Bispecific antibodies against cd3 and cd20

Abstract

The present invention relates to bispecific antibodies (bsAb) and the use of such antibodies in the treatment of a disease in a subject. Furthermore, an advantageous treatment regimen is provided for the treatment of B-cell non-Hodgkin lymphoma (B-NHL).

Description

[0001] This application is a divisional application of Chinese invention patent application No. 202180041298.9, filed on May 7, 2021, entitled "Bispecific Antibody against CD3 and CD20". Technical Field

[0002] This invention relates to bispecific antibodies (bsAbs) targeting both CD3 and CD20, and the use of such antibodies in treating diseases in subjects. Furthermore, advantageous treatment regimens are provided. Background Technology

[0003] Monoclonal antibodies (mAbs) have shown high success in cancer treatment. Another promising approach to improving antibody therapy is through the specific recruitment of T cells to cancer cells expressing antigens. This can be achieved by utilizing bispecific antibodies that target both T cells and antigen-expressing cells. However, initial clinical studies were quite disappointing, primarily due to the low efficacy, severe side effects (cytokine storm), and immunogenicity of bispecific antibodies. Advances in the design and application of bispecific antibodies have partially overcome the initial hurdle of cytokine release syndrome and improved clinical efficacy without dose-limiting toxicities (Garber, 2014, Nat. Rev. Drug Discov. 13: 799-801).

[0004] The CD20 molecule, also known as human B lymphocyte restriction differentiation antigen or Bp35, is found on the surface of over 90% of B cells originating from peripheral blood or lymphoid organs, and is expressed during early pre-B cell development and maintained until plasma cell differentiation. CD20 is present on both normal and malignant B cells. In particular, CD20 is expressed on over 90% of B-cell non-Hodgkin lymphomas (B-NHL), but is not found on hematopoietic stem cells, pre-B cells, normal plasma cells, or other normal tissues. Methods for treating cancer and autoimmune and immune diseases by targeting CD20 are known in the art.

[0005] For example, the chimeric CD20 antibody rituximab has been used or recommended for the treatment of cancers such as B-NHL and chronic lymphocytic leukemia (CLL). The human monoclonal anti-CD20 antibody ofatumumab has been used or recommended for the treatment of various other CLL indications, including follicular lymphoma (FL), neuromyelitis optica (NMO), and diffuse and relapsing-remitting multiple sclerosis (RRMS).

[0006] Currently, bispecific antibodies targeting both CD20 and CD3 are under development. For example, WO2011028952 specifically describes a CD3xCD20 bispecific molecule generated using Xencor's XmAb bispecific Fc domain technology, WO2014047231 describes REGN1979 and other CD3xCD20 bispecific antibodies generated using Regeneron Pharmaceuticals' FcΔAdp technology, and Sun et al. (2015, Science Translational Medicine 7, 287ra70) described an anti-CD20 / CD3 T cell-dependent bispecific antibody targeting B cells constructed using a "knobs-into-holes" technique. These bispecific antibodies are currently being tested in clinical trials for specific indications in humans.

[0007] The bispecific antibodies of particular concern under development are epcoritamab (DuobodyCD3xCD20; GEN3013) (Engelberts et al., 2020, EBioMedicine, Vol. 52, 102625, WO2016110576 and WO2019155008, which are incorporated herein by reference).

[0008] Although treatment options exist for CD20+ cancers, such as B-NHL, further options are needed because some patients relapse or are refractory to currently available therapies. Eccleximab is a candidate drug that could expand the range of treatment options that may benefit patients with cancers like B-NHL.

[0009] Therefore, one object of the present invention is to provide means and methods for treating cancer by using a bispecific antibody (such as icoretuzumab) targeting CD3xCD20 as described herein, and to provide a bispecific antibody (such as icoretuzumab) targeting CD3xCD20 as described herein for the treatment of cancer.

[0010] Specific dose ranges and / or dosing regimens that are advantageous for such methods or uses (e.g., for cancers known or identified as CD20-positive, such as B-NHL) are provided. More specifically, this article provides specific dose ranges and / or dosing regimens that are advantageous for treating patients with diffuse large B-cell lymphoma (DLBCL) or high-grade B-cell lymphoma (FL). The dose ranges and / or dosing regimens provided herein have been evaluated as safe for use in humans and / or highly effective in the treatment of B-NHL. Summary of the Invention

[0011] The present invention provides the following technical solutions.

[0012] 1. A method for treating B-cell non-Hodgkin lymphoma (B-NHL) in a human subject, the method comprising subcutaneously administering a bispecific antibody as a full-length antibody to the human subject in need, the bispecific antibody comprising...

[0013] (i) A first binding arm comprising a first antigen-binding region binding to human CD3ε (epsilon), comprising a variable heavy chain region and a variable light chain region, wherein the variable heavy chain region comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:6; and wherein the variable light chain region comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:7; and

[0014] (ii) A second binding arm comprising a second antigen-binding region that binds to human CD20, comprising a variable heavy chain region and a variable light chain region, wherein the variable heavy chain region comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID No:13; and wherein the variable light chain region comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID No:14; The bispecific antibody is administered at a dose of at least 24 mg.

[0015] 2. The method according to claim 1, wherein the B-NHL is selected from the group consisting of: diffuse large B-cell lymphoma (DLBCL), high-grade B-cell lymphoma (HGBCL), primary mediastinal large B-cell lymphoma (PMBCL), mantle cell lymphoma, follicular lymphoma (FL), marginal zone lymphoma, and small lymphocytic lymphoma.

[0016] 3. The method according to item 1, wherein the B-NHL is FL, HGBCL or DLBCL.

[0017] 4. The method according to any one of claims 1-3, wherein the subject has received treatment with a CD20 monospecific antibody, such as rituximab, prior to treatment with the bispecific antibody.

[0018] 5. The method according to item 4, wherein the cancer recurs during the treatment with the CD20 monospecific antibody.

[0019] 6. The method according to item 4, wherein the cancer is refractory to the treatment during the treatment with the CD20 monospecific antibody.

[0020] 7. The method according to any one of items 4-6, wherein the CD20 monospecific antibody is used in combination therapy.

[0021] 8. The method according to any one of items 1-7, wherein the patient has received a further prior line of treatment for said B-cell NHL.

[0022] 9. The method according to any one of items 1-8, wherein the bispecific antibody is administered at a dose of at least 40 mg.

[0023] 10. The method according to any one of items 1-8, wherein the bispecific antibody is administered in a dose ranging from 40 mg to 100 mg.

[0024] 11. The method according to any one of items 1-8, wherein the bispecific antibody is administered at a dose of at least 48 mg.

[0025] 12. The method according to any one of items 1-8, wherein the bispecific antibody is administered at a dose of at least 60 mg.

[0026] 13. The method according to any one of items 1-8, wherein the bispecific antibody is administered at a dose of 48 mg.

[0027] 14. The method according to any one of items 1-8, wherein the bispecific antibody is administered at a dose of 60 mg.

[0028] 15. The method according to any one of items 1-14, wherein the dose is administered weekly.

[0029] 16. The method according to item 15, wherein the weekly application is performed at least 4 times.

[0030] 17. The method according to item 15 or 16, wherein the antibody is administered every two weeks after the weekly administration.

[0031] 18. The method according to item 17, wherein the bi-weekly application is performed (at least) 6 times.

[0032] 19. The method according to item 17 or 18, wherein the antibody is administered every four weeks after the administration every two weeks.

[0033] 20. The method according to any one of items 15-19, wherein the initiating dose of the bispecific antibody is administered prior to the administration of the weekly dose.

[0034] 21. The method according to item 20, wherein the initiating dose is administered one week prior to the administration of the first dose of the weekly dose.

[0035] 22. The method according to item 20 or 21, wherein the initiating dose is in the range of 50-350 μg.

[0036] 23. The method according to any one of items 20-22, wherein the initiating dose is 160 μg.

[0037] 24. The method according to items 20-23, wherein an intermediate dose of the bispecific antibody is administered after the initiating dose and before the weekly dose.

[0038] 25. The method according to item 24, wherein the initiating dose is administered two weeks prior to the administration of the first dose of the weekly dose, and the intermediate dose is administered one week prior to the administration of the first dose of the weekly dose.

[0039] 26. The method according to any one of items 24 or 25, wherein the intermediate dose is in the range of 600-1200 μg.

[0040] 27. The method according to any one of items 24-26, wherein the intermediate dose is 800 μg.

[0041] 28. The method according to any one of items 24-27, wherein the treatment method comprises subcutaneously administering the bispecific antibody in a 28-day cycle, wherein: a) On days 1, 8, 15 and 22 of the first cycle, the initiating dose is administered on day 1, the intermediate dose on day 8, and a dose of 48 mg is administered on days 15 and 22; for the first cycle; b) Administer a dose of 48 mg on days 1, 8, 15, and 22 of cycle 2-3; c) Administer a dose of 48 mg on days 1 and 15 of cycles 4-9; and d) On day 1 of the subsequent cycle, administer a dose of 48 mg.

[0042] 29. The method according to items 24-27, wherein the treatment method comprises subcutaneous administration of the bispecific antibody in a 28-day cycle, wherein: a) On days 1, 8, 15 and 22 of the first cycle, administer an initiating dose of 160 μg on day 1, an intermediate dose of 800 μg on day 8, and a dose of 48 mg on days 15 and 22; for the first cycle. b) Administer a dose of 48 mg on days 1, 8, 15, and 22 of cycle 2-3; c) Administer a dose of 48 mg on days 1 and 15 of cycles 4-9; and d) On day 1 of the subsequent cycle, administer a dose of 48 mg.

[0043] 30. The method according to any one of items 24-27, wherein the treatment method comprises subcutaneously administering the bispecific antibody in a 28-day cycle, wherein: a) On days 1, 8, 15 and 22 of the first cycle, administer the initiating dose on day 1, the intermediate dose on day 8, and the 60 mg dose on days 15 and 22; for the first cycle; b) Administer a dose of 60 mg on days 1, 8, 15, and 22 of cycle 2-3; c) Administer a dose of 60 mg on days 1 and 15 of cycles 4-9; and d) On day 1 of the subsequent cycle, administer a dose of 60 mg.

[0044] 31. The method according to any one of items 24-27, wherein the treatment method comprises subcutaneous administration of the bispecific antibody in a 28-day cycle, wherein: a) On days 1, 8, 15 and 22 of the first cycle, administer an initiating dose of 160 μg on day 1, an intermediate dose of 800 μg on day 8, and a dose of 60 mg on days 15 and 22; for the first cycle. b) Administer a dose of 60 mg on days 1, 8, 15, and 22 of cycle 2-3; c) Administer a dose of 60 mg on days 1 and 15 of cycles 4-9; d) On day 1 of the subsequent cycle, administer a dose of 60 mg.

[0045] 32. The method according to any one of items 28-31, wherein the B-cell NHL is FL, DLBCL and / or HGBCL.

[0046] 33. The method according to any one of items 1-32, wherein the subject has managed cytokine release syndrome (CRS) at grade 1 or 2 during the period.

[0047] 34. The method according to any one of items 1-33, wherein the subject does not experience tumor lysis syndrome.

[0048] 35. The method according to any one of items 1-34, wherein the subject is treated with prevention and treatment of cytokine release syndrome (CRS).

[0049] 36. The method according to claim 35, wherein the prevention and treatment includes the administration of corticosteroids.

[0050] 37. The method according to any one of items 35-36, wherein the prevention and treatment are administered on the same day as the bispecific antibody.

[0051] 38. The method according to any one of items 37, wherein the treatment is applied on the second to third day and optionally the fourth day, or on the second to fourth day thereafter.

[0052] 39. The method according to any one of items 37-38, wherein when the prevention and treatment are administered on the same day as the bispecific antibody, the prevention and treatment are administered 30-120 minutes before the bispecific antibody is administered.

[0053] 40. The method according to any one of claims 36-39, wherein the corticosteroid is prednisolone, for example, at an intravenous dose of 100 mg, or an equivalent thereof, including an oral dose.

[0054] 41. The method according to any one of items 1-40, wherein the human subject is treated with a pre-treatment drug to reduce the response to injection.

[0055] 42. The method according to claim 41, wherein the precursor medication includes the administration of an antihistamine.

[0056] 43. The method according to any one of claims 41-42, wherein the precursor medication comprises the administration of an antipyretic.

[0057] 44. The method according to any one of items 41-43, wherein the antihistamine is diphenhydramine, for example, in an intravenous or oral dose of 50 mg, or an equivalent thereof.

[0058] 45. The method according to any one of claims 43-44, wherein the antipyretic is acetaminophen, for example, an oral dose of 650-1000 mg, or an equivalent thereof.

[0059] 46. ​​The method according to any one of items 41-45, wherein the precursor drug and the bispecific antibody are administered on the same day.

[0060] 47. The method according to item 46, wherein the precursor drug is administered 30-120 minutes prior to the administration of the bispecific antibody.

[0061] 48. The method according to any one of items 28-47, wherein the prevention and treatment as defined in items 35-40 are applied during the first cycle.

[0062] 49. The method according to any one of items 28-48, wherein the precursor drug as defined in items 41-47 is administered during the first cycle.

[0063] 50. The method according to items 48 and 49, wherein the prophylaxis is administered during the second cycle when the human subject experiences a CRS greater than grade 1 after the fourth administration of the bispecific antibody in cycle 1.

[0064] 51. The method according to claim 50, wherein when the human subject experiences a CRS greater than grade 1 during the last administration of the bispecific antibody in a previous cycle, the prevention continues during subsequent cycles.

[0065] 52. The method according to items 49 and 50, wherein the precursor drug is optionally administered during the second cycle.

[0066] 53. The method of claim 52, wherein the precursor drug is optionally administered during a subsequent cycle.

[0067] 54. The method according to any one of the preceding claims, wherein the bispecific antibody comprises: (i) A first binding arm comprising a first antigen-binding region binding to human CD3ε (epsilon), wherein the first antigen-binding region comprises heavy chain variable (VH) regions CDR1, CDR2, and CDR3 having sequences as listed in SEQ ID NO. 1, 2, and 3, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 having sequences as listed in SEQ ID NO. 4, sequence GTN, and sequences as listed in SEQ ID NO. 5, respectively. (ii) A second binding arm comprising a second antigen-binding region that binds to human CD20, wherein the second antigen-binding region comprises heavy chain variable (VH) regions CDR1, CDR2, and CDR3 having sequences as listed in SEQ ID NO. 8, 9, and 10, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 having sequences as listed in SEQ ID NO. 11, sequence DAS, and sequences as listed in SEQ ID NO. 12, respectively.

[0068] 55. The method according to any one of the preceding claims, wherein the bispecific antibody comprises: (i) a first binding arm comprising a first antigen-binding region binding to human CD3ε (epsilon), comprising a variable heavy chain region and a variable light chain region, wherein the variable heavy chain region comprises SEQ ID NO:6; and wherein the variable light chain region comprises SEQ ID NO:7; and (ii) A second binding arm comprising a second antigen-binding region that binds to human CD20, comprising a variable heavy chain region and a variable light chain region, wherein the variable heavy chain region comprises SEQ ID No:13; and wherein the variable light chain region comprises SEQ ID No:14.

[0069] 56. The method according to any one of the preceding claims, wherein the first binding arm of the bispecific antibody is derived from a humanized antibody, preferably from a full-length IgG1,λ (lambda) antibody.

[0070] 57. The method according to item 56, wherein the bispecific antibody comprises a λ light chain constant region as defined in SEQ ID NO. 22.

[0071] 58. The method according to any one of the preceding claims, wherein the second binding arm of the bispecific antibody is derived from a human antibody, preferably from a full-length IgG1,κ (kappa) antibody.

[0072] 59. The method according to claim 58, wherein the bispecific antibody comprises a constant region of the κ light chain as defined in SEQ ID NO. 23.

[0073] 60. The method according to any one of the preceding items, wherein the bispecific antibody is a full-length antibody having a constant region of human IgG1.

[0074] 61. The method according to any one of the preceding items, wherein the bispecific antibody comprises an inert Fc region.

[0075] 62. The method according to claim 61, wherein the bispecific antibody comprises amino acids F, E and A at positions L234, L235 and D265 in the human IgG1 heavy chain corresponding to both the first and second heavy chains, respectively.

[0076] 63. The method according to any one of the preceding items, wherein the bispecific antibody comprises amino acid L at position F405 in the human IgG1 heavy chain of SEQ ID NO:15 in the first heavy chain, and wherein the second heavy chain comprises amino acid R at position K409 in the human IgG1 heavy chain of SEQ ID NO:15, and vice versa.

[0077] 64. The method according to any one of the preceding claims, wherein the bispecific antibody comprises amino acids F, E, and A at positions L234, L235, and D265 in the human IgG1 heavy chain of SEQ ID NO:15, respectively, corresponding to positions L234, L235, and D265 in both the first and second heavy chains, and wherein the first heavy chain comprises amino acid L at position F405 in the human IgG1 heavy chain of SEQ ID NO:15, and wherein the second heavy chain comprises amino acid R at position K409 in the human IgG1 heavy chain of SEQ ID NO:15, and vice versa.

[0078] 65. The method according to item 64, wherein the bispecific antibody comprises a constant region as defined in SEQ ID NO. 19 and 20.

[0079] 66. The method according to any one of the preceding items, wherein the bispecific antibody comprises a heavy chain and a light chain as defined in SEQ ID NO. 24 and 25, and a heavy chain and a light chain as defined in SEQ ID NO. 26 and 27, respectively.

[0080] 67. The method according to any one of the preceding items, wherein the bispecific antibody comprises a heavy chain and a light chain as defined in SEQ ID NO. 24 and 25, and a heavy chain and a light chain as defined in SEQ ID NO. 26 and 27, respectively.

[0081] 68. The method according to any one of the preceding items, wherein the bispecific antibody is icoretuzumab or a biosimilar thereof. Attached Figure Description

[0082] Figure 1 Population pharmacokinetic (PK) models were used to predict target-mediated drug disposition (TMDD) saturation. For each dose, 100 dummy patients were simulated, and the predicted TMDD saturation was summarized using box-and-whisker plots, representing the median (middle horizontal bar), the 25th and 75th percentiles (lower and upper hinges), and 1.5... Interquartile range (must) and outlier (point).

[0083] Figure 2 Overview of Human PK / PD Modeling and Analysis.

[0084] Figure 3Comparison of observed and calibrated model-predicted response rates in icotropium bacillus dose escalation trials. The observed response rates in icotropium bacillus dose escalation trials and the 90% CI as a function of the tertiles of icotropium bacillus exposure are represented by closely spaced circles and error bars, respectively. The model-predicted trial response rates and 90% prediction intervals are shown by solid lines and shaded areas.

[0085] Figure 4 Trimer formation predicted by the comprehensive PK / PD model of FL. For each dose, the mean number of trimers formed in the first 84 days of the simulation in 100 virtual patients (normalized by tumor cell number) is summarized using cassette plots, representing the median (middle horizontal bar), 25th and 75th percentiles (lower hinge and upper hinge), and 1.5. Interquartile range (must) and outlier (point).

[0086] Figure 5 Trimer formation predicted by a comprehensive pharmacokinetic / pharmacodynamic model for DLBCL / HGBCL. For each dose, 100 dummy patients from the icoretuzumab extension trial were simulated, and the mean number of trimers (normalized by tumor cell number) predicted by the model was summarized using a box-and-whisker plot.

[0087] Figure 6 Response rates of the icoretuzumab extension trial predicted by a comprehensive PK / PD model in FL patients. For each dose, 100 dummy patients in the icoretuzumab extension trial were simulated, and the predicted trial response rates were summarized using a box-and-whisker plot, representing the median (middle horizontal bar), the 25th and 75th percentiles (lower and upper hinges), and 1.5%. Interquartile range (must) and outlier (point).

[0088] Figure 7 Response rates of the icoretuzumab extension trial predicted by a comprehensive PK / PD model in patients with DLBCL / HGBCL. For each dose, 100 dummy patients in the icoretuzumab extension trial were simulated, and the predicted trial response rates were summarized using a box-and-whisker plot, representing the median (middle horizontal bar), the 25th and 75th percentiles (lower and upper hinges), and 1.5%. Interquartile range (must) and outlier (point). Detailed Implementation

[0089] The term "immunoglobulin" used in this article refers to a class of structure-associated glycoproteins composed of two pairs of polypeptide chains: a pair of light (L) low molecular weight chains and a pair of heavy (H) chains, all four linked together by disulfide bonds. The structure of immunoglobulins has been well characterized. See, for example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). In short, each heavy chain typically consists of a heavy chain variable region (abbreviated as VH or V in this article). H ) and heavy chain constant region (abbreviated as CH or C in this article) H The heavy chain constant region typically consists of three domains: CH1, CH2, and CH3. The hinge region is the region between the CH1 and CH2 domains of the heavy chain and is highly flexible. The disulfide bonds in the hinge region are part of the interaction between the two heavy chains in the IgG molecule. Each light chain typically consists of a light chain variable region (abbreviated as VL or VL in this article). L ) and light chain constant region (abbreviated as CL or C in this article) L The light chain constant region typically consists of a single CL domain. The VH and VL regions can be further subdivided into highly variable regions (or highly variable areas, which may be highly variable in the structurally defined ring sequence and / or form), also known as complement-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL typically consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol). 196, 901-917 (1987)). Unless otherwise stated or contradicted by the context, the CDR sequences in this paper are defined according to the IMGT rules (Brochet X., Nucl Acids Res. 2008;36: W503-508 and Lefranc MP., Nucleic Acids Research 1999;27:209-212; see also the Internet http: / / www.imgt.org / ). Unless otherwise stated or contradicted by the context, the amino acid positions in constant regions referenced in this invention are according to EU numbers (Edelman et al., Proc Natl Acad Sci U SA. 1969 May;63(1):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No. 91-3242). For example, SEQ ID NO:15 of this article lists the amino acid positions 118-447 of the constant region of the IgG1 heavy chain according to the EU number.

[0090] As used herein, the term "amino acid corresponding to position..." refers to the amino acid position number in the human IgG1 heavy chain. Corresponding amino acid positions in other immunoglobulins can be identified by comparison with human IgG1. Therefore, the amino acid or fragment in one sequence that "corresponds" to an amino acid or fragment in another sequence is aligned using a standard sequence alignment program such as ALIGN, ClustalW, or similar programs, typically at default settings, and has at least 50%, at least 80%, at least 90%, or at least 95% identity with the human IgG1 heavy chain. How to align sequences or fragments within sequences and thereby determine their corresponding positions in sequences according to the present invention is considered well known in the art.

[0091] In the context of this invention, the term "antibody" (Ab) refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or any derivative thereof, which, under typical physiological conditions, has the ability to specifically bind antigens and has a half-life of a significant time period, 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 longer, about 48 hours or longer, about 3, 4, 5, 6, 7 or more days, or any other relevant functionally defined period (such as the time sufficient to induce, promote, enhance, and / or regulate physiological responses associated with antibody-bound antigens and / or sufficient to activate antibody-recruited effectors). The variable regions of the heavy and light chains of an immunoglobulin molecule contain binding domains that interact with the antigen. As used herein, the term "antibody-binding region" refers to a region that interacts with the antigen and includes both VH and VL regions. The term antibody as used herein includes not only monospecific antibodies but also multispecific antibodies, which contain multiple, such as two or more, for example, three or more different antigen-binding regions. The constant region of an antibody (Ab) can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system, such as C1q, which is the first component in the classical pathway of complement activation. As stated above, unless otherwise stated or clearly contradicted by the context, the term antibody as used herein includes antibody fragments that are antigen-binding segments, i.e., those that retain the ability to bind specifically to antigens. It has been shown that the antigen-binding function of antibodies can be performed by fragments of full-length antibodies. Examples of antigen-binding fragments encompassed within the term "antibody" 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 fragments, bivalent fragments comprising two Fab fragments linked by disulfide bonds in the hinge region; (iii) Fd fragments primarily consisting of VH and CH1 domains; (iv) Fv fragments essentially consisting of the VL and VH domains of a single antibody arm; and (v) dAb fragments (Ward et al.). . Nature 341 , 544-546 (1989)), which is basically composed of the VH domain, also known as domain antibody (Holt et al; Trends Biotechnol. 2003 Nov; 21 (11):484-90); (vi) Camel or nanobody (Revets et al; Expert Opin Biol Ther. 2005 Jan; 5(1):111-24) and (vii) separate complementarity-determining regions (CDRs). Furthermore, although the two domains VL and VH of the Fv fragment are encoded by different genes, they can be linked via synthetic linkers using recombination methods, enabling them to be made into single-protein chains, where the VL and VH regions pair to form monovalent molecules (called single-chain antibodies or single-chain Fv (scFv), see, for example, Bird et al.). . Science 242 , 423-426 (1988) and Huston et al. . PNAS USA 85 , 5879-5883 (1988)). Unless otherwise stated or explicitly indicated by the context, such single-chain antibodies are encompassed within the term antibody. Although such fragments are generally included in the meaning of antibody, they are collectively and independently unique features of the present invention, exhibiting distinct biological properties and utilities. These and other useful antibody fragments in the context of the present invention, as well as bispecific forms of such fragments, are further discussed herein. It should also be understood that the term antibody, unless otherwise stated, also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like peptides, chimeric antibodies, and humanized antibodies, as well as antibody fragments that retain the ability to specifically bind to antigens (antigen-binding fragments) provided by any known technology, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. The resulting antibodies can have any isotype. 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 the heavy chain constant region gene. When this document refers to a specific isotype, such as IgG1, the term is not limited to a specific isotype sequence, such as a specific IgG1 sequence, but is used to indicate that an antibody is sequence-closer to that isotype, such as IgG1, rather than other isotypes. Therefore, for example, the IgG1 antibody of this invention can be a sequence variant of a naturally occurring IgG1 antibody, including variations in constant regions.

[0092] As used herein, the term "monoclonal antibody" refers to a preparation of an antibody molecule consisting of a single molecule. Monoclonal antibody compositions exhibit single-binding specificity and affinity for a specific epitope. Therefore, the term "human monoclonal antibody" refers to an antibody exhibiting single-binding specificity, having variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies can be generated from hybridomas comprising B cells fused to immortalized cells obtained from transgenic or transchromosomal nonhuman animals such as transgenic mice, which have a genome containing human heavy chain transgenes and light chain transgenes.

[0093] In the context of this invention, the term "bispecific antibody," "bs," or "bsAb" refers to an antibody having two distinct antigen-binding regions defined by different antibody sequences. Bispecific antibodies can be in any form.

[0094] When used in this document, the terms “half-molecule,” “Fab arm,” and “arm” refer to a heavy-light chain pair.

[0095] When a bispecific antibody is described as comprising a half-molecule antibody “derived from” a first parent antibody and a half-molecule antibody “derived from” a second parent antibody, the term “derived from” means that the bispecific antibody is produced by any known method by recombination of half-molecules from each of the first and second parent antibodies into the resulting bispecific antibody. In this context, “recombination” is not intended to be limited to any particular recombination method and therefore includes all methods for producing the bispecific antibodies described below, including, for example, recombination by half-molecule exchange (also known as “controlled Fab arm exchange”), and recombination at the nucleic acid level and / or by co-expressing two half-molecules in the same cell.

[0096] When used in the context of antibodies, the term "full-length" means that an antibody is not a fragment, but rather contains all the domains of a specific isotype typically found in nature, such as the VH, CH1, CH2, CH3, hinge, VL, and CL domains of an IgG1 antibody. Full-length antibodies can be engineered. An example of a "full-length" antibody is icoretuzumab.

[0097] When used herein, unless contradicted by context, the term “Fc region” refers to an antibody region consisting of the Fc sequences of the two heavy chains of an immunoglobulin, wherein the Fc sequences contain at least a hinge region, a CH2 domain, and a CH3 domain.

[0098] When used herein, the term “heterodimeric interaction between the first and second CH3 regions” refers to the interaction between the first CH3 region and the second CH3 region in a first CH3 / second CH3 heterodimeric protein.

[0099] When used herein, the term “homodimal interaction between the first and second CH3 regions” refers to the interaction between the first CH3 region and another first CH3 region in the first CH3 / first CH3 homodimer protein, and the interaction between the second CH3 region and another second CH3 region in the second CH3 / second CH3 homodimer protein.

[0100] As used herein, the term "binding" in the context of antibody binding to a predetermined antigen is generally used to correspond to the binding of an antibody as a ligand and an antigen as an analyte, as determined by, for example, BioLayer Interferometry (BLI) techniques in an OctetHTX instrument. -6 M or smaller, such as 10 -7 M or smaller, such as about 10 -8 M or smaller, such as about 10 -9 M or smaller, approximately 10 -10 M or smaller or about 10 -11 M or smaller K D The affinity binding, and in which the antibody corresponds to K D The K-type antigen binds to the predetermined antigen with affinity. D K, compared to its binding to non-specific antigens other than the predetermined antigen or closely related antigens (e.g., BSA, casein), D At least ten times lower, such as at least 100 times lower, such as at least 1,000 times lower, such as at least 10,000 times lower, such as at least 100,000 times lower. Having a lower binding K. D The amount depends on the K of the antibody. D This makes the K of the antibody... D When it is very low, then the K binding to the antigen... D K binding to non-specific antigens D The low amount may be at least 10,000 times (i.e., the antibody is highly specific).

[0101] As used in this article, the term "K" D "(M)" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. As used in this paper, affinity and K0 D It is negatively correlated, meaning that higher affinity refers to lower K. D Lower affinity refers to higher K... D .

[0102] In a preferred embodiment, the antibody of the present invention is isolated. As used herein, "isolated antibody" is intended to mean 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 also substantially free of monospecific antibodies that specifically bind to CD20 or CD3.

[0103] As used herein, the term "CD3" refers to human differentiation cluster 3 protein, which is part of the T-cell co-receptor protein complex and consists of four distinct chains. CD3 is also found in other species; therefore, the term "CD3" is not limited to human CD3 unless contradicted by the context. In mammals, this complex contains a CD3γ (gamma) chain (human CD3γ chain UniProtKB / Swiss-Prot No P09693 or cynomolgus monkey CD3γ UniProtKB / Swiss-Prot No Q95LI7), a CD3δ (delta) chain (human CD3δ UniProtKB / Swiss-Prot No P04234 or cynomolgus monkey CD3δ UniProtKB / Swiss-Prot No Q95LI8), two CD3ε (epsilon) chains (human CD3ε UniProtKB / Swiss-Prot No P07766; cynomolgus monkey CD3ε UniProtKB / Swiss-Prot No Q95LI5; or rhesus monkey CD3ε UniProtKB / Swiss-Prot No G7NCB9) and a CD3ζ (zeta) chain (human CD3ζ UniProtKB / Swiss-Prot No. P20963, cynomolgus monkey CD3ζUniProtKB / Swiss-Prot No Q09TK0). These chains associate with molecules called T cell receptors (TCRs) and generate activation signals in T lymphocytes. TCRs and CD3 molecules together constitute the TCR complex.

[0104] "CD3 antibody" or "anti-CD3 antibody" is an antibody that specifically binds to the antigen CD3, especially human CD3ε (epsilon).

[0105] The term "human CD20" or "CD20" refers to human CD20 (UniProtKB / Swiss-Prot No P11836), including any variant, isotype, and species homolog of CD20 expressed on cells (including tumor cells) naturally or by transfection with the CD20 gene or cDNA. Species homologs include rhesus macaque CD20 (rhesus macaque (macaca mulatta); UniProtKB / Swiss-Prot No H9YXP1) and cynomolgus monkey CD20 (cynomolgus monkey (macaca fascicularis); UniProtKBNoG7PQ03).

[0106] "CD20 antibody" or "anti-CD20 antibody" is an antibody that specifically binds to the antigen CD20, especially human CD20.

[0107] "CD3xCD20 antibody", "anti-CD3xCD20 antibody", "CD20xCD3 antibody" or "anti-CD20xCD3 antibody" are bispecific antibodies that contain two different antigen-binding regions, one of which specifically binds to the antigen CD20 and the other specifically binds to the antigen CD3.

[0108] The term "DuoBody-CD3xCD20" in this document refers to an IgG1 bispecific CD3xCD20 antibody comprising a first heavy chain and light chain pair as defined in SEQ ID NO:24 and SEQ ID NO:25, and a second heavy chain and light chain pair as defined in SEQ ID NO:26 and SEQ ID NO:27. The first heavy chain and light chain pair comprises a binding region for binding to human CD3ε (epsilon), and the second heavy chain and light chain pair comprises a binding region for binding to human CD20. The first binding region comprises VH and VL sequences as defined in SEQ ID NO. 6 and 7, and the second binding region comprises VH and VL sequences as defined in SEQ ID NO. 13 and 14. This bispecific antibody can be prepared as described in WO 2016 / 110576.

[0109] The present invention also provides antibodies comprising the heavy chain, light chain, VL region, VH region, or one or more functional variants of the CDR of the antibodies of the embodiments. The functional variants of the heavy chain, light chain, VL, VH, or CDR used in the context of the antibody still allow the antibody to retain at least a substantial proportion (at least about 90%, 95%, or more) of the functional characteristics of the “reference” and / or “parent” antibody, including affinity and / or specificity / selectivity for specific epitopes of CD20 and / or CD3, Fc inertness, and PK parameters (such as half-life, Tmax, Cmax).

[0110] Such functional variants typically retain considerable sequence identity with the parent antibody and / or substantially similar heavy and light chain lengths. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of nicks and the length of each nick (which is introduced to achieve optimal alignment of the two sequences) (i.e., homology % = number of identical positions / total number of positions x 100). The percentage of 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 4, 11-17 (1988), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weighted residue table, a nick length penalty of 12, and a nick penalty of 4. Furthermore, the percentage of identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970).

[0111] Exemplary variants include variants that differ primarily from the parent antibody sequence in the heavy chain and / or light chain, VH and / or VL and / or CDR regions by conserved substitutions; for example, 10 substitutions in a variant, such as 9, 8, 7, 6, 5, 4, 3, 2 or 1, may be conserved amino acid residue substitutions.

[0112] In the context of this invention, conservative substitution can be defined by substitutions within the amino acid categories reflected in the table below: Class of conserved substituted amino acid residues

[0113] In the context of this invention, unless otherwise stated, the following symbols are used to describe mutations: i) an amino acid substitution at a given position is written as, for example, K409R, meaning that arginine replaces lysine at position 409; ii) for a particular variant, a specific three-letter or one-letter code is used, including codes Xaa and X, to represent any amino acid residue. Thus, replacing lysine with arginine at position 409 is called K409R, while replacing lysine at position 409 with any amino acid residue is called K409X. If lysine at position 409 is missing, then K409 is used. express.

[0114] The term "epitope" refers to a protein determinant that specifically binds to an antibody. Epitopes are typically composed of surface groups of molecules, such as amino acids or sugar side chains, and usually possess specific three-dimensional structural features and specific charge characteristics. The difference between conformational and non-conformational epitopes is that binding to the former, but not the latter, is lost in the presence of denaturing solvents. Epitopes can contain amino acid residues that directly participate in binding and other amino acid residues that do not directly participate in binding, such as amino acid residues that are effectively blocked or covered by the specific antigen-binding peptide (in other words, the amino acid residues are within the footprint of the specific antigen-binding peptide).

[0115] As used herein, the term "chimeric antibody" refers to an antibody in which the variable region is derived from a non-human species (e.g., rodent-derived) and the constant region is derived from a different species, such as humans. Chimeric monoclonal antibodies are developed for therapeutic applications to reduce antibody immunogenicity. The term "variable region" or "variable domain," as used in the context of chimeric antibodies, refers to the region containing both the CDR and framework regions of immunoglobulins, including both the heavy and light chains. Chimeric antibodies can be developed using methods such as those described by Sambrook et al. . The chimeric antibody is produced using the standard DNA techniques described in *Molecular Cloning: A Laboratory Manual*, 1989, New York: Cold Spring Harbor Laboratory Press, Ch. 15. The chimeric antibody can be a genetically or enzymatically engineered recombinant antibody. The production of chimeric antibodies is within the knowledge of those skilled in the art; therefore, the chimeric antibody according to the present invention can be produced by methods other than those described herein.

[0116] As used herein, the term "humanized antibody" refers to a genetically engineered nonhuman antibody containing a human antibody constant domain and a nonhuman variable domain modified to contain a high sequence homology with the human variable domain. This can be achieved by transplanting the six nonhuman antibody complementarity-determining regions (CDRs) that co-form the antigen-binding site onto the homologous human receptor frame region (FR) (see WO92 / 22653 and EP0629240). To fully reconstruct the binding affinity and specificity of the parent antibody, it may be necessary to replace the frame residues from the parent antibody (i.e., the nonhuman antibody) with the human frame region (reversion mutation). Structural homology modeling may help identify amino acid residues in the frame region that are important for antibody binding properties. Therefore, a humanized antibody may contain a nonhuman CDR sequence, primarily a human frame region, optionally containing one or more amino acid reversion mutations relative to the nonhuman amino acid sequence, and a fully human constant region. The VH and VL of the CD3 arm used in Duobody-CD3XCD20 represent the humanized antigen-binding region. Optionally, additional amino acid modifications, which are not necessarily reversion mutations, can be applied to obtain humanized antibodies with preferred characteristics, such as affinity and biochemical properties.

[0117] 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 through random or site-specific mutagenesis in vitro or through somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from another mammalian species, such as a mouse, has been grafted onto a human frame sequence. The VH and VL of the CD3 arm used in Duobody-CD3XCD20 represent human antigen-binding regions. The human monoclonal antibodies of the present invention can be produced using a variety of techniques, including conventional monoclonal antibody methods such as those employed by Kohler and Milstein. Nature The standard somatic cell hybridization technique of 256:495 (1975). Although somatic cell hybridization is preferred, other techniques for producing monoclonal antibodies may, in principle, be used, such as viral or oncogenic transformation of B lymphocytes or phage display using a human antibody gene library. A suitable animal system for preparing hybridomas that secrete human monoclonal antibodies is the mouse system. The production of hybridomas in mice is a well-established process. Immunoimmunization protocols and techniques for fusion of isolated spleen cells are known in the art. Fusion couples (e.g., mouse myeloma cells) and fusion procedures are also known. Thus, human monoclonal antibodies can be produced, for example, using transgenic or transchromosomal mice or rats carrying a portion of the human immune system instead of mouse or rat systems. Thus, in embodiments, human antibodies are obtained from transgenic animals, such as mice or rats, carrying human germline immunoglobulin sequences instead of animal immunoglobulin sequences. In such implementations, the antibody is derived from a human immunoglobulin sequence introduced into the animal, but the final antibody sequence is the result of further modification of the human immunoglobulin sequence through somatic hypermutation and affinity maturation via endogenous animal antibody mechanisms, see, for example, Mendez et al. 1997 Nat Genet. 15(2):146-56. The VH and VL of the CD3 arm used in Duobody-CD3XCD20 represent human antigen-binding regions.

[0118] The term "reduction conditions" or "reduction environment" refers to the conditions or environment under which the substrate, in this case, the cysteine ​​residues in the antibody hinge region, is more likely to be reduced than oxidized.

[0119] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell in which an expression vector has been introduced, such as an expression vector encoding the antibody of the present invention. Recombinant host cells include, for example, transfected tumor cells such as CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6, or NSO cells, as well as lymphocytes.

[0120] The term "treatment" refers to the application of an effective amount of the therapeutically active antibody of the present invention to alleviate, improve, prevent or eradicate (cure) symptoms or disease states.

[0121] The term "effective dose" or "therapeutic effective dose" refers to the amount of antibody that effectively achieves the desired therapeutic outcome within the necessary dosage and time period. Therapeutic effective doses of antibodies can vary depending on individual factors such as disease state, age, sex, weight, and the antibody's ability to elicit the desired response in the individual. Therapeutic effective dose is also the amount of antibody or antibody fraction in which the beneficial therapeutic effect outweighs any toxic or harmful effects.

[0122] As used herein, the term "buffer" refers to a pharmaceutically acceptable buffer. The term "buffer" encompasses those reagents that maintain the pH of a solution within, for example, an acceptable range, and includes, but is not limited to, acetates, histidines, TRIS® (tris(hydroxymethyl)aminomethane), citrates, succinates, glycolic acid salts, etc. Generally, a "buffer" as used herein has a pKa and buffering capacity suitable for a pH range of about 5 to about 6, preferably about 5.5.

[0123] As used herein, a "surfactant" is a compound commonly used in pharmaceutical formulations to prevent drug adsorption to surfaces and / or aggregation. Furthermore, surfactants reduce surface tension (or interfacial tension) between two liquids or between a liquid and a solid. For example, exemplary surfactants can 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 below 0.1% w / v or less, such as 0.04% w / v). Surfactants are amphiphilic, meaning they are typically 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, sodium docusate, phospholipids, polyalkyl ethers, sodium lauryl sulfate, and triaprylin (anionic surfactants); benzalkonium chloride, citrimide, cetylpyridinium chloride, and phospholipids (cationic surfactants); and α-tocopherol, glycerol monooleate, myristyl alcohol, phospholipids, poloxamers, polyoxyethylene alkyl ether, polyoxyethylene castor oil derivative, polyoxyethylene sorbintan fatty acid ester, and polyoxyethylene stearate. Enesterarate, polyoxyl hydroxystearate, polyoxyl glycerides, polysorbates such as polysorbate 20 or polysorbate 80, propylene glycol dilaurate, propylene glycol monolaurate, sorbitan esters sucrose palmitate, sucrose stearate, triaprylin, and TPGS (nonionic and amphoteric surfactants).

[0124] The term "diluent" of interest herein refers to a pharmaceutically acceptable (safe and non-toxic for human administration) diluent that can be used to prepare a pharmaceutical composition. Preferably, such diluents of the compositions of the present invention dilute only the antibody concentration without diluting the buffers and stabilizers. Therefore, in a preferred embodiment, the diluent contains the same concentration of buffers and stabilizers as present in the pharmaceutical compositions of the present invention. Further exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffer solutions (preferably acetate buffers), sterile saline solutions, Ringer's solution, or glucose solutions. In a preferred embodiment, the diluent comprises acetate buffer and sorbitol, or is substantially composed of acetate buffer and sorbitol.

[0125] The terms “pharmaceutical composition” and “pharmaceutical formulation” are used interchangeably in this document.

[0126] Implementation Plan

[0127] In one aspect, the present invention provides a method for treating B-cell non-Hodgkin lymphoma (B-NHL) in a human subject, the method comprising subcutaneous (sc) administration of a bispecific antibody to the subject in need, the bispecific antibody comprising

[0128] (i) A first binding arm comprising a first antigen-binding region binding to human CD3ε (epsilon), comprising a variable heavy chain region and a variable light chain region, wherein the variable heavy chain region comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:6; and wherein the variable light chain region comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:7; and

[0129] (ii) A second binding arm comprising a second antigen-binding region that binds to human CD20, comprising a variable heavy chain region and a variable light chain region, wherein the variable heavy chain region comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID No:13; and wherein the variable light chain region comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID No:14; The bispecific antibody is administered at a dose of at least 24 mg.

[0130] On the other hand, the present invention provides a method for treating B-cell non-Hodgkin lymphoma (B-NHL) in a human subject, the method comprising subcutaneously administering to the subject in need a bispecific antibody as a full-length antibody having an indolent Fc region, the bispecific antibody comprising

[0131] (i) A first binding arm comprising a first antigen-binding region binding to human CD3ε (epsilon), comprising a variable heavy chain region and a variable light chain region, wherein the variable heavy chain region comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:6; and wherein the variable light chain region comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:7; and

[0132] (ii) A second binding arm comprising a second antigen-binding region that binds to human CD20, comprising a variable heavy chain region and a variable light chain region, wherein the variable heavy chain region comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID No:13; and wherein the variable light chain region comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID No:14; The bispecific antibody is administered at a dose of at least 24 mg.

[0133] In another aspect, the present invention provides a method for treating B-cell non-Hodgkin lymphoma (B-NHL) in a human subject, the method comprising subcutaneously administering a bispecific antibody as a full-length antibody to the subject in need, the bispecific antibody comprising...

[0134] (i) A first binding arm comprising a first antigen-binding region binding to human CD3ε (epsilon), comprising a variable heavy chain region and a variable light chain region, wherein the variable heavy chain region comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:6; and wherein the variable light chain region comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:7; and

[0135] (ii) A second binding arm comprising a second antigen-binding region that binds to human CD20, comprising a variable heavy chain region and a variable light chain region, wherein the variable heavy chain region comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID No:13; and wherein the variable light chain region comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID No:14; The bispecific antibody is administered at a dose of at least 24 mg.

[0136] On the other hand, the present invention provides a method for treating B-cell non-Hodgkin lymphoma (B-NHL) in a human subject, the method comprising subcutaneous administration of a bispecific antibody comprising an inert Fc region to the subject in need, the bispecific antibody comprising

[0137] (i) A first binding arm comprising a first antigen-binding region binding to human CD3ε (epsilon), comprising a variable heavy chain region and a variable light chain region, wherein the variable heavy chain region comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:6; and wherein the variable light chain region comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:7; and

[0138] (ii) A second binding arm comprising a second antigen-binding region that binds to human CD20, comprising a variable heavy chain region and a variable light chain region, wherein the variable heavy chain region comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID No:13; and wherein the variable light chain region comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID No:14; The bispecific antibody is administered at a dose of at least 24 mg.

[0139] It is understood that when this document refers to a treatment method, namely a method for treating B-cell non-Hodgkin lymphoma (B-NHL) in a human subject, the method comprising subcutaneously administering a bispecific antibody according to the invention, preferably a full-length antibody and / or containing an inert Fc region, to a human subject in need, the wording also differs, referring to a bispecific antibody according to the invention for treating B-cell non-Hodgkin lymphoma (B-NHL) in a human subject, this use comprising subcutaneously administering a bispecific antibody according to the invention, preferably a full-length antibody and / or containing an inert Fc region, to a human subject in need. Therefore, when this document refers to a method for treating B-NHL with a bispecific antibody targeting CD3 and CD20 as defined herein, it also refers to the use of said bispecific antibody targeting CD3 and CD20 for treating B-NHL in a human subject.

[0140] In a further aspect, the present invention provides a method for treating B-NHL, wherein the bispecific antibody comprises: (i) A first binding arm comprising a first antigen-binding region binding to human CD3ε (epsilon), wherein the first antigen-binding region comprises heavy chain variable (VH) regions CDR1, CDR2, and CDR3 having sequences as listed in SEQ ID NO. 1, 2, and 3, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 having sequences as listed in SEQ ID NO. 4, sequence GTN, and sequences as listed in SEQ ID NO. 5, respectively. (ii) A second binding arm comprising a second antigen-binding region that binds to human CD20, wherein the second antigen-binding region comprises heavy chain variable (VH) regions CDR1, CDR2, and CDR3 having sequences as listed in SEQ ID NO. 8, 9, and 10, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 having sequences as listed in SEQ ID NO. 11, sequence DAS, and sequences as listed in SEQ ID NO. 12, respectively.

[0141] In a further aspect, the present invention provides a method for treating B-NHL, wherein the bispecific antibody comprises: (i) a first binding arm comprising a first antigen-binding region binding to human CD3ε (epsilon), comprising a variable heavy chain region and a variable light chain region, wherein the variable heavy chain region comprises SEQ ID NO:6; and wherein the variable light chain region comprises SEQ ID NO:7; and (ii) A second binding arm comprising a second antigen-binding region that binds to human CD20, comprising a variable heavy chain region and a variable light chain region, wherein the variable heavy chain region comprises SEQ ID No:13; and wherein the variable light chain region comprises SEQ ID No:14.

[0142] Non-Hodgkin's lymphoma (NHL) is a disease entity characterized by malignant transformation of cells originating from lymphoid tissue. B-cell-derived NHL (“B-NHL” or “B-cell NHL”) as defined herein constitutes a distinct group of vegetations within the broader context of NHL. These are diagnosed by pathologists using standard classification criteria (based on tissue biopsy) to differentiate B-NHL subtypes. Standard classification criteria include histological morphological features, surface markers (immunohistochemistry / flow cytometry), and chromosomal abnormalities / translocations (karyotype analysis, etc.). Fluorescence in situ hybridization(FISH) and molecular (genetic mutation) results. Diagnosis and classification of B-NHL are based on the WHO classification, which is incorporated herein by reference (Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised ed. 4th). Lyon, France: IARC Press (2017); Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues (ed. 4th). Lyon, France: IARC Press (2008)).

[0143] In short, B-NHL is generally classified into indolent (slow-growing) and aggressive subtypes. Aggressive B-NHL has high sKi67 expression, while indolent B-NHL has relatively low sKi67 expression. In general, indolent lymphomas respond to treatment and remain under control (remission), resulting in long-term survival for many years but no cure. Aggressive lymphomas usually require intensive treatment, and some have a good prospect of permanent cure. Aggressive B-NHL includes: diffuse large B-cell lymphoma (DLBCL), high-grade B-cell lymphoma (HGBCL), primary mediastinal large B-cell lymphoma (PMBCL), and mantle cell lymphoma (MCL). Indolent B-NHL includes follicular lymphoma (FL), marginal zone lymphoma (MZL), and small lymphocytic lymphoma (SLL). Diffuse large B-cell lymphoma (DLBCL) is the most common type of NHL, accounting for approximately 30%–40% of all NHL diagnoses, followed by FL (accounting for 20%–25% of all NHL diagnoses). Most B-cell lymphomas express B-cell markers such as CD19, CD20, CD22, and CD79b. The biological heterogeneity of B-cell malignancies is reflected in the clinical course and outcomes of individual diseases. Indolent diseases, such as FL, MZL, and SLL, evolve slowly and have a median survival of 8–10 years. Conversely, more aggressive diseases such as DLBCL / HGBCL have a median survival of 6 months if left untreated. The median age at diagnosis of lymphoma is approximately 60–65 years (WHO, 2008).

[0144] Therefore, the method according to the invention further includes treatment of B-NHL, wherein the B-NHL is selected from diffuse large B-cell lymphoma (DLBCL), high-grade B-cell lymphoma (HGBCL), primary mediastinal large B-cell lymphoma (PMBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), and small lymphocytic lymphoma (SLL).

[0145] In another aspect, the method according to the invention provided herein includes the treatment of FL, HGBCL, or DLBCL. In yet another aspect, the method according to the invention provided herein includes the treatment of FL. In another aspect, the method according to the invention provided herein includes the treatment of HGBCL. In another aspect, the method according to the invention provided herein includes the treatment of DLBCL. In yet another aspect, the method according to the invention provided herein includes the treatment of HGBCL and / or DLBCL. In yet another aspect, the method according to the invention provided herein includes the treatment of FL, MCL, HGBCL, or DLBCL. In yet another aspect, the method according to the invention described herein includes the treatment of MCL.

[0146] The terms “DLBCL / HGBCL”, “DLBCL and HGBCL”, or “DLBCL or HGBCL” as used in this article refer to B-NHL classified as diffuse large B-cell lymphoma or high-grade B-cell lymphoma, according to the WHO classification as defined in Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues (ed. 4th). Lyon, France: IARC Press (2008) and Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised ed. 4th). Lyon, France: IARC Press (2017), which are incorporated herein by reference.

[0147] As defined in this article, "FL" (follicular lymphoma) refers to B-NHL originating from a specific type of B cell, called centrocells and centroblasts, which typically form follicles or follicle-like structures. FL usually has a slow course, remaining largely unchanged for several years. FL can be classified according to the WHO classifications defined in Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumors of Haematopoietic and Lymphoid Tissues (ed. 4th). Lyon, France: IARC Press (2008) and Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumors of Haematopoietic and Lymphoid Tissues (Revised ed. 4th). Lyon, France: IARC Press (2017), which are incorporated herein by reference.

[0148] “MCL” stands for mantle cell lymphoma, which includes B-cell lymphomas with chromosomal translocation t(11;14) resulting in cyclin D1 expression, and also includes CD5+. The MCL defined in this article includes B-NHL classified as MCL, according to the WHO classification as defined in Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues (ed. 4th). Lyon, France: IARC Press (2008) and Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised ed. 4th). Lyon, France: IARC Press (2017), which are incorporated herein by reference.

[0149] Regarding the dosage of at least 24 mg of bispecific antibody to be administered, or any other prescribed dosage, it can be understood that the dosage refers to the amount of bispecific antibody representing the full-length antibody, such as icoretuzumab as defined in the Examples section. Therefore, although the terminology differs, the dosage of bispecific antibody administered according to the present invention can be referred to as the dosage of at least 24 mg of bispecific antibody, where the dosage corresponds to at least 24 mg of icoretuzumab. The amount of antibody to be administered can be determined when, for example, the molecular weight of the antibody to be used differs significantly from that of the full-length antibody (such as icoretuzumab). The required amount of antibody as defined herein can therefore be calculated by dividing the molecular weight of the antibody by the weight of the full-length antibody (such as icoretuzumab) and multiplying the result by the specified dosage described herein. Provided that the bispecific antibody has highly similar characteristics to DuobodyCD3xCD20 in terms of plasma half-life, Fc inertness, and / or binding region characteristics to CD3 and CD20 (i.e., in terms of CDR and epitope binding characteristics), such an antibody may be considered according to the present invention and administered at a dose corresponding to that of a full-length antibody (such as icoretuzumab) as defined herein.

[0150] As described, the bispecific antibody binding to CD3 and CD20 is intended for subcutaneous administration. Therefore, the bispecific antibody is formulated in the pharmaceutical composition such that it is compatible with subcutaneous (sc) administration, i.e., having a formulation and / or concentration that allows for pharmaceutically acceptable sc administration at selected doses as defined herein. Subcutaneous administration is preferably by injection. For example, formulations of Duobody CD3xCD20 compatible with conceivable subcutaneous formulations have been described (e.g., as described in WO2019155008, which is incorporated herein by reference). Preferred formulations of the bispecific antibody according to the invention can be prepared using sodium acetate trihydrate, acetic acid, sodium hydroxide, sorbitol, polysorbate 80, and water for injection, having a pH of 5.5.

[0151] Suitable pharmaceutical compositions of bispecific antibodies according to the invention may comprise a bispecific antibody, 20-40 mM acetate, 140-160 mM sorbitol, and a surfactant (such as polysorbate 80), and have a pH of 5.3-5.6. Suitable pharmaceutical formulations for bispecific antibodies according to the invention may comprise an antibody at a concentration ranging from 5-100 mg / mL, for example, a bispecific antibody at 48 or 60 mg / mL, 30 mM acetate, 150 mM sorbitol, 0.04% w / v polysorbate 80, and have a pH of 5.5. Such formulations may be appropriately diluted with, for example, a formulation buffer to allow for appropriate dosage and subcutaneous administration. In any case, the volume of the pharmaceutical composition is appropriately selected to allow for subcutaneous administration of the antibody. For example, the volume to be administered ranges from about 0.3 mL to 3 mL. The volume to be administered can be 0.5 mL, 0.8 mL, 1 mL, 1.2 mL, 1.5 mL, 1.7 mL, 2 mL, or 2.5 mL. The volume to be administered can be 0.5 mL. The volume to be administered can be 0.8 mL. The volume to be administered can be 1.2 mL. The volume to be administered can be 1.5 mL. The volume to be administered can be 1.7 mL. The volume to be administered can be 2 mL. The volume to be administered can be 2.5 mL. In one embodiment, the preferred volume for sc administration is about 1 mL. In another embodiment, the preferred volume for sc administration is 1 mL.

[0152] As described, the method according to the invention (or the use of the CD3xCD20 antibody) is for treating patients with B-NHL. It is understood that the method according to the invention may be a first-time treatment or part of an initial treatment provided to such patients. However, the patient may have already received prior treatment for B-NHL. Prior treatment may include one or more chemotherapy therapies, radiation therapy, immunotherapy, and targeted therapy, or combinations thereof, but is not limited to these. The most common standards of care include treatment with CD20 monoclonal antibodies, alkylating agents, and anthracyclines, alone or in combination. It is understood that the method and use according to the invention may also be used in combination with other suitable treatments.

[0153] For example, the most common chemotherapy combination for initial treatment of aggressive NHL is called CHOP and contains four drugs: cyclophosphamide, doxorubicin, vincristine, and prednisone. Adding an anti-CD20 monoclonal antibody (such as rituximab) has shown improvement compared to CHOP alone (also known as R-CHOP). Induction with R-CHOP is the standard of care for first-line treatment of DLBCL and is one of the more widely available first-line standard of care treatments in FL. In DLBCL, second-line treatment includes intensive salvage therapy (rituximab / dexamethasone / high-dose cytarabine / cisplatin [R-DHAP], rituximab / ifosfamide / carboplatin / etoposide [R-ICE], or rituximab / gemcitabine / dexamethasone / cisplatin [R-GDP]), followed by high-dose chemotherapy with autologous hematopoietic stem cell transplantation (HDT-ASCT) if chemotherapy is sensitive. For DLBCL patients who are ineligible for intensive rescue therapy and HDT-ASCT due to age or comorbidities, second-line treatments include rituximab / gemcitabine / oxaliplatin (R-GemOx) and rituximab / bendamustine (RB). There is no clear standard of care for advanced relapse of DLBCL, but interventions include allogeneic hematopoietic stem cell transplantation, lenalidomide, ibrutinib, and chimeric antigen receptor T (CAR-T) cell therapy (Chavez et al., Best Pract Res Clin Haematol, 2018 Jun;31(2):135-146).

[0154] Therefore, in a further embodiment, in the method according to the invention, the subject with B-NHL has received at least one line of treatment prior to the treatment according to the invention. In another embodiment, the subject with B-NHL has received one line of treatment prior to the treatment according to the invention. In yet another further embodiment, the subject with B-NHL has received two lines of treatment prior to the treatment according to the invention. In yet another further embodiment, the subject with B-NHL has received three lines of treatment prior to the treatment according to the invention. In yet another further embodiment, the subject with B-NHL has received more than three lines of treatment prior to the treatment according to the invention. In yet another further embodiment, the subject with B-NHL has received one, two, three, or more lines of treatment prior to the treatment according to the invention. In a further embodiment, the prior line of treatment includes R-CHOP. In yet another further embodiment, the prior line of treatment includes CAR-T cell therapy.

[0155] Subjects with B-NHL are classified as having CD20-positive cancer. Therefore, prior cancer treatments received by such subjects may include anti-CD20 mAbs or engineered T cells targeting CD20, such as CAR-T therapy (Schuster SJ, Bishop MR, Tam CS, et al. Tisagenlecleucel in adult relapsed orrefractory diffuse large B-cell lymphoma. N Engl J Med. 2019;380(1):45-56). During such treatment or any other treatment, B-NHL may become refractory or relapse. Therefore, in one aspect of the invention, the subject has received treatment with a CD20 monospecific antibody, such as rituximab, prior to treatment with a bispecific antibody. Furthermore, during the aforementioned prior CD20 monospecific antibody treatment, the B-NHL cancer relapses or becomes refractory to the treatment. Such prior CD20 monospecific antibody treatment may be a treatment in which CD20 monospecific antibodies are used in combination.

[0156] In a further embodiment, the bispecific antibody is administered at a dose of at least 40 mg. In another further embodiment, the bispecific antibody is administered at a dose ranging from 30 mg to 100 mg or from 35 mg to 90 mg. More preferably, in another further embodiment, the bispecific antibody is administered at a dose ranging from 40 mg to 70 mg. In another further embodiment, the bispecific antibody is administered at a dose of at least 48 mg. In another further embodiment, the bispecific antibody is administered at a dose of at least 60 mg. Administering the bispecific antibody at a dose of 60 mg is also contemplated. In another embodiment, the bispecific antibody is administered at a dose of 72 mg. In yet another further embodiment, the bispecific antibody is administered at a dose of 84 mg.

[0157] In a further embodiment, the bispecific antibody is administered at a dose of 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, or 69 mg. In a preferred embodiment, the bispecific antibody is administered at a dose of 48 mg.

[0158] The dosage is preferably administered weekly, and preferably administered (at least) four times. After the weekly administration, the administration interval can be reduced to once every two weeks, also known as bi-weekly administration. Such bi-weekly administration can be performed (at least) six times. After the bi-weekly administration, the interval can be further reduced to once every four weeks. Such bi-weekly administration can be continued for an extended period.

[0159] It is understood that, in the above-mentioned circumstances, the dosage described herein as mentioned above may also be referred to as a full dose or a flat dose, wherein, for example, the weekly dose, the bi-weekly dose, and / or the four-week dose are administered at the same level. Therefore, when a 48 mg dose is selected, it is preferable to administer the same 48 mg dose each week, each bi-weekly dose, and each four-weekly dose. Prior to administering the dose (which may be referred to as a full dose or a flat dose), a priming dose or a priming and subsequent intermediate (second priming) dose may be administered. This can be advantageous as it can effectively reduce the risk and severity of CRS, a side effect that may occur during treatment with the bispecific antibody combining CD3 and CD20 according to the invention. Such priming doses or priming and intermediate doses are lower than flat doses or full doses.

[0160] Therefore, in the method or use according to the invention, an initiating dose of the bispecific antibody may be administered prior to the weekly administration. The initiating dose is preferably administered one week before the first dose of the weekly dose. An initiating dose in the range of 20-2000 µg, preferably in the range of 50-1000 µg, and more preferably in the range of 70-350 µg can be selected. Selectable initiating doses are 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 µg. A preferred initiating dose is 160 µg of the full-length bispecific antibody.

[0161] Furthermore, in the method or use according to the invention, an intermediate dose of the bispecific antibody may be administered after the initiating dose and before the weekly dose. The initiating dose is preferably administered for 2 weeks, and the intermediate dose for 1 week, followed by the first dose of the weekly dose. The intermediate dose is typically selected from a range between the initiating dose and the fixed dose or full dose. The intermediate dose may be selected in the range of 200-8000 µg, or preferably in the range of 400-4000 µg, more preferably in the range of 600-2000 µg. Selectable intermediate doses are 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, or 1600 µg. A preferred intermediate dose is 800 µg of the full-length bispecific antibody.

[0162] Therefore, in another embodiment, an advantageous dosing regimen that may be considered in the method or use of the present invention may include subcutaneous administration of the bispecific antibody over a 28-day cycle, wherein

[0163] a) On days 1, 8, 15 and 22 of the first cycle, the initiating dose is administered on day 1, the intermediate dose on day 8, and a dose of 48 mg is administered on days 15 and 22; for the first cycle; b) Administer a dose of 48 mg on days 1, 8, 15 and 22 of cycle 2-3; c) Administer a dose of 48 mg on days 1 and 15 of cycles 4-9; and d) On day 1 of the subsequent cycle, administer a dose of 48 mg.

[0164] In a further embodiment, advantageous dosing regimens that may be considered in the methods and uses of the present invention may include subcutaneous administration of the bispecific antibody over a 28-day cycle, wherein

[0165] a) On days 1, 8, 15 and 22 of the first cycle, administer an initiating dose of 160 μg on day 1, an intermediate dose of 800 μg on day 8, and a dose of 48 mg on days 15 and 22; for the first cycle. b) Administer a dose of 48 mg on days 1, 8, 15 and 22 of cycle 2-3; c) Administer a dose of 48 mg on days 1 and 15 of cycles 4-9; and d) On day 1 of the subsequent cycle, administer a dose of 48 mg.

[0166] In another embodiment, an advantageous dosing regimen that may be considered in the methods and uses of the present invention may include subcutaneous administration of the bispecific antibody over a 28-day cycle, wherein

[0167] a) On days 1, 8, 15 and 22 of the first cycle, administer the initiating dose on day 1, the intermediate dose on day 8, and the 60 mg dose on days 15 and 22; for the first cycle; b) Administer a dose of 60 mg on days 1, 8, 15 and 22 of cycle 2-3; c) Administer a dose of 60 mg on days 1 and 15 of cycles 4-9; and d) On day 1 of the subsequent cycle, administer a dose of 60 mg.

[0168] In yet another further embodiment, an advantageous dosing regimen that may be considered in the methods and uses of the present invention includes subcutaneous administration of the bispecific antibody over a 28-day cycle, wherein

[0169] a) On days 1, 8, 15 and 22 of the first cycle, administer an initiating dose of 160 μg on day 1, an intermediate dose of 800 μg on day 8, and a dose of 60 mg on days 15 and 22; for the first cycle. b) Administer a dose of 60 mg on days 1, 8, 15, and 22 of cycle 2-3; c) Administer a dose of 60 mg on days 1 and 15 of cycles 4-9; d) On day 1 of the subsequent cycle, administer a dose of 60 mg.

[0170] In one embodiment, alternatively, an initiating dose of 80 µg and an intermediate dose of 800 µg are selected on days 1 and 8 of the first cycle. In another embodiment, alternatively, an initiating dose of 80 µg and an intermediate dose of 1200 µg are selected on days 1 and 8 of the first cycle. In yet another embodiment, an initiating dose of 80 µg and an intermediate dose of 1600 µg are selected on days 1 and 8 of the first cycle. In yet another embodiment, an initiating dose of 160 µg and an intermediate dose of 1600 µg are selected on days 1 and 8 of the first cycle.

[0171] As described, such uses and methods are beneficial for the treatment of B-NHL, and more preferably for the treatment of FL, DLBCL, and / or HGBCL. The treatment according to the invention is maintained continuously using the treatment regimen outlined above. Treatment can be discontinued when disease progression occurs, unacceptable toxicity occurs, or the patient dies.

[0172] Cytokine release syndrome (CRS) may occur when methods and approaches utilizing immune cells and bispecific antibodies (by activating immune effector cells, such as by binding CD3) are used in human subjects (Lee et al., Biol Blood Marrow Transplant. 2019 Apr;25(4):625-638, which is incorporated herein by reference). Therefore, CRS relief is preferred in the methods and uses according to the invention. As part of CRS relief, according to the invention, the initiating dose and / or intermediate dose are highly preferred before administering at least 24 mg of the dose (i.e., the full dose or fixed dose). CRS can be classified according to standard practice (e.g., as outlined in Lee et al., Biol Blood Marrow Transplant. 2019 Apr;25(4):625-638, which is incorporated herein by reference). CRS may include the excessive release of cytokines, such as pro-inflammatory cytokines, such as IL-6, TNF-α, or IL-8, which may lead to adverse reactions such as fever, nausea, vomiting, and chills. Therefore, although bispecific antibodies (such as icoretuzumab) possess unique antitumor activity, their immune response may trigger unwanted "side effects," namely, inducing unwanted inflammatory responses. Consequently, patients may further undergo concomitant treatment, prevention, and / or premedication, such as analgesics, antipyretics, and / or anti-inflammatory drugs, to alleviate possible CRS symptoms.

[0173] In one embodiment, the human subject is treated with prevention and treatment of cytokine release syndrome according to the method and use of the invention. Preferably, such prevention and treatment includes the administration of corticosteroids. In one embodiment, the prevention and treatment is administered on the same day as the bispecific antibody. The prevention and treatment may also be administered on the second day, more preferably on the subsequent second and third days. Optionally, the prevention and treatment may be further administered on the fourth day, and it is understood that the second, third, and fourth days are relative to the administration of the bispecific antibody administered on the first day when in relation to further medication (such as prevention and treatment). Thus, when the antibody is administered and the prevention and treatment are administered, for example, on the 15th day of the cycle, the prevention and treatment on the second, third, and fourth days are the 16th, 17th, and 18th days of the cycle. In a further embodiment, the prevention and treatment is administered on the day of the bispecific antibody administration and on the subsequent second and third days, and the fourth day is optional. When the prevention and treatment are administered on the same day as the bispecific antibody, the prevention and treatment is preferably administered 30-120 minutes before the administration of the bispecific antibody. A potentially preferred corticosteroid is prednisolone, for example, at an intravenous dose of 100 mg or its equivalent, including oral doses.

[0174] Furthermore, in one embodiment, the human subject in the method and use according to the invention is treated with a prophylactic medication to reduce the response to injection. In one embodiment, the prophylactic medication includes the administration of an antihistamine. In another embodiment, the prophylactic medication includes the administration of an antipyretic. In a further embodiment, the prophylactic medication includes a systemic antihistamine and an antipyretic. An optional antihistamine is diphenhydramine, for example, at an intravenous or oral dose of 50 mg or an equivalent thereof. An optional antipyretic is acetaminophen, for example, at an oral dose of 650-1000 mg or an equivalent thereof. More preferably, the prophylactic medication is administered on the same day as the bispecific antibody, more preferably before the bispecific antibody injection, such as 30-120 minutes before the bispecific antibody administration.

[0175] It is understood that the prophylactic medication and / or prophylaxis will be administered at least during the initial phase of treatment. More preferably, during the first four administrations of the bispecific antibody. For example, the prophylaxis may be administered as described above during the first 28-day cycle of bispecific antibody administration. The prophylactic medication is preferably administered during the first cycle.

[0176] Typically, the risk of response during the initial treatment period subsides after several administrations, for example, after the first four administrations (first cycle). Therefore, CRS prevention medication can be discontinued when the human subject has not experienced CRS. However, preferably, CRS prevention can be continued when the human subject experiences CRS greater than grade 1. Similarly, prophylactic medication may optionally be continued.

[0177] In a further embodiment, in the method and use according to the invention as described herein, the prophylaxis is administered during a second 28-day cycle when a human subject experiences a CRS greater than grade 1 after the fourth administration of the bispecific antibody in cycle 1. Furthermore, when a human subject experiences a CRS greater than grade 1 during the last administration of the bispecific antibody in a previous cycle, the prophylaxis continues during subsequent cycles. Any prophylactic medication may optionally be administered during cycle 2. Further prophylactic medication may also optionally be administered during subsequent cycles.

[0178] In one embodiment, the administration of prophylactic medication and prevention for CRS includes an antihistamine, such as diphenhydramine, for example, a 50 mg intravenous or oral dose or an equivalent thereof; an antipyretic, such as acetaminophen, for example, a 650-1000 mg oral dose or an equivalent thereof; and the administration of a corticosteroid, such as prednisolone, for example, a 100 mg intravenous dose or an equivalent thereof. The prophylactic medication and prevention are administered 30-120 minutes prior to the administration of the bispecific antibody. Further prevention is administered on subsequent days 2, 3, and optionally 4, including systemic administration of a corticosteroid, such as prednisolone, for example, a 100 mg intravenous dose or an equivalent thereof. Preferably, the further prevention is administered on subsequent days 2, 3, and 4, including systemic administration of a corticosteroid, such as prednisolone, for example, a 100 mg intravenous dose or an equivalent thereof. According to the invention, such prophylactic medication and prophylaxis are preferably administered during the first four administrations of the bispecific antibody. For example, during the administration of the bispecific antibody in the first 28-day cycle as described herein. Furthermore, subsequent cycles, such as those in which a grade greater than 1 CRS occurs during the last administration of a previous cycle, may include the same administration schedule, wherein the prophylactic medication, as part of the administration schedule, is optional.

[0179] Under the favorable dosage and / or treatment regimens provided herein, CRS can be well managed, while allowing for effective control and / or treatment of B-NHL. As shown in the Examples section, in the methods and uses described herein, manageable cytokine release syndrome (CRS) may occur in the human subjects. Human subjects receiving treatment according to the invention may have Grade 1 CRS as defined in standard practice. Human subjects receiving treatment according to the invention may have Grade 2 manageable CRS as defined in standard practice. Therefore, human subjects receiving treatment according to the invention may have Grade 1 or Grade 2 manageable CRS as defined in standard practice. According to the standard classification of CRS, Grade 1 CRS includes fever to at least 38°C, without hypotension, and without hypoxia, and Grade 2 CRS includes fever to at least 38°C plus hypotension, without the need for vasopressors and / or hypoxia, requiring low-flow nasal intubation or inhaled oxygen. Such manageable CRS may occur in the first cycle. Human subjects receiving treatment according to the invention may also have CRS greater than Grade 2 as defined in standard practice during the treatment period. Therefore, human subjects receiving treatment according to the invention may also have Grade 3 CRS as defined in standard practice during the treatment period. Such manageable CRS may occur further during cycle 1 and subsequent cycles.

[0180] The human subjects may experience fever during the methods and uses according to the invention. Fatigue and injection site reactions may also occur. The human subjects may experience neurotoxicity, partial seizures, CRS-related agraphia, or CRS-related confusion. Furthermore, tumor lysis syndrome may not be observed in the human subjects.

[0181] Due to the safety profile of icoretuzumab, hospitalization may not be necessary for its administration. In a further embodiment, the method and use according to the invention for the treatment of B-NHL involves administering icoretuzumab in an outpatient setting. Administration in an outpatient setting does not involve hospitalization. In another embodiment, the patient is hospitalized only for the first full-dose administration. For example, the first full dose is administered on day 15 of cycle 1, with day 1 and day 8 involving dose escalation as described herein. In the methods and uses described herein, preferred doses of icoretuzumab for outpatient use include a full dose, which can be up to 60 mg. More preferably, the dose administered in an outpatient setting is 48 mg. In another embodiment, the method and use according to the invention for the treatment of B-NHL involves administering icoretuzumab solely in an outpatient setting.

[0182] As described herein, in the methods and uses according to the invention, the bispecific antibody according to the invention, intended to target CD3 and CD20, comprises: (i) A first binding arm comprising a first antigen-binding region binding to human CD3ε (epsilon), comprising a variable heavy chain region and a variable light chain region, wherein the variable heavy chain region comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:6; and wherein the variable light chain region comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:7; and (ii) A second binding arm comprising a second antigen-binding region that binds to human CD20, comprising a variable heavy chain region and a variable light chain region, wherein the variable heavy chain region comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID No:13; and wherein the variable light chain region comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID No:14.

[0183] The CDR1, CDR2, and CDR3 regions can be identified from the variable heavy and light chain regions using methods known in the art. The CDR regions of the variable heavy and light chain regions can be annotated according to IMGT (see Lefranc MP. et al., NucleicAcids Research, 27, 209-212, 1999] and Brochet X. Nucl. Acids Res. 36, W503-508 (2008)). Therefore, it is also disclosed in the methods and uses according to the invention that a bispecific antibody according to the invention, intended to target CD3 and CD20, comprises: (i) A first binding arm comprising a first antigen-binding region binding to human CD3ε (epsilon), wherein the first antigen-binding region comprises heavy chain variable (VH) regions CDR1, CDR2, and CDR3 having sequences as listed in SEQ ID NO. 1, 2, and 3, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 having sequences as listed in SEQ ID NO. 4, sequence GTN, and sequences as listed in SEQ ID NO. 5, respectively. (ii) A second binding arm comprising a second antigen-binding region that binds to human CD20, wherein the second antigen-binding region comprises heavy chain variable (VH) regions CDR1, CDR2, and CDR3 having sequences as listed in SEQ ID NO. 8, 9, and 10, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 having sequences as listed in SEQ ID NO. 11, sequence DAS, and sequences as listed in SEQ ID NO. 12, respectively.

[0184] In a further embodiment, in the method and use according to the invention, the bispecific antibody comprises

[0185] (i) a first binding arm comprising a first antigen-binding region binding to human CD3ε (epsilon), comprising a variable heavy chain region and a variable light chain region, wherein the variable heavy chain region comprises SEQ ID NO:6; and wherein the variable light chain region comprises SEQ ID NO:7; and

[0186] (ii) A second binding arm comprising a second antigen-binding region that binds to human CD20, comprising a variable heavy chain region and a variable light chain region, wherein the variable heavy chain region comprises SEQ ID No:13; and wherein the variable light chain region comprises SEQ ID No:14.

[0187] As described, the bispecific antibody according to the invention is most preferably a full-length antibody and may have an inert Fc region. More preferably, the first binding arm of CD3 is derived from a humanized antibody, preferably from a full-length IgG1,λ (lambda) antibody, such as H1L1 described in WO2015001085 (which is incorporated herein by reference), and / or the second binding arm of CD20 is derived from a human antibody, preferably from a full-length IgG1,κ (kappa) antibody, such as clone 7D8 described in WO2004035607 (which is incorporated herein by reference). The bispecific antibody may be generated from two half-molecule antibodies. Each of the two half-molecule antibodies contains, for example, the respective first and second binding arms listed in SEQ ID NO. 6 and 7 and SEQ ID NO. 13 and 14. The half-antibodies may be generated in CHO cells, and the bispecific antibody may be generated by, for example, Fab arm exchange. In one embodiment, the bispecific antibody according to the invention is a functional variant of Duobody CD3xCD20.

[0188] Various constant regions or variations thereof may be considered according to the invention. However, most preferably, the bispecific antibody according to the invention comprises a human IgG1 constant region, such as the human IgG1 constant region defined in SEQ ID NO. 15, or any other suitable IgG1 allotype. As stated, the first binding arm of the bispecific antibody is preferably derived from a humanized antibody, more preferably from a full-length IgG1,λ (lambda) antibody, and therefore comprises a λ light chain constant region. More preferably, the first binding arm as defined herein comprises a λ light chain constant region as defined in SEQ ID NO. 22. Furthermore, the second binding arm of the bispecific antibody is derived from a human antibody, preferably from a full-length IgG1,κ (kappa) antibody, and therefore comprises a κ light chain constant region. More preferably, the second binding arm comprises a κ light chain constant region as defined in SEQ ID NO. 23.

[0189] It is understandable that, because the constant region is part of the bispecific antibody, the constant region may contain modifications to allow for efficient formation / production of the bispecific antibody and / or to provide an inert Fc region. Such modifications are known in the art.

[0190] Different formats and uses of bispecific antibodies are known in the art (reviewed by Kontermann; Drug Discov Today, 2015 Jul;20(7):838-47 and; MAbs, 2012 Mar-Apr;4(2):182-97) and are not necessarily limited to any particular bispecific format or method of production. For example, bispecific antibodies may include, but are not limited to: (i) bispecific antibodies having complementary CH3 domains to force heterodimerization; (ii) so-called knob-in-pore molecules (Genentech, WO9850431), CrossMAbs (Roche, WO2011117329) or electrostatically matched molecules (Amgen, EP1870459 and WO2009089004; Chugai, US201000155133; Oncomed, WO2010129304).

[0191] Preferably, the bispecific antibody of the present invention comprises an Fc region, including 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 between the first and second CH3 regions. Further details regarding these interactions and how these interactions are achieved are provided in WO2011131746 and WO2013060867 (Genmab), which are incorporated herein by reference. In a preferred embodiment, the bispecific antibody of the method and use according to the present invention comprises (i) amino acid L at position F405 in the human IgG1 heavy chain of SEQ ID NO:15 in the first heavy chain, and amino acid R at position K409 in the human IgG1 heavy chain of SEQ ID NO:15 in the second heavy chain, and vice versa.

[0192] According to the present invention, antibodies may contain modifications to the Fc region to render the Fc region of the antibody inert or inactive. Thus, the bispecific antibodies disclosed herein may be modified with one or both heavy chains such that they induce Fc-mediated effector function to a lesser extent compared to bispecific antibodies without such modifications. Fc-mediated effector function can be measured by determining Fc-mediated CD69 expression on T cells (i.e., CD69 expression resulting from CD3 antibody-mediated Fcγ receptor-dependent CD3 crosslinking), by binding to the Fcγ receptor, by binding to C1q, or by inducing Fc-mediated FcγR crosslinking. In particular, the heavy chain constant sequence can 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% compared to wild-type (unmodified) antibodies, wherein said Fc-mediated CD69 expression is determined in a PBMC-based functional assay, such as that described in Example 3 of WO2015001085. Modification of the constant sequences of the heavy and light chains may also result in reduced binding of C1q to the antibody. This reduction can be at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% compared to the unmodified antibody, and can be measured by ELISA for C1q binding. Furthermore, the Fc region can be modified such that Fc-mediated T cell proliferation of the antibody is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% compared to the unmodified antibody, wherein the T cell proliferation is measured in a PBMC-based functional assay. Examples of amino acid positions that can be modified include, for example, positions L234 and L235 in IgG1 isotype antibodies. Therefore, the antibody according to the invention can comprise first and second heavy chains, wherein in the first and second heavy chains, the amino acid residues corresponding to positions L234 and L235 in the human IgG1 heavy chain according to Eu numbering are F and E, respectively. Furthermore, the D265A amino acid substitution can reduce binding to all Fcγ receptors and prevent ADCC (Shields et al., 2001, J. Biol. Chem. (276):6591-604). Therefore, the antibody according to the invention can comprise first and second heavy chains, wherein in the first and second heavy chains, the amino acid residue corresponding to position D265 in the human IgG1 heavy chain according to Eu number is A.

[0193] In the method and use according to the invention, a most preferred bispecific antibody is provided, wherein the first and second heavy chains have amino acids F, E, and A at positions L234, L235, and D265 corresponding to the human IgG1 heavy chain, respectively. Antibodies having these amino acids at these positions are examples of antibodies having an inert or non-activated Fc region.

[0194] In this application, antibodies having a combination of three amino acid substitutions L234F, L235E and D265A, and additionally the K409R or F405L mutations disclosed herein, may be named with the suffixes “FEAR” or “FEAL”, respectively.

[0195] As described herein, the amino acid sequence of the wild-type IgG1 heavy chain constant region is identified as SEQ ID NO: 15. Consistent with the embodiments disclosed above, the antibody of the present invention may comprise an IgG1 heavy chain constant region carrying an F405L substitution, and may have the amino acid sequence listed in SEQ ID NO: 17 and / or an IgG1 heavy chain constant region carrying a K409R substitution, and may have the amino acid sequence listed in SEQ ID NO: 18, and have further substitutions that render the Fc region inert or inactive. Therefore, the most and highly preferred combination of IgG1 heavy chain constant regions included in the bispecific antibody according to the present invention is an amino acid sequence of an IgG1 heavy chain constant region carrying L234F, L235E, D265A and F405L substitutions (as identified herein as SEQ ID NO: 19) and an amino acid sequence of an IgG1 heavy chain constant region carrying L234F, L235E, D265A and K409R substitutions (as identified herein as SEQ ID NO: 20).

[0196] In a further highly preferred embodiment, the bispecific antibody used in the methods and uses described herein comprises a first binding arm having a heavy chain and a light chain as defined in SEQ ID NO. 24 and 25, respectively, and a second binding arm having a heavy chain and a light chain as defined in SEQ ID NO. 26 and 27. Such an antibody may be referred to herein as DuobodyCD3xCD20. Furthermore, variants of such antibodies may be considered for use in the methods and uses described herein. In yet another further embodiment, the bispecific antibody according to the invention is icoretuzumab (CAS 2134641-34-0) or a biosimilar thereof.

[0197] Further implementation plan

[0198] 1. Bispecific antibodies, which contain

[0199] (i) A first binding arm comprising a first antigen-binding region binding to human CD3ε (epsilon), comprising a variable heavy chain region and a variable light chain region, wherein the variable heavy chain region comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:6; and wherein the variable light chain region comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:7; and

[0200] (ii) A second binding arm comprising a second antigen-binding region that binds to human CD20, comprising a variable heavy chain region and a variable light chain region, wherein the variable heavy chain region comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID No:13; and wherein the variable light chain region comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID No:14; It is used to treat B-cell non-Hodgkin lymphoma (B-NHL) in human subjects, wherein the treatment comprises administering the bispecific antibody subcutaneously to the human subject in need at a dose of at least 24 mg.

[0201] 2. A bispecific antibody, which is a full-length antibody, wherein the bispecific antibody comprises...

[0202] (i) A first binding arm comprising a first antigen-binding region binding to human CD3ε (epsilon), comprising a variable heavy chain region and a variable light chain region, wherein the variable heavy chain region comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:6; and wherein the variable light chain region comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:7; and

[0203] (ii) A second binding arm comprising a second antigen-binding region that binds to human CD20, comprising a variable heavy chain region and a variable light chain region, wherein the variable heavy chain region comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID No:13; and wherein the variable light chain region comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID No:14; It is used to treat B-cell non-Hodgkin lymphoma (B-NHL) in human subjects, wherein the treatment comprises administering the bispecific antibody subcutaneously to the human subject in need at a dose of at least 24 mg.

[0204] 3. The bispecific antibody used according to implementation scheme 1 or 2, wherein the B-NHL is selected from diffuse large B-cell lymphoma (DLBCL), high-grade B-cell lymphoma (HGBCL), primary mediastinal large B-cell lymphoma (PMBCL), mantle cell lymphoma, follicular lymphoma (FL), marginal zone lymphoma, and small lymphocytic lymphoma.

[0205] 4. The bispecific antibody used according to embodiment 1 or 2, wherein the B-NHL is FL, HGBCL or DLBCL.

[0206] 5. A bispecific antibody used according to any one of embodiments 1-4, wherein the subject has received CD20 monospecific antibody, such as rituximab, prior to treatment with the bispecific antibody.

[0207] 6. The bispecific antibody used according to embodiment 5, wherein the cancer recurs during treatment with the CD20 monospecific antibody.

[0208] 7. The bispecific antibody used according to embodiment 5, wherein the cancer is refractory to the treatment during the treatment with the CD20 monospecific antibody.

[0209] 8. A bispecific antibody used according to any one of embodiments 5-7, wherein the CD20 monospecific antibody is used in combination therapy.

[0210] 9. A bispecific antibody used according to any one of embodiments 1-8, wherein the patient has received a further prior line of treatment for said B-cell NHL.

[0211] 10. A bispecific antibody used according to any one of embodiments 1-9, wherein the bispecific antibody is administered at a dose of at least 40 mg.

[0212] 11. The bispecific antibody used according to any one of embodiments 1-9, wherein the bispecific antibody is administered at a dose ranging from 40 mg to 100 mg.

[0213] 12. A bispecific antibody used according to any one of embodiments 1-9, wherein the bispecific antibody is administered at a dose of at least 48 mg.

[0214] 13. The bispecific antibody used according to any one of embodiments 1-9, wherein the bispecific antibody is administered at a dose of at least 60 mg.

[0215] 14. The bispecific antibody used according to any one of embodiments 1-9, wherein the bispecific antibody is administered at a dose of 48 mg.

[0216] 15. A bispecific antibody used according to any one of embodiments 1-9, wherein the bispecific antibody is administered at a dose of 60 mg.

[0217] 16. A bispecific antibody used according to any one of embodiments 1-15, wherein the dose is administered weekly.

[0218] 17. The bispecific antibody used according to implementation scheme 16, wherein the weekly administration is performed at least 4 times.

[0219] 18. The bispecific antibody used according to embodiment 16 or 17, wherein the antibody is administered every two weeks after the weekly administration.

[0220] 19. The bispecific antibody used according to embodiment 18, wherein the bi-weekly administration is performed (at least) 6 times.

[0221] 20. The bispecific antibody used according to embodiment 18 or 19, wherein the antibody is administered every four weeks after the administration every two weeks.

[0222] 21. The bispecific antibody used according to any one of embodiments 16-20, wherein the initiation dose of the bispecific antibody is administered prior to the administration of the weekly dose.

[0223] 22. The bispecific antibody used according to embodiment 21, wherein the initiating dose is administered one week prior to the first dose of the weekly dose.

[0224] 23. The bispecific antibody used according to embodiment 21 or 22, wherein the initiating dose is in the range of 50-350 μg.

[0225] 24. The bispecific antibody used according to any one of embodiments 21-23, wherein the initiating dose is 160 μg.

[0226] 25. The bispecific antibody used according to embodiments 21-24, wherein an intermediate dose of the bispecific antibody is administered after the initiating dose and before the weekly dose.

[0227] 26. The bispecific antibody used according to embodiment 25, wherein the initiating dose is administered two weeks prior to the administration of the first dose of the weekly dose, and the intermediate dose is administered one week prior to the administration of the first dose of the weekly dose.

[0228] 27. The bispecific antibody used according to any one of embodiments 25 or 26, wherein the intermediate dose is in the range of 600-1200 μg.

[0229] 28. The bispecific antibody used according to any one of embodiments 25-27, wherein the intermediate dose is 800 μg.

[0230] 29. The bispecific antibody used according to any one of embodiments 25-28, wherein the use includes subcutaneous administration of the bispecific antibody over a 28-day cycle, wherein: a) On days 1, 8, 15 and 22 of the first cycle, the initiating dose is administered on day 1, the intermediate dose on day 8, and a dose of 48 mg is administered on days 15 and 22; for the first cycle; b) Administer a dose of 48 mg on days 1, 8, 15 and 22 of cycle 2-3; c) Administer a dose of 48 mg on days 1 and 15 of cycles 4-9; and d) On day 1 of the subsequent cycle, administer a dose of 48 mg.

[0231] 30. The bispecific antibody used according to any one of embodiments 25-28, wherein the use includes subcutaneous administration of the bispecific antibody over a 28-day cycle, wherein: a) On days 1, 8, 15 and 22 of the first cycle, administer an initiating dose of 160 μg on day 1, an intermediate dose of 800 μg on day 8, and a dose of 48 mg on days 15 and 22; for the first cycle. b) Administer a dose of 48 mg on days 1, 8, 15 and 22 of cycle 2-3; c) Administer a dose of 48 mg on days 1 and 15 of cycles 4-9; and d) On day 1 of the subsequent cycle, administer a dose of 48 mg.

[0232] 31. The bispecific antibody used according to any one of embodiments 25-28, wherein the use includes subcutaneous administration of the bispecific antibody over a 28-day cycle, wherein: a) On days 1, 8, 15 and 22 of the first cycle, administer the initiating dose on day 1, the intermediate dose on day 8, and the 60 mg dose on days 15 and 22; for the first cycle; b) Administer a dose of 60 mg on days 1, 8, 15 and 22 of cycle 2-3; c) Administer a dose of 60 mg on days 1 and 15 of cycles 4-9; and d) On day 1 of the subsequent cycle, administer a dose of 60 mg.

[0233] 32. A bispecific antibody used according to any one of embodiments 25-28, wherein the treatment method includes subcutaneous administration of the bispecific antibody over a 28-day cycle, wherein: a) On days 1, 8, 15 and 22 of the first cycle, administer an initiating dose of 160 μg on day 1, an intermediate dose of 800 μg on day 8, and a dose of 60 mg on days 15 and 22; for the first cycle. b) Administer a dose of 60 mg on days 1, 8, 15, and 22 of cycle 2-3; c) Administer a dose of 60 mg on days 1 and 15 of cycles 4-9; d) On day 1 of the subsequent cycle, administer a dose of 60 mg.

[0234] 33. The bispecific antibody used according to any one of embodiments 29-32, wherein the B-NHL is FL, DLBCL and / or HGBCL.

[0235] 34. A bispecific antibody used according to any one of embodiments 1-33, wherein the subject has grade 1 or 2 manageable cytokine release syndrome (CRS) during the period of said use.

[0236] 35. A bispecific antibody used according to any one of embodiments 1-34, wherein the subject does not experience tumor lysis syndrome.

[0237] 36. A bispecific antibody used according to any one of embodiments 1-35, wherein the subject is treated with prevention and treatment of cytokine release syndrome (CRS).

[0238] 37. The bispecific antibody used according to embodiment 36, wherein the prevention and treatment includes the administration of corticosteroids.

[0239] 38. The bispecific antibody used according to implementation schemes 36-37, wherein the prevention and treatment are administered on the same day as the bispecific antibody.

[0240] 39. The bispecific antibody used according to any of embodiments 38, wherein the prevention is administered on the 2nd-3rd day and optionally the 4th day, or the 2nd-4th day thereafter.

[0241] 40. The bispecific antibody used according to any one of embodiments 38-39, wherein when the prevention and treatment are applied on the same day as the bispecific antibody, the prevention and treatment are applied 30-120 minutes before the application of the bispecific antibody.

[0242] 41. A bispecific antibody used according to any one of embodiments 37-40, wherein the corticosteroid is prednisolone, for example, at an intravenous dose of 100 mg, or an equivalent thereof, including an oral dose.

[0243] 42. A bispecific antibody used according to any one of embodiments 1-41, wherein the human subject is treated with a prophylactic drug to reduce the response to the injection.

[0244] 43. The bispecific antibody used according to embodiment 42, wherein the precursor medication includes the administration of an antihistamine.

[0245] 44. The bispecific antibody used according to any one of embodiments 42-43, wherein the precursor medication includes the administration of an antipyretic.

[0246] 45. A bispecific antibody used according to any one of embodiments 42-44, wherein the antihistamine is diphenhydramine, for example, in an intravenous or oral dose of 50 mg, or an equivalent thereof.

[0247] 46. ​​A bispecific antibody used according to any one of embodiments 43-45, wherein the antipyretic is acetaminophen, for example, an oral dose of 650-1000 mg, or an equivalent thereof.

[0248] 47. A bispecific antibody used according to any one of embodiments 42-45, wherein the precursor drug is administered on the same day as the bispecific antibody.

[0249] 48. The bispecific antibody used according to embodiment 47, wherein the precursor drug is administered 30-120 minutes before the administration of the bispecific antibody.

[0250] 49. A bispecific antibody used according to any one of embodiments 29-48, wherein the prevention and treatment as defined in embodiments 35-41 are administered during the first cycle.

[0251] 50. A bispecific antibody used according to any one of embodiments 29-49, wherein the precursor drug as defined in embodiments 42-48 is administered during the first cycle.

[0252] 51. The bispecific antibody used according to embodiments 49 and 50, wherein the prevention is administered during the second cycle when the human subject experiences a CRS greater than grade 1 after the fourth administration of the bispecific antibody in cycle 1.

[0253] 52. The bispecific antibody used according to embodiment 51, wherein the prevention continues during subsequent cycles when a human subject experiences a CRS greater than grade 1 during the last administration of the bispecific antibody in a previous cycle.

[0254] 53. The bispecific antibody used according to embodiment 51 or 52, wherein the precursor drug is optionally administered during the second cycle.

[0255] 54. The bispecific antibody used according to embodiment 53, wherein the precursor drug is optionally administered during a subsequent cycle.

[0256] 55. The bispecific antibody used according to any of the foregoing embodiments, wherein the bispecific antibody comprises: (i) A first binding arm comprising a first antigen-binding region binding to human CD3ε (epsilon), wherein the first antigen-binding region comprises heavy chain variable (VH) regions CDR1, CDR2, and CDR3 having sequences as listed in SEQ ID NO. 1, 2, and 3, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 having sequences as listed in SEQ ID NO. 4, sequence GTN, and sequences as listed in SEQ ID NO. 5, respectively. (ii) A second binding arm comprising a second antigen-binding region that binds to human CD20, wherein the second antigen-binding region comprises heavy chain variable (VH) regions CDR1, CDR2, and CDR3 having sequences as listed in SEQ ID NO. 8, 9, and 10, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 having sequences as listed in SEQ ID NO. 11, sequence DAS, and sequences as listed in SEQ ID NO. 12, respectively.

[0257] 56. The bispecific antibody used according to any of the foregoing embodiments, wherein the bispecific antibody comprises: (i) a first binding arm comprising a first antigen-binding region that binds to human CD3ε (epsilon), comprising a variable heavy chain region defined by SEQ ID NO:6 and a variable light chain region defined by SEQ ID NO:7; and (ii) A second binding arm comprising a second antigen-binding region that binds to human CD20, comprising a variable heavy chain region defined by SEQ ID No:13 and a variable light chain region defined by SEQ ID No:14.

[0258] 57. The bispecific antibody used according to any of the foregoing embodiments, wherein the first binding arm of the bispecific antibody is derived from a humanized antibody, preferably derived from a full-length IgG1,λ (lambda) antibody.

[0259] 58. The bispecific antibody used according to embodiment 57, wherein the bispecific antibody comprises a λ light chain constant region as defined in SEQ ID NO. 22.

[0260] 59. The bispecific antibody used according to any of the foregoing embodiments, wherein the second binding arm of the bispecific antibody is derived from a human antibody, preferably from a full-length IgG1,κ (kappa) antibody.

[0261] 60. The bispecific antibody used according to embodiment 59, wherein the bispecific antibody comprises a constant region of the κ light chain as defined in SEQ ID NO. 23.

[0262] 61. The bispecific antibody used according to any of the foregoing embodiments, wherein the bispecific antibody is a full-length antibody having a constant region of human IgG1.

[0263] 62. The bispecific antibody used according to any of the foregoing embodiments, wherein the bispecific antibody comprises an inert Fc region.

[0264] 63. The bispecific antibody used according to embodiment 62, wherein the bispecific antibody contains amino acids F, E and A at positions L234, L235 and D265 in the human IgG1 heavy chain of SEQ ID NO:15, respectively, in the first and second heavy chains.

[0265] 64. The bispecific antibody used according to any of the foregoing embodiments, wherein the bispecific antibody contains amino acid L at position F405 in the human IgG1 heavy chain of SEQ ID NO:15 in the first heavy chain, and wherein the second heavy chain contains amino acid R at position K409 in the human IgG1 heavy chain of SEQ ID NO:15, or vice versa.

[0266] 65. The method according to any one of the foregoing embodiments, wherein the bispecific antibody comprises amino acids F, E, and A at positions L234, L235, and D265 of the human IgG1 heavy chain of SEQ ID NO:15 in the first and second heavy chains, respectively, and wherein amino acid L in the first heavy chain corresponds to position F405 of the human IgG1 heavy chain of SEQ ID NO:15, and wherein the second heavy chain comprises amino acid R at position K409 of the human IgG1 heavy chain of SEQ ID NO:15, and vice versa.

[0267] 66. The bispecific antibody used according to embodiment 65, wherein the bispecific antibody comprises a constant region as defined in SEQ ID NO. 19 and 20.

[0268] 67. The bispecific antibody used according to any of the foregoing embodiments, wherein the bispecific antibody comprises a heavy chain and a light chain as defined in SEQ ID NO. 24 and 25, and a heavy chain and a light chain as defined in SEQ ID NO. 26 and 27, respectively.

[0269] 68. The bispecific antibody used according to any of the foregoing embodiments, wherein the bispecific antibody comprises a heavy chain and a light chain as defined in SEQ ID NO. 24 and 25, and a heavy chain and a light chain as defined in SEQ ID NO. 26 and 27, respectively.

[0270] 69. The bispecific antibody used according to any of the foregoing embodiments, wherein the bispecific antibody is icoretuzumab or a biosimilar thereof.

[0271] sequence

[0272] Table 1. Bold and underlined text corresponds to FE; A; L and R at positions 234 and 235; 265; 405 and 409, respectively, according to EU numbering. In the variable area, the CDR area annotated according to the IMGT definition is underlined.

[0273]

[0274]

[0275]

[0276] Example

[0277] DuoBody-CD3xCD20

[0278] DuoBody-CD3xCD20 is a bsAb that recognizes T cell antigen CD3 and B cell antigen CD20. DuoBody-CD3xCD20 triggers T cell-mediated potent killing of CD20-expressing cells. DuoBody-CD3xCD20 has a regular IgG1 structure.

[0279] Two parental antibodies, IgG1-CD3-FEAL (a humanized IgG1λ, CD3ε specific antibody with heavy and light chain sequences as listed in SEQ ID NO. 24 and 25, respectively), and IgG1-CD20-FEAR (derived from human IgG1κCD20 specific antibody 7D8 with heavy and light chain sequences as listed in SEQ ID NO. 26 and 27, respectively), were manufactured as separate biological intermediates. Each parental antibody contained one of the complementary mutations in the CH3 domain required for the generation of the DuoBody molecule (F405L and K409R, respectively). The parental antibodies contained three additional mutations in the Fc region (L234F, L235E, and D265A; FEA). The parental antibodies were generated in the mammalian Chinese hamster ovary (CHO) cell line using standard suspension cell culture and purification techniques. DuoBody-CD3xCD20 was subsequently manufactured via a controlled Fab arm exchange (cFAE) process (Labrijn et al. 2013, Labrijn et al. 2014, Gramer et al. 2013). Parental antibodies were mixed and subjected to controlled reduction conditions. This resulted in the separation of the parental antibodies, which were then reassembled under re-oxidation. In this manner, a high-purity DuoBody-CD3xCD20 preparation (approximately 93-95%) was obtained. Further purification yielded the final product, approaching 100% purity. The theoretical extinction coefficient was used. = 1.597 mL mg -1 cm -1 The concentration of DuoBody-CD3xCD20 was measured by absorbance at 280 nm. The final product was stored at 4°C. The glycosylation profile of DuoBody-CD3xCD20 showed that it mainly contains core fucosylated N-linked bi-antennary glycans with varying degrees of terminal galactose. The produced product has been granted the international patent name, Icarusotumab.

[0280] DuoBody-CD3xCD20 (5 mg / mL or 60 mg / mL) is prepared as a colorless to pale yellow sterile transparent solution and provided as a concentrate for subcutaneous (SC) injection. DuoBody-CD3xCD20 contains buffers and tonicating agents. For the subcutaneous injection product, all excipients (sodium acetate trihydrate, acetic acid, sodium hydroxide, sorbitol, polysorbate 80, water for injection) and their amounts in the formulation are pharmaceutically acceptable. The appropriate dose is reconstituted to approximately 1 mL for subcutaneous injection.

[0281] Clinical trials: dose escalation

[0282] GCT3013-01 (NCT03625037) is an open-label, two-part (dose escalation and expansion) trial in patients aged 18 years or older with documented CD20+ mature B-cell vegetations and relapsed and / or refractory B-cell NHL.

[0283] A fixed dosing regimen was chosen to simplify dosing because the expected number of T cells (i.e., effector cells functioning GEN3013) is independent of patient weight and / or body surface area. The EMA Guideline on the Evaluation of Anti-cancer Medicinal Products in Man (EMA, 2012; EMA, 2017) acknowledges the weak scientific support for body size-based dosing and recommends using ia PK data for modeling and simulation to determine dosing strategies. Therefore, the purpose of the dose escalation section in this article is to establish the maximum tolerated dose (MTD) and / or achieve a dosing strategy for icoretuzumab.

[0284] The dose escalation portion includes accelerated and standard titrations. The initial dose level is for a single subject cohort (accelerated titration), with the option to expand the cohort to up to two additional subjects to obtain additional pharmacokinetic (PK) / pharmacodynamic and biomarker data. The standard titration portion includes a standard cohort of three subjects for dose-limiting toxicity (DLT) assessment, with at least two evaluable subjects enrolled if none experienced ≥ grade 2 toxicity during the DLT assessment period (i.e., cycle 1, day 28). Dose escalations are based on all available data, and the increments in the accelerated titration portion do not exceed half log10 (3.2-fold) and the increments in the standard titration portion do not exceed 100% (2-fold).

[0285] In the dose-escalation portion, 61 subjects are currently receiving treatment (Table 2). In the initial cohorts 1–5, subjects were initially exposed to a full (fixed) dose of icoretuzumab followed by a lower priming dose. In addition to the priming dose, an intermediate dose (1.5 mg) was introduced in cohort 6 to minimize the gap between the priming dose and the continuously escalating full dose, and to reduce the risk and incidence of cytokine release syndrome (CRS). No DLT occurred, and continued enrollment at doses from 0.004 mg (priming dose in cohort 1) to 48 mg (full dose in cohorts 11 and 11A) was reviewed as safe. MTD has not been reached.

[0286] Table 2 lists the planned cycle 1 doses administered to the subject cohorts. For example, in cohort 8, a trigger dose of 0.04 mg was administered on day 1 of the first 28-day cycle, an intermediate dose of 0.5 mg was administered on day 8, followed by a full (fixed) dose of 6 mg on days 15 and 22. Subsequent 28-day cycles (if administered) consist of fixed doses administered on days 1, 8, 15, and 22 of the 28-day cycle in cycle 2; fixed doses administered on days 1 and 15 of the 28-day cycle from cycles 3–6 onwards; and fixed doses administered on day 1 of the 28-day cycle from cycle 7 onwards.

[0287] Table 2. Planned dosing for each cohort in GCT3013-01

[0288] The prophylactic medication for preventing acute injection reactions consists of diphenhydramine 50 mg IV or oral (PO) (or equivalent) administered 30–120 minutes before icostatumab administration during cycle 1, and acetaminophen 650–1000 mg PO (or equivalent). For the first four doses of icostatumab in cycle 1, CRS remission medication consists of prednisolone 100 mg IV (or equivalent oral or IV dose of corticosteroid) administered 30–120 minutes before icostatumab administration, followed by additional prednisolone 100 mg IV (or equivalent, including oral doses) on days 2 and 3 after icostatumab administration (i.e., corticosteroids for a total of 3 consecutive days). If grade CRS higher than 1 occurs after the fourth icostatumab administration, prophylactic medication with the selected prophylactic medication is continued during cycle 2 (days 1, 2, and 3) and thereafter.

[0289] Other CD3-targeting compounds or drugs have been reported to cause neurological symptoms ranging from confusion to fatal cerebral edema. Therefore, in this study, neurological symptoms were closely monitored at each dose, at the end of treatment, and when clinically indicated, using relevant assessment tools originally developed for chimeric antigen receptor T-cell (CAR-T) therapy, the CAR-T cell-related toxicity 10-point neurological assessment (CARTOX-10), and the American Society for Transplantation and Cell Therapy (ASCTC) Immune Effector Cell-Related Neurotoxicity Syndromes (ICANS) assessment. The CRS prevention regimen covers the administration of medications to reduce the risk and severity of ICANS associated with cytokine release, namely, prednisolone 100 mg IV (or equivalent, including oral dose), diphenhydramine 50 mg IV or oral (PO) (or equivalent) and acetaminophen 650-1000 mg PO (or equivalent) on the day of icoretuzumab administration, followed by additional prednisolone 100 mg IV (or equivalent, including oral dose) on days 2 and 3 after icoretuzumab administration (i.e., prednisolone for a total of 3 consecutive days).

[0290] Demography

[0291] Among the various B-cell NHL subtypes enrolled, patients were classified according to the WHO 2008 or WHO 2016 guidelines (Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues (ed 4th). Lyon, France: IARC Press; 2008;Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised ed 4th). Because there is overlap in the classification of DLBCL and HGBCL between the WHO 2016 and WHO 2008 guidelines, patients diagnosed with either DLBCL or HGBCL were defined as belonging to the DLBCL / HGBCL group.

[0292] Of the 61 patients treated for various B-cell NHL subtypes, 45 (73.8%) had disease classified as DLBCL / HGBCL, and 11 (18.0%) had FL. Other B-cell NHL subtypes included MCL (3 patients), MZL (1 patient), and SLL (1 patient).

[0293] Baseline disease characteristics data from 58 patients were available in clinical databases. Most patients were in Ann Arbor stage III (25.9%) or IV (51.7%) at screening, and the majority had extranodal disease (58.6%). The median time from lymphoma diagnosis was 26.7 months (range 6–330 months), and the median time from last relapse or progression was 1.6 months (range 0–88 months). Most patients were refractory to last-line therapy (67.2%) and / or most recent anti-CD20 therapy (63.8%), and 6.9% had prior CAR T therapy. The median number of prior lines of treatment was 3 (range 1–18), most commonly rituximab (100%), alkylating agents (98.3%), and anthracyclines (89.7%); 23 subjects (39.7%) had >3 prior lines of treatment.

[0294] Clinical efficacy

[0295] According to the Lugano criteria (Cheson, et al. (2014). J Clin Oncol 32, 3059-3068), response to treatment is assessed by radiographic imaging at specific time points, with tissue biopsies performed as necessary, including assessment of metabolic activity on FDG-PET, evaluation of a limited number of target lesion sizes by CT / MRI, and the presence of any new lesions. Patients with FDG-high uptake tumors at screening are subsequently evaluated for disease using FDG-PET-CT, for example, with the Deauville 5-point scale (DS) (Barrington, et al. (2014) J Clin Oncol 32, 3048-3058). For patients with non-high uptake or variable FDG-high uptake tumors, CT scans with IV contrast imaging of the neck / chest / abdomen / pelvis / other known lesions, or MRI in some cases, are performed to evaluate response. Response criteria include calculating the sum of the products of the vertical diameters (SPD) of up to six measurable major lesions and comparing changes in this SPD with each subsequent scan, and considering the presence of any new lesions. When performing FDG-PET, total DS is applied based on the target lesion showing the highest uptake. In some cases, a tissue biopsy is required to clarify or confirm the response.

[0296] Table 3 shows the best overall response (BOR) for the evaluable population (n=53), including subjects who received at least one dose of the investigational drug and at least one post-baseline response assessment across all histological and dose profiles, or who died as of May 5, 2020. One subject who underwent coronary artery bypass grafting (CABG) during the study and one subject who contracted COVID-19 and subsequently died were excluded. The overall response rate (ORR) was 34.0%, and the disease control rate (DCR) was 52.8%.

[0297] Table 3. Best overall response of trial GCT3013-01, according to Lugano classification as assessed by the investigator.

[0298]

[0299] Among evaluable subjects with R / R DLBCL / HGBCL who received at least a full dose of 6.0 mg, 8 / 19 (42.1%) responded, with 4 (21.1%) achieving CR, 4 (21.1%) achieving PR, and 4 (21.1%) of the remaining subjects achieving SD. Among evaluable subjects with FL who received at least a full dose of 0.76 mg at the data cutoff, 7 / 8 (87.5%) achieved PR and 1 / 8 (12.5%) achieved SD.

[0300] The table below lists the maximum reduction in target lesions (i.e., the best percentage change in SPD across all target lesions) and the investigator-defined BOR for subjects receiving DLBCL / HGBCL (full dose ≥6 mg) and FL (full dose ≥0.76 mg).

[0301] Table 4. Efficacy results of the dose escalation portion of GCT3013-01

[0302] - Subject ID lists the anonymous patient identifiers.

[0303] - B-cell NHL subtypes include DLBCL / HGBCL and FL.

[0304] - Response categories include complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD), where the response is assessed based on a comparison of radiological imaging with baseline scans according to the widely accepted Lugano criteria as described above, including measuring the SPD of a defined set of representative lesions and any new lesions that appear.

[0305] - When available, report the 5-point PET score of the best % change visit in the SPD; if the best % change is the same in multiple visits in the SPD, report the lowest 5-point PET score of those visits.

[0306] For patients unclassified as DLBCL / HGBCL and FL, MZL patients receiving the full dose of 0.76 mg showed SD on imaging at week 8. For MCL, one patient achieved CR and another achieved SD.

[0307] In summary, subcutaneous administration of icoretuzumab demonstrated antitumor activity in B-NHL, particularly in DLBCL / HGBCL and FL, during dose escalation, including several responses in some patients at very low doses. Most responses (CR, PR) were observed in the DLBCL / HGBCL group at a full dose of at least 6 mg, and all responses were observed in the FL group at a full dose of at least 0.76 mg.

[0308] Clinical safety

[0309] An overview of adverse events that occurred in all treatments for all subjects is listed in Tables 5A and 5B.

[0310] Table 5A. Summary of adverse events occurring during treatment

[0311] Table 5B

[0312] The GCT3013-01 dose escalation safety dataset lists the observed TEAEs.

[0313] Note: For each subject, multiple adverse events with the same AE are counted only once. Percentages are based on N, the number of subjects in each cohort, or the total number of subjects.

[0314] Related = (Possibly) related, including AEs with missing relationships.

[0315] 1. Data from the three patients in Cohort 12 (full dose 60 mg) were not included in the safety analysis described above. All three patients in Cohort 12 received one to two 60 mg doses, and no adverse events were reported after the 60 mg dose.

[0316] 2. Fatal adverse events (AEs) are due to disease progression / euthanasia and are unrelated to treatment.

[0317] Of the 58 subjects evaluated in the safety analysis, 58 (100%) experienced at least one treatment-associated adverse event (TEAE); 69.0% of subjects had a grade 3 or 4 TEAE (Tables 5A and 5B). The four most common TEAEs were fever (69.0%), CRS (56.9%), fatigue (41.4%), and injection site reaction (41.4%). Eleven (19.0%) subjects had TEAEs that led to death, nine (15.5%) experienced malignant growth progression (lymphoma progression), and one (1.7%) subject died due to euthanasia (also in the context of disease progression). One (1.7%) patient died from coronavirus infection (COVID-19). These events were considered unrelated to icoretuzumab.

[0318] Thirty-nine (67.2%) subjects reported serious adverse events (SAEs). The most frequently reported SAEs were fever (25.9%), progression of malignant vegetations (15.5%), and pulmonary infection (6.9%).

[0319] Only 7 subjects (12.1%) experienced TEAEs leading to permanent discontinuation of treatment, and all of these events involved disease progression. 5 subjects (8.6%) reported malignant vegetation progression events, 1 subject (1.7%) experienced partial seizures (in the context of lymphoma that may involve the CNS), and 1 subject (1.7%) experienced dyspnea due to underlying disease.

[0320] Of the 35 (60.3%) subjects, adverse events of particular concern (AESIs) occurred; of these, 33 (56.9%) experienced grade 1 or 2 CRS; no patients discontinued treatment due to CRS. Four (6.9%) subjects experienced neurotoxicity (one [1.7%] subject [0.76 mg] experienced grade 1 partial seizure; one [1.7%] subject [12 mg] had grade 1 CRS-related agraphia; one [1.7%] subject [12 mg] had grade 3 CRS-related confusion; and one [1.7%] subject [12 mg] had grade 3 confusion in the context of influenza and leptomeningeal disease).

[0321] In summary, treatment was well tolerated at doses up to and including 60 mg. No dose-limiting toxicities were observed and therefore the maximum tolerated dose was not reached. There were no serious (grade 3 or higher) CRS events and no treatment-related deaths.

[0322] Evaluation of clinical response and mechanism of action

[0323] Pharmacodynamic (PD) biomarkers were also investigated to evaluate the clinical response and mechanism of action (MOA) of icoretuzumab. Peripheral blood biomarker analyses were performed, and the effects of icoretuzumab on circulating immune cells and cytokines were evaluated using flow cytometry and immunoassays, respectively. Icoretuzumab induced a rapid and sustained reduction in circulating B cells (if present at baseline following prior anti-CD20 therapy). A transient decrease in peripheral CD4+ and CD8+ T cells was observed within 6 hours of the first dose, and subsequent doses induced CD8+ and CD4+ T cell expansion compared to baseline. Furthermore, the activation markers CD69 and CD279 were upregulated on CD4+ and CD8+ T cells. Exploratory analysis of biomarkers associated with clinical response showed a trend towards greater expansion of activated and total T cells (both CD8+ and CD4+) and higher IFNγ levels in patients with partial or complete responses to icoretuzumab.

[0324] Elevated dosing of icoretuzumab and SC administration were performed during dose escalation to alleviate CRS. Moderate increases in CRS-associated cytokines IL-6 and TNF were observed. These changes in T cells and cytokines were consistently observed across different B-NHL classifications. Further biomarker analyses are ongoing and updated data will be provided.

[0325] The mechanism of action of icoretuzumab involves targeting T cell surface antigen CD3 and B cell surface antigen CD20, triggering T cell-mediated killing of CD20-positive cells. DuoBody-CD3xCD20 showed induction of T cell activation and expansion, resulting in potent T cell-mediated cytotoxicity against malignant B cells, and the observed data are consistent with the mechanism of action that confirms the clinical activity of icoretuzumab in the treatment of B-NHL.

[0326] Modeling

[0327] Because the number of patients assessed in each cohort group in our clinical trial was small, we used a modeling approach to analyze patient data to determine the optimal subcutaneous dose of icoretuzumab for the treatment of DLBCL and FL.

[0328] Plasma icostatumab concentration-time pharmacokinetic (PK) data were obtained from patients treated with a full range of doses from 0.0128 mg to 48 mg. A population PK model for subcutaneous administration of icostatumab was developed using available PK data and Monolis population PK modeling software (MonolixSuite). A one-compartment model with first-order absorption, linearity, and Michaelis-Menten elimination was identified as the model best describing the PK data.

[0329] Based on the population PK model, icoretuzumab exhibited slow absorption, with a Tmax of 2.96 (48.7%) days (geometric mean and CV). The model predicted a terminal half-life of icoretuzumab of 9.58 (112%) days (geometric mean and CV). Notably, icoretuzumab administered subcutaneously exhibited flip-flop kinetics, where absorption was slower than elimination. Therefore, the terminal half-life represents the absorption half-life and is dose-independent. At a full dose of 48 mg, the steady-state Cmax was 3.30E6 (75.0%) pg / mL (geometric mean and CV). Population PK analysis showed that icoretuzumab exhibited target-mediated drug disposal (TMDD). Simulations using the population PK model showed that saturation (>90%) of target-mediated drug disposal occurred at dose levels >12 mg, indicating binding and saturation of CD3 and CD20 in plasma (see [link to data]). Figure 1 ).

[0330] However, as predicted by the population PK model, target binding and saturation in plasma are not the most relevant to efficacy assessment. Binding and saturation at the site of action (near the tumor) are more relevant. Furthermore, for bispecific antibodies, saturation of both targets occurs sequentially, with the high-affinity target arm (CD20) reaching saturation first, followed by the low-affinity target arm (CD3). Trimer formation (bispecific antibody crosslinking CD3 and CD20) increases as the bispecific antibody concentration approaches the KD value of the CD20 arm. Trimer concentration eventually plateaus as the bispecific antibody concentration increases above the KD value of the CD20 arm. After the plateau, trimer concentration begins to decline once the bispecific antibody concentration exceeds the KD value of the CD3 arm. Therefore, maximum trimer formation occurs before complete saturation of CD3 and CD20. Thus, the optimal range for bispecific antibodies to achieve maximum trimer formation and thus maximum efficacy exists.

[0331] To quantitatively predict trimer formation in tumors, a semi-mechanistic model was established. Initially, a semi-mechanistic PK / PD model was developed using preclinical monkey data. The study of icoretuzumab in monkeys generated abundant data, including dose ranges for intravenous and subclinical administration (spanning three orders of magnitude, single and multiple doses), dense PK data, blood T cell counts at relative time, blood B cell counts at relative time, and activated T cell subset counts at relative time.

[0332] The developed model utilizes a minimal physiology-based PK model (mPBPK) to provide physiologically relevant predictions of icoretuzumab concentrations in tissues. This model also considers T and B cell transport from the bloodstream to tissues and dorsal cells, the dynamic generation and death of T / B cells, and feedback mechanisms maintaining T / B cell homeostasis. Based on measured KD values ​​of CD3 and CD20, the model incorporates the binding of icoretuzumab (in blood and tissues) to CD3 on T cells and CD20 on B cells, as well as the cross-linking of CD3 and CD20. The model associates trimer formation with T cell activation, followed by T cell expansion. Activated T cells then induce B cell elimination. Using a population-based modeling approach and Monolix software, the developed PK / PD model was simultaneously fitted to PK, T cell count, activated T cell count, and B cell count data.

[0333] Based on model diagnostics, such as observation and prediction, residual and prediction, and residual and time, and with accurate model parameter estimation, the PK / PD model can well describe PK / PD data (plasma concentration, blood T cell count, blood activated T cell count, and blood B cell count data).

[0334] Subsequently, a comprehensive human PK / PD model was developed based on the structure of the PK / PD model developed using monkey data. An overview of the human PK / PD modeling and analysis is shown below. Figure 2 This model utilizes non-clinical and clinical PK and efficacy data to predict trimer formation and efficacy. Models (population PK model and monkey PK / PD model) were fitted using Monolix software. Simulations were performed using R (Microsoft) with the mrgsolve package. The model incorporates an mPBPK model to predict icoretuzumab concentrations in tumor lesions. The model also incorporates T cell, B cell, and tumor cell counts, as well as CD3 and CD20 expression on these cells. Furthermore, the model incorporates the dynamic binding of icoretuzumab to CD3 and CD20 and trimer formation. Finally, the PK / PD model incorporates tumor dynamics and tumor killing resulting from trimer formation and T cell activation.

[0335] The human PK / PD model utilizes several relevant parameters estimated by the monkey PK / PD model. Human PK / PD model parameters are based on human-specific values ​​from available clinical data or literature. PK sub-model parameters are obtained by fitting the mPBPK model to clinical PK data. Trimer formation sub-model parameters are based on patient biomarker data (e.g., T cell count, B cell count, tumor cell count), in vitro measurements (e.g., CD3 and CD20 KD of icostatumab (16 nM and 5.4 nM, respectively)), and / or literature values ​​(e.g., CD3 expression on T cells, CD20 expression on B cells / tumor cells). Tumor dynamics sub-model parameters are estimated based on literature values ​​(e.g., tumor growth rate), patient tumor size data (e.g., baseline tumor size), or by calibrating the response rate predicted by the human PK / PD model to the response rate observed in icostatumab dose escalation trials (e.g., tumor killing rate). The calibrated model well describes the response rate observed in icostatumab dose escalation trials (based on changes in lesion size). Figure 3 Therefore, the model has been validated for use in computer simulation analysis to predict the optimal dose.

[0336] Therefore, a comprehensive human PK / PD model was subsequently used to simulate clinical trials, incorporating individual variability in key model parameters. The variability in model parameters was based on inter-individual variability observed in icoretuzumab dose-escalation trials (e.g., T cell, B cell, and baseline tumor size, PK parameters) or on literature values ​​(e.g., CD3, CD20 expression, tumor growth rate). FL and DLBCL / HGBCL were simulated separately, differentiated by different tumor growth rates. For DLBCL / HGBCL, a 1-month tumor doubling time was used, while for FL, a 6-month tumor doubling time was used.

[0337] Human PK / PD model ( Figure 4 and Figure 5 The results showed that in FL and DLBCL / HGBCL, trimer formation plateaued at icorituzumab doses ≥ 48 mg. Furthermore, in FL and DLBCL / HGBCL, the plateau of efficacy predicted by the PK / PD model (predicted extended-trial response rate) was observed at icorituzumab doses ≥ 48 mg. Figure 6 and 7 This occurs at a time when... Based on model predictions and utilizing available clinical data from patients, the optimal dose of icoretuzumab is 48 mg.

[0338] Further analysis I

[0339] Following the above results, the patient continued to be evaluated and / or treated, and more patients were treated with Icarusumab.

[0340] At this later assessment time point, a total of 67 patients were enrolled, including 45 patients (67%) with diffuse large B-cell lymphoma (DLBCL), 12 patients (18%) with follicular lymphoma (FL), and 4 patients (6%) with mantle cell lymphoma (MCL). Patients had received extensive prior therapy; the median (range) of prior lines of treatment was 3 (1–6) for DLBCL patients, 4.5 (1–18) for FL patients, and 4 (3–5) for MCL patients; 6 patients had received prior CAR-T therapy. More than half of the patients (37 / 67; 55%) were refractory to their most recent systemic therapy, and 35 / 67 (52%) were refractory to their most recent anti-CD20 mAb therapy.

[0341] At a median overall follow-up of 8.3 months, 25 patients (37%) were still receiving treatment. The median follow-up time was 8.3 months for patients with DLBCL and 8.8 months for patients with FL. Icaretimab was well tolerated and there were no discontinuations due to treatment-related adverse events (AEs). The most common treatment-emergent AEs (TEAEs) were fever (70%), injection site reaction (48%), and fatigue (45%). With increasing dose, the TEAEs of particular interest were consistent with previous reports: all CRS events were grade 1 / 2 (58%), with no grade 3 / 4 events, and limited neurotoxicity was observed (6%; grade 1: 3%; grade 3: 3%; all transient).

[0342] There were no dose-limiting toxicities, tumor lysis syndrome, or febrile neutropenia, nor were there any deaths due to treatment-related adverse events (AEs). Antitumor activity in evaluable patients with DLBCL and FL is shown in Table 6. In 18 DLBCL patients receiving ≥12 mg icoretuzumab, the overall response rate (ORR) was 66.7%, with 6 patients achieving a complete response (CR). In 7 patients receiving ≥48 mg icoretuzumab (48 mg RP2D n=4; 60 mg n=3), all achieved a response, including 2 patients achieving CR. All DLBCL patients previously treated with CAR-T therapy achieved a response (4 / 4: 2 CR; 2 partial response [PR]). In all 8 FL patients receiving ≥0.76 mg icoretuzumab, the ORR was 100%, with 2 patients achieving CR.

[0343] Table 6. Antitumor activity of icoretuzumab in evaluable patients with R / RB-NHL

[0344] Based on data snapshots. Response assessment was based on the Lugano 2014 response criteria (modified response evaluable population) as evaluated by researchers.

[0345] a One COVID-19 patient was ruled out.

[0346] b Only 4 patients underwent PET scans (this was not mandatory until the most recent protocol revision).

[0347] In addition, a total of 4 MCL patients received icoretuzumab treatment. One patient (blastoid; 6 mg) died after rapid disease deterioration 1 week after receiving the priming dose (C1D1), one patient (12 mg) developed SD at week 6 assessment but progressed to PD at week 12, one patient (blastoid, 48 mg) developed PR at week 6 assessment but progressed to PD at week 12, and one patient (polymorphic; priming dose 0.16 mg, intermediate dose 0.8 mg, 24 mg) developed deep structural and metabolic CR at week 6 assessment, which persisted at the most recent response assessment (week 24: SPD = 0 cm). 2 (DS=1). Three of the four MCL patients experienced six CRS events in cycle 1, all of which were grade 1 (i.e., fever only) and resolved within 48 hours of onset (#1, trigger dose 40 µg, intermediate dose, full dose 6 mg, one CRS event within 24 hours of trigger dose; #2, trigger dose 0.16 mg, intermediate dose 0.8 mg, full dose 24 mg, two CRS events: the first event occurred 4 days after the trigger dose, and the second event occurred 4 days after the intermediate dose; #3, trigger dose 0.16 mg, intermediate dose 0.8 mg, full dose 48 mg, three CRS events: the first event occurred within 24 hours of the trigger dose, the second event occurred 3 days after the intermediate dose, and the third event occurred 24 hours after the first full dose). None of the CRS events resulted in a delay in the planned next administration of icoretuzumab, a change in the icoretuzumab dose, or discontinuation of the study. In conclusion, icoretuzumab demonstrated activity and good tolerability in MCL.

[0348] These results demonstrate that icoretuzumab continues to exhibit a favorable safety profile, with no ≥ grade 3 CRS events and limited neurotoxicity, supporting outpatient administration. The results also showed significant monotherapy efficacy, including complete responses in severely pre-treated patients with FL, MCL, and DLBCL.

[0349] In addition to assessing safety and efficacy parameters, further data were collected from patients (including those receiving icoretuzumab ranging from 0.0128 to 60 mg) for further PK / PD analysis. This further data analysis showed that icoretuzumab exhibited slow absorption, a Tmax of 2.8 days, a terminal half-life of 8.67 days, and target-mediated disposal (TMD). As shown above, the model predicted TMD saturation at dose levels ≥48 mg, indicating binding and saturation of CD3 and CD20 in the blood. Furthermore, the model was able to describe the exposure-response relationship observed in clinical practice, and clinical trial simulations using the PK / PD model further demonstrated that a dose of 48 mg achieves optimal trimer formation and clinical response in FL and DLBCL. Exposure-adverse event analysis showed a flat relationship between icoretuzumab exposure and the risk of cytokine release syndrome (CRS) within the evaluated dose range.

[0350] Further Analysis II

[0351] In a further follow-up of the results presented above, the patient continued to be evaluated and / or received icoretuzumab treatment.

[0352] At this later assessment time point, a total of 68 patients were enrolled, including 46 patients (68%) with diffuse large B-cell lymphoma (DLBCL), 12 patients (18%) with follicular lymphoma (FL), and 4 patients (6%) with mantle cell lymphoma (MCL). Patients had received extensive prior therapy; the median (range) of prior lines of treatment was 3 (1–6) for DLBCL patients, 4.5 (1–18) for FL patients, and 4 (3–5) for MCL patients; 6 patients (9%) had received prior CAR-T therapy. The majority of patients (59 / 68; 87%) were refractory to their most recent systemic therapy, and 60 / 68 (88%) were refractory to their most recent anti-CD20 mAb therapy.

[0353] At a median follow-up duration of 10 months, 17 patients (25%) were receiving treatment. The median follow-up time was 7 months for patients with DLBCL and 12 months for patients with FL. No dose-limiting toxicities were observed at doses up to 60 mg and the maximum tolerated dose was not reached. Icaretuzumab was well tolerated and there were no discontinuations due to treatment-related adverse events (AEs). The most common treatment-emergent AEs (TEAEs) were fever (69%), injection site reaction (47%), and fatigue (43%). Most adverse events were grade 1–2. With increasing dose, the TEAEs of particular interest were consistent with previous reports: all CRS events were grade 1 / 2 (59%), with no grade 3 / 4 events (see Table 7), and limited neurotoxicity was observed (6%; grade 1: 3%; grade 3: 3%; all transient, median [range] 1.5 [<1–3] days, and managed with standard of care). One patient had tumor lysis syndrome, grade 3, associated with disease progression.

[0354] Most CRS events were observed during cycle 1, with no CRS events observed during the second full dose of 48 mg. Most events occurred and resolved during cycle 1. The median time to resolution was 2 (range 1.0–9.0) days. No grade 3 or higher CRS events occurred despite dose escalation to the recommended 48 mg and 60 mg doses. The risk of CRS was mitigated through route of administration, dose escalation, and pretreatment with corticosteroids.

[0355] Table 7. Adverse events related to CRS

[0356] The antitumor activity in evaluable patients with DLBCL, FL, and MCL is shown in Table 8. In 22 DLBCL patients receiving 12–60 mg icoretuzumab, the overall response rate (ORR) was 68%, with 10 patients achieving complete response (CR). In 11 DLBCL patients receiving 48–60 mg icoretuzumab (48 mg RP2D n=8; 60 mg n=3), 10 patients achieved a response, including 6 CR patients. All DLBCL patients with evaluable response to prior CAR-T therapy achieved a response (4 / 4: 2 CR; 2 partial response [PR]). In all 10 FL patients receiving 0.76–48 mg icoretuzumab, the ORR was 90%, with 5 patients achieving CR. In 5 FL patients receiving 12–48 mg icoretuzumab, the ORR was 80%, with 3 patients achieving CR.

[0357] No other MCL patients received icoretuzumab. In summary, of the four patients, one (Berberidoid; 6 mg) died one week after receiving the priming dose following presumed rapid disease progression, with no response assessment (C1D1); one (12 mg) developed SD at week 6 assessment but PD at week 12; one (Berberidoid, 48 mg) developed PR at week 6 assessment but PD at week 12; and one (Pleomorphic; priming dose 0.16 mg, intermediate dose 0.8 mg, 24 mg) developed deep structural and metabolic CR at week 6 assessment, which persisted at the most recent response assessment (week 24: SPD = 0 cm2; DS = 1).

[0358] Table 8. Antitumor activity of icoretuzumab in evaluable patients with R / RB-NHL

[0359] The response assessment was based on the Lugano 2014 response criteria (modified response evaluable population) as evaluated by researchers. † This includes 3 patients who received a 60 mg dose before RP2D was determined. ‡ Three patients developed blastomorphic / pleomorphic MCL; the histology of one patient was unknown. § One patient who was discontinued before the initial assessment due to COVID-19 was excluded. ‖ One patient who discontinued treatment before the initial evaluation due to postcardiac ablation (CABG) surgery was excluded. ¶ The response rate is based on the number of evaluable patients (defined as patients who died with at least one post-baseline disease assessment or without a post-baseline disease assessment). This includes one patient who died before the response assessment. †† Response evaluation was performed on 6 out of 10 patients via PET scan (this was not mandatory until the most recent protocol revision). The PK / PD model described above is continuously updated using further data obtained from patients. The results obtained are consistent with... Figure 1 and Figure 3-7 The results described were highly similar. For FL and DLBCL, the overall conclusions remained unchanged, with predicted trimer formation plateauing from 48–192 mg and predicted response rates plateauing from 48 mg. Exposure-adverse event analysis was also performed, showing a low overall risk of grade 2 CRS at the initiating dose, intermediate dose, and full dose, and a flat relationship between icoretuzumab Cmax and grade 2 CRS. Based on modeling and clinical trial simulations, the 48 mg full dose was identified as the bioeffective dose, resulting in adequate target modulation and clinical activity in proportion to the risk of adverse events.

[0360] in conclusion

[0361] Based on a favorable safety profile, along with an impressive efficacy profile and high response rates (including complete responses), icoretuzumab has the potential to be a best-in-class treatment, at least in patients with relapsed or refractory DLBCL / HGBCL, FL, and MCL. These data support future outpatient administration of icoretuzumab. Based on clinical efficacy, safety data, and PK / PD modeling, the recommended full dose for DLBCL / HGBCL and FL, as well as general B-NHL, could be at least 48 mg. Based on currently available data, a full dose of 60 mg is anticipated. A full dose of 48 mg can be recommended. Safety data observed at a trigger dose of 0.16 mg and an intermediate dose of 0.80 mg, along with observations of all CRS events being only mild to moderate (≤ gr. 2) severity and resolving after standard CRS management following these doses, suggest that trigger and intermediate doses of 0.16 mg and 0.80 mg, respectively, can be recommended. Therefore, it is recommended that Cycle 1 be administered at 0.16 mg, 0.80 mg, 48 mg, and 48 mg on days 1, 8, 15, and 22 of a 28-day cycle, respectively. Furthermore, cycles following Cycle 1 can be recommended to include full doses on days 1, 8, 15, and 22 of a 28-day cycle in Cycles 2-3; full doses on days 1 and 15 of a 28-day cycle starting from Cycles 4-9; and full doses on day 1 of a 28-day cycle from Cycle 10 onwards. For all four weekly doses of Cycle 1, it is recommended to continue prophylactic and preventative medication for 3-4 consecutive days as outlined herein for CRS remission, and to continue when CRS > gr. 1 is observed after the fourth weekly administration of icoretuzumab in Cycle 1. Currently, this dosing regimen is being used in clinical trials, with a recommended full dose of 48 mg. It is being expanded in ia FL, MCL, and DLBCL / HGBCL, and a phase III trial has been initiated in relapsed, refractory DLBCL who have failed or are not suitable for HDT-ASCT.

[0362] Dose escalation and expansion portions and Phase III trials A total of 169 participants received treatment in three ongoing icoretuzumab trials: GCT3013-01 (total n = 153 participants; 68 participants in the dose escalation portion and 85 participants in the extension portion), GCT3013-04 (n = 14 participants; 7 participants in the dose escalation portion and 7 participants in the extension portion) and GCT3013-05 (n = 2 participants).

[0363] GCT3013-01 is a phase I / II open-label trial investigating the safety and preliminary efficacy of icoretuzumab in subjects with relapsed, progressive, or refractory B-cell lymphoma. Patients have already received treatment in the dose-escalation phase, the results of which have been extensively described above. Further updates to these results are provided below. Following dose-escalation, in the extension phase, patients received subcutaneous icoretuzumab in 28-day cycles: in cycle 1, 0.16 mg on day 1, 0.8 mg on day 8, and 48 mg on days 15 and 22; in cycles 2-3, 48 mg on days 1, 8, 15, and 22; in cycles 4-9, 48 mg on days 1 and 15; and in cycle 10+, 48 mg on day 1. This trial enrolled 153 subjects.

[0364] GCT3013-05 is a randomized, open-label phase III clinical trial of icoretuzumab, in which the first batch of patients has received treatment. Enrolled patients were relapsed / relapsed DLBCL patients who had previously failed ASCT or were unsuitable for ASCT at screening. Efficacy was compared to standard of care, which involved R-GemOx, consisting of rituximab in combination with gemcitabine and oxaliplatin; and rituximab in combination with bendamustine. Icotrutuzumab was administered subcutaneously over a 28-day cycle: 0.16 mg on day 1, 0.8 mg on day 8, and 48 mg on days 15 and 22 in cycle 1; 48 mg on days 1, 8, 15, and 22 in cycles 2-3; 48 mg on days 1 and 15 in cycles 4-9; and 48 mg on day 1 in cycle 10+. Two patients have been enrolled in this trial.

[0365] GCT3013-04 is a phase I / II open-label trial cohort investigating the safety and preliminary efficacy of icoretuzumab in Japanese patients with relapsed or refractory (R / R) B-NHL. Patients have received treatment in dose-escalation and extension phases, with patients receiving subcutaneous icoretuzumab in 28-day cycles: Cycle 1: 0.16 mg on day 1; 0.8 mg on day 8; 48 mg on days 15 and 22; Cycles 2-3: 48 mg on days 1, 8, 15, and 22; Cycles 4-9: 48 mg on days 1 and 15; Cycle 10+: 48 mg on day 1. Fourteen subjects have been enrolled in this trial.

[0366] Table 9: Dose Increment by Cohort - GCT3013-01

[0367] Note: To bridge the gap between initiation and continuous escalation of the full dose, an intermediate dose of icorituzumab was added before administration at the 1.5 mg full dose level.

[0368] The last administration (i.e., the full dose) in cycle 1 is continued in cycle 2 and thereafter.

[0369] a. One subject in cohort 2 was replaced after receiving only the priming dose due to discontinuation of the trial (disease progression) before the end of the dose-limiting toxicity period (i.e., 28 days).

[0370] b. A queue intended for parallel evaluation.

[0371] Of the 68 subjects treated with icoretuzumab in the dose-escalation portion of GCT3013-01, 15 subjects (22.1%) continued to receive icoretuzumab in the 3-60 mg dosing groups at the data cutoff (2 subjects in the 3 mg group, 1 subject in the 6 mg group, 2 subjects in the 12 mg group, 4 subjects in the 24 mg group, 4 subjects in the 48 mg group, and 2 subjects in the 60 mg group). Of the 53 subjects who discontinued treatment, 46 discontinued due to disease progression. Among the 68 subjects treated in the dose-escalation portion of GCT3013-01, the median age was 67.5 years and the majority of subjects (45, 66.2%) were male. The median time from lymphoma diagnosis to first dose was 29.7 months. The median time from most recent recurrence, relapse, or progression was 1.6 months. Of the various B-NHL subtypes enrolled, 46 subjects (67.6%) had DLBCL and 12 subjects (17.6%) had FL. Enrolled patients had the following disease types: diffuse large B-cell lymphoma, i.e., new-onset (28), transformed (17), or unknown (1); high-grade B-cell lymphoma (3), primary mediastinal large B-cell lymphoma (1), follicular lymphoma (12), mantle cell lymphoma (4), small lymphocytic lymphoma (1), and marginal zone lymphoma (1). Patients receiving the full dose included those with diffuse large B-cell lymphoma, i.e., new-onset (9), primary mediastinal large B-cell lymphoma (1), follicular lymphoma (1), and mantle cell lymphoma (1).

[0372] Eighty-five subjects received icoretuzumab in the GCT3013-01 extension, including 61 subjects (71.8%) who continued receiving icoretuzumab at the data cutoff; 50 subjects continued in the aggressive B-cell non-Hodgkin lymphoma (aNHL) cohort, and 11 subjects continued in the indolent B-cell non-Hodgkin lymphoma (iNHL) cohort. Of the 24 subjects (28.2%) who discontinued treatment, 14 discontinued due to disease progression. Among the 85 subjects treated in the GCT3013-01 extension, the median age was 68.0 years and the majority (60, 70.6%) were male. The median time from lymphoma diagnosis to first dose was 24.9 months. The median time from most recent refractory or relapsed treatment was 1.13 months. Of the various B-NHL subtypes enrolled, 71 subjects (83.5%) had DLBCL and 13 subjects (15.3%) had FL. In the aggressive NHL cohort, the majority of subjects had DLBCL, namely new onset (44), transformation (17), inapplicable (5), unknown (3), and missing (1); and one patient had high-grade B-cell lymphoma. In the indolent B-cell non-Hodgkin lymphoma cohort, 12 subjects had follicular lymphoma grade 1–3A and one had an unknown grade.

[0373] Table 10. Subject Distribution - Extended Section of GCT3013-01

[0374] Note: Percentages are calculated based on the number of subjects in the FAS.

[0375] aNHL = Aggressive B-cell non-Hodgkin lymphoma; FAS = Full analysis set; iNHL = Indolent B-cell non-Hodgkin lymphoma.

[0376] a. The FAS and safety set includes all subjects exposed to icoretuzumab.

[0377] b. Disease progression includes both clinical progression and recorded radiographic progression.

[0378] Pharmacodynamics

[0379] Icaretimab induces rapid and sustained reduction of circulating B cells (in a subgroup of patients with detectable B cells, most subjects had no B cells due to prior anti-CD20 therapy). B cells were detected using an antibody against CD19. Subsequent administration at doses greater than 12 mg induced expansion of circulating T cells relative to baseline and a moderate increase in circulating IFNγ, IL-6, and TNFα (by serological immunoassay). A transient reduction in peripheral CD4+ and CD8+ T- cells was observed within 6 hours of the first SC administration, consistent with T-cell marginalization seen in other bispecific antibodies. Importantly, subsequent administration induced T-cell expansion relative to baseline. Icaretimab was administered with escalating doses and SCs to alleviate CRS. SC administration of iccaretimab resulted in a moderate increase in IFNγ, IL-6, and TNFα.

[0380] effect

[0381] In the dose escalation phase, the evaluation of treatment response was based on investigator assessment according to the Lugano criteria (Cheson et al., 2014). Using the full analysis set, 18 / 46 subjects (39.1%) with DLBCL achieved a response (subjects with partial or complete responses were considered responders). Among subjects with FL, 9 / 12 (75.0%) achieved a response (Table 11).

[0382]

[0383]

[0384]

[0385] Safety and tolerability

[0386] All 68 participants in the dose escalation portion of GCT3013-01 experienced at least one treatment-emergent adverse event (TEAE). The four most common TEAEs were fever (69.1%), critical respiratory syndrome (CRS) (58.8%), injection site reaction (47.1%), and fatigue (44.1%). A total of 80.9% of participants had at least one grade 3 or higher TEAE. Thirteen participants experienced TEAEs that led to death (11 with malignant tumor progression, 1 undergoing euthanasia [also in the context of disease progression], and 1 with COVID-19 [Coronavirus Disease 2019] infectious pneumonia). These events were considered unrelated to icoretuzumab. Forty-six (67.6%) participants reported SAEs. The most common SAE considered related to the investigational drug was fever (19 participants, 27.9%), and CRS symptoms were reported (see Section 4.6.2.2). The incidence of TEAEs leading to permanent treatment discontinuation was low (13.2% of participants). Adverse events of particular concern (AESIs) included the following: 40 subjects (58.8%) experienced CRS, 4 subjects (5.9%) experienced neurological symptoms considered immune-mediated by the investigator, and 1 subject (1.5%) experienced clinical tumor lysis syndrome. AESIs were recorded on a separate AESI page in the eCRF. Symptoms related to AESIs meeting the severity criteria were reported as SAEs, with the overall diagnosis being a major event. Of the 68 subjects treated in the dose escalation portion of GCT3013-01, 46 (67.6%) had experienced at least one SAE. A total of 24 subjects (35.3%) experienced SAEs considered to be related to icoretuzumab. The most common SAE considered related to the investigational drug was fever (19 subjects, 27.9%), reported as a symptom of CRS.

[0387] Of the 85 participants in the extended portion of GCT3013-01, 76 (89.4%) had experienced at least one TEAE (Table 12). The three most common TEAEs were CRS (47.1%), fatigue (18.8%), and fever (18.8%) (Table 13). A total of 40.0% of participants had at least one Grade 3 or higher TEAE (Table 14). Seven participants experienced TEAEs that resulted in death. One participant experienced a TEAE that resulted in death, which the investigators believed was related to icoretuzumab (Immune Effector Cell Related Neurotoxicity Syndrome [ICANS]). 55.3% of participants reported SAEs. The most common SAE considered to be related to the investigational drug was CRS (25 participants, 29.4%). The incidence of TEAEs leading to permanent treatment discontinuation was low (5.9% of participants). AESI included the following: 40 subjects (47.1%) experienced CRS, 3 subjects (3.5%) experienced ICANS, and 1 subject (1.2%) experienced clinical tumor lysis syndrome.

[0388] As described, 7 out of 85 subjects (8.2%) treated in the extended portion of GCT3013-01 experienced a fatal TEAE. One subject experienced a fatal TEAE, which the investigators believe was related to icoretuzumab. This subject was a 72-year-old woman with stage IV non-GCB DLBCL and a history of diabetes, hypertension, hyperlipidemia, and a right upper lobectomy for lung cancer. At screening, the subject had involvement of pancreatic, splenic, and para-aortic lymphoma. Two days after the initiating dose of 0.16 mg icoretuzumab, the subject experienced persistent abdominal pain, was subsequently diagnosed with grade 3 pancreatitis, received multiple repeated doses of morphine, and was confirmed to have progressive lymphoma based on imaging. Four days after an intermediate dose of 0.8 mg icoretuzumab, grade 2 ICANS was reported, which subsequently worsened to grade 4. In addition, grade 1 cerebral ischemia was observed on brain imaging. The study drug was withdrawn. The subject's condition deteriorated, and the subject died 17 days after receiving an intermediate dose of 0.8 mg of icoretuzumab, with death reported as due to ICANS. However, in a subject with a history of long-term diabetes, hypertension, hyperlipidemia, possible microangiopathy (laboratory evidence of new-onset multifocal cerebral infarction, splenic infarction, coagulation disorders, and renal dysfunction), metabolic encephalopathy due to hyperammonemia, and accumulation of active metabolites from morphine use (which depend on functional renal clearance), these are likely confounding factors for neurotoxicity. Tocilizumab administration for the treatment of neurological symptoms is known to be unnecessary in the absence of CRS, as it increases the likelihood of neurotoxicity by increasing circulating IL-6 levels and may have contributed to the exacerbation of neurotoxicity in this case.

[0389] Table 12. Summary of adverse events occurring during treatment - GCT3013-01 Extended Section

[0390] Note: Percentages are calculated based on the number of subjects in the safety set.

[0391] Adverse events were classified using the Medical Dictionary of Regulatory Activities (MedDRA) v23.1 and the Common Terminology Standard for Adverse Events (CTCAE) v5.0, with each category counted only once. Cytokine release syndromes were graded according to (Lee et al., 2019), and clinical tumor lysis syndromes were graded according to Cairo-Bishop (Coiffier et al., 2008).

[0392] aNHL = Aggressive B-cell non-Hodgkin lymphoma; ICANS = Immune effector cell-associated neurotoxicity syndrome; iNHL = Indolent B-cell non-Hodgkin lymphoma; TEAE = Treatment-associated adverse events.

[0393] Table 13: Adverse events occurring during treatment in >10% of subjects - GCT3013-01 Extended Section

[0394] Note: Percentages are calculated based on the number of subjects in the safety set.

[0395] Adverse events are categorized using the Medical Dictionary of Regulatory Activities (MedDRA) v23.1, with each preferred term counted at most once.

[0396] Table 14: Grade 3 or higher adverse events during treatment by system organ classification and preferred terminology - GCT3013-01 Extended Section

[0397]

[0398] Note: Percentages are calculated based on the number of subjects in the safety set.

[0399] Adverse events are classified using the Medical Dictionary of Regulatory Activities (MedDRA) v23.1 and the Common Terminology Standard for Adverse Events (CTCAE) v5.0, with each system organ classification counted at most once and each preferred term counted at most once.

[0400] aNHL = Aggressive B-cell non-Hodgkin lymphoma; COVID-19 = Coronavirus disease; iNHL = Indolent B-cell non-Hodgkin lymphoma; TEAE = Adverse events that occur during treatment.

[0401] Of the 85 subjects treated in the extended portion of GCT3013-01, 47 (55.3%) experienced at least one SAE (Table 23). A total of 28 subjects (32.9%) experienced SAEs that they believed were related to icoretuzumab. The most common SAE believed to be related to the investigational drug was CRS (25 subjects, 29.4%) (Table 15).

[0402] Table 15. Serious adverse events associated with icoretuzumab treatment, classified by systemic organ classification and preferred terminology - GCT3013-01 Extended Section

[0403] Note: Percentages are calculated based on the number of subjects in the safety set.

[0404] Adverse events are classified using the Medical Dictionary of Regulatory Activities (MedDRA) v23.1, with each system organ classification counted at most once and each preferred term counted at most once.

[0405] aNHL = Aggressive B-cell non-Hodgkin lymphoma; iNHL = Indolent B-cell non-Hodgkin lymphoma; TEAE = Adverse events that occur during treatment.

[0406] Of the 85 subjects treated in the extended portion of GCT3013-01, 5 (5.9%) experienced treatment-associated adverse events (TEAEs) leading to permanent discontinuation of the investigational drug: one subject experienced pulmonary vegetations, progression of malignant vegetations, abdominal pain, hepatotoxicity, hyperbilirubinemia, progressive multifocal leukoencephalopathy, elevated transaminases, immune effector cell-associated neurotoxicity syndrome, and respiratory failure (Table 16). Two subjects experienced multiple TEAEs leading to permanent discontinuation of treatment: one subject experienced pulmonary vegetations and respiratory failure; the other subject experienced abdominal pain, hepatotoxicity, hyperbilirubinemia, and elevated transaminases due to disease progression.

[0407] Table 16. Adverse events in treatment leading to permanent discontinuation of treatment, categorized by system organ and preferred terminology - Extended section of GCT3013-01.

[0408]

[0409] Note: Percentages are calculated based on the number of subjects in the safety set.

[0410] Adverse events are classified using the Medical Dictionary of Regulatory Activities (MedDRA) v23.1, with each system organ classification counted at most once and each preferred term counted at most once.

[0411] aNHL = Aggressive B-cell non-Hodgkin lymphoma; iNHL = Indolent B-cell non-Hodgkin lymphoma; NHL = Adverse events during treatment.

[0412] Adverse events of particular interest

[0413] The following is a summary of AESI (Advanced Circulatory System Infections): Cytokine release syndrome (CRS) was reported in 86 subjects, including 40 subjects in the dose-escalation portion of GCT3013-01, 40 subjects in the extended portion of GCT3013-01, and 6 subjects in the dose-escalation portion of GCT3013-04. CRS was graded according to (Lee et al., 2019). All CRS events were grade 1 or 2, except for two events in the extended portion of GCT3013-01 and one event in the dose-escalation portion of GCT3013-04, which were grade 3.

[0414] Neurotoxicity was classified as an AESI in the icoretuzumab regimen. Neurological assessments were performed according to the CARTOX-10 scale during the dose-escalation portion of the GCT3013-01 trial (Neelapu et al., 2017). Suspected immune-mediated neurological symptoms conforming to ICANS were recorded as separate AESIs. ICANS cases were reported using the exact ICANS terminology in the extended portions of both the GCT3013-01 and GCT3013-04 trials. Neurological symptoms reported as AESIs were reported in 7 subjects in GCT3013-01, including 4 in the dose-escalation portion and 3 in the extended portion, and none in the dose-escalation portion of GCT3013-04.

[0415] Clinical tumor lysis syndrome was reported in two subjects: one in the dose-escalation portion of GCT3013-01, one in the extended portion of GCT3013-01, and none in the dose-escalation portion of GCT3013-04. Clinical tumor lysis syndrome was graded according to the Cairo-Bishop classification (Coiffier et al., 2008). Both cases of tumor lysis syndrome were grade 3.

[0416] Summary of clinical data

[0417] This summarizes data from GCT3013-01 (safety of the dose escalation and extension portions, efficacy of the dose escalation portion, and pharmacokinetics of the dose escalation portion) and safety data from the dose escalation portion of GCT3013-04. No DLT occurred, MTD was not reached, and the RP2D was declared as the full dose of 48 mg. All 68 subjects in the dose escalation portion of GCT3013-01 experienced at least one TEAE; 80.9% of subjects had at least one Grade 3 or higher TEAE. The most common TEAEs were fever, CRS, injection site reaction, and fatigue. Thirteen subjects had TEAEs that led to death (11 subjects had malignant tumor progression, one underwent euthanasia [also in the context of disease progression], and one subject had COVID-19 infectious pneumonia). 67.6% of subjects reported SAEs. The most common SAEs considered to be related to the investigational drug were fever, reported as symptoms of CRS. 13.2% of subjects reported TEAEs leading to permanent discontinuation of treatment. AESI included the following: 40 subjects experienced CRS, 4 subjects experienced immune-mediated neurological symptoms as determined by the investigators, and 1 subject experienced clinical tumor lysis syndrome.

[0418] In the extended portion of GCT3013-01, 76 subjects (89.4%) experienced at least one TEAE; 40.0% of subjects experienced at least one Grade 3 or higher TEAE. The most common TEAEs were CRS, fatigue, and fever. Seven subjects had TEAEs that led to death (one subject each for disease progression, general deterioration of physical condition, COVID-19, hepatotoxicity, malignant vegetation progression, and immune effector cell-related neurotoxicity syndrome). 55.3% of subjects reported SAEs. The most common SAE considered to be related to the investigational drug was CRS. 5.9% of subjects reported TEAEs that led to permanent discontinuation of treatment. AESIs included: 40 subjects experienced CRS, 3 subjects experienced ICANS, and 1 subject experienced clinical tumor lysis syndrome.

[0419] All seven subjects in the dose escalation portion of GCT3013-04 experienced at least one treatment-associated adverse event (TEAE); 71.4% of subjects had at least one grade 3 or higher TEAE. The most common TEAEs were critical respiratory syndrome (CRS) and injection site erythema. No subjects experienced TEAEs leading to treatment discontinuation, subacute acute exacerbations (SAEs), or death. Two subjects experienced TEAEs requiring dose adjustment. CRS-related adverse events (AESIs) occurred in six subjects (85.7%).

[0420] Aikerituzumab administration induces rapid and sustained reduction of circulating B cells (in a subgroup of subjects with detectable B cells, most patients did not have B cells due to prior anti-CD20 therapy) and an increase in peripheral T cells and circulating IFNγ.

[0421] No significant ADA (titer ≥1) was observed in patients with icoretuzumab.

[0422] In the dose escalation portion of GCT3013-01, the ORR was 44.1% for all combined dose levels and 66.7% for the RP2D level (48 mg).

[0423] Category effect

[0424] Adverse reactions reported for a class of compounds to which icoretuzumab belongs (i.e., bispecific T-cell conjugates) include CRS, neurological symptoms, and infections.

[0425] B-cell depletion may lead to an increased risk of infection. Infections should be managed according to best medical practice. Latent viral infections, such as hepatitis B or cytomegalovirus, should be monitored in at-risk subjects during and after etorutumab treatment. A total of 58 subjects experienced infections in the dose-escalation portions of the GCT3013-01 and GCT3013-04 trials. No etorutumab-related infections were reported in the GCT3013-01 extension or the GCT3013-04 dose-escalation portions.

[0426] Ectometuzumab and other compounds or drugs targeting CD3 and chimeric antigen receptor T cells have been reported to cause CRS. Close monitoring of vital signs, especially temperature, blood pressure, and blood oxygen saturation, as well as laboratory assessments of hematological, liver, and kidney parameters, is crucial to ensuring timely initiation of supportive care as needed. Supportive care based on (Lee et al., 2019) and (Neelapu et al., 2017) includes, but is not limited to: saline infusion Systemic glucocorticoids, antihistamines, and antipyretics Blood pressure support Ventilation support Monoclonal antibodies against IL-6R, IL-6, or IL-1 (e.g., tocilizumab, siltuximab, and / or anakinase) Additional CRS risk reduction measures were implemented, including four consecutive days of prophylactic corticosteroid administration associated with the administration of icoretuzumab in cycle 1. Most CRS events were reported as grade 1 and 2; only 3 subjects reported grade 3 CRS.

[0427] A total of 86 subjects reported at least one CRS event in the dose-escalation portions of GCT3013-01 and GCT3013-04. The majority of cases were Grade 1 or 2 CRS, while Grade 3 CRS occurred in only 3 patients, indicating that Grade 1 or 2 CRS is a very common event, while Grade 3 appears to be quite common. Specifically, 40 subjects (58.8%) (20 subjects - Grade 1; 20 subjects - Grade 2) were in the dose-escalation portion of GCT3013-01, 40 subjects (47.1%) (24 subjects - Grade 1; 14 subjects - Grade 2; 2 subjects - Grade 3) were in the extended portion of GCT3013-01, and 6 subjects (85.7%) (4 subjects - Grade 1; 1 subject - Grade 2; 1 subject - Grade 3) were in the dose-escalation portion of GCT3013-04. Of the 86 subjects who experienced CRS events, 25 reported SAEs. All CRS cases were assessed as being associated with icoretuzumab and resolved / regressed. No life-threatening or fatal CRS cases were reported. No CRS led to treatment discontinuation.

[0428] Other CD3-targeting compounds or drugs have reported neurological symptoms ranging from confusion to fatal cerebral edema. Neurological evaluation should be performed according to ICANS criteria (Lee et al., 2019). ICANS grading requires assessment of a 10-point immune effector cell-related encephalopathy score and evaluation of four other neurological domains: level of consciousness, seizures, motor symptoms, and signs of increased intracranial pressure / cerebral edema, with or without encephalopathy (see protocol). Close monitoring of mental status during treatment is crucial to ensure timely initiation of supportive care as needed. Supportive care includes, but is not limited to: Initiate IV fluid resuscitation Discontinue oral intake Drugs that cause central nervous system depression Start corticosteroids Anti-cytokine therapy Anticonvulsant therapy A total of 7 subjects experienced adverse events related to neurological symptoms (AESI). Three subjects in the extended portion of GCT3013-01 experienced ICANS, one of which was fatal. Four of the 7 subjects were from the dose-escalation portion of GCT3013-01 and experienced 3 severe AESIs (Grade 3 impaired consciousness, Grade 3 hypersomnia, Grade 1 partial seizures) and 2 non-severe AESIs (Grade 1 dysgraphia and Grade 1 agraphia).

Claims

1. A method for treating B-cell non-Hodgkin lymphoma (B-NHL) in a human subject, the method comprising subcutaneously administering a bispecific antibody as a full-length antibody to the human subject in need, the bispecific antibody comprising... (i) A first binding arm comprising a first antigen-binding region binding to human CD3ε (epsilon), comprising a variable heavy chain region and a variable light chain region, wherein the variable heavy chain region comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:6; and wherein the variable light chain region comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:7; and (ii) A second binding arm comprising a second antigen-binding region that binds to human CD20, comprising a variable heavy chain region and a variable light chain region, wherein the variable heavy chain region comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID No:13; and wherein the variable light chain region comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID No:14; The bispecific antibody is administered at a dose of at least 24 mg.

2. The method according to claim 1, wherein the B-NHL is selected from the group consisting of: diffuse large B-cell lymphoma (DLBCL), high-grade B-cell lymphoma (HGBCL), primary mediastinal large B-cell lymphoma (PMBCL), mantle cell lymphoma, follicular lymphoma (FL), marginal zone lymphoma, and small lymphocytic lymphoma.

3. The method according to claim 1, wherein the B-NHL is FL, HGBCL or DLBCL.

4. The method according to any one of claims 1-3, wherein the subject has received treatment with a CD20 monospecific antibody, such as rituximab, prior to treatment with the bispecific antibody.

5. The method of claim 4, wherein the cancer recurs during the treatment with the CD20 monospecific antibody.

6. The method of claim 4, wherein the cancer is refractory to the treatment during the treatment with the CD20 monospecific antibody.

7. The method according to any one of claims 4-6, wherein the CD20 monospecific antibody is used in combination therapy.

8. The method according to any one of claims 1-7, wherein the patient has received a further prior line of treatment for the B-cell NHL.

9. The method according to any one of claims 1-8, wherein the bispecific antibody is administered at a dose of at least 40 mg.

10. The method according to any one of claims 1-8, wherein the bispecific antibody is administered in a dose ranging from 40 mg to 100 mg.

11. The method according to any one of claims 1-8, wherein the bispecific antibody is administered at a dose of at least 48 mg.

12. The method according to any one of claims 1-8, wherein the bispecific antibody is administered at a dose of at least 60 mg.

13. The method according to any one of claims 1-8, wherein the bispecific antibody is administered at a dose of 48 mg.

14. The method according to any one of claims 1-8, wherein the bispecific antibody is administered at a dose of 60 mg.

15. The method according to any one of claims 1-14, wherein the dose is administered weekly.

16. The method of claim 15, wherein the weekly application is performed at least four times.

17. The method of claim 15 or 16, wherein the antibody is administered every two weeks after the weekly administration.

18. The method of claim 17, wherein the bi-weekly application is performed (at least) 6 times.

19. The method of claim 17 or 18, wherein the antibody is administered every four weeks after the administration every two weeks.

20. The method according to any one of claims 15-19, wherein the initiation dose of the bispecific antibody is administered prior to the administration of the weekly dose.

21. The method of claim 20, wherein the triggering dose is administered one week prior to the administration of the first dose of the weekly dose.

22. The method according to claim 20 or 21, wherein the initiating dose is in the range of 50-350 μg.

23. The method according to any one of claims 20-22, wherein the initiating dose is 160 μg.

24. The method of claims 20-23, wherein an intermediate dose of the bispecific antibody is administered after the initiating dose and before the weekly dose.

25. The method of claim 24, wherein the initiating dose is administered two weeks prior to the administration of the first dose of the weekly dose, and the intermediate dose is administered one week prior to the administration of the first dose of the weekly dose.

26. The method according to any one of claims 24 or 25, wherein the intermediate dose is in the range of 600-1200 μg.

27. The method according to any one of claims 24-26, wherein the intermediate dose is 800 μg.

28. The method according to any one of claims 24-27, wherein the treatment method comprises subcutaneously administering the bispecific antibody in a 28-day cycle, wherein: a) On days 1, 8, 15 and 22 of the first cycle, the initiating dose is administered on day 1, the intermediate dose on day 8, and a dose of 48 mg is administered on days 15 and 22; for the first cycle; b) Administer a dose of 48 mg on days 1, 8, 15, and 22 of cycle 2-3; c) Administer a dose of 48 mg on days 1 and 15 of cycles 4-9; as well as d) On day 1 of the subsequent cycle, administer a dose of 48 mg.

29. The method according to claims 24-27, wherein the treatment method comprises subcutaneously administering the bispecific antibody in a 28-day cycle, wherein: a) On days 1, 8, 15 and 22 of the first cycle, administer an initiating dose of 160 μg on day 1, an intermediate dose of 800 μg on day 8, and a dose of 48 mg on days 15 and 22; for the first cycle. b) Administer a dose of 48 mg on days 1, 8, 15, and 22 of cycle 2-3; c) Administer a dose of 48 mg on days 1 and 15 of cycles 4-9; as well as d) On day 1 of the subsequent cycle, administer a dose of 48 mg.

30. The method according to any one of claims 24-27, wherein the treatment method comprises subcutaneous administration of the bispecific antibody in a 28-day cycle, wherein: a) On days 1, 8, 15 and 22 of the first cycle, administer the initiating dose on day 1, the intermediate dose on day 8, and the 60 mg dose on days 15 and 22; for the first cycle; b) Administer a dose of 60 mg on days 1, 8, 15, and 22 of cycle 2-3; c) Administer a dose of 60 mg on days 1 and 15 of cycles 4-9; and d) On day 1 of the subsequent cycle, administer a dose of 60 mg.

31. The method according to any one of claims 24-27, wherein the treatment method comprises subcutaneous administration of the bispecific antibody in a 28-day cycle, wherein: a) On days 1, 8, 15 and 22 of the first cycle, administer an initiating dose of 160 μg on day 1, an intermediate dose of 800 μg on day 8, and a dose of 60 mg on days 15 and 22; for the first cycle. b) Administer a dose of 60 mg on days 1, 8, 15, and 22 of cycle 2-3; c) Administer a dose of 60 mg on days 1 and 15 of cycles 4-9; d) On day 1 of the subsequent cycle, administer a dose of 60 mg.

32. The method according to any one of claims 28-31, wherein the B-cell NHL is FL, DLBCL and / or HGBCL.

33. The method according to any one of claims 1-32, wherein the subject has managed cytokine release syndrome (CRS) at grade 1 or 2 during the period.

34. The method according to any one of claims 1-33, wherein the subject does not experience tumor lysis syndrome.

35. The method according to any one of claims 1-34, wherein the subject is treated with prevention and treatment of cytokine release syndrome (CRS).

36. The method of claim 35, wherein the prevention and treatment includes the administration of corticosteroids.

37. The method according to any one of claims 35-36, wherein the prevention and treatment are administered on the same day as the bispecific antibody.

38. The method according to any one of claims 37, wherein the treatment is applied on the second or third day thereafter and optionally on the fourth day, or on the second or fourth day thereafter.

39. The method according to any one of claims 37-38, wherein when the prevention and treatment are administered on the same day as the bispecific antibody, the prevention and treatment are administered 30-120 minutes before the bispecific antibody is administered.

40. The method according to any one of claims 36-39, wherein the corticosteroid is prednisolone, for example, at an intravenous dose of 100 mg, or an equivalent thereof, including oral doses.

41. The method according to any one of claims 1-40, wherein the human subject is treated with a pre-treatment drug to reduce the response to the injection.

42. The method of claim 41, wherein the precursor medication comprises the administration of an antihistamine.

43. The method according to any one of claims 41-42, wherein the pre-treatment includes the administration of an antipyretic.

44. The method according to any one of claims 41-43, wherein the antihistamine is diphenhydramine, for example, in an intravenous or oral dose of 50 mg, or an equivalent thereof.

45. The method according to any one of claims 43-44, wherein the antipyretic is acetaminophen, for example, an oral dose of 650-1000 mg, or an equivalent thereof.

46. ​​The method according to any one of claims 41-45, wherein the precursor drug and the bispecific antibody are administered on the same day.

47. The method of claim 46, wherein the precursor drug is administered 30-120 minutes prior to the administration of the bispecific antibody.

48. The method according to any one of claims 28-47, wherein the prevention and treatment as defined in claims 35-40 is applied during the first cycle.

49. The method according to any one of claims 28-48, wherein the precursor drug as defined in claims 41-47 is administered during the first cycle.

50. The method according to claims 48 and 49, wherein the prevention and treatment are administered during the second cycle when the human subject experiences a CRS greater than grade 1 after the fourth administration of the bispecific antibody in cycle 1.

51. The method of claim 50, wherein when the human subject experiences a CRS greater than grade 1 during the last administration of the bispecific antibody in a previous cycle, the prevention continues during subsequent cycles.

52. The method according to claims 49 and 50, wherein the precursor drug is optionally administered during the second cycle.

53. The method of claim 52, wherein the precursor drug is optionally administered during a subsequent cycle.

54. The method according to any one of the preceding claims, wherein the bispecific antibody comprises: (i) A first binding arm comprising a first antigen-binding region binding to human CD3ε (epsilon), wherein the first antigen-binding region comprises heavy chain variable (VH) regions CDR1, CDR2, and CDR3 having sequences as listed in SEQ ID NO. 1, 2, and 3, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 having sequences as listed in SEQ ID NO. 4, sequence GTN, and sequences as listed in SEQ ID NO. 5, respectively. (ii) A second binding arm comprising a second antigen-binding region that binds to human CD20, wherein the second antigen-binding region comprises heavy chain variable (VH) regions CDR1, CDR2, and CDR3 having sequences as listed in SEQ ID NO. 8, 9, and 10, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 having sequences as listed in SEQ ID NO. 11, sequence DAS, and sequences as listed in SEQ ID NO. 12, respectively.

55. The method according to any one of the preceding claims, wherein the bispecific antibody comprises: (i) a first binding arm comprising a first antigen-binding region binding to human CD3ε (epsilon), comprising a variable heavy chain region and a variable light chain region, wherein the variable heavy chain region comprises SEQ ID NO:6; and wherein the variable light chain region comprises SEQ ID NO:7; and (ii) A second binding arm comprising a second antigen-binding region that binds to human CD20, comprising a variable heavy chain region and a variable light chain region, wherein the variable heavy chain region comprises SEQ ID No:13; and wherein the variable light chain region comprises SEQ ID No:

14.

56. The method according to any one of the preceding claims, wherein the first binding arm of the bispecific antibody is derived from a humanized antibody, preferably from a full-length IgG1,λ (lambda) antibody.

57. The method of claim 56, wherein the bispecific antibody comprises a constant region of the λ light chain as defined in SEQ ID NO.

22.

58. The method according to any one of the preceding claims, wherein the second binding arm of the bispecific antibody is derived from a human antibody, preferably from a full-length IgG1,κ (kappa) antibody.

59. The method of claim 58, wherein the bispecific antibody comprises a constant region of the κ light chain as defined in SEQ ID NO.

23.

60. The method according to any one of the preceding claims, wherein the bispecific antibody is a full-length antibody having a constant region of human IgG1.

61. The method according to any one of the preceding claims, wherein the bispecific antibody comprises an inert Fc region.

62. The method of claim 61, wherein the bispecific antibody comprises amino acids F, E, and A at positions L234, L235, and D265 in the human IgG1 heavy chain corresponding to both the first and second heavy chains, respectively.

63. The method according to any one of the preceding claims, wherein the bispecific antibody comprises amino acid L at position F405 in the human IgG1 heavy chain of SEQ ID NO:15 in the first heavy chain, and wherein the second heavy chain comprises amino acid R at position K409 in the human IgG1 heavy chain of SEQ ID NO:15, and vice versa.

64. The method according to any one of the preceding claims, wherein the bispecific antibody comprises amino acids F, E, and A at positions L234, L235, and D265 in the human IgG1 heavy chain of SEQ ID NO:15, respectively, corresponding to positions L234, L235, and D265 in both the first and second heavy chains, and wherein the first heavy chain comprises amino acid L at position F405 in the human IgG1 heavy chain of SEQ ID NO:15, and wherein the second heavy chain comprises amino acid R at position K409 in the human IgG1 heavy chain of SEQ ID NO:15, and vice versa.

65. The method of claim 64, wherein the bispecific antibody comprises a constant region as defined in SEQ ID NO. 19 and 20.

66. The method according to any one of the preceding claims, wherein the bispecific antibody comprises a heavy chain and a light chain as defined in SEQ ID NO. 24 and 25, and a heavy chain and a light chain as defined in SEQ ID NO. 26 and 27, respectively.

67. The method according to any one of the preceding claims, wherein the bispecific antibody comprises a heavy chain and a light chain as defined in SEQ ID NO. 24 and 25, and a heavy chain and a light chain as defined in SEQ ID NO. 26 and 27, respectively.

68. The method according to any one of the preceding claims, wherein the bispecific antibody is icoretuzumab or a biosimilar thereof.

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