Multispecific immune cell engagers

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) 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. 69097-701.601MULTISPECIFIC IMMUNE CELL ENGAGERSCROSS 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] Provided herein, in one aspect, an engineered T Cell Engager (TCE) comprising at least four target binding domains configured to bind to at least four non-identical targets, comprising: a first immunoglobulin constant domain Fc region (Fcl) operably linked to at least two non-identical target binding domains and a second immunoglobulin constant domain Fc region (Fc2) operably linked to one or more target binding domain, wherein the one or more target binding domains are non-identical to the at least two non-identical target binding domains linked to the Fcl, and wherein at least one target binding domain operably linked to the Fc2 is an anti- CD3 domain. In some embodiments, the Fcl is on a first polypeptide chain, and the Fc2 is on a second polypeptide chain, and wherein the Fcl dimerizes with the Fc2. In some embodiments, the Fcl and the Fc2 comprise unmodified immunoglobulin constant regions, or comprise one or more amino acid mutations.WSGR Docket No. 69097-701.601

[0005] In one aspect, provided herein is an engineered T Cell Engager (TCE) comprising at least four target binding domains configured to bind to at least four non-identical targets, comprising: a first immunoglobulin constant domain Fc region, Fcl, operably linked to at least two non-identical target binding domains and a second immunoglobulin constant domain Fc region, Fc2, operably linked to one or more target binding domains, wherein the one or more target binding domains are non-identical to the at least two non-identical target binding domains linked to the Fcl, and wherein one of the one or more target binding domains operably linked to the Fc2 is an anti-CD3 domain.

[0006] In some embodiments, the at least four target binding domains comprise an antibody or a domain or a fragment thereof, a Fab, a F(ab')2, an Fv, an scFv, an sdAb, a VHH cam elid antibody, a nanobody and / or a ligand. In some embodiments, the at least four target binding domains comprises (i) at least three binding domains selected from a binding domain selected from an anti-CD19 domain, an anti-CD20 domain, an anti-CD22 domain, and a CD2 binding domain; and (ii) the anti-CD3 domain. In some embodiments, the at least four target binding domains comprises five target binding domains, where the at least four target binding domains are selected from (i) an anti-CD19 domain, (ii) an anti-CD20 domain, (iii) an anti-CD22 domain, (iv) a CD2 binding domain; and (v) the anti-CD3 domain. In some embodiments, the target binding domain of the at least four target binding domains is a CD2 binding domain, 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.

[0007] In some embodiments, the TCE further comprises a linker, a spacer, or a hinge domain between two or more domains of the least four target binding domains, or between the one or more domains and the Fcl or between the one or more domains and the Fc2. In some embodiments, the Fcl is operably linked to a target binding domain at the N terminus or at the C terminus; and wherein the Fc2 is operably linked to a target binding domain at the N terminus or at the C terminus.

[0008] In some embodiments, the TCE comprises a fifth binding domain. In some embodiments, the fifth binding domain is operably linked to Fcl. In some embodiments, provided herein is a TCE wherein (a) the first polypeptide comprising the Fcl is a dual target engager, wherein the Fcl is operably linked to a first target binding domain at the N terminus via a first linker and the Fcl is operably linked to a second target binding domain at the C terminus via a second linker; or (b) the Fcl is a dual target engager, wherein the Fcl is operably linked to a first target binding domain via a first linker and a second target binding domain in tandem wherein the first target binding domain and the second target binding domain are linked by a second linker. InWSGR Docket No. 69097-701.601 some embodiments, the Fc2 is operably linked to a third target binding domain via a third linker. In some embodiments, the Fc2 further comprises a fourth binding domain, wherein the third target binding domain and the fourth binding domain are operably linked to the Fc2 in tandem and the third target binding domain is linked to the fourth target binding domain via a fourth linker. In some embodiments, provided herein is a TCE, wherein, (a) the Fcl is a triple target engager, wherein the Fcl is operably linked to a first target binding domain, a second target binding domain and a fifth target binding domain, wherein the Fcl is operably linked to the first binding domain and the fifth target binding domain in tandem, wherein a first linker links the first target binding domain to Fcl, a second linker links the second target binding domain to Fcl, a fifth linker links the first target binding domain and the fifth target binding domain; or (b) the Fcl is a triple target engager, wherein the Fcl is operably linked to a first target binding domain, a second target binding domain and a fifth target binding domain in tandem, wherein a first linker connects the first target binding domain to Fcl, a second linker connects the first target binding domain to the second target binding domain and a fifth linker connects the second target binding domain to the fifth target binding domain.

[0009] In some embodiments, the CD2 binding domain is on the first polypeptide chain or on the second polypeptide chain. In some embodiments, the second polypeptide comprises the Fc2 operably linked to a first target binding domain that is the CD58 ligand at the N terminus via a linker. In some embodiments, the second polypeptide comprises the Fc2 operably linked to a first target binding domain that is the CD58 ligand at the C terminus via a linker. In some embodiments, the first polypeptide comprises the Fcl operably linked to a CD58 ligand at the N terminus via a linker. 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 an anti-CD19 domain, an anti-CD20 domain and an anti-CD22 domain. In some embodiments, the Fcl or the Fc2 is further linked to a target binding domain via a linker at its C terminus, selected from an anti-CD19 domain, an anti- CD20 domain and an anti-CD22 domain.

[0010] In some embodiments, a target binding domain of the at least four target binding domains is a CD2 binding domain, 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.

[0011] In some embodiments, the engineered TCE further comprises a linker, a spacer, or a hinge domain between two or more domains of the least four target binding domains, or between the one or more domains and the Fcl or the Fc2.WSGR Docket No. 69097-701.601

[0012] In some embodiments, the Fcl is operably linked to a target binding domain at the N terminus or at the C terminus; and wherein the Fc2 is operably linked to a target binding domain at the N terminus or at the C terminus.

[0013] In some embodiments, the Fcl is a dual target engager, wherein the Fcl is operably linked to a first target binding domain and a second target binding domain in tandem wherein the first target binding domain and the second target binding domain is linked by a linker; or, the Fcl is a triple target engager, wherein the Fcl is operably linked to a first target binding domain, a second target binding domain and a third target binding domain in tandem, wherein a first linker connects the first target binding domain to the second target binding domain and a second linker connects the second binding domain to the third binding domain; or, the Fcl is operably linked to a first target binding domain, a second target binding domain and a third target binding domain in tandem, linked to Fcl- N or a C terminus; and a fourth target binding domain linked to the Fcl-C or the N terminus respectively, wherein a first linker connects the first target binding domain to the second target binding domain and a second linker connects the second binding domain to the third binding domain; wherein the first target binding domain, the second target binding domain, the third target binding domain or the fourth target binding domains are selected from an antiCD 19 domain, an anti-CD20 domain, an anti-CD22 domain and the CD58 ligand.

[0014] In some embodiments, the binding domain is on the first polypeptide chain or on the second polypeptide chain. In some embodiments, the second polypeptide comprises the Fc2 operably linked to a first target binding domain that is the CD58 ligand at the N terminus via a linker. In some embodiments, the second polypeptide comprises the Fc2 operably linked to a first target binding domain that is the CD58 ligand at the C terminus via a linker. In some embodiments, the first polypeptide comprises the Fcl operably linked to a CD58 ligand at the N terminus via a linker. 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 an anti-CD19 domain, an anti-CD20 domain and an anti-CD22 domain. In some embodiments, the Fcl or the Fc2 is further linked to a target binding domain via a linker at its C terminus, selected from an anti-CD19 domain, an anti- CD20 domain and an anti-CD22 domain. In some embodiments, the anti-CD19 domain is a single domain antibody fragment (sdAb) comprising a sequence selected from SEQ ID NOs: 1-21, or any sequence having at least 80% sequence identity to SEQ ID NOs. 1-21. 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 is selected from SEQ ID NOs: 23, 26, 28, 30, 32, and 34, or any sequence having at least 80% sequence identity to any of the sequences; and a VL domain selected from SEQ ID NOs: 24, 27, 29, 31, 33 and 35 or any sequence having at least 80% sequence identity to any of the sequences.WSGR Docket No. 69097-701.601In some embodiments, the anti-CD19 domain comprises an scFV selected from SEQ ID NOs: 22 and 25, or any sequence having at least 80% sequence identity to any of the sequences. In some embodiments, the anti-CD19 domain comprises a sequence depicted in Table 1, or any sequence having at least 80% sequence identity to any of the sequences. In some embodiments, the antiCD 19 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 2. In some embodiments, the anti-CD19 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)1, a LCDR2, and a LCDR3 wherein the HCDR1, HCDR2 and the HCDR3 and the LCDR1, LCDR2 and the LCDR3 comprise amino acid sequences as listed in Table 2.

[0015] The engineered TCE of any one of claims 1-21, wherein the anti-CD20 domain is a single domain antibody fragment (sdAb) comprising a sequence selected from SEQ ID NOs: 36- 46, or any sequence having at least 80% sequence identity to any of the sequences. In some embodiments, the anti-CD20 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 is selected from SEQ ID NOs: 47, 49, 51 and 53, or any sequence having at least 80% sequence identity to any of the sequences; and a VL domain selected from SEQ ID NOs: 48, 50, 52 and 54, or any sequence having at least 80% sequence identity to any of the sequences, as listed in Table 3. 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. 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)1, a LCDR2, and a LCDR3 wherein the HCDR1, HCDR2 and the HCDR3 and the LCDR1, LCDR2 and the LCDR3 comprise amino acid sequences as listed in Table 4. In some embodiments, the anti-CD22 domain is a single domain antibody fragment (sdAb) comprising a sequence selected from SEQ ID NOs: 55-74, or any sequence having at least 80% sequence identity to any of the sequences. In some embodiments, the anti-CD22 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 is selected from SEQ ID NOs: 75 and 77, or any sequence having at least 80% sequence identity to any of the sequences; and a VL domain selected from SEQ ID NOs: 76 and 78, or any sequence having at least 80% sequence identity to any of the sequences, as listed in Table 5. In some embodiments, the anti-CD22 domain is an sdAb, comprising a heavy chain complementarity determining region (HCDR)1, an HCDR2,WSGR Docket No. 69097-701.601 and an HCDR3 wherein the HCDR1, HCDR2 and the HCDR3 comprise amino acid sequences as listed in Table 6. 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)1, a LCDR2, and a LCDR3 wherein the HCDR1, HCDR2 and the HCDR3 and the LCDR1, LCDR2 and the LCDR3 comprise amino acid sequences as listed in Table 6. In some embodiments, the CD2 binding domain comprises a sequence listed in Tables 7 or 8. In some embodiments, the CD2 binding domain comprises a sequence listed in Tables 9 or 10. In some embodiments, the Fcl and the Fc2 comprise a knob- and-hole mutation. 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, or any sequence having at least 80% sequence identity to any of the sequences as listed in Table 11; and / or comprises a mutation as listed in Table 12. In some embodiments, the linker comprises a sequence selected from the sequences in Table 13, or any sequence having at least 80% sequence identity to any of the sequences.

[0016] Provided herein is a composition comprising any one of the engineered TCEs, and an acceptable solvent.

[0017] In one aspect, provided herein is a pharmaceutical composition comprising any one of the engineered TCEs and a therapeutically acceptable excipient. In some embodiments, the composition or the pharmaceutical composition further comprises a pH-stabilizing agent, an organic co-solvent, selected from polysorbate 20, polysorbate 80, propylene glycol and polyethylene glycol (PEG). In some embodiments, the composition or the pharmaceutical composition further comprises a stabilizing agent selected from sucrose, sorbitol, glycerol, trehalose, or mannitol. In some embodiments, the composition or the pharmaceutical composition further comprises a tonicity agent, wherein the tonicity agent is a salt that is NaCl or KC1. In some embodiments, the composition or the pharmaceutical composition further comprises a preservative, wherein the preservative is an antimicrobial agent.

[0018] In one aspect, provided herein is a composition comprising a TCE of any one of embodiments discussed above in the manufacture of a medicament for treating a subject with cancer or an autoimmune disease.

[0019] In one aspect, provided herein is a composition, comprising, a TCE, comprising, (i) a first polypeptide chain and (ii) a second polypeptide chain, wherein the first polypeptide chain comprises a sequence selected from SEQ ID NO: 1070-1095, or a sequence having at least 85% amino acid sequence identity to a sequence selected from SEQ ID NO: 1070-1095 SEQ ID NO: 1070; and the second polypeptide chain comprising a sequence selected from SEQ ID NOs: 184WSGR Docket No. 69097-701.601 and 186, or a sequence having at least 85% amino acid sequence identity to any of the sequences set forth in SEQ ID NO: 184 or 186; and a suitable solvent or an excipient.

[0020] In one aspect, provided herein is a method of making the engineered TCE comprising: (i) identifying the one or more target binding domains; (ii) cloning a polynucleic acid sequence encoding a recombinant polypeptide comprising the first immunoglobulin constant domain Fc region and the one or more target binding domains; (iii) cloning a polynucleic acid sequence encoding a recombinant polypeptide comprising the second immunoglobulin constant domain Fc region and the one or more target binding domains; (iv) expressing the cloned polynucleic acid sequences of (ii) or (iii) in an immune cell.

[0021] In some embodiments, the method further comprises confirming that the engineered TCE is present in a solution having physiological pH in absence of substantial aggregation. An aggregation is a phenomena observed in polypeptides where the polypeptides are not present as monomers in a solution, and aggregate in dimer, multimer and even large structures which severely affect purification, dispersion, solubility and results in being undesirable for downstream use, e.g., preparing and administering a pharmaceutical composition. In some embodiments, a substantial aggregation indicates that greater than 20% protein molecules in the solution are aggregated. In some embodiments, it is desired that the aggregation is / involves less than 10%, less than 5% or less than 2% of the protein molecules. In some embodiments, the method further comprises confirming that greater than 98% of the TCE is present in a solution having physiological pH, e.g., in absence of substantial aggregation.

[0022] In one aspect, provided herein is a method of treating a disease or a condition in a subject in need thereof, comprising administering to the subject a composition comprising any one of the TCEs of embodiments discussed above. In some embodiments, the administering is via intravenous, intramuscular, or subcutaneous injection. In some embodiments, the disease or condition is a cancer. In some embodiments, the disease or condition is a B cell cancer. In some embodiments, the disease or condition is an autoimmune disease.

[0023] Disclosed herein, in some aspects, is a T cell engager comprising: a first 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 binding domain that binds to a fourth target molecule, wherein the fourth target molecule is CD3. 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 target binding domain, second target binding domain, third target binding domain and fourth binding domain in one description of a TCE design may vary from a first, second, third, fourth and / or fifth binding domains in another TCE description.WSGR Docket No. 69097-701.601For example, a fifth target binding domain in one design aspect may be the same as a third or fourth or simply other than fifth target binding domain described elsewhere for another design unless in direct comparison with each other.

[0024] In some embodiments, the T cell engager further comprises a second 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 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 binding domain that binds to a fifth target molecule, wherein the fifth target molecule is CD2.

[0025] Disclosed herein, in some aspects, is a T cell engager comprising: a first binding domain that binds to a first target molecule, wherein the first target molecule is CD 19; a second binding domain that binds to a second target molecule, wherein the second target molecule is CD20; a third binding domain that binds to a third target molecule, wherein the third target molecule is CD22; and a fourth binding domain that binds to a fourth target molecule, wherein the fourth target molecule is CD3.

[0026] In some embodiments, the T cell engager further comprises a fifth binding domain. In some embodiments, the fifth 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 binding domain binds to a fifth target molecule, wherein the fifth target molecule is CD2. In some embodiments, the first binding domain is linked to the second binding domain. In some embodiments, the first binding domain is linked to the third binding domain. In some embodiments, the first binding domain is linked to the fifth binding domain. In some embodiments, the second binding domain is linked to the third binding domain. In some embodiments, the second binding domain is linked to the fourth binding domain. In some embodiments, the second binding domain is linked to the fifth binding domain. In some embodiments, the third binding domain is linked to the fifth binding domain. In some embodiments, the fourth binding domain is linked to the fifth binding domain. In some embodiments, the first binding domain is linked to the second binding domain, and the second binding domain is linked to the third 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 polypeptideWSGR Docket No. 69097-701.601 chain are non-contiguous. In some embodiments, the first polypeptide chain comprises the first binding domain, the second binding domain, the third binding domain, or any combination thereof. In some embodiments, the first polypeptide chain comprises the first binding domain, and the first binding domain is linked to the first portion of the dimerization module. In some embodiments, the first polypeptide chain comprises the second binding domain, and the second binding domain is linked to the first portion of the dimerization module. In some embodiments, the first polypeptide chain comprises the third binding domain, and the third binding domain is linked to the first portion of the dimerization module. In some embodiments, the first polypeptide chain comprises the fifth binding domain, and the fifth binding domain is linked to the first portion of the dimerization module. In some embodiments, the second polypeptide chain comprises the second binding domain, and the second binding domain is linked to the second portion of the dimerization module. In some embodiments, the second polypeptide chain comprises the fourth binding domain, and the fourth binding domain is linked to the second portion of the dimerization module. In some embodiments, the second polypeptide chain comprises the fifth binding domain, and the fifth 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 binding domain linked to the second binding domain linked to the first binding domain linked to the first Fc region; and a second polypeptide comprising from N terminus to C terminus the fourth 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 binding domain linked to the second binding domain linked to the first binding domain linked to the first Fc region; and a second polypeptide comprising from N terminus to C terminus the fourth binding domain linked to the fifth 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 binding domain linked to the second binding domain linked to the first binding domain linked to the first Fc region; and a second polypeptide comprising from N terminus to C terminus the fourth binding domain linked to the second Fc region linked to the fifth binding domain. In some embodiments, the T cell engager comprises: a first polypeptide comprising from N terminus to C terminus the third binding domain linked to the second binding domain linked to the first binding domain linked to the fifth binding domain linked to the first Fc region; and a second polypeptide comprising from N terminus to C terminus the fourth binding domain linked to the second Fc region. In some embodiments, the T cellWSGR Docket No. 69097-701.601 engager comprises: a first polypeptide comprising from N terminus to C terminus the third binding domain linked to the second binding domain linked to the fifth binding domain linked to the first binding domain linked to the first Fc region; and a second polypeptide comprising from N terminus to C terminus the fourth 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 binding domain linked to the fifth binding domain linked to second binding domain linked to the first binding domain linked to the first Fc region; and a second polypeptide comprising from N terminus to C terminus the fourth 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 binding domain linked to the third binding domain linked to second binding domain linked to the first binding domain linked to the first Fc region; and a second polypeptide comprising from N terminus to C terminus the fourth 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 binding domain linked to the second binding domain linked to the first binding domain linked to the first Fc region linked to the fifth binding domain; and a second polypeptide comprising from N terminus to C terminus the fourth 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 binding domain linked to the third binding domain linked to the fifth binding domain linked to the first Fc region; and a second polypeptide comprising from N terminus to C terminus the fourth binding domain linked to the second 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 binding domain linked to the third binding domain linked to the first Fc region linked to the second binding domain linked to the fifth binding domain; and a second polypeptide comprising from N terminus to C terminus the fourth binding domain linked to the second Fc region. In some embodiments, the first binding domain is an antibody domain or an antigen binding domain. In some embodiments, the first 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. In some embodiments, the first 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 binding domain comprises a VHWSGR Docket No. 69097-701.601 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 binding domain comprises a VH, wherein the VH comprises a set of heavy chain CDRs from TABLE 2. In some embodiments, the first 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 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 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 binding domain comprises a VL, wherein the VL comprises a set of light chain CDRs from TABLE 2. In some embodiments, the first 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 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 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 binding domain comprises a heavy chain variable region (VH) comprising a heavy chain complementaritydetermining 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 HCDR2 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 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 binding domain comprises a VH, wherein the VH comprises a set of heavy chain CDRs from TABLE 4. In some embodiments, the second binding domain comprises a light chain variable region (VL) comprising a light chain complementarity-determining regionWSGR Docket No. 69097-701.6011 (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 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 binding domain comprises a VL, wherein the VL comprises a set of light chain CDRs from TABLE 4. In some embodiments, the second 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 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 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 binding domain comprises a heavy chain variable region (VH) comprising a heavy chain complementaritydetermining 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 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 of the HCDR3 sequences in TABLE 6. In some embodiments, the third binding domain comprises a VH, wherein the VH comprises a set of heavy chain CDRs from TABLE 6. In some embodiments, the third 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 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 theWSGR Docket No. 69097-701.601LCDR2 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 binding domain comprises a VL, wherein the VL comprises a set of light chain CDRs from TABLE 6. In some embodiments, the third 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 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 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 binding domain comprises a heavy chain variable region (VH) comprising a heavy chain complementaritydetermining 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 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 binding domain comprises a VH, wherein the VH comprises a set of heavy chain CDRs from TABLE 10. In some embodiments, the fourth 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 10, an LCDR2 sequence having at least 90% sequence identity to 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 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 binding domain comprises a VL, wherein the VL comprises a set of light chain CDRs from TABLE 10. In some embodiments, the fourth 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 binding domain comprises: a VH comprising an amino acid sequenceWSGR Docket No. 69097-701.601 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 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 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 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 binding domain comprises a VH, wherein the VH comprises a set of heavy chain CDRs from TABLE 8. In some embodiments, the fifth 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 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 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. In some embodiments, the fifth binding domain comprises a VL, wherein the VL comprises a set of light chain CDRs from TABLE 8. In some embodiments, the fifth 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 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 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 binding domain comprises an amino acid sequence with at least 90% sequence identity to any one of SEQWSGR Docket No. 69097-701.601ID NOs: 79-86. In some embodiments, the fifth 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 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 T cell engager comprises a polypeptide chain with an Fc region that comprises an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 108-177. In some embodiments, the T cell engager comprises a polypeptide chain with an Fc region that comprises an amino acid sequence with at least 95% sequence identity to any one of SEQ ID NOs: 108-177. In some embodiments, the T cell engager comprises a polypeptide chain with an Fc region that comprises the amino acid sequence of any one of SEQ ID NOs: 108-177. 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.

[0027] 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.

[0028] 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.

[0029] 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 binding domain comprises a full length CD58, an extracellular domain of CD58, a CD2-binding domain of CD58, an Ig-like domain of CD58, anWSGR Docket No. 69097-701.601IgV 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.INCORPORATION BY REFERENCE

[0030] 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

[0031] 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:

[0032] 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).

[0033] FIGs. 2A-2E show schematics of single-, dual-, and triple-targeting TCEs without CD2 costimulation (format 0). FIG. 2A illustrates a TCE with a single tumor-associated antigen (TAA) binding domain (aTAA) at position INI, a single CD3 binding domain (aCD3) at position 2N1, and no CD2 costimulation (format 0 single). A binding domain is often referred to as anWSGR Docket No. 69097-701.601 antiftarget] polypeptide / protein / peptide / domain or simply as a(Target). For example, and anti- CD3 domain or aCD3 domain is a domain that binds to CD3. FIG. 2B illustrates a TCE with 2 aTAAs at positions INI and 1C1, a single aCD3 at position 2N1, and no CD2 costimulation (format OA dual). FIG. 2C illustrates a TCE with 2 aTAAs at positions INI and 1N2, a single aCD3 at position 2N1, and no CD2 costimulation (format OB dual). FIG. 2D illustrates a TCE with 3 aTAAs at positions INI, 1N2, and 1C1, a single aCD3 at position 2N1, and no CD2 costimulation (format OA triple). FIG. 2E illustrates a TCE with 3 aTAAs at positions INI, 1N2, and 1N3, a single aCD3 at position 2N1, and no CD2 costimulation (format OB triple). The aTAA and aCD3 binding domains could be composed of various antibody domains or fragments, including sdAbs, nanobodies, VHHs, HCAbs, Fvs, scFvs, Fabs, scFabs, etc. In dual-targeting constructs the two aTAA binding domains could comprise 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. In triple-targeting constructs the three aTAA binding domains 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).

[0034] FIGs. 3A-3E show schematics of single-, dual-, and triple-targeting TCEs with CD2 costimulation (format 1). FIG. 3A illustrates a TCE with a single TAA binding domain (aTAA) at position INI, a single CD3 binding domain (aCD3) at position 2N2, and CD2 costimulation at position 2N1 (format 1 single). FIG. 3B illustrates a TCE with 2 aTAAs at positions INI and 1C1, a single aCD3 at position 2N2, and CD2 costimulation at position 2N1 (format 1A dual). FIG. 3C illustrates a TCE with 2 aTAAs at positions INI and 1N2, a single aCD3 at position 2N2, and CD2 costimulation at position 2N1 (format IB dual). FIG. 3D illustrates a TCE with 3 aTAAs at positions INI, 1N2, and 1C1, a single aCD3 at position 2N2, and CD2 costimulation at position 2N1 (format 1 A triple). FIG. 3E illustrates a TCE with 3 aTAAs at positions INI, 1N2, and 1N3, a single aCD3 at position 2N2, and CD2 costimulation at position 2N 1 (format IB triple). 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. In dual-targeting constructs the two aTAA binding domains could compriseWSGR Docket No. 69097-701.601 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. In triple-targeting constructs the three aTAA binding domains 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).

[0035] FIGs. 4A-4E show schematics of single-, dual-, and triple-targeting TCEs with CD2 costimulation (format 2). FIG. 4A illustrates a TCE with a single TAA binding domain (aTAA) at position INI, a single CD3 binding domain (aCD3) at position 2N1, and CD2 costimulation at position 2C1 (format 2 single). FIG. 4B illustrates a TCE with 2 aTAAs at positions INI and 1C1, a single aCD3 at position 2N1, and CD2 costimulation at position 2C1 (format 2A dual). FIG. 4C illustrates a TCE with 2 aTAAs at positions INI and 1N2, a single aCD3 at position 2N1, and CD2 costimulation at position 2C1 (format 2B dual). FIG. 4D illustrates a TCE with 3 aTAAs at positions INI, 1N2, and 1C1, a single aCD3 at position 2N1, and CD2 costimulation at position 2C1 (format 2 A triple). FIG. 4E illustrates a TCE with 3 aTAAs at positions INI, 1N2, and 1N3, a single aCD3 at position 2N1, and CD2 costimulation at position 2C1 (format 2B triple). 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. In dualtargeting constructs the two aTAA binding domains could comprise 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. In tripletargeting constructs the three aTAA binding domains 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).

[0036] FIGs. 5A-5E show schematics of single-, dual-, and triple-targeting TCEs with CD2 costimulation (format 3). FIG. 5A illustrates a TCE with a single TAA binding domain (aTAA)WSGR Docket No. 69097-701.601 at position 1N2, a single CD3 binding domain (aCD3) at position 2N1, and CD2 costimulation at position INI (format 3 single). FIG. 5B illustrates a TCE with 2 aTAAs at positions 1N2 and 1C1, a single aCD3 at position 2N1, and CD2 costimulation at position INI (format 3 A dual). FIG. 5C illustrates a TCE with 2 aTAAs at positions 1N2 and 1N3, a single aCD3 at position 2N1, and CD2 costimulation at position INI (format 3B dual). FIG. 5D illustrates a TCE with 3 aTAAs at positions INI, 1N2, and 1C1, a single aCD3 at position 2N1, and CD2 costimulation at position INI (format 3 A triple). FIG. 5E illustrates a TCE with 3 aTAAs at positions 1N2, 1N3, and 1N4, a single aCD3 at position 2N1, and CD2 costimulation at position INI (format 3B triple). 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. In dualtargeting constructs the two aTAA binding domains could comprise 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. In tripletargeting constructs the three aTAA binding domains 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).

[0037] FIGs. 6A-6E show schematics of single-, dual-, and triple-targeting TCEs with CD2 costimulation (format 4). FIG. 6A illustrates a TCE with a single TAA binding domain (aTAA) at position INI, a single CD3 binding domain (aCD3) at position 2N1, and CD2 costimulation at position 1N2 (format 4 single). FIG. 6B illustrates a TCE with 2 aTAAs at positions INI and 1C1, a single aCD3 at position 2N1, and CD2 costimulation at position 1N2 (format 4A dual). FIG. 6C illustrates a TCE with 2 aTAAs at positions INI and 1N3, a single aCD3 at position 2N1, and CD2 costimulation at position 1N2 (format 4B dual). FIG. 6D illustrates a TCE with 3 aTAAs at positions INI, 1N3, and 1C1, a single aCD3 at position 2N1, and CD2 costimulation at position 1N2 (format 4A triple). FIG. 6E illustrates a TCE with 3 aTAAs at positions INI, 1N3, and 1N4, a single aCD3 at position 2N1, and CD2 costimulation at position 1N2 (format 4B triple). 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 extracellularWSGR Docket No. 69097-701.601 domain, the CD58 IgV domain, an antibody domain or fragment that binds to CD2, etc. In dualtargeting constructs the two aTAA binding domains could comprise 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. In tripletargeting constructs the three aTAA binding domains 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).

[0038] FIG. 7 illustrates extracellular staining by flow cytometry of single and triple TAA- expressing cell lines of Example 1. Engineered CHO cell lines expressing human CD 19 (CHO huCD19), human CD20 (CHO huCD20), or human CD22 (CHO huCD22) were stained with an aCD19, aCD20, aCD22, or isotype control antibodies to assess cell surface expression of the three human TAAs. Nalm6 CD 19 knockout (KO) and Daudi human B cell lines were similarly stained with an aCD19, aCD20, aCD22, or isotype control antibodies to assess TAA expression.

[0039] FIGs. 8A-8D show schematics of the single-targeting T cell engagers (TCEs) without CD2 costimulation (format 0) of Examples 2 & 3. FIG. 8A illustrates a TCE targeting CD 19 composed of an aCD19 sdAb, an aCD3 scFv, and no CD2 costimulation. FIG. 8B illustrates a TCE targeting CD20 composed of an aCD20 sdAb, an aCD3 scFv, and no CD2 costimulation. FIG. 8C illustrates a TCE targeting CD22 composed of an aCD22 sdAb, an aCD3 scFv, and no CD2 costimulation. FIG. 8D illustrates a TCE targeting green fluorescent protein (GFP) composed of an aGFP sdAb, an aCD3 scFv, and no CD2 costimulation.

[0040] FIG. 9 shows binding of single-targeting TCEs (aCD19, aCD20, and aCD22 plus a non-targeting aGFP control) to single TAA-expressing cell lines (CHO huCD19, CHO huCD20, CHO huCD22, Nalm6 CD 19 KO) as assessed by flow cytometry. The single-targeting TCEs were similarly tested for binding to a Daudi human B cell line that expresses all three TAAs (huCD19, huCD20, and huCD22).

[0041] FIG. 10 outlines a TDCC screen with single-targeting TCEs in format 0 of Example 2. Single-targeting TCEs to aCD19, aCD20, and aCD22, plus a non-targeting aGFP control in format 0 without CD2 costimulation were screened for their ability to kill single and triple TAA- expressing cell lines in a T cell-dependent cellular cytotoxicity (TDCC) assay.

[0042] FIGs. 11A-11F show schematics of the triple-targeting TCEs without CD2 costimulation (format 0A) of Example 3. FIGS. 11A-11F illustrate multispecific TCEs targetingWSGR Docket No. 69097-701.601CD19, CD20, and CD22. The multispecific TCEs are composed of an aCD19 sdAb, an aCD20 sdAb, and an aCD22 sdAb in all possible configurations of format OA, an aCD3 scFv, and no CD2 costimulation.

[0043] FIGs. 12A-12F show schematics of the triple-targeting TCEs with CD2 costimulation (format 1A) of Examples 3. FIGS. 12A-12F illustrate multispecific TCEs targeting CD19, CD20, and CD22 with CD2 costimulation. The multispecific TCEs are composed of an aCD19 sdAb, an aCD20 sdAb, and an aCD22 sdAb in all possible configurations of format 1 A, an aCD3 scFv, and CD2 costimulation (CD58 IgV domain).

[0044] FIGs. 13A-13F show schematics of the triple-targeting TCEs without CD2 costimulation (format OB) of Example 3. FIGS. 13A-13F illustrate multispecific TCEs targeting CD19, CD20, and CD22. The multispecific TCEs are composed of an aCD19 sdAb, an aCD20 sdAb, and an aCD22 sdAb in all possible configurations in format OB, an aCD3 scFv, and no CD2 costimulation.

[0045] FIGs. 14A-14F show schematics of the triple-targeting TCEs with CD2 costimulation (format IB) of Example 3. FIGS. 14A-14F illustrate multispecific TCEs targeting CD 19, CD20, and CD22 with CD2 costimulation. The multispecific TCEs are composed of an aCD19 sdAb, an aCD20 sdAb, and an aCD22 sdAb in all possible configurations of format IB, an aCD3 scFv, and CD2 costimulation (CD58 IgV domain).

[0046] FIGs. 15A-15B outline a TDCC screen with triple-targeting TCEs in format 0A of Example 3. Triple-targeting TCEs to CD 19, CD20, and CD22 in format 0A without CD2 costimulation were screened for their ability to kill single TAA-expressing cell lines in a TDCC assay.

[0047] FIGs. 16A-16B outline a TDCC Screen with triple-targeting TCEs in format 1A of Example 3. Triple-targeting TCEs to CD19, CD20, and CD22 in format 1A with CD2 costimulation were screened for their ability to kill single TAA-expressing cell lines in a TDCC assay.

[0048] FIGs. 17A-17B outline a TDCC screen with triple-targeting TCEs in format 0B of Example 3. Triple-targeting TCEs to CD 19, CD20, and CD22 in format 0B without CD2 costimulation were screened for their ability to kill single TAA-expressing cell lines in a TDCC assay.

[0049] FIGs. 18A-18B outline a TDCC Screen with triple-targeting TCEs in format IB of Example 3. Triple-targeting TCEs to CD 19, CD20, and CD22 in format IB with CD2 costimulation were screened for their ability to kill single TAA-expressing cell lines in a TDCC assay.WSGR Docket No. 69097-701.601

[0050] FIGs. 19A-19B illustrate Daudi TDCC with triple-targeting TCEs in format 0A of Example 3. Triple-targeting TCEs to CD 19, CD20, and CD22 in format 0A without CD2 costimulation were screened for their ability to kill triple TAA-expressing cell lines in a TDCC assay.

[0051] FIGs. 20A-20C show schematics of single targeting format 0 TCEs of Example 4. FIG. 20A illustrates a TCE targeting CD 19 composed of an anti-CD19 sAb (Clone 2), an anti- CD3 scFv and no CD2 co-stimulation. FIG. 20B illustrate a TCE targeting CD22 composed of an anti-CD22 sdAb (clone 2), an anti-CD3 scFv, and no CD2 costimulation. FIG. 20C is an exemplary schematic showing a Format 1 single TAA binder, typical design shown in FIG. 3A, capable of binding CD19. The exemplary design is referred to by a Complex ID PROT328, which was manufactured and tested, among several others described herein.

[0052] FIG. 21 shows binding of additional single-targeting TCEs to single and triple TAA- expressing cell lines of Example 4. Binding of additional single-targeting TCEs (anti-CD19 clone 2 and anti-CD22 clone 2, plus a non-targeting anti-GFP control) to single TAA-expressing cell lines (CHO huCD19, CHO huCD22, Nalm6 CD 19 KO) as assessed by flow cytometry. The single-targeting TCEs were similarly tested for binding to a Daudi human B cell line that expresses all three TAAs (huCD19, huCD20, and huCD22).

[0053] FIGs. 22A-22F show schematics of the set of triple-targeting format 1A TCEs of Examples 4 and 6. FIGs. 22A-22F illustrate multispecific TCEs targeting CD19, CD20, and CD22 with CD2 costimulation. The multispecific TCEs are composed of an aCD19 sdAb (CD 19 clone 2), an aCD20 sdAb, and an aCD22 sdAb (CD22 clone 1) in various configurations of format 1 A, an aCD3 scFv, and CD2 costimulation (CD58 IgV domain).

[0054] FIGs. 23A-23F show schematics of a set of triple targeting format IB TCEs (Examples 4 and 6). The figures illustrate multispecific TCEs targeting CD 19, CD20, and CD22 with CD2 costimulation. The multispecific TCEs are composed of an anti-CD19 sdAb (CD 19 clone 2), an anti-CD20 sdAb, and an anti-CD22 sdAb (CD22 clone 1) in various configurations of format IB, an anti-CD3 scFv, and CD2 costimulation (CD58 IgV domain).

[0055] FIGs. 24A-24F show schematics of the set 2 triple-targeting format 1A TCEs of Examples 4 & 6. These figures illustrate multispecific TCEs targeting CD 19, CD20, and CD22 with CD2 costimulation. The multispecific TCEs are composed of an aCD19 sdAb (CD 19 clone 2), an aCD20 sdAb, and an aCD22 sdAb (CD22 clone 2) in various configurations of format 1 A, an aCD3 scFv, and CD2 costimulation (CD58 IgV domain).

[0056] FIGs. 25A-25F show schematics of the set 2 triple-targeting format IB TCEs of Examples 4 & 6. illustrate multispecific TCEs targeting CD 19, CD20, and CD22 with CD2 costimulation. The multispecific TCEs are composed of an aCD19 sdAb (CD 19 clone 2), anWSGR Docket No. 69097-701.601 aCD20 sdAb, and an aCD22 sdAb (CD22 clone 2) in various configurations of format IB, an aCD3 scFv, and CD2 costimulation (CD58 IgV domain).

[0057] FIG. 26 shows data from extracellular staining by flow cytometry of Daudi-Luc2 WT and Daudi-Luc2 CD20 CD22 double knockout cell lines of Examples 5 & 6. The Daudi-Luc2 parental (WT) and the engineered Duadi-Luc2 CD20 CD22 double knockout (KO) cell lines were stained with anti-CD19, anti-CD20, anti-CD22, anti-CD58, or isotype control antibodies to assess cell surface expression of the three TAAs (CD 19, CD20, and CD22) and the T cell costimulatory ligand (CD58).

[0058] FIG. 27 shows data from extracellular staining by flow cytometry of Raji-Luc2 WT and Raji-Luc2 CD19 CD22 double KO cell lines of Examples 5 & 6. The Raji-Luc2 parental (WT) and the engineered Raji-Luc2 CD 19 CD22 double KO cell lines were stained with anti-CD19, anti-CD20, anti-CD22, anti-CD58, or isotype control antibodies to assess cell surface expression of the three TAAs (CD19, CD20, and CD22) and the T cell costimulatory ligand (CD58).

[0059] FIGs. 28A-28F show TDCC screen data with set 1 triple-targeting TCEs in format 1 A of Example 6. Set 1 triple-targeting CD19, CD20, and CD22 TCEs in format 1A with CD2 costimulation were screened for their ability to kill single TAA-expressing double KO (D-KO) and triple TAA-expressing cell lines in a TDCC assay. Set 1 triple-targeting TCEs in format 1 A (depicted in schematic in FIG. 28C) (PROT337-PROT342) contain anti-CD19 clone 2, anti- CD20, and anti-CD22 clone 1. For reference the single-targeting TCEs with the same clones (PROT281, PROT001, and PROT073, respectively) were also tested along with a non-targeting control TCE (PROT076). FIGs 28A, 28B, 28D and 28E show results for the cell lines as indicated in the figure. FIG. 28F summarizes the results.

[0060] FIGs. 29A-29F show TDCC screen data with set 1 triple-targeting TCEs in format IB of Example 6. Set 1 triple-targeting CD 19, CD20, and CD22 TCEs in format IB with CD2 costimulation were screened for their ability to kill single TAA-expressing D-KO and triple TAA- expressing cell lines in a TDCC assay. Set 1 triple-targeting TCEs in format IB (depicted in FIG. 29C) (PROT343-PROT348) contain anti-CD19 (aCD19 clone 2), aCD20, and aCD22 clone 1. For reference the single-targeting TCEs with the same clones (PROT281, PROT001, and PROT073, respectively) were also tested along with a non-targeting control TCE (PROT076). FIGs. 29A, 29B, 29D-29E show results for the cell lines as indicated in the figure. FIG. 29F summarizes the results.

[0061] FIGs. 30A-30F show TDCC screen data with set 2 triple-targeting TCEs in format 1 A of Example 6. Set 2 triple-targeting CD19, CD20, and CD22 TCEs in format 1A with CD2 costimulation were screened for their ability to kill single TAA-expressing D-KO and triple TAA- expressing cell lines in a TDCC assay. Set 2 triple-targeting TCEs in format 1 A (depicted in FIG.WSGR Docket No. 69097-701.60130C) (PROT361-PROT366) contain aCD19 clone 2, aCD20, and aCD22 clone 2. For reference the single-targeting TCEs with the same clones (PROT281, PROTOOl, and PROT282, respectively) were also tested along with a non-targeting control TCE (PROT076). FIGs. 30A, 3 OB, 30D and 30E show results for the cell lines as indicated in the figure. FIG. 3 OF summarizes the results.

[0062] FIGs. 31A-31F show TDCC screen with set 2 triple-targeting TCEs in format IB of Example 6. Set 2 triple-targeting CD 19, CD20, and CD22 TCEs in format IB with CD2 costimulation were screened for their ability to kill single TAA-expressing D-KO and triple TAA- expressing cell lines in a TDCC assay. Set 2 triple-targeting TCEs in format IB (FIG. 31C) (PROT367-PROT372) contain aCD19 clone 2, aCD20, and aCD22 clone 2. For reference the single-targeting TCEs with the same clones (PROT281, PROTOOl, and PROT282, respectively) were also tested along with a non-targeting control TCE (PROT076). FIGs. 31 A, 3 IB, 3 ID and 3 IE show results for the cell lines as indicated in the figure. FIG. 3 IF summarizes the results.DETAILED DESCRIPTION

[0063] 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.

[0064] 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

[0065] Disclosed herein are multispecific immune cell engagers, such as multispecific T cell engagers. 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 closeWSGR Docket No. 69097-701.601 proximity and facilitates activation of the immune effector cell to induce, for example, target cell killing, activation of the immune effector cell, pro-inflammatory cytokine production, and / or an anti-cancer immune response.

[0066] 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.

[0067] 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 co-stimulation 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.

[0068] 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, the multispecific immune cell engagers presentedWSGR Docket No. 69097-701.601 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 nonaggregated 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

[0069] 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.

[0070] 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. 69097-701.601

[0071] 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, at least one of the binding domains is non-identical compared to other binding domains.

[0072] 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 four binding domains (e.g., a first, second, third, and fourth binding domain), each of which bind to different targets, such as two binding domains that bind target molecules on target cells and two 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 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.

[0073] 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.

[0074] 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, theWSGR Docket No. 69097-701.601 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.

[0075] 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 binding domain that binds CD3 and a second binding domain that binds CD2. In some embodiments, the target molecules can comprise different epitopes on the same target molecule.

[0076] 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.

[0077] 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 fourWSGR Docket No. 69097-701.601 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 steric 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 flexibilityWSGR Docket No. 69097-701.601 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

[0078] 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.

[0079] 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.

[0080] 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, dimeric (diabody), trimeric (triabody), or tetrameric (tetrabody). Minibodies are scFv-CH3 fusion proteins that assemble into bivalent dimers.

[0081] 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 differWSGR Docket No. 69097-701.601 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.

[0082] 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.

[0083] 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.

[0084] 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 substantially 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 areWSGR Docket No. 69097-701.601 grafted onto a suitable human framework (e.g., sdAb CDRs can be grafted onto a human VH framework).

[0085] 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.

[0086] 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.

[0087] 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).

[0088] 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.

[0089] 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, 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 4WSGR Docket No. 69097-701.601 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.

[0090] 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.

[0091] 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.

[0092] 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 or 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.

[0093] 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 / orWSGR Docket No. 69097-701.601 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.

[0094] 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.

[0095] 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.

[0096] 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 acid substitutions, insertions, and / or deletions relative to any one of the CDR sequences disclosed herein.

[0097] 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 oneWSGR Docket No. 69097-701.601 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.

[0098] 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.

[0099] 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.

[0100] 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 or 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 orWSGR Docket No. 69097-701.601 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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

[0105] 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.

[0106] 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-binding 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.

[0107] 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, forWSGR Docket No. 69097-701.601 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.

[0108] 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.

[0109] 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.

[0110] 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. [OHl] 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.

[0112] 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.

[0113] 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 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 and N81 of the mature CD58 polypeptide chain. In some embodiments a binding domain comprises a CD2-binding domainWSGR Docket No. 69097-701.601(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).

[0114] 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.

[0115] 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.

[0116] 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 least 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.WSGR Docket No. 69097-701.601

[0117] 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

[0118] Non-limiting examples of sequences of binding domains and components thereof are provided in TABLES 1-10.

[0119] 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.

[0120] 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.

[0121] 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 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.WSGR Docket No. 69097-701.601

[0122] TABLE 1 : Illustrative CD 19 binding domain sequencesWSGR Docket No. 69097-701.601

[0123] TABLE 2: illustrative anti-CD19 CDRsWSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601

[0124] 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.

[0125] 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.

[0126] 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.

[0127] TABLE 3 : Illustrative anti-CD20 binding domain sequencesWSGR Docket No. 69097-701.601

[0128] TABLE 4: Illustrative anti-CD20 CDRs.WSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601

[0129] TABLE 5 : Illustrative CD22 binding domains.WSGR Docket No. 69097-701.601

[0130] 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.

[0131] 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.

[0132] 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,WSGR Docket No. 69097-701.601 the binding domain comprises a set of CDRs including two or more CDRs corresponding to different antibody IDs, or variants thereof described herein.

[0133] TABLE 6: illustrative anti-CD22 CDRsWSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601

[0134] 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 anWSGR Docket No. 69097-701.601 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.

[0135] 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 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: 79-91. The binding domain can comprise two such sequences, for example, a VH and a VL.

[0136] 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.

[0137] TABLE 7 : Illustrative CD2 binding domainsWSGR Docket No. 69097-701.601

[0138] TABLE 8: illustrative anti-CD2 CDRs

[0139] 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.

[0140] 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%,WSGR Docket No. 69097-701.601 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: 92-107. The binding domain can comprise two such sequences, for example, a VH and a VL.

[0141] 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.

[0142] TABLE 9 : illustrative CD3 binding domainsWSGR Docket No. 69097-701.601

[0143] TABLE 10: Illustrative anti-CD3 CDRs.WSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601B. Target molecules

[0144] 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

[0145] 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).

[0146] 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.

[0147] 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.WSGR Docket No. 69097-701.601

[0148] 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).

[0149] 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.

[0150] 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.

[0151] A target molecule on a target cell can be a cell surface molecule, for example, a membrane protein or a target molecule present on the surface of the target cell.

[0152] 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 is a wild type protein or is 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).

[0153] 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.

[0154] 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 some 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 on target cells comprise two or more of CD 19, CD20, CD22, CD33, CD37, CD38, CD79a, CD79b, CD123, CD138, BAFF-R, BCMA, CS1,WSGR Docket No. 69097-701.601GPRC5D, 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.

[0155] In some embodiments, target molecules on target cells comprise CD 19, CD20, and / or CD22.

[0156] 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.

[0157] In some embodiments, the target molecules can comprise different epitopes on the same target molecule.

[0158] 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

[0159] 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,WSGR Docket No. 69097-701.601 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).

[0160] 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.

[0161] 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.

[0162] 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).

[0163] 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.

[0164] 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.

[0165] 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 CD2. In some embodiments, a multispecific immune cell engager binds CD3 and CD28, CD3 and CD27, CD3 and 4 IBB, CD3 and 0X40, CD3 and a T cell co-stimulation receptor or co-stimulation associated target molecule, or CD3 and another target molecule disclosed herein.WSGR Docket No. 69097-701.601

[0166] 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.

[0167] 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).

[0168] 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.

[0169] 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 (CD15O,SLAMF1), 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, NCR, NKG2D (KLRK1, CD314), NKp30 (NCR3), NKp44 (NCR2), NKp46 (NCR1), NKp80 (KLRF1, CLEC5C), NTB-A (SLAMF6), PSGL1, and SLAMF7 (CRACC, CS1, CD319).

[0170] 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.

[0171] 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.

[0172] 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.WSGR Docket No. 69097-701.601C. Fc region and immunoglobulin constant domains

[0173] 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.

[0174] 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 interactions 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.

[0175] 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.WSGR Docket No. 69097-701.601

[0176] 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.

[0177] 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.

[0178] 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 refer 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.WSGR Docket No. 69097-701.601

[0179] 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.

[0180] 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.

[0181] 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, IgG2, 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).

[0182] 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 doesWSGR Docket No. 69097-701.601 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.

[0183] 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.

[0184] 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.

[0185] 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. 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.

[0186] 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.

[0187] 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 constantWSGR Docket No. 69097-701.601 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).

[0188] 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

[0189] TABLE 11: illustrative immunoglobulin constant domain and Fc region sequences.WSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.6016. Fc modifications

[0190] 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.

[0191] 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 a 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 desirableWSGR Docket No. 69097-701.601(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).

[0192] 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. 69097-701.601D. Dimerization / multimerization module

[0193] 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.

[0194] 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).

[0195] 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.

[0196] 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 mutations identified therefrom, electrostatic optimization / steering and mutations identified therefrom, DNLWSGR Docket No. 69097-701.601(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.

[0197] In some embodiments, knob-in-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.

[0198] 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.

[0199] 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.

[0200] The residue numbering can be according to EU numbering (e.g., as described herein).

[0201] 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. 69097-701.601 thereof) comprises a Y349C substitution and a second polypeptide chain (e.g., Fc region or immunoglobulin constant domain thereof) comprises an E356'C substitution.

[0202] A dimerization module, immunoglobulin constant domain, or a heterodimerization domain can comprise a combination of cysteine replacement residues and heterodimerizationpromoting (e.g., knob-in-hole) modifications, for example, one or more cysteine replacement residues and one or more knob-in-hole modifications in each of a pair of polypeptide chains.

[0203] 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.

[0204] 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.

[0205] 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.

[0206] 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.

[0207] 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. 69097-701.601 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.

[0208] 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).

[0209] 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.

[0210] 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

[0211] 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. 69097-701.601 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.

[0212] 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.

[0213] 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.

[0214] 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. 69097-701.601 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.

[0215] 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.

[0216] 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.

[0217] 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. 69097-701.601 designating a number of repeats. The presence of proline in non-helical linkers can increase stiffness, and allow for effective separation of protein domains.

[0218] 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.

[0219] 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).

[0220] 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.

[0221] 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.

[0222] 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.

[0223] 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. 69097-701.601 di(methylamino)propane, l,4-di(methylamino)butane, l,5-di(methylamino)pentane, 1,6- di(methylamino)hexane, and pipyrizine.

[0224] 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.

[0225] TABLE 13: illustrative linkers, spacers, hinges, and subunits thereof.WSGR Docket No. 69097-701.601F. Configurations

[0226] 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.

[0227] 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.)

[0228] 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. 69097-701.601 chain. Linkers, spacers, or hinges disclosed herein can optionally be interposed between (e.g., used to join) any pair of domains.

[0229] In some embodiments, advantageous properties are observed for multispecific immune cell engagers with a particular orientation or structural format.

[0230] 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 binding 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.

[0231] 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.

[0232] 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.

[0233] 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. 2C), or at positions INI and 1C1 (FIG. 2B). A format 0 immune cell engager can comprise three binding domains thatWSGR Docket No. 69097-701.601 bind to target cell target molecules, for example, at positions INI, 1N2, and 1N3 (FIG. 2E), or at positions INI, 1N2, and 1C1 (FIG. 2D).

[0234] A multispecific immune cell engager (e.g., T cell engager) disclosed herein can comprise or have a first binding domain that binds to a target molecule on an immune effector cell at the 2N1 position, and a second binding domain that binds to a target molecule on an immune effector cell at the 2N2 position, which can be referred to as “format 1 ” The first and second 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. 3A). 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. 3C) or at positions INI and 1C1 (FIG. 3B). 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. 3E) or at positions INI, 1N2, and 1C1 (FIG. 3D).

[0235] A multispecific immune cell engager (e.g., T cell engager) disclosed herein can comprise or have a first binding domain that binds to a target molecule on an immune effector cell at the 2N1 position, and a second 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 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. 4A). 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. 4C) or at positions INI and 1C1 (FIG. 4B). 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. 4E) or at positions INI, 1N2, and 1C1 (FIG. 4D).

[0236] A multispecific immune cell engager (e.g., T cell engager) disclosed herein can comprise or have a first binding domain that binds to a target molecule on an immune effector cell at the INI position, and a second 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 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. 5A). A format 3 immune cell engager can comprise or have two binding domains that bind target cell target molecules, for example, at positions 1N2 and 1N3 (FIG. 5C)WSGR Docket No. 69097-701.601 or at positions 1N2 and 1C1 (FIG. 5B). 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. 5E) or at positions 1N2, 1N3, and 1C1 (FIG. 5D).

[0237] A multispecific immune cell engager (e.g., T cell engager) disclosed herein can comprise or have a first binding domain that binds to a target molecule on an immune effector cell at the 1N2 position, and a second 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 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. 6A). 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. 6C) or at positions INI and 1C1 (FIG. 6B). 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. 6E) or at positions INI, 1N3, and 1C1 (FIG. 6D).

[0238] A multispecific immune cell engager (e.g., T cell engager) disclosed herein can comprise or have a first binding domain that binds to a target molecule on an immune effector cell at the 1N3 position, and a second 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 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. 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, or at positions INI, 1N2, and 1C1.

[0239] A multispecific immune cell engager (e.g., T cell engager) disclosed herein can comprise or have a first binding domain that binds to a target molecule on an immune effector cell at the 1N4 position, and a second 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 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 immune cell engager can comprise or have three binding domains that bind target cell target molecules, for example, at positions INI, 1N2, and 1N3.

[0240] A multispecific immune cell engager (e.g., T cell engager) disclosed herein can comprise or have a first binding domain that binds to a target molecule on an immune effector cellWSGR Docket No. 69097-701.601 at the 1C1 position, and a second 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 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. 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. 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.

[0241] A multispecific immune cell engager (e.g., T cell engager) disclosed herein can comprise or have a first binding domain that binds to a target molecule on an immune effector cell at the INI position, and a second 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 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. 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, 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.

[0242] A multispecific immune cell engager (e.g., T cell engager) disclosed herein can comprise or have a first binding domain that binds to a target molecule on an immune effector cell at the 2N1 position, and a second 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 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. 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, or positions 1C1 and 2N1. A format 9 immune cell engager can comprise or have three binding domains that bind target cell target molecules, for example, at positions INI, 1N2, and 1C1.

[0243] A multispecific immune cell engager can comprise five binding domains, each of which binds to a target molecule (e.g., a first binding domain that binds to a first target molecule, a second binding domain that binds to a second target molecule, a third binding domain that binds to a third target molecule, a fourth binding domain that binds to a fourth target molecule, and aWSGR Docket No. 69097-701.601 fifth 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 binding domains can be tumor-associated antigens or B cell markers disclosed herein, such as CD 19, CD20, and CD22, in any order). Two of the target molecules, for example, the fourth and fifth target molecules bound by the fourth and fifth binding domains, can be on immune effector cells (e.g., CD3 and CD2, in any order).

[0244] In some embodiments the first target molecule is or can be independently selected from any one of CD19, CD20, CD22, CD3, and CD2. In some embodiments the second target molecule is or can be independently selected from any one of CD19, CD20, CD22, CD3, and CD2. In some embodiments the third target molecule is or can be independently selected from any one of CD 19, CD20, CD22, CD3, and CD2. In some embodiments the fourth target molecule is or can be independently selected from any one of CD19, CD20, CD22, CD3, and CD2. In some embodiments the fifth target molecule is or can be independently selected from any one of CD 19, CD20, CD22, CD3, and CD2.

[0245] 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.

[0246] 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.

[0247] 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.

[0248] 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.

[0249] 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.WSGR Docket No. 69097-701.601

[0250] 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.

[0251] 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.

[0252] 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.

[0253] 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,WSGR Docket No. 69097-701.601CD22, 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 are CD19, 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,WSGR Docket No. 69097-701.601CD22, 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 are CD20, 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,WSGR Docket No. 69097-701.601CD3, 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 are CD22, 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,WSGR Docket No. 69097-701.601CD 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 are CD3, 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,WSGR Docket No. 69097-701.601CD22, 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.

[0254] 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 binding domain, and the second polypeptide chain can comprise the second, third, fourth, and fifth binding domains. In some embodiments, the first polypeptide chain comprises the second binding domain the second polypeptide chain comprises the first, third, fourth, and fifth binding domains. In some embodiments, the first polypeptide chain comprises the third binding domain and the second polypeptide chain comprises the first, second, fourth, and fifth binding domains. In some embodiments, the first polypeptide chain comprises the fourth binding domain and the second polypeptide chain comprises the first, second, third, and fifth binding domains. In some embodiments, the first polypeptide chain comprises the fifth binding domain and the second polypeptide chain comprises the first, second, third, and fourth binding domains. The first and second polypeptides can each comprise a portion of a multimerization or dimerization module, for example, an Fc chain / region.

[0255] 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 binding domains, and the second polypeptide chain can comprise the third, fourth, and fifth binding domains. In some embodiments, the first polypeptide chain comprises the first and third binding domains the second polypeptide chain comprises the second, fourth, and fifth binding domains. In some embodiments, the first polypeptide chain comprises the first and fourth binding domains the second polypeptide chain comprises the second, third, and fifth binding domains. In some embodiments, the first polypeptide chain comprises the first and fifth binding domains the second polypeptide chain comprises the second, third, and fourth binding domains. In some embodiments, the first polypeptide chain comprises the second and third binding domains the second polypeptide chain comprises the first, fourth, and fifth binding domains. In some embodiments, the first polypeptide chain comprises the second and fourth binding domains the second polypeptide chain comprises the first, third, and fifth binding domains. In some embodiments, the first polypeptide chain comprises the second and fifth binding domains the second polypeptide chain comprises the first, third, and fourth binding domains. In some embodiments, the first polypeptide chain comprises the third and fourth binding domains the second polypeptide chain comprises the first, second, and fifth binding domains. In some embodiments, the first polypeptide chain comprises the third and fifth binding domains theWSGR Docket No. 69097-701.601 second polypeptide chain comprises the first, second, and fourth binding domains. In some embodiments, the first polypeptide chain comprises the fourth and fifth binding domains the second polypeptide chain comprises the first, second, and third binding domains. The first and second polypeptides can each comprise a portion of a multimerization or dimerization module, for example, an Fc chain / region.

[0256] Disclosed herein, in some aspects, is a T cell engager comprising: a first polypeptide comprising from N terminus to C terminus a first binding domain linked to a first Fc region; and a second polypeptide comprising from N terminus to C terminus a fourth binding domain linked to a fifth binding domain linked to a second Fc region, wherein the first binding domain binds to a first target molecule, and the fourth binding domain binds to a fourth target molecule, wherein the fourth target molecule is CD3, and the fifth binding domain binds to a fifth target molecule, wherein the fifth target molecule is CD2.

[0257] Disclosed herein, in some aspects, is a T cell engager comprising: a first polypeptide comprising from N terminus to C terminus a first binding domain linked to a fifth binding domain linked to a first Fc region; and a second polypeptide comprising from N terminus to C terminus a fourth binding domain linked to a second Fc region, wherein the first binding domain binds to a first target molecule, the fourth binding domain binds to a fourth target molecule, wherein the fourth target molecule is CD3, and the fifth binding domain binds to a fifth target molecule, wherein the fifth target molecule is CD2.

[0258] Disclosed herein, in some aspects, is a T cell engager comprising: a first polypeptide comprising from N terminus to C terminus a fifth binding domain linked to a first binding domain linked to a first Fc region; and a second polypeptide comprising from N terminus to C terminus a fourth binding domain linked to a second Fc region, wherein the first binding domain binds to a first target molecule, the fourth binding domain binds to a fourth target molecule, wherein the fourth target molecule is CD3, and the fifth binding domain binds to a fifth target molecule, wherein the fifth target molecule is CD2.

[0259] Disclosed herein, in some aspects, is a T cell engager comprising: a first polypeptide comprising from N terminus to C terminus a second binding domain linked to a first binding domain linked to a first Fc region; and a second polypeptide comprising from N terminus to C terminus a fourth binding domain linked to a fifth binding domain linked to a second Fc region, wherein the first binding domain binds to a first target molecule, the second binding domain binds to a second target molecule, the fourth binding domain binds to a fourth target molecule, wherein the fourth target molecule is CD3, and the fifth binding domain binds to a fifth target molecule, wherein the fifth target molecule is CD2.WSGR Docket No. 69097-701.601

[0260] Disclosed herein, in some aspects, is a T cell engager comprising: a first polypeptide comprising from N terminus to C terminus a second binding domain linked to a first binding domain linked to a fifth binding domain linked to a first Fc region; and a second polypeptide comprising from N terminus to C terminus a fourth binding domain linked to a second Fc region, wherein the first binding domain binds to a first target molecule, the second binding domain binds to a second target molecule, the fourth binding domain binds to a fourth target molecule, wherein the fourth target molecule is CD3, and the fifth binding domain binds to a fifth target molecule, wherein the fifth target molecule is CD2.

[0261] Disclosed herein, in some aspects, is a T cell engager comprising: a first polypeptide comprising from N terminus to C terminus a second binding domain linked to a fifth binding domain linked to a first binding domain linked to a first Fc region; and a second polypeptide comprising from N terminus to C terminus a fourth binding domain linked to a second Fc region, wherein the first binding domain binds to a first target molecule, the second binding domain binds to a second target molecule, the fourth binding domain binds to a fourth target molecule, wherein the fourth target molecule is CD3, and the fifth binding domain binds to a fifth target molecule, wherein the fifth target molecule is CD2.

[0262] Disclosed herein, in some aspects, is a T cell engager comprising: a first polypeptide comprising from N terminus to C terminus a third binding domain linked to a second binding domain linked to a first binding domain linked to a first Fc region; and a second polypeptide comprising from N terminus to C terminus a fourth binding domain linked to a fifth binding domain linked to a second Fc region, wherein the first binding domain binds to a first target molecule, the second binding domain binds to a second target molecule, the third binding domain binds to a third target molecule, the fourth binding domain binds to a fourth target molecule, wherein the fourth target molecule is CD3, and the fifth binding domain binds to a fifth target molecule, wherein the fifth target molecule is CD2.

[0263] Disclosed herein, in some aspects, is a T cell engager comprising: a first polypeptide comprising from N terminus to C terminus a third binding domain linked to a second binding domain linked to a first binding domain linked to a fifth binding domain linked to a first Fc region; and a second polypeptide comprising from N terminus to C terminus a fourth binding domain linked to a second Fc region, wherein the first binding domain binds to a first target molecule, the second binding domain binds to a second target molecule, the third binding domain binds to a third target molecule, the fourth binding domain binds to a fourth target molecule, wherein the fourth target molecule is CD3, and the fifth binding domain binds to a fifth target molecule, wherein the fifth target molecule is CD2.WSGR Docket No. 69097-701.601

[0264] Disclosed herein, in some aspects, is a T cell engager comprising: a first polypeptide comprising from N terminus to C terminus a third binding domain linked to a second binding domain linked to a fifth binding domain linked to a first binding domain linked to a first Fc region; and a second polypeptide comprising from N terminus to C terminus a fourth binding domain linked to a second Fc region, wherein the first binding domain binds to a first target molecule, the second binding domain binds to a second target molecule, the third binding domain binds to a third target molecule, the fourth binding domain binds to a fourth target molecule, wherein the fourth target molecule is CD3, and the fifth binding domain binds to a fifth target molecule, wherein the fifth target molecule is CD2.

[0265] Disclosed herein, in some aspects, is a T cell engager comprising: a first polypeptide comprising from N terminus to C terminus a first binding domain linked to a second binding domain linked to a first Fc region linked to a third binding domain; and a second polypeptide comprising from N terminus to C terminus a fourth binding domain linked to a fifth binding domain linked to a second Fc region, wherein the first binding domain binds to a first target molecule, the second binding domain binds to a second target molecule, the third binding domain binds to a third target molecule, the fourth binding domain binds to a fourth target molecule, wherein the fourth target molecule is CD3, and the fifth binding domain binds to a fifth target molecule, wherein the fifth target molecule is CD2.

[0266] Disclosed herein, in some aspects, is a T cell engager comprising: a first polypeptide comprising from N terminus to C terminus a first binding domain linked to a second binding domain linked to a fifth binding domain linked to a first Fc region linked to a third binding domain from C terminus to N terminus; and a second polypeptide comprising from N terminus to C terminus a fourth binding domain linked to a second Fc region, wherein the first binding domain binds to a first target molecule, the second binding domain binds to a second target molecule, the third binding domain binds to a third target molecule, the fourth binding domain binds to a fourth target molecule, wherein the fourth target molecule is CD3, and the fifth binding domain binds to a fifth target molecule, wherein the fifth target molecule is CD2.

[0267] Disclosed herein, in some aspects, is a T cell engager comprising: a first polypeptide comprising from N terminus to C terminus a first binding domain linked to a fifth binding domain linked to a second binding domain linked to a first Fc region linked to a third binding domain; and a second polypeptide comprising from N terminus to C terminus a fourth binding domain linked to a second Fc region, wherein the first binding domain binds to a first target molecule, the second binding domain binds to a second target molecule, the third binding domain binds to a third target molecule, the fourth binding domain binds to a fourth target molecule, wherein the fourth targetWSGR Docket No. 69097-701.601 molecule is CD3, and the fifth binding domain binds to a fifth target molecule, wherein the fifth target molecule is CD2.

[0268] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first binding domain, the second binding domain, the third 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 binding domain, the fifth 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.

[0269] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first binding domain, the second binding domain the third 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 binding domain, a second portion of the dimerization module, and the fifth binding domain. The domains of each chain can optionally be joined by linkers, spacers, and / or hinges disclosed herein.

[0270] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first binding domain, the second binding domain, the third binding domain, the fourth 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 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.

[0271] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first binding domain, the second binding domain, the fourth binding domain, the third 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 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.

[0272] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first binding domain, the fourth binding domain, the second binding domain, the third 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 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.

[0273] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the fourth binding domain, the first binding domain, the second binding domain, the third binding domain, and a first portion of a dimerization module (e.g., Fc region), and a second polypeptideWSGR Docket No. 69097-701.601 chain comprises, from N-to-C terminus, the fifth 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.

[0274] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first binding domain, the second binding domain, the third binding domain, a first portion of a dimerization module (e.g., Fc region), and the fourth binding domain, and a second polypeptide chain comprises, from N-to-C terminus, the fifth 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.

[0275] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first binding domain, the second binding domain, the fourth 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 binding domain, the third 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.

[0276] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first binding domain, the second binding domain, a first portion of a dimerization module (e.g., Fc region), the third binding domain, and the fourth binding domain, and a second polypeptide chain comprises, from N-to-C terminus, the fifth 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.

[0277] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first binding domain, the second binding domain, a first portion of a dimerization module (e.g., Fc region), and the third binding domain, and a second polypeptide chain comprises, from N-to- C terminus, the fourth binding domain, the fifth 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.

[0278] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first binding domain, the second binding domain, a first portion of a dimerization module (e.g., Fc region) and the third binding domain, and a second polypeptide chain comprises, from N-to-C terminus, the fourth binding domain, a second portion of the dimerization module (e.g., Fc region), and the fifth binding domain. The domains of each chain can optionally be joined by linkers, spacers, and / or hinges disclosed herein.

[0279] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first binding domain, the second binding domain, the fourth binding domain, a first portion of aWSGR Docket No. 69097-701.601 dimerization module (e.g., Fc region), and the third binding domain, and a second polypeptide chain comprises, from N-to-C terminus, the fifth 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.

[0280] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first binding domain, the fourth binding domain, the second binding domain, a first portion of a dimerization module (e.g., Fc region), and the third binding domain, and a second polypeptide chain comprises, from N-to-C terminus, the fifth 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.

[0281] A multispecific immune cell engager can comprise four binding domains, each of which binds to a target molecule (e.g., a first binding domain that binds to a first target molecule, a second binding domain that binds to a second target molecule, a third binding domain that binds to a third target molecule, and a fourth 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 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 binding domains, can be on immune effector cells (e.g., CD3 and CD2, in any order).

[0282] 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.

[0283] 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.

[0284] 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.

[0285] 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.WSGR Docket No. 69097-701.601

[0286] 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.

[0287] 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.

[0288] 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.

[0289] 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. In 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 moleculesWSGR Docket No. 69097-701.601 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 molecules 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, theWSGR Docket No. 69097-701.601 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 CD 19, 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, 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,WSGR Docket No. 69097-701.601 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, the 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,WSGR Docket No. 69097-701.601 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, CD19, 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.

[0290] 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 binding domain, and the second polypeptide chain can comprise the second, third, and fourth binding domains. In some embodiments, the first polypeptide chain comprises the second binding domain, and the second polypeptide chain comprises the first, third, and fourth binding domains. In some embodiments, the first polypeptide chain comprises the third binding domain, and the second polypeptide chain comprises the first, second, and fourth binding domains. In some embodiments, the first polypeptide chain comprises the fourth binding domain, and the second polypeptide chain comprises the first, second, and third binding domains.

[0291] 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 target binding domain and second target binding domains, and the second polypeptide chain can comprise the third target binding domain and fourth target bindingWSGR Docket No. 69097-701.601 domains. In some embodiments, the first polypeptide chain comprises the first and third binding domains, and the second polypeptide chain can comprise the second and fourth binding domains. In some embodiments, the first polypeptide chain comprises the first and fourth binding domains, and the second polypeptide chain can comprise the second and third binding domains. In some embodiments, the first polypeptide chain comprises the second and third binding domains, and the second polypeptide chain can comprise the first and fourth binding domains. In some embodiments, the first polypeptide chain comprises the second and fourth binding domains, and the second polypeptide chain can comprise the first and third binding domains. In some embodiments, the first polypeptide chain comprises the third and fourth binding domains, and the second polypeptide chain can comprise the first and second binding domains. The first and second polypeptides can each comprise a portion of a multimerization or dimerization module, for example, an Fc chain / region.

[0292] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first binding domain, the second 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 binding domain, the fourth 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.

[0293] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first binding domain, the second 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 binding domain, a second portion of a dimerization module (e.g., Fc region), and the fourth binding domain. The domains of each chain can optionally be joined by linkers, spacers, and / or hinges disclosed herein.

[0294] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first binding domain, the second binding domain, the third 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 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.

[0295] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first binding domain, the third binding domain, the second 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 binding domain, and a second portion of the dimerization module (e.g., FcWSGR Docket No. 69097-701.601 region). The domains of each chain can optionally be joined by linkers, spacers, and / or hinges disclosed herein.

[0296] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the third binding domain, the first binding domain, the second 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 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.

[0297] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first binding domain, the second binding domain, a first portion of a dimerization module (e.g., Fc region), and the third binding domain, and a second polypeptide chain comprises, from N-to- C terminus, the fourth 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.

[0298] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first binding domain, the third 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 binding domain, the second 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.

[0299] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first binding domain, a first portion of a dimerization module (e.g., Fc region), the second binding domain, and the third binding domain, and a second polypeptide chain comprises, from N-to-C terminus, the fourth 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.

[0300] In some embodiments, a first polypeptide chain comprises, from N-to-C terminus, the first binding domain, a first portion of a dimerization module (e.g., Fc region), the second binding domain, and a second polypeptide chain comprises, from N-to-C terminus, the third binding domain, the fourth 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 binding domain, a first portion of a dimerization module (e.g., Fc region), and the second binding domain, and a second polypeptide chain comprises, from N-to-C terminus, the thirdWSGR Docket No. 69097-701.601 binding domain, a second portion of the dimerization module (e.g., Fc region), and the fourth 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 binding domain, the third binding domain, a first portion of a dimerization module (e.g., Fc region), and the second binding domain, and a second polypeptide chain comprises, from N-to-C terminus, the fourth 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 third binding domain, the first binding domain, a first portion of a dimerization module (e.g., Fc region), and the second binding domain, and a second polypeptide chain comprises, from N-to-C terminus, the fourth 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] 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.

[0305] 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.

[0306] 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 aboutWSGR Docket No. 69097-701.60182%, 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.

[0307] 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.

[0308] 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.

[0309] TABLE 14: Illustrative polypeptide chains.WSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601

[0310] 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.

[0311] TABLE 15: Illustrative polypeptide chain combinationsWSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601

[0312] 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.

[0313] 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 any one of SEQ ID NOs: 1070-1095. A multispecific immune cell engager can comprise two such sequences, for example, a first polypeptide chain and a second polypeptide chain.

[0314] 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- 1095.

[0315] 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 leastWSGR Docket No. 69097-701.601 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- 1095.

[0316] In some embodiments, a multispecific immune cell engager or a part thereof (e.g., a first 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 any one of SEQ ID NOs: 1070-1095. A multispecific immune cell engager can comprise two such sequences, for example, a first polypeptide chain and a second polypeptide chain. In some embodiments, a corresponding second polypeptide chain may 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 the sequence set forth in SEQ ID NO: 184 or SEQ ID NO: 186.

[0317] 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. 69097-701.601 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- 1095.

[0318] In some embodiments, a multispecific immune cell engager or a part thereof (e.g., a first polypeptide chain) comprises 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 about76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about95.5%, 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 the sequence set forth in SEQ ID NO: 1070. In some embodiments, a corresponding second polypeptide chain may comprise 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 about79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about94%, 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 the sequence set forth in SEQ ID NO: 186. [Complex ID PROT328],

[0319] In some embodiments, the first polypeptide and the second polypeptides are organized as described in Table 16 and Table 17.

[0320] Table 16 Illustrative polypeptide chainsWSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601

[0321] Illustrative complexes composed of different polypeptide chains described above are further provided in Table 17.

[0322] Table 17 Illustrative polypeptide chain combinationsWSGR Docket No. 69097-701.601WSGR Docket No. 69097-701.601II. Polynucleotides and vectors

[0323] 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.

[0324] In some embodiments, the polynucleotide can be a DNA. In some embodiments, the polynucleotide can be an RNA. The polynucleotide can comprise a modified base, for example, to enhance stability of the polynucleotide upon administration to a subject. 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.WSGR Docket No. 69097-701.601

[0325] 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, RNA 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.

[0326] In some embodiments, a polynucleotide encoding a multispecific immune cell engager is packaged in a lipid-based delivery vehicle, such as a liposome or lipid nanoparticle.

[0327] 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, Cl 2-200 (including variants and derivatives), DLin-MC3-DMA and analogs thereof.II. PHARMACEUTICAL COMPOSITIONS

[0328] Compositions disclosed herein can comprise a polypeptide, polynucleotide, or vector and a pharmaceutically-acceptable excipient, vehicle, carrier, or diluent. For example, in some embodiments the disclosure provides a pharmaceutical composition comprising a multispecific immune cell engager and a pharmaceutically-acceptable excipient, vehicle, carrier, or diluent. The pharmaceutical composition can be in a unit dosage form.

[0329] A pharmaceutical composition disclosed herein can comprise a saline solution. A pharmaceutical composition disclosed herein can comprise a buffered saline solution, for example, PBS, dPBS, HBSS, or the like. A pharmaceutical composition disclosed herein can comprise Ringer's solution, dextrose solution, or Hank's solution.

[0330] A pharmaceutical composition disclosed herein can comprise a buffer, for example, a citrate buffer (e.g., sodium citrate) or a phosphate buffer (e.g., sodium phosphate buffer). A pharmaceutical composition disclosed herein can comprise a pH-stabilizing agent.

[0331] A pharmaceutical composition disclosed herein can comprise an organic co-solvent, e.g., polysorbate 20, polysorbate 80, propylene glycol, or polyethylene glycol (PEG).

[0332] A pharmaceutical composition disclosed herein can comprise a stabilizing agent, e.g., sucrose, sorbitol, glycerol, trehalose, or mannitol.

[0333] A pharmaceutical composition disclosed herein can comprise a tonicity agent, e.g., a salt, such as NaCl or KC1.WSGR Docket No. 69097-701.601

[0334] A pharmaceutical composition disclosed herein can comprise a preservative. A pharmaceutical composition disclosed herein can comprise an antimicrobial gent. A pharmaceutical composition disclosed herein can comprise an antifungal agent.

[0335] In some embodiments, a polypeptide disclosed herein is in an aqueous buffer. In some embodiments, a polypeptide disclosed herein is in a powdered (e.g., lyophilized) form, e.g., for re-constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0336] Parenteral injections can be formulated for bolus injection or continuous infusion. The pharmaceutical compositions can be in a form suitable for parenteral injection as a sterile suspension, solution, or emulsion in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0337] Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. The suspension can also contain suitable stabilizers or agents which increase the solubility and / or reduces aggregation.

[0338] Non-limiting examples of pharmaceutically-acceptable excipients, vehicles, carriers, and diluents can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkinsl999), and updated versions thereof, each of which is incorporated by reference in its entirety.

[0339] Pharmaceutical compositions comprising a multispecific immune cell engager described herein can be manufactured, for example, by expressing the multispecific immune cell engager in a recombinant system, purifying the multispecific immune cell engager, lyophilizing the multispecific immune cell engager, mixing, or dissolving.

[0340] In some embodiments, the disclosure provides method of making a multispecific immune cell engager described herein (e.g., a TCE), the method comprising identifying target one or more target binding domains; cloning a polynucleic acid sequence encoding a recombinant polypeptide comprising a first immunoglobulin constant domain Fc region and the one or more target binding domains; cloning a polynucleic acid sequence encoding a recombinant polypeptide comprising a second immunoglobulin constant domain Fc region and the one or more target binding domains; and expressing cloned polynucleic acid sequences in an immune cell.WSGR Docket No. 69097-701.601III. METHODS

[0341] The disclosure encompasses methods of treating a subject and compositions for use in a method of treating a subject. For example, a multispecific immune cell engager disclosed herein can be useful for treating a condition in a subject in need thereof.

[0342] In some embodiments, provided is a method of treating a condition in a subject in need thereof, the method comprising administering to the subject an effective amount of a multispecific immune cell engager disclosed herein. The effective amount can be a therapeutically-effective amount. In some embodiments, provided is a method of treating a condition in a subject in need thereof, the method comprising administering to the subject an effective amount of a polynucleotide encoding a multispecific immune cell engager disclosed herein, or a vector comprising the polynucleotide.

[0343] The condition can be a cancer. The condition can be a hematologic tumor. The cancer can be an immune cell cancer. The cancer can be a B cell cancer. The cancer can be a T cell cancer. The cancer can be a myeloid cell cancer. The cancer can be a leukemia. The cancer can be a lymphoma. The cancer can be a myeloma. In some embodiments, the target cells are B cells, and the indication is cancer.

[0344] In some embodiments, the condition is mantle cell lymphoma, diffuse large B 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.

[0345] In some embodiments, the condition is large B cell lymphoma.

[0346] In some embodiments, the condition is diffuse large B-cell lymphoma.

[0347] In some embodiments, the condition is a relapsed, refractory, or relapsed / refractory cancer, such as relapsed / refractory large B cell lymphoma or relapsed / refractory diffuse large B- cell lymphoma.

[0348] In some embodiments, the subject has a cancer that exhibits lost or reduced expression of one or more T cell co-stimulation ligands, for example, CD58, 41BBL, OX40L, CD70 (CD27L), CD80, CD86, GITRL, or a combination thereof. Multispecific immune cell engagers disclosed herein that contain a binding domain that binds to a T cell co-stimulation receptor can elicit favorable anti-cancer immune responses for subjects with cancers that have reduced or lost expression of the T cell co-stimulation ligand, for example, by providing a co-stimulation signalWSGR Docket No. 69097-701.601 to enhance activation of immune effector T cells. The cancer that exhibits lost or reduced expression of one or more T cell co-stimulation ligands can be a B cell cancer, for example, relap sed / refractory large B cell lymphoma or relapsed / refractory diffuse large B-cell lymphoma.

[0349] In some embodiments, the subject has a cancer that exhibits lost or reduced expression of CD58. Multispecific immune cell engagers disclosed herein that contain a CD2 binding domain, such as an Ig-like domain of CD58, can elicit favorable anti-cancer immune responses for subjects with cancers that have reduced or lost expression of CD58, for example, by providing a co-stimulation signal to enhance activation of immune effector T cells. The cancer that exhibits lost or reduced expression of CD58 can be a B cell cancer, for example, relapsed / refractory large B cell lymphoma or relapsed / refractory diffuse large B-cell lymphoma.

[0350] In some embodiments, the subject has previously failed therapy, for example, exhibited disease progression despite another treatment, such as chemotherapy, radiotherapy, cell therapy (e.g., CAR-T therapy, engineered T cell therapy, or allogeneic hematopoietic stem cell transplantation (HSCT)), treatment with an alternative immune cell engager or T cell engager, or a combination thereof.

[0351] The condition can be an autoimmune disease. In some embodiments, the target cells are B cells, and the indication is an autoimmune disease, for example, a B cell-mediated autoimmune disease.

[0352] In some embodiments, the condition is systemic lupus erythematosus, scleroderma, Sjogren’s syndrome, multiple sclerosis, rheumatoid arthritis, Hashimoto’s thyroiditis, antiphospholipid syndrome, celiac disease, Grave’s disease, myasthenic syndrome, myasthenia gravis, polyangiitis, dematomy ositis, scleromyositis, or pemphigus vulgaris.

[0353] In some embodiments, the cancer is metastatic. In some embodiments, the cancer is not metastatic.

[0354] The cancer can be a carcinoma. The cancer can be a sarcoma. The cancer can be an adenoma.

[0355] The condition can be a solid tumor. In some embodiments, the cancer is colorectal cancer (CRC), lung cancer, non-small cell lung cancer (NSCLC), small cell lung cancer, lung adenocarcinoma, prostate cancer, breast cancer, pancreatic cancer, glioblastoma, bone cancer, osteosarcoma, astrocytoma, biliary cancer (cholangiocarcinoma), renal cancer, renal cell carcinoma, esophageal cancer, thyroid cancer, cervical cancer, skin cancer, melanoma, hepatocellular carcinoma, gastric cancer, testicular cancer, ovarian cancer, or uterine cancer.

[0356] The condition can be an infectious disease, for example, a chronic or acute infectious disease. The condition can be a bacterial or viral infectious disease, for example, or an infectious disease caused by a eukaryotic parasite.WSGR Docket No. 69097-701.601

[0357] The subject can be a mammalian subject. The subject can be a human subject. In some embodiments, the subject is a murine, rodent, canine, feline, equine, porcine, primate, or bovine subject. In some embodiments, the subject is a non-human and / or non-rodent mammalian subject.

[0358] In practicing the methods of treatment or use provided herein, a therapeutically- effective amount of the multispecific immune cell engager described herein can be administered in pharmaceutical compositions to a subject suffering from a condition. In some instances, the pharmaceutical composition will affect the physiology of the animal, such as the immune system, inflammatory response, or other physiologic effect. In some embodiments, the subject is a mammal such as a human. A therapeutically-effective amount can vary depending on the severity of the disease, the age and relative health of the subject, the potency of the compounds used, and other factors.

[0359] A pharmaceutical composition can be administered in therapeutically-effective amounts by various forms and routes including, for example, intravenous, intratumoral, subcutaneous, intramuscular, aerosol, parenteral, ophthalmic, optic, pulmonary, transdermal, nasal, oral, inhalation, dermal, intradermal, intra-articular, intrathecal, intranasal, or topical administration. A pharmaceutical composition can be administered in a local or systemic manner, for example, via injection of the multispecific immune cell engager described herein directly into a tumor or organ. The administering can be local. The administering can be systemic. The administering can be parenteral.

[0360] In some embodiments, the method comprises administering one dose of the multispecific immune cell engager. In some embodiments, the method comprises administering multiple (e.g., two or more) doses of the multispecific immune cell engager.

[0361] Certain methods described herein comprise administering to the subject an intravenous pharmaceutical composition comprising a multispecific immune cell engager of the present disclosure. Intravenous pharmaceutical compositions of multispecific immune cell engagers include any formulation suitable for administration to a subject via any intravenous method, including a bolus, an infusion which occurs over time, or any other intravenous method.

[0362] The multispecific immune cell engager can promote activation of immune effector cells, such as T cells, in proximity to target cells, such as cancer cells or B cells. The multispecific immune cell engager can enhance effector function (e.g., target cell killing, pro-inflammatory cytokine production, and / or chemotaxis) of immune effector cells, such as T cells or other immune effector cells disclosed herein, in proximity to target cells (e.g., cancer cells or B cells). The multispecific immune cell engager can promote proliferation, survival, and / or persistence of immune effector cells, (e.g., T cells, and / or anti-tumor immune cells).WSGR Docket No. 69097-701.601

[0363] The disclosure also provides a method of inducing target cell killing, the method comprising treating or contacting a multispecific immune cell engager disclosed herein to (i) a target cell, and (ii) an immune effector cell, thereby inducing killing of the target cell by the immune effector cell. The treating or contacting can be in vitro. The treating or contacting can be ex vivo. The treating or contacting can be in vivo (e.g., the multispecific immune cell engager, a polynucleotide encoding the multispecific immune cell engager, a vector comprising the polynucleotide, or a pharmaceutical composition comprising the multispecific immune cell engager, polynucleotide, or vector can be administered to a subject, such that the contacting and target cell killing occurs in vivo).

[0364] For multispecific immune cell engagers that comprise two or more binding domains that bind to target molecules on target cells, in some embodiments the multispecific immune cell engager is configured such that killing of target cells that comprise any one of the target molecules is induced, for example, either of two, or any of three, four, five, or six target molecules on target cells. The multispecific immune cell engager can be configured such that killing of cells lacking all the target cell target molecules is not induced or substantially not induced.

[0365] The multispecific immune cell engager can be configured such that killing is induced with similar potency for multiple target molecules or for each of the target molecules on target cells. For example, in some embodiments, immune effector cell killing of target cells is induced by binding of the multispecific immune cell engager to either of two target molecules, and the multispecific immune cell engager exhibits EC50 values (e.g., half maximal effective concentration) for each of the target molecules that are within about 2-fold, about 3-fold, about 5- fold, about 10-fold, about 20-fold, or about 50-fold of each other, (e.g., as determined by a targetdependent killing assay, such as an ADCC or TDCC assay).

[0366] In some embodiments, immune effector cell killing of target cells is induced by binding of the multispecific immune cell engager to any of three target molecules, and the multispecific immune cell engager exhibits EC50 values (e.g., half maximal effective concentration) for each of the target molecules that are within about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 20-fold, or about 50-fold of each other, (e.g., as determined by a target-dependent killing assay, such as an ADCC or TDCC assay).

[0367] In some embodiments, immune effector cell killing of target cells is induced by binding of the multispecific immune cell engager to any of four or more target molecules, and the multispecific immune cell engager exhibits EC50 values (e.g., half maximal effective concentration) for each of the target molecules that are within about 2-fold, about 3-fold, about 5- fold, about 10-fold, about 20-fold, or about 50-fold of each other, (e.g., as determined by a targetdependent killing assay, such as an ADCC or TDCC assay).WSGR Docket No. 69097-701.601

[0368] A multispecific immune cell engager can exhibit superior properties over a control compound, such as an immune cell engager configured differently (e.g., with different arrangement of domains, or lacking one or more of the binding domains or binding specificities).

[0369] In some embodiments, a multispecific immune cell engager induces killing of target cells at a rate that is 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, or at least about 5000-fold higher than a control compound (for example, an immune cell engager with the same domains arranged differently, or an immune cell engager that lacks one of the binding domains or binding specificities). The control compound be an otherwise corresponding multispecific immune cell engager that lacks, for example, one of the binding domains or binding specificities for target molecules on target cells (e.g., CD 19, CD20, or CD22). The control compound be an otherwise corresponding multispecific immune cell engager that lacks a binding domain that binds to a target molecule on an immune effector cell, for example, lacks a CD2-binding domain, lacks a domain of CD58 (e.g., lacks the Ig-like domain), or lacks a CD3-binding domain. The increased killing can be reflected by, for example, a lower EC50 than the control compound.

[0370] A multispecific immune cell engager disclosed herein can exhibit or be configured to elicit relatively low killing of cells that lack target cell target molecules. For example, in some embodiments, a multispecific immune cell engager exhibits or is configured to exhibit 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, or at least about 5000-fold lower killing of cells that lack target cell target molecules compared to a control compound (for example, an immune cell engager with the same domains arranged differently, or an immune cell engager that lacks one of the binding domains or binding specificities, or has an additional binding domain or binding specificity).

[0371] The disclosure also provides a method of inducing immune effector cell activity (e.g., immune activation, proliferation, effector function, cytolytic activity, pro-inflammatory cytokine production, survival, persistence, and / or differentiation (e.g., memory and / or effector differentiation)), the method comprising treating or contacting a multispecific immune cell engager disclosed herein to (i) a target cell, and (ii) an immune effector cell, thereby inducing theWSGR Docket No. 69097-701.601 activity of the immune effector cell. The treating or contacting can be in vitro. The treating or contacting can be ex vivo. The treating or contacting can be in vivo.

[0372] For multispecific immune cell engagers that comprise two or more binding domains that bind to target molecules on target cells, in some embodiments the multispecific immune cell engager is configured such that immune effector cell activity is induced in response to cells that comprise any one of the target molecules, for example, either of two, or any of three, four, five, or six target molecules on target cells. The multispecific immune cell engager can be configured such that immune effector cell activity is not induced or substantially not induced by cells lacking all the target cell target molecules.

[0373] The multispecific immune cell engager can be configured such that immune effector cell activity (e.g., immune activation, proliferation, effector function, cytolytic activity, pro- inflammatory cytokine production, survival, persistence, and / or differentiation (e.g., memory and / or effector differentiation)) is induced with similar potency for multiple target molecules or for each of the target molecules on target cells. For example, in some embodiments, immune effector cell activity (e.g., immune activation, proliferation, effector function, cytolytic activity, pro-inflammatory cytokine production, survival, persistence, and / or differentiation (e.g., memory and / or effector differentiation)) is induced by binding of the multispecific immune cell engager to either of two target molecules, and the multispecific immune cell engager exhibits EC50 values (e.g., half maximal effective concentration) for each of the target molecules that are within about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 20-fold, or about 50-fold of each other, (e.g., as determined by a suitable assay).

[0374] In some embodiments, immune effector cell activity (e.g., immune activation, proliferation, effector function, cytolytic activity, pro-inflammatory cytokine production, survival, persistence, and / or differentiation (e.g., memory and / or effector differentiation) is induced by binding of the multispecific immune cell engager to any of three target molecules, and the multispecific immune cell eng...

Claims

WSGR Docket No. 69097-701.601CLAIMSWHAT IS CLAIMED IS:

1. An engineered T Cell Engager (TCE) comprising at least four target binding domains configured to bind to at least four non-identical targets, comprising: a first immunoglobulin constant domain Fc region, Fcl, operably linked to at least two nonidentical target binding domains and a second immunoglobulin constant domain Fc region, Fc2, operably linked to one or more target binding domains, wherein the one or more target binding domains are non-identical to the at least two non-identical target binding domains linked to the Fcl, and wherein one of the one or more target binding domains operably linked to the Fc2 is an anti-CD3 domain.

2. The engineered TCE of claim 1, wherein the Fcl is on a first polypeptide chain, and the Fc2 is on a second polypeptide chain, and wherein the Fcl dimerizes with the Fc2.

3. The engineered TCE of claim 1 or 2, wherein the Fcl and the Fc2 comprise unmodified immunoglobulin constant regions or comprise one or more amino acid mutations.

4. The engineered TCE of any one of claims 1-3, wherein each of the at least four target binding domains comprises an antibody or a domain or a fragment thereof, a Fab, a F(ab')2, an Fv, a single chain variable fragment (scFv), single domain antibody fragment (sdAb), a VHH, a camelid antibody, a nanobody and / or a ligand.

5. The engineered TCE of any one of claims 1-4, wherein the at least four target binding domains comprises five target binding domains, where the at least four target binding domains are selected from (i) an anti-CD19 domain, (ii) an anti-CD20 domain, (iii) an anti-CD22 domain, (iv) a CD2 binding domain; and (v) the anti-CD3 domain.

6. The engineered TCE of any one of claims 1-5, wherein a target binding domain of the at least four target binding domains is a CD2 binding domain, 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.

7. The engineered TCE of any one of claims 1-6, further comprising a linker, a spacer, or a hinge domain between two or more domains of the least four target binding domains, or between the one or more target binding domains and the Fcl or between the one or more target binding domains and the Fc2.

8. The engineered TCE of any one of claims 1-7, wherein the Fcl is operably linked to one of the at least two non-identical target binding domains at the N terminus or at the CWSGR Docket No. 69097-701.601 terminus; and wherein the Fc2 is operably linked to at least one of the one or more target binding domain at the N terminus or at the C terminus.

9. The engineered TCE of any one of claims 1-8 comprises a fifth target binding domain.

10. The engineered TCE of claim 9, wherein the fifth target binding domain is operably linked to the Fcl.

11. The engineered TCE of any one of claims 1-10, wherein, (a) the first polypeptide comprising the Fcl is a dual target engager, wherein the Fcl is operably linked to a first target binding domain at the N terminus via a first linker and the Fcl is operably linked to a second target binding domain at the C terminus via a second linker; or (b) the Fcl is a dual target engager, wherein the Fcl is operably linked to a first target binding domain via a first linker and a second target binding domain in tandem wherein the first target binding domain and the second target binding domain are linked by a second linker.

12. The engineered TCE of claim 11, wherein the Fc2 is operably linked to a third target binding domain via a third linker.

13. The engineered TCE of claim 11 or 12, wherein the Fc2 further comprises a fourth binding domain, wherein the third target binding domain and the fourth target binding domain are operably linked to the Fc2 in tandem and the third target binding domain is linked to the fourth target binding domain via a fourth linker.

14. The engineered TCE of any one of claims 1-13, wherein,(a) the Fcl is a triple target engager, wherein the Fcl is operably linked to a first target binding domain, a second target binding domain and a fifth target binding domain, wherein the Fcl is operably linked to the first binding domain and the fifth target binding domain in tandem, wherein a first linker links the first target binding domain to the Fcl, a second linker links the second target binding domain to the Fcl, and a fifth linker links the first target binding domain and the fifth target binding domain; or(b) the Fcl is a triple target engager, wherein the Fcl is operably linked to a first target binding domain, a second target binding domain and a fifth target binding domain in tandem, wherein a first linker links the first target binding domain to Fcl, a second linker links the first target binding domain to the second target binding domain and a fifth linker links the second target binding domain to the fifth target binding domain.

15. The engineered TCE of any one of claims 5-14, wherein the CD2 binding domain is on the first polypeptide chain or on the second polypeptide chain.

16. The engineered TCE of any one of claims 1-15, wherein the second polypeptide comprises the Fc2 operably linked to a first target binding domain that is the CD58 ligand at the N terminus via a linker.WSGR Docket No. 69097-701.60117. The engineered TCE of any one of claims 1-15, wherein the second polypeptide comprises the Fc2 operably linked to a first target binding domain that is the CD58 ligand at the C terminus via a linker.

18. The engineered TCE of any one of claims 1-15, wherein the first polypeptide comprises the Fcl operably linked to a CD58 ligand at the N terminus via a linker.

19. The engineered TCE of claim 16 or 18, wherein the CD58 ligand is in turn operably linked via a linker to a second, third and / or fourth target binding domains selected from an anti-CD3 domain, an anti-CD19 domain, an anti-CD20 domain and an anti-CD22 domain.

20. The engineered TCE of any one of claims 16-18, wherein the Fcl or the Fc2 is further linked to a target binding domain via a linker at its C terminus, selected from an anti- CD19 domain, an anti-CD20 domain and an anti-CD22 domain.

21. The engineered TCE of any one of claims 1-20, wherein at least one of the at least four target binding domains is an anti-CD19 domain, 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.

22. The engineered TCE of any one of claims 1-21, wherein at least one of the at least four target binding domains is an anti-CD19 domain, the anti-CD19 domain is a single chain variable fragment (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.

23. The engineered TCE of claim 22, 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.

24. The engineered TCE of any one of claims 1-23, wherein at least one of the at least four target binding domains is an anti-CD19 domain, 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.WSGR Docket No. 69097-701.60125. The engineered TCE of any one of claims 1-23, wherein the anti-CD19 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 2.

26. The engineered TCE of any one of claims 1-25, wherein the anti-CD19 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 227. The engineered TCE of any one of claims 1-25, wherein at least one of the at least four target binding domains is an anti-CD20 domain, the anti-CD20 domain is a single domain antibody fragment (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 any one of the sequences SEQ ID NOs: 36-46.

28. The engineered TCE of any one of claims 1-25, wherein at least one of the at least four target binding domains is an anti-CD20 domain, the anti-CD20 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 selected from SEQ ID NOs: 47, 49, 51 and 53 or a sequence having at 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.

29. The engineered TCE of any one of claims 1-26, wherein at least one of the at least four target binding domains is an anti-CD20 domain, 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.

30. The engineered TCE of any one of claims 1-28, wherein at least one of the at least four target binding domains is an anti-CD20 domain, 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)1, a LCDR2, and a LCDR3 wherein the HCDR1, the HCDR2 and the HCDR3 and theWSGR Docket No. 69097-701.601LCDR1, the LCDR2 and the LCDR3 comprise amino acid sequences as listed in Table 4.

31. The engineered TCE of any one of claims 1-25, wherein at least one of the at least four target binding domains is an anti-CD22 domain, the anti-CD22 domain is a single domain antibody fragment (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.

32. The engineered TCE of any one of claims 1-25, wherein the anti-CD22 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 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.

33. The engineered TCE of any one of claims 1-31, 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.

34. The engineered TCE of any one of claims 1-32, 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.

35. The engineered TCE of any one of claims 1-34, 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.

36. The engineered TCE of any one of claims 1-35, 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.

37. The engineered TCE of any one of claims 1-36, wherein the Fcl and the Fc2 comprise a knob-and-hole mutation.

38. The engineered TCE of any one of claims 1-37, 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.WSGR Docket No. 69097-701.60139. The engineered TCE of any one of claims 1-38, wherein the TCE comprises a linker, wherein the linker comprises a sequence selected from the sequences in Table 13.

40. A composition, comprising, a TCE, comprising, (i) a first polypeptide chain and (ii) a second polypeptide chain, wherein the first polypeptide chain comprises a sequence of SEQ ID NO: 1070, or a sequence having at least 85% amino acid sequence identity to SEQ ID NO: 1070; and the second polypeptide chain comprising a sequence selected from SEQ ID NOs: 184 and 186, or a sequence having at least 85% amino acid sequence identity to any of the sequences set forth in SEQ ID NO: 184 or 186; and a suitable solvent or an excipient.

41. A composition comprising any one of the engineered TCEs of claim 1-40, and an acceptable solvent; or a composition comprising a nucleic acid encoding any one of the engineered TCEs of claim 1-40.

42. A pharmaceutical composition comprising:(i) any one of the engineered TCEs of claim 1-39;(ii) a nucleic acid encoding any one of the engineered TCEs of (i);(iii) a cell comprising (i) or (ii);(iv) a composition comprising (i), (ii), or (iii); or(v) a composition of claim 40 or 41; and a pharmaceutically acceptable excipient.

43. The composition of claim 40 or 41 or the pharmaceutical composition of claim 42, further comprising a pH-stabilizing agent; an organic co-solvent, selected from polysorbate 20, polysorbate 80, propylene glycol and polyethylene glycol (PEG), and / or a stabilizing agent selected from sucrose, sorbitol, glycerol, trehalose, or mannitol; and / or a tonicity agent, wherein the tonicity agent is a salt that is NaCl or KC1; and / or a preservative, wherein the preservative is an antimicrobial agent.

44. Use of the engineered TCE of any one of claims 1-39, the composition of claim 40, 41, or 43, or the pharmaceutical composition of claim 42 in the manufacture of a medicament for treating a subject with cancer or an autoimmune disease.

45. A method of making the engineered TCE of any one of the claims 1-39, comprising:(i) identifying the one or more target binding domains;(ii) cloning a polynucleic acid sequence encoding a recombinant polypeptide comprising the first immunoglobulin constant domain Fc region and the one or more target binding domains;WSGR Docket No. 69097-701.601(iii) cloning a polynucleic acid sequence encoding a recombinant polypeptide comprising the second immunoglobulin constant domain Fc region and the one or more target binding domains; and(iv) expressing the cloned polynucleic acid sequences in an immune cell.

46. The method of claim 45, wherein the method further comprises confirming that the TCE is present in a solution having physiological pH in absence of substantial aggregation.

47. A method of treating a disease or a condition in a subject in need thereof, comprising administering to the subject a composition comprising any one of the engineered TCEs of claims 1-46; or a nucleic acid encoding the same.

48. The method of treating in accordance to claim 47, wherein the administering is via intravenous, intramuscular, or subcutaneous injection.

49. The method of treating in accordance to claim 47 or 48, wherein the disease or condition is a cancer.

50. The method of treating in accordance to claim 47 or 48, wherein the disease or condition is a B cell cancer.

51. The method of treating in accordance to claim 47 or 48, wherein the disease or condition is an autoimmune disease.

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