Multifunctional molecules that bind to TCRs and uses thereof

Abstract

Provided herein are multifunctional polypeptide molecules comprising a T cell receptor variable alpha binding portion and a cytokine and methods of using same to treat a condition or disease in a subject.

Description

[Technical field]

[0001] Related Applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 292,859, filed December 22, 2021, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Currently available molecules designed to redirect T cells to promote tumor cell lysis for cancer immunotherapy typically target the CD3 epsilon (CD3e) subunit of the T cell receptor (TCR). However, this approach has limitations. Previous studies have shown that, for example, low doses of anti-CD3e monoclonal antibodies (mAbs) can cause T cell dysfunction and produce immunosuppressive effects. In addition, anti-CD3e mAbs bind to all T cells and thus activate large numbers of T cells. Such non-physiological massive activation of T cells by these anti-CD3e mAbs can result in the production of pro-inflammatory cytokines, e.g., IFN-gamma, IL-1-beta, IL-6, IL-10, and TNF-alpha, causing a "cytokine storm," also known as cytokine release syndrome (CRS) and associated with neurotoxicity (NT). Thus, there is a need for improved T cell receptor binding molecules to redirect T cells for cancer immunotherapy. Summary of the Invention [Means for solving the problem]

[0003]

[0003] Provided herein is a composition comprising a multispecific molecule comprising a T cell receptor alpha variable region (TCRαV) binding moiety linked to a targeting moiety, wherein the TCRαV binding moiety binds to TCRαV of a T cell receptor (TCR) expressed by T cells of a T cell population, and the targeting moiety binds to a target molecule other than TCRαV on a target cell, and when contacted with the T cell population, the multispecific molecule redirects the T cells to the target cells, activates the T cells, expands the T cells, or a combination thereof.

[0004]

[0004] Also provided herein is a composition comprising a recombinant T cell receptor or chimeric antigen receptor (CAR) that comprises a domain that binds to TCRαV.

[0005]

[0005] Also provided herein are compositions comprising T cells genetically modified to express the CAR described herein.

[0006]

[0006] Also provided herein is a pharmaceutical composition comprising a composition described herein and a pharma- ceutically acceptable carrier, excipient, or stabilizer.

[0007]

[0007] Also provided herein is a method for treating cancer in a subject in need of such treatment, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein.

[0008] Also provided herein is a method of expanding T cell receptor beta variable region (TCRαV) expressing T cells in a T cell population, comprising contacting the T cell population with a composition comprising an anti-TCRαV molecule, wherein the anti-TCRαV molecule contacts the TCRαV of the T cell receptor (TCR) expressed by the T cells in the T cell population, thereby expanding the T cells in the T cell population. In some embodiments, the T cell population is an in vivo T cell population.

[0009]

[0009] Also provided herein is a method of expanding T cells expressing a T cell receptor beta variable region (TCRαV) in a T cell population, comprising contacting the T cell population with a composition comprising a multispecific molecule, the multispecific molecule comprising a first domain that binds to a first target molecule and a second domain that binds to a second target molecule, the first target molecule being TCRαV, the second target molecule being a target molecule on a target cell different from the first target molecule, and the first domain contacting TCRαV of a T cell receptor (TCR) expressed by T cells in the T cell population, thereby expanding the T cells in the T cell population. In some embodiments, the T cell population is an in vivo T cell population.

[0010]

[0010] A multifunctional polypeptide molecule comprising a first polypeptide, a second polypeptide, and at least one cytokine polypeptide or variant thereof, wherein the first polypeptide and the second polypeptide are non-contiguous, and (i) the first polypeptide comprises (A) a first TCRαV binding portion comprising a first heavy chain variable domain (VH) and a first light chain variable domain (VL), or a single domain antibody, or (B) a first portion of the first TCRαV binding portion comprising the VH of the first TCRαV binding portion, and the first polypeptide comprises the first portion of the first TCRαV binding portion, the multifunctional polypeptide molecule further comprises a third polypeptide comprising a second portion of the first TCRαV binding portion comprising the VL of the first TCRαV binding portion, and the third polypeptide comprises the first polypeptide and the second Also provided herein are multifunctional polypeptide molecules comprising: (a) a first portion of a dimerization module linked to a first portion of a first TCRαV binding portion that is non-contiguous with the polypeptide; and (ii) a second polypeptide comprising a second portion of the dimerization module; (a) the multifunctional polypeptide molecule comprises a single TCRαV binding portion and at least one cytokine polypeptide or variant thereof is covalently linked to the second polypeptide, or (b) the multifunctional polypeptide molecule further comprises a second TCRαV binding portion and at least one cytokine polypeptide or variant thereof is covalently linked to the first polypeptide, the second polypeptide, or, if the multifunctional polypeptide molecule further comprises a third polypeptide, the third polypeptide, or a combination thereof.

[0011]

[0011] In some embodiments, the multifunctional polypeptide molecule comprises a second TCRαV binding portion, and the second portion of the dimerization module is (A) a second TCRαV binding portion comprising a second VH and a second VL, or a single domain antibody, or (B) a first portion of the second TCRαV binding portion comprising the VH of the second TCRαV binding portion, wherein when the second polypeptide comprises the first portion of the second TCRαV binding portion, the multifunctional polypeptide molecule comprises a second TCRαV binding portion comprising the VL of the second TCRαV binding portion. and a fourth polypeptide comprising a second portion of the TCRαV binding moiety, wherein the fourth polypeptide is linked to the first portion of the second TCRαV binding moiety that is non-contiguous with the first polypeptide, the second polypeptide, and the third polypeptide; and at least one cytokine polypeptide or variant thereof is covalently linked to the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide if the multifunctional polypeptide molecule further comprises a fourth polypeptide, or a combination thereof.

[0012]

[0012] In some embodiments, the first portion of the dimerization module and the second portion of the dimerization module are dimerized.

[0013]

[0013] In some embodiments, the first polypeptide comprises: (A) a first TCRαV binding portion comprising a first VH and a first VL, the first TCRαV binding portion further comprising a first heavy chain constant domain 1 (CH1) linked to the first VH; or (B) a first portion of the first TCRαV binding portion comprising the VH of the first TCRαV binding portion, the first portion of the first TCRαV binding portion further comprising a first CH1 linked to the VH of the first TCRαV binding portion.

[0014]

[0014] In some embodiments, the first CH1 is linked to the C-terminus of the first VH or the C-terminus of the VH of the first TCRαV-binding portion.

[0015]

[0015] In some embodiments, the second polypeptide comprises: (A) a second TCRαV binding portion comprising a second VH and a second VL, the second TCRαV binding portion further comprising a second CH1 linked to the second VH; or (B) a first portion of the second TCRαV binding portion comprising the VH of the second TCRαV binding portion, the second TCRαV binding portion further comprising a second CH1 linked to the VH of the second TCRαV binding portion.

[0016]

[0016] In some embodiments, the second CH1 is linked to the C-terminus of the second VH or the C-terminus of the VH of the second TCRαV-binding portion.

[0017]

[0017] In some embodiments, the multifunctional polypeptide molecule comprises: (1) a first polypeptide comprising a first TCRαV binding portion comprising a first VH and a first VL, wherein the first TCRαV binding portion further comprises a first light chain constant domain (CL) linked to the first VL; or (2) a third polypeptide comprising a first polypeptide comprising a first portion of the first TCRαV binding portion and a second portion of the first TCRαV binding portion further comprising a first CL linked to the VL of the first TCRαV binding portion.

[0018]

[0018] In some embodiments, the first CL is linked to the C-terminus of the first VL or the C-terminus of the VL of the first TCRαV binding portion.

[0019]

[0019] In some embodiments, the multifunctional polypeptide molecule comprises: (1) a second polypeptide comprising a second TCRαV binding portion comprising a second VH and a second VL, wherein the second TCRαV binding portion further comprises a second CL linked to the second VL; or (2) a fourth polypeptide comprising a second polypeptide comprising a first portion of the second TCRαV binding portion and a second portion of the second TCRαV binding portion further comprising a second CL linked to the VL of the second TCRαV binding portion.

[0020]

[0020] In some embodiments, the second CL is linked to the C-terminus of the second VL or the C-terminus of the VL of the second TCRαV binding portion.

[0021]

[0021] In some embodiments, the first portion of the dimerization module is linked to (A) the C-terminus of a first TCRαV binding portion comprising a first VH and a first VL or a single domain antibody, or (B) the C-terminus of a first portion of the first TCRαV binding portion comprising the VH of the first TCRαV binding portion.

[0022]

[0022] In some embodiments, the multifunctional polypeptide molecule comprises a second TCRαV binding portion, and the second portion of the dimerization module is linked to (A) the C-terminus of the second TCRαV binding portion comprising a second VH and a second VL or a single domain antibody, or (B) the C-terminus of the first portion of the second TCRαV binding portion comprising the VH of the second TCRαV binding portion.

[0023]

[0023] In some embodiments, the multifunctional polypeptide molecule comprises a single TCRαV binding portion, and at least one cytokine polypeptide or a variant thereof is covalently linked to the N-terminus of a second polypeptide, the C-terminus of a second polypeptide, or a combination thereof.

[0024]

[0024] In some embodiments, at least one cytokine polypeptide or variant thereof is present in a single contiguous polypeptide chain of the second polypeptide.

[0025]

[0025] In some embodiments, (a) the N-terminus of the first polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the first polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (b) the N-terminus of the second polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the second polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (c) the N-terminus of the third polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the third polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (d) the N-terminus of the fourth polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the fourth polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; or (e) a combination thereof.

[0026]

[0026] In some embodiments, (a-1) the N-terminus of the first polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the first polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (a-2) the N-terminus of the second polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the second polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (b-1) the N-terminus of the first polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the first polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (b-2) the N-terminus of the third polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the third polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (c-1) the N-terminus of the first polypeptide is linked to a cytokine polypeptide or a variant thereof; is linked to a cytokine polypeptide or variant thereof; the C-terminus of the first polypeptide is linked to a cytokine polypeptide or variant thereof; or a combination thereof; (c-2) the N-terminus of the fourth polypeptide is linked to a cytokine polypeptide or variant thereof; the C-terminus of the fourth polypeptide is linked to a cytokine polypeptide or variant thereof; or a combination thereof; (d-1) the N-terminus of the second polypeptide is linked to a cytokine polypeptide or variant thereof; the C-terminus of the second polypeptide is linked to a cytokine polypeptide or variant thereof; or a combination thereof; (d-2) the N-terminus of the third polypeptide is linked to a cytokine polypeptide or variant thereof; the C-terminus of the third polypeptide is linked to a cytokine polypeptide or variant thereof; or a combination thereof; (e-1) the N-terminus of the second polypeptide is linked to a cytokine polypeptide or variant thereof;(e-2) the N-terminus of the fourth polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the fourth polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; or (f-1) the N-terminus of the third polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the third polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (f-2) the N-terminus of the fourth polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the fourth polypeptide is linked to a cytokine polypeptide or a variant thereof;

[0027]

[0027] In some embodiments, (a-1) the N-terminus of the first polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the first polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (a-2) the N-terminus of the second polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the second polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (a-3) the N-terminus of the third polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the third polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (b-1) the N-terminus of the first polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the first polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (b-2) the N-terminus of the second polypeptide is (b-3) the N-terminus of the fourth polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the fourth polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; or (c-1) the N-terminus of the second polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the second polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (c-2) the N-terminus of the third polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the third polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (c-3) the N-terminus of the fourth polypeptide is linked to a cytokine polypeptide or a variant thereof;The C-terminus of the fourth polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof;

[0028]

[0028] In some embodiments, the N-terminus of the first polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the first polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; the N-terminus of the second polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the second polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; the N-terminus of the third polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the third polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; the N-terminus of the fourth polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the fourth polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof.

[0029]

[0029] In some embodiments, the cytokine polypeptide or variant thereof is present within a single contiguous polypeptide chain of the first polypeptide, the second polypeptide, the third cytokine polypeptide, or the fourth cytokine polypeptide to which it is linked.

[0030]

[0030] In some embodiments, the multifunctional polypeptide molecule comprises: (i) a linker between a first portion of the dimerization module and a first TCRαV binding portion comprising a first VH and a first VL or a single domain antibody, or a first portion of the first TCRαV binding portion comprising the VH of the first TCRαV binding portion; (ii) a linker between a second portion of the dimerization module and a second TCRαV binding portion comprising a second VH and a second VL or a single domain antibody, or a first portion of the second TCRαV binding portion comprising the VH of the second TCRαV binding portion; (iii) a linker between the first VH and the first VL; (iv) a linker between the second VH and the second VL; (v) a linker between the first CH1 and the first VH, or the VH of the first TCRαV binding portion; (vi) a linker between the second CH1 and the second (vii) a linker between the first CL and the first VL or VL of the first TCRαV binding moiety; (vii) a linker between the second CL and the second VL or VL of the second TCRαV binding moiety; (viii) a linker between at least one cytokine polypeptide or variant thereof and the first polypeptide, a linker between at least one cytokine polypeptide or variant thereof and a second polypeptide, a linker between at least one cytokine polypeptide or variant thereof and a third polypeptide, a linker between at least one cytokine polypeptide or variant thereof and a fourth polypeptide, or a combination thereof; or (ix) a combination thereof.

[0031]

[0031] In some embodiments, the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker.

[0032]

[0032] In some embodiments, the linker is a peptide linker and comprises the sequence of SEQ ID NO:3308 or SEQ ID NO:3643.

[0033]

[0033] In some embodiments, the multifunctional polypeptide molecule is an isolated multifunctional polypeptide molecule.

[0034]

[0034] In some embodiments, the multifunctional polypeptide molecule comprises (i) a first polypeptide comprising a first portion of a dimerization module linked to the C-terminus of a first portion of a first TCRαV binding portion; (ii) a second polypeptide comprising a second portion of the dimerization module; (iii) a third polypeptide comprising the second portion of the first TCRαV binding portion; and (iv) a cytokine polypeptide or a variant thereof covalently linked to the N-terminus of the second polypeptide, and comprises a single TCRαV binding portion.

[0035]

[0035] In some embodiments, the multifunctional polypeptide molecule comprises (i) a first polypeptide comprising a first portion of a dimerization module linked to the C-terminus of the first portion of a first TCRαV binding portion; (ii) a second polypeptide comprising a second portion of a dimerization module linked to the C-terminus of the first portion of a second TCRαV binding portion; (iii) a third polypeptide comprising the second portion of the first TCRαV binding portion; (iv) a fourth polypeptide comprising the second portion of the second TCRαV binding portion; (v) a cytokine polypeptide or a variant thereof covalently linked to the C-terminus of the third polypeptide, and (vi) a cytokine polypeptide or a variant thereof covalently linked to the C-terminus of the fourth polypeptide.

[0036]

[0036] In some embodiments, the multifunctional polypeptide molecule comprises: (i) a first polypeptide comprising a first portion of a dimerization module linked to the C-terminus of the first portion of a first TCRαV binding portion; (ii) a second polypeptide comprising a second portion of a dimerization module linked to the C-terminus of the first portion of a second TCRαV binding portion; (iii) a third polypeptide comprising the second portion of the first TCRαV binding portion; (iv) a fourth polypeptide comprising the second portion of the second TCRαV binding portion; and (v) a cytokine polypeptide or a variant thereof covalently linked to the C-terminus of the third polypeptide or the C-terminus of the fourth polypeptide, but not to both.

[0037]

[0037] In some embodiments, the multifunctional polypeptide molecule comprises: (i) a first polypeptide comprising a first portion of a dimerization module linked to the C-terminus of the first portion of a first TCRαV binding portion; (ii) a second polypeptide comprising a second portion of a dimerization module linked to the C-terminus of the first portion of a second TCRαV binding portion; (iii) a third polypeptide comprising the second portion of the first TCRαV binding portion; (iv) a fourth polypeptide comprising the second portion of the second TCRαV binding portion; and (v)) a cytokine polypeptide or a variant thereof covalently linked to the C-terminus of the first polypeptide or the C-terminus of the second polypeptide (but not to both).

[0038]

[0038] In some embodiments, the first TCRαV binding portion, the second TCRαV binding portion, or a combination thereof, comprises any one selected from the group consisting of Fab, F(ab')2, Fv, single chain Fv (scFv), single domain antibody, diabody (dAb), camelid antibody, and combinations thereof.

[0039]

[0039] In some embodiments, the first TCRαV binding portion, the second TCRαV binding portion, or a combination thereof comprises a Fab or scFv.

[0040]

[0040] In some embodiments, the TCRαV binding portion is the only antigen-binding portion of the multifunctional polypeptide molecule.

[0041]

[0041] In some embodiments, the multifunctional polypeptide molecule comprises two or more of at least one cytokine polypeptide.

[0042]

[0042] In some embodiments, the at least one cytokine polypeptide comprises interleukin-2 (IL-2) or a fragment thereof.

[0043]

[0043] In some embodiments, at least one cytokine polypeptide comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO:2191.

[0044]

[0044] In some embodiments, the variant is an IL-2 variant that includes a substitution mutation.

[0045]

[0045] In some embodiments, the variant is an IL-2 variant that includes a C125A mutation.

[0046]

[0046] In some embodiments, the variant comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO:2270.

[0047]

[0047] In some embodiments, a first portion of the dimerization module includes a first immunoglobulin constant region (Fc region) and a second portion of the dimerization module includes a second Fc region.

[0048]

[0048] In some embodiments, the first Fc region, the second Fc region, or a combination thereof is selected from the group consisting of an IgG1 Fc region or fragment thereof, an IgG2 Fc region or fragment thereof, an IgG3 Fc region or fragment thereof, an IgGA1 Fc region or fragment thereof, an IgGA2 Fc region or fragment thereof, an IgGA4 Fc region or fragment thereof, an IgJ Fc region or fragment thereof, an IgM Fc region or fragment thereof, an IgD Fc region or fragment thereof, and an IgE Fc region or fragment thereof.

[0049]

[0049] In some embodiments, the first Fc region, the second Fc region, or a combination thereof is selected from the group consisting of a human IgG1 Fc region or fragment thereof, a human IgG2 Fc region or fragment thereof, and a human IgG4 Fc region or fragment thereof.

[0050]

[0050] In some embodiments, the first Fc region, the second Fc region, or a combination thereof, comprises an Fc interface having one or more of paired holes and protrusions, electrostatic interactions, or strand exchange, and dimerization of the first Fc region and the second Fc region is enhanced as indicated by a higher ratio of heteromultimer:homomultimer forms compared to dimerization of an Fc region having an unengineered interface.

[0051]

[0051] In some embodiments, the first Fc region, the second Fc region, or a combination thereof, comprises an amino acid substitution listed in Table 14.

[0052]

[0052] In some embodiments, the first Fc region, the second Fc region, or a combination thereof comprises an Asn297Ala (N297A) mutation or a Leu234Ala / Leu235Ala (LALA) mutation.

[0053]

[0053] In some embodiments, the first Fc region, the second Fc region, or a combination thereof comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:3645, SEQ ID NO:3646, SEQ ID NO:3647, SEQ ID NO:3648, or SEQ ID NO:3649.

[0054] In some embodiments, the first TCRαV binding portion, the second TCRαV binding portion, or a combination thereof, is selected from the group consisting of TCRα V1 subfamily, TCRα V2 subfamily, TCRα V3 subfamily, TCRα V4, TCRα V5 subfamily, TCRα V6 subfamily, TCRα V7 subfamily, TCRα V8 subfamily, TCRα V9 subfamily, TCRα V10 subfamily, TCRα V12 subfamily, TCRα V13 subfamily, TCRα V14 subfamily, TCRα V16 subfamily, TCRα V17 subfamily, TCRα V18 subfamily, TCRα V19 subfamily, TCRα V20 subfamily, TCRα V21 subfamily, TCRα V22 subfamily, TCRα V23 subfamily, TCRα V24 subfamily, TCRα V25 subfamily, TCRα V26 subfamily, TCRα It binds to one or more of the TCRαV subfamilies selected from the group consisting of the V27 subfamily, the TCRα V29 subfamily, the TCRα V30 subfamily, the TCRα V34 subfamily, the TCRα V35 subfamily, the TCRα V36 subfamily, the TCRα V38 subfamily, the TCRα V39 subfamily, the TCRα V40 subfamily, the TCRα V41 subfamily, family members of said subfamilies, and variants thereof (e.g., structural or functional variants thereof).

[0055]

[0055] In some embodiments, the first TCRαV binding portion, the second TCRαV binding portion, or a combination thereof binds to one or more of the TCRαV subfamilies selected from the group consisting of TCRαV1-1, TCRαV1-2, and variants thereof.

[0056]

[0056] In some embodiments, the first TCRαV binding portion, the second TCRαV binding portion, or a combination thereof binds to one or more of the TCRαV subfamilies selected from the group consisting of TCRαV8-1, TCRαV8-2, TCRαV8-3, TCRαV8-4, TCRαV8-6, and variants thereof.

[0057]

[0057] In some embodiments, the first TCRαV binding portion, the second TCRαV binding portion, or a combination thereof binds to one or more of the TCRαV subfamilies selected from the group consisting of TCRαV9-1, TCRαV9-2, and variants thereof.

[0058]

[0058] In some embodiments, the first TCRαV binding portion, the second TCRαV binding portion, or a combination thereof binds to one or more of the TCRαV subfamilies selected from the group consisting of TCRαV12-1, TCRαV12-2, TCRαV12-3, and variants thereof.

[0059]

[0059] In some embodiments, the first TCRαV binding portion, the second TCRαV binding portion, or a combination thereof binds to one or more of the TCRαV subfamilies selected from the group consisting of TCRαV13-1, TCRαV13-2, and variants thereof.

[0060]

[0060] In some embodiments, the first TCRαV binding portion, the second TCRαV binding portion, or a combination thereof binds to one or more TCRαV subfamilies selected from the group consisting of TCRαV14 / DV4, and variants thereof.

[0061]

[0061] In some embodiments, the first TCRαV binding portion, the second TCRαV binding portion, or a combination thereof binds to one or more TCRαV subfamilies selected from the group consisting of TCRαV23 / DV6, and variants thereof.

[0062]

[0062] In some embodiments, the first TCRαV binding portion, the second TCRαV binding portion, or a combination thereof binds to one or more of the TCRαV subfamilies selected from the group consisting of TCRαV26-1, TCRαV26-2, and variants thereof.

[0063]

[0063] In some embodiments, the first TCRαV binding portion, the second TCRαV binding portion, or a combination thereof binds to one or more TCRαV subfamilies selected from the group consisting of TCRαV29 / DV5, and variants thereof.

[0064]

[0064] In some embodiments, the first TCRαV binding portion, the second TCRαV binding portion, or a combination thereof binds to one or more TCRαV subfamilies selected from the group consisting of TCRαV236 / DV7, and variants thereof.

[0065]

[0065] In some embodiments, the first TCRαV binding portion, the second TCRαV binding portion, or a combination thereof binds to one or more TCRαV subfamilies selected from the group consisting of TCRαV38-1, TCRαV38-2 / DV8, and variants thereof.

[0066]

[0066] In some embodiments, the multifunctional polypeptide molecule comprises a first TCRαV binding portion and a second TCRαV binding portion, and the first TCRαV binding portion and the second TCRαV binding portion are the same.

[0067]

[0067] In some embodiments, the multifunctional polypeptide molecule comprises a first TCRαV binding portion and a second TCRαV binding portion, and the first TCRαV binding portion and the second TCRαV binding portion are different.

[0068]

[0068] In some embodiments, the first TCRαV binding portion and the second TCRαV binding portion each bind to (i) one or more TCRα V12 subfamily members and one or more different TCRα subfamily members.

[0069]

[0069] In some embodiments, the first TCRαV binding portion, the second TCRαV binding portion, or a combination thereof, comprises: (i) a VH comprising framework regions (FRs) including FR1, FR2, FR3, and FR4 that have at least 75% sequence identity to non-mouse germline framework region 1 (FR1), non-mouse germline framework region 2 (FR2), non-mouse germline framework region 3 (FR3), and non-mouse germline framework region 4 (FR4); (ii) a VL comprising FRs including FR1, FR2, FR3, and FR4 that have at least 75% sequence identity to non-mouse germline FR1, non-mouse germline FR2, non-mouse germline FR3, and non-mouse germline FR4; or (iii) a combination thereof.

[0070]

[0070] In some embodiments, the first polypeptide, the second polypeptide, or a combination thereof comprises an IgM heavy chain constant region or a fragment thereof.

[0071]

[0071] In some embodiments, the IgM heavy chain constant region comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO:73.

[0072]

[0072] In some embodiments, the first polypeptide, the second polypeptide, or a combination thereof comprises an IgJ heavy chain constant region or a fragment thereof.

[0073]

[0073] In some embodiments, the heavy chain constant region of IgJ comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO:76.

[0074]

[0074] In some embodiments, the first polypeptide, the second polypeptide, or a combination thereof comprises a heavy chain constant region of IgGA1 or a fragment thereof.

[0075]

[0075] In some embodiments, the heavy chain constant region of IgGA1 comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO:74.

[0076]

[0076] In some embodiments, the first polypeptide, the second polypeptide, or a combination thereof comprises a heavy chain constant region of IgGA2 or a fragment thereof.

[0077]

[0077] In some embodiments, the heavy chain constant region of IgGA2 comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO:75.

[0078]

[0078] In some embodiments, the first polypeptide, the second polypeptide, or a combination thereof comprises an IgG1 heavy chain constant region or a fragment thereof.

[0079]

[0079] In some embodiments, the IgG1 heavy chain constant region comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO:41 or SEQ ID NO:3645.

[0080]

[0080] In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a light chain constant region of a kappa chain, or a fragment thereof.

[0081]

[0081] In some embodiments, the light chain constant region of the kappa chain comprises a light chain constant region sequence listed in Table 1.

[0082]

[0082] In some embodiments, the light chain constant region of the kappa chain comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO:39 or SEQ ID NO:3644.

[0083]

[0083] In some embodiments, the first TCRαV binding moiety, the second TCRαV binding moiety, or a combination thereof binds to an outward-facing region on the TCRαV protein.

[0084]

[0084] In some embodiments, the outward-facing region on the TCRαV protein comprises a structurally conserved region of TCRαV that has a similar structure across one or more TCRαV subfamilies.

[0085]

[0085] In some embodiments, the third sequence is linked to the N-terminus of the first sequence.

[0086]

[0086] In some embodiments, the third sequence is linked to the C-terminus of the second sequence.

[0087]

[0087] In some embodiments, the third sequence is linked to the N-terminus of the first sequence.

[0088]

[0088] In some embodiments, the third sequence is linked to the C-terminus of the second sequence.

[0089]

[0089] In some embodiments, the third polypeptide, the fourth polypeptide, or a combination thereof further comprises a third sequence, and the third sequence is linked to a fourth sequence, a fifth sequence, or a combination thereof.

[0090]

[0090] In some embodiments, the third sequence is linked to the N-terminus of the fourth sequence.

[0091]

[0091] In some embodiments, the third sequence is linked to the C-terminus of the fifth sequence.

[0092]

[0092] In some embodiments, the third sequence is linked to the N-terminus of the first sequence.

[0093]

[0093] In some embodiments, the third sequence is linked to the C-terminus of the second sequence.

[0094]

[0094] In some embodiments, the third polypeptide, the fourth polypeptide, or a combination thereof further comprises a third sequence, wherein the third sequence is linked to a fourth sequence, a fifth sequence, or a combination thereof.

[0095]

[0095] In some embodiments, the third sequence is linked to the N-terminus of the fourth sequence.

[0096]

[0096] In some embodiments, the third sequence is linked to the C-terminus of the fifth sequence.

[0097]

[0097] In certain embodiments, a nucleic acid molecule comprising a nucleotide sequence encoding a multifunctional polypeptide molecule or a CAR described herein is described herein.

[0098]

[0098] In some embodiments, the nucleic acid molecule is an isolated nucleic acid molecule.

[0099]

[0099] Described herein, in certain embodiments, are vectors that include one or more of the nucleic acid molecules described herein.

[0100]

[0100] In certain embodiments, a cell comprising a nucleic acid molecule described herein, or a vector described herein, is described herein.

[0101]

[0101] In certain embodiments, described herein is a pharmaceutical composition comprising a T cell genetically modified to express a multifunctional polypeptide molecule or a CAR described herein, a nucleic acid molecule described herein, a vector described herein, or a cell described herein, and a pharma- ceutically acceptable carrier, excipient, or diluent.

[0102]

[0102] In certain embodiments, described herein are methods of treating a condition or disease in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of a multifunctional polypeptide molecule described herein, a CAR described herein, a nucleic acid molecule described herein, a vector described herein, a cell described herein (e.g., a T cell genetically modified to express a CAR), a pharmaceutical composition described herein, or a combination thereof, wherein the administering step is effective to treat the condition or disease in the subject.

[0103]

[0103] In some aspects, the condition or disease is cancer.

[0104]

[0104] In some embodiments, the cancer is a solid tumor, a hematological cancer, a metastatic cancer, a soft tissue tumor, or a combination thereof.

[0105]

[0105] In some embodiments, the cancer is a solid tumor, and the solid tumor is selected from the group consisting of melanoma, pancreatic cancer, breast cancer, colorectal cancer, lung cancer, skin cancer, ovarian cancer, liver cancer, and combinations thereof.

[0106]

[0106] In some embodiments, the cancer is a blood cancer, and the blood cancer is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia (AML), chronic myeloid leukemia, myelodysplastic syndrome, multiple myeloma, T-cell lymphoma, acute lymphocytic leukemia, and combinations thereof.

[0107]

[0107] In some embodiments, the non-Hodgkin's lymphoma is selected from the group consisting of B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (B-CLL), mantle cell lymphoma, marginal zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, and combinations thereof.

[0108]

[0108] In some embodiments, the T cell lymphoma is a peripheral T cell lymphoma.

[0109]

[0109] In some embodiments, the cancer is characterized by a cancer antigen present on the cancer.

[0110]

[0110] In some embodiments, the cancer antigen is a tumor antigen, a stromal antigen, or a blood antigen.

[0111] In some embodiments, the cancer antigen is BCMA, CD19, CD20, CD22, FcRH5, PDL1, CD47, gangloside 2 (GD2), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PMSA), prostate specific antigen (PSA), carcinoembryonic antigen (CEA), Ron kinase, c-Met, immature laminin receptor, TAG-72, BING-4, calcium activated chloride channel 2, Sarcopenia 1, sarcoidosis 1, sarcoidosis 2, sarcoidosis 3, sarcoidosis 4, sarcoidosis 5, sarcoidosis 6, sarcoidosis 7, sarcoidosis 8, sarcoidosis 9, sarcoidosis 10, sarcoidosis 11, sarcoidosis 12, sarcoidosis 13, sarcoidosis 14, sarcoidosis 15, sarcoidosis 16, sarcoidosis 17, sarcoidosis 18, sarcoidosis 19, sarcoidosis 20, sarcoidosis 21, sarcoidosis 22, sarcoidosis 23, sarcoidosis 24, sarcoidosis 25, sarcoidosis 26, sarcoidosis 27, sarcoidosis 28, sarcoidosis 29, sarcoidosis 30, sarcoidosis 31, sarcoidosis 32, sarcoidosis 33, sarcoidosis 34, sarcoidosis 35, sarcoidosis 36, sarcoidosis 37, sarcoidosis 38, sarcoidosis 39, sarcoid Icrin-B1, 9D7, Ep-CAM, EphA3, Her2 / neu, telomerase, SAP-1, survivin, NY-ESO-1 / LAGE-1, PRAME, SSX-2, Melan-A / MART-1, Gp100 / pmel17, tyrosinase, TRP-1 / -2, MC1R, β-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, p53, Ras, TGF-β receptor, AFP, ETA, MAGE, MUC-1, CA-125, BAGE, GAGE, NY-ESO-1, β-catenin, CDK4, CDC27, α-actinin-4, TRP1 / gp75, TRP2, gp100, Melan-A / MART1, ganglioside, WT1, EphA3, epidermal growth factor receptor (EGFR), MART-2, MART-1, MUC1, MUC2, MUM1, MUM2, MUM3, NA88-1, NPM , OA1, OGT, RCC, RUI1, RUI2, SAGE, TRG, TRP1, TSTA, folate receptor alpha, L1-CAM, CAIX, gpA33, GD3, GM2, VEGFR, integrins, carbohydrates, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, TGF-beta, hyaluronic acid, collagen, tenascin C, and tenascin W.

[0112] In some embodiments, the method further includes administering a second therapeutic agent or treatment to the subject.

[0113] In some embodiments, the second therapeutic agent or treatment comprises a chemotherapeutic agent, a biological agent, a hormonal therapy, radiation, or surgery.

[0114]

[0114] In some embodiments, the second therapeutic agent or treatment is administered in combination, sequentially, simultaneously, or in parallel with the multifunctional polypeptide molecule described herein, the CAR described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, or the pharmaceutical composition described herein.

[0115]

[0115] Also provided herein are compositions comprising a recombinant T cell receptor or chimeric antigen receptor (CAR) that comprises a domain that binds to TCRαV.

[0116]

[0116] Also provided herein are compositions comprising T cells comprising a recombinant T cell receptor or chimeric antigen receptor (CAR) that includes a domain that binds to TCRαV.

[0117]

[0117] In some embodiments, the CAR comprises an extracellular domain comprising a domain that binds to TCRαV; a transmembrane domain; and an intracellular domain comprising an intracellular signaling domain.

[0118] In some embodiments, the extracellular domain comprises a CD8 or CD28 extracellular domain.

[0119] In some embodiments, the transmembrane domain comprises a CD8 or CD28 transmembrane domain.

[0120]

[0120] In some embodiments, the intracellular domain comprises a CD3 zeta intracellular signaling domain.

[0121] In some embodiments, the domain that binds to TCRαV is selected from the group consisting of TCRα V1 subfamily, TCRα V2 subfamily, TCRα V3 subfamily, TCRα V4, TCRα V5 subfamily, TCRα V6 subfamily, TCRα V7 subfamily, TCRα V8 subfamily, TCRα V9 subfamily, TCRα V10 subfamily, TCRα V12 subfamily, TCRα V13 subfamily, TCRα V14 subfamily, TCRα V16 subfamily, TCRα V17 subfamily, TCRα V18 subfamily, TCRα V19 subfamily, TCRα V20 subfamily, TCRα V21 subfamily, TCRα V22 subfamily, TCRα V23 subfamily, TCRα V24 subfamily, TCRα V25 subfamily, TCRα V26 subfamily, TCRα V27 subfamily, TCRα V29 subfamily, TCRα It binds to one or more of the TCRαV subfamilies selected from the group consisting of the V30 subfamily, the TCRα V34 subfamily, the TCRα V35 subfamily, the TCRα V36 subfamily, the TCRα V38 subfamily, the TCRα V39 subfamily, the TCRα V40 subfamily, or the TCRα V41 subfamily, and family members of said subfamilies, and variants thereof.

[0122] Also provided herein is a pharmaceutical composition comprising a CAR or CAR-T cell composition described herein and a pharma- ceutically acceptable diluent, carrier, excipient, or stabilizer.Also provided herein is a method of treating a disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a CAR or CAR-T cell composition described herein.

[0123]

[0123] In some embodiments, the disease or condition is cancer. In some embodiments, the cancer is breast cancer and the domain that binds to the TCRαV binds to the TCRα V1 subfamily; the cancer is ER+ breast cancer and the domain that binds to the TCRαV binds to the TCRα V1 subfamily; the cancer is ER+ breast cancer and the domain that binds to the TCRαV binds to the TCRα V3 subfamily; the cancer is diffuse large B cell lymphoma and the domain that binds to the TCRαV binds to the TCRα V6 subfamily; the cancer is diffuse large B cell lymphoma and the domain that binds to the TCRαV binds to the TCRα V8 subfamily; the cancer is breast cancer and the domain that binds to the TCRαV binds to the TCRα V9 subfamily; the cancer is diffuse large B cell lymphoma and the domain that binds to the TCRαV binds to the TCRα V12 subfamily; the cancer is melanoma and the domain that binds to the TCRαV binds to the TCRα V12 subfamily; the cancer is multiple myeloma and the domain that binds to the TCRαV binds to the TCRα the cancer is diffuse large B cell lymphoma and the TCRαV binding domain binds to the TCRα V21 subfamily; the cancer is diffuse large B cell lymphoma and the TCRαV binding domain binds to the TCRα V22 subfamily; the cancer is diffuse large B cell lymphoma and the TCRαV binding domain binds to the TCRα V25 subfamily; the cancer is leukemia and the TCRαV binding domain binds to the TCRα V29 subfamily; the cancer is melanoma and the TCRαV binding domain binds to the TCRα V29 subfamily; the cancer is breast cancer and the TCRαV binding domain binds to the TCRα V29 subfamily; the cancer is endometrial cancer and the TCRαV binding domain binds to the TCRα V30 subfamily; the cancer is leukemia and the TCRαV binding domain binds to the TCRα V38 subfamily and / or the cancer is esophageal squamous cell carcinoma and the TCRαV-binding domain binds to the TCRα V39 subfamily.

[0124] In some embodiments, the disease or condition is an autoimmune disease. In some embodiments, the autoimmune disease is multiple sclerosis and the domain binding to TCRαV binds to the TCRα V1 subfamily; the autoimmune disease is Crohn's disease and the domain binding to TCRαV binds to the TCRα V2 subfamily; the autoimmune disease is Sjogren's syndrome and the domain binding to TCRαV binds to the TCRα V13 subfamily; the autoimmune disease is celiac disease and the domain binding to TCRαV binds to the TCRα V20 subfamily; the autoimmune disease is Crohn's disease and the domain binding to TCRαV binds to the TCRα V22 subfamily; the autoimmune disease is celiac disease and the domain binding to TCRαV binds to the TCRα V26 subfamily; and / or the autoimmune disease is Crohn's disease and / or the autoimmune disease Crohn's disease and the domain binding to TCRαV binds to the TCRα V40 subfamily.

[0125]

[0125] In some embodiments, the disease or condition is an infectious disease. In some embodiments, the infection is a S. parathyphi infection and the domain that binds to the TCRαV binds to the TCRα V1 subfamily; the infection is a Bacteroidetes infection and the domain that binds to the TCRαV binds to the TCRα V1 subfamily; the infection is a Proteobacteria infection and the domain that binds to the TCRαV binds to the TCRα V1 subfamily; the infection is a M. tuberculosis infection and the domain that binds to the TCRαV binds to the TCRα V1 subfamily; the infection is a respiratory viral infection and the domain that binds to the TCRαV binds to the TCRα V6 subfamily; the infection is a SARS-CoV-2 infection and the domain that binds to the TCRαV binds to the TCRα V7 subfamily; the infection is a SARS-CoV-2 infection and the domain that binds to the TCRαV binds to the TCRα V9 subfamily; the infection is a SARS-CoV-2 infection and the domain that binds to the TCRαV binds to the TCRα Binds to the V11 subfamily; infection is caused by S. pyogenes (S.the infection is a SARS-CoV-2 infection, and the domain binding to the TCRαV binds to the TCRα V12 subfamily; the infection is yellow fever, and the domain binding to the TCRαV binds to the TCRα V12 subfamily; the infection is influenza, and the domain binding to the TCRαV binds to the TCRα V13 subfamily; the infection is a respiratory viral infection, and the domain binding to the TCRαV binds to the TCRα V16 subfamily; the infection is an HIV infection, and the domain binding to the TCRαV binds to the TCRα V17 subfamily; the infection is a cytomegalovirus infection, and the domain binding to the TCRαV binds to the TCRα V17 subfamily; the infection is a SARS-CoV-2 infection, and the domain binding to the TCRαV binds to the TCRα V18 subfamily; the infection is an HIV infection, and the domain binding to the TCRαV binds to the TCRα V24 subfamily; the infection is influenza and the domain that binds to the TCRαV binds to the TCRα V27 subfamily; the infection is Epstein-Barr virus infection and the domain that binds to the TCRαV binds to the TCRα V38 subfamily.

[0126] Incorporation by Reference

[0126] All publications, patents, and patent applications mentioned in this specification are incorporated by reference into this specification to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0127]

[0127] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings. [Brief description of the drawings]

[0128] [Figure 1]

[0128] Figure 1 shows an exemplary graph showing the average abundance of each indicated TRAV gene compared to all TRAV genes based on sequencing analysis of samples obtained from five healthy individuals. TCR repertoire sequencing was performed from total RNA isolated from purified T cells of healthy humans. The MiXCR pipeline was used for alignment of reads to TCR clonotypes / germlines. Each TRAV gene is plotted against relative abundance (where 1 equals 100% of all TRAV genes). The average relative abundance for each TRAV is shown, with each individual donor represented as (·). n=5. [Figure 2A]

[0129] Figure 2A shows exemplary flow cytometry results showing T cell expansion after stimulation with anti-TRAV 12-1 antibody. Purified human T cells isolated from a healthy individual were stimulated with plate-bound anti-TCR V alpha 12.1 antibody (6D6.6) for 5 days. The FACS plot shows expanded T cells stained with AF647-labeled anti-TCR V alpha 12.1 antibody. Unstimulated T cells were stained for baseline expression. Data from one representative donor is shown. [Figure 2B]

[0130] FIG. 2B shows exemplary flow cytometry results showing T cell expansion after stimulation with anti-TRAV 12-1 antibody. Purified human T cells isolated from healthy individuals were stimulated with 100 nM plate-bound anti-TCR V alpha 12.1 antibody (6D6.6) for 10 days; followed by culture in the presence of 100 U / mL recombinant human IL-2 for an additional 2 days. FACS plot shows expanded T cells stained with AF647-labeled anti-TCR V alpha 12.1 antibody. Unstimulated T cells were stained for baseline expression. Data from two representative donors are shown. [Figure 3A]

[0131] FIG. 3A shows an exemplary dot plot showing the expansion of human CD4+ CD25+ and CD8+ CD25+ T cells after stimulation with anti-TCRαV-19 / IL-2 bispecific antibody. Human PBMCs were treated with 0.001, 0.01, 0.1, 1, 10, or 100 nM of anti-TCRαV-19 / IL-2 for 5 days at 37° C. Cells were stained with anti-CD4, anti-CD25 (IL2RA), and anti-CD8 antibodies, and the percentages of CD4+ CD25+ and CD8+ CD25+ T cell populations were quantified using FACS. Isotype controls were used for baseline comparison. [Figure 3B]

[0132] FIG. 3B shows an exemplary dot plot showing the expansion of mouse CD4+ CD25+ and CD8+ CD25+ T cells after stimulation with anti-TCRαV-14 / IL-2 bispecific antibody. Isolated mouse T cells were treated with 0.001, 0.01, 0.1, 1, 10, or 100 nM of anti-TCRαV-14 / IL-2 for 4 days at 37° C. Cells were stained with anti-CD4, anti-CD25 (IL2RA), and anti-CD8 antibodies, and the percentages of CD4+ CD25+ and CD8+ CD25+ T cell populations were quantified using FACS. Isotype controls were used for baseline comparison. [Figure 3C]

[0133] FIG. 3C shows an exemplary dot plot showing the expansion of mouse CD4+ CD25+ and CD8+ CD25+ T cells after stimulation with anti-TCRαV-12 / IL-2 bispecific antibody. Isolated mouse T cells were treated with 0.001, 0.01, 0.1, 1, 10, or 100 nM of anti-TCRαV-12 / IL-2 for 4 days at 37° C. Cells were stained with anti-CD4, anti-CD25 (IL2RA), and anti-CD8 antibodies, and the percentages of CD4+ CD25+ and CD8+ CD25+ T cell populations were quantified using FACS. Isotype controls were used for baseline comparison. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0129] definition

[0134] Certain details are described herein to provide a thorough understanding of the various embodiments. However, those skilled in the art will understand that the present disclosure may be practiced without these details. In other instances, well-known structures have not been shown or described in detail to avoid unnecessarily obscuring the description of the embodiments.

[0130]

[0135] Unless the context otherwise requires, throughout this specification and the claims which follow, the terms "comprise" and variations thereof, such as "comprises" and "comprising," are to be interpreted in their open, inclusive sense, i.e., "including, but not limited to." Additionally, the headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed disclosure.

[0131]

[0136] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The use of the terms "a" or "an," when used in conjunction with the term "comprising" herein, can mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0132]

[0137] It should also be noted that the term "or" is generally used in its sense including "and / or" unless the context clearly indicates otherwise.

[0133]

[0138] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below.

[0134]

[0139] When referring to a measurable value, such as an amount, duration, etc., the term "about" is meant to encompass a variation of ±20% from the particular value, or in some instances ±10%, or in some instances ±5%, or in some instances ±1%, or in some instances ±0.1%, where such variations are appropriate for carrying out the disclosed methods. As used herein, "about" and "approximately" generally refer to an acceptable degree of error for the amount measured given the nature or precision of the measurement. Exemplary degrees of error are within 20 percent (%), typically within 10%, and more typically within 5% of a given range of values.

[0135]

[0140] The term "obtain" or "obtaining" as used herein refers to obtaining possession of a physical entity (e.g., a sample, a polypeptide, a nucleic acid, or a sequence) or a value, e.g., a numerical value, by "directly obtaining" or "indirectly obtaining" the physical entity or value. "Directly obtaining" means performing a process (e.g., performing a synthetic or analytical method) to obtain the physical entity or value. "Indirectly obtaining" refers to receiving a physical entity or value from another person or source (e.g., a third party that directly obtained the physical entity or value). Directly obtaining a physical entity includes performing a process that involves a physical change of a physical substance, e.g., a starting material. Directly obtaining a value includes performing a process that involves a physical change of a sample or another substance, e.g., performing an analytical process that involves a physical change of a substance, e.g., a sample.

[0136]

[0141] As used herein, an "antibody molecule" refers to a protein, e.g., an immunoglobulin chain or fragment thereof, that comprises at least one immunoglobulin variable domain structure and / or sequence. Antibody molecules encompass antibodies (e.g., full-length antibodies) and antibody fragments. In some embodiments, an antibody molecule comprises an antigen-binding or functional fragment of a full-length antibody, or a full-length immunoglobulin chain. For example, a full-length antibody is an immunoglobulin (Ig) molecule (e.g., an IgG antibody) that occurs naturally or is formed by the recombinant process of normal immunoglobulin gene fragments. In embodiments, an antibody molecule refers to an immunologically active antigen-binding portion of an immunoglobulin molecule, e.g., an antibody fragment. An antibody fragment, e.g., a functional fragment, is a portion of an antibody, e.g., a Fab, Fab', F(ab') 2 , F(ab) 2 , variable fragment (Fv), domain antibody (dAb), or single chain variable fragment (scFv). A functional antibody fragment binds to the same antigen recognized by an intact (e.g., full-length) antibody. The term "antibody fragment" or "functional fragment" also includes isolated fragments consisting of the variable regions, such as an "Fv" fragment consisting of the variable regions of the heavy and light chains, or a recombinant single chain polypeptide molecule in which the variable regions of the light and heavy chains are linked by a peptide linker ("scFv protein"). In some embodiments, an antibody fragment does not include a portion of an antibody that does not have antigen-binding activity, e.g., an Fc fragment or a single amino acid residue. Exemplary antibody molecules include full length antibodies and antibody fragments, e.g., dAbs (domain antibodies), single chains, Fab, Fab', and F(ab'). 2 In some embodiments, the antibody molecule is an antibody mimic. In some embodiments, the antibody molecule is or comprises an antibody-like framework or scaffold, such as fibronectin, ankyrin repeat (e.g., designed ankyrin repeat proteins (DARPins)), avimer, affibody affinity ligand, anticalin, or affilin molecule.

[0137]

[0142] The term "human-like antibody molecule" as used herein refers to a humanized antibody molecule, a human antibody molecule, or an antibody molecule having at least 95% sequence identity to a non-mouse germline framework region, e.g., FR1, FR2, FR3 and / or FR4. In some embodiments, a human-like antibody molecule comprises a framework region having at least 95% sequence identity to a human germline framework region, e.g., FR1, FR2, FR3 and / or FR4 of a human germline framework region. In some embodiments, a human-like antibody molecule is a recombinant antibody. In some embodiments, a human-like antibody molecule is a humanized antibody molecule. In some embodiments, a human-like antibody molecule is a human antibody molecule. In some embodiments, a human-like antibody molecule is a phage-displayed or yeast-displayed antibody molecule. In some embodiments, a human-like antibody molecule is a chimeric antibody molecule. In some embodiments, a human-like antibody molecule is a CDR-grafted antibody molecule.

[0138]

[0143] As used herein, "immunoglobulin variable domain sequence" refers to an amino acid sequence capable of forming the structure of an immunoglobulin variable domain. For example, the sequence may include all or part of the amino acid sequence of a naturally occurring variable domain. For example, the sequence may or may not include one, two or more N-terminal or C-terminal amino acids, or may include other modifications compatible with forming a protein structure.

[0139]

[0144] In embodiments, the antibody molecule is monospecific, e.g., comprises binding specificity for a single epitope. In some embodiments, the antibody molecule is multispecific, e.g., it comprises multiple immunoglobulin variable domain sequences, a first immunoglobulin variable domain sequence has binding specificity for a first epitope, and a second immunoglobulin variable domain sequence has binding specificity for a second epitope. In some embodiments, the antibody molecule is a bispecific antibody molecule. A "bispecific antibody molecule," as used herein, refers to an antibody molecule that has specificity for more than one (e.g., 2, 3, 4, or more) epitope and / or antigen.

[0140]

[0145] As used herein, "antigen" (Ag) refers to a molecule that can elicit an immune response, e.g., involving activation of certain immune cells and / or antibody production. Any macromolecule, including almost any protein or peptide, can be an antigen. Antigens can also be derived from genomic recombinants or DNA. For example, any DNA that includes a nucleotide sequence or partial nucleotide sequence that encodes a protein that can elicit an immune response encodes an "antigen." In embodiments, an antigen need not be encoded only by the full-length nucleotide sequence of a gene, nor need an antigen be encoded by a gene at all. In embodiments, an antigen can be synthesized or derived from a biological sample, e.g., a tissue sample, a tumor sample, a cell, or a fluid having other biological components. As used herein, "tumor antigen" or synonymously, "cancer antigen" includes any molecule present on or associated with a cancer, e.g., a cancer cell, or a tumor microenvironment that can elicit an immune response. As used herein, "immune cell antigen" includes any molecule present on or associated with an immune cell that can elicit an immune response.

[0141]

[0146] An "antigen-binding site" or "binding site" of an antibody molecule refers to the portion of an antibody molecule, e.g., an immunoglobulin (Ig) molecule, that is involved in antigen binding. In an embodiment, the antigen-binding site is formed by amino acid residues of the variable regions (V) of the heavy (H) and light (L) chains. Three highly branched stretches in the variable regions of the heavy and light chains, called hypervariable regions, are located between more conserved adjacent regions called "framework regions" (FR). FRs are amino acid sequences that are naturally found between and adjacent to the hypervariable regions in immunoglobulins. In an embodiment, in an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are positioned relative to each other in three-dimensional space to form an antigen-binding surface that is complementary to the three-dimensional surface of a bound antigen. The three hypervariable regions of each of the heavy and light chains are called "complementarity determining regions" or "CDRs". Framework regions and CDRs are defined and described, for example, in Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., USDepartment of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917. Each variable chain (e.g., variable heavy and variable light chains) typically consists of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following amino acid order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0142]

[0147] As used herein, "immune cells" refers to any of a variety of cells that function in the immune system, e.g., defend against infection and foreign agents. In embodiments, the term includes white blood cells, e.g., neutrophils, eosinophils, basophils, lymphocytes, and monocytes. Innate white blood cells include phagocytes (e.g., macrophages, neutrophils, and dendritic cells), mast cells, eosinophils, basophils, and natural killer cells. Innate white blood cells identify and eliminate pathogens by attacking larger pathogens through contact or by engulfing and killing microorganisms, and are mediators of the activation of adaptive immune responses. Cells of the adaptive immune system are a specialized type of white blood cell called lymphocytes. B cells and T cells are important types of lymphocytes and are derived from hematopoietic stem cells in the bone marrow. B cells are involved in humoral immune responses, and T cells are involved in cellular immune responses. The term "immune cells" includes immune effector cells.

[0143]

[0148] The term "immune effector cell" as used herein refers to a cell that is involved in an immune response, e.g., promoting an immune effector response. Examples of immune effector cells include, but are not limited to, T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T cells, and mast cells.

[0144]

[0149] The term "effector function" or "effector response" refers to a specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including secretion of cytokines.

[0145]

[0150] The terms "polypeptide", "peptide" and "protein" (when single chain) are used interchangeably herein and refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. Polypeptides can be isolated from natural sources, produced by recombinant techniques from eukaryotic or prokaryotic hosts, or can be the product of synthetic techniques.

[0146]

[0151] The terms "nucleic acid", "nucleic acid sequence", "nucleotide sequence", or "polynucleotide sequence", and "polynucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. A polynucleotide can be either single-stranded or double-stranded, and if single-stranded, can be the coding strand or the non-coding (antisense) strand. A polynucleotide can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, for example, by conjugation with a labeling component. A nucleic acid can be a recombinant polynucleotide, or a polynucleotide of genomic, cDNA, semi-synthetic, or synthetic origin that is not naturally occurring or is linked to another polynucleotide in a non-naturally occurring sequence.

[0147]

[0152] The term "isolated" as used herein refers to a material that is removed from its original or natural environment (e.g., the natural environment if it occurs in nature). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but an identical polynucleotide or polypeptide that has been separated by human intervention from some or all of the coexisting materials of the natural system is isolated. Such a polynucleotide may be part of a vector, and / or such a polynucleotide or polypeptide may be part of a composition, and such a vector or composition may still be isolated in that it is not part of the environment in which it is found in nature. An isolated polynucleotide (ribonucleic acid (RNA), deoxyribonucleic acid (DNA)), or polypeptide does not contain the genes / nucleic acids or sequences / amino acids that flank it in its naturally occurring state.

[0148]

[0153] The compositions and methods of the present disclosure encompass polypeptides and nucleic acids having a specific sequence or a substantially identical or similar sequence thereto, for example, a sequence that is at least 80%, 85%, 90%, 95% identical or more identical to a specific sequence.In the context of amino acid sequences, the term "substantially identical" is used herein to refer to a first amino acid sequence that contains a sufficient or minimal number of amino acid residues that are i) identical to a second amino acid sequence, or ii) are conservative substitutions of aligned amino acid residues in a second amino acid sequence, such that the first and second amino acid sequences can have a common structural domain and / or a common functional activity.For example, an amino acid sequence that contains a common structural domain that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% sequence identity to a reference sequence, for example, a sequence provided herein. In the context of nucleotide sequences, the term "substantially identical" is used herein to refer to a first nucleic acid sequence that contains a sufficient number or a minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences code for a polypeptide having a common functional activity or code for a common polypeptide structural domain or common polypeptide functional activity. For example, a nucleotide sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to a reference sequence, such as a sequence provided herein.

[0149]

[0154] The term "variant" refers to a polypeptide having a substantially identical amino acid sequence to a reference amino acid sequence or encoded by a substantially identical nucleotide sequence. In some embodiments, the variant is a functional variant. In some embodiments, the TCRαV variant can bind to TCRα and form a TCRα:β complex.

[0150]

[0155] The term "functional variant" refers to a polypeptide having an amino acid sequence substantially identical to a reference amino acid sequence, or encoded by a substantially identical nucleotide sequence, and which may have one or more activities of the reference amino acid sequence.

[0151]

[0156] Calculation of homology or sequence identity (these terms are used interchangeably herein) between sequences is performed as follows. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., for optimal alignment, gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences can be ignored for comparison purposes). In a preferred embodiment, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. If a position of the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology").

[0152]

[0157] The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences.Comparing sequences and determining the percent identity between two sequences can be achieved using a mathematical algorithm.In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm, which is incorporated in the GAP program of the GCG software package (available at http: / / www.gcg.com), using either a Blossum62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, percent identity between two nucleotide sequences is determined using the GAP program of the GCG software package (available at http: / / www.gcg.com), using a NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and those that should be used unless otherwise specified) is a Blossum62 scoring matrix, with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0153]

[0158] Percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS 4:11-17) as incorporated into the ALIGN program (version 2.0) using a PAM120 residue weighting table, a gap length penalty of 12, and a gap penalty of 4. The nucleic acid and protein sequences described herein can be used as "query sequences" to perform searches against public databases, for example, to identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the present disclosure. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the present disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0154]

[0159] It is understood that the molecules of the present disclosure may have additional conservative or non-essential amino acid substitutions that do not substantially affect their function.

[0155]

[0160] The term "amino acid" is intended to include all molecules, whether natural or synthetic, that contain both amino and acid functionalities and can be included in naturally occurring amino acid polymers. Exemplary amino acids include naturally occurring amino acids; their analogs, derivatives and congeners; amino acid analogs with variant side chains; and all stereoisomers of any of the foregoing. As used herein, the term "amino acid" includes both D or L optical isomers and peptidomimetics.

[0156]

[0161] A "conservative amino acid substitution" refers to an amino acid residue that is replaced with an amino acid residue that has a similar side chain. Families of amino acid residues that have similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0157]

[0162] As used herein, the term "molecule," as used in, e.g., antibody molecules, cytokine molecules, receptor molecules, includes full-length naturally occurring molecules as well as variants, e.g., functional variants (e.g., truncations, fragments, mutations (e.g., substantially similar sequences) or derivatized forms thereof), so long as at least one function and / or activity of the unmodified (e.g., naturally occurring) molecule remains.

[0158]

[0163] As used herein, the term "mutation" refers to an alteration in the nucleotide sequence of the genome of an organism, virus, or extrachromosomal DNA. In some embodiments, the mutation may be a large-scale mutation, such as an amplification (or gene duplication) or repetition of a chromosomal segment, a deletion of a large chromosomal region, a chromosomal rearrangement (e.g., chromosomal translocation, chromosomal inversion, non-homologous chromosomal crossover, and interstitial deletion), and a loss of heterozygosity. In some embodiments, the mutation may be a small-scale mutation, such as an insertion, deletion, or substitution mutation. As used herein, the term "substitution mutation" refers to a transition that exchanges a single nucleotide for another nucleotide.

[0159]

[0164] "Interleukin-2," also known as IL2, IL-2, IL 2, TCGF, ​​lymphokine, and interleukin 2, as referred to herein, includes any recombinant or naturally occurring form of IL-2, or a variant or homolog thereof, that has or maintains IL-2 activity (e.g., at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variant or homolog has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 contiguous amino acid portion) compared to naturally occurring IL-2. In some embodiments, the IL-2 is substantially identical to a protein identified by UniProt reference number P60568, or a variant or homolog having substantial identity thereto. Anti-TCRαV antibody Human T cell receptor (TCR) complex

[0165] TCRs are disulfide-linked membrane-anchored heterodimeric proteins consisting of typically highly variable alpha (α) and beta (β) chains expressed as part of a complex with an invariant CD3 chain molecule. TCRs on αβ T cells are formed by a heterodimer of one alpha and one beta chain. Each alpha or beta chain consists of a constant domain and a highly variable domain classified as an immunoglobulin superfamily (IgSF) fold. TCRαV chains can be further classified into subfamilies (TRAV1-10, 12-14, 16-27, 29, 30, 34-36 and (an) 38-41). Despite their high structural and functional homology, the amino acid sequence homology in the TRAV genes is very low. Nevertheless, TCRs formed between alpha and beta chains of highly diverse sequences exhibit remarkable structural and similar functions, e.g., eliciting T cell activation.

[0160]

[0166] T cell receptors (TCRs) can be found on the surface of T cells. TCRs recognize antigens, e.g., peptides, bound to, e.g., major histocompatibility complex (MHC) molecules, presented on the surface of cells, e.g., antigen-presenting cells. TCRs are heterodimeric molecules and can include alpha, beta, gamma, or delta chains. TCRs that include alpha and beta chains are also referred to as TCRαβ. The TCRα chain consists of the following regions (also known as segments): variable (V), diverse (D), joining (J), and constant (C) (see Mayer G. and Nyland J. (2010) Chapter 10: Major Histocompatibility Complex and T-cell Receptors-Role in Immune Responses. In: Microbiology and Immunology on-line, University of South Carolina School of Medicine). The TCRα chain consists of the V, J, and C regions. Rearrangement of the T cell receptor (TCR) by somatic recombination of the V (variable), D (diverse), J (joining), and C (constant) regions is a critical event in the development and maturation of T cells. TCR gene rearrangement occurs in the thymus.

[0161]

[0167] The TCR may comprise a receptor complex known as a TCR complex, which comprises a TCR heterodimer comprising an alpha chain and a beta chain, and a dimeric signaling molecule, e.g., a CD3 co-receptor, e.g., CD3δ / ε and / or CD3γ / ε.

[0162]

[0168] As used herein, the term "T cell receptor alpha variable chain" or "TCRαV" or "TRAV" refers to the extracellular region of the T cell receptor alpha chain, which may include a portion of the antigen recognition domain of the T cell receptor. The term TCRαV includes isoforms, mammalian, e.g., human TCRαV, human species homologs and analogs that contain at least one common epitope with TCRαV. Human TCRαV includes the following subfamily: TCRα V1 subfamily, TCRα V2 subfamily, TCRα V3 subfamily, TCRα V4, TCRα V5 subfamily, TCRα V6 subfamily, TCRα V7 subfamily, TCRα V8 subfamily, TCRα V9 subfamily, TCRα V10 subfamily, TCRα V12 subfamily, TCRα V13 subfamily, TCRα V14 subfamily, TCRα V16 subfamily, TCRα V17 subfamily, TCRα V18 subfamily, TCRα V19 subfamily, TCRα V20 subfamily, TCRα V21 subfamily, TCRα V22 subfamily, TCRα V23 subfamily, TCRα V24 subfamily, TCRα V25 subfamily, TCRα V26 subfamily, TCRα V27 subfamily, TCRα V29 subfamily, TCRα V30 subfamily, TCRα The gene families include subfamilies including, but not limited to, the V34 subfamily, the TCRα V35 subfamily, the TCRα V36 subfamily, the TCRα V38 subfamily, the TCRα V39 subfamily, the TCRα V40 subfamily, or the TCRα V41 subfamily, as well as family members of said subfamilies, and variants thereof (e.g., structural or functional variants thereof).

[0163]

[0169] In some embodiments, the TCRα V1 subfamily comprises TCRαV1-1 or TCRαV1-2, or a variant thereof.

[0164]

[0170] In some embodiments, the TCRα V8 subfamily includes TCRαV8-1, TCRαV8-2, TCRαV8-3, TCRαV8-4, or TCRαV8-6, or a variant thereof.

[0165]

[0171] In some embodiments, the TCRα V9 subfamily comprises TCRαV9-1 or TCRαV9-2, or a variant thereof.

[0166]

[0172] In some embodiments, the TCRα V12 subfamily comprises TCRαV12-1, TCRαV12-2, or TCRαV12-3, or a variant thereof.

[0167]

[0173] In some embodiments, the TCRα V13 subfamily comprises TCRα V13-1 or TCRα V13-2, or a variant thereof.

[0168]

[0174] In some embodiments, the TCRα V14 subfamily comprises TCRα V14 / DV4, or a variant thereof.

[0169]

[0175] In some embodiments, the TCRα V23 subfamily comprises TCRα V23 / DV6, or a variant thereof.

[0170]

[0176] In some embodiments, the TCRα V26 subfamily comprises TCRα V26-1 or TCRα V26-2, or a variant thereof.

[0171]

[0177] In some embodiments, the TCRα V29 subfamily comprises TCRαV29 / DV5, or a variant thereof.

[0172]

[0178] In some embodiments, the TCRα V36 subfamily comprises TCRα V236 / DV7, or a variant thereof.

[0173]

[0179] In some embodiments, the TCRα V38 subfamily comprises TCRα V38-1 or TCRα V38-2 / DV8, or a variant thereof.

[0174] TCR Alpha V (TCRαV)

[0180] The diversity of the immune system allows for defense against a vast number of pathogens. Because the size of the germline genome is limited, diversity is achieved not only through the process of V(D)J recombination, but also through deletion of nucleotide junctions (the junctions between the VD and DJ segments) and addition of pseudorandom, non-templated nucleotides. The TCR alpha gene undergoes genetic sequencing to generate diversity.

[0175]

[0181] TCR V alpha repertoires vary between individuals and populations due, for example, to seven frequent inactivating polymorphisms in functional gene segments and a large insertion / deletion associated polymorphism encompassing two V alpha gene segments.

[0176]

[0182] Provided herein are, inter alia, antibody molecules and fragments thereof that bind, e.g., specifically bind, e.g., human TCR alpha V chain (TCRαV), e.g., TCRαV gene family (also referred to as group), e.g., TCRαV subfamily (also referred to as subgroup), as described herein. TCR alpha V families and subfamilies are known in the art and described, for example, in Yassai et al. (2009) Immunogenetics 61(7) 493-502; Wei S. and Concannon P. (1994) Human Immunology 41(3) 201-206. The antibodies described herein can be recombinant antibodies, e.g., recombinant non-mouse antibodies, e.g., recombinant human or humanized antibodies.

[0177]

[0183] The terms TCRAV, TCRVA, TRAV, TCRαV, TCRVα or TRαV are used interchangeably herein and refer to a TCR alpha V chain, e.g., as described herein.

[0178]

[0184] In some embodiments, provided herein are anti-TCRαV antibody molecules that bind to a human TCRαV, e.g., a TCRαV family, e.g., a gene family, or a variant thereof.

[0179]

[0185] Exemplary amino acid sequences of TCRαV subfamily members can be found on the ImMunoGeneTics Information System website: http: / / www.imgt.org / , or similar resources.

[0180] Anti-TCRαV antibody

[0186] Current bispecific constructs designed to redirect T cells to promote tumor cell lysis for cancer immunotherapy typically utilize antibody fragments (e.g., Fab, scFv, VH, single domain antibodies) derived from monoclonal antibodies (mAbs) directed against the CD3e subunit of the T cell receptor (TCR). However, this approach has limitations that may prevent the full realization of the therapeutic potential of such bispecific constructs. Previous studies have shown that even low "activating" doses of anti-CD3e mAbs cause long-term T cell dysfunction and exert immunosuppressive effects. Furthermore, anti-CD3e mAbs are associated with side effects due to the massive T cell activation. Large numbers of activated T cells secrete substantial amounts of cytokines, the most important of which is interferon gamma (IFNγ). This excess of IFNγ then activates macrophages, which then overproduce inflammatory cytokines such as IL-1 beta, IL-6, IL-10 and TNF-alpha, causing a "cytokine storm" known as cytokine release syndrome (CRS) (Shimabukuro-Vornhagen et al., J Immunother Cancer. 2018 Jun. 15;6(1):56, incorporated herein by reference in its entirety). Thus, there is a need to develop antibodies that can bind to and activate only a subset of effector T cells, for example to reduce CRS and / or neurotoxicity (NT).

[0181]

[0187] Described herein are molecules and methods that target the TCRαV chain of the TCR. Without wishing to be bound by theory, such molecules can bind, activate, and / or expand only a subset of T cells, avoid or reduce CRS and / or NT, and minimize the potential immunosuppressive effects of anti-CD3 mAbs.

[0182]

[0188] Described herein is a class of antibodies, i.e., the anti-TCRαV antibody molecules described herein, that recognize structurally conserved but sequence variable regions, e.g., domains, on the TCRαV protein and have similar functions (e.g., activation of T cells and similar cytokine profiles as described herein) despite having low sequence similarity (e.g., low sequence identity among different antibody molecules that recognize different TCRαV subfamilies). Thus, the anti-TCRαV antibody molecules described herein share a structure-function relationship.

[0183]

[0189] In some embodiments, the anti-TCRαV antibody molecules described herein do not recognize, e.g., do not bind to, the interface of the TCRβV:TCR alpha complex. In some embodiments, the anti-TCRαV antibody molecules described herein do not recognize, e.g., do not bind to, the constant region of the TCRβV protein. In some embodiments, the anti-TCRαV antibody molecules described herein do not recognize, e.g., do not bind to, one or more (e.g., all) of the complementarity determining regions (e.g., CDR1, CDR2 and / or CDR3) of the TCRβV protein.

[0184]

[0190] In particular, antibody molecules directed to the variable chain of the alpha subunit of the TCR (TCRαV) that bind to and, for example, activate a subset of T cells are provided herein. The anti-TCRαV antibody molecules described herein result in less or no production of cytokines associated with CRS, such as IL-6, IL-1 beta, IL-10 and TNF alpha; and enhanced and / or delayed production of IL-2 and IFNγ. In some embodiments, the anti-TCRαV antibodies described herein have a cytokine profile, e.g., as described herein, that differs from the cytokine profile of T cell engagers that bind to receptors or molecules other than the TCRαV region ("non-TCRαV binding T cell engagers"). In some embodiments, the non-TCRαV binding T cell engagers include antibodies that bind to CD3 molecules (e.g., CD3 epsilon (CD3e) molecules), or TCR alpha (TCRα) molecules. In some embodiments, the non-TCRαV binding T cell engager is the OKT3 antibody or the SP34-2 antibody.

[0185]

[0191] In some embodiments, the anti-TCRαV antibodies described herein result in the expansion of TCRαV+ T cells, e.g., a subset of memory effector T cells known as TEMRA. Without wishing to be bound by theory, it is believed that in some embodiments, TEMRA cells can promote tumor cell lysis but not CRS. Thus, methods of making the anti-TCRαV antibody molecules and uses thereof are provided herein. Also described herein are multispecific, e.g., bispecific, molecules comprising the anti-TCRαV antibody molecules. In some embodiments, compositions comprising the anti-TCRαV antibody molecules of the present disclosure can be used, for example, to (1) activate and redirect T cells to promote tumor cell lysis for cancer immunotherapy; and / or (2) expand TCRαV+ T cells. In some embodiments, compositions comprising the anti-TCRαV antibody molecules described herein limit the adverse side effects of CRS and / or NT, e.g., CRS and / or NT associated with anti-CD3e targeting.

[0186]

[0192] In some embodiments, the anti-TCR α antibody molecule is a full length antibody or a fragment thereof (e.g., Fab, F(ab') 2 , Fv, single domain antibody, or single chain Fv fragment (scFv). In embodiments, the anti-TCRαV antibody molecule is a monoclonal antibody or an antibody with a single specificity. In some embodiments, the anti-TCRαV antibody molecule can also be a humanized, chimeric, camelid, shark, or in vitro generated antibody molecule. In some embodiments, the anti-TCRαV antibody molecule is a humanized antibody molecule. The heavy and light chains of the anti-TCRαV antibody molecule can be full length (e.g., the antibody can comprise at least one, preferably two, complete heavy chains, and at least one, preferably two, complete light chains), or can comprise an antigen-binding fragment (e.g., Fab, F(ab')2, Fv, single chain Fv fragment, single domain antibody, diabody (dAb), bivalent antibody, or bispecific antibody or fragment thereof, single domain variant thereof, or camelid antibody).

[0187]

[0193] In some embodiments, the anti-TCRαV antibody molecule is in the form of a multispecific, e.g., a bispecific, molecule, e.g., as described herein.

[0188]

[0194] In some embodiments, the anti-TCRαV antibody molecule has a heavy chain constant region (Fc) selected from, for example, the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. In some embodiments, the Fc region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fc region is selected from the heavy chain constant regions of IgG1 or IgG2 (e.g., human IgG1, or IgG2). In some embodiments, the heavy chain constant region is human IgG1. In some embodiments, the Fc region comprises an Fc region variant, for example, as described herein.

[0189]

[0195] In some embodiments, the anti-TCRαV antibody molecule has a light chain constant region selected from, for example, a kappa or lambda, preferably a kappa (e.g., human kappa) light chain constant region. In some embodiments, the constant region is altered, e.g., mutated, to modify the properties of the anti-TCRαV antibody molecule (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function). For example, the constant region has been mutated at positions 296 (M to Y), 298 (S to T), 300 (T to E), 477 (H to K) and 478 (N to F) to alter Fc receptor binding, e.g., compared to human IgG1 (e.g., the mutated positions correspond to positions 132 (M to Y), 134 (S to T), 136 (T to E), 313 (H to K) and 314 (N to F) of SEQ ID NO: 212 or 214, or positions 135 (M to Y), 137 (S to T), 139 (T to E), 316 (H to K) and 317 (N to F) of SEQ ID NO: 215, 216, 217 or 218).

[0190]

[0196] Various TCRαV subfamilies and / or subfamily members may be expressed at different levels in individuals, e.g., healthy individuals, as disclosed in Kitaura K. et al., (2016), BMC Immunology vol 17: 38, the entire contents of which are incorporated herein by reference.

[0191]

[0197] In some embodiments, the anti-TCRαV antibody molecule is a non-mouse antibody molecule, such as a human or humanized antibody molecule. In some embodiments, the anti-TCRαV antibody molecule is a human antibody molecule. In some embodiments, the anti-TCRαV antibody molecule is a humanized antibody molecule.

[0192]

[0198] In some embodiments, the anti-TCRαV antibody molecule is isolated or recombinant.

[0193]

[0199] In some embodiments, the anti-TCRαV antibody molecule comprises a heavy chain constant region for IgG4, e.g., human IgG4. In yet another embodiment, the anti-TCRαV antibody molecule comprises a heavy chain constant region for IgG1, e.g., human IgG1. In some embodiments, the heavy chain constant region comprises an amino acid sequence set forth in Table 3, or a sequence substantially identical thereto (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical).

[0194]

[0200] In some embodiments, the anti-TCRαV antibody molecule comprises a kappa light chain constant region, e.g., a human kappa light chain constant region. In some embodiments, the light chain constant region comprises an amino acid sequence set forth in Table 1, or a sequence substantially identical thereto (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical).

[0195]

[0201] In some embodiments, e.g., those comprising variable regions, CDRs (e.g., combination CDRs, Chothia CDRs or Kabat CDRs), or other sequences mentioned herein, the antibody molecule is a monospecific antibody molecule, a bispecific antibody molecule, a bivalent antibody molecule, a biparatopic antibody molecule, or an antibody molecule comprising an antigen-binding fragment of an antibody, e.g., a half antibody or an antigen-binding fragment of a half antibody. In certain embodiments, the antibody molecule comprises a multispecific molecule, e.g., a bispecific molecule, e.g., as described herein.

[0196]

[0202] In some embodiments, the anti-TCRαV antibody molecule is a non-mouse antibody molecule, such as a human or humanized antibody molecule. In some embodiments, the anti-TCRαV antibody molecule is a human antibody molecule. In some embodiments, the anti-TCRαV antibody molecule is a humanized antibody molecule.

[0197]

[0203] In some embodiments, the anti-TCRαV antibody molecule is isolated or recombinant.

[0198]

[0204] In some embodiments, the anti-TCRαV antibody molecule may contain any combination of CDRs or hypervariable loops according to the Kabat and Chothia definition.

[0199]

[0205] In some embodiments, for example, an antibody molecule comprising a variable region, CDR (e.g., a combined CDR, Chothia CDR or Kabat CDR), or other sequence referred to herein, is a monospecific antibody molecule, a bispecific antibody molecule, a bivalent antibody molecule, a biparatopic antibody molecule, or an antibody molecule comprising an antigen-binding fragment of an antibody, e.g., a half antibody or an antigen-binding fragment of a half antibody. In certain embodiments, an antibody molecule comprises a multispecific molecule, e.g., a bispecific molecule, e.g., as described herein.

[0200]

[0206] In some embodiments, the anti-TCRαV antibody molecule comprises a light chain variable domain comprising (a) a framework region 1 (FR1) that comprises an alteration, e.g., a substitution (e.g., a conservative substitution), at one or more (e.g., all) positions described herein according to Kabat numbering, and (b) a framework region 3 (FR3) that comprises an alteration, e.g., a substitution (e.g., a conservative substitution), at one or more (e.g., all) positions described herein according to Kabat numbering. In some embodiments, the substitution is relative to a human germline light chain framework region sequence.

[0201]

[0207] In some embodiments, the anti-TCRαV antibody molecule is a full-length antibody or a fragment thereof (e.g., Fab, F(ab') 2, Fv, or single chain Fv fragment (scFv). In embodiments, the anti-TCRαV antibody molecule is a monoclonal antibody or an antibody with a single specificity. In some embodiments, the anti-TCRαV antibody molecule can also be a humanized, chimeric, camelid, shark, or in vitro generated antibody molecule. In some embodiments, the anti-TCRαV antibody molecule is a humanized antibody molecule. The heavy and light chains of the anti-TCRαV antibody molecule can be full length (e.g., the antibody can comprise at least one, preferably two, complete heavy chains, and at least one, preferably two, complete light chains), or can comprise an antigen-binding fragment (e.g., Fab, F(ab')2, Fv, single chain Fv fragment, single domain antibody, diabody (dAb), bivalent antibody, or bispecific antibody or fragment thereof, single domain variant thereof, or camelid antibody).

[0202]

[0208] In some embodiments, the anti-TCRαV antibody molecule is in the form of a multispecific, e.g., a bispecific, molecule, e.g., as described herein.

[0203]

[0209] In some embodiments, the anti-TCRαV antibody molecule has a heavy chain constant region (Fc) selected from, for example, the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. In some embodiments, the Fc region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fc region is selected from the heavy chain constant regions of IgG1 or IgG2 (e.g., human IgG1, or IgG2). In some embodiments, the heavy chain constant region is human IgG1.

[0204]

[0210] In some embodiments, the anti-TCRαV antibody molecule has a light chain constant region selected from, for example, a kappa or lambda, preferably a kappa (e.g., human kappa) light chain constant region. In some embodiments, the constant region is altered, e.g., mutated, to modify the properties of the anti-TCRαV antibody molecule (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function). For example, the constant region has been mutated at positions 296 (M to Y), 298 (S to T), 300 (T to E), 477 (H to K) and 478 (N to F) to alter Fc receptor binding (e.g., the mutated positions correspond to positions 132 (M to Y), 134 (S to T), 136 (T to E), 313 (H to K) and 314 (N to F) of SEQ ID NO: 212 or 214, or positions 135 (M to Y), 137 (S to T), 139 (T to E), 316 (H to K) and 317 (N to F) of SEQ ID NO: 215, 216, 217 or 218).

[0205] Anti-TCRα V12 antibody

[0211] In one aspect, anti-TCRαV antibody molecules are provided herein that bind to human TCRαV12 subfamily members. In some embodiments, the TCRαV12 subfamily is also known as TCRαV12. In some embodiments, the TCRαV12 subfamily includes TCRαV12-1, TCRαV12-2, or TCRαV12-3, or variants thereof.

[0206] Antibody-Like Frameworks or Scaffolds

[0212] A variety of antibody / immunoglobulin frameworks or scaffolds can be used in the anti-TCRVA antibody molecules or multifunctional formats described herein, as long as the resulting polypeptide contains at least one binding region that specifically binds to a target antigen, such as TCRVA, a tumor antigen, in particular. Such frameworks or scaffolds include human immunoglobulins of the five main idiotypes, or fragments thereof, including immunoglobulins of other animal species, preferably with humanized versions. New frameworks, scaffolds and fragments continue to be discovered and developed by those skilled in the art.

[0207]

[0213] In some embodiments, the anti-TCRαV antibody molecules or multifunctional formats thereof described herein include non-immunoglobulin-based antibodies using non-immunoglobulin scaffolds onto which CDRs can be grafted. Any non-immunoglobulin framework and scaffold can be used as long as it contains a binding region specific for the target antigen (e.g., TCRαV or tumor antigen). Exemplary non-immunoglobulin frameworks or scaffolds include, but are not limited to, fibronectin (Compound Therapeutics, Inc., Waltham, MA), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd., Cambridge, MA, and Ablynx nv, Zwijnaarde, Belgium), lipocalins (Pieris Proteolab AG, Freising, Germany), small modular immunopharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, WA), maxybodies (Avidia, Inc., Mountain View, CA), protein A (Affibody AG, Sweden), and affilin (gamma-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany).

[0208]

[0214] Fibronectin scaffolds are typically based on fibronectin type III domains (e.g., the 10th module of fibronectin type III (10 Fn3 domain)). Fibronectin type III domains have seven or eight beta strands distributed between two beta sheets that pack against each other to form the core of the protein, and further contain loops (similar to CDRs) that connect the beta strands to each other and are solvent exposed. There are at least three such loops at each edge of the beta sheet sandwich, the edges being the boundaries of the protein perpendicular to the direction of the beta strands (see U.S. Pat. No. 6,818,418). Due to this structure, non-immunoglobulin antibodies mimic antigen binding properties that are similar in nature and affinity to antibodies. These scaffolds can be used in loop randomization and in vitro shuffling strategies that are similar to the process of affinity maturation of antibodies in vivo. These fibronectin-based molecules can be used as scaffolds where the loop regions of the molecule can be replaced with the CDRs of the present disclosure using standard cloning techniques.

[0209]

[0215] Ankyrin technology is based on using a protein with ankyrin-derived repeat modules as a scaffold to carry variable regions that can be used for binding to different targets. Ankyrin repeat modules are typically polypeptides of about 33 amino acids consisting of two antiparallel α-helices and a β-turn. The binding of the variable regions can be optimized by using ribosome display.

[0210]

[0216] Avimers are used due to the nature of protein-protein interactions, with over 250 proteins in humans being structurally based on A domains. Avimers consist of a number of different "A domain" monomers (2-10) linked via amino acid linkers. For example, the methodologies described in U.S. Patent Application Publication Nos. 20040175756, 20050053973, 20050048512, and 20060008844 can be used to generate avimers capable of binding to target antigens.

[0211]

[0217] Affibody affinity ligands are small, simple proteins composed of a three-helix bundle based on a scaffold of one of the IgG-binding domains of Protein A. Protein A is a surface protein from the bacterium Staphylococcus aureus. This scaffold domain consists of 58 amino acids, 13 of which are randomized to generate affibody libraries with a large number of ligand variants (see, for example, U.S. Pat. No. 5,831,012). Affibody molecules mimic antibodies and have a molecular weight of 6 kDa, compared to the molecular weight of antibodies, which is 150 kDa. Despite their small size, the binding site of affibody molecules is similar to that of antibodies.

[0212]

[0218] Anticalins are commercially known, e.g. from Pieris ProteoLab AG. They are derived from lipocalins, a widespread group of small, robust proteins usually involved in the physiological transport or storage of chemically sensitive or insoluble compounds. Several natural lipocalins are present in human tissues or body fluids. The organization of the protein is reminiscent of immunoglobulins, with hypervariable loops on top of a rigid framework. However, in contrast to antibodies or their recombinant fragments, lipocalins are composed of a single polypeptide chain with 160-180 amino acid residues, only slightly larger than a single immunoglobulin domain. The set of four loops that make up the binding pocket show significant structural plasticity, tolerating a variety of side chains. The binding site can therefore be reshaped in a unique process to recognize formulated target molecules of different shapes with high affinity and specificity. One protein of the lipocalin family, the bilin-binding protein (BBP) from Pieris Brassicae, has been used to develop anticalins by mutagenizing a set of four loops. One example of a patent application describing anticalins is in PCT International Publication WO199916873.

[0213]

[0219] Affilin molecules are small non-immunoglobulin proteins that are designed for specific affinity to proteins and small molecules. New affilin molecules can be very rapidly selected from two libraries, each based on a different human-derived scaffold protein. Affilin molecules do not show any structural homology to immunoglobulin proteins. Currently, two affilin scaffolds are used, one of which is gamma crystalline, a human structural eye lens protein, and the other is a "ubiquitin" superfamily protein. Both human scaffolds are very small, show high temperature stability, and are almost resistant to pH changes and denaturants. This high stability is mainly due to the extended beta sheet structure of the protein. Examples of gamma crystalline-derived proteins are described in WO200104144, and examples of "ubiquitin-like" proteins are described in WO2004106368.

[0214]

[0220] Protein epitope mimetics (PEMs) are intermediate-sized, cyclic, peptide-like molecules (MW 1-2 kDa) that mimic the beta-hairpin secondary structure of proteins, which is the major secondary structure involved in protein-protein interactions.

[0215]

[0221] Domain antibodies (dAbs) can be used in the anti-TCRVA antibody molecules described herein or in their multifunctional formats, and are small functional binding fragments of antibodies that correspond to the variable regions of either the heavy or light chains of the antibody. Domain antibodies are well expressed in bacteria, yeast, and mammalian cell systems. Further details of domain antibodies and methods for their production are known in the art (see, e.g., U.S. Pat. Nos. 6,291,158, 6,582,915, 6,593,081, 6,172,197, 6,696,245, European Patent Nos. 0368684 & 0616640, WO05 / 035572, WO04 / 101790, WO04 / 081026, WO04 / 058821, WO04 / 003019 and WO03 / 002609). Nanobodies are derived from the heavy chain of an antibody.

[0216]

[0222] Nanobodies typically contain a single variable domain and two constant domains (CH2 and CH3) and retain the antigen-binding ability of the original antibody. Nanobodies can be prepared by methods known in the art (see, for example, U.S. Pat. No. 6,765,087, U.S. Pat. No. 6,838,254, WO06 / 079372). Unibodies consist of one light chain and one heavy chain of an IgG4 antibody. Unibodies can be made by removing the hinge region of an IgG4 antibody. Further details of unibodies and methods of preparing them can be found in WO2007 / 059782.

[0217] Anti-TCRVα antibody effector functions and Fc variants

[0223] In some embodiments, the anti-TCRVα antibodies described herein comprise an Fc region, e.g., as described herein. In some embodiments, the Fc region is a wild-type Fc region, e.g., a wild-type human Fc region. In some embodiments, the Fc region comprises a variant, e.g., an Fc region that comprises an addition, substitution, or deletion of at least one amino acid residue in the Fc region, e.g., that results in reduced or eliminated affinity for at least one Fc receptor.

[0218]

[0224] The Fc region of an antibody interacts with a number of receptors or ligands, including Fc receptors (e.g., FcγRI, FcγRIIA, FcγRIIIA), complement protein CIq, and other molecules such as proteins A and G. These interactions are essential for a variety of effector functions and downstream signaling events, including antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).

[0219]

[0225] In some embodiments, an anti-TCRVα antibody comprising a variant Fc region has reduced, e.g., ablated, affinity for an Fc receptor, e.g., an Fc receptor described herein, in some embodiments, the reduced affinity is compared to an otherwise similar antibody having a wild-type Fc region.

[0220]

[0226] In some embodiments, an anti-TCRVα antibody comprising a variant Fc region has one or more of the following properties: (1) reduced effector function (e.g., reduced ADCC, ADCP, and / or CDC), (2) reduced binding to one or more Fc receptors, and / or (3) reduced binding to C1q complement. In some embodiments, the reduction in any one or all of properties (1)-(3) is compared to an otherwise similar antibody having a wild-type Fc region.

[0221]

[0227] In some embodiments, the anti-TCRVα antibody comprising a variant Fc region has reduced affinity for a human Fc receptor, e.g., FcγRI, FcγRII and / or FcγRIII. In some embodiments, the anti-TCRVα antibody comprising a variant Fc region comprises a human IgG1 region or a human IgG4 region.

[0222]

[0228] In some embodiments, anti-TCRVα antibodies comprising a variant Fc region activate and / or expand T cells, e.g., as described herein. In some embodiments, anti-TCRVα antibodies comprising a variant Fc region have a cytokine profile that differs from the cytokine profile described herein, e.g., the cytokine profile of a T cell engager that binds to a receptor or molecule other than the TCRαV region (a "non-TCRαV binding T cell engager"). In some embodiments, a non-TCRαV binding T cell engager comprises an antibody that binds to a CD3 molecule (e.g., a CD3 epsilon (CD3e) molecule); or a TCR alpha (TCRα) molecule.

[0223]

[0229] Exemplary Fc region variants are provided in Table 6 and are also disclosed in Saunders O, (2019) Frontiers in Immunology; vol 10, article 1296, the entire contents of which are incorporated herein by reference.

[0224]

[0230] In some embodiments, the anti-TCRVα antibodies described herein comprise any one or all, or any combination, of the Fc region variants disclosed in Table 6.

[0225]

[0231] In some embodiments, the anti-TCRV alpha antibodies described herein comprise any one or all, or any combination of, Fc region variants, e.g., mutations, disclosed in Table 6. In some embodiments, the anti-TCRV alpha antibodies described herein comprise an Asn297Ala (N297A) mutation. In some embodiments, the anti-TCRV alpha antibodies described herein comprise a Leu234Ala / Leu235Ala (LALA) mutation.

[0226] multifunctional molecules

[0232] As used herein, a "multifunctional" or "multispecific" molecule refers to a molecule, e.g., a polypeptide, having two or more functionalities, e.g., two or more binding specificities. In some embodiments, the functionalities can include one or more immune cell engagers, one or more tumor-binding molecules, one or more cytokine molecules, one or more stromal modifying agents, and other moieties described herein. In some embodiments, the multifunctional molecule is a multispecific antibody molecule, e.g., a bispecific antibody molecule. In some embodiments, the multispecific molecule includes an anti-TCRVA antibody molecule described herein.

[0227]

[0233] In certain embodiments, a multifunctional polypeptide molecule is provided comprising a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, and at least one cytokine polypeptide or variant thereof, wherein the first polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide are non-contiguous, and (i) the first polypeptide comprises a first dimerization module linked to a first portion of a first T cell receptor variable alpha (TCRαV) binding portion and a first portion of the first TCRαV binding portion; (ii) the second polypeptide comprises Described herein are multifunctional polypeptide molecules comprising: (i) a second portion of the first TCRαV binding portion; (ii) a third polypeptide comprising a second dimerization module linked to a first portion of the second TCRαV binding portion and a first portion of the second TCRαV binding portion; and (iii) a fourth polypeptide comprising a second portion of the second TCRαV binding portion; and at least one cytokine polypeptide or variant thereof is covalently linked to the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof.

[0228]

[0234] In certain embodiments, a multifunctional polypeptide molecule is described herein comprising a first polypeptide, a second polypeptide, a third polypeptide, and at least one cytokine polypeptide or variant thereof, wherein the first polypeptide, the second polypeptide, and the third polypeptide are non-contiguous, and (i) the first polypeptide comprises a first dimerization module linked to a first portion of the first TCRαV binding moiety and the first portion of the first TCRαV binding moiety; (ii) the second polypeptide comprises a second portion of the first TCRαV binding moiety; (iii) the third polypeptide comprises a second dimerization module; and the at least one cytokine polypeptide or variant thereof is covalently linked to the first polypeptide, the second polypeptide, the third polypeptide, or a combination thereof.

[0229]

[0235] In certain embodiments, a multifunctional polypeptide molecule is described herein comprising a first polypeptide, a second polypeptide, a third polypeptide, and at least one cytokine polypeptide or variant thereof, wherein the first polypeptide, the second polypeptide, and the third polypeptide are non-contiguous, and (i) the first polypeptide comprises a first dimerization module linked to a first portion of the first TCRαV binding moiety and a first portion of the first TCRαV binding moiety; (ii) the second polypeptide comprises a second portion of the first TCRαV binding moiety; (iii) the third polypeptide comprises a second dimerization module; the at least one cytokine polypeptide or variant thereof is covalently linked to the first polypeptide, the second polypeptide, the third polypeptide, or a combination thereof; and the multifunctional polypeptide molecule does not comprise any additional TCRαV binding moieties except for the first TCRαV binding moiety.

[0230]

[0236] In some embodiments, the first portion of the first TCRαV binding moiety comprises a first heavy chain variable domain (VH) and a first heavy chain constant domain (CH1) linked to the first VH. In some embodiments, the first CH1 is linked to the C-terminus of the first VH. In some embodiments, the second portion of the first TCRαV binding moiety comprises a first light chain variable domain (VL) and a first light chain constant domain (CL) linked to the first VL. In some embodiments, the first CL is linked to the C-terminus of the first VL. In some embodiments, a first dimerization module is linked to the first portion of the first TCRαV binding moiety. In some embodiments, the first dimerization module is linked to the C-terminus of the first portion of the first TCRαV binding moiety. In some embodiments, the first portion of the second TCRαV binding moiety comprises a second CH1 linked to a second VH and a second VH. In some embodiments, the second CH1 is linked to the C-terminus of the second VH. In some embodiments, the second portion of the second TCRαV binding moiety comprises a second VL and a second CL linked to the second VL. In some embodiments, the second CL is linked to the C-terminus of the second VL. In some embodiments, the second dimerization module is linked to the first portion of the second TCRαV binding moiety. In some embodiments, the second dimerization module is linked to the C-terminus of the first portion of the second TCRαV binding moiety.

[0231]

[0237] In some embodiments, (a) the N-terminus of the first polypeptide is linked to a first cytokine polypeptide or variant thereof; the C-terminus of the first polypeptide is linked to a second cytokine polypeptide or variant thereof; or a combination thereof; (b) the N-terminus of the second polypeptide is linked to a third cytokine polypeptide or variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide or variant thereof; or a combination thereof; (c) the N-terminus of the third polypeptide is linked to a fifth cytokine polypeptide or variant thereof; the C-terminus of the third polypeptide is linked to a sixth cytokine polypeptide or variant thereof; or a combination thereof; (d) the N-terminus of the fourth polypeptide is linked to a seventh cytokine polypeptide or variant thereof; the C-terminus of the fourth polypeptide is linked to an eighth cytokine polypeptide or variant thereof; or a combination thereof; or (e) a combination thereof.

[0232]

[0238] In some embodiments, (a-1) the N-terminus of the first polypeptide is linked to a first cytokine polypeptide or variant thereof; the C-terminus of the first polypeptide is linked to a second cytokine polypeptide or variant thereof; or a combination thereof; (a-2) the N-terminus of the second polypeptide is linked to a third cytokine polypeptide or variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide or variant thereof; or a combination thereof; (b-1) the N-terminus of the first polypeptide is linked to a first cytokine polypeptide or variant thereof; the C-terminus of the first polypeptide is linked to a second cytokine polypeptide or variant thereof; or a combination thereof; (b-2) the N-terminus of the third polypeptide is linked to a fifth cytokine polypeptide or variant thereof; the C-terminus of the third polypeptide is linked to a sixth cytokine polypeptide or variant thereof; or a combination thereof. (c-1) the N-terminus of the first polypeptide is linked to a first cytokine polypeptide or a variant thereof; the C-terminus of the first polypeptide is linked to a second cytokine polypeptide or a variant thereof; or a combination thereof; (c-2) the N-terminus of the fourth polypeptide is linked to a seventh cytokine polypeptide or a variant thereof; the C-terminus of the fourth polypeptide is linked to an eighth cytokine polypeptide or a variant thereof; or a combination thereof; (d-1) the N-terminus of the second polypeptide is linked to a third cytokine polypeptide or a variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide or a variant thereof; or a combination thereof; (d-2) the N-terminus of the third polypeptide is linked to a fifth cytokine polypeptide or a variant thereof; the C-terminus of the third polypeptide is linked to a sixth cytokine polypeptide or a variant thereof; or a combination thereof;(e-1) the N-terminus of the second polypeptide is linked to a third cytokine polypeptide or a variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide or a variant thereof; or a combination thereof; (e-2) the N-terminus of the fourth polypeptide is linked to a seventh cytokine polypeptide or a variant thereof; the C-terminus of the fourth polypeptide is linked to an eighth cytokine polypeptide or a variant thereof; or a combination thereof; or (f-1) the N-terminus of the third polypeptide is linked to a fifth cytokine polypeptide or a variant thereof; the C-terminus of the third polypeptide is linked to a sixth cytokine polypeptide or a variant thereof; or a combination thereof; (f-2) the N-terminus of the fourth polypeptide is linked to a seventh cytokine polypeptide or a variant thereof; the C-terminus of the fourth polypeptide is linked to an eighth cytokine polypeptide or a variant thereof; or a combination thereof;

[0233]

[0239] In some embodiments, (a-1) the N-terminus of the first polypeptide is linked to a first cytokine polypeptide or variant thereof; the C-terminus of the first polypeptide is linked to a second cytokine polypeptide or variant thereof; or a combination thereof; (a-2) the N-terminus of the second polypeptide is linked to a third cytokine polypeptide or variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide or variant thereof; or a combination thereof; (a-3) the N-terminus of the third polypeptide is linked to a fifth cytokine polypeptide or variant thereof; the C-terminus of the third polypeptide is linked to a sixth cytokine polypeptide or variant thereof; or a combination thereof; (b-1) the N-terminus of the first polypeptide is linked to a first cytokine polypeptide or variant thereof; the C-terminus of the first polypeptide is linked to a second cytokine polypeptide or variant thereof; or a combination thereof. (b-2) the N-terminus of the second polypeptide is linked to a third cytokine polypeptide or a variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide or a variant thereof; or a combination thereof; (b-3) the N-terminus of the fourth polypeptide is linked to a seventh cytokine polypeptide or a variant thereof; the C-terminus of the fourth polypeptide is linked to an eighth cytokine polypeptide or a variant thereof; or a combination thereof; or (c-1) the N-terminus of the second polypeptide is linked to a third cytokine polypeptide or a variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide or a variant thereof; or a combination thereof; (c-2) the N-terminus of the third polypeptide is linked to a fifth cytokine polypeptide or a variant thereof; the C-terminus of the third polypeptide is linked to a sixth cytokine polypeptide or a variant thereof; or a combination thereof;(c-3) the N-terminus of the fourth polypeptide is linked to a seventh cytokine polypeptide or a variant thereof; the C-terminus of the fourth polypeptide is linked to an eighth cytokine polypeptide or a variant thereof; or a combination thereof;

[0234]

[0240] In some embodiments, (1) the N-terminus of the first polypeptide is linked to a first cytokine polypeptide or variant thereof; the C-terminus of the first polypeptide is linked to a second cytokine polypeptide or variant thereof; or a combination thereof; (2) the N-terminus of the second polypeptide is linked to a third cytokine polypeptide or variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide or variant thereof; or a combination thereof; (3) the N-terminus of the third polypeptide is linked to a fifth cytokine polypeptide or variant thereof; the C-terminus of the third polypeptide is linked to a sixth cytokine polypeptide or variant thereof; or a combination thereof; (4) the N-terminus of the fourth polypeptide is linked to a seventh cytokine polypeptide or variant thereof; the C-terminus of the fourth polypeptide is linked to an eighth cytokine polypeptide or variant thereof; or a combination thereof.

[0235]

[0241] In some embodiments, a first cytokine polypeptide, a second cytokine polypeptide, or a combination thereof is present in a single contiguous polypeptide chain of a first polypeptide, a third cytokine polypeptide, a fourth cytokine polypeptide, or a combination thereof is present in a single contiguous polypeptide chain of a second polypeptide, a fifth cytokine polypeptide, a sixth cytokine polypeptide, or a combination thereof is present in a single contiguous polypeptide chain of a third polypeptide, a seventh cytokine polypeptide, an eighth cytokine polypeptide, or a combination thereof is present in a single contiguous polypeptide chain of a fourth polypeptide, or a combination thereof.

[0236]

[0242] In some embodiments, (a) the N-terminus of the first polypeptide is linked to a first cytokine polypeptide or variant thereof; the C-terminus of the first polypeptide is linked to a second cytokine polypeptide or variant thereof; or a combination thereof; (b) the N-terminus of the second polypeptide is linked to a third cytokine polypeptide or variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide or variant thereof; or a combination thereof; (c) the N-terminus of the third polypeptide is linked to a fifth cytokine polypeptide or variant thereof; the C-terminus of the third polypeptide is linked to a sixth cytokine polypeptide or variant thereof; or a combination thereof; or (d) a combination thereof.

[0237]

[0243] In some embodiments, (a-1) the N-terminus of the first polypeptide is linked to a first cytokine polypeptide or variant thereof; the C-terminus of the first polypeptide is linked to a second cytokine polypeptide or variant thereof; or a combination thereof; (a-2) the N-terminus of the second polypeptide is linked to a third cytokine polypeptide or variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide or variant thereof; or a combination thereof; (b-1) the N-terminus of the first polypeptide is linked to a first cytokine polypeptide or variant thereof; the C-terminus of the first polypeptide is linked to a second cytokine polypeptide or variant thereof; or a combination thereof. (b-2) the N-terminus of the third polypeptide is linked to a fifth cytokine polypeptide or a variant thereof; the C-terminus of the third polypeptide is linked to a sixth cytokine polypeptide or a variant thereof; or a combination thereof; or (c-1) the N-terminus of the second polypeptide is linked to a third cytokine polypeptide or a variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide or a variant thereof; or a combination thereof; (c-2) the N-terminus of the third polypeptide is linked to a fifth cytokine polypeptide or a variant thereof; the C-terminus of the third polypeptide is linked to a sixth cytokine polypeptide or a variant thereof; or a combination thereof.

[0238]

[0244] In some embodiments, (1) the N-terminus of the first polypeptide is linked to a first cytokine polypeptide or variant thereof; the C-terminus of the first polypeptide is linked to a second cytokine polypeptide or variant thereof; or a combination thereof; (2) the N-terminus of the second polypeptide is linked to a third cytokine polypeptide or variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide or variant thereof; or a combination thereof; (3) the N-terminus of the third polypeptide is linked to a fifth cytokine polypeptide or variant thereof; the C-terminus of the third polypeptide is linked to a sixth cytokine polypeptide or variant thereof; or a combination thereof.

[0239]

[0245] In some embodiments, the first cytokine polypeptide, the second cytokine polypeptide, or a combination thereof is present in a single contiguous polypeptide chain of a first polypeptide, the third cytokine polypeptide, the fourth cytokine polypeptide, or a combination thereof is present in a single contiguous polypeptide chain of a second polypeptide, the fifth cytokine polypeptide, the sixth cytokine polypeptide, or a combination thereof is present in a single contiguous polypeptide chain of a third polypeptide, or a combination thereof.

[0240]

[0246] In some embodiments, the multifunctional polypeptide molecule described herein further comprises a linker between the first portion of the first TCRαV binding moiety and the first dimerization module, a linker between the first portion of the second TCRαV binding moiety and the second dimerization module, a linker between the first VH and the first CH1, a linker between the first VL and the first CL, a linker between the second VH and the second CH1, a linker between the second VL and the second CL, a linker between at least one cytokine polypeptide or variant thereof and the first polypeptide, a linker between at least one cytokine polypeptide or variant thereof and the second polypeptide, a linker between at least one cytokine polypeptide or variant thereof and a third polypeptide, a linker between at least one cytokine polypeptide or variant thereof and a fourth polypeptide, or a combination thereof.

[0241]

[0247] In some embodiments, the multifunctional polypeptide molecule described herein further comprises a linker between the first portion of the first TCRαV binding portion and the first dimerization module, a linker between the first VH and the first CH1, a linker between the first VL and the first CL, a linker between at least one cytokine polypeptide or variant thereof and the first polypeptide, a linker between at least one cytokine polypeptide or variant thereof and the second polypeptide, a linker between at least one cytokine polypeptide or variant thereof and a third polypeptide, or a combination thereof. In some embodiments, the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helix linker, and a non-helical linker. In some embodiments, the linker is a peptide linker and is a GS linker. In some embodiments, the linker is a peptide linker and comprises the sequence of SEQ ID NO: 3308 or SEQ ID NO: 3643.

[0242]

[0248] In certain embodiments, a multifunctional polypeptide molecule is provided comprising a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, a first cytokine polypeptide or a variant thereof, and a second cytokine polypeptide or a variant thereof, wherein the first polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide are non-contiguous, and (i) the first polypeptide comprises a first portion of a first TCRαV binding moiety and a first dimerization module linked to the first portion of the first TCRαV binding moiety; (ii) the second polypeptide comprises a first dimerization module linked to the first portion of the first TCRαV binding moiety; Described herein are multifunctional polypeptide molecules, wherein: (i) a first polypeptide comprises a second portion of the TCRαV binding portion; (ii) a third polypeptide comprises a second dimerization module linked to a first portion of the second TCRαV binding portion and to a first portion of the second TCRαV binding portion; and (iii) a fourth polypeptide comprises a second portion of the second TCRαV binding portion; a first cytokine polypeptide or variant thereof is covalently linked to the C-terminus of the second polypeptide, and a second cytokine polypeptide or variant thereof is covalently linked to the C-terminus of the fourth polypeptide.

[0243]

[0249] In certain embodiments, a multifunctional polypeptide molecule is described herein comprising a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, a cytokine polypeptide or a variant thereof, wherein the first polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide are non-contiguous, and wherein (i) the first polypeptide comprises a first dimerization module linked to a first portion of the first TCRαV binding moiety and a first portion of the first TCRαV binding moiety; (ii) the second polypeptide comprises a second portion of the first TCRαV binding moiety; (iii) the third polypeptide comprises a second dimerization module linked to a first portion of the second TCRαV binding moiety and a first portion of the second TCRαV binding moiety; (iv) the fourth polypeptide comprises a second portion of the second TCRαV binding moiety; and the cytokine polypeptide or a variant thereof is covalently linked to the C-terminus of the second polypeptide or the C-terminus of the fourth polypeptide.

[0244]

[0250] In certain embodiments, a multifunctional polypeptide molecule is described herein comprising a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, a cytokine polypeptide or a variant thereof, wherein the first polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide are non-contiguous, and (i) the first polypeptide comprises a first dimerization module linked to a first portion of the first TCRαV binding moiety and a first portion of the first TCRαV binding moiety; (ii) the second polypeptide comprises a second portion of the first TCRαV binding moiety; (iii) the third polypeptide comprises a second dimerization module linked to a first portion of the second TCRαV binding moiety and a first portion of the second TCRαV binding moiety; (iv) the fourth polypeptide comprises a second portion of the second TCRαV binding moiety; and the cytokine polypeptide or a variant thereof is covalently linked to the C-terminus of the first polypeptide or the C-terminus of the third polypeptide.

[0245]

[0251] In certain embodiments, a multifunctional polypeptide molecule is described herein comprising a first polypeptide, a second polypeptide, a third polypeptide, and a cytokine polypeptide or variant thereof, wherein the first polypeptide, the second polypeptide, and the third polypeptide are non-contiguous, and (i) the first polypeptide comprises a first dimerization module linked to a first portion of the first TCRαV binding moiety and a first portion of the first TCRαV binding moiety; (ii) the second polypeptide comprises a second portion of the first TCRαV binding moiety; (iii) the third polypeptide comprises a second dimerization module; at least one cytokine polypeptide or variant thereof is covalently linked to the N-terminus of the third polypeptide; and the multifunctional polypeptide molecule does not comprise an additional TCRαV binding moiety other than the first TCRαV binding moiety.

[0246]

[0252] In some embodiments, the first portion of the first TCRαV-binding portion comprises a first VH and a first CH1 linked to the first VH. In some embodiments, the first CH1 is linked to the C-terminus of the first VH.

[0247]

[0253] In some embodiments, the second portion of the first TCRαV-binding portion comprises a first VL and a first CL linked to the first VL. In some embodiments, the first CL is linked to the C-terminus of the first VL.

[0248]

[0254] In some embodiments, the first dimerization module is linked to the first portion of the first TCRαV binding moiety. In some embodiments, the first dimerization module is linked to the C-terminus of the first portion of the first TCRαV binding moiety. In some embodiments, the first portion of the second TCRαV binding moiety comprises a second VH and a second CH1 linked to the second VH. In some embodiments, the second CH1 is linked to the C-terminus of the second VH. In some embodiments, the second portion of the second TCRαV binding moiety comprises a second VL and a second CL linked to the second VL. In some embodiments, the second CL is linked to the C-terminus of the second VL. In some embodiments, the second dimerization module is linked to the first portion of the second TCRαV binding moiety. In some embodiments, the second dimerization module is linked to the C-terminus of the first portion of the second TCRαV binding portion.

[0249]

[0255] In some embodiments, the multifunctional polypeptide molecule described herein further comprises a linker between the first portion of the first TCRαV binding moiety and the first dimerization module, a linker between the first portion of the second TCRαV binding moiety and the second dimerization module, a linker between the first VH and the first CH1, a linker between the first VL and the first CL, a linker between the second VH and the second CH1, a linker between the second VL and the second CL, a linker between at least one cytokine polypeptide or variant thereof and the first polypeptide, a linker between at least one cytokine polypeptide or variant thereof and the second polypeptide, a linker between at least one cytokine polypeptide or variant thereof and a third polypeptide, a linker between at least one cytokine polypeptide or variant thereof and a fourth polypeptide, or a combination thereof. In some embodiments, the multifunctional polypeptide molecule described herein further comprises a linker between the first portion of the first TCRαV binding portion and the first dimerization module, a linker between the first VH and the first CH1, a linker between the first VL and the first CL, a linker between at least one cytokine polypeptide or a variant thereof and a third polypeptide, or a combination thereof. In some embodiments, the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helix linker, and a non-helical linker. In some embodiments, the linker is a peptide linker and is a GS linker. In some embodiments, the linker is a peptide linker and comprises the sequence of SEQ ID NO: 3308 or SEQ ID NO: 3643.

[0250]

[0256] In some embodiments, the first TCRαV binding moiety, the second TCRαV binding moiety, or a combination thereof comprises any one selected from the group consisting of a Fab, a F(ab')2, an Fv, a single chain Fv (scFv), a single domain antibody, a diabody (dAb), a camelid antibody, and a combination thereof. In some embodiments, the first TCRαV binding moiety, the second TCRαV binding moiety, or a combination thereof comprises an scFv or a Fab.

[0251]

[0257] In some embodiments, the multifunctional polypeptide molecule does not comprise an additional antigen-binding moiety other than the TCRαV-binding moiety, hi some embodiments, the multifunctional polypeptide molecule further comprises an additional antigen-binding moiety that is not a TCRαV-binding moiety.

[0252]

[0258] In certain embodiments, a multifunctional polypeptide molecule is provided comprising a first polypeptide, a second polypeptide, and at least one cytokine polypeptide or variant thereof, wherein the first polypeptide and the second polypeptide are non-contiguous, and wherein: (i) the first polypeptide comprises a first TCRαV binding moiety comprising a first VL and a first VH and a first dimerization module linked to the C-terminus of the first TCRαV binding moiety; (ii) the second polypeptide comprises a second TCRαV binding moiety and a second VH. Described herein is a multifunctional polypeptide molecule comprising: a second dimerization module linked to the C-terminus of the TCRαV binding portion; at least one cytokine polypeptide or variant thereof is covalently linked to the first polypeptide, the second polypeptide, or a combination thereof; the first TCRαV binding portion, the second TCRαV binding portion, or a combination thereof comprises an scFv; and does not comprise any additional antigen-binding portions except for the first TCRαV binding portion and the second TCRαV binding portion.

[0253]

[0259] In certain embodiments, a multifunctional polypeptide molecule is described herein comprising a first polypeptide, a second polypeptide, and at least one cytokine polypeptide or variant thereof, wherein the first polypeptide and the second polypeptide are non-contiguous, and (i) the first polypeptide comprises a first TCRαV binding portion and a first dimerization module linked to the C-terminus of the first TCRαV binding portion, the first TCRαV binding portion comprising a first VL and a first VH; (ii) the second polypeptide comprises a second dimerization module; the at least one cytokine polypeptide or variant thereof is covalently linked to the first polypeptide, the second polypeptide, or a combination thereof; the first TCRαV binding portion comprises an scFv; no additional antigen binding portion is included except for the first TCRαV binding portion; and no additional TCRαV binding portion is included except for the first TCRαV binding portion.

[0254]

[0260] In some embodiments, (a) the N-terminus of the first polypeptide is linked to a first cytokine polypeptide or variant thereof; the C-terminus of the first polypeptide is linked to a second cytokine polypeptide or variant thereof; or a combination thereof; (b) the N-terminus of the second polypeptide is linked to a third cytokine polypeptide or variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide or variant thereof; or a combination thereof; or (e) a combination thereof.

[0255]

[0261] In some embodiments, the first cytokine polypeptide, the second cytokine polypeptide, or a combination thereof is present in a single contiguous polypeptide chain of the first polypeptide, and the third cytokine polypeptide, the fourth cytokine polypeptide, or a combination thereof is present in a single contiguous polypeptide chain of the second polypeptide, or a combination thereof.

[0256]

[0262] In some embodiments, the multifunctional polypeptide molecule described herein further comprises a linker between the first TCRαV binding portion and the first dimerization module, a linker between the second TCRαV binding portion and the second dimerization module, a linker between at least one cytokine polypeptide or variant thereof and the first polypeptide, a linker between at least one cytokine polypeptide or variant thereof and the second polypeptide, or a combination thereof.

[0257]

[0263] In some embodiments, the multifunctional polypeptide molecule described herein further comprises a linker between the first TCRαV binding moiety and the first dimerization module, a linker between at least one cytokine polypeptide or variant thereof and the first polypeptide, a linker between at least one cytokine polypeptide or variant thereof and the second polypeptide, or a combination thereof. In some embodiments, the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helix linker, and a non-helical linker. In some embodiments, the linker is a peptide linker and is a GS linker. In some embodiments, the linker is a peptide linker and comprises the sequence of SEQ ID NO: 3308 or SEQ ID NO: 3643.

[0258]

[0264] In some embodiments, the multifunctional polypeptide molecule comprises at least two of the cytokine polypeptides. In some embodiments, the multifunctional polypeptide molecule comprises at least three of the cytokine polypeptides. In some embodiments, the multifunctional polypeptide molecule comprises at least four of the cytokine polypeptides. In some embodiments, the multifunctional polypeptide molecule comprises at least five of the cytokine polypeptides. In some embodiments, the multifunctional polypeptide molecule comprises at least six of the cytokine polypeptides. In some embodiments, the multifunctional polypeptide molecule comprises at least seven of the cytokine polypeptides. In some embodiments, the multifunctional polypeptide molecule comprises at least eight of the cytokine polypeptides. In some embodiments, the multifunctional polypeptide molecule comprises two of the cytokine polypeptides. In some embodiments, the multifunctional polypeptide molecule comprises three of the cytokine polypeptides. In some embodiments, the multifunctional polypeptide molecule comprises four of the cytokine polypeptides. In some embodiments, the multifunctional polypeptide molecule comprises five of the cytokine polypeptides. In some embodiments, the multifunctional polypeptide molecule comprises six of the cytokine polypeptides. In some embodiments, the multifunctional polypeptide molecule comprises seven of the cytokine polypeptides. In some embodiments, the multifunctional polypeptide molecule comprises eight of the cytokine polypeptides. In some embodiments, the multifunctional polypeptide molecule comprises two of the cytokine polypeptides, each of which is linked to a first polypeptide and a second polypeptide; a first polypeptide and a third polypeptide; a first polypeptide and a fourth polypeptide; a second polypeptide and a third polypeptide; a second polypeptide and a fourth polypeptide; or a third polypeptide and a fourth polypeptide, respectively.In some embodiments, the multifunctional polypeptide molecule comprises three of the cytokine polypeptides, each of which is linked to a first polypeptide, a second polypeptide, and a third polypeptide; a first polypeptide, a second polypeptide, and a fourth polypeptide; a first polypeptide, a third polypeptide, and a fourth polypeptide; or a second polypeptide, a third polypeptide, and a fourth polypeptide. In some embodiments, the multifunctional polypeptide molecule comprises four of the cytokine polypeptides, each of which is linked to a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, respectively. In some embodiments, the cytokine polypeptide is linked to a polypeptide comprising a first TCRαV binding portion.

[0259]

[0265] In some embodiments, the at least one cytokine polypeptide is selected from the group consisting of interleukin-2 (IL-2) or a fragment or variant thereof, interleukin-7 (IL-7) or a fragment or variant thereof, interleukin-12 (IL-12) or a fragment or variant thereof, interleukin-15 (IL-15) or a fragment or variant thereof, interleukin-18 (IL-18) or a fragment or variant thereof, interleukin-21 (IL-21) or a fragment or variant thereof, or interferon gamma or a variant thereof, or a combination thereof.

[0260]

[0266] In some embodiments, at least one cytokine polypeptide comprises interleukin-2 (IL-2) or a fragment thereof. In some embodiments, at least one cytokine polypeptide is interleukin-2 (IL-2) or a fragment thereof. In some embodiments, at least one cytokine polypeptide comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO:2191. In some embodiments, at least one cytokine polypeptide comprises a sequence of SEQ ID NO:2191. In some embodiments, the sequence of at least one cytokine polypeptide is a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO:2191. In some embodiments, the sequence of at least one cytokine polypeptide is a sequence of SEQ ID NO:2191.

[0261]

[0267] In some embodiments, the variant of at least one cytokine polypeptide comprises an IL-2 variant comprising a mutation. In some embodiments, the mutation comprises an insertion mutation, a deletion mutation, or a substitution mutation. In some embodiments, the mutation comprises a substitution mutation. In some embodiments, the variant comprises an IL-2 variant comprising a C125A mutation. In some embodiments, the variant of at least one cytokine polypeptide is an IL-2 variant comprising a mutation. In some embodiments, the mutation is an insertion mutation, a deletion mutation, or a substitution mutation. In some embodiments, the mutation is a substitution mutation. In some embodiments, the variant is an IL-2 variant comprising a C125A mutation. In some embodiments, the variant comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO:2270. In some embodiments, the variant comprises the sequence of SEQ ID NO:2270. In some embodiments, the variant sequence is a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 2270. In some embodiments, the variant sequence is the sequence of SEQ ID NO: 2270.

[0262]

[0268] In some embodiments, the first dimerization module comprises a first immunoglobulin constant region (Fc region) and the second dimerization module comprises a second Fc region. In some embodiments, the first dimerization module is a first immunoglobulin constant region (Fc region) and the second dimerization module is a second Fc region.

[0263]

[0269] In some embodiments, the first Fc region, the second Fc region, or a combination thereof is selected from an IgG1 Fc region or fragment thereof, an IgG2 Fc region or fragment thereof, an IgG3 Fc region or fragment thereof, an IgGA1 Fc region or fragment thereof, an IgGA2 Fc region or fragment thereof, an IgGA4 Fc region or fragment thereof, an IgJ Fc region or fragment thereof, an IgM Fc region or fragment thereof, an IgD Fc region or fragment thereof, and an IgE Fc region or fragment thereof.

[0264]

[0270] In some embodiments, the first Fc region, the second Fc region, or a combination thereof is selected from a human IgG1 Fc region or fragment thereof, a human IgG2 Fc region or fragment thereof, and a human IgG4 Fc region or fragment thereof.

[0265]

[0271] In some embodiments, the first Fc region, the second Fc region, or a combination thereof comprises an Fc interface having one or more of paired holes and protrusions, electrostatic interactions, or strand exchange, and dimerization of the first Fc region and the second Fc region is enhanced as indicated by a higher ratio of heteromultimer:homomultimer forms compared to dimerization of an Fc region having an unengineered interface. In some embodiments, dimerization of the first Fc region and the second Fc region is at least 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 55 fold, 60 fold, 65 fold, 70 fold, 75 fold, 80 fold, 85 fold, 90 fold, 100 fold, 105 ... fold, 70x, 75x, 80x, 85x, 90x, 95x, 100x, 150x, 200x, 250x, 300x, 250x, 400x, 450x, 500x, 550x, 600x, 650x, 700x, 750x, 800x, 850x, 900x, 950x, 1000x, 2000x, 3000x, 4000x, 5000x, 6000x, 7000x, 8000x, 9000x, or 1000x. In some embodiments, dimerization of the first Fc region and the second Fc region is increased by up to 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 55 fold, 60 fold, 65 fold, The antibody may be enhanced by 70x, 75x, 80x, 85x, 90x, 95x, 100x, 150x, 200x, 250x, 300x, 250x, 400x, 450x, 500x, 550x, 600x, 650x, 700x, 750x, 800x, 850x, 900x, 950x, 1000x, 2000x, 3000x, 4000x, 5000x, 6000x, 7000x, 8000x, 9000x, or 10000x.In some embodiments, dimerization of the first Fc region and the second Fc region is 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 55 fold, 60 fold, 65 ...10 fold, 15 fold, 10 fold, 15 fold, 10 The antibody is enhanced by 0x, 75x, 80x, 85x, 90x, 95x, 100x, 150x, 200x, 250x, 300x, 250x, 400x, 450x, 500x, 550x, 600x, 650x, 700x, 750x, 800x, 850x, 900x, 950x, 1000x, 2000x, 3000x, 4000x, 5000x, 6000x, 7000x, 8000x, 9000x, or 10000x.

[0266]

[0272] In some embodiments, the first Fc region, the second Fc region, or a combination thereof comprises an amino acid substitution listed in Table 6.

[0267]

[0273] In some embodiments, the first Fc region, the second Fc region, or a combination thereof comprises an Asn297Ala (N297A) mutation or a Leu234Ala / Leu235Ala (LALA) mutation.

[0268]

[0274] In some embodiments, the first Fc region, the second Fc region, or a combination thereof comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 3645, SEQ ID NO: 3646, SEQ ID NO: 3647, SEQ ID NO: 3648, or SEQ ID NO: 3649. In some embodiments, the first Fc region, the second Fc region, or a combination thereof comprises the sequence of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 3645, SEQ ID NO: 3646, SEQ ID NO: 3647, SEQ ID NO: 3648, or SEQ ID NO: 3649.

[0269]

[0275] In some embodiments, the sequence of the first Fc region, the second Fc region, or a combination thereof is a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 3645, SEQ ID NO: 3646, SEQ ID NO: 3647, SEQ ID NO: 3648, or SEQ ID NO: 3649. In some embodiments, the sequence of the first Fc region, the second Fc region, or a combination thereof is a sequence of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 3645, SEQ ID NO: 3646, SEQ ID NO: 3647, SEQ ID NO: 3648, or SEQ ID NO: 3649.

[0270]

[0276] In some embodiments, the first TCRαV binding moiety and the second TCRαV binding moiety are the same. In some embodiments, the first TCRαV binding moiety and the second TCRαV binding moiety are different.

[0271]

[0277] In some embodiments, the first TCRαV binding portion, the second TCRαV binding portion, or a combination thereof, has (i) at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the non-mouse germline framework 1 (FR1), the non-mouse germline framework region 2 (FR2), the non-mouse germline framework region 3 (FR3), and the non-mouse germline framework region 4 (FR4). (ii) a VL comprising a FR comprising FR1, FR2, FR3, and FR4 that have at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to a non-mouse germline FR1, a non-mouse germline FR2, a non-mouse germline FR3, and a non-mouse germline FR4; or (iii) a combination thereof. In some embodiments, the first TCRαV binding portion, the second TCRαV binding portion, or a combination thereof, comprises: (i) a VH comprising FRs comprising FR1, FR2, FR3, and FR4 having the sequences of non-mouse germline FR1, non-mouse germline FR2, non-mouse germline FR3, and non-mouse germline FR4; (ii) a VL comprising FRs comprising FR1, FR2, FR3, and FR4 having the sequences of non-mouse germline FR1, non-mouse germline FR2, non-mouse germline FR3, and non-mouse germline FR4; or (iii) a combination thereof.

[0272]

[0278] In some embodiments, the first TCRαV binding portion, the second TCRαV binding portion, or a combination thereof comprises: (i) a VH comprising FR1, FR2, FR3, and FR4 that have at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity with non-mouse germline FR1, non-mouse germline FR2, non-mouse germline FR3, and non-mouse germline FR4, respectively; (ii) a VL comprising FRs comprising FR1, FR2, FR3, and FR4 that have at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity with non-mouse germline FR1, non-mouse germline FR2, non-mouse germline FR3, and non-mouse germline FR4, respectively; or (iii) a combination thereof. In some embodiments, the first TCRαV binding portion, the second TCRαV binding portion, or a combination thereof, comprises: (i) a VH comprising FRs comprising FR1, FR2, FR3, and FR4 having the sequences of non-mouse germline FR1, non-mouse germline FR2, non-mouse germline FR3, and non-mouse germline FR4, respectively; (ii) a VL comprising FRs comprising FR1, FR2, FR3, and FR4 having the sequences of non-mouse germline FR1, non-mouse germline FR2, non-mouse germline FR3, and non-mouse germline FR4, respectively; or (iii) a combination thereof.

[0273]

[0279] In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a heavy chain constant region having a sequence that has at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the sequences listed in Table 1 or a combination thereof. In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a heavy chain constant region having a sequence that has at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the sequences listed in Table 1 or a combination thereof. In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a heavy chain constant region having any one or a combination of the heavy chain constant region sequences listed in Table 1. In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises an IgM heavy chain constant region or a fragment thereof. In some embodiments, the IgM heavy chain constant region comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 73. In some embodiments, the IgM heavy chain constant region comprises the sequence of SEQ ID NO: 73. In some embodiments, the IgM heavy chain constant region sequence is the sequence of SEQ ID NO: 73.

[0274]

[0280] In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a heavy chain constant region of IgJ or a fragment thereof. In some embodiments, the heavy chain constant region of IgJ comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 76. In some embodiments, the heavy chain constant region of IgJ comprises the sequence of SEQ ID NO: 76. In some embodiments, the sequence of the heavy chain constant region of IgJ is the sequence of SEQ ID NO: 76.

[0275]

[0281] In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a heavy chain constant region of IgGA1 or a fragment thereof. In some embodiments, the heavy chain constant region of IgGA1 comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 74. In some embodiments, the heavy chain constant region of IgGA1 comprises the sequence of SEQ ID NO: 74. In some embodiments, the sequence of the heavy chain constant region of IgGA1 is the sequence of SEQ ID NO: 74.

[0276]

[0282] In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a heavy chain constant region of IgGA2 or a fragment thereof. In some embodiments, the heavy chain constant region of IgGA2 comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 75. In some embodiments, the heavy chain constant region of IgGA2 comprises the sequence of SEQ ID NO: 75. In some embodiments, the sequence of the heavy chain constant region of IgGA2 is the sequence of SEQ ID NO: 75.

[0277]

[0283] In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises an IgG1 heavy chain constant region or a fragment thereof. In some embodiments, the IgG1 heavy chain constant region comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 41. In some embodiments, the IgG1 heavy chain constant region comprises a sequence of SEQ ID NO: 41. In some embodiments, the IgG1 heavy chain constant region is a sequence of SEQ ID NO: 41. In some embodiments, the IgG1 heavy chain constant region comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 3645. In some embodiments, the IgG1 heavy chain constant region comprises a sequence of SEQ ID NO: 3645. In some embodiments, the sequence of the heavy chain constant region of IgG1 is the sequence of SEQ ID NO:3645.

[0278]

[0284] In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a light chain constant region having a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the sequences listed in Table 1 or a combination thereof. In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a light chain constant region having any one of the sequences listed in Table 1 or a combination thereof. In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a light chain constant region having any one of the light chain constant region sequences listed in Table 1 or a combination thereof.

[0279]

[0285] In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a light chain constant region of a kappa chain or a fragment thereof. In some embodiments, the light chain constant region of a kappa chain comprises a light chain constant region sequence listed in Table 1.

[0280]

[0286] In some embodiments, the light chain constant region of the kappa chain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 39 or SEQ ID NO: 3644. In some embodiments, the light chain constant region of the kappa chain comprises a sequence of SEQ ID NO: 39 or SEQ ID NO: 3644. In some embodiments, the light chain constant region of the kappa chain is a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 39 or SEQ ID NO: 3644. In some embodiments, the light chain constant region of the kappa chain is a sequence of SEQ ID NO: 39 or SEQ ID NO: 3644.

[0281]

[0287] In some embodiments, the first TCRαV binding moiety, the second TCRαV binding moiety, or a combination thereof binds to an outward-facing region on a TCRαV protein. In some embodiments, the outward-facing region on a TCRαV protein comprises a structurally conserved region of TCRαV that has a similar structure across one or more TCRαV subfamilies.

[0282] Cytokine molecules

[0288] In some embodiments, the multifunctional molecule comprises a cytokine molecule. As used herein, "cytokine molecule" refers to a full length, fragment or variant of a cytokine; a cytokine further comprising a receptor domain, e.g., a cytokine receptor dimerization domain; or an agonist of a cytokine receptor, e.g., an antibody molecule against a cytokine receptor (e.g., an agonist antibody), which induces activation of at least one of the naturally occurring cytokines. In some embodiments, the cytokine molecule is selected from interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-10 (IL-10), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), or interferon gamma, or fragments or variants thereof, or any combination of the aforementioned cytokines. The cytokine molecule may be a monomer or a dimer. In embodiments, the cytokine molecule may further comprise a cytokine receptor dimerization domain. In other embodiments, the cytokine molecule is an agonist of a cytokine receptor, for example, an antibody molecule (eg, an agonist antibody) against a cytokine receptor selected from IL-15Ra or IL-21R.

[0283]

[0289] Cytokines are generally polypeptides that affect cellular activity, for example, through signal transduction pathways. Thus, multispecific or multifunctional polypeptide cytokines are useful and can be associated with receptor-mediated signal transduction that transmits signals from the outside of the cell membrane to modulate responses within the cell. Cytokines are proteinaceous signaling compounds that are mediators of immune responses. They control many different cellular functions, including proliferation, differentiation and cell survival / apoptosis, and cytokines are also involved in several pathophysiological processes, including viral infections and autoimmune diseases. Cytokines are synthesized under various stimuli by various cells of both the innate immune system (monocytes, macrophages, dendritic cells) and the adaptive immune system (T and B cells). Cytokines can be classified into two groups: pro-inflammatory and anti-inflammatory. Pro-inflammatory cytokines, including IFNγ, IL-1, IL-6 and TNF-alpha, are mainly derived from innate and Th1 cells. Anti-inflammatory cytokines, including IL-10, IL-4, IL-13 and IL-5, are synthesized from Th2 immune cells.

[0284]

[0290] In particular, provided herein are multispecific (e.g., bi-, tri-, tetraspecific) or multifunctional molecules that include, e.g., are engineered to contain, one or more cytokine molecules, e.g., immunomodulatory (e.g., pro-inflammatory) cytokines and variants, e.g., functional variants thereof. Thus, in some embodiments, the cytokine molecule is an interleukin or a variant, e.g., functional variant thereof. In some embodiments, the interleukin is a pro-inflammatory interleukin. In some embodiments, the interleukin is selected from interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), interleukin-7 (IL-7), or interferon gamma. In some embodiments, the cytokine molecule is a pro-inflammatory cytokine.

[0285]

[0291] In certain embodiments, the cytokine is a single-chain cytokine. In certain embodiments, the cytokine is a multi-chain cytokine (e.g., the cytokine comprises two or more (e.g., two) polypeptide chains. An exemplary multi-chain cytokine is IL-12.

[0286]

[0292] Examples of useful cytokines include, but are not limited to, GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-21, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNF-α, and TNFβ. In some embodiments, the cytokine of the multispecific or multifunctional polypeptide is a cytokine selected from the group of GM-CSF, IL-2, IL-7, IL-8, IL-10, IL-12, IL-15, IL-21, IFN-α, IFN-γ, MIP-1α, MIP-1β, and TGF-β. In some embodiments, the cytokine of the multispecific or multifunctional polypeptide is a cytokine selected from the group of IL-2, IL-7, IL-10, IL-12, IL-15, IFN-α, and IFN-γ. In certain embodiments, the cytokine is mutated to remove N- and / or O-glycosylation sites. Elimination of glycosylation increases the homogeneity of the product that can be obtained in recombinant production.

[0287]

[0293] In some embodiments, the cytokine of the multispecific or multifunctional polypeptide is IL-2. In a detailed embodiment, the IL-2 cytokine can induce one or more of the following cellular responses selected from the group consisting of proliferation of activated T lymphocyte cells, differentiation of activated T lymphocyte cells, cytotoxic T cell (CTL) activity, proliferation of activated B cells, differentiation of activated B cells, proliferation of natural killer (NK) cells, differentiation of NK cells, cytokine secretion by activated T cells or NK cells, and NK / lymphocyte-activated killer (LAK) anti-tumor cytotoxicity. In another specific embodiment, the IL-2 cytokine is a mutant IL-2 cytokine with reduced binding affinity to the alpha-subunit of the IL-2 receptor. The alpha-subunit (also known as CD25) together with the beta- and gamma-subunits (also known as CD122 and CD132, respectively) form a heterotrimeric high-affinity IL-2 receptor, while a dimeric receptor consisting only of the beta- and gamma-subunits is referred to as the intermediate-affinity IL-2 receptor. As described in PCT Patent Application No. PCT / EP2012 / 051991 (hereby incorporated by reference in its entirety), mutant IL-2 polypeptides having reduced binding to the alpha-subunit of the IL-2 receptor have a reduced ability to induce IL-2 signaling in regulatory T cells, induce less activation-induced cell death (AICD) in T cells, and have a reduced toxicity profile in vivo compared to wild-type IL-2 polypeptides. The use of such cytokines with reduced toxicity is particularly advantageous in multispecific or multifunctional polypeptides according to the present disclosure that have a long serum half-life due to the presence of an Fc domain. In some embodiments, the mutant IL-2 cytokine of the multispecific or multifunctional polypeptide according to the present disclosure comprises at least one amino acid mutation that reduces or eliminates the affinity of the mutant IL-2 cytokine for the alpha-subunit of the IL-2 receptor (CD25) but preserves the affinity of the mutant IL-2 cytokine for the intermediate affinity IL-2 receptor (consisting of the β and γ subunits of the IL-2 receptor) compared to the non-mutated IL-2 cytokine.In some embodiments, the one or more amino acid mutations are amino acid substitutions. In particular embodiments, the mutant IL-2 cytokine comprises one, two or three amino acid substitutions at one, two or three positions selected from positions corresponding to residues 42, 45 and 72 of human IL-2. In more particular embodiments, the mutant IL-2 cytokine comprises three amino acid substitutions at positions corresponding to residues 42, 45 and 72 of human IL-2. In even more particular embodiments, the mutant IL-2 cytokine is human IL-2 comprising amino acid substitutions F42A, Y45A and L72G. In some embodiments, the mutant IL-2 cytokine additionally comprises an amino acid mutation at a position corresponding to position 3 of human IL-2, which eliminates the O-glycosylation site of IL-2. In particular, said additional amino acid mutation is an amino acid substitution replacing a threonine residue with an alanine residue. A particular mutant IL-2 cytokine useful in the present disclosure comprises four amino acid substitutions at positions corresponding to residues 3, 42, 45 and 72 of human IL-2. Specific amino acid substitutions are T3A, F42A, Y45A and L72G. As demonstrated in PCT patent application no. PCT / EP2012 / 051991 and the appended examples, the quadruple mutant IL-2 polypeptide (IL-2 qm) exhibits no detectable binding to CD25, exhibits a reduced ability to induce apoptosis in T cells, exhibits a reduced ability to induce IL-2 signaling in regulatory T cells (T.sub.reg cells), and exhibits a reduced toxicity profile in vivo. However, it retains the ability to activate IL-2 signaling in effector cells, induce the proliferation of effector cells, and produce IFN-γ as a secondary cytokine by NK cells.

[0288]

[0294] The IL-2 or mutant IL-2 cytokine according to any of the above embodiments may contain additional mutations that provide additional advantages, such as increased expression or stability. For example, the cysteine ​​at position 125 may be replaced with a neutral amino acid, such as alanine, to avoid the formation of IL-2 dimers by disulfide bridges. Thus, in certain embodiments, the IL-2 or mutant IL-2 cytokine of the multispecific or multifunctional polypeptide according to the present disclosure contains an additional amino acid mutation at the position corresponding to residue 125 of human IL-2. In some embodiments, the additional amino acid mutation is the amino acid substitution C125A.

[0289]

[0295] In a particular embodiment, the IL-2 cytokine of the multispecific or multifunctional polypeptide is represented by SEQ ID NO: 2270 It contains the polypeptide sequence of [APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT].

[0290]

[0296] In another particular embodiment, the IL-2 cytokine of the multispecific or multifunctional polypeptide is SEQ ID NO: 2280 It contains the polypeptide sequence of [APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFAMPKKATELKHLQCLEEELKPLEEVLNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT].

[0291]

[0297] In another embodiment, the cytokine of the multispecific or multifunctional polypeptide is IL-12. In a particular embodiment, said IL-12 cytokine is a single chain IL-12 cytokine. In an even more particular embodiment, the single chain IL-12 cytokine is selected from the group consisting of SEQ ID NO: 2290 [IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTF SVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKD RVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS. In some embodiments, the IL-12 cytokine is capable of inducing one or more of a cellular response selected from the group consisting of NK cell proliferation, NK cell differentiation, T cell proliferation, and T cell differentiation.

[0292]

[0298] In another embodiment, the cytokine of the multispecific or multifunctional polypeptide is IL-10. In a particular embodiment, said IL-10 cytokine is a single chain IL-10 cytokine. In an even more particular embodiment, the single chain IL-10 cytokine is selected from the group consisting of SEQ ID NO: 2300 The polypeptide sequence of [SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGGGSGGGGSGGGGSGGGGSSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN].

[0293]

[0299] In another particular embodiment, the IL-10 cytokine is a monomeric IL-10 cytokine. In a more particular embodiment, the monomeric IL-10 cytokine is selected from the group consisting of SEQ ID NO: 2310 [SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN]. In some embodiments, the IL-10 cytokine can induce one or more of the cellular responses selected from the group consisting of inhibiting cytokine secretion, inhibiting antigen presentation by antigen presenting cells, reducing oxygen radical release, and inhibiting T cell proliferation. A multispecific or multifunctional polypeptide according to the present disclosure in which the cytokine is IL-10 is particularly useful for downregulation of inflammation, for example, in the treatment of inflammatory disorders.

[0294]

[0300] In another embodiment, the cytokine of the multispecific or multifunctional polypeptide is IL-15. In a detailed embodiment, said IL-15 cytokine is a mutant IL-15 cytokine having reduced binding affinity to the α-subunit of the IL-15 receptor. Without being bound by theory, mutant IL-15 polypeptides having reduced binding to the alpha-subunit of the IL-15 receptor have a reduced ability to bind to fibroblasts throughout the body compared to wild-type IL-15 polypeptides, resulting in improved pharmacokinetic and toxicity profiles. The use of cytokines with reduced toxicity, such as the described mutant IL-2 and mutant IL-15 effector moieties, is particularly advantageous in multispecific or multifunctional polypeptides according to the present disclosure that have a long serum half-life due to the presence of an Fc domain. In some embodiments, the mutant IL-15 cytokine of the multispecific or multifunctional polypeptide according to the present disclosure comprises at least one amino acid mutation that reduces or eliminates the affinity of the mutant IL-15 cytokine for the alpha-subunit of the IL-15 receptor, but preserves the affinity of the mutant IL-15 cytokine for the intermediate affinity IL-15 / IL-2 receptor (consisting of the beta- and gamma-subunits of the IL-15 / IL-2 receptor), compared to the non-mutated IL-15 cytokine. In some embodiments, the amino acid mutation is an amino acid substitution. In a particular embodiment, the mutant IL-15 cytokine comprises an amino acid substitution at a position corresponding to residue 53 of human IL-15. In a more particular embodiment, the mutant IL-15 cytokine is human IL-15 comprising the amino acid substitution E53A. In some embodiments, the mutant IL-15 cytokine additionally comprises an amino acid mutation at a position corresponding to position 79 of human IL-15, which eliminates the N-glycosylation site of IL-15. In particular, said additional amino acid mutation is an amino acid substitution that replaces an asparagine residue with an alanine residue.In even more particular embodiments, the IL-15 cytokine comprises the polypeptide sequence of SEQ ID NO: 2320 [NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLASGDASIHDTVENLIILANNSLSSNGAVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS]. In some embodiments, the IL-15 cytokine is capable of inducing one or more of a cellular response selected from the group consisting of proliferation of activated T lymphocyte cells, differentiation of activated T lymphocyte cells, cytotoxic T cell (CTL) activity, proliferation of activated B cells, differentiation of activated B cells, proliferation of natural killer (NK) cells, differentiation of NK cells, cytokine secretion by activated T cells or NK cells, and NK / lymphocyte-activated killer (LAK) anti-tumor cytotoxicity.

[0295]

[0301] Mutant cytokine molecules useful as effector moieties in multispecific or multifunctional polypeptides can be prepared by deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis of the coding DNA sequence, PCR, gene synthesis, and the like. Correct nucleotide changes can be verified, for example, by sequencing. Substitutions or insertions may involve unnatural amino acid residues in addition to natural amino acid residues. Amino acid modifications include well-known methods of chemical modification, such as adding or removing glycosylation sites or attaching carbohydrates.

[0296]

[0302] In some embodiments, the cytokine of the multispecific or multifunctional polypeptide, particularly the single chain cytokine, is GM-CSF. In detailed embodiments, the GM-CSF cytokine can induce proliferation and / or differentiation in granulocytes, monocytes or dendritic cells. In some embodiments, the cytokine of the multispecific or multifunctional polypeptide, particularly the single chain cytokine, is IFN-α. In detailed embodiments, the IFN-α cytokine can induce one or more of the cellular responses selected from the group consisting of inhibition of viral replication in virally infected cells and upregulation of expression of major histocompatibility complex I (MHC I). In another detailed embodiment, the IFN-α cytokine can inhibit proliferation in tumor cells. In some embodiments, the cytokine of the multispecific or multifunctional polypeptide, particularly the single chain cytokine, is IFNγ. In detailed embodiments, the IFN-γ cytokine can induce one or more of the cellular responses selected from the group of increased macrophage activity, increased expression of MHC molecules, and increased NK cell activity. In some embodiments, the cytokine of the multispecific or multifunctional polypeptide, particularly the single chain cytokine, is IL-7. In a detailed embodiment, the IL-7 cytokine is capable of inducing proliferation of T and / or B lymphocytes. In some embodiments, the cytokine, particularly the single chain cytokine, of the multispecific or multifunctional polypeptide is IL-8. In a detailed embodiment, the IL-8 cytokine is capable of inducing chemotaxis in neutrophils. In some embodiments, the cytokine, particularly the single chain cytokine, of the multispecific or multifunctional polypeptide is MIP-1α. In a detailed embodiment, the MIP-1α cytokine is capable of inducing chemotaxis in monocytes and T lymphocyte cells. In some embodiments, the cytokine, particularly the single chain cytokine, of the multispecific or multifunctional polypeptide is MIP-1β. In a detailed embodiment, the MIP-1β cytokine is capable of inducing chemotaxis in monocytes and T lymphocyte cells. In some embodiments, the cytokine, particularly the single chain cytokine, of the multispecific or multifunctional polypeptide is TGF-β.In particular embodiments, the TGF-β cytokines are capable of inducing one or more of the cellular responses selected from the group consisting of chemotaxis in monocytes, chemotaxis in macrophages, upregulation of IL-1 expression in activated macrophages, and upregulation of IgA expression in activated B cells.

[0297]

[0303] In some embodiments, the multispecific or multifunctional polypeptides of the present disclosure have a dissociation constant (K) that is at least about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times higher than for a control cytokine. D In another embodiment, the multispecific or multifunctional polypeptide binds to a cytokine receptor with a K that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher than for a corresponding multispecific or multifunctional polypeptide that comprises two or more effector moieties. D In another embodiment, the multispecific or multifunctional polypeptide binds to a cytokine receptor with a dissociation constant K that is about 10-fold higher than for a corresponding multispecific or multifunctional polypeptide that comprises two or more cytokines. D It binds to cytokine receptors.

[0298]

[0304] In some embodiments, the multispecific molecules described herein comprise a cytokine molecule. In embodiments, the cytokine molecule comprises a full-length, fragment or variant of a cytokine; a cytokine receptor domain, e.g., a cytokine receptor dimerization domain; or an agonist of a cytokine receptor, e.g., an antibody molecule against a cytokine receptor (e.g., an agonist antibody).

[0299]

[0305] In some embodiments, the cytokine molecule is selected from IL-2, IL-12, IL-15, IL-18, IL-7, IL-21, or interferon gamma, or a fragment or variant thereof, or any combination of the above cytokines. The cytokine molecule may be a monomer or a dimer. In embodiments, the cytokine molecule may further comprise a cytokine receptor dimerization domain.

[0300]

[0306] In other embodiments, the cytokine molecule is an agonist of a cytokine receptor, for example, an antibody molecule (eg, an agonist antibody) against a cytokine receptor selected from IL-15Ra or IL-21R.

[0301]

[0307] In some embodiments, the cytokine molecule is IL-15, e.g., human IL-15 (e.g., the amino acid sequence: NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 2170), a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid change, but no more than 5, 10 or 15 changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), relative to the amino acid sequence of SEQ ID NO: 2170.

[0302]

[0308] In some embodiments, the cytokine molecule comprises a receptor dimerization domain, such as an IL15R alpha dimerization domain. In some embodiments, the IL15R alpha dimerization domain has the amino acid sequence: MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVL (SEQ ID NO: 2180), a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid change, but no more than 5, 10 or 15 changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), relative to the amino acid sequence of SEQ ID NO: 2180. In some embodiments, the multispecific polypeptide comprises a polypeptide having a sequence similar to that of SEQ ID NO: 2180, such as ... The cytokine molecule (e.g., IL-15) and receptor dimerization domain (e.g., IL15R alpha dimerization domain) of the multispecific molecule are covalently linked, e.g., via a linker (e.g., a Gly-Ser linker, e.g., a linker comprising the amino acid sequence SGGSGGGGSGGGSGGGGSLQ (SEQ ID NO: 2190). In other embodiments, the cytokine molecule (e.g., IL-15) and receptor dimerization domain (e.g., IL15R alpha dimerization domain) of the multispecific molecule are not covalently linked, e.g., are non-covalently associated.

[0303]

[0309] In other embodiments, the cytokine molecule is IL-2, e.g., human IL-2 (e.g., the amino acid sequence: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 2191), or a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid change, but no more than 5, 10 or 15 changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: 2191).

[0304]

[0310] In other embodiments, the cytokine molecule is IL-18, e.g., human IL-18 (e.g., the amino acid sequence: YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 2192), or a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid change, but no more than 5, 10 or 15 changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: 2192).

[0305]

[0311] In other embodiments, the cytokine molecule is IL-21, e.g., human IL-21 (e.g., the amino acid sequence: QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 2193), or a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid change, but no more than 5, 10 or 15 changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: 2193).

[0306]

[0312] In yet other embodiments, the cytokine molecule is interferon gamma, e.g., human interferon gamma (e.g., the amino acid sequence: QDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRG (SEQ ID NO: 2194), a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid change, but no more than 5, 10 or 15 changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: 2194).

[0307] Immune Cell Engagers

[0313] In some embodiments, the multifunctional molecule further comprises an immune cell engager. "Immune cell engager" refers to one or more binding specificities that bind and / or activate immune cells, e.g., cells involved in an immune response. In embodiments, the immune cells are selected from T cells, NK cells, B cells, dendritic cells, and / or macrophage cells. The immune cell engager can be an antibody molecule, a receptor molecule (e.g., a full-length receptor, a receptor fragment, or a fusion thereof (e.g., a receptor-Fc fusion)), or a ligand molecule (e.g., a full-length ligand, a ligand fragment, or a fusion thereof (e.g., a ligand-Fc fusion)) that binds to an immune cell antigen (e.g., a T cell, NK cell antigen, B cell antigen, dendritic cell antigen, and / or macrophage cell antigen). In embodiments, the immune cell engager specifically binds to a target immune cell, e.g., preferentially binds to a target immune cell. For example, when the immune cell engager is an antibody molecule, it binds to an immune cell antigen (e.g., a T cell antigen, an NK cell antigen, a B cell antigen, a dendritic cell antigen, and / or a macrophage cell antigen) with a dissociation constant of less than about 10 nM.

[0308]

[0314] The immune cell engagers, e.g., first and / or second immune cell engagers, of the multispecific or multifunctional molecules described herein can mediate binding to and / or activation of immune cells, e.g., immune effector cells. In some embodiments, the immune cells are selected from T cells, NK cells, B cells, dendritic cells, or macrophage cell engagers, or combinations thereof. In some embodiments, the immune cell engagers are selected from one, two, three, or all of T cell engagers, NK cell engagers, B cell engagers, dendritic cell engagers, or macrophage cell engagers, or combinations thereof. The immune cell engagers can be agonists of the immune system. In some embodiments, the immune cell engagers can be antibody molecules, ligand molecules (e.g., ligands further comprising an immunoglobulin constant region, e.g., an Fc region), small molecules, nucleotide molecules.

[0309]

[0315] Natural Killer Cell Engager

[0316] Natural killer (NK) cells recognize and destroy tumor and virus-infected cells in an antibody-independent manner. Regulation of NK cells is mediated by activating and inhibiting receptors on the NK cell surface. One family of activating receptors is the natural cytotoxicity receptors (NCRs), including NKp30, NKp44, and NKp46. NCRs initiate tumor targeting by recognition of heparan sulfate on cancer cells. NKG2D is a receptor that provides both stimulatory and costimulatory innate immune responses in activated killer (NK) cells, leading to cytotoxic activity. DNAM1 is a receptor involved in cell-cell adhesion, lymphocyte signaling, cytotoxicity, and lymphokine secretion mediated by cytotoxic T lymphocytes (CTLs) and NK cells. DAP10 (also known as HCST) is a transmembrane adaptor protein that associates with KLRK1 in lymphoid and myeloid cells to form the activating receptor KLRK1-HCST, which plays a major role in inducing cytotoxicity against target cells expressing cell surface ligands such as MHC class I chain-associated MICA and MICB, and U(optionally L1)6-binding protein (ULBP); the KLRK1-HCST receptor plays a role in immune surveillance against tumors and is required for tumor cell lysis; indeed, melanoma cells that do not express KLRK1 ligands escape immune surveillance mediated by NK cells. CD16 is a receptor for the Fc region of IgG, binding complexed or aggregated IgG, and also monomeric IgG, thereby mediating antibody-dependent cellular cytotoxicity (ADCC) and other antibody-dependent responses such as phagocytosis.

[0310]

[0317] In some embodiments, the NK cell engager is the viral hemagglutinin (HA), which is a glycoprotein found on the surface of influenza viruses. It is responsible for the binding of the virus to cells in the upper respiratory tract or cells that have sialic acid on their membranes, such as red blood cells. HA has at least 18 different antigens. These subtypes are named H1 to H18. NCRs can recognize viral proteins. It has been shown that NKp46 can interact with influenza HA and HA-NA of paramyxoviruses, including Sendai virus and Newcastle disease virus. Besides NKp46, NKp44 can also functionally interact with HA of different influenza subtypes.

[0311]

[0318] In particular, provided herein are multispecific (e.g., bi-, tri-, tetra-specific) or multifunctional molecules that have been engineered to contain one or more NK cell engagers that mediate binding to and / or activation of NK cells. Thus, in some embodiments, the NK cell engager is selected from an antigen-binding domain or ligand that binds to (e.g., activates) NKp30, NKp40, NKp44, NKp46, NKG2D, DNAM1, DAP10, CD16 (e.g., CD16a, CD16b, or both), CRTAM, CD27, PSGL1, CD96, CD100 (SEMA4D), NKp80, CD244 (also known as SLAMF4 or 2B4), SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E, or CD160.

[0312]

[0319] In some embodiments, the NK cell engager is a ligand for NKp30, B7-6, e.g., DLKVEMMAGGTQITPLNDNVTIFCNIFYSQPLNITSMGITTWFWKSLTFDKEVKVFEFFGDHQEAFRPGAIVSPWRLKSGDASLRPGIQLEEAGEYRCEVVVTPLKAQGTVQLEVVASPASRLLLDQVGMKENEDKYMCESSGFYPEAINITWEKQTQKFPHPIEISEDVITGPTIKNMDGTFNVTSCLKLNSSQEDPGTVYQCVVRHASLHTPLRSNFTLTAARHSLSETEKTDNFS (SEQ ID NO: 3291), a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or an amino acid sequence having at least one amino acid change, but not more than 5, 10 or 15 changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), relative to the amino acid sequence of SEQ ID NO: 3291.

[0313]

[0320] In other embodiments, the NK cell engager is a ligand for viral HA, NKp44 or NKp46. Viral hemagglutinin (HA) is a glycoprotein on the surface of a virus. The HA protein allows the virus to bind to the membrane of a cell via sialic acid sugar moieties, which contributes to the fusion of the viral membrane with the cell membrane (see, e.g., Eur J Immunol. 2001 Sep;31(9):2680-9, "Recognition of viral hemagglutinins by NKp44 but not by NKp30"; and Nature 2001 Feb 22;409(6823):1055-60, "Recognition of haemagglutinins on virus-infected cells by NKp46 activates lysis by human NK cells," the contents of each of which are hereby incorporated by reference herein).

[0314]

[0321] In other embodiments, the NK cell engager is a ligand for NKG2D selected from MICA, MICB, or ULBP1, e.g., (i) MICA has the amino acid sequence: EPHSLRYNLTVLSWDGSVQSGFLTEVHLDGQPFLRCDRQKCRAKPQGQWAEDVLGNKTWDRETRDLTGNGKDLRMTLAHIKDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYY DGELFLSQNLETKEWTMPQSSRAQTLAMNVRNFLKEDAMKTKTHYHAMHADCLQELRRYLKSGVVLRRTVPPMVNVTRSEASEGNITVTCRASGFYPWNITLSWRQDGVSLS HDTQQWGDVLPDGNGTYQTWVATRICQGEEQRFTCYMEHSGNHSTHPVPSGKVLVLQSHW (SEQ ID NO: 3292), or a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid change, but no more than 5, 10 or 15 changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), relative to the amino acid sequence of SEQ ID NO: 3292; (ii) MICB has the amino acid sequence: AEPHSLRYNLMVLSQDESVQSGFLAEGHLDGQPFLRYDRQKRRAKPQGQWAEDVLGAKTWDTETEDLTENGQDLRRTLTHIKDQKGGLHSLQEIRVCEIHEDSSTRGSRHFYY DGELFLSQNLETQESTVPQSSRAQTLAMNVTNFWKEDAMKTKTHYRAMQADCLQKLQRYLKSGVAIRRTVPPMVNVTCSEVSEGNITVTCRASSFYPRNITLTWRQDGVSLSHN TQQWGDVLPDGNGTYQTWVATRIRQGEEQRFTCYMEHSGNHGTHPVPSGKVLVLQSQRTD (SEQ ID NO: 3293), or a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid change, but no more than 5, 10 or 15 changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), relative to the amino acid sequence of SEQ ID NO: 3293; or (iii) ULBP1 has the amino acid sequence: GWVDTHCLCYDFIITPKSRPEPQWCEVQGLVDERPFLHYDCVNHKAKAFASLGKKVNVTKTWEEQTETLRDVVDFLKGQLLDIQVENLIPIEPLTLQARMSCEHEAHGHGRGSWQFLFNGQKFLLFDSNNRKWTALHPGAKKMTEKWEKNRDVTMFFQKISLGDCKMWLEEFLMYWEQMLDPTKPPSLAPG (SEQ ID NO: 3294), or a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than 5, 10 or 15 alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), relative to the amino acid sequence of SEQ ID NO: 3294.

[0315]

[0322] In other embodiments, the NK cell engager is a ligand of DNAM1 selected from NECTIN2 or NECL5, e.g., (i) NECTIN2 has the amino acid sequence: QDVRVQVLPEVRGQLGGTVELPCHLLPPVPGLYISLVTWQRPDAPANHQNVAAFHPKMGPSFPSPKPGSERLSFVSAKQSTGQDTEAELQDATLALHGLTVEDEGNYTCEFATFPKGSVRGMTW LRVIAKPKNQAEAQKVTFSQDPTTVALCISKEGRPPARISWLSSLDWEAKETQVSGTLAGTVTVTSRFTLVPSGRADGVTVTCKVEHESFEEPALIPVTLSVRYPPEVSISGYDDNWYLGRTDA TLSCDVRSNPEPTGYDWSTTSGTFPTSAVAQGSQLVIHAVDSLFNTTFVCTVTNAVGMGRAEQVIFVRETPNTAGAGATGG (SEQ ID NO: 3295), a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid change, but no more than 5, 10 or 15 changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), relative to the amino acid sequence of SEQ ID NO: 3295; or (ii) NECL5 has the amino acid sequence: WPPPGTGDVVVQAPTQVPGFLGDSVTLPCYLQVPNMEVTHVSQLTWARHGESGSMAVFHQTQGPSYSESKRLEFVAARLGAELRNASLRMFGLRVEDEGNYTCLFVTFPQGSRSVDIWLRVL AKPQNTAEVQKVQLTGEPVPMARCVSTGGRPPAQITWHSDLGMPNTSQVPGFLSGTVTVTSLWILVPSSQVDGKNVTCKVEHESFEKPQLLTVNLTVYYPPEVSISGYDNNWYLGQNEATL TCDARSNPEPTGYNWSTTMGPLPPFAVAQGAQLLIRPVDKPINTTLICNVTNALGARQAELTVQVKEGPPSEHSGISRN (SEQ ID NO: 3296), or a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but no more than 5, 10 or 15 alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), relative to the amino acid sequence of SEQ ID NO: 3296a.

[0316]

[0323] In yet other embodiments, the NK cell engager is a ligand for DAP10, an adaptor for NKG2D (see, e.g., Proc Natl Acad Sci US A. 2005 May 24;102(21):7641-7646; and Blood, 15 September 2011 Volume 118, Number 11, the entire contents of each of which are hereby incorporated by reference herein).

[0317]

[0324] In other embodiments, the NK cell engager is a ligand of CD16 that is a CD16a / b ligand, e.g., a CD16a / b ligand that further comprises an antibody Fc region (see, e.g., Front Immunol. 2013;4:76, which discusses how antibodies use Fc to engage NK cells through CD16, the contents of which are incorporated herein in their entirety).

[0318]

[0325] In other embodiments, the NK cell engager is a ligand for CRTAM, which is NECL2, for example, NECL2 has the amino acid sequence: QNLFTKDVTVIEGEVATISCQVNKSDDSVIQLLNPNRQTIYFRDFRPLKDSRFQLLNFSSSELKVSLTNVSISDEGRYFCQLYTDPPQESYTTITVLVPPRNLMIDIQKDTAVEGEEIEVNCT AMASKPATTIRWFKGNTELKGKSEVEEWSDMYTVTSQLMLKVHKEDDGVPVICQVEHPAVTGNLQTQRYLEVQYKPQVHIQMTYPLQGLTREGDALELTCEAIGKPQPVMVTWVRVDDEMPQHA VLSGPNLFINNLNKTDNGTYRCEASNIVGKAHSDYMLYVYDPPTTIPPPTTTTTTTTTTTTTILTIITDSRAGEEGSIRAVDH (SEQ ID NO: 3297), or a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid change, but no more than 5, 10 or 15 changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), relative to the amino acid sequence of SEQ ID NO: 3297.

[0319]

[0326] In other embodiments, the NK cell engager is a ligand for CD27, which is CD70, for example, CD70 has the amino acid sequence: QRFAQAQQQLPLESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRP (SEQ ID NO: 3298), or a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than 5, 10 or 15 alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), relative to the amino acid sequence of SEQ ID NO: 3298.

[0320]

[0327] In other embodiments, the NK cell engager is a ligand for PSGL1 that is L-selectin (CD62L), for example, L-selectin has the amino acid sequence: WTYHYSEKPMNWQRARRFCRDNYTDLVAIQNKAEIEYLEKTLPFSRSYYWIGIRKIGGIWTWVGTNKSLTEEAENWGDGEPNNKKNKEDCVEIYIKRNKDAGKWNDDACHKLKA ALCYTASCQPWSCSGHGECVEIINNYTCNCDVGYYGPQCQFVIQCEPLEAPELGTMDCTHPLGNFSFSSQCAFSCSEGTNLTGIEETTCGPFGNWSSPEPTCQVIQCEPLSAPDL GIMNCSHPLASFSFTSACTFICSEGTELIGKKKTICESSGIWSNPSPICQKLDKSFSMIKEGDYN (SEQ ID NO: 3299), or a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid change, but no more than 5, 10 or 15 changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), relative to the amino acid sequence of SEQ ID NO: 3299.

[0321]

[0328] In other embodiments, the NK cell engager is a ligand for CD96, which is NECL5, for example, NECL5 has the amino acid sequence: WPPPGTGDVVVQAPTQVPGFLGDSVTLPCYLQVPNMEVTHVSQLTWARHGESGSMAVFHQTQGPSYSESKRLEFVAARLGAELRNASLRMFGLRVEDEGNYTCLFVTFPQGSRSVDIWLRV LAKPQNTAEVQKVQLTGEPVPMARCVSTGGRPPAQITWHSDLGMPNTSQVPGFLSGTVTVTSLWILVPSSQVDGKNVTCKVEHESFEKPQLLTVNLTVYYPPEVSISGYDNNWYLGQNEAT LTCDARSNPEPTGYNWSTTMGPLPPFAVAQGAQLLIRPVDKPINTTLICNVTNALGARQAELTVQVKEGPPSEHSGISRN (SEQ ID NO: 3296), or a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but no more than 5, 10 or 15 alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), relative to the amino acid sequence of SEQ ID NO: 3296.

[0322]

[0329] In another embodiment, the NK cell engager is a ligand for CD100 (SEMA4D), which is CD72, e.g., CD72 is Amino acid sequence: RYLQVSQQLQQTNRVLEVTNSSLRQQLRLKITQLGQSAEDLQGSRRELAQSQEALQVEQRAHQAAEGQLQACQADRQKTKETLQSEEQQRRALEQKLSNMENRLKPFFTCGSADTCCPSGWIMHQKSCFYISLTSKNWQESQKQCETLSSKLATFSEIYPQSHSYYFLNSLLPNGGSGNSYWTGLSSNKDWKLTDDTQRTRTYAQSSKCNKVHKTWSWWTLESESCRSSLPYICEMTAFRFPD (SEQ ID NO: 3300), or a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid change, but not more than 5, 10 or 15 changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), relative to the amino acid sequence of SEQ ID NO: 3300.

[0323]

[0330] In other embodiments, the NK cell engager is a ligand for NKp80 that is CLEC2B (AICL), for example, CLEC2B (AICL) has the amino acid sequence: KLTRDSQSLCPYDWIGFQNKCYYFSKEEGDWNSSKYNCSTQHADLTIIDNIEEMNFLRRYKCSSDHWIGLKMAKNRTGQWVDGATFTKSFGMRGSEGCAYLSDDGAATARCYTERKWICRKRIH (SEQ ID NO: 3301), or a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but no more than 5, 10 or 15 alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), relative to the amino acid sequence of SEQ ID NO: 3301.

[0324]

[0331] In other embodiments, the NK cell engager is a ligand for CD244, which is CD48, for example, CD48 has the amino acid sequence: QGHLVHMTVVSGSNVTLNISESLPENYKQLTWFYTFDQKIVEWDSRKSKYFESKFKGRVRLDPQSGALYISKVQKEDNSTYIMRVLKKTGNEQEWKIKLQVLDPVPKPVIKIEKIEDMDDNCYLKLSCVIPGESVNYTWYGDKRPFPKELQNSVLETTLMPHNYSRCYTCQVSNSVSSKNGTVCLSPPCTLARS (SEQ ID NO: 3302), or a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid change, but not more than 5, 10 or 15 changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), relative to the amino acid sequence of SEQ ID NO: 3302.

[0325]

[0332] T Cell Engager

[0333] In particular, provided herein are multispecific (e.g., bi-, tri-, tetraspecific) or multifunctional molecules that are engineered to further contain one or more T cell engagers that mediate binding to and / or activation of T cells. In some embodiments, the T cell engager is an antigen-binding domain that binds to, e.g., activates, TCRα, e.g., the TCRαV region, as described herein. In some embodiments, the T cell engager is selected from an antigen-binding domain or ligand that binds to (e.g., in some embodiments, activates) one or more of CD3, TCRα, TCRγ, TCRζ, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226. In other embodiments, the T cell engager is selected from an antigen-binding domain or ligand that binds to and does not activate one or more of CD3, TCR alpha, TCR gamma, TCR zeta, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226.

[0326]

[0334] B cell, macrophage and dendritic cell engagers

[0335] Broadly speaking, B cells, also known as B lymphocytes, are a type of white blood cell of the lymphocyte subtype. They function in the humoral immune component of the adaptive immune system by secreting antibodies. Additionally, B cells present antigens (they are also classified as professional antigen-presenting cells (APCs)) and secrete cytokines. Macrophages are a type of white blood cell that engulf and digest cellular debris, foreign material, microorganisms, and cancer cells through phagocytosis. Besides phagocytosis, they play an important role in non-specific defense (innate immunity) and also help initiate specific defense mechanisms (adaptive immunity) by recruiting other immune cells such as lymphocytes. For example, they are important as antigen presenters to T cells. Besides increasing inflammation and stimulating the immune system, macrophages can also play an important anti-inflammatory role and reduce immune responses through the release of cytokines. Dendritic cells (DCs) are antigen-presenting cells that function in processing antigenic material and presenting it on their cell surface to T cells of the immune system.

[0327]

[0336] In particular, provided herein are multispecific (e.g., bi-, tri-, tetraspecific) or multifunctional molecules that further comprise, e.g., are engineered to contain, one or more B cell, macrophage, and / or dendritic cell engagers that mediate binding to and / or activation of B cells, macrophages, and / or dendritic cells.

[0328]

[0337] Thus, in some embodiments, the immune cell engager comprises a B cell, macrophage, and / or dendritic cell engager selected from one or more of CD40 ligand (CD40L) or CD70 ligand; an antibody molecule that binds to CD40 or CD70; an antibody molecule against OX40; OX40 ligand (OX40L); an agonist of a Toll-like receptor (e.g., those described herein, e.g., TLR4, e.g., constitutively active TLR4 (caTLR4), or a TLR9 agonist); 41BB; CD2; CD47; or a STING agonist, or a combination thereof.

[0329]

[0338] In some embodiments, the B cell engager is a CD40L, OX40L, or CD70 ligand, or an antibody molecule that binds to OX40, CD40, or CD70.

[0330]

[0339] In some embodiments, the macrophage engager is a CD2 agonist. In some embodiments, the macrophage engager is an antigen-binding domain that binds to CD40L or a ligand that binds to CD40, a Toll-like receptor (TLR) agonist (e.g., those described herein), such as TLR9 or TLR4 (e.g., caTLR4 (constitutively active TLR4), CD47, or a STING agonist. In some embodiments, the STING agonist is a cyclic dinucleotide, such as cyclic di-GMP (cdGMP) or cyclic di-AMP (cdAMP). In some embodiments, the STING agonist is biotinylated.

[0331]

[0340] In some embodiments, the dendritic cell engager is a CD2 agonist. In some embodiments, the dendritic cell engager is a ligand, receptor agonist, or antibody molecule that binds to one or more of OX40L, 41BB, a TLR agonist (e.g., those described herein) (e.g., a TLR9 agonist, TLR4 (e.g., caTLR4 (constitutively active TLR4)), CD47, or a STING agonist. In some embodiments, the STING agonist is a cyclic dinucleotide, e.g., cyclic di-GMP (cdGMP) or cyclic di-AMP (cdAMP). In some embodiments, the STING agonist is biotinylated.

[0332]

[0341] In other embodiments, the immune cell engager mediates binding to or activation of one or more of B cells, macrophages, and / or dendritic cells. Exemplary B cell, macrophage, and / or dendritic cell engagers can be selected from one or more of CD40 ligand (CD40L) or CD70 ligand; antibody molecules that bind to CD40 or CD70; antibody molecules against OX40; OX40 ligand (OX40L); Toll-like receptor agonists (e.g., TLR4, e.g., constitutively active TLR4 (caTLR4) or TLR9 agonists); 41BB agonists; CD2; CD47; or STING agonists, or combinations thereof.

[0333]

[0342] In some embodiments, the B cell engager is selected from one or more of CD40L, OX40L, or CD70 ligand, or an antibody molecule that binds to OX40, CD40, or CD70.

[0334]

[0343] In other embodiments, the macrophage cell engager is selected from one or more of a CD2 agonist; CD40L; OX40L; an antibody molecule that binds to OX40, CD40, or CD70; a Toll-like receptor agonist or fragment thereof (e.g., TLR4, e.g., constitutively active TLR4 (caTLR4)); a CD47 agonist; or a STING agonist.

[0335]

[0344] In other embodiments, the dendritic cell engager is selected from one or more of a CD2 agonist, an OX40 antibody, OX40L, a 41BB agonist, a Toll-like receptor agonist or fragment thereof (e.g., TLR4, e.g., constitutively active TLR4 (caTLR4)), a CD47 agonist, or a STING agonist.

[0336]

[0345] In some embodiments, OX40L has the amino acid sequence: QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL (SEQ ID NO: 3303), or a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid change, but no more than 5, 10 or 15 changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), relative to the amino acid sequence of SEQ ID NO: 3303.

[0337]

[0346] In another embodiment, CD40L has the amino acid sequence: MQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL (SEQ ID NO: 3304), or a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid change, but not more than 5, 10 or 15 changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), relative to the amino acid sequence of SEQ ID NO: 3304.

[0338]

[0347] In yet other embodiments, the STING agonist comprises a cyclic dinucleotide, such as cyclic di-GMP (cdGMP), cyclic di-AMP (cdAMP), or a combination thereof, optionally with 2',5' or 3',5' phosphate linkages.

[0339]

[0348] In some embodiments, the immune cell engager comprises a 41BB ligand, for example, the amino acid sequence: ACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE (SEQ ID NO: 3305) or a fragment thereof, or a 41BB ligand comprising an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid change, but not more than 5, 10 or 15 changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), relative to the amino acid sequence of SEQ ID NO: 3305.

[0340]

[0349] Toll-like receptors Toll-like receptors (TLRs) are evolutionarily conserved receptors that are homologs of the Drosophila Toll protein and recognize highly conserved structural motifs known as pathogen-associated microbial patterns (PAMPs), which are exclusively expressed by microbial pathogens, or danger-associated molecular patterns (DAMPs), which are endogenous molecules released from necrotic or dying cells. PAMPs include flagellin, bacterial DNA and viral double-stranded RNA, as well as various bacterial cell wall components such as lipopolysaccharide (LPS), peptidoglycan (PGN) and lipopeptides. DAMPs include protein fragments from the extracellular matrix, as well as intracellular proteins such as heat shock proteins. Stimulation of TLRs by the corresponding PAMPs or DAMPs initiates a signaling cascade leading to the activation of transcription factors such as AP-1, NF-κB and interferon regulatory factors (IRFs). Signaling by TLRs results in a variety of cellular responses, including the production of interferons (IFNs), proinflammatory cytokines and effector cytokines that direct adaptive immune responses. TLRs have been implicated in numerous inflammatory and immune disorders and play a role in cancer (Rakoff-Nahoum S. and Medzhitov R. 2009. Toll-like receptors and cancer. Nat Revs Cancer 9:57-63).

[0341]

[0350] TLRs are type I transmembrane proteins characterized by an extracellular domain containing leucine-rich repeats (LRRs) and a cytoplasmic tail containing a conserved region called the Toll / IL-1 receptor (TIR) ​​domain. Ten human and twelve mouse TLRs have been characterized: TLR1-TLR10 in humans and TLR1-TLR9, TLR11, TLR12 and TLR13 in mice, with the homolog of TLR10 being a pseudogene. TLR2 is essential for the recognition of various PAMPs from Gram-positive bacteria, including bacterial lipoproteins, lipomannan and lipoteichoic acid. TLR3 is linked to virus-derived double-stranded RNA. TLR4 is primarily activated by lipopolysaccharide. TLR5 detects bacterial flagellin and TLR9 is required for the response to unmethylated CpG DNA. Finally, TLR7 and TLR8 recognize small synthetic antiviral molecules and single-stranded RNA has been reported to be their natural ligand. TLR11 has been reported to recognize profilin-like proteins from uropathogenic E. coli and Toxoplasma gondii. The repertoire of TLR specificity appears to be expanded by the ability of TLRs to heterodimerize with each other. For example, dimers of TLR2 and TLR6 are required for responses to diacylated lipoproteins, and TLR2 and TLR1 interact to recognize triacylated lipoproteins. TLR specificity is also influenced by various adaptors and accessory molecules, such as MD-2 and CD14, which form a complex with TLR4 in response to LPS.

[0342]

[0351] TLR signaling consists of at least two distinct pathways: a MyD88-dependent pathway that leads to the production of inflammatory cytokines, and a MyD88-independent pathway that is associated with the stimulation of IFN-β and maturation of dendritic cells. The MyD88-dependent pathway is common to all TLRs except TLR3 (Adachi O et al., 1998, “Targeted disruption of the MyD88 gene results in loss of IL-1-and IL-18-mediated function.” Immunity, 9(1):143-50). Upon activation by PAMPs or DAMPs, TLRs hetero- or homodimerize and induce the recruitment of adaptor proteins through their cytoplasmic TIR domains. Individual TLRs induce distinct signaling responses by the use of different adaptor molecules. TLR4 and TLR2 signaling require the adaptor TIRAP / Mal, which is involved in the MyD88-dependent pathway. TLR3 induces IFN-β production in response to double-stranded RNA in a MyD88-independent manner through the adaptor TRIF / TICAM-1.TRAM / TICAM-2 is another adaptor molecule involved in the MyD88-independent pathway whose function is restricted to the TLR4 pathway.

[0343]

[0352] TLR3, TLR7, TLR8 and TLR9 recognize viral nucleic acids and induce type I IFN. The signaling mechanisms that lead to type I IFN induction differ depending on the TLR that is activated. They involve the interferon regulatory factors, IRFs, a family of transcription factors known to play essential roles in antiviral defense, cell growth and immune regulation. Three IRFs (IRF3, IRF5 and IRF7) function as direct transducers of virus-mediated TLR signaling. TLR3 and TLR4 activate IRF3 and IRF7, whereas TLR7 and TLR8 activate IRF5 and IRF7 (Doyle S et al., 2002, "IRF3 mediates a TLR3 / TLR4-specific antiviral gene program." Immunity, 17(3):251-63). Furthermore, type I IFN production stimulated by the TLR9 ligand CpG-A has been shown to be mediated by PI(3)K and mTOR (Costa-Mattioli M. and Sonenberg N. 2008, "RAPping production of type I interferon in pDCs through mTOR." Nature Immunol. 9:1097-1099).

[0344]

[0353] TLR-9 TLR9 recognizes unmethylated CpG sequences in DNA molecules. CpG sites are relatively rare (approximately 1%) on vertebrate genomes compared to bacterial or viral DNA. TLR9 is expressed by numerous cells of the immune system, such as B lymphocytes, monocytes, natural killer (NK) cells, and plasmacytoid dendritic cells. TLR9 is expressed intracellularly, in endosomal compartments, and functions to alert the immune system to viral and bacterial infections by binding to DNA rich in CpG motifs. TLR9 signals lead to the activation of cells that initiate a proinflammatory response, resulting in the production of cytokines such as type I interferon and IL-12.

[0345]

[0354] TLR agonistsA TLR agonist can agonize one or more TLRs, for example, one or more of human TLR-1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the adjunctive agent described herein is a TLR agonist. In some embodiments, the TLR agonist specifically agonizes human TLR-9. In some embodiments, the TLR-9 agonist is a CpG moiety. As used herein, a CpG moiety is a linear dinucleotide with the sequence: 5'-C-phosphate-G-3', i.e., a cytosine and a guanine separated by only one phosphate. In some embodiments, the CpG portion comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more CpG dinucleotides. In some embodiments, the CpG portion consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 CpG dinucleotides. In some embodiments, the CpG portion has 1-5, 1-10, 1-20, 1-30, 1-40, 1-50, 5-10, 5-20, 5-30, 10-20, 10-30, 10-40, or 10-50 CpG dinucleotides. In some embodiments, the TLR-9 agonist is a synthetic ODN (oligodeoxynucleotide). CpG ODNs are short synthetic single-stranded DNA molecules that contain unmethylated CpG dinucleotides in a specific sequence context (CpG motif). CpG ODNs have a partially or fully phosphorothioated (PS) backbone, in contrast to the natural phosphodiester (PO) backbone found in genomic bacterial DNA. There are three major classes of CpG ODNs, class A, B and C, which differ in their immunostimulatory activity. CpG-A ODNs are characterized by a PO central CpG-containing palindromic motif and a PS-modified 3' poly-G string.They induce high IFN-α production from pDCs but are weak stimulators of TLR9-dependent NF-κB signaling and proinflammatory cytokine (e.g., IL-6) production. CpG-B ODNs contain a full-length PS backbone with one or more CpG dinucleotides. They strongly activate B cells and TLR9-dependent NF-κB signaling but weakly stimulate IFN-α secretion. CpG-C ODNs combine features of both classes A and B. They contain a full PS backbone and a CpG-containing palindromic motif. C class CpG ODNs induce B cell stimulation in addition to strong IFN-α production from pDCs.

[0346] Stroma modification part

[0355] In some embodiments, the multifunctional molecule further comprises a stromal modifying moiety. As used herein, "stromal modifying moiety" refers to an agent, such as a protein (e.g., an enzyme), that can modify, e.g., degrade, a component of stromal. In embodiments, the component of stromal is selected from, for example, ECM components, such as glycosaminoglycans, such as hyaluronic acid (also known as hyaluronic acid or HA), chondroitin sulfate, chondroitin, dermatan sulfate, heparin sulfate, heparin, entactin, tenascin, aggrecan, and keratin sulfate; or extracellular proteins, such as collagen, laminin, elastin, fibrinogen, fibronectin, and vitronectin.

[0347]

[0356] Solid tumors have a structure that includes two distinct but interdependent compartments that mimic those of normal tissues: the parenchyma (neoplastic cells) and the stroma, which is derived from and in which the neoplastic cells are dispersed. All tumors have a stroma and require it for nutritional support and waste removal. In tumors that grow as a cell suspension (e.g., leukemias, ascites tumors), plasma serves as the stroma (Connolly JL et al. Tumor Structure and Tumor Stroma Generation. In: Kufe DW et al., editors. Holland-Frei Cancer Medicine. 6th edition. Hamilton: BC Decker; 2003). The stroma contains various cell types including fibroblasts / myofibroblasts, glial cells, epithelial cells, adipocytes, vascular cells, smooth muscle cells, and immune cells, along with the extracellular matrix (ECM) and extracellular molecules (Li Hanchen et al. Tumor Microenvironment: The Role of the Tumor Stroma in Cancer. J of Cellular Biochemistry 101: 805-815 (2007)).

[0348]

[0357] Stroma-modifying moieties described herein include moieties (e.g., proteins, e.g., enzymes) that can degrade components of the stroma, e.g., ECM components, e.g., glycosaminoglycans, e.g., hyaluronan (also known as hyaluronic acid or HA), chondroitin sulfate, chondroitin, dermatan sulfate, heparin sulfate, heparin, entactin, tenascin, aggrecan, and keratin sulfate, or extracellular proteins, e.g., collagen, laminin, elastin, fibrinogen, fibronectin, and vitronectin.

[0349]

[0358] Stroma-modifying enzymes

[0359] In some embodiments, the stromal modifying moiety is an enzyme. For example, the stromal modifying moiety can include, but is not limited to, hyaluronidase, collagenase, chondroitinase, matrix metalloproteinase (e.g., macrophage metalloelastase).

[0350]

[0360] Hyaluronidase

[0361] Hyaluronidases are a group of neutral and acid-active enzymes found throughout the animal kingdom. Hyaluronidases are diverse with respect to substrate specificity and mechanism of action. There are three general classes of hyaluronidases: (1) mammalian hyaluronidases (EC 3.2.1.35), which are endo-beta-N-acetylhexosaminidases with tetra- and hexasaccharides as the major end products. These have both hydrolytic and transglycosidase activity and can degrade hyaluronan and chondroitin sulfates, and (2) bacterial hyaluronidases (EC 4.2.99.1), which degrade hyaluronan and, to various extents, chondroitin sulfates and dermatan sulfates. These are primarily endo-beta-N-acetylhexosaminidases that operate by beta-elimination reactions resulting in disaccharide end products, and (3) hyaluronidases (EC 3.2.1.36) from leeches, other parasites, and crustaceans are endo-beta-glucuronidases that yield tetra- and hexasaccharide end products through hydrolysis of beta 1-3 linkages.

[0351]

[0362] Mammalian hyaluronidases can be further divided into two groups: (1) neutral-active enzymes and (2) acid-active enzymes. There are six hyaluronidase-like genes in the human genome: HYAL1, HYAL2, HYAL3, HYAL4, HYALP1, and PH20 / SPAM1. HYALP1 is a pseudogene, and HYAL3 has not been shown to have enzymatic activity toward any known substrates. HYAL4 is a chondroitinase and lacks activity toward hyaluronan. HYAL1 is the prototypic acid-active enzyme, and PH20 is the prototypic neutral-active enzyme. Acid-active hyaluronidases, such as HYAL1 and HYAL2, lack catalytic activity at neutral pH. For example, HYAL1 has no catalytic activity in vitro at pH levels above 4.5 (Frost and Stern, "A Microtiter-Based Assay for Hyaluronidase Activity Not Requiring Specialized Reagents", Analytical Biochemistry, vol. 251, pp. 263-269 (1997). HYAL2 is an acid-active enzyme and has very low specific activity in vitro.

[0352]

[0363] In some embodiments, the hyaluronidase is a mammalian hyaluronidase. In some embodiments, the hyaluronidase is a recombinant human hyaluronidase. In some embodiments, the hyaluronidase is a neutral active hyaluronidase. In some embodiments, the hyaluronidase is a neutral active soluble hyaluronidase. In some embodiments, the hyaluronidase is a recombinant PH20 neutral active enzyme. In some embodiments, the hyaluronidase is a recombinant PH20 neutral active soluble enzyme. In some embodiments, the hyaluronidase is glycosylated. In some embodiments, the hyaluronidase has at least one N-linked glycan. Recombinant hyaluronidase can be produced using conventional methods known to those skilled in the art, such as those in U.S. Pat. No. 7,767,429, the entire contents of which are incorporated herein by reference.

[0353]

[0364] In some embodiments, the hyaluronidase is rHuPH20 (also referred to as Hylenex®, currently manufactured by Halozyme and approved by the FDA in 2005 (see, e.g., Scodeller P (2014) Hyaluronidase and other Extracellular Matrix Degrading Enzymes for Cancer Therapy: New Uses and Nano-Formulations. J Carcinog Mutage 5:178, U.S. Patent Nos. 7,767,429, 8,202,517, 7,431,380, 8,450,470, 8,772,246, and 8,580,252, the entire contents of each of which are incorporated herein by reference. rHuPH20 is produced by genetically engineered CHO cells that contain a DNA plasmid encoding a soluble fragment of human hyaluronidase PH20. In some embodiments, the hyaluronidase is glycosylated. In some embodiments, the hyaluronidase has at least one N-linked glycan. Recombinant hyaluronidase can be produced using conventional methods known to those skilled in the art, such as those in U.S. Patent No. 7,767,429, the entire contents of which are incorporated herein by reference.In some embodiments, rHuPH20 is LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRV It has an amino acid sequence that is at least 95% (e.g., at least 96%, 97%, 98%, 99%, 100%) identical to the amino acid sequence of SKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS (SEQ ID NO: 3306).

[0354]

[0365] In any of the methods provided herein, the anti-hyaluronan preparation can be an agent that degrades hyaluronan or an agent that inhibits the synthesis of hyaluronan. For example, the anti-hyaluronan preparation can be a hyaluronan degrading enzyme. In another example, the anti-hyaluronan preparation is an agent that inhibits the synthesis of hyaluronan. For example, the anti-hyaluronan preparation is an agent that inhibits the synthesis of hyaluronan, such as a sense or antisense nucleic acid molecule against HA synthase, or a small molecule drug. For example, the anti-hyaluronan preparation is 4-methylumbelliferone (MU) or a derivative thereof, or leflunomide or a derivative thereof. Such derivatives include, for example, derivatives of 4-methylumbelliferone (MU), which are 6,7-dihydroxy-4-methylcoumarin or 5,7-dihydroxy-4-methylcoumarin.

[0355]

[0366] In further examples of the methods provided herein, the hyaluronan degrading enzyme is hyaluronidase.In some examples, the hyaluronan degrading enzyme is PH20 hyaluronidase or a truncated form thereof that lacks the C-terminal glycosylphosphatidylinositol (GPI) binding site or a portion of the GPI binding site.In certain examples, the hyaluronidase is PH20 selected from human, monkey, bovine, ovine, rat, mouse, or guinea pig PH20. For example, the hyaluronan degrading enzyme is a neutral active, N-glycosylated, human PH20 hyaluronidase, which is: (a) a hyaluronidase polypeptide that is a full-length PH20, or a C-terminal truncated form of PH20, wherein the truncated form contains at least amino acid residues 36-464, e.g., 36-481, 36-482, 36-483 of SEQ ID NO: 139, and the full-length PH20 has the amino acid sequence set forth in SEQ ID NO: 139; or (b) a hyaluronidase polypeptide that is at least 85%, 86%, 87%, 88%, 89%, or 100% identical to the polypeptide or truncated form of the amino acid sequence set forth in SEQ ID NO: 139. or (c) a hyaluronidase polypeptide of (a) or (b) that contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a polypeptide set forth in SEQ ID NO: 139 or its corresponding truncated form. In an illustrative example, the hyaluronan degrading enzyme is PH20, comprising a composition designated rHuPH20.

[0356]

[0367] In other examples, the anti-hyaluronan preparation is a hyaluronan degrading enzyme modified by conjugation to a polymer. The polymer may be PEG, and the anti-hyaluronan preparation is a PEGylated hyaluronan degrading enzyme. Thus, in some examples of the methods provided herein, the hyaluronan degrading enzyme is modified by conjugation to a polymer. For example, the hyaluronan degrading enzyme is conjugated to PEG, and thus the hyaluronan degrading enzyme is PEGylated. In an illustrative example, the hyaluronan degrading enzyme is a PEGylated PH20 enzyme (PEGPH20). In the methods provided herein, the corticosteroid may be a glucocorticoid selected from among cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, and prednisone.

[0357]

[0368] Chondroitinase

[0369] Chondroitinase is an enzyme found throughout the animal kingdom that breaks down glycosaminoglycans, particularly chondroitin and chondroitin sulfate, through endoglycosidase reactions. In some embodiments, the chondroitinase is a mammalian chondroitinase. In some embodiments, the chondroitinase is a recombinant human chondroitinase. In some embodiments, the chondroitinase is HYAL4. Other exemplary chondroitinases include chondroitinase ABC (derived from Proteus vulgaris, JP Patent Publication No. 6-153947, T. Yamagata et al. J. Biol. Chem., 243, 1523 (1968), S. Suzuki et al. J. Biol. Chem., 243, 1543 (1968)), chondroitinase AC (derived from Flavobacterium heparinum, T. Yamagata et al. J. Biol. Chem., 243, 1523 (1968)), chondroitinase AC II (derived from Arthrobacter aurescens, K. Hiyama, and S. Okada, J. Biol. Chem., 250, 1824 (1975), K. Hiyama and S. Okada, J. Biochem. (Tokyo), 80, 1201) and the like. (1976)), hyaluronidase ACIII (derived from Flavobacterium sp. Hp102, Hirofumi Miyazono et al., Seikagaku, 61, 1023 (1989)), chondroitinase B (derived from Flavobacterium heparinum, YM Michelacci and CPDietrich, Biochem. Biophys. Res. Commun., 56, 973 (1974), YM Michelacci and CPDietrich, Biochem. J., 151, 121 (1975), Kenichi Maeyama et al., Seikagaku, 57, 1189 (1985)), and chondroitinase C (derived from Flavobacterium sp. Hp102, Hirofumi Miyazono et al., Seikagaku, 61, 1023 (1939)).

[0358]

[0370] Matrix metalloproteinases

[0371] Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that are the major proteases involved in extracellular matrix (ECM) degradation. MMPs can degrade a wide range of extracellular molecules and some bioactive molecules. In humans, 24 MMP genes have been identified, which are classified into six groups based on domain organization and substrate preference: collagenases (MMP-1, -8, and -13), gelatinases (MMP-2 and MMP-9), stromelysins (MMP-3, -10, and -11), matrilysins (MMP-7 and MMP-26), membrane-type (MT)-MMPs (MMP-14, -15, -16, -17, -24, and -25), and others (MMP-12, -19, -20, -21, -23, -27, and -28). In some embodiments, the stromal modifying moiety is a human recombinant MMP (e.g., MMP-1, -2, -3, -4, -5, -6, -7, -8, -9, 10, -11, -12, -13, -14, 15, -15, -17, -18, -19, 20, -21, -22, -23, or -24).

[0359]

[0372] Collagenase

[0373] The three mammalian collagenases (MMP-1, -8, and -13) are the major secreted endopeptidases that can cleave collagenous extracellular matrix. In addition to fibrillar collagens, collagenases can cleave multiple other matrix and non-matrix proteins, including growth factors. Collagenases are synthesized as inactive alternative forms, and once activated, their activity is inhibited by specific tissue metalloproteinase inhibitors, TIMPs, as well as by non-specific proteinase inhibitors (Ala-aho R et al. Biochimie. Collagenases in cancer. 2005 Mar-Apr;87(3-4):273-86). In some embodiments, the stromal-modifying moiety is collagenase. In some embodiments, the collagenase is human recombinant collagenase. In some embodiments, the collagenase is MMP-1. In some embodiments, the collagenase is MMP-8. In some embodiments, the collagenase is MMP-13.

[0360]

[0374] Macrophage metalloelastase

[0375] Macrophage metalloelastase (MME), also known as MMP-12, is a member of the stromelysin subgroup of MMPs and catalyzes the hydrolysis of a wide range of matrix and non-matrix substrates, including soluble and insoluble elastin, as well as type IV collagen, fibronectin, laminin, vitronectin, entactin, heparan, and chondroitin sulfate (Erja Kerkela et al. Journal of Investigative Dermatology (2000) 114, 1113-1119; doi:10.1046 / j.1523-1747.2000.00993). In some embodiments, the stromal modifying moiety is MME. In some embodiments, the MME is human recombinant MME. In some embodiments, the MME is MMP-12.

[0361]

[0376] Further stromal modification

[0377] In some embodiments, the stromal modifying moiety causes one or more of: a decrease in the level or production of a stromal or extracellular matrix (ECM) component, a decrease in tumor desmoplasia, an increase in stromal tumor trafficking, an improvement in tumor perfusion, an expansion of tumor microvessels, a decrease in interstitial fluid pressure (IFP) in the tumor, or a decrease or increase in penetration or diffusion of an agent, e.g., a cancer therapeutic or cellular therapy, into the tumor or tumor vasculature.

[0362]

[0378] In some embodiments, the stromal or ECM components that are reduced are selected from glycosaminoglycans or extracellular proteins, or combinations thereof. In some embodiments, the glycosaminoglycans are selected from hyaluronan (also known as hyaluronic acid or HA), chondroitin sulfate, chondroitin, dermatan sulfate, heparin, heparin sulfate, entactin, tenascin, aggrecan, and keratin sulfate. In some embodiments, the extracellular proteins are selected from collagen, laminin, elastin, fibrinogen, fibronectin, or vitronectin. In some embodiments, the stromal modifying portion comprises an enzyme molecule that degrades tumor stroma or extracellular matrix (ECM). In some embodiments, the enzyme molecule is selected from a hyaluronidase molecule, a collagenase molecule, a chondroitinase molecule, a matrix metalloproteinase molecule (e.g., macrophage metalloelastase), or a variant (e.g., a fragment) of any of the foregoing. The term "enzyme molecule" includes full-length enzymes, fragments thereof, or variants, eg, enzyme variants that retain at least one functional property of the naturally occurring enzyme.

[0363]

[0379] In some embodiments, the stromal modifying moiety reduces the level or production of hyaluronic acid, hi other embodiments, the stromal modifying moiety comprises a hyaluronan degrading enzyme, an agent that inhibits hyaluronan synthesis, or an antibody molecule against hyaluronic acid.

[0364]

[0380] In some embodiments, the hyaluronan degrading enzyme is a hyaluronidase molecule, e.g., full length or a variant thereof (e.g., a fragment thereof). In some embodiments, the hyaluronan degrading enzyme is active at neutral or acidic pH, e.g., a pH of about 4-5. In some embodiments, the hyaluronidase molecule is a mammalian hyaluronidase molecule, e.g., a recombinant human hyaluronidase molecule, e.g., full length or a variant thereof (e.g., a fragment thereof, e.g., a truncated form). In some embodiments, the hyaluronidase molecule is selected from HYAL1, HYAL2, or PH-20 / SPAM1, or a variant thereof (e.g., a truncated form thereof). In some embodiments, the truncated form lacks a C-terminal glycosylphosphatidylinositol (GPI) attachment site or a portion of the GPI attachment site. In some embodiments, the hyaluronidase molecule is glycosylated, e.g., comprises at least one N-linked glycan.

[0365]

[0381] In some embodiments, the hyaluronidase molecule is LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIW GTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS (SEQ ID NO: 3311), or a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or an amino acid sequence having at least one amino acid change, but not more than 5, 10, or 15 changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), relative to the amino acid sequence of SEQ ID NO: 3311.

[0366]

[0382] In some embodiments, the hyaluronidase molecule is (i) the amino acid sequence of 36 to 464 of SEQ ID NO: 3311; (ii) an amino acid sequence of 36 to 481, 36 to 482, or 36 to 483 of PH20, wherein PH20 has the amino acid sequence set forth in SEQ ID NO: 3311; (iii) an amino acid sequence having at least 95% to 100% sequence identity to the polypeptide of the amino acid sequence set forth in SEQ ID NO: 3311 or a truncated form thereof; or (iv) an amino acid sequence having 30, 20, 10, 5 or fewer amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 3311 In some embodiments, the hyaluronidase molecule comprises an amino acid sequence at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, 100%) identical to the amino acid sequence of SEQ ID NO: 3311. In some embodiments, the hyaluronidase molecule is encoded by a nucleotide sequence at least 95% (e.g., at least 96%, 97%, 98%, 99%, 100%) identical to the nucleotide sequence of SEQ ID NO: 3311.

[0367]

[0383] In some embodiments, the hyaluronidase molecule is PH20, e.g., rHuPH20. In some embodiments, the hyaluronidase molecule is HYAL1, having the amino acid sequence: FRGPLLPNRPFTTVWNANTQWCLERHGVDVDVSVFDVVANPGQTFRGPDMTIFYSSQGTYPYYTPTGEPVFGGLPQNASLIAHLARTFQDILAAIPAPDFSGLAVIDWEAWRPRWAFNWDTKDIYRQRSRALVQAQHPDWPAPQV EAVAQDQFQGAARAWMAGTLQLGRALRPRGLWGFYGFPDCYNYDFLSPNYTGQCPSGIRAQNDQLGWLWGQSRALYPSIYMPAVLEGTGKSQMYVQHRVAEAFRVAVAAGDPNLPVLPYVQIFYDTTNHFLPLDELEHSLGESAA QGAAGVVLWVSWENTRTKESCQAIKEYMDTTLGPFILNVTSGALLCSQALCSGHGRCVRRTSHPKALLLLNPASFSIQLTPGGGPLSLRGALSLEDQAQMAVEFKCRCYPGWQAPWCERKSMW (SEQ ID NO: 3312), or a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid change, but not more than 5, 10, or 15 changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), relative to the amino acid sequence of SEQ ID NO: 3312.

[0368]

[0384] In some embodiments, the hyaluronan degrading enzyme, e.g., hyaluronidase molecule, further comprises a polymer, e.g., is conjugated to a polymer, e.g., PEG. In some embodiments, the hyaluronan degrading enzyme is a PEGylated PH20 enzyme (PEGPH20). In some embodiments, the hyaluronan degrading enzyme, e.g., hyaluronidase molecule, further comprises an immunoglobulin chain constant region (e.g., Fc region), e.g., selected from the heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4, more particularly, the heavy chain constant regions of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the immunoglobulin constant region (e.g., Fc region) is linked, e.g., covalently linked, to the hyaluronan degrading enzyme, e.g., hyaluronidase molecule. In some embodiments, the immunoglobulin chain constant region (e.g., Fc region) is altered, e.g., mutated, to increase or decrease one or more of Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function. In some embodiments, hyaluronan degrading enzymes, e.g., hyaluronidase molecules form dimers.

[0369]

[0385] In some embodiments, the stromal modifying portion comprises an inhibitor of hyaluronan synthesis, for example, HA synthase. In some embodiments, the inhibitor comprises a sense or antisense nucleic acid molecule against HA synthase, or is a small molecule drug. In some embodiments, the inhibitor is 4-methylumbelliferone (MU) or its derivatives (e.g., 6,7-dihydroxy-4-methylcoumarin or 5,7-dihydroxy-4-methylcoumarin), or leflunomide or its derivatives.

[0370]

[0386] In some embodiments, the stromal modifying moiety comprises an antibody molecule against hyaluronic acid.

[0371]

[0387] In some embodiments, the stromal modifying moiety comprises a collagenase molecule, e.g., a mammalian collagenase molecule, or a variant (e.g., fragment) thereof. In some embodiments, the collagenase molecule is, e.g., (SEQ ID NO: 3313), or a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or an amino acid sequence having at least one amino acid alteration, but not more than 5, 10, or 15 alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), relative to the amino acid sequence of SEQ ID NO: 3313.

[0372] Tumor antigen moiety

[0388] In some embodiments, the multifunctional molecule further comprises a tumor antigen moiety. In some embodiments, the tumor targeting moiety is an antigen, e.g., a cancer antigen. In some embodiments, the cancer antigen is a tumor antigen or a stromal antigen, or a blood antigen.

[0373]

[0389] "Cancer", as used herein, can encompass all types of oncogenic processes and / or cancerous growths. In embodiments, cancer includes primary tumors, as well as metastatic tissues, or malignantly transformed cells, tissues, or organs. In embodiments, cancer includes all histopathologies and stages of cancer, e.g., stages of invasiveness / severity. In embodiments, cancer includes recurrent and / or resistant cancers. The terms "cancer" and "tumor" can be used interchangeably. For example, both terms encompass solid and liquid tumors. As used herein, the terms "cancer" or "tumor" include precancers, as well as malignant cancers and tumors.

[0374]

[0390] In some embodiments, the tumor targeting moiety, e.g., a cancer antigen, is selected from the group consisting of BCMA, FcRH5, CD19, CD20, CD22, CD30, CD33, CD38, CD47, CD99, CD123, FcRH5, CLEC12, CD179A, SLAMF7, or NY-ESO1, PDL1, CD47, gangloside 2 (GD2), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PMSA), prostate specific antigen (PSA), carcinoembryonic antigen (CEA), Ron kinase, c-M et, immature laminin receptor, TAG-72, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, Ep-CAM, EphA3, Her2 / neu, telomerase, SAP-1, survivin, NY-ESO-1 / LAGE-1, PRAME, SSX-2, Melan-A / MART-1, Gp100 / pmel17, tyrosinase, TRP-1 / -2, MC1R, β-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, p53, Ra s, TGF-β receptor, AFP, ETA, MAGE, MUC-1, CA-125, BAGE, GAGE, NY-ESO-1, β-catenin, CDK4, CDC27, α-actinin-4, TRP1 / gp75, TRP2, gp100, Melan-A / MART1, ganglioside, WT1, EphA3, epidermal growth factor receptor (EGFR), MART-2, MART-1, MUC1, MUC2, MUM1, MUM2, MUM3, NA88-1, NPM, OA1, OGT, RCC, RU The tumor targeting moiety is selected from I1, RUI2, SAGE, TRG, TRP1, TSTA, folate receptor alpha, L1-CAM, CAIX, gpA33, GD3, GM2, VEGFR, integrins (integrin alpha V beta 3, integrin alpha 5 beta 1), carbohydrate (Le), IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, (FAP), TGF-beta, hyaluronic acid, collagen, e.g., collagen IV, tenascin C, or tenascin W. In some embodiments, the tumor targeting moiety, e.g., cancer antigen, is BCMA. In some embodiments, the tumor targeting moiety, e.g., cancer antigen, is FcRH5.

[0375]

[0391] In some embodiments, the tumor targeting moiety, e.g., a cancer antigen, is selected from the group consisting of CD19, CD123, CD22, CD30, CD171, CS-1, C-type lectin-like molecule-1, CD33, epidermal growth factor receptor variant III (EGFRvIII), ganglioside G2 (GD2), ganglioside GD3, TNF receptor family member B cell maturation (BCMA), Tn antigen (TnAg) or (GalNAcα-Ser / Thr)), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3), tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (CD117), interleukin-13 receptor subunit alpha-2, mesothelin, interleukin-11 receptor alpha (IL-11Ra), prostate stem cell antigen ( PSCA), protease serine 21, vascular endothelial growth factor receptor 2 (VEGFR2), Lewis (Y) antigen, CD24, platelet-derived growth factor receptor beta (PDGFR-beta), stage-specific embryonic antigen-4 (SSEA-4), CD20, folate receptor alpha, receptor tyrosine-protein kinase ERBB2 (Her2 / neu), cell surface-associated mucin 1 (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), prostase, prostatic acid phosphatase (PAP), and mutant elongation factor 2 (ELF2M), ephrinB2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (prosome, macropain) subunit, beta, 9 (LMP2), glycoprotein 100 (gp100), oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl), tyrosinase, ephrin type A receptor 2 (EphA 2), fucosyl GM1, sialyl Lewis adhesion molecule (sLe), ganglioside GM3, transglutaminase 5 (TGS5), high molecular weight melanoma-associated antigen (HMWMAA), o-acetyl-GD2 ganglioside (OAcGD2), folate receptor beta, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), claudin 6 (CLDN6), thyroid-stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5, member D (GPRC5D), chromosome X open reading frame61 (CXORF61), CD97, CD179a, anaplastic lymphoma kinase (ALK), polysialic acid, placenta specific 1 (PLAC1), hexasaccharide moiety of globoH glycoceramide (GloboH), mammary differentiation antigen (NY-BR-1), uroplakin 2 (UPK2), hepatitis A virus cellular receptor 1 (HAVCR1), adrenoceptor beta 3 (ADRB3), pannexin 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex, locus K9 (LY6K), olfactory receptor 51E2 (OR51E2), TCR gamma alternate reading frame protein (TARP), Wilms tumor protein (WT1), cancer / testis antigen 1 (NY-ESO-1), cancer / testis antigen 2 (LAGE-1a), melanoma-associated antigen 1 (MAGE-A1), ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML), sperm protein 17 (SPA17), X antigen family, member 1A (XAGE1), angiopoietin-binding cell surface receptor 2 (Tie 2), melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), Fos-related antigen 1, tumor protein p53 (p53), p53 mutant, prostein, survivin, telomerase, prostate cancer tumor antigen-1, melanoma antigen 1 recognized by T cells, rat sarcoma (Ras) mutant, human telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoint, melanoma apoptosis inhibitor (ML-IA) P), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), N-acetylglucosaminyl-transferase V (NA17), paired box protein Pax-3 (PAX3), androgen receptor, cyclin B1, v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN), Ras homolog family member C (RhoC), tyrosinase-related protein 2 (TRP-2), cytochrome P4501B1 (CYP1B1), CCCTC-binding factor (zinc finger protein)-like, squamous cell carcinoma antigen recognized by T cells 3 (SART3), paired box protein Pax-5 (PAX5), proacrosin-binding protein sp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), A-kinase anchoring protein 4 (AKAP-4), synovial sarcoma X breakpoint 2 (SSX2), receptor for advanced glycation end products (RAGE-1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), legumain, human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), intestinal carboxylesterase, mutated heat shock protein 70-2 (mut hsp70-2), CD79a, CD79b, CD72, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Fc fragment of the IgA receptor (FCAR or CD89), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), CD300 molecule-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), lymphocyte antigen 75 (LY75), glypican-3 (GPC3), Fc receptor-like 5 (FCRL5), or immunoglobulin lambda-like polypeptide 1 (IGLL1).

[0376]

[0392] FcRH5 targeting moiety:

[0393] In some embodiments, the multispecific molecules described herein include a targeting moiety (e.g., an FcRH5 targeting moiety) that binds to FcRH5. The FcRH5 targeting moiety can be selected from an antibody molecule (e.g., an antigen-binding domain described herein), a receptor or receptor fragment, or a ligand or ligand fragment, or a combination thereof. In some embodiments, the FcRH5 targeting moiety associates with, e.g., binds to, a cancer or hematopoietic cell (e.g., a molecule, e.g., an antigen, present on the surface of a cancer or hematopoietic cell). In certain embodiments, the FcRH5 targeting moiety targets, e.g., directs, a multispecific molecule described herein to a cancer or hematopoietic cell. In some embodiments, the cancer is a hematological cancer, e.g., multiple myeloma.

[0377]

[0394] In some embodiments, the multispecific molecule, for example, the FcRH5 targeting portion, binds to the FcRH5 antigen on the surface of cells, for example, cancer or hematopoietic cells.The FcRH5 antigen can be present on primary tumor cells or on metastatic lesions thereof.In some embodiments, the cancer is a blood cancer, for example, multiple myeloma.For example, the FcRH5 antigen can be present on tumor, for example, on a class of tumors that are typed by having one or more of the following: limited tumor perfusion, vascular compression, or fibrous tumor stroma.

[0378]

[0395] Multispecific molecules described herein include FcRH5 targeting moieties comprising anti-FcRH5 antibodies or antigen-binding fragments thereof described in U.S. Patent No. 7,999,077, U.S. Patent Application Publication No. 20150098900, U.S. Patent No. 8,299,220, U.S. Patent No. 7,105,149, U.S. Patent No. 8,362,213, U.S. Patent No. 8,466,260, U.S. Patent No. 8,617,559, U.S. Patent Application Publication No. 20160368985, U.S. Patent Application Publication No. 20150166661, and U.S. Patent Application Publication No. 20080247944 (the entire contents of any of the above publications are hereby incorporated by reference herein).

[0379]

[0396] In some embodiments, the multispecific molecules described herein comprise an FcRH5 targeting moiety comprising an anti-FcRH5 antibody or antigen-binding fragment thereof described in U.S. Pat. No. 7,999,077, the entire contents of which are hereby incorporated by reference herein.

[0380]

[0397] BCMA targeting moiety:

[0398] In certain embodiments, the multispecific molecules described herein include a targeting moiety (e.g., a BCMA targeting moiety) that binds to BCMA. The BCMA targeting moiety can be selected from an antibody molecule (e.g., an antigen-binding domain described herein), a receptor or receptor fragment, or a ligand or ligand fragment, or a combination thereof. In some embodiments, the BCMA targeting moiety associates with, e.g., binds to, a cancer or hematopoietic cell (e.g., a molecule, e.g., an antigen, present on the surface of a cancer or hematopoietic cell). In certain embodiments, the BCMA targeting moiety targets, e.g., directs, a multispecific molecule described herein to a cancer or hematopoietic cell. In some embodiments, the cancer is a hematological cancer, e.g., multiple myeloma.

[0381]

[0399] In some embodiments, the multispecific molecule, e.g., BCMA targeting moiety, binds to the BCMA antigen on the surface of a cell, e.g., cancer or hematopoietic cell. The BCMA antigen can be present on a primary tumor cell or on a metastatic lesion thereof. In some embodiments, the cancer is a blood cancer, e.g., multiple myeloma. For example, the BCMA antigen can be present on a tumor, e.g., on a class of tumors that are typed by having one or more of the following: limited tumor perfusion, vascular compression, or fibrous tumor stroma.

[0382]

[0400] Exemplary BCMA targeting moieties: The multispecific molecules described herein are disclosed in U.S. Pat. Nos. 8,920,776, 9,243,058, 9,340,621, 8,846,042, 7,083,785, 9,545,086, 7,276,241, 9,034,324, 7,799,902, 9,387,237, 8,821, 8,921, 8,341, 8,421, 8,541, 8,621, 8,721, 8,841, 8,821, 8,921, 8,921, 8,921, 8,541, 8,721, 8,841, 8,92 ... 883, U.S. Patent No. 861745, U.S. Patent Application Publication No. 20130273055, U.S. Patent Application Publication No. 20160176973, U.S. Patent Application Publication No. 20150368351, U.S. Patent Application Publication No. 20150376287, U.S. Patent Application Publication No. 20170022284, U.S. Patent Application Publication No. 20160015749, U.S. Patent Application Publication No. 20140242077, U.S. Patent Application Publication No. US Patent Application Publication No. 20170037128, US Patent Application Publication No. 20170051068, US Patent Application Publication No. 20160368988, US Patent Application Publication No. 20160311915, US Patent Application Publication No. 20160131654, US Patent Application Publication No. 20120213768, US Patent Application Publication No. 20110177093, US Patent Application Publication No. 20160297885, EP3137500, The BCMA targeting moiety may comprise an anti-BCMA antibody or antigen-binding fragment thereof as described in EP2699259, EP2982694, EP3029068, EP3023437, WO2016090327, WO2017021450, WO2016110584, WO2016118641, WO2016168149 (the entire contents of which are hereby incorporated by reference).

[0383]

[0401] In some embodiments, the BCMA targeting moiety comprises an antibody molecule (e.g., a Fab or scFv) that binds to BCMA. In some embodiments, the antibody molecule against BCMA comprises one, two, or three CDRs from any of the heavy chain variable domain sequences in Table 7, or a closely related CDR, e.g., a CDR with at least one amino acid change, but no more than two, three or four changes (e.g., a substitution, deletion, or insertion, e.g., a conservative substitution) from any of the CDR sequences in Table 7. In some embodiments, the antibody molecule against BCMA comprises a heavy chain variable domain sequence selected from any of the amino acid sequences in Table 7, or an amino acid sequence substantially identical thereto (e.g., 95%-99.9% identical thereto, or with at least one amino acid change, but no more than five, ten or fifteen changes (e.g., a substitution, deletion, or insertion, e.g., a conservative substitution)).

[0384]

[0402] Alternatively, or in combination with a heavy chain against BCMA described herein, an antibody molecule against BCMA comprises one, two, or three CDRs from any of the light chain variable domain sequences in Table 7, or a closely related CDR, e.g., a CDR with at least one amino acid change but no more than two, three or four changes (e.g., a substitution, deletion, or insertion, e.g., a conservative substitution) from any of the CDR sequences in Table 7. In some embodiments, an antibody molecule against BCMA comprises a light chain variable domain sequence selected from any of the amino acid sequences in Table 7, or an amino acid sequence substantially identical thereto (e.g., 95%-99.9% identical thereto, or with at least one amino acid change but no more than five, ten or fifteen changes (e.g., a substitution, deletion, or insertion, e.g., a conservative substitution)).

[0385] Tumor targeting moiety

[0403] In some embodiments, the multifunctional or multispecific (e.g., bispecific, trispecific, tetraspecific) molecules described herein further comprise, e.g., are engineered to further comprise, one or more tumor-specific targeting moieties that direct the molecule to tumor cells.

[0386]

[0404] In certain embodiments, the multispecific molecule described herein further comprises a tumor targeting moiety. The tumor targeting moiety can be selected from an antibody molecule (e.g., an antigen-binding domain described herein), a receptor or receptor fragment, or a ligand or ligand fragment, or a combination thereof. In some embodiments, the tumor targeting moiety associates with, e.g., binds to, a tumor cell (e.g., a molecule present on the surface of a tumor cell, e.g., an antigen). In certain embodiments, the tumor targeting moiety targets, e.g., directs, the multispecific molecule described herein to a cancer (e.g., a cancer or tumor cell). In some embodiments, the cancer is selected from a hematological cancer, a solid cancer, a metastatic cancer, or a combination thereof.

[0387]

[0405] In some embodiments, the multispecific molecule, e.g., tumor targeting moiety, binds to a solid tumor antigen or stromal antigen. The solid tumor antigen or stromal antigen can be present on a solid tumor or its metastatic lesion. In some embodiments, the solid tumor is selected from one or more of pancreatic cancer (e.g., pancreatic adenocarcinoma), breast cancer, colorectal cancer, lung cancer (e.g., small cell or non-small cell lung cancer), skin cancer, ovarian cancer, or liver cancer. In some embodiments, the solid tumor is a fibrotic or desmoplastic solid tumor. For example, the solid tumor antigen or stromal antigen can be present on a tumor, e.g., on a class of tumors that are typed by having one or more of limited tumor perfusion, vascular compression, or fibrotic tumor stroma.

[0388]

[0406] In certain embodiments, the solid tumor antigen is selected from the group consisting of PDL1, CD47, gangloside 2 (GD2), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PMSA), prostate specific antigen (PSA), carcinoembryonic antigen (CEA), Ron kinase, c-Met, immature laminin receptor, TAG-72, BING-4, calcium activated chloride channel 2, cyclin-B1, 9D7, Ep-CAM, EphA3, Her2 / neu, telomerase, SAP-1, survivin, NY-ESO-1 / LAGE-1, PRAME, SSX-2, Melan-A / MART-1, Gp100 / pmel17, tyrosinase, TRP-1 / -2, MC1R, β-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, p53, Ras, TGF-β receptor, AFP, ETA, MAGE, MUC-1, CA-125, B AGE, GAGE, NY-ESO-1, β-catenin, CDK4, CDC27, CD47, α-actinin-4, TRP1 / gp75, TRP2, gp100, Melan-A / MART1, ganglioside, WT1, EphA3, epidermal growth factor receptor (EGFR), MART-2, MART-1, MUC1, MUC2, MUM1, MUM2, MUM3, NA88-1, NPM, OA1, OGT, RCC, RU Selected from one or more of I1, RUI2, SAGE, TRG, TRP1, TSTA, folate receptor alpha, L1-CAM, CAIX, EGFRvIII, gpA33, GD3, GM2, VEGFR, integrins (integrin alpha V beta 3, integrin alpha 5 beta 1), carbohydrate (Le), IGF1R, EPHA3, TRAILR1, TRAILR2, or RANKL.

[0389]

[0407] In other embodiments, the multispecific molecule, e.g., tumor targeting moiety, binds to a molecule, e.g., an antigen, present on the surface of a blood cancer, e.g., a leukemia or lymphoma. In some embodiments, the blood cancer is a B-cell or T-cell malignancy. In some embodiments, the blood cancer is selected from one or more of Hodgkin's lymphoma, non-Hodgkin's lymphoma (e.g., B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, marginal zone B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, hairy cell lymphoma), acute myeloid leukemia (AML), chronic myeloid leukemia, myelodysplastic syndrome (MDS), multiple myeloma, or acute lymphocytic leukemia. In embodiments, the cancer is other than acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). In embodiments, the blood antigen is selected from CD47, CD99, CD30, CD38, SLAMF7, or NY-ESO1. In some embodiments, the blood antigen is selected from one or more of BCMA, CD19, CD20, CD22, CD33, CD123, FcRH5, CLEC12, or CD179A.

[0390] antibody molecule

[0408] In some embodiments, the antibody molecule binds to a cancer antigen, e.g., a tumor antigen or a stromal antigen. In some embodiments, the cancer antigen is, e.g., a mammalian, e.g., human, cancer antigen. In other embodiments, the antibody molecule binds to an immune cell antigen, e.g., a mammalian, e.g., human, immune cell antigen. For example, the antibody molecule specifically binds to an epitope, e.g., a linear epitope or a conformational epitope, on the cancer antigen or immune cell antigen.

[0391]

[0409] In some embodiments, the antibody molecule is a monospecific antibody molecule and binds to a single epitope, e.g., a monospecific antibody molecule has multiple immunoglobulin variable domain sequences that each bind to the same epitope.

[0392]

[0410] In some embodiments, the antibody molecule is a multispecific or multifunctional antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domain sequences, a first of the plurality of immunoglobulin variable domain sequences having binding specificity for a first epitope, and a second of the plurality of immunoglobulin variable domain sequences having binding specificity for a second epitope. In some embodiments, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In some embodiments, the first and second epitopes overlap. In some embodiments, the first and second epitopes do not overlap. In some embodiments, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In some embodiments, the multispecific antibody molecule comprises a third, fourth, or fifth immunoglobulin variable domain. In some embodiments, the multispecific antibody molecule is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.

[0393]

[0411] In some embodiments, the multifunctional antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In some embodiments, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In some embodiments, the first and second epitopes overlap. In some embodiments, the first and second epitopes do not overlap. In some embodiments, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In some embodiments, the bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for a second epitope. In some embodiments, a bispecific antibody molecule comprises a half antibody having binding specificity for a first epitope and a half antibody having binding specificity for a second epitope. In some embodiments, a bispecific antibody molecule comprises a half antibody, or a fragment thereof, having binding specificity for a first epitope and a half antibody, or a fragment thereof, having binding specificity for a second epitope. In some embodiments, a bispecific antibody molecule comprises an scFv or Fab, or a fragment thereof, having binding specificity for a first epitope and an scFv or Fab, or a fragment thereof, having binding specificity for a second epitope.

[0394]

[0412] In some embodiments, antibody molecules include diabodies, and single chain molecules, as well as antigen-binding fragments of antibodies (e.g., Fab, F(ab') 2, and Fv). For example, an antibody molecule can include a heavy (H) chain variable domain sequence (abbreviated herein as VH), and a light (L) chain variable domain sequence (abbreviated herein as VL). In some embodiments, an antibody molecule comprises or consists of a heavy chain and a light chain (referred to herein as a half antibody). In another example, an antibody molecule includes two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequences, thereby providing two antigen-binding sites, e.g., Fab, Fab', F(ab') and F(ab') sequences. 2 , Fc, Fd, Fd', Fv, single chain antibodies (e.g., scFv), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which may be produced by modification of full-length antibodies or synthesized de novo using recombinant DNA technology. These functional antibody fragments retain the ability to selectively bind to their respective antigens or receptors. The antibodies and antibody fragments may be from any class of antibodies, including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass of antibodies (e.g., IgG1, IgG2, IgG3, and IgG4). Preparations of antibody molecules may be monoclonal or polyclonal. The antibody molecules may also be human, humanized, CDR-grafted, or in vitro generated antibodies. The antibodies may have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. An antibody may also have a light chain chosen from, e.g., kappa or lambda. The term "immunoglobulin" (Ig) is used interchangeably herein with the term "antibody."

[0395]

[0413] Examples of antigen-binding fragments of antibody molecules include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment, which consists of the VH and CH1 domains; (iv) an Fv fragment, which consists of the VL and VH domains of a single arm of an antibody; (v) a diabody (dAb) fragment, which consists of a VH domain; (vi) a camel or camelized variable domain; (vii) a single chain Fv (scFv); see, e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and (viii) a single domain antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0396]

[0414] Antibody molecules include intact molecules as well as functional fragments thereof. The constant regions of antibody molecules can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function).

[0397]

[0415] The antibody molecule may also be a single domain antibody. Single domain antibodies may include antibodies whose complementary determining regions are portions of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional four chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. The single domain antibody may be any in the art or any future single domain antibody. The single domain antibody may be derived from any species including, but not limited to, mouse, human, camel, llama, fish, shark, goat, rabbit, and cow. According to another aspect of the present disclosure, the single domain antibody is a naturally occurring single domain antibody known as a heavy chain antibody devoid of light chains. Such single domain antibodies are disclosed, for example, in WO9404678. For reasons of clarity, this variable domain derived from a heavy chain antibody naturally devoid of light chains is known herein as a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins. Such VHH molecules can be derived from antibodies produced in Camelidae species, such as camel, llama, dromedary, alpaca and guanaco. Other non-Camelidae species may produce heavy chain antibodies that are naturally devoid of light chains, and such VHHs are within the scope of the present disclosure.

[0398]

[0416] The VH and VL regions can be subdivided into regions of hypervariability, called "complementarity determining regions" (CDRs), interspersed with regions that are more conserved, called "framework regions" (FR or FW).

[0399]

[0417] The extent of the framework regions and CDRs have been precisely defined in a number of ways (see Kabat, E. A. et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., USDepartment of Health and Human Services, NIH Publication No. 91-3242; Chothia, C. et al., (1987) J. Mol. Biol. 196:901-917; and the AbM definitions used by Oxford Molecular's AbM antibody modeling software. See generally, e.g., "Protein Sequence and Structure Analysis of Antibody Variable Domains," Antibody Engineering Lab Manual (Duebel, S. and Kontermann, R., eds., Springer-Verlag, Heidelberg)).

[0400]

[0418] The terms "complementarity determining region" and "CDR" as used herein refer to the amino acid sequence in the antibody variable region that confers antigen specificity and binding affinity. Generally, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.

[0401]

[0419] The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of known schemes, including those described in Kabat et al., (1991), "Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme). As used herein, CDRs defined according to the "Chothia" numbering scheme are sometimes referred to as "hypervariable loops".

[0402]

[0420] For example, under Kabat, the CDR amino acid residues of the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); the CDR amino acid residues of the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under Chothia, the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the amino acid residues in the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).

[0403]

[0421] Each VH and VL typically contains three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0404]

[0422] The antibody molecule can be a polyclonal or a monoclonal antibody.

[0405]

[0423] The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Monoclonal antibodies can be made by hybridoma technology or by methods that do not use hybridoma technology (e.g., recombinant methods).

[0406]

[0424] Antibodies can be produced recombinantly, for example by phage display or combinatorial methods, or yeast display.

[0407]

[0425] Phage display and combinatorial methods for generating antibodies are known in the art (see, e.g., Ladner et al., U.S. Pat. No. 5,223,409; Kang et al., International Publication No. WO 92 / 18619; Dower et al., International Publication No. WO 91 / 17271; Winter et al., International Publication No. WO 92 / 20791; Markland et al., International Publication No. WO 92 / 15679; Breitling et al., International Publication No. WO 93 / 01288; McCafferty et al., International Publication No. WO 92 / 01047; Garrard et al., International Publication No. WO 92 / 09690; Ladner et al., International Publication No. WO 90 / 02809; Fuchs et al., (1991) Bio / Technology 9:1370-1372; Hay et al., (1992) Hum Antibod Hybridomas 3:81-85; Huse et al., (1989) Science 246:1275-1281; Griffths et al., (1993) EMBO J 12:725-734; Hawkins et al., (1992) J Mol Biol 226:889-896; Clackson et al., (1991) Nature 352:624-628; Gram et al., (1992) PNAS 89:3576-3580; Garrad et al., (1991) Bio / Technology 9:1373-1377; Hoogenboom et al., (1991) Nuc Acid Res 19:4133-4137; and Barbas et al., (1991) PNAS 88:7978-7982, the entire contents of which are hereby incorporated by reference.

[0408]

[0426] Yeast display methods for generating or identifying antibodies are known in the art, for example, as described in Chao et al. (2006) Nature Protocols 1(2):755-68, the entire contents of which are hereby incorporated by reference herein.

[0409]

[0427] In some embodiments, the antibody is a fully human antibody (e.g., an antibody made in a mouse genetically engineered to produce antibodies from human immunoglobulin sequences), or a non-human antibody, such as a rodent (mouse or rat), goat, primate (e.g., monkey), or camel antibody. Preferably, the non-human antibody is a rodent (mouse or rat antibody). Methods for producing rodent antibodies are known in the art.

[0410]

[0428] Human monoclonal antibodies can be produced using transgenic mice carrying non-mouse human immunoglobulin genes. Spleen cells from these transgenic mice immunized with an antigen of interest are used to produce hybridomas secreting human mAbs with specific affinity for epitopes from human proteins (see, e.g., Wood et al., International Application WO 91 / 00906; Kucherlapati et al., PCT International Publication WO 91 / 10741; Lonberg et al., International Application WO 92 / 03918; Kay et al., International Application 92 / 03917; Lonberg, N. et al., 1994 Nature 368:856-859; Green, LL et al., 1994 Nature Genet. 7:13-21; Morrison, SL et al., 1994 Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al., 1993 Year Immunol 7:33-40; Tuaillon et al., 1993 PNAS 368:856-859). 90:3720-3724; Bruggeman et al., 1991 Eur J Immunol 21:1323-1326).

[0411]

[0429] The antibody molecule may be one in which the variable region, or a part thereof, such as CDR, is generated in a non-human organism, such as a rat or a mouse. Chimeric, CDR-grafted, and humanized antibodies are within the scope of this disclosure. Antibody molecules generated in a non-human organism, such as a rat or a mouse, and then modified, for example, in the variable framework or constant region, to reduce antigenicity in humans, are within the scope of this disclosure.

[0412]

[0430] An "effectively human" protein is one that does not substantially elicit a neutralizing antibody response, e.g., a human anti-mouse antibody (HAMA) response. HAMA can be problematic in many situations, e.g., when an antibody molecule is administered repeatedly, e.g., in the treatment of chronic or recurrent disease states. HAMA responses can render repeated antibody administration potentially ineffective due to increased clearance of the antibody from serum (see, e.g., Saleh et al., Cancer Immunol. Immunother., 32:180-190 (1990)), and also due to potential allergic reactions (see, e.g., LoBuglio et al., Hybridoma, 5:5117-5123 (1986)).

[0413]

[0431] Chimeric antibodies can be produced by recombinant DNA techniques known in the art (Robinson et al., International Publication No. PCT / US86 / 02269; Akira et al., European Patent Application No. 184,187; Taniguchi, M., European Patent Application No. 171,496; Morrison et al., European Patent Application No. 173,494; Neuberger et al., International Publication WO 86 / 01533; Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application No. 125,023; Better et al., (1988 Science 240:1041-1043); Liu et al., (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al., (1987) PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al., (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559).

[0414]

[0432] A humanized antibody or a CDR-grafted antibody will have at least one or two, but generally all three recipient CDRs (of the immunoglobulin heavy chain and / or immunoglobulin light chain) replaced with donor CDRs. The antibody may have at least a portion of a non-human CDR replaced, or only a portion of the CDRs replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding to the antigen. Preferably, the donor is a rodent antibody, e.g., a rat or mouse antibody, and the recipient is a human framework or a human consensus framework. Typically, the immunoglobulin providing the CDRs is called the "donor" and the immunoglobulin providing the framework is called the "acceptor". In some embodiments, the donor immunoglobulin is non-human (e.g., rodent). The acceptor framework is a naturally occurring (e.g., human) framework or consensus framework, or a sequence that is about 85% or higher, preferably 90%, 95%, 99% or more identical thereto.

[0415]

[0433] As used herein, the term "consensus sequence" refers to a sequence formed from the amino acids (or nucleotides) that occur most frequently in a family of related sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family of proteins, each position in the consensus sequence is occupied by the amino acid that occurs most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence. "Consensus framework" refers to the framework region in the consensus immunoglobulin sequence.

[0416]

[0434] Antibody molecules can be humanized by methods known in the art (see, e.g., Morrison, SL, 1985, Science 229:1202-1207; Oi et al., 1986, BioTechniques 4:214; and Queen et al., U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762, the contents of all of which are hereby incorporated by reference).

[0417]

[0435] Humanized or CDR-grafted antibody molecules can be produced by CDR-grafting or CDR-replacement, in which one, two or all CDRs of an immunoglobulin chain can be replaced. See, for example, U.S. Patent No. 5,225,539; Jones et al., 1986 Nature 321:552-525; Verhoeyan et al., 1988 Science 239:1534; Beidler et al., 1988 J.Immunol.141:4053-4060; Winter, U.S. Patent No. 5,225,539, the contents of all of which are hereby expressly incorporated herein by reference. Winter describes a CDR-grafting method that can be used to prepare the humanized antibodies of the present disclosure (UK Patent Application No. 2188638A filed March 26, 1987; Winter, U.S. Patent No. 5,225,539), the contents of which are hereby expressly incorporated herein by reference.

[0418]

[0436] Humanized antibody molecules in which specific amino acids are replaced, deleted or added are also within the scope of the present disclosure. Criteria for selecting amino acids from donors are described in U.S. Patent No. 5,585,089, e.g., U.S. Patent No. 5,585,089, columns 12-16, e.g., U.S. Patent No. 5,585,089, columns 12-16, the contents of which are hereby incorporated by reference. Other techniques for humanizing antibodies are described in Padlan et al., EP 519596 A1, published December 23, 1992.

[0419]

[0437] The antibody molecule can be a single chain antibody. Single chain antibodies (scFV) can be engineered (see, e.g., Colcher, D. et al., (1999) Ann NY Acad Sci 880:263-80; and Reiter, Y., (1996) Clin Cancer Res 2:245-52). Single chain antibodies can be dimerized or multimerized to generate multivalent antibodies with specificity for different epitopes of the same target protein.

[0420]

[0438] In yet other embodiments, the antibody molecule has a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE heavy chain constant regions, in particular, for example, IgG1, IgG2, IgG3, and IgG4 (e.g., human) heavy chain constant regions. In another embodiment, the antibody molecule has a light chain constant region selected from, for example, kappa or lambda (e.g., human) light chain constant regions. The constant region can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, and / or complement function). In some embodiments, the antibody has effector function; and can fix complement. In other embodiments, the antibody does not recruit effector cells and does not fix complement. In another embodiment, the antibody has reduced or no ability to bind to Fc receptors. For example, it may be an isotype or subtype, fragment or other mutant that does not support binding to an Fc receptor, e.g., it has a mutagenized or deleted Fc receptor binding region.

[0421]

[0439] Methods for modifying antibody constant regions are known in the art.Antibodies with modified functions, such as modified affinity for effector ligands, such as FcR on cells, or C1 component of complement, can be produced by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see, for example, EP388,151A1, U.S. Patent No. 5,624,821 and U.S. Patent No. 5,648,260, the contents of all of which are hereby incorporated by reference).Similar types of modifications can be described that reduce or eliminate these functions when applied to mouse, or other species of immunoglobulins.

[0422]

[0440] An antibody molecule may be derivatized or linked to another functional molecule (e.g., another peptide or protein). As used herein, a "derivatized" antibody molecule is one that has been modified. Methods of derivatization include, but are not limited to, the addition of fluorescent moieties, radionucleotides, toxins, enzymes, or affinity ligands, such as biotin. Thus, the antibody molecules of the present disclosure are intended to include derivatized and otherwise modified forms of the antibodies described herein, including immunoadhesion molecules. For example, an antibody molecule may be functionally linked (by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and / or a protein or peptide that can mediate the association of the antibody or antibody portion with another molecule (e.g., a streptavidin core region or a polyhistidine tag).

[0423]

[0441] One type of derivatized antibody molecule is produced by crosslinking two or more antibodies (of the same or different types, e.g., to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, having two separately reactive groups separated by a suitable spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, Ill.

[0424] CDR-grafted scaffolds

[0442] In some embodiments, the antibody molecule is a CDR-grafted scaffold domain. In some embodiments, the scaffold domain is based on a fibronectin domain, for example, a fibronectin type III domain. The overall fold of the fibronectin type III (Fn3) domain is closely related to that of the smallest functional antibody fragment, the variable domain of the antibody heavy chain. There are three loops at the end of Fn3, and the positions of the BC, DE and FG loops roughly correspond to those of CDR1, 2 and 3 of the VH domain of an antibody. Fn3 does not have disulfide bonds, and therefore, unlike antibodies and their fragments, Fn3 is stable under reducing conditions (see, for example, WO98 / 56915, WO01 / 64942, WO00 / 34784). The Fn3 domain can be modified or altered (e.g., using the CDRs or hypervariable loops described herein) to select domains that bind to, for example, antigens / markers / cells described herein.

[0425]

[0443] In some embodiments, the scaffold domain, e.g., the folded domain, is based on a "minibody" scaffold created by deleting three beta strands from the heavy chain variable domain of an antibody, e.g., a monoclonal antibody (see, e.g., Tramontano et al., 1994, J Mol. Recognit. 7:9, and Martin et al., 1994, EMBO J. 13:5303-5309). A "minibody" can be used to display two hypervariable loops. In some embodiments, the scaffold domain is a V-like domain (see, e.g., Coia et al., WO 99 / 45110), or a domain derived from tendamistatin, a 74-residue six-stranded beta sheet sandwich held together by two disulfide bonds (see, e.g., McConnell and Hoess, 1995, J Mol. Biol. 250:460). For example, the loops of tendamistatin can be modified or altered (e.g., using CDRs or hypervariable loops) to select domains that bind to, for example, markers / antigens / cells described herein. Another exemplary scaffold domain is a beta-sandwich structure derived from the extracellular domain of CTLA-4 (see, e.g., WO00 / 60070).

[0426]

[0444] Other exemplary scaffold domains include, but are not limited to, T cell receptors, MHC proteins, extracellular domains (e.g., fibronectin type III repeats, EGF repeats), protease inhibitors (e.g., Kunitz domains, ecotin, and BPTI, etc.), TPR repeats, trifoil structures, zinc finger domains, DNA binding proteins, particularly monomeric DNA binding proteins, RNA binding proteins, enzymes, such as proteases (particularly inactivating proteases), RNases, chaperones, such as thioredoxin, and heat shock proteins, and intracellular signaling domains (e.g., SH2 and SH3 domains). See, e.g., U.S. Patent Application Publication No. 20040009530 and U.S. Patent No. 7,501,121, which are incorporated herein by reference.

[0427]

[0445] In some embodiments, scaffold domains are evaluated and selected, for example, by one or more of the following criteria: (1) amino acid sequence, (2) sequence of several homologous domains, (3) three-dimensional structure, and / or (4) stability data over a range of pH, temperature, salt, organic solvent, and oxidant concentrations. In some embodiments, scaffold domains are small, stable protein domains, e.g., proteins of less than 100, 70, 50, 40, or 30 amino acids. The domains may contain one or more disulfide bonds or may chelate a metal, e.g., zinc.

[0428] Antibody-Based Fusions

[0446] A variety of formats can be generated that contain additional binding entities attached to the N- or C-terminus of the antibody. These fusions with single chain or disulfide-stabilized Fv or Fab result in the generation of tetravalent molecules with bivalent binding specificity for each antigen. The combination of scFv and scFab with IgG allows the production of molecules that can recognize three or more different antigens.

[0429] Antibody-Fab fusion

[0447] Antibody-Fab fusions are bispecific antibodies that contain a traditional antibody against a first target and a Fab against a second target fused to the C-terminus of the antibody heavy chain. Generally, the antibody and Fab have a common light chain. Antibody fusions can be produced by (1) manipulating the DNA sequence of the target fusion and (2) transfecting the target DNA into a suitable host cell to express the fusion protein. As described by Coloma, J et al. (1997) Nature Biotech 15:159, it appears that antibody-scFv fusions can be linked by a (Gly)-Ser linker between the C-terminus of the CH3 domain and the N-terminus of the scFv.

[0430]

[0448] Antibody-scFv fusion

[0449] Antibody-scFv fusions are bispecific antibodies that contain a traditional antibody and a scFv of unique specificity fused to the C-terminus of the antibody heavy chain. The scFv can be fused to the C-terminus through the heavy chain of the scFv directly or through a linker peptide. Antibody fusions can be produced by (1) manipulating the DNA sequence of the target fusion, and (2) transfecting the target DNA into a suitable host cell to express the fusion protein. As described by Coloma, J et al. (1997) Nature Biotech 15:159, it appears that antibody-scFv fusions may be linked by a (Gly)-Ser linker between the C-terminus of the CH3 domain and the N-terminus of the scFv.

[0431] Variable Domain Immunoglobulin DVD

[0450] A related format is the dual variable domain immunoglobulin (DVD), which consists of a VH and VL domain of a second specificity positioned N-terminal to the V domain by a shorter linker sequence.

[0432]

[0451] Other exemplary multispecific antibody formats include, for example, those described in U.S. Patent Application Publication No. 20160114057A1, U.S. Patent Application Publication No. 20130243775A1, U.S. Patent Application Publication No. 20140051833, U.S. Patent Application Publication No. 20130022601, U.S. Patent Application Publication No. 20150017187A1, U.S. Patent Application Publication No. 20120201746A1, U.S. Patent Application Publication No. 20150133638A1, U.S. Patent Application Publication No. 20130266568A1, U.S. Patent Application Publication No. 20160145340A1, WO2015127158A1, U.S. Patent Application Publication No. 20150203591A1, U.S. Patent Application Publication No. 201 No. 40322221A1, U.S. Patent Application Publication No. 20130303396A1, U.S. Patent Application Publication No. 20110293613, U.S. Patent Application Publication No. 20130017200A1, U.S. Patent Application Publication No. 20160102135A1, WO2015197598A2, WO2015197582A1, U.S. Patent No. 9,359,437, U.S. Patent Application Publication No. 20150018529, WO2016115274A1, WO2016087416A1, U.S. Patent Application Publication No. 20080069820A1, U.S. Patent No. 9,145,588B, U.S. Patent No. 7,919,257, and U.S. Patent Application Publication No. 20150232560A1. Exemplary multispecific molecules utilizing full length antibody-Fab / scFab formats include those described in the following: U.S. Patent No. 9,382,323 B2, U.S. Patent Application Publication No. 20140072581 A1, U.S. Patent Application Publication No. 20140308285 A1, U.S. Patent Application Publication No. 20130165638 A1, U.S. Patent Application Publication No. 20130267686 A1, U.S. Patent Application Publication No. 20140377269 A1, U.S. Patent No. 7,741,446 B2, and WO1995009917 A1.Exemplary multispecific molecules utilizing the domain swapping format include those described in the following: US Patent Application Publication No. 20150315296A1, WO2016087650A1, US Patent Application Publication No. 20160075785A1, WO2016016299A1, US Patent Application Publication No. 20160130347A1, US Patent Application Publication No. 20150166670, U.S. Patent No. 8703132B2, US Patent Application Publication No. 20100316645, U.S. Patent No. 8227577B2, US Patent Application Publication No. 20130078249.

[0433] Fc-containing multispecific molecules

[0452] In some embodiments, the multispecific molecules described herein comprise an immunoglobulin constant region (e.g., an Fc region). Exemplary Fc regions can be selected from the heavy chain constant regions of IgG1, IgG2, IgG3, or IgG4; more particularly, the heavy chain constant regions of human IgG1, IgG2, IgG3, or IgG4.

[0434]

[0453] In some embodiments, the immunoglobulin chain constant region (e.g., Fc region) is altered, e.g., mutated, to increase or decrease one or more of Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function.

[0435]

[0454] In other embodiments, the interface of the first and second immunoglobulin chain constant regions (e.g., first and second Fc regions) is altered, e.g., mutated, to increase or decrease dimerization, e.g., compared to an unengineered interface, e.g., a naturally occurring interface. For example, dimerization of the immunoglobulin chain constant regions (e.g., Fc regions) can be enhanced by providing one or more of paired holes and protrusions ("knobs-in-holes"), electrostatic interactions, or strand exchange at the Fc interface of the first and second Fc regions, thereby forming, e.g., a higher ratio of heteromultimers:homomultimers compared to the unengineered interface.

[0436]

[0455] In some embodiments, the multispecific molecule comprises paired amino acid substitutions, e.g., at positions selected from one or more of 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409, of the Fc region of human IgG1. For example, the immunoglobulin chain constant region (e.g., Fc region) can comprise paired amino acid substitutions selected from T366S, L368A, or Y407V (e.g., corresponding to a pore or hole), and T366W (e.g., corresponding to a protuberance or knob).

[0437]

[0456] In other embodiments, the multifunctional molecule comprises a half-life extender, for example, human serum albumin or an antibody molecule against human serum albumin.

[0438]

[0457] In some embodiments, the Fc contains the exemplary Fc modifications listed in Table 6.

[0439] Heterodimerized antibody molecules and methods of production

[0458] Various methods of producing multispecific antibodies have been disclosed to address the problem of incorrect heavy chain pairing. Exemplary methods are described below. Exemplary multispecific antibody formats and methods of making said multispecific antibodies are also disclosed, for example, in Speiss et al., Molecular Immunology 67 (2015) 95-106, and Klein et al., mAb 4:6, 653-663, November / December 2012 (the contents of each of which are hereby incorporated by reference herein).

[0440]

[0459] Heterodimerized bispecific antibodies are based on the natural IgG structure, where the two binding arms recognize different antigens. IgG-derived formats that allow defined monovalent (and simultaneous) antigen binding are generated by forced heavy chain heterodimerization combined with techniques that minimize light chain mispairing (e.g., common light chains). Forced heavy chain heterodimerization can be obtained, for example, using knobs-in-holes or strand-exchange engineered domains (SEEDs).

[0441] Knob-in-Hole

[0460] Knobs-in-holes, as described in U.S. Patent No. 5,731,116, U.S. Patent No. 7,476,724 and Ridgway, J. et al. (1996) Prot. Engineering 9(7):617-621, generally involve (1) mutating the CH3 domain of one or both antibodies to promote heterodimerization, and (2) combining the mutated antibodies under conditions that promote heterodimerization. The "knobs" or "protrusions" are typically created by replacing small amino acids in the parent antibody with larger ones (e.g., T366Y or T366W), and the "holes" or "pores" are created by replacing larger residues in the parent antibody with smaller ones (e.g., Y407T, T366S, L368A and / or Y407V).

[0442]

[0461] For bispecific antibodies that include an Fc domain, the introduction of specific mutations into the constant region of the heavy chain to promote correct heterodimerization of the Fc portion can be utilized. Some such techniques are reviewed in Klein et al. (mAb (2012) 4:6, pp. 1-11), the contents of which are hereby incorporated by reference in their entirety. These techniques include the "knob-into-hole" (KiH) approach, which involves the introduction of a bulky residue into one of the CH3 domains of one of the antibody heavy chains. This bulky residue fits into a complementary "hole" in the other CH3 domain of the paired heavy chain, thereby promoting correct pairing of the heavy chains (see, e.g., U.S. Pat. No. 7,642,228).

[0443]

[0462] Exemplary KiH mutations include S354C, T366W in the "knob" heavy chain and Y349C, T366S, L368A, Y407V in the "hole" heavy chain. Other exemplary KiH mutations, along with additional optional stabilizing Fc cysteine ​​mutations, are provided in Table 2.

[0444]

[0463] Other Fc mutations were provided by Igawa and Tsunoda, who identified three negatively charged residues in the CH3 domain of one chain that pair with three positively charged residues in the CH3 domain of the other chain. These specific pairs of charged residues are E356-K439, E357-K370, D399-K409, and vice versa. By introducing at least two of the following mutations in chain B: K370E, K409D, K439E, as well as three in chain A: E356K, E357K and D399K, alone or in combination with the newly identified disulfide bridges, they could favor highly efficient heterodimerization while simultaneously suppressing homodimerization (Martens T et al., "A novel one-armed antic-Met antibody inhibits glioblastoma growth in vivo." Clin Cancer Res 2006;12:6144-52; PMID:17062691). Xencor defined 41 variant pairs based on a combination of structural calculations and sequence information, which were then screened for maximum heterodimerization, defining the combination S364H, F405A (HA) on chain A and Y349T, T394F (TF) on chain B (Moore GL et al., "A novel bispecific antibody format enables simultaneous bivalent and monovalent co-engagement of distinct target antigens." MAbs 2011;3:546-57; PMID:22123055).

[0445]

[0464] Other exemplary Fc mutations to promote heterodimerization of multispecific antibodies include those described in the following references, the contents of each of which are hereby incorporated by reference: WO2016071377A1, U.S. Patent Application Publication No. 20140079689A1, U.S. Patent Application Publication No. 20160194389A1, U.S. Patent Application Publication No. 20160257763, and U.S. Patent Application Publication No. 20160257763. No. 2016071376A2, WO2015107026A1, WO2015107025A1, WO2015107015A1, U.S. Patent Application Publication No. 20150353636A1, U.S. Patent Application Publication No. 20140199294A1, U.S. Patent No. 7750128B2, U.S. Patent Application Publication No. 20160229915A1, U.S. Patent Application Publication No. 2015034 4570A1, U.S. Patent No. 8003774A1, U.S. Patent Application Publication No. 20150337049A1, U.S. Patent Application Publication No. 20150175707A1, U.S. Patent Application Publication No. 20140242075A1, U.S. Patent Application Publication No. 20130195849A1, U.S. Patent Application Publication No. 20120149876A1, U.S. Patent Application Publication No. 20140200331A1 No. 9,309,311 B2, U.S. Pat. No. 8,586,713, U.S. Patent Application Publication No. 20140037621 A1, U.S. Patent Application Publication No. 20130178605 A1, U.S. Patent Application Publication No. 20140363426 A1, U.S. Patent Application Publication No. 20140051835 A1, and U.S. Patent Application Publication No. 20110054151 A1.

[0446]

[0465] Stabilizing cysteine ​​mutations have also been used in combination with KiH and other Fc heterodimerization-promoting variants (see, e.g., U.S. Patent No. 7,183,076). Other exemplary cysteine ​​modifications include those disclosed, for example, in U.S. Patent Application Publication No. 20140348839A1, U.S. Patent No. 7,855,275B2, and U.S. Patent No. 9,000,130B2.

[0447] Strand exchange engineering domain (SEED)

[0466] A heterodimeric Fc platform is known that supports the design of bispecific and asymmetric fusion proteins by engineering strand-exchange engineered domain (SEED) C(H)3 heterodimers. These derivatives of human IgG and IgA C(H)3 domains create complementary human SEED C(H)3 heterodimers composed of alternating segments of human IgA and IgG C(H)3 sequences. The resulting pair of SEED C(H)3 domains preferentially associates to form heterodimers when expressed in mammalian cells. A SEED body (Sb) fusion protein consists of [IgG1 hinge]-C(H)2-[SEED C(H)3] optionally genetically linked to one or more fusion partners (see, e.g., Davis JH et al., "SEEDbodies: fusion proteins based on strand exchange engineered domain (SEED)CH3 heterodimers in an Fc analogue platform for asymmetric binders or immunofusions and bispecific antibodies." Protein Eng Des Sel 2010;23:195-202; PMID:20299542 and U.S. Pat. No. 8,871,912, the contents of each of which are hereby incorporated by reference).

[0448] Fc-containing entities (mini-antibodies)

[0467] Fc-containing entities, also known as mini-antibodies, can be generated by fusing s...

Claims

1. A multifunctional polypeptide molecule comprising a first polypeptide, a second polypeptide, and at least one cytokine polypeptide or variant thereof, wherein the first polypeptide and the second polypeptide are non-contiguous; (i) a first polypeptide comprising: (A) a first TCRαV-binding portion comprising a first heavy chain variable domain (VH) and a first light chain variable domain (VL), or a single domain antibody; or (B) a first portion of the first TCRαV binding portion comprising a VH of the first TCRαV binding portion, and when the first polypeptide comprises the first portion of the first TCRαV binding portion, the multifunctional polypeptide molecule further comprises a third polypeptide comprising a second portion of the first TCRαV binding portion comprising a VL of the first TCRαV binding portion, wherein the third polypeptide is non-contiguous with the first and second polypeptides. a first portion of a dimerization module linked to (ii) the second polypeptide comprises a second portion of the dimerization module; (a) the multifunctional polypeptide molecule comprises a single TCRαV-binding portion, and at least one cytokine polypeptide or variant thereof is covalently linked to a second polypeptide; or (b) A multifunctional polypeptide molecule, wherein the multifunctional polypeptide molecule further comprises a second TCRαV binding portion, and wherein at least one cytokine polypeptide or variant thereof is covalently linked to the first polypeptide, the second polypeptide, or, if the multifunctional polypeptide molecule further comprises a third polypeptide, the third polypeptide, or a combination thereof.

2. a second TCRαV-binding portion, and a second portion of the dimerization module comprising: (A) a second TCRαV-binding portion comprising a second VH and a second VL, or a single domain antibody; or (B) a first portion of a second TCRαV binding portion comprising a VH of the second TCRαV binding portion, and if the second polypeptide comprises the first portion of the second TCRαV binding portion, the multifunctional polypeptide molecule further comprises a fourth polypeptide comprising a second portion of the second TCRαV binding portion comprising a VL of the second TCRαV binding portion, wherein the fourth polypeptide is non-contiguous with the first polypeptide, the second polypeptide, and the third polypeptide. is connected to; The multifunctional polypeptide molecule of claim 1, wherein at least one cytokine polypeptide or variant thereof is covalently linked to the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide if the multifunctional polypeptide molecule further comprises a fourth polypeptide, or a combination thereof.

3. 3. The multifunctional polypeptide molecule of claim 1, wherein the first portion of the dimerization module and the second portion of the dimerization module are dimerized.

4. The first polypeptide is (A) a first TCRαV binding moiety comprising a first VH and a first VL, the first TCRαV binding moiety further comprising a first heavy chain constant domain 1 (CH1) linked to the first VH; or (B) a first portion of the first TCRαV binding portion comprising a VH of the first TCRαV binding portion, the first portion of the first TCRαV binding portion further comprising a first CH1 linked to the VH of the first TCRαV binding portion. The multifunctional polypeptide molecule of claim 1 , comprising:

5. The multifunctional polypeptide molecule of claim 4, wherein the first CH1 is linked to the C-terminus of the first VH or the C-terminus of the VH of the first TCRαV-binding portion.

6. The second polypeptide is (A) a second TCRαV binding portion comprising a second VH and a second VL, the second TCRαV binding portion further comprising a second CH1 linked to the second VH; or (B) a first portion of a second TCRαV binding portion comprising a VH of the second TCRαV binding portion, the first portion of the second TCRαV binding portion further comprising a second CH1 linked to the VH of the second TCRαV binding portion.

6. The multifunctional polypeptide molecule of claim 2, comprising:

7. The multifunctional polypeptide molecule of claim 6 , wherein the second CH1 is linked to the C-terminus of the second VH or the C-terminus of the VH of the second TCRαV-binding portion.

8. (1) a first polypeptide comprising a first TCRαV binding portion comprising a first VH and a first VL, the first TCRαV binding portion further comprising a first light chain constant domain (CL) linked to the first VL; or (2) a first polypeptide comprising a first portion of a first TCRαV binding moiety and a third polypeptide comprising a second portion of the first TCRαV binding moiety, the third polypeptide further comprising a first CL linked to the VL of the first TCRαV binding moiety. The multifunctional polypeptide molecule of claim 1 , comprising:

9. The multifunctional polypeptide molecule of claim 8 , wherein the first CL is linked to the C-terminus of the first VL or the C-terminus of the VL of the first TCRαV-binding portion.

10. (1) a second polypeptide comprising a second TCRαV binding portion comprising a second VH and a second VL, the second TCRαV binding portion further comprising a second CL linked to the second VL; or (2) a second polypeptide comprising a first portion of a second TCRαV binding portion and a fourth polypeptide comprising a second portion of the second TCRαV binding portion, the second portion further comprising a second CL linked to the VL of the second TCRαV binding portion.

10. The multifunctional polypeptide molecule of claim 2, comprising:

11. The multifunctional polypeptide molecule of claim 10, wherein the second CL is linked to the C-terminus of the second VL or the C-terminus of the VL of the second TCRαV-binding portion.

12. A multifunctional polypeptide molecule described in any one of claims 1 to 11, wherein the first portion of the dimerization module is linked to (A) the C-terminus of a first TCRαV binding portion comprising a first VH and a first VL or a single domain antibody, or (B) the C-terminus of a first portion of the first TCRαV binding portion comprising the VH of the first TCRαV binding portion.

13. A multifunctional polypeptide molecule described in any one of claims 2 to 12, wherein the multifunctional polypeptide molecule comprises a second TCRαV binding portion and the second portion of the dimerization module is linked to (A) the C-terminus of the second TCRαV binding portion comprising a second VH and a second VL or a single domain antibody, or (B) the C-terminus of the first portion of the second TCRαV binding portion comprising the VH of the second TCRαV binding portion.

14. A multifunctional polypeptide molecule according to any one of claims 1 to 13, wherein the multifunctional polypeptide molecule comprises a single TCRαV binding portion and at least one cytokine polypeptide or variant thereof is covalently linked to the N-terminus of a second polypeptide, the C-terminus of a second polypeptide, or a combination thereof.

15. The multifunctional polypeptide molecule of claim 14, wherein at least one cytokine polypeptide or variant thereof is within a single contiguous polypeptide chain of a second polypeptide.

16. (a) the N-terminus of the first polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the first polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (b) the N-terminus of the second polypeptide is linked to the cytokine polypeptide or a variant thereof; the C-terminus of the second polypeptide is linked to the cytokine polypeptide or a variant thereof; or a combination thereof; (c) the N-terminus of the third polypeptide is linked to the cytokine polypeptide or a variant thereof; the C-terminus of the third polypeptide is linked to the cytokine polypeptide or a variant thereof; or a combination thereof; (d) the N-terminus of the fourth polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the fourth polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; or (e) A multifunctional polypeptide molecule according to any one of claims 1 to 13, which is a combination thereof.

17. (a-1) the N-terminus of the first polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the first polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (a-2) the N-terminus of the second polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the second polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (b-1) the N-terminus of the first polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the first polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (b-2) the N-terminus of the third polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the third polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (c-1) the N-terminus of the first polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the first polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (c-2) the N-terminus of the fourth polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the fourth polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (d-1) the N-terminus of the second polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the second polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (d-2) the N-terminus of the third polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the third polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (e-1) the N-terminus of the second polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the second polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (e-2) the N-terminus of the fourth polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the fourth polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; or The multifunctional polypeptide molecule of claim 16, wherein: (f-1) the N-terminus of the third polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the third polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (f-2) the N-terminus of the fourth polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the fourth polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof.

18. (a-1) the N-terminus of the first polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the first polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (a-2) the N-terminus of the second polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the second polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (a-3) the N-terminus of the third polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the third polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (b-1) the N-terminus of the first polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the first polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (b-2) the N-terminus of the second polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the second polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (b-3) the N-terminus of the fourth polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the fourth polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; or (c-1) the N-terminus of the second polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the second polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (c-2) the N-terminus of the third polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the third polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; (c-3) the N-terminus of the fourth polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the fourth polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof. The multifunctional polypeptide molecule of claim 16.

19. 17. The multifunctional polypeptide molecule of claim 16, wherein the N-terminus of the first polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the first polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; the N-terminus of the second polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the second polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; the N-terminus of the third polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the third polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof; the N-terminus of the fourth polypeptide is linked to a cytokine polypeptide or a variant thereof; the C-terminus of the fourth polypeptide is linked to a cytokine polypeptide or a variant thereof; or a combination thereof.

20. 20. The multifunctional polypeptide molecule of any one of claims 16 to 19, wherein the cytokine polypeptide or variant thereof is present within a single continuous polypeptide chain of the first polypeptide, the second polypeptide, the third cytokine polypeptide, or the fourth cytokine polypeptide to which it is linked.

21. (i) a linker between the first portion of the dimerization module and the first TCRαV binding moiety comprising a first VH and a first VL or a single domain antibody, or the first portion of the first TCRαV binding moiety comprising the VH of the first TCRαV binding moiety; (ii) a linker between the second portion of the dimerization module and the second TCRαV binding moiety comprising a second VH and a second VL or a single domain antibody, or the first portion of the second TCRαV binding moiety comprising the VH of the second TCRαV binding moiety; (iii) a linker between the first VH and the first VL; (iv) a linker between the second VH and the second VL; (v) a linker between the first CH1 and the first VH, or the VH of the first TCRαV-binding portion; (vi) a linker between the second CH1 and the second VH, or the VH of the second TCRαV-binding portion; (vii) a linker between the first CL and the first VL, or the VL of the first TCRαV-binding portion; (vii) a linker between the second CL and the second VL, or the VL of the second TCRαV-binding portion; (viii) a linker between at least one cytokine polypeptide or variant thereof and a first polypeptide, a linker between at least one cytokine polypeptide or variant thereof and a second polypeptide, a linker between at least one cytokine polypeptide or variant thereof and a third polypeptide, a linker between at least one cytokine polypeptide or variant thereof and a fourth polypeptide, or a combination thereof; or (ix) A multifunctional polypeptide molecule according to any one of claims 1 to 20, further comprising a combination thereof.

22. 22. The multifunctional polypeptide molecule of claim 21, wherein the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker.

23. The multifunctional polypeptide molecule of claim 22, wherein the linker is a peptide linker and comprises the sequence of SEQ ID NO: 3308 or SEQ ID NO: 3643.

24. 24. A multifunctional polypeptide molecule according to any one of claims 1 to 23, which is an isolated multifunctional polypeptide molecule.

25. (i) a first polypeptide comprising a first portion of a dimerization module linked to the C-terminus of a first portion of a first TCRαV-binding portion; (ii) a second polypeptide comprising a second portion of the dimerization module; (iii) a third polypeptide comprising a second portion of the first TCRαV-binding portion; and (iv) comprising a cytokine polypeptide or variant thereof covalently linked to the N-terminus of a second polypeptide; The multifunctional polypeptide molecule of claim 1 comprising a single TCRαV binding portion.

26. (i) a first polypeptide comprising a first portion of a dimerization module linked to the C-terminus of a first portion of a first TCRαV-binding portion; (ii) a second polypeptide comprising a second portion of a dimerization module linked to the C-terminus of the first portion of the second TCRαV-binding portion; (iii) a third polypeptide comprising a second portion of the first TCRαV-binding portion; (iv) a fourth polypeptide comprising a second portion of the second TCRαV-binding portion; (v) a cytokine polypeptide or variant thereof covalently linked to the C-terminus of a third polypeptide; and (vi) a cytokine polypeptide or a variant thereof covalently linked to the C-terminus of a fourth polypeptide. The multifunctional polypeptide molecule of claim 2 , comprising:

27. (i) a first polypeptide comprising a first portion of a dimerization module linked to the C-terminus of a first portion of a first TCRαV-binding portion; (ii) a second polypeptide comprising a second portion of a dimerization module linked to the C-terminus of the first portion of the second TCRαV-binding portion; (iii) a third polypeptide comprising a second portion of the first TCRαV-binding portion; (iv) a fourth polypeptide comprising a second portion of the second TCRαV-binding portion; and (v) a cytokine polypeptide or variant thereof that is covalently linked to the C-terminus of a third polypeptide or to the C-terminus of a fourth polypeptide, but not to both. The multifunctional polypeptide molecule of claim 2 , comprising:

28. (i) a first polypeptide comprising a first portion of a dimerization module linked to the C-terminus of a first portion of a first TCRαV-binding portion; (ii) a second polypeptide comprising a second portion of a dimerization module linked to the C-terminus of the first portion of the second TCRαV-binding portion; (iii) a third polypeptide comprising a second portion of the first TCRαV-binding portion; (iv) a fourth polypeptide comprising a second portion of the second TCRαV-binding portion; and (v) a cytokine polypeptide or variant thereof that is covalently linked to the C-terminus of a first polypeptide or to the C-terminus of a second polypeptide, but not to both. The multifunctional polypeptide molecule of claim 2 , comprising:

29. 29. The multifunctional polypeptide molecule of any one of claims 1 to 28, wherein the first TCRαV binding portion, the second TCRαV binding portion, or a combination thereof, comprises any one selected from the group consisting of Fab, F(ab')2, Fv, single-chain Fv (scFv), single domain antibody, diabody (dAb), camelid antibody, and combinations thereof.

30. 30. The multifunctional polypeptide molecule of claim 29, wherein the first TCRαV binding portion, the second TCRαV binding portion, or a combination thereof, comprises a Fab or a scFv.

31. 31. The multifunctional polypeptide molecule of claim 1, wherein the TCRαV binding portion is the only antigen-binding portion of the multifunctional polypeptide molecule.

32. 32. A multifunctional polypeptide molecule according to any one of claims 1 to 31, comprising two or more of at least one cytokine polypeptide.

33. 33. The multifunctional polypeptide molecule of any one of claims 1 to 32, wherein at least one cytokine polypeptide comprises interleukin-2 (IL-2) or a fragment thereof.

34. The multifunctional polypeptide molecule of claim 33, wherein at least one cytokine polypeptide comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 2191.

35. 34. The multifunctional polypeptide molecule of claim 33, wherein the variant is an IL-2 variant comprising a substitution mutation.

36. 36. The multifunctional polypeptide molecule of claim 35, wherein the variant is an IL-2 variant comprising a C125A mutation.

37. 34. The multifunctional polypeptide molecule of claim 33, wherein the variant comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 2270.

38. 38. The multifunctional polypeptide molecule of any one of claims 1 to 37, wherein a first portion of the dimerization module comprises a first immunoglobulin constant region (Fc region) and a second portion of the dimerization module comprises a second Fc region.

39. 39. The multifunctional polypeptide molecule of claim 38, wherein the first Fc region, the second Fc region, or a combination thereof is selected from the group consisting of an IgG1 Fc region or fragment thereof, an IgG2 Fc region or fragment thereof, an IgG3 Fc region or fragment thereof, an IgGAl Fc region or fragment thereof, an IgG2 Fc region or fragment thereof, an IgG4 Fc region or fragment thereof, an IgJ Fc region or fragment thereof, an IgM Fc region or fragment thereof, an IgD Fc region or fragment thereof, and an IgE Fc region or fragment thereof.

40. 40. The multifunctional polypeptide molecule of claim 39, wherein the first Fc region, the second Fc region, or a combination thereof is selected from the group consisting of a human IgG1 Fc region or fragment thereof, a human IgG2 Fc region or fragment thereof, and a human IgG4 Fc region or fragment thereof.

41. 41. The multifunctional polypeptide molecule of any one of claims 38 to 40, wherein the first Fc region, the second Fc region, or a combination thereof comprises an Fc interface having one or more of paired holes and protrusions, electrostatic interactions, or strand exchange, and dimerization of the first Fc region and the second Fc region is enhanced as indicated by a higher ratio of heteromultimer:homomultimer forms compared to dimerization of an Fc region having a non-engineered interface.

42. 42. The multifunctional polypeptide molecule of claim 41, wherein the first Fc region, the second Fc region, or a combination thereof comprises an amino acid substitution listed in Table 6.

43. 43. The multifunctional polypeptide molecule of claim 42, wherein the first Fc region, the second Fc region, or a combination thereof comprises an Asn297Ala (N297A) mutation or a Leu234Ala / Leu235Ala (LALA) mutation.

44. The multifunctional polypeptide molecule of claim 41, wherein the first Fc region, the second Fc region, or a combination thereof comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:3645, SEQ ID NO:3646, SEQ ID NO:3647, SEQ ID NO:3648, or SEQ ID NO:3649.

45. The first TCRαV binding portion, the second TCRαV binding portion, or a combination thereof, is selected from the group consisting of the TCRα V1 subfamily, the TCRα V2 subfamily, the TCRα V3 subfamily, the TCRα V4, the TCRα V5 subfamily, the TCRα V6 subfamily, the TCRα V7 subfamily, the TCRα V8 subfamily, the TCRα V9 subfamily, the TCRα V10 subfamily, the TCRα V12 subfamily, the TCRα V13 subfamily, the TCRα V14 subfamily, the TCRα V16 subfamily, the TCRα V17 subfamily, the TCRα V18 subfamily, the TCRα V19 subfamily, the TCRα V20 subfamily, the TCRα V21 subfamily, the TCRα V22 subfamily, the TCRα 45. The multifunctional polypeptide molecule of any one of claims 1 to 44, which binds to one or more of the TCRαV subfamilies selected from the group consisting of the V23 subfamily, the TCRα V24 subfamily, the TCRα V25 subfamily, the TCRα V26 subfamily, the TCRα V27 subfamily, the TCRα V29 subfamily, the TCRα V30 subfamily, the TCRα V34 subfamily, the TCRα V35 subfamily, the TCRα V36 subfamily, the TCRα V38 subfamily, the TCRα V39 subfamily, the TCRα V40 subfamily, or the TCRα V41 subfamily, and family members of said subfamilies, and variants thereof.

46. A multifunctional polypeptide molecule described in any one of claims 1 to 45, wherein the multifunctional polypeptide molecule comprises a first TCRαV binding portion and a second TCRαV binding portion, and the first TCRαV binding portion and the second TCRαV binding portion are the same.

47. A multifunctional polypeptide molecule described in any one of claims 1 to 45, wherein the multifunctional polypeptide molecule comprises a first TCRαV binding portion and a second TCRαV binding portion, and the first TCRαV binding portion and the second TCRαV binding portion are different.

48. The first TCRαV binding portion, the second TCRαV binding portion, or a combination thereof, (i) a VH comprising a framework region (FR) comprising FR1, FR2, FR3, and FR4 that has at least 75% sequence identity to a non-mouse germline framework 1 (FR1), a non-mouse germline framework region 2 (FR2), a non-mouse germline framework region 3 (FR3), and a non-mouse germline framework region 4 (FR4); (ii) a VL comprising a FR comprising a FR1, a FR2, a FR3, and a FR4 that have at least 75% sequence identity to a non-mouse germline FR1, a non-mouse germline FR2, a non-mouse germline FR3, and a non-mouse germline FR4; or (iii) Combinations thereof 48. The multifunctional polypeptide molecule of claim 47, comprising:

49. 49. The multifunctional polypeptide molecule of any one of claims 1 to 48, wherein the first polypeptide, the second polypeptide, or a combination thereof comprises a heavy chain constant region having a sequence having at least 75% sequence identity to any one of the sequences listed in Table 1 or a combination thereof.

50. 50. The multifunctional polypeptide molecule of claim 49, wherein the first polypeptide, the second polypeptide, or a combination thereof comprises an IgM heavy chain constant region or a fragment thereof.

51. 51. The multifunctional polypeptide molecule of claim 50, wherein the IgM heavy chain constant region comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO:

73.

52. 50. The multifunctional polypeptide molecule of claim 49, wherein the first polypeptide, the second polypeptide, or a combination thereof comprises an IgJ heavy chain constant region or a fragment thereof.

53. 53. The multifunctional polypeptide molecule of claim 52, wherein the heavy chain constant region of IgJ comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO:

76.

54. 50. The multifunctional polypeptide molecule of claim 49, wherein the first polypeptide, the second polypeptide, or a combination thereof comprises an IgGA1 heavy chain constant region or a fragment thereof.

55. 55. The multifunctional polypeptide molecule of claim 54, wherein the heavy chain constant region of IgGA1 comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO:

74.

56. 50. The multifunctional polypeptide molecule of claim 49, wherein the first polypeptide, the second polypeptide, or a combination thereof comprises an IgGA2 heavy chain constant region or a fragment thereof.

57. 57. The multifunctional polypeptide molecule of claim 56, wherein the heavy chain constant region of IgGA2 comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO:

75.

58. 50. The multifunctional polypeptide molecule of claim 49, wherein the first polypeptide, the second polypeptide, or a combination thereof comprises an IgG1 heavy chain constant region or a fragment thereof.

59. 59. The multifunctional polypeptide molecule of claim 58, wherein the heavy chain constant region of IgG1 comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 41 or SEQ ID NO: 3645.

60. 60. The multifunctional polypeptide molecule of any one of claims 1 to 59, wherein the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof, comprises a light chain constant region having a sequence having at least 75% sequence identity to any one of the sequences listed in Table 1, or a combination thereof.

61. 61. The multifunctional polypeptide molecule of claim 60, wherein the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a light chain constant region of a kappa chain or a fragment thereof.

62. 62. The multifunctional polypeptide molecule of claim 61, wherein the light chain constant region of the kappa chain comprises a light chain constant region sequence listed in Table 1.

63. 63. The multifunctional polypeptide molecule of claim 62, wherein the light chain constant region of the kappa chain comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 39 or SEQ ID NO: 3644.

64. 64. The multifunctional polypeptide molecule of any one of claims 1 to 63, wherein the first TCRαV binding portion, the second TCRαV binding portion, or a combination thereof binds to an outward-facing region on the TCRαV protein.

65. 65. The multifunctional polypeptide molecule of claim 64, wherein the outward-facing region on the TCRαV protein comprises a structurally conserved region of TCRαV that has a similar structure across one or more TCRαV subfamilies.

66. 60. The multifunctional polypeptide molecule of any one of claims 1 to 59, wherein the third sequence is linked to the N-terminus of the first sequence.

67. 60. The multifunctional polypeptide molecule of any one of claims 1 to 59, wherein the third sequence is linked to the C-terminus of the second sequence.

68. 60. The multifunctional polypeptide molecule of any one of claims 1 to 59, wherein the third polypeptide, the fourth polypeptide, or a combination thereof further comprises a third sequence, and the third sequence is linked to the fourth sequence, the fifth sequence, or a combination thereof.

69. 60. The multifunctional polypeptide molecule of any one of claims 1 to 59, wherein the third sequence is linked to the N-terminus of the fourth sequence.

70. 60. The multifunctional polypeptide molecule of any one of claims 1 to 59, wherein the third sequence is linked to the C-terminus of the fifth sequence.

71. 60. The multifunctional polypeptide molecule of any one of claims 1 to 59, wherein the third sequence is linked to the N-terminus of the first sequence.

72. 60. The multifunctional polypeptide molecule of any one of claims 1 to 59, wherein the third sequence is linked to the C-terminus of the second sequence.

73. 60. The multifunctional polypeptide molecule of any one of claims 1 to 59, wherein the third sequence is linked to the N-terminus of the fourth sequence.

74. 60. The multifunctional polypeptide molecule of any one of claims 1 to 59, wherein the third sequence is linked to the C-terminus of the fifth sequence.

75. 75. A nucleic acid molecule comprising a nucleotide sequence encoding a multifunctional polypeptide molecule according to any one of claims 1 to 74.

76. 76. The nucleic acid molecule of claim 75, which is an isolated nucleic acid molecule.

77. 77. A vector comprising one or more of the nucleic acid molecules of any one of claims 75 to 76.

78. 78. A cell comprising a nucleic acid molecule according to any one of claims 75 to 76, or a vector according to claim 77.

79. A pharmaceutical composition comprising a multifunctional polypeptide molecule described in any one of claims 1 to 74, a nucleic acid molecule described in any one of claims 75 to 76, a vector described in claim 77, or a cell described in claim 78, and a pharma- ceutically acceptable carrier, excipient, or diluent.

80. 10. A method of treating a condition or disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a multifunctional polypeptide molecule according to any one of claims 1 to 74, a nucleic acid molecule according to any one of claims 75 to 76, a vector according to claim 77, a cell according to claim 78, a pharmaceutical composition according to claim 79, or a combination thereof, The method, wherein the administering step is effective to treat a condition or disease in the subject.

81. 81. The method of claim 80, wherein the condition or disease is cancer.

82. 82. The method of claim 81, wherein the cancer is a solid tumor, a hematological cancer, a metastatic cancer, a soft tissue tumor, or a combination thereof.

83. 83. The method of claim 82, wherein the cancer is a solid tumor, and the solid tumor is selected from the group consisting of melanoma, pancreatic cancer, breast cancer, colorectal cancer, lung cancer, skin cancer, ovarian cancer, liver cancer, and combinations thereof.

84. 83. The method of claim 82, wherein the cancer is a hematological cancer, and the hematological cancer is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia (AML), chronic myeloid leukemia, myelodysplastic syndrome, multiple myeloma, T-cell lymphoma, acute lymphocytic leukemia, and combinations thereof.

85. 85. The method of claim 84, wherein the non-Hodgkin's lymphoma is selected from the group consisting of B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (B-CLL), mantle cell lymphoma, marginal zone B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, and combinations thereof.

86. 85. The method of claim 84, wherein the T cell lymphoma is a peripheral T cell lymphoma.

87. 87. The method of any one of claims 81 to 86, wherein the cancer is characterized by a cancer antigen present on the cancer.

88. 88. The method of claim 87, wherein the cancer antigen is a tumor antigen, a stromal antigen, or a blood antigen.

89. Cancer antigens include BCMA, CD19, CD20, CD22, FcRH5, PDL1, CD47, gangloside 2 (GD2), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PMSA), prostate specific antigen (PSA), carcinoembryonic antigen (CEA), Ron kinase, c-Met, immature laminin receptor, TAG-72, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, and Ep-CA. M, EphA3, Her2 / neu, telomerase, SAP-1, survivin, NY-ESO-1 / LAGE-1, PRAME, SSX-2, Melan-A / MART-1, Gp100 / pmel17, tyrosinase, TRP-1 / -2, MC1R, β-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, p53, Ras, TGF-β receptor, AFP, ETA, MAGE, MUC-1, CA-1 25, BAGE, GAGE, NY-ESO-1, β-catenin, CDK4, CDC27, α-actinin-4, TRP1 / gp75, TRP2, gp100, melan-A / MART1, ganglioside, WT1, EphA3, epidermal growth factor receptor (EGFR), MART-2, MART-1, MUC1, MUC2, MUM1, MUM2, MUM3, NA88-1, NPM, OA1, OGT, RCC, RUI1, RUI 2, SAGE, TRG, TRP1, TSTA, folate receptor alpha, L1-CAM, CAIX, gpA33, GD3, GM2, VEGFR, integrins, carbohydrates, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, TGF-beta, hyaluronic acid, collagen, tenascin-C, and tenascin-W.

90. 90. The method of any one of claims 80-89, further comprising the step of administering a second therapeutic agent or treatment to the subject.

91. 91. The method of claim 90, wherein the second therapeutic agent or treatment comprises a chemotherapeutic agent, a biological agent, a hormonal therapy, radiation, or surgery.

92. The method of any one of claims 90 to 91, wherein a second therapeutic agent or treatment is administered in combination, sequentially, simultaneously or in parallel with a multifunctional polypeptide molecule described in any one of claims 1 to 74, a nucleic acid molecule described in any one of claims 75 to 76, a vector described in claim 77, a cell described in claim 78, or a pharmaceutical composition described in claim 79.

93. 1. A composition comprising a multispecific molecule comprising a T cell receptor alpha variable region (TCRαV) binding moiety linked to a targeting moiety, the TCRαV binding portion binds to TCRαV of a T cell receptor (TCR) expressed by a T cell of the T cell population; the targeting moiety binds to a target molecule other than TCRαV on a target cell; A composition, wherein the multispecific molecule, when contacted with a population of T cells, redirects the T cells to target cells, activates the T cells, expands the T cells, or a combination thereof.

94. 94. The composition of claim 93, wherein the targeting moiety comprises a tumor targeting moiety, a cytokine molecule, or a stromal modifying moiety.

95. A multispecific molecule comprises at least two non-contiguous polypeptide chains, 94. The composition of claim 93, wherein a first polypeptide chain of the at least two non-contiguous polypeptide chains comprises a first member of a dimerization module, and a second polypeptide chain of the at least two non-contiguous polypeptide chains comprises a second member of a dimerization module, and the first polypeptide chain and the second polypeptide chain form a complex via the first member of the dimerization module and the second member of the dimerization module.

96. 96. The composition of claim 95, wherein the first member of the dimerization module is a first Fc region and the second member of the dimerization module is a second Fc region.

97. 97. The composition of claim 96, wherein the first Fc region is an engineered Fc region that comprises a knob and the second Fc region is an engineered Fc region that comprises a hole.

98. (a) a first polypeptide chain comprises a TCRαV-binding portion and a second polypeptide chain comprises a targeting portion; (i) a first polypeptide chain comprises a TCRαV-binding portion linked to a first member of a dimerization module, and a second polypeptide chain comprises a targeting portion linked to a second member of the dimerization module; (ii) the first polypeptide chain comprises a first portion of the TCRαV-binding moiety linked to a first member of the dimerization module and the second polypeptide chain comprises a first portion of the targeting moiety linked to a second member of the dimerization module; the at least two non-contiguous polypeptide chains comprise a third polypeptide chain comprising a second portion of the TCRαV-binding moiety and a fourth polypeptide chain comprising a second portion of the targeting moiety; (iii) the first polypeptide chain comprises a first portion of the TCRαV binding moiety linked to a first member of the dimerization module and the second polypeptide chain comprises a targeting moiety linked to a second member of the dimerization module; the at least two non-contiguous polypeptide chains comprise a third polypeptide chain comprising a second portion of the TCRαV binding moiety; or (iv) the first polypeptide chain comprises a TCRαV-binding portion linked to a first member of the dimerization module and the second polypeptide chain comprises a first portion of the targeting moiety linked to a second member of the dimerization module; the at least two non-contiguous polypeptide chains comprise a third polypeptide chain comprising a second portion of the targeting moiety; or (b) a first polypeptide chain comprises a TCRαV-binding portion and a targeting portion, the first polypeptide chain comprising: (i) a TCRαV-binding moiety linked to a first member of a dimerization module linked to a targeting moiety; (ii) a first portion of a TCRαV binding moiety linked to a first member of a dimerization module linked to a first portion of the targeting moiety, wherein the at least two non-contiguous polypeptide chains comprise a third polypeptide chain comprising a second portion of the TCRβV binding moiety and a fourth polypeptide chain comprising a second portion of the targeting moiety; (iii) a first portion of a TCRαV binding moiety linked to a first member of a dimerization module linked to the targeting moiety, wherein the at least two non-contiguous polypeptide chains comprise a third polypeptide chain that comprises a second portion of the TCRαV binding moiety; or (iv) a TCRαV binding moiety linked to a first member of a dimerization module linked to a first portion of the targeting moiety, wherein the at least two non-contiguous polypeptide chains include a third polypeptide chain that comprises a second portion of the targeting moiety.

96. The composition of claim 95, comprising:

99. the multispecific molecule further comprises a linker between the TCRαV binding moiety and the first member of the dimerization module, a linker between the targeting moiety and the second member of the dimerization module, a linker between the first portion of the TCRαV binding moiety and the first member of the dimerization module, a linker between the first portion of the targeting moiety and the second member of the dimerization module, a linker between the first member of the dimerization module and the targeting moiety, a linker between the first member of the dimerization module and the first portion of the targeting moiety, or a combination thereof; 99. The composition of claim 98, wherein the linker is selected from a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker.

100. The multispecific molecule is (i) a TCRαV-binding moiety linked to a targeting moiety; (ii) a first portion of a TCRαV binding moiety linked to a first portion of a targeting moiety, wherein the polypeptide sequence further comprises a second portion of the TCRαV binding moiety and a second portion of the targeting moiety; (iii) a first portion of a TCRαV binding moiety linked to a targeting moiety, wherein the polypeptide sequence further comprises a second portion of a TCRαV binding moiety; or (iv) a TCRαV binding moiety linked to a first portion of a targeting moiety, wherein the polypeptide sequence further comprises a second portion of the targeting moiety.

94. The composition of claim 93, comprising a polypeptide sequence comprising:

101. the polypeptide sequence further comprises a linker between the TCRαV binding portion and the targeting portion, a linker between the first portion of the TCRβV binding portion and the first portion of the targeting portion, a linker between the first portion of the TCRαV binding portion and the targeting portion, a linker between the TCRαV binding portion and the first portion of the targeting portion, or a combination thereof; The composition of claim 100, wherein the linker is selected from a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker.

102. The targeting moiety may be selected from the group consisting of BCMA, FcRH5, CD19, CD20, CD22, CD30, CD33, CD38, CD47, CD99, CD123, FcRH5, CLEC12, CD179A, SLAMF7, or NY-ESO1, PDL1, CD47, gangloside 2 (GD2), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), prostate specific antigen (PSA), carcinoembryonic antigen (CEA), Ron kinase, c-Met, immature laminin receptor. receptor, TAG-72, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, Ep-CAM, EphA3, Her2 / neu, telomerase, SAP-1, survivin, NY-ESO-1 / LAGE-1, PRAME, SSX-2, Melan-A / MART-1, Gp100 / pmel17, tyrosinase, TRP-1 / -2, MC1R, b-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, p53, Ras, TGF-B receptor, AFP, ETA, MAGE, MUC-1, CA-125, BAGE, GAGE, NY-ESO-1, b-catenin, CDK4, CDC27, a-actinin-4, TRP1 / gp75, TRP2, gp100, melan-A / MART1, ganglioside, WT1, EphA3, epidermal growth factor receptor (EGFR), MART-2, MART-1, MUC1, MUC2, MUM1, MUM2, MUM3, NA88-1, NPM, OA1, OG 94. The composition of claim 93, comprising a targeting moiety that binds to an antigen selected from the group consisting of T, RCC, RUI1, RUI2, SAGE, TRG, TRP1, TSTA, folate receptor alpha, L1-CAM, CAIX, gpA33, GD3, GM2, VEGFR, integrins, carbohydrates, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, TGF-beta, hyaluronic acid, collagen, tenascin-C, and tenascin-W.

103. 94. The composition of claim 93, wherein the targeting moiety is an NK cell engager, a T cell engager other than a TCRαV binding moiety, a B cell engager, a dendritic cell engager, or a macrophage cell engager.

104. The composition of claim 103, wherein the targeting moiety comprises a targeting moiety that binds to CD19.

105. The composition of claim 103, wherein the targeting moiety comprises a T cell engager that binds to CD3.

106. The composition of claim 93, wherein the targeting moiety comprises a targeting moiety that binds to CD123.

107. 95. The composition of claim 94, wherein the targeting moiety comprises a tumor targeting moiety that binds to a cancer antigen, wherein the cancer antigen is a blood cancer antigen, a solid tumor antigen, a metastatic cancer antigen, a soft tissue tumor antigen, or a cancer antigen of a metastatic lesion.

108. The composition of claim 107, wherein the cancer antigen is a tumor antigen, a stromal antigen, or a blood antigen.

109. Cancer antigens, (i) a solid tumor antigen, wherein the solid tumor is pancreatic cancer, breast cancer, colorectal cancer, lung cancer, skin cancer, ovarian cancer, or liver cancer; or (ii) A blood cancer antigen, wherein the blood cancer is a B cell malignancy or a T cell malignancy. The composition of claim 107,

110. The composition of claim 109, wherein the cancer antigen is a blood cancer antigen and the B cell or T cell malignancy is Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia (AML), chronic myeloid leukemia, myelodysplastic syndrome, multiple myeloma, or acute lymphocytic leukemia.

111. The composition of claim 110, wherein the cancer is a B cell malignancy, the B cell malignancy is a non-Hodgkin's lymphoma, the non-Hodgkin's lymphoma is a B cell lymphoma, a diffuse large B cell lymphoma, a follicular lymphoma, a chronic lymphocytic leukemia, a mantle cell lymphoma, a marginal zone B cell lymphoma, a Burkitt's lymphoma, a lymphoplasmacytic lymphoma, or a hairy cell leukemia.

112. 95. The composition of claim 94, wherein the targeting moiety comprises a cytokine molecule selected from the group consisting of interleukin-2 (IL-2) or a functional fragment or variant thereof, interleukin-7 (IL-7) or a functional fragment or variant thereof, interleukin-12 (IL-12) or a functional fragment or variant thereof, interleukin-15 (IL-15) or a functional fragment or variant thereof, interleukin-18 (IL-18) or a functional fragment or variant thereof, interleukin-21 (IL-21) or a functional fragment or variant thereof, or interferon gamma or a functional fragment or variant thereof.

113. The composition of claim 93, wherein the target cell is a cancer cell.

114. The composition of claim 93, wherein when contacted with a T cell population, the multispecific molecule promotes T cells of the T cell population to kill cancer cells.

115. The composition of claim 93, wherein the target cell is a non-cancerous cell.

116. 94. The composition of claim 93, wherein the target cell is a T cell of a T cell population.

117. 94. A pharmaceutical composition comprising the composition of claim 93 and a pharma- ceutically acceptable diluent, carrier, excipient, or stabilizer.

118. 120. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 117.

119. 1. A method for expanding T cell receptor beta variable region (TCRαV) expressing T cells in a T cell population, comprising: contacting the T cell population with a composition comprising an anti-TCRαV molecule or a multispecific molecule, the multispecific molecule comprising a first domain that binds to a first target molecule and a second domain that binds to a second target molecule; the first target molecule is TCRαV, and the second target molecule is a target molecule on a target cell that is different from the first target molecule; the first domain contacts a TCRαV of a T cell receptor (TCR) expressed by a T cell in the T cell population, thereby expanding the T cells in the T cell population.

120. 120. The method of claim 119, wherein the T cell population is an in vivo T cell population.

121. 120. The method of claim 119, wherein the second domain comprises a tumor targeting domain, a cytokine molecule, or a stromal modifying domain.

122. A multispecific molecule comprises at least two non-contiguous polypeptide chains, 120. The method of claim 119, wherein a first polypeptide chain of the at least two non-contiguous polypeptide chains comprises a first member of a dimerization module, and a second polypeptide chain of the at least two non-contiguous polypeptide chains comprises a second member of a dimerization module, and the first polypeptide chain and the second polypeptide chain form a complex via the first member of the dimerization module and the second member of the dimerization module.

123. the first polypeptide chain comprises a first domain and the second polypeptide chain comprises a second domain; (i) the first polypeptide chain comprises a first domain linked to a first member of the dimerization module, and the second polypeptide chain comprises a second domain linked to a second member of the dimerization module; (ii) the first polypeptide chain comprises a first portion of a first domain linked to a first member of a dimerization module and the second polypeptide chain comprises a first portion of a second domain linked to a second member of the dimerization module; the at least two non-contiguous polypeptide chains comprise a third polypeptide chain comprising a second portion of the first domain and a fourth polypeptide chain comprising a second portion of the second domain; (iii) the first polypeptide chain comprises a first portion of a first domain linked to a first member of a dimerization module and the second polypeptide chain comprises a second domain linked to a second member of a dimerization module; the at least two non-contiguous polypeptide chains comprise a third polypeptide chain comprising a second portion of the first domain; or (iv) the first polypeptide chain comprises a first domain linked to a first member of the dimerization module, the second polypeptide chain comprises a first portion of the second domain linked to a second member of the dimerization module; and the at least two non-contiguous polypeptide chains comprise a third polypeptide chain comprising a second portion of the second domain.

124. the multispecific molecule further comprises a linker between the first domain and the first member of the dimerization module, a linker between the second domain and the second member of the dimerization module, a linker between a first portion of the first domain and the first member of the dimerization module, a linker between a first portion of the second domain and the second member of the dimerization module, a linker between the first member of the dimerization module and the second domain, a linker between the first member of the dimerization module and the first portion of the second domain, or a combination thereof; 124. The method of claim 123, wherein the linker is selected from a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker.

125. The first polypeptide chain comprises a first domain and a second domain, the first polypeptide chain comprising: (i) a first domain linked to a first member of a dimerization module linked to a second domain; (ii) a first portion of a first domain linked to a first member of a dimerization module linked to a first portion of a second domain, wherein the at least two non-contiguous polypeptide chains comprise a third polypeptide chain comprising the second portion of the first domain and a fourth polypeptide chain comprising the second portion of the second domain; (iii) a first portion of a first domain linked to a first member of a dimerization module linked to a second domain, wherein the at least two non-contiguous polypeptide chains comprise a third polypeptide chain that comprises a second portion of the first domain; or (iv) a first domain linked to a first member of a dimerization module linked to a first portion of a second domain, wherein the at least two non-contiguous polypeptide chains include a third polypeptide chain comprising a second portion of the second domain.

123. The method of claim 122, comprising:

126. the multispecific molecule further comprises a linker between the first domain and the first member of the dimerization module, a linker between the second domain and the second member of the dimerization module, a linker between a first portion of the first domain and the first member of the dimerization module, a linker between a first portion of the second domain and the second member of the dimerization module, a linker between the first member of the dimerization module and the second domain, a linker between the first member of the dimerization module and the first portion of the second domain, or a combination thereof; 126. The method of claim 125, wherein the linker is selected from a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker.

127. The multispecific molecule is (i) a first domain linked to a second domain; (ii) a first portion of a first domain linked to a first portion of a second domain, wherein the polypeptide sequence further comprises a second portion of the first domain and a second portion of the second domain; (iii) a first portion of a first domain linked to a second domain, wherein the polypeptide sequence further comprises a second portion of the first domain; or (iv) a first domain linked to a first portion of a second domain, wherein the polypeptide sequence further comprises a second portion of the second domain.

120. The method of claim 119, comprising a polypeptide sequence comprising:

128. the polypeptide sequence further comprises a linker between the first domain and the second domain, a linker between a first portion of the first domain and a first portion of the second domain, a linker between the first portion of the first domain and the second domain, a linker between the first domain and the first portion of the second domain, or a combination thereof; 128. The method of claim 127, wherein the linker is selected from a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker.

129. The second target molecule is selected from the group consisting of BCMA, FcRH5, CD19, CD20, CD22, CD30, CD33, CD38, CD47, CD99, CD123, FcRH5, CLEC12, CD179A, SLAMF7, or NY-ESO1, PDL1, CD47, gangloside 2 (GD2), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), prostate specific antigen (PSA), carcinoembryonic antigen (CEA), Ron kinase, c-Met, immature Laminin receptor, TAG-72, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, Ep-CAM, EphA3, Her2 / neu, telomerase, SAP-1, survivin, NY-ESO-1 / LAGE-1, PRAME, SSX-2, Melan-A / MART-1, Gp100 / pmel17, tyrosinase, TRP-1 / -2, MC1R, b-catenin, BRCA1 / 2, CDK4, CML66, fibronectin , p53, Ras, TGF-B receptor, AFP, ETA, MAGE, MUC-1, CA-125, BAGE, GAGE, NY-ESO-1, b-catenin, CDK4, CDC27, a-actinin-4, TRP1 / gp75, TRP2, gp100, melan-A / MART1, ganglioside, WT1, EphA3, epidermal growth factor receptor (EGFR), MART-2, MART-1, MUC1, MUC2, MUM1, MUM2, MUM3, NA88- 1, NPM, OA1, OGT, RCC, RUI1, RUI2, SAGE, TRG, TRP1, TSTA, folate receptor alpha, L1-CAM, CAIX, gpA33, GD3, GM2, VEGFR, integrins, carbohydrates, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, TGF-beta, hyaluronic acid, collagen, tenascin C and tenascin W.

130. 120. The method of claim 119, wherein the second domain is a NK cell engager, a T cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager.

131. The method of claim 130, wherein the second domain is a T cell engager and the second target molecule is a TCRαV other than the TCRαV to which the first domain binds.

132. The method of claim 130, wherein the second target molecule is not TCRαV.

133. 131. The method of claim 130, wherein the second target molecule is CD19.

134. The method of claim 130, wherein the second target molecule is CD3.

135. The method of claim 130, wherein the second target molecule is CD123.

136. 122. The method of claim 121, wherein the second domain comprises a tumor targeting domain and the second target molecule is a cancer antigen.

137. 137. The method of claim 136, wherein the cancer antigen is a blood cancer antigen, a solid tumor antigen, a metastatic cancer antigen, a soft tissue tumor antigen, a cancer antigen of a metastatic lesion, or a stromal antigen.

138. Cancer antigens, (i) a solid tumor antigen, wherein the solid tumor is pancreatic cancer, breast cancer, colorectal cancer, lung cancer, skin cancer, ovarian cancer, or liver cancer; or (ii) A blood cancer antigen, wherein the blood cancer is a B cell malignancy or a T cell malignancy. The method of claim 137, wherein:

139. 137. The method of claim 136, wherein the cancer antigen is a blood cancer antigen and the B cell or T cell malignancy is Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia (AML), chronic myelogenous leukemia, myelodysplastic syndrome, multiple myeloma, or acute lymphocytic leukemia.

140. The method of claim 137, wherein the cancer antigen is a blood cancer antigen, the B cell malignancy is Hodgkin's lymphoma, and the non-Hodgkin's lymphoma is B cell lymphoma, diffuse large B cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, marginal zone B cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, or hairy cell leukemia.

141. 122. The method of claim 121, wherein the second domain comprises a cytokine molecule selected from the group consisting of interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), interferon gamma, and functional fragments or variants thereof.

142. The method of claim 119, wherein binding of the first domain to the TCRαV and binding of the second molecule to a target molecule promotes the T cell to kill a cancer cell.

143. 120. The method of claim 119, wherein the target cell is a T cell.

144. 120. The method of claim 119, wherein the target cell is a non-cancerous cell.

145. 120. The method of claim 119, wherein the T cells are expanded in vivo.

146. A composition comprising a recombinant T cell receptor or chimeric antigen receptor (CAR) that comprises a domain that binds to TCRαV.

147. A composition comprising a T cell comprising a recombinant T cell receptor or a chimeric antigen receptor (CAR) comprising a domain that binds to TCRαV.

148. CAR, (a) an extracellular domain comprising a domain that binds to TCRαV; (b) a transmembrane domain; and (c) an intracellular domain comprising an intracellular signaling domain 148. The composition of claim 146 or 147, comprising:

149. The composition of claim 148, wherein the extracellular domain comprises a CD8 or CD28 extracellular domain.

150. The composition of claim 148 or 149, wherein the transmembrane domain comprises a CD8 or CD28 transmembrane domain.

151. The composition of any one of claims 148 to 150, wherein the intracellular domain comprises a CD3 zeta intracellular signaling domain.

152. The domain binding to TCRαV is selected from the group consisting of TCRα V1 subfamily, TCRα V2 subfamily, TCRα V3 subfamily, TCRα V4, TCRα V5 subfamily, TCRα V6 subfamily, TCRα V7 subfamily, TCRα V8 subfamily, TCRα V9 subfamily, TCRα V10 subfamily, TCRα V12 subfamily, TCRα V13 subfamily, TCRα V14 subfamily, TCRα V16 subfamily, TCRα V17 subfamily, TCRα V18 subfamily, TCRα V19 subfamily, TCRα V20 subfamily, TCRα V21 subfamily, TCRα V22 subfamily, TCRα V23 subfamily, TCRα V24 subfamily, TCRα 152. The composition of any one of claims 148 to 151, which binds to one or more of the TCRαV subfamilies selected from the group consisting of the V25 subfamily, the TCRα V26 subfamily, the TCRα V27 subfamily, the TCRα V29 subfamily, the TCRα V30 subfamily, the TCRα V34 subfamily, the TCRα V35 subfamily, the TCRα V36 subfamily, the TCRα V38 subfamily, the TCRα V39 subfamily, the TCRα V40 subfamily, or the TCRα V41 subfamily, and family members of said subfamilies, and variants thereof.

153. 153. A pharmaceutical composition comprising the composition of any one of claims 148 to 152 and a pharma- ceutically acceptable diluent, carrier, excipient, or stabilizer.

154. 154. A method of treating a disease or condition in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 153.

155. 155. The method of claim 154, wherein the disease or condition is cancer.

156. (a) the cancer is breast cancer and the TCRαV-binding domain binds to the TCRα V1 subfamily; (b) the cancer is ER+ breast cancer and the TCRαV binding domain binds to the TCRα V1 subfamily; (c) the cancer is ER+ breast cancer and the TCRαV binding domain binds to the TCRα V3 subfamily; (d) the cancer is diffuse large B-cell lymphoma, and the TCRαV-binding domain binds to the TCRα V6 subfamily; (e) the cancer is diffuse large B-cell lymphoma, and the TCRαV-binding domain binds to the TCRα V8 subfamily; (f) the cancer is breast cancer and the TCRαV-binding domain binds to the TCRα V9 subfamily; (g) the cancer is diffuse large B-cell lymphoma, and the TCRαV-binding domain binds to the TCRα V12 subfamily; (h) the cancer is melanoma and the TCRαV-binding domain binds to the TCRα V12 subfamily; (i) the cancer is multiple myeloma and the TCRαV-binding domain binds to the TCRα V19 subfamily; (j) the cancer is diffuse large B-cell lymphoma, and the TCRαV-binding domain binds to the TCRα V21 subfamily; (k) the cancer is diffuse large B-cell lymphoma, and the TCRαV-binding domain binds to the TCRα V22 subfamily; (l) the cancer is diffuse large B-cell lymphoma, and the TCRαV-binding domain binds to the TCRα V25 subfamily; (m) the cancer is leukemia and the TCRαV-binding domain binds to the TCRα V29 subfamily; (n) the cancer is melanoma and the TCRαV-binding domain binds to the TCRα V29 subfamily; (o) the cancer is breast cancer and the TCRαV-binding domain binds to the TCRα V29 subfamily; (p) the cancer is endometrial cancer and the TCRαV-binding domain binds to the TCRα V30 subfamily; (q) the cancer is leukemia and the TCRαV-binding domain binds to the TCRα V38 subfamily; and / or (r) the cancer is esophageal squamous cell carcinoma, and the TCRαV-binding domain binds to the TCRα V39 subfamily. The method of claim 155.

157. 155. The method of claim 154, wherein the disease or condition is an autoimmune disease.

158. (a) the autoimmune disease is multiple sclerosis and the TCRαV-binding domain binds to the TCRα V1 subfamily; (b) the autoimmune disease is Crohn's disease and the TCRαV-binding domain binds to the TCRα V2 subfamily; (c) the autoimmune disease is Sjogren's syndrome and the TCRαV-binding domain binds to the TCRα V13 subfamily; (d) the autoimmune disease is celiac disease and the TCRαV-binding domain binds to the TCRα V20 subfamily; (e) the autoimmune disease is Crohn's disease and the TCRαV-binding domain binds to the TCRα V22 subfamily; (f) the autoimmune disease is celiac disease and the TCRαV-binding domain binds to the TCRα V26 subfamily; (g) the autoimmune disease is Crohn's disease, and the TCRαV-binding domain binds to the TCRα V40 subfamily; The method of claim 157.

159. 155. The method of claim 154, wherein the disease or condition is an infectious disease.

160. (a) the infection is a S. paratyphi infection and the TCRαV-binding domain binds to the TCRα V1 subfamily; (b) the infection is a Bacteroidetes bacterial infection and the TCRαV-binding domain binds to the TCRα V1 subfamily; (c) the infection is a Proteobacterial infection and the TCRαV-binding domain binds to the TCRα V1 subfamily; (d) the infection is a Mycobacterium tuberculosis infection and the TCRαV-binding domain binds to the TCRα V1 subfamily; (e) the infection is a respiratory viral infection and the TCRαV-binding domain binds to the TCRα V6 subfamily; (f) the infection is a SARS-CoV-2 infection and the TCRαV-binding domain binds to the TCRα V7 subfamily; (g) the infection is a SARS-CoV-2 infection and the TCRαV-binding domain binds to the TCRα V9 subfamily; (h) the infection is a SARS-CoV-2 infection and the TCRαV-binding domain binds to the TCRα V11 subfamily; (i) the infection is a S. pyogenes infection and the TCRαV-binding domain binds to the TCRα V12 subfamily; (j) the infection is a SARS-CoV-2 infection and the TCRαV-binding domain binds to the TCRα V12 subfamily; (k) the infectious disease is yellow fever and the TCRαV-binding domain binds to the TCRα V12 subfamily; (l) the infectious disease is influenza and the TCRαV-binding domain binds to the TCRα V13 subfamily; (m) the infection is a respiratory viral infection and the TCRαV-binding domain binds to the TCRα V16 subfamily; (n) the infection is an HIV infection and the TCRαV-binding domain binds to the TCRα V17 subfamily; (o) the infection is a cytomegalovirus infection and the TCRαV-binding domain binds to the TCRα V17 subfamily; (p) the infection is a SARS-CoV-2 infection and the TCRαV-binding domain binds to the TCRα V18 subfamily; (q) the infection is an HIV infection and the TCRαV-binding domain binds to the TCRα V24 subfamily; (r) the infectious disease is influenza and the TCRαV-binding domain binds to the TCRα V27 subfamily; and / or (s) the infection is an Epstein-Barr virus infection and the TCRαV-binding domain binds to the TCRα V38 subfamily. The method of claim 159.

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