Bispecific hemibodies and trispecific antibody constructs for b cell and plasma cell depletion

Abstract

The present disclosure provides bispecific hemibodies and trispecific antibody constructs for the selective depletion of populations of cells in the B cell lineage and / or its plasma cell sublineage. The bispecific hemibodies may target more than two (e.g., three or four) markers present on target cells in the B cell lineage. Those bispecific hemibodies and trispecific antibody constructs are suitable for, and provide novel treatments for, a variety of autoimmune diseases, allergies, and certain neoplasms involving cells in the B cell lineage and / or its plasma cell sublineage.

Description

Bispecific Hemibodies And Trispecific Antibody Constructs For B Cell And Plasma Cell DepletionI. Incorporation of Sequence Listing

[0001] The sequence listing in ST.26 XML format entitled 3061-7_PCT_ST26.xml, created on March 8, 2025, comprising 182,786 bytes, prepared according to 37 CFR 1.822 to 1.824, and submitted concurrently with the filing of this application, is incorporated herein by reference in its entirety.II. Introduction

[0002] In the mammalian immune system, B cells are the cellular source of antibodies that effect humoral immunity. In addition to antibody secretion, B cells have a myriad of cellular functions that contribute to innate and adaptive immunity, including antigen presentation, cytokine production and maintenance of immunological niches necessary for proper immunological function in various contexts. In their functions as antigen-presenting cells (APCs), B cells internalize antigens that are taken up by the B cells through receptor-mediated endocytosis, especially antigens captured by their cell surface B cell receptors (BCRs). The BCR is a unique receptor on the surface of a B cell that imparts antigen-specific recognition, and which is generated via a developmentally regulated genetic shuffling process of germline-encoded mini gene segments. The genetic shuffling is semi-stochastic and provides that(1) individual B cells and their associated BCRs have a unique antigen-specificity and (2) the aggregate population of B cells (aka the B cell "repertoire”) in the host is able to recognize a large diversity of antigens, including foreign antigens and autoantigens. To prevent autoimmunity, developmental processes normally ensure that self-reactive clones within the B cell repertoire are eliminated or made unresponsive to autoantigens. Antigens endocytosed by the B cells are processed into antigenic peptide fragments which then associate with Class II major histocompatibility complex molecules (MHC II) for presentation on the B cell's extracellular surface to CD4+ T cells (e.g., T helper cells) as an MHC I l-antigen complex. T helper cells that bind to the MHC I l-antigen complex cause activation of the B cell. For classical activation, a B cell must recognize a cognate antigen via its antigen BCR. In most cases, T helper cells are required for full activation and initiation of further B cell differentiation. A minority of B cells are specific for and activated by antigens independently of T cell help.

[0003] B cell stimulation by T cells, typically occurring in germinal centers of secondary lymphoid organs such as the spleen and lymph nodes, results in B cell differentiation. Within germinal centers, B cells may differentiate into memory B cells or plasma cells. B cell differentiation into plasma cells is dependent upon the transcription factors Blimp-1 / PRDM 1 and IRF4. B cells may also undergo affinity maturation wherein the activation and growth of B cell clones that secrete antibodies of higher affinity for the antigen occurs. The majority of B cells become immature plasma cells or "plasmablasts'' that eventually become antibody-secreting plasma cells.

[0004] Plasmablasts are the most immature plasma cells. Once formed, plasmablasts, which can divide rapidly and may internalize and present antigens to T cells, either die or irreversibly differentiate into mature fully differentiated plasma cells generally within a matter of days. Plasmablasts secrete more antibodies than B cells, but less than plasma cells. See, e.g., FIG. 9.

[0005] Autoreactive B cells give rise to autoreactive plasma cells that produce autoreactive antibodies or "autoantibodies” that are pathogenic. Pathologies associated with autoantibodies may result from their binding to a target antigen and thereby impair function (e.g., acetylcholine receptor crosslinking and / or blockade as in myasthenia gravis). Alternatively, autoantibodies may form immune complexes in tissues that result in a local complement cascade activation (e.g., anti-dsDNA as in systemic lupus erythematosus or SLE). B cells may also contribute to pathogenesis associated with autoimmune diseases in an antibody-independent manner. Pathogenic antibodyindependent B cell functions may result from activation of cytotoxic effector cells (e.g., effector T cells, NK cells, and macrophages) via antigen presentation (e.g., as in multiple sclerosis, MS); via cytokine secretion (e.g., as in SLE); and / or by sustaining tertiary lymphoid tissues (e.g., as in rheumatoid arthritis, RA). Ultimately, the contribution of B cells to the pathogenesis of autoimmune disease varies greatly in each disease.

[0006] Cell surface expression of BCRs and other phenotypic markers (e.g., CD20, BCMA) on B cells, plasmablasts, and plasma cells varies by differentiation state. The BCR and phenotypic markers of those cells are targets of current pan B cell depletion therapies (e.g., Rituximab, which binds CD20). Broad ranging B cell depletion therapies (BCDT) that do not discriminate between pathogenic B cells and non-pathogenic B cells are, however, problematic for at least the reason that they result in global suppression of B cell based immune responses. Pathogenic B cells may also escape antigen specific BCDT by altering (e.g., reducing or ending) the expression of the target B cell antigen. Moreover, current antigen-specific approaches requiring surface B cell receptors are not effective against antibody-producing plasma cells, as those cells do not express the BCR.III. Summary

[0007] The present disclosure sets forth bispecific antibodies and their component first and second constructs, trispecific antibody constructs, and compositions comprising any one or more of those molecules (e.g., one or more hemibody or trispecific antibody constructs). The present disclosure also sets forth methods for using those molecules in various methods in vitro and / or in vivo, including in the treatment of antigen-specific immune pathologies including, but not limited to, autoimmunity (autoimmune diseases or disorders), allergies, certain neoplasms, and transplant rejection. The constructs and compositions comprising constructs may act by binding cytotoxic effector cells (e.g., T cells, NK cells, macrophages or monocytes) to cells of the B cell lineage (e.g., B cells and / or cells of the plasma cell sublineage) and stimulating the bound effector cells' cytotoxic response, resulting in depletion of a targeted B cell subset. The constructs may also act through Antibody-Dependent Cell Cytotoxicity (ADCC), Antibody-Dependent Cell Phagocytosis (ADCP), and / or Complement-Dependent Cytotoxicity (CDC) provided they comprise amino acid (aa) sequences capable of inducing those responses. In contrast to current pan B cell depletion therapies, a small subset of cells in the B cell lineage, such as those that are activated and / or those that exist in specific tissues (e.g., marginal zone or follicular B cells), may be selectively depleted from a patient's repertoire. The result is a more defined immunotherapy with fewer side effects as the remaining cells of the patient's B cell lineage (e.g., B cell repertoire) remain substantially intact. The selective targeting of members of the B cell lineage (e.g., non-naive activated B cells) also strikes a balance between overly inclusive pan B cell depletion therapies and therapies that target B cell lineage members specific for a single epitope. Indiscriminate (pan) B celldepletion eliminates pathogenic B cells as well as B cells contributing to host defense, leaving patients prone to opportunistic infection. In addition, pan B cell depletion is associated with adverse events such as neutropenia, leading to potentially life-threatening infections. In contrast, antigen-specific B cell depletion may deplete an ineffectively narrow B cell subset, as patients with autoimmune and related diseases often will have multiple B cell specificities directed to different epitopes of the antigen(s) underlying their disease. This can be seen in conditions such as celiac disease, where multiple related but distinct epitopes reactive with different antibodies are present on glutens, gliadins, and hordeins. See, e.g., Iversen et al., J Immunol., 190(12):5981 -91 (2013) and Caja et al., Cell Mol Immunol, 8(2): 103-9 (2011).

[0008] Exemplary constructs are provided in FIG. 1 A and fall into two categories (I) bispecific hemibodies and (II) trispecific antibody constructs. Potential organizations of the bispecific hemibodies include, but are not limited to, the heterodimeric polypeptide constructs shown in FIG. 1 A at A and the single polypeptide chain structures appearing at B. Potential organizations of the trispecific antibodies include, but are not limited to, the heterodimeric polypeptide constructs shown in FIG 1 as structures C to I, and the single polypeptide chain structures appearing in structures J to M.

[0009] Bispecific hemibodies of the present disclosure comprise a first hemibody construct and a second hemibody construct, each of which may be in the form of a polypeptide heterodimer as in FIG. 1A, structure A, or a single polypeptide as in FIG. 1 A, structure B. The first hemibody construct comprises as elements a first element that binds a first marker (e.g., an activation marker) expressed on a cell in the B cell lineage (e.g., CD69 expressed on a B cell) 1', and a VH or VL aa sequence 4 of a hemibody that binds to a cytotoxic effector cell surface antigen (CEC-SA). The second hemibody construct comprises as elements a second element that binds a second marker (e.g., a lineagespecific marker) expressed on a cell in the B cell lineage (e.g., a B cell) 1", and the corresponding VH or VL aa sequence 4’ of the hemibody that binds to a CEC-SA, such that when the first and second hemibody constructs are brought together the VHand V aa sequences (4 and 4’) form a CEC-SA binding paratope (e.g., a T cell surface antigen binding paratope).

[0010] The trispecific antibodies of the present disclosure comprise as elements (I) a first element that binds a first marker (e.g., an activation marker such as CD69) expressed on a cell in the B cell lineage 1', (II) a second element that binds a second marker (e.g., a lineage-specific marker such as CD19 or CD20) expressed on a cell in the B cell lineage (e.g., a B cell) 1", and (ill) amino acid sequence(s) that bind to a CEC-SA (e.g., comprising VH and VL aa sequence(s) 4 and 4’). The trispecific antibody constructs may be comprised of a heterodimer (see, e.g., FIG. 1A, structures C to I) or a single polypeptide chain that comprises all of the above-mentioned elements exemplified by FIG. 1A, constructs J to M.

[0011] Any of the polypeptides of the bispecific hemibodies or trispecific antibody constructs described herein may comprise one or more scaffold aa sequences (2 or 2’) that may provide, among other things, (I) structural organization of the elements involved in binding to cells of the B cell lineage and of the elements involved in binding to cytotoxic effector cells and (II) increased serum half-life. When the scaffold aa sequence comprises immunoglobulin heavy chain constant region sequences, the scaffold aa sequences may induce ADCC, ADCPand / or CDC that can lead or contribute to depletion of cells in the B cell lineage bound by the bispecific hemibodies or trispecific antibody constructs.

[0012] Disulfide bonds may be employed in the constructs provided herein and may stabilize their structure, provide resistance to thermal denaturation, and / or resistance to proteolysis. Disulfide bonds may be intramolecular, specifically between two cysteines in a single polypeptide (e.g., between the scaffold elements 2 in FIG. 1 A, structures B and J to M (particularly when they are IgFc sequences) or between elements 4 and 4’ in FIG. 1 A, structures F to M). Disulfide bonds may also be intermolecular, specifically between cysteines located on different peptides of a bispecific hemibody, such as in a disulfide bond formed between scaffold elements 2 and 2’ of the peptides in the first or second constructs depicted in FIG. 1 A at A.

[0013] Interactions of the trispecific antibody constructs and / or bispecific hemibodies with activated plasmablasts, plasma cells, or B cells are exemplified schematically for B cells in FIGs. 5 and 6. By forming a complex of those cells and cytotoxic effector cells, the trispecific antibody constructs and / or bispecific hemibodies cause cytotoxic effector cell activation and result in cytotoxic action against the activated plasmablasts, plasma cells, and / or B cells, leading to their ablation. Where the constructs comprise IgFc scaffolds that have sequences associated with CDC, ADCC, and / or ADCP, engagement of the B cells by the trispecific antibody constructs and / or bispecific hemibodies may also elicit one or more of those responses augmenting the ablation of activated B cells.

[0014] The present disclosure includes and provides for trispecific antibody constructs and / or bispecific hemibodies and their preparation, which may be constructed as fusion proteins, through cellular expression or in vitro translation or in coupled transcription translation systems. Accordingly, the disclosure also includes and provides for nucleic acids encoding all or part of the trispecific antibody constructs and / or bispecific hemibodies and the cells expressing one or more (all) polypeptides of a trispecific antibody construct and / or bispecific hemibody.

[0015] This disclosure further includes and provides for compositions comprising bispecific hemibody and trispecific antibody constructs and their use in treating a variety of diseases including, but not limited to, autoimmune diseases, allergies, transplant associated diseases (graft vs host disease, "GVHD,” or host vs graft disease, "HVGD”), and certain cancers. The constructs described herein advantageously permit the treatment of one or more diseases using a single type of construct.IV. Brief Description of the Drawings

[0016] FIG. 1 A at A and B shows the structures of bispecific hemibodies comprising a first (1st) and second (2nd) hemibody construct, with each construct in A comprised of a pair of heterodimeric polypeptides and each construct in B comprised of a single polypeptide (single chain). At C to I of FIG. 1 A, the figure shows the structure of trispecific molecules formed from a heterodimer of polypeptides. FIG. 1 A at J to M shows single chain trispecific antibody constructs. In A to M 1' and 1" are elements that bind to markers (molecules) expressed on the surface of a cell in the B cell lineage. Elements 1' and 1" bind respectively to first and second markers that are selectively or exclusively expressed on a cell in the B cell lineage. Element 1' may bind to, for example, a molecule expressed on activated B cells, plasmablasts, plasma cells and / or long lived plasma cells such as an activation specific marker such as CD69, and element 1" may bind to a lineage-specific marker such as CD 19 or BCM A, which is selectively or exclusivelyexpressed on, e.g., B cells, plasmablasts, plasma cells, and / or long lived plasma cells. Elements 2 and 2’ are scaffold aa sequences, typically immunoglobulin Fc sequences, that may bind as dimers (e.g., a dimer or homodimer of element 2 in the constructs at B and J to M) or heterodimers (e.g., a heterodimer of elements 2 and 2’ in combination) depicted as a knob-into-hole pair in any of constructs A to M. One of elements 4 and 4’ may be an antibody heavy chain variable region (VH) aa sequence and the other of elements 4 and 4’ may be an antibody light chain variable region (V ) aa sequence. Together variable regions 4 and 4’ form a paratope that recognizes and binds to a molecule selectively or exclusively expressed on cytotoxic effector cells (e.g., a surface antigen such as CD3). Alternatively, when adjacent to each other in a single peptide (e.g., as in structures F to M), elements 4 and 4’ may together be a single chain antibody sequence (e.g., an scFv aa sequence) that recognizes a molecule selectively or exclusively expressed on T-cells. The solid lines between elements indicate independently selected optional linker aa sequences, any of which may be present or absent. When absent, the elements they connect are joined into a polypeptide sequence by a peptide bond. Scaffold element pairs (2 and 2’) or interspecific pairs (2 and 2’, shown as a knob-in hole pair) may be joined by one or more disulfide bonds. Similarly, elements 4 and 4’ when present in a trispecific antibody may be joined by one or more disulfide bonds. Disulfide bonds, including intrapeptide (intrapolypeptide) and interpeptide (interpolypeptide) bonds joining elements, such as scaffold sequences, may form spontaneously when the polypeptide(s) are expressed in a cell. Where scaffolds are exemplified as comprising a heterodimeric sequence pair (a 2 and 2’ sequence), the scaffold aa sequences may be interchanged. For example, the sequence comprising a knob in FIG. 1 A, structure A, may be replaced by a sequence comprising a hole, while the sequence comprising a hole is replaced by a sequence comprising a knob. Similarly, the sequences of elements 4 and 4’ may be exchanged provided both are present and can form the required paratope. In FIG. 1 A, the molecules may be considered as being oriented with the N-terminus or C-terminus of the polypeptide chain at the top of the diagram.

[0017] FIG. 1 B depicts exemplary bispecific hemibodies and trispecific antibody constructs having at least one element that binds a marker on the surface of a cell of the B cell lineage. The first and second hemibody construct pairs at A and B depict the hemibodies of FIG. 1 A at A and B in which element 1 and element 2 comprise VH (noted as 1' VH and 1" VH, respectively) and the corresponding VL sequences are provided by additional (further) polypeptides (noted as 1' VLand 1" V , respectively). Those VHand V elements effectively form an antibody Fv-like structure, and may be joined by a disulfide bond. The first and second hemibody construct pairs at C and D depict the hemibodies of FIG. 1A at A and B in which element 1 and element 2 comprise a CH1-VHaa sequence (noted as CH1-T VH and CH1-1 " VH, respectively), and the corresponding VL sequences are provided by additional (further) polypeptides (noted as CK-T VL and CK-1" VL, respectively) numbered as 14 and 15. In structures C and D the elements 1 and 2 along with the CH1 and CK aa sequences effectively form an antibody-like structure, which may be joined by a disulfide bond (e.g., between the CH1 and CK aa sequences). In FIG. 1 B, the molecules may be considered as being oriented with the N-terminus or C-terminus of the polypeptide chain at the top of the diagram.

[0018] The trispecific antibodies of FIG. 1 B at E and F depict the trispecific antibodies of FIG. 1A at C and J in which element 1 or element 1 ” comprises a VH aa sequence (noted as 1 ' VH and 1 " VH, respectively) and the corresponding VL sequences are provided by additional (further) polypeptides 14 (noted as 1' VL and 1" VL,respectively). Those VH and VL elements effectively form an antibody Fv-like structure, which may be joined by a disulfide bond. The trispecific antibodies of FIG. 1B at G and H depict the same trispecific antibodies of FIG. 1A at C and J in which element 7 and element 1” comprise a CH1-VH aa sequence (noted as CH1-T VH and CH1-1" VH, respectively), and the corresponding V sequences are provided by additional (further) polypeptides 14 (noted as CK-T VLand CK-1" V , respectively). In structures G and H the elements 1 and 2 effectively form an antibody Fab fragment structure, which may be joined by a disulfide bond (e.g., between the CH1 and CKaa sequences).

[0019] The structures are only exemplary and the VH and VL aa sequences and / or the CK and CH1 aa sequences may be exchanged. In addition, other interspecific sequences may be utilized to pair corresponding VH and VL aa sequences to form a functional paratope. When CK and CH1 are employed as interspecific sequences, their binding affinity may be enhanced such as by the incorporation of MD13 aa substitutions.

[0020] FIG. 2 at A to F shows six different constructs for the preparation of the bispecific hemibody first and second hemibody construct pairs depicted in FIG. 1 A at A. Dashed lines indicate independently selected linker aa sequences that comprise one or more independently selected site-specific protease aa sequences and that are susceptible to proteases specific for those sequences. The constructs are shown before (e.g., as expressed) and after site-specific protease action on all the susceptible linkers to give rise to the bispecific hemibody first and second hemibody construct pairs. The small solid triangle 5, which may be attached by a cleavable linker, represents an affinity tag sequence (e.g., FLAG or 6X Histidine tags) that may be used for, among other things, purifying the constructs. Constructs shown at D to F repeat those shown at A to C with the addition of cleavable linker 6 and masking VH or VL elements (4° and 4’°) to each polypeptide to increase expression levels. The added VH or VL elements are cleaved by site-specific protease action, giving a bispecific hemibody comprising a bispecific hemibody first and second hemibody construct pair. In FIG. 2, the molecules may be considered as being oriented with the N-terminus or C- terminus of the polypeptide chain at the top of the diagram. In addition, the affinity tags and their cleavable linker may be located at the opposite end of the polypeptide.

[0021] FIG. 3 at A to D shows four different constructs for the preparation of the bispecific hemibody first and second hemibody constructs depicted in FIG. 1 A at B. As in FIG. 2, the dashed lines indicate independently selected linkers that comprise one or more independently selected site-specific protease aa sequences and which are susceptible to proteases specific for those sequences, and the small solid triangle 5 represents an affinity tag sequence, that may be attached by a cleavable linker. The constructs are shown before (e.g., as expressed) and after site specific protease action on all the susceptible linkers to give rise to the bispecific hemibody first and second hemibody constructs of FIG. 1 A at B. Constructs shown at C and D repeat those shown at A and B with the addition of VH or VL elements (4° and 4’°) to each polypeptide to increase expression levels. The added VH and VL elements (4° and 4’°) may be cleaved by site-specific protease action on the linkers 6 attaching them to the remainder of the construct. As indicated for FIG. 1A, the molecules may be considered as being oriented with the N-terminus or C- terminus of the polypeptide chain at the top of the diagram.

[0022] FIG. 4 at A to E shows five exemplary constructs for the preparation of trispecific antibodies of the type depicted in FIG. 1 A at C to M. As in FIG. 2, the dashed lines indicate independently selected linkers that comprise one or more independently selected site-specific protease aa sequences and which are susceptible to proteasesspecific for those sequences, and the small solid triangle 5 represents an affinity tag sequence that may be attached by a cleavable linker. The constructs are shown before (e.g., as expressed) and after site-specific protease action on all the susceptible linkers to give rise to the trispecific antibody constructs. Constructs shown at A and B give rise to the construct shown in FIG. 1 A at C. Constructs shown at C and D give rise to the construct shown in FIG. 1 A at F. The construct shown at E gives rise to the type of constructs shown in FIG. 1 A at J to M, specifically construct J. As indicated by this figure, the cleavable linker and affinity tag shown in the constructs of FIG. 2 can be added to any of constructs A to M to provide those constructs following site-specific protease treatment.

[0023] FIG. 5 at A and B shows the structures of multispecific hemibodies comprising a first and second construct, with each construct in A comprised of a pair of heterodimeric polypeptides, and each construct in B comprised of a single polypeptide (single chain). Elements T, 1", 1"', and 1"" are portions of the constructs (e.g., aa sequences) that bind to markers (molecules) expressed on the surface of cells in the B cell lineage. Three or four of elements 1', 1", 1"', and 1"" are present in the constructs in FIG. 5 at A and B. Element 1' binds a first marker (e.g., a molecule expressed on activated B cells, plasmablasts, plasma cells and / or long lived plasma cells, such as CD69 which may serve as an activation specific marker), and element 1" binds a second marker (e.g., element 1" may be an aa sequence that binds to a molecule that is selectively or exclusively expressed on B cells, plasmablasts, plasma cells and / or long lived plasma cells, such as the lineage-specific markers CD19 or BCMA). Elements 2 and 2’ are scaffold molecules, typically immunoglobulin Fc sequences, that may bind as dimers (e.g., a dimer or homodimer of element 2 in the constructs at B and D) or heterodimers (e.g., a heterodimeric combination of elements 2 and 2’) depicted as a knob-into-hole sequence pair in the constructs at A and C. One of elements 4 and 4’ may be an antibody heavy variable region aa sequence and the other of 4 and 4’ may be, for example, an antibody light chain variable region aa sequence. Together variable regions 4 and 4’ form a paratope that recognizes and binds to a molecule selectively or exclusively expressed on cytotoxic effector cells (e.g., a surface antigen such as CD3). The solid lines indicate independently selected optional linker aa sequences, any of which may be present or absent. When linker sequences are absent, the elements they connect are joined into a polypeptide sequence by a peptide bond.Scaffold element pairs (2 and 2 or 2 and 2’) may be joined by one or more disulfide bonds. When disulfide bonds join such elements, they may form spontaneously as the polypeptide(s) are expressed in a cell. Where scaffolds are exemplified as comprising a heterodimeric sequence pair (a 2 and 2’ sequence), the sequences of those elements may be exchanged. For example, the sequence comprising a knob in FIG. 5, structure A, may be replaced by a sequence comprising a hole, while the sequence comprising a hole is replaced by a sequence comprising a knob. Similarly, the sequences of elements 4 and 4’ may be exchanged provided both are present and can form the required paratope. In C and D, the small solid triangle 5 represents an affinity tag sequence (e.g., FLAG or 6X Histidine tags) that may be used for, among other things, purifying the constructs. Constructs shown at C and D repeat those shown at A and B with the addition of cleavable linker 6 and masking VH or VL elements (4° and 4’°) added to each polypeptide to increase expression levels. The added VH or VL elements are cleaved by site-specific protease action giving a bispecific hemibody comprising a bispecific hemibody first and second hemibody construct pair.

[0024] FIG. 6 illustrates the interaction of a bispecific hemibody with a B cell in the presence of an exemplary cytotoxic effector cell (a T cell) resulting in B cell ablation. Bispecific hemibodies are contacted with the B cell at A, and they bind via and 1" to first and second markers (indicated as filled triangles and pentagons) expressed on a cell in the B cell lineage forming the paratope for a T cell specific marker (e.g., CD3) from elements 4 and 4’ at B. In C the T cell is bound to the B cell by the bispecific hemibody complex via the paratope, leading to T cell activation, and in D the B cell is subjected to the T cell's cytotoxic (e.g., granule dependent and / or granule independent) response.

[0025] FIG. 7 illustrates the interaction of a trispecific antibody construct with a B cell in the presence of an exemplary cytotoxic effector cell (a T cell) resulting in B cell ablation. A trispecific antibody construct is contacted with the B cell at A, and at B binds via 1' and 1" to first and second markers (indicated as filled triangles and pentagons) expressed on a cell in the B cell lineage. In C, the T cell binds to the trispecific antibody construct by T-cell specific sequences 4 and 4’, leading to T cell activation. In D, the B cell is subjected to the T cell's cytotoxic (e.g., granule dependent or granule independent) response.

[0026] FIG. 8A shows the formation of two hemibodies from a first hemibody construct comprising a VH aa sequence 4 that forms part of a paratope binding to an effector T cell surface antigen and two different hemibody constructs comprising a V aa sequence 4’ that forms the remainder of the paratope binding to the effector T cell surface antigen. In the middle of the figure the assembly of the first and second constructs on a target B cell surface is shown as two different hemibodies that each result in a complete paratope for binding to the CEC-SA. The bottom of the figure shows the interaction of the hemibodies with a T cell via the CEC-SA and subsequent cytotoxic action against the target B cell. The elements (e.g., aa sequences) indicated as 1', 1" and 1"' bind to different (nonidentical) markers on one or more cells in the B cell lineage.

[0027] FIG. 8B shows the formation of three hemibodies from a first hemibody construct comprising a VH aa sequence that forms part of a paratope binding to an effector T cell surface antigen and three different second hemibody constructs each comprising a VL aa sequence that forms the remainder of the paratope binding to the effector T cell surface antigen. The three hemibodies can bind one or more cells in the B cell lineage, resulting in cytotoxic / cytolytic destruction of the B cells in a manner parallel to that depicted in FIG 8A. The elements (e.g., aa sequences) indicated as T, 1", 1"' and 1"" bind to different (non-identical) markers on one or more cells in the B cell lineage, with element T binding a marker common to each targeted cell in this instance.

[0028] FIG. 8C shows the formation of four hemibodies from two first hemibody constructs comprising a VH aa sequence that forms part of a paratope binding to an effector T cell surface antigen and two second hemibody constructs comprising a VL aa sequence that forms the remainder of the paratope binding to the effector T cell surface antigen. The four hemibodies can bind one or more cells in the B cell lineage, resulting in cytotoxic / cytolytic destruction of the B cells in a manner parallel to that depicted in FIG 8A. The elements indicated as 1', 1", 7"'and 1"" bind to different (non-identical) markers on one or more cells in the B cell lineage.

[0029] FIG. 9 illustrates the development of B cells through to plasma cells, and some of the markers displayed on B and plasma cells during their development. FIG. 9 also shows certain antibody-dependent and independent effects in SLE, myasthenia gravis and RA along with their association with B cell and plasma cell development stages. Anti-dsDNA is antibodies against double stranded DNA, anti-MuSK is antibodies against muscle specific kinase, anti-CP is antibodies against citrullinated peptides (also called anti-CCP), and RF is rheumatoid factor (anti-IgG Fc antibodies).

[0030] FIG. 10 illustrates a bispecific hemibody of the invention comprised of a first hemibody construct having a BC121 polypeptide chain with an anti-CD69 VHaa sequence and a BC122 polypeptide that pairs with BC121 to form an anti-CD69 Fab binding structure. BC121 also has an anti-CD3 VH aa sequence. The second hemibody construct BC124 has an anti CD19 scFv and an anti-CD3 VL aa sequence. Together the hemibody first and second constructs form a complete paratope for binding a CD3 marker on a cytotoxic effector cell. Also shown is a control bispecific construct comprised of two peptide constructs, BC125 and BC122. BC122 pairs with BC125 to form the same anti- CD69 Fab as in the BC121-BC122 pairing, but the control construct has an anti-CD3 scFv formed by the VH and VL elements 4 and 4’. A pair of disulfide bonds joining the scaffold sequences is shown as two short lines between elements labeled 2 (CH2CH3 aa sequences). The CH1 and OK sequences comprise MD 13 substitutions and are joined by a disulfide bond as indicated.V. Detailed DescriptionA. Definitions

[0031] Hemibody or hemibodies as used herein refers to a pair of polypeptides or polypeptide constructs that each comprise one or more aa sequences (generally antibody complementarity determining regions or "CDRs” optionally in the form of VH and VL domains) that gain antigen-engaging capabilities upon quaternary structural assembly of a paratope from the component polypeptides. Hemibodies can be brought into appropriate molecular apposition for paratope assembly by, for example, targeting the same tumor cell surface (e.g., engaging markers on the cell's surface).

[0032] Trispecific antibody or trispecific antibody construct as used herein refers to a polypeptide or protein that comprises three antigen-binding or antigen-engaging domains (generally derived from antibodies or nanobodies) that are each directed to different (non-identical) antigens.

[0033] The terms "polynucleotide” and "nucleic acid” are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and / or pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0034] Throughout this disclosure the abbreviation aa is used for amino acid or amino acids. Where only the plural is intended, the abbreviation aas may be used. Standard three-letter or single-letter abbreviations for amino acids are employed and refer to L-amino acids unless stated otherwise.

[0035] As used herein, amino acid ("aa” singular or "aas" plural) means the naturally occurring proteinogenic amino acids incorporated into polypeptides and proteins in mammalian cell translation. Unless stated otherwise: L (Leu, leucine), A (Ala, alanine), G (Gly, glycine), S (Ser, serine), V (Vai, valine), F (Phe, phenylalanine), Y (Tyr, tyrosine), H (His, histidine), R (Arg, arginine), N (Asn, asparagine), E (Glu, glutamic acid), D (Asp, asparagine), C (Cys, cysteine),Q (Gin, glutamine), I (lie, isoleucine), M (Met, methionine), P (Pro, proline), T (Thr, threonine), K (Lys, lysine), and W (Trp, tryptophan). The amino acids hydroxyproline and selenocysteine, which appear in some proteins found in mammalian cells, are not understood to be included unless their presence is expressly indicated.

[0036] The terms "polypeptide” and "protein” are used interchangeably herein and refer to a polymeric form of amino acids, which unless stated otherwise are the naturally occurring proteinogenic L-amino acids that are incorporated biosy nthetically into proteins during translation in a mammalian cell. Where the sequence of a wild-type (wt.) polypeptide is altered, either by addition or deletion of one or more amino acids, the specific residue or residue number will refer to the same specific amino acid in the altered polypeptide (e.g., the addition of one amino acid at the N-terminus of a peptide referenced as position A21 will be understood to indicate the amino acid alanine that is now position 22). Substitution of an amino acid at a specific position is denoted by the abbreviated notation indicating, in order, the original amino acid, its numerical position in the protein, and the substituted amino acid, e.g., substituting the alanine at position 21 with a cysteine is denoted as A21C.

[0037] The terms "individual,” "subject,” and "patient” are used interchangeably and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired. Mammals include humans and non-human primates. In addition, mammals include rodents (e.g., rats; mice), lagomorphs (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, and the like), felines, canines, etc.

[0038] The terms "disease" and "disorder" are used interchangeably.

[0039] The term "in vitro” as used herein refers to any process or procedure occurring outside of the body, e.g., the body of a patient.

[0040] The term, "in vivo” as used herein refers to any process or procedure occurring inside of the body, e.g., the body of a patient.

[0041] As used herein, the term "about” used in connection with an amount indicates that the amount can vary by 10%. For example, "about 100” means an amount of from 90-110. Where "about” is used in the context of a range, "about” used in reference to the lower amount of the range means that the lower amount includes an amount that is 10% lower than the lower amount of the range, and "about” used in reference to the higher amount of the range means that the higher amount includes an amount 10% higher than the higher amount of the range. For example, from about 100 to about 1000 means that the range extends from 90 to 1100.

[0042] It is noted that, as used herein and in the appended claims and aspects, the singular forms "a,” "an,” and "the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a B cell” includes a plurality of B cells, reference to "the element" includes reference to one or more elements, and reference to "the aa sequence” includes reference to one or more aa sequences, and so forth. It is further noted that the claims and / or aspects may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely,” "only” and the like in connection with the recitation of a claim and / or aspect element, or use of a "negative” limitation to exclude, for example, an optional element.

[0043] Unless stated otherwise, "expressed on the surface of a cell” with regard to antigens and / or markers means one or more molecules (e.g., proteins such as transmembrane proteins) that are expressed by the cell and displayed either transiently or persistently on the cell surface membrane during at least one stage of the cell's life.

[0044] Selectively expressed on a cell or the surface of a cell with regard to markers (e.g., cell surface antigens) refers to a pattern of expression in which the marker is expressed on a cell at levels higher than the level it is expressed on at least one other cell type. Selective expression may occur substantially or wholly (exclusively) on one type of cell (e.g., substantially or exclusively on a cytotoxic effector cell, B cell, or plasma cell) or even during a specific phase of the cell's life cycle or development cycle.

[0045] The term "substantially” is intended to encompass both "wholly” and "largely but not wholly” unless indicated otherwise.

[0046] Unless stated otherwise, "cells of the B cell lineage” include, but are not limited to, naive B cells, follicular B cells, marginal zone B cells, memory B cells, germinal center B cells, regulatory B cells, and cells of the plasma cell sublineage (e.g., plasmablasts, plasma cells and long-lived plasma cells). B cell subsets, including pro-B cells, pre-B cells, and tissue-resident B cells, also are considered to be within the B cell lineage.

[0047] Unless stated otherwise, B cells include, but are not limited to, naive B cells, follicular B cells, marginal zone B cells, memory B cells, germinal center B cells, and regulatory B cells, each of which form a subset of the cells in the B cell lineage. B cells also include pro-B cells, pre-B cells, and tissue-resident B cells, each of which form a subset of the cells in the B cell lineage.

[0048] Unless stated otherwise, cells of the plasma cell sublineage include plasmablasts, plasma cells, and long lived plasma cells, each of which form a subset of the cells in the B cell lineage.

[0049] For the purpose of this disclosure, "cytotoxic effector cells”, in the broadest sense, are non-B cell lineage leukocytes capable of cytotoxic (e.g., granule dependent and / or granule-independent cytotoxic responses) and / or phagocytic responses, and are not understood to include megakaryocytes or red blood cells. Cytotoxic effector cells may be divided into myeloid and lymphoid cytotoxic effector cells.B. Description

[0050] The present disclosure describes bispecific hemibodies and trispecific antibodies that find use in, among other things, the selective depletion or ablation of cells in the B cell lineage. Depletion may be accomplished using bispecific hemibodies and trispecific antibodies described herein to recruit cells capable of directing the killing of the desired cell(s) in the B cell lineage through mechanisms including the recruitment and induction of cytotoxic action by one or more cytotoxic effector cells (e.g., stimulating the granule dependent and granule independent effector functions of CD8+ cytotoxic T cells). Where the bispecific hemibodies and / or trispecific antibodies comprise an aa sequence capable of inducing antibody-mediated actions (effector functions), the desired cells may also be subject to depletion through any one or more of ADCC, ADCP, and / or CDC.

[0051] The selection of cells subject to the depletion is achieved using at least two different markers (cell surface antigens) on the surface of the cell (s) targeted for depletion. The markers may be transiently expressed or undergo a transient increase in expression as the targeted cell(s) proceed through one or more stages of B cell lineage development. Alternatively, the markers may be expressed (e.g., persistently or continuously) in one or more stages of B cell development or as the cells become resident in specific tissues (e.g., marginal zone B cells or follicular B cells).

[0052] Markers expressed by cells in the B cell lineage as part of a process associated with a change in the stage of their development or function (sometimes referred to as an activation marker or a marker of B cell activation) may be a target of the elements of bispecific hemibodies and / or trispecific antibodies that bind to markers on cells of the B cell lineage. As indicated above, such activation markers may be expressed transiently. For example, CD69 is an activation marker whose expression is transiently elevated upon exposure of naive B cells to antigen.

[0053] Markers selectively expressed by cells in one or more developmental stages of cells in the B cell lineage (sometimes referred to as lineage-specific markers) may be a target of elements of bispecific hemibody and trispecific antibody construct sequences that bind to markers on cells of the B cell lineage. Such lineage-specific markers may be expressed transiently or persistently. CD 19 and B Cell Maturation Antigen (BCM A) represent examples of lineage-specific markers expressed by B cells and plasma cells, respectively. A bispecific hemibody or trispecific antibody construct may comprise an element (e.g., an aa sequence) that binds a lineage-specific marker (e.g. CD19) and an element that binds a marker of B cell activation (e.g., CD69 on memory B cells).

[0054] Any group of cells in the B cell lineage may be subject to depletion depending on the cell markers targeted by the bispecific hemibodies and trispecific antibodies. B cells subject to selective depletion or ablation include naive B cells (B cells that have not been exposed to an antigen). B cells subject to selective depletion or ablation include follicular B cells and / or marginal zone B cells. B cells subject to selective depletion or ablation include memory B cells. Cells of the plasma cell sublineage subject to deletion or ablation include plasmablasts, plasma cells, long-lived plasma cells, and sub-populations thereof. The cells subject to depletion may include naive B cells. The cells subject to depletion may include non-naive B cells and / or plasma cells. The cells subject to depletion may include B memory cells.

[0055] The selectivity of the depletion process may be adjusted by the selection and number of markers employed on the targeted cells. While cell depletion using the bispecific hemibodies and trispecific antibodies described herein employs a minimum of two markers present on cells of the B cell lineage, trispecific, tetraspecific, and other multispecific hemibodies permit the use of more than two markers. Using three or four markers present on the target cell(s) permits enhanced selectivity in the depletion of cells in the B cell lineage relative to the selectivity observed targeting two markers expressed on cells in the B cell lineage. Using two, three, four or more markers permits the selective depletion of cells in the B cell lineage relative to cells expressing, for example, one of the markers or relative to cells expressing low levels of two or more of the markers.

[0056] Both the recruitment of cells capable of ablating the desired cells in the B cell lineage and the induction of cytotoxic / cytolytic responses against those cells are accomplished by incorporating into the bispecific hemibodies and trispecific antibody constructs aa sequences directed against surface antigens of cytotoxic effector cells (e.g., effector T cells such as CD8+ T cells) and optionally aa sequences that induce antibody mediated effector functions including, but not limited to, ADCC, ADCP, and / or CDC. The binding of the bispecific hemibodies and trispecific antibody constructs to target cells in the B cell lineage serves to localize cytotoxic effector cells to the target population of cells in the B cell lineage, and may also act to stimulate their cytotoxic / cytolytic responses. Sequences that bind to cell surface antigens selectively expressed on T cells, such as CD3, may be employed to bind and recruit T cells and to activate T cell cytotoxic / cytolytic responses. Alternatively, aa sequences that bind to antigens found onT cells and / or on other cytotoxic effector cells (e.g., NK cells, macrophages, and / or monocytes) may advantageously be incorporated into the bispecific hemibodies and / or trispecific antibody constructs. Utilizing such aa sequences can lead to the colocalization of T cells and one or more additional cytotoxic effector cell types that can contribute, additively or synergistically, to the cytotoxic / cytolytic attack by T cells on the desired population of cells. For example, aa sequences binding to CD2, CD56 (Neural Cell Adhesion Molecule or NCAM), CD158, CD159 and / or CD247, each of which are found on both T cells and NK cells, may be incorporated into the bispecific hemibody and trispecific antibody constructs described herein. Similarly, aa sequences binding to antigens like CD366 found on T cells, macrophages, dendritic cells, and NK cells may be incorporated into the bispecific hemibody and trispecific antibody constructs described herein.1. Bispecific Hemibodies

[0057] Bispecific hemibodies of the present disclosure comprise a first hemibody construct and a second hemibody construct. The first hemibody construct comprises as elements a first element 1’ that binds a first marker (e.g., an activation marker) expressed on a cell in the B cell lineage (e.g., CD69 expressed on a B cell) and a VHor V aa sequence 4 of a hemibody that binds to a CEC-SA. The second hemibody construct comprises as elements a second element 1” that binds a second marker (e.g., a lineage-specific marker) expressed on a cell in the B cell lineage (e.g., a B cell), and the corresponding VHor V aa sequence 4’ of the hemibody that binds to a CEC-SA, such that when the two constructs are brought together the VH and VL aa sequences 4 and 4’ form a CEC-SA binding paratope (e.g., a T cell surface antigen binding paratope). See e.g., FIG. 1 A at A. Either of the first and second hemibody constructs may be a heterodimer comprising two separate polypeptides (the polypeptide pair 7 and 8, and the pair 9 and 10) optionally linked by one or more disulfide bonds. Alternatively, either the first and / or second hemibody constructs may be comprised of a single polypeptide (see, e.g., FIG. 1 A at B polypeptides 11 and 12) that may comprise one or more stabilizing intrachain disulfide bonds not shown in the accompanying FIGs. 1 A or 1 B. In one instance, the first and second hemibody constructs are heterodimers comprised of two separate polypeptide chains as in FIG. 1A, structure A, and are optionally linked by one or more disulfide bonds (e.g., disulfide bonds between scaffold elements 2 and 2’, not shown). In another instance, the first and second hemibody constructs are each single polypeptide chains as in FIG. 1A, structure B, having one or more disulfide bonds between the scaffold elements marked as 2.

[0058] A bispecific hemibody comprising a first hemibody construct and a second hemibody construct, wherein:(i) the first hemibody construct comprises a first element T that binds a first marker expressed on the surface of a cell of B cell lineage (e.g., CD69 expressed on a B cell) and an antibody variable heavy chain (VH) 4 or variable light chain (VL) 4’ aa sequence of a hemibody that binds to a CEC-SA, wherein(a) the first element T comprises an aa sequence, and / or(b) the first element T comprises a VH aa sequence, VL aa sequence, or the aa sequence(s) of complementarity determining regions (CDRs) of a VH or VL aa sequence,wherein, when the first element comprises a VH or VL aa sequence, or aa sequence(s) of CDRs of a VH or VL aa sequence, the first construct further comprises an additional polypeptide 74 comprising the corresponding VH or VL aa sequence or CDRs of a corresponding VH or VL aa sequence, and the VHand VLaa sequences, or the CDRs of the VHand VLaa sequences, together form a paratope binding to the first marker (under these circumstance the first element and the further peptide form the paratope binding to the first marker); and(ii) the second hemibody construct comprises a second element 1" that binds a second marker expressed on the surface of a cell of B cell lineage (e.g., a B cell), and the corresponding VH or VL (4 or 4’) aa sequence of the hemibody that binds to a CEC-SA, wherein(a) the second element 1” comprises an aa sequence, and / or(b) the second element 1” comprises a VH aa sequence, VL aa sequence, or the CDRs of a VH or VL aa sequence, wherein, when the second element comprises a VH or VL aa sequence, or CDRs of a VH or VL aa sequence, the second construct may further comprise an additional polypeptide 15 comprising the corresponding VHor VLaa sequence or CDRs of a corresponding VHor VLaa sequence, and the VHand VLaa sequences, or the CDRs of the VHand VLaa sequences, together form a paratope binding to the second marker (under these circumstance the second element and the further peptide form the paratope binding to the second marker);(ill) the first and second hemibody constructs together comprise VH and VL aa sequences (4 and 4’) that form a CEC-SA binding paratope; and(iv) the first and / or second constructs optionally comprise one or more independently selected scaffold aa sequences and / or optionally one or more independently selected linker sequences. See, e.g., FIG. 1Aat constructs A and B.

[0059] The bispecific hemibodies described above may be of the form shown in, for example, FIG. 1 A at A, in which: the first hemibody construct comprises a first polypeptide 7 comprising a first scaffold aa sequence 2 and a second polypeptide 8 that comprises a second scaffold aa sequence 2’; and the second hemibody construct comprises a third polypeptide 9 comprising a third scaffold aa sequence 2 and a fourth polypeptide 10 that comprises a fourth scaffold aa sequence 2’; and wherein the aa sequence of each scaffold aa sequence may be selected independently.

[0060] In such bispecific hemibodies of the form shown in, for example, FIG. 1 A at A, the first polypeptide 7 and second polypeptide 8 of the first hemibody construct may associate (bind together) through interactions between a first and a second scaffold aa sequence (elements 2 and 2’ or if identical 2) and are optionally covalently linked together by one or more disulfide bonds (e.g., between the first scaffold 2 and second scaffold 2’ aa sequences); and the third polypeptide 9 and fourth polypeptide 10 of the second hemibody construct may associate (bind together) through interactions between the scaffold aa sequences (2 and 2’) of those peptides. The third and fourthpolypeptides of the second hemibody construct are optionally covalently linked together by one or more disulfide bonds (e.g. between the scaffold aa sequences). See, e.g., FIG. 1 A, structure A.

[0061] The bispecific hemibodies described above may also be of the form shown in, for example, FIG. 1 A at B, wherein:(I) the first hemibody construct comprises a first polypeptide 77 comprising both the first element and either the antibody variable heavy chain (VH) 4 or variable light chain (VL) 4’ aa sequence of the first hemibody construct;(II) the second hemibody construct comprises a second polypeptide 72 comprising both the second element and the corresponding VH or VL (4 or 4’) aa sequence of the hemibody that binds to a CEC- SA of the second hemibody construct;(ill) the first and second constructs together comprise the VH and VL aa sequences (4 and 4’) that form a CEC-SA binding paratope; and(iv) the first and / or second constructs optionally comprise one or more independently selected scaffold aa sequences.

[0062] In such bispecific hemibodies of the form shown in, for example, FIG. 1 A at B, the first polypeptide 77 may comprise a first scaffold aa sequence and a second scaffold aa sequence that may be the same (2) or different (e.g., a 2 and 2’ interspecific pair). Similarly, the second polypeptide 72 may comprise a third scaffold aa sequence and a fourth scaffold aa sequence that may be the same (2) or different (e.g., a 2 and 2’ interspecific pair). Each scaffold aa sequence is optionally selected independently. In the bispecific hemibody of the form shown in FIG. 1 A at B, the first and second scaffold aa sequences of the first polypeptide associate (bind together) and are optionally covalently linked by one or more disulfide bonds (e.g. between the first and second scaffold aa sequences), and the third and fourth scaffold aa sequences of the second polypeptide associate (bind together) and are optionally covalently linked by one or more disulfide bonds (e.g. between the third and fourth scaffold aa sequences.

[0063] Elements of the first and / or second hemibody constructs that bind to markers expressed on the surface of a cell of B cell lineage (any one or more of 7', 1”, 1”’ and 7””) may comprise (I) a VH or VL aa sequence, or CDRs of a VH or VL aa sequence; and (II) an additional polypeptide (e.g., elements 74 and / or 15 in FIG. 1 B at A-D) comprising the corresponding VH or VL aa sequence, or CDRs of a corresponding VH or VL aa sequence, and the VH and VL aa sequences, or the CDRs of the VHand VLaa sequences, such that (I) and (II) together form a paratope binding to the desired marker. See, for example, FIG. 1 B at A and B where the first and second elements comprise a VHaa sequence, and the construct comprises an additional polypeptide comprising a VL aa sequence that together form an Fv structure optionally joined by an interchain disulfide bond. See also, for example, FIG. 1B at C and D where the first and second elements (7' and 1") comprise a CH1-VH and the construct comprises an additional polypeptide comprising a CKVL aa sequence, that together form an Fab structure associated by interactions between the CH1 and CK aa sequences that optionally are joined by interchain disulfide bonds.

[0064] Bispecific hemibody constructs, for example of the form shown in FIG. 1 A at A or B or in FIG. 1 B at A to D, may comprise additional elements (e.g., a third element 7”’, or third and fourth elements 1”’ and 7””) that bind to markers expressed on the surface of a cell of B cell lineage. Where an element that binds to a third markerexpressed on the surface of a cell of B cell lineage is incorporated into a hemibody construct, the hemibody may be considered a trispecific hemibody. Where elements that bind to third and fourth markers expressed on the surface of a cell of B cell lineage are incorporated into a hemibody construct, the hemibody may be considered a tetraspecific hemibody. Trispecific hemibodies, tetraspecific hemibodies, and hemibodies with higher order specificities may be referred to collectively as multispecific hemibodies. Generally, only one of the first and third elements in a hemibody construct will comprise part of the paratope forming aa sequence and an additional polypeptide (74 or 15 comprising an aa sequence forming the remainder of the paratope.

[0065] The VH and / or VL (4 and / or 4’) aa sequences of a hemibody that bind to a CEC-SA may be masked as a mechanism to improve yield in preparation and stability of the constructs. Masking may also improve targeting in vivo where the mask can be removed by a protease in a compartment or tissue where a target population of cells is located.

[0066] As discussed above, compositions comprising hemibody constructs that comprise elements directed against three or four markers on one or more target cell (s) in the B cell lineage may be employed. Utilizing three elements directed against different cell markers permits the targeting of at least two different pairs of markers found on cells in the B cell lineage. Utilizing four elements directed against different cell markers permits the targeting of at least three different pairs of markers found on cells in the B cell lineage.

[0067] All elements of the hemibody binding to markers on cells of the B cell lineage (any one or more of 1', 1”, 1”’ and 1””) may be directed to markers expressed (transiently or persistently) on a single target cell type (e.g., cells in the same stage of development). For example, as shown in in FIG. 8A, a bispecific hemibody composition may comprise a first hemibody construct and two second hemibody constructs (recited below as a second and a third hemibody construct), wherein:(i) the first hemibody construct comprises a first element 1' that binds a first marker expressed on the surface of a cell of B cell lineage (e.g., CD69 expressed on a B cell) and an antibody variable heavy chain (VH) 4 or variable light chain (V ) 4' aa sequence of a hemibody that binds to a CEC-SA;(ii) the second hemibody construct comprises a second element 1" that binds a second marker expressed on the surface of a cell of B cell lineage (e.g., CD25) and the corresponding VH or VL (4 or 4') aa sequence of the hemibody that binds to a CEC-SA;(iii) the third hemibody construct comprises a third element 1"' that binds a third marker expressed on the surface of a cell of B cell lineage (e.g., CD80), and the corresponding VH or VL (4 or 4') aa sequence of the hemibody that binds to a CEC-SA;(iv) the first and second and the first and third hemibody constructs comprise VH and VL aa sequences (4 and 4') that form a CEC-SA binding paratope (e.g., an anti-T cell surface antigen binding paratope); and(v) the first, second, and / or third constructs optionally comprise one or more independently selected scaffold aa sequences.

[0068] By way of example, each of CD69, CD25, and CD80 are expressed on subsets of memory B cells. Hemibody compositions may comprise the first, second, and third constructs in amounts such that the total molar amount of the first construct will be the same as the total amounts of the second and third constructs combined. Inthat manner, when administered there will be an equal number of first and second constructs to combine and form CEC-SA binding paratopes (an equal number of constructs with VH and VL aa sequences that form a paratope binding to a CEC-SA). See, for example, FIG. 8A which shows the formation of two different hemibodies from a composition comprising a first construct combined with either a second or third hemibody construct. For example, such compositions may comprise the first, second, and third hemibody constructs in the ratio of 2: 1 :1 (i.e., two (2) first hemibody constructs: one (1) second hemibody construct: one (1) third hemibody construct). Where the density of the marker proteins on the surface of target cells is significantly different, compositions may employ different molar ratios of the first and second peptides so that approximately equal proportions of the hemibody constructs are present on the surface of the target cells. The individual hemibody constructs, or compositions comprising one or more of the first, second, and / or third hemibody constructs, may be separately administered to obtain any proportion desired in the patient.

[0069] Alternatively, the elements (e.g., aa sequences) of the hemibody binding to markers on cells of the B cell lineage may be directed to markers expressed (transiently or persistently) on at least two different target cell types (e.g., cells in different stages of development). For example, a bispecific hemibody composition may comprise a first hemibody construct comprising an element that binds to the BCR (found on both memory and follicular B cells), a second hemibody construct that comprises an element binding to CD22 (found on follicular B cells), and a third hemibody construct that comprises an element binding to CD25 or CD69 (found on memory B cells). Such compositions permit the selective targeting of two different combinations of markers on two different cell types (follicular and memory B cells) targeted for depletion.

[0070] Hemibody compositions in which elements that bind to four different markers expressed on cells in the B cell lineage may comprise:(I) a first hemibody construct that comprises a first element T that binds a first marker expressed on the surface of a cell of B cell lineage (e.g., the BCR), and an antibody variable heavy chain (VH) 4 or variable light chain (V ) 4' aa sequence of a hemibody that binds to a CEC-SA;(II) a second hemibody construct that comprises a second element 1" that binds a second marker expressed on the surface of a cell of B cell lineage (e.g., CD69), and the corresponding VH or VL (4 or 4') aa sequence of the hemibody that binds to a CEC-SA;(ill) a third hemibody construct that comprises a third element T" that binds a third marker expressed on the surface of a cell of B cell lineage (e.g., CD21 or Notch2), and the corresponding VH or VL (4 or 4') aa sequence of the hemibody that binds to a CEC-SA; and(iv) a fourth hemibody construct that comprises a fourth element 1"" that binds a fourth marker expressed on the surface of a cell of B cell lineage (e.g., CD22), and the corresponding VHor VL(4 or 4') aa sequence of the hemibody that binds to a CEC-SA; wherein the first hemibody construct combined with any of the second, third or fourth hemibody constructs comprises VH and VL aa sequences (4 and 4') that form a CEC-SA binding paratope (e.g., an anti-T cell surface antigen binding paratope), andthe first, second, third, and / or fourth constructs optionally comprise one or more independently selected scaffold aa sequences (e.g., Ig aa sequences optionally with substitutions enhancing or diminishing ADCC ADCP and / or CDC effector functions). That composition gives rise to as many as three different hemibodies that may target one, two, or three different types of cells. See, for example, FIG. 8B.

[0071] As shown in FIG. 8C, an alternative hemibody composition in which elements that bind to four different markers expressed on cells in the B cell lineage may comprise:(I) a first hemibody construct that comprises a first element T that binds a first marker expressed on the surface of a cell of B cell lineage (e.g., BCR), and an antibody variable heavy chain (VH) 4 aa sequence of a hemibody that binds to a CEC-SA;(II) a second hemibody construct that comprises a second element 1” that binds a second marker expressed on the surface of a cell of B cell lineage (e.g., CD19), and the variable heavy chain (VH) 4 aa sequence of a hemibody that binds to a CEC-SA;(ill) a third hemibody construct that comprises a third element T" that binds a third marker expressed on the surface of a cell of B cell lineage (e.g., CD80), and the corresponding V 4' aa sequence of the hemibody that binds to a CEC-SA; and(iv) a fourth hemibody construct that comprises a fourth element 1"" that binds a fourth marker expressed on the surface of a cell of B cell lineage (e.g., CD80), and the corresponding VL 4' aa sequence of the hemibody that binds to a CEC-SA; wherein the first hemibody or second hemibody construct combined with either of the third or fourth hemibody constructs together comprise VHand V aa sequences (4 and 4') that form a CEC-SA binding paratope (e.g., an anti-T cell surface antigen binding paratope), and the first, second, third, and / or fourth constructs optionally comprise one or more independently selected scaffold aa sequences.

[0072] The four constructs of the alternative hemibody composition may give rise to four different hemibodies that may target one, two, three, or four different types of cells, depending on the B cell lineage markers chosen. See, for example, FIG. 8C.

[0073] As with compositions comprising three constructs, compositions comprising four constructs may be provided in amounts such that there are an equal number of constructs with VH and VL aa sequences (elements 4 and 41) that form a paratope binding to a CEC-SA. The constructs may be administered in one or more separate compositions, or even individually, to achieve any molar ratio (stoichiometry) of the constructs that is desired in a patient.

[0074] In compositions comprising hemibody constructs with elements directed against three or four markers on target cell (s) in the B cell lineage, any of the elements binding to those markers may be in the form of an aa sequence (e.g., a VHH or scFV) or Fab comprising two peptides. For example, any one or more of elements 1', 1", 1"', or 1"" may be in the form of a nanobody aa sequence or an Fab as in FIG. 1B.2. Trispecific antibody constructs

[0075] Trispecific antibodies of the present disclosure may comprise a first polypeptide 16 and a second polypeptide 17, wherein the first polypeptide and second polypeptide together comprise as elements:(A) (I) a first element 1' that binds a first marker expressed on the surface of a cell of B cell lineage, and(II) a second element 1" that binds a second marker expressed on the surface of a cell of B cell lineage; and(B) either(I) an aa sequence comprising an antibody variable heavy chain (VH) aa sequence 4 of an antibody that binds a CEC-SA, and an aa sequence comprising an antibody variable light chain (VL) aa sequence 4’ of an antibody that binds a CEC-SA (e.g., a T cell marker) (see, e.g., FIG. 1 A, structures C to I), or(II) a single chain antibody sequence (e.g., an scFv or nanobody aa sequence) that binds a CEC-SA (e.g., 4 and 4’ in FIG. 1 A, structures F to I, together are an scFv or nanobody aa sequence); wherein(a) either the first element or second element comprises an aa sequence (e.g., a VHH or scFV aa sequence), and / or(b) either the first element or second element comprises a VH aa sequence, VL aa sequence, or the complementarity determining regions (CDRs) of a VH or VL aa sequence, wherein, when the first element or second element comprises a VH or VL aa sequence, or CDRs of a VH or VL aa sequence, the trispecific antibody construct further comprises a polypeptide (an additional polypeptide 14 or 15) comprising the corresponding VH or VL aa sequence or CDRs of a corresponding VH or VL, and together the VH and VL, or together the CDRs of the VH and VL, form a paratope binding to the first marker or second marker; and the VHand VLaa sequences that bind a CEC-SA (4 and 4’) together form a CEC-SA binding paratope, and the first and second polypeptides optionally comprise an independently selected scaffold aa sequence that can bind together forming a heterodimer comprising the first polypeptide and second polypeptide, and the first and / or second polypeptides each optionally comprise one or more independently selected linker sequences. See, e.g., FIG. 1 A, constructs C to I.

[0076] A trispecific antibody construct may also comprise a single polypeptide 18 comprising:A (I) a first element that binds to a first marker expressed on the surface of a cell of B cell lineage,(II) a second element that binds to a second marker expressed on the surface of a cell of B cell lineage, (ill) optionally, one or more independently selected scaffold aa sequences 2, and(iv) optionally, one or more independently selected linker sequences; andB either(I) an antibody variable heavy chain (VH) 4 aa sequence of an antibody that binds a CEC-SA and an antibody variable light chain (VL) 4’ aa sequence of an antibody that binds a CEC-SA, or(ii) a single chain antibody sequence (e.g., an scFv or nanobody aa sequence) that binds a T-cell surface antigen (e.g., 4 and 4’ in FIG. 1 A, structures J to M, together form a CEC-SA binding paratope); wherein(I) the VHand V aa sequences binding to a CEC-SA (4 and 4’) together form a CEC-SA binding paratope, (ii) (a) either the first element or second element comprises an antigen binding aa sequence and / or (b) either the first element or second element comprises a VHaa sequence, V aa sequence, or the complementarity determining regions (CDRs) of a VH or VL aa sequence, and(ill) when the first element or second element comprises a VH or VL aa sequence, or CDRs of a VH or VL aa sequence, the trispecific antibody construct further comprises a polypeptide (an additional polypeptide) comprising the corresponding VH or VL aa sequence or CDRs of a corresponding VH or VL, and together the VH and VL, or together the CDRs of the VH and VL, form a paratope binding to the first marker or second marker. See, e.g., FIG. 1A, constructs J to M.

[0077] A first 1’ or second 1" element present in the trispecific antibody construct may comprise (I) a VH or VL aa sequence, or CDRs of a VHor VLaa sequence; and (ii) a polypeptide (an additional polypeptide 14) comprising the corresponding VHor VLaa sequence, or CDRs of a corresponding VHor VL, and the VHand VL, or the CDRs of the VHand VL, wherein (I) and (ii) together form a paratope binding to the desired marker. See, for example, FIG. 1 B at E and F, where the first element comprises VH and the construct comprises an additional peptide comprising a VL aa sequence that together form an Fv structure optionally joined by an interchain disulfide bond. See, also, for example, FIG. 1 B at G and H, where the first element comprises a CH1-VH, and the construct comprises an additional polypeptide comprising a CKVL aa sequence, that together form an Fab structure associated by interactions between the CH1 and CK aa sequences, that is optional ly joined by interchain disulfide bonds. Generally, only one of the first and second elements will comprise part of their paratope forming aa sequence in an additional polypeptide.

[0078] Some potential organizations of the trispecific antibodies include, but are not limited to, the cases that follow. In a first case, each polypeptide of a trispecific antibody construct heterodimer 16 and 17 comprises an element (7 ' or 7") that binds to a marker (e.g., a surface antigen) expressed on a cell in the B cell lineage, and a VHor VLaa sequence (4 or 4’) that together in the heterodimeric construct give rise to a paratope with affinity for a CEC-SA as exemplified in FIG. 1 A at C. In a second case, the first and second polypeptides of a trispecific antibody construct heterodimer each comprise one of the aa sequences that together give rise to a paratope with affinity for a CEC-SA (e.g., VH 4 or VL 4’), and either the first or second polypeptide comprises both of the first and second elements (7' and 1") that bind to the first marker (e.g., a marker of activation) and second marker (e.g. a plasma cell lineage surface antigen) expressed on a cell in the B cell lineage as exemplified in FIG. 1 A at D and E. In a third case, the first polypeptide of a trispecific antibody construct heterodimer comprises both the first and second elements (7' and 7") that bind to the first marker (e.g., a plasma cell marker of activation) and the second marker (e.g., a plasma cell lineage surface antigen) expressed on cells in the B cell lineage, and the second polypeptide comprises the aa sequence(s) that bind to a CEC-SA (e.g., VH and VL aa sequences (4 and 4’) as exemplified in, for example, FIG. 1 A at F (see also FIG. 1 A at G). In a fourth case, the first and second polypeptides of a trispecific antibody construct heterodimer each comprise one of the first and second elements (7' and 1”) that bind to the first and second markersexpressed on a cell in the B cell lineage, and either the first or second polypeptide comprises the aa sequence(s) 4 and 4’ that together give rise to a paratope with affinity for (that binds to) a CEC-SA (e.g., VH and VL aa sequences) as exemplified in FIG 1 at H and I. In a fifth case, each of the first and second elements (f ' and 1") that bind to the first and second markers expressed on a cell in the B cell lineage, and the aa sequence(s) 4 and 4’ that together give rise to a paratope with affinity for (that binds to) a CEC-SA is part of a single polypeptide as exemplified in FIG. 1 A at J to M. In any of the third through fifth cases, where elements 4 and 4’ are adjacent, they may be in the form of VHand V elements joined by a linker aa sequence, an scFv, a nanobody, Ig heavy chain, or CDRs from VHand / or V elements that bind to a CEC-SA.

[0079] The specificity of trispecific antibodies for various cells in the B cell lineage may be affected by a variety of factors. The elements binding to markers expressed on the surface of target cells in the B cell lineage may have sufficiently high affinity for the marker that either or both of the elements can bind a trispecific antibody construct to a potential target cell, but that may lead to off-target binding where the marker is not exclusively expressed by the target cell. Where one or both of the markers targeted by the trispecific antibody construct is not uniquely expressed by the target population, elements with lower affinity for the targeted markers found on cells of the B cell lineage may be included in trispecific antibodies. Utilizing elements with lower affinity for the targeted markers in that manner increases the specificity of the trispecific antibodies as only cells expressing both markers will effectively bind the trispecific antibody construct.3. Elements of Bispecific Hemibody and Trispecific Antibody Constructs

[0080] The bispecific hemibodies and trispecific antibodies described herein employ a number of elements in common. Those elements include, among other things, linker aa sequences, scaffold sequences, and the elements that bind to markers (cell surface antigens) on cells in the B cell lineage (i.e., first, second, third, and fourth elements). In addition, hemibodies and trispecific antibodies may include intra-polypeptide and inter-polypeptide disulfide bonds that, among other things, stabilize their structure and increase their resistance to thermal denaturation. In addition to the above-mentioned elements, both the bispecific hemibodies and trispecific antibodies described herein comprise aa sequences that have affinity for one or more surface antigens found on the surface of cytotoxic effector cells.

[0081] Cytotoxic effector cells within the myeloid immune cells group whose surface antigens can be bound by hemibodies and trispecific antibodies include monocytes, macrophages, dendritic cells, and / or granulocytes. Cytotoxic effector cells within the myeloid immune cells group whose surface antigens can be bound by hemibodies and trispecific antibodies include dendritic cells that are, for example, conventional and / or plasmacytoid dendritic cells. Cytotoxic effector cells within the myeloid immune cells group whose surface antigens can be bound by hemibodies and trispecific antibodies include granulocytes (e.g., neutrophils, eosinophils, basophils and / or mast cells).

[0082] Cytotoxic effector cells whose surface antigens are targeted by the bispecific antibodies and hemibodies described herein may be lymphoid cells. Cytotoxic effector cells of the lymphoid type may be T cells and / or natural killer (NK) cells. Lymphoid cytotoxic T effector cells may be CD4+ and CD8+ T cells, but exclude regulatory cells(e.g. , T regs such as CD4+, F0XP3+, and CD25+ T regs). Cytotoxic effector cells whose surface antigens are targeted by the bispecific antibodies and hemibodies described herein may be NK cells. Cytotoxic effector cells whose surface antigens are targeted by the bispecific antibodies and hemibodies described herein may be T effector cells.

[0083] It will be understood by those familiar with the immunological sciences that cell surface antigens on cytotoxic effector cells may not be unique to a cytotoxic effector cell but may be cell surface antigens whose expression is substantially limited to one or more types of cytotoxic effector cells. The expression of some CEC-SAs may be substantially limited to cells of the myeloid immune system. The expression of some CEC-SAs may be substantially limited to cells of the lymphoid immune system. Some cell surface antigens may be present on two or more myeloid and / or lymphoid cells. For example, the surface antigens targeted by bispecific hemibodies may be one or more antigens expressed upon T cells (e.g., effector T cells) and / or NK cells. The surface antigens targeted may also be one or more antigens substantially expressed only upon T cells (e.g., effector T cells).

[0084] In trispecific antibodies the element(s) (e.g., aa sequences) that bind to effector cells comprise an aa sequence or aa sequences forming a paratope (e.g., a nanobody aa sequence, a pair of VHand V aa sequences such as an scFv) that binds to a surface antigen on a cytotoxic effector cell. In contrast, bispecific hemibodies comprise separate first and second constructs, one of which comprises a VH aa sequence and the other of which comprises its cognate VL aa sequence (or the CDRs of those sequences). Together those sequences form a paratope binding to a surface antigen on a cytotoxic effector cell. The first and second constructs must be brought together, for example, by binding of the hemibody constructs to a common cell type to form the paratope that binds to a surface antigen on a cytotoxic effector cell.

[0085] Elements of bispecific hemibodies and trispecific antibodies (e.g., sequences of aas, or VHor V aa sequence, or CDRs of a VHor V aa sequence) forming all or part of a paratope, and any additional polypeptide that forms the remainder of the paratope, may be immunoglobulin (Ig) aa sequences. VHand V elements and the CDRs of those binding sequences may be obtained from known antibodies that bind to the markers / surface antigens. Where the binding sequences are to be in a single chain format (e.g., an scFv), the VH and VL elements may be brought into a suitable single chain framework, for example by CDR grafting, e.g., utilizing the tools of molecular biology to clone sequences expressing the CDRs into a suitable single chain sequence. Techniques for the development of scFVs are well known and practiced in the art (see, e.g., Olafsen et al., Antibody Eng., 2:68-78, (2010)). Some suitable methods for humanizing antibodies include, but are not limited to: CDR-grafting (EP 0239400, WO 91 / 09967, U.S. Pat. Nos. 5,530,101 and 5,585,089; Kim et al., Meth. Mol. Biol., 907:237-245 (2012), and Gupta et al, J. Biol. Chem. 300(1): 105555 (2024)); and veneering or resurfacing (EP 0592106 and EP 0519596).

[0086] All sequences, including aa sequences within bispecific hemibodies and trispecific antibodies that bind to cell surface markers (antigens), may be deimmunized. For example, aa sequences may be deimmunized by selective substitution of aas without substantially changing their properties using techniques known in the art. See, e.g., Zinsli et al., Computational & Structural Biotech. J., 19: 315-329 (2021). All of the elements in bispecific hemibodies or trispecific antibody constructs that bind to markers (surface antigens) on cells in the B cell lineage and to cytotoxic effector cells may be elements (e.g., deimmunized elements) that bind to extracellular epitopes of themarkers from a single species. When intended for human use, all of the elements in the bispecific hemibodies or trispecific antibody constructs that bind to markers on cells in the B cell lineage and on cytotoxic effector cells may be elements (e.g., deimmunized) that bind to markers present on human cells.

[0087] All of the cells bound by the bispecific hemibodies and trispecific antibodies recited herein may be of human origin. Accordingly, all of the bispecific hemibodies and trispecific antibodies recited herein may bind to markers found on a human cell of the B cell lineage (including its plasma cell sublineage) and / or CEC-SAs found on human cytotoxic effector cells. a) Sequences with Affinity for markers expressed on cells in the B cell lineage

[0088] A variety of markers on cells in the B cell lineage may be employed to selectively target groups of B cells for ablation. Because selectivity is driven by the presence of at least two markers, it is possible to avoid substantial depletion of a patient's immune repertoire by selecting markers that target non-naive cells in the B cell lineage so that after treatment with a bispecific hemibody or trispecific antibody construct the patient may regenerate cells of the B lineage and mount humoral immune responses.

[0089] Hemibodies and trispecific antibody constructs comprise first and second elements that bind to different (non-identical) markers expressed on cells in the B cell lineage targeted for depletion. The markers may be selected from the group consisting of: BCMA (CD269), mlg (e.g., membrane bound immunoglobulins including mlgG, mlgM, mlgA, mlgD and / or mlgE), CD1d, CD5, CD10, CD11c, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD34, CD38, CD40, CD43, CD44, CD45, CD53, CD69, CD71, CD72, CD73, CD78, CD79a, CD79b, CD80, CD85j, CD86, CD95 (FAS), CD98, CD126 (IL6R), CD127 (IL7R), CD138 (SDC1), CD184 (CXCR4), CD185 (CXCR5), CD267 (TACI), CD268 (BAFFR), CD273 (PDL2), CD274 (PDL1), CD289 (TLR9), CD307d, CD319 (SLAMF7), CD365 (TIM1), SCA-1 (Ly6A / E), and HLA-DR. At least two of the selected markers (e.g., at least three of the selected markers) should be found on at least one type of cell in the population of cells targeted for deletion. As discussed above, more than two elements that bind to different (non-identical) markers may be selected for use in the depletion of a set (population) of target cells in the B cell lineage. When the depletion employs multiple hemibodies, or multiple hemibodies formed from a combination of first and second constructs giving rise to a paratope binding to cytotoxic effector cells, three or more or four or more elements that bind to different (non- identical) markers present on the target population of cells may be employed. Similarly, where more than one trispecific antibody construct is employed for the depletion of a target set of cells in the B cell lineage, three or more or four or more markers expressed on the population of cells targeted for depletion may be employed. Where three or more or four or more elements that bind to markers expressed on the population of cells targeted for depletion are employed, they may be selected from the group recited above, and as indicated above, at least two of the selected markers should be found on at least one type of cell in the population of cells targeted for deletion.

[0090] Elements of bispecific hemibodies or trispecific antibodies that bind to markers expressed on cells in the B cell lineage targeted for depletion may bind to one or more markers selected from: CD19, CD20, CD22, CD79a, and / or CD79b. Those markers are expressed upon follicular B cells, germinal center B cells, marginal zone B cells, memory B cells, and / or regulatory B cells. Any of the CD19, CD20, CD22, CD79a, and / or CD79b markers may beutilized in combination with one or more markers selected from the group consisting of CD 1 d, CD5, CD 10, CD11c, CD21, CD22, CD23, CD24, CD25, CD27, CD34, CD38, CD40, CD43, CD44, CD45, CD53, CD69, CD71, CD72, CD73, CD80, CD85j, CD86, CD95 (FAS), CD127 (IL7R), CD138, CD184 (CXCR4), CD185 (CXCR5), CD267 (TACI), CD268 (BAFFR), CD273 (PDL2), CD274 (PDL1), CD289 (TLR9), CD307d, CD365 (TIM1), and HLA-DR.

[0091] The bispecific hemibodies or trispecific antibodies may comprise one or more elements that bind to the pan B cell marker CD19, CD20, CD79a, or CD79b. The bispecific hemibodies or trispecific antibodies may comprise an element that binds to a pan B cell marker (e.g., CD19, CD20, CD79a, and / or CD79b) and an element that binds to a marker selected from the group consisting of CD1d, CD5, CD10, CD11c, CD21, CD22, CD23, CD24, CD25, CD27, CD34, CD38, CD40, CD43, CD44, CD45, CD53, CD69, CD71, CD72, CD73, CD80, CD85j, CD86, CD95 (FAS), CD127 (IL7R), CD138, CD184 (CXCR4), CD185 (CXCR5), CD267 (TACI), CD268 (BAFFR), CD273 (PDL2), CD274 (PDL1), CD289 (TLR9), CD307d, CD365 (TIM1), and HLA-DR. The bispecific hemibodies or trispecific antibodies may comprise an element that binds to a pan B cell marker (e.g., CD19, CD20, CD79a, and / or CD79b) and an element that binds to a marker selected from the group consisting of: CD 10, CD21, CD22, CD23, CD24, CD25, CD27, CD40, CD69, and CD95 (FAS).

[0092] The bispecific hemibodies or trispecific antibodies may comprise one or more elements that bind to CD19, and one or more, or two or more, elements that bind to a marker selected independently from the group consisting of: CD1d, CD5, CD10, CD11c, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD34, CD38, CD40, CD43, CD44, CD45, CD53, CD69, CD71, CD72, CD73, CD79a, CD79b, CD80, CD85, CD86, CD95 (FAS), CD127 (IL7R), CD138, CD184 (CXCR4), CD185 (CXCR5), CD267 (TACI), CD268 (BAFFR), CD273 (PDL2), CD274 (PDL1), CD289 (TLR9), CD307d, CD365 (TIM1), and HLA-DR. Optionally, each of the elements binding to B cell markers may bind to different (non-identical) markers.

[0093] The bispecific hemibodies or trispecific antibodies may comprise one or more elements that bind to CD20, and one or more, or two or more, elements that bind to a marker selected independently from the group consisting of: CD1d, CD5, CD10, CD11c, CD21, CD22, CD23, CD24, CD25, CD27, CD34, CD38, CD40, CD43, CD44, CD45, CD53, CD69, CD71, CD72, CD73, CD79a, CD79b,CD80, CD85j, CD86, CD95 (FAS), CD127 (IL7R), CD138, CD184 (CXCR4), CD 185 (CXCR5), CD267 (TACI), CD268 (BAFFR), CD273 (PDL2), CD274 (PDL1), CD289 (TLR9), CD307d, CD365 (TIM1), and HLA-DR. Optionally, each of the elements binding to B cell markers may bind to different (non-identical) markers.

[0094] The bispecific hemibodies or trispecific antibodies may comprise one or more elements that bind to CD19, and one or more, or two or more, elements that bind to a marker selected independently from the group consisting of: CD20, CD22, CD69, CD79a, CD79b, CD80, CD86, CD95 (FAS), CD267 (TACI), CD268 (BAFFR), and CD274 (PDL1). Optionally, each of the elements binding to B cell markers may bind to different (non-identical) markers.

[0095] The bispecific hemibodies or trispecific antibodies may comprise one or more elements that bind to CD20, and one or more, or two or more, elements that bind to a marker selected independently from the group consisting of: CD22, CD69, CD79a, CD79b, CD80, CD86, CD95 (FAS), CD267 (TACI), CD268 (BAFFR), and CD274 (PDL1). Optionally, each of the elements binding to B cell markers may bind to different (non-identical) markers.

[0096] The bispecific hemibodies or trispecific antibodies may comprise one or more elements that bind to CD22, and one or more, or two or more, elements that bind to a marker selected independently from the group consisting of: CD69, CD80, CD86, CD95 (FAS), CD267 (TACI), CD268 (BAFFR), and CD274 (PDL1). Optionally, each of the elements binding to B cell markers may bind to different (non-identical) markers.

[0097] The bispecific hemibodies or trispecific antibodies may comprise one or more elements that bind to CD19, and one or more, or two or more, elements that bind to a marker selected independently from the group consisting of: CD22, CD79a, CD79b, CD95 (FAS), CD267 (TACI), CD268 (BAFFR), and CD274 (PDL1). Optionally, each of the elements binding to B cell markers may bind to different (non-identical) markers.

[0098] The bispecific hemibodies or trispecific antibodies may comprise one or more elements that bind to CD20, and one or more, or two or more, elements that bind to a marker selected independently from the group consisting of: CD22, CD79a, CD79b, CD95 (FAS), CD267 (TACI), CD268 (BAFFR), and CD274 (PDL1). Optionally, each of the elements binding to B cell markers may bind to different (non-identical) markers.

[0099] The bispecific hemibodies or trispecific antibodies may comprise one or more elements that bind to CD79a, and one or more, or two or more, elements that bind to a marker selected independently from the group consisting of: CD79b, CD80, CD86, CD95 (FAS), CD267 (TACI), CD268 (BAFFR), and CD274 (PDL1). Optionally, each of the elements binding to B cell markers may bind to different (non-identical) markers.

[0100] The bispecific hemibodies or trispecific antibodies may comprise one or more elements that bind to CD79b, and one or more, or two or more, elements that bind to a marker selected independently from the group consisting of: CD80, CD86, CD95 (FAS), CD267 (TACI), CD268 (BAFFR), and CD274 (PDL1). Optionally, each of the elements binding to B cell markers may bind to different (non-identical) markers.

[0101] The bispecific hemibodies or trispecific antibodies may comprise one or more elements that bind to CD95 and one or more, or two or more, elements that bind to a marker selected independently from the group consisting of: CD80, CD86, CD267 (TACI), CD268 (BAFFR), and CD274 (PDL1). Optionally, each of the elements binding to B cell markers may bind to different (non-identical) markers.

[0102] The bispecific hemibodies or trispecific antibodies may comprise one or more elements that bind to CD267 and one or more, or two or more, elements that bind to a marker selected independently from the group consisting of: CD80, CD86, CD268 (BAFFR), and CD274 (PDL1). Optionally, each of the elements binding to B cell markers may bind to different (non-identical) markers.

[0103] The bispecific hemibodies or trispecific antibodies may comprise one or more elements that bind to CD268 and one or more, or two or more, elements that bind to a marker selected independently from the group consisting of: CD80, CD86, and CD274 (PDL1). Optionally, each of the elements binding to B cell markers may bind to different (non-identical) markers.

[0104] Bispecific hemibodies or trispecific antibodies for the depletion of one or more types of B cells, including follicular B cells, may comprise an element that binds a marker selected from CD19, CD20, CD79a, and CD79b and an element that binds to a marker selected from the group consisting of CD10 and CD185 (CXCR5).

[0105] Bispecific hemibodies or trispecific antibodies for the depletion of one or more types of B cells, including marginal zone B cells, may comprise an element that binds a marker selected from CD19, CD20, CD79a, and CD79band an aa sequence that binds to a marker selected from the group consisting of CD1d and CD21. Bispecific hemibodies or trispecific antibodies for the depletion of one or more types of B cells, including marginal zone B cells, may comprise an element that binds a marker selected from CD 19, CD20, CD79a, and CD79b and an element that binds to CD1d. In addition to other markers that may be utilized with an element that binds to CD19, CD20, CD79a, and / or CD79b, elements that bind to CD1c or the transmembrane receptor Notch2 may be incorporated into hemibodies and trispecific antibodies described herein for the depletion of one or more types of B cells including follicular B cells. Troen et al. J. Mol. Diag., 6(4):297-307 (2004).

[0106] Bispecific hemibodies or trispecific antibodies for the depletion of one or more types of B cells, including memory B cells, may comprise an element that binds a marker selected from the group consisting of CD 19, CD20, CD79a, and CD79b, and an element that binds to a marker selected from the group consisting of CD25, C27, CD40, CD69, CD80, CD86, CD95, and CD289 (TLR9). Bispecific hemibodies or trispecific antibodies for the depletion of one or more types of B cells, including memory B cells, may comprise an element that binds CD19, CD20, CD79a, or CD79b, and an element that binds to markers present on activated B cells (e.g., either CD69 and / or CD25). Bispecific hemibodies or trispecific antibodies for the depletion of one or more types of B cells, including memory B cells, may comprise an element that binds CD19 and / or CD20, and an aa sequence that binds to a marker selected from the group consisting of CD25 and / or CD69. Bispecific hemibodies or trispecific antibodies for the depletion of one or more types of B cells, including memory B cells, may comprise one or more elements that bind to CD25 and / or CD69. See, e.g., Ziegler et al. Stem Cells 12(5):456-65 (1994).

[0107] Bispecific hemibodies or trispecific antibodies for the depletion of one or more types of B cells, including regulatory B cells, may comprise an element that binds a marker selected from the group consisting of CD19, CD20, CD79a, and CD79b, and an element that binds to a marker selected from the group consisting of CD1d, CD5, CD21, CD24, CD44, CD38, CD71, and CD365. Bispecific hemibodies or trispecific antibodies for the depletion of one or more types of B cells, including regulatory B cells, may comprise an element that binds to either CD19 or CD20, and an element that binds to a marker selected from the group consisting of CD1 d, CD5, CD21, and CD365. Bispecific hemibodies or trispecific antibodies for the depletion of one or more types of B cells, including regulatory B cells, may comprise elements that bind to one or more (e.g., two or more) markers selected from:CD1d, CD5, CD19, CD24, CD38, CD365 (Tim-1, HAVCR1), and PD-1.

[0108] Bispecific hemibodies or trispecific antibodies for the depletion of one or more types of B cells, including B cells in the germinal center compartment of the spleen, may comprise an element that binds a marker selected from the group consisting of CD 19, CD20, CD79a, and CD79b, and an element that binds to a marker selected from the group consisting of CD95 and CD268.

[0109] Bispecific hemibodies or trispecific antibodies for the depletion of one or more cells, including one or more cell types in the plasma cell sublineage, may comprise markers selective for the targeted plasma cells. A bispecific hemibody or trispecific antibody construct for depleting one or more cells, including one or more cell types in the plasma cell sublineage, may comprise an element that binds to a marker selected from the group consisting of CD269 (BCMA), CD138 (SDC1), CD267 (TACI), CD78, CD98, CD268 (BAFFR), CD319 (SLAMF7), and SCA-1 (Ly6A / E), and an element that binds to a marker selected from the group consisting of CD 1 d, CD5, CD 10, CD11c,CD19, CD21, CD22, CD23, CD24, CD27, CD34, CD38, CD40, CD43, CD44, CD45, CD53, CD69, CD71, CD72, CD73, CD80, CD85j, CD86, CD95 (FAS), CD126 (IL6R), CD127 (IL7R), CD138, CD184 (CXCR4), CD185 (CXCR5), CD267 (TACI), CD268 (BAFFR), CD273 (PDL2), CD274 (PDL1), CD289 (TLR9), CD307d, CD365 (TIM1), and HLA- DR. A bispecific hemibody or trispecific antibody construct for depleting one or more cells, including one or more cell types in the plasma cell sublineage, may comprise an element that binds to either CD269 (BCMA) or CD138 (SDC1) and an element that binds to a marker selected from CD 19, CD 27, and CD38. A bispecific hemibody or trispecific antibody construct for depleting one or more cells, including one or more cell types in the plasma cell sublineage, may comprise an element that binds to either BCMA or CD 138 and an element that binds to a marker selected from CD19 and CD38. CXCR4 has been shown to be constitutively up regulated during T-dependent and T-independent plasma-cell differentiation (see, e.g., Muehlinghaus et al, Blood, 105(10): 3965-3971. (2004)). Accordingly, a bispecific hemibody or trispecific antibody construct for depleting one or more cells, including one or more cell types in the plasma cell sublineage, may comprise an element that binds to either BCMA or CD138 and an element that binds to CXCR4.

[0110] Exemplary combinations of markers on cells of the B cell lineage that may be employed to selectively target at least one type of cell (one subset of cells) in that lineage are listed in Table 1 (provided as Parts A-F below). Combinations of elements binding to markers including a third marker, or third and fourth markers, are specific to trispecific and tetraspecific versions of the multispecific hemibodies described herein. In Table 1 " — " indicates that no element(s) binding to third and fourth markers are present in the hemibody or trispecific antibody. The double arrow, indicates that in trispecific hemibodies the markers recited as third markers may alternatively be in positions recited as being occupied by fourth markers, but only three types of elements binding to markers are present in those hemibodies. In hemibodies having three or four elements binding to cell surface markers, the markers designated as first and second markers may be exchanged. Similarly, in hemibodies having three or four elements binding to cell surface markers, the markers designated as third and fourth markers may be exchanged.Table 1 (Part A) Markers of pre-B cells and immature B cellsTable 1 (Part B) Markers of Follicular B cellsTable 1 (Part C) Markers of Marginal Zone B cellsTable 1 (Part D) Markers of Germinal Center B cellsTable 1 (Part E) Markers of Memory B cellsTable 1 (Part F) Markers of Plasma Blasts, Plasma cells, and Long-lived plasma cells

[0111] Table 2 provides a listing of certain cell surface markers found on human cells within the B cell lineage and its plasma cell sublineage and exemplary antibodies that bind to those markers. The antibodies provide exemplary sources of aa sequences that bind to those markers including, for example, VH, VL, scFv, and CDR sequences, and also sources of Fab or Fv polypeptide aa sequences. In Table 2 antibodies are identified by their specific designationor the designation of the clone producing the antibody. The listing is not comprehensive and the absence of any annotation for a specific marker under an indicated cell type does not indicate its absence or low expression levels. The annotations indicate: + the marker is generally present, - the marker is generally absent, + / - the marker may be present or absent. The annotations + / low, + / mid, and + / high indicate markers are generally present on the indicated cell type at the relative level indicated, and - / low indicates a marker is generally absent (undetectable) or present in relatively low amounts. Those annotations are based on information provided by suppliers of the indicated antibodies and / or associated literature. The antibodies recited in Table 2 bind to the human marker, but may also bind the same protein in other species.Table 2. Markers of Cells in the B Cell Lineageslumbers in brackets “{}” indicate the source of the antibody: 1 - Cell Signaling Technology®; 2 - Santa CruzBiotechnology, Inc.; 3 - ABCAM; 4 - R&D Systems; 5 - Becton Dickinson (BD) Biosciences; and 6 - Bio X Cell.

[0112] In addition to the use of elements that bind to markers expressed on the surface of cells of the B cell lineage to target cells, elements (e.g., aa sequences) that bind to surface complexes present on those cells may be employed to target hemibodies and trispecific antibodies. For example, antibodies that specifically recognize membrane bound forms of immunoglobulins or BCR proteins (CD79a and / or CD79b) when in a complex with an immunoglobulin molecule may also be used to target cells in the B cell lineage. See, e.g., Welt et al., Oncotarget, 7(46)74701-74723 (2016) and U.S. Pat. No. 9,926,381.

[0113] The bispecific hemibodies and trispecific antibodies of the present disclosure may, if desired, comprise two or more elements that bind to the same marker on the surface of a cell in the B cell lineage. Using two or more elements (e.g., 1’ and 1”) directed to a single marker would increase the avidity of the constructs for cells displaying that marker, permitting depletion of those cells. For example, bispecific hemibodies and trispecific antibodies having two elements (1’ and 1”) could be used to accomplish pan B cell depletion. Similar results could be obtained using two elements directed against CD20 or the BCR. Bispecific hemibodies and trispecific antibodies having two elements directed against BCMA could be used to deplete plasma blasts and cells that develop therefrom while leaving B cells.(1) Amino acid sequences with affinity for B cell lineage surface markers

[0114] The sequences of elements binding to markers present on cells in the B cell lineage include, but are not limited to: VH, VL, SCFV, Fab, Fv, or nanobody aa sequences of antibodies binding to markers recited in Table 1, or the CDRs of any thereof, such as the antibodies set forth in Table 2.(2) Amino acid sequences with affinity for CD19

[0115] Single polypeptide chain elements with affinity for the extracellular domain of CD19 that may be incorporated into a bispecific hemibody or trispecific antibody construct include, but are not limited to, anti-CD 19 scFv or nanobody (VHH) sequences, such as those that follow.

[0116] An example of an anti-CD 19 VHH sequence includes, but is not limited to, the anti-CD 19 VHH aa sequence QVQLQESGGGSVQAGGSLRLSCAVRRPTDSRNCMAWFRQAPGEQREAVAGIDIYKTTGYAESVKGRFTISQDNAK NTLFLQMNSLKPEDSGTYYCAAARPCKYGSEWRRSASDFLYWGQGTQVIVSS (SEQ ID NOH) or a sequence with greater than 90% aa sequence identity to SEQ ID NON . Anti-Fc VHH aa sequences also include aa sequences with greater than 95% or greater than 98% sequence identity to SEQ ID NON .

[0117] Another example of an anti-CD 19 VHH sequence is the anti-CD 19 VHH aa sequence QVQLQESGGGSVQT GGSLRLSCAASGFSYTGHHMGWFRQVAAQEREWVGSIYYDPGTTFYADSVKGRFTISQDVAKKMVYLQMNNLKPT DTGIYFCAAEPVADGPPLMSRDRCFGSWGQGTQVIVS (SEQ ID NO:2), or a sequence with greater than 90% aa sequence identity to SEQ ID NO:2. Anti-Fc VHH aa sequences also include aa sequences with greater than 95% or greater than 98% sequence identity to SEQ ID NO:2.

[0118] Single polypeptide chain elements with affinity for the extracellular domain of CD19 may comprise the scFv aa sequence DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGS GSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSV KISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAV YFCARRETTTVGRYYYAMDYWGQGTTVTVSS (SEQ ID NO:3), or a sequence having at least 90% or at least 95% aa sequence identity to that sequence. Alternatively, the sequence having affinity for CD19 may have at least 97% or 98% sequence identity to that sequence.

[0119] A sequence having affinity for CD19 may comprise the VHand V aa sequences of SEQ ID NO:3, the V aa sequence is: DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGS GSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK (SEQ ID NO:4); and the VHaa sequence is: QVQLQ QSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAY MQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS (SEQ ID NO:5). Alternatively, the sequence with affinity for CD 19 may comprise an aa sequence with at least 90% or at least 95% aa sequence identity to either or both of those VH and VL sequences. Sequences with affinity for CD 19 may comprise an aa sequence with one or two aa sequence substitutions in either or both of those VH and VL sequences.

[0120] Amino acid sequences having affinity for the extracellular domain of CD19 also include, but are not limited to, aa sequences comprising the CDRs of the scFv of SEQ ID NO:3 or aa sequences having greater than 98% sequence identity to those CDRs. For example, the CDRs may have one or two aa insertions, deletions or substitutions, such as an insertion, deletion or substitution in any one or any two of the CDRs.(3) Amino acid sequences with affinity for CD20

[0121] Elements that have affinity for the extracellular domain of CD20 that may be incorporated into a bispecific hemibody or trispecific antibody construct include, but are not limited to, antibody VH and VL sequences of, for example, Rituximab, Obinutuzumab, Ofatumumab, Tositumomab, or any of the other anti-CD20 antibodies set forthin Table 2. Elements comprising aa sequence pairs that have affinity for the extracellular domain of CD20 include, but are not limited to, aa sequences having greater than 95% or greater than 98% sequence identity to the VH and / or VL sequences of any of those antibodies. Sequences with affinity for CD20 may comprise one or two aa substitutions, deletions, or insertions into, for example, the VHand / or V aa sequences of those antibodies, or in one or two of their CDRs.

[0122] Single chain aa sequences with affinity for the extracellular domain of CD20 that may be incorporated into a bispecific hemibody or trispecific antibody construct include, but are not limited to, anti-CD20 VHH aa sequences described by Liu et al, Vaccines, 10, 1335 (2022) as part of a biTE molecule. Sequences with affinity for CD20 may comprise one or two aa substitutions, deletions, or insertions into, for example, the VHH and / or scFv sequences.(4) Amino acid sequences with affinity for CD69

[0123] Elements that have affinity for the extracellular domain of CD69 that may be incorporated into a bispecific hemibody or trispecific antibody construct may comprise, for example, the VH and VL sequences of SEQ ID NOs:6 and 7. The VHchain: QVQPGGSLRLSCAASGFTFSNFVMHWVRNAPGKGLEWVSSISGSSSSTYYADSVKGRFTIS RDNSKNTLYLQMNSLRAEDTAVYYCARYYYASFDYWGQGTLVTVSS (SEQ ID NO:6); and the VLchain: DIELTQPP SVSVAPGQTARISCSGDSLGSKYVYWYQQKPGQAPWVIYGDSKRPSGIPERFSGSNSGNTATLTISGTQAEDEADY YCQSYDSNIMVFGGGTKLTV (SEQ ID NOV). Elements comprising aa sequence pairs that have affinity for the extracellular domain of CD69 may comprise aa sequences having greater than 95% or greater than 98% sequence identity to the VH and / or VL sequences of SEQ ID NO:6 or SEQ ID NOV.(5) Amino acid sequences with affinity for CD27

[0124] Elements that have affinity for the extracellular domain of CD27 that may be incorporated into a bispecific hemibody or trispecific antibody construct include, but are not limited to, antibody VH and VL sequences of, for example, Varlilumab or any of the other anti-CD27 antibodies set forth in Table 2. Elements with affinity for the extracellular domain of CD27 also include, but are not limited to, anti-CD27 Fab, scFv, and VHH sequences. Varlilumab's heavy and light chain sequences are: QVQLVESGGGWQPGRSLRLSCAASGFTFSSYDMHWVRQAP GKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSGNWGFFDYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGKGSS (SEQ ID NO:8) and DIQMTQSPSSLSASVGDRVTITCRASQGISRWLAW YQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTYPRTFGQGTKVEIKRTVAAP SVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:9). See, e.g., the National Center for Advanced Translational Services record for Varlilumab Accession No. 0125DUV5XC available on the world wide web at: drugs, neats. io / substance / 0125D U V5XC .

[0125] VH and VL aa sequences that have affinity for the extracellular domain of CD27 include, but are not limited to, aa sequences having greater than 95% or greater than 98% sequence identity to the VH and / or VL sequences of Varlilumab (or any of the other anti-CD27 antibodies in Table 2). Sequences with affinity for CD27 may comprise one or two aa substitutions, deletions, or insertions into the VHand / or V sequences of Varlilumab.(6) Amino acid sequences with affinity for CD38

[0126] Elements that have affinity for the extracellular domain of CD38 that may be incorporated into a bispecific hemibody or trispecific antibody construct include, but are not limited to, antibody VHand V sequences of, for example, Isatuximab or any of the other anti-CD38 antibodies set forth in Table 2. Elements with affinity for the extracellular domain of CD38 include, but are not limited to, anti-CD38 Fab, scFv, and VHH sequences. Isatuximab's heavy and light chain sequences are QVQLVQSGAE VAKPGTSVKL SCKASGYTFT DYWMQWVKQR PGQGLEWIGT IYPGDGDTGY AQKFQGKATL TADKSSKTVY MHLSSLASED SAVYYCARGD YYGSNSLDYW GQGTSVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK (SEQ ID NO: 10) and DIVMTQSHLS MSTSLGDPVS ITCKASQDVS TWAWYQQKP GQSPRRLIYS ASYRYIGVPD RFTGSGAGTD FTFTISSVQA EDLAVYYCQQ HYSPPYTFGG GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SWCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC (SEQ ID NON 1). See, e.g., KEGG Entry No. D11050 available on the world wide web at: / www.genome.jp / entry / D11050.

[0127] Elements that have affinity for the extracellular domain of CD38 include, but are not limited to, aa sequences having greater than 95% or greater than 98% sequence identity to the VH and / or VL sequences or the CDRs of elotuzumab (EMPLICITI®). Elements that have affinity for the extracellular domain of CD38 include, but are not limited to, aa sequences having greater than 95% or greater than 98% sequence identity to the VHand / or VLsequences or the CDRs of isatuximab (SARCLISA®). Elements that have affinity for the extracellular domain of CD38 include, but are not limited to, aa sequences having greater than 95% or greater than 98% sequence identity to the VH and / or VL sequences or the CDRs of daratumumab (DARZALEX®). Sequences with affinity for CD38 may comprise one or two aa substitutions, deletions, or insertions into the VH and / or VL sequences of elotuzumab, isatuximab, or daratumumab, or at least one of the CDRs of elotuzumab, isatuximab, or daratumumab.(7) Amino acid sequences with affinity for BCMA

[0128] Elements with affinity for the extracellular domain of BCMA expressed on cells of the plasma cell sublineage include, but are not limited to, anti-BCMA Fab, scFv, and VHH sequences. An example of an anti-BCMA VHH sequence includes, but is not limited to, the anti-BCMA VHH aa sequence QVKLEESGGGLVQAGRSLRLSCAASEH TFSSHVMGWFRQAPGKERESVAVIGWRDISTSYADSVKGRFTISRDNAKKTLYLQMNSLKPEDTAVYYCAARRIDAA DFDSWGQGTQVTVSS (SEQ ID NO:12) or a sequence with greater than 90% aa sequence identity to SEQ IDNO: 12. Anti-Fc VHH aa sequences also include aa sequences with greater than 95% or greater than 98% sequence identity to SEQ ID NO:12.

[0129] Another example of an anti-BCMA VHH sequence is the anti-BCMA VHH aa sequence QVQLQESGGGLV QAGGSLRLSCAASGRAVGNYIIGWFRQAPGKEREFVATTTRDGGSTFYADSVKGRFTISRDNAKNTVNLQMNSLEPE DTAVYYCAAKSWSVPLRPTSADFDSWGQGTQVTVSS (SEQ ID NO: 13), or a sequence with greater than 90% aa sequence identity to SEQ ID NO:13. Anti-Fc VHH aa sequences also include aa sequences with greater than 95% or greater than 98% sequence identity to SEQ ID NO: 13.

[0130] Single chain aa sequences with affinity for the extracellular domain of BCMA may comprise the scFV BCMA targeting portion of idecabtagene vicleucel (ABECMA™) or the CDRs of that BCMA targeting sequence. For example, an element with affinity for the extracellular domain of BCMA may comprise an aa sequence with greater than 95% or greater than 98% sequence identity to the scFV aa sequence of idecabtagene vicleucel or its CDRs. Such a sequence may comprise one or two aa substitutions, deletions, or insertions into the scFv aa sequence or at least one of the CDRs of idecabtagene vicleucel's BCMA targeting sequence.(8) Amino acid sequences with affinity for CD138

[0131] Elements with affinity for the extracellular domain of CD138 include, but are not limited to, anti-CD 138 Fab, scFv, and VHH sequences. Examples of anti-CD 138 scFv and VHH sequences include VHH sequences. Other single chain aa sequences include scFv aa sequences incorporating the VHand V aa sequences of the monoclonal anti-CD 138 antibody Indatuximab or the monoclonal antibody B-B4 (see, e.g., Anttonen et al, (2001) Lung Cancer, 32(3):297-230 or Wijdenes et al. (1996) Br J Haematol. 94:318-323. b) Sequences with Affinity for Cytotoxic Effector Cells and their Masking Sequences(1) Sequences with Affinity for Cytotoxic Effector Cells

[0132] Surface antigens selected as targets of the aa sequences that bind to cytotoxic effector cells in the hemibodies and / or trispecific antibodies of the present disclosure may be preferentially expressed on cytotoxic effector cells. The surface antigens may be expressed substantially on one or more types of cytotoxic effector cell. The aa sequences binding to the cell surface antigens of cytotoxic effector cells may cause stimulation of a cytotoxic response by their binding, particularly when the cell surface antigens are clustered on the effector cell surface by the binding event.

[0133] The sequences of aas with affinity for cytotoxic effector cells take different forms in trispecific antibodies and bispecific hemibodies. In bispecific hemibodies, the aa sequences with affinity for CEC-SAs are divided into two portions, one of which is located on the first hemibody construct and the other of which is located on the second hemibody construct. See, e.g., FIG. 1 A, structures A and B. When the first and second hemibody constructs are both bound to a cell, in particular a B cell lineage target cell, a complete paratope for binding to a cytotoxic effector cell is formed. The portions of the aa sequences located on each of the first and second hemibody constructs (elements 4 and 4’) may comprise the CDRs of an antibody that binds to the desired CEC-SA. The individual CDRs may bearranged as in the VH and VL aa sequences from which they were derived. Alternatively, they may be separated by linker aa sequences that permit their formation of a complete paratope and their binding to the desired CEC-SA.

[0134] In trispecific antibodies, the aa sequences with affinity for a CEC-SA may be divided into two portions, one of which is located on the first polypeptide and the other of which is located on the second polypeptide of the trispecific antibody construct. See, e.g., FIG. 1 A, structures C to E (elements 4 and 41). When divided into two portions located on the first and second polypeptides, the individual portions may comprise the CDRs of an antibody that binds to the desired CEC-SA. The individual CDRs may be arranged as in the VH and VL aa sequences from which they were derived. Alternatively, the CDRs may be separated by linker aa sequences that permit their formation of a complete paratope and binding to the desired CEC-SA.

[0135] In contrast to bispecific hemibodies, the trispecific antibody constructs described herein may also comprise aa sequences with affinity for a CEC-SA as part of a single polypeptide fusion protein. See, e.g., FIG. 1 A, structures F to M (elements 4 and 4’). The sequences may be divided into two portions that may comprise the CDRs of an antibody that binds to the desired CEC-SA. The individual CDRs may be arranged as in the VH and VL aa sequences from which they were derived optionally separated by a linker sequence. Alternatively, the CDRs may be separated by linker aa sequences that permit their formation of a complete paratope and binding to the desired CEC-SA. When located immediately adjacent to each other as in structures F to M of FIG. 1 A, and separated by at most an optional linker sequence, elements 4 and 4’ may take the form of, for example, an scFv, a nanobody (VHH), the heavy chain of a heavy chain only antibody, or an aa sequence comprising the CDRs of an antibody that binds the desired CEC- SA. In a trispecific antibody, it is also possible for the element with affinity for a CEC-SA to be in the form of an Fab, however, in such a case the elements with affinity for B cell lineage markers will generally not be Fabs.

[0136] The aa sequences (e.g., VH or VL (4 or 4’)) with affinity for the surface antigens of cytotoxic effector cells present in the bispecific hemibodies and trispecific antibody constructs described herein may be derived from antibodies that bind to epitopes expressed on the extracellular face of the cell markers (surface antigens) of the cytotoxic effector cells. Examples of cytotoxic effector cells surface antigens that aa sequences may bind to include, but are not limited to: CD3 (CD3y (gamma chain), CD35 (delta chain), CD3E (epsilon chain), or CD3 3 (zeta chain)), TCR (TCRo (alpha chain) or TCRp (beta chain)), CD28, CD137 (41 BB), CD134 (0X40), CD27, and CD278 (IGOS). Antibodies to each of those are known in the art. See Table 2 for examples.

[0137] The aa sequences with affinity for the surface antigens of cytotoxic effector cells may bind to a protein or protein subunit selected from the group consisting of: CD3y (gamma chain), CD35 (delta chain), CD3E (epsilon chain), CD3 (zeta chain), TCRo (alpha chain) and TCR|3 (beta chain). The TCR polypeptide chains are expressed substantially on T cells (e.g., effector T cells). The aa sequences with affinity for the surface antigens of cytotoxic effector cells may bind to a protein or protein subunit selected from the group consisting of: CD3y (gamma chain), CD35 (delta chain), CD3E (epsilon chain), and CD3 (zeta chain). The aa sequences with affinity for the surface antigens of cytotoxic effector cells may bind to a protein or protein subunit selected from the group consisting of TCRo (alpha chain) and TCR|3 (beta chain). The polypeptides of CD3 are expressed substantially on T cells; however, the presence of some on Purkinje cells has also been noted. See, e.g., Gerloff et al., Neuropathol Appl Neurobiol, 19(4):313-23 (1993).

[0138] The aa sequences with affinity for the surface antigens of cytotoxic effector cells may bind to a protein or protein subunit selected from the group consisting of: CD27, CD28, CD134 (0X40), CD137 (41 BB), and CD278 (IGOS), each of which is expressed on one or more type of T cells. The aa sequences with affinity for the surface antigens of cytotoxic effector cells may bind to a protein or protein subunit selected from the group consisting of: CD27 and CD28. The aa sequences with affinity for the surface antigens of cytotoxic effector cells may bind to a protein or protein subunit selected from the group consisting of: CD134 (0X40), CD137 (41 BB), and CD278 (IGOS). The aa sequences with affinity for the surface antigens of cytotoxic effector cells may bind to a protein or protein subunit selected from the group consisting of: CD 134 (0X40) and CD 137 (41 BB).

[0139] The aa sequences with affinity for the surface antigens of cytotoxic effector cells may bind to a protein or protein subunit selected from the group consisting of: CD16A, NKG2D, NKp30, NKp46, CD2, and CD56, each of which is expressed on at least one type of NK cell. The aa sequences with affinity for the surface antigens of cytotoxic effector cells may bind to a protein or protein subunit selected from the group consisting of: CD16A, NKG2D, NKp30, and NKp46. The aa sequences with affinity for the surface antigens of cytotoxic effector cells may bind to a protein or protein subunit selected from the group consisting of: CD16A and NKG2D. The aa sequences with affinity for the surface antigens of cytotoxic effector cells may bind to a protein or protein subunit selected from the group consisting of:NKp30 and NKp46. The aa sequences with affinity for the surface antigens of cytotoxic effector cells may bind to a protein or protein subunit selected from the group consisting of: CD2, and CD56.Table 3 CEC-SAst See, e.g., Hseun et al., Scientific Reports, 6:34310 (2016) DOI:10.1038 / srep34310 and Kashyap et al., Glycobiol., 30: (7) 427-432 (2020) j See e.g., Acker et al., Front. Immunol., 8: 1-9 (July 24, 2017), doi.org / 10.3389 / fimmu.2017.00892.*Vinay & Kwon, Cell Mol Immunol., 8(4): 281-284 (2011)Numbers in brackets “{}” indicates the source of the antibody: 1 - Novus Biologicals™; 2 - Abnova™; 3 - R&D Systems; 4 - ThermoFisher Scientific (including Invitrogen®, NeoBiotechnologies, and OriGene antibodies)(a) Amino acid sequences with affinity for CD3

[0140] Sequences with affinity for an extracellular domain of a CD3 subunit (e.g., a gamma, delta, epsilon and / or zeta) suitable for use in the bispecific hemibodies and trispecific antibody constructs described herein include, but are not limited to, VH and VL aa sequences, anti-CD3 scFv, and anti-CD3 VHH sequences. Examples of anti-CD3 Fab, scFv, VHH, VH and VL sequences include aa sequences based on, for example, Teplizumab, Muromonab (or OKT3), Otelixizumab (or ChAglyCD3), or the antibody L2K. Sequences with affinity for the extracellular domain of CD3 may comprise the CDRs of, for example, Teplizumab, Muromonab or Otelixizumab, or CDRs of their VH and / or VL regions having greater than 95% or greater than 98% sequence identity to the CDRs of any one of Teplizumab, Muromonab, or Otelixizumab.

[0141] Teplizumab’s heavy chain sequence is: QVQLVQSGGGWQPGRSLRLSCKASGYTFTRYTMHWVRQAPGK GLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:14) (VH(1-119), CH1 (120-217), hinge region (218-232), CH2 (233- 342), and CH3 (343-449)); and Teplizumab’s light chain sequence is: DIQMTQSPSSLSASVGDRVTITCSASSSVSY MNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:14 (VL(1-105), and CK (107-213)).

[0142] An anti-CD3 ScFv based on Teplizumab may comprise the aa sequence: QVQLVQSGGGWQPGRSLRLS CKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQ TPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITR (SEQ ID NO: 15). Alternatively, an anti-CD3 ScFv may comprise an aa sequence having greater than 95% or 98% aa sequence identity to SEQ ID NO: 15.

[0143] Amino acid sequence pairs that have affinity for the extracellular domain of CD3 suitable for use in the bispecific hemibodies and trispecific antibody constructs described herein include, but are not limited to, antibody VH and VL sequences of Teplizumab:VHaa sequence: QVQLVQSGGGWQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKV KDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSS (SEQ ID NO: 16);VLaa sequence: DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSG SGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITR (SEQ ID NO:17). Alternatively, an aa sequence pair with affinity for CD3 may comprise aa sequences wherein one or both sequences have greater than 95% or 98% aa sequence identity to SEQ ID NOs:16 and / or 17.

[0144] Amino acid sequences having affinity for the extracellular domain of CD3 also include, but are not limited to, aa sequences comprising the CDRs of Teplizumab or aa sequences having greater than 98% sequence identity to those CDRs. For example, the CDRs may have one or two aa insertions, deletions or substitutions, such as an insertion, deletion or substitution in any one or any two of the CDRs.

[0145] Muromonab’s heavy chain sequence is: QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPG QGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS AKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPS QSITCNVAHPASSTKVDKKIEPRPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:18); and Muromonab’s light chain sequence is: QIVLTQSPAIMSAS PGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQ WSSNPFTFGSGTKLEINRADTAPTVSIFPPSSEQLTSGGASWCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQD SKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:19).

[0146] An anti-CD3 ScFv based on Muromonab (OKT3) may comprise the VHand V aa sequences of Muromonab.See, e.g., Gall et al., J Immunol Methods 2004 Feb 1 ;285(1):111-27. Alternatively, an aa sequence pair with affinity for CD3 may comprise aa sequences wherein one or both sequences have a 95% or 98% aa sequence identity to the VH and / or VL of Muromonab.

[0147] Amino acid sequences having affinity for the extracellular domain of CD3 also include, but are not limited to, aa sequences comprising the CDRs of Muromonab or aa sequences having greater than 98% sequence identity tothose CDRs. For example, the CDRs may have one or two aa insertions, deletions or substitutions, such as an insertion, deletion or substitution in any one or any two of the CDRs.

[0148] Otelixizumab's heavy chain sequence is: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFPMAWVRQAPGK GLEWVSTISTSGGRTYYRDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKFRQYSGGFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYASTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK (SEQ ID NO:20) (VH(1-119), CH1 (120-217), hinge region (218-232), CH2 (233- 342), and CH3 (343-449)); and Otelixizumab’s light chain sequence is: DIQLTQPNSVSTSLGSTVKLSCTLSSGNIEN NYVHWYQLYEGRSPTTMIYDDDKRPDGVPDRFSGSIDRSSNSAFLTIHNVAIEDEAIYFCHSYVSSFNVFGGGTKLTV LRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPE QWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO:21).

[0149] An anti-CD3 ScFv based on Otelixizumab may comprise the VHand V aa sequences of Otelixizumab. Alternatively, an aa sequence pair with affinity for CD3 may comprise aa sequences wherein one or both sequences have a 95% or 98% aa sequence identity to the VH and / or VL of Otelixizumab.

[0150] Amino acid sequences having affinity for the extracellular domain of CD3 also include, but are not limited to, aa sequences comprising the CDRs of Otelixizumab or aa sequences having greater than 98% sequence identity to those CDRs. For example, the CDRs may have one or two aa insertions, deletions or substitutions, such as an insertion, deletion or substitution in any one or any two of the CDRs.

[0151] An anti-CD3 ScFv based on antibody L2K (see e.g., WO 2004 / 106380) may comprise the aa sequence: DVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWIGYINPSRGYTNYADSVKGRFTITTDKST STAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPATLSLSPGE RATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLEAEDAATYYCQQWSS NPLTFGGGTKVEIK (SEQ ID NO:22); or an aa sequence having at least 90% or 95% aa sequence identity to that sequence. Alternatively, an anti-CD3 ScFv may comprise an aa sequence having at least 97% or 98% aa sequence identity to SEQ ID NO:22.Amino acid sequence pairs that have affinity for the extracellular domain of CD3 include, but are not limited to, aa sequences comprising the VH and VL sequences of antibody L2K. The VH aa sequence comprises the aa sequence: DVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWIGYINPSRGYTNYADSVKGR FTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTVTVSS (SEQ ID NO:23) and the VLaa sequence comprises the aa sequence: DIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRWIYDT SKVASGVPARFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTFGGGTKVEIK (SEQ ID NO:24). Alternatively, an aa sequence with affinity for CD3 may comprise aa sequences having greater than 95% or 98% aa sequence identity to one or both of SEQ ID NOs:23 and / or 24. Amino acid sequences having affinity for the extracellular domain of CD3 also include, but are not limited to, aa sequences comprising the CDRs of the L2K antibody or aa sequences having greater than 98% sequence identity to those CDRs. For example, the CDRs may have one or twoaa insertions, deletions or substitutions, such as an insertion, deletion or substitution in any one or any two of the CDRs.(2) Masking sequences

[0152] The bispecific hemibody first and second hemibody constructs of the present disclosure comprise VH and VL aa sequences 4 and 4’ that form a paratope that binds to a CEC-SA when the first and second constructs are combined. Expressing constructs having either an unpaired VH or VL aa sequence tend to result in lower yields. Moreover, while not forming a complete paratope, the individual VHand V aa sequences tend to display non-specific binding, also resulting in reduced yields and stability. The lower yields and stability may be addressed by incorporation of a cognate binding partner that does not form a paratope binding to a CEC-SA, that is, a nonparatope pairing VH aa sequence for constructs expressing a VL, or a non-paratope pairing VL aa sequence for constructs expressing a VH. Constructs with such masking sequences are exemplified by, for example, the constructs shown in FIG. 2 at D-F, FIG. 3 at D, and FIG. 5 at C and D. In those figures, the non-masking sequences are denoted as elements 4° and 4’°. As VH and VL pairs are noted as 4 and 4’, the masked pairs are noted as 4 and 4’° and 4’ and 4°. Those of skill in the art will recognize that the use of non-paring antibody aa sequences does not mask in the traditional sense of blocking access; instead, the masked antibody sequence and non-paratope pairing (nonparatope forming) antibody sequences form either a heterodimer that is incapable of binding to the targeted CEC-SA or a heterodimer that binds with substantially reduced affinity. That contrasts with linker sequences comprising cleavage sites for site-specific proteases that, in some instances, may mask an element by blocking interaction with its cognate binding partner as discussed below.

[0153] The first or second hemibody constructs comprising masked VHor VLaa sequences (e.g., comprising 4’° and 4 (VH) aa sequences and / or 4° and 4’ (VL) aa sequences) may have an affinity for the target CEC-SA that is at least an order of magnitude less (weaker) than the binding affinity of the unmasked hemibody constructs binding via the paratope formed by the (VH) 4 and (VL) 4’ aa sequences. The affinity may be at least two orders of magnitude less than the fully formed paratope. Measurement of the affinities for the masked construct may be conducted by surface plasmon resonance by immobilizing the individual masked constructs on the probe and binding a portion of the cell surface antigen bearing the relevant epitope to the probe. The affinity of the unmasked constructs comprising both a VH and a VL aa sequence pair may be assessed by immobilizing a mixture of the constructs on the probe and binding the portion of the cell surface antigen bearing the relevant epitope to the probe. Measurements may be made at 37° C in phosphate buffered saline with 0.1 % Tween 20 by weight. In some instances the 4’° and (VH) 4 aa sequence pairs and / or the 4° and (VL) 4’ aa sequence pairs do not form a CEC-SA binding paratope (e.g., a T cell surface antigen binding paratope), which is to say they do not effectively bind the cytotoxic cell surface antigen.

[0154] The masking sequences may be prepared from human VH and VL aa sequences by grafting CDRs from antibodies with specificity different from those found in antibodies binding to a CEC-SA or by mutating the specificity determining residues (SDRs) present in VH or VL aa sequences. See, e.g., Kim et al. Methods Mol Biol., 907:237-45 (2012). doi: 10.1007 / 978-1 -61779-974-7_13. By way of example, CDRs from antibodies can bind to bovine or human serum albumin or nitrophenyl-haptanated albumin. Alternatively, aa sequences of low immunogenicity, comprisingglycine serine polymers (e.g., repeats of GS, GGS, G3S (SEQ ID NO:41) or G4S (SEQ ID NO:42) can be grafted in place of one or more CDRs to obtain masking VH and / or VL aa sequences.

[0155] Masking VH and VL elements (4° and 4’°) may be joined to a first or second hemibody construct by a linker that is cleavable by a site-specific protease. Exemplary linkers and site-specific proteases are described herein. The masking sequence may be cleaved by treatment with the site-specific protease prior to administration to a patient. Alternatively, where the site-specific protease is present in a fluid or tissue of the patient that will contact the hemibody construct, the construct including the masking aa sequence may be administered to the patient. The sitespecific protease may be present in the plasma, on the surface of one or more blood cells, or on the endothelial cells lining arteries, veins, and / or capillaries. The site-specific protease capable of cleaving a masking sequence from a construct may also be present on one or more cells of tissues in which the construct will contact cells of the B cell lineage (e.g., cells in the marginal zone). c) Scaffolds

[0156] Scaffold aa sequences 2 and / or 2’ may be incorporated into the constructs (e.g., bispecific hemibody first and / or second hemibody constructs or trispecific antibody constructs) described herein as a means of structural organization. Scaffolds may also act to improve stability (e.g., thermal stability) and / or to increase the half-life of the molecules in vivo. Where scaffolds comprise aa sequences capable of inducing ADCC, ADCP, and / or GDC, they may also act as a basis for initiating those effector functions. Scaffold aa sequences may be capable of forming dimers and higher order complexes (e.g., trimers). In certain instances, scaffolds may be formed from an aa sequence and its non-identical cognate aa sequence (polypeptide) binding partner that selectively interact to form a specific heterodimer. Such sequences may be referred to as interspecific sequences, and the resulting dimer an interspecific heterodimer. Scaffolds may also be non-dimerizable (non-dimerizing) in which case they do not form dimers or higher order complexes.

[0157] Scaffolds incorporated into the constructs described herein may, for example, comprise non-lg aa sequences (e.g., an XTEN polypeptide, leucine zipper, or albumin polypeptide), or Ig aa sequences that can dimerize, heterodimerize (i.e., form interspecific pairs), or remain monomeric.

[0158] Scaffolds incorporated into the constructs described herein may comprise immunoglobulin constant region aa sequences (e.g., CH2-CH3 aa sequences or a CH1-C sequence pair). Immunoglobulin sequences, such as CH2- CH3 aa sequences, may spontaneously form dimers, particularly when expressed in a cell-based or cell-free expression system. Disulfide bonds between the individual scaffold sequences may also form spontaneously when cysteine residues are located at suitable positions, as in the bridging disulfide bonds formed in human and mouse antibodies. As indicated above, scaffolds, and particularly IgFc scaffolds that comprise CH2-CH3 aa sequences, may provide a variety of functions including spatially organizing elements of the molecule, stabilizing the molecule against denaturation (thermal denaturation), extending half-life (e.g., circulating half-life in blood), and providing other immune system related functions associated with phagocytosis and cytotoxicity (e.g., ADCC, ADCP, and / or CDC). While scaffold sequences may be considered a central element in constructs, with other elements located at their N- terminus and / or C-terminus, they also may be located in an N-terminal or C-terminal position of a polypeptide of a construct.

[0159] In some instances, scaffold sequences comprise an Ig heavy chain constant region (e.g., CH2-CH3) polypeptide aa sequence (e.g., an IgFc aa sequence) wherein any one or more cysteines involved in interchain disulfide bonds that stabilize Ig heavy chain dimers may be substituted (e.g., with an alanine or serine). Such scaffold aa sequences cannot homodimerize, heterodimerize or form stabilizing interchain disulfide bonds.

[0160] A scaffold polypeptide aa sequence may comprise the wt. Homo sapiens lgG1 Fc polypeptide sequence of:1 DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVKFNWYVD61 GVEVHNAKTK PREEQYNSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PI EKTISKAK 121 GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS 181 DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK (SEQ ID NO:25) (see NCBI GenPept ACCESSION 7LBL_A), which may form homodimers stabilized by interchain disulfide bonds during expression. A scaffold may comprise an aa sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO:25. A scaffold may also comprise an aa sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO:25. A scaffold polypeptide aa sequence may comprise an aa sequence having at least 90% or at least 95% sequence identity to at least 210 or at least 220 contiguous aas of the wt. lgG1 of SEQ ID NO:25. A scaffold polypeptide aa sequence may comprise an aa sequence having at least 97% or at least 99% sequence identity to at least 210 or at least 220 contiguous aas of the wt. lgG1 of SEQ ID NO:25.

[0161] A scaffold polypeptide aa sequence may comprise a Homo sapiens lgG1 Fc polypeptide sequence of: APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPCRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:26), which comprises a "LALA” substitution and may form homodimers stabilized by interchain disulfide bonds during expression. A scaffold may comprise an aa sequence having at least 90% or at least 95% sequence identity to SEQ ID NO:26. A scaffold may also comprise an aa sequence having at least 96% or at least 98% sequence identity to SEQ ID NO:26. A scaffold polypeptide aa sequence may comprise an aa sequence having at least 90% or at least 95% sequence identity to at least 200 or at least 210 contiguous aas of the lgG1 Fc of SEQ ID NO:26. A scaffold polypeptide aa sequence may comprise an aa sequence having at least 97% or at least 99% sequence identity to at least 200 or at least 210 contiguous aas of the lgG1 Fc of SEQ ID NO:26.

[0162] Ig Fc aa sequences may comprise CH2 and / or CH3 domains modified to prevent dimerization, with the resulting sequences existing in monomeric form. One set of substitutions that may be included in lgG1 heavy chain constant region aa sequences that substantially or completely blocks dimerization with another lgG1 aa sequence includes L351S, T366R, L368H and P395K (corresponding to L131S, T146R, L148H, and P175K in SEQ ID NO:25). See Ying et al. (2014) mAbs 6(5): 1201 -1210. lgG1 Fc sequences bearing those substitutions remain in soluble monomeric form capable of high affinity binding to FcyRI (dissociation constant (Kd) on the order of 10 nM) and bind to the neonatal Fc receptor (FcRn) that prevents lysosomal degradation of antibodies thereby extending their in vivo half-life. Id. At the same time, monomeric lgG1 sequences bearing those substitutions fail to provide effector functions including Fc- mediated CDC and ADCC by NK cells consistent with their failure to bind to the FcyRI 11 areceptor. Id. An lgG1 sequence comprising those sequence substitutions suitable as a monomeric (non-dimerizing) Fc sequence (mono-Fc) scaffold in the molecules described herein may comprise the aa sequence:1 APELLGGPSV FLFPPKPKDT LMISRTPEVT CWVDVSHED PEVKFNWYVD GVEVHNAKTK61 PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PI EKTISKAK GQPREPQVYT121 SPPSRDELTK NQVSLRCHVK GFYPSDIAVE WESNGQPENN YKTTKPVLDS DGSFFLYSKL181 TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK (SEQ ID NO:27). A scaffold may comprise an aa sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO:27, while retaining the substitutions that cause it to remain monomeric. A scaffold may also comprise an aa sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO:27, while retaining the substitutions that cause it to remain monomeric.

[0163] Stabilizing disulfide bonds may be introduced into the sequence of SEQ ID NO:27 between cysteines substituted for L242 and K334 (indicated as C* in SEQ ID NO:28 and SEQ ID NO:29), and / or between cysteines substituted for P343 and A431 (indicated as C** in SEQ ID NO:29). Accordingly, a monomeric lgG1 Fc scaffold sequence may comprise the aa sequence:1 APELLGGPSV FC*FPPKPKDT LMISRTPEVT CWVDVSHED PEVKFNWYVD GVEVHNAKTK61 PREEQYNSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEC*TISKAK GQPREPQVYT121 SPPSRDELTK NQVSLRCHVK GFYPSDIAVE WESNGQPENN YKTTKPVLDS DGSFFLYSKL181 TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK (SEQ ID NO:28), or the aa sequence:1 APELLGGPSV FC*FPPKPKDT LMISRTPEVT CWVDVSHED PEVKFNWYVD GVEVHNAKTK61 PREEQYNSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEC*TISKAK GQC^REPQVYT121 SPPSRDELTK NQVSLRCHVK GFYPSDIAVE WESNGQPENN YKTTKPVLDS DGSFFLYSKL181 TVDKSRWQQG NVFSCSVMHE C^LHNHYTQKS LSLSPGK (SEQ ID NO:29). A scaffold may comprise an aa sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO:28 or SEQ ID NO:29, while retaining the substitutions that cause it to remain monomeric. A scaffold may also comprise an aa sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO:28 or SEQ ID NO:29, while retaining the substitutions that cause it to remain monomeric.

[0164] Non-dimerizing scaffolds may also be prepared as human single chain Fc (scFc) dimers. See, e.g., Zhou et al., Biomaterials 117:24-31 (2017). Such scFc dimers may be based on lgG1 and comprise the sequence:1 DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CWVDVSHED PEVKFNWYVD61 GVEVHNAKTK PREEQYNSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PI EKTISKAK121 GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS181 DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGGGGG SGGGGSGGGG241 SGGGGSGGGG SGGGGSDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVW301 DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRWS VLTVLHQDWL NGKEYKCKVS361 NKALPAPIEK TISKAKGQPR EPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN421 GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS481 PG (SEQ ID NO:30), in which a G4S (SEQ ID NO:42) linker sequence that is bolded and italicized joins the two IgFc sequences. A scaffold may, for example, comprise an aa sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO:30. The scaffold may also comprise an aa sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NQ:30. The scaffold of SEQ ID NQ:30 is competent for the induction of ADCC, ADCP, and CDC, but substitutions including "LALA” and / or other substitutions which are discussed below may be introduced to selectively alter the ability of the scFc to suppress or induce effector functions.

[0165] Other scIgFc scaffold sequences may comprise an aa sequence of SEQ ID NO:26 or a sequence having at least 90% or at least 95% sequence identity to at least 200 or at least 210 contiguous aas of that sequence. For example, a scIgFc sequence may comprise the aa sequence: APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVCTLPPCRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSAPEAAGGPSVFLFPPK PKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVCTLPPCRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:31), in which the linker represented as a G4S sequence may be replaced by another linker sequence and each of the two Fc sequences (aas 1-216 and 247-462) have at least 90% or at least 95% sequence identity to SEQ ID NO:26. Each of the two Fc sequences may also comprise an aa sequence having at least 96% or at least 98% sequence identity to SEQ ID NO:26. Each of the two Fc sequences in the scFc scaffold may also comprise an aa sequence having least 90% or at least 95% sequence identity to at least 200 or at least 210 contiguous aas of the lgG1 of SEQ ID NO:26. Each of the two Fc sequences in the scFc scaffold may also comprise an aa sequence having least 97% or at least 99% sequence identity to at least 200 or at least 210 contiguous aas of the lgG1 of SEQ ID NO:26.

[0166] A scaffold polypeptide may comprise the wt. Homo sapiens lgG2 Fc polypeptide of SEQ ID NO:32 (see GenBank AAN76044, aas 99-325):1 STKGPSVFPL APCSRSTSES TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG 61 LYSLSSWTV PSSNFGTQTY TCNVDHKPSN TKVDKTVERK CCVECPPCPA PPVAGPSVFL 121 FPPKPKDTLM ISRTPEVTCV WDVSHEDPE VQFNWYVDGV EVHNAKTKPR EEQFNSTFRV 181 VSVLTWHQD WLNGKEYKCK VSNKGLPAPI EKTISKTKGQ PREPQVYTLP PSREEMTKNQ 241 VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPMLDSDG SFFLYSKLTV DKSRWQQGNV 301 FSCSVMHEAL HNHYTQKSLS LSPGK (SEQ ID NO:32), optionally modified to prevent dimerization. Alternatively, the scaffold may, for example, comprise an aa sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO:32. The scaffold may also comprise an aa sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO:32.

[0167] A scaffold polypeptide may comprise the wt. Homo sapiens lgG3 Fc polypeptide of SEQ ID NO:33 (see, e.g., GenBank AAW65947, aas 19-246):1 HKPSNTKVDK RVELKTPLGD TTHTCPPCPA PELLGGPSVF LFPPKPKDTL MISRTPEVTC61 VWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR WSVLTVLHQ DWLNGKEYKC121 KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSRDELTKN QVSLTCLVKG FYPSDIAVEW181 ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL241 SLSPGK, OPTIONALLY MODIFIED TO PREVENT DIMERIZATION. ALTERNATIVELY, THE SCAFFOLD MAY, FOR EXAMPLE, COMPRISE AN AA SEQUENCE HAVING GREATER THAN 90% OR GREATER THAN 95% SEQUENCE IDENTITY TO SEQ ID NO:33. THE SCAFFOLD MAY ALSO COMPRISE AN AA SEQUENCE HAVING GREATER THAN 96% OR GREATER THAN 98% SEQUENCE identity to SEQ ID NO:33.

[0168] A scaffold polypeptide may comprise the wt. Homo sapiens lgG4 Fc polypeptide of SEQ ID NO:34:1 PPCPSCPAPE FLGGPSVFLF PPKPKDTLMI SRTPEVTCW VDVSQEDPEV QFNWYVDGVE61 VHNAKTKPRE EQFNSTYRW SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP121 REPQVYTLPP SQEEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS181 FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SPG, optionally modified to prevent dimerization. Alternatively, the scaffold may comprise an aa sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO:34. The scaffold may also comprise an aa sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO:34.

[0169] Other non-dimerizing (non-multimerizing) scaffold domains include, but are not limited to, peptides that have been subject to XTENylation, PEGylation, and / or lipidation (see pubs.acs.org / doi / 10.1021 / acsmedchemlett. 8b00226 on the world wide web “www”), Human Serum Albumin (HSA) fusions (see, e.g., Mandrup et al., nature.com / articles / s42003-021-01790-2. on the www) and anti HSA binding domains, including but not limited to anti-HSA peptides (see pubs. acs.org / doi / 10.1021 / acs.molpharmaceut.2c00106 on the www), antibody and antibody fragments (e.g. scFvs, FABs, etc.) and VHH domains (see semanticscholar.org / paper / Serum-albumin%E2%80%90 binding-VHHs-with- variable-pH-enable-Faassen-Ryan / d34256a0d39a0ab92db9195210fa0fc7430758b6 on the www).

[0170] Ig K or Ig A light chain constant regions or Ig heavy chain CH1 sequences may also be employed as scaffolds.

[0171] Any one or more cysteines in Ig heavy chain aa sequences used as a scaffold may be substituted (e.g., with an alanine or serine) so that the Ig heavy chain sequence cannot dimerize (e.g., homodimerize) and form interchain disulfide bonds. Alternatively, where it is desirable to form stable disulfide bonds between Ig heavy chain constant region aa sequences, cysteines may be introduced into the sequences or one or more of the hinge region disulfide sequences may be utilized.(1) Interspecific Immunoglobulin Fc Scaffold Polypeptides

[0172] Where an asymmetric pairing between two scaffold sequences is present in a bispecific hemibody or trispecific antibody construct of the present disclosure, aa sequences that selectively form heterodimers with a specific cognate counterpart aa sequence can be employed. Such cognate sequence pairs or "interspecific” sequences may be Ig Fc polypeptide sequence variants. A number of such interspecific polypeptide sequences have been described including, but not limited to, knob-in-hole without (KiH) or with (KiHs-s) a stabilizing disulfide bond, HA-TF, ZW-1, 7.8.60, DD-KK, EW-RVT, EW-RVTs-s, and A107 sequences.

[0173] One interspecific binding pair based on lgG1 Fc sequences comprises a T366Y substitution in the first sequence and a Y407T substitution in the second sequence (or the corresponding residues of other IgGs), which affect the CH3 domain interface. See Ridgway et al., Protein Engineering 9:7, 617-621 (1996).

[0174] A knob-in-hole or KiH interspecific binding pair involves the formation of a knob by a T366W substitution in a first aa sequence, and a hole in the complementary Ig Fc sequence formed by the triple substitutions T366S, L368A and Y407V. See Xu et al. mAbs 7:1, 231-242 (2015). A related KiHs-s interspecific binding pair that includes Cys residues to form a stabilizing interchain disulfide bond comprises a first Ig Fc polypeptide with Y349C, T366S, L368A, and Y407V substitutions and a second Ig Fc polypeptide with S354C and T366W substitutions. The stabilizing disulfide bond may form between the Y349C and the S354C when the sequences are co-expressed. See, e.g., Brinkmann and Konthermann, mAbs 9:2,182-212 (2015). Ig Fc polypeptide sequences with or without knob-in-hole modifications may be stabilized by disulfide bond formation between the Ig Fc polypeptides (e.g., hinge region disulfide bonds).

[0175] Knob-into-hole (KiH), KiHs-s, and other interspecific binding sequence pairs based upon Ig sequences are summarized in Table 4. The table includes cross references to the numbering of the wt. IgGl Fc sequence set forth in SEQ ID NO:25.Table 4. Interspecific immunoglobulin sequences and their cognate counterpart interspecific sequences10.3389 / fimmu.2016.00394. A "C asterisk" (i.e. "C*") indicates the cysteine may form a stabilizing disulfide bond.

[0176] Interspecific "SEED” sequences comprising IgA and lgG1 hybrids may also be used as scaffold sequences. See, e.g., Ha et al., Frontiers in Immunol. Vol. 7, Article 394, pages 1-16 (2016) and citations therein.

[0177] A scaffold polypeptide suitable for use in bispecific hemibody and trispecific antibody constructs described herein may comprise the aa sequence of an interspecific binding sequence and / or its counterpart interspecific binding sequence selected from the group consisting of: KiH; KiHs-s; HA-TF; ZW-1; 7.8.60; DD-KK; EW-RVT; EW- RVTs-s; A107; or SEED sequences. IgFc based scaffolds may comprise substitutions that suppress or enhance effector functions (e.g., ADCC, ADCP, and / or CDC) relative to the effect observed with the wt. sequence under otherwise identical conditions. The effector function affected may be ADCC or ADCP. The effector function affected may be CDC.

[0178] A scaffold polypeptide suitable for use in bispecific hemibody and trispecific antibody constructs described herein may comprise a sequence from an lgG1 having a T146W KiH sequence substitution, and its counterpart interspecific binding partner polypeptide comprises an lgG1 aa sequence having T146S, L148A, and Y187V KiH sequence substitutions, where the scaffold polypeptide comprises a sequence having at least 90% or at least 95% sequence identity to at least 210 (e.g., at least 220, or all 227) contiguous aas of the wt. lgG1 of SEQ ID NO:25. The scaffold polypeptide may comprise a KiH substitution and have at least 96% or at least 97% sequence identity to at least 210 (e.g., at least 220, or all 227) contiguous aas of the wt. lgG1 of SEQ ID NO:25. Such scaffold polypeptides optionally comprise substitutions at one or more of: L234 and L235 (e.g., L234A / L235A "LALA” or L234F / L235E); N297 (e.g., N297A); P331 (e.g. P331S); L351 (e.g., L351 K); T366 (e.g., T366S); P395 (e.g., P395V); F405 (e.g., F405R); Y407 (e.g., Y407A); and K409 (e.g., K409Y), that among other things may limit the ability to stimulate cytotoxic effector functions. Those substitutions appear at: L14 and L15 (e.g., L14A / L15A "LALA” or L14F / L15E); N77 (e.g., N77A); P111 (e.g. P111S) L131 (e.g., L131 K); T146 (e.g., T146S); P175 (e.g., P175V); F185 (e.g., F185R); Y187 (e.g., Y187A); and K189 (e.g., K189Y) in the wt. lgG1 sequence of SEQ ID NO:25. For example, scaffold polypeptide sequence(s) may comprise L14 and / or L15 substitutions (e.g., "LALA” substitutions L234A and L235A) and / or an N77 substitution (e.g., N297A or N297G). IgFc based scaffolds may also comprise substitutions that enhance effector functions.

[0179] A scaffold polypeptide suitable for use in bispecific hemibody and trispecific antibody constructs described herein may comprise an lgG1 Fc sequence with T146W and S134C KiHs-s substitutions, and its counterpart interspecific binding partner polypeptide comprises an lgG1 aa sequence having T146S, L148A, Y187V and Y129C KiHs-s substitutions, where the scaffold polypeptide comprises a sequence having at least 90% or at least 95% sequence identity to at least 210 (e.g., at least 220, or all 227) contiguous aas of the wt. lgG1 of SEQ ID NO:25. The scaffold polypeptide may comprise a KiHs-s substitution and have at least 96% or at least 97% sequence identity to at least 210 (e.g., at least 220, or all 227) contiguous aas of the wt. lgG1 of SEQ ID NO:25. Such scaffold polypeptide sequence(s) may comprise additional substitutions such as L14 and / or L15 substitutions (e.g., "LALA” substitutions L234A and L235A) and / or an N77 substitution (N297 e.g., N297A or N297G) or substitutions that enhance one or more effector functions.

[0180] A scaffold polypeptide suitable for use in bispecific hemibody and trispecific antibody constructs described herein may comprise an lgG1 Fc sequence with S144H and F185A HA-TF substitutions, and its counterpartinterspecific binding partner polypeptide comprises an lgG1 aa sequence having Y129T and T174F HA-TF substitutions, where the scaffold polypeptide comprises a sequence having at least 90% or at least 95% sequence identity to at least 210 (e.g., at least 220, or all 227) contiguous aas of the wt. lgG1 of SEQ ID NO:25. The scaffold polypeptide may comprise an HA-TF substitution and have at least 96% or at least 97% sequence identity to at least 210 (e.g., at least 220, or all 227) contiguous aas of the wt. lgG1 of SEQ ID NO:25. Such scaffold polypeptide sequence(s) may comprise additional substitutions such as L14 and / or L15 substitutions (e.g., "LALA” substitutions L234A and L235A) and / or an N77 substitution (N297 e.g., N297A or N297G) or substitutions that enhance one or more effector functions.

[0181] A scaffold polypeptide suitable for use in bispecific hemibody and trispecific antibody constructs described herein may comprise an lgG1 Fc sequence with T130V, L131Y, F185A, and Y187V ZW1 substitutions, and its counterpart interspecific binding partner polypeptide comprises an lgG1 aa sequence having T130V, T146L, K172L, and T174W ZW1 substitutions, where the scaffold polypeptide comprises a sequence having at least 90% or at least 95% sequence identity to at least 210 (e.g., at least 220, or all 227) contiguous aas of the wt. lgG1 of SEQ ID NO:25. The scaffold polypeptide may comprise a ZW1 substitution and have at least 96% or at least 97% sequence identity to at least 210 (e.g., at least 220, or all 227) contiguous aas of the wt. lgG1 of SEQ ID NO:25. Such scaffold polypeptide sequence(s) may comprise additional substitutions such as L14 and / or L15 substitutions (e.g., "LALA” substitutions L234A and L235A) and / or an N77 substitution (N297 e.g., N297A or N297G) or substitutions that enhance one or more effector functions.

[0182] A scaffold polypeptide suitable for use in bispecific hemibody and trispecific antibody constructs described herein may comprise an lgG1 Fc sequence with K140D, D179M, and Y187A 7.8.60 substitutions, and its counterpart interspecific binding partner polypeptide comprises an lgG1 aa sequence having T130V, E125R, Q127R, T146V, and K189V 7.8.60 substitutions, where the scaffold polypeptide comprises a sequence having at least 90% or at least 95% sequence identity to at least 210 (e.g., at least 220, or all 227) contiguous aas of the wt. lgG1 of SEQ ID NO:25. The scaffold polypeptide may comprise a 7.8.60 substitution and have at least 96% or at least 97% sequence identity to at least 210 (e.g., at least 220, or all 227) contiguous aas of the wt. lgG1 of SEQ ID NO:25. Such scaffold polypeptide sequence(s) may comprise additional substitutions such as L14 and / or L15 substitutions (e.g., "LALA” substitutions L234A and L235A) and / or an N77 substitution (N297 e.g., N297A or N297G) or substitutions that enhance one or more effector functions.

[0183] A scaffold polypeptide suitable for use in bispecific hemibody and trispecific antibody constructs described herein may comprise an lgG1 Fc sequence with K189D and K172D DD-KK substitutions, and its counterpart interspecific binding partner polypeptide comprises an lgG1 aa sequence having T130V, D179K and E136K DD-KK substitutions, where the scaffold polypeptide comprises a sequence having at least 90% or at least 95% sequence identity to at least 210 (e.g., at least 220, or all 227) contiguous aas of the wt. lgG1 of SEQ ID NO:25. The scaffold polypeptide may comprise a DD-KK substitution and have at least 96% or at least 97% sequence identity to at least 210 (e.g., at least 220, or all 227) contiguous aas of the wt. lgG1 of SEQ ID NO:25. Such scaffold polypeptide sequence(s) may comprise additional substitutions such as L14 and / or L15 substitutions (e.g., "LALA” substitutionsL234A and L235A) and / or an N77 substitution (N297 e.g., N297A or N297G) or substitutions that enhance one or more effector functions.

[0184] A scaffold polypeptide suitable for use in bispecific hemibody and trispecific antibody constructs described herein may comprise an lgG1 Fc sequence with K140E and K189W EW-RVT substitutions, and its counterpart interspecific binding partner polypeptide comprises an lgG1 aa sequence having T130V, Q127R, D179V, and F185T EW-RVT substitutions, where the scaffold polypeptide comprises a sequence having at least 90% or at least 95% sequence identity to at least 210 (e.g., at least 220, or all 227) contiguous aas of the wt. lgG1 of SEQ ID NO:25. The scaffold polypeptide may comprise an EW-RTV substitution and have at least 96% or at least 97% sequence identity to at least 210 (e.g., at least 220, or all 227) contiguous aas of the wt. lgG1 of SEQ ID NO:25. Such scaffold polypeptide sequence(s) may comprise additional substitutions such as L14 and / or L15 substitutions (e.g., "LALA” substitutions L234A and L235A) and / or an N77 substitution (N297 e.g., N297A or N297G) or substitutions that enhance one or more effector functions.

[0185] A scaffold polypeptide suitable for use in bispecific hemibody and trispecific antibody constructs described herein may comprise an lgG1 Fc sequence with K140E, K189W, and Y129C EW-RVTs-s substitutions, and its counterpart interspecific binding partner polypeptide comprises an lgG1 aa sequence having T130V, Q127R, D179V, F185T, and S134C EW-RVTs-s substitutions, where the scaffold polypeptide comprises a sequence having at least 90% or at least 95% sequence identity to at least 210 (e.g., at least 220, or all 227) contiguous aas of the wt. lgG1 of SEQ ID NO:25. The scaffold polypeptide may comprise an EW-RTVs-s substitution and have at least 96% or at least 97% sequence identity to at least 210 (e.g., at least 220, or all 227) contiguous aas of the wt. lgG1 of SEQ ID NO:25. Such scaffold polypeptide sequence(s) may comprise additional substitutions such as L14 and / or L15 substitutions (e.g., "LALA” substitutions L234A and L235A) and / or an N77 substitution (N297 e.g., N297A or N297G) or substitutions that enhance one or more effector functions.

[0186] A scaffold polypeptide suitable for use in bispecific hemibody and trispecific antibody constructs described herein may comprise an lgG1 Fc sequence with K150E and K189W A107 substitutions, and its counterpart interspecific binding partner polypeptide comprises an lgG1 aa sequence having T130V, E137N, D179V, and F185T A107 substitutions, where the scaffold polypeptide comprises a sequence having at least 90% or at least 95% sequence identity to at least 210 (e.g., at least 220, or all 227) contiguous aas of the wt. lgG1 of SEQ ID NO:25. The scaffold polypeptide may comprise an A107 substitution and have at least 96% or at least 97% sequence identity to at least 210 (e.g., at least 220, or all 227) contiguous aas of the wt. lgG1 of SEQ ID NO:25. Such scaffold polypeptide sequence(s) may comprise additional substitutions such as L14 and / or L15 substitutions (e.g., "LALA” substitutions L234A and L235A) and / or an N77 substitution (N297 e.g., N297A or N297G) or substitutions that enhance one or more effector functions.

[0187] As an alternative to the use of Ig CH2 and CH3 heavy chain constant regions as interspecific scaffold sequences, immunoglobulin Ig K or Ig A light chain constant regions can be utilized with Ig CH1 sequences as an interspecific sequence pair. A scaffold sequence may comprise an Ig K chain constant region of SEQ ID NO:35 or an Ig A chain constant region of SEQ ID NO:36.1 TVAAPSVFIF PPSDEQLKSG TASWCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS61 KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC, Ig K chain constant region (SEQ ID NO:35); or1 GQPKANPTVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKADGSPVK AGVETTKPSK61 QSNNKYAASS YLSLTPEQWK SHRSYSCQVT HEGSTVEKTV APTECS, Ig A chain constant region (SEQ ID NO:36).

[0188] A light chain constant region scaffold sequence may also comprise a sequence having at least 90% or at least 95% sequence identity to SEQ ID NOs:35 or 36. The CH1 sequence paired with a light chain constant region may comprise the aa sequence:1 FTVRETASTK GPSVFPLAPS SKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP61 AVLQSSGLYS LSSWTVPSS SLGTQTYICN VNHKPSNTKV DKKVEPKSCD KT (SEQ ID NO:37). A CH1 sequence used as a scaffold may also comprise a sequence having at least 90% or at least 95% sequence identity to SEQ ID NO:37.

[0189] Ig CH1 and Ig K sequences may be modified to increase their affinity for each other, and accordingly the stability of any heterodimer formed utilizing them. Substitutions that increase the affinity and stability of CH1 - Ig K heterodimers include those identified as the MD13 combination in Chen et al., MAbs, 8(4):761 -774 (2016). In MD13 variants each of the CH1 and Ig K aa sequences comprises two substitutions. The Ig CH1 sequence is modified to contain S64E and S66V substitutions (S70E and S72V of SEQ ID NO:37). The Ig K sequence is modified to contain S69L and T71S substitutions (S68L and T70S of SEQ ID NO:35).(2) Modification of effector function in Ig Fc scaffolds

[0190] Interactions of the IgG class of antibodies that lead to effector functions, including ADCC and ADCP, occur through Fc region engaging members of the Fey family of receptors (FcyRs). The human protein family is comprised of FcyRI (CD64), FcyRII (CD32, which includes isoforms FcyRlla, FcyRllb, and FcyRllc), and FcyRIII (CD 16, which includes isoforms FcyRyll la and FcyRlllb). See, e.g., Lazar et al. (2006) PNAS:103 (11), 4005-4010. Interactions that lead to CDC may arise through IgFc interactions with complement C1q protein.

[0191] Substitutions that can alter effector functions of Ig sequences may be incorporated into Ig Fc sequences used as scaffolds in the constructs described herein. Both non-interspecific homodimerizing sequences such as wt. IgGs (e.g., SEQ ID NO:25) and interspecific Ig sequences may include substitutions that alter binding to either or both of the FcyR or C1q, and alter ADCC, ADCP, and / or CDC effector function.(3) Diminishing or complete suppression of IgFc effector function

[0192] Ig heavy chain constant region aa sequences used as scaffolds may also comprise one or more substitutions that can substantially diminish the ability to stimulate one or more Ig-mediated effector functions (e.g., ADCP, CDC, and / or ADCC) relative to the wt. sequences. For example, the scaffold may comprise a sequence from an lgG1 having substitutions at one or more of L234, L235, G236, G237, P238, S239 and / or P331 (appearing as L14, L15, G16, G17, P18, S19 and / or P111 in SEQ ID NO:25 or the corresponding positions in any of SEQ ID NOs:26-31). The substitutions that diminish effector mediated functions may also be combined with substitutions giving rise to interspecific pairing between IgFc aa sequences described above.

[0193] A scaffold polypeptide may comprise a Homo sapiens lgG1 Fc aa sequence of SEQ ID NO:26 or 31, which comprises a LALA substitution (L234A, L235A substitutions). lgG1 heavy chain constant regions with LALA substitutions do not effectively bind either FcyR or C1q, and have substantially diminished or completely suppressed ADCC, ADCP, and GDC stimulus functions. The positions of substitutions in wt. lgG1 sequences may be converted to positions provided in SEQ ID NO:25 by deducting 220 aas from the indicated position. Accordingly, the L234A, L235A substitutions appear as bolded and underlined Ala residues at positions 14 and 15, respectively, of SEQ ID NO:38:1 DKTHTCPPCP APE GGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVKFNWYVD61 GVEVHNAKTK PREEQYNSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PI EKTISKAK 121 GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS 181 DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG. The sequence set forth in SEQ ID NO:38 may be optionally modified to prevent dimerization by the addition of L351S, T366R, L368H and P395K substitutions (corresponding to L131S, T146R, L148H, and P175K in SEQ ID NO:25). A scaffold may comprise an aa sequence having greater than 90% or greater than 95% sequence identity to SEQ ID NO:38. The scaffold may also comprise an aa sequence having greater than 96% or greater than 98% sequence identity to SEQ ID NO:38.The scaffold may also comprise an aa sequence having greater than 90% or greater than 95% sequence identity to at least 210 or at least 220 contiguous aas of SEQ ID NO:38. The scaffold may also comprise an aa sequence having greater than 96% or greater than 98% sequence identity to at least 210 or at least 220 contiguous aas of SEQ ID NO:38. Any one or more cysteines in SEQ ID NO:38 may be substituted (e.g., with an alanine or serine) so that the Ig heavy chain sequence cannot homodimerize and form interchain disulfide bonds.

[0194] Substitutions at positions D270, K322, P329 and / or P331 (corresponding to D50, K102, P109 and P111 in SEQ ID NO:25) lead to reduced binding to C1q relative to the wt. lgG1 protein, and hence a reduction in CDC. Any one or more of those substitutions may be included in an lgG1 sequence of SEQ ID NOs:25 to 31 at the corresponding positions. In an embodiment, an lgG1 heavy chain constant region aa sequence used as a scaffold comprises a LALA substitution (L234A, L235A) and a P331S substitution:1 DKTHTCPPCP APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVKFNWYVD61 GVEVHNAKTK PREEQYNSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA SIEKTISKAK 121 GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS 181 DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK (SEQ ID NO:39) or a sequence having at least 90% sequence identity to that sequence. Alternatively, the scaffold comprises an aa sequence having at least 95% or at least 98% sequence identity to SEQ ID NO:39.

[0195] Other substitutions that can substantially diminish one or more antibody related effector functions of lgG1 antibodies (e.g., ADCC, ADCP, and / or CDC responses) include, but are not limited to: Leu235Glu; Ser228Pro / Leu235Glu; Leu234Ala / Leu235Ala / Pro329Gly; Pro331Ser / Leu234Glu / Leu235Phe; Asp265Ala;Gly237Ala; Glu318Ala; Glu233Pro; and Gly236Arg / Leu328Arg. As indicated above, the corresponding location of those substitutions in the lgG1 Fc sequence provided as SEQ ID NO:25 can be obtained by subtracting 220 from the indicated positions. In an embodiment, at least ADCC is substantially diminished by the substitutions. In anembodiment, at least ADCP is substantially diminished by the substitutions. In an embodiment, at least CDC is substantially diminished by the substitutions. In an embodiment, at least complement fixation is substantially diminished by the substitutions. See, e.g., Saunders 2019 Front. Immunol. 10:1296.doi: 10.3389 / fimmu.2019.01296 and citations therein.

[0196] Substitutions in lgG2 and lgG4 antibody sequences that can substantially diminish one or more antibody related effector functions include, but are not limited to: H268N / V309L / A330S / P331S (lgG2m4);V234A / G237A / P238Sr / H268A / V309L / A330S / P331 S (lgG2a); L234A / L235 / G237A / P238S / H268A / A330S / P331 S (IgGIcr); and S228P / F234A / L235A (lgG4PAA). See, e.g., Saunders 2019 and citations therein.(4) Enhancement of Ig Fc effector function

[0197] Among the substitutions that can enhance one or more antibody related effector functions (e.g., ADCC, ADCP, and / or CDC responses) of scaffold sequences comprising an lgG1 aa sequence relative to the corresponding wt. sequence are the individual substitutions S239D and I332E, the double substitution S239D / I332E, and the triple substitution S239D / I332E / A330L in human lgG1. See Lazar et al. 2006. The substitutions corresponding to S239D, I332E, and A330L in the human lgG1 Fc sequence (SEQ ID NO:25) are S19D, 1112E, and A110L. In an embodiment, the substitutions are the double and triple mutants S239D / I332E or S239D / I332E / A330L. In an embodiment, the lgG1 Fc substitutions may be the triple substitution S239D / I332E / A330L, which permits enhancement of ADCC without substantial alteration of CDC function (see Lazar et al. 2006).

[0198] Other substitutions that enhance one or more antibody related effector functions in lgG1 antibodies include, but are not limited to: S298A / E333A / K334A; G236A / S239D / A330L / I332E; G236A; S239D / I332E / G236A;L234Y / G236W / S298A; F243L / R292P / Y300L / V305I / P396L; K326W / E333S; K326A / E333S; K326M / E333S;C221D / D222C; S267E / H268F / S324T; H268F / S324T; and E345R. See Saunders (2019). The corresponding location in the lgG1 Fc sequence of SEQ ID NO:25 can be obtained by subtracting 220 from the indicated positions. In a construct, at least ADCC may be enhanced by the substitutions. In a construct, at least ADCP may be enhanced by the substitutions. In a construct, at least CDC may be enhanced by the substitutions. In a construct, at least complement fixation may be enhanced by the substitutions.

[0199] The substitutions that enhance effector mediated functions may also be combined with substitutions giving rise to interspecific pairing between IgFc aa sequences described above. d) Linkers and Affinity Tags(1) Linker Sequences

[0200] Suitable polypeptide linkers (also referred to as "spacers”) can be of any suitable length. Linkers amenable to cleavage by site-specific protease cleavage need to be of a length sufficient to be recognized and cleaved by the protease (e.g., cleaved at an appreciable rate). For example, the linker is cleaved at a rate one-hundredth or one- thousandth of the rate at which the canonical sequence recognized by the site-specific protease is cleaved under otherwise identical conditions. Those skilled in the art will recognize that a portion of the aa sequence recognized by the site-specific protease may arise from the aa sequence of an element (e.g., a scaffold sequence) immediately adjacent to the linker. Using aas of an element immediately adjacent to the sequence to be cleaved permits thepreparation of more compact constructs. In addition, provided the aas of the adjacent element do not form part of the material cleaved by the protease, a more complete cleavage of a linker sequence with few if any linker aa residues remaining in the product is achieved. Site-specific proteases that act on linker sequences do not cleave the aa sequences of other elements of a trispecific antibody construct or bispecific hemibody construct at an appreciable rate, such that cleavage occurs substantially within the linker bearing the sequence recognized by the protease.

[0201] Linkers may be, for example, from 1 aa to about 50 aa, from 1aa to 5 aa, from 1 aa to 15 aa, from 2 aa to 15 aa, from 2 aa to 25 aa, from 3 aa to 12 aa, from 4 aa to about 10 aa, from 4 aa to about 35 aa, from 5 aa to about 35 aa, from 5 aa to about 10 aa, from 5 aa to 20 aa, from 6 aa to about 25 aa, from 7 aa to about 35 aa, from 8 aa to about 40 aa, from 9 aa to about 45 aa, from about 10aa to about 15 aa, from about 10 aa to about 50 aa, from about 15aa to about 30 aa, from about 20 aa to about 40 aa, or from about 40 aa to about 50 aa. Suitable polypeptide linkers may be in the range from 1 aa to about 15 aas, or from about 15 aas to about 30 aas. Suitable polypeptide linkers may be in the range from about 30 aas to 45 aas. Suitable polypeptide linkers in the range from about 10 aa to about 50 aas in length may be from about 10 aas to about 20 aas, or from about 10 aas about to 25 aas long. Linkers may be from about 15 aas to about 25 aas, or from about 20 aas to about 30 aas long. Linkers may also be from about 25 aas to about 35 aas, or from about 25 aas to about 50 aas long. Linkers may also be from about 30 aas to about 45 aas, from 3 about 5 aas to about 45 aas, or from about 40 aas to about 50 aas. In any embodiment, suitable linkers can be independently selected to be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 aa in length.

[0202] Polypeptide linkers in a construct may have, for example, aa sequences that comprise, consist essentially of, or consist of: I) Gly and / or Ser; II) Ala and Ser; ill) Gly and Ala; iv) Gly, Ala, and Ser; v) Gly, Ser, and Cys (e.g., a single Cys residue); vi) Ala, Ser, and Cys (e.g., a single Cys residue); and vii) Gly, Ala, Ser, and Cys (e.g., a single Cys residue). Glycine and glycine-serine polymers can be used as they are relatively unstructured, and accordingly can serve as a neutral linker between any two elements in a construct. Glycine polymers access significantly more phi-psi space than even alanine polymers, and are much less restricted than residues with longer side chains (see, e.g., Scheraga, Rev. Computational Chem. 11173-142 (1992)).

[0203] Exemplary linkers may be comprised of GSGGS (SEQ ID NQ:40), GGGS (SEQ ID NO:41), GGGGS (also referred to as "G4S”, SEQ ID NO:42), AAAGG (SEQ ID NO:43) and / or other flexible linkers known in the art. Any of those aa sequences may appear from 2-10 times in repetition (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 times).

[0204] In some cases, a linker polypeptide, present in a bispecific hemibody construct or trispecific antibody construct, comprises one or more cysteine residues that may be used, for example, as a site for coupling to the construct a payload such as a label (e.g., a fluorescent dye), or a payload such as a drug (e.g., a drug releasable by ester hydrolysis). The payload may be attached to the cysteine of the linker via, for example, a maleimide containing bifunctional reagent (crosslinker).

[0205] Linkers comprising cysteine residues may be employed in bispecific hemibody and trispecific antibody constructs at locations where, for example, stabilizing disulfide bonds may be formed or where a payload is to be added using, for example, a maleimide functionalized linker. Payloads include, but are not limited to, radiolabeledtags, colorimetric labels, fluorescent labels, radio-opaque labels (e.g., iodinated labels for X ray), lanthanide labels, and conjugated drugs. Exemplary cysteine-containing linkers, prior to conjugation (e.g., with maleimide containing molecules) may comprise an aa sequence comprising Gly and / or Ser with a single Cys, such as in the aa sequence GCGGS (SEQ ID NO:44). Examples of such linkers that also comprise G4S units repeated from 2-10 times (e.g., repeated 2, 3, 4, 5, 6, 7, 8, 9, or 10 times) include GCGGSGGGGSGGGGSGGGGS (SEQ ID NO:45) and GCGGSGGGGSGGGGS (SEQ ID NO:46).

[0206] While flexible linkers may be utilized in the constructs described herein, "rigid linkers” that access less phi- psi space than flexible linkers such as a GGGGS linker sequence may be used in circumstances where it is desirable to maintain a substantially fixed distance / spatial separation between elements joined by the linker. Separation between elements (e.g., aa sequences) by rigid linkers can be advantageous where it is desirable to separate aa sequences binding to, for example, markers on the surface of a cell in a B cell lineage and aa sequences binding to CEC-SAs, for example, to separate any of elements 1', 1", 1'” and / or 1"" from any of elements 4 and / or 4’. See, e.g., FIG. 1 A at A-M and FIG.5 at A and B. Similarly, rigid linkers may be useful to maintain separation between aa sequences having affinity for markers (any two of 1', 1", 1"' and / or 1"") on a cell within the B cell lineage.

[0207] Examples of rigid peptide linkers include, but are not limited to, aa sequences comprising EAAAK (SEQ ID NO:47), Lys-Pro repeats, Glu-Pro repeats, Thr-Pro-Arg repeats, and Ala-Pro repeats, any of which may be repeated from 2-15 times (i.e. repeated 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 times). Non-limiting examples of suitable rigid linkers comprising the aa sequence EAAAK (SEQ ID NO:47) include, but are not limited to, (EAAAK)2 (SEQ ID NO:48), and (EAAAK)3 (SEQ ID NO:49. Examples of suitable rigid linkers also include, but are not limited to, sequences comprising AP repeats including, but not limited to: APAP (SEQ ID NQ:50); APAPAPAP (SEQ ID NO:51); APAPAPAPAPAP (SEQ ID NO:52); APAPAPAPAPAPAPAP (SEQ ID NO:53); and APAPAPAPAPAPAPAPAPAP (SEQ ID NO:54). Examples of suitable rigid linkers that comprise the aa sequence KP include, but are not limited to: KPKP (SEQ ID NO:55); KPKPKPKP (SEQ ID NO:56); KPKPKPKPKPKP (SEQ ID NO:57); KPKPKPKPKPKPKPKP (SEQ ID NO:58); and KPKPKPKPKPKPKPKPKPKP (SEQ ID NO:59). Examples of suitable rigid linkers comprising the aa sequence EP include, but are not limited to, EPEP (SEQ ID NQ:60); EPEPEPEP (SEQ ID NO:61);EPEPEPEPEPEP (SEQ ID NO:62); EPEPEPEPEPEPEPEP (SEQ ID NO:63); and EPEPEPEPEPEPEPEPEPEP (SEQ ID NO:64).(2) Linkers with Site Specific Protease Cleavable Sequences

[0208] Linker aa sequences comprising sites subject to cleavage by one or more site-specific proteases may be utilized in more than one role in the bispecific hemibodies and trispecific antibody constructs described herein. In the simplest case, linkers subject to site-specific protease cleavage may be utilized to join an affinity tag to a construct. Following purification from a cell or cell free expression system utilizing the affinity tag, the affinity tag 5 can be removed by contacting the construct with the site-specific protease to cleave the linker 22.

[0209] Linker aa sequences comprising sites subject to cleavage by one or more site-specific proteases also may be used to join the constructs to elements 4° and / or 4’° that mask VHand / or V aa sequences (e.g., elements 4 and 4’) of the hemibody constructs. See, e.g., element 6 in FIG. 2 at D to F, FIG. 3 at C or D, and FIG. 5 at C and D).Such constructs may be expressed in cells that do not provide a site-specific protease capable of cleaving the linkage such that the constructs are expressed in a masked format. The linker sequence joining the masking element to the construct may be cleaved following expression and prior to administration to a patient, or formulation or use in preparing a medicament. Where the linker may be cleaved by a site-specific protease in vivo, the construct including its masking sequence may be administered to a patient. Action by the site-specific protease in vivo will cleave the masking sequence permitting the VH and / or VL aa sequences to engage CEC-SAs.

[0210] Linker aa sequences comprising sites subject to cleavage by one or more site-specific proteases also may be used to facilitate expression of constructs as fusion proteins in a single cell or cell free expression system, thereby controlling the stoichiometry of expression. Site-specific protease cleavable linkers 20 may be employed to form a fusion protein comprising two peptides of a hemibody construct (see, e.g., FIG.2 at B), or to join two hemibody constructs 27 (see, e.g., FIG. 3 at B and D), or to join to individual peptides and constructs of a hemibody (see, e.g., FIG. 2 at C and F). Similarly, site-specific protease cleavable linkers 20 may be employed to form a fusion protein comprising two polypeptides of trispecific antibody constructs (see, e.g., Fig 4 at B and D). Such constructs may be cleaved by contact with a protease capable of cleaving one or more of the sites in the cleavable linkers prior to administration to a patient or formulation as a medicament. Alternatively, where the linkers comprise sites that may be cleaved by a site-specific protease in vivo, they may be formulated or administered as the fusion protein, relying on proteases present in the patient to cleave the linkers.

[0211] Linkers may comprise more than one site susceptible to site specific proteases. The use of more than one site can ensure the linker is cleaved by at least one protease acting either in vitro or in vivo. Where it is desirable to remove substantially all of a linker, such as to be certain the linker does not block a construct from binding to a cell surface marker or surface antigen, two or more sites (aa sequences) sensitive to site-specific protease cleavage may be placed in the linker. For substantially complete removal of a linker at least one site may be located at or proximal to the N-terminal end of the linker and at least one site may be located at or proximal to the C-terminal end of the linker.

[0212] The site-specific protease sites present in each linker may be selected independently. Each linker may have a site cleavable by proteases with different site specificity. Each site may be cleavable by the same site-specific protease so that only one protease and / or one protease treatment is required. The linker joining the affinity tag 5 to the remainder of the construct may be cleavable by a first site-specific protease recognizing a first protease site, and all other aa sequences comprising a site-specific protease cleave site may be cleavable by a second site-specific protease that recognizes a second protease site.

[0213] Linker sequences sensitive to site-specific proteases may be selected by the types of site-specific proteases a construct may contact following administration to a patient. The protease sensitive aa sequence(s) may, for example, be sensitive to cleavage by site specific proteases ubiquitous in the circulatory system (e.g., furin and / or matripase on endothelial cells). The protease sensitive aa sequence(s) also may be sensitive to cleavage by sitespecific proteases found in a tissue (e.g., marginal zone or germinal center), which can enhance selective depletion of B cell in that tissue. Where the site-specific protease is expressed selectively, or even substantially, in such atissue, cleavage of linkers constraining the constructs, or cleavage of linkers that results in the unmasking of a masked hemibody construct, can increase the selectivity of the cell depletion.

[0214] A linker subject to legumain cleavage may comprise the sequence GGGGS(D / N)GGGGS (SEQ ID NO:65), where (D / N) is aspartic acid or asparagine.

[0215] A linker subject to matriptase-1 cleavage may comprise the sequence GGGGS(A / G)(R / K)GGGGS (SEQ ID NO:66), where (A / G) is alanine or glycine and (R / K) arginine or lysine. A linker subject to matriptase-2 cleavage may comprise the sequence GGGGSEXXDR(K / G)(AA / )XIX(L / Q / P)GGGGS (SEQ ID NO:67), where X is any amino acid, (K / G) is Lys or Gly, (AA / ) is Ala or Vai, and (L / Q / P) is Leu, Gin or Pro.

[0216] A linker subject to cathepsin S cleavage may comprise the sequence GGGGSWRGGGGS (SEQ ID NO:68).

[0217] A linker subject to PreScission® protease (a fusion protein of glutathione S-transferase (GST) and human rhinovirus (HRV) type 14 3C protease) cleavage may comprise the Leu-Glu-Val-Leu-Phe-GIn / Gly-Pro (LEVLFQ / GP) SEQ ID NO:69.

[0218] Linkers subject to MMP cleavage are related in that they often have a P or V at the P3 position, an A or L at the P2 position, a G or N at the P1 position, and an L at the PT' position. Table 5 and the accompanying text provided above lists some specific MMP sites. Linkers subject to MMP cleavage may have the general form linker sequence-(MMP site)-linker sequence where the linker sequence comprises one or more repeats of GGGGS (SEQ ID NO:42) and may incorporate any of the MMP cleavage sites provided above. Specific examples of such MMP cleavable linkers include GGGGSPAGLGGGGS (SEQ ID NQ:70), GGGGSPLGLGGGGS (SEQ ID NO:71), GGGGSVLGLGGGGS (SEQ ID NO:72), and GGGGSVANLGGGGS (SEQ ID NO:73).

[0219] A linker subject to uPA protease cleavage may comprise the sequence GGGGSGRKRKGGGGS (SEQ ID NO:74) or GGGGSS(G / S)(R / K)(L / R / V)X(N / G)GGGGS (SEQ ID NO:75), where (G / S) is Gly or Ser, (R / K) is Arg or Lys, (L / R / V) is Leu, Arg, or Vai, X is any amino acid, and (N / G) is Asn or Gly. See, e.g., www.ebi.ac.uk / merops / cgi- bin / pepsum?id=S01 .231 .

[0220] A linker subject to furin cleavage may comprise the sequence GGGGSRXKRGGGGS (SEQ ID NO:76) or GGGGSRXRRGGGGS (SEQ ID NO:77), where X is any amino acid. In some cases, X may be selected from the group consisting of D, E, F, G, H, K, L, P, Q, R, S, T, and V, or the group consisting of H, K, and R.

[0221] A linker subject to granzyme B cleavage may comprise the sequence GGGGSIEPDGGGGS (SEQ ID NO:78).

[0222] A linker subject to caspase 7 cleavage may comprise the sequence GGGGSDEVDGGGGGS (SEQ ID NO:79) or GGGGSDEVEGGGGGS (SEQ ID NQ:80).

[0223] A linker subject to TEV protease cleavage may comprise the sequence GGGGS ENLYFQS GGGGS (SEQ ID NO:81).(3) Affinity Tags

[0224] At least one polypeptide present in the constructs of the present disclosure may comprise a tag aa sequence that permits purification using affinity chromatography or related affinity techniques. Suitable affinity tag sequences include, but are not limited to, histidine tag, FLAG tag, calmodulin-binding peptide (CBP), S-tag, HA tag,and c-Myc tag. See, e.g., Zhao et al., J. of Anal. Met. in Chem. Volume 2013, Article ID 581093 (8 pages). The His tag may consist of, for example, a series of six histidine residues (HHHHHH, SEQ ID NO:82) or eight histidine residues (HHHHHHHH, SEQ ID NO:83).

[0225] Tags may be part of at least one fusion polypeptide of a hemibody or trispecific antibody construct. Provided the tag sequences are suitably small and do not interfere with the function of the constructs, their removal is not required. Where removal of tags is desired, for example to avoid potential immune responses to the tag, tag removal may be facilitated by attachment of the tag to the construct by a linker bearing an aa sequence cleavable by a sitespecific protease followed by treatment of the construct with that protease.

[0226] Among the site-specific protease cleavable linkers that may be employed are linkers comprising any of the site-specific protease aa sequences described herein. By way of example, a histidine tag or FLAG tag may be joined to a construct by a linker sequence comprising a furin, caspase, Tobacco etchvirus (TEV), ADAM (a disintegrin and metalloproteinase) or PreScission® protease cleavable aa sequence. Tag removal would be facilitated by treatment with the protease corresponding to the sequence in the linker following purification of the construct. e) Disulfide bonds

[0227] The bispecific hemibody constructs and trispecific antibody constructs of the present disclosure may include disulfide bonds in any of their elements, including but not limited to the elements (aa sequences) that bind to markers on cells in the B cell lineage or that bind to CEC-SAs. Other elements, including scaffold sequences and linkers, may comprise disulfide bonds. Disulfide bonds may play a role in folding of the molecules as they are produced, provide thermal stability (e.g., maintain functional folding), provide resistance to proteolysis, and / or function in maintaining structural integrity of trispecific antibody constructs, bispecific hemibodies, and individual hemibody constructs. As disulfide bonds stabilize the molecules, they also play an important role in expression as reflected by expression level and / or accumulation of the expressed molecules in cell and cell-free expression systems.

[0228] Elements involved in binding cell surface markers and antigens that may incorporate intrachain disulfide bonds that can contribute to and stabilize the molecule's secondary and / or tertiary structure include, but are not limited to, Fab polypeptides and scFv aa sequences. Elements, such as Fabs, that comprise more than one polypeptide may comprise interchain disulfide bonds that can join the polypeptides and stabilize the molecule's quaternary structure. Fabs employed in the constructs of the present disclosure may, for example, include an interchain disulfide bond between the CH1 and CL aa sequences found in human and mouse immunoglobulins (e.g., IgG molecules). Substitutions that enhance CH1 and light chain CK interactions, including but not limited to MD13 substitutions, may be employed along with disulfide bonds between those aa sequences, regardless of whether they are used as part of an antigen binding element or purely as a scaffold aa sequence, (see, e.g., Chen et al., MAbs., 8(4)761-774 (2016).

[0229] Scaffold sequences employed in the bispecific hemibodies and trispecific antibody constructs of the present disclosure may include one or more disulfide bonds. Individual immunoglobulin aa sequences employed as scaffold aa sequences (e.g., C , CH1, CH2, or CH3), as well as the heavy chain CH2-CH3 constant regions, may comprise intrachain disulfide bonds. In addition, scaffold structures such as scFc, which have first and second CH2-CH3 constant region sequences in a single polypeptide, may comprise intrachain disulfide bonds that result frominteractions between the cysteines in the first and second CH2-CH3 aa sequences (see, e.g., FIG. 1A at B and J to M).

[0230] Pairs of immunoglobulin sequences employed as scaffolds may also comprise interchain disulfide bonds. Examples of this include the interspecific aa sequences (elements 2 and 2’) depicted in FIG. 1 A at A and C to I, which may be KIHs-s Ig sequences. Interchain disulfide bonds may also be located between C and CH1 immunoglobulin scaffold sequences when they are used as an interspecific scaffold sequence pair.

[0231] Once colocalized on a surface, bispecific hemibody first and second constructs may form interchain disulfide bonds resulting in the formation of a trispecific antibody construct-like construct. Due to the increase in the valency of interaction with the surface, the effective binding constant of the disulfide bonded constructs would increase (i.e., the value of Kd would decrease due to an increase in the free energy of binding). Such disulfide-bridged molecules may arise, for example, on the surface of a patient's B cell following hemibody construct administration to the patient, but is not necessary for therapeutic effectiveness.

[0232] Disulfide bonds within the bispecific hemibody constructs and trispecific antibody constructs generally form spontaneously when the molecules are expressed. When it is necessary to refold constructs following expression, reducing agents such as beta-mercaptoethanol (BME), or dithiothreitol (DTT) may be employed. For reshuffling of disulfide bonds in a misfolded construct (disulfide bond interchange), typically substoichiometric amounts of those agents are employed. Other reducing agents such as tris (2-carboxyethyl) phosphine hydrochloride (TCEP) may be employed, but the lack of a corresponding thiol group on phosphine reagents may render them less effective in disulfide bond exchange / interchange.4. Construct Expression and Preparation

[0233] Bispecific hemibody constructs and trispecific antibody constructs of the present disclosure may be obtained by encoding the constructs or their component polypeptides in one or more nucleic acids and expressing the molecules in a cell or cell-free system. Unless stated otherwise, the constructs are understood to be soluble molecules and not associated with or integral to cell membranes as expressed, formulated and / or administered. The molecules may comprise elements such as affinity tags that allow their capture on, for example, affinity matrices or the surface of instrument probes (e.g., plasmon resonance probes). The constructs, while soluble, may comprise binding sites for cell surface molecules, including but not limited to Fey receptors (e.g., FcyR Illa) involved in antibody mediated ADCC, GDC, and / or ADCP.

[0234] Nucleic acids encoding bispecific hemibody constructs, trispecific antibody constructs, or their component polypeptides may take any suitable form. Any one or more nucleic acids may be in the form of linear DNA or RNA sequences or circular forms thereof (e.g., plasmids). The nucleic acids may be incorporated into vectoring systems including, but not limited to, phages and eukaryotic vectors (e.g., lentivirus, adenovirus, baculovirus, vaccinia virus, and the like). The full complement of polypeptides may be introduced into cells (e.g., by transfection, transduction, transformation) for expression by using single nucleic acid sequences encoding the separate polypeptides or as a fusion protein to be later cleaved by site-specific protease. Alternatively, individual nucleic acids encoding thepolypeptides of a construct may be introduced sequentially or simultaneously by transfection, transduction, transformation, or the like.

[0235] Non-limiting examples of cells suitable for expressing the polypeptides of bispecific hemibody constructs and trispecific antibody constructs include prokaryotic cells (e.g, Escherichia coli strains, Bacillus spp. (e.g, B. subtilis, and the like), and non-mammalian lower eukaryotic cell systems such as yeast or fungi (e.g., S. cerevisiae, Pichia spp., and the like). Host cells of higher organisms suitable for the expression of bispecific hemibody constructs and trispecific antibody constructs such as insect and vertebrate cells, particularly mammalian cells, include but are not limited to HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618 and CRL9096), CHO DG44 cells, CHO-KI cells (ATCC CCL-61), 293 cells (e.g, ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g, ATCC No. CRL-1658), Hnh-7 cells, BHK cells (e.g, ATCC No. CCLIO), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like.

[0236] The present disclosure includes and provides for host cells that are genetically modified with a nucleic acid of the present disclosure (e.g, a nucleic acid encoding one or more polypeptides of a bispecific hemibody construct or trispecific antibody construct, optionally operably linked to a promoter). The nucleic acid encoding those polypeptides may be integrated into the host cell's chromosomal nucleic acids or may be an extrachromosomal element.

[0237] Host cells utilized for expressing bispecific hemibody constructs and trispecific antibody constructs may be genetically modified to provide suitable protease properties. Proteases that produce undesirable cleavages in the constructs, such as cleaving protease sensitive sites in linkers, may be inhibited chemically or may be genetically modified to limit the activity or completely knock out expression of that protease. Where the cleavage of linkers in constructs expressed as fusion polypeptides by site-specific proteases is desired, proteases with specificity for aa sequences in the linkers may be co-expressed or integrated into the host cells so that the proteases are expressed constitutively or as induced gene products. The site-specific protease may be secreted so that it may act on fusion proteins that have been expressed and secreted into the media supporting cell-based expression.

[0238] Accordingly, the present disclosure includes and provides for cells comprising one or more nucleic acids that encode one or more (e.g, all) polypeptides of a bispecific hemibody construct or trispecific antibody construct of the present disclosure. One or more polypeptides of the constructs may be expressed as a fusion protein joined by an aa sequence, such as a linker, that comprises a site recognized by a site-specific protease. The disclosure also includes and provides for separate cells for expressing a first and second hemibody construct, in which case the same pair of scaffolds may be used in each of the constructs without interfering cross binding occurring (e.g, same knob-in-hole combination). Both constructs of bispecific hemibodies comprising first and second polypeptides can be expressed in a single cell if different orthogonal interspecific scaffold pairs are used so the first and second polypeptide of each construct pairs with its proper counterpart.

[0239] The present disclosure includes and provides for the preparation of bispecific hemibody constructs and trispecific antibody constructs or polypeptides. The method of preparation may comprise expressing the constructs or polypeptides using a cell or a cell-free system that comprises a nucleic acid encoding the construct(s) orpoly peptide(s) . A number of suitable host cells are discussed above, any of which may comprise one or more nucleic acid sequences encoding the constructs or polypeptides either as an unintegrated element or as an element integrated into the cell's chromosomal genetic material. The method may further comprise purification of the construct by binding it to an affinity matrix (e.g., affinity chromatography media) that can bind to the construct or polypeptide, or bind to an affinity tag of the construct or polypeptide. After removal of unbound protein and, to the extent possible, non-specifically bound protein by washing, the construct or polypeptide may be eluted by suitable means to yield affinity-purified material. If further purification is required, the affinity-purified material may be subject to, for example, one or more of: size separation chromatography, ion exchange chromatography, isoelectric focusing, and / or chromatofocusing. Constructs or their peptides may also be purified and / or concentrated by precipitation (e.g., ammonium sulfate fractionation) or by the use of membranes with defined molecular weight cutoffs. a) Proteases

[0240] Linker aa sequences of the bispecific hemibody constructs and trispecific antibody constructs, and fusion proteins that comprise one or more polypeptides of those constructs, may comprise aa sequences sensitive to one or more site-specific endoproteases. Such linker sequences include the aa sequences that may be used to join affinity tags 5 to a peptide of the constructs (see, e.g., FIG. 2 at A to F). Site-specific proteases from a variety of species may be used to cleave the polypeptides or fusion proteins releasing, for example, the individual constructs, one or more polypeptides of a construct, or an affinity tag. Different locations in a construct or polypeptide (e.g., fusion protein or fusion polypeptide) may comprise the same or different aa sequences subject to cleavage by one or more site-specific proteases.

[0241] One or more sequences recognized by the site-specific protease that act on a construct or polypeptide (e.g., fusion protein described herein) may be at least 3, or at least 4 aa residues in length (e.g., from 3-10 aa residues in length). Alternatively, the sequence recognized by the protease may be at least 5, or at least 7 aas in length (e.g., from 5-12 aa residues in length). The protease may be a human endoprotease. Where human proteases are used, they may be selected from proteases that have a limited tissue distribution, proteases that are developmentally regulated, and proteases that are expressed at low levels in adult human beings.

[0242] Sequences of aas that may be employed as the protease sensitive element(s) of the constructs or polypeptides described herein include sequences subject to a protease selected from the group consisting of: legumain (asparaginyl endopeptidase (AEP)), matriptase, cathepsin S, matrix metalloprotease (MMP) type-1 or “MMP-1”, MMP-2, MMP-9, urokinase-type plasminogin activator (uPA), furin, caspase, Tobacco etchvirus (TEV), ADAM, and PreScission® (available from Cytiva, formerly GE Healthcare).

[0243] Protease sensitive elements (e.g., aa sequences of linkers) in the constructs, polypeptides, or fusion proteins thereof (see, e.g., FIGs. 2-4) as prepared and / or administered may be conditionally active and require a step of activation in vivo or in vitro. Examples of conditional activation of the constructs provided herein include the use of pro-forms of the constructs, including masked forms of the constructs that may be activated by proteases in vivo (or in vitro). The pro-forms of constructs (including masked and / or fused forms) may be activated by proteases present on endothelial cells lining the patient's circulatory system or by proteases found in cells of tissues where a target population of cells in a B cell lineage are located (e.g., marginal zone or germinal centers). The fusion proteinscomprising one or more polypeptides of a construct described herein and pro-forms of constructs also may be activated, in whole or in part, prior to administration to a patient by cleaving some of the site-specific protease susceptible sequences prior to administration. For example, a fusion protein comprising the polypeptides of a hemibody of FIG 5 at C may undergo partial activation but remain in a pro-from by having the linkers between the two polypeptides (elements 2 and T" and / or 2 and 7"") and / or the linker to the affinity TAG 5 cleaved, but leaving linker(s) 6 uncleaved. In that way the pro-form of the hemibody constructs, still bearing the masking elements 4 °and 4’ ° may be administered and cleaved by endogenous site-specific proteases in the patient.

[0244] Suitable site-specific proteases and aa sequences subject to their action for incorporation into bispecific hemibody constructs and trispecific antibody constructs include, but are not limited to, those that follow. It will be understood that constructs do not contain sites within the elements of the bispecific hemibody constructs or trispecific antibody constructs subject to those proteases other than those intended. Sites in linkers intended to be subject to the action of the site specific proteases may be cleaved at rates 10-100 or 100-1,000 times more quickly than sites within the other elements of the constructs (e.g., in PBS at pH 7.4 and 37° C).(1) Legumain

[0245] Legumain, which has a strict specificity for asparaginyl bonds, is active in the monomeric form and represents a suitable protease whose aa sensitive sequences may be incorporated into the bispecific hemibody or trispecific antibody constructs described herein. See, e.g., Dall et al., Proc Natl Acad Sci U S A, (2013) 110 (27)10940-10945. Isoform 1 of human legumain in it preprotein form has 433 amino acids, with aas 1-17 forming the signal peptide and aas 324-433 the propeptide:MVWKVAVFLSVALGIGAVPIDDPEDGGKHWWIVAGSNGWYNYRHQADACHAYQIIHRNGIPDEQIWMMYDDIAYS EDNPTPGIVINRPNGTDVYQGVPKDYTGEDVTPQNFLAVLRGDAEAVKGIGSGKVLKSGPQDHVFIYFTDHGSTGILV FPNEDLHVKDLNETIHYMYKHKMYRKMVFYIEACESGSMMNHLPDNINVYATTAANPRESSYACYYDEKRSTYLGDW YSVNWMEDSDVEDLTKETLHKQYHLVKSHTNTSHVMQYGNKTISTMKVMQFQGMKRKASSPVPLPPVTHLDLTPSP DVPLTIMKRKLMNTNDLEESRQLTEEIQRHLDARHLIEKSVRKIVSLLAASEAEVEQLLSERAPLTGHSCYPEALLHFRT HCFNWHSPTYEYALRHLYVLVNLCEKPYPLHRIKLSMDHVCLGHY. (SEQ ID NO:80 , see UniProtKB - Q99538 )

[0246] A catalytically active portion of the active legumain protein may comprise the aa sequence: VPI DDPEDGGKHW WIVAGSNGW YNYRHQADAC HAYQIIHRNG IPDEQIWMM YDDIAYSEDN PTPGIVINRP NGTDVYQGVP KDYTGEDVTP QNFLAVLRGD EAVKGIGSG KVLKSGPQDH VFIYFTDHGS TGILVFPNED LHVKDLNETI HYMYKHKMYR KMVFYIEACE SGSMMNHLPD NINVYATTAA NPRESSYACY YDEKRSTYLG DWYSVNWMED SDVEDLTKET LHKQYHLVKS HTNTSHVMQY GNKTISTMKV MQFQGMKRKA SSPVPLPPVT HLDLTPSPDV PLTIMKRKLM NTN (SEQ ID NO:84, residues 18-323 of UniProtKB - Q99538).(2) Matriptase

[0247] Matriptase-1 and / or matriptase-2 represent suitable proteases whose aa sensitive sequences may be incorporated into the bispecific hemibody or trispecific antibody constructs described herein. Matriptases cleave substrates with an Arg or Lys at the P1 position and preferentially substrates having an aa with a small side-chain, such as Ala and Gly, at the P2 position.(a) Matriptase-1

[0248] Human matripase-1 (also known as Suppressor of tumorigenicity 14 protein, and ST 14) cleaves substrates with Arg or Lys as the P1 site. Human matriptase-1 starts as a preproprotein of about 855 aa residues: MGSDRARKGGGGPKDFGAGLKYNSRHEKVNGLEEGVEFLPVNNVKKVEKHGPGRWWLAAVLIGLLLVLLGIGFLV WHLQYRDVRVQKVFNGYMRITNENFVDAYENSNSTEFVSLASKVKDALKLLYSGVPFLGPYHKESAVTAFSEGSVIA YYWSEFSIPQHLVEEAERVMAEERWMLPPRARSLKSFVVTSWAFPTDSKTVQRTQDNSCSFGLHARGVELMRFTT PGFPDSPYPAHARCQWALRGDADSVLSLTFRSFDLASCDERGSDLVTVYNTLSPMEPHALVQLCGTYPPSYNLTFH SSQNVLLITLITNTERRHPGFEATFFQLPRMSSCGGRLRKAQGTFNSPYYPGHYPPNIDCTWNIEVPNNQHVKVRFKF FYLLEPGVPAGTCPKDYVEINGEKYCGERSQFWTSNSNKITVRFHSDQSYTDTGFLAEYLSYDSSDPCPGQFTCRT GRCIRKELRCDGWADCTDHSDELNCSCDAGHQFTCKNKFCKPLFWVCDSVNDCGDNSDEQGCSCPAQTFRCSNG KCLSKSQQCNGKDDCGDGSDEASCPKVNVVTCTKHTYRCLNGLCLSKGNPECDGKEDCSDGSDEKCDCGLRSFTR QARWGGTDADEGEWPWQVSLHALGQGHICGASLISPNWLVSAAHCYIDDRGFRYSDPTQWTAFLGLHDQSQRSA PGVQERRLKRIISHPFFNDFTFDYDIALLELEKPAEYSSMVRPICLPDASHVFPAGKAIWVTGWGHTQYGGTGALILQK GEIRVINQTTCENLLPQQITPRMMCVGFLSGGVDSCQGDSGGPLSSVEADGRIFQAGWSWGDGCAQRNKPGVYTL PLFRDWIKENTGV SEQ ID NO:85. See, e.g., UniProtKB - Q9Y5Y6.

[0249] The mature proteolytically active form of matriptase-1 may comprise aa residues 613-852 of SEQ ID NO:82: WGGTDADEGEWPWQVSLHALGQGHICGASLISPNWLVSAAHCYIDDRGFRYSDPTQWTAFLGLHDQSQRSAPGV QERRLKRIISHPFFNDFTFDYDIALLELEKPAEYSSMVRPICLPDASHVFPAGKAIWVTGWGHTQYGGTGALILQKGEI RVINQTTCENLLPQQITPRMMCVGFLSGGVDSCQGDSGGPLSSVEADGRIFQAGWSWGDGCAQRNKPGVYTRLP LFRDWIKENTG (SEQ IS NO:83). A mature proteolytically active form of matriptase-1 may comprise an aa sequence with greater than 95% or greater than 97% sequence identity to SEQ ID NO:83. Alternatively, mature proteolytic ally active form of matriptase-1 may comprise an aa sequence with greater than 98% or greater than 99% sequence identity to SEQ ID NO:86.(b) Matripase-2

[0250] Human matriptase-2 (transmembrane protease, serine 6, matriptase-2 or TMPRSS6) starts as a preproprotein of about 811 aa residues:MLLLFHSKRMPVAEAPQVAGGQGDGGDGEEAEPEGMFKACEDSKRKARGYLRLVPLFVLLALLVLASAGVLLWYFL GYKAEVMVSQVYSGSLRVLNRHFSQDLTRRESSAFRSETAKAQKMLKELITSTRLGTYYNSSSVYSFGEGPLTCFFW FILQIPEHRRLMLSPEWQALLVEELLSTVNSSAAVPYRAEYEVDPEGLVILEASVKDIAALNSTLGCYRYSYVGQGQV LRLKGPDHLASSCLWHLQGPKDLMLKLRLEWTLAECRDRLAMYDVAGPLEKRLITSVYGCSRQEPWEVLASGAIMA WWKKGLHSYYDPFVLSVQPWFQACEVNLTLDNRLDSQGVLSTPYFPSYYSPQTHCSWHLTVPSLDYGLALWFDA YALRRQKYDLPCTQGQWTIQNRRLCGLRILQPYAERIPWATAGITINFTSQISLTGPGVRVHYGLYNQSDPCPGEFLC SVNGLCVPACDGVKDCPNGLDERNCVCRATFQCKEDSTCISLPKVCDGQPDCLNGSDEEQCQEGVPCGTFTFQCE DRSCVKKPNPQCDGRPDCRDGSDEEHCDCGLQGPSSRIVGGAVSSEGEWPWQASLQVRGRHICGGALIADRWVIT AAHCFQEDSMASTVLWTVFLGKVWQNSRWPGEVSFKVSRLLLHPYHEEDSHDYDVALLQLDHPWRSAAVRPVCL PARSHFFEPGLHCWITGWGALREGGPISNALQKVDVQLIPQDLCSEVYRYQVTPRMLCAGYRKGKKDACQGDSGGPLVCKALSGRWFLAGLVSWGLGCGRPNYFGVYTRITGVISWIQQWT SEQ ID NO:87. See, e.g, UniProtKB - Q8IU80, matripase-1 isoform I Q8IU80-4

[0251] A suitable protease for use with bispecific hemibody and trispecific antibody constructs may comprise the aa sequence of a mature proteolytically active matripase-2 protein comprising aa residues 577-811 of SEQ ID NO:84: IVGGAVSSEGEWPWQASLQVRGRHICGGALIADRWVITAAHCFQEDSMASTVLWTVFLGKVWQNSRWPGEVSFKV SRLLLHPYHEEDSHDYDVALLQLDHPWRSAAVRPVCLPARSHFFEPGLHCWITGWGALREGGPISNALQKVDVQLI PQDLCSEVYRYQVTPRMLCAGYRKGKKDACQGDSGGPLVCKALSGRWFLAGLVSWGLGCGRPNYFGVYTRITGVI SWIQQWT SEQ ID NO:88.(3) Cathepsin S

[0252] Cathepsin S represents a suitable protease whose aa sensitive sequences may be incorporated into the bispecific hemibody or trispecific antibody constructs described herein. Cathepsin S cleaves substrates with an at a - Val-Val-Arg- sequence, particularly when the sequence is imbedded in a polypeptide with at least two aa residues on each side of the cleavage site.

[0253] Human cathepsin S (isoform 1) starts as a preproprotein of about 331 aa residues.MKRLVCVLLVCSSAVAQLHKDPTLDHHWHLWKKTYGKQYKEKNEEAVRRLIWEKNLKFVMLHNLEHSMGMHSYDL GMNHLGDMTSEEVMSLMSSLRVPSQWQRNITYKSNPNRILPDSVDWREKGCVTEVKYQGSCGACWAFSAVGALEA QLKLKTGKLVSLSAQNLVDCSTEKYGNKGCNGGFMTTAFQYIIDNKGIDSDASYPYKAMDQKCQYDSKYRAATCSKY TELPYGREDVLKEAVANKGPVSVGVDARHPSFFLYRSGVYYEPSCTQNVNHGVLWGYGDLNGKEYWLVKNSWGH NFGEEGYIRMARNKGNHCGIASFPSYPEI SEQ ID NO:89. See, e.g., UniProtKB - P25774, cathepsin S isoform I P25774-1.

[0254] A suitable protease for use with bispecific hemibody and trispecific antibody constructs may comprise the aa sequence of a mature proteolytically active cathepsin S protein comprising aa residues 115 — 331of SEQ ID NO: 86: LPDSVDWREKGCVTEVKYQGSCGACWAFSAVGALEAQLKLKTGKLVSLSAQNLVDCSTEKYGNKGCNGGFMTTAF QYIIDNKGIDSDASYPYKAMDQKCQYDSKYRAATCSKYTELPYGREDVLKEAVANKGPVSVGVDARHPSFFLYRSGV YYEPSCTQNVNHGVLWGYGDLNGKEYWLVKNSWGHNFGEEGYIRMARNKGNHCGIASFPSYPEI (SEQ ID NQ:90).(4) Matrix Metalloproteinases

[0255] Matrix metalloproteinases (MMPs) are family of calcium-dependent zinc-containing endopeptidases whose aa sensitive sequences may be incorporated into the bispecific hemibody or trispecific antibody constructs described herein. MMP1, MMP 2, and MMP9 are discussed herein as suitable proteases; however, other MMPs may be employed. Table 5, which follows, provides some tetrapeptide sequences that the MMPs specifically proteolyze at the bond indicated by a based on work by Eckhard et al. Matrix Biol. (2016) 49, 37-60. The cleavage rates of the tetrapeptides are listed in rank order.Table 5. Matrix Metalloprotease cleavage sites(a) MMP-1Human MMP-1 starts as a preproprotein of about 469 aa residues:

[0256] MHSFPPLLLLLFWGWSHSFPATLETQEQDVDLVQKYLEKYYNLKNDGRQVEKRRNSGPWEKLKQMQEF FGLKVTGKPDAETLKVMKQPRCGVPDVAQFVLTEGNPRWEQTHLTYRIENYTPDLPRADVDHAIEKAFQLWSNVTPL TFTKVSEGQADIMISFVRGDHRDNSPFDGPGGNLAHAFQPGPGIGGDAHFDEDERWTNNFREYNLHRVAAHELGHS LGLSHSTDIGALMYPSYTFSGDVQLAQDDIDGIQAIYGRSQNPVQPIGPQTPKACDSKLTFDAITTIRGEVMFFKDRFY MRTNPFYPEVELNFISVFWPQLPNGLEAAYEFADRDEVRFFKGNKYWAVQGQNVLHGYPKDIYSSFGFPRTVKHIDA ALSEENTGKTYFFVANKYWRYDEYKRSMDPGYPKMIAHDFPGIGHKVDAVFMKDGFFYFFHGTRQYKFDPKTKRILT LQKANSWFNCRKN (SEQ ID NO:91). See, e.g., UniProtKB - P03956-1. In addition to the tetrapeptide sequences provided in the table above, the sequences PQA-LVA and PQA-IVA are subject to MMP-1 proteolysis. Id.

[0257] A suitable protease for use with bispecific hemibody and trispecific antibody constructs may comprise the aa sequence of the mature proteolytical ly active 22 kDa or 27 kDa form of MMP-1 (interstitial collagenase) protein. The 22kDa form comprises aa residues 100 - 269 of SEQ ID NO:91 :

[0258] FVLTEGNPRWEQTHLTYRIENYTPDLPRADVDHAIEKAFQLWSNVTPLTFTKVSEGQADIMISFVRGDHRDN SPFDGPGGNLAHAFQPGPGIGGDAHFDEDERWTNNFREYNLHRVAAHELGHSLGLSHSTDIGALMYPSYTFSGDVQ LAQDDIDGIQAIYGRSQNPVQP SEQ ID NO:92.(b) MMP-2

[0259] Human MMP-2 cleaves, among other sequences, the collagen-like sequence Pro-GIn-Gly ~lle-Ala-Gly-Gln at the bond between Gly and lie as indicated by the MMP-2 starts as a preproprotein of about 660 aa residues: MEALMARGALTGPLRALCLLGCLLSHAAAAPSPIIKFPGDVAPKTDKELAVQYLNTFYGCPKESCNLFVLKDTLKKMQ KFFGLPQTGDLDQNTIETMRKPRCGNPDVANYNFFPRKPKWDKNQITYRIIGYTPDLDPETVDDAFARAFQVWSDVT PLRFSRIHDGEADIMINFGRWEHGDGYPFDGKDGLLAHAFAPGTGVGGDSHFDDDELWTLGEGQWRVKYGNADG EYCKFPFLFNGKEYNSCTDTGRSDGFLWCSTTYNFEKDGKYGFCPHEALFTMGGNAEGQPCKFPFRFQGTSYDSC TTEGRTDGYRWCGTTEDYDRDKKYGFCPETAMSTVGGNSEGAPCVFPFTFLGNKYESCTSAGRSDGKMWCATTA NYDDDRKWGFCPDQGYSLFLVAAHEFGHAMGLEHSQDPGALMAPIYTYTKNFRLSQDDIKGIQELYGASPDIDLGTG PTPTLGPVTPEICKQDIVFDGIAQIRGEIFFFKDRFIWRTVTPRDKPMGPLLVATFWPELPEKIDAVYEAPQEEKAVFFA GNEYWIYSASTLERGYPKPLTSLGLPPDVQRVDAAFNWSKNKKTYIFAGDKFWRYNEVKKKMDPGFPKLIADAWNAI PDNLDAWDLQGGGHSYFFKGAYYLKLENQSLKSVKFGSIKSDWLGC (SEQ ID NO:93). See, e.g., UniProtKB - P08253-1. In addition to the tetrapeptide sequences provided in the table above, the sequences PAA-LVG and PAG-LVG are subject to MMP-2 proteolysis. Id.

[0260] A suitable protease for use with bispecific hemibody and trispecific antibody constructs may comprise the aa sequence of the mature proteolytically active 72 kDa form of MMP-2 protein. The 22kDa form comprises aa residues 110 - 450 of SEQ ID NO:93:YNFFPRKPKWDKNQITYRIIGYTPDLDPETVDDAFARAFQVWSDVTPLRFSRIHDGEADIMINFGRWEHGDGYPFDG KDGLLAHAFAPGTGVGGDSHFDDDELWTLGEGQWRVKYGNADGEYCKFPFLFNGKEYNSCTDTGRSDGFLWCST TYNFEKDGKYGFCPHEALFTMGGNAEGQPCKFPFRFQGTSYDSCTTEGRTDGYRWCGTTEDYDRDKKYGFCPETA MSTVGGNSEGAPCVFPFTFLGNKYESCTSAGRSDGKMWCATTANYDDDRKWGFCPDQGYSLFLVAAHEFGHAMG LEHSQDPGALMAPIYTYTKNFRLSQDDIKGIQELYGASPD SEQ ID NO:94.(c) MMP-9Human MMP-9 starts as a preproprotein of about 707 aa residues:

[0261] MSLWQPLVLVLLVLGCCFAAPRQRQSTLVLFPGDLRTNLTDRQLAEEYLYRYGYTRVAEMRGESKSLGPAL LLLQKQLSLPETGELDSATLKAMRTPRCGVPDLGRFQTFEGDLKWHHHNITYWIQNYSEDLPRAVIDDAFARAFALW SAVTPLTFTRVYSRDADIVIQFGVAEHGDGYPFDGKDGLLAHAFPPGPGIQGDAHFDDDELWSLGKGVWPTRFGNA DGAACHFPFIFEGRSYSACTTDGRSDGLPWCSTTANYDTDDRFGFCPSERLYTQDGNADGKPCQFPFIFQGQSYSA CTTDGRSDGYRWCATTANYDRDKLFGFCPTRADSTVMGGNSAGELCVFPFTFLGKEYSTCTSEGRGDGRLWCATT SNFDSDKKWGFCPDQGYSLFLVAAHEFGHALGLDHSSVPEALMYPMYRFTEGPPLHKDDVNGIRHLYGPRPEPEPR PPTTTTPQPTAPPTVCPTGPPTVHPSERPTAGPTGPPSAGPTGPPTAGPSTATTVPLSPVDDACNVNIFDAIAEIGNQ LYLFKDGKYWRFSEGRGSRPQGPFLIADKWPALPRKLDSVFEERLSKKLFFFSGRQVWVYTGASVLGPRRLDKLGL GADVAQVTGALRSGRGKMLLFSGRRLWRFDVKAQMVDPRSASEVDRMFPGVPLDTHDVFQYREKAYFCQDRFYW RVSSRSELNQVDQVGYVTYDILQCPED (SEQ ID NO:95). See, e.g., UniProtKB - P14780-1 . In addition to the tetrapeptide sequences provided in the table above, the sequences PAA-LIG, PAG-LIG, PAA—IIG, and PAG-IIG are subject to MMP-1 proteolysis. Id. MMP9 preproprotein is cleaved into 67 kDa and 82 kDa MMP-9 forms.

[0262] A suitable protease for use with bispecific hemibody and trispecific antibody constructs may comprise the aa sequence of the mature proteolytically active 82 kDa form of MMP-9 protein. The 82 kDa form comprises aa residues 113 - 444 of SEQ ID NO:95:DLKWHHHNITYWIQNYSEDLPRAVIDDAFARAFALWSAVTPLTFTRVYSRDADIVIQFGVAEHGDGYPFDGKDGLLAH AFPPGPGIQGDAHFDDDELWSLGKGVWPTRFGNADGAACHFPFIFEGRSYSACTTDGRSDGLPWCSTTANYDTDD RFGFCPSERLYTQDGNADGKPCQFPFIFQGQSYSACTTDGRSDGYRWCATTANYDRDKLFGFCPTRADSTVMGGN SAGELCVFPFTFLGKEYSTCTSEGRGDGRLWCATTSNFDSDKKWGFCPDQGYSLFLVAAHEFGHALGLDHSSVPEA LMYPMYRFTEGPPLHKDDVNGIRHLYG SEQ ID NO:96.(5) Urokinase-type plasminogen activator (uPA)

[0263] Urokinase-type plasminogen activator converts catalytically inactive high molecular weight plasminogen activator into the catalytically active low molecular weight plasminogen activator. The proteolytically active domain of uPA represents suitable proteases whose aa sensitive sequences may be incorporated into the bispecific hemibody or trispecific antibody constructs described herein. See, e.g., Dall et al., Proc Natl Acad Sci U S A, (2013) 110(27)10940-10945. Isoform 1 of human legumain in it preprotein form has 433 amino acids, with aas 1-17 forming the signal peptide and aas 324-433 the propeptide: The protein as initially transcribed has about 431 aa residues: MRALLARLLLCVLWSDSKGSNELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKKFGGQHCEIDKSKTCYEGNGH FYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQLGLGKHNYCRNPDNRRRPWCYVQVGLKLLVQECMVHD CADGKKPSSPPEELKFQCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVISATHCFIDY PKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQ FGTSCEITGFGKENSTDYLYPEQLKMTWKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSL QGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLAL (SEQ ID NO:97). See, e.g, UniProtKB - P00749.

[0264] A suitable protease for use with the bispecific hemibody and trispecific antibody constructs may comprise the aa sequence of the proteolytically active portion of uPA, which comprises aa residues 179 - 423 of SEQ ID NO:97:IIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVE NLILHKDYSADTLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQLKMTW KLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRV SHFLPWIRSHT (SEQ ID NO:98).(6) Furin

[0265] The proteolytically active portion of furin is a sequence specific endoprotease, whose aa sensitive sequences may be incorporated into the bispecific hemibody or trispecific antibody constructs described herein. Furin displays sequence selectivity for the motif Arg-Xaa-Lys-Arg or Arg-Xaa-Arg-Arg, wherein Xaa is any amino acid. In some cases, Xaa may be selected from the group consisting of D, E, F, G, H, K, L, P, Q, R, S, T, and V, or the group consisting of H, K, and R. See Hosaka et al., J. Bio. Chem., 266(19) 12127-12130 (1991).

[0266] Furin is initially transcribed as a 794 aa preproprotein that includes a signal sequence (aas 1-26) and an inhibition polypeptide (27-107):MELRPWLLWWAATGTLVLLAADAQGQKVFTNTWAVRIPGGPAVANSVARKHGFLNLGQIFGDYYHFWHRGVTKRS LSPHRPRHSRLQREPQVQWLEQQVAKRRTKRDVYQEPTDPKFPQQWYLSGVTQRDLNVKAAWAQGYTGHGIWSI LDDGIEKNHPDLAGNYDPGASFDVNDQDPDPQPRYTQMNDNRHGTRCAGEVAAVANNGVCGVGVAYNARIGGVR MLDGEVTDAVEARSLGLNPNHIHIYSASWGPEDDGKTVDGPARLAEEAFFRGVSQGRGGLGSIFVWASGNGGREH DSCNCDGYTNSIYTLSISSATQFGNVPWYSEACSSTLATTYSSGNQNEKQIVTTDLRQKCTESHTGTSASAPLAAGIIA LTLEANKNLTWRDMQHLWQTSKPAHLNANDWATNGVGRKVSHSYGYGLLDAGAMVALAQNWTTVAPQRKCIIDIL TEPKDIGKRLEVRKTVTACLGEPNHITRLEHAQARLTLSYNRRGDLAIHLVSPMGTRSTLLAARPHDYSADGFNDWAF MTTHSWDEDPSGEWVLEIENTSEANNYGTLTKFTLVLYGTAPEGLPVPPESSGCKTLTSSQACWCEEGFSLHQKSC VQHCPPGFAPQVLDTHYSTENDVETIRASVCAPCHASCATCQGPALTDCLSCPSHASLDPVEQTCSRQSQSSRESP PQQQPPRLPPEVEAGQRLRAGLLPSHLPEWAGLSCAFIVLVFVTVFLVLQLRSGFSFRGVKVYTMDRGLISYKGLPP EAWQEECPSDSEEDEGRGERTAFIKDQSAL, (SEQ ID NO:99). See, e.g., UniProtKB - P09958-1.

[0267] A suitable protease for use with the bispecific hemibody and trispecific antibody constructs may comprise the aa sequence of the proteolytically active portion of furin, which comprises aa residues 108 - 794 of SEQ ID NO:99: DVYQEPTDPKFPQQWYLSGVTQRDLNVKAAWAQGYTGHGIWSILDDGIEKNHPDLAGNYDPGASFDVNDQDPDP QPRYTQMNDNRHGTRCAGEVAAVANNGVCGVGVAYNARIGGVRMLDGEVTDAVEARSLGLNPNHIHIYSASWGPE DDGKTVDGPARLAEEAFFRGVSQGRGGLGSIFVWASGNGGREHDSCNCDGYTNSIYTLSISSATQFGNVPWYSEAC SSTLATTYSSGNQNEKQIVTTDLRQKCTESHTGTSASAPLAAGIIALTLEANKNLTWRDMQHLWQTSKPAHLNANDW ATNGVGRKVSHSYGYGLLDAGAMVALAQNWTTVAPQRKCIIDILTEPKDIGKRLEVRKTVTACLGEPNHITRLEHAQA RLTLSYNRRGDLAIHLVSPMGTRSTLLAARPHDYSADGFNDWAFMTTHSWDEDPSGEWVLEIENTSEANNYGTLTKF TLVLYGTAPEGLPVPPESSGCKTLTSSQACWCEEGFSLHQKSCVQHCPPGFAPQVLDTHYSTENDVETIRASVCAP CHASCATCQGPALTDCLSCPSHASLDPVEQTCSRQSQSSRESPPQQQPPRLPPEVEAGQRLRAGLLPSHLPEWAG LSCAFIVLVFVTVFLVLQLRSGFSFRGVKVYTMDRGLISYKGLPPEAWQEECPSDSEEDEGRGERTAFIKDQSAL, SEQ ID NG:100.(7) Other proteases

[0268] Sequences sensitive to numerous other site-specific proteases may be incorporated into the bispecific hemibody or trispecific antibody constructs described herein. Other site-specific proteases and the aa sequences sensitive cleavage include: Cysteine-aspartic acid protease (caspase) family members such as Caspase 7 (cleaving the sequences DEVDG and DEVEG); Enterokinase (cleaving after the sequence DDDDK or (D / E) (D / E) (D / E)K; Granzyme B (which cleaves after the sequence IEPD); Tobacco etch virus protease or "TEV” protease (cleaving the sequence ENLYFQS); and ADAM proteases (A Disintegrin And Metalloproteinase) family members.5. Compositions

[0269] Bispecific hemibody constructs, trispecific antibody constructs, or nucleic acid constructs encoding them may be formulated into compositions including, but not limited to, pharmaceutical compositions comprising pharmaceutically acceptable excipients. Bispecific hemibody constructs and trispecific antibody constructs may be formulated (e.g., for administration to a patient) in a form wherein at least one aa linker sequence that may be cleaved by an endogenous site-specific protease may be present. Those constructs may even be formulated as compositions (e.g., for administration to a patient as fusion proteins that may require cleavage of one or more linker aa sequences by one or more endogenous proteases. Where the compositions are intended for administration to a patient, they may be formulated for intravenous, intramuscular, intraperitoneal, subcutaneous, and / or intralymphatic administration.

[0270] In some instances the bispecific hemibody constructs and trispecific antibody constructs as formulated (e.g., as a pharmaceutical composition) may comprise elements that mask binding to CEC-SAs and / or markers on cells in the B cell lineage in whole or in part (e.g., by altering affinity). Linker aa sequences joined to aa sequences that bind cell surface markers or cell surface antigens that comprise one or more sites subject to site-specific proteases may mask, wholly or partially, binding to cells. By way of example, the cleavable linker in the constructs shown in FIG. 5 at C between 2 and 1"' or 2 and 1"" may wholly or partially mask the ability of 1"' and or 1"" to bind cell surfacemarkers by interfering with binding interactions as reflected by a reduce their affinity for the markers. See also Fig. 2 at C and F. The same is true of the cleavable linker in the trispecific antibody construct depicted in FIG. 4 at B between elements 4 and 1’. Bispecific hemibody constructs comprising masking VH and / or VL aa sequences (elements 4° and 4’°) may also be formulated as compositions, including pharmaceutical compositions for administration to a patient (see e.g., FIG. 2 at D-F, Fig. 3 at C to D, and Fig. 5 at C and D).

[0271] When the bispecific hemibody constructs and / or trispecific antibody constructs to be administered are separate distinct molecules, such as first and second hemibody constructs, they may be combined into a single formulation for administration or formulated to be administered separately (e.g., sequentially, simultaneously, or in temporally overlapping time frames). For example, a first hemibody construct and second hemibody construct that together form a bispecific hemibody may be prepared as two separate formulations suitable for administration to a patient. Alternatively, the first and second hemibody constructs of a bispecific hemibody may be combined into a single composition suitable for administration to a patient. a) Compositions

[0272] The present disclosure provides compositions, including pharmaceutical compositions, comprising bispecific hemibody constructs trispecific antibody constructs (including any masked or fusion protein forms thereof), or nucleic acids encoding any thereof. Pharmaceutical composition can comprise, in addition to the bispecific hemibody construct(s), trispecific antibody construct(s), or encoding nucleic acids, one or more known carriers, excipients, diluents, buffers, salts, surfactants (e.g., non-ionic surfactants), amino acids (e.g., arginine), etc., a variety of which are known in the art and described in, for example, "Remington: The Science and Practice of Pharmacy”, 19th Ed. (1995), or latest edition, Mack Publishing Co., and not described herein.

[0273] In some cases, a subject pharmaceutical composition will be suitable for administration to a subject, e.g., will be substantially free of pyrogens (i.e., pyrogens are absent or within pharmaceutically acceptable ranges for administration by, for example, intravenous administration) and / or sterile. A subject pharmaceutical composition may be suitable for administration to a human patient where, for example, the composition is sterile and is substantially free of detectable pyrogens and / or other toxins, or pyrogens and / or other toxins are below acceptable limits (e.g., the composition is considered non-pyrogenic when administered at the dose administered).

[0274] Compositions may be in the form of aqueous or other solutions. Compositions may also be formulated as powders, granules, tablets, pills, suppositories, capsules, suspensions, sprays, and the like. The composition may be formulated for intravenous and / or intralymphatic administration. Alternatively, the compositions may formulated for other routes of administration including those recited herein.

[0275] Ready to use aqueous formulations, or a non-aqueous formulations, may be employed to administer bispecific hemibody constructs and trispecific antibody constructs (e.g., as an injectable for administration subcutaneously, intraperitoneally, intramuscularly, intralymphatically, and / or intravenously). Dosage forms may be reconstitutable (e.g., a storage-stable powder) comprising pharmaceutically acceptable carriers and excipients. Bispecific hemibody constructs and trispecific antibody constructs may also be provided in formulations designed to enhance their serum half-life following administration. Proteins may, for example, be provided in a liposome or colloidformulation, or in other formulations utilizing conventional techniques for extending serum half-life. Methods for preparing liposomes, including those described in, e.g.,. 1980 Ann. Rev. Biophys. Bioeng. 9:467, U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028. Preparations may be provided in controlled release or slow-release forms.

[0276] In some cases, compositions comprise one or more bispecific hemibody constructs or one or more trispecific antibody constructs and saline (e.g., 0.9% NaCI). In some cases, compositions comprise one or more bispecific hemibody constructs or one or more trispecific antibody constructs, saline (e.g., up to 0.9% NaCI), and a buffer (e.g., a sodium and / or potassium phosphate buffer). Such composition may be sterile and / or substantially pyrogen free, or the amount of detectable pyrogens and / or other toxins may be below an acceptable limit. In some cases, the composition may be suitable for administration to a human subject, for example, where the composition is sterile and is free of detectable pyrogens and / or other toxins, or the amount of detectable pyrogens and / or other toxins are below an acceptable limit. Thus, the present disclosure provides a composition comprising: a) one or more bispecific hemibody constructs or one or more trispecific antibody constructs; and b) saline (e.g., 0.9% NaCI), where the composition is sterile and is substantially free of detectable pyrogens and / or other toxins, or such detectable pyrogens and / or other toxins are below an acceptable limit.

[0277] Other examples of components suitable for inclusion in formulations suitable for parenteral administration include isotonic sterile injection solutions, anti-oxidants, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. A pharmaceutical composition can be present in a container, e.g., a sterile container, such as a syringe. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.

[0278] The concentration of one or more bispecific hemibody constructs or one or more trispecific antibody constructs in a formulation can vary widely. For example, the concentration of the constructs may be from less than about 0.1% (usually at least about 2%) to as much as 50% or more by weight. Exemplary ranges for the total amount of one or more constructs in a formulation include, but are not limited to from 0.2% to 1% and from 1% to 5% by weight. Other exemplary ranges for the total amount of one or more constructs include, but are not limited to from 5% to 10% and from 10% to 20% by weight. The total amount of one or more constructs may also be from about 20% to about 30%, or from about 30% to about -50% by weight, particularly where the formulation is not a liquid (e.g., is nonaqueous) such as a lyophilized composition. The concentration will usually be selected primarily based on fluid volumes, viscosities, and patient-based factors in accordance with the particular mode of administration selected for a patient's needs.

[0279] The present disclosure provides a container comprising any of the above-mentioned compositions comprising a bispecific hemibody construct or a trispecific antibody construct, e.g., a liquid composition. The container can be, e.g., a syringe, an ampoule, and the like. In some cases, the container is sterile. In some cases, both the container and the composition are sterile and substantially free of detectable pyrogens and / or other toxins, or such detectable pyrogens and / or other toxins are below an acceptable limit. A pharmaceutical composition or acontainer comprising a composition (e.g., pharmaceutical composition) set forth herein may be packaged as a kit. The kit may comprise, for example, the composition or the container comprising a composition along with instructions for use of those materials. Materials packaged as a kit may be sterile and / or substantially free of detectable pyrogens and / or other toxins, or such detectable pyrogens and / or other toxins are below an acceptable limit. b) Compositions Comprising a Nucleic Acid or a Recombinant Expression Vector

[0280] The present disclosure provides compositions (e.g., pharmaceutical compositions) comprising one or more nucleic acids or one or more recombinant expression vectors that comprise one or more nucleic acid sequences encoding any one or more bispecific hemibody constructs, any one or more trispecific antibody constructs, or any one or more polypeptides thereof. As discussed above, a numerous varied pharmaceutically acceptable excipients need not be discussed in detail herein as they are known in the art.

[0281] A nucleic acid or a recombinant expression vector composition can include one or more nucleic acids or one or more recombinant expression vectors comprising a nucleic acid (e.g., DNA or RNA) sequences encoding one or more bispecific hemibody constructs, any one or more trispecific antibody constructs, or any one or more polypeptides thereof. A nucleic acid or a recombinant expression vector composition may include one or more nucleic acids or one or more recombinant expression vectors comprising a nucleic acid (e.g., DNA or RNA) sequences encoding all constructs of a bispecific hemibody (e.g., first and second hemibody construct polypeptides), or all polypeptides of a trispecific antibody construct. Such compositions may further include one or more of: salts (e.g., to render the composition isotonic), a buffer, a surfactant, an antioxidant, a hydrophilic polymer, a dextrin, a chelating agent, a suspending agent, a solubilizer, a thickening agent, a stabilizer, a bacteriostatic agent, a wetting agent, and a preservative.

[0282] A pharmaceutically acceptable formulation may comprise a nucleic acid or recombinant expression vector encoding one or more polypeptides of a bispecific hemibody construct or a trispecific antibody construct (e.g., in an amount of from about 0.01% to about 90% (w / w)). Some suitable ranges include about 0.01% to about 0.1% or about 0.1% to about 1% w / w. Other suitable ranges include about 1% to about 10% or about 10% to about 90% w / w. Such pharmaceutical compositions may be suitable for administration to a patient, e.g., they are sterile and / or substantially free of pyrogens. In some embodiments, the pharmaceutical composition may be suitable for administration to a human subject, e.g., where the composition is sterile and is substantially free of detectable pyrogens and / or other toxins, or such detectable pyrogens and / or other toxins are below their acceptable limits.

[0283] A composition comprising a nucleic acid or a recombinant expression vector encoding one or more polypeptides of a bispecific hemibody construct or a trispecific antibody construct, including pharmaceutically acceptable formulations, may be in the form of a liposomal formulation.

[0284] The compositions comprising a nucleic acid or a recombinant expression vector described herein may include penetration enhancers including, but not limited to, any one or more of: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. See e.g., U.S. Pat. No. 6,287,860.6. Methods of utilizing Bispecific Hemibodies and Trispecific Antibodies a) Cytotoxic Effector Cell Activation and Cytotoxic Responses

[0285] As discussed above, cytotoxic effector cells may be divided into myeloid and lymphoid cytotoxic effector cells. Cytotoxic effector cells of the myeloid immune cell type include, but are not limited to, monocytes, macrophages, dendritic cells, and / or granulocytes. Cytotoxic effector cells of the lymphoid type include, but are not limited to T cells and / or NK cells. Cytotoxic T effector cells may be CD4+ and CD8+. Myeloid and lymphoid cytotoxic responses are triggered by engagement of particular cell surface receptors that vary by cell type. For example, macrophages phagocytose and kill certain pathogens following engagement of certain pathogen recognition receptors (PRR) by cognate ligands on the pathogen cell surface. Whereas T cells may become activated following engagement of their T cell receptor (TCR), leading to a variety of potential functional consequences, including cytotoxicity of cognate targets such as viral ly infected host cells and tumor cells. Besides the engagement of classical activating receptors such as PRRs and TCRs, cytotoxic responses are influenced by an array of potential receptorligand interactions with the cytotoxic target, but also by engagements with soluble, cell surface and extracellular matrix molecules in the environment. Such accessory signals as well as classical activating signals can by mimicked by appropriate affinity reagents such as antibodies against the TCR. For example, the OKT3 antibody, which engages the TCR, may induce the activation, proliferation, and the release of IFN gamma by CD8+ effector T cells. The released IFN gamma may then stimulate macrophages to increase phagocytosis and macrophage production of inflammatory mediators and reactive oxygen and nitrogen intermediates. Dendritic cells, which belong to a different lineage than macrophages may also function in phagocytosis and induce a variety of immune responses.

[0286] Myeloid cell cytotoxic responses, particularly macrophage responses include, but are not limited to, phagocytosis, trogocytosis (i.e. phagosomal pinching off of portions of target cells), generation of reactive oxygen and nitrogen species, cytokine release, presentation of a target cell antigens acting as an APC. Granulocytic myeloid cells including, neutrophils, eosinophils, and basophils, have granules filled with inflammatory mediators that are released upon activation. Neutrophils and macrophages, are capable of killing and degrading phagocytosed, trogocytosed, and membrane apposed pathogens, cells and other materials by releasing degradative enzymes and reactive oxidative species. The molecules released include defensins, serine proteases, neutrophil elastase, proteinase 3, cathepsin, alkaline phosphatase, lysozyme, NADPH oxidase, collagenase, lactoferrin, histaminase, gelatinase, and collagenase. Eosinophils effector functions include the release of granule proteins (e.g., eosinophil peroxidase, ribonuclease, deoxyribonucleases, and lipase, and plasminogen), production of reactive oxygen species such as hypobromite, superoxide, and peroxide, production of lipid mediators (e.g., eicosanoids such as the leukotrienes LTC4, LTD4, LTE4 and / or prostaglandins such as PGE2), production and / or release of elastase, and cytokines such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, and TNF alpha. Activated, basophils release proteoglycans (e.g. heparin and chondroitin), histamine, and proteolytic enzymes (e.g. elastase and lysophospholipase) from their granules. Basophils also secrete leukotrienes (e.g., LTD-4), and several cytokines and appear to be a source of IL-4.

[0287] Effector cell function of certain myeloid cells and lymphoid cells (including, e.g., NK cells and T cells), include the production of various granule dependent and granule independent responses. Cytokine producing innate lymphoid cells (ILCs) produce various cytokines depending on their subtype. ILC1 cells may produce IFN-gamma. ILC2 cells may produce IL-5 and IL-13. ILC3 cells may produce IL-17 and IL-22.

[0288] NK cell effector functions including secretion of IFN-gamma are similar to those of CD8+ cytotoxic T cells (CTLs). Both NK cells and CTLs release granulysin, granzymes, and perforin, each of which are stored in intracellular granules. Granzymes are serine proteases that activate caspases thereby inducing apoptosis of target cells.) Upon activation, NK cells also may secrete cytokines including, but not limited to interferon-y (IFN-y), tumor necrosis factorci (TNF-a), granulocyte macrophage colony-stimulating factor (GM-CSF), and chemokines (CCL1, CCL2, CCL3, CCL4, CCL5, and CXCL8). Those molecules can modulate the function of a variety of innate and adaptive immune cells that may be present. NK and CTL effector responses are discussed generally in, for example Abbas et al. Cellular and Molecular Immunology, 9th Ed. ,(2018) Elsevier, Philadelphia, PA, and Lai, Front Immunol., 8:1124 (2018). CTLs' granule independent effector mechanisms include, but are not limited to, the expression of FAS ligand (FasL) on activated CTLS. FasL bind to the apoptotic Fas receptor expressed on, for example, germinal-center B cells. See, e.g., Hao et al. Immunity. ,29(4): 615-627 (2008). The release of IFN-gamma and IL-17 by CTLs and NK cell, may also stimulate actions by other effector cells including phagocytosis by macrophages.

[0289] Any of the foregoing effector cell mechanisms may be directed against cells in the B cell lineage by the bispecific hemibodies and trispecific antibodies of the present disclosure. Any of the above-mentioned molecules released during cytotoxic effector cell action may also be used to measure the effect of the bispecific hemibodies and trispecific antibodies.

[0290] In addition to the cytotoxic effector cell actions mentioned above, the hemibodies and trispecific antibodies described herein comprising immunoglobulin scaffolds may effect the ablation of cells in the B cell lineage by mechanisms including ADCC, ADCP, and GDC. ADCC is a cell-mediated immune response in which an effector cell kills a target cell whose membrane-surface antigens have been bound by specific antibodies. Bispecific hemibody or trispecific antibody constructs described herein that comprise immunoglobulin constant region (IgFc) aa sequences that can bind and stimulate an FcyRIII receptor (e.g., FcyRIII A, CD16A expressed on macrophages and NK cells) effectively take the place of antibodies in stimulating the cytotoxic response. Because the FcyRI 11 A receptor has of low affinity for the IgFc aa sequences it engages, cell bearing that receptor will only bind to cells coated with antibody, bispecific hemibody constructs, or trispecific antibody constructs as opposed to the individual molecules in solution. Engagement of FcyRIII leads to, among other things, synthesis and release of cytokines, such as interferon gamma IFN-gamma and TNF, and degranulation and release of perforin and granzymes, leading to target cell death.

[0291] Like CD16, CD32, which is expressed on myeloid cells including monocytes, macrophages, and dendritic cells, may engage immunoglobulin constant region sequences leading to an ablation of target cells of the B cell lineage. Whereas an excess of engaged CD16A generally results in granule-mediated events leading to ablation of the target cell, an excess of CD32A (engagement tends to result in phagocytosis (ADCP) of the target cell and stimulation inflammatory cytokine secretion.

[0292] The efficacy of bispecific hemibody constructs and trispecific antibody constructs in eliciting ADCC may be assessed by contacting the construct with target cells in the B cell lineage expressing surface antigens to which the constructs bind to form construct-labeled target cells. The construct-labeled target cells may be preloaded with a reporter. Excess constructs may be removed (e.g., by washing) before cytotoxic effector cells expressing FcyRIII are co-incubated with the antibody-labelled target cells. The co-incubated cells are allowed to form a complex between the construct-labeled cell and the effector cell, which leads to lysis of the construct-labeled target cell. Cytotoxicity often results in cell lysis and / or plasma membrane permeability and / or nuclear membrane permeability. If the target cell was pre-loaded with a radioactive (e.g., chromium-51 or sulfur 35 labeled protein), fluorescent, or luminescent label, release into the solution is proportion to the amount of cell lysis. Some non-radioactive labels (e.g. split luciferase or fluorescent proteins) as well as cellular enzymes like glyceraldehyde 3-phosphate dehydrogenase or lactate dehydrogenase do not require pre-loading if they are cytosol ically expressed and can be quantified using fluorescence, luminescence or absorbance measurements. Multiparameter flow cytometric methods are also available to measure cytotoxicity via dye permeability with the benefit of coordinate assessment of additional cellular markers such as cell surface or intracellular markers of lineage, differentiation and activation state.

[0293] ADCC and ADCP takes place multiple Fc gamma receptors on the same effector cell simultaneously engage IgG constant region domains present in the scaffold sequences of the bispecific hemibodies and trispecific antibodies. The binding of antibody-antigen complexes to the FcyRs induces their cross-linking and subsequent signaling through immunoreceptor tyrosine-based activation motifs (ITAMs). Cytoplasmic signaling includes an increase in intracellular calcium concentration and calcineurin / calmodulin-mediated dephosphorylation of NFAT (nuclear factor of activated T cells), allowing its nuclear translocation and binding to promoter regions of genes related to ADCC and ADCP. Assessment of ADCC and ADCP related functions of the bispecific hemibodies and trispecific antibodies may be made by measurement of any of those cell signaling events. Engineered human cells may also be used to assess ADCC and ADCP effector functions. For example, NFAT linked luciferase reporter constructs in Jurkat cells may be used for assessment of ADCC and ADCP, such as in the case of InvivoGen's Jurkat-Lucia™ NFAT-CD16 Cells for ADCC and Jurkat-Lucia™ NFAT-CD32 Cells for ADCP). Assays may be conducted using the stimulate luminescence upon construct addition to assess the ADCC or ADCP potential of the construct using the manufacturer's conditions. Cytometric and microscopic methods for assessing internalization of labeled and unlabeled target cell material by ADCP effectors also exist.

[0294] Complement-dependent cytotoxicity (CDC) is a potent effector mechanism, engaging both innate and adaptive immune responses. Bispecific hemibodies and trispecific antibodies of the present disclosure comprising IgG or IgM scaffold sequences bound to the surface of a target cell in the B cell lineage can bind complement component 1q (C1q) leading to a CDC response. More specifically scaffolds comprising the CH2 domain of lgG1 or 1 gG3, and to a lesser degree the CH2 domain of lgG2) or the CH3 domain of IgM may interact with C1q and initiate a CDC response. As the CDC response progresses, a cytocidal membrane attack complex is formed that produces, among other things, polymerized C9 protein that results in pores in the membrane of the cell upon which the complex is formed. Those pores, similar to the pores formed by perforin released by CTLs and NK cells allow the exchange of water and ions between the interior and exterior of the cell and may even result in osmotic rupture of the cell. Theability of the bispecific hemibodies and trispecific antibody constructs to induce CDC may be assess by any of a variety of mechanism known in the art including, but not limited to, measurement of (i) radioactive Cr release from labeled cells, (ii) the metabolic activity after incubation of target cells with constructs described herein (e.g., live cell staining), (ill) the release of intracellular enzymes (e.g., by colorimetric, fluorescent, or luminescent assay), and / or (iv) dead cell numbers following incubation with a construct described herein (e.g., utilizing propidium iodide, which binds to DNA of dead cells resulting in a fluorescent complex that can be measured). Unless stated otherwise, the ability of the bispecific hemibodies and trispecific antibodies to effect a CDC response is assessed using luminescence assay as described by ALIGENT Application Note Immuno-Oncology titled ""A Semi-Automated, Nonradioactive

[0295] Assay for the Detection of Antibody-Based Complement-Dependent Cytotoxicity" by Larson and Banks using the bispecific hemibodies or trispecific antibodies as a test agent in place of test antibodies. Where the human B cell lymphoma Daudi cells employed in that assay do not express one or more of the B cell markers the bispecific hemibodies or trispecific antibodies target, those markers may be transiently expressed in the cells employed for the assay. For assays on the plasma cell sublineage multiple myeloma cell lines MM.1S or MM.1R may, for example be employed. As with the Daudi cells, were myeloma cell lines do not express the surface markers to which the bispecific hemibodies or trispecific antibodies bind, those marker may be transiently expressed on the cells. b) Methods of T reatment

[0296] The bispecific hemibody constructs and trispecific antibody constructs or compositions comprising any of those constructs may be prepared for parenteral and / or other forms of administration to a patient. Suitable routes of administration include, but are not limited to, intravenous, intramuscular, subcutaneous, and / or intralymphatic administration

[0297] The present disclosure provides treatment methods comprising administering to a patient an amount of one or more bispecific hemibody constructs, one or more trispecific antibody constructs, or compositions comprising one or more polypeptides of those constructs effective to modulate the activity selectively of a cytotoxic effector cell in a patient and to effect treatment of the patient. The present disclosure provides treatment methods comprising administering to a patient an amount of one or more nucleic acids (e.g., mRNA) or expression vectors encoding one or more bispecific hemibody constructs, one or more trispecific antibody constructs or a polypeptide thereof, effective to selectively modulate the activity of a cytotoxic effector cell in a patient and to effect treatment of the patient. The present disclosure provides treatment methods comprising administering to a patient an amount of one or more bispecific hemibody constructs, one or more trispecific antibody constructs, or compositions comprising one or more polypeptides of those constructs effective to deplete or complete ablate a of populations of cells of the B cell lineage in the patient. The present disclosure provides treatment methods comprising administering to a patient an amount of one or more nucleic acids or expression vectors encoding one or more bispecific hemibody constructs, one or more trispecific antibody constructs or a polypeptide thereof, to deplete or complete ablate a of populations of cells of the B cell lineage in the patient. The ablation or depletion of the target cell population may be accessed by, for example, measuring (quantitating e.g., by flow cytometry) the number of the target cells remaining after an appropriate time period following administration relative to their number prior to the administration. In some cases, a treatment method comprises administering to an patient in need thereof one or more recombinant expression vectors comprisingnucleotide sequences encoding one or more bispecific hemibody constructs, one or more trispecific antibody constructs, or compositions comprising one or more polypeptides of those constructs. In some cases, a treatment method comprises administering to an patient in need thereof one or more mRNA molecules comprising nucleotide sequences encoding one or more bispecific hemibody constructs, one or more trispecific antibody constructs, or compositions comprising one or more polypeptides of those constructs. In some cases, a treatment method comprises administering to an patient in need thereof one or more bispecific hemibody constructs, one or more trispecific antibody constructs, or compositions comprising one or more polypeptides of those constructs.

[0298] Modulation of the cytotoxic effector cells by one or more bispecific hemibody constructs, one or more trispecific antibody constructs, or compositions comprising one or more polypeptides of those constructs, is not antigen specific, but may be directed against specific populations of cells in the B cell lineage by the selection of markers on their surface. Accordingly, the depletion or ablation of a population of B cells by administering one or more bispecific hemibody constructs, one or more trispecific antibody constructs, or nucleic acids encoding them is not antigen specific, but may be directed against a population of cells in the B cell lineage bearing appropriate markers.

[0299] The present disclosure provides a method of treating an autoimmune or other disorder in a patient, the method comprising administering to the patient an effective amount of one or more bispecific hemibody constructs, one or more trispecific antibody constructs, or nucleic acids encoding them. In some cases an "effective amount” of one or more bispecific hemibody constructs, one or more trispecific antibody constructs, or nucleic acids encoding them is an amount that, when administered in one or more doses to a patient in need thereof depletes (decreases) the population of cells in the B cell lineage to which at least one hemibody or trispecific antibody construct binds, by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% in the patient or in a compartment or a tissue of the patient's body. The depletion in the target population of cells may be from about 10% to about 20% or from about 20% to about 40%. The depletion in the target population of cells may be from about 40% to about 60% or from about 60% to about 80%. The percentage depletion is calculated by comparison to number of target cells in the patient or in a compartment or a tissue of the patient's body. An "effective amount” of may be an amount that, when administered in one or more doses to a patient in need thereof, increases production of one or more inflammatory cytokines such as IFN-gamma or TNF relative to the amount produced prior to administration based on measurements in vitro or in vivo in a tissue of a patient. An "effective amount” of may be an amount that, when administered in one or more doses to a patient in need thereof, increase the production and / or release of proteins found in the granules of one or more cytotoxic effector cells (e.g., granulysin, granzymes, or perforin) relative to the amount produced prior to administration based on measurements in vitro or in vivo in a tissue of a patient.. An "effective amount” may be an amount that, when administered in one or more doses to a patient in need thereof, ameliorates one or more symptoms associated with an autoimmune disease in the patient. An "effective amount” may be an amount that, when administered in one or more doses to a patient in need thereof, increases the ration of T reg cells or B reg cells to the cells in the B cell lineage to which the bispecific hemibody or trispecific antibody construct bind.

[0300] As noted above, in some cases, in carrying out a subject treatment method, a trispecific antibody construct or a bispecific hemibody (e.g., or the constructs that comprise a bispecific hemibody) is administered to a patient in need thereof, as the polypeptide(s) per se. In other instances a trispecific antibody construct or a bispecific hemibody (e.g., or the constructs that comprise a bispecific hemibody) is administered to a patient in need thereof, as a fusion protein or in a form comprising one or more linker aa sequences requiring cleavage by at least one endogenous sitespecific protease. In other instances, in carrying out a subject treatment method, one or more nucleic acids comprising nucleotide sequences that encode a trispecific antibody construct or a bispecific hemibody is / are administering to a patient in need thereof.

[0301] A trispecific antibody construct, a bispecific hemibody, or one or more nucleic acids encoding such molecules, may be administered alone or with one or more additional therapeutic agents or drugs. The additional therapeutic agent(s) may be administered before, during, or subsequent to administration of the trispecific antibody construct or bispecific hemibody constructs, or nucleic acids encoding such molecules. When the additional therapeutic agents are administered with a composition or formulation comprising any of those molecules, the additional therapeutic agent may be administered concurrently or may be co-administered as part of a formulation or composition comprising any of those molecules.

[0302] Suitable therapeutic agents or drugs that may be administered with or provided as a payload of a trispecific antibody construct or a bispecific hemibody may be virtually any therapeutic agent, including small molecule therapeutics (e.g., less than 2,000 Daltons in molecular weight) approved by the U.S. Food and Drug Administration, and / or listed in the 2020 U.S. Pharmacopeia or National Formulary. In an embodiment, those therapeutic agents or drugs are less than 1,000 Da in molecular weight. Suitable drugs include antibiotics and various immunosuppressive agents.

[0303] Suitable therapeutic agents that may be administered for the treatment of a variety of disorders with trispecific antibody construct or bispecific hemibody constructs include, but are not limited to, glucocorticoids and / or non-steroidal anti-inflammatory drugs (NSAIDs). Glucocorticoids (e.g., prednisone) are both anti-inflammatory and immunosuppressive, and accordingly may be useful for the treatment of, for example, autoimmune disease, GVHD, HVGD, and / or allergic reactions.

[0304] Other suitable therapeutic agents that may be administered with a trispecific antibody construct or bispecific hemibody constructs include, but are not limited to, one or more agents or antibodies directed against: B lymphocyte antigens, provided they do not compete with bispecific hemibody or trispecific antibody construct binding to the target cells. For example, antibodies against any marker appearing on the surface of a cell in the B cell lineage, including, but not limited to: ibritumomab tiuxetan, obinutuzumab, ofatumumab, and / or rituximab each of which bind to CD20; tafasitamab; ioncastuximab tesirine; loncastuximab; ensomafusp (which binds CD19 and CD137 / 41 BB), which bind to CD19; daratumumab which binds to CD38; and blinatumomab, which binds to both CD19 and CD3. Some additional antibodies binding to markers on the surface of cells in the B cell lineage that may be coadministered with the bispecific hemibodies or trispecific antibodies described herein. As indicted above, the coadministered antibodies should not compete with the binding of a bispecific hemibody construct or a trispecific antibody construct.Coadministered antibodies may be selected to bind a cell surface marker present on one or more cells that arebound by a bispecific hemibody construct or trispecific antibody construct. The presence of aa sequence capable of inducing ADCC, CDC, and / or ADCP in the coadministered antibodies may promote the cytotoxic attack on the target cells.

[0305] In addition to the antibody based biologies recited above, other anti-bodies including antibodies that antagonize B-cell Activating Factor of the Tumor Necrosis Factor Family, or “anti-BAFF”, including belimumab and / or antibodies against APRIL or "anti-APRIL may be coadministered with a bispecific antibody or bispecific hemibody of the present disclosure. Because both BAFF and APRIL support the survival and differentiation of B cells, administration of either or both of those antibodies may potentiate the depletion of cells in the B cell lineage by the bispecific hemibodies and trispecific antibodies described herein. See, e.g., Panzer, S.E., Transplantation, 104(1): e3-e4 (2020), doi: 10.1097 / TP.0000000000002993.

[0306] Diseases (e.g., disorders or conditions) that can be treated include autoimmune diseases resulting from the presence or overabundance of one or more cells of the B cell lineage that produce antibodies to a self-antigen. The disorders (diseases or conditions) that can be treated include allergies. The disorders (diseases or conditions) that can be treated also include transplant compatibility issues including GVHD and / or HVGD. Other disorders that can be treated include neoplasms (benign or malignant) involving clonal expansion of cell of the B cell lineage described in more detail below.

[0307] Autoimmune diseases treatable utilizing the constructs of the present disclosure include, but are not limited to, Addison's disease, autoimmune hepatitis, autoimmune thyroid disease, autoimmune bowel diseases (e.g., autoimmune ulcerative colitis, and Morbus Crohn or Crohn's disease), celiac disease, Crohn's disease, dermatomyositis, encephalomyelitis, Grave's disease, inflammatory bowel disease, juvenile idiopathic arthritis, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, Parkinson's disease, pemphigus, myositis / polymyositis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjogren's Syndrome, systemic lupus erythematosus (SLE), thrombocytopenic purpura, Type 1 Diabetes (T1D), ulcerative colitis, vasculitis, and vitiligo. Additional autoimmune and other diseases are described below. c) Dosages

[0308] Suitable dosages for patients can be determined by an attending physician or other qualified medical personnel, based on various clinical factors. Dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular polypeptide or nucleic acid to be administered, sex of the patient, time, and route of administration, general health, and other drugs being administered concurrently. Dose levels may also vary as a function of the bispecific hemibody or trispecific antibody constructs, the severity of the symptoms and the susceptibility of the patient to side effects. A bispecific hemibody or trispecific antibody construct described herein may be administered in amounts between 1 microgram (pig) / kg body weight and 20 mg / kg body weight per dose. Doses may be from 0.1 pig / kg body weight to 1.0 mg / kg body weight, or from 0.1 mg / kg body weight to 1 .0 mg / kg body weight. Doses may be from 0.5 mg / kg body weight to 5 mg / kg body weight or from 5 mg / kg body weight to 10 mg / kg body weight. Doses may be from 10 mg / kg body weight to 15 mg / kg body weight, or from 15 mg / kg body weight to 20 mg / kg body weight. Doses below 1 pig / kg body weight or above 20 mg / kg are envisioned, especially considering the aforementioned factors.

[0309] In some cases, multiple doses of a bispecific hemibody or trispecific antibody construct are administered. The frequency of administration can vary depending on any of a variety of factors, e.g., severity of the symptoms, patient response, etc. In some instances, a bispecific hemibody or trispecific antibody construct is administered less frequently than once per month, e.g., once every two, three, four, six or more months, once per year, or once per month. Alternatively, doses may be administered more frequently than once per month, e.g, twice per month, three times per month, every other week (qow), one every three weeks, once every four weeks, once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid).

[0310] The duration of administration of a bispecific hemibody or trispecific antibody construct, e.g., the period of time over which administration occurs, can vary, depending on any of a variety of factors, e.g., patient response, etc. For example, a bispecific hemibody or trispecific antibody construct can be administered over a period of time ranging from about one day to about one week, from about two weeks to about four weeks, from about one month to about two months, from about two months to about four months, from about four months to about six months, from about six months to about eight months, from about eight months to about 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years, or more, including continued administration for the patient's life.

[0311] Where treatment is of a finite duration, following successful treatment it may be desirable to have the patient undergo periodic maintenance therapy to prevent the recurrence of the disease state. During maintenance therapy a bispecific hemibody or trispecific antibody construct may be administered in maintenance doses, ranging from those recited above, i.e., 1 pig / kg body weight and 20 mg / kg body weight per dose. Maintenance doses may be from 0.1 pig / kg body weight to 1.0 mg / kg body weight or from 0.1 mg / kg body weight to 1.0 mg / kg body weight. Maintenance doses may be from 0.5 mg / kg body weight to 5 mg / kg body weight or from 5 mg / kg body weight to 10 mg / kg body weight. Maintenance doses may be from 10 mg / kg body weight to 15 mg / kg body weight or from 15 mg / kg body weight to 20 mg / kg body weight. Maintenance doses less than 1 pig / kg body weight or greater than 20 mg / kg of body weight are also possible. Maintenance dose administration may be once per month, once every two months, once every three months, once every four months, once every five months, once every six months, or once a year. d) Routes of Administration

[0312] A bispecific hemibody or trispecific antibody construct, or a nucleic acid or recombinant expression vectors comprising nucleic acids encoding one or more polypeptides of a bispecific hemibody or trispecific antibody construct, may be administered to a patient using suitable and available route for drug delivery. Delivery may be effected by in vivo and / or in vitro methods, as well as systemic and localized routes of administration. A bispecific hemibody or trispecific antibody construct can be administered to a patient using any available conventional methods and routes suitable for delivery of conventional drugs, including systemic or localized routes. In general, routes of administration contemplated for use in a method of treating a patient include, but are not necessarily limited to, parenteral (intravenous, intramuscular, or subcutaneous injection), peritoneal, enteral, and inhalational routes.

[0313] Conventional and pharmaceutically acceptable routes of administration include intravenous, intramuscular, intratracheal, subcutaneous, intradermal, topical application, intravenous, intra-arterial, intralymphatic, rectal, nasal, oral, , and other enteral and parenteral routes of administration. Of these, intravenous, intramuscular andsubcutaneous may be more commonly employed. Biispecific hemibodies, trispecific antibodies, nucleic acids, and expression vectors encoding them may be administered, for example, intravenously. Routes of administration may be combined, if desired, or adjusted depending upon, for example, depending on the bispecific hemibodies, trispecific antibodies, nucleic acids, or expression vectors can be administered in a single dose or in multiple doses.

[0314] A bispecific hemibody or trispecific antibody construct, or one or more nucleic acids or recombinant expression vectors comprising nucleic acid sequences encoding one or more polypeptides of bispecific hemibody or trispecific antibody construct (e.g., all polypeptides), may also be contacted with cells or blood in vitro, and the cells administered or returned to a patient. For example, white blood cells may be separated from a patient's blood by leukapheresis and contacted with a bispecific hemibody or trispecific antibody of the present disclosure. After a period of time the remaining white cells, which should be substantially depleted of cells in the B cell lineage targeted by the bispecific hemibody or trispecific antibody construct, may be returned to the patient. An intermediate step of removing the debris of cells that have been subject to the action of cytotoxic effector cells through the engagement of the bispecific hemibody or trispecific antibody construct may be included in the process. e) Disease states

[0315] As previously indicated, disorders (diseases or conditions) that can be treated employing the bispecific hemibodies or trispecific antibody constructs described herein include diseases resulting from the presence or overabundance of one or more cells of the B cell lineage. Those diseases may be autoimmune disorders, wherein those cells produce antibodies to a self-antigen, allergic reactions when those cells produce antibodies to allergens, or neoplasms (benign or malignant) when the number of those cells increases because they fail to stop reproducing and / or succumb to normal apoptotic processes. Other diseases that can be treated also include those involving tissue transplantation, including graft vs host disease and / or host vs graft disease (GVHD and / or HVGD).

[0316] Because the bispecific hemibodies and trispecific antibodies of the preset disclosure recruit cytotoxic effectors cells and direct the depletion or ablation of cells within the B cell lineage in a manner that is not antigen specific, diverse diseases related to cells in that lineage may be treated with those molecules. Those diseases include autoimmune disease, allergic reactions, neoplasms, and transplant-related graft host interactions.(1) Autoimmune Diseases

[0317] Autoimmune diseases that may be treated with the bispecific hemibodies or trispecific antibody constructs described herein include, but are not limited to: Addison disease (Morbus Addison), Addison's anemia (Morbus Biermer), alopecia areata, autoimmune hemolytic anemia ), antiphospholipid syndrome, autoimmune arthritis, arteriitis temporalis, Takayasu arteriitis, autoimmune chronic gastritis, autoimmune infertility, autoimmune inner ear disease (Al ED), Basedow's disease (Morbus Basedow), Bechterew's disease (ankylosing spondylitis), Behcet's syndrome (Morbus Behcet), bowel diseases (e.g., autoimmune ulcerative colitis, and Morbus Crohn or Crohn's disease), autoimmune cardiomyopathy, bullous pemphigoid, celiac disease, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, chronic polyarthritis, cicatricial pemphigoid, Cogan syndrome, CREST syndrome (syndrome with Calcinosis cutis, Raynaud phenomenon, sclerodactyly and telangiectasia), dermatitis herpetiformis, dermatologic autoimmune diseases, dermatomyositis, eosinophilicgranulomatosis with polyangiitis, essential mixed cryoglobulinemia, fibromyalgia, fibromyositis, Goodpasture syndrome (anti-GBM mediated glomerulonephritis), Guillain-Barre syndrome, hematologic autoimmune diseases, Hashimoto thyroiditis, hemophilia, acquired hemophilia, autoimmune hepatitis, idiopathic pulmonary fibrosis, idiopathic dilated cardiomyopathy, idiopathic thrombocytopenic purpura, immuno-thrombocytopenic purpura (, IgA nephropathy, juvenile rheumatoid arthritis (Morbus Still, Still syndrome), Lambert-Eaton syndrome, systemic lupus erythematosus, discoid lupus erythematosus, Lyme arthritis (Lyme disease, borrelia arthritis), mixed connective tissue disease, multiple sclerosis, myasthenia gravis, myositis, polymyositis, neural autoimmune diseases, Parkinson's disease, pemphigus foliaceus, pemphigus vulgaris, polyglandular (autoimmune) syndrome (PGA syndrome, polymyalgia rheumatica, primary agammaglobulinemia, primary autoimmune cholangitis, progressive systemic sclerosis, Raynaud disease, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, Type 1 diabetes (T1D), autoimmune uveitis, vasculitis, vitiligo, and Wegner's disease.

[0318] Autoimmune diseases that may be treated with the bispecific hemibodies or trispecific antibody constructs described herein include, but are not limited to: Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune encephalomyelitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune-associated infertility, autoimmune polyendocrine syndrome, autoimmune thrombocytopenic purpura, bullous pemphigoid, celiac disease, Crohn's disease, dermatomyositis, Goodpasture's syndrome, glomerulonephritis, Grave's disease, inflammatory bowel disease, juvenile idiopathic arthritis, autoimmune thyroid disease (Hashimoto's thyroiditis), mixed connective tissue disease, multiple sclerosis, myasthenia gravis, Parkinson's disease, pemphigus (e.g., pemphigus vulgaris), pernicious anemia, myositis / poly myositis, psoriasis, psoriatic arthritis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus (SLE), Type 1 diabetes (T1D), uveitis, ulcerative colitis, vasculitis, or vitiligo.

[0319] Autoimmune diseases that may be treated with the bispecific hemibodies or trispecific antibody constructs described herein include but are not limited to: Addison's disease, autoimmune hepatitis, autoimmune thyroid disease, celiac disease, Crohn's disease, dermatomyositis, encephalomyelitis, Grave's disease, inflammatory bowel disease, juvenile idiopathic arthritis, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, Parkinson's disease, pemphigus foliaceus, pemphigus vulgaris, myositis / polymyositis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjogren's Syndrome, systemic lupus erythematosus, thrombocytopenic purpura, Type 1 diabetes (T1D), ulcerative colitis, vasculitis, and vitiligo.

[0320] Autoimmune diseases that may be treated with the bispecific hemibodies or trispecific antibody constructs described herein include, but are not limited to, Type 1 diabetes (T1D), and celiac disease.

[0321] Autoimmune diseases that may be treated with the bispecific hemibodies or trispecific antibody constructs described herein include, but are not limited to, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's Syndrome, or myasthenia gravis.(2) Allergies

[0322] Allergic reactions may arise to numerous allergens and identifying the specific epitopes or group of epitopes responsible for an individual patient's allergic reactions, while possible, is presently beyond the scope of typical personalized medicine. Because cells in the B cell lineage, including plasma cells secreting IgE, can be targeted bythe bispecific hemibodies and trispecific antibody constructs described herein, they are capable of suppressing allergic reactions. Due to the time required for recruitment and activation of cytotoxic effector cells, the bispecific hemibodies and trispecific antibodies of described herein are, by themselves, are not suitable for the treatment of immediately life threatening allergic reactions or anaphylactic shock symptoms. The bispecific hemibodies and trispecific antibodies of described herein may be useful to treat patients susceptible to those types of responses prophylactically. The bispecific hemibodies and trispecific antibody constructs described herein may be administered concurrently or subsequent to agents such as epinephrine to treat immediate life threatening symptoms and / or an antihistamine (e.g., diphenhydramine or cetirizine), a glucocorticoid (e.g., cortisone), and / or or beta-agonist (e.g., albuterol).

[0323] Allergenic reactions to allergens that may be treated include, but are not limited to, reactions to peanuts, tree nuts, plant pollens, latex antigens, and the like. Examples of allergens from peanuts (Arachis hypogaea L.) that many be treated include proteins and epitope presenting peptides of proteins in seven families that have been reported as the major peanut allergens: cupins (Ara h 1, Ara h 3), 2S albumins (Ara h 2, Ara h 6, Ara h 7), profilin (Ara h 5), PR -10 (Ara h 8), nsLTP type 1 and type 2 (Ara h 9, Ara h 16, Ara h 17), oleosins (Ara h 10, Ara h 11, Ara h 14, Ara h 15), and defensins (Ara h 12, Ara h 13). See, e.g., Luparelli et al. Foods. 2022 Mar; 11(5): 728. The allergic reactions may be to a peanut allergen selected from Ara h 1, Ara h 2, Ara h 3, and Ara h 6 h.

[0324] Other allergic reactions that may be treated include allergic reactions to proteins from Hymenoptera antigens such as venom proteins (e.g., those found in bee and wasp venoms such as phospholipase A2, melittin, "antigen 5”, and hyaluronidases).(3) Neoplasms

[0325] Neoplasms involving expansion of one (clonal expansion) or more cell of the B cell lineage include leukemias and lymphomas such as non-Hodgkin lymphomas (NHL), diffuse Large B Cell lymphoma (DLBCL, follicular lymphoma, mantle cell lymphoma and chronic lymphocytic leukemia (CLL). Neoplasms involving expansion (e.g., clonal expansion) of B cells and / or plasma cells include monoclonal gammopathy of undetermined significance (MGUS) plasmacytoma, and multiple myeloma). By way of example, bispecific hemibodies or trispecific antibody constructs may be able to target myelomas by binding to one or more, or two or more cell surface antigens selected from the group consisting of CD200, CD33, CD38, CD81, FCRLS, ICAM1, KIT, and NCAM1. Bispecific hemibodies or trispecific antibody construct elements (e.g., a nanobody or scFv aa sequence) targeting any one or more, or two or more of those of those cell surface markers may be combined with another element targeting plasma cells, such as BCMA, for the targeting and treatment of myelomas. Other noncancerous B cell neoplasms that may be treated using bispecific hemibodies or trispecific antibody constructs include, but are not limited to, lymph adenoma, splenic marginal zone hyperplasia, and Monoclonal Gammopathy of Undetermined Significance (MGUS).(4) GVHD or HVGD

[0326] GVHD and HGVD are complex immune responses that involve recognition of a set of numerous antigens that will be different for each patient and the specific tissue graft. Accordingly, those conditions cannot be treated effectively by antigen specific agents. In contrast to antigen specific agents, the bispecific hemibodies and trispecificantibody constructs described herein have the ability to deplete or ablate populations of cell in the B cell lineage (including its plasma cell sublineage) and permit treatment of disorders such as GVHD and HVGD (and allergies) involving multiple antigens. Bispecific hemibodies and / or trispecific antibody constructs that target, for example, plasma cells and / or activated B cells may be employed in the treatment of HVGE and GVHD. The bispecific hemibodies and / or trispecific antibody constructs may be administered as part of a treatment that includes the mainline drugs used to prevent transplant rejection (e.g., mycophenolate, azathioprine, cyclosporine, prednisone, sirolimus, calcineurin inhibitors, tacrolimus, mTOR inhibitors, and / or everolimus) as a combination therapy. The combination therapy may involve the administration of the bispecific hemibodies and / or trispecific antibody constructs prior to, concurrently (e.g., separately or in combination with), or subsequent to, the mainline drugs. . f) Patients Suitable for Treatment

[0327] Patients suitable for treatment are mammals for whom diagnosis, treatment, or therapy is desired. Mammals include humans and non-human primates. In addition, mammals include rodents (e.g., rats; mice), lagomorphs (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, and the like), felines, canines, etc. In any given trispecific antibody construct, and any given bispecific, trispecific, tetraspecific, or multispecific hemibody use for treatment of a patient, all aa sequences (e.g., antibody sequences) binding to markers on cells of the B cell lineage and all aa sequences binding to CEC-SAs bind to markers and cell surface antigens from the patient's species (e.g., in the patient itself). Another way of saying this is that all paratopes on the hemibody and trispecific antibody constructs disclosed herein may bind to epitopes of antigens found in the patient's species (e.g., in the patient itself).Accordingly, in trispecific antibody constructs, and bispecific, trispecific, tetraspecific, or multispecific hemibodies intended for use with human cells or in methods of treating a human patient, the aa sequences that bind to markers on cells of the B cell lineage and aa sequences that bind to CEC-SAs bind the human markers or human surface antigens.

[0328] Patients suitable for treatment include, but are not limited to, individuals with allergic reactions, GVHD, HVGD, and / or autoimmune diseases. Other patients suitable for treatment include individuals with neoplasms involving cells in the B cell lineage. Patients suitable for treatment include, but are not limited to, patients who have been provided other treatments but who partially, substantially, or wholly failed to respond to the treatment.

[0329] Individuals with disorders that affect cytotoxic effector cell populations including, but not limited to, individuals with HIV, DiGeorge Syndrome (DGS), Chromosomal Breakage Syndromes (CBSs), Ataxia- Telangiectasia, Wiskott-Aldrich Syndrome, or Omenn Syndrome, may not effectively respond to the bispecific hemibodies and / or trispecific antibody constructs described herein.VI. Certain Aspects1 . A bispecific hemibody comprising a first hemibody construct and a second hemibody construct, wherein:(I) the first hemibody construct comprises a first element 1' that binds a first marker expressed on the surface of a cell of B cell lineage (e.g., CD69 expressed on a B cell), and an antibody variable heavy chain (VH) 4 or variable light chain (V ) 4’ aa sequence of a hemibody that binds to a cytotoxic effector cell surface antigen (CEC-SA),wherein(a) the first element comprises an aa sequence (e.g., a VHH or scFV aa sequence), and / or(b) the first element comprises a VH aa sequence, VL aa sequence, or the complementarity determining regions (CDRs) of a VHor V aa sequence, wherein, when the first element comprises a VHor V aa sequence, or CDRs of a VHor V aa sequence, the first construct further comprises a polypeptide comprising the corresponding VH or VL aa sequence or CDR aa sequences of a corresponding VH or VL, and the VH and VL, or the CDR aa sequences of the VH and VL, form a paratope binding to the first marker; and(ii) the second hemibody construct comprises a second element 1" that binds a second marker expressed on the surface of a cell of B cell lineage (e.g., a B cell), and the corresponding VH or VL (4 or 4’) aa sequence of the hemibody that binds to a CEC-SA, wherein(a) the second element comprises an aa sequence (e.g., a VHH or scFV aa sequence), and / or(b) the second element comprises a VHaa sequence, VLaa sequence, or the CDRs of a VHor VLaa sequence, wherein, when the second element comprises a VH or VL aa sequence, or CDRs of a VH or VL aa sequence, the second construct further comprises a polypeptide comprising the corresponding VH or VL aa sequence or CDRs of a corresponding VH or VL, and the VH and VL, or the CDRs of the VH and VL, form a paratope binding to the second marker;(ill) the first and second hemibody constructs together comprise VH and VL aa sequences (4 and 4’) that form a CEC-SA binding paratope; and(iv) the first and / or second hemibody constructs optionally comprise one or more independently selected scaffold aa sequences and / or optionally one or more independently selected linker sequences. See, e.g., FIG. 1 A at constructs A and B and FIG. 1 B at A and B. The bispecific hemibody of aspect 1, wherein: the first hemibody construct comprises a first polypeptide 7 comprising a first scaffold aa 2 sequence and a second polypeptide 8 that comprises a second scaffold aa sequence 2’; and the second hemibody construct comprises a third polypeptide 9 comprising a third scaffold aa sequence 2 and a fourth polypeptide 10 that comprises a fourth scaffold aa sequence 2’; and wherein the aa sequence of each scaffold aa sequence is selected independently. See, e.g., FIG. 1 A, construct A. The bispecific hemibody of aspect 2, wherein:A. the first polypeptide 7 and second polypeptide 8 of the first hemibody construct associate (bind together) through interactions between the first and second scaffold aa sequences and are optionally covalently linked together through one or more disulfide bonds (e.g., between the first and second scaffold aa sequences); andB. the third polypeptide 9 and fourth polypeptide 10 of the second hemibody construct associate (bind together) through interactions between the third and fourth scaffold aa sequences and are optionally covalently linked together through one or more disulfide bonds (e.g. between the third and fourth scaffold aa sequences). See, e.g., FIG. 1 A, construct A. The bispecific hemibody of any of aspects 2-3, wherein:A. in the first hemibody construct:(I) the first polypeptide 7 of the first hemibody construct comprises in the N-terminal to C-terminal direction the first element and the first scaffold aa sequence 2, and(ii) the second polypeptide 8 of the first hemibody construct comprises in the N-terminal to C-terminal direction the second scaffold aa sequence 2’ and one of the variable heavy chain (VH) 4 or variable light chain (VL) 4’ aa sequences of the hemibody that binds to a CEC-SA; andB. in the second hemibody construct:(I) the third polypeptide 9 of the second hemibody construct comprises in the N-terminal to C-terminal direction the second element expressed on the surface of a cell of B cell lineage and the third scaffold aa sequence 2, and(II) the fourth polypeptide 10 of the second hemibody construct comprises in the N-terminal to C-terminal direction a fourth scaffold aa sequence 2’ and the other of the variable heavy chain (VH) 4 or variable light chain (VL) 4’ aa sequences of the hemibody not present in the second polypeptide of the first hemibody. See, e.g., FIG. 1A, construct A. The bispecific hemibody of any of aspects 2-3, wherein:A. in the first hemibody construct:(I) the first polypeptide 7 of the first hemibody construct comprises in the C-terminal to N-terminal direction the first element and the first scaffold aa sequence 2, and(ii) the second polypeptide 8 of the first hemibody construct comprises in the C-terminal to N-terminal direction the second scaffold aa sequence 2' and one of the variable heavy chain (VH) 4 and variable light chain (VL) 4’ aa sequences of the hemibody that binds to a CEC-SA; andB. in the second hemibody construct:(I) the third polypeptide 9 of the second hemibody construct comprises in the C-terminal to N-terminal direction the second element expressed on the surface of a cell of B cell lineage and the third scaffold aa sequence 2, and(ii) the fourth polypeptide 10 of the second hemibody construct comprises in the C-terminal to N-terminal direction the fourth scaffold aa sequence 2’ and the other of the variable heavy chain (VH) 4 and variable light chain (VL) 4’ aa sequences of the hemibody not present in the second polypeptide of the first hemibody. See, e.g., FIG. 1A, construct A. The bispecific hemibody of any of aspects 2-5, further comprising a third element 1"' that binds a third marker expressed on the surface of a cell of B cell lineage as part of the first, second, third or fourth polypeptide, wherein(a) the third element comprises an aa sequence, and / or(b) the third element comprises a VH aa sequence, VL aa sequence, or the CDRs of a VH or VL aa sequence, wherein, when the third element comprises a VH or VL aa sequence, or CDRs of a VH or VL aa sequence, the construct comprising the third element further comprises a polypeptide comprising the corresponding VHor V aa sequence or CDRs of a corresponding VHor V , and the VHand V , or the CDRs of the VH and VL, form a paratope binding to the third marker. The bispecific hemibody of aspect 6, wherein the third element 1”’ is part of (I) the first or second polypeptide, or (ii) the third or fourth polypeptide. The bispecific hemibody of aspect 7, wherein:A. in the first hemibody construct:(I) the first polypeptide 7 of the first hemibody construct comprises in the N-terminal to C-terminal direction the first element and the first scaffold aa sequence; and(ii) the second polypeptide 8 of the first hemibody construct comprises in the N-terminal to C-terminal direction the third element, the second scaffold aa sequence and one of the variable heavy chain (VH) 4 and variable light chain (VL) 4’ aa sequences of the hemibody that binds to a CEC-SA; andB. in the second hemibody construct:(I) the third polypeptide 9 of the second hemibody construct comprises in the N-terminal to C-terminal direction the second element expressed on the surface of a cell of B cell lineage and the third scaffold aa sequence; and(ii) the fourth polypeptide 10 of the second hemibody construct comprises in the C-terminal to N-terminal direction the fourth scaffold aa sequence and the other of the variable heavy chain (VH) 4 and variable light chain (VL) 4’ aa sequences of the hemibody not present in the second polypeptide of the first hemibody (the first and second hemibody constructs together comprise a VH 4 and VL 4' pair that form a CEC-SA paratope). The bispecific hemibody of aspect 7, wherein:A. in the first hemibody construct(I) the first polypeptide 7 of the first hemibody construct comprises in the C-terminal to N-terminal direction the first element and the first scaffold aa sequence; and(ii) the second polypeptide 8 of the first hemibody construct comprises in the C-terminal to N-terminal direction the third element, the second scaffold aa sequence, and one of the variable heavy chain (VH) 4 and variable light chain (VL) 4’ aa sequences of the hemibody that binds to a CEC-SA; andB. in the second hemibody construct:(I) the third polypeptide 9 of the second hemibody construct comprises in the C-terminal to N-terminal direction the second element and the third scaffold aa sequence; and(ii) the fourth polypeptide 10 of the second hemibody construct comprises in the C-terminal to N-terminal direction the fourth scaffold aa sequence and the other of the variable heavy chain (VH) 4 and variablelight chain (VL) 4’ aa sequences of the hemibody not present in the second polypeptide of the first hemibody. e bispecific hemibody of any of aspects 2-5, further comprising: a third element 1"' that binds a third marker expressed on the surface of a cell of B cell lineage as part of the first 7 or second 8 polypeptide; wherein(a) the third element comprises an aa sequence, and / or(b) the third element comprises a VH aa sequence, VL aa sequence, or the CDRs of a VH or VL aa sequence, wherein, when the third element comprises a VH or VL aa sequence, or CDRs of a VH or VL aa sequence, the construct comprising the third element further comprises a polypeptide comprising the corresponding VH or VL aa sequence or CDRs of a corresponding VH or VL, and the VH and VL, or the CDRs of the VH and VL, form a paratope binding to the third marker; and a fourth element 1"" that binds a fourth marker expressed on the surface of a cell of B cell lineage as part of the third 9 or fourth 10 polypeptide, wherein(a) the fourth element comprises an aa sequence, and / or(b) the fourth element comprises a VH aa sequence, VL aa sequence, or the CDRs of a VH or VL aa sequence, wherein, when the fourth element comprises a VH or VL aa sequence, or CDRs of a VH or VL aa sequence, the construct comprising the fourth element further comprises a polypeptide comprising the corresponding VH or VL aa sequence or CDRs of a corresponding VH or VL, and the VH and VL, or the CDRs of the VH and VL, form a paratope binding to the fourth marker. The bispecific hemibody of aspect 10, wherein (I) the third element 1"' is part of the first 7 or second 8 polypeptide; and / or (ii) the fourth element 1"" is part of the third 9 or fourth 10 polypeptide. See, e.g. FIG. 5 at A. The bispecific hemibody of aspect 11, wherein:A. in the first hemibody construct(I) the first polypeptide 7 comprises in the N-terminal to C-terminal direction the first element and the first scaffold aa sequence,(ii) the second polypeptide 8 comprises in the N-terminal to C-terminal direction the third element, the second scaffold aa sequence and one of the variable heavy chain (VH) 4 and variable light chain (VL) 4’ aa sequences of the hemibody that binds to a CEC-SA; andB. in the second hemibody construct(I) the third polypeptide 9 of the second hemibody construct comprises in the N-terminal to C-terminal direction the second element expressed on the surface of a cell of B cell lineage and the third scaffold aa sequence, and(ii) the fourth polypeptide 10 comprises in the N-terminal to C-terminal direction the fourth element, the fourth scaffold aa sequence and the other of the variable heavy chain (VH) 4 and variable light chain (VL) 4’ aa sequences of the hemibody not present in the second polypeptide of the first hemibody. The bispecific hemibody of aspect 11, wherein:A. in the first hemibody construct(I) the first polypeptide 7 comprises in the C-terminal to N-terminal direction the first element and the first scaffold aa sequence,(ii) the second polypeptide 8 comprises in the C-terminal to N-terminal direction the third element, the second scaffold aa sequence and one of the variable heavy chain (VH) 4 and variable light chain (VL) 4’ aa sequences of the hemibody that binds to a CEC-SA; andB. in the second hemibody construct:(I) the third polypeptide 9 of the second hemibody construct comprises in the C-terminal to N-terminal direction the second element expressed on the surface of a cell of B cell lineage and the third scaffold aa sequence, and(ii) the fourth polypeptide 10 of the second hemibody construct comprises in the C-terminal to N-terminal direction the fourth element, the fourth scaffold aa sequence and the other of the variable heavy chain (VH) 4 and variable light chain (V ) 4’ aa sequences of the hemibody not present in the second polypeptide of the first hemibody. See FIG. 5 at A. The bispecific hemibody of any of aspects 2-13, wherein the first scaffold sequence and the second scaffold sequence of the first hemibody construct and / or the third scaffold sequence and the fourth scaffold aa sequence of the second hemibody construct are non-immunoglobulin aa sequences or immunoglobulin aa sequences optionally comprising one or more substitutions enhancing or diminishing antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC) effector functions. See, e.g., FIG. 1A construct A. The bispecific hemibody of any of aspects 2-14, wherein each scaffold aa sequence is selected from the group consisting of: XTEN, leucine zipper, and albumin polypeptide immunoglobulin aa sequences (e.g. immunoglobulin CH2-CH3, C or CH1 sequences). See, e.g., FIG. 1A, construct A. The bispecific hemibody of any of aspects 2-15, wherein the first scaffold sequence and the second scaffold sequence of the first hemibody construct and / or the third scaffold sequence and the fourth scaffold aa sequence of the second hemibody construct are a pair of interspecific aa sequences. See, e.g., FIG. 1 A, construct A. The bispecific hemibody of any of aspects 2-16, wherein: the first 7 and second 8 polypeptides associate through interactions between the first and second scaffold sequences and are optionally covalently bound together through one or more disulfide bonds (e.g., between the scaffold aa sequences); and the third 9 and fourth 10 polypeptides of the second hemibody construct associate through interactions between the third and fourth scaffold sequences and are optionally covalently bound together through one or more disulfide bonds (e.g., between the scaffold aa sequences). See, e.g., FIG. 1A, construct A.The bispecific hemibody of any of aspects 15-17, wherein: the first scaffold aa sequence and second scaffold aa sequence are a first pair of interspecific immunoglobulin aa sequences; and the third scaffold aa sequence and the fourth scaffold aa sequence of the second hemibody construct are a second pair of interspecific immunoglobulin aa sequences. See, e.g., FIG. 1A, construct A. The bispecific hemibody of aspect 18, wherein the first and second pairs of interspecific immunoglobulin aa sequences are selected independently from knob-in-hole without (KiH) or with (KiHs-s) a stabilizing disulfide bond, HA-TF, ZW-1, 7.8.60, DD-KK, EW-RVT, EW-RVTs-s, and A107 sequences. See, e.g., FIG. 1A, construct A. The bispecific hemibody of aspect 18 or 19, wherein the first and second pairs of interspecific immunoglobulin aa sequences are selected independently from KiH or KiHs-s. See, e.g., FIG. 1 A, construct A. The bispecific hemibody of aspect 1 , wherein:(I) the first hemibody construct comprises a first polypeptide 77 comprising both the first element and either the antibody variable heavy chain (VH) 4 or variable light chain (VL) 4’ aa sequence of the first hemibody construct;(ii) the second hemibody construct comprises a second polypeptide 72 comprising both the second element and the corresponding VH or VL (4 or 4’) aa sequence of the hemibody that binds to a CEC-SA of the second hemibody construct;(ill) the first and second constructs together comprise the VH and VL aa sequences (4 and 4’) that form a CEC- SA binding paratope; and(iv) the first and / or second construct optionally comprise one or more independently selected scaffold aa sequences. See, e.g., FIG. 1A, construct B. The bispecific hemibody of aspect 21 , wherein: the first polypeptide 77 comprises a first scaffold aa and a second scaffold aa sequence; the second polypeptide 72 comprises a third scaffold aa sequence and a fourth scaffold aa sequence; and the aa sequence of each scaffold aa sequence is optionally selected independently. See e.g. FIG. 1 A construct B. The bispecific hemibody of aspect 22, wherein:A. the first and second scaffold aa sequences of the first polypeptide 77 associate (bind together) and are optionally covalently linked by one or more disulfide bonds (e.g. between the first and second scaffold aa sequences); andB. the third and fourth scaffold aa sequences of the second polypeptide 72 associate (bind together) and are optionally covalently linked by one or more disulfide bonds (e.g. between the third and fourth scaffold aa sequences). See, e.g., FIG. 1A, construct B. The bispecific hemibody of any of aspects 22-23, wherein:A. the first polypeptide 11 comprises in the N-terminal to C-terminal direction (i) the first element, (ii) the first scaffold aa sequence, (ill) the second scaffold aa sequence, and (iv) one of the variable heavy chain (VH) 4 and variable light chain (VL) 4' aa sequences of the hemibody that binds to a CEC-SA; andB. the second polypeptide 12 comprises in the N-terminal to C-terminal direction (I) the second element,(ii) the third scaffold aa sequence, (ill) the fourth scaffold aa sequence, and the other of the variable heavy chain (VH) 4 and (iv) variable light chain (V ) 4’ aa sequence of the hemibody not present in the first polypeptide. See, e.g., FIG. 1A at B. The bispecific hemibody of any of aspects 22-23, wherein:A. the first polypeptide 11 comprises in the C-terminal to N-terminal direction (I) the first element, (ii) the first scaffold aa sequence, (ill) the second scaffold aa sequence, and (iv) one of the variable heavy chain (VH) 4 and variable light chain (VL) 4’ aa sequences of the hemibody that binds to a CEC-SA; andB. the second polypeptide 12 comprises in the C-terminal to N-terminal direction (I) the second element, (ii) the third scaffold aa sequence, (ill) the fourth scaffold aa sequence, and (iv) the other of the variable heavy chain (VH) 4 and variable light chain (VL) 4’ aa sequences of the hemibody not present in the first polypeptide. See, e.g., FIG. 1A at B. The bispecific hemibody of any of aspects 21-25, further comprising a third element 1"' that binds a third marker as part of the first 11 or second 12 polypeptide, wherein(a) the third element comprises an aa sequence; and / or(b) the third element comprises a VHaa sequence, VLaa sequence, or the CDRs of a VHor VLaa sequence, wherein, when the third element comprises a VH or VL aa sequence, or CDRs of a VH or VL aa sequence, the construct comprising the third element further comprises a polypeptide comprising the corresponding VH or VL aa sequence or CDRs of a corresponding VH or VL, and the VH and VL, or the CDRs of the VH and VL, form a paratope binding to the third marker. The bispecific hemibody of aspect 26, wherein:A. the first polypeptide 11 comprises in the N-terminal to C-terminal direction (I) the first element T, (ii) the first scaffold aa sequence, (i...

Claims

Claims1 . A bispecific hemibody comprising a first hemibody construct and a second hemibody construct, wherein:(i) the first hemibody construct comprises first element that binds a first marker expressed on the surface of a cell of B cell lineage, and an antibody variable heavy chain (VH) 4 or variable light chain (VL) 4’ aa sequence of a hemibody that binds to a cytotoxic effector cell surface antigen (CEC-SA), wherein(a) the first element comprises an aa sequence, and / or(b) the first element comprises a VH aa sequence, VL aa sequence, or the complementarity determining regions (CDRs) of a VHor V aa sequence, wherein when the first element comprises a VHor V aa sequence, or CDRs of a VHor V aa sequence, the first construct further comprises a polypeptide comprising the corresponding VH or VL aa sequence or CDR aa sequences of a corresponding VH or VL, and the VH and VL, or the CDR aa sequences of the VH and VL, form a paratope binding to the first marker;(ii) the second hemibody construct comprises a second element 1" that binds a second marker expressed on the surface of a cell of B cell lineage, and the corresponding VH or VL (4 or 4’) aa sequence of the hemibody that binds to a CEC-SA, wherein(a) the second element comprises an aa sequence, and / or(b) the second element comprises a VHaa sequence, VLaa sequence, or the CDRs of a VHor VLaa sequence, wherein when the second element comprises a VH or VL aa sequence, or CDRs of a VH or VL aa sequence, the second construct further comprises a polypeptide comprising the corresponding VH or VL aa sequence or CDRs of a corresponding VH or VL, and the VH and VL, or the CDRs of the VH and VL, form a paratope binding to the second marker;(ill) the first and second hemibody constructs together comprise VH and VL aa sequences (4 and 4’) that form a CEC-SA binding paratope; and(iv) the first and / or second construct optionally comprise one or more independently selected scaffold aa sequences and / or optionally one or more independently selected linker sequences..

2. The bispecific hemibody of claim 1 , wherein: the first hemibody construct comprises a first polypeptide comprising a first scaffold aa sequence and a second polypeptide that comprises a second scaffold aa sequence; and the second hemibody construct comprises a third polypeptide comprising a third scaffold aa sequence and a fourth polypeptide that comprises a fourth scaffold aa sequence; and wherein the aa sequence of each scaffold aa sequence is selected independently.

3. The bispecific hemibody of claim 2, further comprising a third element 1"' that binds a third marker expressed on the surface of a cell of B cell lineage as part of the first, second, third or fourth polypeptide, wherein(a) the third element comprises an aa sequence, and / or(b) the third element comprises a VH aa sequence, VL aa sequence, or the CDRs of a VH or VL aa sequence, wherein when the third element comprises a VH or VL aa sequence, or CDRs of a VH or VL aa sequence, the construct comprising the third element further comprises a polypeptide comprising the corresponding VHor VLaa sequence or CDRs of a corresponding VHor VL, and the VHand VL, or the CDRs of the VHand VL, form a paratope binding to the third marker.

4. The bispecific hemibody of claim 3, wherein the third element is part of (I) the first or second polypeptide, or (ii) the third or fourth polypeptide.

5. The bispecific hemibody of claim 2, further comprising: a third element 1"' that binds a third marker expressed on the surface of a cell of B cell lineage as part of the first or second polypeptide wherein(a) the third element comprises an aa sequence, and / or(b) the third element comprises a VHaa sequence, VLaa sequence, or the CDRs of a VHor VLaa sequence, and wherein when the third element comprises a VH or VL aa sequence, or CDRs of a VH or VL aa sequence, the construct comprising the third element further comprises a polypeptide comprising the corresponding VH or VL aa sequence or CDRs of a corresponding VH or VL, and the VH and VL, or the CDRs of the VH and VL, form a paratope binding to the third marker; and a fourth element 1"" that binds a fourth marker expressed on the surface of a cell of B cell lineage as part of the third or fourth polypeptide, wherein(a) the fourth element comprises an aa sequence, and / or(b) the fourth element comprises a VHaa sequence, VLaa sequence, or the CDRs of a VHor VLaa sequence, and wherein when the fourth element comprises a VH or VL aa sequence, or CDRs of a VH or VL aa sequence, the construct comprising the fourth element further comprises a polypeptide comprising the corresponding VH or VL aa sequence or CDRs of a corresponding VH or VL, and the VH and VL, or the CDRs of the VH and VL, form a paratope binding to the fourth marker.

6. The bispecific hemibody of claim 5, wherein the third element 1"' is part of (I) the first or second polypeptide; and / or the fourth element 1"" is part of the (I) third or (ii) fourth polypeptide.

7. A trispecific antibody construct comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and second polypeptide together comprise as elements:(A) (I) a first element 1' that binds a first marker expressed on the surface of a cell of B cell lineage, and(ii) a second element 1" that binds a second marker expressed on the surface of a cell of B cell lineage; and(B) either(i) an aa sequence comprising an antibody variable heavy chain (VH) aa sequence 4 of an antibody that binds a CEC-SA, and an aa sequence comprising an antibody variable light chain (VL) aa sequence 4’ of an antibody that binds a CEC-SA, or(ii) a single chain antibody sequence (e.g., an scFv or nanobody aa sequence) that binds a CEC-SA; wherein(a) either the first element or second element comprises an aa sequence, and / or(b) the first element comprises a VH aa sequence, VL aa sequence, or the complementarity determining regions (CDRs) of a VH or VL aa sequence, wherein when the first element or second element comprises a VH or VL aa sequence, or CDRs of a VH or VL aa sequence, the trispecific antibody construct further comprises a polypeptide comprising the corresponding VH or VL aa sequence or CDRs of a corresponding VH or VL, and together the VH and VL, or together the CDRs of the VH and VL, form a paratope binding to the first marker or second marker; and the VHand VLaa sequences that bind a CEC-SA together form a CEC-SA binding paratope, and the first and / or second polypeptides optionally comprise an independently selected scaffold aa sequences that can bind together forming a heterodimer comprising the first polypeptide and second polypeptide, and the first and / or second polypeptides each optionally comprise one or more independently selected linker sequences.

8. The trispecific antibody construct of claim 7, wherein: the first polypeptide comprises a first scaffold aa sequence and the second polypeptide comprises a second scaffold aa sequence.

9. The trispecific antibody construct of claim 8, wherein the first polypeptide and second polypeptide associate (bind together) through interactions between the first and second scaffold aa sequences and are optionally covalently linked together through one or more disulfide bonds between the first and second scaffold aa sequences.

10. The bispecific hemibody or trispecific antibody construct of any of claims 1-9, wherein each scaffold aa sequences is selected from the group consisting of: immunoglobulin (CH2-CH3) constant region aa sequences, XTEN, leucine zipper, and albumin polypeptide.

11. The bispecific hemibody or trispecific antibody construct of claims 1-9, wherein:A)(I) the first hemibody construct and / or second hemibody construct comprise one or more independently selected linkers; or(ii) the trispecific antibody construct comprises one or more independently selected linkers; and / orB)(I) the first hemibody construct and / or second hemibody construct comprise one or more independently selected affinity tags (5) optionally joined to the first hemibody construct and / or second hemibodyconstruct by a linker that may comprise one or more aa sequences cleavable by independently selected site-specific proteases, or(II) the trispecific antibody construct comprises one or more independently selected affinity tags 5 optionally joined to the trispecific antibody construct by a linker that may comprise one or more aa sequences cleavable by independently selected site-specific proteases.

12. The bispecific hemibody or trispecific antibody construct of any of claims 1-9, wherein the first marker is a marker appearing on the surface of a cell in the B cell lineage transiently and / or continuously at an increased level following activation by exposure to an antigen relative to either (I) the level of the first marker prior to antigen exposure or(II) the level of the first marker in the corresponding naive B cells prior to antigen exposure.

13. The bispecific hemibody or trispecific antibody construct of any of claims 1-9 wherein the first element and the second element bind to markers selected from the group consisting of: (I) first marker CD 19 and second marker CD69, (ii) first marker CD19 and second marker CD86, (ill) first marker CD19 and second marker CD19, and (iv) first marker BCMA and second marker BCMA.

14. The bispecific hemibody or trispecific antibody construct of any of claims 1-9 wherein: the first element and the second element bind to first and second markers selected from the group consisting of BCMA (CD269), mlg (membrane associated immunoglobulins), CD1d, CD5, CD10, CD11c, CD19, CD20, CD21 , CD22, CD23, CD24, CD25, CD27, CD34, CD38, CD40, CD43, CD44, CD45, CD53, CD69, CD71 , CD72, CD73, CD78, CD79a, CD79b, CD80, CD85j, CD86, CD95 (FAS), CD98, CD 126 (IL6R), CD 127 (IL7R), CD138 (SDC1), CD184 (CXCR4), CD185 (CXCR5), CD267 (TACI), CD268 (BAFFR), CD273 (PDL2), CD274 (PDL1), CD289 (TLR9), CD307d, CD319 (SLAMF7), CD365 (TIM1), SCA-1 (Ly6A / E), and HLA-DR; and the first and second markers are optionally not the same (are different cell markers / cell surface antigens).

15. The bispecific hemibody or trispecific antibody construct of claim 14, wherein, when any one or more, two or more, three or more, or each of the first and second and, when present, third and fourth element(s) is / are an aa sequence, they are selected independently from aa sequences comprising VH, VL, SCFV, nanobody (VHH), heavy chain antibody variable region aa sequences of antibodies that bind to the marker(s) expressed on the surface of a cell of B cell lineage, or the CDRs of any of the foregoing.

16. The bispecific hemibody or trispecific antibody construct of any of claims 1-9, wherein at least one first hemibody construct and / or second hemibody construct, or the trispecific antibody construct, comprises a VH aa sequence and a VL aa sequence, or the CDRs of a VH or VL aa sequence, and a polypeptide comprising the corresponding VH or VL aa sequence or CDRs of a corresponding VH or VL aa sequence.

17. The bispecific hemibody or trispecific antibody construct of claim 16, wherein (I) the VHaa sequence or the VLaa sequence of the first hemibody construct and / or second hemibody construct, or of the trispecific antibody construct, and (ii) the polypeptide comprising the corresponding VH or VL aa sequence or CDRs of a corresponding VH or VL aa sequence form an Fv or an Fab.

18. The bispecific hemibody or trispecific antibody construct of any of claims 1-9, wherein the cytotoxic effector cells are myeloid and / or lymphoid cytotoxic effector cells.

19. A pharmaceutical composition comprising a bispecific hemibody and / or a trispecific antibody construct of any of claims 1-9, and optionally a pharmaceutically acceptable excipient.

20. A bispecific hemibody and / or trispecific antibody construct of any of claims 1 -9for use in the treatment of a patient).21 . The use of one or more bispecific hemibody and / or trispecific antibody constructs of any of claims 1-9, in the preparation of a medicament for the treatment of a patient suffering from an immune disease, an allergy, a benign neoplasm, a malignant neoplasm, graft versus host disease (GVHD) or host versus graft disease (HVGD).

22. A method of treatment comprising administering to a patient one or more bispecific hemibodies and / or trispecific antibody constructs of any of claims 1-9.23.The use of claim 22, wherein the patient is suffering from an immune disease, an allergy, a benign neoplasm, a malignant neoplasm, GVHD or HVGD.

24. The bispecific hemibody and / or trispecific antibody constructs of any of claims 1-9 for use in the treatment of a patient suffering from an immune disease selected from:(i) Addison's disease, autoimmune hepatitis, autoimmune thyroid disease, Crohn's disease, dermatomyositis, encephalomyelitis, Grave's disease, inflammatory bowel disease, juvenile idiopathic arthritis, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, Parkinson's disease, pemphigus foliaceus, pemphigus vulgaris, myositis / polymyositis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjogren's Syndrome, systemic lupus erythematosus, thrombocytopenic purpura, ulcerative colitis, vasculitis, vitiligo, Type 1 Diabetes (T1D), and celiac disease; or(ii) Type 1 Diabetes (T1D), celiac disease, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's Syndrome, or myasthenia gravis.

25. One or more nucleic acids encoding one or more bispecific hemibodies or trispecific antibody constructs of any of claims 1-9.

26. A cell comprising the one or more nucleic acids of claim 25.

27. A method of producing one or more bispecific hemibodies and / or one or more trispecific antibody constructs comprising culturing a cell of claim 26, and expressing the one or more bispecific hemibodies and / or one or more trispecific antibody constructs.

28. A method of treating a disease involving a B cell lineage cell, or its plasma cell sublineage, comprising contacting one or more cells of B cell lineage with two or more bispecific hemibody constructs, wherein:(i) the two or more bispecific hemibody constructs comprise elements that bind to at least three or at least four different markers expressed on the surface of a cell in the B cell lineage; and(ii) the two or more bispecific hemibody constructs comprise VHand V (4 and 4’) aa sequences on separate hemibody constructs that together form a paratope that binds to a CEC-SA.