CD19-binding molecule and its uses

Abstract

The present disclosure provides CD19-binding molecules that specifically bind to CD19, including monospecific, bispecific, and trispecific binding molecules; conjugates comprising the CD19-binding molecules; and pharmaceutical compositions comprising the CD19-binding molecules and conjugates. The present disclosure further provides methods of using the C19-binding molecules to treat diseases and disorders associated with CD19 expression. The present disclosure also further provides recombinant host cells engineered to express the CD19-binding molecules, and methods of producing the CD19-binding molecules by culturing the host cells under conditions that express the CD19-binding molecules.

Description

[0001] 1. Cross - reference to related applications

[0002] This application claims the benefit of priority of U.S. Provisional Application No. 62 / 850,901, filed May 21, 2019, and U.S. Provisional Application No. 62 / 854,695, filed May 30, 2019, the contents of each of which are hereby incorporated by reference in their entirety.

[0003] 2. Sequence listing

[0004] This application contains a sequence listing that has been electronically submitted in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on May 4, 2020, has the name NOV - 007WO_SL.txt, and is 776,262 bytes in size. 3. Field of technology

[0006] This disclosure generally relates to CD19 - binding molecules that specifically bind to CD19, including monospecific, bispecific, and trispecific binding molecules, and their use in the treatment of diseases and disorders associated with CD19 expression. 4. Background art

[0008] B cells express a variety of cell - surface molecules during differentiation and proliferation. CD19 is a pan - B - cell membrane glycoprotein that is expressed early in pre - B - cell development through terminal differentiation, thereby regulating the development and function of B lymphocytes. Expression of CD19 has been identified in most lymphoid cancers, the vast majority of non - Hodgkin lymphomas (NHLs), and leukemias, including chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), and Waldenström macroglobulinemia (WM).

[0009] Blinatumomab is a CD19 - CD3 bispecific T - cell engager that is approved for the treatment of ALL. However, treatment with blinatumomab lacks a durable response and is characterized by a high relapse rate. Von Stackelberg et al., 2016, Journal of Clinical Oncology 34(36):4381 - 4389. In addition, blinatumomab has a short half - life, which requires continuous drug exposure to achieve sufficient efficacy and controllable toxicity. Porter et al., 2013, Clin Pharmacol. 5(Suppl 1):5 - 11.

[0010] Despite significant progress in cancer treatment, B cell malignancies, such as B cell subtypes of non-Hodgkin lymphoma and chronic lymphocytic leukemia, are a major cause of cancer-related death. Thus, there remains a need for further therapeutic agents to treat B cell malignancies. 5. SUMMARY OF THE INVENTION

[0012] The present disclosure provides CD19 binding molecules (e.g., antibodies, antigen-binding fragments thereof) that specifically bind to human CD19, and multispecific molecules that specifically bind to human CD19.

[0013] In one aspect, the present disclosure provides monospecific CD19 binding molecules (e.g., antibodies and antigen-binding fragments thereof) that comprise a CD19 antigen-binding domain or antigen-binding module ("ABM"). Exemplary CD19 binding molecules that may be monospecific are described in Section 7.2 and Specific Examples 1 to Specific Example 15 below.

[0014] In another aspect, the present disclosure provides multispecific binding molecules ("MBM") that comprise a CD19 ABM of the present disclosure.

[0015] In certain embodiments, the MBM is a bispecific binding molecule ("BBM"). The BBMs of the present disclosure comprise a first ABM ("ABM1" or "CD19 ABM") that specifically binds to human CD19 and a second ABM ("ABM2") that specifically binds to a second antigen, e.g., human CD3 or other components of the T cell receptor (TCR) complex (sometimes referred to herein as "TCR ABM"). The terms ABM1, ABM2, CD19 ABM, and TCR ABM are used for convenience only and are not intended to convey any particular configuration of the BBM. In some embodiments, the TCR ABM binds to CD3 (referred to herein as "CD3 ABM", etc.). Thus, the disclosures relating to ABM2 and TCR ABM also apply to CD3 ABM. Such multispecific molecules can be used to direct CD3+ effector T cells to CD19+ sites, thereby allowing CD3+ effector T cells to attack and lyse CD19+ cells and tumors. The characteristics of exemplary MBMs are described in Sections 7.5 to 7.6 and Specific Examples 16 to Specific Example 1190 below.

[0016] The present disclosure also extends the principle of redirected targeted T cell lysis (RTCC) by providing trispecific binding molecules (“TBMs”) that bind to CD19, CD3 or other components of the TCR complex on T cells, and CD2 or a human tumor-associated antigen (“TAA”), such as a B cell antigen other than CD19. The TBMs of the present disclosure comprise at least three antigen-binding modules (“ABMs”) that can bind (i) CD19 (ABM1), (ii) a component of the TCR complex (ABM2), and (iii) CD2 or a TAA (ABM3). For convenience, TBMs that bind to (1) human CD19, (2) CD3 or other components of the TCR complex, and (3) CD2 are referred to herein as “type 1 TBMs”. For convenience, TBMs that bind to (1) human CD19, (2) CD3 or other components of the TCR complex, and (3) a TAA are referred to herein as “type 2 TBMs”.

[0017] Without being bound by theory, the inventors believe that in type 1 TBMs, CD2- and TCR complex-binding can stimulate both the major signaling pathways that promote T cell-mediated tumor cell lysis (e.g., by clustering the TCR) and secondary costimulatory pathways to induce T cell proliferation and potentially overcome anergy. Similarly without being bound by theory, it is believed that engaging a TAA other than the components of CD19 and the TCR complex, type 2 TBMs will improve the clinical outcome of RTCC treatment of cancer (e.g., B cell malignancies) by targeting a greater number of cancerous B cells compared to using bispecific conjugates that target only the components of CD19 and the TCR complex.

[0018] Thus, in one aspect, the present disclosure provides type 1 TBMs that bind to (1) human CD19, (2) CD3 or other components of the TCR complex, and (3) CD2.

[0019] In another aspect, the present disclosure provides type 2 TBMs that bind to (1) human CD19, (2) CD3 or other components of the TCR complex, and (3) a TAA.

[0020] Unless otherwise expressly indicated or unless the context otherwise requires, references to TBMs in the present disclosure apply to both type 1 and type 2 TBMs.

[0021] In some embodiments, each antigen-binding module of the MBMs of the present disclosure is capable of binding its respective target while each of one or more additional antigen-binding modules binds its respective target. ABM1 is immunoglobulin-based, while ABM2 and ABM3 (when present) can be immunoglobulin-based or non-immunoglobulin-based. Thus, the MBMs can include any combination of immunoglobulin-based ABMs or immunoglobulin-based and non-immunoglobulin-based ABMs. Immunoglobulin-based ABMs that can be used for MBMs are described in Sections 7.3.1 and Specific Examples 17 to 21, 24 to 29 below. Non-immunoglobulin-based ABMs that can be used for MBMs are described in Section 7.3.2 and Specific Examples 22 to 23 below. Other features of exemplary ABMs that bind to human CD19 are described in Section 7.2 and Specific Examples 17 to 21 below. Other features of exemplary ABMs that bind to components of the TCR complex are described in Section 7.7 and Specific Examples 30 to 621 below. Other features of exemplary ABMs that bind to CD2 are described in Section 7.8 and Specific Examples 726 to 775 below. Other features of exemplary ABMs that bind to TAAs are described in Section 7.9 and Specific Examples 776 to 894 below.

[0022] The ABMs (or portions thereof) of the MBMs can be interconnected, for example, by short peptide linkers or by Fc domains. Methods and components for linking ABMs to form MBMs are described in Section 7.4 and Specific Examples 895 to 1190 below.

[0023] The BBMs have at least two ABMs (e.g., the BBMs are at least bivalent), and the TBMs have at least three ABMs (e.g., the TBMs are at least trivalent), but they can have a greater valency. For example, the BBMs can have three, four, or more ABMs (i.e., be trivalent, tetravalent, or have a valency greater than tetravalent). Exemplary bivalent, trivalent, and tetravalent BBM configurations are shown in FIG. 1 and described in Section 7.5 and Specific Examples 624 to 684 below.

[0024] The TBMs can have four ABMs (i.e., tetravalent), five ABMs (i.e., pentavalent), or six ABMs (i.e., hexavalent), provided that the TBMs have at least one ABM that can bind CD19, at least one ABM that can bind a component of the TCR complex, and at least one ABM that can bind CD2 or a TAA. Exemplary trivalent, tetravalent, pentavalent, and hexavalent TBM configurations are shown in FIG. 2 and described in Section 7.6 and Specific Examples 687 to 724 below.

[0025] The present disclosure further provides nucleic acids (in the form of a single nucleic acid or multiple nucleic acids) encoding the CD19-binding molecule, and recombinant host cells and cell lines engineered to express the nucleic acids and CD19-binding molecules of the present disclosure. Exemplary nucleic acids, host cells, and cell lines are described in Section 7.10 and Specific Examples 1241 to 1248 below.

[0026] The present disclosure further provides drug conjugates comprising the CD19-binding molecules of the present disclosure. For convenience, such conjugates are referred to herein as "antibody-drug conjugates" or "ADCs", although some ABMs may be non-immunoglobulin domains. Examples of ADCs are described in Section 7.12 and Specific Examples 1191 to 1230 below.

[0027] Also provided are pharmaceutical compositions comprising the CD19-binding molecule and the ADC. Examples of pharmaceutical compositions are described in Section 7.15 and Specific Example 1231 below.

[0028] The present disclosure further provides methods of using the CD19-binding molecule, the ADC, and the pharmaceutical compositions of the present disclosure, for example, for treating proliferative disorders (e.g., cancer) (on which CD19 is expressed), for treating autoimmune disorders, and for treating other diseases and disorders associated with CD19 expression. Exemplary methods are described in Section 7.16 and Specific Examples 1232 to 1239 below.

[0029] The present disclosure further provides methods of using the CD19-binding molecule, the ADC, and the pharmaceutical compositions in combination with other agents and therapies. Exemplary agents, therapies, and methods for combination therapy are described in Section 7.17 and Specific Example 1240 below. 6. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1A - 1AH : Exemplary BBM configurations. Figure 1A Shows Figure 1B - 1AH the components of the exemplary BBM configuration shown in. Not all regions connecting the different domains of each chain are shown (e.g., the linker connecting the VH and VL domains of the scFv, the hinge connecting the CH2 and CH3 domains of the Fc domain, etc. are omitted). Figure 1B - 1F Shows a bivalent BBM; Figure 1G - 1Z Shows a trivalent BBM; Figure 1AA - 1AH Shows a tetravalent BBM.

[0032] Figure 2A - 2V : Exemplary TBM configurations. Figure 2A Shows Figure 2B - 2VComponents of the exemplary TBM configurations shown. All regions connecting the different domains of each chain are not shown (e.g., the linker connecting the VH and VL domains of the scFv, the hinge connecting the CH2 and CH3 domains of the Fc, etc. are omitted). Figure 2B - 2P A trivalent TBM is shown; Figure 2Q - 2S A tetravalent TBM is shown; Figure 2T A pentavalent TBM is shown, and Figure 2U - 2V A hexavalent TBM is shown.

[0033] Figure 3A - 3B : Bispecificity of Example 1 ( Figure 3A and Figure 3C ) and trispecificity ( Figure 3B ) construct schematic diagrams.

[0034] Figure 4A - 4B : The ability of the CD19 BBM to trigger redirected T cell cytotoxicity (RTCC) activity against CD19+ target cells. Both the NEG258-based and NEG218-based BBMs mediated RTCC activity against CD19+ target cell lines. Nalm6-luc ( Figure 4A ) and Karpas422-luc ( Figure 4B ) cells were co-cultured with expanded T cells in the presence of serially diluted BBMs at an effector cell:target cell (E:T) ratio of 3:1. Luminescence signals were measured after 24 h of incubation.

[0035] Figure 5A - 5B : The ability of the CD19 BBM to trigger T cell proliferation. Both the NEG258-based and NEG218-based BBMs induced T cell proliferation. Karpas422-luc ( Figure 5A ) and Nalm6-luc ( Figure 5B ) cells were co-cultured with expanded T cells in the presence of serially diluted BBMs at an E:T ratio of 1:1. Luminescence signals were measured after 96 h of incubation.

[0036] Figure 6A - 6F : The ability of the CD19 TBM to trigger CD2-dependent T cell activation. CD2 knockout attenuated the advantage of the trispecific construct. Figure 6A - 6B Shows representative flow cytometry analysis of CD2 expression on JNL CD2 WT ( Figure 6A ) and KO ( Figure 6B ) cells. Staining with anti-CD2 mAb (filled dot histograms) overlaps with staining with mIgG1 isotype control (hatched histograms) or no staining (open histograms). Figure 6C - 6F Shows JNL CD2 in the presence of serially diluted BBMs and TBMs at an E:T ratio of 3:1 + (Figure 6C - 6D ) and CD2 - ( Figure 6E - 6F ) co - culture data of cells with CD19 + Target cells. Luminescence signals were measured after 24 h of incubation.

[0037] Figure 7A - 7B : Binding of CD19 TBM to cynomolgus monkey B cells. Figure 7A Data of TBM with CD19 - binding arm based on NEG218 are shown, and Figure 7B Data of TBM with CD19 - binding arm based on NEG258 are shown.

[0038] Figure 8A - 8H : Ability of CD19 TBM to induce T - cell activation after depletion of cynomolgus monkey B cells in PBMC. In Figure 8A , PBMC were isolated from cynomolgus monkey whole blood using ficoll gradient centrifugation and incubated overnight with bispecific or trispecific constructs. Samples were harvested and stained for CD3 and CD20 simultaneously to identify B cells and T cells in the PBMC population. The percentage of B - cell depletion was calculated as described in Section 8.6.1. Figure 8B - 8H Shows FACS analysis results of CD69 and CD25 expression on CD3 + T cells to determine single (CD69 + CD25 - or CD69 - CD25 + ) or double - positive cells (CD69 + CD25 + ). Figure 8B : Untreated (media only); Figure 8C - 8E : CD3hi TSP1L; Figure 8F - 8H : CD3hi TSP1.

[0039] Figure 9A - 9P : Ability of TBMs based on NEG258 and NEG218 to induce redirected T - cell cytotoxicity of human donor cells against Nalm6( Figure 9A - 9H ) and Karpas422( Figure 9I - 9P ) target cells.

[0040] Figure 10A - 10P : Ability of TBMs based on NEG258 and NEG218 with different CD3 affinities to induce redirected T - cell cytotoxicity of human donor cells against Nalm6( Figure 10A - 10H ) and Karpas422( Figure 10I - 10P ) target cells.

[0041] Figure 11A - 11L: The ability of NEG258-based TBMs containing a CD2 binding arm and a control lysozyme binding arm to induce redirected T cell cytotoxicity of human donor cells against Nalm6 ( Figure 11A - 11H ) and Karpas422 ( Figure 11I - 11L ) target cells.

[0042] Figure 12A - 12C : Induction of T cell cytokine release by NEG258-based and NEG218-based TBMs. Figure 12A : IFN-γ; Figure 12B : TNF-α; Figure 12C : IL2.

[0043] Figure 13A - 13C : Binding of NEG258-based and NEG218-based TBMs to murine 300.19 cell lines overexpressing human CD19 ( Figure 13A ) or cynomolgus monkey CD19 ( Figure 13B ). The TBMs showed negligible binding to wild-type 300.19 cell lines ( Figure 13C ).

[0044] Figure 14 : Schematic illustration of CD58.

[0045] Figure 15 : Redirected T cell cytotoxicity of TBMs containing CD58 variant sequences.

[0046] Figure 16 : Antigen-independent T cell activation of TBMs containing CD58 variant sequences. Data are presented as relative light units (RLU).

[0047] Figure 17A - 17H : Expression of CD19 and CD58 on various cell lines: Figure 17A - 17B : Expression of CD19 and CD58 on OCI-LY-19 cells, respectively; Figure 17C - 17D : Expression of CD19 and CD58 on Karpas-422 cells, respectively; Figure 17E - 17F : Expression of CD19 and CD58 on Toledo cells, respectively; Figure 17G - 17H : Expression of CD19 and CD58 on Nalm-6 cells, respectively.

[0048] Figure 18A - 18B : The ability of NEG258-based TBMs and BBMs to induce redirected T cell cytotoxicity of human donor cells against Karpas422 target cells. Figure 18A and Figure 18B show data using T cells from two different donors.

[0049] Figure 19A - 19F: T cell cytokine release induced by NEG258-based TBM and BBM. Figure 19A - 19B : IFN-γ (donor 1 and donor 2 respectively); Figure 19C - 19D : IL-2 (donor 1 and donor 2 respectively); Figure 19E - 19F : TNF-α (donor 1 and donor 2 respectively). Triangles on the x-axis indicate decreasing construct concentration from left to right in the figure.

[0050] Figure 20 : NEG-258-based TBM and BBM bound to T cells.

[0051] Figure 21A - 21C : NEG-258-based TBM and BBM-mediated T cell proliferation. Figure 21A : T cell proliferation in OC-LY-19 co-culture; Figure 21B : T cell proliferation in Karpas422 co-culture; Figure 21C : T cell proliferation in Toledo co-culture.

[0052] Figure 22A - 22B : Ability of NEG258-based TBM and BBM to induce redirected T cell cytotoxicity of human donor cells against Karpas422 target cells. Figure 22A and Figure 22B show data using T cells from two different donors.

[0053] Figure 23A - 23J : Ability of NEG258-based TBM and BBM to induce redirected T cell cytotoxicity of human donor cells against multiple target cells. Figure 23A - 23B : OC-LY-19 (donor 1 and donor 2 respectively); Figure 23C - 23D : Toledo (donor 1 and donor 2 respectively); Figure 23E - 23F : Nalm6 (donor 1 and donor 2 respectively); Figure 23G - 23H : Nalm6 KO (donor 1 and donor 2 respectively); Figure 23I - 23J : K562 (donor 1 and donor 2 respectively).

[0054] Figure 24A - 24J : T cell cytokine release induced by NEG258-based TBM and BBM in multiple target cells. Figure 24A - 24B : TNF-α from OC-LY-19 (donor 1 and donor 2 respectively); Figure 24C - 24D : TNF-α from Toledo (donor 1 and donor 2 respectively); Figure 24E - 24F : TNF-α from Nalm6 (donor 1 and donor 2 respectively); Figure 24G - 24H: TNF-α from Nalm6 KO (Donor 1 and Donor 2, respectively); Figure 24I - 24J : TNF-α from K562 (Donor 1 and Donor 2, respectively).

[0055] Figure 25A - 25H : Re-test the RTCC assay with Karpas 422 and OCI-LY-19 cell lines. Figure 25A : Assay setup. Figure 25B - 25D : Karpas 422 (after the first challenge, the second challenge, and the third challenge, respectively); Figure 25E - 25H OCI-LY-19 (after the first challenge, the second challenge, the third challenge, and the fourth challenge, respectively).

[0056] Figure 26A - 26P : Re-test the T cell phenotype with Karpas 422 and OCI-LY-19 cell lines. Figure 26A - 26H : Karpas 422 phenotype; Figure 26I - 26P : OCI-LY-19 phenotype. Figure 26A and 26I : % IL-2+ CD4 T cells; Figure 26B and 26J : % IFNγ+ CD4 T cells; Figure 26C and 26K : % IL-2+ CD8 T cells; Figure 26D and 26L : % IFNγ+ CD8 T cells; Figure 26E and 26M : CD3 young; Figure 26F and 26N : CD4 old; Figure 26G and 26O : CD8 young; Figure 26H and 26P : CD8 old. The lines in the figure represent different T cell donors.

[0057] Figure 27A - 27D : The ability of CD3hi TSP1 and CD3hi BSP1 to induce T cell proliferation in the presence of CD19+ target cells. In the presence of 1 nM ( Figure 27A - 27B ) or 0.1 nM ( Figure 27C - 27D ) CD3hi TSP1 or CD3hi BSP1 and in the presence of ( Figure 27A and 27C ) or absence of ( Figure 27B and 27D ) irradiated autologous PBMC (T cell depleted), Nalm6-luc cells were co-cultured with sorted CD28 + or CD28 -CD8 T cells were co-cultured at an E:T ratio of 1:3 for 72 h. Proliferation was measured as the percentage of CFSE-diluted cells among live cells.

[0058] Figure 28A - 28L : The ability of CD3hi TSP1 and CD3hi BSP1 to induce T cell cytokine production in the presence of Nalm6 CD19+ target cells (E:T 1:3). Figure 28A - 28B : CD28 was produced when co-cultured in the presence of irradiated PBMC and 1 nM CD3hi TSP1 or 1 nM CD3hi BSP1. - and CD28 + The median fluorescence intensity (MFI) of GzB ( Figure 28A ) and IFN-γ ( Figure 28B ) in CD8 T cells. Figure 28C - 28D : CD28 was produced when co-cultured in the absence of irradiated PBMC and 1 nM CD3hi TSP1 or 1 nM CD3hi BSP1. - and CD28 + The MFI of GzB ( ​ ) and IFN-γ ( ​ ) in CD8 T cells. ​ : CD28 was produced when co-cultured in the presence of irradiated PBMC and 0.1 nM CD3hi TSP1 or 0.1 nM CD3hi BSP1. - and CD28 + The MFI of GzB ( ​ ) and IFN-γ ( ​ ) in CD8 T cells. ​ : CD28 was produced when co-cultured in the absence of irradiated PBMC and 0.1 nM CD3hi TSP1 or 0.1 nM CD3hi BSP1. - and CD28 + The MFI of GzB ( ​ ) and IFN-γ ( ​ ) in CD8 T cells. ​ : The proportion of live T cells when co-cultured in the presence ( ​ and 28K ) or absence ( ​ and 28L ) of irradiated PBMC and 1 nM ( ​ and 28J ) or 0.1 nM ( ​ and ​ ) CD3hi TSP1 or CD3hi BSP1.

[0059] Figures: 29A - 29I: Ability of CD3hi TSP1 and CD3hi BSP1 to induce phenotypic changes in T cells. ​ : CD28 - and CD28 + T cells, representative examples. ​ : In the presence ( ​ ) or absence ( ​ ) of PBMC and in the presence of 1 nM ( ​ and 29F - 29G) or 0.1 nM ( ​ and 29H - 29I) CD3hi TSP1 or CD3hi BSP1, co - cultured (E:T 1:3) for 72 hours, the distribution of different T - cell populations defined by the combined expression of two surface markers CD45RO and CCR7 (naïve, CD45RO - CCR7 + ; central memory (CM), CD45RO + CCR7 + ; effector memory (EM), CD45RO + CCR7 - ; and terminally differentiated (TEMRA), CD45RO - CCR7 - ). Data for proliferating cells (CFSE - ) are shown in ​ , 29D , 29F, and 29H. Data for non - proliferating cells (CSFE + ) are shown in ​ , 29E , 29G, and 29I. Data for CD28 - cells are shown on the left side of each figure, and data for CD28 + cells are shown on the right side of the figure.

[0060] ​ : Ability of CD3hi TSP1 and CD3hi BSP1 to elicit redirected T - cell cytotoxicity activity (RTCC) against CD19 + target cells. In the presence of 1 nM ( ​ and 30C ) or 0.1 nM ( ​ and 30D ) CD3hi BSP1, CD3hiTSP1 or CD3hi TSP1C and in the presence ( ​ and 30B ) or absence ( ​ and 30D ) of irradiated autologous PBMC (T - cell depleted), from sorted CD28 + or CD28 -RTCC results of Nalm6-luc cells co-cultured with CD8 T cells at an E:T ratio of 1:3 for 72 h. (n = 3) Luminescence signals were measured at the end of the co-culture incubation. Results are expressed as fold increase compared to the untreated condition, where no antibody was added to evaluate the background signal given by the control antibody.

[0061] ​ : Antitumor activities of CD3hi TSP1( ​ ) and CD3med TSP1( ​ ) in the adoptive transfer adaptation of human PBMCs in the OCI-LY-19 subcutaneous tumor model.

[0062] ​ : Weight changes after treatment with CD3hi TSP1( ​ ) and CD3med TSP1( ​ ) in the adoptive transfer adaptation of human PBMCs in the OCI-LY-19 subcutaneous tumor model.

[0063] ​ : Schematic diagram of the humanization process of NSG mice.

[0064] ​ : Antitumor activities of CD3 TSP1, CD3hi BSP1, and CD3med TSP1 in the DLBCL subcutaneous tumor model of huCD34+ NSG mice( ​ ), and weight changes after treatment with CD3TSP1, CD3hi BSP1, and CD3med TSP1 in the DLBCL subcutaneous tumor model of huCD34+ NSG mice( ​ ).

[0065] ​ : Antitumor activities( ​ and 35B ) and weight responses( ​ and 35D ) after antibody treatment with CD3hi TSP1( ​ and 35C ) and CD3med TSP1( ​ and 35D ) in the OCI-LY-19 DLBCL subcutaneous tumor model of huCD34+ NSG mice.

[0066] ​ : Antitumor activities of CD3hi BSP1( ​ ), CD3hi TSP1( ​ ), and CD3med TSP1( ​)Antitumor activity in the adoptive transfer adaptation of human PBMCs in the Daudi-Luc subcutaneous tumor model.

[0067] ​ : In the adoptive transfer adaptation of human PBMCs in the Daudi-Luc subcutaneous tumor model, after antibody treatment with CD3hi BSP1( ​ ), CD3hi TSP1( ​ ), or CD3med TSP1( ​ ), the change in body weight.

[0068] ​ : Schematic diagram of the trispecific construct of Example 32. ​ : TBM with the full-length CD58 moiety AB2-1; ​ : TBM with a truncated CD58 moiety containing the IgV-like domain of CD58; ​ : TBM with an scFv corresponding to the anti-CD2 antibody Medi 507.

[0069] ​ : Schematic diagram of the trispecific construct of Example 33. ​ : TBM with the CD58IgV domain from Example 32; ​ : TBM with a "left" half-antibody and a "right" half-antibody, where the "left" half-antibody has a CD58 IgV domain, an anti-CD3 scFab, and an Fc domain in the N-terminal to C-terminal direction, and the "right" half-antibody has an anti-CD19 Fab N-terminal for the Fc domain; ​ : TBM with a "left" half-antibody and a "right" half-antibody, where the "left" half-antibody has a CD58 IgV domain, an anti-CD3 scFv, and an Fc domain in the N-terminal to C-terminal direction, and the "right" half-antibody has an anti-CD19 Fab N-terminal for the Fc domain; ​ : TBM with a "left" half-antibody and a "right" half-antibody, where the "left" half-antibody has an anti-CD3scFv, a CD58 IgV domain, and an Fc domain in the N-terminal to C-terminal direction, and the "right" half-antibody has an anti-CD19 Fab N-terminal for the Fc domain; ​ : TBM with a "left" half-antibody and a "right" half-antibody, where the "left" half-antibody has an anti-CD3 scFv, an Fc domain, and a CD58 IgV domain in the N-terminal to C-terminal direction, and the "right" half-antibody has an anti-CD19 Fab N-terminal for the Fc domain.

[0070] ​: Schematic diagram of the trispecific construct of Example 34. ​ : TBM having the CD58IgV domain from Example 32; ​ : TBM having a "left" half-antibody and a "right" half-antibody, the "left" half-antibody having an anti-CD3 scFv, an Fc domain, and a CD19 scFv domain in the N-terminal to C-terminal direction, and the "right" half-antibody having an N-terminal of the CD58 IgV domain for the Fc domain. Figure 40C : TBM having a "left" half-antibody and a "right" half-antibody, the "left" half-antibody having an anti-CD3 scFv, an Fc domain, and a CD19 Fab domain in the N-terminal to C-terminal direction, and the "right" half-antibody having an N-terminal of the CD58 IgV domain for the Fc domain. 7. Detailed Description

[0072] 7.1. Definitions

[0073] As used herein, the following terms are intended to have the following meanings:

[0074] ABM chain : A single ABM can exist as a single polypeptide chain (e.g., in the case of an scFv) or be formed by the association of more than one polypeptide chain (e.g., in the case of a Fab). As used herein, the term "ABM chain" refers to all or part of an ABM present on a single polypeptide chain. The use of the term "ABM chain" is for convenience only and for descriptive purposes only, and does not imply a particular configuration or production method.

[0075] ADCC : As used herein, "ADCC" or "antibody-dependent cell-mediated cytotoxicity" refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing FcγR recognize a bound antibody on a target cell and subsequently cause lysis of the target cell. ADCC is associated with binding to FcγRIIIa; an increase in binding to FcγRIIIa leads to an increase in ADCC activity.

[0076] ADCP : As used herein, "ADCP" or antibody-dependent cell-mediated phagocytosis refers to a cell-mediated reaction in which non-specific phagocytic cells expressing FcγR recognize a bound antibody on a target cell and subsequently cause phagocytosis of the target cell.

[0077] Additional agent : For convenience, agents used in combination with the antigen-binding molecules of the present disclosure are referred to herein as "additional" agents.

[0078] Antibody:As used herein, the term "antibody" refers to a polypeptide (or group of polypeptides) of the immunoglobulin family that is capable of non-covalently, reversibly, and specifically binding an antigen. For example, a naturally occurring IgG-type "antibody" is a tetramer comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region contains three domains, namely CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). The term "antibody" includes, but is not limited to: monoclonal antibodies, human antibodies, humanized antibodies, camelised antibodies, chimeric antibodies, bispecific or multispecific antibodies, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies directed against the antibodies of the present disclosure). These antibodies can belong to any isotype / type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0079] Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it should be understood that the variable domains of both the light chain (VL) and the heavy chain (VH) determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CH1, CH2, or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. By convention, the constant region domains are numbered with increasing numbers the further they are from the antigen-binding site or the amino terminus of the antibody. In a wild-type antibody, the variable region is at the N-terminus and the constant region is at the C-terminus; the CH3 domain and the CL domain actually contain the carboxyl termini of the heavy and light chains, respectively.

[0080] Antibody fragment: As used herein, the term "antibody fragment" of an antibody refers to one or more portions of an antibody. In some embodiments, these portions are part of one or more of the antibody's contact domains. In some other embodiments, these portions are antigen-binding fragments (which retain the ability to bind an antigen non-covalently, reversibly, and specifically), sometimes referred to herein as "antigen-binding fragments", "its antigen-binding fragments", "antigen-binding portions", etc. Examples of binding fragments include, but are not limited to, single-chain Fv (scFv), Fab fragments, monovalent fragments consisting of the VL, VH, CL, and CH1 domains; F(ab)2 fragments, bivalent fragments containing two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragments consisting of the VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; dAb fragments consisting of the VH domain (Ward et al., 1989, Nature 341:544-546); and isolated complementarity-determining regions (CDRs). Thus, the term "antibody fragment" encompasses proteolytic fragments of an antibody (e.g., Fab and F(ab)2 fragments) and engineered proteins containing one or more portions of an antibody (e.g., scFv).

[0081] Antibody fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, intracellular antibodies, diabodies, triabodies, tetra-bodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, 2005 Nature Biotechnology 23:1126-1136). Antibody fragments can be grafted into scaffolds based on polypeptides such as type III fibronectin (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide monomers).

[0082] Antibody fragments can be incorporated into single-chain molecules containing a pair of tandem Fv fragments (e.g., VH-CH1-VH-CH1), together with complementary light-chain polypeptides (e.g., VL-VC-VL-VC) to form a pair of antigen-binding regions (Zapata et al., 1995, Protein Eng. 8:1057-1062; and U.S. Patent No. 5,641,870).

[0083] Antibody numbering system: In this specification, unless otherwise indicated, references to numbered amino acid residues in antibody domains are based on the EU numbering system (e.g., in Table 1). This system was originally designed by Edelman et al., 1969, Proc. Nat’l Acad. Sci. USA 63:78-85 and was described in detail by Kabat et al., 1991, in Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, NIH, USA.

[0084] Antigen-binding module : As used herein, the term “antigen-binding module” or “ABM” refers to the portion of the MBM that has the ability to non-covalently, reversibly, and specifically bind to an antigen. The ABM can be immunoglobulin-based or non-immunoglobulin-based. As used herein, the terms “ABM1” and “CD19 ABM” (etc.) refer to an ABM that specifically binds to CD19, the terms “ABM2” and “TCR ABM” (etc.) refer to an ABM that specifically binds to a component of the TCR complex, the term “ABM3” refers to an ABM that specifically binds to CD2 or a TAA (depending on the context), the term “CD2 ABM” (etc.) refers to an ABM that specifically binds to CD2, and the term “TAA ABM” (etc.) refers to an ABM that specifically binds to a TAA. The terms ABM1, ABM2, and ABM3 are used for convenience only and are not intended to convey any particular configuration of the MBM. In some embodiments, ABM2 binds to CD3 (referred to herein as “CD3 ABM” etc.). Thus, the disclosures relating to ABM2 and multiple ABM2s also apply to CD3 ABM.

[0085] Antigen-binding fragment : The term “antigen-binding fragment” of an antibody refers to the portion of the antibody that retains the ability to non-covalently, reversibly, and specifically bind to an antigen.

[0086] Antigen-binding molecule: The term "antigen-binding molecule" refers to a molecule that contains one or more antigen-binding domains, such as an antibody. The antigen-binding molecule can contain one or more polypeptide chains, e.g., one, two, three, four, or more polypeptide chains. These polypeptide chains in the antigen-binding molecule can associate directly or indirectly with each other (e.g., a first polypeptide chain can associate with a second polypeptide chain, which in turn can associate with a third polypeptide chain to form an antigen-binding molecule, where the first and second polypeptide chains associate directly with each other, the second and third polypeptide chains associate directly with each other, and the first and third polypeptide chains associate indirectly with each other through the second polypeptide chain).

[0087] Association : In the context of an antigen-binding molecule, the term "associate" refers to a functional relationship between two or more polypeptide chains and / or two or more portions of a single polypeptide chain. In particular, the term "associate" means that two or more polypeptides (or portions of a single polypeptide) associate with each other, e.g., non-covalently associate through molecular interactions and / or covalently associate through one or more disulfide bridges or chemical crosslinks, thereby resulting in a functional antigen-binding molecule, e.g., where the antigen-binding domain can bind the BBM or TBM of its respective target. Examples of associations that may be present in the MBM include (but are not limited to) the association between Fc regions in the Fc domain (such as the homodimer or heterodimer described in Section 7.4.1.5), the association between the VH and VL regions in a Fab or Fv, and the association between CH1 and CL in a Fab.

[0088] B cell: As used herein, the term "B cell" refers to a cell of the B cell lineage, which is a type of white blood cell that is a lymphocyte subtype. Examples of B cells include plasmablasts, plasma cells, lymphoplasmacytoid cells, memory B cells, follicular B cells, marginal zone B cells, B-1 cells, B-2 cells, and regulatory B cells.

[0089] B cell malignancy:As used herein, B cell malignancies refer to the uncontrolled proliferation of B cells. Examples of B cell malignancies include non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, leukemia, and myeloma. For example, B cell malignancies can be, but are not limited to: multiple myeloma, chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), follicular lymphoma, mantle cell lymphoma (MCL), diffuse large B cell lymphoma (DLBCL), marginal zone lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (Waldenström macroglobulinemia), hairy cell leukemia, primary central nervous system (CNS) lymphoma, primary mediastinal large B cell lymphoma, mediastinal gray zone lymphoma (MGZL), splenic marginal zone B cell lymphoma, extranodal marginal zone B cell lymphoma of MALT, nodal marginal zone B cell lymphoma, and primary effusion lymphoma, and plasmacytoid dendritic cell tumors.

[0090] Binding sequence : Referring to Table 1, 12, 13, 14, 16, or 17 (including subparts thereof), the term "binding sequence" means an ABM having the entire set of CDRs, VH-VL pairs, or scFvs listed in that table.

[0091] Bispecific binding molecule : The term "bispecific binding molecule" or "BBM" refers to a molecule that specifically binds to two antigens and contains two or more ABMs. The BBMs of the present disclosure contain at least one antigen-binding domain specific for CD19 and at least one antigen-binding domain specific for a different antigen (e.g., a component of the TCR complex). Representative BBMs are shown in Figure 1B-1AH . BBMs can contain one, two, three, four, or even more polypeptide chains.

[0092] Bivalent : As used herein, in the context of an antigen-binding molecule, the term "bivalent" refers to an antigen-binding molecule having two antigen-binding domains. The domains can be the same or different. Thus, a bivalent antigen-binding molecule can be monospecific or bispecific. A bivalent BBM can contain an ABM that specifically binds to CD19 and another ABM that binds to another antigen (e.g., a component of the TCR complex).

[0093] Cancer: The term "cancer" refers to diseases characterized by the uncontrolled (and often rapid) growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and the examples include but are not limited to: leukemia, multiple myeloma, asymptomatic myeloma, Hodgkin lymphoma, and non-Hodgkin lymphoma, for example, any CD19-positive cancer of any of the foregoing types. The term "cancerous B cell" refers to a B cell that is undergoing or has undergone uncontrolled proliferation.

[0094] CD3 : The term "CD3" or "cluster of differentiation 3" refers to the cluster of differentiation 3 coreceptor of the T cell receptor. CD3 helps to activate cytotoxic T cells (e.g., CD8+ naive T cells) and helper T cells (e.g., CD4+ naive T cells) and is composed of four different chains: one CD3γ chain (e.g., Genbank accession number NM_000073 and MP_000064 (human)), one CD3δ chain (e.g., Genbank accession numbers NM_000732, NM_001040651, NP_00732, and NP_001035741 (human)), and two CD3ε chains (e.g., Genbank accession numbers NM_000733 and NP_00724 (human)). The chains of CD3 are highly related cell surface proteins of the immunoglobulin superfamily that contain a single extracellular immunoglobulin domain. The CD3 molecule associates with the T cell receptor (TCR) and the ζ chain to form the T cell receptor (TCR) complex, the role of which is to generate activation signals in T lymphocytes. Unless expressly stated otherwise, references to CD3 in this application may refer to the CD3 coreceptor, the CD3 coreceptor complex, or any polypeptide chain of the CD3 coreceptor complex.

[0095] CD19The term "CD19" or "cluster of differentiation 19" refers to the cluster of differentiation 19 protein, which is an antigenic determinant detectable on preleukemic cells. The amino acid and nucleic acid sequences of human and murine can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD19 can be found as UniProt / Swiss-Prot accession number P15391, and the nucleotide sequence encoding human CD19 can be found under accession number NM_001178098. CD19 is expressed on most B-lineage cancers, including, for example, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma. Other cells expressing CD19 are provided in the definition of "diseases associated with CD19 expression" below. It is also an early marker of B cell progenitors. See, for example, Nicholson et al., 1997, Mol. Immunol. 34(16-17):1157-1165.

[0096] Chimeric antibody The term "chimeric antibody" (or antigen-binding fragment thereof) is an antibody molecule (or antigen-binding fragment thereof) wherein (a) the constant region or a portion thereof is altered, replaced, or exchanged such that the antigen-binding site (variable region) is linked to a constant region of a different or altered type, effector function, and / or class, or to a completely different molecule (e.g., an enzyme, toxin, hormone, growth factor, drug, etc.) that confers new properties to the chimeric antibody; or (b) the variable region or a portion thereof is altered, replaced, or exchanged with a variable region having a different or altered antigen specificity. For example, a murine antibody can be modified by replacing its constant region with a constant region from a human immunoglobulin. Due to the replacement with a human constant region, the chimeric antibody can retain its antigen-recognition specificity while having reduced antigenicity in the human body compared to the original murine antibody.

[0097] Combination As used herein, "administering in combination" means delivering two (or more) different treatments to a subject during the subject's illness, e.g., delivering two or more treatments after the subject has been diagnosed with a disorder and before the disorder has been cured or cleared or before treatment is terminated for other reasons.

[0098] Complementary determining region:As used herein, the term "complementary determining region" or "CDR" refers to the sequences of amino acids within the variable region of an antibody that confer antigen specificity and binding affinity. For example, generally, there are three CDRs present in each heavy chain variable region (e.g., CDR-H1, CDR-H2, and CDR-H3), and three CDRs present in each light chain variable region (CDR-L1, CDR-L2, and CDR-L3). The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al., 1991, "Sequences of Proteins of Immunological Interest", 5th ed., National Institutes of Health, Public Health Service, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., 1997, JMB 273:927-948 ("Chothia" numbering scheme), and the ImMunoGenTics (IMGT) numbering (Lefranc, 1999, The Immunologist 7:132-136; Lefranc et al., 2003, Dev. Comp. Immunol. 27:55-77 ("IMGT" numbering scheme). For example, for the classical form, according to Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3). According to Chothia, the CDR amino acids in VH are numbered 26-32 (CDR-H1), 52-56 (CDR-H2), and 95-102 (CDR-H3); and the amino acid residues in VL are numbered 26-32 (CDR-L1), 50-52 (CDR-L2), and 91-96 (CDR-L3). By combining the CDR definitions of both Kabat and Chothia, the CDR is composed of amino acid residues 26-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3) in human VH and amino acid residues 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3) in human VL.According to IMGT, the CDR amino acid residues in VH are numbered approximately 26 - 35 (CDR-H1), 51 - 57 (CDR-H2), and 93 - 102 (CDR-H3), and the CDR amino acid residues in VL are numbered approximately 27 - 32 (CDR-L1), 50 - 52 (CDR-L2), and 89 - 97 (CDR-L3) (according to "Kabat" numbering). According to IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[0099] Concurrent The term "parallel" is not limited to administering therapies (e.g., prophylactic or therapeutic agents) at exactly the same time, but rather means that a pharmaceutical composition comprising an antigen-binding molecule of the present disclosure is administered to a subject in a sequence and at time intervals such that the molecule can act together with one or more additional therapies to provide an increased benefit (compared to if they were administered otherwise).

[0100] Conservative sequence modification The term "conservative sequence modification" refers to amino acid modifications that do not significantly affect or alter the binding characteristics of a CD19-binding molecule or its components (e.g., the CD19-binding domain or the Fc region). Such conservative modifications include amino acid substitutions, additions, and deletions. The modifications can be introduced into the binding molecule by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the binding molecule can be replaced with other amino acid residues from the same side chain family, and the altered binding molecule can be tested for, e.g., binding to a target molecule and / or efficient heterodimerization and / or effector function.

[0101] Diabody: As used herein, the term "diabody" refers to a small antibody fragment having two antigen-binding sites, typically formed by the pairing of scFv chains. Each scFv contains a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL, where VH is at the N-terminus or C-terminus of VL). Different from a typical scFv in which VH and VL are separated by a linker that allows VH and VL on the same polypeptide chain to pair and form an antigen-binding domain, a diabody typically contains a linker, but the linker is too short to allow VH and VL domains on the same chain to pair, thus forcing the VH and VL domains to pair with the complementary domains of the other chain and generating two antigen-binding sites. Diabodies are more fully described in the following references: for example, EP 404,097; WO93 / 11161; and Hollinger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448.

[0102] dsFv : The term "dsFv" refers to a disulfide-stabilized Fv fragment. In a dsFv, VH and VL are linked by an interdomain disulfide bond. To generate such a molecule, one amino acid in the framework region of both VH and VL is mutated to cysteine, which in turn forms a stable interchain disulfide bond. Typically, position 44 in VH and position 100 in VL are mutated to cysteine. See Brinkmann, 2010, Antibody Engineering 181-189, DOI:10.1007 / 978-3-642-01147-4_14. The term dsFv encompasses so-called dsFv (molecules in which VH and VL are linked by an interchain disulfide bond rather than a linker peptide) or scdsFv (molecules in which VH and VL are linked by a linker and an interchain disulfide bond).

[0103] Effector function: The term "effector function" refers to the activities of an antibody molecule that are mediated by binding through domains of the antibody other than the antigen-binding domain, typically by binding of effector molecules. Effector functions include complement-mediated effector functions, which are mediated, for example, by the binding of C1 component of complement to the antibody. Activation of complement is important in opsonization and lysis of cellular pathogens. Activation of complement also stimulates an inflammatory response and may be involved in autoimmune hypersensitivity responses. Effector functions also include Fc receptor (FcR)-mediated effector functions, which can be triggered by the binding of the constant domain of an antibody to an Fc receptor (FcR). Binding of an antibody to an Fc receptor on the cell surface triggers many important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (referred to as antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production. The effector functions of an antibody can be altered by changing, for example, enhancing or reducing the affinity of the antibody for effector molecules such as Fc receptors or complement components. Typically, the binding affinity will be altered by modifying the effector molecule binding site, and in such cases, it is appropriate to localize the site of interest and modify at least a portion of the site in a suitable manner. It is also contemplated that altering the binding site on the antibody for an effector molecule need not significantly alter the overall binding affinity, but can alter the geometry of the interaction, resulting in an ineffective effector mechanism, as in non-productive binding. It is further contemplated that effector functions can also be altered by modifying sites that do not directly participate in effector molecule binding but otherwise participate in the properties of effector functions.

[0104] Epitope : An epitope or antigenic determinant is a portion of an antigen that can be recognized by an antibody or other antigen-binding moiety as described herein. Epitopes can be linear or conformational.

[0105] Fab : As used herein, "Fab" or "Fab region" means a polypeptide region that includes the VH, CH1, VL, and CL immunoglobulin domains. These terms can refer to this region alone or to this region in the context of an antigen-binding molecule of the present disclosure.

[0106] The Fab domain is formed by the association of the CH1 domain attached to the VH domain with the CL domain attached to the VL domain. The VH domain pairs with the VL domain to form the Fv region, and the CH1 domain pairs with the CL domain to further stabilize the binding module. A disulfide bond between the two constant domains can further stabilize the Fab domain.

[0107] The Fab region can be produced by proteolytic cleavage of an immunoglobulin molecule (e.g., using an enzyme such as papain) or by recombinant expression. In a native immunoglobulin molecule, the Fab is formed by the association of two different polypeptide chains (e.g., VH-CH1 on one chain associates with VL-CL on another chain). The Fab region is typically recombinantly expressed, typically on two polypeptide chains, although single-chain Fabs are also contemplated herein.

[0108] Fc domain : The term "Fc domain" refers to a pair of associated Fc regions. These two Fc regions dimerize to produce the Fc domain. The two Fc regions in the Fc domain can be the same (such an Fc domain is referred to herein as an "Fc homodimer") or different from each other (such an Fc domain is referred to herein as an "Fc heterodimer").

[0109] Fc region: As used herein, the term "Fc region" or "Fc chain" means a polypeptide comprising the CH2-CH3 domains of an IgG molecule and, in some cases, including the hinge. In the EU numbering of human IgG1, the CH2-CH3 domains comprise amino acids 231 to amino acid 447, and the hinge is amino acids 216 to amino acid 230. Thus, the definition of "Fc region" includes amino acids 231-447 (CH2-CH3) or 216-447 (hinge-CH2-CH3), or a fragment thereof. An "Fc fragment" in this context can contain fewer amino acids from one or both of the N-terminus and C-terminus but still retains the ability to dimerize with another Fc region, which ability can be detected using standard methods (usually based on size) (e.g., non-denaturing chromatography, size exclusion chromatography). The human IgG Fc region has specific uses in this disclosure and can be an Fc region from human IgG1, IgG2, or IgG4.

[0110] Fv: The term "Fv" refers to the smallest antibody fragment that can be derived from an immunoglobulin and that contains the complete target recognition and binding site. This region consists of a dimer of one heavy-chain and one light-chain variable domain (VH-VL dimer) that are tightly and non-covalently associated. In this configuration, the three CDRs of each variable domain interact to define the target binding site on the surface of the VH-VL dimer. Typically, six CDRs confer the binding specificity of the antibody for the target. However, in some cases, even a single variable domain (or half of an Fv that contains only the three CDRs specific for the target) can have the ability to recognize and bind the target. The reference herein to the VH-VL dimer is not intended to convey any particular configuration. By way of example and not limitation, the VH and VL can be joined together in any of the configurations described herein to form a half-antibody, or can each be present on separate half-antibodies and bind together when the separate half-antibodies associate to form an antigen-binding domain, such as the TBM of the present disclosure. When present on a single polypeptide chain (e.g., scFv), the VH is either the N-terminus or the C-terminus of the VL.

[0111] Half antibody : The term "half-antibody" refers to a molecule that contains at least one ABM or an ABM chain and that can associate with another molecule containing an ABM or an ABM chain, for example, by a disulfide bridge or a molecular interaction (e.g., the knob-into-hole structure interaction between Fc heterodimers). A half-antibody can consist of one polypeptide chain or more than one polypeptide chain (e.g., the two polypeptide chains of a Fab). In an embodiment, the half-antibody contains an Fc region.

[0112] Examples of half-antibodies are molecules that contain the heavy and light chains of an antibody (e.g., an IgG antibody). Another example of a half-antibody is a molecule that contains a first polypeptide and a second polypeptide, where the first polypeptide contains a VL domain and a CL domain and the second polypeptide contains a VH domain, a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain, and where the VL and VH domains form an ABM. Yet another example of a half-antibody is a polypeptide that contains an scFv domain, a CH2 domain, and a CH3 domain.

[0113] A half-antibody can include more than one ABM, such as a half-antibody that contains (in order from the N-terminus to the C-terminus) an scFv domain, a CH2 domain, a CH3 domain, and another scFv domain.

[0114] A half-antibody can also include an ABM chain that forms a complete ABM when associated with another ABM chain in another half-antibody.

[0115] Thus, an MBM can contain one, more typically two, or even more than two half-antibodies, and a half-antibody can contain one or more ABMs or one or more ABM chains.

[0116] In some MBMs, the first half-antibody will associate with the second half-antibody, e.g., heterodimerize. In other MBMs, the first half-antibody will be covalently linked to the second half-antibody, e.g., via a disulfide bridge or chemical crosslinking. In still other MBMs, the first half-antibody will associate with the second half-antibody via covalent attachment and non-covalent interactions, e.g., disulfide bridge and knob-into-hole structure interactions.

[0117] The term "half-antibody" is only intended for descriptive purposes and does not denote a particular configuration or production method. The description of a half-antibody as a "first" half-antibody, "second" half-antibody, "left" half-antibody, "right" half-antibody, etc. is merely for convenience and descriptive purposes.

[0118] Hexavalent : In the context of an antigen-binding molecule (e.g., a TBM), the term "hexavalent" as used herein refers to an antigen-binding molecule having six antigen-binding domains. Although different configurations (e.g., three antigen-binding domains that bind to CD19, two antigen-binding domains that bind to components of the TCR complex, and one antigen-binding domain that binds to CD2 or a TAA, or three antigen-binding domains that bind to CD19, two antigen-binding domains that bind to CD2 or a TAA, and one antigen-binding domain that binds to components of the TCR complex) are within the scope of this disclosure, the hexavalent TBMs of this disclosure typically have three pairs of antigen-binding domains that each bind to the same antigen. Examples of hexavalent TBMs are schematically shown in Figure 1U-1V in.

[0119] Knob: In the context of a knob-into-hole structure, a "knob" refers to at least one amino acid side chain that indents into the interface of a first Fc chain and can thus be positioned in a complementary "hole" on the adjacent junction surface of a second Fc chain, thereby stabilizing the Fc heterodimer and thus, for example, favoring Fc heterodimer formation compared to an Fc homodimer.

[0120] Host cell or recombinant host cell: The term "host cell" or "recombinant host cell" refers to, for example, a cell that has been genetically engineered by the introduction of heterologous nucleic acid. It should be understood that such term not only refers to a particular subject cell, but also to progeny of such a cell. Because certain modifications may occur in progeny due to mutation or environmental influences, such progeny may in fact be different from the parental cell, but are still included within the scope of the term "host cell" as used herein. A host cell can transiently carry heterologous nucleic acid on an episomal heterologous expression vector, or stably carry heterologous nucleic acid, for example, by integration of the heterologous nucleic acid into the host cell genome. For the purpose of expressing an antigen-binding molecule, the host cell can be a cell line of mammalian origin or a cell line having mammalian-like characteristics, such as monkey kidney cells (COS, e.g., COS-1, COS-7), HEK293, baby hamster kidney (BHK, e.g., BHK21), Chinese hamster ovary (CHO), NSO, PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., Hep G2), SP2 / 0, HeLa, equine-Darby bovine kidney (MDBK), myeloma and lymphoma cells, or derivatives and / or engineered variants thereof. Engineered variants include, for example, glycan profile-modified and / or site-specific integration site derivatives.

[0121] Human antibody : As used herein, the term "human antibody" includes antibodies having variable regions in which both the framework regions and the CDR regions are derived from sequences of human origin. In addition, if the antibody contains a constant region, the constant region is also derived from such human sequences, such as human germline sequences, or mutant forms of human germline sequences or antibodies containing consensus framework sequences derived from analysis of human framework sequences, for example, as described by Knappik et al., 2000, J Mol Biol [Journal of Molecular Biology] 296, 57-86. The structure and location of immunoglobulin variable domains (e.g., CDRs) can be defined using well-known numbering schemes (e.g., the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia) (see, for example, Lazikani et al., 1997, J. Mol. Bio. [Journal of Molecular Biology] 273:927 948; Kabat et al., 1991, Sequences of Proteins of Immunological Interest [Sequences of Proteins of Immunological Interest], 5th ed., NIH Publication No. 91-3242, U.S. Department of Health and Human Services; Chothia et al., 1987, J. Mol. Biol. [Journal of Molecular Biology] 196:901-917; Chothia et al., 1989, Nature [Nature] 342:877-883).

[0122] Human antibodies can include amino acid residues that are not encoded by human sequences (e.g., introduced by random mutagenesis or site-specific mutagenesis in vitro, or by somatic mutation in vivo, or conservative substitutions to enhance stability or production). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been transplanted onto human framework sequences.

[0123] Humanized The "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody that contains minimal sequence derived from a non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the hypervariable regions of the recipient are replaced by residues from hypervariable regions (donor antibody) of a non-human species (such as a mouse, rat, rabbit, or non-human primate) having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by the corresponding non-human residues. Furthermore, humanized antibodies can contain residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further improve antibody performance. Typically, a humanized antibody will contain substantially all of the following: at least one, and typically two, variable domains in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence. A humanized antibody optionally also contains an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region. For further details, see Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988, Nature 332:323-329; and Presta, 1992, Curr. Op. Struct. Biol. 2:593-596. See also the following review articles and references cited therein: Vaswani and Hamilton, 1998, Ann. Allergy, Asthma & Immunol. 1:105-115; Harris, 1995, Biochem. Soc. Transactions 23:1035-1038; Hurle and Gross, 1994, Curr. Op. Biotech. 5:428-433.

[0124] Pestle: In the context of a knob-in-hole structure, the "knob" refers to at least one amino acid side chain that protrudes from the surface of a first Fc chain and can therefore be positioned in a complementary "hole" on the interface of a second Fc chain, thereby stabilizing the Fc heterodimer and thereby favoring the formation of Fc heterodimers over Fc homodimers, for example.

[0125] Knobs and holes (or knobs-into-holes): One mechanism of Fc heterodimerization is often referred to in the art as "knobs and holes," or "knob-in-holes," or "knobs-into-holes." These terms refer to amino acid mutations that produce steric effects that favor the formation of Fc heterodimers over Fc homodimers, as described, for example, in Ridgway et al., 1996, Protein Engineering 9(7):617; Atwell et al., 1997, J. Mol. Biol. 270:26; and U.S. Pat. No. 8,216,805. Knobs and holes mutations can be combined with other strategies to improve heterodimerization, for example, as described in Section 7.4.1.6.

[0126] Monoclonal antibody As used herein, the term "monoclonal antibody" refers to polypeptides, including antibodies, antibody fragments, molecules (including MBMs), etc., that are derived from the same genetic source.

[0127] Monovalent : As used herein, in the context of antigen binding molecules, the term "monovalent" refers to an antigen binding molecule that has a single antigen binding domain.

[0128] Multispecific binding molecule The term "multispecific binding molecule" or "MBM" refers to a molecule that specifically binds to at least two antigens and comprises two or more antigen-binding domains. Each of the antigen-binding domains can independently be an antibody fragment (e.g., scFv, Fab, nanobody), a ligand, or a non-antibody-derived binder (e.g., fibronectin, Fynomer, DARPin).

[0129] Mutation or modification :In the context of the primary amino acid sequence of a polypeptide, the terms "modification" and "mutation" refer to amino acid substitutions, insertions, and / or deletions relative to a reference polypeptide polypeptide sequence. Additionally, the term "modification" further encompasses changes to amino acid residues, such as by chemical conjugation (e.g., chemical conjugation of a drug or polyethylene glycol moiety) or post-translational modification (e.g., glycosylation).

[0130] Nucleic acid : The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, having similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).

[0131] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences as well as the sequences explicitly indicated. Specifically, as detailed below, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues (Batzer et al., 1991, Nucleic Acid Res. 19:5081; Ohtsuka et al., 1985, J. Biol. Chem. 260:2605-2608; and Rossolini et al., 1994, Mol. Cell. Probes 8:91-98).

[0132] Operably linked : The term "operably linked" refers to a functional relationship between two or more peptide or polypeptide domains or nucleic acid (e.g., DNA) segments. In the context of a fusion protein or other polypeptide, the term "operably linked" means that two or more amino acid segments are joined such that a functional polypeptide is produced. For example, in the context of an antigen-binding molecule, individual ABMs (or chains of an ABM) may be operably linked by a peptide linker sequence. In the context of a nucleic acid encoding a fusion protein, such as a polypeptide chain of an antigen-binding molecule, "operably linked" means that two nucleic acids are joined such that the amino acid sequences encoded by the two nucleic acids remain in-frame. In the context of transcriptional regulation, the term refers to the functional relationship between a transcriptional regulatory sequence and a transcriptional sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if the promoter or enhancer sequence stimulates or modulates transcription of the coding sequence in an appropriate host cell or other expression system.

[0133] Pentavalent: In the context of an antigen-binding molecule (e.g., TBM), as used herein, the term "pentavalent" refers to an antigen-binding molecule having five antigen-binding domains. The pentavalent TBMs of the present disclosure generally have (a) two pairs of antigen-binding domains each binding to the same antigen and a single antigen-binding domain binding to a third antigen, or (b) three antigen-binding domains binding to the same antigen and two antigen-binding domains each binding to a separate antigen. Examples of pentavalent TBMs are schematically shown in Figure 1T in.

[0134] Polypeptides and proteins : The terms "polypeptide" and "protein" are used interchangeably herein and refer to polymers of amino acid residues. The terms encompass amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as amino acid polymers suitable for both naturally occurring and non-naturally occurring amino acid polymers. Additionally, the terms encompass amino acid polymers derivatized, for example, by synthetic derivatization of one or more side chains or termini, glycosylation, polyethylene glycolylation, cyclic permutation, cyclization, attachment of linkers to other molecules, fusion to proteins or protein domains, and addition of peptide tags or peptide labels.

[0135] Recognize : As used herein, the term "recognize" refers to an ABM that finds and interacts (e.g., binds) with its epitope.

[0136] Sequence identity: The sequence identity of two similar sequences (e.g., antibody variable domains) can be measured by algorithms such as the following: Smith, T.F. and Waterman, M.S. (1981) "Comparison Of Biosequences," Adv. Appl. Math. 2:482 [local homology algorithm]; Needleman, S.B. and Wunsch, C.D. (1970) "A General Method Applicable To The Search For Similarities In The Amino Acid Sequence Of Two Proteins," J. Mol. Biol. 48:443 [homology alignment algorithm], Pearson, W.R. and Lipman, D.J. (1988) "Improved Tools For Biological Sequence Comparison," Proc. Natl. Acad. Sci. (U.S.A.) 85:2444 [search for similarity method]; or Altschul, S.F. et al., 1990, "Basic Local Alignment Search Tool," J. Mol. Biol. 215:403-10, the "BLAST" algorithm, see blast.ncbi.nlm.nih.gov / Blast.cgi. When using any of the foregoing algorithms, default parameters (for window length, gap penalty, etc.) are used. In one embodiment, the BLAST algorithm is used with default parameters to calculate sequence identity.

[0137] Optionally, identity is determined over a region of at least about 50 nucleotides (or in the case of a peptide or polypeptide, at least about 10 amino acids), or in some cases, over a region of 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids). In some embodiments, identity is determined over a defined domain (e.g., VH or VL of an antibody). Unless otherwise specified, sequence identity between two sequences is determined over the entire length of the shorter of the two sequences.

[0138] Single-chain Fab or scFab : The terms "single-chain Fab" and "scFab" mean a polypeptide that comprises an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, such that the VH and VL associate with each other and the CH1 and CL associate with each other. In some embodiments, the antibody domains and linker have one of the following orders in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL. The linker can be a polypeptide having at least 30 amino acids, such as between 32 and 50 amino acids. The single-chain Fab is stabilized by a native disulfide bond between the CL domain and the CH1 domain.

[0139] Single-chain Fv or scFv : The term "single-chain Fv" or "scFv" as used herein refers to an antibody fragment that comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide can further comprise a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Plückthun, in The Pharmacology of Monoclonal Antibodies, Volume 113, Rosenberg and Moore eds., 1994, Springer-Verlag, New York, pages 269 - 315.

[0140] Specific (or selective) binding: The term "specifically (or selectively) binds" to an antigen or epitope refers to a binding reaction that determines the presence of a homologous antigen or epitope in a heterogeneous population of proteins and other biologicals. The binding reaction can, but need not, be mediated by an antibody or antibody fragment, but can also be mediated by any type of ABM (such as a ligand, DARPin, etc.) as described in, for example, Section 7.3. An ABM typically also has a dissociation rate constant (KD) (koff / kon) of less than 5x10 -2 M, less than 10 -2 M, less than 5x10 -3 M, less than 10 -3 M, less than 5x10 -4 M, less than 10 -4 M, less than 5x10 -5 M, less than 10 -5 M, less than 5x10 -6 M, less than 10 -6 M, less than 5x10 -7 M, less than 10 -7 M, less than 5x10 -8 M, less than 10 -8 M, less than 5x10 -9 M, or less than 10 -9 M, and binds to the target antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., HSA). Binding affinity can be measured using Biacore, SPR, or BLI assays. The term "specifically binds" does not exclude cross-species reactivity. For example, an antigen-binding module (e.g., an antigen-binding fragment of an antibody) that "specifically binds" to an antigen from one species can also "specifically bind" to the antigen in one or more other species. Thus, such cross-species reactivity per se does not alter the classification of the antigen-binding module as a "specific" binder. In certain embodiments, an antigen-binding module that specifically binds to a human antigen has cross-species reactivity with one or more non-human mammalian species, e.g., one or more of the primate species (including but not limited to Macaca fascicularis, Macaca mulatta, and Macaca nemestrina) or rodent species (e.g., Mus musculus). In other embodiments, the antigen-binding module does not have cross-species reactivity.

[0141] Subject:The term "subject" includes human and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. Unless indicated otherwise, the terms "patient" or "subject" are used interchangeably herein.

[0142] Tandem of VH domains :As used herein, the term "tandem of VH domains (or VH)" refers to a string of VH domains, which consists of a number of identical VH domains of an antibody. The C-terminus of each of the VH domains (except for the last one at the end of the tandem) is linked (with or without a linker) to the N-terminus of another VH domain. The tandem has at least 2 VH domains, and in specific embodiments, the antigen-binding molecule has 3, 4, 5, 6, 7, 8, 9, or 10 VH domains. The tandem of VH can be produced by using recombinant methods (with or without a linker) to link the coding nucleic acids of each VH domain in the desired order (which ensures that the nucleic acids are prepared as a single polypeptide chain) (e.g., as described in Section 7.4.3). The N-terminus of the first VH domain in the tandem is defined as the N-terminus of the tandem, and the C-terminus of the last VH domain in the tandem is defined as the C-terminus of the tandem.

[0143] Tandem of VL domains :As used herein, the term "tandem of VL domains (or VL)" refers to a string of VL domains, which consists of a number of identical VL domains of an antibody. The C-terminus of each of the VL domains (except for the last one at the end of the tandem) is linked (with or without a linker) to the N-terminus of another VL. The tandem has at least 2 VL domains, and in specific embodiments, the antigen-binding molecule has 3, 4, 5, 6, 7, 8, 9, or 10 VL domains. The tandem of VL can be produced by using recombinant methods (with or without a linker) to link the coding nucleic acids of each VL domain in the desired order (which ensures that the nucleic acids are prepared as a single polypeptide chain) (e.g., as described in Section 7.4.3). The N-terminus of the first VL domain in the tandem is defined as the N-terminus of the tandem, and the C-terminus of the last VL domain in the tandem is defined as the C-terminus of the tandem.

[0144] Target antigen :As used herein, "target antigen" means a molecule that is non-covalently, reversibly, and specifically bound by an antigen-binding domain.

[0145] Tetravalent: In the context of an antigen-binding molecule (e.g., BBM or TBM), as used herein, the term "tetravalent" refers to an antigen-binding molecule having four antigen-binding domains. The tetravalent TBMs of the present disclosure generally have two antigen-binding domains that bind to the same antigen (e.g., CD19) and two antigen-binding domains that each bind to a separate antigen (e.g., a component of the TCR complex and CD2 or a TAA). Examples of tetravalent BBMs are schematically shown in Figure 1AA-1AH and examples of tetravalent TBMs are schematically shown in Figure 2Q-2S .

[0146] Therapeutically effective amount : "Therapeutically effective amount" means an amount effective to achieve the desired therapeutic result at the required dosage and duration.

[0147] Treat (Treat, Treatment and Treating) : As used herein, the terms "treat", "treatment" and "treating" refer to a reduction or alleviation in the progression, severity and / or duration of a disease or disorder (e.g., a proliferative disorder) or alleviation of one or more symptoms of the disorder (e.g., one or more distinguishable symptoms) resulting from administration of one or more of the CD19-binding molecules of the present disclosure. In some embodiments, the terms "treat", "treatment" and "treating" refer to an improvement in at least one measurable physical parameter of the disorder, such as tumor growth, which may not necessarily be distinguishable to the patient. In other embodiments, the terms "treat", "treatment" and "treating" refer to inhibiting the progression of the disorder physically, e.g., by stabilizing distinguishable symptoms, physiologically, e.g., by stabilizing physical parameters, or both. In some embodiments, the terms "treat", "treatment" and "treating" may refer to reducing or stabilizing tumor size or cancer cell count.

[0148] Trispecific binding molecule : The term "trispecific binding molecule" or "TBM" refers to a molecule that specifically binds to three antigens and contains three or more antigen-binding domains. The TBMs of the present disclosure contain at least one antigen-binding domain specific for CD19, at least one antigen-binding domain specific for a component of the TCR complex, and at least one antigen-binding domain specific for CD2 or a TAA. The antigen-binding domains can each independently be an antibody fragment (e.g., scFv, Fab, nanobody), a ligand, or a non-antibody-derived binder (e.g., fibronectin, Fynomer, DARPin). Representative TBMs are shown in Figure 1. A TBM can contain one, two, three, four or even more polypeptide chains. For example, Figure 1MThe TBM shown in [ID] comprises a single polypeptide chain, said single polypeptide chain comprising three scFvs and a single polypeptide chain linked by an ABM linker. Figure 1K The TBM shown in [ID] comprises two polypeptide chains, said polypeptide chains comprising three scFvs linked, for example, by an Fc domain. Figure 1J The TBM shown in [ID] comprises three polypeptide chains, forming an scFv, a ligand, and a Fab linked, for example, by an Fc domain. Figure 1C The TBM shown in [ID] comprises four polypeptide chains, forming three Fabs linked, for example, by an Fc domain. Figure 1U The TBM shown in [ID] comprises six polypeptide chains, forming four Fabs and two scFvs linked, for example, by an Fc domain.

[0149] Trivalent : In the context of an antigen-binding molecule (e.g., MBM), as used herein, the term "trivalent" refers to an antigen-binding molecule having three antigen-binding domains. The MBMs of the present disclosure are typically bispecific or trispecific. The bispecific BBM specifically binds to CD19 and a component of the TCR complex. The trispecific TBM specifically binds to CD19, a component of the TCR complex, and CD2 or a TAA. Thus, the trivalent BBM has three antigen-binding domains, two of which bind to CD19 and one of which binds to a component of the TCR, or vice versa. The TBM has three antigen-binding domains each binding to a different antigen. Examples of trivalent BBMs are schematically shown in Figure 1G-1Z and examples of trivalent TBMs are schematically shown in Figure 2B-2V in [ID].

[0150] Tumor : The term "tumor" may be used interchangeably with the term "cancer" herein, e.g., both terms encompass solid and liquid tumors, such as diffuse or circulating tumors. As used herein, the terms "cancer" or "tumor" include pre-malignant as well as malignant cancers and tumors.

[0151] Tumor-associated antigen: The term "tumor-associated antigen" or "TAA" refers to a molecule (typically a protein, carbohydrate, lipid, or some combination thereof) that is expressed either fully or as a fragment (e.g., MHC / peptide) on the surface of cancer cells and that can be used to preferentially target pharmacological agents to cancer cells. In some embodiments, the TAA is a marker expressed by both normal and cancer cells, such as a lineage marker, e.g., CD19 on B cells. In some embodiments, the TAA is a cell surface molecule that is overexpressed in cancer cells compared to normal cells, e.g., 1-fold overexpressed, 2-fold overexpressed, 3-fold overexpressed, or more compared to normal cells. In some embodiments, the TAA is a cell surface molecule that is inappropriately synthesized in cancer cells, e.g., a molecule that contains deletions, additions, or mutations compared to the molecule expressed on normal cells. In some embodiments, the TAA will be expressed either fully or as a fragment (e.g., MHC / peptide) only on the cell surface of cancer cells and will not be synthesized or expressed on the surface of normal cells. Thus, the term "TAA" encompasses antigens specific to cancer cells, sometimes referred to as tumor-specific antigens ("TSA"). Although CD19 has the characteristics of a tumor-associated antigen, the terms "tumor-associated antigen" and "TAA" are used throughout the disclosure to refer to molecules other than CD19.

[0152] Variable region : As used herein, "variable region" or "variable domain" means the region of an immunoglobulin that contains one or more Ig domains encoded by any of the Vκ, Vλ, and / or VH genes (which genes respectively make up the κ, λ, and heavy chain immunoglobulin loci) and contains the CDRs that confer antigen specificity. A "variable heavy domain" can pair with a "variable light domain" to form an antigen-binding domain ("ABD") or antigen-binding module ("ABM"). Additionally, each variable domain contains three hypervariable regions ("complementary determining regions", "CDRs") (CDR-H1, CDR-H2, CDR-H3 for the variable heavy domain and CDR-L1, CDR-L2, CDR-L3 for the variable light domain) and four framework (FR) regions, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0153] Vector: The term "vector" is intended to mean a polynucleotide molecule capable of transporting another polynucleotide to which it is linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of the host cell after being introduced into the host cell and thereby replicate with the host genome. In addition, certain vectors are capable of directing the expression of genes operably linked to them. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful in recombinant DNA techniques are commonly in the form of plasmids. In this specification, "plasmid" and "vector" may be used interchangeably because plasmids are the most commonly used form of vector. However, this disclosure is intended to encompass such other forms of expression vectors such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which have the same function.

[0154] VH : The term "VH" refers to the variable region of the immunoglobulin heavy chain of an antibody (including the heavy chain of Fv, scFv, dsFv, or Fab).

[0155] VL : The term "VL" refers to the variable region of the immunoglobulin light chain (including the light chain of Fv, scFv, dsFv, or Fab).

[0156] VH-VL or VH-VL pair : When referring to a VH-VL pair, the terms "VH-VL" and "VH-VL pair" are used for convenience and are not intended to convey any particular orientation, unless otherwise indicated by the context. Thus, an scFv containing "VH-VL" or "VH-VL pair" can have VH and VL domains in either orientation, e.g., VH at the N-terminus of VL or VL at the N-terminus of VH.

[0157] 7.2. CD19 Binding Molecule

[0158] In one aspect, the present disclosure provides CD19 binding molecules, which include monospecific and multispecific molecules that bind to human CD19. In some embodiments, the CD19 binding molecule is a monospecific binding molecule. For example, the monospecific binding molecule can be an antibody or an antigen-binding fragment thereof (e.g., antibody fragment, scFv, dsFv, Fv, Fab, scFab, (Fab’)2, or single domain antibody (SDAB)). In other embodiments, the CD19 binding molecule is a multispecific (e.g., bispecific) CD19 binding molecule (e.g., bispecific antibody).

[0159] In some embodiments, the CD19 binding molecule is a chimeric or humanized monoclonal antibody. Chimeric and / or humanized antibodies can be engineered to minimize the immune response in a human patient to antibodies produced in a non-human subject or antibodies derived from the expression of non-human antibody genes. A chimeric antibody contains a variable region of a non-human animal antibody and a constant region of a human antibody. Such antibodies retain the epitope-binding specificity of the original monoclonal antibody but can have lower immunogenicity when administered to a human and are thus more likely to be tolerated by the patient. For example, one or all (e.g., one, two, or three) of the variable regions of one or more light chains and / or one or all (e.g., one, two, or three) of the variable regions of one or more heavy chains of a murine antibody (e.g., murine monoclonal antibody) can each be linked to a human constant region, such as but not limited to the IgG1 human constant region. Chimeric monoclonal antibodies can be produced by known recombinant DNA techniques. For example, the gene encoding the constant region of a non-human antibody molecule can be replaced with a gene encoding a human constant region (see Robinson et al., PCT patent publication PCT / US 86 / 02269; Akira et al., European patent application 184,187; or Taniguchi, M., European patent application 171,496). Additionally, other suitable techniques for producing chimeric antibodies are described, for example, in U.S. Patent Nos. 4,816,567, 4,978,775; 4,975,369; and 4,816,397.

[0160] The chimeric or humanized antibodies and antigen-binding fragments thereof of the present disclosure can be prepared based on the sequences of murine monoclonal antibodies. DNA encoding the heavy and light chain immunoglobulins can be obtained from murine hybridomas of interest and engineered using standard molecular biology techniques to contain non-murine (e.g., human) immunoglobulin sequences. For example, to generate a chimeric antibody, known methods can be used to join the murine variable regions to human constant regions (see, e.g., U.S. Patent No. 4,816,567 to Cabilly et al.). To generate a humanized antibody, known methods can be used to insert murine CDR regions into a human framework. See, e.g., U.S. Patent No. 5,225,539 (to Winter) and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 (to Queen et al.).

[0161] A variety of known techniques can be used to generate humanized antibodies, including but not limited to CDR grafting (see, e.g., European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 1994, PNAS, 91:969-973), chain shuffling (see, e.g., U.S. Patent No. 5,565,332), and techniques disclosed, e.g., in U.S. Patent Application Publication No. US 2005 / 0042664, U.S. Patent Application Publication No. US 2005 / 0048617, U.S. Patent No. 6,407,213, U.S. Patent No. 5,766,886, International Publication No. WO 9317105, Tan et al., J. Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16):10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55(23 Suppl):5973s-5977s (1995), Couto et al., Cancer Res., 55(8):1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994). Generally, framework residues in the framework regions will be replaced with the corresponding residues from the CDR donor antibody to alter, e.g., improve antigen binding. These framework substitutions (e.g., conservative substitutions) are identified by known methods, e.g., by modeling the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison, thereby identifying unusual framework residues at specific positions.(See, e.g., Queen et al., U.S. Patent No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323).

[0162] As provided herein, a humanized antibody or antibody fragment can comprise one or more CDRs and framework regions from a non-human immunoglobulin molecule, wherein the amino acid residues constituting the framework are entirely or mostly derived from the human germline. A variety of techniques for humanizing antibodies or antibody fragments are well known and can generally be carried out according to the methods of Winter and colleagues (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), which involve replacing the corresponding sequences of a human antibody with rodent CDRs or CDR sequences, i.e., CDR grafting (EP 239,400; PCT Publication No. WO 91 / 09967; and U.S. Patent Nos. 4,816,567, 6,331,415, 5,225,539, 5,530,101, 5,585,089, 6,548,640). In such humanized antibodies and antibody fragments, substantially less than the complete human variable domain has been replaced with the corresponding sequences from non-human species. A humanized antibody is generally a human antibody in which some CDR residues and possibly some framework (FR) residues have been replaced with residues from analogous sites in a rodent antibody. Humanization of antibodies and antibody fragments can also be achieved by veneering or resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., Protein Engineering, 7(6):805-814 (1994); and Roguska et al., PNAS, 91:969-973 (1994)) or chain shuffling (U.S. Patent No. 5,565,332).

[0163] The selection of human variable domains (both light and heavy chains) for the preparation of humanized antibodies is to reduce antigenicity. According to the so-called "best-fit" method, the sequences of the variable domains of rodent antibodies are screened against the entire library of known human variable domain sequences. Then the human sequence that is closest to the rodent sequence is accepted as the human framework (FR) of the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method employs a specific framework derived from the consensus sequence of all human antibodies having a specific subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (see, for example, Nicholson et al. Mol. Immunol. 34(16-17):1157-1165 (1997); Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993). In some embodiments, the framework regions of the heavy chain variable region (e.g., all four framework regions) are derived from the VH4_4-59 germline sequence. In one embodiment, the framework region may contain one, two, three, four, or five modifications, e.g., substitutions, e.g., conservative substitutions, e.g., substitutions of amino acids from the corresponding murine sequence. In one embodiment, the framework regions of the light chain variable region (e.g., all four framework regions) are derived from the VK3_1.25 germline sequence. In one embodiment, the framework region may contain one, two, three, four, or five modifications, e.g., substitutions, e.g., conservative substitutions, e.g., substitutions of amino acids from the corresponding murine sequence.

[0164] In certain embodiments, the CD19-binding molecule comprises a heavy chain variable region from a specific germline heavy chain immunoglobulin gene and / or a light chain variable region from a specific germline light chain immunoglobulin gene. For example, such antibodies can comprise or consist of a human antibody that comprises a heavy or light chain variable region that is a “product” of or “derived from” a specific germline sequence. A human antibody can be identified as a “product” of or “derived from” a human germline immunoglobulin sequence in this manner by comparing the amino acid sequence of the human antibody to the amino acid sequence of human germline immunoglobulins and selecting the human germline immunoglobulin sequence that is most similar (i.e., greatest % identity) to the human antibody sequence in the alignment (using the methods outlined herein). A human antibody that is a “product” of or “derived from” a specific human germline immunoglobulin sequence can contain amino acid differences compared to the germline sequence due to, for example, naturally occurring somatic mutations or intentionally introduced site-directed mutations. However, a humanized antibody is typically at least 90% identical in amino acid sequence to the amino acid sequence encoded by a human germline immunoglobulin gene and contains amino acid residues that identify the antibody as being derived from a human sequence when compared to the germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences). In some cases, the amino acid sequence of a humanized antibody can be at least 95%, 96%, 97%, 98%, or 99% or even at least 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by a germline immunoglobulin gene. Typically, a humanized antibody derived from a specific human germline sequence will exhibit no more than 10-20 amino acid differences from the amino acid sequence encoded by a human germline immunoglobulin gene (prior to introduction of any skews, pIs, and ablation variants introduced herein; i.e., the number of variants is typically low prior to introduction of the variants disclosed herein). In some cases, a humanized antibody can exhibit no more than 5, or even no more than 4, 3, 2, or 1 amino acid differences from the amino acid sequence encoded by a germline immunoglobulin gene (again, prior to introduction of any skews, pIs, and ablation variants introduced herein; i.e., the number of variants is typically low prior to introduction of the variants disclosed herein).

[0165] In one embodiment, the parental antibody has been affinity matured. Structure-based methods can be used for humanization and affinity maturation, e.g., as described in USSN 11 / 004,590. Selection-based methods can be used to humanize and / or affinity mature the antibody variable regions, including but not limited to the methods described in: Wu et al., 1999, J. Mol. Biol. 294:151-162; Baca et al., 1997, J. Biol. Chem. 272(16):10678-10684; Rosok et al., 1996, J. Biol. Chem. 271(37):22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci. USA 95:8910-8915; Krauss et al., 2003, Protein Engineering 16(10):753-759. Other humanization methods can involve transplantation of only partial CDRs, including but not limited to the methods described in: USSN 09 / 810,510; Tan et al., 2002, J. Immunol. 169:1119-1125; De Pascalis et al., 2002, J. Immunol. 169:3076-3084.

[0166] In some embodiments, the CD19 binding molecule comprises an ABM (which is a Fab). The Fab domain can be produced by proteolytic cleavage of an immunoglobulin molecule, using an enzyme such as papain, or by recombinant expression. The Fab domain typically comprises a CH1 domain attached to the VH domain, and the CH1 domain pairs with a CL domain attached to the VL domain. In a wild-type immunoglobulin, the VH domain pairs with the VL domain to form the Fv region, and the CH1 domain pairs with the CL domain to further stabilize the binding module. A disulfide bond between the two constant domains can further stabilize the Fab domain.

[0167] In some embodiments, the CD19 binding molecule comprises an ABM (which is an scFab). In embodiments, the antibody domains and linker in the scFab fragment have one of the following sequences in the N-terminus to C-terminus direction: a) VH-CH1-linker-VL-CL, or b) VL-CL-linker-VH-CH1. In some cases, VL-CL-linker-VH-CH1 is used.

[0168] In another embodiment, the antibody domains and linker in the scFab fragment have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CL-linker-VL-CH1 or b) VL-CH1-linker-VH-CL.

[0169] Optionally, in the scFab fragment, in addition to the native disulfide bond between the CL domain and the CH1 domain, the variable heavy domain (VH) and the variable light domain (VL) of the antibody are also disulfide stabilized by introducing disulfide bonds between the following positions: i) position 44 of the heavy chain variable domain and position 100 of the light chain variable domain, ii) position 105 of the heavy chain variable domain and position 43 of the light chain variable domain, or iii) position 101 of the heavy chain variable domain and position 100 of the light chain variable domain (according to the Kabat EU index numbering).

[0170] Such further disulfide stabilization of the scFab fragment is achieved by introducing a disulfide bond between the variable domains VH and VL of the single-chain Fab fragment. Techniques for introducing non-natural disulfide bridges to stabilize single-chain Fvs are described in the following documents: for example, WO 94 / 029350, Rajagopal et al., 1997, Prot. Engin. [Protein Engineering] 10:1453-59; Kobayashi et al., 1998, Nuclear Medicine & Biology [Nuclear Medicine and Biology], 25:387-393; and Schmidt, et al., 1999, Oncogene [Oncogene] 18:1711-1721. In one embodiment, the optional disulfide bond between the variable domains of the scFab fragment is between position 44 of the heavy chain variable domain and position 100 of the light chain variable domain. In one embodiment, the optional disulfide bond between the variable domains of the scFab fragment is between position 105 of the heavy chain variable domain and position 43 of the light chain variable domain (numbered according to the Kabat EU index).

[0171] In some embodiments, the CD19 binding molecule comprises an ABM (which is an scFv). The single-chain Fv antibody fragment comprises the VH and VL domains of the antibody in a single polypeptide chain, can be expressed as a single-chain polypeptide, and retains the specificity of the intact antibody from which it is derived. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which can enable the scFv to form a desired structure for target binding. Examples of linkers suitable for connecting the VH and VL chains of scFV are the ABM linkers identified in section 7.4.3, such as any of the linkers designated L1 to L58.

[0172] Unless otherwise specified, as used herein, the scFv can have the VL and VH variable regions in either order relative to the N-terminus and C-terminus of the polypeptide, i.e., the scFv can comprise VL-linker-VH or can comprise VH-linker-VL.

[0173] To generate the scFv-encoding nucleic acid, the VH- and VL-encoding DNA fragments can be operably linked to another fragment encoding a linker, such as any of the linkers described in Section 7.4.3 (e.g., the amino acid sequence (Gly4-Ser)3 (SEQ ID NO:53)), such that the VH and VL sequences can be expressed as a continuous single-chain protein in which the VL and VH regions are joined by a flexible linker (see, e.g., Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990, Nature 348:552-554).

[0174] The CD19-binding molecule can also comprise an ABM, which is an Fv, dsFv, (Fab’)2, single domain antibody (SDAB), VH or VL domain, or a camelid VHH domain (also referred to as a nanobody).

[0175] The CD19-binding molecule can comprise a single domain antibody consisting of a single VH or VL domain, and the single VH or VL domain exhibits sufficient affinity for CD19. In embodiments, the single domain antibody is a camelid VHH domain (see, e.g., Riechmann, 1999, Journal of Immunological Methods 231:25-38; WO 94 / 04678).

[0176] Tables 1A and 1B (collectively referred to as “Table 1”) list the sequences of exemplary CD19-binding sequences that can be included in the CD19-binding molecule. The sequences listed in Table 1A are based on the CD19 antibody NEG258.

[0177]

[0178]

[0179] In some embodiments, the CD19-binding molecule comprises the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 sequences of NEG258 as listed in Table 1A. The CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 sequences may be defined by Kabat (SEQ ID NOs: 17-19 and 4-6, respectively), Chothia (SEQ ID NOs: 20-22 and 7-9, respectively), or IMGT (SEQ ID NOs: 23-25 and 10-12, respectively), or a combination of Chothia and Kabat CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 sequences (SEQ ID NOs: 14-16 and 1-3, respectively). The CD19-binding molecule may further comprise the light chain variable sequence (SEQ ID NO: 26) and / or the heavy chain variable sequence (SEQ ID NO: 13) of the anti-CD19 antibody NEG258 as listed in Table 1A.

[0180] The sequences listed in Table 1B are based on the CD19 antibody NEG218.

[0181]

[0182] In some embodiments, the CD19-binding molecule comprises the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 sequences of NEG218 as listed in Table 1B. The CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 sequences may be defined by Kabat (SEQ ID NOs: 43-45 and 30-32, respectively), Chothia (SEQ ID NOs: 46-48 and 33-35, respectively), or IMGT (SEQ ID NOs: 49-51 and 36-38, respectively), or a combination of Chothia and Kabat CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 sequences (SEQ ID NOs: 40-42 and 27-29, respectively). The CD19-binding molecule may further comprise the light chain variable sequence (SEQ ID NO: 52) and / or the heavy chain variable sequence (SEQ ID NO: 39) of the anti-CD19 antibody NEG218 as listed in Table 1B.

[0183] Other CD19-binding molecules include mutated amino acids, but their CDR regions still have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the CDR sequences described in Table 1. In some embodiments, such CD19-binding molecules include mutant amino acid sequences in which no more than 1, 2, 3, 4, or 5 amino acids have been mutated in the CDR region when compared to the CDR sequences described in Table 1.

[0184] Other CD19-binding molecules include VH and / or VL domains that contain amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the VH and / or VL sequences described in Table 1. In some embodiments, the CD19-binding molecule includes VH and / or VL domains in which no more than 1, 2, 3, 4, or 5 amino acids have been mutated when compared to the VH and / or VL domains described in the sequences in Table 1, while retaining substantially the same therapeutic activity.

[0185] The CD19-binding molecule can be fused or chemically conjugated (including both covalent and non-covalent conjugation) to a heterologous protein or polypeptide (or a fragment thereof, e.g., a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids). For example, the CD19-binding molecule can be directly or indirectly fused to a detectable protein, such as an enzyme or a fluorescent protein such as those described in Section 7.13. Methods for fusing or conjugating proteins, polypeptides, or peptides to antibodies or antibody fragments are known and can be used to fuse or conjugate a protein or polypeptide to the CD19-binding molecules disclosed herein. See, for example, U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; European Patent Nos. EP 307,434 and EP 367,166; International Publication Nos. WO96 / 04388 and WO 91 / 06570; Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88:10535-10539; Zheng et al., 1995, J. Immunol. 154:5590-5600; and Vil et al., 1992, Proc. Natl. Acad. Sci. USA 89:11337-11341.

[0186] Additional CD19 binding molecules can be generated by techniques such as gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling can be used to alter the activity of the molecules or fragments thereof disclosed herein (e.g., molecules or fragments thereof having higher affinity and lower dissociation rates). See generally, U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al., 1997, Curr. Opinion Biotechnol. [Current Biotechnology Opinion] 8:724-33; Harayama, 1998, Trends Biotechnol. [Trends in Biotechnology] 16(2):76-82; Hansson et al., 1999, J. Mol. Biol. [Journal of Molecular Biology] 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques [Biotechnology] 24(2):308-313. The CD19 binding molecules or fragments thereof described herein can be altered by random mutagenesis, such as by error-prone PCR, random nucleotide insertion, or other methods, prior to recombination. Polynucleotides encoding fragments of the CD19 binding molecules described herein can be recombined with one or more components, motifs, segments, portions, domains, fragments, etc. of one or more heterologous molecules.

[0187] In addition, the CD19 binding molecules can be fused to a tag sequence, such as a peptide, to facilitate purification. In some embodiments, the tag amino acid sequence is a hexahistidine peptide (SEQ ID NO:54), such as the tag provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, California 91311), many of which are commercially available. As described by Gentz et al., 1989 Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 86:821-824, for example, hexahistidine (SEQ ID NO:54) provides convenience for the purification of fusion proteins. Other peptide tags that can be used for purification include, but are not limited to, the hemagglutinin ("HA") tag corresponding to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984 Cell [Cell] 37:767) and the "flag" tag.

[0188] 7.3. Antigen-Binding Modules of Multispecific Binding Molecules

[0189] Typically, one or more ABDs of an MBM contain immunoglobulin-based antigen-binding domains, such as sequences of antibody fragments or derivatives. These antibody fragments and derivatives typically include the CDRs of an antibody and may include larger fragments and their derivatives, e.g., Fab, scFab, Fv, and scFv.

[0190] Immunoglobulin-based ABMs may contain modifications to framework residues within VH and / or VL, e.g., to improve the properties of the MBM containing the ABM. For example, framework modifications can be made to reduce the immunogenicity of the MBM. One method for making such framework modifications is to "back-mutate" one or more framework residues of the ABM to the corresponding germline sequence. Such residues can be identified by comparing the framework sequence to the germline sequence from which the ABM was derived. To "match" the framework region sequence to the desired germline configuration, residues can be "back-mutated" to the corresponding germline sequence, e.g., by site-directed mutagenesis. MBMs having such "back-mutated" ABMs are intended to be covered by this disclosure.

[0191] Another type of framework modification involves mutating one or more residues within the framework region or even within one or more CDR regions to remove T cell epitopes, thereby reducing the potential immunogenicity of the MBM. This method is also referred to as "deimmunization" and is further described in detail in U.S. Patent Publication 20030153043 to Carr et al.

[0192] ABMs can also be modified to have altered glycosylation, which can be useful, e.g., to increase the affinity of the MBM for one or more of its antigens. Such carbohydrate modifications can be achieved, e.g., by altering one or more glycosylation sites within the ABM sequence. For example, one or more amino acid substitutions can be made that eliminate one or more variable region framework glycosylation sites, thereby eliminating glycosylation at those sites. Such deglycosylation can increase the affinity of the MBM for the antigen. This method is described, e.g., in U.S. Patent Nos. 5,714,350 and 6,350,861 to Co et al.

[0193] 7.3.1. Immunoglobulin-based ABMs

[0194] 7.3.1.1. Fab

[0195] In certain aspects, the ABM is a Fab domain.

[0196] For the MBMs of this disclosure, it is advantageous to use a Fab heterodimerization strategy to allow proper association of Fab domains belonging to the same ABM and to minimize aberrant pairing of Fab domains belonging to different ABMs. For example, a Fab heterodimerization strategy such as that shown in Table 2 below can be used:

[0197]

[0198] Thus, in some embodiments, correct association between the two polypeptides of the Fab is facilitated by swapping the VL and VH domains of the Fab with each other or swapping the CH1 and CL domains with each other, e.g., as described in WO 2009 / 080251.

[0199] Correct Fab pairing can also be facilitated by introducing one or more amino acid modifications in the CH1 domain and one or more amino acid modifications in the CL domain of the Fab and / or one or more amino acid modifications in the VH domain and one or more amino acid modifications in the VL domain. The modified amino acids are typically part of the VH:VL and CH1:CL interactions such that the Fab components preferentially pair with each other rather than with components of other Fabs.

[0200] In one embodiment, the one or more amino acid modifications are limited to the conserved framework residues of the variable (VH, VL) and constant (CH1, CL) domains, as indicated by the Kabat numbering of the residues. Almagro, 2008, Frontiers In Bioscience 13:1619-1633 provides definitions of framework residues based on the Kabat, Chothia, and IMGT numbering schemes.

[0201] In one embodiment, the modifications introduced in the VH and CH1 and / or VL and CL domains are complementary to each other. Complementarity at the heavy and light chain interface can be achieved based on steric and hydrophobic contacts, electrostatic / charge interactions, or a combination of such interactions. Complementarity between protein surfaces has been extensively described in the literature in terms such as: lock and key fit, mortise and tenon structure, protrusion and cavity, donor and acceptor, etc., all of which imply the nature of the structural and chemical match between two contacting surfaces.

[0202] In one embodiment, the one or more introduced modifications introduce new hydrogen bonds across the interface of the Fab components. In one embodiment, the one or more introduced modifications introduce new salt bridges across the interface of the Fab components. Exemplary substitutions are described in WO 2014 / 150973 and WO 2014 / 082179.

[0203] In some embodiments, the Fab domain comprises a 192E substitution in the CH1 domain and 114A and 137K substitutions in the CL domain, where a salt bridge is introduced between the CH1 and CL domains (see Golay et al., 2016, J Immunol 196:3199 - 211).

[0204] In some embodiments, the Fab domain comprises 143Q and 188V substitutions in the CH1 domain and 113T and 176V substitutions in the CL domain, which serve to exchange the hydrophobic and polar contact regions between the CH1 and CL domains (see Golay et al., 2016, J Immunol 196:3199 - 211).

[0205] In some embodiments, the Fab domain may comprise modifications in some or all of the VH, CH1, VL, and CL domains to introduce an orthogonal Fab interface, which promotes the correct assembly of the Fab domain (Lewis et al., 2014, Nature Biotechnology 32:191 - 198). In an embodiment, 39K and 62E modifications are introduced in the VH domain, H172A and F174G modifications are introduced in the CH1 domain, 1R, 38D, (36F) modifications are introduced in the VL domain, and L135Y and S176W modifications are introduced in the CL domain. In another embodiment, a 39Y modification is introduced in the VH domain and a 38R modification is introduced in the VL domain.

[0206] The Fab domain can also be modified to replace the native CH1:CL disulfide bond with an engineered disulfide bond, thereby enhancing the efficiency of Fab pair assembly. For example, an engineered disulfide bond can be introduced by introducing 126C in the CH1 domain and 121C in the CL domain (see Mazor et al., 2015, MAbs 7:377 - 89).

[0207] The Fab domain can also be modified by replacing the CH1 domain and the CL domain with alternative domains that promote correct assembly. For example, Wu et al., 2015, MAbs 7:364 - 76, describe replacing the CH1 domain with the constant domain of the α T cell receptor and the CL domain with the β domain of the T cell receptor, and pairing these domain replacements with additional charge - charge interactions between the VL and VH domains by introducing a 38D modification in the VL domain and a 39K modification in the VH domain.

[0208] An ABM can comprise a single-chain Fab fragment, which is a polypeptide composed of an antibody heavy-chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light-chain variable domain (VL), an antibody light-chain constant domain (CL), and a linker. In some embodiments, the antibody domains and the linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL. The linker can be a polypeptide having at least 30 amino acids, for example, between 32 and 50 amino acids. The single-chain Fab domain is stabilized by a native disulfide bond between the CL domain and the CH1 domain.

[0209] In an embodiment, the antibody domains and the linker in the single-chain Fab fragment have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, or b) VL-CL-linker-VH-CH1. In some cases, VL-CL-linker-VH-CH1 is used.

[0210] In another embodiment, the antibody domains and the linker in the single-chain Fab fragment have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CL-linker-VL-CH1 or b) VL-CH1-linker-VH-CL.

[0211] Optionally, in the single-chain Fab fragment, in addition to the native disulfide bond between the CL domain and the CH1 domain, the antibody heavy-chain variable domain (VH) and the antibody light-chain variable domain (VL) of the ABM are also disulfide-stabilized by introducing disulfide bonds between the following positions: i) position 44 of the heavy-chain variable domain and position 100 of the light-chain variable domain, ii) position 105 of the heavy-chain variable domain and position 43 of the light-chain variable domain, or iii) position 101 of the heavy-chain variable domain and position 100 of the light-chain variable domain (according to the Kabat EU index numbering).

[0212] In one embodiment, the optional disulfide bond between the variable domains of the single-chain Fab fragment is between position 44 of the heavy-chain variable domain and position 100 of the light-chain variable domain. In one embodiment, the optional disulfide bond between the variable domains of the single-chain Fab fragment is between position 105 of the heavy-chain variable domain and position 43 of the light-chain variable domain (numbered according to the Kabat EU index).

[0213] 7.3.1.2. scFv

[0214] In some aspects, the ABM is a single-chain Fv or "scFv". Examples of linkers suitable for joining the VH and VL chains of an scFV are the ABM linkers identified in Section 7.4.3, such as any of the linkers designated L1 to L54.

[0215] To generate a nucleic acid encoding an scFv, DNA fragments encoding VH and VL are operably linked to another fragment encoding a linker (such as encoding any of the ABM linkers described in Section 7.4.3), such as the amino acid sequence (Gly4-Ser)3 (SEQ ID NO:53)

[0216] 7.3.1.3. Other immunoglobulin-based ABMs

[0217] The MBM can also comprise an ABM in the form of an immunoglobulin, such as an Fv, dsFv, (Fab’)2, single domain antibody (SDAB), VH or VL domain, or camelid VHH domain (also known as a nanobody), other than a Fab or scFv.

[0218] The ABM can be a single domain antibody consisting of a single VH or VL domain, where the single VH or VL domain exhibits sufficient affinity for the target. In an embodiment, the single domain antibody is a camelid VHH domain (see, for example, Riechmann, 1999, Journal of Immunological Methods 231:25-38; WO 94 / 04678).

[0219] 7.3.2. Non-immunoglobulin-based ABMs

[0220] In certain embodiments, the MBM comprises one or more ABMs derived from non-antibody scaffold proteins (including but not limited to: designed ankyrin repeat protein (DARPin), Avimer (short for avidity multimer), Anticalin / lipocalin, Centyrin, Kunitz domain, Adnexin, Affilin, Affitin (also known as Nonfitin), Knottin, Pronectin, Versabody, Duocalin, and Fynomer), ligands, receptors, cytokines or chemokines.

[0221] Non-immunoglobulin scaffolds that can be used for MBM include those listed in the following: Tables 3 and 4 of Mintz and Crea, 2013, Bioprocess International 11(2):40-48; Figure 1, Table 1, and Figure I of Vazquez-Lombardi et al., 2015, Drug Discovery Today 20(10):1271-83; Table 1 and Column 2 of Skrlec et al., 2015, Trends in Biotechnology 33(7):408-18. Tables 3 and 4 of Mintz and Crea, 2013, Bioprocess International 11(2):40-48; Figure 1, Table 1, and Figure I of Vazquez-Lombardi et al., 2015, Drug Discovery Today 20(10):1271-83; Table 1 and Column 2 of Skrlec et al., 2015, Trends in Biotechnology 33(7):408-18 (collectively referred to as the "scaffold disclosures"). In a specific embodiment, the scaffold disclosures related to Adnexin are incorporated by reference. In another embodiment, the scaffold disclosures related to Avimer are incorporated by reference. In another embodiment, the scaffold disclosures related to Affibody are incorporated by reference. In yet another embodiment, the scaffold disclosures related to anticalin are incorporated by reference. In yet another embodiment, the scaffold disclosures related to DARPin are incorporated by reference. In yet another embodiment, the scaffold disclosures related to Kunitz domain are incorporated by reference. In yet another embodiment, the scaffold disclosures related to Knottin are incorporated by reference. In yet another embodiment, the scaffold disclosures related to Pronectin are incorporated by reference. In yet another embodiment, the scaffold disclosures related to Nanofitin are incorporated by reference. In yet another embodiment, the scaffold disclosures related to Affilin are incorporated by reference. In yet another embodiment, the scaffold disclosures related to Adnectin are incorporated by reference. In yet another embodiment, the scaffold disclosures related to ABM are incorporated by reference. In yet another embodiment, the scaffold disclosures related to Adhiron are incorporated by reference. In yet another embodiment, the scaffold disclosures related to Affimer are incorporated by reference. In yet another embodiment, the scaffold disclosures related to Alphabody are incorporated by reference.In yet another embodiment, the disclosure relates to scaffolds of Armadillo Repeat Protein, which are incorporated by reference. In yet another embodiment, the disclosure relates to scaffolds of Atrimer / Tetranectin, which are incorporated by reference. In yet another embodiment, the disclosure relates to scaffolds of Obody / OB-fold, which are incorporated by reference. In yet another embodiment, the disclosure relates to scaffolds of Centyrin, which are incorporated by reference. In yet another embodiment, the disclosure relates to scaffolds of Repebody, which are incorporated by reference. In yet another embodiment, the disclosure relates to scaffolds of anti-carrier proteins, which are incorporated by reference. In yet another embodiment, the disclosure relates to scaffolds of Atrimer, which are incorporated by reference. In yet another embodiment, the disclosure relates to scaffolds of bicyclic peptides, which are incorporated by reference. In yet another embodiment, the disclosure relates to scaffolds of cys-knot, which are incorporated by reference. In yet another embodiment, the disclosure relates to scaffolds of Fn3 scaffolds (including adenectin, Centyrin, Pronectin, and Tn3), which are incorporated by reference.

[0222] In an embodiment, the ABM can be a designed ankyrin repeat protein (“DARPin”). DARPins are antibody-mimicking proteins that typically exhibit high specificity and high affinity for target protein binding. They are generally genetically engineered and derived from natural ankyrin proteins and consist of at least three, typically four or five, repeat motifs of these proteins. For tetra-repeat or penta-repeat DARPins, their molecular masses are approximately 14 or 18 kDa (kilodaltons), respectively. Examples of DARPins can be found, for example, in U.S. Patent No. 7,417,130. Multispecific binding molecules comprising a DARPin binding module and an immunoglobulin-based binding module are disclosed, for example, in U.S. Publication No. 2015 / 0030596 A1.

[0223] In another embodiment, the ABM can be an Affibody. Affibodies are well-known and refer to affinity proteins based on a 58-amino acid residue protein domain derived from an IgG-binding domain of staphylococcal protein A.

[0224] In another embodiment, the ABM can be an anti-carrier protein. Anti-carrier proteins are well-known and refer to another antibody-mimicking technology, wherein the binding specificity is derived from lipocalin. Anti-carrier proteins can also be formatted as dual-targeting proteins, called Duocalin.

[0225] In another embodiment, the ABM can be Versabody. Versabody is well known and refers to another antibody mimicking technology. They are small proteins of 3 - 5 kDa with >15% cysteine that form a high disulfide bond density scaffold, displacing the hydrophobic core of typical proteins.

[0226] Other non - immunoglobulin ABMs include "A" domain oligomers (also known as Avimers) (see, e.g., U.S. Patent Application Publication Nos. 2005 / 0164301, 2005 / 0048512, and 2004 / 017576), Fn3 - based protein scaffolds (see, e.g., U.S. Patent Application Publication 2003 / 0170753), VASP polypeptides, avian pancreatic polypeptide (aPP), tetranectin (based on CTLD3), Affililin (based on γB - crystallin / ubiquitin), knottin, SH3 domain, PDZ domain, tendamistat, neocarzinostatin, protein A domain, lipocalin, transferrin, or Kunitz domain. In one aspect, the ABM for constructing the MBM comprises a fibronectin - based scaffold as shown in WO 2011 / 130324.

[0227] In addition, in certain aspects, the ABM comprises a ligand - binding domain of a receptor or a receptor - binding domain of a ligand.

[0228] 7.4. Linker

[0229] It is contemplated that the CD19 - binding molecule can in some cases include pairs of ABMs or ABM chains directly interconnected with each other (e.g., the VH - CH1 or VL - CL components of a Fab), for example, as a fusion protein without a linker. For example, the CD19 - binding molecule comprises a linker portion that connects individual ABMs or ABM chains. The use of the linker portion can improve target binding, for example, by increasing the flexibility of the ABMs in the CD19 - binding molecule and thus reducing steric hindrance. The ABMs or ABM chains can be interconnected via, for example, Fc domains (each Fc domain represents a pair of associated Fc regions) and / or ABM linkers. The use of Fc domains will typically require the use of a hinge region as a linker for the ABMs or ABM chains for optimal antigen binding. Thus, the term "linker" encompasses but is not limited to Fc regions, Fc domains, and hinge regions.

[0230] The linker can be selected or modified to, for example, increase or decrease the biological half-life of the CD19-binding molecule. For example, to decrease the biological half-life, one or more amino acid mutations can be introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the CD19-binding molecule comprising the fragment has impaired Staphylococcus protein A (SpA) binding compared to the native Fc-hinge domain SpA binding. This method is further described in detail by Ward et al. in U.S. Patent No. 6,165,745. Alternatively, the CD19-binding molecule can be modified to increase its biological half-life. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F as described by Ward in U.S. Patent No. 6,277,375. Alternatively, to increase the biological half-life, the CD19-binding molecule can be altered in the CH1 or CL regions to contain the salvage receptor binding epitope of two loops of the CH2 domain taken from the Fc region of IgG as described in U.S. Patent Nos. 5,869,046 and 6,121,022 by Presta et al.

[0231] Examples of the Fc domain (formed by the pairing of two Fc regions), the hinge region, and the ABM linker are described in Sections 7.4.1, 7.4.2, and 7.4.3, respectively.

[0232] 7.4.1. Fc domain

[0233] The CD19-binding molecule can comprise an Fc domain derived from any suitable species. In one embodiment, the Fc domain is derived from a human Fc domain.

[0234] The Fc domain can be derived from any suitable type of antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In one embodiment, the Fc domain is derived from IgG1, IgG2, IgG3, or IgG4. In one embodiment, the Fc domain is derived from IgG1. In one embodiment, the Fc domain is derived from IgG4.

[0235] The Fc domain comprises two polypeptide chains, each referred to as a heavy chain Fc region. These two heavy chain Fc regions dimerize to produce the Fc domain. The two Fc regions in the Fc domain can be the same as or different from each other. In native antibodies, the Fc regions are typically identical, but for the purpose of generating the multispecific binding molecules of the present disclosure, the Fc regions can advantageously be different to allow heterodimerization as described in Section 7.4.1.5 below.

[0236] Typically, each heavy chain Fc region comprises or consists of two or three heavy chain constant domains.

[0237] In native antibodies, the heavy chain Fc regions of IgA, IgD, and IgG are composed of two heavy chain constant domains (CH2 and CH3), and the Fc regions of IgE and IgM are composed of three heavy chain constant domains (CH2, CH3, and CH4). These antibodies dimerize to produce Fc domains.

[0238] In the present disclosure, the heavy chain Fc region may comprise heavy chain constant domains from one or more different types (e.g., one, two, or three different types) of antibodies.

[0239] In one embodiment, the heavy chain Fc region comprises the CH2 and CH3 domains derived from IgG1. An exemplary sequence of the heavy chain Fc region derived from human IgG1 is given in SEQ ID NO: 1109:

[0240] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO: 1109).

[0241] In some embodiments, the CD19-binding molecule of the present disclosure comprises an Fc region, the amino acid sequence of which comprises the amino acid sequence of SEQ ID NO: 1109 modified with one or more of the substitutions described in Section 7.4.1 and its subsections.

[0242] In one embodiment, the heavy chain Fc region comprises the CH2 and CH3 domains derived from IgG2.

[0243] In one embodiment, the heavy chain Fc region comprises the CH2 and CH3 domains derived from IgG3.

[0244] In one embodiment, the heavy chain Fc region comprises the CH2 and CH3 domains derived from IgG4.

[0245] In one embodiment, the heavy chain Fc region comprises the CH4 domain from IgM. The IgM CH4 domain is typically located at the C-terminus of the CH3 domain.

[0246] In one embodiment, the heavy chain Fc region comprises CH2 and CH3 domains derived from IgG and a CH4 domain derived from IgM.

[0247] It will be appreciated that the heavy chain constant domains used to generate the heavy chain Fc region of the CD19 binding molecules of the present disclosure may include variants of the naturally occurring constant domains as described above. Such variants may contain one or more amino acid variations compared to the wild-type constant domain. In one instance, the heavy chain Fc region of the present disclosure comprises at least one constant domain that is different in sequence from the wild-type constant domain. It will be appreciated that the variant constant domain may be longer or shorter than the wild-type constant domain. For example, the variant constant domain is at least 60% identical or similar to the wild-type constant domain. In another instance, the constant domain is at least 70% identical or similar. In another instance, the constant domain is at least 75% identical or similar. In another instance, the constant domain is at least 80% identical or similar. In another instance, the constant domain is at least 85% identical or similar. In another instance, the constant domain is at least 90% identical or similar. In another instance, the constant domain is at least 95% identical or similar. In another instance, the constant domain is at least 99% identical or similar. Exemplary Fc variants are described in Sections 7.4.1.1 to 7.4.1.5 below.

[0248] IgM and IgA naturally occur in humans as covalent polymers of the common H2L2 antibody unit. When incorporated with the J-chain, IgM exists as a pentamer, or when lacking the J-chain, IgM exists as a hexamer. IgA exists as monomeric and dimeric forms. The heavy chains of IgM and IgA have an 18 amino acid extension to the C-terminal constant domain, which is referred to as the tailpiece. The tailpiece includes cysteine residues that form disulfide bonds between the heavy chains in the polymer and is believed to have an important role in polymerization. The tailpiece also contains glycosylation sites. In certain embodiments, the CD19 binding molecules of the present disclosure do not contain a tailpiece.

[0249] The Fc domain incorporated into the CD19 binding molecules of the present disclosure may contain one or more modifications that alter one or more functional properties of the protein, such as serum half-life, complement fixation, Fc receptor binding, and / or antibody-dependent cell cytotoxicity. Additionally, the CD19 binding molecules may be chemically modified (e.g., one or more chemical moieties may be attached to the CD19 binding molecule) or modified to alter their glycosylation, thereby again altering one or more functional properties of the CD19 binding molecule.

[0250] The effector functions of an antibody molecule include complement-mediated effector functions, which are mediated by, for example, the binding of the C1 component of said complement to said antibody. Activation of the complement is important in the opsonization and direct lysis of pathogens. Additionally, it stimulates an inflammatory response by recruiting and activating phagocytes to the site of complement activation. Effector functions include Fc receptor (FcR)-mediated effector functions, which can be triggered by the binding of the constant domain of an antibody to an Fc receptor (FcR). Cross-linking mediated by the antigen-antibody complex of the Fc receptor on the surface of effector cells triggers a number of important and diverse biological responses, including the phagocytosis and destruction of antibody-coated particles, the clearance of immune complexes, the lysis of antibody-coated target cells by killer cells (referred to as antibody-dependent cell-mediated cytotoxicity, or ADCC), the release of inflammatory mediators, placental transfer, and the control of immunoglobulin production.

[0251] The Fc region can be altered by substituting at least one amino acid residue with a different amino acid residue to change effector function. For example, one or more amino acids can be replaced with different amino acid residues such that the Fc region has an altered affinity for an effector ligand. Effector ligands with altered affinity can be, for example, an Fc receptor or the C1 component of complement. This method is described, for example, by Winter et al. in both U.S. Patent Nos. 5,624,821 and 5,648,260. Modified Fc regions can also alter C1q binding and / or reduce or eliminate complement-dependent cytotoxicity (CDC). This method is described, for example, by Idusogie et al. in U.S. Patent No. 6,194,551. Modified Fc regions can also alter the ability of the Fc region to fix complement. This method is described, for example, by Bodmer et al. in PCT Publication WO 94 / 29351. Allotypic amino acid residues include, but are not limited to, the constant regions of the heavy chains of the IgG1, IgG2, and IgG3 subclasses and the constant region of the light chain of the κ isotype, as described by Jefferis et al., 2009, MAbs, 1:332-338.

[0252] The Fc region can also be modified to “silence” the effector functions, e.g., reducing or eliminating the ability of the CD19-binding molecule to mediate antibody-dependent cell cytotoxicity (ADCC) and / or antibody-dependent cell phagocytosis (ADCP). For example, this can be achieved by introducing mutations in the Fc region. Such mutations have been described in the art: LALA and N297A (Strohl, 2009, Curr. Opin. Biotechnol. 20(6):685-691); and D265A (Baudino et al., 2008, J. Immunol. 181:6664-69; Strohl, ibid.). Examples of silent Fc IgG1 antibodies include the so-called LALA mutant, which contains the L234A and L235A mutations in the IgG1 Fc amino acid sequence. Another example of a silent IgG1 antibody contains the D265A mutation. Another silent IgG1 antibody contains the so-called DAPA mutant, which contains the D265A and P329A mutations in the IgG1 Fc amino acid sequence. Another silent IgG1 antibody contains the N297A mutation, which results in an aglycosylated / non-glycosylated antibody.

[0253] The Fc region can be modified to enhance the ability of a CD19-binding molecule containing the Fc region to mediate antibody-dependent cell cytotoxicity (ADCC) and / or antibody-dependent cell phagocytosis (ADCP). For example, by modifying one or more amino acid residues to increase the affinity of the CD19-binding molecule for activating Fcγ receptors or to decrease the affinity of the CD19-binding molecule for inhibitory Fcγ receptors. Human activating Fcγ receptors include FcγRIa, FcγRIIa, FcγRIIIa, and FcγRIIIb, and human inhibitory Fcγ receptors include FcγRIIb. This method is described, for example, by Presta in PCT Publication WO 00 / 42072. In addition, the binding sites on human IgG1 for FcγRl, FcγRII, FcγRIII, and FcRn have been mapped, and variants with improved binding have been described (see Shields et al., J. Biol. Chem. 276:6591-6604, 2001). Optimization of the Fc-mediated effector functions of monoclonal antibodies, such as enhanced ADCC / ADCP function, has been described (see Strohl, 2009, Current Opinion in Biotechnology 20:685-691). Mutations that can enhance ADCC / ADCP function include one or more mutations selected from the following: G236A, S239D, F243L, P247I, D280H, K290S, R292P, S298A, S298D, S298V, Y300L, V305I, A330L, I332E, E333A, K334A, A339D, A339Q, A339T, and P396L (all positions by EU numbering).

[0254] The Fc region can also be modified to enhance the ability of a CD19-binding molecule to mediate ADCC and / or ADCP, for example, by modifying one or more amino acids to increase the affinity of the CD19-binding molecule for an activating receptor that typically does not recognize the parental CD19-binding molecule, such as FcαRI. This method is described, for example, by Borrok et al., 2015, mAbs. 7(4):743-751.

[0255] Accordingly, in some aspects, the CD19-binding molecules of the present disclosure can include an Fc domain having altered effector functions (such as but not limited to binding to Fc receptors, such as FcRn or leukocyte receptors (such as those described above or in Section 7.4.1.1), binding to complement (such as those described above or in Section 7.4.1.2), modified disulfide bond structure (such as those described above or in Section 7.4.1.3) or altered glycosylation pattern (such as those described above or in Section 7.4.1.4)). The Fc domain can also be altered to include modifications that improve the manufacturability of asymmetric CD19-binding molecules, such as by allowing heterodimerization (wherein heterodimerization is the preferential pairing of different Fc regions relative to the same Fc region). Heterodimerization allows the generation of CD19-binding molecules in which different ABMs are interconnected by Fc domains containing Fc regions with different sequences. Examples of heterodimerization strategies are exemplified in Section 7.4.1.5 (and its subsections).

[0256] It will be appreciated that any of the modifications described in Sections 7.4.1.1 to 7.4.1.5 can be combined in any suitable manner to achieve the desired functional properties and / or any of the modifications described in Sections 7.4.1.1 to 7.4.1.5 can be combined with other modifications to alter the properties of the CD19-binding molecule. In some embodiments, the CD19-binding molecule comprises an IgG1 Fc domain having mutations at 1, 2, 3, 4, 5, 6, or more than 6 positions at positions 233, 234, 235, 236, 237, 239, 265, 266, 267, 268, 269, 297, 299, 322, 327, 328, 329, 330, 331, and 332 (EU numbering). For example, the CD19-binding molecule can comprise the IgG1 sequence of SEQ ID NO: 1109 having mutations at 1, 2, 3, 4, 5, 6, or more than 6 positions at positions 233, 234, 235, 236, 237, 239, 265, 266, 267, 268, 269, 297, 299, 322, 327, 328, 329, 330, 331, and 332.

[0257] 7.4.1.1. Fc domain with altered FcR binding

[0258] Compared to the corresponding native immunoglobulin, the Fc domain of the CD19-binding molecule can exhibit altered binding to one or more Fc receptors (FcRs). Binding to any specific Fc receptor can be enhanced or reduced. In one embodiment, the Fc domain comprises one or more modifications that alter its Fc-receptor binding profile.

[0259] Human cells can express a large number of membrane-bound FcRs, which are selected from FcαR, FcεR, FcγR, FcRn, and glycan receptors. Some cells are also capable of expressing soluble (extracellular domain) FcRs (Fridman et al., 1993, J Leukocyte Biology 54:504-512). FcγRs can be further divided by IgG binding affinity (high / low) and biological function (activation / inhibition). Human FcγRI is widely considered to be the only "high-affinity" receptor, while all others are considered to be medium to low affinity. Due to its intracellular ITIM motif, FcγRIIb is the only receptor with "inhibitory" functionality, while all others are considered "activated" due to the ITAM motif or pairing with the common FcγR-γ chain. FcγRIIIb is also unique in that, although activated, it associates with the cell via a GPI anchor. In summary, humans express six "standard" FcγRs: FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In addition to these sequences, there are a large number of sequences or allotypic variants scattered among these families. Some of these sequences have been found to have important functional consequences and are therefore sometimes considered to be their own receptor subtypes. Examples include FcγRIIa H134R , FcγRIIb I190T , FcγRIIIa F158V , FcγRIIIb NA1 , FcγRIIIb NA2 , and FcγRIII SH . Each receptor sequence has been shown to have different affinities for the 4 subclasses of IgG: IgG1, IgG2, IgG3, and IgG4 (Bruhns, 1993, Blood 113:3716-3725). Other species have slightly different numbers and functions of FcγRs, with the mouse system being the best studied currently and consisting of 4 FcγRs, FcγRI FcγRIIb FcγRIII FcγRIV (Bruhns, 2012, Blood 119:5640-5649). Due to the affinity of human FcγRI on the cell surface for IgG1 / IgG3 / IgG4 (about 10 -8 M) and the concentration of these IgGs in serum (about 10 mg / ml), human FcγRI on the cell surface is generally considered to be "occupied" by monomeric IgG under normal serum conditions. Therefore, cells carrying FcγRI on their surface are considered to be able to "screen" or "sample" their antigenic environment alternatively through the bound multispecific IgG. Other receptors with lower affinities for IgG subclasses (at about 10 -5 -10-7 In the range of M) is generally considered to be "unoccupied". Low-affinity receptors are thus inherently sensitive to the detection of immune complexes involving antibodies and their activation thereby. The increased Fc density in antibody immune complexes results in an increased functional avidity of the binding affinity with low-affinity FcγRs. This has been shown in vitro using many methods (Shields et al., 2001, J Biol Chem 276(9):6591-6604; Lux et al., 2013, J Immunol 190:4315-4323). This is also thought to be one of the main modes of action of using anti-RhD to treat human ITP (Crow, 2008, Transfusion Medicine Reviews 22:103-116).

[0260] Many cell types express multiple types of FcγRs and thus depending on the biological context, binding of IgG or antibody immune complexes to cells carrying FcγRs can have diverse and complex outcomes. Most simply, cells can receive activating, inhibitory, or mixed signals. This can lead to events such as phagocytosis (e.g., macrophages and neutrophils), antigen processing (e.g., dendritic cells), reduced IgG production (e.g., B cells), or degranulation (e.g., neutrophils, mast cells). There is data to support the conclusion that inhibitory signals from FcγRIIb can dominate activating signals (Proulx, 2010, Clinical Immunology 135:422-429).

[0261] A large number of useful Fc substitutions can be prepared to alter binding to one or more of the FcγR receptors. Substitutions that result in enhanced binding and reduced binding can be useful. For example, enhanced binding to FcγRIIIa is generally known to result in enhanced ADCC (antibody-dependent cell-mediated cytotoxicity; a cell-mediated response in which non-specific cytotoxic cells expressing FcγR recognize bound antibody on a target cell and subsequently cause lysis of the target cell). Similarly, reduced binding to FcγRIIb (an inhibitory receptor) can also be beneficial in some cases. Amino acid substitutions useful in the present disclosure include those listed in US 2006 / 0024298 (especially FIG. 41), US 2006 / 0121032, US 2006 / 0235208, US 2007 / 0148170, and US 2019 / 0100587. Specific variants that can be used include, but are not limited to, 236A, 239D, 239E, 332E, 332D, 239D / 332E, 267D, 267E, 328F, 267E / 328F, 236A / 332E, 239D / 332E / 330Y, 239D, 332E / 330L, 243A, 243L, 264A, 264V, 299T, 265A / 297A / 329A, 265N / 297D / 329G, and 265E / 297Q / 329S.

[0262] FcRn plays a key role in maintaining the long-term half-life of IgG in the sera of adults and children. The receptor binds IgG in acidified vesicles (pH < 6.5), protects the IgG molecules from degradation, and then releases them in the blood at a higher pH of 7.4.

[0263] FcRn is distinct from leukocyte Fc receptors and, instead, has a structural similarity to MHC class I molecules. A heterodimer composed of a β2-microglobulin chain is non-covalently attached to a membrane-bound chain that includes three extracellular domains. One of these domains (including a carbohydrate chain) interacts with β2-microglobulin at the site between the CH2 and CH3 domains of Fc. The interaction includes a salt bridge made to a histidine residue on IgG, which is positively charged at pH < 6.5. At higher pH, the His residues lose their positive charge, the FcRn-IgG interaction is weakened and IgG dissociates.

[0264] In one embodiment, the CD19-binding molecule comprises an Fc domain that binds to human FcRn.

[0265] In one embodiment, the Fc domain has one or more (e.g., one or two) Fc regions, the Fc region contains a histidine residue at position 310, and in some cases also contains a histidine residue at position 435. These histidine residues are important for human FcRn binding. In one embodiment, the histidine residues at positions 310 and 435 are native residues, i.e., positions 310 and 435 are unmodified. Alternatively, one or both of these histidine residues may be present due to modification.

[0266] The CD19 binding molecule may comprise one or more Fc regions that alter Fc binding to FcRn. The altered binding may be enhanced binding or reduced binding.

[0267] In one embodiment, the CD19 binding molecule comprises an Fc domain, wherein at least one (and optionally two) Fc regions comprise one or more modifications such that it binds to FcRn with higher affinity and avidity than the corresponding native immunoglobulin.

[0268] Fc substitutions that enhance binding to the FcRn receptor and increase serum half-life are described in US 2009 / 0163699, including but not limited to: 434S, 434A, 428L, 308F, 259I, 428L / 434S, 259I / 308F, 436I / 428L, 436I or V / 434S, 436V / 428L, and 259I / 308F / 428L.

[0269] In one embodiment, the Fc region is modified by substituting the threonine residue at position 250 with a glutamine residue (T250Q).

[0270] In one embodiment, the Fc region is modified by substituting the methionine residue at position 252 with a tyrosine residue (M252Y).

[0271] In one embodiment, the Fc region is modified by substituting the serine residue at position 254 with a threonine residue (S254T).

[0272] In one embodiment, the Fc region is modified by substituting the threonine residue at position 256 with a glutamic acid residue (T256E).

[0273] In one embodiment, the Fc region is modified by substituting the threonine residue at position 307 with an alanine residue (T307A).

[0274] In one embodiment, the Fc region is modified by substituting the threonine residue at position 307 with a proline residue (T307P).

[0275] In one embodiment, the Fc region is modified by substituting the valine residue at position 308 with a cysteine residue (V308C).

[0276] In one embodiment, the Fc region is modified by substituting the valine residue at position 308 with a phenylalanine residue (V308F).

[0277] In one embodiment, the Fc region is modified by substituting the valine residue at position 308 with a proline residue (V308P).

[0278] In one embodiment, the Fc region is modified by substituting the glutamine residue at position 311 with an alanine residue (Q311A).

[0279] In one embodiment, the Fc region is modified by substituting the glutamine residue at position 311 with an arginine residue (Q311R).

[0280] In one embodiment, the Fc region is modified by substituting the methionine residue at position 428 with a leucine residue (M428L).

[0281] In one embodiment, the Fc region is modified by substituting the histidine residue at position 433 with a lysine residue (H433K).

[0282] In one embodiment, the Fc region is modified by substituting the asparagine residue at position 434 with a phenylalanine residue (N434F).

[0283] In one embodiment, the Fc region is modified by substituting the asparagine residue at position 434 with a tyrosine residue (N434Y).

[0284] In one embodiment, the Fc region is modified by substituting the methionine residue at position 252 with a tyrosine residue, substituting the serine residue at position 254 with a threonine residue, and substituting the threonine residue at position 256 with a glutamate residue (M252Y / S254T / T256E).

[0285] In one embodiment, the Fc region is modified by substituting the valine residue at position 308 with a proline residue and substituting the asparagine residue at position 434 with a tyrosine residue (V308P / N434Y).

[0286] In one embodiment, the Fc region is modified by substituting the methionine residue at position 252 with a tyrosine residue, the serine residue at position 254 with a threonine residue, the threonine residue at position 256 with a glutamic acid residue, the histidine residue at position 433 with a lysine residue, and the asparagine residue at position 434 with a phenylalanine residue (M252Y / S254T / T256E / H433K / N434F).

[0287] It will be appreciated that any of the above-listed modifications can be combined to alter FcRn binding.

[0288] In one embodiment, the CD19 binding molecule comprises an Fc domain, wherein one or both Fc regions comprise one or more modifications such that the Fc domain binds to FcRn with lower affinity and avidity than the corresponding native immunoglobulin.

[0289] In one embodiment, the Fc region comprises any amino acid residue except histidine at position 310 and / or position 435.

[0290] The CD19 binding molecule can comprise an Fc domain, wherein one or both Fc regions comprise one or more modifications that enhance its binding to FcγRIIb. FcγRIIb is the only inhibitory receptor in humans and the only Fc receptor found on B cells.

[0291] In one embodiment, the Fc region is modified by substituting the proline residue at position 238 with an aspartic acid residue (P238D).

[0292] In one embodiment, the Fc region is modified by substituting the glutamic acid residue at position 258 with an alanine residue (E258A).

[0293] In one embodiment, the Fc region is modified by substituting the serine residue at position 267 with an alanine residue (S267A).

[0294] In one embodiment, the Fc region is modified by substituting the serine residue at position 267 with a glutamic acid residue (S267E).

[0295] In one embodiment, the Fc region is modified by substituting the leucine residue at position 328 with a phenylalanine residue (L328F).

[0296] In one embodiment, the Fc region is modified by substituting the glutamic acid residue at position 258 with an alanine residue and substituting the serine residue at position 267 with an alanine residue (E258A / S267A).

[0297] In one embodiment, the Fc region is modified by substituting the serine residue at position 267 with a glutamic acid residue and substituting the leucine residue at position 328 with a phenylalanine residue (S267E / L328F).

[0298] It will be appreciated that any of the above-listed modifications can be combined to enhance FcγRIIb binding.

[0299] In one embodiment, there is provided a CD19-binding molecule comprising an Fc domain that exhibits reduced binding to FcγR.

[0300] In one embodiment, the CD19-binding molecule comprises an Fc domain, wherein one or both Fc regions comprise one or more modifications that reduce Fc binding to FcγR.

[0301] The Fc domain can be derived from IgG1.

[0302] In one embodiment, the Fc region is modified by substituting the leucine residue at position 234 with an alanine residue (L234A).

[0303] In one embodiment, the Fc region is modified by substituting the leucine residue at position 235 with an alanine residue (L235A).

[0304] In one embodiment, the Fc region is modified by substituting the glycine residue at position 236 with an arginine residue (G236R).

[0305] In one embodiment, the Fc region is modified by substituting the asparagine residue at position 297 with an alanine residue (N297A) or a glutamine residue (N297Q).

[0306] In one embodiment, the Fc region is modified by substituting the serine residue at position 298 with an alanine residue (S298A).

[0307] In one embodiment, the Fc region is modified by substituting the leucine residue at position 328 with an arginine residue (L328R).

[0308] In one embodiment, the Fc region is modified by substituting the leucine residue at position 234 with an alanine residue and substituting the leucine residue at position 235 with an alanine residue (L234A / L235A).

[0309] In one embodiment, the Fc region is modified by substituting the phenylalanine residue at position 234 with an alanine residue and substituting the leucine residue at position 235 with an alanine residue (F234A / L235A).

[0310] In one embodiment, the Fc region is modified by substituting the glycine residue at position 236 with an arginine residue and substituting the leucine residue at position 328 with an arginine residue (G236R / L328R).

[0311] In one embodiment, the Fc region is modified by substituting the aspartic acid residue at position 265 with an alanine residue, substituting the asparagine residue at position 297 with an alanine residue, and substituting the proline residue at position 329 with an alanine residue (D265A / N297A / P329A).

[0312] In one embodiment, the Fc region is modified by substituting the aspartic acid residue at position 265 with an asparagine residue, substituting the asparagine residue at position 297 with an aspartic acid residue, and substituting the proline residue at position 329 with a glycine residue (D265N / N297D / P329G).

[0313] In one embodiment, the Fc region is modified by substituting the aspartic acid residue at position 265 with a glutamate residue, substituting the asparagine residue at position 297 with a glutamate residue, and substituting the proline residue at position 329 with a serine residue (D265E / N297Q / P329S).

[0314] It will be appreciated that any of the above-listed modifications can be combined to reduce FcγR binding.

[0315] In one embodiment, the CD19 binding molecule comprises an Fc domain, wherein one or both Fc regions comprise one or more modifications that reduce Fc binding to FcγRIIIa without affecting Fc binding to FcγRII.

[0316] In one embodiment, the Fc region is modified by substituting the serine residue at position 239 with an alanine residue (S239A).

[0317] In one embodiment, the Fc region is modified by substituting the glutamate residue at position 269 with an alanine residue (E269A).

[0318] In one embodiment, the Fc region is modified by substituting the glutamate residue at position 293 with an alanine residue (E293A).

[0319] In one embodiment, the Fc region is modified by substituting the tyrosine residue at position 296 with a phenylalanine residue (Y296F).

[0320] In one embodiment, the Fc region is modified by substituting the valine residue at position 303 with an alanine residue (V303A).

[0321] In one embodiment, the Fc region is modified by substituting the alanine residue at position 327 with a glycine residue (A327G).

[0322] In one embodiment, the Fc region is modified by substituting the lysine residue at position 338 with an alanine residue (K338A).

[0323] In one embodiment, the Fc region is modified by substituting the aspartic acid residue at position 376 with an alanine residue (D376A).

[0324] It will be appreciated that any of the above-listed modifications can be combined to reduce FcγRIIIa binding.

[0325] Fc region variants with reduced FcR binding can be referred to as "FcγR ablation variants", "FcγR silent variants", or "Fc knockout (FcKO or KO)" variants. For some therapeutic applications, it is desirable to reduce or eliminate the normal binding of the Fc domain to one or more or all Fcγ receptors (e.g., FcγR1, FcγRIIa, FcγRIIb, FcγRIIIa) to avoid additional mechanisms of action. That is, for example, in many embodiments, particularly in the use of monovalent CD3-binding MBMs, it is generally desirable to ablate FcγRIIIa binding to eliminate or significantly reduce ADCC activity. In some embodiments, at least one Fc region of the MBMs described herein comprises one or more Fcγ receptor ablation variants. In some embodiments, both Fc regions comprise one or more Fcγ receptor ablation variants. These ablation variants are described in Table 3 and each can be independently and optionally included or excluded, where some aspects utilize ablation variants selected from the group consisting of: G236R / L328R,

[0326] E233P / L234V / L235A / G236del / S239K,

[0327] E233P / L234V / L235A / G236del / S267K,

[0328] E233P / L234V / L235A / G236del / S239K / A327G,

[0329] E233P / L234V / L235A / G236del / S267K / A327G,

[0330] E233P / L234V / L235A / G236del, D265A / N297A / P329A,

[0331] D265N / N297D / P329G, and D265E / N297Q / P329S (where "del" indicates deletion, e.g., G236del refers to the deletion of the glycine residue at position 236). It should be noted that the ablation variants cited herein ablate FcγR binding but generally do not ablate FcRn binding.

[0332]

[0333]

[0334] In some embodiments, the MBMs of the present disclosure comprise a first Fc region and a second Fc region. In some embodiments, the first Fc region and / or the second Fc region may comprise the following mutations: E233P, L234V, L235A, G236del, and S267K.

[0335] The Fc domain of human IgG1 has the highest binding to Fcγ receptors, and thus ablation variants can be used when the constant domain (or Fc domain) in the backbone of the heterodimeric antibody is IgG1.

[0336] Alternatively, or in addition to ablation variants in the IgG1 background, mutations at glycosylation position 297, e.g., replacing the asparagine residue at position 297 with an alanine residue (N297A) or with a glutamine residue (N297Q), can significantly ablate binding to, e.g., FcγRIIIa. Human IgG2 and IgG4 have a naturally reduced binding to Fcγ receptors, and thus those backbones can be used with or without ablation variants.

[0337] 7.4.1.2. Fc domains with altered complement binding

[0338] The CD19-binding molecule may comprise an Fc domain, wherein one or both Fc regions comprise one or more modifications that alter Fc binding to complement. The altered complement binding can be enhanced binding or reduced binding.

[0339] In one embodiment, the Fc region comprises one or more modifications that reduce its binding to C1q. The initiation of the classical complement pathway begins with the binding of the hexameric C1q protein to the CH2 domains of IgG and IgM that are bound to an antigen.

[0340] In one embodiment, the CD19-binding molecule comprises an Fc domain, wherein one or both Fc regions comprise one or more modifications that reduce Fc binding to C1q.

[0341] In one embodiment, the Fc region is modified by replacing the leucine residue at position 234 with an alanine residue (L234A).

[0342] In one embodiment, the Fc region is modified by substituting the leucine residue at position 235 with an alanine residue (L235A).

[0343] In one embodiment, the Fc region is modified by substituting the leucine residue at position 235 with a glutamic acid residue (L235E).

[0344] In one embodiment, the Fc region is modified by substituting the glycine residue at position 237 with an alanine residue (G237A).

[0345] In one embodiment, the Fc region is modified by substituting the lysine residue at position 322 with an alanine residue (K322A).

[0346] In one embodiment, the Fc region is modified by substituting the proline residue at position 331 with an alanine residue (P331A).

[0347] In one embodiment, the Fc region is modified by substituting the proline residue at position 331 with a serine residue (P331S).

[0348] In one embodiment, the CD19-binding molecule comprises an Fc domain derived from IgG4. IgG4 has a naturally lower complement activation profile than IgG1 and also has a weaker binding to FcγR. Thus, in one embodiment, the CD19-binding molecule comprises an IgG4 Fc domain and also comprises one or more modifications that enhance FcγR binding.

[0349] It will be appreciated that any of the above-listed modifications can be combined to reduce C1q binding.

[0350] 7.4.1.3. Fc domains with altered disulfide bond structure

[0351] The CD19-binding molecule can include an Fc domain comprising one or more modifications to generate and / or remove cysteine residues. Cysteine residues play an important role in the co-assembly of Fc-based multispecific binding molecules by forming disulfide bridges between pairs of polypeptide monomers. Thus, it is possible to modify the CD19-binding molecule structure by altering the number and / or position of cysteine residues to produce proteins with improved therapeutic properties.

[0352] The CD19-binding molecules of the present disclosure may comprise an Fc domain, wherein one or both Fc regions (e.g., both Fc regions) comprise a cysteine residue at position 309. In one embodiment, the cysteine residue at position 309 is generated by modification, e.g., for an Fc domain derived from IgG1, the leucine residue at position 309 is replaced with a cysteine residue (L309C), and for an Fc domain derived from IgG2, the valine residue at position 309 is replaced with a cysteine residue (V309C).

[0353] In one embodiment, the Fc region is modified by replacing the valine residue at position 308 with a cysteine residue (V308C).

[0354] In one embodiment, two disulfide bonds in the hinge region are removed by mutating the core hinge sequence CPPC (SEQ ID NO:55) to SPPS (SEQ ID NO:56).

[0355] 7.4.1.4. Fc domains with altered glycosylation

[0356] In certain aspects, CD19-binding molecules with improved manufacturability are provided, the CD19-binding molecules comprising fewer glycosylation sites than the corresponding immunoglobulins. These proteins have a less complex post-translational glycosylation pattern and are thus simpler and less costly for manufacturers.

[0357] In one embodiment, the glycosylation site in the CH2 domain is removed by replacing the asparagine residue at position 297 with an alanine residue (N297A) or a glutamine residue (N297Q). In addition to improved manufacturability, these glycosylation mutants also reduce FcγR binding as described above herein.

[0358] In some embodiments, CD19-binding molecules can be prepared that have an altered glycosylation pattern, such as afucosylated antibodies with a reduced amount of fucosyl residues or antibodies with an increased bisecting GlcNAc structure. Such altered glycosylation patterns have been shown to increase the ADCC ability of the antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the CD19-binding molecule in a host cell with an altered glycosylation machinery. Cells with an altered glycosylation machinery have been described in the art and can be used as host cells for expressing the CD19-binding molecule, thereby producing a CD19-binding molecule with altered glycosylation. For example, EP 1,176,195 by Hang et al. describes cell lines with a disrupted FUT8 gene, which encodes fucosyltransferase, such that antibodies expressed in such cell lines exhibit afucosylation. Presta describes in PCT Publication WO 03 / 035835 variant CHO cell line Lec13 cells, which have a reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in afucosylation of antibodies expressed in the host cells (see also Shields et al., 2002, J. Biol. Chem. 277:26733-26740). Umana et al. describe in PCT Publication WO 99 / 54342 cell lines that are engineered to express a glycoprotein-modifying glycosyltransferase (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell lines exhibit an increased bisecting GlcNAc structure, which results in increased ADCC activity of the antibodies (see also Umana et al., Nat. Biotech. 17:176-180, 1999).

[0359] 7.4.1.5. Fc Heterodimerization

[0360] Many multispecific molecular formats require dimerization between two Fc regions, which, unlike native immunoglobulins, are operably linked to non-identical antigen-binding domains (or portions thereof, e.g., VH or VH-CH1 of a Fab). Inadequate heterodimerization of the two Fc regions forming the Fc domain has been an obstacle to improving the production of desired multispecific molecules and represents a purification challenge. A variety of methods available in the art can be used to enhance the dimerization of Fc regions that may be present in CD19-binding molecules (and especially in the MBMs disclosed herein), such as those disclosed in: EP1870459 A1; U.S. Patent No. 5,582,996; U.S. Patent No. 5,731,168; U.S. Patent No. 5,910,573; U.S. Patent No. 5,932,448; U.S. Patent No. 6,833,441; U.S. Patent No. 7,183,076; U.S. Patent Application Publication No. 2006204493A1; and PCT Publication No. WO 2009 / 089004 A1.

[0361] The present disclosure provides CD19-binding molecules comprising an Fc heterodimer, i.e., an Fc domain comprising heterologous, non-identical Fc regions. Heterodimerization strategies are used to enhance the dimerization of Fc regions operably linked to different ABMs (or portions thereof, e.g., VH or VH-CH1 of a Fab) and to reduce the dimerization of Fc regions operably linked to the same ABM or portion thereof. Typically, each Fc region in the Fc heterodimer comprises the CH3 domain of an antibody. The CH3 domain is derived from any isotype, type or subclass, and in some cases the constant region of an antibody of the IgG (IgG1, IgG2, IgG3 and IgG4) type, as described in the foregoing section.

[0362] Typically, in addition to the CH3 domain, the MBM also comprises other antibody fragments, such as the CH1 domain, CH2 domain, hinge domain, one or more VH domains, one or more VL domains, one or more CDRs, and / or antigen-binding fragments as described herein. In some embodiments, the two heteropolypeptides are the two heavy chains that form a bispecific or multispecific molecule. Heterodimerization of two different heavy chains at the CH3 domain results in the desired antibody or antibody-like molecule, while homodimerization of identical heavy chains will reduce the production of the desired antibody or molecule. In an exemplary embodiment, the two or more heteropolypeptide chains comprise two chains that comprise the CH3 domain and form a molecule of any of the multispecific molecular formats described above in the present disclosure. In an embodiment, the two heteropolypeptide chains comprising the CH3 domain comprise modifications (relative to the unmodified chains) that favor heterodimeric association of the polypeptides. A variety of examples of modification strategies are provided in Tables 4 and 7.4.1.5.1 to 7.4.1.5.7 below.

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372] Exemplary heterologous, non-identical Fc sequence pairs that can pair to form an Fc heterodimer and can be included in the CD19-binding molecules of the present disclosure include (i) SEQ ID NO: 1106 and SEQ ID NO: 1107, and (ii) SEQ ID NO: 1106 and SEQ ID NO: 1108.

[0373] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1106)

[0374] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:1107)

[0375] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGK(SEQ ID NO:1108)

[0376] The Fc region having an amino acid sequence of one of SEQ ID NOs: 1106 - 1108 can be modified to include one or more substitutions as described in Section 7.4.1 (including its subsections), such as including one or more substitutions corresponding to the ablation variants listed in Table 3. In some embodiments, the CD19 binding molecule comprises an Fc region having an amino acid sequence of one of SEQ ID NOs: 1106 - 1108 that has mutations at 1, 2, 3, 4, 5, 6, or more than 6 positions corresponding to positions 233, 234, 235, 236, 237, 239, 265, 266, 267, 268, 269, 297, 299, 322, 327, 328, 329, 330, 331, and 332 (EU numbering) (e.g., one or more mutations as described in Section 7.4.1 (including its subsections)). For example, the CD19 binding molecule can comprise an Fc region having an amino acid sequence of SEQ ID NO: 1106 that has mutations at 1, 2, 3, 4, 5, 6, or more than 6 positions corresponding to positions 233, 234, 235, 236, 237, 239, 265, 266, 267, 268, 269, 297, 299, 322, 327, 328, 329, 330, 331, and 332 and / or an Fc region having an amino acid sequence of SEQ ID NO: 1107 that has mutations at 1, 2, 3, 4, 5, 6, or more than 6 positions corresponding to positions 233, 234, 235, 236, 237, 239, 265, 266, 267, 268, 269, 297, 299, 322, 327, 328, 329, 330, 331, and 332 and / or an Fc region having an amino acid sequence of SEQ ID NO: 1108 that has mutations at 1, 2, 3, 4, 5, 6, or more than 6 positions corresponding to positions 233, 234, 235, 236, 237, 239, 265, 266, 267, 268, 269, 297, 299, 322, 327, 328, 329, 330, 331, and 332.

[0377] 7.4.1.5.1. Spatial Variant

[0378] The CD19-binding molecule can comprise one or more, e.g., multiple, modifications to one or more constant domains of the Fc domain, e.g., to the CH3 domain. In one instance, the CD19-binding molecule of the present disclosure comprises two polypeptides, each polypeptide comprising a heavy chain constant domain of an antibody, e.g., a CH2 or CH3 domain. In an instance, the two heavy chain constant domains, e.g., the CH2 or CH3 domains of the CD19-binding molecule, comprise one or more modifications that permit heterodimeric association between the two chains. In one aspect, the one or more modifications are disposed on the CH2 domains of the two heavy chains. In one aspect, the one or more modifications are disposed on the CH3 domains of at least two polypeptides of the CD19-binding molecule.

[0379] One mechanism for Fc heterodimerization is commonly referred to as "knobs and holes" or "knobs-into-holes". These terms refer to amino acid mutations that create steric effects to favor the formation of Fc heterodimers (as compared to Fc homodimers), as described, e.g., by Ridgway et al., 1996, Protein Engineering 9(7):617; Atwell et al., 1997, J. Mol. Biol. 270:26; U.S. Patent No. 8,216,805. Knobs-into-holes mutations can be combined with other strategies to improve heterodimerization.

[0380] In one aspect, one or more modifications to a first polypeptide of a CD19-binding molecule comprising a heavy chain constant domain can create a "pestle" and one or more modifications to a second polypeptide of the CD19-binding molecule create a "mortar" such that heterodimerization of the polypeptides of the CD19-binding molecule comprising the heavy chain constant domain creates a "pestle" to engage an interface (e.g., interact, e.g., the CH2 domain of the first polypeptide interacts with the CH2 domain of the second polypeptide, or the CH3 domain of the first polypeptide interacts with the CH3 domain of the second polypeptide) with the "mortar". The pestle protrudes from an interface of the first polypeptide of the CD19-binding molecule comprising the heavy chain constant domain and can thus be positioned in a complementary "mortar" in an interface with the second polypeptide of the CD19-binding molecule comprising the heavy chain constant domain to stabilize the heteropolymer and thus facilitate heteropolymer formation (e.g., relative to a homopolymer). The pestle can be present in the original interface or can be synthetically introduced (e.g., by altering the nucleic acid encoding the interface). The input residues for forming the pestle are typically naturally occurring amino acid residues and can be selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). In some cases, tryptophan and tyrosine are selected. In the examples, the original residues for forming the protrusion have a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.

[0381] The "mortar" comprises at least one amino acid side chain that is recessed into an interface of the second polypeptide of the CD19-binding molecule comprising the heavy chain constant domain and thus accommodates a corresponding pestle on an adjacent junction surface of the first polypeptide of the CD19-binding molecule comprising the heavy chain constant domain. The mortar can be present in the original interface or can be introduced in a synthetic manner (e.g., by altering the nucleic acid encoding the interface). The input residues for forming the mortar are typically naturally occurring amino acid residues and in some examples are selected from alanine (A), serine (S), threonine (T), and valine (V). In one example, the amino acid residue is serine, alanine, or threonine. In another example, the original residues for forming the mortar have a large side chain volume, such as tyrosine, arginine, phenylalanine, or tryptophan.

[0382] In an embodiment, the first CH3 domain is modified at residue 366, 405, or 407 to produce a "pestle" or "mortar" (as described above), and the second CH3 domain that heterodimerizes with the first CH3 domain is modified at the following to produce a "mortar" or "pestle" complementary to the "pestle" or "mortar" of the first CH3 domain: residue 407 (if residue 366 in the first CH3 domain is modified), residue 394 (if residue 405 in the first CH3 domain is modified), or residue 366 (if residue 407 in the first CH3 domain is modified).

[0383] In another embodiment, the first CH3 domain is modified at residue 366, and the second CH3 domain that heterodimerizes with the first CH3 domain is modified at residues 366, 368, and / or 407 to produce a "mortar" or "pestle" complementary to the "pestle" or "mortar" of the first CH3 domain. In one embodiment, the modification of the first CH3 domain introduces a tyrosine (Y) residue at position 366. In an embodiment, the modification of the first CH3 is T366Y. In one embodiment, the modification of the first CH3 domain introduces a tryptophan (W) residue at position 366. In an embodiment, the modification of the first CH3 is T366W. In some embodiments, the modification of the second CH3 domain that heterodimerizes with the first CH3 domain (modified at position 366 (e.g., having a tyrosine (Y) or tryptophan (W) introduced at position 366, e.g., comprising the modification T366Y or T366W)) comprises a modification at position 366, a modification at position .In some embodiments, the modification at position 366 introduces a serine (S) residue, the modification at position 368 introduces an alanine (A), and the modification at position 407 introduces a valine (V). In some embodiments, the modification comprises T366S, L368A, and Y407V. In one embodiment, the first CH3 domain of the multispecific molecule comprises the modification T366Y, and the second CH3 domain that heterodimerizes with the first CH3 domain comprises the modifications T366S, L368A, and Y407V, and vice versa. In one embodiment, the first CH3 domain of the multispecific molecule comprises the modification T366W, and the second CH3 domain that heterodimerizes with the first CH3 domain comprises the modifications T366S, L368A, and Y407V, and vice versa.

[0384] Additional space or "offset" (e.g., mortise and tenon structure) is modified as described in PCT Publication No. WO 2014 / 145806 (e.g., Figures 3, 4, and 12 of WO 2014 / 145806), PCT Publication No. WO 2014 / 110601, and PCT Publication Nos. WO 2016 / 086186, WO 2016 / 086189, WO 2016 / 086196, and WO 2016 / 182751. Examples of KIH variants include a first constant chain containing L368D and K370S modifications, which pairs with a second constant chain containing S364K and E357Q modifications.

[0385] Additional mortise and tenon structure modifications suitable for use in any of the CD19-binding molecules of the present disclosure are further described in, for example, WO 1996 / 027011, and Merchant et al., 1998, Nat. Biotechnol. [Nature Biotechnology], 16:677-681.

[0386] In further embodiments, the CH3 domain may additionally be modified to introduce a pair of cysteine residues. Without being bound by theory, it is believed that introducing a pair of cysteine residues capable of forming a disulfide bond provides stability to a heterodimerizing CD19-binding molecule (e.g., MBM) comprising paired CH3 domains. In some embodiments, the first CH3 domain comprises a cysteine at position 354 and the second CH3 domain heterodimerizing with the first CH3 domain comprises a cysteine at position 349. In some embodiments, the first CH3 domain comprises a cysteine at position 354 (e.g., comprising the modification S354C) and a tyrosine (Y) at position 366 (e.g., comprising the modification T366Y), and the second CH3 domain heterodimerizing with the first CH3 domain comprises a cysteine at position 349 (e.g., comprising the modification Y349C), a serine at position 366 (e.g., comprising the modification T366S), an alanine at position 368 (e.g., comprising the modification L368A), and a valine at position 407 (e.g., comprising the modification Y407V). In some embodiments, the first CH3 domain comprises a cysteine at position 354 (e.g., comprising the modification S354C) and a tryptophan (W) at position 366 (e.g., comprising the modification T366W), and the second CH3 domain heterodimerizing with the first CH3 domain comprises a cysteine at position 349 (e.g., comprising the modification Y349C), a serine at position 366 (e.g., comprising the modification T366S), an alanine at position 368 (e.g., comprising the modification L368A), and a valine at position 407 (e.g., comprising the modification Y407V).

[0387] Additional mechanisms that can be used to generate heterodimers are sometimes referred to as "electrostatic steering", as described in: Gunasekaran et al., 2010, J. Biol. Chem. [Journal of Biological Chemistry] 285(25):19637. This is sometimes referred to herein as a "charge pair". In this embodiment, electrostatic forces are used to form a bias towards heterodimerization. As those skilled in the art will appreciate, these variants can also have an effect on the pI and thus on purification, and can therefore be considered pI variants in some cases. However, given that these variants are generated to promote heterodimerization and are not used as purification tools, they are classified as "spatial variants". These variants include, but are not limited to, D221E / P228E / L368E paired with D221R / P228R / K409R and C220E / P228E / 368E paired with C220R / E224R / P228R / K409R.

[0388] Additional variants that can be combined with other variants (optionally and independently in any amount), such as the pI variants outlined herein or other spatial variants shown in Figure 37 of US 2012 / 0149876.

[0389] In some embodiments, the spatial variants outlined herein can optionally and independently be incorporated into one or both Fc regions together with any pI variant (or other variants such as Fc variants, FcRn variants), and can independently and optionally be included within or excluded from the CD19-binding molecules of the present disclosure.

[0390] A column of suitable offset variants can be seen in Table 5, which shows some variant pairs that have specific applications in many embodiments. Variant pairs of the following groups that have specific uses in many embodiments include, but are not limited to, S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411T / E360E / Q362E:D401K; L368D / K370S:S364K / E357L; and K370S:S364K / E357Q. In terms of nomenclature, the variant pair "S364K / E357Q:L368D / K370S" means that one of the Fc regions has the double-variant group S364K / E357Q and the other has the double-variant group L368D / K370S.

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397] In some embodiments, the CD19-binding molecule comprises a first Fc region and a second Fc region. In some embodiments, the first Fc region comprises the following mutations: L368D and K370S, and the second Fc region comprises the following mutations: S364K and E357Q. In some embodiments, the first Fc region comprises the following mutations: S364K and E357Q, and the second Fc region comprises the following mutations: L368D and K370S.

[0398] 7.4.1.5.2. Alternative Knob and Hole: IgG heterodimerization

[0399] Heterodimerization of the polypeptide chains of a CD19-binding molecule comprising paired CH3 domains can be enhanced by introducing one or more modifications in the CH3 domain derived from the IgG1 antibody type. In embodiments, the modification comprises a K409R modification of one CH3 domain paired with an F405L modification in a second CH3 domain. Additional modifications can also, or alternatively, be at positions 366, 368, 370, 399, 405, 407, and 409. In some cases, heterodimerization of polypeptides comprising such modifications is achieved under reducing conditions, e.g., at 25-37 °C, e.g., 25 °C or 37 °C, in 10-100 mM 2-MEA (e.g., 25, 50, or 100 mM 2-MEA) for 1-10, e.g., 1.5-5, e.g., 5 hours.

[0400] The amino acid substitutions described herein can be introduced into the CH3 domain using well-known techniques (see, e.g., McPherson, ed., 1991, Directed Mutagenesis: a Practical Approach; Adelman et al., 1983, DNA, 2:183).

[0401] The IgG heterodimerization strategy is further described in, for example, WO 2008 / 119353, WO 2011 / 131746, and WO 2013 / 060867.

[0402] In any of the embodiments described in this section, the CH3 domain can alternatively be modified to introduce a pair of cysteine residues, as described in section 7.4.1.3.

[0403] 7.4.1.5.3. pI (isoelectric point) variants

[0404] Generally, as will be understood by those skilled in the art, there are two general classes of pI variants: those that increase the pI of the protein (basic alterations) and those that decrease the pI of the protein (acidic alterations). As described herein, all combinations of these variants can be made: one Fc region can be wild-type or a variant that does not exhibit a significantly different pI from wild-type, and the other can be more basic or acidic. Alternatively, each Fc region can be altered such that one is more basic and the other is more acidic.

[0405] Exemplary combinations of pI variants are shown in Table 6. As outlined herein and shown in Table 6, these alterations are shown relative to IgG1, but all isotypes, as well as isotype hybrids, can be altered in this manner. In the case where the heavy chain constant domain is from IgG2-4, R133E and R133Q can also be used.

[0406]

[0407] In one embodiment, for example, in Figure 1B-1W , Figure 1Y-1AH , Figure 2B-2L , and Figure 2N-2V forms, the combination of pI variants has one Fc region (negative Fab side) that contains the 208D / 295E / 384D / 418E / 421D variant (N208D / Q295E / N384D / Q418E / N421D when compared to human IgG1) and a second Fc region (positive scFv side) that contains a positively charged scFv linker, e.g., L36 (described in section 7.4.3). However, as will be understood by those skilled in the art, the first Fc region includes the CH1 domain that contains position 208. Thus, in constructs that do not include the CH1 domain (e.g., for MBMs that do not use the CH1 domain as one of the domains, such as in the form described in ​ ), the negative pI variant Fc group can include the 295E / 384D / 418E / 421D variant (Q295E / N384D / Q418E / N421D when compared to human IgG1).

[0408] In some embodiments, the first Fc region has a set of substitutions from Table 6 and the second Fc region is linked to a charged linker (e.g., selected from those described in section 7.4.3).

[0409] In some embodiments, the CD19-binding molecules of the present disclosure comprise a first Fc region and a second Fc region. In some embodiments, the first Fc region comprises the following mutations: N208D, Q295E, N384D, Q418E, and N421D. In some embodiments, the second Fc region comprises the following mutations: N208D, Q295E, N384D, Q418E, and N421D.

[0410] 7.4.1.5.4. Isotope variants

[0411] In addition, many embodiments of the present disclosure rely on "inputting" pI amino acids at specific positions from one IgG isotype into another, thereby reducing or eliminating the likelihood that unwanted immunogenicity is introduced into the variant. Many of these variants are shown in FIG. 21 of U.S. Publication No. 2014 / 0370013. That is, IgG1 is a common isotype for therapeutic antibodies for various reasons (including high effector function). However, the heavy constant region of IgG1 has a higher pI (8.10 vs. 7.31) than IgG2. By introducing IgG2 residues at specific positions into the IgG1 backbone, the pI of the resulting Fc region is decreased (or increased) and additionally exhibits a longer serum half-life. For example, IgG1 has glycine (pI 5.97) at position 137, and IgG2 has glutamate (pI 3.22); inputting glutamate will affect the pI of the resulting protein. As described below, a large number of amino acid substitutions are typically required to significantly affect the pI of the variant antibody. However, as discussed below, it should be noted that even alterations in IgG2 molecules allow for an increase in serum half-life.

[0412] In other embodiments, non-isotype amino acid changes are made to reduce the overall charge state of the resulting protein (e.g., by changing higher pI amino acids to lower pI amino acids), or to allow for structural modulation for stability, as further described below.

[0413] In addition, by pI engineering the heavy and light constant domains of a CD19-binding molecule comprising two half-antibodies, significant changes can be seen in each half-antibody. A difference in pI of at least 0.5 between the two half-antibodies can allow for separation by ion exchange chromatography or isoelectric focusing, or other methods sensitive to the isoelectric point.

[0414] 7.4.1.5.5. Calculating pI

[0415] The pI of a half antibody comprising an Fc region and an ABM or ABM chain can depend on the pI of the variant heavy chain constant domain and the pI of the total half antibody (including the variant heavy chain constant domain and the ABM or ABM chain). Thus, in some embodiments, using the chart in Figure 19 of US Publication 2014 / 0370013, the change in pI is calculated based on the variant heavy chain constant domain. As discussed herein, which half antibody to engineer is typically determined by the inherent pI of the half antibody. Alternatively, the pI of each half antibody can be compared.

[0416] 7.4.1.5.6. pI variants that also confer improved in vivo FcRn binding

[0417] In the case where the pI variant reduces the pI of the Fc region, it can have the additional benefit of improved in vivo serum retention.

[0418] The pI variant Fc region is believed to provide a longer half-life for in vivo antigen-binding molecules because binding to FcRn in the endosome at pH 6 sequesters the Fc (Ghetie and Ward, 1997, Immunol Today. 18(12):592 - 598). The endosomal compartment then recycles the Fc back to the cell surface. Once the compartment opens to the extracellular space, the higher pH, approximately 7.4, induces the release of the Fc back into the blood. In mice, Dall’Acqua et al. showed that Fc mutants with improved FcRn binding at pH 6 and pH 7.4 actually had reduced serum concentrations and the same half-life as wild-type Fc (Dall’Acqua et al 2002, J. Immunol. 169:5171 - 5180). An increased affinity of Fc for FcRn at pH 7.4 is thought to prevent the release of the Fc back into the blood. Thus, the Fc mutants that will increase the in vivo half-life of the Fc will desirably increase FcRn binding at lower pH and still allow the release of the Fc at higher pH. The amino acid histidine changes its charge state in the pH range of 6.0 to 7.4. Thus, it is not surprising to find His residues at important positions in the Fc / FcRn complex.

[0419] It has been proposed that antibodies with variable regions (with a lower isoelectric point) can also have a longer serum half-life (Igawa et al., 2010, PEDS. 23(5):385 - 392). However, the mechanism of this finding is still poorly understood. In addition, the variable regions are different between antibodies. Constant region variants with a reduced pI and an extended half-life would provide a more modular approach to improving the pharmacokinetic properties of CD19-binding molecules as described herein.

[0420] 7.4.1.5.7. Polar bridges

[0421] The heterodimerization of a polypeptide chain of a CD19-binding molecule (e.g., MBM) comprising an Fc domain can be enhanced by introducing modifications based on the "polarity bridge" principle, which is to create residues at the binding interface of two polypeptide chains to interact with residues having similar (or complementary) physical properties in the heterodimer configuration while interacting with residues having different physical properties in the homodimer configuration. In particular, these modifications are designed such that in heterodimer formation, polar residues interact with polar residues and hydrophobic residues interact with hydrophobic residues. In contrast, in homodimer formation, the modified residues are such that polar residues interact with hydrophobic residues. The favorable interactions in the heterodimer configuration and the unfavorable interactions in the homodimer configuration together act to make the formation of heterodimers in the Fc region more likely than the formation of homodimers.

[0422] In an exemplary embodiment, the above-mentioned modifications are made at one or more positions among residues 364, 368, 399, 405, 409, and 411 of the CH3 domain.

[0423] In some embodiments, one or more modifications selected from the group consisting of S364L, T366V, L368Q, N399K, F405S, K409F, and R411K are introduced into one of the two CH3 domains. One or more modifications selected from the group consisting of Y407F, K409Q, and T411N can be introduced into the second CH3 domain.

[0424] In another embodiment, one or more modifications selected from the group consisting of S364L, T366V, L368Q, D399K, F405S, K409F, and T411K are introduced into one CH3 domain, while one or more modifications selected from the group consisting of Y407F, K409Q, and T411D are introduced into the second CH3 domain.

[0425] In an exemplary embodiment, the original residue of threonine at position 366 of one CH3 domain is replaced with valine, while the original residue of tyrosine at position 407 of the other CH3 domain is replaced with phenylalanine.

[0426] In another exemplary embodiment, the original residue of serine at position 364 of one CH3 domain is replaced with leucine, and the original residue of leucine at position 368 of the same CH3 domain is replaced with glutamine.

[0427] In yet another exemplary embodiment, the initial residue of phenylalanine at position 405 of one CH3 domain is replaced with serine and the initial residue of lysine at position 409 of this CH3 domain is replaced with phenylalanine, while the initial residue of lysine at position 409 of another CH3 domain is replaced with glutamine.

[0428] In yet another exemplary embodiment, the initial residue of aspartic acid at position 399 of one CH3 domain is replaced with lysine, and the initial residue of threonine at position 411 of the same CH3 domain is replaced with lysine, while the initial residue of threonine at position 411 of another CH3 domain is replaced with aspartic acid.

[0429] The amino acid substitutions described herein can be introduced into the CH3 domain using well-known techniques (see, for example, McPherson, ed., 1991, Directed Mutagenesis: a Practical Approach; Adelman et al., 1983, DNA, 2:183). The polar bridge strategy is described, for example, in WO 2006 / 106905, WO2009 / 089004, and Gunasekaran et al., 2010, JBC 285:19637-19646.

[0430] Additional polar bridge modifications are described, for example, in PCT Publication No. WO 2014 / 145806 (e.g., FIG. 6 of WO 2014 / 145806), PCT Publication No. WO 2014 / 110601, and PCT Publication Nos. WO 2016 / 086186, WO 2016 / 086189, WO 2016 / 086196, and WO 2016 / 182751. Examples of polar bridge variants include constant chains containing N208D, Q295E, N384D, Q418E, and N421D modifications.

[0431] In any of the embodiments described herein, the CH3 domain can additionally be modified to introduce a pair of cysteine residues, as described in Section 7.4.1.3.

[0432] Additional strategies for enhancing heterodimerization are described, for example, in WO 2016 / 105450, WO 2016 / 086186, WO 2016 / 086189, WO 2016 / 086196, WO 2016 / 141378, and WO 2014 / 145806, and WO2014 / 110601. Any of these strategies can be used in the CD19 binding molecules described herein.

[0433] 7.4.1.6. Combinations of heterodimerization variants and other Fc variants

[0434] As will be understood by those skilled in the art, all of the listed heterodimerization variants (including offset and / or pI variants) can be optionally and independently combined in any manner, provided that the Fc regions of the Fc domains retain their ability to dimerize. Additionally, all of these variants can be combined with any of the heterodimerized forms.

[0435] In the case of pI variants, when embodiments for specific uses are shown in Table 6, other combinations can be generated based on the general principle of changing the pI difference between the two Fc regions in the Fc heterodimer to facilitate purification.

[0436] Additionally, any of the heterodimerization variants, offsets, and pI can also be independently and optionally combined with Fc ablation variants, Fc variants, and FcRn variants, as generally outlined herein.

[0437] In some embodiments, specific combinations of offset and pI variants useful in the present disclosure are T366S / L368A / Y407V:T366W (optionally including a bridging disulfide bond, T366S / L368A / Y407V / Y349C:T366W / S354C), where one Fc region contains Q295E / N384D / Q418E / N481D and the other Fc region contains a positively charged scFv linker (when the form includes an scFv domain). As will be understood by those skilled in the art, the "mortise and tenon" variant does not change the pI and can thus be used on either Fc region in the Fc heterodimer.

[0438] In some embodiments, the first and second Fc regions useful in the present disclosure include the amino acid substitutions S364K / E357Q:L368D / K370S, where the first and / or second Fc region includes an ablation variant substitution of 233P / L234V / L235A / G236del / S267K, and the first and / or second Fc region contains a pI variant substitution of N208D / Q295E / N384D / Q418E / N421D (pl_(-)_isoelectric_A).

[0439] 7.4.2. Hinge region

[0440] The CD19 binding molecule can also include a hinge region, such as a hinge region that connects the antigen-binding domain to the Fc region. The hinge region can be a native or modified hinge region. The hinge region is typically found at the N-terminus of the Fc region.

[0441] A native hinge region is a hinge region that is typically found between the Fab and Fc domains in a naturally occurring antibody. A modified hinge region is any hinge that differs from the native hinge region in length and / or composition. Such hinges can include hinge regions from other species, such as human, mouse, rat, rabbit, shark, pig, hamster, camel, alpaca, or goat hinge regions. Other modified hinge regions can comprise a complete hinge region derived from an antibody of a different type or subclass from the heavy chain Fc region. Alternatively, the modified hinge region can comprise portions of the native hinge or repeating units, where each unit in the repeat is derived from the native hinge region. In a further alternative, the native hinge region can be altered by converting one or more cysteine or other residues to neutral residues, such as serine or alanine, or by converting appropriately placed residues to cysteine residues. In this way, the number of cysteine residues in the hinge region can be increased or decreased. The method is further described in U.S. Patent No. 5,677,425 to Bodmer et al. Altering the number of cysteine residues in the hinge region can, for example, facilitate assembly of the light and heavy chains, or increase or decrease the stability of the CD19 binding molecule. Other modified hinge regions can be entirely synthetic and can be designed to have desired properties such as length, cysteine composition, and flexibility.

[0442] Numerous modified hinge regions are described in the following documents: for example, in U.S. Patent No. 5,677,425, WO9915549, WO 2005003170, WO 2005003169, WO 2005003170, WO 9825971, and WO 2005003171.

[0443] Examples of suitable hinge sequences are shown in Table 7.

[0444]

[0445] In one embodiment, the heavy chain Fc region has a complete hinge region at its N-terminus.

[0446] In one embodiment, the heavy chain Fc region and hinge region are derived from IgG4 and the hinge region comprises the modified sequence CPPC (SEQ ID NO:55). The core hinge region of human IgG4 contains the sequence CPSC (SEQ ID NO:65) as compared to IgG1 containing the sequence CPPC (SEQ ID NO:55). The serine residue present in the IgG4 sequence results in increased flexibility in this region and thus a portion of the molecule forms a disulfide bond within the same protein chain (intra-chain disulfide bond) rather than bridging to other heavy chains in the IgG molecule to form an inter-chain disulfide bond. (Angel et al., 1993, Mol Immunol [Molecular Immunology] 30(1):105-108). Changing the serine residue to a proline to give the same core sequence as IgG1 allows inter-chain disulfide bonds to fully form in the IgG4 hinge region and thus reduces the heterogeneity in the purified product. This altered isotype is designated IgG4P.

[0447] 7.4.3. ABM linker

[0448] In some aspects, the present disclosure provides CD19 binding molecules wherein two or more components of an ABM (e.g., VH and VL of an scFv), two or more ABMs, or an ABM and a non-ABM domain (e.g., a dimerization domain such as an Fc region) are connected to each other by a peptide linker. Such linkers are referred to herein as "ABM linkers" and are distinct from ADC linkers for attaching a drug to a CD19 binding molecule as described, for example, in Section 7.12.2.

[0449] The peptide linker can range from 2 amino acids to 60 or more amino acids, and in some aspects, the peptide linker ranges from 3 amino acids to 50 amino acids, 4 to 30 amino acids, 5 to 25 amino acids, 10 to 25 amino acids, or 12 to 20 amino acids. In specific embodiments, the length of the peptide linker is 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids, 24 amino acids, 25 amino acids, 26 amino acids, 27 amino acids, 28 amino acids, 29 amino acids, 30 amino acids, 31 amino acids, 32 amino acids, 33 amino acids, 34 amino acids, 35 amino acids, 36 amino acids, 37 amino acids, 38 amino acids, 39 amino acids, 40 amino acids, 41 amino acids, 42 amino acids, 43 amino acids, 44 amino acids, 45 amino acids, 46 amino acids, 47 amino acids, 48 amino acids, 49 amino acids, or 50 amino acids.

[0450] Charged and / or flexible linkers can be used.

[0451] Examples of flexible ABM linkers that can be used in CD19-binding molecules include those disclosed in: Chen et al., 2013, Adv Drug Deliv Rev. 65(10):1357-1369 and Klein et al., 2014, Protein Engineering, Design & Selection 27(10):325-330. A particularly useful flexible linker is (GGGGS)n (also known as (G4S)n) (SEQ ID NO:78). In some embodiments, n is any number between 1 and 10, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, or any range with any two of the above numbers as endpoints, e.g., 1 to 5, 2 to 5, 3 to 6, 2 to 4, 1 to 4, etc.

[0452] Other examples of suitable ABM linkers that can be used in the CD19-binding molecules of the present disclosure are shown in Table 8 below:

[0453]

[0454]

[0455]

[0456] In various aspects, the present disclosure provides CD19 binding molecules comprising one or more ABM linkers. Each of the ABM linkers can range in length from 2 amino acids to 60 amino acids, for example, 4 to 30 amino acids, 5 to 25 amino acids, 10 to 25 amino acids, or 12 to 20 amino acids, optionally selected from Table 8 above. In specific embodiments, the CD19 binding molecule comprises two, three, four, five, or six ABM linkers. The ABM linkers can be located on one, two, three, four, or even more polypeptide chains of the CD19 binding molecule.

[0457] 7.5. Bispecific Binding Molecule Configurations

[0458] An exemplary BBM configuration is shown in FIG1 . ​ Shown ​ Components of the BBM configuration shown in . scFv, Fab, scFab, non-immunoglobulin-based ABM, and Fc domains can each have the characteristics described for these components in Sections 7.3 and 7.4. The components of the BBM configuration shown in Figure 1 can be associated with each other by any of the methods described in Sections 7.3 and 7.4 (e.g., by direct bonds, ABM linkers, disulfide bonds, Fc domains modified with knob-to-hole interactions, etc.). The orientations and associations of the various components shown in Figure 1 are merely exemplary; as will be appreciated by the skilled artisan, other orientations and associations may be suitable (e.g., as described in Sections 7.3 and 7.4).

[0459] BBMs are not limited to the configuration shown in FIG1 . Other configurations that can be used are known to those skilled in the art. See, for example, WO 2014 / 145806; WO 2017 / 124002; Liu et al., 2017, Front Immunol. 8:38; Brinkmann & Kontermann, 2017, mAbs 9:2, 182-212; US 2016 / 0355600; Klein et al., 2016, MAbs 8(6):1010-20; and US 2017 / 0145116.

[0460] 7.5.1. Exemplary Bivalent BBMs

[0461] The BBMs can be bivalent, ie, they have two antigen binding domains, one of which binds CD19 (ABM1) and one of which binds a second target antigen (ABM2), e.g., a component of a TCR complex.

[0462] Exemplary bivalent BBM configurations are shown in ​In

[0463] As ​ depicted, the BBM can comprise two half-antibodies, one comprising an ABM and the other comprising an ABM, the two half-antibodies being paired through an Fc domain.

[0464] In ​ embodiments, the first (or left) half-antibody comprises a Fab and an Fc region, and the second (or right) half-antibody comprises a Fab and an Fc region. The first and second half-antibodies associate through the Fc regions that form the Fc domain.

[0465] In ​ embodiments, the first (or left) half-antibody comprises a Fab and an Fc region, and the second (or right) half-antibody comprises a scFv and an Fc region. The first and second half-antibodies associate through the Fc regions that form the Fc domain.

[0466] In ​ embodiments, the first (or left) half-antibody comprises a scFv and an Fc region, and the second (or right) half-antibody comprises a scFv and an Fc region. The first and second half-antibodies associate through the Fc regions that form the Fc domain.

[0467] As ​ described, the bivalent BBM can comprise two ABMs attached to one Fc region of an Fc domain.

[0468] In ​ embodiments, the BBM comprises a Fab, a scFv, and an Fc domain, wherein the scFv is located between the Fab and the Fc domain.

[0469] In ​ embodiments, the BBM (a "one-arm scFv-mAb" configuration) comprises a Fab, a scFv, and an Fc domain, wherein the Fab is located between the scFv and the Fc domain.

[0470] In ​ the depicted configurations, each of X and Y represents either ABM1 or ABM2, provided that the BBM comprises one ABM1 and one ABM2. Thus, the present disclosure provides a bivalent BBM as shown in any of ​ wherein X is ABM1 and Y is ABM2 (for convenience, this configuration of ABMs is designated as "B1"). The present disclosure also provides a bivalent BBM as shown in any of ​ wherein X is ABM2 and Y is ABM1 (for convenience, this configuration of ABMs is designated as "B2").

[0471] 7.5.2. Exemplary Trivalent BBM

[0472] The BBMs can be trivalent, i.e., they have three antigen-binding domains, one or two of which bind CD19 (ABM1) and one or two of which bind a second target antigen (ABM2), e.g., a component of the TCR complex.

[0473] Exemplary trivalent BBM configurations are shown in ​ .

[0474] As ​ , 1Q-1W, 1Y-1Z depict, the BBM can comprise two half-antibodies, one of which comprises two ABMs and the other of which comprises one ABM, the two half-antibodies being paired via an Fc domain.

[0475] In ​ 's embodiment, the first (or left) half-antibody comprises a Fab and an Fc region, and the second (or right) half-antibody comprises a scFv, a Fab and an Fc region. The first and second half-antibodies associate via the Fc region that forms the Fc domain.

[0476] In ​ 's embodiment, the first (or left) half-antibody comprises a Fab and an Fc region, and the second (or right) half-antibody comprises a Fab, a scFv and an Fc region. The first and second half-antibodies associate via the Fc region that forms the Fc domain.

[0477] In ​ 's embodiment, the first (or left) half-antibody comprises a scFv and an Fc region, and the second (or right) half-antibody comprises two Fabs and an Fc region. The first and second half-antibodies associate via the Fc region that forms the Fc domain.

[0478] In ​ 's embodiment, the first (or left) half-antibody comprises two Fabs and an Fc region, and the second (or right) half-antibody comprises a Fab and an Fc region. The first and second half-antibodies associate via the Fc region that forms the Fc domain.

[0479] In ​ 's embodiment, the first (or left) half-antibody comprises a scFv and an Fc region, and the second (or right) half-antibody comprises two scFvs and an Fc region. The first and second half-antibodies associate via the Fc region that forms the Fc domain.

[0480] In ​In embodiments, the first (or left) half - antibody comprises an scFv and an Fc region, and the second (or right) half - antibody comprises an scFv, a Fab, and an Fc region. The first and second half - antibodies associate through the Fc region that forms the Fc domain.

[0481] In ​ In embodiments, the first (or left) half - antibody comprises an scFv and an Fc region, and the second (or right) half - antibody comprises a Fab, an scFv, and an Fc region. The first and second half - antibodies associate through the Fc region that forms the Fc domain.

[0482] In ​ In embodiments, the first (or left) half - antibody comprises a diabody - type binding domain and an Fc region, and the second (or right) half - antibody comprises a Fab and an Fc region. The first and second half - antibodies associate through the Fc region that forms the Fc domain.

[0483] In ​ In embodiments, the first (or left) half - antibody comprises a Fab and an Fc region, and the second (or right) half - antibody comprises a Fab, an Fc region, and an scFv. The first and second half - antibodies associate through the Fc region that forms the Fc domain.

[0484] In ​ In embodiments, the first (or left) half - antibody comprises an scFv and an Fc region, and the second (or right) half - antibody comprises a Fab, an Fc region, and an scFv. The first and second half - antibodies associate through the Fc region that forms the Fc domain.

[0485] In ​ In embodiments, the first (or left) half - antibody comprises an scFv and an Fc region, and the second (or right) half - antibody comprises an scFv, an Fc region, and a second scFv. The first and second half - antibodies associate through the Fc region that forms the Fc domain.

[0486] In ​ In embodiments, the first (or left) half - antibody comprises an scFv, an Fc region, and a Fab, and the second (or right) half - antibody comprises a Fab and an Fc region. The first and second half - antibodies associate through the Fc region that forms the Fc domain.

[0487] In ​ In embodiments, the first (or left) half - antibody comprises two Fabs and an Fc region, and the second (or right) half - antibody comprises a non - immunoglobulin - based ABM and an Fc region. The first and second half - antibodies associate through the Fc region that forms the Fc domain.

[0488] In ​In an embodiment, the first (or left) half - antibody comprises a Fab, scFv, and Fc region, and the second (or right) half - antibody comprises a non - immunoglobulin - based ABM and an Fc region. The first and second half - antibodies associate through the Fc region that forms the Fc domain.

[0489] In ​ an embodiment, the first (or left) half - antibody comprises a Fab and an Fc region, and the second (or right) half - antibody comprises an scFv, a non - immunoglobulin - based ABM, and an Fc region. The first and second half - antibodies associate through the Fc region that forms the Fc domain.

[0490] In ​ an embodiment, the first (or left) half - antibody comprises an scFv and an Fc region, and the second (or right) half - antibody comprises a Fab, an scFv, and an Fc region. The first and second half - antibodies associate through the Fc region that forms the Fc domain.

[0491] In ​ an embodiment, the first (or left) half - antibody comprises a Fab, an Fc region, and an scFab, and the second (or right) half - antibody comprises a Fab and an Fc region. The first and second half - antibodies associate through the Fc region that forms the Fc domain.

[0492] Alternatively, as ​ and 1P described, the trivalent BBM can comprise two half - antibodies, where each comprises one complete ABM ( ​ and 1P the Fab in

[0493] ​ ​ shown. ​ The BBM of

[0494] In ​ the configuration shown, each of X, Y, and A represents ABM1 or ABM2, provided that the BBM comprises at least one ABM1 and at least one ABM2. Thus, the trivalent MBM will include one or two ABM1s and one or two ABM2s. In some embodiments, the trivalent BBM comprises two ABM1s and one ABMxplanation or note.

[0495] Thus, in the present disclosure, there is provided as ​A trivalent BBM as shown in any of them, where X is ABM1, Y is ABM1 and A is ABM2 (for convenience, this configuration of ABM is designated as "T1").

[0496] This disclosure further provides a trivalent BBM as shown in any of ​ them, where X is ABM1, Y is ABM2 and A is ABM1 (for convenience, this configuration of ABM is designated as "T2").

[0497] This disclosure further provides a trivalent BBM as shown in any of ​ them, where X is ABM2, Y is ABM1 and A is ABM1 (for convenience, this configuration of ABM is designated as "T3").

[0498] This disclosure further provides a trivalent BBM as shown in any of ​ them, where X is ABM1, Y is ABM2 and A is ABM2 (for convenience, this configuration of ABM is designated as "T4").

[0499] This disclosure further provides a trivalent BBM as shown in any of ​ them, where X is ABM2, Y is ABM1 and A is ABM2 (for convenience, this configuration of ABM is designated as "T5").

[0500] This disclosure further provides a trivalent BBM as shown in any of ​ them, where X is ABM2, Y is ABM2 and A is ABM1 (for convenience, this configuration of ABM is designated as "T6").

[0501] 7.5.3. Exemplary tetravalent BBM

[0502] The BBMs can be tetravalent, i.e., they have four antigen-binding domains, one, two or three of which bind CD19 (ABM1) and one, two or three of which bind a second target antigen (ABM2), e.g., a component of the TCR complex.

[0503] Exemplary tetravalent BBM configurations are shown in ​ them. [

[0504] As ​ depicted, the tetravalent BBM can comprise two half-antibodies, each of which contains two complete ABMs, and the two half-antibodies are paired by an Fc domain.

[0505] In ​In embodiments, the first (or left) half - antibody comprises a Fab, an Fc region, and a scFv, and the second (or right) half - antibody comprises a Fab, an Fc region, and a scFv. The first and second half - antibodies associate through the Fc region that forms the Fc domain.

[0506] In ​ In embodiments, the first (or left) half - antibody comprises a Fab, a scFv, and an Fc region, and the second (or right) half - antibody comprises a Fab, a scFv, and an Fc region. The first and second half - antibodies associate through the Fc region that forms the Fc domain.

[0507] In ​ In embodiments, the first (or left) half - antibody comprises a scFv, a Fab, and an Fc region, and the second (or right) half - antibody comprises a scFv, a Fab, and an Fc region. The first and second half - antibodies associate through the Fc region that forms the Fc domain.

[0508] In ​ In embodiments, the first (or left) half - antibody comprises a Fab, an Fc region, and a second Fab, and the second (or right) half - antibody comprises a Fab, an Fc region, and a second Fab. The first and second half - antibodies associate through the Fc region that forms the Fc domain.

[0509] In ​ In embodiments, the first (or left) half - antibody comprises a scFv, a second scFv, and an Fc region, and the second (or right) half - antibody comprises a scFv, a second scFv, and an Fc region. The first and second half - antibodies associate through the Fc region that forms the Fc domain.

[0510] In ​ In embodiments, the first (or left) half - antibody comprises a Fab, a scFv, and an Fc region, and the second (or right) half - antibody comprises a Fab, a scFv, and an Fc region. The first and second half - antibodies associate through the Fc region that forms the Fc domain.

[0511] In ​ In embodiments, the first (or left) half - antibody comprises a Fab, an Fc region, and a scFv, and the second (or right) half - antibody comprises a scFv, an Fc region, and a Fab. The first and second half - antibodies associate through the Fc region that forms the Fc domain.

[0512] In ​ In embodiments, the first (or left) half - antibody comprises a scFv, an Fc region, and a Fab, and the second (or right) half - antibody comprises a scFv, an Fc region, and a Fab. The first and second half - antibodies associate through the Fc region that forms the Fc domain.

[0513] In ​ the configuration shown, each of X, Y, A, and B represents either ABM1 or ABM2 (although not necessarily in this order), provided that the BBM comprises at least one ABM1 and at least one ABM2. Thus, the tetravalent ABM will include one, two, or three ABM1s and one, two, or three ABM2s. In some embodiments, the tetravalent BBM comprises three ABM1s and one ABM2. In other embodiments, the tetravalent BBM comprises two ABM1s and two ABM2s. In yet other embodiments, the tetravalent BBM comprises one ABM1 and three ABM2s.

[0514] Accordingly, provided herein is a tetravalent BBM as shown in any one of ​ wherein X is ABM1 and each of Y, A, and B is ABM2 (for convenience, this configuration of ABM is designated "Tv 1").

[0515] The present disclosure further provides a tetravalent BBM as shown in any one of ​ wherein Y is ABM1 and each of X, A, and B is ABM2 (for convenience, this configuration of ABM is designated "Tv 2").

[0516] The present disclosure further provides a tetravalent BBM as shown in any one of ​ wherein A is ABM1 and each of X, Y, and B is ABM2 (for convenience, this configuration of ABM is designated "Tv 3").

[0517] The present disclosure further provides a tetravalent BBM as shown in any one of ​ wherein B is ABM1 and each of X, Y, and A is ABM2 (for convenience, this configuration of ABM is designated "Tv 4").

[0518] The present disclosure further provides a tetravalent BBM as shown in any one of ​ wherein both X and Y are ABM1 and both A and B are ABM2 (for convenience, this configuration of ABM is designated "Tv 5").

[0519] The present disclosure further provides a tetravalent BBM as shown in any one of ​ wherein both X and A are ABM1 and both Y and B are ABM2 (for convenience, this configuration of ABM is designated "Tv 6").

[0520] The present disclosure further provides a tetravalent BBM as shown in any one of ​a tetravalent BBM as shown in any one of

[0521] This disclosure further provides a tetravalent BBM as shown in any one of ​ wherein Y and A are each ABM1 and X and B are each ABM2 (for convenience, this configuration of ABM is designated "Tv 8").

[0522] This disclosure further provides a tetravalent BBM as shown in any one of ​ wherein Y and B are each ABM1 and X and A are each ABM2 (for convenience, this configuration of ABM is designated "Tv 9").

[0523] This disclosure further provides a tetravalent BBM as shown in any one of ​ wherein A and B are each ABM1 and X and Y are each ABM2 (for convenience, this configuration of ABM is designated "Tv 10").

[0524] This disclosure further provides a tetravalent BBM as shown in any one of ​ wherein each of X, Y, and A is ABM1 and B is ABM2 (for convenience, this configuration of ABM is designated "Tv 11").

[0525] This disclosure further provides a tetravalent BBM as shown in any one of ​ wherein each of X, Y, and B is ABM1 and A is ABM2 (for convenience, this configuration of ABM is designated "Tv 12").

[0526] This disclosure further provides a tetravalent BBM as shown in any one of ​ wherein each of X, A, and B is ABM1 and Y is ABM2 (for convenience, this configuration of ABM is designated "Tv 13").

[0527] This disclosure further provides a tetravalent BBM as shown in any one of ​ wherein each of Y, A, and B is ABM1 and X is ABM2 (for convenience, this configuration of ABM is designated "Tv 14").

[0528] 7.6. Trispecific binding molecule configurations

[0529] Exemplary TBM configurations are shown in Figure 2. ​ Shows ​Components of the TBM configuration shown in FIG. . scFv, Fab, non-immunoglobulin-based ABM, and Fc can each have the characteristics described for these components in Sections 7.3 and 7.4. The components of the TBM configuration shown in FIG. 2 can be associated with each other by any of the methods described in Sections 7.3 and 7.4 (e.g., by direct bonds, ABM linkers, disulfide bonds, Fc domains modified with knob-to-hole interactions, etc.). The orientations and associations of the various components shown in FIG. 2 are merely exemplary; as will be appreciated by those skilled in the art, other orientations and associations may be suitable (e.g., as described in Sections 7.3 and 7.4).

[0530] TBM is not limited to the configuration shown in Figure 2. Other configurations that can be used are known to those skilled in the art. See, for example, WO 2014 / 145806; WO 2017 / 124002; Liu et al., 2017, Front Immunol. [Immunology Frontier] 8:38; Brinkmann & Kontermann, 2017, mAbs 9:2, 182-212; US 2016 / 0355600; Klein et al., 2016, MAbs 8(6):1010-20; and US 2017 / 0145116.

[0531] 7.6.1. Exemplary Trivalent TBMs

[0532] The TBMs of the present disclosure can be trivalent, ie, they have three antigen binding domains, one of which binds CD19, one of which binds a component of the TCR complex, and one of which binds CD2 or TAA.

[0533] Exemplary trivalent TBM configurations are shown in ​ middle.

[0534] like ​ As depicted in and 2N-2P, a TBM can comprise two half antibodies, one comprising two ABMs and the other comprising one ABM, the two half antibodies being paired via an Fc domain.

[0535] exist ​ In an embodiment of the present invention, the first (or left) half antibody comprises an scFv and an Fc region, and the second (or right) half antibody comprises an Fab, an scFv, and an Fc region. The first and second half antibodies associate via the Fc region forming an Fc domain.

[0536] exist ​ In an embodiment of the present invention, the first (or left) half antibody comprises two Fab and Fc regions, and the second (or right) half antibody comprises a Fab and an Fc region. The first and second half antibodies associate through the Fc regions forming an Fc domain.

[0537] In ​ the embodiment, the first (or left) half-antibody comprises a Fab, a scFv and an Fc region, and the second (or right) half-antibody comprises a Fab and an Fc region. The first and second half-antibodies associate through the Fc region forming the Fc domain.

[0538] In ​ the embodiment, the first (or left) half-antibody comprises a scFv and an Fc region, and the second (or right) half-antibody comprises two Fabs and an Fc region. The first and second half-antibodies associate through the Fc region forming the Fc domain.

[0539] In ​ the embodiment, the first (or left) half-antibody comprises a scFv, an Fc region, and a Fab, and the second (or right) half-antibody comprises a Fab and an Fc region. The first and second half-antibodies associate through the Fc region forming the Fc domain.

[0540] In ​ the embodiment, the first (or left) half-antibody comprises a scFv and an Fc region, and the second (or right) half-antibody comprises a Fab, an Fc region, and a scFV. The first and second half-antibodies associate through the Fc region forming the Fc domain.

[0541] In ​ the embodiment, the first (or left) half-antibody comprises two Fabs and an Fc region, and the second (or right) half-antibody comprises a non-immunoglobulin-based ABM and an Fc region. The first and second half-antibodies associate through the Fc region forming the Fc domain.

[0542] In ​ the embodiment, the first (or left) half-antibody comprises a Fab, a scFv, and an Fc region, and the second (or right) half-antibody comprises a non-immunoglobulin-based ABM and an Fc region. The first and second half-antibodies associate through the Fc region forming the Fc domain.

[0543] In ​ the embodiment, the first (or left) half-antibody comprises a Fab and an Fc region, and the second (or right) half-antibody comprises a scFv, a non-immunoglobulin-based ABM and an Fc region. The first and second half-antibodies associate through the Fc region forming the Fc domain.

[0544] In ​ the embodiment, the first (or left) half-antibody comprises a scFv and an Fc region, and the second (or right) half-antibody comprises a scFv, an Fc region, and a second scFv. The first and second half-antibodies associate through the Fc region forming the Fc domain.

[0545] In ​ the embodiment of, the first (or left) half - antibody comprises a Fab, an Fc region, and a scFv, and the second (or right) half - antibody comprises a Fab and an Fc region. The first and second half - antibodies associate through the Fc region that forms the Fc domain.

[0546] In ​ the embodiment of, the first (or left) half - antibody comprises a Fab, an Fc region, and a scFab, and the second (or right) half - antibody comprises a Fab and an Fc region. The first and second half - antibodies associate through the Fc region that forms the Fc domain.

[0547] In ​ the embodiment of, the first (or left) half - antibody comprises a Fab, a non - immunoglobulin - based ABM, and an Fc region, and the second (or right) half - antibody comprises a scFv and an Fc region. The first and second half - antibodies associate through the Fc region that forms the Fc domain.

[0548] Alternatively, as ​ described, the trivalent TBM can comprise two half - antibodies, where each contains one complete ABM and a part of another ABM (one is VH and the other is VL). The two half - antibodies pair through the Fc domain, so that VH and VL associate to form a complete antigen - binding Fv domain.

[0549] The TBM can be single - chain, as ​ shown. ​ The TBM of contains three scFv domains connected by a linker.

[0550] In ​ each configuration shown, each domain designated as X, Y, and Z represents ABM1, ABM2, or ABM3, although not necessarily in that order. In other words, X can be ABM1, ABM2, or ABM3, Y can be ABM1, ABM2, or ABM3, and Z can be ABM1, ABM2, or ABM3, as long as the TBM contains one ABM1, one ABM2, and one ABM3.

[0551] Thus, provided in the present disclosure is a trivalent TBM as shown in any of ​ wherein X is ABM1, Y is ABM3 and Z is ABM2 (for convenience, this configuration of ABM is designated as "T1").

[0552] The present disclosure also provides a trivalent TBM as shown in ​A trivalent TBM as shown in any of them, where X is ABM1, Y is ABM2 and Z is ABM3 (for convenience, this configuration of ABM is designated as "T2").

[0553] This disclosure further provides a trivalent TBM as shown in any of ​ them, where X is ABM3, Y is ABM1 and Z is ABM2 (for convenience, this configuration of ABM is designated as "T3").

[0554] This disclosure further provides a trivalent TBM as shown in any of ​ them, where X is ABM3, Y is ABM2 and Z is ABM1 (for convenience, this configuration of ABM is designated as "T4").

[0555] This disclosure further provides a trivalent TBM as shown in any of ​ them, where X is ABM2, Y is ABM1 and Z is ABM3 (for convenience, this configuration of ABM is designated as "T5").

[0556] This disclosure further provides a trivalent TBM as shown in any of ​ them, where X is ABM2, Y is ABM3 and Z is ABM1 (for convenience, this configuration of ABM is designated as "T6").

[0557] 7.6.2. Exemplary tetravalent TBM

[0558] The TBMs of this disclosure can be tetravalent, i.e., they have four antigen-binding domains, one or two of which bind CD19, one or two of which bind components of the TCR complex, and one or two of which bind CD2 or TAA.

[0559] Exemplary tetravalent TBM configurations are shown in ​ them.

[0560] As ​ depicted, the tetravalent TBM can comprise two half-antibodies, each of which contains two complete ABMs, and the two half-antibodies are paired via an Fc domain.

[0561] In ​ the embodiment of, the first (or left) half-antibody comprises a Fab, an Fc region, and a second Fab, and the second (or right) half-antibody comprises a Fab, an Fc region, and a second Fab. The first and second half-antibodies associate via the Fc region that forms the Fc domain.

[0562] In ​In embodiments, the first (or left) half-antibody comprises a Fab, an Fc region, and a scFv, and the second (or right) half-antibody comprises a Fab, an Fc region, and a scFv. The first and second half-antibodies associate through the Fc region that forms the Fc domain.

[0563] In ​ embodiments, the first (or left) half-antibody comprises a Fab, an Fc region, and a scFv, and the second (or right) half-antibody comprises a scFv, an Fc region, and a Fab. The first and second half-antibodies associate through the Fc region that forms the Fc domain.

[0564] In ​ the depicted configuration, each of X, Y, Z, and A represents ABM1, ABM2, or ABM3 (although not necessarily in this order), provided that the TBM comprises at least one ABM1, at least one ABM2, and at least one ABM3. Thus, the tetravalent ABM will include two ABMs against one of CD19, a component of the TCR complex, and CD2 or a TAA. In some cases, the tetravalent TBM has two CD19 ABMs.

[0565] Thus, the present disclosure provides a tetravalent TBM as shown in any of ​ wherein X, Y, Z, and A are ABMs against CD19, a component of the TCR complex, and CD2 or a TAA, as shown in Table 9.

[0566]

[0567] 7.6.3. Exemplary pentavalent TBM

[0568] The TBMs of the present disclosure can be pentavalent, i.e., they have five antigen-binding domains, where one, two, or three bind CD19, one, two, or three bind a component of the TCR complex, and one, two, or three bind CD2 or a TAA.

[0569] Exemplary pentavalent TBM configurations are shown in ​ .

[0570] As ​ depicted, the pentavalent TBM can comprise two half-antibodies, where one comprises two complete ABMs and the other comprises one complete ABM, and the two half-antibodies are paired through the Fc domain.

[0571] In ​In embodiments, the first (or left) half - antibody comprises a Fab, a scFv, and an Fc region, and the second (or right) half - antibody comprises a Fab, an Fc region, and a scFv. The first and second half - antibodies associate through the Fc region that forms the Fc domain.

[0572] In ​ the depicted configuration, each of X, Y, Z, A, and B represents ABM1, ABM2, or ABM3 (although not necessarily in this order), provided that the TBM comprises at least one ABM1, one ABM2, and one ABM3. Thus, the pentavalent TBM can comprise two ABMs against two of CD19, a component of the TCR complex, and CD2 or a TAA, or three ABMs against one of CD19, a component of the TCR complex, and CD2 or a TAA. In some cases, the pentavalent TBM has two or three CD19 ABMs. In some embodiments, the pentavalent TBM has three ABM1s, one ABM2, and one ABM3.

[0573] Thus, provided in this disclosure is a pentavalent TBM as ​ depicted, where X, Y, Z, A, and B are ABMs against CD19, a component of the TCR complex, and CD2 or a TAA, as shown in Table 10.

[0574]

[0575]

[0576]

[0577]

[0578] 7.6.4. Exemplary Hexavalent TBM

[0579] The TBMs of this disclosure can be hexavalent, i.e., they have six antigen - binding domains, where one, two, three, or four bind CD19, where one, two, three, or four bind a component of the TCR complex, and where one, two, three, or four bind CD2 or a TAA.

[0580] Exemplary hexavalent TBM configurations are shown in ​ .

[0581] As ​ depicted, the pentavalent TBM can comprise two half - antibodies, one of which comprises two complete ABMs and the other of which comprises one complete ABM, and the two half - antibodies are paired through the Fc domain.

[0582] In ​In embodiments, the first (or left) half - antibody comprises a Fab, a second Fab, an Fc region, and a scFv, and the second (or right) half - antibody comprises a Fab, a second Fab, an Fc region, and a scFv. The first and second half - antibodies associate through the Fc region that forms the Fc domain.

[0583] In ​ embodiments, the first (or left) half - antibody comprises a first Fv, a second Fv, a third Fv, and an Fc region, and the second (or right) half - antibody comprises a first Fv, a second Fv, a third Fv, and an Fc region. The first and second half - antibodies associate through the Fc region that forms the Fc domain.

[0584] In ​ the configurations shown, each of X, Y, Z, A, B, and C represents an ABM1, ABM2, or ABM3 (although not necessarily in this order), provided that the TBM comprises at least one ABM1, one ABM2, and one ABM3. Thus, the hexavalent TBM can include (i) two ABMs each against CD19, a component of the TCR complex, and CD2 or a TAA, (ii) three ABMs against one of CD19, a component of the TCR complex, and CD2 or a TAA, or (iii) four ABMs against one of CD19, a component of the TCR complex, and CD2 or a TAA. For example, a hexavalent ABM can include three ABMs against CD19, two ABMs against CD2 or a TAA, and one ABM against a component of the TCR complex. As another example, a hexavalent ABM can include three ABMs against CD19, two ABMs against a component of the TCR complex, and one ABM against CD2 or a TAA. In some cases, the hexavalent TBM has two, three, or four CD19 ABMs. In some embodiments, the hexavalent TBM has three CD19 ABMs. In other embodiments, the hexavalent TBM has four CD19 ABMs.

[0585] Thus, provided in this disclosure is a hexavalent TBM as shown in any of ​ wherein X, Y, Z, A, B, and C are ABMs against CD19, a component of the TCR complex, and CD2 or a TAA, as shown in Table 11.

[0586]

[0587]

[0588]

[0589]

[0590]

[0591]

[0592]

[0593]

[0594]

[0595]

[0596]

[0597]

[0598] 7.7.TCR ABM

[0599] The MBM of the present disclosure comprises an ABM that specifically binds to CD19 and an ABM2 that is specific for a different antigen. In the BBM, type 1 TBM, and type 2 TBM of the present disclosure, the ABM2 can bind to components of the TCR complex. The TCR is a disulfide-linked membrane-anchored heterodimeric protein that typically consists of highly variable α and β chains that are expressed as part of a complex with invariant CD3 chain molecules. T cells that express this receptor are referred to as α:β (or αβ) T cells, although a minority of T cells (referred to as γδ T cells) express an alternative receptor (formed by variable γ and δ chains).

[0600] In an embodiment, the MBM comprises an ABM that specifically binds to CD3.

[0601] 7.7.1.CD3 ABM

[0602] The MBM can comprise an ABM that specifically binds to CD3. The term "CD3" refers to the cluster of differentiation 3 co-receptor of the T cell receptor (or co-receptor complex, or polypeptide chains of the co-receptor complex). The amino acid sequences of the polypeptide chains of human CD3 are provided in NCBI Accessions P04234, P07766, and P09693. The CD3 protein can also include variants. The CD3 protein can also include fragments. The CD3 protein also includes post-translational modifications of the CD3 amino acid sequence. Post-translational modifications include, but are not limited to, N-linked and O-linked glycosylation.

[0603] In some embodiments, the MBM can comprise an ABM, which is an anti-CD3 antibody (e.g., as described in US 2016 / 0355600, WO 2014 / 110601, and WO 2014 / 145806) or an antigen-binding domain thereof. Exemplary anti-CD3 VH, VL, and scFV sequences that can be used in the MBM are provided in Table 12A.

[0604]

[0605]

[0606]

[0607]

[0608]

[0609]

[0610]

[0611]

[0612] The CDR sequences of many CD3 binders defined by the Kabat numbering scheme (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD), the Chothia numbering scheme (Al-Lazikani et al., 1997, J. Mol. Biol. 273:927-948), and combinations of Kabat and Chothia numbering are provided in Tables 12B - 12D, respectively.

[0613]

[0614]

[0615]

[0616]

[0617]

[0618]

[0619]

[0620]

[0621]

[0622]

[0623]

[0624]

[0625]

[0626]

[0627]

[0628]

[0629]

[0630]

[0631]

[0632]

[0633]

[0634]

[0635]

[0636] In some embodiments, the MBM can comprise a CD3 ABM, the CD3 ABM comprising a CDR of any one of CD3-1 to CD3-130 as defined by Kabat numbering (e.g., as listed in Table 12B). In other embodiments, the MBM can comprise a CD3 ABM, the CD3 ABM comprising a CDR of any one of CD3-1 to CD3-130 as defined by Chothia numbering (e.g., as listed in Table 12C). In still other embodiments, the MBM can comprise a CD3 ABM, the CD3 ABM comprising a CDR of any one of CD3-1 to CD3-130 as defined by a combination of Kabat and Chothia numberings (e.g., as listed in Table 12D).

[0637] In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-1. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-2. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-3. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-4. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-5. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-6. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-7. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-8. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-9. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-10. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-11. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-12. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-13. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-14. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-15. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-16. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-17. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-18. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-19. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-20. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-21. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-22. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-23. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-24. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-25. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-26. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-27. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-28. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-29. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-30. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-31. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-32. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-33.In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-34. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-35. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-36. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-37. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-38. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-39. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-40. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-41. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-42. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-43. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-44. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-45. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-46. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-47. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-48. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-49. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-50. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-51. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-52. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-53. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-54. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-55. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-56. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-57. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-58. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-59. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-60. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-61. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-62. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-63. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-64. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-65. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-66.In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-67. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-68. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-69. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-70. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-71. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-72. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-73. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-74. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-75. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-76. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-77. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-78. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-79. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-80. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-81. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-82. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-83. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-84. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-85. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-86. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-87. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-88. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-89. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-90. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-91. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-92. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-93. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-94. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-95. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-96. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-97. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-98. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-99.In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-100. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-101. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-102. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-103. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-104. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-105. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-106. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-107. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-108. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-109. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-110. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-111. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-112. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-113. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-114. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-115. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-116. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-117. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-118. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-119. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-120. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-121. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-122. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-123. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-124. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-125. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-126. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-127. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-126. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-127. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-128. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-129.In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-130.

[0638] The MBM can include the complete heavy and light variable sequences of any one of CD3-1 to CD3-130. In some embodiments, the MBM includes a CD3 ABM that includes the VH and VL sequences of CD3-1. In some embodiments, the MBM includes a CD3 ABM that includes the VH and VL sequences of CD3-1. In some embodiments, the MBM includes a CD3 ABM that includes the VH and VL sequences of CD3-2. In some embodiments, the MBM includes a CD3 ABM that includes the VH and VL sequences of CD3-3. In some embodiments, the MBM includes a CD3 ABM that includes the VH and VL sequences of CD3-4. In some embodiments, the MBM includes a CD3 ABM that includes the VH and VL sequences of CD3-5. In some embodiments, the MBM includes a CD3 ABM that includes the VH and VL sequences of CD3-6. In some embodiments, the MBM includes a CD3 ABM that includes the VH and VL sequences of CD3-7. In some embodiments, the MBM includes a CD3 ABM that includes the VH and VL sequences of CD3-8. In some embodiments, the MBM includes a CD3 ABM that includes the VH and VL sequences of CD3-9. In some embodiments, the MBM includes a CD3 ABM that includes the VH and VL sequences of CD3-10. In some embodiments, the MBM includes a CD3 ABM that includes the VH and VL sequences of CD3-11. In some embodiments, the MBM includes a CD3 ABM that includes the VH and VL sequences of CD3-12. In some embodiments, the MBM includes a CD3 ABM that includes the VH and VL sequences of CD3-13. In some embodiments, the MBM includes a CD3 ABM that includes the VH and VL sequences of CD3-14. In some embodiments, the MBM includes a CD3 ABM that includes the VH and VL sequences of CD3-15. In some embodiments, the MBM includes a CD3 ABM that includes the VH and VL sequences of CD3-16. In some embodiments, the MBM includes a CD3 ABM that includes the VH and VL sequences of CD3-17. In some embodiments, the MBM includes a CD3 ABM that includes the VH and VL sequences of CD3-18. In some embodiments, the MBM includes a CD3 ABM that includes the VH and VL sequences of CD3-19. In some embodiments, the MBM includes a CD3 ABM that includes the VH and VL sequences of CD3-20. In some embodiments, the MBM includes a CD3 ABM that includes the VH and VL sequences of CD3-21. In some embodiments, the MBM includes a CD3 ABM that includes the VH and VL sequences of CD3-22. In some embodiments, the MBM includes a CD3 ABM that includes the VH and VL sequences of CD3-23. In some embodiments, the MBM includes a CD3 ABM that includes the VH and VL sequences of CD3-24.In some embodiments, the MBM comprises a CD3 ABM that comprises the VH and VL sequences of CD3-25. In some embodiments, the MBM comprises a CD3 ABM that comprises the VH and VL sequences of CD3-26. In some embodiments, the MBM comprises a CD3 ABM that comprises the VH and VL sequences of CD3-27. In some embodiments, the MBM comprises a CD3 ABM that comprises the VH and VL sequences of CD3-28. In some embodiments, the MBM comprises a CD3 ABM that comprises the VH and VL sequences of CD3-129. In some embodiments, the MBM comprises a CD3 ABM that comprises the VH and VL sequences of CD3-130.

[0639] In addition to the CDR sets described in Tables 12B-12D (i.e., the sets of six CDRs for each of CD3-1 to CD3-130), the present disclosure provides variant CDR sets. In one embodiment, a set of 6 CDRs can have 1, 2, 3, 4, or 5 amino acid changes from the CDR sets described in Tables 12B-12D, so long as the CD3 ABM is still able to bind to the target antigen, as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biomolecular layer interferometry, e.g., Octet assay).

[0640] In addition to the variable heavy and variable light domains disclosed in Table 12A (which form the ABM against CD3), the present disclosure provides variant VH and VL domains. In one embodiment, each of the variant VH and VL domains can have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes from the VH and VL domains listed in Table 12A, so long as the ABM is still able to bind to the target antigen, as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biomolecular layer interferometry, e.g., Octet assay). In another embodiment, the variant VH and VL are at least 90%, 95%, 97%, 98%, or 99% identical to each VH or VL disclosed in Table 12A, so long as the ABM is still able to bind to the target antigen, as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biomolecular layer interferometry, e.g., Octet assay).

[0641] In some embodiments, the MBM can comprise an ABM that is a CD3-binding molecule or an antigen-binding domain thereof as described in WO 2020 / 052692. Tables AA through AJ-2 (collectively "Table A") list the sequences of CD3-binding sequences that can be included in the CD3-binding ABM.

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[0744] The Group C1 CDR sequences in Table AA are based on the Kabat CDR sequences, Chothia CDR sequences, IMGT CDR sequences, and combinations thereof of the CD3-binding molecules NOV292, NOV589, NOV567, and CD3-binding molecules including "sp11a" in the conjugate name. The Group C2 CDR sequences in Table AB are based on the Kabat CDR sequences, Chothia CDR sequences, IMGT CDR sequences, and combinations thereof of the CD3-binding molecules NOV453, NOV229, NOV580, NOV221, and CD3-binding molecules including "sp9a" in the conjugate name. The Group C3 CDR sequences in Table AC are based on the Kabat CDR sequences, Chothia CDR sequences, IMGT CDR sequences, and combinations thereof of the CD3-binding molecules NOV123, sp10b, NOV110, and NOV832.

[0745] The specific CDR sequences of the CD3-binding molecules described in the examples of WO 2020 / 052692 are listed in Tables AB-1 to AH-2. The VH and VL sequences described in WO 2020 / 052692 are listed in Tables AJ-1 and AJ-2, respectively.

[0746] In some embodiments, the CD3 ABM can comprise a heavy chain CDR having an amino acid sequence of any of the CDR consensus sequences listed in Table AA, Table AB, or Table AC. In a specific embodiment, the CD3 ABM can comprise (or alternatively, consist of) one, two, three, or more heavy chain CDRs selected from the heavy chain CDRs described in Table AA, Table AB, or Table AC.

[0747] In some embodiments, the CD3 ABM can comprise a light chain CDR having an amino acid sequence of any of the CDR consensus sequences listed in Table AA, Table AB, or Table AC. In a specific embodiment, the CD3 ABM can comprise (or alternatively, consist of) one, two, three, or more light chain CDRs selected from the light chain CDRs described in Table AA, Table AB, or Table AC.

[0748] In some embodiments, the CD3 ABM can comprise the CDR-H1 sequence, CDR-H2 sequence, CDR-H3 sequence, CDR-L1 sequence, CDR-L2 sequence, and CDR-L3 sequence listed in Table AA.

[0749] In some embodiments, the amino acid designated as X1 in Table AA is T. In some embodiments, the amino acid designated as X1 in Table AA is A. In some embodiments, the amino acid designated as X2 in Table AA is S. In some embodiments, the amino acid designated as X2 in Table AA is R. In some embodiments, the amino acid designated as X3 in Table AA is N. In some embodiments, the amino acid designated as X3 in Table AA is Y. In some embodiments, the amino acid designated as X3 in Table AA is Q. In some embodiments, the amino acid designated as X4 in Table AA is H. In some embodiments, the amino acid designated as X4 in Table AA is S. In some embodiments, the amino acid designated as X5 in Table AA is M. In some embodiments, the amino acid designated as X5 in Table AA is L. In some embodiments, the amino acid designated as X6 in Table AA is K. In some embodiments, the amino acid designated as X6 in Table AA is R. In some embodiments, the amino acid designated as X7 in Table AA is S. In some embodiments, the amino acid designated as X7 in Table AA is K. In some embodiments, the amino acid designated as X 55 in Table AA is F. In some embodiments, the amino acid designated as X 55 in Table AA is Y. In some embodiments, the amino acid designated as X 55 in Table AA is S. In some embodiments, the amino acid designated as X8 in Table AA is W. In some embodiments, the amino acid designated as X8 in Table AA is Y. In some embodiments, the amino acid designated as X8 in Table AA is S. In some embodiments, the amino acid designated as X8 in Table AA is T. In some embodiments, the amino acid designated as X9 in Table AA is W. In some embodiments, the amino acid designated as X9 in Table AA is Y. In some embodiments, the amino acid designated as X9 in Table AA is S. In some embodiments, the amino acid designated as X9 in Table AA is T. In some embodiments, the amino acid designated as X 10 in Table AA is H. In some embodiments, the amino acid designated as X 10 in Table AA is Y. In some embodiments, the amino acid designated as X 11 in Table AA is S. In some embodiments, the amino acid designated as X 11 in Table AA is G. In some embodiments, the amino acid designated as X 12 in Table AA is I. In some embodiments, the amino acid designated as X 12 in Table AA is L. In some embodiments, the amino acid designated as X 13 in Table AA is V. In some embodiments, the amino acid designated as X 13 in Table AA is G. In some embodiments, the amino acid designated as X14 The amino acid is R. In some embodiments, it is designated as X in Table AA 14 The amino acid is N. In some embodiments, it is designated as X in Table AA 15 The amino acid is D. In some embodiments, it is designated as X in Table AA 15 The amino acid is E. In some embodiments, it is designated as X in Table AA 15 The amino acid is L. In some embodiments, it is designated as X in Table AA 16 The amino acid is G. In some embodiments, it is designated as X in Table AA 16 The amino acid is N. In some embodiments, it is designated as X in Table AA 16 The amino acid is E. In some embodiments, it is designated as X in Table AA 17 The amino acid is R. In some embodiments, it is designated as X in Table AA 17 The amino acid is S. In some embodiments, it is designated as X in Table AA 18 The amino acid is V. In some embodiments, it is designated as X in Table AA 18 The amino acid is T. In some embodiments, it is designated as X in Table AA 19 The amino acid is N. In some embodiments, it is designated as X in Table AA 19 The amino acid is T. In some embodiments, it is designated as X in Table AA 20 The amino acid is R. In some embodiments, it is designated as X in Table AA 20 The amino acid is L. In some embodiments, it is designated as X in Table AA 21 The amino acid is F. In some embodiments, it is designated as X in Table AA 21 The amino acid is E. In some embodiments, it is designated as X in Table AA 22 The amino acid is S. In some embodiments, it is designated as X in Table AA 22 The amino acid is Y. In some embodiments, it is designated as X in Table AA 23 The amino acid is S. In some embodiments, it is designated as X in Table AA 23 The amino acid is Y. In some embodiments, it is designated as X in Table AA 24 The amino acid is S. In some embodiments, it is designated as X in Table AA 24 The amino acid is A. In some embodiments, it is designated as X in Table AA 25 The amino acid is H. In some embodiments, it is designated as X in Table AA 25 The amino acid is T. In some embodiments, it is designated as X in Table AA 26 The amino acid is F. In some embodiments, it is designated as X in Table AA26 The amino acid at 27 is W. In some embodiments, it is designated as X in Table AA 27 The amino acid at

[0750] In some embodiments, the CD3 ABM can comprise the CDR-H1 sequence C1-1. In some embodiments, the CD3 ABM can comprise the CDR-H1 sequence C1-2. In some embodiments, the CD3 ABM can comprise the CDR-H1 sequence C1-3. In some embodiments, the CD3 ABM can comprise the CDR-H1 sequence C1-4.

[0751] In some embodiments, the CD3 ABM can comprise the CDR-H2 sequence C1-5. In some embodiments, the CD3 ABM can comprise the CDR-H2 sequence C1-6. In some embodiments, the CD3 ABM can comprise the CDR-H2 sequence C1-7.

[0752] In some embodiments, the CD3 ABM can comprise the CDR-H3 sequence C1-8. In some embodiments, the CD3 ABM can comprise the CDR-H3 sequence C1-9. In some embodiments, the CD3 ABM can comprise the CDR-H3 sequence C1-10. In some embodiments, the CD3 ABM can comprise the CDR-H3 sequence C1-11.

[0753] In some embodiments, the CD3 ABM can comprise the CDR-L1 sequence C1-12. In some embodiments, the CD3 ABM can comprise the CDR-L1 sequence C1-13. In some embodiments, the CD3 ABM can comprise the CDR-L1 sequence C1-14. In some embodiments, the CD3 ABM can comprise the CDR-L1 sequence C1-15. In some embodiments, the CD3 ABM can comprise the CDR-L1 sequence C1-16. In some embodiments, the CD3 ABM can comprise the CDR-L1 sequence C1-17.

[0754] In some embodiments, the CD3 ABM can comprise the CDR-L2 sequence C1-18. In some embodiments, the CD3 ABM can comprise the CDR-L2 sequence C1-19.

[0755] In some embodiments, the CD3 ABM can comprise the CDR-L3 sequence C1-20. In some embodiments, the CD3 ABM can comprise the CDR-L3 sequence C1-21. In some embodiments, the CD3 ABM can comprise the CDR-L3 sequence C1-22. In some embodiments, the CD3 ABM can comprise the CDR-L3 sequence C1-23.

[0756] In some embodiments, the CD3 ABM can comprise the CDR-H1 sequence, CDR-H2 sequence, CDR-H3 sequence, CDR-L1 sequence, CDR-L2 sequence, and CDR-L3 sequence listed in Table AB.

[0757] In some embodiments, the amino acid designated as X 28 in Table AB is V. In some embodiments, the amino acid designated as X 28 in Table AB is I. In some embodiments, the amino acid designated as X 29 in Table AB is F. In some embodiments, the amino acid designated as X 29 in Table AB is Y. In some embodiments, the amino acid designated as X 30 in Table AB is N. In some embodiments, the amino acid designated as X 30 in Table AB is S. In some embodiments, the amino acid designated as X 31 in Table AB is A. In some embodiments, the amino acid designated as X 31 in Table AB is S. In some embodiments, the amino acid designated as X 32 in Table AB is T. In some embodiments, the amino acid designated as X 32 in Table AB is K. In some embodiments, the amino acid designated as X 33 in Table AB is T. In some embodiments, the amino acid designated as X 33 in Table AB is A. In some embodiments, the amino acid designated as X 34 in Table AB is S. In some embodiments, the amino acid designated as X 34 in Table AB is R. In some embodiments, the amino acid designated as X 35 in Table AB is N. In some embodiments, the amino acid designated as X 35 in Table AB is G. In some embodiments, the amino acid designated as X 36 in Table AB is S. In some embodiments, the amino acid designated as X 36 in Table AB is A. In some embodiments, the amino acid designated as X 37 in Table AB is A. In some embodiments, the amino acid designated as X 37 in Table AB is T. In some embodiments, the amino acid designated as X 37 in Table AB is S. In some embodiments, the amino acid designated as X 38 in Table AB is N. In some embodiments, the amino acid designated as X 38 in Table AB is D. In some embodiments, the amino acid designated as X39 The amino acid is N. In some embodiments, it is designated as X in Table AB 39 The amino acid is K. In some embodiments, it is designated as X in Table AB 40 The amino acid is D. In some embodiments, it is designated as X in Table AB 40 The amino acid is N. In some embodiments, it is designated as X in Table AB 41 The amino acid is H. In some embodiments, it is designated as X in Table AB 41 The amino acid is N. In some embodiments, it is designated as X in Table AB 42 The amino acid is Q. In some embodiments, it is designated as X in Table AB 42 The amino acid is E. In some embodiments, it is designated as X in Table AB 43 The amino acid is R. In some embodiments, it is designated as X in Table AB 43 The amino acid is S. In some embodiments, it is designated as X in Table AB 43 The amino acid is G.

[0758] In some embodiments, the CD3 ABM can comprise the CDR-H1 sequence C2-1. In some embodiments, the CD3 ABM can comprise the CDR-H1 sequence C2-2. In some embodiments, the CD3 ABM can comprise the CDR-H1 sequence C2-3. In some embodiments, the CD3 ABM can comprise the CDR-H1 sequence C2-4.

[0759] In some embodiments, the CD3 ABM can comprise the CDR-H2 sequence C2-5. In some embodiments, the CD3 ABM can comprise the CDR-H2 sequence C2-6. In some embodiments, the CD3 ABM can comprise the CDR-H2 sequence C2-7.

[0760] In some embodiments, the CD3 ABM can comprise the CDR-H3 sequence C2-8. In some embodiments, the CD3 ABM can comprise the CDR-H3 sequence C2-9.

[0761] In some embodiments, the CD3 ABM can comprise the CDR-L1 sequence C2-10. In some embodiments, the CD3 ABM can comprise the CDR-L1 sequence C2-11. In some embodiments, the CD3 ABM can comprise the CDR-L1 sequence C2-12.

[0762] In some embodiments, the CD3 ABM can comprise the CDR-L2 sequence C2-13. In some embodiments, the CD3 ABM can comprise the CDR-L2 sequence C2-14. In some embodiments, the CD3 ABM can comprise the CDR-L2 sequence C2-15.

[0763] In some embodiments, the CD3 ABM can comprise the CDR-L3 sequence C2-16. In some embodiments, the CD3 ABM can comprise the CDR-L3 sequence C2-17.

[0764] In some embodiments, the CD3 ABM can comprise the CDR-H1 sequence, CDR-H2 sequence, CDR-H3 sequence, CDR-L1 sequence, CDR-L2 sequence, and CDR-L3 sequence listed in Table AC.

[0765] In some embodiments, the amino acid designated as X 44 in Table AC is G. In some embodiments, the amino acid designated as X 44 in Table AC is A. In some embodiments, the amino acid designated as X 45 in Table AC is H. In some embodiments, the amino acid designated as X 45 in Table AC is N. In some embodiments, the amino acid designated as X 46 in Table AC is D. In some embodiments, the amino acid designated as X 46 in Table AC is G. In some embodiments, the amino acid designated as X 47 in Table AC is A. In some embodiments, the amino acid designated as X 47 in Table AC is G. In some embodiments, the amino acid designated as X 48 in Table AC is N. In some embodiments, the amino acid designated as X 48 in Table AC is K. In some embodiments, the amino acid designated as X 49 in Table AC is V. In some embodiments, the amino acid designated as X 49 in Table AC is A. In some embodiments, the amino acid designated as X 50 in Table AC is N. In some embodiments, the amino acid designated as X 50 in Table AC is V. In some embodiments, the amino acid designated as X 51 in Table AC is A. In some embodiments, the amino acid designated as X 51 in Table AC is V. In some embodiments, the amino acid designated as X 52 in Table AC is Y. In some embodiments, the amino acid designated as X 52The amino acid is F. In some embodiments, it is designated as X in Table AC 53 The amino acid is I. In some embodiments, it is designated as X in Table AC 53 The amino acid is V. In some embodiments, it is designated as X in Table AC 54 The amino acid is I. In some embodiments, it is designated as X in Table AC 54 The amino acid is H.

[0766] In some embodiments, the CD3 ABM can comprise the CDR-H1 sequence C3-1. In some embodiments, the CD3 ABM can comprise the CDR-H1 sequence C3-2. In some embodiments, the CD3 ABM can comprise the CDR-H1 sequence C3-3. In some embodiments, the CD3 ABM can comprise the CDR-H1 sequence C3-4.

[0767] In some embodiments, the CD3 ABM can comprise the CDR-H2 sequence C3-5. In some embodiments, the CD3 ABM can comprise the CDR-H2 sequence C3-6. In some embodiments, the CD3 ABM can comprise the CDR-H2 sequence C3-7.

[0768] In some embodiments, the CD3 ABM can comprise the CDR-H3 sequence C3-8. In some embodiments, the CD3 ABM can comprise the CDR-H3 sequence C3-9.

[0769] In some embodiments, the CD3 ABM can comprise the CDR-L1 sequence C3-10. In some embodiments, the CD3 ABM can comprise the CDR-L1 sequence C3-11. In some embodiments, the CD3 ABM can comprise the CDR-L1 sequence C3-12.

[0770] In some embodiments, the CD3 ABM can comprise the CDR-L2 sequence C3-13. In some embodiments, the CD3 ABM can comprise the CDR-L2 sequence C3-14.

[0771] In some embodiments, the CD3 ABM can comprise the CDR-L3 sequence C3-15. In some embodiments, the CD3 ABM can comprise the CDR-L3 sequence C3-16.

[0772] In some embodiments, the CD3 ABM can comprise the CDR-H1, CDR-H2, and CDR-H3 sequences listed in Table AD-1, and the corresponding CDR-L1, CDR-L2, and CDR-L3 sequences listed in Table AD-2.

[0773] In some embodiments, the CD3 ABM can comprise the CDR-H1, CDR-H2, and CDR-H3 sequences listed in Table AE-1, and the corresponding CDR-L1, CDR-L2, and CDR-L3 sequences listed in Table AE-2.

[0774] In some embodiments, the CD3 ABM can comprise the CDR-H1, CDR-H2, and CDR-H3 sequences listed in Table AF-1, and the corresponding CDR-L1, CDR-L2, and CDR-L3 sequences listed in Table AF-2.

[0775] In some embodiments, the CD3 ABM can comprise the CDR-H1, CDR-H2, and CDR-H3 sequences listed in Table AG-1, and the corresponding CDR-L1, CDR-L2, and CDR-L3 sequences listed in Table AG-2.

[0776] In some embodiments, the CD3 ABM can comprise the CDR-H1, CDR-H2, and CDR-H3 sequences listed in Table AH-1, and the corresponding CDR-L1, CDR-L2, and CDR-L3 sequences listed in Table AH-2.

[0777] In some embodiments, the CD3 ABM can comprise the CDR-H1, CDR-H2, and CDR-H3 sequences listed in Table AI-1, and the corresponding CDR-L1, CDR-L, and CDR-L3 sequences listed in Table AI-2.

[0778] In some embodiments, the CD3 ABM can comprise a heavy chain CDR having an amino acid sequence of any one of the CDRs listed in Table AB-1, Table AC-1, Table AD-1, Table AE-1, Table AF-1, Table AG-1, Table AH-1, or Table AI-1. In a specific embodiment, the CD3 ABM can comprise (or alternatively, consist of) one, two, three, or more heavy chain CDRs selected from the heavy chain CDRs described in Table AB-1, Table AC-1, Table AD-1, Table AE-1, Table AF-1, Table AG-1, Table AH-1, and Table AI-1. [[ID=!6]]

[0779] In some embodiments, the CD3 ABM can comprise a light chain CDR having an amino acid sequence of any of the CDRs listed in Table AB-2, Table AC-2, Table AD-2, Table AE-2, Table AF-2, Table AG-2, Table AH-2, or Table AI-2. In a specific embodiment, the CD3 ABM can comprise (or alternatively, consist of) one, two, three, or more light chain CDRs selected from the light chain CDRs described in Table AB-2, Table AC-2, Table AD-2, Table AE-2, Table AF-2, Table AG-2, Table AH-2, and Table AI-2.

[0780] Other CD3 ABMs include mutated amino acids, but their CDR regions still have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the CDR sequences described in Table A. In some embodiments, such CD3 ABMs include mutant amino acid sequences in which no more than 1, 2, 3, 4, or 5 amino acids have been mutated in the CDR region when compared to the CDR sequences described in Table A.

[0781] In some embodiments, the CD3 ABM can comprise a VH and / or VL domain having an amino acid sequence of any of the VH and / or VL domains described in Table A. Other CD3 ABMs include VH and / or VL domains that comprise amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the VH and / or VL sequences described in Table A. In some embodiments, the CD3 ABM includes a VH and / or VL domain in which no more than 1, 2, 3, 4, or 5 amino acids have been mutated when compared to the VH and / or VL domains described in the sequences described in Table A, while retaining substantially the same therapeutic activity.

[0782] VH and VL sequences (amino acid sequences and nucleotide sequences encoding said amino acid sequences) can be "mixed and matched" to generate other CD3 ABMs. Such "mixed and matched" CD3 ABMs can be tested using binding assays known in the art (e.g., ELISA assays). When the chains are mixed and matched, the VH sequence from a particular VH / VL pairing should be replaced with a structurally similar VH sequence. The VL sequence from a particular VH / VL pairing should be replaced with a structurally similar VL sequence.

[0783] Thus, in one embodiment, the CD3 ABM comprises: a heavy chain variable region (VH) that comprises an amino acid sequence selected from any of the VH sequences described in Table A-J1; and a light chain variable region (VL) that comprises the amino acid sequence described in Table A-J2.

[0784] In some embodiments, the antigen-binding domain that specifically binds to human CD3 is non-immunoglobulin-based and, instead, is derived from a non-antibody scaffold protein, such as one of the non-antibody scaffold proteins described in Section 7.3.2. In an embodiment, the antigen-binding domain that specifically binds to human CD3 comprises Affilin-144160 as described in WO 2017 / 013136. Affilin-144160 has the following amino acid sequence:

[0785] MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQWLWFAGKQLEDGRTLSDYNIQKESTLKLWLVDKAAMQIFVYTRTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNIALESGLHLVLRLRAA(SEQ ID NO:305).

[0786] 7.7.2. TCR-α / β ABM

[0787] The MBM can contain an ABM that specifically binds to the TCR-α chain, the TCR-β chain, or the TCR-αβ dimer. Exemplary anti-TCR-α / β antibodies are known (see, e.g., US 2012 / 0034221; Borst et al., 1990, Hum Immunol. [Human Immunology] 29(3):175-88 (describing the antibody BMA031)). The VH, VL, and Kabat CDR sequences of the antibody BMA031 are provided in Table 13.

[0788]

[0789]

[0790] In an embodiment, the TCR ABM can comprise the CDR sequences of the antibody BMA031. In other embodiments, the TCR ABM can comprise the VH and VL sequences of the antibody BMA031.

[0791] 7.7.3. TCR-γ / δ ABM

[0792] The MBM can contain an ABM that specifically binds to the TCR-γ chain, the TCR-δ chain, or the TCR-γδ dimer. Exemplary anti-TCR-γ / δ antibodies are known (see, e.g., US Patent No. 5,980,892 (describing δTCS1, which is produced by a hybridoma deposited with the ATCC under accession number HB9578)).

[0793] 7.8. CD2 ABM

[0794] 7.8.1 Immunoglobulin-based CD2 ABM

[0795] Type 1 TBM may comprise an ABM that is an anti-CD2 antibody or an antigen-binding domain thereof. Exemplary anti-CD2 antibodies are known (see, e.g., US 6,849,258, CN 102827281 A, US 2003 / 0139579 A1, and US 5,795,572). Table 14 provides exemplary CDR, VH, and VL sequences that may be included in an anti-CD2 antibody or an antigen-binding fragment thereof for use in the MBMs of the present disclosure.

[0796]

[0797]

[0798] In some embodiments, the CD2 ABM comprises the CDR sequences of CD2-1 (SEQ ID NOs: 312-317). In some embodiments, the CD2 ABM comprises the heavy and light chain variable sequences of CD2-1 (SEQ ID NOs: 318 and 319, respectively). In some embodiments, the CD2 ABM comprises the heavy and light chain variable sequences of hu1CD2-1 (SEQ ID NOs: 320 and 321, respectively). In some embodiments, the CD2 ABM comprises the heavy and light chain variable sequences of hu2CD2-1 (SEQ ID NOs: 318 and 321, respectively).

[0799] In other embodiments, the CD2 ABM may comprise the CDR sequences of antibody 9D1 produced by a hybridoma deposited with the China General Microbiological Culture Collection Center on May 16, 2012, under accession number CGMCC 6132 and described in CN 102827281 A. In other embodiments, the CD2 ABM may comprise the CDR sequences of antibody LO-CD2b produced by a hybridoma deposited with the American Type Culture Collection on June 22, 1999, under accession number PTA-802 and described in US 2003 / 0139579 A1. In still other embodiments, the CD2 ABM may comprise the CDR sequences of CD2 SFv-Ig produced by expressing a cloned construct in recombinant E. coli deposited with the ATCC on April 9, 1993, under accession number 69277 and described in US 5,795,572.

[0800] In other embodiments, the CD2 ABM can comprise the VH and VL sequences of antibody 9D1. In other embodiments, the CD2 ABM can comprise the VH and VL sequences of antibody LO-CD2b. In still other embodiments, the CD2 ABM can comprise the VH and VL sequences of CD2 SFv-Ig produced by expressing a cloned construct in recombinant Escherichia coli having ATCC Deposit No. 69277.

[0801] 7.8.2. CD2 ABM Based on CD58

[0802] In some aspects, the present disclosure provides a type 1 TBM that includes CD2ABM as a ligand. The CD2 ABM specifically binds to human CD2, whose natural ligand is CD58, also known as LFA-3. The CD58 / LFA-3 protein is expressed on the surface of a variety of cell types (Dustin et al., 1991, Annu. Rev. Immunol. 9:27) and plays a role in mediating the interaction of T cells with APCs in both antigen-dependent and antigen-independent manners (Wallner et al., 1987, J. Exp. Med. 166:923). Thus, in some aspects, the CD2 ABM is a CD58 moiety. As used herein, a CD58 moiety includes an amino acid sequence having at least 70% sequence identity to the CD2-binding portion of CD58 (e.g., having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the CD2-binding portion of CD58). The sequence of human CD58 has the Uniprot identifier P19256 (www.uniprot.org / uniprot / P19256). It has been determined that a CD58 fragment containing amino acid residues 30-123 of full-length CD58 (i.e., the sequence designated as CD58-6 in Table 15 below) is sufficient to bind to CD2. Wan et al., 1999, Cell 97:791-803. Thus, in some aspects, the CD58 moiety includes an amino acid sequence having at least 70% sequence identity to amino acids 30-123 of CD58 (e.g., having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence designated as CD58-6).

[0803] The interaction between CD58 and CD2 has been mapped by x-ray crystallography and molecular modeling. Substitution of residues E25, K29, K30, K32, D33, K34, E37, D84, and K87 (where numbering refers to the mature polypeptide) reduces binding to CD2. Ikemizu et al., 1999, Proc. Natl. Acad. Sci. USA 96:4289-94. Accordingly, in some embodiments, the CD58 portion retains the wild-type residues at E25, K29, K30, K32, D33, K34, E37, D84, and K87.

[0804] In contrast, the following substitutions (where numbering refers to the full-length polypeptide) do not affect binding to CD2: F29S; V37K; V49Q; V86K; T113S; and L121G. Accordingly, the CD58 portion can include one, two, three, four, five, or all six of the foregoing substitutions.

[0805] In some embodiments, the CD58 portion is engineered to include a pair of cysteine substituents that create a disulfide bridge upon recombinant expression. Exemplary amino acid pairs that can be substituted with cysteine to form a disulfide bridge upon expression (where numbering refers to the full-length polypeptide) are (a) V45C substitution and M105C substitution; (b) V54C substitution and G88C substitution; (c) V45C substitution and M114C substitution; and (d) W56C substitution and L90C substitution.

[0806] Exemplary CD58 portions are provided in Table 15 below:

[0807]

[0808]

[0809] 7.8.3. CD2 ABM Based on CD48

[0810] In some aspects, the present disclosure provides an MBM that includes a CD2ABM as a CD48 moiety. As used herein, the CD48 moiety includes an amino acid sequence having at least 70% sequence identity to the CD2-binding portion of CD48 (e.g., having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the CD2-binding portion of CD48). The sequence of human CD48 has the Uniprot identifier P09326 (www.uniprot.org / uniprot / P09326), which includes a signal peptide (amino acids 1-26) and a GPI anchor (amino acids 221-243). In some aspects, the CD48 moiety includes an amino acid sequence having at least 70% sequence identity to the amino acid sequence consisting of amino acids 27-220 of the sequence having Uniprot identifier P09326 (e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity). Human CD48 has an Ig-like C2 type I domain (amino acids 29-127 of the sequence having Uniprot identifier P09326) and an Ig-like C2 type 2 domain (amino acids 132-212 of the sequence having Uniprot identifier P09326). Thus, in some embodiments, the CD48 moiety includes an amino acid sequence having at least 70% sequence identity to the amino acid sequence consisting of amino acids 29-212 of the sequence having Uniprot identifier P09326, to the C2 type I domain (amino acids 29-127 of the sequence having Uniprot identifier P09326) and / or to the Ig-like C2 type 2 domain (amino acids 132-212 of the sequence having Uniprot identifier P09326) (e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity). In some embodiments, the CD48 moiety can include one or more natural variants relative to the sequence having Uniprot identifier P09326. For example, the CD48 moiety can include an E102Q substitution.Alternatively, the CD48 moiety can comprise an amino acid sequence corresponding to a CD-48 isotype or its CD2-binding portion (e.g., the isotype having Uniprot identifier P09326-2 or its CD2-binding portion).

[0811] 7.9. Tumor-associated antigen ABM

[0812] Type 2 TBM can comprise an ABM that specifically binds to a tumor-associated antigen (TAA). In some embodiments, the TAA is a human TAA. The antigen may or may not be present on normal cells. In certain embodiments, the TAA is preferentially expressed or upregulated on tumor cells compared to normal cells. In other embodiments, the TAA is a lineage marker.

[0813] In certain embodiments, the TAA is expressed or upregulated on cancerous B cells compared to normal B cells. In other embodiments, the TAA is a B cell lineage marker.

[0814] Any type of B cell malignancy is expected to be targetable by the MBMs disclosed herein. Representative types of B cell malignancies that can be targeted include Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), and multiple myeloma. Examples of NHL include diffuse large B cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (Waldenström macroglobulinemia), hairy cell leukemia, primary central nervous system (CNS) lymphoma, primary mediastinal large B cell lymphoma, mediastinal gray zone lymphoma (MGZL), splenic marginal zone B cell lymphoma, extranodal marginal zone B cell lymphoma of MALT, nodal marginal zone B cell lymphoma, and primary effusion lymphoma.

[0815] Examples of TAAs that can be targeted by MBM (e.g., TBM) in addition to CD19 include BCMA, CD20, CD22, CD123, CD33, CLL1, CD138 (also known as Syndecan-1, SDC1), CS1, CD38, CD133, FLT3, CD52, TNFRSF13C (TNF receptor superfamily member 13C, also known in the art as BAFFR: B cell-activating factor receptor), TNFRSF13B (TNF receptor superfamily member 13B, also known in the art as TACI: transmembrane activator and CAML interactor), CXCR4 (C-X-C motif chemokine receptor 4), PD-L1 (programmed death ligand 1), LY9 (lymphocyte antigen 9, also known in the art as CD229), CD200, FCGR2B (Fc fragment of IgG receptor IIb, also known in the art as CD32b), CD21, CD23, CD24, CD40L, CD72, CD79a, and CD79b. In some embodiments, the TAA is BCMA. In some embodiments, the TAA is CD20. In some embodiments, the TAA is CD22. In some embodiments, the TAA is CD123. In some embodiments, the TAA is CD33. In some embodiments, the TAA is CLL1. In some embodiments, the TAA is CD138. In some embodiments, the TAA is CS1. In some embodiments, the TAA is CD38. In some embodiments, the TAA is CD133. In some embodiments, the TAA is FLT3. In some embodiments, the TAA is CD52. In some embodiments, the TAA is TNFRSF13C. In some embodiments, the TAA is TNFRSF13B. In some embodiments, the TAA is CXCR4. In some embodiments, the TAA is PD-L1. In some embodiments, the TAA is LY9. In some embodiments, the TAA is CD200. In some embodiments, the TAA is CD21. In some embodiments, the TAA is CD23. In some embodiments, the TAA is CD24. In some embodiments, the TAA is CD40L. In some embodiments, the TAA is CD72. In some embodiments, the TAA is CD79a. In some embodiments, the TAA is CD79b.

[0816] The TAA-binding ABM can include, for example, an anti-TAA antibody or an antigen-binding fragment thereof. The anti-TAA antibody or antigen-binding fragment can include, for example, the CDR sequences of the antibodies listed in Table 16. In some embodiments, the anti-TAA antibody or its antigen-binding domain has the heavy and light chain variable region sequences of the antibodies listed in Table 16.

[0817]

[0818]

[0819]

[0820] In certain embodiments, the TAA is selected from BCMA and CD20. In some embodiments, the TAA is BCMA. "BCMA" refers to B cell maturation antigen. BCMA (also known as TNFRSF17, BCM or CD269) is a member of the tumor necrosis factor receptor (TNFR) family and is mainly expressed on terminally differentiated B cells (such as memory B cells and plasma cells). Its ligands include B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL). The protein BCMA is encoded by the gene TNFRSF17. Exemplary BCMA sequences are available in the Uniprot database, accession number Q02223.

[0821] In certain aspects, the type 2 TBM comprises ABM3 that specifically binds to BCMA, such as an anti-BCMA antibody or its antigen-binding domain. The anti-BCMA antibody or its antigen-binding domain may comprise, for example, the CDR, VH, VL, or scFV sequences listed in Tables 17A - 17G.

[0822]

[0823]

[0824]

[0825]

[0826]

[0827]

[0828]

[0829]

[0830]

[0831]

[0832]

[0833]

[0834]

[0835]

[0836]

[0837]

[0838]

[0839]

[0840]

[0841]

[0842]

[0843]

[0844]

[0845]

[0846]

[0847]

[0848]

[0849]

[0850]

[0851] In some embodiments, the ABM comprises the CDR sequences of BCMA-1. In some embodiments, the ABM comprises the CDR sequences of BCMA-2. In some embodiments, the ABM comprises the CDR sequences of BCMA-3. In some embodiments, the ABM comprises the CDR sequences of BCMA-4. In some embodiments, the ABM comprises the CDR sequences of BCMA-5. In some embodiments, the ABM comprises the CDR sequences of BCMA-6. In some embodiments, the ABM comprises the CDR sequences of BCMA-7. In some embodiments, the ABM comprises the CDR sequences of BCMA-8. In some embodiments, the ABM comprises the CDR sequences of BCMA-9. In some embodiments, the ABM comprises the CDR sequences of BCMA-10. In some embodiments, the ABM comprises the CDR sequences of BCMA-11. In some embodiments, the ABM comprises the CDR sequences of BCMA-12. In some embodiments, the ABM comprises the CDR sequences of BCMA-13. In some embodiments, the ABM comprises the CDR sequences of BCMA-14. In some embodiments, the ABM comprises the CDR sequences of BCMA-15. In some embodiments, the ABM comprises the CDR sequences of BCMA-16. In some embodiments, the ABM comprises the CDR sequences of BCMA-17. In some embodiments, the ABM comprises the CDR sequences of BCMA-18. In some embodiments, the ABM comprises the CDR sequences of BCMA-19. In some embodiments, the ABM comprises the CDR sequences of BCMA-20. In some embodiments, the ABM comprises the CDR sequences of BCMA-21. In some embodiments, the ABM comprises the CDR sequences of BCMA-22. In some embodiments, the ABM comprises the CDR sequences of BCMA-23. In some embodiments, the ABM comprises the CDR sequences of BCMA-24. In some embodiments, the ABM comprises the CDR sequences of BCMA-25. In some embodiments, the ABM comprises the CDR sequences of BCMA-26. In some embodiments, the ABM comprises the CDR sequences of BCMA-27. In some embodiments, the ABM comprises the CDR sequences of BCMA-28. In some embodiments, the ABM comprises the CDR sequences of BCMA-29. In some embodiments, the ABM comprises the CDR sequences of BCMA-30. In some embodiments, the ABM comprises the CDR sequences of BCMA-31. In some embodiments, the ABM comprises the CDR sequences of BCMA-32. In some embodiments, the ABM comprises the CDR sequences of BCMA-33. In some embodiments, the ABM comprises the CDR sequences of BCMA-34.In some embodiments, the ABM comprises the CDR sequences of BCMA-35. In some embodiments, the ABM comprises the CDR sequences of BCMA-36. In some embodiments, the ABM comprises the CDR sequences of BCMA-37. In some embodiments, the ABM comprises the CDR sequences of BCMA-38. In some embodiments, the ABM comprises the CDR sequences of BCMA-39. In some embodiments, the ABM comprises the CDR sequences of BCMA-40.

[0852] In some embodiments, the CDRs are defined by Kabat numbering as listed in Tables 17B and 17E. In other embodiments, the CDRs are defined by Chothia numbering as listed in Tables 17C and 17F. In still other embodiments, the CDRs are defined by a combination of Kabat and Chothia numbering as listed in Tables 17D and 17G.

[0853] In some embodiments, the type 2 TBM in which ABM3 binds to BCMA can comprise the variable heavy and light chain sequences of any one of BCMA-1 to BCMA-40.

[0854] In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-1, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-2, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-3, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-4, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-5, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-6, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-7, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-8, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-9, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-10, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-11, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-12, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-13, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-14, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-15, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-16, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-17, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-18, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-19, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-20, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-21, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-22, as listed in Table 17A.In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-23, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-24, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-25, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-26, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-27, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-28, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-29, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-30, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-31, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-32, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-33, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-34, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-35, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-36, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-37, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-38, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-39, as listed in Table 17A. In some embodiments, the ABM comprises the heavy and light chain variable sequences of BCMA-40, as listed in Table 17A.

[0855] 7.10. Nucleic Acids and Host Cells

[0856] In another aspect, the present disclosure provides nucleic acids (i.e., polynucleotides) encoding the CD19 binding molecules of the present disclosure. In some embodiments, the CD19 binding molecule is encoded by a single nucleic acid. In other embodiments, the CD19 binding molecule is encoded by multiple (e.g., two, three, four, or more) nucleic acids.

[0857] A single nucleic acid can encode a CD19-binding molecule comprising a single polypeptide chain, a CD19-binding molecule comprising two or more polypeptide chains, or a portion of a CD19-binding molecule comprising more than two polypeptide chains (e.g., a single nucleic acid can encode two polypeptide chains of a CD19-binding molecule comprising three, four, or more polypeptide chains, or three polypeptide chains of a CD19-binding molecule comprising four or more polypeptide chains). For separate control of expression, the open reading frames encoding two or more polypeptide chains can be under the control of separate transcriptional regulatory elements (e.g., promoters and / or enhancers). The open reading frames encoding two or more polypeptides can also be controlled by the same transcriptional regulatory element and separated by an internal ribosome entry site (IRES) sequence to allow translation into different polypeptides.

[0858] In some embodiments, a CD19-binding molecule comprising two or more polypeptide chains is encoded by two or more nucleic acids. The number of nucleic acids encoding the CD19-binding molecule can be equal to or less than the number of polypeptide chains in the CD19-binding molecule (e.g., when more than one polypeptide chain is encoded by a single nucleic acid).

[0859] The nucleic acid can be DNA or RNA (e.g., mRNA).

[0860] In another aspect, the present disclosure provides host cells and vectors containing the nucleic acids of the present disclosure. The nucleic acids can be present in a single vector or different vectors, and the vectors can be present in the same host cell or different host cells, as described in more detail below.

[0861] 7.10.1. Vectors

[0862] The present disclosure provides vectors comprising nucleotide sequences encoding the CD19-binding molecules or CD19-binding molecule components described herein. In one embodiment, the vector comprises nucleotides encoding an immunoglobulin-based ABM described herein. In one embodiment, the vector comprises nucleotides encoding an Fc domain described herein. In one embodiment, the vector comprises nucleotides encoding a recombinant non-immunoglobulin-based ABM described herein. The vector can encode one or more ABMs, one or more Fc domains, one or more non-immunoglobulin-based ABMs, or any combination thereof (e.g., when multiple components or sub-components are encoded as a single polypeptide chain). In one embodiment, the vector comprises the nucleotide sequences described herein. The vectors include, but are not limited to, viruses, plasmids, cosmids, λ phages, or yeast artificial chromosomes (YACs).

[0863] A variety of vector systems can be used. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retroviruses (Rous sarcoma virus, MMTV or MoMLV), or SV40 virus. Another class of vectors utilizes RNA elements derived from RNA viruses such as Semliki Forest virus, Eastern Equine Encephalitis virus, and flavivirus.

[0864] In addition, cells that stably integrate DNA into their chromosomes can be selected by introducing one or more markers that allow for the selection of transfected host cells. The markers can provide, for example, prototropy to auxotrophic hosts, can provide microbicide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker genes can be directly linked to the DNA sequence to be expressed or introduced into the same cells by co-transformation. Optimal synthesis of mRNA may also require additional elements. These elements can include splicing signals, as well as transcriptional promoters, enhancers, and termination signals.

[0865] Once an expression vector or DNA sequence containing the construct is prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection, or other conventional techniques can be used to achieve this goal. Methods and conditions for culturing the resulting transfected cells and recovering the expressed polypeptide are known to those skilled in the art and can be varied or optimized based on the particular expression vector and mammalian host cell used in accordance with this specification.

[0866] 7.10.2. Cells

[0867] The present disclosure also provides host cells comprising the nucleic acids of the present disclosure.

[0868] In one embodiment, the host cell is genetically engineered to comprise one or more of the nucleic acids described herein.

[0869] In one embodiment, the host cell is genetically engineered using an expression cassette. The phrase "expression cassette" refers to a nucleotide sequence that is capable of affecting the expression of a gene in a host ...

Claims

1. A CD19-binding molecule, which is a trispecific binding molecule (TBM) and comprises (a) an antigen-binding module 1 (ABM1) that specifically binds to human CD19 and is an antibody or an antigen-binding fragment thereof; and (b) an antigen-binding module 2 (ABM2) that is an antibody or an antigen-binding fragment thereof and specifically binds to a component of the human T cell receptor (TCR) complex, wherein the component of the TCR complex is CD3; and (c) an antigen-binding module 3 (ABM3), wherein ABM3 specifically binds to the CD58 moiety of human CD2, wherein ABM1 that specifically binds to human CD19 comprises a sequence selected from the following: a) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences of SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively; b) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences of SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, respectively; c) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences of SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22, respectively, or d) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences of SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences of SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25, respectively.

2. The CD19-binding molecule according to claim 1, wherein ABM1 comprises a VH consisting of the amino acid sequence of SEQ ID NO:13 and a VL consisting of the amino acid sequence of SEQ ID NO:

26.

3. The CD19-binding molecule according to claim 1 or 2, wherein ABM1 is a Fab.

4. The CD19-binding molecule according to claim 1 or 2, wherein ABM2 is a scFv.

5. The CD19-binding molecule according to claim 1 or 2, wherein ABM1 is a Fab and ABM2 is a scFv.

6. The CD19-binding molecule according to claim 1 or 2, wherein ABM2 comprises a set of CDR sequences as described in any one of the following: 1) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO: 192, SEQ ID NO: 212, and SEQ ID NO: 236, respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO: 193, SEQ ID NO: 213, and SEQ ID NO: 237, respectively; 2) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO: 194, SEQ ID NO: 214, and SEQ ID NO: 238, respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO: 195, SEQ ID NO: 215, and SEQ ID NO: 239, respectively; 3) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO: 196, SEQ ID NO: 216, and SEQ ID NO: 240, respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO: 197, SEQ ID NO: 217, and SEQ ID NO: 241, respectively; 4) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO: 192, SEQ ID NO: 212, and SEQ ID NO: 236, respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO: 198, SEQ ID NO: 218, and SEQ ID NO: 242, respectively; 5) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO: 192, SEQ ID NO: 219, and SEQ ID NO: 236, respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO: 193, SEQ ID NO: 213, and SEQ ID NO: 237, respectively; 6) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO: 199, SEQ ID NO: 220, and SEQ ID NO: 243, respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO: 200, SEQ ID NO: 221, and SEQ ID NO: 244, respectively; 7) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:222, and SEQ ID NO:245 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:195, SEQ ID NO:215, and SEQ ID NO:239 respectively; 8) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:201, SEQ ID NO:223, and SEQ ID NO:246 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:202, SEQ ID NO:224, and SEQ ID NO:247 respectively; 9) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:203, SEQ ID NO:214, and SEQ ID NO:248 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:202, SEQ ID NO:224, and SEQ ID NO:247 respectively; 10) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239 respectively; 11) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:249 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:250 respectively; 12) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:192, SEQ ID NO:219, and SEQ ID NO:251 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:193, SEQ ID NO:213, and SEQ ID NO:237 respectively; 13) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:205, SEQ ID NO:225, and SEQ ID NO:252 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:206, SEQ ID NO:226, and SEQ ID NO:253 respectively; 14) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:227, and SEQ ID NO:249 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:250 respectively; 15) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:254 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:250 respectively; 16) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:255 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:250 respectively; 17) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:256 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:250 respectively; 18) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:207, SEQ ID NO:223, and SEQ ID NO:249 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:250 respectively; 19) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:208, SEQ ID NO:223, and SEQ ID NO:238 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239 respectively; 20) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:207, SEQ ID NO:223, and SEQ ID NO:238 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239 respectively; 21) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:228, and SEQ ID NO:238 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239 respectively; 22) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:229, and SEQ ID NO:238 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239 respectively; 23) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:230, and SEQ ID NO:238 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239 respectively; 24) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:249 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239 respectively; 25) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:257 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239 respectively; 26) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:258 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239 respectively; 27) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:209, SEQ ID NO:215, and SEQ ID NO:239 respectively; 28) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:210, SEQ ID NO:215, and SEQ ID NO:239 respectively; 29) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:231, and SEQ ID NO:239 respectively; 30) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:232, and SEQ ID NO:239 respectively; 31) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238, respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:233, and SEQ ID NO:239, respectively; 32) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238, respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:234, and SEQ ID NO:239, respectively; 33) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238, respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:259, respectively; 34) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238, respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:250, respectively; 35) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238, respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:195, SEQ ID NO:215, and SEQ ID NO:239, respectively; 36) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238, respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:211, SEQ ID NO:215, and SEQ ID NO:239, respectively; 37) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:227, and SEQ ID NO:249, respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239, respectively; 38) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:256, respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239, respectively; 39) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:207, SEQ ID NO:227, and SEQ ID NO:256, respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:21�, and SEQ ID NO:239, respectively; 40) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:235, and SEQ ID NO:238, respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239, respectively; or 41) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:214, and SEQ ID NO:260, respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:209, SEQ ID NO:215, and SEQ ID NO:239, respectively.

7. The CD19-binding molecule according to claim 1 or 2, wherein: a) ABM1 is a Fab and b) ABM2 is a scFv comprising any one of the following groups of CDR sequences 1) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:192, SEQ ID NO:212, and SEQ ID NO:236, respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:193, SEQ ID NO:213, and SEQ ID NO:237, respectively; 2) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:214, and SEQ ID NO:238 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:195, SEQ ID NO:215, and SEQ ID NO:239 respectively; 3) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:196, SEQ ID NO:216, and SEQ ID NO:240 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:197, SEQ ID NO:217, and SEQ ID NO:241 respectively; 4) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:192, SEQ ID NO:212, and SEQ ID NO:236 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:198, SEQ ID NO:218, and SEQ ID NO:242 respectively; 5) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:192, SEQ ID NO:219, and SEQ ID NO:236 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:193, SEQ ID NO:213, and SEQ ID NO:237 respectively; 6) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:199, SEQ ID NO:220, and SEQ ID NO:243 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:200, SEQ ID NO:221, and SEQ ID NO:244 respectively; 7) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:222, and SEQ ID NO:245 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:195, SEQ ID NO:215, and SEQ ID NO:239 respectively; 8) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:201, SEQ ID NO:223, and SEQ ID NO:246, respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:202, SEQ ID NO:224, and SEQ ID NO:247, respectively; 9) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:203, SEQ ID NO:214, and SEQ ID NO:248, respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:202, SEQ ID NO:224, and SEQ ID NO:247, respectively; 10) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238, respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239, respectively; 11) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:249, respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:250, respectively; 12) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:192, SEQ ID NO:219, and SEQ ID NO:251, respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:193, SEQ ID NO:213, and SEQ ID NO:237, respectively; 13) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:205, SEQ ID NO:225, and SEQ ID NO:252, respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:206, SEQ ID NO:226, and SEQ ID NO:253, respectively; 14) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:227, and SEQ ID NO:249 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:250 respectively; 15) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:254 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:250 respectively; 16) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:255 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:250 respectively; 17) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:256 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:250 respectively; 18) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:207, SEQ ID NO:223, and SEQ ID NO:249 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:250 respectively; 19) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:208, SEQ ID NO:223, and SEQ ID NO:238 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239 respectively; 20) CDR-H1, CDR-H2, and CDR-H3, which are composed of amino acid sequences SEQ ID NO:207, SEQ ID NO:223, and SEQ ID NO:238 respectively, and CDR-L1, CDR-L2, and CDR-L3, which are composed of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239 respectively; 21) CDR-H1, CDR-H2, and CDR-H3, which are composed of amino acid sequences SEQ ID NO:194, SEQ ID NO:228, and SEQ ID NO:238 respectively, and CDR-L1, CDR-L2, and CDR-L3, which are composed of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239 respectively; 22) CDR-H1, CDR-H2, and CDR-H3, which are composed of amino acid sequences SEQ ID NO:194, SEQ ID NO:229, and SEQ ID NO:238 respectively, and CDR-L1, CDR-L2, and CDR-L3, which are composed of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239 respectively; 23) CDR-H1, CDR-H2, and CDR-H3, which are composed of amino acid sequences SEQ ID NO:194, SEQ ID NO:230, and SEQ ID NO:238 respectively, and CDR-L1, CDR-L2, and CDR-L3, which are composed of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239 respectively; 24) CDR-H1, CDR-H2, and CDR-H3, which are composed of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:249 respectively, and CDR-L1, CDR-L2, and CDR-L3, which are composed of amino acid sequences SEQ ID NO:204, SEQ ID NO: ​ 26) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:258 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239 respectively; 27) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:209, SEQ ID NO:215, and SEQ ID NO:239 respectively; 28) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:210, SEQ ID NO:215, and SEQ ID NO:239 respectively; 29) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:231, and SEQ ID NO:239 respectively; 30) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:232, and SEQ ID NO:239 respectively; 31) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:233, and SEQ ID NO:239 respectively; 32) CDR-H1, CDR-H2, and CDR-H3, which are composed of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238, respectively, and CDR-L1, CDR-L2, and CDR-L3, which are composed of amino acid sequences SEQ ID NO:204, SEQ ID NO:234, and SEQ ID NO:239, respectively; 33) CDR-H1, CDR-H2, and CDR-H3, which are composed of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238, respectively, and CDR-L1, CDR-L2, and CDR-L3, which are composed of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:259, respectively; 34) CDR-H1, CDR-H2, and CDR-H3, which are composed of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238, respectively, and CDR-L1, CDR-L2, and CDR-L3, which are composed of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:250, respectively; 35) CDR-H1, CDR-H2, and CDR-H3, which are composed of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238, respectively, and CDR-L1, CDR-L2, and CDR-L3, which are composed of amino acid sequences SEQ ID NO:195, SEQ ID NO:215, and SEQ ID NO:239, respectively; 36) CDR-H1, CDR-H2, and CDR-H3, which are composed of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238, respectively, and CDR-L1, CDR-L2, and CDR-L3, which are composed of amino acid sequences SEQ ID NO:211, SEQ ID NO:215, and SEQ ID NO:239, respectively; 37) CDR-H1, CDR-H2, and CDR-H3, which are composed of amino acid sequences SEQ ID NO:194, SEQ ID NO:227, and SEQ ID NO:249, respectively, and CDR-L1, CDR-L2, and CDR-L3, which are composed of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239, respectively; 38) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:256 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239 respectively; 39) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:207, SEQ ID NO:227, and SEQ ID NO:256 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239 respectively; 40) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:235, and SEQ ID NO:238 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239 respectively; or 41) CDR-H1, CDR-H2, and CDR-H3 consisting of amino acid sequences SEQ ID NO:194, SEQ ID NO:214, and SEQ ID NO:260 respectively, and CDR-L1, CDR-L2, and CDR-L3 consisting of amino acid sequences SEQ ID NO:209, SEQ ID NO:215, and SEQ ID NO:239 respectively.

8. The CD19-binding molecule according to claim 1 or 2, wherein ABM3 is selected from the following sequences: The amino acid sequence of SEQ ID NO:322, The amino acid sequence of SEQ ID NO:323, The amino acid sequence of SEQ ID NO:324, The amino acid sequence of SEQ ID NO:325, The amino acid sequence of SEQ ID NO:326, The amino acid sequence of SEQ ID NO:327, The amino acid sequence of SEQ ID NO:328, The amino acid sequence of SEQ ID NO:329, The amino acid sequence of SEQ ID NO:330, The amino acid sequence of SEQ ID NO:331, and The amino acid sequence of SEQ ID NO:

332.

9. The CD19-binding molecule according to claim 1 or 2, wherein ABM3 is a CD58 moiety, and the amino acid sequence of the CD58 moiety is SEQ ID NO:

327.

10. The CD19-binding molecule according to claim 1 or 2, wherein a) ABM1 is Fab, and b) ABM2 is scFv, which comprises any one of the following groups of CDR sequences: 1) CDR-H1, CDR-H2, and CDR-H3, which are composed of amino acid sequences SEQ ID NO:192, SEQ ID NO:212, and SEQ ID NO:236 respectively, and CDR-L1, CDR-L2, and CDR-L3, which are composed of amino acid sequences SEQ ID NO:193, SEQ ID NO:213, and SEQ ID NO:237 respectively; 2) CDR-H1, CDR-H2, and CDR-H3, which are composed of amino acid sequences SEQ ID NO:194, SEQ ID NO:214, and SEQ ID NO:238 respectively, and CDR-L1, CDR-L2, and CDR-L3, which are composed of amino acid sequences SEQ ID NO:195, SEQ ID NO:215, and SEQ ID NO:239 respectively; 3) CDR-H1, CDR-H2, and CDR-H3, which are composed of amino acid sequences SEQ ID NO:196, SEQ ID NO:216, and SEQ ID NO:240 respectively, and CDR-L1, CDR-L2, and CDR-L3, which are composed of amino acid sequences SEQ ID NO:197, SEQ ID NO:217, and SEQ ID NO:241 respectively; 4) CDR-H1, CDR-H2, and CDR-H3, which are composed of amino acid sequences SEQ ID NO:192, SEQ ID NO:212, and SEQ ID NO:236 respectively, and CDR-L1, CDR-L2, and CDR-L3, which are composed of amino acid sequences SEQ ID NO:198, SEQ ID NO:218, and SEQ ID NO:242 respectively; 5) CDR-H1, CDR-H2, and CDR-H3, which are composed of amino acid sequences SEQ ID NO:192, SEQ ID NO:219, and SEQ ID NO:236 respectively, and CDR-L1, CDR-L2, and CDR-L3, which are composed of amino acid sequences SEQ ID NO:193, SEQ ID NO:213, and SEQ ID NO:237 respectively; 6) CDR-H1, CDR-H2, and CDR-H3, which are composed of amino acid sequences SEQ ID NO:199, SEQ ID NO:220, and SEQ ID NO:243 respectively, and CDR-L1, CDR-L2, and CDR-L3, which are composed of amino acid sequences SEQ ID NO:200, SEQ ID NO:221, and SEQ ID NO:244 respectively; 7) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences SEQ ID NO:194, SEQ ID NO:222, and SEQ ID NO:245, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences SEQ ID NO:195, SEQ ID NO:215, and SEQ ID NO:239; 8) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences SEQ ID NO:201, SEQ ID NO:223, and SEQ ID NO:246, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences SEQ ID NO:202, SEQ ID NO:224, and SEQ ID NO:247; 9) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences SEQ ID NO:203, SEQ ID NO:214, and SEQ ID NO:248, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences SEQ ID NO:202, SEQ ID NO:224, and SEQ ID NO:247; 10) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239; 11) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:249, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:250; 12) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences SEQ ID NO:192, SEQ ID NO:219, and SEQ ID NO:251, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences SEQ ID NO:193, SEQ ID NO:213, and SEQ ID NO:237; 13) CDR-H1, CDR-H2 and CDR-H3 consisting of the amino acid sequences SEQ ID NO:205, SEQ ID NO:225 and SEQ ID NO:252, and CDR-L1, CDR-L2 and CDR-L3 consisting of the amino acid sequences SEQ ID NO:206, SEQ ID NO:226 and SEQ ID NO:253; 14) CDR-H1, CDR-H2 and CDR-H3 consisting of the amino acid sequences SEQ ID NO:194, SEQ ID NO:227 and SEQ ID NO:249, and CDR-L1, CDR-L2 and CDR-L3 consisting of the amino acid sequences SEQ ID NO:204, SEQ ID NO:215 and SEQ ID NO:250; 15) CDR-H1, CDR-H2 and CDR-H3 consisting of the amino acid sequences SEQ ID NO:194, SEQ ID NO:223 and SEQ ID NO:254, and CDR-L1, CDR-L2 and CDR-L3 consisting of the amino acid sequences SEQ ID NO:204, SEQ ID NO:215 and SEQ ID NO:250; 16) CDR-H1, CDR-H2 and CDR-H3 consisting of the amino acid sequences SEQ ID NO:194, SEQ ID NO:223 and SEQ ID NO:255, and CDR-L1, CDR-L2 and CDR-L3 consisting of the amino acid sequences SEQ ID NO:204, SEQ ID NO:215 and SEQ ID NO:250; 17) CDR-H1, CDR-H2 and CDR-H3 consisting of the amino acid sequences SEQ ID NO:194, SEQ ID NO:223 and SEQ ID NO:256, and CDR-L1, CDR-L2 and CDR-L3 consisting of the amino acid sequences SEQ ID NO:204, SEQ ID NO:215 and SEQ ID NO:250; 18) CDR-H1, CDR-H2 and CDR-H3 consisting of the amino acid sequences SEQ ID NO:207, SEQ ID NO:223 and SEQ ID NO:249, and CDR-L1, CDR-L2 and CDR-L3 consisting of the amino acid sequences SEQ ID NO:204, SEQ ID NO:215 and SEQ ID NO:250; 19) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences SEQ ID NO:208, SEQ ID NO:223, and SEQ ID NO:238, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239; 20) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences SEQ ID NO:207, SEQ ID NO:223, and SEQ ID NO:238, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239; 21) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences SEQ ID NO:194, SEQ ID NO:228, and SEQ ID NO:238, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239D NO:239; 22) CDR-HI, CDR-H2, and CDR-H3 consisting of the amino acid sequences SEQ ID NO:194, SEQ ID NO:229, and SEQ ID NO:238, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239; 23) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences SEQ ID NO:194, SEQ ID NO:230, and SEQ ID NO:238, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239; 24) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:249, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239; 25) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:257, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239; 26) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:258, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239; 27) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences SEQ ID NO:209, SEQ ID NO:215, and SEQ ID NO:239; 28) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences SEQ ID NO:210, SEQ ID NO:215, and SEQ ID NO:239; 29) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences SEQ ID NO:204, SEQ ID NO:231, and SEQ ID NO:239; 30) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences SEQ ID NO:204, SEQ ID NO:232, and SEQ ID NO:239; 31) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences SEQ ID NO:204, SEQ ID NO:233, and SEQ ID NO:239; 32) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences SEQ ID NO:204, SEQ ID NO:234, and SEQ ID NO:239; 33) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:259; 34) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:250; 35) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences SEQ ID NO:195, SEQ ID NO:215, and SEQ ID NO:239; 36) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:238, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences SEQ ID NO:211, SEQ ID NO:215, and SEQ ID NO:239; 37) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences SEQ ID NO:194, SEQ ID NO:227, and SEQ ID NO:249, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239; 38) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences SEQ ID NO:194, SEQ ID NO:223, and SEQ ID NO:256, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239; 39) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences SEQ ID NO:207, SEQ ID NO:227, and SEQ ID NO:256, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239; 40) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences SEQ ID NO:194, SEQ ID NO:235, and SEQ ID NO:238, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences SEQ ID NO:204, SEQ ID NO:215, and SEQ ID NO:239; or 41) CDR-H1, CDR-H2, and CDR-H3 consisting of the amino acid sequences SEQ ID NO:194, SEQ ID NO:214, and SEQ ID NO:260, and CDR-L1, CDR-L2, and CDR-L3 consisting of the amino acid sequences SEQ ID NO:209, SEQ ID NO:215, and SEQ ID NO:239; And wherein ABM3 is the CD58 portion comprising the amino acid sequence of SEQ ID NO.

327.

11. The CD19-binding molecule according to claim 1 or 2, wherein ABM1 is a Fab specifically binding to human CD19, which comprises CDR-H1, CDR-H2 and CDR-H3 consisting of the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 respectively, and CDR-L1, CDR-L2 and CDR-L3 consisting of the amino acid sequences of SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19 respectively, and wherein ABM2 specifically binding to human CD3 is a scFv, comprising CDR sequences of VH CDR1: SEQ ID NO.194, VH CDR2: SEQ ID NO.214, VH CDR3: SEQ ID NO.238 and VL CDR1: SEQ ID NO.195, VL CDR2: SEQ ID NO.215 and VL CDR3: SEQ ID NO.239, and wherein ABM3 specifically binding to human CD2 is a CD58 moiety and comprises an Fc domain.

12. The CD19-binding molecule according to claim 11, wherein ABM1 is a Fab and comprises VH consisting of the amino acid sequence of SEQ ID NO:13 and VL consisting of the amino acid sequence of SEQ ID NO:

26.

13. The CD19-binding molecule according to claim 11, wherein ABM2 comprises the heavy chain variable sequence of SEQ ID NO.172 and the light chain variable sequence of SEQ ID NO.

173.

14. The CD19-binding molecule according to claim 1, wherein ABM1 is a Fab, which comprises VH consisting of the amino acid sequence of SEQ ID NO:13 and VL consisting of the amino acid sequence of SEQ ID NO:26, and wherein ABM2 specifically binding to human CD3 is a scFv, which comprises the heavy chain variable sequence SEQ ID NO.172 and the light chain variable sequence SEQ ID NO.173, and wherein ABM3 specifically binding to human CD2 is a CD58 moiety and comprises an Fc domain.

15. The CD19-binding molecule according to claim 11, wherein ABM3 comprises the amino acid sequence of SEQ ID NO:

327.

16. The CD19-binding molecule according to claim 1, wherein ABM1 is a Fab and comprises VH consisting of the amino acid sequence of SEQ ID NO:13 and VL consisting of the amino acid sequence of SEQ ID NO:26, and wherein ABM2 specifically binding to human CD3 is a scFv comprising the heavy chain variable sequence SEQ ID NO.172 and the light chain variable sequence SEQ ID NO.173, and wherein ABM3 specifically binding to human CD2 is a CD58 moiety comprising the amino acid sequence SEQ ID NO:327 and comprises an Fc domain.

17. The CD19-binding molecule according to claim 11, wherein the Fc domain is an Fc heterodimer, and the Fc heterodimer comprises a mortise and tenon structure modification.

18. The CD19-binding molecule according to claim 11, wherein the Fc domain comprises a first variant Fc region and a second variant Fc region that together form an Fc heterodimer.

19. A CD19-binding molecule, comprising: (a) a first half-antibody heavy chain, the amino acid sequence of which comprises the amino acid sequence of SEQ ID NO: 758 and an Fc sequence; (b) a first half-antibody light chain, the amino acid sequence of which is the amino acid sequence of SEQ ID NO: 759; (c) a second half-antibody, the amino acid sequence of which comprises the amino acid sequence of SEQ ID NO: 760 and an Fc sequence.

20. A CD19-binding molecule, comprising: (a) a first polypeptide, the amino acid sequence of which is the amino acid sequence of SEQ ID NO: 1077; (b) a second polypeptide, the amino acid sequence of which is the amino acid sequence of SEQ ID NO: 759; and (c) a third polypeptide, the amino acid sequence of which is the amino acid sequence of SEQ ID NO: 1086.

21. One or more nucleic acids encoding the CD19-binding molecule according to any one of claims 1 to 20.

22. A cell engineered to express the CD19-binding molecule according to any one of claims 1 to 20.

23. A method for producing a CD19-binding molecule, comprising: (a) culturing the cell according to claim 22 under conditions for the expression of the CD19-binding molecule; and (b) recovering the CD19-binding molecule from the cell culture.

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