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WO2025051339A1 - FRAGMENT VARIABLE À CHAÎNE UNIQUE ET MOLÉCULES DÉRIVANT DE CELUI-CI SE LIANT AU FRAGMENT TRVB5-1 DE LA CHAÎNE β DU RÉCEPTEUR DES LYMPHOCYTES T HUMAINS - Google Patents

FRAGMENT VARIABLE À CHAÎNE UNIQUE ET MOLÉCULES DÉRIVANT DE CELUI-CI SE LIANT AU FRAGMENT TRVB5-1 DE LA CHAÎNE β DU RÉCEPTEUR DES LYMPHOCYTES T HUMAINS Download PDF

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WO2025051339A1
WO2025051339A1 PCT/EP2023/025286 EP2023025286W WO2025051339A1 WO 2025051339 A1 WO2025051339 A1 WO 2025051339A1 EP 2023025286 W EP2023025286 W EP 2023025286W WO 2025051339 A1 WO2025051339 A1 WO 2025051339A1
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scfvs
lymphocytes
chain
fragment
cell
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Michele Pitaro
Matteo SORRENTI
Maria Teresa PITARO
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Tiber Biotech Srl
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Tiber Biotech Srl
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • Table 1 shows the nucleotide sequence of fragment TRBV5-1 of the P chain of the human TCR as shown in the prior art (the "International ImMunoGeneTics Information System® Wei S, Charmley P, Robinson MA, Concannon
  • Table 2 shows the aminoacidic sequence of fragment TRBV5-1 of the P chain of the human TCR, obtained from the nucleotide sequence according to table 1.
  • Table 3 shows the aminoacidic sequence of the first antigenic peptide singled out inside fragment TRBV5-1 of the P chain of the human TCR. The segment actually used in the production of the scFv by means of the phage display technique is highlighted in red.
  • Table 4 shows the aminoacidic sequence of the second antigenic peptide singled out in fragment TRBV5-1 of the P chain of the human TCR. Inside, two candidate segments have been individuated for the phage display procedure, respectively indicated in black and in green.
  • Table 5 shows the aminoacidic sequence of the third antigenic peptide singled out in fragment TRBV5-1 of the P chain of the human TCR. This peptide immediately follows the second peptide in C-terminal direction.
  • Table 6 highlights, inside the three candidate antigenic peptides, the problematic amino acids, i.e. those amino acids that may compromise the antigenic characteristics of the peptide during the synthesis process.
  • Table 7 shows the scores obtained by the three candidate antigenic peptides in the simulations carried out with two different bioinformatics tools called “Antigen Profiler Peptide” (TM-score) and “AbDesigner” (Ab-score).
  • Table 8 describes the results of the polyclonal ELISA assay (enzyme- linked immunosorbent assay) for the preliminary detection of the phages that recognize the segment of the first antigenic peptide.
  • Table 9 describes the results of the first monoclonal ELISA assay carried out on 96 single phages obtained in round 4.
  • Table 10 describes the results of the second monoclonal ELISA assay carried out on 96 single phages in round 4.
  • Table 11 describes the results of the confirmatory ELISA assay.
  • Table 12 describes the sequences CDR1, CDR2, CDR.3 of the light chains, sequences CDR.1, CDR2, CDR3 of the heavy chains and the relative FR of the six human scFvs identified by means of the phage display procedure, that recognize the segment of the first antigenic peptide of fragment TRBV5-1 of the 0 chain of the human TCR.
  • Table 13 shows the alignment of the sequences between the primary structure of fragment TRBV5-1 as reported in Uniprot (A0A578) and the sequences FASTA 5BRZ and 5BS0. The intensity of the colouring indicates the identity percentage.
  • Table 14 displays the ranking of scFvs sorted by increasing interface energy score and corresponding number of interacting amino acid residues of the TCR. 0 chain constant region.
  • Table 15 shows the complete sequences of the monoclonal antibody A12 obtained by joining the variable regions of the corresponding scFv to the heavy chain of the human IgGl.
  • Table 16 shows the affinity constants (KD) of the six monoclonal antibodies for the antigenic construct.
  • Figure 1 shows the 3D reconstruction of a human TCR expressing fragment TRBV5-1 in the 0 chain (in red is shown the segment of the first antigenic peptide selected for the scFv production by means of the phage display technique).
  • Figure 2 shows the structure of the TCR. identified by code 5BS0.
  • the mutation is shown in the variable region of the 0 chain, in red the potential bonding surface with the six scFvs; in magenta the loop of the constant region of the 0 chain; in green and in yellow respectively the variable and constant regions of 0 and a chains of the TCR.
  • Figure 3 shows the results of the flow cytometry analysis carried out on the PBMC obtained from a patient suffering from Sezary Syndrome who expresses the fragment TRBV5-1 in the variable region of the 0 chain of the TCR.
  • the cells were preincubated with a complete panel of antibodies (anti-CD3, anti-CD4, anti-CD8, anti-CD45) with the addition of the anti-CD14 antibody for the detection of the monocytes.
  • Panels "A”, “B” and “C” show in red the cell population under study in the form of a dot plot. In panel “D” the same population is shown in the form of a histogram.
  • Panels "E”, “F” and “G” instead refer to the test carried out with the addition of the lead compound A12 (in panel “H” the same population is shown in the form of a histogram). The test highlights the binding of the lead compound A12 with 21% of the cell population under examination.
  • Figure 4 shows the results of the flow cytometry analysis carried out on PBMC obtained from a patient suffering from Sezary Syndrome who expresses in the variable region of the TCR 0 chain one of the three fragments (TRBV6-5; TRBV6-6; TRBV6-9) recognized by the antibody anti-Vbl3.1 of the lOTest® Beta Mark (Beckman Coulter).
  • the cells were preincubated with the complete antibodies panel (anti- CD3, anti-CD4, anti-CD8, anti-CD45) with the addition of the anti- CD14 antibody for the detection of the monocytes.
  • Panels “A, "B” and “C” show in red the cell population under examination in the form of a dot plot. In panel “D” the same population is shown in the form of a histogram.
  • Panels "E, "F” and “G” instead refer to the test carried out with the addition of the lead compound A12 (in panel “H” the same population is shown in the form of a histogram). As can be seen, antibody A12 doesn't bind any cell of the population under examination.
  • the present invention concerns fragments of antibodies called scFvs binding a new TAA, i.e. fragment TRBV5-1 of the 0 chain of the human TCR, and molecules deriving therefrom.
  • such molecules may be Fab (fragment antigenbinding), Fab2, minibodies and monoclonal antibodies of the different classes (IgG, IgM, IgA and IgD), monospecific as well as components of bi-specific molecules.
  • Such molecules may also be CAR. (chimeric antigen receptor).
  • Said molecules may be used for diagnostic as well as for therapeutic purposes in pathologies originated by the clonal expansion of T-lymphocytes expressing fragment TRBV5-1 in the 0 chain of the TCR.
  • T lymphocytes are an essential component of adaptive immunity (Abbas AK, Lichtman AH, Pillai S. 2012. Immunologia cellulare e molecolare. Settima ediée. Elsevier, Milano 2012). They originate from progenitor cells of the bone marrow that migrate to the thymus to complete the maturation process. They have a central role in the regulation of both the humoral and cellular immunity.
  • T-cells can be divided into different subpopulations, in particular naive T-cells (who respond to new antigens), memory T-cells (responsible for maintaining the long-term immune response), and regulatory T-cells that regulate the homeostasis of immune responses (Kumar BV, Connors TJ, Farber DL. Human T Cell Development, Localization, and Function throughout Life. Immunity. 2018 Feb 20;48(2):202-213).
  • helper T lymphocytes Based on the expression of specific antigens on the cell membrane called CD4 and CD8, mature T lymphocytes are divided into helper (CD4+) and cytotoxic T lymphocytes (CD8+) (Saravia J, Chapman NM, Chi H. Helper T cell differentiation. Cell Mol Immunol. 2019 Jul;16(7):634-643). Following to their activation, helper T lymphocytes can differentiate into several subtypes classified according to the production of specific cytokines and to the expression of characteristic surface markers. The subtypes of helper T lymphocytes identified to date are Thl, Th2, Thl7, Th9, Th22, follicular helper T-cells (Tfh), induced regulatory T-cells (iTreg) and regulatory cells of type 1 (Tri).
  • Thl cells produce IFN-y, IL-2 and TNFo and activate the immune response against viruses and intracellular bacteria (Luckheeram RV, Zhou R, Verma AD, Xia B. CD4+T cells: differentiation and functions. Clin Dev Immunol. 2012;2012:925135).
  • Th2 cells produce IL-4, IL-5 and IL-13, and activate the immune response against parasites (they are also responsible for allergic reactions).
  • Thl7 cells produce IL-17A, IL-17F and IL-22 and are involved in the immune response against extracellular bacteria and fungi. They are particularly abundant in the intestine, where they modulate the gut microbiota composition.
  • Follicular helper T lymphocytes Tfh
  • Tfh promote humoral immunity inside the germinal centres (GC), by producing IL-21 (which stimulates B lymphocytes) and IL-4 (which participates to the immunoglobulin isotype switching).
  • CD8 is the membrane antigen characteristic of cytotoxic T lymphocytes (Opferman JT, Ober BT, Ashton-Rickardt PG. Linear differentiation of cytotoxic effectors into memory T lymphocytes. Science. 1999 Mar 12;283(5408): 1745-8). They play a central role in the response against intracellular pathogens and cancer cells and they are involved in the pathogenesis of autoimmune diseases. Cytotoxic T lymphocytes kill cells expressing target antigens (mainly antigenic peptides obtained by the processing of viral proteins or proteins mutated in tumors).
  • target antigens mainly antigenic peptides obtained by the processing of viral proteins or proteins mutated in tumors.
  • CD8+ T lymphocytes Once activated, they release cytotoxic molecules like granzymes and perforin and they secrete cytokines like IFN-y and IFN-o, which amplify innate, as well as adaptive immune responses.
  • cytokines like IFN-y and IFN-o, which amplify innate, as well as adaptive immune responses.
  • CD8+ T lymphocytes undergo apoptosis, but a small portion is transformed in memory T-cells. These lymphocytes respond by proliferating and rapidly transforming into effector cells when newly exposed to the antigen (Henning AN, Roychoudhuri R, Restifo NP. 2018. Epigenetic control of CD8+ T cell differentiation. Nat. Rev. Immunol. 18, 340- 356).
  • T lymphocytes characterized by the expression of CD4 and CD25, are at present considered a third sup-population of T lymphocytes (Gliwihski M, Piotrowska M, Iwaszkiewicz-Grzes D, Urban-Wojciuk Z, Trzonkowski P. Therapy with CD4+CD25+ T Regulatory Cells - Should We Be Afraid of Cancer? Contemp. Oncol. 2019;23: 1-6). They eliminate the autoreactive lymphocytes clones and play a central role in the regulation of immune responses. They are divided in two main subpopulations, i.e.
  • T-cells generated in the thymus- nTreg
  • iTreg naive T- cells during the immune response
  • lymphomas and leukemias The two main kinds of neoplasms originated by T lymphocytes are lymphomas and leukemias.
  • the former originate from the main lymphatic organs, i.e. lymph nodes and spleen, but the presence of lymphatic tissue in different tissues allows the development thereof also in other organs.
  • the latter originate from lymphoid progenitors in the bone marrow, but sometimes also in the blood and in extramedullary sites. In a limited number of cases it is difficult to determine whether the neoplasm arises from the bone marrow or the lymphatic tissue and the diagnose remains uncertain.
  • Non-Hodgkin lymphoma is the most frequent hematological malignancy, accounting for nearly 3% of all cancers (Thandra KC, Barsouk A, Saginala K, Padala SA, Barsouk A, Rawla P. Epidemiology of Non-Hodgkin's Lymphoma. Med Sci (2021) 9: 5). It differs from Hodgkin lymphoma in clinical and histologic features, i.e. the absence of Reed-Sternberg cells and positivity to CD15 and CD30. In 85% of cases it originates from B lymphocytes and in 15% of cases from T lymphocytes.
  • T-cell lymphomas TCL
  • PTCL peripheral T-cell lymphomas
  • CCL cutaneous T-cell lymphomas
  • ACL Anaplastic large-cell lymphomas
  • AITL Angioimmunoblastic T-cell lymphoma
  • PTCL-NOS Extranodal NK/T-Cell Lymphoma (mainly gastrointestinal and cutaneous)”
  • Swerdlow SH Campo E, Pileri SA, Harris NL, Stein H, Siebert R, Advani R, Ghielmini M, Salles GA, Zelenetz AD, Jaffe ES.
  • ALCL accounts for 1-3% of NHL and around 15% of TCL (Anaplastic Large Cell Lymphoma: Molecular Pathogenesis and Treatment. Zhang XR, Chien PN, Nam SY, Heo CY.). It is classified into four categories: ALK-positive ALCL (ALK+ALCL), ALK-negative ALCL (ALK-ALCL), primary cutaneous ALCL (pcALCL), and breast-implant-associated ALCL (BIA-ALCL). It is characterized by large lymphoid cells with copious cytoplasm and pleomorphic characteristics with horseshoeshaped or reniform nuclei.
  • the AITL arises from follicular helper T-cells (TFH) and is one of the most common mature T-cell malignancies (Vose J, Armitage J, Weisenburger D. International TCLP. International peripheral T-cell and natural killer/T-cell lymphoma study: pathology findings and clinical outcomes. J Clin Oncol. 2008;26(25):4124-4130). It is characterized by lymphadenopaty, systemic symptoms and often hypergammaglobulinaemia.
  • PTCL-NOS comprise a heterogeneous group of lymphomas originated by mature T-cells that do not fully meet the diagnostic criteria of the other classes. It is therefore a diagnosis by exclusion and it is at present the most common diagnosed subtype.
  • CTCL is a subtype of Extranodal NK/T-Cell Lymphoma and comprises a wide range of malignancies arising from mature T-lymphocytes in the skin. Their incidence is 7-10 cases per million (Olsen EA, Whittaker S, Kim YH, Duvic M, Prince HM, Lessin SR, et al. Clinical end points and response criteria in mycosis fungoides and Sezary syndrome: a consensus statement of the International Society for Cutaneous Lymphomas, the United States Cutaneous Lymphoma Consortium, and the Cutaneous Lymphoma Task Force of the European Organisation for Research and Treatment of Cancer. J. Clin. Oncol. 2011;29(18):2598-607).
  • CTCL mycosis fungoides
  • SS Sezary Syndrome
  • the ATL prognosis varies according to the type of variant: the "acute” and “lymphoma-type” variants have a poor prognosis with an average survival of less than one year, while in most of the "chronic” and “smoldering” forms the progression is slower (Bazarbachi A, Suarez F, Fields P, Hermine 0. How I treat adult T-cell leukemia/lymphoma. Blood. 2011 Aug 18;118(7):1736-45).
  • T-ALL acute lymphoblastic T-cell leukaemia
  • Radiotherapy may be helpful in early stage lymphomas, but chemotherapy is usually the first therapeutic approach to T-cell lymphomas and leukaemia.
  • Common regimens include the combination of cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) with or without etoposide, or similar regimens that also include L-asparaginase, cytarabine, and methotrexate.
  • the latter product is also administered intrathecally in order to reduce the risk of spreading of the disease to the central nervous system.
  • the failure rate of chemotherapy is high and relapses are frequent.
  • Allogenic hematopoietic stem cell transplantation can be decisive, but most patients are not suitable for this kind of intervention, mainly due to advanced age or failure to achieve remission after chemotherapy.
  • T-cell neoplasms are still burdened with a high mortality rate and represent an important unmet medical need. Therefore, the development of safer and more effective innovative treatments is urgent.
  • TCR T-cell receptor
  • TCR is a membrane protein that recognizes antigenic peptides expressed in association with molecules of the Major Histocompatibility Complex (MHC) (Davis MM, Bjorkman PJ. T-cell antigen receptor genes and T-cell recognition. Nature. 1988 Aug 4;334(6181):395-402). From a structural point of view, it consists of two different subunitsof the a and P or y and 6 types. Structural differences correspond to functional differences as op T lymphocytes have the classical helper and cytotoxic functions in the context of adaptive immunity, while 6y T lymphocytes are on the borderline between adaptive and innate immunity.
  • MHC Major Histocompatibility Complex
  • y6 T lymphocytes undergo V(D)J recombination.
  • the antigens recognized by y6 T lymphocytes do not require processing and presentation in association with MHC molecules.
  • the TCR. of y6 T lymphocytes behaves like a PRR (Pattern Recognition Receptor) which recognizes microbial antigens.
  • y6 T lymphocytes are particularly abundant in the intestinal mucosa, where they are referred to as intraepithelial lymphocytes (lELs).
  • the TCR is associated on cell membrane to a protein complex called CD3 which, on one side stabilizes the heterodimer (op or y6), and on the other side transduces the signal produced by the binding of the TCR with its antigen (Love PE, Hayes SM. ITAM-mediated signaling by the T-Cell Antigen Receptor. Cold Spring Harb Perspect Biol. 2010 Jun; 2(6): a002485).
  • CD3 complexes are formed by four types of different subunits called y, 6, E and Typically a CD3 complex contains a subunit y, a subunit 6, two subunits s and two subunits
  • the cytoplasmatic domains of these subunits contain characteristic sequences called ITAM (Immune-receptor-Tyrosine-based-Activation- Motif).
  • Subunits y, 6 and s contain one ITAM sequence each, while the subunit contains three ITAM sequences (overall, each CD3 complex contains ten ITAM sequences). Under inactive conditions, the tyrosine residues of the ITAM sequences localize inside the hydrophobic core of the lipid bilayer of the cell membrane.
  • the binding of the TCR with its antigen produces a deformation of the TCR/CD3 complex that exposes the tyrosine residues of the ITAM sequences in the cytoplasm, making them accessible to two tyrosine kinases named Lek and Fyn.
  • Phosphorylation of the tyrosine residues of the ITAM sequences allows the binding of an additional tyrosine kinase called ZAP-70, that starts a cascade of events leading to the activation of specific transcription factors and to a series of events responsible for the activation of T lymphocytes.
  • helper T lymphocytes takes place with the binding of TCR with a complex consisting of an antigenic peptide expressed in association to a class II MHC protein on the surface of Antigen Presenting Cells (APC). i.e. B lymphocytes, macrophages and dendritic cells.
  • APC Antigen Presenting Cells
  • cytotoxic T lymphocytes CD8+
  • CD4 and CD8 molecules expressed on the surface of helper and cytotoxic T-lymphocytes bind to MHC class I and class II molecules, respectively, thus stabilizing the entire complex.
  • T lymphocytes require the activation of a certain number of secondary signals.
  • the main signal is represented by the binding of CD28 on the surface of the lymphocyte to B7.1 (CD80) or B7.2 (CD86) on the APC surface.
  • This signal induces clonal expansion of activated helper T lymphocytes that would continue endlessly if the production of CTLA-4 (CD152), competing with CD28 in the binding with B7, did not start.
  • CTLA-4 CD152
  • cytotoxic T lymphocytes depends on CD28 to a minor extent, requiring instead the activation of other signals, in particular CD70 and 4-1BB (CD137).
  • T lymphocytes The survival of T lymphocytes depends on additional molecules on their surface, in particular ICOS, 4-1BB and 0X40, which are stimulated by the corresponding ligands on the APCs' surface. Such molecules are expressed following the recognition of pathogens by innate immunity. This is important in order to prevent inappropriate activations of T lymphocyte clones.
  • cytokines play an important role in directing the type of response of activated T lymphocytes.
  • IL-12 directs T lymphocytes to a Thl type response (cytotoxicity against cells infected by intracellular pathogens like viruses and Mycobacterium tuberculosis mediated by the production of IFN-y);
  • IL-4 directs T lymphocytes to a Th2 type response (allergic reactions and antibody responses against parasites);
  • IL-17 directs T lymphocytes to a Thl7 type response against extracellular pathogens. recombination of TCR.
  • CDR1 Complementarity-Determining Regions
  • CDR2 CDR3 located in the variable region of TCR.
  • the first two are encoded by germinal sequences, while CDR3 (which represents the main recognition site of the pMHC complex) is produced by a V(D)J recombination process similar to the one observed for immunoglobulin production.
  • V(D)J V(D)J recombination process
  • the variable region of the a and y chains is produced by the recombination of the sole fragments V and J
  • the variable region of the 0 and 6 chains is produced by the recombination of fragments V, D and J.
  • junctional diversity In the junctions between segment V and D and between segment D and J occurs the deletion of bases codified in the germinal line and their replacement with different bases. This process is called “junctional diversity" and produces the expansion of the TCR repertoire, allowing the recognition of a greater number ofpMHC complexes.
  • V, D and J The expression of single fragments V, D and J is not random and some combinations occur more frequently than others (Freeman JD, Warren RL, Webb JR, Nelson BH, Holt RA. Profiling the T-cell receptor beta-chain repertoire by massively parallel sequencing. Genome Res. 2009 Oct;19(10): 1817-24). Specifically, the variable region of the TCR 0 chain is generated through the rearrangement of 68 V genes, 2 D genes, and 14 J genes. Each individual can express different alleles of the same gene. So far, 152 V gene alleles, 3 D gene alleles, and 16 J gene alleles have been described in the IMGT database.
  • each of the 68 fragments V of the TCR 0 chain should be expressed with the same frequency, corresponding to 1,5% of cases (1/68). Instead, fragment TRBV20-1 is expressed in about 25% of normal T-lymphocytes; fragment TRBV5-1 is expressed in about 12% of normal T-lymphocytes; fragment TRBV29-1 is expressed in about 10% of normal T-lymphocytes; and finally, fragment TR.BV28 is expressed in about 5% of normal T lymphocytes. The remaining fragments are all expressed with considerably lower frequencies. This depends of many factors, in particular on the presence of transcription enhancers near the exons of the constant regions and on many promoters associated to the segments of the V, D and J genes (Krangel MS.
  • TCR as a diagnostic marker of neoplastic T lymphocyte clones
  • T-cell malignancies result from the neoplastic transformation of one single T lymphocyte.
  • a clone of neoplastic T lymphocytes is therefore characterized by the presence on the cell membrane of one single TCR that uniquely identifies cancer cells from healthy ones.
  • lOTest Beta Mark produced by Beckman Coulter is a diagnostic test to identify neoplastic T lymphocytes clones through flow cytometry. It consists of 24 different monoclonal antibodies (of which 22 are murine and 2 from rats) which recognise specific fragments V of the TCR P chain. These 24 antibodies are distributed in eight vials, each containing three antibodies marked with different fluorophores. In absence of neoplastic clones, the antibodies will bind uniformly to T lymphocytes.
  • neoplastic clones In presence of neoplastic clones, if the corresponding TCR expresses one of the 24 V fragments recognized by the kit, most of the T lymphocytes will be marked by the same fluorophore. If the neoplastic TCR. does not express one of these 24 fragments, most of the T lymphocytes will result in not being marked (so-called "null").
  • the inventors of the present invention have identified six scFvs binding an innovative TAA for the treatment of T-cell malignancies, i.e. fragment TRBV5-1 (formerly known as Vb5.1) of the 0 chain of human TCR.
  • these scFvs bind an epitope contained in the amino acid sequence TQTPRYLIKTRGQQ.
  • these scFvs bind 100% of tumor cells, preserving about 90% of the healthy T lymphocytes.
  • scFvs were obtained by means of the "phage display” technique from a human library called SFMAXTM' This library contains 3.OxlO 10 variants of naive human scFvs obtained by B lymphocytes of 368 donors of five different ethnic groups. Therefore, these scFvs are completely human and perfectly suitable for therapeutic use in humans.
  • the above mentioned scFvs are used for the production of monoclonal antibodies of IgG class (of any of the subclasses IgGl, IgG2, IgG3 or IgG4).
  • the above mentioned scFvs are used for the production of monoclonal antibodies of IgM class (monomeric, pentameric and hexameric).
  • the above described scFvs are used for the production of monoclonal antibodies of IgA class (monomeric or dimeric).
  • the above described scFvs are used for the production of other kinds of monoclonal antibodies not specifically mentioned in this list (for exemplifying and not limiting purposes: monoclonal antibodies conjugated with drugs; monoclonal antibodies conjugated with radioactive isotopes, etc.).
  • scFvs are used for the production of Fab (fragment antigenbinding).
  • scFvs are used for the production of Fab2 (obtained by joining two identical Fab fragments).
  • the above described scFvs are used for the production of bispecific Fab2 (wherein one of the two Fab fragments corresponds to one of the six scFvs according to the present invention).
  • the above described scFvs are used for the production of bispecific monoclonal antibodies (wherein one of the two Fab fragments corresponds to one of the six scFvs according to the present invention).
  • the above described scFvs are used for the production of minibodies (formed by one of the six scFvs according to the present invention joined to the constant CH3 domain of immunoglobulins).
  • the above described scFvs are used as antigen-binding domain for the production of a CAR.
  • the above described scFvs are used as antigen-binding domain for the production of a CAR. to be expressed on autologous CD8+ T lymphocytes (i.e. taken from the same patient).
  • the above described scFvs are used as antigen-binding domain for producing a CAR expressed on allogenic or haploidentical (i.e. taken from a family member sharing 50% of genetic heritage with the patient) CD8+ T lymphocytes.
  • the above described scFvs are used as antigen-binding domain for the production of a CAR expressed on autologous CD4+ T lymphocytes (i.e. taken from the same patient).
  • the above described scFvs are used as antigen-binding domain for the production of a CAR to be expressed on allogenic or haploidentical (i.e. taken from a family member sharing 50% of the genetic heritage with the patient) CD4+ T lymphocytes.
  • the above described scFvs are used as antigen-binding domain for the production of a CAR to be expressed on other kinds of autologous lymphocytes (i.e. taken from the same patient) like - for exemplifying but not limiting purpose - lymphocytes NK, NKT, Ty/6, etc).
  • the above described scFvs are used as antigen-binding domain for the production of a CAR to be expressed on other kinds of allogenic or haploidentical (i.e. taken from a family member that shares 50% of the genetic heritage with the patient) lymphocytes, like - for exemplifying and not limiting purpose - lymphocytes NK, NKT, Ty/6, etc.).
  • An eighteenth preferred embodiment of the present invention concerns a pharmaceutical formulation including any of the molecules described in preferred embodiments one to seventeen.
  • a nineteenth preferred embodiment of the present invention concerns a pharmaceutical formulation for therapeutic use in humans comprising any of the molecules described in the preferred embodiments one to seventeen.
  • a twentieth preferred embodiment of the present invention concerns a pharmaceutical formulation for diagnostic use, comprising any of the molecules described in the preferred embodiments of the invention from one to ten, to be used in patients with T-cell neoplasms whose clones express fragment TR.BV5-1 in the 0 chain of the TCR.
  • a twenty-first preferred embodiment of the present invention concerns a pharmaceutical formulation for therapeutic use comprising any of the molecules described in the preferred embodiments from one to seventeen, to be used in patients with T-cell neoplasms whose clones express fragment TR.BV5-1 in the 0 chain of the TCR.
  • V fragments of the TCR. 0 chain in normal and neoplastic T lymphocytes opens a new way in the immunotherapy of the T-cell neoplasms.
  • Said TAAs may represent the target of specific immunotherapies, as in the case of the epidermal growth factor receptor (EGFR), HER/2 neu protein and CD20, respectively targets of monoclonal antibodies (Antitumor Antibodies cetuximab, trastuzumab and rituximab - Wittrup KD Can Drive Therapeutic T Cell Responses. Trends Cancer. 2017 Sep;3(9):615-
  • EGFR epidermal growth factor receptor
  • HER/2 neu protein and CD20 respectively targets of monoclonal antibodies
  • Antitumor Antibodies cetuximab, trastuzumab and rituximab - Wittrup KD Can Drive Therapeutic T Cell Responses. Trends Cancer. 2017 Sep;3(9):615-
  • CAR-T therapies like tisagenlecleucel and axicabtagene ciloleucel (Brudno JN, Kochenderfer JN. Recent advances in CAR T-cell toxicity: Mechanisms, manifestations and management. Blood Rev. 2019 Mar; 34: 45-55).
  • the fragment In order for these new TAAs to represent innovative targets in immunotherapy of human T-cell malignancies, two main requirements must be met: a) the fragment must be expressed in the neoplastic clones of a significant percentage of patients with T-cell malignancies; b) the fragment must be expressed only in a limited number of healthy T lymphocytes.
  • any specific immunotherapies administered to patients would produce severe immunodepression due to impairment of the T-cell compartment of the immune system.
  • Each antigenic peptide binds preferably specific MHC alleles, thus restricting the repertoire of TCRs capable of recognizing the complexes formed by viral antigenic peptides and the correspondent MHC molecules. Therefore, it is possible that the binding of specific antigenic peptides derived from the processing of viral proteins to specific MHC molecules alleles restricts the TCR repertoire of neoplastic T-lymphocytes. In this sense it should be remembered that V fragments plays a central role in the recognition of antigenic peptides, as they express both CDR1 and CDR2, as well as participating to the expression of CDR3 through V(D)J recombination.
  • Tables 1 and 2 respectively show the nucleotide and amino acid sequences of TRBV5-1 fragment, as described in the prior art (the "International ImMunoGeneTics Information System® http://www.imgt.org; Wei S, Charmley P, Robinson MA, Concannon P. The extent of the human germline T-cell receptor V beta gene segment repertoire. Immunogenetics. 1994;40(l):27-36. doi: 10.1007/BF00163961). The nucleotide and amino acid sequence can be found also on the "Protein Data Bank” (https://www.rcsb.org/ - pdbcodes: 5BRZ[1] and 5BSO[1]).
  • a first candidate peptide whose sequence is shown in table 3, is localized near the N-terminal end. This peptide is easily accessible to antibodies but not completely linear and difficult to synthetize. On the other hand, a shorter segment (shown in red in table 3 and in figure 1) represents a good candidate.
  • a second candidate peptide is localized in the intermediate part of the fragment and its sequence is shown in table 4.
  • This peptide as a whole, is highly structured but two segments can be identified within it having characteristics suitable for the development of antibodies (respectively shown in black and green in table 4).
  • a third candidate peptide follows the second in direction C-terminal and is characterized by the amino acid sequence shown in table 5.
  • Figure 1 shows the 3D structure of a human TCR expressing the TRBV5-1 fragment in the 0 chain (in particular, the segment of the first antigenic peptide is shown in red).
  • thermofisher.com https: //www. thermofisher.com/it/en/home/lifescience/antibodies/cu stom-antibodies/custom-anti
  • Table 7 summarizes the score obtained by each candidate peptide. All peptides obtained good scores, although there were some differences in the scores obtained with the two instruments. However, the lack of problematic amino acids, the optimal length and the excellent score obtained in the Ab Designer test led two independent groups to identify in peptide no. 1 (TQTPRYLIKTR.GQQ sequence) the ideal candidate for the following developing stages of scFvs directed against TR.BV5-1 fragment of the TCR. P chain.
  • the lOTest Beta Mark antibodies are not commercially available individually, but only as mixtures of three antibodies inside the same vial and therefore they cannot be used as a positive control in characterization tests. Furthermore, the lOTest Beta Mark antibodies were produced in mice and rats with the hybridoma technique, described for the first time in 1975 by Kohler e Milstein (G Kohler, C Milstein. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7;256(5517):495-7), and therefore they may be used in human beings only for diagnostic purposes.
  • HAMAs anti-mouse antibodies
  • Their administration to human beings for therapeutic purposes would induce the development of anti-mouse antibodies (HAMAs) that would neutralize the activity thereof (Greiner JW, Guadagni F, Hand PH, Pestka S, Noguchi P, Fisher PB, Schlom J. Augmentation of tumor antigen expression by recombinant human interferons: enhanced targeting of monoclonal antibodies to carcinomas. Cancer Treat Res. 1990;51 :413-32).
  • phage display The most common techniques for the production of fully human monoclonal antibodies are the phage display (Frenzel A, Schirrmann T, Hust M. Phage display-derived human antibodies in clinical development and therapy. MAbs. 2016 Oct;8(7): 1177-1194) and the immunization of transgenic mice that produce human immunoglobulins (Green LL. Transgenic mouse strains as platforms for the successful discovery and development of human therapeutic monoclonal antibodies. Curr Drug DiscovTechnol. 2014 Mar;ll(l):74- 84). Of the two, the phage display technique is faster and ethically more acceptable, since it does not require animal sacrifice.
  • the phage display technique consists in the expression of human antibodies fragments called scFvs on the surface of bacteriophages. These fragments consist in the variable regions of the heavy (VH) and light (VL) chains of human antibodies, kept together by a small flexible peptide called "linker” (Bird RE, Hardman KD, Jacobson JW, Johnson S, Kaufman BM, Lee SM, Pope SH, Riordan GS, Whitlow M. Single chain antigen-binding proteins. Science 1988; 242:423-6).
  • the relative genes are extracted from B lymphocytes obtained by spleen, peripheral blood, tonsils and, in some cases, also from fetal liver (Nagano K, Tsutsumi Y. Phage Display Technology as a Powerful Platform for Antibody Drug Discovery. Viruses. 2021 Jan 25;13(2): 178).
  • the portions of the antibody genes encoding the VH and VL regions are fused with genes encoding bacteriophage coat proteins.
  • the final result is the exposition (display) of scFvs on the surface of the bacteriophages, which are first replicated in a bacterium (usually Escherichia coli) and then transferred on plates in which the antigen of interest has been previously fixed.
  • the bacteriophages that express a scFv binding the antigen of interest remain adherent to the bottom of the plate, while the others are eliminated by washing.
  • the adherent bacteriophages are eluted, producing a mixture of bacteriophages enriched by the sole elements that express scFvs which bind the antigen of interest ("panning").
  • panning cycles select bacteriophages that express progressively more specific scFvs.
  • the induction of mutations is also foreseen with the aim of mimicking the so-called "affinity maturation" of B lymphocytes during the course of immune responses.
  • Human LiAb SFMAXTM a library of human scFvs called Human LiAb SFMAXTM has been successfully used This library contains 3.OxlO 10 variants of human naive scFvs obtained from B lymphocytes of 368 donors from five different ethnic groups, with the aim of amplifying the scFvs repertoire as much as possible.
  • the production process of the six scFvs according to the present invention began with the conjugation of the TQTPRYLIKTRGQQ peptide with three different carrier proteins, i.e. bovine serum albumin (BSA), ovalbumin (OVA) and megathura crenulata hemocyanin (KLH).
  • BSA bovine serum albumin
  • OVA ovalbumin
  • KLH megathura crenulata hemocyanin
  • the concentration of eluted phages was determined adding the eluate to a culture of Escherichia coli TGI and measuring the number of plaques (corresponding to the lysed bacterial cells).
  • the eluted phages were amplified adding the eluate to an Escherichia coli TGI culture on plate in presence of a helper phage.
  • the amplified phages were precipitated with PEG/NaCI, resuspended and used for the subsequent panning cycle.
  • a polyclonal ELISA test was then performed by coating the wells of multiwell plates with the peptide TQTPRYLIKTR.GQQ conjugated to the three carriers BSA, OVA and KLH at a concentration of 10 pg/mL. After washing and fixing, the phages amplified after each panning cycle were incubated as shown in table 8.
  • An anti-phage antibody conjugated to horseradish peroxidase (Horseradish Peroxidase - HR.P) was added together with a specific substrate for the peroxidase called tetramethylbenzidine (TMB). After an appropriate incubation period, the reaction catalysed by peroxidase was blocked and the plates were read spectrophotometrically by selecting the 450nm wavelength.
  • the data obtained confirmed the progressive enrichment, from the first to the fourth round of panning, in phages that specifically recognize the antigenic peptide.
  • the fourth round showed high enrichment and its eluate was used for the subsequent tests.
  • the binding affinity to the unconjugated peptide is lower compared to the conjugated peptide and progressively decreases with the dilution of the phages. It probably depends on the suboptimal coating of the plate with the unconjugated antigenic peptide.
  • BLAST can be found on a dedicated website (http: //blast. ncbi.nlm.nih.gov/Blast.cqi).
  • This algorithm allows the realization of "w-mers", i.e. amino acid (or nucleotide) sequences with a length W that give, on the basis of a replacement matrix, a score >T (threshold) if aligned on the query.
  • T the number of w-mers in the list. This means that a low T-value increases both the run time and the sensitivity of the analysis.
  • the so-called “best alignment” is obtained, i.e. the alignment of two polypeptide or nucleotide sequences, that produces the highest identity percentage, meaning by this term the same amino acid (or the same nucleotide) in the same position after correction for any deletions or insertions.
  • identity percentage is determined, i.e. the number of identical amino acids (or nucleotides) out of the total.
  • the present invention does not concern only the sequences of the six identified clones, but also possible variants that may be easily realized by anyone skilled in the art.
  • the term "variant" means a polypeptide with an amino acid sequence similar to one of the six identified clones and/or that performs the same biologic function (i.e. binding of the TR.BV5-1 fragment of the human TCR. 0 chain).
  • the one skilled in the art knows very well the techniques allowing to replace one or more amino acids of the polypeptide sequence of the six identified clones with similar amino acids (for exemplifying and not limiting purposes: an aliphatic amino acid with another aliphatic amino acid; an aromatic amino acid with another aromatic amino acid; an amino acid containing sulfur with another amino acid containing sulfur, and so on) without substantially modifying their function.
  • the present invention concerns not only the above described six scFvs, but also the molecules that may derive therefrom.
  • a person skilled in the art knows very well the techniques for producing said molecules that include - without being limited to: fragment Fab (consisting of the domains VL, CL, VH and CHI); fragment Fd (consisting of domains VH and CHI); fragment Fv (consisting of domains VH and CHI); fragment dAb (consisting of the sole domain VH); single CDR.
  • CAR may have a variable structure but they always share three characteristics: one or more domains in the extracellular side that recognize the antigen (usually obtained by scFvs); a cytoplasmatic domain responsible for the transduction of the activation signal (usually obtained from CD3 ⁇ ; a co-stimulatory domain responsible for maintaining CAR-T activation (usually obtained from the co-stimulatory molecule 4-1BB).
  • the present invention also concerns any type of CAR exposing on its extracellular side an antigen-binding domain containing any of the six variable regions recognizing the TRBV5-1 fragment of human TCR 0 chain described in table 12, and/or any sequence sharing _>70% identity with them.
  • variable p region of the TCR which is the target of this computational study, is crystallized with the variable and constant regions of the a chain and with the constant region of the p chain, which allows for more precise evaluation of the complexes with antibody variable regions.
  • Both the X-ray structures show an amino acid mutation in the variable p region of the TCR. compared with the primary sequence reported on Uniprot (Table 13). From the structure with PDB ID 5BS0, all components except the constant and variable regions of TCR a and p chains were removed. Next, the structure was subjected to a protein preparation protocol through the application of Modeller
  • the resulting molecular dynamics trajectory was clustered by using the RMSD of the atoms of the amino acid residues of the area of the p chain variable region for which the six scFvs were developed, and the RMSD of the atoms of the loop of the p chain constant region. From this clustering process, the representative structures of the three most populated clusters (78 % of the total population) were extracted to be used in the following ensemble docking step.
  • the scFv identified with the code CIO binds a different region from the one for which it was designed, while in the worst interface energy score complex it binds the aforementioned region. Therefore, it can be concluded that CIO should have as its preferred epitope a region different from the one for which it was designed, specifically the CDRs region of the TCR and, for this reason, it has been excluded from the analysis.
  • Table 14 sorts in descending order of predicted affinity the 5 remaining scFvs that should have as their preferred antigenic epitope the peptide region for which they were designed and several other amino acids of the TCR 0 chain variable and constant regions. Moreover, the number of residues of the TCR.
  • an outsourcing company has produced human monoclonal antibodies of IgGl class starting from the sequences of the variable regions of a and 0 chains of the six scFvs according to the present invention, shown in detail in table 12.
  • Table 15 shows the complete sequences of the heavy and of the light chains of monoclonal antibody obtained from scFV A12 (is was indicated as "lead compound" following the results of the bioinformatics study and the SPR. test).
  • the other monoclonal antibodies consist of the same constant domains joined to the variable regions of the respective scFvs as shown in table 12.
  • CHO cells were cultured in Erlenmeyer flasks at 37° with 5% CO2 in an orbital shaker using a serum-free expression medium. The day before the transfection, CHO cells were seeded in Erlenmeyer flasks with an appropriate density. The day of the transfection, recombinant plasmids encoding the antibodies of interest and the transfection reagent were mixed in an optimal ratio and added to cultured CHO cells.
  • Cell culture supernatants were collected when the cell viability was less than 50%. After centrifugation and filtration, recombinant antibodies were purified by means of affinity chromatography. After washing and elution with appropriate buffers, monoclonal antibodies were concentrated and analyzed with SDS-PAGE (to verify the molecular weight of the protein) and SEC-HPLC (to evaluate the degree of purity).
  • the composition of the reducing buffer for SDS- PAGE was the following: 250 mM Tris-HCI, 10% SDS, 30% glycerol, 0.5% bromophenol blue, 250 mM DTT, pH 6.8.
  • composition of the non-reducing buffer was the following: 250 mM Tris-HCI, 5% SDS, 30% glycerol, 0.5% bromophenol blue, 250 mM IAM, pH 8.2. Finally, endotoxin level was measured and the concentration of monoclonal antibodies was determined by the A280 method.
  • Variable amounts of the six monoclonal antibodies ranging from 8.4 mg to 11.2 mg were obtained.
  • Half antibody was left native, while the other half was conjugated with a fluorophore for flow cytometry (fluorescein-5-isothiocyanate - FITC).
  • a fluorophore for flow cytometry fluorescein-5-isothiocyanate - FITC
  • the antibodies were dialyzed with a sodium bicarbonate buffer.
  • the concentration of the antibodies was determined by micro-ultraviolet- visible spectrophotometer (MUVVS) spectrophotometry.
  • FITC was dissolved in DMSO and added to the buffer containing the antibodies.
  • glycine Upon addition of glycine to the buffer, the antibodies conjugated to FITC were dialyzed and their presence was highlighted by MUVVS. Finally, BSA and ProVlin300 were added.
  • Monoclonal antibodies produced in this way were divided into 1 mg aliquots and stocked in a freezer at -20°.
  • the aliquots of native antibody were used for the Surface Plasmon Resonance (SPR) test, while the aliquots of antibody conjugated with fluorophore were used for flow cytometry analysis.
  • SPR Surface Plasmon Resonance
  • SPR Surface Plasmon Resonance
  • the analysis starts with the detection of the baseline signal, which corresponds to the mass of the ligand fixed on the sensor chip. Subsequently, the analyte in liquid phase flows on the surface of the sensor chip and interacts with the ligand, producing the association phase (corresponding to the formation of analyte-ligand complexes). A plateau phase follows, in which a balance is obtained between the analyte bound to the ligand and the analyte dissociated from it. Finally, a running buffer dissociates the analyte from the ligand (dissociation phase).
  • affinity constant which corresponds to the relationship between the dissociation constant (Kd) and the binding constant (Ka).
  • a sensor chip containing streptavidin has been used.
  • the ligand fixed on the sensor chip was the construct produced by the TQTPRYLIKTRGQQ peptide conjugated with BSA and biotin previously used in the first and in the fourth round of bio panning of the phage display.
  • TQTPRYLIKTRGQQ peptide Due to the binding between streptavidin and biotin, antigenic epitopes on TQTPRYLIKTRGQQ peptide were correctly placed on the chip outward turned. Monoclonal antibodies (analytes) were added to the liquid phase in order to interact with the ligand fixed on the sensor chip.
  • Table 16 summarizes the KDs of the six monoclonal antibodies for the antigenic construct.
  • the results obtained with SPR are substantially in line with the study conducted in silico.
  • Slightly lower values were obtained with antibody D5 (which moreover showed a lower specificity in the in- silico study). Lower, although significant affinities were recorded with the other four antibodies.
  • the CIO antibody has shown a significant affinity for the antigenic construct. Therefore, combining the results of the SPR study with those of the in silico study, monoclonal antibody A12 was identified as the lead compound.
  • ScFvs and all the above described molecules deriving therefrom may be used both for diagnostic and therapeutic purposes.
  • scFVs and all the above described molecules deriving therefrom may be conjugated to any kind of fluorophore to verify in flow cytometry their affinity with clones of neoplastic cells. They may be conjugated to radioisotopes to evaluate, with imaging techniques (scintigraphy, PET, etc.) the spread of the tumor and to exclude a possible non-specific binding to tissues different from the tumor cells. Finally, they may be employed in the evaluation of the minimal residual disease at the end of the therapy.
  • the scFvs and all the above described molecules deriving therefrom may be used as such or conjugated to chemotherapeutics, toxins or radioactive isotopes. They may be also loaded on liposomes or other kinds of nanoparticles to deliver pharmacologically active molecules to cancer cells.
  • scFvs may be used as a domain for the recognition of the antigen in the realization of any kind of chimeric receptor (CAR.), namely: a) typical CAR-Ts on the market or still in development; b) atypical CAR-Ts expressing on the surface of T lymphocytes (autologous, haploidentical, allogenic or stable cell lines) any of the different Fc receptors for immunoglobulins (CD16, CD32, CD64).
  • CAR chimeric receptor
  • monoclonal antibodies for therapeutic purposes bind TAA on the cell membrane of different types of malignancies, producing opsonization of the tumor cell and activating Antidoby-Dependent Cell Citotoxicity (ADCC).
  • ADCC Antidoby-Dependent Cell Citotoxicity
  • monoclonal antibodies bind membrane receptors and interfere with said activation by physiologic ligands.
  • rituximab a chimeric murine/human monoclonal antibody of IgGl class, which recognizes the CD20 molecule expressed on the surface of B lymphocytes, currently used for the treatment of B-cell nonHodgkin lymphomas.
  • rituximab binds both healthy T- lymphocytes and cancer cells. Therefore, the treatment with this antibody produces a complete depletion of the B-cell compartment of the adaptive immunity.
  • trastuzumab Herceptin
  • HER.2 an ErbB family tyrosine kinase receptor
  • trastuzumab Herceptin
  • cetuximab a chimeric monoclonal antibody of IgGl class that binds Epidermal Growth Factor (EGF) receptor, preventing the activation thereof.
  • Flow cytometry is a widespread and consolidated technique in laboratory practice that allows to characterize quality and quantity of a cell suspension. Physical and immunological parameters can be examined simultaneously, with particular reference to the expression of specific antigens on cell membrane that may be detected by preincubating the cell suspension with specific monoclonal antibodies conjugated to fluorophores.
  • a laser source intercepts the single cells flowing into a microfluidic system.
  • the rays scattered by the cell (“scatter") provide two types of information: the "forward scatter” (FSC) provides indications on the cell size; the “side scatter” (SSC) provides information on cytoplasmatic granularity (allowing the distinction between granulocytes and other types of leukocytes).
  • FSC forward scatter
  • SSC side scatter
  • emission fluorescence allows the identification of the antigens expressed on the surface of single cells.
  • PBMC peripheral blood mononuclear cells
  • the combination was enriched with a further monoclonal antibody (CD14) which binds monocytes, allowing their exclusion from the population under study. This was necessary to exclude possible aspecific bindings, as the lead compound is a monoclonal class IgGl antibody which binds the Fc receptor for immunoglobulins on the membrane of the monocytes.
  • CD14 monoclonal antibody
  • the first test was conducted on the PBMC obtained from the patient with Sezary Syndrome who expresses the TRBV5-1 fragment in the variable region of the TCR 0 chain.
  • the results of the test show that the A12 lead compound binds with 21% of the cell population under study.
  • the second test was conducted on the PBMC suspension obtained from the patient with Sezary Syndrome who expresses fragments TRBV6-5, TRBV6-6 or TRBV6-9 recognized by the anti-Vbl3.1 antibody of the lOTest® Beta Mark (Beckman Coulter). The results of the test show that the A12 lead compound does not bind any cell of the population under study.
  • the results of the flow citometry test confirm the results of the SPR test, namely that A12 monoclonal antibody binds selectively and with high affinity the fragment TRBV5-1 expressed on the variable region of the TCR. 0 chain.
  • CARs are membrane receptors that do not exist in nature and are created in laboratory combining segments of one gene with segments of other genes. Once expressed on T lymphocytes (CAR-T) or any other cells of the immune system such as natural killer cells (NK), they redirect their specificity and function. CAR-Ts are considered "living drugs" realized with a particularly complex procedure.
  • normal T lymphocytes are isolated from the patient or from a compatible donor through apheresis. T lymphocytes are then transfected, i.e. the construct (plasmid) which contains the information for the production of the CAR. is transferred inside T lymphocytes by a viral vector or by alternative techniques (like electroporation). Transfected T lymphocytes are then expanded in vitro and infused again to the patient.
  • any molecule on the surface of a tumor cell may become a target of CAR-Ts.
  • CARs recognize the antigen without the restrictions related to interindividual differences of MHC alleles and without the target proteins having to be previously processed into antigenic peptides inside the target cell. Thanks to these characteristics, CAR-Ts may also kill cancer cells with low expression of MHC molecules or low proteasome activity (two mechanisms that may contribute to the lack of recognition of cancer cells by the immune system).
  • CARs recognize only membrane antigens, while TCRs may recognize also antigenic peptides obtained by the processing of intracellular proteins.
  • the CARs antigen recognizing domain may be of three different types, namely scFv, Fab or ligands that recognize their natural receptor (Sadelain M, Brentjens R, Riviere I. The basic principles of chimeric antigen receptor design. Cancer Discov. 2013 Apr;3(4):388-98).
  • scFv single-chain Fv
  • Fab single-chain Fv
  • ligands that recognize their natural receptor
  • This patent concerns a construct (whose nucleotide sequence is identified as SEQ ID NO:8) for the expression of a CAR consisting preferably, but not exclusively, of an antigen-binding domain that recognizes the CD19 on the surface of B lymphocytes (an scFV obtained from an anti-CD19 murine monoclonal antibody called FMC63 and identified in the patent as SEQ ID NO: 14); a hinge domain and a transmembrane domain (both obtained from human CD8 and identified in the patent as SEQ ID NO: 15 and SEQ ID NO.16 respectively); a co-stimulatory region, preferably but not exclusively represented by protein 4-1BB (identified in the patent as SEQ ID NO: 17) that plays a fundamental role in maintaining the CAR-T antitumor response; and finally, a cytoplasmic domain responsible for the transduction of the activation signal in the cytoplasm obtained from CD3 ⁇ (identified in the patent as SEQ ID NO: 18), to which the cytoplasmic domains of the above mentioned co-sti
  • the antigen-recognizing domain is preferably a scFv or a Fab, specific for a certain number of antigens present on lymphocytes membrane.
  • Said scFvs preferably recognizes the CD19 molecule on B lymphocytes membrane, but the inventors claim also other CARs that recognize potential antigens on the membrane of neoplastic cells, such as CD27, CD28, 4-1BB, 0X40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C and B7H3, and many others.
  • said patent does not mention the variable regions of the TCRs and, in particular, the variable regions of the P chain of said TCR.
  • nucleotide/amino acid sequence of the scFv obtained from the anti-CD19 murine monoclonal antibody called FMC63 object of patent no. WO 2012/079000 Al
  • FMC63 object of patent no. WO 2012/079000 Al
  • the nucleotide/amino acid sequences of the six scFVs according to the present patent application can be easily replaced with any of the nucleotide/amino acid sequences of the six scFVs according to the present patent application, or by the nucleotide/amino acid sequences of any of the variants with a homology degree .>70% with any of the six scFVs subject of the present patent application. So well is the technique known, that many companies offer "in outsourcing" the drawing and the synthesis of the constructs starting from the three CDRs and the four FRs of scFvs.
  • the main B cell neoplasms indicated as a potential target of CAR-Ts described in patent no. WO 2012/079000 Al are chronic lymphocytic leukaemia and lymphoma.
  • the construct for the expression of the CAR may be transferred to the cells of the immune system (preferably T lymphocytes) with different techniques, but preferably by means of a lentiviral vector (pELPs 19BBz).
  • the treatment was tested in three patients with chronic lymphocytic leukaemia aged between 50 and 68 years, leading to the complete remission of the disease.
  • a second patent filed subsequentely describes an anti-CD19 humanized scFV, less immunogenic than the previous anti-CD19 murine scFv.
  • These two patents (WO 2012/079000 Al and US 2014/0271635 Al) allowed the development of the drug Kymriah (Tisagenlecleucel), authorized by the main global regulatory bodies for the treatment of B cell acute lymphoblastic leukaemia in paediatric age, and large B cell lymphoma in adults.
  • a third patent that started the industrial development of CARs was filed on 16 th November 2017 by Stuart A. Sievers e Jed J. W. Wiltzius ("Modified chimeric antigen receptors and methods of use" - US2019/0144515 Al).
  • the authors discovered that introducing specific modifications in the amino acid sequences of the hinge, transmembrane and intracellular domains, substantial and unexpected modifications were induced in the activity of the corresponding CAR-Ts.
  • a first modification consists in the removal of the N-linked glycosylation site in the hinge domain that considerably increases CAR-Ts proliferation.
  • a second modification consists in mutations of the transmembrane domain that favour its tendency to dimerization on cell membrane.
  • variable TCR regions and particularly the variable regions of the 0 chain of said TCR, as a target of the CAR.
  • any of the six scFvs according to the present invention would confer to the CARs identified in US patent no. US2019/0144515 Al the property of recognizing and destroying tumor cells in patients with T cell neoplasms that express fragment TRBV5-1 in the TCR 0 chain.
  • variable regions of TCRs as targets of CARs, particularly the variable regions of the 0 chain of said TCR.
  • the addition of any of the six scFvs which are the subject of the present invention, as well as of the nucleotide/amino acid sequences of any of the variants with a homology degree of .>70%, to any of the six scFvs according to the present invention, would confer to the CARs identified in patent no. W02019/090004 Al the property of recognizing and destroying tumor cells in patients suffering from T cell neoplasms expressing fragment TRBV5-1 in the TCR 0 chain.
  • the first patent was filed on 12 th May 2014 by Roman Galletto ed al. ("CD19 specific chimeric antigen receptor and uses thereof" - WO 2014/184143 Al).
  • the authors demonstrated that a CAR produced with a domain that binds the antigen obtained from an anti-CD19 monoclonal antibody, called 4G7, increases the activation state of CAR-Ts with respect to the CAR whose antigen-binding domain was obtained from the scFv called FMC63 described in patent no. WO 2012/079000 Al.
  • the antigen-binding domain obtained from monoclonal antibody 4G7 maintains the activation of CAR-Ts independently from the recognition of the antigen. Therefore, this patent is relevant only to CARs recognizing CD19 and does not consider other targets on the membrane of neoplastic cells.
  • Single CARs have one single antigen-binding domain, connected with one single hinge/transmembrane domain, in turn connected with one single intracytoplasmic domain that contains CD3 ⁇ , as well as the coreceptor (4-1BB or CD28).
  • Split CARs have two different antigen-binding domains n. The first one is connected through a hinge/transmembrane domain to an intracytoplasmic domain containing CD3 ⁇ , while the second is connected (again through a hinge/transmembrane domain) to an intracytoplasmic domain obtained from a co-receptor (4-1BB or CD28).
  • Tandem CARs have two domains binding two different antigens connected to one single hinge/transmembrane domain, in turn connected to one single intracytoplasmic domain containing CD3 ⁇ as well as the co-receptor (4-1BB or CD28).
  • CARs with one single vector consist of two antigen-binding domains connected to two different hinge/transmembrane domains, in turn connected to two different intracytoplasmic domains containing CD3 ⁇ , as well as the co-receptor (4-1BB or CD28).
  • each CAR can be single (i.e. it can have only one antigen-binding domain) or multiple (i.e. it can have two or more antigen-binding domains)
  • the DUOCARs can simultaneously recognize three or more antigens on the surface of tumor cells.
  • DUOCARs could recognize simultaneously CD19, CD20 and CD22, reducing the risk of development of clones which do not express one antigen targeted by immunotherapy, thus allowing cancer to escape from CAR-Ts.
  • variable regions of TCRs as CARs targets and, particularly, the variable regions of the 0 chain of said TCR.
  • the addition of any of the six scFvs subject of the present invention, as well as of the nucleotide/amino acid sequences of any of the variants with a homology degree of >70%, to any of the six scFvs previously described, would confer to the CARs identified in patent no. W02019/090004 Al the property to recognize and kill cancer cells in T-cell neoplasms expressing fragment TRBV5-1 in the TCR 0 chain.
  • CAR.-T failure is the occasional transfection of single neoplastic lymphocytes, erroneously isolated during the apheresis procedure.
  • autologous CD8+ T-cells i.e. obtained from the patient
  • cancer cells are CD4+
  • autologous CD4+ T-cells i.e.
  • hESCs embryonic stem cells
  • iPSCs induced pluripotent stem cells

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Abstract

La présente invention concerne un fragment variable à chaîne unique (scFv) et des molécules dérivant de celui-ci qui se lient au fragment TRBV5-1 de la chaîne β du récepteur des lymphocytes T humain (TCR) et l'utilisation desdites molécules pour un diagnostic ainsi qu'à des fins thérapeutiques, dans le traitement de pathologies provenant de l'expansion clonale de lymphocytes T exprimant un antigène associé à une tumeur innovant (TAA), c'est-à-dire le fragment TRBV5-1 dans la chaîne β du TCR.
PCT/EP2023/025286 2023-09-05 2023-09-05 FRAGMENT VARIABLE À CHAÎNE UNIQUE ET MOLÉCULES DÉRIVANT DE CELUI-CI SE LIANT AU FRAGMENT TRVB5-1 DE LA CHAÎNE β DU RÉCEPTEUR DES LYMPHOCYTES T HUMAINS Pending WO2025051339A1 (fr)

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Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012079000A1 (fr) 2010-12-09 2012-06-14 The Trustees Of The University Of Pennsylvania Utilisation de lymphocytes t modifiés par un récepteur chimérique d'antigènes chimérique pour traiter le cancer
US20140271635A1 (en) 2013-03-16 2014-09-18 The Trustees Of The University Of Pennsylvania Treatment of cancer using humanized anti-cd19 chimeric antigen receptor
WO2014184143A1 (fr) 2013-05-13 2014-11-20 Cellectis Récepteur de l'antigène chimère spécifique de cd19 et ses utilisations
WO2015132598A1 (fr) * 2014-03-05 2015-09-11 Ucl Business Plc Récepteur d'antigène chimérique (car) à domaines de liaison à l'antigène pour la région constante de récepteur bêta du lymphocyte t
WO2018231759A1 (fr) * 2017-06-12 2018-12-20 Obsidian Therapeutics, Inc. Compositions de pde5 et méthodes d'immunothérapie
WO2019090004A1 (fr) 2017-11-01 2019-05-09 Juno Therapeutics, Inc. Procédé pour la production d'une composition de lymphocytes t
US20190144515A1 (en) 2017-11-16 2019-05-16 Kite Pharma, Inc. Modified chimeric antigen receptors and methods of use
WO2020142672A2 (fr) * 2019-01-04 2020-07-09 Elstar Therapeutics, Inc. Molécules d'anticorps anti-tcr et leurs utilisations
US20200231648A1 (en) 2016-09-02 2020-07-23 Lentigen Technology, Inc. Compositions and Methods for Treating Cancer with DuoCARs
WO2021097325A1 (fr) * 2019-11-14 2021-05-20 Elstar Therapeutics, Inc. Molécules d'anticorps anti-tcr et leurs utilisations
WO2021138474A2 (fr) * 2020-01-03 2021-07-08 Marengo Therapeutics, Inc. Molécules d'anticorps anti-tcr et leurs utilisations
WO2022119955A1 (fr) * 2020-12-01 2022-06-09 The Johns Hopkins University Méthodes et matériaux pour traiter des cancers des lymphocytes t

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012079000A1 (fr) 2010-12-09 2012-06-14 The Trustees Of The University Of Pennsylvania Utilisation de lymphocytes t modifiés par un récepteur chimérique d'antigènes chimérique pour traiter le cancer
US20140271635A1 (en) 2013-03-16 2014-09-18 The Trustees Of The University Of Pennsylvania Treatment of cancer using humanized anti-cd19 chimeric antigen receptor
WO2014184143A1 (fr) 2013-05-13 2014-11-20 Cellectis Récepteur de l'antigène chimère spécifique de cd19 et ses utilisations
WO2015132598A1 (fr) * 2014-03-05 2015-09-11 Ucl Business Plc Récepteur d'antigène chimérique (car) à domaines de liaison à l'antigène pour la région constante de récepteur bêta du lymphocyte t
US20200231648A1 (en) 2016-09-02 2020-07-23 Lentigen Technology, Inc. Compositions and Methods for Treating Cancer with DuoCARs
WO2018231759A1 (fr) * 2017-06-12 2018-12-20 Obsidian Therapeutics, Inc. Compositions de pde5 et méthodes d'immunothérapie
WO2019090004A1 (fr) 2017-11-01 2019-05-09 Juno Therapeutics, Inc. Procédé pour la production d'une composition de lymphocytes t
US20190144515A1 (en) 2017-11-16 2019-05-16 Kite Pharma, Inc. Modified chimeric antigen receptors and methods of use
WO2020142672A2 (fr) * 2019-01-04 2020-07-09 Elstar Therapeutics, Inc. Molécules d'anticorps anti-tcr et leurs utilisations
WO2021097325A1 (fr) * 2019-11-14 2021-05-20 Elstar Therapeutics, Inc. Molécules d'anticorps anti-tcr et leurs utilisations
WO2021138474A2 (fr) * 2020-01-03 2021-07-08 Marengo Therapeutics, Inc. Molécules d'anticorps anti-tcr et leurs utilisations
WO2022119955A1 (fr) * 2020-12-01 2022-06-09 The Johns Hopkins University Méthodes et matériaux pour traiter des cancers des lymphocytes t

Non-Patent Citations (41)

* Cited by examiner, † Cited by third party
Title
"Mechanisms, manifestations and management", BLOOD REV., vol. 34, March 2019 (2019-03-01), pages 45 - 55
ABBAS AKLICHTMAN AHPILLAI S.: "Settima edizione.", 2012, ELSEVIER, article "Immunologia cellulare e molecolare"
ALTSCHUL SFGISH WMILLER WMYERS EWLIPMAN DJ.: "Basic localalignmentsearch tool", MOL BIOL, vol. 215, no. 3, 5 October 1990 (1990-10-05), pages 403 - 10
ANONYMOUS: "Datasheet for: TCR V[beta]5.1 Antibody, anti-human, Vio Bright R720, REAfinity(TM); Clone REA1062", MILTENYI BIOTEC B.V. & CO. KG, 23 December 2022 (2022-12-23), XP093125318, Retrieved from the Internet <URL:https://static.miltenyibiotec.com/asset/150655405641/document_39cle6caqh5bta43vr2v3r124m?content-disposition=inline> [retrieved on 20240130] *
BAZARBACHI ASUAREZ FFIELDS PHERMINE O.: "How I treat adult T-cell leukemia/lymphoma", BLOOD., vol. 118, no. 7, 18 August 2011 (2011-08-18), pages 1736 - 45, XP055550097, DOI: 10.1182/blood-2011-03-345702
BIRD REHARDMAN KDJACOBSON JWJOHNSON SKAUFMAN BMLEE SMPOPE SHRIORDAN GSWHITLOW M.: "Single chain antigen-binding proteins", SCIENCE, vol. 242, 1988, pages 423 - 6, XP000575094, DOI: 10.1126/science.3140379
BLOOD., vol. 127, no. 20, 19 May 2016 (2016-05-19), pages 2375 - 90
BOULIANNE GLHOZUMI NSHULMAN MJ.: "Production of functional chimaeric mouse/human antibody", NATURE, vol. 19, no. 312, 13 December 1984 (1984-12-13), pages 643 - 6, XP001084103, DOI: 10.1038/312643a0
CA CANCER J CLIN., vol. 68, no. 6, November 2018 (2018-11-01), pages 394 - 424
COLD SPRING HARB PERSPECT BIOL., vol. 2, no. 6, June 2010 (2010-06-01), pages a002485
DAVIS MMBJORKMAN PJ.: "T-cell antigen receptor genes and T-cell recognition", NATURE, vol. 334, no. 6181, 4 August 1988 (1988-08-04), pages 395 - 402, XP037129140, DOI: 10.1038/334395a0
DOGAN AATTYGALLE ADKYRIAKOU C.: "Angioimmunoblastic T-cell lymphoma.", BR J HAEMATOL., vol. 121, no. 5, June 2003 (2003-06-01), pages 681 - 91
FLEISCHER LCSPENCER HTRAIKAR SS.: "Targeting T cell malignancies using CAR-based immunotherapy: challenges and potential solutions", J HEMATOL ONCOL., vol. 29, no. 12, December 2019 (2019-12-01), pages 141
FREEMAN JDWARREN RLWEBB JRNELSON BHHOLT RA.: "Profiling the T-cell receptor beta-chain repertoire by massively parallel sequencing", GENOME RES., vol. 19, no. 10, October 2009 (2009-10-01), pages 1817 - 24, XP002636496, DOI: 10.1101/gr.092924.109
FRENZEL ASCHIRRMANN THUST M.: "Phage display-derived human antibodies in clinical development and therapy", MABS., vol. 8, no. 7, October 2016 (2016-10-01), pages 1177 - 1194, XP055791147, DOI: 10.1080/19420862.2016.1212149
G KOHLERC MILSTEIN: "Continuous cultures of fused cells secreting antibody of predefined specificity", NATURE, vol. 256, no. 5517, 7 August 1975 (1975-08-07), pages 495 - 7
GLIWINSKI MPIOTROWSKA MIWASZKIEWICZ-GRZES DURBAN-WOJCIUK ZTRZONKOWSKI P: "Therapy with CD4+CD25+ T Regulatory Cells - Should We Be Afraid of Cancer?", CONTEMP. ONCOL., vol. 23, 2019, pages 1 - 6
GREEN LL.: "Transgenic mouse strains as platforms for the successful discovery and development of human therapeutic monoclonal antibodies", CURR DRUG DISCOVTECHNOL., vol. 11, no. 1, March 2014 (2014-03-01), pages 74 - 84
GREINER JWGUADAGNI FHAND PHPESTKA SNOGUCHI PFISHER PBSCHLOM J.: "Augmentation of tumor antigen expression by recombinant human interferons: enhanced targeting of monoclonal antibodies to carcinomas", CANCER TREAT RES., vol. 51, 1990, pages 413 - 32
HENNING ANROYCHOUDHURI RRESTIFO NP.: "Epigenetic control of CD8+ T cell differentiation", NAT. REV. IMMUNOL., vol. 18, 2018, pages 340 - 356, XP055660663, DOI: 10.1038/nri.2017.146
HERMINE ORAMOS JCTOBINAI K. A: "Review of New Findings in Adult T-cell Leukemia-Lymphoma: A Focus on Current and Emerging Treatment Strategies", ADVTHER., vol. 35, no. 2, February 2018 (2018-02-01), pages 135 - 152, XP036434609, DOI: 10.1007/s12325-018-0658-4
J CLIN ONCOL., vol. 26, no. 25, 2008, pages 4124 - 4130
JOHN M. GOLDBERGLEWIS B. SILVERMANDONNA E. LEVYVIRGINIA KIMBALL DALTONRICHARD D. GELBERLESLIE LEHMANNHARVEY J. COHENSTEPHEN E. SAL: "Childhood T-Cell Acute Lymphoblastic Leukemia: The Dana-Farber Cancer Institute Acute Lymphoblastic Leukemia Consortium Experience", JOURNAL OF CLINICAL ONCOLOGY, vol. 21, no. 19, 2003, pages 3616 - 3622
KRANGEL MS.: "Gene segment selection in V(D)J recombination: accessibility and beyond", NAT IMMUNOL., vol. 4, no. 7, July 2003 (2003-07-01), pages 624 - 30
KUMAR BVCONNORS TJFARBER DL.: "Human T Cell Development, Localization, and Function throughout Life", IMMUNITY., vol. 48, no. 2, 20 February 2018 (2018-02-20), pages 202 - 213
LIPMAN DJPEARSON WR.: "Rapid and sensitive protein similarity searches", SCIENCE, vol. 227, no. 4693, 22 March 1985 (1985-03-22), pages 1435 - 41, XP000941106, DOI: 10.1126/science.2983426
LONBERG NHUSZAR D.: "Human antibodies from transgenic mice", INT REVIMMUNOL., vol. 13, no. 1, 1995, pages 65 - 93
LUCKHEERAM RVZHOU RVERMA ADXIA B.: "CD4+T cells: differentiation and functions.", CLIN DEV IMMUNOL, vol. 2012, 2012, pages 925135
LV KYIN TYU MCHEN ZZHOU YLI F.: "Treatment Advances in EBV Related Lymphoproliferative Diseases", FRONT ONCOL., vol. 12, 19 April 2022 (2022-04-19), pages 838817
NAGANO KTSUTSUMI Y: "Phage Display Technology as a Powerful Platform for Antibody Drug Discovery", VIRUSES., vol. 13, no. 2, 25 January 2021 (2021-01-25), pages 178
OLSEN EAWHITTAKER SKIM YHDUVIC MPRINCE HMLESSIN SR ET AL.: "Clinical end points and response criteria in mycosis fungoides and Sezary syndrome", J. CLIN. ONCOL., vol. 29, no. 18, 2011, pages 2598 - 607
OPFERMAN JTOBER BTASHTON-RICKARDT PG: "Linear differentiation of cytotoxic effectors into memory T lymphocytes", SCIENCE., vol. 283, no. 5408, 12 March 1999 (1999-03-12), pages 1745 - 8
SADELAIN MBRENTJENS RRIVIERE I.: "The basic principles of chimeric antigen receptor design", CANCER DISCOV., vol. 3, no. 4, April 2013 (2013-04-01), pages 388 - 98, XP055287277, DOI: 10.1158/2159-8290.CD-12-0548
SAKAGUCHI SYAMAGUCHI TNOMURA TONO M.: "Regulatory T cells and immune tolerance", CELL., vol. 133, no. 5, 30 May 2008 (2008-05-30), pages 775 - 87, XP002582219, DOI: 10.1016/j.cell.2008.05.009
SARAVIA JCHAPMAN NMCHI H.: "Helper T cell differentiation", CELL MOL IMMUNOL., vol. 16, no. 7, July 2019 (2019-07-01), pages 634 - 643, XP036826948, DOI: 10.1038/s41423-019-0220-6
SCARISBRICK JJPRINCE HMVERMEER MHQUAGLINO PHORWITZ SPORCU P ET AL.: "Cutaneous Lymphoma International Consortium Study of Outcome in Advanced Stages of Mycosis Fungoides and Sezary Syndrome: Effect of Specific Prognostic Markers on Survival and Development of a Prognostic Model", J. CLIN. ONCOL., vol. 33, no. 32, 2015, pages 3766 - 73
SHIMOYAMA M.: "Diagnostic criteria and classification of clinical subtypes of adult T-cell leukaemia-lymphoma. A report from the Lymphoma Study Group (1984-87", BR J HAEMATOL., vol. 79, no. 3, November 1991 (1991-11-01), pages 428 - 37
TRENDS CANCER., vol. 3, no. 9, pages 615 - 620
WEI SCHARMLEY PROBINSON MA: "Concannon P. The extent of the human germline T-cell receptor V beta gene segment repertoire", IMMUNOGENETICS., vol. 40, no. 1, 1994, pages 27 - 36
WEI SCHARMLEY PROBINSON MACONCANNON P.: "The extent of the human germline T-cell receptor V beta gene segment repertoire", IMMUNOGENETICS., vol. 40, no. 1, 1994, pages 27 - 36
WILLEMZE RCERRONI LKEMPF WBERTI EFACCHETTI FSWERDLOW SH ET AL.: "The 2018 update of the WHO-EORTC classification for primary cutaneous lymphomas", BLOOD. AMERICAN SOCIETY OF HEMATOLOGY, vol. 133, no. 16, 2019, pages 1703 - 14

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