Immune cell engager formats and methods of use

Abstract

Disclosed herein are multispecific immune cell engagers, such as multispecific T cell engagers. Multispecific immune cell engagers comprise one or more (e.g., two) target binding domains that bind to target molecules on immune effector cells, such as T cells, and one or more (e.g., two or three) binding domains that bind to target molecules on target cells, such as cancer cells or B cells. Specificity for multiple target cell targets can result in prevention of antigen escape, and other favorable properties, such as improved affinity or avidity for target cells. Certain multispecific immune receptors also induce costimulatory signaling in immune effector cells to prevent or reduce antigen-independent relapse.

Description

WSGR Docket No. 69097-702.601IMMUNE CELL ENGAGER FORMATS AND METHODS OF USECROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 716,002, filed on November 4, 2024, and U.S. Provisional Application No. 63 / 716,013, filed on November 4, 2024, each of which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Immunotherapies such as T cell engagers and engineered CAR-T cells have been used to induce or enhance anti -cancer immune responses, resulting in good clinical responses for some patients. However, in other cases such therapies fail to control the cancer or exhibit loss of efficacy over time, for example, due to antigen escape, insufficient stimuli for effective T cell activation, and / or immunosuppressive signals from cancer cells or the tumor microenvironment.

[0003] Antigen-dependent relapse can significantly limit the therapeutic efficacy of immunotherapies. For example, a significant proportion of patients treated with T cell engagers targeting a single cancer antigen exhibit disease progression following loss of expression of the target cancer antigen. Additionally, antigen-independent relapse can also be a significant barrier to durable anti-cancer responses, with poor outcomes frequently associated with mutations or loss of T cell costimulatory ligand expression.SUMMARY

[0004] In one aspect, provided herein is an engineered T cell engager (TCE) comprising three four five or more target binding domains wherein the targets are antigens or molecules expressed on certain cells, e.g., target cells, for example a cancer cell. In one aspect, provided herein is an engineered T cell engager (TCE) comprising at least four target binding domains, a first target binding domain, a second target binding domain, a third target binding domain and a fourth target binding domain, the engineered TCE comprising: (a) a first polypeptide, comprising from C terminus to N terminus: (i) a first Fc region (Fcl) linked to the first target binding domain, or (ii) the first target binding domain linked to the Fcl; and (b) a second polypeptide comprising from N terminus to C terminus the fourth target binding domain linked to a second Fc region (Fc2), wherein the fourth target binding domain is selected from a CD3 binding domain and a CD2 binding domain.

[0005] In one aspect, provided herein is an engineered TCE comprising at least four target binding domains configured to bind to at least three non-identical targets, the engineered TCEWSGR Docket No. 69097-702.601 comprising: (a) a first polypeptide, comprising a first Fc region (Fcl), operably linked to at least a target binding domain, and (b) a second polypeptide, comprising a second Fc region (Fc2), linked to at least two target binding domains; wherein at least one target binding domain of the at least two target binding domains comprises a CD3 binding domain or a CD2 binding domain.

[0006] In one aspect, provided herein is an engineered TCE comprising at least four binding domains configured to bind to at least three non-identical targets, the engineered TCE comprising: a first polypeptide, comprising a first Fc region (Fcl), operably linked to at least one target binding domain, wherein the at least one target binding domain is a CD2 binding domain, and a second polypeptide, comprising a second Fc region (Fc2) operably linked to at least one target binding domain, wherein the at least one target binding domain is a CD3 binding domain.

[0007] In some embodiments, the Fcl and Fc2 dimerize. In some embodiments, the CD2 binding domain is a CD58 ligand comprising a full length CD58, an extracellular domain of CD58, a CD2-binding domain of CD58, an Ig-like domain of CD58, an IgV domain of CD58, or a functional fragment or a functional variant thereof.

[0008] In some embodiments, the second polypeptide comprises a CD58 ligand.

[0009] In some embodiments, the TCE comprises five target binding domains, a first target binding domain, a second target binding domain, a third target binding domain, a fourth target binding domain and a fifth target binding domain, wherein the second polypeptide comprises from N terminus to C terminus the fourth target binding domain linked to the fifth target binding domain linked to the Fc2, wherein the fourth target binding domain binds to CD3, and the fifth target binding domain is the CD58 ligand.

[0010] In some embodiments, the TCE comprises five target binding domains, a first target binding domain, a second target binding domain, a third target binding domain, a fourth target binding domain and a fifth target binding domain, wherein the second polypeptide comprises from N terminus to C terminus a fourth target binding domain linked to the Fc2, and the Fc2 linked to a fifth target binding domain, wherein the fourth target binding domain binds to CD3, and the fifth target binding domain is the CD58 ligand.

[0011] In some embodiments, the first polypeptide comprises the CD58 ligand.

[0012] In some embodiments, the TCE comprises five target binding domains, a first target binding domain, a second target binding domain, a third target binding domain, a fourth target binding domain and a fifth target binding domain, wherein (i) the first polypeptide comprises from C terminus to N terminus the Fcl, operably linked to the CD58 ligand, or (ii) the first polypeptide comprises from N terminus to C terminus the Fcl, operably linked to the CD58 ligand and wherein the fourth target binding domain or the fifth target binding domain is linked to the Fc2.WSGR Docket No. 69097-702.601

[0013] In some embodiments, the Fcl is either directly linked to the CD58 ligand or is indirectly linked to the CD58 ligand via linkage through one or more target binding domains other than a CD3 binding domain.

[0014] In some embodiments, the first polypeptide comprises from C terminus to N terminus the CD58 ligand linked to the Fcl ; and wherein the second polypeptide comprises from N terminus to C terminus the fourth target binding domain or the fifth target binding domain linked to a second Fc region, wherein the fourth target binding domain or the fifth target binding domain is the CD3 binding domain.

[0015] In some embodiments, the first polypeptide comprises from C terminus to N terminus a CD58 ligand linked to a first target binding domain linked to the Fcl; and a second polypeptide comprising from N terminus to C terminus a fourth target binding domain or a fifth target binding domain linked to the Fc2, wherein the fourth target binding domain or the fifth target binding domain is the CD3 binding domain.

[0016] In some embodiments, the first target binding domain, the second target binding domain, the third target binding domain and / or the CD3 binding domain comprises a Fab, a F(ab')2, an Fv, a single chain Fv (scFv), a single domain antibody (sdAb), a diabody (dAb), a VHH, a camelid antibody, a nanobody, or any combination thereof.

[0017] In some embodiments, a target binding domain from the at least four target binding domains binds to a target molecule that is a tumor-associated antigen CD 19, and the target binding domain is an anti-CD19 domain.

[0018] In some embodiments, a target binding domain from the at least four target binding domains binds to a target molecule that is a tumor-associated antigen CD20, and the target binding domain is an anti-CD20 domain.

[0019] In some embodiments, a target binding domain from the at least four target binding domains binds to a target molecule is a tumor-associated antigen CD22, and the target binding domain is an anti-CD22 domain.

[0020] In some embodiments, the fourth target binding domain and the fifth target binding domains are selected from the CD3 binding domain and the CD2 binding domain.

[0021] In some embodiments, the first Fc region (Fcl) and the second Fc region (Fc2) form a heterodimer and comprise one or more modifications or is unmodified.

[0022] In some embodiments, the engineered TCE further comprises one or more linkers, wherein the one or more linkers comprise an amino acid sequence selected from any one of SEQ ID NOs: 146-182 listed in Table 13.WSGR Docket No. 69097-702.601

[0023] In some embodiments, the CD58 ligand is in turn operably linked via a linker to a second, third and / or fourth target binding domains selected from a group consisting of an antiCD 19 domain, an anti-CD20 domain and an anti-CD22 domain.

[0024] In some embodiments, the anti-CD19 domain is a single domain antibody fragment (sdAb) comprising an amino acid sequence set forth in SEQ ID NOs: 1-21 or an amino acid sequence that is at least 80% identical to any one of the sequences set forth in SEQ ID NOs: 1-21.

[0025] In some embodiments, the anti-CD19 domain is a single chain variable fragment domain (scFv), comprising a variable heavy chain (VH) domain, and a variable light chain (VL) domain; wherein the VH domain comprises an amino acid sequence is as set forth in the sequences selected from SEQ ID NOs: 23, 26, 28, 30, 32, and 34, or an amino acid sequence that is at least 80% identical to any one of the sequences set forth in SEQ ID NOs: 23, 26, 28, 30, 32, and 34; and a VL domain comprises an amino acid sequence is as set forth in the sequences selected from SEQ ID NOs: 24, 27, 29, 31, 33 and 35, or an amino acid sequence that is at least 80% identical to any one of the sequences set forth in SEQ ID NOs: 24, 27, 29, 31, 33 and 35.

[0026] In some embodiments, the anti-CD19 domain comprises an scFv comprising an amino acid sequence set forth in SEQ ID NOs: 22 or 25, or a sequence that is at least 80% identical to SEQ ID NO: 22 or 25.

[0027] In some embodiments, the anti-CD19 domain comprises a sequence of any one of the sequences depicted in Table 1, or a sequence that is at least 80% identical to any one of the sequences in Table 1.

[0028] In some embodiments, the anti-CD19 domain is a single domain antibody fragment (sdAb), comprising a heavy chain complementarity determining region (HCDR)1, an HCDR2, and an HCDR3 wherein the HCDR1, HCDR2 and the HCDR3 comprise amino acid sequences as listed in Table 2.

[0029] In some embodiments, the anti-CD19 domain is a single chain variable fragment domain (scFv), comprising a heavy chain complementarity determining region (HCDR)1, an HCDR2, and an HCDR3; and a light chain complementarity determining region (LCDR)l, a LCDR2, and a LCDR3 wherein the HCDR1, the HCDR2 and the HCDR3 and the LCDR1, the LCDR2 and the LCDR3 comprise amino acid sequences as listed in Table 2.

[0030] In some embodiments, the anti-CD20 domain is an sdAb comprising an amino acid sequence as set forth in the sequences selected from SEQ ID NOs: 36-46, or a sequence that is at least 80% identical to the any one of the sequences SEQ ID NOs: 36-46.

[0031] In some embodiments, the anti-CD20 domain is an scFv, comprising a variable heavy chain (VH) domain, and a variable light chain (VL) domain; wherein the VH domain comprises an ammino acid sequence selected from SEQ ID NOs: 47, 49, 51 and 53 or a sequence having atWSGR Docket No. 69097-702.601 least 80% sequence identity to any one of the sequences set forth in SEQ ID NOs: 47, 49, 51 and 53; and a VL domain selected from SEQ ID NOs: 48, 50, 52 and 54, or a sequence having at least 80% sequence identity to any one of the sequences set forth in SEQ ID NOs: 48, 50, 52 and 54 as listed in Table 3.

[0032] In some embodiments, the anti-CD20 domain is an sdAb, comprising a heavy chain complementarity determining region (HCDR)1, an HCDR2, and an HCDR3 wherein the HCDR1, HCDR2 and the HCDR3 comprise amino acid sequences as listed in Table 4.

[0033] In some embodiments, the anti-CD20 domain is an scFv, comprising a heavy chain complementarity determining region (HCDR)1, an HCDR2, and an HCDR3; and a light chain complementarity determining region (LCDR)l, a LCDR2, and a LCDR3 wherein the HCDR1, the HCDR2 and the HCDR3 and the LCDR1, the LCDR2 and the LCDR3 comprise amino acid sequences as listed in Table 4.

[0034] In some embodiments, the anti-CD22 domain is an sdAb comprising an amino acid sequence selected from SEQ ID NOs: 55-74, or a sequence having at least 80% sequence identity to any one of the sequences set forth in SEQ ID NOs: 55-74.

[0035] In some embodiments, the anti-CD22 domain is an scFv, comprising a variable heavy chain (VH) domain, and a variable light chain (VL) domain; wherein the VH domain comprises an amino acid sequence selected from SEQ ID NOs: 75 and 77; and a VL domain comprises an amino acid sequence selected from SEQ ID NOs: 76 and 78, as listed in Table 5, or any sequence having at least 80% sequence identity to the same.

[0036] In some embodiments, the anti-CD22 domain is an sdAb, comprising a heavy chain complementarity determining region (HCDR)1, an HCDR2, and an HCDR3 wherein the HCDR1, HCDR2 and the HCDR3 comprise amino acid sequences as listed in Table 6.

[0037] In some embodiments, the anti-CD22 domain is an scFv, comprising a heavy chain complementarity determining region (HCDR)1, an HCDR2, and an HCDR3; and a light chain complementarity determining region (LCDR)l, a LCDR2, and a LCDR3 wherein the HCDR1, the HCDR2 and the HCDR3 and the LCDR1, the LCDR2 and the LCDR3 comprise amino acid sequences as listed in Table 6.

[0038] In some embodiments, the engineered TCE comprises a target binding domain that binds to a tumor antigen, wherein the tumor antigen is DLL3. In some embodiments, the target binding domain that binds to the tumor antigen DLL3 is an sdAb, comprising an amino acid sequence:EVQLVESGGGL VQPGGSLTLSC AAS S S S VSLLSLAWYRQAPGKKRELVAGISDDGSIVY MDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYYCYAYSWITRSPYWGQGTLVTVS S, or a sequence having at least 90% sequence identity to the amino acid sequence.WSGR Docket No. 69097-702.601

[0039] In some embodiments, the CD2 binding domain comprises a sequence listed in Tables 7 or 8 or a sequence having at least 80% sequence identity to any of the sequences listed in Table 7 or 8.

[0040] In some embodiments, the CD3 binding domain comprises a sequence listed in Tables 9 or 10 or a sequence having at least 80% sequence identity to any of the sequences listed in Table 9 or 10.

[0041] In some embodiments, the Fcl and the Fc2 comprise knobs-into-holes mutations.

[0042] In some embodiments, the Fcl and the Fc2 comprise a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 108-145 as listed in Table 11; and / or comprise a mutation as listed in Table 12.

[0043] In some embodiments, the second polypeptide comprising the Fc2 comprises the CD3 binding domain linked to the N terminus of the Fc2, and the CD2 binding domain comprising the CD58 ligand linked to the C terminus of the Fc2.

[0044] Provided herein is an engineered TCE comprising: (a) a first polypeptide, comprising, (i) an anti-CD19 domain comprising an sdAb or an scFv; linked via a first linker with (ii) a first Fc domain (Fcl), and (b) a second polypeptide, comprising, (i) an anti-CD3 scFv linked via a second linker with (ii) a CD58 ligand, which is linked via a third linker with (iii) a second Fc domain (Fc2); and wherein Fcl and Fc2 dimerize and comprise a modification constituting a knobs-into-holes configuration; wherein the first linker, the second linker and / or the third linker may comprise the same or different amino acid sequence. In some embodiments, the engineered TCE comprises a first polypeptide having an amino acid sequence that is at least 90% identical to SEQ ID NO: 1070; and a second polypeptide having an amino acid sequence that is at least 90% identical to SEQ ID NO: 186. In some embodiments, the engineered TCE comprises a first polypeptide having an amino acid sequence of SEQ ID NO: 1070 and a second polypeptide having an amino acid sequence of SEQ ID NO: 186.

[0045] Provided herein is an engineered TCE comprising: (a) a first polypeptide, comprising, (i) an anti-CD19 domain comprising an sdAb or an scFv; linked via a first linker with (ii) a first Fc domain (Fcl), and (b) a second polypeptide, comprising, (i) an anti-CD3 scFv linked via a second linker with (ii) a second Fc domain (Fc2), which is linked via a third linker with (iii) a CD58 ligand; and wherein the Fcl and the Fc2 dimerize and comprise a modification constituting a knob-into-hole configuration; and wherein the first linker, the second linker and / or the third linker may comprise the same or different amino acid sequence. In some embodiments, the engineered TCE comprises the first polypeptide having an amino acid sequence that is at least 90% identical to SEQ ID NO: 1070; and the second polypeptide having an amino acid sequence that is at least 90% identical to SEQ ID NO: 187. In some embodiments the engineered TCEWSGR Docket No. 69097-702.601 comprises the first polypeptide having an amino acid sequence of SEQ ID NO: 1070 and the second polypeptide having an amino acid sequence of SEQ ID NO: 187.

[0046] Provided herein is an engineered TCE comprising: (a) a first polypeptide, comprising, (i) an anti-CD19 domain comprising an sdAb or an scFv; linked via a first linker with (ii) a CD58 ligand; which in turn is linked via a second linker with a first Fc domain (Fcl), and (b) a second polypeptide, comprising, (i) an anti-CD3 scFv linked via a third linker with (ii) a second Fc domain (Fc2), wherein the Fcl and the Fc2 dimerize and comprise a modification constituting a knob-into-hole configuration; and wherein the first linker, the second linker and / or the third linker may comprise the same or different amino acid sequence. In some embodiments, the engineered TCE comprises the first polypeptide having an amino acid sequence that has at least 90% sequence identity to the sequence of SEQ ID NO: 1071 and the second polypeptide having an amino acid sequence that has at least 90% sequence identity to the sequence of SEQ ID NO: 184. In some embodiments, the engineered TCE comprises the first polypeptide having an amino acid sequence of SEQ ID NO: 1071 and the second polypeptide having an amino acid sequence of SEQ ID NO: 184.

[0047] Provided herein is an engineered TCE comprising at least three or at least four target binding domains, the engineered TCE comprising a first polypeptide chain comprising a first Fc region (Fcl) and a second polypeptide chain comprising the second Fc region (Fc2), wherein the Fcl or the Fc2 is operably linked to a target binding domain that binds to a CD2 target molecule, and wherein the Fcl or the Fc2 is operably linked to a target binding domain that binds to a CD3 target molecule, wherein the engineered TCE induces low or negligible levels of targetindependent T cell activation when a target cell for the engineered TCE is not present in proximity of a T cell.

[0048] Provided herein is an engineered TCE comprising at least four target binding domains configured to bind to at least three non-identical targets, having any one of the configurations of any one of the TCEs described above.

[0049] Provided herein is an engineered TCE comprising at least four target binding domains configured to bind to at least two non-identical targets, having any one of the configurations of any one of the TCEs described above.

[0050] Provided herein is a use of the engineered TCEs described above in preparing a medicament for treating a disease in a human subject in need thereof, wherein the disease is a cancer or an autoimmune disease.

[0051] In one aspect, provided herein is an engineered TCE comprising at least four target binding domains, comprising a first polypeptide chain comprising a first Fc region (Fcl) and a second polypeptide chain comprising the second Fc region (Fc2), wherein the at least four targetWSGR Docket No. 69097-702.601 binding domains wherein the Fcl or the Fc2 is operably linked to a target binding domain that binds to a CD2 target molecule, wherein the TCE induces negligible fratricide under resting conditions where a target cell for a T cell is not present in proximity. In one aspect, provided herein is a method of selection of an improved T cell engager comprising one, two, three four or more target binding domains wherein one of the target binding domain binds to a CD3 and optionally, another target binding domain binds to a CD2 co-stimulatory molecule, wherein the selection method involves selecting a TCE with a the collective selection of individual binding domains, the positioning and spatial orientation, arrangement or specific design parameters related to the binding domains that render the TCE safe and efficacious over another design, and that induces at least 2-fold, at least 5-fold or at least 10-fold higher IFN-gamma release by a T cell engaging the TCE, in presence of a cell expressing a target molecule to which the TCE is designed to bind, or a target molecule to which a TCR of the T cells binds; in comparison to a different TCE, that does not comprise any one of the domains of the TCE, or a TCE that has the binding domains in an orientation non-identical to the TCE being selected. In some embodiments, the selection method further involves selecting a TCE that induces at least 2-fold, at least 5-fold or at least 10-fold higher TNF-alpha release by a T cell engaging the TCE, when the T cell is in presence of a cell expressing a target molecule to which the TCE is designed to bind, or a target molecule to which a TCR of the T cell binds; in comparison to a different TCE, that does not comprise any one of the domains of the TCE, or a TCE that has the binding domains in an orientation nonidentical to the TCE being selected. In another embodiment, the selection method involves selecting a TCE that induces at least 2-fold, at least 5-fold or at least 10-fold higher target cell cytotoxicity (TDCC) by a T cell than a different TCE, that does not comprise any one of the domains of the TCE, or a TCE that has the binding domains in an orientation non-identical to the TCE being selected. In some embodiments, the desired TCE induces reduced or negligible fratricide under resting conditions compared to a different TCE that does not comprise any one of the domains of the TCE, or does not contain a TCE that has the binding domains in an orientation identical to the TCE being selected. As is well known, fratricide of T cells is a critical problem for CART cells, when activated T cells induce cytotoxicity to surrounding T cells, often when the T cells do not encounter the target cells (e.g., resting conditions). Under resting conditions, when no target cell (cell presenting the target antigens to which a T cell receptor or a TCE binds) is in proximity T cells could still be induced or activated for example, by inflammatory surroundings, presence of growth factors, cytokines or aberrant intracellular signaling. In the instant case TCEs themselves can activate T cells. Specific designs that allow the possibility to keep fratricide at a lower rate than other designs while allowing the activation of T cells to mount cytotoxic response specific to a target cell could therefore be beneficial for downstream therapeutic development.WSGR Docket No. 69097-702.601

[0052] In one aspect, provided herein is a engineered TCE comprising at least four target binding domains, comprising a first polypeptide chain comprising a first Fc region (Fcl) and a second polypeptide chain comprising the second Fc region (Fc2), wherein the Fcl or the Fc2 is operably linked to a target binding domain that binds to a CD2 target molecule, and wherein the Fcl or the Fc2 is operably linked to a target binding domain that binds to a CD3 target molecule, wherein the TCE induces low or negligible levels of target-independent T cell activation when a target cell for the TCE is not present in proximity of a T cell. In one aspect, an engineered TCE is provided herein, comprising at least four target binding domains configured to bind to at least three non-identical targets, having any one of the configurations described above. In one embodiment, provided herein is an engineered TCE comprising at least four target binding domains configured to bind to at least two non-identical targets, having any one of the configurations described above.

[0053] Provided herein is a use of the engineered TCE of any one of the embodiments in preparing a medicament for treating a disease in a human subject in need thereof, wherein the disease is a cancer or an autoimmune disease.

[0054] As disclosed herein the numbering of the binding domains may be understood in the context of the molecule discussed and the feature may not be specifically associated to the number associated but to orient a reader to a certain structural and spatial arrangement. For example, a first, second, third and fourth binding domains in one description of a TCE design may vary from a first, second, third and fourth binding domains in another TCE description. For example, a fifth binding domain in one design aspect may be the same as a third or fourth or simply other than fifth binding domain described elsewhere for another design unless in direct comparison with each other.

[0055] Disclosed herein, in some aspects, is a T cell engager comprising: a first polypeptide comprising from N terminus to C terminus a first target binding domain linked to a first Fc region; and a second polypeptide comprising from N terminus to C terminus a fourth target binding domain linked to a fifth target binding domain linked to a second Fc region, wherein the first target binding domain binds to a first target molecule, and the fourth target binding domain binds to a fourth target molecule, wherein the fourth target molecule is CD3, and the fifth target binding domain binds to a fifth target molecule, wherein the fifth target molecule is CD2. When describing positional aspects of a TCE, description of certain positions may be omitted for efficiency, for example, in one paragraph, a first and a fourth binding domain may be described, omitting the description of second and third binding domains. However, with the inclusion of a description for a fourth binding domain, for example, it is understood that a third and a second domain exists, andWSGR Docket No. 69097-702.601 their description is saved for description later or is otherwise silent as not material in the immediate description.

[0056] Disclosed herein, in some aspects, is a T cell engager comprising: a first polypeptide comprising from N terminus to C terminus a first target binding domain linked to a fifth target binding domain linked to a first Fc region; and a second polypeptide comprising from N terminus to C terminus a fourth target binding domain linked to a second Fc region, wherein the first target binding domain binds to a first target molecule, the fourth target binding domain binds to a fourth target molecule, wherein the fourth target molecule is CD3, and the fifth target binding domain binds to a fifth target molecule, wherein the fifth target molecule is CD2.

[0057] Disclosed herein, in some aspects, is a T cell engager comprising: a first polypeptide comprising from N terminus to C terminus a fifth target binding domain linked to a first target binding domain linked to a first Fc region; and a second polypeptide comprising from N terminus to C terminus a fourth target binding domain linked to a second Fc region, wherein the first target binding domain binds to a first target molecule, the fourth target binding domain binds to a fourth target molecule, wherein the fourth target molecule is CD3, and the fifth target binding domain binds to a fifth target molecule, wherein the fifth target molecule is CD2.

[0058] Disclosed herein, in some aspects, is a T cell engager comprising: a first polypeptide comprising from N terminus to C terminus a second target binding domain linked to a first target binding domain linked to a first Fc region; and a second polypeptide comprising from N terminus to C terminus a fourth target binding domain linked to a fifth target binding domain linked to a second Fc region, wherein the first target binding domain binds to a first target molecule, the second target binding domain binds to a second target molecule, the fourth target binding domain binds to a fourth target molecule, wherein the fourth target molecule is CD3, and the fifth target binding domain binds to a fifth target molecule, wherein the fifth target molecule is CD2.

[0059] Disclosed herein, in some aspects, is a T cell engager comprising: a first polypeptide comprising from N terminus to C terminus a second target binding domain linked to a first target binding domain linked to a fifth target binding domain linked to a first Fc region; and a second polypeptide comprising from N terminus to C terminus a fourth target binding domain linked to a second Fc region, wherein the first target binding domain binds to a first target molecule, the second target binding domain binds to a second target molecule, the fourth target binding domain binds to a fourth target molecule, wherein the fourth target molecule is CD3, and the fifth target binding domain binds to a fifth target molecule, wherein the fifth target molecule is CD2.

[0060] Disclosed herein, in some aspects, is a T cell engager comprising: a first polypeptide comprising from N terminus to C terminus a second target binding domain linked to a fifth target binding domain linked to a first target binding domain linked to a first Fc region; and a secondWSGR Docket No. 69097-702.601 polypeptide comprising from N terminus to C terminus a fourth target binding domain linked to a second Fc region, wherein the first target binding domain binds to a first target molecule, the second target binding domain binds to a second target molecule, the fourth target binding domain binds to a fourth target molecule, wherein the fourth target molecule is CD3, and the fifth target binding domain binds to a fifth target molecule, wherein the fifth target molecule is CD2.

[0061] Disclosed herein, in some aspects, is a T cell engager comprising: a first polypeptide comprising from N terminus to C terminus a third target binding domain linked to a second target binding domain linked to a first target binding domain linked to a first Fc region; and a second polypeptide comprising from N terminus to C terminus a fourth target binding domain linked to a fifth target binding domain linked to a second Fc region, wherein the first target binding domain binds to a first target molecule, the second target binding domain binds to a second target molecule, the third target binding domain binds to a third target molecule, the fourth target binding domain binds to a fourth target molecule, wherein the fourth target molecule is CD3, and the fifth target binding domain binds to a fifth target molecule, wherein the fifth target molecule is CD2.

[0062] Disclosed herein, in some aspects, is a T cell engager comprising: a first polypeptide comprising from N terminus to C terminus a third target binding domain linked to a second target binding domain linked to a first target binding domain linked to a fifth target binding domain linked to a first Fc region; and a second polypeptide comprising from N terminus to C terminus a fourth target binding domain linked to a second Fc region, wherein the first target binding domain binds to a first target molecule, the second target binding domain binds to a second target molecule, the third target binding domain binds to a third target molecule, the fourth target binding domain binds to a fourth target molecule, wherein the fourth target molecule is CD3, and the fifth target binding domain binds to a fifth target molecule, wherein the fifth target molecule is CD2.

[0063] Disclosed herein, in some aspects, is a T cell engager comprising: a first polypeptide comprising from N terminus to C terminus a third target binding domain linked to a second target binding domain linked to a fifth target binding domain linked to a first target binding domain linked to a first Fc region; and a second polypeptide comprising from N terminus to C terminus a fourth target binding domain linked to a second Fc region, wherein the first target binding domain binds to a first target molecule, the second target binding domain binds to a second target molecule, the third target binding domain binds to a third target molecule, the fourth target binding domain binds to a fourth target molecule, wherein the fourth target molecule is CD3, and the fifth target binding domain binds to a fifth target molecule, wherein the fifth target molecule is CD2.

[0064] Disclosed herein, in some aspects, is a T cell engager comprising: a first polypeptide comprising from N terminus to C terminus a first target binding domain linked to a second targetWSGR Docket No. 69097-702.601 binding domain linked to a first Fc region linked to a third target binding domain; and a second polypeptide comprising from N terminus to C terminus a fourth target binding domain linked to a fifth target binding domain linked to a second Fc region, wherein the first target binding domain binds to a first target molecule, the second target binding domain binds to a second target molecule, the third target binding domain binds to a third target molecule, the fourth target binding domain binds to a fourth target molecule, wherein the fourth target molecule is CD3, and the fifth target binding domain binds to a fifth target molecule, wherein the fifth target molecule is CD2.

[0065] Disclosed herein, in some aspects, is a T cell engager comprising: a first polypeptide comprising from N terminus to C terminus a first target binding domain linked to a second target binding domain linked to a fifth target binding domain linked to a first Fc region linked to a third target binding domain; and a second polypeptide comprising from N terminus to C terminus a fourth target binding domain linked to a second Fc region, wherein the first target binding domain binds to a first target molecule, the second target binding domain binds to a second target molecule, the third target binding domain binds to a third target molecule, the fourth target binding domain binds to a fourth target molecule, wherein the fourth target molecule is CD3, and the fifth target binding domain binds to a fifth target molecule, wherein the fifth target molecule is CD2.

[0066] Disclosed herein, in some aspects, is a T cell engager comprising: a first polypeptide comprising from N terminus to C terminus a first target binding domain linked to a fifth target binding domain linked to a second target binding domain linked to a first Fc region linked to a third target binding domain; and a second polypeptide comprising from N terminus to C terminus a fourth target binding domain linked to a second Fc region, wherein the first target binding domain binds to a first target molecule, the second target binding domain binds to a second target molecule, the third target binding domain binds to a third target molecule, the fourth target binding domain binds to a fourth target molecule, wherein the fourth target molecule is CD3, and the fifth target binding domain binds to a fifth target molecule, wherein the fifth target molecule is CD2.

[0067] In some embodiments, the first target molecule is a first tumor-associated antigen (e.g., CD 19). In some embodiments, the second target molecule is a second tumor-associated antigen (e.g., CD20). In some embodiments, the third target molecule is a third tumor-associated antigen (e.g., CD22). In some embodiments, the fifth target binding domain comprises a full length CD58, an extracellular domain of CD58, a CD2-binding domain of CD58, an Ig-like domain of CD58, an IgV domain of CD58, or a functional fragment or functional variant thereof. In some embodiments, the first Fc region and the second Fc region form a heterodimer. In some embodiments, the first polypeptide chain and the second polypeptide chain are non-contiguous. In some embodiments, the first target binding domain is an antibody domain or an antigen binding domain. In some embodiments, the first target binding domain is an antigen binding domain,WSGR Docket No. 69097-702.601 wherein the antigen binding domain comprises a Fab, a F(ab')2, an Fv, a single chain Fv (scFv), a single domain antibody, a diabody (dAb), a VHH, a camelid antibody, a nanobody, or any combination thereof. In some embodiments, the first target binding domain comprises a heavy chain variable region (VH) comprising a heavy chain complementarity-determining region 1 (HCDR1) sequence having at least 90% sequence identity to any one of the HCDR1 sequences in TABLE 2, an HCDR2 sequence having at least 90% sequence identity to any one of the HCDR2 sequences in TABLE 2, and an HCDR3 sequence comprising a sequence at least 90% sequence identity to any one of the HCDR3 sequences in TABLE 2. In some embodiments, the first target binding domain comprises a VH comprising an HCDR1 sequence comprising the sequence of any one of the HCDR1 sequences in TABLE 2, an HCDR2 sequence comprising the sequence of any one of the HCDR2 sequences in TABLE 2, and an HCDR3 sequence comprising the sequence of any one of the HCDR3 sequences in TABLE 2. In some embodiments, the first target binding domain comprises a VH, wherein the VH comprises a set of heavy chain CDRs from TABLE 2. In some embodiments, the first target binding domain comprises a light chain variable region (VL) comprising a light chain complementarity-determining region 1 (LCDR1) sequence having at least 90% sequence identity to any one of the LCDR1 sequences in TABLE 2, an LCDR2 sequence having at least 90% sequence identity to any one of the LCDR2 sequences in TABLE 2, and an LCDR3 sequence comprising at least 90% sequence identity to any one the LCDR3 sequences in TABLE 2. In some embodiments, the first target binding domain comprises a VL comprising an LCDR1 sequence comprising the sequence of any one of the LCDR1 sequences in TABLE 2, an LCDR2 sequence comprising the sequence of any one of the LCDR2 sequences in TABLE 2, and an LCDR3 sequence comprising the sequence of any one of the LCDR3 sequences in TABLE 2. In some embodiments, the first target binding domain comprises a VL, wherein the VL comprises a set of light chain CDRs from table 2. In some embodiments, the first target binding domain comprises: a VH comprising a set of heavy chain CDRs from TABLE 2; and a VL comprising a set of light chain CDRs from TABLE 2. In some embodiments, the first target binding domain comprises: a VH comprising an amino acid sequence with at least 90% sequence identity to any one of the VH sequences in TABLE 1; and a VL comprising an amino acid sequence with at least 90% sequence identity to any one of the VL sequences in TABLE 1. In some embodiments, the first target binding domain comprises: a VH comprising the amino acid sequence of any one of the VH sequences in TABLE 1; and a VL comprising the amino acid sequence of any one of the VL sequences in TABLE 1. In some embodiments, the second target binding domain comprises a heavy chain variable region (VH) comprising a heavy chain complementarity-determining region 1 (HCDR1) sequence having at least 90% sequence identity to any one of the HCDR1 sequences in TABLE 4, an HCDR2 sequence having at least 90% sequence identity to any one of the HCDR2WSGR Docket No. 69097-702.601 sequences in TABLE 4, and an HCDR3 sequence comprising a sequence at least 90% sequence identity to any one of the HCDR3 sequences in TABLE 4. In some embodiments, the second target binding domain comprises a VH comprising an HCDR1 sequence comprising the sequence of any one of the HCDR1 sequences in TABLE 4, an HCDR2 sequence comprising the sequence of any one of the HCDR2 sequences in TABLE 4, and an HCDR3 sequence comprising the sequence of any one of the HCDR3 sequences in TABLE 4. In some embodiments, the second target binding domain comprises a VH, wherein the VH comprises a set of heavy chain CDRs from TABLE 4. In some embodiments, the second target binding domain comprises a light chain variable region (VL) comprising a light chain complementarity-determining region 1 (LCDR1) sequence having at least 90% sequence identity to any one of the LCDR1 sequences in TABLE 4, an LCDR2 sequence having at least 90% sequence identity to any one of the LCDR2 sequences in TABLE 4, and an LCDR3 sequence comprising at least 90% sequence identity to any one the LCDR3 sequences in TABLE 4. In some embodiments, the second target binding domain comprises a VL comprising an LCDR1 sequence comprising the sequence of any one of the LCDR1 sequences in TABLE 4, an LCDR2 sequence comprising the sequence of any one of the LCDR2 sequences in TABLE 4, and an LCDR3 sequence comprising the sequence of any one of the LCDR3 sequences in TABLE 4. In some embodiments, the second target binding domain comprises a VL, wherein the VL comprises a set of light chain CDRs from TABLE 4. In some embodiments, the second target binding domain comprises: a VH comprising a set of heavy chain CDRs from TABLE 4; and a VL comprising a set of light chain CDRs from TABLE 4. In some embodiments, the second target binding domain comprises: a VH comprising an amino acid sequence with at least 90% sequence identity to any one of the VH sequences in TABLE 3; and a VL comprising an amino acid sequence with at least 90% sequence identity to any one of the VL sequences in TABLE 3. In some embodiments, the second target binding domain comprises: a VH comprising the amino acid sequence of any one of the VH sequences in TABLE 3; and a VL comprising the amino acid sequence of any one of the VL sequences in TABLE 3. In some embodiments, the third target binding domain comprises a heavy chain variable region (VH) comprising a heavy chain complementarity-determining region 1 (HCDR1) sequence having at least 90% sequence identity to any one of the HCDR1 sequences in TABLE 6, an HCDR2 sequence having at least 90% sequence identity to any one of the HCDR2 sequences in TABLE 6, and an HCDR3 sequence comprising a sequence at least 90% sequence identity to any one of the HCDR3 sequences in TABLE 6. In some embodiments, the third target binding domain comprises a VH comprising an HCDR1 sequence comprising the sequence of any one of the HCDR1 sequences in TABLE 6, an HCDR2 sequence comprising the sequence of any one of the HCDR2 sequences in TABLE 6, and an HCDR3 sequence comprising the sequence of any one ofWSGR Docket No. 69097-702.601 the HCDR3 sequences in TABLE 6. In some embodiments, the third target binding domain comprises a VH, wherein the VH comprises a set of heavy chain CDRs from TABLE 6. In some embodiments, the third target binding domain comprises a light chain variable region (VL) comprising a light chain complementarity-determining region 1 (LCDR1) sequence having at least 90% sequence identity to any one of the LCDR1 sequences in TABLE 6, an LCDR2 sequence having at least 90% sequence identity to any one of the LCDR2 sequences in TABLE 6, and an LCDR3 sequence comprising at least 90% sequence identity to any one the LCDR3 sequences in TABLE 6. In some embodiments, the third target binding domain comprises a VL comprising an LCDR1 sequence comprising the sequence of any one of the LCDR1 sequences in TABLE 6, an LCDR2 sequence comprising the sequence of any one of the LCDR2 sequences in TABLE 6, and an LCDR3 sequence comprising the sequence of any one of the LCDR3 sequences in TABLE 6. In some embodiments, the third target binding domain comprises a VL, wherein the VL comprises a set of light chain CDRs from TABLE 6. In some embodiments, the third target binding domain comprises: a VH comprising a set of heavy chain CDRs from TABLE 6; and a VL comprising a set of light chain CDRs from TABLE 6. In some embodiments, the third target binding domain comprises: a VH comprising an amino acid sequence with at least 90% sequence identity to any one of the VH sequences in TABLE 5; and a VL comprising an amino acid sequence with at least 90% sequence identity to any one of the VL sequences in TABLE 5. In some embodiments, the third target binding domain comprises: a VH comprising the amino acid sequence of any one of the VH sequences in TABLE 5; and a VL comprising the amino acid sequence of any one of the VL sequences in TABLE 5. In some embodiments, the fourth target binding domain comprises a heavy chain variable region (VH) comprising a heavy chain complementarity-determining region 1 (HCDR1) sequence having at least 90% sequence identity to any one of the HCDR1 sequences in TABLE 10, an HCDR2 sequence having at least 90% sequence identity to any one of the HCDR2 sequences in TABLE 10, and an HCDR3 sequence comprising a sequence at least 90% sequence identity to any one of the HCDR3 sequences in TABLE 10. In some embodiments, the fourth target binding domain comprises a VH comprising an HCDR1 sequence comprising the sequence of any one of the HCDR1 sequences in TABLE 10, an HCDR2 sequence comprising the sequence of any one of the HCDR2 sequences in TABLE 10, and an HCDR3 sequence comprising the sequence of any one of the HCDR3 sequences in TABLE 10. In some embodiments, the fourth target binding domain comprises a VH, wherein the VH comprises a set of heavy chain CDRs from TABLE 10. In some embodiments, the fourth target binding domain comprises a light chain variable region (VL) comprising a light chain complementaritydetermining region 1 (LCDR1) sequence having at least 90% sequence identity to any one of the LCDR1 sequences in TABLE 10, an LCDR2 sequence having at least 90% sequence identity toWSGR Docket No. 69097-702.601 any one of the LCDR2 sequences in TABLE 10, and an LCDR3 sequence comprising at least 90% sequence identity to any one the LCDR3 sequences in TABLE 10. In some embodiments, the fourth target binding domain comprises a VL comprising an LCDR1 sequence comprising the sequence of any one of the LCDR1 sequences in TABLE 10, an LCDR2 sequence comprising the sequence of any one of the LCDR2 sequences in TABLE 10, and an LCDR3 sequence comprising the sequence of any one of the LCDR3 sequences in TABLE 10. In some embodiments, the fourth target binding domain comprises a VL, wherein the VL comprises a set of light chain CDRs from TABLE 10. In some embodiments, the fourth target binding domain comprises: a VH comprising a set of heavy chain CDRs from TABLE 10; and a VL comprising a set of light chain CDRs from TABLE 10. In some embodiments, the fourth target binding domain comprises: a VH comprising an amino acid sequence with at least 90% sequence identity to any one of the VH sequences in TABLE 9; and a VL comprising an amino acid sequence with at least 90% sequence identity to any one of the VL sequences in TABLE 9. In some embodiments, the fourth target binding domain comprises: a VH comprising the amino acid sequence of any one of the VH sequences in TABLE 9; and a VL comprising the amino acid sequence of any one of the VL sequences in TABLE 9. In some embodiments, the fifth target binding domain comprises a heavy chain variable region (VH) comprising a heavy chain complementarity-determining region 1 (HCDR1) sequence having at least 90% sequence identity to any one of the HCDR1 sequences in TABLE 8, an HCDR2 sequence having at least 90% sequence identity to any one of the HCDR2 sequences in TABLE 8, and an HCDR3 sequence comprising a sequence at least 90% sequence identity to any one of the HCDR3 sequences in TABLE 8. In some embodiments, the fifth target binding domain comprises a VH comprising an HCDR1 sequence comprising the sequence of any one of the HCDR1 sequences in TABLE 8, an HCDR2 sequence comprising the sequence of any one of the HCDR2 sequences in TABLE 8, and an HCDR3 sequence comprising the sequence of any one of the HCDR3 sequences in TABLE 8. In some embodiments, the fifth target binding domain comprises a VH, wherein the VH comprises a set of heavy chain CDRs from TABLE 8. In some embodiments, the fifth target binding domain comprises a light chain variable region (VL) comprising a light chain complementarity-determining region 1 (LCDR1) sequence having at least 90% sequence identity to any one of the LCDR1 sequences in TABLE 8, an LCDR2 sequence having at least 90% sequence identity to any one of the LCDR2 sequences in TABLE 8, and an LCDR3 sequence comprising at least 90% sequence identity to any one the LCDR3 sequences in TABLE 8. In some embodiments, the fifth target binding domain comprises a VL comprising an LCDR1 sequence comprising the sequence of any one of the LCDR1 sequences in TABLE 8, an LCDR2 sequence comprising the sequence of any one of the LCDR2 sequences in TABLE 8, and an LCDR3 sequence comprising the sequence of any one of the LCDR3 sequences in TABLE 8.WSGR Docket No. 69097-702.601In some embodiments, the fifth target binding domain comprises a VL, wherein the VL comprises a set of light chain CDRs from TABLE 8. In some embodiments, the fifth target binding domain comprises: a VH comprising a set of heavy chain CDRs from TABLE 8; and a VL comprising a set of light chain CDRs from TABLE 8. In some embodiments, the fifth target binding domain comprises: a VH comprising an amino acid sequence with at least 90% sequence identity to any one of the VH sequences in TABLE 7; and a VL comprising an amino acid sequence with at least 90% sequence identity to any one of the VL sequences in TABLE 7. In some embodiments, the fifth target binding domain comprises: a VH comprising the amino acid sequence of any one of the VH sequences in TABLE 7; and a VL comprising the amino acid sequence of any one of the VL sequences in TABLE 7. In some embodiments, the fifth target binding domain comprises an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 79-86. In some embodiments, the fifth target binding domain comprises an amino acid sequence with at least 95% sequence identity to any one of SEQ ID NOs: 79-86. In some embodiments, the fifth target binding domain comprises the amino acid sequence of any one of SEQ ID NOs: 79-86. In some embodiments, the CDRs are according to the Kabat definition. In some embodiments, the CDRs are according to the Chothia definition. In some embodiments, the CDRs are according to the IMGT definition. In some embodiments, the first Fc region or the second Fc region comprises an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 108-117. In some embodiments, the first Fc region or the second Fc region comprises an amino acid sequence with at least 95% sequence identity to any one of SEQ ID NOs: 108-117. In some embodiments, the first Fc region or the second Fc region comprises the amino acid sequence of any one of SEQ ID NOs: 108-117. In some embodiments, the T cell engager comprises a linker, hinge, or spacer comprising an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 146-182. In some embodiments, the T cell engager comprises a linker, hinge, or spacer comprising the amino acid sequence of any one of SEQ ID NOs: 146-182.

[0068] In some embodiments, provided herein is a T cell engager of any one of the preceding embodiments, comprising a first polypeptide chain is a CD19(sdAb)-Fc(hole) having a sequence of SEQ ID NO: 1070, and a second polypeptide chain a CD3 (scFv)-CD58(WT)-Fc(knob)having a sequence of SEQ ID NO: 186.

[0069] In some embodiments, provided herein T cell engager of any one of the preceding embodiments, wherein the first polypeptide is a CD19(sdAb)-Fc(hole) having a sequence of SEQ ID NO: 1070, and the second polypeptide is a aCD3(scFv)- Fc(knob)-CD58(WT) having a sequence of SEQ ID NO: 187.

[0070] In some embodiments, provided herein is a T cell engager of any one of the preceding embodiments, wherein the first polypeptide is a CD19(sdAb)-CD58 IgV WT-Fc(hole) having aWSGR Docket No. 69097-702.601 sequence of SEQ ID NO: 1071, the second polypeptide is a aCD3(scFv)-Fc(knob)-CD58(WT) having a sequence of SEQ ID NO: 184.

[0071] Disclosed herein, in some aspects, is a method of treating a condition in a subject in need thereof, the method comprising administering to the subject the T cell engager of any one of the preceding embodiments.

[0072] In some embodiments the condition is an autoimmune disease. In some embodiments, the autoimmune disease is a B cell related autoimmune disease. In some embodiments, the subject in need thereof suffers from an autoimmune condition characterized by a B cell hyperactivity. For example, the autoimmune disease is SLE.

[0073] In some embodiments, the condition is a cancer. In some embodiments, the condition is a b cell cancer. In some embodiments, the condition is lymphoma. In some embodiments, the condition is large B cell lymphoma. In some embodiments, the condition is diffuse large B-cell lymphoma. In some embodiments, the condition is mantle cell lymphoma, follicular lymphoma, lymphoplasmacytic lymphoma, marginal zone b-cell lymphoma, small-cell lymphocytic lymphoma, burkitt lymphoma, primary central nervous system lymphoma, primary intraocular lymphoma, hodgkin lymphoma, non-hodgkin lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, hairy cell leukemia, chronic myeloid leukemia, myelodysplastic syndrome, myeloproliferative disorder, myeloma, or multiple myeloma. In some embodiments, the condition is a relap sed / refractory cancer. In some embodiments, the cancer exhibits low expression of CD58. In some embodiments, the condition is an autoimmune disease.

[0074] Disclosed herein, in some aspects, is a T cell engager comprising: a first target binding domain that binds to a first target molecule, wherein the first target molecule is a first tumor- associated antigen (e.g., CD 19); and a fourth target binding domain that binds to a fourth target molecule, wherein the fourth target molecule is CD3.

[0075] In some embodiments, the T cell engager further comprises a second target binding domain that binds to a second target molecule, wherein the second target molecule is a second tumor-associated antigen (e.g., CD20). In some embodiments, the T cell engager further comprises a third target binding domain that binds to a third target molecule, wherein the third target molecule is a third tumor-associated antigen (e.g., CD22). In some embodiments, the T cell engager further comprises a fifth target binding domain that binds to a fifth target molecule, wherein the fifth target molecule is CD2.

[0076] Disclosed herein, in some aspects, is a T cell engager comprising: a first target binding domain that binds to a first target molecule, wherein the first target molecule is CD 19; a second target binding domain that binds to a second target molecule, wherein the second target molecule is CD20; a third target binding domain that binds to a third target molecule, wherein the thirdWSGR Docket No. 69097-702.601 target molecule is CD22; and a fourth target binding domain that binds to a fourth target molecule, wherein the fourth target molecule is CD3.

[0077] In some embodiments, the T cell engager further comprises a fifth target binding domain. In some embodiments, the fifth target binding domain comprises a full length CD58, an extracellular domain of CD58, a CD2-binding domain of CD58, an Ig-like domain of CD58, an IgV domain of CD58, or a functional fragment or functional variant thereof. In some embodiments, the fifth target binding domain binds to a fifth target molecule, wherein the fifth target molecule is CD2. In some embodiments, the first target binding domain is linked to the second target binding domain. In some embodiments, the first target binding domain is linked to the third target binding domain. In some embodiments, the first target binding domain is linked to the fifth target binding domain. In some embodiments, the second target binding domain is linked to the third target binding domain. In some embodiments, the second target binding domain is linked to the fourth target binding domain. In some embodiments, the second target binding domain is linked to the fifth target binding domain. In some embodiments, the third target binding domain is linked to the fifth target binding domain. In some embodiments, the fourth target binding domain is linked to the fifth target binding domain. In some embodiments, the first target binding domain is linked to the second target binding domain, and the second target binding domain is linked to the third target binding domain. In some embodiments, the T cell engager further comprises a first portion of a dimerization module and a second portion of the dimerization module. In some embodiments, the T cell engager comprises: a first polypeptide chain comprising the first portion of a dimerization module, and a second polypeptide chain comprising a second portion of the dimerization module, wherein the first polypeptide chain and the second polypeptide chain are non-contiguous. In some embodiments, the first polypeptide chain comprises the first target binding domain, the second target binding domain, the third target binding domain, or any combination thereof. In some embodiments, the first polypeptide chain comprises the first target binding domain, and the first target binding domain is linked to the first portion of the dimerization module. In some embodiments, the first polypeptide chain comprises the second target binding domain, and the second target binding domain is linked to the first portion of the dimerization module. In some embodiments, the first polypeptide chain comprises the third target binding domain, and the third target binding domain is linked to the first portion of the dimerization module. In some embodiments, the first polypeptide chain comprises the fifth target binding domain, and the fifth target binding domain is linked to the first portion of the dimerization module. In some embodiments, the second polypeptide chain comprises the second target binding domain, and the second target binding domain is linked to the second portion of the dimerization module. In some embodiments, the second polypeptide chain comprises the fourth target bindingWSGR Docket No. 69097-702.601 domain, and the fourth target binding domain is linked to the second portion of the dimerization module. In some embodiments, the second polypeptide chain comprises the fifth target binding domain, and the fifth target binding domain is linked to the second portion of the dimerization module. In some embodiments, the first portion of the dimerization module and the second portion of the dimerization module are dimerized. In some embodiments, the first portion of the dimerization module comprises a first Fc region and the second portion of the dimerization module comprises a second Fc region. In some embodiments, the T cell engager comprises: a first polypeptide comprising from N terminus to C terminus the third target binding domain linked to the second target binding domain linked to the first target binding domain linked to the first Fc region; and a second polypeptide comprising from N terminus to C terminus the fourth target binding domain linked to the second Fc region. In some embodiments, the T cell engager comprises: a first polypeptide comprising from N terminus to C terminus the third target binding domain linked to the second target binding domain linked to the first target binding domain linked to the first Fc region; and a second polypeptide comprising from N terminus to C terminus the fourth target binding domain linked to the fifth target binding domain linked to the second Fc region. In some embodiments, the T cell engager comprises: a first polypeptide comprising from N terminus to C terminus the third target binding domain linked to the second target binding domain linked to the first target binding domain linked to the first Fc region; and a second polypeptide comprising from N terminus to C terminus the fourth target binding domain linked to the second Fc region linked to the fifth target binding domain. In some embodiments, the T cell engager comprises: a first polypeptide comprising from N terminus to C terminus the third target binding domain linked to the second target binding domain linked to the first target binding domain linked to the fifth target binding domain linked to the first Fc region; and a second polypeptide comprising from N terminus to C terminus the fourth target binding domain linked to the second Fc region. In some embodiments, the T cell engager comprises: a first polypeptide comprising from N terminus to C terminus the third target binding domain linked to the second target binding domain linked to the fifth target binding domain linked to the first target binding domain linked to the first Fc region; and a second polypeptide comprising from N terminus to C terminus the fourth target binding domain linked to the second Fc region. In some embodiments, the T cell engager comprises: a first polypeptide comprising from N terminus to C terminus the third target binding domain linked to the fifth target binding domain linked to second target binding domain linked to the first target binding domain linked to the first Fc region; and a second polypeptide comprising from N terminus to C terminus the fourth target binding domain linked to the second Fc region. In some embodiments, the T cell engager comprises: a first polypeptide comprising from N terminus to C terminus the fifth target binding domain linked to the third targetWSGR Docket No. 69097-702.601 binding domain linked to second target binding domain linked to the first target binding domain linked to the first Fc region; and a second polypeptide comprising from N terminus to C terminus the fourth target binding domain linked to the second Fc region. In some embodiments, the T cell engager comprises: a first polypeptide comprising from N terminus to C terminus the third target binding domain linked to the second target binding domain linked to the first target binding domain linked to the first Fc region linked to the fifth target binding domain; and a second polypeptide comprising from N terminus to C terminus the fourth target binding domain linked to the second Fc region. In some embodiments, the T cell engager comprises: a first polypeptide comprising from N terminus to C terminus the first target binding domain linked to the third target binding domain linked to the fifth target binding domain linked to the first Fc region; and a second polypeptide comprising from N terminus to C terminus the fourth target binding domain linked to the second target binding domain linked to the second Fc region. In some embodiments, the T cell engager comprises: a first polypeptide comprising from N terminus to C terminus the first target binding domain linked to the third target binding domain linked to the first Fc region linked to the second target binding domain linked to the fifth target binding domain; and a second polypeptide comprising from N terminus to C terminus the fourth target binding domain linked to the second Fc region. In some embodiments, the first target binding domain is an antibody domain or an antigen binding domain. In some embodiments, the first target binding domain is an antigen binding domain, wherein the antigen binding domain comprises a Fab, a F(ab')2, an Fv, a single chain Fv (scFv), a single domain antibody, a diabody (dAb), a VHH, a camelid antibody, a nanobody, or any combination thereof. The structure, sequences and complex IDs are as provided in the figures, Tables and the specification throughout.INCORPORATION BY REFERENCE

[0078] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0079] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:WSGR Docket No. 69097-702.601

[0080] FIG. 1 is a schematic of a T cell engager (TCE) with up to 16 binding domains and an Fc. The location of the binding domains are named based on their position relative to the two chains of the Fc chains (Fc-1 and Fc-2). For example, position INI is the first binder N-terminal to Fc-1, position 1N2 is the second binder N-terminal to Fc-1, position 1N3 is the third binder N- terminal to Fc-1, and position 1N4 is the fourth binder N-terminal to Fc-1. Similarly, position 1C1 is the first binder C-terminal to Fc-1, position 1C2 is the second binder C-terminal to Fc-1, position 1C3 is the third binder C-terminal to Fc-1, and position 1C4 is the fourth binder C-terminal to Fc- 1. The same naming convention was applied to describe binders fused to Fc-2. The binding domains can be composed of various antibody domains or fragments, including single-domain antibodies (sdAb), nanobodies, VHHs, heavy chain-only antibodies (HCAb), fragment variable regions (Fv), single-chain fragment variable regions (scFv), fragment antigen binding regions (Fab), single-chain fragment antigen binding regions (scFab), etc. The binding domains can also be composed of a natural ligand of a target of interest, the extracellular domain of the natural ligand, or another fragment of the natural ligand, etc. For simplicity, not all regions of the molecular components are illustrated (e.g., the disulfides connecting the hinge region of the Fc, the CH2 and CH3 domains of the Fc, heterodimerization mutations in the Fc, VH and VL domains and linkers of an scFv, etc., are omitted).

[0081] FIGs. 2A-2H provide schematics of different TCE formats targeting a single tumor- associated antigen (TAA). FIG. 2A illustrates a TCE with a single TAA binding domain (aTAA), a single CD3 binding domain (aCD3), and no CD2 costimulation. FIGS. 2B-2H illustrate TCEs with a single aTAA, a single aCD3, and CD2 costimulation (CD58). The aTAA and aCD3 binding domains can be composed of various antibody domains or fragments, including sdAbs, nanobodies, VHHs, HCAbs, Fvs, scFvs, Fabs, scFabs, etc. The CD2 costimulation module can be composed of a CD2-binding domain, such as the CD58 extracellular domain, the CD58 immunoglobulin-like variable (IgV) domain, an antibody domain or fragment that binds to CD2, etc. For simplicity, not all regions of the molecular components are illustrated (e.g., the disulfides connecting the hinge region of the Fc, the CH2 and CH3 domains of the Fc, heterodimerization mutations in the Fc, VH and VL domains and linkers of an scFv, etc., are omitted).

[0082] FIGs. 3A-3I are schematics of different TCE formats targeting one or two TAAs. FIG. 3A illustrates a TCE with two aTAAs, a single aCD3, and no CD2 costimulation. FIGS. 3B-3I illustrate TCEs with two aTAAs, a single aCD3, and CD2 costimulation (CD58). The aTAA and aCD3 binding domains can be composed of various antibody domains or fragments, including sdAbs, nanobodies, VHHs, HCAbs, Fvs, scFvs, Fabs, scFabs, etc. The CD2 costimulation module can be composed of a CD2-binding domain, such as the CD58 extracellular domain, the CD58 IgV domain, an antibody domain or fragment that binds to CD2, etc. The two aTAAs can compriseWSGR Docket No. 69097-702.601 or consist of two copies of the same binding domain to a single TAA, two binding domains that recognize different epitopes within the same TAA, or two binding domains that recognize two different TAAs. For simplicity, not all regions of the molecular components are illustrated (e.g., the disulfides connecting the hinge region of the Fc, the CH2 and CH3 domains of the Fc, heterodimerization mutations in the Fc, VH and VL domains and linkers of an scFv, etc., are omitted).

[0083] FIGs. 4A-4J are schematics of different TCE formats targeting one, two, or three TAAs. FIG. 4A illustrates a TCE with three aTAAs, a single aCD3, and no CD2 costimulation. FIGs. 4B-4J illustrate TCEs with three aTAAs, a single aCD3, and CD2 costimulation (CD58). The aTAA and aCD3 binding domains can be composed of various antibody domains or fragments, including sdAbs, nanobodies, VHHs, HCAbs, Fvs, scFvs, Fabs, scFabs, etc. The CD2 costimulation module can be composed of a CD2-binding domain, such as the CD58 extracellular domain, the CD58 IgV domain, an antibody domain or fragment that binds to CD2, etc. The three aTAAs could comprise or consist of three copies of the same binding domain to a single TAA, three binding domains that recognize different epitopes within the same TAA, three binding domains that recognize three different TAAs, or some combination thereof. For simplicity, not all regions of the molecular components are illustrated (e.g., the disulfides connecting the hinge region of the Fc, the CH2 and CH3 domains of the Fc, heterodimerization mutations in the Fc, VH and VL domains and linkers of an scFv, etc., are omitted).

[0084] FIG. 5 is a schematic of the non-targeting control TCE without CD2 costimulation (format 0) of Examples 1-3. The non-targeting control TCE is composed of an anti -green fluorescent protein (aGFP) sdAb, an aCD3 scFv, and no CD2 costimulation.

[0085] FIGs. 6A-6J show schematics of the TCE formats 0-9 targeting three TAAs (CD 19, CD20, and CD22) of Examples 1-3. FIG. 6A illustrates a TCE in format 0 composed of an aCD19 sdAb, an aCD20 sdAb, an aCD22 sdAb, an aCD3 scFv, and no CD2 costimulation. FIGS. 6B-6J illustrate TCEs in formats 1-9 composed of an aCD19 sdAb, an aCD20 sdAb, an aCD22 sdAb, an aCD3 scFv, and CD2 costimulation (CD58 IgV domain) in various configurations.

[0086] FIGs. 7A-7C illustrate T cell-dependent cellular cytotoxicity (TDCC) assays with TCE formats 0-9 and a non-targeting control TCE using CHO huCD19 target cells. FIG. 7B: Cytokine production (IFN-g, IL-2, and TNF-a) in the TDCC assay in FIG. 7A at all concentrations of TCEs tested (0.095 pM to 100 nM). The limit of quantification (LOQ) for IFN-g was 7.3 pg / mL and 18.3 pg / mL for IL-2 and TNF-a. For samples with results below the LOQ (BLQ), the cytokine concentrations are null and have been omitted from the graph. FIG 7C: Cytokine production (IFN- g, IL-2, and TNF-a) in the TDCC assay in FIG. 7A at the highest concentration of TCE tested (100 nM).WSGR Docket No. 69097-702.601

[0087] FIGs. 8A-8B illustrate target cell-independent cytokine production with TCE formats 0-9 of Example 3. FIG. 8A shows cytokine production (IFN-g, IL-2, and TNF-a) in the absence of target cells with TCE formats 0-9 tested at a range of concentrations (0.095 pM to 100 nM). IFN-g production was assessed from a separate experiment. The LOQ for IFN-g was 7.3 pg / mL and 18.3 pg / mL for IL-2 and TNF-a. For samples with results BLQ, the cytokine concentrations are null and have been omitted from the graph. FIG 8B shows cytokine production (IFN-g, IL-2, and TNF-a) in the absence of target cells at the highest concentration of TCEs tested (100 nM). For samples with results BLQ, the cytokine concentrations are null and have been omitted from the graph.

[0088] FIGs. 9A-9B illustrate fratricide with rested T cells and TCE formats 0-9 of Example 3. FIG. 9A outlines a fratricide assay with rested T cells in the absence of target cells and TCE formats 0-9 at 100 nM. Cells were co-stained with Annexin V and SYTOXTM AADvancedTM and analyzed by flow cytometry to distinguish apoptotic, necrotic, and viable cell populations. The percentage of T cells that fall into each quadrant (Q1-Q4) of the two-parameter density plots is reporter for each TCE. FIG. 9B shows fratricide with rested T cells in the absence of target cells and TCE formats 0-9 at a range of TCE concentrations (0.095 pM to 100 nM). The percent of necrotic cells (sum of the percent of cells in QI and Q2 in FIG. 9A) is plotted as a function of TCE concentration.

[0089] FIG. 10 shows Jurkat NF AT reporter activation (TCR signaling) in the absence of target cells with TCE formats 0-9 tested at a range of concentrations (0.004 pM to 200 nM).

[0090] FIG. 11 provides a summary of safety and activity assessments of Examples 2 & 3.

[0091] FIG. 12 illustrates crystal structure of the human CD58 IgV domain bound to the human CD2 IgV domain (PDB 1QA9). The three N-linked glycosylation sites in the CD58 IgV domain (N12, N66, N81) are shown as sticks and labeled.

[0092] FIGs. 13A-13B summarize the expression of CD58 IgV glycovariants. Wild-type (WT) or CD58 IgV glycovariants were produced recombinantly as a single-targeting TCEs in either format 1 (FIG. 13A) or format 2 (FIG. 13B). Proteins were analyzed by SDS-PAGE under reduced (R) or non-reduced (NR) conditions. The yield after Protein A purification for each TCE is reported.

[0093] FIGs. 14A-14B illustrate the binding of the CD58 IgV single glycovariants (FIG. 14A) and double glycovariants (FIG. 14B) in single-targeting TCE format 1 (Fl) and format 2 (F2) to primary human T cells by flow cytometry. A single-targeting TCE in format 0 (F0, no CD58) as well as single-targeting TCEs in Fl and F2 containing WT CD58 IgV were included as controls. All TCEs were tested at a range of concentrations (0.024 ng / mL to 100 pg / mL).WSGR Docket No. 69097-702.601

[0094] FIGs. 15A-15D show schematic diagrams of single-targeting CD 19 TCE Formats 0- 3. FIG. 15A illustrates a TCE in format 0 composed of an aCD19 sdAb, an aCD3 scFv, and no CD2 costimulation. FIGs. 15B-15D illustrate TCEs in Formats 1-3 composed of an aCD19 sdAb, an aCD3 scFv, and CD2 costimulation (CD58 IgV domain) in various configurations.

[0095] FIG. 16 shows data indicating short-term TDCC with single-targeting CD 19 TCE Formats 0-3. Short-term TDCC assay results shown here are with single-targeting CD 19 TCE Formats 0-3 and a non-targeting control TCE using Nalm6 target cells.

[0096] FIGs. 17A-17B shows data indicating target cell-independent cytokine production with single-targeting CD19 TCE Formats 0-3. FIG. 17A: Cytokine production (IFN-y and TNF- a) in the absence of target cells with single-targeting CD 19 TCE formats 0-3 tested at a range of concentrations (1.7 pM to 100 nM). FIG. 17B: Cytokine production (IFN-g and TNF-a) in the absence of target cells at the highest concentration of TCEs tested (100 nM). The LOQ for IFN-y was 7.3 pg / mL and 18.3 pg / mL for TNF-a. For samples with results BLQ, the cytokine concentrations are null and have been omitted from the graphs.

[0097] FIG. 18 shows data indicating fratricide with rested T cells and single-targeting CD 19 TCE Formats 0-3 at a range of TCE concentrations (24.4 pM to 100 nM). The percent of necrotic cells is plotted as a function of TCE concentration.

[0098] FIG. 19 shows data from Jurkat NF AT reporter assay with single-targeting CD 19 TCE formats 0-3. Jurkat NF AT reporter activation (TCR signaling) in the absence of target cells with single-targeting CD19 TCE Formats 0-3 tested at a range of concentrations (0.4 fM to 100 nM).

[0099] FIG. 20 shows data indicating serial TDCC with single-targeting CD 19 TCE Formats 1-3. Serial TDCC assay was carried out with single-targeting CD19 TCE Formats 1-3 using JeKo- 1 target cells. The EC50s for target cell killing are plotted across the serial TDCC assay challenge number. When the EC50 could no longer be calculated due to complete loss of target cell killing activity, 1000 pM was used as the EC50 for the graph, but denoted as not determined (ND).

[0100] FIGs. 21A-21E Schematics of single-targeting DLL3 TCE formats 0-4. FIG. 21A illustrates a TCE in format 0 composed of an aDLL3 sdAb, an aCD3 scFv, and no CD2 costimulation. FIGs. 21B-21E illustrate TCEs in formats 1-4 composed of an aDLL3 sdAb, an aCD3 scFv, and CD2 costimulation (CD58 IgV domain) in various configurations.

[0101] FIG. 22 shows data indicating short-term TDCC with single-targeting DLL3 TCE Formats 0-4. Short-term TDCC assay with single-targeting DLL3 TCE formats 0-4 and a nontargeting control TCE using SHP-77 target cell.

[0102] FIGs. 23A-23B shows data indicating target cell-independent cytokine production with single-targeting DLL3 TCE Formats 0-4. FIG. 23A: Cytokine production (IFN-y and IL-2)WSGR Docket No. 69097-702.601 in the absence of target cells with single-targeting DLL3 TCE Formats 0-4 tested at a range of concentrations (0.095 pM to 100 nM). FIG. 23B: Cytokine production (IFN-y and IL-2) in the absence of target cells at the highest concentration of TCEs tested (100 nM). The LOQ for IFN-y was 7.3 pg / mL and 18.3 pg / mL for IL-2. For samples with results BLQ, the cytokine concentrations are plotted as zero in the graphs.

[0103] FIG. 24 shows data indicating fratricide with rested T cells and single-targeting DLL3 TCE Formats 0-4 at a range of TCE concentrations (24.4 pM to 400 nM). The percent of necrotic cells is plotted as a function of TCE concentration.

[0104] FIG. 25 shows data indicating JurkatNFAT reporter assay with single-targeting DLL3 TCE Formats 0-4. Jurkat NF AT reporter activation (TCR signaling) in the absence of target cells with single-targeting DLL3 TCE formats 0-4 tested at a range of concentrations (0.4 fM to 100 nM).DETAILED DESCRIPTION

[0105] Immunotherapies that harness the host immune system, such as T cell engagers and T cells engineered to express chimeric antigen receptors, have in some cases induced robust anticancer immune responses that lead to improved outcomes for patients. However, in other cases such therapies fail to control the cancer or exhibit loss of efficacy over time, for example, due to antigen escape (e.g., mutation or loss of expression of the target antigen), insufficient stimuli for effective T cell activation (e.g., reduced levels of T cell costimulatory ligands), and / or immunosuppressive signals from cancer cells or the tumor microenvironment.

[0106] Antigen-dependent relapse can significantly limit the therapeutic efficacy of such immunotherapies. For example, a significant proportion of patients treated with CD19-targeting CAR-T cells exhibit loss of CD 19 expression, and a significant proportion of patients treated with CD20-targeting T cell engagers exhibit loss of CD20 expression. Antigen-independent relapse can also be a significant barrier to durable anti-cancer responses, with poor outcomes frequently associated with mutations or loss of T cell costimulatory ligand expression.I. MULTISPECIFIC IMMUNE CELL ENGAGERS

[0107] Disclosed herein are multispecific immune cell engagers, such as multispecific T cell engagers (TCE). Multispecific immune cell engagers comprise one or more (e.g., two) binding domains that bind to target molecules on immune effector cells, such as T cells, and one or more (e.g., two or three) binding domains that bind to target molecules on target cells, such as cancer cells or B cells. Binding to both cells brings the target cell and immune effector cell together into close proximity and facilitates activation of the immune effector cell to induce, for example, targetWSGR Docket No. 69097-702.601 cell killing, activation of the immune effector cell, pro-inflammatory cytokine production, and / or an anti-cancer immune response.

[0108] Multispecific immune cell engagers of the disclosure can comprise multiple binding domains that bind to target molecules on target cells (e.g., on cancer cells or B cells). Such specificity for multiple target cell targets can facilitate retention of binding to target cells and maintained immune responses against the target cells, even if expression of one or more of the target molecules is reduced or lost. For example, if one or more target molecules are mutated or expression lost in a cancer, binding to other target molecules can be retained, thereby circumventing antigen escape. In an illustrative example, multispecific immune cell engagers comprise binding domains that bind CD 19, CD20, and CD22, such that even if cancerous B cells lose expression of one or even two of the target molecules, binding to the cancerous B cells can be retained, along with the corresponding immune effector cell response against the cancerous B cells. Specificity for multiple target cell targets can also confer other advantages, such as improved affinity or avidity for the target cells.

[0109] Multispecific immune cell engagers of the disclosure can comprise multiple binding domains that bind to target molecules on immune effector cells, such as T cells. Binding to one or more of the target molecules on the immune effector cells can enhance activation of the immune effector cell, for example, in the presence of the target cell. For example, the multispecific immune cell engager can comprise a CD3 binding domain (e.g., to bind T cells and / or induce CD3 signaling), and can further comprise a binding domain that binds to a T cell costimulation receptor to induce costimulatory signaling and improve activation of the T cell. In an illustrative example, multispecific immune cell engagers of the disclosure comprise a CD3 binding domain and a CD2 binding domain (e.g., an Ig-like domain of CD58). Costimulatory signaling induced by the immune cell engager can help to prevent or reduce antigen-independent relapse, for example, by providing a costimulatory ligand that remains present even if the cancer cells or cells in the tumor microenvironment lose expression of immune costimulatory ligands, and / or to counteract immune inhibitory ligands that may be present on the cancer cells or in the tumor microenvironment. Specificity for multiple immune effector cell targets can also confer other advantages, such as improved affinity or avidity for the immune effector cells.

[0110] Multispecific immune cell engagers of various configurations are disclosed herein, including configurations exhibiting favorable properties such as robust induction of targetdependent activity (e.g., cytotoxicity or immune effector cell activation, facilitating efficacy), and / or low levels of background / target-independent activity (e.g., associated with improved safety or reduced toxicity). In some embodiments, a multispecific immune cell engager disclosed herein is substantially free of stearic hindrance. In some embodiments, the multispecific immune cellWSGR Docket No. 69097-702.601 engagers presented herein are specifically designed to overcome the technical challenge of protein aggregation. A TCE generated herein can be maintained in a suitable physiologically relevant solution in a substantially non-aggregated form. For example, at least greater than 80%, greater than 90%, greater than 95%, greater than 97%, greater than 98% of the TCE molecules remain in non-aggregated monomeric form. In some embodiments, the multispecific immune cell engagers presented herein are designed for increased safety in vivo. In some embodiments, the multispecific immune cell engagers presented herein are designed to have extended half-life in circulation in vivo. In some embodiments, the multispecific immune cell engagers presented herein are designed to be efficacious in vivo.A. Binding domains

[0111] Multispecific immune cell engagers disclosed herein, such as T cell engagers (TCE), comprise binding domains to bind multiple target molecules, facilitating binding to an immune effector cell (e.g., cytotoxic immune cell such as T cell), and a target cell (e.g., B cell or cancer cell). Binding the immune effector cell and target cell can bring the two into close proximity to facilitate interaction between the two cells, for example, inducing activation of the immune effector cell and killing of the target cell. The multispecific immune cell engagers can comprise one or multiple binding domains that bind target molecules on target cells, for example, two, three, or more target molecules on target cells, facilitating more efficient recognition of target cells and maintained activity if expression of one of the target molecules is reduced or lost (e.g., to help avoid antigen escape if one target molecule is mutated or expression of the target molecule lost in a cancer). The multi specific immune cell engagers can comprise two or more binding domains that bind target molecules on immune effector cells, for example, to facilitate improved activation of the immune effector cell (e.g., via inducing signaling by one or more of the target molecules) and / or improved binding to the immune effector cell.

[0112] A multispecific immune cell engager disclosed herein can comprise any suitable number of binding domains. For example, a multispecific immune cell engager can comprise 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 binding domains. In some embodiments, a multispecific immune cell engager has or comprises at least 1, at least 2, at least3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 binding domains. In some embodiments, a multispecific immune cell engager has or comprises at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, or at most 20 binding domains.WSGR Docket No. 69097-702.601

[0113] In some embodiments, a multispecific immune cell engager has or comprises two binding domains. In some embodiments, a multispecific immune cell engager has or comprises three binding domains. In some embodiments, a multispecific immune cell engager has or comprises four binding domains. In some embodiments, a multispecific immune cell engager has or comprises five binding domains. In some embodiments, a multispecific immune cell engager has or comprises six binding domains. In some embodiments, all binding domains are nonidentical. In some embodiments, at least two of the binding domains are identical. In some embodiments, the at least two identical binding domains are operably linked to Fcl. In some embodiments, the at least two identical binding domains are operably linked to Fc2. In some embodiments, of the two at least identical binding domains, a first identical binding domain is operably linked to Fcl and a second identical binding domain is operably linked to Fc2. In some embodiments, at least one of the binding domains is non-identical compared to other binding domains.

[0114] The multispecific immune cell engager can comprise binding domains that bind to multiple target molecules, which can be a combination of binding domains that bind target molecules on target cells and binding domains that bind target molecules on immune effector cells. For example, a multispecific immune cell engager can comprise five binding domains (e.g., a first, second, third, fourth, and fifth target binding domain), each of which bind to different targets, such as three binding domains that bind target molecules on target cells and two binding domains that bind target molecules on immune effector cells.

[0115] A multispecific immune cell engager disclosed herein can comprise any suitable number of binding domains that bind to target molecules on target cells (e.g., B cells or cancer cells). For example, a multispecific immune cell engager can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 binding domains that bind to target molecules on target cells. In some embodiments, a multispecific immune cell engager has or comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 binding domains that bind to target molecules on target cells. In some embodiments, a multispecific immune cell engager has or comprises at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, or at most 16 binding domains that bind to target molecules on target cells. The target molecules can be the same or different (e.g., mutually exclusive). For instance, the target molecules on target cells can comprise multiple (e.g., two or three) distinct tumor-associated antigens, such as CD 19, CD20, and / or CD22. In some embodiments, the target molecules can comprise different epitopes on the same target molecule.WSGR Docket No. 69097-702.601

[0116] In some embodiments, the multispecific immune cell engager has or comprises one binding domain that binds to target molecules on target cells. In some embodiments, the multispecific immune cell engager has or comprises two binding domains that bind to target molecules on target cells. In some embodiments, the multispecific immune cell engager has or comprises three binding domains that bind to target molecules on target cells. In some embodiments, the multispecific immune cell engager has or comprises four binding domains that bind to target molecules on target cells. In some embodiments, the multispecific immune cell engager has or comprises five binding domains that bind to target molecules on target cells. In some embodiments, the multispecific immune cell engager has or comprises six binding domains that bind to target molecules on target cells.A multispecific immune cell engager disclosed herein can comprise any suitable number of binding domains that bind to target molecules on immune effector cells (e.g., T cells). For example, a multispecific immune cell engager can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 binding domains that bind to target molecules on immune effector cells. In some embodiments, a multispecific immune cell engager has or comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 binding domains that bind to target molecules on immune effector cells. In some embodiments, a multispecific immune cell engager has or comprises at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, or at most 16 binding domains that bind to target molecules on immune effector cells. The target molecules can be the same or different (e.g., mutually exclusive). For example, in some embodiments a multispecific immune cell engager comprises a first target binding domain that binds CD3 and a second target binding domain that binds CD2. In some embodiments, the target molecules can comprise different epitopes on the same target molecule.

[0117] In some embodiments, the multispecific immune cell engager has or comprises one binding domain that binds to target molecules on immune effector cells. In some embodiments, the multispecific immune cell engager has or comprises two binding domains that bind to target molecules on immune effector cells. In some embodiments, the multispecific immune cell engager has or comprises three binding domains that bind to target molecules on immune effector cells. In some embodiments, the multispecific immune cell engager has or comprises four binding domains that bind to target molecules on immune effector cells. In some embodiments, the multispecific immune cell engager has or comprises five binding domains that bind to target molecules on immune effector cells. In some embodiments, the multispecific immune cell engager has or comprises six binding domains that bind to target molecules on immune effector cells.WSGR Docket No. 69097-702.601

[0118] In some embodiments, a multi-specific immune cell engager described herein comprises at least four binding domains arranged in a specific geometric format. In some embodiments, a multi-specific immune cell engager described herein comprises at least four binding domains arranged in a specific geometric format has increased target specificity compared to a multi-specific immune cell engager comprising identical binding domains but arranged in a different geometric format. In some embodiments, a multi-specific immune cell engager described herein comprises at least four binding domains arranged in a specific geometric format has increased target accessibility compared to a multi-specific immune cell engager comprising identical binding domains but arranged in a different geometric format. In some embodiments, a multi-specific immune cell engager described herein comprises at least four binding domains arranged in a specific geometric format has improved synapse formation between target cells and T-cells compared to a multi-specific immune cell engager comprising identical binding domains but arranged in a different geometric format. In some embodiments, a multi-specific immune cell engager described herein comprises at least four binding domains arranged in a specific geometric format has reduced target-independent T-cell activation compared to a multi-specific immune cell engager comprising identical binding domains but arranged in a different geometric format. In some embodiments, a multi-specific immune cell engager described herein comprises at least four binding domains arranged in a specific geometric format has increased T-cell persistence compared to a multi-specific immune cell engager comprising identical binding domains but arranged in a different geometric format. In some embodiments, a multi-specific immune cell engager described herein comprises at least four binding domains arranged in a specific geometric format has enhanced target-dependent T-cell activation compared to a multi-specific immune cell engager comprising identical binding domains but arranged in a different geometric format. In some embodiments, a multi-specific immune cell engager described herein comprises at least four binding domains arranged in a specific geometric format has enhanced potency in TDCC assays compared to a multi-specific immune cell engager comprising identical binding domains but arranged in a different geometric format. In some embodiments, a multi-specific immune cell engager described herein comprises at least four binding domains arranged in a specific geometric format shows lower aggregation compared to a multi-specific immune cell engager comprising identical binding domains but arranged in a different geometric format. In some embodiments, a multi-specific immune cell engager described herein comprises at least four binding domains arranged in a specific geometric format shows better cytotoxicity compared to a multi-specific immune cell engager comprising identical binding domains but arranged in a different geometric format. In some embodiments, a multi-specific immune cell engager described herein comprises at least four binding domains arranged in a specific geometric format shows lower stericWSGR Docket No. 69097-702.601 interference in target binding compared to a multi-specific immune cell engager comprising identical binding domains but arranged in a different geometric format. In some embodiments, the respective linkers are designed for improvement of the spatial arrangement, increasing flexibility or providing adequate support for the individual binders and the Fc regions. In some embodiments, the engager designs are being optimized for length and composition of the linkers that would allow greater flexibility, for example by increasing the number of amino acids in the linker peptide, or conversely, increasing rigidity and support as needed, with respect to the adjoining binding domains. Likewise, the linkers are being optimized for composition of the amino acids, length of the linkers in consideration of individual binding domains that the linkers are linked to. In some embodiments, a multi-specific immune cell engager described herein comprises at least four binding domains arranged in a specific geometric format shows higher therapeutic efficacy compared to a multi-specific immune cell engager comprising identical binding domains but arranged in a different geometric format.1. Antigen-binding domains

[0119] A binding domain disclosed herein can be or can comprise an antigen-binding domain, such as an antibody domain (for example, an antibody -based binding domain), an antigen-binding fragment of an antibody, or a derivative thereof.

[0120] An antigen-binding domain, antibody-based binding domain, or antigen-binding fragment can comprise a portion of an antibody, for example, the antigen-binding or variable region of the intact antibody. Non-limiting examples of antigen-binding domains, antibody-based binding domains, or antigen-binding fragments that can be used include Fab, Fab', F(ab')2, dimers and trimers of Fab conjugates, fragment variable region (Fv), single chain variable fragment (scFv), single chain Fab (scFab), single variable domain (sVD), minibodies, dia-, tria-, and tetrabodies, single domain antibodies (sdAb) heavy chain only antibodies (HCAbs), VHH, nanobodies, and linear antibodies.

[0121] Fab and Fab' are antigen-binding fragments that can comprise the VH and CHI domains of the heavy chain linked to the VL and CL domains of the light chain via a disulfide bond. A F(ab')2 can comprise two Fab or Fab' that are joined by disulfide bonds. An Fv can comprise the VH and VL domains, e.g., held together by non-covalent interactions. An scFv (single-chain variable fragment) is a fusion protein that can comprise VH and VL domains connected by a peptide linker. Manipulation of the orientation of the VH and VL domains and the linker length can be used to create different forms of molecules that can be monomeric, dimericWSGR Docket No. 69097-702.601(diabody), trimeric (triabody), or tetrameric (tetrabody). Minibodies are scFv-CH3 fusion proteins that assemble into bivalent dimers.

[0122] The variable (V) domain(s) of an antibody can mediate antigen binding and define the specificity of a particular antibody for an antigen. The variable domain can comprise relatively invariant sequences called framework regions, and hypervariable regions, which differ considerably in sequence among antibodies of different binding specificities. The variable domain can comprise four framework regions separated by three hypervariable regions. The variable domains can fold in a manner that brings the hypervariable regions together in close proximity to create an antigen binding site. The four framework regions can largely adopt an f -sheet configuration, while the three hypervariable regions form loops connecting, and in some cases forming part of, the f3 -sheet structure. In some embodiments, framework regions of a VH and / or VL comprise one or more engineered mutations. For instance, a VH or VHH domain can comprise an R19T mutation in framework 1 to aid in purification of heterodimers as disclosed herein.

[0123] Within hypervariable regions are amino acid residues that primarily determine the binding specificity of the antibody in most cases. Sequences comprising these residues are known as complementarity determining regions (CDRs). One antigen binding site of an antibody with heavy and light chains or variable domains therefrom can comprise six CDRs, three in the hypervariable regions of the light chain variable domain, and three in the hypervariable regions of the heavy chain variable domain. The CDRs in the light chain are designated LI, L2, and L3, while the CDRs in the heavy chain are designated Hl, H2, and H3. CDRs can also be designated LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3, or LC-CDR-1, LC-CDR-2, LC-CDR- 3, HC-CDR-1, HC-CDR-2, and HC-CDR-3, respectively. The contribution of each CDR to antigen binding varies among antibodies, but in some embodiments heavy chain CDRs, in particular HCDR3, can contribute most to antigen-specific binding. CDRs can vary in length. For example, CDRs are often 5 to 14 residues in length, but CDRs as short as 0, 1, 2, or 3 residues and / or as long as 25 residues or longer exist.

[0124] Certain antibodies or antigen-binding domains contain less than six CDRs. For example, certain antibodies lack a light chain, and can be referred to as heavy chain only antibodies (HCAbs). HCAbs have three CDRs in a variable region that can be referred to as VHH. A single domain antibody, or nanobody, can be generated from such a VHH region of a heavy chain only antibody, for example. In some embodiments, HCAbs, VHH, and / or sdAb are used interchangeably herein.

[0125] A binding domain of the disclosure can comprise complementarity determining regions (CDRs). For example, an antibody, antigen-binding fragment thereof, or antigen-binding domain can comprise CDRs. In some embodiments, the CDRs determine or substantiallyWSGR Docket No. 69097-702.601 determine binding specificity and / or affinity for the target molecule. For example, the CDRs can be grafted onto a different suitable framework, or the framework region can be altered (e.g., via amino acid substitutions, deletions, and / or insertions), the antigen-binding fragment or domain can retain binding for the target, and the binding domain remains functional despite the alterations outside of the CDRs. For example, in some embodiments CDRs from an antibody or sdAb are grafted onto a suitable human framework (e.g., sdAb CDRs can be grafted onto a human VH framework).

[0126] In some embodiments, one or more framework regions or amino acid sequences therein do not contribute to binding specificity and / or affinity. In some embodiments, one or more framework regions or amino acid sequences therein contribute to binding specificity and / or affinity.

[0127] CDRs in or for use in a binding domain or multispecific immune cell engager can be identified by various methods, including but not limited to the Kabat method, the Chothia method, the IMGT method, the AHO method, the AbM method, the contact method, and the Paratome method. For example, CDRs can be identified from any VH, VL, and in some embodiments VHH domains disclosed herein using the Kabat method, the Chothia method, the IMGT method, the AHO method, the AbM method, the contact method, and / or the Paratome method.

[0128] Single domain antibodies can in some embodiments have longer CDR Hl and H3 loops compared with the respective classical CDRs, and different methods can be used to identify to identify CDRs in, for example, VHH, sdAb, or HCAbs. Single domain antibody CDRs can be identified, for example, using the single domain antibody database (SAbDab), based on common sequence elements, or based on a sequence alignment to the Chothia numbering scheme (e.g., as described by Wilton, et al. (2018). sdAb-DB: the single domain antibody database. ACS Synthetic Biology 2018 7 (11), 2480-2484 DOI: 10.1021 / acssynbio.8b00407).

[0129] A subset of residues within CDRs contacts an antigen. These residues that contact antigen can be referred to as specificity-determining residues (SDRs). In some embodiments residues other than SDRs can contribute to binding activity, for example, by helping to maintain the conformation of the binding site. The number of SDRs in an antibody can vary based on the size and type of antigen that is recognized, for example, between 0-14 SDRs can be found within a CDR. SDRs can be enriched in some residues, such as tyrosine, serine, tryptophan, and asparagine.

[0130] A CDR of a sequence or binding domain herein can be, for example, between 0 and 91 residues in length, between 0 and 25 residues in length, between 5 and 14 residues in length, between 2 and 20 residues in length, between 2 and 15 residues in length, between 2 and 12 residues in length, between 2 and 10 residues in length, between 2 and 8 residues in length,WSGR Docket No. 69097-702.601 between 2 and 6 residues in length, between 2 and 5 residues in length, between 2 and 4 residues in length, between 3 and 20 residues in length, between 3 and 15 residues in length, between 3 and 12 residues in length, between 3 and 10 residues in length, between 3 and 8 residues in length, between 3 and 6 residues in length, between 3 and 5 residues in length, between 3 and 4 residues in length, between 4 and 20 residues in length, between 4 and 15 residues in length, between 4 and 12 residues in length, between 4 and 10 residues in length, between 4 and 8 residues in length, between 4 and 6 residues in length, between 4 and 5 residues in length, between 5 and 20 residues in length, between 5 and 15 residues in length, between 5 and 12 residues in length, between 5 and 10 residues in length, between 5 and 8 residues in length, between 5 and 6 residues in length, between 6 and 20 residues in length, between 6 and 15 residues in length, between 6 and 12 residues in length, between 6 and 10 residues in length, or between 6 and 8 residues in length.

[0131] In some embodiments, a CDR is about 0 residues in length, about 1 residue in length, about 2 residues in length, about 3 residues in length, about 4 residues in length, about 5 residues in length, about 6 residues in length, about 7 residues in length, about 8 residues in length, about 9 residues in length, about 10 residues in length, about 11 residues in length, about 12 residues in length, about 13 residues in length, about 14 residues in length, about 15 residues in length, about 16 residues in length, about 17 residues in length, about 18 residues in length, about 19 residues in length, about 20 residues in length, about 21 residues in length, about 22 residues in length, about 23 residues in length, about 24 residues in length, or about 25 residues in length.

[0132] A binding domain can comprise the CDR amino acid sequence(s) of any one or more of the CDR sequences disclosed herein. In some embodiments, a binding domain comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequence selected from the CDR sequences disclosed herein. In some embodiments, a binding domain comprises an HCDR1, HCDR2, and HCDR3 amino acid sequence selected from the CDR sequences disclosed herein.

[0133] A binding domain can comprise one or more variant CDR sequences, e.g., each with at most one amino acid substitution, insertion, or deletion relative to any one of the CDR sequences disclosed herein. In some embodiments, a binding domain comprises one or more variant CDR sequences each with at most two amino acid substitutions, insertions, and / or deletions relative to any one of the CDR sequences disclosed herein. In some embodiments, a binding domain comprises one or more variant CDR sequences each with at most three amino acid substitutions, insertions, and / or deletions relative to any one of the CDR sequences disclosed herein. In some embodiments, a binding domain comprises one or more variant CDR sequences each with at most four amino acid substitutions, insertions, and / or deletions relative to any one of the CDR sequences disclosed herein. In some embodiments, a binding domain comprises one orWSGR Docket No. 69097-702.601 more variant CDR sequences each with at most five amino acid substitutions, insertions, and / or deletions relative to any one of the CDR sequences disclosed herein.

[0134] In some embodiments, a binding domain comprises one or more variant CDR sequences with at least one amino acid substitution, insertion, and / or deletion relative to any one of the CDR sequences disclosed herein. In some embodiments, a binding domain comprises one or more variant CDR sequences with at least two amino acid substitutions, insertions, and / or deletions relative to any one of the CDR sequences disclosed herein. In some embodiments, a binding domain comprises one or more variant CDR sequences with at least three amino acid substitutions, insertions, and / or deletions relative to any one of the CDR sequences disclosed herein. The binding domain can further comprise additional CDRs with no substitutions relative to CDR sequences disclosed herein, for example, one CDR or two variant CDRs can be mutated, and the remaining CDRs can remain unchanged.

[0135] In some embodiments, a binding domain comprises one or more CDR sequences each with 0-1 amino acid substitutions, insertions, and / or deletions relative to any one of the CDR sequences disclosed herein. In some embodiments, a binding domain comprises one or more CDR sequences each with 0-2 amino acid substitutions, insertions, and / or deletions relative to any one of the CDR sequences disclosed herein. In some embodiments, a binding domain comprises one or more CDR sequences each with 0-3 amino acid substitutions, insertions, and / or deletions relative to any one of the CDR sequences disclosed herein.

[0136] In some embodiments, a binding domain comprises one or more variant CDR sequences each with 1-2 amino acid substitutions, insertions, and / or deletions relative to any one of the CDR sequences disclosed herein. In some embodiments, a binding domain comprises one or more variant CDR sequences each with 1-3 amino acid substitutions, insertions, and / or deletions relative to any one of the CDR sequences disclosed herein. In some embodiments, a binding domain comprises one or more variant CDR sequences each with 1-4 amino acid substitutions, insertions, and / or deletions relative to any one of the CDR sequences disclosed herein. In some embodiments, a binding domain comprises one or more variant CDR sequences each with 1-5 amino acid substitutions, insertions, and / or deletions relative to any one of the CDR sequences disclosed herein.

[0137] In some embodiments, a binding domain comprises one or more variant CDR sequences each with 2-3 amino acid substitutions, insertions, and / or deletions relative to any one of the CDR sequences disclosed herein. In some embodiments, a binding domain comprises one or more variant CDR sequences each with 2-4 amino acid substitutions, insertions, and / or deletions relative to any one of the CDR sequences disclosed herein. In some embodiments, a binding domain comprises one or more variant CDR sequences each with 2-5 amino acidWSGR Docket No. 69097-702.601 substitutions, insertions, and / or deletions relative to any one of the CDR sequences disclosed herein.

[0138] In some embodiments, a binding domain comprises one or more variant CDR sequences each with 3-4 amino acid substitutions, insertions, and / or deletions relative to any one of the CDR sequences disclosed herein. In some embodiments, a binding domain comprises one or more variant CDR sequences each with 3-5 amino acid substitutions, insertions, and / or deletions relative to any one of the CDR sequences disclosed herein.

[0139] In some embodiments, a binding domain comprises an HCDR3 amino acid sequence disclosed herein, and comprises HCDR1, HCDR2, LCDR1, LCDR2, and LCDR3 (or, e.g., HCDR1 and HCDR2) sequences each with at most one amino acid substitutions, insertions, and / or deletions relative to sequences disclosed herein. In some embodiments, a binding domain comprises an HCDR3 amino acid sequence disclosed herein, and comprises HCDR1, HCDR2, LCDR1, LCDR2, and LCDR3 (or, e.g., HCDR1 and HCDR2) sequences each with at most two amino acid substitutions, insertions, and / or deletions relative to sequences disclosed herein. In some embodiments, a binding domain comprises an HCDR3 amino acid sequence disclosed herein, and comprises HCDR1, HCDR2, LCDR1, LCDR2, and LCDR3 (or, e g., HCDR1 and HCDR2) sequences each with at most three amino acid substitutions, insertions, and / or deletions relative to sequences disclosed herein. In some embodiments, a binding domain comprises an HCDR3 amino acid sequence disclosed herein, and comprises HCDR1, HCDR2, LCDR1, LCDR2, and LCDR3 (or, e.g., HCDR1 and HCDR2) sequences each with at most four amino acid substitutions, insertions, and / or deletions relative to sequences disclosed herein.

[0140] The binding domain can be or can comprise a single domain antibody. The single domain antibody can be or can comprise a variable region of a heavy chain only antibody. Such a single domain antibody can also be known as a nanobody or VHH. The single domain antibody can be, for example, a variable region from or derived from a heavy chain only antibody from a camelid (e.g., camels: one-humped Camelus dromedaries and two-humped Camelus bactrianus; llamas: Lama glama, Lama guanicoe, and Lama vicugna; and alpacas: Vicugna pacos), a shark (e.g., a nurse shark), a wobbegong, or a spotted ratfish. Such animals have a special type of antibody called heavy chain Abs (HCAbs), that lack the entire light chain and the first heavy chain C region (CHI) compared to regular antibodies.

[0141] A binding domain can comprise an antigen-binding domain or fragment of a chimeric, humanized, or fully human antibody. A binding domain can comprise CDRs grafted onto a humanized or fully human framework sequence. A binding domain can comprise a chimeric antibody wherein a portion of the heavy and / or light chain (e.g., variable region) is identical to or homologous to a corresponding sequence in an antibody derived from a particular species orWSGR Docket No. 69097-702.601 belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, or an antigen-binding fragment of such an antibody. In some embodiments, a binding domain comprises an antigen-binding domain or fragment that is not of a chimeric, humanized, or fully human antibody, for example, from a nonhuman mammalian antibody, a camelid, or another species disclosed herein.

[0142] For human administration, monoclonal antibodies or fragments thereof generated from non-human species that will be used in a binding domain or multispecific immune cell engager can be further refined by a humanization process to reduce the likelihood of immunogenicity while preserving target specificity. Humanization processes can involve the incorporation of human sequences in the amino acid sequences of isolated antibodies (e.g., via manipulation of encoding nucleic acids), and / or the removal of predicted epitopes, such as T cell epitopes.

[0143] A binding domain can bind to an epitope of a target molecule. Non-limiting examples of epitopes include amino acids / amino acid sequences, peptides, sugars, lipids, phosphoryl, and sulfonyl groups. An epitope can have specific three-dimensional structural characteristics, and / or specific charge characteristics. Epitopes can be conformational or linear.

[0144] Additional non-limiting examples of antigen-binding elements that a binding domain can comprise include ankyrin proteins, ankyrin repeat proteins, designed ankyrin repeat proteins (DARPins), affibodies, avimers, adnectins, anticalins, Fynomers, Kunitz domains, knottins, P- hairpin mimetics, and receptors and derivatives thereof.

[0145] In some embodiments, a multispecific immune cell engager disclosed herein can comprise a binding domain that can be or can comprise an antigen-binding domain, such as an antibody domain (for example, an antibody-based binding domain), an antigen-binding fragment of an antibody, or a derivative thereof and / or a binding domain that can be or can comprise a ligand-based domain or a receptor-based domain, for example, from a receptor-ligand pair.2. Ligand-based and receptor-based binding domains

[0146] A binding domain disclosed herein can be or can comprise a ligand-based domain or a receptor-based domain, for example, from a receptor-ligand pair. For example, a binding domain can comprise a receptor-binding domain of a ligand, or a ligand-binding domain of a receptor. Such binding domains in some cases utilize the naturally-occurring specificity of a receptor-ligand pair.

[0147] A binding domain that binds to a target molecule on an immune effector cell can be a receptor-based or ligand-based domain. For example, a binding domain disclosed herein can comprise a CD2-binding domain of CD58, a CD2-binding domain of CD48, a CD28-bindingWSGR Docket No. 69097-702.601 domain of B7H1 or B7H2, a 41BB binding domain of 41BBL, an 0X40 binding domain of OX40L, a 2B4-binding domain of CD48, or a CD27-binding domain of CD70.

[0148] A binding domain disclosed herein can comprise CD58, or a CD2-binding domain of CD58 (Lymphocyte function-associated antigen 3 / LFA3). The binding domain can comprise, for example, an extracellular domain of CD58 (e.g., a full extracellular domain or a CD2-binding domain thereof). The binding domain can comprise, for example, an Ig-like domain or IgV domain of CD58, or a CD2-binding domain thereof. In some embodiments, the binding domain comprises an IgC domain or IgC and IgV domains of CD58. Illustrative sequences of CD58 and CD2-binding domains of CD58 are provided in SEQ ID NOs: 79-88 in TABLE 7.

[0149] The CD58 or CD2-binding domain thereof (e.g., extracellular domain, Ig-like domain, or IgV domain of CD28) can be a variant of CD58, for example, comprising one or more substitutions, deletions or insertions. The CD58 or CD2-binding domain thereof can comprise one or more mutation(s) to remove glycosylation sites. Such glycoengineering of a domain from CD58 can confer advantageous properties to the expression titer, immune effector cell / T cell / CD2- binding, and / or homogeneity of a multispecific immune cell engager disclosed herein.

[0150] For example, where the N-linked glycosylation sites at residues N12, N66, N81, N107, 141, and / or 167 of the mature polypeptide chain are present in the binding domain (i.e., residues N40, N94, N109, N135, N169, and / or N195 of CD58 if the signal peptide is included), one or more of those residues can be mutated to remove the glycosylation site. In some embodiments, residues corresponding to the N-linked glycosylation sites at residues N12, N66, and / or N81 of the mature polypeptide chain are present in the binding domain (i.e., residues 40, 94, and / or 109 of CD58 if the signal peptide is included), and one or more are mutated to remove the N-linked glycosylation site.

[0151] In some embodiments a binding domain comprises a CD2-binding domain (e.g., extracellular domain, Ig-like domain, or IgV domain) of CD58, and comprises a substitution or deletion of the asparagine corresponding to residue N12 of the mature CD58 polypeptide chain.

[0152] In some embodiments a binding domain comprises a CD2-binding domain (e.g., extracellular domain, Ig-like domain, or IgV domain) of CD58, and comprises a substitution or deletion of the asparagine corresponding to residue N66.

[0153] In some embodiments a binding domain comprises a CD2-binding domain (e.g., extracellular domain, Ig-like domain, or IgV domain) of CD58, and comprises a substitution or deletion of the asparagine corresponding to residue N81.

[0154] In some embodiments a binding domain comprises a CD2-binding domain (e.g., extracellular domain, Ig-like domain, or IgV domain) of CD58, and comprises substitutions or deletions of the asparagines corresponding to residues N12 and N66 of the mature CD58WSGR Docket No. 69097-702.601 polypeptide chain. In some embodiments a binding domain comprises a CD2-binding domain (e.g., extracellular domain, Ig-like domain, or IgV domain) of CD58, and comprises substitutions or deletions of the asparagines corresponding to residues N12 and N81 of the mature CD58 polypeptide chain. In some embodiments a binding domain comprises a CD2-binding domain (e.g., extracellular domain, Ig-like domain, or IgV domain) of CD58, and comprises substitutions or deletions of the asparagines corresponding to residues N66 and N81 of the mature CD58 polypeptide chain. In some embodiments a binding domain comprises a CD2-binding domain (e.g., extracellular domain, Ig-like domain, or IgV domain) of CD58, and comprises substitutions or deletions of the asparagines corresponding to residues N12, N66, and N81 of the mature CD58 polypeptide chain. The amino acids can be substituted to any suitable residue, in some embodiments glutamine (e.g., N12Q, N66Q, and / or N81Q mutations).

[0155] In some embodiments a binding domain comprises a CD2-binding domain (e.g., extracellular domain, Ig-like domain, or IgV domain) of CD58, and contains a wild type N12 residue relative to the mature CD58 polypeptide chain. In some embodiments a binding domain comprises a CD2-binding domain (e.g., extracellular domain, Ig-like domain, or IgV domain) of CD58, and comprises a wild type N66 residue relative to the mature CD58 polypeptide chain. In some embodiments a binding domain comprises a CD2-binding domain (e.g., extracellular domain, Ig-like domain, or IgV domain) of CD58, and comprises a wild type residue N81 residue relative to the mature CD58 polypeptide chain.

[0156] In some embodiments, a multispecific immune cell engager comprising a CD2-binding domain of CD58 with one or more glycosylation sites removed as disclosed herein exhibits improved yield in an expression system, for example, at least about 10%, at least about 25%, at least about 50%, at least about 2-fold, at least about 3-fold, at least about 5-fold, at least about 10- fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold at least about 500-fold, at least about 750-fold, at least about 1000-fold, at least about 2500-fold, at least about 5000-fold improved yield compared to a corresponding multispecific immune cell engager comprising a CD2-binding domain of CD58 that includes the one or more glycosylation sites.

[0157] In some embodiments, a multispecific immune cell engager comprising a CD2-binding domain of CD58 with one or more glycosylation sites removed as disclosed herein exhibits improved binding to target molecule(s) (for example, on immune effector cells, on target cells, or a combination thereof), immune effector cells, and / or target cells. For example, at least about 10%, at least about 25%, at least about 50%, at least about 2-fold, at least about 3-fold, at least about 5- fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold at least about 500-fold, at leastWSGR Docket No. 69097-702.601 about 750-fold, at least about 1000-fold, at least about 2500-fold, or at least about 5000-fold improved binding to one or more target molecules, target cells, or immune effector cells as compared to a corresponding multispecific immune cell engager comprising a CD2-binding domain of CD58 that includes the one or more glycosylation sites.

[0158] A binding domain that binds to a target molecule on a target cell can be a receptorbased or ligand-based domain. For example, a binding domain can comprise a receptor-binding or ligand-binding domain of APRIL, BAFF, B7H6, CD16, CD27, CTLX, DNAM-1, E13Y IL13, adnectin, EPHRINB2, FLT3L, FSH, GMCSF, ICAM-I, IL10, IL11, LFA-1, MICA, MICB, MPL, Nectin-2, or NKG2D.3. Illustrative binding domain sequences

[0159] Non-limiting examples of sequences of binding domains and components thereof are provided in TABLES 1-10.

[0160] A multispecific immune cell engager can comprise a binding domain that binds to CD19 as a target molecule, e.g., on target cells. Illustrative, non-limiting examples of binding domains that bind to CD19 are provided in TABLE 1. For example, SEQ ID NOs: 1-21 provide sequences of sdAbs that bind to CD 19, SEQ ID NOs: 22 and 25 provide illustrative scFvs that bind to CD 19, and SEQ ID NOs: 23, 24, and 26-35 provide illustrative VH and VL sequences of CD19-binding antibodies. The VH and VL sequences can be adapted into, for example, an scFv format by joining a suitable VH and VL pair, e.g., via a flexible peptide linker.

[0161] In some embodiments, a binding domain that binds to CD 19 comprises, consists essentially of, or consists of an amino acid sequence with at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or sequence similarity to any one of SEQ ID NOs: 1-35. The binding domain can comprise two such sequences, for example, a VH and a VL.

[0162] Illustrative examples of CDRs of antibodies that bind CD 19 that can be used in a multispecific immune cell engager disclosed herein are provided in TABLE 2. A multispecific immune cell engager can comprise a binding domain that binds to CD 19 and comprises CDRs (e.g., a set of three or six CDRs) selected from the CDRs in TABLE 2, or variants thereof asWSGR Docket No. 69097-702.601 disclosed herein. In some embodiments, the binding domain comprises a set of CDRs corresponding to a given antibody ID, or variants thereof described herein. In some embodiments, the binding domain comprises a set of CDRs including two or more CDRs corresponding to different antibody IDs, or variants thereof described herein.

[0163] TABLE 1 : Illustrative CD 19 binding domain sequencesWSGR Docket No. 69097-702.601

[0164] TABLE 2: illustrative anti-CD19 CDRsWSGR Docket No. 69097-702.601WSGR Docket No. 69097-702.601WSGR Docket No. 69097-702.601WSGR Docket No. 69097-702.601WSGR Docket No. 69097-702.601WSGR Docket No. 69097-702.601

[0165] A multispecific immune cell engager can comprise a binding domain that binds to CD20 as a target molecule, e.g., on target cells. Illustrative, non-limiting examples of binding domains that bind to CD20 are provided in TABLE 3. For example, SEQ ID NOs: 36-46 provide sequences of sdAbs that bind to CD20, and SEQ ID NOs: 47-54 provide illustrative VH and VL sequences of CD20-binding antibodies. The VH and VL sequences can be adapted into, for example, an scFv format by joining a suitable VH and VL pair, e.g., via a flexible peptide linker.

[0166] In some embodiments, a binding domain that binds to CD20 comprises, consists essentially of, or consists of an amino acid sequence with at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or sequence similarity to any one of SEQ ID NOs: 36-54. The binding domain can comprise two such sequences, for example, a VH and a VL.

[0167] Illustrative examples of CDRs of antibodies that bind CD20 that can be used in a multispecific immune cell engager disclosed herein are provided in TABLE 4. A multispecific immune cell engager can comprise a binding domain that binds to CD20 and comprises CDRs (e.g., a set of three or six CDRs) selected from the CDRs in TABLE 4, or variants thereof as disclosed herein. In some embodiments, the binding domain comprises a set of CDRs corresponding to a given antibody ID, or variants thereof described herein. In some embodiments, the binding domain comprises a set of CDRs including two or more CDRs corresponding to different antibody IDs, or variants thereof described herein.

[0168] TABLE 3 : Illustrative anti-CD20 binding domain sequencesWSGR Docket No. 69097-702.601

[0169] TABLE 4: Illustrative anti-CD20 CDRs.WSGR Docket No. 69097-702.601WSGR Docket No. 69097-702.601WSGR Docket No. 69097-702.601

[0170] TABLE 5 : Illustrative CD22 binding domains.WSGR Docket No. 69097-702.601WSGR Docket No. 69097-702.601

[0171] A multispecific immune cell engager can comprise a binding domain that binds to CD22 as a target molecule, e.g., on target cells. Illustrative, non-limiting examples of binding domains that bind to CD22 are provided in TABLE 5. For example, SEQ ID NOs: 55-74 provide sequences of sdAbs that bind to CD22, and SEQ ID NOs: 75-78 provide illustrative VH and VL sequences of CD22-binding antibodies. The VH and VL sequences can be adapted into, for example, an scFv format by joining a suitable VH and VL pair, e.g., via a flexible peptide linker.

[0172] In some embodiments, a binding domain that binds to CD22 comprises, consists essentially of, or consists of an amino acid sequence with at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or sequence similarity to any one of SEQ ID NOs: 55-78. The binding domain can comprise two such sequences, for example, a VH and a VL.

[0173] Illustrative examples of CDRs of antibodies that bind CD22 that can be used in a multispecific immune cell engager disclosed herein are provided in TABLE 6. A multispecific immune cell engager can comprise a binding domain that binds to CD22 and comprises CDRs (e.g., a set of three or six CDRs) selected from the CDRs in TABLE 6, or variants thereof as disclosed herein. In some embodiments, the binding domain comprises a set of CDRs corresponding to a given antibody ID, or variants thereof described herein. In some embodiments, the binding domain comprises a set of CDRs including two or more CDRs corresponding to different antibody IDs, or variants thereof described herein.

[0174] TABLE 6: illustrative anti-CD22 CDRsWSGR Docket No. 69097-702.601WSGR Docket No. 69097-702.601WSGR Docket No. 69097-702.601WSGR Docket No. 69097-702.601

[0175] A multispecific immune cell engager can comprise a binding domain that binds to CD2 as a target molecule, e.g., on immune effector cells. Illustrative, non-limiting examples of binding domains that bind to CD2 are provided in TABLE 7. For example, SEQ ID NOs: 79-86 provide illustrative sequences of IgV domains of CD58, SEQ ID NOs: 87 and 88 provide the full length and full extracellular domain sequence of CD58, respectively, SEQ ID NOs: 89 provides an illustrative scFv that binds to CD2, and SEQ ID NOs: 90 & 91 provide illustrative VH and VL sequences of a CD2-binding antibody. The VH and VL sequences can be adapted into, for example, an scFv format by joining a suitable VH and VL pair, e.g., via a flexible peptide linker.WSGR Docket No. 69097-702.601

[0176] In some embodiments, a binding domain that binds to CD2 comprises, consists essentially of, or consists of an amino acid sequence with at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or sequence similarity to any one of SEQ ID NOs: 79-91. The binding domain can comprise two such sequences, for example, a VH and a VL.

[0177] Illustrative examples of CDRs of antibodies that bind CD2 that can be used in a multispecific immune cell engager disclosed herein are provided in TABLE 8. A multispecific immune cell engager can comprise a binding domain that binds to CD2 and comprises CDRs (e.g., a set of three or six CDRs) selected from the CDRs in TABLE 8, or variants thereof as disclosed herein. In some embodiments, the binding domain comprises a set of CDRs corresponding to a given antibody ID, or variants thereof described herein.

[0178] TABLE 7 : Illustrative CD2 binding domainsWSGR Docket No. 69097-702.601

[0179] TABLE 8: illustrative anti-CD2 CDRs

[0180] A multispecific immune cell engager can comprise a binding domain that binds to CD3 as a target molecule, e.g., on immune effector cells. Illustrative, non-limiting examples of binding domains that bind to CD3 are provided in TABLE 9. For example, SEQ ID NOs: 92, 95, 98, & 101 provide illustrative scFvs that bind to CD3, and SEQ ID NOs: 93, 94, 96, 97, 99, 100, and 102-107 provide illustrative VH and VL sequences of CD3-binding antibodies. The VH and VL sequences can be adapted into, for example, an scFv format by joining a suitable VH and VL pair, e.g., via a flexible peptide linker.

[0181] In some embodiments, a binding domain that binds to CD3 comprises, consists essentially of, or consists of an amino acid sequence with at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%,WSGR Docket No. 69097-702.601 at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or sequence similarity to any one of SEQ ID NOs: 92-107. The binding domain can comprise two such sequences, for example, a VH and a VL.

[0182] Illustrative examples of CDRs of antibodies that bind CD3 that can be used in a multispecific immune cell engager disclosed herein are provided in TABLE 10. A multispecific immune cell engager can comprise a binding domain that binds to CD3 and comprises CDRs (e.g., a set of three or six CDRs) selected from the CDRs in TABLE 10, or variants thereof as disclosed herein. In some embodiments, the binding domain comprises a set of CDRs corresponding to a given antibody ID, or variants thereof described herein. In some embodiments, the binding domain comprises a set of CDRs including two or more CDRs corresponding to different antibody IDs, or variants thereof described herein.

[0183] TABLE 9 : illustrative CD3 binding domainsWSGR Docket No. 69097-702.601

[0184] TABLE 10: Illustrative anti-CD3 CDRs.WSGR Docket No. 69097-702.601WSGR Docket No. 69097-702.601WSGR Docket No. 69097-702.601WSGR Docket No. 69097-702.601WSGR Docket No. 69097-702.601B. Target molecules

[0185] Multispecific immune cell engagers (such as T cell engagers) disclosed herein bind to target molecules on immune effector cells and target cells, thereby promoting interaction between the two cells, for example, inducing activation of the immune effector cell and killing of the target cell. The multispecific immune cell engagers can bind to multiple target molecules on target cells and / or immune cell engagers as disclosed herein.4. Target cells

[0186] Multispecific immune cell engagers can bind one or multiple target molecules on target cells, for example, two, three, or more target molecules on target cells. Binding to multiple target molecules on target cells can allow more effective recognition of target cells and can facilitate, for example, retained activity if expression of one of the target molecules is reduced or lost (e.g., if one or more target molecules are mutated or expression lost in a cancer, binding to other target molecules can be retained, thereby circumventing antigen escape).

[0187] A multispecific immune cell engager can bind to multiple target molecules on target cells as disclosed herein. For example, an immune cell engager can bind to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 target molecules on target cells, at least at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 target molecules on target cells, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 target molecules on target cells.WSGR Docket No. 69097-702.601

[0188] In some embodiments, a multispecific immune cell engager binds to one target molecule on target cells. In some embodiments, a multispecific immune cell engager binds to two target molecules on target cells. In some embodiments, a multispecific immune cell engager binds to three target molecules on target cells. In some embodiments, a multispecific immune cell engager binds to four target molecules on target cells. In some embodiments, a multispecific immune cell engager binds to five target molecules on target cells.

[0189] In an illustrative example, a multispecific immune cell engager binds to five target molecules, including three target molecules on target cells (e.g., first, second, and third target molecules, or third, fourth, and fifth target molecules of the total five).

[0190] Target molecules on target cells can comprise multiple (e.g., two or three) distinct tumor-associated antigens, such as CD 19, CD20, and CD22.

[0191] A target cell can be a cell that is associated with a disease or condition. A target cell can be a cancer cell. A target cell can be an immune cell. A target cell can be a hematologic cancer cell. A target cell can be a solid tumor cell. A target cell can be a leukemia cell. A target cell can be a lymphoma cell. A target cell can be a myeloma cell. A target cell can be a B cell. A target cell can be a CD19+ cell. A target cell can be a cell that is associated with an autoimmune disease. A target cell can be a cell that is associated with an inflammatory disease.

[0192] A target molecule on a target cell can a cell surface molecule, for example, a membrane protein or a target molecule present on the surface of the target cell.

[0193] A target molecule on a target cell can be or can comprise a cancer-associated target molecule, for example, a tumor-associated antigen (TAA). In some embodiments, a TAA is an antigen present in a wild type protein or encoded by a wild type genome. The TAA in some embodiments is abnormally expressed (e.g., expressed more highly) in a cancerous cell. In some embodiments, a TAA is expressed on both normal and cancerous cells but can serve as a target for eliminating cancerous cells (e.g., for targeting cancerous B cells via expression of CD19 and other B cell markers).

[0194] In some embodiments, a TAA comprises a mutation, for example, a neoantigen or neoepitope. In some cases, the target molecule on a target cell comprises a neoepitope. Cancerspecific mutations can arise, for example, as a result of missense, splice-site, frameshift or read- through point mutations, or from the fusion of two genes (or within the same gene). Splice-site, frameshift, and read-through mutations and gene fusions can generate novel stretches of amino acids that are normally not translated, but now are expressed and translated as a result of mutation. Missense mutations can lead to neoepitopes with single amino acid changes.

[0195] In some cases, target molecule on a target cell comprises a B cell marker, for example, a factor that is expressed by B cells and / or associated with B cell lineage. For example, in someWSGR Docket No. 69097-702.601 embodiments target molecules on target cells comprise one or more of CD 19, CD20, CD22, CD33, CD37, CD38, CD79a, CD79b, CD123, CD138, BAFF-R, BCMA, CS1, GPRC5D, and TACI. In some embodiments target molecules comprise two or more of CD 19, CD20, CD22, CD33, CD37, CD38, CD79a, CD79b, CD123, CD138, BAFF-R, BCMA, CS1, GPRC5D, and TACI. In some embodiments target molecules comprise three or more of CD 19, CD20, CD22, CD33, CD37, CD38, CD79a, CD79b, CD123, CD138, BAFF-R, BCMA, CS1, GPRC5D, and TACI. In some embodiments target molecules comprise four or more of CD 19, CD20, CD22, CD33, CD37, CD38, CD79a, CD79b, CD123, CD138, BAFF-R, BCMA, CS1, GPRC5D, and TACI. In some embodiments target molecules comprise five or more of CD 19, CD20, CD22, CD33, CD37, CD38, CD79a, CD79b, CD123, CD138, BAFF-R, BCMA, CS1, GPRC5D, and TACI.

[0196] In some embodiments, target molecules on target cells comprise CD 19, CD20, and / or CD22.

[0197] In some embodiments, the target molecule comprises one or more B cell markers expressed at one or multiple stages of B cell development, for example, a B lymphocyte progenitor, Pre-Pro B Cell, Pro-B Cell, Pre-B cell, immature B cell, mature B cell, plasma cell, memory B cell, or a combination thereof. In some embodiments, a target molecule is a B cell marker that is not expressed at one or more stages of B cell development, for example, not expressed or lowly expressed by a common lymphoid progenitor, B lymphocyte progenitor, Pre- Pro B Cell, Pro-B Cell, Pre-B cell, immature B cell, mature B cell, plasma cell, memory B cell or a combination thereof.

[0198] In some embodiments, a target molecule on a target cell is or comprises ACE2, an Fc domain, APRIL, BAFFR, B7H6, B7H3, BCMA, CA9, CAIX, carcinoembryonic antigen, CD133, CD16, CD174, CD22, CD23, CD27, CD274, CD276, CD33, CD38, CD44, CD5, CD70, CEACAM5, CSPG4, CTLX, DLL3, DNAM-1, Dsg3, E13 Y IL13, E3 adnectin, EGFR, EGFRvIII, Envs, EPCAM, EPHA2, EPHB4, EPHRIN B2, ErbB, ERBB2, fibroblast activation protein, FLT3, FLT3L, FOLH1, FOLR1, FSH, FSHR, GD2, glycoprotein B, glycoprotein E2, GMCSF, GMR, gpl20, gp41, GPC3, GPNMB, HBsAg, HER2, ICAM-I, IL10, IL10R, IL11, ILl lRa, IL13Ra2, IL1RAP, IL3RA, Insulin-B chain, Islet-specific glucose-6-phosphatase catalytic subunit-related protein, KDR, L1CAM, LFA-1, M2e, mesothelin, MET, MICA, MICB, MPL, MS4A1, MUC1, myelin oligodendrocyte glycoprotein, NCAM1, Nectin-2, NKG2D, NKp30, PDCD1, PSCA, PSMA, PVR, ROR1, SARS-CoV2 S protein, SDC1, SLAMF7, SSTR, STEAP1, STEAP2, TIE, TACI, TEM1, TNFRSF17, TNFRSF8, TPO, transmembrane form of IgE, TriPRIL, ULBP1, ULBP1-6, ULBP2, or VEGFR2.WSGR Docket No. 69097-702.601

[0199] In some embodiments, the target molecules can comprise different epitopes on the same target molecule.

[0200] In some embodiments, a multispecific immune cell engager can bind to multiple target molecules that are part of a receptor-ligand pair. In some cases, the multiple target molecules are bound by one binding domain each. In some embodiments, multiple target molecules are bound by a single binding domain, for example, all BAFF receptors (e.g., BAFF-R, BCMA, and TACI) can be bound by BAFF or a domain therefrom; all APRIL receptors (e.g., BCMA and TACI) can be bound by APRIL or a domain therefrom. In some embodiments, one or more binding domains bind a single target molecule each, and one or more other binding domains bind multiple target molecules each.5. Immune effector cells

[0201] Multispecific immune cell engagers can bind target molecules on immune effector cells (e.g., T cells), for example, to induce activation of the immune effector cells in proximity to target cells. Both immune effector cells and target cells can be immune cells. For example, immune effector cells can be cytotoxic cells and / or T cells, and target cells can be immune cells that are cancerous or associated with an autoimmune disease (e.g., B cells).

[0202] A multispecific immune cell engager can bind to multiple target molecules on immune effector cells as disclosed herein. For example, an immune cell engager can bind to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 target molecules on immune effector cells, at least at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 target molecules on immune effector cells, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 target molecules on immune effector cells.

[0203] In some embodiments, a multispecific immune cell engager binds to one target molecule on immune effector cells. In some embodiments, a multispecific immune cell engager binds to two target molecules on immune effector cells. In some embodiments, a multispecific immune cell engager binds to three target molecules on immune effector cells. In some embodiments, a multispecific immune cell engager binds to four target molecules on immune effector cells. In some embodiments, a multispecific immune cell engager binds to five target molecules on immune effector cells.

[0204] In an illustrative example, a multispecific immune cell engager binds to five target molecules, including two target molecules on immune effector cells (e.g., first and second target molecules, or fourth and fifth target molecules from the total five).WSGR Docket No. 69097-702.601

[0205] Multispecific immune cell engagers disclosed herein can bind two or more target molecules on immune effector cells, for example, to facilitate improved binding to and / or activation of the immune effector cell.

[0206] In some embodiments, one or more of the binding domains that binds to a target molecule on an immune effector cell induces signaling facilitated by the target molecule, for example, to promote immune cell activation, cytolytic activity, killing of target cells (e.g., cancer cells or B cells), proliferation, survival, persistence, cytokine (e.g., pro-inflammatory cytokine) production, chemokine production, chemotaxis, cytolytic activity, reduced exhaustion, enhanced immune effector functions, enhanced anti-cancer immune response, cellular differentiation (e.g., memory and / or effector differentiation), or a combination thereof.

[0207] A multispecific immune cell engager disclosed herein can bind to one or multiple target molecules on immune effector cells (e.g., T cells). For example, in some embodiments a multispecific immune cell engager binds CD3 and / or CD2. In some embodiments, a multispecific immune cell engager binds CD3 and / or CD28, CD3 and / or CD27, CD3 and / or 4 IBB, CD3 and / or 0X40, CD3 and / or a T cell costimulation receptor, CD3 and / or a costimulation associated target molecule, or CD3 and / or another target molecule disclosed herein.

[0208] A target molecule on an immune effector cell can be a T cell-associated target molecule. Non-limited examples of T cell associated target molecules include CD2, CD3, CD4, CD8, TCR (e.g., alpha, beta, gamma, or delta chain, such as a constant or conserved part of a variable region thereof), CD28, 4 IBB, and 0X40.

[0209] In some embodiments, a target molecule on an immune effector cell is associated with CD4+ T cells (e.g., CD4), CD8+ T cells (e.g., CD8), cytotoxic T lymphocytes, alpha-beta T cells (e.g., alpha or beta chain of TCR), or gamma-delta T cells (e.g., gamma or delta chain of TCR).

[0210] A target molecule on an immune effector cell can be a costimulation-associated target molecule. Non-limited examples of costimulation associated target molecules include CD2, CD28, 4 IBB, 0X40, and other molecules disclosed herein.

[0211] Non-limiting examples of target molecules that can be bound on immune effector cells include CD2, CD3, CD27, CD28, 41BB, 0X40, CD21, CD226 (DNAM1), CD30 (TNFRSF8), CD4, CD40, CD8, CD84 (SLAMF5), CRACC (CD319, BLAME), CRTAM (CD355), DcR3, DR3 (TNFRSF25), GITR (CD357), HVEM (CD270), ICOS (CD278), LIGHT, LTpR (TNFRSF3), Lyl08 (NTBA,CD352,SLAMF6), Ly9 (CD229,SLAMF3), SLAM (CD150,SLAMFl), TIM1 (HAVCR1,KIM1), TIM2, CD 100 (SEMA4D), CD 16 (FcgRIIIA), CD 160 (BY55), CD244 (2B4, SLAMF4), CD94-NKG2C, CD94-NKG2E, CD94-NKG2H, CD96, CRTAM, DAP12, DNAM1 (CD226), KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, Ly49,WSGR Docket No. 69097-702.601NCR, NKG2D (KLRK1, CD314), NKp30 (NCR3), NKp44 (NCR2), NKp46 (NCR1), NKp80 (KLRF1, CLEC5C), NTB-A (SLAMF6), PSGL1, and SLAMF7 (CRACC, CS1, CD319).

[0212] In some embodiments, a target molecule on an immune effector cell is associated with (for example, immune effector cells can be of the cell type): Natural killer cells (NKs), Thl cells, Th2 cells, Thl7 cells, Th9 cells, naive T cells, memory T cells, effector T cells, effector-memory T cells (TEM), central memory T cells (TCM), resident memory T cells (TRM), follicular helper T cells (TFH), Natural killer T cells (NKTs), tumor-infiltrating lymphocytes (TILs), Innate Lymphoid Cells (ILCs), myeloid cells, monocytes, macrophages, Ml macrophages, neutrophils, dendritic cells, plasmacytoid dendritic cells, or combinations thereof.

[0213] A target molecule on an immune effector cell can be associated with NK or NKT cells.Non-limited examples of NK or NKT associated target molecules include CD 16, 2B4, NKG2D, NKp30, NKp44, NKp46, and other markers disclosed herein.

[0214] In some embodiments, a target molecule on an immune effector cell is associated with an immune effector cell type that is not a T cell.C. Fc region and immunoglobulin constant domains

[0215] A multispecific immune cell engager can comprise an immunoglobulin constant domain, such as an Fc region. Linking binding domain(s) disclosed herein to an immunoglobulin constant domain or Fc region can facilitate, for example, a simpler dosing regimen and / or longer half-life of the multispecific immune cell engager within a subject, and can further facilitate heterodimerization of two polypeptide chains as disclosed herein.

[0216] An immunoglobulin constant domain can be described with reference to the basic four chain antibody unit, which comprises two heavy chain (H) polypeptide sequences and two light chain (L) polypeptide sequences. Each of the heavy chains of the basic four chain antibody unit can comprise one N-terminal heavy chain variable domain (VH) and three or four C-terminal constant domains (CHI, CH2, and CH3, and in some cases CH4). Each of the light chains of the basic four chain antibody unit can comprise one N-terminal light chain variable domain (VL) and one C-terminal constant (CL) domain. The light chain variable domain in the basic four chain antibody unit is aligned with the heavy chain variable domain and the light chain constant domain is aligned with heavy chain constant domain CHI. Each light chain is linked to a heavy chain by one covalent disulfide bond. The two heavy chains are linked to each other by one or more disulfide bonds depending on the heavy chain isotype. Each heavy and light chain also comprises regularly-spaced intrachain disulfide bridges. The C-terminal constant domains of the heavy chains (e.g., CH2 and CH3, or CH2, CH3, and CH4) comprise the Fc region, Fc domain, or Fc fragment of the antibody, which can mediate effector functions, for example, through interactionsWSGR Docket No. 69097-702.601 with Fc receptors or complement proteins. The terms “VH” and “HV” can be used interchangeably herein to refer to a heavy chain variable domain. The terms “VL” and “LV” can be used interchangeably herein to refer to a light chain variable domain.

[0217] The light chain can be designated kappa or lambda based on the amino acid sequence of the constant region. The heavy chain can be designated alpha, delta, epsilon, gamma, or mu based on the amino acid sequence of the constant region. Antibodies can be categorized into five immunoglobulin classes, or isotypes, based on the heavy chain. IgA comprises alpha heavy chains, IgD comprises delta heavy chains, IgE comprises epsilon heavy chains, IgG comprises gamma heavy chains, and IgM comprises mu heavy chains. Antibodies of the IgG, IgD, and IgE classes comprise monomers of the four chain unit described above (two heavy and two light chains), while the IgM and IgA classes can comprise multimers of the four chain unit. The alpha and gamma classes are further divided into subclasses on the basis of differences in the sequence and function of the heavy chain constant region. Subclasses of IgA and IgG expressed by humans include IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.

[0218] Generally, the constant regions of an antibody can mediate various effector functions, while the variable regions primarily mediate antigen binding. Different IgG isotypes or subclasses can be associated with different effector functions or therapeutic characteristics, for example, because of interactions with different Fc receptors and / or complement proteins. Multispecific immune cell engagers comprising immunoglobulin constant domains or Fc regions that engage activating Fc receptors can, for example, participate in antibody-dependent cell-mediated cytotoxicity (ADCC), T cell-dependent cellular cytotoxicity (TDCC), and / or in some embodiments, antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), induction of signaling through immunoreceptor tyrosine-based activation motifs (ITAMs), and induction of cytokine secretion. In some embodiments, immunoglobulin constant domains in a multispecific immune cell engager are modified to reduce or modulate such effector functions.

[0219] Different antibody subclasses comprise varying abilities to elicit immune effector functions, which can be utilized or modified in multispecific immune cell engagers disclosed herein. For example, wild type IgGl and IgG3 can effectively recruit complement to activate CDC, and IgG2 elicits minimal ADCC. IgG4 has a lesser ability to trigger immune effector functions and can be used, e.g., where reduced immune effector functions triggered by the immunoglobulin constant domain are desired.

[0220] Multispecific immune cell engagers disclosed herein can comprise an immunoglobulin constant domain from a heavy chain and / or light chain of an antibody isotype, class, or subclass disclosed herein. As used herein, “immunoglobulin constant domain” does not necessarily referWSGR Docket No. 69097-702.601 to the full constant region of an immunoglobulin chain. Immunoglobulin constant domain can describe at least one domain from the full immunoglobulin constant region. For example, immunoglobulin constant domain can describe a CHI domain only or a variant, derivative or fragment thereof; a CH2 domain only or a variant, derivative, or fragment thereof; a CH3 domain only or a variant, derivative, or fragment thereof; a CH2 and CH3 domain without CHI; a CH2, CH3, and a hinge or fragment thereof without a CHI domain; or a CHI, CH2, and CH3, with or without a hinge. In each case, the CHI, CH2, CH3, or hinge, may be a variant, derivative, or fragment thereof. In some embodiments, an immunoglobulin constant domain is a CH2 and CH3, for example of an IgG, IgGl, IgG2, IgG3, IgG4, IgA, IgAl, IgA2, IgM, IgD, or IgE. In some embodiments, an immunoglobulin constant domain is a CH2, CH3, and a hinge or fragment thereof, for example of an IgG, IgGl, IgG2, IgG3, IgG4, IgA, IgAl, IgA2, IgM, IgD, or IgE. In some embodiments, an immunoglobulin constant domain is a CHI, hinge, CH2, and CH3, for example of an IgG, IgGl, IgG2, IgG3, IgG4, IgA, IgAl, IgA2, IgM, IgD, or IgE. In some embodiments, an immunoglobulin constant domain is a CL.

[0221] A hinge region of an antibody can be subdivided into three domains, an upper hinge domain, core hinge domain, and lower hinge domain. A core hinge domain can comprise one or more cysteine residues that can form a disulfide bond, for example, with a corresponding core hinge region of a second immunoglobulin constant region. In some embodiments, a hinge or fragment thereof in an immunoglobulin constant domain comprises, consists essentially of, or consists of the upper hinge domain, core hinge domain, and / or lower hinge domain. In some embodiments, an immunoglobulin constant domain can lack an upper hinge region, for example, to reduce susceptibility to proteolysis. In some embodiments, a hinge or a part thereof (e.g., an upper hinge region) can be replaced by a linker disclosed herein. In some embodiments, an immunoglobulin constant domain comprises a first hinge domain or hinge fragment from a first immunoglobulin isotype or subclass, and a second hinge domain or hinge fragment from a second immunoglobulin isotype or subclass. For example, in some embodiments an immunoglobulin constant domain comprises a core hinge domain of IgGl and a lower hinge domain of IgG2, for example, to enhance resistance to proteases and / or reduce effector function.

[0222] A multispecific immune cell engager can comprise an immunoglobulin constant domain that is a heavy chain constant domain, for example, a CHI, CH2, CH3, and / or CH4 domain, or a variant, derivative, or fragment thereof. The heavy chain constant domain can be a mammalian heavy chain constant domain. The heavy chain constant domain can be a human heavy chain constant domain.

[0223] The immunoglobulin constant domain can be or can comprise any one or more domains from any suitable immunoglobulin isotype(s), class(es), or subclass(es) (e.g., IgG, IgGl,WSGR Docket No. 69097-702.601IgG2, IgG3, IgG4, IgA, IgAl, IgA2, IgM, IgD, or IgE). In some embodiments, an immunoglobulin heavy chain constant domain can be a CHI, CH2, and / or CH3 of IgGl or IgG4 (e.g., mammalian, human, or other IgGl or IgG4).

[0224] A multispecific immune cell engager can comprise an immunoglobulin constant domain of IgG, IgGl, IgG2, IgG3, IgG4, IgA, IgAl, IgA2, IgM, IgD, or IgE, for example, mammalian or human IgG, IgGl, IgG2, IgG3, IgG4, IgA, IgAl, IgA2, IgM, IgD, or IgE. In some embodiments, a multispecific immune cell engager comprises a CHI, hinge, CH2, CH3, fragment thereof, or a combination thereof, of IgG, IgGl, IgG2, IgG3, IgG4, IgA, IgAl, IgA2, IgM, IgD, or IgE. In some embodiments, a multispecific immune cell engager comprises a CH2 and CH3 of IgG, IgGl, IgG2, IgG3, IgG4, IgA, IgAl, IgA2, IgM, IgD, or IgE. In some embodiments, a multispecific immune cell engager comprises a CH2, CH3, and hinge or fragment thereof of IgG, IgGl, IgG2, IgG3, IgG4, IgA, IgAl, IgA2, IgM, IgD, or IgE. In some embodiments, a multispecific immune cell engager comprises an Fc domain of IgG, IgGl, IgG2, IgG3, IgG4, IgA, IgAl, IgA2, IgM, IgD, or IgE. In some embodiments, a multispecific immune cell engager does not include a CHI domain of IgG, IgGl, IgG2, IgG3, IgG4, IgA, IgAl, IgA2, IgM, IgD, or IgE. The immunoglobulin constant domain can comprise a modification, e.g., to induce, promote, or favor heterodimerization, increase resistance to proteases, alter Fc receptor binding, and / or alter effector function. In some embodiments, an immunoglobulin constant domain comprises a single chain Fc domain, for example, parts of two heavy chains within the one open reading frame.

[0225] A multispecific immune cell engager can comprise an immunoglobulin constant domain of IgG, for example, mammalian or human IgG. In some embodiments, a multispecific immune cell engager comprises a CHI, hinge, CH2, CH3, fragment thereof, or a combination thereof, of IgG. In some embodiments, a multispecific immune cell engager comprises a CH2 and CH3 of IgG. In some embodiments, a multispecific immune cell engager comprises a CH2, CH3, and hinge or fragment thereof of IgG. In some embodiments, a multi specific immune cell engager comprises an Fc domain of IgG. In some embodiments, a multispecific immune cell engager does not include a CHI domain of IgG.

[0226] SEQ ID NOs: 108-117 provide illustrative sequences of Fc regions of polypeptide chains. SEQ ID NOs: 118-122 provide illustrative sequences of full constant regions of human IgG, which comprise a CHI domain, hinge, CH2 domain, and CH3 domain, and SEQ ID NOs: 123-127 provide additional immunoglobulin full constant region sequences. SEQ ID NOs: 128- 143 provide illustrative sequences of IgG CHI, CH2, CH3, hinge, and Fc domains.

[0227] The immunoglobulin constant domain can comprise a modification, e.g., to induce heterodimerization, increase resistance to proteases, alter Fc receptor binding, and / or alter effectorWSGR Docket No. 69097-702.601 function. In some embodiments, an immunoglobulin constant domain comprises a single chain Fc domain, for example, parts of two heavy chains within the one open reading frame.

[0228] A multispecific immune cell engager can comprise an immunoglobulin constant domain of IgA, for example, mammalian or human IgAl or IgA2. In some embodiments, a multispecific immune cell engager comprises a CHI, hinge, CH2, CH3, fragment thereof, or a combination thereof, of IgA. In some embodiments, a multispecific immune cell engager comprises CH2 and CH3 of IgA. In some embodiments, a multispecific immune cell engager comprises a CH2, CH3, and hinge or fragment thereof of IgA. In some embodiments, a multispecific immune cell engager comprises an Fc domain of IgA. In some embodiments, a multispecific immune cell engager does not include a CHI domain of IgA. The immunoglobulin constant domain can comprise a modification, e.g., to induce heterodimerization, increase resistance to proteases, alter Fc receptor binding, and / or alter effector function.

[0229] In some embodiments, an immunoglobulin constant domain can comprise a light chain constant domain, for example, a CL domain. The light chain constant domain can be a mammalian light chain constant domain. The light chain constant domain can be a human light chain constant domain. The immunoglobulin light chain constant domain can be or can comprise a domain from any suitable immunoglobulin isotype, class, or subclass. For example, an immunoglobulin light chain constant domain can be a lambda (IgL) or kappa (IgK) CL domain (e.g., mammalian, human, or other CL).

[0230] A multispecific immune cell engager or a chain, domain, or region thereof (for example, a first polypeptide chain, a second polypeptide chain, an Fc region, an immunoglobulin constant domain, a dimerization module or portion thereof, other domain disclosed herein, or combination thereof) can comprise, consist essentially of, or consist of an amino acid sequence with at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or sequence similarity to any one of SEQ ID NOs: 108-145

[0231] TABLE 11: illustrative immunoglobulin constant domain and Fc region sequences.WSGR Docket No. 69097-702.601WSGR Docket No. 69097-702.601WSGR Docket No. 69097-702.601WSGR Docket No. 69097-702.6016. Fc modifications

[0232] Modifications to the constant domains can affect characteristics of an antibody or multispecific immune cell engager (e.g., T cell engager) that comprises the constant domain, for example, for enhancement or reduction of Fc receptor ligation, enhancement or reduction of ADCC or TDCC, enhancement or reduction of ADCP, enhancement or reduction of CDC, enhancement or reduction of signaling through ITAMs, enhancement or reduction of cytokine induction, enhancement or reduction of signaling through ITIMs, or enhancement or reduction of susceptibility to protease-mediated degradation, or enhancement or reduction of half-life. Modifications can include, for example, amino acid mutations, altering post-translational modifications (e.g., glycosylation), combining domains from different isotypes or subclasses, or a combination thereof. A multispecific immune cell engager disclosed herein can comprise an immunoglobulin constant domain or Fc region that is modified to achieve desirable characteristics, for example, reduced binding to one or more particular Fc receptors, reduced induction of immune effector functions, increased resistance to proteases, and enhanced half-life in vivo. Binding to one or more particular Fc receptors can be increased or decreased, while binding to one or more other Fc receptors is not substantially altered. Binding to one or more particular Fc receptors can be increased, while binding to one or more other Fc receptors is not substantially altered. Binding to one or more particular Fc receptors can be decreased, while binding to one or more other Fc receptors is not substantially altered. Binding to one or more particular Fc receptors can be increased, while binding to one or more other Fc receptors is decreased. A particular Fc receptor can be, for example, a single chain IgG receptor (e.g., FcyRIIA, FcyRIIB, FcyRIIC, and FcyRIIIB), IgE receptor (e g., FcsRII), IgM receptor (e.g., FcpR), or IgA / IgM receptors (e.g., plgR and FcapR). A particular Fc receptor can be, for example, a multichain receptor of IgA (e.g., FcaRI), IgE (e.g., FcsRI), or IgG (e.g., FcyRI, FcyRIIIA, FcyRIV, and FcRn). In some embodiments, an immunoglobulin constant domain or Fc region comprises modifications that enhance or reduce recycling via the FcRn receptor.

[0233] A multispecific immune cell engager disclosed herein can comprise an immunoglobulin constant domain or Fc region that is selected or modified to provide suitable characteristics, for example, suitable characteristics for treating a disease or condition as disclosed herein. In some embodiments, IgGl can be used, for example, to promote immune activation effector functions (e.g., ADCC, TDCC, ADCP, CDC, IT AM signaling, cytokine induction, or aWSGR Docket No. 69097-702.601 combination thereof). In some embodiments, IgG4 can be used, for example, in cases where reduced immune effector functions based on antibody binding to a target molecule are desirable (e.g., when bound to a target molecule, but not bound to both a target molecule on a target cell plus a target molecule on an immune effector cell).

[0234] Non-limiting examples of immunoglobulin constant domain modifications and their effects are provided in TABLE 12. The numbering used can be EU numbering. For example, for IgGl, numbering of the constant region according to EU numbering starts with residue number 118, and accordingly, a mutation at residue “L234” in the table below will be at residue LI 17 in SEQ ID NO: 118, residue “N434” will be residue N317 in SEQ ID NO: 118, etc. Similarly, numbering can be adjusted to the EU numbering of constant region sequences of other isotypes.WSGR Docket No. 69097-702.601D. Dimerization / multimerization module

[0235] Multispecific immune cell engagers disclosed herein can optionally be multimers, for example, dimers, such as heterodimers. Multispecific immune cell engagers can utilize heterodimeric heavy chains, Fc regions, or immunoglobulin constant regions as a dimerization or multimerization domain. For example, a multispecific immune cell engager can comprise a two polypeptide chains (which can be referred to, e.g., as a first and second polypeptide chain). Each of the first and second polypeptide chains can comprise a portion of a dimerization or multimerization module, facilitating dimerization or multimerization. The portions of the dimerization module can be or comprise, for example, heavy chains or Fc regions thereof (e.g., with CH2 and CH3 domains, or CH2, CH3, and CH4 domains). For general description of the structural designs the expressions “composed of’, “comprised of’ or “consist of’ and their grammatical or semantic equivalents (for example, composed of anti-CD 19 binding domains) may be interchangeably used. However, the expressions may be read narrowly in suitable contexts to specify open or closed meanings. For example, in specific contexts, such as description of formats (e.g., format 0A, 1 A or IB), “composed of’ may be read in its closed meaning, indicating that the binding domain component(s) is / are as listed in the description or shown in the figures.

[0236] Heterodimerization can be induced using a number of methods. Various techniques can be used to promote pairing of desirable heavy chain combinations, rather than random chain associations. Dimerization modules disclosed herein can comprise, utilize, or be heterodimerization domains to facilitate formation of the heterodimer. A heterodimerization domain can be, for example, an immunoglobulin constant domain or Fc chain with one or more modifications that promote heterodimer formation. A heterodimerization domain can be or can comprise, for example, one or more modifications in an immunoglobulin constant domain or Fc chain that facilitates heterodimer formation (e.g., promotes preferential heterodimer formation versus homodimer formation, or a mix of homo- and hetero-dimerization).

[0237] In some embodiments, engineering strategies are used to introduce mutations into the CH2 and / or CH3 domains to promote heterodimerization based on steric and / or electrostatic complementarity.

[0238] Non-limiting examples of heterodimerization domains and / or strategies to induce heterodimerization of polypeptides (e.g., immunoglobulin constant domains disclosed herein, such as Fc domains) include knobs-in-holes, SEEDbody, biochemical optimization and mutationsWSGR Docket No. 69097-702.601 identified therefrom, electrostatic optimization / steering and mutations identified therefrom, DNL (natural association of 2 antibodies or antibody fragments anchored with DDD (dimerization and docking domain) from PKA (protein kinase A) and AD (anchoring domain) from A-kinase anchor protein (AKAP), respectively), CrossMab, LUZ-Y (e.g., leucine zipper tethered at the C-termini of HC and later proteolytically removed, plus point mutation), quadroma (e.g., somatic fusion of hybridomas each encoding a monoclonal antibody), and strand exchange.

[0239] In some embodiments, knob-into-hole modifications of immunoglobulin constant domains or Fc regions of heavy chains are used to promote formation of heterodimers between the first polypeptide chain (e.g., heavy / Fc chain) and the second polypeptide chain (e.g., heavy / Fc chain). The “knobs in holes” approach allows the generation of complementary interacting interfaces by manipulating key amino acid residues that participate in the Fc dimeric interaction. Amino acids with small side chain are replaced by ones with larger side chains, thereby creating a knob or protrusion in one chain, and vice versa to create a hole or socket in the partner chain.

[0240] The “knob” heavy chain can contain a mutation of threonine at a position equivalent to 366 in CH3 of IgG, such as a T366W or T366Y mutation. The “knob” heavy chain can also contain, for example, an F405A mutation.

[0241] The “hole” heavy chain can contain multiple mutations, e.g., T366S, L368A, T394W, F405A, and / or Y407V / T). In some embodiments, the “hole” heavy chain comprises T366S, L368A, and Y407V substitutions. In some embodiments, the “hole” heavy chain comprises T366S, L368A, and Y407V substitutions.

[0242] The residue numbering can be according to EU numbering (e.g., as described herein).

[0243] In some embodiments, a polypeptide chain (e.g., Fc region or immunoglobulin constant domain thereof) comprises one or more cysteine replacement residues, for example, to facilitate formation of a disulfide bond with another polypeptide chain. The polypeptide chain (e.g., Fc region or immunoglobulin constant domain thereof) can comprise a cysteine replacement, for example, at residue Y349, L351, S354, E356, E357, K392, T394, V397, D399, or a combination thereof. A first polypeptide chain (e.g., Fc region or immunoglobulin constant domain thereof) and a second polypeptide chain (e.g., Fc region or immunoglobulin constant domain thereof) can each comprise residues replaced with cysteines, for example, the pair can comprise K392C and D399'C; S354C and Y349'C; E356C and Y349'C; or E357C and Y349'C mutations (where the ' indicates the mutation is in the second polypeptide chain). In some embodiments, a first polypeptide chain (e.g., Fc region or immunoglobulin constant domain thereof) comprises an S354C substitution and a second polypeptide chain (e.g., Fc region or immunoglobulin constant domain thereof) comprises a Y349'C substitution. In some embodiments, a first polypeptide chain (e.g., Fc region or immunoglobulin constant domainWSGR Docket No. 69097-702.601 thereof) comprises a Y349C substitution and a second polypeptide chain (e.g., Fc region or immunoglobulin constant domain thereof) comprises an E356'C substitution.

[0244] A dimerization module, immunoglobulin constant domain, or a heterodimerization domain can comprise a combination of cysteine replacement residues and heterodimerizationpromoting (e.g., knob-into-hole) modifications, for example, one or more cysteine replacement residues and one or more knob-into-hole modifications in each of a pair of polypeptide chains.

[0245] In some embodiments, a first polypeptide chain (e.g., Fc region or immunoglobulin constant domain thereof) comprises substitutions at positions S354 and T366, and a second polypeptide chain (e.g., Fc region or immunoglobulin constant domain thereof) comprises substitutions at positions Y349, T366, L368, and Y407. In some embodiments, a first polypeptide chain (e.g., Fc region or immunoglobulin constant domain thereof) comprises S354C and T366W substitutions, and a second polypeptide chain (e.g., Fc region or immunoglobulin constant domain thereof) comprises Y349C, T366S, L368A, and Y407V substitutions. In some embodiments, knob and hole mutations are introduced into the Fc chain as described in or based on the disclosure in Merchant (1998) An efficient route to human bispecific IgG. Nature Biotechnology 16, 677-681.

[0246] In some embodiments, pairs of a heterodimer comprise mutations in CH2 domain residues (e.g., F241R / F243S or F241S / F243R) that remain solvent exposed in aglycosylated IgG molecules to avoid covalent association of knob / knob or hole / hole monomers.

[0247] A SEEDbody (Strand-Exchange Engineered Domain) approach can involve creating alternating human IgG and IgA fragments in CH3 to guide heavy chain heterodimerization. For example, patches of IgG and IgA CH3 can be mutually replaced in a heterodimerization domain to facilitate heterodimerization.

[0248] In some embodiments, a heterodimerization domain developed using biochemical optimization is used to facilitate heterodimer formation. An illustrative heterodimerization domain comprises mutations in CH3 domains, for example, S364H and F405A in the first chain CH3, and Y349T and T394F in the second chain CH3.

[0249] In some embodiments, an electrostatic heterodimerization domain is used to promote heterodimer formation. A multispecific immune cell engager can comprise an electrostatic steering modification that favors heterodimeric interaction between a first polypeptide chain (e.g., Fc region or immunoglobulin constant domain thereof) and a second polypeptide chain (e.g., Fc region or immunoglobulin constant domain thereof). For example, charged pair based attraction / repulsion can be tailored in different Fc chains. Mutations in certain residues of the chains can favor heterodimer formation, e.g., (K409D-D399'K, K409D-D399'R, K409E- D399'K, K409E-D399'R, D399'K-E356'K, or K392D-E356'K). In some embodiments, charged amino acids from the core of the CH3 domain are substituted with hydrophobic residues to perturbWSGR Docket No. 69097-702.601 the structural symmetry, and long-range electrostatic attraction is engineered at the edge of the CH3 domain to promote heterodimer formation. Illustrative examples include a K409W and D399'V / F405'T pair; and K360E and Q347'R pair. In some embodiments, heterodimer formation is promoted by mutations (T350V / L351Y / F405A / Y407V) in a first chain heterodimerization domain and (T350V / T366L / K393L / T394W) in a second chain heterodimerization domain. In some embodiments, heterodimer formation is promoted by mutations (K409D / K392D) in a first chain heterodimerization domain and (D399'K / D357'K) in a second chain heterodimerization domain. In some embodiments, heterodimer formation is promoted by mutations (K409D / K370D) in a first chain heterodimerization domain and (D399'K / D357'K) in a second chain heterodimerization domain. In some embodiments, heterodimer formation is promoted by mutations (K409D / K392D) in a first chain heterodimerization domain and (D339'K / E356'K) in a second chain heterodimerization domain. In some embodiments, heterodimer formation is promoted by mutations (K409D / K392D) in a first chain heterodimerization domain and (D399'K / E356'K) in a second chain heterodimerization domain.

[0250] In some embodiments, an oxidation-reduction methodology or heterodimerization domain can be used for chain pairing, for example, K409 and L368 can be mutated in the CH3 domain (e.g., of IgGl / IgG2), and the chains can be co-expressed, or purified monomers can be mixed under mild reducing conditions. The K409 / L368 mutations can be introduced alone or in combination with IgGl -hinge or IgG2-hinge mutations (if the hinge domain or a fragment thereof is present).

[0251] In some embodiments, H435R and Y436F (“RF”) mutations are used to aid in preferential recovery of the heterodimeric species during purification with Protein A. In some embodiments, variable domains can also comprise one or more mutations (e.g., in framework regions) to aid preferential recovery of heterodimers. For example, one of the two polypeptide chains can comprise one or more VH or VHH domain(s) with an R19T mutation in framework 1 to reduce or prevent binding of homodimers to protein A during purification and / or to enhance heterodimer binding to Protein A, e.g., for use with a l-step purification process.

[0252] In some embodiments the two polypeptide chains are not contiguous, i.e., are separate polypeptide chains. In some embodiments the two polypeptide chains are contiguous, e.g., can be expressed as one polypeptide with a flexible linker joining them.E. Linkers, spacers, & hinges

[0253] A polypeptide disclosed herein, such as a multispecific immune cell engager, can comprise one or more linkers or spacers, for example, between different domains of the polypeptide. A linker or spacer can be a chemical bond, for example, a covalent bond or a non-WSGR Docket No. 69097-702.601 covalent bond. A linker or spacer as described herein can include a flexible or rigid linker. A linker or spacer can be a peptide linker or spacer. In some embodiments, a linker or spacer comprises a hinge or a domain thereof (e.g., an immunoglobulin, such as IgG, hinge or domain thereof). The terms linker and spacer can be used interchangeably herein.

[0254] A linker or spacer can comprise a sequence, for example, an amino acid sequence. The length and composition of a linker or spacer can be adjusted to allow for proper folding or to increase or decrease biological activity of multispecific immune cell engager, or, e.g., to facilitate binding of all binding domains to target molecules, and / or to facilitate low background activity in the absence of an immune effector cell and induction of a suitable level of activity in the presence of an immune effector cell.

[0255] A linker can perform the function of joining, connecting, linking, structurally and / or functionally two fragments of a polypeptide, e.g., two domains described herein, wherein the polypeptide for the description herein is a synthetic or recombinant polypeptide. Two discrete structural or functional domains may be held in a continuum, e.g. a chain, an amino acid chain, a peptide chain via the linkage effected by a linker, e.g., an amino acid or a peptide linker comprising one or more amino acids held by amino acid bonds. Two fragments or domains are often referred to as functionally or operably linked within a single polypeptide chain, even if said two domains are not next to each other linked by a linker. For example, a polypeptide chain may consist of four domains A, B, C, D, linked by short peptide linkers a, b, and c respectively, each peptide linker is 4 amin acids long, for example, having an amino acid sequence GGGG. The polypeptide may be visualized thus: A-a-B-b-C-c-D. In this case domain A and domain C are understood to be operably linked, even though they are not juxtaposed. Similarly, A and D domains are operably linked with each other, via linkers and other domains, in this case domains B and C. Of note, it is understood that two juxtaposed domains linked by one linker are also functionally linked, i.e., operably linked. Whereas all domains in a single polypeptide may be operably linked, two operably linked domains on a single polypeptide may not be linked by a single linker. In an analogous matter, for example, a receptor may comprise a multidomain polypeptide chain, e.g., comprising an extracellular domain (usually comprising a target binding domain), a transmembrane domain and an intracellular domain (usually comprising an intracellular signaling domain). When an extracellular domain is engaged with its target, a functional change may be effected through the intracellular domain, e.g., phosphorylation of the intracellular signaling domain, and therefore the extracellular domain and the intracellular domain are operably linked even though they are structurally intervened by the transmembrane domain.

[0256] A linker or spacer can be, for example, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15,WSGR Docket No. 69097-702.601 about 20, about 25, about 30, about 40, about 50, about 60, or about 70 amino acid residues in length. In some cases, a linker can be, for example at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, or at least about 50 amino acids in length. In some cases, a linker can be, for example at most about 2, at most about 3, at most about 4, at most about 5, at most about 6, at most about 7, at most about 8, at most about 9, at most about 10, at most about 15, at most about 20, at most about 30, at most about 40, at most about 50, at most about 60, at most about 70, at most about 80, or at most about 100 amino acids in length.

[0257] In some cases, a linker is about 2-20 amino acids in length. In some cases, a linker is about 2-15 amino acids in length. In some cases, a linker is about 2-12 amino acids in length. In some cases, a linker is about 2-10 amino acids in length. In some cases, a linker is about 2-8 amino acids in length. In some cases, a linker is about 2-6 amino acids in length. In some cases, a linker is about 3-20 amino acids in length. In some cases, a linker is about 3-15 amino acids in length. In some cases, a linker is about 3-12 amino acids in length. In some cases, a linker is about 3-10 amino acids in length. In some cases, a linker is about 3-8 amino acids in length. In some cases, a linker is about 3-6 amino acids in length. In some cases, a linker is about 4-20 amino acids in length. In some cases, a linker is about 4-15 amino acids in length. In some cases, a linker is about 4-12 amino acids in length. In some cases, a linker is about 4-10 amino acids in length. In some cases, a linker is about 4-8 amino acids in length. In some cases, a linker is about 4-6 amino acids in length. In some cases, a linker is about 5-20 amino acids in length. In some cases, a linker is about 5-15 amino acids in length. In some cases, a linker is about 5-12 amino acids in length. In some cases, a linker is about 5-10 amino acids in length. In some cases, a linker is about 5-8 amino acids in length. In some cases, a linker is about 5-6 amino acids in length.

[0258] A flexible linker can have a sequence containing glycine residues. The small size of the glycine residues can provide flexibility, and allow for mobility of the connected protein domains. The incorporation of serine or threonine can maintain the stability of the linker in aqueous conditions by forming hydrogen bonds with the water molecules, thereby reducing unfavorable interactions between the linker and protein moieties. In some cases flexible linkers can also contain additional amino acids, such as threonine and alanine, to maintain flexibility, and / or polar amino acids such as lysine and glutamine, to improve solubility.

[0259] A rigid linker can have, for example, an alpha helix-structure. An alpha-helical rigid linker can act as a spacer between protein domains. A rigid linker can have a proline-rich sequence, (XP)n, with X designating alanine, lysine, glutamine, or any amino acid, and nWSGR Docket No. 69097-702.601 designating a number of repeats. The presence of proline in non-helical linkers can increase stiffness, and allow for effective separation of protein domains.

[0260] A linker can comprise a hinge region, for example an amino acid sequence derived from a hinge region of an antibody or immune receptor. In some embodiments, a linker comprises a hinge region from CD8a, IgGl, or IgG4. In some embodiments, a multispecific immune cell engager lacks a hinge or hinge region.

[0261] Examples of linkers include, but are not limited to, those disclosed in TABLE 13 and SEQ ID NOs: 146-182, repeats thereof (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats), and combinations thereof, which can be used to link any portion (e.g., domain) of a polypeptide to any other portion (e.g., domain).

[0262] A linker, spacer, or hinge used in a multispecific immune cell engager disclosed herein, or a subunit thereof, can comprise an amino acid sequence with at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% sequence identity or sequence similarity to any one of SEQ ID NOs: 146-182.

[0263] In some embodiments, a linker used in a multispecific immune cell engager is a non- cleavable linker, for example, does not contain a protease cleavage site or lacks a human protease cleavage site.

[0264] A non-cleavable linker can also include a non-proteolytically cleavable peptide. A non- proteolytically cleavable peptide can be inert to proteases present in a given sample or organism. For example, a peptide may be inert to all human protease cleavage sequences, and thereby can comprise a high degree of stability within humans and human samples. Such a peptide can also comprise a secondary structure which renders a protease cleavage site inert or inaccessible to a protease. A non-cleavable linker of the present disclosure can include a chemical linker that is stable. Examples of non-cleavable linkers can include a thioether linker, an alkyl linker, a polymeric linker. A linker can be an SMCC linker or a PEG linker.

[0265] A linker of the disclosure can include a chemical linker. For example, two compounds (e.g., amino acid sequences) of the disclosure can be connected together by a chemical linker. Each chemical linker of the disclosure can be alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene, any of which is optionally substituted. In some embodiments, a chemical linker of the disclosure can be an ester, ether, amide, thioether, or polyethyleneglycol (PEG). Non-limiting examples of such linkers include diesters of dicarboxylic acids, such as oxalyl diester, malonyl diester, succinyl diester, glutaryl diester, adipyl diester, pimetyl diester, fumaryl diester, maleyl diester, phthalyl diester, isophthalyl diester, and terephthalyl diester. Non-limiting examples of such linkers include diamides of diamino linkers, such as ethylene diamine, l,2-di(methylamino)ethane, 1,3 -diaminopropane, 1,3-WSGR Docket No. 69097-702.601 di(methylamino)propane, l,4-di(methylamino)butane, l,5-di(methylamino)pentane, 1,6- di(methylamino)hexane, and pipyrizine.

[0266] A polynucleotide encoding a multispecific immune cell engager of the disclosure can be designed to encode two or more components of a protein (e.g., first and second polypeptide chains) linked by one or more cleavable linkers which can be processed into separate polypeptides (e.g., 2A linkers that can be cleaved co-translationally or after translation). Inclusion of a 2A linker can increase the likelihood that an appropriate ratio of components are produced (e.g., a 1 : 1, 1 :2, 1 :3, 1 :4, or 1 :5 ratio of two components), such as a first polypeptide chain and a second polypeptide chain.

[0267] TABLE 13: illustrative linkers, spacers, hinges, and subunits thereof.WSGR Docket No. 69097-702.601F. Configurations

[0268] Multispecific immune cell engagers (e.g., T cell engagers) disclosed herein can comprise domains arranged in a number of formats and configurations. A multispecific immune cell engager can comprise two chains, as illustrated schematically in FIG. 1 for up to 16 binding domains. The first polypeptide chain can comprise a first portion of a multimerization or dimerization module, for example, a first Fc region, depicted as Fc-1. The second polypeptide chain can comprise a second portion of a multimerization or dimerization module, for example, a second Fc region, depicted as Fc-2.

[0269] The location of the binding domains can be named based on their position relative to the two Fc chains (Fc-1 and Fc-2). For example, INI can indicate the position of the first binder N-terminal to Fc-1, position 1N2 the second binder N-terminal to Fc-1, position 1N3 the third binder N-terminal to Fc-1, and position 1N4 the fourth binder N-terminal to Fc-1. Similarly, 1C1 can indicate the position of the first binder C-terminal to Fc-1, position 1C2 the second binder C- terminal to Fc-1, position 1C3 the third binder C-terminal to Fc-1, and position 1C4 the fourth binder C-terminal to Fc-1. The same naming convention can be applied to describe binders fused to Fc-2 (e.g., 2N 1 to indicate the first binder N-terminal to Fc-2, 2N2 to indicate the second binder N-terminal to Fc-2, etc.)

[0270] Any set of between 2-16 binding domains disclosed herein can be arranged in the positions indicated in FIG. 1 (INI, 1N2, 1N3, 1N4, 1C1, 1C2, 1C3, 1C4, 2N1, 2N2, 2N3, 2N4, 2C1, 2C2, 2C3, and / or 2C4). For example, for a multispecific immune cell engager that has two binding domains, the two binding domains can be present at positions INI and 1N2, 1C1 and 1C2, 2N1 and 2N2, 2C1 and 2C2, INI and 1C1, INI and 2C1, INI and 2N1, 1C1 and 2C1, 1C1 and 2N1, or 2C1 and 2N1, and the remaining positions indicated in FIG. 1 can be absent. Accordingly, a multispecific immune cell engager can comprise 0, 1, 2, 3, or 4 binding domains joined to the N-terminus of a first Fc chain, 0, 1, 2, 3, or 4 binding domains joined to the C- terminus of the first Fc chain, 0, 1, 2, 3, or 4 binding domains joined to the N-terminus of the second Fc chain, and / or 0, 1, 2, 3, or 4 binding domains joined to the C-terminus of the second FcWSGR Docket No. 69097-702.601 chain. Linkers, spacers, or hinges disclosed herein can optionally be interposed between (e.g., used to join) any pair of domains.

[0271] In some embodiments, advantageous properties are observed for multispecific immune cell engagers with a particular orientation or structural format. It has been observed that in an attempt to design specific T cell engagers in order to establish highly specific binding to a particular target cell to reduce any off-target effect, a skilled artisan is faced with various challenges with regards to the ultimate efficacy. For instance, the multi-target binding domains may pose structural hindrance with respect to each other. It has been understood during the course of the instant endeavor that there may arise challenges with solubility, and dispersion. An ideal TCE design ought to be safe and effective. Off-target effects and tonic signaling, which is understood to be an intracellular signaling cascade triggered even in absence of a target or in resting conditions, often associate with toxicity of a TCE in vivo. Reduced dispersion and aggregation in physiological solution or a physiological condition negatively affects bioavailability and overall efficacy. While highly effective binders with high affinity towards its target could be a good thing, it may have to be weighed against the abundance of the target in vivo, such that a slightly reduced affinity may be considered effective with a longer serum halflife for reaching the target destination, such as a specific organ or to a solid tumor. Additionally, a highly sensitive binder with highly effective and specific binding at low concentrations of the effectortarget, may be associated with tonic signaling and fratricide. Therefore, a large number factors interplay in resulting to a selection of few therapeutically viable designs to pursue, starting from a large number of putitive TCE designs, which may first be generated, the prototypes manufactured, and then tested or screened for establishing at least a few candidates that fulfill the criteria for safety and effectivity in general. For example, in this particular matter of engineering the TCEs as disclosed herein, because at least one of the binders act as co-stimulatory molecule (e.g., a CD2 binder that binds and activates CD2 on a T cell), there is a substantial possibility of non-target intracellular signal firing and activation of T cells under resting conditions, where the T cells attack each other (fratricide) and therefore may culminate in reduced T cell (effector) viability and toxicity. In some embodiments, the instant disclosure provides platform scaffolds that have been found to be safe or efficacious at least for a certain orientation of binders at certain positions. Additionally, in some embodiments the instant disclosure provides a suitable geometrical scaffold for one or more admissible or permissible target binding domains on a TCE, that allows for further development by adding or replacing one or more binding domains keeping other positions stable or invariable, to preserve the indicated safety or efficacy feature.

[0272] For example, in some embodiments multispecific immune cell engagers with a binding domain in position 2N1 that binds an immune effector cell target molecule (e.g., a CD3 bindingWSGR Docket No. 69097-702.601 domain or CD2 binding domain) exhibit favorable properties. In some embodiments multispecific immune cell engagers with a binding domain in position 2N2 that binds an immune effector cell target molecule (e.g., a CD3 binding domain or CD2 binding domain) exhibit favorable properties. In some embodiments multispecific immune cell engagers with binding domains in positions 2N1 and 2N2 that bind immune effector cell target molecules (e.g., a CD3 binding domain and a CD2 binding domain) exhibit favorable properties.

[0273] In some embodiments multispecific immune cell engagers with a binding domain in position INI that binds an immune effector cell target molecule (e.g., a CD3 binding domain or CD2 binding domain) exhibit favorable properties. In some embodiments multispecific immune cell engagers with a binding domain in position 2N1 that binds an immune effector cell target molecule (e.g., a CD3 binding domain or CD2 binding domain) exhibit favorable properties. In some embodiments multispecific immune cell engagers with binding domains in positions INI and 2N1 that bind immune effector cell target molecules (e.g., a CD3 binding domain and a CD2 binding domain) exhibit favorable properties.

[0274] In some embodiments multispecific immune cell engagers with a binding domain in position 1N2 that binds an immune effector cell target molecule (e.g., a CD3 binding domain or CD2 binding domain) exhibit favorable properties. In some embodiments multispecific immune cell engagers with a binding domain in position 2N1 that binds an immune effector cell target molecule (e.g., a CD3 binding domain or CD2 binding domain) exhibit favorable properties. In some embodiments multispecific immune cell engagers with binding domains in positions 1N2 and 2N1 that bind immune effector cell target molecules (e.g., a CD3 binding domain and a CD2 binding domain) exhibit favorable properties.

[0275] A multispecific immune cell engager (e.g., T cell engager) disclosed herein can comprise or have a binding domain that binds to a target molecule on an immune effector cell at the 2N1 position, which can be referred to as “format 0.” A format 0 immune cell engager can comprise one binding domain that binds to a target cell target molecule, for example, at position INI (FIG. 2A). A format 0 immune cell engager can comprise two binding domains that bind to target cell target molecules, for example, at positions INI and 1N2 (FIG. 3A), or at positions INI and 1C1. A format 0 immune cell engager can comprise three binding domains that bind to target cell target molecules, for example, at positions INI, 1N2, and 1N3 (FIG. 4A). Typically, a format 0 immune cell engager or TCE comprises a CD3 binding domain, and does not comprise a costimulatory domain, e.g. a CD2 binder or a CD58 ligand.

[0276] A multispecific immune cell engager (e.g., T cell engager) disclosed herein can comprise or have a first target binding domain that binds to a target molecule on an immune effector cell at the 2N 1 position, and a second target binding domain that binds to a target moleculeWSGR Docket No. 69097-702.601 on an immune effector cell at the 2N2 position, which can be referred to as “format 1.” The first and second target binding domains can comprise, for instance, a CD3 binding domain and a CD2 binding domain (e.g., CD58 IgV domain), each in either of the two positions. A format 1 immune cell engager can comprise or have one binding domain that binds to a target cell target molecule, for example, at position INI (FIG. 2B). A format 1 immune cell engager can comprise or have two binding domains that bind target cell target molecules, for example, at positions INI and 1N2 (FIG. 3B) or at positions INI and 1C1. A format 1 immune cell engager can comprise or have three binding domains that bind target cell target molecules, for example, at positions INI, 1N2, and 1N3 (FIG. 4B) or at positions INI, 1N2, and 1C1. Typically, a format 1 engager may comprise a CD3 binding domain and a CD2 binding domain (e.g., CD58 IgV domain), both on the N terminal side of the Fcl or the Fc2.

[0277] A multispecific immune cell engager (e.g., T cell engager) disclosed herein can comprise or have a first target binding domain that binds to a target molecule on an immune effector cell at the 2N 1 position, and a second target binding domain that binds to a target molecule on an immune effector cell at the 2C1 position, which can be referred to as “format 2.” The first and second target binding domains can comprise, for instance, a CD3 binding domain and a CD2 binding domain (e.g., CD58 IgV domain), each in either of the two positions. A format 2 immune cell engager can comprise or have one binding domain that binds to a target cell target molecule, for example, at position INI (FIG. 2C). A format 2 immune cell engager can comprise or have two binding domains that bind target cell target molecules, for example, at positions INI and 1N2 (FIG. 3C) or at positions INI and 1C1. A format 2 immune cell engager can comprise or have three binding domains that bind target cell target molecules, for example, at positions INI, 1N2, and 1N3 (FIG. 4C) or at positions INI, 1N2, and 1C1. Typically, a format 2 engager may comprise at least the two engagers, a CD3 binding domain and a CD2 binding domain (e.g., CD58 IgV domain), where one of the two engagers are positioned on the opposing sides of the Fcl and the Fc2, e.g., on the N terminal side of the Fcl and the Fc2, and the other is on the C terminal side of the Fcl and the Fc2.

[0278] A multispecific immune cell engager (e.g., T cell engager) disclosed herein can comprise or have a first target binding domain that binds to a target molecule on an immune effector cell at the IN 1 position, and a second target binding domain that binds to a target molecule on an immune effector cell at the 2N1 position, which can be referred to as “format 3.” The first and second target binding domains can comprise, for instance, a CD3 binding domain and a CD2 binding domain (e.g., CD58 IgV domain), each in either of the two positions. A format 3 immune cell engager can comprise or have one binding domain that binds to a target cell target molecule, for example, at position 1N2 (FIG. 2D). A format 3 immune cell engager can comprise or haveWSGR Docket No. 69097-702.601 two binding domains that bind target cell target molecules, for example, at positions 1N2 and 1N3 (FIG. 3D) or at positions 1N2 and 1C1. A format 3 immune cell engager can comprise or have three binding domains that bind target cell target molecules, for example, at positions 1N2, 1N3, and 1N4, (FIG. 4D) or at positions 1N2, 1N3, and 1C1. Typically, a format 2 engager may comprise a CD2 binding domain and an anti-CD3 binding domain on different polypeptides, e.g., the CD2 binding domain on the first polypeptide comprising the Fcl region and the CD3 binding domain on the second polypeptide comprising the Fc2 region.

[0279] A multispecific immune cell engager (e.g., T cell engager) disclosed herein can comprise or have a first target binding domain that binds to a target molecule on an immune effector cell at the 1N2 position, and a second target binding domain that binds to a target molecule on an immune effector cell at the 2N1 position, which can be referred to as “format 4.” The first and second target binding domains can comprise, for instance, a CD3 binding domain and a CD2 binding domain (e.g., CD58 IgV domain), each in either of the two positions. A format 4 immune cell engager can comprise or have one binding domain that binds to a target cell target molecule, for example, at position INI (FIG. 2E). A format 4 immune cell engager can comprise or have two binding domains that bind target cell target molecules, for example, at positions INI and 1N3 (FIG. 3E) or at positions INI and 1C1. A format 4 immune cell engager can comprise or have three binding domains that bind target cell target molecules, for example, at positions INI, 1N3, and 1N4, (FIG. 4E) or at positions INI, 1N3, and 1C1.

[0280] A multispecific immune cell engager (e.g., T cell engager) disclosed herein can comprise or have a first target binding domain that binds to a target molecule on an immune effector cell at the 1N3 position, and a second target binding domain that binds to a target molecule on an immune effector cell at the 2N1 position, which can be referred to as “format 5.” The first and second target binding domains can comprise, for instance, a CD3 binding domain and a CD2 binding domain (e.g., CD58 IgV domain), each in either of the two positions. A format 5 immune cell engager can comprise or have two binding domains that bind target cell target molecules, for example, at positions INI and 1N2 (FIG. 3F). A format 5 immune cell engager can comprise or have three binding domains that bind target cell target molecules, for example, at positions INI, 1N2, and 1N4, (FIG. 4F) or at positions INI, 1N2, and 1C1.

[0281] A multispecific immune cell engager (e.g., T cell engager) disclosed herein can comprise or have a first target binding domain that binds to a target molecule on an immune effector cell at the 1N4 position, and a second target binding domain that binds to a target molecule on an immune effector cell at the 2N1 position, which can be referred to as “format 6.” The first and second target binding domains can comprise, for instance, a CD3 binding domain and a CD2 binding domain (e.g., CD58 IgV domain), each in either of the two positions. A format 6 immuneWSGR Docket No. 69097-702.601 cell engager can comprise or have three binding domains that bind target cell target molecules, for example, at positions INI, 1N2, and 1N3, (FIG. 4G).

[0282] A multispecific immune cell engager (e.g., T cell engager) disclosed herein can comprise or have a first target binding domain that binds to a target molecule on an immune effector cell at the 1 C 1 position, and a second target binding domain that binds to a target molecule on an immune effector cell at the 2N1 position, which can be referred to as “format 7.” The first and second target binding domains can comprise, for instance, a CD3 binding domain and a CD2 binding domain (e.g., CD58 IgV domain), each in either of the two positions. A format 7 immune cell engager can comprise or have a single binding domain that binds to a target cell target molecule, for example, at position INI (FIG. 2F). A format 7 immune cell engager can comprise or have two binding domains that bind target cell target molecules, for example, at positions INI and 1N2 (FIG. 3G). A format 7 immune cell engager can comprise or have three binding domains that bind target cell target molecules, for example, at positions INI, 1N2, and 1N3, (FIG. 4H).

[0283] A multispecific immune cell engager (e.g., T cell engager) disclosed herein can comprise or have a first target binding domain that binds to a target molecule on an immune effector cell at the IN 1 position, and a second target binding domain that binds to a target molecule on an immune effector cell at the 2N2 position, which can be referred to as “format 8.” The first and second target binding domains can comprise, for instance, a CD3 binding domain and a CD2 binding domain (e.g., CD58 IgV domain), each in either of the two positions. A format 8 immune cell engager can comprise or have a single binding domain that binds to a target cell target molecule, for example, at position 2N1 (FIG. 2G). A format 8 immune cell engager can comprise or have two binding domains that bind target cell target molecules, for example, at positions 1N2 and 2N1 (FIG. 3H), or positions 1C1 and 2N1. A format 8 immune cell engager can comprise or have three binding domains that bind target cell target molecules, for example, at positions 1N2, 1N3, and 2N1 (FIG. 41).

[0284] A multispecific immune cell engager (e.g., T cell engager) disclosed herein can comprise or have a first target binding domain that binds to a target molecule on an immune effector cell at the 2N 1 position, and a second target binding domain that binds to a target molecule on an immune effector cell at the 1C2 position, which can be referred to as “format 9.” The first and second target binding domains can comprise, for instance, a CD3 binding domain and a CD2 binding domain (e.g., CD58 IgV domain), each in either of the two positions. A format 9 immune cell engager can comprise or have a single binding domain that binds to a target cell target molecule, for example, at position 1C1 (FIG. 2H). A format 9 immune cell engager can comprise or have two binding domains that bind target cell target molecules, for example, at positions INI and 1C1 (FIG. 31), or positions 1C1 and 2N1. A format 9 immune cell engager can comprise orWSGR Docket No. 69097-702.601 have three binding domains that bind target cell target molecules, for example, at positions INI, 1N2, and 1C1 (FIG. 4 J)

[0285] In some embodiments, a multispecific immune cell engager disclosed herein can comprise five binding domains, each of which binds to a target molecule arranged as INI, 1N2, 1N3, and 1N4. In some embodiments, a multispecific immune cell engager disclosed herein can comprise four binding domains, each of which binds to a target molecule arranged as INI, 1N2, 1N3, and 2C1. In some embodiments, a multispecific immune cell engager disclosed herein can comprise four binding domains, each of which binds to a target molecule arranged as INI, 1N2, 2C1, and 2C2. In some embodiments, a multispecific immune cell engager disclosed herein can comprise four binding domains, each of which binds to a target molecule arranged as INI, 2C1, 2C2, and 2C3. In some embodiments, a multispecific immune cell engager disclosed herein can comprise four binding domains, each of which binds to a target molecule arranged as 2C1, 2C2, 2C3, and 2C4.

[0286] In some embodiments, a multispecific immune cell engager disclosed herein can comprise five binding domains, each of which binds to a target molecule arranged as INI, 1N2, 1N3, 1N4, and 1N5. In some embodiments, a multispecific immune cell engager disclosed herein can comprise five binding domains, each of which binds to a target molecule arranged as INI, 1N2, 1N3, 1N4, and 2C1. In some embodiments, a multispecific immune cell engager disclosed herein can comprise five binding domains, each of which binds to a target molecule arranged as INI, 1N2, 1N3, 2C1, and 2C2. In some embodiments, a multispecific immune cell engager disclosed herein can comprise five binding domains, each of which binds to a target molecule arranged as INI, 1N2, 2C1, 2C2, and 2C3. In some embodiments, a multispecific immune cell engager disclosed herein can comprise five binding domains, each of which binds to a target molecule arranged as INI, 2C1, 2C2, 2C3, and 2C4. In some embodiments, a multispecific immune cell engager disclosed herein can comprise five binding domains, each of which binds to a target molecule arranged as 2C1, 2C2, 2C3, 2C4, and 2C5.

[0287] A multispecific immune cell engager can comprise five binding domains, each of which binds to a target molecule (e.g., a first target binding domain that binds to a first target molecule, a second target binding domain that binds to a second target molecule, a third target binding domain that binds to a third target molecule, a fourth target binding domain that binds to a fourth target molecule, and a fifth target binding domain that binds to a fifth target molecule). Three of the target molecules can be on target cells (e.g., the first, second, and third target molecules bound by the first, second, and third target binding domains can be tumor-associated antigens or B cell markers disclosed herein, such as CD 19, CD20, and CD22, in any order). TwoWSGR Docket No. 69097-702.601 of the target molecules, for example, the fourth and fifth target molecules bound by the fourth and fifth target binding domains, can be on immune effector cells (e.g., CD3 and CD2, in any order).

[0288] In some embodiments the first target molecule is or can be independently selected from any one of CD19, CD20, CD22, CD3, DLL3, and CD2. In some embodiments the second target molecule is or can be independently selected from any one of CD19, CD20, CD22, CD3, DLL3, and CD2. In some embodiments the third target molecule is or can be independently selected from any one of CD19, CD20, CD22, CD3, DLL3, and CD2. In some embodiments the fourth target molecule is or can be independently selected from any one of CD19, CD20, CD22, CD3, DLL3, and CD2. In some embodiments the fifth target molecule is or can be independently selected from any one of CD19, CD20, CD22, CD3, DLL3, and CD2.

[0289] In some embodiments the first target molecule is CD 19, the second target molecule is CD20, and the third target molecule is CD22. In some embodiments the first target molecule is CD22, the second target molecule is CD 19, and the third target molecule is CD20. In some embodiments the first target molecule is CD20, the second target molecule is CD22, and the third target molecule is CD 19.

[0290] In some embodiments the fourth target molecule is CD2 and the fifth target molecule is CD3. In some embodiments the fourth target molecule is CD3 and the fifth target molecule is CD2.

[0291] In some embodiments, the first target molecule is CD 19. In some embodiments, the first target molecule is CD20. In some embodiments, the first target molecule is CD22. In some embodiments, the first target molecule is CD3. In some embodiments, the first target molecule is CD2.

[0292] In some embodiments, the second target molecule is CD 19. In some embodiments, the second target molecule is CD20. In some embodiments, the second target molecule is CD22. In some embodiments, the second target molecule is CD3. In some embodiments, the second target molecule is CD2.

[0293] In some embodiments, the third target molecule is CD 19. In some embodiments, the third target molecule is CD20. In some embodiments, the third target molecule is CD22. In some embodiments, the third target molecule is CD3. In some embodiments, the third target molecule is CD2.

[0294] In some embodiments, the fourth target molecule is CD 19. In some embodiments, the fourth target molecule is CD20. In some embodiments, the fourth target molecule is CD22. In some embodiments, the fourth target molecule is CD3. In some embodiments, the fourth target molecule is CD2.WSGR Docket No. 69097-702.601

[0295] In some embodiments, the fifth target molecule is CD 19. In some embodiments, the fifth target molecule is CD20. In some embodiments, the fifth target molecule is CD22. In some embodiments, the fifth target molecule is CD3. In some embodiments, the fifth target molecule is CD2.

[0296] In some embodiments, the first, second, third, fourth, and fifth target molecules are each independently selected from the group consisting of CD19, CD20, CD22, CD3, and CD2.

[0297] In some embodiments, the first, second, third, fourth, and fifth target molecules are CD19, CD20, CD22, CD3, and CD2, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD19, CD20, CD22, CD2, and CD3, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD 19, CD20, CD3, CD22, and CD2, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD19, CD20, CD3, CD2, and CD22, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD 19, CD20, CD2, CD22, and CD3, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD19, CD20, CD2, CD3, and CD22, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD19, CD22, CD20, CD3, and CD2, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD 19, CD22, CD20, CD2, and CD3, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD19, CD22, CD3, CD20, and CD2, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD 19, CD22, CD3, CD2, and CD20, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD19, CD22, CD2, CD20, and CD3, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD19, CD22, CD2, CD3, and CD20, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD 19, CD3, CD20, CD22, and CD2, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD19, CD3, CD20, CD2, and CD22, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD 19, CD3, CD22, CD20, and CD2, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD19, CD3, CD22, CD2, and CD20, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD19, CD3, CD2, CD20, and CD22, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD 19, CD3, CD2, CD22, and CD20, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD19, CD2, CD20, CD22, and CD3, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD 19, CD2, CD20, CD3, and CD22, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules areWSGR Docket No. 69097-702.601CD19, CD2, CD22, CD20, and CD3, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD19, CD2, CD22, CD3, and CD20, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD 19, CD2, CD3, CD20, and CD22, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD19, CD2, CD3, CD22, and CD20, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD20, CD 19, CD22, CD3, and CD2, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD20, CD19, CD22, CD2, and CD3, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD20, CD19, CD3, CD22, and CD2, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD20, CD 19, CD3, CD2, and CD22, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD20, CD19, CD2, CD22, and CD3, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD20, CD 19, CD2, CD3, and CD22, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD20, CD22, CD19, CD3, and CD2, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD20, CD22, CD19, CD2, and CD3, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD20, CD22, CD3, CD 19, and CD2, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD20, CD22, CD3, CD2, and CD19, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD20, CD22, CD2, CD 19, and CD3, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD20, CD22, CD2, CD3, and CD19, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD20, CD3, CD19, CD22, and CD2, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD20, CD3, CD 19, CD2, and CD22, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD20, CD3, CD22, CD19, and CD2, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD20, CD3, CD22, CD2, and CD 19, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD20, CD3, CD2, CD19, and CD22, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD20, CD3, CD2, CD22, and CD19, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD20, CD2, CD 19, CD22, and CD3, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD20, CD2, CD19, CD3, and CD22, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD20, CD2, CD22, CD 19, and CD3, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules areWSGR Docket No. 69097-702.601CD20, CD2, CD22, CD3, and CD19, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD20, CD2, CD3, CD19, and CD22, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD20, CD2, CD3, CD22, and CD 19, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD22, CD19, CD20, CD3, and CD2, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD22, CD 19, CD20, CD2, and CD3, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD22, CD19, CD3, CD20, and CD2, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD22, CD19, CD3, CD2, and CD20, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD22, CD 19, CD2, CD20, and CD3, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD22, CD19, CD2, CD3, and CD20, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD22, CD20, CD 19, CD3, and CD2, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD22, CD20, CD19, CD2, and CD3, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD22, CD20, CD3, CD19, and CD2, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD22, CD20, CD3, CD2, and CD 19, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD22, CD20, CD2, CD19, and CD3, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD22, CD20, CD2, CD3, and CD 19, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD22, CD3, CD19, CD20, and CD2, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD22, CD3, CD19, CD2, and CD20, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD22, CD3, CD20, CD 19, and CD2, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD22, CD3, CD20, CD2, and CD19, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD22, CD3, CD2, CD 19, and CD20, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD22, CD3, CD2, CD20, and CD19, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD22, CD2, CD19, CD20, and CD3, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD22, CD2, CD 19, CD3, and CD20, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD22, CD2, CD20, CD19, and CD3, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD22, CD2, CD20, CD3, and CD 19, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules areWSGR Docket No. 69097-702.601CD22, CD2, CD3, CD19, and CD20, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD22, CD2, CD3, CD20, and CD19, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD3, CD 19, CD20, CD22, and CD2, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD3, CD19, CD20, CD2, and CD22, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD3, CD 19, CD22, CD20, and CD2, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD3, CD19, CD22, CD2, and CD20, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD3, CD19, CD2, CD20, and CD22, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD3, CD 19, CD2, CD22, and CD20, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD3, CD20, CD19, CD22, and CD2, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD3, CD20, CD 19, CD2, and CD22, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD3, CD20, CD22, CD19, and CD2, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD3, CD20, CD22, CD2, and CD19, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD3, CD20, CD2, CD 19, and CD22, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD3, CD20, CD2, CD22, and CD19, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD3, CD22, CD 19, CD20, and CD2, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD3, CD22, CD19, CD2, and CD20, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD3, CD22, CD20, CD19, and CD2, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD3, CD22, CD20, CD2, and CD 19, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD3, CD22, CD2, CD19, and CD20, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD3, CD22, CD2, CD20, and CD 19, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD3, CD2, CD19, CD20, and CD22, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD3, CD2, CD19, CD22, and CD20, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD3, CD2, CD20, CD 19, and CD22, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD3, CD2, CD20, CD22, and CD19, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD3, CD2, CD22, CD 19, and CD20, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules areWSGR Docket No. 69097-702.601CD3, CD2, CD22, CD20, and CD19, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD2, CD 19, CD20, CD22, and CD3, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD2, CD 19, CD20, CD3, and CD22, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD2, CD19, CD22, CD20, and CD3, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD2, CD 19, CD22, CD3, and CD20, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD2, CD19, CD3, CD20, and CD22, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD2, CD19, CD3, CD22, and CD20, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD2, CD20, CD 19, CD22, and CD3, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD2, CD20, CD19, CD3, and CD22, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD2, CD20, CD22, CD 19, and CD3, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD2, CD20, CD22, CD3, and CD19, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD2, CD20, CD3, CD19, and CD22, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD2, CD20, CD3, CD22, and CD 19, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD2, CD22, CD19, CD20, and CD3, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD2, CD22, CD 19, CD3, and CD20, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD2, CD22, CD20, CD19, and CD3, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD2, CD22, CD20, CD3, and CD19, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD2, CD22, CD3, CD 19, and CD20, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD2, CD22, CD3, CD20, and CD19, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD2, CD3, CD 19, CD20, and CD22, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD2, CD3, CD19, CD22, and CD20, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD2, CD3, CD20, CD19, and CD22, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD2, CD3, CD20, CD22, and CD 19, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD2, CD3, CD22, CD19, and CD20, respectively. In some embodiments, the first, second, third, fourth, and fifth target molecules are CD2, CD3, CD22, CD20, and CD 19, respectively.WSGR Docket No. 69097-702.601

[0298] A first polypeptide chain can comprise one of the binding domains and the second polypeptide chain can comprise four of the binding domains. For example, the first polypeptide chain can comprise the first target binding domain, and the second polypeptide chain can comprise the second, third, fourth, and fifth target binding domains. In some embodiments, the first polypeptide chain comprises the second target binding domain the second polypeptide chain comprises the first, third, fourth, and fifth target binding domains. In some embodiments, the first polypeptide chain comprises the third target binding domain and the second polypeptide chain comprises the first, second, fourth, and fifth target binding domains. In some embodiments, the first polypeptide chain comprises the fourth target binding domain and the second polypeptide chain comprises the first, second, third, and fifth target binding domains. In some embodiments, the first polypeptide chain comprises the fifth target binding domain and the second polypeptide chain comprises the first, second, third, and fourth target binding domains. The first and second polypeptides can each comprise a portion of a multimerization or dimerization module, for example, an Fc chain / region.

[0299] A first polypeptide chain can comprise two of the binding domains and the second polypeptide chain can comprise three of the binding domains. For example, the first polypeptide chain can comprise the first and second target binding domains, and the second polypeptide chain can comprise the third, fourth, and fifth target binding domains. In some embodiments, the first polypeptide chain comprises the first and third target binding domains the second polypeptide chain comprises the second, fourth, and fifth target binding domains. In some embodiments, the first polypeptide chain comprises the first and fourth target binding domains the second polypeptide chain comprises the second, third, and fifth target binding domains. In some embodiments, the first polypeptide chain comprises the first and fifth target binding domains the second polypeptide chain comprises the second, third, and fourth target binding domains. In some embodiments, the first polypeptide chain comprises the second and third target binding domains the second polypeptide chain comprises the first, fourth, and fifth target binding domains. In some embodiments, the first polypeptide chain comprises the second and fourth target binding domains the second polypeptide chain comprises the first, third, and fifth target binding domains. In some embodiments, the first polypeptide chain comprises the second and fifth target binding domains the second polypeptide chain comprises the first, third, and fourth target binding domains. In some embodiments, the first polypeptide chain comprises the third and fourth target binding domains the second polypeptide chain comprises the first, second, and fifth target binding domains. In some embodiments, the first polypeptide chain comprises the third and fifth target binding domains the second polypeptide chain comprises the first, second, and fourth target binding domains. In some embodiments, the first polypeptide chain comprises the fourth and fifth target binding domainsWSGR Docket No. 69097-702.601 the second polypeptide chain comprises the first, second, and third target binding domains. The first and second polypeptides can each comprise a portion of a multimerization or dimerization module, for example, an Fc chain / region.

[0300] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first target binding domain, the second target binding domain, the third target binding domain, and a first portion of a dimerization module (e.g., Fc region) and a second polypeptide chain comprises, from N-to-C terminus, the fourth target binding domain, the fifth target binding domain, and a second portion of the dimerization module (e.g., Fc region). The domains of each chain can optionally be joined by linkers, spacers, and / or hinges disclosed herein.

[0301] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first target binding domain, the second target binding domain the third target binding domain, and a first portion of a dimerization module (e.g., Fc region), and a second polypeptide chain comprises, from N-to-C terminus, the fourth target binding domain, a second portion of the dimerization module, and the fifth target binding domain. The domains of each chain can optionally be joined by linkers, spacers, and / or hinges disclosed herein.

[0302] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first target binding domain, the second target binding domain, the third target binding domain, the fourth target binding domain, and a first portion of a dimerization module (e.g., Fc region), and a second polypeptide chain comprises, from N-to-C terminus, the fifth target binding domain and a second portion of the dimerization module (e.g., Fc region). The domains of each chain can optionally be joined by linkers, spacers, and / or hinges disclosed herein.

[0303] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first target binding domain, the second target binding domain, the fourth target binding domain, the third target binding domain, and a first portion of a dimerization module (e.g., Fc region), and a second polypeptide chain comprises, from N-to-C terminus, the fifth target binding domain, and a second portion of the dimerization module (e.g., Fc region). The domains of each chain can optionally be joined by linkers, spacers, and / or hinges disclosed herein.

[0304] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first target binding domain, the fourth target binding domain, the second target binding domain, the third target binding domain, and a first portion of a dimerization module (e.g., Fc region), and a second polypeptide chain comprises, from N-to-C terminus, the fifth target binding domain, and a second portion of the dimerization module (e.g., Fc region). The domains of each chain can optionally be joined by linkers, spacers, and / or hinges disclosed herein.

[0305] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the fourth target binding domain, the first target binding domain, the second target binding domain,WSGR Docket No. 69097-702.601 the third target binding domain, and a first portion of a dimerization module (e.g., Fc region), and a second polypeptide chain comprises, from N-to-C terminus, the fifth target binding domain and a second portion of the dimerization module (e.g., Fc region). The domains of each chain can optionally be joined by linkers, spacers, and / or hinges disclosed herein.

[0306] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first target binding domain, the second target binding domain, the third target binding domain, a first portion of a dimerization module (e.g., Fc region), and the fourth target binding domain, and a second polypeptide chain comprises, from N-to-C terminus, the fifth target binding domain and a second portion of the dimerization module (e.g., Fc region). The domains of each chain can optionally be joined by linkers, spacers, and / or hinges disclosed herein.

[0307] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first target binding domain, the second target binding domain, the fourth target binding domain, and a first portion of a dimerization module (e.g., Fc region), and a second polypeptide chain comprises, from N-to-C terminus, the fifth target binding domain, the third target binding domain, and a second portion of the dimerization module (e.g., Fc region). The domains of each chain can optionally be joined by linkers, spacers, and / or hinges disclosed herein.

[0308] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first target binding domain, the second target binding domain, a first portion of a dimerization module (e.g., Fc region), the third target binding domain, and the fourth target binding domain, and a second polypeptide chain comprises, from N-to-C terminus, the fifth target binding domain and a second portion of the dimerization module (e.g., Fc region). The domains of each chain can optionally be joined by linkers, spacers, and / or hinges disclosed herein.

[0309] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first target binding domain, the second target binding domain, a first portion of a dimerization module (e.g., Fc region), and the third target binding domain, and a second polypeptide chain comprises, from N-to-C terminus, the fourth target binding domain, the fifth target binding domain, and a second portion of the dimerization module (e.g., Fc region). The domains of each chain can optionally be joined by linkers, spacers, and / or hinges disclosed herein.

[0310] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first target binding domain, the second target binding domain, a first portion of a dimerization module (e.g., Fc region) and the third target binding domain, and a second polypeptide chain comprises, from N-to-C terminus, the fourth target binding domain, a second portion of the dimerization module (e.g., Fc region), and the fifth target binding domain. The domains of each chain can optionally be joined by linkers, spacers, and / or hinges disclosed herein.WSGR Docket No. 69097-702.601

[0311] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first target binding domain, the second target binding domain, the fourth target binding domain, a first portion of a dimerization module (e.g., Fc region), and the third target binding domain, and a second polypeptide chain comprises, from N-to-C terminus, the fifth target binding domain and a second portion of the dimerization module (e.g., Fc region). The domains of each chain can optionally be joined by linkers, spacers, and / or hinges disclosed herein.

[0312] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first target binding domain, the fourth target binding domain, the second target binding domain, a first portion of a dimerization module (e.g., Fc region), and the third target binding domain, and a second polypeptide chain comprises, from N-to-C terminus, the fifth target binding domain and a second portion of the dimerization module (e.g., Fc region). The domains of each chain can optionally be joined by linkers, spacers, and / or hinges disclosed herein.

[0313] A multispecific immune cell engager can comprise four binding domains, each of which binds to a target molecule (e.g., a first target binding domain that binds to a first target molecule, a second target binding domain that binds to a second target molecule, a third target binding domain that binds to a third target molecule, and a fourth target binding domain that binds to a fourth target molecule). Two of the target molecules can be on target cells (e.g., the first and second target molecules bound by the first and second target binding domains can be tumor- associated antigens or B cell markers disclosed herein, such as two of CD 19, CD20, and CD22). Two of the target molecules, for example, the third and fourth target molecules bound by the third and fourth target binding domains, can be on immune effector cells (e.g., CD3 and CD2, in any order).

[0314] In some embodiments the first target molecule is CD 19 and the second target molecule is CD20. In some embodiments the first target molecule is CD 19 and the second target molecule is CD22. In some embodiments the first target molecule is CD20 and the second target molecule is CD22.

[0315] In some embodiments the third target molecule is CD2 and the fourth target molecule is CD3. In some embodiments the third target molecule is CD3 and the fourth target molecule is CD2.

[0316] In some embodiments, the first target molecule is CD19. In some embodiments, the first target molecule is CD20. In some embodiments, the first target molecule is CD22. In some embodiments, the first target molecule is CD3. In some embodiments, the first target molecule is CD2.

[0317] In some embodiments, the second target molecule is CD 19. In some embodiments, the second target molecule is CD20. In some embodiments, the second target molecule is CD22. InWSGR Docket No. 69097-702.601 some embodiments, the second target molecule is CD3. In some embodiments, the second target molecule is CD2.

[0318] In some embodiments, the third target molecule is CD 19. In some embodiments, the third target molecule is CD20. In some embodiments, the third target molecule is CD22. In some embodiments, the third target molecule is CD3. In some embodiments, the third target molecule is CD2.

[0319] In some embodiments, the fourth target molecule is CD19. In some embodiments, the fourth target molecule is CD20. In some embodiments, the fourth target molecule is CD22. In some embodiments, the fourth target molecule is CD3. In some embodiments, the fourth target molecule is CD2.

[0320] In some embodiments, the first, second, third, and fourth target molecules are each independently selected from the group consisting of CD 19, CD20, CD22, CD3, and CD2.

[0321] In some embodiments, the first, second, third, and fourth target molecules are CD 19, CD20, CD22, and CD3, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD 19, CD20, CD22, and CD2, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD19, CD20, CD3, and CD22, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD 19, CD20, CD3, and CD2, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD 19, CD20, CD2, and CD22, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD19, CD20, CD2, and CD3, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD19, CD22, CD20, and CD3, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD 19, CD22, CD20, and CD2, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD19, CD22, CD3, and CD20, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD19, CD22, CD3, and CD2, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD 19, CD22, CD2, and CD20, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD19, CD22, CD2, and CD3, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD19, CD3, CD20, and CD22, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD19, CD3, CD20, and CD2, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD 19, CD3, CD22, and CD20, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD19, CD3, CD22, and CD2, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD 19, CD3, CD2, and CD20, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD19, CD3, CD2, and CD22, respectively. InWSGR Docket No. 69097-702.601 some embodiments, the first, second, third, and fourth target molecules are CD 19, CD2, CD20, and CD22, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD19, CD2, CD20, and CD3, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD 19, CD2, CD22, and CD20, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD19, CD2, CD22, and CD3, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD 19, CD2, CD3, and CD20, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD19, CD2, CD3, and CD22, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD20, CD19, CD22, and CD3, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD20, CD 19, CD22, and CD2, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD20, CD19, CD3, and CD22, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD20, CD19, CD3, and CD2, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD20, CD 19, CD2, and CD22, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD20, CD 19, CD2, and CD3, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD20, CD22, CD 19, and CD3, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD20, CD22, CD 19, and CD2, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD20, CD22, CD3, and CD 19, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD20, CD22, CD3, and CD2, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD20, CD22, CD2, and CD 19, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD20, CD22, CD2, and CD3, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD20, CD3, CD 19, and CD22, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD20, CD3, CD19, and CD2, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD20, CD3, CD22, and CD19, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD20, CD3, CD22, and CD2, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD20, CD3, CD2, and CD 19, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD20, CD3, CD2, and CD22, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD20, CD2, CD 19, and CD22, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD20, CD2, CD19, and CD3, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD20, CD2, CD22, and CD 19, respectively. In some embodiments, the first, second, third, and fourth target moleculesWSGR Docket No. 69097-702.601 are CD20, CD2, CD22, and CD3, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD20, CD2, CD3, and CD19, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD20, CD2, CD3, and CD22, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD22, CD 19, CD20, and CD3, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD22, CD 19, CD20, and CD2, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD22, CD19, CD3, and CD20, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD22, CD 19, CD3, and CD2, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD22, CD 19, CD2, and CD20, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD22, CD 19, CD2, and CD3, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD22, CD20, CD19, and CD3, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD22, CD20, CD 19, and CD2, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD22, CD20, CD3, and CD19, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD22, CD20, CD3, and CD2, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD22, CD20, CD2, and CD 19, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD22, CD20, CD2, and CD3, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD22, CD3, CD19, and CD20, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD22, CD3, CD19, and CD2, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD22, CD3, CD20, and CD 19, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD22, CD3, CD20, and CD2, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD22, CD3, CD2, and CD19, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD22, CD3, CD2, and CD20, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD22, CD2, CD 19, and CD20, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD22, CD2, CD19, and CD3, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD22, CD2, CD20, and CD 19, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD22, CD2, CD20, and CD3, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD22, CD2, CD3, and CD 19, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD22, CD2, CD3, and CD20, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD3, CD19, CD20, and CD22, respectively. In some embodiments,WSGR Docket No. 69097-702.601 the first, second, third, and fourth target molecules are CD3, CD19, CD20, and CD2, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD3, CD 19, CD22, and CD20, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD3, CD19, CD22, and CD2, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD3, CD19, CD2, and CD20, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD3, CD19, CD2, and CD22, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD3, CD20, CD 19, and CD22, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD3, CD20, CD 19, and CD2, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD3, CD20, CD22, and CD19, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD3, CD20, CD22, and CD2, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD3, CD20, CD2, and CD 19, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD3, CD20, CD2, and CD22, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD3, CD22, CD19, and CD20, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD3, CD22, CD19, and CD2, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD3, CD22, CD20, and CD 19, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD3, CD22, CD20, and CD2, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD3, CD22, CD2, and CD19, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD3, CD22, CD2, and CD20, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD3, CD2, CD 19, and CD20, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD3, CD2, CD19, and CD22, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD3, CD2, CD20, and CD19, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD3, CD2, CD20, and CD22, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD3, CD2, CD22, and CD 19, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD3, CD2, CD22, and CD20, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD2, CD 19, CD20, and CD22, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD2, CD19, CD20, and CD3, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD2, CD 19, CD22, and CD20, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD2, CD19, CD22, and CD3, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD2, CD19, CD3, and CD20, respectively. In some embodiments, theWSGR Docket No. 69097-702.601 first, second, third, and fourth target molecules are CD2, CD19, CD3, and CD22, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD2, CD20, CD 19, and CD22, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD2, CD20, CD 19, and CD3, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD2, CD20, CD22, and CD 19, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD2, CD20, CD22, and CD3, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD2, CD20, CD3, and CD 19, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD2, CD20, CD3, and CD22, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD2, CD22, CD 19, and CD20, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD2, CD22, CD 19, and CD3, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD2, CD22, CD20, and CD 19, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD2, CD22, CD20, and CD3, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD2, CD22, CD3, and CD19, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD2, CD22, CD3, and CD20, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD2, CD3, CD 19, and CD20, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD2, CD3, CD19, and CD22, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD2, CD3, CD20, and CD19, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD2, CD3, CD20, and CD22, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD2, CD3, CD22, and CD 19, respectively. In some embodiments, the first, second, third, and fourth target molecules are CD2, CD3, CD22, and CD20, respectively.

[0322] A first polypeptide chain can comprise one of the binding domains and the second polypeptide chain can comprise three of the binding domains. For example, the first polypeptide chain can comprise the first target binding domain, and the second polypeptide chain can comprise the second, third, and fourth target binding domains. In some embodiments, the first polypeptide chain comprises the second target binding domain, and the second polypeptide chain comprises the first, third, and fourth target binding domains. In some embodiments, the first polypeptide chain comprises the third target binding domain, and the second polypeptide chain comprises the first, second, and fourth target binding domains. In some embodiments, the first polypeptide chain comprises the fourth target binding domain, and the second polypeptide chain comprises the first, second, and third target binding domains.WSGR Docket No. 69097-702.601

[0323] A first polypeptide chain can comprise two of the binding domains and the second polypeptide chain can comprise two of the binding domains. For example, the first polypeptide chain can comprise the first and second target binding domains, and the second polypeptide chain can comprise the third and fourth target binding domains. In some embodiments, the first polypeptide chain comprises the first and third target binding domains, and the second polypeptide chain can comprise the second and fourth target binding domains. In some embodiments, the first polypeptide chain comprises the first and fourth target binding domains, and the second polypeptide chain can comprise the second and third target binding domains. In some embodiments, the first polypeptide chain comprises the second and third target binding domains, and the second polypeptide chain can comprise the first and fourth target binding domains. In some embodiments, the first polypeptide chain comprises the second and fourth target binding domains, and the second polypeptide chain can comprise the first and third target binding domains. In some embodiments, the first polypeptide chain comprises the third and fourth target binding domains, and the second polypeptide chain can comprise the first and second target binding domains. The first and second polypeptides can each comprise a portion of a multimerization or dimerization module, for example, an Fc chain / region.

[0324] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first target binding domain, the second target binding domain, and a first portion of a dimerization module (e.g., Fc region), and a second polypeptide chain comprises, from N-to-C terminus, the third target binding domain, the fourth target binding domain, and a second portion of the dimerization module (e.g., Fc region). The domains of each chain can optionally be joined by linkers, spacers, and / or hinges disclosed herein.

[0325] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first target binding domain, the second target binding domain, and a first portion of a dimerization module (e.g., Fc region), and a second polypeptide chain comprises, from N-to-C terminus, the third target binding domain, a second portion of a dimerization module (e.g., Fc region), and the fourth target binding domain. The domains of each chain can optionally be joined by linkers, spacers, and / or hinges disclosed herein.

[0326] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first target binding domain, the second target binding domain, the third target binding domain, and a first portion of a dimerization module (e.g., Fc region), and a second polypeptide chain comprises, from N-to-C terminus, the fourth target binding domain, and a second portion of a dimerization module (e.g., Fc region). The domains of each chain can optionally be joined by linkers, spacers, and / or hinges disclosed herein.WSGR Docket No. 69097-702.601

[0327] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first target binding domain, the third target binding domain, the second target binding domain, and a first portion of a dimerization module (e.g., Fc region), and a second polypeptide chain comprises, from N-to-C terminus, the fourth target binding domain, and a second portion of the dimerization module (e.g., Fc region). The domains of each chain can optionally be joined by linkers, spacers, and / or hinges disclosed herein.

[0328] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the third target binding domain, the first target binding domain, the second target binding domain, and a first portion of a dimerization module (e.g., Fc region), and a second polypeptide chain comprises, from N-to-C terminus, the fourth target binding domain and a second portion of the dimerization module (e.g., Fc region). The domains of each chain can optionally be joined by linkers, spacers, and / or hinges disclosed herein.

[0329] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first target binding domain, the second target binding domain, a first portion of a dimerization module (e.g., Fc region), and the third target binding domain, and a second polypeptide chain comprises, from N-to-C terminus, the fourth target binding domain and a second portion of a dimerization module (e.g., Fc region). The domains of each chain can optionally be joined by linkers, spacers, and / or hinges disclosed herein.

[0330] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first target binding domain, the third target binding domain, and a first portion of a dimerization module (e.g., Fc region) and a second polypeptide chain comprises, from N-to-C terminus, the fourth target binding domain, the second target binding domain, and a second portion of the dimerization module (e.g., Fc region). The domains of each chain can optionally be joined by linkers, spacers, and / or hinges disclosed herein.

[0331] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first target binding domain, a first portion of a dimerization module (e.g., Fc region), the second target binding domain, an the third target binding domain, and a second polypeptide chain comprises, from N-to-C terminus, the fourth target binding domain and a second portion of the dimerization module (e.g., Fc region). The domains of each chain can optionally be joined by linkers, spacers, and / or hinges disclosed herein.

[0332] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first target binding domain, a first portion of a dimerization module (e.g., Fc region), the second target binding domain, and a second polypeptide chain comprises, from N-to-C terminus, the third target binding domain, the fourth target binding domain, and a second portion of the dimerizationWSGR Docket No. 69097-702.601 module (e.g., Fc region). The domains of each chain can optionally be joined by linkers, spacers, and / or hinges disclosed herein.

[0333] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first target binding domain, a first portion of a dimerization module (e.g., Fc region), and the second target binding domain, and a second polypeptide chain comprises, from N-to-C terminus, the third target binding domain, a second portion of the dimerization module (e.g., Fc region), and the fourth target binding domain. The domains of each chain can optionally be joined by linkers, spacers, and / or hinges disclosed herein.

[0334] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first target binding domain, the third target binding domain, a first portion of a dimerization module (e.g., Fc region), and the second target binding domain, and a second polypeptide chain comprises, from N-to-C terminus, the fourth target binding domain and a second portion of the dimerization module (e.g., Fc region). The domains of each chain can optionally be joined by linkers, spacers, and / or hinges disclosed herein.

[0335] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the third target binding domain, the first target binding domain, a first portion of a dimerization module (e.g., Fc region), and the second target binding domain, and a second polypeptide chain comprises, from N-to-C terminus, the fourth target binding domain and a second portion of the dimerization module (e.g., Fc region). The domains of each chain can optionally be joined by linkers, spacers, and / or hinges disclosed herein.

[0336] Illustrative, non-limiting examples of polypeptide chains that can be used in multispecific immune cell engagers (e.g., T cell engagers) are provided in TABLE 14.

[0337] In some embodiments, a multispecific immune cell engager or a part thereof (e.g., a first polypeptide chain or a second polypeptide chain) comprises, consists essentially of, or consists of an amino acid sequence with at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or sequence similarity to any one of SEQ ID NOs: 183-222. A multispecific immune cell engager can comprise two such sequences, for example, a first polypeptide chain and a second polypeptide chain.WSGR Docket No. 69097-702.601

[0338] In some embodiments, a multispecific immune cell engager or a part thereof (e.g., a first polypeptide chain or a second polypeptide chain) comprises, consists essentially of, or consists of an amino acid sequence with at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or sequence similarity to at least 250 consecutive amino acids of any one of SEQ ID NOs: 183-222.

[0339] In some embodiments, a multispecific immune cell engager or a part thereof (e.g., a first polypeptide chain or a second polypeptide chain) comprises, consists essentially of, or consists of an amino acid sequence with at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or sequence similarity to at least 400 consecutive amino acids of any one of SEQ ID NOs: 183-222.

[0340] In some embodiments, a multispecific immune cell engager or a part thereof (e.g., a first polypeptide chain or a second polypeptide chain) comprises, consists essentially of, or consists of an amino acid sequence with at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or sequence similarity to at least 500 consecutive amino acids of any one of SEQ ID NOs: 183-222.

[0341] TABLE 14: Illustrative polypeptide chains.| TABLE 14WSGR Docket No. 69097-702.601WSGR Docket No. 69097-702.601WSGR Docket No. 69097-702.601WSGR Docket No. 69097-702.601WSGR Docket No. 69097-702.601WSGR Docket No. 69097-702.601WSGR Docket No. 69097-702.601WSGR Docket No. 69097-702.601WSGR Docket No. 69097-702.601

[0342] Illustrative, non-limiting examples of combinations of the polypeptide chains to form multispecific immune cell engagers (e.g., T cell engagers) and control constructs are provided in TABLE 15. A “complex” as denoted herein is a polypeptide chain complex (namely, a construct), composed of the polypeptide chains as described in the columns to the right. Each such complex, e.g., composed of a first polypeptide chain and a second polypeptide chain is provided an identification number, e.g., a complex ID.

[0343] TABLE 15: Illustrative polypeptide chain combinationsWSGR Docket No. 69097-702.601WSGR Docket No. 69097-702.601WSGR Docket No. 69097-702.601

[0344] Additional illustrative, non-limiting examples of polypeptide chains that can be used in multispecific immune cell engagers (e.g., T cell engagers) are provided in Table 16. In some embodiments, exemplary chain 1 sequences are as follows in Table 16. In several instances platform structures have been generated, purified and tested as exemplified herein, which can provide information on positional candidate binder domains, (e.g., scaffolds), while leaving the opportunity open for extending an engager to include one or more additional domains at some positions.

[0345] In some embodiments, a multispecific immune cell engager or a part thereof (e.g., a first polypeptide chain or a second polypeptide chain) comprises, consists essentially of, or consists of an amino acid sequence with at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or sequence similarity to any one of SEQ ID NOs: 1070-1074. A multispecific immune cell engagerWSGR Docket No. 69097-702.601 can comprise two such sequences, for example, a first polypeptide chain and a second polypeptide chain.

[0346] In some embodiments, a multispecific immune cell engager or a part thereof (e.g., a first polypeptide chain or a second polypeptide chain) comprises, consists essentially of, or consists of an amino acid sequence with at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or sequence similarity to at least 250 consecutive amino acids of any one of SEQ ID NOs: 1070- 1074

[0347] In some embodiments, a multispecific immune cell engager or a part thereof (e.g., a first polypeptide chain or a second polypeptide chain) comprises, consists essentially of, or consists of an amino acid sequence with at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or sequence similarity to at least 400 consecutive amino acids of any one of SEQ ID NOs: 1070- 1074.

[0348] In some embodiments, a multispecific immune cell engager or a part thereof (e.g., a first polypeptide chain or a second polypeptide chain) comprises, consists essentially of, or consists of an amino acid sequence with at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, atWSGR Docket No. 69097-702.601 least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or sequence similarity to at least 500 consecutive amino acids of any one of SEQ ID NOs: 1070- 1074.

[0349] In some embodiments, a multispecific immune cell engager or a part thereof (e.g., a first polypeptide chain or a second polypeptide chain) comprises, consists essentially of, or consists of an amino acid sequence with at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or sequence similarity to any one of SEQ ID NOs: 1075-1076. A multispecific immune cell engager can comprise two such sequences, for example, a first polypeptide chain and a second polypeptide chain.

[0350] Table 16. Exemplary non-limiting polypeptide chain sequences.WSGR Docket No. 69097-702.601

[0351] Table 17. Additional illustrative, non-limiting examples of polypeptide chains that can be used in multispecific immune cell engagers (e.g., T cell engagers) are provided in Table 17. In some embodiments, exemplary chain 2 sequences are as follows in Table 17.WSGR Docket No. 69097-702.601

[0352] Table 18 Illustrative polypeptide chain combinations (complexes, or constructs as described herein).

[0353] In some embodiments, an exemplary multispecific immune cell engager (e.g., TCE) polypeptide chains comprise a first polypeptide (chain 1) and a second polypeptide (chain 2), wherein the first polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 1070; and a second polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 184. In some embodiments, the polypeptide chains comprise a first polypeptide (chain 1) and a second polypeptide (chain 2), wherein the first polypeptide comprises a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: 1070; and a second polypeptide comprises a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: 184. In some embodiments, the first polypeptide has an amino acid sequence set forth in SEQ ID NO: 1070; and a second polypeptide has an amino acid sequence set forth in SEQ ID NO: 184. In some embodiments, the first polypeptide has an amino acid sequence set forth in SEQ ID NO: 1070, and the second polypeptide has an amino acid sequenceWSGR Docket No. 69097-702.601 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: 184. In some embodiments, the first polypeptide has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: SEQ ID NO: 1070; and the second polypeptide has an amino acid sequence set forth in SEQ ID NO: 184.

[0354] In some embodiments, the polypeptide chains comprise a first polypeptide (chain 1) and a second polypeptide (chain 2), wherein the first polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 1070; and a second polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 186. In some embodiments, the polypeptide chains comprise a first polypeptide (chain 1) and a second polypeptide (chain 2), wherein the first polypeptide comprises a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: 1070; and a second polypeptide comprises a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: 186. In some embodiments, the first polypeptide has an amino acid sequence set forth in SEQ ID NO: 1070; and a second polypeptide has an amino acid sequence set forth in SEQ ID NO: 186. In some embodiments, the first polypeptide has an amino acid sequence set forth in SEQ ID NO: 1070, and the second polypeptide has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: 186. In some embodiments, the first polypeptide has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: SEQ ID NO: 1070; and the second polypeptide has an amino acid sequence set forth in SEQ ID NO: 186.

[0355] In some embodiments, the polypeptide chains comprise a first polypeptide (chain 1) and a second polypeptide (chain 2), wherein the first polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 1070; and a second polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 187. In some embodiments, the polypeptide chains comprise a first polypeptide (chain 1) and a second polypeptide (chain 2), wherein the first polypeptide comprises a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: 1070; and a second polypeptide comprises a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: 187. In some embodiments, the first polypeptide has an amino acid sequence set forth in SEQ ID NO: 1070; and a second polypeptide has an amino acid sequence set forth in SEQ ID NO: 187. In some embodiments, the first polypeptide has an amino acid sequence set forth in SEQ ID NO: 1070, and the second polypeptide has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: 187. In some embodiments, the first polypeptide has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99%WSGR Docket No. 69097-702.601 identical to SEQ ID NO: SEQ ID NO: 1070; and the second polypeptide has an amino acid sequence set forth in SEQ ID NO: 187.

[0356] In some embodiments, the polypeptide chains comprise a first polypeptide (chain 1) and a second polypeptide (chain 2), wherein the first polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 1071; and a second polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 184. In some embodiments, the polypeptide chains comprise a first polypeptide (chain 1) and a second polypeptide (chain 2), wherein the first polypeptide comprises a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: 1071; and a second polypeptide comprises a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: 184. In some embodiments, the first polypeptide has an amino acid sequence set forth in SEQ ID NO: 1071; and a second polypeptide has an amino acid sequence set forth in SEQ ID NO: 184. In some embodiments, the first polypeptide has an amino acid sequence set forth in SEQ ID NO: 1071, and the second polypeptide has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: 184. In some embodiments, the first polypeptide has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: SEQ ID NO: 1071; and the second polypeptide has an amino acid sequence set forth in SEQ ID NO: 184.

[0357] In some embodiments, the polypeptide chains comprise a first polypeptide (chain 1) and a second polypeptide (chain 2), wherein the first polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 1072; and a second polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 184. In some embodiments, the polypeptide chains comprise a first polypeptide (chain 1) and a second polypeptide (chain 2), wherein the first polypeptide comprises a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: 1072; and a second polypeptide comprises a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: 184. In some embodiments, the first polypeptide has an amino acid sequence set forth in SEQ ID NO: 1072; and a second polypeptide has an amino acid sequence set forth in SEQ ID NO: 184. In some embodiments, the first polypeptide has an amino acid sequence set forth in SEQ ID NO: 1072, and the second polypeptide has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: 184. In some embodiments, the first polypeptide has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: SEQ ID NO: 1072; and the second polypeptide has an amino acid sequence set forth in SEQ ID NO: 184.WSGR Docket No. 69097-702.601

[0358] In some embodiments, the polypeptide chains comprise a first polypeptide (chain 1) and a second polypeptide (chain 2), wherein the first polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 1072; and a second polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 1075. In some embodiments, the polypeptide chains comprise a first polypeptide (chain 1) and a second polypeptide (chain 2), wherein the first polypeptide comprises a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: 1072; and a second polypeptide comprises a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: 1075. In some embodiments, the first polypeptide has an amino acid sequence set forth in SEQ ID NO: 1072; and a second polypeptide has an amino acid sequence set forth in SEQ ID NO: 1075. In some embodiments, the first polypeptide has an amino acid sequence set forth in SEQ ID NO: 1072, and the second polypeptide has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: 1075. In some embodiments, the first polypeptide has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: SEQ ID NO: 1072; and the second polypeptide has an amino acid sequence set forth in SEQ ID NO: 1075.

[0359] In some embodiments, the polypeptide chains comprise a first polypeptide (chain 1) and a second polypeptide (chain 2), wherein the first polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 1072; and a second polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 1076. In some embodiments, the polypeptide chains comprise a first polypeptide (chain 1) and a second polypeptide (chain 2), wherein the first polypeptide comprises a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: 1072; and a second polypeptide comprises a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: 1076. In some embodiments, the first polypeptide has an amino acid sequence set forth in SEQ ID NO: 1072; and a second polypeptide has an amino acid sequence set forth in SEQ ID NO: 1076. In some embodiments, the first polypeptide has an amino acid sequence set forth in SEQ ID NO: 1072, and the second polypeptide has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: 1076. In some embodiments, the first polypeptide has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: SEQ ID NO: 1072; and the second polypeptide has an amino acid sequence set forth in SEQ ID NO: 1076.

[0360] In some embodiments, the polypeptide chains comprise a first polypeptide (chain 1) and a second polypeptide (chain 2), wherein the first polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 1073; and a second polypeptide comprises a sequence that isWSGR Docket No. 69097-702.601 at least 90% identical to SEQ ID NO: 184. In some embodiments, the polypeptide chains comprise a first polypeptide (chain 1) and a second polypeptide (chain 2), wherein the first polypeptide comprises a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: 1073; and a second polypeptide comprises a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: 184. In some embodiments, the first polypeptide has an amino acid sequence set forth in SEQ ID NO: 1073; and a second polypeptide has an amino acid sequence set forth in SEQ ID NO: 184. In some embodiments, the first polypeptide has an amino acid sequence set forth in SEQ ID NO: 1073, and the second polypeptide has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: 184. In some embodiments, the polypeptide chains comprise a first polypeptide (chain 1) and a second polypeptide (chain 2), wherein the first polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 1074; and a second polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 184. In some embodiments, the polypeptide chains comprise a first polypeptide (chain 1) and a second polypeptide (chain 2), wherein the first polypeptide comprises a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: 1074; and a second polypeptide comprises a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: 184. In some embodiments, the first polypeptide has an amino acid sequence set forth in SEQ ID NO: 1074; and a second polypeptide has an amino acid sequence set forth in SEQ ID NO: 184. In some embodiments, the first polypeptide has an amino acid sequence set forth in SEQ ID NO: 1074, and the second polypeptide has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: 184. In some embodiments, the first polypeptide has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to SEQ ID NO: SEQ ID NO: 1074; and the second polypeptide has an amino acid sequence set forth in SEQ ID NO: 184.

[0361] In some embodiments, provided herein is a polypeptide complex, (e.g., a T cell engager, or TCE) comprising a first polypeptide comprising a CD19 binder, e.g., aCD19(sdAb), linked to a first Fc region, e.g., a Fc(hole) having a sequence of SEQ ID NO: 1070 (an exemplary first polypeptide designated as C232, Table 18); and a second polypeptide comprising from N terminus to C terminus a fourth target binding domain linked to a fifth target binding domain linked to a second Fc region, wherein the first target binding domain binds to a first target molecule, and the fourth target binding domain binds to a fourth target molecule, wherein the fourth target molecule is CD3, and the fifth target binding domain binds to a fifth target molecule, wherein the fifth target molecule is CD2. Accordingly, in some embodiments, the second polypeptide is aCD3(scFv)-CD58(WT)-Fc(knob). In some embodiments, the second polypeptideWSGR Docket No. 69097-702.601 is C007; comprising or consisting of SED ID NO: 186. In some embodiments, the polypeptide complex is a Format 1 complex, having the complex ID of PROT328 (Table 16, and Table 18), comprising a first polypeptide chain having a sequence of SEQ ID NO: 1070, and a second polypeptide chain having a sequence of SEQ ID NO: 186. In some embodiments, the polypeptide complex is a Format 2 complex, having a complex ID ofPROT330, (Table 16, Table 18), wherein the first polypeptide is an anti-CD19sdAb-Fc-Hole (aCD19(sdAb)-Fc(hole)) (chain 1), having a sequence of SEQ ID NO: 1070; and the second polypeptide is an anti-CD3scFv-Fc-Knob- CD58(WT), (aCD3(scFv)- Fc(knob)-CD58(WT)) (chain 2), having a sequence of SEQ ID NO: 187. Often, a short form of a binding domain is denoted by the prefix, “a”- followed by the target molecule, which denotes “anti”- (target denoted). For example, an anti-CD19 sdAb may be denoted as aCD19sdAb. In some embodiments, the polypeptide complex is a Format 3 complex, having a complex ID of PROT331, (Table 16, Table 18), wherein the first polypeptide is a aCD19(sdAb)-CD58 IgV WT-Fc(hole) having a sequence of SEQ ID NO: 1071, the second polypeptide is a aCD3(scFv)-Fc(knob)-CD58(WT) having a sequence of SEQ ID NO: 184.

[0362] Additional constituent domains or fragments or parts of some of the polypeptides whose sequences are disclosed above are exemplified below, where each part with its respective amino acid sequence is depicted in Table 19.

[0363] Table 19. Exemplary constituent parts of exemplary polypeptide chainsWSGR Docket No. 69097-702.601WSGR Docket No. 69097-702.601II. POLYNUCLEOTIDES AND VECTORS

[0364] In some embodiments the disclosure provides a polynucleotide that encodes a multispecific immune cell engager disclosed herein. The polynucleotide can be prepared by standard molecular biology techniques. The polynucleotide can be prepared by molecular cloning. The polynucleotide can be synthesized de novo. The polynucleotide can comprise a nucleotide sequence encoding the multispecific immune cell engager, operably linked to transcription regulatory sequences such as a promoter, and optionally a 3' untranslated region. A constitutive, inducible, or tissue-specific promoter can be used.

[0365] The polynucleotide can be a DNA. The polynucleotide can be an RNA, e.g., a mRNA. The polynucleotide can comprise a modified base, for example, to enhance stability of the polynucleotide upon administration to a subject. In some embodiments, a polynucleotide disclosed herein can be codon optimized. A polynucleotide provided can include a recombinant, artificial, or synthetic polynucleotide. The polynucleotide can be single stranded. The polynucleotide can be double stranded. The polynucleotide can be recombinant and / or isolated.

[0366] The polynucleotide can be inserted into or part of a vector, such as an expression vector, such that the genes are operatively linked to transcriptional and / or translational control sequences. The vector can comprise a selectable marker for selection of a vector-carrying host cell. The vector can lack a selectable marker. The vector can comprise an origin of replication or can lack an origin of replication. The vector can be a plasmid, for example, a nanoplasmid. The vector can be a minicircle. The vector can be a liner polynucleotide phagemid, cosmid, RNAWSGR Docket No. 69097-702.601 vector, viral vector or the like. Non-limiting examples of viral vectors include a retrovirus (e.g., lentivirus), an adenovirus, and an adeno-associated virus. The vector can be a nonviral vector.

[0367] In some embodiments, a polynucleotide encoding a multispecific immune cell engager is packaged in a lipid-based delivery vector, such as a liposome or lipid nanoparticle. In some embodiments, the lipid based delivery vehicle can comprise a polar lipid, a non-polar lipid, a neutral lipid, and / or polyethylene glycol. In some embodiments, the lipid based delivery vehicle can comprise cholesterol. In some embodiments, the lipid components are selected from, but are not limited to, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200 (including variants and derivatives), DLin-MC3-DMA and analogs thereof.

[0368] In one aspect, a TCE described herein when expressed in a cell, e.g. a lymphocyte, more specifically a T cell, activates the cell. In some embodiments, activation of a cell expressing the TCE is detectable by enhanced cytokine secretion, e.g., proinflammatory cytokines, e.g., IFN- gamma (IFN-g), Tumor Necrosis Factor- alpha (TNF-a), interleukin-2 (IL-2) and others. In some embodiments, the cellular activation upon expression of the TCE in the cell activates the cell to exhibit cytotoxicity towards a target cell that expresses at least one of the target molecules to which the target binding domains of the TCE are designed to bind. Cytotoxicity is measured by cell lysis. In some embodiments, the TCE when expressed in a T cell, induces T cell dependent cell cytotoxicity (TDCC) which can be used to drive cancer cell death when a cancer cell is the target cell.

[0369] In some embodiments, a TCE is designed such that it can induce higher TDCC when expressed in a T cell compared to a T cell that does not express the TCE or expresses a different TCE. The different TCE may comprise one or more binding domain that is different that the TCE disclosed herein. Additionally, or alternatively, the different TCE may comprise a TCE that comprises the same binding domains which are not in the exact same orientation as the TCE designed herein. In some embodiments, the TCE designed and selected herein is a TCE exhibiting superior functionality when expressed in a T cell, when such T cell is in the presence of a target cell expressing the target molecule to which at least one of the TCE binders is designed to bind; comprising one or more of: enhanced cytokine production by the T cell in presence of the target cell, comprising increased release of IFN-g, TNF-a and / or IL-2 in the presence of a target cell; enhanced lysis of a target cell, increased NF AT activation, or activation of transcription factor associated with T cell activation and proliferation. In some embodiments, a TCE designed and selected herein when expressed in a T cell induces at least 2-fold, 5-fold, 10 -fold, 20-fold, 30- fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 1000-fold more IFN-g release in the presence of the target cell than when the T cell expresses a different TCE that does not express one or two or any of the binding domains of the selected TCE. In some embodiments, aWSGR Docket No. 69097-702.601TCE designed and selected herein when expressed in a T cell induces at least 50-fold, 100-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold 7000-fold, 8000-fold, 9000- fold, 10000-fold more IFN-g release in the presence of a target cell, than when the T cell expresses a different TCE that does not express any of the binding domains of the selected TCE, all other conditions being identical. In some embodiments, a TCE designed and selected herein when expressed in a T cell induces in the presence of a target cell at least 2-fold, 5-fold, 10 -fold, 20- fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 1000-fold more TNF- a release in the presence of a target cell, than when the T cell expresses a different TCE that does not express one or two or any o...

Claims

WSGR Docket No. 69097-702.601CLAIMSWHAT IS CLAIMED IS:

1. An engineered T cell engager (TCE) comprising at least four target binding domains, a first target binding domain, a second target binding domain, a third target binding domain and a fourth target binding domain, the engineered TCE comprising:(a) a first polypeptide, comprising from C terminus to N terminus:(i) a first Fc region (Fcl) linked to the first target binding domain, or(ii) the first target binding domain linked to the Fcl; and(b) a second polypeptide comprising from N terminus to C terminus the fourth target binding domain linked to a second Fc region (Fc2), wherein the fourth target binding domain is selected from a CD3 binding domain and a CD2 binding domain.

2. An engineered TCE comprising at least four target binding domains configured to bind to at least three non-identical targets, the engineered TCE comprising:(a) a first polypeptide, comprising a first Fc region (Fcl), operably linked to at least a target binding domain, and(b) a second polypeptide, comprising a second Fc region (Fc2), linked to at least two target binding domains; wherein at least one target binding domain of the at least two target binding domains comprises a CD3 binding domain or a CD2 binding domain.

3. An engineered TCE comprising at least four binding domains configured to bind to at least three non-identical targets, the engineered TCE comprising:(a) a first polypeptide, comprising a first Fc region (Fcl), operably linked to at least one target binding domain, wherein the at least one target binding domain is a CD2 binding domain, and(b) a second polypeptide, comprising a second Fc region (Fc2) operably linked to at least one target binding domain, wherein the at least one target binding domain is a CD3 binding domain.

4. The engineered TCE of any one of claims 1-3, wherein the Fcl and Fc2 dimerize.

5. The engineered TCE of any one of claims 1-4, wherein the CD2 binding domain is a CD58 ligand comprising a full length CD58, an extracellular domain of CD58, a CD2- binding domain of CD58, an Ig-like domain of CD58, an IgV domain of CD58, or a functional fragment or a functional variant thereof.

6. The engineered TCE of any one of claims 1, 2, 4 or 5, wherein the second polypeptide comprises a CD58 ligand.WSGR Docket No. 69097-702.6017. The engineered TCE of claims 1, 2 or 4-6, wherein the TCE comprises five target binding domains, a first target binding domain, a second target binding domain, a third target binding domain, a fourth target binding domain and a fifth target binding domain, wherein the second polypeptide comprises from N terminus to C terminus the fourth target binding domain linked to the fifth target binding domain linked to the Fc2, wherein the fourth target binding domain binds to CD3, and the fifth target binding domain is the CD58 ligand.

8. The engineered TCE of any one of claims 1, 2, or 4-6, wherein the TCE comprises five target binding domains, a first target binding domain, a second target binding domain, a third target binding domain, a fourth target binding domain and a fifth target binding domain, wherein the second polypeptide comprises from N terminus to C terminus a fourth target binding domain linked to the Fc2, and the Fc2 linked to a fifth target binding domain, wherein the fourth target binding domain binds to CD3, and the fifth target binding domain is the CD58 ligand.

9. The engineered TCE of any one of claims 1-5, wherein the first polypeptide comprises the CD58 ligand.

10. The engineered TCE of any one of claims 1, 4-6 or 9, wherein the TCE comprises five target binding domains, a first target binding domain, a second target binding domain, a third target binding domain, a fourth target binding domain and a fifth target binding domain, wherein (i) the first polypeptide comprises from C terminus to N terminus the Fcl, operably linked to the CD58 ligand, or (ii) the first polypeptide comprises from N terminus to C terminus the Fcl, operably linked to the CD 58 ligand and wherein the fourth target binding domain or the fifth target binding domain is linked to the Fc2.

11. The engineered TCE of claim 10, wherein the Fcl is either directly linked to the CD58 ligand or is indirectly linked to the CD58 ligand via linkage through one or more target binding domains other than a CD3 binding domain.

12. The engineered TCE of any one of claims 1-5 or 9, wherein the first polypeptide comprises from C terminus to N terminus the CD58 ligand linked to the Fcl; and wherein the second polypeptide comprises from N terminus to C terminus the fourth target binding domain or the fifth target binding domain linked to a second Fc region, wherein the fourth target binding domain or the fifth target binding domain is the CD3 binding domain.

13. The engineered TCE of any one of claims 1-5, or 9, wherein the first polypeptide comprises from C terminus to N terminus a CD58 ligand linked to a first target binding domain linked to the Fcl; and a second polypeptide comprising from N terminus to CWSGR Docket No. 69097-702.601 terminus a fourth target binding domain or a fifth target binding domain linked to the Fc2, wherein the fourth target binding domain or the fifth target binding domain is the CD3 binding domain.

14. The engineered TCE of any one of claims 1-13 wherein the first target binding domain, the second target binding domain, the third target binding domain and / or the CD3 binding domain comprises a Fab, a F(ab')2, an Fv, a single chain Fv (scFv), a single domain antibody (sdAb), a diabody (dAb), a VHH, a camelid antibody, a nanobody, or any combination thereof.

15. The engineered TCE of any one of claims 1-14, wherein a target binding domain from the at least four target binding domains binds to a target molecule that is a tumor- associated antigen CD 19, and the target binding domain is an anti-CD19 domain.

16. The engineered TCE of any one of claims 1-15, wherein a target binding domain from the at least four target binding domains binds to a target molecule that is a tumor- associated antigen CD20, and the target binding domain is an anti-CD20 domain.

17. The engineered TCE of any one of claims 1-16, wherein a target binding domain from the at least four target binding domains binds to a target molecule is a tumor- associated antigen CD22, and the target binding domain is an anti-CD22 domain.

18. The engineered TCE of any one of claims 1-17, wherein the fourth target binding domain and the fifth target binding domains are selected from the CD3 binding domain and the CD2 binding domain.

19. The engineered TCE of any one of claims 1-18, wherein the first Fc region and the second Fc region form a heterodimer and comprise one or more modifications or is unmodified.

20. The engineered TCE of any one of claims 1-19, wherein the engineered TCE further comprises one or more linkers, wherein the one or more linkers comprise an amino acid sequence selected from any one of SEQ ID NOs: 146-182 listed in Table 13.

21. The engineered TCE of any one of claims 1-20, wherein the CD58 ligand is in turn operably linked via a linker to a second, third and / or fourth target binding domains selected from a group consisting of an anti-CD19 domain, an anti-CD20 domain and an anti-CD22 domain.

22. The engineered TCE of any one of claims 15-21, wherein the anti-CD19 domain is a single domain antibody fragment (sdAb) comprising an amino acid sequence set forth inWSGR Docket No. 69097-702.601SEQ ID NOs: 1-21 or an amino acid sequence that is at least 80% identical to any one of the sequences set forth in SEQ ID NOs: 1-21.

23. The engineered TCE of any one of claims 15-22, wherein the anti-CD19 domain is a single chain variable fragment domain (scFv), comprising a variable heavy chain (VH) domain, and a variable light chain (VL) domain; wherein the VH domain comprises an amino acid sequence is as set forth in the sequences selected from SEQ ID NOs: 23, 26, 28, 30, 32, and 34, or an amino acid sequence that is at least 80% identical to any one of the sequences set forth in SEQ ID NOs: 23, 26, 28, 30, 32, and 34; and a VL domain comprises an amino acid sequence is as set forth in the sequences selected from SEQ ID NOs: 24, 27, 29, 31, 33 and 35, or an amino acid sequence that is at least 80% identical to any one of the sequences set forth in SEQ ID NOs: 24, 27, 29, 31, 33 and 35.

24. The engineered TCE of claim 23, wherein the anti-CD19 domain comprises an scFv comprising an amino acid sequence set forth in SEQ ID NOs: 22 or 25, or a sequence that is at least 80% identical to SEQ ID NO: 22 or 25.

25. The engineered TCE of any one of claims 15-24, wherein the anti-CD19 domain comprises a sequence of any one of the sequences depicted in Table 1, or a sequence that is at least 80% identical to any one of the sequences in Table 1.

26. The engineered TCE of any one of claims 15-25, wherein the anti-CD19 domain is a single domain antibody fragment (sdAb), comprising a heavy chain complementarity determining region (HCDR)l, an HCDR2, and an HCDR3 wherein the HCDR1, HCDR2 and the HCDR3 comprise amino acid sequences as listed in Table 2.

27. The engineered TCE of any one of claims 15-24, wherein the anti-CD19 domain is a single chain variable fragment domain (scFv), comprising a heavy chain complementarity determining region (HCDR)1, an HCDR2, and an HCDR3; and a light chain complementarity determining region (LCDR)1, a LCDR2, and a LCDR3 wherein the HCDR1, the HCDR2 and the HCDR3 and the LCDR1, the LCDR2 and the LCDR3 comprise amino acid sequences as listed in Table 2.

28. The engineered TCE of any one of claims 16-27, wherein the anti-CD20 domain is an sdAb comprising an amino acid sequence as set forth in the sequences selected from SEQ ID NOs: 36-46, or a sequence that is at least 80% identical to the any one of the sequences SEQ ID NOs: 36-46.

29. The engineered TCE of any one of claims 16-27, wherein the anti-CD20 domain is an scFv, comprising a variable heavy chain (VH) domain, and a variable light chain (VL) domain; wherein the VH domain comprises an ammino acid sequence selected from SEQ ID NOs: 47, 49, 51 and 53 or a sequence having at least 80% sequence identity toWSGR Docket No. 69097-702.601 any one of the sequences set forth in SEQ ID NOs: 47, 49, 51 and 53; and a VL domain selected from SEQ ID NOs: 48, 50, 52 and 54, or a sequence having at least 80% sequence identity to any one of the sequences set forth in SEQ ID NOs: 48, 50, 52 and 54 as listed in Table 3.

30. The engineered TCE of any one of claims 16-27, wherein the anti-CD20 domain is an sdAb, comprising a heavy chain complementarity determining region (HCDR)l, an HCDR2, and an HCDR3 wherein the HCDR1, HCDR2 and the HCDR3 comprise amino acid sequences as listed in Table 4.

31. The engineered TCE of any one of claims 16-27, wherein the anti-CD20 domain is an scFv, comprising a heavy chain complementarity determining region (HCDR)1, an HCDR2, and an HCDR3; and a light chain complementarity determining region (LCDR)l, a LCDR2, and a LCDR3 wherein the HCDR1, the HCDR2 and the HCDR3 and the LCDR1, the LCDR2 and the LCDR3 comprise amino acid sequences as listed in Table 4.

32. The engineered TCE of any one of claims 17-31, wherein the anti-CD22 domain is an sdAb comprising an amino acid sequence selected from SEQ ID NOs: 55-74, or a sequence having at least 80% sequence identity to any one of the sequences set forth in SEQ ID NOs: 55-74.

33. The engineered TCE of any one of claims 17-31, wherein the anti-CD22 domain is an scFv, comprising a variable heavy chain (VH) domain, and a variable light chain (VL) domain; wherein the VH domain comprises an amino acid sequence selected from SEQ ID NOs: 75 and 77; and a VL domain comprises an amino acid sequence selected from SEQ ID NOs: 76 and 78, as listed in Table 5, or any sequence having at least 80% sequence identity to the same.

34. The engineered TCE of any one of claims 17-31, wherein the anti-CD22 domain is an sdAb, comprising a heavy chain complementarity determining region (HCDR)l, an HCDR2, and an HCDR3 wherein the HCDR1, HCDR2 and the HCDR3 comprise amino acid sequences as listed in Table 6.

35. The engineered TCE of any one of claims 17-31, wherein the anti-CD22 domain is an scFv, comprising a heavy chain complementarity determining region (HCDR)1, an HCDR2, and an HCDR3; and a light chain complementarity determining region (LCDR)l, a LCDR2, and a LCDR3 wherein the HCDR1, the HCDR2 and the HCDR3 and the LCDR1, the LCDR2 and the LCDR3 comprise amino acid sequences as listed in Table 6.WSGR Docket No. 69097-702.60136. The engineered TCE of any one of claims 1-14, comprising a target binding domain that binds to a tumor antigen, wherein the tumor antigen is DLL3.

37. The engineered TCE of claim 36, wherein the target binding domain that binds to the tumor antigen DLL3 is an sdAb, comprising an amino acid sequence:EVQLVESGGGL VQPGGSLTLSC AAS S S S VSLLSLAWYRQAPGKKRELVAGISDD GSIVYMDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYYCYAYSWITRSPYW GQGTLVTVSS, or a sequence having at least 90% sequence identity to the amino acid sequence.

38. The engineered TCE of any one of claims 1-37, wherein the CD2 binding domain comprises a sequence listed in Tables 7 or 8 or a sequence having at least 80% sequence identity to any of the sequences listed in Table 7 or 8.

39. The engineered TCE of any one of claims 1-38, wherein the CD3 binding domain comprises a sequence listed in Tables 9 or 10 or a sequence having at least 80% sequence identity to any of the sequences listed in Table 9 or 10.

40. The engineered TCE of any one of claims 1-39, wherein the Fcl and the Fc2 comprise knobs-into-holes mutations.

41. The engineered TCE of any one of claims 1-40, wherein the Fcl and the Fc2 comprise a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 108-145 as listed in Table 11; and / or comprise a mutation as listed in Table 12.

42. The engineered TCE of any one of claims 1-6, 14-41, wherein the second polypeptide comprising the Fc2 comprises the CD3 binding domain linked to the N terminus of the Fc2, and the CD2 binding domain comprising the CD58 ligand linked to the C terminus of the Fc2.

43. An engineered TCE comprising: (a) a first polypeptide, comprising, (i) an anti-CD19 domain comprising an sdAb or an scFv; linked via a first linker with (ii) a first Fc domain (Fcl), and (b) a second polypeptide, comprising, (i) an anti-CD3 scFv linked via a second linker with (ii) a CD58 ligand, which is linked via a third linker with (iii) a second Fc domain (Fc2); and wherein Fcl and Fc2 dimerize and comprise a modification constituting a knobs-into-holes configuration; wherein the first linker, the second linker and / or the third linker comprise the same or different amino acid sequence.

44. The engineered TCE of claim 43, comprising a first polypeptide having an amino acid sequence that is at least 90% identical to SEQ ID NO: 1070; and a second polypeptide having an amino acid sequence that is at least 90% identical to SEQ ID NO: 186.WSGR Docket No. 69097-702.60145. The engineered TCE of claim 44, comprising a first polypeptide having an amino acid sequence of SEQ ID NO: 1070 and a second polypeptide having an amino acid sequence of SEQ ID NO: 186.

46. An engineered TCE comprising: (a) a first polypeptide, comprising, (i) an anti-CD19 domain comprising an sdAb or an scFv; linked via a first linker with (ii) a first Fc domain (Fcl), and (b) a second polypeptide, comprising, (i) an anti-CD3 scFv linked via a second linker with (ii) a second Fc domain (Fc2), which is linked via a third linker with (iii) a CD58 ligand; and wherein the Fcl and the Fc2 dimerize and comprise a modification constituting a knob-into-hole configuration; and wherein the first linker, the second linker and / or the third linker comprise the same or different amino acid sequence.

47. The engineered TCE of claim 46, comprising the first polypeptide having an amino acid sequence that is at least 90% identical to SEQ ID NO: 1070; and the second polypeptide having an amino acid sequence that is at least 90% identical to SEQ ID NO: 187.

48. The engineered TCE of claim 47, comprising the first polypeptide having an amino acid sequence of SEQ ID NO: 1070 and the second polypeptide having an amino acid sequence of SEQ ID NO: 187.

49. An engineered TCE comprising: (a) a first polypeptide, comprising, (i) an anti-CD19 domain comprising an sdAb or an scFv; linked via a first linker with (ii) a CD58 ligand; which in turn is linked via a second linker with a first Fc domain (Fcl), and (b) a second polypeptide, comprising, (i) an anti-CD3 scFv linked via a third linker with (ii) a second Fc domain (Fc2), wherein the Fcl and the Fc2 dimerize and comprise a modification constituting a knob-into-hole configuration; and wherein the first linker, the second linker and / or the third linker comprise the same or different amino acid sequence.

50. The engineered TCE of claim 49, comprising the first polypeptide having an amino acid sequence that has at least 90% sequence identity to the sequence of SEQ ID NO: 1071 and the second polypeptide having an amino acid sequence that has at least 90% sequence identity to the sequence of SEQ ID NO: 184.

51. The engineered TCE of claim 49, comprising the first polypeptide having an amino acid sequence of SEQ ID NO: 1071 and the second polypeptide having an amino acid sequence of SEQ ID NO: 184.

52. An engineered TCE comprising at least three or at least four target binding domains, the engineered TCE comprising a first polypeptide chain comprising a first Fc region (Fcl) and a second polypeptide chain comprising the second Fc region (Fc2), wherein the Fcl or the Fc2 is operably linked to a target binding domain that binds to a CD2 target molecule, and wherein the Fcl or the Fc2 is operably linked to a target binding domainWSGR Docket No. 69097-702.601 that binds to a CD3 target molecule, wherein the engineered TCE induces low or negligible levels of target-independent T cell activation when a target cell for the engineered TCE is not present in proximity of a T cell.

53. An engineered TCE comprising at least four target binding domains configured to bind to at least three non-identical targets, having any one of the configurations of any one of claims 1-52.

54. An engineered TCE comprising at least four target binding domains configured to bind to at least two non-identical targets, having any one of the configurations of any one of claims 1-52.

55. Use of the engineered TCE of any one of claims 1-54 in preparing a medicament for treating a disease in a human subject in need thereof, wherein the disease is a cancer or an autoimmune disease.

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