CD19 binding molecules and uses thereof

Abstract

The present disclosure provides CD19 binding molecules, including monospecific, bispecific, and trispecific binding molecules, that specifically bind to CD19; a conjugate comprising the CD19 binding molecule; and pharmaceutical compositions comprising the CD19 binding molecules and conjugates. The disclosure further provides methods of treating diseases and disorders associated with CD19 expression using the C19 binding molecules. The disclosure 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] This application is a divisional application of Chinese application 202080037829.2, filed on May 19, 2020, entitled "CD19 binding molecule and its use".

[0002] 1. Cross-references to related applications

[0003] This application claims priority to 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 which are incorporated herein by reference in their entirety.

[0004] 2. Sequence List

[0005] This application contains a sequence list that has been electronically filed in ASCII format, and the entire sequence list is hereby incorporated by reference. The ASCII copy was created on May 4, 2020, named NOV-007WO_SL.txt, and is 776,262 bytes in size. Technical Field

[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 treating diseases and disorders associated with CD19 expression. Background Technology

[0007] 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 B lymphocyte development and function. CD19 expression has been identified in most lymphoblastic cancers, the vast majority of non-Hodgkin's lymphomas (NHL), and leukemias (including chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), and Waldenström macroglobulinemia (WM)).

[0008] Bonatumab is a CD19-CD3 bispecific T-cell conjugate approved for the treatment of ALL. However, treatment with bonatumab lacks durable responses and is characterized by a high relapse rate. (Von Stackelberg et al., 2016, Journal of Clinical Oncology, 34(36):4381-4389.) Furthermore, bonatumab has a short half-life, requiring sustained drug exposure to achieve adequate efficacy and manageable toxicity. (Porter et al., 2013, Clin Pharmacol., 5(Supplement 1):5-11.)

[0009] Despite significant advances in cancer treatment, B-cell malignancies, such as the B-cell subtype of non-Hodgkin's lymphoma and chronic lymphocytic leukemia, remain a leading cause of cancer-related death. Therefore, further therapeutic agents are still needed to treat B-cell malignancies. Summary of the Invention

[0010] This disclosure provides CD19-binding molecules (e.g., antibodies, their antigen-binding fragments) that specifically bind to human CD19, and multispecific molecules that specifically bind to human CD19.

[0011] In one aspect, this disclosure provides single-specific CD19 binding molecules (e.g., antibodies and their antigen-binding fragments) that include a CD19 antigen-binding domain or antigen-binding module (“ABM”). Exemplary CD19 binding molecules that may be single-specific are described in Section 7.2 below and in Specific Examples 1 through Specific Examples 15.

[0012] On the other hand, this disclosure provides a multispecific binding molecule (“MBM”) comprising the CD19 ABM disclosed herein.

[0013] In some embodiments, the MBM is a bispecific binding molecule (“BBM”). The BBM disclosed herein comprises 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, such as human CD3 or other components of the T-cell receptor (TCR) complex (sometimes referred to herein as “TCRABM”). The terms ABM1, ABM2, CD19 ABM, and TCR ABM are used merely for convenience and are not intended to convey any particular conformation of the BBM. In some embodiments, the TCR ABM binds to CD3 (referred to herein as “CD3 ABM”, etc.). Therefore, the disclosure relating to ABM2 and TCR ABM also applies to CD3 ABM. Such multispecific molecules can be used to guide CD3+ effector T cells to CD19+ sites, thereby allowing CD3+ effector T cells to attack and lyse CD19+ cells and tumors. Exemplary MBMs are characterized in Sections 7.5 through 7.6 below and in Specific Examples 16 through Specific Examples 1190.

[0014] This disclosure also extends the principle of redirected targeted T cell lysis (RTCC) by providing a trispecific binding molecule (“TBM”) that binds to additional components of the TCR complex on CD19, CD3, or T cells, as well as CD2 or human tumor-associated antigens (“TAAs”), such as B cell antigens other than CD19. The TBM of this disclosure comprises at least three antigen-binding modules (“ABM”) that can bind (i) CD19 (ABM1), (ii) a component of the TCR complex (ABM2), and (iii) CD2 or TAA (ABM3). For convenience, a TBM binding to (1) human CD19, (2) CD3 or other components of the TCR complex, and (3) CD2 is referred to herein as a “type 1 TBM”. For convenience, a TBM binding to (1) human CD19, (2) CD3 or other components of the TCR complex, and (3) TAA is referred to herein as a “type 2 TBM”.

[0015] Unbound by theory, the inventors believe that conjugating the CD2- and TCR complex in type 1 TBM can stimulate both the primary signaling pathway promoting T cell-mediated tumor cell lysis (e.g., by TCR aggregation) and the secondary co-stimulatory pathway to induce T cell proliferation and potentially overcome unresponsiveness. Also unbound by theory, it is believed that conjugating a TAA other than the components of the CD19 and TCR complex, compared to using a bispecific conjugate targeting only the components of the CD19 and TCR complex, will improve clinical outcomes for RTCC treatment of cancers (e.g., B-cell malignancies) by targeting a greater number of cancerous B cells.

[0016] Therefore, in one aspect, this disclosure provides for additional components that bind to (1) human CD19, (2) CD3 or TCR complexes, and (3) CD2 type 1 TBM.

[0017] On the other hand, this disclosure provides additional components that bind to (1) human CD19, (2) CD3 or TCR complexes, and (3) TAA type 2 TBM.

[0018] Unless otherwise expressly stated or unless the context otherwise requires, references to TBM in this disclosure apply to both Type 1 and Type 2 TBMs.

[0019] In some embodiments, each antigen-binding module of the MBM disclosed herein is capable of binding its respective target simultaneously with each of one or more additional antigen-binding modules. ABM1 is immunoglobulin-based, while ABM2 and ABM3 (when present) may be immunoglobulin-based or non-immunoglobulin-based. Thus, an MBM may comprise an immunoglobulin-based ABM or any combination of immunoglobulin-based and non-immunoglobulin-based ABMs. Immunoglobulin-based ABMs that may be used in MBMs are described in Section 7.3.1 below and in Specific Examples 17 to 21, 24 to 29. Non-immunoglobulin-based ABMs that may be used in MBMs are described in Section 7.3.2 below and in Specific Examples 22 to 23. Further features of exemplary ABMs binding to human CD19 are described in Section 7.2 below and in Specific Examples 17 to 21. Further features of exemplary ABMs binding to components of the TCR complex are described in Section 7.7 below and in Specific Examples 30 to 621. Other features of the exemplary ABM incorporated into CD2 are described below in Section 7.8 and in specific embodiments 726 to 775. Other features of the exemplary ABM incorporated into TAA are described below in Section 7.9 and in specific embodiments 776 to 894.

[0020] The ABMs (or portions thereof) of MBM can be interconnected, for example, through short peptide linkers or through Fc domains. Methods and components for linking ABMs to form MBMs are described below in Section 7.4 and in specific examples 895 to 1190.

[0021] BBM has at least two ABMs (e.g., BBM is at least divalent), and TBM has at least three ABMs (e.g., TBM is at least trivalent), but they may have greater valences. For example, BBM may have three, four, or more ABMs (i.e., it is trivalent, tetravalent, or has a valence greater than tetravalent). Exemplary divalent, trivalent, and tetravalent BBM configurations are shown in Figure 1 and described in Section 7.5 below and in specific embodiments 624 to 684.

[0022] A TBM can have four ABMs (i.e., tetravalent), five ABMs (i.e., pentavalent), or six ABMs (i.e., hexavalent), provided that the TBM has at least one ABM capable of binding CD19, at least one ABM capable of binding a component of the TCR complex, and at least one ABM capable of binding CD2 or TAA. Exemplary trivalent, tetravalent, pentavalent, and hexavalent TBM configurations are shown in Figure 2 and described in Section 7.6 below and in specific examples 687 to 724.

[0023] This disclosure further provides nucleic acids (in the form of a single nucleic acid or multiple nucleic acids) encoding the CD19 binding molecule described herein, and recombinant host cells and cell lines engineered to express the nucleic acids and CD19 binding molecules of this disclosure. Exemplary nucleic acids, host cells, and cell lines are described in Section 7.10 and Specific Examples 1241 to 1248 below.

[0024] This disclosure further provides pharmaceutical conjugates comprising the CD19 binding molecule disclosed herein. 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.

[0025] Pharmaceutical compositions comprising the CD19 binding molecule and the ADC are also provided. Examples of pharmaceutical compositions are described below in Section 7.15 and Specific Example 1231.

[0026] This document further provides methods for using the CD19 binding molecule, the ADC, and the pharmaceutical compositions disclosed herein, for example, to treat proliferative disorders (e.g., cancer) (on which CD19 is expressed), to treat autoimmune disorders, and to treat other diseases and conditions associated with CD19 expression. Exemplary methods are described in Section 7.16 and Specific Examples 1232 to 1239 below.

[0027] This disclosure further provides methods for using the CD19 binding molecule, the ADC, and the pharmaceutical composition in combination with other agents and therapies. Exemplary agents, therapies, and methods for combination therapies are described in Section 7.17 and Specific Example 1240 below. Attached Figure Description

[0028] Figure 1A-1AH Example BBM configuration. Figure 1A It shows Figure 1B-1AH The components of the exemplary BBM configuration are shown in the figure. Not all regions connecting the different structural domains of each chain are shown (e.g., the joints connecting the VH and VL structural domains of scFv, the hinges connecting the CH2 and CH3 structural domains of Fc, etc., are omitted). Figure 1B-1F Divalent BBM was shown; Figure 1G-1Z Trivalent BBM was shown; Figure 1AA-1AH The tetravalent BBM is shown.

[0029] Figure 2A-2V Example TBM configuration. Figure 2A It shows Figure 2B-2VThe components of the exemplary TBM configuration shown are not shown. All regions connecting the different structural domains of each chain are not shown (e.g., the joints connecting the VH and VL structural domains of scFv, the hinges connecting the CH2 and CH3 structural domains of Fc, etc., are omitted). Figure 2B-2P Trivalent TBM is shown; Figure 2Q-2S The tetravalent TBM is shown; Figure 2T The pentavalent TBM was shown, and Figure 2U-2V The hexavalent TBM is shown.

[0030] Figures 3A-3B : Bispecificity of Example 1 ( Figure 3A and Figure 3C ) and trispecific ( Figure 3B A schematic diagram of the construct.

[0031] Figures 4A-4B CD19 BBM has the ability to induce redirected T cell cytotoxicity (RTCC) against CD19+ target cells. Both NEG258-based and NEG218-based BBMs mediated RTCC activity against CD19+ target cell lines. In the presence of serially diluted BBMs with an effector cell:target cell (E:T) ratio of 3:1, Nalm6-luc ( Figure 4A ) and Karpas422-luc( Figure 4B Cells were co-cultured with expanded T cells. The luminescence signal was measured after 24 hours of incubation.

[0032] Figures 5A-5B CD19 BBM's ability to induce T cell proliferation. Both NEG258-based and NEG218-based BBMs induce T cell proliferation. In the presence of serially diluted BBMs at an E:T ratio of 1:1, Karpas422-luc( Figure 5A ) and Nalm6-luc( Figure 5B Cells were co-cultured with expanded T cells. The luminescence signal was measured after 96 hours of incubation.

[0033] Figures 6A-6F The ability of CD19 TBM to induce CD2-dependent T cell activation. CD2 knockout weakens the advantage of the trispecific construct. Figures 6A-6B JNL CD2 WT( Figure 6A ) and KO( Figure 6B Representative flow cytometry analysis of CD2 expression on cells. Staining of anti-CD2 mAb (dot-filled histogram) overlaps with staining of mIgG1 isotype controls (diagonal-filled histogram) or unstained (open histogram). Figure 6C-6F The results show that in the presence of BBM and TBM at a series of dilutions with an E:T ratio of 3:1, JNL CD2 + ( Figures 6C-6D ) and CD2 - ( Figure 6E-6F Cells and CD19 + Data from target cell co-culture. Luminescence signal was measured after 24 hours of incubation.

[0034] Figures 7A-7B : Binding of CD19 TBM to cynomolgus monkey B cells. Figure 7A Data for a TBM with a CD19 bonding arm based on NEG218 are shown, and Figure 7B Data for a TBM with a CD19-based NEG 258-based coupling arm are shown.

[0035] Figures 8A-8H The ability of CD19 TBM to induce T cell activation after depletion of cynomolgus monkey B cells in PBMCs. Figure 8A In this study, PBMCs were isolated from whole blood of cynomolgus monkeys using Ficoll gradient centrifugation and incubated overnight with bispecific or trispecific constructs. Samples were harvested and simultaneously stained for CD3 and CD20 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 CD3 is shown + FACS analysis results of CD69 and CD25 expression on T cells were used to determine the expression of CD69 and CD25 on individual T cells. + CD25 - Or CD69 - CD25 + ) or double-positive cells (CD69) + CD25 + ). Figure 8B Unprocessed (media only); Figure 8C-8E :CD3hi TSP1L; Figure 8F-8H :CD3hi TSP1.

[0036] Figures 9A-9P TBM-induced human donor cells targeting Nalm6 based on NEG258 and NEG218 (NEG258 and NEG218) Figures 9A-9H ) and Karpas422 ( Figure 9I-9P The ability of T cells to redirect target cells to act cytotoxically.

[0037] Figure 10A-10P TBM-induced human donor cells with different CD3 affinities, based on NEG258 and NEG218, targeting Nalm6 (… Figures 10A-10H ) and Karpas422 ( Figure 10I-10P The ability of T cells to redirect target cells to act cytotoxically.

[0038] Figure 11A-11L: NEG258-based TBM-induced human donor cells targeting Nalm6 containing a CD2 binding arm and a control lysozyme binding arm ( Figure 11A-11H ) and Karpas422 ( Figure 11I-11L The ability of T cells to redirect target cells to act cytotoxically.

[0039] Figures 12A-12C T cell cytokine release induced by NEG258-based and NEG218-based TBM. Figure 12A :IFN-γ; Figure 12B TNF-α; Figure 12C :IL2.

[0040] Figures 13A-13C TBM based on NEG258 and NEG218 and overexpression of human CD19 ( Figure 13A ) or cynomolgus monkey CD19 ( Figure 13B The binding of the TBM to the wild-type 300.19 cell line was negligible. Figure 13C ).

[0041] Figure 14 : A schematic diagram of CD58.

[0042] Figure 15 : Redirected T cell cytotoxicity of TBM containing CD58 variant sequences.

[0043] Figure 16 Antigen-independent T cell activation by TBM containing the CD58 variant sequence. Data expressed in relative luminescent units (RLU).

[0044] Figures 17A-17H Expression of CD19 and CD58 in multiple cell lines: Figures 17A-17B : Expression of CD19 and CD58 in OCI-LY-19 cells; Figure 17C-17D Expression of CD19 and CD58 in Karpas-422 cells; Figure 17E-17F : Expression of CD19 and CD58 in Toledo cells; Figure 17G-17H Expression of CD19 and CD58 in Nalm-6 cells.

[0045] Figures 18A-18B The ability of NEG258-based TBM and BBM to induce redirected T cell cytotoxicity in human donor cells against Karpas422 target cells. Figure 18A and Figure 18B Data using T cells from two different donors is shown.

[0046] Figures 19A-19FT cell cytokine release was induced by NEG258-based TBM and BBM. Figures 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). The triangles on the X-axis represent the decreasing concentration of the constructs from left to right in the figure.

[0047] Figure 20 : NEG-258-based TBM and BBM that bind to T cells.

[0048] Figures 21A-21C T cell proliferation mediated by NEG-258-based TBM and BBM. Figure 21A T cell proliferation in OC-LY-19 co-culture; Figure 21B T cell proliferation in co-culture with Karpas422; Figure 21C T cell proliferation in Toledo co-culture.

[0049] Figures 22A-22B The ability of NEG258-based TBM and BBM to induce redirected T cell cytotoxicity in human donor cells against Karpas422 target cells. Figure 22A and Figure 22B Data using T cells from two different donors is shown.

[0050] Figure 23A-23J The ability of NEG258-based TBM and BBM to induce T cell retargeting cytotoxicity in human donor cells against a variety of target cells. Figures 23A-23B : OC-LY-19 (for 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 (representing donor 1 and donor 2 respectively).

[0051] Figure 24A-24J T cell cytokine release was induced in a variety of target cells by NEG258-based TBM and BBM. Figures 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-24HTNF-α from Nalm6 KO (donor 1 and donor 2, respectively); Figure 24I-24J TNF-α from K562 (donor 1 and donor 2, respectively).

[0052] Figures 25A-25H RTCC assays were retested using Karpas 422 and OCI-LY-19 cell lines. Figure 25A Measurement settings. 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, after the second challenge, after the third challenge, and after the fourth challenge).

[0053] Figures 26A-26P : Retest T cell phenotypes using Karpas 422 and OCI-LY-19 cell lines. Figures 26A-26H :Karpas 422 phenotype; Figures 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 is young; Figure 26F and 26N CD4 is old; Figure 26G and 26O CD8 is young; Figure 26H and 26P CD8 is old. The lines in the diagram represent different T cell donors.

[0054] Figures 27A-27D CD3hi TSP1 and CD3hi BSP1 have the ability to induce T cell proliferation in the presence of CD19+ target cells. In the presence of 1 nM ( Figures 27A-27B ) or 0.1nM ( Figure 27C-27D CD3hi TSP1 or CD3hi BSP1 and exists ( Figure 27A and 27C ) or does not exist ( Figure 27B and 27D In the case of autologous PBMCs (T cell depletion) after irradiation, Nalm6-luc cells were mixed with sorted CD28 cells. + 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 in the viable cell count.

[0055] 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). Figures 28A-28B CD28 is produced when PBMCs are co-cultured with irradiated PBMCs and 1 nM CD3hi TSP1 or 1 nM CD3hi BSP1. - and CD28 + GzB in CD8 T cells ( Figure 28A ) and IFN-γ Figure 28B The median fluorescence intensity (MFI) of ). Figure 28C-28D CD28 is produced when co-cultured in the absence of irradiated PBMCs and 1 nM CD3hi TSP1 or 1 nM CD3hi BSP1. - and CD28 + GzB in CD8 T cells ( Figure 28C ) and IFN-γ Figure 28D MFI. Figure 28E-28F CD28 is produced when PBMCs are co-cultured in the presence of irradiated PBMCs and 0.1 nM CD3hi TSP1 or 0.1 nM CD3hi BSP1. - and CD28 + GzB in CD8 T cells ( Figure 28E ) and IFN-γ Figure 28F MFI. Figure 28G-28H CD28 is produced when co-cultured in the absence of irradiated PBMCs and 0.1 nM CD3hi TSP1 or 0.1 nM CD3hi BSP1. - and CD28 + GzB in CD8 T cells ( Figure 28G ) and IFN-γ Figure 28H MFI. Figure 28I-28L When there is ( Figure 28I and 28K ) or does not exist ( Figure 28J and 28L ) Irradiated PBMCs and 1 nM ( Figure 28I and 28J ) or 0.1nM ( Figure 28K and Figure 28L The proportion of live T cells when co-cultured with CD3hi TSP1 or CD3hi BSP1.

[0056] Figure: 29A-29I: The ability of CD3hi TSP1 and CD3hi BSP1 to induce T cell phenotypic changes. Figure 29A CD28 sorting targeting CCR7 and CD45RO expression - and CD28 + A representative example of T cells. Figure 29B-29I : In existence ( Figures 29B-29E ) or does not exist ( Figure 29F-29I )PBMC and the existence of 1nM ( Figures 29B-29C (and 29F-29G) or 0.1nM ( Figures 29D-29E After co-culturing with 29H-29I)CD3hi TSP1 or CD3hi BSP1 (E:T 1:3) for 72 hours, the two surface markers CD45RO and CCR7 (naïve, CD45RO) were used. - CCR7 + Central memory (CM), CD45RO + CCR7 + Effect memory (EM), CD45RO + CCR7 - ; and terminal differentiation (TEMRA), CD45RO - CCR7 - The distribution of different T cell populations as defined by the combined expression of ) is shown. Data on proliferating cells (CFSE-) are presented in Figure 29B , 29D Data for non-proliferating cells (CSFE+) in 29F and 29H are shown in [data missing]. Figure 29C , 29E The data for CD28- cells are displayed on the left side of each graph, while the data for CD28+ cells are displayed on the right side.

[0057] Figures 30A-30D CD3hi TSP1 and CD3hi BSP1 have the ability to induce redirected T cell cytotoxicity (RTCC) against CD19+ target cells. In the presence of 1 nM ( Figure 30A and 30C ) or 0.1nM ( Figure 30B and 30D CD3hi BSP1, CD3hiTSP1, or CD3hi TSP1C and the presence of ( Figure 30A and 30B ) or does not exist ( Figure 30C and 30D In the case of irradiated autologous PBMCs (T cell depletion), CD28 cells derived from sorted cells... + 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) Luminescent signal was measured at the end of co-culture incubation. Results are expressed as a fold increase compared to untreated conditions, where no antibody was added to assess the background signal given by the control antibody.

[0058] Figures 31A-31B :CD3hi TSP1( Figure 31A ) and CD3med TSP1 ( Figure 31B Antitumor activity in human PBMC adoptive transfer adaptation in the OCI-LY-19 subcutaneous tumor model.

[0059] Figures 32A-32B In the adoptive metastasis adaptation of human PBMCs in the OCI-LY-19 subcutaneous tumor model, CD3hi TSP1 was used ( Figure 32A ) and CD3med TSP1 ( Figure 32B Weight changes after treatment.

[0060] Figure 33 : Schematic diagram of the humanization process of NSG mice.

[0061] Figures 34A-34B Antitumor activity of CD3 TSP1, CD3hi BSP1 and CD3med TSP1 in a DLBCL subcutaneous tumor model in huCD34+NSG mice ( Figure 34A ), and weight changes after treatment with CD3TSP1, CD3hi BSP1 and CD3med TSP1 in a DLBCL subcutaneous tumor model in huCD34+NSG mice ( Figure 34B ).

[0062] Figures 35A-35D In the OCI-LY-19DLBCL subcutaneous tumor model of huCD34+NSG mice, CD3hi TSP1 ( Figure 35A and 35B ) and CD3med TSP1 ( Figure 35C and 35D Antitumor activity after antibody therapy Figure 35A and 35C ) and weight response ( Figure 35B and 35D ).

[0063] Figures 36A-36C CD3hi BSP1 ( Figure 36A ), CD3hi TSP1 ( Figure 36B ) and CD3med TSP1 ( Figure 36CAntitumor activity in human PBMC adoptive metastasis adaptation in the Daudi-Luc subcutaneous tumor model.

[0064] Figures 37A-37C In the adoptive metastasis adaptation of human PBMCs in the Daudi-Luc subcutaneous tumor model, CD3hi BSP1 was used ( Figure 37A ), CD3hi TSP1 ( Figure 37B ) or CD3med TSP1 ( Figure 37C Weight changes after antibody treatment.

[0065] Figures 38A-38C : Schematic diagram of the three-specific construct of Example 32. Figure 38A : A TBM with a full-length CD58 section AB2-1; Figure 38B : TBM having a truncated CD58 portion containing an IgV-like structural domain of CD58; Figure 38C : TBM with scFv corresponding to anti-CD2 antibody Medi 507.

[0066] Figures 39A-39E : Schematic diagram of the three-specific construct of Example 33. Figure 39A : TBM with CD58IgV structure domain from Instance 32; Figure 39B TBM having a "left" half antibody and a "right" half antibody, wherein the "left" half antibody has a CD58IgV domain, an anti-CD3 scFab domain and an Fc domain in the direction from the N-terminus to the C-terminus, and the "right" half antibody has an anti-CD19 Fab N-terminus with respect to the Fc domain; Figure 39C TBM having a "left" half antibody and a "right" half antibody, wherein the "left" half antibody has a CD58IgV domain, an anti-CD3 scFv domain and an Fc domain in the direction from the N-terminus to the C-terminus, and the "right" half antibody has an anti-CD19 Fab N-terminus with respect to the Fc domain; Figure 39D TBM having a "left" half antibody and a "right" half antibody, wherein the "left" half antibody has an anti-CD3scFv, CD58 IgV and Fc domains in the direction from the N-terminus to the C-terminus, and the "right" half antibody has an anti-CD19 Fab N-terminus with respect to the Fc domain; Figure 39E TBM having a "left" half antibody and a "right" half antibody, wherein the "left" half antibody has an anti-CD3 scFv, an Fc domain and a CD58 IgV domain in the direction from the N-terminus to the C-terminus, and the "right" half antibody has an anti-CD19 Fab N-terminus with respect to the Fc domain.

[0067] Figure 40A-40C: Schematic diagram of the three-specific construct of Example 34. Figure 40A : TBM with CD58IgV structure domain from Instance 32; Figure 40B TBM having a "left" half antibody and a "right" half antibody, wherein the "left" half antibody has an anti-CD3scFv, an Fc domain and a CD19 scFv domain in the direction from the N-terminus to the C-terminus, and the "right" half antibody has a CD58 IgV domain at the N-terminus of the Fc domain. Figure 40C TBM having a "left" half antibody and a "right" half antibody, wherein the "left" half antibody has an anti-CD3scFv, an Fc domain and a CD19 Fab domain in the direction from the N-terminus to the C-terminus, and the "right" half antibody has a CD58 IgV domain at the N-terminus of the Fc domain. Detailed Implementation

[0068] 7.1. Definition

[0069] As used herein, the following terms are intended to have the following meanings:

[0070] ABM Chain A single ABM can exist as a single polypeptide chain (e.g., in the case of scFv) or be formed by the association of more than one polypeptide chain (e.g., in the case of Fab). As used herein, the term "ABM chain" refers to all or part of the ABM present on a single polypeptide chain. The use of the term "ABM chain" is for convenience and descriptive purposes only and does not imply a particular configuration or method of production.

[0071] ADCC As used herein, “ADCC” or “antibody-dependent cell-mediated cytotoxicity” refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize binding antibodies on target cells and subsequently cause lysis of said target cells. ADCC is associated with binding to FcγRIIIa; increased binding to FcγRIIIa leads to increased ADCC activity.

[0072] ADCP As used herein, “ADCP” or antibody-dependent cell-mediated phagocytosis refers to a cell-mediated response in which nonspecific phagocytes expressing FcγR recognize binding antibodies on target cells and subsequently cause phagocytosis of said target cells.

[0073] Other medicines For convenience, agents used in combination with the antigen-binding molecules disclosed herein are referred to herein as “other” agents.

[0074] AntibodyAs used herein, the term "antibody" refers to a polypeptide (or group of polypeptides) belonging to the immunoglobulin family that binds to an antigen non-covalently, reversibly, and specifically. For example, a naturally occurring IgG-type "antibody" is a tetramer comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated VH herein) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated VL herein) and a light chain constant region. The light chain constant region consists of one domain (abbreviated CL herein). 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 consists 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 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 (Clq) of the classical complement system. The term "antibody" includes, but is not limited to: monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, bispecific or multispecific antibodies, and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies against the antibodies disclosed herein). These antibodies can belong to any isotype / type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subtype (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0075] Both the light and heavy chains are divided into structural homology regions and functional homology regions. The terms "constant" and "variable" are used functionally. In this respect, it should be understood that the variable domains of both the light chain (VL) and heavy chain (VH) portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and heavy chain (CH1, CH2, or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, and complement binding. By convention, the farther the constant domain is from the antibody's antigen-binding site or N-terminus, the higher its number. In wild-type antibodies, the variable region is at the N-terminus and the constant region is at the C-terminus; the CH3 and CL domains actually contain the C-termini of the heavy and light chains, respectively.

[0076] antibody fragmentsAs used herein, the term "antibody fragment" refers to one or more portions of an antibody. In some embodiments, these portions are part of one or more contact domains of the antibody. In some other embodiments, these portions are antigen-binding fragments (which retain the ability to bind antigens non-covalently, reversibly, and specifically), sometimes referred to herein as "antigen-binding fragments," "their 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 VL, VH, CL, and CH1 domains; F(ab)2 fragments, bivalent fragments comprising two Fab fragments connected by a disulfide bridge at the hinge region; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of VL and VH domains of a single arm of the antibody; dAb fragments consisting of VH domains (Ward et al., 1989, Nature 341:544-546); and separated complementarity-determining regions (CDRs). Therefore, the term "antibody fragment" encompasses proteolytic fragments of antibodies (e.g., Fab and F(ab)2 fragments) and engineered proteins containing one or more portions of an antibody (e.g., scFv).

[0077] Antibody fragments can also be incorporated into single-domain antibodies, macrobodies, minibodies, intracellular antibodies, bisomal antibodies, tripoisome antibodies, tetrasomal antibodies, v-NARs, and bis-scFvs (see, for example, Hollinger and Hudson, 2005 Nature Biotechnology 23:1126-1136). Antibody fragments can be grafted into peptide-based scaffolds such as type III fibronectin (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin peptide monomers).

[0078] Antibody fragments can be incorporated into single-chain molecules containing a pair of tandem Fv fragments (e.g., VH-CH1-VH-CH1) to form a pair of antigen-binding regions together with complementary light chain peptides (e.g., VL-VC-VL-VC) (Zapata et al., 1995, Protein Eng. 8:1057-1062; and U.S. Patent No. 5,641,870).

[0079] Antibody numbering systemIn this specification, unless otherwise stated, references to the numbered amino acid residues in the 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 [Proceedings of the National Academy of Sciences] 63:78-85 and described in detail by Kabat et al., 1991, in Sequences of Proteins of Immunological Interest, US National Institutes of Health, Department of Health and Human Services (NIH, USA).

[0080] Antigen binding module As used herein, the term "antigen-binding module" or "ABM" refers to a portion of an MBM that has the ability to bind non-covalently, reversibly, and specifically to an antigen. An 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 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 TAA. The terms ABM1, ABM2, and ABM3 are used merely for convenience and are not intended to convey any particular conformation of the MBM. In some embodiments, ABM2 binds to CD3 (hereinafter referred to as "CD3 ABM," etc.). Therefore, disclosures involving ABM2 and multiple ABM2s also apply to CD3 ABM.

[0081] antigen-binding fragments The term "antigen-binding fragment" in the context of an antibody refers to the portion of the antibody that retains the ability to bind nonvalently, reversibly, and specifically to an antigen.

[0082] antigen-binding moleculesThe term "antigen-binding molecule" refers to a molecule containing one or more antigen-binding domains, such as an antibody. The antigen-binding molecule may contain one or more polypeptide chains, for example, one, two, three, four, or more polypeptide chains. These polypeptide chains in the antigen-binding molecule may associate directly or indirectly with each other (e.g., a first polypeptide chain may associate with a second polypeptide chain, which in turn may associate with a third polypeptide chain to form an antigen-binding molecule, wherein 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).

[0083] association In the context of antigen-binding molecules, the term "association" refers to a functional relationship between two or more polypeptide chains and / or two or more portions of a single polypeptide chain. Specifically, the term "association" means that two or more polypeptides (or portions of a single polypeptide) associate with each other, for example, through non-covalent association via molecular interactions and / or through covalent association via one or more disulfide bridges or chemical crosslinks, thereby creating a functional antigen-binding molecule, for example, where the antigen-binding domain can bind to its respective target BBM or TBM. Examples of association that may be present in an MBM include (but are not limited to) association between Fc regions in the Fc domain (as described in Section 7.4.1.5 for homodimers or heterodimers), association between VH and VL regions in Fab or Fv, and association between CH1 and CL in Fab.

[0084] B cells: As used herein, the term "B cell" refers to a cell in the B cell lineage, which is a type of leukocyte subtype of lymphocytes. Examples of B cells include plasmablasts, plasma cells, lymphoplasmacytic cells, memory B cells, follicular B cells, marginal zone B cells, B-1 cells, B-2 cells, and regulatory B cells.

[0085] B-cell malignant tumors:As used in this article, 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, nodular marginal zone B-cell lymphoma, and primary exudative lymphoma, and plasmacytoid dendritic cell tumor.

[0086] Combining sequences Referring to Tables 1, 12, 13, 14, 16, or 17 (including their subsections), the term "combination sequence" means an ABM having a complete set of CDR, VH-VL pairs, or scFv listed in that table.

[0087] Bispecific binding molecules 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 disclosed herein contain at least one antigen-binding domain specific to CD19 and at least one antigen-binding domain specific to different antigens (e.g., components of the TCR complex). Representative BBMs are shown in... Figure 1B-1AH In the middle. BBM can contain one, two, three, four or even more polypeptide chains.

[0088] Bivalent As used herein, in the context of antigen-binding molecules, the term "bivalent" refers to an antigen-binding molecule having two antigen-binding domains. These domains may be the same or different. Therefore, a bivalent antigen-binding molecule may be monospecific or bispecific. A bivalent BBM may comprise an ABM that specifically binds to CD19 and another ABM that binds to another antigen (e.g., a component of the TCR complex).

[0089] cancerThe term "cancer" refers to a disease characterized by the uncontrolled (and often rapid) growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers are described herein, including but not limited to: leukemia, multiple myeloma, asymptomatic myeloma, Hodgkin's lymphoma, and non-Hodgkin's lymphoma, such as any of the aforementioned types of CD19-positive cancers. The term "cancerous B cell" refers to B cells that are undergoing or have undergone uncontrolled proliferation.

[0090] CD3 The term "CD3" or "differentiation cluster 3" refers to the differentiation cluster 3 co-receptor of T cell receptors. CD3 contributes to the activation of cytotoxic T cells (e.g., CD8+ naive T cells) and helper T cells (e.g., CD4+ naive T cells) and consists of four distinct chains: one CD3γ chain (e.g., Genbank accession numbers 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 CD3 chains are highly correlated cell surface proteins of the immunoglobulin superfamily containing 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, which functions to generate an activation signal in T lymphocytes. Unless explicitly stated otherwise, reference to CD3 in this application may refer to the CD3 co-receptor, the CD3 co-receptor complex, or any polypeptide chain of the CD3 co-receptor complex.

[0091] CD19The term "CD19" or "differentiation cluster 19" refers to the differentiation cluster 19 protein, an antigenic determinant detectable on leukemia progenitor cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and SwissProt. For example, the amino acid sequence of human CD19 can be found as UniProt / SwissProt accession number P15391, and the nucleotide sequence encoding human CD19 can be found as accession number NM_001178098. CD19 is expressed in most B-lineage cancers, including, for example, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma. Other cell types expressing CD19 are provided in the definition of "Diseases Associated with CD19 Expression" below. It is also an early marker of B-cell progenitor cells. See, for example, Nicholson et al., 1997, Mol. Immun. [Molecular Immunology] 34(16-17):1157-1165.

[0092] chimeric antibodies The term "chimeric antibody" (or its antigen-binding fragment) refers to an antibody molecule (or its antigen-binding fragment) in which (a) the constant region or a portion thereof is altered, replaced, or replaced such that the antigen-binding site (variable region) is linked to a constant region of a different or altered type, effector function, and / or kind, or to a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug, etc.) that confers novel properties to the chimeric antibody; or (b) the variable region or a portion thereof is altered, replaced, or replaced with a variable region having a different or altered antigen specificity. For example, mouse antibodies can be modified by replacing their constant region with a constant region derived from human immunoglobulins. Due to the replacement with a human constant region, the chimeric antibody can retain its antigen-recognition specificity while exhibiting reduced antigenicity in humans compared to the original mouse antibody.

[0093] combination As used herein, “combination” means delivering two (or more) different treatments to a subject during the course of the illness, such as after the subject has been diagnosed with a disorder and before the disorder is cured or cleared, or before treatment is terminated for other reasons.

[0094] Complementarity Determinant RegionAs used herein, the term "complementarity-determining region" or "CDR" refers to the sequence of amino acids within an antibody variable region that confers antigen specificity and binding affinity. For example, generally, three CDRs exist in each heavy chain variable region (e.g., CDR-H1, CDR-H2, and CDR-H3), and three CDRs exist 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 many well-known schemes, including those described in the following literature: Kabat et al., 1991, "Sequences of Proteins of Immunological Interest," 5th edition, National Institutes of Health, Department of Public Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., 1997, JMB 273:927-948 (“Josiah” numbering scheme), and 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 Cabat and Jothia, the CDR consists 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). The CDR regions of the antibody can be determined using the IMGT / DomainGap Align procedure according to IMGT.

[0095] parallel The term “parallel” is not limited to administering a therapy (e.g., a prophylactic or therapeutic agent) at exactly the same time, but rather means administering a pharmaceutical composition containing an antigen-binding molecule disclosed herein to a subject in a sequence and at time intervals such that the molecule can work in conjunction with one or more other therapies to provide an increased benefit (compared to if they were administered in other ways).

[0096] Conservative sequence modification The term "conserved 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 Fc region). Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the binding molecule using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conserved amino acid substitution is a substitution in which an amino acid residue is replaced by 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 with 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). Therefore, one or more amino acid residues in a binding molecule can be replaced with other amino acid residues from the same side chain family, and the altered binding molecule's binding to a target molecule and / or efficient heterodimerization and / or effector function can be tested.

[0097] bisomal antibodiesAs used herein, the term "bimeric antibody" 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) on the same polypeptide chain (VH-VL, where VH is located at the N-terminus or C-terminus of VL). Unlike typical scFvs (where 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), bimeric antibodies typically contain a linker, but this linker is too short to allow the VH and VL domains on the same chain to pair, thus forcing the VH and VL domains to pair with a complementary domain of another chain, resulting in two antigen-binding sites. More comprehensive descriptions of bisomatic antibodies can be found in the following literature: for example, EP 404,097; WO 93 / 11161; and Hollinger et al., 1993, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 90:6444-6448.

[0098] dsFv The term "dsFv" refers to a disulfide-stabilized Fv fragment. In dsFv, VH and VL are linked by interdomain disulfide bonds. To generate such molecules, one amino acid in each of the framework regions of VH and VL is mutated to a cysteine ​​residue, which in turn forms a stable interchain disulfide bond. Typically, position 44 in VH and position 100 in VL are mutated to cysteine ​​residues. 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 interchain disulfide bonds rather than linker peptides) or scdsFv (molecules in which VH and VL are linked by both linkers and interchain disulfide bonds).

[0099] Effector FunctionThe term "effective function" refers to the activity of an antibody molecule mediated by binding through the antibody's domain rather than the antigen-binding domain, typically mediated by the binding of effector molecules. Effector functions include complement-mediated effector functions, mediated by, for example, the binding of the C1 component of the complement to the antibody. Complement activation is important in the opsonization and lysis of cellular pathogens. Complement activation also stimulates inflammatory responses and can participate in autoimmune hypersensitivity responses. Effector functions also include Fc receptor (FcR)-mediated effector functions, which can be triggered by the binding of the antibody's constant domain to the Fc receptor (FcR). Antibody binding to Fc receptors on cell surfaces 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 cytotoxic cells (referred to as antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production. The effector function of an antibody can be altered, for example, by increasing or decreasing the antibody's affinity for effector molecules such as Fc receptors or complement components. Binding affinity is typically altered by modifying the binding site of the effector molecule, and in this case, it is appropriate to target the site of interest and modify at least a portion of the site in a suitable manner. It is also envisioned that altering the binding site on an antibody against an effector molecule does not need to significantly change the overall binding affinity, but can alter the geometry of the interaction, leading to the disenfranchisement of the effector mechanism, as in nonproductive binding. Further envisioning is that effector function can also be altered by modifying sites that do not directly participate in effector molecule binding but participate in effector function in other ways.

[0100] Epitope An epitope, or antigenic determinant, is a portion of an antigen that can be recognized by an antibody or other antigen-binding motif as described herein. Epitopes can be linear or conformational.

[0101] Fab As used herein, “Fab” or “Fab region” refers to a polypeptide region containing the VH, CH1, VL, and CL immunoglobulin domains. These terms may refer to this region alone or in the context of the antigen-binding molecules disclosed herein.

[0102] The Fab domain is formed through the association of the CH1 domain attached to the VH domain and 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. Disulfide bonds between the two constant domains can further stabilize the Fab domain.

[0103] Fab regions can be generated through proteolytic cleavage of immunoglobulin molecules (e.g., using enzymes such as papain) or through recombinant expression. In native immunoglobulin molecules, Fab regions are formed by the association of two distinct polypeptide chains (e.g., VH-CH1 on one chain with VL-CL on another). These Fab regions are typically recombinantly expressed, typically on both polypeptide chains, although single-chain Fab regions are also considered herein.

[0104] Fc structural domain The term "Fc domain" refers to a pair of associated Fc regions. These two Fc regions dimerize to produce an Fc domain. The two Fc regions in an Fc domain can be identical (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").

[0105] Fc area As used herein, the term "Fc region" or "Fc chain" refers to a polypeptide containing the CH2-CH3 domain of an IgG molecule, and in some cases, includes a hinge. In the EU designation of human IgG1, the CH2-CH3 domain contains amino acids 231 to 447, and the hinge is amino acids 216 to 230. Therefore, the definition of "Fc region" includes amino acids 231-447 (CH2-CH3) or 216-447 (hinge-CH2-CH3), or fragments thereof. In this context, an "Fc fragment" may contain fewer amino acids from one or both of the N-terminus and C-terminus, but still retains the ability to form a dimer with another Fc region, an ability detectable using standard methods (typically size-based) (e.g., non-denaturing chromatography, size exclusion chromatography). The human IgG Fc region has a specific use in this disclosure and may be an Fc region derived from human IgG1, IgG2, or IgG4.

[0106] FvThe term "Fv" refers to the smallest antibody fragment that can be derived from an immunoglobulin and contains a complete target recognition and binding site. This region consists of a dimer (VH-VL dimer) consisting of a heavy chain and a light chain variable domain tightly, 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 target binding specificity to the antibody. However, in some cases, even a single variable domain (or half of an Fv containing only the three CDRs specific to the target) can have the ability to recognize and bind to the target. The reference to the VH-VL dimer herein is not intended to convey any particular configuration. By way of example, but not limitation, the VH and VL can combine in any configuration described herein to form a hapten, or they can exist separately on individual haptens and combine together when the individual haptens associate to form an antigen-binding domain, such as the TBM disclosed herein. When present on a single polypeptide chain (e.g., scFv), the VH is the N-terminus or C-terminus of the VL.

[0107] Haptop The term "half-antibody" refers to a molecule that contains at least one ABM or an ABM chain and can associate with another molecule containing ABM or an ABM chain via, for example, disulfide bridging or molecular interactions (e.g., the mortar-and-mortar interaction between Fc heterodimers). A half-antibody may consist of one or more polypeptide chains (e.g., two polypeptide chains of Fab). In embodiments, the half-antibody contains an Fc region.

[0108] Examples of haptens are molecules comprising both a heavy chain and a light chain of an antibody (e.g., an IgG antibody). Another example of a hapten is a molecule comprising a first polypeptide and a second polypeptide, the first polypeptide comprising a VL domain and a CL domain, and the second polypeptide comprising a VH domain, a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain, wherein the VL and VH domains form an ABM. Yet another example of a hapten is a polypeptide comprising an scFv domain, a CH2 domain, and a CH3 domain.

[0109] A half antibody may include more than one ABM, such as a half antibody containing (in order from N-terminus to C-terminus) an scFv domain, a CH2 domain, a CH3 domain, and another scFv domain.

[0110] The hapten may also include an ABM chain, which, when associated with another ABM chain in another hapten, forms a complete ABM.

[0111] Therefore, an MBM can contain one, more typically two, or even more than two halves, and a halves can contain one or more ABMs or one or more ABM chains.

[0112] In some MBMs, the first hapten will associate with the second hapten, for example, through heterodimerization. In other MBMs, the first hapten will be covalently linked to the second hapten, for example, through disulfide bridges or chemical cross-linking. In still other MBMs, the first hapten will associate with the second hapten through covalent attachment and non-covalent interactions, such as disulfide bridges and mortise-and-tenon interactions.

[0113] The term "half-antibody" is intended for descriptive purposes only and does not indicate a specific configuration or method of manufacture. The descriptions of half-antibodies as "first" half-antibody, "second" half-antibody, "left" half-antibody, "right" half-antibody, etc., are merely for convenience and descriptive purposes.

[0114] hexavalent In the context of antigen-binding molecules (e.g., TBMs), 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 bound to CD19, two antigen-binding domains bound to a component of the TCR complex, and one antigen-binding domain bound to CD2 or TAA, or three antigen-binding domains bound to CD19, two antigen-binding domains bound to CD2 or TAA, and one antigen-binding domain bound to a component of the TCR complex) are within the scope of this disclosure, the hexavalent TBMs of this disclosure generally have three pairs of antigen-binding domains, each bound to the same antigen. Examples of hexavalent TBMs are schematically illustrated in… Figure 1U-1V middle.

[0115] In the context of a mortar and pestle structure, a "mortar" refers to at least one amino acid side chain that is recessed into the interface of the first Fc chain and thus can be positioned in a complementary "pestle" on the adjacent interface surface of the second Fc chain, thereby stabilizing the Fc heterodimer and thus being more conducive to the formation of the Fc heterodimer, for example, compared to the Fc homodimer.

[0116] Host cells or recombinant host cellsThe term "host cell" or "recombinant host cell" refers, for example, a genetically engineered cell through the introduction of heterologous nucleic acids. It should be understood that this term refers not only to a specific subject cell but also to its progeny. Because certain modifications can occur in offspring due to mutations or environmental influences, such progeny may differ in fact from the parent cell but are still included within the scope of the term "host cell" as used herein. Host cells can transiently carry heterologous nucleic acids, for example, on an extrachromosomal heterologous expression vector, or stably carry heterologous nucleic acids, for example, by integrating them into the host cell genome. For the purpose of expressing antigen-binding molecules, the host cell can be a mammalian-derived cell line or a cell line with mammalian-like characteristics, such as monkey kidney cells (COS, e.g., COS-1, COS-7), HEK293, juvenile rat kidney (BHK, e.g., BHK21), Chinese hamster ovary (CHO), NSO, PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., Hep G2), SP2 / 0, HeLa, Martha's bovine kidney (MDBK), myeloma and lymphoma cells, or derivatives thereof and / or engineered variants. Engineered variants include, for example, glycan-profile modified and / or site-specific integration site derivatives.

[0117] Human antibodies As used herein, the term "human antibody" includes antibodies having a variable region, wherein both the frame region and the CDR region are derived from human-derived sequences. Furthermore, 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 a common frame sequence derived from human frame sequence analysis, for example, as described in 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., Kabat numbering scheme, Josiah numbering scheme, or a combination of Kabat and Josiah numbering schemes) (see, for example, Lazikani et al., 1997, J. Mol. Bio. 273:927-948; Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition, NIH Publication No. 91-3242; Chothia et al., 1987, J. Mol. Bio. 196:901-917; Chothia et al., 1989, Nature 342:877-883).

[0118] Human antibodies may include amino acid residues not encoded by human sequences (e.g., by introducing mutations to promote stability or production through random or site-specific mutagenesis in vitro, or through somatic mutations or conserved substitutions in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies whose CDR sequences derived from a lineage of another mammalian species (such as a mouse) have been grafted onto a human frame sequence.

[0119] Humanization The “humanized” form of the term nonhuman (e.g., mouse) antibody is a chimeric antibody containing a minimal sequence derived from a nonhuman immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (receptor antibody) in which residues from the receptor hypervariable region are replaced by residues from a hypervariable region (donor antibody) of a nonhuman species (such as a mouse, rat, rabbit, or nonhuman primate) with the desired specificity, affinity, and capacity. In some cases, framework region (FR) residues of a human immunoglobulin are replaced by corresponding nonhuman residues. Furthermore, humanized antibodies may contain residues not found in the receptor or 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, typically two, variable domains, wherein all or substantially all hypervariable loops correspond to those of nonhuman immunoglobulins, and all or substantially all FRs are those of the human immunoglobulin lo sequence. Optionally, a humanized antibody also contains an immunoglobulin constant region (Fc), typically at least a portion of the 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 commentaries and their cited references: 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.

[0120] pestleIn the context of the mortar and pestle structure, a "mortar" refers to at least one amino acid side chain that protrudes from the surface of the first Fc chain and is thus positioned in a complementary "mortar" at the interface of the second Fc chain, thereby stabilizing the Fc heterodimer and thus favoring the formation of the Fc heterodimer, for example, compared to the Fc homodimer.

[0121] Knobs and holes (or knocks-in-holes): A mechanism of Fc heterodimerization is commonly referred to in the art as “knobs and holes,” “knobs-in-holes,” or “knobs-in-holes.” These terms refer to amino acid mutations that produce spatial effects that favor the formation of Fc heterodimers (as opposed to Fc homodimers), as described below, for example, Ridgway et al., 1996, Protein Engineering 9(7):617; Atwell et al., 1997, J. Mol. Biol. 270:26; and U.S. Patent 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.

[0122] Monoclonal antibodies As used herein, the term "monoclonal antibody" refers to polypeptides derived from the same genetic source, including antibodies, antibody fragments, molecules (including MBM), etc.

[0123] unit price As used herein, in the context of antigen-binding molecules, the term "monovalent" refers to an antigen-binding molecule having a single antigen-binding domain.

[0124] Multispecific binding molecules The term "multispecific binding molecule" or "MBM" refers to a molecule that specifically binds to at least two antigens and contains two or more antigen-binding domains. These antigen-binding domains can each be independently an antibody fragment (e.g., scFv, Fab, nanobody), a ligand, or a non-antibody-derived conjugate (e.g., fibronectin, Fynomer, DARPin).

[0125] Mutation or modification In the context of the primary amino acid sequence of a polypeptide, the terms "modification" and "mutation" refer to amino acid substitution, insertion, and / or deletion relative to the reference polypeptide sequence. Additionally, the term "modification" further encompasses alterations to amino acid residues, such as through chemical conjugation (e.g., chemical conjugation of a pharmaceutical or polyethylene glycol moiety) or post-translational modifications (e.g., glycosylation).

[0126] Nucleic Acids The term "nucleic acid" is used interchangeably with the term "polynucleotide" herein and refers to deoxyribonucleotides or ribonucleotides and their polymers in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, or non-natural, have similar binding properties to a reference nucleic acid, and are metabolized in a manner similar to that of a reference nucleotide. Examples of such analogs include, but are not limited to, thiophosphates, aminophosphates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide-nucleic acids (PNAs).

[0127] Unless otherwise stated, a particular nucleic acid sequence also implicitly encompasses its conserved variants (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly indicated sequences. Specifically, as detailed below, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of 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).

[0128] Operable connection 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 fusion proteins or other polypeptides, "operably linked" means linking two or more amino acid segments to produce a functional polypeptide. For example, in the context of antigen-binding molecules, individual ABMs (or chains of ABMs) can be operably linked via peptide linker sequences. In the context of nucleic acids encoding fusion proteins, such as polypeptide chains of antigen-binding molecules, "operably linked" means linking two nucleic acids such that the amino acid sequences encoded by the two nucleic acids remain within the frame. In the context of transcriptional regulation, the term refers to a functional relationship between a transcriptional regulatory sequence and a transcriptional sequence. For example, if a promoter or enhancer sequence stimulates or regulates transcription of a coding sequence in a suitable host cell or other expression system, the promoter or enhancer sequence is operably linked to the coding sequence.

[0129] PentavalentIn the context of antigen-binding molecules (e.g., TBMs), the term "pentavalent" as used herein refers to an antigen-binding molecule having five antigen-binding domains. The pentavalent TBMs disclosed herein typically 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 illustrated in… Figure 1T middle.

[0130] polypeptides and proteins The terms “peptide” and “protein” are used interchangeably herein and refer to polymers of amino acid residues. The term encompasses 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 both naturally occurring and non-naturally occurring amino acid polymers. Additionally, the term encompasses amino acid polymers derivatized, for example, by synthetic derivatization, glycosylation, PEGylation, cyclic arrangement, cyclization, linker attachment to other molecules, fusion into proteins or protein domains, and addition of peptide tags or peptide labels.

[0131] Identification As used herein, the term “identification” refers to the discovery of and interaction (e.g., binding) with an epitope of an ABM.

[0132] Sequence identityThe sequence identity of two similar sequences (e.g., antibody variable domains) can be measured by algorithms such as those of Smith, TF, and Waterman, MS (1981), "Comparison of Biosequences," Adv. Appl. Math. 2:482; Needleman, SB, and Wunsch, CD. (1970), "A General Method Applicable to the Search for Similarities in the Amino Acid Sequence of Two Proteins," J. Mol. Biol. 48:443; and Pearson, WR, and Lipman, DJ (1988), "Improved Tools for Biological Sequence." Comparison [An Improved Biological Sequence Alignment Tool], Proc. Natl. Acad. Sci. (USA) 85:2444 [A Search for Similarity Method]; or Altschul, SF 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 aforementioned 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.

[0133] Optionally, identity is determined over a region of at least about 50 nucleotides (or at least about 10 amino acids in the case of a peptide or polypeptide), 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., the VH or VL of an antibody). Unless otherwise stated, sequence identity between two sequences is determined over the entire length of the shorter of the two sequences.

[0134] Single-chain Fab or scFab The terms "single-chain Fab" and "scFab" refer to a polypeptide comprising 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 direction from the N-terminus to the C-terminus: 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 may be a polypeptide having at least 30 amino acids, for example, between 32 and 50 amino acids. The single-chain Fab is stabilized by a native disulfide bond between the CL and CH1 domains.

[0135] Single-chain Fv or scFv As used herein, the term "single-chain Fv" or "scFv" refers to an antibody fragment containing both VH and VL domains of the antibody, wherein these domains are contained within a single polypeptide chain. The Fv polypeptide may further include 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*, Vol. 113, edited by Rosenburg and Moore, 1994, Springer-Verlag, New York, pp. 269–315.

[0136] Specific (or selective) bindingThe term "specific (or selective) binding" 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 biological products. This binding reaction may, but does not need to, be mediated by an antibody or antibody fragment, but may also be mediated by any type of ABM (such as ligands, DARPin, etc.) as described in Section 7.3. ABMs typically also have a size of less than 5 x 10⁻⁶. -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 has a dissociation rate constant (KD) (koff / kon) and binds to the target antigen with an affinity at least twice that of binding to a nonspecific antigen (e.g., HSA). Binding affinity can be measured using a Biacore, SPR, or BLI assay. The term "specific binding" 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 said antigen in one or more other species. Therefore, such cross-species reactivity does not, in itself, alter the classification of the antigen-binding module as a "specific" binder. In some embodiments, an antigen-binding module that specifically binds to a human antigen exhibits cross-species reactivity with one or more non-human mammalian species, such as primate species (including, but not limited to, one or more of the following: *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.

[0137] SubjectsThe term "subject" includes both humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise specified, the terms "patient" or "subject" are used interchangeably herein.

[0138] cascading of VH domains As used herein, the term "tandem of VH domains (or VHs)" refers to a string of VH domains consisting of many identical VH domains of an antibody. The C-terminus of each of the VH domains (except 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 two VH domains, and in specific embodiments, the antigen-binding molecule has 3, 4, 5, 6, 7, 8, 9, or 10 VH domains. Tandems of VHs can be generated by using a recombination method (with or without a linker) to link the coding nucleic acids of each VH domain in the desired order (this 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.

[0139] Serialization of VL domains As used herein, the term "tandem of VL domains (or VLs)" refers to a string of VL domains consisting of many identical VL domains of an antibody. The C-terminus of each VL domain (except 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 two VL domains, and in specific embodiments, the antigen-binding molecule has 3, 4, 5, 6, 7, 8, 9, or 10 VL domains. Tandems of VLs can be generated by using a recombination method (with or without a linker) to link the coding nucleic acids of each VL domain in a desired order (this 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.

[0140] target antigen As used in this article, “target antigen” refers to a molecule that is non-covalently, reversibly and specifically bound by an antigen-binding domain.

[0141] QuadrivalentIn the context of antigen-binding molecules (e.g., BBM or TBM), the term "tetravalent" as used herein refers to an antigen-binding molecule having four antigen-binding domains. The tetravalent TBMs disclosed herein typically 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 a TCR complex and CD2 or TAA). Examples of tetravalent BBMs are schematically illustrated in… Figure 1AA-1AH In the middle, and an example of tetravalent TBM is schematically shown in Figure 2Q-2S middle.

[0142] Therapeutic effective dose "Therapeutic effective dose" refers to the amount that effectively achieves the desired therapeutic outcome at the required dose and duration.

[0143] Treatment (Treat, Treatment, and Treating) As used herein, the terms "treat," "treatment," and "treating" refer to a reduction or relief of the progression, severity, and / or duration of a disease or disorder (e.g., a proliferative disorder) resulting from administration of one or more CD19-binding molecules disclosed herein, or relief of one or more symptoms of the disorder (e.g., one or more identifiable symptoms). In some embodiments, the terms "treat," "treatment," and "treating" refer to improving at least one measurable physical parameter of the disorder, such as tumor growth, which is not necessarily identifiable by the patient. In other embodiments, the terms "treat," "treatment," and "treating" refer to inhibiting the progression of the disorder physically, for example, by stabilizing identifiable symptoms, physiologically, for example, by stabilizing physical parameters, or by both. In some embodiments, the terms "treat," "treatment," and "treating" may refer to reducing or stabilizing tumor size or cancer cell count.

[0144] Trispecific binding molecules The term "triple-specific binding molecule" or "TBM" refers to a molecule that specifically binds to three antigens and contains three or more antigen-binding domains. The TBM disclosed herein contains at least one antigen-binding domain specific to CD19, at least one antigen-binding domain specific to a component of the TCR complex, and at least one antigen-binding domain specific to CD2 or TAA. The antigen-binding domains can each independently be an antibody fragment (e.g., scFv, Fab, nanobody), a ligand, or a non-antibody-derived conjugate (e.g., fibronectin, Fynomer, DARPin). Representative TBMs are shown in Figure 1. TBMs can contain one, two, three, four, or even more polypeptide chains. For example, Figure 1MThe TBM shown contains a single polypeptide chain, which includes three scFvs and a single polypeptide chain linked by an ABM linker. Figure 1K The TBM shown contains two polypeptide chains, each containing three scFvs linked by Fc domains, etc. Figure 1J The TBM shown contains three polypeptide chains forming scFv, ligands, and Fab connected by Fc domains, etc. Figure 1C The TBM shown contains four polypeptide chains, forming three Fabs connected by Fc domains and other structures. Figure 1U The TBM shown contains six polypeptide chains, forming four Fabs and two scFvs connected by Fc domains, etc.

[0145] Trivalent In the context of antigen-binding molecules (e.g., MBMs), the term "trivalent" as used herein refers to an antigen-binding molecule having three antigen-binding domains. The MBMs disclosed herein are typically bispecific or trispecific. Bispecific BBMs specifically bind to components of the CD19 and TCR complexes. Trispecific TBMs specifically bind to CD19, components of the TCR complex, and CD2 or TAA. Thus, a trivalent BBM has three antigen-binding domains, two of which bind to CD19 and one to a component of the TCR, or vice versa. A TBM has three antigen-binding domains, each binding to a different antigen. Examples of trivalent BBMs are schematically shown in [illustration / illustration]. Figure 1G-1Z In the middle, and an example of trivalent TBM is schematically shown in Figure 2B-2V middle.

[0146] tumor The term "tumor" may be used interchangeably with the term "cancer" as used herein, for example, both terms cover solid and liquid tumors, such as diffuse or circulating tumors. As used herein, the terms "cancer" or "tumor" include both pre-existing and malignant cancers and tumors.

[0147] Tumor-associated antigensThe term "tumor-associated antigen" or "TAA" refers to a molecule (typically a protein, carbohydrate, lipid, or some combination thereof) expressed fully or as a fragment (e.g., MHC / peptide) on the surface of cancer cells, and it can be used to preferentially target pharmacological agents to cancer cells. In some embodiments, a TAA is a marker expressed by both normal cells and cancer cells, such as a lineage marker, such as CD19 on B cells. In some embodiments, a TAA is a cell surface molecule overexpressed in cancer cells compared to normal cells, for example, 1-fold, 2-fold, 3-fold, or more overexpression compared to normal cells. In some embodiments, a TAA is a cell surface molecule inappropriately synthesized in cancer cells, for example, a molecule containing deletions, additions, or mutations compared to molecules expressed on normal cells. In some embodiments, a TAA will be expressed 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. Therefore, the term "TAA" encompasses antigens specific to cancer cells, sometimes referred to as tumor-specific antigens ("TSA"). Although CD19 is characterized by tumor-associated antigens, the terms “tumor-associated antigen” and “TAA” are used throughout the disclosure to refer to molecules other than CD19.

[0148] variable region As used herein, “variable region” or “variable domain” refers to a region of an immunoglobulin that contains one or more Ig domains encoded by essentially any one of the Vκ, Vλ, and / or VH genes (which respectively constitute the κ, λ, and heavy chain immunoglobulin loci) and contains a CDR that confers antigen specificity. A “variable heavy domain” can pair with a “variable light domain” to form an antigen-binding domain (“ABD”) or an antigen-binding module (“ABM”). Additionally, each variable domain contains three hypervariable regions (“complementarity-determining regions”, “CDRs”) (CDR-H1, CDR-H2, CDR-H3 for variable heavy domains and CDR-L1, CDR-L2, CDR-L3 for variable light domains) and four frame (FR) regions arranged in the following order from the N-terminus to the C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0149] carrierThe term "vector" is intended to refer to a polynucleotide molecule capable of transporting another polynucleotide linked to it. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop in which an additional DNA segment can be attached. Another type of vector is a viral vector, in which the additional DNA segment can be attached to a viral genome. Some vectors are capable of autonomous replication in the host cell to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and attachable mammalian vectors). Other vectors (e.g., non-attached mammalian vectors) can integrate into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome. Furthermore, some vectors are capable of directing the expression of genes operatively linked to them. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful in recombinant DNA technology are typically in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used form of vector. However, this disclosure is intended to include other forms of expression vectors such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses) that have the same function.

[0150] VH The term "VH" refers to the variable region of the immunoglobulin heavy chain of the antibody (including the heavy chains of Fv, scFv, dsFv, or Fab).

[0151] VL The term "VL" refers to the variable region of an immunoglobulin light chain (including light chains of Fv, scFv, dsFv, or Fab).

[0152] VH-VL or VH-VL to When referring to VH-VL pairs, whether located on the same or different polypeptide chains, the terms "VH-VL" and "VH-VL pair" are used for convenience and are not intended to convey any particular orientation unless otherwise specified in the context. Therefore, an scFv containing "VH-VL" or "VH-VL pair" can have VH and VL domains in any orientation, for example, VH at the N-terminus of VL or VL at the N-terminus of VH.

[0153] 7.2. CD19 binding molecules

[0154] In one aspect, this disclosure provides CD19 binding molecules comprising single-specific and multi-specific molecules that bind to human CD19. In some embodiments, the CD19 binding molecule is a single-specific binding molecule. For example, a single-specific binding molecule may be an antibody or an antigen-binding fragment thereof (e.g., antibody fragment, scFv, dsFv, Fv, Fab, scFab, (Fab')2, or a single-domain antibody (SDAB)). In other embodiments, the CD19 binding molecule is a multi-specific (e.g., bispecific) CD19 binding molecule (e.g., a bispecific antibody).

[0155] 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 of human patients to antibodies generated in non-human subjects or antibodies derived from the expression of non-human antibody genes. Chimeric antibodies comprise 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 may have lower immunogenicity when administered to humans and are therefore more likely to be tolerated by patients. For example, one or all (e.g., one, two, or three) of the variable regions of one or more light chains of a mouse antibody (e.g., a mouse monoclonal antibody) and / or one or all (e.g., one, two, or three) of the variable regions of one or more heavy chains 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 using known recombinant DNA techniques. For example, the gene encoding the constant region of a non-human antibody molecule can be replaced with the gene encoding the 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). Other suitable techniques that can be used to generate chimeric antibodies are described, for example, in U.S. patent numbers 4,816,567, 4,978,775; 4,975,369; and 4,816,397.

[0156] The chimeric or humanized antibodies and their antigen-binding fragments disclosed herein can be prepared based on sequences of mouse monoclonal antibodies. DNA encoding heavy and light chain immunoglobulins can be obtained from mouse hybridomas of interest and engineered using standard molecular biology techniques to contain non-mouse (e.g., human) immunoglobulin sequences. For example, to generate chimeric antibodies, known methods can be used to link mouse variable regions with human constant regions (see, for example, U.S. Patent No. 4,816,567 to Cabilly et al.). To generate humanized antibodies, known methods can be used to insert mouse CDR regions into human frames. See, for example, U.S. Patent No. 5,225,539 (belonging to Winter) and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 (belonging to Queen et al.).

[0157] Humanized antibodies can be generated using a variety of known techniques, including but not limited to CDR transplantation (see, for example, European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and US Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneer or surface resurfacing (see, for example, 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 truncation (see, for example, U.S. Patent No. 5,565,332), and techniques disclosed in, for example, the following: U.S. Patent Application Publication No. US2005 / 0042664, U.S. Patent Application Publication No. US2005 / 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. [Protein Engineering], 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 Supplement):5973s-5977s (1995), Couto et al., Cancer Res., 55(8):1717-22 (1995), Sandhu JS, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994). Typically, framework residues in the framework region are replaced by corresponding residues from a CDR donor antibody to alter, for example, improve antigen binding. These framework substitutions (e.g., conserved substitutions) are identified by known methods, such as by modeling the interaction between the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison, thereby identifying aberrant framework residues at specific locations.(See, for example, Queen et al., U.S. Patent No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323).

[0158] As provided in this article, humanized antibodies or antibody fragments may contain one or more CDRs and framework regions derived from non-human immunoglobulin molecules, wherein the amino acid residues constituting the framework are wholly or mostly derived from human lineages. A variety of techniques for humanizing antibodies or antibody fragments are well known and can be performed in a manner consistent with that 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)). This method involves replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence, i.e., CDR transplantation (EP 239,400; PCT Publication No. WO 91 / 09967; and US 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 fewer than the complete human variable domain has been replaced by corresponding sequences from non-human species. Humanized antibodies are typically human antibodies in which some CDR residues and possibly some framework (FR) residues have been replaced by residues from similar sites in rodent antibodies. Humanization of antibodies and antibody fragments can also be achieved by varnishing or remodeling (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 tampering (US Patent No. 5,565,332).

[0159] The selection of human variable domains (both light and heavy chains) used for preparing humanized antibodies is to reduce antigenicity. According to the so-called "best fit" method, sequences of variable domains for rodent antibodies are screened against an entire library of known human variable domain sequences. The human sequence closest to the rodent sequence is then accepted as the human frame (FR) for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Another approach uses a specific frame derived from a common sequence of all human antibodies with 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. Immun. [Molecular Immunology] 34(16-17):1157-1165 (1997); Carter et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences], 89:4285 (1992); Presta et al., J. Immunol. [Journal of Immunology], 151:2623 (1993). In some embodiments, the framework region of the heavy chain variable region (e.g., the whole...) The four frame regions (e.g., all four frame regions) are derived from the VH4_4-59 germline sequence. In one embodiment, the frame regions may contain one, two, three, four, or five modifications, such as substitutions, such as conserved substitutions, such as substitutions of amino acids from the corresponding mouse sequence. In one embodiment, the frame regions of the light chain variable region (e.g., all four frame regions) are derived from the VK3_1.25 germline sequence. In one embodiment, the frame regions may contain one, two, three, four, or five modifications, such as substitutions, such as conserved substitutions, such as substitutions of amino acids from the corresponding mouse sequence.

[0160] In some 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 may comprise or be composed of human antibodies containing heavy or light chain variable regions that are “products” or “derived from” a specific germline sequence. Human antibodies can be identified as “products” or “derived from” human germline immunoglobulin sequences by comparing the amino acid sequence of a human antibody with that of a human germline immunoglobulin and selecting the human germline immunoglobulin sequence that is closest in sequence to the human antibody sequence (i.e., has the highest percentage of identity) (using the methods outlined herein). Human antibodies that are “products” or “derived from” a specific human germline immunoglobulin sequence may contain amino acid differences compared to the germline sequence due to, for example, naturally occurring somatic mutations or intentionally introduced site-directed mutations. However, humanized antibodies are typically at least 90% identical in amino acid sequence to the amino acid sequence encoded by human germline immunoglobulin genes and contain amino acid residues that, when compared with germline immunoglobulin amino acid sequences from other species (e.g., mouse germline sequences), identify the antibody as derived from a human sequence. In some cases, the amino acid sequence of a humanized antibody may be at least 95%, 96%, 97%, 98%, or 99% identical, or even at least 96%, 97%, 98%, or 99%, to the amino acid sequence encoded by germline immunoglobulin genes. Typically, humanized antibodies derived from a specific human germline sequence will exhibit a difference of no more than 10-20 amino acids from the amino acid sequence encoded by human germline immunoglobulin genes (prior to the introduction of any skew, pI, and ablation variants herein; i.e., prior to the introduction of the variants disclosed herein, the number of variants is typically low). In some cases, humanized antibodies can exhibit differences of no more than 5, or even no more than 4, 3, 2, or 1 amino acid from the amino acid sequence encoded by germline immunoglobulin genes (again, prior to the introduction of any offsets, pI, and ablation variants herein; i.e., prior to the introduction of the variants disclosed herein, the number of variants is generally low).

[0161] In one embodiment, the parent antibody has been affinity-matured. Structure-based methods can be used for humanization and affinity maturation, for example, as described in USSN 11 / 004,590. Selection-based methods can be used to humanize and / or mature the affinity of antibody variable regions, including but not limited to those described in the following: Wu et al., 1999, J. Mol. Biol. [Journal of Molecular Biology] 294:151-162; Baca et al., 1997, J. Biol. Chem. [Journal of Biochemistry] 272(16):10678-10684; Rosok et al., 1996, J. Biol. Chem. [Journal of Biochemistry] 271(37):22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 95:8910-8915; Krauss et al., 2003, Protein Engineering [Proceedings of the National Academy of Sciences of the United States of America] 16(10):753-759. Other humanization methods may involve the transplantation of only a portion of the CDR, including but not limited to the methods described below: USSN 09 / 810,510; Tan et al., 2002, J. Immunol. [Journal of Immunology] 169:1119-1125; De Pascalis et al., 2002, J. Immunol. [Journal of Immunology] 169:3076-3084.

[0162] In some embodiments, the CD19 binding molecule comprises an ABM (which stands for Fab). The Fab domain can be generated by proteolytic cleavage of the immunoglobulin molecule, using an enzyme such as papain, or through recombinant expression. The Fab domain typically comprises a CH1 domain attached to a VH domain, which pairs with a CL domain attached to a VL domain. In wild-type immunoglobulins, 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.

[0163] In some embodiments, the CD19 binding molecule comprises ABM (which is scFab). In embodiments, the antibody domain and adapter in the scFab fragment have one of the following orders in the direction from the N-terminus to the C-terminus: a) VH-CH1-adaptor-VL-CL, or b) VL-CL-adaptor-VH-CH1. In some cases, VL-CL-adaptor-VH-CH1 is used.

[0164] In another embodiment, the antibody domain and adapter in the scFab fragment have one of the following orders in the direction from the N-terminus to the C-terminus: a) VH-CL-adaptor-VL-CH1 or b) VL-CH1-adaptor-VH-CL.

[0165] Optionally, in the scFab fragment, in addition to the native disulfide bond between the CL and CH1 domains, the antibody heavy chain variable domain (VH) and antibody light chain variable domain (VL) are also stabilized by disulfide bonds introduced between: i) heavy chain variable domain position 44 and light chain variable domain position 100, ii) heavy chain variable domain position 105 and light chain variable domain position 43, or iii) heavy chain variable domain position 101 and light chain variable domain position 100 (according to the Cabat EU index numbering).

[0166] Such further disulfide bond stabilization of the scFab fragment is achieved by introducing disulfide bonds between the variable domains VH and VL of the single-chain Fab fragment. Techniques for introducing non-natural disulfide bridges to stabilize single-chain Fv are described in, for example, WO 94 / 029350, Rajagopal et al., 1997, Prot. Engin. 10:1453-59; Kobayashi et al., 1998, Nuclear Medicine & Biology 25:387-393; and Schmidt et al., 1999, Oncogene 18:1711-1721. In one embodiment, the optional disulfide bond between the variable domains of the scFab fragment is located between position 44 of the heavy chain variable domain and position 100 of the light chain variable domain. In one embodiment, optional disulfide bonds between the variable domains of the scFab fragment are located between the heavy chain variable domain position 105 and the light chain variable domain position 43 (numbered according to the Cabat EU index).

[0167] In some embodiments, the CD19-binding molecule comprises ABM (which is scFv). The single-chain Fv antibody fragment comprises the VH and VL domains of the antibody in a single polypeptide chain, is capable of being expressed as a single-chain polypeptide, and retains the specificity of the complete antibody from which it is derived. Typically, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the 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 linker designated L1 to L58.

[0168] Unless otherwise stated, as used herein, scFv may have VL and VH variable regions in either order relative to the N-terminus and C-terminus of the polypeptide, i.e., the scFv may contain VL-connector-VH or may contain VH-connector-VL.

[0169] To generate scFv-encoded nucleic acids, the VH and VL-encoded DNA fragments can be operatively linked to another fragment encoding a linker, such as any linker 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-stranded protein (with its VL and VH regions linked by a flexible linker) (see, for example, 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).

[0170] CD19 binding molecules may also contain ABM (which are Fv, dsFv, (Fab')2, single-domain antibodies (SDAB), VH or VL domains, or camel VHH domains (also known as nanobodies)).

[0171] CD19 binding molecules may comprise a single-domain antibody consisting of a single VH or VL domain, which exhibits sufficient affinity for CD19. In an example, the single-domain antibody is a camel VHH domain (see, for example, Riechmann, 1999, Journal of Immunological Methods 231:25-38; WO 94 / 04678).

[0172] Tables 1A and 1B (collectively, “Table 1”) list exemplary CD19 binding sequences that may be included in CD19 binding molecules. The sequences listed in Table 1A are based on the CD19 antibody NEG258.

[0173]

[0174]

[0175] 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 as those by Kabat (SEQ ID NO: 17-19 and 4-6, respectively), Josiah (SEQ ID NO: 20-22 and 7-9, respectively), or IMGT (SEQ ID NO: 23-25 ​​and 10-12, respectively), or combinations of Josiah and Kabat CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 sequences (SEQ ID NO: 14-16 and 1-3, respectively). The CD19 binding molecule may also include a light chain variable sequence (SEQ ID NO: 26) and / or a heavy chain variable sequence (SEQ ID NO: 13) of the anti-CD19 antibody NEG258 as listed in Table 1A.

[0176] The sequences listed in Table 1B are based on the CD19 antibody NEG218.

[0177]

[0178] 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 as those by Kabat (SEQ ID NO: 43-45 and 30-32, respectively), Josiah (SEQ ID NO: 46-48 and 33-35, respectively), or IMGT (SEQ ID NO: 49-51 and 36-38, respectively), or combinations of Josiah and Kabat CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 sequences (SEQ ID NO: 40-42 and 27-29, respectively). The CD19 binding molecule may also include a light chain variable sequence (SEQ ID NO: 52) and / or a heavy chain variable sequence (SEQ ID NO: 39) of the anti-CD19 antibody NEG218 as listed in Table 1B.

[0179] Other CD19-binding molecules include mutated amino acids, but their CDR regions still possess 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, wherein no more than 1, 2, 3, 4, or 5 amino acids in the CDR region are mutated when compared to the CDR sequences described in Table 1.

[0180] Other CD19-binding molecules include VH and / or VL domains, which 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 a VH and / or VL domain wherein 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 1, while retaining substantially the same therapeutic activity.

[0181] The CD19 binding molecule can be fused or chemically conjugated (including both covalent and non-covalent conjugation) to a heterologous protein or polypeptide (or fragment thereof, e.g., to a polypeptide of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids). For example, the CD19 binding molecule can be fused directly or indirectly to a detectable protein, such as an enzyme or a fluorescent protein as described in Section 7.13. Methods for fusion or conjugation of proteins, polypeptides, or peptides to antibodies or antibody fragments are known and can be used to fusion or conjugate proteins or polypeptides to the CD19 binding molecule 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 Publications Nos. WO96 / 04388 and WO 91 / 06570; Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 88:10535-10539; Zheng et al., 1995, J. Immunol. [Journal of Immunology] 154:5590-5600; and Vil et al., 1992, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 89:11337-11341.

[0182] Additional CD19-binding molecules can be generated using techniques such as gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively, “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 with higher affinity and lower dissociation rates). See also 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 Perspectives on Biotechnology] 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 molecule or fragments thereof described herein can be altered by random mutagenesis prior to recombination using error-prone PCR, random nucleotide insertion, or other methods. The polynucleotide encoding the fragment of the CD19 binding molecule described herein can recombine with one or more components, motifs, segments, parts, domains, fragments, etc. of one or more heterologous molecules.

[0183] Furthermore, CD19-binding molecules can be fused with marker sequences (such as peptides) to facilitate purification. In some embodiments, the marker amino acid sequence is a hexahistine peptide (SEQ ID NO:54), such as the markers provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA 91311), many of which are commercially available. As described in Gentz ​​et al., 1989 Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 86:821-824, hexahistine (SEQ ID NO:54) facilitates the purification of fusion proteins. Other peptide tags that can be used for purification include, but are not limited to, hemagglutinin (“HA”) tags and “flag” tags corresponding to epitopes derived from influenza hemagglutinin proteins (Wilson et al., 1984 Cell [Cell] 37:767).

[0184] 7.3. Antigen-binding module of multispecific binding molecules

[0185] Typically, one or more ABDs of an MBM contain sequences of immunoglobulin-based antigen-binding domains, such as antibody fragments or derivatives. These antibody fragments and derivatives typically include the CDR of an antibody and may include larger fragments and their derivatives, such as Fab, scFab, Fv, and scFv.

[0186] Immunoglobulin-based ABMs may contain modifications to framework residues within the VH and / or VL regions, for example, to improve the properties of MBMs containing ABM. For instance, framework modifications can be performed to reduce the immunogenicity of the MBM. One method for performing such framework modifications is to “reverse mutate” one or more framework residues of the ABM to the corresponding germline sequence. Such residues can be identified by comparing the framework sequence with the germline sequence from which the ABM is derived. To make the framework region sequence “match” the desired germline conformation, residues can be “reverse mutated” to the corresponding germline sequence, for example, through site-directed mutagenesis. MBMs with such “reverse mutated” ABMs are intended to be covered by this disclosure.

[0187] Another type of framework modification involves mutating one or more residues within the framework region or even one or more CDR regions to remove T-cell epitopes, thereby reducing the potential immunogenicity of MBM. This method is also known as "deimmunization" and is described in further detail in U.S. Patent Publication 20030153043 by Carr et al.

[0188] ABM can also be modified to have altered glycosylation, which can be useful, for example, to increase the affinity of MBM for one or more antigens. Such carbohydrate modification can be achieved, for example, by altering one or more glycosylation sites within the ABM sequence. For example, one or more amino acid substitutions can be made, which eliminates one or more variable region framework glycosylation sites, thereby deglycosylating said sites. This glycosylation-free process can increase the affinity of MBM for antigens. This method is described, for example, in U.S. Patent Nos. 5,714,350 and 6,350,861 to Co et al.

[0189] 7.3.1. Immunoglobulin-based ABM

[0190] 7.3.1.1.Fab

[0191] In some respects, ABM is a Fab domain.

[0192] For the MBM disclosed herein, it is advantageous to use a Fab heterodimerization strategy to allow the correct association of Fab domains belonging to the same ABM and minimize anomalous pairings of Fab domains belonging to different ABMs. For example, the Fab heterodimerization strategies shown in Table 2 below can be used:

[0193]

[0194] Therefore, in some embodiments, proper association between two peptides of Fab is facilitated by exchanging the VL and VH domains of Fab with each other or by exchanging the CH1 and CL domains with each other, for example, as described in WO 2009 / 080251.

[0195] Proper Fab pairing can also be facilitated by introducing one or more amino acid modifications into the CH1 domain and into the CL domain of the Fab, and / or into the VH domain and into the VL domain. The modified amino acids are typically part of the VH:VL and CH1:CL interactions, causing Fab components to preferentially pair with each other rather than with components of other Fabs.

[0196] In one embodiment, the one or more amino acid modifications are limited to conserved framework residues with 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, Josiah, and IMGT numbering schemes.

[0197] In one embodiment, the modifications introduced into the VH and CH1 and / or VL and CL domains are complementary to each other. The complementarity of the heavy-chain and light-chain interfaces can be achieved based on spatial and hydrophobic contacts, electrostatic / charge interactions, or a combination of these interactions. Complementarity between protein surfaces is extensively described in the literature using terms such as lock-and-key fit, club-and-socket structure, protrusions and cavities, donor and acceptor, all of which imply the nature of the structural and chemical matching between two contacting surfaces.

[0198] In one embodiment, the one or more introduced modifications introduce novel hydrogen bonds across the interface of the Fab component. In one embodiment, the one or more introduced modifications introduce novel salt bridges across the interface of the Fab component. Exemplary alternatives are described in WO 2014 / 150973 and WO 2014 / 082179.

[0199] In some embodiments, the Fab domain comprises a 192E substitution in the CH1 domain and 114A and 137K substitutions in the CL domain, wherein a salt bridge is introduced between the CH1 and CL domains (see Golay et al., 2016, JImmunol 196:3199-211).

[0200] In some embodiments, the Fab domain includes 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, JImmunol [Journal of Immunology] 196:3199-211).

[0201] In some embodiments, the Fab domains may include modifications in some or all of the VH, CH1, VL, and CL domains to introduce orthogonal Fab interfaces, which facilitate proper assembly of the Fab domains (Lewis et al., 2014 Nature Biotechnology 32:191-198). In one embodiment, 39K and 62E modifications are introduced in the VH domain, H172A and F174G modifications are introduced in the CH1 domain, 1R, 38D, and (36F) modifications are introduced in the VL domain, and L135Y and S176W modifications are introduced in the CL domain. In another embodiment, 39Y modification is introduced in the VH domain and 38R modification is introduced in the VL domain.

[0202] The Fab domain can also be modified to replace the natural CH1:CL disulfide bonds with engineered disulfide bonds, thereby improving the pairing efficiency of Fab components. For example, engineered disulfide bonds can be introduced by introducing 126C into the CH1 domain and 121C into the CL domain (see Mazor et al., 2015, MAbs 7:377-89).

[0203] The Fab domain can also be modified by replacing the CH1 and CL domains with alternative domains that promote proper assembly. For example, Wu et al., 2015, MAbs 7:364-76, described replacing the CH1 domain with the constant domain of the αT cell receptor and the CL domain with the β domain of the T cell receptor, and further replacing these domains with additional charge-charge interaction pairings between the VL and VH domains by introducing 38D modification into the VL domain and 39K modification into the VH domain.

[0204] The antibody-binding membrane (ABM) may 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 direction from the N-terminus to the C-terminus: 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 may 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.

[0205] In the embodiments, the antibody domains and adapters in the single-chain Fab fragments have one of the following orders in the direction from the N-terminus to the C-terminus: a) VH-CH1-adaptor-VL-CL, or b) VL-CL-adaptor-VH-CH1. In some cases, VL-CL-adaptor-VH-CH1 is used.

[0206] In another embodiment, the antibody domain and adapter in the single-chain Fab fragment have one of the following orders in the direction from the N-terminus to the C-terminus: a) VH-CL-adaptor-VL-CH1 or b) VL-CH1-adaptor-VH-CL.

[0207] Optionally, in the single-chain Fab fragment, in addition to the native disulfide bond between the CL and CH1 domains, the antibody heavy chain variable domain (VH) and antibody light chain variable domain (VL) ABM are also stabilized by disulfide bonds introduced between: i) heavy chain variable domain position 44 and light chain variable domain position 100, ii) heavy chain variable domain position 105 and light chain variable domain position 43, or iii) heavy chain variable domain position 101 and light chain variable domain position 100 (according to the Cabart EU index numbering).

[0208] In one embodiment, optional disulfide bonds between the variable domains of a single-chain Fab fragment are located between heavy chain variable domain position 44 and light chain variable domain position 100. In another embodiment, optional disulfide bonds between the variable domains of a single-chain Fab fragment are located between heavy chain variable domain position 105 and light chain variable domain position 43 (numbered according to the Cabat EU index).

[0209] 7.3.1.2.scFv

[0210] In some respects, ABM is a single-chain Fv or “scFv”. Examples of joints suitable for connecting VH and VL chains of scFV are the ABM joints identified in Section 7.4.3, such as any joint designated as L1 to L54.

[0211] In order to generate nucleic acids encoding scFv, DNA fragments encoding VH and VL are operatively linked to another fragment encoding a adapter (e.g., any ABM adapter described in Section 7.4.3) such as the amino acid sequence (Gly4–Ser)3 (SEQ ID NO: 53).

[0212] 7.3.1.3. Other immunoglobulin-based ABMs

[0213] MBM may also include ABM in the form of an immunoglobulin, such as Fv, dsFv, (Fab')2, single-domain antibody (SDAB), VH or VL domain, or camel VHH domain (also known as nanobody) in addition to Fab or scFv.

[0214] An ABM can be a single-domain antibody consisting of a single VH or VL domain, which exhibits sufficient affinity for the target. In an example, the single-domain antibody is a camel VHH domain (see, for example, Riechmann, 1999, Journal of Immunological Methods 231:25-38; WO 94 / 04678).

[0215] 7.3.2. ABM based on non-immunoglobulins

[0216] In some 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 (abbreviation for affinity multimer), anti-carrier protein (Anticalin) / lipocarrier protein, Centyrin, Kunitz domain, Adnexin, Affilin, Affitin (also known as Nonfitin), Knottin, Pronectin, Versabody, Duocalin, and Fynomer), ligands, receptors, cytokines, or chemokines).

[0217] Non-immunoglobulin scaffolds that can be used for MBM include those listed below: Mintz and Crea, 2013, Bioprocess International, 11(2):40-48, Tables 3 and 4; Vazquez-Lombardi et al., 2015, Drug Discovery Today, 20(10):1271-83, Figures 1, Table 1 and Figure I; Skrlec et al., 2015, Trends in Biotechnology, 33(7):408-18, Tables 1 and Column 2. 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, “Scaffold Disclosures”). In specific embodiments, disclosures relating to Adnexin scaffold disclosures are incorporated by reference. In another embodiment, disclosures relating to Avimer scaffold disclosures are incorporated by reference. In another embodiment, disclosures relating to Affibody scaffold disclosures are incorporated by reference. In yet another embodiment, disclosures relating to anticarrier proteins scaffold disclosures are incorporated by reference. In yet another embodiment, disclosures relating to DARPin scaffold disclosures are incorporated by reference. In yet another embodiment, disclosures relating to Kunitz domain scaffolds are incorporated by reference. In yet another embodiment, disclosures relating to knottin scaffolds are incorporated by reference. In yet another embodiment, disclosures relating to pronectin scaffolds are incorporated by reference. In yet another embodiment, disclosures relating to nanofitin scaffolds are incorporated by reference. In yet another embodiment, disclosures relating to affilin scaffolds are incorporated by reference. In yet another embodiment, disclosures relating to adnectin scaffolds are incorporated by reference. In yet another embodiment, disclosures relating to ABM scaffolds are incorporated by reference. In yet another embodiment, disclosures relating to adhiron scaffolds are incorporated by reference. In yet another embodiment, disclosures relating to affimer scaffolds are incorporated by reference. In yet another embodiment, disclosures relating to Alphabody scaffolds are incorporated by reference.In yet another embodiment, disclosures relating to the Armadillo Repeat Protein scaffold are incorporated by reference. In yet another embodiment, disclosures relating to the Atrimer / Tetranectin scaffold are incorporated by reference. In yet another embodiment, disclosures relating to the Obody / OB-fold scaffold are incorporated by reference. In yet another embodiment, disclosures relating to the Centyrin scaffold are incorporated by reference. In yet another embodiment, disclosures relating to the Repebody scaffold are incorporated by reference. In yet another embodiment, disclosures relating to the anti-carrier protein scaffold are incorporated by reference. In yet another embodiment, disclosures relating to the Atrimer scaffold are incorporated by reference. In yet another embodiment, disclosures relating to the bicyclic peptide scaffold are incorporated by reference. In yet another embodiment, disclosures relating to the cys-knot scaffold are incorporated by reference. In yet another embodiment, disclosures relating to the Fn3 scaffold (including adenonectin, Centyrin, Pronectin, and Tn3) are incorporated by reference.

[0218] In embodiments, the ABM may be a designed anchor protein repeat sequence protein (“DARPin”). DARPin is an antibody mimic protein that typically exhibits high specificity and high affinity for target protein binding. They are generally genetically engineered and derived from natural anchor proteins and consist of at least three, typically four, or five repeat motifs from these proteins. For tetra- or penta-repeating DARPin, their molecular weights are approximately 14 or 18 kDa (kilodaltons), respectively. Examples of DARPin 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 / 0030596A1.

[0219] In another embodiment, ABM can be an Affibody. An Affibody is a well-known affinity protein derived from an IgG-binding domain of staphylococcal protein A, based on a 58-amino acid residue protein domain.

[0220] In another embodiment, the ABM can be an anticarrier protein. Anticarrier proteins are well-known and refer to another antibody mimicry technique where the binding specificity is derived from a lipid carrier protein. Anticarrier proteins can also be formatted as dual-targeting proteins, called duocalin.

[0221] In another embodiment, ABM can be a Versabody. Versabody is well known and refers to another antibody mimicry technology. They are small proteins of 3-5 kDa with >15% cysteine, forming a high disulfide bond density scaffold that replaces the hydrophobic core of a typical protein.

[0222] Other non-immunoglobulin ABMs include “A” domain oligomers (also known as Avimers) (see, for example, U.S. Patent Application Publications 2005 / 0164301, 2005 / 0048512, and 2004 / 017576), Fn3-based protein scaffolds (see, for example, U.S. Patent Application Publication 2003 / 0170753), VASP peptides, avian pancreatic polypeptides (aPP), tetraconnectins (CTLD3-based), affililin (γB-based crystal protein / ubiquitin), knotting proteins, SH3 domains, PDZ domains, tendamistat, neocarzinostatin, protein A domains, lipid transport proteins, transferrin, or Kunitz domains. In one aspect, ABMs used to construct MBMs comprise fibronectin-based scaffolds as shown in WO 2011 / 130324.

[0223] In addition, in some respects, ABM contains a ligand-binding domain of the receptor or a receptor-binding domain of the ligand.

[0224] 7.4. Connector

[0225] It is anticipated that the CD19 binding molecule may, in some cases, comprise directly interconnected pairs of ABM or ABM chains (e.g., the VH-CH1 or VL-CL components of Fab), for example, as a fusion protein without a linker. For example, the CD19 binding molecule may contain a linker portion connecting individual ABM or ABM chains. The use of a linker portion can improve target binding, for example, by increasing the flexibility of ABM in the CD19 binding molecule and thus reducing steric hindrance. ABM or ABM chains can be interconnected via, for example, Fc domains (each Fc domain representing a pair of associated Fc regions) and / or ABM linkers. The use of Fc domains will typically require the use of hinge regions as linkers of ABM or ABM chains for optimal antigen binding. Therefore, the term "linker" encompasses, but is not limited to, Fc regions, Fc domains, and hinge regions.

[0226] Linkers can be selected or modified to, for example, increase or decrease the biological half-life of CD19-binding molecules. 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 containing said fragment has impaired staphylococcal protein A (SpA) binding compared to 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, CD19-binding molecules can be modified to increase their biological half-life. For example, one or more of the following mutations can be introduced: T252L, T254S, and 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 modified within the CH1 or CL region to contain a salvage receptor binding epitope with two loops containing the CH2 domain of the Fc region derived from IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 to Presta et al.

[0227] Examples of Fc structural domains (formed by pairing two Fc regions), hinge regions, and ABM joints are described in Sections 7.4.1, 7.4.2, and 7.4.3, respectively.

[0228] 7.4.1. Fc structural domain

[0229] The CD19 binding molecule may include an Fc domain derived from any suitable species. In one embodiment, the Fc domain is derived from a human Fc domain.

[0230] 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.

[0231] The Fc domain comprises two polypeptide chains, each referred to as a heavy chain Fc region. These two heavy chain Fc regions dimerize to generate the Fc domain. These two Fc regions within the Fc domain may be identical or different from each other. In natural antibodies, the Fc regions are typically identical, but for the purpose of generating the multispecific binding molecules disclosed herein, the Fc regions may advantageously be different to allow heterodimerization, as described in Section 7.4.1.5 below.

[0232] Typically, each heavy chain Fc region contains or consists of two or three heavy chain constant structural domains.

[0233] In natural antibodies, the heavy chain Fc region of IgA, IgD, and IgG consists of two heavy chain constant domains (CH2 and CH3), while the Fc region of IgE and IgM consists of three heavy chain constant domains (CH2, CH3, and CH4). These antibodies dimerize to produce the Fc domain.

[0234] In this disclosure, the heavy chain Fc region may contain heavy chain constant domains from one or more different types of antibodies (e.g., one, two, or three different types).

[0235] In one embodiment, the heavy chain Fc region comprises 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:

[0236] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP(SEQ ID NO:1109).

[0237] In some embodiments, the CD19 binding molecule disclosed herein comprises an Fc region whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:1109 modified with one or more substitutions as described in Section 7.4.1 and its subsections.

[0238] In one embodiment, the heavy chain Fc region includes CH2 and CH3 domains derived from IgG2.

[0239] In one embodiment, the heavy chain Fc region includes CH2 and CH3 domains derived from IgG3.

[0240] In one embodiment, the heavy chain Fc region includes CH2 and CH3 domains derived from IgG4.

[0241] In one embodiment, the heavy chain Fc region contains a CH4 domain from IgM. The IgM CH4 domain is typically located at the C-terminus of the CH3 domain.

[0242] In one embodiment, the heavy chain Fc region includes CH2 and CH3 domains derived from IgG and a CH4 domain derived from IgM.

[0243] It will be appreciated that the heavy chain constant domain used to generate the heavy chain Fc region of the CD19 binding molecule of this disclosure may include variants of the naturally occurring constant domain 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 this disclosure contains at least one constant domain that differs sequentially 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 structural domain is at least 99% identical or similar. Exemplary Fc variants are described in sections 7.4.1.1 through 7.4.1.5 below.

[0244] IgM and IgA are naturally occurring covalent polymers of common H2L2 antibody units in humans. IgM exists as a pentamer when a J-chain is incorporated, or as a hexamer when a J-chain is absent. IgA exists as both a monomer and a dimer. The heavy chains of IgM and IgA have an 18-amino acid extension to a constant C-terminal domain, referred to as a tailpiece. The tailpiece includes cysteine ​​residues that form disulfide bonds between the heavy chains in the polymer and is believed to play an important role in polymerization. The tailpiece also contains glycosylation sites. In some embodiments, the CD19-binding molecules disclosed herein do not include a tailpiece.

[0245] The Fc domain incorporated into the CD19 binding molecule disclosed herein may include 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 antigen-dependent cytotoxicity. Furthermore, the CD19 binding molecule may be chemically modified (e.g., one or more chemical moieties may be attached to the CD19 binding molecule) or modified to alter its glycosylation, thereby again altering one or more functional properties of the CD19 binding molecule.

[0246] Effector functions of antibody molecules include complement-mediated effector functions, which are mediated by, for example, the binding of the C1 component of complement to the antibody. Complement activation is important in opsonization and direct lysis of pathogens. Additionally, it stimulates inflammatory responses by recruiting and activating phagocytes to sites of complement activation. Effector functions also include Fc receptor (FcR)-mediated effector functions, which can be triggered by the binding of the constant domain of the antibody to the Fc receptor (FcR). Antigen-antibody complex-mediated crosslinking of Fc receptors on the surface of effector cells triggers a wide range of important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, cytotoxic cell lysis of antibody-coated target cells (referred to as antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transport, and control of immunoglobulin production.

[0247] The Fc region can be modified by replacing at least one amino acid residue with a different amino acid residue to alter the effector function. For example, one or more amino acids can be replaced with different amino acid residues, giving the Fc region an altered affinity for effector ligands. The effector ligand with altered affinity can be, for example, an Fc receptor or a C1 component of complement. This method is described, for example, by Winter et al. in 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. Allotype amino acid residues include, but are not limited to, constant regions of the heavy chains of IgG1, IgG2, and IgG3 subclasses and constant regions of the light chains of the κ allotype, as described in Jefferis et al., 2009, MAbs, 1:332-338.

[0248] The Fc region can also be modified to “silence” the effector functions, for example, reducing or eliminating the ability of CD19-binding molecules to mediate antibody-dependent cytotoxicity (ADCC) and / or antibody-dependent phagocytosis (ADCP). This can be achieved, for example, by introducing mutations into the Fc region. Such mutations have been described in the art: LALA and N297A (Strohl, 2009, Curr. Opin. Biotechnol. [Current Biotechnol] 20(6):685-691); and D265A (Baudino et al., 2008, J. Immunol. [Journal of Immunology] 181:6664-69; Strohl, ibid.). Examples of Fc-silencing IgG1 antibodies include the so-called LALA mutant, which contains L234A and L235A mutations in the IgG1 Fc amino acid sequence. Another example of silencing IgG1 antibodies includes the D265A mutation. Another silenced IgG1 antibody contains a so-called DAPA mutant, which includes the D265A and P329A mutations in the IgG1 Fc amino acid sequence. Another silenced IgG1 antibody contains the N297A mutation, which results in a glycosylated / non-glycosylated antibody.

[0249] The Fc region can be modified to enhance the ability of CD19-binding molecules containing the Fc region to mediate antibody-dependent cytotoxicity (ADCC) and / or antibody-dependent phagocytosis (ADCP), for example, by modifying one or more amino acid residues to increase the affinity of CD19-binding molecules for activated Fcγ receptors or decrease the affinity of CD19-binding molecules for inhibitory Fcγ receptors. Human activated Fcγ receptors include FcγRIa, FcγRIIa, FcγRIIIa, and FcγRIIIb, and human inhibitory Fcγ receptors include FcγRIIb. This approach is described, for example, by Presta in PCT Publication WO 00 / 42072. Furthermore, binding sites on human IgG1 for FcγR1, 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 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 are numbered via EU).

[0250] The Fc region can also be modified to enhance the ability of CD19-binding molecules to mediate ADCC and / or ADCP, for example, by modifying one or more amino acids to increase the affinity of CD19-binding molecules for activated receptors that typically do not recognize the parental CD19-binding molecule, such as FcαRI. This method is described, for example, in Borrok et al., 2015, mAbs.7(4):743-751.

[0251] Therefore, in some aspects, the CD19 binding molecules disclosed herein may include Fc domains having altered effector functions (e.g., but not limited to binding to Fc receptors, such as FcRn or leukocyte receptors (e.g., as described above or in Section 7.4.1.1), binding to complement (e.g., as described above or in Section 7.4.1.2), modified disulfide bond structures (e.g., as described above or in Section 7.4.1.3), or altered glycosylation patterns (e.g., as described above or in Section 7.4.1.4)). The Fc domains may also be modified to include modifications that improve the manufacturability of asymmetric CD19 binding molecules, for example by allowing heterodimerization (where heterodimerization is the preferential pairing of different Fc regions relative to the same Fc region). Heterodimerization allows for the generation of CD19 binding molecules in which different ABMs are interconnected through Fc domains containing Fc regions with different sequences. Examples of heterodimerization strategies are illustrated in Section 7.4.1.5 (and its subsections).

[0252] 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 includes mutated IgG1 Fc domains at positions 1, 2, 3, 4, 5, 6, or more than 6 of positions 233, 234, 235, 236, 237, 239, 265, 266, 267, 268, 269, 297, 299, 322, 327, 328, 329, 330, 331, and 332 (EU numbers). For example, a CD19 binding molecule may contain an IgG1 sequence of SEQ ID NO:1109 with mutations at positions 1, 2, 3, 4, 5, 6, or more than 6 of positions 233, 234, 235, 236, 237, 239, 265, 266, 267, 268, 269, 297, 299, 322, 327, 328, 329, 330, 331, and 332.

[0253] 7.4.1.1. Fc domains with modified FcR binding

[0254] Compared to the corresponding innate 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 increased or decreased. In one embodiment, the Fc domain contains one or more modifications that alter its Fc-receptor binding profile.

[0255] Human cells can express a wide range of membrane-bound FcRs, 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 categorized by IgG binding affinity (high / low) and biological function (activation / inhibition). Human FcγRI is widely considered the only "high affinity" receptor, while all others are considered intermediate to low affinity. FcγRIIb is the only receptor with "inhibitory" function due to its intracellular ITIM motif, while all others are considered "activating" due to the ITIM 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. Besides these sequences, numerous other sequences or allotypes are scattered throughout these families. Some of these sequences have been found to have important functional consequences and are therefore sometimes considered 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 four subclasses of IgG: IgG1, IgG2, IgG3, and IgG4 (Bruhns, 1993, Blood 113:3716-3725). Other species have slightly different numbers and functions of FcγRs, among which the mouse system is the most well-studied and consists of 4 FcγRs, FcγRI, FcγRIIb, FcγRIII, and FcγRIV (Bruhns, 2012, Blood 119:5640-5649). Due to the high affinity of human FcγRIs on cells for IgG1 / IgG3 / IgG4 (approximately 10...), the affinity of human FcγRIs for IgG1 / IgG3 / IgG4 is significantly higher. -8 Given the concentrations of these IgGs in serum (approximately 10 mg / ml), human FcγRIs on cells are generally considered to be "occupied" by monomeric IgGs under normal serum conditions. Therefore, cells carrying FcγRIs on their surfaces are thought to be able to alternatively "screen" or "sample" their antigenic environment by binding multispecific IgGs. Other receptors with lower affinity for IgG subclasses (at approximately 10 mg / ml) -5 -10-7 Within the range of M, receptors are generally considered "unoccupied." Low-affinity receptors are therefore intrinsically sensitive to the detection of and activation by antibody-mediated immune complexes. The increased Fc density in antibody-mediated immune complexes results in enhanced functional affinity for binding to low-affinity FcγR. This has been demonstrated in vitro using numerous methods (Shields et al., 2001, J Biol Chem 276(9):6591-6604; Lux et al., 2013, J Immunol 190:4315-4323). This is also considered one of the main mechanisms of action for treating human ITP with anti-RhD therapy (Crow, 2008, Transfusion Medicine Reviews 22:103-116).

[0256] Many cell types express multiple types of FcγR, and therefore, depending on the biological context, the binding of IgG or antibody immune complexes to FcγR-carrying cells can have a variety of complex outcomes. At its simplest, 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). Data support the conclusion that inhibitory signals from FcγRIIb can dominate activating signals (Proulx, 2010, Clinical Immunology 135:422-429).

[0257] Numerous useful Fc substitutions can be prepared to alter binding to one or more of the FcγR receptors. Substitutions that result in both increased and decreased binding can be useful. For example, increased binding to FcγRIIIa is known to generally lead to increased ADCC (antibody-dependent cell-mediated cytotoxicity; a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize a bound antibody on a target cell and subsequently cause lysis of the target cell). Similarly, decreased binding to FcγRIIb (an inhibitory receptor) can also be beneficial in some cases. Amino acid substitutions that can be used in this disclosure include those listed in US2006 / 0024298 (especially Figure 41), US2006 / 0121032, US2006 / 0235208, US2007 / 0148170, and US2019 / 0100587. Specific variants that may 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.

[0258] FcRn plays a crucial role in maintaining the long half-life of IgG in the serum of adults and children. The receptor binds to IgG in acidified vesicles (pH < 6.5), protecting IgG molecules from degradation, and then releases them into the bloodstream at higher pH levels (7.4).

[0259] FcRn differs from the leukocyte Fc receptor and, conversely, exhibits structural similarity to MHC class I molecules. A heterodimer composed of β2-microglobulin chains is non-covalently attached to a membrane-bound chain comprising three extracellular domains. One of these domains (including the carbohydrate chain) interacts with the site between the CH2 and CH3 domains of Fc, along with β2-microglobulin. This interaction involves a salt bridge constructed targeting histidine residues on IgG, which are positively charged at pH < 6.5. At higher pH values, these histidine residues lose their positive charge, the FcRn-IgG interaction is weakened, and IgG dissociates.

[0260] In one embodiment, the CD19 binding molecule includes an Fc domain that binds to human FcRn.

[0261] In one embodiment, the Fc domain has one or more (e.g., one or two) Fc regions containing a histidine residue at position 310, and in some cases also containing 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.

[0262] The CD19-binding molecule may include one or more Fc regions that alter the binding of Fc to FcRn. This altered binding may be an enhanced or degraded binding.

[0263] In one embodiment, the CD19 binding molecule comprises an Fc domain, wherein at least one (and optionally both) Fc region contains one or more modifications such that it binds to FcRn with higher affinity and affinity than the corresponding natural immunoglobulin.

[0264] 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.

[0265] In one embodiment, the Fc region is modified by replacing the threonine residue at position 250 with a glutamine residue (T250Q).

[0266] In one embodiment, the Fc region is modified by replacing the methionine residue at position 252 with a tyrosine residue (M252Y).

[0267] In one embodiment, the Fc region is modified by replacing the serine residue at position 254 with a threonine residue (S254T).

[0268] In one embodiment, the Fc region is modified by replacing the threonine residue at position 256 with a glutamic acid residue (T256E).

[0269] In one embodiment, the Fc region is modified by replacing the threonine residue at position 307 (T307A) with an alanine residue.

[0270] In one embodiment, the Fc region is modified by replacing the threonine residue at position 307 with a proline residue (T307P).

[0271] In one embodiment, the Fc region is modified by replacing the valine residue at position 308 with a cysteine ​​residue (V308C).

[0272] In one embodiment, the Fc region is modified by replacing the valine residue at position 308 with a phenylalanine residue (V308F).

[0273] In one embodiment, the Fc region is modified by replacing the valine residue at position 308 (V308P) with a proline residue.

[0274] In one embodiment, the Fc region is modified by replacing the glutamine residue at position 311 with an alanine residue (Q311A).

[0275] In one embodiment, the Fc region is modified by replacing the glutamine residue at position 311 with an arginine residue (Q311R).

[0276] In one embodiment, the Fc region is modified by replacing the methionine residue at position 428 (M428L) with a leucine residue.

[0277] In one embodiment, the Fc region is modified by replacing the histidine residue at position 433 (H433K) with a lysine residue.

[0278] In one embodiment, the Fc region is modified by replacing the asparagine residue at position 434 (N434F) with a phenylalanine residue.

[0279] In one embodiment, the Fc region is modified by replacing the asparagine residue at position 434 (N434Y) with a tyrosine residue.

[0280] In one embodiment, the Fc region is modified by replacing the methionine residue at position 252 with a tyrosine residue, the serine residue at position 254 with a threonine residue, and the threonine residue at position 256 with a glutamic acid residue (M252Y / S254T / T256E).

[0281] In one embodiment, the Fc region is modified by replacing the valine residue at position 308 with a proline residue and the asparagine residue at position 434 with a tyrosine residue (V308P / N434Y).

[0282] In one embodiment, the Fc region is modified by replacing 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).

[0283] It will be recognized that any of the modifications listed above can be combined to alter the binding of FcRn.

[0284] In one embodiment, the CD19 binding molecule includes an Fc domain, one or both of which contain one or more modifications such that the Fc domain binds to the FcRn with lower affinity and affinity than the corresponding natural immunoglobulin.

[0285] In one embodiment, the Fc region contains any amino acid residues except for histidine at position 310 and / or position 435.

[0286] The CD19 binding molecule may contain an Fc domain, one or both of which contain 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.

[0287] In one embodiment, the Fc region is modified by replacing the proline residue at position 238 with an aspartic acid residue (P238D).

[0288] In one embodiment, the Fc region is modified by replacing the glutamic acid residue at position 258 (E258A) with an alanine residue.

[0289] In one embodiment, the Fc region is modified by replacing the serine residue at position 267 (S267A) with an alanine residue.

[0290] In one embodiment, the Fc region is modified by replacing the serine residue at position 267 with a glutamic acid residue (S267E).

[0291] In one embodiment, the Fc region is modified by replacing the leucine residue at position 328 with a phenylalanine residue (L328F).

[0292] In one embodiment, the Fc region is modified by replacing the glutamic acid residue at position 258 with an alanine residue and the serine residue at position 267 with an alanine residue (E258A / S267A).

[0293] In one embodiment, the Fc region is modified by replacing the serine residue at position 267 with a glutamic acid residue and the leucine residue at position 328 with a phenylalanine residue (S267E / L328F).

[0294] It will be recognized that any of the modifications listed above can be combined to improve FcγRIIb binding.

[0295] In one embodiment, a CD19-binding molecule comprising an Fc domain is provided, the Fc domain exhibiting binding with reduced FcγR.

[0296] In one embodiment, the CD19 binding molecule comprises an Fc domain, wherein one or both Fc regions contain one or more modifications that reduce Fc binding to FcγR.

[0297] The Fc domain can be derived from IgG1.

[0298] In one embodiment, the Fc region is modified by replacing the leucine residue (L234A) at position 234 with an alanine residue.

[0299] In one embodiment, the Fc region is modified by replacing the leucine residue at position 235 (L235A) with an alanine residue.

[0300] In one embodiment, the Fc region is modified by replacing the glycine residue at position 236 with an arginine residue (G236R).

[0301] In one embodiment, the Fc region is modified by replacing the asparagine residue at position 297 with an alanine residue (N297A) or a glutamine residue (N297Q).

[0302] In one embodiment, the Fc region is modified by replacing the serine residue at position 298 with an alanine residue (S298A).

[0303] In one embodiment, the Fc region is modified by replacing the leucine residue at position 328 (L328R) with an arginine residue.

[0304] In one embodiment, the Fc region is modified by replacing the leucine residue at position 234 with an alanine residue and replacing the leucine residue at position 235 with an alanine residue (L234A / L235A).

[0305] In one embodiment, the Fc region is modified by replacing the phenylalanine residue at position 234 with an alanine residue and the leucine residue at position 235 with an alanine residue (F234A / L235A).

[0306] In one embodiment, the Fc region is modified by replacing the glycine residue at position 236 with an arginine residue and the leucine residue at position 328 with an arginine residue (G236R / L328R).

[0307] In one embodiment, the Fc region is modified by replacing the aspartic acid residue at position 265 with an alanine residue, replacing the asparagine residue at position 297 with an alanine residue, and replacing the proline residue at position 329 with an alanine residue (D265A / N297A / P329A).

[0308] In one embodiment, the Fc region is modified by replacing the aspartic acid residue at position 265 with an asparagine residue, replacing the asparagine residue at position 297 with an aspartic acid residue, and replacing the proline residue at position 329 with a glycine residue (D265N / N297D / P329G).

[0309] In one embodiment, the Fc region is modified by replacing the aspartic acid residue at position 265 with a glutamate residue, replacing the asparagine residue at position 297 with a glutamate residue, and replacing the proline residue at position 329 with a serine residue (D265E / N297Q / P329S).

[0310] It will be recognized that any of the modifications listed above can be combined to reduce FcγR binding.

[0311] In one embodiment, the CD19 binding molecule includes an Fc domain, and one or both Fc regions contain one or more modifications that reduce Fc binding to FcγRIIIa without affecting Fc binding to FcγRII.

[0312] In one embodiment, the Fc region is modified by replacing the serine residue at position 239 (S239A) with an alanine residue.

[0313] In one embodiment, the Fc region is modified by replacing the glutamic acid residue at position 269 (E269A) with an alanine residue.

[0314] In one embodiment, the Fc region is modified by replacing the glutamic acid residue at position 293 (E293A) with an alanine residue.

[0315] In one embodiment, the Fc region is modified by replacing the tyrosine residue at position 296 (Y296F) with a phenylalanine residue.

[0316] In one embodiment, the Fc region is modified by replacing the valine residue at position 303 (V303A) with an alanine residue.

[0317] In one embodiment, the Fc region is modified by replacing the alanine residue at position 327 (A327G) with a glycine residue.

[0318] In one embodiment, the Fc region is modified by replacing the lysine residue at position 338 (K338A) with an alanine residue.

[0319] In one embodiment, the Fc region is modified by replacing the aspartic acid residue at position 376 (D376A) with an alanine residue.

[0320] It will be recognized that any of the modifications listed above can be combined to reduce FcγRIIIa binding.

[0321] Fc region variants with reduced FcR binding may be referred to as “FcγR ablation variants,” “FcγR silencing 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 MBM described herein contains one or more Fcγ receptor ablation variants. In some embodiments, both Fc regions contain one or more Fcγ receptor ablation variants. These ablation variants are described in Table 3, and each may be included or excluded independently and optionally. Some aspects utilize ablation variants selected from the group consisting of: G236R / L328R, E233P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G236del / S267K, E233P / L234V / L235A / G236del / S 239K / A327G, E233P / L234V / L235A / G236del / S267K / A327G, E233P / L234V / L235A / G236del, D265A / N297A / P329A, D265N / N297D / P329G, and D265E / N297Q / P329S (“del” indicates deletion; for example, G236del refers to the deletion of glycine at position 236). It should be noted that the ablation variants cited herein ablate FcγR binding but generally do not ablate FcRn binding.

[0322]

[0323]

[0324] In some embodiments, the MBM disclosed herein includes a first Fc region and a second Fc region. In some embodiments, the first Fc region and / or the second Fc region may contain the following mutations: E233P, L234V, L235A, G236del, and S267K.

[0325] The Fc domain of human IgG1 has the highest binding affinity to the Fcγ receptor, and therefore, when the constant domain (or Fc domain) in the backbone of a heterodimeric antibody is IgG1, an ablation variant can be used.

[0326] Alternatively, or in addition to ablation variants in the IgG1 background, a mutation at glycosylation position 297, for example, replacing the asparagine residue at position 297 with an alanine residue (N297A) or a glutamine residue (N297Q), can significantly ablate binding to, for example, FcγRIIIa. Human IgG2 and IgG4 naturally bind less to the Fcγ receptor, and therefore those backbones can be used with or without ablation variants.

[0327] 7.4.1.2. Fc domains with altered complement binding

[0328] The CD19-binding molecule may contain an Fc domain, one or both Fc regions of which contain one or more modifications that alter the binding of the Fc domain to complement. The altered complement binding may be an enhanced or degraded binding.

[0329] In one embodiment, the Fc region contains one or more modifications that reduce its binding to C1q. The classical complement pathway is initiated by binding the hexameric C1q protein to the CH2 domain of antigen-binding IgG and IgM.

[0330] In one embodiment, the CD19 binding molecule comprises an Fc domain, wherein one or both Fc regions contain one or more modifications that reduce the binding of Fc to C1q.

[0331] In one embodiment, the Fc region is modified by replacing the leucine residue (L234A) at position 234 with an alanine residue.

[0332] In one embodiment, the Fc region is modified by replacing the leucine residue at position 235 (L235A) with an alanine residue.

[0333] In one embodiment, the Fc region is modified by replacing the leucine residue at position 235 with a glutamic acid residue (L235E).

[0334] In one embodiment, the Fc region is modified by replacing the glycine residue at position 237 (G237A) with an alanine residue.

[0335] In one embodiment, the Fc region is modified by replacing the lysine residue at position 322 (K322A) with an alanine residue.

[0336] In one embodiment, the Fc region is modified by replacing the proline residue at position 331 (P331A) with an alanine residue.

[0337] In one embodiment, the Fc region is modified by replacing the proline residue at position 331 with a serine residue (P331S).

[0338] 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 exhibits weaker binding to FcγR. Therefore, in one embodiment, the CD19 binding molecule comprises an IgG4 Fc domain and further comprises one or more modifications that enhance FcγR binding.

[0339] It will be recognized that any of the modifications listed above can be combined to reduce C1q binding.

[0340] 7.4.1.3. Fc domain with modified disulfide bond structure

[0341] The CD19 binding molecule may include an Fc domain comprising one or more modifications to generate and / or remove cysteine ​​residues. Cysteine ​​residues play an important role in the simultaneous assembly of Fc-based multispecific binding molecules by forming disulfide bridges between single pairs of polypeptide monomers. Therefore, it is possible to modify the structure of the CD19 binding molecule to generate proteins with improved therapeutic properties by changing the number and / or position of cysteine ​​residues.

[0342] The CD19 binding molecule disclosed herein may include an Fc domain, wherein one or both Fc regions (e.g., two Fc regions) contain a cysteine ​​residue at position 309. In one embodiment, the cysteine ​​residue at position 309 is generated by modification, for example, for an Fc domain derived from IgG1, a cysteine ​​residue (L309C) is used to replace the leucine residue at position 309, and for an Fc domain derived from IgG2, a cysteine ​​residue (V309C) is used to replace the valine residue at position 309.

[0343] In one embodiment, the Fc region is modified by replacing the valine residue at position 308 with a cysteine ​​residue (V308C).

[0344] In one embodiment, two disulfide bonds in the hinge region are removed by mutating the core hinge sequence CPCC (SEQ ID NO:55) to SPPS (SEQ ID NO:56).

[0345] 7.4.1.4. Fc domain with modified glycosylation

[0346] In some respects, CD19-binding molecules with improved manufacturability are provided, which contain fewer glycosylation sites than the corresponding immunoglobulins. These proteins have less complex post-translational glycosylation patterns and are therefore simpler and less expensive for manufacturers.

[0347] 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 herein.

[0348] In some embodiments, CD19 binding molecules with altered glycosylation patterns can be prepared, such as antibodies with low fucosylation having reduced amounts of fucosylation residues or antibodies with enhanced bipartite GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC ability of antibodies. This type of carbohydrate modification can be achieved, for example, by expressing CD19 binding molecules in host cells with altered glycosylation mechanisms. Cells with altered glycosylation mechanisms have been described in the art, and said cells can be used as host cells for expressing CD19 binding molecules, thereby producing CD19 binding molecules with altered glycosylation. For example, EP 1,176,195 by Hang et al. describes a cell line with a dysfunctional FUT8 gene encoding a fucosylation enzyme, such that antibodies expressed in this cell line exhibit low fucosylation. Presta described the variant CHO cell line Lecl3 in PCT Publication WO 03 / 035835, which has a reduced ability to attach fucose to Asn(297) linked carbohydrates and also results in hypofucosylation of antibodies expressed in the host cells (see also Shields et al., 2002, J. Biol. Chem. [Journal of Biochemistry] 277:26733-26740). Umana et al. described a cell line in PCT Publication WO 99 / 54342 that was engineered to express a glycoprotein-modified glycosyltransferase (e.g., β(1,4)-N-acetylglucosamine transferase III (GnTIII)) such that antibodies expressed in the engineered cell line exhibited an increased bipartite GlcNac structure, which resulted in increased ADCC activity of the antibody (see also Umana et al., Nat. Biotech. [Nature Biotechnology] 17:176-180, 1999).

[0349] 7.4.1.5. Fc heterodimerization

[0350] Many multispecific molecular forms require dimerization between two Fc regions, unlike native immunoglobulins, where the two Fc regions are operatively linked to a non-identical antigen-binding domain (or a portion thereof, e.g., Fab's VH or VH-CH1). Inadequate heterodimerization of the two Fc regions forming the Fc domain has been an obstacle to improving the production of the desired multispecific molecule and represents a challenge for purification. Various methods available in the art can be used to enhance the dimerization of the Fc region that may be present in CD19-binding molecules (and especially in the MBM 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.

[0351] This disclosure provides CD19-binding molecules comprising Fc heterodimers, i.e., Fc domains containing heterologous, non-identical Fc regions. Heterodimerization strategies are used to enhance dimerization of Fc regions operatively linked to different ABMs (or portions thereof, e.g., VH or VH-CH1 of Fab) and reduce dimerization of Fc regions operatively linked to the same ABM or portions thereof. Typically, each Fc region in an Fc heterodimer contains a CH3 domain of the antibody. This CH3 domain is derived from any isotype, type, or subclass, and in some cases, constant regions of antibodies in the IgG (IgG1, IgG2, IgG3, and IgG4) types, as described in the preceding sections.

[0352] Typically, in addition to the CH3 domain, the MBM also includes 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 described herein. In some embodiments, the two heteropeptides are two heavy chains forming a bispecific or multispecific molecule. Heterodimerization of two different heavy chains at the CH3 domain produces the desired antibody or antibody-like molecule, while homodimerization of the same heavy chain reduces the production of the desired antibody or molecule. In an exemplary embodiment, the two or more heteropeptide chains comprise two chains that contain the CH3 domain and form a molecule in any of the multispecific molecular forms described above in this disclosure. In embodiments, the two heteropeptide chains containing the CH3 domain contain modifications (relative to the unmodified chain) that facilitate heterodimer association of the peptide.

[0353] Several examples of embellishment strategies are provided in Table 4 and Sections 7.4.1.5.1 through 7.4.1.5.7.

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363] Exemplary heterologous, non-identical Fc sequence pairs that can be paired to form Fc heterodimers and can be included in the CD19 binding molecules disclosed herein include (i) SEQ ID NO:1106 and SEQ ID NO:1107, and (ii) SEQ ID NO:1106 and SEQ ID NO:1108.

[0364] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:1106)

[0365] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:1107)

[0366] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGK(SEQ ID NO:1108)

[0367] The Fc region having an amino acid sequence of one of SEQ ID NO: 1106-1108 can be modified to include one or more substitutions as described in Section 7.4.1 (including its subsections), for example, including one or more substitutions corresponding to the ablation variants listed in Table 3. In some embodiments, the CD19 binding molecule includes an Fc region having a mutation (e.g., one or more mutations as described in Section 7.4.1 (including its subsections)) at positions 1, 2, 3, 4, 5, 6, or more than 6 of the amino acid sequence of one of SEQ ID NO: 1106-1108 at positions 233, 234, 235, 236, 237, 239, 265, 266, 267, 268, 269, 297, 299, 322, 327, 328, 329, 330, 331, and 332 (EU numbers). For example, a CD19 binding molecule may be contained in the Fc region of the amino acid sequence of SEQ ID NO:1106 with mutations at positions 1, 2, 3, 4, 5, 6, or more than 6 at positions 233, 234, 235, 236, 237, 239, 265, 266, 267, 268, 269, 297, 299, 322, 327, 328, 329, 330, 331, and 332, and / or in the SEQ ID NO:1106 with mutations at positions 1, 2, 3, 4, 5, 6, or more than 6 at positions 233, 234, 235, 236, 237, 239, 265, 266, 267, 268, 269, 297, 299, 322, 327, 328, 329, 330, 331, and 332. The Fc region of the amino acid sequence of NO:1107 and / or the Fc region of the amino acid sequence of SEQ ID NO:1108 with mutations at positions 1, 2, 3, 4, 5, 6, or more than 6 at positions 233, 234, 235, 236, 237, 239, 265, 266, 267, 268, 269, 297, 299, 322, 327, 328, 329, 330, 331, and 332.

[0368] 7.4.1.5.1. Spatial Variations

[0369] CD19 binding molecules may contain one or more, for example, multiple, modifications to one or more constant domains of the Fc domain, such as modifications to the CH3 domain. In one example, the CD19 binding molecule disclosed herein comprises two peptides, each peptide containing a heavy chain constant domain of an antibody, such as a CH2 or CH3 domain. In one example, the two heavy chain constant domains, such as the CH2 or CH3 domain of the CD19 binding molecule, contain one or more modifications that allow 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 another aspect, the one or more modifications are disposed on the CH3 domains of at least two peptides of the CD19 binding molecule.

[0370] One mechanism of Fc heterodimerization is often referred to as “knobs and holes” or “knobs-into-holes.” These terms refer to amino acid mutations that produce spatial effects that favor the formation of Fc heterodimers (compared to Fc homodimers), as described below, for example, Ridgway et al., 1996, Protein Engineering 9(7):617; Atwell et al., 1997, J. Mol. Biol. 270:26; US Patent No. 8,216,805. Knobs-into-holes mutations can be combined with other strategies to improve heterodimerization.

[0371] In one aspect, one or more modifications to a first polypeptide containing a CD19-binding molecule with a heavy chain constant domain can produce a "pestle" and one or more modifications to a second polypeptide containing a CD19-binding molecule can produce a "mortar," such that heterodimerization of the polypeptide containing the heavy chain constant domain of the CD19-binding molecule produces a "pestle" to engage with a "mortar" interface (e.g., an interaction, such as the CH2 domain of the first polypeptide interacting with the CH2 domain of the second polypeptide, or the CH3 domain of the first polypeptide interacting with the CH3 domain of the second polypeptide). The pestle protrudes from the interface of the first polypeptide containing the heavy chain constant domain of the CD19-binding molecule and can therefore be localized in a complementary "mortar" at the interface with the second polypeptide containing the heavy chain constant domain of the CD19-binding molecule to stabilize the heteropolymer and thus facilitate heteropolymer formation (e.g., relative to homopolymers). The pestle may be present in the initial interface or may be synthetically introduced (e.g., by altering the nucleic acid encoding the interface). The input residues used to form 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 chosen. In the examples, the initial residues used to form the protrusion have small side chain volumes, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.

[0372] The "pot" comprises at least one amino acid side chain that is recessed into the interface of a second polypeptide containing a CD19-binding molecule with a heavy chain constant domain and thus accommodates a corresponding pot on the adjacent interface surface of a first polypeptide containing a CD19-binding molecule with a heavy chain constant domain. The pothole may be present in the initial interface or may be introduced synthetically (e.g., by altering the nucleic acid encoding the interface). The input residues used to form the pothole are typically naturally occurring amino acid residues and are selected from alanine (A), serine (S), threonine (T), and valine (V) in some embodiments. In one embodiment, the amino acid residue is serine, alanine, or threonine. In another embodiment, the initial residues used to form the pothole have a large side chain volume, such as tyrosine, arginine, phenylalanine, or tryptophan.

[0373] In an embodiment, the first CH3 domain is modified at residues 366, 405, or 407 to produce a "mortar" or "pestle" (as described above), and a second CH3 domain heterodimerized with the first CH3 domain is modified at residues 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).

[0374] In another embodiment, a first CH3 domain is modified at residue 366, and a second CH3 domain heterodimerized with the first CH3 domain is modified at residues 366, 368, and / or 407 to produce a "mortar" or "stick" complementary to the "mortar" or "stick" of the first CH3 domain. In one embodiment, the modification of the first CH3 domain introduces a tyrosine (Y) residue at position 366. In this 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 this embodiment, the modification of the first CH3 is T366W. In some embodiments, the modification of the second CH3 domain, which is heterodimerized with the first CH3 domain (modified at position 366 (e.g., having a tyrosine (Y) or tryptophan (W) introduced at position 366, e.g., containing the modification T366Y or T366W)), includes a modification at position 366, a modification at position 368, and a modification at position 407. In some embodiments, the modification at position 366 introduces a serine (S) residue, the modification at position 368 introduces alanine (A), and the modification at position 407 introduces valine (V). In some embodiments, the modification comprises T366S, L368A, and Y407V. In one embodiment, the first CH3 domain of the multispecific molecule contains the modification T366Y, and the second CH3 domain heterodimerized with the first CH3 domain contains the modifications T366S, L368A, and Y407V, and vice versa. In one embodiment, the first CH3 domain of the multispecific molecule comprises a modification of T366W, and the second CH3 domain heterodimerized with the first CH3 domain comprises a modification of T366S, L368A, and Y407V, and vice versa.

[0375] Additional spatial or "offset" modifications (e.g., pestle-and-mortar structure) are described in PCT Publication WO 2014 / 145806 (e.g., Figures 3, 4, and 12 of WO 2014 / 145806), PCT Publication WO 2014 / 110601, and PCT Publication WO 2016 / 086186, WO 2016 / 086189, WO 2016 / 086196, and WO 2016 / 182751. Examples of KIH variants comprise a first constant chain containing L368D and K370S modifications, paired with a second constant chain containing S364K and E357Q modifications.

[0376] Further mortar-and-stick structure modifications suitable for use in any CD19-binding molecule disclosed herein are further described, for example, in WO 1996 / 027011 and Merchant et al., 1998, Nat. Biotechnol. [Nature Biotechnology], 16:677-681.

[0377] In a further embodiment, the CH3 domain may be additionally 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 heterodimerized CD19-binding molecules (e.g., MBM) containing paired CH3 domains. In some embodiments, the first CH3 domain contains a cysteine ​​residue at position 354, and the second CH3 domain, heterodimerized with the first CH3 domain, contains a cysteine ​​residue at position 349. In some embodiments, the first CH3 domain comprises cysteine ​​at position 354 (e.g., comprising modification S354C) and tyrosine (Y) at position 366 (e.g., comprising modification T366Y), and the second CH3 domain heterodimerized with the first CH3 domain comprises cysteine ​​at position 349 (e.g., comprising modification Y349C), serine at position 366 (e.g., comprising modification T366S), alanine at position 368 (e.g., comprising modification L368A), and valine at position 407 (e.g., comprising modification Y407V). In some embodiments, the first CH3 domain comprises cysteine ​​at position 354 (e.g., comprising modification S354C) and tryptophan (W) at position 366 (e.g., comprising modification T366W), and the second CH3 domain heterodimerized with the first CH3 domain comprises cysteine ​​at position 349 (e.g., comprising modification Y349C), serine at position 366 (e.g., comprising modification T366S), alanine at position 368 (e.g., comprising modification L368A), and valine at position 407 (e.g., comprising modification Y407V).

[0378] Another mechanism that can be used to generate heterodimers is sometimes referred to as “electrostatic guidance,” as described in Gunasekaran et al., 2010, J. Biol. Chem. [Journal of Biochemistry] 285(25):19637. This is sometimes referred to herein as “charge pair.” In this embodiment, electrostatics are used to form a shift toward heterodimerization. As those skilled in the art will understand, these variants can also have an effect on pI, and therefore on purification, and thus can be considered pI variants in some cases. However, given that these variants are generated to promote heterodimerization and that these 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.

[0379] Additional variants, such as the pI variant outlined herein or other spatial variants shown in Figure 37 of US2012 / 0149876, may be combined with other variants (optionally and independently in any amount).

[0380] In some embodiments, the spatial variants outlined herein may optionally and independently be incorporated into one or two Fc regions along with any pI variant (or other variants such as Fc variants, FcRn variants), and may independently and optionally be included within or excluded from the CD19 binding molecules disclosed herein.

[0381] Table 5 shows a list of suitable offset variants, illustrating some variant pairs that are specifically used in many embodiments. Variant pairs that are specifically used 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 naming, the variant pair “S364K / E357Q:L368D / K370S” means that one of the Fc regions has the dual variant group S364K / E357Q and the others have the dual variant group L368D / K370S.

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388] 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 mutations L368D and K370S, and the second Fc region comprises the mutations S364K and E357Q. In some embodiments, the first Fc region comprises the mutations S364K and E357Q, and the second Fc region comprises the mutations L368D and K370S.

[0389] 7.4.1.5.2. Alternative Knob and Hole: IgG Heterodimerization

[0390] Heterodimerization of a polypeptide chain containing a paired CH3 domain of a CD19-binding molecule can be enhanced by introducing one or more modifications into the CH3 domain derived from an IgG1 antibody type. In an example, the modification comprises a K409R modification of one CH3 domain, which is paired with an F405L modification in a second CH3 domain. Additional modifications may also be, or alternatively, at positions 366, 368, 370, 399, 405, 407, and 409. In some cases, heterodimerization of polypeptides containing such modifications is achieved under reducing conditions, for example, at 25-37°C, e.g., 25°C or 37°C, for 1-10 hours, e.g., 1.5-5 hours, e.g., 5 hours.

[0391] 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).

[0392] The IgG heterodimerization strategy is further described, for example, in WO 2008 / 119353, WO 2011 / 131746, and WO 2013 / 060867.

[0393] In any of the embodiments described in this section, the CH3 domain may be further modified to introduce a pair of cysteine ​​residues, as described in section 7.4.1.3.

[0394] 7.4.1.5.3. pI (isoelectric point) variant

[0395] Generally, as those skilled in the art will understand, pI variants fall into two general categories: those that increase protein pI (basicity alteration) and those that decrease protein pI (acidity alteration). As described herein, all combinations of these variants are possible: one Fc region can be wild-type, or a variant that does not exhibit a pI significantly different from wild-type, and the other can be more basic or more acidic. Alternatively, each Fc region can be altered, with one being more basic and the other more acidic.

[0396] Exemplary combinations of pI variants are shown in Table 6. As outlined herein and as shown in Table 6, these modifications are shown relative to IgG1, but all isotypes and isotype hybrids can be modified in this manner. R133E and R133Q can also be used in cases where the heavy chain constant domain is derived from IgG2-4.

[0397]

[0398] In one embodiment, for example in Figure 1B-1W , Figure 1Y-1AH , Figure 2B-2L ,and Figure 2N-2V In this form, the combination of pI variants has an Fc region (negative Fab side) comprising 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) comprising a positively charged scFv connector, such as L36 (described in Section 7.4.3). However, as those skilled in the art will understand, the first Fc region includes a CH1 domain comprising position 208. Therefore, in constructs that do not include the CH1 domain (e.g., for MBMs that do not use the CH1 domain as one of their domains, e.g., for...) Figure 2K (as described in the document), the negative pI variant Fc group can include the 295E / 384D / 418E / 421D variants (Q295E / N384D / Q418E / N421D when relative to human IgG1).

[0399] In some embodiments, the first Fc region has a set of replacements from Table 6 and the second Fc region is connected to a charged connector (e.g., selected from those described in Section 7.4.3).

[0400] In some embodiments, the CD19 binding molecule disclosed herein comprises 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.

[0401] 7.4.1.5.4. Isotope variants

[0402] Furthermore, many embodiments of this disclosure rely on “introducing” pI amino acids at specific positions from one IgG isotype to another, thereby reducing or eliminating the possibility of unwanted immunogenicity being introduced into the variant. Many of these variants are shown in Figure 21 of U.S. Publication No. 2014 / 0370013. That is, IgG1 is a common isotype of therapeutic antibodies for various causes, including high-effects functions. However, the recurrent region of IgG1 has a higher pI than IgG2 (8.10 vs. 7.31). By introducing IgG2 residues at specific positions into the IgG1 backbone, the resulting Fc region has a lower (or higher) pI and additionally exhibits a longer serum half-life. For example, IgG1 has glycine at position 137 (pI 5.97), and IgG2 has glutamate (pI 3.22); introducing glutamate will affect the pI of the resulting protein. As described below, substantial 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 changes in the IgG2 molecule can allow for an increase in serum half-life.

[0403] In other embodiments, non-isotype amino acid changes are produced 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 adjustments for stability, as further described below.

[0404] Furthermore, significant variations in each hapten can be observed by engineering the heavy and light constant domains of the CD19-binding molecule containing both haptens using pI. A pI difference of at least 0.5 between the two haptens allows for separation by ion-exchange chromatography, isoelectronic focusing, or other methods sensitive to the isoelectric point.

[0405] 7.4.1.5.5. Calculate pI

[0406] The pI of a half antibody containing an Fc region and ABM or ABM chain can depend on the pI of the variable heavy chain constant domain and the pI of the total half antibody (including the variable heavy chain constant domain and ABM or ABM chain). Therefore, in some embodiments, the pI variation is calculated based on the variable heavy chain constant domain using the graph in Figure 19 of U.S. Publication 2014 / 0370013. As discussed herein, which half antibody is engineered is typically determined by the inherent pI of the half antibody. Alternatively, the pI of each half antibody can be compared.

[0407] 7.4.1.5.6. Also endows the pI variant with better in vivo FcRn binding.

[0408] In cases where the pI variant reduces the pI in the Fc region, it may have the additional benefit of improved in vivo serum retention.

[0409] The pI variant's Fc region is believed to provide a longer half-life for the antigen-binding molecule in vivo, since at pH 6, binding to FcRn in the endosome isolates Fc (Ghetie and Ward, 1997, Immunol Today, 18(12):592-598). The endosome chamber then allows Fc to recirculate to the cell surface. Once the chamber opens to the extracellular space, a higher pH, around 7.4, induces the release of Fc back into the bloodstream. In mice, Dall'Acqua et al. showed that Fc mutants with enhanced 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). The increased affinity of Fc for FcRn at pH 7.4 is thought to prevent the release of Fc back into the bloodstream. Therefore, the Fc mutation that increases the in vivo half-life of Fc will ideally enhance FcRn binding at lower pH and still allow Fc release at higher pH. The amino acid histidine alters its charge state in the pH range of 6.0 to 7.4. Therefore, the presence of His residues at key positions in the Fc / FcRn complex is not surprising.

[0410] It has been proposed that antibodies with variable regions (having lower isoelectric points) can also have longer serum half-lives (Igawa et al., 2010, PEDS.23(5):385-392). However, the mechanisms underlying this finding remain poorly understood. Furthermore, the variable regions differ between antibodies. Constant-region variants with lower pI and longer half-lives would offer a more modular approach to improving the pharmacokinetic properties of CD19-binding molecules as described herein.

[0411] 7.4.1.5.7. Polarity Bridge

[0412] Heterodimerization of polypeptide chains containing Fc domain-binding molecules (e.g., MBM) can be enhanced by introducing modifications based on the fundamental principle of "polar bridging." This principle involves creating residues at the binding interface of the two polypeptide chains to interact with residues in the heterodimeric configuration that have similar (or complementary) physical properties, while simultaneously interacting with residues in the homodimeric configuration that have different physical properties. Specifically, these modifications are designed so that, in heterodimer formation, polar residues interact with polar residues, and hydrophobic residues interact with hydrophobic residues. In contrast, in homodimer formation, residues are modified so that polar residues interact with hydrophobic residues. The combined effect of favorable interactions in the heterodimeric configuration and unfavorable interactions in the homodimeric configuration makes heterodimer formation in the Fc region more likely than homodimer formation.

[0413] In an exemplary embodiment, the above modifications are made at one or more of residues 364, 368, 399, 405, 409, and 411 of the CH3 domain.

[0414] 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 may be introduced into the second CH3 domain.

[0415] In another embodiment, one or more modifications selected from the group consisting of S364L, T366V, L368Q, D399K, F405S, K409F and T411K are introduced into a CH3 domain, while one or more modifications selected from the group consisting of Y407F, K409Q and T411D are introduced into a second CH3 domain.

[0416] In one exemplary embodiment, the initial residue of threonine at position 366 of one CH3 domain is replaced by valine, while the initial residue of tyrosine at position 407 of another CH3 domain is replaced by phenylalanine.

[0417] In another exemplary embodiment, the initial residue of serine at position 364 of a CH3 domain is replaced by leucine, while the initial residue of leucine at position 368 of the same CH3 domain is replaced by glutamine.

[0418] In yet another exemplary embodiment, the initial residue of phenylalanine at position 405 of one CH3 domain is replaced by serine and the initial residue of lysine at position 409 of the same CH3 domain is replaced by phenylalanine, while the initial residue of lysine at position 409 of another CH3 domain is replaced by glutamine.

[0419] In yet another exemplary embodiment, the initial residue of aspartic acid at position 399 of one CH3 domain is replaced by lysine, and the initial residue of threonine at position 411 of the same CH3 domain is replaced by lysine, while the initial residue of threonine at position 411 of another CH3 domain is replaced by aspartic acid.

[0420] 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 bridging strategy is described, for example, in WO 2006 / 106905, WO2009 / 089004, and Gunasekaran et al., 2010, JBC 285:19637-19646.

[0421] Other polar bridge modifications are described, for example, in PCT Publication WO 2014 / 145806 (e.g., Figure 6 of WO 2014 / 145806), PCT Publication WO 2014 / 110601, and PCT Publications WO 2016 / 086186, WO 2016 / 086189, WO 2016 / 086196, and WO 2016 / 182751. Examples of polar bridge variants include constant chains containing modifications of N208D, Q295E, N384D, Q418E, and N421D.

[0422] In any of the embodiments described herein, the CH3 domain may be additionally modified to introduce a pair of cysteine ​​residues, as described in Section 7.4.1.3.

[0423] Other strategies for enhancing heterodimerization are described, for example, in WO 2016 / 105450, WO 2016 / 086186, WO 2016 / 086189, WO 2016 / 086196, WO 2016 / 141378, WO 2014 / 145806, and WO2014 / 110601. Any of these strategies may be used in the CD19-binding molecules described herein.

[0424] 7.4.1.6. Combinations of heterodimer variants and other Fc variants

[0425] As those skilled in the art will understand, all the listed heterodimerization variants (including offset and / or pI variants) can be optionally and independently combined in any way, provided that the Fc regions of the Fc domains retain their dimerization capabilities. Furthermore, all these variants can be combined with any of the heterodimerization forms.

[0426] In the case of pI variants, when specific examples are shown in Table 6, other combinations can be produced based on the fundamental principle of altering the pI difference between the two Fc regions in the Fc heterodimer to facilitate purification.

[0427] In addition, any of the heterodimerization variants, offsets, and pI can also be combined independently and optionally with the Fc ablation variant, Fc variant, or FcRn variant, as generally outlined herein.

[0428] In some embodiments, a specific combination of offset and pI variants available for use in this disclosure is T366S / L368A / Y407V:T366W (optionally including bridging disulfide bonds, 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 junction (when the form includes an scFv structural domain). As those skilled in the art will understand, the "mortar and pestle" variant does not alter the pI and can therefore be used on either Fc region in the Fc heterodimer.

[0429] In some embodiments, the first and second Fc regions disclosed herein may include amino acid-substituted S364K / E357Q:L368D / K370S, wherein the first and / or second Fc regions include ablation variant-substituted 233P / L234V / L235A / G236del / S267K, and the first and / or second Fc regions contain pI variant-substituted N208D / Q295E / N384D / Q418E / N421D(pl_(-)_isoelectric_A).

[0430] 7.4.2. Hinge Area

[0431] The CD19 binding molecule may also include a hinge region, such as a hinge region connecting the antigen-binding domain to the Fc region. The hinge region may be natural or modified. Hinge regions are typically found at the N-terminus of the Fc region.

[0432] A natural hinge region is a hinge region typically found between the Fab and Fc domains in naturally occurring antibodies. A modified hinge region is any hinge that differs from a natural hinge region in length and / or composition. Such hinges may include hinge regions derived from other species, such as those of humans, mice, rats, rabbits, sharks, pigs, hamsters, camels, alpacas, or goats. Other modified hinge regions may comprise complete hinge regions derived from antibodies of a different type or subclass than the heavy chain Fc region. Alternatively, the modified hinge region may comprise portions of a natural hinge or repeating unit, wherein each unit in the repeat is derived from a natural hinge region. In a further alternative, the natural hinge region may be modified 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 manner, the number of cysteine ​​residues in the hinge region can be increased or decreased. This 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, promote the assembly of light and heavy chains, or increase or decrease the stability of CD19-binding molecules. Other modified hinge regions can be entirely synthetic and can be designed to have desired properties such as length, cysteine ​​composition, and flexibility.

[0433] Many modified hinge areas are described in the following documents: for example, in U.S. Patent Nos. 5,677,425, WO9915549, WO 2005003170, WO 2005003169, WO 2005003170, WO 9825971 and WO 2005003171.

[0434] Examples of suitable hinge sequences are shown in Table 7.

[0435]

[0436] In one embodiment, the heavy chain Fc region has a complete hinge region at its N-end.

[0437] In one embodiment, the heavy chain Fc region and hinge region are derived from IgG4, and the hinge region contains the modified sequence CPCC (SEQ ID NO: 55). Compared to IgG1 containing the sequence CPCC (SEQ ID NO: 55), the core hinge region of human IgG4 contains the sequence CPSC (SEQ ID NO: 65). Serine residues present in the IgG4 sequence result in increased flexibility in this region, and thus a portion of the molecule forms disulfide bonds (intra-chain disulfide bonds) within the same protein chain rather than bridging to other heavy chains in the IgG molecule to form inter-chain disulfide bonds. (Angel et al., 1993, Mol lmmunol [Molecular Immunology] 30(1):105-108). Changing the serine residues to proline to give the same core sequence as IgG1 allows for complete formation of inter-chain disulfide bonds in the IgG4 hinge region, thus reducing heterogeneity in the purified product. This modified isotype is named IgG4P.

[0438] 7.4.3. ABM connector

[0439] In some respects, this disclosure provides CD19-binding molecules in which two or more components of ABM (e.g., VH and VL of scFv), two or more ABMs, or ABM and non-ABM domains (e.g., dimerization domains, such as the Fc region) are interconnected via peptide linkers. Such linkers are referred to herein as “ABM linkers”, and are distinct from ADC linkers used for attaching drugs to CD19-binding molecules as described, for example, in Section 7.12.2.

[0440] The range of peptide linkers can be from 2 amino acids to 60 or more amino acids, and in some respects, the range of peptide linkers is 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.

[0441] Charged and / or flexible connectors can be used.

[0442] Examples of flexible ABM linkers that can be used for 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.

[0443] Other examples of suitable ABM linkers that can be used in the CD19 binding molecules disclosed herein are shown in Table 8 below:

[0444]

[0445]

[0446]

[0447] In several aspects, this disclosure provides a CD19 binding molecule comprising one or more ABM linkers. Each of the ABM linkers may be of a length ranging from 2 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 may be located on one, two, three, four, or even more polypeptide chains of the CD19 binding molecule.

[0448] 7.5. Bispecific binding molecular configuration

[0449] An exemplary BBM configuration is shown in Figure 1. Figure 1A It shows Figure 1B-1AH The components of the BBM conformation shown in Figure 1, including scFv, Fab, scFab, non-immunoglobulin-based ABM, and the Fc domain, can each have the features described for these components in Sections 7.3 and 7.4. The components of the BBM conformation shown in Figure 1 can associate with each other by any of the methods described in Sections 7.3 and 7.4 (e.g., via direct bonds, ABM linkers, disulfide bonds, Fc domains modified with mortar-and-mortar interactions, etc.). The orientations and associations of the various components shown in Figure 1 are merely exemplary; as those skilled in the art will understand, other orientations and associations may be suitable (e.g., as described in Sections 7.3 and 7.4).

[0450] BBM is not limited to the configuration shown in Figure 1. Other configurations that may 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 Frontiers] 8:38; Brinkmann & Kontermann, 2017, mAbs 9:2,182-212; US2016 / 0355600; Klein et al., 2016, MAbs8(6):1010-20; and US2017 / 0145116.

[0451] 7.5.1. Exemplary bivalent BBM

[0452] The BBMs can be bivalent, meaning they have two antigen-binding domains, one of which binds to CD19 (ABM1) and the other binds to a second target antigen (ABM2), such as a component of the TCR complex.

[0453] An exemplary bivalent BBM configuration is shown in Figure 1B-1F middle.

[0454] like Figure 1B-1D As described, BBM may contain two halves, one containing an ABM and the other containing an ABM, the two halves being paired through an Fc domain.

[0455] exist Figure 1B In one embodiment, the first (or left) hemibody comprises Fab and Fc regions, and the second (or right) hemibody comprises Fab and Fc regions. The first and second hemibody associate by forming Fc regions of Fc domains.

[0456] exist Figure 1C In one embodiment, the first (or left) hemibody comprises Fab and Fc regions, and the second (or right) hemibody comprises scFv and Fc regions. The first and second hemibody associate by forming Fc regions of Fc domains.

[0457] exist Figure 1D In one embodiment, the first (or left) hemibody comprises scFv and Fc regions, and the second (or right) hemibody comprises scFv and Fc regions. The first and second hemibody associate by forming Fc regions of Fc domains.

[0458] like Figure 1E-1F As described, a bivalent BBM can contain two ABMs attached to an Fc region of an Fc structural domain.

[0459] exist Figure 1E In one embodiment, the BBM includes Fab, scFv, and Fc structural domains, wherein the scFv is located between the Fab and Fc structural domains.

[0460] exist Figure 1F In one embodiment, the ("one-arm scFv-mAb" configuration) BBM includes Fab, scFv and Fc structural domains, wherein Fab is located between the scFv and Fc structural domains.

[0461] exist Figure 1B-1F In the configuration shown, each of X and Y represents either ABM1 or ABM2, provided that the BBM comprises one ABM1 and one ABM2. Therefore, this disclosure provides as follows: Figures 1B to 1F Any of the bivalent BBMs shown in the diagram, where X is ABM1 and Y is ABM2 (for convenience, this configuration of ABM is designated "B1"). This disclosure also provides, as Figures 1B to 1F Any of the two-valent BBMs shown in the diagram, where X is ABM2 and Y is ABM1 (for convenience, this configuration of ABM is designated as "B2").

[0462] 7.5.2. Example Trivalent BBM

[0463] The BBMs can be trivalent, meaning 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), such as a component of the TCR complex.

[0464] An exemplary trivalent BBM configuration is shown in Figure 1G-1Z middle.

[0465] like Figure 1G-1N As described in 1Q-1W and 1Y-1Z, BBM can contain two halves, one containing two ABMs and the other containing one ABM, the two halves being paired via an Fc domain.

[0466] exist Figure 1G In one embodiment, the first (or left) hemibody comprises Fab and Fc regions, and the second (or right) hemibody comprises scFv, Fab, and Fc regions. The first and second hemibody associate by forming Fc regions of Fc domains.

[0467] exist Figure 1H In one embodiment, the first (or left) hemibody comprises Fab and Fc regions, and the second (or right) hemibody comprises Fab, scFv, and Fc regions. The first and second hemibody associate by forming Fc regions of Fc domains.

[0468] exist Figure 1I In one embodiment, the first (or left) hemibody comprises an scFv and an Fc region, and the second (or right) hemibody comprises two Fab and Fc regions. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0469] exist Figure 1J In one embodiment, the first (or left) hemibody comprises two Fav and Fc regions, and the second (or right) hemibody comprises a Fab and an Fc region. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0470] exist Figure 1K In one embodiment, the first (or left) hemibody comprises scFv and Fc regions, and the second (or right) hemibody comprises two scFv and Fc regions. The first and second hemibody associate by forming Fc regions of Fc domains.

[0471] exist Figure 1L In one embodiment, the first (or left) hemibody comprises an scFv and an Fc region, and the second (or right) hemibody comprises an scFv, a Fab, and an Fc region. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0472] exist Figure 1M In one embodiment, the first (or left) hemibody comprises an scFv and an Fc region, and the second (or right) hemibody comprises a Fab, an scFv, and an Fc region. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0473] exist Figure 1N In one embodiment, the first (or left) hemibody comprises a bispecific antibody-type binding domain and an Fc region, and the second (or right) hemibody comprises a Fab region and an Fc region. The first and second hemibody associate by forming the Fc region of the Fc domain.

[0474] exist Figure 1Q In one embodiment, the first (or left) hemibody comprises a Fab and an Fc region, and the second (or right) hemibody comprises a Fab, an Fc region, and an scFv. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0475] exist Figure 1R In one embodiment, the first (or left) hemibody comprises an scFv and an Fc region, and the second (or right) hemibody comprises a Fab, an Fc region, and an scFv. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0476] exist Figure 1S In one embodiment, the first (or left) hemibody comprises an scFv and an Fc region, and the second (or right) hemibody comprises an scFv, an Fc region, and a second scFv. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0477] exist Figure 1T In one embodiment, the first (or left) hemibody comprises scFv, an Fc region, and Fab, and the second (or right) hemibody comprises Fab and an Fc region. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0478] exist Figure 1U In one embodiment, the first (or left) hemibody comprises two Fab and Fc regions, and the second (or right) hemibody comprises a non-immunoglobulin-based ABM and Fc region. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0479] exist Figure 1V In one embodiment, the first (or left) hemibody comprises a Fab, scFv, and an Fc region, and the second (or right) hemibody comprises a non-immunoglobulin-based ABM and an Fc region. The first and second hemibody associate by forming an Fc region with an Fc domain.

[0480] exist Figure 1W In one embodiment, the first (or left) hemibody comprises a Fab and an Fc region, and the second (or right) hemibody comprises scFv, a non-immunoglobulin-based ABM, and an Fc region. The first and second hemibody associate by forming an Fc region with an Fc domain.

[0481] exist Figure 1Y In one embodiment, the first (or left) hemibody comprises an scFv and an Fc region, and the second (or right) hemibody comprises a Fab, an scFv, and an Fc region. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0482] exist Figure 1Z In one embodiment, the first (or left) hemibody comprises a Fab, an Fc region, and scFab, and the second (or right) hemibody comprises a Fab and an Fc region. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0483] Alternatively, such as Figure 10 and 1P As described, trivalent BBM can contain two half-antibodies, each of which contains a complete ABM. Figure 10 and 1P The two halves of the antibody are Fab and another part of an ABM (one is VH and the other is VL). The two halves pair through the Fc domain, so VH and VL associate to form the complete antigen-binding Fv domain.

[0484] The BBM can be a single chain, such as... Figure 1X As shown. Figure 1X The BBM contains three scFv structural domains connected by connectors.

[0485] exist Figure 1G-1Z In the configurations shown, each of X, Y, and A represents either ABM1 or ABM2, provided that the BBM comprises at least ABM1 and at least one ABM2. Therefore, the trivalent BBM will comprise one or two ABM1s and one or two ABM2s. In some embodiments, the trivalent BBM comprises two ABM1s and one ABM2. In other embodiments, the trivalent BBM disclosed herein comprises one ABM1 and two ABM2s.

[0486] Therefore, this disclosure provides, as follows Figures 1G to 1Z Any of the trivalent BBMs shown in the diagram, where X is ABM1, Y is ABM1 and A is ABM2 (for convenience, this configuration of ABM is designated as "T1").

[0487] This disclosure further provides, as follows Figures 1G to 1Z Any of the trivalent BBMs shown in the diagram, where X is ABM1, Y is ABM2 and A is ABM1 (for convenience, this configuration of ABM is designated as "T2").

[0488] This disclosure further provides, as follows Figures 1G to 1Z Any of the trivalent BBMs shown in the diagram, where X is ABM2, Y is ABM1 and A is ABM1 (for convenience, this configuration of ABM is designated as "T3").

[0489] This disclosure further provides, as follows Figures 1G to 1Z Any of the trivalent BBMs shown in the diagram, where X is ABM1, Y is ABM2 and A is ABM2 (for convenience, this configuration of ABM is designated as "T4").

[0490] This disclosure further provides, as follows Figures 1G to 1Z Any of the trivalent BBMs shown in the diagram, where X is ABM2, Y is ABM1 and A is ABM2 (for convenience, this configuration of ABM is designated as "T5").

[0491] This disclosure further provides, as follows Figures 1G to 1Z Any of the trivalent BBMs shown in the diagram, where X is ABM2, Y is ABM2 and A is ABM1 (for convenience, this configuration of ABM is designated as "T6").

[0492] 7.5.3. Example of tetravalent BBM

[0493] The BBMs can be tetravalent, meaning 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), such as a component of the TCR complex.

[0494] An exemplary tetravalent BBM configuration is shown in Figure 1AA-1AH middle.

[0495] like Figure 1AA-1AH The described tetravalent BBM may comprise two halves, each containing two complete ABMs, the two halves being paired via an Fc domain.

[0496] exist Figure 1AA In one embodiment, the first (or left) hemibody comprises Fab, an Fc region, and scFv, and the second (or right) hemibody comprises Fab, an Fc region, and scFv. The first and second hemibody associate by forming the Fc region of the Fc domain.

[0497] exist Figure 1ABIn one embodiment, the first (or left) hemibody comprises a Fab, scFv, and Fc region, and the second (or right) hemibody comprises a Fab, scFv, and Fc region. The first and second hemibody associate by forming the Fc region of the Fc domain.

[0498] exist Figure 1AC In one embodiment, the first (or left) hemibody comprises scFv, Fab, and Fc regions, and the second (or right) hemibody comprises scFv, Fab, and Fc regions. The first and second hemibody associate by forming Fc regions of Fc domains.

[0499] exist Figure 1AD In one embodiment, the first (or left) hemibody comprises a Fab, an Fc region, and a second Fab, and the second (or right) hemibody comprises a Fab, an Fc region, and a second Fab. The first and second hemibody associate by forming the Fc region of the Fc domain.

[0500] exist Figure 1AE In one embodiment, the first (or left) hemibody comprises an scFv, a second scFv, and an Fc region, and the second (or right) hemibody comprises an scFv, a second scFv, and an Fc region. The first and second hemibody associate by forming the Fc region of the Fc domain.

[0501] exist Figure 1AF In one embodiment, the first (or left) hemibody comprises a Fab, scFv, and Fc region, and the second (or right) hemibody comprises a Fab, scFv, and Fc region. The first and second hemibody associate by forming the Fc region of the Fc domain.

[0502] exist Figure 1AG In one embodiment, the first (or left) hemibody comprises Fab, an Fc region, and scFv, and the second (or right) hemibody comprises scFv, an Fc region, and Fab. The first and second hemibody associate by forming the Fc region of the Fc domain.

[0503] exist Figure 1AH In one embodiment, the first (or left) hemibody comprises scFv, an Fc region, and Fab, and the second (or right) hemibody comprises scFv, an Fc region, and Fab. The first and second hemibodies associate by forming the Fc region of the Fc domain.

[0504] exist Figure 1AA-1AHIn the configurations shown, each of X, Y, A, and B represents either ABM1 or ABM2 (though not necessarily in such an order), provided that the BBM contains at least one ABM1 and at least one ABM2. Therefore, the tetravalent ABM will comprise 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 another embodiment, the tetravalent BBM comprises one ABM1 and three ABM2s.

[0505] Therefore, this disclosure provides, as follows Figure 1AA-1AH Any of the tetravalent BBMs shown in the diagram, where X is ABM1 and each of Y, A, and B is ABM2 (for convenience, this configuration of ABM is designated as "Tv 1").

[0506] This disclosure further provides, as follows Figure 1AA-1AH Any of the tetravalent BBMs shown in the diagram, where Y is ABM1 and each of X, A, and B is ABM2 (for convenience, this configuration of ABM is designated as "Tv 2").

[0507] This disclosure further provides, as follows Figure 1AA-1AH Any of the tetravalent BBMs shown in the diagram, where A is ABM1 and each of X, Y, and B is ABM2 (for convenience, this configuration of ABM is designated as "Tv 3").

[0508] This disclosure further provides, as follows Figure 1AA-1AH Any of the tetravalent BBMs shown in the diagram, where B is ABM1 and each of X, Y, and A is ABM2 (for convenience, this configuration of ABM is designated as "Tv 4").

[0509] This disclosure further provides, as follows Figure 1AA-1AH Any of the tetravalent BBMs shown in the diagram, where X and Y are both ABM1 and A and B are both ABM2 (for convenience, this configuration of ABM is designated as "Tv 5").

[0510] This disclosure further provides, as follows Figure 1AA-1AH Any of the tetravalent BBMs shown in the diagram, where X and A are both ABM1 and Y and B are both ABM2 (for convenience, this configuration of ABM is designated as "Tv 6").

[0511] This disclosure further provides, as follows Figure 1AA-1AHAny of the tetravalent BBMs shown in the diagram, where X and B are both ABM1 and Y and A are both ABM2 (for convenience, this configuration of ABM is designated as "Tv 7").

[0512] This disclosure further provides, as follows Figure 1AA-1AH Any of the tetravalent BBMs shown in the diagram, where Y and A are both ABM1 and X and B are both ABM2 (for convenience, this configuration of ABM is designated as "Tv 8").

[0513] This disclosure further provides, as follows Figure 1AA-1AH Any of the tetravalent BBMs shown in the diagram, where Y and B are both ABM1 and X and A are both ABM2 (for convenience, this configuration of ABM is designated as "Tv 9").

[0514] This disclosure further provides, as follows Figure 1AA-1AH Any of the tetravalent BBMs shown in the diagram, where A and B are both ABM1 and X and Y are both ABM2 (for convenience, this configuration of ABM is designated as "Tv 10").

[0515] This disclosure further provides, as follows Figure 1AA-1AH Any of the tetravalent BBMs shown in the diagram, where each of X, Y, and A is ABM1 and B is ABM2 (for convenience, this configuration of ABM is designated as "Tv 11").

[0516] This disclosure further provides, as follows Figure 1AA-1AH Any of the tetravalent BBMs shown in the diagram, where each of X, Y, and B is ABM1 and A is ABM2 (for convenience, this configuration of ABM is designated as "Tv 12").

[0517] This disclosure further provides, as follows Figure 1AA-1AH Any of the tetravalent BBMs shown in the diagram, where each of X, A, and B is ABM1 and Y is ABM2 (for convenience, this configuration of ABM is designated as "Tv 13").

[0518] This disclosure further provides, as follows Figure 1AA-1AH Any one of the tetravalent BBMs shown in the diagram, where each of Y, A, and B is ABM1 and X is ABM2 (for convenience, this configuration of ABM is designated as "Tv 14").

[0519] 7.6. Trispecific binding molecular configuration

[0520] An exemplary TBM configuration is shown in Figure 2. Figure 2A It shows Figure 2B-1VThe components of the TBM conformation shown in Figure 2, scFv, Fab, non-immunoglobulin-based ABM, and Fc, can each have the features described for these components in Sections 7.3 and 7.4. The components of the TBM conformation shown in Figure 2 can associate with each other by any of the methods described in Sections 7.3 and 7.4 (e.g., via direct bonds, ABM linkers, disulfide bonds, Fc domains modified with mortar-and-mortar interactions, etc.). The orientations and associations of the various components shown in Figure 2 are merely exemplary; as those skilled in the art will understand, other orientations and associations may be suitable (e.g., as described in Sections 7.3 and 7.4).

[0521] The TBM is not limited to the configuration shown in Figure 2. Other configurations that may 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 Frontiers] 8:38; Brinkmann & Kontermann, 2017, mAbs 9:2,182-212; US2016 / 0355600; Klein et al., 2016, MAbs8(6):1010-20; and US2017 / 0145116.

[0522] 7.6.1. Exemplary Trivalent TBM

[0523] The TBMs disclosed herein may be trivalent, meaning 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.

[0524] An exemplary trivalent TBM configuration is shown in Figures 2B to 2P middle.

[0525] like Figure 2B-2K As depicted in 2N-2P, TBM may contain two halves, one containing two ABMs and the other containing one ABM, the two halves being paired via an Fc domain.

[0526] exist Figure 2B In one embodiment, the first (or left) hemibody comprises an scFv and an Fc region, and the second (or right) hemibody comprises a Fab, an scFv, and an Fc region. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0527] exist Figure 2C In one embodiment, the first (or left) hemibody comprises two Fab and Fc regions, and the second (or right) hemibody comprises Fab and Fc regions. The first and second hemibody associate by forming Fc regions of Fc domains.

[0528] exist Figure 2D In one embodiment, the first (or left) hemibody comprises a Fab, scFv, and Fc region, and the second (or right) hemibody comprises a Fab and an Fc region. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0529] exist Figure 2E In one embodiment, the first (or left) hemibody comprises an scFv and an Fc region, and the second (or right) hemibody comprises two Fab and Fc regions. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0530] exist Figure 2F In one embodiment, the first (or left) hemibody comprises scFv, an Fc region, and Fab, and the second (or right) hemibody comprises Fab and an Fc region. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0531] exist Figure 2G In one embodiment, the first (or left) hemibody comprises scFv and an Fc region, and the second (or right) hemibody comprises Fab, an Fc region, and scFV. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0532] exist Figure 2H In one embodiment, the first (or left) hemibody comprises two Fab and Fc regions, and the second (or right) hemibody comprises a non-immunoglobulin-based ABM and Fc region. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0533] exist Figure 2I In one embodiment, the first (or left) hemibody comprises a Fab, scFv, and an Fc region, and the second (or right) hemibody comprises a non-immunoglobulin-based ABM and an Fc region. The first and second hemibody associate by forming an Fc region with an Fc domain.

[0534] exist Figure 2J In one embodiment, the first (or left) hemibody comprises a Fab and an Fc region, and the second (or right) hemibody comprises scFv, a non-immunoglobulin-based ABM, and an Fc region. The first and second hemibody associate by forming an Fc region with an Fc domain.

[0535] exist Figure 2K In one embodiment, the first (or left) hemibody comprises an scFv and an Fc region, and the second (or right) hemibody comprises an scFv, an Fc region, and a second scFv. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0536] exist Figure 2N In one embodiment, the first (or left) hemibody comprises Fab, an Fc region, and scFv, and the second (or right) hemibody comprises Fab and an Fc region. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0537] exist Figure 2O In one embodiment, the first (or left) hemibody comprises a Fab, an Fc region, and scFab, and the second (or right) hemibody comprises a Fab and an Fc region. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0538] exist Figure 2P In one embodiment, the first (or left) hemibody comprises Fab, a non-immunoglobulin-based ABM, and an Fc region, and the second (or right) hemibody comprises scFv and an Fc region. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0539] Alternatively, such as Figure 2L As described, a trivalent TBM may comprise two half-antibodies, each containing a complete ABM and a portion of another ABM (one being VH and the other VL). The two half-antibodies pair via an Fc domain, thus VH and VL associate to form a complete antigen-binding Fv domain.

[0540] The TBM can be a single chain, such as Figure 2M As shown. Figure 2M The TBM contains three scFv structural domains connected by connectors.

[0541] exist Figure 2B-2P In each configuration shown, each structural 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 TBM contains one ABM1, one ABM2, and one ABM3.

[0542] Therefore, this disclosure provides, as follows Figures 2B to 2P Any of the trivalent TBMs shown in the diagram, where X is ABM1, Y is ABM3 and Z is ABM2 (for convenience, this configuration of ABM is designated as "T1").

[0543] This disclosure also provides, as well as Figures 2B to 2PAny of the trivalent TBMs shown in the diagram, where X is ABM1, Y is ABM2 and Z is ABM3 (for convenience, this configuration of ABM is designated as "T2").

[0544] This disclosure further provides, as follows Figures 2B to 2P Any of the trivalent TBMs shown in the diagram, where X is ABM3, Y is ABM1 and Z is ABM2 (for convenience, this configuration of ABM is designated as "T3").

[0545] This disclosure further provides information such as Figures 2B to 2P Any of the trivalent TBMs shown in the diagram, where X is ABM3, Y is ABM2 and Z is ABM1 (for convenience, this configuration of ABM is designated as "T4").

[0546] This disclosure further provides information such as Figures 2B to 2P Any of the trivalent TBMs shown in the diagram, where X is ABM2, Y is ABM1 and Z is ABM3 (for convenience, this configuration of ABM is designated as "T5").

[0547] This disclosure further provides information such as Figures 2B to 2P Any of the trivalent TBMs shown in the diagram, where X is ABM2, Y is ABM3 and Z is ABM1 (for convenience, this configuration of ABM is designated as "T6").

[0548] 7.6.2. Exemplary tetravalent TBM

[0549] The TBMs disclosed herein may be tetravalent, that is, 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.

[0550] An exemplary tetravalent TBM configuration is shown in Figure 2Q-2S middle.

[0551] like Figure 2Q-2S The depicted tetravalent TBM may contain two halves, each containing two complete ABMs, the two halves being paired via an Fc domain.

[0552] exist Figure 2Q In one embodiment, the first (or left) hemibody comprises a Fab, an Fc region, and a second Fab, and the second (or right) hemibody comprises a Fab, an Fc region, and a second Fab. The first and second hemibody associate by forming the Fc region of the Fc domain.

[0553] exist Figure 2RIn one embodiment, the first (or left) hemibody comprises Fab, an Fc region, and scFv, and the second (or right) hemibody comprises Fab, an Fc region, and scFv. The first and second hemibody associate by forming the Fc region of the Fc domain.

[0554] exist Figure 2S In one embodiment, the first (or left) hemibody comprises Fab, an Fc region, and scFv, and the second (or right) hemibody comprises scFv, an Fc region, and Fab. The first and second hemibody associate by forming the Fc region of the Fc domain.

[0555] exist Figure 2Q-2S In the configurations shown, each of X, Y, Z, and A represents ABM1, ABM2, or ABM3 (though not necessarily in such an order), provided that the TBM contains at least one ABM1, at least one ABM2, and at least one ABM3. Therefore, the tetravalent ABM will comprise two ABMs targeting the CD19, TCR complex, and one of CD2 or TAA. In some cases, the tetravalent TBM has two CD19 ABMs.

[0556] Therefore, this disclosure provides as follows Figure 2Q-2S The tetravalent TBMs shown in Table 9 are as follows, where X, Y, Z, and A are components of the CD19, TCR complex and ABMs of CD2 or TAA.

[0557]

[0558]

[0559] 7.6.3. Exemplary pentavalent TBM

[0560] The TBMs disclosed herein may be pentavalent, that is, they have five antigen-binding domains, one, two, or three of which bind CD19, one, two, or three of which bind components of the TCR complex, and one, two, or three of which bind CD2 or TAA.

[0561] An exemplary pentavalent TBM configuration is shown in Figure 2T middle.

[0562] like Figure 2T As described, a pentavalent TBM may contain two halves, one containing two complete ABMs and the other containing one complete ABM, the two halves being paired via an Fc domain.

[0563] exist Figure 2TIn one embodiment, the first (or left) hemibody comprises Fab, scFv, and an Fc region, and the second (or right) hemibody comprises Fab, an Fc region, and scFv. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0564] exist Figure 2T In the configurations shown, each of X, Y, Z, A, and B represents ABM1, ABM2, or ABM3 (although not necessarily in such an order), provided that the TBM contains at least one ABM1, one ABM2, and one ABM3. Therefore, the pentavalent TBM can comprise two ABMs targeting the CD19, TCR complex component and two of CD2 or TAA, or three ABMs targeting the CD19, TCR complex component and one of CD2 or 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.

[0565] Therefore, this disclosure provides, as follows Figure 2T The pentavalent TBMs shown are X, Y, Z, A, and B, which are components for the CD19, TCR complex, and ABMs for CD2 or TAA, as shown in Table 10.

[0566]

[0567]

[0568]

[0569]

[0570] 7.6.4. Exemplary Hexavalent TBM

[0571] The TBMs disclosed herein may be hexavalent, that is, they have six antigen-binding domains, one, two, three, or four of which bind CD19, one, two, three, or four of which bind components of the TCR complex, and one, two, three, or four of which bind CD2 or TAA.

[0572] An exemplary hexavalent TBM configuration is shown in Figure 2U-2V middle.

[0573] like Figure 2U-2V As described, a pentavalent TBM may contain two halves, one containing two complete ABMs and the other containing one complete ABM, the two halves being paired via an Fc domain.

[0574] exist Figure 2UIn one embodiment, the first (or left) hemibody comprises Fab, a second Fab, an Fc region, and scFv, and the second (or right) hemibody comprises Fab, a second Fab, an Fc region, and scFv. The first and second hemibody associate by forming the Fc region of the Fc domain.

[0575] exist Figure 2V In one embodiment, the first (or left) hemibody comprises a first Fv, a second Fv, a third Fv, and an Fc region, and the second (or right) hemibody comprises a first Fv, a second Fv, a third Fv, and an Fc region. The first and second hemibody associate by forming the Fc region of the Fc domain.

[0576] exist Figure 2U-2V In the configurations shown, each of X, Y, Z, A, B, and C represents ABM1, ABM2, or ABM3 (although not necessarily in such an order), provided that the TBM contains at least one ABM1, one ABM2, and one ABM3. Therefore, the hexavalent TBM can include (i) two ABMs targeting each of the components of the CD19, TCR complex, and CD2 or TAA; (ii) three ABMs targeting the components of the CD19, TCR complex, and one of CD2 or TAA; or (iii) four ABMs targeting the components of the CD19, TCR complex, and one of CD2 or TAA. For example, a hexavalent ABM can include three ABMs targeting CD19, two ABMs targeting CD2 or TAA, and one ABM targeting the component of the TCR complex. As another example, a hexavalent ABM can include three ABMs targeting CD19, two ABMs targeting the component of the TCR complex, and one ABM targeting CD2 or 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.

[0577] Therefore, this disclosure provides, as follows Figure 2U-2V Any of the hexavalent TBMs shown in Table 11, where X, Y, Z, A, B, and C are components for the CD19, TCR complex, and CD2 or TAA ABMs, are shown in Table 11.

[0578]

[0579]

[0580]

[0581]

[0582]

[0583]

[0584]

[0585]

[0586]

[0587]

[0588]

[0589] 7.7.TCR ABM

[0590] The MBM disclosed herein contains ABM that specifically binds to CD19 and ABM2 that is specific to different antigens. In the BBM, type 1 TBM, and type 2 TBM disclosed herein, ABM2 can bind to components of the TCR complex. The TCR is a disulfide-linked membrane-anchored heterodimer protein, typically composed of highly variable α and β chains expressed as a portion of a complex with an invariant CD3 chain molecule. T cells expressing this receptor are called α:β (or αβ) T cells, although a minority of T cells (called γδ T cells) express alternative receptors (formed by variable γ and δ chains).

[0591] In the embodiments, MBM contains ABM that specifically binds to CD3.

[0592] 7.7.1.CD3 ABM

[0593] The MBM may contain ABM that specifically binds to CD3. The term "CD3" refers to the cluster 3 co-receptor of the T cell receptor (or a co-receptor complex, or a polypeptide chain of the co-receptor complex). The amino acid sequence of the human CD3 polypeptide chain is provided in NCBI accessions P04234, P07766, and P09693. CD3 proteins may also include variants. CD3 proteins may also include fragments. CD3 proteins also include post-translational modifications to the CD3 amino acid sequence. Post-translational modifications include, but are not limited to, N-linked and O-linked glycosylation.

[0594] In some embodiments, the MBM may comprise an ABM, which is an anti-CD3 antibody (e.g., as described in US2016 / 0355600, WO 2014 / 110601, and WO 2014 / 145806) or its antigen-binding domain. Exemplary anti-CD3 VH, VL, and scFV sequences that can be used in the MBM are provided in Table 12A.

[0595]

[0596]

[0597]

[0598]

[0599]

[0600]

[0601]

[0602]

[0603] The CDR sequences of many CD3 binding compounds defined by the Kabat numbering scheme (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition, Department of Public Health, National Institutes of Health, Bethesda, MD), the Josiah numbering scheme (Al-Lazikani et al., 1997, J. Mol. Biol, 273: 927-948), and combinations of Kabat and Josiah numbering are provided in Tables 12B-12D.

[0604]

[0605]

[0606]

[0607]

[0608]

[0609]

[0610]

[0611]

[0612]

[0613]

[0614]

[0615]

[0616]

[0617]

[0618]

[0619]

[0620]

[0621]

[0622]

[0623]

[0624]

[0625]

[0626] In some embodiments, the MBM may include a CD3 ABM containing a CDR such as any one of CD3-1 to CD3-130 as defined by the Kabat numbering (e.g., as listed in Table 12B). In other embodiments, the MBM may include a CD3 ABM containing a CDR such as any one of CD3-1 to CD3-130 as defined by the Josiah numbering (e.g., as listed in Table 12C). In still other embodiments, the MBM may include a CD3 ABM containing a CDR such as any one of CD3-1 to CD3-130 as defined by a combination of Kabat and Josiah numbering (e.g., as listed in Table 12D).

[0627] In some embodiments, a CD3 ABM contains a CDR sequence of CD3-1. In some embodiments, a CD3 ABM contains a CDR sequence of CD3-2. In some embodiments, a CD3 ABM contains a CDR sequence of CD3-3. In some embodiments, a CD3 ABM contains a CDR sequence of CD3-4. In some embodiments, a CD3 ABM contains a CDR sequence of CD3-5. In some embodiments, a CD3 ABM contains a CDR sequence of CD3-6. In some embodiments, a CD3 ABM contains a CDR sequence of CD3-7. In some embodiments, a CD3 ABM contains a CDR sequence of CD3-8. In some embodiments, a CD3 ABM contains a CDR sequence of CD3-9. In some embodiments, a CD3 ABM contains a CDR sequence of CD3-10. In some embodiments, a CD3 ABM contains a CDR sequence of CD3-11. In some embodiments, a CD3 ABM contains a CDR sequence of CD3-12. In some embodiments, a CD3 ABM contains a CDR sequence of CD3-13. In some embodiments, a CD3 ABM contains a CDR sequence of CD3-14. In some embodiments, a CD3 ABM contains a CDR sequence of CD3-15. In some embodiments, a CD3 ABM contains CDR sequences of CD3-16. In some embodiments, a CD3 ABM contains CDR sequences of CD3-17. In some embodiments, a CD3 ABM contains CDR sequences of CD3-18. In some embodiments, a CD3 ABM contains CDR sequences of CD3-19. In some embodiments, a CD3 ABM contains CDR sequences of CD3-20. In some embodiments, a CD3 ABM contains CDR sequences of CD3-21. In some embodiments, a CD3 ABM contains CDR sequences of CD3-22. In some embodiments, a CD3 ABM contains CDR sequences of CD3-23. In some embodiments, a CD3 ABM contains CDR sequences of CD3-24. In some embodiments, a CD3 ABM contains CDR sequences of CD3-25. In some embodiments, a CD3 ABM contains CDR sequences of CD3-26. In some embodiments, a CD3 ABM contains CDR sequences of CD3-27. In some embodiments, a CD3 ABM contains CDR sequences of CD3-28. In some embodiments, a CD3 ABM contains CDR sequences of CD3-29. In some embodiments, a CD3 ABM contains CDR sequences of CD3-30. In some embodiments, a CD3 ABM contains CDR sequences of CD3-31. In some embodiments, a CD3 ABM contains CDR sequences of CD3-32. In some embodiments, a CD3 ABM contains CDR sequences of CD3-33.In some embodiments, a CD3 ABM contains CDR sequences of CD3-34. In some embodiments, a CD3 ABM contains CDR sequences of CD3-35. In some embodiments, a CD3 ABM contains CDR sequences of CD3-36. In some embodiments, a CD3 ABM contains CDR sequences of CD3-37. In some embodiments, a CD3 ABM contains CDR sequences of CD3-38. In some embodiments, a CD3 ABM contains CDR sequences of CD3-39. In some embodiments, a CD3 ABM contains CDR sequences of CD3-40. In some embodiments, a CD3 ABM contains CDR sequences of CD3-41. In some embodiments, a CD3 ABM contains CDR sequences of CD3-42. In some embodiments, a CD3 ABM contains CDR sequences of CD3-43. In some embodiments, a CD3 ABM contains CDR sequences of CD3-44. In some embodiments, a CD3 ABM contains CDR sequences of CD3-45. In some embodiments, a CD3 ABM contains CDR sequences of CD3-46. In some embodiments, a CD3 ABM contains CDR sequences of CD3-47. In some embodiments, a CD3 ABM contains CDR sequences of CD3-48. In some embodiments, a CD3 ABM contains CDR sequences of CD3-49. In some embodiments, a CD3 ABM contains CDR sequences of CD3-50. In some embodiments, a CD3 ABM contains CDR sequences of CD3-51. In some embodiments, a CD3 ABM contains CDR sequences of CD3-52. In some embodiments, a CD3 ABM contains CDR sequences of CD3-53. In some embodiments, a CD3 ABM contains CDR sequences of CD3-54. In some embodiments, a CD3 ABM contains CDR sequences of CD3-55. In some embodiments, a CD3 ABM contains CDR sequences of CD3-56. In some embodiments, a CD3 ABM contains CDR sequences of CD3-57. In some embodiments, a CD3 ABM contains CDR sequences of CD3-58. In some embodiments, a CD3 ABM contains CDR sequences of CD3-59. In some embodiments, a CD3 ABM contains CDR sequences of CD3-60. In some embodiments, a CD3 ABM contains CDR sequences of CD3-61. In some embodiments, CD3 ABM comprises CDR sequences of CD3-62. In some embodiments, CD3 ABM comprises CDR sequences of CD3-63. In some embodiments, CD3 ABM comprises CDR sequences of CD3-64. In some embodiments, CD3 ABM comprises CDR sequences of CD3-65. In some embodiments, CD3 ABM comprises CDR sequences of CD3-66.In some embodiments, CD3ABM comprises CDR sequences of CD3-67. In some embodiments, CD3 ABM comprises CDR sequences of CD3-68. In some embodiments, CD3 ABM comprises CDR sequences of CD3-69. In some embodiments, CD3 ABM comprises CDR sequences of CD3-70. In some embodiments, CD3 ABM comprises CDR sequences of CD3-71. In some embodiments, CD3ABM comprises CDR sequences of CD3-72. In some embodiments, CD3 ABM comprises CDR sequences of CD3-73. In some embodiments, CD3 ABM comprises CDR sequences of CD3-74. In some embodiments, CD3ABM comprises CDR sequences of CD3-75. In some embodiments, CD3 ABM comprises CDR sequences of CD3-76. In some embodiments, CD3ABM comprises CDR sequences of CD3-77. In some embodiments, CD3 ABM comprises CDR sequences of CD3-78. In some embodiments, CD3 ABM comprises CDR sequences of CD3-79. In some embodiments, CD3 ABM comprises CDR sequences of CD3-80. In some embodiments, a CD3 ABM contains CDR sequences of CD3-81. In some embodiments, a CD3 ABM contains CDR sequences of CD3-82. In some embodiments, a CD3 ABM contains CDR sequences of CD3-83. In some embodiments, a CD3 ABM contains CDR sequences of CD3-84. In some embodiments, a CD3 ABM contains CDR sequences of CD3-85. In some embodiments, a CD3 ABM contains CDR sequences of CD3-86. In some embodiments, a CD3 ABM contains CDR sequences of CD3-87. In some embodiments, a CD3 ABM contains CDR sequences of CD3-88. In some embodiments, a CD3 ABM contains CDR sequences of CD3-89. In some embodiments, a CD3 ABM contains CDR sequences of CD3-90. In some embodiments, a CD3 ABM contains CDR sequences of CD3-91. In some embodiments, a CD3 ABM contains CDR sequences of CD3-92. In some embodiments, a CD3 ABM contains CDR sequences of CD3-93. In some embodiments, a CD3 ABM contains CDR sequences of CD3-94. In some embodiments, CD3ABM comprises CDR sequences of CD3-95. In some embodiments, CD3 ABM comprises CDR sequences of CD3-96. In some embodiments, CD3ABM comprises CDR sequences of CD3-97. In some embodiments, CD3 ABM comprises CDR sequences of CD3-98. In some embodiments, CD3 ABM comprises CDR sequences of CD3-99.In some embodiments, a CD3 ABM contains CDR sequences of CD3-100. In some embodiments, a CD3 ABM contains CDR sequences of CD3-101. In some embodiments, a CD3 ABM contains CDR sequences of CD3-102. In some embodiments, a CD3 ABM contains CDR sequences of CD3-103. In some embodiments, a CD3 ABM contains CDR sequences of CD3-104. In some embodiments, a CD3 ABM contains CDR sequences of CD3-105. In some embodiments, a CD3 ABM contains CDR sequences of CD3-106. In some embodiments, a CD3 ABM contains CDR sequences of CD3-107. In some embodiments, a CD3 ABM contains CDR sequences of CD3-108. In some embodiments, a CD3 ABM contains CDR sequences of CD3-109. In some embodiments, a CD3 ABM contains CDR sequences of CD3-110. In some embodiments, a CD3 ABM contains CDR sequences of CD3-111. In some embodiments, a CD3 ABM contains CDR sequences of CD3-112. In some embodiments, a CD3 ABM contains CDR sequences of CD3-113. In some embodiments, a CD3 ABM contains CDR sequences of CD3-114. In some embodiments, a CD3 ABM contains CDR sequences of CD3-115. In some embodiments, a CD3 ABM contains CDR sequences of CD3-116. In some embodiments, a CD3 ABM contains CDR sequences of CD3-117. In some embodiments, a CD3 ABM contains CDR sequences of CD3-118. In some embodiments, a CD3 ABM contains CDR sequences of CD3-119. In some embodiments, a CD3 ABM contains CDR sequences of CD3-120. In some embodiments, a CD3 ABM contains CDR sequences of CD3-121. In some embodiments, a CD3 ABM contains CDR sequences of CD3-122. In some embodiments, a CD3 ABM contains CDR sequences of CD3-123. In some embodiments, a CD3 ABM contains CDR sequences of CD3-124. In some embodiments, a CD3 ABM contains CDR sequences of CD3-125. In some embodiments, a CD3 ABM contains CDR sequences of CD3-126. In some embodiments, a CD3 ABM contains CDR sequences of CD3-127. In some embodiments, a CD3 ABM contains CDR sequences of CD3-126. In some embodiments, a CD3 ABM contains CDR sequences of CD3-127. In some embodiments, a CD3 ABM contains CDR sequences of CD3-128. In some embodiments, a CD3 ABM contains CDR sequences of CD3-129.In some embodiments, a CD3 ABM contains a CD3-130 CDR sequence.

[0628] MBM may contain complete heavy and light variable sequences of any one of CD3-1 to CD3-130. In some embodiments, MBM contains CD3 ABM, which contains VH and VL sequences of CD3-1. In some embodiments, MBM contains CD3 ABM, which contains VH and VL sequences of CD3-1. In some embodiments, MBM contains CD3 ABM, which contains VH and VL sequences of CD3-2. In some embodiments, MBM contains CD3 ABM, which contains VH and VL sequences of CD3-3. In some embodiments, MBM contains CD3ABM, which contains VH and VL sequences of CD3-4. In some embodiments, MBM contains CD3 ABM, which contains VH and VL sequences of CD3-5. In some embodiments, MBM contains CD3 ABM, which contains VH and VL sequences of CD3-6. In some embodiments, MBM contains CD3 ABM, which contains VH and VL sequences of CD3-7. In some embodiments, MBM contains CD3 ABM, which contains VH and VL sequences of CD3-8. In some embodiments, the MBM comprises a CD3 ABM containing VH and VL sequences of CD3-9. In some embodiments, the MBM comprises a CD3 ABM containing VH and VL sequences of CD3-10. In some embodiments, the MBM comprises a CD3 ABM containing VH and VL sequences of CD3-11. In some embodiments, the MBM comprises a CD3 ABM containing VH and VL sequences of CD3-12. In some embodiments, the MBM comprises a CD3 ABM containing VH and VL sequences of CD3-13. In some embodiments, the MBM comprises a CD3 ABM containing VH and VL sequences of CD3-14. In some embodiments, the MBM comprises a CD3 ABM containing VH and VL sequences of CD3-15. In some embodiments, the MBM comprises a CD3 ABM containing VH and VL sequences of CD3-16. In some embodiments, the MBM comprises a CD3 ABM containing VH and VL sequences of CD3-17. In some embodiments, the MBM comprises a CD3 ABM containing VH and VL sequences of CD3-18. In some embodiments, the MBM comprises a CD3 ABM containing VH and VL sequences of CD3-19. In some embodiments, the MBM comprises a CD3 ABM containing VH and VL sequences of CD3-20. In some embodiments, the MBM comprises a CD3 ABM containing VH and VL sequences of CD3-21. In some embodiments, the MBM comprises a CD3 ABM containing VH and VL sequences of CD3-22. In some embodiments, the MBM comprises a CD3 ABM containing VH and VL sequences of CD3-23. In some embodiments, the MBM comprises a CD3 ABM containing VH and VL sequences of CD3-24.In some embodiments, the MBM comprises a CD3 ABM containing VH and VL sequences of CD3-25. In some embodiments, the MBM comprises a CD3 ABM containing VH and VL sequences of CD3-26. In some embodiments, the MBM comprises a CD3 ABM containing VH and VL sequences of CD3-27. In some embodiments, the MBM comprises a CD3 ABM containing VH and VL sequences of CD3-28. In some embodiments, the MBM comprises a CD3 ABM containing VH and VL sequences of CD3-129. In some embodiments, the MBM comprises a CD3 ABM containing VH and VL sequences of CD3-130.

[0629] In addition to the CDR groups described in Tables 12B-12D (i.e., groups of six CDRs for each of CD3-1 to CD3-130), this disclosure provides variant CDR groups. In one embodiment, a group of six CDRs may have 1, 2, 3, 4, or 5 amino acid variations, as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biofilm layer interferometry, e.g., Octet assay), provided that the CD3 ABM remains capable of binding to the target antigen.

[0630] In addition to the variable heavy and variable light domains disclosed in Table 12A (which form the CD3-targeting ABM), this disclosure provides variant VH and VL domains. In one embodiment, the variant VH and VL domains may each have a variation of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biofilm layer interferometry, e.g., Octet assay), as long as the ABM remains capable of binding to the target antigen. In another embodiment, the variant VH and VL are at least 90%, 95%, 97%, 98%, or 99% identical to the respective VH or VL disclosed in Table 12A, as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biofilm layer interferometry, e.g., Octet assay), as long as the ABM remains capable of binding to the target antigen.

[0631] In some embodiments, the MBM may comprise an ABM, which is a CD3-binding molecule or its antigen-binding domain as described in WO 2020 / 052692. Tables AA through AJ-2 (collectively, “Table A”) list sequences of CD3-binding sequences that may be included in a CD3-binding ABM.

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[0734] The CDR sequences for group C1 in Table AA are based on the Kabat CDR sequences, Josiah CDR sequences, IMGT CDR sequences, and combinations thereof of CD3-binding molecules NOV292, NOV589, NOV567, and CD3-binding molecules whose names include "sp11a". The CDR sequences for group C2 in Table AB are based on the Kabat CDR sequences, Josiah CDR sequences, IMGT CDR sequences, and combinations thereof of CD3-binding molecules NOV453, NOV229, NOV580, NOV221, and CD3-binding molecules whose names include "sp9a". The CDR sequences for group C3 in Table AC are based on the Kabat CDR sequences, Josiah CDR sequences, IMGT CDR sequences, and combinations thereof of CD3-binding molecules NOV123, sp10b, NOV110, and NOV832.

[0735] 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.

[0736] In some embodiments, a CD3 ABM may comprise a heavy chain CDR having an amino acid sequence having a common sequence of any of the CDRs listed in Table AA, Table AB, or Table AC. In specific embodiments, a CD3 ABM may comprise (or alternatively, consist of) one, two, three, or more heavy chain CDRs selected from those described in Table AA, Table AB, or Table AC.

[0737] In some embodiments, a CD3 ABM may comprise a light chain CDR having an amino acid sequence having a common sequence of any of the CDRs listed in Table AA, Table AB, or Table AC. In specific embodiments, a CD3 ABM may comprise (or alternatively, consist of) one, two, three, or more light chain CDRs selected from those described in Table AA, Table AB, or Table AC.

[0738] In some embodiments, the CD3 ABM may include the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences listed in Table AA.

[0739] 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 The amino acid is F. In some embodiments, it is specified as X in Table AA. 55 The amino acid is Y. In some embodiments, it is specified as X in Table AA. 55 The amino acid 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 X9 in Table AA is W. 10 The amino acid is H. In some embodiments, it is specified as X in Table AA. 10 The amino acid is Y. In some embodiments, it is specified as X in Table AA. 11 The amino acid is S. In some embodiments, it is specified as X in Table AA. 11 The amino acid is G. In some embodiments, it is specified as X in Table AA. 12 The amino acid is I. In some embodiments, it is specified as X in Table AA. 12 The amino acid is L. In some embodiments, it is specified as X in Table AA. 13 The amino acid is V. In some embodiments, it is specified as X in Table AA. 13 The amino acid is G. In some embodiments, it is specified as X in Table AA.14 The amino acid is R. In some embodiments, it is specified as X in Table AA. 14 The amino acid is N. In some embodiments, it is specified as X in Table AA. 15 The amino acid is D. In some embodiments, it is specified as X in Table AA. 15 The amino acid is E. In some embodiments, it is specified as X in Table AA. 15 The amino acid is L. In some embodiments, it is specified as X in Table AA. 16 The amino acid is G. In some embodiments, it is specified as X in Table AA. 16 The amino acid is N. In some embodiments, it is specified as X in Table AA. 16 The amino acid is E. In some embodiments, it is specified as X in Table AA. 17 The amino acid is R. In some embodiments, it is specified as X in Table AA. 17 The amino acid is S. In some embodiments, it is specified as X in Table AA. 18 The amino acid is V. In some embodiments, it is specified as X in Table AA. 18 The amino acid is T. In some embodiments, it is specified as X in Table AA. 19 The amino acid is N. In some embodiments, it is specified as X in Table AA. 19 The amino acid is T. In some embodiments, it is specified as X in Table AA. 20 The amino acid is R. In some embodiments, it is specified as X in Table AA. 20 The amino acid is L. In some embodiments, it is specified as X in Table AA. 21 The amino acid is F. In some embodiments, it is specified as X in Table AA. 21 The amino acid is E. In some embodiments, it is specified as X in Table AA. 22 The amino acid is S. In some embodiments, it is specified as X in Table AA. 22 The amino acid is Y. In some embodiments, it is specified as X in Table AA. 23 The amino acid is S. In some embodiments, it is specified as X in Table AA. 23 The amino acid is Y. In some embodiments, it is specified as X in Table AA. 24 The amino acid is S. In some embodiments, it is specified as X in Table AA. 24 The amino acid is A. In some embodiments, it is specified as X in Table AA. 25 The amino acid is H. In some embodiments, it is specified as X in Table AA. 25 The amino acid is T. In some embodiments, it is specified as X in Table AA. 26 The amino acid is F. In some embodiments, it is specified as X in Table AA.26 The amino acid is Y. In some embodiments, it is specified as X in Table AA. 27 The amino acid is W. In some embodiments, it is specified as X in Table AA. 27 The amino acid is Y.

[0740] In some embodiments, a CD3 ABM may contain a CDR-H1 sequence C1-1. In some embodiments, a CD3 ABM may contain a CDR-H1 sequence C1-2. In some embodiments, a CD3 ABM may contain a CDR-H1 sequence C1-3. In some embodiments, a CD3 ABM may contain a CDR-H1 sequence C1-4.

[0741] In some embodiments, a CD3 ABM may contain CDR-H2 sequences C1-5. In some embodiments, a CD3 ABM may contain CDR-H2 sequences C1-6. In some embodiments, a CD3 ABM may contain CDR-H2 sequences C1-7.

[0742] In some embodiments, a CD3 ABM may contain CDR-H3 sequences C1-8. In some embodiments, a CD3 ABM may contain CDR-H3 sequences C1-9. In some embodiments, a CD3 ABM may contain CDR-H3 sequences C1-10. In some embodiments, a CD3 ABM may contain CDR-H3 sequences C1-11.

[0743] In some embodiments, a CD3 ABM may contain CDR-L1 sequences C1-12. In some embodiments, a CD3 ABM may contain CDR-L1 sequences C1-13. In some embodiments, a CD3 ABM may contain CDR-L1 sequences C1-14. In some embodiments, a CD3 ABM may contain CDR-L1 sequences C1-15. In some embodiments, a CD3 ABM may contain CDR-L1 sequences C1-16. In some embodiments, a CD3 ABM may contain CDR-L1 sequences C1-17.

[0744] In some embodiments, a CD3 ABM may contain a CDR-L2 sequence C1-18. In some embodiments, a CD3 ABM may contain a CDR-L2 sequence C1-19.

[0745] In some embodiments, a CD3 ABM may include CDR-L3 sequences C1-20. In some embodiments, a CD3 ABM may include CDR-L3 sequences C1-21. In some embodiments, a CD3 ABM may include CDR-L3 sequences C1-22. In some embodiments, a CD3 ABM may include CDR-L3 sequences C1-23.

[0746] In some embodiments, the CD3 ABM may include the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences listed in Table AB.

[0747] In some embodiments, X is specified in table AB. 28 The amino acid is V. In some embodiments, it is specified as X in Table AB. 28 The amino acid is I. In some embodiments, it is specified as X in Table AB. 29 The amino acid is F. In some embodiments, it is specified as X in Table AB. 29 The amino acid is Y. In some embodiments, it is specified as X in Table AB. 30 The amino acid is N. In some embodiments, it is specified as X in Table AB. 30 The amino acid is S. In some embodiments, it is specified as X in Table AB. 31 The amino acid is A. In some embodiments, it is specified as X in Table AB. 31 The amino acid is S. In some embodiments, it is specified as X in Table AB. 32 The amino acid is T. In some embodiments, it is specified as X in Table AB. 32 The amino acid is K. In some embodiments, it is specified as X in Table AB. 33 The amino acid is T. In some embodiments, it is specified as X in Table AB. 33 The amino acid is A. In some embodiments, it is specified as X in Table AB. 34 The amino acid is S. In some embodiments, it is specified as X in Table AB. 34 The amino acid is R. In some embodiments, it is specified as X in Table AB. 35 The amino acid is N. In some embodiments, it is specified as X in Table AB. 35 The amino acid is G. In some embodiments, it is specified as X in Table AB. 36 The amino acid is S. In some embodiments, it is specified as X in Table AB. 36 The amino acid is A. In some embodiments, it is specified as X in Table AB. 37 The amino acid is A. In some embodiments, it is specified as X in Table AB. 37 The amino acid is T. In some embodiments, it is specified as X in Table AB. 37 The amino acid is S. In some embodiments, it is specified as X in Table AB. 38 The amino acid is N. In some embodiments, it is specified as X in Table AB. 38 The amino acid is D. In some embodiments, it is specified as X in Table AB.39 The amino acid is N. In some embodiments, it is specified as X in Table AB. 39 The amino acid is K. In some embodiments, it is specified as X in Table AB. 40 The amino acid is D. In some embodiments, it is specified as X in Table AB. 40 The amino acid is N. In some embodiments, it is specified as X in Table AB. 41 The amino acid is H. In some embodiments, it is specified as X in Table AB. 41 The amino acid is N. In some embodiments, it is specified as X in Table AB. 42 The amino acid is Q. In some embodiments, it is specified as X in Table AB. 42 The amino acid is E. In some embodiments, it is specified as X in Table AB. 43 The amino acid is R. In some embodiments, it is specified as X in Table AB. 43 The amino acid is S. In some embodiments, it is specified as X in Table AB. 43 The amino acid in it is G.

[0748] In some embodiments, a CD3 ABM may contain a CDR-H1 sequence C2-1. In some embodiments, a CD3 ABM may contain a CDR-H1 sequence C2-2. In some embodiments, a CD3 ABM may contain a CDR-H1 sequence C2-3. In some embodiments, a CD3 ABM may contain a CDR-H1 sequence C2-4.

[0749] In some embodiments, a CD3 ABM may contain the CDR-H2 sequence C2-5. In some embodiments, a CD3 ABM may contain the CDR-H2 sequence C2-6. In some embodiments, a CD3 ABM may contain the CDR-H2 sequence C2-7.

[0750] In some embodiments, a CD3 ABM may contain CDR-H3 sequences C2-8. In some embodiments, a CD3 ABM may contain CDR-H3 sequences C2-9.

[0751] In some embodiments, a CD3 ABM may include a CDR-L1 sequence C2-10. In some embodiments, a CD3 ABM may include a CDR-L1 sequence C2-11. In some embodiments, a CD3 ABM may include a CDR-L1 sequence C2-12.

[0752] In some embodiments, a CD3 ABM may include a CDR-L2 sequence C2-13. In some embodiments, a CD3 ABM may include a CDR-L2 sequence C2-14. In some embodiments, a CD3 ABM may include a CDR-L2 sequence C2-15.

[0753] In some embodiments, a CD3 ABM may contain a CDR-L3 sequence C2-16. In some embodiments, a CD3 ABM may contain a CDR-L3 sequence C2-17.

[0754] In some embodiments, the CD3 ABM may include the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences listed in Table AC.

[0755] In some embodiments, X is specified in table AC. 44 The amino acid is G. In some embodiments, it is specified as X in table AC. 44 The amino acid is A. In some embodiments, it is specified as X in table AC. 45 The amino acid is H. In some embodiments, it is specified as X in table AC. 45 The amino acid is N. In some embodiments, it is specified as X in Table AC. 46 The amino acid is D. In some embodiments, it is specified as X in table AC. 46 The amino acid is G. In some embodiments, it is specified as X in table AC. 47 The amino acid is A. In some embodiments, it is specified as X in table AC. 47 The amino acid is G. In some embodiments, it is specified as X in table AC. 48 The amino acid is N. In some embodiments, it is specified as X in Table AC. 48 The amino acid is K. In some embodiments, it is specified as X in table AC. 49 The amino acid is V. In some embodiments, it is specified as X in table AC. 49 The amino acid is A. In some embodiments, it is specified as X in table AC. 50 The amino acid is N. In some embodiments, it is specified as X in Table AC. 50 The amino acid is V. In some embodiments, it is specified as X in table AC. 51 The amino acid is A. In some embodiments, it is specified as X in table AC. 51 The amino acid is V. In some embodiments, it is specified as X in table AC. 52 The amino acid is Y. In some embodiments, it is specified as X in table AC. 52The amino acid is F. In some embodiments, it is specified as X in Table AC. 53 The amino acid is I. In some embodiments, it is specified as X in Table AC. 53 The amino acid is V. In some embodiments, it is specified as X in table AC. 54 The amino acid is I. In some embodiments, it is specified as X in Table AC. 54 The amino acid is H.

[0756] In some embodiments, a CD3 ABM may contain a CDR-H1 sequence C3-1. In some embodiments, a CD3 ABM may contain a CDR-H1 sequence C3-2. In some embodiments, a CD3 ABM may contain a CDR-H1 sequence C3-3. In some embodiments, a CD3 ABM may contain a CDR-H1 sequence C3-4.

[0757] In some embodiments, a CD3 ABM may contain the CDR-H2 sequence C3-5. In some embodiments, a CD3 ABM may contain the CDR-H2 sequence C3-6. In some embodiments, a CD3 ABM may contain the CDR-H2 sequence C3-7.

[0758] In some embodiments, a CD3 ABM may contain the CDR-H3 sequence C3-8. In some embodiments, a CD3 ABM may contain the CDR-H3 sequence C3-9.

[0759] In some embodiments, a CD3 ABM may include a CDR-L1 sequence C3-10. In some embodiments, a CD3 ABM may include a CDR-L1 sequence C3-11. In some embodiments, a CD3 ABM may include a CDR-L1 sequence C3-12.

[0760] In some embodiments, a CD3 ABM may contain a CDR-L2 sequence C3-13. In some embodiments, a CD3 ABM may contain a CDR-L2 sequence C3-14.

[0761] In some embodiments, a CD3 ABM may contain a CDR-L3 sequence C3-15. In some embodiments, a CD3 ABM may contain a CDR-L3 sequence C3-16.

[0762] In some embodiments, the CD3 ABM may include 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.

[0763] In some embodiments, the CD3 ABM may include 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.

[0764] In some embodiments, the CD3 ABM may include 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.

[0765] In some embodiments, the CD3 ABM may include 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.

[0766] In some embodiments, the CD3 ABM may include 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.

[0767] In some embodiments, a CD3 ABM may include the CDR-H1, CDR-H2 and CDR-H3 sequences listed in Table AI-1, and the corresponding CDR-L1, CDR-L2 and CDR-L3 sequences listed in Table AI-2.

[0768] In some embodiments, a CD3 ABM may comprise a heavy chain CDR having an amino acid sequence of any of the CDRs listed in Tables AB-1, AC-1, AD-1, AE-1, AF-1, AG-1, AH-1, or AI-1. In specific embodiments, a CD3 ABM may comprise (or alternatively, consist of) one, two, three, or more heavy chain CDRs selected from those described in Tables AB-1, AC-1, AD-1, AE-1, AF-1, AG-1, AH-1, and AI-1.

[0769] In some embodiments, a CD3 ABM may comprise a light chain CDR having an amino acid sequence of any of the CDRs listed in Tables AB-2, AC-2, AD-2, AE-2, AF-2, AG-2, AH-2, or AI-2. In specific embodiments, a CD3 ABM may comprise (or alternatively, consist of) one, two, three, or more light chain CDRs selected from those described in Tables AB-2, AC-2, AD-2, AE-2, AF-2, AG-2, AH-2, and AI-2.

[0770] Other CD3 ABMs include mutated amino acids, but their CDR regions still possess 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 wherein no more than 1, 2, 3, 4, or 5 amino acids in the CDR region are mutated when compared to the CDR sequences described in Table A.

[0771] In some embodiments, a CD3 ABM may comprise a VH and / or VL domain having an amino acid sequence having any of the VH and / or VL domains described in Table A. Other CD3 ABMs include VH and / or VL domains comprising an amino acid sequence 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, a CD3 ABM includes a VH and / or VL domain wherein 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.

[0772] VH and VL sequences (amino acid sequences and nucleotide sequences encoding said amino acid sequences) can be "mixed and matched" to produce additional CD3 ABMs. Such "mixed and matched" CD3 ABMs can be tested using binding assays known in the art (e.g., ELISA assays). When the strands are mixed and matched, the VH sequence from a particular VH / VL pair should be replaced with a structurally similar VH sequence. The VL sequence from a particular VH / VL pair should be replaced with a structurally similar VL sequence.

[0773] Therefore, in one embodiment, the CD3 ABM comprises: a heavy chain variable region (VH) containing an amino acid sequence selected from any of the VH sequences described in Tables A-J1; and a light chain variable region (VL) containing an amino acid sequence described in Tables A-J2.

[0774] In some embodiments, the antigen-binding domain that specifically binds to human CD3 is based on a non-immunoglobulin and, conversely, 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:

[0775] MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQWLWFAGKQLEDGRRTLSDYNIQKESTLKLWLVDKAAMQIFVYTRTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRRTLSDYNIALESGLHLVLRLRAA (SEQ ID NO: 305).

[0776] 7.7.2. TCR-α / βABM

[0777] MBMs can contain ABMs that specifically bind to the TCR-α chain, TCR-β chain, or TCR-αβ dimer. Exemplary anti-TCR-α / β antibodies are known (see, for example, US 2012 / 0034221; Borst et al., 1990, Hum Immunol. [Human Immunology] 29(3):175-88 (describes antibody BMA031)). The VH, VL, and cabat CDR sequences of antibody BMA031 are provided in Table 13.

[0778]

[0779]

[0780] In one embodiment, the TCR ABM may contain the CDR sequence of antibody BMA031. In other embodiments, the TCR ABM may contain the VH and VL sequences of antibody BMA031.

[0781] 7.7.3.TCR-γ / δABM

[0782] MBMs may contain ABMs that specifically bind to the TCR-γ chain, TCR-δ chain, or TCR-γδ dimer. Exemplary anti-TCR-γ / δ antibodies are known (see, for example, U.S. Patent No. 5,980,892 (which describes δTCS1, which is produced by a hybridoma deposited in ATCC with accession number HB 9578)).

[0783] 7.8.CD2 ABM

[0784] 7.8.1. Immunoglobulin-based CD2 ABM

[0785] Type 1 TBMs may comprise ABMs that are anti-CD2 antibodies or their antigen-binding domains. Exemplary anti-CD2 antibodies are known (see, for example, US 6,849,258, CN 102827281 A, US2003 / 0139579A1, and US 5,795,572). Table 14 provides exemplary CDR, VH, and VL sequences that may be included in anti-CD2 antibodies or their antigen-binding fragments for use in the MBMs disclosed herein.

[0786]

[0787]

[0788] In some embodiments, the CD2 ABM comprises the CDR sequence of CD2-1 (SEQ ID NO: 312-317). In some embodiments, the CD2 ABM comprises variable sequences of the heavy and light chains of CD2-1 (SEQ ID NO: 318 and 319, respectively). In some embodiments, the CD2 ABM comprises variable sequences of the heavy and light chains of hu1CD2-1 (SEQ ID NO: 320 and 321, respectively). In some embodiments, the CD2 ABM comprises variable sequences of the heavy and light chains of hu2CD2-1 (SEQ ID NO: 318 and 321, respectively).

[0789] In other embodiments, the CD2 ABM may comprise the CDR sequence of antibody 9D1 generated by a hybridoma deposited on May 16, 2012, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC6132, and described in CN 102827281A. In other embodiments, the CD2 ABM may comprise the CDR sequence of antibody LO-CD2b generated by a hybridoma deposited on June 22, 1999, at the United States Type Culture Collection (USC) with accession number PTA-802, and described in US 2003 / 0139579A1. In yet another embodiment, the CD2 ABM may comprise the CDR sequence of CD2 SFv-Ig produced by a construct expressing a clone in recombinant Escherichia coli, deposited on April 9, 1993 at ATCC with accession number 69277, and described in US 5,795,572.

[0790] In other embodiments, the CD2 ABM may comprise the VH and VL sequences of antibody 9D1. In other embodiments, the CD2 ABM may comprise the VH and VL sequences of antibody LO-CD2b. In yet another embodiment, the CD2 ABM may comprise the VH and VL sequences of CD2 SFv-Ig generated from a construct of a clone expressed in recombinant E. coli having ATCC accession number 69277.

[0791] 7.8.2. CD2 ABM based on CD58

[0792] In some respects, this disclosure provides a type 1 TBM comprising 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 a glycoprotein expressed on the surface of multiple cell types (Dustin et al., 1991, Annu. Rev. Immunol. [Annual Review of Immunology] 9:27) and plays a role in mediating T cell-APC interactions in both antigen-dependent and antigen-independent modes (Wallner et al., 1987, J. Exp. Med. [Journal of Experimental Medicine] 166:923). Therefore, in some respects, the CD2 ABM is a CD58 moiety. As used herein, the CD58 portion comprises an amino acid sequence having at least 70% sequence identity with the CD2-binding portion of CD58 (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 with 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 the CD58 fragment containing amino acid residues 30-123 of the 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. Therefore, in some respects, the CD58 portion contains an amino acid sequence that has at least 70% sequence identity with amino acids 30-123 of CD58 (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 with the amino acid sequence designated as CD58-6).

[0793] The interaction between CD58 and CD2 has been mapped using X-ray crystallography and molecular modeling. Substitution of residues E25, K29, K30, K32, D33, K34, E37, D84, and K87 (where the numbers refer to those in the mature polypeptide) reduces binding to CD2. Ikemizu et al., 1999, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 96:4289-94. Therefore, in some embodiments, the CD58 portion retains wild-type residues at E25, K29, K30, K32, D33, K34, E37, D84, and K87.

[0794] In contrast, the following substitutions (where the numbers refer to the full-length polypeptide) do not affect binding to CD2: F29S; V37K; V49Q; V86K; T113S; and L121G. Therefore, the CD58 moiety can include one, two, three, four, five, or all six of the aforementioned substitutions.

[0795] In some embodiments, the CD58 portion is engineered to include a pair of cysteine ​​substituents that generate a disulfide bridge upon recombinant expression. Exemplary amino acid pairs (where the numbers refer to the full-length polypeptide) that can be cysteine-substituted to form a disulfide bridge upon expression are (a) V45C and M105C substitution; (b) V54C and G88C substitution; (c) V45C and M114C substitution; and (d) W56C and L90C substitution.

[0796] An exemplary CD58 portion is provided in the following Table 15:

[0797]

[0798]

[0799] 7.8.3. CD2 ABM based on CD48

[0800] In some respects, this disclosure provides an MBM comprising a CD2ABM as a CD48 portion. As used herein, the CD48 portion comprises an amino acid sequence having at least 70% sequence identity with 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 with the CD2-binding portion of CD48). The human CD48 sequence 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 contains an amino acid sequence that has at least 70% sequence identity with the amino acid sequence consisting of amino acids 27-220 having the 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 with Uniprot identifier P09326) and an Ig-like C2 type II domain (amino acids 132-212 with Uniprot identifier P09326). Therefore, in some embodiments, the CD48 portion comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence consisting of amino acids 29-212 having the Uniprot identifier P09326, with the C2 type I domain (amino acids 29-127 having the Uniprot identifier P09326), and / or with the Ig-like C2 type II domain (amino acids 132-212 having the 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 portion may contain one or more natural variants relative to a sequence having the Uniprot identifier P09326. For example, the CD48 portion may include an E102Q substitution.For example, the CD48 portion may contain an amino acid sequence corresponding to the CD-48 isotype or its CD2 binding portion (e.g., the isotype with Uniprot identifier P09326-2 or its CD2 binding portion).

[0801] 7.9. Tumor-associated antigen ABM

[0802] Type 2 TBMs may comprise ABMs that specifically bind to tumor-associated antigens (TAAs). In some embodiments, the TAA is a human TAA. The antigen may or may not be present on normal cells. In some embodiments, the TAA is preferentially expressed or upregulated on tumor cells compared to normal cells. In other embodiments, the TAA is a lineage marker.

[0803] In some 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.

[0804] It is anticipated that any type of B-cell malignancy can be targeted by the MBM disclosed herein. Typical 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, nodular marginal zone B-cell lymphoma, and primary exudative lymphoma.

[0805] Besides CD19, examples of TAAs that can be targeted by MBM (e.g., TBM) 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 13C), and TNFRSF13B (TNF receptor superfamily member 13C). Family member 13B, also referred to in the art as TACI (transmembrane activator and CAML interaction factor), CXCR4 (CXC motif chemokine receptor 4), PD-L1 (programmed death ligand 1), LY9 (lymphocyte antigen 9, also referred to in the art as CD229), CD200, FCGR2B (Fc fragment of IgG receptor IIb, also referred to 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.

[0806] TAA-binding ABMs may comprise, for example, anti-TAA antibodies or antigen-binding fragments thereof. The anti-TAA antibody or antigen-binding fragment may comprise, for example, the CDR sequence of the antibodies listed in Table 16. In some embodiments, the anti-TAA antibody or its antigen-binding domain has the heavy chain and light chain variable region sequences of the antibodies listed in Table 16.

[0807]

[0808]

[0809]

[0810] In some embodiments, the TAA is selected from BCMA and CD20. In some embodiments, the TAA is BCMA. "BCMA" refers to the B cell maturation antigen. BCMA (also known as TNFRSF17, BCM, or CD269) is a member of the tumor necrosis receptor (TNFR) family and is primarily expressed on terminally differentiated B cells, such as memory B cells and plasma cells. Its ligands include B cell activating factor (BAFF) and 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.

[0811] In some respects, type 2 TBM includes 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 include, for example, CDR, VH, VL, or scFV sequences listed in Tables 17A-17G.

[0812]

[0813]

[0814]

[0815]

[0816]

[0817]

[0818]

[0819]

[0820]

[0821]

[0822]

[0823]

[0824]

[0825]

[0826]

[0827]

[0828]

[0829]

[0830]

[0831]

[0832]

[0833]

[0834]

[0835]

[0836]

[0837]

[0838]

[0839]

[0840]

[0841] In some embodiments, the ABM contains a CDR sequence of BCMA-1. In some embodiments, the ABM contains a CDR sequence of BCMA-2. In some embodiments, the ABM contains a CDR sequence of BCMA-3. In some embodiments, the ABM contains a CDR sequence of BCMA-4. In some embodiments, the ABM contains a CDR sequence of BCMA-5. In some embodiments, the ABM contains a CDR sequence of BCMA-6. In some embodiments, the ABM contains a CDR sequence of BCMA-7. In some embodiments, the ABM contains a CDR sequence of BCMA-8. In some embodiments, the ABM contains a CDR sequence of BCMA-9. In some embodiments, the ABM contains a CDR sequence of BCMA-10. In some embodiments, the ABM contains a CDR sequence of BCMA-11. In some embodiments, the ABM contains a CDR sequence of BCMA-12. In some embodiments, the ABM contains a CDR sequence of BCMA-13. In some embodiments, the ABM contains a CDR sequence of BCMA-14. In some embodiments, the ABM contains a CDR sequence of BCMA-15. In some embodiments, the ABM contains a CDR sequence of BCMA-16. In some embodiments, the ABM contains a CDR sequence of BCMA-17. In some embodiments, the ABM contains a CDR sequence of BCMA-18. In some embodiments, the ABM contains a CDR sequence of BCMA-19. In some embodiments, the ABM contains a CDR sequence of BCMA-20. In some embodiments, the ABM contains a CDR sequence of BCMA-21. In some embodiments, the ABM contains a CDR sequence of BCMA-22. In some embodiments, the ABM contains a CDR sequence of BCMA-23. In some embodiments, the ABM contains a CDR sequence of BCMA-24. In some embodiments, the ABM contains a CDR sequence of BCMA-25. In some embodiments, the ABM contains a CDR sequence of BCMA-26. In some embodiments, the ABM contains a CDR sequence of BCMA-27. In some embodiments, the ABM contains a CDR sequence of BCMA-28. In some embodiments, the ABM contains a CDR sequence of BCMA-29. In some embodiments, the ABM contains a CDR sequence of BCMA-30. In some embodiments, the ABM contains a CDR sequence of BCMA-31. In some embodiments, the ABM contains a CDR sequence of BCMA-32. In some embodiments, the ABM contains a CDR sequence of BCMA-33. In some embodiments, the ABM contains a CDR sequence of BCMA-34.In some embodiments, the ABM contains a CDR sequence of BCMA-35. In some embodiments, the ABM contains a CDR sequence of BCMA-36. In some embodiments, the ABM contains a CDR sequence of BCMA-37. In some embodiments, the ABM contains a CDR sequence of BCMA-38. In some embodiments, the ABM contains a CDR sequence of BCMA-39. In some embodiments, the ABM contains a CDR sequence of BCMA-40.

[0842] In some embodiments, the CDR is defined by a Kabat number, as listed in Tables 17B and 17E. In other embodiments, the CDR is defined by a Josiah number, as listed in Tables 17C and 17F. In still other embodiments, the CDR is defined by a combination of Kabat and Josiah numbers, as listed in Tables 17D and 17G.

[0843] In some embodiments, the type 2 TBM in which ABM3 is combined with BCMA may contain heavy chains of any one of BCMA-1 to BCMA-40 and the light chains may have variable sequences.

[0844] In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-1, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-2, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-3, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-4, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-5, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-6, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-7, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-8, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-9, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-10, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-11, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-12, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-13, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-14, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-15, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-16, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-17, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-18, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-19, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-20, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-21, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-22, as listed in Table 17A.In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-23, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-24, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-25, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-26, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-27, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-28, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-29, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-30, as listed in Table 17A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-31, as listed in Table 17A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-32, as listed in Table 17A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-33, as listed in Table 17A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-34, as listed in Table 17A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-35, as listed in Table 17A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-36, as listed in Table 17A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-37, as listed in Table 17A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-38, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-39, as listed in Table 17A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-40, as listed in Table 17A.

[0845] 7.10. Nucleic Acids and Host Cells

[0846] On the other hand, this disclosure provides nucleic acids (i.e., polynucleotides) encoding the CD19-binding molecule of this 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.

[0847] A single nucleic acid can encode a CD19-binding molecule containing a single polypeptide chain, a CD19-binding molecule containing two or more polypeptide chains, or a portion of a CD19-binding molecule containing more than two polypeptide chains (e.g., a single nucleic acid can encode two polypeptide chains of a CD19-binding molecule containing three, four, or more polypeptide chains, or three polypeptide chains of a CD19-binding molecule containing four or more polypeptide chains). For individual control of expression, read frames encoding two or more polypeptide chains can be under the control of individual transcriptional regulatory elements (e.g., promoters and / or enhancers). Read frames encoding two or more polypeptides can also be controlled by the same transcriptional regulatory element and separated by internal ribosome entry site (IRES) sequences to allow translation into different polypeptides.

[0848] 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 a CD19 binding molecule may 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).

[0849] The nucleic acid can be DNA or RNA (e.g., mRNA).

[0850] On the other hand, this disclosure provides a host cell and a vector containing the nucleic acid disclosed herein. The nucleic acid may be present in a single vector or in different vectors, and the vectors may be present in the same host cell or different host cells, as described in more detail below.

[0851] 7.10.1. Carrier

[0852] This disclosure provides vectors comprising nucleotide sequences encoding the CD19-binding molecule or CD19-binding molecule components described herein. In one embodiment, the vector comprises nucleotides encoding the immunoglobulin-based ABM described herein. In one embodiment, the vector comprises nucleotides encoding the Fc domain described herein. In one embodiment, the vector comprises nucleotides encoding the recombinant non-immunoglobulin-based ABM described herein. The vector may 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 sequence described herein. The vector includes, but is not limited to, viruses, plasmids, viscera, λ phages, or yeast artificial chromosomes (YACs).

[0853] A variety of vector systems can be used. For example, one type of vector utilizes DNA elements derived from animal viruses, such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrotranscriptovirus (Raoul's sarcoma virus, MMTV, or MOMLV), or SV40 virus. Another type of vector utilizes RNA elements derived from RNA viruses, such as Semliki Forest virus, Eastern Equine Encephalitis virus, and flaviviruses.

[0854] Furthermore, cells that stably integrate DNA into their chromosomes can be selected by introducing one or more markers that allow for selection of the transfected host cells. These markers can provide proton shifts to, for example, auxotrophic hosts, provide resistance to antimicrobial agents (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be directly linked to the DNA sequence to be expressed or introduced into the same cells via co-transformation. Optimal mRNA synthesis may also require additional elements. These elements may include splicing signals, as well as transcription promoters, enhancers, and termination signals.

[0855] Once an expression vector or DNA sequence containing the construct is prepared for expression, the expression vector can be transfected or introduced into suitable host cells. This can be achieved using 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. The methods and conditions used to culture the resulting transfected cells and recover the expressed peptide are known to those skilled in the art and can be modified or optimized based on the specific expression vector and mammalian host cells used in this specification.

[0856] 7.10.2. Cells

[0857] This disclosure also provides host cells containing the nucleic acids disclosed herein.

[0858] In one embodiment, the host cell is genetically engineered to contain one or more nucleic acids as described herein.

[0859] In one embodiment, host cell genetic engineering is achieved by using an expression cassette. The phrase "expression cassette" refers to a nucleotide sequence capable of influencing gene expression in a host compatible with such a sequence. Such cassettes may include promoters, read frames with or without introns, and termination signals. Other factors necessary or helpful in achieving expression, such as inducible promoters, may also be used.

[0860] This disclosure also provides host cells containing the vectors described herein.

[0861] The cells may be, but are not limited to, eukaryotic cells, bacterial cells, insect cells, or human cells. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells.

[0862] 7.11. CD19-binding molecules with extended in vivo half-life

[0863] The CD19 binding molecule disclosed herein can be modified to have an extended in vivo half-life.

[0864] A variety of strategies can be used to extend the half-life of the CD19 binding molecules disclosed herein. For example, through chemical linking with polyethylene glycol (PEG), reCODE PEG, antibody scaffolds, polysialic acid (PSA), hydroxyethyl starch (HES), albumin-binding ligands, and carbohydrate shielding; through genetic fusion and transfer with proteins that bind serum pr...

Claims

1. A CD19 binding molecule that specifically binds to human CD19 and comprises CDR-H1, CDR-H2, and CDR-H3 having amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and CDR-L1, CDR-L2, and CDR-L3 having amino acid sequences of SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:

16.

2. A CD19 binding molecule that specifically binds to human CD19 and comprises CDR-H1, CDR-H2, and CDR-H3 having amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and CDR-L1, CDR-L2, and CDR-L3 having amino acid sequences of SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:

19.

3. A CD19 binding molecule that specifically binds to human CD19 and comprises CDR-H1, CDR-H2, and CDR-H3 having amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, and CDR-L1, CDR-L2, and CDR-L3 having amino acid sequences of SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:

22.

4. A CD19 binding molecule that specifically binds to human CD19 and comprises CDR-H1, CDR-H2, and CDR-H3 having amino acid sequences of SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, and CDR-L1, CDR-L2, and CDR-L3 having amino acid sequences of SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:

25.

5. A CD19 binding molecule that specifically binds to human CD19 and comprises CDR-H1, CDR-H2, and CDR-H3 having amino acid sequences of SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29, and CDR-L1, CDR-L2, and CDR-L3 having amino acid sequences of SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:

42.

6. A CD19 binding molecule that specifically binds to human CD19 and comprises CDR-H1, CDR-H2, and CDR-H3 having amino acid sequences of SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, and CDR-L1, CDR-L2, and CDR-L3 having amino acid sequences of SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:

45.

7. A CD19 binding molecule that specifically binds to human CD19 and comprises CDR-H1, CDR-H2, and CDR-H3 having amino acid sequences of SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, and CDR-L1, CDR-L2, and CDR-L3 having amino acid sequences of SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:

48.

8. A CD19 binding molecule that specifically binds to human CD19 and comprises CDR-H1, CDR-H2, and CDR-H3 having amino acid sequences of SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38, and CDR-L1, CDR-L2, and CDR-L3 having amino acid sequences of SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:

51.

9. A conjugate comprising (a) a CD19 binding molecule as described in any one of claims 1 to 8, and (b) a pharmaceutical agent.

10. A pharmaceutical composition comprising (a) a CD19 binding molecule as claimed in any one of claims 1 to 8 or a conjugate as claimed in claim 9, and (b) an excipient.

11. A method of treating a subject suffering from a CD19-related disease or disorder, comprising administering to the subject an effective amount of a CD19 binding molecule as described in any one of claims 1 to 8, a conjugate as described in claim 9, or a pharmaceutical composition as described in claim 10.

12. One or more nucleic acids, said nucleic acids encoding a CD19 binding molecule as described in any one of claims 1 to 8.

13. A cell engineered to express the CD19 binding molecule as described in any one of claims 1 to 8.

14. A cell transfected with one or more expression vectors under the control of one or more promoters, the expression vectors comprising one or more nucleic acid sequences encoding a CD19 binding molecule as described in any one of claims 1 to 8.

15. A method for generating CD19-binding molecules, comprising: (a) The cells as described in any one of claims 13 to 14 are cultured under the conditions of CD19 binding molecule expression; as well as (b) The CD19 binding molecule was recovered from the cell culture.

16. A CD19-binding molecule comprising the following trispecific binding molecules (TBMs): (a) An antigen-binding module 1 (ABM1) that specifically binds to CD19 and comprises CDR-H1, CDR-H2, and CDR-H3 having amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and CDR-L1, CDR-L2, and CDR-L3 having amino acid sequences of SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19; (b) Antigen-binding module 2 (ABM2) that specifically binds to a component of the human T-cell receptor (TCR) complex; and (c) Antigen-binding module 3 (ABM3) specifically binds to human CD2.

17. A CD19-binding molecule comprising the following trispecific binding molecule (TBM): (a) Antigen-binding module 1 (ABM1), which specifically binds to CD19 and comprises the following Fab: (i) CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19; or (ii) CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, and CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45; (b) Antigen-binding module 2 (ABM2), which specifically binds to CD3 and contains the amino acid sequence of scFv designated as CD3-21 in Table 12A; (c) Antigen-binding module 3 (ABM3), which specifically binds to human CD2 and contains the amino acid sequence of CD58-6 as listed in Table 15; and (d) Fc structural domain.

18. A CD19-binding molecule comprising a trispecific binding molecule (TBM): (a) Antigen-binding module 1 (ABM1), which specifically binds to CD19 and comprises the following Fab: (i) CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19; or (ii) CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, and CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45; (b) Antigen-binding module 2 (ABM2), which specifically binds to CD3 and contains the amino acid sequence of scFv designated as CD3-129 in Table 12A; (c) Antigen-binding module 3 (ABM3), which specifically binds to human CD2 and contains the amino acid sequence of CD58-6 as listed in Table 15; and (d) Fc structural domain.

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

20. A CD19-binding molecule comprising: (a) A first polypeptide whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:1077; (b) A second polypeptide whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:759; and (c) A third polypeptide whose amino acid sequence contains the amino acid sequence of SEQ ID NO:1078 or SEQ ID NO:1086.

21. A CD19-binding molecule comprising: (a) A first polypeptide whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:1079; (b) A second polypeptide whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:759; and (c) A third polypeptide whose amino acid sequence contains the amino acid sequence of SEQ ID NO:1078 or SEQ ID NO:1086.

22. A CD19-binding molecule comprising: (a) A first polypeptide whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:1077; (b) A second polypeptide whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:759; and (c) A third polypeptide whose amino acid sequence includes the amino acid sequence of SEQ ID NO:1086.

23. A CD19-binding molecule comprising the sequence of the construct shown in Table 19-A1, 19A-2, 19-B, or 19C, preferably the sequence shown in Table 19C.

24. A combination comprising the CD19 binding molecule as claimed in any one of claims 16 to 23 and at least one additional therapeutic agent.

25. A pharmaceutical composition comprising (a) a CD19 binding molecule as described in any one of claims 16 to 23 or a combination as described in claim 24, and (b) a pharmaceutically acceptable excipient.

26. The CD19 binding molecule as described in any one of claims 16 to 23, or the combination as described in claim 24, or the composition as described in claim 25, as a pharmaceutical preparation.

27. The CD19 binding molecule of any one of claims 16 to 23, or the combination of claims 24, or the composition of claims 25, for the treatment of CD19-related diseases or disorders.

28. Use of the antibody or antigen-binding fragment thereof as described in any one of claims 16 to 23, or a combination thereof as described in claim 24, or a composition as described in claim 25, for the manufacture of a medicament for the treatment of CD19-related diseases or disorders.

29. A method of treating a subject suffering from a CD19-related disease or disorder, comprising administering to the subject an effective amount of a CD19 binding molecule as described in any one of claims 16 to 23, or a combination as described in claim 24, or a composition as described in claim 25.

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