Anti-TRBC1 antigen binding domains

Abstract

To provide new methods for targeted treatment of T cell malignancy.SOLUTION: The invention provides agents, such as chimeric antigen receptors (CARs) which selectively bind TRBC1. Such agents are useful in methods for treating a T-cell lymphoma or leukemia in a subject. T cell malignancies are clonal, so they either express TRBC1 or TRBC2. By administering a TCRB1 selective agent to the subject, the agent causes selective depletion of the TRBC1-expressing malignant T-cells, together with TRBC1-expressing normal T-cells, but does not cause depletion of TRBC2-expressing normal T-cells.SELECTED DRAWING: None

Description

Technical Field

[0001] Field of the Invention The present invention relates to an agent useful in the treatment of T cell lymphoma or leukemia.

Background Art

[0002] Background of the Invention Lymphoid malignancies can be broadly divided into those derived from T cells or those derived from B cells. T cell malignancies are a group of clinically and biologically heterogeneous disorders, together accounting for 10-20% of non-Hodgkin lymphomas and 20% of acute leukemias. The most commonly identified histological subtypes are peripheral T cell lymphoma, not otherwise specified (PTCL-NOS); angioimmunoblastic T cell lymphoma (AITL) and anaplastic large cell lymphoma (ALCL). Twenty percent of all acute lymphoblastic leukemias (ALL) have a T cell phenotype.

[0003] These conditions generally behave more aggressively, for example, compared to B cell malignancies, and the estimated 5-year survival rate is only 30%. In the case of T cell lymphoma, it is associated with a high proportion of patients with disseminated disease, an unfavorable International Prognostic Indicator (IPI) score, and a high incidence of extranodal disease. Chemotherapy alone is usually not effective, and less than 30% of patients are cured with current treatments.

[0004] Furthermore, unlike B cell malignancies, whose outcome is dramatically improved by immunotherapy such as the anti-CD20 monoclonal antibody rituximab, there are currently no immunotherapeutic drugs available for treating T cell malignancies that are equally effective and have minimal toxicity. An important difficulty in the development of immunotherapy for T cell disorders is that the marker expression of clonal T cells and normal T cells overlaps considerably, and there is no single antigen that can clearly identify clonal (malignant) cells.

[0005] The same problem exists when targeting pan-B cell antigens for treating B cell malignancies. However, in this case, simultaneous depletion of the B cell compartment results in relatively mild immunosuppression that is more readily tolerated by the majority of patients. Furthermore, in therapies where depletion of the normal B compartment occurs, particularly over a long period, administration of pooled immunoglobulin can significantly suppress the loss of the normal B compartment. The situation is quite different when targeting T cell malignancies. In this case, simultaneous depletion of the T cell compartment results in severe immunosuppression and severe toxicity. Furthermore, there is no satisfactory way to reduce the loss of the T cell compartment.

[0006] Toxicity is exemplified in part by the clinical effect of the therapeutic monoclonal antibody alemtuzumab. This agent lyses cells expressing CD52 and has some efficacy in T cell malignancies. The usefulness of this agent is severely limited by the profound cellular immunodeficiency largely resulting from T cell depletion and the markedly increased risk of infection.

[0007] Therefore, there is a need for new methods for the targeted treatment of T cell malignancies that do not have the above disadvantages. Summary of the Invention Means for Solving the Problems

[0008] Summary of Aspects of the Invention The inventors have developed a series of humanized antigen-binding domains that bind to human TRBC1. The antigen-binding domains can be used in various therapeutic formats including chimeric antigen receptors (CARs), therapeutic antibodies, antibody-drug conjugates (ADCs), and bispecific T cell engagers (BiTEs) to deplete malignant TRBC1-expressing T cells in a subject without affecting healthy TRBC2-expressing T cells.

[0009] Accordingly, in a first aspect, the invention provides a) A VH domain having an amino acid sequence selected from SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18; and b) A VL domain having an amino acid sequence selected from SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 Provided is an anti-TRBC1 antigen-binding domain comprising the same.

[0010] In a second aspect, the present invention provides a chimeric antigen receptor (CAR) comprising an anti-TRBC1 antigen-binding domain according to the first aspect of the present invention.

[0011] In a third aspect, the present invention provides an antibody comprising an anti-TRBC1 antigen-binding domain according to the first aspect of the present invention.

[0012] In a fourth aspect, the present invention provides a bispecific T cell engager (BiTE) comprising an anti-TRBC1 antigen-binding domain according to the first aspect of the present invention.

[0013] In a fifth aspect, the present invention provides an antibody-drug conjugate comprising an anti-TRBC1 antigen-binding domain according to the first aspect of the present invention.

[0014] In a sixth aspect, the present invention provides a nucleic acid sequence encoding a CAR according to the second aspect of the present invention.

[0015] In a seventh aspect, the present invention provides a vector comprising the nucleic acid sequence according to the sixth aspect of the present invention.

[0016] In an eighth aspect, the present invention provides a cell comprising a CAR according to the second aspect of the present invention.

[0017] In a ninth aspect, the present invention provides a method for producing cells according to the eighth aspect of the present invention, the method comprising the step of introducing a nucleic acid according to the sixth aspect of the present invention or a vector according to the seventh aspect of the present invention into a cell.

[0018] In a tenth aspect, the present invention provides a pharmaceutical composition comprising a plurality of cells according to the eighth aspect of the present invention, an antibody according to the third aspect of the present invention, a BiTE according to the fourth aspect of the present invention, or an antibody-drug conjugate according to the fifth aspect of the present invention.

[0019] In an eleventh aspect, the present invention provides a pharmaceutical composition according to the tenth aspect of the present invention for use in the treatment of TRBC1-expressing T cell lymphoma or leukemia in a subject.

[0020] In a twelfth aspect, the present invention provides a method for treating TRBC1-expressing T cell lymphoma or leukemia in a subject, the method comprising the step of administering to the subject a pharmaceutical composition according to the tenth aspect of the present invention.

[0021] In a thirteenth aspect, the present invention provides the use of a pharmaceutical composition according to the tenth aspect of the present invention in the manufacture of a medicament for the treatment of TRBC1-expressing T cell lymphoma or leukemia in a subject.

[0022] TRBC1-expressing T cell lymphoma or leukemia may be selected from peripheral T cell lymphoma not otherwise specified (PTCL-NOS); angioimmunoblastic T cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), enteropathy-associated T cell lymphoma (EATL), hepatosplenic T cell lymphoma (HSTL), extranodal NK / T cell lymphoma, nasal type, cutaneous T cell lymphoma, primary cutaneous ALCL, T cell prolymphocytic leukemia, and T cell acute lymphoblastic leukemia. In certain embodiments, for example, the following are provided: (Item 1) a) A VH domain having an amino acid sequence selected from SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18; and b) A VL domain having an amino acid sequence selected from SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 An anti-TRBC1 antigen-binding domain comprising the above. (Item 2) A chimeric antigen receptor (CAR) comprising the anti-TRBC1 antigen-binding domain according to Item 1. (Item 3) An antibody comprising the anti-TRBC1 antigen-binding domain according to Item 1. (Item 4) A bispecific T cell engager (BiTE) comprising the anti-TRBC1 antigen-binding domain according to Item 1. (Item 5) An antibody-drug conjugate comprising the anti-TRBC1 antigen-binding domain according to Item 1. (Item 6) A nucleic acid sequence encoding the CAR according to Item 2. (Item 7) A vector comprising the nucleic acid sequence according to Item 6. (Item 8) A cell comprising the CAR according to Item 2. (Item 9) A method for producing the cell according to Item 8, the method comprising introducing the nucleic acid according to Item 6 or the vector according to Item 7 into the cell. (Item 10) A pharmaceutical composition comprising the plurality of cells according to Item 8, the antibody according to Item 3, the BiTE according to Item 4, or the antibody-drug conjugate according to Item 15. (Item 11) The pharmaceutical composition according to Item 10 for use in the treatment of TRBC1-expressing T cell lymphoma or leukemia in a subject. (Item 12) A method for treating TRBC1-expressing T cell lymphoma or leukemia in a subject, the method comprising administering the pharmaceutical composition according to item 10 to the subject. (Item 13) Use of the pharmaceutical composition according to item 10 in the manufacture of a medicament for the treatment of TRBC1-expressing T cell lymphoma or leukemia in a subject. (Item 14) The pharmaceutical composition for use according to item 11, the method according to item 12, or the use according to item 13, wherein the TRBC1-expressing T cell lymphoma or leukemia is selected from peripheral T cell lymphoma not otherwise specified (PTCL-NOS); angioimmunoblastic T cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), enteropathy-associated T cell lymphoma (EATL), hepatosplenic T cell lymphoma (HSTL), extranodal NK / T cell lymphoma nasal type, cutaneous T cell lymphoma, primary cutaneous ALCL, T cell prolymphocytic leukemia, and T cell acute lymphoblastic leukemia.

Brief Description of the Drawings

[0023]

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Mode for Carrying Out the Invention

[0024] Detailed Description The present invention provides an agent such as a chimeric antigen receptor (CAR) that selectively binds to TRBC1. Such an agent is useful in a method for treating T cell lymphoma or leukemia in a subject. T cell malignancies are clonal and thus express TRBC1 or TRBC2. By administering a TCRB1-selective agent to a subject, the agent causes selective depletion of malignant T cells expressing TRBC1 and normal T cells expressing TRBC1, but does not cause depletion of normal T cells expressing TRBC2.

[0025] TCRβ constant region (TRBC) The T cell receptor (TCR) is expressed on the surface of T lymphocytes and is involved in the recognition of antigens bound to major histocompatibility complex (MHC) molecules. When the TCR associates with an antigenic peptide and MHC (peptide / MHC), the T lymphocyte is activated through a series of biochemical events mediated by associated enzymes, co-receptors, special adapter molecules, and activated or released transcription factors.

[0026] The TCR is a disulfide-linked membrane-anchored heterodimer, usually consisting of highly variable alpha (α) and beta (β) chains that are expressed as part of a complex with invariant CD3 chain molecules. T cells expressing this receptor are called α:β (or αβ) T cells (about 95% of total T cells). A minority of T cells express an alternative receptor formed by variable gamma (γ) and delta (δ) chains, and these are called γδ T cells (about 5% of total T cells).

[0027] Each α and β chain is composed of two extracellular domains: a variable (V) region and a constant (C) region, both of which are immunoglobulin superfamily (IgSF) domains that form antiparallel β-sheets. The constant region is proximal to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail, and the variable region binds to the peptide / MHC complex (see Figure 1). The constant region of the TCR consists of a short connecting sequence where cysteine residues form disulfide bonds, thereby forming a link between the two chains.

[0028] The variable domains of the TCR α and β chains both have three hypervariable or complementarity-determining regions (CDRs). The variable region of the β chain also has additional hypervariability in region HV4, which usually does not contact the antigen and is therefore not considered a CDR.

[0029] The TCR also includes up to five invariant chains γ, δ, ε (collectively called CD3) and ζ. The CD3 and ζ subunits mediate TCR signaling through specific cytoplasmic domains that interact with second messengers and adapter molecules after antigen recognition by αβ or γδ. Prior to cell surface expression of the TCR complex, the subunits assemble in pairs, where both the transmembrane and extracellular domains of TCR α and β and CD3 γ and δ play roles.

[0030] Thus, the TCR generally consists of the CD3 complex, the TCR α and β chains, which in turn are composed of variable and constant regions (Figure 1).

[0031] The locus (Chr7:q34) that supplies the TCR β constant region (TRBC) has been duplicated during the course of evolution, giving rise to two nearly identical and functionally equivalent genes: TRBC1 and TRBC2 (Figure 2), which differ by only 4 amino acids within the mature proteins arising from each gene (Figure 3). Each TCR contains either TRBC1 or TRBC2 mutually exclusively, and thus, each αβ T cell expresses either TRBC1 or TRBC2 mutually exclusively.

[0032] Despite the similarity between the sequences of TRBC1 and TRBC2, it is possible to distinguish them. The amino acid sequences of TRBC1 and TRBC2 can be discriminated in situ on the surface of cells, such as T cells.

[0033] Antigen-binding domain The present invention provides a humanized anti-TRBC1 antigen-binding domain having a variable heavy chain (VH) and a variable light chain (VL) comprising the following complementarity-determining regions (CDRs): VH CDR1: GYTFTGY (SEQ ID NO: 1); VH CDR2: NPYNDD (SEQ ID NO: 2); VH CDR3: GAGYNFDGAYRFFDF (SEQ ID NO: 3); VL CDR1: RSSQRLVHSNGNTYLH (SEQ ID NO: 4); VL CDR2: RVSNRFP (SEQ ID NO: 5); and VL CDR3: SQSTHVPYT (SEQ ID NO: 6).

[0034] The antigen-binding domain includes a human framework region, or a human framework region having one or more mutations. For example, the framework region(s) may include one or more substitutions as compared to the human framework region sequence. The substitution(s) may be "back mutations" in which one or more amino acids are substituted with equivalent residues from a mouse antibody sequence. The mouse antibody variable heavy chain (VH) sequence is shown below as SEQ ID NO: 7, and the variable light chain (VL) sequence is shown as SEQ ID NO: 8. In both sequences, the CDR sequences are shown in bold and underlined.

Chemical formula

[0035] The humanized VH sequence containing the mouse JOVI-1 CDRs shown in SEQ ID NOs: 1, 2 and 3 may have 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2, or 1 mutation as compared to the wild-type human framework region sequence.

[0036] The humanized VL sequence containing the mouse JOVI-1 CDRs shown in SEQ ID NOs: 4, 5 and 6 may have 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2, or 1 mutation as compared to the wild-type human framework region sequence.

[0037] The VH sequence may include the JOVI-1 VH CDR having the human framework H-AF062256. This sequence is shown as SEQ ID NO: 9. The CDR sequence is underlined.

Chemical formula

[0038] The VH sequence may include the JOVI-1 VH CDR having the human framework H-EF177999. This sequence is shown as SEQ ID NO: 10. The CDR sequence is underlined.

Chem.

[0039] The VH sequence may include the JOVI-1 VH CDR having the human framework H-KF688165. This sequence is shown by SEQ ID NO: 11. The CDR sequences are underlined.

Chem.

[0040] The VH sequence may include the sequences shown by SEQ ID NO: 9, 10 or 11 having one or more mutations such as revertant mutations. The VH sequence may have 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2, or 1 mutation compared to the wild-type human framework region sequence. For example, the VH sequence may include the sequence shown by SEQ ID NO: 9 having one of the sets of revertant mutations shown in Table 1 of the Examples.

[0041] The VH sequence may include one of the sequences shown by SEQ ID NO: 12 - 18. The CDR sequences are underlined and the revertant mutations are shown in bold.

Chem.

Chem.

[0042] The VL sequence may include the JOVI-1 VL CDR having the human framework 3aaz. This sequence is shown by SEQ ID NO: 19. The CDR sequences are underlined.

Chem.

[0043] The VL array may include the array shown in SEQ ID NO: 19, which has one or more mutations such as revertant mutations. The VL array may have 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2, or 1 mutation compared to the wild-type human framework region array. For example, the VL array may include one of the arrays shown in SEQ ID NOs: 20-34. The CDR sequences are underlined and the revertant mutations are shown in bold. [Chemical formula] [Chemical formula] [Chemical formula]

[0044] The anti-TRBC1 antigen-binding domain may a) a VH domain comprising a JOVI-1 VH CDR having a human framework H-AF062256 or a variant thereof; and b)) a VH domain comprising a JOVI-1 VL CDR having a human framework 3aaz or a variant thereof and may include.

[0045] The variant may have 5 or fewer, 4 or fewer, 3 or fewer, 2 or 1 mutation compared to the wild-type human framework region array.

[0046] The VH domain may comprise the sequence shown by SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18. The VL domain may comprise the sequence shown by SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34.

[0047] The anti-TRBC1 antigen-binding domain a) a VH domain comprising the sequence shown by SEQ ID NO: 9; and b) a VL domain comprising the sequence shown by SEQ ID NO: 19 may comprise.

[0048] antibody The antigen-binding domain of the first aspect of the present invention may be an antibody or a functional fragment thereof. The antibody may be a therapeutic antibody such as a depleting antibody. The antibody may be a bispecific antibody that binds to TRBC1 and another antigen. The antibody may be, for example, a bispecific affinity retargeting antibody.

[0049] The term "depleting antibody" is used in its conventional meaning and relates to an antibody that binds to an antigen present on target T cells (i.e., TRBC1) and mediates the death of the target T cells. Thus, administration of a depleting antibody to a subject results in a decrease / reduction in the number of cells expressing the target antigen within the subject.

[0050] As used herein, "antibody" means a polypeptide having an antigen-binding site that includes at least one complementarity determining region (CDR). An antibody can include three CDRs and have an antigen-binding site equivalent to that of a domain antibody (dAb). An antibody can include six CDRs and have an antigen-binding site equivalent to that of a classical antibody molecule. The remainder of the polypeptide can be any sequence that provides a suitable scaffold for the antigen-binding site and displays it in a manner suitable for binding to an antigen. An antibody can be an entire immunoglobulin molecule or a portion thereof that retains the antigen specificity of the whole antibody, such as a Fab, F(ab)'2, Fv, single-chain Fv (ScFv) fragment, and scFv-Fc fusion or diabody, triabody or nanobody. An antibody can be a bifunctional antibody. An antibody can be a non-human antibody, a chimeric antibody, a humanized antibody or a fully human antibody.

[0051] Conjugate An antibody can be an antibody or a conjugate of an antibody with another effector, for example, the conjugate can be a detectable entity or an entity for chemotherapy.

[0052] The detectable entity can be a fluorescent moiety, such as a fluorescent peptide. "Fluorescent peptide" refers to a polypeptide that emits light at a detectable wavelength after excitation. Examples of fluorescent proteins include, but are not limited to, fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), green fluorescent protein (GFP), enhanced GFP, red fluorescent protein (RFP), blue fluorescent protein (BFP) and mCherry.

[0053] As used herein, a chemotherapeutic entity refers to an entity that is destructive to cells, i.e., the viability of the cells is reduced by the entity. A chemotherapeutic entity may be a cytotoxic drug. Intended chemotherapeutic agents include, but are not limited to, alkylating agents, nitrosoureas, ethyleneimine / methylmelamine, alkyl sulfonates, antimetabolites, pyrimidine analogs, epipodophylotoxins, enzymes such as L-asparaginase; biological response modifiers such as IFNα, IL-2, G-CSF, and GM-CSF; platinum coordination complexes such as cisplatin and carboplatin, substituted ureas such as anthraquinone and hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine, adrenocortical suppressants such as mitotane (o,p'-DDD) and aminoglutethimide; hormones and antagonists including adrenocortical steroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate; estrogens such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogen drugs such as tamoxifen; androgens including testosterone propionate and fluoxymesterone / equivalents; antiandrogen drugs such as flutamide, gonadotropin-releasing hormone analogs, and leuprolide; and nonsteroidal antiandrogen drugs such as flutamide.

[0054] A TRBC1-specific antibody-drug conjugate enables targeted delivery of a chemotherapeutic entity to cells expressing TRBC1.

[0055] Bispecific T cell engager A variety of molecules have been developed based on the basic concept of having two antibody-like binding domains.

[0056] Bispecific T-cell engaging molecule is a class of bispecific antibody-type molecules that have been developed mainly for use as anti-cancer drugs. These direct the cytotoxic activity of the host immune system, more specifically T cells, towards target cells such as cancer cells. In these molecules, one binding domain binds to T cells via the CD3 receptor, while the other binds to target cells such as tumor cells (via tumor-specific molecules). Since the bispecific molecule binds to both the target cell and the T cell, it thereby brings the target cell closer to the T cell, enabling the T cell to exert its effects, for example, the cytotoxic effect on cancer cells. Formation of the T cell:bispecific Ab:cancer cell complex induces signal transduction in the T cell, which in turn leads to, for example, the release of cytotoxic mediators. It is desirable that the agent induces only the desired signal transduction in the presence of the target cell, leading to selective killing.

[0057] Bispecific T-cell engaging molecules have been developed in several different configurations, one of the most common being a fusion consisting of two single-chain variable fragments (scFv) of different antibodies. These are sometimes known as BiTE (bispecific T-cell engagers).

[0058] Accordingly, the present invention provides a bispecific molecule that selectively recognizes TRBC1 and is capable of activating T cells. For example, the agent may be a BiTE. The agent may comprise (i) a first domain that binds to TRBC1 and has an antigen-binding domain as defined above; and (ii) a second domain that is capable of activating T cells.

[0059] The bispecific molecule may comprise a signal peptide to assist in its production. The signal peptide enables the bispecific molecule to be secreted from the host cell, thereby allowing the bispecific molecule to be recovered from the host cell supernatant.

[0060] The signal peptide may be at the amino terminus of the molecule. The bispecific molecule may have the general formula: signal peptide - first domain - second domain.

[0061] The bispecific molecule may include a spacer sequence that connects the first domain to the second domain and spatially separates the two domains.

[0062] The spacer sequence may include, for example, an IgG1 hinge or a CD8 stalk. Alternatively, the linker may include an alternative linker sequence whose length and / or the nature of the domain spacing is similar to that of an IgG1 hinge or a CD8 stalk.

[0063] Chimeric antigen receptor (CAR) The present invention provides a CAR that selectively recognizes TRBC1.

[0064] Chimeric antigen receptors (CARs), also known as chimeric T cell receptors, artificial T cell receptors, and chimeric immunoreceptors, are engineered receptors that transfer any specificity to immune effector cells. In classical CARs, the specificity of a monoclonal antibody is transferred to T cells. Nucleic acids encoding CARs can be introduced into T cells using, for example, retroviral vectors. In this way, a large number of cancer-specific T cells for adoptive cell transfer can be generated. Efficacy has been shown in a Phase I clinical trial of this approach.

[0065] The target antigen-binding domain of a CAR is generally fused to an endodomain that includes or associates with an intracellular T cell signaling domain via a spacer and a transmembrane domain. When a CAR binds to a target antigen, an activation signal is transmitted to the T cells expressing it.

[0066] A CAR may also include a transmembrane domain that spans the membrane. A CAR may include a hydrophobic alpha helix. The transmembrane domain may be derived from CD28, which provides good receptor stability.

[0067] The end domain is the part of the CAR that is involved in signal transduction. The end domain contains or associates with an intracellular T cell signal transduction domain. After antigen recognition, the receptors cluster and signals are transmitted to the cell. The most commonly used T cell signal transduction component is that of CD3-zeta, which contains three ITAMs. Thereby, after the antigen binds, an activation signal is transmitted to the T cell. A fully competent activation signal is not provided by CD3-zeta alone, and additional co-stimulatory signals may be required. For example, chimeric CD28 and OX40 can be used together with CD3-zeta to transmit proliferation / survival signals, or all three can be used together.

[0068] The end domain of the CAR may include the CD28 end domain and OX40 and the CD3-zeta end domain.

[0069] Alternatively, the CAR of the second aspect of the present invention may lack an intracellular signal transduction domain but can be conjugated to another molecule that provides signal transduction functionality.

[0070] A CAR signaling system has been previously described that includes two parts: a CAR that includes an antigen-binding domain and a transmembrane domain; and an intracellular signal transduction component that includes an intracellular signal transduction domain. One or more co-stimulatory domains may be located on the CAR and / or the intracellular signal transduction component.

[0071] Heterodimer formation between the CAR and intracellular signaling components results in a functional CAR system. Heterodimer formation may occur spontaneously as described in WO2016 / 124930, or may only occur in the presence of a chemical inducer of dimerization (CID) as described in WO2015 / 150771. In a third option, heterodimer formation is disrupted by the presence of an agent such as a specific small molecule, and thus CAR-mediated signaling only occurs in the absence of the agent. Such systems are described in WO2016 / 030691.

[0072] The CAR may include a signal peptide, such that when the CAR is expressed inside a cell such as a T cell, the nascent protein is directed to the endoplasmic reticulum and then to the cell surface where it is expressed.

[0073] The CAR may connect the TRBC binding domain to the transmembrane domain and may include a spacer sequence to spatially separate the TRBC binding domain from the membrane. The flexible spacer allows the TRBC binding domain to be oriented in different directions to enable binding to TRBC.

[0074] The spacer sequence may include, for example, an IgG1 Fc region, an IgG1 hinge or CD8 stalk, or a combination thereof.

[0075] Nucleic acid The present invention further provides a nucleic acid encoding a BiTE or CAR as defined above.

[0076] As used herein, the terms "polynucleotide", "nucleotide", and "nucleic acid" shall be synonymous with each other.

[0077] As a result of the degeneracy of the genetic code, one of ordinary skill in the art will understand that a number of different polynucleotides and nucleic acids may encode the same polypeptide. Furthermore, one of ordinary skill in the art should understand that nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein can be made using conventional techniques to reflect the codon usage of any particular host organism in which the polypeptide is expressed.

[0078] The nucleic acids according to the invention may comprise DNA or RNA. The nucleic acid may be single-stranded or double-stranded. It may be a polynucleotide that contains synthetic or modified nucleotides therein. Several different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, the addition of acridine or polylysine chains to the 3' and / or 5' ends of the molecule. It should be understood that the polynucleotides can be modified by any method available in the art for use as described herein. Such modifications can be made to enhance the in vivo activity or lifespan of the polynucleotide of interest.

[0079] The terms "variant," "homologue," or "derivative" in relation to a nucleotide sequence include any substitution, variation, modification, replacement, deletion, or addition of one (or more) nucleic acids from or to the sequence.

[0080] The invention also provides a nucleic acid construct comprising a first nucleic acid encoding a CAR as defined above; and a second nucleic acid encoding a suicide gene.

[0081] Suitable suicide genes for use in the CAR-expressing cells of the invention include RQR8 as described in WO2013 / 153391; and RapCasp9 as described in WO2016 / 135470.

[0082] In the above nucleic acid construct, the first nucleic acid sequence and the second nucleic acid sequence may be in either order.

[0083] Vector The present invention also provides a vector or a kit of vectors comprising one or more nucleic acid sequences or nucleic acid constructs of the present invention. Such vectors can be used to introduce the nucleic acid sequence(s) or construct(s) into host cells, for example, to express a CAR having an antigen-binding domain according to the first aspect of the present invention.

[0084] The vector may be, for example, a plasmid, a viral vector such as a retroviral vector or a lentiviral vector, or a transposon-based vector or synthetic mRNA.

[0085] The vector may be one that can transfect or transduce T cells or NK cells.

[0086] Cell The present invention also relates to cells such as immune cells comprising a CAR according to the first aspect of the present invention.

[0087] The cell may comprise the nucleic acid, nucleic acid construct or vector of the present invention.

[0088] The cell may be a T cell or a natural killer (NK) cell.

[0089] The T cell may be a T cell or T lymphocyte, a type of lymphocyte that plays a central role in cell-mediated immunity. These can be distinguished from other lymphocytes such as B cells and natural killer cells (NK cells) by the presence of a T cell receptor (TCR) on the cell surface. As summarized below, there are various types of T cells.

[0090] Helper T cells (TH cells) assist other white blood cells in immunological processes, including the maturation of B cells into plasma cells and memory B cells, as well as the activation of cytotoxic T cells and macrophages. TH cells express CD4 on their surface. TH cells are activated when peptide antigens are presented by MHC class II molecules on the surface of antigen-presenting cells (APCs). These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, Th9, or TFH, which secrete different cytokines that promote different types of immune responses.

[0091] Cytotoxic T cells (TC cells, or CTLs) destroy virus-infected cells and tumor cells and are also implicated in transplant rejection. CTLs express CD8 on their surface. These cells recognize their targets by binding to antigens associated with MHC class I, which is present on the surface of all nucleated cells. Through IL-10, adenosine, and other molecules secreted by regulatory T cells, CD8+ cells can be inactivated and put into a state of anergy, thereby preventing autoimmune diseases such as experimental autoimmune encephalomyelitis.

[0092] Memory T cells are a subset of antigen-specific T cells that persist for long periods after an infection has resolved. When re-exposed to the homologous antigen, they immediately expand into a large number of effector T cells, thereby providing an immune system with "memory" for past infections. Memory T cells include three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells can be either CD4+ or CD8+. Memory T cells generally express the cell surface protein CD45RO.

[0093] Regulatory T cells (Treg cells), formerly known as suppressor T cells, are extremely important for maintaining immune tolerance. Their main roles are to shut down T cell-mediated immunity and drive the end of an immune response, and to suppress autoreactive T cells that have escaped the process of negative selection in the thymus.

[0094] Two major classes of CD4+ Treg cells have been described - naturally occurring Treg cells and adaptive Treg cells.

[0095] Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) arise in the thymus and are associated with the interaction between developing T cells and bone marrow dendritic cells (CD11c+) and plasmacytoid dendritic cells (CD123+) activated by TSLP. Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutations in the FOXP3 gene can prevent the development of regulatory T cells, which can thereby cause the lethal autoimmune disease IPEX.

[0096] Adaptive Treg cells (also known as Tr1 cells or Th3 cells) can arise during a normal immune response.

[0097] The cell may be a natural killer cell (or NK cell). NK cells form part of the innate immune system. NK cells provide a rapid response to innate signals from virus-infected cells in an MHC-independent manner.

[0098] NK cells (which belong to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGL) and constitute a third type of cell that differentiates from a common lymphoid progenitor cell that gives rise to B lymphocytes and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils and thymus and then enter the circulation from there.

[0099] The CAR cells of the present invention may be any of the above cell types.

[0100] T cells or NK cells expressing a CAR according to the first aspect of the present invention can be prepared ex vivo from any of the patient's own peripheral blood (first party), or in the context of hematopoietic stem cell transplantation from donor peripheral blood (second party), or peripheral blood from an unrelated donor (third party).

[0101] Alternatively, T cells or NK cells expressing a CAR according to the first aspect of the present invention may be induced from the ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells into T cells or NK cells. Alternatively, an immortalized T cell line that retains its lytic function and can function as a therapeutic agent can be used.

[0102] In all of these embodiments, the CAR cells are generated by introducing DNA or RNA encoding the CAR by one of many means including transduction using a viral vector, transfection using DNA or RNA.

[0103] The CAR-expressing cells of the present invention may be ex vivo T cells or NK cells derived from a subject. The T cells or NK cells may be derived from a peripheral blood mononuclear cell (PBMC) sample. The T cells or NK cells can be activated and / or expanded by treatment, for example, with an anti-CD3 monoclonal antibody, prior to transduction with a nucleic acid encoding a CAR according to the first aspect of the present invention.

[0104] The T cells or NK cells of the present invention (i) isolation of a sample containing T cells or NK cells from a subject or other source listed above; and (ii) transduction or transfection of a nucleic acid sequence encoding the CAR of the present invention into the T cells or NK cells can be prepared by.

[0105] Next, T cells or NK cells can be purified, for example, selected based on the expression of the antigen-binding domain of the antigen-binding polypeptide.

[0106] The present invention also provides a kit comprising T cells or NK cells comprising a CAR according to a first aspect of the present invention.

[0107] Pharmaceutical composition The present invention also relates to a pharmaceutical composition containing a therapeutic entity such as a CAR-expressing cell, a therapeutic antibody or conjugate thereof, or a bispecific T cell engager of the present invention. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition may optionally contain one or more additional pharmaceutically active polypeptides and / or compounds. Such formulations may be in a form suitable for, for example, intravenous infusion.

[0108] T cell lymphoma and / or leukemia The present invention relates to agents, cells and methods for treating T cell lymphoma and / or leukemia.

[0109] A method for treating T cell lymphoma and / or leukemia relates to the therapeutic use of an agent. As used herein, an agent can be administered to a subject having an existing disease of T cell lymphoma and / or leukemia to alleviate, reduce or improve at least one symptom associated with the disease and / or to slow, reduce or block the progression of the disease.

[0110] The method of the present invention can be used to treat any lymphoma and / or leukemia associated with the clonal expansion of cells expressing a T cell receptor (TCR) comprising TRBC1.

[0111] The method of the present invention can be used to treat T cell lymphomas in which malignant T cells express a TCR containing TRBC1. As used herein, "lymphoma" is used according to its standard meaning and generally refers to a cancer that occurs in lymph nodes but can also affect the spleen, bone marrow, blood, and other organs. Lymphomas generally exist as solid tumors of lymphoid cells. The primary symptom associated with lymphoma is lymph node swelling, but secondary (B) symptoms can include fever, night sweats, weight loss, loss of appetite, fatigue, shortness of breath, and pruritus.

[0112] The method of the present invention can be used to treat T cell leukemias in which malignant T cells express a TCR containing TRBC1. As used herein, "leukemia" is used according to its standard meaning and refers to a cancer of the blood or bone marrow.

[0113] The following is an exemplary, non-inclusive list of diseases that can be treated by the method of the present invention.

[0114] Peripheral T cell lymphoma Peripheral T cell lymphomas are relatively rare lymphomas, accounting for less than 10% of all non-Hodgkin lymphomas (NHL). However, they are associated with an aggressive clinical course, and the cause and exact cell origin of the majority of T cell lymphomas have not yet been fully defined.

[0115] Lymphomas usually first appear as swelling in the neck, under the armpits, or groin. Additional swelling can occur in other locations where lymph nodes are located, such as within the spleen. Generally, enlarged lymph nodes can invade blood vessels, nerves, or the space of the stomach, resulting in swelling of the arms and legs, tingling and numbness, or a feeling of fullness, respectively. Symptoms of lymphoma can also include non-specific symptoms such as fever, chills, unexplained weight loss, night sweats, lethargy, and pruritus.

[0116] In the WHO classification, to elaborate on prognostically and therapeutically meaningful categorizations for peripheral T-cell lymphomas, morphological and immunophenotypic features are used in conjunction with clinical aspects and, in some cases, genetics (Swerdlow et al.; WHO classification of tumours of haematopoietic and lymphoid tissues. 4th ed; Lyon: IARC Press; 2008). The anatomical localization of neoplastic T cells, in part, parallels their proposed normal cell counterparts and functions, and thus, T-cell lymphomas are associated with lymph nodes and peripheral blood. This approach enables a better understanding of some aspects of the manifestation of T-cell lymphomas, including cell distribution, some aspects of morphology, and even the associated clinical findings.

[0117] The most common T-cell lymphoma is peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), which accounts for 25% of the total, followed by angioimmunoblastic T-cell lymphoma (AITL) (18.5%).

[0118] Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) PTCL-NOS accounts for more than 25% of all peripheral T-cell lymphomas and NK / T-cell lymphomas and is the most common subtype. It is determined by exclusion diagnosis and does not correspond to any of the specific mature T-cell lymphoma entities listed in the current WHO 2008. Therefore, it is similar to diffuse large B-cell lymphoma, not otherwise specified (DLBCL-NOS).

[0119] The majority of patients are adults, with a median age of 60 years and a male-to-female ratio of 2:1. The majority of cases originate from nodules, but extranodal manifestations occur in approximately 13% of patients, most commonly involving the skin and gastrointestinal tract.

[0120] The cytological spectrum is very broad, ranging from polymorphic to monomorphic. Three morphologically defined variants have been described, including the lymphoepithelial (Lennert) variant, the T-zone variant, and the follicular variant. The lymphoepithelial variant of PTCL contains abundant background lymphoepithelial histiocytes and is generally positive for CD8. It is associated with a better prognosis. The follicular variant of PTCL-NOS has begun to emerge as a potentially distinct clinicopathological entity.

[0121] Most PTCL-NOS have a mature T-cell phenotype, and the majority of cases are CD4 positive. Loss of variability of at least one pan-T-cell marker (CD3, CD2, CD5, or CD7) is shown in 75% of cases, and CD7 and CD5 are most often downregulated. CD30 and rarely CD15 may be expressed, and CD15 is a feature of poor prognosis. CD56 expression is also rare but has a negative prognostic impact. Additional adverse pathological prognostic factors include a proliferation rate exceeding 25% based on KI-67 expression and the presence of more than 70% transformed cells. The insights into their biology provided by immunophenotypic analysis of these lymphomas are limited.

[0122] Angioimmunoblastic T-cell lymphoma (AITL) AITL is a systemic disease characterized by polymorphic infiltrates involving lymph nodes, prominent high endothelial venules (HEVs), and perivascular expansion of the follicular dendritic cell (FDC) network. AITL is thought to be a de novo T-cell lymphoma derived from follicular helper-type αβ T cells (TFH), which are usually found in germinal centers.

[0123] AITL is the second most common entity among peripheral T-cell lymphomas and NK / T-cell lymphomas, accounting for approximately 18.5% of cases. It occurs in middle-aged to elderly adults, with a median age of 65 years, and the incidence is approximately equal between men and women. Clinically, patients usually have advanced disease with generalized lymphadenopathy, hepatosplenomegaly, and prominent constitutional symptoms. Pruritic skin rash is commonly present. Polyclonal hypergammaglobulinemia associated with autoimmune phenomena is often present.

[0124] Three different morphological patterns have been described in AITL. In the early lesions of AITL (pattern I), a preserved architecture with characteristic hyperplastic follicles is usually shown. Tumoral proliferation is localized to the periphery of the follicles. In pattern II, the nodular architecture is partially lost, and slightly regressed follicles are retained. Subcapsular sinuses are preserved and even enlarged. The paracortex contains branched HEVs, and FDCs proliferate beyond B-cell follicles. Neoplastic cells are small to medium-sized, with minimal cytological atypia. It often has clear to hazy cytoplasm and may show a distinct cell membrane. A polymorphic inflammatory background is usually evident.

[0125] AITL is a T-cell malignancy, but B cells and plasma cells characteristically expand, which is thought to reflect the function of neoplastic cells as TFH cells. Both EBV-positive B cells and EBV-negative B cells are present. Occasionally, atypical B cells may be morphologically and immunophenotypically similar to Hodgkin / Reed-Sternberg-like cells, and at times, diagnostic confusion with that entity occurs. B-cell proliferation in AITL can be extensive, and in some patients, secondary EBV-positive diffuse large B-cell lymphoma (DLBCL) or more rarely EBV-negative B-cell tumors occur, often with plasmacytic differentiation.

[0126] Tumoral CD4-positive T cells in AITL show strong expression of CD10 and CD279 (PD-1), and are positive for CXCL13. CXCL13 leads to increased B-cell mobilization to lymph nodes via adhesion to HEVs, B-cell activation, plasmacytic differentiation, and increased FDC meshworks, all of which contribute to the morphological and clinical features of AITL. Strong PD-1 expression in the perifollicular tumor cells is particularly useful for differentiating AITL pattern I from reactive follicles and paracortical hyperplasia.

[0127] The follicular variant of PTCL-NOS is a distinct entity with a TFH phenotype. In contrast to AITL, it does not have prominent extracapsular expansion of HEVs or FDC meshworks. Neoplastic cells may form intrafollicular aggregates mimicking B-cell follicular lymphoma, but may also have an interfollicular growth pattern or be associated with mantle zone expansion. Clinically, the follicular variant of PTCL-NOS is distinct from AITL as patients more frequently present with early disease with partial lymph node involvement and may not have the constitutional symptoms associated with AITL.

[0128] Anaplastic large cell lymphoma (ALCL) ALCL can be subdivided into ALCL-“anaplastic lymphoma kinase” (ALK)+ or ALCL-ALK-.

[0129] ALCL-ALK+ is one of the best-defined entities among peripheral T-cell lymphomas, has a horseshoe-shaped nucleus, and has characteristic “hallmark cells” that express ALK and CD30. It accounts for approximately 7% of all peripheral T-cell and NK-cell lymphomas and is most common in the first 30 years of life. Patients often present with lymph node swelling, but involvement of extranodal sites (skin, bone, soft tissue, lung, liver) and B symptoms are common.

[0130] ALCL, ALK+ shows a wide morphological spectrum, and five different patterns have been described, but all variants contain some characteristic cells. The characteristic cells have eccentric horseshoe or kidney-shaped nuclei and a prominent perinuclear eosinophilic Golgi region. Tumor cells grow in an adhesive pattern with a preference for sinus involvement. In the small cell variant, smaller tumor cells are dominant, and in the lymphohistiocytic variant, the presence of tumor cells is masked by abundant histiocytes, many of which are small.

[0131] By definition, ALK and CD30 positivity is shown in all cases, and the expression is usually weaker in smaller tumor cells. In many cases, pan-T cell markers are absent, and surface expression of CD3 is absent in 75% of cases.

[0132] ALK expression is the result of a characteristic recurrent genetic alteration consisting of a rearrangement of the ALK gene on chromosome 2p23 to one of many partner genes, which results in the expression of a chimeric protein. The most common partner gene, occurring in 75% of cases, is nucleophosmin (NPM1) on chromosome 5q35, resulting in t(2;5)(p23;q35). The cellular distribution of ALK in different translocation variants can vary depending on the partner gene.

[0133] ALCL-ALK- is included as a provisional category in the 2008 WHO classification. It is defined as a CD30-positive T cell lymphoma that is morphologically indistinguishable from ALCL-ALK+ but lacks ALK protein expression, with an adhesive growth pattern and the presence of characteristic cells.

[0134] Patients are usually adults between 40 and 65 years old, which is in contrast to ALCL-ALK+, which is more common in children and young adults. In ALCL-ALK-, both lymph nodes and extranodal tissues can be involved, but the latter is less common than that seen in ALCL-ALK+. In the majority of cases of ALCL-ALK-, the destruction of lymph node architecture by sheets of cohesive neoplastic cells with typical "hallmark" features is demonstrated. In contrast to ALCL-ALK+, small cell morphological variants are not recognized.

[0135] In ALCL-ALK-, in contrast to its ALK+ counterpart, greater conservation of surface T cell marker expression is shown, but the expression of cytotoxic markers and epithelial membrane antigen (EMA) is less likely. Gene expression signatures and recurrent chromosomal imbalances differ between ALCL-ALK- and ALCL-ALK+, confirming that they are distinct entities at the molecular and genetic levels.

[0136] ALCL-ALK- is clinically distinct from both ALCL-ALK+ and PTCL-NOS, and there are significant differences in prognosis among these three different entities. The 5-year overall survival rate of ALCL-ALK- has been reported to be 49%, which is not as good as the 5-year overall survival rate of ALCL-ALK+ (70%), but at the same time, it is significantly better than the 5-year overall survival rate of PTCL-NOS (32%).

[0137] Enteropathy-associated T cell lymphoma (EATL) EATL is an aggressive neoplasm thought to originate from intraepithelial T cells of the intestine. In 2008 In the WHO classification, two morphologically, immunohistochemically and genetically distinct types of EATL are recognized: type I (representing the majority of EATL) and type II (accounting for 10-20% of cases).

[0138] Type I EATL is usually associated with overt or clinically asymptomatic gluten-sensitive enteropathy and is more frequently seen in patients of Nordic descent, which is due to the high prevalence of celiac disease in this population.

[0139] Most commonly, EATL lesions are found in the jejunum or ileum (90% of cases) and rarely occur in the duodenum, colon, stomach, or areas outside the gastrointestinal tract. Intestinal lesions are usually multifocal and associated with mucosal ulceration. The clinical course of EATL is aggressive, and the majority of patients die within 1 year from the disease or its complications.

[0140] The cytological spectrum of EATL type I is broad, and some cases may contain undifferentiated cells. In some cases, there is a polymorphic inflammatory background that can obscure the neoplastic component. The intestinal mucosa within the area adjacent to the tumor often shows features of celiac disease, with villous blunting and an increase in the number of intraepithelial lymphocytes (IELs), which may represent dysplastic precursor cells.

[0141] By immunohistochemistry, neoplastic cells are often CD3+CD4-CD8-CD7+CD5-CD56-βF1+ and contain cytotoxic granule-associated proteins (TIA-1, granzyme B, perforin). CD30 is partially expressed in almost all cases. CD103, a mucosal homing receptor, may be expressed in EATL.

[0142] Type II EATL, also termed monomorphic CD56+ intestinal T-cell lymphoma, is defined as an intestinal tumor composed of small to medium-sized monomorphic T cells that express both CD8 and CD56. In most cases, the tumor spreads laterally within the mucosa and there is no inflammatory background. γδ TCR is expressed in the majority of cases, but there are also cases with αβ TCR.

[0143] Type II EATL is more globally distributed than type I EATL and is often seen in Asian or Latin American populations where celiac disease is rare. In individuals of European descent, EATL, type II represents approximately 20% of intestinal T-cell lymphomas and is associated with a history of celiac disease in at least a subset of cases. The clinical course is aggressive.

[0144] Hepatosplenic T-cell lymphoma (HSTL) HSTL is generally an aggressive systemic neoplasm derived from the γδ cytotoxic T cells of the innate immune system, although in rare cases it can also be derived from αβ T cells. It is one of the rarest T-cell lymphomas and generally affects young and young adults (median age, 35 years) and is strongly male-predominant.

[0145] Extranodal NK / T-cell lymphoma, nasal type Extranodal NK / T-cell lymphoma, nasal type is an aggressive disease and often presents with destructive midline lesions and necrosis. The majority of cases are derived from NK cells, although some cases are derived from cytotoxic T cells. It is universally associated with Epstein-Barr virus (EBV).

[0146] Cutaneous T-cell lymphoma The methods of the present invention can also be used to treat cutaneous T-cell lymphoma.

[0147] Cutaneous T-cell lymphoma (CTCL) is characterized by the migration of malignant T cells to the skin, resulting in the appearance of various lesions. These lesions change in shape as the disease progresses and generally begin as what appears to be a rash and ultimately form plaques and tumors before metastasizing to other parts of the body.

[0148] Examples of cutaneous T-cell lymphomas include, but are not limited to, those listed in the following exemplary, non-exhaustive list: mycosis fungoides, Pagetoid reticulosis, Sézary syndrome, granulomatous slack skin, lymphomatoid papulosis, chronic pityriasis lichenoides, CD30+ cutaneous T-cell lymphoma, secondary cutaneous CD30+ large cell lymphoma, non-mycosis fungoides CD30− cutaneous large T-cell lymphoma, polymorphic T-cell lymphoma, Lennert lymphoma, subcutaneous T-cell lymphoma, and angiocentric lymphoma.

[0149] The signs and symptoms of CTCL vary depending on the specific disease, and the two most common types are mycosis fungoides and Sézary syndrome. Classic mycosis fungoides has three stages: Patch (atrophic or non-atrophic): non-specific dermatitis, patches on the lower trunk and buttocks; minimal or no pruritus; Plaque: intensely pruritic plaques, lymph node swelling; and Tumor: tendency to ulcerate which are divided.

[0150] Sézary syndrome is defined by erythroderma and leukemia. Signs and symptoms include edematous skin, lymph node swelling, palm and / or sole hyperkeratosis, alopecia, nail dystrophy, ectropion, and hepatosplenomegaly.

[0151] Among all primary cutaneous lymphomas, 65% are of the T-cell type. The most common immunophenotype is CD4 positive. Since the term cutaneous T-cell lymphoma encompasses a diverse range of disorders, there is no common pathophysiology among these diseases.

[0152] The primary etiologic mechanism for the development of cutaneous T-cell lymphoma (i.e., mycosis fungoides) is not understood. Mycosis fungoides may be preceded by a T-cell-mediated chronic inflammatory skin disease, which, in some cases, may progress to a lethal lymphoma.

[0153] Primary cutaneous ALCL (C-ALCL) C-ALCL often cannot be distinguished morphologically from ALC-ALK-. It is defined as a cutaneous tumor of large cells in an undifferentiated, pleomorphic, or immunoblastic form in which more than 75% of the cells express CD30. Together with lymphomatoid papulosis (LyP), C-ALCL belongs to the spectrum of primary cutaneous CD30-positive T-cell lymphoproliferative disorders, which as a group include the second most common group of cutaneous T-cell lymphoproliferations after mycosis fungoides.

[0154] The immunohistochemical staining profile is quite similar to ALCL-ALK- and a larger proportion of cases stain positive for cytotoxic markers. At least 75% of the tumor cells should be positive for CD30. CD15 is also expressed and, if nodal involvement occurs, it may be difficult to distinguish from classical Hodgkin lymphoma. Rare cases of ALCL-ALK+ may present with localized skin lesions and may be similar to C-ALCL.

[0155] T-cell acute lymphoblastic leukemia T-cell acute lymphoblastic leukemia (T-ALL) accounts for approximately 15% of ALL in pediatric cohorts and approximately 25% of ALL in adult cohorts. Patients usually have a high white blood cell count and may show organomegaly, particularly mediastinal enlargement and CNS involvement.

[0156] The method of the present invention can be used to treat T-ALL associated with malignant T cells expressing a TCR comprising TRBC1.

[0157] T-cell prolymphocytic leukemia T-cell prolymphocytic leukemia (T-PLL) is a mature T-cell leukemia with an aggressive behavior and a predilection for involvement of blood, bone marrow, lymph nodes, liver, spleen, and skin. T-PLL mainly affects adults over 30 years of age. Other names include T-cell chronic lymphocytic leukemia, "warty" type T-cell leukemia, and T-prolymphocytic leukemia / T-cell lymphocytic leukemia.

[0158] In peripheral blood, T-PLL consists of medium-sized lymphocytes with a single nucleolus and basophilic cytoplasm, sometimes with blebs or projections. The nucleus is usually round to oval in shape, and in some patients, there are cells with a more irregular nuclear contour, similar to the cerebriform nuclear shape seen in Sézary syndrome. The small cell variant accounts for 20% of all T-PLL cases, and the Sézary cell-like (cerebriform) variant is seen in 5% of cases.

[0159] T-PLL has the immunophenotype of mature (post-thymic) T lymphocytes, and neoplastic cells are generally positive for the pan-T antigens CD2, CD3, and CD7, and negative for TdT and CD1a. The immunophenotype CD4+ / CD8- is present in 60% of cases, the CD4+ / CD8+ immunophenotype is present in 25% of cases, and the CD4- / CD8+ immunophenotype is present in 15% of cases.

[0160] Pharmaceutical composition The method of the present invention may include the step of administering the agent in the form of a pharmaceutical composition.

[0161] The agent can be administered with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The choice of pharmaceutical carrier, excipient or diluent is selected with respect to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may include, as (or in addition to) a carrier, excipient or diluent, any suitable binding substance, lubricant, suspending agent, coating agent, solubilizing agent, and other carrier agents.

[0162] Administration The administration of the agent can be achieved using any of various routes such that the active ingredient becomes bioavailable to the organism. For example, the agent can be administered intraperitoneally, intravenously, subcutaneously, transdermally, intramuscularly, by local delivery, e.g., by catheter or stent, via oral and parenteral routes.

[0163] Generally, the actual dosage most suitable for an individual subject is determined by a physician and will vary depending on the age, weight, and response of the particular patient. The dosage is such that it is sufficient to reduce or deplete the number of clonal T cells expressing TRBC1.

[0164] The invention will now be further described by way of examples, which are meant to assist those skilled in the art in carrying out the invention and are not intended to limit the scope of the invention in any way.

Examples

[0165] (Example 1) Heavy and light chain graft selection Human VH frameworks: H-AF062256, H-EF177999, H-KF A humanized VH domain having the JOVI-1 VH CDR after 688165 was constructed. A humanized VL domain having the 3aaz human framework was constructed.

[0166] Antibodies were made using either a humanized VH domain and a mouse JOVI-1 VL domain; a mouse VH domain and a humanized VL domain; or a humanized VH domain and a humanized VL domain (see Figure 4).

[0167] Binding to TRBC1 was tested by ELISA. The results are shown in Figure 5.

[0168] It was found that all combinations of chimeric and humanized binding substances were able to bind to TRBC1, and the binding was similar to that of a chimeric antibody having mouse VH and VL domains.

[0169] (Example 2) Generation and testing of revertant mutants A series of binding substances were generated that were reverted for the human framework H-AF062256 as shown in Table 1. In Table 1, the revertant mutations are shown in bold.

[0170]

Table 1-1

Table 1-2

[0171] The conjugate substance had a humanized VL domain containing a 3aaz framework.

[0172] Binding to TRBC1 and TRBC2 was tested by ELISA. The results are shown in Figure 6. All constructs bound to TRBC1 but not to TRBC2 and showed an EC50 similar to that of the chimeric antibody (Jovi-Mu) with mouse VH and VL domains. This indicates that after CDR grafting, the specificity and affinity of the mouse antibody are retained in the humanized antibody.

[0173] (Example 3) Investigation of the stability of humanized mAb and scFv The stability of the conjugate substance described in Example 2 was tested by differential scanning fluorimetry. After storing the protein at 150 μg / ml in PBS, sypro orange dye was added at a protein:dye ratio of 5000:1. The solution was mixed and placed in a qPCR machine and run in FRET mode. The solution was held at 15 °C for 10 minutes, and then the temperature gradient was increased in 0.5 °C steps up to 95 °C, holding for 30 seconds at each step. Fluorescence readings were obtained after each step. To obtain the Tm value (equilibrium constant; unfolded protein = folded protein), the first derivative of the fluorescence change (ΔRFU / Δ°C) was plotted against the temperature change (Δ°C).

[0174] The results are shown in Figure 7. It was found that the use of the human framework increased the stability of the conjugate substance in the mAb format.

[0175] Similar experiments were conducted to investigate the stability of the equivalent binding substances in scFv format. For this test, differential scanning calorimetry was used. The experiments were carried out using a CAP DSC system. The protein in the storage buffer (1×PBS) was placed in the calorimeter sample cell, and the reference cell was filled with only the storage buffer. After stabilizing the cell inside the calorimeter at 25°C for 1 hour, it was heated at a rate of 200°C per hour up to a final temperature of 100°C. The denaturation temperature, Tm, corresponding to the maximum transition peak was determined by performing at least 2 repeated runs, and it was a variation of 0.25°C or less.

[0176] The results of one binding substance scFv comparison are shown in Figure 8. The melting temperature of the mouse Jovi-1 scFv was 61°C, and the melting temperature of the humanized scFv (H-AF1, 3aaz) was 65°C.

[0177] (Example 4) Generation of a chimeric antigen receptor (CAR) with a humanized anti-TRBC1 antigen-binding domain

[0178] As schematically illustrated in Figure 9, a second-generation CAR with an antigen-binding domain containing the 41BB and CD3 zeta end domains and the humanized JOVI-1 scFv (H-AF1, 3aaz) was designed. Primary human T cells from normal donors were transduced with a retroviral vector expressing the anti-TRBC1 CAR or an unrelated EGFRvIII as a negative control CAR-expressing retroviral vector. The ability of the cells to kill TRBC1-expressing target cells or TRBC2-expressing target cells was investigated using flow cytometry.

[0179] The results are shown in Figure 10. T cells expressing the humanized TRBC1 CAR killed the TRBC1-expressing target cells but did not kill the TRBC2-expressing target cells.

[0180] T cell proliferation was measured after co-culturing with TRBC1-expressing target cells or TRBC2-expressing target cells for 72 hours. The results are shown in Fig. 11. T cells expressing either mouse Jovi-1 CAR or humanized CAR showed increased proliferation when co-cultured with TRBC1-expressing target cells, but did not show increased proliferation when co-cultured with TRBC2-expressing target cells.

[0181] Cytokine release was measured after co-culturing with TRBC1-expressing target or TRBC2-expressing target for 24 hours. The results are shown in Fig. 12. T cells expressing either mouse Jovi-1 CAR or humanized CAR showed increased release of IFNγ and IL-2 when co-cultured with TRBC1-expressing target cells, but did not show increased release of IFNγ and IL-2 when co-cultured with TRBC2-expressing target cells.

[0182] (Example 5) Tumors are removed by humanized αTRBC1 CAR in NSG mouse model Female NSG mice at 7 - 8 weeks of age were implanted with Jurkat cells transduced to express CD19-Fluc (3×10 6 cells per animal, in 0.1 ml of PBS). On day 7, mice (n = 8 / group) were injected intravenously with 1.0×106 aTRBC1 CAR T cells or mock-transduced cells (NT). Tumor growth was confirmed by bioluminescence imaging (BLI) on days 6, 9, 12, and 15 of the study. Briefly, 150 mg / kg of D-luciferin was injected (s.c.) into the mice 15 minutes before imaging. Ten minutes after D-luciferin administration, the mice were anesthetized and placed in an imaging chamber, and imaged for luminescence (ventral and dorsal views; up to 5 mice were laid side by side in cage order). The duration of image acquisition and binning (sensitivity) were captured and processed using Living Image 4.3.1 software.

[0183] The results are shown in Fig. 13. By day 12, tumors were eliminated by humanized anti-TRBC1 CAR-T cells in all animals.

[0184] (Example 6) Comparison of the effects of mouse anti-TRBC1 CAR T cells and humanized anti-TRBC1 CAR T cells on exhaustion Activated PBMCs were transduced with a vector expressing a mouse Jovi-1 CAR or a humanized Jovi-1 CAR containing CDRs from JOVI-1 with an H-AF062256 VH framework region and a 3aaz VL framework region. Cells were collected 2 days later and maintained for an additional 2 days in culture medium with 50 U / mL of IL-2. The EasySep CD56 positive selection kit was used to deplete CD56-expressing cells from the transduced T cells. Co-culture of the TRBC1-TCR-expressing radiolabeled target and CAR T cells was performed as follows: Target cells were plated at 50,000 cells per well in a 96-well U-bottom plate at an effector:target ratio of 1:1. After 96 hours, the co-cultures were harvested, and the cells were stained with anti-PD1 antibody, anti-LAG3 antibody, and anti-Tim3 antibody and then analyzed by flow cytometry.

[0185] The results are shown in Fig. 14. T cells expressing humanized anti-TRBC1 CAR expressed all three exhaustion markers at lower levels than T cells expressing mouse CAR. This was true for both CD4+ T cells and CD8+ T cells. Thus, surprisingly, T cells expressing humanized CAR are less exhausted than T cells expressing CAR with an scFv derived from a mouse Jovi-1 antibody during exposure to target cells.

[0186] T cell exhaustion is a state of T cell dysfunction that occurs during many chronic infections and cancers. T cell exhaustion is defined by poor effector function. To be efficient in killing target cells, it is advantageous to avoid T cell exhaustion or reduce the rate of T cell exhaustion.

[0187] All publications mentioned in the above specification are hereby incorporated by reference into this specification. Without departing from the scope and spirit of the present invention, various modifications and variations of the described method and the system of the present invention will be apparent to those skilled in the art. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described manner of carrying out the invention which are obvious to those skilled in the art of molecular biology or related fields are intended to be within the scope of the following claims.

Claims

【Claim 1】 The invention described in the drawings.