in culture.

[0109] In this case it is necessary, before adding the reagent, to remove the lymphocytes by washing with PBS, so that they do not interfere with the reading.

[0110] The reagent is then added after washing each well three times, and reading is carried out with a luminescence analyzer (TECAN). The tests were ended 72 or 120 hours after CAR-T addition.

[0111] As it is possible to observe in Figure 9, the proliferation rate of all the tested tumor lines cultured together with the NT (non-transduced) cells is equal to the proliferation rate of the tumor cells cultured alone. On the contrary, the addition of the CAR-T cells expressing the CAR against the antigen Globo H is capable of causing a decrease of cell proliferation in all the E:T ratios considered (Fig.9a, 9b, 9c, and 9d). Contextually, the specificity of CAR-T cells to the antigen was assayed, repeating the experiment with the cell line of the vulvar tumor, A431. In this case, the presence of CAR-T cells according to the invention does not influence the proliferation of tumor cells, showing no difference in terms of cell growth (Fig. 9e).

[0112] Finally, using the same protocol described previously, the test was performed by co-culturing CAR-T cells, VL_VH_41BB and VH_VL_CD28 (designated as CAR A and CAR B in Figure 10, respectively), with N87 and EGI- 1 cell lines to evaluate the in vitro cytotoxic activity of the CAR- T cells according to the invention. Specifically, CAR-T cells expanded for 7 days were compared with CAR-T cells expanded for 14 days, which are commonly used in clinical practice (along with the usual comparison to controls). As can be seen, the cytotoxic effect at 7 days is very similar to that of CAR-T at 14 days, which provides an advantage both in terms of reduced production costs since the time to obtain them is reduced and in terms of greater feasibility, accessibility and speed of clinical use of the CAR-T of the invention in therapy for patients.

[0113] 1.14 Phenotypic characterization- Staining protocol

[0114] 1.0 x 106CAR-T cells and non-transduced (NT) controls were incubated with FcR Blocking Reagent and 5 pl monoclonal anti-CCR7 PE-Dazzle 594 antibody for 30 minutes at room temperature. After the first incubation, 20 pl anti-CD45RA FITC antibody, lOpl CD95 PE, 3pl CD8 APC-H7, 2pl CD4 Alexa Fluor 700 and 2.5 pl anti-FLAG tag APC antibody were added. The incubation was then continued for 30 min at 4°C. The samples were acquired through a 13 -color cytofluorimeter (CytoFLEX, Beckman Coulter) and analyzed with the software FlowJo (v. 10.9.0). Specifically, living lymphocytes were selected and debris were discarded (plot of SSC-A vs FSC-A), afterwards, doublets were excluded (plot of FSC- H vs FSC-A) and the subpopulations CD4 and CD8 were selected (plot of CD8 APC-H7 vs CD4- AlexaFluor700) . The CAR-T cells (FLAG-APC+) were then selected within the two subpopulations. The CAR-T cells were characterized using the CCR7 vs CD45RA model (4) which allowed the identification of four T-cell types T: naive T cells (TN) , central memory T cells (TCM), effector memory T cells (TEM), terminally differentiated T cells (TEMRA) . Afterwards, within the gate of the naive T cells, stem cell-like memory T cells (TSCM) were identified using the plot of CD62L vs CD95.

[0115] Among the subsets considered for engineering with CAR constructs, there are central memory T cells (TCM) and effector memory T cells (TEM), thanks to their ability to expand and persist for long periods after administration. A further subset was identified in the stem cell-like memory T cells (TSCM) which, in addition to having a proliferation potential and in-vivo persistence characteristics, like the previously described subsets, also have characteristics that are similar to stem cells. For this reason, said stem cell-like memory T cells are capable of regenerating and differentiating, thereby acquiring greater effector abilities, thus bringing about a great advantage in terms of in-vivo persistence6. In order to understand the potentiality in terms of persistence and efficacy of the CAR-T cells produced, their phenotypic characteristics during expansion were analyzed. In particular, the subsets TN, TCM, TEM and TEMRA were analyzed, using the plot of CCR7 vs CD45RA, which reflects the degree of differentiation of the populations within the pool of CAR-T cells according to the invention. As can be observed in Figure 11, during the first week of expansion, the subset TN is almost totally absent, which is consistent with the in-vitro activation following treatment with anti-CD3 and anti- CD28 antibodies, whereas the most numerous populations are TCM e TEM. Throughout the expansion, during the first week, the population TCM is more numerous than the population TEM. During the second week, the two populations are evening out in terms of frequency, however, with a prevalence of TCM. There is, instead, a trend reversal during the third week. Based on these data, the time window for the use is in the second week.

[0116] BIBLIOGRAPHY

[0117] 1. Bear A. S., Fraietta J. A., Naranyan V. K., O’Hara M and

[0118] Haas N. B, Adoptive cellular therapy for solid tumors, ASCO Educational Book 41, 57-65 (2021)

[0119] 2. Ramos C. A and Dotti G, Chimeric antigen receptor (CAR)- engineered lymphocytes for cancer therapy. Expert Opin on Biol Ther, 11:7, 855-873 (2011).

[0120] 3. Eshar Z., Waks T., Grosst G. and Shindler D.G., Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunit of the immunoglobulin and T- cell receptors. Proc Natl Acad Sci U S A, 90(2):720-724 (1993)

[0121] 4. Sallusto F., Lenig D., Forster R., Lipp M and Lanzavecchia A, Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401, 708-712(1999)

[0122] 5. Rea, I. M., Mcnerlan, S. E. & Alexander, H. D. CD69, CD25, and HLA-DR activation antigen expression on CD3+ lymphocytes and relationship to serum TNF-, IFN-, and sIL-2R levels in aging. Exp Gerontol, 31(4): 79-93 (1999).

[0123] 6. Gattinoni, L., Klebanoff, C. A. & Restifo, N. P. Paths to sternness: building the ultimate antitumour T cell. Nat Rev Cancer 12(10):671-684 (2012).

[0124] SEQUENCES

[0125] SEQUENCE 1

[0126] PRT

[0127] Artificial Sequence

[0128] Variable heavy chain (VH)

[0129] QVQLQQSGTELASPGASVTLSCKASGYTFTDHIINWVKKRPGQGLEWIGRI

[0130] YPVSGVTNYNQKFMGKATFSVDRSSNTVYMVLNSLTSEDPAVYYCGRGFD

[0131] FDYWGQGTTVTVSS

[0132] SEQUENCE 2

[0133] PRT

[0134] Artificial Sequence

[0135] Variable light chain (VL)

[0136] DIQLTQSPPSLTVSVGERVTISCKSNQNLLWSGNRRYCLGWHQWKPGQTP

[0137] TPLITWTSDRFSGVPDRFIGSGSVTDFTLTISSVQAEDVAVYFCQQHLDLPY

[0138] TFGGGTKLEIKRER

[0139] SEQUENCE 3

[0140] PRT

[0141] Artificial Sequence scFv VH-linker-VL format QVQLQQSGTELASPGASVTLSCKASGYTFTDHIINWVKKRPGQGLEWIGRI

[0142] YPVSGVTNYNQKFMGKATFSVDRSSNTVYMVLNSLTSEDPAVYYCGRGFD

[0143] FDYWGQGTTVTVSSGGGGSGGGGSGGGGSTDIQLTQSPPSLTVSVGERV

[0144] TISCKSNQNLLWSGNRRYCLGWHQWKPGQTPTPLITWTSDRFSGVPDRFI

[0145] GSGSVTDFTLTISSVQAEDVAVYFCQQHLDLPYTFGGGTKLEIKRER

[0146] SEQUENCE 4

[0147] PRT

[0148] Artificial Sequence scFv VL-linker-VH format

[0149] DIQLTQSPPSLTVSVGERVTISCKSNQNLLWSGNRRYCLGWHQWKPGQTP

[0150] TPLITWTSDRFSGVPDRFIGSGSVTDFTLTISSVQAEDVAVYFCQQHLDLPY

[0151] TFGGGTKLEIKREVSSGGGGSGGGGSGGGGSTQVQLQQSGTELASPGAS

[0152] VTLSCKASGYTFTDHIINWVKKRPGQGLEWIGRIYPVSGVTNYNQKFMGKA

[0153] TFSVDRSSNTVYMVLNSLTSEDPAVYYCGRGFDFDYWGQGTTVTVSS

[0154] SEQUENCE 5

[0155] PRT

[0156] Artificial Sequence

[0157] CD8

[0158] IYIWAPLAGTCGVLLLSLVITLYC

[0159] SEQUENCE 6 PRT

[0160] Artificial Sequence

[0161] CD28

[0162] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0163] SEQUENCE 7

[0164] PRT

[0165] Artificial Sequence

[0166] 4 IBB

[0167] RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0168] SEQUENCE 8

[0169] PRT

[0170] Artificial Sequence

[0171] CD3-zeta

[0172] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP

[0173] RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK

[0174] DTYDALHMQALPPR

[0175] SEQUENCE 9 (CD3_CD28_CD8_VL_linker_VH_FLAG)

[0176] PRT

[0177] Artificial Sequence CARscFv

[0178] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP

[0179] RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK

[0180] DTYDALHMQALPPRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFA

[0181] AYRSIYIWAPLAGTCGVLLLSLVITLYCDIQLTQSPPSLTVSVGERVTISCKSN

[0182] QNLLWSGNRRYCLGWHQWKPGQTPTPLITWTSDRFSGVPDRFIGSGSVTD

[0183] FTLTISSVQAEDVAVYFCQQHLDLPYTFGGGTKLEIKREGGGGSGGGGSG

[0184] GGGSGGGGSQVQLQQSGTELASPGASVTLSCKASGYTFTDHIINWVKKRP

[0185] GQGLEWIGRIYPVSGVTNYNQKFMGKATFSVDRSSNTVYMVLNSLTSEDP

[0186] AVYYCGRGFDFDYWGQGTTVTVSSGGSDYKDDDDK

[0187] SEQUENCE 10 (CD3_41BB_CD8_VL_linker_VH_FLAG)

[0188] PRT

[0189] Artificial Sequence

[0190] CARscFv

[0191] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP

[0192] RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK

[0193] DTYDALHMQALPPRRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE

[0194] GGCELIYIWAPLAGTCGVLLLSLVITLYCDIQLTQSPPSLTVSVGERVTISCKS

[0195] NQNLLWSGNRRYCLGWHQWKPGQTPTPLITWTSDRFSGVPDRFIGSGSVT

[0196] DFTLTISSVQAEDVAVYFCQQHLDLPYTFGGGTKLEIKREGGGGSGGGGS

[0197] GGGGSGGGGSQVQLQQSGTELASPGASVTLSCKASGYTFTDHIINWVKKR PGQGLEWIGRIYPVSGVTNYNQKFMGKATFSVDRSSNTVYMVLNSLTSED

[0198] PAVYYCGRGFDFDYWGQGTTVTVSSGGSDYKDDDDK

[0199] SEQUENCE 11 (CD3_CD28_CD8_VH_linker_VL_FLAG)

[0200] PRT

[0201] Artificial Sequence

[0202] CARscFv

[0203] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP

[0204] RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK

[0205] DTYDALHMQALPPRRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDF

[0206] AAYRSIYIWAPLAGTCGVLLLSLVITLYCQVQLQQSGTELASPGASVTLSCKA

[0207] SGYTFTDHIINWVKKRPGQGLEWIGRIYPVSGVTNYNQKFMGKATFSVDRS

[0208] SNTVYMVLNSLTSEDPAVYYCGRGFDFDYWGQGTTVTVSSGGGGSGGGG

[0209] SGGGGSTDIQLTQSPPSLTVSVGERVTISCKSNQNLLWSGNRRYCLGWHQ

[0210] WKPGQTPTPLITWTSDRFSGVPDRFIGSGSVTDFTLTISSVQAEDVAVYFCQ

[0211] QHLDLPYTFGGGTKLEIKRERGSDYKDDDDK

[0212] SEQUENCE 12 (CD3_41BB_CD8_VH_linker_VL_FLAG)

[0213] PRT

[0214] Artificial Sequence

[0215] CARscFv

[0216] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP

[0217] RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPRRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE

[0218] GGCELIYIWAPLAGTCGVLLLSLVITLYCQVQLQQSGTELASPGASVTLSCK

[0219] ASGYTFTDHIINWVKKRPGQGLEWIGRIYPVSGVTNYNQKFMGKATFSVDR

[0220] SSNTVYMVLNSLTSEDPAVYYCGRGFDFDYWGQGTTVTVSSGGGGSGGG GSGGGGSTDIQLTQSPPSLTVSVGERVTISCKSNQNLLWSGNRRYCLGWH

[0221] QWKPGQTPTPLITWTSDRFSGVPDRFIGSGSVTDFTLTISSVQAEDVAVYFC

[0222] QQHLDLPYTFGGGTKLEIKRERGSDYKDDDDK

Claims

CLAIMS1. A chimeric antigen receptor (CAR) comprising: a first endodomain CD3, and a single-chain antibody fragment (scFv) that recognizes the Globo-H antigen, wherein the CAR comprises an amino acid sequence having a sequence identity between 80% and 100%, preferably between 85% and 95%, with respect to SEQ ID No: 9, 10, 11, or 12.

2. The chimeric antigen receptor (CAR) according to claim 1, wherein said scFv comprises an amino acid sequence having a sequence identity between 80% and 100%, preferably between 85% and 95%, with respect to SEQ ID No: 3 or 4.

3. The chimeric antigen receptor (CAR) according to claim 1 or 2, wherein the first endodomain CD3 is selected from CD3-zeta or CD3gamma.

4. The chimeric antigen receptor (CAR) according to claim 3, wherein the chimeric antigen receptor comprises a first endodomain CD3-zeta having an amino acid sequence that has a sequence identity between 80% and 100%, preferably between 85% and 95%, with respect to SEQ ID No: 8.

5. The chimeric antigen receptor (CAR) according to any one of claims 1-4. comprising a hinge region and a transmembrane domain that is CD8 having an amino acid sequence that has a sequence identity between 80% and 100%, preferably between 85% and 95%, with respect to SEQ ID No:5.

6. The chimeric antigen receptor (CAR) according to any one of claims 1- 5, wherein said CAR further comprises at least one second endodomain selected from CD28, 4 IBB, 0X40, ICOS, CD27, CD40, CD40L or TLR, wherein scFv is fused to the second endodomain and the second endodomain is fused to the first endodomain.

7. The chimeric antigen receptor (CAR) according to claim 6, wherein the chimeric antigen receptor (CAR) comprises a second endodomain CD28 having an amino acid sequence that has a sequence identity between 80% and 100%, preferably between 85% and 95%, with respect to SEQ ID No:6.

8. The chimeric antigen receptor (CAR) according to claim 6, wherein the chimeric antigen receptor (CAR) comprises a second endodomain 4 IBB having an amino acid sequence that has a sequence identity between 80% and 100%, preferably between 85% and 95%, with respect to SEQ ID No:7.

9. A process for obtaining a cell population expressing the chimeric antigen receptor according to any one of claims 1-8, comprising the following steps: a) providing a cell population; b) providing a vector that includes a nucleic acid sequence encoding said chimeric antigen receptor (CAR); c) generating a cell population expressing the chimeric antigen receptor by transducing the cell population with the aforementioned vector, thus obtaining a cell population expressing the chimeric antigen receptor (CAR).

10. The process according to claim 9, wherein in step b) the vector is selected from lentiviruses, retroviruses, or adeno-associated viruses.

11. The process according to claim 9 or 10, wherein step b) comprises:- assembly of the VL and VH variable regions in VH-linker-VL and / or VL- linker-VH conformation; and- cloning within retroviral vectors in order to obtain said CAR constructs.

12. A cell population expressing a chimeric antigen receptor according toany one of claims 1-8.

13. The cell population according to claim 12, wherein the cell population is selected from T lymphocytes, gamma delta T lymphocytes (Ty5), Natural Killer cells (NK), Natural Killer T lymphocytes (NKT), and macrophages generated by monocytes.

14. A cell population of the chimeric antigen receptor according to any one of claims 1-8 for use in the treatment of solid tumors expressing the Globo series antigens.

15. A pharmaceutical composition for the treatment of solid tumors comprising the cell population according to claim 12 or 13.

Images