CD74 CAR-T therapy and its method of use

Abstract

This disclosure provides a chimeric antigen receptor (CAR) polypeptide comprising a CD74 antigen-binding domain, a transmembrane domain, an intracellular signaling domain, and a co-stimulatory signaling region. Furthermore, this specification provides isolated nucleic acids encoding the recombinant polypeptide disclosed herein, vectors containing the isolated nucleic acids, and cells containing the vectors. This specification also provides a method for treating lymphoma in a subject of interest, comprising administering a therapeutically effective amount of the chimeric antigen receptor polypeptide disclosed herein to the subject. Furthermore, this specification describes a method for reducing tumor activity in a lymphoma subject, comprising administering to the subject a therapeutically effective amount of a chimeric antigen receptor polypeptide as disclosed herein.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of priority and the privileges of U.S. Provisional Patent Application No. 63 / 521,467 filed June 16, 2023, which is expressly incorporated herein by reference in its entirety.

[0002] Sequence listing reference This sequence listing, created as an XML file named "103361-486WO1_ST26.xml" on 13 June 2024 and submitted on 14 June 2024, with a file size of 153,628 bytes, is incorporated herein by reference in accordance with Section 1.52(e)(5) of the Code of Federal Regulations.

[0003] field This disclosure relates to chimeric antigen receptors and their uses. [Background technology]

[0004] Mantle cell lymphoma (MCL) is a refractory subtype of B-cell non-Hodgkin lymphoma (NHL) characterized by an increasing incidence over the past 20 years. MCL is conventionally classified into three morphological variants: classical, blastoid, and pleomorphic, the latter two being more invasive and associated with a poorer prognosis. Immunophenotypicly, MCL cells express pan-B-cell markers such as CD19 and CD20, and are typically CD5+, CD10-, CD23-, and FMC7+, although variability has been reported in subsets of patients. Molecularly, MCL is characterized by a translocation (11;14), which leads to overexpression of cyclin D1 and dysregulation of the cell cycle. Furthermore, secondary chromosomal alterations targeting genes involved in the regulation of the cell cycle, DNA damage response, and cell survival pathways are frequently reported in this disease. These include deletions of TP53, BCL2L11, RB1, and ATM, and amplification of BCL2 and c-MYC.

[0005] Younger MCL patients are treated with integrated therapy, including autologous hematopoietic stem cell transplantation, after intensive chemoimmunotherapy regimens. However, the majority of older patients are treated with less intense approaches. Apart from allogeneic stem cell transplantation, treatment for relapsed or refractory MCL is typically palliative, primarily involving covalent or non-covalent Bruton's tyrosine kinase (BTK) inhibitors and Bcl2 antagonists. While the disease responds to initial treatment, relapses are inevitable, and the overall prognosis is poor. In 2020, the first and only anti-CD19 CAR-T therapy (Tecartus KTE-X19) was approved for the treatment of relapsed and refractory MCL. A multicenter Phase II clinical trial enrolled 74 patients with a history of relapsed or refractory MCL who had previously received ibrutinib (a covalent BTK inhibitor) and up to five other therapies. Patients received 2 × 10⁶ doses. 6 A single dose of KTE-X19 CAR-T cells at a dose of / kg was administered. After a median follow-up of 35.6 months, the overall response rate remained high at 91%, with 68% of the 68 treated patients achieving complete remission. However, progression-free survival and overall survival were only 25.8 months and 46.6 months, respectively, suggesting that the majority of these patients require additional treatment.

[0006] CD74 is a non-polymorphic type II transmembrane glycoprotein with a cytosolic-facing N-terminal domain that functions as an MHC class II chaperone, facilitating the processing and presentation of exogenous antigens. In the endoplasmic reticulum (ER), the CD74 trimer binds to three MHCII alpha-beta dimers to form a nonamer structure, which then evacuates the ER and migrates to the cell surface. CD74 also functions as a receptor for the inflammatory cytokine macrophage migration inhibitor (MIF) and has been previously shown to promote normal B cell proliferation and survival by activating downstream pathways such as NF-κB and B cell receptor / PI3K / Akt signaling. CD74 is produced in molar excess compared to MHCII, resulting in abundant free-load expression on the cell surface, and is degraded via a proteolytic mechanism initiated by cytoplasmic cleavage of its N-terminal domain by signal peptide peptidase-like 2A (SPPL2a). Consistent with its role in antigen presentation, CD74 is expressed in specialized antigen-presenting cells such as macrophages, dendritic cells, and B cells, as well as in epithelial cells under inflammatory conditions. In addition to solid tumors of the gastrointestinal tract, CD74 is expressed at much higher levels in various malignant hematological disorders, including B and T cell lymphomas, multiple myeloma (MM), and chronic lymphocytic leukemia (CLL). In a phase I clinical trial conducted in 22 patients with relapsed / refractory B-cell NHL, milatuzumab was generally well-tolerated, with the most common toxicities being infusion reactions and cytopenia, but clinical activity was very limited and no objective responses were reported. In a phase I-II clinical trial conducted in 8 patients with relapsed / refractory CLL, milatuzumab was generally well-tolerated, with improvement in performance status observed in most patients, and hematological parameters showed moderate responses, but no patients met the iwCLL criteria for partial or complete remission.

[0007] The compositions and methods disclosed herein address these and other needs. [Overview of the project]

[0008] According to the purposes of the disclosed materials and methods embodied and broadly described herein, the disclosed subject matter, in one aspect, relates to chimeric antigen receptor polypeptides and methods related thereto.

[0009] Thus, in one example, a chimeric antigen receptor (CAR) polypeptide is provided that includes a CD74 antigen-binding domain, a transmembrane domain, an intracellular signaling domain, and a co-stimulatory signaling region.

[0010] In a further example, an isolated nucleic acid encoding a recombinant polypeptide is provided. Further provided is a method of treating lymphoma in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a chimeric antigen receptor polypeptide as disclosed herein.

[0011] Further provided is a method of reducing tumor activity in a subject with lymphoma, the method comprising administering to the subject a therapeutically effective amount of a chimeric antigen receptor polypeptide as disclosed herein.

[0012] Additional advantages will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the aspects described below. The advantages described below will be realized and achieved by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive.

[0013] The accompanying drawings, which are incorporated herein and constitute a part of this specification, illustrate several aspects described below. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] [Figure 1A]Figures 1A-1G show that scFV sequence optimization results in superiorly functional anti-CD74 CAR(74bbz)-T cells. Figure 1A shows the design of the CD74 CAR lentiviral vector. CD74 scFV: anti-CD74 scFV terminated at the CD8α hinge region; TM: CD8α transmembrane domain; 4-1BB: signaling domain of the 4-1BB molecule; CD3ζ: CD3ζ chain; T2A: self-cleaving peptide derived from signalovirus 2A; reporter: GFP or tEGFR reporter gene. [Figure 1B] Figures 1A–1G show that scFV sequence optimization results in superiorly functional anti-CD74 CAR (74bbz)-T cells. Figure 1B shows the distribution of mutations in the VH / VL chain (right) and framework (FR) / complementarity-determining region (CDR) of scFV on the 74bbz CAR. [Figure 1C] Figures 1A–1G show that scFV sequence optimization results in superiorly functional anti-CD74 CAR (74bbz)-T cells. Figure 1C shows the binding affinity of the 74bbz CAR mutant to CD74 ECD-Fc presented in Jurkat cells. Jurkat CAR mutant cells were reacted with CD74-ECD-Fc and anti-Fc-APC by flow cytometry. The red bars represent the parent 74bbz. All numbers on the x-axis represent the number of CAR clones. [Figure 1D] Figures 1A–1G show that scFV sequence optimization results in superiorly functional anti-CD74 CAR(74bbz)-T cells. Figure 1D shows the expression of the early T cell activation marker CD69 in Jurkat CAR mutant cells. Jurkat CAR mutant cells were incubated with Mino cells in a 1:2 effector-to-target (ET) ratio for 6 hours, and then stained for CD69. [Figure 1E]Figures 1A-1G show that scFV sequence optimization results in superiorly functional anti-CD74 CAR(74bbz)-T cells. Figure 1E shows a repeated antigen stimulation assay for Jurkat CAR mutant cells. Jurkat CAR mutant cells were stimulated weekly with gamma-irradiated Mino cells for 3 weeks and cultured under suboptimal conditions. Cell proliferation in Jurkat CAR mutant cells was assayed, and the percentage increase in cell number was normalized as a multiplier compared to Jurkat cells with parental 74bbz cells. [Figure 1F] Figures 1A–1G show that scFV sequence optimization results in superiorly functional anti-CD74 CAR (74bbz)-T cells. Figure 1F shows specific lysis of Mino cells by Jurkat CAR mutant cells. Jurkat CAR mutant cells were co-cultured with Mino cells at a 5:1 ET ratio for 24 hours, and specific lysis was analyzed. Figure 1G shows a four-directional Venn diagram identifying two clones of 74bbz CAR that excel in four aspects of CAR-T cells: CD69 activation (yellow), cytotoxicity (green), proliferation (red), and CD74 binding (blue). [Figure 2A] Figures 2A-2B show that the 42105-74bbz CAR-T cells induced the highest specific lysis among the mutants against Mino and JeKo-1 cells. Figure 2A shows that three 74bbz CAR mutants were expressed in primary CD4 / 8 T cells (n=3) and used as effector cells in co-culture with Mino (left) and JeKo-1 (right). Mutants 543, 5311, and 42105 were compared to parental 74bbz CAR-T cells. [Figure 2B]Figures 2A-2B show that 42105-74bbz CAR-T cells induced the highest specific lysis among the mutants against Mino and JeKo-1 cells. Figure 2B shows that 42105-74bbz CAR-T cells had the best signal-to-noise specific lysis compared to CD74-SUDHL-1 cells, as determined by specific lysis in CD74+ Mino cells. Ratios from all three mutants were compared to the parent ratio. Data are mean ± SEM from three independent experiments. *p<0.05; **p<0.01; ***p<0.001. [Figure 3A] Figures 3A-3C: Cytotoxicity and IFN-γ production of optimized 42105-74bbz CAR-T cells against MCL cells. Figure 3A shows CD74 expression in the MCL cell line. Number in upper right corner: Surface antigen density. The data shown is representative of one of three independent experiments. [Figure 3B] Figures 3A-3C: Cytotoxicity and IFN-γ production of optimized 42105-74bbz CAR-T cells against MCL cells. Figure 3B shows cytotoxicity assays of optimized 42105-74bbz CAR-T cells against six different MCL cell lines. Transduced T cells (UTT, red circles) or optimized 42105-74bbz CAR-T cells (blue triangles) were co-cultured with target cells at an ET ratio of od5 for 24 hours, after which specific lysis was determined. The CD74-T cell lymphoma cell line SUDHL-1 was used as a negative control. Each dot represents one experimental result from each healthy donor CAR-T cell. [Figure 3C] Figures 3A–3C: Cytotoxicity and IFN-γ production of optimized 42105-74bbz CAR-T cells against MCL cells. Figure 3C shows IFN-γ production from 42105-74bbz CAR-T cells after co-culture with the cells shown for 24 hours. Effector cells alone were used as negative controls. Results are shown as mean ± SEM from three independent experiments. *p<0.05; **p<0.01; ***p<0.001. [Figure 4A]Figures 4A–4C show the activity of optimized 42105-74bbz CAR-T cells against primary MCL patient lymphoma cells. Figure 4A shows CD74 expression on MCL patient lymphoma cells identified by the CD5+CD19+MCL subset. The numbers in the upper corner represent antigen density. The data shown are representative of one of three independent experiments. [Figure 4B] Figures 4A–4C show the activity of optimized 42105-74bbz CAR-T cells against primary MCL patient lymphoma cells. Figure 4B shows the sensitivity of three MCL patient lymphoma cells to 42105-74bbz CAR-T cells. MCL patient cells were used as target cells in co-culture of optimized 74bbz CAR-T cells at a 5:1 ET ratio for 24 hours. Results are shown as mean ± SEM from three independent experiments. **p<0.01; ***p<0.001. [Figure 4C] Figures 4A–4C show the activity of optimized 42105-74bbz CAR-T cells against primary MCL patient lymphoma cells. Figure 4C shows the antigen dependence of CD74 on the specific lysis of 42105-74bbz CAR-T cells. Correlation calculations were performed for the correlation coefficient R² and p-value using the CD74 surface antigen density MESF and specific lysis rate for JeKo-1, Mino, UPN, Granta-519, Z138, SUDHL-1 (left), and patients 1–5 (right). [Figure 5A] Figures 5A–5G show that CD74 expression remains on the CD33+ bone marrow subset of normal PBMCs. Figure 5A shows that the CD74 antigen density on MCL cells (Table 1) was significantly higher than that of PBMCs from healthy blood donors, but no statistically significant difference was found between PBMCs and negative control SUDHL-1. [Figure 5B] Figures 5A–5G show that CD74 expression remains on the CD33+ bone marrow subset of normal PBMCs. Figure 5B shows histogram plots of CD74 expression in CD33+ and CD33- cells (gray shading) compared to isotype staining control (dotted line). CD74 density on CD33+ and CD33- cells is summarized from three independent donors (right). [Figure 5C] Figures 5A–5G show that CD74 expression remains on the CD33+ bone marrow subset of normal PBMCs. Figure 5C shows the specific lysis of normal CD33+ cells when used as target cells against 42105-74bbz CAR-T cells. Mino cells were used as a positive control target. [Figure 5D] Figures 5A–5G show that CD74 expression remains on the CD33+ bone marrow subset of normal PBMCs. Figure 5D shows CD74 expression in CD14+ monocytes (gray shading) compared to isotype staining control (dotted line). A summary plot of CD74 density in monocytes from three independent donors is shown (right). [Figure 5E] Figures 5A–5G show that CD74 expression remains on the CD33+ bone marrow subset of normal PBMCs. Figure 5E shows the sensitivity of monocytes to 42105-74bbz CAR-T cells (white) and UTT (black). [Figure 5F] Figures 5A–5G show that CD74 expression remains on a CD33+ bone marrow subset of normal PBMCs. Figure 5F shows that subsets of TH cells (CD4+ T cells), TCYTO cells (CD8+ T cells), B cells, and NK cells expressed CD74. The data is representative of one of three independent experiments. The CD74 density on B cells, TH cells, TCYTO cells, and NK cells was all less than 10,000 molecules per cell. The numbers represent the median number of molecules per cell. The data are from three independent donors (below). [Figure 5G] Figures 5A–5G show that CD74 expression remains on the CD33+ bone marrow subset of normal PBMCs. Figure 5G shows the sensitivity of quiescent and activated T / B cells to 42105-74bbz CAR-T cells (white) and UTT cells (black). Results are mean ± SEM from three independent experiments. *p<0.05; **p<0.01; ***p<0.001. [Figure 6A]Figures 6A and 6B show that 42105-74bbz CAR-T cells did not deplete normal immune cells. Figure 6A shows the number of autologous 42105-74bbz CAR-T cells that peaked 18 days after CAR-T cell injection in humanized NSG mice. [Figure 6B] Figures 6A and 6B show that 42105-74bbz CAR-T cells did not deplete normal immune cells. Figure 6B shows that there were no significant changes in the absolute cell counts of B cells, monocytes, granulocytic myeloid suppressor cells (G-MDSCs), monocytic myeloid suppressor cells (M-MDSCs), and NK cells. All human cells were identified by human CD45+. B: CD19+; Monocytes: LIN-CD14+; G-MDSCs: HLA-DR+CD14-CD33+; M-MDSCs: HLA-DR+CD14+CD33+; NK: CD3-CD56+. Mice were administered either UTT cells (n=5) or 42105-74bbz (n=7). The bars indicate the median cell count. [Figure 7A] Figures 7A and 7B show that 42105-74bbz CAR-T cells extended survival time compared to the parent in a xenograft MCL NSG model derived from CD19 CAR-T relapse patients. Figure 7A shows Kaplan-Meier curves for untreated mice (blue) and mice treated with parental 74bbz CAR-T cells (green) and 42105-74bbz CAR-T cells (red). Eight mice were used in each group. Lymphoma-specific survival rates were determined by the presence of tumor cells in situ in any organ. *p<0.05; **p<0.01; ***p<0.001. [Figure 7B] Figures 7A and 7B show that 42105-74bbz CAR-T cells extended survival time compared to the parent in a xenograft MCL NSG model derived from CD19 CAR-T relapse patients. Figure 7B shows that 42105-74bbz CAR-T cells were more abundant in the ERC spleen of mice treated with parental 74bbz CAR-T cells than in mice treated with parental 74bbz CAR-T cells. The line represents the median log-absolute number of cells in 100 μL of blood or 1 × 10⁶ splenocytes. [Figure 8A]Figures 8A-8B show that 42105-74bbz CAR-T cells are not inferior to 19bbz CAR-T cells in extending survival in a preclinical MCL NSG mouse model. Figure 8A shows that mice were subcutaneously transplanted with Mino cells and randomized into 10 groups: tumor alone (blue), tumor + UTT cells (green), tumor + 19bbz CAR-T cells (black), and tumor + 42105-74bbz CAR-T cells (red). The table (below) summarizes the p-values ​​for survival comparisons across all groups. [Figure 8B] Figures 8A-8B show that 42105-74bbz CAR-T cells are not inferior to 19bbz CAR-T cells in extending survival in a preclinical MCL NSG mouse model. Figure 8B shows that mice treated with 42105-74bbz CAR-T cells had the lowest median log-absolute number of tumor cells in blood and spleen collected when the mice reached ERC. The median log-absolute number of circulating CAR-T cells in mice treated with 74bbz CAR-T cells was higher than that in mice treated with 19bbz CAR-T cells, but similar numbers of CAR-T cells were found in mice treated with both 19bbz and 74bbz CAR-T cells. [Figure 9A] Figures 9A–9B show in silico modeling of the CD74-anti-CD74 scFV interaction. (A) Figure 9A shows the best-generated model of the CD74-anti-CD74 scFV interaction, indicated by the lowest HADDOCK score as a function of RMSD. The blue cluster was selected for further in silico mutation introduction. [Figure 9B] Figures 9A-9B show in silico modeling of the CD74-anti-CD74 scFV interaction. (B)9B shows visualization of the CD74-anti-CD74 scFV interaction. Red: CD74 trimer; Blue: anti-CD74 scFV. [Figure 10A] Figures 10A-10C show the generation of 74bbz mutant clones. Figure 10A shows that GFP+ cells from the 74bbz mutant and parental CAR-expressing Jurkat cells were sorted by flow cytometry to the same intensity. [Figure 10B] Figures 10A-10C show the generation of 74bbz mutant clones. Figure 10B shows the immunoblot of CD3ζ to indicate the expression of the parental, 543, 5311, and 42105-74bbz clones. Endogenous CD3ζ was detected at 15 kDa, while chimeric CD3ζ on CAR was detected at 55 kDa. [Figure 10C] Figures 10A-10C show the generation of 74bbz mutant clones. (C) Figure 10C shows the CD74-ECD-Fc fusion protein stained with Coomassie blue. [Figure 11] This shows the expression of CD74 after activation in T cells and B cells. T cells and B cells isolated from PBMCs of three healthy blood donors were either untreated or activated with CD3 / CD28 soluble antibody and IL-2 for T cells, and LPS (10 ng / mL) / anti-IgM (10 ug / mL) for B cells. The numbers in the upper right corner represent the MFI (Multiple First Intake) of CD74 expression in resting (red) and activated (blue) cells. One representative donor out of the three healthy blood donors is shown. [Figure 12] This table shows the absolute cell counts of B cells, monocytes, granulocytic myeloid suppressor cells, monocytic myeloid suppressor cells, and NK cells in humanized NSG mice at 3, 11, and 23 days post-grafting of UTT or 74bbz CAR-T cells. All human cells were identified by human CD45+. B:CD19+; Monocytes:LIN-CD14+; G-MDSC:HLA-DR+CD14-CD33+; M-MDSC:HLA-DR+CD14+CD33+; NK:CD3-CD56+. Mice were administered either UTT cells (n=5) or 42105-74bbz (n=7). The bar represents the median cell count. [Figure 13A] Figures 13A-13B show cHL malignant cells (HRS) with abundant CD74 expression. Figure 13A shows the surface expression of CD74 compared to isotypes in cHL cell lines and HRS cells derived from primary cHL samples, using flow cytometry. [Figure 13B]Figures 13A–13B show cHL malignant cells (HRS) with abundant CD74 expression. Figure 13B shows immunohistochemical (IHC) staining (200x and 600x) of lymph nodes involved in cHL, showing strong surface and cytoplasmic CD74 expression (brown) in HRS cells (black arrows), as well as mild to moderate positivity in a background subset of normal immune cells. [Figure 14] This study demonstrates that 74bbz CAR-T cells effectively kill cHL cells in vivo. Figure 14 shows the engraftment of 1 × 10⁶ KMH2 cells in NSG mice via tail vein injection. Once bioluminescence signals became detectable by IVIS (2 weeks after engraftment), mice were randomized into five groups: tumor alone (blue), UTT cells (5 × 10⁶, red), and 74bbz CAR-T cells (5 × 10⁶, green). Mice treated with 74bbz CAR-T cells showed no evidence of disease by peripheral blood IVIS or flow cytometry at day 70, when the experiment was terminated due to early signs of GVHD. 50% of mice in the UTT group survived at day 70 (p=0.03). [Figure 15A] Figures 15A-15B show that 74bbz CAR-T efficiently kills TAM2. Figure 15A shows the surface expression of CD74 compared to isotypes on peripheral blood monocyte-derived TAMs stimulated with the M2-polarizing cytokine M-CSF. [Figure 15B] Figures 15A–15B show that 74bbz CAR-T cells efficiently kill TAM2 cells. Figure 15B shows TAM2 cells co-cultured with 74bbz CAR-T cells (or UTT control) at a 5:1 ET ratio for 24 hours. Cell death was measured by an AK-releasing cytotoxicity assay. Results are shown as mean ± SEM from three independent experiments. **p<0.01;****p<0.0001. [Figure 16A] Figures 16A-16B show cell lines and primary malignant cells derived from T-cell lymphoma patients exhibiting abnormal CD74 expression. Figure 16A shows T-cell lymphoma cell lines stained with isotype control (green) or anti-CD74 (red) antibody. [Figure 16B] Figures 16A-16B show cell lines and primary malignant cells derived from T-cell lymphoma patients with abnormal CD74 expression. Figure 16B shows that cells from primary T-cell lymphoma patients with abnormal CD2+ and CD7- (gated in red) are CD74+. [Modes for carrying out the invention]

[0015] In accordance with the purpose of the disclosed materials and methods, as embodied and broadly described herein, the subject matter disclosed relates to CD74 chimeric antigen receptor polypeptides and methods relating thereto.

[0016] Many modifications and other embodiments disclosed herein relate to the disclosed compositions and methods and will be conceivable to those skilled in the art who benefit from the teachings presented in the foregoing description and accompanying drawings. Therefore, it should be understood that this disclosure is not limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the accompanying claims. Those skilled in the art will recognize many variations and adaptations of the embodiments described herein. These variations and adaptations are included within the teachings of this disclosure and are intended to be covered by the claims herein.

[0017] Certain terms are used herein, but they are used only in their general and descriptive sense and not for limiting purposes.

[0018] As will become apparent to those skilled in the art upon reading this disclosure, each individual embodiment described and illustrated herein has discontinuous components and characteristics which can be readily separated from or combined with the characteristics of any of the other embodiments without departing from the scope or spirit of this disclosure.

[0019] Any enumerated method may be performed in the order of the enumerated events or in any other logically possible order. That is, unless otherwise expressly stated, no method or embodiment described herein is intended to be construed as requiring its steps to be performed in a particular order. Accordingly, unless a method claim expressly states in the claim or description that the steps should be limited to a particular order, no order is implied in any way. This also applies to any possible ambiguity for interpretation, such as logical issues relating to the arrangement of steps or operational flows, clear meanings arising from grammatical construction or punctuation, or the number or type of embodiments described in the specification.

[0020] All publications referenced herein are incorporated herein by reference to disclose and describe the manner and / or material by which such publications are cited. Publications considered herein are provided only for their disclosure prior to the filing date of this application. Nothing herein should be construed as an acknowledgment that the present invention has no prior rights to such publications due to prior inventions. Furthermore, publication dates provided herein may differ from actual publication dates and may require independent verification.

[0021] It should be understood that the terms used herein are intended solely to describe and not to limit specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in the field to which the disclosed compositions and methods belong. It should be understood that terms (such as those defined in commonly used dictionaries) should be interpreted as having meanings consistent with their meanings in the context of the specification and related art, and should not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0022] Prior to describing the various aspects of this disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in this disclosure.

[0023] definition When used herein, “comprising” is construed to mean the existence of a specified characteristic, integer, step, or component being referenced, but not to exclude the existence or addition of one or more characteristics, integers, steps, or components, or groups thereof. Furthermore, “by,” “comprising,” “comprises,” “comprised of,” “including,” “includes,” “including,” “involve,” “involves,” “involved,” and “such as” are used in their open, non-restrictive sense and may be used interchangeably. In addition, the term “comprising” is intended to include examples and aspects covered by the terms “consisting essentially of” and “consisting of.” Similarly, the term “consisting essentially of” is intended to include examples covered by the term “consisting of.”

[0024] When used herein and in the appended claims, the singular indefinite articles “a,” “an,” and “the” encompass multiple references unless the context clearly indicates otherwise. Thus, for example, references to “a compound,” “a composition,” or “a disorder” include, but are not limited to, two or more such compounds, compositions, or disorders.

[0025] It should be noted that ratios, concentrations, quantities, and other numerical data may be expressed herein in a range format. It may be further understood that each endpoint of this range is significant both in relation to and independently of the other endpoints. It may also be understood that there are numerous values ​​disclosed herein, and that each value is disclosed herein not only as the value itself, but also "about" its specific value. For example, if the value "10" is disclosed, then "about 10" is also disclosed. Ranges may be expressed herein as "about" a particular value and / or "about" another particular value. Similarly, it may be understood that a particular value forms further aspects when the value is expressed as an approximation by the use of the antecedent "about". For example, if the value "about 10" is disclosed, then "10" is also disclosed.

[0026] Where a range is expressed, further embodiments include encompassing a range from one specific value and / or to another specific value. For example, if an expressed range includes one or both of the limits, a range that excludes either or both of the limits that it includes is also encompassed in this disclosure (e.g., the phrase "x~y" includes the range from "x" to "y", as well as the ranges greater than "x" and less than "y"). This range may also be expressed as an upper limit (e.g., "about x, y, z, or less") and should be interpreted to include the ranges less than "x", less than "y", and less than "z", as well as the specific ranges about "x", about "y", and about "z". Similarly, the phrase "about x, y, z, or greater" should be interpreted to include the ranges greater than "x", greater than "y", and greater than "z", as well as the specific ranges about "x", about "y", and about "z". In addition, the phrase "'x' ~ 'y'" includes "approximately 'x' ~ approximately 'y'" when "x" and "y" are numerical values.

[0027] It should be understood that such range formats are used for convenience and conciseness, and therefore should be interpreted flexibly to include not only the numbers explicitly listed as range limits, but also all individual numbers or subranges encompassed within that range, as if each number and subrange were explicitly listed. For example, the numerical range "approximately 0.1% to 5%" should be interpreted to include not only the explicitly listed values ​​of approximately 0.1% to approximately 5%, but also individual values ​​within the indicated range (e.g., approximately 1%, approximately 2%, approximately 3%, approximately 4%) and subranges (e.g., approximately 0.5% to approximately 1.1%; approximately 5% to approximately 2.4%; approximately 0.5% to approximately 3.2%; and approximately 0.5% to approximately 4.4%, as well as other possible subranges). When used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the quantity or value in question may be an exact value or a value that provides equivalent results or effects to those enumerated in the claims or taught herein. That is, quantities, sizes, formulations, parameters, and other quantities and characteristics are not and do not need to be exact, but may be approximate and / or greater or less, reflecting tolerances, conversion factors, rounding, measurement errors, etc., as well as other factors known to those skilled in the art, that result in equivalent results or effects. In some situations, a value that provides equivalent results or effects cannot be logically determined. In such cases, when used herein, unless otherwise indicated or implied, “about” and “at or about” generally mean a variation of ±10% of the nominal value shown. In general, a certain quantity, size, proportion, parameter, or other quantity or characteristic is such whether or not it is explicitly stated that it is “about,” “approximately,” or “is or is about.” When “about,” “approximately,” or “is or is about” is used before a quantitative value, it is understood that the parameter also includes the specific quantitative value itself, unless otherwise specifically stated. As used herein, when used in the context of a composition or component of a composition that is substantially nonexistent, the term “substantially nonexistent” is intended to mean an amount less than about 1% by weight of the material described, based on the total weight of the composition, for example, less than about 0.5% by weight, less than about 0.1% by weight, less than about 0.05% by weight, or less than about 0.01% by weight.

[0028] The term “subject” preferably refers to humans. However, the term “subject” may also refer to non-human animals, preferably mammals such as dogs, cats, horses, cattle, pigs, sheep, and non-human primates, among others.

[0029] The words "reduce," or other forms such as "reducing" or "reduction," mean to decrease an event or characteristic (e.g., tumor growth). This is usually understood to be relative to some standard or expected value, in other words, but the standard or relative value does not necessarily have to be referenced. For example, "to reduce tumor growth" means to reduce the rate of tumor growth compared to a standard or control (e.g., an untreated tumor).

[0030] The term “treating” refers to the medical management of a patient with the aim of curing, improving, stabilizing, or preventing a disease, pathological condition, or disorder. This term includes active treatment, i.e., treatment specifically aimed at improving the disease, condition, or disorder, and causal treatment, i.e., treatment aimed at eliminating the cause of the associated disease, condition, or disorder. Furthermore, this term includes palliative treatment, i.e., treatment aimed at reducing symptoms rather than curing the disease, pathological condition, or disorder; preventive treatment, i.e., treatment aimed at minimizing, partially or completely inhibiting, the onset of the associated disease, pathological condition, or disorder; and supportive treatment, i.e., treatment employed to complement other specific therapies aimed at improving the associated disease, pathological condition, or disorder.

[0031] The term "therapeutically effective" means that the amount of the composition used is sufficient to restore one or more causes or symptoms of a disease or disorder. Such restoration requires only reduction or modification, and does not necessarily require elimination.

[0032] The term "pharmaceutically acceptable" refers to a compound, material, composition, and / or dosage form that is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, within the bounds of safe medical judgment, and that has a reasonable benefit-risk ratio.

[0033] The terms "peptide," "protein," and "polypeptide" are used interchangeably and refer to natural or synthetic molecules containing two or more amino acids linked to the alpha-amino group of another amino acid by the carboxyl group of one amino acid.

[0034] The term "nucleic acid" refers to a natural or synthetic molecule containing a single nucleotide, or two or more nucleotides linked by a phosphate group from the 3' position of one nucleotide to the 5' end of another. Since this nucleic acid is not limited by length, it may include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0035] The abbreviations for amino acids used herein are the conventional single-letter codes for amino acids, and are represented as follows: A, alanine; B, asparagine or aspartic acid; C, cysteine; D, aspartic acid; E, glutamate, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine; Z, glutamine or glutamic acid. The term "antibody" refers to a natural or synthetic antibody that selectively binds to a target antigen. This term encompasses both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, the term "antibody" also includes fragments or polymers of those immunoglobulin molecules, as well as humanized or humanized forms of immunoglobulin molecules that selectively bind to a target antigen.

[0036] The term "fusion protein" refers to a polypeptide formed by joining two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. A fusion protein may be formed by chemical coupling of the component polypeptides or may be expressed as a single polypeptide from a nucleic acid sequence encoding a single continuous fusion protein. A single-chain fusion protein is a fusion protein having a single continuous polypeptide backbone. Fusion proteins can be prepared using conventional techniques in molecular biology by ligating two genes in-frame into a single nucleic acid and then expressing the nucleic acid in a suitable host cell under conditions in which the fusion protein is produced.

[0037] As used herein, the term "specifically binds" when referring to a polypeptide (including an antibody) or a receptor refers to a binding reaction that determines the presence of a protein or polypeptide or receptor in a heterogeneous population of proteins and other biological agents. Thus, under specified conditions (e.g., immunoassay conditions in the case of an antibody), a particular ligand or antibody "specifically binds" to a particular "target" if it does not bind significantly to other proteins present in the sample or to other proteins to which the ligand or antibody may come into contact in an organism (e.g., an antibody specifically binds to an endothelial antigen). Generally, a first molecule that "specifically binds" to a second molecule has an affinity constant (Ka) greater than about 10 5 M -1 greater (e.g., 10 6 M -1 、10 7 M -1 、10 8 M -1 、10 9 M -1 、10 10 M -1 、10 11 M -1 、and 10 12 M -1 or greater).

[0038] The term "variant" refers to conserved amino acid substitutions, non-conserved amino acid substitutions (i.e., degenerate variants), substitutions within the wobble position of each codon (i.e., DNA and RNA) encoding a particular amino acid, amino acids added to the C-terminus and / or N-terminus of a peptide, or peptides having 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to a reference sequence.

[0039] The term "vector" refers to a nucleic acid sequence capable of transporting another nucleic acid to which this vector sequence is bound into a cell. The term "expression vector" includes any vector (e.g., plasmid, cosmid, or phage chromosome) that contains a gene construct in a form suitable for expression by cells (e.g., linked to a transcription factor).

[0040] The term "domain" refers to a specific physical region or amino acid sequence of a protein that is related to a particular function of DNA or a corresponding segment.

[0041] As used herein, the term "spacer," also referred to herein as "linker," refers to a peptide that links proteins, including a fusion protein, together. Generally, spacers have no specific biological activity other than linking proteins together or maintaining a minimum distance or other spatial relationship between proteins. However, the constituent amino acids of a spacer may be selected to influence some molecular property, such as molecular folding, net charge, or hydrophobicity.

[0042] composition Chimeric antigen receptor polypeptide Provided herein are chimeric antigen receptor (CAR) polypeptides comprising a CD74 antigen-binding domain, a transmembrane domain, an intracellular signaling domain, and a co-stimulatory signaling region.

[0043] Chimeric antigen receptors (CARs) are receptor proteins that have been engineered to confer the ability of T cells to target specific antigens. These receptors are chimeric in that they integrate both antigen-binding and T-cell activation functions into a single receptor.

[0044] CD74 is a protein encoded in humans by the CD74 gene. The CD74 gene is a protein-coding gene and is associated with diseases including, but not limited to, gastrointestinal lymphomas and solid tumors. Related pathways include, but are not limited to, the innate immune system and the presentation of antigens by major histocompatibility complex (MHC) class II.

[0045] The antigen-binding domain refers to the region of an antibody that binds to an antigen. This may include one constant domain and one variable domain in both the heavy and light chains.

[0046] A transmembrane domain is a transmembrane protein domain.

[0047] Hinge domains are plastic amino acid stretches present in some immunoglobulins. They provide segmental plasticity, potentially enabling cross-linking of two antigens on the same antigen molecule or the binding of two antigenic determinants.

[0048] Intracellular signaling domains transmit signals via protein-protein interactions with effector proteins, which then transmit the signals to their destinations. The co-stimulatory signaling region refers to the CAR portion of a co-stimulatory molecule that includes its intracellular domain. A co-stimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for the efficient response of lymphocytes to an antigen.

[0049] In some embodiments, this CD74 antigen-binding domain is a single-chain variable fragment (scFv) of an antibody that specifically binds to CD74.

[0050] Single-chain variable fragments (scFv) are fusion proteins of the variable regions of the heavy and light chains of immunoglobulins, linked by short linker peptides.

[0051] In further embodiments, this co-stimulatory region includes the cytoplasmic domain of the co-stimulatory molecule 4-1BB.

[0052] In some embodiments, this cytoplasmic domain includes CD28, 4-1BB, CD278, CD134, CD27, CD40, CD40L, TLR, or any combination thereof. In further embodiments, the co-stimulatory region includes one, two, three, or four cytoplasmic domains from one or more cytoplasmic molecules.

[0053] 4-1BB is a costimulatory glycoprotein receptor that is part of the tumor necrosis factor superfamily. It is an inducible cell surface receptor expressed in the presence of activating stimuli and functions in cellular signaling during T cell activation and proliferation.

[0054] In certain embodiments, this intracellular signaling domain includes a CD3 zeta (CD3ζ) signaling domain.

[0055] The T cell surface glycoprotein CD3 zeta (CD3ζ) chain, also known as the T cell receptor T3 zeta chain or CD247 (differentiation cluster 247), is a protein encoded in humans by the CD247 gene.

[0056] In a specific example, a CAR polypeptide is defined by the following formula: SP-CD74-HG-TM-CSR-ISD; or SP-CD74-HG-TM-ISD-CSD In the formula, "SP" represents the signal peptide, "CD74" represents the CD74 binding domain, "HG" represents an optional hinge domain, "CSR" represents the co-stimulatory signaling domain, "ISD" represents the intracellular signaling domain, and "-" represents an optional divalent linker.

[0057] Signal peptides are short amino acid sequences that control protein secretion and translocation. A divalent linker is a single chemical substance composed of two pharmacophores covalently bonded by a spacer of variable size.

[0058] In some embodiments, the scFv of an antibody that specifically binds to CD74 contains a sequence having at least 60% identity (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 100%) with SEQ ID NOs. Further embodiments include a sequence having at least 60% identity (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 100%) with SEQ ID NOs.

[0059] In certain embodiments, the scFv of an antibody that specifically binds to CD74 contains a sequence having at least 60% identity (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 100%) with SEQ ID NOs.

[0060] In some embodiments, the scFv of an antibody that specifically binds to CD74 contains a sequence having at least 60% identity (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 100%) with SEQ ID NOs. 13-14 or a fragment thereof. In further embodiments, the scFv of an antibody that specifically binds to CD74 contains a sequence having at least 60% identity (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 100%) with SEQ ID NOs. 13 or a fragment thereof.

[0061] In certain embodiments, the scFv of an antibody that specifically binds to CD74 contains a sequence that has at least 60% identity (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 100%) with respect to the sequence numbers in Table 4.

[0062] Complementarity-determining regions (CDRs) are parts of the variable chain in immunoglobulins (antibodies) and T cell receptors. CDRs are the most variable parts of a molecule and are therefore crucial for the diversity of antigen specificity produced by lymphocytes. Three CDRs (CDR1, CDR2, and CDR3) exist, discontinuously positioned on the amino acid sequence of the variable domain of the antigen receptor. In some embodiments, the antigen receptor consists of two variable domains (on two different polypeptide chains: a heavy chain variable region and a light chain variable region), and therefore, there are six CDRs for each antigen receptor.

[0063] The heavy chain variable region (VH) is a large polypeptide subunit of an antibody.

[0064] The light chain variable region (VL) is a small polypeptide subunit of an antibody.

[0065] In further embodiments, the antibody comprises a heavy chain variable region (VH) containing a sequence having at least 60% identity (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 100%) with SEQ ID NOs: 74, 75, and 76, and a light chain variable region (VL) containing a sequence having at least 60% identity with SEQ ID NOs: 77, 78, and 79.

[0066] In some embodiments, the antibody comprises CDRs derived from the heavy chain variable region (VH) including SEQ ID NO: 74 (CDR1), SEQ ID NO: 75 (CDR2), and SEQ ID NO: 76 (CDR3), as well as CDRs derived from the light chain variable region (VL) including SEQ ID NO: 77 (CDR1), SEQ ID NO: 78 (CDR2), and SEQ ID NO: 79 (CDR3).

[0067] In some embodiments, the antibody comprises a CDR derived from a heavy chain variable region (VH) containing sequences having at least 60% identity (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 100%) to SEQ ID NOs. 80, 81, and 82, and a CDR derived from a light chain variable region (VL) containing sequences having at least 60% identity to SEQ ID NOs. 83, 84, and 85.

[0068] In some embodiments, the antibody comprises CDRs derived from the heavy chain variable region (VH) including SEQ ID NO: 80 (CDR1), SEQ ID NO: 81 (CDR2), and SEQ ID NO: 82 (CDR3), as well as CDRs derived from the light chain variable region (VL) including SEQ ID NO: 83 (CDR1), SEQ ID NO: 84 (CDR2), and SEQ ID NO: 85 (CDR3).

[0069] In some embodiments, the antibody comprises a CDR derived from a heavy chain variable region (VH) containing sequences having at least 60% identity (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 100%) identity to SEQ ID NOs. 86, 87, and 88, and a CDR derived from a light chain variable region (VL) containing sequences having at least 60% identity (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 100%) identity to SEQ ID NOs. 89, 90, and 91.

[0070] In some embodiments, the antibody comprises CDRs derived from the heavy chain variable region (VH) including SEQ ID NO: 86 (CDR1), SEQ ID NO: 87 (CDR2), and SEQ ID NO: 88 (CDR3), as well as CDRs derived from the light chain variable region (VL) including SEQ ID NO: 89 (CDR1), SEQ ID NO: 90 (CDR2), and SEQ ID NO: 91 (CDR3).

[0071] In some embodiments, the antibody includes a heavy chain variable region containing sequences having at least 60% identity (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 100%) to the SEQ ID NOs in Table 1, and a light chain variable region containing sequences having at least 60% identity (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 100%) to the SEQ ID NOs in Table 1. [Table 1]

[0072] Isolated nucleic acid sequences, vectors, and cells Furthermore, this specification provides isolated nucleic acid sequences encoding any one of the recombinant polypeptides disclosed herein.

[0073] In some embodiments, the peptide contains an scFv coding nucleotide having a sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 100%) identical to SEQ ID NOs.

[0074] In further embodiments, the peptide comprises an scFv coding nucleotide having a sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 100%) identical to SEQ ID NOs.45. In some embodiments, the peptide comprises an scFv coding nucleotide having a sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 100%) identical to SEQ ID NOs.45.

[0075] In certain embodiments, the peptide contains an scFv coding nucleotide having a sequence that has at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 100%) identity with SEQ ID NOs. In some embodiments, the peptide contains an scFv coding nucleotide having a sequence that has at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 100%) identity with SEQ ID NOs.

[0076] In further embodiments, the peptide comprises an scFv coding nucleotide having a sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 100%) identical to the sequence numbers in Table 5.

[0077] Similarly, provided herein are vectors containing isolated nucleic acid sequences, as disclosed herein.

[0078] Furthermore, what is provided herein are cells containing the vectors disclosed herein. In some examples, cells exhibit reduced tumor activity when the antigen-binding domain of a CAR binds to CD74.

[0079] In further examples, the cells include those shown in Table 2. [Table 2]

[0080] method Lymphoma treatment methods In one aspect, the present disclosure provides a method for treating lymphoma in a subject of interest, comprising administering to the subject a therapeutically effective dose of a chimeric antigen receptor polypeptide, as disclosed herein.

[0081] Lymphoma refers to a disease characterized by blood cell tumors that originate from lymphocytes. These tumors may include CD74 tumors. In some examples, lymphoma may include Hodgkin lymphoma or non-Hodgkin lymphoma (NHL).

[0082] NHL can affect either B lymphocytes or T lymphocytes, which produce antibodies to fight bacteria and viruses, while T lymphocytes destroy bacteria or abnormal cells in the body and / or enhance or reduce the activity of other immune system cells.

[0083] In further embodiments, non-Hodgkin lymphoma includes mantle cell lymphoma (MCL).

[0084] MCL is caused by the malignant transformation of B lymphocytes located at the outer edge of lymph node follicles, known as the "mantle zone." These transformed B lymphocytes proliferate uncontrollably, leading to an accumulation of lymphoma cells, which causes the lymph nodes to enlarge. MCL cells can enter lymphatic channels and the bloodstream, spreading to other lymph nodes or tissues such as the bone marrow, liver, and gastrointestinal tract. Diagnosis of MCL is made by examining lymphoma cells obtained from a lymph node biopsy under a microscope if they possess B cell surface markers (e.g., CD20), overexpress cyclin D1 protein intracellularly, and contain translocations 11;14. Blood tests and physical imaging scans may be performed to determine the extent of the disease. MCL may contain rare spore variants in which cells are larger, divide more rapidly, and are more aggressive compared to common types of MCL, making treatment increasingly difficult.

[0085] MCL includes disease in stages I, II, III, or IV.

[0086] Stage I MCL corresponds to the presence of disease in one lymph node area or a single organ above the diaphragm. Stage II MCL corresponds to the presence of disease in two or more lymph node areas on the same side of the diaphragm. Stage III MCL corresponds to the presence of disease in two or more lymph node areas above and below the diaphragm. Stage IV corresponds to the presence of disease in the lymph nodes above and below the diaphragm and / or other parts of the body.

[0087] In some embodiments, this method further includes administering a CD74 expression regulator. In some embodiments, this method further includes administering a therapeutically effective dose of an autophagy inhibitor.

[0088] Autophagy is a self-degradation process in which damaged proteins and organelles are taken up by autophagosomes for digestion and ultimately recycled for cellular metabolism to maintain cellular homeostasis. Once cancer has formed, autophagy can protect cancer cells by providing them with additional nutrients or by preventing them from being destroyed by anticancer drugs or other substances. Autophagy can also influence the body's immune response to viruses, bacteria, and cancer cells. Therefore, cancer treatment may include administering autophagy inhibitors as a means of therapeutically treating cancer. Examples of autophagy inhibitors include, but are not limited to, chloroquine and hydroxychloroquine.

[0089] In further embodiments, the method further comprises administering a therapeutically effective dose of a histone deacetylase (HDAC) inhibitor.

[0090] HDAC inhibitors are a class of anticancer agents that play a role in epigenetic or non-epidadic regulation, inducing cancer cell death, apoptosis, and cell cycle arrest. HDAC inhibition can affect tumor cell survival by blocking tumor angiogenesis and by inhibiting intracellular stress response pathways. Examples of HDAC inhibitors, but not limited to, include vorinostat, deshipeptides, romidepsin, panobinostat, and bellinostat.

[0091] In further examples, the chimeric antigen receptor is administered in combination with at least one cancer treatment.

[0092] In certain embodiments, additional cancer treatments include surgery, chemotherapy, immunotherapy, ionizing radiation, or a combination thereof.

[0093] In certain cases, the procedure may include stem cell transplantation. Stem cell transplantation (SCT), also known as bone marrow transplantation, is a procedure in which a recipient receives a transplant of healthy stem cells to replace damaged stem cells. This may include autologous transplantation, in which the transplant uses the recipient's own stem cells. SCT may also include allogeneic transplantation, in which stem cells derived from a donor are used.

[0094] In autologous transplantation, target stem cells are collected and stored. The cells are frozen and then returned to the subject after intensive high-dose chemotherapy, with or without radiotherapy. This procedure can be used for clinically symptomatic subjects who are healthy, young, and have few or no comorbidities.

[0095] In some cases, chemotherapy has included R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone), VcR-CAP (bortezomib, rituximab, cyclophosphamide, doxorubicin, and prednisone), R-hyperCVAD (alternating administration of rituximab, cyclophosphamide, vincristine, doxorubicin, and dexamethasone with high doses of cytarabine and methotrexate), B Examples include +R (bendamustine and rituximab), R-FCM (rituximab, fludarabine, cyclophosphamide, and mitoxantrone), R-DHAP (rituximab, dexamethasone, cytarabine, and cisplatin), R-CVP (rituximab, cyclophosphamide, vincristine, and prednisone), or R-CBP (rituximab, cyclophosphamide, bortezomib, and prednisone), or any combination thereof. These chemotherapy regimens can be administered intravenously or orally.

[0096] In further embodiments, cancer treatment may include R-CHOP followed by autologous SCT, R-CHOP followed by a high dose of cytarabine and further autologous stem cell transplantation, or R-hyperCVAD followed by autologous SCT, or any combination thereof.

[0097] Methods to reduce tumor activity In one aspect, the present disclosure provides a method for reducing tumor activity in a lymphoma patient, comprising administering to the patient a therapeutically effective dose of a chimeric antigen receptor polypeptide, as disclosed herein.

[0098] In further embodiments, non-Hodgkin lymphoma includes mantle cell lymphoma (MCL). In certain embodiments, mantle cell lymphoma includes disease of stage I, stage II, stage III, or stage IV.

[0099] In some embodiments, this method further includes administering a CD74 expression regulator.

[0100] In some embodiments, this method further includes administering a therapeutically effective dose of an autophagy inhibitor.

[0101] Autophagy is a self-degradation process in which damaged proteins and organelles are taken up by autophagosomes for digestion and ultimately recycled for cellular metabolism to maintain cellular homeostasis. Once cancer has formed, autophagy can protect cancer cells by providing them with additional nutrients or by preventing them from being destroyed by anticancer drugs or other substances. Autophagy can also influence the body's immune response to viruses, bacteria, and cancer cells. Therefore, cancer treatment may include administering autophagy inhibitors as a means of therapeutically treating cancer. Examples of autophagy inhibitors include, but are not limited to, chloroquine and hydroxychloroquine.

[0102] In further embodiments, the method further comprises administering a therapeutically effective dose of a histone deacetylase (HDAC) inhibitor.

[0103] HDAC inhibitors are a class of anticancer agents that play a role in epigenetic or non-epidadic regulation, inducing cancer cell death, apoptosis, and cell cycle arrest. HDAC inhibition can affect tumor cell survival by blocking tumor angiogenesis and by inhibiting intracellular stress response pathways. Examples of HDAC inhibitors, but not limited to, include vorinostat, deshipeptides, romidepsin, panobinostat, and bellinostat.

[0104] In further examples, the chimeric antigen receptor is administered in combination with at least one cancer treatment.

[0105] In certain embodiments, additional cancer treatments include surgery, chemotherapy, immunotherapy, ionizing radiation, or a combination thereof.

[0106] In further embodiments, the Disclosure provides, in one aspect, a method for reducing tumor activity in a subject with classical Hodgkin lymphoma, T-cell lymphoma, or a combination thereof, comprising administering to the subject a therapeutically effective dose of a chimeric antigen receptor polypeptide, as disclosed herein.

[0107] In some embodiments, the Disclosure provides, in one aspect, a method for treating classical Hodgkin lymphoma, T-cell lymphoma, or a combination thereof, in a subject of interest, comprising administering to the subject a therapeutically effective amount of a chimeric antigen receptor polypeptide, as disclosed herein.

[0108] In certain embodiments, the chimeric antigen receptor polypeptide administered to the subject targets the immunosuppressive lymphoma microenvironment (LME). Immunosuppressive cells are part of the LME and promote tumor growth. Since these immunosuppressive cells within the LME express CD74, in some embodiments, the CAR polypeptides disclosed herein are used to target them.

[0109] Several embodiments of this disclosure have been described. Needless to say, it is understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are described in the following "Claims".

[0110] As non-limiting examples, the following are examples of certain embodiments of the present disclosure. [Examples]

[0111] The following examples illustrate the methods and results of the subject matter disclosed herein. These examples are not intended to include all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the invention that would be apparent to those skilled in the art.

[0112] While we strive to ensure accuracy in numerical values ​​(e.g., quantities, temperatures, etc.), please allow for a certain degree of error and deviation. Unless otherwise specified, parts are measured in parts by weight, temperatures are in °C or ambient temperature, and pressure is atmospheric pressure or near atmospheric pressure. Numerous variations and combinations of component concentrations, temperatures, pressures, and other reaction ranges and conditions exist that can be used to optimize reaction conditions, such as the purity and yield of the product obtained from the described process. Only reasonable and routine experiments are necessary to optimize such process conditions.

[0113] Example 1: Novel CAR-T therapy targeting CD74 that provides a sustained response in mantle cell lymphoma Mantle cell lymphoma (MCL) is a refractory non-Hodgkin lymphoma, with an estimated 5,000 new cases diagnosed annually in the United States, highlighting the need for new therapeutic approaches. Miratuzumab is an IgG1 monoclonal antibody that targets CD74, but its rapid internalization and short half-life result in minimal clinical efficacy. It has been hypothesized that targeting CD74 using chimeric antigen receptor T cells in MCL and binding to CD74-overexpressing MCL cells would exhibit potent and durable antitumor activity.

[0114] We engineered a second-generation anti-CD74 CAR (74bbz) possessing 4-1BB and CD3z signaling domains. Random in silico mutagenesis of the scFV domain functionally optimized the 74bbz CAR for superior antigen binding, proliferation, and cytotoxicity upon encounter with MCL cells in vitro and in vivo.

[0115] Clone 42105 74bbz (42105-74bbz) CAR-T cells exhibited specific cytolysis against a panel of MCL cell lines and primary patient samples, but not against the CD74-negative cell line SUDHL-1. CD74 expression was eccentrically limited, primarily in the CD33+ immune population derived from healthy donors, while expression was minimal in lymphocytes excluding B cells. More importantly, relatively low levels of CD74 on normal immune cells did not induce significantly specific lysis in vitro or in vivo. NOD-SCIDγc - / - In a (NSG) mouse model using lymphoma cells derived from patients with aggressive lymphoma that relapsed after CD19 CAR-T therapy, mice treated with 42105-74bbz CAR-T had a higher absolute number of persistent CAR T cells than the parental 74bbz CAR-T-treated group. In a subcutaneous MCL NSG xenograft model, survival was significantly extended in 42105-74bbz CAR-T-treated mice using more persistent CAR-T cells, comparable to CD19bbz CAR-T-treated mice.

[0116] CD74 on MCL was better targeted by 74bbz CAR-T therapy, and 42105-74bbz CAR-T cells were able to provide durable and potent antitumor activity to improve MCL clearance.

[0117] To overcome the limitations of milatuzumab, we developed an anti-CD74 CAR-T cell product named 42105-74bbz, which possesses a 4-1BB+CD3ζ co-stimulatory domain. We functionally optimized this product and investigated its activity in a preclinical MCL model. We hypothesized that anti-CD74 CAR-T cell therapy would provide a safe and long-lasting anti-lymphoma effect. Our findings showed that 42105-74bbz CAR-T cells exhibited cytotoxicity against both MCL cell lines and primary MCL patient samples, and their activity was positively correlated with target antigen density. In vivo, 42105-74bbz CAR-T cells extended survival in a patient-derived xenograft (PDX) mouse model established from cells of MCL patients who had relapsed after CD19 CAR-T therapy, and also extended survival in a subcutaneous human cell line xenograft MCL model. This approach was no less effective than in vivo CD19 CAR-T cell therapy. Furthermore, in humanized mice using CD34+ stem cells, no significant changes were observed in the absolute cell counts of B cells, monocytes, myeloid suppressor cells, and NK cells after treatment with 42105-74bbz CAR-T cells.

[0118] method Construction and expression of the CD74 CAR-T vector A second-generation lentiviral carrier (CAR) was designed by conjugating a single-chain variable fragment (scFv) of mouse anti-human CD74 via the 4-1BB and CD3ζ chains, CD8 hinge region, and transmembrane domain. A CMV promoter was added before the entire sequence for constitutive expression. The sequence was then codon-optimized, synthesized (Twitst Bioscience, OR), and subcloned into the lentiviral vector pCDH (SBI Bioscience, CA). Lentiviral viruses were generated by transfecting Lenti-X 293T cells (Clontech, Takara Bio USA Inc, CA) with the CAR construct packaging plasmids containing PsPax (Addgene #12260) and pMD2.G (Addgene #12259) via Lipofectamine 2000 (Invitrogen MA). 48 hours after transfection, the viral supernatant was collected, filtered through 0.22 μm, and then rapidly frozen at -80°C until use.

[0119] In silico SCFv optimization and mutagenesis The amino acid paratopes within the complementarity-determining regions (CDRs) 1, 2, and 3 were initially identified using ProABC-2. The scFV was then reconstructed in silico as a PDB using ABodyBuiler2 (Leem, 2019) and prepared for the antigen (CD74, PDB number 1IIE). The scFV was then docked to the antigen using HADDOCK2.4 (Abrosetti, 2023). To refine this docking model, alanine scanning was performed on the amino acid residues of each CDR. Among all candidate model clusters, the best model with the lowest HADDOCK score was selected for computational mutagenesis analysis (Figures 9A-9B). Candidate binding residues were substituted with all 20 possible amino acids and docked to the CD74 antigen. (Yin, 2007) mutations were introduced into the scFV region on these binding residues using the Eris molecular suite. The estimated free energy of the mutant conformations was compared to the wild type, and lead mutants were selected for site-directed mutagenesis and downstream function assays. Mutagenesis on the CDR region was performed using the GeneMorph II EZClone Domain Mutagenesis Kit (Agilent, CA) according to the manufacturer's instructions. Three CAR constructs with anti-CD74 scFV (named 543, 553, and 563) derived from the public domain were also developed and used for comparison. Mutant plasmids were transformed, and lentiviral production was induced in each CAR mutant. To facilitate screening, each mutant was displayed on the T-ALL cell line Jurkat cells, and CAR expression levels were quantified by sorting GFP+ cells from each mutant by flow cytometry, and CD3ζ was shown by immunoblotting (Figures 10A-10B). The performance of the mutant was evaluated and compared with the parent 74bbz CAR using the following four parameters: 1) functional binding affinity to the chimeric CD74 extracellular domain (ECD)-Fc fusion protein (Figure 10C); 2) expression of the CD69 activation marker upon binding to CD74+ target cells; 3) repeated antigen proliferation assay; and 4) in vitro cytotoxic assay against CD74+ target Mino cells.

[0120] Cell culture and isolation MCL cell lines JeKo-1, Mino, Sp53, UPN-1, Granta-519, and Z-138 were obtained from ATCC (Manassas, VA) and cultured under the manufacturer's instructions. All cell lines used were regularly tested for mycoplasma using MycoAlert (Lonza, MA), and passaged for no more than two months. Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy blood donors by gradient density using Ficoll-Paque Plus (GE Healthcare Life Science, PA) with the approval of the Ohio State University Institutional Review Board. Human T cells were isolated from peripheral blood using CD4 and CD8 microbeads in a 1:1 ratio according to the manufacturer's instructions (Miltenyi Biotech, CA). To determine whether activation in T cells and B cells induces CD74 upregulation, T cells were treated overnight with CD3 and CD28 soluble antibodies (10 ng / mL, BioLegend, CA) and 250 U / mL of IL-2. B cells were isolated from PBMCs using the EasySep human B cell isolation kit (StemCell Technologies, MA), and these B cells were treated with LPS (10 ng / mL) and anti-IgM antibody (10 ug / mL). The activation status of the cells was confirmed by flow cytometry.

[0121] Antibodies and flow cytometry The antibodies used in this study included anti-CD3 (clones SK7 and 145-2C11), CD56 (clone N901), CD14 (clone MφP9), human CD45 (clone 2D1), mouse CD45 (clone 30-F11), human NKG2D (clone 1D11), CD4 (clone RPA-T4), CD69 (clone FN50), and CD25 (clone BC96). All antibodies were from BioLegend, CA, except for CD74 (clone MB741), which was from BD Biosciences, CA. Cells were washed once with PBS, blocked with Trustin human Fc blocker (BioLegend), stained with antibody at room temperature for 20 minutes, and analyzed using an LSRII flow cytometer (BD Biosciences, CA, USA). For the detection of parental or mutant CAR-T cells in primary T cells, a shortened EGFR (tEGFR) tag was used in the CAR construct and detected by an anti-EGFR antibody (AY13, BioLegend). For the determination of CD74 antigen density, a calibration curve correlated with the instrumental detection channel value and standardized fluorescence intensity units of Equivalent Soluble Fluorochrome (Molecules of Soluble Fluorochrome) (MESF) FITC-5 premix beads (Bangs Laboratory Inc, IN) was used with a R of 0.9995. 2 The formula was constructed using this method, and the antigen density of CD74 on MCL cells was calculated from the average fluorescence intensity obtained on the same day and under the same conditions, according to the manufacturer's instructions. In the flow cytometry-based functional binding affinity assay, the measurement of CD74 scFV on Jurkat cells was modified to measure against the CD74 extracellular domain (ECD)-Fc. Briefly, 1 × 10⁶ cells with parental or mutant scFV were measured. 6Jurkat cells (viability >90%) were washed twice with cold PBS and blocked with an Fc blocker (Trustain human Fc blocker, BioLegend) at room temperature for 10 minutes. These cells were then stained in excess with 5 μg / mL CD74-ECD (Sino Biological US Inc., PA) on ice for 45 minutes, washed twice with PBS, and stained at room temperature for 15 minutes with an anti-Fc flow cytometry grade antibody and SYTOX blue dead cell stain (Invitrogen). After washing twice with cold PBS, the cells were immediately analyzed by flow cytometry. CD69 and CD25 expression were determined by flow cytometry to confirm activation of Jurkat cells or primary T cells.

[0122] Repeated antigen stimulation assay The stimulation and proliferation methods were carried out with modifications as previously reported (Smith, 2018). Jurkat cells or primary T cell clones were irradiated with Mino cells (stimulator) at an effector-to-target ratio (ET ratio) of 1:2, with a total cell density of 3 × 10⁶. 5 The cells were co-cultured in RPMI 1640 medium containing a low concentration of 1% FBS at a concentration of / mL. The cultures were refreshed with medium every 3 days, and after a total of 3 restimulations with freshly irradiated Mino cells, the absolute cell count was determined by trypan blue exclusion assay. Untransduced Jurkat or T cells, with or without parental CD74bbz CAR and stimulating factors, were used as negative controls.

[0123] Cytotoxic assay The cytotoxicity of CAR-T cells was performed using the ToxiLight® Non-Destructive Cytotoxic Bioassay Kit (Lonza) according to the manufacturer's instructions (29) as described. Briefly, MCL cell lines or primary MCL patient samples (lymphoma % ranging from 73.7% to 98.7%) were co-cultured for 24 hours with 74 bbz CAR-T cells or untransduced T cell control (UTT) cells at an ET ratio of 5:1. After 24 hours, 100% lysis buffer (Lonza) was added to the wells for maximum lysis at room temperature for 10 minutes. The volumes of the other wells were adjusted with prepared Tris AC buffer. Next, the cell supernatant was collected from each well and reacted with the provided substrate for 5 minutes, after which the bioluminescence of the plate was read using a Synergy HT microplate reader (Biotek, VT).

[0124] ELISA Human IFN-γ levels in the culture supernatant were measured using the ELISAMAX® Deluxe Set Human IFN-γ Kit (BioLegend) according to the manufacturer's instructions. Cell-free supernatant was collected from the cell culture 24 hours after co-culture with effectors. The plates were then washed, incubated with tetramethylbenzidine substrate (Agilent Technologies, CA), and read at 450 nm using a Synergy HT microplate reader (Biotek, VT).

[0125] In vivo experiment All animal studies were approved by the Institutional Animal Care and Use Committee at Ohio State University. NOD-SCID (non-organic dwarf staghorn fowl) aged 4-6 weeks. IL2γ- / - Human PDX was established using (NSG) mice and MCL cells obtained from patients who relapsed after CD19 CAR-T therapy. PDX was established from four consecutive adoptive transplants. PDX MCL cells were cryopreserved and banked for future use. If used, 1 × 10⁶ 6PDX MCL cells were injected intravenously. Three days later, mice were randomized to the indicated groups, and 5 × 10⁶ cells were administered. 6 Each cell was treated with 74bbz CAR-T cells (either parental or mutant cells were injected). Disease progression was monitored weekly by flow cytometry. A subcutaneous tumor model using CD74+ Mino cells was also employed for survival studies. -On day 1, 1 × 10⁶ cells were used. 6 Mino cells were injected subcutaneously (sc). Three days later, when the tumor became palpable, 5 x 10 6 Individual 74bbz CAR-T cells or UTT cell controls were injected intratumorically. All human T cells used were allogeneic to Mino cells. Disease progression was monitored and survival data were recorded. Mice were sacrificed when they reached the endpoints of body conditioning score <2, paralysis, and 20% weight loss. To measure the activity of 74bbz CAR-T cells against a subset of human immune cells in vivo, humanized mice were established from the aforementioned human umbilical cord blood (Verma, 2020). Briefly, 4-week-old female NSG mice were irradiated with 125 cGy (RS-2000, Rad Source Technologies, GA), and then 5 × 10⁶ cells isolated per mouse by the human CD34 microbeads ultra-pure kit (Miltenyi Biotech) were administered. 5 Human CD34+ cells were injected intravenously. The purity of the human CD34+ cells was over 85% (n=3). Mice were intraperitoneally injected with SCF, GM-CSF, and IL-3 at 2-4 ng / mL every 3 weeks to promote myeloid development. Ten weeks after transplantation of human CD34+ cells, human chimerism in circulating cells was identified by facial / submandibular venous blood sampling and flow cytometry. The absolute number of human immune cell subsets was collected from the mice, and 5 × 10⁶ mice were subjected to the procedure. 5 Individual UTTs or 5 × 10 5Participants were randomized to receive 42105-74bbz CAR-T cells. The absolute numbers of human B cells (human CD45+CD33-CD19+), monocytes (human CD45+CD11b+CD33+CD14+ cells), granulocytic myeloid suppressor cells (G-MDSC, human CD45+CD11b+CD33+CD14-HLA-DR-), monocytic myeloid suppressor cells (M-MDSC, human CD45+CD11b+CD33+CD14-HLA-DR-), and NK cells (human CD45+CD33-CD3-CD56+) were measured on days 3, 11, 18, and 23 after UTT / CAR-T injection. UTT cells and CAR-T cells were traced and identified as human CD45+CD3+ and CD45+CD3+EGFR+, respectively.

[0126] statistical analysis Two independent groups or two matched groups were compared using Student's t-tests or unpaired t-tests for data following a normal distribution or a transformed normal distribution. Multiple independent groups were compared using linear models. Linear mixed models were applied to multiple group comparisons under a variance-covariance structure by repeating measurements. For survival data, the Kaplan-Meier method was applied to estimate survival function, and survival between two groups was compared using log-rank tests. P-values ​​were adjusted for multiple comparisons using Holm's procedure. A P-value of ≤0.05 was considered statistically significant.

[0127] result CD74bbz CAR scFV optimization identifies nearby CAR-T cells with maximum cytotoxicity and target-induced proliferation. A second-generation CAR construct with 4-1BB and CD3ζ chain signaling domains was designed by conjugating scFVs that specifically target human CD74 to the CD8 hinge / transmembrane domain and CD3ζ (Figure 1A). The goal was to optimize the CDR region of the scFVs and select the most functional CD74bbz CAR-T cells using both in silico approaches and functional assays. An anti-CD74 scFV-CD74 antigen docking model was created and refined by alanine scanning (Figures 9A-9C).H In this case, four positions of CDR1, ten positions of CDR2, and eight positions of CDR3 were found to be useful for interaction. L In this case, the 5th position of CDR1, the 2nd position of CDR2, and the 6th position of CDR3 were the important interaction residues. Next, using mCSM-AB2, a computational application that accurately and rationally evaluates the effect of single point mutations on the binding affinity between antibody and antigen, V H 22 residues in the region, V L Thirteen amino acid residues were mutated in the region (Myung, 2020). A total of 741 mutants were successfully tested, and the potential energy difference between the wild type and the mutants was ranked as ΔΔG (kcal / mol). Based on the positive energy obtained from the wild type, a total of 17 mutants were selected. Of the 17 mutants, 64.7% were V H It is located above, and 35.3% is V LIt was located above (Figure 1B). Furthermore, of the 17 mutants, 35.3% were located on the CDR3 region, 17.65% on CDR1, and 5.86% on CDR2 (Figure 1B). The acute lymphoblastic leukemia cell line (Jurkat) was manipulated to express the selected mutants. CAR expression in each mutant clone was normalized by selecting each Jurkat clone at the same intensity as GFP, and confirmed by immunoblotting (Figures 10A-10B). Flow cytometry (Geuijen, 2005) and the binding affinity of each mutant CAR to the chimeric CD74 ECD-Fc of four clones (5311, 4218, 42105, and 543) showed increased binding affinity to the CD74 antigen compared to the parent (Figure 1C). The expression of the T cell activation marker CD69 was evaluated, and five clones (429, 4218, 5310, 5311, and 42105) were identified that showed significantly increased CD69 expression 8 hours after co-incubation with the MCL cell line Mino at an ET ratio of 1:2 (Figure 1D). After repeated antigen stimulation assays, clones 42105, 5311, and 5310 showed a significantly higher cell number increase compared to the parental CAR (Figure 1E). Similarly, clones 543, 563, 5311, 532, 42105, and 553 induced significantly higher specific lysis compared to parental CAR-T cells (Figure 1F). By including all clones with enhanced functionality compared to the parental CAR-T cells in the Benn analysis, clones 42105, 5311, and 543 were identified as three lead candidates for further screening, as they all met the four proposed criteria: CD69 activation, binding affinity, proliferation, and cytotoxicity (Figure 1G). When primary T cells (n=3) were used as effectors and the MCL cell lines Mino (CD74 high-expression strain) and JeKo-1 (CD74 low-expression strain) were used as target cells, all three mutants induced significantly higher specific lysis compared to the parental 74bbz CAR-T cells (Mino, Figure 2A; JeKo-1, Figure 2A).To determine whether the mutant clones were CD74-specific, specific lysis was measured in CD74-positive Mino cells (on-target signal) over CD74-negative SUDHL-1 cells (off-target noise) and compared among three mutant clones that outperformed the parent. Clones 5311 and 42105 showed higher signal-to-noise ratios than 543 (Figure 2B).

[0128] CD74+ MCL cells are sensitive to 42105-74bbz CAR-T cells. To test the sensitivity of MCL cells to 42105-74bbz CAR-T cells, cytotoxic assays were performed on six CD74-positive MCL cell lines (JeKo-1, Mino, UPN-1, Granta-519, Z138, and Sp53) (Figure 3A). 42105-74bbz CAR-T cells were generated using purified T cells from healthy donors with a mean transduction efficiency of 73.03 ± 11.4% over 3 days. The MCL cell lines were sensitive to 42105-74bbz CAR-T cells and showed significantly more specific lysis compared to UTT controls. JeKo-1, Mino, UPN, Granta-519, Z138, and Sp53 lysed at median rates of 28.1%, 62.4%, 43.3%, 43.6%, 35.8%, and 45% in 24 hours, respectively, with an ET ratio of 5:1 (Figure 3B). Binding of the 42105-74bbz CAR-T cell line with the target MCL cell line induced T cell activation with a significant increase in IFN-γ production compared to UTT and effector T cell controls (Figure 3C). Five primary CD74+ / CD19+ / CD5+ MCL patient samples were used for further validation (the clinical characteristics of these five MCL patients are summarized in Table 3). (Figure 4A) [Table 3]

[0129] When 42105-74bbz CAR-T cells and primary MCL patient cells (n=5) were co-cultured for 24 hours at a 5:1 ET ratio, significant lysis occurred in all tested samples compared to the UTT control (Figure 4B). Further analysis of MCL cell lines (Figure 4C) and primary MCL patient samples (Figure 4C) showed a positive correlation between target antigen expression and cytotoxicity (R, respectively). 2 (The coefficients were 0.855 and 0.806). These results indicate that MCL is sensitive to the effector function of 42105-74bbz CAR-T cells.

[0130] 42105-74bbz CAR-T cells exhibit minimal cytotoxicity against a subset of normal immune cells. To differentiate the activity of 42105-74bbz CAR-T cells against MCL and non-malignant immune cells, CD74 density was measured in PBMCs derived from healthy blood donors (n=3) and primary MCL patient samples (n=5). PBMCs from healthy blood donors had fewer than 10,000 molecules per cell, significantly less than MCL cells, which ranged from 26,752 to 34,561 molecules per cell (Figure 5A, p=0.0019). There was no statistically significant difference between normal cells and negative control SUDHL1 cells (p=0.656). Within PBMCs, CD33+ bone marrow cells had a higher percentage of positivity and CD74 molecules per cell than the CD33- subset (95% vs. 22% CD74+, Figure 5B). 42105-74bbz CAR-T cells exhibited minimal cytotoxicity against CD33+ cells (Figure 5C, p=0.456). Within the CD33+ population, the majority of monocytes (83.6%) were CD74+, with a median CD74 molecule count of 13,830 per cell (Figure 5D). When purified monocytes were used as target cells in co-culture with 42105-74bbz CAR-T cells, a significant increase in specific lysis from UTT was observed, indicating that monocytes are sensitive to cytolysis by 42105-74bbz CAR-T cells (Figure 5E). Among lymphoid cells, the majority of normal B cells (93.1%) were CD74+, with a median density of 7315 molecules per cell, but CD8+ cytotoxic T(T) cells were not. CYTO) A subset of cells (16.7% CD74+, median density 2687 molecules per cell), and CD4+ T helper (T H Only CAR-T cells (10.4% CD74+, median density of 56 molecules per cell) expressed CD74 (Figure 5F). Notably, physiological activation of T cells (CD3 / CD28 soluble antibody and IL-2) and B cells (LPS, 10 ng / mL, and anti-IgM, 10 ug / mL) did not affect CD74 expression or CD74-specific lysis (Figure 5G, Figure 11). To characterize the activity of 42105-74bbz CAR-T cells against normal immune cells in vivo, a humanized mouse model of CD34+ hematopoietic stem cells was developed and used (Verma, 2020). Human CD34+ stem cells were isolated from umbilical cord blood and transplanted into NSG mice for engraftment. Mice were intraperitoneally injected with stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-3 to promote myeloid differentiation, as previously reported (Verma, 2020). Ten weeks after human CD45+ cell rearrangement, as determined in peripheral blood, the mice were randomized and 5×10⁶ cells generated from the same umbilical cord blood donor were selected. 6 Either autologous 42105-74bbz CAR-T cells or UTT cells were administered intravenously. As shown in Figure 6A, 74bbz CAR-T cells, which peaked on day 18 after CAR-T injection, did not significantly affect human B cells, monocytes, granulocytic myeloid suppressor cells, monocytic myeloid suppressor cells, and NK cells (Figure 6B; Figure 12). In summary, these in vitro and in vivo results indicate that while CD74 is expressed at varying levels in resting and activated immune cell subsets, 42105-74bbz CAR-T cells are significantly cytotoxic only to normal B cells.

[0131] 42105-74bbz CAR-T cells are PDX cells generated from MCL patients whose disease progressed after commercially available CD19 CAR-T therapy, and they exhibit potent antitumor activity. The activity of the optimized 42105-74bbz CAR-T compared to the parent construct was determined in vivo. A newly established PDX generated from an MCL patient (Patient #3, Table 3) whose condition progressed after commercially available CD19 CAR-T therapy was used. NSG mice were subjected to 1 × 10⁶ passages from passage 4. 6 Individual MCL cells were engrafted and divided into eight groups: control (tumor alone) and 5 × 10⁶ cells. 6 42105-74bbz CAR-T, or 5x10 6 Participants were randomized to receive parental-74bbz CAR-T cells, which were administered intravenously on day 3. MCL cell engraftment was confirmed by weekly peripheral blood sampling and flow cytometry (human CD5+ / CD19+ cells). In the presence of tumor cells in the blood or spleen, all mice died from MCL. Both animals treated with parental and 42105-74bbz CAR-T cells survived significantly longer than control mice (median survival 47 days, p<0.0001, n=8). Mice treated with 42105-74bbz CAR-T cells survived significantly longer than mice treated with parental-74bbz CAR-T cells (median survival 94 days vs. 81.5 days, p=0.005, n=8) (Figure 7A). Supporting the advantage in survival rate, mice treated with CAR-T cells of 42105-74bbz according to the early removal criteria (ERC) had a significantly higher number of CAR-T cells in their spleen compared to controls and parental CAR-T cells (Figure 7B).

[0132] To determine whether CAR-T therapy with 42105-74bbz is inferior to standard CD19 CAR-T therapy, a subcutaneous MCL xenograft model using NSG mice engrafted with Mino cells was employed. Ten groups of animals were randomized to receive either tumor alone or tumor + 5 × 10⁶ treatments. 6 Individual UTT cells, tumor + 5 × 10 6 Individual 19bbz CAR-T cells, and tumor + 5 × 10 6 Individual 42105-74bbz CAR-T cells, 1 × 10 6Mino cells were administered intratumorally three days after transplantation and when a palpable tumor was detected. As shown in Figure 8A, treatment with both 19bbz CAR-T cells and 42105-74bbz CAR-T cells significantly extended mouse survival compared to tumor alone (p<0.0001, n=10) and the UTT group (p<0.0001 compared to the untreated group, n=10). Furthermore, the median survival of mice treated with 42105-74bbz CAR-T cells was 56 days (n=10) compared to 50 days (n=10) for mice treated with 19bbz CAR-T cells, although the survival advantage did not reach statistical significance (p=0.0675) (Figure 8B), despite the fact that mice treated with 42105-74bbz CAR-T cells had the lowest number of MCL cells in both peripheral blood and spleen. In mice treated with 42105-74bbz CAR-T cells, circulating CAR-T cells and tumor-infiltrating CAR-T cells remained high and comparable between the two CAR-T cell treatment groups (Figure 8B, p>0.05). In summary, these data provide evidence that 42105-74bbz CAR-T cells are active and comparable to 19bbz CAR-T cells in PDX generated from MCL patients whose condition progressed after commercially available CD19 CAR-T cell treatment.

[0133] Consideration Currently, the development of CAR-T therapies for MCL patients primarily focuses on CD19 as a target. In 2020, the U.S. Food and Drug Administration (FDA) approved brexucabtagene autoleucel (Tecartus), a CD19-targeted CAR-T cell therapy for the treatment of MCL, based on the results of the ZUMA-2 clinical trial (Wang, 2016), which offers a promising treatment option for this refractory disease. This specification discusses CAR-T cell therapy targeting CD74, an invariant MHC class II HLA-DR chain overexpressed in MCL cells. 42105-74bbz CAR-T cells demonstrated significant cytotoxicity against MCL cells in vitro and in vivo, including MCL PDX derived from patients who had relapsed after commercially available CD19 CAR-T cell therapy. Although CD74 is expressed at low levels even in a subset of normal immune cells, in this context, the cytotoxicity of 42105-74bbz CAR-T cells was surprisingly minimal both in vitro and in vivo.

[0134] Current treatments for MCL include a multimodal approach encompassing chemotherapy, stem cell transplantation, and targeted therapy (Campo, 2015). NCCN guidelines recommend autologous stem cell transplantation and maintenance rituximab, following an intensive chemotherapy regimen, for physically healthy patients under 65 years of age. For the majority of MCL patients over 65 years of age, less invasive treatment approaches are employed (Al-Mansour, 2022). Small molecule inhibitors of BTK, including ibrutinib, acalabrutinib, zanubrutinib, and pirtobrutinib, as well as Bcl2 such as venetoclax, are FDA-approved for the treatment of relapsed / refractory MCL and provide significant clinical benefits over a period of time. Unfortunately, the majority of MCL patients treated with targeted therapy eventually progress to more invasive diseases, resulting in lower response rates to other treatment approaches and shorter overall survival. Anti-CD19 KTE-X19 CAR-T cells (Tecartus) offer a promising cell therapy option for relapsed and refractory MCL. While the overall response rate and complete remission rate after Tecartus treatment were high (91% and 68%, respectively) in MCL patients, progression-free survival and overall survival at approximately 3 years of follow-up were 25.8 months and 46.6 months, respectively, suggesting that a significant portion of these patients require additional treatment.

[0135] As reported by Wang et al., their early studies showed that patients treated with KTE-X19 CAR-T cells experienced predictable CAR-T therapy adverse events, such as cytopenia (grade 3 or higher in 94% of patients), cytokine release syndrome (91% of patients, 15% of which were grade 3–4), and neurotoxicity (63% of patients, 31% of which were grade 3–4), which may be mediated by CD19 expression in brain pericytes (Parker, 2020). This observation was supported by analysis of multiple single-cell RNA sequencing datasets from adult samples originating from different brain regions and validated by immunohistochemistry using CD19 antibodies (Parker, 2020). Interestingly, analysis of the same datasets revealed that CD74 was the gene with the highest differential expression between B cells and brain pericytes, as confirmed by flow cytometry. This suggests that CAR-T cells with 74bbz may not induce pericyte depletion, endothelial activation, and cerebral edema. While milatuzumab monotherapy has shown significant activity in preclinical models of B-cell malignancies, its rapid internalization of antibody-antigen complexes and antigen sinks, coupled with its extremely short half-life, has prevented it from providing meaningful clinical benefit to patients with CLL, MM, and B-cell NHL (Haran, 2018; Meartin, 2015). Relying on CD74 as a novel therapeutic target and to overcome the limitations of milatuzumab, we designed a second-generation CAR construct with 4-1 BB and CD3ζ chain signaling domains by ligating scFVs that specifically target human CD74 using CD8 hinge / transmembrane domains and CD3ζ. This construct was then transduced into CD74-negative central memory T cells to minimize the risk of CAR-T fratricide effects. Furthermore, the CAR-T cell construct contained humanized scFVs to minimize the risks of immunogenicity and anti-CAR immunity (Parker, 2020).

[0136] A strategy utilizing various assays, including flow cytometry-based antigen binding assays, T cell activation assays, repeated antigen stimulation assays (Smith, 2018), and cytotoxicity assays, was employed to investigate 74bbz CAR-T cells. In silico modeling of the binding of the CD74 antigen to the anti-CD74 scFV variant was used to identify V H Mutations on the chain and CDR3 were found to produce mutants with the highest docking scores and higher T cell activation. However, increased binding affinity and T cell activation do not necessarily lead to improved cytotoxicity and cell proliferation (Figure 1).

[0137] The main limitation of targeting CD74 with either naked monoclonal antibodies or CAR-T cell products is its expression in a subset of normal immune cells (Barrera, 2005; Martin, 2015). In summary, CD74 is expressed at varying levels on antigen-presenting cells such as B cells, monocytes, and activated T cells, in addition to a subset of bone marrow cells. Interestingly, milatuzumab demonstrated a desirable safety profile, with the most common adverse event being the infusion reaction. In phase I-II clinical trials in patients with relapsed / refractory CLL, treatment with milatuzumab did not result in significant cytopenia. Interestingly, a significant increase in non-malignant lymphocyte counts was observed after each infusion of milatuzumab in patients who showed a transient response to milatuzumab. (Haran, 2018) In a Phase I dose-escalation clinical trial using milatuzumab monotherapy in patients with relapsed / refractory MM, no significant changes from baseline were observed in routine blood tests (Kaufman, 2013). In a Phase I study of milatuzumab monotherapy in patients with previously treated B-cell NHL, the most common treatment-related grade 3–4 hematological toxicities were neutropenia (9%) and thrombocytopenia (5%), but lymphopenia was not observed (Martin, 2015).

[0138] Overall CD74 expression is significantly lower in the normal immune cell subset compared to MCL cells. Among the normal immune cell subset, T H and TCYTO Compared to normal cells, bone marrow and B cells expressed the highest levels of CD74. Interestingly, 42105-74bbz CAR-T cells failed to induce significant cytotoxicity in quiescent and activated normal T cells and CD33+ bone marrow cells, while cytotoxicity was significant against quiescent and activated B cells. Notably, in the humanized mouse model, there was no significant depletion of peripheral blood immune cell subsets, including B cells, monocytes, and NK cells.

[0139] The distinct cytotoxic effects of 42105-74bbz CAR-T cells on immune cell subsets can be partially explained by the pro-survival function of CD74 in normal B cells (Starlets, 2006). CD74 is essential for proper B cell development and, together with CD44, promotes B cell proliferation and survival by activating downstream pathways such as NF-κB and PI3K / Akt signaling upon MIF binding (Schruder, 2016). CD74 can also be expressed by gastrointestinal (GI) tract epithelial cells under inflammatory conditions (Barrera, 2005; Beswick, 2009), which is clearly a concern regarding potential toxicity after treatment with 42105-74bbz CAR-T cells. CD74 is post-translationally glycosylated and exists in five isoforms in humans (Schruder, 2016).

[0140] An interesting and relatively unexplored aspect of CD74 as a therapeutic target is that its surface expression can be pharmacologically regulated by inhibiting its degradation in the autophagosome / lysosomal compartment (Alinari, FTY720 increases CD74 expression, 2011; Alinari, 2012). Enhanced CD74 expression was positively correlated with milatuzumab-mediated MCL cell death (Alinari, FTY720 increases CD74 expression, 2011; Alinari, 2012). The data presented here demonstrate a positive correlation between CAR-T cell activity and target antigen density, providing a rationale for combination strategies to maximize the therapeutic potential of 42105-74bbz CAR-T cells. Important from a toxicity perspective is that inhibition of CD74 degradation affects CD74 expression on lymphoma cells but remains unaffected on its expression on normal immune cells. This is likely because lymphoma cells rely far more on autophagy for survival than normal immune cells (Alinari, 2017).

[0141] conclusion This is the first study to describe and investigate the development of CD74bbz CAR-T cell products. These findings provide evidence of significant activity of 42105-74bbz CAR-T cells in an MCL model with remarkably low toxicity in a subset of normal immune cells.

[0142] Other advantages will be apparent to those skilled in the art. It will be understood that certain properties and partial combinations are beneficial and can be used regardless of other properties and partial combinations. This is intended and within the scope of the claims. Since many possible embodiments of the present invention can be carried out without departing from its scope, it will be understood that all matters in this specification described or shown in the accompanying drawings should be interpreted as illustrative rather than restrictive. [Table 4-1] Table 4-2 Table 4-3 Table 4-4 Table 4-5 Table 4-6 Table 5-1 Table 5-2 Table 5-3 Table 5-4 Table 5-5 Table 5-6 Table 5-7 Table 5-8 Table 5-9 Table 5-10 Table 5-11 Table 5-12

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Table 5-19

[0143] References Armitage JO, Longo DL. Mantle-Cell Lymphoma. N Engl J Med. 2022;386(26):2495-506. Navarro A, Bea S, Jares P, Campo E. Molecular Pathogenesis of Mantle Cell Lymphoma. Hematol Oncol Clin North Am. 2020;34(5):795-807. Jain P, Wang M. Mantle cell lymphoma: 2019 update on the diagnosis, pathogenesis, prognostication, and management. Am J Hematol. 2019;94(6):710-25. Hill HA,Qi X,Jain P,Nomie K,Wang Y,Zhou S,et al.Genetic mutations and features of mantle cell lymphoma:a systematic review and meta-analysis. Blood Adv.2020;4(13):2927-38. Cheah CY,Chihara D,Romaguera JE,Fowler NH,Seymour JF,Hagemeister FB,et al.Patients with mantle cell lymphoma failing ibrutinib are unlikely to respond to salvage chemotherapy and have poor outcomes. Ann Oncol.2015;26(6):1175-9. Campo E,Rule S.Mantle cell lymphoma:evolving management strategies. Blood.2015;125(1):48-55. Kluin-Nelemans HC,Hoster E,Hermine O,Walewski J,Geisler CH,Trneny M,et al.Treatment of Older Patients With Mantle Cell Lymphoma(MCL):Long-Term Follow-Up of the Randomized European MCL Elderly Trial.J Clin Oncol.2020;38(3):248-56. Wang ML,Rule S,Martin P,Goy A,Auer R,Kahl BS,et al.Targeting BTK with ibrutinib in relapsed or refractory mantle-cell lymphoma. N Engl J Med.2013;369(6):507-16. Tam CS,Anderson MA,Pott C,Agarwal R,Handunnetti S,Hicks RJ,et al.Ibrutinib plus Venetoclax for the Treatment of Mantle-Cell Lymphoma. N Engl J Med.2018;378(13):1211-23. Wang M,Munoz J,Goy A,Locke FL,Jacobson CA,Hill BT,et al.KTE-X19 CAR T-Cell Therapy in Relapsed or Refractory Mantle-Cell Lymphoma. N Engl J Med.2020;382(14):1331-42. Wang M,Munoz J,Goy A,Locke FL,Jacobson CA,Hill BT,et al.Three-Year Follow-Up of KTE-X19 in Patients With Relapsed / Refractory Mantle Cell Lymphoma,Including High-Risk Subgroups,in the ZUMA-2 Study. J Clin Oncol.2023;41(3):555-67. Schroder B.The multifaceted roles of the invariant chain CD74--More than just a chaperone. Biochim Biophys Acta.2016;1863(6 Pt A):1269-81. Basha G,Omilusik K,Chavez-Steenbock A,Reinicke AT,Lack N,Choi KB,et al.A CD74-dependent MHC class I endolysosomal cross-presentation pathway. Nat Immunol.2012;13(3):237-45. Starlets D,Gore Y,Binsky I,Haran M,Harpaz N,Shvidel L,et al.Cell-surface CD74 initiates a signaling cascade leading to cell proliferation and survival.Blood.2006;107(12):4807-16. Barrera CA,Beswick EJ,Sierra JC,Bland D,Espejo R,Mifflin R,et al.Polarized expression of CD74 by gastric epithelial cells. J Histochem Cytochem.2005;53(12):1481-9. Ong GL,Goldenberg DM,Hansen HJ,Mattes MJ.Cell surface expression and metabolism of major histocompatibility complex class II invariant chain(CD74)by diverse cell lines. Immunology.1999;98(2):296-302. Stein R,Mattes MJ,Cardillo TM,Hansen HJ,Chang CH,Burton J,et al.CD74:a new candidate target for the immunotherapy of B-cell neoplasms. Clin Cancer Res.2007;13(18 Pt 2):5556s-63s. Haran M,Mirkin V,Braester A,Harpaz N,Shevetz O,Shtreiter M,et al.A phase I-II clinical trial of the anti-CD74 monoclonal antibody milatuzumab in frail patients with refractory chronic lymphocytic leukaemia:A patient based approach. Br J Haematol.2018;182(1):125-8. Alinari L,Mahoney E,Patton J,Zhang X,Huynh L,Earl CT,et al.FTY720 increases CD74 expression and sensitizes mantle cell lymphoma cells to milatuzumab-mediated cell death. Blood.2011;118(26):6893-903. Alinari L,Yu B,Christian BA,Yan F,Shin J,Lapalombella R,et al.Combination anti-CD74(milatuzumab)and anti-CD20(rituximab)monoclonal antibody therapy has in vitro and in vivo activity in mantle cell lymphoma. Blood.2011;117(17):4530-41. Alinari L,Baiocchi RA,Praetorius-Ibba M.FTY720-induced blockage of autophagy enhances anticancer efficacy of milatuzumab in mantle cell lymphoma:is FTY720 the next autophagy-blocking agent in lymphoma treatment? Autophagy.2012;8(3):416-7. Stein R,Qu Z,Cardillo TM,Chen S,Rosario A,Horak ID,et al.Antiproliferative activity of a humanized anti-CD74 monoclonal antibody,hLL1,on B-cell malignancies. Blood.2004;104(12):3705-11. Martin P,Furman RR,Rutherford S,Ruan J,Ely S,Greenberg J,et al.Phase I study of the anti-CD74 monoclonal antibody milatuzumab(hLL1)in patients with previously treated B-cell lymphomas. Leuk Lymphoma.2015;56(11):3065-70. Leem J,Deane CM. High-Throughput Antibody Structure Modeling and Design Using ABodyBuilder. Methods Mol Biol.2019;1851:367-80. Ambrosetti F,Jandova Z,Bonvin AMJJ.Information-Driven Antibody-Antigen Modelling with HADDOCK. Methods Mol Biol.2023;2552:267-82. Yin S,Ding F,Dokholyan NV. Eris:an automated estimator of protein stability. Nat Methods.2007;4(6):466-7. Geuijen CA,Clijsters-van der Horst M,Cox F,Rood PM,Throsby M,Jongeneelen MA,et al.Affinity ranking of antibodies using flow cytometry:application in antibody phage display-based target discovery. J Immunol Methods.2005;302(1-2):68-77. Smith EL,Staehr M,Masakayan R,Tatake IJ,Purdon TJ,Wang X,et al.Development and Evaluation of an Optimal Human Single-Chain Variable Fragment-Derived BCMA-Targeted CAR T Cell Vector. Mol Ther.2018;26(6):1447-56. Chan WK,Suwannasaen D,Throm RE,Li Y,Eldridge PW,Houston J,et al.Chimeric antigen receptor-redirected CD45RA-negative T cells have potent antileukemia and pathogen memory response without graft-versus-host activity. Leukemia.2014. Verma B,Wesa A.Establishment of Humanized Mice from Peripheral Blood Mononuclear Cells or Cord Blood CD34+ Hematopoietic Stem Cells for Immune-Oncology Studies Evaluating New Therapeutic Agents. Curr Protoc Pharmacol.2020;89(1):e77. Myung Y,Rodrigues CHM,Ascher DB,Pires DEV.mCSM-AB2:guiding rational antibody design using graph-based signatures. Bioinformatics.2020;36(5):1453-9. Al-Mansour M.Treatment Landscape of Relapsed / Refractory Mantle Cell Lymphoma:An Updated Review. Clin Lymphoma Myeloma Leuk.2022;22(11):e1019-e31. Parker KR,Migliorini D,Perkey E,Yost KE,Bhaduri A,Bagga P,et al.Single-Cell Analyses Identify Brain Mural Cells Expressing CD19 as Potential Off-Tumor Targets for CAR-T Immunotherapies. Cell.2020;183(1):126-42.e17. Kaufman JL,Niesvizky R,Stadtmauer EA,Chanan-Khan A,Siegel D,Horne H,et al.Phase I,multicentre,dose-escalation trial of monotherapy with milatuzumab(humanized anti-CD74 monoclonal antibody)in relapsed or refractory multiple myeloma. Br J Haematol.2013;163(4):478-86. Beswick EJ,Reyes VE. CD74 in antigen presentation,inflammation,and cancers of the gastrointestinal tract. World J Gastroenterol.2009;15(23):2855-61. Rodriguez-Marquez P,Calleja-Cervantes ME,Serrano G,Oliver-Caldes A,Palacios-Berraquero ML,Martin-Mallo A,et al.CAR density influences antitumoral efficacy of BCMA CAR T cells and correlates with clinical outcome. Sci Adv.2022;8(39):eabo0514. Majzner RG,Rietberg SP,Sotillo E,Dong R,Vachharajani VT,Labanieh L,et al.Tuning the Antigen Density Requirement for CAR T-cell Activity. Cancer Discov.2020;10(5):702-23. Alinari L.Toward autophagy-targeted therapy in lymphoma. Blood.2017;129(13):1740-2.

Claims

1. A chimeric antigen receptor (CAR) polypeptide comprising a CD74 antigen-binding domain, a transmembrane domain, an intracellular signaling domain, and a co-stimulatory signaling region.

2. The polypeptide according to claim 1, wherein the CD74 antigen-binding domain is a single-chain variable fragment (scFv) of an antibody that specifically binds to CD74.

3. The polypeptide according to any one of claims 1 to 2, wherein the costimulatory signaling region includes the cytoplasmic domain of costimulatory molecule 4-1BB.

4. The polypeptide according to any one of claims 1 to 3, wherein the intracellular signaling domain includes a CD3 zeta (CD3δ) signaling domain.

5. A polypeptide according to any one of claims 1 to 4, wherein the CAR polypeptide is of formula: SP-CD74-HG-TM-CSR-ISD; or Defined by SP-CD74-HG-TM-ISD-CSR, In the formula, "SP" represents signal peptide. In the formula, "CD74" represents the CD74 binding region. In the formula, "HG" represents an optional hinge domain. In the formula, "CSR" represents the co-stimulus signaling region. In the formula, "ISD" represents the intracellular signal transduction domain. The polypeptide wherein "-" represents an optional divalent linker.

6. A polypeptide according to any one of claims 1 to 5, wherein the CAR polypeptide is of formula: SP-(VL-VH) ｎ -HG-TM-CSR-ISD; or SP-(VH-VL) ｎ -HG-TM-CSR-ISD;SP-(VL-VH) ｎ -HG-TM-ISD-CSR; or SP-(VH-VL) ｎ - Defined by HG-TM-CSR-ISD, In the formula, "SP" represents signal peptide. In the formula, "VL" represents the light chain variable region. In the formula, "VH" represents the heavy chain variable region. In the formula, n is ≥ 1, In the formula, "HG" represents an optional hinge domain. In the formula, "CSR" represents the co-stimulus signaling region. In the formula, "ISD" represents the intracellular signal transduction domain. The polypeptide wherein "-" represents an optional divalent linker.

7. The polypeptide according to any one of claims 2 to 6, wherein the scFv of the antibody that specifically binds to the CD74 comprises a sequence having at least 60% identity with SEQ ID NOs. 2 to 7 or a fragment thereof.

8. The polypeptide according to claim 6, wherein the scFv of the antibody that specifically binds to the CD74 comprises a sequence having at least 60% identity with SEQ ID NO: 2 or a fragment thereof.

9. The polypeptide according to any one of claims 2 to 6, wherein the scFv of the antibody that specifically binds to the CD74 comprises a sequence having at least 60% identity with SEQ ID NOs. 8 to 12 or a fragment thereof.

10. The polypeptide according to claim 9, wherein the scFv of the antibody that specifically binds to CD74 comprises a sequence having at least 60% identity with SEQ ID NO: 8 or a fragment thereof.

11. The polypeptide according to any one of claims 2 to 6, wherein the scFv of the antibody that specifically binds to the CD74 comprises a sequence having at least 60% identity with SEQ ID NOs. 13 to 14 or a fragment thereof.

12. The polypeptide according to claim 11, wherein the scFv of the antibody that specifically binds to the CD74 comprises a sequence having at least 60% identity with SEQ ID NO: 13 or a fragment thereof.

13. The polypeptide according to claim 8, wherein the antibody comprises a heavy chain variable region (VH) having at least 60% identity with sequences of sequence numbers 74, 75, and 76, and a light chain variable region (VL) having at least 60% identity with sequences of sequence numbers 77, 78, and 79.

14. The polypeptide according to claim 10, wherein the antibody comprises a heavy chain variable region (VH) having at least 60% identity with sequences of sequence numbers 80, 81, and 82, and a light chain variable region (VL) having at least 60% identity with sequences of sequence numbers 83, 84, and 85.

15. The polypeptide according to claim 12, wherein the antibody comprises a heavy chain variable region (VH) having at least 60% identity with sequence numbers 86, 87, and 88, and a light chain variable region (VL) having at least 60% identity with sequence numbers 89, 90, and 91.

16. An isolated nucleic acid sequence encoding a recombinant polypeptide according to any one of claims 1 to 15.

17. A vector comprising the isolated nucleic acid described in claim 16.

18. A cell comprising the vector according to claim 17.

19. The cell according to claim 18, wherein the cell reduces tumor activity when the antigen-binding domain of CAR binds to CD74.

20. A method for treating lymphoma in a subject requiring treatment, comprising administering to the subject a therapeutically effective amount of a chimeric antigen receptor polypeptide according to any one of claims 1 to 15.

21. The method according to claim 20, wherein the lymphoma includes non-Hodgkin lymphoma.

22. The method according to claim 21, wherein the non-Hodgkin lymphoma includes mantle cell lymphoma (MCL).

23. The method according to claim 22, wherein the mantle cell lymphoma includes mantle cell lymphoma of stage I, stage II, stage III, or stage IV.

24. The method according to any one of claims 20 to 23, further comprising administering a therapeutically effective amount of a CD74 expression regulator.

25. The method according to any one of claims 20 to 24, further comprising administering a therapeutically effective amount of an autophagy inhibitor.

26. The method according to any one of claims 20 to 25, further comprising administering a therapeutically effective amount of a histone deacetylase (HDAC) inhibitor.

27. The method according to any one of claims 20 to 26, wherein the chimeric antigen receptor is administered in combination with at least one cancer treatment.

28. The method according to claim 27, wherein the additional cancer treatment includes surgery, chemotherapy, immunotherapy, ionizing radiation, stem cell transplantation (SCT), or a combination thereof.

29. A method for reducing tumor activity in a subject with lymphoma, comprising administering to the subject a therapeutically effective amount of a chimeric antigen receptor polypeptide according to any one of claims 1 to 15.

30. The method according to claim 29, wherein the lymphoma includes non-Hodgkin lymphoma.

31. The method according to claim 30, wherein the non-Hodgkin lymphoma includes mantle cell lymphoma (MCL).

32. The method according to any one of claims 29 to 31, wherein the mantle cell lymphoma includes mantle cell lymphoma of stage I, stage II, stage III, or stage IV.

33. The method according to any one of claims 29 to 32, further comprising administering a therapeutically effective amount of a CD74 expression regulator.

34. The method according to any one of claims 29 to 33, further comprising administering a therapeutically effective amount of an autophagy inhibitor.

35. The method according to any one of claims 29 to 34, further comprising administering a therapeutically effective amount of an HDAC inhibitor.

36. The method according to any one of claims 29 to 35, wherein the chimeric antigen receptor is administered in combination with at least one cancer treatment.

37. The method according to claim 36, wherein the additional cancer treatment includes surgery, chemotherapy, immunotherapy, ionizing radiation, stem cell transplantation (SCT), or a combination thereof.

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