Composition of Zebrutinib, anti-CD20 antibody and lenalidomide and application of composition in preparation of drug for combined treatment of diffuse large B-cell lymphoma

Abstract

The invention relates to a composition of Zebrutinib, an anti-CD20 antibody and lenalidomide and application of the composition in preparation of a drug for combined treatment of diffuse large B-cell lymphoma. According to the scheme, aiming at the group of old DLBCL patients, a chemotherapy-free immunotherapy with dual targeting molecules and microenvironment change is adopted as a first-line therapy, and the combined treatment medicine for the diffuse large B-cell lymphoma is prepared by utilizing the combination of the Zobutinib, the anti-CD20 antibody and the lenalidomide. Side effects can be reduced, and the living quality of patients is improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a composition of zanubrutinib, an anti-CD20 antibody and lenalidomide and use thereof in preparing a combined therapeutic drug for diffuse large B-cell lymphoma. Background Art

[0002] Diffuse large B-cell lymphoma (DLBCL) is the most important subtype of non-Hodgkin's lymphoma, with different clinical, immunophenotypes and genetic characteristics. Recent studies have shown that the incidence of DLBCL increases with age, and patients over 75 years old account for about 30% of DLBCL cases. It is worth noting that age is an important influencing factor of the established International Prognostic Index (IPI) and one of the most relevant risk factors for DLBCL.

[0003] For elderly patients without severe comorbidities, the anti-CD20 monoclonal antibody rituximab combined with reduced-dose cyclophosphamide, doxorubicin, vincristine, and prednisone (R-miniCHOP) is the standard treatment. The complete response rate (CR) of this treatment regimen is approximately 60%, while the 2-year progression-free survival (PFS) and overall survival (OS) are 47% and 59%, respectively. However, since elderly DLBCL patients often have comorbidities and are intolerant to standard immunochemotherapy, there is an urgent need to explore new effective and safe treatments to meet the needs of this population.

[0004] Elderly DLBCL patients are a unique biological entity with unfavorable molecular features, such as age-related oncogenic mutations in MYD88 and CD79B, and an immunosuppressive tumor microenvironment, such as reduced cytotoxic activity and increased exhausted T cells. This microenvironment significantly affects the therapeutic effect, resulting in reduced efficacy of traditional treatments.

[0005] Since elderly patients with DLBCL have unique biological characteristics and treatment challenges, there is an urgent need to develop new treatment strategies. Summary of the invention

[0006] The purpose of the present invention is to provide a composition of zanubrutinib, anti-CD20 antibody and lenalidomide and its use in the preparation of a combined therapeutic drug for diffuse large B-cell lymphoma in order to overcome the defects of the above-mentioned prior art. The composition of zanubrutinib, anti-CD20 antibody and lenalidomide provided in the present application shows good clinical efficacy and safety in elderly patients with newly treated DLBCL by targeting changes in the tumor microenvironment.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] The present invention first provides a composition of zanubrutinib or a pharmaceutically acceptable salt thereof, an anti-CD20 antibody and lenalidomide.

[0009] In one embodiment of the present invention, the dosage relationship of zanubrutinib or a pharmaceutically acceptable salt thereof, anti-CD20 antibody and lenalidomide is: 320 mg: 375 mg / m 2 BSA: 20-25mg. BSA is body surface area, BSA (m 2 )=(√(height(cm)xweight(kg) / 3600))

[0010] In one embodiment of the present invention, the anti-CD20 antibody is selected from one of rituximab, ibritumomab tiuxetan, tositumomab, ofatumumab or obinutuzumab. Preferably, the anti-CD20 antibody is selected from rituximab.

[0011] The present invention further provides a use of a combination of zanubrutinib or a pharmaceutically acceptable salt thereof, an anti-CD20 antibody and lenalidomide in the preparation of a combined therapeutic drug for diffuse large B-cell lymphoma. In the combined therapeutic drug for diffuse large B-cell lymphoma, the zanubrutinib or a pharmaceutically acceptable salt thereof, the anti-CD20 antibody and lenalidomide are stored separately.

[0012] In one embodiment of the present invention, the diffuse large B-cell lymphoma is diffuse large B-cell lymphoma in patients over 75 years old.

[0013] In one embodiment of the present invention, the diffuse large B-cell lymphoma is de novo DLBCL.

[0014] In one embodiment of the present invention, the newly diagnosed diffuse large B-cell lymphoma (DLBCL) is activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL), germinal center B-cell diffuse large B-cell lymphoma (GCB-DLBCL), non-germinal center B-cell diffuse large B-cell lymphoma (non-GCB DLBCL) or DLBCL of uncertain subtype.

[0015] The present invention further provides a combined treatment kit for diffuse large B-cell lymphoma, comprising zanubrutinib or a pharmaceutically acceptable salt thereof, an anti-CD20 antibody and lenalidomide.

[0016] In one embodiment of the present invention, in the method for using the kit:

[0017] a) Subjects were given 160 mg of zanubrutinib twice daily or 320 mg of zanubrutinib once daily; on the first day of each treatment cycle, subjects were given rituximab 375 mg / m2 intravenously or subcutaneously. 2 BSA; and administering lenalidomide 20-25 mg orally daily to the subject from the first day or second day of each treatment cycle to approximately the first 1 / 2 to 3 / 4 of each cycle; or,

[0018] b) During each treatment cycle, subjects were given 160 mg of zanubrutinib twice daily or 320 mg of zanubrutinib once daily; subjects were given rituximab 375 mg / m2 BSA intravenously or subcutaneously on the first day of the first treatment cycle, and 500 mg / m2 BSA intravenously or subcutaneously on the first day of each subsequent treatment cycle. 2 BSA; and administering lenalidomide 20-25 mg orally daily to subjects from the first or second day of each treatment cycle to approximately the first 1 / 2 to 3 / 4 of each cycle.

[0019] In one embodiment of the present invention, in the method for using the kit: the treatment of diffuse large B-cell lymphoma is the first-line treatment.

[0020] In one embodiment of the present invention, in the method of using the kit: zanubrutinib or a pharmaceutically acceptable salt thereof is administered once a day (QD) in an amount of 320 mg zanubrutinib or twice a day (BID) in an amount of 160 mg zanubrutinib.

[0021] In one embodiment of the present invention, in the method for using the kit: the treatment includes an induction period, wherein the induction period includes one or more treatment cycles.

[0022] In one embodiment of the present invention, in the method for using the kit: the induction period includes 1 to 6 treatment cycles.

[0023] In one embodiment of the present invention, in the method for using the kit: the treatment cycle is 21 days or 28 days.

[0024] In one embodiment of the present invention, in the method for using the kit: the treatment further includes a maintenance period after the induction period.

[0025] In one embodiment of the present invention, in the method for using the kit: the maintenance period begins after the subject achieves complete remission (CR) or partial remission (PR).

[0026] In one embodiment of the present invention, in the method for using the kit: the maintenance period includes one or more cycles, wherein the subject is administered 20-25 mg of lenalidomide per day for approximately the first 1 / 2 to 3 / 4 of each cycle.

[0027] In one embodiment of the present invention, in the method for using the kit: the maintenance period is 21 days or 28 days.

[0028] In one embodiment of the present invention, in the method of using the kit: the maintenance period lasts for up to 2 years.

[0029] The present invention further provides a method for treating diffuse large B-cell lymphoma, which comprises the combined use of zanubrutinib or a pharmaceutically acceptable salt thereof, an anti-CD20 antibody and lenalidomide.

[0030] In one embodiment of the present invention, zanubrutinib or a pharmaceutically acceptable salt thereof is administered orally. In an embodiment of each of the above aspects, zanubrutinib or a pharmaceutically acceptable salt thereof is administered in the form of a capsule or tablet.

[0031] In one embodiment of the present invention, the anti-CD20 antibody is administered by intravenous injection or subcutaneous injection.

[0032] In one embodiment of the present invention, zanubrutinib or a pharmaceutically acceptable salt thereof is administered once a day (QD) in an amount of 320 mg zanubrutinib or twice a day (BID) in an amount of 160 mg zanubrutinib.

[0033] In one embodiment of the invention, the treatment comprises an induction period, wherein the induction period comprises one or more treatment cycles.

[0034] In one embodiment of the present invention, the induction period includes 1-6 treatment cycles.

[0035] In one embodiment of the present invention, the treatment cycle is 21 days or 28 days.

[0036] In one embodiment of the present invention, a) in each treatment cycle, the subject is administered 160 mg of zanubrutinib twice a day or 320 mg of zanubrutinib once a day; on the first day of each treatment cycle, the subject is intravenously or subcutaneously injected with rituximab 375 mg / m2 BSA; and on the first day or second day of each treatment cycle to about the first 1 / 2 to 3 / 4 of each cycle, the subject is administered lenalidomide 20-25 mg orally daily; or,

[0037] b) During each treatment cycle, the subjects were administered 160 mg of zanubrutinib twice a day or 320 mg of zanubrutinib once a day; rituximab 375 mg / m2 BSA was injected intravenously or subcutaneously on the first day of the first treatment cycle, and rituximab 500 mg / m2 BSA was injected intravenously or subcutaneously on the first day of each subsequent treatment cycle; and lenalidomide 20-25 mg was administered orally daily from the first or second day of each treatment cycle to approximately the first 1 / 2 to 3 / 4 of each cycle.

[0038] In one embodiment of the present invention, a) the treatment cycle is 21 days, and in each treatment cycle, the subject is administered 160 mg of zanubrutinib twice a day or 320 mg of zanubrutinib once a day; on the first day of each treatment cycle, the subject is intravenously or subcutaneously injected with rituximab 375 mg / m2 BSA; and on the first day or the second day to the 11th day of each treatment cycle, the subject is administered lenalidomide 20-25 mg orally per day; or,

[0039] b) The treatment cycle is 28 days. During each treatment cycle, the subjects are administered 160 mg of zanubrutinib twice a day or 320 mg of zanubrutinib once a day; on the first day of each treatment cycle, the subjects are injected intravenously or subcutaneously with rituximab 375 mg / m2 BSA; and from the first or second day to the 15th or 21st day of each treatment cycle, the subjects are administered 20-25 mg of lenalidomide orally every day.

[0040] In one embodiment of the present invention, the treatment further comprises a maintenance period after the induction period, and the maintenance period begins after the subject achieves complete remission (CR) or partial remission (PR).

[0041] In one embodiment of the present invention, the maintenance period comprises one or more cycles, wherein the subject is administered 20-25 mg of lenalidomide per day for approximately the first 1 / 2 to 3 / 4 of each cycle.

[0042] In one embodiment of the present invention, the maintenance period has a period of 21 to 28 days and the maintenance period lasts for up to 2 years.

[0043] As an irreversible and selective Bruton's tyrosine kinase (BTK) inhibitor, zanubrutinib can effectively disrupt chronic B cell receptor (BCR) signaling, leading to downregulation of the nuclear factor κB (NF-κB) pathway and apoptosis of lymphoma cells. In addition, BTK inhibition also promotes the differentiation of monocytic dendritic cells (DCs) and enhances the immunity of anti-tumor T cells.

[0044] Lenalidomide induces NF-κB inactivation by targeting IRF4 downstream of the BCR / MYD88 cascade and co-stimulates CD4+ and CD8+ T cells.

[0045] The present application scheme targets the group of elderly DLBCL patients, adopts chemotherapy-free immunotherapy with dual-targeted molecules and microenvironment changes as first-line therapy, and utilizes a combination of zanubrutinib, anti-CD20 antibodies and lenalidomide to prepare a combined therapeutic drug for diffuse large B-cell lymphoma. The scheme of the present invention not only helps to improve the therapeutic effect, but also reduces side effects and improves the quality of life of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1. Flowchart, survival, and mutational profile of patients with diffuse large B-cell lymphoma.

[0047] (a) Patient flow chart of this trial.

[0048] (b) Kaplan Meier curves of progression-free survival and overall survival of patients.

[0049] (c) Waterfall plot of the patient's mutated genes.

[0050] Figure 2. Intertumor heterogeneity in diffuse large B-cell lymphoma is associated with the efficacy of ZR2.

[0051] (a) Flowchart of sample collection and analysis by single-cell RNA sequencing.

[0052] (b) UMAP of malignant and non-malignant B cells, with each cell type stained for sample origin.

[0053] (c) UMAP of malignant and non-malignant B cells, each cell type is colored for sample subsets according to efficacy and time point. Samples from responders (n=12) and non-responders (n=5) were collected before treatment, and one paired sample was collected from one of the non-responders at the time of disease progression.

[0054] (d) UMAP of malignant and non-malignant B cells, each cell is stained for malignant and normal clusters.

[0055] (e) Inferred single-cell CNVs are shown for single cells (rows) and chromosomal regions (columns) for each malignant cell cluster and reference immune cells.

[0056] (f) Inferred CNV scores for each malignant cell cluster and reference immune cells are shown.

[0057] (g) Upregulated pathways in malignant B cells of responders (n=12) compared with non-responders (n=5) (analyzed by GSEA). The color and size of the dots represent the normalized P value of the upregulated pathways in the two groups, respectively.

[0058] (h) Heat map of differentially expressed genes in malignant B cells between responders (n=12) and non-responders (n=5).

[0059] (i) Immunohistochemical detection of HLA-I and HLA-II in tumor samples from responders (n=10) and non-responders (n=7).

[0060] Figure 3. Myeloid cell infiltration in diffuse large B-cell lymphoma is associated with the efficacy of ZR2.

[0061] (a) UMAP of myeloid cells, each stained for a specific cell type.

[0062] (b) Heat map showing the expression of marker genes in myeloid cell types identified by single-cell RNA sequencing.

[0063] (c) Comparison of the percentage of myeloid cells in each sample according to the efficacy of ZR2.

[0064] (d) Expression levels of four gene signatures (M1-like, M2-like, angiogenesis, and antigen presentation signatures) in myeloid cell clusters.

[0065] (e) Volcano plot showing the difference in cDC1 gene expression between responders (n=12) and non-responders (n=5).

[0066] (f) cDC1 pathway up-regulated in responders (n=12) compared to non-responders (n=5) (by GSEA analysis). The color and size of the dots represent the normalized P value of the up-regulated pathway in the two groups, respectively.

[0067] (g) Comparison of the expression of cDC1 signature at baseline according to the efficacy of treatment without chemotherapy (ZR2) by bulk RNA sequencing.

[0068] (h) Kaplan Meier curves of progression-free survival in patients treated without chemotherapy (ZR2) according to the expression of the cDC1 signature. In the chemotherapy-free cohort, group cutoff was determined by disease progression within 2 years.

[0069] (i) Expression of maturation and activation markers of monocyte-derived DCs treated with zanubrutinib alone, lenalidomide alone, and the combination in three healthy donors. Z: zanubrutinib 25 nM; L: lenalidomide 2 μM.

[0070] Figure 4. T cell and NK cell infiltration in diffuse large B-cell lymphoma is associated with the efficacy of ZR2.

[0071] (a) UMAP of T and NK cells, each stained for a specific cell type.

[0072] (b) Comparison of the percentages of T and NK cells in each sample according to the efficacy of ZR2.

[0073] (c) Scatterplot of the correlation between the number of T cell clonotypes (i.e., the number of T cells contributing to the clonotype) and the size of the clonotype according to the efficacy of ZR2.

[0074] (d) Distribution of the most abundant T cell clonotypes in each sample according to the efficacy of ZR2.

[0075] (e) Single-cell trajectory inference of CD8+ T cells, colored according to their corresponding pseudotime, cell type, and expression levels of cytotoxicity and exhaustion signatures.

[0076] (f) Heat map of overlapping clonotypes between different cell types according to the efficacy of ZR2.

[0077] (g) Comparison of the expression levels of cytotoxic and exhaustion signatures of CD8+ T cells according to the efficacy of ZR2.

[0078] (h) Comparison of the expression of cytotoxic and exhaustion signatures at baseline according to the efficacy of treatment without chemotherapy (ZR2) by bulk RNA sequencing.

[0079] (i) Kaplan Meier curves for progression-free survival in patients treated without chemotherapy (ZR2) according to expression of cytotoxic or exhaustion signatures. Group cutoffs were determined by disease progression within two years.

[0080] Figure 5. Subgroup analysis of treatment outcomes according to clinicopathological characteristics and genetic subtypes.

[0081] (a) Subgroup analysis of complete response rate.

[0082] (b) Subgroup analysis of progression-free survival.

[0083] (c) Subgroup analysis of overall survival.

[0084] Figure 6. Study design and annotation of major cell clusters in diffuse large B-cell lymphoma by single-cell RNA sequencing.

[0085] (a) UMAP shows the sample origin of major cell clusters and sample subsets according to treatment efficacy and time point. All 17 samples from 12 responders and 5 non-responders were analyzed before treatment. A paired sample from one non-responder at the time of disease progression was also analyzed.

[0086] (b) UMAP shows the UMIs of detected genes for all cells.

[0087] (c) UMAP shows the number of detected genes for all cells.

[0088] (d) Dot plot showing the expression of marker genes in major cell clusters identified by single-cell RNA sequencing.

[0089] (e) UMAP of the major cell clusters identified in diffuse large B-cell lymphoma.

[0090] (f) Distribution of major cell types according to efficacy and time points.

[0091] Figure 7. Distribution of malignant B cells associated with the efficacy of ZR2.

[0092] (a) Distribution of malignant B cell clusters in each sample.

[0093] (b) Distribution of sample origins for each malignant B cell cluster.

[0094] (c) Comparison of CNV scores of malignant B cells between responders (n=12) and non-responders (n=5).

[0095] (d) Volcano plot showing the differential expression of malignant B cell genes between responders (n=12) and non-responders (n=5).

[0096] Figure 8. Stromal cells in diffuse large B-cell lymphoma are associated with the efficacy of ZR2.

[0097] (a) UMAP of fibroblasts, with each cell stained for a specific cell type.

[0098] (b) UMAP of fibroblasts, with each cell stained for its sample origin.

[0099] (c) UMAP of fibroblasts, with each cell type colored for sample subsets according to efficacy and time point. Samples from responders (n=12) and non-responders (n=5) were collected before treatment, and one paired sample was collected from one of the non-responders at the time of disease progression.

[0100] (d) Dot plot showing the expression of marker genes in fibroblasts identified by single-cell RNA sequencing.

[0101] (e) Comparison of the percentage of fibroblasts in each sample according to the efficacy of ZR2.

[0102] (f) UMAP of endothelial cells, each cell type stained for a specific cell type.

[0103] (g) UMAP of endothelial cells, with each cell type stained for its sample origin.

[0104] (h) UMAP of endothelial cells, with each cell type colored for sample subsets according to efficacy and time point. Samples from responders (n=12) and non-responders (n=5) were collected before treatment, and a paired disease progression sample was collected from one of the non-responders at the time of disease progression.

[0105] (i) Dot plots showing the expression of marker genes in endothelial cells identified by single-cell RNA sequencing.

[0106] (j) Comparison of the endothelial cell percentage in each sample according to the efficacy of ZR2.

[0107] Figure 9. Sample origin and upregulated genes of myeloid cells according to ZR2 efficacy, and cDC1 signature associated with diffuse large B-cell lymphoma prognosis.

[0108] (a) UMAP of myeloid cells, with each cell type stained for its sample origin.

[0109] (b) UMAP of myeloid cells, with each cell type colored for sample subsets according to efficacy and time point. Samples from responders (n=12) and non-responders (n=5) were collected before treatment, and one paired sample was collected from one of the non-responders at the time of disease progression.

[0110] (c) Violin plot showing the difference in cDC1 gene expression between responders (n=12) and non-responders (n=5).

[0111] (d) Comparison of the expression of cDC1 signature at baseline according to the efficacy of RCHOP-based therapy by bulk RNA sequencing.

[0112] (e) Kaplan Meier curves of progression-free survival in patients receiving RCHOP-based therapy according to expression of the cDC1 signature. In the chemotherapy-free (ZR2) cohort, group cutoff was determined by disease progression within two years.

[0113] (f) Expression of maturation and activation markers in monocyte-derived DCs treated with zanubrutinib alone, lenalidomide alone, and the combination in three healthy donors. Figure 3g Related. Z: zanubrutinib 25nM; L: lenalidomide 2μM.

[0114] Figure 10. Comparison of T and NK cells in each sample according to the efficacy of ZR2.

[0115] (a) Dot plots showing the expression of marker genes in T cells and NK cells identified by single-cell RNA sequencing.

[0116] (b) UMAP of T cells and NK cells, each cell type is stained for sample origin according to the efficacy of ZR2.

[0117] (c) UMAP of T cells and NK cells, each cell type is colored for sample subsets according to efficacy and time point. Samples from responders (n=12) and non-responders (n=5) were collected before treatment, and one paired sample was collected from one of the non-responders at the time of disease progression.

[0118] (d) UMAP of T cells and NK cells, each cell type stained for cell cycle status.

[0119] (e) Heat map of selected cell cycle marker genes of T cell clusters.

[0120] (f) Diversity of T cell clonotypes according to the efficacy of ZR2.

[0121] (g) Distribution of the most abundant T cell clonotypes in each cell type according to the efficacy of ZR2.

[0122] Figure 11. Cytotoxicity and exhaustion characteristics of CD8+ T cells correlate with the efficacy of ZR2.

[0123] (a) Expression of cytotoxic and exhaustion signatures according to cell type of CD8+ T cells.

[0124] (b) Correlation between the expression of cytotoxic and exhaustion signatures obtained by single-cell RNA sequencing.

[0125] (c) Correlation between the expression of cytotoxic and exhaustion signatures in the chemotherapy-free (ZR2) cohort by bulk RNA sequencing.

[0126] (d) Correlation between the expression of cytotoxic and exhaustion signatures in the RCHOP-based cohort by bulk RNA sequencing.

[0127] (e) Comparison of the expression of cytotoxic and exhaustion signatures of CD8+ T subsets according to the efficacy of ZR2.

[0128] (f) Comparison of the expression of cytotoxic and exhaustion signatures at baseline according to the efficacy of RCHOP-based therapy by bulk RNA sequencing.

[0129] (g) Kaplan Meier curves of progression-free survival in patients receiving RCHOP-based therapy according to expression of cytotoxic or exhaustion signatures. Figure 4i same.

[0130] (h) Interaction of T cell activation and inhibitory signals with CD8+ T cells from malignant B cells and myeloid cells according to the efficacy of ZR2.

[0131] Figure 12. Recruitment, proliferation, and activation of cDC1 in diffuse large B-cell lymphoma are associated with the efficacy of ZR2.

[0132] (a) Interaction of recruitment, proliferation, and activation signals with cDC1 in diffuse large B-cell lymphoma according to the efficacy of ZR2.

[0133] (b) Correlation between the expression of cDC1 signature and the expression of genes involved in cDC1 recruitment, proliferation, and activation in the chemotherapy-free (ZR2) cohort. P values ​​and R values ​​are indicated in each figure.

[0134] (c) Correlation between the expression of cDC1 signatures and the expression of genes involved in cDC1 recruitment, proliferation, and activation in the RCHOP-based cohort. P values ​​and R values ​​are indicated in each figure.

[0135] Figure 13. Longitudinal single-cell analysis of diffuse large B-cell lymphoma before treatment and at progression.

[0136] (a) UMAP shows malignant B cells from patient ZR209, with samples collected before treatment and at disease progression. Each cell is stained for a specific B cell receptor clonotype.

[0137] (b) UMAP shows malignant B cells from patient ZR209, with samples collected before treatment and at disease progression. Each cell is stained for a specific clone.

[0138] (c) Dynamic changes of malignant B cells in patient ZR209 during treatment.

[0139] (d) UMAP showing malignant clones from patient ZR209, samples collected before treatment and at disease progression. Each cell is stained for a specific clone.

[0140] (e) Dynamic changes of malignant clones during treatment in patient ZR209.

[0141] (f) Bar graph showing pathway enrichment analysis of malignant B cells between sensitive clones and resistant or selected clones by GSEA.

[0142] (g) Volcano plot showing the differences in gene expression of malignant B cells between sensitive clones and resistant or selected clones.

[0143] (h) UMAP shows malignant B cells from patient ZR209, samples were collected before treatment and at disease progression. Each cell is colored according to its cell cycle state.

[0144] (i) Pie chart showing the percentage of different cell cycle states in the malignant clone of patient ZR209.

[0145] (j) Interaction of T cell activation signals with T and NK cells from the malignant clone of patient ZR209.

[0146] (k) Interaction of T cell inhibitory signals with T and NK cells from the malignant clone of patient ZR209. DETAILED DESCRIPTION

[0147] Unless specifically defined otherwise in this application, all technical and scientific terms used in this application have the same meanings as commonly understood by one of ordinary skill in the art.

[0148] The terms "administration" and "treatment" of the present application mean that an exogenous agent, therapeutic agent, diagnostic agent or composition contacts the animal, human, subject, cell, tissue, organ or biological fluid when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid. The treatment of cells covers reagent contacting the cell, and reagent contacting fluid, wherein the fluid contacts the cell. The terms "administration" and "treatment" also mean, for example, in vitro and ex vivo treatment of cells by reagents, diagnosis, binding compounds or other cells. The term "subject" of the present application includes any organism, preferably animal, more preferably mammal (e.g., rat, mouse, dog, cat, rabbit), and most preferably people. In one aspect, treating any disease or disorder refers to improving the disease or disorder (i.e., slowing down or preventing or alleviating the development of the disease or at least one of its clinical symptoms). In another aspect, "treatment" refers to alleviating or improving at least one physical parameter, including those that the patient may not be able to distinguish. In another aspect, "treatment" refers to either physically (e.g., the stability of discernible symptoms), physiologically (e.g., the stability of physical parameters), or regulating the disease or disorder in these two aspects. In yet another aspect, "treating" or "treating" refers to preventing or delaying the onset or development or progression of the disease or disorder.

[0149] As used in the present application, the term "therapeutically effective amount" refers to the amount of an inhibitor of such treatment that is sufficient to affect the disease, disorder, or symptom when administered to a subject for the treatment of at least one clinical symptom of a disease or disorder. "Therapeutically effective amount" can vary with age factors: the severity of the symptom of a medicament, disease, disorder, and / or disease or disorder, disease, disorder, and / or disease or disorder, the age of the subject to be treated, and / or the weight of the subject to be treated. In any given case, an appropriate amount can be apparent to those skilled in the art, or can be determined by routine experiments. In the case of combination therapy, "therapeutically effective amount" refers to the total amount of the composition for the effective treatment of a disease, disorder, or condition.

[0150] As used herein, the term "ZR2" refers to the combination therapy of zanubrutinib, rituximab, and lenalidomide.

[0151] Zanubrutinib

[0152] WO2014 / 173289A discloses (S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (hereinafter referred to as zanubrutinib). Zanubrutinib can be used to treat cancers in which there are aberrations in the B cell receptor (BCR) and FcR signaling pathways in which BTK plays an important role, and has been shown to have potent and irreversible inhibitory activity against BTK.

[0153]

[0154] Anti-CD20 antibody

[0155] "Anti-CD20 antibodies" include, but are not limited to, rituximab, ibritumomab tiuxetan, tositumomab, ofatumumab, or obinutuzumab.

[0156] Combination therapy

[0157] Zanubrutinib, anti-CD20 antibody and lenalidomide may be administered in a simultaneous or separate or sequential regimen. When administered sequentially, the combination may be administered in two or more administrations. Combined administration includes co-administration using separate formulations and sequential administration in any order, wherein preferably there is a period of time during which both (or all) active agents simultaneously exert their biological activities.

[0158] The 2-year PFS and OS rates of patients treated with the combination therapy of the present invention can reach 67.1% (95% CI: 50.1-79.4) and 82.4% (95% CI: 66.5-91.2), respectively.

[0159] In some embodiments, the combination therapy of the present application provides a synergistic and / or statistically improved effect relative to any use of a single drug or dual therapy. For example, the combination therapy of the present application can also significantly improve the patient's prognosis for the treatment of DLBCL. For example, in some embodiments, the combination therapy prolongs patient survival and progression-free survival, while having a manageable toxicity profile in patients with DLBCL.

[0160] In one embodiment of each of the above aspects, the amounts of zanubrutinib, anti-CD20 antibody, and lenalidomide disclosed herein and the relative timing of administration are determined by the individual need of the patient to be treated, the route of administration, the severity of the disease or affliction, the dosing schedule, and the evaluation and judgment of the prescribing physician.

[0161] Zanubrutinib, anti-CD20 antibodies and lenalidomide disclosed in the present application can be administered in a variety of known ways, such as orally, topically, rectally, parenterally, by inhalation spray or via an implanted reservoir, although the most appropriate route in any given case will depend on the specific host and the nature and severity of the condition for which the active ingredient is administered. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

[0162] The drug combination of zanubrutinib, anti-CD20 antibody, and lenalidomide enhances the anti-tumor effect through different mechanisms. Zanubrutinib inhibits the B cell signaling pathway, reducing B cell proliferation and survival; anti-CD20 antibody directly targets and eliminates B cells; lenalidomide enhances the immune system's anti-tumor response and inhibits tumor cell proliferation. The combination of these three drugs comprehensively inhibits the malignant transformation and spread of B cells through different pathways.

[0163] At the same time, the immunomodulatory effect of lenalidomide can enhance the ADCC activity of rituximab, while zanubrutinib reduces immune evasion by inhibiting BTK, making lenalidomide and anti-CD20 antibodies more effective. In addition, in our study, cell sequencing results showed that the combination of zanubrutinib and lenalidomide significantly enhanced the anti-tumor effect of dendritic cells (DCs), which are an important bridge between innate and acquired immunity and play a core role in anti-tumor immune responses.

[0164] In summary, the combination of zanubrutinib, anti-CD20 antibodies, and lenalidomide can jointly inhibit the growth and survival of tumor cells through multiple mechanisms and enhance the body's immune response, thereby playing a synergistic role in the treatment of DLBCL.

[0165] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0166] The present invention is further illustrated by the following examples which illustrate the invention but are not limited thereto.

[0167] Example 1: Efficacy and safety of ZR2 in elderly patients with newly diagnosed DLBCL

[0168] method

[0169] Study design, ethics, and oversight

[0170] This is a single-arm, open-label, phase 2 study that enrolled patients with DLBCL from August 2020 to August 2021. The study was approved by the Ethics Committee and Institutional Review Board of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, and was conducted in accordance with the ethical principles of the International Conference on Harmonization guidelines on Good Clinical Practice (ICH E6), Chinese regulatory requirements, and the Declaration of Helsinki. The trial was supervised by a steering committee and an independent data monitoring committee. Written informed consent was obtained from all patients before enrollment. This trial has been registered at ClincalTrials.gov (NCT04460248).

[0171] Participants

[0172] According to the World Health Organization classification, patients with previously untreated, histologically confirmed DLBCL in the study met the following criteria: age ≥ 75 years; disagreement with systemic chemotherapy or unsuitability for standard chemotherapy; the longest and shortest dimensions of measurable lesions in one or more sites determined by radiological examination were not less than 1.5 cm; life expectancy > 3 months; written informed consent. Exclusion criteria were as follows: history of systemic or local treatment including chemotherapy within 3 weeks before enrollment; uncontrolled cardiovascular and cerebrovascular diseases, coagulation diseases, connective tissue diseases or serious infectious diseases; neutrophil count < 1.5 × 10 9 / L, platelet count <80×10 9 / L, alanine aminotransferase or aspartate aminotransferase level>2× upper limit of normal (ULN), alkaline phosphatase or bilirubin>1.5×ULN, creatinine>1.5×ULN; unable to comply with the protocol due to mental or other unknown reasons; human immunodeficiency virus (HIV) infection; hepatitis B virus (HBV) DNA positive; other conditions that may affect the study.

[0173] Research Process

[0174] Eligible patients underwent pretreatment clinical examinations, such as complete blood count; serum biochemical tests, including lactate dehydrogenase (LDH), coagulation tests, β-2-microglobulin, C-reactive protein, and N-terminal pro-brain natriuretic peptide; viral diagnosis, including HBV and HIV; urinalysis; routine stool tests; bone marrow biopsy; electrocardiogram; color Doppler echocardiogram; and positron emission tomography (PET-CT). IPI was calculated and treatment response was assessed according to the 2014 Lugano classification.

[0175] Pathological data were reviewed independently by two experienced pathologists (HMY and BSOO). GCB or non-GCB subgroups were determined according to the Hans classification. The cutoff values ​​for BCL-2 and MYC were 50% and 40%, respectively, and the cutoff values ​​for CD10, BCL-6, and MUM-1 were 30%. BCL-2, BCL-6, and MYC rearrangements were detected by fluorescence in situ hybridization in all patients.

[0176] Treatment and dose adjustments

[0177] The dosage and administration regimen of ZR2 includes an induction phase and a maintenance phase. During the induction phase, the dosage of zanubrutinib is 160 mg, orally, twice a day, from cycle 1 to cycle 6, with each 21-day treatment cycle; rituximab 375 mg / m2 was injected intravenously on day 1. 2 , from cycle 1 to cycle 6; oral lenalidomide 25 mg from day 2 to day 11, from cycle 1 to cycle 6. Patients who achieve CR or PR after induction take lenalidomide 25 mg orally every day from day 1 to day 10, with each 21-day treatment cycle for a maximum of 2 years. If there is evidence of local disease, local radiotherapy is performed after cycle 6.

[0178] Patients with grade 3 or 4 non-hematologic AEs, grade 3 or 4 neutropenia with infection or fever, or grade 4 hematologic AEs should discontinue zanubrutinib and lenalidomide until the AE improves to grade 1 or normal levels. If drug-related AEs recur, reduce the dose of zanubrutinib and lenalidomide. For patients with heavy tumor burden (bulky tumors or LDH ≥ 500 U / L), ECOG score ≥ 2, or gastrointestinal lesions, use allopurinol, sodium bicarbonate, and oral prednisone during the first cycle to prevent tumor lysis syndrome or gastrointestinal perforation. Provide comprehensive supportive care such as blood transfusions and blood products, antibiotics, antiemetics, and granulocyte colony-stimulating factor when appropriate.

[0179] end

[0180] The primary endpoint was the CR rate after six cycles or at the end of induction therapy. Secondary endpoints were PFS (measured from diagnosis to the date of documented disease progression or death) and OS (measured from diagnosis to the date of documented death) rates at 2 years. Adverse events were evaluated according to the National Cancer Institute Common Terminology Criteria of Adverse Events (version 4.0). Exploratory endpoints were the analysis of potential prognostic biomarkers.

[0181] result

[0182] Patient characteristics

[0183] From July 8, 2020, to August 9, 2021, a total of 40 patients with newly diagnosed DLBCL were enrolled. 27 (67.5%) patients were male and 13 (32.5%) were female. The mean age of the patients was 78 years (range: 75-91), and 12 (30.0%) patients had an ECOG score ≥ 2. The IPI score of 26 (65.0%) patients was high-intermediate or high risk. 17 (42.5%) patients were identified as germinal center B-cell-like (GCB) subtype; 13 (32.5%) patients were classified as BCL-2 / MYC double-expressing lymphoma (DEL), and 2 (5%) patients had MYC and BCL-6 rearrangements. All participants completed the Comprehensive Geriatric Assessment (CGA) scale. Six (15.0%) patients were classified as fit, sixteen (40.0%) were classified as unfit, and eighteen (45.0%) were classified as frail. Table 1 summarizes all baseline characteristics.

[0184] Table 1 Clinical and pathological characteristics of the patients

[0185]

[0186]

[0187] Unless otherwise stated, values ​​represent n (%) of patients.

[0188] Patient disposition and dose intensity

[0189] Of the 40 patients enrolled, 6 patients discontinued treatment due to stable disease (SD) or progressive disease (PD), and 1 patient withdrew consent for personal reasons after 3 cycles of treatment. After discontinuation and withdrawal of treatment, 33 patients (72.5%) completed six cycles of induction therapy. In addition, 22 of the 33 patients who completed induction therapy (66.6%) completed maintenance therapy with lenalidomide. Eleven patients did not complete all cycles of maintenance therapy, of which 4 patients developed SD or PD after induction therapy, 1 patient died unexpectedly, and 6 patients had disease relapse during maintenance therapy ( Figure 1a The median dose intensity of zanubrutinib, rituximab, and lenalidomide during induction was 95.8% (interquartile range [IQR] = 93.7%-97.7%), 100% (IQR = 100%-100%), and 97.0% (IQR = 95.7%–98.1%), respectively.

[0190] Treatment Response and Survival

[0191] All 40 patients underwent at least one response assessment during the induction phase. The CR rate at the end of induction was 65.0% (n=26, 95% CI:48.3-78.9), and the objective response rate was 75.0% (n=30, 95% CI:58.5-86.8). Five patients (12.5%) experienced SD, and five patients (12.5%) experienced PD. One patient who withdrew from the study early had a PR after three cycles of induction therapy and subsequently received radiotherapy, and the patient was still alive at the last follow-up. The median follow-up time was 34.8 months (range: 6.0-45.3), and the 2-year PFS and OS rates were 67.1% (95% CI:50.1-79.4) and 82.4% (95% CI:66.5-91.2), respectively ( Figure 1b ). None of the factors, including IPI, cell of origin (COO), or DEL, were associated with clinical outcomes (Figure 5).

[0192] Adverse Events

[0193] Table 2 summarizes the hematologic and non-hematologic adverse events (AEs) during induction therapy. No treatment-related mortality was observed. The most common grade 3 and 4 hematologic adverse reactions were neutropenia (n=14; 35.0%). The most common grade 3 and 4 non-hematologic AEs were increased alanine aminotransferase levels (n=5; 12.5%), increased aspartate aminotransferase levels (n=5; 12.5%), and lung infections (n=5; 12.5%). No events of atrial fibrillation were observed.

[0194] Table 2 Adverse events during induction in patients (n=40)

[0195]

[0196] Unless otherwise stated, values ​​represent n (%) of patients.

[0197] discuss

[0198] The trial met the pre-specified endpoints. The trial demonstrated the efficacy and safety of ZR2 in elderly patients with previously untreated DLBCL. The 2-year PFS and OS rates were 67.1% and 82.4%, respectively, compared with 47% and 59%, respectively, in R-miniCHOP. Of the 30 patients who achieved CR or PR during induction, 23 maintained durable remission for more than two years. The incidence of AEs was lower than that of R-miniCHOP, especially in terms of hematological toxicity, and could be controlled with G-CSF prevention and supportive care. For non-hematological toxicity, lung infections were well controlled and atrial fibrillation was not observed. No fatal or unexpected adverse events occurred during treatment. This is a new, effective, and safe chemotherapy-free regimen that can replace traditional DLBCL immunochemotherapy and is simple in clinical practice.

[0199] Example 2 Single Cell Analysis

[0200] DNA sequencing and genotyping

[0201] Formalin-fixed paraffin-embedded (FFPE) tumor samples were collected from 39 patients. Genomic DNA was extracted using the GeneRead DNA FFPE Tissue Kit (Qiagen, Hilden, Germany). A targeted sequencing panel was evaluated using a custom SureSelect library (Agilent Technologies), which included 55 genes that were associated with DLBCL pathogenesis or with significant changes in gene expression shown by RNA sequencing.

[0202] Collection of tumor samples and single-cell RNA sequencing

[0203] Eighteen tumor samples were collected from 17 DLBCL patients recruited for this study for scRNA-seq analysis. Tumor samples were placed in individual containers containing Tissue Storage Solution (Miltenyi Biotec) (on regular ice) and immediately transported to the laboratory. After mechanical dissociation into small pieces, tumor samples were enzymatically dissociated into single cells at 37°C, then filtered through a 40 μm filter and washed once with phosphate-buffered saline (PBS). Single cells were then cryopreserved in liquid nitrogen until the experiment was performed. For scRNA-seq, cells were immediately thawed and washed, and then processed using the Dead Cell Removal Kit (Miltenyi Biotec). Preparation of single-cell suspensions and synthesis of complementary DNA and gene expression libraries were performed according to the manufacturer's instructions. Two samples used the Chromium Single Cell 3'Kit v2 (10× Genome), while 16 samples used the Chromium Single Cell 5'Kit v2 (10× Genome), scBCR-seq, and scTCR-seq. The 3' gene expression library was sequenced on a Novasek 6000 (Illumina), while the 5' gene expression library, enriched T cell library, and B cell library were sequenced using an MGI-2000 sequencer. scRNA-seq FASTQ files were preprocessed using Cell Ranger v6.0.1 (10x Genomics) with default parameters, and detailed bioinformatics analysis is described in the Extended Methods.

[0204] Quality Control, Dimensionality Reduction, and Clustering (Scanpy)

[0205] Before downstream analysis, environmental RNA contamination was removed using Soup X (version 1.6.2). Quality control, dimensionality reduction, and clustering were performed using Scanpy (version 1.8.1) under Python 3.7. For each sample dataset, we filtered the expression matrix according to the following criteria: 1) exclude cells with gene counts less than 200 or in the top 2% of gene counts; 2) exclude cells in the top 2% of UMI counts; 3) exclude cells with mitochondrial content >10%; 4) exclude genes expressed in less than 5 cells. The raw count matrix was normalized by the total counts per cell and log-transformed into a normalized data matrix. The top 2000 variable genes were selected by setting flavor = 'seurat'. Principal component analysis (PCA) was performed on the scaled variable gene matrix, and the first 20 principal components were used for clustering and dimensionality reduction. Cell clusters were visualized using Uniform Manifold Approximation and Projection (UMAP, https: / / doi.org / 10.48550 / arXiv.1802.03426). Cell doublets were estimated based on the expression patterns of typical cell markers. Any clusters enriched for multiple cell type-specific markers were excluded for downstream analysis.

[0206] Cell type identification and annotation

[0207] Cell ID is a multivariate method that extracts gene signatures for each individual cell and uses a hypergeometric test (HGT) for cell identity. Dimensionality reduction of the normalized gene expression matrix was performed by multiple correspondence analysis, projecting both cells and genes into the same low-dimensional space. Then, the gene ranking for each cell was calculated to obtain the most representative gene set. These gene sets were HGTed with reference to the cell types in lymph nodes from the SynEcoSys database, which contains representative gene sets for all cell types in lymph nodes. The identity of each cell was determined as the cell type with the smallest HGT P value. For cluster annotation, the frequency of each cell type in each cluster was calculated, and the one with the highest frequency was selected as the identity of the cluster. The cell type annotation of each cluster was further validated based on the expression of typical markers in the reference database SynEcoSys.

[0208] Phenotyping of major cell types and removal of batch effects

[0209] To obtain high-resolution images of B cells, T and NK cells, myeloid cells, fibroblasts, and endothelial cells, cells from specific cell types were extracted and re-clustered for more detailed analysis, following the same procedure as above, with cluster resolutions set to 0.8, 1.2, 1.2, 0.5, and 1.2, respectively. The first 20 principal components from PCA of T and NK cells, myeloid cells, fibroblasts, and endothelial cells were used by Harmony (version 1.0) to remove batch effects between samples.

[0210] Differentially expressed gene analysis

[0211] To identify differentially expressed genes (DEGs), we used the Seurat FindMarkers function based on the Wilcoxon rank sum test with default parameters and selected genes expressed in more than 10% of cells in both cell comparison groups with an average log(fold change) value greater than 0.25 as DEGs. Adjusted P values ​​were calculated using the Bonferroni correction, and statistical significance was evaluated at a value of 0.05.

[0212] Pathway enrichment analysis

[0213] To investigate the potential functions of specific gene sets among the differentially expressed genes, gene set enrichment analysis was performed using the R package clusterProfiler (version 3.16.1) with reference gene sets from Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Pathways with adjusted P values ​​less than 0.05 were considered significantly enriched. For specific gene set scoring of individual cells, the irGSEA (version 1.1.2) package ( https: / / github.com / chuiqin / irGSEA / The Wilcoxon rank sum test was used to calculate the significance of gene set enrichment differences between groups.

[0214] CNV detection based on scRNA-seq

[0215] CNVs in malignant cells were detected using the InferCNV package. CNVs in malignant cells were assessed using myeloid, T, and NK cells as baseline references. Genes expressed in more than 20 cell types were categorized based on their location on each chromosome. Relative expression values ​​were centered around 1, using 1.5 standard deviations from the residual normalized expression value as lower and upper limits. A sliding window size of 101 genes was used to smooth the relative expression on each chromosome to eliminate the effects of gene-specific expression. Inferred CNVs on each short or long arm or full length of the chromosome were plotted using the R pheatmap package ( https: / / cran.r- project.org / web / packages / pheatmap / index.html) to visualize the heatmap generated.

[0216] VDJ Analysis

[0217] TCR and BCR clonotype assignment was performed using the Cell Ranger (10x Genomics, v6.0.1) vdj pipeline, with GRCh38 as reference. Briefly, TCR diversity metrics containing clonotype frequencies and barcode information were obtained. For TCR analysis, only cells with one productive TCRα chain (TRA) and one productive TCRβ chain (TRB) were retained for further analysis, while for BCR analysis, cells with one productive IGH chain and one productive IGK or IGL chain were retained for further analysis. Unique TRA-TRB pairs or IGH-IGK / IGL pairs were defined as TCR or BCR clonotypes, respectively. Cells with the same clonotype were considered clonal, and the number of cells was used to indicate the degree of clonality of the clonotype. The inverse of the Simpson index was used to quantify clonal diversity.

[0218] Cell-cell interaction analysis

[0219] Cell-cell interactions between cell types were predicted based on known ligand-receptor pairs by Cellphone DB (version 2.1.0). The number of permutations to calculate the null distribution of mean ligand-receptor pair expression in random cell identities was set to 1000. Thresholds for individual ligand or receptor expression were determined by cutoffs based on the mean log gene expression distribution of all genes in each cell type. Predicted interacting pairs with P values ​​< 0.05 and mean log expression > 0.1 were considered significant and visualized by heatmaps and dot plots from CellphoneDB.

[0220] Pseudo-time trajectory analysis

[0221] Monocle2 (version 2.10.0) was used to construct trajectory analysis of naive T cells and CD8+ T cells. To construct trajectory branches, Seurat (version 3.1.2) FindVairableFeatures was used to select the top 1000 highly variable genes, and dimensionality reduction was performed using DDRTree. Trajectories were visualized using the plot_cell_trajectory function in Monocle2.

[0222] Immunohistochemistry

[0223] Immunohistochemistry of patient tumor tissues was performed on 5-μm paraffin sections using antibodies against HLA-I molecules (abcam, ab70328, 0.2 μg / mL) and HLA-II molecules (abcam, ab55152, 1:50).

[0224] DC differentiation and cell treatment

[0225] CD14+ monocytes were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors using a CD14 positive selection kit (Miltenyi) according to the manufacturer's instructions. For immature monocyte-derived DC (moDC) differentiation studies, CD14+ cells (2×10 5 / mL) were suspended in cell culture medium (10% heat-activated FBS + 1% Pen / Strep + 1‰ 2-mercaptoethanol + RPMI-1640) supplemented with recombinant human GM-CSF (50ng / mL) and IL-4 (25ng / mL), and the culture medium was replenished with cytokines and drugs every 3 days. On the 6th day, the cells were collected and characterized by flow cytometry for DC maturation-related surface markers using Cytek Aurora, and the cells were analyzed using Analyze the data.

[0226] RNA sequencing analysis

[0227] We collected RNA sequencing data from 346 patients from previous studies, of whom 328 received RCHOP-based therapy and 18 received iR2 therapy. In addition, RNA sequencing was newly performed on available frozen tumor samples from 18 patients from this study. RNA was extracted by Trizol and RNeasy Mini Kit (Qiagen) and purified by RiboZeror RNA Removal Kits (Illumina). The concentration and integrity of RNA were assessed on NanoDrop and Agilent 2100 Bioanalyzer, respectively. RNA libraries were generated using TruSeq RNA Library Preparation Kit (Illumina) according to the manufacturer's instructions. The concentration and quality of RNA libraries were assessed by Qubit and BioAnalyzer 2100 systems. Paired-end sequencing was performed on an Illumina HiSeq sequencer according to the protocol provided by Illumina. Read pairs were aligned to Refseq hg19 by Burrows-Wheeler Aligner version 0.7.13-r1126. Transcript count table files were generated by HTSeq. Potential false positive results were excluded by visual inspection. Batch effects were eliminated by using the R package “sva” via R 4.0.3. Specific gene set scoring was performed by “ssgsea” using the R package GSVA (version 1.38.2).

[0228] Sample size and statistical analysis

[0229] This study used Simon's two-stage design to evaluate the CR rate of the ZR2 regimen, with a one-sided alpha of 0.05 and a power of 0.95. The null hypothesis of the CR rate is <30%. This refers to a previous study that reported that the iR2 regimen resulted in a CR rate of approximately 30% in patients with relapsed or refractory DLBCL (Goy, A., et al. Ibrutinib plus lenalidomide and rituximab has promising activity in relapsed / refractory non-germinal center B-cell-like DLBCL. Blood 134, 1024-1036 (2019)). Based on the R-miniCHOP data of patients with DLBCL who were >80 years old and had just been treated, we conservatively assumed that the CR rate of ZR2 in elderly DLBCL patients was 60%. According to these calculations, a total of 34 patients were required. In Phase 1, 13 patients were recruited and then subjected to a futility analysis. If 4 or fewer patients achieve CR, the trial must be stopped early. The remaining patients were included in phase 2 because they did not meet the futility criteria. If 14 or fewer CRs were observed, the null hypothesis was rejected. Considering a dropout rate of 20%, 40 patients were required. The sample size was calculated using PASS software (version 11.0.10, NCSS, Kaysville, UT).

[0230] When the data followed a normal distribution and equal variance, the means of continuous variables between the two groups were compared using a two-tailed unpaired t-test. When the conditions were not met, a nonparametric test (Mann-Whitney U test) was used. Fisher's exact test was used for non-ordered categorical variables. Survival estimates were calculated using the Kaplan-Meier method, and survival curves were compared using the log-rank test. Univariate Cox proportional hazards models were used to estimate hazard ratios and associated 95% confidence intervals to assess the effects on PFS and OS. A bilateral P value of <0.05 was considered significant. All analyses were performed using SPSS statistical software, version 25.0 (SPSS, Chicago, IL, USA).

[0231] Single-cell RNA sequencing (scRNA-seq) analysis of tumor samples before treatment and at progression

[0232] Pre-treatment tumor biopsies from 17 patients and paired samples from one patient at the time of disease progression were collected. These samples were processed for scBRCR-seq along with 16 paired samples using single-cell BCR sequencing (scBCR-seq) and single-cell T-cell receptor (TCR) sequencing (scTCR-seq). Figure 2a ; Figure 6a ). According to the first-line clinical efficacy, the 17 pre-treatment samples were divided into two groups: responders (n = 12) and non-responders (n = 5). After quality control, a total of 124,866 high-quality cells were obtained for subsequent analysis, with an average of 1006 genes and 6503 UMIs per cell ( Figure 2b -2c). We evaluated and performed unsupervised cluster analysis, which revealed seven major cell compartments based on typical cell type markers, including B cells (non-malignant and malignant B cells), plasma cells, T cells, natural killer (NK) cells, myeloid cells, fibroblasts, and endothelial cells ( Figure 6d -6e). These cell types were shared between samples, with no significant differences in the proportions of immune or stromal cells in pre-treatment samples from responders and non-responders ( Figure 6f ).

[0233] Tumor human leukocyte antigen (HLA) expression as a reactive biomarker

[0234] Extraction of B cells for further subclustering analysis showed that non-malignant B cells from different tumor samples clustered together into commonly shared clusters, while malignant B cells tended to cluster individually into tumor-specific clusters or tumor-shared clusters ( Figure 2b -2d; Figure 7a -7b). In addition, InferCNV analysis (a method for inferring tumor copy number variation) confirmed the identification of malignant B cells through interpatient heterogeneity, as malignant B cells have more copy number variation (CNV) than T cells or myeloid cells ( Figure 2e -2f). CNV scores were significantly higher in malignant B cells in non-responders compared with responders ( Figure 7c ).

[0235] To explore adverse features associated with ZR2 efficacy, we performed GSEA analysis and direct comparison of DEGs in malignant B cells from non-responders and responders. Immunostimulatory pathways (i.e., antigen presentation, cell adhesion, and interferon-γ signaling) were significantly enriched in responders and were associated with a favorable response to ZR2 ( Figure 2g We observed increased expression of HLA-I molecules (HLA-A, HLA-B, HLA-C, HLA-E, and HLA-F) and HLA-II molecules (HLA-DQB1, HLA-DRB1, HLA-DQA1, HLA-DRA, HLA-DQA1, and CD74) ( Figure 2h ; Figure 7d ). Immunohistochemistry results showed that the expression of HLA-I and HLA-II in responders was significantly higher than that in non-responders ( Figure 2i ).

[0236] cDC1 as a microenvironmental biomarker

[0237] The changes in the tumor microenvironment between responders and non-responders were also analyzed to investigate the mechanism of ZR2 resistance. The proportion of stromal cells, including fibroblasts and endothelial cells, was low, and there was no significant difference in specific cell types (Figure 8). Myeloid cells showed significant heterogeneity. After batch correction and unsupervised clustering, a total of 7 myeloid cell subclusters were identified, including monocytes, two macrophage subclusters (macrophage c1, macrophage c2), monocytes, DC subclusters (conventional DC 1 [cDC1], conventional DC 2 [cDC2], mature DC and plasmacytoid DC [pDC]) ( Figure 3a -3b).

[0238] The percentage of cDC1 in responders was significantly increased (P = 0.048, Figure 3c ; Figure 9a -9b), higher expression of antigen presenting cell (APC) gene signature, and lower expression of M2-like and angiogenic gene signatures, indicating enhanced anti-tumor ability ( Figure 3d ). DEG analysis showed that HLA molecules and their transcriptional regulators of HLA-I and HLA-II genes (NLRC5 and CIITA, respectively) were significantly increased in responders. At the same time, upregulation of DC maturation markers IDO1 and ITGB7, DC development and maintenance-related factor FLT3, DC activation marker CD83, and key factor of antigen cross-presentation MPEG1 were also found in responders, indicating a higher antigen cross-presentation ability ( Figure 3e ; Fig.9c Similarly, pathways including antigen presentation, endocytosis, tumor necrosis factor-α (TNF-α) signaling via NF-κB, and response to IFNγ were significantly enriched in cDC1s of responders ( Figure 3f ).

[0239] Using bulk RNA-seq data, a higher cDC1 signature was also observed in responders treated with ZR2 (n=18) and was associated with improved PFS ( Figure 3g -3h), but not with the efficacy of RCHOP-based therapy (n=328) ( Figure 9d -9e). We detected markers involved in DC maturation and activation, such as HLA-I, HLA-DR, CD80, CD40, and CD83, by flow cytometry and found that zanubrutinib and lenalidomide enhanced the expression of these markers in DCs derived from monocytes of healthy donors ( Figure 3i ; Figure 9f ).

[0240] Activated CD8+ T cells and clonal TCR repertoires correlate with favorable responses after strong antigen presentation

[0241] After batch correction and unsupervised clustering, a total of 20 infiltrating T and NK cell clusters were identified across all samples ( Figure 4a , Fig.10a These T and NK cells were then classified into naive T cells (naive T_IL7R, naive T_FOS, and naive T_FOSB), CD4+ T follicular helper (Tfh) cells (CD4 Tfh_TCF7 and CD4 Tfh_CD200), CD4+ regulatory T cells (CD4 Treg_FOXP3), CD8+ effector T cells (CD8 Teff_GZMK, CD8 Teff_GZMM, CD8 Teff_IFNG, CD8 Teff_MT1F, CD8 Teff_NKG7, CD8 Teff_MCM5, and CD8 Teff_MKI67), CD8+ exhausted T cells (CD8Tex_CCL4L2, CD8 Tex_LAG3, and CD8 Tex_XCL1), GDT cells (GDT_TRDV2), NKT cells (NKT_TYROBP), and NK cells (NK_CD56 and NK_CD16). Among these cell types, CD8+ effector T cells were the major component of infiltrating T cells and NK cells, followed by CD4+ T cells and NK cells.

[0242] Compared with non-responders, the percentage of CD8 Teff_MCM5 cells in responders was significantly increased (P = 0.048), and CD8 Teff_MKI67 cells showed the same trend (P = 0.064, Figure 4b Higher percentages of S and G2M phases of the cell cycle were observed in CD8 Teff_MCM5 and CD8 Teff_MKI67 cells, indicating proliferation characteristics ( Fig.10d ). Similarly, they also highly expressed genes related to the cell cycle ( Fig.10e Clonotype size (expressed as the number of cells expressing the same TCR sequence) was higher in responders, but diversity was lower, indicating that responders had higher T cell expansion than nonresponders ( Figure 4c -4d; Fig.10f -10g).

[0243] Cytotoxicity and exhaustion of CD8+ T cells predict favorable responses to ZR2

[0244] We observed a positive correlation between cytotoxicity and exhaustion signatures in CD8+ T cells at both single-cell and bulk RNA-seq levels ( Fig.11a-11d). Intermediate levels of cytotoxicity and exhaustion signatures were observed in CD8 Teff_MCM5 and CD8 Teff_MKI67 cells. By pseudo-time analysis, CD8 Teff_MCM5 and CD8 Teff_MKI67 cells can be generated from CD8+ exhausted T cells and the terminal branches of the trajectory, which together indicate that they are well transitioned to the CD8+ exhausted state. Consistently, both branches had high cytotoxicity and exhaustion scores ( Figure 4e ). In addition, lineage transitions were investigated by the fraction of clonotypes shared between T cell lineages and other T cell lineages. Overlap was mainly observed between CD8+ subtypes, which was increased in responders compared to non-responders, indicating a rich transition of CD8+ cells between different activation and exhaustion states in responders. Specifically, there was an increased overlap between CD8 Teff_MCM5 and CD8 Teff_MKI67 cells and other CD8+ subtypes, especially with CD8+ exhausted T cells (CD8 Tex_CCL4L2, CD8 Tex_LAG3, and CD8Tex_XCL1), which are well-established transitioners to the CD8+ exhausted state ( Figure 4f Notably, CD8+ T cells from responders had higher cytotoxicity and exhaustion scores, suggesting stronger antitumor capacity through efficient antigen presentation and chronic TCR stimulation ( Figure 4g ; Fig.11e ). In addition, using bulk RNA-seq data, higher cytotoxicity and exhaustion scores and better prognosis were observed in the chemotherapy-free cohort, which is consistent with the results of scRNA-seq data but not in the RCHOP-based cohort ( Figure 4h -4i; Fig.11f -11g).

[0245] We performed cell-cell interaction analysis using CellPhoneDB to better understand the activation and exhaustion of CD8+ T cells regulated by APCs in DLBCL, which is mainly composed of malignant B cells and myeloid cells. Stronger interactions of TNF superfamily members and TNF-TNFRSF1B receptors were stronger in DLBCL than in non-responders ( Fig.11h ).

[0246] Effects of tumor microenvironment on the migration and development of cDC1

[0247] The abundance of cDC1 in tumors involves chemokine-mediated recruitment and retention, as well as the local availability of DC growth factors in the tumor microenvironment. Cell-cell interaction analysis analyzed potential ligand-receptor interactions involved in cDC1 recruitment and maintenance. Interaction pairs including CXCL19-CXCR3, CXCL10-CXCR3, and CXCL11-CXCR3 were observed in responders for cDC1 recruitment compared to non-responders. In addition, FLT3LG-FLT3 and CD40LG-LC40 were increased in the tumor microenvironment of responders, which are critical for maintaining cDC1 viability and participating in cDC1 activation, respectively ( Fig.12a To validate the interaction between cDC1 and the tumor microenvironment in DLBCL, we analyzed the correlation between the cDC1 signature and the expression of the above-mentioned chemokines and regulatory factors in two bulk RNA-seq data cohorts. The cDC1 signature was positively correlated with the expression of other genes except CXCL14 ( Figure 12b -12c).

[0248] Longitudinal single-cell clonal dynamics associated with treatment response

[0249] To elucidate the characteristics of resistant and progressive clones, we performed longitudinal single-cell analysis of malignant cells before treatment and at progression, combined with single-cell BCR analysis. The data showed that each transcriptional clone had a different BCR clonotype, indicating that different malignant cell sources have similar transcriptional features ( Fig.13a ). The transcriptional signature correlated with ZR2 efficacy, as distinct clonal dynamics were observed between samples before treatment and at progression ( Fig.13b -13c). The percentage of sensitive clones decreased, while the percentage of resistant clones remained unchanged, and the percentage of selective clones increased significantly as the dominant clones ( Fig.13d -13e). By GSEA analysis, BCR signaling pathway, MAPK signaling pathway, apoptosis, and immune stimulation pathways (such as antigen presentation, cell adhesion, and interferon γ signaling) were significantly enriched in sensitive clones, while tumor progression-related pathways (including cell cycle, MYC target, glycolysis, and oxidative phosphorylation pathways) were upregulated in resistant and selected clones ( Fig.13f Similar to the enriched genes in malignant cells of responders, antigen-presenting molecules were overexpressed in sensitive clones; however, cell cycle-related genes (e.g., MKI67, PCNV, CDK4, MCM7, HMGA1, MYC) and metabolic genes involved in glucose metabolism (e.g., LDHA, LDHB) were upregulated in resistant or selected clones ( Figure 13g Consistently, the percentages of S and G2M phases were increased in drug-resistant or selected clones compared with sensitive clones ( Fig.13h-13i). By cell-cell interaction analysis, we found that the selected clones rarely interacted with T and NK cells and had no co-stimulatory or co-inhibitory interactions with T and NK cells compared with the selected clones and the resistant clones ( Fig.13j Sensitive clones had stronger co-inhibitory interactions with T and NK cells, including CD86-CTLA4, AD5RE5, and LGALS9-HAVCR2, whereas resistant clones interacted with ADORA2A-ENTPD1.

[0250] discuss

[0251] The efficacy of ZR2 is largely dependent on the tumor microenvironment. Antigen presentation by tumor cells is key to T cell activation, recognition, and killing of tumor cells by cytotoxic T cells. Previous studies have shown that tumor cells can evade immune recognition through a variety of escape mechanisms, including modulation of antigen presentation, impaired expression of HLA molecules, and altered antigen processing and presentation mechanisms. HLA deficiency has been associated with poor outcomes in immunotherapy for recurrent solid tumors as PD-1 inhibitors. Here, we found that lymphoma cells in responders presented features of antigenic tumors with high expression of HLA-I and HLA-II, suggesting a novel microenvironment-dependent mechanism of sensitivity to immunotherapy as ZR2, which was not observed in RCHOP-based immunochemotherapy.

[0252] cDC1 is a key APC for activating antitumor immunity due to its ability to process and prime CD4+ and CD8+ T cells for antigens. In our cohort, a higher proportion of cDC1 and its upregulated antitumor function were associated with a better response to ZR2. Although cDC1 is ‘rare’ in the immune population, it is considered an important factor in predicting tumor sensitivity to immune checkpoint blockade. Similarly, unlike RCHOP-based immunotherapy, the abundance of cDC1 has prognostic value for immunotherapy with ZR2. Zanubrutinib and lenalidomide increase DC cells and their maturation. In vitro, we confirmed that the combination of zanubrutinib and lenalidomide further enhanced DC maturation in healthy donors compared with single-agent therapy alone. These results again explain why changes in the tumor microenvironment are attributed to the response to ZR2. Therefore, cDC1 may be a potential biomarker for good response to immunotherapy, associated with increased antigen presentation and T cell activation in DLBCL.

[0253] The pre-existing expansion of tumor-specific T cell clones and their high proliferative potential in responders are consistent with upregulation of antigen presentation caused by increased HLA-I and HLA-II molecules on tumors and increased number and function of cDC1. By trajectory analysis and TCR-seq analysis, we found that proliferating CD8+ T cells had the same TCR clonotypes and similar exhaustion characteristics as exhausted T cells, indicating that there is a simultaneous proliferative potential in exhausted T cells to generate progeny with exhaustion characteristics. In fact, the exhausted T cell characteristics are accompanied by cytotoxic characteristics, which is consistent with previous studies that exhausted T cells still retain cytotoxicity. Therefore, the increase in T cell cytotoxicity and tumor-reactive exhaustion is closely related to the good response to ZR2. Considering the higher cytotoxicity observed in ZR2 responders but not in RCHOP-based regimens, the efficacy of ZR2 depends largely on the activated immune response.

[0254] Finally, longitudinal analysis of tumor samples reflected dynamic changes before treatment and at progression. Selective and resistant clones became dominant clones at progression, with characteristics that were masked by sensitive clones before treatment. Sensitive clones had similar biological characteristics to responder samples, such as high expression of HLA molecules and abundant cell-cell interactions to promote T cell toxicity. Selective and resistant clones showed proliferation characteristics of non-responders and exhibited inhibitory cell-cell interactions with T cells. Together, these findings suggest that drugs that inhibit the cell cycle (such as cyclin-dependent kinase inhibitors), drugs that block the MAX-MYC interaction, and those that target metabolic activity as inhibitors of oxidative phosphorylation or glycolysis may be potential ways to restore DLBCL immunogenicity and make DLBCL more sensitive to immunotherapy.

[0255] Single-cell analysis confirmed the underlying mechanism of ZR2. ZR2 showed good clinical efficacy and safety in elderly patients with newly treated DLBCL by targeting microenvironmental changes. This strategy of reprogramming the tumor microenvironment as a first-line treatment for DLBCL provides a paradigm shift for this patient category in the era of mechanism-based targeted therapy.

[0256] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A composition of zanubrutinib or a pharmaceutically acceptable salt thereof, an anti-CD20 antibody and lenalidomide.

2. The composition according to claim 1, characterized in that The dosage relationship of zanubrutinib or a pharmaceutically acceptable salt thereof, anti-CD20 antibody and lenalidomide is: 320 mg: 375 mg / m 2 BSA: 20-25mg.

3. The composition according to claim 1, characterized in that The anti-CD20 antibody is selected from one of rituximab, ibritumomab tiuxetan, tositumomab, ofatumumab or obinutuzumab.

4. Use of a combination of zanubrutinib or a pharmaceutically acceptable salt thereof, an anti-CD20 antibody and lenalidomide in the preparation of a combined therapeutic drug for diffuse large B-cell lymphoma, characterized in that: In the combined therapeutic drug for diffuse large B-cell lymphoma, the zanubrutinib or a pharmaceutically acceptable salt thereof, the anti-CD20 antibody and lenalidomide are stored separately.

5. The use according to claim 4, characterized in that: Select one or more of the following conditions: The diffuse large B-cell lymphoma is diffuse large B-cell lymphoma in patients aged 75 years or above; The diffuse large B-cell lymphoma is newly diagnosed diffuse large B-cell lymphoma; The newly diagnosed diffuse large B-cell lymphoma is activated B-cell diffuse large B-cell lymphoma, germinal center B-cell diffuse large B-cell lymphoma, non-germinal center B-cell diffuse large B-cell lymphoma or DLBCL of uncertain subtype.

6. A combined treatment kit for diffuse large B-cell lymphoma, characterized in that: The invention comprises zanubrutinib or a pharmaceutically acceptable salt thereof, an anti-CD20 antibody and lenalidomide.

7. The combined therapy kit according to claim 6, characterized in that: In the method for using the kit: a) Subjects were given 160 mg of zanubrutinib twice daily or 320 mg of zanubrutinib once daily; on the first day of each treatment cycle, subjects were given rituximab 375 mg / m2 intravenously or subcutaneously. 2 BSA; and administering lenalidomide 20-25 mg orally daily to the subject from the first day or second day of each treatment cycle to approximately the first 1 / 2 to 3 / 4 of each cycle; or, b) In each treatment cycle, subjects were given 160 mg of zanubrutinib twice a day or 320 mg of zanubrutinib once a day; subjects were given rituximab 375 mg / m2 BSA intravenously or subcutaneously on the first day of the first treatment cycle, and 500 mg / m2 BSA intravenously or subcutaneously on the first day of each subsequent treatment cycle. 2 BSA; and administering lenalidomide 20-25 mg orally daily to subjects from the first or second day of each treatment cycle to approximately the first 1 / 2 to 3 / 4 of each cycle.

8. The combined therapy kit according to claim 6, characterized in that: In the method of using the kit: the treatment of diffuse large B-cell lymphoma is the first-line treatment, or, In the method of using the kit: zanubrutinib or a pharmaceutically acceptable salt thereof is administered once a day in an amount of 320 mg zanubrutinib or twice a day in an amount of 160 mg zanubrutinib.

9. The combined therapy kit according to claim 6, characterized in that: In the method of using the kit: the treatment includes an induction period, wherein the induction period includes one or more treatment cycles; The induction phase includes 1 to 6 treatment cycles, and each treatment cycle is 21 days or 28 days.

10. The combined therapy kit according to claim 6, characterized in that: In the method for using the kit: the treatment also includes a maintenance period after the induction period, and the maintenance period begins when the subject achieves complete remission or partial remission, The maintenance period includes one or more cycles, wherein the subject is administered 20-25 mg of lenalidomide per day during the first 1 / 2 to 3 / 4 of each cycle; In the method for using the kit: the maintenance period is 21 days or 28 days, and the maintenance period lasts for up to 2 years.

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