CN116457013A

CN116457013A - Bispecific antibodies against CD3 and CD20 in combination therapy for the treatment of diffuse large B-cell lymphoma

Info

Publication number: CN116457013A
Application number: CN202180069862.8A
Authority: CN
Inventors: B·埃利奥特; T·艾哈迈德; P·加里多卡斯特罗
Original assignee: Jian Mabao
Current assignee: Jian Mabao
Priority date: 2020-09-10
Filing date: 2021-09-10
Publication date: 2023-07-18

Abstract

Methods of clinical treatment of diffuse large B-cell lymphoma (DLBCL) (e.g., recurrent and/or refractory DLBCL that does not meet autologous stem cell transplant conditions) in a human subject using a bispecific antibody that binds CD3 and CD20 in combination with a standard of care regimen (GemOx) of gemcitabine and oxaliplatin are provided.

Description

Bispecific antibodies against CD3 and CD20 in combination therapy for the treatment of diffuse large B-cell lymphoma

Technical Field

The present invention relates to bispecific antibodies targeting both CD3 and CD20, and the use of such antibodies in combination with standard of care regimens (gemcitabine) and oxaliplatin (GemOx) for the treatment of Diffuse Large B Cell Lymphomas (DLBCL), such as recurrent (recurrent) and/or recurrent (R/R) DLBCL (e.g., R/R DLBCL that do not meet autologous Hematopoietic Stem Cell Transplant (HSCT) conditions due to age, PS, or complications. Advantageous treatment regimens are also provided.

Background

DLBCL is the most common non-hodgkin lymphoma (NHL), and the standard first-line therapy is R-CHOP. For the total population of newly diagnosed DLBCL, the cure rate of this combination is between 60% and 70% (Sehn et al, blood 2007; 109:1867-61). Attempts to improve the outcome of first line treatment, including boosting doses and the addition of other agents to boost the treatment regimen, have failed to provide adequate evidence to alter the standard of care.

Risk factors affecting first-line treatment CR rate, disease recurrence, and OS are included in the international prognostic index (International Prognostic Index, IPI) or revised IPI (R-IPI): age >60 years, ECOG >1 or KPS <60, ldh > uln; extrinsic disease >1 (2 or more), and stage 3 or 4 disease (Project et al, N Engl J Med 1993;329:987-994; sehn et al, supra). Although patients in the good risk group (1-2 IPI factors) had 80% of 4-year PFS after standard first-line R-CHOP, patients in the 45% of the bad risk (high risk) group (3-5 IPI factors) achieved only 55% of 4-year PFS and OS (Sehn et al, supra).

About 35% of DLBCL patients are either primary refractory to standard front line chemoimmunotherapy or relapse after standard front line chemoimmunotherapy. In this group, the only choice for long-term survival is rescue chemotherapy, such as rituximab in combination with DHAX (dexamethasone, cytarabine, and oxaliplatin), followed by high-dose therapy (HDT) with ASCT (Tixier et al, hematol Oncol 2017; 35:584-90). However, only half of the R/R DLBCL patients are eligible to receive HDT-ASCT, and among those eligible for transplantation, some are not sensitive to salvage therapy, interfering with the ASCT procedure. Finally, a substantial proportion of patients relapse after HDT-ASCT treatment, with approximately 45% progressing within 3 years (Gisselbrecht et al, J Clin Oncol 2010; 28:4184-90). Overall, less than 10% of R/RDLBCL patients may be expected to be cured with standard secondary therapy. For patients who relapse or are ill-conditioned after HDT-ASCT, there is a palliative treatment option with the goal of achieving remission and prolonged survival. However, there is no consistent gold standard, and patients will typically be provided with non-potentiation (e.g., R-GemOx, BR) or other palliative intervention (sequential single agent chemotherapy, local radiation therapy for focal symptoms). Recently approved CAR-T cell therapies have shown a sustained response in only a small fraction of patients (Locke et al, lancet Oncol 2019;20:31-42; schuster et al, N Engl J Med 2019; 380:45-56). However, obtaining such highly specific interventions is limited.

Given the limited efficacy and response of subjects to currently available therapies, particularly those subjects who have relapsed or are refractory to currently available therapies, new and effective therapies are needed.

Summary of The Invention

Provided herein are methods of treating human subjects with DLBCL, e.g., refractory and/or recurrent (R/R) DLBCL (e.g., R/R DLBCL that does not meet autologous HSCT conditions), by administering a bispecific antibody that binds to CD3 and CD20, such as escoritamab, in combination with a standard of care regimen (GemOx) of gemcitabine and oxaliplatin, particularly an advantageous clinical treatment regimen.

In one aspect, provided herein are methods of treating DLBCL in a human subject, the methods comprising administering to the subject an combination of icorituximab with gemcitabine and oxaliplatin, e.g., the methods comprising administering to the subject an effective amount of gemcitabine, oxaliplatin, and icorituximab.

In one aspect, provided herein is a method of treating diffuse large B-cell lymphoma (DLBCL), comprising administering to a subject a bispecific antibody and an effective amount of gemcitabine and oxaliplatin, wherein the bispecific antibody comprises:

(i) A first binding arm comprising a first antigen binding region that binds to human CD3 epsilon (epsilon) and comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences in the VH region sequence of SEQ ID No. 6, and the VL region comprises CDR1, CDR2, and CDR3 sequences in the VL region sequence of SEQ ID No. 7; and

(ii) A second binding arm comprising a second antigen binding region that binds to human CD20 and comprises a VH region and a VL region, wherein the VH region comprises CDR1, CDR2 and CDR3 sequences in the VH region sequence of SEQ ID NO:13 and the VL region comprises CDR1, CDR2 and CDR3 sequences in the VL region sequence of SEQ ID NO:14,

wherein the bispecific antibody is administered at a dose of 24mg or 48mg, and wherein the gemcitabine, oxaliplatin, and bispecific antibody are administered at a 28 day period.

In some embodiments, the bispecific antibody is administered at a dose of 24mg (or a dose of about 24 mg). In some embodiments, the bispecific antibody is administered at a dose of 48mg (or a dose of about 48 mg).

In one embodiment, the bispecific antibody is administered once per week (weekly administration) at a dose of 24mg or 48mg, for example for 2.5 28 days periods. In some embodiments, the bispecific antibody is administered once every two weeks (bi-weekly administration) after weekly administration, for example for six 28-day periods. In some embodiments, the bispecific antibody is administered once every four weeks after a bi-weekly administration, e.g., for at least two 28-day periods or until disease progression or unacceptable toxicity. In further embodiments, a priming dose of bispecific antibody (e.g., 0.16mg or about 0.16 mg) is administered two weeks before the first weekly dose of 24mg or 48 mg. In some embodiments, an intermediate dose (e.g., 0.8mg or about 0.8 mg) of bispecific antibody is administered after administration of the priming dose and before administration of a weekly dose of 24mg or 48 mg. In some embodiments, the priming dose is administered one week prior to the intermediate dose, and the intermediate dose is administered one week prior to the first weekly dose of 24mg or 48 mg.

In some embodiments, gemcitabine is administered every two weeks with a 28 day period, e.g., for four 28 day periods. In some embodiments, gemcitabine is at 1000mg/m ² Is administered at a dose of (a).

In some embodiments, oxaliplatin is administered once every two weeks with a 28 day period, e.g., for 4 28 day periods. In some embodiments, oxaliplatin is present at 100mg/m ² Is administered at a dose of (a).

In some embodiments, gemcitabine, oxaliplatin, and bispecific antibody are administered on the same day (e.g., on days 1 and 15 of cycles 1-4), e.g., as shown in table 2.

In some embodiments, the administration is performed with a 28 day period, wherein

(a) Bispecific antibodies were administered as follows:

(i) In cycle 1, a dose of 0.16mg of priming was administered on day 1, an intermediate dose of 0.8mg on day 8, and a dose of 24mg on days 15 and 22;

(ii) In cycles 2 and 3, a dose of 24mg was administered on days 1, 8, 15 and 22;

(iii) In cycles 4-9, a dose of 24mg was administered on days 1 and 15; and

(iv) In cycle 10 and subsequent cycles, a dose of 24mg was administered on day 1;

(b) Gemcitabine is administered on days 1 and 15 of cycles 1-4; and

(c) Oxaliplatin is administered on days 1 and 15 of cycles 1-4.

(a) Bispecific antibodies were administered as follows:

(i) In cycle 1, a dose of 0.16mg of priming was administered on day 1, an intermediate dose of 0.8mg on day 8, and a dose of 48mg on days 15 and 22;

(ii) In cycles 2 and 3, a dose of 48mg was administered on days 1, 8, 15 and 22;

(iii) In cycles 4-9, a dose of 48mg was administered on days 1 and 15; and

(iv) In cycle 10 and subsequent cycles, a dose of 48mg was administered on day 1;

(b) Gemcitabine is administered on days 1 and 15 of cycles 1-4; and

(c) Oxaliplatin is administered on days 1 and 15 of cycles 1-4.

In some embodiments, gemcitabine, oxaliplatin, and the bispecific antibody elcuritumumab are administered on the same day (e.g., on days 1 and 15 of cycles 1-4), e.g., as shown in table 2.

(a) The bispecific antibody ectronimumab was administered as follows:

(iii) In cycles 4-9, a dose of 24mg was administered on days 1 and 15; and

(b) Gemcitabine is administered on days 1 and 15 of cycles 1-4; and

(c) Oxaliplatin is administered on days 1 and 15 of cycles 1-4.

(a) The bispecific antibody ectronimumab was administered as follows:

(iii) In cycles 4-9, a dose of 48mg was administered on days 1 and 15; and

(b) Gemcitabine is administered on days 1 and 15 of cycles 1-4; and

(c) Oxaliplatin is administered on days 1 and 15 of cycles 1-4.

In some embodiments, the bispecific antibody is administered subcutaneously. In some embodiments, the gemcitabine is administered intravenously. In some embodiments, oxaliplatin is administered intravenously.

In some embodiments, the bispecific antibody, gemcitabine, and oxaliplatin are administered sequentially. If administered on the same day, gemcitabine is administered first, oxaliplatin is administered second, and bispecific antibody is administered last. In some embodiments, oxaliplatin is administered first, gemcitabine is administered second, and bispecific antibody is administered last if administered on the same day.

In some embodiments, the DLBCL is double-hit (double-hit) or triple-hit (triple-hit) DLBCL. In some embodiments, DLBCL is grade 3B follicular lymphoma. In some embodiments, the subject has relapsed after at least one prior therapy. In some embodiments, the subject is refractory to at least one prior therapy. In further embodiments, the subject has failed a previous autologous HSCT. In yet further embodiments, the subject does not meet autologous HSCT conditions due to age, performance status, complications, and/or insufficient response to previous treatments.

In some embodiments, the subject is treated with control for Cytokine Release Syndrome (CRS). In some embodiments, controlling comprises administering a corticosteroid (e.g., prednisolone, at a dose of, e.g., 100 mg/day or equivalent thereof, including oral doses), e.g., on the same day as the bispecific antibody. In some embodiments, the corticosteroid is further administered the second, third, and fourth days after administration of the bispecific antibody.

In some embodiments, a precursor drug (pre-treatment) such as an antihistamine (e.g., diphenhydramine), intravenously or orally, e.g., at a dose of 50mg or equivalent thereof, and/or an antipyretic (e.g., at a dose of acetaminophen, e.g., 560-1000 mg) is administered to the subject to reduce the response to the injection. In some embodiments, the prodrug is administered on the same day as the bispecific antibody.

In some embodiments, the control and the prodrug are administered during cycle 1. In some embodiments, control is administered during cycle 2 when the subject experiences CRS greater than grade 1 after the last administration of the bispecific antibody of cycle 1. In some embodiments, control continues in subsequent cycles when the subject experiences CRS greater than grade 1 in the last administration of bispecific antibody in the previous cycle. In a further embodiment, the prodrug is administered during cycle 2. In yet further embodiments, the precursor drug is administered during a subsequent cycle.

In some embodiments, if the subject develops grade 1 CRS, the subject is administered an antibiotic. In some embodiments, if the subject develops grade 2 or grade 3 CRS, the subject is administered a vasopressor. In some embodiments, if the subject develops grade 4 CRS, then at least two vasopressors are administered to the subject.

In some embodiments, the subject is administered tobalizumab (tobalizumab) if the subject develops grade 2, grade 3, or grade 4 CRS. In some embodiments, the subject is further administered a steroid (e.g., dexamethasone or methylprednisolone). In some embodiments, if the subject is refractory to tolizumab, tolizumab is exchanged for an anti-IL-6 antibody (e.g., siltuximab) or an IL-1R antagonist (e.g., anakinra).

In some embodiments, the subject is administered a control for Tumor Lysis Syndrome (TLS). In some embodiments, control against TLS comprises administering one or more uric acid lowering agents prior to administering the bispecific antibody. In some embodiments, a rasburicase (ras) and/or an allopurinol (allopurinol) is administered as a uric acid lowering agent. In some embodiments, supportive therapy, such as labyrinthine, may be used when the subject exhibits signs of TLS.

In some embodiments, a subject treated with the methods described herein achieves a complete response, a partial response, or disease stabilization, e.g., as defined by the Lugano standard or LYRIC.

In some embodiments, the first antigen binding region of the bispecific antibody comprises VHCDR1, VHCDR2 and VHCDR3 comprising the amino acid sequences set forth in SEQ ID NOs 1, 2 and 3, respectively, and VLCDR1, VLCDR2 and VLCDR3 comprising the amino acid sequences set forth in SEQ ID NOs 4, sequence GTN and 5, respectively; and the second antigen binding region comprises VHCDR1, VHCDR2 and VHCDR3 comprising the amino acid sequences set forth in SEQ ID NO. 8, 9 and 10, respectively, and VLCDR1, VLCDR2 and VLCDR3 comprising the amino acid sequences set forth in SEQ ID NO. 11, sequence DAS and SEQ ID NO. 12, respectively.

In some embodiments, the first antigen-binding region of the bispecific antibody comprises a VH region comprising the amino acid sequence of SEQ ID No. 6 and a VL region comprising the amino acid sequence of SEQ ID No. 7; and the second antigen binding region comprises a VH region comprising the amino acid sequence of SEQ ID NO. 13 and a VL region comprising the amino acid sequence of SEQ ID NO. 14.

In some embodiments, the first binding arm of the bispecific antibody is derived from a humanized antibody, preferably from a full length IgG1, lambda (lambda) antibody (e.g., SEQ ID NO: 22). In some embodiments, the second binding arm of the bispecific antibody is derived from a human antibody, preferably from a full length IgG1, kappa (kappa) antibody (e.g., SEQ ID NO: 23). In some embodiments, the bispecific antibody is a full length antibody having a human IgG1 constant region.

In some embodiments, the bispecific antibody comprises an inert Fc region, e.g., an Fc region wherein the amino acids in positions L234, L235 and D265 in the human IgG1 heavy chain constant region corresponding to SEQ ID NO. 15 are F, E and A, respectively. In some embodiments, the bispecific antibody comprises a substitution that promotes bispecific antibody formation, e.g., wherein in the first heavy chain the amino acid in position F405 in the human IgG1 heavy chain constant region corresponding to SEQ ID No. 15 is L, and wherein in the second heavy chain the amino acid in position K409 in the human IgG1 heavy chain constant region corresponding to SEQ ID No. 15 is R, or vice versa. In some embodiments, the bispecific antibody has both an inert Fc region (e.g., substitutions at L234, L235, and D265 (e.g., L234F, L235E and D265A)) and a substitution that promotes bispecific antibody formation (e.g., F405L and K409R). In a further embodiment, the bispecific antibody comprises a heavy chain constant region comprising the amino acid sequences of SEQ ID NOs 19 and 20.

In some embodiments, the bispecific antibody comprises (or consists of) a first heavy chain and a first light chain comprising (or consisting of) the amino acid sequences set forth in SEQ ID NOS: 24 and 25, respectively, and a second heavy chain and a second light chain comprising (or consisting of) the amino acid sequences set forth in SEQ ID NOS: 26 and 27, respectively. In some embodiments, the bispecific antibody is elcatuzumab or a biological analog thereof.

Brief Description of Drawings

FIG. 1 is a schematic illustration of an overall clinical trial design.

Fig. 2 is a schematic illustration of a dose escalation design.

Figures 3A-3D show the activation of T cells induced by elctritumomab (Epco) in the presence of gemcitabine (Gem), oxaliplatin (Ox), or elctritumomab alone or in combination. T cells were incubated with Raji (left panel) or SU-DHL-4 (right panel) cells in the presence of either elcatuzumab alone or elcatuzumab and gemcitabine, oxaliplatin, or a combination. The data shown are the percentage of activated CD8+ T cells (FIG. 3A: CD69; FIG. 3B: CD25; FIG. 3C: PD-1; FIG. 3D: LAMP-1). Data are expressed as mean ± SD of replicates from a representative donor of the 4 donors tested.

Figure 4 shows the T cell mediated cytotoxicity induced by elcatuzumab (Epco) in the presence of gemcitabine (Gem), oxaliplatin (Ox), or elcatuzumab in combination or alone. T cells were incubated with Raji (left panel) or SU-DHL-4 (right panel) cells in the presence of either elcatuzumab alone or elcatuzumab and gemcitabine, oxaliplatin, or a combination. The data shown are percent T cell mediated cytotoxicity. Data are expressed as mean ± SD of replicates from a representative donor of the 4 donors tested.

Detailed Description

As used herein, the term "immunoglobulin" refers to a class of structurally related glycoproteins that consist of two pairs of polypeptide chains, a pair of light (L) low molecular weight chains and a pair of heavy (H) chains, all four being linked to each other by disulfide bonds. The structure of immunoglobulins has been well characterized (see, e.g., fundamental immunoch.7 (Paul, W., eds., 2 nd edition Raven Press, N.Y. (1989)). In short, each heavy chain is typically composed of a heavy chain variable region (abbreviated herein as VH or V) _H ) And a heavy chain constant region (abbreviated herein as CH or C _H ) Composition is prepared. The heavy chain constant region is typically composed of three domains, CH1, CH2 and CH 3. The hinge region is the region between the heavy chain CH1 and CH2 domains and is highly flexible. Disulfide bonds in the hinge region are part of the interaction between the two heavy chains in an IgG molecule. Each light chain is typically composed of a light chain variable region (abbreviated herein as VL or V _L ) And a light chain constant region (abbreviated herein as CL or C _L ) Composition is prepared. The light chain constant region typically consists of one domain CL. VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions, which may be hypervariable in the form of sequence and/or structure defining loops), also known as Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, known as Framework Regions (FR). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J Mol Biol 1987; 196:90117). Unless otherwise indicated or contradicted by context, CDR sequences herein are identified according to the IMGT rules (Brochet X., nucl Acids Res2008;36:W503-508; lefranc MP.; nucl Acids Res 1999;27:209-12; www.imgt. Org /). Unless otherwise indicated or contradicted by context, references to amino acid positions in a constant region are according to EU numbering (Edelman et al, PNAS.1969;63:78-85; kabat et al, sequences of Proteins of Immunological Interest, fifth edition 1991NIH Publication No.91-3242). For example, SEQ ID NO. 15 shows amino acid positions 118-447 of the IgG1 heavy chain constant region according to EU numbering.

As used herein, the term "amino acid corresponding to position. The corresponding amino acid positions in other immunoglobulins can be found by alignment with human IgG 1. Thus, an amino acid or segment in one sequence that "corresponds to" an amino acid or segment in another sequence is one that is aligned with the other amino acid or segment, typically by default, using standard sequence alignment procedures (such as ALIGN, clustalW or similar procedures), and has at least 50%, at least 80%, at least 90% or at least 95% identity to a human IgG1 heavy chain. It is within the ability of one of ordinary skill in the art to align sequences or segments of sequences and thereby determine the position in the sequence corresponding to the amino acid position according to the invention.

As used herein, the term "antibody" (Ab) in the context of the present invention refers to an immunoglobulin molecule that has the ability to specifically bind to an antigen under typical physiological conditions, has a half-life of a significant period of time, such as at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 days or more, etc., or any other relevant functionally defined period of time (such as a time sufficient to induce, promote, enhance, and/or modulate a physiological reaction associated with an antibody binding antigen and/or a time sufficient to cause an antibody to recruit effector activity). The variable regions of the heavy and light chains of immunoglobulin molecules contain binding domains that interact with antigens. Unless otherwise indicated, the term antibody also encompasses polyclonal antibodies, monoclonal antibodies (mabs), antibody-like polypeptides, chimeric antibodies, and humanized antibodies.

As used herein, the term "antibody fragment" or "antigen binding fragment" refers to an immunoglobulin molecule that retains the ability to specifically bind an antigenFragments, and may be generated by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. Examples of antibody fragments include (i) Fab' or Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains, or monovalent antibodies as described in WO2007059782 (Genmab); (ii) F (ab') ₂ A fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bond at the hinge region; (iii) an Fd fragment consisting essentially of VH and CH1 domains; (iv) Fv fragments consisting essentially of the VL and VH domains of the single arm of the antibody; (v) dAb fragments (Ward et al Nature1989; 341:54446) consisting essentially of VH domains and also referred to as domain antibodies (Holt et al; trends Biotechnol 2003; 21:484-90); (vi) Camelid or nanobodies (Revets et al; expert Opin Biol Ther 2005; 5:111-24) and (vii) isolated Complementarity Determining Regions (CDRs). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker, enabling them to be made into a single protein chain, in which the VL and VH regions pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv), see, e.g., bird et al, science1988; 24242326 and Huston et al, PNAS 1988; 85:587983). Such single chain antibodies are encompassed within the term antibody fragment unless otherwise indicated or clearly indicated by context.

As used herein, the term "antibody binding region" or "antigen binding region" refers to a region that interacts with an antigen and comprises both VH and VL regions. As used herein, the term antibody refers not only to monospecific antibodies, but also to multispecific antibodies comprising a plurality, such as two or more, e.g., three or more, different antigen-binding regions. Unless otherwise indicated or clearly contradicted by context, the term antigen-binding region includes antibody fragments as antigen-binding fragments, i.e., retaining the ability to specifically bind an antigen.

As used herein, the term "isotype" refers to the class of immunoglobulins (e.g., igG1, igG2, igG3, igG4, igD, igA, igE, or IgM) encoded by heavy chain constant region genes. When referring to a particular isotype, e.g., igG1, the term is not limited to a particular isotype sequence, e.g., a particular IgG1 sequence, but is used to indicate that the antibody is closer in sequence to that isotype, e.g., igG1, than to other isotypes. Thus, for example, an IgG1 antibody may be a sequence variant of a naturally occurring IgG1 antibody, which may include variations in the constant region.

As used herein, the term "bispecific antibody" or "bs" or "bsAb" refers to an antibody having two different antigen binding regions defined by different antibody sequences. Bispecific antibodies can be in any form.

As used herein, the terms "half molecule", "Fab-arm" and "arm" refer to a heavy chain-light chain pair.

When a bispecific antibody is described as comprising a half-molecular antibody "derived from" a first parent antibody and a half-molecular antibody "derived from" a second parent antibody, the term "derived from" means that the half-molecules from each of the first and second parent antibodies are recombined into the resulting bispecific antibody by any known method to generate the bispecific antibody. In this context, "recombinant" is not intended to be limited to any particular method of recombination, and thus includes all methods for producing bispecific antibodies described herein, including, for example, recombination by half-molecule exchange (also referred to as "controlled Fab-arm exchange"), and recombination at the nucleic acid level and/or by co-expression of two half-molecules in the same cell.

As used herein, the term "full length" in the context of an antibody means that the antibody is not a fragment, but contains all domains of a particular isotype that are commonly found in nature, e.g., VH, CH1, CH2, CH3, hinge, VL, and CL domains of an IgG1 antibody. Full length antibodies may be engineered. An example of a "full length" antibody is elcuritumumab.

As used herein, the term "Fc region" refers to an antibody region consisting of the Fc sequences of two heavy chains of an immunoglobulin, wherein the Fc sequences comprise at least a hinge region, a CH2 domain, and a CH3 domain.

As used herein, the term "heterodimeric interaction between the first and second CH3 regions" refers to an interaction between the first CH3 region and the second CH3 region in the first CH 3/second CH3 heterodimeric protein.

As used herein, the term "homodimeric interaction of a first and a second CH3 region" refers to an interaction between a first CH3 region and another first CH3 region in a first CH 3/first CH3 homodimeric protein and an interaction between a second CH3 region and another second CH3 region in a second CH 3/second CH3 homodimeric protein.

As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities. In a preferred embodiment, the isolated bispecific antibody that specifically binds to CD20 and CD3 is additionally substantially free of monospecific antibodies that specifically bind to CD20 or CD 3.

As used herein, the term "CD3" refers to a human cluster of differentiation 3 protein that is part of a T cell co-receptor protein complex and is composed of four distinct chains. CD3 is also present in other species, and thus the term "CD3" is not limited to human CD3 unless contradicted by context. In mammals, the complex contains a CD3 gamma (gamma) chain (human CD3 gamma chain UniProtKB/Swiss-Prot No P09693, or cynomolgus monkey CD3 gamma UniProtKB/Swiss-Prot No Q95LI 7), a CD3 delta (delta) chain (human CD3 delta UniProtKB/Swiss-Prot No P04234, or cynomolgus monkey CD3 delta UniProtKB/Swiss-Prot No Q95LI 8), two CD3 epsilon (epsilon) chains (human CD3 epsilon UniProtKB/Swiss-Prot No P07766, SEQ ID NO: 28); cynomolgus monkey CD3 εUniProtKB/Swiss-Prot No Q95LI5; or rhesus CD3 εUniProtKB/Swiss-Prot No G7NCB 9) and CD3 zeta chain (zeta) chain (human CD3 ζ UniProtKB/Swiss-Prot No P20963, cynomolgus CD3 ζ UniProtKB/Swiss-Prot No Q09TK 0). These chains associate with molecules called T Cell Receptors (TCRs) and generate activation signals in T lymphocytes. The TCR and CD3 molecules together constitute the TCR complex.

As used herein, the term "CD3 antibody" or "anti-CD 3 antibody" refers to an antibody that specifically binds the antigen CD3, particularly human CD3 epsilon (epsilon).

The term "human CD20" or "CD20" refers to human CD20 (UniProtKB/Swiss-Prot No P11836, SEQ ID NO: 29) and includes any variant, isoform and species homolog of CD20 that is naturally expressed by a cell (including tumor cells) or expressed on cells transfected with the CD20 gene or cDNA. Species homologs include rhesus CD2 (rhesus (macaca mulatta); uniProtKB/Swiss-Prot No H9YXP 1) and cynomolgus CD20 (cynomolgus monkey (macaca fascicularis); uniProtKB No G7PQ 03).

As used herein, the term "CD20 antibody" or "anti-CD 20 antibody" refers to an antibody that specifically binds the antigen CD20, particularly human CD 20.

As used herein, the terms "CD3xCD20 antibody", "anti-CD 3xCD20 antibody", "CD20xCD3 antibody" or "anti-CD 20xCD3 antibody" refer to a bispecific antibody comprising two different antigen binding regions, one of which specifically binds to antigen CD20 and one of which specifically binds to CD 3.

As used herein, the term "DuoBody-CD3xCD20" refers to an IgG1 bispecific CD3xCD20 antibody comprising a first heavy and light chain pair as defined in SEQ ID No. 24 and SEQ ID No. 25, respectively, and a second heavy and light chain pair as defined in SEQ ID No. 26 and SEQ ID No. 27. The first heavy and light chain pair comprises a region that binds human CD3 epsilon (epsilon) and the second heavy and light chain pair comprises a region that binds human CD 20. The first binding region comprises the VH and VL sequences as defined in SEQ ID nos. 6 and 7, and the second binding region comprises the VH and VL sequences as defined in SEQ ID nos. 13 and 14. Such bispecific antibodies can be prepared as described in WO 2016/110576.

Also provided herein are antibodies comprising functional variants of the heavy chain, light chain, VL region, VH region, or one or more CDRs of the example antibodies. Functional variants of the heavy chain, light chain, VL, VH or CDR used in the context of antibodies still allow the antibodies to retain at least a substantial proportion (at least about 90%, 95% or more) of the functional characteristics of the "reference" and/or "parent" antibodies, including affinity and/or specificity/selectivity for a particular epitope of CD20 and/or CD3, fc inertness and PK parameters such as half-life, tmax, cmax. Such functional variants typically retain significant sequence identity and/or heavy and light chains of substantially similar length to the parent antibody. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent homology = # of identical positions/total # x 100 of positions) taking into account the number of gaps that need to be introduced to achieve optimal alignment of the two sequences and the length of each gap. The percent identity between two nucleotide or amino acid sequences can be determined, for example, using the algorithm of E.Meyers and W.Miller, comput.Appl.Biosci, 11-17 (1988) that have been incorporated into the ALIGN program (version 2.0), using the PAM120 weight residual table, gap length penalty of 12 and gap penalty of 4. Furthermore, needleman and Wunsch, J Mol Biol 1970 can be used; the 48:444-453 algorithm determines the percent identity between two amino acid sequences. Exemplary variants include variants that differ primarily by conservative substitutions from the heavy and/or light chain, VH and/or VL and/or CDR regions of the parent antibody sequence; for example, 10, e.g., 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in a variant may be conservative amino acid residue substitutions.

Conservative substitutions may be defined by substitutions within the amino acid categories reflected in the following table:

table 1: conservatively substituted amino acid residue classes

Unless otherwise indicated, mutations were described using the following nomenclature: i) An amino acid substitution at a given position is written, for example, as K409R, which means that the lysine at position 409 is replaced with arginine; ii) for a particular variant, a particular three-letter or one-letter code is used, including codes Xaa and X to indicate any amino acid residue. Thus, substitution of lysine with arginine at position 409 is referred to as: K409R, and substitution of lysine with any amino acid residue at position 409 is referred to as K409X. If the lysine at position 409 is missing, it is denoted by K409.

As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody that contains a human antibody constant domain and a non-human variable domain modified to contain a high degree of sequence homology to a human variable domain. This can be achieved by grafting six non-human antibody CDRs together forming an antigen binding site onto a cognate human acceptor Framework Region (FR) (see WO92/22653 and EP 0629240). In order to fully reestablish the binding affinity and specificity of the parent antibody, it may be necessary to replace the framework residues of the parent antibody (i.e., the non-human antibody) with human framework regions (back mutations). Structural homology modeling may help identify amino acid residues in the framework regions that are important for antibody binding properties. Thus, a humanized antibody may comprise non-human CDR sequences, predominantly human framework regions, optionally comprising one or more amino acid back mutations relative to the non-human amino acid sequence, as well as fully human constant regions. VH and VL of the CD3 arm used herein in DuoBody-CD3xCD20 represent humanized antigen binding regions. Optionally, additional amino acid modifications that are not necessarily back-mutated can be applied to obtain humanized antibodies with preferred characteristics such as affinity and biochemical properties.

As used herein, the term "human antibody" refers to an antibody having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto human framework sequences. VH and VL of the CD20 arm used in DuoBody-CD3xCD20 represent human antigen binding regions. The human monoclonal antibodies of the invention can be produced by a variety of techniques, including conventional monoclonal antibody methods, such as standard somatic hybridization techniques of Kohler and Milstein, nature 256:495 (1975). Although the somatic hybridization protocol is preferred, in principle, other techniques for producing monoclonal antibodies may be employed, such as viral or oncogenic transformation of B lymphocytes or phage display techniques using human antibody gene libraries. A suitable animal system for preparing hybridomas secreting human monoclonal antibodies is the murine system. The production of hybridomas in mice is a very well established protocol. Immunization protocols and techniques for isolating immunized spleen cells for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion protocols are also known. Thus, for example, a transgenic or transchromosomal mouse or rat carrying a part of the human immune system other than a mouse or rat system may be used to generate monoclonal antibodies. Thus, in one embodiment, the human antibody is obtained from a transgenic animal, such as a mouse or rat, that carries human germline immunoglobulin sequences instead of animal immunoglobulin sequences. In such embodiments, the antibodies are derived from human germline immunoglobulin sequences introduced into the animal, but the final antibody sequences are the result of further modification of said human germline immunoglobulin sequences by somatic hypermutation and affinity maturation of endogenous animal antibody mechanisms (see, e.g., mendez et al Nat Genet 1997; 15:146-56). The VH and VL regions of the CD20 arm used in DuoBody-CD3xCD20 represent human antigen binding regions.

As used herein, the term "biosimilar" of (e.g., approved reference product/biopharmaceutical) refers to a biosubstance that is similar to the reference product based on data from: (a) Analytical studies, which show that biological products are highly similar to reference products, despite subtle differences in clinically inactive components; (b) animal studies (including toxicity assessment); and/or (c) one or more clinical studies (including evaluation of immunogenicity and pharmacokinetics or pharmacodynamics) sufficient to demonstrate safety, purity, and efficacy under one or more appropriate conditions for approval and intended use of the reference product and for which approval is sought (e.g., no clinically significant differences in safety, purity, and efficacy of the product between the biologic and the reference product). In some embodiments, the biosimilar biologicals and reference products use the same mechanism or mechanisms of action for one or more conditions of use specified, recommended, or suggested in the suggested tags, but are limited to the range of known mechanism or mechanisms of action for the reference products. In some embodiments, one or more conditions of use specified, recommended, or suggested in the label proposed for the biological product have been previously approved for reference products. In some embodiments, the route of administration, dosage form, and/or specification (strength) of the biologic is the same as the reference product. The biological analogue may be, for example, a presently known antibody having the same primary amino acid sequence as the marketed antibody, but may be prepared in a different cell type or by a different production, purification or formulation method.

As used herein, the term "reducing conditions" or "reducing environment" refers to conditions or environments in which a substrate (here, a cysteine residue in the antibody hinge region) is more likely to be reduced than oxidized.

As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell into which an expression vector, e.g., an expression vector encoding an antibody described herein, has been introduced. Recombinant host cells include, for example, transfectomas such as CHO, CHO-S, HEK, HEK293, HEK-293F, expi293F, per.c6 or NSO cells, and lymphocytes.

As used herein, the term "diffuse large B-cell lymphoma" or "DLBCL" refers to neoplasms of germinal center B-lymphocytes having a diffuse pattern of growth and a high intermediate proliferation index. DLBCL accounts for about 30% of all lymphomas. The subtype of DLBCL appears to have different prospects (prognosis) and responses to treatment. DLBCL can affect any age group but occurs mainly in elderly (average age 60 years). "double-hit" and "triple-hit" DLBCL refer to DLBCL with MYC and BCL2 and/or BCL6 translocations, classified according to WHO 2016, belonging to the class of high-grade B-cell lymphomas (HGBCL) with MYC and BCL2 and/or BCL6 translocations (Swerdlow SH, campo E, harris NL et al, WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues (revised fourth edition). Lyon, france: IARC Press (2017), which is incorporated herein by reference). Grade 3B follicular lymphoma is also often considered equivalent to DLBCL and is thus treated as such.

As used herein, the term "recurrent DLBCL" refers to DLBCL that progresses after achieving a Partial Response (PR) or a Complete Response (CR) to a previous treatment with an anti-neoplastic therapy.

As used herein, the term "refractory DLBCL" refers to DLBCL treated with at least one previous anti-neoplastic therapy but failing to achieve at least a partial response to the therapy.

As used herein, unless otherwise indicated, the term "R/R DLBCL" is intended to refer to recurrent and/or refractory DLBCL.

As used herein, the term "GemOx" refers to a combination of gemcitabine and oxaliplatin.

"gemcitabine" is a nucleoside analog with antitumor activity that belongs to the group of common chemotherapeutic drugs known as antimetabolites. Gemcitabine inhibits DNA synthesis by inhibiting thymidylate synthase, resulting in cell death. Gemcitabine may be referred to, for example, by IUPAC name: 4-amino-1- [3, 3-difluoro-4-hydroxy-5- (hydroxymethyl) oxacyclopent-2-yl]-1, 2-dihydropyrimidin-2-one, or cytidine, 2' -deoxy-2 ',2' -difluoro-, and having the formula C ₉ H ₁₁ F ₂ N ₃ O ₄ And CAS number 95058-81-4. In some embodiments, the gemcitabine is gemcitabine hydrochloride (CAS number 122111-03-9). Gemcitabine is, for example, under trade name And->And the like are commercially available. The term "gemcitabine" is also intended to encompass branded or imitated (imitated equivalents) versions of gemcitabine, as well as pharmaceutically acceptable salts, isomers, racemates, solvates, complexes and hydrates, anhydrous forms thereof, as well as any polymorphic or amorphous forms or combinations thereof.

"oxaliplatin" refers to a platinum-based drug that acts as a DNA cross-linking agent to effectively inhibit DNA replication and transcription, thereby producing cytotoxicity that is a non-specific cell cycle. Oxaliplatin can be referred to as, for example, [ SP-4-2- (1R-trans-)]- (1, 2-cyclohexanediamine-N, N ') [ oxalic acid (2- -) -O, O ]']Platinum; [ (1R, 2R) -cyclohexane-1, 2-diamine](oxalic acid-O, O') platinum (II). Oxaliplatin has the formula C ₈ H ₁₄ N ₂ O ₄ Pt (CAS No. 61825-94-3) and under the trade name, for exampleAnd Oxaliptin->And the like are commercially available. Surgery (operation)The term "oxaliplatin" is also intended to encompass branded and imitated versions (imitated equivalents) of oxaliplatin, as well as pharmaceutically acceptable salts, isomers, racemates, solvates, complexes and hydrates, anhydrous forms of oxaliplatin, as well as any polymorphic or amorphous forms thereof or combinations thereof.

As used herein, the term "autologous stem cell transplant" or "ASCT" refers to stem cells that are collected from and fed back to an individual.

The term "treating" refers to administering an effective amount of a therapeutically active antibody described herein for the purpose of alleviating, ameliorating, preventing, or eradicating (curing) a symptom or disease state, such as DLBCL. Treatment may result in Complete Response (CR), partial Response (PR), or disease Stabilization (SD), e.g., as defined by the Lugano standard and/or LYRIC. Treatment may continue, for example, until disease progression or unacceptable toxicity.

As used herein, the term "administering" refers to physically introducing a composition (or formulation) comprising a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those of skill in the art. Preferred routes of administration of the antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional (intralesional), intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. Alternatively, the therapeutic agents described herein may be administered via a non-parenteral route, such as a topical, epidermal, or mucosal route of administration, e.g., intranasal, oral, vaginal, rectal, sublingual, or topical administration. Administration may also be performed, for example, once, multiple times, and/or over one or more extended periods of time. In the methods described herein, a bispecific antibody (e.g., elcuritumumab) is administered subcutaneously. Other agents used in combination with bispecific antibodies, such as GemOx, cytokine release syndrome control, and/or Tumor Lysis Syndrome (TLS) control, may be administered via other routes, e.g., intravenously or orally.

The term "effective amount" or "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic result over the necessary dosage and period of time. For example, a dose for subcutaneous administration of a bispecific antibody (e.g., elcuritumumab), as defined herein, i.e., 24mg or 48mg, may be defined as such "effective amount" or "therapeutically effective amount". The therapeutically effective amount of the antibody may vary depending on factors such as the disease state, age, sex and weight of the individual, the ability of the antibody to elicit a desired response in the individual, and the like. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the antibody or antibody portion are offset by a therapeutically beneficial effect. In some embodiments, a patient treated with the methods described herein will exhibit an improvement in ECOG performance status. A therapeutically effective amount or dose of a drug includes a "therapeutically effective amount" or a "therapeutically effective dose" that is any amount of a drug that inhibits the progression or recurrence of a disease when the drug is administered alone or in combination with another therapeutic agent to a subject at risk of developing the disease or disorder (e.g., cytokine release syndrome) or at risk of having a recurrence of the disease.

As used herein, the term "inhibiting tumor growth" includes any measurable reduction in tumor growth, such as inhibiting tumor growth by at least about 10%, such as at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 99%, or 100%.

As used herein, the term "subject" refers to a human patient, e.g., a human patient with DLBCL. The terms "subject" and "patient" are used interchangeably herein.

As used herein, the term "buffer" refers to a pharmaceutically acceptable buffer. The term "buffer" encompasses those reagents that maintain the pH of the solution within, for example, an acceptable range, and includes, but is not limited to, acetate, histidine,(tris (hydroxymethyl) aminomethane), citrate, succinate, glycolate, and the like. Typically, as used herein, a "buffer" has a pKa and a buffering capacity suitable for a pH range of about 5 to about 6, preferably about 5.5.

As used herein, "disease progression" or "PD" refers to a situation in which one or more indices of DLBCL show that the disease is still progressing despite treatment. In one embodiment, disease progression is defined based on the Lugano response standard for malignant lymphoma ("Lugano standard") and/or the response of lymphoma to immunomodulatory therapy standard (LYRIC). Cheson et al J Clin Oncol 2014;32:3059-68 provides detailed information about the Lugano standard/classification system, including definitions of Complete Response (CR), partial Response (PR), no response/disease stabilization (NR/SD), and disease Progression (PD), the contents of which are incorporated herein by reference (see in particular Table 3 of Cheson et al, 2014). Detailed information about LYRIC is provided in Table 8.

As used herein, a "surfactant" is a compound commonly used in pharmaceutical formulations to prevent adsorption of a drug to a surface and/or aggregation. In addition, surfactants reduce the surface tension (or interfacial tension) between two liquids or between a liquid and a solid. For example, the exemplary surfactant may significantly reduce surface tension when present at very low concentrations (e.g., 5% w/v or less, such as 3% w/v or less, such as 1% w/v or less, such as 0.4% w/v or less, such as less than 0.1% w/v or less, such as 0.04% w/v). Surfactants are amphiphilic, meaning that they are generally composed of hydrophilic and hydrophobic or lipophilic groups, and are therefore capable of forming micelles or similar self-assembled structures in aqueous solutions. Known pharmaceutical surfactants include glycerol monooleate, benzethonium chloride, docusate sodium, phospholipids, polyvinyl alkyl ether (polyethylene alkyl ethers), sodium lauryl sulfate and trioctyl (anionic surfactant); benzalkonium chloride, citric acid imide, cetylpyridinium chloride (cetylpyridinium chloride), and phospholipids (cationic surfactant); and alpha-tocopherol, glyceryl monooleate, myristyl alcohol, phospholipids, poloxamers, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, polyethylene glycol hydroxystearates (polyoxyl hydroxystearate), polyethylene glycol glycerides (polyoxylglycerides), polysorbates such as polysorbate 20 or polysorbate 80, propylene glycol dilaurate, propylene glycol monolaurate, sorbitan sucrose palmitate, sucrose stearate, trioctyl and TPGS (nonionic and zwitterionic surfactants).

As used herein, a "diluent" is a diluent that is pharmaceutically acceptable (safe and non-toxic for human administration) and can be used to prepare a pharmaceutical composition or pharmaceutical formulation (the terms "composition" and "formulation" are used interchangeably herein). Preferably, such dilutions of the composition only dilute the antibody concentration and not the buffer and stabilizer. Thus, in one embodiment, the diluent contains the same concentration of buffer and stabilizer as is present in the pharmaceutical composition of the invention. Further exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (preferably acetate buffer), sterile saline solutions such as water for injection, ringer's solution or dextrose solution. In one embodiment, the diluent comprises or consists essentially of acetate buffer and sorbitol.

As used herein, the term "about" refers to a value that is no more than 10% above the specified value and no more than 10% below the specified value.

DLBCL treatment regimen

Provided herein are methods of treating DLBCL in a human subject using bispecific antibodies that bind to CD3 and CD20 ("anti-CD 3xCD20 antibodies"), e.g., isolated anti-CD 3xCD20 antibodies that bind to human CD3 and human CD20, such as ectropin, in combination with standard of care regimens of gemcitabine and oxaliplatin (also referred to herein as "GemOx"). The methods are useful for treating, for example, recurrent and/or refractory (R/R) DLBCL (e.g., R/R DLBCL that does not meet autologous HSCT conditions). It is to be understood that methods of treating DLBCL (e.g., R/R DLBCL, such as R/R DLBCL that does not meet autologous HSCT conditions) with the bispecific antibodies described herein that bind to both CD3 and CD20 also encompass the corresponding use of the bispecific antibodies for treating DLBCL (e.g., R/R DLBCL, such as R/R DLBCL that does not meet autologous HSCT conditions) in a human subject.

Thus, in one aspect, provided herein is a method of treating DLBCL in a human subject, the method comprising administering a bispecific antibody and an effective amount of gemcitabine and oxaliplatin, wherein the bispecific antibody comprises:

(i) A first binding arm comprising a first antigen binding region that binds human CD3 epsilon (epsilon) and comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences in the VH region sequence of SEQ ID No. 6, and the VL region comprises CDR1, CDR2, and CDR3 sequences in the VL region sequence of SEQ ID No. 7; and

(ii) A second binding arm comprising a second antigen binding region that binds human CD20 and comprises a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences in the VH region sequence of SEQ ID NO:13, and the VL region comprises CDR1, CDR2, and CDR3 sequences in the VL region sequence of SEQ ID NO: 14;

In some embodiments, the bispecific antibody is a full length antibody. In other embodiments, the bispecific antibody is an antibody having an inert Fc region. In yet other embodiments, the bispecific antibody is a full length antibody having an inert Fc region.

With respect to a 24mg or 48mg dose (or about 24mg or 48mg dose) of bispecific antibody to be administered, or any other prescribed dose, it is understood that this amount refers to the amount of bispecific antibody that represents a full length antibody, such as the elcuritumumab defined in the examples section. Thus, the dose at which 24mg of the bispecific antibody is administered may be referred to as the dose at which the bispecific antibody described herein is administered, wherein the dose corresponds to the dose of 24mg of the elcatuzumab. When, for example, the molecular weight of the antibody used is significantly different from the molecular weight of a full length antibody such as elcuritumumab, one of ordinary skill in the art can readily determine the amount of antibody to be administered. For example, the amount of antibody can be calculated by dividing the molecular weight of the antibody by the weight of a full length antibody, such as elcatuzumab, and multiplying the result by a particular dose, as described herein. So long as the bispecific antibody (e.g., a functional variant of DuoBody CD3xCD 20) has highly similar characteristics to DuoBody CD3xCD20 in terms of plasma half-life, fc inertness, and/or binding characteristics to CD3 and CD20 (i.e., in terms of CDR and epitope binding characteristics), such antibodies are suitable for use in the methods provided herein at dosages described for full-length antibodies such as ectrituximab.

In some embodiments, the dose of bispecific antibody is administered once per week (weekly administration) at a 28 day period. In one embodiment, a weekly dose of 24mg or 48mg is administered for 2.5 28 day periods (i.e., 10 times; days 15 and 22 on cycle 1, and days 1, 8, 15 and 22 on cycles 2-3). In other embodiments, the interval between administrations may be reduced to once every two weeks (bi-weekly administration) after weekly administration. In one embodiment, the two week administration is performed for six 28 day periods (i.e., 12 times). In some embodiments, after a two week administration, the interval of administration may be reduced to once every four weeks. In one embodiment, administration once every four weeks may be for a longer period of time, for example at least 1 cycle, at least 2 cycles, at least 3 cycles, at least 4 cycles, at least 5 cycles, at least 6 cycles, at least 7 cycles, at least 8 cycles, at least 9 cycles, at least 10 cycles, at least 11 cycles, at least 12 cycles, at least 13 cycles, at least 14 cycles, at least 15 cycles, at least 16 cycles, at least 17 cycles, such as between 1-20 cycles, 1-19 cycles, 1-18 cycles, 1-17 cycles, 1-16 cycles, 1-15 cycles, 1-14 cycles, 1-13 cycles, 1-12 cycles, 1-10 cycles, 1-5 cycles, 5-20 cycles, 5-15 cycles, or 5-10 cycles. In some embodiments, the elcurizumab is administered as monotherapy (i.e., without GemOx) from cycle 10 of the 28 day cycle. In some embodiments, the elcatuzumab is administered as monotherapy from cycle 10 to cycle 26 of the 28-day cycle. In some embodiments, the elcatuzumab is administered as monotherapy from cycle 7 of the 28-day cycle until disease progression (e.g., as defined by the Lugano standard or LYRIC) or unacceptable toxicity.

In one embodiment, the weekly dose of bispecific antibody is administered at a 28 day cycle at cycles 1-3 (which may include priming and intermediate doses, as described below), the bi-weekly dose of bispecific antibody is administered at cycles 4-9, and once every four weeks the dose is administered starting at cycle 10, e.g., at cycles 10-15, 10-20, 10-25, 10-30 or more, e.g., until disease progression or unacceptable toxicity is observed in the subject. In some embodiments, a dose of once every four weeks is administered at cycles 10-26.

It will be appreciated that the dosages referred to herein may also be referred to as full or fixed dosages (flat dose) in the above cases, wherein, for example, weekly, bi-weekly and/or weekly dosages are administered at the same level. Thus, when a selected 48mg dose is preferably administered every week, every two weeks, and every four weeks, the same 48mg dose should be administered. Before the dose is administered, a priming or priming and subsequent intermediate (secondary) dose may be administered. This may be advantageous because it may help to reduce the risk and severity of Cytokine Release Syndrome (CRS), a side effect that may occur during treatment with the bispecific anti-CD 3xCD20 antibodies described herein. Such priming or priming and intermediate doses are at lower doses than fixed or full doses.

Thus, in some embodiments, a priming dose of bispecific antibody may be administered prior to the administration of a weekly dose of 24mg or 48 mg. In one embodiment, the priming dose is administered two weeks before the first weekly dose of 24mg or 48mg administered in cycle 1. In one embodiment, the amount of priming is 0.16mg (or about 0.16 mg) of full length bispecific antibody.

In some embodiments, an intermediate dose of the bispecific antibody is administered after administration of the priming dose and before administration of a weekly dose of 24mg or 48 mg. In one embodiment, the priming dose is administered one week prior to the intermediate dose (i.e., on day 1 of cycle 1) and the intermediate dose is administered one week prior to the first weekly dose of 24mg or 48mg (i.e., on day 8 of cycle 1). In one embodiment, the intermediate dose is 800 μg (0.8 mg) or about 800 μg (0.8 mg) of full length bispecific antibody.

The methods described herein relate to treating a human subject with DLBCL with a bispecific antibody that binds to CD3 and CD20 in combination with a standard of care regimen (GemOx) of gemcitabine and oxaliplatin.

In some embodiments, gemcitabine and oxaliplatin are administered at standard of care doses for GemOx, e.g., as supported by clinical studies, according to local guidelines, and/or related local labels.

In some embodiments, gemcitabine is administered according to a product label or product property profile (see, e.g., for injectionPrescription information available at www.accessdata.fda.gov/drugsatfda_docs/label/2014/020509s077pdf; />Prescription information, available at www.accessdata.fda.gov/drugsatfda_docs/label/2018/208313 original 1s000lbl. In some embodiments, the gemcitabine is gemcitabine hydrochloride. />

In some embodiments, oxaliplatin is administered according to an associated local product label or product property profile (see, e.g.Prescription information, available at www.accessdata.fda.gov/drugsatfda_docs/label/2020/021759s023lbl.

In one embodiment, gemcitabine is administered according to local guidelines and local markers. In some embodiments, gemcitabine is at 1000mg/m ² Is administered (or about 1000 mg/m) ² Is administered at a dose). In some embodiments, the gemcitabine is administered intravenously.

In one embodiment, gemcitabine is administered every two weeks (Q2W) with a 28 day period. In some embodiments, gemcitabine is administered once every two weeks for four 28-day cycles (i.e., 8 times).

In one embodiment, oxaliplatin is administered according to local guidelines and local markers. In some embodiments, oxaliplatin is present at 100mg/m ² Is administered (or about 100 mg/m) ² Is administered at a dose). In some embodiments, oxaliplatin is administered intravenously.

In one embodiment, oxaliplatin is administered once every two weeks (Q2W) with a 28 day period. In some embodiments, oxaliplatin is administered once every two weeks for four 28-day cycles (i.e., 8 times).

The dose of oxaliplatin can be reduced when the subject develops neuropathy (worsening from baseline). In some embodiments, oxaliplatin is stopped if the subject develops an abnormal outcome of the neurological examination, or the subject experiences a significant paresthesia that lasts 14 days or more. Once symptoms improve, oxaliplatin can be present at 75mg/m ² Is restarted.

In certain embodiments, gemcitabine, oxaliplatin, and a bispecific antibody are administered simultaneously.

In some embodiments, the gemcitabine, oxaliplatin, and bispecific antibody are administered sequentially (e.g., on the same day). For example, in one embodiment, when gemcitabine, oxaliplatin, and a bispecific antibody are administered on the same day, the gemcitabine is administered first, the oxaliplatin is administered second, and the bispecific antibody is administered last. In some embodiments, when gemcitabine, oxaliplatin, and a bispecific antibody are administered on the same day, oxaliplatin is administered first, gemcitabine is administered second, and the bispecific antibody is administered last.

In some embodiments, the precursor drugs and/or controls for CRS are administered to the subject prior to the administration of gemcitabine, oxaliplatin, and bispecific antibodies.

In some embodiments, gemcitabine (e.g., intravenous), oxaliplatin (e.g., intravenous), and a bispecific antibody (e.g., subcutaneous) are administered in a 28 day cycle, wherein:

(a) The bispecific antibody is administered as follows:

(iii) In cycles 4-9, a dose of 24mg was administered on days 1 and 15; and

(iv) In cycle 10 and subsequent cycles (e.g., cycles 10-15, 10-20, 10-25, 10-30 or more), a dose of 24mg is administered on day 1;

(b) Gemcitabine is administered on days 1 and 15 of cycles 1-4; and

(c) Oxaliplatin is administered on days 1 and 15 of cycles 1-4.

(a) The bispecific antibody is administered as follows:

(iii) In cycles 4-9, a dose of 48mg was administered on days 1 and 15; and

(iv) In cycle 10 and subsequent cycles (e.g., cycles 10-15, 10-20, 10-25 or more), a dose of 48mg is administered on day 1;

(b) Gemcitabine is administered on days 1 and 15 of cycles 1-4; and

(c) Oxaliplatin is administered on days 1 and 15 of cycles 1-4.

In some embodiments, gemcitabine (e.g., intravenous), oxaliplatin (e.g., intravenous), and the bispecific antibody ectrituximab (e.g., subcutaneous) are administered in a 28 day cycle, wherein:

(a) The bispecific antibody ectronimumab was administered as follows:

(iii) In cycles 4-9, a dose of 24mg was administered on days 1 and 15; and

(b) Gemcitabine is administered on days 1 and 15 of cycles 1-4; and

(c) Oxaliplatin is administered on days 1 and 15 of cycles 1-4.

(a) The bispecific antibody ectronimumab was administered as follows:

(iii) In cycles 4-9, a dose of 48mg was administered on days 1 and 15; and

(b) Gemcitabine is administered on days 1 and 15 of cycles 1-4; and

(c) Oxaliplatin is administered on days 1 and 15 of cycles 1-4.

(a) The bispecific antibody is administered as follows:

(iii) In cycles 4-9, a dose of 24mg was administered on days 1 and 15; and

(b) Gemcitabine is administered at a dose of 1000mg/m2 on days 1 and 15 of cycles 1-4; and

(c) Oxaliplatin is administered at a dose of 100mg/m2 on days 1 and 15 of cycles 1-4.

(a) The bispecific antibody is administered as follows:

(iii) In cycles 4-9, a dose of 48mg was administered on days 1 and 15; and

(a) The bispecific antibody ectronimumab was administered as follows:

(iii) In cycles 4-9, a dose of 24mg was administered on days 1 and 15; and

(a) The bispecific antibody ectronimumab was administered as follows:

(iii) In cycles 4-9, a dose of 48mg was administered on days 1 and 15; and

In one embodiment, subjects undergoing treatment with the methods described herein suffer from recorded DLBCL (new or histologically transformed by indolent lymphoma, except CLL) according to WHO classification of 2016 (Swerdlow SH, campo E, harris NL et al, WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues (revised 4 th edition). Lyon, france: IARC Press (2017), the contents of which are incorporated herein by reference). In some embodiments, the subject has DLBCL, NOS (not otherwise specified). In some embodiments, the subject has a "double hit" or "triple hit" DLBCL, which is classified as HGBCL in WHO 2016, with MYC and BCL2 and/or BCL6 translocation. In some embodiments, the subject has grade 3B follicular lymphoma. In further embodiments, the subject has relapsed after or is refractory to at least one prior therapy. In yet further embodiments, the subject has failed a previous autologous HSCT. In yet further embodiments, the subject fails to meet autologous HSCT conditions, e.g., due to age, performance status (performance status), complications, and/or insufficient response to previous treatments.

In some embodiments, the subject has an eastern tumor cooperative group (Eastern Cooperative Oncology Group, ECOG) performance status (ECOG PS) of 0, 1, or 2. Information about ECOG PS scores can be found, for example, in OKEN et al, am J Clin Oncol 1982Dec;5 (6) found in 649-55.

In some embodiments, the subject has a measurable disease, as defined as (a) 1 or more measurable nodular lesions (major axis >1.5cm and minor axis >1.0 cm) or 1 or more measurable extranodal lesions (major axis >1 cm) on CT or MRI.

In some embodiments, the subject has acceptable organ function, as defined as: (a) ANC is more than or equal to 1.0X10 ⁹ /L, (b) platelet count>75x 10 ⁹ /L, or 50x 10 or more if bone marrow infiltrates or splenomegaly ⁹ L, (c) ULN having ALT level of 2.5 times or less, (d) total bilirubin level of 2 XULN, and (e) eGFR>50mL/min (by the Cockcroft-Gault formula), and (f) PT, INR and aPTT.ltoreq.1.5 xULN (unless anticoagulants are received).

In some embodiments, the subject is not severely allergic or allergic to the anti-CD 20 antibody therapy, gemcitabine, oxaliplatin, or a bispecific antibody, or is not known to be allergic or intolerant to any component or excipient of gemcitabine, oxaliplatin, and/or bispecific antibodies.

In some embodiments, the subject has no clinically significant heart disease, including (a) myocardial infarction within one year prior to the first dose of bispecific antibody, or an unstable or uncontrolled disease/disorder that is associated with or affects cardiac function (e.g., unstable angina, congestive heart failure, NYHA class III-IV), arrhythmia (CTCAE, version 2 or higher), or clinically significant ECG abnormalities, and/or (b) a 12-lead ECG exhibiting a baseline QTcF >470 msec.

In some embodiments, the subject does not have a contraindication to oxaliplatin or gemcitabine.

The human subject receiving the treatment described herein may be a patient with one or more inclusion criteria as set forth in example 1, or without one or more exclusion criteria as set forth in example 1.

The methods described herein are advantageous for treating DLBCL, such as R/R DLBCL (e.g., R/R DLBCL that does not meet autologous HSCT conditions). Treatment is maintained using, for example, the treatment regimens described herein, continually until progressive disease is developed or unacceptable toxicity occurs.

The response of a subject with DLBCL to treatment using the methods described herein can be assessed according to the Lugano response criteria for malignant lymphoma (also referred to herein as "Lugano criteria") and/or the response of lymphoma to immunomodulatory treatment criteria (also referred to herein as "LYRIC"), as described in example 1. In one embodiment, complete Response (CR), partial Response (PR), and disease Stabilization (SD) are assessed using the Lugano standard. In some embodiments, patients exhibiting disease progression, also known as Progressive Disease (PD), according to the Lugano standard are further evaluated according to LYRIC. Cheson et al J Clin Oncol2014;32:3059-68 provides detailed information about the Lugano standard/classification system, including definitions of complete response, partial response, no response/disease stabilization, and disease progression (see in particular Cheson et al, table 3 of 2014). Detailed information about LYRIC is provided in Table 8

In some embodiments, the subject is treated with the methods described herein until they exhibit disease Progression (PD), e.g., as defined by the Lugano standard and/or LYRIC. In one embodiment, subjects are treated with the methods described herein until they exhibit disease Progression (PD) as defined by both the Lugano standard and LYRIC.

The subject treated according to the methods described herein preferably experiences an improvement in at least one sign of DLBCL. In one embodiment, the improvement is measured by a decrease in the number and/or size of measurable neoplastic lesions. In some embodiments, lesions may be measured on CT, PET-CT or MRI slices. In some embodiments, cytology or histology may be used to assess responsiveness to therapy. In some embodiments, bone marrow aspirate and bone marrow biopsy may be used to evaluate response to therapy.

In one embodiment, the treated subject exhibits a Complete Response (CR), partial Response (PR), or disease Stabilization (SD), as defined by the Lugano standard or LYRIC (see, e.g., table 8). In some embodiments, the methods described herein produce at least one therapeutic effect selected from prolonged survival (such as progression free survival or overall survival), optionally compared to another therapy or placebo.

In some embodiments, T cell activity (e.g., cd4+ and/or cd8+ T cell activity) is increased in a subject treated with a combination of bispecific antibody, gemcitabine, and oxaliplatin. In some embodiments, CD69, CD25, PD-1 and/or LAMP-1 expression is increased in cd4+ and/or cd8+ T cells from a subject treated with a combination of bispecific antibody, gemcitabine and oxaliplatin.

In some embodiments, the anti-tumor activity (e.g., B cell cytotoxicity) is increased in a subject treated with a combination of a bispecific antibody, gemcitabine, and oxaliplatin, e.g., relative to a subject treated with a bispecific antibody alone or a combination of a bispecific antibody and gemcitabine or oxaliplatin.

Cytokine Release Syndrome (CRS) can occur when a method is used in a human subject that utilizes an immune cell and bispecific antibody based approach that works by activating immune effector cells, such as by engaging CD3 (Lee et al Biol Blood Marrow Transplant2019;25:625-38, which is incorporated herein by reference). Thus, in some embodiments, CRS mitigation is performed with the methods described herein. As part of CRS mitigation, the selection of the priming and/or intermediate doses is performed prior to administration of the full dose (e.g., 24 or 48 mg), as described herein. CRS can be categorized according to standard practice (e.g., as outlined in Lee et al, biol Blood Marrow Transplant, 2019;25:625-38, which is incorporated herein by reference). CRS may include cytokines, such as pro-inflammatory cytokines, e.g., excessive release of IL-6, TNF- α, or IL-8, which may cause adverse effects such as fever, nausea, vomiting, and chills. Thus, although bispecific antibodies such as elcatuzumab have unique anti-tumor activity, their immunological mode of action may trigger unwanted "side effects," i.e., induce unwanted inflammatory responses. Thus, the patient may further undergo concomitant treatment, control and/or prodrug administration with, for example, analgesics, antipyretics and/or anti-inflammatory agents to alleviate possible CRS symptoms.

Thus, in one embodiment, a human subject in the methods described herein is treated with CRS control. In some embodiments, the control includes administration of a corticosteroid. In one embodiment, the control is administered on the same day as the bispecific antibody. Control may also be applied on subsequent days, more preferably on subsequent days 2, 3 and 4. It will be appreciated that days 2, 3 and 4 are relative to the administration of bispecific antibody administered on day 1 when further administration (such as control) is involved. For example, when antibodies are applied on day 15 of the cycle and control is also applied, control corresponding to days 2, 3, and 4 is days 16, 17, and 18 of the cycle. In some embodiments, control is administered on the day of bispecific antibody administration and on days 2-4 thereafter. When the control is administered on the same day as the bispecific antibody, control is preferably administered 30-120 minutes prior to the bispecific antibody administration. An exemplary corticosteroid suitable for use in the methods and uses described herein is prednisolone. In some embodiments, prednisolone is administered at an intravenous dose of 100mg or an equivalent thereof (including an oral dose). Exemplary corticosteroid equivalents of prednisolone, as well as dose equivalents, that may be used for CRS control are shown in table 5.

Furthermore, in some embodiments, the human subject in the methods described herein is treated with a prodrug to reduce the response to injection. In one embodiment, the precursor drug comprises administration of an antihistamine. In some embodiments, the precursor drug comprises administration of an antipyretic. In a further embodiment, the prodrug comprises systemic administration of an antihistamine and an antipyretic.

An exemplary antihistamine suitable for use in the precursor is diphenhydramine. In one embodiment, diphenhydramine is administered at an intravenous or oral dose of 50mg or equivalent thereof. An exemplary antipyretic suitable for use in the precursor formulation is acetaminophen. In one embodiment, acetaminophen is administered at an oral dosage of 650-1000mg or an equivalent thereof. In some embodiments, the precursor drug is administered on the same day as the bispecific antibody, e.g., prior to injection of the bispecific antibody, e.g., 30-120 minutes prior to administration of the bispecific antibody.

The prodrugs and/or controls for CRS may be administered at least during the initial stages of treatment. In some embodiments, the prodrug and/or control is administered during the first four administrations of the bispecific antibody. For example, control may be administered as described herein during the first 28-day period of bispecific antibody administration. In some embodiments, a precursor drug is administered during the first period.

Typically, the risk of response during initial treatment will subside after several administrations, e.g., after the first four administrations (first period). Thus, control of CRS may be stopped when the human subject does not experience CRS after the fourth administration. However, CRS control may continue, particularly when human subjects experience CRS of greater than grade 1. Likewise, the precursor dosing may optionally continue. CRS classification may be performed as described in table 6 and table 7.

In further embodiments, in the methods described herein, when the human subject experiences CRS of greater than grade 1 after the fourth administration of the bispecific antibody in cycle 1, control for CRS is administered during the second 28 day cycle. Furthermore, when a human subject experiences CRS of greater than grade 1 in the last administration of a bispecific antibody of a previous cycle, control may continue during the subsequent cycle. Any precursor drug may optionally be administered during the second cycle. Further precursor medications may also optionally be administered in subsequent cycles.

In one embodiment, prodrugs and controls for CRS are administered, including antihistamines such as diphenhydramine (e.g., at an intravenous or oral dose of 50mg, or an equivalent thereof), antipyretics such as acetaminophen (e.g., at an oral dose of 650-1000mg, or an equivalent thereof), and corticosteroids such as prednisolone (e.g., at an intravenous dose of 100mg, or an equivalent thereof). In some embodiments, the pre-drug and control are administered 30-120 minutes prior to the bispecific antibody. On subsequent days 2, 3 and optionally 4, further control is administered, including systemic administration of a corticosteroid such as prednisolone (e.g., at an intravenous dose of 100mg or equivalent thereof). In some embodiments, it is preferred to administer the pre-drugs and control schedule during the first four administrations of the bispecific antibody, e.g., during the first 28 day period of bispecific antibody administration described herein. Furthermore, in the event that CRS, for example greater than level 1, occurs during the last administration of a previous cycle, a subsequent cycle may include the same administration schedule, with the precursor medication as part of the administration schedule being optional.

CRS may be well managed while effectively controlling and/or treating DLBCL during treatment of human subjects with DLBCL using the dosages and treatment regimens described herein. As described in the examples, subjects treated with the methods described herein may experience manageable CRS. In some cases, a subject receiving the treatment described herein may develop CRS of class 1 defined according to standard practice. In other cases, the subject may develop a manageable CRS of class 2 defined according to standard practice. Thus, a subject receiving the treatment described herein may have a manageable CRS of grade 1 or grade 2 defined according to standard practice. According to the standard classification of CRS, class 1 CRS includes fever to at least 38 ℃, no hypotension, no hypoxia, and class 2 CRS includes fever to at least 38 ℃ plus hypotension without vasopressors and/or hypoxia requiring oxygen inhalation or air leakage through low flow nasal cannulae. Such manageable CRS may occur at cycle 1. Human subjects receiving the treatment described herein may also have CRS of greater than grade 2 as defined according to standard practice during treatment. Thus, a human subject receiving the treatment described herein may also have class 3 CRS defined according to standard practice during the treatment. Such manageable CRS may further occur during cycle 1 and subsequent cycles.

Human subjects treated according to the methods described herein may also experience fever, fatigue, and injection site reactions. They may also experience neurotoxicity, partial seizures, CRS-related write loss, or CRS-related confusion.

As described above, although CRS control has been accepted, subjects may develop CRS during treatment with the methods described herein. CRS ranking criteria are described in table 6 and table 7.

In one embodiment, subjects who develop CRS grade 1 are treated with antibiotics if an infection occurs. In some embodiments, the antibiotic is continued until neutropenia (if present) subsides. In some embodiments, a subject with CRS grade 1 exhibiting systemic symptoms is treated with an NSAID.

In one embodiment, subjects who develop grade 2 CRS are treated with intravenous fluid bolus (fluid bolus) and/or oxygenation. In some embodiments, a subject who develops grade 2 CRS is treated with a vasopressor. In some embodiments, subjects with grade 2 CRS complications are treated with tobrazumab (humanized antibodies to IL-6 receptor, e.g., as an antigenCommercially available) and/or steroid (e.g., dexamethasone or its methylprednisolone equivalent). In a further embodiment, dexamethasone is administered to a subject presenting with concurrent ICANS. In yet another embodiment, the second dose of tolizumab is administered with a dose of corticosteroid if the subject does not show improvement in CRS symptoms for, e.g., 6 hours, or if the subject begins to worsen after initial improvement. In some embodiments, if the subject is refractory to tolizumab after three administrations, additional cytokine therapies, such as anti-IL-6 antibodies (e.g., cetuximab) or IL-1R antagonists (e.g., anakinra), are administered to the subject.

In one embodiment, a subject who develops grade 3 CRS is supported and/or supplemented with a vasopressor (e.g., norepinephrine). In some embodiments, a subject with grade 3 CRS is treated with tolizumab, or tolizumab in combination with a steroid (e.g., dexamethasone or methylprednisolone equivalent thereof). In some embodiments, dexamethasone is administered to a subject that is present in concurrent ICANS. In further embodiments, if the subject is refractory to tolizumab after three administrations, additional cytokine therapies, such as anti-IL-6 antibodies (e.g., cetuximab) or IL-1R antagonists (e.g., anakinra), are administered to the subject.

In one embodiment, a subject developing grade 4 CRS is treated with vasopressor support and/or oxygenation (e.g., via positive airway pressure, such as CPAP, biPAP, cannula, or mechanical ventilation). In some embodiments, at least two vasopressors are administered to a subject. In some embodiments, the subject is administered tobrazumab and a steroid. In a further embodiment, dexamethasone is administered to a subject presenting with concurrent ICANS. In yet further embodiments, if the subject is refractory to tolizumab after three administrations, additional cytokine therapies, such as anti-IL-6 antibodies (e.g., cetuximab) or IL-1R antagonists (e.g., anakinra), are administered to the subject.

In some embodiments, the human subject is receiving prophylactic treatment of Tumor Lysis Syndrome (TLS). Classification and stratification of oncolytic syndromes can be performed using methods known in the art, for example, as in Howard et al N Engl J Med 2011;364:1844-54 and Coiffier et al, J Clin Oncol2008; 26:2767-78. In some embodiments, prophylactic treatment of TLS comprises administering a uric acid lowering agent prior to administration of the bispecific antibody. Exemplary uric acid lowering agents include labyrinase and allopurinol. Thus, in one embodiment, the prophylactic treatment against TLS comprises administering a labyrine enzyme prior to the administration of the bispecific antibody. In some embodiments, supportive therapies such as labyrinthine may be used when the subject exhibits signs of TLS.

In one embodiment, the bispecific antibody is administered subcutaneously and thus formulated in a pharmaceutical composition to render it compatible for subcutaneous (s.c.) administration, i.e., with a formulation and/or concentration that allows for pharmaceutically acceptable s.c. administration at the dosages described herein. In some embodiments, the subcutaneous administration is by injection. For example, formulations of DuoBody CD3xCD20 that are compatible with subcutaneous formulations and useful in the methods described herein have been previously described (see, e.g., WO2019155008, which is incorporated herein by reference). In some embodiments, bispecific antibodies can be formulated using sodium acetate trihydrate, acetic acid, sodium hydroxide, sorbitol, polysorbate 80, and water for injection, and have a pH of 5.5 or about 5.5. In some embodiments, the bispecific antibody is provided as a 5mg/mL or 60mg/mL concentrate. In other embodiments, the desired dose of bispecific antibody is reconstituted to a volume of about 1mL for subcutaneous injection.

In one embodiment, a suitable pharmaceutical composition for a bispecific antibody may comprise a bispecific antibody, 20-40mM acetate, 140-160mM sorbitol, and a surfactant such as polysorbate 80, and have a pH of 5.3-5.6. In some embodiments, the pharmaceutical formulation may comprise an antibody concentration in the range of 5-100mg/mL, e.g., 48 or 60mg/mL of bispecific antibody, 30mM acetate, 150mM sorbitol, 0.04% w/v polysorbate 80, and have a pH of 5.5. Such formulations may be diluted with, for example, a formulation buffer to allow for proper administration and subcutaneous administration.

The volume of the pharmaceutical composition is suitably selected to allow subcutaneous administration of the antibody. For example, the volume to be administered is in the range of about 0.3mL to about 3mL, such as 0.3mL to 3mL. The volume to be administered may be 0.5mL, 0.8mL, 1mL, 1.2mL, 1.5mL, 1.7mL, 2mL, or 2.5mL, or about 0.5mL, about 0.8mL, about 1mL, about 1.2mL, about 1.5mL, about 1.7mL, about 2mL, or about 2.5mL. Thus, in one embodiment, the volume to be administered is 0.5mL or about 0.5mL. In some embodiments, the volume to be administered is 0.8mL or about 0.8mL. In some embodiments, the volume to be administered is 1mL or about 1mL. In some embodiments, the volume to be administered is 1.2mL or about 1.2mL. In some embodiments, the volume to be administered is 1.5mL or about 1.5mL. In some embodiments, the volume to be administered is 1.7mL or about 1.7mL. In some embodiments, the volume to be administered is 2mL or about 2mL. In some embodiments, the volume to be administered is 2.5mL or about 2.5mL.

In one embodiment, the gemcitabine and oxaliplatin are formulated separately in a pharmaceutical composition comprising a pharmaceutically acceptable excipient for administration (e.g., intravenous administration) according to local standard of care practice (e.g., as specified by local guidelines or local product labels) or as directed by the manufacturer. In some embodiments, gemcitabine and oxaliplatin are diluted from a stock solution, or if in lyophilized form, reconstituted according to instructions in, for example, a product label (e.g., with a 0.9% saline solution).

In one embodiment, a bispecific antibody for use in the methods described herein comprises:

(i) A first binding arm comprising a first antigen binding region that binds human CD3 epsilon (epsilon) and comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences within the amino acid sequence of SEQ ID NO:6, and the VL region comprises CDR1, CDR2, and CDR3 sequences within the amino acid sequence of SEQ ID NO: 7; and

(ii) A second binding arm comprising a second antigen binding region that binds human CD20 and comprises a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences within the amino acid sequence of SEQ ID NO:13, and the VL region comprises CDR1, CDR2, and CDR3 sequences within the amino acid sequence of SEQ ID NO: 14.

CDR1, CDR2, and CDR3 regions can be identified from variable heavy and variable light chain regions using methods known in the art. CDR regions from the variable heavy and variable light chain regions can be annotated according to IMGT (see Lefranc et al Nucleic Acids Research 1999;27:209-12,1999) and brochet.nucleic Acids Res 2008; 36:w503-8).

In some embodiments, the bispecific antibody comprises:

(i) A first binding arm comprising a first antigen binding region that binds human CD3 epsilon (epsilon) and comprises VHCDR1, VHCDR2, and VHCDR3 comprising the amino acid sequences set forth in SEQ ID NOs 1, 2, and 3, respectively, and VLCDR1, VLCDR2, and VLCDR3 comprising the amino acid sequences set forth in SEQ ID NOs 4, GTN, and 5, respectively; and

(ii) A second binding arm comprising a second antigen binding region that binds human CD20 and comprises VHCDR1, VHCDR2, and VHCDR3 comprising the amino acid sequences shown in SEQ ID nos. 8, 9, and 10, respectively, and VLCDR1, VLCDR2, and VLCDR3 comprising the amino acid sequences shown in SEQ ID nos. 11, DAS, and 12, respectively.

In some embodiments, the bispecific antibody comprises:

(i) A first binding arm comprising a first antigen binding region that binds human CD3 epsilon (epsilon) and comprises a VH region comprising the amino acid sequence of SEQ ID No. 6 and a VL region comprising the amino acid sequence of SEQ ID No. 7; and

(ii) A second binding arm comprising a second antigen binding region that binds human CD20 and comprises a VH region comprising the amino acid sequence of SEQ ID No. 13 and a VL region comprising the amino acid sequence of SEQ ID No. 14.

In one embodiment, the bispecific antibody is a full length antibody and may have an inert Fc region. In some embodiments, the first binding arm to CD3 is derived from a humanized antibody, e.g., derived from a full length IgG1, lambda (lambda) antibody, such as H1L1 described in WO2015001085 (which is incorporated herein by reference), and/or the second binding arm to CD20 is derived from a human antibody, e.g., derived from a full length IgG1, kappa (kappa) antibody, such as clone 7D8 described in WO2004035607 (which is incorporated herein by reference). Bispecific antibodies can be produced from two half-molecular antibodies. Each of the two half-molecule antibodies comprises a respective first and second binding arm, e.g., as shown in SEQ ID NOS: 24 and 25 and SEQ ID NOS: 26 and 27. The half antibodies may be produced in CHO cells and the bispecific antibodies may be produced by Fab arm exchange, for example. In one embodiment, the bispecific antibody is a functional variant of Duobody CD3xCD 20.

Thus, in some embodiments, a bispecific antibody comprises (i) a first binding arm comprising a first antigen binding region that binds human CD3 epsilon (epsilon) and comprises a VH region comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID No. 6 or a VH region comprising an amino acid sequence of SEQ ID No. 6 but having 1, 2, or 3 mutations (e.g., amino acid substitutions), and a VL region comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID No. 7 or a VL region comprising an amino acid sequence of SEQ ID No. 7 but having 1, 2, or 3 mutations (e.g., amino acid substitutions); and

(ii) A second binding arm comprising a second antigen binding region that binds human CD20 and comprises a VH region comprising an amino acid sequence at least 85%, 90%, 95%, 98% or 99% identical to SEQ ID No. 13 or a VH region comprising an amino acid sequence of SEQ ID No. 13 but having 1, 2 or 3 mutations (e.g., amino acid substitutions), and a VL region comprising an amino acid sequence at least 85%, 90%, 95%, 98% or 99% identical to SEQ ID No. 14 or a VL region comprising an amino acid sequence of SEQ ID No. 14 but having 1, 2 or 3 mutations (e.g., amino acid substitutions).

In one embodiment, the bispecific antibody comprises:

(i) A first binding arm comprising a first antigen binding region that binds human CD3 epsilon (epsilon) and comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 24 and a light chain comprising the amino acid sequence of SEQ ID No. 25; and

(ii) A second binding arm comprising a second antigen binding region that binds human CD20 and comprises a VH region comprising the amino acid sequence of SEQ ID No. 26 and a VL region comprising the amino acid sequence of SEQ ID No. 27.

In some embodiments, the bispecific antibody comprises (i) a first binding arm comprising a first antigen binding region that binds human CD3 epsilon (epsilon) and comprises a heavy chain comprising at least 85%, 90%, 95%, 98% or 99% identical amino acid sequence to SEQ ID No. 24 or a heavy chain comprising the amino acid sequence of SEQ ID No. 24 but having 1, 2, or 3 mutations (e.g., amino acid substitutions), and a light chain comprising at least 85%, 90%, 95%, 98% or 99% identical amino acid sequence to SEQ ID No. 25 or a light chain comprising the amino acid sequence of SEQ ID No. 25 but having 1, 2, or 3 mutations (e.g., amino acid substitutions); and

(ii) A second binding arm comprising a second antigen binding region that binds human CD20 and comprises a heavy chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 98% or 99% identical to SEQ ID No. 26 or a heavy chain comprising an amino acid sequence of SEQ ID No. 26 but having 1, 2 or 3 mutations (e.g., amino acid substitutions), and a light chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 98% or 99% identical to SEQ ID No. 27 or a light chain comprising an amino acid sequence of SEQ ID No. 27 but having 1, 2 or 3 mutations (e.g., amino acid substitutions).

Various constant regions or variants thereof may be used for bispecific antibodies. In one embodiment, the antibody comprises an IgG constant region, such as a human IgG1 constant region, e.g., a human IgG1 constant region as defined in SEQ ID NO. 15, or any other suitable IgG1 isotype. In one embodiment, the first binding arm of the bispecific antibody is derived from a humanized antibody, e.g. from a full length IgG1, lambda (lambda) antibody, and thus comprises a lambda light chain constant region. In some embodiments, the first binding arm comprises a lambda light chain constant region as defined in SEQ ID NO. 22. In some embodiments, the second binding arm of the bispecific antibody is derived from a human antibody, preferably a full length IgG1, kappa (kappa) antibody, and thus may comprise a kappa light chain constant region. In some embodiments, the second binding arm comprises a kappa light chain constant region as defined in SEQ ID NO. 23.

It will be appreciated that the constant region portion of the bispecific antibody may comprise modifications that allow for efficient formation/production of the bispecific antibody and/or provide an inert Fc region. Such modifications are well known in the art.

Bispecific antibodies of different formats are known in the art (reviewed by Kontermann, drug Discov Today 2015;20:838-47; MAbs,2012; 4:182-97). Thus, the bispecific antibodies used in the methods and uses described herein are not limited to any particular bispecific format or method of producing the same. For example, bispecific antibodies may include, but are not limited to, bispecific antibodies with complementary CH3 domains to force heterodimerization, knob-in-Hole molecules (Genntech, WO 9850431), crossMAbs (Roche, WO 2011117329), or electrostatic matching molecules (Amgen, EP1870459 and WO2009089004; chugai, US201000155133; oncomed, WO 2010129304).

Preferably, the bispecific antibody comprises an Fc region comprising a first heavy chain having a first Fc sequence comprising a first CH3 region and a second heavy chain having a second Fc sequence comprising a second CH3 region, wherein the sequences of the first and second CH3 regions are different and such that the heterodimeric interaction between the first and second CH3 regions is stronger than each of the homodimeric interactions of the first and second CH3 regions. More details on these interactions and how they are achieved are provided, for example, in WO2011131746 and WO2013060867 (Genmab), which are incorporated herein by reference. In one embodiment, the bispecific antibody comprises in the first heavy chain (i) amino acid L in a position corresponding to F405 in the human IgG1 heavy chain constant region of SEQ ID NO:15, and in the second heavy chain (ii) amino acid R in a position corresponding to K409 in the human IgG1 heavy chain constant region of SEQ ID NO:15, or vice versa.

Bispecific antibodies may contain modifications in the Fc region to render the Fc region inert or inactive. Thus, in the bispecific antibodies disclosed herein, one or both heavy chains can be modified such that the antibodies induce Fc-mediated effector function to a lesser extent relative to the bispecific antibodies without modification. Fc-mediated effector function may be measured by assaying Fc-mediated CD69 expression on T cells (i.e., CD69 expression by CD3 antibody-mediated, fcγ receptor-dependent CD3 crosslinking), by binding to fcγ receptors, by binding to Clq, or by inducing Fc-mediated fcγr crosslinking. In particular, the heavy chain constant region sequence may be modified such that Fc-mediated CD69 expression is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% or 100% when compared to a wild-type (unmodified) antibody, wherein said Fc-mediated CD69 expression is determined in a PBMC-based functional assay, e.g. as described in example 3 of WO 2015001085. Modification of the heavy and light chain constant region sequences can also result in reduced binding of Clq to the antibody. The decrease may be at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% compared to the unmodified antibody, and Clq binding may be determined, for example, by ELISA. Furthermore, the Fc region may be modified such that the antibody mediates at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% or 100% reduction in Fc-mediated T cell proliferation as measured in a PBMC-based functional assay as compared to the unmodified antibody. Examples of amino acid positions that may be modified include positions L234 and L235, for example, in IgG1 isotype antibodies. Thus, in one embodiment, a bispecific antibody may comprise a first heavy chain and a second heavy chain, and wherein in the first heavy chain and the second heavy chain the amino acid residues in positions L234 and L235 in the heavy chain of human IgG1 corresponding to Eu numbering are F and E, respectively. In addition, D265A amino acid substitutions may reduce binding to all Fc gamma receptors and prevent ADCC (Shields et al, JBC 2001; 276:6591-604). Thus, a bispecific antibody may comprise a first heavy chain and a second heavy chain, wherein in the first heavy chain and the second heavy chain the amino acid residue in position D265 in the human IgG1 heavy chain corresponding to Eu numbering is a.

In one embodiment, the amino acids in the first and second heavy chains of the bispecific antibody in positions L234, L235 and D265 corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and a, respectively. Antibodies having these amino acids at these positions are examples of antibodies having an inert Fc region or a non-activated Fc region.

With respect to the bispecific antibodies described herein, those having a combination of three amino acid substitutions L234F, L235E and D265A and additionally a K409R or F405L mutation, as described above, may be represented by the suffix "FEAR" or "FEAL", respectively.

The amino acid sequence of the wild-type IgG1 heavy chain constant region can be identified herein as SEQ ID NO. 15. Consistent with the embodiments disclosed above, bispecific antibodies may comprise an IgG1 heavy chain constant region carrying an F405L substitution and may have the amino acid sequence shown in SEQ ID No. 17 and/or an IgG1 heavy chain constant region carrying a K409R substitution and may have the amino acid sequence shown in SEQ ID No. 18, and have further substitutions that render the Fc region inactive or inactive. Thus, in one embodiment, the bispecific antibody comprises a combination of IgG1 heavy chain constant regions, wherein the amino acid sequence of one of the IgG1 heavy chain constant regions carries L234F, L235E, D265A and F405L substitutions (e.g., as shown in SEQ ID NO: 19) and the amino acid sequence of the other IgG1 heavy chain constant region carries L234F, L235E, D265A and K409R substitutions (e.g., as shown in SEQ ID NO: 20).

In some embodiments, bispecific antibodies for use in the methods and uses described herein comprise a first binding arm comprising a heavy chain and a light chain as defined in SEQ ID NOS: 24 and 25, respectively, and a second binding arm comprising a heavy chain and a light chain as defined in SEQ ID NOS: 26 and 27, respectively. Such antibodies are referred to herein as DuoBody CD3xCD20. Furthermore, variants of such antibodies are contemplated for use in the methods and uses described herein. In some embodiments, the bispecific antibody is elcatuzumab (CAS 2134641-34-0) or a biological analog thereof.

Kit for detecting a substance in a sample

Also provided herein are kits comprising a pharmaceutical composition containing a bispecific antibody that binds to CD3 and CD20 according to the invention, such as DuoBody CD3xCD20 or elcatuzumab, in a therapeutically effective amount suitable for use in the methods described herein, and a pharmaceutically acceptable carrier. The kit may also include a pharmaceutical composition containing gemcitabine (e.g., for intravenous administration) and/or a separate pharmaceutical composition containing oxaliplatin (e.g., for intravenous administration). The kit optionally may also include instructions, for example, including an administration schedule, to allow a practitioner (e.g., physician, nurse, or patient) to administer one or more compositions contained therein to a patient having DLBCL. The kit may also include one or more syringes.

Optionally, the kit comprises a plurality of packaged single dose pharmaceutical compositions, each containing an effective amount of a bispecific antibody for single administration according to the methods described herein. They may also include a plurality of packaged single dose pharmaceutical compositions containing doses of gemcitabine and/or oxaliplatin according to standard of care protocols. The necessary instruments or devices for administering the pharmaceutical composition may also be included in the kit.

Further embodiments

1. A bispecific antibody comprising:

(i) A first binding arm comprising a first antigen binding region that binds human CD3 epsilon (epsilon) and comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences in the VH region sequence of SEQ ID NO:6, and the VL region comprises CDR1, CDR2, and CDR3 sequences in the VL region sequence of SEQ ID NO: 7; and

The bispecific antibody is for use in treating Diffuse Large B Cell Lymphoma (DLBCL) in a human subject, wherein the treatment comprises administering the bispecific antibody and an effective amount of gemcitabine and oxaliplatin to the human subject, wherein the bispecific antibody is administered at a dose of 24mg or 48mg, and wherein the bispecific antibody, gemcitabine, and oxaliplatin are administered at a 28 day cycle.

2. The bispecific antibody of embodiment 1, wherein the bispecific antibody is administered at a dose of 24 mg.

3. The bispecific antibody of embodiment 1, wherein the bispecific antibody is administered at a dose of 48 mg.

4. The bispecific antibody of any one of embodiments 1-3, wherein the bispecific antibody is administered once weekly (weekly administration).

5. The bispecific antibody of embodiment 4, wherein 24mg or 48mg of the weekly administration is performed for 2.5 28 days of period.

6. The bispecific antibody of embodiments 4 or 5, wherein the bispecific antibody is administered once every two weeks (bi-weekly administration) after the weekly administration.

7. The bispecific antibody of embodiment 6, wherein the two week administration is performed for six 28 day periods.

8. The bispecific antibody of embodiments 6 or 7, wherein the bispecific antibody is administered once every four weeks after the bi-weekly administration.

9. The bispecific antibody of embodiment 8, wherein the administration is performed once every four weeks for at least two 28 day periods.

10. The bispecific antibody of any one of embodiments 4-9, wherein the amount of a trigger of the bispecific antibody is administered in cycle 1 of the 28 day cycle prior to 24mg or 48mg of the weekly administration.

11. The bispecific antibody of embodiment 10, wherein the priming dose is administered two weeks prior to the administration of the first weekly dose of 24mg or 48 mg.

12. The bispecific antibody of embodiments 10 or 11, wherein the amount of the priming agent is 0.16mg.

13. The bispecific antibody of any one of embodiments 10-12, wherein an intermediate dose of the bispecific antibody is administered after the administration of the priming dose and before the administration of the first weekly dose of 24mg or 48 mg.

14. The bispecific antibody of embodiment 13, wherein the priming dose is administered on day 1 of cycle 1 and the intermediate dose is administered on day 8, followed by a first weekly dose of 24mg or 48mg on days 15 and 22.

15. The bispecific antibody of embodiments 13 or 14, wherein the intermediate dose is 0.8mg.

16. The bispecific antibody of any one of embodiments 1-15, wherein gemcitabine is administered once every two weeks.

17. The bispecific antibody of embodiment 16, wherein the administration of gemcitabine once every two weeks is for four 28 day cycles.

18. The bispecific antibody of any one of embodiments 1-17, wherein gemcitabine is at 1000mg/m ² Is administered at a dose or equivalent thereof.

19. The bispecific antibody of any one of embodiments 1-18, wherein oxaliplatin is administered once every two weeks.

20. The bispecific antibody of any one of embodiments 1-19, wherein the administration of oxaliplatin once every two weeks is performed for four 28 day cycles.

21. The bispecific antibody of any one of embodiments 1-20, wherein oxaliplatin is present at 100mg/m ² Is administered at a dose of (a).

22. The bispecific antibody of any one of embodiments 1-21, wherein gemcitabine, oxaliplatin, and the bispecific antibody are administered on the same day (e.g., on days 1 and 15 of cycles 1-4).

23. The bispecific antibody of any one of embodiments 1-22, wherein the dosing schedule of gemcitabine, oxaliplatin, and the bispecific antibody is shown in table 2.

24. The bispecific antibody of any one of embodiments 1, 2 and 4-23, wherein administration is performed with a 28 day period, and wherein:

(a) The bispecific antibody is administered as follows:

(iii) In cycles 4-9, a dose of 24mg was administered on days 1 and 15; and

(b) Gemcitabine is administered on days 1 and 15 of cycles 1-4; and

(c) Oxaliplatin is administered on days 1 and 15 of cycles 1-4.

25. The bispecific antibody of any one of embodiments 1 and 3-23, wherein administration is performed with a 28 day period, and wherein:

(a) The bispecific antibody is administered as follows:

(iii) In cycles 4-9, a dose of 48mg was administered on days 1 and 15; and

(b) Gemcitabine is administered on days 1 and 15 of cycles 1-4; and

(c) Oxaliplatin is administered on days 1 and 15 of cycles 1-4.

26. The bispecific antibody of any one of embodiments 1-25, wherein the bispecific antibody is administered subcutaneously.

27. The bispecific antibody of any one of embodiments 1-26, wherein gemcitabine is administered intravenously.

28. The bispecific antibody of any one of embodiments 1-27, wherein oxaliplatin is administered intravenously.

29. The bispecific antibody of any one of embodiments 1-28, wherein the bispecific antibody, gemcitabine, and oxaliplatin are administered sequentially.

30. The bispecific antibody of any one of embodiments 1-29, wherein when gemcitabine, oxaliplatin, and the bispecific antibody are administered on the same day, the gemcitabine is administered first, the oxaliplatin is administered second, and the bispecific antibody is administered last.

31. The bispecific antibody of any one of embodiments 1-30, wherein the DLBCL is double hit or triple hit DLBCL.

32. The bispecific antibody of any one of embodiments 1-31, wherein the DLBCL is grade 3B follicular lymphoma.

33. The bispecific antibody of any one of embodiments 1-32, wherein the subject has relapsed after at least one prior therapy.

34. The bispecific antibody of any one of embodiments 1-33, wherein the subject is refractory to at least one prior therapy.

35. The bispecific antibody of any one of embodiments 1-34, wherein the subject has failed a prior autologous hematopoietic stem cell transplantation.

36. The bispecific antibody of any one of embodiments 1-35, wherein the subject does not meet autologous hematopoietic stem cell transplantation conditions due to age, performance status, complications, and/or insufficient response to prior therapy.

37. The bispecific antibody of any one of embodiments 1-36, wherein the subject is treated with a control for cytokine release syndrome.

38. The bispecific antibody of embodiment 37, wherein the controlling comprises administering to the subject a corticosteroid.

39. The bispecific antibody of embodiment 38, wherein the corticosteroid is administered on the same day as the bispecific antibody.

40. The bispecific antibody of embodiment 39, wherein the corticosteroid is further administered the second, third, and fourth days after administration of the bispecific antibody.

41. The bispecific antibody of any of embodiments 38-40, wherein the corticosteroid is prednisolone.

42. The bispecific antibody of embodiment 41, wherein the prednisolone is administered at an intravenous dose of 100mg or an equivalent thereof (including an oral dose).

43. The bispecific antibody of any one of embodiments 1-42, wherein a prodrug is administered to the subject to reduce the response to an injection.

44. The bispecific antibody of embodiment 43, wherein the prodrug comprises an antihistamine.

45. The bispecific antibody of embodiment 44, wherein the antihistamine is diphenhydramine.

46. The bispecific antibody of embodiment 45, wherein the diphenhydramine is administered in an intravenous or oral dose or equivalent thereof of 50 mg.

47. The bispecific antibody of any of embodiments 43-46, wherein the prodrug comprises an antipyretic.

48. The bispecific antibody of embodiment 47, wherein the antipyretic is acetaminophen.

49. The bispecific antibody of embodiment 48, wherein the acetaminophen is administered at an oral dose of 650mg to 1000mg or an equivalent thereof.

50. The bispecific antibody of any one of embodiments 43-49, wherein the prodrug is administered on the same day as the bispecific antibody.

51. The bispecific antibody of any one of embodiments 37-50, wherein the control is administered in cycle 1 of the 28 day cycle.

52. The bispecific antibody of any one of embodiments 43-51, wherein the prodrug is administered in cycle 1 of the 28 day cycle.

53. The bispecific antibody of any one of embodiments 37-52, wherein the control is administered during cycle 2 of the 28 day cycle when the subject experiences CRS of greater than grade 1 following the last administration of the bispecific antibody in cycle 1 of the 28 day cycle.

54. The bispecific antibody of embodiment 53, wherein the control is continued in a subsequent cycle when the subject experiences CRS of greater than grade 1 in the last administration of the bispecific antibody of a previous cycle.

55. The bispecific antibody of any one of embodiments 43-54, wherein the prodrug is administered during cycle 2 of the 28 day cycle.

56. The bispecific antibody of embodiment 55, wherein the prodrug is administered during a subsequent cycle.

57. The bispecific antibody of any one of embodiments 1-56, wherein an antibiotic is administered to the subject if the subject develops CRS grade 1.

58. The bispecific antibody of any one of embodiments 1-56, wherein a vasopressor is administered to the subject if the subject develops CRS grade 2 or grade 3.

59. The bispecific antibody of any one of embodiments 1-56, wherein at least two vasopressors are administered to the subject if the subject develops grade 4 CRS.

60. The bispecific antibody of any one of embodiments 1-59, wherein the subject is administered tobrazumab if the subject develops a CRS of grade 2, grade 3, or grade 4.

61. The bispecific antibody of embodiment 60, wherein the subject is further administered a steroid.

62. The bispecific antibody of embodiment 61, wherein the steroid is dexamethasone.

63. The bispecific antibody of embodiment 61, wherein the steroid is methylprednisolone.

64. The bispecific antibody of any of embodiments 60-63, wherein the tobrazumab is converted to an anti-IL-6 antibody (e.g., rituximab) if the subject is refractory to tobrazumab.

65. The bispecific antibody of any one of embodiments 60-63, wherein the tobrazumab is converted to an IL-1R antagonist (e.g., anakinra) if the subject is refractory to tobrazumab.

66. The bispecific antibody of any one of embodiments 1-65, wherein the subject is treated with a control for Tumor Lysis Syndrome (TLS).

67. The bispecific antibody of embodiment 66, wherein the control for TLS comprises administration of one or more uric acid lowering agents prior to administration of the bispecific antibody.

68. The bispecific antibody of embodiment 67, wherein the one or more uric acid lowering agents comprise a labyrinase and/or allopurinol.

69. The bispecific antibody of any one of embodiments 1-68, wherein the subject achieves a complete response, a partial response, or disease stabilization.

70. The bispecific antibody of any one of embodiments 1-69, wherein:

(i) The first antigen binding region of the bispecific antibody comprises a VHCDR1, VHCDR2 and VHCDR3 comprising the amino acid sequences shown in SEQ ID NOs 1, 2 and 3, respectively, and a VLCDR1, VLCDR2 and VLCDR3 comprising the amino acid sequences shown in SEQ ID NO 4, sequence GTN and SEQ ID NO 5, respectively; and

(ii) The second antigen binding region of the bispecific antibody comprises a VHCDR1, VHCDR2 and VHCDR3 comprising the amino acid sequences shown in SEQ ID NO. 8, 9 and 10, respectively, and a VLCDR1, VLCDR2 and VLCDR3 comprising the amino acid sequences shown in SEQ ID NO. 11, sequence DAS and SEQ ID NO. 12, respectively.

71. The bispecific antibody of any one of embodiments 1-70, wherein:

(i) The first antigen-binding region of the bispecific antibody comprises a VH region comprising the amino acid sequence of SEQ ID No. 6 and a VL region comprising the amino acid sequence of SEQ ID No. 7; and is also provided with

(ii) The second antigen-binding region of the bispecific antibody comprises a VH region comprising the amino acid sequence of SEQ ID No. 13 and a VL region comprising the amino acid sequence of SEQ ID No. 14.

72. The bispecific antibody of any one of embodiments 1-71, wherein the first binding arm of the bispecific antibody is derived from a humanized antibody, preferably a full length IgG1, lambda (lambda) antibody.

73. The bispecific antibody of embodiment 72, wherein the first binding arm of the bispecific antibody comprises a lambda light chain constant region comprising the amino acid sequence as set forth in SEQ ID No. 22.

74. The bispecific antibody of any one of embodiments 1-73, wherein the second binding arm of the bispecific antibody is derived from a human antibody, preferably a full length IgG1, kappa (kappa) antibody.

75. The bispecific antibody of embodiment 74, wherein the second binding arm comprises a kappa light chain constant region comprising the amino acid sequence depicted in SEQ ID No. 23.

76. The bispecific antibody of any one of embodiments 1-75, wherein the bispecific antibody is a full length antibody having a human IgG1 constant region.

77. The bispecific antibody of any one of embodiments 1-76, wherein the bispecific antibody comprises an inert Fc region.

78. The bispecific antibody of any one of embodiments 1-77, wherein the bispecific antibody comprises a first heavy chain and a second heavy chain, wherein in both the first heavy chain and the second heavy chain the amino acids in positions L234, L235 and D265 in the human IgG1 heavy chain constant region corresponding to SEQ ID No. 15 are F, E and a, respectively.

79. The bispecific antibody of any one of embodiments 1-78, wherein the bispecific antibody comprises a first heavy chain and a second heavy chain, wherein in the first heavy chain the amino acid in position F405 in the human IgG1 heavy chain constant region corresponding to SEQ ID No. 15 is L, and wherein in the second heavy chain the amino acid in position K409 in the human IgG1 heavy chain constant region corresponding to SEQ ID No. 15 is R, or vice versa.

80. The bispecific antibody of any one of embodiments 1-79, wherein the bispecific antibody comprises a first heavy chain and a second heavy chain, wherein

(i) In both the first and second heavy chains, the amino acids in positions L234, L235 and D265 in the constant region of the human IgG1 heavy chain corresponding to SEQ ID NO. 15 are F, E and A, respectively, and

(ii) In the first heavy chain, the amino acid in the position corresponding to F405 in the human IgG1 heavy chain constant region of SEQ ID NO. 15 is L, and wherein in the second heavy chain, the amino acid in the position corresponding to K409 in the human IgG1 heavy chain constant region of SEQ ID NO. 15 is R, or vice versa.

81. The bispecific antibody of embodiment 80, wherein the bispecific antibody comprises a heavy chain constant region comprising the amino acid sequences of SEQ ID NOs 19 and 20.

82. The bispecific antibody of any one of embodiments 1-81, wherein the bispecific antibody comprises a heavy chain and a light chain comprising the amino acid sequences shown in SEQ ID NOs 24 and 25, respectively, and a heavy chain and a light chain comprising the amino acid sequences shown in SEQ ID NOs 26 and 27, respectively.

83. The bispecific antibody of any one of embodiments 1-82, wherein the bispecific antibody comprises a heavy chain and a light chain consisting of the amino acid sequences of SEQ ID NOs 24 and 25, respectively, and a heavy chain and a light chain consisting of the amino acid sequences of SEQ ID NOs 26 and 27, respectively.

84. The bispecific antibody of any one of embodiments 1-83, wherein the bispecific antibody is elcuritumumab or a biological analogue thereof.

A method of treating diffuse large B-cell lymphoma (DLBCL) in a human subject, the method comprising administering to the subject a bispecific antibody and an effective amount of gemcitabine and oxaliplatin, wherein the bispecific antibody comprises:

wherein the bispecific antibody is administered at a dose of 24mg or 48mg, and wherein gemcitabine, oxaliplatin, and the bispecific antibody are administered at a 28 day period.

The method of embodiment 1a, wherein the bispecific antibody is administered at a dose of 24 mg.

3a. The method of embodiment 1a, wherein the bispecific antibody is administered at a dose of 48 mg.

4a. The method of any one of embodiments 1a-3a, wherein the bispecific antibody is administered once weekly (weekly administration).

5a. The method of embodiment 4a, wherein 24mg or 48mg of the weekly administration is performed for 2.5 28 days.

The method of embodiment 4a or 5a, wherein the bispecific antibody is administered once every two weeks (biweekly administration) after the weekly administration.

The method of embodiment 6a, wherein the two week administration is performed for six 28 day periods.

The method of embodiment 6a or 7a, wherein the bispecific antibody is administered once every four weeks after the bi-weekly administration.

9a. The bispecific antibody of embodiment 8a, wherein the administration is performed once every four weeks for at least two 28 day periods.

The method of any one of embodiments 4a-9a, wherein the priming amount of the bispecific antibody is administered at cycle 1 of the 28 day cycle prior to 24mg or 48mg of the weekly administration.

The method of embodiment 10a, wherein the priming dose is administered two weeks prior to the administration of the first weekly dose of 24mg or 48 mg.

The method of embodiment 10a or 11a, wherein the initiator amount is 0.16mg.

The method of any one of embodiments 10a-12a, wherein an intermediate dose of the bispecific antibody is administered after administration of the priming dose and before administration of a first weekly dose of 24mg or 48 mg.

The method of embodiment 13a, wherein the priming dose is administered on day 1 of cycle 1 and the intermediate dose is administered on day 8, followed by a first weekly dose of 24mg or 48mg on days 15 and 22.

The method of embodiment 13a or 14a, wherein the intermediate dose is 0.8mg.

The method of any one of embodiments 1a-15a, wherein gemcitabine is administered once every two weeks.

The method of embodiment 16a, wherein the administering gemcitabine once every two weeks is for four 28-day cycles.

The method of any one of embodiments 1a-17a, wherein gemcitabine is at 1000mg/m ² Is administered at a dose or equivalent thereof.

The method of any one of embodiments 1a-18a, wherein oxaliplatin is administered once every two weeks.

The method of any one of embodiments 1a-19a, wherein the administering oxaliplatin once every two weeks is performed for four 28 day periods.

The method of any one of embodiments 1a-20a, wherein oxaliplatin is present at 100mg/m ² Is administered at a dose of (a).

The bispecific antibody of embodiments 1a-21a, wherein gemcitabine, oxaliplatin, and the bispecific antibody are administered on the same day (e.g., on days 1 and 15 of cycles 1-4).

The method of any one of embodiments 1a-22a, wherein the dosing schedule of gemcitabine, oxaliplatin, and the bispecific antibody is shown in table 2.

The method of any one of embodiments 1a, 2a, and 4a-23a, wherein the administering is performed over a 28 day period, and wherein:

(a) The bispecific antibody is administered as follows:

(iii) In cycles 4-9, a dose of 24mg was administered on days 1 and 15; and

(b) Gemcitabine is administered on days 1 and 15 of cycles 1-4; and

(c) Oxaliplatin is administered on days 1 and 15 of cycles 1-4.

The method of any one of embodiments 1a and 3a-23a, wherein the administering is performed over a 28 day period, and wherein:

(a) The bispecific antibody is administered as follows:

(iii) In cycles 4-9, a dose of 48mg was administered on days 1 and 15; and

(b) Gemcitabine is administered on days 1 and 15 of cycles 1-4; and

(c) Oxaliplatin is administered on days 1 and 15 of cycles 1-4.

The method of any one of embodiments 1a-25a, wherein the bispecific antibody is administered subcutaneously.

The method of any one of embodiments 1a-26a, wherein the bispecific antibody is administered intravenously.

The method of any one of embodiments 1a-27a, wherein oxaliplatin is administered intravenously.

The method of any one of embodiments 1a-28a, wherein the bispecific antibody, gemcitabine, and oxaliplatin are administered sequentially.

The method of any one of embodiments 1a-29a, wherein when gemcitabine, oxaliplatin, and the bispecific antibody are administered on the same day, then gemcitabine is administered first, oxaliplatin is administered second, and the bispecific antibody is administered last.

The method of any of embodiments 1a-30a, wherein the DLBCL is double-hit or triple-hit DLBCL.

The method of embodiment 31a, wherein the DLBCL is grade 3B follicular lymphoma.

The method of embodiment 31a or 32a, wherein the subject has relapsed after at least one prior therapy.

The method of any one of embodiments 1a-33a, wherein the subject is refractory to at least one prior therapy.

The bispecific antibody of any one of embodiments 1a-34a, wherein the subject has failed prior autologous hematopoietic stem cell transplantation.

36. The bispecific antibody of any one of embodiments 1a-35a, wherein the subject is not eligible for autologous hematopoietic stem cell transplantation due to age, performance status, complications, and/or inadequate response to previous treatments.

The bispecific antibody of any one of embodiments 1a-36a, wherein the subject is treated with a control for cytokine release syndrome.

The method of embodiment 37a, wherein said controlling comprises administering a corticosteroid to said subject.

39a the method of embodiment 38a, wherein the corticosteroid is administered on the same day as the bispecific antibody.

The method of embodiment 39a, wherein the corticosteroid is further administered the second, third, and fourth days after administration of the bispecific antibody.

The method of any one of embodiments 38a-40a, wherein the corticosteroid is prednisolone.

The method of embodiment 41a, wherein the prednisolone is administered at an intravenous dose of 100mg or an equivalent thereof (including an oral dose).

The method of any one of embodiments 1a-42a, wherein a prodrug is administered to the subject to reduce the response to an injection.

44a the method of embodiment 43a, wherein said prodrug comprises an antihistamine.

45a the method of embodiment 44a, wherein said antihistamine is diphenhydramine.

46a. The method of embodiment 45a, wherein the diphenhydramine is administered in an intravenous or oral dose of 50mg or equivalent thereof.

The method of any one of embodiments 43a-46a, wherein the precursor drug comprises an antipyretic.

48a the method of embodiment 47a, wherein the antipyretic is acetaminophen.

49a the method of embodiment 48a, wherein the acetaminophen is administered at an oral dose of 650mg to 1000mg or its equivalent.

The method of any one of embodiments 43a-49a, wherein the prodrug is administered on the same day as the bispecific antibody.

The method of any one of embodiments 37a-50a, wherein the controlling is applied in cycle 1 of the 28 day cycle.

The method of any one of embodiments 43a-51a, wherein the prodrug is administered in cycle 1 of the 28 day cycle.

The method of any one of embodiments 37a-52a, wherein the control is administered during cycle 2 of the 28-day cycle when the subject experiences CRS of greater than grade 1 following the last administration of the bispecific antibody in cycle 1 of the 28-day cycle.

54a. The method of embodiment 53a, wherein the controlling is continued in a subsequent cycle when the subject experiences CRS of greater than grade 1 in the last administration of the bispecific antibody of a previous cycle.

The method of any one of embodiments 43a-54a, wherein the prodrug is administered during cycle 2 of the 28 day cycle.

56a the method of embodiment 55a, wherein the prodrug is administered during a subsequent cycle.

57a the method of any one of embodiments 1a-56a, wherein if the subject develops CRS grade 1, then an antibiotic is administered to the subject.

The method of any one of embodiments 1a-56a, wherein if the subject develops CRS grade 2 or grade 3, then a vasopressor is administered to the subject.

59a. the method of any one of embodiments 1a-56a, wherein if the subject develops grade 4 CRS, then at least two vasopressors are administered to the subject.

The method of any one of embodiments 1a-59a, wherein the subject is administered tobrazumab if the subject develops CRS grade 2, grade 3 or grade 4.

The method of embodiment 60a, wherein the subject is further administered a steroid.

The method of embodiment 61a, wherein the steroid is dexamethasone.

63a. The method of embodiment 61a, wherein the steroid is methylprednisolone.

The method of any one of embodiments 60a-63a, wherein, if the subject is refractory to tolizumab, the tolizumab is converted to an anti-IL-6 antibody (e.g., rituximab).

The method of any one of embodiments 60a-63a, wherein, if the subject is refractory to tolizumab, tolizumab is converted to an IL-1R antagonist (e.g., anakinra).

The method of any one of embodiments 1a-65a, wherein the subject is treated with a control for Tumor Lysis Syndrome (TLS).

The method of embodiment 66a, wherein said controlling against TLS comprises administering one or more uric acid lowering agents prior to administering said bispecific antibody.

The method of embodiment 67a, wherein the one or more uric acid lowering agents comprise a labyrinase and/or allopurinol.

69a. the method of any one of embodiments 1a-68a, wherein the subject achieves a complete response, a partial response, or disease stabilization.

The method of any one of embodiments 1a-69a, wherein:

The method of any one of embodiments 1a-70a, wherein:

The method of any one of embodiments 1a-71a, wherein the first binding arm of the bispecific antibody is derived from a humanized antibody, preferably a full length IgG1, lambda (lambda) antibody.

The method of embodiment 72a, wherein the first binding arm of the bispecific antibody comprises a lambda light chain constant region comprising the amino acid sequence as set forth in SEQ ID No. 22.

74a. The method of any one of embodiments 1a-73a, wherein the second binding arm of the bispecific antibody is derived from a human antibody, preferably a full length IgG1, kappa (kappa) antibody.

75a. The method of embodiment 74a, wherein said second binding arm comprises a kappa light chain constant region comprising the amino acid sequence set forth in SEQ ID NO. 23.

76a. The method of any of embodiments 1a-75a, wherein the bispecific antibody is a full length antibody having a human IgG1 constant region.

77a. The method of any one of embodiments 1a-76a, wherein the bispecific antibody comprises an inert Fc region.

The method of any one of embodiments 1a-77a, wherein the bispecific antibody comprises a first heavy chain and a second heavy chain, wherein in both the first heavy chain and the second heavy chain the amino acids in positions L234, L235 and D265 in the human IgG1 heavy chain constant region corresponding to SEQ ID No. 15 are F, E and a, respectively.

79a. The method of any one of embodiments 1a-78a, wherein the bispecific antibody comprises a first heavy chain and a second heavy chain, wherein in the first heavy chain the amino acid in position F405 in the human IgG1 heavy chain constant region corresponding to SEQ ID No. 15 is L, and wherein in the second heavy chain the amino acid in position K409 in the human IgG1 heavy chain constant region corresponding to SEQ ID No. 15 is R, or vice versa.

80a. The method of any one of embodiments 1a-79a, wherein the bispecific antibody comprises a first heavy chain and a second heavy chain, wherein

81a. The method of embodiment 80a, wherein said bispecific antibody comprises a heavy chain constant region comprising the amino acid sequences of SEQ ID NOs 19 and 20.

82a. The method of any one of embodiments 1a-81a, wherein the bispecific antibody comprises a heavy chain and a light chain comprising the amino acid sequences shown in SEQ ID NOs 24 and 25, respectively, and a heavy chain and a light chain comprising the amino acid sequences shown in SEQ ID NOs 26 and 27, respectively.

83a. The method of any one of embodiments 1a-82a, wherein the bispecific antibody comprises a heavy chain and a light chain consisting of the amino acid sequences of SEQ ID NOs 24 and 25, respectively, and a heavy chain and a light chain consisting of the amino acid sequences of SEQ ID NOs 26 and 27, respectively.

The method of any one of embodiments 1a-83a, wherein the bispecific antibody is elcuritumumab or a biological analog thereof.

The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and all references, genbank sequences, journal publications, patents and published patent applications cited in this application are expressly incorporated herein by reference.

Examples

DuoBody-CD3xCD20

DuoBody-CD3xCD20 is bsAb that recognizes the T cell antigen CD3 and the B cell antigen CD 20. DuoBody-CD3xCD20 triggers T cell mediated potent killing of CD20 expressing cells. DuoBody-CD3xCD20 has a regular IgG1 structure.

Preparation of two parent antibodies, igG1-CD3-FEAL (humanized IgG1 lambda, CD3 epsilon specific antibody with heavy and light chain sequences as set forth in SEQ ID NOS: 24 and 25, respectively) and IgG1-CD20-FEAR (derived from human IgG1 kappa CD 20-specific antibody 7D8 with heavy and light chain sequences as set forth in SEQ ID NOS: 26 and 27, respectively)Is a single biological intermediate. Each parent antibody contains one of the complementary mutations in the CH3 domain (F405L and K409R, respectively) required to generate a DuoBody molecule. The parent antibody contains three additional mutations in the Fc region (L234F, L235E and D265A; FEA). Parent antibodies were produced in mammalian Chinese Hamster Ovary (CHO) cell lines using standard suspension cell culture and purification techniques. Subsequently, duoBody-CD3xCD20 was prepared by a controlled Fab arm exchange (cFAE) procedure (Labrijn et al 2013, labrijn et al 2014, gramer et al 2013). The parent antibodies are mixed and subjected to controlled reducing conditions. This results in isolation of the parent antibody that reassembles under reoxidation. Thus, a high purity preparation (about 93% -95%) of DuoBody-CD3xCD20 was obtained. After further purification (poling)/purification, the final product was obtained in nearly 100% purity. DuoBody-CD3xCD20 concentration Using the theoretical extinction coefficient ε= 1.597 mL.mg ^-1 cm ^-1 Measured by absorbance at 280 nm. The final product was stored at 4 ℃. The international proprietary name of this product was elcatuzumab.

The elcatuzumab was prepared as a sterile clear colorless to pale yellow solution (5 mg/mL or 60 mg/mL) provided as a concentrate of a solution for Subcutaneous (SC) injection. The elcuritumumab contained a buffer and a strengthening agent (tonicifying agents). All excipients and amounts thereof in the formulated product are pharmaceutically acceptable for subcutaneous injection products. The appropriate dose is reconstituted to a volume of about 1mL for subcutaneous injection.

Example 1: ecuritumumab combined standard of care GemOx for treatment of recurrent or refractory (R/R) DLBCL phase 1b, open label, safety and efficacy studies that do not meet Autologous Stem Cell Transplantation (ASCT) conditions

Open label, two-part (dose escalation and extension), multinational, multicenter intervention studies were performed to evaluate safety, tolerability, PK, pharmacodynamics/biomarkers, immunogenicity, and primary efficacy of the standard of care regimen of elcuritumumab in combination with gemcitabine and oxaliplatin (GemOx) in R/R DLBCL patients who did not meet ASCT conditions due to age, PS, or complications.

Pooling of ongoing clinical trials with elcatuzumab Total (S)

The use of elcurizumab as monotherapy is currently in clinical trials for recurrent/refractory (R/R) B-NHL treatment (ClinicalTrials gov identifier: NCT 03625037). Preliminary data indicate that in R/R B-NHL patients, the drug is tolerated at doses up to at least 48mg (including 60 mg), with no dose limiting toxicity reported.

Target object

Dose escalation

The main objective of the dose escalation section was to evaluate the safety and tolerability of the combination of elcurizumab and GemOx (endpoint: incidence of Dose Limiting Toxicity (DLT), incidence and severity of Adverse Events (AEs), incidence and severity of laboratory value changes, and incidence of dose interruption and delay).

Secondary objectives of the dose escalation portion include characterizing PK properties of the ectrituximab (endpoints: PK parameters, including clearance, distribution volume, AUC0-last, AUC0-x, cmax, tmax, pre-dose values and half-life), evaluating pharmacodynamic markers related to efficacy and mechanism of action of the ectrituximab (endpoints: pharmacodynamic markers in blood samples and within tumors), evaluating immunogenicity (endpoints: incidence of anti-drug antibodies (ADA) against the ectrituximab), and evaluating primary anti-tumor activity of the ectrituximab in combination with GemOx (endpoints: overall Response Rate (ORR) according to the luxano standard and LYRIC, duration of response (DOR) according to the luxano standard and LYRIC, time to pre-response (TTR) according to the luxano standard and LYRIC, progression Free Survival (PFS) according to the luxano standard and LYRIC, overall Survival (OS), time to next anti-lymphoma therapy (TTNT), and rate and duration of negative for minimal residual disease (minimal residual disease, MRD).

Exploratory targets for the dose escalation portion included evaluation of potential biomarkers that predicted clinical responses to elcurizumab (endpoints: CD3, CD20 and other molecular/phenotypic markers, DNA mutation status and gene profile before and during treatment).

Expansion of

The main purpose of the extension was to evaluate the primary anti-tumor activity of elcuritumumab in combination with GemOx (endpoint: ORR according to Lugano standard).

Secondary objectives of the extension include evaluating primary anti-tumor activity of the elctritumumab in combination with GemOx (endpoint: DOR according to Lugano standard and LYRIC, TTR according to Lugano standard and LYRIC, PFS according to Lugano standard and LYRIC, ORR, OS, TTNT according to LYRIC and ratio and duration of Minor Residual Disease (MRD) negatives), further evaluating safety and tolerability of the elctritumumab in combination with GemOx (endpoint: incidence and severity of laboratory value changes, and incidence of dose interruption and delay), characterizing PK properties of the elctritumumab (PK parameters including clearance, distribution volume, AUC0-last, AUC0-x, cmax, tmax, pre-dosing values and half-life), evaluating pharmacodynamic markers related to efficacy and mechanism of the elctritumumab (endpoint: in blood samples and intratumoral pharmacodynamic markers), and evaluating immunogenicity (endpoint: incidence of adam against the elctritumumab).

Exploratory targets for the extension include evaluating potential biomarkers that predict clinical response to elcatuzumab (endpoints: expression of CD20 in tumors, assessment of molecular and genetic tumor markers, immune populations, phenotypes and functions in tumors and blood, and DNA mutation status and gene profile), and evaluating Patient Report Outcome (PRO) (endpoints: changes in lymphoma symptoms and general health status assessed by FACT-Lym).

Overview of study design

The test was performed in two parts: dose escalation (part 1) and expansion (part 2). The subject is engaged in only one part. A schematic of the overall experimental design is shown in fig. 1. Both parts consist of a screening period, a treatment period, a safety follow-up period and a survival follow-up period.

Dose escalation (first portion) and expansion (second portion)

Part 1 dose escalation evaluation of initial safety, tolerability and clinical activity of ectricity in combination with GemOx. The elcuritumumab was initially administered in combination with GemOx in a cohort of 3 subjects. DLT was evaluated during the first 28 days. Based on the number of DLTs observed in the first 3 subjects, the other 3 subjects were administered with elcuritumumab (full dose: 48mg or 24 mg) in combination with GemOx as shown in fig. 2.

In part 2, elcuritumumab was administered in combination with GemOx (dosing regimen determined with the dose escalation part). In addition to safety, tolerability, PK, pharmacodynamics and immunogenicity data, the extension will include 20 subjects in order to evaluate the primary clinical activity of the combination.

In both the first and second fractions, elcatuzumab was administered as Subcutaneous (SC) injections (24 mg or 48mg; increasing dose) in combination with GemOx as follows until disease progression or unacceptable toxicity:

table 2: dosing regimen

QW: once weekly (days 1, 8, 15 and 22), Q2W: once every 2 weeks (days 1 and 8), Q4W: once every 4 weeks (day 1).

Ascending dosing was used for the ectrituximab to mitigate the likelihood of CRS: the initiator dose on day 1 of cycle 1 (0.16 mg), followed by the intermediate dose on day 8 of cycle 1 (0.8 mg), the full dose on days 15 and 22 of cycle 1 (24 mg or 48 mg), and the full dose in the subsequent cycles (QW for cycles 2-3, Q2W for cycles 4-9, and Q4W for the subsequent cycles). Gemcitabine (1000 mg/m) ² ) Intravenous administration (Q2W) was performed once every two weeks for 1-4 th cycle. Oxaliplatin (100 mg/m) ² ) Intravenous administration (Q2W) was performed once every two weeks for 1-4 th cycle.

The treatment sequence is as follows:

TABLE 3 therapeutic administration sequence

Inclusion criteria

1. The subject must be at least 18 years old

Ecog PS score of 0, 1 or 2

CD20 Positive NHL representative tumor biopsies

4. Measurable disease, defined as ≡1 measurable nodular lesions (> 1.5cm long axis and >1.0cm short axis) or ≡1 measurable extranodal lesions (> 1.0cm long axis) on CT or MRI.

5. Acceptable organ function at screening, defined as:

a.ANC≥1.0×10 ⁹ l (allowing the use of growth factors)

b. Platelet count if bone marrow infiltrates or splenomegaly>75x 10 ⁹ L, or ≡50x10 ⁹ /L

ALT level is less than or equal to 2.5 times ULN

d. Total bilirubin level is less than or equal to 2 xULN

eGFR >50mL/min (according to the Cockcroft-Gault formula)

PT, INR and aPTT are less than or equal to 1.5 xULN unless anticoagulants are received

6. According to the WHO classification of 2016, there was recorded DLBCL (new or histologically transformed by indolent lymphomas, except CLL), comprising:

a.DLBCL，NOS

"double-hit" or "triple-hit" DLBCL (technically classified as HGBCL in WHO 2016 with MYC and BCL2 and/or BCL6 translocation) -other double-hit/triple-hit lymphomas are not eligible

c.3B stage FL

7. Recurrence or refractory to at least one prior therapy

8. Failure of previous autologous Hematopoietic Stem Cell Transplantation (HSCT), or failure to meet autologous HSCT conditions due to age, performance status, complications and/or inadequate response to previous treatments

9. Qualification to accept GemOx.

Exclusion criteria

1. Contraindications to any of the individual drugs in a GemOx treatment regimen

2. History of severe allergic or anaphylactic reaction to CD20 mAb therapy or known allergic or intolerance to any component or excipient of elcatuzumab

3. Previous treatment using bispecific antibodies targeting CD3 and CD20

4. Chemotherapy, radiotherapy or major surgery within 4 weeks prior to the first dose of elcatuzumab

5. Treatment with the test drug within the first 4 weeks or 5 half-lives (whichever is longer) of the first dose of elcatuzumab

6. Treatment with CAR-T therapy within 30 days prior to the first dose of elcatuzumab

7. The cumulative dose of corticosteroid is greater than or equal to 140mg prednisone or equivalent within the first 2 weeks of the first dose of elcatuzumab

8. Vaccination with live vaccine within 28 days prior to the first dose of elcatuzumab

9. Clinically significant heart disease, including:

a. the first dose of elcorerituximab is myocardial infarction over the first 1 year, or an unstable or uncontrolled disease/disorder associated with or affecting cardiac function (e.g., unstable angina, congestive heart failure, new york heart association (New York Heart Association) grade III-IV), arrhythmia (CTCAE 4 th edition 2 or higher), or clinically significant ECG abnormalities

b. Screening 12-lead ECG, showing baseline QTcF >470msec

10. Evidence of significant uncontrolled concomitant diseases that can affect protocol compliance or outcome interpretation

11. Active bacterial, viral, fungal, mycobacterial, parasitic or other infections (excluding fungal nail bed infections) or significant infections within 2 weeks prior to the first dose of elcatuzumab, known at the time of trial into the group

Known CNS involvement of CNS lymphomas or lymphomas at screening, as confirmed by brain MRI/CT scan, and if clinically indicated, by lumbar puncture

13. Activity positive detection of hepatitis B virus or hepatitis C virus indicative of acute or chronic infection

History of HIV antibody positivity, or positive HIV detection at screening

Positive test results for HTLV-1

16. Suspected active or latent tuberculosis

17. Unlike past or current malignancy that is included in a diagnosis, the following are excluded:

cervical cancer at stage a.1B or below

b. Non-invasive basal cell or squamous cell skin carcinoma

c. Non-invasive superficial bladder cancer

d. Prostate cancer with current PSA level <0.1ng/mL

e. CR duration >2 years of any curable cancer

18. Neuropathy > grade 1

19. Women pregnant, lactating or scheduled to become pregnant at the time of inclusion in the group trial or within 12 months after the last dose of elcatuzumab

20. Men who were father were scheduled to go into the group at the time of the trial or within 12 months after the last dose of elcatuzumab

21. A subject with any condition that participates in an assessment that does not meet the subject's maximum benefit (e.g., impairs well-being) or that may prevent, limit or confuse the regimen.

CRS control

Four days of corticosteroid administration were performed to reduce/prevent the severity of symptoms from potential CRS of each dose of elcatuzumab. For the administration of elcatuzumab at and after cycle 2, CRS control with corticosteroids is optional. Corticosteroid administration may be intravenous or oral route, using recommended dosages or equivalents.

TABLE 4 prodrugs and CRS control

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* 30 minutes to 2 hours prior to administration of elcatuzumab

Annotation: if the dose of elcatuzumab is administered more than 24 hours after the start of GemOx, the prodrug is administered before the dose of elcatuzumab and corticosteroid control is continued for 3 days after the administration of elcatuzumab.

Table 5: corticosteroid dose equivalent-conversion table

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Supportive care for cytokine release syndrome

CRS was graded according to ASTCT grading of CRS (tables 6 and 7), and for treatment of CRS, subjects should receive supportive care. Supportive care may include, but is not limited to

Saline infusion

Systemic glucocorticoid, antihistamine and antipyretic (antipyrexia)

Support of blood pressure (vasopressin, vasopressor)

Support for low and high flow oxygen and positive pressure ventilation

IV administration of monoclonal antibodies, e.g., tobulab, to IL-6R

If repeated tobrazumab is not responded, then it is a monoclonal antibody against IL-6, e.g. IV rituximab.

Table 6: grading and management of cytokine release syndrome

According to the American Society of Transplantation and Cell Therapy (ASTCT), previously the american society of blood and bone marrow transplantation (ASBMT), the unified definition and grading criteria for CRS appear below.

Fractionation of cytokine release syndrome

Abbreviations: biPAP, bi-level positive airway pressure (bilevel positive airway pressure); CPAP, continuous positive airway pressure (continuous positive airway pressure); CRS, cytokine release syndrome; IV, intravenous.

Annotation: organ toxicity or constitutional symptoms associated with CRS may be ranked according to CTCAE, but they do not affect CRS ranking.

1. Fever is defined as the temperature not attributable to any other cause ∈38.0 ℃, with or without constitutional symptoms (e.g. myalgia, arthralgia, debilitation). In CRS subjects receiving antipyretics, anti-cytokine therapy and/or corticosteroids, fever is no longer required to rank the subsequent CRS severity. In this case, CRS classification is driven by hypotension and/or hypoxia.

Crs ranking is determined by more severe events: not due to hypotension or hypoxia of any other cause. For example, subjects with a temperature of 39.5 ℃, hypotension requiring 1 vascular pressurizer, and hypoxia requiring a low flow nasal cannula were classified as class 3 CRS. Both systolic and mean arterial pressure are acceptable for blood pressure measurement. Without specific limitation, hypotension should be determined on a case-by-case basis, i.e., a blood pressure lower than the normal value expected for an individual in a given environment, taking into account the age and individual baseline of the subject.

3. Intubation of subjects without hypoxia is not by definition grade 4 CRS for possible neurological damage or procedure of an patent airway alone.

The source is as follows: adapted from Lee et al Biol Blood Marrow Transplant 2019;25:625-638

Table 7: grading and management of cytokine release syndrome

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Tumor lysis syndrome prevention and management

For prophylactic treatment of oncolytic syndrome, subjects received hydration and uric acid lowering agents prior to administration of elcatuzumab. If signs of Tumor Lysis Syndrome (TLS) occur, supportive therapy including a labyrine enzyme is used.

Dose modification guidance and safety management

The elcatuzumab will not be dose-modified (see fig. 2 for exceptions in the dose expansion cohort), although it may be paused or suspended depending on any toxicity (and toxicity level) that the subject develops during their use.

Dose reduction for oxaliplatin against neuropathy (worsening compared to baseline):

for paresthesias lasting 1 to 6 days after each administration, the dose is not reduced.

In the case of significant paresthesia lasting 7 to 13 days after each administration (increase in severity from baseline), the dose was reduced to 75mg/m ² . If abnormal results were obtained by neurological examination or if the subject experienced significant paresthesia for 14 days or more, oxaliplatin should be stopped until symptoms improved, then at 75mg/m ² Is restarted. If throat dysesthesia occurs, the duration of oxaliplatin infusion should be extended from 2 hours to 6 hours.

Study evaluation

Demographics and demographicsBaseline assessment

Demographic details of the subject are collected, such as information such as lymphoma diagnosis date, ann Arbor stage at diagnosis (including constitutional symptoms (B symptoms)), and prior evidence of CD20 positivity. Information about previous and concomitant medications, concomitant protocols, and previous cancer therapies and surgery (including previous anti-cancer therapies for NHL such as surgery, radiation therapy, chemo-radiation therapy, and systemic treatment regimens) is also collected.

Efficacy assessment

A eligible subject has at least 1 measurable disease site (as indicated by inclusion criteria) for disease assessment. The measurable site of lymphoma is defined as a lymph node, lymph node tumor, or extranodal site. The measurements were determined by imaging evaluation, tracking up to 6 measurable sites as target lesions for each subject. An unmeasurable site as defined above is considered to be assessed by objective evidence of disease (i.e. radiological imaging, physical examination or other procedure). Examples of evaluable diseases include, for example, bone marrow involvement, bone lesions, effusion (fusion), or intestinal wall thickening.

Tumor and bone marrow biopsy

For all subjects with accessible tumors, two fresh core tumor biopsies were collected prior to treatment with elcuritumumab (during the screening period) and 2 fresh core tumor biopsies were collected at the beginning of cycle 2, day 15 (±1 week). If no fresh biopsies could be collected at screening, an archived tumor biopsy at the time of collection within 3 months prior to group entry is acceptable. The biopsy may be a whole lymph node or core biopsy. The tumor biopsy should be FFPE. For MRD evaluation and exploratory biomarkers, tumor biopsies were examined.

Radiography evaluation

FDG PET-CT scan (or CT/MRI and FDG PET when PET-CT scan is not available) is performed during the screening. For subjects with FDGavid tumors at the time of screening, all subsequent disease assessments included the use of the 5-score scale FDGPET described by Barrington et al (J Clin Oncol2014;32:3048-58; 1; no uptake; 2; uptake. Ltoreq. Mediastinum; 3; renewal > mediastinum but. Ltoreq. Liver; 4; uptake moderately higher than liver; 5; uptake significantly higher than liver and/or new lesions; X; newer new regions are unlikely to be associated with lymphomas). For subjects with non-avid or variable FDG-avid tumors, CT scans with IV contrast can be performed on neck/chest/abdomen/pelvis/other known lesions. If the CT assembly has similar diagnostic qualities as a contrast enhanced CT performed without PET, the CT assembly of PET-CT may be used in place of a stand-alone CT/MRI. If contrast enhanced PETCT is not available, then a stand alone diagnostic CT/MRI and standard FDGPET are performed. Subjects intolerant of IV CT contrast agents undergo CT scans of oral contrast agents.

MRI can be used to evaluate disease sites that cannot be adequately imaged using CT or for subjects that are intolerant to CT contrast agents. If MRI is the imaging modality of choice, MRI is obtained at the time of screening and at the time of all subsequent response evaluations.

Bone marrow assessment

Bone marrow biopsies (archived or fresh) were obtained for all patients at screening, with or without aspirate, to record bone marrow involvement of lymphomas. Bone marrow biopsies obtained as conventional SOC can be used if collected up to 42 days before the first dose of elcatuzumab. If bone marrow aspirate is obtained, an assay of bone marrow involvement may be determined by flow cytometry. Bone marrow biopsy was collected as follows: (1) screening; (2) For subjects with bone marrow involvement at screening but later CR achieved by imaging-bone marrow assessment including morphological examination and flow cytometry or IHC (if necessary) to confirm the presence or absence of lymphoma (complete remission); (3) For subjects with bone marrow involvement recorded at screening and later CR achieved by imaging-a fraction of aspirate collected to confirm CR will be used for MRD assessment.

Minimal residual disease assessment

MRD is assessed by following the presence of DNA encoding B Cell Receptors (BCR) specifically expressed by cancer cells. The DNA sequence of the BCR was identified by tumor biopsy submitted at the time of screening. After initiation of treatment, blood samples were taken at fixed time points and at CR to assess whether the amount of cancer DNA was decreasing, as a potential measure of (early) response, and to assess MRD. As an exploratory analysis, when the subject reached metabolic/radiological CR and had bone marrow involvement recorded at the time of screening, a fraction of aspirate collected to confirm CR was used for MRD assessment.

Disease response and progressive disease assessment

Disease response was assessed according to both the Lugano standard (described in Cheson et al, J Clin Oncol 2014;32:3059-68 (see Table 3 in Cheson et al, 2014, in particular)) and LYRIC (Table 8) to inform the decision to continue treatment.

The endpoint is defined as follows:

overall Response Rate (ORR) is defined as the proportion of subjects who reached a response to PR or CR before starting subsequent therapy.

Time To Response (TTR) is defined between responders as the time between the first dose of elcatuzumab (from cycle 1, day 1) to the initial recording of PR or CR.

Response Duration (DOR) is defined between responders as the time from the initial recording of PR or CR to the date of disease progression or death (whichever occurs earlier).

Progression Free Survival (PFS) is defined as the time from the date of first administration of elcatuzumab (cycle 1, day 1) to the date of disease progression or death (whichever occurs earlier).

Overall Survival (OS) is defined as the time from the date of first administration of elcatuzumab (cycle 1, day 1) to the date of death.

The time to next anti-lymphoma therapy (TTNT) is defined as the number of days from day 1 of cycle 1 to the first recorded administration of the subsequent anti-lymphoma therapy.

MRD negative rate is defined as the proportion of subjects with at least 1 undetectable MRD result according to a particular threshold before starting subsequent therapy.

Lugano standard(see, e.g., cheson et al, J Clin Oncol 2014;32:3059-68 for complete response, partDefinition of partial response, no response/disease stability and progressive disease).

(a) Target lesions and non-target lesions

The target lesions of the Lugano standard include up to 6 of the largest apparent nodules (domino nodes), nodular masses, or other lymphoma lesions measurable in two diameters, and preferably are from different body areas representing the overall disease burden of the subject, including mediastinal and retroperitoneal lesions, where applicable. At baseline, the longest diameter of the nodule (LDI) >15mm may be measured. Measurable extranodal disease can be included in six representative target lesions. At baseline, the LDi of the measurable extranodal lesions should be greater than 10mm.

All other lesions (including nodules, extranodal, and evaluable diseases) can be tracked as non-target lesions (e.g., skin, GI, bone, spleen, liver, kidney, pleural or pericardial effusions, ascites, bone marrow).

(b) Split lesions (split lesions) and fusion lesions (confuent lesions)

Over time, the lesions may divide or may become fused. In the case of a split lesion, the individual perpendicular diameter products (PPDs) of the nodules should be added together to represent the PPD of the split lesion; this PPD was added to the sum of PPDs of the remaining lesions to measure the response. If subsequent growth of any or all of these discrete nodules occurs, the nadir of each individual nodule is used to determine progression. In the case of a fusogenic lesion, the sum of the PPD of the fusogenic mass and the PPD of the individual nodules should be compared, and the PPD of the fusogenic mass must be increased by more than 50% compared to the sum of the individual nodules to indicate Progressive Disease (PD). LDi and minimum diameter (SDi) are no longer required to determine progress.

LYRIC

Clinical studies have shown that cancer immunotherapy may lead to early apparent radiological imaging advances (including the appearance of new lesions), followed by delayed responses. Since this initial increase in tumor size may be caused by immune cell infiltration in the context of T cell responses, this progression may not be indicative of actual disease progression and is therefore referred to as "pseudo-progression" (Wolchok et al Clin Cancer Res2009; 15:7412-20).

The current Lugano response assessment standard (Cheson et al, J Clin Oncol 2014; 32:3059-68) does not take into account spurious progress and there is a significant risk of premature deactivation of potentially effective immunomodulatory drugs after an atypical response is observed. Atypical responses are characterized by early progression of an existing lesion followed by progression of either the response or a new lesion with or without tumor shrinkage elsewhere.

LYRIC is a modification of the Lugano response assessment criteria that has been applied to immunization-based therapies and which carries out a new class of remission response: "uncertain response" (IR) name (Cheson et al Blood2016; 128:2489-96). This IR name was introduced to potentially identify "atypical response" cases until confirmed by biopsy or subsequent imaging as scintillation (flare)/false progression or true PD.

Subjects exhibiting PD according to the Lugano standard/classification will be considered to have IR in one or more of the following 3 cases:

IR (1): the overall tumor burden of up to 6 target lesions increased (estimated by the sum of the diameter products [ SPD ]) by 50% over the first 12 weeks of therapy without clinical deterioration.

IR (2): the appearance of new lesions or the growth of one or more existing lesions at any time during the treatment is greater than or equal to 50%; in the absence of overall progression of overall tumor burden (SPD <50% increase), as measured by SPD of up to 6 lesions at any time during treatment.

IR (3): FDG uptake of 1 or more lesions increased without concomitant increase in lesion size or number.

It is possible that at a single point in time, the subject may meet the criteria for IR (1) or both IR (2) and IR (3): for example, a new FDG-avid lesion (IR 2) may exist without global progression, while FDG uptake of individual lesions increases (IR 3). In this case, IR (1) or IR (2) (e.g., IR [2] in the above embodiment) should be specified preferentially.

LYRIC

Clinical studies have shown that cancer immunotherapy may lead to early apparent imaging progression (including the appearance of new lesions) followed by delayed responses. Since this initial increase in tumor size may be caused by immune cell infiltration in the context of a T cell response, this progression may not be indicative of actual disease progression and is therefore referred to as "pseudo-progression" (Wolchok et al Clin Cancer Res 2009; 15:7412-20).

IR (2): the appearance of new lesions or the growth of one or more existing lesions at any time during the treatment is greater than or equal to 50%; occurs without overall progression of overall tumor burden (SPD <50% increase), as measured by SPD for up to 6 lesions at any time during treatment.

It is possible that at one point in time, the subject may meet the criteria for IR (1) or both IR (2) and IR (3): for example, there may be new FDG-avid lesions (IR 2) without global progression, while FDG uptake of individual lesions increases (IR 3). In this case, IR (1) or IR (2) (e.g., IR [2] in the above example) should be specified preferentially.

After a further 12 weeks (or earlier if there is a clinical indication), subjects classified as having any of the IR types received repeated imaging. At that point, the response should be re-evaluated and the subject considered to have true PD taking into account the following factors:

follow-up IR (1):in the case of IR (1), a comparison should be made between the first IR (1) and the current SPD. If (a) SPD increases by ≡10% from the first IR1 and (b) for lesions of ≡2cm ≡1 lesions increases by ≡5mm (any dimension), and for lesions of No. 1>2cm lesions, increased by ≡10mm to conform to the Lugano standard, IR (1) will become PD.

Follow-up IR (2):in the case of IR (2), new or growing lesions are added to the target lesions until no more than 6 total lesions total. If (a) SPD (the newly defined target lesion group) increases by 50% or more from the lowest value, IR (2) will become PD.

Follow-up IR (3):if lesions with increased FDG uptake also show an increase in size, IR (3) will become PD.

TABLE 8 LYRIC

Clinical safety assessment

Safety was assessed by measuring adverse events, laboratory test results, ECG, vital sign measurements, physical examination results, and ECOG performance status. Immune effector cell-related neurotoxicity syndromes (e.g., as described in Lee et al, biol Blood Marrow Transplant 2019; 25:625-638), systemic symptoms (B symptoms), tumor scintillation responses, and survival were also assessed.

Patient reporting outcome

Patient report outcomes were assessed using the FACT-Lym health-related quality of life (QOL) questionnaire, which assessed the QOL of lymphoma patients.

Further analysis

As outlined above, patients have been treated with a maximum dose of 24mg or 48mg of elcatuzumab and GemOx, and some patients show signs of response to combination therapy.

Preliminary results

By day 8 of 9 of 2021, a total of 26 patients had been dosed. An expansion phase of 48mg was opened at day 5 and 9 of 2021. 5 responders were observed in the increment phase and 9 were observed in the extension phase. The most common related AEs were CRS, thrombocytopenia, fatigue and anemia. All CRSs are of class 1/2. An event of class 3 immune effector cell-associated neurotoxic syndrome (ICANS) was reported in which patients had recovered.

Example 2: in vitro antitumor Activity of Ecuritumumab in combination with gemcitabine/oxaliplatin

Combinations of gemcitabine (an antimetabolite) and oxaliplatin (a platinum-based alkylating agent) are used to treat elderly and/or non-graft-eligible B-NHL patients (Sarkozy et al Annals of Lymphoma2019; 3). To determine whether this chemotherapy regimen affected the anti-tumor activity of ectropin, in vitro T cell activation and cytotoxicity assays were performed.

Briefly, human Burkitt lymphoma (Raji) and DLBCL (SU-DHL-4) cell lines were used as target cells. At 37℃with 5% CO ₂ In culture medium (RPMI 1640, HEPES and L-glutamine supplemented with 10% heat inactivated donor bovine serum and iron, and 1% [ v/v ]]Penicillin/streptomycin), to which 2mM L-glutamine and 1mM sodium pyruvate were added. T cells are used as effector cells by using Rosetteep ^TM Human T cell enriched mixture (Stemcell Technologies) was negative selected and then density centrifuged on Ficoll gradient (all according to manufacturer's instructions) from human healthThe donor buffy coat (Sanquin) was isolated. Isolated cells were washed twice in Phosphate Buffered Saline (PBS) and stained with acridine orange/propidium iodide (AO/PI) viability staining solution (Nexcelom Bioscience) inThe Auto 2000 cell viability counter was counted. T cells (100,000 cells/well) were incubated with Raji or SU-DHL-4 cells (50,000 cells/well) in the presence of elcatuzumab (0.01 pg/mL-100 ng/mL), gemcitabine (1 nM) and oxaliplatin (0.3. Mu.M) in medium at 37℃with 5% CO ₂ Incubate for 48 hours. B cell viability and T cell activation were measured by flow cytometry (CD 22 positive cell count; T cell activation markers CD69, CD25, programmed cell death protein 1 (PD-1) and CD107 on CD4+ and CD8+ T cells [ lysosomal associated membrane protein 1; LAMP-1 ] ]Expression of (c). The antibodies used are shown in table 9. Absolute cell numbers were determined by adding Accucheck Counting beads (100 μl/well) (thermosusher) and calculated as follows:

correction factor= ([ bead count) ₁ +bead count ₂ ]Average bead count _{Board board} )

Absolute cell number= [ cell count ] _{Sample of} Correction factor]

The percent cytotoxicity was calculated as follows:

% cytotoxicity = 100- ([ absolute cell number) _{Sample of} Absolute cell number _{Antibody-free} ]×100％)

As shown in FIG. 3A-3D, elcuritumumab induced concentration-dependent activation of CD4+ (data not shown) and CD8+ T cells, as shown by up-regulation of CD69, CD25, PD-1 and LAMP-1 expression (left panel: raji; right panel: SU-DHL-4). In the presence of all concentrations of gemcitabine, oxaliplatin, or a combination tested, the T cell activation induced by elcuritumumab was not affected. The elcuritumumab also induced concentration-dependent T cell mediated cytotoxicity of Raji and SU-DHL-4 cells as measured by a decrease in the number of surviving B cells (fig. 4, left panel: raji; right panel: SU-DHL-4). With gemcitabine or oxaliplatin alone (to target on each cell lineEC determined for each drug ₃₀ Concentration) together with incubation induced cytotoxicity of B cells (open symbols in fig. 4). The combination of gemcitabine and oxaliplatin induces more cytotoxicity than a single agent. Addition of gemcitabine and oxaliplatin to the elcatuzumab enhances the cytotoxicity of B cells compared to elcatuzumab alone, or gemcitabine and oxaliplatin induced cytotoxicity. Taken together, these in vitro data demonstrate that the combination of elcatuzumab with gemcitabine and oxaliplatin can result in improved cytotoxicity of B cells compared to elcatuzumab or gemcitabine/oxaliplatin alone. The combination of elcoreactant and GemOx is being evaluated in a clinical trial (NCT 04663347). The first patient group had been admitted to the group and treated.

Table 9: antibodies for flow cytometry

¹ APC: allophycocyanin; PE: phycoerythrin; cy: cyanine dyes (cyanine dye); FITC: fluorescein isothiocyanate; BV: bright purple (brilliant violet)

Table 10: summary of sequences

/>

Bold and underline are for positions 234 and 235, respectively; 265; FE of 405 and 409; a, A is as follows; l and R, said positions being numbered according to EU. In the variable region, the CDR regions annotated according to the IMGT definition are underlined.

Sequence listing

<110> Jianma protection

<120> bispecific antibodies against CD3 and CD20 in combination therapy for the treatment of diffuse large B-cell lymphoma

<130> P/0162-WO-PCT

<160> 29

<170> PatentIn version 3.5

<210> 1

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> CDR sequence

<400> 1

Gly Phe Thr Phe Asn Thr Tyr Ala

1 5

<210> 2

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> CDR sequence

<400> 2

Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr

1 5 10

<210> 3

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> CDR sequence

<400> 3

Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr

1 5 10 15

<210> 4

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> CDR sequence

<400> 4

Thr Gly Ala Val Thr Thr Ser Asn Tyr

1 5

<210> 5

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> CDR sequence

<400> 5

Ala Leu Trp Tyr Ser Asn Leu Trp Val

1 5

<210> 6

<211> 125

<212> PRT

<213> artificial sequence

<220>

<223> heavy chain variable region sequence l

<400> 6

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 7

<211> 109

<212> PRT

<213> artificial sequence

<220>

<223> light chain variable region sequence

<400> 7

Gln Ala Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Gln Thr Pro Gly Gln Ala Phe Arg Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Ala Asp Asp Glu Ser Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 8

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> CDR sequence

<400> 8

Gly Phe Thr Phe His Asp Tyr Ala

1 5

<210> 9

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> CDR sequence

<400> 9

Ile Ser Trp Asn Ser Gly Thr Ile

1 5

<210> 10

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> CDR sequence

<400> 10

Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Val

1 5 10 15

<210> 11

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> CDR sequence

<400> 11

Gln Ser Val Ser Ser Tyr

1 5

<210> 12

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> CDR sequence

<400> 12

Gln Gln Arg Ser Asn Trp Pro Ile Thr

1 5

<210> 13

<211> 122

<212> PRT

<213> artificial sequence

<220>

<223> heavy chain variable region sequence

<400> 13

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Asp Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe His Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Trp Asn Ser Gly Thr Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 14

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> light chain variable region sequence

<400> 14

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 15

<211> 329

<212> PRT

<213> Chile person

<400> 15

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210> 16

<211> 329

<212> PRT

<213> artificial sequence

<220>

<223> heavy chain constant region

<400> 16

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210> 17

<211> 329

<212> PRT

<213> artificial sequence

<220>

<223> heavy chain constant region

<400> 17

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Leu

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210> 18

<211> 329

<212> PRT

<213> artificial sequence

<220>

<223> heavy chain constant region

<400> 18

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210> 19

<211> 329

<212> PRT

<213> artificial sequence

<220>

<223> heavy chain constant region

<400> 19

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Leu

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210> 20

<211> 329

<212> PRT

<213> artificial sequence

<220>

<223> heavy chain constant region

<400> 20

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210> 21

<211> 106

<212> PRT

<213> Chile person

<400> 21

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

1 5 10 15

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

35 40 45

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

65 70 75 80

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

100 105

<210> 22

<211> 106

<212> PRT

<213> Chile person

<400> 22

Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser

1 5 10 15

Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp

20 25 30

Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro

35 40 45

Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn

50 55 60

Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys

65 70 75 80

Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val

85 90 95

Glu Lys Thr Val Ala Pro Thr Glu Cys Ser

100 105

<210> 23

<211> 107

<212> PRT

<213> Chile person

<400> 23

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 24

<211> 454

<212> PRT

<213> artificial sequence

<220>

<223> antibody heavy chain

<400> 24

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

325 330 335

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys

355 360 365

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Leu Leu Tyr Ser

405 410 415

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Pro Gly

450

<210> 25

<211> 215

<212> PRT

<213> artificial sequence

<220>

<223> antibody light chain

<400> 25

Gln Ala Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Gln Thr Pro Gly Gln Ala Phe Arg Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Ala Asp Asp Glu Ser Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro

100 105 110

Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu

115 120 125

Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro

130 135 140

Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala

145 150 155 160

Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala

165 170 175

Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg

180 185 190

Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr

195 200 205

Val Ala Pro Thr Glu Cys Ser

210 215

<210> 26

<211> 451

<212> PRT

<213> artificial sequence

<220>

<223> antibody heavy chain

<400> 26

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Asp Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe His Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Trp Asn Ser Gly Thr Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ala Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly

450

<210> 27

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> antibody light chain

<400> 27

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 28

<211> 207

<212> PRT

<213> Chile person

<400> 28

Met Gln Ser Gly Thr His Trp Arg Val Leu Gly Leu Cys Leu Leu Ser

1 5 10 15

Val Gly Val Trp Gly Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr

20 25 30

Gln Thr Pro Tyr Lys Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr

35 40 45

Cys Pro Gln Tyr Pro Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys

50 55 60

Asn Ile Gly Gly Asp Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp

65 70 75 80

His Leu Ser Leu Lys Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr

85 90 95

Val Cys Tyr Pro Arg Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu

100 105 110

Tyr Leu Arg Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp Val Met

115 120 125

Ser Val Ala Thr Ile Val Ile Val Asp Ile Cys Ile Thr Gly Gly Leu

130 135 140

Leu Leu Leu Val Tyr Tyr Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys

145 150 155 160

Pro Val Thr Arg Gly Ala Gly Ala Gly Gly Arg Gln Arg Gly Gln Asn

165 170 175

Lys Glu Arg Pro Pro Pro Val Pro Asn Pro Asp Tyr Glu Pro Ile Arg

180 185 190

Lys Gly Gln Arg Asp Leu Tyr Ser Gly Leu Asn Gln Arg Arg Ile

195 200 205

<210> 29

<211> 297

<212> PRT

<213> Chile person

<400> 29

Met Thr Thr Pro Arg Asn Ser Val Asn Gly Thr Phe Pro Ala Glu Pro

1 5 10 15

Met Lys Gly Pro Ile Ala Met Gln Ser Gly Pro Lys Pro Leu Phe Arg

20 25 30

Arg Met Ser Ser Leu Val Gly Pro Thr Gln Ser Phe Phe Met Arg Glu

35 40 45

Ser Lys Thr Leu Gly Ala Val Gln Ile Met Asn Gly Leu Phe His Ile

50 55 60

Ala Leu Gly Gly Leu Leu Met Ile Pro Ala Gly Ile Tyr Ala Pro Ile

65 70 75 80

Cys Val Thr Val Trp Tyr Pro Leu Trp Gly Gly Ile Met Tyr Ile Ile

85 90 95

Ser Gly Ser Leu Leu Ala Ala Thr Glu Lys Asn Ser Arg Lys Cys Leu

100 105 110

Val Lys Gly Lys Met Ile Met Asn Ser Leu Ser Leu Phe Ala Ala Ile

115 120 125

Ser Gly Met Ile Leu Ser Ile Met Asp Ile Leu Asn Ile Lys Ile Ser

130 135 140

His Phe Leu Lys Met Glu Ser Leu Asn Phe Ile Arg Ala His Thr Pro

145 150 155 160

Tyr Ile Asn Ile Tyr Asn Cys Glu Pro Ala Asn Pro Ser Glu Lys Asn

165 170 175

Ser Pro Ser Thr Gln Tyr Cys Tyr Ser Ile Gln Ser Leu Phe Leu Gly

180 185 190

Ile Leu Ser Val Met Leu Ile Phe Ala Phe Phe Gln Glu Leu Val Ile

195 200 205

Ala Gly Ile Val Glu Asn Glu Trp Lys Arg Thr Cys Ser Arg Pro Lys

210 215 220

Ser Asn Ile Val Leu Leu Ser Ala Glu Glu Lys Lys Glu Gln Thr Ile

225 230 235 240

Glu Ile Lys Glu Glu Val Val Gly Leu Thr Glu Thr Ser Ser Gln Pro

245 250 255

Lys Asn Glu Glu Asp Ile Glu Ile Ile Pro Ile Gln Glu Glu Glu Glu

260 265 270

Glu Glu Thr Glu Thr Asn Phe Pro Glu Pro Pro Gln Asp Gln Glu Ser

275 280 285

Ser Pro Ile Glu Asn Asp Ser Ser Pro

290 295

Claims

1. A method of treating diffuse large B-cell lymphoma (DLBCL) in a human subject, the method comprising administering to the subject a bispecific antibody and an effective amount of gemcitabine and oxaliplatin, wherein the bispecific antibody comprises:

(i) A first binding arm comprising a first antigen binding region that binds to human CD3 epsilon (epsilon) and comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences in the VH region sequence of SEQ ID NO:6, and the VL region comprises CDR1, CDR2, and CDR3 sequences in the VL region sequence of SEQ ID NO: 7; and

(ii) A second binding arm comprising a second antigen binding region that binds to human CD20 and comprises a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences in the VH region sequence of SEQ ID No. 13, and the VL region comprises CDR1, CDR2, and CDR3 sequences in the VL region sequence of SEQ ID No. 14;

2. The method of claim 1, wherein the bispecific antibody is administered at a dose of 24 mg.

3. The method of claim 1, wherein the bispecific antibody is administered at a dose of 48 mg.

4. The method of any one of claims 1-3, wherein the bispecific antibody is administered once weekly (weekly administration).

5. The method of claim 4, wherein 24mg or 48mg of said weekly administration is for 2.5 28 day cycles.

6. The method of claim 4 or 5, wherein the bispecific antibody is administered once every two weeks (biweekly administration) after the weekly administration.

7. The method of claim 6, wherein the two week administration is performed for six 28 day periods.

8. The method of claim 6 or 7, wherein the bispecific antibody is administered once every four weeks after the two-week administration.

9. The method of claim 8, wherein said administering is performed once every four weeks for at least two 28 day periods.

10. The method of any one of claims 4-9, wherein a priming dose of the bispecific antibody is administered in cycle 1 of the 28 day cycle prior to 24mg or 48mg of the weekly administration.

11. The method of claim 10, wherein the trigger amount is administered two weeks before the first weekly dose of 24mg or 48 mg.

12. The method of claim 10 or 11, wherein the initiator amount is 0.16mg.

13. The method of any one of claims 10-12, wherein an intermediate dose of the bispecific antibody is administered after administration of the priming dose and before administration of the first weekly dose of 24mg or 48 mg.

14. The method of claim 13, wherein the initiator dose is administered on day 1 of cycle 1 and the intermediate dose is administered on day 8, followed by a first weekly dose of 24mg or 48mg on days 15 and 22.

15. The method of claim 13 or 14, wherein the intermediate dose is 0.8mg.

16. The method of any one of claims 1-15, wherein gemcitabine is administered once every two weeks.

17. The method of claim 16, wherein the administration of gemcitabine once every two weeks is for four 28 day cycles.

18. The method of any one of claims 1-17, wherein gemcitabine is at 1000mg/m ² Is administered at a dose or equivalent thereof.

19. The method of any one of claims 1-18, wherein oxaliplatin is administered once every two weeks.

20. The method of any one of claims 1-19, wherein the administration of oxaliplatin once every two weeks is performed for four 28 day cycles.

21. The method of any one of claims 1-20, wherein oxaliplatin is at 100mg/m ² Is administered at a dose of (a).

22. The method of any one of claims 1-21, wherein gemcitabine, oxaliplatin, and the bispecific antibody are administered on the same day (e.g., on days 1 and 15 of cycles 1-4).

23. The method of any one of claims 1-22, wherein the dosing schedule of gemcitabine, oxaliplatin, and the bispecific antibody is as shown in table 2.

24. The method of any one of claims 1, 2, and 4-23, wherein the administering is performed with a 28 day period, and wherein:

(a) The bispecific antibody is administered as follows:

(iii) In cycles 4-9, a dose of 24mg was administered on days 1 and 15; and

(b) Gemcitabine is administered on days 1 and 15 of cycles 1-4; and

(c) Oxaliplatin is administered on days 1 and 15 of cycles 1-4.

25. The method of any one of claims 1 and 3-23, wherein administration is performed over a 28 day period, and wherein:

(a) The bispecific antibody is administered as follows:

(iii) In cycles 4-9, a dose of 48mg was administered on days 1 and 15; and

(b) Gemcitabine is administered on days 1 and 15 of cycles 1-4; and

(c) Oxaliplatin is administered on days 1 and 15 of cycles 1-4.

26. The method of any one of claims 1-25, wherein the bispecific antibody is administered subcutaneously.

27. The method of any one of claims 1-26, wherein gemcitabine is administered intravenously.

28. The method of any one of claims 1-27, wherein oxaliplatin is administered intravenously.

29. The method of any one of claims 1-28, wherein the bispecific antibody, gemcitabine, and oxaliplatin are administered sequentially.

30. The method of any one of claims 1-29, wherein when gemcitabine, oxaliplatin, and the bispecific antibody are administered on the same day, gemcitabine is administered first, oxaliplatin is administered second, and the bispecific antibody is administered last.

31. The method of any one of claims 1-30, wherein the DLBCL is double-hit or triple-hit DLBCL.

32. The method of any one of claims 1-31, wherein the DLBCL is grade 3B follicular lymphoma.

33. The method of any one of claims 1-32, wherein the subject has relapsed after at least one prior therapy.

34. The method of any one of claims 1-33, wherein the subject is refractory to at least one prior therapy.

35. The method of any one of claims 1-34, wherein the subject has failed a prior autologous hematopoietic stem cell transplant.

36. The method of any one of claims 1-35, wherein the subject is not eligible for autologous hematopoietic stem cell transplantation due to age, performance status, complications, and/or insufficient response to prior treatments.

37. The method of any one of claims 1-36, wherein:

38. The method of any one of claims 1-37, wherein:

39. The method of any one of claims 1-38, wherein the first binding arm of the bispecific antibody is derived from a humanized antibody, preferably from a full length IgG1, lambda (lambda) antibody.

40. The method of claim 39, wherein the first binding arm of the bispecific antibody comprises a lambda light chain constant region comprising the amino acid sequence as set forth in SEQ ID NO. 22.

41. The method of any one of claims 1-40, wherein the second binding arm of the bispecific antibody is derived from a human antibody, preferably from a full length IgG1, kappa (kappa) antibody.

42. The method of claim 41, wherein the second binding arm comprises a kappa light chain constant region comprising the amino acid sequence set forth in SEQ ID NO. 23.

43. The method of any one of claims 1-42, wherein the bispecific antibody is a full length antibody having a human IgG1 constant region.

44. The method of any one of claims 1-43, wherein the bispecific antibody comprises an inert Fc region.

45. The method of any one of claims 1-44, wherein the bispecific antibody comprises a first heavy chain and a second heavy chain, wherein in both the first heavy chain and the second heavy chain the amino acids in positions L234, L235 and D265 in the human IgG1 heavy chain constant region corresponding to SEQ ID No. 15 are F, E and a, respectively.

46. The method of any one of claims 1-46, wherein the bispecific antibody comprises a first heavy chain and a second heavy chain, wherein in the first heavy chain the amino acid in position F405 in the human IgG1 heavy chain constant region corresponding to SEQ ID No. 15 is L, and wherein in the second heavy chain the amino acid in position K409 in the human IgG1 heavy chain constant region corresponding to SEQ ID No. 15 is R, or vice versa.

47. The method of any one of claims 1-46, wherein the bispecific antibody comprises a first heavy chain and a second heavy chain, wherein

48. The method of claim 47, wherein the bispecific antibody comprises a heavy chain constant region comprising the amino acid sequences of SEQ ID NOs 19 and 20.

49. The method of any one of claims 1-48, wherein the bispecific antibody comprises a heavy chain and a light chain comprising the amino acid sequences set forth in SEQ ID nos. 24 and 25, respectively, and a heavy chain and a light chain comprising the amino acid sequences set forth in SEQ ID nos. 26 and 27, respectively.

50. The method of any one of claims 1-49, wherein the bispecific antibody comprises a heavy chain and a light chain consisting of the amino acid sequences of SEQ ID NO. 24 and 25, respectively, and a heavy chain and a light chain consisting of the amino acid sequences of SEQ ID NO. 26 and 27, respectively.

51. The method of any one of claims 1-50, wherein the bispecific antibody is elcuritumumab or a biological analogue thereof.