Predictive biomarkers for patients with follicular lymphoma and diffuse large B-cell lymphoma

Abstract

This disclosure provides a method for treating lymphoma, the method comprising administering a bispecific CD20xCD3 antibody to a patient in need, wherein the patient is selected based on an improvement in the level of circulating tumor (ct) DNA. In some embodiments, this disclosure provides a method for identifying lymphoma patients who may respond well to a therapy comprising a bispecific CD20xCD3 antibody.

Description

[0001] References to sequence lists

[0002] This application incorporates a computer-readable sequence list in ST.26 XML format, created on October 31, 2024, containing 26,269 bytes and titled 11667WO01_Sequence. Technical Field

[0003] This invention belongs to the medical field and relates to the use of predictive biomarkers to select patients for treatment or continued treatment with bispecific anti-CD3 x anti-CD20 antibodies for lymphoma. Background Technology

[0004] Molecular characterization of B-cell non-Hodgkin lymphoma, identifying various mutations and subtypes, is widely used in clinical decision-making for patients with lymphoma (de Leval et al., *Hematology*). Blood (140(21):2193-2227, 2022). Although CD20 x CD3 bispecific antibodies, including odronextamab, have been shown to produce profound and durable responses in patients with relapsed or refractory lymphoma, specific biomarkers that can predict the efficacy of such therapies or help identify and select patient subgroups that respond better to such therapies have not yet been determined. Summary of the Invention

[0005] On one hand, this disclosure provides a method for treating lymphoma, the method comprising administering a bispecific antibody to a subject in need, the bispecific antibody comprising a first antigen-binding region binding to human CD20 and a second antigen-binding region binding to human CD3, wherein the subject is selected based on exhibiting a decrease in circulating tumor (ct) DNA levels relative to a reference level of ctDNA after an initial period of treatment with the bispecific antibody.

[0006] On one hand, this disclosure provides a method for selecting a subject to treat lymphoma with a bispecific antibody, the bispecific antibody comprising a first antigen-binding region binding to human CD20 and a second antigen-binding region binding to human CD3, the method comprising: (a) measuring the level of circulating tumor (ct) DNA in the subject after an initial period of treatment with the bispecific antibody; and (b) comparing the measured ctDNA level of the subject with a reference level of ctDNA, wherein if the measured ctDNA level of the subject is lower than the reference level of ctDNA, the bispecific antibody is continued to be administered to the subject during a maintenance period.

[0007] In various embodiments, the reference level of ctDNA is the baseline level of ctDNA measured prior to the initial time period of treatment with the bispecific antibody.

[0008] In various embodiments, the initial treatment period comprises four treatment cycles, each cycle comprising weekly administration of a dose of the bispecific antibody. In some embodiments, each cycle lasts for three weeks. In some cases, the weekly administration of the dose during the first cycle comprises escalating dosing and / or fractionated dosing, wherein two dose portions of the dose are administered over several consecutive days.

[0009] In each embodiment, a decrease in ctDNA levels corresponds to a negative minimum residual disease (MRD) result.

[0010] In various embodiments, the lymphoma is a follicular lymphoma or a diffuse large B-cell lymphoma.

[0011] In various embodiments, treatment of lymphoma includes further administration of the bispecific antibody during a maintenance period. In some cases, the bispecific antibody is administered once every week during the maintenance period.

[0012] On one hand, this disclosure provides a method for treating lymphoma, the method comprising administering a bispecific antibody to a subject in need, the bispecific antibody comprising a first antigen-binding region binding to human CD20 and a second antigen-binding region binding to human CD3, wherein the subject is selected based on the absence of a tumor protein p53 mutation, as measured by circulating tumor (ct) DNA. In some cases, the lymphoma is follicular lymphoma or diffuse large B-cell lymphoma.

[0013] On one hand, this disclosure provides a method for treating diffuse large B-cell lymphoma (DLBCL), the method comprising administering a bispecific antibody to a subject in need, the bispecific antibody comprising a first antigen-binding region binding to human CD20 and a second antigen-binding region binding to human CD3, wherein the subject is selected based on genetic modifications exhibiting an EZB subtype, as measured by circulating tumor (ct) DNA. In some cases, the DLBCL is a germinal center B-cell-like subtype. In some cases, the DLBCL is a non-germinal center B-cell-like subtype.

[0014] In each embodiment, the subject suffers from a relapsed or refractory disease.

[0015] In various embodiments, the first antigen-binding region of the bispecific antibody comprises: three heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 7, 8, and 9; and three light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 13, 14, and 15; and the second antigen-binding region of the bispecific antibody comprises: three heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 10, 11, and 12; and three light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 13, 14, and 15. In some cases, the first antigen-binding region of the bispecific antibody comprises: a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 4; and a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 6, and the second antigen-binding region of the bispecific antibody comprises: an HCVR containing the amino acid sequence of SEQ ID NO: 5; and an LCVR containing the amino acid sequence of SEQ ID NO: 6. In some cases, the bispecific antibody comprises a human IgG heavy chain constant region, optionally isotype IgG1 or IgG4. In some embodiments, the bispecific antibody comprises: a first heavy chain containing amino acid residues 1-452 of SEQ ID NO: 1 paired with a common light chain containing the amino acid sequence of SEQ ID NO: 3; and a second heavy chain containing amino acid residues 1-448 of SEQ ID NO: 2 paired with a common light chain containing the amino acid sequence of SEQ ID NO: 3. In some embodiments, the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 1, which is paired with a common light chain comprising the amino acid sequence of SEQ ID NO: 3; and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 2, which is paired with a common light chain comprising the amino acid sequence of SEQ ID NO: 3. In some cases, the bispecific antibody is onitolumab.

[0016] In various embodiments of the method: (a) the subject has been diagnosed with follicular lymphoma; (b) the subject has been diagnosed with grade 1-3a follicular lymphoma; (c) the subject has been diagnosed with relapsed or refractory follicular lymphoma after receiving at least two prior lines of systemic therapy; (d) the subject has been diagnosed with follicular lymphoma and has not previously received systemic anti-lymphoma therapy; (e) the subject has been diagnosed with follicular lymphoma and is receiving a full dose of 80 mg; and / or (f) the subject has been diagnosed with follicular lymphoma and is receiving a maintenance dose of 160 mg or 320 mg.

[0017] In various embodiments of the method: (a) the subject has been diagnosed with diffuse large B-cell lymphoma (DLBCL); (b) the subject has been diagnosed with DLBCL, wherein the DLBCL is newly diagnosed or transformed from a low-grade tumor; (c) the subject has been diagnosed with DLBCL and is refractory to at least two lines of prior systemic therapy; (d) the subject has been diagnosed with relapsed or refractory DLBCL after receiving at least two lines of prior systemic therapy (including CAR-T therapy); (e) the subject has been diagnosed with DLBCL and has not previously received systemic anti-lymphoma therapy; (f) the subject has been diagnosed with DLBCL and is receiving a full dose of 160 mg; and / or (g) the subject has been diagnosed with DLBCL and is receiving a maintenance dose of 320 mg.

[0018] In various embodiments of the method, prior to treatment with the bispecific antibody, the subject's tumor cell sample has detectable levels of CD20, as measured by mRNA expression and / or immunohistochemistry. In other embodiments of the method, prior to treatment with the bispecific antibody, the subject's tumor cell sample has undetectable levels of CD20, as measured by mRNA expression and / or immunohistochemistry.

[0019] On one hand, this disclosure provides a method for treating lymphoma, the method comprising administering a bispecific CD20xCD3 antibody to a patient in need, wherein the patient is predicted to respond to the therapy, and wherein the patient exhibits a level of circulating tumor DNA (ctDNA) that indicates minimal residual disease.

[0020] On one hand, this disclosure provides a method for treating lymphoma, the method comprising administering a bispecific CD20xCD3 antibody to a patient in need, wherein the patient is predicted to respond to the therapy, and wherein the patient does not exhibit a TP53 mutation.

[0021] This disclosure also considers the use of antiCD20 x antiCD3 antibodies (e.g., onitumumab) in the methods discussed above or herein, and the use of antiCD20 x antiCD3 antibodies (e.g., onitumumab) in the preparation of medicaments for treating subjects (e.g., FL or DLBCL) with B-cell cancer according to the methods discussed above or herein.

[0022] In various embodiments, any feature or component of the embodiments discussed above or herein may be combined, and such combinations are covered within the scope of this disclosure. Any particular value discussed above or herein may be combined with another related value discussed above or herein to enumerate a range of values ​​having an upper and lower end representing a range, and such ranges are covered within the scope of this disclosure. The use of therapeutic proteins in any of the methods discussed herein, or the use of therapeutic proteins in the manufacture of medicaments used in any of the methods discussed herein, is covered within the scope of this disclosure.

[0023] Other embodiments will become apparent upon reading the following detailed description. Attached Figure Description

[0024] Figure 1A and 1B This demonstrates follicular lymphoma (FL) with circulating tumor (ct) DNA detected after 4 cycles of treatment with onitumumab. Figure 1A ) and diffuse large B-cell lymphoma (DLBCL) Figure 1B The survival probability of patients relative to those whose ctDNA was undetectable after 4 cycles of onintuzumab treatment was discussed in Example 1. As shown in the KM curves, in both FL and DLBCL, patients with detectable MRD at day 15 of cycle 4 (C4D15) had a longer median progression-free survival (PFS) compared to patients who were still negative for minimal residual disease (MRD) at that time point. The hazard ratios were 0.26 and 0.34, respectively.

[0025] Figure 2A , 2B 2C and 2D demonstrate FL patients based on a combination of MRD and PET-CT complete response (CR) status. Figure 2A and 2B ) and DLBCL patients ( Figure 2C and 2D The survival probability of FL patients is as discussed in Example 1. MRD status predicts PFS benefit, even in patients who achieve CR at C4D15. Figure 2AThe risk ratio is 0.30. In DLBCL patients who did not achieve CR at C4D15, MRD status can predict PFS ( Figure 2D Its risk ratio is 0.25.

[0026] Figure 3A and 3B This demonstrates how survival probability changes with baseline TP53 mutations in ctDNA, as discussed in Example 1. TP53 is the most common mutation observed at baseline in evaluable patients and indicates reduced PFS in patients with FL and DLBCL.

[0027] Figure 4 This demonstrates the survival probabilities of DLBCL patients classified as MCD subtype relative to EZB subtype relative to other subtypes, as determined by ctDNA, as discussed in Example 1. Lymphgen classification was performed on 84 DLBCL patients. The most common subtypes were MCD and EZB. EZB patients had a longer median PFS compared to MCD patients. "Other" patients included those classified as ST2 or other Lymphgen subtypes. EZB, including... EZH2 Mutations and BCL2 Translocation; MCD, including MYD88L265P and CD79B Mutation; ST2, SGK1 and TET2 Mutant.

[0028] Figure 5 This demonstrates how the survival probability of DLBCL patients changes with baseline cell origin, as discussed in Example 1. Cell origin is a known prognostic factor in DLBCL and was assessable in 85 patients. Figure 5 As shown, the median PFS was improved in the subgroup of patients with germinal center B-like (or GCB) disease compared with patients with non-GCB subtypes.

[0029] Figure 6A and 6B The relationship between PFS and CD20 expression in FL patients is shown, as discussed in Example 2.

[0030] Figure 7A and 7B The relationship between duration of response (DOR) and CD20 expression in FL patients is shown, as discussed in Example 2. Detailed Implementation

[0031] Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to be limiting, as the scope of the invention will be limited only by the appended claims.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. When used to refer to a specific listed numerical value, the term “about” means that the value may differ from the listed value by no more than 1%. For example, the expression “about 100” includes 99 and 101 and all values ​​in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0033] Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, preferred methods and materials are now described. All patents, applications, and non-patent publications referenced in this specification are incorporated herein by reference in their entirety.

[0034] definition

[0035] All references to proteins, peptides, and protein fragments in this article are intended to refer to the human version of the corresponding protein, peptide, or protein fragment, unless explicitly stated to be from a non-human species. Therefore, the expressions “CD3” and “CD20” refer to human CD3 and human CD20, respectively, unless otherwise specified to be from a non-human species.

[0036] The term "CD3" refers to an antigen expressed on T cells as part of a multimolecular T cell receptor (TCR), and the antigen consists of a homodimer or heterodimer formed by the association of two of the following four receptor chains: CD3-ε, CD3-δ, CD3-ζ, and CD3-γ.

[0037] "Antigen-binding domain that binds to CD3", "antigen-binding region that binds to CD3", "antibody that binds to CD3", or "anti-CD3 antibody" are antibodies and their antigen-binding fragments that specifically recognize a single CD3 subunit (e.g., ε, δ, γ, or ζ), and antibodies and their antigen-binding fragments that specifically recognize a dimer complex of two CD3 subunits (e.g., γ / ε, δ / ε, and ζ / ζ CD3 dimers). The antibodies and antigen-binding fragments of the present invention can bind to soluble CD3 and / or CD3 expressed on the cell surface. Soluble CD3 includes native CD3 protein and recombinant CD3 protein variants such as monomeric and dimer CD3 constructs that lack a transmembrane domain or otherwise do not associate with the cell membrane.

[0038] The term "CD20" refers to a non-glycosylated phosphoprotein expressed on the cell membrane of mature B cells. Because CD20 is expressed in more than 95% of B-cell non-Hodgkin lymphomas (NHL) and other B-cell malignancies, but is absent on precursor B cells, dendritic cells, and plasma cells, it is considered a B-cell tumor-associated antigen.

[0039] "Antigen-binding structural region that binds to CD20", "antigen-binding region that binds to CD20", "antibody that binds to CD20" or "anti-CD20 antibody" includes antibodies that specifically recognize CD20 and their antigen-binding fragments.

[0040] Non-Hodgkin lymphoma can be divided into two major prognostic groups: indolent (low-grade; slow-growing) lymphoma and aggressive (high-grade; rapidly growing) lymphoma. Based on the World Health Organization classification, "aggressive lymphoma" is a lymphoma characterized by one of the following subtypes: diffuse large B-cell lymphoma (DLBCL) not otherwise specified by the WHO classification (NOS); germinal center B-cell type; activated B-cell type; primary mediastinal (thymic) large B-cell lymphoma; T-cell / histocyte-rich large B-cell lymphoma; Epstein-Barr virus (EBV) + DLBCL, NOS; high-grade B-cell lymphoma with MYC and BCL2 and / or BCL6 rearrangements; high-grade B-cell lymphoma, NOS; B-cell lymphoma, unclassifiable, with characteristics intermediate between DLBCL and typical Hodgkin lymphoma; and follicular lymphoma, grade 3b. Follicular lymphoma, grades 1-3a, is the most common form of indolent lymphoma.

[0041] "Antibody" means any antigen-binding molecule or molecular complex containing at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., CD20 or CD3). The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains linked together by disulfide bonds, and their polymers (e.g., IgM). The term "antibody" also includes immunoglobulin molecules consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or V). H The heavy-chain constant region contains three structural domains C. H 1. C H 2 and C H 3. Each light chain contains a light chain variable region (abbreviated as LCVR or V in this document). L ) and the light chain constant region. The light chain constant region contains a structural domain (C L 1). V can be... H District and V LThe region is further subdivided into highly variable regions known as complementary determinant regions (CDRs), interspersed with more conservative regions known as frame regions (FRs). Each V H and V L It consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the FRs of the antibody (or its antigen-binding portion) may be identical to the human germline sequence, or may be natural or artificially modified. The common amino acid sequence can be defined based on a side-by-side analysis of two or more CDRs. Unless otherwise stated, the term "antibody" includes "bispecific antibody". The antibodies of this disclosure may include the human IgG heavy chain. In various embodiments, the heavy chain constant region may be an isotype of IgG1, IgG2, IgG3, or IgG4. In some cases, the heavy chain constant region belongs to isotype IgG1. In some cases, the heavy chain constant region belongs to isotype IgG4. In some embodiments, the antibody or bispecific antibody is a human antibody. The term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.

[0042] In some embodiments, the antibodies of the present invention may be recombinant human antibodies. The term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, generated, or isolated by a recombinant manner, such as antibodies expressed using a recombinant expression vector transfected into host cells (further described below), antibodies isolated from a recombinant combined human antibody library (further described below), antibodies isolated from transgenic animals (e.g., mice) containing human immunoglobulin genes (see, for example, Taylor et al. (1992) Nucleic Acids Res. 20:6287-6295), or antibodies prepared, expressed, generated, or isolated by any other means involving splicing a human immunoglobulin gene sequence onto another DNA sequence. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies undergo in vitro mutagenesis (or, when using animals with transgenic human Ig sequences, in vivo somatic cell mutagenesis), and therefore the V of the recombinant antibody... H District and V L The amino acid sequence of the region is as follows: Although it originates from human lineage V H Sequence and V L The sequence is related to it, but it may not be naturally present in human antibody germline libraries.

[0043] Biomarkers that can predict the efficacy of lymphoma treatment

[0044] This disclosure includes identifying biomarkers that can predict the efficacy of lymphoma treatment. In some cases, patients being evaluated have follicular lymphoma (FL) or diffuse large B-cell lymphoma (DLBCL). In embodiments, the biomarker may be the measured level of circulating tumor (ct) DNA, which may indicate minimal residual disease (MRD) negativity, or may be a specific mutation identified within the ctDNA (e.g., a mutation in the tumor protein p53), or may be a gene modification identified within the ctDNA corresponding to a specific lymphoma subtype (e.g., the EZB subtype of DLBCL). Using ctDNA to identify biomarkers predicting treatment efficacy provides a non-invasive method for molecular characterization of lymphoma patients without available tissue and can be used to select patients for treatment with an anti-CD20 x CD3 bispecific antibody or for continued treatment. ctDNA measurements can be performed, as discussed, for example, in the following literature: Newman et al., *Nature Medicine* (…). Nat. Med. )》, 20:548-554, 2014; Newman et al., Nature Biotechnology ( Nat. Biotechnol. )》, 34:547-555, 2016; or Kurtz et al., Journal of Clinical Oncology ( Journal of Clinical Oncology )》, 36(28):2845-2853,2018.

[0045] In some cases, ctDNA is measured at baseline, i.e., before the patient receives treatment with a CD20 x CD3 bispecific antibody (as discussed herein). In other cases, ctDNA is measured after one or more cycles of treatment with a CD20 x CD3 bispecific antibody (e.g., after four 21-day cycles) to measure an early response to the therapy. In embodiments, the measured ctDNA level may be compared to a reference level of ctDNA, which may be a baseline measurement (e.g., if the measured level is after one or more treatment cycles) or a measurement taken at an earlier time within the treatment regimen. In some embodiments, the reference level may be a level representing a population of healthy individuals or a level known to be associated with MRD negativity. As shown in the following examples, FL and DLBCL patients with ctDNA levels associated with MRD negativity after four cycles (three weeks per cycle, administered weekly) treated with an anti-CD20 x CD3 bispecific antibody (e.g., onitumumab) demonstrated significantly longer progression-free survival.

[0046] In various embodiments, the reference level of ctDNA is the baseline level of ctDNA measured prior to the initial treatment period with the bispecific antibody. In some cases, ctDNA is measured after the initial treatment period (which may comprise one or more treatment cycles). The initial treatment period may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) treatment cycles, wherein each cycle lasts for 1-7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks or longer. In some embodiments, the initial treatment period is one cycle (e.g., lasting three or four weeks). In some embodiments, the initial treatment period is two cycles (e.g., lasting three or four weeks). In some embodiments, the initial treatment period is three cycles (ideally, lasting three or four weeks). In some embodiments, the initial treatment period is four cycles (e.g., lasting three or four weeks). In some embodiments, the initial treatment period is two, three, or four cycles (e.g., each cycle lasts three weeks). During each cycle, the bispecific antibody may be administered daily, every other day, every three days, weekly, twice a week, three times a week, four times a week, every other week, every three weeks, etc. In some embodiments, the bispecific antibody is administered weekly.

[0047] In some cases, ctDNA is measured to identify specific mutations or modifications present in tumor DNA, thereby determining whether an individual patient has mutations associated with an increased or decreased probability of treatment efficacy, or whether an individual patient has one or more mutations associated with a specific lymphoma subtype (e.g., by LymphGen classification) that are associated with an increased or decreased probability of treatment efficacy. In some cases, ctDNA is measured at baseline and / or during treatment to determine whether a patient has a mutation in the tumor protein p53. In some cases, ctDNA is measured at baseline and / or during treatment to determine whether a patient has gene modifications associated with the EZB subtype of DLBCL. As shown in the following examples, when treated with anti-CD20 x CD3 bispecific antibodies (e.g., onitumumab), FL and DLBCL patients without tumor protein p53 mutations, DLBCL patients with EZB subtypes, DLBCL patients with germinal center B-cell-like (GCB) cell origins, and FL patients with CD20+ tumors (by mRNA or IHC; e.g., those with ≥10% CD20+ cells, H-score ≥ 50, or median mRNA CD20 TPM (million transcripts) ≥ 1664.8) exhibited significantly longer progression-free survival.

[0048] Therapeutic uses of bispecific antibodies

[0049] This invention includes a method of treating lymphoma in a subject selected based on the predictive biomarkers discussed above. Bispecific antibodies may be included in a composition comprising a pharmaceutically acceptable carrier or diluent. The terms "subject" or "subject in need" refer to a human or non-human animal exhibiting one or more symptoms or signs of cancer (e.g., a subject expressing a tumor or suffering from any of the cancers mentioned below).

[0050] In some embodiments, bispecific anti-CD3 x anti-CD20 antibodies can be used to treat B-cell malignancies, including non-Hodgkin lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, small lymphocytic lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, Waldenström macroglobulinemia, primary mediastinal B-cell lymphoma, lymphoblastic lymphoma, or Burkitt lymphoma. In some embodiments, the cancer is follicular lymphoma. In some embodiments, the cancer is diffuse large B-cell lymphoma (DLBCL). In some embodiments, the cancer is mantle cell lymphoma. In some embodiments, the cancer is marginal zone lymphoma.

[0051] Non-Hodgkin's lymphoma (NHL) is the most common hematologic malignancy. Within a heterogeneous group of NHL, 85-90% are of B-cell origin and include follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and several other B-NHL types. Anti-CD20 antibody in combination with chemotherapy is the standard of care for B-NHL; however, despite an initial response, many patients relapse, and the duration of response typically diminishes with subsequent lines of therapy, resulting in a poor prognosis. Therefore, in some embodiments, the antigen-binding molecule is a bispecific anti-CD3 x anti-CD20 that binds to both CD3+ T cells and CD20+ B cells targeting CD20+ tumor cells via T-cell-mediated cytotoxicity. In some cases, anti-CD3 x CD20 bispecific antibodies are used to treat B-cell cancers (e.g., NHL) in subjects who have failed previous therapy with anti-CD20 monospecific antibodies.

[0052] For patients who do not fully respond to CAR-T therapy, the prognosis is generally poor, and there are no standard care treatment options. Therefore, in some cases, the anti-CD3 x CD20 bispecific antibody of the present invention is used to treat B-cell cancers (e.g., NHL, such as DLBCL) in subjects who have failed previous CAR-T therapy or are unresponsive to previous CAR-T therapy (e.g., anti-CD19 CAR-T therapy).

[0053] In one embodiment, onintuzumab is used to direct treatment in an adult patient with relapsed or refractory follicular lymphoma (FL) who has undergone at least two prior systemic therapies.

[0054] In one embodiment, onituzumab is used to direct treatment in an adult patient diagnosed with follicular lymphoma or DLBCL who has not previously received any systemic anti-lymphoma therapy.

[0055] For relapsed or refractory follicular lymphoma (R / R FL), it can be administered via intravenous (IV) infusion. Treatment consists of the following: escalating dosing in cycle 1, weekly dosing in cycles 2–4, followed by maintenance dosing every 2 weeks until disease progression, as shown in Table 1 below.

[0056] A single treatment cycle (of FL) consists of 21 days.

[0057] Cycle 1: Escalation – Administer onitumumab via 4-hour infusion. The recommended starting dose of onitumumab is 0.2 mg on day 1. If tolerated, administer 0.5 mg on day 2. If tolerated, administer 2 mg on day 8 and 2 mg on day 9. If tolerated, administer 10 mg on day 15 and 10 mg on day 16. If tolerated, proceed to Cycle 2.

[0058] 2-4 cycles: 80 mg weekly - Administer 80 mg via 4-hour infusion on day 1 of cycle 2. If tolerated, the infusion time for all subsequent doses can be reduced to 1 hour. Administer 80 mg doses on days 1, 8, and 15.

[0059] Maintenance: 160 mg every 2 weeks - After cycle 4, administer 160 mg of onitumumab every two weeks via 1-hour infusion. If the patient achieves complete remission for 9 months, administer 160 mg every 4 weeks, or 320 mg every 8 weeks.

[0060] Table 1: Dosage and schedule of onitumumab for treating R / R FL

[0061] a. The dose for day 2 can be administered on day 2, day 3, or day 4.

[0062] b. The dose for day 9 can be administered on day 9, day 10, or day 11.

[0063] c. The dose for day 16 can be administered on day 16, day 17, or day 18.

[0064] In one embodiment, onintuzumab is used to direct treatment in an adult patient with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) who has undergone at least two prior systemic therapies.

[0065] For diffuse large B-cell lymphoma (DLBCL), it can be administered via intravenous (IV) infusion. Treatment consists of the following: escalating dosing in cycle 1, weekly dosing in cycles 2–4, followed by maintenance dosing every 2 weeks until disease progression, as shown in Table 2 below.

[0066] A single treatment cycle for DLBCL consists of 21 days.

[0067] Cycle 1: Escalation – Administer onitumumab via 4-hour infusion. The recommended starting dose of onitumumab is 0.2 mg on day 1. If tolerated, administer 0.5 mg on day 2. If tolerated, administer 2 mg on day 8 and 2 mg on day 9. If tolerated, administer 10 mg on day 15 and 10 mg on day 16. If tolerated, proceed to Cycle 2.

[0068] Cycles 2-4: 160 mg weekly - Administer 160 mg via 4-hour infusion on day 1 of cycle 2. If tolerated, the infusion time for all subsequent doses can be reduced to 1 hour. Administer 160 mg on days 1, 8, and 15.

[0069] Maintenance: 320 mg every 2 weeks - After cycle 4, administer 320 mg of onitumumab every two weeks via 1-hour infusion. If the patient achieves complete remission for 9 months, administer 320 mg every 4 weeks.

[0070] Table 2: Dosage and schedule of onitolimab for treating R / R DLBCL

[0071] a. The dose for day 2 can be administered on day 2, day 3, or day 4.

[0072] b. The dose for day 9 can be administered on day 9, day 10, or day 11.

[0073] c. The dose for day 16 can be administered on day 16, day 17, or day 18.

[0074] Table 3 below shows a summary of the sequences cited herein and their corresponding SEQ ID NOs. Anti-CD3 x anti-CD20 antibodies containing the heavy chain and common light chain of SEQ ID NOs: 1-3, HCVR and LCVR of SEQ ID NOs: 4-6, and CDR of SEQ ID NOs: 7-15 are also referred to herein as onituzumab.

[0075] Table 3: Summary of Sequences

[0076] Example

[0077] The following examples are provided to offer a complete disclosure and description to those skilled in the art regarding how to prepare and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors consider to be their invention. Efforts have been made to ensure the accuracy of the figures used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should still be taken into account. Unless otherwise specified, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

[0078] Example 1: Circulating tumor DNA (ctDNA) analysis predicts progression-free survival (PFS) for patients with relapsed / refractory (R / R) follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) treated with onitumumab monotherapy: identification of minimal residual disease (MRD) status and high-risk subgroups.

[0079] Background: Molecular characterization of B-cell non-Hodgkin lymphoma (B-NHL) via ctDNA MRD assessment or subtype classification can serve as a tool for predicting clinical outcomes. The CD20×CD3 bispecific antibody, onintuzumab, demonstrated profound and durable responses in patients with relapsed / relapsed FL (ORR 80.5%) or DLBCL (ORR 52%) in the phase 2 ELM-2 study (NCT03888105; Kim TM et al. and Kim WS et al. ASH. 2022). In the overall population, the 12-month progression-free survival (PFS) rates were 64% and 29%, respectively. Using tumor biopsies and ctDNA from ELM-2, this example demonstrates that these assessments can predict response to onintuzumab treatment.

[0080] Methods: In ELM-2, patients received IV onintuzumab in 21-day cycles, escalating in cycle (C)1, 80 mg (FL) / 160 mg (DLBCL) QW in C2-4, followed by 160 mg (FL) / 320 mg (DLBCL) Q2W until disease progression or unacceptable toxicity. Baseline (BL) ctDNA and tumor biopsies were used for molecular characterization. BL and treatment-interventional ctDNA were used for MRD assays of the biomarker population (BP; patients required ≥1 available plasma biomarker sample to be included in BP). The first post-BL ctDNA sample was collected on day 15 (D) of C4 during positron emission tomography (PET-CT). An improved AVENIO ctDNA analysis workflow and pipeline (Roche; for research only) was used for next-generation sequencing based on personalized cancer characterization via deep sequencing technology (Kurtz et al., Journal of Clinical Oncology). J Clin Oncol (2018). Whole blood precipitate was used to filter germline allele variants. When the p-value for variant allele frequency was >0.005, an MRD negative was reported.

[0081] Results: Baseline characteristics were similar in the overall population and the BP population. BP included 70 FL patients and 93 DLBCL patients; at BL, 64 FL patients and 83 DLBCL patients had MRD(+). Patients who continued the study until C4D15 had similar PFS, regardless of whether they were in BP or the overall population (FL, n = 128; DLBCL, n = 160). Patients with MRD(-) at C4D15 had significantly longer PFS than those who remained MRD(+) (FL: HR 0.26 [95% CI 0.10–0.66], [ Figure 1A ]; DLBCL: HR 0.34 [95% CI 0.18-0.63], [ Figure 1B ]).like Figures 2A-2D As shown, MRD status predicts the PFS benefit in FL patients who achieve CR at C4D15. Figure 2A Furthermore, MRD status predicts PFS benefit in DLBCL patients who have not achieved CR at C4D15. Figure 2D ].

[0082] Mutation analysis and identification of BL ctDNA TP53 It is the most common mutation in both FL and DLBCL (FL, n = 29 / 64 [45%]; DLBCL, n = 45 / 85 [53%]). For example... Figure 3A and 3B As shown, TP53Mutations indicate reduced progression-free survival (PFS) in patients with FL and DLBCL.

[0083] LymphGen classification of BL ctDNA in DLBCL patients (Wright et al., Cancer Cells) Cancer Cell (2023) mainly identified MCD and EZB subtypes (MCD, n = 20; EZB, n = 22; ST2, n = 5; others, n = 37). For example... Figure 4 As shown, EZB subtype patients treated with onitumumab had significantly longer PFS compared to MCD subtype patients. The cell origin of 85 DLBCL patients was determined using BLctDNA. Figure 5 As shown, germinal center B-cell-like (GCB) cell origin, compared to non-GCB, was associated with improved PFS. Further molecular assessment focused on gene fusions in DLBCL patients with BP.

[0084] Conclusion: The study presented in this example is one of the first prospective analyses of ctDNA in patients with relapsed / relapsed FL and DLBCL in a pivotal trial setting. This non-invasive approach allows for molecular characterization of patients without available tissue, enabling the identification of high-risk subgroups. The CtDNA MRD status at C5D1 in patients treated with onitumumab is a strong predictor of progression-free survival (PFS) in both FL and DLBCL patients and could form the basis for a response-guided treatment paradigm.

[0085] Example 2: Association analysis of CD20 expression and clinical outcomes in patients with follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL)

[0086] Background: CD20 is an effective therapeutic target for B-NHL; however, little is known about the impact of CD20 expression levels on the efficacy of bispecific antibodies (BsAbs). The CD20×CD3 BsAb, onintuzumab, demonstrated profound and durable responses and generally manageable safety in patients (pts) with relapsed / relapsed follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) in the ELM-2 study (Villasboas et al. and Ayyappan et al., ASH 2023). This case report presents a post-hoc analysis of the association between CD20 expression and clinical outcomes in FL and DLBCL patients from the ELM-2 study.

[0087] Methods: ELM-2 recruited adult patients with B-NHL who had experienced remission (R / R) after ≥ 2 lines of prior therapy. IV onintuzumab was administered in 21-day cycles until disease progression / unacceptable toxicity, as discussed in Example 1. The primary endpoint was objective response rate (ORR). CD20 expression in baseline biopsy or archived tissues was assessed by mRNA levels and immunohistochemistry (IHC). Fully automated chromogenic IHC was performed centrally, and H-scores were calculated (McCarty et al., Archives of Pathology and Laboratory Medicine, 1985). CD20 expression was categorized into tertiles, and its relationship with outcomes was determined.

[0088] Results: At baseline, tissues from 127 patients (FL n=62; DLBCL n=65) were available for mRNA analysis, and tissues from 111 patients (FL n=71; DLBCL n=40) were available for IHC. The median sample age at baseline was 25 days. Baseline CD20 expression was higher in FL patients than in DLBCL patients by IHC and mRNA analysis, but responses were even observed in some CD20- patients. Tertile analysis of FL patients showed that higher CD20 H-scores and higher CD20 mRNA expression were associated with longer PFS. Specifically, in the mRNA analysis, the high tertile of CD20 expression was most significantly associated with PFS benefit compared to the low tertile. PFS benefit was observed in FL patients with ≥10% CD20+ (predefined), H-score ≥50 (predefined), or median mRNA CD20 TPM (transcripts per million) ≥1664.8. Figure 6A and 6B Regardless of whether IHC or mRNA analysis was performed, PFS in DLBCL patients was not related to CD20 expression.

[0089] Tertiary analysis of FL patients showed that higher CD20 expression was associated with a longer duration of response (DOR), which was more pronounced in mRNA and IHC analyses. Figure 7A and 7BUnivariate analysis of FL patients revealed a marginal association between baseline LDH levels or time since the last treatment and CD20 expression (via H-score and mRNA TPM). Multivariate analysis of FL patients showed that mRNA CD20 TPM remained significantly associated with PFS after adjusting for refractory status at the last treatment, age at biopsy, LDH levels, time since the last treatment, and POD24 (disease progression within 2 years) at baseline, and CD20 H-score was marginally associated with PFS, but this association decreased after adjusting for LDH levels or time since the last treatment.

[0090] Conclusion: This is one of the first studies to report the association between baseline CD20 expression and clinical response to onatuzumab in R / R FL and DLBCL. The data indicate that patients benefit from onatuzumab regardless of the analytical method used or their baseline CD20 expression. An association between CD20 expression and DOR / PFS was observed in FL.

[0091] This invention is not limited to the specific embodiments described herein. In fact, various modifications to the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0092] sequence

Claims

1. A method for treating lymphoma, the method comprising administering a bispecific antibody to a subject in need, the bispecific antibody comprising a first antigen-binding region binding to human CD20 and a second antigen-binding region binding to human CD3, wherein the subject is selected based on exhibiting a decrease in circulating tumor (ct) DNA levels relative to a reference level of ctDNA after an initial period of treatment with the bispecific antibody.

2. A method for selecting a subject for treatment of lymphoma with a bispecific antibody, the bispecific antibody comprising a first antigen-binding region binding to human CD20 and a second antigen-binding region binding to human CD3, the method comprising: (a) measuring the level of circulating tumor (ct) DNA in the subject after an initial period of treatment with the bispecific antibody; and (b) comparing the measured ctDNA level of the subject with a reference level of ctDNA, wherein if the measured ctDNA level of the subject is lower than the reference level of ctDNA, the bispecific antibody is continued to be administered to the subject during a maintenance period.

3. The method of claim 1 or 2, wherein the reference level of ctDNA is the baseline level of ctDNA measured prior to the initial time period of treatment with the bispecific antibody.

4. The method according to any one of claims 1 to 3, wherein the initial treatment period comprises four treatment cycles, wherein each cycle comprises weekly administration of a certain dose of the bispecific antibody for three weeks.

5. The method of claim 4, wherein the weekly administration of the dose during the first cycle comprises incremental administration and / or fractional administration, wherein two dose portions of the dose are administered over several consecutive days.

6. The method according to any one of claims 1 to 5, wherein a decrease in ctDNA levels corresponds to a negative minimum residual disease (MRD) result.

7. The method according to any one of claims 1 to 6, wherein the lymphoma is a follicular lymphoma or a diffuse large B-cell lymphoma.

8. The method of claim 1, wherein treating lymphoma comprises further administration of the bispecific antibody during a maintenance period.

9. The method of claim 2 or 8, wherein the bispecific antibody is administered once a week during the maintenance period.

10. A method of treating lymphoma, the method comprising administering a bispecific antibody to a subject in need, the bispecific antibody comprising a first antigen-binding region binding to human CD20 and a second antigen-binding region binding to human CD3, wherein the subject is selected based on the absence of a tumor protein p53 mutation as measured by circulating tumor (ct) DNA.

11. The method of claim 10, wherein the lymphoma is a follicular lymphoma or a diffuse large B-cell lymphoma.

12. A method for treating diffuse large B-cell lymphoma (DLBCL), the method comprising administering a bispecific antibody to a subject in need, the bispecific antibody comprising a first antigen-binding region binding to human CD20 and a second antigen-binding region binding to human CD3, wherein the subject is selected based on genetic modifications exhibiting an EZB subtype, as measured by circulating tumor (ct) DNA.

13. The method of claim 12, wherein the DLBCL is a germinal center B-cell-like subtype.

14. The method of claim 12, wherein the DLBCL is a non-germinal center B-cell-like subtype.

15. The method according to any one of claims 1 to 14, wherein the subject suffers from a relapsed or refractory disease.

16. The method according to any one of claims 1 to 15, wherein the first antigen-binding region of the bispecific antibody comprises: three heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, each of the three heavy chain complementarity-determining regions comprising the amino acid sequences of SEQ ID NO: 7, 8, and 9; and three light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, each of the three light chain complementarity-determining regions comprising the amino acid sequences of SEQ ID NO: 13, 14, and 15; and wherein the second antigen-binding region of the bispecific antibody comprises: three heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, each of the three heavy chain complementarity-determining regions comprising the amino acid sequences of SEQ ID NO: 10, 11, and 12; and three light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, each of the three light chain complementarity-determining regions comprising the amino acid sequences of SEQ ID NO: 13, 14, and 15.

17. The method of claim 16, wherein the first antigen-binding region of the bispecific antibody comprises: a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 4; and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 6, and the second antigen-binding region of the bispecific antibody comprises: an HCVR comprising the amino acid sequence of SEQ ID NO: 5; and an LCVR comprising the amino acid sequence of SEQ ID NO:

6.

18. The method of claim 17, wherein the bispecific antibody comprises a constant region of the human IgG heavy chain, optionally isotype IgG1 or IgG4.

19. The method of claim 17, wherein the bispecific antibody comprises: a first heavy chain comprising amino acid residues 1-452 of SEQ ID NO: 1 paired with a common light chain comprising the amino acid sequence of SEQ ID NO: 3; and a second heavy chain comprising amino acid residues 1-448 of SEQ ID NO: 2 paired with a common light chain comprising the amino acid sequence of SEQ ID NO:

3.

20. The method of claim 17, wherein the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 1 paired with a common light chain comprising the amino acid sequence of SEQ ID NO: 3; and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 2 paired with a common light chain comprising the amino acid sequence of SEQ ID NO:

3.

21. The method according to any one of claims 1 to 20, wherein the bispecific antibody is odronextamab.

22. The method according to any one of claims 1 to 21, wherein prior to treatment with the bispecific antibody, the subject's tumor cell sample has a detectable level of CD20, such as as measured by mRNA expression and / or immunohistochemistry.

23. The method according to any one of claims 1 to 21, wherein prior to treatment with the bispecific antibody, the subject's tumor cell sample has undetectable levels of CD20, such as those measured by mRNA expression and / or immunohistochemistry.

24. A method of treating lymphoma, the method comprising administering a bispecific CD20 x CD3 antibody to a patient in need, wherein the patient is predicted to respond to the therapy, and wherein the patient exhibits a level of circulating tumor DNA (ct DNA) that indicates minimal residual disease.

25. A method of treating lymphoma, the method comprising administering a bispecific CD20 x CD3 antibody to a patient in need, wherein the patient is predicted to respond to the therapy, and wherein the patient does not exhibit a TP53 mutation.

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