Combination therapy of Anti-cd20 / Anti-cd3 bispecific antibodies with chemical therapy in ctdna high

Abstract

The present invention relates to methods of treating previously untreated diffuse large B-cell lymphoma (DLBCL), which is defined as high risk according to circulating tumor DNA (ctDNA), by administering gphentumab and in combination with chemotherapy.

Description

Technical Field

[0001] This invention relates to a method for treating previously untreated diffuse large B-cell lymphoma (DLBCL) defined as high risk based on circulating tumor DNA (ctDNA) by administering glimetuzumab in combination with R-CHOP. Background Technology

[0002] Diffuse large B-cell lymphoma (DLBCL) is the most common histological subtype of non-Hodgkin lymphoma (NHL), affecting approximately 25,000 new patients annually in the United States and 1.5 million new patients annually worldwide (Rodriguez-Abreu et al. 2007).

[0003] This disease exhibits significant biological and clinical heterogeneity. Although first-line treatment with immunochemotherapy using a combination of rituximab with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) (with or without radiotherapy) is curative in approximately 60% of cases, a subset of patients is refractory to this standard of care (SOC) approach (10%–15%) or relapses after this approach (20%–30%) (Chaganti et al. 2016).

[0004] According to the International Prognostic Index (IPI), the five-year survival rate for patients with DLBCL ranges from 90% for low-risk patients to less than 40% to 50% for high-risk patients (Mauer et al. 2014; Zhou et al. 2014). Treatment for patients with relapsed / refractory (R / R) DLBCL remains challenging and typically involves therapy with anti-CD20 antibodies and chemotherapy.

[0005] IPIs are widely used to stratify the risk of patients with DLBCL based on age, biochemical parameters, performance status, and disease stage. Existing prognostic scoring systems are unable to identify all high-risk patients with sufficient accuracy (International Non-Hodgkin's Lymphoma Prognostic Factors Project 1993; Sehn et al. 2007; Zhou et al. 2014). Furthermore, patients with DLBCL who respond poorly to first-line SOC therapy or relapse after first-line SOC therapy can only be identified after treatment completion, at which point their potential for salvage is limited; therefore, it is necessary to optimize effective drugs and rational treatment combinations for all patient subgroups in a front-line setting.

[0006] Up to one-third of patients with 1L LBCL treated with R-CHOP do not achieve a long-term response or cure based on disease stage. Outcome prediction beyond the International Prognostic Index (IPI) score is difficult due to the lack of reproducibility of common biomarkers. ctDNA, as a sensitive biomarker, has the potential to identify newly diagnosed patients with poor responses to chemoimmunotherapy. Decreased ctDNA levels after 1–2 therapy cycles in patients with 1L diffuse LBCL (DLBCL) have been associated with improved survival (Kurtz et al., J Clin Oncol 2018). Whether early intervention improves outcomes in patients with elevated ctDNA levels during treatment remains unclear. Griffithomab, a CD20xCD3 bispecific antibody that binds to and redirects T cells to eliminate B cells, has been approved by the FDA and EMA as a monotherapy for relapsed / refractory DLBCL after ≥2 lines of prior therapy. Glofit + R-CHOP has favorable safety and efficacy in 1L DLBCL (Topp et al. ASH 2022).

[0007] Glofit + R-CHOP demonstrated a manageable safety profile in 1L high-risk LBCL as defined by ctDNA and induced high response rates at interim analysis and EOT. Dynamic treatment-interim risk assessment using ctDNA offers the potential to identify high-risk patients with LBCL independently of baseline characteristics. Summary of the Invention

[0008] The present invention is characterized by a method for treating previously untreated patients with diffuse large B-cell lymphoma who are at high risk of circulating tumor DNA (ctDNA), the method comprising administering a combination of a bispecific antibody targeting CD3 and CD20 containing the VH and VL sequences of glimetuzumab with chemotherapy, wherein the patient has been identified as being at high risk of ctDNA if the ctDNA decreases by less than 2.5 log after one or two cycles of chemotherapy.

[0009] In one aspect, the bispecific antibody targeting CD3 and CD20 comprises the heavy chain of SEQ ID NO: 17, the heavy chain of SEQ ID NO: 18, the two light chains of SEQ ID NO: 20, and the light chain of SEQ ID NO: 19; and wherein the heavy and light chains are assembled to form a first Fab molecule that specifically binds to CD20, a second Fab molecule that specifically binds to CD3, and a third Fab molecule that specifically binds to CD20, and an Fc domain consisting of a first subunit and a second subunit capable of stable association.

[0010] In one embodiment, the chemotherapy therapy is selected from: rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP); rituximab, cyclophosphamide, doxorubicin and prednisone (R-CHP); or rituximab, ifosfamide, carboplatin and etoposide phosphate (R-ICE).

[0011] In one embodiment, the bispecific antibody targeting CD3 and CD20 is glimetuzumab, and the chemotherapy is rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), and the patient is identified as high-risk for ctDNA if the ctDNA decreases by less than 2.5 log after one or two R-CHOP cycles.

[0012] In one aspect, the invention is characterized by a method for treating a previously untreated patient with diffuse large B-cell lymphoma at high risk of circulating tumor DNA (ctDNA), the method comprising administering glimetuzumab in combination with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), wherein the patient is identified as at high risk of ctDNA if the ctDNA decreases by less than 2.5 log after one or two R-CHOP cycles.

[0013] In one embodiment, ctDNA reduction is determined by measuring ctDNA levels before or on the day of the first treatment (baseline) and after at least one R-CHOP treatment cycle. In one embodiment, ctDNA levels are determined on or before day 1 of the first R-CHOP treatment cycle (baseline) and day 1 of the second R-CHOP treatment cycle. In another embodiment, ctDNA levels are determined on day 1 of the first R-CHOP treatment cycle (baseline) and day 1 of the second R-CHOP treatment cycle. In one embodiment, a patient is identified as high-risk for ctDNA if the reduction in ctDNA is less than 2 log.

[0014] In another aspect of the invention, a method for treating previously untreated diffuse large B-cell lymphoma is provided, the method comprising:

[0015] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0016] b. Determine the amount of ctDNA at or before cycle 1 (baseline) and after at least one R-CHOP treatment cycle; and

[0017] c. Select patients whose ctDNA reduction is <2.5-log between baseline and at least one R-CHOP treatment cycle and continue treatment with R-CHOP and glimetuzumab.

[0018] In one embodiment, step a) includes applying R-CHOP on day 1 of cycles 1, 2, and optionally cycle 3. In another embodiment, step a) includes applying R-CHOP on day 1 of cycles 1 and 2.

[0019] In one embodiment, step b) includes determining the amount of ctDNA on or before day 1 of cycle 1 (baseline) and day 1 of cycle 2 or day 1 of cycle 3 of the R-CHOP treatment in step a). In another embodiment, step b) includes determining the amount of ctDNA on day 1 of cycle 1 (baseline) and day 1 of cycle 2 of the R-CHOP treatment in step a).

[0020] In one embodiment, step c) includes selecting patients with a ctDNA reduction of <2-log. In another embodiment, step c) includes continuing R-CHOP treatment for a total of six cycles (cycles 1 to 6) and adding glimetuzumab for a total of eight cycles (cycles 3 to 10).

[0021] In one embodiment, glimetuzumab is administered at a dose of 2.5 mg on day 8 of cycle 3, at a dose of 10 mg on day 15, at a dose of 30 mg on day 8 of cycles 4 through 6, and at a dose of 30 mg on day 1 of cycles 7 through 10.

[0022] In any of the foregoing aspects of the invention, ctDNA is circulating tumor DNA in plasma. In one such embodiment, the amount of ctDNA is measured in a blood sample from a patient. In one embodiment, the amount of ctDNA is measured in genomic DNA isolated from peripheral mononuclear cells or plasma-depleted whole blood (PDWB) or in cell-free DNA (cfDNA) in plasma. In one embodiment, ctDNA is measured in cell-free DNA in plasma. In one embodiment, ctDNA is determined by targeted next-generation sequencing (NGS).

[0023] In any of the above aspects of the present invention, rituximab is administered at 375 mg / m². 2 Cyclophosphamide was administered intravenously at a dose of 750 mg / m². 2 Doxorubicin was administered intravenously at a dose of 50 mg / m². 2 Vincristine was administered intravenously at a dose of 1.4 mg / m². 2 The dose was administered via IV bolus, and prednisone was administered at a dose of 100 mg / day from day 1 to day 5.

[0024] In any of the above aspects of the invention, the length of each cycle is 21 days.

[0025] In any of the foregoing aspects of the invention, administration of such treatment to multiple human patients at high risk of ctDNA results in an overall response rate (ORR) of at least 80%, at least 85%, at least 90%, or at least 95%. In one embodiment, the ORR is at least 95%.

[0026] In one embodiment of any of the foregoing aspects of the invention, administration of such treatment to multiple human patients at high risk of ctDNA results in a complete response (CR) rate of at least 75%, at least 80%, or at least 85%. In one embodiment, the CR rate is at least 85%.

[0027] In one embodiment, the ORR or CR rate is determined at the end of treatment. In one embodiment, the ORR or CR rate is determined by PET-CT scan. In one embodiment, the ORR or CR is determined according to the 2014 Lugano response criteria.

[0028] One aspect of the invention relates to a bispecific antibody targeting CD3 and CD20 comprising the VH and VL sequences of glimetuzumab, in a method for treating previously untreated diffuse large B-cell lymphoma at high (ct)DNA risk, the method comprising administering glimetuzumab in combination with chemotherapy to a patient, wherein the patient has been identified as at high ctDNA risk if the ctDNA level decreases by less than 2.5 log after one cycle of chemotherapy.

[0029] One aspect of the present invention relates to glimetuzumab, in a method of treating previously untreated diffuse large B-cell lymphoma with high (ct)DNA risk, the method comprising:

[0030] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0031] b. Determine the amount of ctDNA at or before cycle 1 (baseline) and after at least one R-CHOP treatment cycle; and

[0032] c. Select patients whose ctDNA reduction is <2.5-log between baseline and at least one R-CHOP treatment cycle and continue treatment with R-CHOP and glimetuzumab.

[0033] One aspect of the invention relates to the use of a bispecific antibody targeting CD3 and CD20 comprising the VH and VL sequences of glimetuzumab in the manufacture of a medicament for treating a subject with previously untreated diffuse large B-cell lymphoma at high risk of (ct)DNA, wherein the patient is administered a combination of glimetuzumab with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), wherein the patient has been identified as at high risk of ctDNA if the ctDNA decreases by less than 2.5 log after one R-CHOP cycle.

[0034] One aspect of the invention relates to the use of glimetuzumab in the manufacture of a medicament for treating subjects with previously untreated diffuse large B-cell lymphoma at high risk of (ct)DNA, comprising:

[0035] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0036] b. Determine the amount of ctDNA at or before cycle 1 (baseline) and after at least one R-CHOP treatment cycle; and

[0037] c. Select patients whose ctDNA reduction is <2.5-log between baseline and at least one R-CHOP treatment cycle and continue treatment with R-CHOP and glimetuzumab.

[0038] Another aspect of the present invention relates to the invention as described herein.

[0039] Unless the context clearly indicates otherwise, each embodiment may be incorporated. Unless the context clearly indicates otherwise, each embodiment may be applied to various aspects of the invention.

[0040] Certain embodiments of the invention will become apparent from the following more detailed description of some preferred embodiments and claims. Attached Figure Description

[0041] Figure 1 This is a schematic diagram showing the structure of gfinitusumab.

[0042] Figure 2 This is a schematic diagram illustrating an overview of the study design as described in Example 1. ctDNA = circulating tumor DNA; R-CHOP = rituximab combined with cyclophosphamide, doxorubicin, vincristine, and prednisone. Patients at high ctDNA risk are those who do not achieve a ≥ 2-log (100×-) decrease in ctDNA levels between Day 1 of Cycle 1 and Day 1 of Cycle 2. Patients showing a ≥ 2-log-fold decrease in ctDNA levels during the same time period are considered low ctDNA risk patients and are considered unqualified for continued participation for the purposes of this study.

[0043] Figure 3 This is a schematic diagram illustrating the dosing regimen used in the study described in Example 1. ctDNA = circulating tumor DNA; R-CHOP = rituximab combined with cyclophosphamide, doxorubicin, vincristine, and prednisone.

[0044] Figure 4 Patient response rates (n=15) in interim analyses conducted at the end of cycle 2 and at the end of treatment (EOT). CR, complete response; EOT, end of treatment; ORR, overall response rate; PR, partial response; SD, stable disease. Detailed Implementation

[0045] This invention provides a method for treating previously untreated diffuse large B-cell lymphoma (DLBCL) defined as high risk based on circulating tumor DNA (ctDNA) by administering glimetuzumab in combination with R-CHOP.

[0046] (i) General Technology

[0047] Unless otherwise indicated, the present invention will be practiced using conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the scope of the art. Such techniques are well explained in the following references, such as "Molecular Cloning: A Laboratory Manual," 2nd edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (edited by MJ Gait, 1984); "Animal Cell Culture" (edited by RI Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (edited by FM Ausubel et al., 1987, and periodically updated); "PCR: The Polymerase Chain Reaction" (edited by Mullis et al., 1994); "A Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988); and "Phage Display: A Laboratory Manual" (Barbas et al., 2001).

[0048] (ii) Definition

[0049] Unless otherwise defined below, the terminology used herein is generally as it is used in the art.

[0050] Unless otherwise specified, the terms “differentiation cluster 20” or “CD20” as used herein refer to any natural CD20 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). CD20 (also known as B-lymphocyte antigen CD20, B-lymphocyte surface antigen B1, Leu-16, Bp35, BM5, and LF5; human protein described in UniProt database registry number P11836) is a hydrophobic transmembrane protein with a molecular weight of approximately 35 kD, expressed on pre-B and mature B lymphocytes. (Valentine, MA et al., J. Biol. Chem. 264 (1989) 11282-11287; Tedder, TF et al., Proc. Natl. Acad. Sci. USA 85 (1988) 208-212; Stamenkovic, I. et al., J. Exp. Med. 167 (1988) 1975-1980; Einfeld, DA, et al., EMBO J. 7 (1988) 711-717; Tedder, TF et al., J. Immunol. 142 (1989) 2560-2568). The corresponding human gene is transmembrane 4 domain, subfamily A member 1, also known as MS4A1. This gene encodes a member of the transmembrane 4A gene family. Members of this neonatal protein family are characterized by common structural features and similar intron / exon splicing boundaries, and exhibit distinct expression patterns in hematopoietic cells and non-lymphoid tissues. This gene encodes a B lymphocyte surface molecule that plays a role in B cell development and differentiation into plasma cells. This family member is located at 11q12 in the family member cluster. The term encompasses “full-length”, unprocessed CD20, as well as any form of CD20 produced by intracellular processing. The term also encompasses naturally occurring variants of CD20, such as splice variants or allelic variants. Alternative splicing of this gene produces two transcript variants encoding the same protein. In one embodiment, CD20 is human CD20.

[0051] An example of an anti-CD20 / anti-CD3 bispecific antibody is glimetuzumab (WHO Drug Information (International Nonproprietary Name of the drug), Recommended INN: List 83, 2020, Vol. 34, No. 1, p. 39; Proposed INN: List 121 WHO Drug Information, Vol. 33, No. 2, 2019, p. 276, also known as CD20-TCB, RO7082859, or RG6026; CAS #: 2229047-91-8). Gglimetuzumab is a novel T-cell binding bispecific (TCB) full-length antibody with a 2:1 molecular conformation for bivalent binding to CD20 on B cells and monovalent binding to CD3 (particularly the CD3 ε chain (CD3e)) on T cells. Its CD3 binding region is fused to one of the CD20 binding regions in a head-to-tail manner via a flexible linker. This structure endows glimetuzumab with superior in vitro potency compared to other CD20-CD3 bispecific antibodies with a 1:1 configuration, and it exhibits significant antitumor efficacy in a preclinical DLBCL model. The CD20 bivalent nature maintains this potency in the presence of competitive anti-CD20 antibodies, providing opportunities for pre-treatment or co-treatment with these agents. Gglimetuzumab contains an engineered heterodimeric Fc region whose binding to FcgR and C1q is completely eliminated. By simultaneously binding to CD3e of the T cell receptor (TCR) complex on tumor cells expressing human CD20 and T cells, it also induces tumor cell lysis in addition to T cell activation, proliferation, and cytokine release. Gglimetuzumab-mediated B cell lysis is CD20-specific and does not occur in the absence of CD20 expression or in the absence of T cells simultaneously binding to (crosslinking) CD20-expressing cells. In addition to killing, T cells are also activated by CD3 cross-linking, as detected by increased T cell activation markers (CD25 and CD69), cytokine release (IFNγ, TNFα, IL-2, IL-6, IL-10), cytotoxic granule release (granzyme B), and T cell proliferation. A schematic diagram of the molecular structure of glimetuzumab is shown below. Figure 1 The sequences of glimetuzumab are summarized in Table 1.

[0052] Table 1. Sequence IDs of Gifitumab

[0053]

[0054] As used herein, the term "glucantumab" refers to any bispecific anti-CD3 / CD20 antibody having the VH and VL sequences depicted in the table above, and includes any variants with post-translational modifications. Furthermore, it should be understood that, as used herein, the term "glucantumab" also encompasses any biosimilar version approved by a regulatory agency, such as the US FDA or the European EMA. In one embodiment, glucantumab is an antibody described in the following literature: WHO Drug Information, proposed INN: List 83, 2020, Vol. 34, No. 1, p. 39; proposed INN: List 121 WHO Drug Information, Vol. 33, No. 2, 2019, p. 276, also known as CD20-TCB, RO7082859, or RG6026; CAS #: 2229047-91-8.

[0055] As used herein, the term “cytokine release” or “cytokine release” is synonymous with “cytokine storm” or “cytokine release syndrome” (abbreviated as “CRS”) and refers to an increase in the levels of cytokines, particularly tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-2 (IL-2), and / or interleukin-8 (IL-8), in the blood of a subject during or shortly thereafter (e.g., within 1 day) of treatment administration, leading to adverse symptoms. Cytokine release is defined as a supraphysiological response resulting in or involving the activation of endogenous or infused T cells and / or other immune effector cells following administration of any immunotherapy. Symptoms may be progressive, always include fever at the onset, and may include hypotension, capillary leakage (hypoxia), and peripheral organ dysfunction (Lee et al. 2019). In some cases, such as after CAR-T cell administration, CRS may also occur several days later during CAR-T cell expansion. The incidence and severity usually decrease with subsequent infusions. Symptoms can range from mild discomfort to fatal events and may include fever, chills, dizziness, high blood pressure, low blood pressure, dyspnea, restlessness, sweating, flushing, rash, tachycardia, tachypnea, headache, tumor pain, nausea, vomiting, and / or organ failure.

[0056] The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0057] It is known in the art that glutamine and glutamate at the N-terminus of recombinant monoclonal antibodies can spontaneously cyclize to pyroglutamic acid in vitro. In some embodiments, for each sequence disclosed herein containing N-terminal glutamine and / or glutamate, a corresponding sequence having an N-terminal pyroglutamic acid residue is also considered.

[0058] A "human antibody" is an antibody whose amino acid sequence corresponds to that of an antibody produced by a human or human cell, or to a non-human antibody derived from a complete library of human antibodies or other antibody-encoding sequences. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues.

[0059] "Humanized" antibodies refer to chimeric antibodies that contain amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, the humanized antibody will contain substantially all of at least one (usually two) variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to the HVRs of the non-human antibody, and all or substantially all of the FRs correspond to the FRs of the human antibody. Optionally, the humanized antibody may contain at least a portion of the antibody constant region derived from the human antibody. Antibodies in a "humanized form," such as non-human antibodies, refer to antibodies that have undergone humanization.

[0060] As used herein, the term "hypervariant region" or "HVR" refers to each of the following: a region of antibody variable domain that is hypervariable in sequence ("complementarity-determining regions" or "CDRs") and / or forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). Typically, an antibody contains six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs in this document include:

[0061] (a) Highly variable rings appearing at the following amino acid residues: 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

[0062] (b) CDRs appearing at the following amino acid residues: 24 to 34 (L1), 50 to 56 (L2), 89 to 97 (L3), 31 to 35b (H1), 50 to 65 (H2), and 95 to 102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, MD (1991)).

[0063] (c) Antigen contact sites located at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and

[0064] (d) Combinations of (a), (b) and / or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3) and 94-102 (H3).

[0065] Unless otherwise specified, HVR residues and other residues (e.g., FR residues) in the variable domains are referenced in this paper to Kabat et al., with the same reference numbering above.

[0066] "Frame" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences usually appear in the VH (or VL) as follows: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0067] The “human common framework” is a framework that represents the most frequently occurring amino acid residues in the selection of the human immunoglobulin VL or VH framework sequence. Generally, the selection of the human immunoglobulin VL or VH sequence is derived from a subgroup of variable domain sequences. Typically, the sequence subgroup is as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In one embodiment, for VL, the subgroup is subgroup κI as described in Kabat et al., ibid. In one embodiment, for VH, the subgroup is subgroup III as described in Kabat et al. (ibid.).

[0068] For the purposes of this document, a “recipient human frame” is a frame that comprises an amino acid sequence derived from a light chain variable domain (VL) frame or a heavy chain variable domain (VH) frame of a human immunoglobulin frame or a human common frame as defined below. A recipient human frame “derived from” a human immunoglobulin frame or a human common frame may contain the same amino acid sequence as that human immunoglobulin frame or human common frame, or it may contain amino acid sequence variations. In some embodiments, the number of amino acid variations is 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer. In some embodiments, the VL recipient human frame is sequence-identical to the VL human immunoglobulin frame sequence or the human common frame sequence.

[0069] An antibody's "class" refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these antibodies can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0070] As used herein, the term IgG “isotype” or “subclass” refers to any subclass of immunoglobulins defined by the chemical and antigenic characteristics of the immunoglobulin constant region.

[0071] The term "Fc domain" or "Fc region" used herein is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes both native sequence Fc regions and variant Fc regions. Although the boundaries of the IgG heavy chain Fc region may vary slightly, the human IgG heavy chain Fc region is generally defined as extending from Cys226 or Pro230 to the C-terminus of the heavy chain. However, antibodies produced by host cells can undergo post-translational cleavage of one or more (particularly one or two) amino acids from the C-terminus of the heavy chain. Therefore, antibodies produced by host cells by expressing a specific nucleic acid molecule encoding the full-length heavy chain can include the full-length heavy chain, or the antibody can include a cleaved variant of the full-length heavy chain (also referred to herein as a "cleaved variant heavy chain"). This could be the case where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU number). Therefore, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (K447) in the Fc region may or may not be present. Unless otherwise stated herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see above). As used herein, a “subunit” of the Fc domain refers to one of two polypeptides that form a dimer Fc domain, namely a polypeptide that includes the C-terminal constant region of the immunoglobulin heavy chain and is capable of stable self-association. For example, the subunit of the IgG Fc domain contains the IgG CH2 and IgG CH3 constant domains.

[0072] "Modification that promotes association between the first and second subunits of the Fc domain" refers to manipulation of the peptide backbone or post-translational modification of the Fc domain subunits that reduces or prevents the association of a polypeptide containing an Fc domain subunit with the same polypeptide to form a homodimer. As used herein, association-promoting modifications specifically include individual modifications to each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) to which association is desired, wherein the modifications are complementary to each other to promote association between the two Fc domain subunits. For example, association-promoting modifications may alter the structure or charge of one or both of the Fc domain subunits to make their association sterically or electrostatically favorable, respectively. Thus, (hetero)dimerization occurs between a polypeptide containing the first Fc domain subunit and a polypeptide containing the second Fc domain subunit, which may differ in the sense of inconsistency in the additional components (e.g., antigen-binding moieties) fused to each subunit. In some embodiments, association-promoting modifications include amino acid mutations, particularly amino acid substitutions, in the Fc domain. In one particular embodiment, the association-promoting modification includes individual amino acid mutations, particularly amino acid substitutions, in each of the two subunits of the Fc domain.

[0073] "Activated Fc receptors" are Fc receptors that, upon binding to the Fc region of an antibody, trigger signal transduction events that stimulate receptor-carrying cells to perform effector functions. Activated Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0074] When discussing antibody use, the term "effective function" refers to those biological activities attributable to the Fc region of the antibody, which vary across antibody isotypes. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0075] As used herein, the term "effective cells" refers to a population of lymphocytes that display effector portion receptors (e.g., cytokine receptors) and / or Fc receptors on their surface. These receptors allow them to bind to the Fc region of effector portions (e.g., cytokines) and / or antibodies, contributing to the destruction of target cells, such as tumor cells. Effector cells can, for example, mediate cytotoxic or phagocytic responses. Effector cells include, but are not limited to, effector T cells, such as CD8+.+ Cytotoxic T cells, CD4 + Helper T cells, γδ T cells, NK cells, lymphokine-activated killer (LAK) cells, and macrophages / monocytes.

[0076] The terms "multispecific" and "bispecific" refer to the ability of an antigen-binding molecule to specifically bind to at least two different antigenic determinants. Typically, a bispecific antigen-binding molecule contains two antigen-binding sites, each of which is specific to a different antigenic determinant. In some embodiments, a bispecific antigen-binding molecule can bind to two antigenic determinants simultaneously, particularly two antigenic determinants expressed on two distinct cell types.

[0077] As used herein, the term "valence" or "valence state" indicates the presence of a specified number of antigen-binding sites in an antigen-binding molecule. Therefore, the term "monovalently bound to antigen" indicates the presence of one (and no more than one) antigen-binding site in an antigen-binding molecule that is specific to the antigen.

[0078] An "antigen binding site" is a site on an antigen-binding molecule that provides an interaction with an antigen; it is typically one or more amino acid residues. For example, the antigen binding site of an antibody contains amino acid residues from its complementarity-determining region (CDR). Natural immunoglobulin molecules usually have two antigen binding sites, while Fab molecules usually have a single antigen binding site.

[0079] A “Fab molecule” is a protein composed of the VH and CH1 domains of the heavy chain (“Fab heavy chain”) and the VL and CL domains of the light chain (“Fab light chain”) of an immunoglobulin.

[0080] "Fusion" means that components (e.g., Fab molecules and Fc domain subunits) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0081] The “effective amount” of a drug refers to the amount required to produce physiological changes in the cells or tissues to which it is applied.

[0082] A "therapeuticly effective amount" of a pharmaceutical agent (e.g., a pharmaceutical composition) refers to the amount that effectively achieves the desired therapeutic or preventative outcome at the necessary dosage and time period. A therapeutically effective amount of a pharmaceutical agent may eliminate, reduce, delay, minimize, or prevent adverse reactions to a disease.

[0083] "Therapeutic agent" refers to, for example, the active ingredient of a pharmaceutical composition, administered to a subject in an attempt to alter the natural course of a disease in the treated subject, and may be used for prevention or to be performed during a clinicopathological process. "Immunotherapy agent" refers to a therapeutic agent administered to a subject in an attempt to restore or enhance the subject's immune response (e.g., an immune response against a tumor).

[0084] The term "pharmaceutical composition" refers to a preparation in a form that makes the bioactivity of the active ingredient contained therein effective, and that the preparation does not contain any additional components that would have unacceptable toxicity to a subject who would administer the composition.

[0085] "Pharmaceutical carrier" refers to a component of a pharmaceutical composition that is non-toxic to the subject, excluding the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffer solutions, excipients, stabilizers, or preservatives.

[0086] The terms “packaging insert” or “instructions for use” are used to refer to instructions that are typically included in the commercial packaging of a therapeutic product, which include information on the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings for using the therapeutic product.

[0087] The term "combination therapy" as used herein encompasses combined administration (where two or more therapeutic agents are included in the same or separate formulations); and separate administration, in which case the administration of an antibody as reported herein may occur before, simultaneously with, and / or after the administration of one or more additional therapeutic agents (preferably one or more antibodies).

[0088] A “crossfab” molecule (also called a “Crossfab”) refers to a Fab molecule in which the variable or constant domains of the Fab heavy chain and light chain are exchanged (i.e., replaced). Specifically, a crossfab molecule comprises a peptide chain consisting of a light chain variable domain VL and a heavy chain constant domain 1 CH1 (VL-CH1, N-terminal to C-terminal direction), and a peptide chain consisting of a heavy chain variable domain VH and a light chain constant domain CL (VH-CL, N-terminal to C-terminal direction). For clarity, in a crossfab molecule in which the variable domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain containing the heavy chain constant domain 1 CH1 is referred to herein as the “heavy chain” of the (cross)fab molecule. Conversely, in a crossfab molecule in which the constant domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain containing the heavy chain variable domain VH is referred to herein as the “heavy chain” of the (cross)fab molecule.

[0089] In contrast, "conventional" Fab molecules refer to Fab molecules in their natural form, namely, heavy chains consisting of variable and constant domains in the heavy chain (VH-CH1, in the direction from the N-terminus to the C-terminus), and light chains consisting of variable and constant domains in the light chain (VL-CL, in the direction from the N-terminus to the C-terminus).

[0090] As used herein, the term "about" refers to a common range of error for a corresponding value that is readily known to those skilled in the art. References to "about" values ​​or parameters herein include (and describe) embodiments involving that value or parameter itself.

[0091] In the context of this invention, "DLBCL" refers to previously untreated CD20-positive diffuse large B-cell lymphoma (DLBCL), and includes nonspecific (NOS) type DLBCL, including germinal center B-cell-like (GCB) and activated B-cell-like (ABC) / non-GCB types, as well as dual-expressing lymphoma (MYC and BCL2 co-expression), high-grade B-cell lymphoma (HGBCL) with MYC and BCL2 and / or BCL6 translocations, high-grade B-cell lymphoma (HGBCL; e.g., HGBCL NOS, double-hit HGBCL, and triple-hit HGBCL), and new-onset transformed follicular lymphoma.

[0092] "Individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the individual or subject is a human. In one case, each subject in the subject population is a human. In another case, each subject in the subject reference population is a human. As used herein, the term "patient" refers to a human individual or subject requiring treatment.

[0093] As used herein, “treatment” (and its grammatical variations such as treat or treating) refers to an attempt to alter the natural course of the disease in the treated individual and is a clinical intervention that can be performed for prevention or may be performed during a clinicopathological process. In one embodiment, the term “treatment” refers to treatment with R-CHOP and glimetuzumab. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and mitigating or improving prognosis. In some embodiments, the methods of the present invention are used to delay the development of disease or slow the progression of disease. In one embodiment, the disease treated is previously untreated DLBCL as defined herein.

[0094] As used herein, “delayed progression” of a condition or disease means the slowing, hindering, mitigating, delaying, stabilizing, and / or postponing the development of a disease or condition (e.g., DLBCL). Such delay can vary in length depending on the individual’s medical history and / or treatment. It will be apparent to those skilled in the art that a sufficient or significant delay can effectively encompass prevention, as the individual will not develop the disease. For example, in advanced cancer, the development of central nervous system (CNS) metastases may be delayed.

[0095] As used herein, “administration” means the method of giving a subject a dose of a compound (e.g., glimetuzumab) or a composition (e.g., a pharmaceutical composition, such as a pharmaceutical composition comprising glimetuzumab). The compounds and / or compositions used in the methods described herein may be administered intravenously (e.g., by intravenous infusion).

[0096] In this article, "target dose" refers to the dose of glimetuzumab required to achieve the therapeutic effect, i.e., the desired clinical efficacy. The potential target doses for glimetuzumab are found to be 16 mg or 30 mg.

[0097] "Using the same or predetermined dose of the target dose" and "treatment regimens without using an escalation dosing regimen" refer to a dosing regimen that uses the same dose in the first and second cycles and optionally any subsequent treatment cycles. This is different from escalation dosing, which uses a lower dose in the first few treatment cycles and only reaches the target dose in the second or subsequent treatment cycles.

[0098] As used herein, the term "treatment cycle" or "cycle" (abbreviated as "C") refers to a process of repeating one or more doses of a therapeutic agent (e.g., R-CHOP and / or glimetuzumab) at a regular schedule, optionally with rest periods (no treatment). In one aspect of the invention, a third treatment cycle comprises a first and a second dose of glimetuzumab, followed by a rest period. In one such embodiment, the third treatment cycle comprises a first dose of glimetuzumab on day 8 of the third cycle and a second dose of glimetuzumab on day 15 of the third cycle, followed by a 6-day rest. In one embodiment, the subsequent two cycles (cycles 4 to 6) comprise a dose of glimetuzumab administered on day 8 of that cycle, followed by a 13-day rest. In one embodiment, a treatment cycle comprises 21 days. In another embodiment, a treatment cycle comprises 14 days. Treatment regimens according to the invention may comprise 6 or more treatment cycles, particularly 10 treatment cycles. In some embodiments, a treatment cycle is referred to as a "dosing cycle".

[0099] "Individual response" or "response" may be assessed using any endpoint that indicates benefit to the subject, including but not limited to (1) inhibiting disease progression (e.g., DLBCL progression) to some extent, including slowing and completely blocking it; (2) reducing tumor size; (3) inhibiting (i.e., reducing, slowing or completely stopping) cancer cell infiltration into adjacent organs and / or tissues; (4) inhibiting (i.e., reducing, slowing or completely stopping) metastasis; (5) alleviating one or more symptoms associated with DLBCL to some extent; (6) increasing or prolonging survival, including overall survival and progression-free survival; and / or (7) reducing mortality at a given time point after treatment.

[0100] As used in this article, “End of Treatment” or “EOT” is defined as the time when a patient has completed all study treatment cycles, namely 2 cycles of R-CHOP, 4 cycles of R-CHOP and glimetuzumab, and 4 cycles of glimetuzumab treatment (i.e., all 10 cycles depicted in Figures 2 and 3).

[0101] As used herein, “complete remission” or “CR” refers to the disappearance of all target lesions. In one embodiment, the NHL remission criteria are used to determine CR. (Lugano Classification, Cheson et al. J Clin Oncol. 20 Sep 2014; 32(27): 3059–3067.). In one embodiment, the CR rate is defined as the proportion of participants who achieve CR at the end of treatment (i.e., within the 10 cycles depicted in Figures 2 and 3), determined by the investigator according to the Lugano criteria. The CR rate is defined as the percentage of patients with CR at EOT.

[0102] In one embodiment, CR is defined as complete metabolic remission, as determined by PET / CT of lymph nodes and extralymphatic sites, wherein a score of 1, 2, or 3 is given, with or without residual mass at 5PS, where PET 5PS: 1 = no uptake above background; 2 = uptake > mediastinum; 3 = uptake > mediastinum, but ≤ liver; 4 = adequate uptake > liver; 5 = significantly higher uptake than liver and / or new lesions; X = new uptake area unlikely to be associated with lymphoma. In one embodiment, CR is defined as complete radiological remission determined by CT of lymph nodes and extralymphatic sites, wherein the target nodule / nodular mass must shrink to ≤ 1.5 cm in LDi (longest transverse diameter of the lesion) and no extralymphatic disease sites remain.

[0103] As used herein, “partial response” or “PR” refers to a partial metabolic response and is defined according to Lugano Classification, Cheson et al. J Clin Oncol. 20 Sep 2014; 32(27): 3059–3067. In one embodiment, PR is determined by PET / CT of lymph nodes and extralymphatic sites, and / or partial remission is determined by CT of lymph nodes and extralymphatic sites. In one embodiment, partial metabolic remission is defined by a score 4 or 5b, a reduction in uptake compared to baseline, and residual mass of any size is determined by PET / CT of lymph nodes and extralymphatic sites, where PET 5PS: 1 = no uptake above background; 2 = uptake > mediastinum; 3 = uptake > mediastinum, but ≤ liver; 4 = adequate uptake > liver; 5 = significantly higher uptake than liver and / or new lesions; X = new uptake area unlikely to be associated with lymphoma. In one embodiment, partial relief is defined as a reduction of at least 50% in the diameter product (SPD) of up to six measurable nodules and extranodal sites, with baseline SPD as a reference.

[0104] As used herein, “objective response rate (ORR)” is defined as the sum of the partial response (PR) rate and the complete response (CR) rate at the end of treatment (EOT). In one embodiment, ORR is assessed based on the Lugano classification (Cheson et al. J Clin Oncol. 20 Sep 2014; 32(27): 3059-3067). In one embodiment, ORR is measured by PET-CT scan. In one embodiment, the analysis of ORR is the same as that described for the primary efficacy endpoint of CR rate at EOT measured by PET / CT scan.

[0105] “Progression-free survival” (PFS) is defined as the time from the first treatment with glimetuzumab to the first occurrence of disease progression, relapse, or death from any cause (whichever occurs first). In one embodiment, PFS is assessed based on the Lugano classification (Cheson et al. J Clin Oncol. 20 Sep 2014; 32(27): 3059–3067.). In one embodiment, the distribution of PFS, median (if analytically feasible) 6-month and 1-year PFS will be estimated using the Kaplan-Meier method (Kaplan and Meier 1958), where the 95% CI for median PFS is constructed using the Brookmeyer-Crowley method (Brookmeyer and Crowley 1982).

[0106] Overall survival (OS) is defined as the time from the first use of glimetuzumab treatment to death from any cause.

[0107] The “duration of objective response (DOR)” is defined as the time from the first recorded objective response (CR or PR) to disease progression, relapse, or death from any cause (whichever occurs first), as determined by the investigator according to the Lugano criteria (Cheson et al. J Clin Oncol. 20 Sep 20, 2014; 32(27): 3059–3067.). In one embodiment, DOR is measured by PET / CT scan.

[0108] As used in this article, "stable disease" or "SD" refers to the minimum SLD since the start of treatment, where the target lesion has neither shrunk sufficiently to meet PR nor increased sufficiently to meet PD.

[0109] As used in this article, “disease progression” or “PD” means an increase of at least 20% in the target lesion’s SLD relative to the minimum SLD recorded since the start of treatment, or an increase of at least 50% in the target lesion’s SPD relative to the minimum SPD recorded since the start of treatment, or the appearance of one or more new lesions.

[0110] As used herein, “infusion-related reaction,” “IRR,” or infusion-related adverse event refers to an adverse event that occurs in a patient or subject during or within 24 hours after administration of a drug (e.g., glimetuzumab or rituximab). IRR can be graded from 1 to 5 according to, for example, NCI CTCAE v.4.

[0111] As used herein, the term "R-CHOP" refers to rituximab plus ifosfamide, carboplatin, etoposide, or etoposide phosphate.

[0112] As used herein, the term "ctDNA" refers to circulating tumor DNA in the bloodstream that is not cellular. It refers to fragments of DNA derived from cancer cells and tumors and can be found in plasma or serum. In one embodiment, ctDNA is plasma ctDNA.

[0113] As used herein, “high-risk ctDNA” refers to a patient with high levels of residual circulating tumor DNA following initial first-line treatment of DLBCL using the standard of care (R-CHOP). In one embodiment, a patient is identified as high-risk ctDNA if the reduction in ctDNA is less than 2.5 log at or before treatment initiation (i.e., at or before cycle 1, baseline) and after at least one R-CHOP treatment cycle. In another embodiment, a patient is identified as high-risk ctDNA if the reduction in ctDNA is less than 2 log at or before treatment initiation (i.e., at or before cycle 1, baseline) and after at least one R-CHOP treatment cycle. In the context of this invention, “at or before cycle 1” means that the baseline ctDNA level is preferably determined on day 1 of cycle 1 of R-CHOP treatment or shortly before day 1 of cycle 1 of R-CHOP treatment (e.g., up to 1, 2, 3, 4, or 5 days prior, preferably up to 1 or 2 days prior).

[0114] In one embodiment, a high-risk ctDNA patient is a patient who fails to achieve a ≥ 2 log-fold reduction in ctDNA levels between Day 1 of Cycle 1 and Day 1 of Cycle 2 of R-CHOP treatment.

[0115] As used in this article, "reduction of 2.5 log" and "reduction of 2 log" refer to the reduction in ctDNA levels measured in the patient's plasma before the start of treatment and after at least one R-CHOP treatment cycle. For example, a "reduction of 2 log" for ctDNA means a 100-fold reduction in plasma ctDNA levels.

[0116] As used in this article, the “2014 Lugano response criteria” refers to the 2014 Lugano response criteria for malignant lymphomas described in Cheson BD, Fisher RI, Barrington SF, et al., Recommendations for initial evaluation, staging and response assessment of Hodgkin and non-Hodgkin lymphoma: the Lugano classification. J Clin Oncol 2014;32:1-9.

[0117] (iii) Combination therapy of anti-CD20 / anti-CD3 bispecific antibody with anti-CD20 antibody and chemotherapy in patients at high risk of ctDNA

[0118] The present invention is characterized by a method for treating previously untreated patients with diffuse large B-cell lymphoma who are at high risk of circulating tumor DNA (ctDNA), the method comprising administering a combination of a bispecific antibody targeting CD3 and CD20 containing the VH and VL sequences of glimetuzumab with chemotherapy, wherein the patient has been identified as being at high risk of ctDNA if the ctDNA decreases by less than 2.5 log after one or two cycles of chemotherapy.

[0119] In one aspect, the bispecific antibody targeting CD3 and CD20 comprises the heavy chain of SEQ ID NO: 17, the heavy chain of SEQ ID NO: 18, the two light chains of SEQ ID NO: 20, and the light chain of SEQ ID NO: 19; and wherein the heavy and light chains are assembled to form a first Fab molecule that specifically binds to CD20, a second Fab molecule that specifically binds to CD3, and a third Fab molecule that specifically binds to CD20, and an Fc domain consisting of a first subunit and a second subunit capable of stable association.

[0120] In one embodiment, the chemotherapy therapy is selected from: rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP); rituximab, cyclophosphamide, doxorubicin and prednisone (R-CHP); or rituximab, ifosfamide, carboplatin and etoposide phosphate (R-ICE).

[0121] In one embodiment, ctDNA reduction is determined by measuring ctDNA levels before or on the day of the first treatment (baseline) and after at least one chemotherapy cycle. In one embodiment, ctDNA levels are determined on or before day 1 of the first chemotherapy cycle (baseline) and day 1 of the second chemotherapy cycle. In another embodiment, ctDNA levels are determined on day 1 of the first chemotherapy cycle (baseline) and day 1 of the second chemotherapy cycle. In one embodiment, a patient is identified as high-risk for ctDNA if the reduction in ctDNA is less than 2 log.

[0122] In one embodiment, the bispecific antibody targeting CD3 and CD20 is glucentumab.

[0123] In another aspect of the invention, a method for treating previously untreated diffuse large B-cell lymphoma is provided, the method comprising:

[0124] a. Administer chemotherapy for at least two cycles;

[0125] b. Determine the amount of ctDNA at or before cycle 1 (baseline) and after at least one chemotherapy cycle; and

[0126] c. Select patients whose ctDNA reduction is <2.5-log between baseline and at least one cycle of chemotherapy, and continue chemotherapy and glimetuzumab treatment.

[0127] In one embodiment, step a) includes administering chemotherapy on day 1 of cycles 1, 2, and optionally cycle 3. In one embodiment, step a) includes administering chemotherapy on day 1 of cycles 1 and 2.

[0128] In one embodiment, step b) includes determining the amount of ctDNA on or before day 1 of cycle 1 (baseline) and day 1 of cycle 2 or day 1 of cycle 3 of the chemotherapy treatment in step a). In another embodiment, step b) includes determining the amount of ctDNA on day 1 of cycle 1 (baseline) and day 1 of cycle 2 of the chemotherapy treatment in step a).

[0129] In one embodiment, step c) includes selecting patients with a ctDNA reduction of <2-log. In another embodiment, step c) includes continuing chemotherapy for a total of six cycles (cycles 1 to 6) and adding glimepiride for a total of eight cycles (cycles 3 to 10).

[0130] In one embodiment, glimetuzumab is administered at a dose of 2.5 mg on day 8 of cycle 3, at a dose of 10 mg on day 15, at a dose of 30 mg on day 8 of cycles 4 through 6, and at a dose of 30 mg on day 1 of cycles 7 through 10.

[0131] In any of the foregoing aspects of the invention, ctDNA is circulating tumor DNA in plasma. In one such embodiment, the amount of ctDNA is measured in a blood sample from a patient. In one embodiment, the amount of ctDNA is measured in genomic DNA isolated from peripheral mononuclear cells or plasma-depleted whole blood (PDWB) or in cell-free DNA (cfDNA) in plasma. In one embodiment, ctDNA is measured in cell-free DNA in plasma. In one embodiment, ctDNA is determined by targeted next-generation sequencing (NGS).

[0132] In any of the above aspects of the invention, the length of each cycle is 21 days.

[0133] In one embodiment, the bispecific antibody targeting CD3 and CD20 is glimetuzumab, and the chemotherapy is rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), wherein the patient has been identified as high-risk for ctDNA if the ctDNA decreases by less than 2.5 log after one or two R-CHOP cycles.

[0134] In one aspect, the invention is characterized by a method for treating previously untreated patients with diffuse large B-cell lymphoma at high risk of circulating tumor DNA (ctDNA), the method comprising administering glimepiride in combination with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), wherein the patient is identified as high-risk for ctDNA if the ctDNA decreases by less than 2.5 log after one or two R-CHOP cycles. ctDNA is a novel biomarker that offers the possibility of early and sensitive identification of patients with DLBCL at high risk of first-line treatment failure. In the first-line setting, a subset of patients with DLBCL has a poor prognosis after treatment with R-CHOP immunochemotherapy. The inventors of this invention have discovered that treatment of previously untreated patients with high-risk ctDNA diffuse large B-cell lymphoma using a combination of glimetuzumab with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) produces unexpectedly high overall response and complete response rates.

[0135] In one embodiment, ctDNA reduction is determined by measuring ctDNA levels before or on the day of the first treatment (baseline) and after at least one R-CHOP treatment cycle. In one embodiment, ctDNA levels are determined on or before day 1 of the first R-CHOP treatment cycle (baseline) and day 1 of the second R-CHOP treatment cycle. In another embodiment, ctDNA levels are determined on day 1 of the first R-CHOP treatment cycle (baseline) and day 1 of the second R-CHOP treatment cycle. In one embodiment, a patient is identified as high-risk for ctDNA if the reduction in ctDNA is less than 2 log. In one embodiment, a patient is identified as high-risk for ctDNA if the reduction in ctDNA levels between day 1 of cycle 1 and day 1 of cycle 2, as determined by the AOA-NHL test, is less than 2 log-fold.

[0136] In another aspect of the invention, a method for treating previously untreated diffuse large B-cell lymphoma is provided, the method comprising:

[0137] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0138] b. Determine the amount of ctDNA at or before cycle 1 (baseline) and after at least one R-CHOP treatment cycle; and

[0139] c. Select patients whose ctDNA reduction is <2.5-log between baseline and at least one R-CHOP treatment cycle and continue treatment with R-CHOP and glimetuzumab.

[0140] In another aspect of the invention, a method for treating previously untreated diffuse large B-cell lymphoma is provided, the method comprising:

[0141] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0142] b. Determine the amount of ctDNA after cycle 1 (baseline) and at least one R-CHOP treatment cycle; and

[0143] c. Select patients whose ctDNA reduction is <2.5-log between baseline and at least one R-CHOP treatment cycle and continue treatment with R-CHOP and glimetuzumab.

[0144] In one embodiment, step a) includes applying R-CHOP on day 1 of cycles 1, 2, and optionally cycle 3. In another embodiment, step a) includes applying R-CHOP on day 1 of cycles 1 and 2.

[0145] In one embodiment, step b) includes determining the amount of ctDNA on or before day 1 of cycle 1 (baseline) and day 1 of cycle 2 or day 1 of cycle 3 of the R-CHOP treatment in step a). In another embodiment, step b) includes determining the amount of ctDNA on day 1 of cycle 1 (baseline) and day 1 of cycle 2 of the R-CHOP treatment in step a).

[0146] In one embodiment, step c) includes selecting patients with a ctDNA reduction of <2-log. In another embodiment, step c) includes continuing R-CHOP treatment for a total of six cycles (cycles 1 to 6) and adding glimetuzumab for a total of eight cycles (cycles 3 to 10).

[0147] In one aspect of the invention, the third treatment cycle comprises a first dose and a second dose of glimetuzumab, followed by a rest period. In one such embodiment, the third treatment cycle comprises a first dose of glimetuzumab on day 8 of the third cycle and a second dose of glimetuzumab on day 15 of the third cycle, followed by a 6-day rest period. In one embodiment, the subsequent two cycles (cycles 4 to 6) comprise a dose of glimetuzumab administered on day 8 of that cycle, followed by a 13-day rest period. In one embodiment, the remaining subsequent cycles (cycles 7 to 10) comprise a dose of glimetuzumab administered on day 1 of that cycle, followed by a 20-day rest period.

[0148] In one embodiment, a treatment cycle comprises 21 days. In another embodiment, a treatment cycle comprises 14 days. The treatment regimen according to the invention may comprise 6 or more treatment cycles, particularly 10 treatment cycles.

[0149] In one embodiment, glimetuzumab is administered at a dose of 2.5 mg on day 8 of cycle 3, at a dose of 10 mg on day 15 of cycle 3, at a dose of 30 mg on day 8 of cycles 4 through 6, and at a dose of 30 mg on day 1 of cycles 7 through 10.

[0150] In another embodiment, glucentumab is administered at a dose of 0.5 mg on day 4 of cycle 3, at a dose of 2.5 mg on day 8 of cycle 3, at a dose of 10 mg on day 15 of cycle 3, at a dose of 30 mg on day 8 of cycles 4 through 6, and at a dose of 30 mg on day 1 of cycles 7 through 10.

[0151] In another embodiment, glucantuzumab is administered at a dose of 0.5 mg on day 8 of cycle 3, at a dose of 2 mg on day 9, at a dose of 10 mg on day 15 of cycle 3, at a dose of 30 mg on day 8 of cycles 4 through 6, and at a dose of 30 mg on day 1 of cycles 7 through 10.

[0152] One aspect of the invention relates to a bispecific antibody targeting CD3 and CD20 comprising the VH and VL sequences of glimetuzumab, in a method for treating previously untreated diffuse large B-cell lymphoma at high (ct)DNA risk, the method comprising administering glimetuzumab in combination with chemotherapy to a patient, wherein the patient has been identified as at high ctDNA risk if the ctDNA level decreases by less than 2.5 log after one cycle of chemotherapy.

[0153] In one aspect, the bispecific antibody targeting CD3 and CD20 comprises the heavy chain of SEQ ID NO: 17, the heavy chain of SEQ ID NO: 18, the two light chains of SEQ ID NO: 20, and the light chain of SEQ ID NO: 19; and wherein the heavy and light chains are assembled to form a first Fab molecule that specifically binds to CD20, a second Fab molecule that specifically binds to CD3, and a third Fab molecule that specifically binds to CD20, and an Fc domain consisting of a first subunit and a second subunit capable of stable association.

[0154] In one embodiment, the chemotherapy therapy is selected from: rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP); rituximab, cyclophosphamide, doxorubicin and prednisone (R-CHP); or rituximab, ifosfamide, carboplatin and etoposide phosphate (R-ICE).

[0155] In one embodiment, glimetuzumab is provided in a method of treating previously untreated diffuse large B-cell lymphoma with high (ct)DNA risk, the method comprising administering glimetuzumab in combination with chemotherapy to a patient, wherein the patient has been identified as high-risk for ctDNA if the ctDNA level decreases by less than 2.5 log after one cycle of chemotherapy.

[0156] In one embodiment, the patient's ctDNA decreased by less than 2-log.

[0157] In one embodiment, the bispecific antibody targeting CD3 and CD20 is glimetuzumab, and the chemotherapy is rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), wherein the patient has been identified as high-risk for ctDNA if the ctDNA decreases by less than 2.5 log after one or two R-CHOP cycles.

[0158] One aspect of the invention relates to glimetuzumab, in a method of treating previously untreated diffuse large B-cell lymphoma with high (ct)DNA risk, the method comprising administering glimetuzumab to a patient in combination with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), wherein the patient has been identified as high-risk for ctDNA if the ctDNA decreases by less than 2.5 log after one R-CHOP cycle.

[0159] In one embodiment, ctDNA reduction is determined by measuring ctDNA levels before or on the day of the first treatment (baseline) and after at least one R-CHOP treatment cycle. In one embodiment, ctDNA levels are determined on or before day 1 of the first R-CHOP treatment cycle (baseline) and day 1 of the second R-CHOP treatment cycle. In another embodiment, ctDNA levels are determined on day 1 of the first R-CHOP treatment cycle (baseline) and day 1 of the second R-CHOP treatment cycle. In one embodiment, a patient is identified as high-risk for ctDNA if the reduction in ctDNA is less than 2 log. In one embodiment, a patient is identified as high-risk for ctDNA if the reduction in ctDNA levels between day 1 of cycle 1 and day 1 of cycle 2, as determined by the AOA-NHL test, is less than 2 log-fold.

[0160] One aspect of the invention relates to glimetuzumab, in a method of treating previously untreated diffuse large B-cell lymphoma with high (ct)DNA risk, the method comprising administering glimetuzumab to a patient in combination with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), wherein the patient has been identified as high-risk for ctDNA if the patient's ctDNA reduction is less than 2-log, wherein the reduction in ctDNA is measured on or before day 1 of the first R-CHOP treatment cycle (baseline) and day 1 of the second R-CHOP treatment cycle. In one such embodiment, the reduction in ctDNA is measured on day 1 of the first R-CHOP treatment cycle (baseline) and day 1 of the second R-CHOP treatment cycle.

[0161] One aspect of the present invention relates to glimetuzumab, in a method of treating previously untreated diffuse large B-cell lymphoma, the method comprising:

[0162] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0163] b. Determine the amount of ctDNA at or before cycle 1 (baseline) and after at least one R-CHOP treatment cycle; and

[0164] c. Select patients whose ctDNA reduction is <2.5-log between baseline and at least one R-CHOP treatment cycle and continue treatment with R-CHOP and glimetuzumab.

[0165] One aspect of the present invention relates to glimetuzumab, in a method of treating previously untreated diffuse large B-cell lymphoma with high (ct)DNA risk, the method comprising:

[0166] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0167] b. Determine the amount of ctDNA at or before cycle 1 (baseline) and after at least one R-CHOP treatment cycle; and

[0168] c. Select patients whose ctDNA reduction is <2.5-log between baseline and at least one R-CHOP treatment cycle and continue treatment with R-CHOP and glimetuzumab.

[0169] In another aspect of the invention, glimetuzumab is provided in a method of treating previously untreated diffuse large B-cell lymphoma, the method comprising:

[0170] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0171] b. Determine the amount of ctDNA after cycle 1 (baseline) and at least one R-CHOP treatment cycle; and

[0172] c. Select patients whose ctDNA reduction is <2.5-log between baseline and at least one R-CHOP treatment cycle and continue treatment with R-CHOP and glimetuzumab.

[0173] In one embodiment, step a) includes applying R-CHOP on day 1 of cycles 1, 2, and optionally cycle 3. In another embodiment, step a) includes applying R-CHOP on day 1 of cycles 1 and 2.

[0174] In one embodiment, step b) includes determining the amount of ctDNA on or before day 1 of cycle 1 (baseline) and day 1 of cycle 2 or day 1 of cycle 3 of the R-CHOP treatment in step a). In another embodiment, step b) includes determining the amount of ctDNA on day 1 of cycle 1 (baseline) and day 1 of cycle 2 of the R-CHOP treatment in step a).

[0175] In one embodiment, step c) includes selecting patients with a ctDNA reduction of <2-log. In another embodiment, step c) includes continuing R-CHOP treatment for a total of six cycles (cycles 1 to 6) and adding glimetuzumab for a total of eight cycles (cycles 3 to 10).

[0176] In one aspect of the invention, a method of using glimetuzumab for treating previously untreated diffuse large B-cell lymphoma is provided, the method comprising:

[0177] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0178] b. Determine the amount of ctDNA on day 1 of cycle 1 (baseline) and day 1 of cycle 2; and

[0179] c. Select patients whose ctDNA decrease is <2-log between baseline and one R-CHOP treatment cycle and continue treatment with R-CHOP and glimetuzumab.

[0180] In another aspect of the invention, glimetuzumab is provided in a method of treating previously untreated diffuse large B-cell lymphoma, the method comprising:

[0181] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0182] b. Determine the amount of ctDNA on day 1 of cycle 1 (baseline) and day 1 of cycle 2; and

[0183] c. Select patients whose ctDNA decreased by <2-log between baseline and one R-CHOP treatment cycle, and continue R-CHOP treatment for a total of six cycles (cycles 1 to 6), and add glimetuzumab for a total of eight cycles (cycles 3 to 10).

[0184] In another aspect of the invention, glimetuzumab is provided in a method of treating previously untreated diffuse large B-cell lymphoma, the method comprising:

[0185] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for 2 cycles;

[0186] b. Determine the amount of ctDNA on day 1 of cycle 1 (baseline) and day 1 of cycle 2; and

[0187] c. Select patients whose ctDNA decreased by <2-log between baseline and one R-CHOP treatment cycle, and continue R-CHOP treatment for a total of six cycles (cycles 1 to 6), and add glimetuzumab for a total of eight cycles (cycles 3 to 10).

[0188] In one aspect of the invention, the third treatment cycle comprises a first dose and a second dose of glimetuzumab, followed by a rest period. In one such embodiment, the third treatment cycle comprises a first dose of glimetuzumab on day 8 of the third cycle and a second dose of glimetuzumab on day 15 of the third cycle, followed by a 6-day rest period. In one embodiment, the subsequent two cycles (cycles 4 to 6) comprise a dose of glimetuzumab administered on day 8 of that cycle, followed by a 13-day rest period. In one embodiment, the remaining subsequent cycles (cycles 7 to 10) comprise a dose of glimetuzumab administered on day 1 of that cycle, followed by a 20-day rest period.

[0189] In one embodiment, a treatment cycle comprises 21 days. In another embodiment, a treatment cycle comprises 14 days. The treatment regimen according to the invention may comprise 6 or more treatment cycles, particularly 10 treatment cycles.

[0190] In one embodiment, glimetuzumab is administered at a dose of 2.5 mg on day 8 of cycle 3, at a dose of 10 mg on day 15 of cycle 3, at a dose of 30 mg on day 8 of cycles 4 through 6, and at a dose of 30 mg on day 1 of cycles 7 through 10.

[0191] In another embodiment, glucentumab is administered at a dose of 0.5 mg on day 4 of cycle 3, at a dose of 2.5 mg on day 8 of cycle 3, at a dose of 10 mg on day 15 of cycle 3, at a dose of 30 mg on day 8 of cycles 4 through 6, and at a dose of 30 mg on day 1 of cycles 7 through 10.

[0192] In another embodiment, glucantuzumab is administered at a dose of 0.5 mg on day 8 of cycle 3, at a dose of 2 mg on day 9, at a dose of 10 mg on day 15 of cycle 3, at a dose of 30 mg on day 8 of cycles 4 through 6, and at a dose of 30 mg on day 1 of cycles 7 through 10.

[0193] In one aspect of the invention, glimetuzumab is provided for use in a method of treating previously untreated diffuse large B-cell lymphoma, the method comprising:

[0194] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0195] b. Determine the amount of ctDNA on day 1 of cycle 1 (baseline) and day 1 of cycle 2; and

[0196] c. Select patients whose ctDNA reduction is <2-log between baseline and one R-CHOP treatment cycle and continue R-CHOP treatment for a total of six cycles (cycles 1 to 6), and add glimetuzumab for a total of eight cycles (cycles 3 to 10), wherein glimetuzumab is administered at doses of 2.5 mg and 10 mg on different days of cycle 3, and at doses of 30 mg on cycles 4 to 10.

[0197] In one aspect of the invention, glimetuzumab is provided for use in a method of treating previously untreated diffuse large B-cell lymphoma, the method comprising:

[0198] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for 2 cycles;

[0199] b. Determine the amount of ctDNA on day 1 of cycle 1 (baseline) and day 1 of cycle 2; and

[0200] c. Select patients whose ctDNA reduction is <2-log between baseline and one R-CHOP treatment cycle and continue R-CHOP treatment for a total of six cycles (cycles 1 to 6), and add glimetuzumab for a total of eight cycles (cycles 3 to 10), wherein glimetuzumab is administered at doses of 2.5 mg and 10 mg on different days of cycle 3, and at doses of 30 mg on cycles 4 to 10.

[0201] In one aspect of the invention, glimetuzumab is provided for use in a method of treating previously untreated diffuse large B-cell lymphoma, the method comprising:

[0202] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0203] b. Determine the amount of ctDNA on day 1 of cycle 1 (baseline) and day 1 of cycle 2; and

[0204] c. Select patients whose ctDNA reduction is <2-log between baseline and one R-CHOP treatment cycle and continue R-CHOP treatment for a total of six cycles (cycles 1 to 6), and add glimetuzumab for a total of eight cycles (cycles 3 to 10), wherein glimetuzumab is administered at a dose of 2.5 mg on day 8 of cycle 3, at a dose of 10 mg on day 15 of cycle 3, at a dose of 30 mg on day 8 of cycles 4 to 6, and at a dose of 30 mg on day 1 of cycles 7 to 10.

[0205] In one aspect of the invention, glimetuzumab is provided for use in a method of treating previously untreated diffuse large B-cell lymphoma, the method comprising:

[0206] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for 2 cycles;

[0207] b. Determine the amount of ctDNA on day 1 of cycle 1 (baseline) and day 1 of cycle 2; and

[0208] c. Select patients whose ctDNA reduction is <2-log between baseline and one R-CHOP treatment cycle and continue R-CHOP treatment for a total of six cycles (cycles 1 to 6), and add glimetuzumab for a total of eight cycles (cycles 3 to 10), wherein glimetuzumab is administered at a dose of 2.5 mg on day 8 of cycle 3, at a dose of 10 mg on day 15 of cycle 3, at a dose of 30 mg on day 8 of cycles 4 to 6, and at a dose of 30 mg on day 1 of cycles 7 to 10.

[0209] In one embodiment, glimetuzumab is administered over 4 hours (±15 minutes) on days 8 and 15 of cycle 3, day 8 of cycles 4 to 6, and day 1 of cycles 7 to 10. In another embodiment, the infusion time is extended to a maximum of 8 hours. In another embodiment, the infusion time is shortened to 2 hours (±15 minutes). In one embodiment, glimetuzumab is administered over 2 hours (±15 minutes) on day 8 of cycles 5 to 6. In one embodiment, glimetuzumab is administered over 4 hours (±15 minutes) on days 8 and 15 of cycle 3, day 8 of cycle 4, and day 8 of cycles 5 to 6. In one embodiment, glimetuzumab is administered over 4 hours (±15 minutes) on days 8 and 15 of cycle 3, day 8 of cycle 4, and day 8 of cycles 5 to 6 and day 1 of cycles 7 to 10. In another embodiment, the infusion time is reduced to 90 minutes (±30 minutes). In one embodiment, glucintumab is administered over 90 minutes (±30 minutes) on day 1 of cycles 7, 8, 9, and 10. In one embodiment, glucintumab is administered over 4 hours (±15 minutes) on days 8 and 15 of cycle 3, and on day 8 of cycles 4 through 6, and over 90 minutes (±30 minutes) on day 1 of cycles 7, 8, 9, and 10. In one embodiment, glucintumab is administered over 4 hours (±15 minutes) on days 8 and 15 of cycle 3, and on day 8 of cycle 4, and over 2 hours (±15 minutes) on day 8 of cycles 5 through 6, and over 90 minutes (±30 minutes) on day 1 of cycles 7, 8, 9, and 10.

[0210] In any of the foregoing aspects of the invention, ctDNA is circulating tumor DNA in plasma. In one such embodiment, the amount of ctDNA is measured in a blood sample from a patient. In one embodiment, the amount of ctDNA is measured in genomic DNA isolated from peripheral mononuclear cells or plasma-depleted whole blood (PDWB) or in cell-free DNA (cfDNA) in plasma. In one embodiment, ctDNA is measured in cell-free DNA in plasma.

[0211] In one embodiment, ctDNA is determined by targeted next-generation sequencing (NGS). In another embodiment, immunoglobulin loci (i.e., IGH, IGL, and IGK) are assessed. In yet another embodiment, ctDNA is determined by high-throughput amplicon sequencing (IgHTS; or clonoSEQ assay) of immunoglobulin loci. IgHTS allows for sensitive detection of lymphoma-specific clonal types from cellular DNA or cfDNA by amplifying only rearranged VDJ sequences using shared primers followed by sequencing library construction. In another embodiment, ctDNA levels are assessed using multiple genomic alterations. In one embodiment, multiple genomic alterations are defined for NHL or DLBCL. In yet another embodiment, ctDNA levels are assessed using a targeted ultra-deep NGS platform. These platforms detect multiple mutations in each individual case. In one embodiment, the targeted ultra-deep NGS platform is based on a hybridization capture method (CAPP-SEQ, as described in Newman AM, Bratman SV, To J et al., An ultrasensitive method for quantitating circulating tumor DNA with broadpatient coverage. Nat Med. 2014;20(5):548-554, the entirety of which is incorporated herein by reference). In another embodiment, ctDNA is evaluated according to an amplicon-based method (i.e., Lymphopanel, as described in Bohers E, Viailly PJ, Becker S et al., Non-invasive monitoring of diffuse large B-cell lymphoma bycell-free DNA high-throughput targeted sequencing: analysis of a prospective cohort. Blood Cancer J. 2018;8(8):74, the entirety of which is incorporated herein by reference). Hybridization capture and amplicon-based methods utilize ultra-high sequencing depth by focusing on relatively small portions of the genome. By sequencing thousands of unique molecules at each location, while utilizing PCR-induced error suppression, these methods enable sensitive detection of residual disease from plasma DNA. Monitoring ctDNA levels via targeted sequencing first requires identifying tumor-specific alterations, typically through sequencing of tumor DNA or pre-treatment plasma DNA, as well as matching germline DNA used to remove primary variants.This generates a series of tumor-specific alterations that can then be investigated in future samples to measure ctDNA levels. By targeting multiple recurrently mutated regions of the genome, including those affected by single nucleotide variants (SNVs), insertions / deletions, gene rearrangements, and copy number variations (CNVs), these methods enable deeper insights into tumor biology and ultrasensitive disease detection. Compared to IgHTS, the initial approach using CAPP-Seq identified >100 independent mutations that enabled tumor surveillance, thereby increasing disease detection in pretreatment ctDNA samples from 23 DLBCL patients from 74% to 100% (Scherer F, Kurtz DM, Newman AM et al. Distinct biological subtypes and patterns of genome evolution in lymphoma revealed by circulating tumor DNA. Sci Transl Med. 2016;8(364):364ra155, which is incorporated herein by reference).

[0212] In one embodiment, ctDNA is measured using a Cancer Personalized Deep Sequencing Analysis (CAPPseq) assay (tumor-targeted NGS), an AVENIO NHL assay, an assay measuring SNVs (single nucleotide variants), an immunoglobulin (Ig) NGS assay, a Clonoseq assay (Adaptive), a PhaseSeq assay (tumor-targeted NGS), or a Foresight assay measuring PVs (phase variants, multiple mutations on a single DNA molecule).

[0213] The AVENIO Oncology Assay for Non-Hodgkin Lymphoma (AOA-NHL) test uses targeted NGS and sequencing of genomic DNA isolated from peripheral mononuclear cells or plasma-depleted whole blood (PDWB) and plasma cell-free DNA (cfDNA) to qualitatively detect ctDNA as an indicator of minimal residual disease (MRD). The AOA-NHL test can be used to determine the ctDNA status of patients with DLBCL (Herrera et al. Blood Adv 2022, incorporated herein by reference). In one embodiment, plasma is used to determine ctDNA using the AVENIO Oncology Assay for Non-Hodgkin Lymphoma (AOA-NHL) test.

[0214] In one embodiment, SNV mutations in ctDNA at baseline are analyzed using an AVENIO NHL CAPP-Seq assay. In another embodiment, ctDNA is determined by identifying tumor-specific variants (TSVs) by subtracting sample variants present in a normal PDWB sample from those variants detected in a plasma sample.

[0215] In one aspect of the invention, a method of using glimetuzumab for treating previously untreated diffuse large B-cell lymphoma is provided, the method comprising:

[0216] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0217] b. Determine the amount of ctDNA from plasma samples on day 1 of cycle 1 (baseline) and day 1 of cycle 2; and

[0218] c. Select patients whose ctDNA decrease is <2-log between baseline and one R-CHOP treatment cycle and continue treatment with R-CHOP and glimetuzumab.

[0219] In one aspect of the invention, a method of using glimetuzumab for treating previously untreated diffuse large B-cell lymphoma is provided, the method comprising:

[0220] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0221] b. Determine the amount of ctDNA from plasma samples on day 1 of cycle 1 (baseline) and day 1 of cycle 2; and

[0222] c. Select patients whose ctDNA decreased by <2-log between baseline and one R-CHOP treatment cycle, and continue R-CHOP treatment for a total of six cycles (cycles 1 to 6), and add glimetuzumab for a total of eight cycles (cycles 3 to 10).

[0223] In one aspect of the invention, a method of using glimetuzumab for treating previously untreated diffuse large B-cell lymphoma is provided, the method comprising:

[0224] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0225] b. Determine the amount of ctDNA from plasma samples on day 1 of cycle 1 (baseline) and day 1 of cycle 2; and

[0226] c. Select patients whose ctDNA reduction is <2-log between baseline and one R-CHOP treatment cycle and continue R-CHOP treatment for a total of six cycles (cycles 1 to 6), and add glimetuzumab for a total of eight cycles (cycles 3 to 10), wherein glimetuzumab is administered at doses of 2.5 mg and 10 mg on different days of cycle 3, and at doses of 30 mg on cycles 4 to 10.

[0227] In one aspect of the invention, a method of using glimetuzumab for treating previously untreated diffuse large B-cell lymphoma is provided, the method comprising:

[0228] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0229] b. Determine the amount of ctDNA from plasma samples on day 1 of cycle 1 (baseline) and day 1 of cycle 2; and

[0230] c. Select patients whose ctDNA reduction is <2-log between baseline and one R-CHOP treatment cycle and continue R-CHOP treatment for a total of six cycles (cycles 1 to 6), and add glimetuzumab for a total of eight cycles (cycles 3 to 10), wherein glimetuzumab is administered at a dose of 2.5 mg on day 8 of cycle 3, at a dose of 10 mg on day 15 of cycle 3, at a dose of 30 mg on day 8 of cycles 4 to 6, and at a dose of 30 mg on day 1 of cycles 7 to 10.

[0231] In one aspect of the invention, a method of using glimetuzumab for treating previously untreated diffuse large B-cell lymphoma is provided, the method comprising:

[0232] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0233] b. The amount of ctDNA was determined using the AVENIO Oncology Assay (AOA-NHL) for non-Hodgkin's lymphoma in plasma on day 1 of cycle 1 (baseline) and day 1 of cycle 2; and

[0234] c. Select patients whose ctDNA decrease is <2-log between baseline and one R-CHOP treatment cycle and continue treatment with R-CHOP and glimetuzumab.

[0235] In one aspect of the invention, a method of using glimetuzumab for treating previously untreated diffuse large B-cell lymphoma is provided, the method comprising:

[0236] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0237] b. The amount of ctDNA was determined using the AVENIO Oncology Assay (AOA-NHL) for non-Hodgkin's lymphoma in plasma on day 1 of cycle 1 (baseline) and day 1 of cycle 2; and

[0238] c. Select patients whose ctDNA decreased by <2-log between baseline and one R-CHOP treatment cycle, and continue R-CHOP treatment for a total of six cycles (cycles 1 to 6), and add glimetuzumab for a total of eight cycles (cycles 3 to 10).

[0239] In one aspect of the invention, a method of using glimetuzumab for treating previously untreated diffuse large B-cell lymphoma is provided, the method comprising:

[0240] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0241] b. The amount of ctDNA was determined using the AVENIO Oncology Assay (AOA-NHL) for non-Hodgkin's lymphoma in plasma on day 1 of cycle 1 (baseline) and day 1 of cycle 2; and

[0242] c. Select patients whose ctDNA reduction is <2-log between baseline and one R-CHOP treatment cycle and continue R-CHOP treatment for a total of six cycles (cycles 1 to 6), and add glimetuzumab for a total of eight cycles (cycles 3 to 10), wherein glimetuzumab is administered at doses of 2.5 mg and 10 mg on different days of cycle 3, and at doses of 30 mg on cycles 4 to 10.

[0243] In any of the above aspects of the present invention, rituximab is administered at 375 mg / m². 2 The dose is administered intravenously. In one embodiment, rituximab is administered every 21 days. In one embodiment, rituximab is administered at a dose of 375 mg / m² every 21 days. 2 The dose was administered intravenously to participants along with CHOP on day 1 of cycles 1 through 6.

[0244] In one embodiment, CHOP is administered over six 21-day cycles. In one embodiment, CHOP is administered as follows: cyclophosphamide at 750 mg / m² 2 Doxorubicin was administered intravenously at a dose of 50 mg / m². 2 Vincristine was administered intravenously at a dose of 1.4 mg / m². 2 The dose is administered via IV bolus, and prednisone or prednisolone is administered at a dose of 100 mg / day. In one embodiment, cyclophosphamide is administered at a dose of 750 mg / m² on day 1. 2 Doxorubicin was administered intravenously at a dose of 50 mg / m² on day 1. 2 The dose was administered intravenously, vincristine at 1.4 mg / m² on day 1. 2 The dose is administered via IV bolus, and prednisone or prednisolone is administered at a dose of 100 mg / day from day 1 to day 5. In one embodiment, the maximum dose of vincristine per cycle is 2 mg. In one embodiment, for subjects ≥70 years of age, the maximum dose of vincristine per cycle is 1.5 mg. In one embodiment, prednisone or prednisolone is administered intravenously on day 1, and the remaining doses on days 2 to 5 are administered orally. In one embodiment, all doses of prednisone or prednisolone are administered orally. In one embodiment, prednisone or prednisolone is replaced with 80 mg of methylprednisolone.

[0245] In one embodiment, prednisone (100 mg) or prednisolone (100 mg) is administered prior to rituximab.

[0246] In any of the above aspects of the present invention, rituximab is administered at 375 mg / m². 2 Cyclophosphamide was administered intravenously at a dose of 750 mg / m². 2 Doxorubicin was administered intravenously at a dose of 50 mg / m², and vincristine at a dose of 1.4 mg / m². 2 The dose was administered via IV bolus, and prednisone was administered at a dose of 100 mg / day from day 1 to day 5.

[0247] In any of the above aspects of the invention, the length of each cycle is 21 days.

[0248] In any of the foregoing aspects of the invention, administration of such treatment to multiple human patients at high risk of ctDNA results in an overall response rate (ORR) of at least 80%, at least 85%, at least 90%, or at least 95%. In one embodiment, the ORR is at least 90%. In another embodiment, the ORR is at least 95%.

[0249] In one embodiment, ORR is determined at the end of treatment. In one embodiment, ORR is determined by PET-CT scan. In one embodiment, ORR is determined according to the 2014 Lugano response criteria. In one embodiment, ORR is determined between day 15 and day 21 of cycle 2. In one embodiment, ORR is measured by PET / CT scan and determined at the end of treatment by an automated response (aLugano) according to the 2014 Lugano response criteria. In one embodiment, aLugano analysis is performed in a PET / CT-evaluable population defined as all patients with available PET / CT scans at baseline and during treatment.

[0250] In one embodiment of any of the foregoing aspects of the invention, administration of such treatment to multiple human patients at high risk of ctDNA results in a complete response (CR) rate of at least 75%, at least 80%, or at least 85%. In one embodiment, the CR rate is at least 80%. In another embodiment, the CR rate is at least 85%.

[0251] In one embodiment, a method of using glimetuzumab for the treatment of previously untreated diffuse large B-cell lymphoma at high risk of ctDNA includes administering glimetuzumab in combination with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) to a patient, wherein the patient is identified as high-risk for ctDNA if the ctDNA decreases by less than 2-log after one R-CHOP cycle, and wherein administration of such treatment to multiple human patients at high risk of ctDNA produces an overall response rate (ORR) of at least 80%, at least 85%, at least 90%, or at least 95%. In one embodiment, the ORR is at least 90%. In another embodiment, the ORR is at least 95%.

[0252] In one embodiment, a method of treating previously untreated diffuse large B-cell lymphoma at high risk of ctDNA with glucentumab is provided, comprising administering glucentumab to a patient in combination with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), wherein the patient is identified as at high risk of ctDNA if the ctDNA decreases by less than 2-log after one R-CHOP cycle, wherein administration of such treatment to multiple human patients at high risk of ctDNA produces a complete response (CR) rate of at least 75%, at least 80%, or at least 85%. In one embodiment, the CR rate is at least 80%. In another embodiment, the CR rate is at least 85%.

[0253] In one embodiment, ctDNA reduction is determined by measuring ctDNA levels before or on the day of the first treatment (baseline) and after at least one R-CHOP treatment cycle. In one embodiment, ctDNA levels are determined on or before day 1 of the first R-CHOP treatment cycle (baseline) and day 1 of the second R-CHOP treatment cycle. In another embodiment, ctDNA levels are determined on day 1 of the first R-CHOP treatment cycle (baseline) and day 1 of the second R-CHOP treatment cycle. In one embodiment, a patient is identified as high-risk for ctDNA if the reduction in ctDNA is less than 2 log. In one embodiment, a patient is identified as high-risk for ctDNA if the reduction in ctDNA levels between day 1 of cycle 1 and day 1 of cycle 2, as determined by the AOA-NHL test, is less than 2 log-fold.

[0254] One aspect of the invention relates to glimetuzumab for the treatment of previously untreated diffuse large B-cell lymphoma at high (ct)DNA risk, the method comprising administering glimetuzumab to a patient in combination with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), wherein the patient is identified as high-ctDNA risk if the patient's ctDNA reduction is less than 2-log, wherein the reduction in ctDNA is measured on or before day 1 of the first R-CHOP treatment cycle (baseline) and day 1 of the second R-CHOP treatment cycle, wherein administration of such treatment to multiple high-ctDNA risk human patients produces an overall response rate (ORR) of at least 80%, at least 85%, at least 90%, or at least 95%. In one embodiment, the ORR is at least 90%. In one embodiment, the ORR is at least 95%. In one such embodiment, the reduction in ctDNA is measured on day 1 of the first R-CHOP treatment cycle (baseline) and day 1 of the second R-CHOP treatment cycle.

[0255] One aspect of the invention relates to glimetuzumab, in a method of treating previously untreated diffuse large B-cell lymphoma at high (ct)DNA risk, the method comprising administering glimetuzumab to a patient in combination with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), wherein the patient has been identified as ctDNA high-risk if the patient's ctDNA reduction is less than 2-log, wherein the ctDNA reduction is measured on or before day 1 of the first R-CHOP treatment cycle (baseline) and day 1 of the second R-CHOP treatment cycle, wherein administration of such treatment to multiple ctDNA high-risk human patients produces a complete response (CR) rate of at least 75%, at least 80%, or at least 85%. In one embodiment, the CR rate is at least 80%. In another embodiment, the CR rate is at least 85%.

[0256] In one such embodiment, the reduction in ctDNA was measured on day 1 of the first R-CHOP treatment cycle (baseline) and day 1 of the second R-CHOP treatment cycle.

[0257] One aspect of the invention relates to glimetuzumab, in a method of treating previously untreated diffuse large B-cell lymphoma to produce a complete response (CR) rate of at least 75%, at least 80%, or at least 85%, the method comprising:

[0258] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0259] b. Determine the amount of ctDNA at or before cycle 1 (baseline) and after at least one R-CHOP treatment cycle; and

[0260] c. Select patients whose ctDNA reduction is <2.5-log between baseline and at least one R-CHOP treatment cycle, and continue treatment with R-CHOP and glimetuzumab, where

[0261] In one embodiment, the CR rate is at least 80%. In another embodiment, the CR rate is at least 85%.

[0262] One aspect of the invention relates to glimetuzumab, in a method of treating previously untreated diffuse large B-cell lymphoma to produce an overall response rate (ORR) of at least 80%, at least 85%, at least 90%, or at least 95%, the method comprising:

[0263] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0264] b. Determine the amount of ctDNA at or before cycle 1 (baseline) and after at least one R-CHOP treatment cycle; and

[0265] c. Select patients whose ctDNA reduction is <2.5-log between baseline and at least one R-CHOP treatment cycle, and continue treatment with R-CHOP and glimetuzumab, where

[0266] In one embodiment, the ORR is at least 90%. In another embodiment, the ORR is at least 95%.

[0267] One aspect of the invention relates to glimetuzumab, in a method of treating previously untreated diffuse large B-cell lymphoma with high (ct)DNA risk to produce a complete response (CR) rate of at least 75%, at least 80%, or at least 85%, the method comprising:

[0268] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0269] b. Determine the amount of ctDNA at or before cycle 1 (baseline) and after at least one R-CHOP treatment cycle; and

[0270] c. Select patients whose ctDNA reduction is <2.5-log between baseline and at least one R-CHOP treatment cycle and continue treatment with R-CHOP and glimetuzumab.

[0271] In one embodiment, the CR rate is at least 80%. In another embodiment, the CR rate is at least 85%.

[0272] In another aspect of the invention, glimetuzumab is provided in a method for treating previously untreated diffuse large B-cell lymphoma to produce a complete response (CR) rate of at least 75%, at least 80%, or at least 85%, the method comprising:

[0273] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0274] b. Determine the amount of ctDNA after cycle 1 (baseline) and at least one R-CHOP treatment cycle; and

[0275] c. Select patients whose ctDNA reduction is <2.5-log between baseline and at least one R-CHOP treatment cycle and continue treatment with R-CHOP and glimetuzumab.

[0276] In one embodiment, the CR rate is at least 80%. In another embodiment, the CR rate is at least 85%.

[0277] In one embodiment, step a) includes applying R-CHOP on day 1 of cycles 1, 2, and optionally cycle 3. In another embodiment, step a) includes applying R-CHOP on day 1 of cycles 1 and 2.

[0278] In one embodiment, step b) includes determining the amount of ctDNA on or before day 1 of cycle 1 (baseline) and day 1 of cycle 2 or day 1 of cycle 3 of the R-CHOP treatment in step a). In another embodiment, step b) includes determining the amount of ctDNA on day 1 of cycle 1 (baseline) and day 1 of cycle 2 of the R-CHOP treatment in step a).

[0279] In one embodiment, step c) includes selecting patients with a ctDNA reduction of <2-log. In another embodiment, step c) includes continuing R-CHOP treatment for a total of six cycles (cycles 1 to 6) and adding glimetuzumab for a total of eight cycles (cycles 3 to 10).

[0280] In one aspect of the invention, glimetuzumab is provided in a method of treating previously untreated diffuse large B-cell lymphoma to produce a complete response (CR) rate of at least 75%, at least 80%, or at least 85%, the method comprising:

[0281] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0282] b. Determine the amount of ctDNA on day 1 of cycle 1 (baseline) and day 1 of cycle 2; and

[0283] c. Select patients whose ctDNA decrease is <2-log between baseline and one R-CHOP treatment cycle and continue treatment with R-CHOP and glimetuzumab.

[0284] In one embodiment, the CR rate is at least 80%. In another embodiment, the CR rate is at least 85%.

[0285] In another aspect of the invention, glimetuzumab is provided in a method for treating previously untreated diffuse large B-cell lymphoma to produce a complete response (CR) rate of at least 75%, at least 80%, or at least 85%, the method comprising:

[0286] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0287] b. Determine the amount of ctDNA on day 1 of cycle 1 (baseline) and day 1 of cycle 2; and

[0288] c. Select patients whose ctDNA decreased by <2-log between baseline and one R-CHOP treatment cycle, and continue R-CHOP treatment for a total of six cycles (cycles 1 to 6), and add glimetuzumab for a total of eight cycles (cycles 3 to 10).

[0289] In one embodiment, the CR rate is at least 80%. In another embodiment, the CR rate is at least 85%.

[0290] In another aspect of the invention, glimetuzumab is provided in a method for treating previously untreated diffuse large B-cell lymphoma to produce a complete response (CR) rate of at least 75%, at least 80%, or at least 85%, the method comprising:

[0291] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for 2 cycles;

[0292] b. Determine the amount of ctDNA on day 1 of cycle 1 (baseline) and day 1 of cycle 2; and

[0293] c. Select patients whose ctDNA decreased by <2-log between baseline and one R-CHOP treatment cycle, and continue R-CHOP treatment for a total of six cycles (cycles 1 to 6), and add glimetuzumab for a total of eight cycles (cycles 3 to 10).

[0294] In one embodiment, the CR rate is at least 80%. In another embodiment, the CR rate is at least 85%.

[0295] In one aspect of the invention, the third treatment cycle comprises a first dose and a second dose of glimetuzumab, followed by a rest period. In one such embodiment, the third treatment cycle comprises a first dose of glimetuzumab on day 8 of the third cycle and a second dose of glimetuzumab on day 15 of the third cycle, followed by a 6-day rest period. In one embodiment, the subsequent two cycles (cycles 4 to 6) comprise a dose of glimetuzumab administered on day 8 of that cycle, followed by a 13-day rest period. In one embodiment, the remaining subsequent cycles (cycles 7 to 10) comprise a dose of glimetuzumab administered on day 1 of that cycle, followed by a 20-day rest period.

[0296] In one embodiment, a treatment cycle comprises 21 days. In another embodiment, a treatment cycle comprises 14 days. The treatment regimen according to the invention may comprise 6 or more treatment cycles, particularly 10 treatment cycles.

[0297] In one embodiment, glimetuzumab is administered at a dose of 2.5 mg on day 8 of cycle 3, at a dose of 10 mg on day 15 of cycle 3, at a dose of 30 mg on day 8 of cycles 4 through 6, and at a dose of 30 mg on day 1 of cycles 7 through 10.

[0298] In another embodiment, glucentumab is administered at a dose of 0.5 mg on day 4 of cycle 3, at a dose of 2.5 mg on day 8 of cycle 3, at a dose of 10 mg on day 15 of cycle 3, at a dose of 30 mg on day 8 of cycles 4 through 6, and at a dose of 30 mg on day 1 of cycles 7 through 10.

[0299] In another embodiment, glucantuzumab is administered at a dose of 0.5 mg on day 8 of cycle 3, at a dose of 2 mg on day 9, at a dose of 10 mg on day 15 of cycle 3, at a dose of 30 mg on day 8 of cycles 4 through 6, and at a dose of 30 mg on day 1 of cycles 7 through 10.

[0300] In one aspect of the invention, glimetuzumab is provided in a method of treating previously untreated diffuse large B-cell lymphoma to produce a complete response (CR) rate of at least 75%, at least 80%, or at least 85%, wherein the method comprises:

[0301] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0302] b. Determine the amount of ctDNA on day 1 of cycle 1 (baseline) and day 1 of cycle 2; and

[0303] c. Select patients whose ctDNA reduction is <2-log between baseline and one R-CHOP treatment cycle and continue R-CHOP treatment for a total of six cycles (cycles 1 to 6), and add glimetuzumab for a total of eight cycles (cycles 3 to 10), wherein glimetuzumab is administered at doses of 2.5 mg and 10 mg on different days of cycle 3, and at doses of 30 mg on cycles 4 to 10.

[0304] In one embodiment, the CR rate is at least 80%. In another embodiment, the CR rate is at least 85%.

[0305] In one aspect of the invention, glimetuzumab is provided in a method of treating previously untreated diffuse large B-cell lymphoma to produce a complete response (CR) rate of at least 75%, at least 80%, or at least 85%, wherein the method comprises:

[0306] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for 2 cycles;

[0307] b. Determine the amount of ctDNA on day 1 of cycle 1 (baseline) and day 1 of cycle 2; and

[0308] c. Select patients whose ctDNA reduction is <2-log between baseline and one R-CHOP treatment cycle and continue R-CHOP treatment for a total of six cycles (cycles 1 to 6), and add glimetuzumab for a total of eight cycles (cycles 3 to 10), wherein glimetuzumab is administered at doses of 2.5 mg and 10 mg on different days of cycle 3, and at doses of 30 mg on cycles 4 to 10.

[0309] In one embodiment, the CR rate is at least 80%. In another embodiment, the CR rate is at least 85%.

[0310] In one aspect of the invention, glimetuzumab is provided in a method of treating previously untreated diffuse large B-cell lymphoma to produce a complete response (CR) rate of at least 75%, at least 80%, or at least 85%, wherein the method comprises:

[0311] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0312] b. Determine the amount of ctDNA on day 1 of cycle 1 (baseline) and day 1 of cycle 2; and

[0313] c. Select patients whose ctDNA reduction is <2-log between baseline and one R-CHOP treatment cycle and continue R-CHOP treatment for a total of six cycles (cycles 1 to 6), and add glimetuzumab for a total of eight cycles (cycles 3 to 10), wherein glimetuzumab is administered at a dose of 2.5 mg on day 8 of cycle 3, at a dose of 10 mg on day 15 of cycle 3, at a dose of 30 mg on day 8 of cycles 4 to 6, and at a dose of 30 mg on day 1 of cycles 7 to 10.

[0314] In one embodiment, the CR rate is at least 80%. In another embodiment, the CR rate is at least 85%.

[0315] In one aspect of the invention, glimetuzumab is provided in a method of treating previously untreated diffuse large B-cell lymphoma to produce a complete response (CR) rate of at least 75%, at least 80%, or at least 85%, wherein the method comprises:

[0316] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for 2 cycles;

[0317] b. Determine the amount of ctDNA on day 1 of cycle 1 (baseline) and day 1 of cycle 2; and

[0318] c. Select patients whose ctDNA reduction is <2-log between baseline and one R-CHOP treatment cycle and continue R-CHOP treatment for a total of six cycles (cycles 1 to 6), and add glimetuzumab for a total of eight cycles (cycles 3 to 10), wherein glimetuzumab is administered at a dose of 2.5 mg on day 8 of cycle 3, at a dose of 10 mg on day 15 of cycle 3, at a dose of 30 mg on day 8 of cycles 4 to 6, and at a dose of 30 mg on day 1 of cycles 7 to 10.

[0319] In one embodiment, the CR rate is at least 80%. In another embodiment, the CR rate is at least 85%.

[0320] One aspect of the invention relates to glimetuzumab, in a method of treating previously untreated diffuse large B-cell lymphoma with high (ct)DNA risk to produce an overall response rate (ORR) of at least 80%, at least 85%, at least 90%, or at least 95%, the method comprising:

[0321] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0322] b. Determine the amount of ctDNA at or before cycle 1 (baseline) and after at least one R-CHOP treatment cycle; and

[0323] c. Select patients whose ctDNA reduction is <2.5-log between baseline and at least one R-CHOP treatment cycle and continue treatment with R-CHOP and glimetuzumab.

[0324] In one embodiment, the ORR is at least 90%. In another embodiment, the ORR is at least 95%.

[0325] In another aspect of the invention, a method of using glimetuzumab to treat previously untreated diffuse large B-cell lymphoma to produce an overall response rate (ORR) of at least 80%, at least 85%, at least 90%, or at least 95%, comprising:

[0326] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0327] b. Determine the amount of ctDNA after cycle 1 (baseline) and at least one R-CHOP treatment cycle; and

[0328] c. Select patients whose ctDNA reduction is <2.5-log between baseline and at least one R-CHOP treatment cycle and continue treatment with R-CHOP and glimetuzumab.

[0329] In one embodiment, the ORR is at least 90%. In another embodiment, the ORR is at least 95%.

[0330] In one embodiment, step a) includes applying R-CHOP on day 1 of cycles 1, 2, and optionally cycle 3. In another embodiment, step a) includes applying R-CHOP on day 1 of cycles 1 and 2.

[0331] In one embodiment, step b) includes determining the amount of ctDNA on or before day 1 of cycle 1 (baseline) and day 1 of cycle 2 or day 1 of cycle 3 of the R-CHOP treatment in step a). In another embodiment, step b) includes determining the amount of ctDNA on day 1 of cycle 1 (baseline) and day 1 of cycle 2 of the R-CHOP treatment in step a).

[0332] In one embodiment, step c) includes selecting patients with a ctDNA reduction of <2-log. In another embodiment, step c) includes continuing R-CHOP treatment for a total of six cycles (cycles 1 to 6) and adding glimetuzumab for a total of eight cycles (cycles 3 to 10).

[0333] In one aspect of the invention, a method of using glimetuzumab to treat previously untreated diffuse large B-cell lymphoma to produce an overall response rate (ORR) of at least 80%, at least 85%, at least 90%, or at least 95% is provided, the method comprising:

[0334] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0335] b. Determine the amount of ctDNA on day 1 of cycle 1 (baseline) and day 1 of cycle 2; and

[0336] c. Select patients whose ctDNA decrease is <2-log between baseline and one R-CHOP treatment cycle and continue treatment with R-CHOP and glimetuzumab.

[0337] In one embodiment, the ORR is at least 90%. In another embodiment, the ORR is at least 95%.

[0338] In another aspect of the invention, a method of using glimetuzumab to treat previously untreated diffuse large B-cell lymphoma to produce an overall response rate (ORR) of at least 80%, at least 85%, at least 90%, or at least 95% is provided, the method comprising:

[0339] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0340] b. Determine the amount of ctDNA on day 1 of cycle 1 (baseline) and day 1 of cycle 2; and

[0341] c. Select patients whose ctDNA decreased by <2-log between baseline and one R-CHOP treatment cycle, and continue R-CHOP treatment for a total of six cycles (cycles 1 to 6), and add glimetuzumab for a total of eight cycles (cycles 3 to 10).

[0342] In one embodiment, the ORR is at least 90%. In another embodiment, the ORR is at least 95%.

[0343] In another aspect of the invention, a method of using glimetuzumab to treat previously untreated diffuse large B-cell lymphoma to produce an overall response rate (ORR) of at least 80%, at least 85%, at least 90%, or at least 95% is provided, the method comprising:

[0344] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for 2 cycles;

[0345] b. Determine the amount of ctDNA on day 1 of cycle 1 (baseline) and day 1 of cycle 2; and

[0346] c. Select patients whose ctDNA decreased by <2-log between baseline and one R-CHOP treatment cycle, and continue R-CHOP treatment for a total of six cycles (cycles 1 to 6), and add glimetuzumab for a total of eight cycles (cycles 3 to 10).

[0347] In one embodiment, the ORR is at least 90%. In another embodiment, the ORR is at least 95%.

[0348] In one aspect of the invention, the third treatment cycle comprises a first dose and a second dose of glimetuzumab, followed by a rest period. In one such embodiment, the third treatment cycle comprises a first dose of glimetuzumab on day 8 of the third cycle and a second dose of glimetuzumab on day 15 of the third cycle, followed by a 6-day rest period. In one embodiment, the subsequent two cycles (cycles 4 to 6) comprise a dose of glimetuzumab administered on day 8 of that cycle, followed by a 13-day rest period. In one embodiment, the remaining subsequent cycles (cycles 7 to 10) comprise a dose of glimetuzumab administered on day 1 of that cycle, followed by a 20-day rest period.

[0349] In one embodiment, a treatment cycle comprises 21 days. In another embodiment, a treatment cycle comprises 14 days. The treatment regimen according to the invention may comprise 6 or more treatment cycles, particularly 10 treatment cycles.

[0350] In one embodiment, glimetuzumab is administered at a dose of 2.5 mg on day 8 of cycle 3, at a dose of 10 mg on day 15 of cycle 3, at a dose of 30 mg on day 8 of cycles 4 through 6, and at a dose of 30 mg on day 1 of cycles 7 through 10.

[0351] In another embodiment, glucentumab is administered at a dose of 0.5 mg on day 4 of cycle 3, at a dose of 2.5 mg on day 8 of cycle 3, at a dose of 10 mg on day 15 of cycle 3, at a dose of 30 mg on day 8 of cycles 4 through 6, and at a dose of 30 mg on day 1 of cycles 7 through 10.

[0352] In another embodiment, glucantuzumab is administered at a dose of 0.5 mg on day 8 of cycle 3, at a dose of 2 mg on day 9, at a dose of 10 mg on day 15 of cycle 3, at a dose of 30 mg on day 8 of cycles 4 through 6, and at a dose of 30 mg on day 1 of cycles 7 through 10.

[0353] In one aspect of the invention, glimetuzumab is provided in a method of treating previously untreated diffuse large B-cell lymphoma to produce an overall response rate (ORR) of at least 80%, at least 85%, at least 90%, or at least 95%, comprising:

[0354] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0355] b. Determine the amount of ctDNA on day 1 of cycle 1 (baseline) and day 1 of cycle 2; and

[0356] c. Select patients whose ctDNA reduction is <2-log between baseline and one R-CHOP treatment cycle and continue R-CHOP treatment for a total of six cycles (cycles 1 to 6), and add glimetuzumab for a total of eight cycles (cycles 3 to 10), wherein glimetuzumab is administered at doses of 2.5 mg and 10 mg on different days of cycle 3, and at doses of 30 mg on cycles 4 to 10.

[0357] In one embodiment, the ORR is at least 90%. In another embodiment, the ORR is at least 95%.

[0358] In one aspect of the invention, glimetuzumab is provided in a method of treating previously untreated diffuse large B-cell lymphoma to produce an overall response rate (ORR) of at least 80%, at least 85%, at least 90%, or at least 95%, comprising:

[0359] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for 2 cycles;

[0360] b. Determine the amount of ctDNA on day 1 of cycle 1 (baseline) and day 1 of cycle 2; and

[0361] c. Select patients whose ctDNA reduction is <2-log between baseline and one R-CHOP treatment cycle and continue R-CHOP treatment for a total of six cycles (cycles 1 to 6), and add glimetuzumab for a total of eight cycles (cycles 3 to 10), wherein glimetuzumab is administered at doses of 2.5 mg and 10 mg on different days of cycle 3, and at doses of 30 mg on cycles 4 to 10.

[0362] In one embodiment, the ORR is at least 90%. In another embodiment, the ORR is at least 95%.

[0363] In one aspect of the invention, glimetuzumab is provided in a method of treating previously untreated diffuse large B-cell lymphoma to produce an overall response rate (ORR) of at least 80%, at least 85%, at least 90%, or at least 95%, comprising:

[0364] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0365] b. Determine the amount of ctDNA on day 1 of cycle 1 (baseline) and day 1 of cycle 2; and

[0366] c. Select patients whose ctDNA reduction is <2-log between baseline and one R-CHOP treatment cycle and continue R-CHOP treatment for a total of six cycles (cycles 1 to 6), and add glimetuzumab for a total of eight cycles (cycles 3 to 10), wherein glimetuzumab is administered at a dose of 2.5 mg on day 8 of cycle 3, at a dose of 10 mg on day 15 of cycle 3, at a dose of 30 mg on day 8 of cycles 4 to 6, and at a dose of 30 mg on day 1 of cycles 7 to 10.

[0367] In one embodiment, the ORR is at least 90%. In another embodiment, the ORR is at least 95%.

[0368] In one aspect of the invention, glimetuzumab is provided in a method of treating previously untreated diffuse large B-cell lymphoma to produce an overall response rate (ORR) of at least 80%, at least 85%, at least 90%, or at least 95%, comprising:

[0369] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for 2 cycles;

[0370] b. Determine the amount of ctDNA on day 1 of cycle 1 (baseline) and day 1 of cycle 2; and

[0371] c. Select patients whose ctDNA reduction is <2-log between baseline and one R-CHOP treatment cycle and continue R-CHOP treatment for a total of six cycles (cycles 1 to 6), and add glimetuzumab for a total of eight cycles (cycles 3 to 10), wherein glimetuzumab is administered at a dose of 2.5 mg on day 8 of cycle 3, at a dose of 10 mg on day 15 of cycle 3, at a dose of 30 mg on day 8 of cycles 4 to 6, and at a dose of 30 mg on day 1 of cycles 7 to 10.

[0372] In one embodiment, the ORR is at least 90%. In another embodiment, the ORR is at least 95%.

[0373] In one embodiment, the CR rate is determined at the end of treatment. In one embodiment, the CR rate is determined by PET-CT scan. In one embodiment, the CR rate is determined according to the 2014 Lugano Response Criteria. In one embodiment, the CR rate is determined between day 15 and day 21 of cycle 2. In one embodiment, the CR rate is measured by PET / CT scan and determined at the end of treatment by an automated response (aLugano) according to the 2014 Lugano Response Criteria. In one embodiment, the aLugano analysis is performed in a PET / CT-evaluable population, defined as all patients with available PET / CT scans at baseline and during treatment.

[0374] In one embodiment, tumor assessment is performed using 18FFDG-PET / CT and diagnostic CT scans before the first treatment (on or before day 1 of cycle 1), during the intermediate response assessment (between day 15 and day 21 of cycle 2), and at EOT.

[0375] In one embodiment, the presence and extent of lymphadenopathy, hepatomegaly, and splenomegaly are determined by physical examination. In another embodiment, the response is assessed based on the results of physical examination, CT scan, and FDG-PET / CT scan.

[0376] In one embodiment of any of the foregoing aspects of the invention, administration of such treatment to multiple human patients at high risk of ctDNA resulted in an improved duration of response (DOR) rate compared to R-CHOP treatment alone. In one embodiment, the DOR rate was determined according to the 2014 Lugano Response Criteria. In one embodiment, the DOR rate was measured by PET / CT scans and determined by an automated response (aLugano) according to the 2014 Lugano Response Criteria. In one embodiment, the aLugano analysis was performed in a PET / CT-evaluable population defined as all patients with available PET / CT scans at baseline and during treatment. In one embodiment, DOR was defined as the time from the first occurrence of a documented objective response (CR or PR) to disease progression, relapse, or death from any cause, whichever occurred first. In one embodiment, the DOR analysis included only participants with an objective response (CR or PR).

[0377] In one embodiment of any of the foregoing aspects of the invention, administration of such treatment to multiple human patients at high risk of ctDNA resulted in improved progression-free survival (PFS) compared to R-CHOP treatment alone.

[0378] In one embodiment of any of the foregoing aspects of the invention, administration of such treatment to multiple human patients at high risk of ctDNA resulted in improved overall survival (OS) compared to R-CHOP treatment alone.

[0379] The International Prognostic Index (IPI) is widely used to stratify the risk of patients with DLBCL based on their age, biochemical parameters, performance status, and disease stage. In one embodiment, the performance status of the subjects to be treated is 0-2 (Eastern Cooperative Oncology Group, see Oken MM, Creech RH, Tormey DC et al. Toxicity and response criteria of the Eastern Cooperative Oncology Group. Am J Clin Oncol 1982;5:649-55.). The International Prognostic Index (IPI, Shipp MA, Harrington DP, Anderson JR et al. A predictive model for aggressive Non-Hodgkin's Lymphoma. N Engl J Med 1993;329:987-94.) is also widely used to stratify the risk of patients with DLBCL based on their age, biochemical parameters, performance status, and disease stage. In one embodiment, the IPI of the subjects to be treated is 1-5 or 2-5. In one embodiment, the subject to be treated has an IP of 0 and a large lesion (> 6 cm). In another embodiment, the subject to be treated has at least one two-dimensional FDG-avid measurable lymphoma lesion on a PET / CT scan, defined as having a longest diameter > 1.5 cm on a CT scan.

[0380] In one aspect, previously untreated DLBCL is previously untreated CD20-positive diffuse large B-cell lymphoma (DLBCL). In one embodiment, previously untreated DLBCL includes nonspecific (NOS) type DLBCL, germinal center B-cell-like (GCB) and activated B-cell-like (ABC) / non-GCB types, as well as dual-expressing lymphoma (MYC and BCL2 co-expression), high-grade B-cell lymphoma (HGBCL) with MYC and BCL2 and / or BCL6 translocations, high-grade B-cell lymphoma (HGBCL; e.g., HGBCL NOS, double-hit HGBCL, and triple-hit HGBCL), and new-onset transformed follicular lymphoma.

[0381] In one respect, previously untreated DLBCL does not include unclassifiable B-cell lymphomas (gray zone lymphomas) with intermediate features between DLBCL and classical Hodgkin lymphoma, primary mediastinal (thymic) large B-cell lymphomas, T-cell / histiocytic abundant large B-cell lymphomas, Burkitt lymphomas, CNS lymphomas (primary or secondary involvement), primary exudative DLBCL, and primary cutaneous DLBCL.

[0382] In some embodiments, the method and use characterized by the present invention further includes administering one or more additional therapeutic agents to a subject. In some embodiments, the method characterized by the present invention further includes administering one or more additional therapeutic agents to a subject. In some embodiments, the one or more additional therapeutic agents are tocilizumab. In some embodiments, the one or more additional therapeutic agents are corticosteroids. In some embodiments, corticosteroids include prednisone, prednisolone, methylprednisolone, or dexamethasone. In some embodiments, the one or more additional therapeutic agents are antihistamines. In some embodiments, the antihistamine is diphenhydramine. In some embodiments, the one or more additional therapeutic agents include allopurinol and raburicase. In some embodiments, the one or more additional therapeutic agents are antipyretics. In some embodiments, the one or more additional therapeutic agents include granulocyte colony-stimulating factor (G-CSF). In some embodiments, the one or more additional therapeutic agents are mesna.

[0383] In some embodiments, one or more additional therapeutic agents are corticosteroids. In some embodiments, corticosteroids include prednisone, prednisolone, methylprednisolone, or dexamethasone. In one embodiment, dexamethasone is administered intravenously at a dose of about 20 mg (e.g., 20 mg ± 0.1 mg, ± 0.25 mg, ± 0.5 mg, ± 1 mg, ± 1.5 mg, or ± 2 mg) at least one hour (i.e., at least one hour ± 6 minutes; for example, at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36, 48 hours or more) before administration of any dose of the bispecific antibody (i.e., glimetuzumab). In one embodiment, dexamethasone is administered at a dose of 20 mg before any dose of glimetuzumab. In one embodiment, dexamethasone is administered at a dose of 20 mg one hour before any dose of glimetuzumab. In one embodiment, dexamethasone is administered at a dose of 20 mg prior to the first and second doses of glimetuzumab. In one embodiment, dexamethasone is administered at a dose of 20 mg one hour prior to the first and second doses of glimetuzumab. In one embodiment, the dexamethasone prophylaxis is administered only during cycle 3 (i.e., the first treatment cycle using glimetuzumab). In one embodiment, dexamethasone is administered intravenously at a dose of about 20 mg (e.g., 20 mg ± 0.1 mg, ± 0.25 mg, ± 0.5 mg, ± 1 mg, ± 1.5 mg, or ± 2 mg) at least one hour (i.e., at least one hour ± 6 minutes; for example, at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36, 48 hours or more) prior to the administration of any dose of R-CHOP. In one embodiment, methylprednisolone is administered intravenously at a dose of about 80 mg (e.g., 80 mg ± 0.5 mg, ± 1 mg, ± 1.5 mg, ± 2 mg, ± 4 mg, ± 6 mg, or ± 8 mg) at least one hour (i.e., at least one hour ± 6 minutes; for example, at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36, 48 hours or more) before any dose of the bispecific antibody (i.e., glucuronidumab).In one embodiment, methylprednisolone is administered intravenously at a dose of about 80 mg (e.g., 80 mg ± 0.5 mg, ± 1 mg, ± 1.5 mg, ± 2 mg, ± 4 mg, ± 6 mg, or ± 8 mg) at least about one hour (i.e., at least one hour ± 6 minutes; for example, at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36, 48 hours or more) before any dose of R-CHOP is administered. In one embodiment, prednisone is administered orally at a dose of about 100 mg (e.g., 100 mg ± 0.5 mg, ± 1 mg, ± 1.5 mg, ± 2 mg, ± 4 mg, ± 6 mg, ± 8 mg, or ± 10 mg) at least about one hour (i.e., at least one hour ± 6 minutes; for example, at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36, 48 hours or more) before any dose of the bispecific antibody (i.e., glimetuzumab). In one embodiment, prednisolone is administered intravenously at a dose of about 100 mg (e.g., 100 mg ± 0.5 mg, ± 1 mg, ± 1.5 mg, ± 2 mg, ± 4 mg, ± 6 mg, ± 6 mg, ± 8 mg, or ± 10 mg) at least one hour (i.e., at least one hour ± 6 minutes; for example, at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36, 48 hours or more) before any dose of the bispecific antibody (i.e., glimetuzumab).

[0384] In some embodiments, one or more additional therapeutic agents are antihistamines. In some embodiments, the antihistamine is diphenhydramine. In one embodiment, diphenhydramine is administered orally or intravenously at a dose of about 50 mg (e.g., 50 mg ± 0.5 mg, ± 1 mg, ± 1.5 mg, ± 2 mg, ± 3 mg, ± 4 mg, or ± 5 mg) at least 30 minutes (i.e., at least 30 minutes ± 3 minutes; for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36, 48 hours or more) before administration of any dose of the bispecific antibody (i.e., glimetuzumab). In one embodiment, diphenhydramine is administered at a dose of 50 mg before administration of any dose of glimetuzumab. In one embodiment, diphenhydramine is administered at a dose of 50 mg about 30 minutes before administration of any dose of glimetuzumab.

[0385] In some embodiments, one or more additional therapeutic agents are antipyretics. In one embodiment, the antipyretic is acetaminophen or paracetamol. In one embodiment, acetaminophen or paracetamol is administered orally at a dose between about 500 mg and about 1000 mg (e.g., at least 30 minutes ± 3 minutes; for example, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36, 48 hours or more) before administration of any dose of the bispecific antibody (i.e., glimepiride). In one embodiment, acetaminophen or paracetamol is administered orally at a dose between about 500 mg and about 1000 mg (e.g., at least 30 minutes ± 3 minutes; for example, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36, 48 hours or more) before any dose of R-CHOP. In one embodiment, acetaminophen or paracetamol is administered at a dose of 500 or 1000 mg before any dose of glimepiride. In another embodiment, acetaminophen or paracetamol is administered at a dose of 500 or 1000 mg about 30 minutes before any dose of glimepiride.

[0386] In one embodiment, one or more additional therapeutic agents are tocilizumab. In one embodiment, tocilizumab is administered at a dose of about 8 mg / kg (e.g., 8 mg / kg ± 0.05 mg / kg, ± 0.1 mg / kg, ± 0.25 mg / kg, ± 0.5 mg / kg, or ± 0.8 mg / kg), or if the subject weighs less than 30 kg, tocilizumab is administered at a dose of about 12 mg / kg (e.g., 12 mg / kg ± 0.05 mg / kg, ± 0.1 mg / kg, ± 0.25 mg / kg, ± 0.5 mg / kg, ± 0.75 mg / kg, ± 1 mg / kg, or ± 1.2 mg / kg), and wherein the maximum dose is about 800 mg (e.g., 800 mg ± 10 mg, ± 25 mg, ± 50 mg, or ± 80 mg).

[0387] In some embodiments, one or more additional therapeutic agents are methotrexate. In one embodiment, intrathecal methotrexate is administered as CNS prophylaxis. In one embodiment, intrathecal methotrexate is administered to a subject who has four or more of the following risk factors: age >60 years, stage III or IV disease, more than two extranodal sites, longest diameter greater than the upper limit of normal, or kidney or adrenal gland involvement.

[0388] In one embodiment, one or more additional therapeutic agents comprise granulocyte colony-stimulating factor (G-CSF). In one embodiment, G-CSF is administered during cycles 1 to 6. In one embodiment, G-CSF is administered during cycles 3 to 6. In one embodiment, the G-CSF is filgrastim. In one embodiment, filgrastim is administered subcutaneously once daily at a dose of approximately 5 µg / kg, up to 48 hours after administration of R-CHOP. In one embodiment, the dose of filgrastim is rounded to 300 µg or 400 µg. In one embodiment, treatment with filgrastim lasts for 7 days. In one embodiment, treatment with filgrastim lasts until two consecutive days of ANC > 1.5 × 10⁻⁶. 9 / L. In one embodiment, filgrastim is administered subcutaneously once daily at a dose of about 5 µg / kg, up to 48 hours after R-CHOP administration. In one embodiment, filgrastim is administered on day 1 of cycle 1 to 6.

[0389] In one embodiment, G-CSF is glycolated filgrastim. In one embodiment, glycolated filgrastim is administered subcutaneously at a dose of approximately 6 mg 48 hours after administration of R-CHOP. In one embodiment, glycolated filgrastim is administered on day 1 of cycles 1 to 6.

[0390] In another embodiment, G-CSF is levofloxacin.

[0391] In one embodiment, G-CSF is administered approximately one to two days after any dose of R-CHOP (e.g., 24, 26, 28, 30, 32, 36, 38, 40, 42, 44, 46, or 48 hours). In one embodiment, filgrastim is administered intravenously or subcutaneously at a dose of about 5 µg / kg / day (e.g., 5 µg / kg / day ± 0.05 µg / kg / day, ± 0.1 µg / kg / day, ± 0.2 µg / kg / day, ± 0.3 µg / kg / day, ± 0.4 µg / kg / day, ± 0.5 µg / kg / day) or about 10 µg / kg / day (e.g., 10 µg / kg / day ± 0.1 µg / kg / day, ± 0.2 µg / kg / day, ± 0.4 µg / kg / day, ± 0.6 µg / kg / day, ± 0.8 µg / kg / day, ± 1 µg / kg / day). In one embodiment, filgrastim is administered at a dose of about 5 µg / kg / day in the first dosing cycle (e.g., 5 µg / kg / day ± 0.05 µg / kg / day, ± 0.1 µg / kg / day, ± 0.2 µg / kg / day, ± 0.3 µg / kg / day, ± 0.4 µg / kg / day, ± 0.5 µg / kg / day) in the second dosing cycle and / or each additional dosing cycle at a dose of about 10 µg / kg / day (e.g., 10 µg / kg / day ± 0.1 µg / kg / day, ± 0.2 µg / kg / day, ± 0.4 µg / kg / day, ± 0.6 µg / kg / day, ± 0.8 µg / kg / day, ± 1 µg / kg / day).

[0392] One aspect of the invention relates to the use of a bispecific antibody targeting CD3 and CD20 comprising the VH and VL sequences of glimetuzumab in the manufacture of a medicament for treating a subject with previously untreated diffuse large B-cell lymphoma at high risk of (ct)DNA, wherein the patient is administered a combination of glimetuzumab and chemotherapy, wherein the patient has been identified as at high risk of ctDNA if the ctDNA level decreases by less than 2.5 log after one cycle of chemotherapy.

[0393] In one embodiment, the patient's ctDNA decreased by less than 2-log.

[0394] In one embodiment, the use of glimetuzumab in manufacturing a medicament for treating a subject with previously untreated diffuse large B-cell lymphoma at high risk of (ct)DNA is provided, wherein the patient is administered glimetuzumab in combination with chemotherapy, wherein the patient has been identified as at high risk of ctDNA if the ctDNA decreases by less than 2.5 log after one cycle of chemotherapy.

[0395] One aspect of the invention relates to the use of glimetuzumab in the manufacture of a medicament for treating a subject with previously untreated diffuse large B-cell lymphoma at high risk of (ct)DNA, wherein the patient is administered a combination of glimetuzumab with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), wherein the patient has been identified as at high risk of ctDNA if the ctDNA decreases by less than 2.5 log after one R-CHOP cycle.

[0396] One aspect of the invention relates to the use of glimetuzumab according to any of the above embodiments in the manufacture of a medicament for treating a subject with previously untreated diffuse large B-cell lymphoma at high risk of (ct)DNA.

[0397] One aspect of the invention relates to the use of glimetuzumab in the manufacture of a medicament for treating subjects with previously untreated diffuse large B-cell lymphoma at high risk of (ct)DNA, comprising:

[0398] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0399] b. Determine the amount of ctDNA at or before cycle 1 (baseline) and after at least one R-CHOP treatment cycle; and

[0400] c. Select patients whose ctDNA reduction is <2.5-log between baseline and at least one R-CHOP treatment cycle and continue treatment with R-CHOP and glimetuzumab.

[0401] Another aspect of the present invention relates to the invention as described herein.

[0402] Unless the context clearly indicates otherwise, each embodiment may be incorporated. Unless the context clearly indicates otherwise, each embodiment may be applied to various aspects of the invention.

[0403] Certain embodiments of the invention will become apparent from the following more detailed description of some preferred embodiments and claims.

[0404] (iv) CRS risk mitigation strategies

[0405] This invention relates to a method for treating previously untreated patients with high-risk circulating tumor DNA (ctDNA) diffuse large B-cell lymphoma, the method comprising administering glimetuzumab in combination with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), wherein the patient is identified as high-risk for ctDNA if the ctDNA decreases by less than 2.5 log after one or two R-CHOP cycles.

[0406] Bispecific antibody therapies involving T-cell activation, such as glimepiride, have been associated with cytokine release syndrome (CRS). CRS is a potentially life-threatening complex caused by the excessive release of cytokines by immune effectors or target cells during an excessive and sustained immune response. CRS can be triggered by a variety of factors, including infection with toxic pathogens, or by agents that activate or enhance the immune response to produce a pronounced and sustained immune response.

[0407] Regardless of the triggering factor, severe or life-threatening CRS constitutes a medical emergency. If not successfully managed, it can lead to significant disability or death. Current clinical management focuses on treating individual signs and symptoms, providing supportive care, and attempting to suppress the inflammatory response with high-dose corticosteroids. However, this approach is not always successful, especially in cases of late intervention. Furthermore, steroids may negatively affect T-cell function, potentially diminishing the clinical benefits of immunomodulatory therapies in cancer treatment.

[0408] A. CRS Symptoms and Grading

[0409] CRS is graded according to the modified cytokine release syndrome grading system established by Lee et al., Blood, 124: 188-195, 2014 or Lee et al., Biol BloodMarrow Transplant, 25(4): 625-638, 2019, as described in Table 2. In addition to the diagnostic criteria, recommended CRS management based on severity (including early intervention with corticosteroids and / or anti-cytokine therapy) is also provided and mentioned in Tables 2 and 3.

[0410] Table 2. Grading system for cytokine release syndrome

[0411]

[0412] Lee 2014 Criterion: Lee et al., Blood, 124: 188-195, 2014.

[0413] ASTCT consensus classification: Lee et al., Biol Blood Marrow Transplant, 25(4): 625-638, 2019.

[0414] a Low-dose vasopressors: Single vasopressors at doses lower than those shown in Table 4.

[0415] b High-dose vasopressors: as defined in Table 4.

[0416] Fever is defined as a body temperature ≥ 38°C that is not attributable to any other cause. In CRS patients who subsequently receive antipyretics or anticytokine therapy (such as tocilizumab or steroids), fever is no longer required for grading the severity of subsequent CRS. In this case, CRS grading is driven by hypotension and / or hypoxia.

[0417] CRS grade is determined by more serious events: hypotension or hypoxia that cannot be attributed to any other cause. For example, a patient with a body temperature of 39.5°C, hypotension requiring one vasopressor, and hypoxia requiring a low-flow nasal cannula is classified as grade 3 CRS.

[0418] Low-flow nasal cannula is defined as delivering oxygen at a rate of ≤ 6 L / min. Low flow also includes blowing oxygen, sometimes used in pediatrics. High-flow nasal cannula is defined as delivering oxygen at a rate of > 6 L / min.

[0419] Table 3. High-dose vasopressors

[0420]

[0421] min = minutes; VASST = Vasopressin and Septic Shock Test.

[0422] a VASST vasopressor equivalence equation: Norepinephrine equivalent dose = [Norepinephrine (µg / min)] + [Dopamine (µg / kg / min) ÷ 2] + [Epinephrine (µg / min)] + [Phenylephrine (µg / min) ÷ 10].

[0423] Mild to moderate CRS and / or infusion-related reactions (IRR) may include symptoms such as fever, headache, and myalgia, and can be treated symptomatically with analgesics, antipyretics, and antihistamines as needed. Severe or life-threatening CRS and / or IRR, such as hypotension, tachycardia, dyspnea, or chest discomfort, should be aggressively treated with supportive and resuscitation measures as directed, including high-dose corticosteroids, intravenous fluids, admission to the intensive care unit, and other supportive measures. Severe CRS may be associated with other clinical sequelae such as disseminated intravascular coagulation, capillary leak syndrome, or macrophage activation syndrome (MAS). Standards of care for severe or life-threatening CRS resulting from immunotherapy have not been established; case reports and recommendations on the use of anticytokine therapies (such as tocilizumab) have been published (Teachey et al., Blood, 121: 5154-5157, 2013; Lee et al., Blood, 124: 188-195, 2014; Maude et al., New Engl J Med, 371: 1507-1517, 2014).

[0424] B. Pretreatment or management of CRS-related symptoms with tocilizumab

[0425] CRS is associated with high IL-6 levels (Panelli et al., J Transl Med, 2: 17, 2004; Lee et al., Blood, 124: 188-195, 2014; Doessegger and Banholzer, Clin Transl Immunology, 4: e39, 2015), and IL-6 is associated with the severity of CRS, with patients experiencing severe or life-threatening CRS (NCI CTCAE grade 4 or 5) having much higher IL-6 levels compared to patients who did not experience CRS or experienced a milder CRS response (NCI CTCAE grade 0-3) (Chen et al., J Immunol Methods, 434: 1-8, 2016).

[0426] Tocilizumab (ACTEMRA® / ROACTEMRA®) is a recombinant humanized anti-human monoclonal antibody against soluble and membrane-bound IL-6R, which inhibits IL-6-mediated signaling (see, for example, WO 1992 / 019579, which is incorporated herein by reference in its entirety). Tocilizumab has been approved by the U.S. Food and Drug Administration for the treatment of severe or life-threatening CAR-T cell-induced CRS in adults and pediatric patients aged 2 years and older. Initial clinical data (Locke et al., Blood, 130: 1547, 2017) suggest that tocilizumab prophylaxis can reduce the severity of CAR-T cell-induced CRS by blocking IL-6 receptor signaling prior to cytokine release. Therefore, tocilizumab prodrugation may also reduce the frequency or severity of CRS associated with bispecific antibody therapy. Other anti-IL-6R antibodies that can be used in combination with tocilizumab include sarilumab, vobarilizumab (ALX-0061), SA-237 and its variants.

[0427] In some respects, an effective amount of tocilizumab is administered as a prodrug, for example, before administration of glimetuzumab to the subject. Administration of tocilizumab as a prodrug may reduce the frequency or severity of CRS. In some respects, tocilizumab is administered as a prodrug in cycle 1, for example, before the first dose (C1D1), second dose (C1D2), and / or third dose (C1D3) of glimetuzumab. In some respects, tocilizumab is administered intravenously to the subject in a single dose of about 1 mg / kg to about 15 mg / kg (e.g., about 4 mg / kg to about 10 mg / kg, for example, about 6 mg / kg to about 10 mg / kg, for example, about 8 mg / kg). In some respects, tocilizumab is administered intravenously to the subject in a single dose of about 8 mg / kg. Other anti-IL-6R antibodies that can be used in combination with tocilizumab include sarilumab, vobarilizumab (ALX-0061), SA-237 and its variants.

[0428] For example, in one embodiment, glimetuzumab is co-administered with tocilizumab (ACTEMRA® / ROACTEMRA®), wherein tocilizumab (ACTEMRA® / ROACTEMRA®) is first administered to the subject, followed by administration of the bispecific antibody alone (e.g., the subject has been pre-treated with tocilizumab (ACTEMRA® / ROACTEMRA®)).

[0429] On the other hand, in subjects treated with glimetuzumab, tocilizumab is administered to treat or alleviate symptoms associated with CRS. If a subject has a grade 2 or higher CRS event with extensive comorbidity after glimetuzumab administration, the method may further include administering a first dose of an IL-6R antagonist (e.g., an anti-IL-6R antibody, such as tocilizumab (ACTEMRA® / ROACTEMRA®)) to the subject to manage the grade 2 or higher CRS event while discontinuing treatment with glimetuzumab. In some cases, the first dose of tocilizumab is administered intravenously to the subject at a dose of approximately 8 mg / kg. Other anti-IL-6R antibodies that can be used in combination with tocilizumab include sarilumab, vobarilizumab (ALX-0061), SA-237, and variants thereof. In some cases, if a Grade 2 or higher CRS event resolves to a ≤ Grade 1 CRS event within two weeks, the method further includes resuming treatment with glimetuzumab at a reduced dose. In some cases, if the event occurs during or within 24 hours of infusion, the smaller dose is 50% of the initial infusion rate of the previous cycle. On the other hand, if a Grade 2 or higher CRS event does not resolve or worsens to a ≥ Grade 3 CRS event within 24 hours of treatment for the Grade 2 or higher CRS event, the method may further include administering one or more (e.g., one, two, three, four, or five or more) additional doses of an IL-6R antagonist (e.g., an anti-IL-6R antibody, such as tocilizumab) to the subject to manage the Grade 2 or ≥ Grade 3 CRS event. In certain specific cases, grade 2 or higher CRS events do not resolve or worsen to ≥ grade 3 CRS events within 24 hours of treatment for grade 2 or higher CRS events, and the method may further include administering one or more additional doses of tocilizumab to the subject to manage grade 2 or ≥ grade 3 CRS events. In some cases, one or more additional doses of tocilizumab may be administered intravenously to the subject at a dose of about 1 mg / kg to about 15 mg / kg (e.g., about 4 mg / kg to about 10 mg / kg, for example, about 6 mg / kg to about 10 mg / kg, for example, about 8 mg / kg).

[0430] C. Other preventative treatments for CRS risk mitigation

[0431] In one embodiment, the treatment regimen provided herein further includes administering a prophylactic medication prior to administration of glimepiride. In one embodiment, the prophylactic medication includes a corticosteroid (such as, for example, prednisolone, dexamethasone, or methylprednisolone), acetaminophen / paracetamol, and / or an antihistamine (such as, for example, diphenhydramine). In one embodiment, the prophylactic medication is administered at least 60 minutes prior to administration of glimepiride (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36, 48 hours, or longer). In one embodiment, the treatment regimen further includes administering a prophylactic medication prior to administration of glimepiride. In the embodiment, the prophylactic medication includes a corticosteroid (such as, for example, prednisolone, dexamethasone, or methylprednisolone), an antipyretic (such as, for example, acetaminophen / paracetamol), and / or an antihistamine (such as, for example, diphenhydramine). In one embodiment, the subject receives a corticosteroid pre-treatment prior to glucintumab. It has been demonstrated that, compared to methylprednisolone, pre-treatment with dexamethasone reduces the levels of glucintumab-induced cytokines in mice pretreated with dexamethasone. Therefore, in one embodiment, the corticosteroid is dexamethasone. In one embodiment, the pre-treatment is administered at least 60 minutes prior to glucintumab administration (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36, 48 hours or longer). In another embodiment, the pre-treatment is administered at least 60 minutes prior to each glucintumab administration (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36, 48 hours or longer). In another embodiment, a prophylactic dose of corticosteroids is administered before the first dose (C1D1) of the first cycle and the second dose (C1D2) of the first cycle, and before the first dose (C2D1) of the second cycle and the first dose (C3D1) of the third cycle, and this prophylactic dose may be optional for subsequent cycles in which the target dose has been reached and tolerated by two doses for patients who did not have CRS in the previous cycles.

[0432] In one embodiment, a prophylactic drug is administered at least 60 minutes (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36, 48 hours or longer) prior to the administration of pretreatment with an anti-CD20 antibody (particularly olibutuzumab).

[0433] In one embodiment, corticosteroids are administered to manage any associated adverse events following administration of an anti-CD20 / anti-CD3 bispecific antibody (e.g., glimepiride).

[0434] (v) Administration of anti-CD20 / anti-CD3 bispecific antibodies

[0435] Methods may involve administering glimetuzumab (and / or any additional therapeutic agent) by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral infusion includes intravenous, subcutaneous, intramuscular, intra-arterial, and intraperitoneal routes. In some embodiments, glimetuzumab is administered via intravenous infusion.

[0436] In one embodiment, the infusion time of the anti-CD20 / anti-CD3 bispecific antibody (particularly glimetuzumab) is at least 4 hours (e.g., about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours, or about 6 hours). In a specific embodiment, the infusion duration of glimetuzumab is about 4 hours. In one embodiment, the infusion time of glimetuzumab may be shortened or lengthened. In one embodiment (e.g., in the absence of infusion-related adverse events), the infusion time of glimetuzumab in subsequent cycles is reduced to 2 hours ± 15 minutes. In one embodiment, the infusion time is increased to a maximum of 8 hours (e.g., about 4 hours, about 5 hours, about 6 hours, about 7 hours, or about 8 hours) (e.g., for high-risk subjects with a history of CRS). In one embodiment, for example, for patients at increased risk of CRS, patients who have experienced IRR or CRS with their previous dose of glimetuzumab, or patients at increased risk of recurrence of IRR / CRS with subsequent doses, the infusion time of glimetuzumab may be extended to a maximum of 8 hours.

[0437] For all methods described herein, gifentumab is formulated, administered, and given in accordance with good medical practice. Factors to be considered in this context include the specific condition being treated, the specific mammal being treated, the individual subject's clinical condition, the cause of the condition, the site of delivery, the method of administration, the administration regimen, and other factors known to the physician. Gifentumab is not mandatory but optional, co-formulated with one or more formulations currently used for the prevention or treatment of the discussed condition. The effective amount of such other formulations depends on the amount of gifentumab present in the formulation, the type of condition or treatment, and other factors discussed above. Gifentumab can be appropriately administered to the subject through a series of treatments.

[0438] Another aspect of the present invention relates to the invention described above.

[0439] Example

[0440] Some embodiments of the technology described herein can be defined according to any of the following numbered embodiments:

[0441] 1. A method for treating a previously untreated patient with diffuse large B-cell lymphoma at high risk of circulating tumor DNA (ctDNA), the method comprising administering a combination of a bispecific antibody targeting CD3 and CD20 comprising the VH and VL sequences of glimetuzumab with chemotherapy, wherein the patient has been identified as at high risk of ctDNA if the ctDNA level decreases by less than 2.5 log after one or two cycles of chemotherapy.

[0442] 2. The method according to Example 1, wherein the bispecific antibody targeting CD3 and CD20 comprises the heavy chain of SEQ ID NO: 17, the heavy chain of SEQ ID NO: 18, the two light chains of SEQ ID NO: 20, and the light chain of SEQ ID NO: 19; and wherein the heavy chain and the light chain are assembled to form a first Fab molecule specifically binding to CD20, a second Fab molecule specifically binding to CD3, and a third Fab molecule specifically binding to CD20, and

[0443] The Fc domain is composed of a first subunit and a second subunit that can stably associate.

[0444] 3. The method according to Example 1 or 2, wherein the chemotherapy is selected from: rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP); rituximab, cyclophosphamide, doxorubicin, and prednisone (R-CHP); or rituximab, ifosfamide, carboplatin, and etoposide phosphate (R-ICE).

[0445] 4. The method according to any one of Examples 1 to 3, wherein the bispecific antibody targeting CD3 and CD20 is glimetuzumab, and the chemotherapy is rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), and wherein the patient has been identified as high-risk for ctDNA if the patient's ctDNA decreases by less than 2.5 log after one or two R-CHOP cycles.

[0446] 5. The method according to Example 4, wherein the reduction in ctDNA is determined by measuring the amount of ctDNA before or on the day of the first treatment (baseline) and after at least one R-CHOP treatment cycle.

[0447] 6. The method according to Example 4 or 5, wherein the amount of ctDNA is determined on or before day 1 of the first R-CHOP treatment cycle (baseline) and on day 1 of the second R-CHOP treatment cycle.

[0448] 7. The method according to any one of Examples 4 to 6, wherein if the patient's ctDNA decreases by less than 2 log, the patient is identified as high-risk for ctDNA.

[0449] 8. A method for treating previously untreated diffuse large B-cell lymphoma, the method comprising:

[0450] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0451] b. Determine the amount of ctDNA at or before cycle 1 (baseline) and after at least one R-CHOP treatment cycle; and

[0452] c. Select patients whose ctDNA reduction is <2.5-log between baseline and at least one R-CHOP treatment cycle and continue treatment with R-CHOP and glimetuzumab.

[0453] 9. The method according to Example 8, wherein step a) includes applying R-CHOP on day 1 of cycles 1, 2 and optionally cycle 3.

[0454] 10. The method according to Example 9, wherein step a) includes applying R-CHOP on day 1 of cycles 1 and 2.

[0455] 11. The method according to any one of Examples 8 to 10, wherein step b) comprises determining the amount of ctDNA on or before day 1 of cycle 1 (baseline) and day 1 of cycle 2 or day 1 of cycle 3 of the R-CHOP treatment in step a).

[0456] 12. The method according to Example 11, wherein step b) includes determining the amount of ctDNA on day 1 (baseline) of cycle 1 and day 1 of cycle 2 of the R-CHOP treatment in step a).

[0457] 13. The method according to any one of Examples 8 to 12, wherein step c) includes selecting patients with a ctDNA reduction of <2-log.

[0458] 14. The method according to any one of Examples 8 to 13, wherein step c) includes continuing treatment with R-CHOP for a total of six cycles (cycles 1 to 6) and adding glimetuzumab for a total of eight cycles (cycles 3 to 10).

[0459] 15. The method according to any one of Examples 8 to 14, wherein glucantuma is administered at a dose of 2.5 mg on day 8 of cycle 3 and at a dose of 10 mg on day 15, at a dose of 30 mg on day 8 of cycles 4 to 6, and at a dose of 30 mg on day 1 of cycles 7 to 10.

[0460] 16. The method according to any one of the foregoing embodiments, wherein the ctDNA is circulating tumor DNA in plasma.

[0461] 17. The method according to any one of the foregoing embodiments, wherein the amount of ctDNA is measured in a blood sample from the patient.

[0462] 18. The method according to Example 17, wherein the amount of ctDNA is measured in genomic DNA isolated from peripheral mononuclear cells or plasma-depleted whole blood (PDWB) or in cell-free DNA (cfDNA) in plasma.

[0463] 19. The method according to Example 18, wherein the ctDNA is measured in cell-free DNA in plasma.

[0464] 20. The method according to any one of the foregoing embodiments, wherein the ctDNA is determined by targeted next-generation sequencing (NGS).

[0465] 21. The method according to any one of Examples 4 to 20, wherein rituximab is administered at 375 mg / m². 2 Cyclophosphamide was administered intravenously at a dose of 750 mg / m². 2 Doxorubicin was administered intravenously at a dose of 50 mg / m². 2 Vincristine was administered intravenously at a dose of 1.4 mg / m². 2 The dose was administered via IV bolus, and prednisone was administered at a dose of 100 mg / day from day 1 to day 5.

[0466] 22. The method according to any one of Examples 1 to 21, wherein the length of each cycle is 21 days.

[0467] 23. The method according to any one of Examples 4 to 22, wherein administration of such treatment to multiple human patients at high risk of ctDNA produces an overall response rate (ORR) of at least 80%, at least 85%, at least 90%, or at least 95%.

[0468] 24. The method according to Example 23, wherein the ORR is at least 95%.

[0469] 25. The method according to any one of Examples 1 to 21, wherein administration of such treatment to multiple human patients at high risk of ctDNA results in a complete response (CR) rate of at least 75%, at least 80%, or at least 85%.

[0470] 26. The method according to Example 25, wherein the CR rate is at least 85%.

[0471] 27. The method according to Examples 23 and 24 or Examples 25 and 26, wherein the ORR or CR rate is determined at the end of treatment.

[0472] 28. The method according to Example 27, wherein the ORR or CR is determined by PET-CT scan.

[0473] 29. The method according to Example 27 or 28, wherein the ORR or CR is determined according to the 2014 Lugano Reaction Standard.

[0474] 30. Glimetuzumab, in a method of treating previously untreated diffuse large B-cell lymphoma with high (ct)DNA risk, the method comprising administering a combination of glimetuzumab and chemotherapy to a patient, wherein the patient is identified as high-risk for ctDNA if the ctDNA level decreases by less than 2.5 log after one cycle of chemotherapy.

[0475] 31. Glimepiride, in a method of treating previously untreated diffuse large B-cell lymphoma with high (ct)DNA risk, the method comprising:

[0476] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0477] b. Determine the amount of ctDNA at or before cycle 1 (baseline) and after at least one R-CHOP treatment cycle; and

[0478] c. Select patients whose ctDNA reduction is <2.5-log between baseline and at least one R-CHOP treatment cycle and continue treatment with R-CHOP and glimetuzumab.

[0479] 32. Use of glimetuzumab in the manufacture of a medicament for treating a subject with previously untreated diffuse large B-cell lymphoma at high risk of (ct)DNA, wherein the patient is administered glimetuzumab in combination with chemotherapy, wherein the patient has been identified as at high risk of ctDNA if the ctDNA level decreases by less than 2.5 log after one cycle of chemotherapy.

[0480] 33. Use of glimetuzumab in the manufacture of a medicament for the treatment of subjects with previously untreated diffuse large B-cell lymphoma at high risk of (ct)DNA, including:

[0481] a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles;

[0482] b. Determine the amount of ctDNA at or before cycle 1 (baseline) and after at least one R-CHOP treatment cycle; and

[0483] c. Select patients whose ctDNA reduction is <2.5-log between baseline and at least one R-CHOP treatment cycle and continue treatment with R-CHOP and glimetuzumab.

[0484] 34. The present invention as described above.

[0485] Example

[0486] The following are examples of the methods and compositions of the present invention. It should be understood that various other embodiments can be implemented in light of the general description provided above.

[0487] Example 1. A phase II study evaluating the safety and efficacy of glimepiride in combination with rituximab (R) plus cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) in patients with untreated diffuse large B-cell lymphoma at high risk of circulating tumor (ct) DNA.

[0488] Research rationale

[0489] The aim of this study was to evaluate the safety, efficacy, and pharmacokinetics of the combination of glimepiride and R-CHOP in patients at high risk of ctDNA. Patients at high risk of ctDNA were defined as those who failed to achieve a ≥2 log-fold reduction in ctDNA levels between Day 1 of Cycle 1 (pre-treatment) and Day 1 of Cycle 2. ctDNA is a novel biomarker that offers the possibility of a method for early and sensitive identification of patients with DLBCL at high risk of first-line treatment failure. In the first-line setting, a subset of patients with DLBCL has a poor prognosis after treatment with R-CHOP immunochemotherapy. Nevertheless, R-CHOP remains a state-of-the-art (SOC) treatment for DLBCL. To date, efforts to define baseline prognostic scores and develop biomarkers to improve the International Prognostic Index (IPI) and guide treatment decisions have been unsuccessful. Compared to current baseline prognostic factors, ctDNA offers a significant advantage by allowing for dynamic assessment early in the patient’s treatment process and, in doing so, identifying newly diagnosed individuals who are poorly responsive to immunochemotherapy but may progress without altering their treatment strategy and thus potentially benefit from therapy intensification using glimetuzumab.

[0490] Purpose

[0491] This Phase II, open-label, multicenter study evaluated the safety, efficacy, and pharmacokinetics of the combination of glimetuzumab and R-CHOP in individuals at high risk of ctDNA-mediated DLBCL in a first-line setting. The specific objectives and corresponding endpoints of the study are outlined in Table 4 below. In this protocol, “study treatment” refers to the combination of treatments (i.e., R-CHOP, with or without glimetuzumab) assigned to participants as part of this study.

[0492] Table 4. Goals and End Points

[0493]

[0494] ADA = Anti-drug antibody; aLugano = Automated response according to the 2014 Lugano Response Criteria; ASTCT = American Society for Transplantation and Cell Therapy; aTMTV = Automated total metabolic tumor volume; AUC = Area under the concentration-time curve; Cmax = Maximum concentration; CR = Complete response; CRS = Cytokine release syndrome; CT = Computed tomography; CTCAE = Common Terminology Criteria for Adverse Events; ctDNA = Circulating tumor DNA; DLBCL = Diffuse large B-cell lymphoma; DOR = Duration of response; EOT = End of treatment; NCI = National Cancer Institute; ORR = Objective response rate; OS = Overall survival; PET = Positron emission tomography; PFS = Progression-free survival; PK = Pharmacokinetics; R-CHOP = Rituximab in combination with cyclophosphamide, doxorubicin, vincristine, and prednisone; SUVmax = Maximum standardized uptake value; TMTV = Total metabolic tumor volume.

[0495] Overall research design

[0496] This is a phase II, multicenter study evaluating the safety and efficacy of glimetuzumab in combination with R-CHOP immunochemotherapy in individuals with treatment-naïve DLBCL who are assessed as high-risk based on ctDNA. Patients were classified as high-risk for R-CHOP treatment failure if they demonstrated a <2-log (100 × 100) decrease in ctDNA levels between baseline (before day 1 of cycle 1) and after one cycle of R-CHOP treatment (before day 1 of cycle 2) using the experimental AOA-NHL test.

[0497] Patients at high ctDNA risk were enrolled before day 8 of cycle 3 and received glimetuzumab at escalating doses (2.5 mg, 10 mg, and 30 mg) starting on day 8 of cycle 3. For the purposes of this study, individuals whose ctDNA levels decreased by at least 2-log-fold between baseline (before day 1 of cycle 1) and after one cycle of R-CHOP treatment (before day 1 of cycle 2) were considered to be at lower risk of R-CHOP treatment failure and were ineligible for enrollment or rescreening. For those enrolled in cycle 3, eligible patients were treated with standard of care (SOC) doses of R-CHOP starting in cycle 1. Participants received combination therapy with R-CHOP and glimetuzumab during cycles 3 through 6 and received glimetuzumab monotherapy as consolidation during cycles 7 through 10. PET / CT scans were performed at the end of treatment (EOT), and early-mid PET / CT response assessments were conducted after cycle 2; results were used to evaluate the correlation between imaging findings and ctDNA levels. To reduce the risk of CRS, glimetuzumab was administered using an escalating dosing regimen consisting of 2.5 mg and 10 mg doses administered on days 8 and 15 of cycle 3, respectively. The total dose administered in cycle 3 did indeed exceed the dose for which safety was established in the NP40126 study. For subsequent cycles, 30 mg of glimetuzumab was administered on day 8 of cycles 5 and 6, and on day 1 of cycles 7 through 10. All participants were closely monitored for adverse events throughout the study and for at least 90 days after the last dose of study treatment. Adverse events were graded according to the National Cancer Institute Common Criteria for Adverse Events version 5.0 (NCI CTCAE v5.0), and CRS was graded according to the American Society for Transplantation and Cell Therapy (ASTCT) 2019 CRS Consensus Classification. An internal monitoring committee (IMC) was established to monitor patient safety during the study and to make recommendations on study practices based on trial safety data to ensure patient safety during the study treatment.

[0498] Participants' radiation-induced tumor response was assessed via PET / CT and CT scans at the end of Cycle 2 (ctDNA screening), after treatment completion, and during the study follow-up period. Tumor response was assessed according to the investigator's 2014 Lugano Response Criteria for malignant lymphomas (hereinafter referred to as the 2014 Lugano Response Criteria).

[0499] The research plan is in Figure 2 Provided in, and the dosing regimen is in Figure 3As shown in the image.

[0500] Research on treatment

[0501] The investigational drugs (IMPs) used in this study were glimetuzumab and tocilizumab. R-CHOP and any pre-treatment with corticosteroids were considered non-investigational drugs. CNS prophylaxis using intrathecal methotrexate, neutropenia prophylaxis using granulocyte colony-stimulating factors (G-CSFs), and dexamethasone were also considered non-investigational drugs.

[0502] Glucetuzumab

[0503] Glimetumab is a human IgG1 bispecific antibody that targets CD20 expressed on the surface of B cells and CD3ε expressed on the surface of T cells. Glimetumab induces tumor cell lysis by binding to CD20 on tumor cells, and simultaneously activates, proliferates, and releases cytokines by binding to CD3ε on the T cell receptor complex. Glimetumab has a unique structure containing two CD20-binding domains and one CD3-binding domain (2:1 design), significantly enhancing its affinity for CD20 compared to the 1:1 form. This novel antibody has demonstrated a manageable safety profile, and its use in NHL, both as a monotherapy and as a combination partner with other therapies, is currently under rigorous investigation.

[0504] Glucophage is a bispecific antibody containing two Fab molecules that specifically bind to CD20. Each Fab molecule contains six hypervariable regions (HVRs):

[0505] HVR-H1: YSWIN (SEQ ID NO: 1); HVR-H2: RIFPGDGDTDYNGKFKG (SEQ ID NO: 2); HVR-H3: NVFDGYWLVY (SEQ ID NO: 3); HVR-L1: RSSSKSLLHSNGITYLY (SEQ ID NO: 4); HVR-L2: QMSNLVS (SEQ ID NO: 5); and HVR-L3: AQNLELPYT (SEQ ID NO: 6)

[0506] Griffithomab further comprises at least one Fab molecule that specifically binds to CD3, the Fab molecule comprising the following six hypervariable regions (HVR): HVR-H1: TYAMN (SEQ ID NO: 9); HVR-H2: RIRSKYNNYATYYADSVKG (SEQ ID NO: 10); HVR-H3: HGNFGNSYVSWFAY (SEQ ID NO: 11); HVR-L1: GSSTGAVTTSNYAN (SEQ ID NO: 12); HVR-L2: GTNKRAP (SEQ ID NO: 13); and HVR-L3: ALWYSNLWV (SEQ ID NO: 14).

[0507] Intravenous administration of glimetuzumab is administered using an escalating dosing regimen, starting with 2.5 mg on day 8 of cycle 3 and 10 mg on day 15 of cycle 3, followed by 30 mg on day 8 of cycles 4 through 6 and day 1 of cycles 7 through 10, with each cycle lasting 21 days (i.e., every 3 weeks). Additional escalating doses may also be introduced (e.g., on day 4 of cycle 3) or divided doses (e.g., 0.5 mg on day 8 of cycle 3 and 2 mg on day 9 of cycle 3).

[0508] Administer 20 mg IV dexamethasone as a pre-treatment one hour before administering gifostumab; administer oral acetaminophen or paracetamol (500 mg or 1000 mg) and antihistamines such as diphenhydramine (50 mg) as a pre-treatment approximately 30 minutes before starting the infusion.

[0509] Initially, glimetuzumab was administered to participants over 4 hours (±15 minutes) on days 8 and 15 of cycle 3, day 8 of cycles 4–6, and day 1 of cycles 7–10. For participants with increased CRS risk, or those who experienced CRS at previous glimetuzumab doses or, according to the investigator's judgment, had an increased risk of recurrent CRS at subsequent doses, the infusion time was extended to a maximum of 8 hours. In the absence of infusion-related adverse events or CRS, the infusion time of glimetuzumab in cycles 5 and 6 could be reduced to 2 hours (±15 minutes) according to the investigator's judgment. For cycles 7, 8, 9, and 10, in the absence of infusion-related adverse events or CRS, the infusion time of glimetuzumab could be reduced to 90 minutes (±30 minutes) according to the investigator's judgment.

[0510] For participants who tolerate two 30 mg doses of glimetuzumab without CRS, prophylactic administration of dexamethasone during subsequent cycles is optional, depending on investigator assessment. However, if a subject experiences CRS, prophylactic administration of dexamethasone is necessary for subsequent glimetuzumab doses until no additional CRS events are observed.

[0511] Tocilizumab

[0512] Tocilizumab is a recombinant humanized anti-human monoclonal antibody that targets the soluble and membrane-bound IL-6 receptor, inhibiting IL-6-mediated signaling. Tocilizumab may be administered as an investigational rescue drug (IMP) to participants experiencing CRS events when necessary. Tocilizumab is administered at a dose of 8 mg / kg to participants ≥ 30 kg and at a dose of 12 mg / kg to participants < 30 kg. A dose exceeding 800 mg per infusion is not recommended.

[0513] Rituximab

[0514] Rituximab every 21 days at 375 mg / m² 2 The dose was administered intravenously to participants along with CHOP on day 1 of cycles 1 through 6.

[0515] Rituximab preparation and administration were performed according to local prescribing information, including prophylactic medication. Subcutaneous administration of rituximab was not permitted in this study. The use of locally approved standard-of-care biosimilar rituximab was permitted.

[0516] CHOP chemotherapy

[0517] CHOP chemotherapy consists of IV cyclophosphamide, IV doxorubicin, vincristine administered via IV bolus, and oral prednisone or prednisolone. Standard CHOP administration is as follows, for six 21-day cycles (including cycles 1 and 2 during ctDNA screening):

[0518] Cyclophosphamide: 750 mg / m² administered intravenously on day 1. 2

[0519] Doxorubicin: Administer intravenously on day 1 or according to institutional guidelines at a dose of 50 mg / m². 2

[0520] Vincristine: Administered via IV bolus on day 1 at a dose of 1.4 mg / m². 2 The recommended upper limit is 2 mg.

[0521] Prednisone: 100 mg orally per day from day 1 to day 5 (prednisone on day 1 can be administered intravenously, and the remaining doses on days 2 to 5 can be administered orally).

[0522] In countries where prednisone is unavailable or not the preferred treatment, prednisone is replaced by prednisolone (1:1 conversion; 100 mg); in countries or regions where prednisone is unavailable, 80 mg methylprednisolone is used instead of prednisone.

[0523] Based on the researchers' judgment, the upper limit of the permissible vincristine dose is 2 mg. For participants ≥ 70 years of age, the upper limit of the permissible vincristine dose is 1.5 mg. According to institutional standards, the upper limit of the permissible body surface area is 2 m². 2 .

[0524] When rituximab is administered on the same day as the CHOP program, it is recommended to administer prednisone (100 mg) before the rituximab infusion.

[0525] When rituximab and the CHOP schedule are administered on the same day, it is recommended to administer prednisone (100 mg) before the rituximab infusion. If the rituximab dose for Day 1 of Cycle 1 is split, both infusions must be administered with appropriate prophylaxis, following the infusion guidelines described in the pharmacy manual or product characteristics summary. If CHOP starts later than Day 1 of the cycle, the planned Day 1 of the next cycle is calculated from the start date of CHOP to maintain the standard 21-day chemotherapy interval.

[0526] Intrathecal methotrexate

[0527] For patients with high-risk CNS IPI (defined as having four or more of the following risk factors: age >60 years, stage III or IV disease, more than two extranodal sites, longest diameter greater than the upper limit of normal, or kidney or adrenal involvement), intrathecal methotrexate is recommended (but not mandatory) for CNS prophylaxis and may be included as part of the patient's treatment plan at the investigator's discretion. If CNS prophylaxis is administered, the dosage and regimen should be clearly documented and administered according to local guidelines.

[0528] Granulocyte colony-stimulating factor

[0529] To minimize the incidence, severity, and duration of neutropenia-related complications, all participants received primary prophylaxis with G-CSF during cycles 1 through 6.

[0530] G-CSF must be used during periods 3 to 6, i.e., any of the following:

[0531] Figexidine: Administer subcutaneously at a rate of 5 μg / kg (rounded to 300 μg or 480 μg) once daily for up to 48 hours after R-CHOP administration (day 1 of cycles 1 to 6) for 7 days or until two consecutive days of ANC > 1.5 × 10⁻⁶. 9 / L

[0532] Ethylene glycolated filgrastim: 6 mg subcutaneously administered up to 48 hours after R-CHOP administration (day 1 of cycles 1 to 6).

[0533] Legasil is an acceptable alternative.

[0534] If preferred or clinically indicated, G-CSF shall be administered intravenously.

[0535] Dexamethasone

[0536] Dexamethasone is administered 1 hour before the administration of glimetuzumab; oral acetaminophen or paracetamol (500 mg or 1000 mg) and antihistamines such as diphenhydramine (50 mg) are administered approximately 30 minutes before the start of the infusion.

[0537] AVENIO Oncology Assay for Non-Hodgkin Lymphoma

[0538] The AVENIO Oncology Assay (AOA-NHL) for non-Hodgkin's lymphoma uses targeted next-generation sequencing (NGS) to sequence genomic DNA and cell-free plasma DNA (cfDNA) isolated from peripheral mononuclear cells or plasma-depleted whole blood (PDWB) to qualitatively detect ctDNA, an indicator of minimal residual disease (MRD). The AOA-NHL test can be used to determine the ctDNA status of patients with DLBCL.

[0539] The AOA-NHL test is an NGS tumor profiling and monitoring assay that identifies somatic DNA mutations in individuals with DLBCL by sequencing cfDNA isolated from plasma samples at different time points during treatment. cfDNA consists of DNA fragments released into the bloodstream due to cell death in both normal and tumor tissues. The presence of somatic variants (non-individually inherited variants) in tumor-derived DNA is presumed and referred to as ctDNA.

[0540] The AOA-NHL test is a laboratory-developed assay validated at Roche Molecular Systems (San Jose, California, USA). The AOA-NHL test provides a streamlined end-to-end NGS solution from DNA extraction to bioinformatics data analysis and reporting. The test is based on iDES-enhanced CAPP-Seq (Integrated Digital Error Suppression Enhanced Cancer Personalized Deep Sequencing Analysis) technology, described in detail by Newman et al. (2014, 2016). Multiple studies have demonstrated that ctDNA deep sequencing can detect somatic variants with very low allele fractions (Schwarzenbach et al. 2011; Narayan et al. 2012; Diehn et al. 2017). This makes ctDNA assays suitable for monitoring disease burden in patients receiving treatment (Chin et al. 2019).

[0541] This test aims to report the presence of 466 tumor-derived DNA variants spanning approximately 314 kilobases across the genome associated with disease presence and to assess changes in the levels of such variants from the patient's peripheral blood throughout treatment. The AOA-NHL test uses a proprietary bioinformatics workflow to identify tumor-specific variants (TSVs) by subtracting sample variants present in a matching normal PDWB sample from those variants detected in plasma samples. The workflow also monitors for the same TSVs from plasma samples collected at subsequent time points. The mean allele fraction of all detected TSVs is calculated to estimate the ctDNA level per milliliter of plasma. The detection of these TSVs or a subset of these TSVs in a plasma sample indicates the presence of ctDNA in the patient's blood. Statistical significance of TSVs is expressed as a p-value, calculated using a Monte Carlo statistical method (Newman et al. 2016) to account for background variance.

[0542] The use of the AOA-NHL test is experimental and is intended to identify high-risk patients with untreated DLBCL who may be eligible to participate in this study. The test is limited to centralized analysis at RocheMolecular Systems in San Jose, California, a company accredited by the Clinical Laboratory Improvement Act of 1988 (CLIA) to perform highly complex clinical tests.

[0543] Research duration

[0544] The total duration of each individual's participation in the study is expected to be approximately 24 months.

[0545] research group

[0546] Approximately 40 participants with untreated DLBCL who were at high risk of ctDNA were enrolled in this study.

[0547] Prospective approval of deviations from recruitment and inclusion criteria is not permitted; this is referred to as a protocol exemption or waiver.

[0548] Inclusion criteria

[0549] Participants are eligible to be included in the study only if they meet all of the following criteria:

[0550] Participants aged 18 and above

[0551] Life expectancy ≥ 6 months

[0552] Patients with previously untreated CD20-positive DLBCL, including those diagnosed according to one of the following diagnoses based on the 2016 WHO classification of lymphomas (Swerdlow et al. 2016):

[0553] – Non-specific types of DLBCL, including GCB and ABC / non-GCB types, as well as dual-expression lymphomas (MYC and BCL2 co-expression).

[0554] – High-grade B-cell lymphoma (HGBCL) with MYC and BCL2 and / or BCL6 translocations.

[0555] The maximum number of participants with HGBCL was 15% of the screened population.

[0556] - Patients with newly diagnosed transformed follicular lymphoma

[0557] Eastern Cooperative Oncology Group (ECOG) performance status 0-2

[0558] IPI 1-5,

[0559] Patients with an IPI of 0 and a large lesion (> 6 cm) are eligible.

[0560] Before starting R-CHOP on Day 1 of Cycle 1 and on Day 1 of Cycle 2, submit sufficient biomarkers for screening to determine ctDNA status.

[0561] At least one two-dimensional FDG-avid measurable lymphoma lesion on a PET / CT scan, defined as having a longest diameter > 1.5 cm on the CT scan.

[0562] Left ventricular ejection fraction (LVEF) ≥ 50%, determined by multi-gated cardiac acquisition (MUGA) scan or echocardiography (ECHO).

[0563] Adequate hematological function (unless there is evidence of extensive bone marrow infiltration or hypersplenism secondary to underlying lymphoma, in which case blood product transfusion is permitted) is defined as follows:

[0564] – Hemoglobin ≥ 9.0 g / dL (≥ 90 g / L), and no RBC concentrate infusion in the 7 days prior to the first study treatment.

[0565] – ANC ≥ 1.0 × 10 9 / L (≥ 1000 / µL)

[0566] – Platelet count ≥ 75 × 10⁻⁶ 9 / L (≥ 75,000 / μL)

[0567] Additional inclusion criteria for high-risk participants using ctDNA

[0568] After two R-CHOP cycles, participants are eligible to continue enrollment in the study only if they meet the following inclusion criteria:

[0569] Plasma samples assessed as high-risk for ctDNA using the experimental AOA-NHL test were defined as those with a < 2-log-fold decrease in ctDNA levels between Day 1 of Cycle 1 and Day 1 of Cycle 2, as determined by a central laboratory.

[0570] Exclusion criteria

[0571] Participants will be excluded from the study if any of the following criteria are met:

[0572] The following have been diagnosed: unclassifiable B-cell lymphoma (grey zone lymphoma) with intermediate features between DLBCL and classical Hodgkin's lymphoma; primary mediastinal (thymic) large B-cell lymphoma; T-cell / histiocytic abundant large B-cell lymphoma; Burkitt lymphoma; CNS lymphoma (primary or secondary involvement); primary exudative DLBCL; and primary cutaneous DLBCL.

[0573] Contraindications to any of the individual components of R-CHOP include prior anthracycline therapy.

[0574] Those with a history of severe allergy or allergic reaction to mouse monoclonal antibodies, or those with known sensitivity or allergy to mouse products.

[0575] Previous treatment for indolent lymphoma

[0576] Prior use of any monoclonal antibody within 4 weeks or 5 half-lives (whichever is shorter) prior to the start of Cycle 1, and / or prior use of any investigational therapy for cancer treatment within 28 days prior to the start of Cycle 1.

[0577] Previous solid organ or allogeneic stem cell transplantation

[0578] Administer a live vaccine within 28 days before the start of Cycle 1 (currently available SARS-CoV-2 vaccines, including mRNA vaccines and viral vector vaccines, are not live vaccines).

[0579] Prior therapy for DLBCL and HGBCL, excluding palliative short-term corticosteroid therapy.

[0580] Patients who need to control lymphoma symptoms during screening can receive steroids in the following ways:

[0581] Following a diagnostic biopsy, prednisone or an equivalent (up to 100 mg / day) can be administered for up to 7 days during the screening period to control lymphoma symptoms. For doses >1 mg / kg, consult a medical monitor.

[0582] Patients receiving ≤ 30 mg / day of prednisone or an equivalent corticosteroid for reasons other than controlling lymphoma symptoms (e.g., rheumatoid arthritis) must have documented evidence of using a stable dose for at least 4 weeks prior to starting cycle 1.

[0583] Orthostatic hypotension and adrenal insufficiency can be managed separately using mineralocorticoids and corticosteroids.

[0584] Receive systemic immunosuppressive drugs (including but not limited to cyclophosphamide, imidazoline, methotrexate, thalidomide, and anti-tumor necrosis factor agents) within 4 weeks prior to the first dose of R-CHOP.

[0585] Blood transfusions should be administered within 7 days prior to screening, unless there is evidence of lymphoma involving the bone marrow or spleen (in which case blood product transfusions are permitted).

[0586] Any of the following laboratory test values:

[0587] – Serum creatinine clearance <40 mL / min (calculated according to the Cockcroft-Gault formula) – AST or ALT > 2.5 × Upper Limit of Normal (ULN)

[0588] – Total bilirubin > 1.5 × ULN (participants with documented Gilbert's disease are eligible for inclusion if their total bilirubin is ≤ 3 × ULN)

[0589] – In the absence of therapeutic anticoagulation therapy, INR or PT > 1.5 times ULN

[0590] – In the absence of therapeutic anticoagulation or lupus anticoagulation therapy, PTT or aPTT > 1.5 times ULN.

[0591] I recently underwent major surgery (within 6 weeks prior to the start of day 1 of cycle 1), except for diagnoses.

[0592] A history of severe or long-term cardiovascular disease, such as New York Heart Association Class III or IV heart disease or objective assessment Class C or D, myocardial infarction within the past 6 months, unstable arrhythmia, or unstable angina.

[0593] No history of other cancers within 2 years prior to screening, except for appropriately treated cervical carcinoma in situ, non-melanoma skin cancer, or stage I uterine cancer.

[0594] Patients with malignant tumors treated with radical surgery alone are not eligible unless they have a history of cancer remission for ≥2 years prior to enrollment and have not received treatment.

[0595] Patients with current low-grade early prostate cancer (Gleason score ≤ 6, stage 1 or 2) who did not require treatment at any time prior to the study are eligible.

[0596] Any active infection that could affect patient safety within 7 days prior to Day 1 of Cycle 1 or at enrollment.

[0597] – In the event of a pandemic or epidemic, active infection screening should be considered in accordance with local or institutional guidelines or guidelines of applicable professional societies (such as the American Society of Clinical Oncology [ASCO] or the European Society for Medical Oncology [ESMO]).

[0598] The hepatitis B surface antigen (HBsAg) test result was positive during screening.

[0599] – If HBV DNA is undetectable, patients with occult or prior hepatitis B virus (HBV) infection (defined as HBsAg negative and total hepatitis B core antibody [HBcAb] positive) may be included, provided they are willing to undergo monthly DNA testing. Patients with a protective titer of hepatitis B surface antibody (HBsAb) after vaccination or those with prior hepatitis B but now cured are eligible.

[0600] The hepatitis C virus (HCV) antibody test result was positive during screening.

[0601] – Patients who are positive for other HCV antibodies are eligible for inclusion only if their HCV RNA polymerase chain reaction (PCR) results are negative.

[0602] Suspected or latent tuberculosis

[0603] History of progressive multifocal leukoencephalopathy

[0604] Confirmed or suspected chronic active Estane-Barr virus infection

[0605] Known or suspected HLH history

[0606] A clinically significant history of liver disease, including viral hepatitis or other types of hepatitis, or cirrhosis.

[0607] Substance abuse within the 12 months prior to screening, including over-the-counter drugs and alcohol dependence.

[0608] Any serious medical condition or abnormality in clinical laboratory tests that, in the investigator's judgment, would prevent a patient from safely participating in and completing the study, including severe psychosis requiring hospitalization and antipsychotic medication.

[0609] Patients with a history of autoimmune diseases, including but not limited to: myasthenia gravis, myositis, autoimmune hepatitis, systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, vascular thrombosis associated with antiphospholipid syndrome, Wegener's granulomatosis, Sjögren's syndrome, Guillain-Barré syndrome, multiple sclerosis, vasculitis, or glomerulonephritis, may be excluded in the following cases:

[0610] - Patients with a history of autoimmune-related hypothyroidism requiring stable doses of thyroid replacement hormone.

[0611] Patients with type 1 diabetes who are receiving an insulin regimen are eligible for this study.

[0612] Patients with a history of disease-related immune thrombocytopenic purpura, autoimmune hemolytic anemia, or other stable autoimmune diseases.

[0613] – Participants with eczema, psoriasis, chronic simple lichen simplex, or vitiligo with only skin manifestations (e.g., excluding participants with psoriatic arthritis) are eligible for this study if they meet all of the following criteria:

[0614] The rash must cover less than 10% of the body surface area.

[0615] The disease is well controlled at baseline and only requires the use of ineffective topical corticosteroids.

[0616] No acute exacerbations of underlying conditions requiring psoralen plus UVA radiation, methotrexate, retinoids, biologics, oral calcineurin inhibitors, or potent or oral corticosteroids have occurred in the past 12 months.

[0617] Malabsorption syndrome or other conditions that affect intestinal absorption

[0618] Pregnancy or breastfeeding, or planning to become pregnant during the study period or within 12 months after the last dose of R-CHOP, within 3 months after the last dose of tocilizumab (if applicable), or within 2 months after the last dose of glimetuzumab.

[0619] end

[0620] The primary efficacy endpoint was the EOT CR rate, measured by PET-CT scans and determined by the investigator according to the 2014 Lugano Response Criteria. The CR rate was estimated as the percentage of participants who achieved CR at EOT, and a corresponding Clopper-Pearson precision 95% CI was constructed for the study cohort.

[0621] Secondary endpoint

[0622] Secondary efficacy endpoints are EOT ORR, PFS, and OS.

[0623] ORR is defined as CR or PR at EOT, measured by PET-CT scan and determined by the investigator. The analysis of ORR is the same as that described above for the primary efficacy endpoint of CR rate at EOT measured by PET / CT scan.

[0624] PFS was defined as the time from the first treatment in the study to the first occurrence of disease progression, relapse, or death from any cause (whichever occurs first). For participants who did not experience disease progression, relapse, or death, PFS was reviewed on the date of the last evaluable oncology assessment. For participants without a baseline evaluable oncology assessment, PFS was reviewed one day after the date of the first treatment in the study. PFS was assessed by the investigator using the 2014 Lugano Response Criteria (Cheson et al. 2014). The distribution of PFS, median (if analytically feasible) 6-month and 1-year PFS were estimated using the Kaplan-Meier method (Kaplan and Meier 1958), with the 95% CI for median PFS constructed using the Brookmeyer-Crowley method (Brookmeyer and Crowley 1982). The standard errors and corresponding 95% CIs for 6-month and 1-year PFS were calculated using the Greenwood formula.

[0625] OS is defined as the time from the first treatment in the study to death from any cause. The analysis of OS is the same as that described above for PFS.

[0626] Relevant laboratory data, vital signs (pulse rate, respiratory rate, blood pressure and body temperature), and ECG data are displayed over time, with grades indicated where appropriate. Additionally, a transpose table of selected laboratory tests is used to summarize the severity grades at baseline and after maximum baseline. Changes in vital signs and ECG are summarized.

[0627] Safety was assessed by summarizing study treatment exposures, the occurrence and severity of adverse events, changes in laboratory test results, and changes in vital signs and ECG. Study treatment exposures (such as duration of treatment, total dose received, number of cycles, and number of dose modifications) were summarized using descriptive statistics. All verbatim adverse event terms were mapped to MedDRA terminology and severity was graded according to NCI CTCAE v5.0. For CRS, severity was determined according to the ASTCT CRS consensus grading criteria. All adverse events occurring during or after the first study treatment administration, critical adverse events, adverse events leading to death, adverse events of particular concern, and adverse events leading to termination of study treatment (i.e., adverse events occurring during treatment) were summarized using mapped terms, appropriate terminology levels, and severity grades. For events of different severity, the highest grade was used in the summary. Deaths and causes of death were summarized.

[0628] Exploratory endpoint

[0629] The exploratory efficacy endpoints were DOR and EOT CR rate, measured by PET / CT scans and determined by automated response (aLugano) according to the 2014 Lugano Response Criteria. DOR was defined as the time from the first recorded objective response (CR or PR) to disease progression or death from any cause, prior to the first occurrence. For participants who did not die or experience disease progression, DOR was reviewed at the date of the last evaluable tumor assessment. DOR was calculated by the investigator using the 2014 Lugano Response Criteria (Cheson et al. 2014). DOR analysis included only participants who achieved an objective response (CR or PR) at any time during the study. The distribution of DOR and median DOR was estimated using the Kaplan-Meier method (Kaplan and Meier 1958) (where analytically feasible), and the 95% CI for median DOR was constructed using the Brookmeyer-Crowley method (Brookmeyer and Crowley 1982). The DOR is summarized for the subset of participants whose best objective response is PR and the subset of participants whose best objective response is CR.

[0630] The aLugano analysis was conducted in a PET / CT-evaluable cohort, defined as all patients with available PET / CT scans at baseline and during treatment. The analysis of EOT CR rates determined by aLugano was identical to the analysis described above for the investigator-determined primary efficacy endpoint of CR rate at EOT.

[0631] A contingency matrix was calculated for aLugano and responses assessed according to the 2014 Lugano Response Criteria. The percentages of positive concordance, negative concordance, overall concordance, sensitivity, and specificity for CR, CR / PR, and DOR were calculated. Automated total metabolic tumor volume (aTMTV) was calculated for an evaluable patient population with baseline available PET / CT scans. The overall distribution of aTMTV at baseline and during treatment could be plotted, and values ​​for the evaluable population could be summarized using descriptive statistics such as mean, standard deviation, median, and range. The concordance between aTMTV and TMTV assessed according to the sponsor's radiologist could be assessed using Spearman correlation analysis, and visualized using a scatter plot. The relationship between aTMTV and CR rate, ORR, PFS, OS, and DOR could be further evaluated to determine whether the characteristic is prognostic or predictive. Additional analyses of quantitative measures (e.g., ΔSUVmax) and / or exploratory biomarkers could be performed within this study and after summarizing data from other studies.

[0632] Other security analyses

[0633] Assess the incidence of ADA response (i.e., the presence of anti-glucantuma antibody in serum) and its potential relevance to PK, pharmacodynamic and safety parameters.

[0634] Efficacy evaluation

[0635] Tumor and response assessment

[0636] Tumor assessments were performed on participants using 18FFDG-PET / CT and diagnostic CT scans at screening, interim response assessment (between days 15 and 21 of cycle 2), and EOT. Response was assessed according to the 2014 Lugano criteria for response. During survival follow-up, response was assessed every 6 months until any time the participant experienced disease progression, discontinued the study, suspected disease progression, or up to one year after the EOT response assessment, whichever occurred first. Tumor assessments were repeated at the investigator's discretion if disease progression was suspected.

[0637] Clinical response assessment includes physical examination to evaluate the presence and extent of lymphadenopathy, hepatomegaly, and splenomegaly. All measurable and / or evaluable lesions are assessed and documented at screening. Tumor assessments performed as SOCs before informed consent is obtained and within 28 days prior to the initiation of study treatment do not need to be repeated at screening, provided they meet the following criteria.

[0638] Radiographic assessment

[0639] FDG-PET / CT scans combined with diagnostic-quality CT scans are required at screening and EOT. Interim efficacy assessment (days 15-21 of cycle 2) requires only FDG-PET / CT scans. Throughout the study period, any diagnosis of disease progression based on clinical examination must be confirmed as soon as possible (within 30 days) by imaging (e.g., CT scan, FDG PET / CT scan) before initiating non-protocol-prescribed anticancer therapy. Bone marrow biopsy is not a routine requirement for response assessment but may be indicated in specific circumstances according to the 2014 Lugano Response Criteria (e.g., when response assessment is based solely on CT or when residual uptake is observed on PET / CT scans). All measurable and / or evaluable disease must be documented at screening and reassessed at each subsequent tumor assessment. Response is assessed by the investigator based on the results of physical examination, CT scans, and FDG-PET / CT scans.

[0640] Physical examination

[0641] A full physical examination includes assessments of the cardiovascular, cutaneous, musculoskeletal, respiratory, gastrointestinal, and nervous systems; a genitourinary examination may be performed if clinically indicated. Any abnormalities identified at baseline should be recorded on the general history and baseline status eCRF. As part of the full physical examination, the presence and extent of lymphadenopathy, hepatomegaly, and splenomegaly should be recorded on the appropriate tumor assessment eCRF. A targeted, symptom-oriented physical examination includes assessments of the cutaneous, respiratory, cardiovascular, and abdominal (liver and spleen) systems, and should be performed at the designated post-baseline visit and as clinically indicated. Investigators should pay particular attention to clinical signs associated with previous serious illness. Changes in abnormalities relative to baseline should be recorded in participants' notes. New or worsening clinically significant abnormalities should be recorded as adverse events on the adverse events eCRF. As part of tumor assessment, a targeted physical examination, including assessment of the presence and extent of lymphadenopathy, hepatomegaly, splenomegaly, or other findings associated with lymphoma, should be recorded on the appropriate tumor assessment eCRF.

[0642] Sample quantity determination

[0643] The aim of this study was to evaluate the safety and efficacy of the combination of glimepiride and R-CHOP in a first-line setting in participants with previously untreated DLBCL at high risk of ctDNA. This study is ongoing. Based on previously reported high-risk ctDNA rates of approximately 20% in patients with DLBCL after one cycle of R-CHOP therapy in a first-line setting, approximately 200 participants will be screened to enroll 40 evaluable participants for study treatment (Kurtz et al. 2018). The primary efficacy endpoint was the CR rate at EOT as assessed by the investigator according to the 2014 Lugano response criteria. For 40 patients, the margin of error for the exact Clopper-Pearson (Clopper and Pearson 1934) 95% CI used to estimate the true CR rate at EOT was no more than ±17%. Based on a sample size of 40, the exact Clopper-Pearson 95% CI corresponded to observed CR rates ranging from 40% to 80%. It has been previously reported that the optimal complete response (CR) rate is 40% for patients with high-risk ctDNA DLBCL receiving SOC R-CHOP therapy in a first-line setting (Kurtz et al. 2018). With 40 participants and an observed CR rate of at least 60%, true CR rates below 43% or above 75% can be excluded with 95% confidence.

[0644] result

[0645] We report preliminary safety and efficacy from a phase II open-label prospective study (NCT04980222) evaluating glofit + R-CHOP in patients with 1L LBCL who were defined as high risk based on ctDNA kinetics. Eligible patients had 1L CD20+ LBCL, with an Eastern Oncology Cooperative Group performance status of 0 to 2 and an IPI of 0 to 5 (IPI of 0 for patients with larger lesions; IPI of 2 to 5 for US patients). During ctDNA screening, patients received R-CHOP (21-day cycle) on day 1 (D) of both cycle (C)1 and cycle C2. In patients defined as high-risk based on ctDNA (plasma ctDNA reduction <2 log after one R-CHOP cycle), R-CHOP continued to C6, with glimetuzumab administered in escalating doses at C3 (D8, 2.5 mg; D15, 10 mg), and at the target dose (30 mg) at D8 in C4–6 and D1 in C7–10 (21-day cycle). Hospitalization for cytokine release syndrome (CRS) was determined at the investigator's discretion based on the first dose of glimetuzumab; granulocyte colony-stimulating factor was recommended for neutropenia prophylaxis. Real-time ctDNA analysis was performed centrally (AVENIO oncology assay for non-Hodgkin lymphoma) (Stokowski et al. ASH 2022). The primary endpoint was complete response (CR) at the end of treatment (EOT). Responses were assessed by investigators via PET according to the Lugano criteria (Cheson et al., J Clin Oncol 2014) after C2 and at EOT. CRS was graded according to the ASTCT criteria (Lee et al., Biol Blood Marrow Transplant 2019); other adverse events (AEs) were assessed according to CTCAE v5.0. At the data cutoff date (May 25, 2023), of the 121 screened patients, 24 (19.8%) were defined as high-risk based on ctDNA (47 low-risk patients [38.8%]; 49 non-evaluable patients [40.5%]) and received ≥1 dose of the study drug from C3D1 (safety cohort). Of the 24 high-risk patients, 15 reached EOT assessment (14 patients completed treatment; 1 patient discontinued due to disease progression [PD]), and 9 patients were still receiving treatment.Of the 24 patients, none had nonspecific high-grade B-cell lymphoma by histological examination; two had double-hit and four had triple-hit cytogenetic patterns; the median age was 61.5 years (range: 39–77); 14 (58.3%) patients had an IPI of 1–2 (IPI 1: 12.5%, IPI 2: 45.8%), and nine (37.5%) patients had an IPI of 3–5 (IPI 3: 29.2%, IPI 4: 8.3%, IPI 5: 4.2%). In the safety cohort, 20 (83.3%) patients had any grade of adverse events (AE); 15 (62.5%) patients had grade 3 / 4 AEs, including neutropenia (11 patients [45.8%]); no febrile neutropenia was observed (Table 5). The most common (>20%) treatment-related adverse events (AEs) of any grade were neutropenia (13 patients [54.2%]) and diarrhea (6 patients [25.0%]). No grade 5 AEs were reported. Nine patients (37.5%) experienced 17 serious AEs (14 of which were related to glimetuzumab), including 7 CRS events. Eight patients (33.3%) experienced ≥ grade 3 glimetuzumab-related AEs. Five patients (20.8%) experienced CRS events: four (16.7%) had grade 1 fever, and one (4.2%) had grade 2 fever and hypotension due to hypotension. CRS events occurred early and subsided at the data cutoff; tocilizumab and dexamethasone were administered only for grade 2 CRS events. One patient (4.2%) experienced a non-immunotoxic Grade 3 neurological adverse event (apnea, unrelated to glimetuzumab), which resolved by the data cutoff. One patient died of disease progression (PD) during treatment follow-up after discontinuation of glimetuzumab. The interim CR rate was 46.7% (95% CI: 21.3–73.4); the CR rate at EOT was 80.0% (95% CI: 51.9–95.7), and the overall response rate was 93.3% (95% CI: 68.1–99.8) (Figure 4). Glofit+ R-CHOP demonstrated a manageable safety profile in 1L high-risk LBCL as defined by ctDNA and induced high response rates at both the interim analysis and EOT. Dynamic treatment-intermediate risk assessment using ctDNA offers the potential to identify high-risk patients with LBCL independently of baseline characteristics.

[0646] Table 5: Summary of AEs by the highest NCI CTCAE level (>10%)

[0647]

[0648] * Neutropenia: A combined result of neutropenia and a decreased neutrophil count; multiple occurrences of the same AE in the same individual are counted once for the highest grade of that AE.

[0649] AE, Adverse Event; CTCAE, Common Terminology Standard for Adverse Events; NCI, National Cancer Institute.

[0650] Update results

[0651] Table 6 below reports updated End-of-Treatment (EOT) results after another data cutoff date (September 18, 2023). The overall response rate was 95% (see “Responders” percentage). The EoT secondary efficacy cohort includes all patients who received the first dose of their study treatment (post-enrollment) early enough for protocol scheduling to be assessed for end-of-treatment response before the data cutoff date.

[0652]

[0653] sequence list

[0654] Serial ID No:1

[0655] Length: 5

[0656] Molecular type: protein

[0657] Source: Artificial Sequence

[0658] Species: Synthetic Constructs

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[0660]

[0661] Serial ID No:2

[0662] Length: 17

[0663] Molecular type: protein

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[0665] Species: Synthetic Constructs

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[0668] Serial ID No:3

[0669] Length: 10

[0670] Molecular type: protein

[0671] Source: Artificial Sequence

[0672] Species: Synthetic Constructs

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[0675] Serial ID No:4

[0676] Length: 16

[0677] Molecular type: protein

[0678] Source: Artificial Sequence

[0679] Species: Synthetic Constructs

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[0682] Serial ID No:5

[0683] Length: 7

[0684] Molecular type: protein

[0685] Source: Artificial Sequence

[0686] Species: Synthetic Constructs

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[0688]

[0689] Serial ID No:6

[0690] Length: 9

[0691] Molecular type: protein

[0692] Source: Artificial Sequence

[0693] Species: Synthetic Constructs

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[0695]

[0696] Serial ID No:7

[0697] Length: 119

[0698] Molecular type: protein

[0699] Source: Artificial Sequence

[0700] Species: Synthetic Constructs

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[0702]

[0703] Serial ID No:8

[0704] Length: 112

[0705] Molecular type: protein

[0706] Source: Artificial Sequence

[0707] Species: Synthetic Constructs

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[0710] Serial ID No:9

[0711] Length: 5

[0712] Molecular type: protein

[0713] Source: Artificial Sequence

[0714] Species: Synthetic Constructs

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[0717]

[0718] Serial ID No:10

[0719] Length: 19

[0720] Molecular type: protein

[0721] Source: Artificial Sequence

[0722] Species: Synthetic Constructs

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[0724]

[0725] Serial ID No:11

[0726] Length: 14

[0727] Molecular type: protein

[0728] Source: Artificial Sequence

[0729] Species: Synthetic Constructs

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[0731]

[0732] Serial ID No:12

[0733] Length: 14

[0734] Molecular type: protein

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[0736] Species: Synthetic Constructs

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[0738]

[0739] Serial ID No:13

[0740] Length: 7

[0741] Molecular type: protein

[0742] Source: Artificial Sequence

[0743] Species: Synthetic Constructs

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[0746] Serial ID No:14

[0747] Length: 9

[0748] Molecular type: protein

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[0750] Species: Synthetic Constructs

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[0753] Serial ID No:15

[0754] Length: 125

[0755] Molecular type: protein

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[0760] Serial ID No:16

[0761] Length: 109

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[0767] Serial ID No:17

[0768] Length: 672

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[0774] Serial ID No:18

[0775] Length: 447

[0776] Molecular type: protein

[0777] Source: Artificial Sequence

[0778] Species: Synthetic Constructs

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[0780]

[0781] Serial ID No:19

[0782] Length: 232

[0783] Molecular type: protein

[0784] Source: Artificial Sequence

[0785] Species: Synthetic Constructs

[0786] sequence:

[0787]

[0788] Serial ID No:20

[0789] Length: 219

[0790] Molecular type: protein

[0791] Source: Artificial Sequence

[0792] Species: Synthetic Constructs

[0793] sequence:

[0794]

[0795] Other embodiments

[0796] Although the invention has been described in considerable detail above by way of demonstration and example for purposes of clarity, such description and example should not be construed as limiting the scope of the invention. All disclosures of patents and scientific literature cited herein are expressly incorporated in their entirety by reference.

Claims

1. A method for treating a previously untreated patient with diffuse large B-cell lymphoma at high risk of circulating tumor DNA (ctDNA), the method comprising administering a combination of a bispecific antibody targeting CD3 and CD20, comprising the VH and VL sequences of glimetuzumab, with chemotherapy, wherein the patient has been identified as at high risk of ctDNA if the ctDNA level decreases by less than 2.5 log after one or two cycles of chemotherapy.

2. The method according to claim 1, wherein the bispecific antibody targeting CD3 and CD20 comprises the heavy chain of SEQ ID NO: 17, the heavy chain of SEQ ID NO: 18, the two light chains of SEQ ID NO: 20, and the light chain of SEQ ID NO: 19; and wherein the heavy chain and the light chain are assembled to form a first Fab molecule specifically binding to CD20, a second Fab molecule specifically binding to CD3, and a third Fab molecule specifically binding to CD20, and an Fc domain composed of a first subunit and a second subunit capable of stable association.

3. The method according to claim 1 or 2, wherein the chemotherapy is selected from: rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP); rituximab, cyclophosphamide, doxorubicin and prednisone (R-CHP); or rituximab, ifosfamide, carboplatin and etoposide phosphate (R-ICE).

4. The method according to any one of claims 1 to 3, wherein the bispecific antibody targeting CD3 and CD20 is glimetuzumab, and the chemotherapy is rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), and wherein the patient has been identified as high-risk for ctDNA if the patient's ctDNA decreases by less than 2.5 log after one or two R-CHOP cycles.

5. The method of claim 4, wherein the reduction in ctDNA is determined by measuring the amount of ctDNA before or on the day of the first treatment (baseline) and after at least one R-CHOP treatment cycle.

6. The method of claim 4 or 5, wherein the amount of ctDNA is determined on or before day 1 of the first R-CHOP treatment cycle (baseline) and on day 1 of the second R-CHOP treatment cycle.

7. The method according to any one of claims 4 to 6, wherein if the patient's ctDNA decreases by less than 2 log, the patient is identified as high-risk for ctDNA.

8. A method for treating previously untreated diffuse large B-cell lymphoma, the method comprising: a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles; b. Determine the amount of ctDNA at or before cycle 1 (baseline) and after at least one R-CHOP treatment cycle; and c. Select patients whose ctDNA reduction is <2.5-log between baseline and at least one R-CHOP treatment cycle and continue treatment with R-CHOP and glimetuzumab.

9. The method of claim 8, wherein step a) comprises applying R-CHOP on day 1 of cycles 1, 2 and optionally cycle 3.

10. The method of claim 9, wherein step a) comprises applying R-CHOP on day 1 of cycles 1 and 2.

11. The method according to any one of claims 8 to 10, wherein step b) comprises determining the amount of ctDNA on or before day 1 of cycle 1 (baseline) and day 1 of cycle 2 or day 1 of cycle 3 of the R-CHOP treatment in step a).

12. The method of claim 11, wherein step b) comprises determining the amount of ctDNA on day 1 (baseline) of cycle 1 and day 1 of cycle 2 of the R-CHOP treatment in step a).

13. The method according to any one of claims 8 to 12, wherein step c) comprises selecting patients with a ctDNA reduction of <2-log.

14. The method according to any one of claims 8 to 13, wherein step c) comprises continuing treatment with R-CHOP for a total of six cycles (cycles 1 to 6) and adding glimetuzumab for a total of eight cycles (cycles 3 to 10).

15. The method according to any one of claims 8 to 14, wherein glucentumab is administered at a dose of 2.5 mg on day 8 of cycle 3 and at a dose of 10 mg on day 15, at a dose of 30 mg on day 8 of cycles 4 to 6, and at a dose of 30 mg on day 1 of cycles 7 to 10.

16. The method according to any one of the preceding claims, wherein the ctDNA is circulating tumor DNA in plasma.

17. The method according to any one of the preceding claims, wherein the amount of ctDNA is measured in a blood sample from the patient.

18. The method of claim 17, wherein the amount of ctDNA is measured in genomic DNA isolated from peripheral mononuclear cells or plasma-depleted whole blood (PDWB) or in cell-free DNA (cfDNA) in plasma.

19. The method of claim 18, wherein the ctDNA is measured in cell-free DNA in plasma.

20. The method according to any one of the preceding claims, wherein the ctDNA is determined by targeted next-generation sequencing (NGS).

21. The method according to any one of claims 4 to 20, wherein rituximab is administered at 375 mg / m². 2 Cyclophosphamide was administered intravenously at a dose of 750 mg / m². 2 Doxorubicin was administered intravenously at a dose of 50 mg / m². 2 Vincristine was administered intravenously at a dose of 1.4 mg / m². 2 The dose was administered via IV bolus, and prednisone was administered at a dose of 100 mg / day from day 1 to day 5.

22. The method according to any one of claims 1 to 21, wherein the length of each cycle is 21 days.

23. The method according to any one of claims 4 to 22, wherein administration of such treatment to multiple human patients at high risk of ctDNA results in an overall response rate (ORR) of at least 80%, at least 85%, at least 90%, or at least 95%.

24. The method of claim 23, wherein the ORR is at least 95%.

25. The method according to any one of claims 1 to 21, wherein administration of such treatment to multiple human patients at high risk of ctDNA results in a complete remission (CR) rate of at least 75%, at least 80%, or at least 85%.

26. The method of claim 25, wherein the CR rate is at least 85%.

27. The method according to claims 23 and 24 or claims 25 and 26, wherein the ORR or CR rate is determined at the end of treatment.

28. The method of claim 27, wherein the ORR or CR is determined by PET-CT scan.

29. The method of claim 27 or 28, wherein the ORR or CR is determined according to the 2014 Lugano Reaction Standard.

30. A bispecific antibody targeting CD3 and CD20 comprising the VH and VL sequences of glimetuzumab, for use in a method of treating previously untreated diffuse large B-cell lymphoma at high risk of (ct)DNA, the method comprising administering glimetuzumab in combination with chemotherapy to a patient, wherein the patient has been identified as at high risk of ctDNA if the ctDNA level decreases by less than 2.5 log after one cycle of chemotherapy.

31. Glimepiride, in a method of treating previously untreated diffuse large B-cell lymphoma with high (ct)DNA risk, the method comprising: a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles; b. Determine the amount of ctDNA at or before cycle 1 (baseline) and after at least one R-CHOP treatment cycle; and c. Select patients whose ctDNA reduction is <2.5-log between baseline and at least one R-CHOP treatment cycle and continue treatment with R-CHOP and glimetuzumab.

32. Use of a bispecific antibody targeting CD3 and CD20 comprising the VH and VL sequences of glimetuzumab in the manufacture of a medicament for treating a subject with previously untreated diffuse large B-cell lymphoma at high risk of (ct)DNA, wherein the patient is administered glimetuzumab in combination with chemotherapy, wherein the patient has been identified as at high risk of ctDNA if the ctDNA decreases by less than 2.5 log after one cycle of chemotherapy.

33. Use of glimetuzumab in the manufacture of a medicament for the treatment of subjects with previously untreated diffuse large B-cell lymphoma at high risk of (ct)DNA, including: a. Administer rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for at least 2 cycles; b. Determine the amount of ctDNA at or before cycle 1 (baseline) and after at least one R-CHOP treatment cycle; and c. Select patients whose ctDNA reduction is <2.5-log between baseline and at least one R-CHOP treatment cycle and continue treatment with R-CHOP and glimetuzumab.

34. The present invention as described above.

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