Trispecific antibody targeting CD79b, CD20, and CD3

Abstract

Provided herein are trispecific antibodies and fragments thereof that bind to CD79b, CD20 and CD3 and methods of treating cancer using the same. Also described are related polynucleotides capable of encoding the provided trispecific antibodies, cells expressing the provided trispecific antibodies, as well as associated vectors.

Description

TRISPECIFIC ANTIBODY TARGETING CD79b, CD20, AND CD3CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 769,534, filed 10 March 2025 and to U.S. Provisional Application No. 63 / 882,127, filed 15 September 2025, each of which are incorporated herein by reference in their entirety.SEQUENCE LISTING

[0002] The instant application contains a sequence listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on February 19, 2026, is named JBI6983-SL.xml and is 95,299 bytes in size.BACKGROUND OF THE INVENTION

[0003] Non-Hodgkin lymphoma (NHL) accounts for about 4% of all cancers.Worldwide, DLBCL represents the most common subtype of B-cell NHL, accounting for 30% to 40% of all newly diagnosed cases. Outcomes in DLBCL have improved dramatically over the last 2 decades with the addition of rituximab to cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), which remains the current standard of care for upfront therapy. However, about 30% to 40% of patients with DLBCL treated with R-CHOP will have relapsed or refractory (R / R) disease. Despite improvements in available therapies, relap sed / refractory (R / R, sometimes referred to as r / r) NHLs are characterized by uniformly poor prognosis.

[0004] Patients with primary refractory disease or patients who relapse within 12 months of first-line therapy may be eligible for chimeric antigen receptor (CAR) T-cell therapy, but real-world evidence shows that many patients in whom CAR-T-cell therapy is indicated, have several barriers that limit access to treatment.

[0005] Stem cell transplantation (SCT) is indicated in patients with refractory disease or patients who relapse after 12 months of front-line therapy. However, about 50% of patients willbe ineligible due to comorbidities or other factors, and less than half of patients who are eligible, will be cured.

[0006] More recently, bispecific T-cell engager therapies have been approved in the third-line setting in R / RDLBCL with promising results. However serious and life-threatening cytokine release syndrome (CRS) and life-threatening or fatal immune effector cell-associated neurotoxicity syndrome (ICANS) remain a concern. In addition, hospitalization is required for certain doses and may be a barrier to treatment for some patients. There continues to be a need for novel treatments that are effective, accessible, and less toxic than the current options, including treatments that avoid the use of cytotoxic chemotherapy.

[0007] The B-cell receptor (BCR) is a multicomponent receptor located on B cells that is composed of a transmembrane immunoglobulin molecule (mlg) and a disulfide linked heterodimer of CD79a (Iga) and CD79b (IgP). CD79b is highly expressed in a wide range of B-cell lymphomas. Its expression plays a role in cancer cell viability of most DLBCL tumor models.

[0008] The CD20 molecule (also called human B -lymphocyte-restricted differentiation antigen or Bp35) is a hydrophobic transmembrane protein that is over-expressed on most hematological malignancies in B cell lineages. CD20 is found on the surface of greater than 90% of B cells from peripheral blood or lymphoid organs and is expressed during early pre-B cell development and remains until plasma cell differentiation. CD20 is present on both normal B cells as well as malignant B cells. In particular, CD20 is expressed on greater than 90% of B cell non-Hodgkin's lymphomas (NHL).

[0009] Targeting more than one B-cell antigens CD79b and CD20 could lead to enhance tumor binding specificity leading to more potent T-cell mediated cytotoxicity, maximize tumor eradication in the presence of heterogeneous cell population, and prevent resistance via antigen escape. Accordingly, there is a need for therapeutic antibodies that target both CD79b and CD20 for the treatment of B-cell malignancies such as B-cell lymphomas and non-Hodgkin's lymphomas.SUMMARY OF THE INVENTION

[0010] Disclosed herein is a CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprising:a) a first antigen-binding domain that binds to CD79b, wherein the first antigen-binding domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5 and 6, respectively;b) a second antigen-binding domain that binds to CD20, wherein the second antigenbinding domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 comprising the amino acid sequences of SEQ ID NOs: 9, 10, 11, 12, 13 and 14, respectively; andc) a third antigen -binding domain that binds to CD3, wherein the third antigen-binding domain comprises the HCDR1, the HCDR2,the HCDR3, the LCDR1, the LCDR2 and the LCDR3 comprising the amino acid sequences of SEQ ID NOs: 17, 18, 19, 20, 21, and 22, respectively;wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are according to the AbM delineation.

[0011] The disclosure also provides a CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprisinga) a first antigen-binding domain that binds to CD79b comprising a VH1 having an amino acid sequence of SEQ ID NO: 7 and comprising a VL1 having an amino acid sequence of SEQ ID NO: 8;b) a second antigen-binding domain that binds to CD20 comprising a VH2 having an amino acid sequence of SEQ ID NO: 15 and comprising a VL2 having an amino acid sequence of SEQ ID NO: 16; andc) a third antigen-binding domain that binds to CD3 comprising a VH3 having an amino acid sequence of SEQ ID NO: 23 and comprising a VL3 having an amino acid sequence of SEQ ID NO: 24.

[0012] In one embodiment, a trispecific antibody or a fragment thereof comprises first binding domain that binds to CD79b, a second binding domain that binds to CD20, and a third binding domain that binds to CD3, wherein:a) the first binding domain is a Fab comprising a VH1 and a VL1 having an amino acid sequence of SEQ ID NO: 7 and 8, respectively;b) the second binding domain is a spFv comprising a VL2-linkerl-VH2, wherein VH2 and VL2 comprise an amino acid sequence of SEQ ID NO: 15 and 16, respectively, and wherein the linkerl comprises an amino acid sequence of SEQ ID NO: 72; and c) the third binding domain is a spFv comprising a VL3-linkerl-VH3, wherein VH3 and VL3 comprise an amino acid sequence of SEQ ID NO: 23 and 24, respectively, and wherein the linkerl comprises an amino acid sequence of SEQ ID NO: 72.

[0013] In another embodiment, a trispecific antibody or a fragment thereof comprises first binding domain that binds to CD79b, a second binding domain that binds to CD20, and a third binding domain that binds to CD3, wherein:a) the first binding domain is a Fab comprising a VH1 and VL1 having an amino acid sequence of SEQ ID NO: 7 and 8, respectively;b) the second binding domain is a spFv having an amino acid sequence of SEQ ID NO: 70; andc) the third binding domain is a spFv having an amino acid sequence of SEQ ID NO: 71.

[0014] The disclosure also provides a CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprising a first heavy chain (HC1) having an amino acid sequence of SEQ ID NO: 25, a second heavy chain (HC2) having an amino acid sequence of SEQ ID NO: 26, and a light chain (LC) having an amino acid sequence of SEQ ID NO: 27.

[0015] The disclosure also provides a method of killing cancer cells and / or a method or treating cancer in a subject comprising administering to the subject a trispecific antibody or a fragment thereof that comprises a first antigen-binding domain that binds CD79b, a second antigen-binding domain that binds CD20 and a third antigen-binding domain that binds CD3, wherein(a) the first antigen-binding domain comprises:(i) the HCDRs of a VH1 comprising an amino acid sequence of SEQ ID NO: 7; and the LCDRs of a VL1 comprising an amino acid sequence of SEQ ID NO: 8;(ii) a VH1 comprising a HCDR1 having an amino acid sequence of SEQ ID NO: 1, a HCDR2 having an amino acid sequence of SEQ ID NO: 2, and a HCDR3 having an amino acid sequence of SEQ ID NO: 3; and a VL1 comprising a LCDR1 having an amino acid sequence of SEQ ID NO: 4, a LCDR2 having an amino acid sequence of and SEQ ID NO: 5, and a LCDR3 having an amino acid sequence of SEQ ID NO: 6; and / or(iii) a VH1 having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 7, and a VL1 having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 8;(a) the second antigen-binding domain comprises:(i) the HCDRs of a VH2 comprising an amino acid sequence of SEQ ID NO: 15; and the LCDRs of a VL2 comprising an amino acid sequence of SEQ ID NO: 16;(ii) a VH2 comprising a HCDR1 having an amino acid sequence of SEQ ID NO: 9, a HCDR2 having an amino acid sequence of SEQ ID NO: 10, and a HCDR3 having an amino acid sequence of SEQ ID NO: 11; and a VL2 comprising a LCDR1 having an amino acid sequence of SEQ ID NO: 12, a LCDR2 having an amino acid sequence of and SEQ ID NO: 13, and a LCDR3 having an amino acid sequence of SEQ ID NO: 14;(iii) a VH2 having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 15, and a VL2 having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 16; and(iv) a first spFv having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 70;(a) the third antigen-binding domain comprises:(i) the HCDRs of a VH3 comprising an amino acid sequence of SEQ ID NO: 23; and the LCDRs of a VL3 comprising an amino acid sequence of SEQ ID NO: 24;(ii) a VH3 comprising a HCDR1 having an amino acid sequence of SEQ ID NO: 17, a HCDR2 having an amino acid sequence of SEQ ID NO: 18, and a HCDR3 having an amino acid sequence of SEQ ID NO: 19; and a VL3 comprising a LCDR1 having an amino acid sequence of SEQ ID NO: 20, a LCDR2 having an amino acid sequence of and SEQ ID NO: 21, and a LCDR3 having an amino acid sequence of SEQ ID NO: 22;(iii) a VH3 having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 23, and / or a VL3 having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 24; and(iv) a second spFv having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 71.

[0016] In some embodiments, the disclosure also provides a method of killing cancer cells and / or a method or treating cancer in a subject comprising administering to the subject a trispecific antibody or a fragment thereof that comprises a first antigen-binding domain that binds CD79b, a second antigen-binding domain that binds CD20 and a third antigen-binding domain that binds CD3, wherein the antibody comprisesa) a HC1 having at least at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 25;b) a HC2 having at least at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 26; andc) a LC having at least at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 27.DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 shows the different CD79bxCD20xCD3 trispecific antibody formats evaluated.

[0018] FIG. 2 shows binding of CD79bxCD20xCD3 trispecific antibody formats to Camaval cells following 1 hour incubation at 37°C.

[0019] FIG. 3A and FIG. 3B shows binding of CD79bxCD20xCD3 trispecific antibody formats to human T cells following 1 hour incubation at 37°C. DN1, Donor 1; DN2, Donor 2.

[0020] FIG. 4A - 4D shows the therapeutic efficacy (in vitro cytotoxicity) of various CD79bxCD20xCD3 trispecific antibody formats in single and double antigen expressing K562 isogenic cell line models. FIG. 4A shows in vitro cytotoxicity of CD79bxCD20xCD3 trispecific antibody formats in antigen negative (K562) cells. FIG. 4B shows in vitro cytotoxicity of CD79bxCD20xCD3 trispecific antibody formats in CD79b positive cell lines (K562 CD79b cells). FIG. 4C shows in vitro cytotoxicity of CD79bxCD20xCD3 trispecific antibody formats in CD20 positive cell lines (K562 CD20 cells). FIG. 4D shows in vitro cytotoxicity of CD79bxCD20xCD3 trispecific antibody formats in CD79b and CD20 positive cell lines (K562 CD79b / CD20 cells). Graphing of data was done in GraphPad Prism 10.

[0021] FIG. 5A - 5D shows the therapeutic efficacy (in vitro cytotoxicity) of various CD79bxCD20xCD3 trispecific antibody formats in B-NHL models displaying a wide range of CD79b and CD20 endogenous expression levels. FIG. 5A shows in vitro cytotoxicity of CD79bxCD20xCD3 trispecific antibody formats in Carnaval cells. FIG. 5B shows in vitro cytotoxicity of CD79bxCD20xCD3 trispecific antibody formats in WILL-2 cells. FIG. 5C shows in vitro cytotoxicity of CD79bxCD20xCD3 trispecific antibody formats in OCI-LY10 cells. FIG. 5D shows in vitro cytotoxicity of CD79bxCD20xCD3 trispecific antibody formats in HT cells. Graphing of data was done in GraphPad Prism 10.

[0022] FIG. 6A- 6D shows T-cell activation by various CD79bxCD20xCD3 trispecific antibody formats in antigen positive cell lines (K562 CD20, K562 CD79b or K562 CD79b / CD20) cell lines or antigen-negative (K562) cancer cell line. FIG. 6A shows T cell activation by CD79bxCD20xCD3 trispecific antibody formats in antigen negative (K562) cells. FIG. 6B shows T cell activation by CD79bxCD20xCD3 trispecific antibody formats in CD79bpositive cell lines (K562 CD79b cells). FIG. 6C shows T cell activation by CD79bxCD20xCD3 trispecific antibody formats in CD20 positive cell lines (K562 CD20 cells). FIG. 6D shows T cell activation by CD79bxCD20xCD3 trispecific antibody formats in CD79b / CD20 positive cell lines (K562 CD79b / CD20 cells). Graphing of data was done in GraphPad Prism 10.

[0023] FIG. 7A- 7D shows T-cell activation by various CD79bxCD20xCD3 trispecific antibody formats in endogenous DLBCL cancer cell lines. FIG. 7A shows T cell activation by CD79bxCD20xCD3 trispecific antibody formats in Carnaval cells. FIG. 7B shows T cell activation by CD79bxCD20xCD3 trispecific antibody formats in WIL1-2. FIG. 7C shows T cell activation by CD79bxCD20xCD3 trispecific antibody formats in OCI-LylO cells. FIG. 7D shows T cell activation of CD79bxCD20xCD3 trispecific antibody formats in HT cells.Graphing of data was done in GraphPad Prism 10.

[0024] FIG. 8A- 8D shows binding of CD79bxCD20xCD3 trispecific antibody (C923B387) and CD3xNullxNull control to endogenous tumor cell lines expressing CD79b, CD20 or CD79b and CD79b following 1 hour incubation at 37°C. FIG. 8A shows binding of CD79bxCD20xCD3 trispecific antibody to Camaval WT (CD79b+, CD20+) cells. FIG. 8B shows binding of CD79bxCD20xCD3 trispecific antibody to Carnaval-CD20-KO cells (CD79b+, CD20-) cells (clone P1E5). FIG. 8C shows binding of CD79bxCD20xCD3 trispecific antibody to K562-CD79b-GFP target cells (CD79b+, CD20"). FIG. 8D shows binding of CD79bxCD20xCD3 trispecific antibody to K562-CD20-mKate (CD79b~, CD20+) cells (clone H1B9) .

[0025] FIG. 9 shows CD79bxCD20xCD3 trispecific antibody (C923B387) binding on human T cells. CD, cluster of differentiation; MFI, mean fluorescence intensity. The graph shows T-cell binding of CD79bxCD20xCD3 trispecific antibody and NullxCD79b-Ofa null control following a 1-hour 37°C incubation. The graph includes two technical replicates at each concentration.

[0026] FIG. 10A - 10E shows the therapeutic efficacy (in vitro cytotoxicity assay) of CD79bxCD20xCD3 trispecific antibody (C923B387) in a panel of cell line models showing differential antigen expression levels in comparison to the Reference Trispecific Antibody. FIG.10A shows in vitro cytotoxicity of CD79bxCD20xCD3 trispecific antibody in Carnaval cells. FIG. 10B shows in vitro cytotoxicity of CD79bxCD20xCD3 trispecific antibody in OCI-LylOcells. FIG. IOC shows in vitro cytotoxicity of CD79bxCD20xCD3 trispecific antibody in HT cells. FIG. 10D shows in vitro cytotoxicity of CD79bxCD20xCD3 trispecific antibody in WILL-2 cells. FIG. 10E shows in vitro cytotoxicity of CD79bxCD20xCD3 trispecific antibody in K562 cells. Graphing of data was done in GraphPad Prism 10. Data from 3 to 4 independent experiments were pooled and represented as mean ± SEM.

[0027] FIG. 11A- 1 IE shows CD79bxCD20xCD3 trispecific antibody(C923B387) -induced T-cell activation in the presence of different endogenous DLBCL cancer cell lines in comparison with a Reference Trispecific Ab. FIG. 11 A shows CD79bxCD20xCD3 trispecific antibody-induced T-cell activation in Camaval cells. FIG. 1 IB shows CD79bxCD20xCD3 trispecific antibody-induced T-cell activation in OCI-LylO cells. FIG. 11C shows CD79bxCD20xCD3 trispecific antibody-induced T-cell activation in HT cells. FIG. 11D shows CD79bxCD20xCD3 trispecific antibody-induced T-cell activation in WILL-2 cells. FIG. HE shows CD79bxCD20xCD3 trispecific antibody-induced T-cell activation inK562 cells.

[0028] FIG. 12 shows the effect of Reference Trispecific Antibody or CD79bxCD20xCD3 trispecific antibody (C923B387) on growth of established SC OCI-LylO DLBCL xenografts in T-cell-humanized NSG mice. DPBS, Dulbecco’s phosphate-buffered saline. Group tumor volumes are graphed as mean ± SEM (n=9 / group). Tumor cells (1 x 106in matrigel [1:1]) were implanted subcutaneous (SC) on Day 0, T cells were injected IP on Day 13, and dosing occurred on Days 14, 17, 20, 24, 28, 32, 35, 38, 41, and 45 (dosing period indicated by the bar below the X-axis) Data are displayed when at least two thirds of animals remained in each group. * Denotes significant differences (p<0.05) compared to DPBS-treated controls on Day 35.

[0029] FIG. 13 shows the effect of CD79bxCD20xCD3 (C923B387) on growth of established SC OCI-LylO DLBCL xenografts in T-cell-humanized NSG MHC Eli dKO mice. Group tumor volumes are graphed as mean ± SEM (n=10 / group). Tumor cells (1 * 106 in matrigel [1:1]) were implanted SC on Day 0, T cells were injected IP on Day 13, and dosing occurred on Days 14, 17, 20, 23, 27, 30, 34, 37, 41, and 44 for all dosing groups and additionally on Days 48, 51, 55, and 58 for remaining animals in groups dosed with CD79bxCD20xCD3 trispecific antibody at 0.15 and 0.05 mg / kg (dosing period indicated by the bar below the X-axis). Data are displayed when at least two thirds of animals remained in eachgroup. * Denotes significant differences (p<0.05) compared to DOBS-treated controls on Day 30.

[0030] FIG. 14A and FIG. 14B compared therapeutic efficacy (in vitro cytotoxicity) of CD79bxCD20xCD3 trispecific antibody C923B387 and CD79bxCD20xCD3 trispecific antibody C923B413 in Carnaval cells at effector target ratio of 3:1 and 1:1, respectively. FIG.14A also compared in vitro cytotoxicity of C923B387 and C923B413 to reference CD79bxCD20xCd3 trispecific antibody.

[0031] FIG. 15 compares the expression of CD79bxCD20xCD3 trispecific antibody C923B387 and CD79bxCD20xCD3 trispecific antibody C923B413 in ExpiCHO cells at different HC1:HC2 transfection ratios.

[0032] FIG. 16A and FIG.16B show the effect of CD79bxCD20xCD3(C923B387), Reference Trispecific Antibody and germline NullxNullxCD3 Control Antibodies on the Cytotoxicity of CARNAVAL or Autologous B-cells in a Whole Blood T-cell Functional Assay Conducted at a 1 : 1 E:T Ratio. Whole blood from 8 healthy donors was tested in a flow cytometry assay at 72 hours. Co-culture with CD79b+ / CD20+ Camaval cells was set up to achieve an E:T ratio of 1:1. (FIG. 16A) Carnaval cell cytotoxicity and (FIG. 16B) autologous B-cell cytotoxicity were assessed upon treatment with indicated antibodies. Data are averaged and mean + SEM are graphed.

[0033] FIG. 17A - 17H shows the effect of CD79bxCD20xCD3 (C923B387) and Reference Trispecific Antibody on cytokine release in a whole-blood T-cell functional assay conducted at a 1:1 E:T Ratio.DETAILED DESCRIPTION OF THE INVENTION

[0034] Provided herein are CD79bxCD20xCD3 trispecific antibodies that bind CD79b, CD20 and CD3 and antigen binding fragments thereof, polynucleotides and expression vectors encoding the antibodies, host cells containing the vectors, and compositions comprising the antibodies. Methods of making the antibodies, and methods of using the antibodies to treat diseases are also provided.

[0035] The trispecific CD79bxCD20xCD3 antibodies or a fragment thereof disclosed herein possess one or more desirable functional properties, including but not limited to highspecificity and high affinity to CD79b, CD20 and CD3. In certain embodiments, the antibodies disclosed herein possess the ability to treat or prevent a disease or disorder when administered to a subject.

[0036] The disclosed CD79bxCD20xCD3 trispecific antibodies or a fragment thereof, polynucleotides, vectors, cells, compositions, and methods may be understood more readily by reference to the following detailed description which forms a part of this disclosure. It is to be understood that the disclosed antibodies, antigen binding domains, antibody fragments, polynucleotides, vectors, cells, compositions, and methods are not limited to those specifically described and / or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed antibody, antigen binding domains, antibody fragments, polynucleotides, vectors, cells, compositions, and methods.

[0037] Throughout this text, where a range of numerical values is recited or established herein, the range includes the endpoints thereof and all the individual integers and fractions within the range, and also includes each of the narrower ranges therein formed by all the various possible combinations of those endpoints and internal integers and fractions to form subgroups of the larger group of values within the stated range to the same extent as if each of those narrower ranges was explicitly recited. Where a range of numerical values is stated herein as being greater than a stated value, the range is nevertheless finite and is bounded on its upper end by a value that is operable within the context of the embodiment as described herein. Where a range of numerical values is stated herein as being less than a stated value, the range is nevertheless bounded on its lower end by a non-zero value. It is not intended that the scope of the embodiment be limited to the specific values recited when defining a range. All ranges are inclusive and combinable.Definitions

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure pertains. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

[0039] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

[0040] It should also be understood that the terms “approximately,” “generally,” “substantially” and like terms, used herein when referring to a dimension or characteristic of a component of the preferred embodiment, indicate that the described dimension / characteristic is not a strict boundary or parameter and does not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one having ordinary skill in the art. At a minimum, such references that include a numerical parameter would include variations that, using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.), would not vary the least significant digit. It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (for example, bodies of the appended claims) are generally intended as “open” terms (for example, the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc ).

[0041] The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of up to ±10% from the specified value, as such variations are appropriate to perform the disclosed methods. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present embodiment. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0042] Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize orbe able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the embodiments described herein. Such equivalents are intended to be encompassed by the embodiment.

[0043] “ Antigen binding fragment”, “antigen binding domain”, “antibody fragment” or “binding domain” refers to a portion of the protein that binds an antigen, or to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen (e. g., CD79b, CD20, CD3). Antigen binding domain may be synthetic, enzymatically obtainable or genetically engineered polypeptides and include portions of an immunoglobulin that bind an antigen, such as a heavy chain variable region (VH), a light chain variable region (VL), a VH and a VL, a Fab, a Fab’, a F(ab')2 , a Fd and a Fv fragments, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv1), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), or a disulfide stabilized single-chain antibody molecule (spFv or stapled scFv), a single domain antibody (sdab) an scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a domain antibody (dAb) consisting of one VH domain or one VL domain, a shark variable IgNAR domain, a camelized VH domain, a VHH domain, a minimal recognition unit consisting of the amino acid residues that mimic the complementarity determining regions (CDRs) of an antibody, such as a FR3-CDR3-FR4 portion, the HCDR1, the HCDR2 and / or the HCDR3 and the LCDR1, the LCDR2 and / or the LCDR3, an alternative scaffold that bind an antigen, a bivalent domain antibody, a multispecific protein comprising the antibody or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure.

[0044] The term “antibody” refers to all isotypes of immunoglobulins (IgG, IgA, IgE, IgM, IgD, and IgY) including various monomeric, polymeric and chimeric forms, unless otherwise specified. Specifically encompassed by the term “antibody” are polyclonal antibodies, monoclonal antibodies (mAbs), and antibody-like polypeptides, such as chimeric antibodies and humanized antibodies. The term antibody also includes monospecific, bispecific, trispecific, and multispecific antibodies, and fragment thereof. Examples of suitable antibody fragments include, without limitation diabodies and single-chain molecules as well as Fab, F(ab’)2, Fc, Fabc, and Fv molecules, scFv, single chain (Sc) antibodies, individual antibody light chains,individual antibody heavy chains, chimeric fusions between antibody chains or CDRs and other proteins, protein scaffolds, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy and one light chain, a monovalent fragment consisting of the VL, VH, CL (light chain constant domain) and CHI (heavy chain first constant domain) domains, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, a Fd fragment consisting essentially of the VH and CH I domains; a Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody, a dAb fragment which consists essentially of a VH domain, camelid or nanobodies, an isolated complementarity determining region (CDR), and the like.

[0045] The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the antibody is administered. Such vehicles may be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. For example, 0.4% saline and 0.3% glycine may be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating and coloring agents, etc. The concentration of the antibodies in such pharmaceutical formulation may vary from less than about 0.5%, usually to at least about 1% to as much as 15 or 20% by weight and may be selected primarily based on required dose, fluid volumes, viscosities, etc., according to the mode of administration selected.

[0046] The transitional terms “comprising,” “consisting essentially of,” and “consisting of’ are intended to connote their generally accepted meanings in the patent vernacular; that is, (i) “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; (ii) “consisting of’ excludes any element, step, or ingredient not specified in the claim; and (iii) “consisting essentially of’ limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel character! stic(s)” of the claimed disclosure. Embodiments described in terms of the phrase “comprising” (or its equivalents) also provide as embodiments those independently described in terms of “consisting of’ and“consisting essentially of.” Embodiments described in terms of the phrase “consisting essentially of’ (or its equivalents) also provide as embodiments those independently described in terms of “consisting of.”

[0047] As used herein, “Cluster of Differentiation CD79B protein” or “CD79b” refers to a B-cell antigen receptor (BCR) signaling component Ig|3. The amino acid sequences of the various isoforms are retrievable from GenBank accession numbers AAH32651.1, EAW94232.1, AAH02975.2, NP_000617.1, and NP_001035022.1. Unless specified, as used herein, “CD79b” refers to human CD79b. An exemplary human CD79b comprises the amino acid sequence of SEQ ID NO: 76. The sequence includes the extracellular domain (residues 29-159) and the cytoplasmic domain (residues 181-229).SEQ ID NO: 76 (human CD79b)MARL ALSP VP SHWMVALLLLL S AEP VPAARSEDRYRNPKGS AC SRIWQ SPRFIARKRGF TVKMHCYMNSASGNVSWLWKQEMDENPQQLKLEKGRMEESQNESLATLTIQGIRFED NGIYFCQQKCNNTSEVYQGCGTELRVMGFSTLAQLKQRNTLKDGIIMIQTLLIILFIIVPIF LLLDKDDSKAGMEEDHTYEGLDIDQTATYEDIVTLRTGEVKWSVGEHPGQE

[0048] As used herein “Cluster of Differentiation 20” or “CD20” refers to an antigenic determinant known to be detectable on B cells. Human CD20 is also called membrane-spanning 4-domains, subfamily A, member 1 (MS4A1). The CD20 protein consists of 4 hydrophobic transmembrane (TM) domains, one intracellular domain, and 2 extracellular domains (ECDs; large and small loops) with both N- and C-termini residing within the cytosol. The human and murine amino acid and nucleic acid sequences can be found in a public database, such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequence of human CD20 can be found at Accession Nos. NP_690605.1 and NP_068769.2, and the nucleic acid sequence encoding transcript variants 1 and 3 of the human CD20 can be found at Accession No.NM 152866.2 and NM 021950.3, respectively. All references to proteins, polypeptides and protein fragments herein are intended to refer to the human version of the respective protein, polypeptide or protein fragment unless explicitly specified as being from a non-human species. Thus, “CD20” means human CD20 unless specified as being from a non-human species, e.g.,“mouse CD20” “monkey CD20,” etc. An exemplary human CD20 comprises the amino acid sequence of SEQ ID NO: 77.SEQ ID NO: 77 (human CD20) MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGAVQIM NGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIISGSLLAATEKNSRKCLVKGKMI MNSLSLFAAISGMILSIMDILNIKISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPST QYCYSIQSLFLGILSVMLIFAFFQELVIAGIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEI KEE V VGLTET S S QPKNEEDIEIIPIQEEEEEETETNFPEPPQDQES SPIEND S SP

[0049] The term “CD3” refers to an antigen that is expressed on T cells as part of the multimeric T cell receptor (TCR) complex and which consists of a homodimer or heterodimer formed from the association of two or four receptor chains: CD3 epsilon, CD3 delta, CD3 zeta and CD3 gamma. In some embodiments, CD3 antibodies provided herein bind to the CD3-epsilon polypeptide, which together with CD3-gamma, -delta and -zeta, and the T cell receptor alpha / beta and gamma / delta heterodimers, forms the T cell receptor-CD3 complex. This complex plays an important role in coupling antigen recognition to several intracellular signaltransduction pathways. The CD3 complex mediates signal transduction, resulting in T cell activation and proliferation. CD3 is required for the immune response. The term “CD3” includes any CD3 variant, isoform, and species homolog, which is naturally expressed by cells (including T cells) or can be expressed on cells transfected with genes or cDNA encoding the polypeptide. In specific embodiments, the CD3 is a human CD3. All references to proteins, polypeptides and protein fragments herein are intended to refer to the human version of the respective protein, polypeptide or protein fragment unless explicitly specified as being from a non-human species. Thus, “CD3” means human CD3 unless specified as being from a nonhuman species, e.g., “mouse CD3” “monkey CD3,” etc. An exemplary human CD3 epsilon comprises the amino acid sequence of SEQ ID NO: 78.SEQ ID NO: 78 (human CD3 epsilon) MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEIL WQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRA RVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQR

[0050] “ Chimeric antibodies” refer to antibodies having a variable region or part of variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals (e.g., a non-human mammal such as mouse, rabbit, and rat), while the constant portions are homologous to the sequences in antibodies derived from another mammal such as human. In some embodiments, amino acid modifications can be made in the variable region and / or the constant region.Techniques developed for the production of “chimeric antibodies” are well known in the art..

[0051] “ dAb” or “dAb fragment” refers to an antibody fragment composed of a VH domain.

[0052] An “effective amount” refers to an amount effective, at doses and for periods of time necessary, to achieve a desired therapeutic result. Such concentrations can be routinely determined by those of skilled in the art. An effective amount may vary depending on factors such as the disease state, age, sex, and weight of the individual, the physical condition of the patient, the duration of the treatment, the nature of concurrent therapy (if any), the specific formulations employed, the structure of the antibody or antibody fragment or of its variants and the ability of a therapeutic or a combination of therapeutics to elicit a desired response in the individual. An effective amount of the administered antibody or antibody fragment may depend on the type and severity of the cancer being treated, and the route of administration of the antibody polypeptide or the pharmaceutical composition of the antibody.

[0053] “Fab” or “Fab fragment” refers to an antibody fragment composed of VH, CHI, VL and CL domains.

[0054] “F(ab')2” or “F(ab')2 fragment” refers to an antibody fragment containing two Fab fragments connected by a disulfide bridge in the hinge region.

[0055] “Fd” or “Fd fragment” refers to an antibody fragment composed of VH and CHI domains.

[0056] “Fv” or “Fv fragment” refers to an antibody fragment composed of the VH and the VL domains from a single arm of the antibody. Fv fragments lack the constant regions of Fab (CHI and CL) regions. The VH and VL in Fv fragments are held together by non-covalent interactions.

[0057] The term “humanized” antibody (or antigen-binding fragment thereof), as used herein, refers to an antibody in which at least one CDR is derived from non-human species and at least one framework is derived from human immunoglobulin sequences. Humanized antibody may include substitutions in the frameworks so that the frameworks may not be exact copies of expressed human immunoglobulin or human immunoglobulin germline gene sequences. An humanized antibody (or antigen-binding fragment thereof) retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for instance, by retaining the non-human CDR regions and replacing the remaining parts of the antibody with their human counterparts (. e.g., the constant region as well as the framework portions of the variable region). Additional framework region modifications may be made within the human framework sequences as well as within the CDR sequences derived from the germline of another mammalian species. The humanized antibodies of the disclosure may include amino acid residues not encoded by human sequences (e. g., mutations introduced by random or sitespecific mutagenesis in vitro or by somatic mutation in vivo, or a conservative substitution to promote stability or manufacturing).

[0058] The term “immunoglobulin Fc region”, or “Fc region”, as used herein, is used to define a C-terminal region of an immunoglobulin heavy chain constant region, i. e. the CH2 and CH3 domains of the heavy chain constant regions. The term “Fc region” may include nativesequence Fc regions and variant Fc regions, i. e. Fc regions that are engineered to exhibit certain desired properties, such as for example altered Fc receptor binding function, reduced or suppressed arm exchange, and / or increased serum half-life. “Fc polypeptide” of a dimeric Fc refers to one of the two polypeptides forming the dimeric Fc domain. For example, an Fc polypeptide of a dimeric IgG Fc comprises an IgG CH2 and an IgG CH3 constant domain sequence).

[0059] The term “IgG region”, as used herein, refers to the heavy and light chain of an immunoglobulin G, i. e. the Fc region, as defined above, and the Fab region, consisting of the VL, VH, CL and CHI domains. The term “IgG region” includes native-sequence Ig regions, such as human IgGl, IgG2 (IgG2A, IgG2B), IgG3 and IgG4, as well as engineered IgG regions, which exhibit certain desired properties, as for example the properties define above for the Fc region. The term “functional IgG region”, as used herein, refers to an IgG region comprising afunctional Fc region. The “IgG constant region” consists of the CHI, CH2 and CH3 of the immunoglobulin heavy chain and / or the CL domain of the immunoglobulin light chain of an IgG.

[0060] In the context of the present disclosure, “linkerl” specifically refers to the linker used to connect the CD20 variable heavy and light chains (VH2 and VL2) or CD3 variable heavy and light chains (VH3 and VL3). For example, linkerl refers to a linker connecting VH2 to the VL2 in the VH2-Linkerl-VL2 orientation, or the V2L to the VH2 in the VL2 -Linker 1-VH2 orientation, or the VH3 to the VL3 in the VH3 -Linkerl -VL3 orientation or the VL3 to the VH3 in the VL3-Linkerl-VH3 orientation to create an spFv. Linkerl may comprise, consist of and / or consist essentially of the amino acid sequence of SEQ ID NO: 72, 79, 80, 81 or 82. Linkerl may comprise, consist of and / or consist essentially of the amino acid sequence of SEQ ID NO: 72 (GGGSGGSGGCPPCGGSGG). Linkerl may comprise, consist of and / or consist essentially of the amino acid sequence of SEQ ID NO: 79 (GGGSGGCPPCGGGSGG). Linkerl may comprise, consist of and / or consist essentially of the amino acid sequence of SEQ ID NO: 80 (GGSGGSGGCPPCGSGG). Linkerl may comprise, consist of and / or consist essentially of the amino acid sequence of SEQ ID NO: 81 (GGGSGGSGGCPPCGSGG). Linkerl may comprise, consist of and / or consist essentially of the amino acid sequence of SEQ ID NO: 82 (GGGSGGGSGCPPCGGGG).

[0061] In the context of the present disclosure, “linker2” refers to a linker that further connects the spFv of the CD20 antigen-binding domain to the VH1 of the CD79b binding domain to form a VL2-linkerl-VH2-linker2-VHl. Linker2 may comprise, consist of and / or consist essentially of the amino acid sequence of SEQ ID NO: 73 (GGGGSGGGGS).

[0062] In the context of the present disclosure, “linker3” refers to a linker that further connects the CD79b and CD20 binding domains to an IgG constant region or a fragment thereof to form a VL2-linkerl-VH2-linker2-VHl-linker3-Fc. Linker3 may comprise, consist of and / or consist essentially of the amino acid sequence of SEQ ID NO: 75 (EPKSCDKTHTCPPCP).

[0063] As used herein, “linker4” refers to a linker that further connects the spFv of the CD3 binding domain to an IgG constant region or the fragment of an IgG constant region to form a VL3-linkerl-VH3-linker4-Fc. Linker4 may comprise, consist of and / or consist essentially of the amino acid sequence of SEQ ID NO: 74 (EPKSSDKTHTCPPCP).

[0064] The term “operably linked”, “fused together” or “linked” as used herein, indicates that two molecules (e. g., polypeptides, domains, binding domains) are attached in a way that each molecule retains functional activity. Two molecules can be “operably linked” whether they are attached directly or indirectly (e. g., via a linker, via a moiety, via a linker to a moiety). The term “linker” refers to a peptide or other moiety that is optionally located between binding domains, portions of binding domains or antibody fragments in the disclosure. A number of strategies may be used to covalently link molecules together. These include, but are not limited to, polypeptide linkages between N- and C-termini of proteins or protein domains, linkage via disulfide bonds, and linkage via chemical cross-linking reagents. In one aspect of this embodiment, the linker is a peptide bond, generated by recombinant techniques or peptide synthesis. Choosing a suitable linker for a specific case where two polypeptide chains are to be connected depends on various parameters, including but not limited to the nature of the two polypeptide chains (e. g., whether they naturally oligomerize), the distance between the N- and the C-termini to be connected if known, and / or the stability of the linker towards proteolysis and oxidation. Furthermore, the linker may contain amino acid residues that provide flexibility.

[0065] In the context of the present disclosure, the term “polypeptide linker”, “peptide linker”, “linker”, or “linker peptide” refers to a linker consisting of a chain of amino acid residues linked by peptide bonds that is connecting two domains, each being attached to one end of the linker. The polypeptide linker should have a length that is adequate to link two molecules in such a way that they assume the correct conformation relative to one another so that they retain the desired activity. In particular embodiments, the polypeptide linker has a continuous chain of between 2 and 30 amino acid residues (e. g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues). In addition, the amino acid residues selected for inclusion in the polypeptide linker should exhibit properties that do not interfere significantly with the activity of the polypeptide. Thus, the linker peptide on the whole should not exhibit a charge that would be inconsistent with the activity of the polypeptide, or interfere with internal folding, or form bonds or other interactions with amino acid residues in one or more of the monomers that would seriously impede the binding of receptor monomer domains. In particular embodiments, the polypeptide linker is non- structured polypeptide. Useful linkers include glycine-serine, or GS linkers. By “Gly-Ser” or “GS” linkersis meant a polymer of glycines and serines in series (including, for example, (Gly-Ser)n, (GSGGS)n (GGGGS)n and (GGGS)n, where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers such as the tether for the shaker potassium channel, and a large variety of other flexible linkers, as will be appreciated by those in the art. Glycine-serine polymers are preferred since oligopeptides comprising these amino acids are relatively unstructured, and therefore may be able to serve as a neutral tether between components. Secondly, serine is hydrophilic and therefore able to solubilize what could be a globular glycine chain. Third, similar chains have been shown to be effective in joining subunits of recombinant proteins such as single-chain antibodies.

[0066] “Single chain Fv” or “scFv” refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region (VL) and at least one antibody fragment comprising a heavy chain variable region (VH), wherein the VL and the VH are contiguously linked via a polypeptide linker, and capable of being expressed as a single chain polypeptide. Unless specified, as used herein, a scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL -linker- VH or may comprise VH-linker-VL.

[0067] “(scFv)2” or “tandem scFv” or “bis-scFv” fragments refers to a fusion protein comprising two light chain variable region (VL) and two heavy chain variable region (VH), wherein the two VL and the two VH regions are contiguously linked via polypeptide linkers, and capable of being expressed as a single chain polypeptide. The two VL and two VH regions fused by peptide linkers form a bivalent molecule VLA-linker-VHA-linker-VLB-linker-VHB to form two binding sites, capable of binding two different antigens or epitopes concurrently.

[0068] “Stapled single chain Fv”, “stapled scFv” or “spFv” refers to a scFv that comprises one or more disulfide bonds between the VH and the linker or the VL and the linker. Typically, the spFv may comprise one disulfide bond between the VH and the linker, and one disulfide bond between the VL and the linker, or two disulfide bonds between the VH and the linkerl and the VL and the linker. Typically, the spFv linker may contain one or two cysteine residues that are capable of forming a disulfide bond with the cysteine residue of the VH and / or the VH. Exemplary linker that contain two Cys residues are listed in Table 1.Table 1 : spFv linkers with two Cys residues

[0069] “Subject” includes any human or non-human animal. “Non-human animal” includes all vertebrates, e.g., mammals and non-mammals. The term “mammal” as used herein, encompasses any mammal. Examples of mammals include, but are not limited to, non-human primates, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc. The terms “subject” and “patient” can be used interchangeably herein. In some embodiments, the subject or patient is human.

[0070] “Treat,” “treating,” or “treatment” of a disease or disorder such as cancer refers to accomplishing one or more of the following: reducing the severity and / or duration of the disorder, delaying the progression of the disorder, slowing the progression of the disorder, inhibiting worsening of symptoms characteristic of the disorder being treated, limiting or preventing recurrence of the disorder in subjects that have previously had the disorder, or limiting or preventing recurrence of symptoms in subjects that were previously symptomatic for the disorder. As used herein, the terms “delaying the progression of’ or “slowing the progression of’ shall include (a) delaying or slowing the development of one or more symptoms or complications of the disease, condition or disorder; (b) delaying or slowing the development of one or more new / additional symptoms or complications of the disease, condition or disorder; and / or (c) delaying or slowing the progression of the disease, condition or disorder to a later stage or more serious form of said disease, condition or disorder.

[0071] Any reference in the description to a method of treatment equally refers to the compound, pharmaceutical composition, medicament, etc. for use in the method for treatment or for use in the manufacture of the medicament. For example, every embodiment or combination of embodiments, which has been described herein for methods for treating a disease, disorder or condition (comprising the administration of a compound, composition, medicament,combination, association etc ), is also applicable to the same compound, composition, medicament, combination, association, etc. for use in treating the same disease, disorder or condition as well as to the use of the same compound, composition, medicament, combination, association, etc. in the manufacture of a medicament for the treatment of the same disease, disorder or condition.

[0072] In an attempt to help the reader of the application, the description has been separated in various paragraphs or sections or is directed to various embodiments of the application. These separations should not be considered as disconnecting the substance of a paragraph or section or embodiments from the substance of another paragraph or section or embodiments. To the contrary, one skilled in the art will understand that the description has broad application and encompasses all the combinations of the various sections, paragraphs and sentences that can be contemplated. The discussion of any embodiment is meant only to be exemplary and is not intended to suggest that the scope of the disclosure, including the claims, is limited to these examples.

[0073] It is to be appreciated that certain features of the embodiment which are, for clarity, described herein in the context of separate embodiments may also be provided in combination in a single embodiment. That is, unless obviously incompatible or specifically excluded, each individual embodiment is deemed to be combinable with any other embodiment(s) and such a combination is considered to be another embodiment. Conversely, various features of the embodiments that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any sub-combination. Finally, although an embodiment may be described as part of a series of steps or part of a more general structure, each said step may also be considered an independent embodiment in itself, combinable with others.CD79BXCD20XCD3 ANTIBODIESAntigen-binding domains

[0074] Provided herein are trispecific antibodies that bind CD79b, CD20 and CD3.

[0075] Disclosed herein are trispecific antibodies or a fragment thereof comprising a binding domain that specifically binds to CD79b, a binding domain that specifically binds to CD20 and a binding domain that specifically binds to CD3.

[0076] In some embodiments, the disclosure provides a trispecific antibody or a fragment thereof, that specifically binds to CD79b, CD20 and CD3.

[0077] As used herein, “CD79bxCD20xCD3 antibody”, “CD79bxCD20xCD3 trispecific antibody”, or ‘trispecific antibody” are used interchangeably and refer to the trispecific antibody of the disclosure that specifically binds to CD79b, CD20 and CD3. Suitably, in the context of the present disclosure, the CD79bxCD20xCD3 tri specific antibody comprises a CD79b binding domain that specifically binds to CD79b. Suitably, in the context of the present disclosure the CD79bxCD20xCD3 trispecific antibody comprises a CD20 binding domain that specifically binds to CD20. Suitably, in the context of the present disclosure the CD79bxCD20xCD3 trispecific antibody comprises a CD3 binding domain that specifically binds to CD3. Suitably, in the context of the present disclosure the CD79bxCD20xCD3 trispecific antibody comprises a CD79b binding domain, a CD20 binding domain, and a CD3 binding domain that specifically binds to CD79b, CD20 and CD3, respectively. Suitably, in the context of the present disclosure, the CD79bxCD20xCD3 trispecific antibody disclosed herein comprises a first binding domain that specifically binds CD79b, a second binding domain that specifically binds CD20 and a third binding domain that specifically binds CD3. The domains specifically binding to CD79b are typically referred herein as VH1 / VL1 pairs. The domains specifically binding to CD20 are typically referred herein as VH2 / VL2 pairs. The domains specifically binding to CD3 are typically referred herein as VH3 / VL3 pairs.

[0078] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof, comprisesa) a first antigen-binding domain comprising a first heavy chain complementarity determining region (HCDR) 1, a first HCDR2, and a first HCDR3 of a first heavy chain variable region (VH1) of SEQ ID NO: 7 and a first light chain complementarity determining region (LCDR) 1, a first LCDR2, and a first LCDR3 of a first light chain variable region (VL1) having an amino acid sequence of SEQ ID NO: 8;b) a second antigen-binding domain comprising a second heavy chain complementarity determining region (HCDR) 1, a second HCDR2, and a second HCDR3 of a second heavy chain variable region (VH2) of SEQ ID NO: 15 and a second light chain complementarity determining region (LCDR) 1, a second LCDR2, and a second LCDR3 of a second light chain variable region (VL2) having an amino acid sequence of SEQ ID NO: 16;c) a third antigen-binding domain comprising a third heavy chain complementarity determining region (HCDR) 1, a third HCDR2, and a third HCDR3 of a third heavy chain variable region (VH3) of SEQ ID NO: 23 and a third light chain complementarity determining region (LCDR) 1, a third LCDR2, and a third LCDR3 of a third light chain variable region (VL3) having an amino acid sequence of SEQ ID NO: 24,wherein the first antigen-binding domain binds to an epitope on cluster of differentiation 79b protein (CD79b), the second antigen-binding domain binds to an epitope on cluster of differentiation 20 (CD20), and the third antigen-binding domain binds to an epitope on cluster of differentiation 3 (CD3).

[0079] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof, comprisesa) a first antigen-binding domain comprising a first heavy chain complementarity determining region (HCDR) 1, a first HCDR2, and a first HCDR3 of a first heavy chain variable region (VH1) having an amino acid sequence of SEQ ID NO: 7, and a first light chain complementarity determining region (LCDR) 1, a first LCDR2, and a first LCDR3 of a first light chain variable region (VL1) having an amino acid sequence of SEQ ID NO: 8;b) a second antigen-binding domain comprising a second heavy chain complementarity determining region (HCDR) 1, a second HCDR2, and a second HCDR3 of a second heavy chain variable region (VH2) having an amino acid sequence of SEQ ID NO: 15, and a second light chain complementarity determining region (LCDR) 1, a second LCDR2, and a second LCDR3 of a second light chain variable region (VL2) having an amino acid sequence of SEQ ID NO: 16;c) a third antigen-binding domain comprising a third heavy chain complementarity determining region (HCDR) 1, a third HCDR2, and a third HCDR3 of a third heavy chain variable region (VH3) having an amino acid sequence of SEQ ID NO: 23, and a third light chain complementarity determining region (LCDR) 1, a third LCDR2, and a third LCDR3 of a third light chain variable region (VL3) having an amino acid sequence of SEQ ID NO: 24,wherein the first antigen-binding domain binds to an epitope on cluster of differentiation 79b protein (CD79b), the second antigen-binding domain binds to an epitope on cluster of differentiation 20 (CD20), and the third antigen-binding domain binds to an epitope on cluster of differentiation 3 (CD3); and wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences are according to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the Contact numbering system or the IMGT numbering system or a combination thereof.

[0080] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof, comprises a first antigen-binding domain that binds to CD79b comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of:a) amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5 and 6, respectively, according to the AbM numbering system;b) amino acid sequences of SEQ ID NOs: 28, 29, 3, 4, 5, and 6, respectively, according to the Kabat numbering system;c) amino acid sequences of SEQ ID NOs: 30, 31, 3, 4, 5, and 6, respectively, according to the Chothia numbering system;d) amino acid sequences of SEQ ID NOs: 32, 33, 34, 35, amino acid residues TLS and amino acid sequence of SEQ ID NO: 6 respectively, according to the IMGT numbering system; ore) amino acid sequences of SEQ ID NOs: 36, 37, 38, 39, 40 and 41, respectively, according to the Contact numbering system.

[0081] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof, comprises a VH1 comprising an amino acid sequence which is at least 90%, or at least 95%, or at least 98%, at least 99%, or 100% identical to SEQ ID NO: 7 andcomprises a VL1 comprising an amino acid sequence which is at least 90%, or at least 95%, or at least 98%, at least 99%, or 100% identical to SEQ ID NO: 8.

[0082] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a VH1 comprising an amino acid sequence of SEQ ID NO: 7 and comprises a VL1 comprising an amino acid sequence of SEQ ID NO: 8.

[0083] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a second antigen-binding domain that binds CD20 comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of:a) amino acid sequences of SEQ ID NOs: 9, 10, 11, 12, 13, and 14, respectively, according to the AbM numbering system;b) amino acid sequences of SEQ ID NOs: 42, 43, 11, 12, 13 and 14, respectively, according to the Kabat numbering system;c) amino acid sequences of SEQ ID NOs: 44, 45, 11, 12, 13, and 14, respectively, according to the Chothia numbering system;d) amino acid sequences of SEQ ID NOs: 46, 47, 48, 49, amino acids DAS and SEQ ID NO: 14, according to the IMGT numbering system; ore) amino acid sequences of SEQ ID NO: 50, 51, 52, 53, 54 and 55, respectively, according to the Contact numbering system.

[0084] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a VH2 comprising an amino acid sequence which is at least 90%, or at least 95%, or at least 98%, at least 99%, or 100% identical to SEQ ID NO: 15 and a VL2 comprising an amino acid sequence which is at least 90%, or at least 95%, or at least 98%, at least 99%, or 100% identical to SEQ ID NO: 16.

[0085] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a VH2 comprising an amino acid sequence of SEQ ID NO: 15 and comprises a VL2 comprising an amino acid sequence of SEQ ID NO: 16.

[0086] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a third antigen-binding domain that binds CD3 comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of:a) amino acid sequences of SEQ ID NOs: 17, 18, 19, 20, 21, and 22, respectively, according to the AbM numbering system;b) amino acid sequences of SEQ ID NOs: 56, 57, 19, 20, 21 and 22, respectively, according to the Kabat numbering system;c) amino acid sequences of SEQ ID NOs: 58, 59, 19, 20, 21 and 22, respectively, according to the Chothia numbering system;d) amino acid sequences of SEQ ID NOs: 60, 61, 62, 63, amino acids YAS and SEQ ID NO:22, respectively, according to the IMGT numbering system; ore) amino acid sequences of SEQ ID NO: 64, 65, 66, 67, 68 and 69, respectively, according to the Contact numbering system.

[0087] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a VH3 comprising an amino acid sequence which is at least 90%, or at least 95%, or at least 98%, at least 99%, or 100% identical to SEQ ID NO: 23 and comprises a VL3 comprising an amino acid sequence which is at least 90%, or at least 95%, or at least 98%, at least 99%, or 100% identical to SEQ ID NO: 24.

[0088] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment comprises a VH3 comprising an amino acid sequence of SEQ ID NO: 23 and comprises a VL3 comprising an amino acid sequence of SEQ ID NO: 24.

[0089] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises:a) a first antigen-binding domain that binds to CD79b comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 having an amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5 and 6, respectively;b) a second antigen-binding domain that binds to CD20 comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 having an amino acid sequences of SEQ ID NOs: 9, 10, 11, 12, 13 and 14, respectively; andc) a third antigen-binding domain that binds CD3 comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 having an amino acid sequences of SEQ ID NOs: 17, 18, 19, 20, 21, and 22, respectively;wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are according to the AbM delineation.

[0090] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprisesa) a first antigen-binding domain that binds CD79b and comprises the VH1 having an amino acid sequence of SEQ ID NO: 7 and the VL1 having an amino acid sequence of SEQ ID NO: 8;b) a second antigen-binding domain that binds CD20 and comprises the VH2 having an amino acid sequence of SEQ ID NO: 15 and the VL2 having an amino acid sequence of SEQ ID NO: 16; andc) a third antigen -binding domain that binds CD3 and comprises the VH3 having an amino acid sequence of SEQ ID NO: 23 and the VL3 having an amino acid sequence of SEQ ID NO: 24.Format of the CD79bxCD20xCD3 trispecific antibody construct

[0091] The trispecific antibodies of the present disclosure include, but are not limited to, full length antibodies, antigen binding fragments, chimeric, human and humanized antibodies.

[0092] In some embodiments, the trispecific antibodies of the present disclosure comprises antibody fragments.

[0093] In some embodiments, the trispecific antibody of the disclosure or the fragment thereof may comprise a diabody, a Fab, Fab’, a F(ab’)2, a variable fragment (Fv), a single chain variable fragment (scFv), a (ScFv)2, a Fd fragment, a single chain disulfide bond stabilized fragments, stapled scFv fragment (spFv), a disulfide stabilized Fv fragment (dsFv), or a disulfide stabilized diabody (ds diabody).

[0094] In some embodiments, the trispecific antibody or the fragment thereof comprises a first binding domain that binds to CD79b, a second binding domain that binds to CD20 and a third binding domain that binds to CD3, wherein the first binding domain, the second binding domain and the third binding domain may comprise a diabody, a Fab, Fab’, a F(ab’)2, a variable fragment (Fv), a single chain variable fragment (scFv), a (ScFv)2, a Fd fragment, asingle chain disulfide bond stabilized fragments, stapled scFv fragment (spFv), a disulfide stabilized Fv fragment (dsFv), or a disulfide stabilized diabody (ds diabody).

[0095] In some embodiments, the antibody fragments of the CD79bXCD20xCD3 trispecific antibody of the disclosure (such as VH and VL) may be linked together via a synthetic linker to form various types of single antibody designs where the VH / VL domains may pair intramolecularly, or intermolecularly in those cases when the VH and VL domains are expressed by separate single chains, to form a monovalent antigen binding domain, such as single chain Fv (scFv) or diabody.

[0096] Any of the VH and the VL domains identified herein that bind CD79b, CD20 or CD3 may be engineered into scFv format in either VH-linker-VL or VL -linker- VH orientation. Any of the VH and the VL domains identified herein may also be used to generate sc(Fv)2 structures, such as VH-linker-VL-linker-VL-linker-VH, VH-linker-VL-linker- VH-linker-VL, VH-linker- VH-linker-VL -linker- VL, VL-linker- VH-linker- VH-linker-VL, VL-linker- VH-linker-VL-linker-VH or VL-linker-VL-linker- VH-linker- VH.

[0097] Any of the VH and the VL domains identified herein (e.g. those that bind CD79b, those that bind CD20, or those that bind CD3) may be engineered into scFv format in either VH-linker-VL or VL-linker-VH orientation. In some embodiments, the scFv format is, from the N- to C-terminus, in the VH-linker-VL orientation. In other embodiments, the scFv format is, from the N- to C-terminus, in the VL-linker-VH orientation.

[0098] Any of the VH and the VL domains identified herein can also be used to generate sc(Fv)2 structures. In some embodiments, the sc(Fv)2 structure is VH-linker- VL-linker-VL-linker- VH. In some embodiments, the sc(Fv)2 structure is VH-linker-VL-linker- VH-linker-VL. In some embodiments, the sc(Fv)2 structure is VH-linker- VH-linker- VL-linker-VL. In some embodiments, the sc(Fv)2 structure is VL-linker- VH-linker- VH-linker-VL. In some embodiments, the sc(Fv)2 structure is VL-linker- VH-linker- VL-linker-VH.

[0099] In some embodiments, the scFv may comprise a linker peptide, comprising about two to about eight glycine or other amino acid residues, which connects the variable region of the heavy chain and the variable region of the light chain. In some embodiments, the linker is a peptide linker. In some embodiments, the liker comprises a naturally occurring amino acid. Exemplary amino acids that can be included into the linker are Gly, Ser Pro, Thr, Glu, Lys, Arg,He, Leu, His and Phe. In some embodiments, the linker has a length that is adequate to link the VH and the VL in such a way that they form the correct conformation relative to one another so that they retain the desired activity, such as binding to the target (e.g., CD79b, CD20 or CD3).

[0100] In some embodiments, the antibody fragments of the CD79bXCD20xCD3 trispecific antibody of the disclosure (such as VH and VL) that are linked together via a synthetic linker to form a scFv may be stabilized by the introduction of two disulfide bonds between the linker and the variable domains of the scFv to form a stabilized scFv molecule (herein referred to as spFv (or stapled scFv). The generation of stabilized scFv molecules (herein referred to as spFv (or stapled scFv) is described in W02021 / 030657 filed August 14, 2020, which is incorporated herein by reference in its entirety.

[0101] In some embodiments, the spFv may comprise one disulfide bond between the VH and the spFv linker (herein referred to as linkerl), and one disulfide bond between the VL and linkerl, or two disulfide bonds between the VH and linkerl and the VL and linkerl.

[0102] In some embodiments, the spFv linker (linkerl) contains one or two cysteine residues that are capable of forming a disulfide bond with the cysteine residue of the VH and / or the VH. Exemplary linkers that contain two Cys residues are listed in Table 1.

[0103] In some embodiments, the antibody fragments of the CD79bXCD20xCD3 trispecific antibody of the disclosure (such as VH and VL) is a spFv comprising a VH comprising a Cys at H105; a VL comprising a Cys at L42; and a linkerl comprising an amino acid sequence of SEQ ID NOs: 72, 79, 80, 81 or 82; wherein the spFv is in the VL-Linkerl-VH orientation.

[0104] Any of the VH and the VL domains identified herein that bind CD79b, CD20 or CD3 may be engineered into spFv format in either VH-linkerl-VL or VL-linkerl-VH orientation.

[0105] The binding domains of the CD79bxCD20xC20 trispecific antibodies in the present disclosure may be independently of each other selected from a Fab fragment, an Fv fragment, a dsFv fragment and a single-chain Fv fragment (scFv) or a disulfide stabilized single-chain antibody molecule (spFv or stapled scFv). In particular embodiments, the binding domains of the trispecific antibodies in the present disclosure are independently of each otherselected from a Fab fragment or a disulfide stabilized single-chain antibody molecule (spFv or stapled scFv).[001061 In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a first binding domain that binds to CD79b, wherein the first binding domain is or comprises a diabody, a Fab, Fab’, a F(ab’)2, a variable fragment (Fv), a single chain variable fragment (scFv), a (ScFv)2, a Fd fragment, a single chain disulfide bond stabilized fragments, stapled scFv fragment (spFv), a disulfide stabilized Fv fragment (dsFv), or a disulfide stabilized diabody (ds diabody), preferably a Fab.

[0107] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a first binding domain that binds to CD79b, wherein the first binding domain is or comprises a Fab.

[0108] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a first binding domain that binds CD79b, wherein the first binding domain is or comprises a Fab comprising the VH1 and VL1 and having amino acid sequences of SEQ ID NOs: 7 and 8, respectively.

[0109] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a second binding domain that binds to CD20, wherein the second antigen-binding domain that binds to CD20 is or comprises a diabody, a Fab, Fab’, a F(ab’)2, a variable fragment (Fv), a single chain variable fragment (scFv), a (ScFv)2, a Fd fragment, a single chain disulfide bond stabilized fragments, stapled scFv fragment (spFv), a disulfide stabilized Fv fragment (dsFv), or a disulfide stabilized diabody (ds diabody), optionally a spFv.

[0110] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a second binding domain that binds to CD20, wherein the second antigen-binding domain is or comprises a spFv comprising the VH2 and VL2 and having amino acid sequences of SEQ ID NOs: 15 and 16, respectively.

[0111] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a second binding domain that binds to CD20, wherein the second binding domain is or comprises a spFv in an “VH2-linkerl-VL2” orientation or in a “VL2-linkerl-VH2” orientation.

[0112] The linkerl may comprises, consists of and / or consists essentially of the amino acid sequence of SEQ ID NO: 72 (GGGSGGSGGCPPCGGSGG), the amino acid sequence of SEQ ID NO: 79 (GGGSGGCPPCGGGSGG), the amino acid sequence of SEQ ID NO: 80 (GGSGGSGGCPPCGSGG), the amino acid sequence of SEQ ID NO: 81 (GGGSGGSGGCPPCGSGG), or the amino acid sequence of SEQ ID NO: 82 (GGGSGGGSGCPPCGGGG).

[0113] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a second binding domain that binds to CD20, wherein the second binding domain is or comprises a spFv in a “VL2-linkerl-VH2” orientation, and wherein the linkerl is selected from the group consisting of SEQ ID NOs: 72, 79, 80, 81 and 82.

[0114] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a second binding domain that binds to CD20, wherein the second binding domain is or comprises a spFv in a “VL2-linkerl-VH2” orientation, and wherein the linkerl has an amino acid sequence of SEQ ID NO: 72 (GGGSGGSGGCPPCGGSGG).

[0115] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a second binding domain that binds to CD20, wherein the second binding domain is or comprises a spFv in a “VL2-linkerl-VH2” orientation, and wherein the linkerl has an amino acid sequence of SEQ ID NO: 79 (GGGSGGCPPCGGGSGG).

[0116] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a second binding domain that binds to CD20, wherein the second binding domain is or comprises a spFv in a “VL2-linkerl-VH2” orientation, and wherein the linkerl has an amino acid sequence of SEQ ID NO: 80 (GGSGGSGGCPPCGSGG).

[0117] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a second binding domain that binds to CD20, wherein the second binding domain is or comprises a spFv in a “VL2-linkerl-VH2” orientation, and wherein the linkerl has an amino acid sequence of SEQ ID NO: 81 (GGGSGGSGGCPPCGSGG).

[0118] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a second binding domain that binds to CD20, wherein the secondbinding domain is or comprises a spFv in a “VL2-linkerl-VH2” orientation, and wherein the linkerl has an amino acid sequence of SEQ ID NO: 82 (GGGSGGGSGCPPCGGGG).

[0119] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a second binding domain that binds to CD20, wherein the second binding domain is or comprises a spFv which is at least 90%, or at least 95%, or at least 98%, at least 99%, or 100% identical to SEQ ID NO: 70.

[0120] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a second binding domain that binds to CD20, wherein the second binding domain is or comprises a spFv comprising the amino acid sequence of SEQ ID NO: 70.

[0121] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a third binding domain that binds to CD3, wherein the third antigenbinding domain that binds CD3 is or comprises a diabody, a Fab, Fab’, a F(ab’)2, a variable fragment (Fv), a single chain variable fragment (scFv), a (ScFv)2, a Fd fragment, a single chain disulfide bond stabilized fragments, stapled scFv fragment (spFv), a disulfide stabilized Fv fragment (dsFv), or a disulfide stabilized diabody (ds diabody), optionally a spFv.

[0122] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a third binding domain that binds to CD3, wherein the third antigenbinding domain is or comprises a spFv comprising the VH3 and VL3 and having amino acid sequences of SEQ ID NOs: 23 and 24, respectively.

[0123] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a third binding domain that binds to CD3, wherein the third binding domain is or comprises a spFv in an “VH3-linkerl-VL3” orientation or in a “VL3-linkerl-VH3” orientation.

[0124] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a third binding domain that binds to CD3, wherein the third binding domain is or comprises a spFv in a “VL3-linkerl-VH3” orientation, and wherein the linkerl is selected from the group consisting of SEQ ID NOs: 72, 79, 80, 81 and 82.

[0125] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a third binding domain that binds to CD3, wherein the third bindingdomain is or comprises a spFv in a “VL3-linkerl-VH3” orientation, and wherein the linkerl has an amino acid sequence of SEQ ID NO: 72 (GGGSGGSGGCPPCGGSGG).

[0126] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a third binding domain that binds to CD3, wherein the third binding domain is or comprises a spFv in a “VL3-linkerl-VH3” orientation, and wherein the linkerl has an amino acid sequence of SEQ ID NO: 79 (GGGSGGCPPCGGGSGG).

[0127] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a third binding domain that binds to CD3, wherein the third binding domain is or comprises a spFv in a “VL3-linkerl-VH3” orientation, and wherein the linkerl has an amino acid sequence of SEQ ID NO: 80 (GGSGGSGGCPPCGSGG).

[0128] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a third binding domain that binds to CD3, wherein the third binding domain is or comprises a spFv in a “VL3-linkerl-VH3” orientation, and wherein the linkerl has an amino acid sequence of SEQ ID NO: 81 (GGGSGGSGGCPPCGSGG).

[0129] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a third binding domain that binds to CD3, wherein the third binding domain is or comprises a spFv in a “VL3-linkerl-VH3” orientation, and wherein the linkerl has an amino acid sequence of SEQ ID NO: 82 (GGGSGGGSGCPPCGGGG).

[0130] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a third binding domain that binds to CD3, wherein the third binding domain is or comprises a spFv which is at least 90%, or at least 95%, or at least 98%, at least 99%, or 100% identical to SEQ ID NO: 71.

[0131] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a third binding domain that binds CD3, wherein the third binding domain is or comprises a spFv comprising the amino acid sequence of SEQ ID NO: 71.

[0132] In some embodiments, the CD79bxCD20xCD3 tri specific antibody of the disclosure or a fragment thereof comprises first binding domain that binds CD79b, a second binding domain that binds CD20 and a third binding domain that binds CD3, whereina) a first binding domain that binds CD79b is a Fab comprising the VH1 and VL1 having the amino acid sequences of SEQ ID NO: 7 and 8, respectively;b) the second binding domain that binds CD20 is a spFv comprising a VL2-linkerl- VH2, wherein VH2 and VL2 comprise the amino acid sequences of SEQ ID NO: 15 and 16, respectively and wherein the linker 1 comprises the amino acid sequence of SEQ ID NO: 72; and / orc) the third binding domain that binds CD3 is a spFv comprising a VL3 -linker 1-VH3, wherein VH3 and VL3 comprise the amino acid sequences of SEQ ID NO: 23 and 24, respectively and wherein the linkerl comprises the amino acid sequence of SEQ ID NO: 72.

[0133] In some embodiments, the CD79bxCD20xCD3 trispecific antibody of the disclosure or a fragment thereof comprises first binding domain that binds CD79b, a second binding domain that binds CD20 and a third binding domain that binds CD3, whereina) a first binding domain that binds CD79b is or comprises a Fab comprising the VH1 and VL1 having the amino acid sequences of SEQ ID NO: 7 and 8, respectively; b) the second binding domain that binds CD20 is a spFv having the amino acid sequence of SEQ ID NO: 70; andc) the third binding domain that binds CD3 is a spFv having the amino acid sequence of SEQ ID NO: 71.Antigen binding domains may be operably linked

[0134] The antigen binding domains or portions of the antigen binding domains of the CD79bxCD20xCD3 trispecific antibody may be operably linked or fused together.

[0135] In some embodiments, at least one portion of the binding domain that binds CD79b and at least one portion of the binding domain that binds CD20 are fused together (or operably linked), or at least one portion of the binding domain that binds CD3 and at least one portion of the binding domain that binds CD20 are fused together (or operably linked), or at least one portion of the binding domain that binds CD79b and at least one portion of the binding domain that binds CD3 are fused together (or operably linked).

[0136] In a particular embodiment, the trispecific antibody comprises at least one portion of the binding domain that binds CD79b fused to the N-terminus of at least one portion of the binding domain that binds CD20. In another embodiment, the trispecific antibody comprises at least one portion of the binding domain that binds CD20 fused to the N-terminus of at least one portion of the binding domain that binds CD79b.

[0137] In another embodiment, the trispecific antibody comprises at least one portion of the binding domain that binds CD3 fused to the N-terminus of at least one portion of the binding domain that binds CD20. In another embodiment, the trispecific antibody comprises at least one portion of the binding domain that binds CD20 fused to the N-terminus of at least one portion of the binding domain that binds CD3.

[0138] In another embodiment, the trispecific antibody comprises at least one portion of the binding domain that binds CD79b fused to the N-terminus of at least one portion of the binding domain that binds CD3. In another embodiment, the trispecific antibody comprises at least one portion of the binding domain that binds CD3 fused to the N-terminus of at least one portion of the binding domain that binds CD79b.

[0139] In some embodiments, the trispecific antibody comprises a CD20 antigenbinding domain, wherein the CD20 antigen-binding domain is or comprises a spFv in the VL2-linkerl-VH2 orientation and wherein the C-terminus of the spFv is fused to the N-terminus of the Fab heavy chain (VH1) of the CD79b antigen-binding domain via a linker2.

[0140] In some embodiments, the linker2 comprises, consists of and / or consists essentially of the amino acid sequence of SEQ ID NO: 73 (GGGGSGGGGS).

[0141] In some embodiments, the trispecific antibody comprises a CD20 antigenbinding domain, wherein the CD20 antigen-binding domain is or comprises a spFv in the VL2-linkerl-VH2 orientation and wherein the C-terminus of the spFv is fused to the N-terminus of the Fab heavy chain (VH1) of the CD79b binding domain in a VL2-linkerl-VH2-linker2-VHl orientation, wherein the linkerl comprises the amino acid sequence of SEQ ID NO: 72 (GGGSGGSGGCPPCGGSGG) and the linker2 comprises the amino acid sequence of SEQ ID NO: 73 (GGGGSGGGGS).

[0142] In some embodiments, the trispecific antibody comprises a CD20 antigenbinding domain, wherein the CD20 antigen-binding domain is or comprises a spFv in the VL2-linkerl-VH2 orientation and wherein the C-terminus of the spFv is fused to the N-terminus of the Fab heavy chain (VH1) of the CD79b binding domain in a VL2-linkerl-VH2-linker2-VHl orientation, wherein the linkerl comprises the amino acid sequence of SEQ ID NO: 79 (GGGSGGCPPCGGGSGG) and the linker2 comprises the amino acid sequence of SEQ ID NO: 73 (GGGGSGGGGS).

[0143] In some embodiments, the trispecific antibody comprises a CD20 antigenbinding domain, wherein the CD20 antigen-binding domain is or comprises a spFv in the VL2-linkerl-VH2 orientation and wherein the C-terminus of the spFv is fused to the N-terminus of the Fab heavy chain (VH1) of the CD79b binding domain in a VL2-linkerl-VH2-linker2-VHl orientation, wherein the linkerl comprises the amino acid sequence of SEQ ID NO: 80 (GGSGGSGGCPPCGSGG) and the linker2 comprises the amino acid sequence of SEQ ID NO: 73 (GGGGSGGGGS)

[0144] In some embodiments, the trispecific antibody comprises a CD20 antigenbinding domain, wherein the CD20 antigen-binding domain is or comprises a spFv in the VL2-linkerl-VH2 orientation and wherein the C-terminus of the spFv is fused to the N-terminus of the Fab heavy chain (VH1) of the CD79b binding domain in a VL2-linkerl-VH2-linker2-VHl orientation, wherein the linkerl comprises the amino acid sequence of SEQ ID NO: 81 (GGGSGGSGGCPPCGSGG) and the linker2 comprises the amino acid sequence of SEQ ID NO: 73 (GGGGSGGGGS).

[0145] In some embodiments, the trispecific antibody comprises a CD20 antigenbinding domain, wherein the CD20 antigen-binding domain is or comprises a spFv in the VL2-linkerl-VH2 orientation and wherein the C-terminus of the spFv is fused to the N-terminus of the Fab heavy chain (VH1) of the CD79b binding domain in a VL2-linkerl-VH2-linker2-VHl orientation, wherein the linkerl comprises the amino acid sequence of SEQ ID NO: 82 (GGGSGGGSGCPPCGGGG) and the linker2 comprises the amino acid sequence of SEQ ID NO: 73 (GGGGSGGGGS).Fc and TgG constant region

[0146] In some embodiments, the CD79bxCD20xCD3 trispecific antibodies, or a fragment thereof further comprise a Fc region, an IgG constant region or the fragment of an IgG constant region.

[0147] The binding domains of the CD79bxCD20xCD3 trispecific antibody may be operably linked to a Fc region, an IgG constant region or the fragment of an IgG constant region through direct fusion or through fusion with a linker polypeptide.

[0148] In a particular embodiment, binding domains of the CD79bxCD20xCD3 trispecific antibody may be fused (or operably linked) to a Fc region, an IgG constant region or the fragment of an IgG constant region via a linker, e.g., a peptide linker.

[0149] In some embodiments, the trispecific antibody or a fragment thereof of the disclosure comprises a diabody, a Fab, Fab’, a F(ab’)2, a variable fragment (Fv), a single chain variable fragment (scFv), a (ScFv)2, a Fd fragment, a single chain disulfide bond stabilized fragments, stapled scFv fragment (spFv), a disulfide stabilized Fv fragment (dsFv), or a disulfide stabilized diabody (ds diabody), operably linked to a Fc region.

[0150] In some embodiments, the trispecific antibody or a fragment thereof of the disclosure comprises a diabody, a Fab, Fab’, a F(ab’)2, a variable fragment (Fv), a single chain variable fragment (scFv), a (ScFv)2, a Fd fragment, a single chain disulfide bond stabilized fragments, stapled scFv fragment (spFv), a disulfide stabilized Fv fragment (dsFv), or a disulfide stabilized diabody (ds diabody), operably linked an IgG constant region or the fragment of an IgG constant region.

[0151] In some embodiments, the trispecific antibody or a fragment thereof of the disclosure comprises an anti-CD79b Fab, an anti-CD20 spFv and an anti-CD3 spFv operably linked to an IgG constant region or the fragment of an IgG constant region,

[0152] In some embodiments, the CD79bxCD20xCD3 trispecific antibody comprises a CD20 binding domain and a CD79b binding domain in tandem and operably linked to an IgG constant region or the fragment of an IgG constant region.

[0153] In some embodiments, the CD79bxCD20xCD3 trispecific antibody comprises a CD20 binding domain and a CD79b binding domain in tandem and fused (or operably linked)to an IgG constant region or the fragment of an IgG constant region, wherein the IgG constant region or the fragment of an IgG constant region is an IgGl, an IgG2, an IgG3, or an IgG4 isotype.

[0154] In some embodiments, the CD79bxCD20xCD3 trispecific antibody comprises a CD20 binding domain and a CD79b binding domain in tandem and fused (or operably linked) to an IgG constant region or the fragment of an IgG constant region, wherein the fusion comprises direct fusion or fusion through a linker polypeptide.

[0155] In some embodiments, the CD79bxCD20xCD3 trispecific antibody comprises a CD20 binding domain and a CD79b binding domain in tandem and fused (or operably linked) to the CHI, CH2 and CH3 of a human IgGl constant region as follows: VL2-linkerl-VH2-Iinker2-VH1-Iinker3-CH1-CH2-CH3, wherein the linkerl has an amino acid sequence of SEQ ID NO: 72 (GGGSGGSGGCPPCGGSGG), linker2 has an amino acid sequence of SEQ ID NO: 73 (GGGGSGGGGS) and linker3 has an amino acid sequence of SEQ ID NO: 75 (EPKSCDKTHTCPPCP)

[0156] In some embodiments, linker3 comprises, consists of and / or consists essentially of amino acid sequence of SEQ ID NO: 75 (EPKSCDKTHTCPPCP).

[0157] In some embodiments, the CD79bxCD20xCD3 trispecific antibody comprises a CD3 binding domain in the form of stapled scFv (VL3 -linker 1-VH3) fused (or operably linked) to an IgG constant region or a fragment of an IgG constant region.

[0158] In some embodiments, CD79bxCD20xCD3 trispecific antibody comprises a CD3 binding domain in the form of stapled scFv (VL3-linkerl-VH3) fused (or operably linked) to an IgG constant region or the fragment of an IgG constant region, wherein the IgG constant region or the fragment of an IgG constant region is an IgGl, an IgG2, an IgG3, or an IgG4 isotype.

[0159] In some embodiments, CD79bxCD20xCD3 trispecific antibody comprises a CD3 binding domain in the form of stapled scFv (VL3-linkerl-VH3) fused (or operably linked) to an IgG constant or the fragment of an IgG constant region, wherein the fusion comprises direct fusion or fusion through a linker polypeptide.

[0160] In some embodiments, CD79bxCD20xCD3 trispecific antibody comprises a CD3 binding domain in the form of stapled scFv (VL3-linkerl-VH3) fused (or operably linked)to an IgGl constant region or the fragment of an IgGl constant region, with a linker polypeptide (linker4) of SEQ ID NO: 74 (EPKSSDKTHTCPPCP).

[0161] In some embodiments, linker4 comprises, consists of and / or consists essentially of amino acid sequence of SEQ ID NO: 74 (EPKSSDKTHTCPPCP).

[0162] In some embodiments, CD79bxCD20xCD3 trispecific antibody comprises a CD3 binding domain as VL3 -linker! -VH3 fused (or operably linked) to the CH2 and CH3 of a human IgGl constant region as follows: VL3-linkerl-VH3-linker4-CH2-CH3, wherein linkerl has an amino acid sequence of SEQ ID NO: 72 (GGGSGGSGGCPPCGGSGG), and linker4 has an amino acid sequence of SEQ ID NO: 74 (EPKSSDKTHTCPPCP)

[0163] In some embodiments, CD79bxCD20xCD3 trispecific antibody comprises a CD3 binding domain as VL3-linkerl-VH3 fused (or operably linked) to the CH2 and CH3 of a human IgGl constant region as follows: VL3-linkerl-VH3-linker4-CH2-CH3, wherein linkerl has an amino acid sequence of SEQ ID NO: 79 (GGGSGGCPPCGGGSGG), and linker4 has an amino acid sequence of SEQ ID NO: 74 (EPKSSDKTHTCPPCP).

[0164] In some embodiments, CD79bxCD20xCD3 trispecific antibody comprises a CD3 binding domain as VL3-linkerl-VH3 fused (or operably linked) to the CH2 and CH3 of a human IgGl constant region as follows: VL3-linkerl-VH3-linker4-CH2-CH3, wherein linkerl has an amino acid sequence of SEQ ID NO: 80 (GGSGGSGGCPPCGSGG), and linker4 has an amino acid sequence of SEQ ID NO: 74 (EPKSSDKTHTCPPCP).

[0165] In some embodiments, CD79bxCD20xCD3 trispecific antibody comprises a CD3 binding domain as VL3-linkerl-VH3 fused (or operably linked) to the CH2 and CH3 of a human IgGl constant region as follows: VL3-linkerl-VH3-linker4-CH2-CH3, wherein linkerl has an amino acid sequence of SEQ ID NO: 81 (GGGSGGSGGCPPCGSGG), and linker4 has an amino acid sequence of SEQ ID NO: 74 (EPKSSDKTHTCPPCP).

[0166] In some embodiments, CD79bxCD20xCD3 trispecific antibody comprises a CD3 binding domain as VL3-linkerl-VH3 fused (or operably linked) to the CH2 and CH3 of a human IgGl constant region as follows: VL3-linkerl-VH3-linker4-CH2-CH3, wherein linkerl has an amino acid sequence of SEQ ID NO: 82 (GGGSGGGSGCPPCGGGG), and linker4 has an amino acid sequence of SEQ ID NO: 74 (EPKSSDKTHTCPPCP).

[0167] In some embodiments, CD79bxCD20xCD3 trispecific antibody comprises a VL1 fused (or operably linked) to the CL of a human IgGl constant region.Fc mutations

[0168] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises an IgG constant region or to the fragment of an IgG constant region that modulates the antibody effector function and / or promotes antibody dimerization.

[0169] For example, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises an IgG constant region or a fragment of an IgG constant region that modulates the antibody effector functions such as ADCC, ADCP and / or ADCP and / or pharmacokinetic properties. This may be achieved by introducing mutation(s) into the Fc that modulate binding of the mutated Fc to activating FcyRs (FcyRI, FcyRIIa, FcyRIII), and / or to inhibitory FcyRIIb.

[0170] In some embodiments, the trispecific antibody or a fragment thereof comprises an IgG constant region or a fragment of the IgG constant region comprising at least one mutation that reduces binding of the protein to an activating Fey receptor (FcyR) and / or reduces Fc effector functions such as Clq binding, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) or phagocytosis (ADCP).

[0171] In some embodiments, the trispecific antibody or a fragment thereof comprises an IgG constant region, or the fragment of an IgG constant region, comprising at least one mutation that results in reduced binding of the antibody or antigen binding fragment thereof to a Fey receptor (FcyR).

[0172] Fc positions that may be mutated to reduce binding to the activating FcyR and subsequently reduce effector function include positions 214, 233, 234, 235, 236, 237, 238, 265, 267, 268, 270, 295, 297, 309, 327, 328, 329, 330, 331 and 365. Exemplary mutations that may be made singularly or in combination are mutations K214T, E233P, L234V, L234A, deletion of G236, V234A, F234A, L235A, G237A, P238A, P238S, D265A, S267E, H268A, H268Q, Q268A, N297A, A327Q, P329A, D270A, Q295A, V309L, A327S, L328F, A33OS and P331S in IgGl, IgG2, IgG3 or IgG4. Exemplary combination mutations that result in proteins with reduced ADCC are mutations L234A / L235A on IgGl, L234A / L235A / D265S on IgGl, V234A / G237A / P238S / H268A / V309L / A330S / P331S on IgG2, F234A / L235A on IgG4,S228P / F234A / L235A on IgG4, N297A on all Ig isotypes, V234A / G237A on IgG2, K214T / E233P / L234V / L235A / G236-deleted / A327G / P331A / D365E / L358M on IgGl, H268Q / V309L / A330S / P331S on IgG2, S267E / L328F on IgGl, L234F / L235E / D265A on IgGl, L234A / L235A / G237A / P238S / H268A / A330S / P331S on IgGl, S228P / F234A / L235A / G237A / P238S on IgG4, and S228P / F234A / L235A / G236-deleted / G237A / P238S on IgG4. Hybrid IgG2 / 4 Fc domains may also be used, such as Fc with residues 117-260 from IgG2 and residues 261-447 from IgG4.

[0173] In some embodiments, the trispecific antibody or a fragment thereof comprises an IgG constant region or a fragment of an IgG constant region comprising the F234A / L235A, L234A / L235A, L234A / L235A / D265S, V234A / G237A / P238S / H268A / V309L / A330S / P331S, F234A / L235A, S228P / F234A / L235A, N297A, V234A / G237A, K214T / E233P / L234 V / L235 A / G236-deleted / A327G / P331 A / D365E / L358M, H268Q / V309L / A330S / P331 S, S267E / L328F, L234F / L235E / D265A, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238S, and S228P / F234A / L235A / G236-deleted / G237A / P238S mutations, wherein residue numbering is according to the EU index.

[0174] In some embodiments, the trispecific antibody or a fragment thereof comprises an IgG constant region or a fragment of the IgG constant region comprising the L234A / L235A / D265S.

[0175] In some embodiments, the trispecific antibody or a fragment comprises an IgG constant region or a fragment of the IgG constant region comprising at least one mutation that results in reduced binding of the antibody or antigen binding fragment thereof to the FcyR, wherein the mutation is the L234A / L235A / D265S, wherein residue numbering is according to the EU index.

[0176] In some embodiments, the trispecific antibody or a fragment comprises an IgG constant region or a fragment of the IgG constant region comprising at least one mutation that results in reduced binding of the antibody or antigen binding fragment thereof to the FcyR, wherein the FcyR is FcyRI, FcyRIIA, FcyRIIB, FcyRIII, or any combination thereof.

[0177] In addition, Fc mutation may include CH3 mutations that promote dimerization to generate the CD79bxCD20xCD3 trispecific antibody of the disclosure or their antigen binding fragments thereof. Co-expression of unique polypeptides to make a multispecificantibody, such as a bispecific (or trispecific) antibody may lead to multiple mis-paired species that are difficult to separate from a target multispecific antibody due to, for example, their similar biophysical properties. In addition, mis-paired polypeptides fail to secrete from the cell, resulting in unprecedented purity of secreted multispecific antibodies, such as the trispecific antibodies (bsAbs) of the present disclosure.

[0178] Substitutions that promote heterodimerization may be introduced within the CH3 domain of the IgG constant region to promote Fab arm exchange in vitro.

[0179] CH3 mutations that may be used include technologies such as Knob-in-Hole mutations (Genentech), electrostatically-matched mutations (Chugai, Amgen, NovoNordisk, Oncomed), the Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono), Duobody® mutations (Genmab), and other asymmetric mutations (e.g., Zymeworks).

[0180] Knob-in-hole mutations are disclosed for example in WO 1996 / 027011 and include mutations on the interface of CH3 region in which an amino acid with a small side chain (hole) is introduced into the first CH3 region and an amino acid with a large side chain (knob) is introduced into the second CH3 region, resulting in preferential interaction between the first CH3 region and the second CH3 region. Exemplary CH3 region mutations forming a knob and a hole are T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S and T366W / T366S_L368A_Y407V.

[0181] Heavy chain heterodimer formation may be promoted by using electrostatic interactions by substituting positively charged residues on the first CH3 region and negatively charged residues on the second CH3 region as described in US2010 / 0015133,US2009 / 0182127, US2010 / 028637 or US2011 / 0123532.

[0182] Other asymmetric mutations that can be used to promote heavy chain heterodimerization are L351Y_F405A_Y407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F, or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W as described in US2012 / 0149876 or US2013 / 0195849 (Zymeworks).

[0183] SEEDbody mutations involve substituting select IgG residues with IgA residues to promote heavy chain heterodimerization as described in US20070287170.

[0184] Other exemplary mutations that may be used are R409D_K370E / D399K_E357K, S354C_T366W / Y349C_ T366S_L368A_Y407V, Y349C T366W / S354C T366S L368A Y407V, T366K / L351D, L351K / Y349E, L351K / Y349D, L351K / L368E, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V_K409F, K392D / D399K, K392D / E356K, K253E_D282K_K322D / D239K_E240K_K292D, K392D_K409D / D356K_D399K as described in W02007 / 147901, WO 2011 / 143545, WO2013157954, WO2013096291 andUS2018 / 0118849.

[0185] Duobody® mutations (Genmab) are disclosed for example in U. S. Pat. No. 9,150,663 and US2014 / 0303356 and include mutations F405L / K409R, wild-type / F405L_R409K, T350I_K370T_F405L / K409R, K370W / K409R, D399AFGHILMNRSTVWY / K409R, T366ADEFGHILMQVY / K409R, L368ADEGHNRSTVQ / K409AGRH, D399FHKRQ / K409AGRH, F405IKLSTVW / K409AGRH and Y407LWQ / K409AGRH.

[0186] Additional bispecific or multispecific structures into which the antigen binding regions of the CD79bxCD20xCD3 antibody can be incorporated include Dual Variable Domain Immunoglobulins (DVD) (Int. Pat. Publ. No. WO2009 / 134776), structures that include various dimerization domains to connect the two antibody arms with different specificity, such as leucine zipper or collagen dimerization domains (Int. Pat. Publ. No. W02012 / 022811; U.S. Pat. No. 5,932,448; U.S. Pat. No. 6,833,441), two or more domain antibodies (dAbs) conjugated together, diabodies, heavy chain only antibodies such as camelid antibodies and engineered camelid antibodies, Dual Targeting (DT)-Ig (GSK / Dom antis), Two-in-one Antibody (Genentech), Cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star) and CovX-body (CovX / Pfizer), IgG-like Bispecific (InnClone / Eli Lilly), Ts2Ab (Medlmmune / AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idee) and TvAb (Roche), ScFv / Fc Fusions (Academic Institution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS), Dual Affinity Retargeting Technology (Fc-DART) (MacroGenics) and Dual(ScFv)2-Fab (National Research Center for Antibody Medicine— China), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol) and Fab-Fv (UCB-Celltech). ScFv-, diabody -based, and domain antibodies, include but are not limited to, Bispecific T Cell Engager(BiTE) (Micromet), Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART) (MacroGenics), Single-chain Diabody (Academic), TCR-like Antibodies (AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack) and COMBODY (Epigen Biotech), dual targeting nanobodies (Ablynx), dual targeting heavy chain only domain antibodies.

[0187] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises an IgG constant region or a fragment of an IgG constant region comprising a mutation that promotes dimerization.

[0188] In some embodiments, the IgG constant region or the fragment of the IgG constant region that is fused to the arm comprising CD20 and CD79b binding domains (VL2-Iinkerl-VH2-Linker2-VH1) comprises the T366S, L368A, and Y407V mutations. In some embodiments, the IgG constant region or the fragment of the IgG constant region that is fused to the arm comprising the CD3 binding domain (VL3 -linker 1-VH3) comprises the T366W mutation (numbering is according to EU index). In some embodiments, the IgG constant region or the fragment of the IgG constant region that is fused to the to the arm comprising CD20 and CD79b binding domains (VL2-linkerl-VH2-Linker2-VHl) comprises the T366W mutation. In some embodiments, the IgG constant region or the fragment of the IgG constant region fused to the arm comprising the CD3 binding domain (VL3-linkerl-VH3) comprises the T366S, L368A, and Y407V mutations (numbering is according to EU index).

[0189] In some embodiments, the IgG constant region or the fragment of the IgG constant region fused to the CD20 and CD79b binding domains (VL2-linkerl-VH2-linker2-VH1) comprises the T366S, L368A, and Y407V mutations, and the IgG constant region or the fragment of the IgG constant region fused to the CD3 binding domain (VL3-linkerl-VH3) comprises the T366W mutation (numbering is according to EU index).

[0190] In some embodiments, the Fc domain of CD79bxCD20xCD3 trispecific antibody may comprise proline to alanine (ProAla) mutations. ProAla IgG designs address the risk of generating mis-paired species during co-expression of unique polypeptides to generate a trispecific antibody such as the CD79bxCD20xCD3 trispecific antibody of the disclosure, and comprise a set of mutations which exploits a unique mechanism based on natural antibody folding. Included are ProAla heavy chains engineered to increase their folding energy barrierssuch that only the cognate light and heavy chains can for example induce folding, chaperone release, and secretion. In some embodiments, CD79bxCD20xCD3 trispecific antibody has an enhanced pairing between a first heavy chain and a second heavy chain. In some embodiments, the Fc domain of CD79bxCD20xCD3 trispecific antibody comprises a mutation at P374 (Eu numbering) in the CH3 domain of the first heavy chain and the second heavy chain. In some embodiments, the P374 mutation is an amino acid substitution P374X, wherein X comprises any amino acid. In some embodiments, the P374 mutation is an amino acid substitution P374X, wherein X comprises A, G or Q. Further descriptions and teachings of ProAla mutations are disclosed in WO / 2025 / 158376 which is incorporated herein in its entirety.

[0191] In some embodiments, the Fc domain of one of the heavy chain portions of the CD79bxCD20xCD3 antibody described herein further comprises one or more mutations which reduce Fc binding to protein A. In some embodiments, the IgG constant region or the fragment of the IgG constant region fused to the to the arm comprising CD20 and CD79b binding domains (VL2-linkerl-VH2-linker2-VHl) comprises the H435R and / or Y436F. In some embodiments, the IgG constant region or the fragment of the IgG constant region fused to the arm comprising CD3 binding domain (VL3 -linker 1-VH3) comprises the H435R and / or Y436F (numbering is according to EU index).

[0192] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises three polypeptide chains.

[0193] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises the Fab that specifically binds CD79b, a first spFv that specifically binds CD20 and a second spFv that specifically binds CD3.

[0194] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises:a) a first heavy chain (HC1) comprising from N-terminus to C-terminus a VL3-linkerl-VH3-linker4-Fc, wherein the Fc comprises the CH2 and CH3 domain of a human IgGl constant region;b) a second heavy chain (HC2) comprising from N-terminus to C-terminus a VL2-linkerl-VH2-linker2-VHl-linker3-Fc, wherein the Fc comprises the CHI, CH2 and CH3 domain of a human IgGl constant region; andc) a light chain (LC) comprising from N-terminus to C-terminus a VL1-CL, wherein the CL is the CL of a human kappa light chain.[001951 In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprisesa) a first heavy chain (HC1) comprising from the N-terminus to the C-terminus a second spFv-linker4-Fc, wherein the Fc comprises CH2 and CH3 of a human IgGl constant region; b) a second heavy chain (HC2) comprising from the N-terminus to the C-terminus a first spFv-linker2-VHl-linker3-Fc, wherein the Fc comprises the CHI, CH2 and CH3 of a human IgGl constant region; andc) a light chain (LC) comprising from the N-terminus to the C -terminus a VLl-linker4- CL, wherein the CL is the CL of a human kappa light chain.

[0196] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a HC1 comprising an amino acid sequence at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 25.

[0197] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a HC1 comprising an amino acid sequence of SEQ ID NO: 25.

[0198] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a HC2 comprising an amino acid sequence at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 26.

[0199] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a HC2 comprising an amino acid sequence of SEQ ID NO: 26.

[0200] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a LC comprising an amino acid sequence at least 90%, at least 95%, at least 98%, at least 99% or 100 % identical to SEQ ID NO: 27.

[0201] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a LC comprising a LC comprising an amino acid of SEQ ID NO: 27.

[0202] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a HC1 comprising an amino acid sequence at least 90%, at least 95%, at least 98%, at least 99% or 100 % identical to SEQ ID NO: 25, a HC2 comprising anamino acid sequence at least 90%, at least 95%, at least 98%, at least 99% or 100 % identical to SEQ ID NO: 26, and a LC comprising an amino acid sequence at least 90%, at least 95%, at least 98%, at least 99% or 100 % identical to SEQ ID NO: 27.

[0203] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a HC1 having an amino acid sequence of SEQ ID NO: 25, a HC2 having an amino acid sequence of SEQ ID NO: 26, and a LC having an amino acid sequence of SEQ ID NO: 27.

[0204] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a HC1 comprising an amino acid sequence at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 94.

[0205] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a HC1 comprising an amino acid sequence of SEQ ID NO: 94.

[0206] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a HC2 comprising an amino acid sequence at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 95.

[0207] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a HC2 comprising an amino acid sequence of SEQ ID NO: 95.

[0208] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a HC1 comprising an amino acid sequence at least 90%, at least 95%, at least 98%, at least 99% or 100 % identical to SEQ ID NO: 94, a HC2 comprising an amino acid sequence at least 90%, at least 95%, at least 98%, at least 99% or 100 % identical to SEQ ID NO: 95, and a LC comprising an amino acid sequence at least 90%, at least 95%, at least 98%, at least 99% or 100 % identical to SEQ ID NO: 27.

[0209] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or a fragment thereof comprises a HC1 having an amino acid sequence of SEQ ID NO: 94, a HC2 having an amino acid sequence of SEQ ID NO: 95, and a LC having an amino acid sequence of SEQ ID NO: 27POLYNUCLEOTIDES ENCODING THE ANTIBODIES OF THE DISCLOSURE

[0210] Further disclosed herein are polynucleotides encoding the CD79bxCD20xCD3 trispecific antibody of the disclosure and fragments thereof.

[0211] In some embodiments, the disclosure provides polynucleotide sequences encoding polypeptide sequences of SEQ ID NOs: 7 and 8.

[0212] In some embodiments, the disclosure provides polynucleotide sequences encoding polypeptide sequences of SEQ ID NOs: 15 and 16.

[0213] In some embodiments, the disclosure provides polynucleotide sequences encoding polypeptide sequences of SEQ ID NOs: 23 and 24.

[0214] In some embodiments, the disclosure provides polynucleotide sequences encoding polypeptide sequences of SEQ ID NOs: 71.

[0215] In some embodiments, the disclosure provides polynucleotide sequences encoding polypeptide sequences of SEQ ID NOs: 70.

[0216] In some embodiments, the disclosure provides polynucleotide sequences encoding polypeptide sequences of SEQ ID NOs: 25, 26 and 27.

[0217] In some embodiments, the disclosure provides polynucleotide sequences encoding polypeptide sequences of SEQ ID NOs: 94, 95, and 27.

[0218] In some embodiments, the disclosure provides a polynucleotide having a nucleic acid sequence of SEQ ID NO: 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 and 93.

[0219] The polynucleotides encoding the CD79bxCD20xCD3 antibody and a fragment thereof, include polynucleotides with nucleic acid sequences that are substantially the same as the nucleic acid sequences of the polynucleotide of the disclosure. “Substantially the same” nucleic acid sequence is defined herein as a sequence with at least 80% identity to another nucleic acid sequence when the two sequences are aligned. Two nucleic acid sequences are substantially identical if the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0220] Modified nucleotides may be used to generate the polynucleotides of the disclosure. Exemplary modified nucleotides are 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil, carboxymethylaminomethyl -2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, N6-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2 -thiouracil, beta-D-mannosylqueosine, 5 "-methoxy carboxymethyluracil, 5-methoxyuracil, 2- methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queuosine, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3 -methylcytosine, 5-methylcytosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine.VECTORS COMPRISING THE POLYNUCLEOTIDES ENCODING THE CD79BXCD20XCD3 ANTIBODIES[00221J Vectors comprising DNA encoding the CD79bxCD20xCD3 antibodies or fragments thereof of the disclosure are also provided. The disclosed vectors can be used, for example, to generate any of the above disclosed CD79bxCD20xCD3 antibodies, or antigen binding fragments thereof. Polynucleotides encoding any of the CD79bxCD20xCD3 antibody or antigen binding fragment thereof of the disclosure may be incorporated into vectors using standard molecular biology methods.

[0222] In some embodiments, the disclosure provides an expression vector comprising the polynucleotide encoding the amino acid sequences of the CD79bxCD20xCD3 antibody. Such vectors may be plasmid vectors, viral vectors, vectors for baculovirus expression, transposon-based vectors or any other vector suitable for introduction of the synthetic polynucleotide into a given organism or genetic background by any means. The vector of the disclosure may be an expression vector for the efficient synthesis and expression of the CD79bxCD20xCD3 antibody polypeptide or CD79bxCD20xCD3 antibody polypeptide of the disclosure in prokaryotic and eukaryotic systems, including but not limited to yeast andmammalian cell culture. Also provided is a vector comprising, consisting of and / or consisting essentially of a nucleic acid encoding an CD79bxCD20xCD3 antibody or a fragment thereof.

[0223] Any vector known to those skilled in the art in view of the present disclosure can be used, such as a plasmid, a cosmid, a phage vector or a viral vector. In some embodiments, the vector is a recombinant expression vector such as a plasmid. The vector can include any element to establish a conventional function of an expression vector, for example, a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication. The promoter can be a constitutive, inducible or repressible promoter. A number of expression vectors capable of delivering nucleic acids to a cell are known in the art and can be used herein for production of an antibody or antigen-binding fragment thereof in the cell.Conventional cloning techniques or artificial gene synthesis can be used to generate a recombinant expression vector according to certain embodiments. Such techniques are well known to those skilled in the art in view of the present disclosure.

[0224] Recombinant expression vectors within the scope of the description include synthetic, genomic, or cDNA-derived nucleic acid fragments that encode at least one recombinant protein which can be operably linked to suitable regulatory elements. Such regulatory elements can include a transcriptional promoter, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation. Expression vectors, especially mammalian expression vectors, can also include one or more non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, other 5’ or 3’ flanking non-transcribed sequences, 5’ or 3’ non-translated sequences (such as necessary ribosome binding sites), a polyadenylation site, splice donor and acceptor sites, or transcriptional termination sequences. An origin of replication that confers the ability to replicate in a host can also be incorporated.

[0225] Exemplary vectors that may be used are Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia), pEE6.4 (Lonza) and pEE12.4 (Lonza). Additional vectors include the pUC series (Fermentas Life Sciences, Glen Bumie, Md.), the pBluescript series (Stratagene, LaJolla,Calif.), the pET series (Novagen, Madison, Wis ), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors, such as XGT10, XGT11, EMBL4, and ANM1149, XZapII (Stratagene) can be used. Exemplary plant expression vectors include pBIOl, pBI01.2, pBI121, pBI101.3, and pBIN19 (Clontech). Exemplary animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The expression vector may be a viral vector, e.g, a retroviral vector, e.g, a gamma retroviral vector.

[0226] The vector of the disclosure may contain a promoter and an enhancer sequence. Polynucleotides encoding the CD79bxCD20xCD3 binding proteins of the disclosure may be operably linked to control sequences in the expression vector(s) that ensure the expression of the CD79bxCD20xCD3 binding proteins. Such regulatory elements may include a transcriptional promoter, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation. Expression vectors may also include one or more non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, other 5' or 3' flanking non-transcribed sequences, 5' or 3' non-translated sequences (such as necessary ribosome binding sites), a polyadenylation site, splice donor and acceptor sites, or transcriptional termination sequences. An origin of replication that confers the ability to replicate in a host may also be incorporated.

[0227] Vectors of the disclosure may also contain one or more Internal Ribosome Entry Site(s) (IRES). Inclusion of an IRES sequence into fusion vectors may be beneficial for enhancing expression of some proteins. In some embodiments, the vector system will include one or more polyadenylation sites (e.g., SV40), which may be upstream or downstream of any of the aforementioned nucleic acid sequences. Vector components may be contiguously linked or arranged in a manner that provides optimal spacing for expressing the gene products (i.e., by the introduction of “spacer” nucleotides between the ORFs) or positioned in another way.Regulatory elements, such as the IRES motif, may also be arranged to provide optimal spacing for expression.

[0228] Vectors of the disclosure may be circular or linear. They may be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. Replicationsystems can be derived, e.g., from ColEl, SV40, 2p plasmid, , bovine papilloma vims, and the like.

[0229] The recombinant expression vectors can be designed for either transient expression, for stable expression, or for both. Also, the recombinant expression vectors can be made for constitutive expression or for inducible expression.

[0230] The vectors may also comprise selection markers, which are well known in the art. Selection markers include positive and negative selection marker. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like. Exemplary marker genes include antibiotic resistance genes (e.g., neomycin resistance gene, a hygromycin resistance gene, a kanamycin resistance gene, a tetracycline resistance gene, a penicillin resistance gene, histidinol resistance gene, histidinol x resistance gene), glutamine synthase genes, HSV-TK, E1SV-TK derivatives for ganciclovir selection, or bacterial purine nucleoside phosphorylase gene for 6-methylpurine selection. A nucleic acid sequence encoding a selection marker or the cloning site may be upstream or downstream of a nucleic acid sequence encoding a polypeptide of interest or cloning site.

[0231] Numerous techniques are known in the art for the introduction of foreign genes into cells and can be used to construct the recombinant cells for purposes of carrying out the described methods, in accordance with the various embodiments described and exemplified herein. The technique used should provide for the stable transfer of the heterologous gene sequence to the host cell, such that the heterologous gene sequence is heritable and expressible by the cell progeny, and so that the necessary development and physiological functions of the recipient cells are not disrupted. Techniques which can be used include but are not limited to chromosome transfer (e.g., cell fusion, chromosome mediated gene transfer, micro cell mediated gene transfer), physical methods (e.g., transfection, spheroplast fusion, microinjection, electroporation, liposome carrier), viral vector transfer (e.g., recombinant DNA viruses, recombinant RNA viruses) and the like. Calcium phosphate precipitation and polyethylene glycol (PEG)-induced fusion of bacterial protoplasts with mammalian cells can also be used to transform cells.HOST CELLS

[0232] The disclosure also provides for a host cell comprising any of the vectors of the disclosure. “Host cell” refers to a cell into which a vector has been introduced. It is understood that the term host cell is intended to refer not only to the particular subject cell but to the progeny of such a cell, and also to a stable cell line generated from the particular subject cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not be identical to the parent cell but are still included within the scope of the term “host cell” as used herein. Such host cells may be eukaryotic cells, prokaryotic cells, plant cells or archeal cells. Escherichia coli, bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species are examples of prokaryotic host cells. Other microbes, such as yeast, are also useful for expression. Saccharomyces (e.g., S. cerevisiae) and Pichia are examples of suitable yeast host cells. Exemplary eukaryotic cells may be of mammalian, insect, avian or other animal origins. Mammalian eukaryotic cells include immortalized cell lines such as hybridomas or myeloma cell lines such as SP2 / 0 (American Type Culture Collection (ATCC), Manassas, VA, CRL-1581), NSO (European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646) and Ag653 (ATCC CRL-1580) murine cell lines. An exemplary human myeloma cell line is U266 (ATTC CRL-TIB-196). Other useful cell lines include those derived from Chinese Hamster Ovary (CHO) cells such as CHO-K1SV (Lonza Biologies, Walkersville, MD), CHO-K1 (ATCC CRL-61) orDG44.

[0233] Nucleic acids encoding CD79bxCD20xCD3 antibody or fragments thereof can be used for transformation of a suitable mammalian host cell. Host cell transformation, culture, antibody expression and purification are done using well known methods. Also provided is a host cell comprising, consisting of and / or consisting essentially of a vector comprising, consisting of and / or consisting essentially of a nucleic acid encoding a CD79bxCD20xCD3 antibody provided herein.

[0234] Cell lines may be selected based on high level of expression of the CD79bxCD20xCD3 antibody of interest and minimal contamination from host cell proteins. Mammalian cell lines available as host cells for expression are well known in the art andinclude but are not limited to from Chinese Hamster Ovary (CHO) cells such as CHO-K1SV (Lonza Biologies, Walkersville, MD), CH0-K1 (ATCC CRL-61), or CHO DG44, and Baby Hamster Kidney (BHK) cells. These cell lines can be used to produce any of the CD79bxCD20xCD3 antibody or antibody fragment thereof of the disclosure by culturing the cells under conditions suitable for expression of the antibody and purifying the antibody from the host cell or medium surrounding the host cell.

[0235] The disclosure also provides a method of producing the CD79bxCD20xCD3antibody of the disclosure comprising culturing the host cell of the disclosure in conditions that the CD79bxCD20xCD3 antibody is expressed, and recovering the CD79bxCD20xCD3 antibody peptides produced by the host cell using well known methods in the art. A subject protein may be substantially pure, e.g., at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or at least about 98% to 99%, or more, pure, e.g., free from contaminants such as cell debris, macromolecules, etc. other than the subject protein.PHARMACEUTICAL COMPOSITIONS

[0236] Further disclosed herein are pharmaceutical compositions comprising the disclosed CD79bxCD20xCD3 antibody and fragments thereof, for the preparation of a medicament for treating a CD79b and CD20 related condition, such as cancer.

[0237] For therapeutic use, the CD79bxCD20xCD3 antibody and fragments thereof of the disclosure may be prepared as pharmaceutical compositions containing an effective amount of the antibody as an active ingredient in a pharmaceutically acceptable carrier.

[0238] A pharmaceutically acceptable carrier can include a buffer, excipient, stabilizer, or preservative. The term “pharmaceutically acceptable,” as used herein with regard to pharmaceutical compositions, means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals and / or in humans.

[0239] Examples of pharmaceutically acceptable carriers are solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents,and the like that are physiologically compatible, such as salts, buffers, antioxidants, saccharides, aqueous or non-aqueous carriers, preservatives, wetting agents, surfactants or emulsifying agents, or combinations thereof. The amounts of pharmaceutically acceptable carrier(s) in the pharmaceutical compositions may be determined experimentally based on the activities of the carrier(s) and the desired characteristics of the formulation, such as stability and / or minimal oxidation.

[0240] Pharmaceutical compositions may comprise buffers such as acetic acid, citric acid, formic acid, succinic acid, phosphoric acid, carbonic acid, malic acid, aspartic acid, histidine, boric acid, Tris buffers, HEPPSO, HEPES, neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (c.g, aluminum hydroxide); antibacterial and antifungal agents; and preservatives.

[0241] In some embodiments, the CD79bxCD20xCD3 antibody or fragments thereof of the disclosure may be formulated in a composition comprising 10 mM sodium acetate, 7.5% sucrose, and 0.04% polysorbate 20 at pH5.5.

[0242] Pharmaceutical compositions of the present disclosure can be formulated for a variety of means of parenteral or non-parenteral administration. In one embodiment, the compositions can be formulated for infusion or intravenous administration. Pharmaceutical compositions disclosed herein can be provided, for example, as sterile liquid preparations, e.g, isotonic aqueous solutions, emulsions, suspensions, dispersions, or viscous compositions, which may be buffered to a desirable pH. Formulations suitable for oral administration can include liquid solutions, capsules, sachets, tablets, lozenges, and troches, powders liquid suspensions in an appropriate liquid and emulsions.METHOD OF TREATMENT

[0243] Further disclosed is the use of any of the disclosed CD79bxCD20xCD3 trispecific antibody or a fragment thereof and pharmaceutical compositions for the treatment of cancer or for the killing of cancer cells expressing CD79b and / or CD20 in a subject.

[0244] The disclosure provides methods comprising administering an effective amount of any the antibody, antibody fragment and pharmaceutical composition disclosed herein to the subject, thereby killing the cancer cells.

[0245] In some embodiments, the cancer is hematological cancer. In some embodiments, the cancer is B-cell lymphoma, non-Hodgkin lymphoma, large B-cell lymphoma (LBCL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or Waldenstrom macroglobulinemia (WM).

[0246] In some embodiments, the cancer is relapsed, refractory, or malignant cancer, or any combination thereof.

[0247] In some embodiments, the cancer is early-stage cancer, non-metastatic cancer, primary cancer, advanced cancer, locally advanced cancer, metastatic cancer, cancer in remission, recurrent cancer, cancer in an adjuvant setting, cancer in a neoadjuvant setting, or cancer substantially refractory to a therapy.

[0248] In some embodiments, routes of administration include intravenous, intramuscular, intratumoral, intraperitoneal, or subcutaneous.

[0249] In some embodiments, the disclosure provides a method of killing cancer cells and / or a method or treating cancer in a subject comprising administering to the subject a trispecific antibody or a fragment thereof that comprises a first binding domain that binds to CD79b, a second binding domain that binds to CD20 and a third binding domain that binds to CD3, wherein(a) the first binding domain comprises:(i) the HCDRs of a VH1 comprising an amino acid sequence of SEQ ID NO: 7; and the LCDRs of a VL1 comprising an amino acid sequence of SEQ ID NO: 8; (ii) a VH1 comprising a HCDR1 having an amino acid sequence of SEQ ID NO: 1, a HCDR2 having an amino acid sequence of SEQ ID NO: 2, and a HCDR3 having an amino acid sequence of SEQ ID NO: 3; and a VL1 comprising a LCDR1 having an amino acid sequence of SEQ ID NO: 4, a LCDR2 having an amino acid sequenceof and SEQ ID NO: 5, and a LCDR3 having an amino acid sequence of SEQ ID NO: 6; and / or(iii) a VH1 having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 7, and / or a VL1 having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 8;(a) the second binding domain comprises:(i) the HCDRs of a VH2 comprising an amino acid sequence of SEQ ID NO: 15; and the LCDRs of a VL2 comprising an amino acid sequence of SEQ ID NO: 16;(ii) a VH2 comprising a HCDR1 having an amino acid sequence of SEQ ID NO: 9, a HCDR2 having an amino acid sequence of SEQ ID NO: 10, and a HCDR3 having an amino acid sequence of SEQ ID NO: 11 ; and a VL2 comprising a LCDR1 having an amino acid sequence of SEQ ID NO: 12, a LCDR2 having an amino acid sequence of and SEQ ID NO: 13, and a LCDR3 having an amino acid sequence of SEQ ID NO: 14;(iii) a VH2 having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 15, and / or a VL2 having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 16; and / or(iv) a spFv having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 70;(a) the third binding domain comprises:(i) the HCDRs of a VH3 comprising an amino acid sequence of SEQ ID NO: 23; and the LCDRs of a VL3 comprising an amino acid sequence of SEQ ID NO: 24;(ii) a VH3 comprising a HCDR1 having an amino acid sequence of SEQ ID NO: 17, a HCDR2 having an amino acid sequence of SEQ ID NO: 18, and a HCDR3 having an amino acid sequence of SEQ ID NO: 19; and a VL3 comprising a LCDR1 having an amino acid sequence of SEQ ID NO: 20, a LCDR2 having an amino acid sequence of and SEQ ID NO: 21, and a LCDR3 having an amino acid sequence of SEQ ID NO: 22;(iii) a VH3 having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 23, and / or a VL3 having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 24; and / or (iv) a spFv having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 71.

[0250] In some embodiments, a method of treating cancer in a subject comprises administering to the subject a trispecific antibody or a fragment thereof comprising:(i) a first antigen-binding domain that binds to CD79b comprising a VH1 comprising a HCDR1 having an amino acid sequence of SEQ ID NO: 1, a HCDR2 having an amino acid sequence of SEQ ID NO: 2, and a HCDR3 having an amino acid sequence of SEQ ID NO: 3; and a VL1 comprising a LCDR1 having an amino acid sequence of SEQ ID NO: 4, a LCDR2 having an amino acid sequence of and SEQ ID NO: 5, and a LCDR3 having an amino acid sequence of SEQ ID NO: 6; (ii) a second antigen-binding domain that binds to CD20 comprising a VH2 comprising a HCDR1 having an amino acid sequence of SEQ ID NO: 9, a HCDR2 having an amino acid sequence of SEQ ID NO: 10, and a HCDR3 having an amino acid sequence of SEQ ID NO: 11; and a VL2 comprising a LCDR1 having an amino acid sequence of SEQ ID NO: 12, a LCDR2 having an amino acid sequence of and SEQ ID NO: 13, and a LCDR3 having an amino acid sequence of SEQ ID NO: 14; and(iii) a third antigen-binding domain that binds to CD3 comprising a VH3 comprising a HCDR1 having an amino acid sequence of SEQ ID NO: 17, a HCDR2 having an amino acid sequence of SEQ ID NO: 18, and a HCDR3 having an amino acid sequence of SEQ ID NO: 19; and a VL3 comprising a LCDR1 having an amino acid sequence of SEQ ID NO: 20, a LCDR2 having an amino acid sequence of and SEQ ID NO: 21, and a LCDR3 having an amino acid sequence of SEQ ID NO: 22.

[0251] In some embodiments, a method of treating cancer in a subject comprises administering to the subject a trispecific antibody or a fragment thereof comprising:a) a first antigen-binding domain that binds CD79b comprising the VH1 having an amino acid sequence of SEQ ID NO: 7 and the VL1 having an amino acid sequence of SEQ ID NO: 8;b) a second antigen-binding domain that binds CD20 comprising the VH2 having an amino acid sequence of SEQ ID NO: 15 and the VL2 having an amino acid sequence of SEQ ID NO: 16; andc) a third antigen-binding domain that binds CD3 comprising the VH3 having an amino acid sequence of SEQ ID NO: 23 and the VL3 having an amino acid sequence of SEQ ID NO: 24.

[0252] In some embodiments, the disclosure provides a method of killing cancer cells and / or a method or treating cancer in a subject comprising administering to the subject a trispecific antibody or a fragment thereof that comprises a first binding domain that binds CD79b, a second binding domain that binds CD20 and a third binding domain that binds CD3, wherein the antibody comprises:a) a HC1 having at least at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 25;b) a HC2 having at least at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 26; and / orc) a LC having at least at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 27.

[0253] In some embodiments, a method of treating cancer in a subject comprises administering to the subject a trispecific antibody or a fragment thereof comprising a HC1 having an amino acid sequence of SEQ ID NO: 25, a HC2 having an amino acid sequence of SEQ ID NO: 26, and a LC having an amino acid sequence of SEQ ID NO: 27.

[0254] In some embodiments, the disclosure provides a method of killing cancer cells and / or a method or treating cancer in a subject comprising administering to the subject a trispecific antibody or a fragment thereof that comprises a first binding domain that binds CD79b, a second binding domain that binds CD20 and a third binding domain that binds CD3, wherein the antibody comprises:a) a HC1 having at least at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 94;b) a HC2 having at least at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 95; and / or c) a LC having at least at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 27.

[0255] In some embodiments, a method of treating cancer in a subject comprises administering to the subject a trispecific antibody or a fragment thereof comprising a HC1 having an amino acid sequence of SEQ ID NO: 94, a HC2 having an amino acid sequence of SEQ ID NO: 95, and a LC having an amino acid sequence of SEQ ID NO: 27.

[0256] In some embodiments, the subject has relapsed after one or more lines of prior therapy or refractory to one or more lines of prior therapy.

[0257] In some embodiments, the subject has received at least 2 prior lines of therapy including an aCD20 monoclonal antibody containing chemotherapy combination schedule.

[0258] In some embodiments, the subject has received at least one prior line of therapy but are not eligible or do not have access to standard second line therapies, such as CAR-T.

[0259] In some embodiments, the disclosure provides a CD79bxCD20xCD3 trispecific antibody or a fragment thereof, or a pharmaceutical composition for use in combination therapy. In some embodiments, CD79bxCD20xCD3 trispecific antibody of the disclosure may be used in combination with a supplemental therapy. In some embodiments, the supplemental therapy is surgery. In some embodiments, the supplemental therapy is radiation. In some embodiments, the supplemental therapy is chemotherapy. In some embodiments, the CD79bxCD20xCD3 trispecific antibody of the disclosure is administered in combination with one or more pharmaceutically acceptable ingredient(s), active principle(s) or composition(s).EMBODIMENTS1. A trispecific antibody or a fragment thereof, comprising:a) a first antigen-binding domain comprising a first heavy chain complementarity determining region (HCDR) 1, a first HCDR2, and a first HCDR3 of a first heavy chain variable region (VH1) having an amino acid sequence of SEQ ID NO: 7 and a first lightchain complementarity determining region (LCDR) 1, a first LCDR2, and a first LCDR3 of a first light chain variable region (VL1) having an amino acid sequence of SEQ ID NO: 8;b) a second antigen-binding domain comprising a second heavy chain complementarity determining region (HCDR) 1, a second HCDR2, and a second HCDR3 of a second heavy chain variable region (VH2) having an amino acid sequence of SEQ ID NO: 15 and a second light chain complementarity determining region (LCDR) 1, a second LCDR2, and a second LCDR3 of a second light chain variable region (VL2) having an amino acid sequence of SEQ ID NO: 16;c) a third antigen-binding domain comprising a third heavy chain complementarity determining region (HCDR) 1, a third HCDR2, and a third HCDR3 of a third heavy chain variable region (VH3) having an amino acid sequence of SEQ ID NO: 23 and a third light chain complementarity determining region (LCDR) 1, a third LCDR2, and a third LCDR3 of a third light chain variable region (VL3) having an amino acid sequence of SEQ ID NO: 24,wherein the first antigen-binding domain binds to an epitope on cluster of differentiation 79b protein (CD79b), the second antigen-binding domain binds to an epitope on cluster of differentiation 20 (CD20), and the third antigen-binding domain binds to an epitope on cluster of differentiation 3 (CD3); andwherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences are according to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the Contact numbering system or the IMGT numbering system or a combination thereof.2. The trispecific antibody or the fragment thereof of embodiment 1, whereinthe first antigen-binding domain that binds CD79b comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprising:a) the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5 and 6, respectively according to the AbM numbering system;b) the amino acid sequences of SEQ ID NOs: 28, 29, 3, 4, 5, and 6, respectively according to the Kabat numbering system;c) the amino acid sequences of SEQ ID NOs: 30, 31, 3, 4, 5, and 6, respectively according to the Chothia numbering system;d) the amino acid sequences of SEQ ID NOs: 32, 33, 34, 35, amino acid residues TLS and SEQ ID NO: 6 respectively according to the IMGT numbering system; ore) the amino acid sequences of SEQ ID NOs: 36, 37, 38, 39, 40 and 41 according to the Contact numbering system, respectively.3. The trispecific antibody or the fragment thereof of embodiment 1 or 2, wherein the first antigen-binding domain that bind CD79b comprises a VH1 comprising an amino acid sequence which is at least 90%, or at least 95%, or at least 98%, at least 99%, or 100% identical to SEQ ID NO: 7 and a VL1 comprising an amino acid sequence which is at least 90%, or at least 95%, or at least 98%, at least 99%, or 100% identical to SEQ ID NO: 8.4. The trispecific antibody or the fragment thereof of any one of embodiments 1-3, wherein the first antigen-binding domain that bind CD79b comprises a VH1 comprising an amino acid sequence of SEQ ID NO: 7 and a VL1 comprising an amino acid sequence of SEQ ID NO: 8.5. The trispecific antibody or the fragment thereof of any one of embodiments 1-4, wherein the second antigen-binding domain that binds CD20 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 comprising:a) the amino acid sequences of SEQ ID NOs: 9, 10, 11, 12, 13, and 14, respectively according to the AbM numbering system;b) the amino acid sequences of SEQ ID NOs: 42, 43, 11, 12, 13 and 14, respectively according to the Kabat numbering system;c) the amino acid sequences of SEQ ID NOs: 44, 45, 11, 12, 13, and 14, respectively according to the Chothia numbering system;d) the amino acid sequences of SEQ ID NOs: 46, 47, 48, 49, amino acid residues DAS and SEQ ID NO: 14 according to the IMGT numbering system; ore) the amino acid sequences of SEQ ID NO: 50, 51, 52, 53, 54 and 55, respectively according to the Contact numbering system.6. The trispecific antibody or the fragment thereof of any one of embodiments 1-5, wherein the second antigen-binding domain that binds CD20 comprises a VH2 comprising an amino acid sequence which is at least 90%, or at least 95%, or at least 98%, at least 99%, or 100% identical to SEQ ID NO: 15 and a VL2 comprising an amino acid sequence which is at least 90%, or at least 95%, or at least 98%, at least 99%, or 100% identical to SEQ ID NO: 16.7. The trispecific antibody or the fragment thereof of any one of embodiments 1-6, wherein the second antigen-binding domain that binds CD20 comprises a VH2 comprising an amino acid sequence of SEQ ID NO: 15 and a VL2 comprising an amino acid sequence of SEQ ID NO: 16.8. The trispecific antibody or the fragment thereof of any one of embodiments 1-7, wherein the third antigen-binding domain that binds CD3 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 comprising:a) the amino acid sequences of SEQ ID NOs: 17, 18, 19, 20, 21, and 22, respectively according to the AbM numbering system;b) the amino acid sequences of SEQ ID NOs: 56, 57, 19, 20, 21 and 22, respectively according to the Kabat numbering system;c) the amino acid sequences of SEQ ID NOs: 58, 59, 19, 20, 21 and 22, respectively according to the Chothia numbering system;d) the amino acid sequences of SEQ ID NOs: 60, 61, 62, 63, amino acid residues YAS and SEQ ID NO: 22, respectively according to the IMGT numbering system; ore) the amino acid sequences of SEQ ID NO: 64, 65, 66, 67, 68 and 69, respectively according to the Contact numbering system.9. The trispecific antibody or the fragment thereof of any one of embodiments 1-8, wherein the third antigen-binding domain that binds CD3 comprises a VH3 comprising an amino acid sequence which is at least 90%, or at least 95%, or at least 98%, at least 99%, or 100% identicalto SEQ ID NO: 23 and a VL3 comprising an amino acid sequence which is at least 90%, or at least 95%, or at least 98%, at least 99%, or 100% identical to SEQ ID NO: 24.10. The trispecific antibody or the fragment thereof of any one of embodiments 1-9, wherein the third antigen-binding domain that binds CD3 comprises a VH3 comprising an amino acid sequence of SEQ ID NO: 23 and a VL3 comprising an amino acid sequence of SEQ ID NO: 24.11. The trispecific antibody or the fragment thereof of any one of embodiments 1-10, wherein a) the first antigen-binding domain that binds CD79b comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 having an amino acid sequence of SEQ ID NOs: 1, 2, 3, 4, 5 and 6, respectively;b) the second antigen-binding domain that binds CD20 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 having an amino acid sequence of SEQ ID NOs: 9, 10, 11, 12, 13 and 14, respectively; andc) the third antigen-binding domain that binds CD3 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 having an amino acid sequence of SEQ ID NOs: 17, 18, 19, 20, 21, and 22, respectively;wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are according to the AbM delineation.12. The trispecific antibody or the fragment thereof of any one of embodiments 1-11, wherein a) the first antigen -binding domain that binds CD79b comprises the VH1 having an amino acid sequence of SEQ ID NO: 7 and the VL1 having an amino acid sequence of SEQ ID NO: 8;b) the second antigen-binding domain that binds CD20 comprises the VH2 having an amino acid sequence of SEQ ID NO: 15 and the VL2 having an amino acid sequence of SEQ ID NO: 16; andc) the third antigen-binding domain that binds CD3 comprises the VH3 having an amino acid sequence of SEQ ID NO: 23 and the VL3 having an amino acid sequence of SEQ ID NO: 24.13. The trispecific antibody or the fragment thereof of any one of embodiments 1-12, wherein the first antigen-binding domain that binds CD79b comprises a diabody, a Fab, Fab’, a F(ab’)2, a variable fragment (Fv), a single chain variable fragment (scFv), a (ScFv)2, a Fd fragment, a single chain disulfide bond stabilized fragments, stapled scFv fragment (spFv), a disulfide stabilized Fv fragment (dsFv), or a disulfide stabilized diabody (ds diabody).14. The trispecific antibody or the fragment thereof of embodiment 13, wherein the first antigen-binding domain is a Fab.15. The trispecific antibody or the fragment thereof of any one of embodiments 1-14, wherein the second antigen-binding domain that binds CD20 comprises a diabody, a Fab, Fab’, a F(ab’)2, a variable fragment (Fv), a single chain variable fragment (scFv), a (ScFv)2, a Fd fragment, a single chain disulfide bond stabilized fragments, stapled scFv fragment (spFv), a disulfide stabilized Fv fragment (dsFv), or a disulfide stabilized diabody (ds diabody).16. The trispecific antibody or the fragment thereof of embodiment 15, wherein the second antigen-binding domain is a spFv.17. The trispecific antibody or the fragment thereof of any one of embodiments 1-16, wherein the third antigen-binding domain that binds CD3 comprises a diabody, a Fab, Fab’, a F(ab’)2, a variable fragment (Fv), a single chain variable fragment (scFv), a (ScFv)2, aFd fragment, a single chain disulfide bond stabilized fragments, stapled scFv fragment (spFv), a disulfide stabilized Fv fragment (dsFv), or a disulfide stabilized diabody (ds diabody).18. The trispecific antibody or the fragment thereof of embodiment 17, wherein the third antigen-binding domain is a spFv.19. The trispecific antibody or the fragment thereof of any one of embodiments 1-18, wherein the first antigen binding domain that binds CD79b is Fab, the second antigen binding domain that binds CD20 is a spFv and the third antigen binding domain that binds CD3 is a spFv.20. The trispecific antibody or the fragment thereof of any one of embodiments 1-19, wherein the spFv of the second binding domain is in an “ VH2 -linker 1-VL2” orientation or in a “VL2-linkerl-VH2” orientation.21. The trispecific antibody or the fragment thereof of any one of embodiments 1-20, wherein the spFv of the third binding domain is in an “VH3 -linker 1-VL3” orientation or in a “VL3-linkerl-VH3” orientation.22. The trispecific antibody or the fragment thereof of embodiment 20 or 21, wherein the linkerl is selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 72, 79, 80, 81 and 82.23. The trispecific antibody or the fragment thereof of any one of embodiments 1-22, wherein the spFv of the second binding domain is in the “VL2-linkerl-VH2” orientation and wherein the linkerl has an amino acid sequence of SEQ ID NO: 72 (GGGSGGSGGCPPCGGSGG).24. The trispecific antibody or the fragment thereof of any one of embodiments 1-23, wherein the spFv of the third binding domain is in the “VL3-linkerl-VH3” orientation and the linkerl has an amino acid sequence of SEQ ID NO: 72 (GGGSGGSGGCPPCGGSGG).25. The trispecific antibody or the fragment thereof of embodiment 23, wherein the spFv of the second binding domain comprises the amino acid sequence of SEQ ID NO: 70.26. The trispecific antibody or the fragment thereof of embodiment 24, wherein the spFv of the third binding domain comprises the amino acid sequence of SEQ ID NO: 71.27. The trispecific antibody or the fragment thereof of any one of embodiments 1-26, wherein the second binding domain comprising the VL2-linkerl-VH2 is linked to the N-terminus of the VH1 of the first binding domain in a VL2-linkerl-VH2-linker2-VHl orientation, and wherein the linker2 comprises the amino acid sequence of SEQ ID NO: 73 (GGGGSGGGGS).28. The trispecific antibody or the fragment thereof of any one of embodiments 1-27, further comprises an IgG constant region or the fragment of an IgG constant region.29. The trispecific antibody or the fragment thereof of embodiment 28, wherein the IgG constant region or the fragment of an IgG constant region is an IgGl, an IgG2, an IgG3, or an IgG4 isotype.30. The trispecific antibody or the fragment thereof of embodiment 29, wherein the IgG constant region or the fragment of an IgG constant region is an IgGl .31. The trispecific antibody or the fragment thereof of any one of embodiments 28-30, wherein the IgG constant region or the fragment of the IgG constant region comprises at least one mutation that results in reduced binding of the antibody or the fragment thereof to a Fey receptor (FcyR).32. The trispecific antibody or the fragment thereof of embodiment 31, wherein the mutation that results in reduced binding of the antibody or the fragment thereof to the FcyR is selected from F234A / L235A, L234A / L235A, L234A / L235A / D265S, V234A / G237A / P238S / H268A / V309L / A330S / P33 IS, F234A / L235A, S228P / F234A / L235A, N297A, V234A / G237A, K214T / E233P / L234V / L235A / G236-deleted / A327G / P331A / D365E / L358M, H268Q / V309L / A330S / P331S, S267E / L328F, L234F / L235E / D265A, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238S, and S228P / F234AZL235A / G236-deleted / G237A / P238S, wherein residue numbering is according to the EU index.33. The trispecific antibody or the fragment thereof of embodiment 32, wherein the mutation that results in reduced binding of the antibody or the fragment thereof to the FcyR are L234AZL235AZD265S.34. The trispecific antibody or the fragment thereof of any one of embodiments 31-33, wherein the FcyR is FcyRI, FcyR.II A, FcyRIIB, FcyRIII, or any combination thereof.35. The trispecific antibody or the fragment thereof of any one of embodiments 28-34, wherein the IgG constant region or the fragment of the IgG constant region comprises the T366W, T366S, L368A, and Y407V mutations according to EU index numbering.36. The trispecific antibody or the fragment thereof of embodiment 35, wherein the IgG constant region or the fragment of the IgG constant region further comprises the P374A mutation according to EU index numbering.37. A trispecific antibody or a fragment thereof comprising a first binding domain that binds to CD79b, a second binding domain that binds to CD20, and a third binding domain that binds to CD3, wherein:a) the first antigen-binding domain comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2 and a LCDR3 having an amino acid sequence of SEQ ID NOs: 1, 2, 3, 4, 5 and 6, respectively;b) the second antigen-binding domain comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, aLCDR2 and aLCDR3 having an amino acid sequence of SEQ ID NOs: 9, 10, 11, 12, 13 and 14, respectively; andc) the third antigen-binding domain comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2 and a LCDR3 having an amino acid sequence of SEQ ID NOs: 17, 18, 18, 20, 21, and 22, respectively; andwherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are according to the AbM delineation.38. A trispecific antibody or a fragment thereof comprising a first binding domain that binds to CD79b, a second binding domain that binds to CD20, and a third binding domain that binds to CD3, wherein:a) the first antigen-binding domain comprises a VH1 having an amino acid sequence of SEQ ID NO: 7 and a VL1 having an amino acid sequence of SEQ ID NO: 8;b) the second antigen-binding domain comprises a VH2 having an amino acid sequence of SEQ ID NO: 15 and a VL2 having an amino acid sequence of SEQ ID NO: 16; andc) the third antigen-binding domain comprises a VH3 having an amino acid sequence of SEQ ID NO: 23 and a VL3 having an amino acid sequence of SEQ ID NO: 24.39. A trispecific antibody or a fragment thereof comprising:a) a first heavy chain (HC1) comprising from N-terminus to C-terminus a VL3-linkerl- VH3-linker4-Fc, wherein the Fc comprises the CH2 and CH3 domain of a human IgGl constant region and the linker4 comprises the amino acid sequence of SEQ ID NO: 74 (EPKSSDKTHTCPPCP);b) a second heavy chain (HC2) comprising from N-terminus to C-terminus a VL2- linkerl-VH2-linker2-VHl -linker3 -Fc, wherein the Fc comprises the CHI, CH2 and CH3 domain of a human IgGl constant region and the linker 3 comprises the amino acid sequence of SEQ ID NO: 75 (EPKSCDKTHTCPPCP); andc) a light chain (LC) comprising from N-terminus to C-terminus a VL1-CL, wherein the CL is the CL of a human kappa light chain.40. The trispecific antibody or the fragment thereof of embodiment 39, wherein:the VH1 comprises an amino acid sequence of SEQ ID NO: 7 and the VL1 comprises an amino acid sequence of SEQ ID NO: 8;the VH2 comprises an amino acid sequence of SEQ ID NO: 15 and the VL2 comprises an amino acid sequence of SEQ ID NO: 16; andthe VH3 comprises an amino acid sequence of SEQ ID NO: 23 and the VL3 comprises an amino acid sequence of SEQ ID NO: 24.41. The trispecific antibody or the fragment thereof of any one of embodiments 39-40, wherein the HC1 comprises an amino acid sequence at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 25.42. The trispecific antibody or the fragment thereof of embodiment 41, wherein the HC1 comprises an amino acid sequence of SEQ ID NO: 25.43. The trispecific antibody or the fragment thereof of any one of embodiments 39-42, wherein the HC2 comprises an amino acid sequence at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 26.44. The trispecific antibody or the fragment thereof of embodiment 43, wherein the HC2 comprises an amino acid sequence of SEQ ID NO: 26.45. The trispecific antibody or the fragment thereof of any one of embodiments 39-40, wherein the HC1 comprises an amino acid sequence at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 94.46. The trispecific antibody or the fragment thereof of embodiment 45, wherein the HC1 comprises an amino acid sequence of SEQ ID NO: 94.47. The trispecific antibody or the fragment thereof of embodiment 45 or 46, wherein the HC2 comprises an amino acid sequence at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 95.48. The trispecific antibody or the fragment thereof of embodiment 47, wherein the HC2 comprises an amino acid sequence of SEQ ID NO: 95.49. The trispecific antibody or the fragment thereof of any one of embodiments 39-48, wherein the LC comprises an amino acid sequence at least 90%, at least 95%, at least 98%, at least 99% or 100 % identical to SEQ ID NO: 27.500. The trispecific antibody or the fragment thereof of embodiment 49, wherein the LC comprises an amino acid of SEQ ID NO: 27.51. The trispecific antibody or the fragment thereof of any one of embodiments 39-44 and 49- 50, wherein the trispecific antibody or the fragment thereof comprises the HC1 comprising an amino acid sequence of SEQ ID NO: 25, the HC2 comprising an amino acid sequence of SEQ ID NO: 26, and the LC comprising an amino acid sequence of SEQ ID NO: 27.52. The trispecific antibody or the fragment thereof of any one of claims 39-40 and 45-48, wherein the trispecific antibody or the fragment thereof comprises the HC1 comprising an amino acid sequence of SEQ ID NO: 94, the HC2 comprising an amino acid sequence of SEQ ID NO: 95, and the LC comprising an amino acid sequence of SEQ ID NO: 27.53. A trispecific antibody or a fragment thereof comprising first binding domain that binds to CD79b, a second binding domain that binds to CD20, and a third binding domain that binds to CD3, wherein:a) the first binding domain is a Fab comprising a VH1 and a VL1 having an amino acid sequence of SEQ ID NO: 7 and 8, respectively;b) the second binding domain is a spFv comprising a VL2-linkerl-VH2, wherein VH2 and VL2 comprise an amino acid sequence of SEQ ID NO: 15 and 16, respectively, and wherein the linkerl comprises an amino acid sequence of SEQ ID NO: 72; andc) the third binding domain is a spFv comprising a VL3 -linker 1-VH3, wherein VH3 and VL3 comprise an amino acid sequence of SEQ ID NO: 23 and 24, respectively, and wherein the linkerl comprises an amino acid sequence of SEQ ID NO: 72.54. A trispecific antibody or a fragment thereof comprising first binding domain that binds to CD79b, a second binding domain that binds to CD20, and a third binding domain that binds to CD3, wherein:a) the first binding domain is a Fab comprising a VH1 and VL1 having an amino acid sequence of SEQ ID NO: 7 and 8, respectively;b) the second binding domain is a spFv having an amino acid sequence of SEQ ID NO: 70; andc) the third binding domain is a spFv having an amino acid sequence of SEQ ID NO: 71.55. Atrispecific antibody or a fragment thereof comprises a first heavy chain (HC1) comprising an amino acid sequence of SEQ ID NO: 25, a second heavy chain (HC2) comprising an amino acid sequence of SEQ ID NO: 26, and a light chain (LC) comprising an amino acid sequence of SEQ ID NO: 27.56. Atrispecific antibody or a fragment thereof comprises a first heavy chain (HC1) comprising an amino acid sequence of SEQ ID NO: 94, a second heavy chain (HC2) comprising an amino acid sequence of SEQ ID NO: 95, and a light chain (LC) comprising an amino acid sequence of SEQ ID NO: 27.57. A polynucleotide encoding a trispecific antibody or a fragment thereof of any one of embodiments 1-56.58. An expression vector comprising a polynucleotide of embodiment 57.59. A host cell expressing a trispecific antibody or a fragment thereof of any one of embodiments 1-56.60. A pharmaceutical composition comprising a trispecific antibody or a fragment thereof of any one of embodiment 1-56, a polynucleotide of embodiment 57, a vector of embodiment 58, or a host cell of embodiment 59, and a pharmaceutical carrier.61. A method of treating cancer in a subject comprising administering to the subject a trispecific antibody or a fragment thereof of any one of embodiments 1-56, or the pharmaceutical composition of embodiment 60.62. The method of embodiment 61, wherein the subject is a human subject.63. The method of any one of embodiments 61 or 62, wherein the cancer is hematological cancer.64. The method of any one of embodiments 61-63, wherein the cancer is B-cell lymphoma, non-Hodgkin lymphoma, large B-cell lymphoma (LBCL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or Waldenstrom macroglobulinemia (WM).65. The method of embodiment 64, wherein the cancer is relapsed, refractory, or malignant cancer, or any combination thereof.66. A method of treating cancer in a subject comprising administering to the subject a pharmaceutical composition comprising a trispecific antibody or a fragment thereof comprising first binding domain that binds to CD79b, a second binding domain that binds to CD20, and a third binding domain that binds to CD3, wherein:a) the first binding domain is a Fab comprising a VH1 and a VL1 having an amino acid sequence of SEQ ID NO: 7 and 8, respectively;b) the second binding domain is a spFv comprising a VL2 -linker 1-VH2, wherein VH2 and VL2 comprise an amino acid sequence of SEQ ID NO: 15 and 16, respectively, and wherein the linkerl comprises an amino acid sequence of SEQ ID NO: 72; andc) the third binding domain is a spFv comprising a VL3-linkerl-VH3, wherein VH3 and VL3 comprise an amino acid sequence of SEQ ID NO: 23 and 24, respectively, and wherein the linkerl comprises an amino acid sequence of SEQ ID NO: 72.67. The method of embodiment 66, wherein the cancer is B-cell lymphoma, non-Hodgkin lymphoma, large B-cell lymphoma (LBCL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or Waldenstrom macroglobulinemia (WM).68. A method of treating cancer in a subject comprising administering to the subject a pharmaceutical composition comprising a trispecific antibody or a fragment thereof comprising a first heavy chain (HC1) comprising an amino acid sequence of SEQ ID NO: 25, a second heavy chain (HC2) comprising an amino acid sequence of SEQ ID NO: 26, and a light chain (LC) comprising an amino acid sequence of SEQ ID NO: 27.69. A method of treating cancer in a subject comprising administering to the subject a pharmaceutical composition comprising a trispecific antibody or a fragment thereof comprising a first heavy chain (HC1) comprising an amino acid sequence of SEQ ID NO: 94, a second heavy chain (HC2) comprising an amino acid sequence of SEQ ID NO: 95, and a light chain (LC) comprising an amino acid sequence of SEQ ID NO: 27.70. The method of embodiments 68 or 69, wherein the cancer is B-cell lymphoma, nonHodgkin lymphoma, large B-cell lymphoma (LBCL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or Waldenstrom macroglobulinemia (WM).71. Use of a trispecific antibody or a fragment thereof in the manufacture of a medicament for treating cancer in a subject comprising administering to the subject a pharmaceuticalcomposition comprising the trispecific antibody or a fragment thereof comprising first binding domain that binds to CD79b, a second binding domain that binds to CD20, and a third binding domain that binds to CD3, wherein:a) the first binding domain is a Fab comprising a VH1 and a VL1 having an amino acid sequence of SEQ ID NO: 7 and 8, respectively;b) the second binding domain is a spFv comprising a VL2-linkerl-VH2, wherein VH2 and VL2 comprise an amino acid sequence of SEQ ID NO: 15 and 16, respectively, and wherein the linkerl comprises an amino acid sequence of SEQ ID NO: 72; andc) the third binding domain is a spFv comprising a VL3-linkerl-VH3, wherein VH3 and VL3 comprise an amino acid sequence of SEQ ID NO: 23 and 24, respectively, and wherein the linkerl comprises an amino acid sequence of SEQ ID NO: 72.72. The use of embodiment 71, wherein the cancer is B-cell lymphoma, non-Hodgkin lymphoma, large B-cell lymphoma (LBCL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or Waldenstrom macroglobulinemia (WM).73. Use of a trispecific antibody or a fragment thereof in the manufacture of a medicament for treating cancer in a subject comprising administering to the subject a pharmaceutical composition comprising the trispecific antibody or a fragment thereof comprising a first heavy chain (HC1) comprising an amino acid sequence of SEQ ID NO: 25, a second heavy chain (HC2) comprising an amino acid sequence of SEQ ID NO: 26, and a light chain (LC) comprising an amino acid sequence of SEQ ID NO: 27.74. Use of a trispecific antibody or a fragment thereof in the manufacture of a medicament for treating cancer in a subject comprising administering to the subject a pharmaceutical composition comprising the trispecific antibody or a fragment thereof comprising a first heavy chain (HC1) comprising an amino acid sequence of SEQ ID NO: 94, a second heavy chain(HC2) comprising an amino acid sequence of SEQ ID NO: 95, and a light chain (LC) comprising an amino acid sequence of SEQ ID NO: 27.

[0260] Various embodiments or subject matter have been described. It will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the following examples are intended to illustrate but not limit the scope of inventions described in the claims.EXAMPLES EXAMPLE 1: SCREENING FOR OPTIMAL CD79BXCD20XCD3 TRISPECIFIC ANTIBODY FORMATS

[0261] Different CD79bxCD20xCD3 trispecific antibody formats were explored to identify the format that provides optimal in vitro cell binding, in vitro cytotoxicity and T cell activation. CD79bxCD20xCD3 trispecific antibodies are immunoglobin (Ig) G1 trispecific molecules comprising a CD3 binding domain that binds to the epsilon subunit of the cluster of differentiation 3 receptor complex (CD3e) on T lymphocytes (T cells), a CD79b binding domain that binds to CD79b (B-cell antigen receptor complex-associated protein B chain), and a CD20 binding domain that to CD20 (B -lymphocyte antigen CD20) on tumor cells.

[0262] The anti-CD79b variable region of the CD79bxCD20xCD3 trispecific antibodies was discovered by immunizing transgenic humanized mice (Ablexis mice) with recombinant CD79b. The anti-CD20 variable region of the CD79bxCD20xCD3 trispecific antibodies was derived from an approved therapeutic antibody, Ofatumumab. The anti-CD3e binding domain was of the CD79bxCD20xCD3 trispecific antibodies was discovered by immunizing human antibody transgenic mice (Ablexis mice) with recombinant CD3e protein.

[0263] The anti-CD79b variable region, the anti-CD20 variable region and the anti-CD3 variable region were engineered into Fab or spFv in the VL-linkerl-VH or VH-linkerl-VL orientation. The variable regions were further engineered into a Heavy Chain (HC1 or HC2) and expressed as IgGl comprising the Fc silencing mutation L234A / L235A / D265S and theheterodimerization (“knob” mutation T366W) in the Fc domain or fused to the CL of a kappa light chain and engineered into a Light Chain (LC). The study evaluated the position and orientation of each antigen binding domain within the trispecific antibody format. The CD3 and CD20 binding domains were engineered as either a “wedged” or a “free” spFv in the VL-linker-VH or the VH-linker-VL orientation. FIG. 1 and Table 2 show the different CD79bxCD20xCD3 trispecific antibody formats evaluated. Format C and E featured a “wedged” CD3 binding domain while format A, B and D featured a “free” CD3 binding domain. Format B also featured a “wedged” CD20 binding domain.

[0264] All CD79bxCD20xCD3 trispecific antibody formats evaluated (at the exception of C923B397) comprised the same CD79b heavy chain and light chain variable regions (VH1 and VL1), the same CD20 ofatumumab derived heavy chain and light chain variable regions (VH2 and VL2), and the same high affinity CD3 heavy chain and light chain variable regions (VH3 and VL3). C923B397 comprised a lower affinity CD3 binding domain but the same CD79b and CD20 binding domains as the other CD79bxCD20xCD3 trispecific antibodies and was used as a control antibody.Table 2. Summary of CD79bxCD20xCD3 trispecific antibody formats evaluatedspFv LH, spFv VL-linkerl-VH; spFv HL, spFv VH-linkerl-VLBinding of the different CD79bxCD20xCD3 trispecific antibody formats to CD79b+ and CD20+ Camaval cancer cells

[0265] Flow cytometry was used to evaluate binding of the various CD79bxCD20xCD3 trispecific antibody formats to CD79b+, CD20+ Camaval cancer cells.Binding of the CD79bxCD20xCD3 trispecific antibodies was tested at a physiologically relevant temperature of 37°C for 1 hour. The EC50 values for binding to CD79b+, CD20+ Camaval cancer cells of all the CD79bxCD20xCD3 trispecific antibody formats are listed in Table 3. All tested trispecific antibodies showed a dose-dependent binding to Carnaval cells (FIG. 2) with C923B390 (Format E), C923B387 (Format D), and C923B385 (Format B) showing high affinity binding to CD79b+, CD20+ Carnaval cancer cells.Table 3. Summary of ECso values for binding of the various CD79bxCD20xCD3 trispecific antibody formats to Carnaval CD79b+, CD20+cells.Binding to T cells

[0266] Flow cytometry was used to evaluate the binding affinity of the CD3 binding domain of the various CD79bxCD20xCD3 trispecific antibody formats to human pan T cells.The readout was done after 1 hour. All CD79bxCD20xCD3 trispecific antibody formats displayed a dose-dependent binding to human T cells (FIG. 3A and FIG. 3B). The EC50 values are shown in Table 4.Table 4. Summary of ECso values (in nM) for binding of various CD79bxCD20xCD3 trispecific antibody formats to pan T cells.DN1, Donor 1; DN2, Donor 2.LH, VL-Linkerl-VH; HL, VH-Linkerl-VL.T cell binding potency differed based on the orientation of the CD3 spFv domain (VH-linker-VL or VL-linker-VH) and whether the CD3 binding domain was “wedged” (Format C and E) or “free” (Format A, B and D). As shown in Table 4, two of the “wedged” CD3 formats C923B388 and C923B390 displayed higher affinity when compared to “free” CD3 formats C923B385, C923B384, and C923B387. In contrast, C923B389 which has a “wedged” CD3 format showed weak binding, and was comparable to control C923B397 (antibody with low affinity CD3 binding domain).CD79bxCD20xCD3 trispecific antibodies induce cancer cell cytotoxicity in single and double antigen expressing K562 isogenic models

[0267] To validate their cytotoxic potential, the CD79bxCD20xCD3 trispecific antibodies were tested in single and double antigen expressing K562 isogenic models (K562 cells expressing CD20, K562 cells expressing CD79b or K562 cells expressing both CD20 and CD79b) by flow cytometry.

[0268] Healthy human pan-T cells from 1 to 3 donors were used as a source of effector cells and cocultured at an E:T ratio of 1:1. T-cell-mediated cytotoxicity was assessed in the presence of different CD79bxCD20xCD3 trispecific antibody formats. Coculture of pan CD3+ T cells with the indicated antigen-positive or -negative cancer cell lines were set up to achieve an E:T ratio of 1 : 1. Cell cytotoxicity was analyzed after 72 hours incubation and normalized to the untreated cells.

[0269] All tested trispecific antibodies showed T-cell mediated cytotoxicity in both single and dual antigen expressing cancer cells (Table 5, FIG. 4A-FIG. 4D), although the EC50 values varied among the different formats tested.Table 5. Summary of ECso values (in nM) in CD79bxCD20xCD3 trispecific antibody induced cancer cell cytotoxicity in single and double antigen expressing K562 isogenic models.CD79bxCD20xCD3 trispecific antibodies induce cancer cell cytotoxicity in B-NHL models

[0270] CD79bxCD20xCD3 trispecific antibodies were also tested in B-NHL models displaying a wide range of CD79b and CD20 endogenous expression levels. The CD79b and CD20 antigen density of the hematologic tested cell lines are described Table 6.Table 6. CD79b and CD20 antigen densityABC, antibody binding capacity; DLBCL, diffuse large B-cell lymphoma

[0271] Healthy human pan-T cells from 1 to 3 donors were used as a source of effector cells and cocultured at an E:T ratio of 1:1. T-cell-mediated cytotoxicity was assessed in the presence of different CD79bxCD20xCD3 trispecific antibody formats. Coculture of pan CD3+T cells with the indicated cancer cell lines were set up to achieve an E:T ratio of 1 : 1. Cell cytotoxicity was analyzed after 72 hours incubation and normalized to the untreated cells.

[0272] All tested CD79bxCD20xCD3 trispecific antibodies showed efficacy in the different B-NHL cell lines (Table 7, FIG. 5A - FIG. 5D), albeit exhibiting different EC50 values. The measured activity correlated with levels of antigen expression on each cell line.Table 7. Summary of ECso values (in nM) in CD79bxCD20xCD3 trispecific antibody induced cancer cell cytotoxicity in B-NHL models.T-cell activation (%CD8+ / CD25+) in K562 isogenic models

[0273] To assess the effect of CD79bxCD20xCD3 trispecific antibody formats on T-cell activation, the expression of late activation marker (CD25) on CD8+ T cells was monitored in the cytotoxicity assays described above. T-cell activation was assessed by flow cytometry at 72 hours upon coculture with a panel of antigen-positive (K562 CD20, K562 CD79b and K562 CD79b / CD20) or antigen-negative (K562) cancer cell lines at an E:T ratio of 1 : 1. After 72 hours of treatment of healthy-donor pan-T cells with CD79bxCD20xCD3 trispecific antibody, a concentration-dependent T-cell activation was induced in CD8+T-cells. The expression (%) of late T-cell activation marker CD25 was used to identify the activated T cells. Graphing of data was done in GraphPad Prism 10. Data from 3 independent experiments for all molecules except for C923B384 and C923B388 (N=l) were pooled and represented as mean ± SEM. All tested CD79bxCD20xCD3 trispecific antibody formats induced dose-dependent T-cell activation (FIG. 6A - 6D). Table 8 shows the EC50 values measured.Table 8. Summary of EC50 values (in nM) in CD79bxCD20xCD3 trispecific antibody induced cancer T-cell activation assayT-cell activation (%CD8+ / CD25+) in CD79b and CD20 endogenously expressing cell lines

[0274] CD79bxCD20xCD3 trispecific antibody induced T-cell activation was also assessed in cell lined expressing endogenous CD20 and / or CD79b. T cell activation was measured by flow cytometry at 72 hours upon coculture at an E:T ratio of 1 : 1. Data from 3 independent experiments for all molecules except for C923B388 (N=3 for CARNAVAL and OCI-Ly 10 but N=1 for HT and WILL-2) and C923B384 (N=l) were pooled and represented as mean ± SEM. All tested CD79bxCD20xCD3 trispecific antibody formats induced dosedependent T-cell activation (FIG. 7A - 7D). Table 9 shows the EC50 values measured.Table 9. Summary of EC50 values (in nM) in CD79bxCD20xCD3 induced cancer T-cell activation assay.Expression and stability of CD79bxCD20xCD3 trispecific antibody formatsThe expression and stability of C923B385, C923B387 and C923B388 were evaluated to identify the format and Heavy Chain 1 : Heavy Chain 2: Light Chain (HC1 :HC2:LC) ratio thatprovides the highest expression yields and purity. The CD79bxCD20xCD3 trispecific antibodies were expressed in ExpiCHPO cells in various HC1 :HC2:LC ration and purified through Protein A chromatography. Purity was assessed by size exclusion chromatography (SEC) and mass spectrometry (MS). Table 10 describes the purification yields and the % purity of the main peak for each CD79bxCD20xCD3 tripsecific HC1:HC2:LC ratios tested.Table 10. Summary of expression and stability analysis of C923B385, C923B387 and C923B388.

[0275] C923B387 gave the highest yields (159 mg / L) and highest purity levels (> 90%). C923B387 performed well in all HC1:HC2:LC ratios tested. C923B387 was selected for further evaluation.EXAMPLE 2: STRUCTURE OF CD79BXCD20XCD3 TRISPECIFIC ANTIBODY (C923B387)

[0276] CD79bxCD20xCD3 trispecific antibody (C923B387) is an immunoglobin (Ig) G1 trispecific molecule comprising a CD3 binding domain that binds to the epsilon subunit of the cluster of differentiation 3 receptor complex (CD3e) on T lymphocytes (T cells), a CD79b binding domain that binds to CD79b (B-cell antigen receptor complex-associated protein B chain), and a CD20 binding domain that to CD20 (B-lymphocyte antigen CD20,) on tumor cells.CD79b binding domain

[0277] The anti-CD79b variable region of CD79bxCD20xCD3 trispecific antibody was discovered by immunizing transgenic humanized mice (Ablexis mice) with recombinant CD79b. The sequences of the CD79b binding domain are shown in Table 11-17.Table 11. Amino acid sequences of HCDRs and LCDRs of CD79b binding domain under the AbM delineation.Table 12. Amino acid sequences of HCDRs and LCDRs of CD79b binding domain under the Kabat delineation.Table 13. Amino acid sequences of HCDRs and LCDRs of CD79b binding domain under the Chothia delineation.Table 14. Amino acid sequences of HCDRs and LCDRs of CD79b binding domain under the IMGT delineation.Table 15. Amino acid sequences of HCDRs and LCDRs of CD79b binding domain under the Contact delineation.Table 16. Amino acid sequences of VH1 and VL1 of CD79b binding domainTable 17. Nucleotide sequences of VH1 and VL1 of CD79b binding domainCD20 binding domain

[0278] The anti-CD20 variable region of CD79bxCd20xCD3 trispecific antibody was derived from Ofatumumab and was engineered as an spFv in the VL2-Linkerl-VH2 orientation. The sequences of the CD20 binding domain are shown in Table 18-24.Table 18. Amino acid sequences of HCDRs and LCDRs of CD20 binding domain under the AbM delineation.Table 19. Amino acid sequences ofHCDRs and LCDRs of CD20 binding domain under the Kabat delineation.Table 20. Amino acid sequences of HCDRs and LCDRs of CD20 binding domain under the Chothia delineation.Table 21. Amino acid sequences of HCDRs and LCDRs of CD20 binding domain under the IMGT delineation.Table 22. Amino acid sequences of HCDRs and LCDRs of CD20 binding domain under the Contact delineation.Table 23. Amino acid sequences of VH2 and VL2 of CD20 binding domainTable 24. Nucleotide sequences of VH2 and VL2 of CD20 binding domainCD3 binding domain

[0279] The anti-CD3e variable region of CD79bxCD20xCD3 trispecific antibody was discovered by immunizing human antibody transgenic mice (Ablexis mice) with recombinant CD3E protein and was engineered as an spFv in the VL3 -Linker 1-VH3 orientation. The amino acid sequences of the CD3 binding domain are shown in Table 25-31.Table 25. Amino acid sequences of HCDRs and LCDRs of CD3 binding domain under the AbM delineation.Table 26. Amino acid sequences of HCDRs and LCDRs of CD3 binding domain under the Kabat delineation.Table 27. Amino acid sequences of HCDRs and LCDRs of CD3 binding domain under the Chothia delineation.Table 28. Amino acid sequences of HCDRs and LCDRs of CD3 binding domain under the IMGT delineation.Table 29. Amino acid sequences of HCDRs and LCDRs of CD3 binding domain under the Contact delineation.Table 30. Amino acid sequences of VH3 and VL3 of CD3 binding domainTable 31. Nucleotide sequences of VH3 and VL3 of CD3 binding domainCD79bxCD20xCD3 Heavy Chains and Light chain sequences

[0280] The stapled scFv of the CD3 binding domain in the VL3 -Linker 1-VH3 orientation was further engineered into a Heavy Chain 1 (HC1) and expressed as IgGl comprising the Fc silencing mutation L234A / L235A / D265S and the heterodimerization (“knob” mutation T366W) in the Fc domain. The C-terminus of the stapled scFv of the CD20 bindingdomain in the VL2-linkerl-VH2 orientation was fused to N-terminus of the VH1 of the CD79b binding domain in the VL2-linkerl-VH2-linker2-VHl orientation and further engineered into a Heavy Chain 2 (HC2) and expressed as IgGl comprising the Fc silencing mutation L234AZL235AZD265S, the H435R / Y436F mutations and the heterodimerization (“hole” mutation T366S / L368A / Y407V) in the Fc domain. The VL1 of the CD79b binding domain was fused to the CL of a kappa light chain and engineered as a Light Chain (LC). The HC 1, HC2 and LC were paired using the knob-in-hole heterodimerization technology to generate the CD790bxCD20xCD3 trispecific antibody. The amino acid sequences of the CD79bxCD20xCD3 trispecific antibody heavy chains (HC1 and HC2) and light chain (LC) are shown in Table 32. The nucleotide sequences of the CD79bxCD20xCD3 trispecific antibody (C923B387) heavy chains (HC1 and HC2) and light chain (LC) are shown in Table 33.Table 32. Amino acid sequences of full-length CD79bxCD20xCD3 antibody (C923B387)Table 33. Nucleotide sequences of full-length CD79bxCD20xCD3 antibodyEXAMPLE 3: TARGET AND CD3 BINDING CHARACTERIZATION OF CD79BXCD20XCD3 TRISPECIFIC ANTIBODY (C923B387)Endogenous tumor cell binding

[0281] Flow cytometry was used to evaluate the CD79b and CD20 arm binding of the CD79bxCD20xCD3 trispecific antibody C923B387 in vitro. CD79bxCD20xCD3 trispecific antibody and isotype controls were tested on a panel of cell lines of varying CD79b and CD20 receptor densities at a physiologically relevant temperature of 37°C for 1 hour.CD79bxCD20xCD3 trispecific antibody displayed dose-dependent binding to Camaval WT (CD79b+, CD20+), Carnaval-CD20-KO (clone P1E5; CD79b+, CD20"), K562-CD20-mKate(clone H1B9; CD79b" CD20+), and K562-CD79b-GFP (CD79b+, CD20") cells (FIG. 8A-8D). The EC50 values for cell binding by CD79bxCD20xCD3 trispecific antibody are listed in Table 34. The CD3 x Null x Null control did not bind to any of the listed cell lines.Table 34. Summary of ECXvalues for binding of CD79bxCD20xCD3 trispecific antibody to Camaval WT, Carnaval-CD20-KO (clone P1E5), K562-CD20-mKate (clone H1B9), and K562-CD79b-GFP cells.4PL, 4-parameter logistic; CD, cluster of differentiation; ECX, x% effective concentration; Emax, maximal efficacy; GFP, green fluorescent protein; KO, knockout; MFI, mean fluorescence intensity; WT, wild type. ECXvalues were calculated using a 4PL curve-fitting model in Graphpad Prism.Primary human T-cell binding

[0282] Flow cytometry was used to evaluate the CD3 binding of CD79bxCD20xCD3 trispecific antibody and the CD3 null control NullxCD79b-Ofa to human T cells.CD79bxCD20xCD3 trispecific antibody displayed dose-dependent binding to human T cells with an EC50 value of 34.81 nM (FIG. 9, Table 35). The NullxCD79b-Ofa control did not bind to human T cells.Table 35. Summary of ECXvalues for binding of CD79bxCD20xCD3 trispecific antibody to human T cells.4PL, 4-parameter logistic; ECX, x% effective concentration; Emax, maximal efficacy; MFI, mean fluorescence intensity. ECXvalues were calculated using a 4PL curve-fitting model in Graphpad Prism.EXAMPLE 4: EFFECT OF CD79BXCD20XCD3 TRISPECIFIC ANTIBODY (C923B387) IN T-CELL CYTOTOXICITY AND ACTIVATION ASSAYS USING PAN-T CELLS AND B-CELL LYMPHOMA CELL LINESCytotoxicity

[0283] To validate its cytotoxic potential, CD79bxCD20xCD3 trispecific antibody (C923B387) was tested in a panel of B-cell lymphoma lines (i.e., CARNAVAL, OCI-LylO, HT, and WILL-2) that endogenously express varying levels of CD79b and / or CD20. As a negative control, the K562 chronic myelogenous leukemia (CML) cell line was included as a CD79b-CD20- model. Healthy human pan-T cells from 3 to 4 donors were used as a source of effector cells and cocultured at an E:T ratio of 1:1. T-cell-mediated cytotoxicity was assessed in the presence of CD79bxCD20xCD3 trispecific antibody, or corresponding matched null control antibody. A reference CD79bxCD20xCD3 trispecific antibody (Reference Trispecific Antibody) with SEQ ID NOs: 170, 172, and 175, disclosed in US Patent No. 12,103,977 was included as a benchmark. Cancer cell cytotoxicity was assessed by flow cytometry at 72 hours. Coculture of pan CD3+ T cells with the indicated antigen-positive or -negative cancer cell lines was set up to achieve an E:T ratio of 1 : 1. Cancer cell cytotoxicity was analyzed using FlowJo (vlO) and normalized to the untreated cells. Treatment with CD79bxCD20xCD3 trispecific antibody resulted in antigen-dependent cancer cell cytotoxicity (FIG. 10A-FIG. 10E, Table 36).

[0284] The observed potency correlated with the levels of CD79b and / or CD20 expression. A consistent increase in potency was observed in comparison to the Reference Trispecific Antibody across the 4 models tested. Either no or a minimal effect was observed after treatment with the NullxNullxCD3 control, with the exception of the highest concentrations (mainly in the presence of the CARNAVAL cell line). No cytotoxicity wasinduced in the presence of the TAA-negative cell line K562. These results demonstrate that CD79bxCD20xCD3 trispecific antibody can induce antigen-specific cytotoxicity of tumor cell lines expressing a broad range of target antigens.Table 36. CD79bxCD20xCD3 trispecific antibody-induced cytotoxicity of antigen-positive endogenous models: ECx and max cytotoxicity values from T-cell cytotoxicity assays at 72 hours.4PL, 4-parameter logistic; CD, cluster of differentiation; ECx, x% effective concentration; max, maximal; NLME, non-linear mixed effects; SE, standard error. Values are representative of 3 to 4 individual healthy donors. The data that presented a sigmoidal pattern were modeled using a NLME model. The model had the 4 parameters of the standard 4PL regression model as the fixed effects (minimum, maximum, logEC50, and slope) and donor as a random effect. EC 20, EC50, and EC90 values were estimated using post-hoc estimation from the fitted model parameters. All analyses were performed in R Version 4.3.0.* Conditions with large estimates of SE. Note: as the dose range had to be adjusted to fit the full dose range for CD79bxCD20xCD3 trispecific antibody, EC50 estimates for the Reference Trispecific Antibody could not be reliably calculated.T-cell activation

[0285] To assess the effect of CD79bxCD20xCD3 trispecific antibody (C923B387) on T-cell activation, the expression of the early (CD69, data not shown) and late activation marker(CD25) was monitored in the cytotoxicity assays above. T-cell activation was assessed by flow cytometry at 72 hours upon coculture with a panel of antigen-positive or antigen-negative cancer cell lines at an E:T ratio of 1 : 1. After 72 hours of treatment of healthy-donor pan-T cells with CD79bxCD20xCD3 trispecific antibody, a concentration-dependent T-cell activation was induced in both CD4+and CD8+T-cell subsets. T-cell activation was determined using FlowJo (vlO) for both CD41(not shown) and CD81T-cell subpopulations. The expression (%) of late T-cell activation marker CD25 was used to identify the activated T cells. Graphing of data was done in GraphPad Prism 10. Data from 3 to 4 independent experiments were pooled and represented as mean ± SEM (FIG. 11 A-FIG. 1 IE, Table 37).

[0286] CD25 expression on T cells could only be detected in the presence of antigenexpressing cells, and no activation was induced upon coculture with the antigen-negative K562 model, thus demonstrating the antigen specificity of its activity. In co-culture with WILL-2 cancer cells, despite the no / low amount of cancer cell cytotoxicity observed with both the Reference Trispecific Antibody and CD79bxCD20xCD3 trispecific antibody, a robust induction of CD25 expression was detected. T-cell activation in this case could be amplified by the coculture time (72 hours), allowing a large time window for interaction. Similar to the activity seen in cytotoxicity assays, CD79bxCD20xCD3 trispecific antibody treatment led to higher levels of T-cell activation when compared to the Reference Trispecific Antibody. Again, the negative control antibody NullxNullxCD3 showed signs of activation only at the highest concentration, correlating with the previously observed increase in cancer cell cytotoxicity.Table 37. CD79bxCD20xCD3 trispecific antibody (C923B387) induced T-cell activation: ECx and maximal values from T-cell cytotoxicity assays with different cancer cell lines at 72 hours.4PL, 4-parameter logistic; CD, cluster of differentiation; ECx, x% effective concentration; SE, standard error. Values are representative of 3 to 4 individual healthy donors The data that presented a sigmoidal pattern were modeled using a non-linear mixed effects (NLME) model. The model had the 4 parameters of the standard 4PL regression model as the fixed effects (minimum, maximum, logECso, and slope) and donor as a random effect. EC20, EC50, and EC90 values were estimated using post-hoc estimation from the fitted model parameters. All analyses were performed in R Version 4.3.0. * Conditions with large estimates of SEEXAMPLE 5: T-CELL-MEDIATED TUMOR GROWTH INHIBITION OF B-CELL LYMPHOMA XENOGRAFTS IN VIVO

[0287] The antitumor efficacy of CD79bxCD20xCD3 trispecific antibody (C923B387) was evaluated in SC human ABC DLBCL OCI-LylO in T-cell-humanized mice. For OCI-LylO, xenografts female non-obese diabetic (NOD) severe combined immunodeficiency (scid) or NOD.Cg(NSG) mice (Charles River Labs, Lyon, France) were used to provide a suitable host for reconstituting a human immune system using human donor CD3+ pan-T cells. T-cell-humanized mice were given Fc block and intravenous immunoglobulin (IVIG) intraperitoneally (IP) at least 30 minutes prior to Reference Tri specific Antibody or CD79bxCD20xCD3 trispecific antibody dosing to correct for the low Ig environment in NSG or NSGMHC Eli dKO mice. The tolerability of CD79bxCD20xCD3 trispecific antibody could not be assessed with respect to CD20 or CD79b binding to host tissues due to the lack of crossreactivity to corresponding mouse antigens. In all studies, treatment with CD79bxCD20xCD3 trispecific antibody did not cause body weight loss or other signs of toxicity. No signs of graft-versus-host disease (GvHD)-related morbidity due to the humanization with T cells was observed in these studies.

[0288] CD79bxCD20xCD3 trispecific antibody was compared to the Reference Trispecific Ab in the study. NSG mice were randomized into groups of 9 animals with starting tumor volumes averaging 102 mm3, humanized with pan-T cells on Day 13, and treated with Reference Trispecific Antibody or CD79bxCD20xCD3 trispecific antibody IP starting on Day 14 at 0.3 and 1 mg / kg twice weekly for a total of 10 doses. Percent A tumor growth inhibition (TGI) of OCI-Ly 10 xenografts was calculated on Day 35 post tumor implantation, when at least two thirds of animals in all groups remained on study. Statistically significant ATGI of 60.2% and 94.5%, respectively, as compared with Dulbecco’s phosphate-buffered saline (DPBS)-treated control animals was observed with Reference Trispecific Antibody at 0.3 and 1 mg / kg (FIG. 12). More profound significant antitumor effects were observed upon treatment with CD79bxCD20xCD3 trispecific antibody at 0.3 and 1 mg / kg resulting in 105% and 106% ATGI, respectively, as compared to DPBS-treated animals. Tumor regression (TR) as compared to initial tumor burden of OCI-Ly 10 xenografts was evaluated on Day 48 post tumor cell implantation resulting in 48.2% TR in animals treated with Reference Trispecific Antibody at 1 mg / kg, with 4 of 9 partial regressions (PRs) and 3 of 9 complete regressions (CRs). Treatment with CD79bxCD20xCD3 trispecific antibody at 0.3 and 1 mg / kg resulted in 100% and 100%> TR, respectively, with CRs in all animals showing superior antitumor effects over Reference Trispecific Antibody.

[0289] In dose escalation efficacy study, NSG MHC I / I I dKO mice were randomized into groups of 10 animals and humanized with T cells at 13 days post OCI-LylO cell implantation with starting tumor volumes averaging 107 mm3. On Day 14, IP treatment with CD79bxCD20xCD3 trispecific antibody at 0.05, 0.15, and 0.5 mg / kg was initiated twice weekly for a total of 14, 14, and 10 doses, respectively. In the CD79bxCD20xCD3 trispecific antibody 0.5 mg / kg treatment group, 1 animal showed no antitumor response due to limited T-cell engraftment and was removed from the study. Percent ATGI of OCI-LylO xenografts was calculated on Day 30 post tumor implantation, when at least two thirds of animals in all groups remained on study. Upon treatment with CD79bxCD20xCD3 trispecific antibody at 0.05, 0.15, and 0.5 mg / kg, statistically significant ATGI of 37.2%, 106%, and 107%, respectively, ascompared with vehicle-treated controls (DPBS) was observed (FIG. 13). TR as compared to initial tumor burden of OCI-LylO xenografts was evaluated on Day 62 and resulted in 100% and 100% TR in animals treated with CD79bxCD20xCD3 trispecific antibody at 0.15 and 0.5 mg / kg, with 10 of 10 and 9 of 9 CRs, respectively. Dosing with CD79bxCD20xCD3 trispecific antibody at 0.05 mg / kg resulted in 1 of 10 PR and 3 of 10 CRs. Taken together, these results indicate that the minimal efficacious dose for CD79bxCD20xCD3 trispecific antibody is 0.15 mg / kg in this model.EXAMPLE 6: BIOPHYSICAL ASSESSEMENT OF CD79BXCD20XCD3 TRISPECIFIC ANTIBODY

[0290] Expression of CD79bxCD20xCD3 trispecific antibody (C923B387) was compared to Reference Trispecific Antibody. The CD79bxCD20xCD3 trispecific antibody C923B387 was expressed in ExpiCHO cells and purified. Purity was assessed by size exclusion chromatography (SEC) and mass spectrometry (MS). CD79bxCD20xCD3 trispecific antibody C923B387 gave higher yields than Reference Trispecific Antibody (Table 38).Table 38. Expression yields of CD79bxCD20xCD3 trispecific antibody C923B387 compared to Reference Trispecific Antibody.*Only one step purification protocol needed to achieve high purity**Multiple step purification required to achieve high purity

[0291] The stability of CD79bxCD20xCD3 trispecific antibody (C923B387) and Reference CD79bxCD20xCD3 trispecific Ab was also evaluated after incubation of the antibodies under high concentrations and stress conditions at high concentration.CD79bxCD20xCD3 trispecific antibody (C923B387) and Reference Trispecific Ab were concentrated to ~ 162 mg / ml in 10 mM Histidine buffer at pH 6.5 and 10 mM Histidine buffer at pH 6.5 with 0.04% PS20, respectively and incubated for two weeks at 4° Celsius or 40°Celsius. The antibodies were then analyzed by analytical SEC (aSEC). CD79bxCD20xCD3 trispecific antibody C923B387 showed higher stability at both 4° C and 40°C compared to Reference Trispecifc Antibody.Table 39. Stability of CD79bxCD20xCD3 trispecific antibody C923B387 at high concentration compared to Reference Trispecific Antibody.*In histidine pH 6.0**In histidine pH 6.0 with 0.04% PS20aSEC, analytical Size Exclusion ChromatographyEXAMPLE 7: STRUCTURE AND ASSESSMENT OE CD79BXCD20XCD3 TRISPECIFCI ANTIBODY C923B413

[0292] The Fc domain of the C923B387 trispecific antibody was modified to comprise a proline to alanine mutation (P374A) to further address the risk of generating multiple mispaired species that are difficult to separate. The amino acid sequences of the C923B413 trispecific antibody generated are shown in Table 40.Table 40. Amino acid sequences of full-length CD79bxCD20xCD3 antibody C923B413

[0293] To validate its cytotoxic potential, CD79bxCD20xCD3 trispecific antibody (C923413) was compared to CD79bxCD20xCD3 trispecific antibody (C923B387) and Reference Trispecific antibody in CARNAVAL cells (a DLBCL cell line that endogenously express CD79b and CD20). Healthy human pan-T cells were used as a source of effector cells and cocultured at an E:T ratio of 1:1 or 1:3. T-cell-mediated cytotoxicity was assessed in the presence of CD79bxCD20xCD3 trispecific antibody (C923B387 or C923B413), or corresponding matched null control antibody. Treatment with CD79bxCD20xCD3 trispecific antibody C923B387 and C923B413 resulted in similar antigen-dependent cancer cell cytotoxicity (FIG. 14A and 14B).

[0294] To compare expression of CD79bxCD20xCD3 trispecific antibody (C923B413) and CD79bxCD20xCD3 trispecific antibody (C923B387), the two antibodies were expressed in ExpiCHO cells at different transfection ratios (HCEHC2) as indicated in FIG. 15. The two antibodies showed similar expression profiles in ExpiCHO cells (FIG. 15).EXAMPLE 8: CD79BXCD20XCD3 TRISPECIFIC ANTIBODY (C923B387) CYTOTOXICITY AND CYTOKINE SECRETION IN WHOLE BLOOD T-CELL COCULTUREASSAY

[0295] To evaluate the impact of CD79bxCD20xCD3 trispecific antibody (C923B387) in a more physiologically relevant setting, T-cell co-culture assays were conducted using healthy human whole blood co-cultured with fluorescently-labeled CD79b+ / CD20+ CARNAVAL tumor cells and primary B cells at a 1:1 E:T ratio. C923B387 elicited concentration-dependent cytotoxicity of CARNAVAL cells, reaching an averaged maximal cytotoxicity of -90% after 72 hours (FIG. 7A, Table 41). Autologous B-cell cytotoxicity was also induced in a dose-dependent manner, with maximal levels of cytotoxicity at 72 hours of -40% (FIG. 16B). Treatment with a germline NullxNullxCD3 control antibody did not result in T-cell cytotoxicity at any concentration evaluated. Similar experiments were performed with Reference Trispecific Antibody and EC50 values are shown in Table 41.Table 41 : ECx (nM) and Maximal % Cytotoxicity Values for C923B387 and Reference Trispecific Antibody Observed on CARNAVAL cells in a Whole-blood Assay at 48 and 72 Hours.Values are representative of 8 individual healthy donors. Data from independent experiments were pooled and represented as means. A nonlinear regression model was used for ECx and maximum cytotoxicity value estimation across the donors. Model-estimate values are reported.

[0296] To characterize the effect of C923B387 on T-cell-mediated cytokine release, cytokine levels were measured using the MSD Proinflammatory Panel 1 in the supernatants of the whole-blood co-culture collected at 48 hours. A concentration-dependent induction of major pro-inflammatory cytokines was observed for all the treatments, with exception for the NullxNullxCD3 antibody. Consistent with the activity on T-cell activation, C923B387 T-cell engagement resulted in similar levels of cytokine production, while reduced cytokines levels were detected upon Reference Trispecific Antibody treatment (see FIG. 17A-17H).EXAMPLE 9: PHARMACOKINETICS

[0297] Serum PK parameters of CD79bxCD20xCD3 trispecific antibody (C923B387) were characterized in male cynomolgus monkeys (n=3 / group) following 0.2 and 1 mg / kg IV injection administered as a solution at 1 mL / kg. All animals had quantifiable serum exposures of CD79bxCD20xCD3 trispecific antibody (C923B387) up to 10 days after dosing and concentrations beyond 10 days were below the LLOQ in most samples, indicating likely ADA impact. Following single IV dosing of CD79bxCD20xCD3 trispecific antibody (C923B387) at 0.2 and 1 mg / kg, serum exposure increased more than dose-proportionally, suggestive of target-mediated drug disposition. The mean Cmax was 3.84 and 30.4 pg / mL with a mean AUCo-7d of 8.84 and 88.3 pg*day / mL, respectively, at 0.2 and 1 mg / kg (Table 42).Table 42: PK Parameters of CD79bxCD20xCD3 trispecific antibody (C923B387) following a Single IV Dose at 0.2 and 1 mg / kg in Male Cynomolgus MonkeysEXAMPLE 10: A PHASE 1 FIRST IN HUMAN STUDY OF CD79BXCD20XCD3 TRISPECIFIC ANTIBODY IN B-CELL NON-HODGKIN LYMPHOMA MALIGNANCIES

[0298] CD79bxCD20xCD3 trispecific antibody (C923B387) is a novel, fully human IgGl trispecific T-cell engaging antibody that binds to CD79b and CD20 on the surface of B cells and the CD3 receptor on the surface of T cells. CD79bxCD20><CD3 is being developed for the treatment of B-cell non-Hodgkin lymphoid malignancies (NHL).

[0299] The primary objective is to characterize safety and to determine the recommended Phase 2 dose(s) (RP2D[s]) and recommended Phase 2 regimens (RP2R[s]) of CD79bxCD20xCD3 trispecific antibody in participants with B-cell NHL. Key secondary objectives are to assess pharmacokinetics (PK), immunogenicity, and clinical activity of CD79bxCD20xCD3 trispecific antibody in the participant population(s) identified.

[0300] This study will be conducted in 2 parts: Part 1 (dose escalation) followed by Part 2 (dose expansion). Part 1 is designed to determine the putative RP2D(s) and dose schedule(s) of CD79bxCD20xCD3 trispecific antibody as a single agent based on safety, PK, preliminary pharmacodynamics, and preliminary assessment of clinical activity. The RP2D(s) and dose schedule(s) determined in Part 1 may be studied in participants in Part 2 to furtherassess safety and clinical activity and, if necessary, to characterize PK / pharmacodynamics and clinical activity relationships in participants with B-cell NHL. The study will be initiated with biweekly treatment doses. Multiple dose levels and other dose schedules may be explored after approval by the Study Evaluation Team (SET).

[0301] The total number of participants enrolled in the study, including both in Part 1 and Part 2, is approximately 80 participants.

[0302] CD79bxCD20*CD3 trispecific antibody will be administered subcutaneously at a dose determined by the sponsor according to the dose escalation or cohort expansion strategy. Dose escalation will be initiated at a dose of 0.05 mg. Dosing will continue until disease progression or unacceptable toxicity.

[0303] The safety of CD79bxCD20xCD3 trispecific antibody will be assessed by adverse event (AE) monitoring, clinical laboratory tests, electrocardiograms (ECGs), vital sign measurements, physical examination findings, and Eastern Cooperative Oncology Group (ECOG) performance status score. The severity of AEs will be assessed using the National Cancer Institute Common Terminology Criteria for Adverse Events (NCLCTCAE) (Version 6.0) or American Society for Transplantation and Cellular Therapy (ASTCT) guidelines.Concomitant medication usage will be recorded.

[0304] Dose escalation or de-escalation decisions will be guided by Bayesian Optimal Interval Design with the intention to select an recommended Phase 2 dose (RP2D) for which less or equal than 28% of participants experience dose-limiting toxicities.Study Rationale

[0305] NHL is a heterogeneous group of malignancies, from B- or T-cell origin, accounting for about 4% of all malignancies in the United States. Approximately 80% of NHLs are derived from the B-cell lineage and express B-cell differentiation antigens, including CD 19, CD20, CD22, and CD79b. These surface antigens represent pivotal targets for current standard of care treatment. However, despite improvements in available therapies, B-cell NHLs carry a poor prognosis with 30% to 50% of DLBCL patients relapsing after initial therapy, and only 40% of patients with relapsed / refractory disease achieving long-term complete remission upon second- or later-line CD 19 CAR-T therapy.

[0306] Recent approvals of bispecific CD20*CD3 TCEs mosunetuzumab (LUNSUMIO), glofitamab (COLUMVI), and epcoritamab (EPKINLY) clinically validate immune cell engagement as a promising treatment strategy for B-cell malignancies; this treatment approach results in durable antitumor response in patients with indolent or aggressive lymphoma following relapse / progression on standard chemo- or immunotherapies. Despite these treatment advances, not all patients derive clinical benefit from bispecific TCEs, and others develop resistance after initial response resulting from mechanisms including, but not limited to TAA loss / escape. One strategy to circumvent bispecific TCE resistance and improve first generation TCE antitumor efficacy relies on simultaneous targeting of 2 TAAs with a trispecific TCE. New treatment options are needed to address the urgent unmet need of this growing population whose disease no longer responds to standard chemo- or immunotherapies.

[0307] This clinical study is designed to collect the necessary data (including, but not limited to, safety, PK, and biomarkers) to determine if CD79bxCD20xCD3 trispecific antibody is safe at the doses administered. The protocol outlines the potential risks associated with the administration of the study treatment and the mitigation strategies. Additional monitoring and mitigation strategies will be implemented as appropriate if unanticipated toxicities are observed.Objectives and endpoints

[0308] The overall primary objective of the study will be to characterize the safety and to determine the RP2D(s), Recommended Phase 2 Regiments (RP2R(s)) and optimal dosing schedule(s) of CD79bxCD20xCD3 trispecific antibody in participants with relapsed / refractory B-cell NHL (Table 43).Table 43.Dose Limiting Toxicology (DLT)

[0309] A DLT is defined as any of the toxicities listed below that occur during the DLT evaluation period. The DLT window will start at the time of first dose and will end prior to administration of the scheduled second target dose. In addition, the cumulative safety data (all AEs and applicable data) will be reviewed by the SET (study evaluation team) as part of the continued safety.

[0310] Criteria for DLT are outlined below:1. Generala. Any toxicity that would require discontinuation of treatment.b. Fatal toxicity.2. Non-hematologic toxicitya. Grade >3 toxicityb. Exceptions to Non-hematologic Toxicity Criteria:i. First occurrence of Grade 3 CRS (cytokine release syndrome) or sARR (systemic administration related reactions) after any dose in the DLT window that improves to Grade <2 within 72h.ii. TLS Grade <4 that recovers in <5 daysb.iii. Grade 3 constipation, vomiting, diarrhea that improves to Grade <2 within 72h with best supportive care.iv. Grade 3 fatigue, fever or nausea, that improves to Grade <2 within <7 days with best supportive care.v. Grade 3 AE (adverse event) that that after discussion and approval by the SET (study evaluation team) is deemed related to tumor flare or pseudoprogression. vi. Grade 3 hypertension that can be controlled by oral medical management and improve to Grade <2 within 72h.vii. Grade >3 ALT (alanine aminotransferase) or AST (aspartate aminotransferase) that resolves to Grade <1 or baseline within 7 days unless criteria for Hy’s Law are metc.viii. Grade >3 lipase or amylase increase not associated with clinical or radiological evidence of pancreatitis.ix. Grade >3 alkaline phosphatase, GGT, uric acid or electrolyte abnormalities that resolve spontaneously to Grade <2 or baseline within 7 days, or that respond to best supportive care.3. Hematologic Toxicityi. Grade 4 neutrophil count decrease for >7 days despite supportive care according to institutional standards.ii. Grade 4 febrile neutropenia.iii. Grade 3 febrile neutropenia that does not recover with best supportive care within 7 days.iv. Grade 4 platelet count decrease for >7 days or Grade >3 with Grade >2 bleeding.v. Grade 4 anemia.Study population

[0311] The inclusion and exclusion criteria for enrolling participants in this study are described below.Inclusion CriteriaAge1. Be >18 years of age (or the legal age of majority in the jurisdiction in which the study is taking place, whichever is greater) at the time of informed consent. Type of Participant and Disease Characteristics2. B-cell NHL according to WHO 2022 with relapsed or refractory disease and no other approved therapies available that would be more appropriate in the investigator’s judgment.Histologic documentation of DLBCL according to the WHO 2022 classification including high-Grade B-cell lymphoma and DLBCL arising from indolent lymphoma.• Participants must have received at least 2 prior lines of therapy including an aCD20 monoclonal antibody containing chemotherapy combination schedule.• Participants who have received at least one prior line of therapy but are not eligible or do not have access to standard second line therapies, such as CAR-T, will be allowed to enroll.Other B-cell NHL may be enrolled based on emerging data in specific cohorts as stipulated by SET.3. Participants must have the following hematologic values without transfusions or growth factors for at least 7 days prior to the first dose of study treatment:a. Hemoglobin >8 g / dLb. Platelets >75x 109 / L (in case of bone marrow involvement of disease >50X109 / L)c. Absolute neutrophil count >1x109 / L (in case of bone marrow involvement of disease >0.75xl09 / L).4. Participants must have the following chemistry values:a. AST and ALT <3xULN, or <5xULN if there is documented liver involvement by diseaseb. Serum total bilirubin <1.5xULN (participants with congenital non-hemolytic hyperbilirubinemia such as Gilbert’s disease can enroll if conjugated (direct) bilirubin is within normal limits)c. Calculated or measured eGFR >30 mL / min, using the MDRD equation.5. Have an ECOG performance status of 0 to 1.6. Participants must have measurable disease as defined by the disease criteria (Lugano criteria).Exclusion criteria

[0312] Any potential participant who meets any of the following criteria will be excluded from participating in the study:(1) Known CNS or leptomeningeal involvement.(2) Any of the following regarding prior therapies within stipulated days before the first dose of study treatmentwithin 14 days: Chemotherapy, targeted therapy, immunotherapy or radiotherapy. within 30 days: T-cell redirecting antibodies.within 90 days: Gene-modified adoptive cell therapy (eg, CAR modified T-cells, natural killer cells), Autologous stem cell transplant.within 180 days: Allogeneic stem cell transplant; must be stable off all immunosuppressant medications without signs of graft versus host disease for at least 28 days.(3) Prior solid-organ transplantation.(4) Malignancy diagnosis other than the disease under study within 1 year prior to the first dose of the study treatment; exceptions are squamous and basal cell carcinoma of the skin, carcinoma in situ of the cervix and any malignancy that is considered cured or has minimal risk of recurrence within 1 year of first dose of the study treatment in the opinion of both the investigator and sponsor’s medical monitor. Participant with concurrent malignancy (eg, lower Grade lymphoproliferative disorder) that are unlikely to interfere with any study endpoints may be allowed after discussion with the study responsible physician. Co-existence of 2 histologies of B-cell NHL is not excluded. (5) Use of >30 mg daily prednisone equivalents at the time of study treatment initiation. A short tapering course of corticosteroids (eg, 5 to 30 mg of prednisone equivalents for less than 21 days) is permitted if used to treat disease-related symptoms. If corticosteroids were used to treat immune-related AEs associated with prior therapy, >7 days must have elapsed since the last dose of corticosteroid.(6) Autoimmune or inflammatory disease requiring systemic steroids or other immunosuppressive agents (eg, methotrexate or tacrolimus) within 3 months prior to first dose of study treatment.(7) Toxicity from prior anti cancer therapy that has not resolved to baseline levels or to Grade <1 (except alopecia, vitiligo, peripheral neuropathy, or Grade <2 endocrinopathies that are stable on hormone replacement).(8) Participants with a known history of IEC-HS / HLH(9) Any of the following within 6 months prior to first dose of study treatment: severe or unstable angina, myocardial infarction, major thromboembolic events (eg, pulmonary embolism, cerebrovascular accident), clinically significant ventricular arrhythmias, heartfailure New York Heart Association functional classification Class III-IV, pericarditis, myocarditis, or endocarditis. Deep vein thrombosis is not exclusionary.(10) Uncontrolled (despite optimal treatment) and persistent hypertension (ie, on multiple and repeated measurements) defined as systolic blood pressure >160 mm Hg or diastolic blood pressure >100 mm Hg.(11) Corrected QTc intervals using Fridericia’s formula >480 milliseconds based on the average of triplicate ECG assessments performed in less than 4 (±2) minutes apart.(12) Clinically significant pulmonary compromised defined as the need for supplemental oxygen to maintain adequate oxygenation.(13) Evidence of clinically significant and / or symptomatic infection (viral, bacterial, or fungal) at the time of study treatment initiation. Anti-microbial treatment for infection must be discontinued at least 7 days before the first dose of study treatment.(14) Active Hepatitis B or C infection. Participants who test positive for Hepatitis C antibody with a detectable HCV RNA load are not eligible.(15) Human immunodeficiency virus-positive participants are not eligible if they meet any of the following:(a) Detectable viral load (ie, >50 copies / mL) at screening.(b) CD4+ count <300 cells / mm3 at screening.(c ) AIDS-defining opportunistic infection within 6 months of screening.(d) Not receiving HAART. Any changes in HAART due to resistance / progression should occur at least 3 months prior to screening. A change in HAART due to toxicity is allowed up to 4 weeks prior to screening.( e) Note: HAART that could interfere with study treatment is excluded (consult the sponsor for a review of medications prior to enrollment).(16) Known allergies, hypersensitivity, or intolerance to excipients of CD79bXCD20XCD3. (17) Trauma or major surgery within 28 days prior to the first dose of study treatment.(18) Any serious underlying medical or psychiatric condition (eg, alcohol or drug use), dementia or altered mental status; or any issue that would impair the ability of the participant to receive or tolerate the planned treatment at the study site, to understand informed consent, or that in the opinion of the investigator would contraindicate the participation in the study or confound the protocol-specified assessments or results of the study.(19) Vaccination with a live, attenuated vaccine within 4 weeks before the first administration of study treatment.(20) Body weight <40 kg at screening or at the time of the first administration of study treatment.

Claims

CLAIMSWhat is claimed is:

1. A trispecific antibody or a fragment thereof comprising a first binding domain that binds to CD79b, a second binding domain that binds to CD20, and a third binding domain that binds to CD3, wherein:a) the first antigen-binding domain comprises a HCDR1 having an amino acid sequence GASISSFY (SEQ ID NO: 32), a HCDR2 having an amino acid sequence ISPSGKT (SEQ ID NO: 33), a HCDR3 having an amino acid sequence ARGEYSGTYSYSFDV (SEQ ID NO: 34), a LCDR1 having an amino acid sequence ESLLDSEDGNTY (SEQ ID NO: 35), a LCDR2 having an amino acid sequence TLS, and a LCDR3 having an amino acid sequence MQRMEFPLT (SEQ ID NO: 6) ;b) the second antigen-binding domain comprises a HCDR1 having an amino acid sequence GFTFNDYA (SEQ ID NO: 46), a HCDR2 having an amino acid sequence ISWNSGSI (SEQ ID NO: 47), a HCDR3 having an amino acid sequence AKDIQYGNYYYGMDV (SEQ ID NO: 48), a LCDR1 having an amino acid sequence QSVSSY (SEQ ID NO: 49), a LCDR2 having an amino acid sequence DAS, and a LCDR3 having an amino acid sequence QQRSNWPIT (SEQ ID NO: 14); andc) the third antigen-binding domain comprises aHCDRl having an amino acid sequence GFTFSRYN (SEQ ID NO: 60), a HCDR2 having an amino acid sequence ISTSSNYI (SEQ ID NO: 61), a HCDR3 having an amino acid sequence TRGWGPFDY (SEQ ID NO: 62), a LCDR1 having an amino acid sequence QSIGTA (SEQ ID NO: 63), a LCDR2 having an amino acid sequence YAS, and a LCDR3 having an amino acid sequence QQSGSWPYT (SEQ ID NO: 22)and wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are according to the IMGT delineation.

2. The trispecific antibody or the fragment thereof of claim 1, wherein the first antigen-binding domain comprises a first heavy chain variable region (VH1) comprising an amino acid sequence which is at least 90%, or at least 95%, or at least 98%, at least 99%, or 100% identical to SEQ ID NO: 7, and a first light chain variable region (VL1) comprising an amino acid sequence which is at least 90%, or at least 95%, or at least 98%, at least 99%, or 100% identical to SEQ ID NO: 8.

3. The trispecific antibody or the fragment thereof of any one of claims 1-2, wherein the first antigen-binding domain comprises a VH1 comprising an amino acid sequence of SEQ ID NO: 7 and a VL1 comprising an amino acid sequence of SEQ ID NO: 8.

4. The trispecific antibody or the fragment thereof of any one of claims 1-3, wherein the second antigen-binding domain comprises a second heavy chain variable region (VH2) comprising an amino acid sequence which is at least 90%, or at least 95%, or at least 98%, at least 99%, or 100% identical to SEQ ID NO: 15, and a second light chain variable region (VL2) comprising an amino acid sequence which is at least 90%, or at least 95%, or at least 98%, at least 99%, or 100% identical to SEQ ID NO: 16.

5. The trispecific antibody or the fragment thereof of any one of claims 1-4, wherein the second antigen-binding domain comprises a VH2 comprising an amino acid sequence of SEQ ID NO: 15 and a VL2 comprising an amino acid sequence of SEQ ID NO: 16.

6. The trispecific antibody or the fragment thereof of any one of claims 1-5, wherein the third antigen-binding domain comprises a third heavy chain variable region (VH3) comprising an amino acid sequence which is at least 90%, or at least 95%, or at least 98%, at least 99%, or 100% identical to SEQ ID NO: 23, and a third light chain variable region (VL3) comprising an amino acid sequence which is at least 90%, or at least 95%, or at least 98%, at least 99%, or 100% identical to SEQ ID NO: 24.

7. The trispecific antibody or the fragment thereof of any one of claims 1-6, wherein the third antigen-binding domain comprises a VH3 comprising an amino acid sequence of SEQ ID NO: 23 and a VL3 comprising an amino acid sequence of SEQ ID NO: 24.

8. The trispecific antibody or the fragment thereof of any one of claims 1-7, whereina) the first antigen-binding domain comprises the VH1 having an amino acid sequence of SEQ ID NO: 7 and the VL1 having an amino acid sequence of SEQ ID NO: 8; b) the second antigen-binding domain comprises the VH2 having an amino acid sequence of SEQ ID NO: 15 and the VL2 having an amino acid sequence of SEQ ID NO: 16; andc) the third antigen-binding domain comprises the VH3 having an amino acid sequence of SEQ ID NO: 23 and the VL3 having an amino acid sequence of SEQ ID NO: 24.

9. The trispecific antibody or the fragment thereof of any one of claims 1-8, wherein the first antigen-binding domain comprises a diabody, a Fab, Fab’, a F(ab’)2, a variable fragment (Fv), a single chain variable fragment (scFv), a (ScFv)2, a Fd fragment, a single chain disulfide bond stabilized fragments, stapled scFv fragment (spFv), a disulfide stabilized Fv fragment (dsFv), or a disulfide stabilized diabody (ds diabody).

10. The trispecific antibody or the fragment thereof of claim 9, wherein the first antigen-binding domain is a Fab.

11. The tri specific antibody or the fragment thereof of any one of claims 1-8, wherein the second antigen-binding domain comprises a diabody, a Fab, Fab’, a F(ab’)2, a variable fragment (Fv), a single chain variable fragment (scFv), a (ScFv)2, a Fd fragment, a single chain disulfide bond stabilized fragments, stapled scFv fragment (spFv), a disulfide stabilized Fv fragment (dsFv), or a disulfide stabilized diabody (ds diabody).

12. The trispecific antibody or the fragment thereof of claim 11, wherein the second antigenbinding domain is a spFv.

13. The trispecific antibody or the fragment thereof of any one of claims 1-8, wherein the third antigen-binding domain comprises a diabody, a Fab, Fab’, a F(ab’)2, a variable fragment (Fv), a single chain variable fragment (scFv), a (ScFv)2, a Fd fragment, a single chain disulfide bond stabilized fragments, stapled scFv fragment (spFv), a disulfide stabilized Fv fragment (dsFv), or a disulfide stabilized diabody (ds diabody).

14. The trispecific antibody or the fragment thereof of claim 13, wherein the third antigenbinding domain is a spFv.

15. The trispecific antibody or the fragment thereof of any one of claims 1-14, wherein the first antigen binding domain is Fab, the second antigen binding domain is a spFv and the third antigen binding domain is a spFv.

16. The trispecific antibody or the fragment thereof of any one of claims 1-15, wherein the spFv of the second binding domain is in an “ VH2 -linker 1-VL2” orientation or in a “VL2-linkerl-VH2” orientation.

17. The trispecific antibody or the fragment thereof of any one of claims 1-16, wherein the spFv of the third binding domain is in an “VH3-linkerl-VL3” orientation or in a “VL3-linkerl-VH3” orientation.

18. The trispecific antibody or the fragment thereof of claim 16 or 17, wherein the linkerl is selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 72, 79, 80, 81 and 82.

19. The trispecific antibody or the fragment thereof of any one of claims 1-18, wherein the spFv of the second binding domain is in the “VL2-linkerl-VH2” orientation and wherein the linkerl has an amino acid sequence of SEQ ID NO: 72 (GGGSGGSGGCPPCGGSGG).

20. The trispecific antibody or the fragment thereof of any one of claims 1-19, wherein the spFv of the third binding domain is in the “VL3 -linker 1-VH3” orientation and the linkerl has an amino acid sequence of SEQ ID NO: 72 (GGGSGGSGGCPPCGGSGG).

21. The trispecific antibody or the fragment thereof of claim 19, wherein the spFv of the second binding domain comprises the amino acid sequence of SEQ ID NO: 70.

22. The trispecific antibody or the fragment thereof of claim 20, wherein the spFv of the third binding domain comprises the amino acid sequence of SEQ ID NO: 71.

23. The trispecific antibody or the fragment thereof of any one of claims 1-22, wherein the second binding domain comprising the VL2-linkerl-VH2 is linked to the N-terminus of the VH1 of the first binding domain in a VL2-linkerl-VH2-linker2-VHl orientation, and wherein the linker2 comprises the amino acid sequence of SEQ ID NO: 73 (GGGGSGGGGS).

24. The trispecific antibody or the fragment thereof of any one of claims 1-23, further comprises an IgG constant region or the fragment of an IgG constant region.

25. The trispecific antibody or the fragment thereof of claim 24, wherein the IgG constant region or the fragment of an IgG constant region is an IgGl, an IgG2, an IgG3, or an IgG4 isotype.

26. The trispecific antibody or the fragment thereof of claim 25, wherein the IgG constant region or the fragment of an IgG constant region is an IgGl.

27. The trispecific antibody or the fragment thereof of any one of claims 24-26, wherein the IgG constant region or the fragment of the IgG constant region comprises at least one mutation that results in reduced binding of the antibody or the fragment thereof to a Fey receptor (FcyR).

28. The trispecific antibody or the fragment thereof of claim 27, wherein the mutation that results in reduced binding of the antibody or the fragment thereof to the FcyR is selected from F234A / L235A, L234A / L235A, L234A / L235A / D265S, V234A / G237A / P238S / H268A / V309L / A330S / P331S, F234A / L235A, S228P / F234A / L235A, N297A, V234A / G237A, K214T / E233P / L234V / L235A / G236-deleted / A327G / P331 A / D365E / L358M, H268Q / V309L / A330S / P331S, S267E / L328F, L234F / L235E / D265A, L234AZL235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238S, and S228P / F234A / L235A / G236-deleted / G237A / P238S, wherein residue numbering is according to the EU index.

29. The trispecific antibody or the fragment thereof of claim 28, wherein the mutation that results in reduced binding of the antibody or the fragment thereof to the FcyR are L234A / L235A / D265S.

30. The trispecific antibody or the fragment thereof of any one of claims 27-29, wherein the FcyR is FcyRI, FcyRIIA, FcyRIIB, FcyRIII, or any combination thereof.3E The trispecific antibody or the fragment thereof of any one of claims 24-30, wherein the IgG constant region or the fragment of the IgG constant region comprises the T366W, T366S, L368A, and Y407V mutations according to EU index numbering.

32. The trispecific antibody or the fragment thereof of claim 31, wherein the IgG constant region or the fragment of the IgG constant region further comprises the P374A mutation according to EU index numbering33. A trispecific antibody or a fragment thereof comprising a first binding domain that binds to CD79b, a second binding domain that binds to CD20, and a third binding domain that binds to CD3, wherein:a) the first antigen-binding domain comprises a VH1 having an amino acid sequence of SEQ ID NO: 7 and a VL1 having an amino acid sequence of SEQ ID NO: 8;b) the second antigen-binding domain comprises a VH2 having an amino acid sequence of SEQ ID NO: 15 and a VL2 having an amino acid sequence of SEQ ID NO: 16; andc) the third antigen-binding domain comprises a VH3 having an amino acid sequence of SEQ ID NO: 23 and a VL3 having an amino acid sequence of SEQ ID NO: 24.

34. A trispecific antibody or a fragment thereof comprising:a) a first heavy chain (HC1) comprising from N-terminus to C-terminus a VL3-linkerl- VH3-linker4-Fc, wherein the Fc comprises the CH2 and CH3 domain of a human IgGl constant region and the linker4 comprises the amino acid sequence of SEQ ID NO: 74 (EPKSSDKTHTCPPCP);b) a second heavy chain (HC2) comprising from N-terminus to C-terminus a VL2- linkerl-VH2-linker2-VHl-linker3-Fc, wherein the Fc comprises the CHI, CH2 and CH3 domain of a human IgGl constant region and the linker 3 comprises the amino acid sequence of SEQ ID NO: 75 (EPKSCDKTHTCPPCP); andc) a light chain (LC) comprising from N-terminus to C-terminus a VL1-CL, wherein the CL is the CL of a human kappa light chain.

35. The trispecific antibody or the fragment thereof of claim 34, wherein:the VH1 comprises an amino acid sequence of SEQ ID NO: 7 and the VL1 comprises an amino acid sequence of SEQ ID NO: 8;the VH2 comprises an amino acid sequence of SEQ ID NO: 15 and the VL2 comprises an amino acid sequence of SEQ ID NO: 16; andthe VH3 comprises an amino acid sequence of SEQ ID NO: 23 and the VL3 comprises an amino acid sequence of SEQ ID NO: 24.

36. The trispecific antibody or the fragment thereof of any one of claims 34-35, wherein the HC1 comprises an amino acid sequence at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 25.

37. The trispecific antibody or the fragment thereof of claim 36, wherein the HC1 comprises an amino acid sequence of SEQ ID NO: 25.

38. The trispecific antibody or the fragment thereof of any one of claims 34-37, wherein the HC2 comprises an amino acid sequence at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 26.

39. The trispecific antibody or the fragment thereof of claim 38, wherein the HC2 comprises an amino acid sequence of SEQ ID NO: 26.

40. The trispecific antibody or the fragment thereof of any one of claims 34-35, wherein the HC1 comprises an amino acid sequence at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 94.

41. The trispecific antibody or the fragment thereof of claim 40, wherein the HC1 comprises an amino acid sequence of SEQ ID NO: 94.

42. The trispecific antibody or the fragment thereof of claims 40 or 41, wherein the HC2 comprises an amino acid sequence at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 95.

43. The trispecific antibody or the fragment thereof of claim 42, wherein the HC2 comprises an amino acid sequence of SEQ ID NO: 95.

44. The trispecific antibody or the fragment thereof of any one of claims 34-43, wherein the LC comprises an amino acid sequence at least 90%, at least 95%, at least 98%, at least 99% or 100 % identical to SEQ ID NO: 27.

45. The trispecific antibody or the fragment thereof of claim 39, wherein the LC comprises an amino acid of SEQ ID NO: 27.

46. The trispecific antibody or the fragment thereof of any one of claims 34-39 and 44-45, wherein the trispecific antibody or the fragment thereof comprises the HC1 comprising an amino acid sequence of SEQ ID NO: 25, the HC2 comprising an amino acid sequence of SEQ ID NO: 26, and the LC comprising an amino acid sequence of SEQ ID NO: 27.

47. The trispecific antibody or the fragment thereof of any one of claims 34-35 and 40-45, wherein the trispecific antibody or the fragment thereof comprises the HC1 comprising an amino acid sequence of SEQ ID NO: 94, the HC2 comprising an amino acid sequence of SEQ ID NO: 95, and the LC comprising an amino acid sequence of SEQ ID NO: 27.

48. A trispecific antibody or a fragment thereof comprising first binding domain that binds to CD79b, a second binding domain that binds to CD20, and a third binding domain that binds to CD3, wherein:a) the first binding domain is a Fab comprising a VH1 and a VL1 having an amino acid sequence of SEQ ID NO: 7 and 8, respectively;b) the second binding domain is a spFv comprising a VL2 -linker 1-VH2, wherein VH2 and VL2 comprise an amino acid sequence of SEQ ID NO: 15 and 16, respectively, and wherein the linkerl comprises an amino acid sequence of SEQ ID NO: 72; andc) the third binding domain is a spFv comprising a VL3-linkerl-VH3, wherein VH3 and VL3 comprise an amino acid sequence of SEQ ID NO: 23 and 24, respectively, and wherein the linkerl comprises an amino acid sequence of SEQ ID NO: 72.

49. A trispecific antibody or a fragment thereof comprising first binding domain that binds to CD79b, a second binding domain that binds to CD20, and a third binding domain that binds to CD3, wherein:a) the first binding domain is a Fab comprising a VH1 and VL1 having an amino acid sequence of SEQ ID NO: 7 and 8, respectively;b) the second binding domain is a spFv having an amino acid sequence of SEQ ID NO: 70; andc) the third binding domain is a spFv having an amino acid sequence of SEQ ID NO: 71.

50. A trispecific antibody or a fragment thereof comprises a first heavy chain (HC1) comprising an amino acid sequence of SEQ ID NO: 25, a second heavy chain (HC2) comprising an amino acid sequence of SEQ ID NO: 26, and a light chain (LC) comprising an amino acid sequence of SEQ ID NO: 27.

51. A trispecific antibody or a fragment thereof comprises a first heavy chain (HC1) comprising an amino acid sequence of SEQ ID NO: 94, a second heavy chain (HC2) comprising an amino acid sequence of SEQ ID NO: 95, and a light chain (LC) comprising an amino acid sequence of SEQ ID NO: 27.

52. A polynucleotide encoding a trispecific antibody or a fragment thereof of any one of claims 1-51.

53. An expression vector comprising a polynucleotide of claim 52.

54. A host cell expressing a trispecific antibody or a fragment thereof of any one of claims 1-51.

55. A pharmaceutical composition comprising a trispecific antibody or a fragment thereof of any one of claims 1-51, a polynucleotide of claim 52, a vector of claim 53, or a host cell of claim 54 and a pharmaceutical carrier.

56. A method of treating cancer in a subject comprising administering to the subject a trispecific antibody or a fragment thereof of any one of claims 1-51, or the pharmaceutical composition of claim 55.

57. The method of claim 56, wherein the subject is a human subject.

58. The method of any one of claim 56 or 57, wherein the cancer is hematological cancer.

59. The method of any one of claims 56-58, wherein the cancer is B-cell lymphoma, nonHodgkin lymphoma, large B-cell lymphoma (LBCL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or Waldenstrom macroglobulinemia (WM).

60. The method of claim 59, wherein the cancer is relapsed, refractory, or malignant cancer, or any combination thereof.

61. A method of treating cancer in a subject comprising administering to the subject a pharmaceutical composition comprising a trispecific antibody or a fragment thereof comprising first binding domain that binds to CD79b, a second binding domain that binds to CD20, and a third binding domain that binds to CD3, wherein:a) the first binding domain is a Fab comprising a VH1 and a VL1 having an amino acid sequence of SEQ ID NO: 7 and 8, respectively;b) the second binding domain is a spFv comprising a VL2 -linker LVH2, wherein VH2 and VL2 comprise an amino acid sequence of SEQ ID NO: 15 and 16, respectively, and wherein the linkerl comprises an amino acid sequence of SEQ ID NO: 72; and c) the third binding domain is a spFv comprising a VL3 -linker 1-VH3, wherein VH3 and VL3 comprise an amino acid sequence of SEQ ID NO: 23 and 24, respectively, and wherein the linkerl comprises an amino acid sequence of SEQ ID NO: 72.

62. The method of claim 61, wherein the cancer is B-cell lymphoma, non-Hodgkin lymphoma, large B-cell lymphoma (LBCL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or Waldenstrom macroglobulinemia (WM).

63. A method of treating cancer in a subject comprising administering to the subject a pharmaceutical composition comprising a trispecific antibody or a fragment thereof comprising a first heavy chain (HC1) comprising an amino acid sequence of SEQ ID NO: 25, a second heavy chain (HC2) comprising an amino acid sequence of SEQ ID NO: 26, and a light chain (LC) comprising an amino acid sequence of SEQ ID NO: 27.

64. A method of treating cancer in a subject comprising administering to the subject a pharmaceutical composition comprising a trispecific antibody or a fragment thereof comprising a first heavy chain (HC1) comprising an amino acid sequence of SEQ ID NO: 94, a second heavy chain (HC2) comprising an amino acid sequence of SEQ ID NO: 95, and a light chain (LC) comprising an amino acid sequence of SEQ ID NO: 27.

65. The method of claim 63 and 64, wherein the cancer is B-cell lymphoma, non-Hodgkin lymphoma, large B-cell lymphoma (LBCL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or Waldenstrom macroglobulinemia (WM).

Images