Diagnostic and therapeutic methods for lymphoma

Abstract

To provide the use of an anti-CD20 antibody in the manufacture of a medicament for treating lymphoma, and a medicament comprising the anti-CD20 antibody.SOLUTION: The present invention provides the use of an anti-CD20 antibody for treating a patient with an amount or a level of a macrophage biomarker exceeding an amount or a level of a reference macrophage biomarker in a sample from the patient, in the manufacture of a medicament for treating lymphoma. The reference macrophage biomarker amount or level may be a pre-assigned macrophage biomarker amount or level.SELECTED DRAWING: Figure 9

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. This ASCII copy, created on August 3, 2021, is named 50474_212WO2_Sequence_Listing_08_03_21_ST25 and is 15,672 bytes in size.

[0002] FIELD OF THE INVENTION The present invention relates to diagnostic and therapeutic methods for treating lymphoma using anti-CD20 antibodies, and related assays and kits are also provided. [Background technology]

[0003] Background of the Invention In the United States, lymphoma causes approximately 20,000 deaths and is predicted to account for approximately 85,000 new cancer cases in 2020. Although significant advances have been made in the treatment of lymphoma, the 5-year survival rate is only approximately 73%.

[0004] Diffuse large B-cell lymphoma (DLBCL) is the most common type of aggressive non-Hodgkin's lymphoma (NHL). Immunochemotherapy using the type I anti-CD20 monoclonal antibody, rituximab, in combination with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) is the standard of care treatment for previously untreated patients presenting with advanced-stage disease. While first-line treatment of DLBCL is potentially curative, many patients do not respond or ultimately relapse.

[0005] Thus, there is an unmet need for improved diagnostic and therapeutic methods for the treatment of DLBCL patients. Summary of the Invention

[0006] Summary of the Invention The present invention relates to the use of macrophage biomarkers or Th2 biomarkers in methods of identifying, diagnosing, or predicting the therapeutic efficacy of treating a lymphoma (e.g., a B cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B cell lymphoma (e.g., germinal center B cell-like or activated B cell-like diffuse large B cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)) with an anti-CD20 antibody (e.g., a Type II (e.g., obinutuzumab) or Type I (e.g., rituximab) anti-CD20 antibody).

[0007] In a first aspect, the invention features a method for identifying, diagnosing, and / or predicting whether a patient having lymphoma could benefit from a treatment comprising an anti-CD20 antibody, the method comprising measuring a macrophage biomarker in a sample from the patient, wherein an amount or level of the macrophage biomarker that exceeds an amount or level of a reference macrophage biomarker in the sample identifies, diagnoses, and / or predicts the patient as one who could benefit from a treatment comprising an anti-CD20 antibody.

[0008] In a second aspect, the invention features a method of selecting a therapy for a patient having lymphoma, the method including measuring a macrophage biomarker in a sample from the patient, wherein an amount or level of the macrophage biomarker that exceeds an amount or level of a reference macrophage biomarker in the sample identifies the patient as one who may benefit from a therapy including an anti-CD20 antibody.

[0009] In some embodiments of the first or second aspect, the patient has an amount or level of a macrophage biomarker in the sample that exceeds the amount or level of a reference macrophage biomarker, and the method further comprises administering to the patient an effective amount of an anti-CD20 antibody.

[0010] In a third aspect, the invention provides a method of treating a patient having lymphoma, the method comprising: (a) measuring a macrophage biomarker in a sample from the patient, wherein the amount or level of the macrophage biomarker in the sample exceeds the amount or level of a reference macrophage biomarker; and (b) administering to the patient an effective amount of an anti-CD20 antibody based on the macrophage biomarker measured in step (a).

[0011] In a fourth aspect, the invention features a method of treating a patient having lymphoma, the method including administering to the patient an effective amount of an anti-CD20 antibody, wherein prior to treatment, the amount or level of a macrophage biomarker in a sample from the patient has been determined to exceed the amount or level of a reference macrophage biomarker.

[0012] In a fifth aspect, the invention features a method of treating a patient having lymphoma and having an amount or level of a macrophage biomarker in a sample from the patient that exceeds the amount or level of a reference macrophage biomarker, the method comprising administering to the patient an effective amount of an anti-CD20 antibody.

[0013] In some embodiments of any of the first, second, third, fourth and fifth aspects, the reference macrophage biomarker amount or level is a pre-assigned macrophage biomarker amount or level.

[0014] In some embodiments of any of the first, second, third, fourth, and fifth aspects, the reference macrophage biomarker amount or level is the amount or level of the macrophage biomarker in a reference population. In some embodiments, the amount or level of the macrophage biomarker in the reference population is the median amount or level of the macrophage biomarker in the reference population. In some embodiments, the reference macrophage biomarker amount or level is the amount or level of the macrophage biomarker at the 25th percentile of the reference population. In some embodiments, the reference macrophage biomarker amount or level is the amount or level of the macrophage biomarker at the 50th percentile of the reference population. In some embodiments, the reference macrophage biomarker amount or level is the amount or level of the macrophage biomarker at the 75th percentile of the reference population. In some embodiments, the reference population is a population of patients with lymphoma. In some embodiments, the population of patients with lymphoma has previously been treated with an anti-CD20 antibody. In some embodiments, the reference macrophage biomarker amount or level is the amount or level of the macrophage biomarker in the reference population before initiation of treatment with an anti-CD20 antibody. In some embodiments, the amount or level of the reference macrophage biomarker significantly separates the reference population into a first set of patients who have benefited from treatment with an anti-CD20 antibody and a second set of patients who have not benefited from treatment with an anti-CD20 antibody.

[0015] In some embodiments of any of the first, second, third, fourth, and fifth aspects, the amount or level of the reference macrophage biomarker is the amount of macrophages as measured by gene expression. In some embodiments, the amount of macrophages is between about 0% and about 30.7%.

[0016] In some embodiments of the first or second aspect, the benefit is an increase in progression-free survival (PFS).

[0017] In some embodiments of the first or second aspect, the benefit is increased overall survival (OS).

[0018] In some embodiments of any of the third, fourth, and fifth aspects, the method further comprises achieving an improvement in PFS or OS.

[0019] In some embodiments of any of the first, second, third, fourth, and fifth aspects, the macrophage biomarker is an average M1 macrophage gene signature set score of one or more M1 macrophage gene signature sets. In some embodiments, each M1 macrophage gene signature set score is an average of the expression levels of one or more genes in the M1 macrophage gene signature set. In some embodiments, each M1 macrophage gene signature set score is an average of the normalized expression levels of one or more genes in the M1 macrophage gene signature set. In some embodiments, the one or more M1 macrophage gene signature sets are: (a) ACP2, ABCD1, C1QA, FDX1, CCL22, CD163, SCAMP2, ADAMDECl, ARL8B, and HAMP; (b) ACP2, ABCD1, FDX1, CCL8, CCL22, CD163, ADAMDECl, TREM2, and HAMP;(c) ACP2, ADRA2B, ALCAM, ABCD1, ATOX1, ATP6V0C, ATP6V1E1, BLVRA, C1QA, CD48, CD63, CLCN7, TPP1, CLTC, CCR1, CMKLR1, SLC31A1, COX5B, FCER1G, FDX1, FOLR2, FPR3, FTL, HEXB, HK3, IL10, IL12B, ITGAE, LAIR1, CXCL9, MMP19, NARS, NDUFS2, P2RX7, PDCL, MAPK13, PTGIR, PTPRA, RELA, CCL7, CCL8, CCL19, CCL22, SRC, STX4, TCEB1, TFRC, AGPS, MARCO, SNX3, CD84, USP14, ITGB1BP1, ATP6V1F, TRIP4, CD163, CIAO1, WTAP, ARHGEF11, ABI1, SCAMP2, ACTR2, BCAP31, ZMPSTE24, BCKDK, EXOC5, STIP1, UQCR11, SDS, LILRB4, OGFR, TFEC, FKBP15, DNAJC13, TDRD7, STX12, IL17RA, ABTB2, FAM32A, SIGLEC7, SIGLEC9, ADAMDEC1, CECR5, SLC25A24, NRBP1, MS4A4A, TREM2, OTUD4, PQLC2, HAUS2, ARL8B, NECAP2, WDR11, ZC3H15, CCDC47, UTP3, MRS2, HAMP, MRPL40, VPS33A, CORO7, LIMD2, TMX1, DOT1L, ADO, and ADCK2; (d) ACP2, ADRA2B, ALCAM, TSPO, C3AR1, DAGLA, CALR, CHIT1, CYBB, CYC1, CYP19A1, DLAT, FCER1G, GP1BA, GPD1, IFNAR1, IL10, KCNJ5, KIFC3, MT2A, MYBPH, MYH11, MYO7A, P2RX7, PRDX1, RAB3IL1, RNH1, MRPL12, CCL1, CCL7, CCL8, CCL24, SRC, VIM, RRP1, MARCO, S1PR2, AP1M2, ACTR3, LILRB1, AFG3L2, SDS, LILRB4, EMILIN1, VSIG4, HSPB7, COQ2, ADAMDEC1, CECR5, WSB2, SLAMF8, DNASE2B, CLPB, MFSD7, and ADCK2;(e) ACP2, ADCY3, ADRA2B, ALCAM, TSPO, C1QA, C1QB, C3AR1, DAGLA, CD63, CHIT1, CMKLR1, SLC31A1, CSF1, CSF1R, CYBB, CYC1, CYP19A1, FANCE, FCER1G, FDX1, FPR3, FTL, GP1BA, GPD1, HEXB, IL10, KCNJ1, KCNJ5, KIFC3, LAMP1, MMP19, MSR1, MT2A, MYBPH, MYO7A, P2RX7, PRDX1, RAB3IL1, MRPL12, CCL1, CCL7, CCL8, CCL18, CCL19, CCL24, SLC6A12, SPR, SRC, RRP1, MARCO, PKD2L1, S1PR2, CD163, LONP1, AP1M2, IGSF6, LILRB1, SDS, LILRB4, EMILIN1, VSIG4, TFEC, PHLDB1, CYFIP1, FKBP15, NCAPH, MYOF, HSPB7, ADAMDEC1, GLRX2, NDUFAF1, SPG21, MS4A4A, ATP6V1D, ATP6V1H, TREM2, PQLC2, TMEM70, PLEKHB2, TMEM33, SLAMF8, HAMP, DNASE2B, MYOZ1, LONRF3, CLPB, MFSD7, and ADCK2;and / or (f) ACP2, ADCY3, ADRA2B, ALCAM, ABCD1, ANXA2, ATP6V1A, C1QA, C1QB, C3AR1, DAGLA, CD80, CD63, CHIT1, CMKLR1, SLC31A1, CSF1, CSF1R, CYBB, CYC1, CYP19A1, FANCE, FDX1, FPR2, FPR3, GPD1, HEXB, KCNJ1, KCNJ5, KIFC3, MMP19, MSR1, MT2A, MYBPH, P2RX7, MAPK13, S100A11, CCL1, CCL7, CCL8, CCL18, CCL19, CCL22 , CCL24, SLC1A2, SLC6A12, SLC11A1, SIGLEC1, SRC, TIE1, MARCO, HYAL2, CD163, LONP1, IGSF6, LILRB1, CD300C, SDS, LILRB4, EMILIN1, VSIG4, PHLDB1, NCAPH, CLEC4E, MYOF, HSPB7, ADAMDEC1, GLRX2, MS4A4A, ATP6V1H, TREM2, TMEM70, TMEM33, KCNK13, SLAMF8, HAMP, DNASE2B, MYOZ1, MFSD7, ADO, ADCK2, and TBC1D16.

[0020] In some embodiments of any of the first, second, third, fourth, and fifth aspects, the macrophage biomarker is an average macrophage gene signature set score of one or more macrophage gene signature sets. In some embodiments, each macrophage gene signature set score is an average of the expression levels of one or more genes in the macrophage gene signature set. In some embodiments, each macrophage gene signature set score is an average of the normalized expression levels of one or more genes in the macrophage gene signature set. In some embodiments, the one or more macrophage gene signature sets are any of the macrophage gene signature sets in Table 2.

[0021] In some embodiments of any of the first, second, third, fourth, and fifth aspects, the macrophage biomarker is a gene expression value. In some embodiments, the gene expression value is a median gene expression value. In some embodiments, the gene expression value is measured using a gene signature matrix. In some embodiments, the gene signature matrix includes the following genes: CD200, KLHL14, TCL1A, NRG1, EOMES, PPP2R2B, RNF165, WNT7A, CCR4, PDGFD, EBF1, FCGBP, PCDH9, MLC1, TSHZ2, S1PR5, NCALD, LAYN, GCNT4, FASLG, TRAT1, ADAM6, GUCY1A3, LRRC4, TSPAN18, SBK1, ICOS, BTNL8, WNT5B, AUTS 2, SH2D2A, ADGRG3, PNOC, SPIB, VPREB3, DPEP3, MME, ZBTB16, FOXP3, SEMA3G, CD8A, TOGARAM2, COLGALT2, ABCB1, STAP1, SAMD3, FAM 46C, BLK, CTLA4, CD19, REPS2, RTKN2, POU2AF1, DAPK2, PYHIN1, NLRC3, GATM, KLRD1, AFF3, FCRLA, AATBC, REM2, YPEL1, TXK, CD8B, P2 RX5, CEACAM1, BCL11A, ABCB4, CD5, HPGD, BLNK, PLCL1, HPSE, SLFN13, HOPX, CD1D, GNG7, TCF4, BANK1, FHIT, FCMR, GNG2, GFRA2, KBT BD11, RALGPS2, TSPOAP1, PLEKHF1, MEF2C, MAOA, TTYH2, HLA-DOB, DGAT2, FXYD6, TMCC3, MGAM, TTC38, LRRC32, ARHGAP24, STAT4, SLC 7A8, CD72, FZD1, GK5, DYSF, PLTP, SMARCD3, FAM160B1, PDPN, AKAP2, ACVRL1, KCNJ15, ALDH1A2, ENPP2, COLEC12, PTGS1, TMEM170B, TREM2, ECM1, SLC1A3, ABHD5, MS4A4A, CLIC2, IL1R1, SLC2A6, GAS7, RNF144B, SLC6A12, FPR2, ADAM28, GRK3, KDM1B, MATK, LMO2, CFB,CCRL2, CLEC4A, LILRA2, ACE, NUPR1, CISH, EREG, ADAMDEC1, RNASE6, CXCL3, VSIG4, CXCL2, CD86, LILRB4, SERPING1, SQOR, INHBA, and ICAM1; or CD200, KLHL14, TCL1A, NRG1, CYP4F3, EOMES, PPP2R2B, RNF165, WNT7A, CCR4, PDGFD, EBF1, FCGBP, PCDH9, MLC1, TSHZ2, S1PR5, NCALD, LAYN, CD248, GCNT4, FASLG, TRAT1, ADAM6, GUCY1A3, LRRC4, TSPAN18, SBK1, ICOS, BTNL8, WNT5B, AUTS2, SH2D2A, ADGRG3, PNOC, SPIB, VPREB3, DPEP3, MME, ZBTB16, FOXP3, SEMA3G, CD8A, TOGARAM2, COLGALT2, ABCB1, STAP1, SAMD3, FAM46C, BLK, CTLA4, CD19, REPS2, RTKN2, POU2AF1, DAPK2, PYHIN1, NLRC3, GATM, KLRD1, AFF3, FCRLA, AATBC, REM2, YPEL1, TXK, CD8B, P2RX5, CEACAM1, BCL11A, NINJ2, ABCB4, CD5, HAL, HPGD, BLNK, PLCL1, CEP19, HPSE, SLFN13, HOPX, CD1D, GNG7, TMEM154, TCF4, BANK1, FHIT, FCMR, GNG2, GFRA2, KBTBD11, TECPR2, RALGPS2, TSPOAP1, PLEKHF1, MEF2C, MAOA, TTYH2, HLA-DOB, NRGN, DGAT2, FXYD6, TMCC3, MGAM, TTC38, LRRC32, ARHGAP24, PPP1R3B, STAT4, SLC7A8, CD72, FZD1, GK5, DYSF, PLTP, SMARCD3, FAM160B1, PDPN, AKAP2, ACVRL1, KCNJ15, CD36, ALDH1A2, ENPP2, COLEC12, PTGS1, TMEM170B, DOCK5, TREM2, C5AR2, ECM1, SLC1A3, ABHD5, MS4A4A, CLIC2, IL1R1, SLC2A6, GAS7, RNF144B, SLC6A12, FPR2, ADAM28, GRK3, KDM1B,These include MATK, LMO2, CFB, CCRL2, CLEC4A, TLR4, LILRA2, ACE, TLR1, LRRK2, LY96, NUPR1, CISH, CSTA, EREG, ADAMDEC1, RNASE6, CXCL3, VSIG4, CXCL2, CD86, LILRB4, SERPING1, SQOR, INHBA, and ICAM1. In some embodiments, the gene signature matrix includes the following genes: CD200, KLHL14, TCL1A, NRG1, EOMES, PPP2R2B, RNF165, WNT7A, CCR4, PDGFD, EBF1, FCGBP, PCDH9, MLC1, TSHZ2, S1PR5, NCALD, LAYN, GCNT4, FASLG, TRAT1, ADAM6, GUCY1A3, LRRC4, TSPAN18, SBK1, ICOS, BTNL8, WNT5B , AUTS2, SH2D2A, ADGRG3, PNOC, SPIB, VPREB3, DPEP3, MME, ZBTB16, FOXP3, SEMA3G, CD8A, TOGARAM2, COLGALT2, ABCB1, STAP1, SA MD3, FAM46C, BLK, CTLA4, CD19, REPS2, RTKN2, POU2AF1, DAPK2, PYHIN1, NLRC3, GATM, KLRD1, AFF3, FCRLA, AATBC, REM2, YPEL1, T XK, CD8B, P2RX5, CEACAM1, BCL11A, ABCB4, CD5, HPGD, BLNK, PLCL1, HPSE, SLFN13, HOPX, CD1D, GNG7, TCF4, BANK1, FHIT, FCMR, GN G2, GFRA2, KBTBD11, RALGPS2, TSPOAP1, PLEKHF1, MEF2C, MAOA, TTYH2, HLA-DOB, DGAT2, FXYD6, TMCC3, MGAM, TTC38, LRRC32, ARH GAP24, STAT4, SLC7A8, CD72, FZD1, GK5, DYSF, PLTP, SMARCD3, FAM160B1, PDPN, AKAP2, ACVRL1, KCNJ15, ALDH1A2, ENPP2, COLEC1 2, PTGS1, TMEM170B, TREM2, ECM1, SLC1A3, ABHD5, MS4A4A, CLIC2, IL1R1, SLC2A6, GAS7, RNF144B, SLC6A12, FPR2, ADAM28, GRK3,In some embodiments, a gene signature matrix is ​​used to determine the number of macrophages. In some embodiments, a gene signature matrix is ​​used to determine the number of M1 macrophages.

[0022] In some embodiments of any of the first, second, third, fourth, and fifth aspects, the macrophage biomarker is the amount of M1 macrophages. In some embodiments, the amount of M1 macrophages is measured directly or indirectly. In some embodiments, the amount of M1 macrophages is measured directly using flow cytometry, spatial transcriptomics, spatial proteomics, or a combination thereof. In some embodiments, the amount of M1 macrophages is measured indirectly using nucleic acids or proteins. In some embodiments, the nucleic acids are measured using RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technology, ISH, or a combination thereof. In some embodiments, the amount of M1 macrophages is measured using a marker gene approach or a deconvolution approach. In some embodiments, the marker gene approach uses xCell. In some embodiments, the deconvolution approach uses quanTIseq.

[0023] In some embodiments of any of the first, second, third, fourth, and fifth aspects, the macrophage biomarkers in the sample from the patient are measured using nucleic acids or proteins. In some embodiments, the macrophage biomarkers in the sample from the patient are determined using nucleic acid expression levels. In some embodiments, the nucleic acid expression levels are determined by RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technology, ISH, or a combination thereof. In some embodiments, the nucleic acid expression levels are mRNA expression levels. In some embodiments, the mRNA expression levels are determined by RNA-seq.

[0024] In some embodiments of any of the first, second, third, fourth, and fifth aspects, the sample is a tissue sample, a tumor sample, a whole blood sample, a plasma sample, a serum sample, or a combination thereof. In some embodiments, the sample is a tissue sample. In some embodiments, the tissue sample is a tumor tissue sample. In some embodiments, the tumor tissue sample comprises tumor cells, tumor-infiltrating immune cells, stromal cells, normal adjacent tissue (NAT) cells, or a combination thereof. In some embodiments, the tumor tissue sample is a biopsy. In some embodiments, the sample is an archived sample, a fresh sample, or a frozen sample.

[0025] In some embodiments of any of the first, second, third, fourth and fifth aspects, the lymphoma is an indolent lymphoma.

[0026] In some embodiments of any of the first, second, third, fourth, and fifth aspects, the lymphoma is a B-cell lymphoma. In some embodiments, the B-cell lymphoma is a germinal center-derived B-cell lymphoma. In some embodiments, the B-cell lymphoma is a non-Hodgkin's lymphoma (NHL). In some embodiments of any of the first, second, third, fourth, and fifth aspects, the lymphoma is a diffuse large B-cell lymphoma (DLBCL), a follicular lymphoma (FL), a chronic lymphocytic leukemia (CLL), or a marginal zone lymphoma (MZL). In some embodiments, the lymphoma is a DLBCL. In some embodiments, the DLBCL is a germinal center B-cell-like (GCB) cell-of-origin subgroup or an activated B-cell-like (ABC) cell-of-origin subgroup of DLBCL. In some embodiments of any of the first, second, third, fourth, and fifth aspects, the lymphoma is a CD20-positive lymphoma. In some embodiments of any of the first, second, third, fourth, and fifth aspects, the anti-CD20 antibody is a type I anti-CD20 antibody or a type II anti-CD20 antibody. In some embodiments, the anti-CD20 antibody is a type II anti-CD20 antibody. In some embodiments, the type II anti-CD20 antibody comprises the following complementarity determining regions (CDRs): (a) CDR-H1 having the amino acid sequence of SEQ ID NO: 1; (b) CDR-H2 having the amino acid sequence of SEQ ID NO: 2; (c) CDR-H3 having the amino acid sequence of SEQ ID NO: 3; (d) CDR-L1 having the amino acid sequence of SEQ ID NO: 4; (e) CDR-L2 having the amino acid sequence of SEQ ID NO: 5; and (f) CDR-L3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, the type II anti-CD20 antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the type II anti-CD20 antibody comprises the following CDRs: (a) CDR-H1 having the amino acid sequence of SEQ ID NO: 27; (b) CDR-H2 having the amino acid sequence of SEQ ID NO: 28; (c) CDR-H3 having the amino acid sequence of SEQ ID NO: 3; (d) CDR-L1 having the amino acid sequence of SEQ ID NO: 4; (e) CDR-L2 having the amino acid sequence of SEQ ID NO: 5; and (f) CDR-L3 having the amino acid sequence of SEQ ID NO: 6.In some embodiments, the type II anti-CD20 antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:8. In some embodiments, the type II anti-CD20 antibody is obinutuzumab. In some embodiments, the anti-CD20 antibody is a type I anti-CD20 antibody. In some embodiments, the type I anti-CD20 antibody comprises the following CDRs: (a) CDR-H1 having the amino acid sequence of SEQ ID NO:11; (b) CDR-H2 having the amino acid sequence of SEQ ID NO:12; (c) CDR-H3 having the amino acid sequence of SEQ ID NO:13; (d) CDR-L1 having the amino acid sequence of SEQ ID NO:14; (e) CDR-L2 having the amino acid sequence of SEQ ID NO:15; and (f) CDR-L3 having the amino acid sequence of SEQ ID NO:16. In some embodiments, the type I anti-CD20 antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO:25 and a VL domain comprising the amino acid sequence of SEQ ID NO:26. In some embodiments, the type I anti-CD20 antibody is rituximab.

[0027] In some embodiments of any of the third, fourth, and fifth aspects, the method further comprises administering to the patient an effective amount of an additional therapeutic agent. In some embodiments, the additional therapeutic agent is one or more of a chemotherapeutic agent, an anti-neoplastic agent, a growth inhibitory agent, an anti-angiogenic agent, radiation therapy, a cytotoxic agent, or a combination thereof. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is cyclophosphamide, doxorubicin, vincristine, or prednisone. In some embodiments, the chemotherapeutic agent is cyclophosphamide, doxorubicin, vincristine, and prednisone.

[0028] In some embodiments of any of the first, second, third, fourth, and fifth aspects, the patient has not been previously treated for lymphoma.

[0029] In some embodiments of any of the first, second, third, fourth and fifth aspects, the patient has not previously been administered an anti-CD20 antibody.

[0030] In a sixth aspect, the invention features the use of an anti-CD20 antibody to treat a patient having an amount or level of a macrophage biomarker that exceeds the amount or level of a reference macrophage biomarker in a sample from the patient in the manufacture of a medicament for the treatment of lymphoma.

[0031] In some embodiments of the sixth aspect, the reference macrophage biomarker amount or level is a pre-assigned macrophage biomarker amount or level.

[0032] In some embodiments of the sixth aspect, the reference macrophage biomarker amount or level is the amount or level of the macrophage biomarker in a reference population. In some embodiments, the amount or level of the macrophage biomarker in the reference population is the median amount or level of the macrophage biomarker in the reference population. In some embodiments, the reference macrophage biomarker amount or level is the amount or level of the macrophage biomarker at the 25th percentile of the reference population. In some embodiments, the reference macrophage biomarker amount or level is the amount or level of the macrophage biomarker at the 50th percentile of the reference population. In some embodiments, the reference macrophage biomarker amount or level is the amount or level of the macrophage biomarker at the 75th percentile of the reference population. In some embodiments, the reference population is a population of patients with lymphoma. In some embodiments, the population of patients with lymphoma has previously been treated with an anti-CD20 antibody. In some embodiments, the reference macrophage biomarker amount or level is the amount or level of the macrophage biomarker in the reference population before initiation of treatment with the anti-CD20 antibody. In some embodiments, the amount or level of the reference macrophage biomarker significantly separates the reference population into a first set of patients who have benefited from treatment with an anti-CD20 antibody and a second set of patients who have not benefited from treatment with an anti-CD20 antibody, hi some embodiments, the amount of macrophages is between about 0% and about 30.7%.

[0033] In some embodiments of the sixth aspect, the treatment achieves an improvement in PFS or OS.

[0034] In some embodiments of the sixth aspect, the macrophage biomarker is an average of M1 macrophage gene signature set scores of one or more M1 macrophage gene signature sets. In some embodiments, each M1 macrophage gene signature set score is an average of the expression levels of one or more genes in the M1 macrophage gene signature set. In some embodiments, each M1 macrophage gene signature set score is an average of the normalized expression levels of one or more genes in the M1 macrophage gene signature set. In some embodiments, the one or more M1 macrophage gene signature sets are: (a) ACP2, ABCD1, C1QA, FDX1, CCL22, CD163, SCAMP2, ADAMDECl, ARL8B, and HAMP; (b) ACP2, ABCD1, FDX1, CCL8, CCL22, CD163, ADAMDECl, TREM2, and HAMP;(c) ACP2, ADRA2B, ALCAM, ABCD1, ATOX1, ATP6V0C, ATP6V1E1, BLVRA, C1QA, CD48, CD63, CLCN7, TPP1, CLTC, CCR1, CMKLR1, SLC31A1, COX5B, FCER1G, FDX1, FOLR2, FPR3, FTL, HEXB, HK3, IL10, IL12B, ITGAE, LAIR1, CXCL9, MMP19, NARS, NDUFS2, P2RX7, PDCL, MAPK13, PTGIR, PTPRA, RELA, CCL7, CCL8, CCL19, CCL22, SRC, STX4, TCEB1, TFRC, AGPS, MARCO, SNX3, CD84, USP14, ITGB1BP1, ATP6V1F, TRIP4, CD163, CIAO1, WTAP, ARHGEF11, ABI1, SCAMP2, ACTR2, BCAP31, ZMPSTE24, BCKDK, EXOC5, STIP1, UQCR11, SDS, LILRB4, OGFR, TFEC, FKBP15, DNAJC13, TDRD7, STX12, IL17RA, ABTB2, FAM32A, SIGLEC7, SIGLEC9, ADAMDEC1, CECR5, SLC25A24, NRBP1, MS4A4A, TREM2, OTUD4, PQLC2, HAUS2, ARL8B, NECAP2, WDR11, ZC3H15, CCDC47, UTP3, MRS2, HAMP, MRPL40, VPS33A, CORO7, LIMD2, TMX1, DOT1L, ADO, and ADCK2; (d) ACP2, ADRA2B, ALCAM, TSPO, C3AR1, DAGLA, CALR, CHIT1, CYBB, CYC1, CYP19A1, DLAT, FCER1G, GP1BA, GPD1, IFNAR1, IL10, KCNJ5, KIFC3, MT2A, MYBPH, MYH11, MYO7A, P2RX7, PRDX1, RAB3IL1, RNH1, MRPL12, CCL1, CCL7, CCL8, CCL24, SRC, VIM, RRP1, MARCO, S1PR2, AP1M2, ACTR3, LILRB1, AFG3L2, SDS, LILRB4, EMILIN1, VSIG4, HSPB7, COQ2, ADAMDEC1, CECR5, WSB2, SLAMF8, DNASE2B, CLPB, MFSD7, and ADCK2;(e) ACP2, ADCY3, ADRA2B, ALCAM, TSPO, C1QA, C1QB, C3AR1, DAGLA, CD63, CHIT1, CMKLR1, SLC31A1, CSF1, CSF1R, CYBB, CYC1, CYP19A1, FANCE, FCER1G, FDX1, FPR3, FTL, GP1BA, GPD1, HEXB, IL10, KCNJ1, KCNJ5, KIFC3, LAMP1, MMP19, MSR1, MT2A, MYBPH, MYO7A, P2RX7, PRDX1, RAB3IL1, MRPL12, CCL1, CCL7, CCL8, CCL18, CCL19, CCL24, SLC6A12, SPR, SRC, RRP1, MARCO, PKD2L1, S1PR2, CD163, LONP1, AP1M2, IGSF6, LILRB1, SDS, LILRB4, EMILIN1, VSIG4, TFEC, PHLDB1, CYFIP1, FKBP15, NCAPH, MYOF, HSPB7, ADAMDEC1, GLRX2, NDUFAF1, SPG21, MS4A4A, ATP6V1D, ATP6V1H, TREM2, PQLC2, TMEM70, PLEKHB2, TMEM33, SLAMF8, HAMP, DNASE2B, MYOZ1, LONRF3, CLPB, MFSD7, and ADCK2;and / or (f) ACP2, ADCY3, ADRA2B, ALCAM, ABCD1, ANXA2, ATP6V1A, C1QA, C1QB, C3AR1, DAGLA, CD80, CD63, CHIT1, CMKLR1, SLC31A1, CSF1, CSF1R, CYBB, CYC1, CYP19A1, FANCE, FDX1, FPR2, FPR3, GPD1, HEXB, KCNJ1, KCNJ5, KIFC3, MMP19, MSR1, MT2A, MYBPH, P2RX7, MAPK13, S100A11, CCL1, CCL7, CCL8, CCL18, CCL19, CCL22 , CCL24, SLC1A2, SLC6A12, SLC11A1, SIGLEC1, SRC, TIE1, MARCO, HYAL2, CD163, LONP1, IGSF6, LILRB1, CD300C, SDS, LILRB4, EMILIN1, VSIG4, PHLDB1, NCAPH, CLEC4E, MYOF, HSPB7, ADAMDEC1, GLRX2, MS4A4A, ATP6V1H, TREM2, TMEM70, TMEM33, KCNK13, SLAMF8, HAMP, DNASE2B, MYOZ1, MFSD7, ADO, ADCK2, and TBC1D16.

[0035] In some embodiments of the sixth aspect, the macrophage biomarker is an average macrophage gene signature set score of one or more macrophage gene signature sets. In some embodiments, each macrophage gene signature set score is an average of the expression levels of one or more genes in the macrophage gene signature set. In some embodiments, each macrophage gene signature set score is an average of the normalized expression levels of one or more genes in the macrophage gene signature set. In some embodiments, the one or more macrophage gene signature sets are any of the macrophage gene signature sets in Table 2.

[0036] In some embodiments of the sixth aspect, the macrophage biomarker is a gene expression value. In some embodiments, the gene expression value is a median gene expression value. In some embodiments, the gene expression value is measured using a gene signature matrix. In some embodiments, the gene signature matrix includes the following genes: CD200, KLHL14, TCL1A, NRG1, EOMES, PPP2R2B, RNF165, WNT7A, CCR4, PDGFD, EBF1, FCGBP, PCDH9, MLC1, TSHZ2, S1PR5, NCALD, LAYN, GCNT4, FASLG, TRAT1, ADAM6, GUCY1A3, LRRC4, TSPAN18, SBK1, ICOS, BTNL8, WNT5B, AUTS2, SH2 D2A, ADGRG3, PNOC, SPIB, VPREB3, DPEP3, MME, ZBTB16, FOXP3, SEMA3G, CD8A, TOGARAM2, COLGALT2, ABCB1, STAP1, SAMD3, FAM46C, BLK, CTLA4, CD19, REPS2, RTKN2, POU2AF1, DAPK2, PYHIN1, NLRC3, GATM, KLRD1, AFF3, FCRLA, AATBC, REM2, YPEL1, TXK, CD8B, P2RX5, CEACAM 1, BCL11A, ABCB4, CD5, HPGD, BLNK, PLCL1, HPSE, SLFN13, HOPX, CD1D, GNG7, TCF4, BANK1, FHIT, FCMR, GNG2, GFRA2, KBTBD11, RALGPS2, TSPOAP1, PLEKHF1, MEF2C, MAOA, TTYH2, HLA-DOB, DGAT2, FXYD6, TMCC3, MGAM, TTC38, LRRC32, ARHGAP24, STAT4, SLC7A8, CD72, FZD1, G K5, DYSF, PLTP, SMARCD3, FAM160B1, PDPN, AKAP2, ACVRL1, KCNJ15, ALDH1A2, ENPP2, COLEC12, PTGS1, TMEM170B, TREM2, ECM1, SLC1A3, ABHD5, MS4A4A, CLIC2, IL1R1, SLC2A6, GAS7, RNF144B, SLC6A12, FPR2, ADAM28, GRK3, KDM1B, MATK, LMO2, CFB, CCRL2, CLEC4A, LILRA2,ACE, NUPR1, CISH, EREG, ADAMDEC1, RNASE6, CXCL3, VSIG4, CXCL2, CD86, LILRB4, SERPING1, SQOR, INHBA, and ICAM1; or CD200, KLHL14, TCL1A, NRG1, CYP4F3, EOMES, PPP2R2B, RNF165, WNT7A, CCR4, PDGFD, EBF1, FCGBP, PCDH9, MLC1, TSHZ2, S1PR5, NCALD, LAYN, CD248, GCNT4, FASLG, TRAT1, ADAM6, GUCY1A3, LRRC4, TSPAN18, SBK1, ICOS, BTNL8, WNT5B, AUTS2, SH2D2A, ADGRG3, PNOC, SPIB, VPREB3, DPEP3, MME, ZBTB16, FOXP3, SEMA3G, CD8A, TOGARAM2, COLGALT2, ABCB1, STAP1, SAMD3, FAM46C, BLK, CTLA4, CD19, REPS2, RTKN2, POU2AF1, DAPK2, PYHIN1, NLRC3, GATM, KLRD1, AFF3, FCRLA, AATBC, REM2, YPEL1, TXK, CD8B, P2RX5, CEACAM1, BCL11A, NINJ2, ABCB4, CD5, HAL, HPGD, BLNK, PLCL1, CEP19, HPSE, SLFN13, HOPX, CD1D, GNG7, TMEM154, TCF4, BANK1, FHIT, FCMR, GNG2, GFRA2, KBTBD11, TECPR2, RALGPS2, TSPOAP1, PLEKHF1, MEF2C, MAOA, TTYH2, HLA-DOB, NRGN, DGAT2, FXYD6, TMCC3, MGAM, TTC38, LRRC32, ARHGAP24, PPP1R3B, STAT4, SLC7A8, CD72, FZD1, GK5, DYSF, PLTP, SMARCD3, FAM160B1, PDPN, AKAP2, ACVRL1, KCNJ15, CD36, ALDH1A2, ENPP2, COLEC12, PTGS1, TMEM170B, DOCK5, TREM2, C5AR2, ECM1, SLC1A3, ABHD5, MS4A4A, CLIC2, IL1R1, SLC2A6, GAS7, RNF144B, SLC6A12, FPR2, ADAM28, GRK3, KDM1B, MATK, LMO2, CFB, CCRL2,In some embodiments, the gene signature matrix comprises the following genes: CLEC4A, TLR4, LILRA2, ACE, TLR1, LRRK2, LY96, NUPR1, CISH, CSTA, EREG, ADAMDEC1, RNASE6, CXCL3, VSIG4, CXCL2, CD86, LILRB4, SERPING1, SQOR, INHBA, and ICAM1. In some embodiments, the gene signature matrix comprises the following genes: CD200, KLHL14, TCL1A, NRG1, EOMES, PPP2R2B, RNF165, WNT7A, CCR4, PDGFD, EBF1, FCGBP, PCDH9, MLC1, TSHZ2, S1PR5, NCALD, LAYN, GCNT4, FASLG, TRAT1, ADAM6, GUCY1A3, LRRC4, TSPAN18, SBK1, ICOS, BTNL8, WNT5B, AUTS 2, SH2D2A, ADGRG3, PNOC, SPIB, VPREB3, DPEP3, MME, ZBTB16, FOXP3, SEMA3G, CD8A, TOGARAM2, COLGALT2, ABCB1, STAP1, SAMD3, FAM 46C, BLK, CTLA4, CD19, REPS2, RTKN2, POU2AF1, DAPK2, PYHIN1, NLRC3, GATM, KLRD1, AFF3, FCRLA, AATBC, REM2, YPEL1, TXK, CD8B, P2 RX5, CEACAM1, BCL11A, ABCB4, CD5, HPGD, BLNK, PLCL1, HPSE, SLFN13, HOPX, CD1D, GNG7, TCF4, BANK1, FHIT, FCMR, GNG2, GFRA2, KBT BD11, RALGPS2, TSPOAP1, PLEKHF1, MEF2C, MAOA, TTYH2, HLA-DOB, DGAT2, FXYD6, TMCC3, MGAM, TTC38, LRRC32, ARHGAP24, STAT4, SLC 7A8, CD72, FZD1, GK5, DYSF, PLTP, SMARCD3, FAM160B1, PDPN, AKAP2, ACVRL1, KCNJ15, ALDH1A2, ENPP2, COLEC12, PTGS1, TMEM170B, TREM2, ECM1, SLC1A3, ABHD5, MS4A4A, CLIC2, IL1R1, SLC2A6, GAS7, RNF144B, SLC6A12, FPR2, ADAM28, GRK3, KDM1B, MATK, LMO2, CFB,In some embodiments, a gene signature matrix is ​​used to determine the number of macrophages. In some embodiments, a gene signature matrix is ​​used to determine the number of M1 macrophages.

[0037] In some embodiments of the sixth aspect, the macrophage biomarker is the amount of M1 macrophages. In some embodiments, the amount of M1 macrophages is measured directly or indirectly. In some embodiments, the amount of M1 macrophages is measured directly using flow cytometry, spatial transcriptomics, spatial proteomics, or a combination thereof. In some embodiments, the amount of M1 macrophages is measured indirectly using nucleic acids or proteins. In some embodiments, the nucleic acids are measured using RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technology, ISH, or a combination thereof. In some embodiments, the amount of M1 macrophages is measured using a marker gene approach or a deconvolution approach. In some embodiments, the marker gene approach uses xCell. In some embodiments, the deconvolution approach uses quanTIseq.

[0038] In some embodiments of the sixth aspect, the macrophage biomarkers in the sample from the patient are measured using nucleic acids or proteins. In some embodiments, the macrophage biomarkers in the sample from the patient are determined using nucleic acid expression levels. In some embodiments, the nucleic acid expression levels are determined by RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technology, ISH, or a combination thereof. In some embodiments, the nucleic acid expression levels are mRNA expression levels. In some embodiments, the mRNA expression levels are determined by RNA-seq.

[0039] In some embodiments of the sixth aspect, the sample is a tissue sample, a tumor sample, a whole blood sample, a plasma sample, a serum sample, or a combination thereof. In some embodiments, the sample is a tissue sample. In some embodiments, the tissue sample is a tumor tissue sample. In some embodiments, the tumor tissue sample comprises tumor cells, tumor-infiltrating immune cells, stromal cells, NAT cells, or a combination thereof. In some embodiments, the tumor tissue sample is a biopsy. In some embodiments, the sample is an archived sample, a fresh sample, or a frozen sample.

[0040] In some embodiments of the sixth aspect, the lymphoma is an indolent lymphoma.

[0041] In some embodiments of the sixth aspect, the lymphoma is a B-cell lymphoma. In some embodiments, the B-cell lymphoma is NHL.

[0042] In some embodiments of the sixth aspect, the lymphoma is DLBCL, FL, CLL, or MZL. In some embodiments, the lymphoma is DLBCL. In some embodiments, the DLBCL is the GCB cell of origin subgroup or the ABC cell of origin subgroup of DLBCL.

[0043] In some embodiments of the sixth aspect, the lymphoma is a CD20-positive lymphoma.

[0044] In some embodiments of the sixth aspect, the anti-CD20 antibody is a type I anti-CD20 antibody or a type II anti-CD20 antibody. In some embodiments, the anti-CD20 antibody is a type II anti-CD20 antibody. In some embodiments, the type II anti-CD20 antibody comprises the following complementarity determining regions (CDRs): (a) CDR-H1 having the amino acid sequence of SEQ ID NO: 1; (b) CDR-H2 having the amino acid sequence of SEQ ID NO: 2; (c) CDR-H3 having the amino acid sequence of SEQ ID NO: 3; (d) CDR-L1 having the amino acid sequence of SEQ ID NO: 4; (e) CDR-L2 having the amino acid sequence of SEQ ID NO: 5; and (f) CDR-L3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, the type II anti-CD20 antibody comprises the following CDRs: (a) CDR-H1 having the amino acid sequence of SEQ ID NO:27; (b) CDR-H2 having the amino acid sequence of SEQ ID NO:28; (c) CDR-H3 having the amino acid sequence of SEQ ID NO:3; (d) CDR-L1 having the amino acid sequence of SEQ ID NO:4; (e) CDR-L2 having the amino acid sequence of SEQ ID NO:5; and (f) CDR-L3 having the amino acid sequence of SEQ ID NO:6. In some embodiments, the type II anti-CD20 antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:8. In some embodiments, the type II anti-CD20 antibody is obinutuzumab. In some embodiments, the anti-CD20 antibody is a type I anti-CD20 antibody. In certain embodiments, the anti-CD20 antibody comprises the following CDRs: (a) CDR-H1 having the amino acid sequence of SEQ ID NO: 11; (b) CDR-H2 having the amino acid sequence of SEQ ID NO: 12; (c) CDR-H3 having the amino acid sequence of SEQ ID NO: 13; (d) CDR-L1 having the amino acid sequence of SEQ ID NO: 14; (e) CDR-L2 having the amino acid sequence of SEQ ID NO: 15; and (f) CDR-L3 having the amino acid sequence of SEQ ID NO: 16. In some embodiments, the type I anti-CD20 antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 25 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the anti-CD20 antibody is rituximab.

[0045] In some embodiments of the sixth aspect, the medicament is administered to the patient in combination with an effective amount of an additional therapeutic agent. In some embodiments, the additional therapeutic agent is one or more of a chemotherapeutic agent, an anti-neoplastic agent, a growth inhibitory agent, an anti-angiogenic agent, radiation therapy, a cytotoxic agent, or a combination thereof. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is cyclophosphamide, doxorubicin, vincristine, or prednisone. In some embodiments, the chemotherapeutic agent is cyclophosphamide, doxorubicin, vincristine, and prednisone.

[0046] In some embodiments of the sixth aspect, the patient has not been previously treated for lymphoma.

[0047] In some embodiments of the sixth aspect, the patient has not previously been administered an anti-CD20 antibody.

[0048] In a seventh aspect, the invention features an anti-CD20 antibody for use in treating a patient having lymphoma and having an amount or level of a macrophage biomarker in a sample from the patient that exceeds a reference amount or level of a macrophage biomarker. In some embodiments, the reference amount or level of the macrophage biomarker is a pre-assigned amount or level of the macrophage biomarker.

[0049] In some embodiments of the seventh aspect, the reference macrophage biomarker amount or level is the amount or level of the macrophage biomarker in a reference population. In some embodiments, the amount or level of the macrophage biomarker in the reference population is the median amount or level of the macrophage biomarker in the reference population. In some embodiments, the reference macrophage biomarker amount or level is the amount or level of the macrophage biomarker at the 25th percentile of the reference population. In some embodiments, the reference macrophage biomarker amount or level is the amount or level of the macrophage biomarker at the 50th percentile of the reference population. In some embodiments, the reference macrophage biomarker amount or level is the amount or level of the macrophage biomarker at the 75th percentile of the reference population. In some embodiments, the reference population is a population of patients with lymphoma. In some embodiments, the population of patients with lymphoma has previously been treated with an anti-CD20 antibody. In some embodiments, the reference macrophage biomarker amount or level is the amount or level of the macrophage biomarker in the reference population before initiation of treatment with the anti-CD20 antibody. In some embodiments, the amount or level of the reference macrophage biomarker significantly separates the reference population into a first set of patients who have benefited from treatment with an anti-CD20 antibody and a second set of patients who have not benefited from treatment with an anti-CD20 antibody.

[0050] In some embodiments of the seventh aspect, the amount or level of the reference macrophage biomarker is the amount of macrophages as measured by gene expression. In some embodiments, the amount of macrophages is between about 0% and about 30.7%.

[0051] In some embodiments of the seventh aspect, the treatment achieves an improvement in PFS or OS.

[0052] In some embodiments of the seventh aspect, the macrophage biomarker is an average of M1 macrophage gene signature set scores of one or more M1 macrophage gene signature sets. In some embodiments, each M1 macrophage gene signature set score is an average of the expression levels of one or more genes in the M1 macrophage gene signature set. In some embodiments, each M1 macrophage gene signature set score is an average of the normalized expression levels of one or more genes in the M1 macrophage gene signature set. In some embodiments, the one or more M1 macrophage gene signature sets are: (a) ACP2, ABCD1, C1QA, FDX1, CCL22, CD163, SCAMP2, ADAMDECl, ARL8B, and HAMP; (b) ACP2, ABCD1, FDX1, CCL8, CCL22, CD163, ADAMDECl, TREM2, and HAMP;(c) ACP2, ADRA2B, ALCAM, ABCD1, ATOX1, ATP6V0C, ATP6V1E1, BLVRA, C1QA, CD48, CD63, CLCN7, TPP1, CLTC, CCR1, CMKLR1, SLC31A1, COX5B, FCER1G, FDX1, FOLR2, FPR3, FTL, HEXB, HK3, IL10, IL12B, ITGAE, LAIR1, CXCL9, MMP19, NARS, NDUFS2, P2RX7, PDCL, MAPK13, PTGIR, PTPRA, RELA, CCL7, CCL8, CCL19, CCL22, SRC, STX4, TCEB1, TFRC, AGPS, MARCO, SNX3, CD84, USP14, ITGB1BP1, ATP6V1F, TRIP4, CD163, CIAO1, WTAP, ARHGEF11, ABI1, SCAMP2, ACTR2, BCAP31, ZMPSTE24, BCKDK, EXOC5, STIP1, UQCR11, SDS, LILRB4, OGFR, TFEC, FKBP15, DNAJC13, TDRD7, STX12, IL17RA, ABTB2, FAM32A, SIGLEC7, SIGLEC9, ADAMDEC1, CECR5, SLC25A24, NRBP1, MS4A4A, TREM2, OTUD4, PQLC2, HAUS2, ARL8B, NECAP2, WDR11, ZC3H15, CCDC47, UTP3, MRS2, HAMP, MRPL40, VPS33A, CORO7, LIMD2, TMX1, DOT1L, ADO, and ADCK2; (d) ACP2, ADRA2B, ALCAM, TSPO, C3AR1, DAGLA, CALR, CHIT1, CYBB, CYC1, CYP19A1, DLAT, FCER1G, GP1BA, GPD1, IFNAR1, IL10, KCNJ5, KIFC3, MT2A, MYBPH, MYH11, MYO7A, P2RX7, PRDX1, RAB3IL1, RNH1, MRPL12, CCL1, CCL7, CCL8, CCL24, SRC, VIM, RRP1, MARCO, S1PR2, AP1M2, ACTR3, LILRB1, AFG3L2, SDS, LILRB4, EMILIN1, VSIG4, HSPB7, COQ2, ADAMDEC1, CECR5, WSB2, SLAMF8, DNASE2B, CLPB, MFSD7, and ADCK2;(e) ACP2, ADCY3, ADRA2B, ALCAM, TSPO, C1QA, C1QB, C3AR1, DAGLA, CD63, CHIT1, CMKLR1, SLC31A1, CSF1, CSF1R, CYBB, CYC1, CYP19A1, FANCE, FCER1G, FDX1, FPR3, FTL, GP1BA, GPD1, HEXB, IL10, KCNJ1, KCNJ5, KIFC3, LAMP1, MMP19, MSR1, MT2A, MYBPH, MYO7A, P2RX7, PRDX1, RAB3IL1, MRPL12, CCL1, CCL7, CCL8, CCL18, CCL19, CCL24, SLC6A12, SPR, SRC, RRP1, MARCO, PKD2L1, S1PR2, CD163, LONP1, AP1M2, IGSF6, LILRB1, SDS, LILRB4, EMILIN1, VSIG4, TFEC, PHLDB1, CYFIP1, FKBP15, NCAPH, MYOF, HSPB7, ADAMDEC1, GLRX2, NDUFAF1, SPG21, MS4A4A, ATP6V1D, ATP6V1H, TREM2, PQLC2, TMEM70, PLEKHB2, TMEM33, SLAMF8, HAMP, DNASE2B, MYOZ1, LONRF3, CLPB, MFSD7, and ADCK2;and / or (f) ACP2, ADCY3, ADRA2B, ALCAM, ABCD1, ANXA2, ATP6V1A, C1QA, C1QB, C3AR1, DAGLA, CD80, CD63, CHIT1, CMKLR1, SLC31A1, CSF1, CSF1R, CYBB, CYC1, CYP19A1, FANCE, FDX1, FPR2, FPR3, GPD1, HEXB, KCNJ1, KCNJ5, KIFC3, MMP19, MSR1, MT2A, MYBPH, P2RX7, MAPK13, S100A11, CCL1, CCL7, CCL8, CCL18, CCL19, CCL22 , CCL24, SLC1A2, SLC6A12, SLC11A1, SIGLEC1, SRC, TIE1, MARCO, HYAL2, CD163, LONP1, IGSF6, LILRB1, CD300C, SDS, LILRB4, EMILIN1, VSIG4, PHLDB1, NCAPH, CLEC4E, MYOF, HSPB7, ADAMDEC1, GLRX2, MS4A4A, ATP6V1H, TREM2, TMEM70, TMEM33, KCNK13, SLAMF8, HAMP, DNASE2B, MYOZ1, MFSD7, ADO, ADCK2, and TBC1D16.

[0053] In some embodiments of the seventh aspect, the macrophage biomarker is an average macrophage gene signature set score of one or more macrophage gene signature sets. In some embodiments, each macrophage gene signature set score is an average of the expression levels of one or more genes in the macrophage gene signature set. In some embodiments, each macrophage gene signature set score is an average of the normalized expression levels of one or more genes in the macrophage gene signature set. In some embodiments, the one or more macrophage gene signature sets are any of the macrophage gene signature sets in Table 2.

[0054] In some embodiments of the seventh aspect, the macrophage biomarker is a gene expression value. In some embodiments, the gene expression value is a median gene expression value. In some embodiments, the gene expression value is measured using a gene signature matrix. In some embodiments, the gene signature matrix includes the following genes: CD200, KLHL14, TCL1A, NRG1, EOMES, PPP2R2B, RNF165, WNT7A, CCR4, PDGFD, EBF1, FCGBP, PCDH9, MLC1, TSHZ2, S1PR5, NCALD, LAYN, GCNT4, FASLG, TRAT1, ADAM6, GUCY1A3, LRRC4, TSPAN18, SBK1, ICOS, BTNL8, WNT5B, AUTS2, SH2 D2A, ADGRG3, PNOC, SPIB, VPREB3, DPEP3, MME, ZBTB16, FOXP3, SEMA3G, CD8A, TOGARAM2, COLGALT2, ABCB1, STAP1, SAMD3, FAM46C, BLK, CTLA4, CD19, REPS2, RTKN2, POU2AF1, DAPK2, PYHIN1, NLRC3, GATM, KLRD1, AFF3, FCRLA, AATBC, REM2, YPEL1, TXK, CD8B, P2RX5, CEACAM 1, BCL11A, ABCB4, CD5, HPGD, BLNK, PLCL1, HPSE, SLFN13, HOPX, CD1D, GNG7, TCF4, BANK1, FHIT, FCMR, GNG2, GFRA2, KBTBD11, RALGPS2, TSPOAP1, PLEKHF1, MEF2C, MAOA, TTYH2, HLA-DOB, DGAT2, FXYD6, TMCC3, MGAM, TTC38, LRRC32, ARHGAP24, STAT4, SLC7A8, CD72, FZD1, G K5, DYSF, PLTP, SMARCD3, FAM160B1, PDPN, AKAP2, ACVRL1, KCNJ15, ALDH1A2, ENPP2, COLEC12, PTGS1, TMEM170B, TREM2, ECM1, SLC1A3, ABHD5, MS4A4A, CLIC2, IL1R1, SLC2A6, GAS7, RNF144B, SLC6A12, FPR2, ADAM28, GRK3, KDM1B, MATK, LMO2, CFB, CCRL2, CLEC4A, LILRA2,ACE, NUPR1, CISH, EREG, ADAMDEC1, RNASE6, CXCL3, VSIG4, CXCL2, CD86, LILRB4, SERPING1, SQOR, INHBA, and ICAM1; or CD200, KLHL14, TCL1A, NRG1, CYP4F3, EOMES, PPP2R2B, RNF165, WNT7A, CCR4, PDGFD, EBF1, FCGBP, PCDH9, MLC1, TSHZ2, S1PR5, NCALD, LAYN, CD248, GCNT4, FASLG, TRAT1, ADAM6, GUCY1A3, LRRC4, TSPAN18, SBK1, ICOS, BTNL8, WNT5B, AUTS2, SH2D2A, ADGRG3, PNOC, SPIB, VPREB3, DPEP3, MME, ZBTB16, FOXP3, SEMA3G, CD8A, TOGARAM2, COLGALT2, ABCB1, STAP1, SAMD3, FAM46C, BLK, CTLA4, CD19, REPS2, RTKN2, POU2AF1, DAPK2, PYHIN1, NLRC3, GATM, KLRD1, AFF3, FCRLA, AATBC, REM2, YPEL1, TXK, CD8B, P2RX5, CEACAM1, BCL11A, NINJ2, ABCB4, CD5, HAL, HPGD, BLNK, PLCL1, CEP19, HPSE, SLFN13, HOPX, CD1D, GNG7, TMEM154, TCF4, BANK1, FHIT, FCMR, GNG2, GFRA2, KBTBD11, TECPR2, RALGPS2, TSPOAP1, PLEKHF1, MEF2C, MAOA, TTYH2, HLA-DOB, NRGN, DGAT2, FXYD6, TMCC3, MGAM, TTC38, LRRC32, ARHGAP24, PPP1R3B, STAT4, SLC7A8, CD72, FZD1, GK5, DYSF, PLTP, SMARCD3, FAM160B1, PDPN, AKAP2, ACVRL1, KCNJ15, CD36, ALDH1A2, ENPP2, COLEC12, PTGS1, TMEM170B, DOCK5, TREM2, C5AR2, ECM1, SLC1A3, ABHD5, MS4A4A, CLIC2, IL1R1, SLC2A6, GAS7, RNF144B, SLC6A12, FPR2, ADAM28, GRK3, KDM1B, MATK, LMO2, CFB, CCRL2,In some embodiments, the gene signature matrix comprises the following genes: CLEC4A, TLR4, LILRA2, ACE, TLR1, LRRK2, LY96, NUPR1, CISH, CSTA, EREG, ADAMDEC1, RNASE6, CXCL3, VSIG4, CXCL2, CD86, LILRB4, SERPING1, SQOR, INHBA, and ICAM1. In some embodiments, the gene signature matrix comprises the following genes: CD200, KLHL14, TCL1A, NRG1, EOMES, PPP2R2B, RNF165, WNT7A, CCR4, PDGFD, EBF1, FCGBP, PCDH9, MLC1, TSHZ2, S1PR5, NCALD, LAYN, GCNT4, FASLG, TRAT1, ADAM6, GUCY1A3, LRRC4, TSPAN18, SBK1, ICOS, BTNL8, WNT5B, AUTS 2, SH2D2A, ADGRG3, PNOC, SPIB, VPREB3, DPEP3, MME, ZBTB16, FOXP3, SEMA3G, CD8A, TOGARAM2, COLGALT2, ABCB1, STAP1, SAMD3, FAM 46C, BLK, CTLA4, CD19, REPS2, RTKN2, POU2AF1, DAPK2, PYHIN1, NLRC3, GATM, KLRD1, AFF3, FCRLA, AATBC, REM2, YPEL1, TXK, CD8B, P2 RX5, CEACAM1, BCL11A, ABCB4, CD5, HPGD, BLNK, PLCL1, HPSE, SLFN13, HOPX, CD1D, GNG7, TCF4, BANK1, FHIT, FCMR, GNG2, GFRA2, KBT BD11, RALGPS2, TSPOAP1, PLEKHF1, MEF2C, MAOA, TTYH2, HLA-DOB, DGAT2, FXYD6, TMCC3, MGAM, TTC38, LRRC32, ARHGAP24, STAT4, SLC 7A8, CD72, FZD1, GK5, DYSF, PLTP, SMARCD3, FAM160B1, PDPN, AKAP2, ACVRL1, KCNJ15, ALDH1A2, ENPP2, COLEC12, PTGS1, TMEM170B, TREM2, ECM1, SLC1A3, ABHD5, MS4A4A, CLIC2, IL1R1, SLC2A6, GAS7, RNF144B, SLC6A12, FPR2, ADAM28, GRK3, KDM1B, MATK, LMO2, CFB,In some embodiments, a gene signature matrix is ​​used to determine the number of macrophages. In some embodiments, a gene signature matrix is ​​used to determine the number of M1 macrophages.

[0055] In some embodiments of the seventh aspect, the macrophage biomarker is the amount of M1 macrophages. In some embodiments, the amount of M1 macrophages is measured directly or indirectly. In some embodiments, the amount of M1 macrophages is measured directly using flow cytometry, spatial transcriptomics, spatial proteomics, or a combination thereof. In some embodiments, the amount of M1 macrophages is measured indirectly using nucleic acids or proteins. In some embodiments, the nucleic acids are measured using RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technology, ISH, or a combination thereof. In some embodiments, the amount of M1 macrophages is measured using a marker gene approach or a deconvolution approach. In some embodiments, the marker gene approach uses xCell. In some embodiments, the deconvolution approach uses quanTIseq.

[0056] In some embodiments of the seventh aspect, the macrophage biomarkers in the sample from the patient are measured using nucleic acids or proteins. In some embodiments, the macrophage biomarkers in the sample from the patient are determined using nucleic acid expression levels. In some embodiments, the nucleic acid expression levels are determined by RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technology, ISH, or a combination thereof. In some embodiments, the nucleic acid expression levels are mRNA expression levels. In some embodiments, the mRNA expression levels are determined by RNA-seq.

[0057] In some embodiments of the seventh aspect, the sample is a tissue sample, a tumor sample, a whole blood sample, a plasma sample, a serum sample, or a combination thereof. In some embodiments, the sample is a tissue sample. In some embodiments, the tissue sample is a tumor tissue sample. In some embodiments, the tumor tissue sample comprises tumor cells, tumor-infiltrating immune cells, stromal cells, NAT cells, or a combination thereof. In some embodiments, the tumor tissue sample is a biopsy. In some embodiments, the sample is an archived sample, a fresh sample, or a frozen sample.

[0058] In some embodiments of the seventh aspect, the lymphoma is an indolent lymphoma.

[0059] In some embodiments of the seventh aspect, the lymphoma is a B-cell lymphoma. In some embodiments, the B-cell lymphoma is NHL.

[0060] In some embodiments of the seventh aspect, the lymphoma is DLBCL, FL, CLL, or MZL. In some embodiments, the lymphoma is DLBCL. In some embodiments, the DLBCL is the GCB cell of origin subgroup or the ABC cell of origin subgroup of DLBCL.

[0061] In some embodiments of the seventh aspect, the lymphoma is a CD20-positive lymphoma.

[0062] In some embodiments of the seventh aspect, the anti-CD20 antibody is a type I anti-CD20 antibody or a type II anti-CD20 antibody. In some embodiments, the anti-CD20 antibody is a type II anti-CD20 antibody. In some embodiments, the type II anti-CD20 antibody comprises the following complementarity determining regions (CDRs): (a) CDR-H1 having the amino acid sequence of SEQ ID NO: 1; (b) CDR-H2 having the amino acid sequence of SEQ ID NO: 2; (c) CDR-H3 having the amino acid sequence of SEQ ID NO: 3; (d) CDR-L1 having the amino acid sequence of SEQ ID NO: 4; (e) CDR-L2 having the amino acid sequence of SEQ ID NO: 5; and (f) CDR-L3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, the type II anti-CD20 antibody comprises the following CDRs: (a) CDR-H1 having the amino acid sequence of SEQ ID NO:27; (b) CDR-H2 having the amino acid sequence of SEQ ID NO:28; (c) CDR-H3 having the amino acid sequence of SEQ ID NO:3; (d) CDR-L1 having the amino acid sequence of SEQ ID NO:4; (e) CDR-L2 having the amino acid sequence of SEQ ID NO:5; and (f) CDR-L3 having the amino acid sequence of SEQ ID NO:6. In some embodiments, the type II anti-CD20 antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:8. In some embodiments, the type II anti-CD20 antibody is obinutuzumab. In some embodiments, the anti-CD20 antibody is a type I anti-CD20 antibody. In certain embodiments, the anti-CD20 antibody comprises the following CDRs: (a) CDR-H1 having the amino acid sequence of SEQ ID NO: 11; (b) CDR-H2 having the amino acid sequence of SEQ ID NO: 12; (c) CDR-H3 having the amino acid sequence of SEQ ID NO: 13; (d) CDR-L1 having the amino acid sequence of SEQ ID NO: 14; (e) CDR-L2 having the amino acid sequence of SEQ ID NO: 15; and (f) CDR-L3 having the amino acid sequence of SEQ ID NO: 16. In some embodiments, the type I anti-CD20 antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 25 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the anti-CD20 antibody is rituximab.

[0063] In some embodiments of the seventh aspect, the treatment further comprises use of an effective amount of an additional therapeutic agent. In some embodiments, the additional therapeutic agent is one or more of a chemotherapeutic agent, an anti-neoplastic agent, a growth inhibitory agent, an anti-angiogenic agent, radiation therapy, a cytotoxic agent, or a combination thereof. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is cyclophosphamide, doxorubicin, vincristine, or prednisone. In some embodiments, the chemotherapeutic agent is cyclophosphamide, doxorubicin, vincristine, and prednisone.

[0064] In some embodiments of the seventh aspect, the patient has not been previously treated for lymphoma.

[0065] In some embodiments of the seventh aspect, the patient has not previously been administered an anti-CD20 antibody.

[0066] In an eighth aspect, the invention features a method for identifying, diagnosing, and / or predicting whether a patient having lymphoma could benefit from a treatment comprising an anti-CD20 antibody, the method including measuring a Th2 biomarker in a sample from the patient, wherein an amount or level of a Th2 biomarker in the sample that exceeds an amount or level of a reference Th2 biomarker identifies, diagnoses, and / or predicts the patient as one who could benefit from a treatment comprising an anti-CD20 antibody.

[0067] In a ninth aspect, the invention features a method of selecting a therapy for a patient having lymphoma, the method including measuring a Th2 biomarker in a sample from the patient, wherein an amount or level of the Th2 biomarker in the sample that exceeds an amount or level of a reference Th2 biomarker identifies the patient as one who may benefit from a therapy including an anti-CD20 antibody.

[0068] In some embodiments of the eighth or ninth aspect, the patient has a Th2 biomarker in the sample that exceeds an amount or level of a reference Th2 biomarker, and the method further comprises administering to the patient an effective amount of an anti-CD20 antibody.

[0069] In a tenth aspect, the present invention provides a method of treating a patient having lymphoma, the method comprising: (a) measuring a Th2 biomarker in a sample from the patient, wherein the amount or level of the Th2 biomarker in the sample exceeds the amount or level of a reference Th2 biomarker; and (b) administering to the patient an effective amount of an anti-CD20 antibody based on the Th2 biomarker measured in step (a).

[0070] In an eleventh aspect, the invention features a method of treating a patient having lymphoma, the method including administering to the patient an effective amount of an anti-CD20 antibody, wherein prior to treatment, the amount or level of a Th2 biomarker in a sample from the patient has been determined to exceed the amount or level of a reference Th2 biomarker.

[0071] In a twelfth aspect, the invention features a method of treating a patient having lymphoma and having an amount or level of a Th2 biomarker in a sample from the patient that exceeds a reference amount or level of a Th2 biomarker, the method comprising administering to the patient an effective amount of an anti-CD20 antibody.

[0072] In some embodiments of any of the eighth, ninth, tenth, eleventh and twelfth aspects, the reference Th2 biomarker amount or level is a pre-assigned Th2 biomarker amount or level.

[0073] In some embodiments of any of the eighth, ninth, tenth, eleventh, and twelfth aspects, the reference Th2 biomarker amount or level is the amount or level of the Th2 biomarker in a reference population. In some embodiments, the amount or level of the Th2 biomarker in the reference population is the median amount or level of the Th2 biomarker in the reference population. In some embodiments, the reference Th2 biomarker amount or level is the amount or level of the Th2 biomarker at the 25th percentile of the reference population. In some embodiments, the reference Th2 biomarker amount or level is the amount or level of the Th2 biomarker at the 50th percentile of the reference population. In some embodiments, the reference Th2 biomarker amount or level is the amount or level of the Th2 biomarker at the 75th percentile of the reference population. In some embodiments, the reference population is a population of patients with lymphoma. In some embodiments, the population of patients with lymphoma has previously been treated with an anti-CD20 antibody. In some embodiments, the reference Th2 biomarker amount or level is the amount or level of the Th2 biomarker in the reference population before initiating treatment with the anti-CD20 antibody. In some embodiments, the amount or level of the reference Th2 biomarker significantly separates the reference population into a first set of patients who have benefited from treatment with an anti-CD20 antibody and a second set of patients who have not benefited from treatment with an anti-CD20 antibody.

[0074] In some embodiments of any of the eighth, ninth, tenth, eleventh, and twelfth aspects, the amount or level of the reference Th2 biomarker is the amount of Th2 cells as measured by gene expression.

[0075] In some embodiments of the eighth or ninth aspect, the benefit is prolonged PFS.

[0076] In some embodiments of the eighth or ninth aspect, the benefit is increased OS.

[0077] Some embodiments of any of the tenth, eleventh, and twelfth aspects include achieving an improvement in PFS or OS.

[0078] In some embodiments of any of the eighth, ninth, tenth, eleventh, and twelfth aspects, the Th2 biomarker is an average Th2 gene signature set score of one or more Th2 gene signature sets. In some embodiments, each Th2 gene signature set score is an average of the expression levels of one or more genes in the Th2 gene signature set. In some embodiments, each Th2 gene signature set score is an average of the normalized expression levels of one or more genes in the Th2 gene signature set. In some embodiments, the one or more Th2 gene signature sets are any of the Th2 gene signature sets in Table 5.

[0079] In some embodiments of any of the eighth, ninth, tenth, eleventh, and twelfth aspects, the Th2 biomarker is the amount of Th2 cells. In some embodiments, the amount of Th2 cells is measured directly or indirectly. In some embodiments, the amount of Th2 cells is measured directly using flow cytometry, spatial transcriptomics, spatial proteomics, or a combination thereof. In some embodiments, the amount of Th2 cells is measured indirectly using nucleic acids or proteins. In some embodiments, the nucleic acids are measured using RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technology, ISH, or a combination thereof. In some embodiments, the amount of Th2 cells is measured using a marker gene approach or a deconvolution approach. In some embodiments, the marker gene approach uses xCell. In some embodiments, the deconvolution approach uses quanTIseq.

[0080] In some embodiments of any of the eighth, ninth, tenth, eleventh, and twelfth aspects, the Th2 biomarkers in the sample from the patient are measured using nucleic acids or proteins. In some embodiments, the Th2 biomarkers in the sample from the patient are determined using nucleic acid expression levels. In some embodiments, the nucleic acid expression levels are determined by RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technology, ISH, or a combination thereof. In some embodiments, the nucleic acid expression levels are mRNA expression levels. In some embodiments, the mRNA expression levels are determined by RNA-seq.

[0081] In some embodiments of any of the eighth, ninth, tenth, eleventh, and twelfth aspects, the sample is a tissue sample, a tumor sample, a whole blood sample, a plasma sample, a serum sample, or a combination thereof. In some embodiments, the sample is a tissue sample. In some embodiments, the tissue sample is a tumor tissue sample. In some embodiments, the tumor tissue sample comprises tumor cells, tumor-infiltrating immune cells, stromal cells, NAT cells, or a combination thereof. In some embodiments, the tumor tissue sample is a biopsy. In some embodiments, the sample is an archived sample, a fresh sample, or a frozen sample.

[0082] In some embodiments of any of the eighth, ninth, tenth, eleventh, and twelfth aspects, the lymphoma is an indolent lymphoma.

[0083] In some embodiments of any of the eighth, ninth, tenth, eleventh, and twelfth aspects, the lymphoma is a B-cell lymphoma. In some embodiments, the B-cell lymphoma is a germinal center-derived B-cell lymphoma. In some embodiments, the B-cell lymphoma is NHL.

[0084] In some embodiments of any of the eighth, ninth, tenth, eleventh, and twelfth aspects, the lymphoma is DLBCL, FL, CLL, or MZL. In some embodiments, the lymphoma is DLBCL. In some embodiments, the DLBCL is the GCB cell of origin subgroup or the ABC cell of origin subgroup of DLBCL.

[0085] In some embodiments of any of the eighth, ninth, tenth, eleventh, and twelfth aspects, the lymphoma is a CD20-positive lymphoma.

[0086] In some embodiments of any of the eighth, ninth, tenth, eleventh, and twelfth aspects, the anti-CD20 antibody is a type I anti-CD20 antibody or a type II anti-CD20 antibody. In some embodiments, the anti-CD20 antibody is a type II anti-CD20 antibody. In some embodiments, the type II anti-CD20 antibody comprises the following complementarity determining regions (CDRs): (a) CDR-H1 having the amino acid sequence of SEQ ID NO: 1; (b) CDR-H2 having the amino acid sequence of SEQ ID NO: 2; (c) CDR-H3 having the amino acid sequence of SEQ ID NO: 3; (d) CDR-L1 having the amino acid sequence of SEQ ID NO: 4; (e) CDR-L2 having the amino acid sequence of SEQ ID NO: 5; and (f) CDR-L3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, the type II anti-CD20 antibody comprises the following complementarity determining regions (CDRs): (a) CDR-H1 having the amino acid sequence of SEQ ID NO:27; (b) CDR-H2 having the amino acid sequence of SEQ ID NO:28; (c) CDR-H3 having the amino acid sequence of SEQ ID NO:3; (d) CDR-L1 having the amino acid sequence of SEQ ID NO:4; (e) CDR-L2 having the amino acid sequence of SEQ ID NO:5; and (f) CDR-L3 having the amino acid sequence of SEQ ID NO:6. In some embodiments, the type II anti-CD20 antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:8. In some embodiments, the type II anti-CD20 antibody is obinutuzumab. In some embodiments, the anti-CD20 antibody is a type I anti-CD20 antibody. In certain embodiments, the anti-CD20 antibody comprises the following CDRs: (a) CDR-H1 having the amino acid sequence of SEQ ID NO: 11; (b) CDR-H2 having the amino acid sequence of SEQ ID NO: 12; (c) CDR-H3 having the amino acid sequence of SEQ ID NO: 13; (d) CDR-L1 having the amino acid sequence of SEQ ID NO: 14; (e) CDR-L2 having the amino acid sequence of SEQ ID NO: 15; and (f) CDR-L3 having the amino acid sequence of SEQ ID NO: 16. In some embodiments, the type I anti-CD20 antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 25 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the anti-CD20 antibody is rituximab.

[0087] In some embodiments of any of the tenth, eleventh, and twelfth aspects, the method further comprises administering to the patient an effective amount of an additional therapeutic agent. [Brief explanation of the drawings]

[0088] [Figure 1] Figure 1 is a schematic diagram showing the study design of a global, open-label, randomized, phase III clinical trial (NCT01287741) in patients with first-line (1L) DLBCL. G-CHOP is obinutuzumab (G) plus cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), and R-CHOP is rituximab (R) plus CHOP. [Figure 2] Figure 2 is a pair of graphs showing improved survival in dichotomized patient subgroups of marker gene-derived macrophage biomarkers. Investigator progression-free survival (PFS) (left) and overall survival (OS) (right) Kaplan-Meier curves are shown for the xCell-based estimated high / low M1 macrophage signature samples. Black indicates the high M1 group, gray indicates low. [Figure 3] Figure 3 is a table showing hazard ratios for marker gene-derived (xCell) and deconvolution-derived (Quantiseq) biomarkers in dichotomized patient subgroups. Biomarker cell subpopulations include macrophages, M1 macrophages, M2 macrophages, mast cells, and memory B cells. Univariate and multivariate forest plots are shown for macrophage-high / low groups, including hazard ratios and 95% confidence intervals. The left column shows relevant statistics for investigator-assessed PFS. The right column shows relevant statistics for OS. [Figure 4] Figure 4 is a graph showing the association between lymphoma marker gene scores and PFS in xCell. [Figure 5]Figure 5 shows the M1 macrophage composition present in patient samples as estimated by quanTIseq. Each bar represents the number of patient samples (y-axis) with an M1 macrophage composition within a range (x-axis). The dashed line indicates the median M1 macrophage composition (0.03346). [Figure 6] Figure 6 is a heatmap showing immune deconvolution scores (scaled by columns) generated by quanTIseq on 604 de novo DLBCL biopsies from patients treated with R / G-CHOP. [Figure 7] Figure 7 is a pair of graphs showing improved survival in dichotomized patient subgroups of deconvolution-derived macrophage biomarkers. Investigator PFS (left) and OS (right) Kaplan-Meier curves are shown for the putative high / low M1 macrophage signature samples utilizing quanTIseq. Black indicates the high M1 group, gray indicates low. [Figure 8] Figure 8 is a graph showing the association between lymphoma infiltration deconvolution score in quanTIseq and PFS. [Figure 9] FIG. 9 is a graph showing PFS for patients with high and low M1 macrophage enrichment based on quanTIseq. DETAILED DESCRIPTION OF THE INVENTION

[0089] Detailed Description of the Invention I. General techniques The techniques and procedures described or referenced herein are generally well understood and may be found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (F.M.A.usubel, et al. eds., (2003)); Methods in Enzymology series (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(JECellis,ed.,1998)Academic Press;Animal Cell Culture(RIFreshney),ed.,1987);Introduction to Cell and Tissue Culture(JPMather and PERoberts,1998)Plenum Press;Cell and Tissue Culture:Laboratory Procedures(A.Doyle,JBGriffiths,and DGNewell,eds.,1993-8)J.Wiley and Sons;Handbook of Experimental Immunology(DMWeir and CCBlackwell, eds.);Gene Transfer Vectors for Mammalian Cells(JMMiller and MPCalos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (JEColigan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D.Lane(Cold Spring Harbor Laboratory Press,1999);The Antibodies(M.Zanetti and These methods are commonly employed by those skilled in the art using conventional methodologies such as the widely used methodologies described in JD Capra, eds., Harwood Academic Publishers, 1995; and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., J.B. Lippincott Company, 1993).

[0090] II. Definition It is understood that aspects and embodiments of the invention described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments. As used herein, the singular forms "a," "an," and "the" include plurals unless otherwise indicated.

[0091] As used herein, the term "about" refers to a normal error range for the respective value, as would be readily understood by one of ordinary skill in the art. Reference herein to a value or parameter preceded by "about" includes (and describes) embodiments directed to the value or parameter itself. For example, a statement referring to "about X" includes a statement of "X." In some embodiments, "about" can refer to ±15%, ±10%, ±5%, or ±1%, as would be understood by one of ordinary skill in the art.

[0092] As used interchangeably herein, the "amount," "level," or "expression level" of a biomarker refers to a detectable level in a biological sample (e.g., a blood sample or biopsy). "Expression" generally refers to the process by which information (e.g., genetic coding information and / or epigenetic information) is converted into structures present and functional in a cell. Thus, as used herein, "expression" can refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and / or polypeptide modification (e.g., post-translational modification of a polypeptide). Fragments of a transcribed polynucleotide, a translated polypeptide, or a polynucleotide and / or polypeptide modification (e.g., post-translational modification of a polypeptide) should also be considered expressed, regardless of whether they are derived from a transcript generated by alternative splicing or a degraded transcript, or from post-translational processing of a polypeptide, for example, by proteolysis. "Expressed genes" include those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, and also those that are transcribed into RNA but not translated into a polypeptide (e.g., transfer and ribosomal RNA). Expression levels can be measured by methods known to those skilled in the art and also disclosed herein. The expression level or amount of a biomarker can be used to identify / characterize subjects with lymphoma (e.g., B-cell lymphoma, e.g., non-Hodgkin's lymphoma, e.g., diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)) who are likely to respond to or benefit from a particular therapy (e.g., a therapy comprising one or more dosing cycles of an anti-CD20 antibody (e.g., obinutuzumab or rituximab)).

[0093] The presence and / or expression levels / amounts of the various biomarkers described herein in a sample (e.g., a blood sample or biopsy) can be analyzed by several methodologies, many of which are known in the art and understood by those of skill in the art, including immunohistochemistry ("IHC"), Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, flow cytometry, fluorescence activated cell sorting ("FACS"), spatial transcriptomics, spatial proteomics, MassARRAY, proteomics, quantitative blood-based assays (e.g., serum ELISA), biochemical enzyme activity assays, in situ hybridization (ISH), fluorescence in situ hybridization (FACS), and the like. These include, but are not limited to, any one of a wide variety of assays that can be performed by in situ hybridization (FISH), Southern analysis, Northern analysis, whole genome sequencing, massively parallel DNA sequencing (e.g., next-generation sequencing), NANOSTRING®, polymerase chain reaction (PCR) including quantitative real-time PCR (qRT-PCR) and other amplification-based detection methods such as branched DNA, SISBA, TMA, RNA-Seq, microarray analysis, gene expression profiling, and / or serial analysis of gene expression ("SAGE"), as well as protein, gene, and / or tissue array analysis. Exemplary protocols for assessing the status of genes and gene products can be found, for example, in Parts 2 (Northern blotting), 4 (Southern blotting), 15 (immunoblotting), and 18 (PCR analysis) of Ausubel et al., eds., 1995, Current Protocols in Molecular Biology. Multiplexed immunoassays, such as those available from Rules Based Medicine or Meso Scale Discovery ("MSD"), may also be used.

[0094] The term "antagonist" is used in the broadest sense and includes any molecule that partially or completely blocks, inhibits, or neutralizes the biological activity of a native polypeptide disclosed herein. Suitable antagonist molecules specifically include antagonist antibodies or antibody fragments (e.g., antigen-binding fragments), fragments or amino acid sequence variants of native polypeptides, peptides, antisense oligonucleotides, small organic molecules, and the like. Methods for identifying antagonists of a polypeptide may include contacting the polypeptide with a candidate antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the polypeptide.

[0095] The meanings of "type I" and "type II" anti-CD20 antibodies are well known in the art. Generally, anti-CD20 monoclonal antibodies are classified into two different categories based on their mechanism of action in eradicating lymphoma cells. "Type I" anti-CD20 antibodies primarily utilize complement to kill target cells, while "type II" anti-CD20 antibodies act primarily through a different mechanism, apoptosis. Rituximab (e.g., U.S. Pat. No. 5,736,137, incorporated herein by reference in its entirety) and 1F5 are examples of type I anti-CD20 antibodies, while obinutuzumab (e.g., see WO 2005 / 044859 and U.S. Patent Application Publication No. 2005 / 0123546, incorporated herein by reference in their entirety) and B1 are examples of type II antibodies. See, for example, Cragg (Blood 103(7), 2004, 2738-2743); Teeling (Blood 104(6), 2004, 1793-1800); EP 2380910, and WO 2005 / 044859, the entire contents of which are incorporated herein by reference.

[0096] As used herein, "administering" refers to a method of providing a subject with a dose of a compound (e.g., an anti-CD20 antibody (e.g., obinutuzumab or rituximab)) or composition (e.g., a pharmaceutical composition, e.g., a pharmaceutical composition comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab)). The compounds and / or compositions used in the methods described herein can be administered, for example, intravenously (e.g., by intravenous infusion), subcutaneously, intramuscularly, intradermally, transcutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intraperitoneally, subconjunctivally, intravesicularly, transmucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation, injection, infusion, continuous infusion, localized perfusion directly into target cells, by catheter, lavage, cream, or in a lipid composition. The method of administration can vary depending on various factors (e.g., the compound or composition being administered and the severity of the condition, disease, or disorder being treated).

[0097] A "fixed" or "flat" dose of a therapeutic agent (e.g., an anti-CD20 antibody (e.g., obinutuzumab or rituximab)) herein refers to a dose administered to a patient without regard to the patient's weight or body surface area (BSA). Thus, this fixed or constant dose may be expressed as a mg / kg dose or a mg / m 2 It is not provided as a dose, but rather as an absolute amount (eg, mg) of therapeutic agent.

[0098] As used herein, the term "treatment" or "treating" refers to a clinical intervention designed to alter the natural history of the individual or cell being treated during the course of a clinical lesion. Desirable effects of treatment include slowing or reducing the rate of disease progression, ameliorating or alleviating the disease state, and ameliorating or improving prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with cancer (e.g., lymphoma, e.g., B-cell lymphoma, e.g., non-Hodgkin's lymphoma, e.g., diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)) are reduced or eliminated, including, but not limited to, reducing the proliferation (or destruction) of cancerous cells, reducing symptoms resulting from the disease, improving the quality of life of those suffering from the disease, reducing the dose of other medications required to treat the disease, slowing disease progression, and / or extending the individual's survival.

[0099] As used herein, "in combination with" or "in conjunction with" refers to the administration of one therapeutic modality with another. Thus, "in combination with" or "in conjunction with" refers to the administration of one therapeutic modality to an individual before, during, or after the administration of another therapeutic modality. Co-administration can be simultaneous or sequential, and preferably there is a period of time during which both or all active agents simultaneously exert their biological activities. The antibody and the additional agent(s) are co-administered simultaneously or sequentially (e.g., intravenously (iv)), e.g., by continuous infusion. When both therapeutic agents are co-administered sequentially, the doses can be administered on the same day in two separate administrations, or one of the agents can be administered on day 1 and the second can be co-administered on days 2-7, preferably days 2-4. Thus, in one embodiment, the term "sequentially" refers to within (about) 7 days after administration of the first component, preferably within (about) 4 days after administration of the first component, and the term "concurrently" refers to the same time. The term "co-administration" with respect to maintenance doses of an antibody and / or additional agent means that the maintenance doses can be co-administered simultaneously when a treatment cycle is appropriate for both drugs, e.g., weekly, or when the additional agent is administered, e.g., every 1 to 3 days, and the antibody is administered weekly. Alternatively, the maintenance doses are co-administered consecutively, either within one day or within several days. In preferred embodiments, the anti-CD20 antibodies described herein (e.g., obinutuzumab, rituximab, or functional equivalents thereof) can be administered in combination with chemotherapy, such as CHOP chemotherapy or variants of CHOP chemotherapy (e.g., CHOEP chemotherapy, CHOP-14 chemotherapy, or ACVBP chemotherapy (see, e.g., the Examples and EP 2380910 B1, WO 2005 / 044859, and Scott, 2014 and 2015, loc.cit.)). Thus, in a preferred embodiment, the co-administered additional chemotherapeutic agent is selected from the group consisting of cyclophosphamide, hydroxydaunorubicin, carcinomatosis, prednisone or prednisolone, and optionally etoposide.

[0100] A "disorder" or "disease" is any condition that would benefit from treatment, including, but not limited to, disorders associated with some degree of abnormal cell proliferation, eg, cancer, eg, lymphoma.

[0101] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, lymphoma, carcinoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, multiple myeloma and B-cell lymphomas (low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaving cell NHL; bulky mass disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myogenic leukemia (AML); hairy cell leukemia; chronic myeloblastic leukemia (CML); post-transplant lymphoproliferative disorder (PTLD); and myelodysplastic syndromes (MDS), and associated metastases. In some embodiments, the cancer is a lymphoma (e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)). In some embodiments, the lymphoma is a low-grade lymphoma. In some embodiments, the lymphoma is a B-cell lymphoma. In some embodiments, the B-cell lymphoma is a germinal center-derived B-cell lymphoma. In some embodiments, the B-cell lymphoma is an NHL. In some embodiments, the lymphoma is a diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), or marginal zone lymphoma (MZL). In some embodiments, the lymphoma is DLBCL. In some embodiments, the DLBCL is of the germinal center B-cell-like (GCB) cell of origin subgroup or the activated B-cell-like (ABC) cell of origin subgroup of DLBCL. In some embodiments, the lymphoma is a CD20-positive lymphoma. In some embodiments, the lymphoma is a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma).

[0102] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein.

[0103] As used herein, "metastasis" means the spread of a cancer (e.g., a lymphoma, e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)) from its primary site to another location in the body. Cancer cells may break off from the primary tumor, infiltrate lymphatic and blood vessels, circulate through the bloodstream, and grow at a distant focus within normal tissue elsewhere in the body (metastasize). Metastasis can be local or distant. Metastasis is a sequential process in which tumor cells break off from the primary tumor, travel through the bloodstream, and arrest at a distant site. At the new site, the cells establish a blood supply and may grow to form a life-threatening mass. Both stimulatory and inhibitory molecular pathways within tumor cells control this behavior, and interactions between tumor cells and host cells at distant sites are also important.

[0104] The term "anti-cancer therapy" refers to a therapy useful for treating cancer (e.g., a lymphoma, e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)). Examples of anti-cancer therapeutic agents include, but are not limited to, for example, immunomodulatory agents or agents that increase or activate one or more immune co-stimulatory receptors, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, and other agents for treating cancer. Combinations of these are also encompassed by the present invention. In some embodiments, the anticancer therapy includes cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) or variations thereof (e.g., CHOEP chemotherapy, CHOP-14 chemotherapy, or ACVBP chemotherapy (see, e.g., the Examples and EP 2380910 B1, WO 2005 / 044859, and Scott, 2014 and 2015, loc.cit.)).

[0105] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction. Cytotoxic agents include radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212, and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof, e.g., nucleases; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin (including fragments and / or variants thereof); and various anti-tumor or anti-cancer agents disclosed below.

[0106] A "chemotherapeutic agent" includes chemical compounds useful in the treatment of cancer. Examples of chemotherapeutic agents include erlotinib (TARCEVA®, Genentech / OSI Pharm), bortezomib (VELCADE®, Millennium), and erythropoietinib (ERT). Pharm.), disulfiram, epigallocatechin gallate, salinosporamide A, carfilzomib, 17-AAG (geldanamycin), Radicol, lactate dehydrogenase A (LDH-A), fulvestrant (FASLODEX®, AstraZeneca), sunitib (SUTENT®, Pfizer / Sugen)), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), finasteride (VATALANIB®, Novartis), oxaliplatin (ELOXATIN®, Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, GlaxoSmithKline), Kline), lonafamib (SCH 66336), sorafenib (NEXAVAR®, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), AG1478, thiotepa, and CYTOXAN® cyclophosphamide; alkylating agents such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carbocuron, metoledopa, and uredopa; ethylenimines such as altretamine, triethylenemelamine, triethylenephosphamide, triethylenethiophosphamide, and trimethylmelamine; and methylmelamines; acetogenins (especially bullatacin and bullatacinone); camptothecins (topotecan and irinotecan); bryostatin; kallystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); corticosteroids (including prednisone and prednisolone); cyproterone acetate; 5α-reductase inhibitors, including finasteride and dutasteride;vorinostat, romidepsin, panobinostat, valproic acid, mocetinostat dolastatins; aldesleukin, talc duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); eletarobin; pancratistatin; sarcodictine; spongistatin, chlorambucil, chromafazine, chlorophosphamide, estramustine, ifosfamide, mechlorestamine, mechlorestamine oxide hydrochloride, melphalan, nobembine, phenesterine, prednimustine, trophosfamide, nitrogen mustards such as uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as endene antibiotics (e.g., calicheamicins, particularly calicheamicin γ1I and calicheamicin ω1I (Angew Chem. Intl. Ed. Engl. 1994 33:183-186); dynemicins, including dynemicin A; bisphosphonates such as clodronate; espermycin; as well as neocarzinostatin chromophores and related enediyne antibiotic chromophores), aclacinomycins, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, Adriamycin® (doxorubicin), morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), mitomycins such as epirubicin, esorubicin, idarubicin, marcelomycin, and mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; metabolic antagonists such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine;Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiosteine, and testolactone; antiadrenal agents such as aminoglutethimide, mitotane, and trilosteine; folic acid supplements such as floric acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; and bestravsil ;Bisantrene;Edatraxate;Defofamine;Demecolcine;Diaziquione;Elfomitin;Elliptinium acetate;Epothilone;Etoglucide;Gallium nitrate;Hydroxyurea;Lentinan;Lonidynin;Maytansinoids such as maytansine and ansamitocin;Mitoguazone;Mitoxantrone;Mopidanmol;Nitraerin;Pentostatin;Fenameth;Pirarubicin;Losoxantrone;Podophyllic acid;2-Ethylhydrazide;Procarbazine;PSK(R) polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; schizofuran; spirogermanium; tenuazonic acid; triaziquione; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veraculin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as taxol (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE® (cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® (paclitaxel; Sanofi-Aventis): chlorambucil, GEMZAR® (gemcitabine), 6-thioguanine, mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16);Ifosfamide; mitoxantrone; vincristine; NAVELBINE® (vinorelbine); nobandrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®); ibandronate; CPT-11; the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid, and pharmaceutically acceptable salts, acids, and derivatives of any of the above;

[0107] Chemotherapeutic agents also include: (i) antihormonal agents that act to regulate or inhibit hormone action on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), for example, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene, droxifene, iodoxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifine citrate); ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, for example, antiandrogens such as 4(5)-imidazole, aminoglutethimide, MEGASE® (megstrol acetate), AROMASIN® (exemestane; Pfizer), formestany, fadrozole, RIVISOR® (borzole), FEMARA® (letrozole; Novartis), and ARIMIDEX® (anastrozole; AstraZeneca); i) antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; buserelin, triplerin, medroxyprogesterone acetate, diethylstilbestrol, premarin, fluoxymesterone, all-trans retinoic acid, fenretinide, as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors (e.g., anaplastic lymphoma kinase (Alk) inhibitors, such as AF-802 (also known as CH-5424802 or alectinib) (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation, such as PKC-α, Ralf, and H-Ras; (vii) ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors; (viii) gene therapy vaccines, such as ALLOVECTIN®, LEUVECTIN®, and VAXID®; PROLEUKIN®, rIL-2;Topoisomerase 1 inhibitors such as LURTOTECAN®; ABARELIX® rmRH; and (ix) pharmaceutically acceptable salts, acids, and derivatives of any of the above.

[0108] Chemotherapeutic agents also include antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech), cetuximab (ERBITUX®, Imclone), panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech / Biogen Idec), pertuzumab (OMNITARG®, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), and the antibody-drug conjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth). Additional humanized monoclonal antibodies with therapeutic potential as agents in combination with the described compounds include apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, celizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, nat ... Talizumab, nimotuzumab, norobizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pexelizumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, reslizumab, rovelizumab, lupizumab, sibrotuzumab, siplizumab, sontuzumab, tacatatuzumab tetraxetan, tadoxizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tucotuzumab selmoreukin, tuxituuzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab, and interleukin-12 and anti-interleukin-12 (ABT-874 / J695, Wyeth Research and Abbott Laboratories), a human-sequence only, full-length IgG1λ antibody genetically engineered to recognize the p40 protein.

[0109] Chemotherapeutic agents also include "EGFR inhibitors," alternatively referred to as "EGFR antagonists," which refer to compounds that bind to or otherwise directly interact with EGFR and block or reduce its signaling activity. Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies that bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB 8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see U.S. Pat. No. 4,943,533, Mendelsohn et al.) and variants thereof, such as chimerized 225 (C225 or cetuximab; ERBUTIX®) and reshaped human 225 (H225) (see WO 96 / 40210, Imclone Systems, Inc.). Inc.); the fully human EGFR-targeting antibody IMC-11F8 (Imclone); antibodies that bind type II mutant EGFR (U.S. Pat. No. 5,212,290); humanized and chimeric antibodies that bind EGFR, such as those described in U.S. Pat. No. 5,891,996; and human antibodies that bind EGFR, such as ABX-EGF or Panitumumab (WO 98 / 50433, Abgenix / Amgen); EMD 55900 (Stragliotto et al. Eur. J. Cancer 32A:636-640 (1996); EMD7200 (matuzumab), a humanized EGFR antibody against EGFR that competes with both EGF and TGF-α for EGFR binding (EMD / Merck); the human EGFR antibody, HuMax-EGFR (GenMab); the fully human antibodies known as E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3, and E7.6.3 and described in U.S. Pat. No. 6,235,883; MDX-447 (Medarex Inc); and mAb 806 or humanized mAb 806 (Johns et al., J. Biol. Chem. 279(29):30375-30384 (2004)).Anti-EGFR antibodies can be conjugated to cytotoxic agents to form immunoconjugates (see, e.g., European Patent Application Publication No. 659,439 A2, Merck Patent GmbH). EGFR antagonists include those described in U.S. Patent Nos. 5,616,582, 5,457,105, 5,475,001, 5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726, 6,713,484, 5,770,599, 6,140,332, and 5,866. ,572, 6,399,602, 6,344,459, 6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008, and 5,747,498, and the following PCT publications: WO 98 / 14451, WO 98 / 50038, WO 99 / 09016, and WO 99 / 24037.Specific small molecule EGFR antagonists include OSI-774 (CP-358774, erlotinib, TARCEVA®, Genentech / OSI Pharmaceuticals), PD183805 (CI1033, 2-propenamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-, dihydrochloride, Pfizer Inc.), ZD1839, gefitinib (IRESSA®) 4-(3'-chloro-4'-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline, AstraZeneca), ZM105180 ((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca), BIBX-1382 (N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine, Boehringer Ingelheim), Ingelheim), PKI-166 ((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol), (R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine), CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide), EKB-569 (N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano- 7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-butynamide) (Wyeth), AG1478 (Pfizer), AG1571 (SU5271, Pfizer), and dual EGFR / HER2 tyrosine kinase inhibitors such as lapatinib (TYKERB®, GSK572016 or N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6[5[[[2methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-quinazolinamine).

[0110] Chemotherapeutic agents include "tyrosine kinase inhibitors," such as the EGFR-targeted drugs described in the preceding paragraph; inhibitors of insulin receptor tyrosine kinase, such as anaplastic lymphoma kinase (Alk) inhibitors, such as AF-802 (also known as CH-5424802 or alectinib), ASP3026, X396, LDK378, AP26113, crizotinib (XALKORI®), and ceritinib (ZYKADIA®); small molecule HER2 tyrosine kinase inhibitors, such as TAK165 available from Takeda; oral selectivity inhibitors of ErbB2 receptor tyrosine kinase, such as HER2 inhibitors ... dual HER inhibitors, such as EKB-569 (available from Wyeth), which preferentially binds to EGFR but inhibits both HER2 and EGFR overexpressing cells; lapatinib (GSK572016, available from Glaxo-SmithKline); oral HER2 and EGFR tyrosine kinase inhibitors; PKI-166 (available from Novartis); pan-HER inhibitors, such as canertinib (CI-1033, Pharmacia); Raf-1 inhibitors, such as ISIS, which inhibits Raf-1 signaling antisense drug ISIS-5132 available from Pharmaceuticals; non-HER-targeted TK inhibitors such as imatinib mesylate (GLEEVEC®, available from GlaxoSmithKline); multi-targeted tyrosine kinase inhibitors such as sunitinib (SUTENT®, available from Pfizer); VEGF receptor tyrosine kinase inhibitors such as vatalanib (PTK787 / ZK222584, available from Novartis / Schering AG); MAPK extracellular regulated kinase I inhibitor CI-1040 (available from Pharmacia); quinazolines such as PD 153035, 4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines such as CGP 59326, CGP 60261, and CGP 62706;Pyrazolopyrimidine, 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidine;Curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide);Tyrphostins containing a nitrothiophene moiety; PD-0183805 (Warner-Lambert); antisense molecules (e.g., those that bind to HER-encoding nucleic acids); quinoxalines (U.S. Pat. No. 5,804,396); tryphostin (U.S. Pat. No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis / Schering AG); pan-HER inhibitors, e.g., CI-1033 (Pfizer); Affinitac (ISIS 3521; Isis / Lilly); imatinib mesylate (GLEEVEC®); PKI 166 (Novartis); GW2016 (Glaxo) SmithKline; CI-1033 (Pfizer); EKB-569 (Wyeth); semaxinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); INC-1C11 (Imclone), rapamycin (sirolimus, RAPAMUNE®); or the following patent publications: U.S. Pat. No. 5,804,396, WO 1999 / 09016 (American Cyanamid), WO 1998 / 43960 (American Cyanamid), WO 1997 / 38983 (Warner Lambert), WO 1999 / 06378 (Warner Lambert), WO 1999 / 06396 (Warner Lambert), 1996 / 30347 (Pfizer, Inc.), 1996 / 33978 (Zeneca), 1996 / 3397 (Zeneca), and 1996 / 33980 (Zeneca).

[0111] Chemotherapeutic agents also include dexamethasone, interferon, colchicine, metoprine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, live BCG, bevacuzimab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, erotinib, filgrastim, histrelin acetate, ibritumomab, interferon alfa-2a, and interferon alfa -2b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, oprelvekin, palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium, plicamycin, porfimer sodium, quinacrine, rasburicase, sargramostim, temozolomide, VM-26, 6-TG, toremifene, tretinoin, ATRA, valrubicin, zoledronate, and zoledronic acid, as well as pharmaceutically acceptable salts thereof.

[0112] Chemotherapeutic agents include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, and hydrocortisone-17. -butyrate, hydrocortisone-17-valerate, aclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasone-17-propionate, fluocortolone caproate, fluocortolone pivalate, and fluprednidene acetate; phenylalanine-glutamine-glycine (FEG) and its D-form (feG) (IMULAN) Immunoselective anti-inflammatory peptides (ImSAIDs) such as those from BioTherapeutics, LLC; antirheumatic drugs such as azathioprine, cyclosporine (cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine, leflunomide, minocycline, and sulfasalazine; etanercept (Enbrel), infliximab (Remicade), adalimumab (Humira), cetolithumab pegol (Cimzia), and golimumab (Sympo); tumor necrosis factor alpha (TNFα) blockers such as lebrikizumab (Lyskin), interleukin 1 (IL-1) blockers such as anakinra (Kineret), T-cell costimulation blockers such as abatacept (Orencia), interleukin 6 (IL-6) blockers such as tocilizumab (ACTEMERA®); interleukin 13 (IL-13) blockers such as lebrikizumab; interferon alpha (IFN) blockers such as rontalizumab; rhuMAb beta7 integrin blockers such as Beta7; IgE pathway blockers such as anti-M1 prime; secreted homotrimeric LTa3 and membrane-bound heterotrimeric LTa1 / β2 blockers such as anti-lymphotoxin alpha (LTa); radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and radioactive isotopes of Lu);Various investigational drugs such as thioplatin, PS-341, phenylbutyrate, ET-18-OCH3, and farnesyltransferase inhibitors (L-739749, L-744832); polyphenols such as quercetin, resveratrol, piceatannol, epigallocatechin gallate, theaflavins, flavanols, procyanidins, betulinic acid and its derivatives; autophagy inhibitors such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol ; colchicine; betulinic acid; acetylcamptothecin, scopolectin, and 9-aminocamptothecin; podophyllotoxin; tegafur (UFTORAL®); bexarotene (TARGRETIN®); clodronate (e.g., BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDI®), A (registered trademark), bisphosphonates such as tiludronate (SKELID (registered trademark), or risedronate (ACTONEL (registered trademark); and epidermal growth factor receptor (EGF-R); vaccines such as the THERATOPE (registered trademark) vaccine; B inhibitors such as perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); CCI-779; tipifarnib (R11577); orafenib, ABT510; oblimersen sodium (GENASENSE (registered trademark)); Cl-2 inhibitors; pixantrone; farnesyltransferase inhibitors such as lonafarnib (SCH6636, SARASARTM); and pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of CHOP, which is an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone (prednisone), and FOLFOX, which is an abbreviation for the treatment regimen using oxaliplatin in combination with 5-FU and leucovorin (ELOXATIN™);

[0113] Chemotherapeutic agents also include nonsteroidal anti-inflammatory drugs (NSAIDs) with analgesic, antipyretic, and anti-inflammatory effects. NSAIDs include nonselective inhibitors of the enzyme cyclooxygenase. Specific examples of NSAIDs include propionic acid derivatives such as aspirin, ibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin, and naproxen; acetic acid derivatives such as indomethacin, sulindac, etodolac, and diclofenac; enolic acid derivatives such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, and isoxicam; fenamic acid derivatives such as mefenamic acid, meclofenamic acid, flufenamic acid, and tolfenamic acid; and COX-2 inhibitors such as celecoxib, etoricoxib, lumiracoxib, parecoxib, rofecoxib, and valdecoxib. NSAIDs may be indicated for the symptomatic relief of conditions such as rheumatoid arthritis, osteoarthritis, inflammatory arthropathy, ankylosing spondylosis, psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhea, metastatic bone pain, headache and migraine, post-operative pain, mild to moderate pain due to inflammation and tissue injury, fever, intestinal obstruction, and renal colic.

[0114] An "effective amount" of a compound, e.g., an anti-CD20 antibody (e.g., obinutuzumab or rituximab) or composition thereof (e.g., pharmaceutical composition) is at least the minimum amount required to achieve a desired therapeutic result, e.g., a measurable increase in overall survival or progression-free survival of a particular disease or disorder (e.g., a lymphoma, e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)). An effective amount herein may vary depending on factors such as the patient's disease state, age, sex, and weight, as well as the ability of the antibody to elicit a desired response in the subject. An effective amount is also one in which any toxic or adverse effects of the treatment are outweighed by the therapeutically beneficial effects. Beneficial or desired results for prophylactic use include results such as elimination or reduction of the risk of, reduction in severity of, or delay in the onset of disease, including biochemical, histological and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes manifest during the development of the disease. For therapeutic use, beneficial or desired results include clinical outcomes such as a reduction in one or more symptoms attributable to the disease (EORT CQLQ-C30, e.g., fatigue, nausea, vomiting, pain, dyspnea, insomnia, loss of appetite, constipation, diarrhea, or general level of somatic, emotional, cognitive, or social functioning), an increase from baseline in the Functional Assessment of Cancer Therapy Lymphoma (FACT-Lym) subscale score, a reduction in the dose of other medications needed to treat the disease, enhancing the effect of another medication by targeting, etc., a delay in disease progression (e.g., progression-free survival), a delay in overt clinical progression (e.g., progression of cancer-related pain, worsening of Eastern Cooperative Oncology Group (ECOG) performance status (PS) (e.g., how the disease affects a patient's ability to do daily activities), and / or initiation of subsequent systemic anti-cancer therapy, and / or an increase in survival time.In the case of cancer or tumors, an effective amount of a drug may have the effect of reducing the number of cancer cells, reducing tumor size, inhibiting (i.e., slowing or preferably stopping to some extent) the infiltration of cancer cells into peripheral organs, inhibiting (i.e., slowing or preferably stopping to some extent) tumor metastasis, inhibiting tumor growth to some extent, and / or alleviating to some extent one or more symptoms associated with the disorder. An effective amount may be administered in one or more administrations. For purposes of this invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve prophylactic or therapeutic treatment. As understood in the clinical field, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of the administration of one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desired result can be achieved or is achieved.

[0115] "Immunogenicity" refers to the ability of a particular substance to induce an immune response. Tumors are immunogenic, and enhancing tumor immunogenicity helps the immune response eliminate tumor cells. Examples of enhancing tumor immunogenicity include, but are not limited to, treatment with anti-CD20 antibodies (e.g., obinutuzumab or rituximab).

[0116] "Individual response" or "response" can be assessed using any endpoint that indicates benefit to a subject, including, but not limited to, (1) inhibition to some extent (including slowing and complete halting) of disease progression (e.g., progression of a cancer, e.g., lymphoma); (2) reduction in tumor size; (3) suppression (i.e., reduction, slowing, or complete halt) of cancer cell infiltration into adjacent peripheral organs and / or tissues; (4) inhibition (i.e., reduction, slowing, or complete halt) of metastasis; (5) alleviation to some extent of one or more symptoms associated with a disease or disorder (e.g., lymphoma, e.g., B-cell lymphoma, e.g., non-Hodgkin's lymphoma, e.g., diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)); (6) increase or prolongation of survival, including overall survival and progression-free survival; and / or (9) reduction in mortality at a given time point after treatment.

[0117] A subject's "effective response" or subject "responsiveness" to treatment with a pharmaceutical agent, and similar terms, refers to a clinical or therapeutic benefit conferred on a subject at risk for or suffering from a disease or disorder, such as cancer. In one embodiment, such benefit includes any one or more of extending survival (including overall survival and progression-free survival), producing an objective response (including a complete or partial response), or ameliorating the signs or symptoms of cancer.

[0118] A subject who "does not respond effectively" to treatment is one who does not have any of the following: an extension of survival (including overall survival and progression-free survival), an objective response (including a complete or partial response), or an improvement in the signs or symptoms of cancer.

[0119] As used herein, the term "survival" refers to the patient being alive, and includes overall survival and progression-free survival.

[0120] As used herein, "overall survival" (OS) refers to the time from enrollment in a study to death from any cause. As used herein, "overall survival rate" refers to the proportion of subjects in a group who are alive at a specific time, for example, 6 months, 1 year, or 5 years from the time of diagnosis or treatment.

[0121] As used herein, "complete response" or "CR" refers to the disappearance of all evidence of disease.

[0122] As used herein, "partial response" or "PR" refers to a measurable reduction in symptoms, a diminution in the direct or indirect pathological consequences of lymphoma, a slowing of the rate of disease progression, an improvement or palliation of the disease state, or prevention of metastases without eliminating all evidence of the disease.

[0123] As used herein, "progression-free survival" (PFS) refers to the length of time during and after treatment that the disease being treated (e.g., a cancer, e.g., a lymphoma, e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)) does not worsen (e.g., lymphoma progression or death as a result of any cause). PFS can include the time a patient experiences a complete response or a partial response, and the time a patient experiences stable disease.

[0124] As used herein, "prolonging survival" refers to increasing overall survival or progression-free survival of a treated patient compared to untreated patients (e.g., compared to patients not treated with a pharmaceutical agent), or compared to patients who do not express a specified level of a biomarker, and / or compared to patients treated with an approved anti-tumor agent. Objective response refers to a measurable response, including a complete response (CR) or partial response (PR).

[0125] Those skilled in the art can readily determine whether a given clinical outcome is improved in accordance with the present invention (e.g., improved compared to treatment without an anti-CD20 antibody (e.g., obinutuzumab or rituximab)). For example, "improved" in this context means that the clinical outcome (resulting from treatment with an anti-CD20 antibody (e.g., obinutuzumab / obinutuzumab or a functional equivalent of rituximab), particularly in combination with chemotherapy, particularly in combination with CHOP chemotherapy) is at least 3% higher, at least 5% higher, at least 7% higher, at least 10% higher, at least 15% higher, at least 20% higher, at least 25% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 75% higher, at least 100% higher, or at least 120% higher compared to the clinical outcome obtained from the equivalent treatment without an anti-CD20 antibody (e.g., obinutuzumab or rituximab), particularly in combination with chemotherapy, particularly in combination with CHOP chemotherapy.

[0126] The time at which clinical outcomes / clinical endpoints are evaluated can be easily determined by those skilled in the art. In principle, it is determined when the difference in clinical outcomes / clinical endpoints between two treatments (e.g., obinutuzumab treatment vs. rituximab treatment) becomes apparent. This time can be, for example, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 30 months, at least 36 months, at least 42 months, or at least 48 months after the start of treatment.

[0127] As used herein, "delaying progression" of a disorder or disease means postponing, hindering, delaying, stabilizing, and / or postponing the onset of the disease or disorder (e.g., a cancer, e.g., a lymphoma, e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)). The delay can be of varying duration depending on the history of the disease and / or the subject being treated. As will be apparent to one of skill in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the subject does not develop the disease.

[0128] As used herein, the term "reducing or inhibiting cancer recurrence" means reducing or inhibiting the recurrence of a tumor or cancer, or the progression of a tumor or cancer.

[0129] "Reduce or inhibit" refers to the ability to result in an overall decrease of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more. Reduction or inhibition can refer to symptoms of the disorder being treated (e.g., a lymphoma, e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)), the presence or size of metastases, or the size of the primary tumor.

[0130] As used herein, "subject" or "individual" means a mammal, including, but not limited to, a human or non-human mammal, such as a cow, horse, dog, sheep, or cat. In some embodiments, the subject is a human. A patient, as used herein, is also a human.

[0131] The terms "detect" and "detection" are used herein in the broadest sense to include both qualitative and quantitative measurements of a target molecule. Detection includes simply identifying the presence of a target molecule in a sample, as well as determining whether the target molecule is present in a sample at detectable levels. Detection can be direct or indirect.

[0132] "Tumor-infiltrating immune cells," as used herein, refer to any immune cells present in a tumor or its tissue. Tumor-infiltrating immune cells include, but are not limited to, intratumoral immune cells, peritumoral immune cells, other tumor stromal cells (e.g., fibroblasts), or any combination thereof. Such tumor-infiltrating immune cells may be, for example, macrophages (e.g., M1 macrophages or M2 macrophages), monocytes, T lymphocytes (e.g., CD8+ T lymphocytes and / or CD4+ T lymphocytes), B lymphocytes, or other myeloid cells, including granulocytes (e.g., neutrophils, eosinophils, and basophils), dendritic cells (e.g., interdigitating dendritic cells), histiocytes, and natural killer cells.

[0133] As used herein, the term "biomarker" refers to an indicator, e.g., predictive, diagnostic, and / or prognostic, that can be detected in a sample (e.g., a tumor tissue sample (e.g., a lymphoma tumor tissue sample, e.g., a B-cell lymphoma tumor tissue sample, e.g., a non-Hodgkin's lymphoma tumor tissue sample, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) tumor tissue sample), a blood sample, or a biopsy). A biomarker can serve as an indicator of a particular subtype of a disease or disorder (e.g., diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma)) characterized by particular molecular, pathological, histological, and / or clinical features. In some aspects, a biomarker is a gene (e.g., any of the genes described herein). Biomarkers include, but are not limited to, polypeptide, polynucleotide (e.g., DNA, and / or RNA), alterations in polynucleotide copy number (e.g., DNA copy number), polypeptide and polynucleotide modifications (e.g., post-translational modifications), carbohydrate, and / or glycolipid-based molecular markers. In some embodiments, the biomarker is a gene expression value. In some embodiments, the biomarker is an M1 macrophage gene signature set score. In some embodiments, the biomarker is a cell (e.g., an immune cell, e.g., a macrophage, e.g., M1 macrophage or M2 macrophage). In some embodiments, the biomarker is macrophage abundance. In some embodiments, the biomarker is M1 macrophage abundance.

[0134] As used herein, the term "macrophage biomarker" refers to a biomarker that indicates the quantity, level, characteristic, or phenotype of macrophages within a sample (e.g., a tumor tissue sample (e.g., a lymphoma tumor tissue sample, e.g., a B-cell lymphoma tumor tissue sample, e.g., a non-Hodgkin's lymphoma tumor tissue sample, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) tumor tissue sample), a blood sample, or a biopsy). In some aspects, a macrophage biomarker is a gene (e.g., any of the genes described herein). In some aspects, a macrophage biomarker is a polypeptide, polynucleotide (e.g., DNA, and / or RNA), polynucleotide copy number alteration (e.g., DNA copy number), polypeptide and polynucleotide modification (e.g., post-translational modification), carbohydrate, and / or glycolipid-based molecular marker. In some embodiments, a macrophage biomarker is a gene expression value that can reflect one or more genes (e.g., one or more genes described herein). In some embodiments, the macrophage biomarker is an M1 macrophage gene signature set score. In some embodiments, the macrophage biomarker is a cell (e.g., an immune cell, e.g., a macrophage, e.g., M1 macrophages or M2 macrophages). In some embodiments, the macrophage biomarker is an amount of macrophages (e.g., an amount of M1 macrophages or an amount of M2 macrophages). In some embodiments, the macrophage biomarker is an amount of M1 macrophages.

[0135] As used herein, the term "Th2 biomarker" refers to a biomarker that indicates the amount, level, characteristic, or phenotype of CD4+ type 2 helper T cells in a sample (e.g., a tumor tissue sample (e.g., a lymphoma tumor tissue sample, e.g., a B-cell lymphoma tumor tissue sample, e.g., a non-Hodgkin's lymphoma tumor tissue sample, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) tumor tissue sample), a blood sample, or a biopsy). In some embodiments, a Th2 biomarker is a gene. In some embodiments, a Th2 biomarker is a polypeptide, polynucleotide (e.g., DNA, and / or RNA), polynucleotide copy number alteration (e.g., DNA copy number), polypeptide and polynucleotide modification (e.g., post-translational modification), carbohydrate, and / or glycolipid-based molecular marker. In some embodiments, a Th2 biomarker is a gene expression value that can reflect one or more genes. In some embodiments, a Th2 biomarker is a gene signature set score. In some embodiments, the Th2 biomarker is a cell (e.g., an immune cell, e.g., a T cell, e.g., a helper T cell, e.g., a Th2 cell). In some embodiments, the Th2 biomarker is the amount of T cells (e.g., the amount of Th2 cells). In some embodiments, the Th2 biomarker is the amount of Th2 cells.

[0136] A biomarker is "predictive" according to the present invention if it can be used to identify patients as defined herein (optionally in combination with one or more other biomarkers), e.g., patients who will respond to treatment with an anti-CD20 antibody (e.g., obinutuzumab or rituximab) (particularly in combination with chemotherapy, particularly in combination with CHOP chemotherapy). In some embodiments, a biomarker is predictive if the treatment effect differs between biomarker-defined patient subgroups. In this regard, it is preferred that the predictive biomarker is a biomarker defined elsewhere herein. A specific example of a predictive biomarker evaluated in the context of the present invention is the macrophage biomarker described herein.

[0137] The term "antibody" includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), and antibody fragments, including antigen-binding fragments such as Fab, F(ab'), and Fv. The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein.

[0138] The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). IgM antibodies consist of five basic heterotetrameric units plus an additional polypeptide called a J chain, which contains 10 antigen-binding sites, whereas IgA antibodies consist of two to five basic four-chain units, which can polymerize to form multivalent aggregates with the J chain. In the case of IgG, the four-chain unit is generally approximately 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, whereas the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced interchain disulfide bridges. Each H chain contains a variable domain (V) at its N-terminus. H ), followed by three constant domains (C H ), and four C for μ and ε isotypes H Each L chain has a variable domain (V L ) followed by a constant domain at the opposite end. L is V H It is aligned with C L is the first constant domain of the heavy chain (C H 1). Particular amino acid residues are thought to form an interface between the light-chain variable domain and the heavy-chain variable domain. V H and V Land pair together to form a single antigen-binding site. For the structure and properties of various classes of antibodies, see, for example, page 71 and chapter 6 of Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, CT, 1994. Light chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domain. Depending on the amino acid sequence of the constant domain of their heavy chain (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each with a heavy chain designated α, δ, ε, γ, and μ. The gamma and alpha classes are further divided into subclasses based on relatively minor differences in CH sequence and function; for example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0139] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence and / or forms structurally defined loops. Generally, antibodies contain six HVRs: three in the VH (H1, H2, and H3) and three in the VL (L1, L2, and L3). In natural antibodies, H3 and L3 exhibit the highest diversity among the six HVRs, and H3 in particular is thought to play a unique role in conferring superior specificity to antibodies. See, e.g., Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable in the absence of light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0140] Several HVR delineations are used and encompassed herein. Kabat complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia instead refers to the location of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). AbM HVRs represent a compromise between Kabat HVRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. "Contact" HVRs are based on analysis of available complex crystal structures. Residues from each of these HVRs are listed below. Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B (Kabat numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102 H96-H101 H93-H101

[0141] HVRs may include the following "extended HVRs": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH. The variable domain residues are numbered according to Kabat et al. (supra) for each of these definitions.

[0142] The phrases "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat," and variations thereof, refer to the numbering system used for the heavy or light chain variable domains in the organization of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FRs or HVRs of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat, etc.). The Kabat numbering of residues can be determined for a given antibody by alignment of the antibody's sequence with the "standard" Kabat-numbered sequence at the regions of homology.

[0143] The term "variable" refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed throughout the variable domains. Rather, it is concentrated in three segments called hypervariable regions (HVRs) in both the light- and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Natural heavy- and light-chain variable domains each contain four FR regions that largely adopt a beta-sheet configuration, connected by three HVRs that form loops that connect, and in some cases form part of, the beta-sheet structure. The HVRs within each chain are held in close proximity by the FR regions and, together with the HVRs of the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institutes of Health, Bethesda, MD (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0144] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The heavy and light chain variable domains may be referred to as "VH" and "VL," respectively. These domains are generally the most variable parts of an antibody (relative to other antibodies of the same class) and contain the antigen-binding site.

[0145] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0146] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically, a whole antibody includes one having heavy and light chains, including an Fc region. The constant domains may be native-sequence constant domains (e.g., human native-sequence constant domains) or amino acid sequence variants thereof. In some cases, an intact antibody may have one or more effector functions. An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding region and / or the variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2; diabodies; linear antibodies (see Example 2 of U.S. Pat. No. 5,641,870; Zapata et al., Protein Eng. 8(10):1057-1062 (1995)).

[0147] see, for example, ); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies yields two identical antigen-binding fragments called "Fab" fragments and a residual "Fc" fragment, a designation reflecting its ability to readily crystallize. The Fab fragment contains the entire L chain along with the variable region domain of the H chain (V H ), and the first constant domain of one heavy chain (C H1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with different antigen-binding activities and is still capable of cross-linking antigen. Fab' fragments contain one or more cysteines from the antibody hinge region, and the remaining C(ab')2 fragments contain one or more cysteines from the antibody hinge region. H F(ab')2 antibody fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of one domain. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0148] The Fc fragment contains the carboxy termini of both H chains held together by disulfides. The effector functions of the antibody are determined by sequences in the Fc region, which is also recognized by Fc receptors (FcRs) found on certain cell types.

[0149] "Functional fragments" of the described antibodies include portions of the intact antibody, generally including the antigen-binding or variable region of the intact antibody, or the Fc region of the antibody that retains or has modified FcR binding ability. Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0150] An "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This fragment consists of a dimer of one heavy-chain variable region domain and one light-chain variable region domain in tight, non-covalent association. The folding of these two domains generates six hypervariable loops (three loops from each H chain and L chain) that provide the amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific HVRs) has the ability to recognize and bind to antigen, although with lower affinity than the entire binding site.

[0151] "Single-chain Fv," also abbreviated as "sFv" or "scFv," is a Fv consisting of V fragments joined together into a single polypeptide chain. H and V L Preferably, the sFv polypeptide is an antibody fragment containing the V H Domains and V L The sFv further comprises a polypeptide linker between the domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFvs, see, for example, Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0152] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the Fc region of a human IgG heavy chain is usually defined to stretch from the amino acid residue at position Cys226 or from the amino acid residue at position Pro230 to its carboxyl-terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during antibody production or purification or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, intact antibody compositions may include antibody populations with all K447 residues removed, antibody populations without the removed K447 residue, and antibody populations having a mixture of antibodies with and without the K447 residue. Native-sequence Fc regions suitable for use in the described antibodies include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0153] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. A preferred FcR is a native-sequence human FcR. Furthermore, a preferred FcR is one that binds IgG antibodies (gamma receptors), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses (including allelic variants and alternatively spliced ​​forms of these receptors). FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein.

[0154] The term "diabody" refers to a diabody that combines V domains to achieve inter-chain but not intra-chain V domain pairing, thereby resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. H Domains and V L This refers to small antibody fragments prepared by constructing sFv fragments (see previous paragraph) with a short linker (approximately 5-10 residues) between the domains. Bispecific diabodies are small antibody fragments prepared by constructing sFv fragments (see previous paragraph) with a short linker (approximately 5-10 residues) between the domains. H Domain and V LDiabodies are heterodimers of two "crossover" sFv fragments in which the domains are present on different polypeptide chains. Diabodies are described in more detail in, for example, EP 404,097, WO 93 / 11161, and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

[0155] The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include PRIMATIZED® antibodies, in which the antigen-binding region of the antibody is derived from an antibody produced, for example, by immunizing macaque monkeys with the antigen of interest. As used herein, "humanized antibodies" is used as a subset of "chimeric antibodies."

[0156] The "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0157] "Affinity" refers to the strength of the total non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen, e.g., CD20). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D ) Affinity can be measured by methods common in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0158] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or an antibody produced using any of the techniques for producing human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, such as phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). The methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991) can also be used to prepare human monoclonal antibodies. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., immunized xenomouse, that have been engineered to produce such antibodies in response to antigen challenge but whose endogenous gene loci have been disabled (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). Also, for human antibodies produced by human B cell hybridoma technology, see, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006).

[0159] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from an HVR (defined below) of the recipient are replaced by residues from an HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and / or capacity. In some instances, framework ("FR") residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications may be made to further refine antibody performance, such as binding affinity. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence and all or substantially all of the FR regions correspond to those of a human immunoglobulin sequence, although the FR regions may include one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FRs typically will be no more than six in the H chain and no more than three in the L chain. The humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409.

[0160] The term "isolated antibody," as used to describe various antibodies disclosed herein, refers to an antibody that has been identified, separated, and / or recovered from the cell or cell culture in which it is expressed. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified to greater than 95% or greater than 99% purity, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007). In preferred embodiments, antibodies are purified (1) sufficiently to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the polypeptide's natural environment will not be present. Ordinarily, however, isolated polypeptides will be prepared by at least one purification step.

[0161] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies within the population are identical except for naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation), which may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by a hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in accordance with the present invention can be produced by, for example, hybridoma techniques (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2002) nded.1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, eg, US Patent No. 4,816,567), phage-display technologies (see, eg, Clackson et al. al.,Nature,352:624-628(1991);Marks et al.,J.Mol.Biol.222:581-597(1992);Sidhu et al.,J.Mol.Biol.338(2):299-310(2004);Lee et al. al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004), and techniques for producing human or human-like antibodies in animals that have some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (e.g., WO 1998 / 24893, WO 1996 / 34096, WO 1996 / 33735, WO 1991 / 10741, Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551(1993);Jakobovits et al.,Nature 362:255-258(1993);Bruggemann et al.,Year in Immunol.7:33 (1993), U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, Marks et al.,Bio / Technology 10:779-783(1992);Lonberg et al.,Nature 368:856-859(1994);Morrison,Nature 368:812-813(1994);Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995)).

[0162] As used herein, the terms "bind," "specifically bind to," or "specific for" refer to a measurable and reproducible interaction, such as binding between a target and an antibody, that is determinative of the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically binds to a target (which may be an epitope) is one that binds to this target with higher affinity, avidity, more readily, and / or with a longer duration than it binds to other targets. In one embodiment, the extent to which an antibody binds to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (K) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, or ≦0.1 nM. D In certain embodiments, the antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In another embodiment, specific binding can include, but is not required to be, exclusive binding. As used herein, the term refers to, for example, binding to a target. -4 M or less, or 10 -5 M or less, or 10 -6 M or less, or 10 -7 M or less, or 10 -8 M or less, or 10 -9 M or less, or 10 -10 M or less, or 10 -11 M or less, or 10 -12 K below M D , or 10 -4 M~10 -6 M or 10 -6 M~10 -10 M or 10-7 M~10 -9 M range K D As will be appreciated by those skilled in the art, affinity and K D The values ​​are inversely correlated: a high affinity for the antigen corresponds to a low K D In one embodiment, the term "specific binding" refers to binding when a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptides or polypeptide epitopes.

[0163] The terms "substantially reduced" or "substantially different," as used herein, refer to a sufficiently high difference between two numerical values ​​(typically one associated with a molecule and the other associated with a reference / comparator molecule) such that one of ordinary skill in the art can easily determine that the difference between these two values ​​is sufficient to differentiate such values ​​(e.g., K D A difference between two such values ​​will be considered statistically significant with respect to a biological characteristic measured by a reference / comparator molecule (value). The difference between such two values ​​can be, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and / or greater than about 50% as a function of the value of the reference / comparator molecule.

[0164] The terms "substantially similar" or "substantially the same," as used herein, refer to a sufficiently high similarity between two numerical values ​​(typically one associated with a molecule and the other associated with a reference / comparison molecule) such that one of ordinary skill in the art would recognize the difference between these two values ​​as a significant difference between such values ​​(e.g., K D In some cases, a difference between two such values ​​may be considered to have little or no biological and / or statistical significance with respect to a biological characteristic measured by a reference / comparator value (e.g., a difference of less than about 50%, less than about 40%, less than about 30%, less than about 20%, and / or less than about 10%).

[0165] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with those in the reference polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximal alignment over the full length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code, together with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California) or can be compiled from its source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0166] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (alternatively, it may be written as a given amino acid sequence A having or comprising a particular % amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 × fraction X / Y

[0167] where X is the number of amino acid residues scored as identical by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is different from the length of amino acid sequence B, the % amino acid sequence identity of A to B will differ from the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​as used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0168] As used herein, the term "sample" refers to a composition obtained or derived from a subject and / or individual of interest that contains cellular and / or other molecular entities to be characterized and / or identified, e.g., based on physical, biochemical, chemical, and / or physiological properties. For example, the phrases "tumor sample," "disease sample," and variations thereof refer to any sample (e.g., a biopsy or blood sample) obtained from a subject of interest that is expected to contain or known to contain the cellular and / or molecular entities to be characterized. In some embodiments, the sample is a tumor tissue sample (e.g., a lymphoma tumor tissue sample, e.g., a B-cell lymphoma tumor tissue sample, e.g., a non-Hodgkin's lymphoma tumor tissue sample, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) tumor tissue sample). Other samples include, but are not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymphatic fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, stool, tumor lysates, and tissue culture media, tissue extracts, e.g., homogenized tissue, cell extracts, and combinations thereof. Samples may be fresh or processed for preservation (e.g., frozen, fixed, or formalin-fixed, paraffin-embedded (FFPE)).

[0169] A "tissue sample" or "cell sample" refers to a collection of similar cells obtained from the tissue of a subject or individual. The source of the tissue or cell sample may be solid tissue, such as from fresh, frozen, and / or preserved organs, tissue samples, biopsies, and / or aspirates; blood or any blood constituents, such as plasma; bodily fluids, such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; or cells from any stage in a subject's pregnancy or development. A tissue sample may also be primary cells or cultured cells or cell lines. Optionally, a tissue or cell sample is obtained from a diseased tissue / organ. A tissue sample may contain compounds not naturally mixed with natural tissue, such as preservatives, anticoagulants, buffers, fixatives, waxes, nutrients, or antibiotics.

[0170] As used herein, a "reference sample," "reference cell," "reference tissue," "control sample," "control cell," or "control tissue" refers to a sample, cell, tissue, standard, or level used for comparison purposes. In one embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and / or unaffected part of the body (e.g., tissue or cells) of the same subject. For example, a healthy and / or unaffected cell or tissue adjacent to a diseased cell or tissue (e.g., a cell or tissue adjacent to a tumor). In another embodiment, the reference sample is obtained from an untreated tissue and / or cells of the same subject's body. In yet another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and / or unaffected part of the body (e.g., tissue or cells) of a subject that is not the subject. In yet another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from an untreated tissue and / or cells of the body of an individual that is not the subject.

[0171] Generally, non-limiting examples of "controls" as used in the context of the present invention are preferably "non-responder" controls, e.g., samples / cells / tissues obtained from one or more patients who are not afflicted with (are not "patients as defined herein") a particular lymphoma as defined herein (e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal or splenic marginal zone lymphoma)) and who are known not to respond favorably to an anti-CD20 antibody according to the present invention (e.g., obinutuzumab or rituximab) (particularly in combination with chemotherapy, more particularly in combination with CHOP chemotherapy). Another example of a "non-responder" control is a cell line / sample / cell / tissue that does not show an improved response to an anti-CD20 antibody (e.g., obinutuzumab or rituximab) in ex vivo tests (especially in combination with chemotherapy, in particular in combination with CHOP chemotherapy). Another non-limiting example of a "control" is an "internal standard", such as a purified or synthetically produced protein, peptide, DNA and / or RNA, or a mixture thereof, the amount of each protein / peptide / DNA / RNA being measured using the "non-responder" control described herein. In principle, the patient to be treated in the context of the present invention is assumed to be a patient with lymphoma (e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal or splenic marginal zone lymphoma)). In other words, the patient is a patient having / afflicted with lymphoma (e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., a germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., an extranodal, nodal or splenic marginal zone lymphoma)).Thus, it is particularly envisaged that the patients defined with respect to any of the aspects / embodiments are also patients with lymphoma (e.g., B-cell lymphoma, e.g., non-Hodgkin's lymphoma, e.g., diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or marginal zone lymphoma (e.g., extranodal, nodal or splenic marginal zone lymphoma)), and patients having / suffering from lymphoma (e.g., B-cell lymphoma, e.g., non-Hodgkin's lymphoma, e.g., diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or marginal zone lymphoma (e.g., extranodal, nodal or splenic marginal zone lymphoma)). However, it is not necessary that a given patient be diagnosed as a lymphoma patient before (or after) being determined / identified / diagnosed as, for example, a patient as defined herein, particularly as defined in one or more aspects / embodiments. However, it is preferred that a patient to be treated according to the present invention is diagnosed as a lymphoma patient in a first step and determined / identified / diagnosed as a patient as defined herein, particularly as defined in one or more aspects / embodiments, in a second step. In principle, according to the present invention, a given patient can be determined / identified / diagnosed as a patient as defined herein in a first step and diagnosed as a lymphoma patient in a second step. However, the latter option is less preferred, and as mentioned above, the (previous or subsequent) step of diagnosing whether the patient to be treated is a lymphoma (e.g., DLBCL) patient can also be omitted.

[0172] As used herein, the term "protein," unless otherwise indicated, refers to any naturally occurring protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term also includes "full-length," unprocessed proteins and any form of a protein that results from processing within a cell. The term also encompasses naturally occurring variants of the protein, such as splice variants or allelic variants.

[0173] As used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Thus, for example, polynucleotides as defined herein include, but are not limited to, single- and double-stranded DNA, DNA containing single- and double-stranded regions, single- and double-stranded RNA, and RNA containing single- and double-stranded regions, and hybrid molecules containing DNA and RNA that may contain single strands or, more typically, double strands, or may contain single- and double-stranded regions. Additionally, as used herein, the term "polynucleotide" refers to triple-stranded regions containing RNA or DNA, or both RNA and DNA. The strands within such regions may be from the same molecule or from different molecules. These regions may include all of one or more of these molecules, but more typically include only some regions of these molecules. One of the molecules in the triple helix region is often an oligonucleotide. The terms "polynucleotide" and "nucleic acid" specifically include mRNA and cDNA.

[0174] A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after synthesis, such as by conjugation with a label. Other types of modifications include substitution of one or more analogs of naturally occurring nucleotides, internucleotide modifications such as those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those containing intercalating agents (e.g., acridine, psoralen, etc.), those containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylating agents, those with modified linkages (e.g., alpha-anomeric nucleic acids, etc.), and unmodified forms of polynucleotide(s). Additionally, any of the hydroxyl groups normally present in the sugar may be replaced by, for example, phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to provide for additional linkage to additional nucleotides, or conjugated to a solid or semi-solid support. The 5' and 3' terminal OH groups can be phosphorylated or substituted with amines or organic capping group moieties of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized with standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl-, 2'-fluoro-, or 2'-azido-ribose; carbocyclic sugar analogs; α-anomeric sugars; epimeric sugars such as arabinose, xylose, or lyxose; pyranose sugars; furanose sugars; sedoheptulose; acrylic acid analogs; and abasic nucleoside analogs, such as methyl riboside. One or more phosphodiester linkages can be replaced by alternative linking groups.These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), "(O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H or substituted or unsubstituted alkyl (1-20C) (optionally containing an ether (-O-) linkage), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0175] As used herein, "carrier" includes pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to cells or mammals exposed thereto at the dosages and concentrations employed. Often, physiologically acceptable carriers are pH-buffered aqueous solutions. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0176] The phrase "pharmaceutically acceptable" indicates that a substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal being treated therewith.

[0177] The term "pharmaceutical formulation" refers to a preparation in which the biological activity of the active ingredients contained in the preparation is in a form such that it is effective, and which does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered.

[0178] An "article of manufacture" is any product (e.g., package or container) or kit that includes at least one reagent, e.g., a pharmaceutical agent for treating a disease or disorder (e.g., a lymphoma, e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)), and a package insert. In certain embodiments, the article of manufacture or kit is promoted, distributed, or sold as a unit for performing the methods described herein.

[0179] "Package insert" means the written instructions customarily included in the commercial packaging of a drug product that contain information regarding the indications, dosage, administration, contraindications, other drugs that may be combined with the packaged product, and / or warnings regarding the use of such drugs.

[0180] III. Diagnostic Methods and Assays Provided herein are methods and assays for identifying, diagnosing, and / or predicting whether a patient having a lymphoma (e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)) will benefit from a treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab). The methods and assays described herein are based on the discovery that macrophage biomarkers (e.g., gene expression values ​​(e.g., gene expression values ​​from any of the gene signature sets described herein (e.g., any of the exemplary gene signature sets in Tables 1 and 2)) or macrophage abundance (e.g., M1 macrophages)), or the amount or level of Th2 biomarkers (e.g., Th2 cell abundance) in a sample (e.g., a tissue sample, e.g., a tumor tissue sample, e.g., a biopsy, etc.) from a patient can be used to identify, diagnose, and / or predict a patient as one that may benefit from a treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab). Any of the methods provided herein may further include administering an anti-CD20 antibody (e.g., obinutuzumab or rituximab) to the patient.

[0181] A. Macrophage Biomarkers In certain examples, the methods and assays provided herein can be used to determine the amount or level of a macrophage biomarker. Various diagnostic methods based on determining the amount or level of a macrophage biomarker are further described below.

[0182] In one aspect, provided herein are methods for identifying, diagnosing, and / or predicting whether a patient having lymphoma will benefit from a treatment comprising an anti-CD20 antibody, the method comprising measuring a macrophage biomarker in a sample from the patient, wherein an amount or level of the macrophage biomarker that exceeds the amount or level of a reference macrophage biomarker in the sample identifies, diagnoses, and / or predicts the patient as one who will benefit from a treatment comprising an anti-CD20 antibody. In some examples, the method further comprises administering the anti-CD20 antibody. Alternatively, an amount or level of the macrophage biomarker in the sample that is below the amount or level of the reference macrophage biomarker identifies, diagnoses, and / or predicts the patient as one who is likely not to benefit from a treatment comprising an anti-CD20 antibody.

[0183] In another aspect, provided herein is a method for selecting a therapy for a patient with lymphoma, the method comprising measuring a macrophage biomarker in a sample from the patient, wherein an amount or level of the macrophage biomarker that exceeds the amount or level of a reference macrophage biomarker in the sample identifies the patient as one who may benefit from a therapy comprising an anti-CD20 antibody. In some examples, the method further comprises administering an anti-CD20 antibody. Alternatively, an amount or level of the macrophage biomarker in the sample that is below the amount or level of the reference macrophage biomarker identifies the patient as one who is unlikely to benefit from a therapy comprising an anti-CD20 antibody.

[0184] (i) Increased macrophage biomarkers An amount or level of a macrophage biomarker in a sample from a patient having a lymphoma (e.g., a B cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B cell lymphoma (e.g., germinal center B cell-like or activated B cell-like diffuse large B cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)) that exceeds an amount or level of a reference macrophage biomarker can identify, diagnose, and / or predict the patient as one who may benefit from a treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0185] For example, in some instances, the upper about 99th percentile (prevalence level of about 1% or greater), upper about 95th percentile (prevalence level of about 5% or greater), upper about 90th percentile (prevalence level of about 10% or greater), upper about 85th percentile (prevalence level of about 15% or greater), upper about 80th percentile (prevalence level of about 20% or greater), upper about 75th percentile (prevalence level of about 25% or greater), upper about 70th percentile (prevalence level of about 30% or greater), upper about 65th percentile (prevalence level of about 35% or greater), upper about 60th percentile (prevalence level of about 40% or greater), upper about 55th percentile (prevalence level of about 10% or greater), upper about 50th percentile (prevalence level of about 50% or greater), upper about 45th percentile of the amount or level of macrophage biomarker in a reference population. (a prevalence level of about 55% or greater), the top about 40th percentile (a prevalence level of about 60% or greater), the top about 35th percentile (a prevalence level of about 65% or greater), the top about 30th percentile (a prevalence level of about 70% or greater), the top about 25th percentile (a prevalence level of about 75% or greater), the top about 20th percentile (a prevalence level of about 80% or greater), the top about 15th percentile (a prevalence level of about 85% or greater), the top about 10th percentile (a prevalence level of about 90% or greater), the top about 5th percentile (a prevalence level of about 95% or greater), or the top about 1st percentile (a prevalence level of about 99% or greater) identifies the individual as one who may benefit from a treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0186] In some examples, an amount or level of a macrophage biomarker in a sample that is in the top about 10 to about 90 percentile, the top about 20 to about 80 percentile, the top about 30 to about 70 percentile, the top about 40 to about 60 percentile, the top about 45 to about 55 percentile, the top about 48 to about 52 percentile, the top about 49.5 to about 50.5 percentile, the top about 49.9 to about 50.1 percentile, or the top about 50 percentile of the amount or level of the macrophage biomarker in a reference population identifies the individual as one that may benefit from a treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab). For example, in some instances, an amount or level of a macrophage biomarker in a sample that is between about 10% and about 90% prevalence, between about 15% and about 85% prevalence, between about 20% and about 80% prevalence, between about 25% and about 75% prevalence, between about 30% and about 70% prevalence, between about 35% and about 65% prevalence, between about 40% and about 60% prevalence, between about 45% and about 55% prevalence, between about 48% and about 52% prevalence, between about 49.5% and about 50.5% prevalence, between about 49.9% and about 50.1% prevalence, or about 50% prevalence in a reference population identifies the individual as one who would benefit from a treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0187] In some examples, an amount or level of a macrophage biomarker in a sample that is in the top 80 percentile of a reference population (i.e., a prevalence level of 20% or greater) identifies an individual as one who could benefit from a treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab). In some examples, an amount or level of a macrophage biomarker in a sample that is in the top 75 percentile of a reference population (i.e., a prevalence level of 25% or greater) identifies an individual as one who could benefit from a treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab). In some examples, an amount or level of a macrophage biomarker in a sample that is in the top 50 percentile of a reference population (i.e., a prevalence level of 50% or greater) identifies an individual as one who could benefit from a treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab). In some examples, an amount or level of a macrophage biomarker in a sample that is in the top about 25th percentile of a reference population (i.e., a prevalence level of 75% or greater) identifies an individual as one who could benefit from a treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab). In some examples, an amount or level of a macrophage biomarker in a sample that is in the top about 20th percentile of a reference population (i.e., a prevalence level of 80% or greater) identifies an individual as one who could benefit from a treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0188] In some examples, an amount or level of a macrophage biomarker that exceeds the amount or level of a reference macrophage biomarker refers to an overall increase of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more in the amount or level of the macrophage biomarker as detected by standard art-known methods, such as those described herein, compared to the amount or level of the macrophage biomarker in a reference sample, cell, tissue, control sample, cell, or tissue. In particular examples, an amount or level of a macrophage biomarker that exceeds the amount or level of a reference macrophage biomarker refers to an increase in the amount or level of the macrophage biomarker in a sample, which increase is at least about 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, or 100-fold the amount or level of the macrophage biomarker in the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some examples, an amount or level of a macrophage biomarker that exceeds the amount or level of a reference macrophage biomarker refers to an overall increase in the amount or level of the macrophage biomarker that is greater than about 1.5-fold, about 1.75-fold, about 2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3.0-fold, or about 3.25-fold compared to the amount or level of the macrophage biomarker in the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.

[0189] In some examples, an amount or level of a macrophage biomarker that exceeds the amount or level of a reference macrophage biomarker refers to an overall increase of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more in the amount or level of the macrophage biomarker detected by standard art-known methods, such as those described herein, compared to the pre-assigned amount or level of the macrophage biomarker. In particular examples, an amount or level of a macrophage biomarker that exceeds the amount or level of a reference macrophage biomarker refers to an increase in the amount or level of the macrophage biomarker in a sample, where the increase is at least about 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, or 100-fold the pre-assigned amount or level of the macrophage biomarker. In some examples, an amount or level of a macrophage biomarker that exceeds the amount or level of a reference macrophage biomarker refers to an overall increase in the amount or level of the macrophage biomarker of more than about 1.5-fold, about 1.75-fold, about 2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3.0-fold, or about 3.25-fold compared to a pre-assigned amount or level of the macrophage biomarker.

[0190] (ii) Decreased macrophage biomarkers An amount or level of a macrophage biomarker in a sample from a patient having a lymphoma (e.g., a B cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B cell lymphoma (e.g., germinal center B cell-like or activated B cell-like diffuse large B cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)) that is below the amount or level of a reference macrophage biomarker can identify, diagnose, and / or predict the patient as not benefiting from a treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0191] In some examples, the lower percentile of the amount or level of the macrophage biomarker in the reference population may be about the lower 99th percentile (a prevalence level of about 99% or less), lower 95th percentile (a prevalence level of about 95% or less), lower 90th percentile (a prevalence level of about 90% or less), lower 85th percentile (a prevalence level of about 85% or less), lower 80th percentile (a prevalence level of about 80% or less), lower 75th percentile (a prevalence level of about 75% or less), lower 70th percentile (a prevalence level of about 70% or less), lower 65th percentile (a prevalence level of about 65% or less), lower 60th percentile (a prevalence level of about 60% or less), lower 55th percentile (a prevalence level of about 55% or less), lower 50th percentile (a prevalence level of about 50% or less), lower 45th percentile. An amount or level of a macrophage biomarker in the sample that is in the lower percentile (a prevalence level of about 45% or less), lower about 40th percentile (a prevalence level of about 40% or less), lower about 35th percentile (a prevalence level of about 35% or less), lower about 30th percentile (a prevalence level of about 30% or less), lower about 25th percentile (a prevalence level of about 25% or less), lower about 20th percentile (a prevalence level of about 20% or less), lower about 15th percentile (a prevalence level of about 15% or less), lower about 10th percentile (a prevalence level of about 10% or less), lower about 5th percentile (a prevalence level of about 5% or less), or lower about 1 percentile (a prevalence level of about 1% or less) identifies the individual as one that is unlikely to benefit from a treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0192] In some examples, an amount or level of a macrophage biomarker in a sample that is at about the bottom 10 to about the bottom 90 percentile, about the bottom 20 to about the bottom 80 percentile, about the bottom 30 to about the bottom 70 percentile, about the bottom 40 to about the bottom 60 percentile, about the bottom 45 to about the bottom 55 percentile, about the bottom 48 to about the bottom 52 percentile, about the bottom 49.5 to about the bottom 50.5 percentile, about the bottom 49.9 to about the bottom 50.1 percentile, or about the bottom 50 percentile of the amount or level of a macrophage biomarker in a reference population identifies the individual as one that is unlikely to benefit from a treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab). For example, in some instances, an amount or level of a macrophage biomarker in a sample that is between about 10% and about 90% prevalence, between about 15% and about 85% prevalence, between about 20% and about 80% prevalence, between about 25% and about 75% prevalence, between about 30% and about 70% prevalence, between about 35% and about 65% prevalence, between about 40% and about 60% prevalence, between about 45% and about 55% prevalence, between about 48% and about 52% prevalence, between about 49.5% and about 50.5% prevalence, between about 49.9% and about 50.1% prevalence, or about 50% prevalence in a reference population identifies an individual as one that is unlikely to benefit from a treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0193] In some examples, an amount or level of a macrophage biomarker that is lower than the reference amount or level of a macrophage biomarker refers to about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or greater decrease in the amount or level of the macrophage biomarker as detected by standard art-known methods, such as those described herein, compared to the amount or level of the macrophage biomarker in a reference sample, cell, tissue, control sample, cell, or tissue. In particular examples, an amount or level of a macrophage biomarker that is lower than a reference amount or level of the macrophage biomarker refers to a decrease in the amount or level of the macrophage biomarker in a sample, where the decrease is at least about 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, or 100-fold the amount or level of the macrophage biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some examples, an amount or level of a macrophage biomarker that is lower than a reference amount or level of the macrophage biomarker refers to a decrease in the amount or level of the macrophage biomarker that is greater than about 1.5-fold, about 1.75-fold, about 2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3.0-fold, or about 3.25-fold compared to the amount or level of the macrophage biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.

[0194] In some examples, an amount or level of a macrophage biomarker that is lower than a reference amount or level of the macrophage biomarker refers to an overall decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more in the amount or level of the macrophage biomarker detected by standard art-known methods, such as those described herein, compared to a pre-assigned amount or level of the macrophage biomarker. In particular examples, an amount or level of a macrophage biomarker that is lower than a reference amount or level of the macrophage biomarker refers to a decrease in the amount or level of the macrophage biomarker in a sample, wherein the decrease is at least about 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, or 100-fold the pre-assigned amount or level of the macrophage biomarker. In some examples, an amount or level of a macrophage biomarker that is lower than the reference amount or level of a macrophage biomarker refers to an overall decrease in the amount or level of the macrophage biomarker of more than about 1.5-fold, about 1.75-fold, about 2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3.0-fold, or about 3.25-fold compared to a pre-assigned amount or level of the macrophage biomarker.

[0195] (iii) Reference macrophage biomarkers The reference macrophage biomarker amount or level can be a pre-assigned amount or level of the macrophage biomarker. In some examples, the amount or level of the macrophage biomarker in the reference population is the median amount or level of the macrophage biomarker in the reference population. In some examples, the amount or level of the macrophage biomarker in the reference population is the mean amount or level of the macrophage biomarker in the reference population.

[0196] In some examples, the pre-assigned amount or level of a macrophage biomarker is a percentage of a cell subtype within a sample. In some examples, the percentage of a cell subtype within a sample is between about 0% and 40% (e.g., 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40%). In some examples, the percentage of a cell subtype within a sample is between about 0% and 10% (e.g., 0%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%). In some examples, the percentage of a cell subtype within a sample is less than 10% (e.g., 0%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%). In some examples, the percentage of a cell subtype within a sample is about 6%. In some examples, the percentage of cell subtypes in the sample is about 5%. In some examples, the percentage of cell subtypes in the sample is about 4.74%. In some examples, the percentage of cell subtypes in the sample is about 4%. In some examples, the percentage of cell subtypes in the sample is about 3.35%. In some examples, the percentage of cell subtypes in the sample is about 3%. In some examples, the percentage of cell subtypes in the sample is about 2.5%. In some examples, the percentage of cell subtypes in the sample is about 2%. In some examples, the percentage of cell subtypes in the sample is about 1.67%. In some examples, the percentage of cell subtypes in the sample is about 1%. In some examples, the percentage of cell subtypes in the sample is about 0%.

[0197] The reference amounts or levels of macrophage biomarkers described herein may be based on the amounts or levels of the macrophage biomarkers in a reference population. In some examples, the reference macrophage biomarkers described herein are amounts or levels of macrophage biomarkers in a reference population that includes two or more (e.g., two or more, three or more, four or more, or five or more) subsets of patients.

[0198] In some examples, the reference macrophage biomarker is the amount or level of the macrophage biomarker in a reference population, and the reference population includes at least a subset of patients with lymphoma (e.g., a B cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B cell lymphoma (e.g., germinal center B cell-like or activated B cell-like diffuse large B cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)).

[0199] In some examples, the reference macrophage biomarker is the amount or level of the macrophage biomarker in a reference population, and the reference population includes at least a subset of patients with lymphoma (e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)) who have received one or more doses (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more doses) of an anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0200] In some examples, the reference macrophage biomarker is the amount or level of the macrophage biomarker in a reference population, and the reference population includes at least a subset of patients with lymphoma (e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)) who have been treated with an anti-CD20 antibody (e.g., obinutuzumab or rituximab) as monotherapy.

[0201] In some examples, the reference macrophage biomarker is the amount or level of the macrophage biomarker in a reference population, and the reference population includes at least a subset of patients with lymphoma (e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)) who have been treated with an anti-CD20 antibody (e.g., obinutuzumab or rituximab) as a combination therapy (e.g., a combination therapy including an anti-CD20 antibody (e.g., obinutuzumab or rituximab) and an additional therapeutic agent (e.g., an anti-cancer therapy (e.g., a cytotoxic agent, a growth inhibitory agent, radiation therapy, an anti-angiogenic agent, or a combination thereof), e.g., CHOP).

[0202] In some examples, the reference macrophage biomarker is the amount or level of a macrophage biomarker in a reference population, and the reference population includes at least a subset of patients with lymphoma (e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)) who have been treated with a therapy that does not include an anti-CD20 antibody (e.g., obinutuzumab or rituximab) and includes an anti-cancer therapy (e.g., a cytotoxic agent, a growth inhibitory agent, radiation therapy, an anti-angiogenic agent, or a combination thereof), e.g., CHOP.

[0203] For example, in some instances, the reference population includes a first subset of patients who have been treated with an anti-CD20 antibody (e.g., obinutuzumab or rituximab) and a second subset of patients who have not been treated with an anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0204] In some examples, the baseline amount or level of the macrophage biomarker significantly separates each of the first and second subsets of patients based on a significant difference between the patient's responsiveness (e.g., PFS or OS) to treatment with an anti-CD20 antibody (e.g., obinutuzumab or rituximab) and the patient's responsiveness to treatment without the anti-CD20 antibody (e.g., obinutuzumab or rituximab) over the baseline macrophage biomarker, wherein the patient's responsiveness to treatment with the anti-CD20 antibody (e.g., obinutuzumab or rituximab) is significantly improved compared to the patient's responsiveness to treatment without the anti-CD20 antibody (e.g., obinutuzumab or rituximab). For example, in some instances, the baseline amount or level of the macrophage biomarker optimally separates each of the first and second subsets of patients based on the greatest difference between the patient's responsiveness (e.g., PFS or OS) to treatment with an anti-CD20 antibody (e.g., obinutuzumab or rituximab) and the patient's responsiveness to treatment without the anti-CD20 antibody (e.g., obinutuzumab or rituximab) over the baseline macrophage biomarker, wherein the patient's responsiveness to treatment with the anti-CD20 antibody (e.g., obinutuzumab or rituximab) is significantly improved compared to the patient's responsiveness to treatment without the anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0205] In some examples, the baseline amount or level of the macrophage biomarker significantly separates each of the first and second subsets of patients based on a significant difference between the patient's responsiveness (e.g., PFS or OS) to treatment with an anti-CD20 antibody (e.g., obinutuzumab or rituximab) and the patient's responsiveness to treatment without the anti-CD20 antibody (e.g., obinutuzumab or rituximab) below the baseline macrophage biomarker, and the patient's responsiveness to treatment without the anti-CD20 antibody (e.g., obinutuzumab or rituximab) is significantly improved compared to the patient's responsiveness to treatment with the anti-CD20 antibody (e.g., obinutuzumab or rituximab). For example, in some instances, the baseline amount or level of the macrophage biomarker optimally separates each of the first and second subsets of patients based on the maximum difference between the patient's responsiveness (e.g., PFS or OS) to treatment with an anti-CD20 antibody (e.g., obinutuzumab or rituximab) and the patient's responsiveness to treatment without the anti-CD20 antibody (e.g., obinutuzumab or rituximab) below the baseline macrophage biomarker, and the patient's responsiveness to treatment without the anti-CD20 antibody (e.g., obinutuzumab or rituximab) is significantly improved compared to the patient's responsiveness to treatment with the anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0206] In some instances, optimal or significant separation can be based on a hazard ratio (HR) determined from an analysis of the amount or level of a macrophage biomarker in a first and second subset of patients, where the HR is less than 1, e.g., an HR of about 0.95, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, about 0.1, or less. For example, in certain instances, optimal or significant separation can be based on a hazard ratio (HR) determined from an analysis of the amount or level of a macrophage biomarker in a first and second subset of patients, where the upper limit of the 95% confidence interval of the HR is less than 1, e.g., an upper limit of the 95% confidence interval of the HR of about 0.95, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, about 0.1, or less.

[0207] Additionally or alternatively, the reference macrophage biomarker may be the amount or level of the macrophage biomarker in a reference population, the reference population including at least a subset of patients who do not have a lymphoma (e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)), or who have a lymphoma but are treatment-naive.

[0208] (iv) Indications The methods described herein are useful for predicting the therapeutic response of an individual having a lymphoma (e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)) to treatment with an anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0209] In some examples, the lymphoma can be a low-grade lymphoma. In some examples, the lymphoma can be a B-cell lymphoma, such as a non-Hodgkin's lymphoma, such as a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma). In some examples, the lymphoma can be a follicular lymphoma (FL). In some examples, the lymphoma can be a chronic lymphocytic leukemia (CLL). In some examples, the lymphoma can be a CD20-positive lymphoma.

[0210] In certain instances, the cancer may be a B-cell lymphoma. For example, the B-cell lymphoma may be a non-Hodgkin's lymphoma, including, but not limited to, diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma). For example, the methods described herein can be used to identify, diagnose, and / or predict whether a patient with a B-cell lymphoma (e.g., a non-Hodgkin's lymphoma (e.g., a diffuse large B-cell lymphoma (e.g., a germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., an extranodal, nodal, or splenic marginal zone lymphoma))) will benefit from a treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab), the method comprising measuring a macrophage biomarker in a sample from the patient, wherein an amount or level of the macrophage biomarker in the sample that exceeds an amount or level of a reference macrophage biomarker identifies, diagnoses, and / or predicts the patient as one who will benefit from a treatment comprising an anti-CD20 antibody. Alternatively, an amount or level of a macrophage biomarker in the sample that is below the amount or level of a reference macrophage biomarker identifies, diagnoses, and / or predicts the patient as one who is unlikely to benefit from a treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0211] In some instances, an individual having a lymphoma (e.g., a B cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B cell lymphoma (e.g., germinal center B cell-like or activated B cell-like diffuse large B cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)) has not been previously treated for the lymphoma (treatment naive). For example, in some instances, an individual having a lymphoma has not previously received an anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0212] In some examples, an individual with a lymphoma (e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)) has previously received treatment for the lymphoma. In some examples, an individual with a lymphoma has previously received treatment including an anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0213] (v) Therapeutic benefits A patient who benefits from treatment with an anti-CD20 antibody (e.g., obinutuzumab or rituximab) may, for example, experience a delay or prevention of the onset or recurrence of a lymphoma (e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)), alleviation of symptoms, a reduction in the direct or indirect pathological consequences of the cancer, prevention of metastasis, a slowing of disease progression, an improvement or palliation of the condition, or a remission or improved prognosis. In some examples, the treatments described herein are used to delay the onset of cancer or delay the progression of a lymphoma (e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)). In some examples, the benefit can be an increase in overall survival (OS), progression-free survival (PFS), complete remission (CR), partial remission (PR), or a combination thereof.

[0214] In some examples, an amount or level of a macrophage biomarker that exceeds the amount or level of a reference macrophage biomarker (e.g., the amount or level of a macrophage biomarker in a reference population) identifies the individual as one who may benefit from a treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab), where the benefit is an increase in OS (e.g., 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) compared to a treatment that does not include an anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0215] In some examples, an amount or level of a macrophage biomarker that exceeds a reference amount or level of a macrophage biomarker (e.g., the amount or level of a macrophage biomarker in a reference population) identifies the individual as one who may benefit from a treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab), where the benefit is an increase in PFS (e.g., 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) compared to a treatment that does not include an anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0216] B. Th2 biomarkers In certain examples, the methods and assays provided herein can be used to determine the amount or level of a Th2 biomarker. Various diagnostic methods based on determining the amount or level of a Th2 biomarker are further described below.

[0217] In one aspect, provided herein are methods for identifying, diagnosing, and / or predicting whether a patient having lymphoma will benefit from a treatment comprising an anti-CD20 antibody, the method comprising measuring a Th2 biomarker in a sample from the patient, wherein an amount or level of the Th2 biomarker in the sample that exceeds a reference amount or level of the Th2 biomarker identifies, diagnoses, and / or predicts the patient as one who will benefit from a treatment comprising an anti-CD20 antibody. In some examples, the method further comprises administering the anti-CD20 antibody. Alternatively, an amount or level of the Th2 biomarker in the sample that is below a reference amount or level of the Th2 biomarker identifies, diagnoses, and / or predicts the patient as one who is likely not to benefit from a treatment comprising an anti-CD20 antibody.

[0218] In another aspect, provided herein is a method for selecting a therapy for a patient with lymphoma, the method comprising measuring a Th2 biomarker in a sample from the patient, wherein an amount or level of the Th2 biomarker in the sample that exceeds a reference amount or level of the Th2 biomarker identifies the patient as one who may benefit from a therapy comprising an anti-CD20 antibody. In some examples, the method further comprises administering an anti-CD20 antibody. Alternatively, an amount or level of the Th2 biomarker in the sample that is below a reference amount or level of the Th2 biomarker identifies the patient as one who is unlikely to benefit from a therapy comprising an anti-CD20 antibody.

[0219] (i) Increase in Th2 biomarkers An amount or level of a Th2 biomarker in a sample from a patient with a lymphoma (e.g., a B cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B cell lymphoma (e.g., germinal center B cell-like or activated B cell-like diffuse large B cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)) that exceeds an amount or level of a reference Th2 biomarker can identify, diagnose, and / or predict the patient as one that may benefit from a treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0220] For example, in some instances, the upper about 99th percentile (prevalence level of about 1% or greater), upper about 95th percentile (prevalence level of about 5% or greater), upper about 90th percentile (prevalence level of about 10% or greater), upper about 85th percentile (prevalence level of about 15% or greater), upper about 80th percentile (prevalence level of about 20% or greater), upper about 75th percentile (prevalence level of about 25% or greater), upper about 70th percentile (prevalence level of about 30% or greater), upper about 65th percentile (prevalence level of about 35% or greater), upper about 60th percentile (prevalence level of about 40% or greater), upper about 55th percentile (prevalence level of about 55%) of the amount or level of the Th2 biomarker in a reference population. about 10% or higher prevalence level), about the top 50th percentile (about 50% or higher prevalence level), about the top 45th percentile (about 55% or higher prevalence level), about the top 40th percentile (about 60% or higher prevalence level), about the top 35th percentile (about 65% or higher prevalence level), about the top 30th percentile (about 70% or higher prevalence level), about the top 25th percentile (about 75% or higher prevalence level), about the top 20th percentile (about 80% or higher prevalence level), about the top 15th percentile (about 85% or higher prevalence level), about the top 10th percentile (about 90% or higher prevalence level), about the top 5th percentile (about 95% or higher prevalence level), or about the top 1 stAn amount or level of a Th2 biomarker in a sample that is in a percentile (a prevalence level of about 99% or greater) identifies the individual as one who may benefit from treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0221] In some examples, an amount or level of a Th2 biomarker in a sample that is in the top about 10 to about 90 percentile, the top about 20 to about 80 percentile, the top about 30 to about 70 percentile, the top about 40 to about 60 percentile, the top about 45 to about 55 percentile, the top about 48 to about 52 percentile, the top about 49.5 to about 50.5 percentile, the top about 49.9 to about 50.1 percentile, or the top about 50 percentile of the amount or level of the Th2 biomarker in a reference population identifies the individual as one that may benefit from a treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab). For example, in some instances, an amount or level of a Th2 biomarker in a sample that is between about 10% and about 90% prevalence, between about 15% and about 85% prevalence, between about 20% and about 80% prevalence, between about 25% and about 75% prevalence, between about 30% and about 70% prevalence, between about 35% and about 65% prevalence, between about 40% and about 60% prevalence, between about 45% and about 55% prevalence, between about 48% and about 52% prevalence, between about 49.5% and about 50.5% prevalence, between about 49.9% and about 50.1% prevalence, or about 50% prevalence in a reference population identifies the individual as one who would benefit from treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0222] In some examples, an amount or level of a Th2 biomarker in a sample that is in the top about 80 percentile of a reference population (i.e., a prevalence level of 20% or greater) identifies an individual as one who could benefit from a treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab). In some examples, an amount or level of a Th2 biomarker in a sample that is in the top about 75 percentile of a reference population (i.e., a prevalence level of 25% or greater) identifies an individual as one who could benefit from a treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab). In some examples, an amount or level of a Th2 biomarker in a sample that is in the top about 50 percentile of a reference population (i.e., a prevalence level of 50% or greater) identifies an individual as one who could benefit from a treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab). In some examples, an amount or level of a Th2 biomarker in a sample that is in the top 25 percentile of a reference population (i.e., a prevalence level of 75% or greater) identifies an individual as one who could benefit from a treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab). In some examples, an amount or level of a Th2 biomarker in a sample that is in the top approximately 20 percentile of a reference population (i.e., a prevalence level of 80% or greater) identifies an individual as one who could benefit from a treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0223] In some examples, an amount or level of a Th2 biomarker that exceeds the amount or level of a reference Th2 biomarker refers to an overall increase of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more in the amount or level of the Th2 biomarker as detected by standard art-known methods, such as those described herein, compared to the amount or level of the Th2 biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In certain examples, an amount or level of a Th2 biomarker that exceeds a reference amount or level of a Th2 biomarker refers to an increase in the amount or level of the Th2 biomarker in a sample, which increase is at least about 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, or 100-fold the amount or level of the Th2 biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some examples, an amount or level of a Th2 biomarker that exceeds a reference amount or level of a Th2 biomarker refers to an overall increase in the amount or level of the Th2 biomarker that is greater than about 1.5-fold, about 1.75-fold, about 2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3.0-fold, or about 3.25-fold compared to the amount or level of the Th2 biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.

[0224] In some examples, an amount or level of a Th2 biomarker that exceeds a reference amount or level of a Th2 biomarker refers to an overall increase of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more in the amount or level of the Th2 biomarker detected by standard art-known methods, such as those described herein, compared to a pre-assigned amount or level of the Th2 biomarker. In particular examples, an amount or level of a Th2 biomarker that exceeds a reference amount or level of a Th2 biomarker refers to an increase in the amount or level of the Th2 biomarker in a sample, wherein the increase is at least about 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, or 100-fold the pre-assigned amount or level of the Th2 biomarker. In some examples, an amount or level of a Th2 biomarker that exceeds the amount or level of a reference Th2 biomarker refers to an overall increase in the amount or level of the Th2 biomarker of more than about 1.5-fold, about 1.75-fold, about 2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3.0-fold, or about 3.25-fold compared to a pre-assigned amount or level of the Th2 biomarker.

[0225] (ii) Reference Th2 biomarkers The reference Th2 biomarker amount or level can be a pre-assigned Th2 biomarker amount or level. In some examples, the Th2 biomarker amount or level in the reference population is the median amount or level of the Th2 biomarker in the reference population. In some examples, the Th2 biomarker amount or level in the reference population is the mean amount or level of the Th2 biomarker in the reference population.

[0226] In some examples, the pre-assigned amount or level of a Th2 biomarker is a percentage of a cell subtype within a sample. In some examples, the percentage of a cell subtype within a sample is between about 0% and 40% (e.g., 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40%). In some examples, the percentage of a cell subtype within a sample is between about 0% and 10% (e.g., 0%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%). In some examples, the percentage of a cell subtype within a sample is less than 10% (e.g., 0%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%). In some examples, the percentage of a cell subtype within a sample is about 0%.

[0227] The reference amounts or levels of Th2 biomarkers described herein may be based on the amounts or levels of the Th2 biomarkers in a reference population. In some examples, the reference Th2 biomarkers described herein are amounts or levels of Th2 biomarkers in a reference population that includes two or more (e.g., two or more, three or more, four or more, or five or more) subsets of patients.

[0228] In some examples, the reference Th2 biomarker is the amount or level of a Th2 biomarker in a reference population, and the reference population includes at least a subset of patients with lymphoma (e.g., a B cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B cell lymphoma (e.g., germinal center B cell-like or activated B cell-like diffuse large B cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)).

[0229] In some examples, the reference Th2 biomarker is the amount or level of a Th2 biomarker in a reference population, and the reference population includes at least a subset of patients with lymphoma (e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)) who have received one or more doses (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more doses) of an anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0230] In some examples, the reference Th2 biomarker is the amount or level of a Th2 biomarker in a reference population, and the reference population includes at least a subset of patients with lymphoma (e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)) who have been treated with an anti-CD20 antibody (e.g., obinutuzumab or rituximab) as monotherapy.

[0231] In some examples, the reference Th2 biomarker is the amount or level of a Th2 biomarker in a reference population, and the reference population includes at least a subset of patients with lymphoma (e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)) who have been treated with an anti-CD20 antibody (e.g., obinutuzumab or rituximab) as a combination therapy (e.g., a combination therapy including an anti-CD20 antibody (e.g., obinutuzumab or rituximab) and an additional therapeutic agent (e.g., an anti-cancer therapy (e.g., a cytotoxic agent, a growth inhibitory agent, radiation therapy, an anti-angiogenic agent, or a combination thereof), e.g., CHOP).

[0232] In some examples, the reference Th2 biomarker is the amount or level of a Th2 biomarker in a reference population, and the reference population includes at least a subset of patients with lymphoma (e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)) who have been treated with a therapy that does not include an anti-CD20 antibody (e.g., obinutuzumab or rituximab) and includes an anti-cancer therapy (e.g., a cytotoxic agent, a growth inhibitory agent, radiation therapy, an anti-angiogenic agent, or a combination thereof), e.g., CHOP.

[0233] For example, in some instances, the reference population includes a first subset of patients who have been treated with an anti-CD20 antibody (e.g., obinutuzumab or rituximab) and a second subset of patients who have not been treated with an anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0234] In some examples, the reference amount or level of the Th2 biomarker significantly separates each of the first and second subsets of patients based on a significant difference between the patient's responsiveness (e.g., PFS or OS) to treatment with an anti-CD20 antibody (e.g., obinutuzumab or rituximab) and the patient's responsiveness to treatment without the anti-CD20 antibody (e.g., obinutuzumab or rituximab) over the reference Th2 biomarker, wherein the patient's responsiveness to treatment with the anti-CD20 antibody (e.g., obinutuzumab or rituximab) is significantly improved compared to the patient's responsiveness to treatment without the anti-CD20 antibody (e.g., obinutuzumab or rituximab). For example, in some instances, the reference amount or level of the Th2 biomarker optimally separates each of the first and second subsets of patients based on the greatest difference between the patient's responsiveness (e.g., PFS or OS) to treatment with an anti-CD20 antibody (e.g., obinutuzumab or rituximab) and the patient's responsiveness to treatment without the anti-CD20 antibody (e.g., obinutuzumab or rituximab) over the reference Th2 biomarker, wherein the patient's responsiveness to treatment with the anti-CD20 antibody (e.g., obinutuzumab or rituximab) is significantly improved compared to the patient's responsiveness to treatment without the anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0235] In some examples, the reference amount or level of the Th2 biomarker significantly separates each of the first and second subsets of patients based on a significant difference between the patient's responsiveness (e.g., PFS or OS) to treatment with an anti-CD20 antibody (e.g., obinutuzumab or rituximab) and the patient's responsiveness to treatment without the anti-CD20 antibody (e.g., obinutuzumab or rituximab) below the reference Th2 biomarker, and the patient's responsiveness to treatment without the anti-CD20 antibody (e.g., obinutuzumab or rituximab) is significantly improved compared to the patient's responsiveness to treatment with the anti-CD20 antibody (e.g., obinutuzumab or rituximab). For example, in some instances, the reference amount or level of the Th2 biomarker optimally separates each of the first and second subsets of patients based on the maximum difference between the patient's responsiveness (e.g., PFS or OS) to treatment with an anti-CD20 antibody (e.g., obinutuzumab or rituximab) and the patient's responsiveness to treatment without an anti-CD20 antibody (e.g., obinutuzumab or rituximab) below the reference Th2 biomarker, and the patient's responsiveness to treatment without an anti-CD20 antibody (e.g., obinutuzumab or rituximab) is significantly improved compared to the patient's responsiveness to treatment with an anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0236] In some instances, optimal or significant separation can be based on a hazard ratio (HR) determined from an analysis of the amount or level of a Th2 biomarker in a first and second subset of patients, where the HR is less than 1, e.g., an HR of about 0.95, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, about 0.1, or less. For example, in certain instances, optimal or significant separation can be based on a hazard ratio (HR) determined from an analysis of the amount or level of a Th2 biomarker in a first and second subset of patients, where the upper limit of the 95% confidence interval of the HR is less than 1, e.g., an upper limit of the 95% confidence interval of the HR of about 0.95, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, about 0.1, or less.

[0237] Additionally or alternatively, the reference Th2 biomarker may be the amount or level of the Th2 biomarker in a reference population, the reference population including at least a subset of patients who do not have a lymphoma (e.g., a B cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B cell lymphoma (e.g., germinal center B cell-like or activated B cell-like diffuse large B cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)), or who have a lymphoma but are treatment-naive.

[0238] (iii) Indications The methods described herein are useful for predicting the therapeutic response of an individual having a lymphoma (e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)) to treatment with an anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0239] In some examples, the lymphoma can be a low-grade lymphoma. In some examples, the lymphoma can be a B-cell lymphoma, such as a non-Hodgkin's lymphoma, such as a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma). In some examples, the lymphoma can be a follicular lymphoma (FL). In some examples, the lymphoma can be a chronic lymphocytic leukemia (CLL). In some examples, the lymphoma can be a CD20-positive lymphoma.

[0240] In certain instances, the cancer may be a B-cell lymphoma. For example, the B-cell lymphoma may be a non-Hodgkin's lymphoma, including, but not limited to, diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma). For example, the methods described herein can be used to identify, diagnose, and / or predict whether a patient with a B-cell lymphoma (e.g., a non-Hodgkin's lymphoma (e.g., a diffuse large B-cell lymphoma (e.g., a germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., an extranodal, nodal, or splenic marginal zone lymphoma))) will benefit from a treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab), the method comprising measuring a Th2 biomarker in a sample from the patient, wherein an amount or level of the Th2 biomarker in the sample that exceeds a reference amount or level of the Th2 biomarker identifies, diagnoses, and / or predicts the patient as one who will benefit from a treatment comprising an anti-CD20 antibody. Alternatively, an amount or level of a Th2 biomarker in a sample that is below the amount or level of a reference Th2 biomarker identifies, diagnoses, and / or predicts the patient as one who is unlikely to benefit from treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0241] In some instances, an individual having a lymphoma (e.g., a B cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B cell lymphoma (e.g., germinal center B cell-like or activated B cell-like diffuse large B cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)) has not been previously treated for the lymphoma (treatment naive). For example, in some instances, an individual having a lymphoma has not previously received an anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0242] In some examples, an individual with a lymphoma (e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)) has previously received treatment for the lymphoma. In some examples, an individual with a lymphoma has previously received treatment including an anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0243] (iv) therapeutic benefit A patient who benefits from treatment with an anti-CD20 antibody (e.g., obinutuzumab or rituximab) may, for example, experience a delay or prevention of the onset or recurrence of a lymphoma (e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)), alleviation of symptoms, a reduction in the direct or indirect pathological consequences of the cancer, prevention of metastasis, a slowing of disease progression, an improvement or palliation of the condition, or a remission or improved prognosis. In some examples, the treatments described herein are used to delay the onset of cancer or delay the progression of a lymphoma (e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)). In some examples, the benefit can be an increase in overall survival (OS), progression-free survival (PFS), complete remission (CR), partial remission (PR), or a combination thereof.

[0244] In some examples, an amount or level of a Th2 biomarker that exceeds a reference amount or level of a Th2 biomarker (e.g., the amount or level of a Th2 biomarker in a reference population) identifies the individual as one who may benefit from a treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab), where the benefit is an increase in OS (e.g., 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) compared to a treatment that does not include an anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0245] In some examples, an amount or level of a Th2 biomarker that exceeds a reference amount or level of a Th2 biomarker (e.g., the amount or level of a Th2 biomarker in a reference population) identifies the individual as one who may benefit from a treatment comprising an anti-CD20 antibody (e.g., obinutuzumab or rituximab), where the benefit is an increase in PFS (e.g., 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) compared to a treatment that does not include an anti-CD20 antibody (e.g., obinutuzumab or rituximab).

[0246] C. Determination of Macrophage Biomarkers (i) Detection method The macrophage biomarkers described herein may be based on the amount or expression level of nucleic acids (e.g., mRNA), proteins, or cells (e.g., macrophages, e.g., M1 macrophages). The presence and / or expression level / amount of the genes described herein (see, e.g., Tables 1-4) can be qualitatively and / or quantitatively determined based on any suitable criteria known in the art, including, but not limited to, DNA, mRNA, cDNA, protein, protein fragments, and / or gene copy number. The presence and / or level / amount of the cells described herein can be qualitatively and / or quantitatively determined based on any suitable criteria known in the art, including, but not limited to, microscopy, cytometry, DNA, mRNA, cDNA, protein, protein fragments, and / or gene copy number. [Table 1] [Table 2] JPEG2024178150000004.jpg251170JPEG2024178150000005.jpg251170 [Table 3] JPEG2024178150000007.jpg251170JPEG2024178150000008.jpg252170JPEG202 4178150000009.jpg251170JPEG2024178150000010.jpg251170JPEG20241781500 00011.jpg251170JPEG2024178150000012.jpg255170JPEG2024178150000013.j pg255170JPEG2024178150000014.jpg255170JPEG2024178150000015.jpg255170 [Table 4] JPEG2024178150000017.jpg255170JPEG2024178150000018.jpg255170JPEG20241781500 00019.jpg255170JPEG2024178150000020.jpg255170JPEG2024178150000021.jpg255170 JPEG2024178150000022.jpg255170JPEG2024178150000023.jpg255170JPEG20241781500 00024.jpg255170JPEG2024178150000025.jpg255170JPEG2024178150000026.jpg255170

[0247] In some examples, the nucleic acid expression levels of the genes described herein can be measured by polymerase chain reaction (PCR)-based assays, such as quantitative PCR, real-time PCR, quantitative real-time PCR (qRT-PCR), reverse transcriptase PCR (RT-PCR), and reverse transcriptase quantitative PCR (RT-qPCR). Platforms for performing quantitative PCR assays include Fluidigm (e.g., BIOMARK™ HD System). Other amplification-based methods include, for example, transcription-mediated amplification (TMA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), and signal amplification methods, such as bDNA.

[0248] In some examples, nucleic acid expression levels of the genes described herein can also be measured by sequencing-based techniques such as, for example, RNA-seq, serial analysis of gene expression (SAGE), high-throughput sequencing techniques (e.g., massively parallel sequencing), and Sequenom MassARRAY® technology. Nucleic acid expression levels can also be measured by, for example, NanoString nCounter and high-coverage expression profiling (HiCEP). Additional protocols for assessing the status of genes and gene products can be found, for example, in Part 2 (Northern blotting), Part 4 (Southern blotting), Part 15 (immunoblotting), and Part 18 (PCR analysis) of Ausubel et al., eds., 1995, Current Protocols In Molecular Biology.

[0249] Other methods for detecting nucleic acid levels of the genes described herein include protocols for testing or detecting mRNA, such as target mRNA, in tissue or cell samples using microarray technology. Using nucleic acid microarrays, test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to generate cDNA probes. The probes are then hybridized to an array of nucleic acids immobilized on a solid support. The array is configured so that the sequence and location of each member of the array is known. Hybridization of a labeled probe with a specific array member indicates that the sample from which the probe was derived expresses that gene.

[0250] Primers and probes may be labeled with a detectable marker, such as, for example, a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator, or an enzyme. Such probes and primers can be used to detect the presence of expressed genes (e.g., genes described herein). As one of skill in the art will appreciate, many different primers and probes can be prepared based on the sequences provided herein (or, in the case of genomic DNA, their flanking sequences) and used to effectively amplify, clone, and / or determine the presence and / or expression levels of the genes described herein.

[0251] Other methods for detecting nucleic acid expression levels of the genes described herein include electrophoresis, Northern and Southern blot analysis, in situ hybridization (e.g., single or multiplex nucleic acid in situ hybridization), RNAse protection assays, and microarrays (e.g., Illumina BEADARRAY™ technology; bead arrays for detecting gene expression (BADGE)).

[0252] In some examples, macrophage biomarkers can be analyzed by many methodologies, including, but not limited to, RNA-seq, PCR, RT-qPCR, qPCR, multiplex qPCR, multiplex RT-qPCR, NANOSTRING® nCOUNTER® gene expression assays, microarray analysis, serial analysis of gene expression (SAGE), Northern blot analysis, MassARRAY, ISH, whole genome sequencing, FACS, spatial transcriptomics, spatial proteomics, Western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blotting, immunodetection, surface plasmon resonance, optical spectroscopy, mass spectrometry, and HPLC, or a combination thereof.

[0253] (ii) RT-qPCR In some examples, the nucleic acid expression levels of the genes described herein (e.g., genes in the M1 macrophage gene signature set or genes in the gene signature matrix) can be detected using reverse transcription quantitative polymerase chain reaction (RT-qPCR). The RT-qPCR technique is a form of PCR in which the nucleic acid to be amplified is RNA, which is first reverse-transcribed into cDNA, and the amount of PCR product is measured at each step of the PCR reaction. Because RNA cannot be used as a template for PCR, the first step in gene expression profiling by PCR is the reverse transcription of the RNA template into cDNA, followed by its amplification in a PCR reaction. For example, reverse transcriptases can include avilo myeloblastosis virus reverse transcriptase (AMY-RT) or Moloney murine leukemia virus reverse transcriptase (MMLV-RT). The reverse transcription step is typically primed using specific primers, random hexamers, or oligo-dT primers, depending on the situation and the goal of expression profiling. For example, extracted RNA can be reverse transcribed using the GENEAMP™ RNA PCR kit (Perkin Elmer, California, USA) according to the manufacturer's instructions. The resulting cDNA can then be used as a template in a subsequent PCR reaction.

[0254] One variation of the PCR technique is quantitative real-time PCR (qRT-PCR), which measures the accumulation of PCR products through dual-labeled fluorescent probes (i.e., TAQMAN® probes). Quantitative real-time polymerase chain reaction technology refers to a form of PCR in which the amount of PCR product is measured at each step of the PCR reaction. This technique has been described in various publications, including Cronin et al., Am. J. Pathol. 164(1):35-42 (2004); and Ma et al., Cancer Cell 5:607-616 (2004). Real-time PCR is compatible with quantitative competitive PCR, which uses internal competitors for each target sequence for normalization, and / or quantitative comparative PCR, which uses normalization genes contained within the sample or housekeeping genes for PCR. For further details, see, for example, Held et al., Genome Research 6:986-994 (1996).

[0255] Representative protocol steps for profiling gene expression using fixed, paraffin-embedded tissue as an RNA source, such as mRNA isolation, purification, primer extension, and amplification, are provided in various published journal articles (e.g., Godfrey et al., Malec. Diagnostics 2:84-91 (2000); Specht et al., Am. J. Pathol. 158:419-29 (2001)). Briefly, a representative process begins with cutting sections (e.g., 10 microgram sections) of a paraffin-embedded tumor tissue sample. RNA is then extracted and protein and DNA are removed. After analyzing the RNA concentration, RNA repair and / or amplification steps may be included as needed, in which the RNA is reverse transcribed using a gene-specific promoter, followed by PCR.

[0256] Nucleic acid expression levels determined by amplification-based methods (e.g., RT-qPCR) can be expressed as cycle threshold (Ct). From this value, the normalized expression level of each gene can be determined, for example, using the delta Ct (dCt) method as follows: Ct(control / reference gene) - Ct(gene of interest / target gene) = dCt(gene of interest / target gene). Those skilled in the art will understand that the obtained dCt value can be a negative dCt value or a positive dCt value. As defined herein, a higher dCt value indicates a higher expression level of the gene of interest compared to the control gene. Conversely, a lower dCt value indicates a lower expression level of the gene of interest compared to the control gene. When the expression levels of multiple genes are determined, for example, the expression levels of each gene expressed as a dCt value can be used to determine a single value representing the aggregate or composite expression level of the multiple genes (e.g., genes in an M1 macrophage gene signature set). The expression level of the aggregate or complex may be the mean or median dCt determined for each target gene / gene of interest. As defined herein, a higher mean dCt or median dCt indicates a higher aggregate expression level of the target genes compared to the control gene(s). A lower mean dCt or median dCt indicates a lower aggregate expression level of the target genes compared to the control gene(s). The expression level may be compared to a reference level.

[0257] In one particular example, the nucleic acid expression levels described herein can be determined using a method including: (a) obtaining or providing a sample from an individual (samples include tumor tissue samples (e.g., paraffin-embedded, formalin-fixed tumor tissue samples); (b) isolating mRNA from the sample; (c) reverse transcribing the mRNA into cDNA (e.g., for at least one of the genes described herein (e.g., a gene in the M1 macrophage gene signature set or a gene in the gene signature matrix)); (d) using PCR to amplify cDNA (e.g., for at least one of the genes described herein (e.g., a gene in the M1 macrophage gene signature set or a gene in the gene signature matrix)); and (e) quantifying nucleic acid expression levels (e.g., for at least one of the genes described herein (e.g., a gene in the M1 macrophage gene signature set or a gene in the gene signature matrix)).

[0258] Depending on the primers or probes used, one or more genes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more genes (e.g., 55, 82, 89, 106, 153, or 170 genes)) can be detected in a single assay. Furthermore, assays can be performed across one or more tubes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more tubes (e.g., 55, 82, 89, 106, 153, or 170 tubes)).

[0259] In some examples, the method further includes (f) normalizing the nucleic acid expression level of a gene (e.g., at least one of the genes described herein (e.g., a gene in an M1 macrophage gene signature set or a gene in a gene signature matrix)) in the sample to the expression level of one or more reference genes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or more reference genes, e.g., housekeeping genes (e.g., β-actin)). For example, RT-qPCR can be used to analyze the expression levels of a gene described herein (e.g., at least one of the genes described herein (e.g., a gene in an M1 macrophage gene signature set or a gene in a gene signature matrix)) to generate an expression level that reflects a normalized average dCT value for the analyzed genes.

[0260] (iii) RNA-seq and microarray In some examples, the nucleic acid expression levels of genes described herein (e.g., genes in the M1 macrophage gene signature set or genes in the gene signature matrix) can be detected using RNA-seq. RNA-seq, also known as whole transcriptome shotgun sequencing (WTSS), refers to the use of high-throughput sequencing technology to sequence and / or quantify cDNA to obtain information about the RNA content of a sample. Publications describing RNA-seq include Wang et al., "RNA-Seq: a revolutionary tool for transcriptomics," Nature Reviews Genetics 10(1):57-63 (January 2009); Ryan et al., BioTechniques 45(1):81-94 (2008); and Maher et al., "Transcriptome sequencing to detect gene fusions in cancer," Nature 458(7234):97-101 (January 2009). In some examples, quality control of the sequencing is performed. In some instances, counts are normalized to transcripts per million (TPM).

[0261] (a) Marker gene approach The marker gene approach uses the expression of one or more genes within a gene signature set (see, e.g., Tables 1 and 2) to determine a macrophage biomarker (e.g., a high number of M1 macrophages in a sample). In some examples, the marker gene approach uses xCell (see, e.g., Aran et al. Genome Biol. 18(1):220(2017)).

[0262] The exemplary gene signature sets in Table 1 may be modified to remove, replace, or add genes. In some examples, the number of genes in any of the exemplary gene signature sets may be increased or decreased by one or more genes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more genes). In some examples, the number of genes in any of the exemplary gene signature sets may be increased or decreased by between about 5% and about 20% (e.g., 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%). In some examples, genes from any of the exemplary gene signature sets may be replaced with genes in the same signaling pathway. In some examples, genes from any of the example gene signature sets can be added to a different example gene signature set (e.g., genes from gene signature set 1 (e.g., C1QA) can be added to gene signature set 2) to generate a gene signature set. In some examples, a gene signature set can include genes present in all of the example gene signature sets (i.e., ACP2 and ADAMDEC1) or most of the example gene signature sets (e.g., FDX1, CD163, HAMP, ABCD1, C1QA, CCL22, and TREM2). In some examples, a gene signature set includes ACP2 and ADAMDEC1. In some examples, a gene signature set includes ACP2, ADAMDEC1, and FDX1. In some examples, a gene signature set includes ACP2, ADAMDEC1, and CD163. In some examples, the gene signature set includes ACP2, ADAMDECl, and HAMP. In some examples, the gene signature set includes ACP2, ADAMDECl, FDX1, and CD163. In some examples, the gene signature set includes ACP2, ADAMDECl, FDX1, and HAMP. In some examples, the gene signature set includes ACP2, ADAMDECl, CD163, and HAMP.In some examples, the gene signature set includes ACP2, ADAMDECl, FDX1, CD163, and HAMP. In some examples, the gene signature set includes ACP2, ADAMDECl, FDX1, CD163, HAMP, and ABCD1. In some examples, the gene signature set includes ACP2, ADAMDECl, FDX1, CD163, HAMP, and CCL22. In some examples, the gene signature set includes ACP2, ADAMDECl, FDX1, CD163, HAMP, and C1QA. In some examples, the gene signature set includes ACP2, ADAMDECl, FDX1, CD163, HAMP, and TREM2. In some examples, the gene signature set includes ACP2, ADAMDECl, FDX1, CD163, HAMP, ABCD1, and CCL22.

[0263] The exemplary gene signature sets in Table 2 may be modified to remove, replace, or add genes. In some examples, the number of genes in any of the exemplary gene signature sets may be increased or decreased by one or more genes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more genes). In some examples, the number of genes in any of the exemplary gene signature sets may be increased or decreased by between about 5% and about 20% (e.g., 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%). In some examples, genes from any of the exemplary gene signature sets may be replaced with genes in the same signaling pathway. In some examples, genes from any of the exemplified gene signature sets can be added to a different exemplified gene signature set (e.g., genes from gene signature set 1 (e.g., CD48) can be added to gene signature set 4) to generate a gene signature set. In some examples, a gene signature set can include genes present in the most exemplified gene signature sets (e.g., ACP2, FDX1, HK3, MSR1, CD84, SDS, VSIG4, CLEC5A, ADAMDEC1, HAMP, DNASE2B, and CYBB). In some examples, a gene signature set includes ACP2. In some examples, a gene signature set includes FDX1. In some examples, a gene signature set includes HK3. In some examples, a gene signature set includes MSR1. In some examples, a gene signature set includes CD84. In some examples, a gene signature set includes SDS. In some examples, the gene signature set includes VSIG4. In some examples, the gene signature set includes CLEC5A. In some examples, the gene signature set includes ADAMDEC1. In some examples, the gene signature set includes HAMP. In some examples, the gene signature set includes DNASE2B. In some examples, the gene signature set includes CYBB.In some examples, the gene signature set includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of ACP2, FDX1, HK3, MSR1, CD84, SDS, VSIG4, CLEC5A, ADAMDEC1, HAMP, DNASE2B, and CYBB. In some examples, the gene signature set includes ACP2, FDX1, HK3, MSR1, CD84, SDS, VSIG4, CLEC5A, ADAMDEC1, HAMP, DNASE2B, and CYBB.

[0264] Representative protocol steps for determining macrophage numbers (e.g., M1 macrophages) using an RNA-seq marker gene approach can be found in Aran et al. Genome Biol. 18(1):220 (2017). Briefly, sequence scores obtained from a sample (e.g., preprocessed raw sequence reads) are transformed based on a power function derived from a synthetic mixture of a cell type (e.g., M1 macrophages) and a control cell type (e.g., multipotent progenitor cells or endothelial cells) within a range (e.g., 0.8%-25.6%) based on the expected abundance of the cell type present in the sample. The transformed scores are further adjusted using a spillover compensation matrix (constrained to 0.5 off the diagonal) derived from a synthetic mixture of 25% of the cell type (e.g., M1 macrophages) and 75% of the control cell type (e.g., multipotent progenitor cells or endothelial cells). The final adjusted score represents the proportion of the cell type present in the sample.

[0265] (b) Deconvolution approach The deconvolution approach uses the expression of one or more genes in a gene signature matrix (see, e.g., Tables 3 and 4) to determine a macrophage biomarker (e.g., a high number of M1 macrophages in a sample). In some examples, the deconvolution approach uses quanTIseq (see, e.g., Finotello et al. Genome Med. 11(1):34(2019)).

[0266] Representative protocol steps for determining the number of M1 macrophages using an RNA-seq deconvolution approach can be found in Finotello et al. Genome Med. 11(1):34 (2019). Briefly, sequence scores (e.g., preprocessed raw sequence reads) obtained from a sample are normalized and deconvolved using a gene signature matrix (see, e.g., Tables 3 and 4) to identify cell types present in the sample (e.g., M1 macrophages, M2 macrophages, B cells, monocytes, neutrophils, NK cells, non-regulatory CD4 + T cells, CD8 + Calculate the percentage of cells (e.g., T cells, regulatory T cells, dendritic cells, or other cell types) that are present in the sera.

[0267] (iv) Immunohistochemistry In some examples, macrophages (e.g., M1 macrophages) can be detected using immunohistochemistry (IHC). In some examples, any of the genes described herein (e.g., CD68, a gene in the M1 macrophage gene signature set, or a gene in the gene signature matrix) can be used to identify macrophages (e.g., M1 macrophages) or distinguish macrophages from other cell types. In some examples, an antibody specific for any of the genes described herein (e.g., CD68, a gene in the M1 macrophage gene signature set, or a gene in the gene signature matrix) is used as a primary antibody in an IHC assay. In some examples, a horseradish peroxidase (HRP)-conjugated secondary antibody is used in an IHC assay. In some examples, the signal from an IHC assay is compared to an IHC assay performed using a negative control antibody. In some examples, macrophages (e.g., M1 macrophages) can be detected using an antibody that binds to CD68.

[0268] (v) Flow cytometry In some examples, macrophages (e.g., M1 macrophages) can be detected using flow cytometry. In some examples, any of the genes described herein (e.g., genes in an M1 macrophage gene signature set or genes in a gene signature matrix) can be used to identify macrophages (e.g., M1 macrophages) or distinguish macrophages from other cell types. In some examples, an antibody specific for any of the genes described herein (e.g., genes in an M1 macrophage gene signature set or genes in a gene signature matrix) is used to label macrophages (e.g., M1 macrophages).

[0269] (vi) Sample Samples can be taken from individuals suspected of having or diagnosed with lymphoma and therefore likely to require treatment, or from healthy individuals not suspected of having lymphoma or who do not have lymphoma but have a family history of lymphoma. For the assessment of gene expression, samples such as those containing cells or proteins or nucleic acids produced by these cells may be used in the methods of the present invention. The expression level of a gene can be determined by assessing the amount (e.g., absolute amount or concentration) of a marker in a sample (e.g., a tissue sample, e.g., a tumor tissue sample such as a biopsy). Additionally, the level of a gene can be assessed in a bodily fluid or secretion containing a detectable level of the gene. Examples of bodily fluids or secretions useful as samples in the present invention include, for example, blood, urine, saliva, stool, pleural effusion, lymph, sputum, ascites, prostatic fluid, cerebrospinal fluid (CSF), or any other bodily secretion or derivative thereof. The term "blood" is meant to include whole blood, plasma, serum, or any blood derivative. Such assessment of genes in bodily fluids or secretions may be preferred in situations where invasive sampling methods are inappropriate or inconvenient. In other embodiments, tumor tissue samples are preferred.

[0270] Samples can be frozen, fresh, fixed (e.g., formalin-fixed), centrifuged, and / or embedded (e.g., paraffin-embedded), etc. Cell samples can be subjected to a variety of well-known post-collection preparation and storage techniques (e.g., nucleic acid and / or protein extraction, fixation, storage, freezing, ultrafiltration, concentration, evaporation, centrifugation, etc.) prior to assessing the amount of marker in the sample. Similarly, biopsies can be subjected to post-collection preparation and storage techniques, e.g., fixation, such as formalin fixation.

[0271] In a specific example, the sample is a clinical sample. In another example, the sample is used in a diagnostic assay, such as a diagnostic assay or method of the present invention. In some examples, the sample is obtained from a primary tumor or a metastatic tumor. Tissue biopsy is often used to obtain a representative piece of tumor tissue. Alternatively, tumor cells can be obtained indirectly in the form of tissue or fluid known or believed to contain the tumor cells of interest. For example, a sample of a lymphoma lesion can be obtained by resection, fine needle aspiration, pleural effusion, or blood. Genes or gene products can be detected from cancer or tumor tissue or from other body samples, such as urine, sputum, serum, or plasma. The same techniques discussed above for detecting target genes or gene products in cancerous samples can be applied to other body samples. Cancer cells can shed from cancer lesions and appear in such body samples. By screening such body samples, simple and early diagnosis of these cancers can be achieved. In addition, the progress of therapeutic approaches can be more easily monitored by testing such body samples for target genes or gene products.

[0272] In some examples, the sample from the individual is a tissue sample, a whole blood sample, a plasma sample, a serum sample, or a combination thereof. In some examples, the sample is a tissue sample. In some examples, the sample is a tumor tissue sample. In some examples, the sample is obtained before treatment. In some examples, the tissue sample is a formalin-fixed and paraffin-embedded (FFPE) sample, an archived sample, a fresh sample, or a frozen sample. In some examples, the sample from the individual is a tissue sample. In some examples, the tissue sample is a tumor tissue sample (e.g., biopsy tissue). In some examples, the tumor tissue sample includes tumor cells, tumor-infiltrating immune cells, stromal cells, normal adjacent tissue (NAT) cells, or a combination thereof. In some examples, the tissue sample is a biopsy material. In some examples, the tissue sample is blood cells, lymph nodes, or bone / bone marrow.

[0273] In some examples, the tumor tissue sample is extracted from a malignant cancerous tumor (i.e., cancer). In some examples, the cancer is a solid tumor or a non-solid or soft tissue tumor. In some examples, the tumor tissue sample is a lymphoma (e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)) sample.

[0274] (vii)RNA extraction Prior to detecting the level of nucleic acid, mRNA can be isolated from the target sample. In some instances, the mRNA is total RNA isolated from a tumor or tumor cell line, or alternatively, from a normal tissue or cell line. When the source of mRNA is a primary tumor, mRNA can be extracted, for example, from frozen or archived paraffin-embedded and fixed (e.g., formalin-fixed) tissue samples. General methods for mRNA extraction are well known in the art and are disclosed in standard molecular biology textbooks, including Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997). Methods for RNA extraction from paraffin-embedded tissue are disclosed, for example, in Rupp and Locker, Lab Invest. 56:A67 (1987), and De Andres et al., BioTechniques 18:42044 (1995). In particular, RNA isolation can be performed using purification kits, buffer sets, and proteases from commercial manufacturers such as Qiagen, according to the manufacturer's instructions. For example, total RNA from cells in culture can be isolated using Qiagen RNeasy mini-columns. Other commercially available RNA isolation kits include MASTERPURE® Complete DNA and RNA Purification Kit (EPICENTRE®, Madison, Wisconsin) and Paraffin Block RNA Isolation Kit (Ambion, Inc.). Total RNA from tissue samples can be isolated, for example, by using RNA Stat-60 (TelTest). RNA prepared from tumor tissue samples can also be isolated, for example, by cesium chloride density gradient centrifugation.

[0275] (viii) Expression level The expression level may reflect the expression level of one or more genes described herein (e.g., one or more genes in the M1 macrophage gene signature set or one or more genes in the gene signature matrix). In certain examples, the detected expression level of each gene is normalized using any one of standard normalization methods known in the art. Those skilled in the art will understand that the normalization method used may depend on the gene expression methodology used (e.g., one or more housekeeping genes may be used for normalization in terms of RT-qPCR methodology, while the whole genome or substantially the whole genome may be used as the normalization baseline in terms of RNA-seq methodology). For example, the detected expression level of each assayed gene may be normalized for both differences in the amount of the assayed gene(s), variations in the quality of the samples used, and / or variations between assay runs.

[0276] In some examples, normalization can be achieved by detecting the expression of one or more specific normalizing genes, including reference gene(s) (e.g., housekeeping genes (e.g., β-actin)). For example, in some examples, nucleic acid expression levels detected using the methods described herein (e.g., for at least one of the genes described herein (e.g., genes in the M1 macrophage gene signature set or genes in the gene signature matrix)) can be normalized to the expression levels of one or more reference genes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or more reference genes, e.g., housekeeping genes (e.g., β-actin)). Alternatively, normalization can be performed based on the mean or median signal of all assayed genes. For each gene, the measured normalized mRNA amount can be compared to the amount found at a reference expression level. The measured presence and / or expression level / amount in a particular subject sample being analyzed is expressed as a percentile within this range, which can be determined by methods well known in the art.

[0277] In other instances, the detected expression levels of each assayed gene are not normalized to determine the expression level.

[0278] The expression level may reflect a single gene or an aggregate or complex expression level of multiple genes described herein (e.g., for at least one of the genes described herein (e.g., a gene in the M1 macrophage gene signature set or a gene signature matrix)). Any statistical method known in the art can be used to determine the expression level.

[0279] For example, the expression level may reflect a median expression level, a mean expression level, or a number reflecting an aggregated Z-score expression level for the combination of genes assayed (e.g., for at least one of the genes described herein (e.g., a gene in an M1 macrophage gene signature set or a gene signature matrix)).

[0280] In some examples, the expression level reflects a median normalized expression level, a mean normalized expression level, or a number reflecting an aggregated Z-score normalized expression level for a combination of genes assayed (e.g., for at least one of the genes described herein (e.g., genes in an M1 macrophage gene signature set or genes in a gene signature matrix)).

[0281] D. Determination of Th2 biomarkers (i) Detection method The Th2 biomarkers described herein can be based on the amount or expression level of nucleic acids (e.g., mRNA), proteins, or cells (e.g., T cells, e.g., Th2 cells). The presence and / or expression level / amount of the genes described herein (see, e.g., Table 5) can be qualitatively and / or quantitatively determined based on any suitable criteria known in the art, including, but not limited to, DNA, mRNA, cDNA, protein, protein fragments, and / or gene copy number. The presence and / or level / amount of the cells described herein can be qualitatively and / or quantitatively determined based on any suitable criteria known in the art, including, but not limited to, microscopy, cytometry, DNA, mRNA, cDNA, protein, protein fragments, and / or gene copy number. [Table 5]

[0282] In some examples, the nucleic acid expression levels of the genes described herein can be measured by polymerase chain reaction (PCR)-based assays, such as quantitative PCR, real-time PCR, quantitative real-time PCR (qRT-PCR), reverse transcriptase PCR (RT-PCR), and reverse transcriptase quantitative PCR (RT-qPCR). Platforms for performing quantitative PCR assays include Fluidigm (e.g., BIOMARK™ HD System). Other amplification-based methods include, for example, transcription-mediated amplification (TMA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), and signal amplification methods, such as bDNA.

[0283] In some examples, nucleic acid expression levels of the genes described herein can also be measured by sequencing-based techniques such as, for example, RNA-seq, serial analysis of gene expression (SAGE), high-throughput sequencing techniques (e.g., massively parallel sequencing), and Sequenom MassARRAY® technology. Nucleic acid expression levels can also be measured by, for example, NanoString nCounter and high-coverage expression profiling (HiCEP). Additional protocols for assessing the status of genes and gene products can be found, for example, in Part 2 (Northern blotting), Part 4 (Southern blotting), Part 15 (immunoblotting), and Part 18 (PCR analysis) of Ausubel et al., eds., 1995, Current Protocols in Molecular Biology.

[0284] Other methods for detecting nucleic acid levels of the genes described herein include protocols for testing or detecting mRNA, such as target mRNA, in tissue or cell samples using microarray technology. Using nucleic acid microarrays, test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to generate cDNA probes. The probes are then hybridized to an array of nucleic acids immobilized on a solid support. The array is configured so that the sequence and location of each member of the array is known. Hybridization of a labeled probe with a specific array member indicates that the sample from which the probe was derived expresses that gene.

[0285] Primers and probes may be labeled with a detectable marker, such as, for example, a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator, or an enzyme. Such probes and primers can be used to detect the presence of expressed genes (e.g., genes described herein). As one of skill in the art will appreciate, many different primers and probes can be prepared based on the sequences provided herein (or, in the case of genomic DNA, their flanking sequences) and used to effectively amplify, clone, and / or determine the presence and / or expression levels of the genes described herein.

[0286] Other methods for detecting nucleic acid expression levels of the genes described herein include electrophoresis, Northern and Southern blot analysis, in situ hybridization (e.g., single or multiplex nucleic acid in situ hybridization), RNAse protection assays, and microarrays (e.g., Illumina BEADARRAY™ technology; bead arrays for detecting gene expression (BADGE)).

[0287] In some examples, Th2 biomarkers can be analyzed by many methodologies, including, but not limited to, RNA-seq, PCR, RT-qPCR, qPCR, multiplex qPCR, multiplex RT-qPCR, NANOSTRING® nCOUNTER® gene expression assays, microarray analysis, serial analysis of gene expression (SAGE), Northern blot analysis, MassARRAY, ISH, whole genome sequencing, FACS, spatial transcriptomics, spatial proteomics, Western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blotting, immunodetection, surface plasmon resonance, optical spectroscopy, mass spectrometry, and HPLC, or a combination thereof.

[0288] (ii) RT-qPCR In some examples, the nucleic acid expression levels of the genes described herein (e.g., genes in the Th2 cell gene signature set or genes in the gene signature matrix) can be detected using reverse transcription quantitative polymerase chain reaction (RT-qPCR). The RT-qPCR technique is a form of PCR in which the nucleic acid to be amplified is RNA, which is first reverse transcribed into cDNA, and the amount of PCR product is measured at each step of the PCR reaction. Because RNA cannot be used as a template for PCR, the first step in gene expression profiling by PCR is the reverse transcription of the RNA template into cDNA, followed by its amplification in a PCR reaction. For example, reverse transcriptases can include avilo myeloblastosis virus reverse transcriptase (AMY-RT) or Moloney murine leukemia virus reverse transcriptase (MMLV-RT). The reverse transcription step is typically primed using specific primers, random hexamers, or oligo-dT primers, depending on the situation and the goal of expression profiling. For example, extracted RNA can be reverse transcribed using the GENEAMP™ RNA PCR kit (Perkin Elmer, California, USA) according to the manufacturer's instructions. The resulting cDNA can then be used as a template in a subsequent PCR reaction.

[0289] One variation of the PCR technique is quantitative real-time PCR (qRT-PCR), which measures the accumulation of PCR products through dual-labeled fluorescent probes (i.e., TAQMAN® probes). Quantitative real-time polymerase chain reaction technology refers to a form of PCR in which the amount of PCR product is measured at each step of the PCR reaction. This technique has been described in various publications, including Cronin et al., Am. J. Pathol. 164(1):35-42 (2004); and Ma et al., Cancer Cell 5:607-616 (2004). Real-time PCR is compatible with quantitative competitive PCR, which uses internal competitors for each target sequence for normalization, and / or quantitative comparative PCR, which uses normalization genes contained within the sample or housekeeping genes for PCR. For further details, see, for example, Held et al., Genome Research 6:986-994 (1996).

[0290] Representative protocol steps for profiling gene expression using fixed, paraffin-embedded tissue as an RNA source, such as mRNA isolation, purification, primer extension, and amplification, are provided in various published journal articles (e.g., Godfrey et al., Malec. Diagnostics 2:84-91 (2000); Specht et al., Am. J. Pathol. 158:419-29 (2001)). Briefly, a representative process begins with cutting sections (e.g., 10 microgram sections) of a paraffin-embedded tumor tissue sample. RNA is then extracted and protein and DNA are removed. After analyzing the RNA concentration, RNA repair and / or amplification steps may be included as needed, in which the RNA is reverse transcribed using a gene-specific promoter, followed by PCR.

[0291] Nucleic acid expression levels determined by amplification-based methods (e.g., RT-qPCR) can be expressed as cycle threshold (Ct). From this value, the normalized expression level of each gene can be determined, for example, using the delta Ct (dCt) method as follows: Ct(control / reference gene) - Ct(gene of interest / target gene) = dCt(gene of interest / target gene). Those skilled in the art will understand that the obtained dCt value can be a negative dCt value or a positive dCt value. As defined herein, a higher dCt value indicates a higher expression level of the gene of interest compared to the control gene. Conversely, a lower dCt value indicates a lower expression level of the gene of interest compared to the control gene. When the expression levels of multiple genes are determined, for example, the expression level of each gene expressed as a dCt value can be used to determine a single value representing the aggregate or complex expression level of the multiple genes (e.g., genes in a Th2 cell gene signature set). The aggregate or complex expression level can be the average or median dCt value determined for each target gene / gene of interest. As defined herein, a higher mean dCt or median dCt indicates a higher aggregate expression level of the target genes compared to the control gene(s). A lower mean dCt or median dCt indicates a lower aggregate expression level of the target genes compared to the control gene(s). Expression levels can be compared to a reference level.

[0292] In one particular example, the nucleic acid expression levels described herein can be determined using a method including: (a) obtaining or providing a sample from an individual (samples include tumor tissue samples (e.g., paraffin-embedded, formalin-fixed tumor tissue samples); (b) isolating mRNA from the sample; (c) reverse transcribing the mRNA into cDNA (e.g., for at least one of the genes described herein (e.g., a gene in a Th2 cell gene signature set or a gene in a gene signature matrix)); (d) using PCR to amplify cDNA (e.g., for at least one of the genes described herein (e.g., a gene in a Th2 cell gene signature set or a gene in a gene signature matrix)); and (e) Quantifying nucleic acid expression levels (e.g., for at least one of the genes described herein (e.g., a gene in a Th2 cell gene signature set or a gene in a gene signature matrix)).

[0293] Depending on the primers or probes used, one or more genes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more genes (e.g., 55, 82, 89, 106, 153, or 170 genes)) can be detected in a single assay. Furthermore, assays can be performed across one or more tubes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more tubes (e.g., 55, 82, 89, 106, 153, or 170 tubes)).

[0294] In some examples, the method further includes (f) normalizing the nucleic acid expression level of a gene (e.g., at least one of the genes described herein (e.g., a gene in a Th2 cell gene signature set or a gene in a gene signature matrix)) in the sample to the expression level of one or more reference genes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or more reference genes, e.g., housekeeping genes (e.g., β-actin)). For example, RT-qPCR can be used to analyze the expression levels of a gene described herein (e.g., at least one of the genes described herein (e.g., a gene in a Th2 cell gene signature set or a gene in a gene signature matrix)) to generate an expression level that reflects a normalized average dCT value for the analyzed genes.

[0295] (iii) RNA-seq and microarray In some examples, the nucleic acid expression levels of the genes described herein (e.g., genes in a Th2 cell gene signature set or genes in a gene signature matrix) can be detected using RNA-seq. RNA-seq, also known as whole transcriptome shotgun sequencing (WTSS), refers to the use of high-throughput sequencing technology to sequence and / or quantify cDNA to obtain information about the RNA content of a sample. Publications describing RNA-seq include Wang et al., "RNA-Seq: a revolutionary tool for transcriptomics," Nature Reviews Genetics 10(1):57-63 (January 2009); Ryan et al., BioTechniques 45(1):81-94 (2008); and Maher et al., "Transcriptome sequencing to detect gene fusions in cancer," Nature 458(7234):97-101 (January 2009). In some examples, quality control of the sequencing is performed. In some instances, counts are normalized to transcripts per million (TPM).

[0296] (c) Marker gene approach The marker gene approach uses the expression of one or more genes in a gene signature set to determine a Th2 biomarker (e.g., the number of Th2 cells in a sample). In some examples, the marker gene approach uses xCell (see, e.g., Aran et al. Genome Biol. 18(1):220(2017)).

[0297] The exemplary gene signature sets in Table 5 may be modified to remove, replace, or add genes. In some examples, the number of genes in any of the exemplary gene signature sets may be increased or decreased by one or more genes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more genes). In some examples, the number of genes in any of the exemplary gene signature sets may be increased or decreased by between about 5% and about 20% (e.g., 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%). In some examples, genes from any of the exemplary gene signature sets may be replaced with genes in the same signaling pathway. In some examples, genes from any of the exemplified gene signature sets can be added to a different exemplified gene signature set (e.g., genes from gene signature set 1 (e.g., GZMK) can be added to gene signature set 2) to generate a gene signature set. In some examples, a gene signature set can include genes present in the most exemplified gene signature set (e.g., IL5, IL13, BAG2, and CXCR6). In some examples, a gene signature set includes IL5 and IL13. In some examples, a gene signature set includes BAG2 and IL13. In some examples, a gene signature set includes BAG2 and CXCR6. In some examples, a gene signature set includes IL5, IL13, and BAG2. In some examples, a gene signature set includes IL5, IL13, and CXCR6. In some examples, a gene signature set includes IL13, BAG2, and CXCR6. In some examples, the gene signature set includes IL13, BAG2, and CXCR6. In some examples, the gene signature set includes IL5, IL13, BAG2, and CXCR6. In some examples, the gene signature set includes IL5, IL13, MAD2L1, BAG2, CXCR6, and CEP55.In some examples, the gene signature set includes IL5, IL13, MAD2L1, RRM2, BAG2, CXCR6, and CEP55. In some examples, the gene signature set includes GZMK, IL5, IL13, MAD2L1, BAG2, CXCR6, and CEP55.

[0298] Representative protocol steps for determining the number of Th2 cells using an RNA-seq marker gene approach can be found in Aran et al. Genome Biol. 18(1):220 (2017). Briefly, sequence scores obtained from a sample (e.g., preprocessed raw sequence reads) are transformed based on a power function derived from a synthetic mixture of a cell type (e.g., Th2 cells) and a control cell type (e.g., multipotent progenitor cells or endothelial cells) within a range (e.g., 0.8%-25.6%) based on the expected abundance of the cell type present in the sample. The transformed scores are further adjusted using a spillover compensation matrix (constrained to 0.5 off the diagonal) derived from a synthetic mixture of 25% of the cell type (e.g., Th2 cells) and 75% of the control cell type (e.g., multipotent progenitor cells or endothelial cells). The final adjusted score represents the proportion of the cell type present in the sample.

[0299] (d) Deconvolution approach The deconvolution approach uses the expression of one or more genes in the gene signature matrix to determine a Th2 biomarker (e.g., the number of Th2 cells in a sample). In some examples, the deconvolution approach uses quanTIseq (see, e.g., Finotello et al. Genome Med. 11(1):34(2019)).

[0300] Representative protocol steps for determining the number of Th2 cells using an RNA-seq deconvolution approach can be found in Finotello et al. Genome Med. 11(1):34 (2019). Briefly, sequence scores obtained from a sample (e.g., preprocessed raw sequence reads) are normalized and deconvolved using a gene signature matrix to identify the cell types present in the sample (e.g., Th2 cells, macrophages, M2 macrophages, B cells, monocytes, neutrophils, NK cells, non-regulatory CD4 + T cells, CD8 + Calculate the percentage of cells (e.g., T cells, regulatory T cells, dendritic cells, or other cell types) that are present in the sera.

[0301] (iv) Immunohistochemistry In some examples, T cells (e.g., Th2 cells) can be detected using immunohistochemistry (IHC). In some examples, any of the genes described herein (e.g., genes in a Th2 cell gene signature set or genes in a gene signature matrix) can be used to identify T cells (e.g., Th2 cells) or distinguish T cells from other cell types. In some examples, an antibody specific for any of the genes described herein is used as a primary antibody in an IHC assay. In some examples, a horseradish peroxidase (HRP)-conjugated secondary antibody is used in an IHC assay. In some examples, the signal from an IHC assay is compared to an IHC assay performed using a negative control antibody.

[0302] (v) Flow cytometry In some examples, flow cytometry can be used to detect T cells (e.g., Th2 cells). In some examples, any of the genes described herein (e.g., genes in a Th2 cell gene signature set or genes in a gene signature matrix) can be used to identify T cells (e.g., Th2 cells) or distinguish T cells from other cell types. In some examples, antibodies specific for any of the genes described herein are used to label T cells (e.g., Th2 cells).

[0303] (vi) Sample Samples can be taken from individuals suspected of having or diagnosed with lymphoma and therefore likely to require treatment, or from healthy individuals not suspected of having lymphoma or who do not have lymphoma but have a family history of lymphoma. For the assessment of gene expression, samples such as those containing cells or proteins or nucleic acids produced by these cells may be used in the methods of the present invention. The expression level of a gene can be determined by assessing the amount (e.g., absolute amount or concentration) of a marker in a sample (e.g., a tissue sample, e.g., a tumor tissue sample such as a biopsy). Additionally, the level of a gene can be assessed in a bodily fluid or secretion containing a detectable level of the gene. Bodily fluids or secretions useful as samples in the present invention include, for example, blood, urine, saliva, stool, pleural effusion, lymph, sputum, ascites, prostatic fluid, cerebrospinal fluid (CSF), or any other bodily secretion or derivative thereof. The term "blood" is meant to include whole blood, plasma, serum, or any blood derivative. Such assessment of genes in bodily fluids or secretions may be preferred in situations where invasive sampling methods are inappropriate or inconvenient. In other embodiments, tumor tissue samples are preferred.

[0304] Samples can be frozen, fresh, fixed (e.g., formalin-fixed), centrifuged, and / or embedded (e.g., paraffin-embedded), etc. Cell samples can be subjected to a variety of well-known post-collection preparation and storage techniques (e.g., nucleic acid and / or protein extraction, fixation, storage, freezing, ultrafiltration, concentration, evaporation, centrifugation, etc.) prior to assessing the amount of marker in the sample. Similarly, biopsies can be subjected to post-collection preparation and storage techniques, e.g., fixation, such as formalin fixation.

[0305] In a specific example, the sample is a clinical sample. In another example, the sample is used in a diagnostic assay, such as a diagnostic assay or method of the present invention. In some examples, the sample is obtained from a primary tumor or a metastatic tumor. Tissue biopsy is often used to obtain a representative piece of tumor tissue. Alternatively, tumor cells can be obtained indirectly in the form of tissue or fluid known or believed to contain the tumor cells of interest. For example, a sample of a lymphoma lesion can be obtained by resection, fine needle aspiration, pleural effusion, or blood. Genes or gene products can be detected from cancer or tumor tissue or from other body samples, such as urine, sputum, serum, or plasma. The same techniques discussed above for detecting target genes or gene products in cancerous samples can be applied to other body samples. Cancer cells can shed from cancer lesions and appear in such body samples. By screening such body samples, simple and early diagnosis of these cancers can be achieved. In addition, the progress of therapeutic approaches can be more easily monitored by testing such body samples for target genes or gene products.

[0306] In some examples, the sample from the individual is a tissue sample, a whole blood sample, a plasma sample, a serum sample, or a combination thereof. In some examples, the sample is a tissue sample. In some examples, the sample is a tumor tissue sample. In some examples, the sample is obtained before treatment. In some examples, the tissue sample is a formalin-fixed and paraffin-embedded (FFPE) sample, an archived sample, a fresh sample, or a frozen sample. In some examples, the sample from the individual is a tissue sample. In some examples, the tissue sample is a tumor tissue sample (e.g., biopsy tissue). In some examples, the tumor tissue sample includes tumor cells, tumor-infiltrating immune cells, stromal cells, normal adjacent tissue (NAT) cells, or a combination thereof. In some examples, the tissue sample is a biopsy material. In some examples, the tissue sample is blood cells, lymph nodes, or bone / bone marrow.

[0307] In some examples, the tumor tissue sample is extracted from a malignant cancerous tumor (i.e., cancer). In some examples, the cancer is a solid tumor or a non-solid or soft tissue tumor. In some examples, the tumor tissue sample is a lymphoma (e.g., a B-cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B-cell lymphoma (e.g., germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)) sample.

[0308] (vii) RNA extraction Prior to detecting the level of nucleic acid, mRNA can be isolated from the target sample. In some instances, the mRNA is total RNA isolated from a tumor or tumor cell line, or alternatively, from a normal tissue or cell line. When the source of mRNA is a primary tumor, mRNA can be extracted, for example, from frozen or archived paraffin-embedded and fixed (e.g., formalin-fixed) tissue samples. General methods for mRNA extraction are well known in the art and are disclosed in standard molecular biology textbooks, including Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997). Methods for RNA extraction from paraffin-embedded tissue are disclosed, for example, in Rupp and Locker, Lab Invest. 56:A67 (1987), and De Andres et al., BioTechniques 18:42044 (1995). In particular, RNA isolation can be performed using purification kits, buffer sets, and proteases from commercial manufacturers such as Qiagen, according to the manufacturer's instructions. For example, total RNA from cells in culture can be isolated using Qiagen RNeasy mini-columns. Other commercially available RNA isolation kits include MASTERPURE® Complete DNA and RNA Purification Kit (EPICENTRE®, Madison, Wisconsin) and Paraffin Block RNA Isolation Kit (Ambion, Inc.). Total RNA from tissue samples can be isolated, for example, by using RNA Stat-60 (TelTest). RNA prepared from tumor tissue samples can also be isolated, for example, by cesium chloride density gradient centrifugation.

[0309] (viii) Expression level The expression level may reflect the expression level of one or more genes (e.g., one or more genes in a Th2 cell gene signature set or one or more genes in a gene signature matrix) described herein. In a specific example, the detected expression level of each gene is normalized using any one of standard normalization methods known in the art. Those skilled in the art will understand that the normalization method used may depend on the gene expression methodology used (e.g., one or more housekeeping genes may be used for normalization in terms of RT-qPCR methodology, while the whole genome or substantially the whole genome may be used as the normalization baseline in terms of RNA-seq methodology). For example, the detected expression level of each assayed gene may be normalized for both differences in the amount of the assayed gene(s), variations in the quality of the samples used, and / or variations between assay runs.

[0310] In some examples, normalization can be achieved by detecting the expression of one or more specific normalizing genes, including reference gene(s) (e.g., housekeeping genes (e.g., β-actin)). For example, in some examples, nucleic acid expression levels detected using the methods described herein (e.g., for at least one of the genes described herein (e.g., genes in a Th2 cell gene signature set or genes in a gene signature matrix)) can be normalized to the expression levels of one or more reference genes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or more reference genes, e.g., housekeeping genes (e.g., β-actin)). Alternatively, normalization can be performed based on the mean or median signal of all assayed genes. For each gene, the measured normalized mRNA amount can be compared to the amount found at a reference expression level. The measured presence and / or expression level / amount in a particular subject sample being analyzed is expressed as a percentile within this range, which can be determined by methods well known in the art.

[0311] In other instances, the detected expression levels of each assayed gene are not normalized to determine the expression level.

[0312] The expression level may reflect a single gene or an aggregate or complex expression level of multiple genes described herein (e.g., for at least one of the genes described herein (e.g., a gene in a Th2 cell gene signature set or a gene in a gene signature matrix)). Any statistical method known in the art may be used to determine the expression level.

[0313] For example, the expression level may reflect a median expression level, a mean expression level, or a number reflecting an aggregated Z-score expression level for the combination of genes assayed (e.g., for at least one of the genes described herein (e.g., a gene in a Th2 cell gene signature set or a gene signature matrix)).

[0314] In some examples, the expression level reflects a median normalized expression level, a mean normalized expression level, or a number reflecting an aggregated Z-score normalized expression level for a combination of genes assayed (e.g., for at least one of the genes described herein (e.g., genes in a Th2 cell gene signature set or genes in a gene signature matrix)).

[0315] IV. Treatment Methods, Compositions and Uses Provided herein are methods, compositions, and uses thereof for treating a patient having a lymphoma (e.g., a B cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B cell lymphoma (e.g., germinal center B cell-like or activated B cell-like diffuse large B cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)), the method comprising administering to the patient an effective amount of an anti-CD20 antibody (e.g., obinutuzumab or rituximab) based on a macrophage biomarker (e.g., a gene expression value (e.g., a gene expression value from any of the gene signature sets described herein (e.g., any of the exemplary gene signature sets of Table 1 and Table 2)), or a macrophage abundance (e.g., M1 macrophages)) or a Th2 biomarker (e.g., a Th2 cell abundance) determined in a sample (e.g., a tissue sample, e.g., a tumor tissue sample, such as a biopsy) from the patient.

[0316] In some instances, the anti-CD20 antibody may be administered as a first line therapy. Alternatively, the anti-CD20 antibody may be administered as a second line therapy.

[0317] In any of the following sections, the lymphoma can be a B-cell lymphoma. In some examples, the B-cell lymphoma is a non-Hodgkin's lymphoma. In some examples, the non-Hodgkin's lymphoma is DLBCL. In some examples, the DLBCL is a germinal center B-cell-like or activated B-cell-like diffuse large B-cell lymphoma. In some examples, the non-Hodgkin's lymphoma is a marginal zone lymphoma. In some examples, the marginal zone lymphoma is an extranodal, nodal, or splenic marginal zone lymphoma. In some examples, the lymphoma is a low-grade lymphoma. In some examples, the lymphoma is a follicular lymphoma (FL). In some examples, the lymphoma is a chronic lymphocytic leukemia (CLL). In some examples, the lymphoma is a CD20-positive lymphoma.

[0318] In any of the following sections, the patient is a human. In some instances, the patient has not been previously treated. In some instances, the patient has been previously treated. In some instances, the patient has been previously treated with an anti-CD20 antibody. In some instances, the patient has not been previously treated with an anti-CD20 antibody.

[0319] In any of the following sections, the sample may be a tissue sample, a tumor sample, a whole blood sample, a plasma sample, or a serum sample. In some examples, the tissue sample is a tumor tissue sample. In some examples, the tumor tissue sample includes tumor cells, tumor-infiltrating immune cells, stromal cells, normal adjacent tissue (NAT) cells, or a combination thereof. In some examples, the tumor tissue sample is a biopsy. In some examples, the sample is an archived sample, a fresh sample, or a frozen sample.

[0320] In any of the following sections, macrophage biomarkers can be measured directly or indirectly. In some examples, the macrophage biomarker is a cell, a nucleic acid, a protein, a lipid, or a carbohydrate. In some examples, the macrophage biomarker is a gene expression value. In some examples, the macrophage biomarker is macrophage abundance. In some examples, the macrophage abundance is M1 macrophage abundance.

[0321] In any of the following sections, the Th2 biomarker can be measured directly or indirectly. In some examples, the Th2 biomarker is a cell, a nucleic acid, a protein, a lipid, or a carbohydrate. In some examples, the Th2 biomarker is a gene expression value. In some examples, the Th2 biomarker is the amount of T cells (e.g., the amount of Th2 cells). In some examples, the amount of T cells is the amount of Th2 cells.

[0322] A. Macrophage Biomarkers for Use in Therapeutic Methods In particular examples, methods of treating a patient having a lymphoma (e.g., a B cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B cell lymphoma (e.g., germinal center B cell-like or activated B cell-like diffuse large B cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)) with an effective amount of an anti-CD20 antibody (e.g., obinutuzumab or rituximab) are based on a macrophage biomarker (e.g., a gene expression value (e.g., a gene expression value from any of the gene signature sets described herein (e.g., any of the exemplary gene signature sets in Table 1 and Table 2)), or an amount of macrophages (e.g., M1 macrophages)) determined in a sample (e.g., a tissue sample, e.g., a tumor tissue sample, such as a biopsy) from the patient.

[0323] In one aspect, provided herein are methods of treating a patient having lymphoma (e.g., a B cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B cell lymphoma (e.g., germinal center B cell-like or activated B cell-like diffuse large B cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)), the method comprising: (a) detecting a macrophage biomarker (e.g., a gene expression level ... and (b) measuring a gene expression value from any of the gene signature sets described in Table 1 and Table 2 (e.g., any of the exemplary gene signature sets in Table 1 and Table 2), or an amount of macrophages (e.g., M1 macrophages), wherein the amount or level of the macrophage biomarker in the sample exceeds a reference macrophage biomarker amount or level; and (b) administering to the patient an effective amount of an anti-CD20 antibody based on the macrophage biomarker measured in step (a).

[0324] In another aspect, provided herein are methods of treating a patient having a lymphoma (e.g., a B cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B cell lymphoma (e.g., germinal center B cell-like or activated B cell-like diffuse large B cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)), the method comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein prior to treatment, it has been determined that the amount or level of a macrophage biomarker (e.g., a gene expression value (e.g., a gene expression value from any of the gene signature sets described herein (e.g., any of the exemplary gene signature sets in Table 1 and Table 2)), or the amount of macrophages (e.g., M1 macrophages)) in a sample (e.g., a tissue sample, e.g., a tumor tissue sample, such as a biopsy) from the patient exceeds the amount or level of a reference macrophage biomarker.

[0325] In another aspect, provided herein is a method of treating a patient having a lymphoma (e.g., a B cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B cell lymphoma (e.g., germinal center B cell-like or activated B cell-like diffuse large B cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)) and having an amount or level of a macrophage biomarker (e.g., a gene expression value (e.g., a gene expression value from any of the gene signature sets described herein (e.g., any of the exemplary gene signature sets in Table 1 and Table 2)), or an amount of macrophages (e.g., M1 macrophages)) in a sample (e.g., a tissue sample, e.g., a tumor tissue sample, such as a biopsy) from the patient that exceeds the amount or level of a reference macrophage biomarker, the method comprising administering to the patient an effective amount of an anti-CD20 antibody.

[0326] The amounts or levels of macrophage biomarkers that are determined in the various methods described herein are further described below.

[0327] (i) Increased macrophage biomarkers An amount or level of a macrophage biomarker in a sample from a patient with lymphoma (e.g., a B cell lymphoma, e.g., a non-Hodgkin's lymphoma, e.g., a diffuse large B cell lymphoma (e.g., germinal center B cell-like or activated B cell-like diffuse large B cell lymphoma) or a marginal zone lymphoma (e.g., extranodal, nodal, or splenic marginal zone lymphoma)) that exceeds the amount or level of a reference macrophage biomarker can determine to administer an anti-CD20 antibody (e.g., obinutuzumab or rituximab) to the patient.

[0328] For example, in some instances, the amount or level of a macrophage biomarker in a reference population may be in the upper 99th percentile (a prevalence level of about 1% or greater), the upper 95th percentile (a prevalence level of about 5% or greater), the upper 90th percentile (a prevalence level of about 10% or greater), the upper 85th percentile (a prevalence level of about 15% or greater), the upper 80th percentile (a prevalence level of about 20% or greater), the upper 75th percentile (a prevalence level of about 25% or greater), the upper 70th percentile (a prevalence level of about 30% or greater), the upper 65th percentile (a prevalence level of about 35% or greater), the upper 60th percentile (a prevalence level of about 40% or greater), the upper 55th percentile (a prevalence level of about 10% or greater), the upper 50th percentile (a prevalence level of about 50% or greater), the upper 45th percentile (a prevalence level of about 45 ...55th percentile (a prevalence level of about 10% or greater), the upper 50th percentile (a prevalence level of about 50% or greater), the upper 55th percentile (a prevalence level of about 10% or greater), the upper 50th percentile (a prevalence level of about 50% or greater), the upper 55 A method of treating a patient having an amount or level of a macrophage biomarker in a sample that is in the upper 50th percentile (a prevalence level of about 55% or greater), about 40th percentile (a prevalence level of about 60% or greater), about 35th percentile (a prevalence level of about 65% or greater), about 30th percentile (a prevalence level of about 70% or greater), about 25th percentile (a prevalence level of about 75% or greater), about 20th percentile (a prevalence level of about 80% or greater), about 15th percentile (a prevalence level of about 85% or greater), about 10th percentile (a prevalence level of about 90% or greater), about 5th percentile (a prevalence...

Claims

1. 1. A method for identifying, diagnosing, and / or predicting whether a patient having lymphoma may benefit from a treatment comprising an anti-CD20 antibody, the method comprising measuring a macrophage biomarker in a sample from the patient, wherein an amount or level of the macrophage biomarker that exceeds an amount or level of a reference macrophage biomarker in the sample identifies, diagnoses, and / or predicts the patient as one who may benefit from a treatment comprising an anti-CD20 antibody.

2. 1. A method for selecting a therapy for a patient having lymphoma, the method comprising measuring a macrophage biomarker in a sample from the patient, wherein an amount or level of the macrophage biomarker that exceeds an amount or level of a reference macrophage biomarker in the sample identifies the patient as one who may benefit from a therapy comprising an anti-CD20 antibody.

3. 3. The method of claim 1 or 2, wherein the patient has an amount or level of a macrophage biomarker in the sample that exceeds the amount or level of a reference macrophage biomarker, and the method further comprises administering to the patient an effective amount of an anti-CD20 antibody.

4. 1. A method of treating a patient having lymphoma, the method comprising: (a) measuring a macrophage biomarker in a sample from a patient, wherein the amount or level of the macrophage biomarker in the sample exceeds the amount or level of a reference macrophage biomarker; and (b) administering to the patient an effective amount of an anti-CD20 antibody based on the macrophage biomarker measured in step (a). A method comprising:

5. 1. A method of treating a patient having lymphoma, the method comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein prior to treatment, the amount or level of a macrophage biomarker in a sample from the patient has been determined to exceed the amount or level of a reference macrophage biomarker.

6. 1. A method of treating a patient having lymphoma and having an amount or level of a macrophage biomarker that exceeds the amount or level of a reference macrophage biomarker in a sample from the patient, comprising administering to the patient an effective amount of an anti-CD20 antibody.

7. The method of any one of claims 1 to 6, wherein the reference macrophage biomarker amount or level is a pre-assigned macrophage biomarker amount or level.

8. The method of any one of claims 1 to 7, wherein the amount or level of the reference macrophage biomarker is the amount or level of the macrophage biomarker in a reference population.

9. 9. The method of claim 8, wherein the amount or level of the macrophage biomarker in the reference population is the median amount or level of the macrophage biomarker in the reference population.

10. 9. The method of claim 8, wherein the reference macrophage biomarker amount or level is the amount or level of the macrophage biomarker that is at the 25th percentile of a reference population.

11. 9. The method of claim 8, wherein the reference macrophage biomarker amount or level is the amount or level of the macrophage biomarker that is at the 50th percentile of a reference population.

12. 9. The method of claim 8, wherein the reference macrophage biomarker amount or level is the amount or level of the macrophage biomarker that is at the 75th percentile of a reference population.

13. The method according to any one of claims 8 to 12, wherein the reference population is a population of patients with lymphoma.

14. The method of claim 13, wherein the population of patients having lymphoma have been previously treated with an anti-CD20 antibody.

15. 15. The method of claim 14, wherein the reference macrophage biomarker amount or level is the amount or level of the macrophage biomarker in a reference population before initiating treatment with an anti-CD20 antibody.

16. 16. The method of claim 14 or 15, wherein the amount or level of the reference macrophage biomarker significantly separates the reference population into a first set of patients who have benefited from treatment with an anti-CD20 antibody and a second set of patients who have not benefited from treatment with an anti-CD20 antibody.

17. The method of any one of claims 1 to 16, wherein the amount or level of a reference macrophage biomarker is the amount of macrophages measured by gene expression.

18. 18. The method of claim 17, wherein the amount of macrophages is between about 0% and about 30.7%.

19. The method of any one of claims 1 to 3, wherein the benefit is an increase in progression-free survival (PFS).

20. The method of any one of claims 1 to 3, wherein the benefit is increased overall survival (OS).

21. The method of any one of claims 4 to 6, further comprising achieving improved PFS or OS.

22. The method of any one of claims 1 to 21, wherein the macrophage biomarker is an average of the M1 macrophage gene signature set scores of one or more M1 macrophage gene signature sets.

23. 23. The method of claim 22, wherein each M1 macrophage gene signature set score is an average of the expression levels of one or more genes in the M1 macrophage gene signature set.

24. 24. The method of claim 23, wherein each M1 macrophage gene signature set score is an average of normalized expression levels of one or more genes in the M1 macrophage gene signature set.

25. One or more M1 macrophage gene signature sets, (a) ACP2, ABCD1, C1QA, FDX1, CCL22, CD163, SCAMP2, ADAMDEC1, ARL8B, and HAMP; (b) ACP2, ABCD1, FDX1, CCL8, CCL22, CD163, ADAMDEC1, TREM2, and HAMP; (c) ACP2, ADRA2B, ALCAM, ABCD1, ATOX1, ATP6V0C, ATP6V1E1, BLVRA, C1QA, CD48, CD63, CLCN7, TPP1, CLTC, CCR1, CMKLR1, SLC31A1, COX5B, FCER1G, FDX1, FOLR2, FPR3, FTL, HEXB, HK3, IL10, IL12B, ITGAE, LAIR1, CXCL9, MMP19, NARS, NDUFS2, P2RX7, PDCL, MAPK13, PTGIR, PTPRA, RELA, CCL7, CCL8, CCL19, CCL22, SRC, STX4, TCEB1, TFRC, AGPS, MARCO, SNX3, CD84, USP14, ITGB1BP1, ATP6V1F, TRIP4, CD163, CIAO1, WTAP, ARHGEF11, ABI1, SCAMP2, ACTR2, BCAP31, ZMPSTE24, BCKDK, EXOC5, STIP1, UQCR11, SDS, LILRB4, OGF, TFE, FKBP15, DNAJC13, TDRD7, STX12, IL17RA, ABTB2, FAM32A, SIGLEC7, SIGLEC9, ADAMDEC1, CECR5, SLC25A24, NRBP1, MS4A4A, TREM2, OTUD4, PQLC2, HAUS2, ARL8B, NECAP2, WDR11, ZC3H15, CCDC47, UTP3, MRS2, HAMP, MRPL40, VPS33A, CORO7, LIMD2, TMX1, DOT1L, ADO, and ADCK2; (d) ACP2, ADRA2B, ALCAM, TSPO, C3AR1, DAGLA, CALR, CHIT1, CYBB, CYC1, CYP19A1, DLAT, FCER1G, GP1BA, GPD1, IFNAR1, IL10, KCNJ5, KIFC3, MT2A, MYBPH, MYH11, MYO7A, P2RX7, PRDX1, RAB3IL1, RNH1, MRPL12, CCL1, CCL7, CCL8, CCL24, SRC, VIM, RRP1, MARCO, S1PR2, AP1M2, ACTR3, LILRB1, AFG3L2, SDS, LILRB4, EMILIN1, VSIG4, HSPB7, COQ2, ADAMDEC1, CECR5, WSB2, SLAMF8, DNASE2B, CLPB, MFSD7, and ADCK2; (SAM2、SAMA33、SAMA20、SAMA2、SAS1 FAMOUS、FAMOUS、FAMOUS、FAMOUS33 C1、SYS11、SYS1、SYS3、SYS1 1101、CHY10、CHY1、CHY3、CHY 100001000000000000000000000000000000 33、HAR10、HAR10、HYS1、DY20、DYS 20207、00000000000000000000000000000000000000000000000. ROSE7、ROSE8、ROSE18、ROSE19、ROSE24、ROSE1 、SAKA、SAX、SAS1、SASHA、SHA201、SAS22、S 4. 163、LOVE1、DY102、CHSIS、LOVES10、SHR、4 LOVE 4、 LOVE1 1 LOVE1 CHEEK1、HARSH15、HARSHY、HARHYH、HARSHAR4、HARSH 11、DYS2、DYSYS1、DYSYS1、DYSYS 10、SHAS10、SHAS2、SHAS20、SHAS70、SHAS C2、DY33、SYSY8、SYSY、DYSYSY、F1 11. LOVE3, LOVE, LOVE, LOVE, LOVE, LOVE, LOVE, (f) ACP2, ADCY3, ADRA2B, ALCAM, ABCD1, ANXA2, ATP6V1A, C1QA, C1QB, C3AR1, DAGLA, CD80, CD63, CHIT1, CMKLR1, SLC31A1, CSF1, CSF1R, CYBB, CYC1, CYP19A 1, FANCE, FDX1, FPR2, FPR3, GPD1, HEXB, KCNJ1, KCNJ5, KIFC3, MMP19, MSR1, M T2A, MYBPH, P2RX7, MAPK13, S100A11, CCL1, CCL7, CCL8, CCL18, CCL19, CCL22, CCL24, SLC1A2, SLC6A12, SLC11A1, SIGLEC1, SRC, TIE1, MARCO, HYAL2, CD163 , LONP1, IGSF6, LILRB1, CD300C, SDS, LILRB4, EMILIN1, VSIG4, PHLDB1, NCAPH , CLEC4E, MYOF, HSPB7, ADAMDEC1, GLRX2, MS4A4A, ATP6V1H, TREM2, TMEM70, T MEM33, KCNK13, SLAMF8, HAMP, DNASE2B, MYOZ1, MFSD7, ADO, ADCK2, and TBC1D16 The method according to claim 23 or 24, wherein

26. The method of any one of claims 1 to 21, wherein the macrophage biomarker is a gene expression value.

27. 27. The method of claim 26, wherein the gene expression value is a median gene expression value.

28. 28. The method of claim 26 or 27, wherein the gene expression values ​​are measured using a gene signature matrix.

29. The gene signature matrix contains the following genes: (a) CD200, KLHL14, TCL1A, NRG1, EOMES, PPP2R2B, RNF165, WNT7A, CCR4 , PDGFD, EBF1, FCGBP, PCDH9, MLC1, TSHZ2, S1PR5, NCALD, LAYN, GCNT4, F ASLG, TRAT1, ADAM6, GUCY1A3, LRRC4, TSPAN18, SBK1, ICOS, BTNL8, WNT5 B, AUTS2, SH2D2A, ADGRG3, PNOC, SPIB, VPREB3, DPEP3, MME, ZBTB16, FOX P3, SEMA3G, CD8A, TOGARAM2, COLGALT2, ABCB1, STAP1, SAMD3, FAM46C, B LK, CTLA4, CD19, REPS2, RTKN2, POU2AF1, DAPK2, PYHIN1, NLRC3, GATM, K LRD1, AFF3, FCRLA, AATBC, REM2, YPEL1, TXK, CD8B, P2RX5, CEACAM1, BCL 11A, ABCB4, CD5, HPGD, BLNK, PLCL1, HPSE, SLFN13, HOPX, CD1D, GNG7, TC F4, BANK1, FHIT, FCMR, GNG2, GFRA2, KBTBD11, RALGPS2, TSPOAP1, PLEKH F1, MEF2C, MAOA, TTYH2, HLA-DOB, DGAT2, FXYD6, TMCC3, MGAM, TTC38, LR RC32, ARHGAP24, STAT4, SLC7A8, CD72, FZD1, GK5, DYSF, PLTP, SMARCD3, FAM160B1, PDPN, AKAP2, ACVRL1, KCNJ15, ALDH1A2, ENPP2, COLEC12, PTG S1, TMEM170B, TREM2, ECM1, SLC1A3, ABHD5, MS4A4A, CLIC2, IL1R1, SLC2 A6, GAS7, RNF144B, SLC6A12, FPR2, ADAM28, GRK3, KDM1B, MATK, LMO2, CF B, CCRL2, CLEC4A, LILRA2, ACE, NUPR1, CISH, EREG, ADAMDEC1, RNASE6, C XCL3, VSIG4, CXCL2, CD86, LILRB4, SERPING1, SQOR, INHBA, and ICAM1; or <h2 style=";text-align:left;direction:ltr">(b)CD200、KLHL14、TCL1A、NRG1、CYP4 F3、EOMES、PPP2R2B、RNF165、WNT7A、C CR4、PDGFD、EBF1、FCGBP、PCDH9、MLC1 、TSHZ2、S1PR5、NCALD、LAYN、CD248、GC NT4、FASLG、TRAT1、ADAM6、GUCY1A3、L RRC4、TSPAN18、SBK1、ICOS、BTNL8、WN T5B、AUTS2、SH2D2A、ADGRG3、PNOC、SPIB、VPREB3、DPEP3、MME、ZBTB16、FOXP3 、SEMA3G、CD8A、TOGARAM2、COLGALT2、 ABCB1、STAP1、SAMD3、FAM46C、BLK、CT LA4、CD19、REPS2、RTKN2、POU2AF1、DA PK2、PYHIN1、NLRC3、GATM、KLRD1、AFF3 、FCRLA、AATBC、REM2、YPEL1、TXK、CD8 B、P2RX5、CEACAM1、BCL11A、NINJ2、AB CB4、CD5、HAL、HPGD、BLNK、PLCL1、CEP 19、HPSE、SLFN13、HOPX、CD1D、GNG7、TM EM154、TCF4、BANK1、FHIT、FCMR、GNG2 、GFRA2、KBTBD11、TECPR2、RALGPS2、T SPOAP1、PLEKHF1、MEF2C、MAOA、TTYH2 、HLA-DOB、NRGN、DGAT2、FXYD6、TMCC3、 MGAM、TTC38、LRRC32、ARHGAP24、PPP1 R3B、STAT4、SLC7A8、CD72、FZD1、GK5、 DYSF、PLTP、SMARCD3、FAM160B1、PDPN 、AKAP2、ACVRL1、KCNJ15、CD36、ALDH1A 2、ENPP2、COLEC12、PTGS1、TMEM170B、 DOCK5、TREM2、C5AR2、ECM1、SLC1A3、A BHD5、MS4A4A、CLIC2、IL1R1、SLC2A6、 GAS7、RNF144B、SLC6A12、FPR2、ADAM28 、GRK3、KDM1B、MATK、LMO2、CFB、CCRL2 、CLEC4A、TLR4、LILRA2、ACE、TLR1、LRR K2、LY96、NUPR1、CISH、CSTA、EREG、AD AMDEC1、RNASE6、CXCL3、VSIG4、CXCL2、CD86, LILRB4, SERPING1, SQOR, INHBA, and ICAM1; 30. The method of claim 28, comprising:

30. The gene signature matrix contains the following genes: CD200, KLHL14, TCL1A, NRG1, EOMES, PPP2R2B, RNF165, WNT7A, CCR4, PDGFD, EBF1, FCGBP, PCDH9, MLC1, TSHZ2, S1PR5, NCALD, LAYN, GCNT4, FASLG, TRAT1, ADAM6, GUCY1A3, LRRRC4, TSPAN18, SBK1, ICOS, BTNL8, WNT5B, AUTS2, SH2D2A, ADGRG3, PNOC, SPIB, VPREB3, DPEP3, MME, ZBTB16, FOXP3, SEMAG3, CD8A, TOGARAM2, COLGAALT2, ABCB1, STAP1, SAMD3, FAM46C, BLK, CTLA4, CD19, REPS2, RTKN2, POU2AF1, DAPK2, PYHIN1, NLRRC3, GATM, KLRD1, AFF3, FCRLA, AATBC, REM2, YPEL1, TXK, CD8B, P2RX5, CEACAM1, BCL11A, ABCB4, CD5, HPGD, BLNK, PLC1, HPSE, SLFN13, HOPX, CD1D, GNG7, TCF4, BANK1, FHIT, FCMR, GNG2, GFR2, KBTBD11, RALGPS2, TSPOAP1, PLEKH1, MEF2C, MAOA, TTYH2, HLA - DOB, DGAT2, FXYD6, TMCC3, MGAM, TTC38, LRRRC32, ARHGAP24, STAT4, SLC7A8, CD72, FZD1, GK5, DYSF, PLTP, SMARCD3, FAM160B1, PDPN, AKAP2, ACVR1, KCNJ15, ALDH1A2, ENPP2, COLEC12, PTGS1, TMEM170B, TRE2, ECM1, SLC1A3, ABHD5, MS4A4A, CLIC2, IL1R1, SLC2A6, GAS7, RNF144B, SLC6A12, FPR2, ADAM28, GRK3, KDM1B, MATK, LMO2, CFB, CCRL2, CLEC4A, LILRA2, ACE, NUPR1, CISH, EREG, ADAMDEC1, RNASE6, CXCL3, VSIG4, CXCL2, CD86, LILRB4, SERPING1, SQR, INHBA, and ICAM1 The method according to claim 29, comprising

31. The method of any one of claims 28 to 30, wherein a gene signature matrix is ​​used to determine the number of M1 macrophages.

32. The method of any one of claims 1 to 31, wherein the macrophage biomarker is the amount of M1 macrophages.

33. 33. The method of claim 32, wherein the amount of M1 macrophages is measured directly or indirectly.

34. 34. The method of claim 33, wherein the amount of M1 macrophages is measured directly using flow cytometry, spatial transcriptomics, spatial proteomics, or a combination thereof.

35. 34. The method of claim 33, wherein the amount of M1 macrophages is measured indirectly using nucleic acids or proteins.

36. 36. The method of claim 35, wherein the nucleic acid is measured using RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technology, ISH, or a combination thereof.

37. 37. The method of claim 36, wherein the amount of M1 macrophages is measured using a marker gene approach or a deconvolution approach.

38. The method of claim 37, wherein the marker gene approach uses xCell.

39. 38. The method of claim 37, wherein the deconvolution approach uses quanTIseq.

40. The method of any one of claims 1 to 21, wherein macrophage biomarkers in a sample from a patient are measured using nucleic acids or proteins.

41. 41. The method of claim 40, wherein the macrophage biomarker in the sample from the patient is determined using nucleic acid expression levels.

42. 42. The method of claim 41, wherein the nucleic acid expression level is determined by RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technology, ISH, or a combination thereof.

43. 43. The method of claim 41 or 42, wherein the nucleic acid expression level is an mRNA expression level.

44. The method of claim 43, wherein the mRNA expression level is determined by RNA-seq.

45. The method of any one of claims 1 to 44, wherein the sample is a tissue sample, a tumor sample, a whole blood sample, a plasma sample, a serum sample, or a combination thereof.

46. 46. ​​The method of claim 45, wherein the sample is a tissue sample.

47. 47. The method of claim 46, wherein the tissue sample is a tumor tissue sample.

48. 48. The method of claim 47, wherein the tumor tissue sample comprises tumor cells, tumor-infiltrating immune cells, stromal cells, normal adjacent tissue (NAT) cells, or a combination thereof.

49. 49. The method of claim 47 or 48, wherein the tumor tissue sample is a biopsy.

50. 50. The method of any one of claims 45 to 49, wherein the sample is an archived sample, a fresh sample, or a frozen sample.

51. 51. The method of any one of claims 1 to 50, wherein the lymphoma is an indolent lymphoma.

52. The method of any one of claims 1 to 51, wherein the lymphoma is a B-cell lymphoma.

53. 53. The method of claim 52, wherein the B cell lymphoma is germinal center-derived B cell lymphoma.

54. 54. The method of claim 52 or 53, wherein the B cell lymphoma is non-Hodgkin's lymphoma (NHL).

55. 55. The method of any one of claims 1 to 54, wherein the lymphoma is diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), or marginal zone lymphoma (MZL).

56. 56. The method of claim 55, wherein the lymphoma is DLBCL.

57. 57. The method of claim 56, wherein the DLBCL is of the germinal center B cell-like (GCB) cell of origin or the activated B cell-like (ABC) cell of origin subgroup of DLBCL.

58. The method of any one of claims 1 to 57, wherein the lymphoma is a CD20 positive lymphoma.

59. The method of any one of claims 1 to 58, wherein the anti-CD20 antibody is a type I anti-CD20 antibody or a type II anti-CD20 antibody.

60. 60. The method of claim 59, wherein the anti-CD20 antibody is a type II anti-CD20 antibody.

61. Type II anti-CD20 antibodies have the following complementarity determining regions (CDRs): (a) CDR-H1 having the amino acid sequence of SEQ ID NO:27; (b) CDR-H2 having the amino acid sequence of SEQ ID NO:28; (c) a CDR-H3 having the amino acid sequence of SEQ ID NO:3; (d) a CDR-L1 having the amino acid sequence of SEQ ID NO:4; (e) a CDR-L2 having the amino acid sequence of SEQ ID NO:5; and (f) CDR-L3 having the amino acid sequence of SEQ ID NO:

6.

61. The method of claim 60, comprising:

62. The method of claim 61 , wherein the type II anti-CD20 antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:

8.

63. 63. The method of claim 62, wherein the type II anti-CD20 antibody is obinutuzumab.

64. 60. The method of claim 59, wherein the anti-CD20 antibody is a type I anti-CD20 antibody.

65. A type I anti-CD20 antibody has the following CDRs: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO:11; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 12; (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 13; (d) CDR-L1 having the amino acid sequence of SEQ ID NO: 14; (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 15; and (f) CDR-L3 having the amino acid sequence of SEQ ID NO:

16.

65. The method of claim 64, comprising:

66. 66. The method of claim 65, wherein the type I anti-CD20 antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO:25 and a VL domain comprising the amino acid sequence of SEQ ID NO:

26.

67. 67. The method of claim 66, wherein the type I anti-CD20 antibody is rituximab.

68. 68. The method of any one of claims 3 to 67, further comprising administering to the patient an effective amount of an additional therapeutic agent.

69. 69. The method of claim 68, wherein the additional therapeutic agent is one or more of a chemotherapeutic agent, antineoplastic agent, a growth inhibitory agent, an anti-angiogenic agent, radiation therapy, a cytotoxic agent, or a combination thereof.

70. 70. The method of claim 69, wherein the additional therapeutic agent is a chemotherapeutic agent.

71. 71. The method of claim 69 or 70, wherein the chemotherapeutic agent is cyclophosphamide, doxorubicin, vincristine or prednisone.

72. 71. The method of claim 69 or 70, wherein the chemotherapeutic agents are cyclophosphamide, doxorubicin, vincristine and prednisone.

73. 73. The method of any one of claims 1 to 72, wherein the patient has not been previously treated for lymphoma.

74. The method of any one of claims 1 to 73, wherein the patient has not previously been administered an anti-CD20 antibody.

75. 20. The use of an anti-CD20 antibody to treat a patient having an amount or level of a macrophage biomarker that exceeds an amount or level of a reference macrophage biomarker in a sample from the patient in the manufacture of a medicament for the treatment of lymphoma.

76. 76. The use of claim 75, wherein the reference macrophage biomarker amount or level is a pre-assigned macrophage biomarker amount or level.

77. 77. The use of claim 75 or 76, wherein the amount or level of the reference macrophage biomarker is the amount or level of the macrophage biomarker in a reference population.

78. 78. The use of claim 77, wherein the amount or level of the macrophage biomarker in the reference population is the median amount or level of the macrophage biomarker in the reference population.

79. 78. The use of claim 77, wherein the reference macrophage biomarker amount or level is an amount or level of the macrophage biomarker that is at the 25th percentile of a reference population.

80. 78. The use of claim 77, wherein the reference macrophage biomarker amount or level is the amount or level of the macrophage biomarker that is at the 50th percentile of a reference population.

81. 78. The use of claim 77, wherein the reference macrophage biomarker amount or level is the amount or level of the macrophage biomarker that is at the 75th percentile of a reference population.

82. The use according to any one of claims 77 to 81, wherein the reference population is a population of patients with lymphoma.

83. 83. The use of claim 82, wherein the patient population having lymphoma has been previously treated with an anti-CD20 antibody.

84. 84. The use of claim 83, wherein the reference macrophage biomarker amount or level is the amount or level of the macrophage biomarker in a reference population before initiating treatment with an anti-CD20 antibody.

85. 85. The use of claim 83 or 84, wherein the amount or level of the reference macrophage biomarker significantly separates the reference population into a first set of patients who have benefited from treatment with an anti-CD20 antibody and a second set of patients who have not benefited from treatment with an anti-CD20 antibody.

86. 86. The use according to any one of claims 75 to 85, wherein the amount or level of the reference macrophage biomarker is the amount of macrophages measured by gene expression.

87. 87. The use of claim 86, wherein the amount of macrophages is between about 0% and about 30.7%.

88. 76. The use of claim 75, wherein the treatment achieves an improvement in PFS or OS.

89. The use according to any one of claims 75 to 88, wherein the macrophage biomarker is an average of the M1 macrophage gene signature set scores of one or more M1 macrophage gene signature sets.

90. 90. The use of claim 89, wherein each M1 macrophage gene signature set score is the average of the expression levels of one or more genes in the M1 macrophage gene signature set.

91. The use of claim 90, wherein each M1 macrophage gene signature set score is the average of the normalized expression levels of one or more genes in the M1 macrophage gene signature set.

92. One or more M1 macrophage gene signature sets, (a) ACP2, ABCD1, C1QA, FDX1, CCL22, CD163, SCAMP2, ADAMDEC1, ARL8B, and HAMP; (b) ACP2, ABCD1, FDX1, CCL8, CCL22, CD163, ADAMDEC1, TREM2, and HAMP; (c) ACP2, ADRA2B, ALCAM, ABCD1, ATOX1, ATP6V0C, ATP6V1E1, BLVRA, C1QA, CD48, CD63, CLCN7, TPP1, CLTC, CCR1, CMKLR1, SLC31A1, COX5B, FCER1G, FDX1, FOLR2, FPR3, FTL, HEXB, HK3, IL10, IL12B, ITGAE, LAIR1, CXCL9, MMP19, NARS, NDUFS2, P2RX7, PDCL, MAPK13, PTGIR, PTPRA, RELA, CCL7, CCL8, CCL19, CCL22, SRC, STX4, TCEB1, TFRC, AGPS, MARCO, SNX3, CD84, USP14, ITGB1BP1, ATP6V1F, TRIP4, CD163, CIAO1, WTAP, ARHGEF11, ABI1, SCAMP2, ACTR2, BCAP31, ZMPSTE24, BCKDK, EXOC5, STIP1, UQCR11, SDS, LILRB4, OGF, TFE, FKBP15, DNAJC13, TDRD7, STX12, IL17RA, ABTB2, FAM32A, SIGLEC7, SIGLEC9, ADAMDEC1, CECR5, SLC25A24, NRBP1, MS4A4A, TREM2, OTUD4, PQLC2, HAUS2, ARL8B, NECAP2, WDR11, ZC3H15, CCDC47, UTP3, MRS2, HAMP, MRPL40, VPS33A, CORO7, LIMD2, TMX1, DOT1L, ADO, and ADCK2; (d) ACP2, ADRA2B, ALCAM, TSPO, C3AR1, DAGLA, CALR, CHIT1, CYBB, CYC1, CYP19A1, DLAT, FCER1G, GP1BA, GPD1, IFNAR1, IL10, KCNJ5, KIFC3, MT2A, MYBPH, MYH11, MYO7A, P2RX7, PRDX1, RAB3IL1, RNH1, MRPL12, CCL1, CCL7, CCL8, CCL24, SRC, VIM, RRP1, MARCO, S1PR2, AP1M2, ACTR3, LILRB1, AFG3L2, SDS, LILRB4, EMILIN1, VSIG4, HSPB7, COQ2, ADAMDEC1, CECR5, WSB2, SLAMF8, DNASE2B, CLPB, MFSD7, and ADCK2; (SAM2、SAMA33、SAMA20、SAMA2、SAS1 FAMOUS、FAMOUS、FAMOUS、FAMOUS33 C1、SYS11、SYS1、SYS3、SYS1 1101、CHY10、CHY1、CHY3、CHY 100001000000000000000000000000000000 33、HAR10、HAR10、HYS1、DY20、DYS 20207、00000000000000000000000000000000000000000000000. ROSE7、ROSE8、ROSE18、ROSE19、ROSE24、ROSE1 、SAKA、SAX、SAS1、SASHA、SHA201、SAS22、S 4. 163、LOVE1、DY102、CHSIS、LOVES10、SHR、4 LOVE 4、 LOVE1 1 LOVE1 CHEEK1、HARSH15、HARSHY、HARHYH、HARSHAR4、HARSH 11、DYS2、DYSYS1、DYSYS1、DYSYS 10、SHAS10、SHAS2、SHAS20、SHAS70、SHAS C2、DY33、SYSY8、SYSY、DYSYSY、F1 11. LOVE3, LOVE, LOVE, LOVE, LOVE, LOVE, LOVE, (f) ACP2, ADCY3, ADRA2B, ALCAM, ABCD1, ANXA2, ATP6V1A, C1QA, C1QB, C3AR1, DAGLA, CD80, CD63, CHIT1, CMKLR1, SLC31A1, CSF1, CSF1R, CYBB, CYC1, CYP19A 1, FANCE, FDX1, FPR2, FPR3, GPD1, HEXB, KCNJ1, KCNJ5, KIFC3, MMP19, MSR1, M T2A, MYBPH, P2RX7, MAPK13, S100A11, CCL1, CCL7, CCL8, CCL18, CCL19, CCL22, CCL24, SLC1A2, SLC6A12, SLC11A1, SIGLEC1, SRC, TIE1, MARCO, HYAL2, CD163 , LONP1, IGSF6, LILRB1, CD300C, SDS, LILRB4, EMILIN1, VSIG4, PHLDB1, NCAPH , CLEC4E, MYOF, HSPB7, ADAMDEC1, GLRX2, MS4A4A, ATP6V1H, TREM2, TMEM70, T MEM33, KCNK13, SLAMF8, HAMP, DNASE2B, MYOZ1, MFSD7, ADO, ADCK2, and TBC1D16 92. The use according to claim 90 or 91,

93. The use according to any one of claims 75 to 88, wherein the macrophage biomarker is a gene expression value.

94. 94. The use of claim 93, wherein the gene expression value is the median gene expression value.

95. 95. The use of claim 93 or 94, wherein the gene expression values ​​are measured using a gene signature matrix.

96. The gene signature matrix contains the following genes: (a) CD200, KLHL14, TCL1A, NRG1, EOMES, PPP2R2B, RNF165, WNT7A, CCR4 , PDGFD, EBF1, FCGBP, PCDH9, MLC1, TSHZ2, S1PR5, NCALD, LAYN, GCNT4, F ASLG, TRAT1, ADAM6, GUCY1A3, LRRC4, TSPAN18, SBK1, ICOS, BTNL8, WNT5 B, AUTS2, SH2D2A, ADGRG3, PNOC, SPIB, VPREB3, DPEP3, MME, ZBTB16, FOX P3, SEMA3G, CD8A, TOGARAM2, COLGALT2, ABCB1, STAP1, SAMD3, FAM46C, B LK, CTLA4, CD19, REPS2, RTKN2, POU2AF1, DAPK2, PYHIN1, NLRC3, GATM, K LRD1, AFF3, FCRLA, AATBC, REM2, YPEL1, TXK, CD8B, P2RX5, CEACAM1, BCL 11A, ABCB4, CD5, HPGD, BLNK, PLCL1, HPSE, SLFN13, HOPX, CD1D, GNG7, TC F4, BANK1, FHIT, FCMR, GNG2, GFRA2, KBTBD11, RALGPS2, TSPOAP1, PLEKH F1, MEF2C, MAOA, TTYH2, HLA-DOB, DGAT2, FXYD6, TMCC3, MGAM, TTC38, LR RC32, ARHGAP24, STAT4, SLC7A8, CD72, FZD1, GK5, DYSF, PLTP, SMARCD3, FAM160B1, PDPN, AKAP2, ACVRL1, KCNJ15, ALDH1A2, ENPP2, COLEC12, PTG S1, TMEM170B, TREM2, ECM1, SLC1A3, ABHD5, MS4A4A, CLIC2, IL1R1, SLC2 A6, GAS7, RNF144B, SLC6A12, FPR2, ADAM28, GRK3, KDM1B, MATK, LMO2, CF B, CCRL2, CLEC4A, LILRA2, ACE, NUPR1, CISH, EREG, ADAMDEC1, RNASE6, C XCL3, VSIG4, CXCL2, CD86, LILRB4, SERPING1, SQOR, INHBA, and ICAM1; or <h2 style=";text-align:left;direction:ltr">(b)CD200、KLHL14、TCL1A、NRG1、CYP4 F3、EOMES、PPP2R2B、RNF165、WNT7A、C CR4、PDGFD、EBF1、FCGBP、PCDH9、MLC1 、TSHZ2、S1PR5、NCALD、LAYN、CD248、GC NT4、FASLG、TRAT1、ADAM6、GUCY1A3、L RRC4、TSPAN18、SBK1、ICOS、BTNL8、WN T5B、AUTS2、SH2D2A、ADGRG3、PNOC、SPIB、VPREB3、DPEP3、MME、ZBTB16、FOXP3 、SEMA3G、CD8A、TOGARAM2、COLGALT2、 ABCB1、STAP1、SAMD3、FAM46C、BLK、CT LA4、CD19、REPS2、RTKN2、POU2AF1、DA PK2、PYHIN1、NLRC3、GATM、KLRD1、AFF3 、FCRLA、AATBC、REM2、YPEL1、TXK、CD8 B、P2RX5、CEACAM1、BCL11A、NINJ2、AB CB4、CD5、HAL、HPGD、BLNK、PLCL1、CEP 19、HPSE、SLFN13、HOPX、CD1D、GNG7、TM EM154、TCF4、BANK1、FHIT、FCMR、GNG2 、GFRA2、KBTBD11、TECPR2、RALGPS2、T SPOAP1、PLEKHF1、MEF2C、MAOA、TTYH2 、HLA-DOB、NRGN、DGAT2、FXYD6、TMCC3、 MGAM、TTC38、LRRC32、ARHGAP24、PPP1 R3B、STAT4、SLC7A8、CD72、FZD1、GK5、 DYSF、PLTP、SMARCD3、FAM160B1、PDPN 、AKAP2、ACVRL1、KCNJ15、CD36、ALDH1A 2、ENPP2、COLEC12、PTGS1、TMEM170B、 DOCK5、TREM2、C5AR2、ECM1、SLC1A3、A BHD5、MS4A4A、CLIC2、IL1R1、SLC2A6、 GAS7、RNF144B、SLC6A12、FPR2、ADAM28 、GRK3、KDM1B、MATK、LMO2、CFB、CCRL2 、CLEC4A、TLR4、LILRA2、ACE、TLR1、LRR K2、LY96、NUPR1、CISH、CSTA、EREG、AD AMDEC1、RNASE6、CXCL3、VSIG4、CXCL2、CD86, LILRB4, SERPING1, SQOR, INHBA, and ICAM1; 96. The use of claim 95, comprising:

97. The gene signature matrix contains the following genes: CD200, KLHL14, TCL1A, NRG1, EOMES, PPP2R2B, RNF165, WNT7A, CCR4, PDGFD, EBF1, FCGBP, PCDH9, MLC1, TSHZ2, S1PR5, NCALD, LAYN, GCNT4, FASLG, TRAT1, ADAM6, GUCY1A3, LRRRC4, TSPAN18, SBK1, ICOS, BTNL8, WNT5B, AUTS2, SH2D2A, ADGRG3, PNOC, SPIB, VPREB3, DPEP3, MME, ZBTB16, FOXP3, SEMAG3, CD8A, TOGARAM2, COLGAALT2, ABCB1, STAP1, SAMD3, FAM46C, BLK, CTLA4, CD19, REPS2, RTKN2, POU2AF1, DAPK2, PYHIN1, NLRRC3, GATM, KLRD1, AFF3, FCRLA, AATBC, REM2, YPEL1, TXK, CD8B, P2RX5, CEACAM1, BCL11A, ABCB4, CD5, HPGD, BLNK, PLCL1, HPSE, SLFN13, HOPX, CD1D, GNG7, TCF4, BANK1, FHIT, FCMR, GNG2, GFRRA2, KBTBD11, RALGPS2, TSPOAP1, PLEKHFI, MEF2C, MAOA, TTYH2, HLA-DOB, DGAT2, FXYD6, TMCC3, MGAM, TTC38, LRRRC32, ARHGAP24, STAT4, SLC7A8, CD72, FZD1, GK5, DYSF, PLTP, SMARCD3, FAM160B1, PDPN, AKAP2, ACVR1, KCNJ15, ALDH1A2, ENPP2, COLEC12, PTGS1, TMEM170B, TREMB2, ECM1, SLC1A3, ABHD5, MS4A4A, CLIC2, IL1R1, SLC2A6, GAS7, RNF144B, SLC6A12, FPR2, ADAM28, GRK3, KDM1B, MATK, LMO2, CFB, CCRL2, CLEC4A, LILRA2, ACE, NUPR1, CISH, EREG, ADAMDEC1, RNASE6, CXCL3, VSIG4, CXCL2, CD86, LILRB4, SERPING1, SQR, INHBA, and ICAM1 The use according to claim 96, comprising the above. [

98. ] The use according to any one of claims 95 to 97, wherein the gene signature matrix is ​​used to determine the number of M1 macrophages.

99. The use according to any one of claims 75 to 98, wherein the macrophage biomarker is the amount of M1 macrophages.

100. 100. The use according to claim 99, wherein the amount of M1 macrophages is measured directly or indirectly.

101. The use of claim 100, wherein the amount of M1 macrophages is measured directly using flow cytometry, spatial transcriptomics, spatial proteomics, or a combination thereof.

102. The use according to claim 100, wherein the amount of M1 macrophages is measured indirectly using nucleic acids or proteins.

103. 103. The use according to claim 102, wherein the nucleic acid is measured using RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technology, ISH, or a combination thereof.

104. The use according to claim 103, wherein the amount of M1 macrophages is measured using a marker gene approach or a deconvolution approach.

105. The use described in claim 104, wherein the marker gene approach uses xCell.

106. The use of claim 104, wherein the deconvolution approach uses quanTIseq.

107. The use according to any one of claims 75 to 88, wherein macrophage biomarkers in a sample from a patient are measured using nucleic acids or proteins.

108. The use of claim 107, wherein macrophage biomarkers in a sample from a patient are determined using nucleic acid expression levels.

109. 109. The use according to claim 108, wherein the nucleic acid expression level is determined by RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technology, ISH, or a combination thereof.

110. 110. The use of claim 108 or 109, wherein the nucleic acid expression level is an mRNA expression level.

111. The use of claim 110, wherein the mRNA expression level is determined by RNA-seq.

112. The use according to any one of claims 75 to 111, wherein the sample is a tissue sample, a tumor sample, a whole blood sample, a plasma sample, a serum sample, or a combination thereof.

113. 113. The use according to claim 112, wherein the sample is a tissue sample.

114. 114. The use according to claim 113, wherein the tissue sample is a tumor tissue sample.

115. The use of claim 114, wherein the tumor tissue sample comprises tumor cells, tumor-infiltrating immune cells, stromal cells, NAT cells, or a combination thereof.

116. 116. The use according to claim 114 or 115, wherein the tumor tissue sample is a biopsy.

117. The use according to any one of claims 112 to 116, wherein the sample is an archived sample, a fresh sample or a frozen sample.

118. The use according to any one of claims 75 to 117, wherein the lymphoma is a low-grade lymphoma.

119. The use according to any one of claims 75 to 118, wherein the lymphoma is a B-cell lymphoma.

120. 120. The use of claim 118 or 119, wherein the B cell lymphoma is NHL.

121. The use according to any one of claims 75 to 120, wherein the lymphoma is DLBCL, FL, CLL or MZL.

122. The use of claim 121, wherein the lymphoma is DLBCL.

123. The use according to claim 122, wherein the DLBCL is the GCB cell of origin or the ABC cell of origin subgroup of DLBCL.

124. The use according to any one of claims 75 to 123, wherein the lymphoma is a CD20 positive lymphoma.

125. The use according to any one of claims 75 to 124, wherein the anti-CD20 antibody is a type I anti-CD20 antibody or a type II anti-CD20 antibody.

126. The use according to claim 125, wherein the anti-CD20 antibody is a type II anti-CD20 antibody.

127. A type II anti-CD20 antibody has the following CDRs: (a) CDR-H1 having the amino acid sequence of SEQ ID NO:27; (b) CDR-H2 having the amino acid sequence of SEQ ID NO:28; (c) a CDR-H3 having the amino acid sequence of SEQ ID NO:3; (d) a CDR-L1 having the amino acid sequence of SEQ ID NO:4; (e) a CDR-L2 having the amino acid sequence of SEQ ID NO:5; and (f) CDR-L3 having the amino acid sequence of SEQ ID NO:

6. The use of claim 126, comprising:

128. The use according to claim 127, wherein the type II anti-CD20 antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO:

8.

129. The use according to claim 128, wherein the type II anti-CD20 antibody is obinutuzumab.

130. The use according to claim 125, wherein the anti-CD20 antibody is a type I anti-CD20 antibody.

131. A type I anti-CD20 antibody has the following CDRs: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO:11; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 12; (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 13; (d) CDR-L1 having the amino acid sequence of SEQ ID NO: 14; (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 15; and (f) CDR-L3 having the amino acid sequence of SEQ ID NO:

16. The use of claim 130, comprising:

132. The use according to claim 131, wherein the type I anti-CD20 antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 25 and a VL domain comprising the amino acid sequence of SEQ ID NO:

26.

133. 133. The use according to claim 132, wherein the type I anti-CD20 antibody is rituximab.

134. The use according to any one of claims 75 to 133, wherein the medicament is administered to the patient in combination with an effective amount of an additional therapeutic agent.

135. 135. The use of claim 134, wherein the additional therapeutic agent is one or more of a chemotherapeutic agent, antineoplastic agent, a growth inhibitory agent, an anti-angiogenic agent, radiation therapy, a cytotoxic agent, or a combination thereof.

136. 136. The use of claim 135, wherein the additional therapeutic agent is a chemotherapeutic agent.

137. 137. The use of claim 135 or 136, wherein the chemotherapeutic agent is cyclophosphamide, doxorubicin, vincristine or prednisone.

138. 137. The use of claim 135 or 136, wherein the chemotherapeutic agents are cyclophosphamide, doxorubicin, vincristine and prednisone.

139. The use according to any one of claims 75 to 138, wherein the patient has not been previously treated for lymphoma.

140. The use according to any one of claims 75 to 139, wherein the patient has not previously been administered an anti-CD20 antibody.

141. An anti-CD20 antibody for use in treating a patient with lymphoma, the patient having an amount or level of a macrophage biomarker that exceeds the amount or level of a reference macrophage biomarker in a sample from the patient.

142. 142. The anti-CD20 antibody for use according to claim 141, wherein the reference macrophage biomarker amount or level is a pre-assigned macrophage biomarker amount or level.

143. 143. An anti-CD20 antibody for use according to claim 141 or 142, wherein the amount or level of the reference macrophage biomarker is the amount or level of the macrophage biomarker in a reference population.

144. The anti-CD20 antibody for use according to claim 143, wherein the amount or level of the macrophage biomarker in the reference population is the median amount or level of the macrophage biomarker in the reference population.

145. The anti-CD20 antibody for use according to claim 143, wherein the reference macrophage biomarker amount or level is an amount or level of a macrophage biomarker that is at the 25th percentile of a reference population.

146. The anti-CD20 antibody for use according to claim 143, wherein the reference macrophage biomarker amount or level is an amount or level of a macrophage biomarker that is at the 50th percentile of a reference population.

147. The anti-CD20 antibody for use according to claim 143, wherein the reference macrophage biomarker amount or level is an amount or level of a macrophage biomarker that is at the 75th percentile of a reference population.

148. The anti-CD20 antibody for use according to any one of claims 143 to 147, wherein the reference population is a population of patients with lymphoma.

149. The anti-CD20 antibody for use according to claim 148, wherein the patient population having lymphoma has been previously treated with the anti-CD20 antibody.

150. 150. The anti-CD20 antibody for use according to claim 149, wherein the amount or level of the reference macrophage biomarker is the amount or level of the macrophage biomarker in a reference population prior to initiating treatment with the anti-CD20 antibody.

151. 151. The anti-CD20 antibody for use according to claim 149 or 150, wherein the amount or level of the reference macrophage biomarker significantly separates the reference population into a first set of patients who have benefited from treatment with an anti-CD20 antibody and a second set of patients who have not benefited from treatment with an anti-CD20 antibody.

152. The CD20 antibody for use according to any one of claims 141 to 151, wherein the amount or level of a reference macrophage biomarker is the amount of macrophages measured by gene expression.

153. The anti-CD20 antibody for use according to claim 152, wherein the amount of macrophages is between about 0% and about 30.7%.

154. The anti-CD20 antibody for use according to claim 141, wherein the treatment achieves improvement in PFS or OS.

155. The anti-CD20 antibody for use according to any one of claims 141 to 154, wherein the macrophage biomarker is the average of the M1 macrophage gene signature set scores of one or more M1 macrophage gene signature sets.

156. The anti-CD20 antibody for use according to claim 155, wherein each M1 macrophage gene signature set score is the average of the expression levels of one or more genes in the M1 macrophage gene signature set.

157. The anti-CD20 antibody for use according to claim 156, wherein each M1 macrophage gene signature set score is the average of the normalized expression levels of one or more genes in the M1 macrophage gene signature set.

158. One or more M1 macrophage gene signature sets, (a) ACP2, ABCD1, C1QA, FDX1, CCL22, CD163, SCAMP2, ADAMDEC1, ARL8B, and HAMP; (b) ACP2, ABCD1, FDX1, CCL8, CCL22, CD163, ADAMDEC1, TREM2, and HAMP; (c) ACP2, ADRA2B, ALCAM, ABCD1, ATOX1, ATP6V0C, ATP6V1E1, BLVRA, C1QA, CD48, CD63, CLCN7, TPP1, CLTC, CCR1, CMKLR1, SLC31A1, COX5B, FCER1G, FDX1, FOLR2, FPR3, FTL, HEXB, HK3, IL10, IL12B, ITGAE, LAIR1, CXCL9, MMP19, NARS, NDUFS2, P2RX7, PDCL, MAPK13, PTGIR, PTPRA, RELA, CCL7, CCL8, CCL19, CCL22, SRC, STX4, TCEB1, TFRC, AGPS, MARCO, SNX3, CD84, USP14, ITGB1BP1, ATP6V1F, TRIP4, CD163, CIAO1, WTAP, ARHGEF11, ABI1, SCAMP2, ACTR2, BCAP31, ZMPSTE24, BCKDK, EXOC5, STIP1, UQCR11, SDS, LILRB4, OGF, TFEC, FKBP15, DNAJC13, TDRD7, STX12, IL17RA, ABTB2, FAM32A, SIGLEC7, SIGLEC9, ADAMDEC1, CECR5, SLC25A24, NRBP1, MS4A4A, TREM2, OTUD4, PQLC2, HAUS2, ARL8B, NECAP2, WDR11, ZC3H15, CCDC47, UTP3, MRS2, HAMP, MRPL40, VPS33A, CORO7, LIMD2, TMX1, DOT1L, ADO, and ADCK2; (d) ACP2, ADRA2B, ALCAM, TSPO, C3AR1, DAGLA, CALR, CHIT1, CYBB, CYC1, CYP19A1, DLAT, FCER1G, GP1BA, GPD1, IFNAR1, IL10, KCNJ5, KIFC3, MT2A, MYBPH, MYH11, MYO7A, P2RX7, PRDX1, RAB3IL1, RNH1, MRPL12, CCL1, CCL7, CCL8, CCL24, SRC, VIM, RRP1, MARCO, S1PR2, AP1M2, ACTR3, LILRB1, AFG3L2, SDS, LILRB4, EMILIN1, VSIG4, HSPB7, COQ2, ADAMDEC1, CECR5, WSB2, SLAMF8, DNASE2B, CLPB, MFSD7, and ADCK2; (SAM2、SAMA33、SAMA20、SAMA2、SAS1 FAMOUS、FAMOUS、FAMOUS、FAMOUS33 C1、SYS11、SYS1、SYS3、SYS1 1101、CHY10、CHY1、CHY3、CHY 100001000000000000000000000000000000 33、HAR10、HAR10、HYS1、DY20、DYS 20207、00000000000000000000000000000000000000000000000. ROSE7、ROSE8、ROSE18、ROSE19、ROSE24、ROSE1 、SAKA、SAX、SAS1、SASHA、SHA201、SAS22、S 4. 163、LOVE1、DY102、CHSIS、LOVES10、SHR、4 LOVE 4、 LOVE1 1 LOVE1 CHEEK1、HARSH15、HARSHY、HARHYH、HARSHAR4、HARSH 11、DYS2、DYSYS1、DYSYS1、DYSYS 10、SHAS10、SHAS2、SHAS20、SHAS70、SHAS C2、DY33、SYSY8、SYSY、DYSYSY、F1 11. LOVE3, LOVE, LOVE, LOVE, LOVE, LOVE, LOVE, (f) ACP2, ADCY3, ADRA2B, ALCAM, ABCD1, ANXA2, ATP6V1A, C1QA, C1QB, C3AR1, DAGLA, CD80, CD63, CHIT1, CMKLR1, SLC31A1, CSF1, CSF1R, CYBB, CYC1, CYP19A 1, FANCE, FDX1, FPR2, FPR3, GPD1, HEXB, KCNJ1, KCNJ5, KIFC3, MMP19, MSR1, M T2A, MYBPH, P2RX7, MAPK13, S100A11, CCL1, CCL7, CCL8, CCL18, CCL19, CCL22, CCL24, SLC1A2, SLC6A12, SLC11A1, SIGLEC1, SRC, TIE1, MARCO, HYAL2, CD163 , LONP1, IGSF6, LILRB1, CD300C, SDS, LILRB4, EMILIN1, VSIG4, PHLDB1, NCAPH , CLEC4E, MYOF, HSPB7, ADAMDEC1, GLRX2, MS4A4A, ATP6V1H, TREM2, TMEM70, T MEM33, KCNK13, SLAMF8, HAMP, DNASE2B, MYOZ1, MFSD7, ADO, ADCK2, and TBC1D16 158. An anti-CD20 antibody for use according to claim 156 or 157,

159. The anti-CD20 antibody for use according to any one of claims 141 to 154, wherein the macrophage biomarker is a gene expression value.

160. 160. An anti-CD20 antibody for use as described in claim 159, wherein the gene expression value is the median gene expression value.

161. 161. An anti-CD20 antibody for use according to claim 159 or 160, wherein gene expression values ​​are measured using a gene signature matrix.

162. The gene signature matrix contains the following genes: (a) CD200, KLHL14, TCL1A, NRG1, EOMES, PPP2R2B, RNF165, WNT7A, CCR4 , PDGFD, EBF1, FCGBP, PCDH9, MLC1, TSHZ2, S1PR5, NCALD, LAYN, GCNT4, F ASLG, TRAT1, ADAM6, GUCY1A3, LRRC4, TSPAN18, SBK1, ICOS, BTNL8, WNT5 B, AUTS2, SH2D2A, ADGRG3, PNOC, SPIB, VPREB3, DPEP3, MME, ZBTB16, FOX P3, SEMA3G, CD8A, TOGARAM2, COLGALT2, ABCB1, STAP1, SAMD3, FAM46C, B LK, CTLA4, CD19, REPS2, RTKN2, POU2AF1, DAPK2, PYHIN1, NLRC3, GATM, K LRD1, AFF3, FCRLA, AATBC, REM2, YPEL1, TXK, CD8B, P2RX5, CEACAM1, BCL 11A, ABCB4, CD5, HPGD, BLNK, PLCL1, HPSE, SLFN13, HOPX, CD1D, GNG7, TC F4, BANK1, FHIT, FCMR, GNG2, GFRA2, KBTBD11, RALGPS2, TSPOAP1, PLEKH F1, MEF2C, MAOA, TTYH2, HLA-DOB, DGAT2, FXYD6, TMCC3, MGAM, TTC38, LR RC32, ARHGAP24, STAT4, SLC7A8, CD72, FZD1, GK5, DYSF, PLTP, SMARCD3, FAM160B1, PDPN, AKAP2, ACVRL1, KCNJ15, ALDH1A2, ENPP2, COLEC12, PTG S1, TMEM170B, TREM2, ECM1, SLC1A3, ABHD5, MS4A4A, CLIC2, IL1R1, SLC2 A6, GAS7, RNF144B, SLC6A12, FPR2, ADAM28, GRK3, KDM1B, MATK, LMO2, CF B, CCRL2, CLEC4A, LILRA2, ACE, NUPR1, CISH, EREG, ADAMDEC1, RNASE6, C XCL3, VSIG4, CXCL2, CD86, LILRB4, SERPING1, SQOR, INHBA, and ICAM1; or <h2 style=";text-align:left;direction:ltr">(b)CD200、KLHL14、TCL1A、NRG1、CYP4 F3、EOMES、PPP2R2B、RNF165、WNT7A、C CR4、PDGFD、EBF1、FCGBP、PCDH9、MLC1 、TSHZ2、S1PR5、NCALD、LAYN、CD248、GC NT4、FASLG、TRAT1、ADAM6、GUCY1A3、L RRC4、TSPAN18、SBK1、ICOS、BTNL8、WN T5B、AUTS2、SH2D2A、ADGRG3、PNOC、SPIB、VPREB3、DPEP3、MME、ZBTB16、FOXP3 、SEMA3G、CD8A、TOGARAM2、COLGALT2、 ABCB1、STAP1、SAMD3、FAM46C、BLK、CT LA4、CD19、REPS2、RTKN2、POU2AF1、DA PK2、PYHIN1、NLRC3、GATM、KLRD1、AFF3 、FCRLA、AATBC、REM2、YPEL1、TXK、CD8 B、P2RX5、CEACAM1、BCL11A、NINJ2、AB CB4、CD5、HAL、HPGD、BLNK、PLCL1、CEP 19、HPSE、SLFN13、HOPX、CD1D、GNG7、TM EM154、TCF4、BANK1、FHIT、FCMR、GNG2 、GFRA2、KBTBD11、TECPR2、RALGPS2、T SPOAP1、PLEKHF1、MEF2C、MAOA、TTYH2 、HLA-DOB、NRGN、DGAT2、FXYD6、TMCC3、 MGAM、TTC38、LRRC32、ARHGAP24、PPP1 R3B、STAT4、SLC7A8、CD72、FZD1、GK5、 DYSF、PLTP、SMARCD3、FAM160B1、PDPN 、AKAP2、ACVRL1、KCNJ15、CD36、ALDH1A 2、ENPP2、COLEC12、PTGS1、TMEM170B、 DOCK5、TREM2、C5AR2、ECM1、SLC1A3、A BHD5、MS4A4A、CLIC2、IL1R1、SLC2A6、 GAS7、RNF144B、SLC6A12、FPR2、ADAM28 、GRK3、KDM1B、MATK、LMO2、CFB、CCRL2 、CLEC4A、TLR4、LILRA2、ACE、TLR1、LRR K2、LY96、NUPR1、CISH、CSTA、EREG、AD AMDEC1、RNASE6、CXCL3、VSIG4、CXCL2、CD86, LILRB4, SERPING1, SQOR, INHBA, and ICAM1; 162. An anti-CD20 antibody for use according to claim 161, comprising:

163. The gene signature matrix contains the following genes: CD200, KLHL14, TCL1A, NRG1, EOMES, PPP2R2B, RNF165, WNT7A, CCR4, PD GFD, EBF1, FCGBP, PCDH9, MLC1, TSHZ2, S1PR5, NCALD, LAYN, GCNT4, FASL G, TRAT1, ADAM6, GUCY1A3, LRRC4, TSPAN18, SBK1, ICOS, BTNL8, WNT5B, AUTS2, SH2D2A, ADGRG3, PNOC, SPIB, VPREB3, DPEP3, MME, ZBTB16, FOXP3 , SEMA3G, CD8A, TOGARAM2, COLGALT2, ABCB1, STAP1, SAMD3, FAM46C, BL K, CTLA4, CD19, REPS2, RTKN2, POU2AF1, DAPK2, PYHIN1, NLRC3, GATM, KL RD1, AFF3, FCRLA, AATBC, REM2, YPEL1, TXK, CD8B, P2RX5, CEACAM1, BCL 11A, ABCB4, CD5, HPGD, BLNK, PLCL1, HPSE, SLFN13, HOPX, CD1D, GNG7, TC F4, BANK1, FHIT, FCMR, GNG2, GFRA2, KBTBD11, RALGPS2, TSPOAP1, PLEK HF1, MEF2C, MAOA, TTYH2, HLA-DOB, DGAT2, FXYD6, TMCC3, MGAM, TTC38, L RRC32, ARHGAP24, STAT4, SLC7A8, CD72, FZD1, GK5, DYSF, PLTP, SMARCD 3, FAM160B1, PDPN, AKAP2, ACVRL1, KCNJ15, ALDH1A2, ENPP2, COLEC12, P TGS1, TMEM170B, TREM2, ECM1, SLC1A3, ABHD5, MS4A4A, CLIC2, IL1R1, S LC2A6, GAS7, RNF144B, SLC6A12, FPR2, ADAM28, GRK3, KDM1B, MATK, LMO2 , CFB, CCRL2, CLEC4A, LILRA2, ACE, NUPR1, CISH, EREG, ADAMDEC1, RNASE 6, CXCL3, VSIG4, CXCL2, CD86, LILRB4, SERPING1, SQOR, INHBA, and ICAM1 163. An anti-CD20 antibody for use according to claim 162, comprising:

164. The anti-CD20 antibody for use according to any one of claims 161 to 163, wherein a gene signature matrix is ​​used to determine the number of M1 macrophages.

165. The anti-CD20 antibody for use according to any one of claims 141 to 164, wherein the macrophage biomarker is the amount of M1 macrophages.

166. The anti-CD20 antibody for use according to claim 165, wherein the amount of M1 macrophages is measured directly or indirectly.

167. The anti-CD20 antibody for use according to claim 166, wherein the amount of M1 macrophages is measured directly using flow cytometry, spatial transcriptomics, spatial proteomics, or a combination thereof.

168. The anti-CD20 antibody for use according to claim 166, wherein the amount of M1 macrophages is measured indirectly using nucleic acid or protein.

169. The anti-CD20 antibody for use according to claim 168, wherein the nucleic acid is measured using RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technology, ISH, or a combination thereof.

170. The anti-CD20 antibody for use according to claim 169, wherein the amount of M1 macrophages is measured using a marker gene approach or a deconvolution approach.

171. The anti-CD20 antibody for use according to claim 170, wherein the marker gene approach uses xCell.

172. The anti-CD20 antibody for use according to claim 170, wherein the deconvolution approach uses quanTIseq.

173. An anti-CD20 antibody for use according to any one of claims 141 to 154, wherein macrophage biomarkers in a sample from a patient are measured using nucleic acids or proteins.

174. An anti-CD20 antibody for use as described in claim 173, wherein macrophage biomarkers in a sample from a patient are determined using nucleic acid expression levels.

175. The anti-CD20 antibody for use according to claim 174, wherein the nucleic acid expression level is determined by RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technology, ISH, or a combination thereof.

176. 176. An anti-CD20 antibody for use according to claim 174 or 175, wherein the nucleic acid expression level is an mRNA expression level.

177. The anti-CD20 antibody for use according to claim 176, wherein the mRNA expression level is determined by RNA-seq.

178. The anti-CD20 antibody for use according to any one of claims 141 to 177, wherein the sample is a tissue sample, a tumor sample, a whole blood sample, a plasma sample, a serum sample, or a combination thereof.

179. The anti-CD20 antibody for use according to claim 178, wherein the sample is a tissue sample.

180. 180. The anti-CD20 antibody for use according to claim 179, wherein the tissue sample is a tumor tissue sample.

181. The anti-CD20 antibody for use according to claim 180, wherein the tumor tissue sample comprises tumor cells, tumor infiltrating immune cells, stromal cells, NAT cells, or a combination thereof.

182. 182. An anti-CD20 antibody for use according to claim 180 or 181, wherein the tumor tissue sample is a biopsy.

183. The anti-CD20 antibody for use according to any one of claims 178 to 182, wherein the sample is an archived sample, a fresh sample or a frozen sample.

184. The anti-CD20 antibody for use according to any one of claims 141 to 183, wherein the lymphoma is a low-grade lymphoma.

185. The anti-CD20 antibody for use according to any one of claims 141 to 184, wherein the lymphoma is a B-cell lymphoma.

186. 186. The anti-CD20 antibody for use according to claim 184 or 185, wherein the B cell lymphoma is NHL.

187. The anti-CD20 antibody for use according to any one of claims 141 to 186, wherein the lymphoma is DLBCL, FL, CLL or MZL.

188. The anti-CD20 antibody for use according to claim 187, wherein the lymphoma is DLBCL.

189. The anti-CD20 antibody for use according to claim 188, wherein the DLBCL is of the GCB cell of origin subgroup or the ABC cell of origin subgroup of DLBCL.

190. The anti-CD20 antibody for use according to any one of claims 141 to 189, wherein the lymphoma is a CD20 positive lymphoma.

191. The anti-CD20 antibody for use according to any one of claims 141 to 190, wherein the anti-CD20 antibody is a type I anti-CD20 antibody or a type II anti-CD20 antibody.

192. The anti-CD20 antibody for use according to claim 191, wherein the anti-CD20 antibody is a type II anti-CD20 antibody.

193. A type II anti-CD20 antibody has the following CDRs: (a) CDR-H1 having the amino acid sequence of SEQ ID NO:27; (b) CDR-H2 having the amino acid sequence of SEQ ID NO:28; (c) a CDR-H3 having the amino acid sequence of SEQ ID NO:3; (d) a CDR-L1 having the amino acid sequence of SEQ ID NO:4; (e) a CDR-L2 having the amino acid sequence of SEQ ID NO:5; and (f) CDR-L3 having the amino acid sequence of SEQ ID NO:

6.

193. An anti-CD20 antibody for use according to claim 192, comprising:

194. The anti-CD20 antibody for use according to claim 193, wherein the type II anti-CD20 antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:

8.

195. The anti-CD20 antibody for use according to claim 194, wherein the type II anti-CD20 antibody is obinutuzumab.

196. The anti-CD20 antibody for use according to claim 191, wherein the anti-CD20 antibody is a type I anti-CD20 antibody.

197. A type I anti-CD20 antibody has the following CDRs: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO:11; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 12; (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 13; (d) CDR-L1 having the amino acid sequence of SEQ ID NO: 14; (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 15; and (f) CDR-L3 having the amino acid sequence of SEQ ID NO:

16.

197. An anti-CD20 antibody for use according to claim 196, comprising:

198. An anti-CD20 antibody for use according to claim 197, wherein the type I anti-CD20 antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO:25 and a VL domain comprising the amino acid sequence of SEQ ID NO:

26.

199. 199. The anti-CD20 antibody for use according to claim 198, wherein the type I anti-CD20 antibody is rituximab.

200. 200. The anti-CD20 antibody for use according to any one of claims 141 to 199, wherein the treatment further comprises the use of an effective amount of an additional therapeutic agent.

201. The anti-CD20 antibody for use according to claim 200, wherein the additional therapeutic agent is one or more of a chemotherapeutic agent, an anti-neoplastic agent, a growth inhibitory agent, an anti-angiogenic agent, radiation therapy, a cytotoxic agent, or a combination thereof.

202. The anti-CD20 antibody for use according to claim 201, wherein the additional therapeutic agent is a chemotherapeutic agent.

203. 203. An anti-CD20 antibody for use according to claim 201 or 202, wherein the chemotherapeutic agent is cyclophosphamide, doxorubicin, vincristine or prednisone.

204. 203. An anti-CD20 antibody for use according to claim 201 or 202, wherein the chemotherapeutic agents are cyclophosphamide, doxorubicin, vincristine and prednisone.

205. The anti-CD20 antibody for use according to any one of claims 141 to 204, wherein the patient has not been previously treated for lymphoma.

206. The anti-CD20 antibody for use according to any one of claims 141 to 205, wherein the patient has not previously been administered an anti-CD20 antibody.

207. A method for identifying, diagnosing, and / or predicting whether a patient having lymphoma may benefit from a treatment comprising an anti-CD20 antibody, the method comprising measuring a Th2 biomarker in a sample from the patient, wherein an amount or level of the Th2 biomarker in the sample that exceeds an amount or level of a reference Th2 biomarker identifies, diagnoses, and / or predicts the patient as one who may benefit from a treatment comprising an anti-CD20 antibody.

208. A method for selecting a therapy for a patient having lymphoma, the method comprising measuring a Th2 biomarker in a sample from the patient, wherein an amount or level of the Th2 biomarker in the sample that exceeds an amount or level of a reference Th2 biomarker identifies the patient as one who may benefit from a treatment comprising an anti-CD20 antibody.

209. The method of claim 207 or 208, wherein the patient has a Th2 biomarker in the sample that exceeds an amount or level of a reference Th2 biomarker, the method further comprising administering to the patient an effective amount of an anti-CD20 antibody.

210. 1. A method of treating a patient having lymphoma, the method comprising: (a) measuring a Th2 biomarker in a sample from a patient, wherein the amount or level of the Th2 biomarker in the sample exceeds the amount or level of a reference Th2 biomarker; and (b) administering to the patient an effective amount of an anti-CD20 antibody based on the Th2 biomarker measured in step (a). A method comprising:

211. A method of treating a patient having lymphoma, the method comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein prior to treatment, the amount or level of a Th2 biomarker in a sample from the patient has been determined to exceed the amount or level of a reference Th2 biomarker.

212. A method of treating a patient having lymphoma and having an amount or level of a Th2 biomarker in a sample from the patient that exceeds the amount or level of a reference Th2 biomarker, the method comprising administering to the patient an effective amount of an anti-CD20 antibody.

213. The method of any one of claims 207 to 212, wherein the reference Th2 biomarker amount or level is a pre-assigned Th2 biomarker amount or level.

214. The method of any one of claims 207 to 213, wherein the amount or level of the reference Th2 biomarker is the amount or level of the Th2 biomarker in a reference population.

215. The method of claim 214, wherein the amount or level of the Th2 biomarker in the reference population is the median amount or level of the Th2 biomarker in the reference population.

216. The method of claim 214, wherein the reference Th2 biomarker amount or level is the amount or level of the Th2 biomarker that is at the 25th percentile of a reference population.

217. The method of claim 214, wherein the reference Th2 biomarker amount or level is the amount or level of the Th2 biomarker that is at the 50th percentile of a reference population.

218. The method of claim 214, wherein the reference Th2 biomarker amount or level is the amount or level of the Th2 biomarker that is at the 75th percentile of a reference population.

219. The method of any one of claims 214 to 218, wherein the reference population is a population of patients with lymphoma.

220. 220. The method of claim 219, wherein the population of patients having lymphoma have been previously treated with an anti-CD20 antibody.

221. The method of claim 220, wherein the reference Th2 biomarker amount or level is the amount or level of the Th2 biomarker in a reference population before initiating treatment with an anti-CD20 antibody.

222. The method of claim 220 or 221, wherein the amount or level of the reference Th2 biomarker significantly separates the reference population into a first set of patients who have benefited from treatment with an anti-CD20 antibody and a second set of patients who have not benefited from treatment with an anti-CD20 antibody.

223. 223. The method of any one of claims 207 to 222, wherein the amount or level of the reference Th2 biomarker is the amount of Th2 cells as measured by gene expression.

224. The method of any one of claims 207 to 209, wherein the benefit is prolongation of PFS.

225. 210. The method of any one of claims 207 to 209, wherein the benefit is increased OS.

226. 213. The method of any one of claims 210-212, further comprising achieving improved PFS or OS.

227. The method of any one of claims 207 to 226, wherein the Th2 biomarker is the amount of Th2 cells.

228. The method of claim 227, wherein the amount of Th2 cells is measured directly or indirectly.

229. The method of claim 228, wherein the amount of Th2 cells is measured directly using flow cytometry, spatial transcriptomics, spatial proteomics, or a combination thereof.

230. The method of claim 228, wherein the amount of Th2 cells is measured indirectly using nucleic acids or proteins.

231. 231. The method of claim 230, wherein the nucleic acid is measured using RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technology, ISH, or a combination thereof.

232. The method of claim 231, wherein the amount of Th2 cells is measured using a marker gene approach or a deconvolution approach.

233. The method of claim 232, wherein the marker gene approach uses xCell.

234. The method of claim 232, wherein the deconvolution approach uses quanTIseq.

235. A method according to any one of claims 207 to 226, wherein the Th2 biomarker in a sample from a patient is measured using nucleic acid or protein.

236. The method of claim 235, wherein the Th2 biomarker in a sample from a patient is determined using nucleic acid expression levels.

237. 237. The method of claim 236, wherein the nucleic acid expression level is determined by RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technology, ISH, or a combination thereof.

238. The method of claim 236 or 237, wherein the nucleic acid expression level is an mRNA expression level.

239. The method of claim 238, wherein the mRNA expression level is determined by RNA-seq.

240. 240. The method of any one of claims 207 to 239, wherein the sample is a tissue sample, a tumor sample, a whole blood sample, a plasma sample, a serum sample, or a combination thereof.

241. 241. The method of claim 240, wherein the sample is a tissue sample.

242. 242. The method of claim 241, wherein the tissue sample is a tumor tissue sample.

243. 243. The method of claim 242, wherein the tumor tissue sample comprises tumor cells, tumor-infiltrating immune cells, stromal cells, NAT cells, or a combination thereof.

244. 244. The method of claim 242 or 243, wherein the tumor tissue sample is a biopsy.

245. 245. The method of any one of claims 240 to 244, wherein the sample is an archived sample, a fresh sample, or a frozen sample.

246. The method of any one of claims 207 to 245, wherein the lymphoma is an indolent lymphoma.

247. The method of any one of claims 207 to 246, wherein the lymphoma is a B cell lymphoma.

248. The method of claim 247, wherein the B cell lymphoma is germinal center-derived B cell lymphoma.

249. The method of claim 247 or 248, wherein the B cell lymphoma is NHL.

250. The method of any one of claims 207 to 249, wherein the lymphoma is DLBCL, FL, CLL or MZL.

251. The method of claim 250, wherein the lymphoma is DLBCL.

252. The method of claim 251, wherein the DLBCL is the GCB cell of origin subgroup or the ABC cell of origin subgroup of DLBCL.

253. The method of any one of claims 207 to 252, wherein the lymphoma is a CD20 positive lymphoma.

254. The method of any one of claims 207 to 253, wherein the anti-CD20 antibody is a type I anti-CD20 antibody or a type II anti-CD20 antibody.

255. The method of claim 254, wherein the anti-CD20 antibody is a type II anti-CD20 antibody.

256. A type II anti-CD20 antibody has the following CDRs: (a) CDR-H1 having the amino acid sequence of SEQ ID NO:27; (b) CDR-H2 having the amino acid sequence of SEQ ID NO:28; (c) a CDR-H3 having the amino acid sequence of SEQ ID NO:3; (d) a CDR-L1 having the amino acid sequence of SEQ ID NO:4; (e) a CDR-L2 having the amino acid sequence of SEQ ID NO:5; and (f) CDR-L3 having the amino acid sequence of SEQ ID NO:

6. The method of claim 255, comprising:

257. The method of claim 256, wherein the type II anti-CD20 antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:

8.

258. The method of claim 257, wherein the type II anti-CD20 antibody is obinutuzumab.

259. The method of claim 254, wherein the anti-CD20 antibody is a type I anti-CD20 antibody.

260. A type I anti-CD20 antibody has the following CDRs: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO:11; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 12; (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 13; (d) CDR-L1 having the amino acid sequence of SEQ ID NO: 14; (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 15; and (f) CDR-L3 having the amino acid sequence of SEQ ID NO:

16.

260. The method of claim 259, comprising:

261. The method of claim 260, wherein the type I anti-CD20 antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO:25 and a VL domain comprising the amino acid sequence of SEQ ID NO:

26.

262. The method of claim 261, wherein the type I anti-CD20 antibody is rituximab.

263. 263. The method of any one of claims 209-262, further comprising administering to the patient an effective amount of an additional therapeutic agent.

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