Usutuzumab obabine treatment of DLBCL patient subgroup

Abstract

The present invention relates to Usutuzumab octobabine (or its functional equivalents) for use in the treatment of DLBCL patients defined by specific biomarkers and a new subgroup of DLBCL patients, respectively. The present invention further relates to a method for treating DLBCL with Usutuzumab octobabine (or a functional equivalent thereof) in a patient in need thereof, wherein the patient is a specific biomarker defined DLBCL patient or belongs to a new biomarker defined DLBCL patient subset. The present invention further relates to the use of Usutuzumab octobabine (or its functional equivalents) for the preparation of a pharmaceutical composition for the treatment of DLBCL in specific biomarker defined DLBCL patients / new subsets of DLBCL patients. The invention further relates, respectively, to a method for identifying a specific DLBCL patient / a new DLBCL patient subset and to a method for diagnosing a new form of DLBCL and a specific DLBCL patient / a new DLBCL patient subset.

Description

[0001] This application is a divisional application of an invention patent application with an application date of August 8, 2018, application number 201880051243.4, and invention name “A treatment of a subgroup of DLBCL patients with obinutuzumab”. Field of the Invention

[0002] The present invention relates to obinutuzumab (or its functional equivalent) for use in the treatment of DLBCL patients and a new subgroup of DLBCL patients defined by specific biomarkers, respectively. The present invention further relates to a method for treating DLBCL with obinutuzumab (or its functional equivalent) in a patient in need, wherein the patient is a DLBCL patient defined by a specific biomarker or belongs to a new subgroup of DLBCL patients defined by a biomarker. The present invention further relates to the use of obinutuzumab (or its functional equivalent) to prepare a pharmaceutical composition for treating DLBCL in DLBCL patients / new subgroups of DLBCL patients defined by specific biomarkers. The present invention further relates to a method for identifying specific DLBCL patients / new subgroups of DLBCL patients and a method for diagnosing a new form of DLBCL and specific DLBCL patients / new subgroups of DLBCL patients, respectively. Background of the Invention

[0003] Diffuse large B-cell lymphoma (DLBCL) is the most common type of aggressive non-Hodgkin's lymphoma (NHL). Immunochemotherapy with the anti-CD20 monoclonal antibody (mAb) rituximab (R) plus cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) is the standard of care treatment for previously untreated patients presenting with advanced stage disease (Coiffier, N. Engl. J. Med. 346, 2002, 235-242; Tilly, Ann. Oncol. 26, 2015, v116-v125 (suppl 5); NCCN Clincal Practice Guidelines in Oncology: Non-Hodgkin's Lymphomas, Version 3, 2016; also see www.NCCN.org). Studies have shown a 76% complete and unconfirmed complete response (CR / CRu) rate (GELA trial) (Coiffier, supra), and a 2-year failure-free survival rate of 77% (Habermann, J. Clin. Oncol. 24, 2006, 3121-3127). Although first-line (1L) treatment for DLBCL is curative for many patients (Maurer, J. Clin. Oncol. 32, 2014, 1066-1073), there is still a need to improve outcomes for the 20-40% of patients who fail to achieve regression or relapse, and the outcomes of salvage therapy are still poor (Sehn, Blood 125, 2015, 22-32).

[0004] Obinutuzumab (Gazyva TM / Gazyvaro TM and GA101; G) is a glycoengineered type II anti-CD20 mAb with greater direct cell death induction than R, and antibody-dependent cellular cytotoxicity and phagocytosis (Herter, Mol. Cancer Ther. 12, 2013, 2031-2042; Blood 115, 2010, 4393-4402; EP-B1 2380910; WO 2005 / 044859; Illidge, Expert Opin. Biol. Ther. 12(5), 2012, 543-5). In a phase 3 study of previously untreated patients with chronic lymphocytic leukemia (CLL) and coexisting conditions (CLL11), or follicular lymphoma (FL; GALLIUM), G proved to be more effective than R (Goede, N. Engl. J. Med. 370, 2014, 1101-1110; Marcus, N. Engl. J. Med. Accepted May 2017, in press). In smaller studies, G monotherapy and G-CHOP have shown promise in aggressive forms of non-Hodgkin lymphoma (NHL), including DLBCL (Morschhauser, J. Clin. Oncol. 31, 2013, 2912-2919; Zelenetz, Blood 122, 2013, 1820). Also, Owen (Expert Opin. Biol. Ther. 12 (3), 2012, 343-51) discussed the use of obinutuzumab for the treatment of lymphoproliferative disorders. A multicenter, open-label, randomized, phase 3 clinical study (GOYA; see below for more details) compared the efficacy and safety of G-CHOP with R-CHOP in previously untreated patients with DLBCL. However, in GOYA, G-CHOP did not improve clinical outcomes (e.g., progression-free survival (PFS)) relative to R-CHOP in previously untreated DLBCL (1L DLBCL) for the entire group of 1L DLBCL patients originally intended to be treated in the setting of GOYA.

[0005] Scott (Blood 123(8), 2014, 1214-7; JCO 33(26), 2015, 2848-57; Am. Soc. Clin. Oncol. Educ. Book 2015, 35:e458-66) and others (Nowakowski, Am. Soc. Clin. Oncol. Educ. Book 2015, 35:e449-57) performed gene expression-based determination of cell of origin (COO) subtypes of DLBCL using the NanoString Lymph2Cx assay (Scott, 2014 and 2015, supra). In particular, Scott (2014 and 2015, supra) assigned the COO subtypes of DLBCL, germinal center B-cell-like DLBCL (GCB DLBCL), activated B-cell-like DLBCL (ABC DLBCL), and unclassified DLBCL based on a 20-gene gene expression assay and linear predictor scores (LPS) of about <1900, about 1900 to about 2450, and about >2450, respectively (Scott 2014, supra, Figure 1 ). Scott (2014 and 2015, supra) also evaluated the therapeutic effect of R-CHOP on these COO subtypes of DLBCL. Punnoose (Blood 126, 2015, 3971; see also http: / / www.bloodjournal.org / content / 126 / 23 / 3971) described the prevalence and prognostic value of BCL2 and MYC protein expression in patients with previously untreated DLBCL from MAIN, a phase III trial evaluating bevacizumab plus R-CHOP (NCT00486759) within the ABC and GCB COO subtypes. Challa-Malladi (Cancer Cell 20, 2011, 728-40) disclosed that combined genetic inactivation of β2-microglobulin and CD58 revealed frequent escape from immune recognition in DLBCL.

[0006] Despite previous successful treatments for DLBCL (eg, due to advances in R, particularly R-CHOP), some DLBCL patients (NCCN Clinical Practice Guidelines in Oncology; Non-Hodgkin's Lymphoma, v 2.2015) still have a high unmet medical need for improved treatments (Sehn, supra).

[0007] The technical problem underlying the present invention is therefore to provide an improved treatment of DLBCL in certain patients.

[0008] The solution to the technical problem is provided herein below and characterized in the appended claims.

[0009] Description of the invention

[0010] In the context of the present invention, it was surprisingly found that among all DLBCL patients, some patients responded to treatment with obinutuzumab (particularly in combination with chemotherapy, more particularly in combination with CHOP chemotherapy) by improved clinical outcomes, in particular compared to treatment with rituximab (particularly in combination with chemotherapy, more particularly in combination with CHOP chemotherapy). In the context of the present invention, it was also surprisingly found that a subgroup of DLBCL patients who responded to treatment with obinutuzumab (particularly in combination with chemotherapy, more particularly in combination with CHOP chemotherapy) by improved clinical outcomes, in particular compared to treatment with rituximab (particularly in combination with chemotherapy, more particularly in combination with CHOP chemotherapy) can be identified / determined. In the context of the present invention, it was also surprisingly found that there are DLBCL patients who responded to treatment with obinutuzumab (particularly in combination with chemotherapy, more particularly in combination with CHOP chemotherapy) by improved clinical outcomes, in particular compared to treatment with rituximab (particularly in combination with chemotherapy, more particularly in combination with CHOP chemotherapy). The main point of the present invention is that such specific DLBCL patients and subgroups of DLBCL patients can indeed be defined; also referred to herein as "patients defined herein". Yet another point of the present invention is that such patients can be defined by biomarkers (also referred to herein as "predictive biomarkers" and "predictive biomarker-defined patients", respectively).

[0011] In particular, GOYA-based exploratory analyses demonstrate in the context of the present invention, and exemplified by the accompanying examples, that obinutuzumab is superior to rituximab (both in combination with CHOP chemotherapy) in one or more subsets of GCB DLBCL patients (e.g., in a novel molecular follicular lymphoma (FL)-like GCB DLBCL patient subgroup) and / or in DLBCL patients with mutations in CD58 and / or with low expression of CD58. This is the first time that obinutuzumab has been identified to benefit over rituximab for certain patients, particularly for the patients defined herein.

[0012] For example, in the context of the present invention, the following biomarker-defined DLBCL patients who benefit from treatment with G (e.g., G-CHOP) over treatment with R (e.g., R-CHOP) are identified / determined:

[0013] Patients with BCL2 translocation (see e.g. Figure 4 );

[0014] Patients with positive BCL2 protein expression (see e.g. Figure 5 );

[0015] Patients with BCL2 translocation who are positive for BCL2 protein expression (see e.g. Figure 6 );

[0016] One or more subsets of GCB DLBCL patients. These can be identified, for example, as follows:

[0017] o Classification of GCB patients into a subgroup of "strong GCB" patients by cutoff of linear predictor score (LPS) (COO) classification (patients with LPS < cutoff; e.g., compared to the general GCB DBLCL cutoff of LPS of about 1900) (see e.g., Figures 7, 10 and 12);

[0018] o GCB patients with high BCL2 gene expression;

[0019] ο GCB patients with positive BCL2 protein expression;

[0020] ο GCB patients with BCL2 translocation;

[0021] o GCB patients with BCL2 translocation who are positive for BCL2 protein expression (see e.g. Figure 8 );

[0022] Patients with CD58 mutations and / or patients with low expression of CD58 (see e.g. Fig. 9 ).

[0023] More specifically, exploratory analyses based on GOYA demonstrated in the context of the present invention, and exemplified by the accompanying Examples, that assessment of LPS as a continuous variable identified a subgroup of GCB patients who benefited from G (particularly G-CHOP) over R (particularly R-CHOP). Even more specifically, weighted expression of gene expression (GE) array profiles (measured by LPS) was observed to be associated with outcome benefit of G therapy (e.g., G-CHOP) over R therapy (e.g., R-CHOP) in GCB patients in GOYA.

[0024] On this basis, new LPS cutoffs were identified that define a subgroup of patients with strong GCB DLBCL who benefit from G therapy over R therapy. These new cutoffs were substantially lower than the LPS cutoffs typically assigned to GCB DLBCL (approximately <1900). For example, a new LPS cutoff of ≤749 was identified in multivariate simulation analysis (see Fig.10 ). According to this example, 'strong GCB' patients, defined as patients with an LPS of ≤749, represented 25% (233 / 933) of the evaluable DLBCL patients and 43% (233 / 540) of the evaluable GCB patients in GOYA. As another example, a new LPS cutoff of ≤725 was identified in the multivariate simulation analysis (see Fig.10,12). Following this example, 'strong GCB' patients were more strictly defined as those with an LPS of ≤725. These patients represented 25% (229 / 933) of evaluable DLBCL patients and 43% (229 / 540) of evaluable GCB patients in GOYA. The LPS cutoff around 725 was shown to reflect exceptional robustness and high generalizability of the results to an independent cohort ('patients defined herein'), namely strong GCB DLBCL patients. This was demonstrated by bootstrap simulations.

[0025] Strong GCB patients treated with G (G-CHOP) achieved significantly better clinical outcomes than those treated with R (R-CHOP), such as in terms of progression-free survival (PFS), event-free survival (EFS), and overall survival (OS) (see Table 4). The high level of safety was similar for both treatment regimens.

[0026] In the right In gene set enrichment analysis of Heme (FOH) data, strong GCB patients were further characterized as significantly enriched for FL somatic mutation signatures compared to other GCB patients referred to as "weak GCB" patients (e.g., false discovery rate, FDR, 3.54e-9). In particular, mutations / mutation rates in BCL2 translocations and several m7-FLIPI genes (BCL2, BCL6, CD70, CDKN2A, CREBBP, EP300, IGH, MEF2B, MYC, MYD88, PCLO, TNFAIP3, TNFRSF14) were highly enriched in strong GCB patients, and / or in DLBCL patients with BCL2 translocations and / or with high BCL2 expression compared to other DLBCL patients (with <5% FDR; Fig.11 ). Central pathology review did not show strong evidence of transformed indolent NHL in the GCB subset.

[0027] In summary, new clinical and molecularly distinct subtypes of DLBCL, particularly GCB DLBCL, have been identified, particularly a subtype referred to as 'strong GCB'. The identified subtypes represent completely new DLBCL. These exhibit molecular features of FL, such as FL typical mutations (see Morin, Nature 476 (7360), 2011, 298-303), however, they are clinically different from FL. In these new subsets of (GCB) DLBCL patients ("patients defined herein"), particularly 1L (GCB) DLBCL, treatment with G (e.g., G-CHOP) confers substantial clinical benefit over treatment with R (e.g., R-CHOP).

[0028] Thus, the present invention provides means and methods for identifying / determining / diagnosing one or more subsets of (GCB) DLBCL patients who respond favorably to obinutuzumab ("patients as defined herein"), in particular who respond more favorably to obinutuzumab than R. The identification / determination / diagnosis can be performed in several ways, for example by determining whether there is a BCL2 translocation and / or overexpression of BCL2 protein, whether there is one or more genetic mutations in CD58 and / or there is reduced CD58 expression, or by gene expression profiling / determination of weighted gene expression (e.g. by employing the NanoString COO assay) and using a novel LPS cutoff (as described elsewhere herein).

[0029] More particularly, the present invention relates to a method for identifying DLBCL patients (patients having / suffering from DLBCL) who respond to treatment with obinutuzumab (particularly in combination with chemotherapy, more particularly in combination with CHOP chemotherapy) by achieving an improved clinical outcome compared to treatment with rituximab (particularly in combination with chemotherapy, more particularly in combination with CHOP chemotherapy). The method comprises determining (e.g. by using a (tumor) sample of the patient) whether the patient is (a step of) a patient as defined herein.

[0030] The present invention further relates to a method for diagnosing in a patient a form of DLBCL that can be treated with obinutuzumab (particularly in combination with chemotherapy, more particularly in combination with CHOP chemotherapy) to achieve an improved clinical outcome compared to treatment with rituximab (particularly in combination with chemotherapy, more particularly in combination with CHOP chemotherapy). The method comprises determining (e.g. by using a (tumor) sample of the patient) whether the patient is a patient as defined herein. The method (further) comprises diagnosing the form of DLBCL if the patient is a patient as defined herein.

[0031] The present invention also relates to medical intervention / treatment of patients as defined herein by obinutuzumab or by its functional equivalents. In principle, as used herein, the term "obinutuzumab" also encompasses its functional equivalents (see below for further explanation / definition).

[0032] In one aspect, the invention relates to obinutuzumab for use in the treatment of DLBCL in a patient as defined herein.

[0033] In the context of such use, for example, it is encompassed that (i) it has been determined (e.g. by using a (tumor) sample from the patient) whether the patient to be treated is a patient as defined herein, (ii) the patient to be treated has been identified according to the identification method of the invention, or (iii) a form of DLBCL has been diagnosed in the patient to be treated according to the diagnostic method of the invention.

[0034] In another aspect, the invention relates to obinutuzumab for use in the treatment of DLBCL in a patient as defined herein, wherein, for example, it is contemplated that the treatment comprises the following steps: (i) determining (e.g. by using a (tumor) sample from the patient) whether the (DLBCL) patient to be treated is a patient as defined herein, (ii) identifying a DLBCL patient according to the identification method of the invention, or (iii) diagnosing a form of DLBCL in a patient according to the diagnostic method of the invention.

[0035] In the context of determining / identifying / diagnosing (the step of), a sample of a (DLBCL) patient, such as a tumor sample of a (DLBCL) patient, may be employed. The assay may be in a sample of a (DLBCL) patient, such as a tumor sample of a (DLBCL) patient. A non-limiting example of a specific sample to be employed in accordance with the present invention is a sample of a tumor tissue / tumor biopsy, more particularly a formalin-fixed, paraffin-embedded tumor tissue / tumor biopsy. Such samples may be prepared, for example, as described in Scott (2014 and 2015, supra). Other suitable samples are described elsewhere herein.

[0036] In another aspect, the present invention relates to a method for treating DLBCL in a patient in need thereof, wherein the patient is a patient as defined herein. The method may comprise the steps of obtaining a sample of a patient for whom a DLBCL therapy is envisaged and / or testing a sample of a patient to determine whether the patient is a patient as defined herein. Contemplated are steps of the treatment methods of the present invention comprising administering a pharmaceutically effective amount of obinutuzumab to the patient to be treated. In the context of these steps, in particular the testing steps, a (tumor) sample of a (DLBCL) patient may be employed. The test may be in a (tumor) sample of a (DLBCL) patient. What is described herein above and elsewhere with respect to the "sample" to be employed also applies mutatis mutans here.

[0037] The identification or diagnosis method according to the invention may also be employed in the context of these steps, in particular in the context of a testing step (or instead of this step).

[0038] In another aspect, the present invention relates to the use of obinutuzumab for preparing a pharmaceutical composition for treating DLBCL in a patient as defined herein. The treatment may comprise a step of determining / identifying / diagnosing whether the (DLBCL) patient to be treated is a patient as defined herein in accordance with the present invention. In the context of the determination / identification / diagnosis step, a sample of a (DLBCL) patient may be used, such as a tumor sample of a (DLBCL) patient. The determination may be in a sample of a (DLBCL) patient, such as a tumor sample of a (DLBCL) patient. The references described elsewhere herein with respect to the "sample" to be used apply here. Similarly, the references described above with respect to use and treatment apply here.

[0039] The patient to be treated according to the present invention ("patient as defined herein") is a patient, in particular a patient having / suffering from DLBCL, who responds to treatment with obinutuzumab (in particular in combination with chemotherapy, more particularly in combination with CHOP chemotherapy) by achieving an improved clinical outcome compared to treatment with rituximab (in particular in combination with chemotherapy, more particularly in combination with CHOP chemotherapy).

[0040] In one aspect / embodiment of the invention (Aspect / Embodiment A), the patient as defined herein (to be treated with obinutuzumab) is a predictive biomarker defined patient.

[0041] 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 (predictive) biomarkers), i.e. patients who respond more favorably to treatment with obinutuzumab (particularly in combination with chemotherapy, more particularly in combination with CHOP chemotherapy) than to treatment with rituximab (particularly in combination with chemotherapy, more particularly in combination with CHOP chemotherapy). More particularly, a biomarker is predictive if the treatment effect (i.e. G treatment compared to R treatment) differs between patients (subgroups) defined by the biomarker. It is preferred in this context that a predictive biomarker is a biomarker as defined elsewhere herein. Specific examples of predictive biomarkers to be assessed in the context of the present invention are CD58 (e.g. one or more genetic mutations therein and / or its underexpression), BCL2 (e.g. its translocation and / or overexpression) and one or more (preferably all) of the genes TNFRSF13B, LIMD1, IRF4, CREB3L2, PIM2, CYB5R2, RAB7L1, and CCDC50; and MME, SERPINA9, ASB13, MAML3, ITPKB, MYBL1, and S1PR2 (e.g. its weighted expression produces an LPS substantially below about 1900). In this context, reference is also made to what is disclosed herein below, in particular in the context of aspects / embodiments B to G below.

[0042] In one aspect / embodiment of the invention (Aspect / Embodiment B), the patient as defined herein (to be treated with obinutuzumab) is a patient having / suffering from molecular follicular lymphoma (FL)-like germinal center B-cell (GCB) DLBCL.

[0043] "Molecular" in this context means that the patient is similar to FL patients at the molecular level (see Morin, supra). However, it is contemplated that the patient is not clinically similar to FL patients at the clinical level / clinically.

[0044] According to the present invention, the molecular FL-like GCB DLBCL according to this aspect / embodiment is preferably characterized as a strong GCB DLBCL according to the present invention and / or a DLBCL in a patient with a BCL2 translocation and / or high BCL2 expression (see aspects / embodiments D and E below).

[0045] Patients suffering from molecular FL-like GCB DLBCL according to the present invention may also be characterized as patients having one or (preferably) multiple mutations in one or (preferably) multiple genes selected from the group consisting of BCL2, BCL6, CD70, CDKN2A, CREBBP, EP300, IGH, MEF2B, MYC, MYD88, PCLO, TNFAIP3 and TNFRSF14. Although less preferred, patients suffering from molecular FL-like GCB DLBCL according to the present invention may also be characterized as patients having one or (preferably) multiple mutations in one or (preferably) multiple genes selected from the group consisting of BCL2, CREBBP, EP300, EZH2, MEF2B, PCLO, and TNFRSF14, having one or (preferably) multiple mutations in one or (preferably) multiple genes selected from the group consisting of CREBBP, EP300, EZH2, MEF2B and TNFRSF14, or having one or (preferably) multiple mutations in one or (preferably) multiple genes selected from the group consisting of EZH2, MEF2B and TNFRSF14.

[0046] A specific but non-limiting example of a mutation that can be used in this regard is a BCL2 mutation, in particular a BCL2 translocation (see below for details).

[0047] Mutations can be identified, for example, by relying on the accompanying examples. In this regard, Foundation Medicine next generation sequencing assays can be used, for example. Heme (as per distributor's manual).

[0048] In one aspect / embodiment of the invention (Aspect / Embodiment C), the patient as defined herein (to be treated with obinutuzumab) is a patient having one or more genetic mutations in CD58 and / or having low expression of CD58.

[0049] CD58 (see also Challa-Malladi, supra) is known to be involved in immune recognition of tumor cells and is expressed on tumor cells. CD58 binds to CD2 on effector CTL and NK cells (thereby providing an activation signal for immune effector cells). The presence of genetic abnormalities in CD58 is associated with lost or abnormal CD58 surface expression (e.g., detectable by immunohistochemistry, IHC). 67% of DLBCL cases show abnormal CD58 protein expression; the same proportions in the GCB and ABC COO subgroups.

[0050] Nucleotide sequences encoding CD58 (particularly human (Homo sapiens) CD58) and the amino acid sequence of CD58 are well known in the art. They can be downloaded, for example, via the following uniform resource locator (URL): https: / / www.ncbi.nlm.nih.gov / search / ?term=Homo+sapiens+CD58&utm_expid=.fAeHyO5JTBGxnObh2WlrCA.0&utm_referrer=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fsearch%2F%3Fterm%3DHomo%2Bsapiens%2BCD58. Nucleotide sequences encoding CD58, in particular human CD58, are available, for example, via NCBI accession numbers XM_017002869.2 (variant X1); NR_026665.1 (variant 3); NM_001779.2 (variant 1); NM_001144822.1 (variant 2). Amino acid sequences of CD58, in particular human CD58, are available, for example, via NCBI accession numbers XP_016858358.1 (isoform X1); NP_001138294.1 (isoform 2); NP_001770.1 (isoform 1). An example of a nucleotide sequence encoding human CD58 is depicted in SEQ ID NO: 11. An example of an amino acid sequence of human CD58 is depicted in SEQ ID NO: 12.

[0051] In principle, "expression" in the context of the present invention encompasses both gene expression, ie the occurrence of (mainly) mRNA (transcription level), and protein expression, ie the occurrence of protein (translation level).

[0052] The presence of (mainly) mRNA can be measured / detected, for example, by in situ hybridization (ISH) techniques, for example by fluorescent ISH (FISH). The respective means and methods are known in the art and are described, for example, in Zhang (Chin. J. Cancer. Res. 23 (2), 2011, 160-4; see also https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC3587538 / ).

[0053] Gene expression / (mainly) mRNA appearance, especially CD58 gene expression / (mainly) CD58 mRNA appearance can also be measured by using RNA sequencing to assess (depending on the distributor( Inc.) manual).

[0054] The presence of the protein can be measured / detected, for example, by IHC. The respective means and methods are known in the art and are described, for example, in Punnoose (supra; see also http: / / www.bloodjournal.org / content / 126 / 23 / 3971).

[0055] Protein expression / presence of proteins, in particular CD58 protein expression, can also be measured / detected as described in Challa-Malladi (supra).

[0056] Generally, means and methods for assessing CD58 expression and CD58 mutations are known in the art (see, e.g., Challa-Malladi, supra). Furthermore, the skilled person can easily assess whether a given CD58 expression is "low" according to the invention or whether there are one or more CD58 mutations according to the invention. Moreover, the skilled person can easily select a suitable control, compared to which a given CD58 expression is considered "low" according to the invention or compared to which there are one or more CD58 mutations according to the invention. In this context, the skilled person can also, for example, rely on Challa-Malladi (supra).

[0057] In the context of the present invention, "low expression of CD58" means that CD58 is expressed at a substantially lower level, especially compared to a suitable control. Generally, "low expression of CD58" according to the present invention means that CD58 expression is as low (e.g. ±10% or less, ±7.5% or less, ±5% or less, ±3% or less, ±2% or less, ±1% or less, or even ±0%) as CD58 expression in responders according to the present invention (i.e. patients who respond to treatment with obinutuzumab (in particular in combination with chemotherapy, more particularly in combination with CHOP chemotherapy) by achieving an improved clinical outcome compared to treatment with rituximab (in particular in combination with chemotherapy, more particularly in combination with CHOP chemotherapy); "patients as defined herein") and / or lower than CD58 expression in non-responders according to the present invention (i.e. patients who respond to treatment with obinutuzumab (in particular in combination with chemotherapy, more particularly in combination with CHOP chemotherapy) by not achieving an improved clinical outcome compared to treatment with rituximab (in particular in combination with chemotherapy, more particularly in combination with CHOP chemotherapy); not "patients as defined herein"). The skilled person is readily able to assess when CD58 expression is "low" in this regard and to apply an appropriate control. For example, the control in this regard may be a general DLBCL population, more particularly a DLBCL patient who is not classified as belonging to a patient subgroup classified according to the present invention (not a "patient as defined herein").

[0058] "Low expression of CD58" may be CD58 expression that is lower than the median CD58 expression in a control group, such as such control group described above. For example, "low expression of CD58" and "lower level" of CD58 expression according to the present invention may be CD58 expression that is lower than the median CD58 expression in observed GOYA patients, respectively.

[0059] As a unit of CD58 (gene) expression, the unit log2 (nRPKM) can be used, which is normalized reads per kilobase per million. The median CD58 expression in GOYA patients using this unit is about 5.3. Thus, "low expression of CD58" and "lower level" of CD58 expression according to the present invention can be a CD58 expression lower than the CD58 expression in the control patient (group) (see above) corresponding to the unit log2 (nRPKM) 5.3 (median CD58 expression in the control patient (group)). In other words, the CD58 expression in the patient to be treated according to the present invention can be substantially lower than the CD58 expression in the control patient (group) corresponding to the unit log2 (nRPKM) 5.3. That is, the CD58 expression in the patient to be treated according to the present invention can be a CD58 expression corresponding to the unit log2 (nRPKM) ≤ 5.2, ≤ 5.1, ≤ 5.0, ≤ 4.5, ≤ 4.0, ≤ 3.5, ≤ 3.0, ≤ 2.5 or ≤ 2.0.

[0060] As mentioned above, CD58 is expressed on tumor cells and on the surface of B cells. Thus, in a specific aspect, "low expression of CD58" and "lower level" of CD58 expression according to the present invention means that CD58 is expressed on tumor cells and / or B cells at a level substantially lower than CD58 expression on common DLBCL tumor cells and / or common DLBCL B cells, respectively. "Common DLBCL" in this context means, for example, that tumor cells and B cells, respectively, are derived from a non-responder according to the present invention, preferably from a DLBCL tumor cell and a DLBCL B cell, respectively, derived from a DLBCL patient who is not classified as a patient as defined herein.

[0061] For example, "low expression of CD58" and (substantially) "lower levels" of CD58 expression according to the present invention mean that CD58 is expressed at a level that is at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 75% lower, or at least 100% lower, respectively, in particular compared to CD58 expression in a suitable control (e.g., normal DLBCL patients / population; not "patients as defined herein"). This applies to both gene expression and protein expression.

[0062] Mutations, in particular CD58 mutations, can be detected, for example, by using Heme (FOH) kit (see, e.g., He, Blood 127(24), 2017, 3004-14; see also the accompanying Examples).

[0063] Examples of genetic mutations in CD58 that may be present (and detected) in a patient as defined herein are short variant mutations and / or copy number variants.

[0064] The skilled person will be readily able to select an appropriate sample (either as a test sample or as a control sample) to be used when assessing / detecting CD58 expression or one or more CD58 mutations in accordance with the present invention.

[0065] A specific example of a sample to be used (either as a test sample or as a control sample) for assessing / detecting whether there is low CD58 expression in the context of the present invention is a sample (e.g. a biopsy) of a (CD58-expressing) tumor and / or a sample (e.g. a biopsy) containing (CD58-expressing) B cells.

[0066] A specific example of a sample to be used (either as a test sample or as a control sample) for assessing / detecting the presence of one or more CD58 mutations in the context of the present invention is a DNA sample.

[0067] In one aspect / embodiment of the invention (Aspect / Embodiment D), the patient as defined herein (to be treated with obinutuzumab) is a patient having / suffering from GCB-strong DLBCL.

[0068] According to the present invention, patients with / suffering from strong GCB DLBCL can be identified by determining the (weighted) expression of one or more (preferably all) of the genes TNFRSF13B, LIMD1, IRF4, CREB3L2, PIM2, CYB5R2, RAB7L1, and CCDC50 (genes overexpressed in ABC DLBCL); and MME, SERPINA9, ASB13, MAML3, ITPKB, MYBL1, and S1PR2 (genes overexpressed in GCB DLBCL) (a gene set comprising the same).

[0069] More particularly, a patient having / suffering from strong GCB DLBCL can be defined as a patient having a tumor having a certain (weighted) expression of the genes TNFRSF13B, LIMD1, IRF4, CREB3L2, PIM2, CYB5R2, RAB7L1, and CCDC50; and one or more, preferably all, of MME, SERPINA9, ASB13, MAML3, ITPKB, MYBL1, and S1PR2; and optionally one or more, preferably all, of (a gene set comprising) the genes R3HDM1, WDR55, ISY1, UBXN4, and TRIM56 (housekeeping genes).

[0070] Specifically contemplated in the context of this aspect / embodiment of the invention is the assessment of weighted gene expression.

[0071] In particular, a GCB DLBCL is considered a "strong GCB DLBCL" when the linear predictor score (LPS) derived from the weighted expression of the gene set disclosed herein is below a certain cutoff, i.e. substantially below the LPS cutoff typically assigned to GCB DLBCL (approximately <1900). Likewise, a GCB DLBCL is considered a "strong GCB DLBCL" when the weighted expression of the gene set disclosed herein corresponds to the weighted expression of the gene set disclosed herein from which an LPS below a certain cutoff is generated, i.e. substantially below the LPS cutoff typically assigned to GCB DLBCL (approximately <1900). Examples of specific cut-offs applicable according to the present invention, i.e. defining "strong GCBDL BCL", i.e. resulting LPS, are cut-offs of (approximately) ≤ 1200, (approximately) ≤ 1141, (approximately) ≤ 1100, (approximately) ≤ 756, (approximately) ≤ 750, (approximately) ≤ 749, (approximately) ≤ 745, (approximately) ≤ 725 or (approximately) ≤ 699. Preferred cut-offs are (approximately) ≤ 750, (approximately) ≤ 749 and (approximately) ≤ 725. Particularly preferred cut-offs are (approximately) ≤ 750 and (approximately) ≤ 725.

[0072] According to the present invention, the gene set to be used for determination / identification / diagnosis of strong GCB DLBCL may further comprise one or more housekeeping genes, such as one or more (preferably all) of the housekeeping genes R3HDM1, WDR55, ISY1, UBXN4, and TRIM56.

[0073] The expression of one or more of the other genes to be employed may be normalized relative to the expression of one or more housekeeping genes, for example housekeeping genes as defined herein. The skilled person is readily able to normalize the expression of one or more of these other genes (and one or more other genes that may be included in the gene set to be employed according to the invention) based on one or more housekeeping genes, for example based on one or more of the housekeeping genes mentioned above. For example, at least 1, 2, 3, 4 or 5 (of these) housekeeping genes may be employed in this regard. For respective guidance, the skilled person may rely on, for example, Scott (2014 and 2015, supra).

[0074] In principle, a gene set comprising only a subset of the gene sets mentioned above can also be used according to the present invention. For example, such a subset may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 of the entire gene set mentioned above. However, the more such genes are evaluated, the more preferred.

[0075] Examples of such gene subsets that can be used according to the present invention are gene subsets comprising at least 1, 2, 3, 4, 5, 6 or 7 of the genes TNFRSF13B, LIMD1, IRF4, CREB3L2, PIM2, CYB5R2, RAB7L1, and CCDC50, at least 1, 2, 3, 4, 5 or 6 of the genes MME, SERPINA9, ASB13, MAML3, ITPKB, MYBL1, and S1PR2, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the genes of these two gene subsets. In principle, a higher number of genes is preferred.

[0076] It is also contemplated in the context of the present invention that not only a specific gene set or gene subset as mentioned herein may be assessed according to the present invention; but also a gene (sub)set comprising one or more further genes (e.g. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 150 or more, 170 or more, or 180 or more further genes) may be assessed. This / these further genes may be, for example, one or more of the genes (about 180) known to separate / distinguish GCB from ABC (and unclassified) DLBCL based on their (enhanced) expression (see in particular Lenz, N. Engl. J. Med. 359 (2), 2008, 2313-23 and also Geiss, Nature Biotechnology 26 (3), 2008, 317-25).

[0077] For example, a gene set to be assessed in accordance with the present invention may comprise (at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19) genes of any of the specific gene sets or gene subsets mentioned above and one or more additional genes (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 150 or more, 170 or more, or 180 or more additional genes), for example (about 180) genes that are known to separate / distinguish GCB from ABC (and unclassified) DLBCL based on their (weighted) expression (see Lenz and Geiss, supra).

[0078] For example, one (or more) other genes can be added to one of the gene bases or subsets described above, such as one of the 20, 15, 8 or 7 gene groups mentioned above, or one (or more) genes of one of the gene bases or subsets described above, such as one of the 20, 15, 8 or 7 gene groups mentioned above can be replaced with one (or more) other genes.

[0079] A non-limiting but preferred example of another gene to be employed in accordance with Aspect / Embodiment D is the BCL2 gene.

[0080] NanoString (NanoString Technologies, Inc., Seattle, WA, USA; see also Lenz, supra and Geiss, supra) provides an example of means and methods that can be used in accordance with the present invention to determine the expression, particularly weighted expression, of the genes described above. A non-limiting specific example of a method that can determine the (weighted) expression of the genes described above is the NanoString Research Use Only LST Assay. Another example is RNA Tools ( Inc.). Further examples are described in Wright, PNAS 100(17), 2003, 9991-6. Also described in Wright (supra) is the general applicability of any suitable panel for (weighted) gene expression analysis.

[0081] According to the mentioned examples of means and methods that can be used to determine (weighted) expression, a patient having / suffering from strong GCB DLBCL can also be defined as a patient having a tumor with a (weighted) gene expression that results in an LPS lower than the LPS typically assigned to GCB DLBCL (approximately <1900; see above for examples of respective cutoffs), wherein the LPS is derived from a NanoString LST, such as the NanoString Research Use Only LST (NanoString Technologies, Inc., Seattle, WA, USA) as the (weighted) gene expression of genes in a Nanostring panel (or a tumor with a (weighted) gene expression corresponding to such a (weighted) gene expression). For example, the specific genes mentioned above can be in the Nanostring panel.

[0082] Patients with / suffering from strong GCB DLBCL may also be defined as patients having a tumor with a (weighted) gene expression having / resulting in an LPS lower than the LPS typically assigned to GCB DLBCL (approximately <1900; see above for examples of respective cut-offs), wherein the LPS is derived from the COO classification by (weighted) gene expression profiling (e.g. by using NanoString LST as described herein) (or a tumor with a (weighted) gene expression corresponding to such a (weighted) gene expression).

[0083] In principle, COO classification (e.g., classification into strong, unclassified, and weak GCB DCBCLs) can be based on gene expression profiling, in particular weighted gene expression profiling (e.g., using NanoString LST (such as, for example, NanoString Research Use Only LST) (NanoString Technologies, Inc., Seattle, WA, USA).

[0084] In principle, in the context of the present invention, the meaning of "LPS" is known in the art and is thus understood by the skilled person. In particular, the LPS according to the present invention is a continuous variable (weighted average of gene expression; for example it can be of the genes mentioned herein in Nanostring LST). In the sum of patients evaluated in GOYA, this LPS has a range of -1138 to 4504. Under normal circumstances, as mentioned above, this LPS is used to classify patients into the COO subgroups GCB DLBCL, ABC DLBCL, and unclassified DLBCL (see above and Scott, 2014 and 2015, supra). The default COO algorithm uses a Bayesian approach, with the GCB / ABC classification based on a ≥90% cutoff of the likelihood of being GCB or ABC (unclassified as a buffer).

[0085] More particularly, it is contemplated that the LPS according to the present invention is a weighted sum of the expression of the genes to be employed in the gene expression profiling analysis (e.g. the genes mentioned above contained in the mentioned gene (sub)sets). The weighted sum of gene expression can be calculated according to the following formula (Formula 1):

[0086] LPS(X)=Σ j a j X j ,

[0087] Where X represents each sample, X j is the gene expression of gene j and a j is the coefficient for gene j (see also Wright, supra; in particular, the subsections "Gene Expression Data" and "Formula for DLBCL Subgroup Predictors"; incorporated herein by reference).

[0088] In general, according to the teachings of the present invention, one skilled in the art can determine the expression of genes, in particular the weighted expression of genes. Respective means and methods are known in the art and are described, for example, in Wright, supra, in particular in the subsections "Gene Expression Data" and "Formula for DLBCL Subgroup Predictors"; incorporated herein by reference. For example, Wright (supra) also outlines how the weighted gene expression algorithm can be used and how it can be transferred between gene expression platforms, like NanoString LST and others. Comparable guidance is also found in Lenz, supra.

[0089] In the context of a strong GCB DLBCL classification according to the present invention, in particular as described above, the following may be further considered:

[0090] Biomarker treatment effects can be evaluated using multivariate Cox regression and / or elastic net penalty regression (α=0.5). Cross-validation and / or bootstrapping can be used to implement simulations for treatment effects to identify optimal cutoffs, such as based on NanoString LST and respective LPS. Multiple testing adjustments can be made by evaluating the false discovery rate (FDR), such as using the Benjamini-Hochberg procedure (e.g., significance <5% FDR). Pathway enrichment analysis can be performed by gene set enrichment, such as by using gene sets defined by MSigDB markers and / or curated FL somatic mutation marker gene sets.

[0091] In particular, LPS retention may be determined in one or more simulation analyses, preferably in one or more multivariate simulation analyses (eg as described elsewhere herein).

[0092] The robustness of LPS retention (eg, as described elsewhere herein) can be demonstrated by bootstrap simulations.

[0093] Furthermore, instead of using a specific weighting algorithm for LPS, such as that from NanoString LST, one could apply a weighting algorithm from, for example, another expression analysis panel (e.g. RNA Tools ( Inc.)) by evaluating gene expression, such as the genes described above (e.g., one or more of the approximately 180 genes known to separate GCB and ABC). In accordance with the present invention, a high correlation between LPS (derived from NanoString LST) and the first principal component was also shown.

[0094] In one aspect / embodiment of the invention (Aspect / Embodiment E), the patient as defined herein (to be treated with obinutuzumab) is a patient with a BCL2 translocation and / or high BCL2 expression. Preferably, patients with a BCL2 translocation with high BCL2 expression are encompassed in the context of this aspect / embodiment.

[0095] BCL2 (see also Zhang, supra; Punnoose, supra; Iqbal, Clin Cancer Res 17(24), 2011, 7785-95; Iqbal, JCO 24(6), 2006, 961-8; Hu, Blood 121(20), 2013, 4021-31; Johnson, JCO 30(28), 2012, 3452-67; Green, JCO 30(28), 2012, 3460-67) is generally known to be an anti-apoptotic protein whose overexpression resists the mitochondrial apoptotic pathway. BCL2 is known to be expressed in tumors of patients with DLBCL.

[0096] Nucleotide sequences encoding BCL2 (particularly human (Homo sapiens) BCL2) and amino acid sequences of BCL2 are well known in the art. They can be downloaded, for example, via the following uniform resource locator (URL): https: / / www.ncbi.nlm.nih.gov / search / ?term=Homo+sapiens+BCL2&utm_expid=.fAeHyO5JTBGxnObh2WlrCA.0&utm_referrer=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fsearch%2F%3Fterm%3DHomo%2Bsapiens%2BBCL2. The nucleotide sequence encoding BCL2 (particularly human (Homo sapiens) BCL2) is available, for example, via NCBI accession number XM_017025917.2 (variant X3); XM_011526135.3 (variant X2); XR_935248.3 (variant X1); NM_000657.2 (variant β); NM_000633.2 (variant α). The amino acid sequence of BCL2 (particularly human (Homo sapiens) BCL2) is available, for example, via NCBI accession number XP_016881406.1 (isoform X2); XP_011524437.1 (isoform X1); NP_000648.2 (isoform β); NP_000624.2 (isoform α). An example of a nucleotide sequence encoding human (Homo sapiens) BCL2 is depicted in SEQ ID NO: 13. An example of an amino acid sequence of human (Homo sapiens) BCL2 is depicted in SEQ ID NO:14.

[0097] As generally described herein above with regard to "expression", the measurement / detection of (mainly) mRNA and the measurement / detection of protein apply mutatis mutandis here.

[0098] In general, means and methods for measuring / detecting BCL2 expression and BCL2 translocations are known in the art and are described, for example, in Zhang (supra) and Puunoose (supra). Furthermore, the skilled person can easily assess whether a given BCL2 expression is "high" according to the invention or whether there are one or more BCL2 translocations according to the invention. Moreover, the skilled person can easily select a suitable control, compared to which a given BCL2 expression is considered to be "higher" according to the invention or compared to which there are one or more BCL2 translocations according to the invention. In this context, the skilled person can also, for example, rely on Zhang (supra) and Puunoose (supra).

[0099] BCL2 expression can be assessed, for example, by a Ventana immunohistochemistry (IHC) assay, such as by the Ventana Investigational IHC Assay (BCL2 Antibody Clone 124) (by consulting the supplier's manual). For example, high BCL2 expression in this context can be defined as moderate or strong staining in ≥50% of tumor cells (see below for more details).

[0100] BCL2 protein expression / presence of BCL2 protein may also be measured / detected as described in Punnoose (supra), Iqbal (2011 and 2006, supra), Hu (supra), Johnson (supra), Green (supra).

[0101] It can be used, for example, as described in Zhang (supra) or by following the distributor's manual. RNA sequencing ( Inc.) to assess gene expression / (mainly) mRNA occurrence, in particular BCL2 gene expression / (mainly) BCL2 mRNA occurrence.

[0102] In the context of the present invention, "high expression of BCL2" means that BCL2 is expressed at a substantially higher level, especially compared to a suitable control. Generally, "high expression of BCL2" according to the present invention means that the BCL2 expression is as high (e.g. ±10% or less, ±7.5% or less, ±5% or less, ±3% or less, ±2% or less, ±1% or less or even ±0%) as the BCL2 expression in a responder according to the present invention (i.e. a patient who responds to treatment with obinutuzumab (in particular in combination with chemotherapy, more particularly in combination with CHOP chemotherapy) by achieving an improved clinical outcome compared to treatment with rituximab (in particular in combination with chemotherapy, more particularly in combination with CHOP chemotherapy); a "patient as defined herein") and / or higher than the BCL2 expression in a non-responder according to the present invention (i.e. a patient who responds to treatment with obinutuzumab (in particular in combination with chemotherapy, more particularly in combination with CHOP chemotherapy) by not achieving an improved clinical outcome compared to treatment with rituximab (in particular in combination with chemotherapy, more particularly in combination with CHOP chemotherapy); a non-"patient as defined herein"). The skilled person is readily able to assess when BCL2 expression is "high" in this regard and to apply appropriate controls. For example, controls in this regard may be a common DLBCL population, more particularly DLBCL patients who are not classified as belonging to a patient subgroup classified according to the present invention (non-"patients as defined herein"). "High expression of BCL2" may be BCL2 expression that is higher than the median BCL2 expression in a control group, such as such a control group described above. For example, "high expression of BCL2" and "higher levels" of BCL2 expression according to the present invention may be BCL2 expression that is higher than the median BCL2 expression in patients assessed in GOYA, respectively.

[0103] An example of a control based on which it can be considered whether BCL2 expression is "high" in the context of the present invention is normal, i.e. non-tumor tissue, more particularly normal, i.e. non-tumor lymphoid tissue. The tissue can be from a DLBCL patient. For example, it can be from a DLBCL patient to be treated. However, in principle, the tissue can also be from a normal / healthy subject.

[0104] A preferred example of a control based on which BCL2 expression can be considered "high" in the context of the present invention is tumor tissue, more particularly lymphoid tumor tissue, from a non-responder according to the present invention (not a "patient as defined herein"). Preferably, the tissue is from a non-responder DLBCL patient (not a "patient as defined herein" who is a DLBCL patient).

[0105] BCL2 expression is considered "high" if, for example, ≥30%, ≥40%, ≥50% or ≥60% of the tumor cells express BCL2 (e.g., show BCL2 staining in an IHC assay), particularly show moderate to strong BCL2 expression (e.g., show moderate to strong BCL2 staining in an IHC assay).

[0106] It is preferred in the context of the present invention that BCL2 expression, in particular "high" BCL2 refers to both the percentage of tumor cells expressing BCL2 and the intensity of BCL2 expression in these cells.

[0107] In assessing whether a given BCL2 expression is "high" in accordance with the present invention, one may also rely on Iqbal (2011 and 2006, supra), Hu (supra), Johnson (supra) and Green (supra).

[0108] More generally, "high expression of BCL2" and (substantially) "higher levels" of BCL2 expression according to the present invention respectively mean that BCL2 is expressed at a level that is at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 75% higher, or at least 100% higher, in particular compared to BCL2 expression in a suitable control (e.g., normal DLBCL patients / populations; not "patients as defined herein"). This applies to both gene expression and protein expression.

[0109] The meaning of "(one or more) BCL2 translocation" is well known in the art. Typically, a "BCL2 translocation" is a gene fusion between BCL2 and IgH (involving chromosomes 14 and 18). BCL2 translocations are described, for example, in Zhang (supra). BCL2 translocations can be assessed / detected, for example, by using BCL2 dual-color separation technology (Vysis, Abbott Molecular), in particular by using VysisLSI dual-color separation FISH probes (e.g., with a (FISH) cutoff of 5% (typically used) or 50%); by consulting the supplier's manual. BCL2 translocations can also be assessed / detected using Foundation Medicine next-generation sequencing assays, Heme (by consulting the supplier's manual; see also He, supra). Means and methods for assessing / detecting BCL2 translocation are known in the art and are described, for example, in Zhang (supra) and He (supra).

[0110] The skilled person will be readily able to select an appropriate sample (either as a test sample or as a control sample) to be used when assessing / detecting BCL2 translocation(s) or BCL2 expression according to the present invention.

[0111] A specific example of a sample to be employed (either as a test sample or as a control sample) for assessing / detecting whether there is high BCL2 expression in the context of the present invention is a sample (eg a biopsy) of a (BCL2 expressing) tumor.

[0112] A specific example of a sample to be employed (either as a test sample or as a control sample) for assessing / detecting the presence or absence of BCL2 translocation(s) in the context of the present invention is a DNA sample.

[0113] In one aspect / embodiment of the invention (Aspect / Embodiment F), the patient as defined herein (to be treated with obinutuzumab) is defined by a combination / intersection of any 2, any 3, any 4 or any 5 of the patient definitions mentioned in Aspects / Embodiments A, B, C, D and E above. That is, the patient may be defined by a combination / intersection of the patient definitions mentioned in Aspects / Embodiments A and B; A and C; A and D; A and E; B and C; B and D; B and E; C and D; C and E; A, B and C; A, C and D; A, D and E; A, B and D; A, B and E; A, C and E; B, C and D; B, C and E; B, D and E; C, D and E; A, B, C and D; A, C, D and E; B, C, D and E; and A, B, D and E. Preferred are combinations / intersections comprising the definitions according to Aspects / Embodiments D and E.

[0114] In one aspect / embodiment of the invention (Aspect / Embodiment G), the patient defined herein (to be treated with obinutuzumab) is defined by a combination / crossover of the patient definitions mentioned in Aspects / Embodiments D and E (or A, D and E) above. That is, the patient defined herein according to this aspect / embodiment is a patient who (i) has / suffers from strong GCB DLBCL; and (ii) has a BCL2 translocation and / or high BCL2 expression. This combination / crossover of patient definitions defines a preferred patient defined herein.

[0115] Generally, as used in the context of the present invention, a non-limiting example of a "control" is preferably a "non-responder" control, such as a sample / cell / tissue obtained from one or more patients who do not suffer from a specific DLBCL as defined herein (not a "patient as defined herein") and are known not to respond favorably to obinutuzumab (particularly in combination with chemotherapy, more particularly in combination with CHOP chemotherapy) compared to rituximab (particularly in combination with chemotherapy, more particularly in combination with CHOP chemotherapy) according to the present invention. Another example of a "non-responder" control is a cell line / sample / cell / tissue that does not show an improved response to obinutuzumab (particularly in combination with chemotherapy, more particularly in combination with CHOP chemotherapy) compared to rituximab (particularly in combination with chemotherapy, more particularly in combination with CHOP chemotherapy) in an ex vivo test. 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, wherein the amount of each protein / peptide / DNA / RNA is measured by using a "non-responder" control as described herein.

[0116] In principle, it is encompassed that the patient to be treated in the context of the present invention is a DLBCL patient. In other words, the patient is a patient with / suffering from DLBCL. Thus, it is particularly encompassed that the patient defined with respect to any of the above aspects / embodiments A, B, C, D, E, F, G is also a DLBCL patient and a patient with / suffering from DLBCL, respectively. However, it is not necessarily required that a given patient is diagnosed as a DLBCL patient, e.g. before (or after) determining / identifying / diagnosing as a patient as defined herein, in particular as defined in one (or more) of aspects / embodiments A to G above. However, it is preferred that the patient to be treated according to the present invention is diagnosed as a DLBCL patient, or at least a lymphoma patient, in a first step, and is determined / identified / diagnosed as a patient as defined herein, in particular as defined in one (or more) of aspects / embodiments A to G above, in a second step. In principle, according to the present invention, a given patient may also be determined / identified / diagnosed as a patient as defined herein in a first step, and diagnosed as a DLBCL patient, or at least a lymphoma patient, in a second step. However, the latter option is less preferred and, as mentioned, the (prior or subsequent) step of diagnosing whether the patient to be treated is a (DLBC)L patient may also be omitted.

[0117] The following provides a non-limiting example of how an attending physician might choose whether to treat a given patient in accordance with the present invention:

[0118] A (tumor) sample (e.g., a (tumor) biopsy) may be obtained, e.g., from a patient with an abnormality (e.g., enlarged lymph nodes) that raises a clinical suspicion of lymphoma. The (tumor) sample may be diagnosed as (DLBC)L positive (e.g., by a pathologist). This may be one of the two steps mentioned above.

[0119] As mentioned, this step can be omitted.

[0120] Protein, RNA (e.g. (mainly) mRNA) and / or DNA may be extracted from (the remainder of) a (tumor) sample (e.g. (tumor) tissue / biopsy), or from another (tumor) sample of the same or another patient, or from another tumor of the same or another patient. A patient as defined herein may then be determined / identified / diagnosed, i.e. the sampled protein, RNA (e.g. (mainly) mRNA) and / or DNA may then be subjected to one or more biomarker analyses according to the invention. For example, the sample may be analyzed with a weighted gene expression assay (e.g. by using NanoString LST) to obtain LPS, tested for (one or more) genetic mutations in CD58 and / or low expression of CD58 and / or tested for BCL2 translocations and / or high BCL2 expression. The results of the one or more analyses then allow the patient to be classified into a DLBCL subgroup as defined according to the invention. In other words, the results of the one or more analyses then allow the classification of whether the patient is a "patient as defined herein".

[0121] This can be the other (ie mandatory) of the two steps mentioned above.

[0122] The following are non-limiting examples of one or more biomarker analyses that may be employed in accordance with the present invention:

[0123] Tumor samples, such as diagnostic tumor samples (e.g., tissue biopsies), such as formalin-fixed and / or paraffin-embedded, can be obtained from the patient. RNA (or protein or DNA) can be extracted and gene expression can be analyzed for strong GCB classification, CD58 translocation / low expression and / or BCL2 mutation / low expression. DNA can be extracted to assess CD58 mutation (one or more). Tissue sections, particularly tumor tissue sections, can be cut and embedded, such as for IHC and / or (F) ISH analysis.

[0124] As mentioned, encompassed in the context of the present invention is the use of obinutuzumab, or a functional equivalent of obinutuzumab, to treat a patient as defined herein.

[0125] Obinutuzumab itself is well known in the art and is described, for example, in EP-B1 2380910 and WO 2005 / 044859. More details on obinutuzumab itself are given below.

[0126] The meaning of "functional equivalents of obinutuzumab" is also clear to the skilled person. In particular, the term "functional equivalents of obinutuzumab" refers to antibodies, in particular humanized type II anti-CD20 antibodies, that are more suitable for treating (in particular in combination with chemotherapy, more particularly in combination with CHOP chemotherapy) patients as defined herein than rituximab (in particular in combination with chemotherapy, more particularly in combination with CHOP chemotherapy). In other words, this term refers to antibodies, in particular human type II anti-CD20 antibodies, that have characteristics and modes of action (MOA) that enable the antibody to treat patients as defined herein, thereby responding by achieving an improved clinical outcome compared to treatment with rituximab. More particularly, the term "functional equivalents of obinutuzumab" refers to antibodies, in particular type II anti-CD20 antibodies, that have the same characteristics and biological functions as obinutuzumab itself, in particular the same biological functions as obinutuzumab itself, that make the antibody more suitable than rituximab for treating patients as defined herein.

[0127] Examples of the most relevant features and MOA of equivalents of obinutuzumab according to the invention (and of obinutuzumab itself) are defined elsewhere herein. The skilled person can easily determine them.

[0128] In principle, it is contemplated in the context of the present invention that the term "functional equivalent of obinutuzumab" also encompasses biosimilars of obinutuzumab. In particular, it is contemplated that the meaning of the term also encompasses any biosimilar of obinutuzumab that is more suitable than rituximab for treating patients as defined herein. In other words, a "functional equivalent of obinutuzumab" may be a biosimilar of obinutuzumab that is able to treat patients as defined herein, thereby responding by achieving an improved clinical outcome compared to treatment with rituximab, in particular in combination with chemotherapy, more particularly in combination with CHOP chemotherapy.

[0129] Generally, the meaning of "biosimilar" is well known in the art. In this context, "biosimilar" is known to be a biological medical product that is an almost identical copy of the original biological medical product and is also known to be a follow-on biological product or a subsequent entry biological product. A biosimilar is an officially approved version of the original "innovative" product. In this context, reference may be made, for example, to the EMEA guidelines on similar biological medical products (CHMP / 437 / 04 London, 2005).

[0130] In the context of the present invention, obinutuzumab, in particular functional equivalents of obinutuzumab, are antibodies, in particular humanized type II anti-CD20 antibodies comprising:

[0131] (a) a heavy chain variable region as depicted in SEQ ID NO: 1 and a light chain variable region as depicted in SEQ ID NO: 2 (herein

[0132] The light chain variable region is also called the KV1 light chain variable region; "KV1" represents the humanized light chain variable region of the murine B-Lyl monoclonal antibody.

[0133] Variable area; see EP-B1 2380910);

[0134] (b) a heavy chain variable region having the specificity determining residues of the heavy chain variable region of (a) and a light chain variable region having the specificity determining residues of (a)

[0135] or

[0136] (c) at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 3

[0137] A nucleic acid sequence encoding a heavy chain variable region and having at least 80%, at least 85%, at least 90%,

[0138] The light chain variable region is encoded by a nucleic acid sequence that is at least 95%, at least 98% or at least 99% identical (higher values ​​are preferred).

[0139] Preferably, obinutuzumab, in particular a functional equivalent of obinutuzumab to be employed in the context of the present invention is a monoclonal antibody, in particular a monoclonal humanized type II anti-CD20 antibody.

[0140] It is particularly preferred that the antibody to be employed according to the present invention is a type II anti-CD20 antibody, in particular a humanized type II anti-CD20 monoclonal antibody, or preferably an antibody comprising a glycoengineered Fc region, in particular a glycoengineered Fc region as defined herein below. It is further preferred that, according to the present invention, such an antibody or any other antibody to be employed according to the present invention shows a substantially higher level of ADCC activity, in particular compared to a comparable type I anti-CD20 antibody and / or compared to a non-glycoengineered antibody (e.g. rituximab).

[0141] The meaning of "Type II" anti-CD20 antibodies is well known in the art. In general, anti-CD20 monoclonal antibodies fall into two distinct categories based on the mechanism of action by which they eliminate lymphoma cells. Type I anti-CD20 antibodies primarily utilize complement to kill target cells, while Type II antibodies operate through a different mechanism, primarily apoptosis. Rituximab and 1F5 are examples of Type I anti-CD20 antibodies, while B1 is an example of a Type II antibody. See, e.g., Cragg (Blood 103(7), 2004, 2738-2743); Teeling (Blood 104(6), 2004, 1793-1800), the entire contents of which are hereby incorporated by reference. Obitutuzumab itself is also a Type II antibody. See, e.g., EP-B1 2380910 and WO 2005 / 044859, the entire contents of which are hereby incorporated by reference.

[0142] The skilled person knows but is at least easily able to determine the relevant specificity determining residues of the heavy and light chain variable regions of Obinutuzumab. For respective guidance, the skilled person can, for example, rely on EP-B1 2380910 and WO 2005 / 044859.

[0143] In one aspect, obinutuzumab / functional equivalents of obinutuzumab as employed in the context of the present invention, in particular as defined in (b) and (c) above, encompass inter alia having one or more of the following characteristics:

[0144] (i) Induction when incubated with CD20-positive human cells relative to controls under the same conditions with rituximab

[0145] Higher levels of apoptosis capacity;

[0146] (ii) Causes increased CD20 compared with rituximab + The ability of tumor B cells to kill;

[0147] (iii) the ability to cause increased direct cell death compared to rituximab (without being bound by theory, this is due to alternative mechanisms

[0148] composite geometry (e.g. elbow hinge modifications);

[0149] (iv) the ability to induce reduced complement-dependent cytotoxicity (CDC) compared to rituximab (without being bound by theory, this

[0150] is due to alternative bonding geometries (e.g. elbow hinge modifications);

[0151] (v) Ability to induce increased antibody-dependent cellular cytotoxicity (ADCC) compared to rituximab (not limited by theory)

[0152] tethering, which is due to the glycoengineered Fc region);

[0153] (vi) the ability to induce increased antibody-dependent cellular phagocytosis (ADCP) compared to rituximab (without being bound by theory,

[0154] This is due to the glycoengineered Fc region);

[0155] (vii) increased affinity for the FcγRIII receptor compared to rituximab (without being bound by theory, this is due to glycoengineered

[0156] Modified Fc region);

[0157] (viii) The ability to trigger less internalization of surface CD20 compared to rituximab upon binding to CD20.

[0158] In another aspect, obinutuzumab / functional equivalents of obinutuzumab as employed in the context of the present invention, in particular as defined in (b) and (c) above, encompass inter alia showing one or more of the following MOAs:

[0159] (i) Induction when incubated with CD20-positive human cells relative to controls under the same conditions with rituximab

[0160] Higher levels of apoptosis capacity;

[0161] (iii) the ability to cause increased direct cell death compared to rituximab (without being bound by theory, this is due to alternative mechanisms

[0162] composite geometry (e.g. elbow hinge modifications);

[0163] (iv) the ability to induce reduced complement-dependent cytotoxicity (CDC) compared to rituximab (without being bound by theory, this

[0164] is due to alternative bonding geometries (e.g. elbow hinge modifications);

[0165] (v) Ability to induce increased antibody-dependent cellular cytotoxicity (ADCC) compared to rituximab (not limited by theory)

[0166] tethering, which is due to the glycoengineered Fc region);

[0167] (vi) the ability to induce increased antibody-dependent cellular phagocytosis (ADCP) compared to rituximab (without being bound by theory,

[0168] This is due to the glycoengineered Fc region);

[0169] (vii) increased affinity for the FcγRIII receptor compared to rituximab (without being bound by theory, this is due to glycoengineered

[0170] Modified Fc region);

[0171] (viii) The ability to trigger less internalization of surface CD20 compared to rituximab upon binding to CD20.

[0172] Means and methods that can be used to determine the relevant characteristics (eg biological function, MOA) of the antibodies to be employed in accordance with the present invention are well known in the art and can be readily applied by the skilled person.

[0173] Means and methods that can be used to determine the level of apoptosis, in particular whether a given antibody is able to induce a higher level of apoptosis when incubated with CD20-positive human cells relative to a control under the same conditions using rituximab, are known in the art and are described, for example, in EP-B1 2380910 and WO 2005 / 044859.

[0174] "Higher levels of apoptosis" according to the present invention means, for example, at least 1.2 times higher, at least 1.5 times higher, at least 2 times higher, at least 3 times higher, at least 4 times higher, at least 5 times higher, or at least 10 times higher than the level of apoptosis resulting from a comparable application of rituximab.

[0175] Can be used to measure CD20 + Tumor B cell killing, especially whether there is an increase in CD20 compared with rituximab + Means and methods for tumor B cell killing are known in the art and are disclosed, for example, in EP-B1 2380910 and WO 2005 / 044859.

[0176] According to the present invention, if, for example, the CD20 derived from rituximab is used + If the tumor B cell killing is at least 1.2 times higher, at least 1.5 times higher, at least 2 times higher, at least 3 times higher, at least 4 times higher, at least 5 times higher, or at least 10 times higher, CD20 + Tumor B cell killing is "elevated".

[0177] Means and methods that can be used to determine direct cell death, in particular whether there is increased direct cell death compared to rituximab, are known in the art and are disclosed, for example, in EP-B1 2380910 and WO 2005 / 044859.

[0178] According to the present invention, direct cell death is "elevated" if it is at least 1.2-fold higher, at least 1.5-fold higher, at least 2-fold higher, at least 3-fold higher, at least 4-fold higher, at least 5-fold higher or at least 10-fold higher than direct cell death resulting from a comparable application of rituximab.

[0179] Means and methods that can be used to determine CDC, in particular whether there is reduced CDC compared to rituximab, are known in the art and disclosed, for example, in Herter (supra), (ibid.), EP-B1 238090, WO 2005 / 044859, WO 2015 / 067586 and WO 2016 / 207312.

[0180] According to the present invention, a CDC is "reduced" if, for example, it is at least 1.2-fold lower, at least 1.5-fold lower, at least 2-fold lower, at least 3-fold lower, at least 4-fold lower, at least 5-fold lower, or at least 10-fold lower than the CDC derived from a comparable application of rituximab.

[0181] The term "complement dependent cytotoxicity (CDC)" refers to the lysis of human tumor target cells by the antibodies to be employed according to the invention in the presence of complement. CDC is preferably measured by treating a preparation of CD20 expressing cells with an anti-CD20 antibody to be employed according to the invention in the presence of complement. CDC is detected if the antibody, e.g. at a concentration of 100 nM, induces, e.g. 20% or more tumor cell lysis (cell death) after, e.g. 4 hours. Preferably, CDC is measured by treating a preparation of CD20 expressing cells with an anti-CD20 antibody to be employed according to the invention in the presence of complement. CDC is detected if the antibody, e.g. at a concentration of 100 nM, induces, e.g. 20% or more tumor cell lysis (cell death) after, e.g. 4 hours. 51 Cr or Eu labeled tumor cells and measurement of released 51 The assay is performed with Cr or Eu. Controls include incubating tumor target cells with complement but with rituximab, optionally without the antibody.

[0182] The skilled person is easily able to adapt this specific example of a CDC assay and thus be able to test whether the CDC activity is reduced when applying the antibody to be used according to the invention compared to applying rituximab, as might be the case.

[0183] Means and methods that can be used to determine ADCC, in particular whether there is increased ADCC compared to rituximab, are known in the art and disclosed, for example, in Herter (supra), (supra), Tobinai (Adv. Ther. 34, 2017, 324-56), EP-B1 2380910, WO 2005 / 044859, WO 2015 / 067596 and WO 2016 / 207312.

[0184] According to the invention, more generally, ADCC is "increased" if, for example, it is at least 1.2-fold higher, at least 1.5-fold higher, at least 2-fold higher, at least 3-fold higher, at least 4-fold higher, at least 5-fold higher, or at least 10-fold higher than the ADCC from a comparable application of rituximab.

[0185] A non-limiting, generally accepted in vitro ADCC assay is as follows:

[0186] 1) The assay uses target cells known to express a target antigen (CD20) recognized by the antigen binding region of the antibody;

[0187] 2) The assay uses human peripheral blood mononuclear cells (PBMCs) isolated from the blood of randomly selected healthy donors as effector cells;

[0188] 3) Perform the assay according to the following protocol:

[0189] i) PBMCs were isolated using standard density centrifugation protocols and cultured at 5 x 10 6 cells / ml suspension;

[0190] ii) Target cells were cultured by standard tissue culture methods, harvested from the exponential growth phase with a viability of more than 90%, washed in RPMI cell culture medium, and incubated with 100 μg of Cu 51 Cr labeling, the cells were washed twice with cell culture medium,

[0191] And 10 5 Resuspend in cell culture medium at a density of 10 cells / ml;

[0192] iii) transferring 100 μL of the final target cell suspension to each well of a 96-well microtiter plate;

[0193] iv) serially diluting the antibody from 4000 ng / ml to 0.04 ng / ml in cell culture medium and adding 50 μL of the resulting antibody solution to target cells in a 96-well microtiter plate, testing multiple antibody concentrations covering the entire concentration range in triplicate;

[0194] v) For the maximum release (MR) control, an additional 3 wells of the plate containing labeled target cells received 50 μL of 2%

[0195] (VN) non-ionic detergent aqueous solution (Nonidet, Sigma, St. Louis) replaced the antibody solution (point iv above);

[0196] vi) For spontaneous release (SR) controls, three additional wells of the plate containing labeled target cells received 50 μL of RPMI cell culture medium instead of the antibody solution (point iv above);

[0197] vii) the 96-well microtiter plate was then centrifuged at 50 x g for 1 minute and incubated at 4°C for 1 hour;

[0198] viii) 50 μL of PBMC suspension (point i above) was added to each well to produce an effector:target cell ratio of 25:1 and the plate was placed in an incubator at 37°C in a 5% CO2 atmosphere for 4 hours;

[0199] ix) harvesting the cell-free supernatant from each well and quantifying the experimentally released radioactivity (ER) using a gamma counter;

[0200] x) calculating the percentage of specific lysis for each antibody concentration according to the formula (ER-MR) / (MR-SR) x 100, where ER is the mean radioactivity quantified for that antibody concentration (see point ix above), MR is the mean radioactivity quantified for the MR control (see point v above) (see point ix above), and SR is the mean radioactivity quantified for the SR control (see point vi above) (see point ix above);

[0201] 4) "Increased ADCC" is defined as an increase in the maximum percentage of specific lysis observed within the antibody concentration range tested above, and / or a decrease in the antibody concentration required to achieve half of the maximum percentage of specific lysis observed within the antibody concentration range tested above. The increase in ADCC is relative to ADCC measured by the above assay by the same antibody, produced by the same type of host cell using the same standard production, purification, formulation and storage methods known to those skilled in the art, but not by a host cell engineered to overexpress

[0202] Host cell production of GnTIII.

[0203] The skilled person will easily be able to adapt this specific example of an ADCC assay so as to be able to test whether ADCC activity is increased when using the antibody to be used according to the invention compared to using rituximab, as might be the case.

[0204] Means and methods that can be used to determine ADCP, in particular whether there is increased ADCP compared to rituximab, are known in the art and disclosed, for example, in Herter (supra) and (Same as above).

[0205] According to the present invention, ADCP is "elevated" if, for example, it is at least 1.2-fold higher, at least 1.5-fold higher, at least 2-fold higher, at least 3-fold higher, at least 4-fold higher, at least 5-fold higher, or at least 10-fold higher than the ADCP derived from a comparable application of rituximab.

[0206] Means and methods that can be used to determine affinity for the FcγRIII receptor, in particular whether there is increased affinity for the FcγRIII receptor compared to rituximab, are known in the art and disclosed, for example, in Tobinai (supra).

[0207] According to the present invention, affinity for the FcγRIII receptor is "increased" if, for example, it is at least 1.2-fold higher, at least 1.5-fold higher, at least 2-fold higher, at least 3-fold higher, at least 4-fold higher, at least 5-fold higher, or at least 10-fold higher than the affinity for the FcγRIII receptor derived from a comparable application of rituximab.

[0208] Means and methods that can be used to determine the ability to trigger surface CD20 internalization (when bound to an anti-CD20 antibody), in particular whether there is the ability to trigger less surface CD20 internalization (when bound to obinutuzumab) than rituximab, are known in the art and are disclosed, for example, in Lim (Blood 118(9), 2011, 2530-40).

[0209] According to the present invention, surface CD20 internalization is "less" if, for example, the ability to trigger surface CD20 internalization is at least 1.2-fold lower, at least 1.5-fold lower, at least 2-fold lower, at least 3-fold lower, at least 4-fold lower, at least 5-fold lower, or at least 10-fold lower than that resulting from a comparable application of rituximab.

[0210] As mentioned, it is preferred in the context of the present invention that the functional equivalents of Obinutuzumab to be employed in the context of the present invention comprise a glycoengineered Fc region. In this context, reference is also made to EP-B 12380910 and WO 2005 / 044859; the entire contents of which are incorporated herein by reference.

[0211] It is particularly preferred in the context of the present invention that the Fc region of the antibody to be employed is glycoengineered so that the antibody has one or more of the above-mentioned features and MOA, more particularly in subsections (v), (vi) and (vii) above. The features / MOA of subsection (v) are most preferred in this regard (increase in ADCC).

[0212] Obinutuzumab / functional equivalents of Obinutuzumab to be employed in the context of the present invention may have an increase in the fraction of non-fucosylated oligosaccharides attached to the glycoengineered Fc region.

[0213] Obinutuzumab / functional equivalents of Obinutuzumab to be employed in the context of the present invention may have an increase in the fraction of bisected non-fucosylated oligosaccharides attached to the glycoengineered Fc region.

[0214] Obinutuzumab / functional equivalents of Obinutuzumab to be employed in the context of the present invention may have a significantly higher level of binding to the human FcγRIII receptor relative to the non-glycoengineered antibody, and / or relative to rituximab.

[0215] As mentioned, it is preferred in the context of the present invention that the obinutuzumab / functional equivalents of obinutuzumab to be employed in the context of the present invention, in particular as defined in (b) and (c) above, exhibit significantly higher levels of ADCC activity, in particular relative to non-glycoengineered antibodies, and / or relative to rituximab. Without being bound by theory, the significantly higher levels of ADCC activity result from the glycoengineered Fc region (see above).

[0216] The skilled person is readily able to glycoengineer the Fc region of an antibody so as to achieve the antibody to be employed according to the present invention, e.g. as mentioned above and in such a way as to obtain the relevant features / MOA. Furthermore, the skilled person is readily able to deduce what the fractionally increased non-fucosylated oligosaccharides and the fractionally increased bifucosylated oligosaccharides are according to the present invention. In this context, the skilled person can particularly rely on the guidance provided by EP-B1 2380910 and WO 2005 / 044859; the contents of which are incorporated herein by reference. Non-limiting examples of such increases are at least 1.2-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold or at least 10-fold increases (relative to the non-glycoengineered antibody).

[0217] According to the present invention, the term "identity" or "identical / same" or "percent identity" or "percent identity" or "sequence identity" in the context of two (or more) nucleic acid sequences refers to two (or more) sequences or subsequences that are identical or have a certain percentage of identical nucleotides (preferably at least 80% identity, more preferably at least 85%, 90%, 95%, 96%, 97% or 98% identity, most preferably at least 99% identity) when compared and aligned for maximum correspondence over a comparison window or over a specified region, as measured using a sequence comparison algorithm as known in the art, or by manual alignment and visual inspection. Sequences with, for example, 80% to 90% or greater sequence identity can be considered to be substantially identical. Such definitions also apply to the complement of the test sequence. The described identity can exist over a region of at least about 15 to 25 nucleotides in length, over a region of at least about 50 to 100 nucleotides in length, or over a region of at least about 800 to 1200 nucleotides in length (or over the entire length of the sequence). One skilled in the art will know how to determine the percent identity between two or more sequences using, for example, algorithms as known in the art, such as those based on the CLUSTALW computer program (Thompson, Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB (Brutlag, Comp. App. Biosci. 6 (1990), 237-245).

[0218] Although the FASTDB algorithm typically does not take into account internal non-matching deletions or additions, i.e., gaps, in the sequence in its calculations, this can be manually corrected to avoid overestimating % identity. However, CLUSTALW does take into account sequence gaps in its identity calculations. Also available to those skilled in the art are the BLAST and BLAST 2.0 algorithms (Altschul (1997) Nucl. Acids Res. 25:3389-3402; Altschul (1993) J. Mol. Evol. 36:290-300; Altschul (1990) J. Mol. Biol. 215:403-410). The BLASTN program for nucleic acid sequences uses as default a word length (W) of 11, an expectation (E) of 10, M=5, N=4, and both chains are compared. The BLOSUM62 scoring matrix (Henikoff (1989) PNAS 89:10915) uses alignments (B) 50, expectations (E) 10, M=5, N=4, and both strands are compared.

[0219] To determine whether a nucleotide residue in a nucleic acid sequence corresponds to a position in a given nucleotide sequence, a skilled person can use means and methods known in the art, such as algorithms, either manually or by using computer programs, such as those mentioned herein. For example, BLAST 2.0 (Altschul (1997), supra; Altschul (1993), supra; Altschul (1990), supra), which stands for Basic Local Alignment Search Tool BLAST, can be used to search for local sequence alignments. As discussed above, BLAST generates alignments of nucleotide sequences to determine sequence similarity. Due to the local nature of the alignment, BLAST is particularly useful in determining exact matches or in identifying similar sequences. The basic unit of the BLAST algorithm output is a high-scoring segment pair (HSP). An HSP consists of two sequence fragments of arbitrary but equal length, whose alignment is the local maximum and whose alignment score meets or exceeds a threshold or cutoff score set by the user. The BLAST approach is to search for HSPs between the query sequence and the database sequence to assess the statistical significance of any matches found, and to report only those matches that meet the threshold of significance selected by the user. The parameter E establishes a statistically significant threshold for reporting database sequence matches. E is interpreted as an upper limit on the expected frequency of occurrence of an HSP (or HSP set) in the context of the entire database search. Any database sequence whose match satisfies E is reported in the program output.

[0220] A similar computer technique using BLAST (Altschul (1997), supra; Altschul (1993), supra; Altschul (1990), supra) is used to search nucleotide databases such as GenBank or EMBL for identical or related molecules. This analysis is much faster than multiple membrane-based hybridization. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is classified as exact or similar. The basis for the search is a cumulative score defined as follows:

[0221] % sequence identity x % maximum BLAST score 100

[0223] Moreover, it considers both the degree of similarity between the two sequences and the length of the sequence match. For example, for a score of 40, the match would be exact within 1-2% error; and at 70, the match would be exact. Similar molecules are usually identified by selecting those that show a score between 15 and 40, although lower scores may identify related molecules. Another example of a program that can generate a sequence alignment is the CLUSTALW computer program (Thompson (1994) Nucl. Acids Res. 2:4673-4680) or FASTDB (Brutlag (1990) Comp. App. Biosci. 6:237-245), as known in the art.

[0224] Generally, as used herein, the term "antibody" or "functional equivalent thereof" is a term recognized in the art and is understood to refer to a molecule or active fragment of a molecule that binds a known antigen, in particular an immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule, i.e., a molecule that contains a binding site that immunospecifically binds to an antigen. An immunoglobulin can in principle be of any type (IgG, IgM, IgD, IgE, IgA and IgY) or class (IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of an immunoglobulin molecule.

[0225] In the context of the present invention, "antibodies" are intended to include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, single chain antibodies, bispecific antibodies, simian antibodies, human antibodies and humanized antibodies, as well as active fragments thereof. Examples of active fragments of molecules that bind to known antigens include Fab and F(ab')2 fragments, including the products of Fab immunoglobulin expression libraries and epitope binding fragments of any of the antibodies and fragments mentioned above.

[0226] These active fragments can be derived from a particular antibody (e.g., obinutuzumab) by a variety of techniques. For example, purified monoclonal antibodies can be cleaved with an enzyme, such as pepsin, and submitted to HPLC gel filtration. The appropriate fractions containing the Fab fragments can then be collected and concentrated by membrane filtration, etc. For further description of general techniques for isolating active fragments of antibodies, see, for example, Khaw, BA et al., J. Nucl. Med. 23: 1011-1019 (1982); Rousseaux et al., Methods Enzymology, 121: 663-69, Academic Press, 1986.

[0227] A "humanized antibody" refers to a type of engineered antibody that has its CDRs derived from a non-human donor immunoglobulin, the remaining immunoglobulin-based portions of the molecule being derived from one (or more) human immunoglobulins.

[0228] Humanized antibodies may further refer to antibodies having variable regions in which one or more of its framework regions have human (or primate) amino acids. In addition, framework support residues may be altered to retain binding affinity. Methods for obtaining "humanized antibodies" are well known to those skilled in the art (see, e.g., Queen et al., Proc. Natl. Acad. Sci. USA, 86: 10029-10032 (1989); Hodgson et al., Bio / Technoloy, 9: 421 (1991)).

[0229] "Humanized antibodies" can also be obtained by a new genetic engineering approach that enables the production of affinity-matured human-like polyclonal antibodies in large animals, such as, for example, rabbits (http: / / www.rctech.com / bioventures / therapeutic.php).

[0230] The term "monoclonal antibody" is also art-recognized and refers to an antibody that is produced in large quantities in the laboratory from a single clone and that recognizes only one antigen. Monoclonal antibodies are typically produced by fusing normally short-lived antibody-producing B cells with rapidly growing cells, such as cancer cells (sometimes referred to as "immortal" cells). The resulting hybrid cell, or hybridoma, multiplies rapidly, producing clones that produce large quantities of the antibody.

[0231] The term "antigen" refers to an entity or fragment thereof that is capable of inducing an immune response in an organism, particularly an animal, more particularly a mammal, including a human. The term includes immunogens and regions or antigenic determinants that are responsible for antigenicity.

[0232] As used herein, the term "soluble" means partially or completely soluble in aqueous solution.

[0233] Also as used herein, the term "immunogenic" refers to a substance that elicits or enhances the production of antibodies, T cells and other reactive immune cells to the immunogenic agent and contributes to an immune response in a human or animal.

[0234] The term "hybridoma" is art-recognized and understood by those of ordinary skill in the art to refer to cells that produce antibodies by fusion and immortal cells, such as cells produced by multiple myeloma cells. Such hybrid cells are capable of producing a continuous supply of antibodies. See the definition of "monoclonal antibody" above for a more detailed description of fusion methods.

[0235] In one embodiment, the functional equivalent of Obinutuzumab encompasses a constant heavy chain region comprising Obinutuzumab itself (e.g., amino acid positions 120 to 449 of SEQ ID NO.5) or a constant light chain region of Obinutuzumab itself (e.g., amino acid positions 116 to 219 of SEQ ID NO.6) or both, constant heavy and light chain regions of Obinutuzumab itself. The amino acid sequence of the constant heavy and / or light chain region to be included in the functional equivalent of Obinutuzumab may be 100% identical to the amino acid sequence of the constant heavy and / or light chain region of Obinutuzumab itself. However, it may also vary to some extent from this / these amino acid sequence. For example, it may be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical to the respective amino acid sequence of Obinutuzumab itself. However, it is contemplated that such variant constant heavy and / or light chain regions still contribute to the relevant characteristics of Ureza and functional equivalents of Ureza, respectively (see above for details), in particular to the characteristics of being glycoengineered as defined herein and to significantly higher levels of ADCC activity as defined herein, respectively.

[0236] Most preferably, the antibody to be used according to the present invention is obinutuzumab itself (also known as Gazyva TM / Gazyvaro TM and GA101; WHO Drug Information 27(1), 2013, 90, Recommended INN: List 69). As mentioned, obinutuzumab is well known in the art and is described, for example, in EP-B12380910 and WO 2005 / 044859. Obinutuzumab has the following structure:

[0237] Heavy chain

[0238]

[0239] Light chain

[0240]

[0241] Disulfide bridge position

[0242] 22-96 22″-96″ 23′-93′ 23″′-93″′ 139'-199' 139″′-199″′ 146-202 146"-202″

[0243] 219′-222 219″′-222″ 228-228″ 231-231″ 263-323 263″-323″ 369-427 369″-427″

[0244] Glycosylation site

[0245] H CH2 N84.4

[0246] 299,299" (enriched in bifucosylated oligosaccharides)

[0247] Antibody Rituximab (Medical Product Name: Also known as ) is also known in the art. It is described, for example, in EP-B11005870 (e.g. Figure 4 and 5 ). The amino acid sequence of the heavy chain of rituximab is depicted in SEQ ID NO. 9. The amino acid sequence of the light chain of rituximab is depicted in SEQ ID NO. 10.

[0248] According to the present invention, the skilled person is easily able to assess whether a patient responds favorably to treatment with obinutuzumab (particularly in combination with chemotherapy, more particularly in combination with CHOP chemotherapy) compared to treatment with rituximab (particularly in combination with chemotherapy, more particularly in combination with CHOP chemotherapy). In particular, the clinical outcome or clinical endpoint of the treatment can be assessed in this regard. Clinical outcomes / clinical endpoints that can be assessed in this regard are available to the skilled person and are described, for example, in Goede (supra), Owen (Expert Opin. Biol. Ther. 12 (3), 2012, 343-51) and Illidge (Expert Opin. Biol. Ther. 12 (5), 2012, 543-5) and the accompanying examples.

[0249] Preferred examples of clinical outcomes to be assessed according to the present invention are progression-free survival (PFS), overall survival (OS) and / or event-free survival (EFS). The superiority of obinutuzumab over rituximab according to the present invention may also be determined based on one or more clinical endpoints. In principle, according to the present invention, the term clinical outcome encompasses the time during treatment, while the term clinical endpoint encompasses the time when (or after) treatment ends. According to the present invention, the clinical endpoint may be a primary clinical endpoint. Specific but non-limiting clinical outcomes and clinical endpoints are described in the accompanying examples.

[0250] The skilled person is readily able to determine whether a given clinical outcome is improved according to the invention, i.e. improved compared to treatment with rituximab. For example, "improved" in this context means that the clinical outcome (deriving from treatment with obinutuzumab / a functional equivalent of obinutuzumab (particularly in combination with chemotherapy, more 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 than the clinical outcome resulting from treatment with comparable rituximab (particularly in combination with chemotherapy, more particularly in combination with CHOP chemotherapy).

[0251] The skilled person can easily determine the time to assess the clinical outcome / clinical endpoint. In principle, it is determined to be the time point when the difference in clinical outcome / clinical endpoint between the two treatments (obinutuzumab treatment vs. rituximab treatment) becomes (significantly) apparent. Such a time may 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.

[0252] Preferably, the (DLBCL) patient to be treated according to the present invention is a human patient / human.

[0253] Most preferably, the (DLBCL) patient to be treated according to the present invention is a 1L DLBCL human patient. This means that the DLBCL patient is a previously untreated DLBCL patient.

[0254] However, in principle, other patients, such as non-human patients, such as pets (e.g. dogs, cats, rabbits, rats or mice), livestock (e.g. cows, pigs, sheep), horses or ponies or birds (e.g. chickens, turkeys, parrots) can also be treated according to the present invention. Other warm-blooded animals can also be treated according to the present invention.

[0255] As mentioned, it is particularly contemplated in the context of the present invention that the patients defined herein respond to treatment with obinutuzumab (particularly in combination with chemotherapy, more particularly in combination with CHOP chemotherapy) by achieving an improved clinical outcome compared to treatment with rituximab (particularly in combination with chemotherapy, more particularly in combination with CHOP chemotherapy).

[0256] The antibody to be used in the context of the present invention (i.e., obinutuzumab or a functional equivalent thereof) may be administered in combination with other agents. For example, one or more additional cytotoxic or chemotherapeutic agents, or ionizing radiation that enhances the effect of such agents, may be co-administered; respective examples are described, for example, in EP-B1 2380910, WO 2005 / 044859, WO 2015 / 067586 and WO 2016 / 207312.

[0257] The term "administered in combination with" or "co-administered", "combination therapy" or "combination treatment" refers to the administration of an antibody as described herein, and an additional agent as described herein, e.g., as separate formulations / applications (or as a single formulation / one application). Co-administration can be simultaneous or sequential in either order, wherein preferably there is a period of time while all active agents exert their biological activities simultaneously. The antibody and the additional agent are co-administered simultaneously or sequentially (e.g., intravenously (iv)), e.g., via continuous infusion. When two therapeutic agents are co-administered sequentially, either the administrations are administered on the same day in two separate administrations, or one of the agents may be administered on day 1 and the second agent may be co-administered on day 2 to day 7, preferably on day 2 to day 4. Thus, in one embodiment, the term "sequentially" means within (approximately) 7 days after administration of the first component, preferably within (approximately) 4 days after administration of the first component; whereas the term "simultaneously" means at the same time. The term "co-administered" with respect to maintenance doses of an antibody and / or an additional agent means that the maintenance doses may be co-administered simultaneously, if the treatment cycle is appropriate for both agents, e.g., weekly, or, e.g., every first to third day for the additional agent and weekly for the antibody. Alternatively, the maintenance doses may be co-administered sequentially, either within a single day or over several days.

[0258] In addition to the antibody (optionally in combination with other agents), one or more chemotherapeutic agents or targeted therapies may be administered.

[0259] Such additional chemotherapeutic agents that may be co-administered include, but are not limited to, anti-tumor agents, including alkylating agents, including nitrogen mustards such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan, and chlorambucil; nitrosoureas such as carmustine (BCNU), lomustine (CCNU), and semustine (methyl-CCNU); Temodal TM(temozolomide), ethyleneimine / methylmelamines such as triethylenemelamine (TEM), triethylenethiophosphoramide (thiotepa), hexamethylmelamine (HMM, hexamethylmelamine); alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC); antimetabolites including folic acid analogs such as methotrexate and trimetrexate, pyrimidine analogs such as 5-fluorouracil (5FU), fluorodeoxyuridine, gemcitabine, cytarabine (AraC , cytosine arabinoside), 5-azacytidine, 2,2'-difluorodeoxycytidine, purine analogs such as 6-mercaptopurine, 6-thioguanine, azathioprine, T-deoxyco-synuclein (pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and 2-chlorodeoxyadenosine (cladribine, 2-CdA); natural products, including antimitotic drugs such as paclitaxel, vinca alkaloids, including vinblastine (VLB) , vincristine, and vinorelbine, taxotere, estramustine, and estramustine phosphate; epipodophyllotoxins such as etoposide and teniposide; antibiotics such as dactinomycin D, daunomycin (rubromycin), doxorubicin, mitoxantrone, idarubicin, bleomycin, plicamycin (mithramycin), mitomycin C, and actinomycin; enzymes such as L-asparaginase; biological response modifiers such as interferon-α, IL-2, G-CSF and GM-CSF; Miscellaneous agents, including platinum coordination complexes such as oxaliplatin, cisplatin and carboplatin, anthracenediones such as mitoxantrone, substituted ureas such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH) and phenylcarbazine, adrenocortical suppressants such as mitotane (o,p-DDD) and aminoglutethimide; Hormones and antagonists, including adrenocortical hormone antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; Gemzar TM(gemcitabine), progestogens such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogens such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogens such as tamoxifen; androgens including testosterone propionate and fluoxymesterone / equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; and nonsteroidal antiandrogens such as flutamide. Therapies targeting epigenetic mechanisms can also be combined with antigen binding proteins, including but not limited to histone deacetylase inhibitors, demethylating agents (e.g., Vidaza) and release transcriptional repression (ATRA) therapy. In one embodiment, the chemotherapeutic agent is selected from the group consisting of taxanes (such as, for example, paclitaxel (Taxol), docetaxel (Taxotere), modified paclitaxel (such as Abraxane and Opaxio)), doxorubicin, sunitinib (Sutent), sorafenib (Nexavar), and other multikinase inhibitors, oxaliplatin, cisplatin and carboplatin, etoposide, gemcitabine, and vinblastine. In one embodiment, the chemotherapeutic agent is selected from the group consisting of taxanes (such as, for example, paclitaxel (Taxol), docetaxel (Taxotere), modified paclitaxel (such as Abraxane and Opaxio)). In one embodiment, the additional chemotherapeutic agent is selected from 5-fluorouracil (5-FU), folinic acid, irinotecan, or oxaliplatin. In one embodiment, the chemotherapeutic agent is 5-fluorouracil, folinic acid and irinotecan (FOLFIRI). In one embodiment, the chemotherapeutic agent is 5-fluorouracil and oxaliplatin (FOLFOX).

[0260] In a preferred embodiment, the antibodies defined herein (i.e., obinutuzumab and its functional equivalents) can be administered in combination with chemotherapy, such as CHOP chemotherapy (more preferably) or variants of CHOP chemotherapy, such as CHOEP chemotherapy, CHOP-14 chemotherapy or ACVBP chemotherapy (see, e.g., the Examples described below, and also EP-B12380910, WO2005 / 044859 and Scott, 2014 and 2015, supra). Thus, in a preferred embodiment, the additional chemotherapeutic agent to be co-administered is selected from the group consisting of cyclophosphamide, hydroxydaunorubicin, Oncovein, prednisone or prednisolone and optionally etoposide.

[0261] The antibodies to be used in the context of the present invention may be contained in a composition, in particular in a pharmaceutical composition. The pharmaceutical composition may contain a pharmaceutically acceptable carrier.

[0262] Suitable pharmaceutical compositions and pharmaceutically acceptable carriers are known in the art and are described, for example, in EP-B12380910, WO 2005 / 044859, WO 2015 / 067586 and WO 2016 / 207312.

[0263] Thus, in another aspect, contemplated are compositions, such as pharmaceutical compositions, containing an antibody or antigen-binding portion thereof as defined herein, optionally formulated together with a pharmaceutically acceptable carrier to be used according to the present invention.

[0264] As used herein, "pharmaceutically acceptable carriers" include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption / resorption delaying agents, etc. that are physiologically compatible. Preferably, the pharmaceutical composition and carrier are suitable for injection or infusion, respectively.

[0265] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for preparing sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. In addition to water, the carrier can be, for example, an isotonic buffered saline solution.

[0266] Acceptable carriers, excipients, or stabilizers are encompassed that are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) (c) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN®. TM ,PLURONICS TM or polyethylene glycol (PEG).

[0267] The compositions / antibodies of the invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and / or mode of administration will vary depending on the desired results.

[0268] Regardless of the route of administration selected, the compound of the present invention, which may be used in a suitable hydrated form, and / or the pharmaceutical composition of the present invention are formulated into a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art.

[0269] WO98 / 56418 describes exemplary antibody formulations that can also be used according to the present invention after appropriate adjustments. This publication describes a liquid multi-dose formulation comprising 40 mg / mL antibody, 25 mM acetate, 150 mM trehalose, 0.9% benzyl alcohol, 0.02% polysorbate 20, at pH 5.0, with a minimum shelf life of two years stored at 2-8°C. Another antibody formulation comprises 10 mg / mL antibody in 9.0 mg / mL sodium chloride, 7.35 mg / mL sodium citrate dihydrate, 0.7 mg / mL polysorbate 80, and sterile water for injection, pH 6.5.

[0270] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient (effective amount). The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the present invention employed, or its ester, salt or amide, the route of administration, the time of administration, the excretion rate of the particular compound employed, other drugs, compounds and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar (other) factors well known in the medical field.

[0271] The term "treatment method" or its equivalent, when applied to, for example, DLBCL and a patient as defined herein, respectively, refers to, for example, a procedure or course of action designed to reduce or eliminate the number of DLBCL tumor cells in a patient, or to alleviate the symptoms of a DLBCL tumor. However, a "treatment method" for DLBCL may not necessarily mean that DLBCL tumor cells will actually be eliminated, that the number of cells will actually be reduced, or that the symptoms of a DLBCL tumor will actually be alleviated. Generally, a treatment method for DLBCL will be implemented even if it has a lower probability of success, but given the patient's medical history and estimated survival expectancy, it is still considered to induce an overall beneficial course of action, especially compared to rituximab treatment.

[0272] It is self-evident that the antibody is administered to a patient in a "therapeutically effective amount" (or simply "effective amount"), which is the amount of the respective compound or combination that will elicit the biological or medical response, e.g., of a tissue, system, animal or human, that is being sought by the researcher, veterinarian, medical doctor or other clinician.

[0273] The amount (co)administered and the timing of (co)administration will depend on the type (species, sex, age, weight, etc.) and condition of the patient being treated and the severity of the disease or condition being treated. The antibody and optionally the further agent(s) are suitably (co)administered to the patient at one time or over a series of treatments, e.g. on the same day or on subsequent days.

[0274] Depending on the type and severity of the disease, about 0.1 mg / kg to 50 mg / kg (eg, 0.1-20 mg / kg) of an antibody defined herein is an initial candidate dosage for co-administration to a patient.

[0275] The accompanying examples (particularly Examples 1 and 2 detailing the study design and treatment used in the context of the GOYA study) describe and provide a specific but non-limiting example of an administration regimen (including administration routes and dosages) of obinutuzumab / functional equivalents of obinutuzumab (more particularly G-CHOP). If necessary, the skilled person will readily be able to adapt this example of a G administration regimen to any other G administration regimen that may be suitable in accordance with the present invention.

[0276] The antibodies and pharmaceutical compositions to be used according to the present invention, respectively, can be provided with a manual or a leaflet. The manual / leaflet can include instructions for technicians / attending physicians on how to treat DLBCL and patients defined herein according to the present invention. For example, the manual / leaflet can include instructions on the mode of administration / administration regimen (e.g., route of administration, dosage regimen, time of administration, frequency of administration) described herein. In particular, the manual / leaflet can include information about the patient to be treated, i.e., the patient defined herein. In principle, what is described elsewhere herein with respect to obinutuzumab, the patient to be treated, the mode of administration / administration regimen (including dosage, etc.) can be included in the manual / leaflet.

[0277] A preferred sample to be employed in the context of the present invention is derived from a patient's tumor tissue (e.g. as a biopsy). For example, formalin-fixed or preferably paraffin-embedded tumor tissue (e.g. a section of tumor tissue on a slide) may be employed. However, it is also contemplated that other samples are employed in the context of the present invention, such as sections / biopsies of other tissues, blood samples, serum samples, or other body fluid samples, etc.

[0278] Some documents, including patents / patent applications, are cited in this specification. Although the disclosures of these documents are not considered relevant to the patentability of the present invention, they are incorporated herein in their entirety by reference. More specifically, all cited documents are incorporated by reference as if each individual document was specifically and individually indicated to be incorporated by reference.

[0279] The present invention will now be described by reference to the accompanying drawings and the following examples, which should not be construed as limiting the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0280] Figure 1 :Description of patients in GOYA

[0281] *Discontinued refers to patients discontinuing study (antibody) treatment. The median observation time in each group was 29 months; treatment completion refers to patients completing study (antibody) treatment.

[0282] Patients were stratified at randomization by IPI score (low / low-intermediate, high-intermediate, and high risk), planned number of CHOP cycles (8 vs 6), and geographic region (Western Europe, Eastern Europe, Central and South America, North America, Asia, and other).

[0283] G-CHOP, obinutuzumab plus cyclophosphamide, doxorubicin, vincristine, and prednisone / prednisolone; R-CHOP, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone / prednisolone.

[0284] Figure 2 :Kaplan-Meier estimates of PFS and OS in GOYA

[0285] (A) Investigator-assessed PFS by treatment (primary endpoint), intent-to-treat population, (B) OS by treatment, intent-to-treat population, (C) Investigator-assessed PFS by cell-of-origin subtype (regardless of study treatment) in patients with cell-of-origin data.

[0286] ABC, activated B-cell-like; CI, confidence interval; GCB, germinal center B-cell-like; G-CHOP, obinutuzumab plus cyclophosphamide, doxorubicin, vincristine, and prednisone; HR, hazard ratio; OS, overall survival; PFS, progression-free survival; R-CHOP, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone.

[0287] Figure 3 : Unstratified hazard ratios for investigator-assessed PFS in GOYA patient subgroups

[0288] (A) Randomization stratification factors and (B) baseline characteristics.

[0289] ABC, activated B-cell-like; CI, confidence interval; DLBCL, diffuse large B-cell lymphoma; ECOGPS, Eastern Cooperative Oncology Group performance status; GCB, germinal center B-cell-like; G-CHOP, obinutuzumab plus cyclophosphamide, doxorubicin, vincristine, and prednisone; IPI, international prognostic index; KM, Kaplan–Meier; PFS, progression-free survival; R-CHOP, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone.

[0290] *Cases with 'yes' ticked in the eCRF regarding extranodal involvement; 14 patients with 0 extranodal sites were incorrectly ticked.

[0291] Figure 4 Gazyva Benefit in Patients with BCL2 Translocations in GOYA

[0292] Figure 5 Gazyva Benefit in Patients with Positive BCL2 Protein Expression in GOYA

[0293] Figure 6 Gazyva benefit in patients with BCL2 translocations who are positive for BCL2 protein expression in GOYA

[0294] Evaluation of treatment efficacy in quadrants defined by BCL2 IHC (positive / negative) and BCL2 FISH (positive / negative) in GOYA. Gazyva superiority was shown to be specific for BCL2 IHC+ / FISH+ patients.

[0295] Figure 7: Gazyva benefit in GCB subgroups defined by various cutoffs of linear predictor scores in GOYA

[0296] A: Forest plot 25%-36% interception (%)

[0297] B: Forest plot 25%-50% interception (%)

[0298] C: Gazyva benefit for the primary endpoint (INV-PFS) in the strong GCB subgroup at an LPS cutoff of <749.

[0299] □HR* (95% CI) in patients with strong GCB GOYA

[0300] - PFS: 0.33 [0.18-0.63], p value = 0.0007

[0301] □When LPS < 749, strong GCB patients represented 25% (n = 233 / 933) of all DLBCL patients in GOYA.

[0302] □ Strong GCB patients constituted 43% (n=233 / 540) of GCB patients in GOYA.

[0303] Figure 8 Gazyva benefit in GCB patients with BCL2 translocation in GOYA and positive BCL2 protein expression

[0304] Fig. 9 Gazyva benefit in patients with CD58 mutations and / or those with low CD58 gene expression in GOYA

[0305] Fig.10 : Multivariate simulation optimization of LPS cutoff with COO analysis to assess the benefit of G-CHOP over R-CHOP on progression-free survival in patients (N=xx).

[0306] Optimization of LPS retention for strong GCB treatment effect in GOYA raw data.

[0307] *Multivariate HRs were adjusted for treatment, International Prognostic Index, number of chemotherapy cycles (6 or 8), and geographic region. Blue line, point estimate of HR, yellow line, 95% CI; CI, confidence interval; COO, cell of origin; HR, hazard ratio; LPS, linear predictor score.

[0308] Fig.11 : Patients with strong GCB in GOYA # Molecular characterization of

[0309] Strong GCB patients had a significantly higher prevalence of FL somatic mutation signatures.

[0310] (A) Prevalence of FL somatic mutation signatures* (any mutation type)

[0311] (B) Prevalence of BCL2 translocations

[0312] # Other biomarkers evaluated to characterize strong versus weak GCB, for which no significant differences in prevalence were identified, were: by gene expression, the stromal-1 / 2 gene signature, the immune response 1 / 2 gene signature, CD20, and PTEN; by protein expression, BCL2, MYC, and BCL2 / MYC double expressors; and by gene translocation, MYC translocation and BCL2 / MYC double hits. ABC, activated B cell; DLBCL, diffuse large B-cell lymphoma; FDR, false discovery rate; FISH, fluorescence in situ hybridization; FL, follicular lymphoma; GCB, germinal center B cell; NGS, next-generation sequencing.

[0313] Fig.12 : Distribution of optimal LPS retention among bootstrapped samples

[0314] □ Bootstrapped multivariate simulations were used to further test the robustness and feasibility of the optimal LPS identified using the “minimum HR rule”.

[0315] Promotional

[0316] □The extreme peak between bootstrap samples is located at LPS=725

[0317] □The LPS distribution with its unique peak supports the robustness of the therapeutic effect signal

[0318] The optimal LPS retention recommended for the new potential confirmatory study is LPS ≤ 725

[0319] - All historical GOYA biomarker analyses had a strong GCB defined as <749 (25% of patients in GOYA), including the biomarker analyses presented in this OBRF

[0320] - n = 4 patients had 725 < LPS < 749, all on G-CHOP (1 event)

[0321] Fig.13 : Kaplan-Meier assessment of time to next anti-lymphoma treatment (secondary endpoint) in the intention-to-treat population

[0322] CI, confidence interval; G-CHOP, obinutuzumab plus cyclophosphamide, doxorubicin, vincristine, and prednisone / prednisolone; HR, hazard ratio; R-CHOP, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone / prednisolone.

[0323] Fig.14 : Kaplan-Meier assessment of PFS by investigator assessment stratified by treatment arm in patients with COO data, subgrouped by COO subtype

[0324] (A) GCB; (B) ABC; (C) unclassified.

[0325] ABC, activated B-cell-like; CI, confidence interval; COO, cell of origin; GCB, germinal center B-cell-like; G-CHOP, obinutuzumab plus cyclophosphamide, doxorubicin, vincristine, and prednisone / prednisolone; HR, hazard ratio; R-CHOP, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone / prednisolone.

[0326] The present invention also relates to the following table.

[0327] Table 1: Baseline patient and disease characteristics (intention-to-treat population)

[0328]

[0329]

[0330] ABC, activated B-cell-like (subgroup); ECOGPS, Eastern Cooperative Oncology Group performance status; G-CHOP, obinutuzumab plus cyclophosphamide, doxorubicin, vincristine, and prednisone / prednisolone; GCB, germinal center B-cell-like (subgroup); IPI, international prognostic index; LDH, lactate dehydrogenase; PET, positron emission tomography; R-CHOP, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone / prednisolone.

[0331] *Unless otherwise noted, n=706 for G-CHOP, n=712 for R-CHOP for all parameters.

[0332] Cases with 'yes' ticked in the eCRF regarding extranodal involvement; 14 patients with 0 extranodal sites were incorrectly ticked.

[0333] 485 patients (G-CHOP, 235; R-CHOP, 250) lacked COO subtype classification; including samples from China, which could not be analyzed due to lack of export licenses – analysis of these samples is planned in the near future.

[0334] Table 2: Summary of Efficacy Endpoints (Intent-to-Treat Population)

[0335]

[0336]

[0337] CI, confidence interval; CR, complete response; DFS, disease-free survival; EFS, event-free survival; G-CHOP, obinutuzumab plus cyclophosphamide, doxorubicin, vincristine, and prednisone / prednisolone; HR, hazard ratio; IRC, independent review committee; ORR, overall response rate; OS, overall survival; PET, positron emission tomography; PFS, progression-free survival; R-CHOP, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone / prednisolone.

[0338] * Stratification factors were the International Prognostic Index score and the planned number of CHOP cycles (6 or 8).

[0339] In accordance with the revised response standards. 13

[0340] Table 3: Summary of AEs reported by ≥5% of patients in either group, including Grade 3-5 and serious AEs (at the preferred level; safety population)

[0341]

[0342]

[0343] AE, adverse event; G-CHOP, obinutuzumab plus cyclophosphamide, doxorubicin, vincristine, and prednisone / prednisolone; R-CHOP, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone / prednisolone. *Fatal AEs reported in more than one patient in either group, listed as preferred, were: septic shock (six [0.9%] patients), pneumonia (five [0.7%]), death (cause unknown; three [0.4%]), pulmonary embolism (two [0.3%]), and cerebrovascular accident (two [0.3%]) in the G-CHOP group and pneumonia (six [0.9%]), sepsis (three [0.4%]), cerebrovascular accident (two [0.3%]), and death (cause unknown; two [0.3%]) in the R-CHOP group.

[0344] Table 4: Effect of G-CHOP and R-CHOP on clinical outcomes in strong GCB and all other patients

[0345]

[0346] *Adjusted for treatment arm, International Prognostic Index, number of chemotherapy cycles (6 or 8), and geographic region. CI, confidence interval; EFS, event-free survival; GCB, germinal center B cells; HR, hazard ratio; OS, overall survival; PFS, progression-free survival (investigator-assessed); yr, year

[0347] Table 5: Study Drug Exposure

[0348]

[0349] G-CHOP, obinutuzumab plus cyclophosphamide, doxorubicin, vincristine, and prednisone; R-CHOP, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone.

[0350] *Includes interruption of infusion and reduction of infusion rate.

[0351] Table 6: Number (and %) of patients reporting AEs of any grade with an incidence of at least 10% in any treatment arm (at the preferred term level), listed by system organ class and preferred term (safety population)

[0352]

[0353]

[0354] AE, adverse event; G-CHOP, obinutuzumab plus cyclophosphamide, doxorubicin, vincristine, and prednisone / prednisolone; R-CHOP, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone / prednisolone.

[0355] Table 7: Summary of AEs of particular interest as preferred terms or predefined categories (safety population)

[0356]

[0357]

[0358] AE, adverse event; G-CHOP, obinutuzumab plus cyclophosphamide, doxorubicin, vincristine, and prednisone / prednisolone; HBV, hepatitis B infection; MedDRA, Medical Dictionary for Regulatory Activities; R-CHOP, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone / prednisolone.

[0359] *Any preference in System Organ Class Infection and Infestation.

[0360] Standardized MedDRA queries.

[0361] Neutropenia and related complications were reported as AEs, excluding abnormal laboratory values.

[0362] § Related to any infused treatment and occurring during / within 24 hours of infusion.

[0363] Any preference in the System Organ Class Cardiac Disorders.

[0364] **Any preference among system organ class benign, malignant and unspecified neoplasms (including cysts and polyps) beginning 6 months after first study drug intake.

[0365] At least one of the following AEs: increase in HBV DNA level ≥10 IU / ml or hepatitis B reactivation.

[0366] Standardized MedDRA query including perforation events (preferred terms in the system organ class Gastrointestinal disorders) and abscesses and other events (preferred terms in other system organ classes).

[0367] §§ Standardized MedDRA query covering hemorrhagic cerebrovascular conditions, and hemorrhage (laboratory and non-laboratory items). Example

[0368] Example 1 - General Materials and Methods

[0369] Description of the NanoString LST Assay

[0370] If not indicated to the contrary, the NanoString LST assay was performed in the context of the present invention according to the manual of the distributor (NanoString Technologies, Inc., Seattle, WA, USA). For further guidance on the assay, see Scott (2014 and 2015, supra).

[0371] Overview of COO Assay

[0372] NanoString's LST gene expression assay is designed to The assay system was developed to identify COO isoforms. Gene expression assays together with NanoString technology provide a super-sensitive and highly multiplexed method for detecting mRNA with molecular barcodes called nCounter reporter probes without the use of reverse transcription or amplification (Geiss, supra). Detection of mRNA is based on digital detection and direct molecular barcoding of target molecules via the use of color-coded probe pairs. The probe pair consists of a reporter probe carrying a signal at its 5' end and a capture probe. The color code carries six positions and each position can be one of four colors, thereby allowing multiple labels that can be mixed together in a single well for direct hybridization with the target and still be individually resolved and identified during data collection. NanoString reporter and capture probe technology employs reporter probes carrying color-coded barcodes and capture probes that allow immobilized complexes for data collection.

[0373] Custom LST probe pairs are mixed with sample RNA in large excess over target mRNA to ensure that each target finds one probe pair. After hybridization, excess unbound probe is washed away and the target / probe complex is immobilized in a cartridge for data collection. Data collection was performed in a digital analyzer. Digital images were processed and barcode counts were tabulated in comma separated value (CSV) format for sample normalization and data analysis.

[0374] Prior to data analysis, each sample was normalized to reference counts generated from synthetic RNA reference control transcripts and housekeeping genes.

[0375] Determination of COO status in patient samples

[0376] The LST assay generates gene expression data for 20 genes per patient (see Table 5). Five of these genes are housekeeping reference genes, while the other 15 genes distinguish GCB from ABC, specifically, 8 genes are known to be overexpressed in ABC-like DLBCL, while 7 genes are known to be overexpressed in GCB-like DLBCL (based on Scott, 2014, supra).

[0377] Table 5: List of 20 genes in the NanoString LST assay

[0378]

[0379] A linear predictor score (LPS) was calculated for each patient based on gene expression data. The LPS is a weighted sum of the gene expression of the 20 genes in the LST assay:

[0380] LPS(X)=Σ j a j X j ,

[0381] Where X j is the gene expression of gene j and a j is the coefficient of gene j.

[0382] Each patient's LPS is then compared against a predefined threshold to determine the patient's DLBCL COO subtype. For a single LPS score, a probability is determined as to whether the tumor is likely to be part of the ABC subtype or the GCB subtype. Tumors with a probability of being ABC greater than 90% are considered ABC, while those with a probability of being GCB greater than 90% are considered GCB. Tumors with a probability of being ABC or GCB less than 90% are considered unclassified (Scott, 2014 and 2015, supra).

[0383] G-CHOP and R-CHOP

[0384] Patients will be treated with one of two immunochemotherapy regimens:

[0385] G-CHOP (investigational arm): CHOP chemotherapy combined with obinutuzumab;

[0386] R-CHOP (control arm): CHOP chemotherapy combined with rituximab.

[0387] For the purposes of this regimen, obinutuzumab and rituximab are considered investigational medical products.

[0388] In the investigational arm, obinutuzumab will be administered by IV infusion at an absolute (flat) dose of 1000 mg on Day 1 of each 21-day cycle for 8 cycles. During Cycle 1, obinutuzumab will also be infused on Days 8 and 15. On days when both obinutuzumab and CHOP are to be given, administration of obinutuzumab should be completed at least 30 minutes prior to the start of chemotherapy administration.

[0389] If the duration of obinutuzumab infusion requires a next-day CHOP infusion, CHOP chemotherapy may be given the day after obinutuzumab administration. CHOP chemotherapy will be given for up to 6 or 8 cycles, as described in Section 4.5.3.a and Table 5. If only 6 cycles of CHOP chemotherapy are to be administered (see Table 5), cycles 7 and 8 of obinutuzumab will be given as monotherapy on a 21-day schedule.

[0390] In the control arm of the study, 375 mg / m2 was administered by IV infusion on Day 1 of each 21-day cycle. 2 Rituximab is administered at a dose of 100 mg / dL for 8 cycles. Rituximab will be administered before CHOP, and the infusion should be completed and the patient observed for at least 30 minutes before starting CHOP. CHOP can be given on day 2 if the duration of the rituximab infusion requires a CHOP infusion the next day. If only 6 cycles of CHOP chemotherapy are to be administered (see Table 5), rituximab will be given as monotherapy on a 21-day schedule for cycles 7 and 8.

[0391] The dose of rituximab and chemotherapy should be calculated based on the patient's weight at the screening assessment (Day -14 to Day -1). For all subsequent doses, for weight changes >10% from screening, the dose of rituximab and chemotherapy should be adjusted accordingly. The weight that triggered the dose adjustment will serve as the new reference weight for future dose adjustments.

[0392] As described in section 3.6.1.c, G-CSF is recommended for patients ≥ 60 years of age and for those with comorbidities according to ASCO, EORTC, and ESMO guidelines. It is strongly recommended for all patients treated with G-CHOP during cycle 1.

[0393] Obinutuzumab (technical data)

[0394] aPreparation

[0395] Obituzumab is provided as a single-dose, sterile liquid formulation in a 50 mL pharmaceutical grade glass vial containing a nominal 1000 mg of Obituzumab (G3 material). The formulated drug product consists of 25 mg / mL drug substance (G3) formulated in histidine, trehalose, and poloxamer 188. The vial contains 41 mL (2.5% overfill).

[0396] For further details, see the Obituzumab Investigator's Brochure.

[0397] bHandling and storage

[0398] The recommended storage conditions for the obinutuzumab drug product are between 2°C and 8°C, protected from light. The chemical and physical in-use stability of obinutuzumab dilutions in 0.9% sodium chloride (NaCl) has been demonstrated at 2°C-8°C and ambient temperature and ambient room lighting for 24 hours. The prepared diluted product should be used immediately. If not used immediately, the in-use storage time and conditions prior to use are the responsibility of the user and will not normally be longer than 24 hours at 2°C-8°C. Obinutuzumab should not be frozen or shaken. Mix gently. All transfer procedures require strict adherence to aseptic technique. Do not use additional in-line filters due to potential adsorption.

[0399] For further details, see the Obituzumab Investigator's Brochure.

[0400] cObinutuzumab dosage and schedule

[0401] Obinutuzumab will be administered at an absolute (flat) dose of 1000 mg by IV infusion on Day 1 of each 21-day cycle for 8 cycles. Obinutuzumab will be administered before CHOP, and patients should be observed for 30 minutes before starting CHOP. If CHOP is not completed on Day 1 due to the long duration of obinutuzumab therapy, CHOP chemotherapy may be administered on Day 2. During Cycle 1, obinutuzumab will also be infused on Days 8 and 15. Obinutuzumab will be administered as a monotherapy if CHOP chemotherapy is not given in Cycles 7 and 8.

[0402] d Preparation of Obinutuzumab

[0403] Prepare the obinutuzumab drug product intended for IV infusion by diluting the drug product into an infusion bag containing 0.9% NaCl to a final drug concentration of 4 mg / mL. Using a 250 mL infusion bag containing 0.9% NaCl, remove and discard 40 mL of NaCl. Remove 40 mL of obinutuzumab from a single glass vial and inject into the infusion bag (discard any unused portion of obinutuzumab remaining in the vial). Gently invert the infusion bag to mix the solution; do not shake.

[0404] Administration sets having polyvinyl chloride (PVC), polyurethane (PUR), or polyethylene as product contact surfaces and IV bags having polyolefins, polypropylene (PP), PVC, or polyethylene as product contact surfaces are compatible and may be used.

[0405] Do not use obinutuzumab beyond the expiration date printed on the carton.

[0406] e Administration of Obinutuzumab

[0407] Obinutuzumab should be administered to patients in a clinical setting (inpatient or outpatient) where complete emergency resuscitation equipment is immediately available and patients should be under close supervision of the investigator at all times. Do not administer as an IV push or bolus. The IV line or central venous catheter should remain in place for ≥2 hours after the end of the first infusion to allow for administration of IV medications if necessary. If no adverse events occur after 2 hours, the IV line may be removed or the central venous catheter may be removed. For subsequent infusions, access (either via an IV line or central venous catheter) should remain in place for at least 1 hour from the end of the infusion, and if no adverse events occur after 1 hour, the IV access may be removed.

[0408] Before administering obinutuzumab, refer to section 4.3.5, General Precautions (for guidance on the use of premedication and prophylaxis for tumor lysis syndrome). Instructions for the first and subsequent infusions of obinutuzumab are listed in Table 8.

[0409] Table 8: Administration of the first and subsequent infusions of obinutuzumab

[0410]

[0411] Obinutuzumab should be given as a slow IV infusion via a dedicated line. An IV infusion pump should be used to control the infusion rate of obinutuzumab. Do not administer as an IV push or bolus.

[0412] On days when both obinutuzumab and CHOP are given, obinutuzumab will be administered before CHOP and patients should be observed for 30 minutes before starting CHOP. CHOP chemotherapy may be administered the following day if it cannot be given on the same day as obinutuzumab. Prior to each infusion of obinutuzumab given in combination with CHOP (Day 1 of Cycles 1-6 or Cycles 1-8), patients should take the Day 1 dose of oral prednisone (100 mg) prescribed for each cycle of the CHOP regimen. Prophylactic use of corticosteroids (e.g., 100 mg IV prednisolone or equivalent) may also be considered for patients considered at high risk for IRR, if deemed appropriate by the investigator, and should also be administered prior to the obinutuzumab infusion.

[0413] For the management of IRRs and allergic reactions, see Section 4.3.6.a and Table 3.

[0414] CHOP chemotherapy

[0415] CHOP is considered the standard of care for the treatment of DLBCL. By center choice, sites will choose before the start of the study whether they will administer 8 or 6 cycles of CHOP chemotherapy.

[0416] a Dosage and administration

[0417] CHOP chemotherapy consists of IV cyclophosphamide, IV doxorubicin, vincristine administered by IV bolus, and oral prednisone or prednisolone. Six to eight 21-day cycles of standard CHOP will be administered. Cycles 7 and 8 of CHOP chemotherapy will be administered according to the center's prospective choice of 6 versus 8 planned cycles.

[0418] Cyclophosphamide 750 mg / m intravenously on day 1 2

[0419] Doxorubicin 50 mg / m intravenously on day 1 2

[0420] Vincristine 1.4 mg / m by IV bolus on day 1 2 , recommended upper limit 2.0mg

[0421] Prednisone 100 mg / day orally (PO) on days 1-5

[0422] Note: Dosing of prednisone follows the recommendations of the National Comprehensive Cancer Network (2010), which are based on Cruzman et al., 1999; Hiddemann et al., 2005. Prednisone can be substituted with prednisolone (1:1 switch, 100 mg) in countries where prednisone / prednisolone is not available or is not a treatment option, or it can be substituted with 80 mg of methylprednisolone in countries or sites where prednisone / prednisolone is not available.

[0423] CHOP will be administered at each investigational site following the infusion of rituximab or obinutuzumab according to standard preparation and infusion protocols. Refer to the specific package insert for preparation, administration, and storage instructions. The dose of vincristine may be capped at 2 mg at the discretion of the investigator. For patients ≥ 70 years of age, the dose of vincristine may be capped at 1.5 mg. BSA may be capped at 2 mg per institutional criteria. 2 is the upper limit.

[0424] When obinutuzumab or rituximab and CHOP are scheduled for administration on the same day, it is recommended that prednisone (100 mg) be administered prior to the obinutuzumab or rituximab infusion. Administration of obinutuzumab or rituximab should be completed at least 30 minutes prior to administration of the CHOP (cyclophosphamide, vincristine, and doxorubicin) infusion. Administration of rituximab or obinutuzumab the day before CHOP and premedication (defined in subsection 4.3.5) is permitted if the investigator or site strongly prefers (e.g., for logistical reasons). Splitting the antibody (rituximab or obinutuzumab) infusion over 2 days is also permitted if the patient is at increased risk for IRR (high tumor burden, high peripheral lymphocyte count). Also, in patients who experience adverse events during the obinutuzumab or rituximab infusion, administration of obinutuzumab may be continued the next day if necessary. If the Cycle 1 Day 1 dose of obinutuzumab is split, both infusions must be given with appropriate premedication and at the first infusion rate (see Table 2). If CHOP is started later than Cycle Day 1, the planned Day 1 of the next cycle should be calculated from the day CHOP was actually started, thus maintaining a regular 21-day chemotherapy interval.

[0425] Example 2 - Obinutuzumab or rituximab (plus CHOP) in previously untreated DLBCL (Description of the Roche GOYA (B021005) Phase 3 study)

[0426] patient

[0427] Eligible patients were aged ≥18 years with: previously untreated, histologically proven, CD20-positive DLBCL (as assessed by the local pathology laboratory); ≥1 two-dimensionally measurable lesion (>1.5 cm in greatest dimension on computed tomography [CT] scan); Eastern Cooperative Oncology Group performance status 0-2; adequate hematologic, hepatic, and renal function; left ventricular ejection fraction ≥50%; and International Prognostic Index (IPI) score ≥2 (and patients with IPI score 1, age ≤60 years, with or without bulky disease, and patients with IPI score 0 and bulky disease, i.e., one lesion ≥7.5 cm). Full inclusion / exclusion criteria are detailed below. All patients provided informed consent.

[0428] Complete inclusion and exclusion criteria

[0429] Patients must meet the following criteria to enter the study:

[0430] 1. Informed consent form.

[0431] 2. Previously untreated CD20-positive diffuse large B-cell lymphoma (DLBCL) with histological confirmation as follows:

[0432] a. Pathology reports must be available for review and tissue blocks sent for retrospective central confirmation.

[0433] b. Formalin-fixed, paraffin-embedded tissue blocks are preferred; however, in countries where different fixatives are used, any available tissue blocks including a note on the type of fixative will be accepted.

[0434] c. If tissue blocks are not available, 15 unstained slides (3-5 μm thickness) will be accepted.

[0435] d. Optional Roche clinical repository samples and requested exploratory biomarker samples will be obtained from the same tissue block. Diagnostic stained slides may also be requested if central validation cannot be performed on the submitted material.

[0436] 3. Patients in one of the following International Prognostic Index (IPI) disease risk groups: high, high-intermediate, or low-intermediate.

[0437] a. Patients in the low-risk group are eligible but must have an IPI score of 1 regardless of bulky disease, or an IPI score of 0 and bulky disease, defined as one lesion ≥7.5 cm.

[0438] b. NOTE: Patients without bulky disease who have an IPI of 1 solely due to age (ie, patients >60 years with no other risk factors) are not eligible for this trial.

[0439] 4. At least one two-dimensionally measurable lesion, defined as >1.5 cm in its largest dimension on computed tomography scan.

[0440] 5. Ability and willingness to comply with the study protocol.

[0441] 6. Age ≥ 18 years old.

[0442] 7. Eastern Cooperative Oncology Group performance status 0, 1, or 2.

[0443] 8. Left ventricular ejection fraction ≥ 50% on cardiac multi-gated acquisition scan or cardiac echocardiography.

[0444] 9. Adequate hematologic function (unless due to underlying disease, such as hypersplenism established by extensive bone marrow involvement or secondary to splenic involvement due to DLBCL, as determined by the investigator), defined as follows:

[0445] a. Hemoglobin ≥ 9 g / dl;

[0446] b. Absolute neutrophil count ≥ 1.5 x 10 9 / l;

[0447] c. Platelet count ≥ 75 x 10 9 / l.

[0448] 10. For males who are not surgically infertile: agree to use a barrier method of contraception during the treatment period and until ≥ 3 months after the last dose of obinutuzumab or rituximab, or in accordance with institutional guidelines for CHOP chemotherapy, whichever is longer, and agree to require their partner to use an additional method of contraception, such as oral contraceptives, intrauterine devices, barrier methods, or spermicide.

[0449] 11. For females of reproductive potential who are not surgically infertile: Agree to use two appropriate methods of contraception, such as oral contraceptives, intrauterine devices, or barrier contraceptive methods in conjunction with spermicide during the treatment period and until ≥12 months after the last dose of obinutuzumab or rituximab, or in accordance with institutional guidelines for CHOP chemotherapy, whichever is longer.

[0450] Patients meeting any of the following criteria were excluded from entering the study:

[0451] 1. History of severe allergic or anaphylactic reaction to humanized or murine monoclonal antibodies or known sensitivity or allergy to murine products or any component of CHOP or obinutuzumab.

[0452] 2. Contraindications to any individual component of CHOP, including prior reception of anthracyclines.

[0453] 3. Patients with transformed lymphoma and patients with stage 3b follicular lymphoma.

[0454] 4. Prior therapy for DLBCL, with the exception of node biopsy or local irradiation.

[0455] 5. Prior treatment with cytotoxic drugs or rituximab for other conditions (e.g., rheumatoid arthritis) or prior use of anti-CD20 antibodies.

[0456] 6. Prior use of any monoclonal antibody within 3 months from the start of cycle 1.

[0457] 7. Use of corticosteroids >30 mg / day of prednisone or equivalent for purposes other than controlling lymphoma symptoms.

[0458] a. Patients receiving ≤ 30 mg / day of prednisone or equivalent corticosteroids must have

[0459] (Cycle 1, Day 1) Record of stable dose for at least 4 weeks prior.

[0460] b. If glucocorticoid therapy is urgently needed to control lymphoma symptoms before starting study treatment, prednisone 100 mg or equivalent may be given for a maximum of 5 days, but all tumor assessments must be completed before starting glucocorticoid therapy.

[0461] 8. Primary central nervous system (CNS) lymphoma and secondary CNS involvement due to lymphoma, mantle cell lymphoma, or histological evidence of transformation to Burkitt lymphoma, primary mediastinal DLBCL, primary effusion lymphoma, plasmablastic lymphoma, and primary cutaneous DLBCL.

[0462] 9. Vaccination with a live vaccine within 28 days prior to randomization.

[0463] 10. Any investigational therapy within 28 days prior to starting Cycle 1.

[0464] 11. History of other malignancies that could affect compliance with the regimen or interpretation of results.

[0465] a. Patients with a history of curatively treated basal or squamous cell carcinoma, or cutaneous melanoma or cervical carcinoma in situ at any time prior to the study are eligible.

[0466] b. Patients with any other malignancy that has been treated with surgery alone with curative intent and that has been in remission without treatment for ≥5 years prior to enrollment are eligible.

[0467] 12. Significant, uncontrolled concomitant illness that could affect compliance with the protocol or interpretation of results, including evidence of significant cardiovascular disease (such as New York Heart Association class III or IV heart disease, myocardial infarction within the last 6 months, unstable arrhythmia, or unstable angina) or pulmonary disease (including history of obstructive pulmonary disease and bronchospasm).

[0468] 13. Recent major surgery (within 4 weeks prior to starting Cycle 1) other than for diagnostic purposes.

[0469] 14. Any of the following abnormal laboratory values ​​(unless any of these abnormalities are due to underlying lymphoma):

[0470] a. Creatinine > 1.5 times the upper limit of normal (ULN; unless creatinine clearance is normal), or calculated creatinine clearance < 40

[0471] ml / min (using Cockcroft-Gault formula).

[0472] b. Aspartate aminotransferase or alanine aminotransferase > 2.5 times ULN.

[0473] c. Total bilirubin ≥1.5 times ULN: Patients with documented Gilbert's disease may enroll if total bilirubin is ≤3.0 times ULN.

[0474] d. In the absence of therapeutic anticoagulation, the international normalized ratio is >1.5 times ULN.

[0475] e. Partial thromboplastin time or activated partial thromboplastin time >1.5 times ULN in the absence of lupus anticoagulant.

[0476] 15. Known active bacterial, viral, fungal, mycobacterial, parasitic, or other infection (excluding fungal infection of the nail bed) within 4 weeks prior to the start of Cycle 1 or any major infectious episode requiring treatment with intravenous antibiotics or hospitalization (related to completion of the antibiotic course, excluding tumor fever).

[0477] 16. Patients suspected of having active or latent tuberculosis.

[0478] a. Latent TB needs to be confirmed by a positive interferon-γ release assay.

[0479] 17. Positive test result for chronic hepatitis B infection (defined as positive HBsAg serology).

[0480] a. If HBV DNA is undetectable, this may include cases with occult or prior hepatitis B infection

[0481] Patients with positive total HBsAg and negative HBsAg were also considered eligible. These patients had to be willing to undergo monthly DNA testing.

[0482] 18. Positive test result for hepatitis C (HCV antibody serology test).

[0483] a. Patients who are positive for HCV antibodies are eligible only if they are PCR negative for HCV RNA.

[0484] 19. Known history of HIV seropositive status.

[0485] a. For patients with unknown HIV status, HIV testing will be performed at screening if required by local regulations.

[0486] 20. Positive result for human T-lymphotropic virus 1 (HTLV).

[0487] a. HTLV testing is required for patients at sites in endemic countries (Japan and Melanesia, and countries in the Caribbean Basin, South America, Central America, and sub-Saharan Africa).

[0488] 21. Patients with a history of confirmed progressive multifocal leukoencephalopathy.

[0489] 22. Pregnancy or breast-feeding.

[0490] 23. Life expectancy <12 months.

[0491] Study Design and Treatment

[0492] GOYA was a multicenter, open-label, randomized, phase 3 study. Patients were randomized (1:1 ratio) to receive eight 21-day cycles of G (1000 mg intravenous [IV] on days 1, 8, and 15 of cycle 1 and on day 1 of cycles 2-8) or R (375 mg / m on day 1 of cycles 1-8). 2 IV), plus 6 or 8 cycles of CHOP at the following doses: cyclophosphamide 750 mg / m 2 IV (Day 1); doxorubicin 50 mg / m 2 IV (Day 1); vincristine 1.4 mg / m 2IV (day 1, maximum 2.0 mg); and prednisone 100 mg / day orally (days 1-5). The number of CHOP cycles in both arms was agreed upon in advance by each study site; if only 6 CHOP cycles were administered, the antibody was administered as monotherapy during cycles 7-8. Preplanned radiation therapy to the initial site of bulky or extranodal disease was permitted within 8 weeks of day 1 of the last antibody cycle and after completion of the end-of-treatment assessment. Details of prior medications, permitted concomitant therapy, and permitted reasons for dose delays / reductions are given in the Supplementary Appendix. Randomization was by interactive voice-response system, stratified according to the number of planned chemotherapy cycles (6 / 8 cycles of CHOP), IPI score, and geographic region (Western Europe, Eastern Europe, Central and South America, North America, and Asia and Other).

[0493] GOYA was conducted in accordance with the European Clinical Trials Guidelines (for European centers) and the International Conference on Harmonization guidelines on Good Clinical Practice. The protocol was approved by the ethics committees of participating centers and registered with ClinicalTrials.gov (NCT01287741).

[0494] Prior medications, dose changes, and permitted therapies

[0495] All patients received premedication with oral acetaminophen and antihistamines prior to administration of obinutuzumab (G) or rituximab (R). It was recommended that administration of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) be initiated at least 30 minutes after administration of G or R. In each cycle of G or R given with CHOP, the Day 1 dose of oral prednisone was administered prior to antibody infusion. Additional glucocorticoid therapy may also be administered to patients considered at high risk for infusion-related reactions. Tumor lysis prevention (adequate hydration and allopurinol) is recommended for patients with high tumor burden and considered at risk for tumor lysis. Treatment doses were delayed for up to 2 weeks in the case of grade 3 or 4 hematologic toxicity or grade 2-4 non-hematologic toxicity; patients were withdrawn from study treatment if toxicity did not resolve. Dose reductions were allowed for chemotherapy, but not for G or R.

[0496] Prophylaxis with G-CSF is recommended for patients aged ≥60 years and / or with comorbidities and for all patients in the G-CHOP arm during Cycle 1. G-CSF is also allowed for the treatment of neutropenia. The use of chemotherapy (except CHOP), immunotherapy, hormonal therapy (except birth control pills, hormone replacement therapy, or megestrol acetate), and any therapy intended to treat lymphoma are prohibited (for radiation therapy, see text).

[0497] Study End Points and Assessments

[0498] The primary study endpoint was investigator-assessed progression-free survival (PFS), defined as the time from the date of randomization to the first occurrence of disease progression, relapse, or death from any cause. To exclude bias and support the primary analysis, PFS was also assessed by an independent review committee (IRC). Secondary endpoints included overall survival (OS), event-free survival (EFS), CR rate, overall response rate (ORR, including complete response (CR) and partial response (PR)), disease-free survival (DFS), duration of response, time to next antilymphoma treatment (TTNT), and safety. PFS was also analyzed in DLBCL cell of origin (COO) subgroups (germinal center B-cell-like [GCB], activated B-cell-like [ABC], and unclassified) (exploratory analysis). COO classification was based on gene expression profiling using the NanoString Research Use Only Lymphoma Subtype Test (NanoString Technologies, Inc., Seattle, WA, USA).

[0499] Tumor response and progression were assessed by investigators using regular clinical and laboratory examinations and CT scans according to the revised response criteria for malignant lymphoma (Cheson, J. Clin. Oncol. 25, 2007, 579-586). 18 For those patients who had a F-fluorodeoxyglucose positron emission tomography (FDG-PET) scan (mandatory at sites with PET scanners), an additional response assessment was performed, incorporating the FDG-PET results. The primary endpoint analysis was based on the assessment of all patients using conventional CT scans. Response was assessed 4-8 weeks (CT) or 6-8 weeks (FDG-PET) after the last study treatment, or earlier in the case of premature discontinuation.

[0500] Safety was assessed by monitoring and recording all adverse events (AEs) and serious AEs (SAEs), including abnormalities identified from laboratory assessments, vital sign measurements, and physical examinations. AEs were graded using the National Cancer Institute Common Terminology Criteria for Adverse Events v4.0. Laboratory safety assessments included routine testing of hematology and blood chemistry, and immunology parameters. An independent data monitoring committee (IDMC) performed regular safety reviews.

[0501] Statistical analysis

[0502] The sample size was calculated to allow detection of a 25% reduction in the risk of disease progression, relapse, or death with G-CHOP versus R-CHOP (i.e., a PFS hazard ratio [HR] of 0.75 with G-CHOP versus R-CHOP), with a two-sided alpha level of 0.05 and 80% power. To achieve this and allow for a 5% annual withdrawal rate, 405 PFS events were required for the primary analysis, requiring enrollment of 1,400 patients over 3 years.

[0503] Efficacy assessments were performed on the intention-to-treat (ITT) population, which included all randomized patients. The safety analysis population included all patients who received any study drug (antibody or CHOP). Treatment comparisons of PFS were performed using a two-sided log-rank test at a 0.05 level. The Kaplan-Meier method was used to estimate the distribution of PFS for each treatment arm. Estimates of treatment effects were expressed as HRs, including 95% confidence intervals (CIs), using stratified Cox proportional-hazards analysis.

[0504] The IDMC evaluated efficacy and safety in three formal interim analyses; two for futility and one for efficacy. Preplanned subgroup analyses evaluated the impact of selected baseline patient characteristics (including COO subtype) on PFS.

[0505] result

[0506] Overview

[0507] After a median observation of 29 months, the number of investigator-assessed PFS events was similar for G (201, 28.5%) and R (215, 30.2%); the stratified hazard ratio was 0.92 (95% confidence interval, 0.76 to 1.11; P = .39); and the 3-year PFS rates were 70% and 67%, respectively. Secondary endpoints of PFS by independent review, time to other event endpoints, and tumor response rate were similar between the two arms. In exploratory subgroup analyses, patients with the germinal center B-cell-like subtype had better PFS than those with the activated B-cell-like subtype, regardless of treatment. The frequencies of grade 3-5 adverse events (AEs; 73.7% vs 64.7%) and serious AEs (42.6% vs 37.6%) were higher with G-CHOP. The frequency of fatal AEs was 5.8% with G-CHOP and 4.3% with R-CHOP. The most common AEs were neutropenia (G-CHOP, 48.3%; R-CHOP, 40.7%), infusion-related reactions (36.1%; 23.5%), nausea (29.4%; 28.3%), and constipation (23.4%; 24.5%).

[0508] Patient Characteristics and Treatment

[0509] Patients were enrolled at 207 centers in 29 countries. A total of 1,418 patients were randomized to receive G-CHOP (n=706) or R-CHOP (n=712) between July 2011 and June 2014, and 1,188 patients (G-CHOP, 587; R-CHOP, 601) completed the planned treatment ( Figure 1 AEs were the main reason for discontinuation of study (antibody) treatment in both arms and were reported more frequently in the G-CHOP arm. Progressive disease led to discontinuation of study (antibody) treatment approximately twice as often in the R-CHOP arm as in the G-CHOP arm.

[0510] Demographics and baseline disease characteristics were well balanced between the two arms (Table 1). COO subgroup information was available for 933 patients; the distribution of subtypes was well balanced and there were no clinically relevant differences between the two arms within COO subtypes. The reasons for missing COO information were: restricted Chinese sample export licenses precluding biomarker assessment (n = 252), CD20-positive DLBCL without central laboratory confirmation (n = 102; note: these patients were balanced between the two treatment arms: G-CHOP, n = 53; R-CHOP, n = 49), and missing / insufficient tissue (n = 131).

[0511] Median duration of exposure was 25.3 (range, 1-32) weeks for G and 25.3 (0-32) weeks for R. Dose intensity for G and R exceeded 90% for 95.3% and 99.1% of patients, respectively. Most patients (>88%) in both arms received more than 90% of the planned doses of each CHOP component. Antibody dose delays were more common in the G-CHOP arm: at least one delay of ≤7 days (G-CHOP, 34.9%; R-CHOP, 30.0%) and >7 days (G-CHOP, 13.1%; R-CHOP, 9.1%) (Table 5). One hundred and three patients before disease progression (G-CHOP, 49; R-CHOP, 54) (including RT in 23 patients with evidence of residual disease after completion of study treatment (G-CHOP, 9; R-CHOP, 14)) and 227 patients after disease progression (G-CHOP, 102; R-CHOP, 125) received new (unplanned) antilymphoma therapy.

[0512] effect

[0513] As of April 30, 2016, and after a median observation of 29 months, the number of investigator-assessed PFS events in the ITT population was similar for G-CHOP (201, 28.5%) and R-CHOP (215, 30.2%), with a stratified HR of 0.92 (95% CI, 0.76 to 1.11; P = .3868). The estimated 3-year PFS rates were 69.6% and 66.9%, respectively ( Figure 2 A; Table 2).

[0514] Secondary endpoints were consistent with the primary endpoint, with no clinically meaningful differences in IRC-assessed PFS or any other time to event endpoints (OS, EFS, DFS, and TTNT) between the two treatment arms ( Figure 2 B,Table 2, Fig.13 ).

[0515] Subgroup and Exploratory Analyses

[0516] The efficacy of G-CHOP compared with R-CHOP (unstratified HR for investigator-assessed PFS) was generally similar across selected patient subgroups, including patients who received 6 versus 8 cycles of CHOP ( Figure 3 ).

[0517] Kaplan-Meier analysis of PFS in patients with different COO subtypes (regardless of study treatment) suggested that the GCB subtype was associated with better outcomes than the ABC or unclassified subtypes. The HRs for PFS were 1.71 (95% CI, 1.31 to 2.23) for the ABC-GCB comparison, 1.57 (95% CI, 1.14 to 2.15) for the unclassified-GCB comparison, and 1.08 (95% CI, 0.77 to 1.52) for the ABC-unclassified comparison ( Figure 2C); 3-year PFS rates were 75%, 59%, and 63% for GCB, ABC, and unclassified subtypes, respectively. In an exploratory analysis of investigator-assessed PFS, the stratified HR for G-CHOP versus R-CHOP was 0.82 (95% CI, 0.64 to 1.04) for 933 patients with available COO data, suggesting a potential selection bias compared with the ITT population. The stratified HR for the 540 patients in the GCB COO subgroup was 0.72 (95% CI, 0.51 to 1.03; 3-year PFS, 79% [G-CHOP] vs 71% [R-CHOP]); no clinically meaningful differences in PFS between the 2 treatment groups were seen in the 243 patients with the ABC subtype (HR, 0.86 [95% CI, 0.57 to 1.29]; 3-year PFS, 61% vs 58%]) or in the 150 patients with the unclassified COO subtype (HR, 1.02 [95% CI, 0.60 to 1.75]; 3-year PFS, 62% vs 64%). Fig.14 ).

[0518] Security

[0519] In the safety population, the proportion of patients who experienced at least 1 AE of any grade was similar in the G-CHOP and R-CHOP arms (97.0% [683 / 704] and 93.5% [657 / 703], respectively) (Table 3). The most common AEs in both arms were neutropenia (G-CHOP, 48.3%; R-CHOP, 40.7%), infusion-related reactions (IRRs; 36.1%; 23.5%), nausea (29.4%; 28.3%), and constipation (23.4%; 24.5%) (Table 6). Grade 3-5 AEs were more common in the G-CHOP arm (73.7% [519 / 704] vs 64.7% [455 / 703]), as were SAEs (42.6% [300 / 704] vs 37.6% [264 / 703]). The most common grade 3-5 AEs in both arms were neutropenia (G-CHOP, 46.2%; R-CHOP, 38.1%), infections (19.2%; 15.5%), febrile neutropenia (17.5%; 15.2%), and leukopenia (13.6%; 10.1%) ( Table 3 ).

[0520] Analysis of AEs of special interest showed that infections of any grade, neutropenia, IRR, cardiac events, thrombocytopenia, and bleeding events (as well as grade 3-5 AEs and SAEs) were more common in G-CHOP than in R-CHOP (Table 7). Of note, the rate of hepatitis B reactivation was higher in G-CHOP (2.3%) than in R-CHOP (0.9%), with most events being grade 1 or 2, and grade 3 or 4 events being well balanced between the two arms (G-CHOP 0.3% vs R-CHOP 0.3%). All other AE groups of special interest, i.e., opportunistic infections, tumor lysis syndrome, secondary malignancies, and gastrointestinal perforations (excluding abscesses), occurred with similar frequencies in both arms (Table 7).

[0521] A similar proportion of patients in each arm received at least 1 dose of G-CSF during the study period (G-CHOP, 611 [86.5%]; R-CHOP, 586 [82.3%]).

[0522] A higher proportion of patients in the G-CHOP arm discontinued ≥1 component of study treatment due to AEs than in the R-CHOP arm (84 [11.9%] vs 60 [8.5%]). 71 patients (G-CHOP, 5.8% [41 / 704]; R-CHOP, 4.3% [30 / 703]) experienced fatal AEs, which are detailed in Table 3.

[0523] discuss

[0524] In the current study of patients with previously untreated DLBCL, G-CHOP and R-CHOP demonstrated similar efficacy for all time-to-event endpoints, and the primary study endpoint of investigator-assessed PFS was not reached. The lack of superiority of G-CHOP over R-CHOP in a population with aggressive NHL contrasts with the results of studies evaluating G in CLL and FL. In the GALLIUM study, G-based induction and maintenance therapy significantly improved investigator-assessed PFS relative to R-based therapy in 1,202 previously untreated FL patients (Marcus, supra). G also prolonged PFS relative to R in untreated CLL patients (n=663) when both were combined with chlorambucil in the Phase 3 CLL11 study (Goede, supra).

[0525] Given the advantages of G-based therapies in FL and CLL patients, the lack of benefit of G-CHOP in GOYA in DLBCL patients is unexpected. It may simply stem from the differences in the biological and clinical profiles between indolent lymphoproliferative diseases, such as FL and CLL, and aggressive diseases, such as DLBCL (Lenz, N. Engl. J. Med. 362, 2010, 1417-1429; Lim, supra). Indeed, obinutuzumab may be more beneficial in lymphomas that are less aggressive or derived from germinal centers like FL. The trend in GOYA pointing to a benefit of G-CHOP over R-CHOP for the GCB subtype, which is derived from germinal centers and is known to be more similar to FL than other DLBCL subtypes (Morin, Nature 476, 2011, 298-303; Shaffer, Nat. Rev. Immunol. 2, 2002, 920-932), with a more favorable prognosis and a different immune microenvironment than the ABC and unclassified subtypes, seems to support this finding. The different modes of action of obinutuzumab and rituximab may also play a role in the differential benefit of these agents in FL and DLBCL, however, no data are available to support this claim. Ongoing analysis of GOYA biomarker data will provide further insight into these differences. Of note, dose interruptions and dose skipping in cycle 1 were more frequent for G-CHOP, reflecting a higher rate of AEs (IRRs and cytopenias); this may contribute to the lack of efficacy benefit compared with R-CHOP.

[0526] Since the dramatic improvement in outcomes after rituximab was first added to CHOP (Coiffier, supra), no major advances have been made in the management of patients with DLBCL. Randomized trials have failed to show a benefit from shortening the intercycle interval (Cunningham, Lancet 381, 2013, 1817-1826), or from combining high-dose chemotherapy and autologous stem cell transplantation (Stiff, N. Engl. J. Med. 369, 2013, 1681-1690; Schmitz, Lancet Oncol. 13, 2012, 1250-1259); adding bortezomib to R-CHOP in a randomized trial of patients with non-GCB DLBCL also failed to improve outcomes (Leonard, Blood 126, 2015, 811), and maintenance with lenalidomide did not improve OS (Thieblemont, Blood 128, 2016, 471). Given the aggressive behavior of DLBCL, replacing rituximab with new anti-CD20 antibodies with different modes of action may not be sufficient to overcome refractory to chemotherapy. Combinations of drug-conjugated antibodies or anti-BCL2 agents with R-CHOP may hold greater promise, as shown by preliminary results from recent phase 1 / 2 studies (Zelenetz, Blood 128, 2016, 3032; Tilly, Blood 128, 2016, 1853).

[0527] Using gene expression profiling to determine COO status has identified biologically distinct DLBCL subtypes, including GCB and ABC origin subtypes (Lenz, Proc Natl Acad Sci USA 105, 2008, 13520-13525; Scott, J. Clin. Oncol. 33, 2015, 2848-2856). These molecular subtypes are of great significance for carcinogenesis and treatment outcomes, as reflected by their inclusion in the current World Health Organization DLBCL classification (Swerdlow, Blood 127, 2016, 2375-2390). Patients with the GCB subtype typically have a more favorable outcome, while the ABC subtype is associated with inferior outcomes after chemotherapy or immunochemotherapy (including R-CHOP), and may represent a subset of poor-risk patients with unmet medical needs (as shown in retrospective studies) (Lenz, Proc. Natl. Acad. Sci. USA, supra; Scott, 2015, supra). GOYA is the largest prospective study evaluating the impact of COO on clinical outcomes. PFS comparisons between COO subtypes are consistent with better outcomes in GCB DLBCL, with the HR indicating a 70% increased risk of disease progression in patients with ABC relative to the GCB subtype. Outcomes in the unclassified subgroup were similar to those in the ABC subgroup, in contrast to reports in some prior studies (Scott, 2015, supra). Interestingly, COO classification was not associated with ORR and / or PFS primary assessments in other prospectively defined studies, such as REMoDL-B (Davies, Blood 126, 2015, 812) or PYRAMID (Leonard, supra), although these studies used different COO assays. Specific treatments for the COO subtype of DLBCL may provide an alternative strategy to improve outcomes. For example, selective targeting of the B cell receptor or NF-κB pathway may prove beneficial in DLBCL subtypes (ABC or non-GCB), as suggested by the results of phase 2 studies evaluating lenalidomide or ibrutinib and R-CHOP (Nowakowski, J. Clin. Oncol. 33, 2015, 251-257; Vitolo, Lancet Oncol. 15 2014, 730-737; Younes, Lancet Oncol. 15, 2014, 1019-1026). These strategies are currently being evaluated in randomized phase 3 studies.

[0528] The profile and nature of AEs reported in G-CHOP-treated patients were as expected, with no new safety signals. The incidence of grade 3-5 AEs, SAEs, and treatment discontinuation due to AEs was slightly higher in the G-CHOP group than in the R-CHOP group, consistent with reports from other studies. These deviations may be due to the different structural and biological properties of G and R.

[0529] In conclusion, the current study demonstrated that G-CHOP does not improve PFS compared with R-CHOP, which remains the standard of care for these patients, in a large population of patients with previously untreated DLBCL. No new safety signals were identified.

[0530] Example 3 - Superiority of Obinutuzumab over Rituximab in a Novel DLBCL Subgroup (Defined by Predictive Biomarkers) (Description and Results of Exploratory Analysis of the GOYA Phase 3 Study)

[0531] Example 3.1 - Gazyva in biomarker-defined DLBCL subsets TM -CHOP is superior to rituximab -CHOP - Results from Roche GOYA (BO21005) Phase 3 clinical trial

[0532] Overview

[0533] Rituximab (R) plus CHOP chemotherapy is the standard of care for previously untreated diffuse large B-cell lymphoma (DLBCL). Obituzumab (G) is a glycoengineered type II anti-CD20 monoclonal antibody. GOYA is a randomized phase 3 study comparing G-CHOP and R-CHOP in previously untreated advanced DLBCL.

[0534] The GOYA trial in 1L DLBCL did not meet its primary endpoint: stratified HR, PFS: 0.92 (95% CI 0.76-1.12), but the GALLIUM trial demonstrated superiority of Gazyva over rituximab in 1L FL (HR 0.66, 95% CI 0.51-0.85; currently under submission).

[0535] In an exploratory analysis of GOYA, benefit of Gazyva was seen in a subset of patients with GCB DLBCL and / or also in patients with mutations in CD58 and / or low expression of CD58. TM Superiority over rituximab: This is the first time that a Gazyva benefit has been identified in a biomarker-defined subgroup of DLBCL.

[0536] The results suggest that one or more subsets of GCB DLBCL patients who respond to Gazyva can be identified in several ways, such as by measuring BCL2 translocations and BCL2 protein overexpression, and also by measuring gene expression profiling, such as the novel truncated Nanostring Cell of Origin (COO) assay using the Linear Predictor Score (LPS).

[0537] method

[0538] Classification into germinal center B cell (GCB), activated B cell (ABC), and cell of origin (COO) subgroups was based on gene expression profiling using the NanoString Research Use Only Lymphoma Subtype Test (NanoString Technologies, Inc., Seattle, WA, USA).

[0539] The linear predictor score (LPS) is a continuous variable (weighted average of gene expression of genes in the Nanostring lymphoma subtyping assay) with a range of -1138 to 4504 in GOYA. LPS is normally used to classify patients into COO subgroups GCB, ABC, and unclassified. The default COO algorithm uses a Bayesian approach with GCB / ABC classification based on a ≥90% cutoff of the likelihood of being GCB or ABC (unclassified as a buffer).

[0540] The first direct analysis of LPS with respect to clinical outcomes. LPS was treated as a continuous variable in an exploratory unspecified analysis of the GOYA trial to assess the treatment effect (efficacy of G-CHOP versus R-CHOP).

[0541] BCL-2 translocation was assessed using Bcl-2 dual-color separation (Vysis, Abbott Molecular), and Foundation Medicine next-generation sequencing assay (FoundationOne Heme). BCL-2 protein expression was assessed using Ventana investigative IHC assay (BCL2 antibody clone, 124). RNA Access Library Preparation Kit evaluates transcriptome-wide gene expression in the GOYA trial. CD58 mutations are identified using the FoundationOne heme gene panel.

[0542] result

[0543] Identification of biomarker-defined DLBCL subgroups that benefit from G (G-CHOP) over R (R-CHOP):

[0544] Patients with BCL2 translocation (see Figure 4 )

[0545] Patients with positive BCL2 protein expression (see Figure 5 )

[0546] Patients with BCL2 translocation who are positive for BCL2 protein expression (see Figure 6 )

[0547] Subsets of GCB DLBCL patients. These can be identified as follows:

[0548] o New subgroup classification of GCB patients into a new subgroup "Strong GCB" by a new cutoff of linear predictor scores (patients with LPS < cutoff) (see Figure 7)

[0549] o or GCB patients with high BCL2 gene expression

[0550] GCB patients with positive expression of BCL2 protein

[0551] o or GCB patients with BCL2 translocation

[0552] o or BCL2 protein positive GCB patients with BCL2 translocation (see Figure 8 )

[0553] In general, patients with CD58 mutations, and / or patients with low expression of CD58 (see Fig. 9 )

[0554] Example 3.2 - Superiority of obinutuzumab over rituximab in a novel molecular FL-like DLBCL subgroup - Results from the Phase 3 GOYA trial

[0555] method

[0556] GOYA was an open-label, randomized, phase 3 study comparing 1L G-CHOP with R-CHOP in 1418 patients with DLBCL. Biomarker testing was performed on formalin-fixed, paraffin-embedded tumor tissue collected before treatment and retrospectively tested in a central laboratory. COO was assessed in 933 patients using the NanoString Research Use Only LST (NanoString Technologies Inc., Seattle, WA, USA). Additional biomarker analyses for molecular characterization included the use of The Heme (FOH) group performed targeted DNA sequencing of 467 genes (n = 499 patients) and used RNA sequencing assessed transcriptome-wide gene expression in 552 patients. BCL2 translocations were identified using Vysis LSI dual-color break-apart FISH probes (n = 644 patients; FISH cutoff, 50%), and BCL2 expression was assessed using the Ventana investigative IHC assay (BCL2 antibody clone, 124); BCL2+IHC was defined as moderate / strong staining in ≥50% of tumor cells. Biomarker treatment effects were assessed using multivariate Cox regression and elastic net penalty regression (α = 0.5). In addition, bootstrap simulations were performed to identify the best LPS (NanoString LST) to reflect the robustness of the treatment effects observed in GOYA and the generalizability of the treatment effects to independent study populations. Multiple testing adjustments were made by assessing FDR using the Benjamini-Hochberg procedure (significance, <5% FDR). Gene set enrichment was performed using the gene set defined by MSigDB Hallmarks and a curated FL somatic mutation signature gene set based on a recently published review; pathway enrichment analysis was performed using the hypergeometric test. All patients consented for biomarker analysis.

[0557] result

[0558] Evaluation of LPS as a continuous variable identified a subgroup of GCB patients who benefited from G-CHOP. In particular, strong expression of the germinal center gene expression profile (by LPS) was associated with outcome benefit from G-CHOP treatment compared with R-CHOP treatment in patients in GOYA. Bootstrap simulations identified an optimal LPS cutoff (≤725) for predicting benefit from G-CHOP, with 25% (233 / 933) of GOYA patients having the lowest LPS score. These patients were specifically termed 'strong GCB' patients, and comprised 43% (233 / 540) of evaluable GCB patients in GOYA. Strong GCB patients treated with G-CHOP achieved significantly better clinical outcomes in terms of investigator-assessed progression-free (HR=0.33, p=0.0007), event-free (HR=0.47, p=0.003), and overall (HR=0.41, p=0.019) survival than patients treated with R-CHOP (see Fig.10,12; Table 4). In multivariate analysis, the observed benefit was independent of baseline demographics and disease characteristics. The safety profile of the two regimens was similar. Molecular characterization of strong-GCB patients was performed using FOH, TruSeq RNA, and BCL2 IHC / FISH GOYA data. In gene set analysis of FOH data, strong-GCB patients were significantly enriched for mutations characteristic of FL patients ('weak-GCB': FDR, 3.54e-9) compared with other GCB patients. In particular, BCL2 translocations and mutations in several m7-FLIPI genes were highly enriched in strong-GCB patients compared with other DLBCL subgroups ( Fig.11 ). Central pathology review did not have strong evidence of transformed indolent NHL in the GCB subset.

[0559] in conclusion

[0560] By analyzing data from the GOYA trial, a new clinically and molecularly distinct subgroup of GCB DLBCL was identified, comprising at least about 25% of all DLBCL patients, termed 'strong GCB'. This unique subgroup was identifiable by gene expression profiling (using, e.g., an LPS cutoff of ≤725 for the Nanostring LST assay) and is characterized by mutations that are also commonly identified in FL patients (see Morin, supra). G-CHOP treatment conferred substantial clinical benefit over R-CHOP in this new subset of 1L DLBCL patients.

[0561] The present invention refers to the following nucleotide and amino acid sequences:

[0562] SEQ ID NO:1

[0563] Amino acid sequence of the heavy chain variable region of obinutuzumab

[0564] Mouse-human chimeric polypeptide

[0565] Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

[0566] Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser

[0567] Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

[0568] Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

[0569] Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

[0570] Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

[0571] Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

[0572] Thr Leu Val Thr Val Ser Ser

[0573] SEQ ID NO:2

[0574] Amino acid sequence of the KV1 light chain variable region of obinutuzumab

[0575] Mouse-human chimeric polypeptide

[0576] Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

[0577] Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

[0578] Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

[0579] Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val Ser Gly Val Pro

[0580] Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

[0581] Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn

[0582] Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

[0583] Arg Thr Val

[0584] SEQ ID NO:3

[0585] Nucleic acid sequence encoding the heavy chain variable region of Obinutuzumab

[0586] Mouse-human chimeric DNA.

[0587] caggtgcaat tggtgcagtc tggcgctgaa gttaagaagc ctgggagttc agtgaaggtc 60

[0588] tcctgcaagg cttccggata cgccttcagc tattcttgga tcaattgggt gcggcaggcg 120

[0589] cctggacaag ggctcgagtg gatgggacgg atctttcccg gcgatgggga tactgactac 180

[0590] aatgggaaat tcaagggcag agtcacaatt accgccgaca aatccactag cacagcctat 240

[0591] atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc aagaaatgtc 300

[0592] tttgatggtt actggcttgt ttactggggc cagggaaccc tggtcaccgt ctcctca 357

[0593] SEQ ID NO:4

[0594] Nucleic acid sequence encoding the KV1 light chain variable region of Obinutuzumab

[0595] Mouse-human chimeric DNA

[0596] gatatcgtga tgacccagac tccactctcc ctgcccgtca cccctggaga gcccgccagc 60

[0597] attagctgca ggtctagcaa gagcctcttg cacagcaatg gcatcactta tttgtattgg 120

[0598] tacctgcaaa agccagggca gtctccacag ctcctgattt atcaaatgtc caaccttgtc 180

[0599] tctggcgtcc ctgaccggtt ctccggatcc gggtcaggca ctgatttcac actgaaaatc 240

[0600] agcagggtgg aggctgagga tgttggagtt tattactgcg ctcagaatct agaacttcct 300

[0601] tacaccttcg gcggagggac caaggtggag atcaaacgta cggtg 345

[0602] SEQ ID NO:5

[0603] Amino acid sequence of the heavy chain of obinutuzumab

[0604] The variable region comprises amino acid positions 1 to 119.

[0605]

[0606] SEQ ID NO:6

[0607] The amino acid sequence of the KV1 light chain variable region of obinutuzumab comprises amino acid positions 1 to 115

[0608]

[0609] SEQ ID NO:7

[0610] Nucleic acid sequence encoding the heavy chain variable region of Obinutuzumab (B-HH6)

[0611]

[0612] SEQ ID NO:8

[0613] Nucleic acid sequence encoding the KV1 light chain variable region of Obinutuzumab

[0614] SEQ ID NO:9

[0615] Amino acid sequence of the heavy chain of rituximab

[0616] Rituximab heavy chain chimerism

[0617] QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSSLTSEDSAVYYCARSTYYGGDWYFNVWG AGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0618] SEQ ID NO:10

[0619] Amino acid sequence of the light chain of rituximab

[0620] Rituximab light chain chimerism

[0621] QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0622] SEQ ID NO:11

[0623] Example of the nucleotide sequence encoding human (Homo sapiens) CD58 (variant 1)

[0624] 1 gggccgccgg ctgccagccc agggcggggc ggagccctac ttctggccga ccgcgtaggc

[0625] 61 ggtgcttgaa cttagggctg cttgtggctg ggcactcgcg cagaggccgg cccgacgagc

[0626] 121 catggttgct gggagcgacg cggggcgggc cctgggggtc ctcagcgtgg tctgcctgct

[0627] 181 gcactgcttt ggtttcatca gctgtttttc ccaacaaata tatggtgttg tgtatgggaa

[0628] 241 tgtaactttc catgtaccaa gcaatgtgcc tttaaaagag gtcctatgga aaaaacaaaa

[0629] 301 ggataaagtt gcagaactgg aaaattctga attcagagct ttctcatctt ttaaaaatag

[0630] 361 ggtttattta gacactgtgt caggtagcct cactatctac aacttaacat catcagatga

[0631] 421 agatgagtat gaaatggaat cgccaaatat tactgatacc atgaagttct ttctttatgt

[0632] 481 gcttgagtct cttccatctc ccacactaac ttgtgcattg actaatggaa gcattgaagt

[0633] 541 ccaatgcatg ataccagagc attacaacag ccatcgagga cttataatgt actcatggga

[0634] 601 ttgtcctatg gagcaatgta aacgtaactc aaccagtata tattttaaga tggaaaatga

[0635] 661 tcttccacaa aaaatacagt gtactcttag caatccatta tttaatacaa catcatcaat

[0636] 721 cattttgaca acctgtatcc caagcagcgg tcattcaaga cacagatatg cacttatacc

[0637] 781 cataccatta gcagtaatta caacatgtat tgtgctgtat atgaatggta ttctgaaatg

[0638] 841 tgacagaaaa ccagacagaa ccaactccaa ttgattggta acagaagatg aagacaacag

[0639] 901 cataactaaa ttattttaaa aactaaaaag ccatctgatt tctcatttga gtattacaat

[0640] 961 ttttgaacaa ctgttggaaa tgtaacttga agcagctgct ttaagaagaa atacccacta

[0641] 1021 acaaagaaca agcattagtt ttggctgtca tcaacttatt atatgactaggtgcttgctt

[0642] 1081 tttttgtcag taaattgttt ttactgatga tgtagatact tttgtaaataaatgtaaata

[0643] 1141 tgtacacaag tga

[0644] SEQ ID NO:12:

[0645] Example of the amino acid sequence of human (Homo sapiens) CD58 (isoform 1)

[0646] 1 mvagsdagra lgvlsvvcll hcfgfiscfs qqiygvvygn vtfhvpsnvp lkevlwkkqk

[0647] 61 dkvaelense frafssfknr vyldtvsgsl tiynltssde deyemespni tdtmkfflyv

[0648] 121 leslpsptlt caltngsiev qcmipehyns hrglimyswd cpmeqckrns tsiyfkmend

[0649] 181 lpqkiqctls nplfnttssi ilttcipssg hsrhryalip iplavittci vlymngilkc

[0650] 241 drkpdrtnsn

[0651] SEQ ID NO:13

[0652] Example of a nucleotide sequence encoding human (Homo sapiens) BCL2 (variant α)

[0653] 1 tttctgtgaa gcagaagtct gggaatcgat ctggaaatcc tcctaatttt tactccctct

[0654] 61 ccccgcgact cctgatcat tgggagtttt caatcagct atactggag agtgctgaag

[0655] 121 attgatggga tcgttgcctt atgcattgt ttggtttta caaaaggaa acttgacaga

[0656] 181 ggatcatgct gtacttaaaaatacacat cacagaggaa gtagactgat attackacata

[0657] 241 cttactaata ataacgtgcc tcatgaata aagatccgaa aggaattgga aaaaattt

[0658] 301 cctgcatctc atgccaaggg ggaacacca gatcaagtg ttccgcgtga ttgagacac

[0659] 361 cccctcgtcc aagaatgcaa agcacatcca ataaatagc tggattataa ctcctctctct

[0660] 421 ttctctgggg gccgtggggt gggagctggg gcgagaggtg ccgttggcccc ccgttgctt

[0661] 481 tcctctggga aggatggcgc acgctgggag aacaggtac gataacgggg agatagtgat

[0662] 541 gaagtacatc cattaagc tgtcgcagag gggctacgag tgggatgcgg gagatgtgg

[0663] 601 cgccgcgccc ccggggccg cccccgcacc gggcatctc tcccccagc ccggcacac

[0664] 661 gccccatcca gccgcatccc gggacccggt cgccaggacc tcgccgctgc agaccccggc

[0665] 721 tgcccccggc gccgccgcgg ggcctgcgct cagcccggtg ccacctgtgg tccacctgac

[0666] 781 cctccgccag gccggcgacg acttctcccg ccgctaccgc cgcgacttcg ccgagatgtc

[0667] 841 cagccagctg cacctgacgc ccttcaccgc gcggggacgc tttgccacgg tggtggagga

[0668] 901 gctcttcagg gacggggtga actgggggag gattgtggcc ttctttgagt tcggtggggt

[0669] 961 catgtgtgtg gagagcgtca accgggagat gtcgcccctg gtggacaaca tcgccctgtg

[0670] 1021 gatgactgag tacctgaacc ggcacctgca cacctggatc caggataacggaggctggga

[0671] 1081 tgcctttgtg gaactgtacg gccccagcat gcggcctctg tttgatttctcctggctgtc

[0672] 1141 tctgaagact ctgctcagtt tggccctggt gggagcttgc atcaccctgggtgcctatct

[0673] 1201 gggccacaag tgaagtcaac atgcctgccc caaacaaata tgcaaaaggttcactaaagc

[0674] 1261 agtagaata atgcattg tcagtgt accatgaac aaagctgcaggctgtttaag

[0675] 1321 aaaaaataac

[0676] 1381 aacagcttc aggcaaacg tcgaatcagc tattactgc aaagggaaatcatttat

[0677] 1441 ttttacatt attagaaaa aagatttat tttattaga cagtcccatcaaaactccctg

[0678] 1501 tcttggaa tcgaccact aattgccag caccgctcg tgtgctccacctgtgtt

[0679] 1561 ctgtgctgt aacatagat tcgctttcca tgttgttgc cggatcaccatctgaagagc

[0680] 1621 agacggatgg aaaaaggacc tgatcattgg ggagctggc ttctggctgctgaggctg

[0681] 1681 gggagaaggt gttcattcac ttgcattct tgccctggg ggctgtgatattaacagagg

[0682] 1741 gaggttcct gtgggggaa gtccatgcct ccctggctg aagagagactctttgcata

[0683] 1801 tgactcacat gatgcatacc tggtgggagg aaagagttg ggaacttcagatgacctag

[0684] 1861 tacccactga gatttccacg ccgaaggaca gcgatgggaa aaatgcccttaaatcatagg

[0685] 1921 aaagtatttt tttaagctac caattgtgcc gagaaaagca ttttagcaatttatacaata

[0686] 1981 tcatccagta ccttaagccc tgattgtgta tattcatata ttttggatacgcacccccca

[0687] 2041 actcccaata ctggctctgt ctgagtaaga aacagaatcc tctggaacttgaggaagtga

[0688] 2101 acatttcggt gacttccgca tcaggaaggc tagagttacc cagagcatcaggccgccaca

[0689] 2161 agtgcctgct tttaggagac cgaagtccgc agaacctgcc tgtgtcccagcttggaggcc

[0690] 2221 tggtcctgga actgagccgg ggccctcact ggcctcctcc agggatgatcaacagggcag

[0691] 2281 tgtggtctcc gaatgtctgg aagctgatgg agctcagaat tccactgtcaagaaagagca

[0692] 2341 gtagaggggt gtggctgggc ctgtcaccct ggggccctcc aggtaggcccgttttcacgt

[0693] 2401 ggagcatggg agccacgacc cttcttaaga catgtatcac tgtagagggaaggaacagag

[0694] 2461 gccctgggcc cttcctatca gaaggacatg gtgaaggctg ggaacgtgaggaggcaat

[0695] 2521 ggccacggcc cattttggct gtagcacatg gcacgttggc tgtgtggccttggccacct

[0696] 2581 gtgagtttta agcaggctt taatgactt tggaggtt cacaatcctaaaagaagca

[0697] 2641 ttgaagtgag gtgtcatgga ttaattgacc cctgtctatg gatttacatgtaaaacatta

[0698] 2701 tcttgtcact gtagttggt tttattgaa aacctgacaa aaaaaagttccaggtgtgg

[0699] 2761 atatggggg t atctgtac atcctggggc attaaaaaaa aaatcaatggtgggaacta

[0700] 2821 taagaagta aaagag tgacatctc agcaataa ctaggaatttttttcttt

[0701] 2881 ccagtttaga atcagccttg aacattgat ggaataactc tgtggcattattgcattata

[0702] 2941 taccattat ctgtattaac tttggaatgt actctgttca atgtttaatgctgtggttga

[0703] 3001 tattcgaaa gctgctttaaaaaatacat gcatctcagc gttttttgtttttaattgt

[0704] 3061 atttagttat ggcctataca ctatttgtga gcaaaggtga tcgttttctgtttgagattt

[0705] 3121 ttatctcttg attcttcaaa agcattctga gaaggtgaga taagccctgagtctcagcta

[0706] 3181 cctaagaaaa acctggatgt cactggccac tgaggagctt tgtttcaaccaagtcatgtg

[0707] 3241 catttccacg tcaacagaat tgtttattgt gacagttata tctgttgtccctttgacctt

[0708] 3301 gtttcttgaa ggtttcctcg tccctgggca attccgcatt taattcatggtattcaggat

[0709] 3361 tacatgcatg tttggttaaa cccatgagat tcattcagtt aaaaatccagatggcaaatg

[0710] 3421 accagcagat tcaaatctat ggtggtttga cctttagaga gttgctttacgtggcctgtt

[0711] 3481 tcaacacaga cccacccaga gccctcctgc cctccttccg cgggggctttctcatggctg

[0712] 3541 tccttcaggg tcttcctgaa atgcagtggt gcttacgctc caccaagaaagcaggaaacc

[0713] 3601 tgtggtatga agccagacct ccccggcggg cctcagggaa cagaatgatcagacctttga

[0714] 3661 atgattctaa ttttaagca aaatattatt tttgaaagg tttacattgtcaaagtgatg

[0715] 3721 aatatggaat atccaatcct gtgctgctat cctgccaaaa tcattttaatggagtcagtt

[0716] 3781 tgcagtatgc tccacgtggt aagatcctcc aagctgcttt agaagtaacaatgaagaacg

[0717] 3841 tggacgtttt tatataaag cctgttttgt cttttgttgt tgttcaaacgggattcacag

[0718] 3901 agtatttgaa aaatgtatat atattaagag gtcacggggg ctaattgctggctggctgcc

[0719] 3961 ttttgctgtg gggttttgtt acctggtttt aataacagta aatgtgcccagcctcttggc

[0720] 4021 cccagaactg tacagtattg tggctgcact tgctctaaga gtagttgatgttgcatttc

[0721] 4081 cttattgtta aaaacatgtt agaagcaatg aatgtatata aaagcctcaactagtcattt

[0722] 4141 ttttctcctc ttcttttttt tcattatatc taattatttt gcagttgggcaacagagaac

[0723] 4201 catccctatt ttgtattgaa gagggattca catctgcatc ttaactgctctttatgaatg

[0724] 4261 aaaaaacagt cctctgtatg tactcctctt tacactggcc agggtcagagttaaatagag

[0725] 4321 tatatgcact ttccaaattg gggacaaggg ctctaaaaaa agccccaaaaggagaagaac

[0726] 4381 atctgagaac ctcctcggcc ctcccagtcc ctcgctgcac aaatactccgcaagagaggc

[0727] 4441 cagaatgaca gctgacaggg tctatggcca tcgggtcgtc tccgaagatttggcaggggc

[0728] 4501 agaaaactct ggcaggctta agatttggaa taaagtcaca gaattaaggaagcacctcaa

[0729] 4561 tttagttcaa acaagacgcc aacattctct ccacagctca cttacctctctgtgttcaga

[0730] 4621 tgtggccttc catttatatg tgatctttgt tttattagta aatgcttatcatctaaagat

[0731] 4681 gtagctctgg cccagtggga aaaattagga agtgattata aatcgagaggagttataata

[0732] 4741 atcaagatta aatgtaaata atcagggcaa tcccaacaca tgtctagctttcacctccag

[0733] 4801 gatctattga gtgaacagaa ttgcaaatag tctctatttg taattgaacttatcctaaaa

[0734] 4861 caatagttt aaatgtga acttaaactc taattattc caacgtactttttaggcag

[0735] 4921 tggctgtttt tagacttct tatcacttat agttagtaat gtacacctactctatcagag

[0736] 4981 aaaaacagga aaggctcgaa atacaagcca ttctaggaa attaggtcagttgaat

[0737] 5041 tctattctga tcttattctg tgtgtctt tgcagcccag acaaatgtggttacacactt

[0738] 5101 tttagaat acaatctac attgtcaagc tttagaggt tccaatcagatctttattgt

[0739] 5161 tattcaattt ggatctttca gggatttt ttttaatta tattgggacaaggacattt

[0740] 5221 gttggagggg tgggagggag gagaattttt taatgtaa acatcccaagttgattca

[0741] 5281 gggagttgga agttttcaga ataccagaa ctaagggtat gaaggacctgtattggggtc

[0742] 5341 gatgtgatgc ctctgcgaag aaccttgtgt gaacatgag aacattttgaagtttgtgg

[0743] 5401 tacgaccttt agatccaga gatacagca tggctcaag tgcagctccgtttggcagtg

[0744] 5461 caatggtata aatttcaagc tggatatgtc taatgggtat ttaaacaataaatgtgcagt

[0745] 5521 tttaactaac aggatatta atgacaacct tctggttggt aggacatctgtttctaaat

[0746] 5581 gtttattatg take aaaaaaattt father agcaatgtgaaactgaattg

[0747] 5641 gagagtgata atacaagtcc tttagtctta cccagtgat cattctgttccatgtctttg

[0748] 5701 gacaaccatg accttggaca atcatgaat atgcatctca ctggatgcaaagaaaatcag

[0749] 5761 atggagcatg aatggtactg taccggttca tctggactgc cccagaaaaataacttcaag

[0750] 5821 caaacatcct atcaacaaca aggttgttct gcataccaag ctgagcacagaagatgggaa

[0751] 5881 cactggtgga ggatggaag gctcgctcaa tcaagaaaat tctgagactattaataa

[0752] 5941 agactgtagt gtagatactg agtaaatcca tgcacctaaa ccttttggaaaatctgccgt

[0753] 6001 gggccctcca gatagctcat ttcattaagt ttttccctcc aaggtagaattgcaagagt

[0754] 6061 gacagtggat tgcatttctt ttggggaagc tttctttgg tggttttgtttattacct

[0755] 6121 tcttaagttt tcaaccaagg tttgctttg ttttgagtta ctggggttattttgtttta

[0756] 6181 aataaaaata agtgtacaat aagtgttttt gtattgaaag cttttgttatcaagattttc

[0757] 6241 atactttac cttccatggc tctttttaag attgatactt ttaagaggtggctgatattc

[0758] 6301 tgcaacactg tacacataaa aatacggta aggatacttt acatggttaaggtaaagtaa

[0759] 6361 gtctccagtt ggccaccatt agctataatg gcactttgtt tgtgttgttggaaaaagtca

[0760] 6421 cattgccatt aaactttcct tgtctgcta gttaatattg tgaagaaaaataaagtacag

[0761] 6481 tgtgagatac tg

[0762] SEQ ID NO:14

[0763] (Homo sapiens) BCL2 has a cellular function.

[0764] 1 mahagrtgyd nreivmkyih yklsqrgyew dagdvgaapp gaapapgifs sqpghtphpa

[0765] 61 asrdpvarts plqtpaapga aagpalspvp pvvhltlrqa gddfsrryrr dfaemssqlh

[0766] 121 ltpftargrf atvveelfrd gvnwgrivaf fefggvmcve svnremsplv dnialwmtey

[0767] 181 lnrhlhtwiq dnggwdafve lygpsmrplf dfswlslktl lslalvgaci tlgaylghk

Claims

1. A humanized type II anti-CD20 antibody comprising (a) a heavy chain variable region as depicted in SEQ ID NO: 1 or as contained in SEQ ID NO: 5 (amino acid residues 1 to 119; SEQ ID NO: 40 as disclosed in EP-B1 2380910, see the specification) and a light chain variable region as depicted in SEQ ID NO: 2 or as contained in SEQ ID NO: 6 (amino acid residues 1 to 115; SEQ ID NO: 76 as disclosed in EP-B1 2380910, see the specification); (b) a heavy chain variable region having the specificity determining residues of the heavy chain variable region of (a) and a light chain variable region having the specificity determining residues of the light chain variable region of (a); or (c) a heavy chain variable region encoded by a nucleic acid sequence at least 80% identical to SEQ ID NO: 3 or SEQ ID NO: 7 (such as SEQ ID NO: 39 disclosed in EP-B1 2380910, see the specification) and a light chain variable region encoded by a nucleic acid sequence at least 80% identical to SEQ ID NO: 4 or SEQ ID NO: 8 (such as SEQ ID NO: 75 disclosed in EP-B1 2380910, see the specification), for use in the treatment of diffuse large B-cell lymphoma (DLBCL) in patients who respond to treatment with obinutuzumab by achieving an improved clinical outcome compared to treatment with rituximab.

2. An antibody for use according to claim 1, wherein the clinical outcome is progression-free survival (PFS), overall survival (OS) and / or event-free survival (EFS).

3. An antibody for use according to claim 1 or 2, wherein the patient is a patient defined by a predictive biomarker.

4. An antibody for use according to any one of claims 1 to 3, wherein the patient is: (i) patients with molecular follicular lymphoma (FL)-like germinal center B cell (GCB) DLBCL; and / or (ii) Patients with one or more genetic mutations in CD58 and / or with low expression of CD58.

5. An antibody for use according to any one of claims 1 to 4, in particular according to claim 4(i), wherein the patient is (i) patients with GCB-intensive DLBCL; and / or (ii) DLBCL patients with BCL2 translocation and / or high BCL2 expression.

6. An antibody for use according to any one of claims 1 to 5, in particular according to claim 5(i), wherein the patient (with strong GCB DLBCL) is identified by determining the expression of a gene set comprising one or more (preferably all) of the genes TNFRSF13B, LIMD1, IRF4, CREB3L2, PIM2, CYB5R2, RAB7L1, and CCDC50; and MME, SERPINA9, ASB13, MAML3, ITPKB, MYBL1, and S1PR2.

7. An antibody for use according to claim 5(i) or 6, wherein the patient with strong GCB DLBCL is a patient with a tumor having a weighted expression of a gene set comprising one or more (preferably all) of the genes TNFRSF13B, LIMD1, IRF4, CREB3L2, PIM2, CYB5R2, RAB7L1, and CCDC50; and MME, SERPINA9, ASB13, MAML3, ITPKB, MYBL1, and S1PR2 resulting in a linear predictor score (LPS) <1141.

8. An antibody for use according to claim 6 or 7, wherein the gene set further comprises one or more (preferably all) of the genes R3HDM1, WDR55, ISY1, UBXN4, and TRIM56.

9. An antibody for use according to any one of claims 6 to 8, wherein the expression of the one or more genes as defined in claim 6 or 7 is normalized to the expression of one or more (preferably all) genes as defined in claim 8.

10. An antibody for use according to any one of claims 7 to 9, wherein the LPS is a weighted sum of the expression of the genes calculated according to the following formula (Formula 1): LPS(X)=Σ j a j X j , Where X j is the gene expression of gene j and a j is the coefficient of gene j.

11. An antibody for use according to any one of claims 6 to 10, wherein the expression of the gene is determined by the NanoString Research Use Only Lymphoma Subtype Test (LST) (NanoString Technologies, Inc., Seattle, WA, USA).

12. An antibody for use according to any one of claims 7 to 11, wherein the LPS is ≤ 1100.

13. An antibody for use according to any one of claims 7 to 12, wherein the LPS is ≤749.

14. An antibody for use according to any one of claims 7 to 13, wherein the LPS is ≤725.

15. An antibody for use according to any one of claims 1 to 14, wherein the antibody comprises a glycoengineered Fc region.

16. The antibody according to claim 15, wherein said antibody has an increase in the fraction of non-fucosylated oligosaccharides attached to said glycoengineered Fc region.

17. The antibody according to claim 15 or 16, wherein the antibody has an increase in the fraction of bisected non-fucosylated oligosaccharides attached to the glycoengineered Fc region.

18. The antibody according to any one of claims 1 to 17, in particular according to claim 15, wherein the antibody has a significantly higher level of binding to the human FcγRIII receptor relative to a non-glycoengineered antibody and / or relative to rituximab.

19. The antibody according to any one of claims 1 to 18, in particular according to claim 15, wherein the antibody has a significantly higher level of ADCC activity relative to a non-glycoengineered antibody and / or relative to rituximab.

20. An antibody for use according to any one of claims 1 to 19, wherein the antibody is Gazyvastatin TM / Gazyvaro TM ; GA101).

21. An antibody for use according to any one of claims 1 to 20, wherein one or more additional other cytotoxic or chemotherapeutic agents or ionizing radiation which enhances the effect of such agents is to be administered.

22. An antibody for use according to any one of claims 1 to 21, wherein the antibody is to be administered in combination with CHOP chemotherapy.

23. An antibody for use according to any one of claims 1 to 22, wherein the antibody is contained in a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

24. A method for identifying a DLBCL patient (a patient having / suffering from DLBCL) who responds to treatment with obinutuzumab by achieving an improved clinical outcome compared to treatment with rituximab, The method comprises (the step of determining) whether the patient is a patient as defined in any one of claims 1 to 14 (eg by using a (tumour) sample from the patient).

25. A method for diagnosing a form of DLBCL in a patient that can be treated with obinutuzumab to achieve an improved clinical outcome compared to treatment with rituximab, the method comprising determining (e.g. by using a (tumor) sample from the patient) whether the patient is a patient as defined in any one of claims 1 to 14, and diagnosing said form of DLBCL if the patient is a patient as defined in any one of claims 1 to 14.

26. An antibody for use according to any one of claims 1 to 23, wherein (i) it has been determined (e.g. by using a (tumor) sample from the patient) whether the patient is a patient as defined in any one of claims 1 to 14, wherein (ii) the patient has been identified according to the method of claim 24, or wherein (iii) a form of DLBCL has been diagnosed in the patient according to the method of claim 25.

27. An antibody for use according to any one of claims 1 to 23, wherein the treatment comprises the steps of: (i) determining (e.g. by using a (tumour) sample from the patient) whether the patient to be treated is a patient as defined in any one of claims 1 to 14, (ii) identifying a DLBCL patient according to the method of claim 24, or (iii) diagnosing a form of DLBCL in the patient according to the method of claim 25.

28. A method for treating DLBCL in a patient in need thereof, wherein the patient is a patient as defined in any one of claims 1 to 14, the method comprising the steps of: determining (e.g. by using a (tumor) sample from the patient) whether the patient is a patient as defined in any one of claims 1 to 14, and A pharmaceutically effective amount of an antibody as defined in any one of claims 1 and 15 to 23 is administered to the patient.

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