WO2024165024A1 - Chimeric antigen receptor and use thereof - Google Patents

Abstract

Provided is a chimeric antigen receptor (CAR). The CAR comprises a CD30 binding domain and a hinge region; the CD30 binding domain comprises a heavy chain complementarity determining region 1 (HCDR1), a heavy chain complementarity determining region 2 (HCDR2), a heavy chain complementarity determining region 3 (HCDR3), a light chain complementarity determining region 1 (LCDR1), a light chain complementarity determining region 2 (LCDR2) and a light chain complementarity determining region 3 (LCDR3); and the hinge region is derived from CD28.

Description

A chimeric antigen receptor and its application Technical Field

The present application belongs to the field of immune cell therapy, and specifically, relates to a chimeric antigen receptor and its application.

Background Art

Chimeric antigen receptors (CARs) specifically recognize antigens expressed on the surface of tumor cells to guide CAR-expressing immune cells to eliminate tumors. For example, when antigen-positive cells targeted by CAR exist, cells expressing the CAR can recognize and kill these antigen-positive cells.

However, the existing CAR in the art still has the problem of low activity and weak killing ability of cells expressing the CAR. Therefore, the art needs a CAR with a further optimized structure to achieve the effect of improving the killing ability of cells expressing the CAR.

Summary of the invention

The present application provides a chimeric antigen receptor, which can have one or more effects selected from the following: (1) excellent expression efficiency; (2) cells expressing the chimeric antigen receptor have significant anti-tumor activity; (3) cells expressing the chimeric antigen receptor have sustained anti-tumor activity; (4) cells expressing the chimeric antigen receptor have significant cell activation ability; and (5) cells expressing the chimeric antigen receptor have significant cytokine secretion ability.

Specifically, the CAR efficiently transduced healthy human T lymphocytes in vitro and produced a strong killing effect on CD30-positive target cells; and in in vivo experiments, it also showed a strong killing effect.

On the one hand, the present application provides a chimeric antigen receptor (CAR), which comprises: a CD30 binding domain and a hinge region, wherein the CD30 binding domain comprises a heavy chain complementary determining region 1 (HCDR1), a heavy chain complementary determining region 2 (HCDR2), a heavy chain complementary determining region 3 (HCDR3), a light chain complementary determining region 1 (LCDR1), a light chain complementary determining region 2 (LCDR2) and a light chain complementary determining region 3 (LCDR3), the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2 and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 5 (the amino acid sequence represented by SEQ ID NO: 5 is SAS), the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 6, and the hinge region is derived from CD28.

In certain embodiments, the CD30 binding domain comprises a heavy chain variable region VH, the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 7 or a functional variant thereof.

In certain embodiments, the CD30 binding domain comprises a light chain variable region VL, and the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 8 or a functional variant thereof.

In certain embodiments, the CD30 binding domain is a single chain antibody.

In certain embodiments, the CD30 binding domain is a single-chain scFv antibody, wherein the single-chain scFv comprises a VL domain-linker-VH domain or a VH domain-linker-VL domain from N-terminus to C-terminus.

In certain embodiments, the CD30 binding domain comprises the amino acid sequence shown in SEQ ID NO: 9 or a functional variant thereof.

In certain embodiments, the hinge region comprises a CD28 hinge region or a functional variant thereof.

In certain embodiments, the hinge region comprises an amino acid sequence as shown in SEQ ID NO: 11 or a functional variant thereof.

In certain embodiments, the CD30 binding domain is directly or indirectly linked to the hinge region derived from CD28.

In certain embodiments, the CAR comprises an amino acid sequence as shown in SEQ ID NO: 15 and its functional variants.

In certain embodiments, the CAR further comprises a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular signaling domain.

In certain embodiments, the CAR comprises a transmembrane domain, which comprises a transmembrane domain derived from one or more proteins in the following group: CD8, CD28, CD3ε (CD3e), 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L (CD154), TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, SLAM and their variants.

In certain embodiments, wherein the CAR comprises a transmembrane domain, the transmembrane domain is derived from CD28.

In certain embodiments, the CAR comprises a transmembrane domain, and the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 12 or a functional variant thereof.

In certain embodiments, the CAR further comprises one or more intracellular co-stimulatory domains, wherein the co-stimulatory domain comprises a co-stimulatory domain of one or more proteins derived from the following group: CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKG2C, SLP76, TRIM and ZAP70.

In certain embodiments, the CAR comprises an intracellular co-stimulatory domain, and the co-stimulatory domain is derived from CD28.

In certain embodiments, the CAR comprises an intracellular co-stimulatory domain, and the co-stimulatory domain comprises an amino acid sequence as shown in SEQ ID NO: 13 or a functional variant thereof.

In certain embodiments, the CAR comprises one or more intracellular signaling domains, the intracellular signaling domains comprising intracellular signaling domains derived from one or more proteins of the following group: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FceRIγ, FceRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, DAP10, DAP-12 and a domain comprising at least one ITAM.

In certain embodiments, wherein the CAR comprises an intracellular signaling domain, the intracellular signaling domain is CD3ζ.

In certain embodiments, the CAR comprises an intracellular signaling domain, and the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO: 14 or a functional variant thereof.

In certain embodiments, wherein the CAR further comprises a signal peptide, the signal peptide comprises a signal peptide derived from CD8 or CD45.

In certain embodiments, the CAR further comprises a signal peptide, and the signal peptide comprises a CD8α signal peptide.

In certain embodiments, the CAR further comprises a signal peptide, wherein the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 10 or a functional variant thereof.

In certain embodiments, from N-terminus to C-terminus, the CAR comprises, in sequence, a signal peptide, a CD30 binding domain, a hinge region derived from CD28, a transmembrane domain, an intracellular co-stimulatory signaling domain, and an intracellular signaling domain.

In certain embodiments, from N-terminus to C-terminus, the CAR comprises, in sequence, a CD8α signal peptide, a CD30 binding domain, a hinge region derived from CD28, a CD28 transmembrane domain, a CD28 intracellular co-stimulatory signaling domain, and a CD3ζ intracellular signaling domain.

In certain embodiments, the CAR comprises an amino acid sequence as described in SEQ ID NO: 16.

On the other hand, the present application provides an isolated nucleic acid molecule comprising the nucleic acid sequence of the CAR. The nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 17.

On the other hand, the present application provides a vector comprising the nucleic acid molecule.

In certain embodiments, the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenovirus Vector and retroviral vector: The vector comprises the nucleotide sequence shown in SEQ ID NO:18.

On the other hand, the present application provides a cell comprising the CAR, the nucleic acid molecule and/or the vector.

In certain embodiments, the cell is an immune cell.

In certain embodiments, the immune cells are selected from the group consisting of T cells, natural killer (NK) cells, NKT cells, and macrophages.

On the other hand, the present application provides a method for preparing a cell, which comprises introducing the CAR, the nucleic acid molecule and/or the vector into the cell.

In certain embodiments, the cell is an immune cell.

In certain embodiments, the immune cell is selected from the group consisting of T cells, natural killer (NK) cells, NKT cells, and macrophages.

In certain embodiments, the immune cell is a T cell.

On the other hand, the present application provides a composition comprising the CAR, the nucleic acid molecule, the vector and/or the cell, and optionally a pharmaceutically acceptable carrier.

On the other hand, the present application provides a use of the CAR, the nucleic acid molecule, the vector and/or the cell in the preparation of a medicament for treating a CD30-related disorder.

On the other hand, the present application provides a method for preventing and/or treating CD30-related disorders, comprising administering an effective amount of the CAR, the nucleic acid molecule, the vector and/or the cell.

In certain embodiments, the CD30-related disorder is a tumor expressing CD30.

In certain embodiments, the tumor is a lymphoma.

In certain embodiments, the tumor is Hodgkin's lymphoma or non-Hodgkin's lymphoma.

In certain embodiments, the CD30-related disorder is selected from the group consisting of Hodgkin lymphoma, anaplastic large cell lymphoma (ALCL), cutaneous T-cell lymphoma, adult T-cell lymphoma (ATL) and angioimmunoblastic T-cell lymphoma (AITL).

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1B show the detection of the CD30 CAR described in the present application on the 6th day after viral transduction of T cells. Figure 1A shows CAR-T cells after viral transduction, and Figure 1B shows T cells that have not been transduced with the virus.

FIG. 2 shows the results of detecting the activity of T cells expressing the CD30 CAR described in the present application in killing tumor cells.

Figure 3 shows the results of IFN-γ release detected by enzyme-linked immunosorbent assay (ELISA) after T cells expressing the CD30 CAR described in the present application were incubated with target cells.

Figure 4 shows the results of IL-2 release detected by enzyme-linked immunosorbent assay (ELISA) after T cells expressing the CD30 CAR described in the present application were incubated with target cells.

Figure 5 shows the results of in vivo efficacy evaluation of T cells expressing the CD30 CAR described in this application in tumor xenograft mice.

Figure 6 shows the molecular schematic diagrams of the CD30 CAR (P037), P038 CAR, and P050 CAR described in the present application.

Figure 7 shows the expression of the CD30 CAR (P037) and P038 CAR, P050 CAR described in this application detected on the 5th day after viral transduction of T cells.

Figures 8A-8B show the results of in vitro killing experiments of T cells expressing the CD30 CAR (P037) described in the present application and T cells expressing P050 CAR. Figure 8A shows the in vitro killing effect on tumor cells L540, and Figure 8B shows the results of enzyme-linked immunosorbent assay (ELISA) detection of IFN-γ release after incubation with target cells L540 cells.

Figures 9A-9C show the results of in vitro killing experiments of T cells expressing the CD30 CAR (P037) described in the present application and T cells expressing P038 CAR. Figure 9A shows the in vitro killing effect on tumor cells L540, Figure 9B shows the results of IL-2 release detected by enzyme-linked immunosorbent assay (ELISA) after incubation with target cells L540 cells, and Figure 9C shows the results of IFN-γ release detected by enzyme-linked immunosorbent assay (ELISA) after incubation with target cells L540 cells.

Figures 10A-10B show the results of in vivo efficacy evaluation of T cells expressing the CD30 CAR (P037) described in this application and T cells expressing P050 CAR in tumor xenograft mice. Figure 10A shows the changes in tumor volume, and Figure 10B shows the mouse survival curve.

Figures 11A-11B show the results of in vivo efficacy evaluation of T cells expressing the CD30 CAR (P037) and T cells expressing P038 CAR described in the present application in tumor xenograft mice. Figure 11A shows the changes in tumor volume, and Figure 11B shows the mouse survival curve.

DETAILED DESCRIPTION

The following is an explanation of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

Definition of terms

In this application, the term "CD30" generally refers to the CD30 protein or the gene encoding it. The CD30 protein, also known as TNFRSF8, is a cell membrane protein of the tumor necrosis factor receptor family (see Cell 68:421, 1992). For example, human CD30 protein The Genbank accession number of the complete amino acid sequence of is NP_001234, and the Genbank accession number of the complete cDNA sequence encoding human CD30 protein is NM_001243.

In this application, the term "Chimeric Antigen Receptor (CAR)" generally refers to an artificial cell receptor engineered to express and specifically bind to an antigen on an immune effector cell. CAR can be used as a therapy for adoptive cell transfer. CAR can include an extracellular domain, a transmembrane domain, and an intracellular domain.

In the present application, the term "signal peptide" generally refers to a propeptide present as an N-terminal peptide in the form of a protein precursor. The function of the signal peptide is to promote the translocation of the expressed polypeptide connected to the endoplasmic reticulum and to express the target protein on the cell membrane, and the signal peptide can usually be removed in the process. For example, the antigen-binding protein of the present application may not have a signal peptide and may not affect its biological activity. The signal peptide may be heterologous or homologous to the organism used to produce the polypeptide.

In the present application, the term "antigen binding domain" generally refers to a domain that can bind to a target antigen. The antigen binding domain may include a chimeric antigen receptor and a fragment thereof, an antibody or an antigen binding fragment thereof that can specifically bind to an antigen. The antigen binding domain may be of natural origin, synthetic origin, semisynthetic origin or recombinant origin. For example, the antigen binding domain may include a single-chain antibody.

In this application, the term "antigen binding fragment" generally refers to a portion of an antibody that includes a region that specifically binds to and is complementary to part or all of an antigen. The antigen binding domain can be provided by, for example, one or more antibody variable domains (also referred to as antibody variable regions). In some cases, the antigen binding domain may include an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

In the present application, the term "CD30 binding domain" generally refers to a domain that can specifically bind to CD30. The CD30 binding domain may include an anti-CD30 antibody or an antigen-binding fragment thereof that can specifically bind to CD30. For example, the CD30 binding domain may include an anti-CD30 single-chain antibody.

In the present application, the term "antibody" generally refers to an immunoglobulin or a fragment thereof or a derivative thereof, covering any polypeptide comprising an antigen binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated and transplanted antibodies. The term "antibody" may also include antibody fragments, such as Fab, F(ab')2, Fv, scFv, Fd, dAb and other antibody fragments that retain antigen binding function.

In this application, the term "heavy chain variable region" or "VH" generally refers to the region of the immunoglobulin heavy chain structure that includes the heavy chain complementary determining regions CDR1, CDR2, CDR3 and the framework regions FR1, FR2, FR3, FR4. The heavy chain variable region contains a binding domain that interacts with an antigen.

In this application, the term "light chain variable region" or "VL" generally refers to the immunoglobulin light chain structure containing the light chain interactions. The light chain variable region is the region of complement determining region CDR1, CDR2, CDR3 and framework region FR2, FR3. In some embodiments, the light chain variable region also includes FR1 and/or FR4. In this application, the term "CDR" generally refers to the complementarity determining region, and CDR is mainly responsible for binding to the antigen epitope. The CDR of the heavy chain is generally referred to as HCDR1, HCDR2 and HCDR3, and is numbered sequentially from the N-terminus. In this application, CDRs can be defined or identified by conventional methods. For example, according to the IMGT numbering method of Lefranc et al. (Lefranc M P, C Pommié, Ruiz M, et al. Developmental & Comparative Immunology, 2003, 27 (1): 55-77.). For example, according to the method of Kabat et al. (Wu, TT and Kabat, EA, J Exp Med. 132(2):211-50, (1970); Borden, P. and Kabat EA, PNAS, 84:2440-2443 (1987), or by the method of Chothia et al. (Chothia, C. and Lesk, AM, J Mol. Biol., 196(4):901-917 (1987). For example, the method for defining or identifying CDRs in the present application is the IMGT numbering method.

In the present application, the term "single-chain antibody (scFv)" generally refers to a fusion protein comprising at least one antibody fragment including a light chain variable region (VH) and at least one antibody fragment including a heavy chain variable region (VL), wherein the light chain and heavy chain variable regions are adjacent (e.g., via a synthetic linker such as a short flexible polypeptide linker), and can be expressed in a single-chain polypeptide form, and wherein the scFv retains the specificity of the complete antibody from which it is derived. Unless otherwise specified, as used herein, scFv may have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and scFv may include VL-linker-VH or may include VH-linker-VL. For example, scFv may be an antibody formed by connecting the heavy chain variable region (VH) and the light chain variable region (VL) of the antibody. For example, the VH and the VL may be connected by a linker. For example, the linker may be a polypeptide.

In the present application, the term "hinge region" generally refers to a binding domain that plays a role in positioning the binding domain away from the effector cell surface in a chimeric antigen receptor to enable appropriate cell/cell contact, antigen binding and activation. In the present application, the hinge region may be located between the binding domain and the transmembrane domain. The hinge region may be derived from natural sources, synthetic sources, semi-synthetic sources or recombinant sources.

In this application, the term "CD28" generally refers to T-cell-specific surface glycoprotein CD28. For example, CD28 can be found under the UniProt number P10747. CD28 can include its variants, homologs, and functionally active fragments thereof.

In the present application, the term "CD28 hinge region" generally refers to any polypeptide comprising an amino acid sequence having sequence identity or similarity to a naturally occurring CD28 hinge region sequence. For example, the CD28 hinge region may comprise an amino acid sequence from position 114 to position 152 of an amino acid sequence having a UniProt number of P10747. For example, the CD28 hinge region may comprise a functional variant of a CD28 hinge region sequence.

In this application, the term "transmembrane domain" can be used interchangeably with "transmembrane region (TM)" to refer to CAR A part of the CAR that fuses the extracellular binding part and the intracellular signal transduction domain and anchors the CAR to the plasma membrane of the immune effector cell. The transmembrane region can be obtained from natural proteins or can be synthesized, semi-synthesized or recombinantly obtained. The TM domain may include at least the transmembrane domain of the following proteins: CD8, CD28, CD3ε (CD3e), 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L (CD154), TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, SLAM and their variants.

In the present application, the term "CD28 transmembrane domain" generally refers to any polypeptide comprising an amino acid sequence having sequence identity or similarity to a naturally occurring CD28 transmembrane sequence. For example, the CD28 transmembrane domain may comprise an amino acid sequence from position 153 to position 179 of the amino acid sequence of UniProt number P10747. For example, the CD28 transmembrane domain may comprise a functional variant of the CD28 transmembrane sequence.

In this application, the term "intracellular domain" is used interchangeably with "intracellular domain" and generally refers to the inner intracellular part of a molecule.

In the present application, the term "intracellular signaling domain" is generally a part of a CAR that is involved in transducing information of an effective anti-BCMA CAR binding human BCMA polypeptide into the interior of an immune effector cell to trigger the function of the effector cell, such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors to CAR-bound target cells or other cellular responses triggered by antigens bound to an extracellular CAR domain. In the present application, the intracellular signaling domain may include a signaling domain of CD3ζ.

In the present application, the term "costimulatory domain" generally refers to the intracellular signal transduction domain of a costimulatory molecule. For example, a costimulatory molecule can be a cell surface molecule other than an antigen receptor or an Fc receptor, which can provide a second signal required for the efficient activation and function of T lymphocytes when the antigen binds. For example, the costimulatory domain can be selected from the following group: CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM and ZAP70.

In this application, the term "CD3ζ" generally refers to CD247 or T-cell surface glycoprotein CD3 zeta chain. CD3ζ can be found under UniProt number P20963. CD3ζ can include variants, homologs, and functionally active fragments thereof.

In the present application, the term "directly or indirectly linked" refers to direct linkage via a peptide bond, or indirect linkage via a linker or via a non-peptide linkage.

In the present application, the term "functional variant" generally refers to an amino acid sequence that has substantially the same function (e.g., can have the properties of the chimeric antigen receptor) and has at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity with the amino acid sequence. In certain embodiments, the variant of the amino acid sequence is an amino acid sequence that has substantially the same function (e.g., can have the properties of the chimeric antigen receptor) and comprises one or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10 or more) amino acid substitutions, deletions or additions thereto.

In the present application, the term "vector" generally refers to a nucleic acid molecule capable of transporting another nucleic acid connected to it. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which other DNA segments can be connected. Another type of vector is a viral vector, in which other DNA segments can be connected to a viral genome. Some vectors can replicate autonomously in the host cells they introduce (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell, thereby replicating together with the host genome, such as naked RNA polynucleotides that cannot replicate autonomously, naked DNA polynucleotides, polynucleotides composed of DNA and RNA in the same chain, poly-lysine-coupled DNA or RNA, peptide-coupled DNA or RNA, liposome-coupled DNA, etc. In addition, some vectors can guide the expression of genes effectively connected to them. This type of vector is referred to as a "recombinant expression vector" (or simply "expression vector") in the present application. Generally speaking, the expression vector used in recombinant DNA technology is usually in the form of a plasmid. In this specification, "plasmid" and "vector" are used interchangeably, as plasmid is the most commonly used form of vector.

In the present application, the term "viral vector" generally refers to a non-wild-type recombinant viral particle that acts as a gene delivery vector and comprises a recombinant viral genome packaged in a viral capsid. Animal virus species used as vectors can include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses (AAV), herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (e.g., SV40).

In this application, the term "immune cell" generally refers to cells that participate in an immune response, such as cells that promote immune effector responses. Examples of immune cells include, but are not limited to, T cells, B cells, natural killer (NK) cells, NKT cells, mast cells, granulocytes, monocytes, lymphocytes, and macrophages. The term also includes engineered immune cells, such as immune cells that are genetically modified by adding exogenous genetic material in the form of DNA or RNA to the total genetic material of the cell.

In this application, the terms "T lymphocyte" and "T cell" can be used interchangeably, and generally refer to thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes or activated T lymphocytes. T cells can be helper T (Th) cells, such as helper T1 (Th1) cells or helper T2 (Th2) cells. T cells can be The T lymphocytes may be helper T cells (HTL; CD4+T cells), CD4+T cells, cytotoxic T cells (CTL; CD8+T cells), CD4+CD8+T cells or CD4-CD8-T cells. Alternatively, the T lymphocytes may include naive T cells and memory T cells.

In this application, the term "natural killer (NK) cell" generally refers to a subtype of leukocytes, which is a component of the innate immune system. NK cells play a major role in host rejection of tumors and virally infected cells. NK cells are cytotoxic and induce apoptosis. NK cells can be used to inhibit viral infections and produce antigen-specific cytotoxic T cells through adaptive immune responses to clear infections.

In the present application, the term "killing ability" generally refers to killing cells by contacting the cells with an effective amount of antibodies, immunoconjugates, bispecific/multispecific molecules or compositions. The method may include killing cells that express positive antigens, optionally in the presence of effector cells, such as by CDC, apoptosis, ADCC, phagocytosis or by a combination of two or more of these mechanisms.

In this application, the term "cytokine" generally refers to a protein released by a cell population that acts as an intercellular regulator on another cell. For example, cytokines can include interleukins (IL), interferons (IFN), chemokines, tumor necrosis factors (TNF) and transforming growth factors (TGF).

In the present application, the term "CD30-related disorder" generally refers to a disorder associated with CD30 expression. For example, a CD30-related disorder may include a tumor associated with CD30 expression. For example, CD30 expression in a tumor is determined using immunohistochemical staining of tumor cell membranes, wherein any immunohistochemical staining above background levels in a tumor sample indicates that the tumor is a tumor expressing CD30. Methods for detecting CD30 expression in cells are known in the art and include immunohistochemical assays. CD30-related disorders may include Hodgkin's lymphoma, anaplastic large cell lymphoma (ALCL), cutaneous T-cell lymphoma, adult T-cell lymphoma (ATL), angioimmunoblastic T-cell lymphoma (AITL).

In this application, the term "tumor" generally refers to or describes the physiological condition in mammals that is generally characterized by unregulated cell growth. Examples of tumors include, but are not limited to, carcinomas, lymphomas, blastomas (including medulloblastomas and retinoblastomas), sarcomas (including liposarcoma and synovial cell sarcomas), neuroendocrine tumors (including carcinoid tumors, gastrinomas, and islet cell carcinomas), mesotheliomas, schwannomas (including acoustic neuromas), meningiomas, adenocarcinomas, and melanomas.

In this application, the term "cancer" generally refers to a neoplasm formed when a cell in a local tissue loses normal regulation of its growth at the genetic level under the action of various carcinogenic factors, resulting in its clonal abnormal proliferation. Because this neoplasm is mostly a space-occupying mass protrusion, it is also called a neoplasm. In this application, the cancer may include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer or gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, Liver cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, head and neck cancer, B-cell lymphoma (including low-grade/follicular non-Hodgkin lymphoma NHL), small lymphocytic (SL) NHL, intermediate/follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphocytic NHL, high-grade small non-cleaved cell NHL, AIDS-related lymphoma, Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), hairy cell leukemia chronic myeloblastic leukemia and post-transplant lymphoproliferative disease (PTLD)). In the present application, the cancer may include cancer or malignancy associated with the expression of CD30.

In the present application, the term "carrier" refers to a diluent, adjuvant or excipient with which the polypeptide of the present disclosure is applied. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. The carrier can be a saline solution, gum arabic, gelatin, starch paste, mica, keratin, colloidal silica, urea, etc. In addition, adjuvants, stabilizers, thickeners, lubricants and colorants can also be used. Saline solutions and aqueous glucose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients, such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, mica, sodium chloride, dry skim milk, glycerol, propylene, ethylene glycol, water, ethanol, etc. If necessary, the composition of the present invention can also contain a small amount of wetting agent or emulsifier or pH buffer.

In the present application, the term "prevention and/or treatment" includes not only the prevention and/or treatment of a disease, but also generally includes preventing the onset of the disease, slowing down or reversing the progression of the disease, preventing or slowing down the onset of one or more symptoms associated with the disease, reducing and/or alleviating one or more symptoms associated with the disease, reducing the severity and/or duration of the disease and/or any symptoms associated therewith, and/or preventing further increase in the severity of the disease and/or any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by the disease and any pharmacological effects that are generally beneficial to the treatment of patients.

In this application, the term "subject" generally refers to an animal, usually a mammal, such as a human, non-human primate (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), livestock (dogs and cats), farm animals (poultry such as chickens and ducks, horses, cattle, goats, sheep, pigs), and laboratory animals (mice, rats, rabbits, guinea pigs). Human subjects may include fetuses, newborns, infants, adolescents, and adult subjects. Subjects may include animal disease models.

In the present application, the term "therapeutically effective amount" or "effective amount" generally refers to an amount of the present invention's antibody sufficient to prevent or alleviate symptoms associated with a disease or disorder (e.g., cancer). The therapeutically effective amount is related to the disease being treated, wherein those skilled in the art can easily discern the actual effective amount.

In this application, the term "drug" generally refers to a substance that, when properly administered to a patient, is capable of inducing a desired therapeutic effect. A chemical compound or composition.

In the present application, the term "composition" means a mixture containing one or more compounds described herein or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote administration to an organism, facilitate the absorption of the active ingredient and thus exert biological activity. The therapeutic composition should generally be sterile and stable under manufacturing and storage conditions. The composition can be formulated as a solution, microemulsion, dispersant, liposome or other ordered structure suitable for high antibody concentration. The active compound (i.e., antibody or antibody portion) can be incorporated in a suitable solvent in the required amount together with one of the components or component combinations listed above, and then filtered and sterilized as needed to prepare a sterile injectable solution.

In the present application, the term "anti-tumor activity" refers to a decrease in the proliferation rate, viability or metastatic activity of tumor cells. For example, anti-tumor activity can be shown by a decrease in the growth rate of abnormal cells that occur during treatment, or a stable or reduced tumor size, or a longer survival period due to treatment compared to a control without treatment. This activity can be evaluated using recognized in vitro or in vivo tumor models, including but not limited to xenograft models, allograft models, MMTV models, and other known models known in the art for studying anti-tumor activity.

In this application, the term "adjuvant" generally refers to any substance that assists or modulates the action of a drug, including but not limited to immunological adjuvants, which enhance or diversify the immune response to an antigen.

In this application, the term "comprises", "comprising" or "including" generally means including the features explicitly stated, but not excluding other elements.

In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below a specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

DETAILED DESCRIPTION OF THE INVENTION

Without intending to be bound by any theory, the following embodiments are merely intended to illustrate the methods and uses of the present application and are not intended to limit the scope of the invention of the present application.

The present application provides a chimeric antigen receptor (CAR), the CAR comprising: a CD30 binding domain and a hinge region, wherein the CD30 binding domain comprises a heavy chain complementary determining region 1 (HCDR1), a heavy chain complementary determining region 2 (HCDR2), a heavy chain complementary determining region 3 (HCDR3), a light chain complementary determining region 1 (LCDR1), a light chain complementary determining region 2 (LCDR2) and a light chain complementary determining region 3 (LCDR3), the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 2 and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 3, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 4, and the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 5. The amino acid sequence shown in SEQ ID NO: 5, the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 6, and the hinge region is derived from CD28.

In the present application, the CD30 binding domain may comprise an anti-CD30 antibody or an antigen-binding fragment thereof. For example, the CD30 binding domain may comprise a heavy chain variable region. For example, the heavy chain variable region comprises HCDR1 to 3 of the sequence shown in SEQ ID NO: 1 to 3. For example, the heavy chain variable region may comprise the sequence shown in SEQ ID NO: 7 or a functional variant thereof. For example, the CD30 binding domain may comprise a light chain variable region. For example, the light chain variable region comprises LCDR1 to 3. For example, the light chain variable region comprises the sequence LCDR1 to 3 shown in SEQ ID NO: 4 to 6. For example, the light chain variable region may comprise the sequence shown in SEQ ID NO: 8 or a functional variant thereof. For example, a linker is included between the light chain variable region and the heavy chain variable region. For example, the linker may comprise the amino acid sequence shown in SEQ ID NO: 21.

In the present application, the CD30 binding domain may be a single-chain antibody. For example, the CD30 binding domain is a single-chain scFv antibody, wherein the single-chain scFv comprises a VL domain-linker-VH domain or a VH domain-linker-VL domain from the N-terminus to the C-terminus. For example, the CD30 binding domain comprises but is not limited to the amino acid sequence shown in SEQ ID NO: 9 or a functional variant thereof. The functional variant is selected from the following group: a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the CD30 binding domain; and a protein or polypeptide having a sequence homology of more than 90% (e.g., at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) with the CD30 binding domain.

In the present application, the CD30 binding domain can have a Ka (i.e., the equilibrium association constant for the binding interaction, which is ^{1 / M} ) of greater than or equal to about 10 ⁵ M ^-1 (e.g., greater than or equal to about 10 ⁵ M ^-1 , greater than or equal to about 10 ⁶ M ^-1 , greater than or equal to about 10 ⁷ M ^-1 , greater than or equal to about 10 ⁸ M ^-1 , greater than or equal to about 10 ⁹ M ^-1 , greater than or equal to about 10 ¹⁰ M ^-1 , greater than or equal to about 10 ¹¹ M -1, greater than or equal to about 10 ¹² M ^-1 , greater than or equal to about 10 ¹³ M ^-1 , or ^higher ); or, have a Ka of less than or equal to about ^{10 -5} M (e.g., less than or equal to about 10 ^-5 M, less than or equal to about 10 ^-6 M, less than or equal to about 10 ^-7 M, less than or equal to about 10 ^-8 M, less than or equal to about 10 ^-9 M, less than or equal to about 10 ^-10 The present invention relates to a polynucleotide that binds or associates with CD30 with an equilibrium dissociation constant ^, Kd, of about 10-14 M, about ^10-11 M or less, about ^10-12 M or less, about ^10-13 M or less.

In the present application, the affinity of the CD30 binding domain and CD30 can be determined using conventional techniques in the art, for example, by competitive ELISA (enzyme-linked immunosorbent assay), or by binding association, or by displacement assay using a labeled ligand, or by using a surface plasmon resonance device (such as Biacore T100, which can be obtained from Biacore, Inc., Piscataway, NJ) or optical biosensor technology.

In the present application, the CD30 binding domain may be connected to the transmembrane domain via a hinge region. For example, the CD30 binding domain may be directly or indirectly connected to a hinge region derived from CD28. The hinge region may include one or more hinge regions between the CD30 binding domain and the transmembrane domain. For example, the hinge region may include mutations or substitutions of some amino acids, for example, mutations or substitutions of proline residues by other amino acid residues (e.g., serine residues). In the present application, the hinge region may include an amino acid sequence as shown in SEQ ID NO: 11 or a functional variant thereof. The functional variant is selected from the following groups: a protein or polypeptide having one or more amino acids substituted, deleted or added in the hinge region; and a protein or polypeptide having a sequence homology of more than 90% (e.g., at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) with the hinge region.

In the present application, the CAR may include a transmembrane domain. In the present application, the transmembrane domain may include a polypeptide selected from the following proteins: CD8, CD28, CD3ε (CD3e), 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L (CD154), TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, SLAM. In the present application, the transmembrane domain may be derived from the transmembrane domain of CD28. In the present application, the transmembrane domain may comprise an amino acid sequence as shown in SEQ ID NO: 12 or a functional variant thereof. The functional variant is selected from the following groups: a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the transmembrane domain; and a protein or polypeptide having a sequence homology of more than 90% (e.g., at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) with the transmembrane domain.

In the present application, the CAR may include one or more intracellular costimulatory domains. For example, the costimulatory domain may be connected to the C-terminus of the transmembrane domain. In the present application, the costimulatory domain may include a costimulatory domain selected from: CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKG2C, SLP76, TRIM and ZAP70. For example, the CAR includes a costimulatory domain. In the present application, the intracellular costimulatory domain may include a costimulatory domain derived from CD28. For example, the costimulatory domain may comprise an amino acid sequence as shown in SEQ ID NO: 13 or a functional variant thereof. The functional variant is selected from the following groups: a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the intracellular costimulatory domain; and a protein or polypeptide having a sequence homology of more than 90% (e.g., at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) with the intracellular costimulatory domain.

In the present application, the CAR may comprise one or more intracellular signal transduction domains. The signal transduction domain may include an intracellular signal transduction domain selected from the following CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FceRIγ, FceRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, DAP10, DAP-12 and a domain comprising at least one ITAM. For example, the CAR comprises an intracellular signal transduction domain. In the present application, the intracellular signal transduction domain may include a CD3ζ intracellular signal transduction domain. For example, the intracellular signal transduction domain may include an amino acid sequence as shown in SEQ ID NO: 14 or a functional variant thereof. The functional variant is selected from the following groups: a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the intracellular signal transduction domain; and a protein or polypeptide having a sequence homology of more than 90% (e.g., at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) with the intracellular signal transduction domain.

In the present application, the CAR may include a signal peptide. For example, the signal peptide may include a signal peptide selected from CD8 and CD45. For example, the signal may include a CD8α signal peptide. For example, the signal peptide may include an amino acid sequence as shown in SEQ ID NO: 10 or a functional variant thereof. Wherein the functional variant is selected from the following groups: a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the signal peptide; and a protein or polypeptide having a sequence homology of more than 90% (e.g., at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) with the signal peptide.

In the present application, the CAR may include a linker between the domains. For example, a linker added for proper spacing and conformation between the domains. In the present application, the linker may include the following amino acid sequences: GGG (SEQ ID NO: 22), (GGGGS)n (SEQ ID NO: 23) and GGRRGGGS (SEQ ID NO: 24).

In the CAR of the present application, the transmembrane domain may be derived from CD28, the co-stimulatory domain may be derived from CD28, and the intracellular signaling domain may be derived from CD3ζ.

In the present application, the CAR may comprise a signal peptide sequence comprising an amino acid sequence as shown in SEQ ID NO:10, a HCDR1 comprising an amino acid sequence as shown in SEQ ID NO:1, a HCDR2 comprising an amino acid sequence as shown in SEQ ID NO:2, a HCDR3 comprising an amino acid sequence as shown in SEQ ID NO:3, a LCDR1 comprising an amino acid sequence as shown in SEQ ID NO:4, a LCDR2 comprising an amino acid sequence as shown in SEQ ID NO:5, a CD30 binding domain comprising an amino acid sequence as shown in SEQ ID NO:6, a hinge region comprising an amino acid sequence as shown in SEQ ID NO:11, a transmembrane domain comprising an amino acid sequence as shown in SEQ ID NO:12, a costimulatory domain comprising an amino acid sequence as shown in SEQ ID NO:13, and an intracellular signal transduction domain comprising an amino acid sequence as shown in SEQ ID NO:14.

In the present application, the CAR may comprise a signal peptide sequence comprising an amino acid sequence as shown in SEQ ID NO: 10, a CD30 binding domain comprising an amino acid sequence as shown in SEQ ID NO: 9, a CD30 binding domain comprising an amino acid sequence as shown in SEQ ID NO: 11, The hinge region comprises an amino acid sequence as shown in SEQ ID NO: 12, a transmembrane domain comprises an amino acid sequence as shown in SEQ ID NO: 13, and an intracellular signal transduction domain comprises an amino acid sequence as shown in SEQ ID NO: 14.

In the present application, the CAR may comprise an amino acid sequence as shown in SEQ ID NO: 15 or a functional variant thereof. In the present application, the CAR may comprise an amino acid sequence as shown in SEQ ID NO: 16 or a functional variant thereof. Wherein the functional variant is selected from the following groups: a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the CAR; and a protein or polypeptide having a sequence homology of more than 90% (e.g., at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) with CAR.

The present application provides an isolated nucleic acid molecule that can encode the CAR. The nucleic acid molecule described in the present application can contain a nucleotide sequence as shown in SEQ ID NO: 17.

The present application provides a vector, which may contain the nucleic acid molecule.

In the present application, the vector may contain one or more of a replication origin, a selection box, a promoter, an enhancer, a translation initiation signal (Shine Dalgarno sequence or Kozak sequence), an intron, a polyadenylation sequence, and 5' and 3' untranslated regions.

In the present application, the vector may be selected from a plasmid, a bacteriophage, an artificial chromosome (e.g., a yeast artificial chromosome YAC) and an animal virus. For example, the vector may be selected from a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector and a retroviral vector. For example, the vector described in the present application may comprise a nucleotide sequence as shown in SEQ ID NO: 18.

The present application provides a cell, which comprises the CAR, the nucleic acid molecule and/or the vector.

In the present application, the cell may comprise an immune cell. For example, the immune cell may comprise a cell selected from a T cell, a natural killer (NK) cell, or a NKT cell. For example, the immune cell is a T cell.

The present application provides a method for preparing cells, which comprises introducing the CAR, the nucleic acid molecule and/or the vector into the cells.

In the present application, the cells may include immune cells. For example, the immune cells may include cells selected from T cells, natural killer (NK) cells, and NKT cells. For example, the immune cells are T cells. In the present application, the method may include the step of obtaining the immune effector cells from the subject. For example, the T lymphocytes can be obtained from peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In addition, the method may further include the step of selecting specific cell subsets from the immune effector cells. For example, specific expression of CD3, CD28, CD4, CD8, CD45RA, and CD45RO can be used to identify the specific cell subsets. In certain situations, select specific T cell subsets.

The present application provides a composition, which may include the CAR, the nucleic acid molecule, the vector and/or the cell, and optionally a pharmaceutically acceptable carrier.

In the present application, the pharmaceutically acceptable carrier can be selected from the following groups: excipients, glidants, sweeteners, diluents, preservatives, dyes/colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, surfactants and emulsifiers.

In the present application, the composition may further comprise other active ingredients, such as one or more of cytokines, growth factors, hormones, small molecule chemical drug active ingredients, prodrugs and antibodies.

In the present application, the amount of the cells in the composition can be an effective amount. The effective amount can be the minimum dose required to achieve a beneficial or desired preventive or therapeutic effect. For example, the effective amount can be affected by factors such as the degree of disease, age, weight, gender, etc. of the subject.

The present application provides the use of the CAR, the nucleic acid molecule, the vector, or the cell for preparing a drug, wherein the drug is used to treat a disease or condition associated with the expression of CD30.

The present application provides the CAR, the nucleic acid molecule, the vector, or the cell, which is used to treat a disease or condition related to the expression of CD30.

The present application provides a method for preventing and/or treating a disease or condition associated with the expression of CD30, comprising the following steps: administering the CAR, the nucleic acid molecule, the vector, or the cell to a subject.

In the present application, the disease or disorder associated with the expression of CD30 may include cancer or malignant tumor. For example, the tumor may include lymphoma. For example, the tumor may include Hodgkin's lymphoma or non-Hodgkin's lymphoma. For example, the tumor may include Hodgkin's lymphoma, anaplastic large cell lymphoma (ALCL), cutaneous T-cell lymphoma, adult T-cell lymphoma (ATL), angioimmunoblastic T-cell lymphoma (AITL).

In the present application, the administration of the drug may include aerosol inhalation, injection, ingestion, infusion, implantation or transplantation. For example, the injection may include intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection, or may be directly injected into a tumor or lymph node.

In the present application, the CAR may have one or more effects selected from the following: (1) excellent expression efficiency; (2) cells expressing the chimeric antigen receptor have significant anti-tumor activity; (3) cells expressing the chimeric antigen receptor have sustained anti-tumor activity; (4) cells expressing the chimeric antigen receptor have significant cell activation ability; and (5) cells expressing the chimeric antigen receptor have significant cytokine secretion ability.

For example, the effect may refer to a CAR targeting CD30 other than the CAR described in the present application. For example, the effect may refer to a CAR targeting CD30 comprising a hinge region other than the CD28 hinge region. For example, the effect may refer to a CAR targeting CD30 comprising a hinge region other than the CD28 hinge region. For example, the effect may refer to a CAR targeting P050. For example, the effect may refer to a CAR comprising a HCDR1 having an amino acid sequence as shown in SEQ ID NO: 1, a HCDR2 having an amino acid sequence as shown in SEQ ID NO: 2, a HCDR3 having an amino acid sequence as shown in SEQ ID NO: 3, a LCDR1 having an amino acid sequence as shown in SEQ ID NO: 4, a LCDR2 having an amino acid sequence as shown in SEQ ID NO: 5, a LCDR3 having an amino acid sequence as shown in SEQ ID NO: 6, and a hinge region other than the CD28 hinge region. For example, the effect may refer to a CAR comprising a heavy chain variable region VH having an amino acid sequence as shown in SEQ ID NO: 7, a light chain variable region VL having an amino acid sequence as shown in SEQ ID NO: 8, and a hinge region other than the CD28 hinge region. For example, the effect may be relative to P038 CAR. For example, the effect may be relative to a CAR comprising a CD30 binding domain having an amino acid sequence as shown in SEQ ID NO: 9 and a hinge region other than a CD28 hinge region.

For example, the CAR may have excellent expression efficiency. For example, the expression efficiency may refer to CARs other than the CAR described in the present application. For example, the expression efficiency may refer to CARs targeting CD30 other than the CAR described in the present application. For example, cells expressing the CAR may have excellent expression efficiency. For example, the expression efficiency may refer to cells that do not express CAR. For example, the expression efficiency may refer to cells before expressing the CAR. For example, the expression efficiency may refer to cells expressing CARs other than the CAR described in the present application. For example, the expression efficiency may refer to cells expressing CARs targeting CD30 other than the CAR described in the present application. For example, the expression efficiency may refer to the CAR positive rate of cells expressing the CAR. For example, the expression efficiency may be detected by technical means known in the art. For example, the expression efficiency may be detected by flow cytometry. For example, the expression efficiency of the CAR can be greater than at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300% or more.

For example, cells expressing the CAR may have significant anti-tumor activity. For example, the anti-tumor activity may be manifested as a killing effect on tumor cells. For example, the anti-tumor activity may be manifested as an inhibitory effect on tumor cell growth. For example, the anti-tumor activity may be manifested as a stabilization or reduction of tumor size. For example, the anti-tumor activity may be detected by technical means known in the art. For example, the anti-tumor activity may be detected by in vitro experiments. For example, the anti-tumor activity may be detected by in vivo experiments. For example, the anti-tumor activity may refer to cells that do not express CAR. For example, the anti-tumor activity may refer to cells before expressing the CAR. For example, the anti-tumor activity may refer to cells that express CAR other than the CAR described in the present application. For example, the anti-tumor activity It may refer to cells expressing CAR targeting CD30 other than the CAR described herein. For example, the anti-tumor activity may refer to the anti-tumor activity exhibited by administering cells expressing the CAR. For example, the anti-tumor activity may refer to cells expressing the CAR that are not administered. For example, the anti-tumor activity may refer to cells that do not express the CAR. For example, the anti-tumor activity may refer to cells expressing the CAR before administration. For example, the anti-tumor activity of cells expressing the CAR may be higher than at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300% or more.

For example, the cell expressing the CAR may have sustained anti-tumor activity. For example, the sustained anti-tumor activity may be manifested as the cell expressing the CAR can still be detected after a period of time after the cell expressing the CAR is administered. For example, the sustained anti-tumor activity can be detected by technical means known in the art. For example, the cell expressing the CAR can be detected by technical means known in the art. For example, the sustained anti-tumor activity may refer to cells that do not express CAR. For example, the sustained anti-tumor activity may refer to cells before expressing the CAR. For example, the sustained anti-tumor activity may refer to cells expressing CAR other than the CAR described in the present application. For example, the sustained anti-tumor activity may refer to cells expressing CD30 targeting CAR other than the CAR described in the present application. For example, the sustained anti-tumor activity may refer to the anti-tumor activity exhibited by administering cells expressing the CAR. For example, the sustained anti-tumor activity may refer to cells that do not express the CAR. For example, the sustained anti-tumor activity may refer to cells that do not express the CAR. For example, the anti-tumor activity may refer to cells before administering the CAR. For example, the anti-tumor activity of cells expressing the CAR can last for at least 1 day, 7 days, 21 days, 28 days, 1 month, 3 months, 6 months, 12 months, 18 months, 24 months, 30 months, 36 months or longer.

For example, the cell expressing the CAR has significant cell activation ability. For example, the cell activation ability may include the ability to activate immune cells. For example, the cell activation ability may include the ability to activate T cells. For example, the cell expressing CAR can significantly activate T cells. For example, the cell activation ability may refer to an increase in the measurable activity of activated cells. For example, the cell activation ability can be detected by technical means known in the art. For example, the measurable activity of activated cells can be detected by technical means known in the art. For example, the cell activation ability may refer to cells that do not express CAR. For example, the anti-tumor activity may refer to cells before expressing the CAR. For example, the cell activation ability may refer to cells expressing CARs other than the CAR described in this application. For example, the cell activation ability may refer to cells expressing CD30-targeted CARs other than the CAR described in this application. For example, the cell activation ability may refer to the expression of cells expressing the CAR. Anti-tumor activity. For example, the cell activation ability may refer to cells expressing the CAR that are not administered. For example, the cell activation ability may refer to cells that do not express the CAR. For example, the cell activation ability may refer to cells that are relative to the administration of cells that do not express the CAR. For example, the cell activation ability may refer to cells expressing the CAR before administration. For example, the cell activation ability of the CAR expressing the CAR may be higher than at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300% or more.

For example, the cell expressing the CAR has a significant cytokine secretion capacity. For example, the cytokine secretion capacity can be expressed as the amount of cytokine secretion. For example, the cytokine secretion capacity can be detected by technical means known in the art. For example, the measurable activity of activated cells can be detected by technical means known in the art. For example, the cytokine secretion capacity can refer to cells that do not express CAR. For example, the cytokine secretion capacity can refer to cells before expressing the CAR. For example, the cytokine secretion capacity can refer to cells expressing CAR other than the CAR described in the present application. For example, the cytokine secretion capacity can refer to cells expressing CD30 targeting CAR other than the CAR described in the present application. For example, the cytokine secretion capacity can refer to the cytokine secretion capacity of cells expressing the CAR ... For example, the cytokine secretion capacity of the cells expressing the CAR can be higher than at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300% or more.

The present application is further described below through detailed examples. It should be understood that the following examples are only used to illustrate the present application and do not limit the content of the invention.

Example

Example 1 Preparation of CD30 CAR virus and CD30 CAR-T cells

1) CD30 CAR retroviral packaging

HEK293T cells with good growth status were digested and centrifuged for medium replacement. 2×10 ⁷ cells per dish were inoculated in 15 cm culture dishes and cultured for 24 hours. CD30 CAR plasmid (inserted into the vector after full gene synthesis, the vector sequence is shown in SEQ ID NO: 18) and auxiliary plasmid gag-pol plasmid (purchased from NovoPro, catalog number V006593) and GALV plasmid (purchased from NovoPro, catalog number V000819) were pre-mixed with polyetherimide (PEI) at a ratio of 4:3:1, and then 0.5 ml of the transfection mixture was added dropwise to HEK293T cells (about 4×10 ⁷ ) and gently mixed. Cultured at 37°C and 5% CO ₂ , and collected after 48 hours. Supernatant.

2) Preparation of CD30 CAR-T cells

Fresh blood from healthy individuals was collected and peripheral blood mononuclear cells (PBMC) were separated using lymphocyte separation medium. The separated cells were resuspended in T cell culture medium (X-VIVO15 culture medium containing 300 IU/mL IL-2), T Cell TransAct ^TM (Miltenyi) reagent was added, and the cells were placed in a cell culture incubator for activation for 48 hours.

Novonectin protein (nearshore) was diluted to 20 ng/mL with DPBS and added to an untreated 12-well plate at 1 mL/well for overnight coating. The next day, the coating solution was removed and 2 mL/well of CD30 CAR virus supernatant was added. The cells were centrifuged at 32°C and 2000 g for 120 min. The activated T cells for 2 days were centrifuged and resuspended in T cell culture medium for counting and diluted to 2.5×10 ⁵ cells/mL. The cells were added to a 12-well plate at 1 mL/well and centrifuged at 32°C and 400 g for 10 min. The cells were placed in an incubator and cultured overnight. The cells were transferred to a new 12-well plate the next day and the culture medium was changed every 3 days.

On the 6th day after the virus transfection of T cells, the expression of the CAR was detected. The results are shown in Figure 1A-Figure 1B. The CD30 CAR described in the present application can be expressed in T cells.

Example 2 CD30 CAR-T in vitro killing experiment

This example uses a luciferase method to validate the function of CD30 CAR effector cells in killing target cells. The experimental results are shown in Figure 2. This example shows through in vitro experiments that T cells expressing the CAR described in this application can significantly kill target cells and have significant anti-tumor activity.

The lentiviral packaging kit (Aikangde Biotechnology, catalog number LVP2MIX) was mixed with a plasmid expressing luciferase (purchased from NovoPro, catalog number V006878) and then transfected into HEK293T cells. The virus preparation method was the same as described in Example 1. After the human Hodgkin's lymphoma cell line L-428 cells were transduced with the virus, they were cultured for 3-5 days, and monoclonal cells expressing luciferase were sorted by flow cytometry and continued to be cultured for subsequent experiments.

L-428 expressing luciferase was used as target cells. The target cells were diluted to 2×10 ⁵ cells/mL and inoculated into 96-well plates at 50 μL/well. The cells in the CAR-T and blank control groups were counted according to the effector-target ratio (E:T) of 8:1, 4:1, and 2:1, and added to the corresponding 96-well plates. After incubation for 16 hours, 50 μL of luciferase substrate (Promega) was added, and the remaining luciferase activity in each well was evaluated to quantify the remaining live target cells in each well.

Example 3 In vitro functional verification of CD30 CAR-T cells

This example analyzes the release of IL-2 and IFN-γ by T cells expressing the CD30 CAR described in this application under tumor cell stimulation by ELISA test. The experimental results are shown in Figures 3 and 4. This example shows that T cells expressing the CD30 CAR described in this application have significant cytokine secretion ability.

CAR-T cells were co-cultured with CD30-positive L-428 cells (purchased from DSMZ, catalog number: ACC 197). The effector-target ratio (E:T) was 8:1, 4:1, and 2:1 for incubation for 16 hours, and then the supernatant was collected by centrifugation. The cytokines in the supernatant were quantified using the Human IL-2 ELISA Kit II (BD Biosciences) and the Human IFN-γ ELISA Set (BD Biosciences). The absorbance of each well of the sample to be tested was read at 450 nm by a multi-mode microplate reader (Thermoscientific#Varioskan LUX), and the cytokine concentration corresponding to the absorbance of the sample to be tested was calculated based on the standard curve of the absorbance of the standard. For specific operations, refer to the instructions of the above kits.

Example 4 In vivo efficacy evaluation of CD30 CAR-T cells in tumor xenograft mice

This example tests the killing/clearing ability of CD30 CAR-T cells on mouse xenografted Hodgkin lymphoma in NOG mice. The experimental results are shown in Figure 5. This example illustrates through in vivo experiments that cells expressing the CAR described in this application have significant anti-tumor activity.

NOG mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) that passed the adaptive observation were selected, and 5×10 ⁶ L-428 cells (purchased from DSMZ, item number: ACC 197) were injected subcutaneously into each mouse. When the tumor volume was about 50 cubic millimeters after 5 days of feeding, the mice were randomly divided into groups and injected with 1×10 ⁷ CAR-T cells (prepared according to the method of Example 1, the solvent was PBS buffer) and non-virus-infected control T cells (the solvent was PBS buffer) through the tail vein. The tumor diameter was measured with a vernier caliper twice a week. The tumor volume was calculated as follows: V=0.5×a×b ² , where a and b are the length and width of the tumor, respectively.

Example 5 Comparison of T cells expressing different CD30 CARs

According to the preparation method in Example 1, T cells expressing P038 CAR and P050 CAR were prepared. As shown in Figure 6, P038 CAR and the CD30 CAR (P037 CAR) described in this application can bind to CD30, but the antigen binding domain sequence is different, which is 5F11scFv, wherein the amino acid sequence of the heavy chain variable region of 5F11 is shown in SEQ ID NO: 19, and the amino acid sequence of the light chain variable region of 5F11 is shown in SEQ ID NO: 20, and the heavy chain variable region and the light chain variable region are connected by the linker shown in SEQ ID NO: 21. The hinge region of P050 CAR is different from that of the CD30 CAR described in this application, and the hinge region of P050 CAR is derived from IgG4. Figure 7 shows the expression of different CARs after viral transduction of T cells, and the results show that different CD30 CARs can be expressed in T cells.

According to the methods in Examples 2 and 3, the function of CAR-T cells expressing different CD30 CARs in killing target cells in vitro (human Hodgkin's lymphoma cell line L540 cells, DSMZ German Collection of Microorganisms and Cell Culture GmbH, catalog number ACC72) and the release of IL-2 factors and IFN-γ under the stimulation of tumor cells (human Hodgkin's lymphoma cell line L540 cells) were detected. As shown in Figure 8A, the killing effect of T cells expressing the CD30 CAR described in this application on target cells is significantly stronger than that of T cells expressing P050 CAR. As shown in Figure 8B, the release ability of IFN-γ after incubation of T cells expressing CD30 CAR described in this application with L540 cells is stronger than that of T cells expressing P050 CAR. As shown in Figure 9A, the killing effect of T cells expressing CD30 CAR described in this application on target cells is stronger than that of T cells expressing As shown in Figures 9B-9C, the release of IL-2 and IFN-γ by T cells expressing the CD30 CAR described in the present application after incubation with L540 cells was significantly stronger than that by T cells expressing P038 CAR.

According to the method in Example 4, the in vivo efficacy of CAR-T cells expressing different CD30 CARs in mice with tumor xenografts (human Hodgkin lymphoma cell line L540 cells) was detected. As shown in Figures 10A-10B, the T cells expressing the CD30 CAR described in the present application have a stronger ability to inhibit tumor volume growth than the T cells expressing P037 CAR, and show good safety. As shown in Figures 11A-11B, the T cells expressing the CD30 CAR described in the present application have a stronger ability to inhibit tumor volume growth than the T cells expressing P038 CAR, and the survival time of the mice is longer, showing good safety.

Claims (47)

1. A chimeric antigen receptor (CAR), the CAR comprising: a CD30 binding domain and a hinge region, wherein the CD30 binding domain comprises a heavy chain complementary determining region 1 (HCDR1), a heavy chain complementary determining region 2 (HCDR2), a heavy chain complementary determining region 3 (HCDR3), a light chain complementary determining region 1 (LCDR1), a light chain complementary determining region 2 (LCDR2) and a light chain complementary determining region 3 (LCDR3), the HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 1, the HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 2 and the HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 3, the LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 4, the LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 5, the LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 6, and the hinge region is derived from CD28.
2. The CAR according to claim 1, wherein the CD30 binding domain comprises a heavy chain variable region VH, and the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 7 or a functional variant thereof.
3. The CAR according to any one of claims 1-2, wherein the CD30 binding domain comprises a light chain variable region VL, and the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 8 or a functional variant thereof.
4. The CAR according to any one of claims 1-3, wherein the CD30 binding domain is a single-chain antibody.
5. The CAR according to any one of claims 1-4, wherein the CD30 binding domain is a single-chain scFv antibody, wherein the single-chain scFv comprises, from N-terminus to C-terminus, a VL domain-linker-VH domain or a VH domain-linker-VL domain.
6. The CAR according to any one of claims 1-5, wherein the CD30 binding domain comprises the amino acid sequence shown in SEQ ID NO: 9 or a functional variant thereof.
7. The CAR according to any one of claims 1-6, wherein the hinge region comprises a CD28 hinge region or a functional variant thereof.
8. A CAR according to any one of claims 1-7, wherein the hinge region comprises an amino acid sequence as shown in SEQ ID NO: 11 or a functional variant thereof.
9. The CAR according to any one of claims 1-8, wherein the CD30 binding domain is directly or indirectly connected to the hinge region derived from CD28.
10. The CAR according to any one of claims 1-9, wherein the CAR comprises the amino acid sequence shown in SEQ ID NO: 15 and its functional variants.
11. The CAR of any one of claims 1-10, wherein the CAR further comprises a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular signaling domain.
12. The CAR according to any one of claims 1 to 11, wherein the CAR comprises a transmembrane domain, the transmembrane domain comprising a transmembrane domain derived from one or more proteins in the following group: CD8, CD28, CD3ε (CD3e), 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L (CD154), TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, SLAM and their variants.
13. The CAR of any one of claims 1-12, wherein the CAR comprises a transmembrane domain derived from CD28.
14. The CAR according to any one of claims 1-13, wherein the CAR comprises a transmembrane domain, and the transmembrane domain comprises an amino acid sequence as shown in SEQ ID NO: 12 or a functional variant thereof.
15. The CAR according to any one of claims 1-14, wherein the CAR further comprises one or more intracellular co-stimulatory domains, wherein the co-stimulatory domain comprises a co-stimulatory domain of one or more proteins derived from the following group: CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKG2C, SLP76, TRIM and ZAP70.
16. The CAR according to any one of claims 1-15, wherein the CAR comprises an intracellular co-stimulatory domain, and the co-stimulatory domain is derived from CD28.
17. The CAR according to any one of claims 1-16, wherein the CAR comprises an intracellular co-stimulatory domain, and the co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO: 13 or a functional variant thereof.
18. The CAR according to any one of claims 1-17, wherein the CAR comprises one or more intracellular signaling domains, and the intracellular signaling domain comprises an intracellular signaling domain derived from one or more proteins of the following group: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FceRIγ, FceRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, DAP10, DAP-12 and a domain comprising at least one ITAM.
19. The CAR of any one of claims 1-18, wherein the CAR comprises an intracellular signaling domain, and the intracellular signaling domain is CD3ζ.
20. The CAR according to any one of claims 1-19, wherein the CAR comprises an intracellular signaling domain, and the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO: 14 or a functional variant thereof.
21. The CAR according to any one of claims 1-20, wherein the CAR further comprises a signal peptide, and the signal peptide comprises a signal peptide derived from CD8 or CD45.
22. The CAR according to any one of claims 1-21, wherein the CAR further comprises a signal peptide, the signal peptide comprising Contains CD8α signal peptide.
23. The CAR according to any one of claims 1-22, wherein the CAR further comprises a signal peptide, and the signal peptide comprises an amino acid sequence as shown in SEQ ID NO: 10 or a functional variant thereof.
24. The CAR according to any one of claims 1 to 23, wherein the CAR comprises a signal peptide, a CD30 binding domain, a hinge region derived from CD28, a transmembrane domain, an intracellular co-stimulatory signaling domain, and an intracellular signaling domain.
25. The CAR according to any one of claims 1-24, wherein the CAR comprises a CD8α signal peptide, a CD30 binding domain, a hinge region derived from CD28, a CD28 transmembrane domain, a CD28 intracellular co-stimulatory signaling domain, and a CD3ζ intracellular signaling domain in sequence.
26. The CAR according to any one of claims 1-25, wherein the CAR comprises the amino acid sequence as described in SEQ ID NO: 16.
27. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the CAR according to any one of claims 1 to 26.
28. According to claim 27, the nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO: 17.
29. A vector comprising the nucleic acid molecule according to any one of claims 27-28.
30. The vector according to claim 29, which is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector and a retroviral vector.
31. According to any one of claims 29-30, the vector comprises a nucleotide sequence as shown in SEQ ID NO: 18.
32. A cell comprising the CAR of any one of claims 1-26, the nucleic acid molecule of any one of claims 27-28 and/or the vector of any one of claims 29-31.
33. The cell according to claim 32, wherein the cell is an immune cell.
34. The cell according to any one of claims 32-33, wherein the immune cell is selected from the group consisting of T cells, natural killer (NK) cells, NKT cells and macrophages.
35. The cell according to any one of claims 32-34, wherein the cell is a T cell.
36. A method for preparing a cell, comprising introducing into the cell the CAR of any one of claims 1-26, the nucleic acid molecule of any one of claims 27-28 and/or the vector of any one of claims 29-31.
37. The method of claim 36, wherein the cell is an immune cell.
38. The method according to any one of claims 36-37, wherein the immune cells are selected from the group consisting of T cells, natural killer (NK) cells and NKT cells.
39. The method according to any one of claims 36-38, wherein the cell is a T cell.
40. A composition comprising the CAR of any one of claims 1-26, the nucleic acid molecule of any one of claims 27-28, the vector of any one of claims 29-31 and/or the cell of any one of claims 32-35, and optionally a pharmaceutically acceptable carrier.
41. Use of the CAR according to any one of claims 1 to 26, the nucleic acid molecule according to any one of claims 27 to 28, the vector according to any one of claims 29 to 31 and/or the cell according to any one of claims 32 to 35 in the preparation of a medicament for treating a CD30-related disorder.
42. A method for preventing and/or treating a CD30-related disorder, comprising administering an effective amount of the CAR of any one of claims 1-26, the nucleic acid molecule of any one of claims 27-28, the vector of any one of claims 29-31 and/or the cell of any one of claims 32-35.
43. The CAR of any one of claims 1-26, the nucleic acid molecule of claim 27, the vector of any one of claims 27-28 and/or the cell of any one of claims 29-31, for preventing and/or treating a CD30-related disorder.
44. The use according to claim 41 or the method according to any one of claims 42-43, wherein the CD30-related disorder is a tumor expressing CD30.
45. The use according to claim 41 or the method according to any one of claims 42-43, wherein the CD30-related disorder is lymphoma.
46. The use according to claim 41 or the method according to any one of claims 42-43, wherein the CD30-related disorder is Hodgkin's lymphoma or non-Hodgkin's lymphoma.
47. The use according to claim 41 or the method according to any one of claims 42-43, wherein the CD30-related disorder is selected from the group consisting of Hodgkin lymphoma, anaplastic large cell lymphoma (ALCL), cutaneous T-cell lymphoma, adult T-cell lymphoma (ATL) and angioimmunoblastic T-cell lymphoma (AITL).