Single-chain variable region fragments that bind to TRBV5-1, a fragment of the β-chain of the human T cell receptor, and molecules derived therefrom.

Abstract

The present invention relates to a single-chain variable region fragment (scFv) that binds to TRBV5-1, a fragment of the β-chain of the human T cell receptor (TCR), and molecules derived therefrom, as well as the use of said molecules for diagnostic and therapeutic purposes in the treatment of innovative tumor-associated antigens (TAAs), namely conditions resulting from the clonal proliferation of T lymphocytes expressing the TRBV5-1 fragment in the β-chain of the TCR.

Description

[[Technical Field]]

[0001] The present invention relates to a novel TAA, that is, an antibody fragment called scFv that binds to TRBV5-1, a fragment of the β chain of human TCR, and molecules derived therefrom. By way of illustration and not limitation, such molecules can be components of Fab (antigen-binding fragment), Fab2, minibodies, monoclonal antibodies of various classes (IgG, IgM, IgA, IgD), monospecific and bispecific molecules. These molecules may also be chimeric antigen receptors (CAR). Said molecules can be used for diagnostic and therapeutic purposes in pathological conditions caused by clonal expansion of T lymphocytes that express the TRBV5-1 fragment in the β chain of TCR. [[Background Art]]

[0002] T lymphocytes are an integral component of adaptive immunity (Non-Patent Document 1). T lymphocytes are derived from progenitor cells in the bone marrow, migrate to the thymus and complete the maturation process. T lymphocytes play a central role in the regulation of both humoral and cellular immunity.

[0003] T cells are classified into different subpopulations, particularly naive T cells (which respond to new antigens), memory T cells (which are responsible for maintaining long-term immune responses), and regulatory T cells that regulate the homeostasis of the immune response (Non-Patent Document 2).

[0004] Based on the expression of specific antigens on the cell membrane called CD4 and CD8, mature T lymphocytes are classified into helper T lymphocytes (CD4+) and cytotoxic T lymphocytes (CD8+) (Non-Patent Literature 3). After activation, helper T lymphocytes can differentiate into several subtypes based on the production of specific cytokines and the expression of characteristic surface markers. The helper T lymphocyte subtypes identified to date are Th1, Th2, Th17, Th9, Th22, follicular helper T cells (Tfh), induced regulatory T cells (iTreg), and type 1 regulatory T cells (Tr1).

[0005] Th1 cells produce IFN-γ, IL-2, and TNFα, activating the immune response against viruses and intracellular bacteria (Non-Patent Literature 4). Th2 cells produce IL-4, IL-5, and IL-13, activating the immune response against parasites (these can also cause allergic reactions). Th17 cells produce IL-17A, IL-17F, and IL-22, which are involved in the immune response against extracellular bacteria and fungi. These are particularly abundant in the intestinal tract, where they regulate the composition of the gut microbiota. Follicular helper T lymphocytes (Tfh) promote humoral immunity within germinal centers (GCs) by producing IL-21 (which stimulates B lymphocytes) and IL-4 (which is involved in immunoglobulin isotype switching).

[0006] On the other hand, CD8 is a membrane antigen characteristic of cytotoxic T lymphocytes (Non-Patent Literature 5). Cytotoxic T lymphocytes play a central role in responses to intracellular pathogens and cancer cells and are also involved in the pathogenesis of autoimmune diseases. Cytotoxic T lymphocytes kill cells that express target antigens (mainly antigen peptides obtained by processing viral proteins or mutant proteins in tumors). When activated, cytotoxic T lymphocytes release cytotoxic molecules such as granzymes and perforins, and secrete cytokines such as IFN-γ and IFN-α, which amplify innate and adaptive immune responses. At the end of the primary response, most CD8+ T lymphocytes undergo apoptosis, but a small number are converted into memory T cells. These lymphocytes respond by proliferating and rapidly converting into effector cells when newly exposed to the antigen (Non-Patent Literature 6).

[0007] Regulatory T lymphocytes (Tregs), characterized by the expression of CD4 and CD25, are now considered a third subpopulation of T lymphocytes (Non-Patent Literature 7). They eliminate autoreactive lymphocyte clones and play a central role in regulating the immune response. They are mainly classified into two major subpopulations: natural regulatory T-cells (-nTregs) (produced in the thymus) and those generated from naive T cells during the immune response (iTregs) (Non-Patent Literature 8).

[0008] T cell-derived tumors The two main types of neoplasms (or tumors) derived from T lymphocytes are lymphoma and leukemia. Lymphoma originates from the major lymphoid organs, namely the lymph nodes and spleen, but can occur in other organs as lymphoid tissue is present in various tissues. Leukemia originates from lymphoid progenitor cells in the bone marrow, but can sometimes originate from cells in the blood or extramedullary tissue. In a very small number of cases, it is difficult to determine whether the tumor originated from the bone marrow or lymphoid tissue, and a definitive diagnosis may not be made.

[0009] Non-Hodgkin lymphoma (NHL) is the most common hematological malignancy, accounting for approximately 3% of all cancers (Non-Patent Literature 9). It differs from Hodgkin lymphoma in its clinical and histological features, namely the absence of Reed-Sternberg cells and positivity for CD15 and CD30. In 85% of cases, it originates from B lymphocytes, and in 15% of cases, from T lymphocytes. Its incidence in developed and developing countries is 7.8 cases / 100,000 and 4.3 cases / 100,000 in men, and 5.6 cases / 100,000 and 2.9 cases / 100,000 in women, respectively (Non-Patent Literature 10). It is more frequently seen in men over 65 years of age, patients with autoimmune diseases, and those with a family history of hematological malignancies. NHL arising from mature T cells is called T-cell lymphoma (TCL), and is usually classified into peripheral T-cell lymphoma (PTCL) and cutaneous T-cell lymphoma (CTCL). According to the WHO classification, these are classified into "anaplastic large-cell lymphoma (ALCL)," "angioimmunoblastic T-cell lymphoma (AITL)," "Peripheral T-cell Lymphoma, Not Otherwise Specified (PTCL-NOS)," and "Extranodal NK / T-cell Lymphoma (mainly gastrointestinal and cutaneous)" (Non-patent Literature 11). Most subtypes of TCL have a poor prognosis, worse than most subtypes of B-cell non-Hodgkin lymphoma (NHL).

[0010] ALCL accounts for 1-3% of NHL and approximately 15% of TCL (Non-Patent Literature 12). ALCL 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 lymphocytes with abundant cytoplasm, exhibiting pleomorphism and possessing horseshoe-shaped or kidney-shaped nuclei.

[0011] AITLs arise from follicular helper T cells (TFHs) and are one of the most common mature T-cell malignancies (Non-Patent Literature 13). AITLs are characterized by lymphadenopathy, systemic symptoms, and often hypergammaglobulinemia. Histologically, they show pleomorphic immunoinfiltration, consisting not only of cancer cells but also of a rich microenvironment containing small reactive lymphocytes, histiocytes, eosinophils, follicular dendritic cells, prominent endothelial venules, and EBV-positive B immunoblasts (Non-Patent Literature 14).

[0012] PTCL-NOS includes a heterogeneous group of lymphomas derived from mature T cells that do not fully meet the diagnostic criteria for other classes. Therefore, it is a diagnosis of exclusion and is currently the most frequently diagnosed subtype.

[0013] CTCL is a subtype of extranodal NK / T-cell lymphoma, encompassing a wide range of malignant tumors arising from mature T-lymphocytes in the skin. Its incidence is 7 to 10 cases per million people (Non-patent Literature 15).

[0014] The most common forms of CTCL are mycosis fungoides (MF) and Sézary syndrome (SS) (Non-Patent Literature 16). Generally, MF follows a slow course, but in 30% of cases it progresses rapidly, with a median survival time of 13 to 56 months (Non-Patent Literature 17). Sézary syndrome (SS) is the rarest form of CTCL, but it is the most aggressive, with a 5-year survival rate of 15% to 40%.

[0015] Adult T-cell leukemia (ATL) is a rare tumor associated with HTLV-1 infection. It is estimated that approximately 10 million people worldwide are infected with this virus (Non-Patent Literature 18). There are four clinical subtypes of ATL: acute, lymphoma, chronic, and a subtype called "smoldering" (Non-Patent Literature 19). The prognosis of ATL differs depending on the subtype. The acute and lymphoma subtypes have a poor prognosis, with an average survival time of less than one year, while the chronic and subtypes progress slowly in most cases (Non-Patent Literature 20).

[0016] As far as childhood age is concerned, acute T-cell lymphoblastic leukemia (T-ALL) accounts for 10-15% of all cases of acute lymphoblastic leukemia (Non-patent Literature 21). Generally, patients with T-ALL have a worse prognosis than patients with B-ALL.

[0017] While radiotherapy may be effective for early-stage lymphoma, chemotherapy is usually the first-line treatment for T-cell lymphoma and leukemia. Common regimens include combination therapy with or without cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), or similar regimens that also include L-asparaginase, cytarabine, and methotrexate. The latter drugs are also administered intrathecally to reduce the risk of lesion spread to the central nervous system. Chemotherapy has a high failure rate, and relapses are frequent.

[0018] Allogeneic hematopoietic stem cell transplantation (allo-HSCT) can be a definitive treatment, but most patients are not suitable for this type of treatment, mainly due to their advanced age or their inability to achieve remission after chemotherapy.

[0019] In recent years, four new drugs—pralatrexate, brentuximab vedotin, romidepsin, and bellinostat—have been approved by major regulatory authorities for the treatment of relapsed / refractory PTCL. Further drugs registered for the treatment of the above tumors are bortezomib, alemtuzumab, mogamulizumab, and denileukin difutitox.

[0020] Despite recent therapeutic advances through the development of new targeted therapies and monoclonal antibodies, T-cell tumors still suffer from high mortality rates and represent a significant unmet medical need. Therefore, the development of safer and more effective innovative treatments is urgently needed.

[0021] Antigens expressed to a greater degree on the surface of malignant T lymphocytes than normal lymphocytes, particularly CD5, CD7, CD37, CD30, TRBC1, CD3, and CD1a, have been identified as novel targets for the treatment of T-cell malignancies (Non-Patent Literature 22). Monoclonal antibodies and CAR-Ts against these targets could represent innovative therapeutic approaches in the treatment of these serious diseases. However, the results obtained so far have been extremely limited, and their actual efficacy in the treatment of T-cell malignancies has not yet been established. In particular, because these antigens can only be used in specific subsets of T-cell malignancies, they do not offer the opportunity for a comprehensive approach to the treatment of these serious neoplastic diseases.

[0022] T cell receptor (TCR) TCRs are membrane proteins that recognize antigen peptides expressed in association with major histocompatibility complex (MHC) molecules (Non-Patent Literature 23). From a structural standpoint, TCRs are composed of two different subunits: α and β, or γ and δ. These structural differences correspond to functional differences; αβ T lymphocytes possess classical helper and cytotoxic functions in the context of adaptive immunity, while δγ T lymphocytes occupy the boundary between adaptive and innate immunity.

[0023] Both αβT lymphocytes and γδT lymphocytes undergo V(D)J gene rearrangement (recombination). However, the antigens recognized by γδT lymphocytes do not require processing and presentation associated with MHC molecules. In this sense, the TCR of γδT lymphocytes behaves like a PRR (pattern recognition receptor) that recognizes microbial antigens. γδT lymphocytes are particularly abundant in the intestinal mucosa, where they are called intraepithelial lymphocytes (IELs). The TCR associates with a protein complex called CD3 on the cell membrane. CD3 stabilizes heterodimers (αβ or γδ) on the one hand, and transmits signals resulting from the binding of the TCR to its antigen on the other hand (Non-patent Literature 24). The CD3 complex is formed by four different subunits called γ, δ, ε, and ζ. Typically, the CD3 complex consists of one γ subunit, one δ subunit, two ε subunits, and two ζ subunits. The cytoplasmic domains of these subunits contain a characteristic sequence called ITAM (Immune-receptor-Tyrosine-based-Activation-Motif). The γ, δ, and ε subunits each contain one ITAM sequence, and the ζ subunit contains three ITAM sequences (totaling 10 ITAM sequences in each CD3 complex). In the inactive state, the tyrosine residues of the ITAM sequences are located within the hydrophobic core of the lipid bilayer of the cell membrane. When the TCR binds to an antigen, the TCR / CD3 complex deforms, exposing the tyrosine residues of the ITAM sequences into the cytoplasm and making them accessible to two tyrosine kinases called Lck and Fyn. Phosphorylation of the tyrosine residues of the ITAM sequences allows further binding of a tyrosine kinase called ZAP-70, which initiates a cascade of events, leading to a series of events involving the activation of specific transcription factors and T lymphocyte activation.

[0024] Activation of helper T lymphocytes (CD4+) occurs when TCR binds to a complex consisting of an antigenic peptide expressed in association with MHC class II proteins on the surface of antigen-presenting cells (APCs) (i.e., B lymphocytes, macrophages, and dendritic cells). In contrast, activation of cytotoxic T lymphocytes (CD8+) occurs in peripheral tissues and is initiated when TCR binds to an antigenic peptide expressed in association with MHC class I proteins on the surface of any cell in the body. CD4 and CD8 molecules expressed on the surface of helper T lymphocytes and cytotoxic T lymphocytes bind to MHC class II and class I molecules, respectively, thereby stabilizing the entire complex.

[0025] Full activation of T lymphocytes requires activation of a certain number of secondary signals. The main signal is represented by the binding of CD28 on the surface of lymphocytes to B7.1 (CD80) or B7.2 (CD86) on the surface of antigen-presenting cells (APCs). This signal induces clonal proliferation of activated helper T lymphocytes, but this proliferation would continue indefinitely unless production of CTLA-4 (CD152), which competes with CD28 for binding to B7, is initiated. Proliferation (expansion) of cytotoxic T lymphocytes is less dependent on CD28, and instead requires activation of other signals, particularly CD70 and 4-1BB (CD137).

[0026] Survival of T lymphocytes depends on additional molecules on their surface, particularly ICOS, 4-1BB, and OX40, which are stimulated by corresponding ligands on the surface of APCs. These molecules are expressed after pathogen recognition by innate immunity. This is important for preventing inappropriate activation of T lymphocyte clones.

[0027] Certain cytokines play an important role in determining the type of response of activated T lymphocytes. In particular, IL-12 induces T lymphocytes to differentiate into a Th1-type response, which is mediated by the production of IFN-γ and is cytotoxic against cells infected with intracellular pathogens such as viruses and *Mycobacterium tuberculosis*, while IL-4 induces T lymphocytes to differentiate into a Th2-type response, which corresponds to allergic reactions and antibody responses against parasites; IL-17 induces T lymphocytes to differentiate into a Th17-type response against extracellular pathogens.

[0028] TCR V(D)J gene rearrangement Recognition of an antigen peptide (pMHC) associated with an MHC molecule depends on the three "complementarity determining regions (CDRs)" in the variable region of the T cell receptor (TCR), namely CDR1, CDR2, and CDR3. The first two are encoded by germinal sequences, while CDR3, which represents the main recognition site of the pMHC complex, is generated by a V(D)J gene rearrangement process similar to that observed in immunoglobulin production. More specifically, the variable regions of the α and γ chains are generated by gene rearrangement involving only V and J fragments, whereas the variable regions of the β and δ chains are generated by gene rearrangement of V, D and J. At the junctions between the V and D segments, and between the D and J segments, deletion of bases encoded in the germline and their substitution with different bases occur. This process is called "junctional diversity", which leads to the expansion of the TCR repertoire and enables recognition of more pMHC complexes.

[0029] The expression of single fragments V, D, and J is not random; some combinations occur more frequently than others (Non-Patent Literature 25). Specifically, the variable region of the TCRβ chain is made up of rearrangements of 68 V genes, 2 D genes, and 14 J genes. Each individual can express different alleles for the same gene. To date, 152 V gene alleles, 3 D gene alleles, and 16 J gene alleles have been listed in the IMGT database. Theoretically, each of the 68 V fragments of the TCRβ chain should be expressed at the same frequency, corresponding to 1.5% (1 / 68) of cases. However, in reality, 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 TRBV28 is expressed in about 5% of normal T lymphocytes. All remaining fragments are expressed at fairly low frequencies. This depends on many factors, but particularly on the presence of transcriptional enhancers near the exons of the constant region and on the numerous promoters associated with the segments of the V, D, and J genes (Non-Patent Literature 26). The stage of differentiation and the activity of enhancers and promoters induce covalent modifications of histones, particularly in the form of acetylation of lysine residues at the N-terminus of histones H3 and H4, resulting in hyperacetylation of the active reconstitution region. Since even methylation of a single CpG dinucleotide appears to block cleavage by the RAG protein, the CpG dinucleotide methylation step is thought to play a crucial role in the V(D)J gene reconstitution mechanism.

[0030] TCR as a diagnostic marker for neoplastic T lymphocyte clones T-cell malignancies result from the neoplastic transformation of a single T lymphocyte. Therefore, clones of neoplastic T lymphocytes are characterized by the presence of a single TCR on the cell membrane that clearly distinguishes them from healthy cells and cancer cells.

[0031] IOTest Beta Mark, manufactured by Beckman Coulter, is a diagnostic test that identifies neoplastic T lymphocyte clones using flow cytometry. This product consists of 24 monoclonal antibodies (22 mouse-derived and 2 rat-derived) that recognize specific V fragments of the TCR β chain. These 24 antibodies are divided into eight vials, each containing three antibodies labeled with different fluorescent dyes. If no neoplastic clones are present, the antibodies bind uniformly to T lymphocytes. If neoplastic clones are present, and the corresponding TCR expresses one of the 24 V fragments recognized by this kit, most T lymphocytes will be labeled with the same fluorescent dye. If the neoplastic TCR does not express any of these 24 fragments, most T lymphocytes will remain unlabeled (resulting in a so-called "null" result). [Prior art documents] [Non-patent literature]

[0032] [Non-Patent Document 1] Abbas AK, Lichtman AH, Pillai S. 2012. Immunologia cellulare e molecolare. Settima edizione. Elsevier, Milano 2012 [Non-Patent Document 2] Kumar BV, Connors TJ, Farber DL. Human T Cell Development, Localization, and Function throughout Life. Immunity. 2018 Feb 20;48(2):202-213 [Non-Patent Document 3] Saravia J, Chapman NM, Chi H. Helper T cell differentiation. Cell Mol Immunol. 2019 Jul;16(7):634-643 [Non-Patent Document 4] Luckheeram RV, Zhou R, Verma AD, Xia B. CD4+T cells: differentiation and functions. Clin Dev Immunol. 2012;2012:925135

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[0033] The inventors of this invention have identified six scFvs that bind to TRBV5-1 (formerly known as Vb5.1), a fragment of the β-chain of the human TCR, as an innovative TAA for the treatment of T-cell malignancies. In particular, these scFvs bind to an epitope contained in the amino acid sequence TQTPRYLIKTRGQQ. In patients with T-cell malignancies in which the tumor clone expresses the fragment TRBV5-1 on the β-chain of the TCR, these scFvs bind to 100% of tumor cells and maintain approximately 90% of healthy T lymphocytes. These scFvs were obtained from a human library called SFMAX® using "phage display" technology. This library contains 3.0 × 10⁶ naive human scFvs obtained from B lymphocytes of 368 donors from five different ethnic groups. 10 It contains several variants. Therefore, these scFv are entirely human-derived and fully suitable for therapeutic use in humans.

[0034] In a preferred embodiment of the present invention, the above-described scFv is used as is.

[0035] In a second preferred embodiment of the present invention, the scFv described above is used to produce monoclonal antibodies of the IgG class (any of the subclasses of IgG1, IgG2, IgG3, or IgG4).

[0036] In a third preferred embodiment of the present invention, the above-mentioned scFv is used to produce monoclonal antibodies of the IgM class (monomer, pentamer, and hexamer).

[0037] In a fourth preferred embodiment of the present invention, the scFv described above is used to produce monoclonal antibodies of the IgA class (monomer or dimer).

[0038] In a fifth preferred embodiment of the present invention, the scFv described above is used to produce other types of monoclonal antibodies not specifically described in this list (for illustrative purposes only, but not limited to, monoclonal antibodies conjugated with a drug; monoclonal antibodies conjugated with a radioisotope, etc.).

[0039] In a sixth preferred embodiment of the present invention, the scFv described above is used to produce a Fab (antigen-binding fragment).

[0040] In a seventh preferred embodiment of the present invention, the scFv described above is used to produce Fab2 (obtained by combining two identical Fab fragments).

[0041] In an eighth preferred embodiment of the present invention, the above-described scFv is used to produce a bispecific Fab2 (one of the two Fab fragments corresponds to one of the six scFvs according to the present invention).

[0042] In a ninth preferred embodiment of the present invention, the scFv described above is used to produce a bispecific monoclonal antibody (one of the two Fab fragments corresponds to one of the six scFvs according to the present invention).

[0043] In a tenth preferred embodiment of the present invention, the scFv described above is used to create a minibody (formed by conjugating one of the six scFvs according to the present invention to the constant CH3 domain of an immunoglobulin).

[0044] In an eleventh preferred embodiment of the present invention, the scFv described above is used as an antigen-binding domain for the production of a CAR.

[0045] In a twelfth preferred embodiment of the present invention, the scFv described above is used as an antigen-binding domain for the creation of a CAR expressed on autologous (i.e., taken from the same patient) CD8+ T lymphocytes.

[0046] In a thirteenth preferred embodiment of the present invention, the scFv described above is used as an antigen-binding domain for the creation of a CAR expressed on allogeneic or hemimitable (i.e., taken from a family member that shares 50% of the patient's genetic heritage) CD8+ T lymphocytes.

[0047] In a 14th preferred embodiment of the present invention, the scFv described above is used as an antigen-binding domain for the creation of a CAR expressed on autologous (i.e., taken from the same patient) CD4+ T lymphocytes.

[0048] In a 15th preferred embodiment of the present invention, the scFv described above is used as an antigen-binding domain for the creation of a CAR expressed on allogeneic or hemismatched (i.e., taken from a family member that shares 50% of the genetic heritage with the patient) CD4+ T lymphocytes.

[0049] In a sixteenth preferred embodiment of the present invention, the scFv described above is used as an antigen-binding domain for the creation of CARs expressed on other types of autologous (i.e., taken from the same patient) lymphocytes (for illustrative purposes only and not limited to, but such as NK lymphocytes, NKT, Tγ / δ, etc.).

[0050] In a 17th preferred embodiment of the present invention, the scFv described above is used as an antigen-binding domain for the creation of CARs expressed on other types of allogeneic or hemimitable (i.e., taken from a family member that shares 50% of the genetic heritage with the patient) lymphocytes (for illustrative purposes only and not limited to the present, such as NK lymphocytes, NKT lymphocytes, Tγ / δ lymphocytes).

[0051] A preferred embodiment of the 18th present invention relates to a pharmaceutical formulation comprising any of the molecules described in the 1st to 17th preferred embodiments.

[0052] A 19th preferred embodiment of the present invention relates to a pharmaceutical formulation for therapeutic use in humans, comprising any of the molecules described in the 1st to 17th preferred embodiments.

[0053] A 20th preferred embodiment of the present invention relates to a pharmaceutical formulation for diagnostic use comprising any of the molecules described in the 1st to 10th preferred embodiments of the present invention, wherein the clone thereof is used in patients having a T-cell tumor in which the fragment TRBV5-1 is expressed in the β-chain of the TCR.

[0054] A 21st preferred embodiment of the present invention relates to a pharmaceutical formulation for therapeutic use comprising any of the molecules described in the 1st to 17th preferred embodiments, wherein the clone thereof is used in patients having a T-cell tumor in which the fragment TRBV5-1 is expressed in the β-chain of the TCR.

[0055] Also provided are: specifications for a method used to obtain human scFv from a phage library; the amino acid sequence of the scFv; specifications for an in silico method for identifying the epitope recognized by the scFv; specifications for a method for measuring the affinity of a monoclonal antibody obtained from the scFv to an antigen; and finally, specifications for a method used to demonstrate the ability of a monoclonal antibody obtained from the scFv to bind to a clone of a neoplastic T lymphocyte expressing the fragment TRBV5-1 in the variable region of the β-chain of the TCR. [Brief explanation of the drawing]

[0056] [Figure 1] This image shows a 3D reconstruction of a human TCR expressing the fragment TRBV5-1 on its β-chain (the segment of the first antigen peptide selected for scFv generation by phage display is shown in red). [Figure 2] The structure of the TCR identified by code 5BS0 is shown. Mutations in the variable region of the β chain are shown in blue, potential binding surfaces with the six scFvs are shown in red, the loops in the constant region of the β chain are shown in magenta, and the variable and constant regions of the β and α chains of the TCR are shown in green and yellow, respectively. [Figure 3] The results of flow cytometry analysis performed on PBMCs (Primary Biomedical Cells) collected from patients with Sézary syndrome expressing the fragment TRBV5-1 in the variable region of the β-chain of the TCR are shown. Cells were pre-incubated with a complete antibody panel (anti-CD3, anti-CD4, anti-CD8, anti-CD45) with anti-CD14 antibody added for monocyte detection. Panels "A," "B," and "C" show the cell population during testing in red dot plot format. Panel "D" shows the same cell population in histogram format. Meanwhile, panels "E," "F," and "G" show the results of tests performed with lead compound A12 (panel "H" shows the same cell population in histogram format). This test highlights that lead compound A12 binds to 21% of the cell population during testing. [Figure 4]The results of flow cytometry analysis performed on PBMCs (Primary Bacterial Cells) collected from patients with Sézary syndrome expressing one of three fragments (TRBV6-5; TRBV6-6; TRBV6-9) recognized by the IOTest® Beta Mark (Beckman Coulter) anti-Vb13.1 antibody in the variable region of the TCRβ chain are shown. Cells were pre-incubated with a complete antibody panel (anti-CD3, anti-CD4, anti-CD8, anti-CD45) with anti-CD14 antibody added for monocyte detection. Panels "A," "B," and "C" show the cell population under test in red dot plot format. Panel "D" shows the same population in histogram format. Panels "E," "F," and "G" show tests performed with lead compound A12 added (panel "H" shows the same population in histogram format). As can be seen from the figures, antibody A12 does not bind to any of the cells in the cell population under test. [Modes for carrying out the invention]

[0057] A brief explanation of the table Table 1 shows the nucleotide sequence of TRBV5-1, a fragment of the β-chain of the human TCR, as shown in the prior art (International ImMunoGeneTics Information System; 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(1):27-36. doi: 10.1007 / BF00163961).

[0058] Table 2 shows the amino acid sequence of TRBV5-1, a fragment of the β-chain of the human TCR, obtained from the nucleotide sequences listed in Table 1.

[0059] Table 3 shows the amino acid sequence of the first antigen peptide selected within the β-chain fragment TRBV5-1 of the human TCR. The segment actually used in the production of scFv by phage display is highlighted in red.

[0060] Table 4 shows the amino acid sequence of the second antigen peptide selected within the β-chain fragment TRBV5-1 of the human TCR. Within this sequence, two candidate segments were individualized for phage display and are shown in black and green, respectively.

[0061] Table 5 shows the amino acid sequence of the third antigenic peptide selected within the human TCR β-chain fragment TRBV5-1. This peptide is located immediately after the second peptide in the C-terminal direction.

[0062] Table 6 highlights the problematic amino acids within the three candidate antigen peptides, i.e., those that may impair the antigenic properties of the peptides during the synthesis process.

[0063] Table 7 shows the scores obtained for three candidate antigen peptides in simulations performed using two different bioinformatics tools called "Antigen Profiler Peptide" (TM score) and "AbDesigner" (Ab score).

[0064] Table 8 shows the results of a polyclonal ELISA assay (enzyme-linked immunosorbent assay) for preliminary detection of phages that recognize the segment of the first antigen peptide.

[0065] Table 9 shows the results of the first monoclonal ELISA assay performed on 96 single phages obtained in the fourth round.

[0066] Table 10 shows the results of the second monoclonal ELISA assay performed on 96 single phages obtained in the fourth round.

[0067] Table 11 shows the results of the confirmatory ELISA assay.

[0068] Table 12 shows the light chain CDR1, CDR2, and CDR3 sequences, heavy chain CDR1, CDR2, and CDR3 sequences, and associated framework regions (FRs) of six human scFvs that recognize the first antigen peptide segment of the β-chain fragment of human TCR, TRBV5-1, as identified by phage display.

[0069] Table 13 shows the sequence alignment between the primary structure (A0A578) of fragment TRBV5-1 reported in UniProt and the sequences of FASTA 5BRZ and 5BS0. The shades of color indicate the identity percentage.

[0070] Table 14 shows the ranking of scFv sorted in descending order by interface energy score, and the number of interacting amino acid residues in the constant region of the β-chain of the corresponding TCR.

[0071] Table 15 shows the complete sequence of monoclonal antibody A12 obtained by conjugating the variable region of the corresponding scFv to the heavy chain of human IgG1.

[0072] Table 16 shows the affinity constants (KD) of six monoclonal antibodies against the antigen construct.

[0073] Preliminary flow cytometry study Using the IOTest Beta Mark diagnostic kit from Beckman Coulter, flow cytometry analysis was performed on the expression of different V fragments of the TCRβ chain in a consecutive series of 103 patients with T-cell lymphoma. Surprisingly, the expression frequencies of these fragments in tumor cells differed from those reported by Freeman et al. in healthy T lymphocytes. In particular, fragment 29-1 was not detected at all in neoplastic lymphocytes (whereas it was expressed in 10% of healthy lymphocytes). Of the most frequently expressed V fragments in normal T lymphocytes, only TRBV5-1 was expressed at the same frequency (approximately 12%) in both normal and neoplastic T lymphocytes. The expression frequency of the TRBV5-1 fragment in T-cell lymphoma patients was significantly higher than that of other fragments (p<0.0001).

[0074] The discovery of differences in the expression frequency distribution of the V fragment of the TCR β chain between normal T lymphocytes and neoplastic T lymphocytes opens new avenues for immunotherapy of T-cell malignancies. The V fragment, which is most frequently expressed in the TCR of neoplastic T lymphocytes and less frequently expressed in normal T lymphocytes, represents a new class of tumor-associated antigens (TAAs) in T-cell malignancies. These TAAs can be targeted by specific immunotherapies, similar to epidermal growth factor receptor (EGFR), HER2 / neu protein, and CD20, which are targets of monoclonal antibodies (antitumor antibodies cetuximab, trastuzumab, and rituximab; Wittrup KD Can Drive Therapeutic T Cell Responses. Trends Cancer. 2017 Sep;3(9):615-620); or similar to CD19, which is a target of CAR-T therapies such as 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).

[0075] For these novel TAAs to become innovative targets in immunotherapy for human T-cell malignancies, they must meet the following two key requirements: a) The fragment is expressed in a significant proportion of neoplastic clones in patients with T-cell malignancies; b) The fragment is expressed only in a limited number of normal T lymphocytes.

[0076] If the first condition is not met, the number of treatable patients will be limited, making the industrial development of specific immunotherapy economically unprofitable. On the other hand, if the second condition is not met, the specific immunotherapy administered to patients will cause severe immunosuppression due to dysfunction of the T cell compartment of the immune system.

[0077] Specific immunotherapy against the TRBV5-1 fragment of the TCRβ chain satisfies both conditions. In fact, they may be effective in 12% of T-cell malignancy patients, making their development interesting from an industrial standpoint. Furthermore, they result in 100% destruction of tumor T lymphocytes while preserving approximately 90% of normal T lymphocytes.

[0078] The reason for the difference in the expression frequency of the V fragment of the TCR β chain between normal T lymphocytes and neoplastic T lymphocytes is unknown. A possible explanation can be found in the role played by viruses such as EBV and HTLV-1 in the pathogenesis of these diseases (Lv K, Yin T, Yu M, Chen Z, Zhou Y, Li F. Treatment Advances in EBV Related Lymphoproliferative Diseases. Front Oncol. 2022 Apr 19;12:838817). T lymphocyte clones that recognize viral antigen peptides expressed in association with MHC molecules can, for unknown reasons, evade physiological regulatory mechanisms and proliferate unchecked. Since each antigen peptide preferentially binds to a specific MHC allele, it limits the repertoire of TCRs that can recognize the complex formed by the viral antigen peptide and the corresponding MHC molecule. Therefore, the binding of specific antigen peptides to specific MHC molecule alleles, derived from the processing of viral proteins, may be limiting the TCR repertoire of neoplastic T lymphocytes. In this regard, it should be kept in mind that the V fragment plays a central role in antigen peptide recognition because it not only expresses both CDR1 and CDR2, but also participates in the expression of CDR3 through V(D)J gene rearrangement.

[0079] Research on the target peptide of the TRBV5-1 fragment The development of scFvs that bind to the TRBV5-1 fragment of the TCRβ chain began with the analysis of the amino acid sequence constituting the fragment. Tables 1 and 2 show the nucleotide and amino acid sequences of the TRBV5-1 fragment described in the prior art, respectively (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(1):27-36. doi: 10.1007 / BF00163961). These nucleotide and amino acid sequences can also be found in the Protein Data Bank (https: / / www.rcsb.org / - pdbcodes: 5BRZ[1] and 5BS0[1]).

[0080] Using 3D modeling and docking techniques with MHC class II molecules and TCRα chains, we were able to identify three candidate peptides for antibody development.

[0081] The first candidate peptide, whose sequence is shown in Table 3, is located near the N-terminus. This peptide is easily accessible to the antibody, but it is not perfectly linear and is difficult to synthesize. On the other hand, shorter segments (shown in red in Table 3 and Figure 1) represent superior candidates.

[0082] The second candidate peptide is located in the middle portion of the fragment, and its sequence is shown in Table 4. Although this peptide as a whole is highly structured, two segments with features suitable for antibody development can be identified within it (shown in black and green, respectively, in Table 4).

[0083] The third candidate peptide follows the second peptide in the C-terminal direction and is characterized by the amino acid sequence shown in Table 5.

[0084] Figure 1 shows the 3D structure of a human TCR expressing the TRBV5-1 fragment on its β-chain (the segment of the first antigen peptide is shown in red).

[0085] The selection of peptides for subsequent scFv development was based on a priority list. Table 6 lists the presence of "problematic" amino acids in each candidate peptide. Segment 2 of peptide number 2 contains two asparagine residues and one methionine. The N-terminal asparagine residue is difficult to remove during cleavage, and the methionine can be irreversibly oxidized to methionine sulfoxide and methionine sulfone. Peptide number 3 contains aspartic acid, which can form a cyclic intermediate that causes cleavage under acidic conditions. On the other hand, peptide number 1 and segment 1 of peptide number 2 do not contain any problematic amino acids.

[0086] Next, candidate peptide sequences were analyzed using two different bioinformatics tools: "Antigen Profiler Peptide" (https: / / www.thermofisher.com / it / en / home / lifescience / antibodies / custom-antibodies / custom-antibodyproduction / antigen-profiler-antigen-preparation.html) and "AbDesigner" (https: / / esbl.nhlbi.nih.gov / AbDesigner / ). Table 7 summarizes the scores obtained for each candidate peptide. Although there were some differences in the scores obtained by the two tools, all peptides received good scores. However, due to the absence of problematic amino acids, optimal length, and excellent scores obtained in the AbDesigner test, two independent groups identified peptide number 1 (TQTPRYLIKTRGQQ sequence) as the ideal candidate for the next stage of scFv development for the TRBV5-1 fragment of the TCRβ chain.

[0087] Production of scFv using phage display method IOTest Beta Mark antibodies are not commercially available individually, but only as a mixture of three antibodies in the same vial; therefore, they cannot be used as a positive control in characterization tests. Furthermore, IOTest Beta Mark antibodies were produced in mice and rats using the hybridoma method (G Koehler, C Milstein. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7;256(5517):495-7) first reported by Koehler and Milstein in 1975, and therefore can only be used in humans for diagnostic purposes. When administered to humans for therapeutic purposes, it induces the production of anti-mouse antibodies (HAMAs) that neutralize its activity (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).

[0088] The problem of immunogenicity in mouse antibodies has been partially solved by biotechnological techniques that have led to the development of chimeric and humanized antibodies. Chimeric antibodies are obtained by replacing mouse Fc fragments with human Fc fragments (Boulianne GL, Hozumi N, Shulman MJ. Production of functional chimaeric mouse / human antibody. Nature. 1984 Dec 13-19;312(5995):643-6), while humanized antibodies are obtained by inserting mouse CDRs into the structure of human antibodies (Lonberg N, Huszar D. Human antibodies from transgenic mice. Int RevImmunol. 1995;13(1):65-93). The use of these techniques has made it possible to produce monoclonal antibodies such as rituximab, infliximab, and trastuzumab, which are currently used to treat important diseases such as B-cell lymphoma, rheumatoid arthritis, and certain types of breast cancer. However, chimeric antibodies and humanized antibodies retain some mouse-derived sequences that are recognized as non-self by the human immune system, resulting in the production of neutralizing antibodies. In any case, the development and humanization of chimeric antibodies of anti-TRBV5-1 mouse antibodies would have resulted in products that do not meet current quality standards (fully human antibodies).

[0089] The most common techniques for producing fully human monoclonal antibodies are 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 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;11(1):74-84). Of these two methods, phage display is faster and more ethically acceptable because it does not require animal sacrifice.

[0090] Phage display is a method of expressing human antibody fragments called scFvs on the surface of bacteriophages. These fragments consist of variable regions of the heavy (VH) and light (VL) chains of human antibodies linked by small, flexible peptides called "linkers" (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). To obtain a bacteriophage library expressing a broad repertoire of human scFvs, relevant genes are extracted from B lymphocytes collected from the spleen, peripheral blood, tonsils, and sometimes 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 VH and VL regions encoding the antibody gene are fused with the gene encoding the bacteriophage's coat protein. As a final result, scFv is displayed on the surface of the bacteriophage. These bacteriophages are first replicated in bacteria (usually E. coli) and then transferred to a plate pre-fixed with the target antigen. Bacteriophages expressing scFv that bind to the target antigen remain attached to the bottom of the plate, while others are removed by washing. The attached bacteriophages are eluted, resulting in a mixture of bacteriophages concentrated with only the elements expressing scFv that bind to the target antigen ("panning"). By repeating the panning cycle, phages expressing more specific scFv are gradually selected. Some phage display protocols also anticipate the induction of mutations, aiming to mimic the so-called "affinity mutation" of B lymphocytes during the immune response.

[0091] In the process of producing six scFvs according to the present invention, a human scFv library called "Human LiAb SFMAX®" was successfully used. This library was designed to amplify the scFv repertoire as much as possible, and it contains 3.0 × 10⁶ human naive scFvs obtained from B lymphocytes collected from 368 donors belonging to five different ethnic groups. 10 Includes variants of .

[0092] The process for producing the six scFvs according to the present invention began with the conjugation of the peptide TQTPRYLIKTRGQQ with three carrier proteins: bovine serum albumin (BSA), ovalbumin (OVA), and keyhole limpet hemocyanin (Megathura crenulata hemocyanin: KLH). The panning cycle was carried out in special test tubes coated with the conjugated antigen peptide. The library was pre-depleted of phages that recognized only the carrier without the antigen peptide. After further washing, phages attached to the coating were eluted by adding glycine HCl and then neutralized.

[0093] The eluate was added to the culture medium of E. coli TG1, and the plaque count (corresponding to lysed bacterial cells) was measured to determine the concentration of eluted phages. The eluate was added to the E. coli TG1 culture medium on a plate in the presence of helper phages to amplify the eluted phages. The amplified phages were precipitated with PEG / NaCl, resuspended, and then used in the next panning cycle.

[0094] Next, the wells of a multi-well plate were coated with the peptide TQTPRYLIKTRGQQ (concentration 10 μg / mL) conjugated to three carriers (BSA, OVA, and KLH), and a polyclonal ELISA test was performed. After washing and fixation, the amplified phages were incubated after each panning cycle as shown in Table 8. Horseradish peroxidase (HRP)-conjugated anti-phage antibody was added together with a peroxidase-specific substrate (tetramethylbenzidine (TMB)). After an appropriate incubation time, the peroxidase-catalyzed reaction was stopped, and the plate was read using a spectrophotometer at a wavelength of 450 nm.

[0095] The data obtained supported the gradual enrichment of phages that specifically recognized the antigen peptide from the first to the fourth round of panning. The fourth round, in particular, showed high enrichment, and its eluate was used in subsequent tests.

[0096] Two monoclonal ELISA tests yielded the results shown in Tables 9 and 10. The first test identified 18 clearly positive clones expressing six different sequences (shown in bold in Table 9). The second test identified 27 clearly positive clones expressing five sequences (shown in bold in Table 10), all of which were identical to the sequences already identified in the first test.

[0097] Finally, confirmatory monoclonal ELISA testing yielded the results shown in Table 11. The results indicate that all six identified clones specifically bind to the TRBV5-1 fragment peptide TQTPRYLIKTRGQQ. Binding affinity to the non-conjugate peptide was lower than that to the conjugate peptide and gradually decreased with phage dilution. This is likely due to suboptimal coating of the plates with the non-conjugate antigen peptide.

[0098] Sequencing of the six clones identified by monoclonal ELISA yielded the six sequences shown in Table 12. Some of these show a high degree of identity with one another, measurable by so-called "alignment algorithms."

[0099] Alignment algorithms emerged in the late 1980s with the aim of performing rapid searches on nucleotide or polypeptide sequence databases. The two most commonly used programs are FASTA and BLAST. FASTA (http: / / www.ebi.ac.uk / Tools / fasta33 / index.html) splits polypeptides (or polynucleotides) into sequences of user-selected lengths using the "ktup" parameter. A higher ktup value results in faster searches but lower accuracy. This algorithm creates a so-called "offset table" in which the diagonal with the highest score is highlighted. For a detailed explanation of this technique, see prior art (Lipman DJ, Pearson WR. Rapid and sensitive protein similarity searches. Science 1985:Mar 22;227(4693):1435-41).

[0100] BLAST is available on its dedicated website (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi). This algorithm enables the realization of "w-mers," i.e., amino acid (or nucleotide) sequences of length W that, when aligned to a query sequence, give a score > T (threshold) based on the permutation matrix. Note that a smaller T value results in a larger number of w-mers in the list. This means that a lower T value increases both execution time and the sensitivity of the analysis. For a detailed explanation of this method, please refer to the prior art (Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic localalignmentsearch tool. Mol Biol 1990 Oct 5;215(3):403-10).

[0101] These algorithms yield the so-called "best alignment," that is, the alignment of two polypeptide or nucleotide sequences that yields the highest identity percentage. Identity means that after correcting for any deletions or insertions, the same amino acids (or the same nucleotides) are in the same positions. Once the best alignment is obtained, the so-called "identity percentage," that is, the number of identical amino acids (or nucleotides) out of the total, is determined.

[0102] Regarding the sequences of the six identified clones, those designated A2 and A5 exhibit identical VH regions, and their VL regions are also very similar (95% identity), differing by only two amino acids in their respective CDR3 sequences. The VH sequences of the other clones differ significantly from each other, with an identity percentage not exceeding 71%. Clone C10 shares a similar VL sequence to clones A2 and A5, but exhibits a completely different VH region. The differences observed in these six clones may be due to them binding to different epitopes of the peptide TQTPRYLIKTRGQQ, but more likely, they are due to them binding to the same epitope with different affinities.

[0103] This invention relates not only to the sequences of the six identified clones, but also to possible variants that can be readily realized by those skilled in the art. In this invention, “variant” means a polypeptide having an amino acid sequence similar to any of the six identified clones and / or exhibiting the same biological function (i.e., binding of the human TCRβ chain to the TRBV5-1 fragment). Those skilled in the art are familiar with the technique of replacing one or more amino acids in the polypeptide sequences of the six identified clones with similar amino acids (exemplary but not limited to aliphatic amino acids with other aliphatic amino acids, aromatic amino acids with other aromatic amino acids, sulfur-containing amino acids with other sulfur-containing amino acids, etc.) without substantially altering their function. This invention relates not only to the sequences of the six identified clones, but also to possible variants having an identity percentage of ≥70% as measured by BLAST (with parameters of score=50 and word length=3 for amino acid sequences (or score=100 and word length=12 for nucleotide sequences)).

[0104] The present invention relates not only to the six scFv described above, but also to molecules that may be derived from them. Those skilled in the art are well familiar with the techniques for producing these molecules, and these molecules include, but are not limited to, the following: Fab fragments (consisting of VL, CL, VH, and CH1 domains); Fd fragments (consisting of VH and CH1 domains); Fv fragments (consisting of VH and CH1 domains); dAb fragments (consisting of VH domains only); single CDRs (light chain CDR1, CDR2, or CDR3, or heavy chain CDR1, CDR2, or CDR3); F(ab)2 fragments (linked by hinge regions) Consists of two Fab fragments; diabodies (polypeptides that recognize an antigen using the VL domain of one polypeptide and the VH domain of another polypeptide); various classes and subclasses of human monoclonal antibodies (monomer or dimer IgA; IgD; IgE; IgG, subclasses 1, 2, 3, or 4); IgM (monomer, pentamer, or hexamer); and various types of CARs described below (single CAR, tandem CAR, multiple CAR using one single vector, and multiple CAR using one or more vectors). Different types of CARs may have variable structures, but always share the following three characteristics: - One or more extracellular domains that recognize an antigen (usually obtained by scFv); - The cytoplasmic domain responsible for transmitting activation signals (usually derived from CD3ζ); - A co-stimulatory domain responsible for maintaining CAR-T activation (usually obtained from the co-stimulatory molecule 4-1BB).

[0105] Therefore, the present invention relates to any and / or thereto any of the six variable regions that recognize the TRBV5-1 fragment of the human TCRβ chain as described in Table 12. ≧ This also applies to all types of CARs that expose an antigen-binding domain containing any sequence sharing 70% identity to the extracellular space.

[0106] In silico identification of antigen epitopes In silico studies were conducted to identify antigen epitopes recognized by six scFvs and to evaluate their affinity and specificity for the antigens. The primary sequence of fragment TRBV5-1 is registered in the Uniprot database as code A0A578. Two crystal structures, 5BRZ and 5BS0 (with resolutions of 2.62 Å and 2.40 Å, respectively), are associated with it. Both X-ray structures show the constant and variable regions of the α and β chains of the TCR, which are complexed with the HLA class I histocompatibility antigen, β-2-microglobulin, and a 9-amino acid antigen peptide. The variable β region of the TCR, the target of this computational study, was crystallized together with the variable and constant regions of the α chain, as well as the constant region of the β chain, allowing for a more precise evaluation of the complex with the antibody variable region. Both X-ray structures show amino acid mutations in the variable region of the β chain of the TCR compared to the primary sequence reported in Uniprot (Table 13). From the structure of PDB ID 5BS0, all components except the constant and variable regions of the α and β chains of the TCR were removed. Next, using Modeller (https: / / doi.org / 10.1006 / jmbi.1993.1626), PDB2PQR (https: / / doi.org / 10.1093 / nar / gkm276), and Rosetta software (https: / / doi.org / 10.1371 / journal.pone.0059004), hydrogen atoms, side chains, and missing loops were added, bond orders were assigned, the hydrogen bond network was optimized at pH 7.4, and the energy of the system was further minimized.

[0107] To evaluate the effect of the aforementioned loop in the constant region of the TCR β-chain on the binding of the six scFvs to their targeted regions, conventional molecular dynamics simulations (300 ns simulations) were performed on antigen structures obtained from the protein preparation protocol using Amber software (https: / / doi.org / 10.1002 / wcms.1121). The resulting molecular dynamics trajectories were clustered using the RMSD of amino acid residues in the β-chain variable region area targeted for the development of the six scFvs, and the RMSD of atoms in the loop in the β-chain constant region. From this clustering process, representative structures of the three clusters with the largest number of individuals (78% of the total number of individuals) were extracted and used in the subsequent ensemble docking step.

[0108] Modeling of the six scFvs was performed using the multi-mer version of Alphafold software (https: / / doi.org / 10.1038 / s41586-021-03819-2), starting from the provided primary structures. The best model obtained for each scFv was then subjected to protein preparation protocols using PDB2PQR and Rosetta software for hydrogen bond network optimization and energy minimization at pH 7.4.

[0109] After obtaining the structures of each scFv and the antigen ensemble structures, local ensemble docking (targeting the antigen regions targeted for the development of the six scFvs) was performed between each scFv and the three antigen structures using the Rosetta suite's Snugdock software (https: / / doi.org / 10.1371 / journal.pcbi.1000644). Finally, the complex with the best interfacial energy score was extracted from each docking run (three times for each scFv) and visually confirmed.

[0110] In the two complexes with the best interfacial energy scores, the scFv identified by code C10 binds to a region different from the region targeted for design, whereas in the complex with the worst interfacial energy score, it binds to the aforementioned region. Therefore, it was concluded that C10 should have a region different from the region targeted for design, specifically the CDR region of the TCR, as its preferred epitope, and for this reason, it was excluded from this analysis. Table 14 shows the remaining five scFvs, sorted in descending order by predicted affinity, which should have the targeted peptide region as well as several other amino acids in the variable and constant regions of the TCRβ chain as their preferred antigen epitopes. Furthermore, the number of amino acid residues in the TCRβ chain constant region that should be involved in binding to these five scFvs according to binding interface calculations and which may introduce selectivity issues is also shown. The results indicate that the two best scFv candidates are D5 and A12, with D5 showing the highest predicted binding affinity and A12 showing the highest predicted binding selectivity.

[0111] Synthesis of monoclonal antibodies Techniques for producing monoclonal antibodies starting from scFv sequences are widely described in the prior art, and many companies offer rapid and low-cost contract synthesis services. More specifically, those skilled in the art can easily produce scFv itself; or obtain IgG-class human monoclonal antibodies (any subclass of IgG1, IgG2, IgG3, or IgG4); or obtain IgM-class human monoclonal antibodies (monomer, pentamer, and hexamer); or obtain IgA-class human monoclonal antibodies (monomer or dimer); or obtain various types of chimeric receptors as described below; or, for illustrative purposes only and not limiting, obtain other types of molecules such as drug-conjugated monoclonal antibodies; radioisotopes-conjugated monoclonal antibodies; Fab (antigen-binding fragment); Fab2 (obtained by conjugating two identical Fab fragments); bispecific Fab2; bispecific monoclonal antibodies; and minibodies.

[0112] In this case, the outsourcing company produced IgG1-class human monoclonal antibodies starting from the sequences of the variable regions of the α and β chains of the six scFvs according to the present invention, which are detailed in Table 12. Table 15 shows the complete heavy and light chain sequences of the monoclonal antibody obtained from scFV A12 (designated as the "lead compound" based on the results of bioinformatics studies and SPR testing). The other monoclonal antibodies consist of the same constant domain ligated to the variable region of each scFv shown in Table 12.

[0113] We began by designing, optimizing, and synthesizing the DNA sequence obtained by adding the sequence of the constant region of a human IgG1 class antibody to the sequence of the variable region (scFv). The complete sequence was cloned into the optimized vector, and plasmids were prepared for transfection into CHO cells.

[0114] CHO cells were cultured in Erlenmeyer flasks at 5% CO2 and 37°C in an orbital shaker using serum-free expression medium. The day before transfection, CHO cells were seeded into Erlenmeyer flasks at an appropriate density. On the day of transfection, the recombinant plasmid encoding the target antibody and the transfection reagent were mixed in an optimal ratio and added to the cultured CHO cells.

[0115] The cell culture supernatant was collected when the cell viability fell below 50%. After centrifugation and filtration, the recombinant antibody was purified by affinity chromatography. After washing and elution with appropriate buffers, the monoclonal antibody was concentrated and analyzed using SDS-PAGE (to verify protein molecular weight) and SEC-HPLC (to evaluate purity). The composition of the reducing buffer for SDS-PAGE was as follows: 250 mM Tris-HCl, 10% SDS, 30% glycerol, 0.5% bromophenol blue, 250 mM DTT, pH 6.8. The composition of the non-reducing buffer was as follows: 250 mM Tris-HCl, 5% SDS, 30% glycerol, 0.5% bromophenol blue, 250 mM IAM, pH 8.2. Finally, endotoxin levels were measured and the concentration of the monoclonal antibody was determined by the A280 method.

[0116] Six types of monoclonal antibodies were obtained in variable amounts ranging from 8.4 mg to 11.2 mg. Half of the antibodies were left unmodified, and the other half were conjugated with a fluorescent dye (fluorescein-5-isothiocyanate-FITC) for flow cytometry. To perform conjugation, the antibodies were dialyzed with sodium bicarbonate buffer. The antibody concentration was measured using a micro-ultraviolet-visible spectrophotometer (MUVVS). FITC was dissolved in DMSO and added to the buffer containing the antibodies. After adding glycine to the buffer, the antibodies conjugated to FITC were dialyzed, and their presence was confirmed using MUVVS. Finally, BSA and ProVlin300 were added.

[0117] The monoclonal antibodies prepared in this manner were divided into 1 mg aliquots and stored in a freezer at -20°C. Aliquots of unmodified antibodies were used for surface plasmon resonance (SPR) testing, and aliquots of antibodies labeled with fluorescent dyes were used for flow cytometry analysis.

[0118] Surface plasmon resonance (SPR) testing and identification of lead compounds The first experience with SPR dates back to the 1960s, but the first devices appeared on the market in 1990. These devices typically consist of a monochromatic polarized light source, a glass prism, a thin metal film (sensor chip) in contact with the base of the prism, and a photodetector. A ligand (e.g., antigen) is immobilized on the sensor chip, and an analyte (e.g., antibody) in a liquid phase is flowed over the sensor chip. The SPR signal generated by the monochromatic polarized beam that passes through the prism to the sensor chip is directly proportional to the mass on the sensor surface.

[0119] The analysis begins with the detection of a baseline signal corresponding to the mass of the ligand immobilized on the sensor tip. Subsequently, the analyte in the liquid phase flows over the surface of the sensor tip, interacting with the ligand to produce an association phase (corresponding to the formation of an analyte-ligand complex). This is followed by a plateau phase in which equilibrium is achieved between the analyte bound to the ligand and the analyte dissociating from the ligand. Finally, a running buffer dissociates the analyte from the ligand (dissociation phase). To allow the sensor tip to be reused for a second analyte, an acid or alkaline solution is injected to detach the remaining analyte from the ligand and enable tip regeneration. The result is expressed as an affinity constant (KD) corresponding to the relationship between the dissociation constant (Kd) and the binding constant (Ka).

[0120] In the experiments according to the present invention, a sensor chip containing streptavidin was used. The ligand immobilized on the sensor chip was a construct made of the peptide TQTPRYLIKTRGQQ conjugated with BSA and biotin, which had been previously used in the first and fourth rounds of biopanning for phage display.

[0121] The binding of streptavidin and biotin correctly positioned the antigenic epitope of the peptide TQTPRYLIKTRGQQ on the chip so that it faced outwards. A monoclonal antibody (analyte) was added to the liquid phase and interacted with the ligand immobilized on the sensor chip.

[0122] Table 16 summarizes the KD values ​​of six monoclonal antibodies against the antigen construct. The results obtained in SPR are substantially consistent with those obtained in in silico studies. When antibody A12 challenged the peptide TQTPRYLIKTRGQQGQQ, high affinity was recorded (KD = 3.10 × 10⁻¹⁰). -8 ). A slightly lower value was obtained for antibody D5 (this antibody also showed lower specificity in the in silico study). Significant but lower affinity was recorded for the other four antibodies. Contrary to the results obtained in the in silico study, the C10 antibody showed significant affinity for the antigen construct. Therefore, combining the results of the SPR study and the in silico study, monoclonal antibody A12 was identified as the lead compound. [Examples]

[0123] ScFv and all the molecules derived therefrom (especially monoclonal antibodies) can be used for both diagnostic and therapeutic purposes.

[0124] From a diagnostic standpoint, scFv and all the molecules derived therefrom (especially monoclonal antibodies) can be conjugated with any type of fluorescent dye to verify their affinity to tumor cell clones in flow cytometry. Furthermore, by conjugating them with radioisotopes, tumor extent can be evaluated using imaging techniques (scintigraphy, PET, etc.) to eliminate the possibility of nonspecific binding to tissues other than tumor cells. Finally, these can be used to evaluate minimal residual disease at the end of treatment.

[0125] From a therapeutic standpoint, scFv and all the molecules derived therefrom (especially monoclonal antibodies) may be used as is, or conjugated with chemotherapeutic agents, toxins, or radioisotopes. Alternatively, they may be loaded onto liposomes or other types of nanoparticles to deliver pharmacologically active molecules to cancer cells.

[0126] Finally, scFv is any type of chimeric antigen receptor (CAR), namely: a) Typical CAR-T models that are commercially available or under development; b) Atypical CAR-T cells that express one of several Fc receptors (CD16, CD32, CD64) for immunoglobulins on the surface of T lymphocytes (autologous, haploid, allogeneic, or stable cell lines). In achieving this, it can be used as a domain for antigen recognition.

[0127] The following examples are for illustrative purposes only and do not limit the application areas of the six scFvs whose CDR and FR sequences are listed in Table 12, and the aforementioned molecules derived therefrom (in particular, monoclonal antibodies and CARs).

[0128] Example 1 - Monoclonal antibody Typically, monoclonal antibodies used for therapeutic purposes bind to tumor-associated antigens (TAAs) on the cell membranes of various types of malignant tumors, inducing opsonization of tumor cells and activating antibody-dependent cell-mediated cytotoxicity (ADCC). In other cases, monoclonal antibodies bind to membrane receptors, interfering with activation by physiological ligands.

[0129] One example of the first type is rituximab (MabThera), an IgG1-class chimeric mouse / human monoclonal antibody that recognizes the CD20 molecule expressed on the surface of B lymphocytes and is currently used to treat B-cell non-Hodgkin lymphoma. Unfortunately, rituximab binds to both healthy T lymphocytes and cancer cells. Therefore, treatment with this antibody results in the complete depletion of the adaptive immune B-cell compartment.

[0130] A second example is represented by trastuzumab (Herceptin), which is a humanized monoclonal antibody of the IgG1 class that recognizes HER2, a tyrosine kinase receptor (HER) of the ErbB family that is overexpressed in 25–30% of breast cancer patients. Yet another example is cetuximab (Erbitux), which is a chimeric monoclonal antibody of the IgG1 class that binds to the epidermal growth factor (EGF) receptor and inhibits its activation.

[0131] To verify that the lead compounds identified by SPR effectively recognize tumor cells from TCL patients expressing the TRBV5-1 fragment on the TCRβ chain, we conducted in-depth flow cytometry studies.

[0132] Flow cytometry is a widely used and established technique in laboratory practice that allows for the characterization of the quality and quantity of cell suspensions. In particular, it allows for the simultaneous examination of physical and immunological parameters regarding the expression of specific antigens on the cell membrane, which can be detected by pre-incubating the cell suspension with a specific monoclonal antibody conjugated with a fluorescent dye. A laser light source illuminates single cells flowing into a microfluidic system. The light scattered by the cells ("scattered light") provides two types of information: "forward scattered light" (FSC) provides an indicator of cell size, and "side scattered light" (SSC) provides information about the granularity of the cytoplasm (allowing for the distinction between granulocytes and other types of leukocytes). Finally, fluorescence emission (depending on the fluorescent dye conjugated to the monoclonal antibody used for pre-incubation of the cell suspension) allows for the identification of antigens expressed on the surface of single cells.

[0133] Multi-parameter flow cytometry was performed on peripheral blood mononuclear cell (PBMC) samples collected from two patients with Sézary syndrome, a severe form of CTCL. One patient expressed the TRBV5-1 fragment in the variable region of the TCRβ chain, while the other patient expressed one of three fragments (TRBV6-5, TRBV6-6, or TRBV6-9) recognized by the IOTest® Beta Mark (Beckman Coulter) anti-Vb13.1 antibody. A "gating" strategy (i.e., composite analysis of specific cell populations identified by monoclonal antibodies) was used to identify tumor cells in Sézary syndrome, namely CD3+, CD4+, CD8-, and CD45+ cells. This combination was enriched with an additional monoclonal antibody (CD14) that binds to monocytes, allowing for the exclusion of monocytes from the population under study. This was necessary to eliminate the possibility of nonspecific binding, as the lead compound is an IgG1 class monoclonal antibody that binds to Fc receptors against immunoglobulins on the monocyte membrane.

[0134] In the first stage of the experiment, the dilution of lead compound A12 was gradually increased and tested (5 μl, 1 μl, and 0.5 μl of antibody were added to 1 ml of cell suspension at a concentration of 1 mg / ml). The results of this first stage showed that the addition of 0.5 μl of A12 antibody produced an optimal signal, but higher amounts (1 μl and 5 μl) produced abnormally high signals.

[0135] After determining the optimal A12 antibody concentration, two tests were performed using the fluorescence minus one (FMO) method, which involves comparing the entire antibody panel with the A12 lead compound added to the entire antibody panel without the A12 lead compound added.

[0136] The first trial was conducted using PBMCs (Primary Biomedical Cells) isolated from patients with Sézary syndrome expressing the TRBV5-1 fragment in the variable region of the TCRβ chain. The results showed that the A12 lead compound bound to 21% of the cell population in the trial.

[0137] The second trial was conducted using PBMC suspensions collected from Sézary syndrome patients expressing TRBV6-5, TRBV6-6, or TRBV6-9 fragments recognized by the IOTest® Beta Mark (Beckman Coulter) anti-Vb13.1 antibody. The results of the trial showed that the A12 lead compound did not bind to any of the cells in the cell population under study.

[0138] The flow cytometry results support the SPR results, which indicate that the A12 monoclonal antibody selectively and with high affinity binds to the TRBV5-1 fragment expressed in the variable region of the TCRβ chain.

[0139] Example 2 - CAR CARs are membrane receptors that do not exist naturally and are created in the laboratory by combining segments of one gene with segments of another. When expressed in T lymphocytes (CAR-T) or any other cell in the immune system, such as natural killer cells (NK), they redirect the specificity and function of those cells. CAR-Ts are considered "living drugs" realized through an extremely complex procedure. First, normal T lymphocytes are isolated from the patient or a suitable donor by apheresis. Next, the T lymphocytes are transfected, meaning that a construct (plasmid) containing the information necessary for CAR production is introduced into the T lymphocytes using a viral vector or an alternative technique (such as electroporation). Subsequently, the transfected T lymphocytes are grown (expanded) in vivo and then injected back into the patient.

[0140] In principle, any molecule on the surface of tumor cells can be a target for CAR-T receptors. Compared to TCRs, CARs recognize antigens without the limitations associated with individual differences in MHC alleles, and without the need for the target protein to be pre-processed into antigen peptides within the target cell. Thanks to these characteristics, CAR-T receptors can kill cancer cells with low MHC molecule expression or low proteasome activity (two mechanisms that may contribute to the lack of recognition of cancer cells by the immune system). On the other hand, CARs recognize only membrane antigens, while TCRs can also recognize antigen peptides obtained through the processing of intracellular proteins.

[0141] As prior art has shown, the antigen-recognition domain of CARs can be of three different types: scFv, Fab, or ligands that recognize intrinsic receptors (Sadelain M, Brentjens R, Riviere I. The basic principles of chimeric antigen receptor design. Cancer Discov. 2013 Apr;3(4):388-98). Initially, the use of mouse scFv resulted in immunogenic CARs that induced specific immune responses in the host, impairing their efficacy. Nevertheless, the introduction of phage libraries expressing fully human scFv has made it possible to overcome this problem.

[0142] The first patent relating to the industrial development of CARs was filed on December 9, 2010, by Carl June et al. ("Use of chimeric antigen receptor-modified T cells to treat cancer" - WO2012 / 079000A1). The authors reported that CD19 had previously been identified as a potential target for immunotherapy of malignancies caused by B lymphocytes. Although several anti-CD19CARs were developed, they did not proliferate (expand) in vivo, rapidly disappeared after injection into patients, and their clinical activity was unsatisfactory.

[0143] The patent relates to a construct for CAR expression (its nucleotide sequence is identified as SEQ ID NO: 8), which preferably comprises, but not limited to, an antigen-binding domain that recognizes CD19 on the surface of B lymphocytes (an scFV obtained from an anti-CD19 mouse monoclonal antibody called FMC63, identified as SEQ ID NO: 14 in the patent); a hinge domain and a transmembrane domain (both obtained from human CD8, identified as SEQ ID NO: 15 and SEQ ID NO: 16 in the patent, respectively); a co-stimulatory region preferably represented by protein 4-1BB (identified as SEQ ID NO: 17 in the patent), which plays a fundamental role in maintaining the CAR-T antitumor response; and finally, a cytoplasmic domain (identified as SEQ ID NO: 18 in the patent) obtained from CD3ζ, which is responsible for transmitting the intracellular activation signal (to which the cytoplasmic domain of the aforementioned co-stimulatory molecule may be added). The position of the antigen-recognition domain can be optimized by adding a "spacer" domain to these domains.

[0144] As explicitly stated in the patent, the antigen-recognition domain is preferably an scFv or Fab specific to a certain number of antigens present on the lymphocyte membrane. The scFv preferably recognizes the CD19 molecule on the B lymphocyte membrane, but the inventors also claim other CARs that recognize potential antigens on the tumor cell membrane, such as CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, and B7H3. However, the patent does not mention the variable region of the TCR, particularly the variable region of the β chain of the TCR. As will be apparent to those skilled in the art, the nucleotide / amino acid sequence of scFv obtained from an anti-CD19 mouse monoclonal antibody called FMC63, which is the subject of WO2012 / 079000A1, can be replaced with any of the nucleotide / amino acid sequences of the six scFVs covered by this patent application, or with any of the nucleotide / amino acid sequences of any of the variants having 70% or more homology to any of the six scFVs covered by this patent application. This technique is widely known, and many companies offer the design and synthesis of constructs starting from three CDRs and four FRs of scFv on a contract (outsourced) basis.

[0145] The primary B-cell tumors identified as potential targets for CAR-T as described in WO2012 / 079000A1 are chronic lymphocytic leukemia and lymphoma. The construct for expressing CAR is transferred to immune system cells (preferably T lymphocytes) by various techniques, but preferably by lentiviral vectors (pELPs 19BBz). This therapy was tested in three patients with chronic lymphocytic leukemia aged 50 to 68 years and resulted in complete remission of the disease.

[0146] A second patent filed subsequently (US2014 / 0271635A1) describes a humanized anti-CD19 scFv with lower immunogenicity than the previous anti-CD19 mouse scFv. These two patents (WO2012 / 079000A1 and US2014 / 0271635A1) enabled the development of Kymriah (Tisagenlecleucel), a drug approved by major global regulatory bodies for the treatment of childhood acute lymphoblastic leukemia (B cell acute lymphoblastic leukemia) and adult large B cell lymphoma.

[0147] The third patent that initiated the industrial development of CARs was filed on November 16, 2017, by A. Sievers and Jed JW Wiltzius ("Modified chimeric antigen receptors and methods of use" - US2019 / 0144515A1). The authors discovered that introducing specific modifications to the amino acid sequences of the hinge domain, transmembrane domain, and intracellular domain induces substantial and unexpected changes in the activity of the corresponding CAR-T receptors. The first modification is the removal of an N-linked glycosylation site in the hinge domain, which significantly increases CAR-T proliferation. The second modification is a mutation in the transmembrane domain that favors dimerization on the cell membrane. Data presented by the inventors show that CAR-T proliferation is greater when the dimeric construct is used instead of the monomeric construct. The third modification is a mutation in the transmembrane domain that regulates cytokine production. Overall, these findings constitute a groundbreaking leap that led to the granting of patents beyond the aforementioned patents WO2012 / 079000A1 and US2014 / 0271635A1. This patent formed the basis for the development of Yescarta (axicabtagene ciloleucel), approved as a treatment for large B-cell lymphoma in adults, and Tecartus (brexucabtagene autoleucel), approved as a treatment for mantle cell lymphoma and large B-cell lymphoma. It should be noted that this patent does not mention the possibility of using the variable region of the TCR, particularly the variable region of the β-chain of the TCR, as a target for CAR. As described above, it will be apparent to those skilled in the art that adding any of the six scFvs according to the present invention, and any variant having 70% or more homology to any of the six scFvs according to the present invention, confers the properties of recognizing and destroying tumor cells in patients with T-cell tumors expressing the TRBV5-1 fragment in the TCRβ chain to the CAR identified in US2019 / 0144515A1.

[0148] A third patent that facilitated the industrial production of CARs was filed on November 1, 2017, by Archana Brahmandam et al. ("Process for producing a T cell composition" - WO2019 / 090004A1). The authors begin with the consideration that existing CAR-T based therapies needed further optimization. Their invention involves adding an mTOR (mammalian target of rapamycin) inhibitor to the CAR and / or co-stimulatory molecule, which, when administered to a patient, enhances the survival and activity of CAR-T cells. This patent forms the basis of Breyanzi (lisocabtagene maraleucel), which was recently approved by the FDA as a treatment for large B-cell lymphoma. As with the aforementioned cases, the possibility of using the variable region of the TCR, particularly the variable region of the β-chain of the TCR, as a target for CARs is not mentioned. As already emphasized, it will be apparent to those skilled in the art that adding the nucleotide / amino acid sequence of any of the six scFvs that are the subject of the present invention, and any variant having 70% or more homology to any of the six scFvs according to the present invention, confers the properties to the CARs identified in WO2019 / 090004A1 that enable them to recognize and destroy tumor cells in patients with T-cell tumors expressing the TRBV5-1 fragment in the TCRβ chain.

[0149] Two further patents describe compositions and methods for producing CAR-T cells, but to our knowledge, neither of them has been registered as a pharmaceutical product.

[0150] The first patent was filed on May 12, 2014, by Roman Galletto et al. ("CD19 specific chimeric antigen receptor and uses thereof" - WO2014 / 184143A1). The authors demonstrated that a CAR constructed using an antigen-binding domain obtained from an anti-CD19 monoclonal antibody called 4G7 increased the activation state of CAR-T compared to a CAR having an antigen-binding domain obtained from an scFv called FMC63, as described in WO2012 / 079000A1. In particular, the antigen-binding domain obtained from the monoclonal antibody 4G7 maintained CAR-T activation independently of antigen recognition. Therefore, this patent relates only to CD19-recognizing CARs and does not consider other targets on the tumor cell membrane.

[0151] The second of these two patents was filed on November 22, 2019, by Rimas J. Orentas et al. ("Compositions and methods for treating cancer with DuoCARs" - US2020 / 0231648A1). The authors describe in detail the different types of CARs developed to date, distinguishing between single CARs, split CARs, tandem CARs, and multiple CARs using a single vector.

[0152] A single CAR has one single antigen-binding domain, which is connected to a single hinge / transmembrane domain, and this hinge / transmembrane domain is further connected to a single intracellular domain containing CD3ζ and a co-receptor (4-1BB or CD28).

[0153] Split CARs have two distinct antigen-binding domains. The first antigen-binding domain is connected via a hinge / transmembrane domain to an intracellular domain containing CD3ζ, while the second antigen-binding domain is connected (similarly via a hinge / transmembrane domain) to an intracellular domain derived from a costimulatory molecule (4-1BB or CD28).

[0154] A tandem CAR has two domains that bind to two different antigens, each connected to a single hinge / transmembrane domain, and the hinge / transmembrane domain is further connected to a single intracellular domain containing CD3ζ and a costimulatory molecule (4-1BB or CD28).

[0155] Multiple CARs with one single vector consist of a structure in which two antigen-binding domains are connected to two different hinge / transmembrane domains, which are further connected to two different intracellular domains containing CD3ζ and a co-stimulatory molecule (4-1BB or CD28).

[0156] The inventors found that transfecting autologous T lymphocytes from tumor patients with two or three vectors expressing multiple CARs was far more effective than previously reported CARs. Since each CAR can be single (i.e., having only one antigen-binding domain) or multiple (i.e., having two or more antigen-binding domains), DuoCARs can simultaneously recognize three or more antigens on the surface of tumor cells. In the treatment of B-cell malignancies, DuoCARs can simultaneously recognize CD19, CD20, and CD22, reducing the risk of clones developing that do not express one of the antigens targeted by immunotherapy, thereby preventing cancer from escaping CAR-T therapy. This patent also does not mention the possibility of using the variable region of the TCR, particularly the variable region of the β-chain of the TCR, as a target for CARs. As already stated, it will be apparent to those skilled in the art that adding the nucleotide / amino acid sequence of any of the six scFvs that are the subject of the present invention, and any variant having 70% or more homology to any of the aforementioned six scFvs, confers the properties of recognizing and killing cancer cells in T-cell tumors expressing the TRBV5-1 fragment in the TCRβ chain to the CAR identified in WO2019 / 090004A1.

[0157] One of the most common reasons for CAR-T therapy failure is the accidental transfection of a single tumor lymphocyte that is mistakenly isolated during apheresis. Administering a CAR-T containing a single tumor lymphocyte expressing a CAR to a patient impairs treatment outcomes. To avoid this risk (which is even greater in the treatment of T-cell malignancies, as tumor T lymphocytes can be readily isolated alongside normal T lymphocytes), the following can be done: a) If the cancer cells are CD4+, use autologous (i.e., patient-derived) CD8+ T cells; b) If the cancer cells are CD8+, use autologous (i.e., CD4+ T cells taken from the patient); c) Use T lymphocytes (CD4+ or CD8+) collected from a haploidentical donor (parent or sibling); d) Use autologous NK lymphocytes (which spontaneously express low affinity immunoglobulin Fc receptor CD16) and those obtained from haploidentical donors; e) Use any other type of lymphocyte derived from the patient's own body or from a haploid donor (e.g., NKT lymphocytes or Tγ / δ lymphocytes, etc.); f) Use lymphocytes obtained by maturation from embryonic stem cells (hESCs) or iPS cells (induced pluripotent stem cells): g) Use cells obtained from the umbilical cord; h) For example, but not limited to, commercially available stable cell lines such as NK-92, KHYG-1, NKL, NKG, and YT cells may be used.

[0158] [Table 1]

[0159] [Table 2]

[0160] [Table 3]

[0161] [Table 4]

[0162] [Table 5]

[0163] [Table 6]

[0164] [Table 7]

[0165] Table 8

[0166] Table 9

[0167] Table 10

[0168] Table 11

[0169] Table 12

[0170] Table 13

[0171] Table 14

[0172] Table 15

[0173] Table 16

Claims

1. A single-chain variable region fragment (scFv) comprising complementarity-determining regions (CDR1, CDR2, CDR3) and framework regions (FR1, FR2, FR3, FR4) that can bind to the peptide TQTPRYLIKTRGQQ located within the β-chain fragment TRBV5-1 of human TCR, and which can be used for diagnostic or therapeutic purposes in the treatment of pathological conditions caused by clonal proliferation of T lymphocytes.

2. The complementarity determination regions (CDR1, CDR2, CDR3) and the framework regions (FR1, FR2, FR3, FR4) have the following configurations for the heavy chain (VH) and light chain (VL): A single-chain variable region fragment according to claim 1, having the above characteristics.

3. A single-chain variable region fragment (scFv) comprising complementarity-determining regions (CDR1, CDR2, CDR3) and framework regions (FR1, FR2, FR3, FR4) that can bind to the peptide TQTPRYLIKTRGQQ located within the β-chain fragment TRBV5-1 of human TCR, wherein the scFv comprises a nucleotide / amino acid sequence that is at least 70% identical to the amino acid / nucleotide sequence of the scFv described in claim 1 or 2.

4. A peptide / protein such as a Fab fragment, Fd fragment, Fv fragment, dAb fragment, a heavy chain single CDR (CDR1, CDR2, or CDR3), or a light chain single CDR (CDR1, CDR2, or CDR3), an F(ab)2 fragment, a diabody, or various subclasses of human monoclonal antibodies, i.e., IgA (monomer or dimer); IgD; IgE; IgG (subclasses IgG1, IgG2, IgG3, or IgG4); IgM (monomer, pentamer, or hexamer), comprising the amino acid / nucleotide sequence of scFv as described in any one of claims 1 to 3.

5. A chimeric antigen receptor (CAR) comprising an amino acid / nucleotide sequence of an antigen-binding domain containing the amino acid / nucleotide sequence of the scFv described in any one of claims 1 to 3.

6. A chemotherapy agent, toxin, or radioisotope conjugated with a peptide / protein containing the amino acid / nucleotide sequence of scFv as described in any one of claims 1 to 3.

7. Nanoparticles, nanospheres, nanocapsules, nanocarriers, and liposomes carrying peptides / proteins containing the amino acid / nucleotide sequence of scFv described in any one of claims 1 to 3.

8. A product for diagnostic and / or therapeutic use in human pathological conditions resulting from clonal proliferation of T lymphocytes, comprising at least one of claims 1 to 7, the scFv and / or peptides / proteins and / or chimeric antigen receptors (CARs) and / or chemotherapeutic agents and / or toxins and / or radioisotopes and / or nanoparticles.

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