Baffr×CD3 bispecific antibodies and methods of use

Abstract

To provide compositions useful for treating B cell lymphoma by using human BAFFR / human CD3 bispecific antibodies.SOLUTION: Disclosed herein is a bispecific antibody that specifically binds to human BAFFR (hBAFFR) and human CD3 (hCD3), where the bispecific antibody comprises a first antigen binding domain that specifically binds to hBAFFR and a second antigen binding domain that specifically binds to hCD3, where the first antigen binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1), where the VH1 comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and the VL1 comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, where HCDR1, HCDR2 and HCDR3 each comprise a specific amino acid sequence, and the LCDR1, LCDR2 and LCDR3 each comprise a specific amino acid sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to antibodies that specifically bind to human BAFFR, bispecific antibodies that specifically bind to both human BAFFR and human CD3, compositions comprising such antibodies, and methods of using such antibodies for the treatment of B-cell cancers and other diseases. [Background technology]

[0002] The number of newly diagnosed lymphoma and leukemia cases in the United States in 2024 is projected to be approximately 89,190 and 62,770, respectively (Siegel, R. et al., Cancer Statistics Jan 2024). There are currently five commercially available T Cell Engager (TCE) therapies to address some of these diseases: Blincyto®-CD19 / CD3, blinatumomab (Amgen), Lunsumio®-CD20 / CD3, mosunetuzumab (Genentech-Roche), Epkinly®-CD20 / CD3, epcolitamab (Genmab-Abbvie), Columvi®-CD20 / CD3, glofitamab (Roche), and Tecvayli®-BCMA / CD3, teclistamab (Janssen) (see Tapia-Galisteo, A. et al., J Hematol Oncol 2023).

[0003] One of the earliest approved TCE therapies is the CD19 / CD3 BiTE blinatumomab (Blincyto®), which is indicated for R / RB cell precursor acute lymphocytic leukemia (ALL) (see Tapia-Galisteo 2023). However, this therapy has drawbacks, such as toxicity and a short serum half-life. Severe cytokine release syndrome (CRS) and neurological adverse events are the main reasons for discontinuation of blinatumomab therapy, which are thought to be due to the aberrant activation of induced effector T cells and macrophages (see Jain, T. and Litzow, M., Ther Adv Hematol v.11 2020). Another limitation is that blinatumomab's short half-life requires continuous infusion over 6-8 weeks, which is a major obstacle to its clinical use; see Viardot A. et al., Ann Hematol. 2020 and Bukhari Al and Lee ST, Expert Rev Hematol. 2019.

[0004] Similarly, mosunetuzumab (Lunsumio®) is a CD20 / CD3 T cell-dependent bispecific antibody (TDB) approved for relapsed or refractory (R / R) follicular lymphoma (FL) in 2022. This TDB is a humanized IgG1-based bispecific antibody, generated using knobs-in-holes technology, and has a non-glycosylated, non-functional Fc domain. In non-human primates (NHPs), mosunetuzumab potently depleted B cells but exhibited pharmacokinetics similar to those of conventional antibodies. In a pivotal phase II study, mosunetuzumab showed CRS as the most common adverse event (AE) (44% of patients), although only two patients (2%) showed CRS of grade 3 or higher (Budde, L. et al., Lancet Oncol 2022).

[0005] Another challenge with both CD19 and CD20 therapy is the phenomenon of antigen loss after treatment, which can lead to relapse in patients (see Mejstrikova, E. et al., Blood Cancer J 2017 Dec;7(12):659). Approximately 20% of patients with B-cell acute lymphoblastic leukemia (B-ALL) experienced relapse after their last treatment with blinatumomab (see Zhou, T. and Wang, HW. Clin Lab Med 2021 Sep;41(3)). In a retrospective study, the authors investigated CD20 expression before, during, and at the time of progression in R / R NHL with mosunetuzumab in a phase 1 / 2 clinical trial; CD20 expression was prevalent in 95% of patients. Upon disease progression, 34% of relapsed patients showed CD20 loss (Schuster, S. et al., Blood 2024;143(9):822-832).

[0006] Thus, there is a need to identify alternative targets, improve efficacy, and alleviate CRS. The present disclosure represents an advancement in the art by providing compositions and methods useful in the treatment of B-cell cancers using human BAFFR / human CD3 bispecific antibodies. Summary of the Invention

[0007] The present invention describes bispecific T cell engager (TCE) antibodies for the treatment of various B cell cancers. One arm of the TCE specifically binds to human CD3, a human immune effector cell, and the other arm specifically binds to human BAFFR.

[0008] Compared to monoclonal antibodies, bispecific antibodies offer several advantages, such as a superior cytotoxic effect on cancer cells, due to a lower rate of resistance resulting from their ability to target two different antigens. Bispecific immune engager antibodies, such as those that target a general marker of T cell cluster of differentiation 3 (CD3) in one arm and a tumor-specific antigen in the other arm, are known as T cell engagers (TCEs). When both a T cell and a tumor cell bind via the TCE, a cytolytic synapse is formed, which activates the T cell and releases pore-forming perforin and cytotoxic granzyme-B, resulting in the death of the targeted tumor cell.

[0009] TCEs can be divided into two categories: immunoglobulin G (IgG)-based antibodies, such as Lunsumio®-mozunetuzumab, and variable fragment (Fv)-based bispecific antibodies, such as Blincyto®-blinatumomab. Bispecific antibodies based on IgG structures exhibit a structure similar to that of natural antibodies. Generally, compared with Fv-based bispecific antibodies, IgG-based bispecific antibodies have a longer in vivo half-life due to their larger size and therefore more difficult clearance by the kidney. In addition, the solubility and stability of IgG-based bispecific antibodies are improved over Fv-based bispecific antibodies due to the presence of a fragment crystallizable (Fc) domain. As defined herein, reference to the term TCE refers to an IgG-based TCE unless otherwise indicated.

[0010] Early methods for producing IgG-based TCEs involved combining half-molecules from heterologous parent antibodies. However, such techniques are prone to homodimer mismatching; for example, co-expressing two heavy chains and two light chains to produce an IgG TCE can result in some misassembly and unwanted by-products (Lewis SM et al., Nature Biotechnology 2014;32:191-202; Leaver-Fay A, et al., Structure 2016;24:641-651). Subsequent techniques to recombine functional half molecules to produce IgG-based TCEs include orthogonal Fab interfaces (Lewis, S. et al., Nature Biotech, 2014;32:191-198), DuoBody®-Genmab, XmAb®-Xencor, CrossMab (Schaefer, W. et al., PNAS 2011;108:11187-92), Knobs-into-Holes (KiH) (Ridgway, J. et al., Protein Eng. 1996;9:617-21 and Atwell, S. et al., J Mol Biol 1997;270:26-35), and Biclonics® (De Nardis, C. et al., JBC 2017;292(35):14706-14717), all of which increase the likelihood of proper heterodimerization.

[0011] Another challenge is that TCE requires the precise pairing of two distinct light chains. Otherwise, random light chain association results in a mixture of species, with correctly assembled antibodies occurring only 25% of the time. Because specific binding of antibodies to antigens is primarily provided by the heavy chain, such specific antigen binding can be preserved when such antibodies have non-cognate light chains. The use of a common light chain (cLC) combined with two distinct heavy chains avoids Fab mispairing. Therefore, only three peptide chains, rather than four, need to be expressed, which is a clear advantage in terms of manufacturability and simplified purification processes for typical biopharmaceutical production. Common light chain antibodies have been isolated from phage scFv display libraries with limited light chain diversity (Merchant et al., Nature Biotech, 1998, 16:677-681) or phage Fab libraries with unique light chains (Jackman et al., J Biol Chem 2010 Jul 2;285(27)). In an attempt to eliminate the engineering process for identifying cLCs, McWhirter et al. (WO 2011 / 097603) generated transgenic mice with fixed cLCs using human germline VK1-39JK5 or VK3-20JK light chains.

[0012] Thus, the present disclosure provides a human BAFFR / human CD3 bispecific TCE that facilitates proper heterodimerization assembly and exhibits in vivo efficacy in at least one preclinical model of B-cell cancer while minimizing CRS.

[0013] Cluster of differentiation 3 (CD3) is a protein complex and a T cell coreceptor involved in the activation of both cytotoxic T cells (naive CD8+ T cells) and T helper cells (naive CD4+ T cells). CD3 is composed of four distinct chains. In mammals, the complex contains the CD3γ chain (SwissProt P09693), the CD3δ chain (SwissProt P04234), and two CD3ε chains (SwissProt P07766). These chains associate with the T-cell receptor (TCR) and CD3 zeta (ζ chain) to generate activation signals in T lymphocytes. The TCR, CD3 zeta, and other CD3 molecules together comprise the TCR complex.

[0014] The B cell activating factor receptor (BAFF-R, also known as TNFRSF13C or CD268) is a member of the TNF receptor superfamily (TNFRSF) involved in B cell development and survival. BAFF-R expression is restricted to B cells and is found to be expressed at various stages of their maturation and differentiation, except during early B cell development.

[0015] The NCBI reference amino acid sequence for human BAFFR is NP 443177.1. BAFF-R has an extracellular domain (ECD) of 78 amino acids containing a single cysteine-rich domain (CRD) with four cysteine ​​residues. Two disulfide bridges formed by these four cysteine ​​residues are required for proper conformation of the CRD and, therefore, for ligand binding (Kim et al., Nat Structural Bio, 2003).

[0016] BAFF-R expression has been demonstrated in various human B-cell lymphomas, including pre-B ALL, CLL, and NHL, although information regarding expression levels is limited (Rodig et al. Human Pathol 2005, Parameswaran et al. Cancer Res 2010). Mechanistically, BAFF-R signaling activates the NF-κB pathway to promote tumor survival and proliferation, and increased BAFF-R expression correlates with disease progression in patients with B-cell lymphoma and pre-B ALL (Qin, H. et al., Sci Trans Med 2019 v.11, no. 511). Furthermore, mouse strains expressing mutant BAFF-R exhibit reduced B cell lifespan associated with a substantially reduced peripheral B cell compartment, and BAFF-R null mice exhibit greatly reduced B cell numbers and essentially lack marginal zone B cells (Hildebrand, J. et al., J Exp Med 2010, 207:2569-2579). Collectively, these reports suggest that BAFF-R signaling is a driver of B cell growth and survival. This characteristic may also limit the ability of B cell tumors to escape BAFFR-directed therapy by downregulating BAFF-R expression (Novak, A. et al., Blood 2004 104:2247-2253). BAFF-R expression is independent of CD19 expression on malignant B cells, which means that the observed downregulation of CD19 antigen on malignant B cells could still be targeted by BAFFR CAR-T cell therapy (Qin, H. et al., Sci Transl Med. 2019: Sep 25; 11(511)). [Brief explanation of the drawings]

[0017] [Figure 1A]Figure 1A shows the binding activity of hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE3, TCE4, and TCE5, as well as negative controls Control A, B, C, TTx38E4v1, and positive controls C90x38E4v1 and Lunsumio® to the human BAFFR-expressing Jeko-1 cell line, and the relative EC50 in nM for each antibody tested (Figure 1B). "NA" indicates that the antibody tested showed minimal binding, and thus a relative EC50 was not applicable. See Example 2. [Figure 1B] Figure 1A shows the binding activity of hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE3, TCE4, and TCE5, as well as negative controls Control A, B, C, TTx38E4v1, and positive controls C90x38E4v1 and Lunsumio® to the human BAFFR-expressing Jeko-1 cell line, and the relative EC50 in nM for each antibody tested (Figure 1B). "NA" indicates that the antibody tested showed minimal binding, and thus a relative EC50 was not applicable. See Example 2. [Figure 2A] Figure 2A shows the binding activity of the same hBAFFRxhCD3 bispecific antibodies and the same controls described in Figures 1A-1B to a human CD3-expressing Jurkat cell line, and shows the relative EC50 in nM for each antibody tested (Figure 2B). "NA" indicates that the antibody tested showed minimal binding, and therefore a relative EC50 was not applicable. See Example 2. [Figure 2B] Figure 2A shows the binding activity of the same hBAFFRxhCD3 bispecific antibodies and the same controls described in Figures 1A-1B to a human CD3-expressing Jurkat cell line, and shows the relative EC50 in nM for each antibody tested (Figure 2B). "NA" indicates that the antibody tested showed minimal binding, and therefore a relative EC50 was not applicable. See Example 2. [Figure 3A]ELISA results for binding activity of hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE3, TCE4, and TCE5, as well as negative controls Control A, B, C, TTx38E4v1, and positive controls C90x38E4v1 and Lunsumio® to human recombinant BAFFR-his (FIG. 3A), and the corresponding EC50 (nM) (FIG. 3B). "NA" indicates that the antibody tested showed minimal binding, and thus a relative EC50 was not applicable. See Example 3. [Figure 3B] ELISA results for binding activity of hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE3, TCE4, and TCE5, as well as negative controls Control A, B, C, TTx38E4v1, and positive controls C90x38E4v1 and Lunsumio® to human recombinant BAFFR-his (FIG. 3A), and the corresponding EC50 (nM) (FIG. 3B). "NA" indicates that the antibody tested showed minimal binding, and thus a relative EC50 was not applicable. See Example 3. [Figure 4A] ELISA results for the binding activity of the hBAFFR×hCD3 bispecific antibodies and controls described in FIGS. 3A-B to the human CD3 heterodimer εδ His ( FIG. 4A ) and the corresponding EC50 (nM) ( FIG. 4B ) are shown; “NC” indicates that the antibody tested was not saturated at the highest concentration, and thus an EC50 was not calculable. See Example 3. [Figure 4B] ELISA results for the binding activity of the hBAFFR×hCD3 bispecific antibodies and controls described in FIGS. 3A-B to the human CD3 heterodimer εδ His ( FIG. 4A ) and the corresponding EC50 (nM) ( FIG. 4B ) are shown; “NC” indicates that the antibody tested was not saturated at the highest concentration, and thus an EC50 was not calculable. See Example 3. [Figure 5A]ELISA results for binding activity of the hBAFFR×hCD3 bispecific antibodies and controls described above in FIGS. 3A-B to the human CD3 heterodimer εγ His (FIG. 5A) and the corresponding EC50 (nM) (FIG. 5B) are shown; "NC" indicates that the antibody tested was not saturated at the highest concentration, and thus an EC50 was not calculable. See Example 3. [Figure 5B] ELISA results for binding activity of the hBAFFR×hCD3 bispecific antibodies and controls described above in FIGS. 3A-B to the human CD3 heterodimer εγ His (FIG. 5A) and the corresponding EC50 (nM) (FIG. 5B) are shown; "NC" indicates that the antibody tested was not saturated at the highest concentration, and thus an EC50 was not calculable. See Example 3. [Figure 6]

[0033] Figure 1 shows intracellular antibody accumulation (IAA) of hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE3, TCE4, and TCE5, as well as negative controls Control A, B, and C, in BAFFR-expressing Jeko-1 cells at 24 hours. See Example 4. [Figure 7] Intracellular antibody accumulation (IAA) of the above hBAFFRxhCD3 bispecific antibodies and controls in Figure 6 is shown at 24 hours in CD3-expressing Jurkat cells. See Example 4. [Figure 8A] Figure 8A shows the relative levels of IFNγ (Figure 8A) and TNFα (Figure 8B) cytokine induction by hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5, and negative controls Controls A, B, and C, at 48 hours in BAFFR-expressing Jeko-1 cells co-cultured with a representative healthy donor PBMC. See Example 5. [Figure 8B]Figure 8A shows the relative levels of IFNγ (Figure 8A) and TNFα (Figure 8B) cytokine induction by hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5, and negative controls Controls A, B, and C, at 48 hours in BAFFR-expressing Jeko-1 cells co-cultured with a representative healthy donor PBMC. See Example 5. [Figure 9A] Figure 9 shows the relative levels of IFNγ (Figure 9A) and TNFα (Figure 9B) cytokine induction by the hBAFFRxhCD3 bispecific antibodies described in Figure 8 and controls at 48 hours in BAFFR-expressing Z-138 cells co-cultured with a representative healthy donor PBMC. See Example 5. [Figure 9B] Figure 9 shows the relative levels of IFNγ (Figure 9A) and TNFα (Figure 9B) cytokine induction by the hBAFFRxhCD3 bispecific antibodies described in Figure 8 and controls at 48 hours in BAFFR-expressing Z-138 cells co-cultured with a representative healthy donor PBMC. See Example 5. [Figure 10A] Figure 10 shows the relative levels of IFNγ (Figure 10A) and TNFα (Figure 10B) cytokine induction by the hBAFFRxhCD3 bispecific antibodies and controls described in Figure 8 at 48 hours in BAFFR-expressing Nalm-6 cells co-cultured with a representative healthy donor PBMC. See Example 5. [Figure 10B] Figure 10 shows the relative levels of IFNγ (Figure 10A) and TNFα (Figure 10B) cytokine induction by the hBAFFRxhCD3 bispecific antibodies and controls described in Figure 8 at 48 hours in BAFFR-expressing Nalm-6 cells co-cultured with a representative healthy donor PBMC. See Example 5. [Figure 11A] Figures 11A-11B show the relative levels of IFNγ (Figure 11A) and TNFα (Figure 11B) cytokine induction by the above hBAFFRxhCD3 bispecific antibodies and controls at 48 hours in BAFFR knockout in BAFFR-expressing Nalm-6 cells co-cultured with a representative healthy donor PBMC. See Example 5. [Figure 11B]Figures 11A-11B show the relative levels of IFNγ (Figure 11A) and TNFα (Figure 11B) cytokine induction by the above hBAFFRxhCD3 bispecific antibodies and controls at 48 hours in BAFFR knockout in BAFFR-expressing Nalm-6 cells co-cultured with a representative healthy donor PBMC. See Example 5. [Figure 12A] Figures 12A-C show T cell killing by hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5, as well as negative controls Control A, B, and C, in autologous B cells (PBMC donor RV504) at 48 hours. The positive control is Lunsumio® (Figure 12E), and the positive and negative controls C90x38E4v1 and TTx38E4v1 (Figure 12D). The relative EC50s for the study are summarized in Figure 12F, with "NA" indicating that the TCE or control tested showed minimal binding, and thus the relative EC50 was not applicable. See Example 6. [Figure 12B] Figures 12A-C show T cell killing by hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5, as well as negative controls Control A, B, and C, in autologous B cells (PBMC donor RV504) at 48 hours. The positive control is Lunsumio® (Figure 12E), and the positive and negative controls C90x38E4v1 and TTx38E4v1 (Figure 12D). The relative EC50s for the study are summarized in Figure 12F, with "NA" indicating that the TCE or control tested showed minimal binding, and thus the relative EC50 was not applicable. See Example 6. [Figure 12C]Figures 12A-C show T cell killing by hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5, as well as negative controls Control A, B, and C, in autologous B cells (PBMC donor RV504) at 48 hours. The positive control is Lunsumio® (Figure 12E), and the positive and negative controls C90x38E4v1 and TTx38E4v1 (Figure 12D). The relative EC50s for the study are summarized in Figure 12F, with "NA" indicating that the TCE or control tested showed minimal binding, and thus the relative EC50 was not applicable. See Example 6. [Figure 12D] Figures 12A-C show T cell killing by hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5, as well as negative controls Control A, B, and C, in autologous B cells (PBMC donor RV504) at 48 hours. The positive control is Lunsumio® (Figure 12E), and the positive and negative controls C90x38E4v1 and TTx38E4v1 (Figure 12D). The relative EC50s for the study are summarized in Figure 12F, with "NA" indicating that the TCE or control tested showed minimal binding, and thus the relative EC50 was not applicable. See Example 6. [Figure 12E] Figures 12A-C show T cell killing by hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5, as well as negative controls Control A, B, and C, in autologous B cells (PBMC donor RV504) at 48 hours. The positive control is Lunsumio® (Figure 12E), and the positive and negative controls C90x38E4v1 and TTx38E4v1 (Figure 12D). The relative EC50s for the study are summarized in Figure 12F, with "NA" indicating that the TCE or control tested showed minimal binding, and thus the relative EC50 was not applicable. See Example 6. [Figure 12F]Figures 12A-C show T cell killing by hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5, as well as negative controls Control A, B, and C, in autologous B cells (PBMC donor RV504) at 48 hours. The positive control is Lunsumio® (Figure 12E), and the positive and negative controls C90x38E4v1 and TTx38E4v1 (Figure 12D). The relative EC50s for the study are summarized in Figure 12F, with "NA" indicating that the TCE or control tested showed minimal binding, and thus the relative EC50 was not applicable. See Example 6. [Figure 13A] Figures 13A-C show T cell killing by hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5 at 48 hours in autologous B cells (PBMC donor #110044241). The positive control, Lunsumio® (Figure 13E), and the positive and negative controls, C90x38E4v1 and TTx38E4v1 (Figure 13D). The relative EC50s for the study are summarized in Figure 13F, with "NA" indicating that the TCE or control tested showed minimal binding, and thus the relative EC50 was not applicable. See Example 6. [Figure 13B] Figures 13A-C show T cell killing by hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5 at 48 hours in autologous B cells (PBMC donor #110044241). The positive control, Lunsumio® (Figure 13E), and the positive and negative controls, C90x38E4v1 and TTx38E4v1 (Figure 13D). The relative EC50s for the study are summarized in Figure 13F, with "NA" indicating that the TCE or control tested showed minimal binding, and thus the relative EC50 was not applicable. See Example 6. [Figure 13C]Figures 13A-C show T cell killing by hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5 at 48 hours in autologous B cells (PBMC donor #110044241). The positive control, Lunsumio® (Figure 13E), and the positive and negative controls, C90x38E4v1 and TTx38E4v1 (Figure 13D). The relative EC50s for the study are summarized in Figure 13F, with "NA" indicating that the TCE or control tested showed minimal binding, and thus the relative EC50 was not applicable. See Example 6. [Figure 13D] Figures 13A-C show T cell killing by hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5 at 48 hours in autologous B cells (PBMC donor #110044241). The positive control, Lunsumio® (Figure 13E), and the positive and negative controls, C90x38E4v1 and TTx38E4v1 (Figure 13D). The relative EC50s for the study are summarized in Figure 13F, with "NA" indicating that the TCE or control tested showed minimal binding, and thus the relative EC50 was not applicable. See Example 6. [Figure 13E] Figures 13A-C show T cell killing by hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5 at 48 hours in autologous B cells (PBMC donor #110044241). The positive control, Lunsumio® (Figure 13E), and the positive and negative controls, C90x38E4v1 and TTx38E4v1 (Figure 13D). The relative EC50s for the study are summarized in Figure 13F, with "NA" indicating that the TCE or control tested showed minimal binding, and thus the relative EC50 was not applicable. See Example 6. [Figure 13F]Figures 13A-C show T cell killing by hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5 at 48 hours in autologous B cells (PBMC donor #110044241). The positive control, Lunsumio® (Figure 13E), and the positive and negative controls, C90x38E4v1 and TTx38E4v1 (Figure 13D). The relative EC50s for the study are summarized in Figure 13F, with "NA" indicating that the TCE or control tested showed minimal binding, and thus the relative EC50 was not applicable. See Example 6. [Figure 14A] Figures 14A-C show T cell killing by hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5 at 48 hours in autologous B cells (PBMC donor #3464). The positive control, Lunsumio® (Figure 14E), and the positive and negative controls, C90x38E4v1 and TTx38E4v1 (Figure 14D). The relative EC50s for the study are summarized in Figure 14F, with "NA" indicating that the TCE or control tested showed minimal binding, and thus the relative EC50 was not applicable. See Example 6. [Figure 14B] Figures 14A-C show T cell killing by hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5 at 48 hours in autologous B cells (PBMC donor #3464). The positive control, Lunsumio® (Figure 14E), and the positive and negative controls, C90x38E4v1 and TTx38E4v1 (Figure 14D). The relative EC50s for the study are summarized in Figure 14F, with "NA" indicating that the TCE or control tested showed minimal binding, and thus the relative EC50 was not applicable. See Example 6. [Figure 14C]Figures 14A-C show T cell killing by hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5 at 48 hours in autologous B cells (PBMC donor #3464). The positive control, Lunsumio® (Figure 14E), and the positive and negative controls, C90x38E4v1 and TTx38E4v1 (Figure 14D). The relative EC50s for the study are summarized in Figure 14F, with "NA" indicating that the TCE or control tested showed minimal binding, and thus the relative EC50 was not applicable. See Example 6. [Figure 14D] Figures 14A-C show T cell killing by hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5 at 48 hours in autologous B cells (PBMC donor #3464). The positive control, Lunsumio® (Figure 14E), and the positive and negative controls, C90x38E4v1 and TTx38E4v1 (Figure 14D). The relative EC50s for the study are summarized in Figure 14F, with "NA" indicating that the TCE or control tested showed minimal binding, and thus the relative EC50 was not applicable. See Example 6. [Figure 14E] Figures 14A-C show T cell killing by hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5 at 48 hours in autologous B cells (PBMC donor #3464). The positive control, Lunsumio® (Figure 14E), and the positive and negative controls, C90x38E4v1 and TTx38E4v1 (Figure 14D). The relative EC50s for the study are summarized in Figure 14F, with "NA" indicating that the TCE or control tested showed minimal binding, and thus the relative EC50 was not applicable. See Example 6. [Figure 14F]Figures 14A-C show T cell killing by hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5 at 48 hours in autologous B cells (PBMC donor #3464). The positive control, Lunsumio® (Figure 14E), and the positive and negative controls, C90x38E4v1 and TTx38E4v1 (Figure 14D). The relative EC50s for the study are summarized in Figure 14F, with "NA" indicating that the TCE or control tested showed minimal binding, and thus the relative EC50 was not applicable. See Example 6. [Figure 15A] The ability of hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5, as well as positive controls Lunsumio® and C90x38E4v1, and negative controls Controls A, B, C, and TTx38E4v1 to activate T cells is shown at 24 hours using Jurkat-NFAT-RE luciferase reporter cells cultured with Jeko-1 cells (Figures 15A-B). The relative EC50 and maximum activity in RLU for this study are summarized in Figure 15C, with "NA" indicating that the antibody tested showed minimal binding, and thus the relative EC50 was not applicable. See Example 7. [Figure 15B] The ability of hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5, as well as positive controls Lunsumio® and C90x38E4v1, and negative controls Controls A, B, C, and TTx38E4v1 to activate T cells is shown at 24 hours using Jurkat-NFAT-RE luciferase reporter cells cultured with Jeko-1 cells (Figures 15A-B). The relative EC50 and maximum activity in RLU for this study are summarized in Figure 15C, with "NA" indicating that the antibody tested showed minimal binding, and thus the relative EC50 was not applicable. See Example 7. [Figure 15C]The ability of hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5, as well as positive controls Lunsumio® and C90x38E4v1, and negative controls Controls A, B, C, and TTx38E4v1 to activate T cells is shown at 24 hours using Jurkat-NFAT-RE luciferase reporter cells cultured with Jeko-1 cells (Figures 15A-B). The relative EC50 and maximum activity in RLU for this study are summarized in Figure 15C, with "NA" indicating that the antibody tested showed minimal binding, and thus the relative EC50 was not applicable. See Example 7. [Figure 16A] The ability of the hBAFFRxhCD3 bispecific antibody and controls described in Figure 15 to activate T cells is shown at 24 hours using Jurkat-NFAT-RE luciferase reporter cells cultured with Z-138 cells (Figures 16A-B). The relative EC50 and maximum activity in RLU from this study are summarized in Figure 16C, with "NA" indicating that the antibody tested showed minimal binding, and therefore the relative EC50 was not applicable. See Example 7. [Figure 16B] The ability of the hBAFFRxhCD3 bispecific antibody and controls described in Figure 15 to activate T cells is shown at 24 hours using Jurkat-NFAT-RE luciferase reporter cells cultured with Z-138 cells (Figures 16A-B). The relative EC50 and maximum activity in RLU from this study are summarized in Figure 16C, with "NA" indicating that the antibody tested showed minimal binding, and therefore the relative EC50 was not applicable. See Example 7. [Figure 16C]The ability of the hBAFFRxhCD3 bispecific antibody and controls described in Figure 15 to activate T cells is shown at 24 hours using Jurkat-NFAT-RE luciferase reporter cells cultured with Z-138 cells (Figures 16A-B). The relative EC50 and maximum activity in RLU from this study are summarized in Figure 16C, with "NA" indicating that the antibody tested showed minimal binding, and therefore the relative EC50 was not applicable. See Example 7. [Figure 17A] The ability of the hBAFFRxhCD3 bispecific antibody and controls described in Figure 15 to activate T cells is shown at 24 hours using Jurkat-NFAT-RE luciferase reporter cells cultured with Nalm-6 (DSMZ) cells (Figures 17A-B). The relative EC50 and maximum activity in RLU from this study are summarized in Figure 17C, with "NA" indicating that the antibody tested showed minimal binding, and therefore the relative EC50 was not applicable. See Example 7. [Figure 17B] The ability of the hBAFFRxhCD3 bispecific antibody and controls described in Figure 15 to activate T cells is shown at 24 hours using Jurkat-NFAT-RE luciferase reporter cells cultured with Nalm-6 (DSMZ) cells (Figures 17A-B). The relative EC50 and maximum activity in RLU from this study are summarized in Figure 17C, with "NA" indicating that the antibody tested showed minimal binding, and therefore the relative EC50 was not applicable. See Example 7. [Figure 17C] The ability of the hBAFFRxhCD3 bispecific antibody and controls described in Figure 15 to activate T cells is shown at 24 hours using Jurkat-NFAT-RE luciferase reporter cells cultured with Nalm-6 (DSMZ) cells (Figures 17A-B). The relative EC50 and maximum activity in RLU from this study are summarized in Figure 17C, with "NA" indicating that the antibody tested showed minimal binding, and therefore the relative EC50 was not applicable. See Example 7. [Figure 18-1]Figures 18A and 18C show CFSE-labeled isolated effector T cells co-cultured with PKH-26-labeled target Jeko-1 cells at a 5:1 E:T ratio in the presence of hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5, negative controls Control A, B, and C, and positive controls Mosunetuzumab and C90x38E4v1, for 48 hours (Figures 18A and 18C) or 96 hours (Figures 18B and 18D). Percent (%) activity data are presented as means with standard deviations. See Example 8. [Figure 18-2] Figures 18A and 18C show CFSE-labeled isolated effector T cells co-cultured with PKH-26-labeled target Jeko-1 cells at a 5:1 E:T ratio in the presence of hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5, negative controls Control A, B, and C, and positive controls Mosunetuzumab and C90x38E4v1, for 48 hours (Figures 18A and 18C) or 96 hours (Figures 18B and 18D). Percent (%) activity data are presented as means with standard deviations. See Example 8. [Figure 19-1] Figure 19 shows CFSE-labeled T cells co-cultured with Jeko-1 cells (E:T = 5:1) in the presence of hBAFFRxhCD3 bispecific antibodies and controls described in Figure 18 for 48 hours (Figures 19A, 19C, 19E, and 19G) or 96 hours (Figures 19B, 19D, 19F, and 19H). Percent proliferation was obtained from the CFSE population of live CD4+ (Figures 19A, 19B, 19E, and 19F) or CD8+ (Figures 19C, 19D, 19G, and 19H) T cells and plotted in GraphPad Prism. Data are presented as means with standard deviations. See Example 8. [Figure 19-2]Figure 19 shows CFSE-labeled T cells co-cultured with Jeko-1 cells (E:T = 5:1) in the presence of hBAFFRxhCD3 bispecific antibodies and controls described in Figure 18 for 48 hours (Figures 19A, 19C, 19E, and 19G) or 96 hours (Figures 19B, 19D, 19F, and 19H). Percent proliferation was obtained from the CFSE population of live CD4+ (Figures 19A, 19B, 19E, and 19F) or CD8+ (Figures 19C, 19D, 19G, and 19H) T cells and plotted in GraphPad Prism. Data are presented as means with standard deviations. See Example 8. [Figure 20-1] Figure 2 shows CFSE-labeled T cells co-cultured with Jeko-1 cells (E:T = 5:1) in the presence of hBAFFRxhCD3 bispecific antibodies and controls described in Figure 18 for 48 hours (Figures 20A, 20C, 20E, and 20G) or 96 hours (Figures 19C, 19D, 19G, and 19H). The percentage of the early T cell activation marker CD69 was obtained from live CD4+ (Figures 20A, 20B, 20E, and 20F) or CD8+ (Figures 20C, 20D, 20G, and 20H) T cells and plotted in GraphPad Prism. Data are presented as means with standard deviations. See Example 8. [Figure 20-2] Figure 2 shows CFSE-labeled T cells co-cultured with Jeko-1 cells (E:T = 5:1) in the presence of hBAFFRxhCD3 bispecific antibodies and controls described in Figure 18 for 48 hours (Figures 20A, 20C, 20E, and 20G) or 96 hours (Figures 19C, 19D, 19G, and 19H). The percentage of the early T cell activation marker CD69 was obtained from live CD4+ (Figures 20A, 20B, 20E, and 20F) or CD8+ (Figures 20C, 20D, 20G, and 20H) T cells and plotted in GraphPad Prism. Data are presented as means with standard deviations. See Example 8. [Figure 21-1]Figures 21A, 21C, 21E, and 21G show CFSE-labeled T cells co-cultured with Jeko-1 cells (E:T = 5:1) in the presence of the hBAFFRxhCD3 bispecific antibody and controls described in Figure 18 for 48 hours (Figures 21A, 21C, 21E, and 21G) or 96 hours (Figures 21C, 21D, 21G, and 21H). The percentage of the late T cell activation marker CD25 was obtained from live CD4+ (Figures 21A, 21B, 21E, and 21F) or CD8+ (Figures 21C, 21D, 21G, and 21H) T cells and plotted in GraphPad Prism. Data are presented as means with standard deviations. See Example 8. [Figure 21-2] Figures 21A, 21C, 21E, and 21G show CFSE-labeled T cells co-cultured with Jeko-1 cells (E:T = 5:1) in the presence of the hBAFFRxhCD3 bispecific antibody and controls described in Figure 18 for 48 hours (Figures 21A, 21C, 21E, and 21G) or 96 hours (Figures 21C, 21D, 21G, and 21H). The percentage of the late T cell activation marker CD25 was obtained from live CD4+ (Figures 21A, 21B, 21E, and 21F) or CD8+ (Figures 21C, 21D, 21G, and 21H) T cells and plotted in GraphPad Prism. Data are presented as means with standard deviations. See Example 8. [Figure 22A] Figure 22 shows the cytotoxic activity of hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5, as well as negative controls Controls A, B, and C, on highly hBAFFR-expressing Jeko-1 cells co-cultured with representative healthy donor PBMCs at effector-to-target ratios of 10:1 (Figure 22A) and 5:1 (Figure 22B) for 48 hours. Graphs show the mean values ​​of quadruplicate dose-response curves with standard deviations. See Example 9. [Figure 22B]Figure 22 shows the cytotoxic activity of hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5, as well as negative controls Controls A, B, and C, on highly hBAFFR-expressing Jeko-1 cells co-cultured with representative healthy donor PBMCs at effector-to-target ratios of 10:1 (Figure 22A) and 5:1 (Figure 22B) for 48 hours. Graphs show the mean values ​​of quadruplicate dose-response curves with standard deviations. See Example 9. [Figure 23A] Figure 23 shows the cytotoxic activity of the hBAFFRxhCD3 bispecific antibodies described in Figure 22 and controls on high hBAFFR-expressing Z-138 cells co-cultured with representative healthy donor PBMCs at effector-to-target ratios of 10:1 (Figure 23A) and 5:1 (Figure 23B) for 48 hours. Graphs show the mean values ​​of quadruplicate dose-response curves with standard deviations. See Example 9. [Figure 23B] Figure 23 shows the cytotoxic activity of the hBAFFRxhCD3 bispecific antibodies described in Figure 22 and controls on high hBAFFR-expressing Z-138 cells co-cultured with representative healthy donor PBMCs at effector-to-target ratios of 10:1 (Figure 23A) and 5:1 (Figure 23B) for 48 hours. Graphs show the mean values ​​of quadruplicate dose-response curves with standard deviations. See Example 9. [Figure 24A] Figure 24 shows the cytotoxic activity of the hBAFFRxhCD3 bispecific antibody described in Figure 22 and a control on low hBAFFR-expressing Nalm-6 cells co-cultured with representative healthy donor PBMCs at effector-to-target ratios of 10:1 (Figure 24A) and 5:1 (Figure 24B) for 48 hours. Graphs show the mean values ​​of quadruplicate dose-response curves with standard deviations. See Example 9. [Figure 24B] Figure 24 shows the cytotoxic activity of the hBAFFRxhCD3 bispecific antibody described in Figure 22 and a control on low hBAFFR-expressing Nalm-6 cells co-cultured with representative healthy donor PBMCs at effector-to-target ratios of 10:1 (Figure 24A) and 5:1 (Figure 24B) for 48 hours. Graphs show the mean values ​​of quadruplicate dose-response curves with standard deviations. See Example 9. [Figure 25A]Figure 25 shows the cytotoxic activity of hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5, as well as negative controls Controls A, B, and C, on BAFFR knockout Nalm-6 cells co-cultured with representative healthy donor PBMCs at effector-to-target ratios of 10:1 (Figure 25A) and 5:1 (Figure 25B) for 48 hours. Graphs show the mean values ​​of quadruplicate dose-response curves with standard deviations. See Example 9. [Figure 25B] Figure 25 shows the cytotoxic activity of hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5, as well as negative controls Controls A, B, and C, on BAFFR knockout Nalm-6 cells co-cultured with representative healthy donor PBMCs at effector-to-target ratios of 10:1 (Figure 25A) and 5:1 (Figure 25B) for 48 hours. Graphs show the mean values ​​of quadruplicate dose-response curves with standard deviations. See Example 9. [Figure 26] Figure 1 summarizes the % Maximum Activity and EC50 in nM of the cytotoxic activity of the tested hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5, and negative controls Control A, B, and C, on target cells Jeko-1, Z-138, or Nalm-6 co-cultured with representative healthy donor PBMCs at effector-to-target ratios of 10:1 and 5:1 for 48 hours. Maximum Activity (Max.act.) and Half Maximal Effective Concentration (EC50) were calculated in Graphpad Prism 10. See Example 9. [Figure 27A]Figure 27 shows 10-plex ex vivo human cytokine serum levels produced by humanized mice treated with hBAFFRxhCD3 bispecific antibodies in the Jeko-1 luciferase tumor model. Figure 27A shows, from top to bottom, hIFNg, hIL4, hIL8, and hIL22; Figure 27B shows, from top to bottom, hIL1b, hIL5, hIL10, and hTNFa; and Figure 27C shows, from top to bottom, hIL2, hIL6, and hIL12p70. Standard curves for each analyte were manually and minimally adjusted to ensure that TCE1, TCE2, TCE4, and TCE5, as well as the negative controls Controls A, B, and C, and the positive controls C90x38E4v1 and Lunsumio®, had curve fits close to 1 and error bars representing one standard deviation of the mean concentration per group. See Example 10. [Figure 27B] Figure 27 shows 10-plex ex vivo human cytokine serum levels produced by humanized mice treated with hBAFFRxhCD3 bispecific antibodies in the Jeko-1 luciferase tumor model. Figure 27A shows, from top to bottom, hIFNg, hIL4, hIL8, and hIL22; Figure 27B shows, from top to bottom, hIL1b, hIL5, hIL10, and hTNFa; and Figure 27C shows, from top to bottom, hIL2, hIL6, and hIL12p70. Standard curves for each analyte were manually and minimally adjusted to ensure that TCE1, TCE2, TCE4, and TCE5, as well as the negative controls Controls A, B, and C, and the positive controls C90x38E4v1 and Lunsumio®, had curve fits close to 1 and error bars representing one standard deviation of the mean concentration per group. See Example 10. [Figure 27C]Figure 27 shows 10-plex ex vivo human cytokine serum levels produced by humanized mice treated with hBAFFRxhCD3 bispecific antibodies in the Jeko-1 luciferase tumor model. Figure 27A shows, from top to bottom, hIFNg, hIL4, hIL8, and hIL22; Figure 27B shows, from top to bottom, hIL1b, hIL5, hIL10, and hTNFa; and Figure 27C shows, from top to bottom, hIL2, hIL6, and hIL12p70. Standard curves for each analyte were manually and minimally adjusted to ensure that TCE1, TCE2, TCE4, and TCE5, as well as the negative controls Controls A, B, and C, and the positive controls C90x38E4v1 and Lunsumio®, had curve fits close to 1 and error bars representing one standard deviation of the mean concentration per group. See Example 10. [Figure 28A] Figure 28 shows 10-plex ex vivo human cytokine levels produced by humanized mice treated with the hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5, negative controls Control A, B, and C, and positive controls C90x38E4v1 and Lunsumio® in the Z-138 xenograft tumor model, 2 hours after treatment. Figure 28A shows, from top to bottom, hIFNg, hIL4, hIL8, and hIL22; Figure 28B shows, from top to bottom, hIL1b, hIL5, hIL10, and hTNFa; and Figure 28C shows, from top to bottom, hIL2, hIL6, and hIL12p70. Standard curves for each analyte were manually and minimally adjusted to ensure a curve fit close to 1, and error bars representing one standard deviation of the mean concentration per group. See Example 11. [Figure 28B]Figure 28 shows 10-plex ex vivo human cytokine levels produced by humanized mice treated with the hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5, negative controls Control A, B, and C, and positive controls C90x38E4v1 and Lunsumio® in the Z-138 xenograft tumor model, 2 hours after treatment. Figure 28A shows, from top to bottom, hIFNg, hIL4, hIL8, and hIL22; Figure 28B shows, from top to bottom, hIL1b, hIL5, hIL10, and hTNFa; and Figure 28C shows, from top to bottom, hIL2, hIL6, and hIL12p70. Standard curves for each analyte were manually and minimally adjusted to ensure a curve fit close to 1, and error bars representing one standard deviation of the mean concentration per group. See Example 11. [Figure 28C] Figure 28 shows 10-plex ex vivo human cytokine levels produced by humanized mice treated with the hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5, negative controls Control A, B, and C, and positive controls C90x38E4v1 and Lunsumio® in the Z-138 xenograft tumor model, 2 hours after treatment. Figure 28A shows, from top to bottom, hIFNg, hIL4, hIL8, and hIL22; Figure 28B shows, from top to bottom, hIL1b, hIL5, hIL10, and hTNFa; and Figure 28C shows, from top to bottom, hIL2, hIL6, and hIL12p70. Standard curves for each analyte were manually and minimally adjusted to ensure a curve fit close to 1, and error bars representing one standard deviation of the mean concentration per group. See Example 11. [Figure 29A]Figure 29 shows 10-plex ex vivo human cytokine levels 24 hours after treatment produced by humanized mice treated with the hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5, negative controls Control A, B, and C, and positive controls C90x38E4v1 and Lunsumio® in the Z-138 xenograft tumor model. Figure 29A shows, from top to bottom, hIFNg, hIL4, hIL8, and hIL22; Figure 29B shows, from top to bottom, hIL1b, hIL5, hIL10, and hTNFa; and Figure 29C shows, from top to bottom, hIL2, hIL6, and hIL12p70. Standard curves for each analyte were manually and minimally adjusted to ensure a curve fit close to 1, and error bars representing one standard deviation of the mean concentration per group. See Example 11. [Figure 29B] Figure 29 shows 10-plex ex vivo human cytokine levels 24 hours after treatment produced by humanized mice treated with the hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5, negative controls Control A, B, and C, and positive controls C90x38E4v1 and Lunsumio® in the Z-138 xenograft tumor model. Figure 29A shows, from top to bottom, hIFNg, hIL4, hIL8, and hIL22; Figure 29B shows, from top to bottom, hIL1b, hIL5, hIL10, and hTNFa; and Figure 29C shows, from top to bottom, hIL2, hIL6, and hIL12p70. Standard curves for each analyte were manually and minimally adjusted to ensure a curve fit close to 1, and error bars representing one standard deviation of the mean concentration per group. See Example 11. [Figure 29C]Figure 29 shows 10-plex ex vivo human cytokine levels 24 hours after treatment produced by humanized mice treated with the hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5, negative controls Control A, B, and C, and positive controls C90x38E4v1 and Lunsumio® in the Z-138 xenograft tumor model. Figure 29A shows, from top to bottom, hIFNg, hIL4, hIL8, and hIL22; Figure 29B shows, from top to bottom, hIL1b, hIL5, hIL10, and hTNFa; and Figure 29C shows, from top to bottom, hIL2, hIL6, and hIL12p70. Standard curves for each analyte were manually and minimally adjusted to ensure a curve fit close to 1, and error bars representing one standard deviation of the mean concentration per group. See Example 11. [Figure 30] Tumor growth inhibition in an established Jeko-1 luciferase xenograft tumor model is shown via the kinetics of quantified luciferase signal over time, shown as the logarithm of total flux (p / sec) for hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5 dosed at either 0.15 mg / kg, 0.14 mg / kg, or 1.5 mg / kg on days 6, 13, and 20 as indicated by the arrows in the Jeko-1 luciferase-expressing xenograft tumor model, as well as negative controls Control A, B, and C, and positive controls C90x38E4v1 and Lunsumio®. Data represent the mean ± SEM for all groups (n=5). See Example 12. [Figure 31]Figure 1 shows in vivo tumor growth inhibition in the Z-138 xenograft tumor model using hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE4, and TCE5, negative controls Control A, B, and C, and positive controls C90x38E4v1 and Lunsumio® in an established humanized Z-138 xenograft model. Tumor cells and PBMCs were co-implanted on day 0, and dosing began on day 6 and was weekly, as indicated by the arrows. Tumor volumes are shown as geometric mean ± SEM, n=6 per group. See Example 13. [Figure 32]

[0033] Figure 1 shows in vivo antitumor efficacy in an immunocompetent syngeneic BAFFR+ tumor model using the hBAFFRxhCD3 bispecific antibody TCE1 at two doses and negative control A. Data represent the mean ± SEM for all groups (n=4). See Example 14. [Figure 33]

[0039] Figure 1 shows dose-dependent in vivo tumor growth inhibition in a patient-derived xenograft (PDX) model of B-ALL using the hBAFFRxhCD3 bispecific antibody TCE1 with negative controls (vehicle and control A) and the positive control mosunetuzumab. Data represent the mean ± SEM for all groups (n=6). See Example 15. DETAILED DESCRIPTION OF THE INVENTION

[0018] Embodiment Embodiments of the present invention are contemplated, including, but not limited to, the following.

[0019] Embodiment 1. A bispecific antibody that specifically binds to human B cell-activating factor receptor (hBAFFR) and human CD3 (hCD3), comprising: (a) a first antigen-binding domain that specifically binds to hBAFFR, the first antigen-binding domain comprising a first heavy chain variable region (VH1) and a first light chain variable region (VL1), wherein VH1 comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and VL1 comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3; (a) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6, or (b) a first antigen-binding domain, wherein HCDR1 comprises SEQ ID NO: 29, HCDR2 comprises SEQ ID NO: 30, HCDR3 comprises SEQ ID NO: 31, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6. and a second antigen-binding domain that specifically binds to human CD3.

[0020] Embodiment 2. The bispecific antibody of embodiment 1, wherein the second antigen-binding domain that specifically binds to hCD3 comprises a second heavy chain variable region (Second Heavy Chain Variable Region, VH2) and a second light chain variable region (Second Light Chain Variable Region, VL2), wherein VH2 comprises heavy chain complementarity determining regions (HCDRs) HCDR4, HCDR5, and HCDR6, and VL2 comprises light chain complementarity determining regions (LCDRs) LCDR4, LCDR5, and LCDR6.

[0021] Embodiment 3. A bispecific antibody that specifically binds to hBAFFR and hCD3, comprising: (a) a first antigen-binding domain that specifically binds to hBAFFR; and (b) a second antigen-binding domain that specifically binds to hCD3, the second antigen-binding domain comprising a heavy chain variable region (VH2) and a light chain variable region (VL2), wherein VH2 comprises heavy chain complementarity-determining regions (HCDRs) HCDR4, HCDR5, and HCDR6, and VL2 comprises light chain complementarity-determining regions (LCDRs) LCDR4, LCDR5, and LCDR6; a) HCDR4 comprises SEQ ID NO:7, HCDR5 comprises SEQ ID NO:8, HCDR6 comprises SEQ ID NO:9, LCDR4 comprises SEQ ID NO:4, LCDR5 comprises SEQ ID NO:5, and LCDR6 comprises SEQ ID NO:6; or b) HCDR4 comprises SEQ ID NO: 19, HCDR5 comprises SEQ ID NO: 20, HCDR6 comprises SEQ ID NO: 21, LCDR4 comprises SEQ ID NO: 4, LCDR5 comprises SEQ ID NO: 5, and LCDR6 comprises SEQ ID NO: 6, or c) a second antigen-binding domain, wherein HCDR4 comprises SEQ ID NO: 24, HCDR5 comprises SEQ ID NO: 25, HCDR6 comprises SEQ ID NO: 26, LCDR4 comprises SEQ ID NO: 4, LCDR5 comprises SEQ ID NO: 5, and LCDR6 comprises SEQ ID NO: 6.

[0022] Embodiment 4. The bispecific antibody of any one of Embodiments 1 to 3, wherein the first antigen-binding domain has a binding affinity (KD) for hBAFFR that is about 5 to about 50 times greater than the binding affinity for hCD3 of the second antigen-binding domain, as measured by surface plasmon resonance (SPR). In another embodiment, the first antigen-binding domain that binds to hBAFFR has a binding affinity (KD) to hBAFFR that is about 1.5 to about 2 times greater, or 2 to about 5 times greater, or about 5 to about 10 times greater, or about 5 to about 20 times greater, or about 5 to about 30 times greater, or about 5 to about 40 times greater, or about 5 to about 50 times greater, than the binding affinity to human CD3 of the second antigen-binding domain, as measured by surface plasmon resonance (SPR). 4. The hBAFFR×hCD3 bispecific antibody of any one of embodiments 1 to 3, having a binding affinity (KD), or about 10- to about 200-fold greater than the binding affinity (KD), or about 20- to about 200-fold greater than the binding affinity (KD), or about 30- to about 200-fold greater than the binding affinity (KD), or about 40- to about 200-fold greater than the binding affinity (KD), or about 50- to about 200-fold greater than the binding affinity (KD), or about 10- to about 100-fold greater than the binding affinity (KD), or about 10- to about 50-fold greater than the binding affinity (KD), or about 10- to about 20-fold greater than the binding affinity (KD).

[0023] Embodiment 5. The bispecific antibody of any one of Embodiments 1 to 4, wherein the bispecific antibody demonstrates in vivo tumor growth inhibition of at least 10% or more. In another embodiment, the hBAFFRxhCD3 bispecific antibody of any one of Embodiments 1 to 4 demonstrates in vivo tumor growth inhibition of at least 10% or more, at least 20% or more, at least 30% or more, at least 40% or more, at least 50% or more, at least 60% or more, at least 70% or more, or at least 80% or more. In yet another embodiment, the hBAFFRxhCD3 bispecific antibody described herein demonstrates in vivo tumor growth inhibition of 2% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100%.

[0024] Embodiment 6. The bispecific antibody of any one of embodiments 1 to 5, wherein the bispecific antibody induces an in vivo IFNg concentration of 6000 fg / ml or less. In another embodiment, the hBAFFRxhCD3 bispecific antibody of any one of embodiments 1 to 5 induces an in vivo IFNg concentration of 6000 fg / ml or less, or 5000 fg / ml or less, or 4000 fg / ml or less, or 3000 fg / ml or less, or 2000 fg / ml or less, or 1000 fg / ml or less, or 900 fg / ml or less, or 800 fg / ml or less, or 700 fg / ml or less, or 600 fg / ml or less, or 500 fg / ml or less, or 400 fg / ml or less, or 300 fg / ml or less, or 200 fg / ml or less, or 100 fg / ml or less, or 50 fg / ml or less. In another embodiment, the hBAFFRxhCD3 bispecific antibodies described herein induce in vivo IFNg concentrations of 50-100 fg / ml, 100-200 fg / ml, 200-300 fg / ml, 300-400 fg / ml, 400-500 fg / ml, 500-600 fg / ml, 600-700 fg / ml, 700-800 fg / ml, 800-900 fg / ml, 900-1000 fg / ml, 1000-2000 fg / ml, 2000-3000 fg / ml, 3000-4000 fg / ml, 4000-5000 fg / ml, or 5000-6000 fg / ml.

[0025] Embodiment 7. The bispecific antibody of any one of embodiments 1 to 6, wherein the bispecific antibody inhibits binding of hBAFFR to hBAFF.

[0026] Embodiment 8. The bispecific antibody of any one of embodiments 1 to 7, wherein the bispecific antibody inhibits antibody-induced shedding of the hBAFFR extracellular domain.

[0027] Embodiment 9. (a) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, LCDR3 comprises SEQ ID NO: 6, HCDR4 comprises SEQ ID NO: 7, HCDR5 comprises SEQ ID NO: 8, HCDR6 comprises SEQ ID NO: 9, LCDR4 comprises SEQ ID NO: 4, LCDR5 comprises SEQ ID NO: 5, and LCDR6 comprises SEQ ID NO: 6; or (b) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, LCDR3 comprises SEQ ID NO: 6, HCDR4 comprises SEQ ID NO: 19, HCDR5 comprises SEQ ID NO: 20, HCDR6 comprises SEQ ID NO: 21, LCDR4 comprises SEQ ID NO: 4, LCDR5 comprises SEQ ID NO: 5, and LCDR6 comprises SEQ ID NO: 6; or (c) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, LCDR3 comprises SEQ ID NO: 6, HCDR4 comprises SEQ ID NO: 24, HCDR5 comprises SEQ ID NO: 25, HCDR6 comprises SEQ ID NO: 26, LCDR4 comprises SEQ ID NO: 4, LCDR5 comprises SEQ ID NO: 5, and LCDR6 comprises SEQ ID NO: 6; or (d) HCDR1 comprises SEQ ID NO:29, HCDR2 comprises SEQ ID NO:30, HCDR3 comprises SEQ ID NO:31, LCDR1 comprises SEQ ID NO:4, LCDR2 comprises SEQ ID NO:5, LCDR3 comprises SEQ ID NO:6, HCDR4 comprises SEQ ID NO:7, HCDR5 comprises SEQ ID NO:8, HCDR6 comprises SEQ ID NO:9, LCDR4 comprises SEQ ID NO:4, LCDR5 comprises SEQ ID NO:5, and LCDR6 comprises SEQ ID NO:6; or (e) The bispecific antibody of embodiment 2, wherein HCDR1 comprises SEQ ID NO: 29, HCDR2 comprises SEQ ID NO: 30, HCDR3 comprises SEQ ID NO: 31, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, LCDR3 comprises SEQ ID NO: 6, HCDR4 comprises SEQ ID NO: 19, HCDR5 comprises SEQ ID NO: 20, HCDR6 comprises SEQ ID NO: 21, LCDR4 comprises SEQ ID NO: 4, LCDR5 comprises SEQ ID NO: 5, and LCDR6 comprises SEQ ID NO: 6.

[0028] Embodiment 10. (a) VH1 comprises SEQ ID NO: 10 and VL1 comprises SEQ ID NO: 12 wherein VH2 comprises SEQ ID NO: 11 and VL2 comprises SEQ ID NO: 12; or (b) VH1 comprises SEQ ID NO: 10 and VL1 comprises SEQ ID NO: 12 wherein VH2 comprises SEQ ID NO: 22 and VL2 comprises SEQ ID NO: 12; or (c) VH1 comprises SEQ ID NO: 10 and VL1 comprises SEQ ID NO: 12 wherein VH2 comprises SEQ ID NO: 27 and VL2 comprises SEQ ID NO: 12; or (d) VH1 comprises SEQ ID NO: 32 and VL1 comprises SEQ ID NO: 12 wherein VH2 comprises SEQ ID NO: 11 and VL2 comprises SEQ ID NO: 12; or (e) VH1 comprises SEQ ID NO: 32 and VL1 comprises SEQ ID NO: 12 10. The bispecific antibody of any one of embodiments 1 to 9, comprising: VH2 comprising SEQ ID NO: 22; and VL2 comprising SEQ ID NO: 12.

[0029] Embodiment 11. The bispecific antibody of any one of embodiments 1 to 10, wherein the bispecific antibody further comprises an Fc region, the Fc region comprising (a) a BAFFR heavy chain constant region comprising a first CH2 and a first CH3 domain, and (b) a CD3 heavy chain constant region comprising a second CH2 and a second CH3 domain, wherein the first CH3 domain has amino acid substitutions L351D and L368E, and the second CH3 domain has amino acid substitutions L351K and T366K. 11. The bispecific antibody of any one of embodiments 1 to 10, wherein the bispecific antibody further comprises an Fc region, the Fc region comprising (a) a BAFFR heavy chain constant region comprising a first CH2 and a first CH3 domain, and (b) a CD3 heavy chain constant region comprising a second CH2 and a second CH3 domain, wherein the first CH3 domain has amino acid substitutions L351K and T366K, and the second CH3 domain has amino acid substitutions L351D and L368E, and wherein mutation numbering is according to Kabat.

[0030] Embodiment 12. The bispecific antibody of any one of embodiments 1 to 10, wherein the bispecific antibody further comprises an Fc region, the Fc region comprising (a) a BAFFR heavy chain constant region comprising a first CH2 and a first CH3 domain, and (b) a CD3 heavy chain constant region comprising a second CH2 and a second CH3 domain, wherein the first CH3 domain has the amino acid substitutions T366S / L368A / Y407V and the second CH3 domain has the amino acid substitution T366W. 11. The bispecific antibody of any one of embodiments 1 to 10, wherein the bispecific antibody further comprises an Fc region, the Fc region comprising (a) a BAFFR heavy chain constant region comprising a first CH2 and a first CH3 domain, and (b) a CD3 heavy chain constant region comprising a second CH2 and a second CH3 domain, wherein the first CH3 domain has the amino acid substitution T366W and the second CH3 domain has the amino acid substitutions T366S / L368A / Y407V, and wherein the mutation numbering is according to Kabat.

[0031] Embodiment 13. The bispecific antibody of any one of embodiments 11 or 12, wherein the Fc region comprises an Fc-silencing mutation.

[0032] Embodiment 14. The bispecific antibody of embodiment 13, wherein the Fc silencing mutations are (a) L235G and G236R, or (b) L234A, L235A, or (c) L234A, L235A, and D265A, or (d) L234A, L235A, and P329G, or (e) N297A, or (f) N297A and K322A, or (g) L234A, L235A, and D265S, wherein the mutation numbering is according to Kabat.

[0033] Embodiment 15. The antibody comprises a first heavy chain (HC1) and a common light chain (cLC) that specifically binds to hBAFFR, and a second heavy chain (HC2) and the common light chain (cLC) that specifically binds to human CD3; (a) HC1 comprises SEQ ID NO: 16, HC2 comprises SEQ ID NO: 17, and cLC comprises SEQ ID NO: 18; or (b) HC1 comprises SEQ ID NO: 16, HC2 comprises SEQ ID NO: 23, and cLC comprises SEQ ID NO: 18; or (c) HC1 comprises SEQ ID NO: 16, HC2 comprises SEQ ID NO: 28, and cLC comprises SEQ ID NO: 18; or (d) HC1 comprises SEQ ID NO: 33, HC2 comprises SEQ ID NO: 17, and cLC comprises SEQ ID NO: 18; or (e) The bispecific antibody of any one of embodiments 1 to 14, wherein HC1 comprises SEQ ID NO: 33, HC2 comprises SEQ ID NO: 23, and cLC comprises SEQ ID NO: 18.

[0034] Embodiment 16. The bispecific antibody of any one of embodiments 1 to 15, wherein the antibody is of the human IgG1 or IgG4 isotype.

[0035] Embodiment 17. The bispecific antibody of embodiment 16, wherein the antibody is of the human IgG1 isotype.

[0036] Embodiment 18. A nucleic acid encoding the amino acid sequence of embodiment 15.

[0037] Embodiment 19. a) a first vector comprising a nucleic acid encoding SEQ ID NO: 16 and a second vector comprising a nucleic acid encoding SEQ ID NO: 17; b) a first vector comprising a nucleic acid encoding SEQ ID NO: 16 and a second vector comprising a nucleic acid encoding SEQ ID NO: 23; c) a first vector comprising a nucleic acid encoding SEQ ID NO: 16 and a second vector comprising a nucleic acid encoding SEQ ID NO: 28; d) a first vector comprising a nucleic acid encoding SEQ ID NO: 33 and a second vector comprising a nucleic acid encoding SEQ ID NO: 17; or A host cell transfected with (e) a first vector comprising a nucleic acid encoding SEQ ID NO: 33 and (j) a second vector comprising a nucleic acid encoding SEQ ID NO: 23.

[0038] Embodiment 20. The host cell of embodiment 19, further transfected with a third vector comprising a nucleic acid encoding SEQ ID NO: 18.

[0039] Embodiment 21 The host cell of embodiment 19, wherein the host cell is a mammalian host cell.

[0040] Embodiment 22. A process for producing a bispecific antibody, comprising culturing a cell according to embodiment 20 or 21 in a culture medium under conditions such that the bispecific antibody is expressed and then recovered from the culture medium.

[0041] Embodiment 23. A pharmaceutical composition comprising the hBAFFR×hCD3 bispecific antibody of any one of Embodiments 1 to 17 and 22, and a pharmaceutically acceptable excipient, diluent, or carrier.

[0042] Embodiment 24. An isolated antibody that specifically binds to hBAFFR, wherein the antibody comprises a heavy chain variable region (Heavy Chain Variable Region, VH) and a light chain variable region (Light Chain Variable Region, VL), wherein the VH comprises heavy chain complementarity determining regions (HCDRs): HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions (LCDRs): LCDR1, LCDR2, and LCDR3; (a) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6, or (b) An antibody in which HCDR1 comprises SEQ ID NO: 29, HCDR2 comprises SEQ ID NO: 30, HCDR3 comprises SEQ ID NO: 31, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6.

[0043] Embodiment 25. a) the VH comprises SEQ ID NO: 10 and the VL comprises SEQ ID NO: 12, or b) The antibody of embodiment 24, wherein the VH comprises SEQ ID NO: 32 and the VL comprises SEQ ID NO: 12.

[0044] Embodiment 26. The antibody comprises a heavy chain (HC) and a light chain (LC), a) HC comprises SEQ ID NO: 34 and LC comprises SEQ ID NO: 18, or b) An antibody according to embodiment 24 or 25, wherein the HC comprises SEQ ID NO: 35 and the LC comprises SEQ ID NO: 18.

[0045] Embodiment 27. The antibody of any one of embodiments 24 to 26, wherein the antibody is of the human IgG1 or IgG4 isotype.

[0046] Embodiment 28. The antibody of embodiment 27, wherein the antibody is of the human IgG1 isotype.

[0047] Embodiment 29. The antibody of any one of embodiments 24 to 28, wherein the antibody is an antibody fragment or an antigen-binding fragment.

[0048] Embodiment 30. The antibody of embodiment 29, wherein the antibody fragment or antigen-binding fragment is a Fab, Fab', F(ab')2, single-chain variable fragment (scFv), Fv, disulfide-linked Fv (sdFv), Fd fragment, or single-chain Fab (scFab).

[0049] Embodiment 31. The antibody of any one of embodiments 24 to 30, wherein the antibody is a multispecific antibody.

[0050] Embodiment 32. The antibody of embodiment 31, wherein the multispecific antibody is a bispecific antibody, or a trispecific antibody, or a tetraspecific antibody, or a diabody, or a tandem scFv, or a tandem VHH, or a tandem scFab.

[0051] Embodiment 33. An antibody-drug conjugate (ADC) comprising the antibody of any one of embodiments 24 to 32 and a drug moiety.

[0052] Embodiment 34. The ADC of embodiment 33, wherein the drug moiety is selected from the group consisting of an auristatin, an N-acetyl-gamma calicheamicin, a maytansinoid, a pyrrolobenzodiazepine, exatecan, and SN-38.

[0053] Embodiment 35: An immunocytokine comprising the antibody according to any one of embodiments 24 to 32 and a cytokine.

[0054] Embodiment 36. The immunocytokine of embodiment 35, wherein the cytokine is selected from the group consisting of IL-2, IL-4, IL-10, IL-12, IL-15, TNF, and IFNα.

[0055] Embodiment 37. A chimeric antigen receptor (CAR) comprising the antibody of any one of embodiments 24 to 32, a transmembrane domain, and an intracellular signaling domain.

[0056] Embodiment 38. A pharmaceutical composition comprising an hBAFFR antibody according to any one of embodiments 24 to 32, or an ADC according to any one of embodiments 33 to 34, or an immunocytokine according to any one of embodiments 35 to 36, or a CAR according to embodiment 37, and a pharmaceutically acceptable excipient, diluent, or carrier.

[0057] Embodiment 39. A method of treating a B-cell cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a bispecific antibody according to any one of embodiments 1 to 17 and 23, or an antibody according to any one of embodiments 24 to 32, or an ADC according to embodiments 33 to 34, or an immunocytokine according to embodiments 35 to 36, or a CAR according to embodiment 37.

[0058] Embodiment 40. The method of embodiment 39, wherein the B-cell cancer is relapsed or refractory.

[0059] Embodiment 41. The B-cell cancer is selected from the group consisting of B-cell acute lymphoblastic leukemia (B-ALL); Hodgkin lymphoma (HL); non-Hodgkin lymphoma (NHL); diffuse large B-cell lymphoma (DLBCL), a subtype of DLBCL, and primary mediastinal B-cell lymphoma; follicular lymphoma (FL); chronic lymphocytic leukemia (CLL); small lymphocytic lymphoma (SLL); mantle cell lymphoma (MCL); marginal zone lymphoma and extranodal marginal zone B-cell lymphoma (also known as mucosa-associated lymphoid tissue (MALT) lymphoma); mediastinal gray zone lymphoma Lymphoma (MGZL); Nodal Marginal Zone B-cell Lymphoma; Splenic Marginal Zone B-cell Lymphoma (SMZL); Splenic Diffuse Red Pulp Small B-cell Lymphoma (SDRPL); High-Grade B-Cell Lymphoma (HGBCL); Burkitt Lymphoma (BL) and Burkitt-Like Lymphoma (BLL); Lymphoplasmacytic Lymphoma (or Waldenstrom's Macroglobulinemia); B-cell Prolymphocytic Leukemia (B-PLL); Hairy Cell Leukemia (HCL); Multiple Myeloma (MM) Myeloma (MM): plasma cell neoplasm; primary central nervous system (CNS) lymphoma; and primary intraocular lymphoma.

[0060] Embodiment 42. A method of treating an autoimmune disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a bispecific antibody of any one of embodiments 1 to 17 and 23, or an antibody of any one of embodiments 24 to 32, or an ADC of embodiments 33 to 34, or an immunocytokine of embodiments 35 to 36, or a CAR of embodiment 37.

[0061] Embodiment 43. The method of claim 45, wherein the autoimmune disorder is Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE), Multiple Sclerosis (MS), Celiac Disease (CD), Type 1 Diabetes, Primary Sjogren's Syndrome (pSS), Guillain-Barré Syndrome, Inflammatory Bowel Disease (IBD), and psoriasis.

[0062] Embodiment 44. A bispecific antibody according to any one of embodiments 1 to 17 and 23, or an antibody according to any one of embodiments 24 to 32, or an ADC according to embodiments 33 to 34, or an immunocytokine according to embodiments 35 to 36, or a CAR according to embodiment 37, for use in the treatment of B-cell cancer.

[0063] Embodiment 45. The use of embodiment 44, wherein the B-cell cancer is relapsed or refractory.

[0064] Embodiment 46. The B-cell cancer is selected from the group consisting of B-cell acute lymphoblastic leukemia (B-ALL); Hodgkin's lymphoma (HL); non-Hodgkin's lymphoma (NHL); diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, which is a subtype of DLBCL; follicular lymphoma (FL); chronic lymphocytic leukemia (CLL); small lymphocytic lymphoma (SLL); mantle cell lymphoma (MCL); marginal zone lymphoma and extranodal marginal zone B-cell lymphoma (also known as mucosa-associated lymphoid tissue (MALT) lymphoma); mediastinal gray zone lymphoma (MGZL); nodal marginal zone The use of embodiment 44 or 45, wherein the tumor is B-cell lymphoma; splenic marginal zone B-cell lymphoma (SMZL); splenic diffuse small red pulp B-cell lymphoma (SDRPL); high-grade B-cell lymphoma (HGBCL); Burkitt's lymphoma (BL) and Burkitt-like lymphoma (BLL); lymphoplasmacytic lymphoma (or Waldenstrom's macroglobulinemia); B-cell prolymphocytic leukemia (B-PLL); hairy cell leukemia (HCL); multiple myeloma (MM): plasma cell neoplasm; primary central nervous system (CNS) lymphoma; and primary intraocular lymphoma.

[0065] Embodiment 47. A bispecific antibody according to any one of embodiments 1 to 17 and 23, or an antibody according to any one of embodiments 24 to 32, or an ADC according to embodiments 33 to 34, or an immunocytokine according to embodiments 35 to 36, or a CAR according to embodiment 37, for use in treating an autoimmune disorder.

[0066] Embodiment 48. The use of embodiment 47, wherein the autoimmune disorder is rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), celiac disease (CD), type 1 diabetes, primary Sjogren's syndrome (pSS), Guillain-Barré syndrome, inflammatory bowel disease (IBD), and psoriasis.

[0067] Embodiment 49. A pharmaceutical composition comprising a bispecific antibody according to any one of embodiments 1 to 17 and 23, or an antibody according to any one of embodiments 24 to 32, or an ADC according to embodiments 33 to 34, or an immunocytokine according to embodiments 35 to 36, or a CAR according to embodiment 37, for use in the treatment of B-cell cancer.

[0068] Embodiment 50. The pharmaceutical composition of embodiment 49, wherein the B-cell cancer is relapsed or refractory.

[0069] Embodiment 51. The B-cell cancer is selected from the group consisting of B-cell acute lymphoblastic leukemia (B-ALL); Hodgkin's lymphoma (HL); non-Hodgkin's lymphoma (NHL); diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, which is a subtype of DLBCL; follicular lymphoma (FL); chronic lymphocytic leukemia (CLL); small lymphocytic lymphoma (SLL); mantle cell lymphoma (MCL); marginal zone lymphoma and extranodal marginal zone B-cell lymphoma (also known as mucosa-associated lymphoid tissue (MALT) lymphoma); mediastinal gray zone lymphoma (MGZL); nodal marginal zone B-cell lymphoma. The pharmaceutical composition of embodiment 49 or 50, wherein the tumor is selected from the group consisting of: cell lymphoma; splenic marginal zone B-cell lymphoma (SMZL); splenic diffuse red pulp small B-cell lymphoma (SDRPL); high-grade B-cell lymphoma (HGBCL); Burkitt's lymphoma (BL) and Burkitt-like lymphoma (BLL); lymphoplasmacytic lymphoma (or Waldenstrom's macroglobulinemia); B-cell prolymphocytic leukemia (B-PLL); hairy cell leukemia (HCL); multiple myeloma (MM): plasma cell neoplasm; primary central nervous system (CNS) lymphoma; and primary intraocular lymphoma.

[0070] Embodiment 52. A pharmaceutical composition comprising a bispecific antibody according to any one of embodiments 1 to 17 and 23, or an antibody according to any one of embodiments 24 to 32, or an ADC according to embodiments 33 to 34, or an immunocytokine according to embodiments 35 to 36, or a CAR according to embodiment 37, for use in the treatment of an autoimmune disorder.

[0071] Embodiment 53. The pharmaceutical composition of embodiment 52, wherein the autoimmune disorder is rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), celiac disease (CD), type 1 diabetes, primary Sjogren's syndrome (pSS), Guillain-Barré syndrome, inflammatory bowel disease (IBD), and psoriasis.

[0072] Embodiment 54. Use of a bispecific antibody according to any one of embodiments 1 to 17 and 23, or an antibody according to any one of embodiments 24 to 32, or an ADC according to embodiments 33 to 34, or an immunocytokine according to embodiments 35 to 36, or a CAR according to embodiment 37, in the manufacture of a medicament for the treatment of B-cell cancer.

[0073] Embodiment 55. The use of embodiment 54, wherein the B-cell cancer is relapsed or refractory.

[0074] Embodiment 56. The B-cell cancer is selected from the group consisting of B-cell acute lymphoblastic leukemia (B-ALL); Hodgkin's lymphoma (HL); non-Hodgkin's lymphoma (NHL); diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, which is a subtype of DLBCL; follicular lymphoma (FL); chronic lymphocytic leukemia (CLL); small lymphocytic lymphoma (SLL); mantle cell lymphoma (MCL); marginal zone lymphoma and extranodal marginal zone B-cell lymphoma (also known as mucosa-associated lymphoid tissue (MALT) lymphoma); mediastinal gray zone lymphoma (MGZL); nodal marginal zone The use of embodiment 54 or 55, wherein the tumor is B-cell lymphoma; splenic marginal zone B-cell lymphoma (SMZL); splenic diffuse small red pulp B-cell lymphoma (SDRPL); high-grade B-cell lymphoma (HGBCL); Burkitt's lymphoma (BL) and Burkitt-like lymphoma (BLL); lymphoplasmacytic lymphoma (or Waldenstrom's macroglobulinemia); B-cell prolymphocytic leukemia (B-PLL); hairy cell leukemia (HCL); multiple myeloma (MM): plasma cell neoplasm; primary central nervous system (CNS) lymphoma; and primary intraocular lymphoma.

[0075] Embodiment 57. Use of a bispecific antibody according to any one of embodiments 1 to 17 and 23, or an antibody according to any one of embodiments 24 to 32, or an ADC according to embodiments 33 to 34, or an immunocytokine according to embodiments 35 to 36, or a CAR according to embodiment 37, in the manufacture of a medicament for the treatment of an autoimmune disorder.

[0076] Embodiment 58. The use of embodiment 57, wherein the autoimmune disorder is rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), celiac disease (CD), type 1 diabetes, primary Sjogren's syndrome (pSS), Guillain-Barré syndrome, inflammatory bowel disease (IBD), and psoriasis.

[0077] definition As used herein, the term "antibody" refers to an isolated immunoglobulin molecule that specifically binds to an antigen, e.g., a tumor antigen. Antibody embodiments include monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific or multispecific antibodies, or conjugated antibodies. Unless otherwise specified, the antibody can be of any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3, IgG4).

[0078] An exemplary bispecific antibody or T cell engager (TCE) of the present disclosure is an immunoglobulin G (IgG) type antibody, which is composed of four polypeptide chains: two heavy chains (HC) and two light chains, with two common LCs (cLCs) cross-linked via interchain disulfide bonds. The amino-terminal portion of each of the four polypeptide chains contains a variable region of about 100 to 125 amino acids or more that is primarily responsible for antigen recognition. The carboxy-terminal portion of each of the four polypeptide chains contains a constant region that is primarily responsible for effector function. Each heavy chain (HC) contains a heavy chain variable region (VH), a heavy chain constant region (C), and a heavy chain constant region (C). H1), hinge region, heavy chain constant region 2 (C H 2), and Heavy Chain Constant Region 3, C H IgG isotypes are further divided into subclasses (e.g., IgG1, IgG2, IgG3, and IgG4).

[0079] The VH and VL regions can be further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). The CDRs are exposed on the surface of the protein and are critical regions of the antibody for antigen-binding specificity. Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Herein, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and HCDR3," and the three CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3." The CDRs contain most of the residues that form specific interactions with the antigen.The assignment of amino acid residues to CDRs can be performed according to schemes well known to those skilled in the art, such as those of Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991)), Chothia (Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins", Journal of Molecular Biology, 196, 901-917 (1987), Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), North (North et al., "A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 406, 228-256 (2011)), or IMGT (the international (See, e.g., Lefranc et al., Nucleic Acids Res. 1999;27:209-212; ImMunoGeneTics database, available at www.imgt.org). Unless otherwise specified, the CDR sequences of this disclosure use the North numbering convention herein.

[0080] Antibody fragments or antigen-binding fragments are also contemplated and as used herein, an antibody fragment or antigen-binding fragment comprises at least a portion of an antibody that retains the ability to specifically interact with an antigen or an epitope of an antigen, e.g., Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, scFab, disulfide-linked Fvs (sdFv), Fd fragments.

[0081] As used herein, the term "antigen-binding domain" refers to a portion of an antibody, antibody fragment, bispecific antibody, multispecific binding protein, antibody-drug conjugate, immunocytokine, or chimeric antigen receptor (CAR) that specifically binds to an antigen or an epitope of an antigen.

[0082] As used herein, the term "bispecific" refers to a molecule that contains two different antigen-binding domains. A bispecific binding molecule can bind to two different antigens or two different epitopes of the same antigen. Exemplary embodiments of bispecific molecules include the TCEs disclosed herein.

[0083] As used herein, the term "multispecific" refers to a molecule comprising two or more different antigen-binding domains. A multispecific binding molecule can bind to two or more different antigens or two or more different epitopes of the same antigen. Exemplary embodiments of multispecific binding molecules include bispecific, trispecific, or tetraspecific binding molecules known in the art, and single-chain multispecific binding molecules, such as diabodies, tandem scFvs, tandem VHHs, or tandem scFabs.

[0084] As used herein, the term "antibody drug conjugate" (ADC) refers to a class of compounds that include (a) an antibody or antibody fragment, (2) a payload, e.g., a cytotoxic agent, immunomodulator, or therapeutic peptide, and (3) a linker that conjugates the antibody to the payload.

[0085] The term "chimeric antigen receptor" (CAR) refers to a recombinant polypeptide construct that comprises at least an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

[0086] As used herein, the term "Fc silencing mutation" includes mutations in the amino acid sequence of the Fc region of an antibody that result in human Fc gamma receptors on effector cells binding to the antibody. These mutations reduce or eliminate effector function while retaining binding to neonatal Fc receptors, which are important for normal antibody pharmacokinetics.

[0087] As used herein, the term "immunocytokine" refers to a molecule comprising an antigen-binding domain connected to a cytokine by chemical conjugation. In some embodiments, the antigen-binding domain is connected to the cytokine by fusion to the cytokine. The immunocytokine may further comprise an Fc domain connected to the antigen-binding domain.

[0088] As used interchangeably herein, the terms "nucleic acid" or "polynucleotide" refer to a polymer of nucleotides, including single- and / or double-stranded nucleotide-containing molecules, e.g., DNA, cDNA, and RNA molecules, that incorporate naturally occurring nucleotides, modified nucleotides, and / or nucleotide analogs. A polynucleotide of the present disclosure can also include substrates incorporated therein, for example, by a DNA or RNA polymerase or a synthetic reaction.

[0089] The polynucleotides of the present disclosure can be expressed in host cells, for example, after the polynucleotide is operably linked to an expression control sequence. Expression control sequences capable of expressing an operably linked polynucleotide are well known in the art. For example, an expression vector can include a sequence encoding one or more signal peptides that facilitate secretion of the polypeptide from a host cell. The signal peptide can be, for example, an immunoglobulin signal peptide or a heterologous signal peptide. An expression vector containing a polynucleotide of interest (e.g., a polynucleotide encoding an antibody polypeptide) can be transferred into host cells by well-known methods. Additionally, the expression vector can include one or more selectable markers, such as, for example, tetracycline, neomycin, and dihydrofolate reductase, to facilitate detection of host cells transformed with the desired polynucleotide sequence.

[0090] Host cells include cells stably or transiently transfected, transformed, transduced, or infected with one or more expression vectors expressing all or a portion of an antibody of the present disclosure. According to some embodiments, host cells may be stably or transiently transfected, transformed, transduced, or infected with an expression vector expressing the HC polypeptide and an expression vector expressing the LC polypeptide of an antibody of the present disclosure. In some embodiments, host cells may be stably or transiently transfected, transformed, transduced, or infected with expression vectors expressing the HC and LC polypeptides of an antibody of the present disclosure. The antibodies of the present disclosure may be produced in mammalian cells, such as CHO, NS0, HEK293, or COS cells, according to techniques well known in the art.

[0091] The medium into which the antibody of the present invention is secreted can be purified by conventional techniques, such as mixed-mode methods of ion exchange and hydrophobic interaction chromatography. For example, the medium can be applied to and eluted from a Protein A or Protein G column using conventional methods; mixed-mode methods of ion exchange and hydrophobic interaction chromatography can also be used. Soluble aggregates and multimers can be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The product can be immediately frozen, e.g., at -70°C, refrigerated, or lyophilized. Various methods of protein purification can be used, and such methods are known in the art and are described, for example, in Deutscher, Methods in Enzymology 182:83-89 (1990), and Scopes, Protein Purification: Principles and Practice, 3rd Edition, Springer, NY (1994).

[0092] As used herein, the terms "recurrence" or "recurrent" refer to when a disease, e.g., cancer, returns after successful treatment. A synonymous term for relapse is "recurrence."

[0093] As used herein, the term "refractory" refers to a disease, such as cancer, that is unable to respond to medical treatment from the beginning of treatment. A synonym for refractory is "resistance." Refractory diseases, such as refractory cancer, are different from recurrent diseases, such as recurrent cancer, as defined above.

[0094] As used herein, unless otherwise indicated, the terms "specifically bind" and "specifically binds" are intended to mean the ability of an antibody or molecule to form a chemical bond or attractive interaction with another protein or molecule, which chemical bond or attractive interaction brings the antibody into proximity with the other protein or molecule, as determined by common methods known in the art.

[0095] As used herein, the term "T cell engager" (TCE) refers to a specific type of bispecific antibody in which one antibody arm specifically binds to a tumor-associated antigen (TAA) and the other antibody arm specifically binds to CD3, thereby allowing the TCE to engage cytotoxic T cells via the CD3 arm and redirect the T cells to tumor cells expressing the TAA, whereupon the TCE forms a cytolytic synapse and the engaged T cells release pore-forming perforin and cytotoxic granzyme-B into the cytolytic synapse, resulting in the killing of the targeted tumor cells. In one embodiment, the TCE disclosed herein is a human BAFFR x human CD3 bispecific antibody of the invention. As used herein, the terms "BAFFR TCE" and "BAFFR x CD3 bispecific antibody" or "BAFFR x CD3 bispecific antibody" are synonymous.

[0096] As used herein, the term "treating" or "treating" in the context of a disease refers to reducing the severity and / or frequency of one or more symptoms, eliminating one or more symptoms and / or the underlying cause of said symptoms, reducing the frequency or likelihood of one or more symptoms and / or their underlying causes, delaying, preventing, and / or slowing the progression of the disease and / or disorder, and ameliorating or reversing damage caused directly or indirectly by the disease and / or disorder. For clarity, treating a disease does not include, and is distinct from, preventing the disease.

[0097] The sequences of the BAFFR antibodies and BAFFRxCD3 bispecific antibodies of the invention are numbered according to the sequence identification numbers listed in Tables 1 and 2, respectively. The sequences in both Tables 1 and 2 are amino acid sequences, and CDR sequences use the North numbering convention unless otherwise indicated (see North, B. et al., J Mol Biol. 2011 Feb 18;406(2):228-256).

[0098] [Table 1-1]

[0099] [Table 1-2]

[0100] [Table 2-1]

[0101] [Table 2-2]

[0102] [Table 2-3]

[0103] [Table 2-4]

[0104] [Table 2-5]

[0105] [Table 2-6]

[0106] [Table 2-7] [Example]

[0107] Example 1: Generation of BAFFRxCD3 bispecific antibodies Immunization of MeMo® mice A diverse panel of cLC-based BAFF-R-targeting monoclonal antibodies was generated by selecting and characterizing antigen-specific molecules from MeMo® mice, which generate single light chain or common light chain (cLC) fully human antibodies in combination with diverse heavy chains (see WO 2009 / 157771). MeMo® mice were immunized with BAFF-R antigenic moieties, and immunizations included the use of different forms of DNA, protein, and cell-based delivery, as appropriate.

[0108] A diverse panel of cLC-based human CD3 monoclonal antibodies was obtained using MeMo® mice (see, e.g., WO 2020 / 204708). MeMo® mice were immunized with BAFF-R antigenic moieties, and immunizations optionally included the use of different forms of DNA, protein, and cell-based delivery.

[0109] The BAFF-R and CD3 binding domain sequences herein, once characterized and sequenced via the techniques provided herein, can then be obtained by any method known in the art.

[0110] Production and screening of hBAFFR×hCD3 bispecific antibodies Fifteen hBAFFR arms were selected and paired with 23 hCD3 arms. The hBAFFR arms were cloned into an expression vector that generates specific mutations L351D and L368E in the CH3 region of the hBAFFR antibody, and the hCD3 arms were cloned into an expression vector that generates specific mutations L351K and T366K in the CH3 region of the hCD3 antibody, thereby enabling the two arms to heterodimerize properly.

[0111] The resulting 363 hBAFFRxhCD3 bispecific antibodies were screened for T cell activation using Jeko-1 and Jurkat-NFAT-RE reporter assays (Promega) according to the manufacturer's instructions, resulting in the selection of 64 hBAFFRxhCD3 bispecific antibodies. Subsequent screening for cytotoxicity and cytokine release in Jeko-1 and donor T cell co-culture assays was performed on the 64 bispecific antibodies, resulting in the selection of 12 based on cytotoxicity, affinity, and cytokine release. These 12 bispecific antibodies were then screened for cell binding and cytotoxicity and cytokine release in Jeko-1 or Z-138 cell line and donor PBMC co-culture assays, resulting in the selection of 6 bispecific antibodies.

[0112] Reformatting of hBAFFR×hCD3 Bispecific Antibody Six hBAFFRxhCD3 bispecific antibodies were reformatted from a DEKK backbone to a knobs-in-holes backbone, which contained a T366W substitution in the hCD3 arm ("knob") and T366S / L368A / Y407V in the hBAFFR arm ("hole"), and the Fc-silencing mutations LALADS, L234A / L235A / D265S, were introduced by conventional cloning techniques.

[0113] For many of the examples, the following TCEs and controls were used:

[0114] [Table 3]

[0115] Example 2: In vitro characterization of cell binding activity to BAFFR and CD3 This study was performed to test the cell binding activity of huBAFFRxhuCD3 bispecific antibodies (TCE1, TCE2, TCE3, TCE4, and TCE5) in the human BAFFR+ cell line Jeko-1 (ATCC CRL-3006) and the human CD3+ cell line Jurkat clone E6-1 (ATCC CRL-TIB-152). Controls used were: Control A, B, C, C90x38E4v1, TTx38E4v1, and Lunsumio®.

[0116] Jeko-1 and Jurkat cells were counted and resuspended in cold staining buffer with 20 μg of human Fc block. Cells were plated at 5×10 4Cells were seeded at 100 cells / well (volume 25 μl / well) and incubated on ice for 30 minutes. Titrated antibodies were added to the cells at a 1:1 volume and incubated on ice for 2 hours. All antibodies were diluted to a starting final concentration of 15 μg / ml or 100 nM except for TCE4, which was diluted to a starting final concentration of 13.7 μg / ml or 91.8 nM. The plate was centrifuged at 2000 rpm for 2 minutes at 4°C to pellet the cells. The cells were washed three times by centrifugation with 100 μl of staining buffer. A PE-conjugated mouse anti-human IgG secondary antibody was prepared at a 1:25 dilution in staining buffer. The cells were incubated with 50 μl / well of diluted secondary antibody on ice for 30 minutes. Zombie Violet™ viability dye was prepared at a 1:1000 dilution in PBS. Cells were resuspended in 50 μl / well of viability dye and incubated on ice for 20 minutes. Cells were washed once and resuspended in 100 μl of staining buffer for analysis on an Attune NxT flow cytometer.

[0117] Plates were run on an Attune NxT flow cytometer (ThermoScientific), and analysis was performed using FlowJo (version 10.9). Doublets and dead cells were excluded in the gating strategy in FlowJo. PE-MFI (geometric mean) was obtained for each sample based on singlets and live cells. The following gating strategy was used: cells (FSC-A vs. SSC-A) → single cells (SSC-A vs. SSC-H) → live cells gated on Zombie Violet™ negativity (SSC-A vs. Zombie Violet™) → PE-MFI (geometric mean). MFI was transferred to Microsoft Excel for further analysis. Background (buffer-only wells) MFI was subtracted from all wells. Normalization across all plates was performed using plate control wells, in which "maximum activity" wells contained 15 μg / ml C90x38E4v1 and "minimum activity" wells contained 15 μg / ml LSN3985061. Percent activity was calculated as % = ((sample MFI - minimum MFI) / (maximum MFI - minimum MFI)) x 100. Graph generation and relative EC50 calculations were performed using GraphPad Prism. Antibody concentrations (nM) were transformed to the logarithm 10 of the concentration. Curves were fitted by a nonlinear regression log (agonist) vs. response - variable slope (four parameters) model in GraphPad Prism.

[0118] Summary: All tested hBAFFRxhCD3 bispecific antibodies bind to human BAFF-R-expressing Jeko-1 cells. See Figure 1A. Bispecific antibodies (clone A or clone B) with the same BAFF-R arm had comparable relative EC50s (less than a 3-fold difference) on Jeko-1 cells, but different maximum binding values ​​were observed. See Figure 1B. CD3 binding on the surface of Jurkat cells was observed with all bispecific antibodies. See Figure 2A. TCE and controls A, B, and C, which have the same CD3 arm, had comparable relative EC50s on Jurkat cells. See Figure 2B.

[0119] Example 3: Evaluation of hBAFFR and hCD3 binding by ELISA This study was carried out to test the protein binding activity of hBAFFRxhCD3 bispecific antibodies TCE1, TCE2, TCE3, TCE4, and TCE5 with human BAFF-R recombinant protein, human CD3 heterodimer epsilon delta recombinant protein, and human CD3 heterodimer epsilon gamma recombinant protein in ELISA. Controls used: Control A, B, C, C90x38E4v1, TTx38E4v1, and Lunsumio®.

[0120] Immulon 4HBX 96-well plates were coated with 1 μg / ml of recombinant protein (in PBS) at a volume of 100 μl / well. The plates were sealed and incubated overnight at 4° C. on a plate shaker. The next day, the plates were washed three times with 1× PBS-Tween (containing 0.05% Tween) using a plate washer (ELx-405 wash program IMM_300_3). The plates were then blocked with 300 μl / well of blocking buffer (3% BSA in PBS) for 2 hours at room temperature with orbital shaking. Antibody dilutions in assay buffer (1% BSA in PBS) were prepared manually in 10-point, 3-fold dilutions with a starting concentration of 15 μg / ml or 100 nM (except for TCE4, for which starting concentrations of 13.7 μg / ml or 91.8 nM were used). Antibodies used for plate normalization were also prepared at 15 μg / ml. The blocking buffer was decanted, and antibody dilutions (including plate control or assay buffer wells) were added to the plate at 100 μl / well and incubated for 1 hour at room temperature with orbital shaking. Peroxidase-conjugated anti-human IgG F(ab')2 secondary antibody was prepared at a dilution of 1:10,000 in assay buffer. The plate was washed three times. Anti-human secondary antibody was added to the plate at 100 μl / well and incubated for 1 hour at room temperature with orbital shaking. The plate was then washed three times. TMB substrate was added to the plate at 100 μl / well for 5 minutes. 100 μl / well of stop solution was then added and the plate was read at 450 nm in a SpectraMax M5e plate reader using a SoftMax The readings were taken using the Pro program. Background (values ​​from assay buffer-only wells) was subtracted from all values. Normalization across the entire plate was performed using plate control wells, in which "maximum activity" wells contained 15 μg / ml C90×38E4v1 and "minimum activity" wells contained 15 μg / ml LSN3985061. Percent activity was calculated as % = ((sample value - minimum) / (maximum - minimum)) x 100. Graph generation and EC50 calculations were performed using GraphPad Prism.Titrated antibody concentrations (nM) were converted to the logarithm of the concentration. Curves were fitted by a nonlinear regression log(agonist) vs. response-variable slope (four parameters) model in Graphpad Prism.

[0121] Summary: All hBAFFRxhCD3 bispecific antibodies tested showed binding to human BAFFR recombinant protein. See Figure 3A. TCEs with the same BAFFR arms had very similar EC50s. See Figure 3B. CD3 binding activity of all bispecific antibodies was observed with both human CD3 heterodimeric epsilon delta and heterodimeric epsilon gamma recombinant proteins. See Figures 4A and 5A. TCEs with the same CD3 arms had comparable EC50s. See Figures 4B and 5B.

[0122] Example 4: In vitro characterization of bispecific antibody internalization This study was performed to evaluate the internalization of the hBAFFR x hCD3 bispecific antibodies TCE1, TCE2, TCE3, TCE4, and TCE5, as well as negative controls Control A, B, and C, in the BAFFR-expressing Jeko-1 cell line and the CD3-expressing Jurkat cell line. TCEs and negative controls were conjugated with Alexa Fluor 488 (AF488) by combining 0.5 mg of antibody in amine-free buffer with 1.25–2 μL of 10 mM AF488 NHS ester dissolved in DMSO and sodium bicarbonate (10% v / v) for 1 hour at room temperature, protected from light. The labeled TCEs were then transferred to a 30K MW protein concentrator. Excess dye was removed by adding PBS, and centrifugation was repeated at 14,000 RPM for 10 minutes at room temperature until the flow-through was colorless. The protein and label concentrations of each AF488-conjugated TCE were assessed using a NanoDrop spectrophotometer. The degree of labeling (DOL) was calculated by dividing the molar concentration of AF488 by the molar concentration of the protein.

[0123] Jeko-1 cells or CD3-expressing Jurkat cells, incubated with purified azide-free Fc Block according to the manufacturer's instructions, were added to a 96-well polypropylene plate at 50–100,000 cells per well in 50 μL of assay buffer. AF488-conjugated TCE was serially titrated to generate a 10-point, 4-fold dilution series with a starting concentration of 32 μg / mL in assay buffer. 50 μL was added to the cells in duplicate. The plate was covered with a breathable seal and incubated at 37°C for 24 hours. To terminate the assay, the plate was centrifuged at 800 × g for 2 minutes at 4°C, and the cell pellet was resuspended in cold FACS buffer. After two washes, the cells were resuspended in 1:1000 Zombie Violet™ Live / Dead dye in 1x PBS and incubated on ice for 20 minutes. Plates were washed again, and cells were resuspended in 1:50 anti-AF488 antibody in FACs buffer to quench the signal from surface-bound AF488-TCE. Samples were acquired on an Attune NxT flow cytometer with Quantum AF488 MESF beads from Bangs Laboratories according to the manufacturer's instructions.

[0124] Single live cells were identified using FlowJo version 10 software using sequential gating: FSC-A vs. SSC-A, FSC-H vs. FSC-A, and Zombie Violet™ Live / Dead dye exclusion. The geometric mean fluorescent intensity (gMFI) of AF488 in single live cells was then assessed. Using the bead run-specific gMFI values ​​and the MESF values ​​provided by the manufacturer, a log-log plot and linear fit model were generated in Graphpad Prism 10. This was used to interpolate sample MESF values, which were converted to intracellular antibody accumulation (IAA) by dividing by the DOL for each TCE.

[0125] Summary: Intracellular antibody accumulation (IAA) of hBAFFR×hCD3 bispecific antibodies at 24 hours in BAFFR-expressing Jeko-1 cells was consistent with the affinity of the BAFFR arms, with the BAFFR clone A bispecific antibody having a higher IAA than the BAFFR clone B bispecific antibody (Figure 6). In Jeko-1 cells treated with the CD3 negative control, little or no IAA was observed at 24 hours. See Figure 6. Additionally, IAA of hBAFFR×hCD3 bispecific antibodies at 24 hours in CD3-expressing Jurkat cells was observed at the highest concentrations of 27 nM and 107 nM (Figure 7). In addition, the IAA of the tested bispecific antibodies corresponded to the potency observed in the cytotoxicity assay (data not shown). Although some internalization was observed, TCE-mediated cytotoxic activity occurred at concentrations much lower than those with substantial IAA. The IAA of hBAFFRxhCD3 bispecific antibodies on BAFFR-expressing Jeko-1 and CD3-expressing Jurkats was consistent with the affinity of the BAFFR and CD3 arms, demonstrating target-specific internalization of these bispecific antibodies.

[0126] Example 5: In vitro cytokine release characterization This study was conducted to evaluate the levels of pro-inflammatory cytokines induced by TCE 1-5 and negative controls A, B, and C in healthy donor PBMCs cocultured with human malignant B cell lines. Z-138 (CRL-3001) and Jeko-1 (CRL-3006) were originally sourced from ATCC. Nalm-6 (ACC 128) was originally sourced from DSMZ. DiscoverX KILR Retroparticles were purchased from Eurofins and used to transduce reporter cells used in cytotoxicity assays according to the manufacturer's instructions. BAFFR was deleted from the genome of Nalm-6 cells by lentiviral introduction of a single guide RNA using the CRISPR / Cas9 system (TNFRSF13C CRISPR guide RNA_pLentiCRISPR v2 from Genscript), and then a KILR reporter cell line was generated.

[0127] Supernatants were removed from the cytotoxicity assay at 48 hours. See Example 9. 10 μL from quadruplicate wells were combined into a single well per sample. Samples were stored at -80°C and thawed at room temperature at the start of the assay. BD Cytometric Bead Array lyophilized standards were reconstituted in assay diluent and titrated according to the manufacturer's instructions. Duplicate standard curves and samples were transferred to the assay plate at 10 μL per well. The capture beads were vortexed and 2 μL of each capture bead per test was combined with 12 μL of PE detection reagent per test. The capture bead / PE detection reagent solution was added to all wells at 20 μL per well. The assay plate was incubated for 2 hours at room temperature in the dark with agitation (500 RPM).

[0128] The assay plate was washed twice with 200 μL of wash buffer per well and centrifuged at 400 × g for 5 minutes. Standards and samples were resuspended in 100 μL of wash buffer and acquired on an Attune NxT flow cytometer. Analyte-specific bead populations were identified using FlowJo version 10 software with sequential gating: FSC-H vs. SSC-H and APC vs. FSC-H. The geometric mean fluorescence intensity (gMFI) of PE in each bead population was then assessed. Using the standard curve and blank gMFI values, a log-log plot and nonlinear fit model for a sigmoidal dose-response (variable slope) were generated in Graphpad Prism 10. This was used to interpolate the analyte concentrations of the samples.

[0129] Summary: The pro-inflammatory cytokine levels induced by the tested hBAFFR×hCD3 bispecific antibodies in the highly BAFF-R-expressing cell lines Jeko-1 (Figures 8A-8B) and Z-138 (Figures 9A-9B) were relatively low, corresponding to the affinity of the BAFFR arms overall. TCEs using the BAFFR clone A arm had greater cytokine release than those using the BAFFR clone B arm. This trend was also observed in the Nalm-6 cell line, which has lower surface expression of BAFF-R and, correspondingly, lower cytokine levels (Figures 10A-10B). In Nalm-6 BAFF-R-KO target cells lacking BAFF-R expression, cytokine release was observed only at the highest TCE concentrations, confirming that hBAFFR×hCD3 results in target-specific cytokine release (Figures 11A-11B).

[0130] Example 6: Evaluation of endogenous B cell depletion in healthy donor PBMCs This study was performed to evaluate T cell-mediated endogenous B cell depletion induced by the hBAFFRxhCD3 bispecific antibodies TCE 1-5 in healthy donor PBMCs. Controls used were negative controls Control A, B, C, and TTx38E4v1, and positive controls Lunsumio® and C90x38E4v1.

[0131] Healthy PBMCs were thawed in culture medium and kept in a 37°C incubator for 2 hours before use. Antibody dilutions in PBS were prepared manually in the plate at 8-point 4-fold dilutions starting at a final concentration of 100x. PBMCs were plated at 1x10 in 160 μl / well in a 96-well plate. 5 Cells were plated at a density of 100 cells / well using a Thermo MultiDrop Combi reagent dispenser. Antibody dilution series were diluted to 5x final concentrations in assay medium and transferred to the cells at 40 μl / well in a final volume of 200 μl / well, starting at a concentration of 1600 ng / ml except for TCE4, which started at 1469 ng / ml. After incubation with antibodies for 48 hours at 37°C in 5% CO2, cells were harvested into 96-well conical polypropylene plates and pelleted by centrifugation. Human Fc block was diluted 1:100 in staining buffer and added to the cells (20 μl / well) on ice for 10 minutes. Diluted APC-CD19 (1:10) and Zombie Violet™ viability dye (1:500) were simultaneously added (20 μl) to the cells and incubated on ice for 20 minutes. Cells were washed once with PBS by centrifugation and resuspended in 100 μl / well of staining buffer prior to flow cytometer analysis.

[0132] Plates were run on an Attune NxT flow cytometer (ThermoScientific), and analysis was performed using FlowJo (version 10.9). Doublets and dead cells were excluded using the gating strategy in FlowJo. The following gating strategy was used: cells (FSC-A vs. SSC-A) → single cells (SSC-A vs. SSC-H) → live cells (SSC-A vs. ZombieViolet™) gating on ZombieViolet™-negative cells → APC count. APC counts (number of CD19+ cells) were obtained for each sample based on singlets and live cells. Counts were transferred to Microsoft Excel for further analysis. Percent cytotoxicity (activity) was calculated by 100 × (number of live CD19+ cells in treated samples / average number of live CD19+ cells in untreated samples), where untreated sample = PBS only.

[0133] Normalization across all plates was performed using plate control wells, where "maximum activity" wells contained 1.6 μg / ml C90×38E4v1 and "minimum activity" wells contained 1.6 μg / ml LSN3985061 control. Percent cytotoxicity was then normalized and calculated as: normalized % cytotoxicity = 100 × ((% cytotoxicity of sample - mean % cytotoxicity of LSN3985061) / (mean % cytotoxicity of C90×38E4v1 - mean % cytotoxicity of LSN3985061)).

[0134] Graph generation and relative EC50 calculations were performed using GraphPad Prism. Antibody concentrations (nM) were converted to the logarithm 10 of the concentration. Curves were fitted by a nonlinear regression log(agonist) vs. response-variable slope (four parameters) model in Graphpad Prism.

[0135] Summary: All hBAFF-R x hCD3 bispecific antibodies tested induced T cell killing of autologous B cells in all three healthy donor PBMCs (Figures 12A-12C, 12F; Figures 13A-13C, 13F; Figures 14A-14C, 14F). Across all three PBMC donors, TCEs with BAFFR arm clone A had more potent killing than those with BAFFR arm clone B (Figures 12A, 12C, 12F; Figures 13A, 13C, 13F; Figures 14A, 14C, 14F).

[0136] Example 7: In vitro T cell activation A study was performed to evaluate T cell activation induced by the tested hBAFFRxhCD3 bispecific antibodies, TCE 1-5. Controls used were: Control A, B, C, C90x38E4v1, TTx38E4v1, and Lunsumio®.

[0137] Jurkat NFAT-RE luciferase reporter cells were plated in 384-well plates at a density of 5 × 10 cells / well in assay medium using a Thermo MultiDrop Combi reagent dispenser. For coculture assays, target cells were added to Jurkat-NFAT effector cells at a density of 5 × 10 cells / well in assay medium at a 1:1 target:effector cell ratio. Antibody dilutions in PBS were manually prepared in the plate at 10-point, 4-fold dilutions starting at a 50x final concentration. The antibody dilution series was diluted to a 5x final concentration in assay medium and transferred to the cells. After 24 hours of incubation with antibody at 37°C in 5% CO2, the cells were processed using the Bio-Glo Luciferase Assay System and a Thermo MultiDrop Combi reagent dispenser. Luminescence signals were measured using a Molecular Devices Spectramax M5e. Raw luminescence values ​​were plotted in GraphPad Prism. Curves were fitted by a nonlinear regression log(agonist) vs. response-variable slope (four parameters) model in Graphpad Prism.

[0138] Luminescence (0.5 ms) values ​​were measured on a SpectraMax M5e plate reader using the SoftMax Pro program. Raw luminescence values ​​were plotted in GraphPad Prism for graph generation and relative EC50 calculations.

[0139] Summary: T cell activation was observed in the presence of hBAFFR-R-expressing B cell tumor cell lines with all hBAFFR×hCD3 bispecific antibodies tested. The highest T cell activation activity was observed in the highest hBAFFR-expressing Jeko-1 cells (Figures 15A-C), whereas lower T cell activation activity was observed in Z-138 cells (Figures 16A-C) and Nalm-6 cells (Figures 17A-C), the latter two expressing lower levels of hBAFFR than Jeko-1. T cell activation was not observed in Nalm-6 BAFF-R KO cells or in the absence of target cells (data not shown). This indicates that BAFFR must be present for hBAFFR×hCD3 to activate T cells.

[0140] Example 8: In vitro T cell activation, proliferation, and tumor cell cytotoxicity This study was conducted to evaluate the ability of the hBAFFR x hCD3 bispecific antibodies TCE 1-5, negative controls Control A, B, and C, and positive controls Lunsumio® and C90x38E4v1 to induce T cell activation and proliferation and T cell-mediated tumor cell cytotoxicity using human healthy donor PBMCs cocultured with Jeko-1 cells. PBMCs were thawed in a 37°C water bath, transferred to a 15 mL centrifuge tube containing 7 mL of complete medium (RPMI + 10% HI FBS + 1x Glutamax) and washed once at 300 x g for 5 minutes. Cells were resuspended in 5 mL of RoboSep buffer and counted using a Cellometer cell viability counter. Cells were transferred to a 14 mL polystyrene tube, washed, and resuspended in 1 mL of RoboSep buffer for T cell isolation. T cell isolation was performed manually using a Stemcell Human T Cell Isolation Kit and a "Big Easy" EasySep™ magnet. T cells were washed, resuspended in RoboSep buffer, and counted for CFSE labeling. Isolated T cells (effector cells) were washed once with 10 mL of 1x PBS, resuspended in 10 mL of 1x PBS containing 10 μl of diluted CFSE, and incubated at 37°C, 5% CO for 20 minutes. 30 mL of complete medium was added, allowed to stand at room temperature for 5 minutes, and then washed. Cells were resuspended in 10 mL of complete medium, allowed to stand at room temperature for 10 minutes, and then counted. The cell concentration was 0.625 x 10 in complete medium. 6CFSE-labeled T cells were adjusted to 1000 cells / mL and 80 μl (50,000 cells / well) were plated into a 96-well TC-treated U-bottom plate. Thirty million Jeko-1 cells (target cells) were washed twice in 10 mL of 1x PBS and resuspended in 1 mL of diluent C from the PKH26 kit. A 2x dye solution was prepared, and 1 mL of cells was added to 1 mL of 2x dye solution and mixed by pipetting. The cells were incubated for 1-5 minutes with periodic mixing. Five mL of complete medium was added and washed at 300 x g for 10 minutes, and the supernatant was carefully removed. The cells were resuspended in 10 mL of complete medium, transferred to a new tube, and washed three times for 5 minutes. The cells were counted after the final wash, and the cell concentration was adjusted to 0.125 x 10 in complete medium. 6 The antibody dilutions were adjusted to 100 cells / mL, and 80 μL (10,000 target cells / well) was plated into 96-well wells containing CFSE-labeled T cells. Antibody dilutions were prepared in PBS in 10-point, 4-fold dilutions starting at a final concentration of 50x in a 96-well deep plate. The antibody dilution series was then diluted to a final concentration of 5x in complete medium and transferred to the cells. Cells were incubated for 48 or 96 hours at 37°C and 5% CO2. On the day of harvest, cells were washed once, resuspended in 25 μl of staining buffer containing 1:100 human Fc block, and incubated on ice for 30 minutes. The staining antibody mix (CD4, CD8, CD25, and CD69 at 1:50 per antibody) was prepared 2x in brilliant staining buffer, and 25 μl of the mix was added and incubated for 1 hour on ice, protected from light. Cells were washed three times at 700 xg for 3 minutes, resuspended in 80 μl of staining buffer containing helix blue (1:1000 dilution), and 40 μl was collected on an Attune flow cytometer.

[0141] Data were analyzed using FlowJo version 10.10.0, using the following gating strategy: T cell activation: cells (FSC-A vs. SSC-A), single cells (FSC-A vs. FSC-H), live cells (FSC-A vs. BV421-Helix Blue), CD4 or CD8+ (FSC-A vs. BV650-CD4 or FSC-A vs. BV605-CD8), %CD25 or %CD69 (APC-CD25 or PerCpCy5.5-CD69 histograms). T cell proliferation: cells (FSC-A vs. SSC-A), single cells (FSC-A vs. FSC-H), live cells (FSC-A vs. BV421-Helix Blue), CD4 or CD8+ (FSC-A vs. BV650-CD4 or FSC-A vs. BV605-CD8), %CFSE (FITC-CFSE histogram) PKH-26+: Cells (FSC-A vs. SSC-A), Single Cells (FSC-A vs. FSC-H), Live Cells (FSC-A vs. BV421-Helix Blue), PKH-26+ (FSC-A vs. PE-PKH-26), Number of Cells

[0142] Percent cytotoxicity (activity) was calculated by 100-100 x (number of viable PKH26+ cells in treated samples / average number of viable PKH26+ cells in untreated samples), where untreated sample = T cells + Jeko-1, PBS only. Tested antibody concentrations were transformed to log10 (conventional) and data were plotted in GraphPad Prism version 10.1.2 as a nonlinear regression curve fit (sigmoidal, 4PL, X = log(concentration)).

[0143] Summary: In both donors 1 and 2, by 48 hours, the tested hBAFFR×hCD3 TCEs induced low levels of cytotoxicity (Figures 18A and 18C) and T cell activation via the early T cell activation marker CD69+ (Figures 20A, 20C, 20E, and 20G). By 96 hours, the tested TCEs induced higher levels of cytotoxicity (Figures 18B and 18D) and T cell activation via the late T cell activation marker CD25+ (Figures 21B, 21D, 21F, and 21H) in both donors. The majority of CD4+ and CD8+ T cells treated with the tested TCEs reached maximal levels of CD25 activation (Figures 21B, 21D, 21F, and 21H) and proliferation (Figures 19B, 19D, 19F, and 19H) by 96 hours for both donors. T cell activation (Figures 20A-20H and 21A-21H), T cell proliferation (Figures 19A-19H), and T cell cytotoxicity (Figures 18A-18D) were not observed in the negative CD3 control, suggesting that the cytotoxicity, proliferation, and activation observed in the hBAFFR x hCD3 treatment group were due to specific engagement of the anti-BAFFR arm to BAFFR+ cells.

[0144] Example 9: In vitro T cell cytotoxicity in malignant B cell lines This study was conducted to evaluate the T cell cytotoxicity of hBAFFR×hCD3 TCEs 1-5 in malignant B cell lines. High-expressing hBAFFR cells, Z-138 and JeKo-1, and the KILR target cells, Nalm-6 and Nalm-6 BAFF-R knockout (KO) cells, were added to 384-well assay plates at 2,500 cells per well in 20 μL of assay buffer. Normal healthy donor PBMCs were added as effector cells at two effector-to-target cell (E:T) ratios: 25,000 PBMCs in 20 μL of assay buffer were added to wells with an E:T ratio of 10:1, and 12,500 PBMCs in 20 μL of assay buffer were added to wells with an E:T ratio of 5:1. The tested TCEs, hBAFFR x hCD3 bispecific antibodies TCE1, TCE2, TCE3, TCE4, and TCE5, as well as negative controls Controls A, B, and C, were serially titrated to generate a 10-point, 4-fold dilution series with a starting concentration of 80 μg / mL in assay buffer. 10 μL was added to the cells in quadruplicate. PBS or lysis buffer was added to minimum or maximum signal control wells, respectively. The plate was covered with a breathable seal and incubated at 37°C for 48 hours. At the end of the assay, DiscoverX Detection Kit Reagents 1, 2, and 3 were combined in a 4:1:1 ratio according to the manufacturer's instructions. The assay plate was removed from the 37°C incubator, and 10 μL of supernatant was collected from each well for downstream cytokine analysis. See Example 5. Detection reagent was added at 30 μL per well, and the plate was incubated at room temperature for 45 minutes. Luminescence was detected using a SpectraMax M5e plate reader with standard settings.

[0145] Luminescence values ​​were converted to percent activity using a two-point normalization, in which the average value from the lysis buffer control wells determined the maximum signal and the average value from the PBS control wells determined the minimum signal. Maximum activity and relative EC50 values ​​for dose-response curves were calculated in Graphpad Prism 10. Curves were fitted by a nonlinear regression log(agonist) vs. response-variable slope model in Graphpad Prism.

[0146] The cytotoxic activity of the tested hBAFFR×hCD3 bispecific antibodies in the highly BAFF-R-expressing cell lines Jeko-1 (Figures 22A-B) and Z-138 (Figures 23A-B) appeared to correspond to the affinity of the hBAFFR arm, with hBAFFR clone A having higher maximal activity and potency than those with hBAFFR clone B (Figures 23A-B, 24A-B, 25A-B, and 26). Additionally, the affinity of the hCD3 arm distinguished the activity of the hBAFFR×hCD3 bispecific antibodies, with TCE1 having the highest maximal activity and potency. This trend was observed at both the 5:1 and 10:1 effector-to-target cell ratios tested. Despite lower surface expression of hBAFFR in the Nalm-6 cell line, the tested hBAFFRxhCD3 bispecific antibody maintains cytotoxic activity at both E:T ratios (Figures 24A-B and 26). The reduced cytotoxic activity in Nalm-6 BAFF-R-KO cells confirms the target specificity of the tested hBAFFRxhCD3 (Figures 25A-B).

[0147] Summary: As summarized in Figure 26, cytotoxic activity of hBAFFRxhCD3 TCEs was observed in the high BAFFR-expressing cell lines Jeko-1 and Z-138, corresponding to the affinity of the BAFFR and CD3 arms. The tested TCEs maintained cytotoxic activity in the Nalm-6 cell line, despite lower surface expression of BAFFR. This activity was target-specific, as evidenced by reduced cytotoxic activity in Nalm-6 BAFF-R-KO target cells.

[0148] Example 10: Ex vivo analysis of serum cytokine levels in the Jeko-1 luciferase model Serum cytokine levels produced by humanized mice treated with hBAFFRxhCD3 bispecific antibodies and controls were evaluated in a Jeko-1 luciferase tumor model. 50 μl of serum per mouse was collected from mice treated as described in Example 12 and stored at -80°C until cytokine detection was performed. Serum was used once without any freeze-thaw cycles. Two assays, an IL2 singleplex and a complex cytokine 10-plex, were used for each time point in this study, thus two sets of assays were used. The two sets of assays were performed on separate days. Assays were performed according to the manufacturer's instructions. Briefly, samples were prepared by adding 22 μl of serum to 33 μl of diluent. Each assay plate was washed five times with 300 μl of 1x wash buffer before loading the calibrators and samples. 50 μl of either calibrator or sample was added to the assay plate and shaken at approximately 525 rpm on a benchtop shaker for 2 hours. Each assay plate was washed five times with 300 μl of 1x wash buffer, after which 50 μl of biotinylated detection reagent was added and shaken for 30 minutes. Each plate was washed an additional five times with 300 μl of 1x wash buffer, after which 50 μl of streptavidin-HRP was added and shaken for 30 minutes. Each assay plate was washed 10 times with 300 μl of 1x wash buffer, after which SuperSignal reagent was added and immediately scanned on an SP-X imager at the ultra-high sensitivity setting.

[0149] Assays were read on an SP-X imager, and data were analyzed using SP-X analysis software version 2.2.8789. Assay product information and sample plate layout with calibrator and sample dilution factors were loaded into the software, after which data analysis was performed. Standard curves for each analyte were manually and minimally adjusted to ensure a curve fit close to 1. After validating the standard curve for each analyte, quantification of each sample was calculated by the software. Sample concentrations were exported to Excel and graphed in Spotfire with error bars representing the Lower Limit of Quantification (LLoQ), Limit of Detection (LoD), and one standard deviation of the mean concentration per group.

[0150] Summary: Induced levels of IL2 and IFNg were highest with TCE1, followed by TCE2, TCE4, and TCE5 (Figures 27A and 27C). TCE1 induced a slight increase in IL-8 levels (Figure 27A). Levels of other 10-plex analytes (IL4, IL5, IL6, TNFα, IL1b, IL10, IL12p70, IL22) induced by the hBAFFRxhCD3 bispecific antibody were low and below the LLoQ (Figures 27A, 27B, 27C). For almost all cytokines measured, Lunsumio® induced higher levels than the TCEs tested (Figures 27A, 27B, 27C).

[0151] Example 11: Ex vivo analysis of serum cytokine levels in the Z-138 xenograft model Serum cytokine levels produced by humanized mice treated with hBAFFRxhCD3 bispecific antibodies and controls were evaluated in a Z-138 xenograft tumor model performed as described in Example 13. 50 μl of serum per mouse was collected 2 and 24 hours after treatment and stored at -80°C until cytokine detection was performed. Serum was used once without any freeze-thaw cycles. Two assays, an IL2 singleplex and a complex cytokine 10-plex, were used for each time point in this study, thus two sets of assays were used. The two sets of assays were performed on separate days. Assays were performed according to the manufacturer's instructions. Briefly, samples were prepared by adding 22 μl of serum to 33 μl of diluent. Each assay plate was washed five times with 300 μl of 1x wash buffer before loading the calibrators and samples. 50 μl of either calibrator or sample was added to the assay plate and shaken at approximately 525 rpm on a benchtop shaker for 2 hours. Each assay plate was washed five times with 300 μl of 1x wash buffer, after which 50 μl of biotinylated detection reagent was added and shaken for 30 minutes. Each plate was washed an additional five times with 300 μl of 1x wash buffer, after which 50 μl of streptavidin-HRP was added and shaken for 30 minutes. Each assay plate was washed 10 times with 300 μl of 1x wash buffer, after which SuperSignal reagent was added and immediately scanned on an SP-X imager at the ultra-high sensitivity setting.

[0152] Assays were read on an SP-X imager, and data were analyzed using SP-X analysis software version 2.2.8789. Assay product information and sample plate layout with calibrator and sample dilution factors were loaded into the software, after which data analysis was performed. Standard curves for each analyte were manually and minimally adjusted to ensure a curve fit close to 1. After validating the standard curve for each analyte, quantitation of each sample was calculated by the software. Sample concentrations were exported to Excel and graphed in Spotfire with error bars representing the lower limit of quantitation (LLoQ), limit of detection (LoD), and one standard deviation of the mean concentration per group.

[0153] Summary: Serum cytokine levels induced by treatment with the tested hBAFF-R x hCD3 bispecific antibodies in an established humanized mantle cell lymphoma Z-138 xenograft tumor model were assessed 2 and 24 hours after the first treatment.

[0154] In the 2-hour samples, IL-2 was induced by TCE1, followed by TCE2 (Figure 28C). TCE2 induced a slight increase in IL-8, IL-10, and IL-22 levels (Figures 28A and 28B). For many cytokines measured, Lunsumio® induced higher cytokine release compared to the TCEs tested. See Figures 28A, 28B, and 28C. Overall, higher induction of IFNg and IL-8 than other cytokines was observed across multiple samples (Figure 28A).

[0155] In the 24-hour samples, IL-2 was induced by TCE2, followed by TCE1 (Figure 29C). IFNg was induced by TCE1, followed by TCE2 (Figure 29A). IL-6 was induced by TCE1, followed by TCE2 and TCE5 (Figure 29C). TCE1 and TCE2 induced slight increases in IFNg, IL-5, IL-8, IL-10, and IL-22 levels. See Figures 29A, 29B, and 29C. As with the 2-hour time point, for many cytokines measured, Lunsumio® induced higher cytokine release at 24 hours compared to the TCEs tested. See Figures 29A, 29B, and 29C. Overall, induction of IFNg, IL-5, IL-8, IL-10, and IL-22 was higher than other cytokines across samples (Figures 29A and 29B). Levels of other 10-plex analytes (IL-1b, IL-4, IL-12p70, TNFa) by the hBAFFRxhCD3 bispecific antibody tested were low and below the LLoQ (Figures 29A, 29B, and 29C).

[0156] Example 12: In vivo analysis in the Jeko-1 xenograft model This study was conducted to evaluate the antitumor activity of the hBAFFR x hCD3 bispecific antibodies TCE1-5, negative controls Controls A, B, and C, and positive controls C90x38E4v1 and Lunsumio® in a humanized systemic mantle cell lymphoma Jeko-1-luciferase xenograft mouse model. Jeko-1 luciferase-expressing (Jeko-1 Luciferase, Jeko1-luc) single clone 2B5 cells (Jeko1-luc single clone 2B5, Jeko1-luc-cl2B5) were derived from the human mantle cell lymphoma Jeko-1 cell line by stable integration of a constitutive firefly luciferase expression construct (Systembio, catalog number LL250PA-1). The parental Jeko-1 was obtained from the American Type Culture Collection, CRL-3006™. Jeko1-luc-cl2B5 cells were maintained in ATCC-formulated RPMI-1640 medium, catalog number 30-2001, supplemented with 20% fetal bovine serum (catalog number SH300070.03 from CYTIVA) and 1 μg / ml puromycin (catalog number A1113803 from ThermoFisher). All cultures were maintained in a humidified incubator at 37°C under 5% CO2 / 95% air. Low cell passages (maximum 4) were used in experiments after recovery from frozen stocks. Human PBMCs were isolated from whole blood (donor number 8068, AllCell, lot number 3029827) using Ficoll-Hypaque density gradient centrifugation and then frozen at -196°C until use.

[0157] All test articles were freshly diluted to a final concentration of 0.015 mg / ml or 0.15 mg / ml in PBS for each dose and animals were dosed intravenously by body weight (10 μl / g).

[0158] Female NOD SCID Gamma (NSG) mice (strain number 005557) were obtained from the Jackson Laboratory. Jeko-1 luciferase-expressing cells were cultured at 2.5 × 10 in PBS. 6 / ml (single cell suspension with >90% viability) and 200 ul was injected iv (tail vein) into each mouse (0.5 x 10 6 Tumor growth was identified by bioluminescence imaging on day 5 after inoculation, and mice were randomized into different groups and started on weekly treatment for 3 weeks.

[0159] 0.5 × 10 6 Luciferase-expressing Jeko-1 cells were inoculated via the tail vein on day 0. Mice were randomized into groups of 12 on day 5 based on luciferase signal intensity. Mice were inoculated with 4 × 10 6 Human PBMCs (donor no. 8068) were injected i.v. (tail vein) on day 6. Two hours after PBMC injection, 0.15 mg / kg or 1.5 mg / kg of the indicated test article or vehicle was administered i.v. according to body weight (10 μl / g) once a week for 3 weeks. Bioluminescence images were taken before treatment and on days 5, 12, 19, and 26 to monitor tumor growth (data not shown). Group 01: Jeko-1-luc only, vehicle, QW x 3 doses Group 02: Jeko-1 + PBMC, vehicle, QW x 3 doses Group 03: 0.15 mg / kg TCE1, QW x 3 doses Group 04: 0.15 mg / kg TCE3, QW x 3 doses Group 05: 0.15 mg / kg TCE2 QW x 3 doses Group 06: 0.14 mg / kg TCE4 QW x 3 doses Group 07: 1.5 mg / kg TCE5 QW x 3 doses Group 08: Control A, 0.15 mg / kg QW x 3 doses Group 09: Control C, 0.15 mg / kg QW x 3 doses Group 10: Control B, 0.15 mg / kg QW x 3 doses Group 11: 0.15 mg / kg C90×38E4v1 QW×3 doses Group 12: 0.15 mg / kg Lunsumio® QW x 3 doses

[0160] Bioluminescence imaging (BLI) is based on the highly sensitive detection of visible light produced when an enzyme (luciferase) is expressed in vivo as a molecular reporter during enzyme-mediated oxidation of a molecular substrate. BLI in this study was performed using an IVIS 200 system (Xenogen). Before each imaging session, animals were injected intraperitoneally with luciferin solution (VivoGlo™ Luciferin, In Vivo Grade, Catalog No. P1043) prepared at a 15 mg / ml stock in PBS, at a dose of 150 mg / kg body weight (10 μl / g). Luciferin was allowed to distribute for 10 minutes in awake animals. Under the luminescence imaging model, mice were imaged dorsally under 2% isoflurane gas anesthesia. Image acquisition times ranged from 5 seconds to 1 minute to obtain unsaturated images in a small-binning model. BLI signals were quantified in regions of interest (ROIs) drawn on the whole body, and the signals were expressed as photons per second, representing the flux emanating in all directions from the user-defined region. Images were analyzed using Living Image 4.7.4 (PerkinElmer, Waltham, MA) software. Data were analyzed using GraphPad Prism 9.3.1 software (Graph-Pad Software, USA). Statistical differences were assessed using unpaired t-tests and two-way ANOVA tests, as indicated. Differences were considered significant at p<0.05. Body weight was measured twice weekly. The percent change in body weight was calculated by the formula: (body weight on observation day - body weight on first day) / body weight on first day × 100%.

[0161] Summary: Six days after the first dose on day 12, Jeko-1 tumor regression was observed in mice treated with TCE1 and TCE2, and their antitumor activity was comparable to that of Lunsumio® (p<0.001). See Figure 30. In all three treatment groups (TCE1, TCE2, and Lunsumio®), tumors began to regrow after day 19. TCE3, TCE4, and TCE5 significantly inhibited tumor growth by day 19. The third dose did not maintain tumor growth inhibition in any group. The three sham-arm controls did not demonstrate any in vivo activity compared with the vehicle group. No significant weight loss was observed in any group (data not shown).

[0162] Example 13: In vivo analysis in an established Z-138 xenograft model This study was conducted to evaluate the antitumor activity of the hBAFFR x hCD3 bispecific antibodies TCE1-5, as well as the negative controls Controls A, B, and C, and the positive controls C90x38E4v1 and Lunsumio®, in an established humanized mantle cell lymphoma Z-138 xenograft model in NSG mice. The Z-138 human lymphoma cell line was obtained from the American Type Culture Collection (ATCC, CRL-3001™, Lot No. 57634322). Z-138 cells were maintained in ATCC-formulated Iscove's Modified Dulbecco's Medium (IMEM), Catalog No. 30-2005, supplemented with 10% fetal bovine serum (Cat. No. SH300070.03). All cultures were maintained in a humidified incubator at 37°C under 5% CO2 / 95% air. Low cell passages (maximum 4) were used in the experiments after recovery from frozen stocks. Human PBMCs were isolated from whole blood (donor B001000834, Miltenyi, lot number 031908290118999) using Ficoll-Hypaque density gradient centrifugation and then frozen at -196°C until use.

[0163] All test articles were freshly diluted to a final concentration of 0.05 mg / ml in Phosphate-Buffered Saline (PBS) for each dose. * Note: The concentration for TCE4 was incorrectly calculated as 7.7 mg / ml when preparing the mouse dose. Therefore, the final concentration of TCE4 is 0.046 mg / ml.

[0164] Female NOD SCID gamma (NSG) mice (strain number 005557) were obtained from Jackson Laboratory and allowed to acclimate for one week before the start of the experiment. Mice were housed in microisolator cages under pathogen-free conditions in a 12-hour light / dark cycle facility and provided with standard laboratory chow and water ad libitum. Prepared Z-138 cells [6 × 10 in 100 μL] were added to the 100 μL culture. 5 10 x 10 PBMCs 6 A single cell suspension with >90% viability in Hank's balanced saline mixed with an equal volume of Matrigel (100 μL) was injected subcutaneously into the right hind flank of each mouse.

[0165] On day 0, 5 x 10 6 0.3 × 10 Z-138 human lymphoma cells 6 The cells were mixed with healthy human PBMCs and injected subcutaneously into the right flank of mice (200 μl / mouse in Hank's balanced saline with an equal volume of Matrigel). Tumors grew to approximately 200 mm on day 6 after cell implantation. 3 When the tumor reached 100 mg / kg, mice were randomized into 8 groups (6 mice / group). All treatment groups were dosed at 0.5 mg / kg by intravenous injection (IV) once a week for 3 weeks. Tumor volume (TV) and body weight were measured twice a week. TV was calculated as TV (mm 3 ) = π / 6 × length × width 2. 3 If the growth exceeds 2000mm before the next measurement, 3 If the GI profiling exceeded 100 mg / kg, the animals were sacrificed due to progressive disease. Group 1: Z-138 + PBMC, vehicle Group 2: Z-138 + PBMC, 0.5 mg / kg TCE1, QW x 3 doses Group 3: Z-138 + PBMC, 0.5 mg / kg TCE3, QW x 3 doses Group 4: Z-138 + PBMC, 0.5 mg / kg TCE2, QW x 3 doses Group 5: Z-138 + PBMC, 0.5 mg / kg TCE4, QW x 3 doses Group 6: Z-138 + PBMC, 0.5 mg / kg TCE5, QW x 3 doses Group 7: Z-138 + PBMC, 0.5 mg / kg control A, QW x 3 doses Group 8: Z-138 + PBMC, control C, 0.5 mg / kg, QW x 3 doses Group 9: Z-138 + PBMC, control B at 0.5 mg / kg, QW x 3 doses Group 10: Z-138 + PBMC, 0.5 mg / kg C90x38E4v1, QW x 3 doses Group 11: Z-138 + PBMC, 0.5 mg / kg Lunsumio®, QW x 3 doses

[0166] Blood samples were collected from the retro-orbital cavity 2 and 24 hours after the first dose (3 mice / group per time point), and cytokine levels (included in a separate study report) were measured in serum separator tubes (BD sku no. 365967). An appropriate time (approximately 20 minutes) was allowed for blood to clot at room temperature, and then the blood was centrifuged at 10,000 x g for 10 minutes in a refrigerated centrifuge. Serum was collected, aliquoted, and stored at -80°C until cytokine detection experiments.

[0167] Tumor volumes were transformed to the log10 scale to equalize variance across time and treatment. Log10 volumes and body weights were analyzed separately using a two-way repeated measures analysis of variance (RM ANOVA) model consisting of time, treatment, and the interaction between time and treatment using the MIXED procedure in the SAS software package (version 9.4). A spatial power covariance structure was used to model the correlation of observations on the same subject over time. Tests of fixed effects were performed using the Kenward and Roger (1997) Denominator Degrees of Freedom (DDFM) calculation. Post-hoc pairwise t-tests were used to compare tumor volumes and body weights of treatment groups with the control group on summarized days, with p-values ​​of ≤0.05 considered statistically significant. Least squares means (LS Means) and standard errors for each time point were also calculated using the MIXED procedure separately for each treatment group for plotting and inclusion purposes.

[0168] Efficacy Calculation: Efficacy was calculated at the end of treatment if the number of remaining control subjects was at least half the baseline sample size or greater than 4. Otherwise, efficacy was calculated at the most recent observation date before the end of treatment on which these conditions were met. Percent Treatment / Control (%T / C) values ​​were calculated using the following formula: If ΔT>0, %T / C=100×ΔT / ΔC If ΔT<0, %Regression=100×ΔT / T 初日 , in the formula, T = mean tumor volume of the drug treatment group on the last day of the study; ΔT = mean tumor volume of the drug treatment group on the last day of the study - mean tumor volume of the drug treatment group on the first day of dosing, T 初日 = mean tumor volume of drug treatment group on the first day of dosing, C = mean tumor volume in the control group on the last day of the study; ΔC = mean tumor volume of the control group on the last day of the study - mean tumor volume of the control group on the first day of dosing. The % change in body weight was calculated as follows: (Current BW - First day of BW) / (First day of BW) x 100

[0169] Data were calculated as percent body weight change from the start of treatment.

[0170] Summary: The tested hBAFFR×hCD3 bispecific antibodies demonstrated antitumor efficacy in an established human mantle cell lymphoma Z-138 xenograft mouse model co-transplanted with human PBMCs. TCE1-5 significantly (P<0.001) inhibited Z-138 tumor growth compared to hPBMC vehicle (Group 1) at day 32. See Figure 31. Of the five hBAFFR×hCD3 bispecific antibodies tested, TCE2 exhibited the lowest T / C ratio. Tumors in all three sham-arm control groups had growth rates very similar to those in the vehicle group. No significant weight loss was observed in any group (data not shown).

[0171] Example 14: In vivo antitumor activity in an immunocompetent syngeneic BAFFR+ tumor model EO771 mouse tumor cells expressing human BAFFR were subcutaneously implanted in humanized CD3EDG mice. TCE1 and isotype control A were intravenously injected three times at two doses, 0.5 mg / kg and 0.05 mg / kg, on days 1, 9, and 15 after EO771 cell implantation.

[0172] Data are means ± SEM, N=4.

[0173] Summary: TCE1 exhibits potent antitumor activity against a human BAFFR-expressing mouse tumor model in humanized CD3EDG mice with competent immune systems at doses as low as 0.05 mg / kg, demonstrating the efficacy of this TCE. See Figure 32.

[0174] Example 15: In vivo antitumor activity in a patient-derived B-ALL xenograft model The patient-derived B-ALL model BP2101 is homogeneously double-positive for human CD19 and human BAFFR. Approximately 30% of BP2101 cells are positive for human CD20 and simultaneously positive for human CD19 and human BAFFR. BP2101 cells were intravenously transplanted into NSG mice. After 20 days, 3 × 10 cells per mouse were transplanted. 6 Human donor PBMCs were transplanted, followed by treatment with TCE1 at different doses of 2.0 mg / kg, 0.5 mg / kg, or 0.15 mg / kg 24 hours later. An isotype negative control (Control A) was tested at 2.0 mg / kg, and a positive control (mosunetuzumab) was tested at 0.5 mg / kg. All test articles and controls were administered IV once weekly. Peripheral blood from all mice was subjected to flow analysis for hCD19+ and hCD3+ T cells 24 hours after each dose.

[0175] Statistical analysis was performed using one-way ANOVA. All data were compared to vehicle. * P<0.0001, ** P=0.0003. Data are means±SEM.

[0176] Summary: TCE1 dose-dependently reduces B cells in a PBMC-humanized patient-derived B-ALL model 24 hours after the third TCE dose. Furthermore, TCE1 demonstrated complete depletion of patient-derived B-ALL B cells at 2 mg / kg. See Figure 33.

Claims

1. A bispecific antibody that specifically binds to human B-cell activating factor receptor (hBAFFR) and human CD3 (hCD3), comprising: (a) a first antigen-binding domain that specifically binds to hBAFFR, the first antigen-binding domain comprising a first heavy chain variable region (VH1) and a light chain variable region (VL1), wherein the VH1 comprises heavy chain complementarity-determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the VL1 comprises light chain complementarity-determining regions (LCDRs) LCDR1, LCDR2, and LCDR3; (a) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6; or (b) a first antigen-binding domain, wherein HCDR1 comprises SEQ ID NO:29, HCDR2 comprises SEQ ID NO:30, HCDR3 comprises SEQ ID NO:31, LCDR1 comprises SEQ ID NO:4, LCDR2 comprises SEQ ID NO:5, and LCDR3 comprises SEQ ID NO:

6. and a second antigen-binding domain that specifically binds to human CD3.

2. 2. The bispecific antibody of claim 1 , wherein the second antigen-binding domain that specifically binds to hCD3 comprises a second heavy chain variable region (VH2) and a light chain variable region (VL2), wherein the VH2 comprises heavy chain complementarity-determining regions (HCDRs) HCDR4, HCDR5, and HCDR6, and the VL2 comprises light chain complementarity-determining regions (LCDRs) LCDR4, LCDR5, and LCDR6.

3. A bispecific antibody that specifically binds to hBAFFR and hCD3, comprising: (a) a first antigen-binding domain that specifically binds to hBAFFR; and (b) a second antigen-binding domain that specifically binds to hCD3, the second antigen-binding domain comprising a heavy chain variable region (VH2) and a light chain variable region (VL2), wherein the VH2 comprises heavy chain complementarity-determining regions (HCDRs) HCDR4, HCDR5, and HCDR6, and the VL2 comprises light chain complementarity-determining regions (LCDRs) LCDR4, LCDR5, and LCDR6; a) HCDR4 comprises SEQ ID NO:7, HCDR5 comprises SEQ ID NO:8, HCDR6 comprises SEQ ID NO:9, LCDR4 comprises SEQ ID NO:4, LCDR5 comprises SEQ ID NO:5, and LCDR6 comprises SEQ ID NO:6; or b) HCDR4 comprises SEQ ID NO: 19, HCDR5 comprises SEQ ID NO: 20, HCDR6 comprises SEQ ID NO: 21, LCDR4 comprises SEQ ID NO: 4, LCDR5 comprises SEQ ID NO: 5, and LCDR6 comprises SEQ ID NO: 6; or c) a second antigen-binding domain wherein HCDR4 comprises SEQ ID NO:24, HCDR5 comprises SEQ ID NO:25, HCDR6 comprises SEQ ID NO:26, LCDR4 comprises SEQ ID NO:4, LCDR5 comprises SEQ ID NO:5, and LCDR6 comprises SEQ ID NO:

6.

4. 4. The bispecific antibody of claim 1, wherein the first antigen-binding domain has a binding affinity (KD) to hBAFFR that is about 5 to about 50 times greater than the binding affinity to hCD3 of the second antigen-binding domain, as measured by surface plasmon resonance (SPR).

5. 5. The bispecific antibody of any one of claims 1 to 4, wherein the bispecific antibody demonstrates in vivo tumor growth inhibition of at least 10% or more.

6. The bispecific antibody of any one of claims 1 to 5, wherein the bispecific antibody induces an in vivo IFNg concentration of 6000 fg / ml or less.

7. The bispecific antibody of any one of claims 1 to 6, wherein the bispecific antibody inhibits binding of hBAFFR to hBAFF.

8. The bispecific antibody of any one of claims 1 to 7, wherein the bispecific antibody inhibits antibody-induced shedding of the hBAFFR extracellular domain.

9. (a) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, LCDR3 comprises SEQ ID NO: 6, HCDR4 comprises SEQ ID NO: 7, HCDR5 comprises SEQ ID NO: 8, HCDR6 comprises SEQ ID NO: 9, LCDR4 comprises SEQ ID NO: 4, LCDR5 comprises SEQ ID NO: 5, and LCDR6 comprises SEQ ID NO: 6; or (b) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, LCDR3 comprises SEQ ID NO: 6, HCDR4 comprises SEQ ID NO: 19, HCDR5 comprises SEQ ID NO: 20, HCDR6 comprises SEQ ID NO: 21, LCDR4 comprises SEQ ID NO: 4, LCDR5 comprises SEQ ID NO: 5, and LCDR6 comprises SEQ ID NO: 6; or (c) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, LCDR3 comprises SEQ ID NO: 6, HCDR4 comprises SEQ ID NO: 24, HCDR5 comprises SEQ ID NO: 25, HCDR6 comprises SEQ ID NO: 26, LCDR4 comprises SEQ ID NO: 4, LCDR5 comprises SEQ ID NO: 5, and LCDR6 comprises SEQ ID NO: 6; or (d) HCDR1 comprises SEQ ID NO:29, HCDR2 comprises SEQ ID NO:30, HCDR3 comprises SEQ ID NO:31, LCDR1 comprises SEQ ID NO:4, LCDR2 comprises SEQ ID NO:5, LCDR3 comprises SEQ ID NO:6, HCDR4 comprises SEQ ID NO:7, HCDR5 comprises SEQ ID NO:8, HCDR6 comprises SEQ ID NO:9, LCDR4 comprises SEQ ID NO:4, LCDR5 comprises SEQ ID NO:5, and LCDR6 comprises SEQ ID NO:6; or (e) The bispecific antibody of claim 2, wherein HCDR1 comprises SEQ ID NO: 29, HCDR2 comprises SEQ ID NO: 30, HCDR3 comprises SEQ ID NO: 31, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, LCDR3 comprises SEQ ID NO: 6, HCDR4 comprises SEQ ID NO: 19, HCDR5 comprises SEQ ID NO: 20, HCDR6 comprises SEQ ID NO: 21, LCDR4 comprises SEQ ID NO: 4, LCDR5 comprises SEQ ID NO: 5, and LCDR6 comprises SEQ ID NO:

6.

10. (a) the VH1 comprises SEQ ID NO: 10 and the VL1 comprises SEQ ID NO: 12; the VH2 comprises SEQ ID NO: 11 and the VL2 comprises SEQ ID NO: 12; or (b) the VH1 comprises SEQ ID NO: 10 and the VL1 comprises SEQ ID NO: 12; the VH2 comprises SEQ ID NO: 22 and the VL2 comprises SEQ ID NO: 12; or (c) the VH1 comprises SEQ ID NO: 10 and the VL1 comprises SEQ ID NO: 12; the VH2 comprises SEQ ID NO:27 and the VL2 comprises SEQ ID NO:12; or (d) the VH1 comprises SEQ ID NO: 32 and the VL1 comprises SEQ ID NO: 12; the VH2 comprises SEQ ID NO: 11 and the VL2 comprises SEQ ID NO: 12; or (e) the VH1 comprises SEQ ID NO: 32 and the VL1 comprises SEQ ID NO: 12; 10. The bispecific antibody of claim 1, wherein the VH2 comprises SEQ ID NO: 22 and the VL2 comprises SEQ ID NO:

12.

11. 11. The bispecific antibody of claim 1 , further comprising an Fc region, the Fc region comprising (a) a BAFFR heavy chain constant region comprising a first CH2 and a first CH3 domain, and (b) a CD3 heavy chain constant region comprising a second CH2 and a second CH3 domain, wherein the first CH3 domain has amino acid substitutions L351D and L368E, and the second CH3 domain has amino acid substitutions L351K and T366K.

12. 11. The bispecific antibody of claim 1 , further comprising an Fc region, the Fc region comprising (a) a BAFFR heavy chain constant region comprising a first CH2 and a first CH3 domain, and (b) a CD3 heavy chain constant region comprising a second CH2 and a second CH3 domain, wherein the first CH3 domain has the amino acid substitutions T366S / L368A / Y407V and the second CH3 domain has the amino acid substitution T366W.

13. 13. The bispecific antibody of claim 11 or 12, wherein the Fc region comprises an Fc-silencing mutation.

14. 14. The bispecific antibody of claim 13, wherein the Fc silencing mutations are (a) L235G and G236R, or (b) L234A, L235A, or (c) L234A, L235A, and D265A, or (d) L234A, L235A, and P329G, or (e) N297A, or (f) N297A and K322A, or (g) L234A, L235A, and D265S.

15. the antibody comprises a first heavy chain (HC1) and a common light chain (cLC) that specifically binds to hBAFFR, and a second heavy chain (HC2) and the common light chain (cLC) that specifically binds to human CD3; (a) HC1 comprises SEQ ID NO: 16, HC2 comprises SEQ ID NO: 17, and cLC comprises SEQ ID NO: 18; or (b) HC1 comprises SEQ ID NO: 16, HC2 comprises SEQ ID NO: 23, and cLC comprises SEQ ID NO: 18; or (c) HC1 comprises SEQ ID NO: 16, HC2 comprises SEQ ID NO: 28, and cLC comprises SEQ ID NO: 18; or (d) HC1 comprises SEQ ID NO: 33, HC2 comprises SEQ ID NO: 17, and cLC comprises SEQ ID NO: 18; or (e) The bispecific antibody of any one of claims 1 to 14, wherein HC1 comprises SEQ ID NO: 33, HC2 comprises SEQ ID NO: 23, and cLC comprises SEQ ID NO:

18.

16. 16. The bispecific antibody of any one of claims 1 to 15, wherein the antibody is of the human IgG1 or IgG4 isotype.

17. 17. The bispecific antibody of claim 16, wherein the antibody is of the human IgG1 isotype.

18. A nucleic acid encoding the amino acid sequence of claim 15.

19. a) a first vector comprising a nucleic acid encoding SEQ ID NO: 16 and a second vector comprising a nucleic acid encoding SEQ ID NO: 17; b) a first vector comprising a nucleic acid encoding SEQ ID NO: 16 and a second vector comprising a nucleic acid encoding SEQ ID NO: 23; c) a first vector comprising a nucleic acid encoding SEQ ID NO: 16 and a second vector comprising a nucleic acid encoding SEQ ID NO: 28; d) a first vector comprising a nucleic acid encoding SEQ ID NO: 33 and a second vector comprising a nucleic acid encoding SEQ ID NO: 17; or (e) a host cell transfected with a first vector comprising a nucleic acid encoding SEQ ID NO:33 and (j) a second vector comprising a nucleic acid encoding SEQ ID NO:

23.

20. 20. The host cell of claim 19, further transfected with a third vector comprising a nucleic acid encoding SEQ ID NO:

18.

21. 20. The host cell of claim 19, wherein the host cell is a mammalian host cell.

22. 22. A process for producing a bispecific antibody, comprising culturing a cell according to claim 20 or 21 in a culture medium under conditions such that the bispecific antibody is expressed and then recovered from the culture medium.

23. A pharmaceutical composition comprising the hBAFFRxhCD3 bispecific antibody of any one of claims 1 to 17 and 22, and a pharmaceutically acceptable excipient, diluent, or carrier.

24. An isolated antibody that specifically binds to hBAFFR, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions (HCDRs): HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions (LCDRs): LCDR1, LCDR2, and LCDR3; (a) HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6; or (b) An antibody in which HCDR1 comprises SEQ ID NO: 29, HCDR2 comprises SEQ ID NO: 30, HCDR3 comprises SEQ ID NO: 31, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO:

6.

25. a) the VH comprises SEQ ID NO: 10 and the VL comprises SEQ ID NO: 12; or b) the antibody of claim 24, wherein the VH comprises SEQ ID NO: 32 and the VL comprises SEQ ID NO: 12

26. the antibody comprises a heavy chain (HC) and a light chain (LC); a) the HC comprises SEQ ID NO: 34 and the LC comprises SEQ ID NO: 18; or b) The antibody of claim 24 or 25, wherein the HC comprises SEQ ID NO: 35 and the LC comprises SEQ ID NO:

18.

27. The antibody of any one of claims 24 to 26, wherein the antibody is of the human IgG1 or IgG4 isotype.

28. 28. The antibody of claim 27, wherein the antibody is a human IgG1 isotype.

29. The antibody of any one of claims 24 to 28, wherein the antibody is an antibody fragment or an antigen-binding fragment.

30. 30. The antibody of claim 29, wherein the antibody fragment or antigen-binding fragment is a Fab, Fab', F(ab')2, single-chain variable fragment (scFv), Fv, disulfide-linked Fv (sdFv), Fd fragment, or single-chain Fab (scFab).

31. The antibody of any one of claims 24 to 30, wherein the antibody is a multispecific antibody.

32. 32. The antibody of claim 31 , wherein the multispecific antibody is a bispecific antibody, or a trispecific antibody, or a tetraspecific antibody, or a diabody, or a tandem scFv, or a tandem VHH, or a tandem scFab.

33. An antibody-drug conjugate (ADC) comprising the antibody of any one of claims 24 to 32 and a drug moiety.

34. 34. The ADC of claim 33, wherein the drug moiety is selected from the group consisting of an auristatin, an N-acetyl-gamma calicheamicin, a maytansinoid, a pyrrolobenzodiazepine, exatecan, and SN-38.

35. An immunocytokine comprising the antibody according to any one of claims 24 to 32 and a cytokine.

36. 36. The immunocytokine of claim 35, wherein the cytokine is selected from the group consisting of IL-2, IL-4, IL-10, IL-12, IL-15, TNF, and IFNα.

37. A chimeric antigen receptor (CAR) comprising the antibody of any one of claims 24 to 32, a transmembrane domain, and an intracellular signaling domain.

38. A pharmaceutical composition comprising the hBAFFR antibody of any one of claims 24 to 32, or the ADC of claim 33 or 34, or the immunocytokine of claim 35 or 36, or the CAR of claim 37, and a pharmaceutically acceptable excipient, diluent, or carrier.

39. A method of treating B-cell cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the bispecific antibody of any one of claims 1 to 17 and 23, or the antibody of any one of claims 24 to 32, or the ADC of claim 33 or 34, or the immunocytokine of claim 35 or 36, or the CAR of claim 37.

40. 40. The method of claim 39, wherein the B-cell cancer is relapsed or refractory.

41. The B-cell cancer may be B-cell acute lymphoblastic leukemia (B-ALL); Hodgkin's lymphoma (HL); non-Hodgkin's lymphoma (NHL); diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, which is a subtype of DLBCL; follicular lymphoma (FL); chronic lymphocytic leukemia (CLL); small lymphocytic lymphoma (SLL); mantle cell lymphoma (MCL); marginal zone lymphoma and extranodal marginal zone B-cell lymphoma (also known as mucosa-associated lymphoid tissue (MALT) lymphoma); mediastinal gray zone lymphoma (MGZL); nodal marginal zone B-cell lymphoma.

41. The method of claim 39 or 40, wherein the tumor is selected from the group consisting of: splenic marginal zone B-cell lymphoma (SMZL); splenic diffuse small red pulp B-cell lymphoma (SDRPL); high-grade B-cell lymphoma (HGBCL); Burkitt's lymphoma (BL) and Burkitt-like lymphoma (BLL); lymphoplasmacytic lymphoma (or Waldenstrom's macroglobulinemia); B-cell prolymphocytic leukemia (B-PLL); hairy cell leukemia (HCL); multiple myeloma (MM): plasma cell neoplasm; primary central nervous system (CNS) lymphoma; and primary intraocular lymphoma.

42. A method of treating an autoimmune disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the bispecific antibody of any one of claims 1 to 17 and 23, or the antibody of any one of claims 24 to 32, or the ADC of claim 33 or 34, or the immunocytokine of claim 35 or 36, or the CAR of claim 37.

43. 46. ​​The method of claim 45, wherein the autoimmune disorder is rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), celiac disease (CD), type 1 diabetes, primary Sjogren's syndrome (pSS), Guillain-Barré syndrome, inflammatory bowel disease (IBD), and psoriasis.

44. The bispecific antibody of any one of claims 1 to 17 and 23, or the antibody of any one of claims 24 to 32, or the ADC of claim 33 or 34, or the immunocytokine of claim 35 or 36, or the CAR of claim 37, for use in the treatment of B-cell cancer.

45. 45. The use of claim 44, wherein the B-cell cancer is relapsed or refractory.

46. The B-cell cancer may be B-cell acute lymphoblastic leukemia (B-ALL); Hodgkin's lymphoma (HL); non-Hodgkin's lymphoma (NHL); diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, which is a subtype of DLBCL; follicular lymphoma (FL); chronic lymphocytic leukemia (CLL); small lymphocytic lymphoma (SLL); mantle cell lymphoma (MCL); marginal zone lymphoma and extranodal marginal zone B-cell lymphoma (also known as mucosa-associated lymphoid tissue (MALT) lymphoma); mediastinal gray zone lymphoma (MGZL); nodal marginal zone B-cell lymphoma.

46. ​​The use of claim 44 or 45, wherein the tumor is selected from the group consisting of: splenic marginal zone B-cell lymphoma (SMZL); splenic diffuse small red pulp B-cell lymphoma (SDRPL); high-grade B-cell lymphoma (HGBCL); Burkitt's lymphoma (BL) and Burkitt-like lymphoma (BLL); lymphoplasmacytic lymphoma (or Waldenstrom's macroglobulinemia); B-cell prolymphocytic leukemia (B-PLL); hairy cell leukemia (HCL); multiple myeloma (MM): plasma cell neoplasm; primary central nervous system (CNS) lymphoma; and primary intraocular lymphoma.

47. A bispecific antibody according to any one of claims 1 to 17 and 23, or an antibody according to any one of claims 24 to 32, or an ADC according to claim 33 or 34, or an immunocytokine according to claim 35 or 36, or a CAR according to claim 37, for use in the treatment of an autoimmune disorder.

48. 48. The use of claim 47, wherein the autoimmune disorder is rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), celiac disease (CD), type 1 diabetes, primary Sjogren's syndrome (pSS), Guillain-Barré syndrome, inflammatory bowel disease (IBD), and psoriasis.

49. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 17 and 23, or the antibody of any one of claims 24 to 32, or the ADC of claim 33 or 34, or the immunocytokine of claim 35 or 36, or the CAR of claim 37, for use in the treatment of B-cell cancer.

50. 50. The pharmaceutical composition of claim 49, wherein the B-cell cancer is relapsed or refractory.

51. The B-cell cancer may be B-cell acute lymphoblastic leukemia (B-ALL); Hodgkin's lymphoma (HL); non-Hodgkin's lymphoma (NHL); diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, which is a subtype of DLBCL; follicular lymphoma (FL); chronic lymphocytic leukemia (CLL); small lymphocytic lymphoma (SLL); mantle cell lymphoma (MCL); marginal zone lymphoma and extranodal marginal zone B-cell lymphoma (also known as mucosa-associated lymphoid tissue (MALT) lymphoma); mediastinal gray zone lymphoma (MGZL); nodal marginal zone B-cell The pharmaceutical composition of claim 49 or 50, which is selected from the group consisting of lymphomas; splenic marginal zone B-cell lymphoma (SMZL); splenic diffuse small red pulp B-cell lymphoma (SDRPL); high-grade B-cell lymphoma (HGBCL); Burkitt's lymphoma (BL) and Burkitt-like lymphoma (BLL); lymphoplasmacytic lymphoma (or Waldenstrom's macroglobulinemia); B-cell prolymphocytic leukemia (B-PLL); hairy cell leukemia (HCL); multiple myeloma (MM): plasma cell neoplasm; primary central nervous system (CNS) lymphoma; and primary intraocular lymphoma.

52. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 17 and 23, or the antibody of any one of claims 24 to 32, or the ADC of claim 33 or 34, or the immunocytokine of claim 35 or 36, or the CAR of claim 37, for use in the treatment of an autoimmune disorder.

53. 53. The pharmaceutical composition of claim 52, wherein the autoimmune disorder is rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), celiac disease (CD), type 1 diabetes, primary Sjogren's syndrome (pSS), Guillain-Barré syndrome, inflammatory bowel disease (IBD), and psoriasis.

54. Use of the bispecific antibody of any one of claims 1 to 17 and 23, or the antibody of any one of claims 24 to 32, or the ADC of claim 33 or 34, or the immunocytokine of claim 35 or 36, or the CAR of claim 37, in the manufacture of a medicament for the treatment of B-cell cancer.

55. 55. The use of claim 54, wherein the B-cell cancer is relapsed or refractory.

56. The B-cell cancer may be B-cell acute lymphoblastic leukemia (B-ALL); Hodgkin's lymphoma (HL); non-Hodgkin's lymphoma (NHL); diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, which is a subtype of DLBCL; follicular lymphoma (FL); chronic lymphocytic leukemia (CLL); small lymphocytic lymphoma (SLL); mantle cell lymphoma (MCL); marginal zone lymphoma and extranodal marginal zone B-cell lymphoma (also known as mucosa-associated lymphoid tissue (MALT) lymphoma); mediastinal gray zone lymphoma (MGZL); nodal marginal zone B-cell lymphoma.

56. The use of claim 54 or 55, wherein the tumor is selected from the group consisting of: splenic marginal zone B-cell lymphoma (SMZL); splenic diffuse small red pulp B-cell lymphoma (SDRPL); high-grade B-cell lymphoma (HGBCL); Burkitt's lymphoma (BL) and Burkitt-like lymphoma (BLL); lymphoplasmacytic lymphoma (or Waldenstrom's macroglobulinemia); B-cell prolymphocytic leukemia (B-PLL); hairy cell leukemia (HCL); multiple myeloma (MM): plasma cell neoplasm; primary central nervous system (CNS) lymphoma; and primary intraocular lymphoma.

57. Use of the bispecific antibody of any one of claims 1 to 17 and 23, or the antibody of any one of claims 24 to 32, or the ADC of claim 33 or 34, or the immunocytokine of claim 35 or 36, or the CAR of claim 37, in the manufacture of a medicament for the treatment of an autoimmune disorder.

58. 58. The use of claim 57, wherein the autoimmune disorder is rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), celiac disease (CD), type 1 diabetes, primary Sjogren's syndrome (pSS), Guillain-Barré syndrome, inflammatory bowel disease (IBD), and psoriasis.

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